KR102232319B1 - Composition for preventing or treating cerebrovascular diseases comprising a telomerase peptide as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cerebrovascular diseases comprising a telomerase peptide as an active ingredient. It was confirmed that the composition inhibited the death of brain cells due to insufficient blood and oxygen supply, increased cell proliferation and activity, and did not show toxicity in neural stem cells. Therefore, it has high stability in vivo and is expected to be very useful as a composition for preventing or treating cerebrovascular diseases.

Description

Composition for preventing or treating cerebrovascular diseases comprising a telomerase peptide as an active ingredient}

The present invention relates to a composition for preventing or treating cerebrovascular diseases comprising a telomerase peptide as an active ingredient.

According to data from the National Statistical Office, it is estimated that about 56.5 deaths per 100,000 cerebrovascular diseases per year, more than 60,000 stroke patients occur annually, and more than 200,000 patients are fighting the disease. Stroke is a disease with a high incidence in the elderly, but due to excessive stress from daily work and an unrestrained lifestyle such as alcohol, tobacco, lack of exercise, and meat-oriented diet, the incidence of stroke accounts for a quarter of all stroke patients. To this extent, the age of onset is spreading to the younger and is becoming a problem.

Such a stroke is a disease caused by a blockage or burst of cerebrovascular blood vessels, and is divided into two types: cerebral infarction and cerebral hemorrhage. Cerebral infarction is called an ischemic cerebrovascular disease, and it is a disease that occurs when the blood and oxygen supply to the brain is not smooth due to the blockage of the blood vessels in the brain. On the other hand, cerebral hemorrhage is called a hemorrhagic cerebrovascular disease, and it is a case of brain damage due to bleeding and accumulation of cerebrovascular blood vessels. The cerebral infarction and cerebral hemorrhage result in loss of brain function, resulting in hemiplegia, speech impairment, and consciousness disorder, and in severe cases, death.

The ischemic cerebrovascular disease is a disease that occurs when the blood flow to the brain is reduced and oxygen and glucose are not supplied to the brain cells, causing delayed neuronal death in the CAI region of the hippocampus. Kirino, Brain Res., 239, p57-69, 1982). In addition, ischemic brain disease is subdivided into thrombosis, embolism, transient ischemic attack, and lacume.

The molecular biological mechanism of ischemic injury such as stroke is that when the blood supply to a specific part of the brain parenchyma is blocked, the supply of oxygen and glucose is interrupted, making it impossible to supply sufficient ATP to maintain the ion channel in the cell membrane. acid) receptors, AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole-propinoic acid), and metabotropic glutamate receptors. Increase (Science, 244, p1360-1362, 1989). It is known that the influx of calcium into cells causes excessive release of glutamate and asphaltate to generate cytotoxic molecules including free radicals, destroying cellular structures such as DNA and cell membranes, thereby promoting neuronal cell death.

Therefore, development of an excellent therapeutic agent having a prophylactic or therapeutic effect of cerebrovascular disease to treat or prevent cerebrovascular disease is a very urgent situation.

Brain Res., 239, p57-69, 1982 Science, 244, p1360-1362, 1989

The present invention in order to solve the above problems, the present inventors to provide a composition for preventing or treating cerebrovascular disease.

An object according to an aspect of the present invention is to provide a composition for preventing or treating cerebrovascular disease, which is effective in preventing or treating cerebrovascular disease, and has excellent safety in which cytotoxicity does not appear.

In order to achieve the above object, in one aspect of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a composition comprising a peptide that is a fragment thereof Can be provided.

In the composition according to an aspect of the present invention, the fragment may be a fragment composed of three or more amino acids.

In the composition according to an aspect of the present invention, the composition may be for the prevention and treatment of cerebrovascular diseases.

According to another aspect of the present invention, a method for treating and preventing cerebrovascular disease, characterized in that administering the composition for preventing and treating cerebrovascular disease to a subject in need, may be provided.

It is effective in the prevention and treatment of cerebrovascular diseases comprising the telomerase peptide according to the present invention as an active ingredient, and has excellent safety without cytotoxicity, so it can be very useful in preventing or treating cerebrovascular diseases.

1 is a view showing a Western blot result confirming the expression level of HIF-1а, which is a hypoxic environmental marker, and is a result diagram confirming that the expression level of HIF-1а increases as the exposure time to the hypoxic environment increases.
FIG. 2A is a diagram illustrating a decrease in apoptosis through absorbance by treating a telomerase peptide with 1, 10, and 50 μM after exposure to a hypoxic environment for 8 hours.
Figure 2b is a diagram confirming the reduction of apoptosis through Annexin V staining and propidium iodide by treating the telomerase peptide with 1, 10, and 50 μM after exposure in a hypoxic environment for 8 hours.
Figure 2c is a view confirming the reduction of apoptosis through the Tunnel assay by treating the telomerase peptide with 1, 10, and 50 μM after exposure to a hypoxic environment for 8 hours.
Figure 2d is a diagram confirming the reduction of apoptosis by treating with 1, 10, and 50 μM of telomerase peptide after 8 hours of exposure in a hypoxic environment.
3 is a diagram confirming that cell proliferation is increased by treatment with 1, 10, and 50 μM of telomerase peptide after 8 hours of exposure in a hypoxic environment.
FIG. 4 is a diagram confirming that cell migration is increased by treatment with 1, 10, and 50 μM of telomerase peptide after 8 hours of exposure in a hypoxic environment.
5A is a diagram illustrating the protective effect of a telomerase peptide against oxidative damage caused by a hypoxic environment.
Figure 5b is a view confirming that the reduction of active oxygen by treating oxidative damage caused by a hypoxic environment with telomerase peptides 1, 10, and 50 μM.
6 is a diagram illustrating a decrease in apoptosis by exposing cortical neurons in a hypoxic environment for 8 hours and then treating telomerase peptides with 1, 10, and 50 μM.
7 is a view confirming that the oxidative damage of cortical neurons in a hypoxic environment is treated with telomerase 1, 10, and 50 μM to reduce free radicals.
8 is a diagram illustrating mitochondrial DNA damage by treating with 1, 10, and 50 μM of telomerase peptide after 8 hours exposure in a hypoxic environment.
FIG. 9 is a diagram illustrating a signal that demonstrates the neuroprotective effect in spots related to inflammatory response, TCA cycle, ATP cycle related signal, cell migration, GPCR, etc. and confirms the activation mechanism of cellular amyloid beta and telomerase peptide.
10 shows apoptosis, cell cycle, neurobiology, cytoskeleton through an antibody microarray by treating telomerase peptide with 1, 10, and 50 μM after 8 hours exposure in a hypoxic environment, This is a diagram confirming the signal transduction and nuclear protein.
11 is a Western blot result diagram confirming the expression level of a cerebrovascular disease-related protein. Representative of inflammatory reaction with GSK3β caspase-3, apoptosis signal, and increase in the expression levels of pAKT and HSTF-1, which are cell survival signals, according to the increase of telomerase peptide (μM) after exposure to hypoxic environment for 1 hour or 8 hours. A decrease in the expression level of the signal COX-2 was confirmed.
FIG. 12 is a diagram illustrating a location where an infarct volume is formed in a brain image and calculates an infarct volume.
13 is a result of a behavioral test conducted to check the symptoms of an ischemic animal model before administration of a telomerase peptide.

The present invention is a bar that can apply a variety of transformations and can have various embodiments, the present invention will be described in more detail below. However, this is not intended to limit the present invention to a specific embodiment, it should be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

Telomere (telomere) is a genetic material repetitively present at the end of a chromosome, and is known to prevent damage to the corresponding chromosome or binding to other chromosomes. Each time a cell divides, the length of the telomeres decreases little by little. If there are more than a certain number of cell divisions, the telomeres become very short, and the cells stop dividing and die. On the other hand, it is known that lengthening telomeres extends the lifespan of cells. For example, cancer cells secrete an enzyme called telomerase to prevent shortening of telomeres, so it is known that cancer cells can continue to proliferate without dying.

In one aspect of the present invention, the peptide of SEQ ID NO: 1, the peptide that is a fragment of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence is a telomerase, specifically a human ( Homo sapiens) telomerase. It includes derived peptides.

The peptides disclosed herein may include peptides having sequence homology of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. In addition, the peptide disclosed herein is a peptide comprising SEQ ID NO: 1 or fragments thereof and one or more amino acids, two or more amino acids, three or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids. Or it may include a peptide in which 7 or more amino acids have been changed.

In one aspect of the present invention, amino acid changes belong to properties that cause the physicochemical properties of the peptides to be altered. For example, amino acid changes such as improving the thermal stability of the peptide, changing the substrate specificity, changing the optimal pH, and the like can be performed.

The term "amino acid" as used herein includes the 22 standard amino acids that are naturally incorporated into a peptide, as well as D-isomers and modified amino acids. Accordingly, in one aspect of the present invention, the peptide may be a peptide containing D-amino acid. Meanwhile, in another aspect of the present invention, the peptide may include a non-standard amino acid that has been modified after translation (post-translational modification). Examples of post-translational modifications include phosphorylation, glycosylation, acylation (e.g., including acetylation, myristoylation and palmitoylation), alkylation. ), carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-removal deimidation) , Deamidation) and structural changes (eg, formation of disulfide bridges). It also includes changes in amino acids, such as changes in amino groups, carboxyl groups, or side chains, caused by chemical reactions occurring in the process of bonding with crosslinkers to form peptide conjugates.

The peptides disclosed herein may be wild-type peptides identified and isolated from native sources. Meanwhile, the peptide disclosed in the present specification may be an artificial variant comprising an amino acid sequence in which one or more amino acids are substituted, deleted, and/or inserted compared to the peptides of SEQ ID NO: 1. Amino acid changes in wild-type polypeptides as well as in artificial variants include conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein. Examples of conservative substitutions include basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). In general, amino acid substitutions that do not alter a specific activity are known in the art. The most common exchanges 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, and Asp/Gly, and vice versa. Other examples of conservative substitution are shown in the following table.

Original amino acids Exemplary residue substitution Preferred residue substitution Ala (A) val; leu; ile Val Arg (R) lys; gln; asn Lys Asn (N) gln; his; asp, lys; arg Gln Asp (D) glu; asn Glu Cys (C) ser; ala Ser Gln (Q) asn; glu Asn Glu (E) asp; gln Asp Gly (G) ala Ala His (H) asn; gln; lys; arg Arg Ile (I) leu; val; met; ala; phe; norleucine Leu Leu (L) norleucine; ile; val; met; ala; phe Ile Lys (K) arg; gln; asn Arg Met (M) leu; phe; ile Leu Phe (F) leu; val; ile; ala; tyr Tyr Pro (P) ala Ala Ser (S) thr Thr Thr (T) ser Ser Trp (W) tyr; phe Tyr Tyr (Y) trp; phe; thr; ser Phe Val (V) ile; leu; met; phe; ala; norleucine Leu

Substantial modifications in the biological properties of peptides include (a) their effect in maintaining the structure of the polypeptide backbone in the substitution region, e.g., a sheet or helical conformation, and (b) the charge of the molecule at the target site. Alternatively, their effect in maintaining hydrophobicity, or (c) their effect in maintaining the bulk of the side chain, is performed by selecting substituents which differ considerably. Natural residues are divided into the following groups based on their usual side chain properties:

(1) hydrophobicity: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilicity: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; And

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will be made by exchanging a member of one of these classes for another class. Any cysteine residue not related to maintaining the proper conformational structure of the peptide can generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine bond(s) can be added to the peptide to improve its stability.

Another type of amino acid variant of the peptide is a change in the glycosylation pattern of the antibody. The meaning of change refers to the deletion of one or more carbohydrate moieties found in the peptide and/or the addition of one or more glycosylation sites that are not present in the peptide.

Glycosylation of peptides is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of the asparagine moiety. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatically attaching carbohydrate residues to the asparagine side chain. Thus, the presence of one of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to attaching one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine You can also use

The addition of the glycosylation site to the peptide is conveniently carried out by changing the amino acid sequence to contain one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). These changes may also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

In addition, a peptide having the sequence of SEQ ID NO: 1 according to an aspect of the present invention, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence has low toxicity in cells and It has the advantage of high stability.

The peptides listed in SEQ ID NO: 1 are shown in Table 2 below. The "name" in Table 2 below is named to distinguish the peptide. In one aspect of the invention, the peptide set forth in SEQ ID NO: 1 represents the entire peptide of human telomerase. In another aspect of the present invention, a peptide having the sequence of SEQ ID NO: 1, a peptide that is a fragment of the sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the peptide sequence corresponds to the peptide included in telomerase It includes a "synthetic peptide" obtained by selecting and synthesizing the peptide at the position. SEQ ID NO: 2 shows the amino acid sequence of all telomerase.

Sequence number name Telomerase phase position order Length One pep1, GV1001 [611-626] EARPALLTSRLRFIPK 16 aa 2 [1-1132] MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLPVCRPGALFGLVGRLGALPRPGALFGLVGRAQLPRR
GAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGT TLTALEAAANPALPSDFKTILD ?? 1132 aa

In one aspect of the composition for preventing or treating cerebrovascular diseases according to an aspect of the present invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% or more with the amino acid sequence, or a fragment thereof The peptide may be included in an amount of 0.1 μg/mg to 1 mg/mg, specifically 1 μg/mg to 0.5 mg/mg, and more specifically 10 μg/mg to 0.1 mg/mg. When included in the above range, it is appropriate not only to exhibit the intended effect of the present invention, but also can satisfy both the stability and safety of the composition, and it may be appropriate to include it in the above range in terms of cost-effectiveness.

The composition according to an aspect of the present invention can be applied to all animals including humans, dogs, chickens, pigs, cows, sheep, guinea pigs or monkeys.

In one aspect of the present invention, the composition comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having 80% or more sequence homology with the amino acid sequence, or a telomerase-derived peptide as an active ingredient. It provides a composition for preventing or treating cerebrovascular disease.

The pharmaceutical composition according to an aspect of the present invention may be administered orally, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intramedullary, intrathecal or subcutaneous.

Formulations for oral administration may be tablets, pills, soft or hard capsules, granules, powders, liquids, or emulsions, but are not limited thereto. Formulations for parenteral administration may be injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays, but are not limited thereto.

In addition to the active ingredients, each formulation may be appropriately selected and blended by a person skilled in the art according to the formulation or purpose of use without difficulty, and synergistic effects may occur when applied simultaneously with other raw materials.

The determination of the dosage of the active ingredient is within the level of those skilled in the art, and the daily dosage thereof is, for example, 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, but is not limited thereto, and various factors such as age, health condition, and complications of the subject to be administered It can be different.

The terms used in this specification are intended only for the purpose of describing specific embodiments, but not for limiting the present invention. The term in which the number is omitted before the noun is not intended to limit the quantity, but rather to indicate the existence of one or more of the mentioned noun items. The terms “comprising”, “having”, and “including” are interpreted as open terms (ie, the meaning of “comprising”).

Remarks to a range of values are merely in lieu of referring individually to each separate value falling within that range, and unless otherwise stated, each numerical value is used herein as if it were individually recited in the specification. Apply. Limit values of all ranges are included within that range and can be combined independently.

All methods mentioned herein can be performed in any suitable order unless otherwise specified or clearly contradicted by context. The use of any one and all embodiments or illustrative language (eg, "such as") is only intended to facilitate the description of the present invention, unless included in the claims, but the scope of the present invention. I am not trying to limit it. No language in the specification should be construed as essential to the practice of the present invention for any unclaimed element. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the present invention belongs.

Preferred embodiments of the invention include the most optimal mode known to the inventor for carrying out the invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the preceding description. The inventors expect those skilled in the art to make appropriate use of such variations, and the inventors expect the invention to be practiced in a manner different from that described herein. Accordingly, the present invention includes equivalents and all variations of the subject matter of the invention mentioned in the appended claims, as permitted by the patent law. Moreover, within all possible variations, any combination of the above-mentioned elements is included in the present invention unless otherwise indicated herein or clearly contradicted by context. While the present invention has been specifically shown and described with reference to exemplary embodiments, those skilled in the art will appreciate that various changes may be made in form and detail without departing from the spirit and scope of the invention as defined by the following claims .

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples and experimental examples. However, the following examples and experimental examples are provided only for purposes of illustration to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

Example 1: Synthesis of Peptide

The peptide of SEQ ID NO: 1 (hereinafter referred to as "PEP 1") was prepared according to a known solid-phase peptide synthesis method. Specifically, the peptides were synthesized by coupling one amino acid from the C-terminus through the Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). As follows, it was used that the first amino acid of the C-terminus of the peptides was attached to the resin. For example:

NH ₂ -Lys(Boc)-2-chloro-Trityl Resin

NH ₂ -Ala-2-chloro-Trityl Resin

NH ₂ -Arg(Pbf)-2-chloro-Trityl Resin

All amino acid raw materials used for peptide synthesis are Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6, where the N-term is protected by Fmoc and all residues are removed from the acid). 7-pentamethyl dihydro-benzofuran-5-sulfonyl) was used. For example:

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-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt [N-Hydroxxybenzotriazole] /NMM [4-Methylmorpholine] is used as the coupling reagent. I did. Fmoc removal was performed using piperidine in DMF of 20%. Cleavage Cocktail [TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / H ₂ O=92.5/2.5/2.5/2.5] is used to separate the synthesized peptide from the resin and remove the protecting group from the residue. I did.

Each peptide was synthesized by reacting each of the amino acids to the starting amino acid to which the amino acid protecting group was bound to the solid support, washing with a solvent, and repeating the process of deprotection. The synthesized peptide was cleaved from the resin and purified by HPLC, and the synthesis was confirmed by MS and freeze-dried.

As a result of high performance liquid chromatography for the peptides used in this example, the purity of all peptides was 95% or higher.

A detailed process for manufacturing PEP 1 will be described as follows.

1) coupling

After _{dissolving amino acid protected in NH 2} -Lys(Boc)-2-chloro-Trityl Resin (8 equivalents) and coupling reagent HBTU (8 equivalents)/HOBt (8 equivalents)/NMM (16 equivalents) in DMF , Reacted at room temperature for 2 hours, and washed in the order of DMF, MeOH, and DMF.

2) Fmoc deprotection

20% of piperidine in DMF was added and reacted twice for 5 minutes at room temperature, followed by washing in the order of DMF, MeOH, and DMF.

_{3) Peptide basic skeleton NH 2} -E(OtBu)-AR(Pbf)-PALLT(tBu)-S(tBu)-R(Pbf)LR(Pbf)-FIPK(Boc) by repeating the reaction of 1 and 2 -2-chloro-Trityl Resin).

4) Cleavage: The peptide was separated from the resin by adding a cleavage cocktail to the synthesized peptide resin.

5) After cooling diethyl ether was added to the obtained mixture, the peptide obtained by centrifugation was precipitated.

6) After purification by Prep-HPLC, the molecular weight was confirmed by LC/MS and frozen to prepare a powder form.

Example 2: Culture of neural stem cells and cortical neurons

Neural stem cells are treated with basic fibroblast growth factor (bFGF) (R&D Systems, Minneapolis, MN) for a week after separating the cerebral cortex from the head of a rat embryo on the 13th day of pregnancy. Stem cells were cultured. Cortical neurons were isolated from the cerebral cortex of rat (spraguee.Dawley, Orient Bio, Kyungki, Korea) embryos on the 16th day of pregnancy and cultured in complete PMEM medium. Two days after culture, non-neuronal cells were isolated by treatment with 5LM cytosine arabinoside for 24 hours. Only mature culture cultured for 7 days was used in the experiment, and the population of neurons obtained from the cerebral cortex was approximately 79.7%.

Example 3: Analysis of neurotoxicity by hypoxia and OGD (Oxygen Glucose Deprivation) environment and protective effect of neural stem cells and cortical neurons by treatment with telomerase peptide

To create an in-vitro hypoxic environment that can confirm the cytotoxicity of neural stem cells due to a hypoxic environment, 5 mol% CO _{2 and} 0.2 mol% O ₂ and 94.8 mol% N ₂ are used. A hypoxic chamber was constructed, and neural stem cells were cultured in the hypoxic chamber for 0 to 24 hours to confirm cell viability. Whether or not the hypoxic chamber is well formed in a hypoxic environment was confirmed by Western blot using HIF-1а, a marker of the hypoxic environment. The hypoxic environment was confirmed from the fact that the expression level of HIF-1а increased as the time for the cells to be cultured in the chamber increased (see FIG. 1). As a result, it was confirmed that cell death, which was increased by hypoxic environment, was effectively reduced by the telomerase peptide, and the effect was shown from the concentration of 1 μM of the telomerase peptide.

After that, in order to check the neurotoxicity of neural stem cells due to the hypoxic environment, various concentrations of GV1001 were treated in a hypoxic chamber for 8 hours, and the degree of hypoxic neurotoxicity was determined by LDH assay, DAPI staining assay and It was evaluated by TUNEL staining assay, BrdU assay, and cell migration assay analysis.

Example 3-1. TBS (trypan Blue Staining) staining and LDH (Lactate Dehydrogenase Release) assay

The survival rate of neural stem cells was confirmed by TBS staining assay. 10 µl neural stem cells were incubated in 10 µl TBS solution for 2 minutes. The number of non-stained living cells was counted using a haemocytometer, and the amount of LDH was measured using a colorimetric assay kit (Roche Boehringer-mannheim, In, USA) (see FIG. 2A). . Cell activity was performed at 490 nm using an ELISA reader, and the comparative wavelength was normalized using 690 nm.

Example 3-2. Conducting BrdU Assay

The proliferation rate of neural stem cells was confirmed by BRdU assay. After culturing neural stem cells in 10 μM BrdU medium for 2 hours, the cell proliferation rate was measured using a BrdU Labeling and Detection kit (Roche Boehringer-Mannheim, In. USA) (see FIG. 3). Cell proliferation was performed at 370 nm using an ELISA reader, and the comparative wavelength was normalized using 492 nm. It was confirmed that the cell proliferation rate, which was reduced by 40-50% due to the hypoxic environment, decreased and increased in a concentration-dependent manner in the group pretreated with GV1001.

Example 3-3. Cell migration assay

The mobility of neural stem cells was confirmed by a cell migration assay. After dispensing neural stem cells into the upper chamber and incubating them in a low oxygen environment for 8 hours, neural stem cells that migrated through the cell membrane were measured using a QCM 24-well colorimetric assay kit (Chemicon, Temecula, CA). It was confirmed that the cell migration reduced by the low oxygen environment was increased by the GV1001 treatment (see FIG. 4).

Example 3-4. Annexin V staining and propidium iodide performed

Annexin V and propidium iodide were measured using the FITC Annexin V Apoptosis Detection Kit (Becton Dickinson, Franklin Lakes, NJ, USA) (see Fig. 2b). Cortical neurons were exposed to Oxygen Glucose Deprivation and treated with various concentrations of GV1001 (1, 10, 50mM) for 8 hours. After washing the cells, they were resuspended in 1x binding buffer solution, and then 5 mL FITC Annexin V and 10 mL propidium iodide were treated. After incubation for 15 minutes, the samples were analyzed using flow cytometry (Accuri C6 Flow cytometer, BD Biosciences).

Example 3-4. Free Radical Generation Measurement

To measure free radical generation, neural stem cells were exposed to a hypoxic environment for 30 minutes, incubated with a fluorescent probe 2,7'-dichlorodihydrofluorescein diacetate (DCHF_DA), and treated with various concentrations of GV1001. The accumulation amount of DCF in cells was performed at 90 nm using an ELISA reader (Synergy H1 Hybrid reader, BioTek Instruments, Winnoski, USA), and the comparative wavelength was standardized using 490 nm. It was confirmed that active oxygen was evidenced by the hypoxic environment, and increased active oxygen was decreased in the group treated with GV1001 (see FIGS. 5A and 5B).

Example 3-5. Oxidative mitochondrial DNA damage assay

Oxidative mitochondrial DNA damage was measured using the OxiSelect Oxidative DNA Damage ELISA kit. After mitochondrial DNA was extracted using the Purelink Genomic DNA Mini Kit (Invitrogen), anti-OHdH monoclonal antibody and horseradish peroxidase (HRP)-conjugated secondary antibody were treated. The treated samples were quantified by default using authentic 8?OHdG (Cell Biotics), and then mitochondrial DNA damage was measured at 450 nm using an ELISA plate reader (Synergy H1, BioTek Instruments) (see FIG. 8).

Example 3-6. Conducted mitochondrial membrane potential assay

^{5x10 5} /mL per well in a 96 well plate After dispensing neural stem cells so that they were treated with various concentrations of GV1001 (1, 10, 50mM) in a hypoxic environment, they were cultured for 8 hours. Mitochondrial membrane potential was measured using a JC-1 mitochondrial membrane potential assay kit (Abnova) and analyzed using an ELISA plate reader (Synergy H1 Hybrid reader, BioTek Instruments). Healthy cells aggregated with JC-1 J appeared as rhodamine (excitation/emsission = 540/570 nm) or texas red in the fluorescence setting. Conversely, dead or unhealthy cells appeared in the form of JC-1 monomers at FITC setting 485/535nm.

Example 3-7. Conducting Immunocytochemistry

To confirm the expression of neural stem cells in the cell, 1x10 After dispensing neural stem cells into chamber wells, they were exposed to 20 mM Oxygen Glucose Deprivation environment, and then treated with different concentrations of GV1001 (1, 10, 50 mM) for 8 hours. Each of the cells was fixed, permeated, and inhibited, and then incubated for 60 minutes. After incubation, 2% normal serum and mouse anti-nestin (1:100; Abcam), rabbit anti-Ki67 (1:100; Abcam), rabbit antidoublecortin (1 mg/mL; Abcam), rabbit anti-glial fibrillary acidic protein ( Primary antibodies such as GFAP) (1:5000; Abcam), rabbit anti-Tuj1 (1 mg/mL; Abcam), and mouse anti-GalC (1 mg/mL; Millipore) were treated and incubated for 24 hours. The next day, secondary antibodies such as goat-anti-rabbit Alexa Fluor 488 (Molecular Probes), anti-rabbit tetramethylrhodamine (Molecular Probes), and goat-anti-mouse Alexa Fluor 488 (Molecular Probes) were treated and incubated for 60 minutes. After washing the cells, after fixing the cells on the slide with a mounting medium, the cells were observed using an Olympus fluorescence microscope or a confocal laser microscope (Leica).

Example 4. Amyloid beta and Hypoxic Neurotoxicity About Telomerase Peptide neuroprotective effect mechanism PI3K Analysis of the impact on the route

Phosphatidylinositol-3-kinase (PI3K)/AKT-induced effect of telomerase peptide was confirmed. The PI3K pathway is activated by various growth factors and regulators and is involved in the normal regulation of neuronal growth and survival. The AKT signaling pathway inactivates several proapoptotic factors and inhibits the activity of GSK3β, a well-known representative apoptosis signal. Therefore, in this experiment, phosphorylated Akt (pAkt) (Ser473), phosphorylated glycogen synthase kinase-3β (GSK-3β) (Ser9), and B cell lymphoma to confirm the effect of hypoxic environment and telomerase peptide on the PI3K pathway. The expression levels of -2 (Bcl-2) and cleaved caspase-3 (Asp175) were confirmed by proteomics and western blot experiments (see FIGS. 9 and 11).

Example 4-1. Proteomics

Experimental Example 4-1-1. Protein sample preparation

Cultured neural stem cells were washed with PBS and then digested solution (7M urea, 2M Thiourea containing 4%(w/v) 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate(CHAPS), 1%(w/v) After treatment with dithiothreitol (DTT), 2% (v/v) pharmalyte, and 1mM benzamidine), ultrasonic treatment was performed using Sonoplus (Brandelin electronic, Germany) for 10 seconds. After protein extraction, centrifugation was performed at 15,000 xg for 1 hour. The dissolved samples were subjected to 2D gel electrophoresis, and the protein concentration was carried out by Bradford.

Experimental Example 4-1-2. 2D polyacrylamide gel electrophoresis (PAGE)

IPG dry strips (4-10 NL IPG, 24cm, Genomine, Korea) with 7M urea, 2M thiourea containing 2% 3-[(3-cholamidopropy)dimethyammonio]-1-propanesulfonate(CHAPS), 1% dithiothreitol (DTT), and 1% pharmalyte for 12 to 16 hours and then treated with 200 ug of sample. Isoelectric focusing was performed using a Multiphor ll electrophoresis unit and an EPS 3500 XL power supply (Amersham Bioscence). Before performing the second dimention, the strips were incubated with equilibrium buffer (50mM Tris-Cl, pH6.8 containing 6M urea, 2% SDS and 30% glycerol) for 10 minutes and normalized to 1% DTT and 2.5% iodoacetamide. The standardized strip was inserted into an SDS-PAGE gel (20 x 24cm, 10-16%), and then gel electrophoresis was performed using a Hoefer DALT 2D system (Amersham Biosciences).

Experimental Example 4-1-3. Image analysis

Quantitative analysis of digitized images was performed using PDQuest (version 7.0, BioRad) software. The quantification of each spot was standardized using total valid spot intensity, and only spots in which the expression pattern and the expression difference of the control group deviate more than two times were selected (see FIG. 9).

Example 4-2. Peptide Mass Fingerprinting

To identify the protein, peptide mass fingerprinting was used. Protein spots were dissected and digested with trypsin (Promega, Madison, WI), mixed with 50% acetonitrile/0.1% TFA alpha cyano-4-hydroxycinnamic acid, as described in Fernandez J et al. Likewise, MALDI-TOF analysis (Microflex LRF 20, BrukerDaltonics) was performed. Spectra were collected at 300 shots per spectrum, and were calibrated by two point internal calibration using Trypsin auto-digestion peak (m/z 842.5099, 2211.1046). A peak list was created using Flex Analysis 3.0, and protein identification was performed using the MASCOT (Matrixscience) peptide mass fingerprinting method.

Example 4-3. Western Blot implementation

Ki67, p85α phosphatidylinositol 3-kinase (PI3K), phosphorylated Akt (pAkt) (Ser473), phosphorylated glycogen synthase kinase-3β (pGSK-3β) (Ser9), COX-2, cytosolic cytochrome c and cleaved caspase-3 (Asp175) The level of was analyzed by Western blot (see Fig. 11). After washing 5x10 ⁶ cells with PBS solution for 30 minutes, lysis buffer buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.2% SDS, 100 μg/ml phenyl methyl sulfonyl fluoride (PMSF) ), 50 μl/ml aprotinin, 1% Igepal 630, 100 mM NaF, 0.5% sodium deoxy choate, 0.5 mM EDTA, 0.1 mM EGTA]. Cell nuclei and unbroken cells were separated by centrifugation. Mitochondria and cytoplasm were isolated using a Mitochondria/Cytosol Fraction Kit (Abcam, UK) for cytosolic cytochrome C level analysis. Neural stem cells cultured in Oxygen Glucose Deprivation environmental conditions after 8 hours of treatment with various concentrations of GV1001 were washed with cold PBS solution, and then 1.0 mL of 1 x cytoplasmic extraction buffer mixed solution containing dithiothreitol (DTT) and protease inhibitors. Was resuspended in. After incubation on ice for 10 minutes, the cell suspension was homogenized 30 to 50 times and centrifuged at 4° C. for 10 minutes at 3,000 rpm. The supernatant was centrifuged at 13,000 rpm for 30 minutes to separate mitochondrial nuclei and cytoplasmic supernatants. The mitochondrial pellet was washed once with an isolation buffer solution, and then dissolved in a mitochondrial extraction buffer containing DTT and a protease inhibitor. Samples containing the same amount (30 μg) of protein were digested using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). Cell membranes were blocked with 5% skim milk and anti-Ki67 (1:200, Abcam, UK), anti-p85α PI3K (1:1000, Millipore), anti-pAkt (1:500, Cell Signaling, Beverly, MA, USA) ), anti-pGSK-3β (Ser9) (1:500, Cell Signaling, Beverly, MA, USA), anti-cytochrome c (1:200, Cell Signaling), and anti-cleaved caspase-3 (Asp 175) ( 1:1000, Cell signaling, Beverly, MA, USA). Nitrocellulose membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBST), treated with HRP-conjugated rabbit antibody or anti-mouse antibody (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and then ECL detection ( Amersham Pharmacia Biotech). The blot was quantified using an image analyzer (GE Healthcare Image Quant LAS 4000).

Example 4-4. Antibody microarray

Antibody microarrays were performed using the Panorama Antibody Microarray Signaling Kit (Sigma-Aldrich) (see Fig. 10). 1.5x10 ⁷ cells were dispensed into a 100 mm culture dish, treated with 1 μM of GV1001, and cultured for 8 hours under Oxygen Glucose Deprivation conditions. After collecting the cultured cells, labeling with Cy3 and Cy5, the antibody microarray (Sigma-Aldrich) was analyzed. The array slide was skinned using a GenePix Personal 4100A scanner (Molecular Devices), and data analysis was performed using GenePix Pro 5.0 (Molecular Devices).

Example 5: Analysis of neuronal protective effect by treatment with telomerase peptide in ischemic animal model

Example 5-1. Ischemic injury animal model production

A 9-week-old male SD rat (Orient Bio, Korea) was anesthetized with isofluran using FORANE. In the gusseted rat, the hair was removed from the neck and the epidermis was cut. The salivary glands and muscles were lifted and the common carotid artery (CCA) was fixed using Silk. External carotid artery (ECA) was also tied with silk to block the artery, and then internal carotid artery (ICA) was fixed with silk. After making a hole between the ECA and the ICA, 2cm coated nylon was added and the ICA was ligation. The time the nylon was inserted was recorded as the occlusion time. Group 1 was administered with a control group, group 2 with 6um/ml, group 3 with 30uM/ml, and group 4 with 60uM/ml. In order to check the symptoms before administration of GV1001, a neurobehavioral function test was performed, and the model without symptoms after Stroke Surgery was excluded from each group (see FIG. 13).

Example 5-2. Conduct TTC staining

After 24 hours of surgery, the rat was sacrificed and the brain was isolated. The separated rat brain was placed on a brain mold, cut at 2 mm intervals, and immersed in 2% TTC solution for about 20 minutes. After 20 minutes, it was replaced with a 3% formaldehyde solution. The six brain slices obtained as described above were imaged using a scanner. One brain infarct volume was calculated by measuring the location where the infarct volume was formed in the brain image using the Image J program (see FIG. 12).

<110> KAEL-GEMVAX CO., LTD. KIM, Sangjae <120> Composition for preventing or treating cerebrovascular diseases comprising a telomerase peptide as an active ingredient <130> 14p261/ind <160> 2 <170> PatentIn version 3.2 <210> 1 <211> 16 <212> PRT <213> Homo sapiens <400> 1 Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys 1 5 10 15 <210> 2 <211> 1132 <212> PRT <213> Homo sapiens <400> 2 Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser 1 5 10 15 His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly 20 25 30 Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg 35 40 45 Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro 50 55 60 Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu 65 70 75 80 Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val 85 90 95 Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro 100 105 110 Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr 115 120 125 Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val 130 135 140 Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val 145 150 155 160 Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr 165 170 175 Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly 180 185 190 Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg 195 200 205 Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg 210 215 220 Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg 225 230 235 240 Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp 245 250 255 Ala His Pro Gly Arg Thr Arg Gly Pro Ser Asp Arg Gly Phe Cys Val 260 265 270 Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala 275 280 285 Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His 290 295 300 Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro 305 310 315 320 Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly 325 330 335 Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro 340 345 350 Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser 355 360 365 Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln 370 375 380 Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His 385 390 395 400 Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg 405 410 415 Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln 420 425 430 Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu 435 440 445 Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe 450 455 460 Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser 465 470 475 480 Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser 485 490 495 Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met 500 505 510 Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys 515 520 525 Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe 530 535 540 Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe 545 550 555 560 Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr 565 570 575 Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His 580 585 590 Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln 595 600 605 His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile 610 615 620 Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val 625 630 635 640 Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser 645 650 655 Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg 660 665 670 Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg 675 680 685 Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro 690 695 700 Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile 705 710 715 720 Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln 725 730 735 Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His 740 745 750 Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp 755 760 765 Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser 770 775 780 Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu 785 790 795 800 Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His 805 810 815 Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro 820 825 830 Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp 835 840 845 Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu 850 855 860 Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala 865 870 875 880 Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys 885 890 895 Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu 900 905 910 Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe 915 920 925 Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser 930 935 940 Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe 945 950 955 960 Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly 965 970 975 Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn 980 985 990 Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln 995 1000 1005 Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln 1010 1015 1020 Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala 1025 1030 1035 1040 Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu 1045 1050 1055 Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp 1060 1065 1070 Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr 1075 1080 1085 Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser 1090 1095 1100 Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn 1105 1110 1115 1120 Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp 1125 1130

Claims (6)

A composition for preventing or treating cerebral hemorrhage comprising a telomerase peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. The method of claim 1,
The telomerase peptide is a composition for preventing or treating cerebral hemorrhage, characterized in that it regulates the expression of Ki67, pAKT, HSTF-1 or phopho-GSK-3 β protein. The method of claim 1,
The content of the telomerase peptide is 0.1 μg/ml to 1 mg/ml of a composition for preventing or treating cerebral hemorrhage. The method of claim 1,
In addition, a composition for preventing or treating cerebral hemorrhage comprising at least one selected from a pharmaceutically acceptable carrier, diluent or excipient. delete delete

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