KR20160140147A - Compositions and methods for treating and preventing injury by reperfusion after ischemia in heart - Google Patents

Abstract

The present invention relates to a composition for preventing and treating cardiac disorder caused by injury of reperfusion after ischemia in a heart and, more specifically, to a composition for preventing and treating cardiac disorder caused by injury of reperfusion after ischemia in a heart, which is effective in preventing and treating cardiac disorder caused by injury of reperfusion after ischemia in a heart by comprising peptide derived from telomerase. The peptide shows an effect of reducing levels of inflammation of cardiac disorder caused by injury of reperfusion after ischemia in a heart, thereby providing a method for preventing and treating cardiac disorder caused by injury of reperfusion after ischemia in a heart.

Description

Technical Field [0001] The present invention relates to a composition for treating and preventing heart disease caused by reperfusion injury after cardiac ischemia,

The present invention relates to a peptide having an effect of preventing and treating cardiovascular diseases caused by reperfusion injury after cardiac ischemia, and a composition containing the same, and more specifically, it relates to a peptide comprising a telomerase-derived peptide, The present invention relates to a composition for preventing and treating heart disease caused by reperfusion injury after cardiac ischemia, which is effective for treating cardiac tissue damage including myocardial infarction through antiinflammatory activity.

Ischemic injury is a disorder that occurs when blood circulation is blocked (heart, cerebral infarction, nerve color) in organs requiring blood supply such as heart, brain, kidney, etc., Damage can be said. Ischemic injury causes lethal complications in the heart, brain, and kidneys, and slows the recovery of the transplant organ, increases acute rejection and decreases survival of the transplant organ in the long term.

Substantial reduction of oxygen delivery due to ischemia leads to conditions known as hypoxia. Persistent ischemia and hypoxia can result in loss of function of the tissue and even death. There are a number of conditions that cause ischemia and hypoxia, including, but not limited to, obstructive vascular disease, coronary artery thrombosis, cerebrovascular thrombosis, ruptured aneurysm, systemic bleeding, crush injury, sepsis, (Eg, spinal cord ischemia in the course of a thoracic aneurysm), cardiopulmonary bypass, organ transplantation, cardiopulmonary resuscitation (acute cardiac arrest), and asphyxia.

Conventional treatment for ischemia and thereby hypoxia is to restore blood flow and oxygen delivery to normal levels either by increasing the systemic oxygen supply or by eliminating the cause of vascular occlusion. Compared with a situation where ischemia or hypoxia is maintained for a long time, restoring blood flow can provide improved results. However, there is a problem that restoration of blood flow and oxygen transfer may result in additional cell death or loss of function, irrespective of the damage caused by ischemia or hypoxia.

Additional damage caused by restoration of blood flow and oxygen transfer is known as reperfusion injury. Paradoxical tissue damage caused by reperfusion injury appears to be similar to acute inflammatory conditions resulting from adherence of inflammatory cells to reperfused tissue, activation of these inflammatory cells, and subsequent formation of free radicals [Granger et al. Ann. Rev. Physiol., 57, 311-332, (1995)]. Generation of free radicals and other cytotoxic biomolecules in reperfused tissue may lead to cell death by activation of necrosis or apoptotic pathways.

Cardiac arrest and myocardial protection, which are essential for cardiac surgery, can cause damage to the myocardium during reperfusion. In order to prevent such reperfusion injury, there is a method of controlling cardiac fluid perfusion pressure, containing calcium, adenosine, or oxygen in the perfusion solution, and free radical scavenger There is disagreement.

WO 2012148200 A2

Under these circumstances, the present inventors have made intensive efforts to develop a peptide composition for prevention and treatment of heart disease caused by reperfusion injury after cardiac ischemia with little or no side effects and completed the present invention.

It is an object of the present invention to provide a composition for the prevention and treatment of heart disease caused by reperfusion injury after cardiac ischemia, which is effective and has no side effects.

According to an aspect of the present invention, there is provided a method for producing 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 peptide thereof, There is provided a composition for preventing and treating heart disease caused by reperfusion injury.

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

In the composition according to the present invention, the reperfusion injury after cardiac ischemia may be due to cardiac surgery or heart disease, including one or more of heart extraction, heart attack, cardiac arrest, and heart transplantation.

In the composition according to the present invention, the cardiac disease caused by the post-ischemic reperfusion may be selected from the group consisting of tachyarrhythmia or myocardial infarction, tachyarrhythmia, arrhythmia, heart disease event, and cardiac death.

In the composition according to the present invention, the composition may be a pharmaceutical composition.

According to another aspect of the present invention, there is provided a method for preventing and treating heart disease caused by reperfusion injury after cardiac ischemia, comprising administering a pharmaceutically effective amount of the composition to a subject suffering from heart disease caused by reperfusion injury after ischemia Can be provided.

In the method for the prevention and treatment of heart disease caused by cardiac ischemic reperfusion injury according to the present invention, administration of the composition may be performed by administering a concentration of the peptide of 0.1 mg / kg or more based on the weight of the individual.

In the method for the prevention and treatment of heart disease caused by cardiac ischemic reperfusion injury according to the present invention, administration of the composition may be performed by administering the concentration of the peptide based on the weight of the individual at a concentration of 0.1 mg / kg to 10 mg / kg .

According to another aspect of the present invention, there is provided 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 peptide thereof, And applications for use in the manufacture of pharmaceutical compositions for therapeutic purposes.

The peptide having the sequence of SEQ ID NO: 1 or the peptide having the sequence having the homology of 80% with the above sequence according to the present invention has an anti-inflammatory effect against cardiac ischemia reperfusion injury, It is expected to provide prevention or treatment of heart disease due to damage.

FIG. 1 is a graph showing the results of the administration of a control group (physiological saline, normal saline) and a peptide according to the present invention (hereinafter referred to as PEP1) at a concentration of 0.01, 0.01, 0.1, 1, 5 and 10 mg / kg . The H & E staining was performed to compare the degree of hemorrhage in the heart of the experimental animal model group.
FIG. 2 is a photograph of H & E staining performed to compare the degree of inflammation among the hearts of the experimental animal model group divided into the control group and the administration group.
FIG. 3 is a photograph of TUNEL staining to compare the degree of apoptosis of the hearts of experimental animal models divided into the control group and the administration group.
FIG. 4 is a graph showing quantitative results of TUNEL staining for comparing the degree of apoptosis.
FIG. 5 is a graph showing numerical values obtained by performing MPO (Myeloperoxidase) analysis in order to quantify the degree of inflammation in heart tissue by extracting the heart of an experimental animal model group divided into the control group and the administration group.
FIG. 6 is a graph showing the expression levels of the inflammatory cytokine TNF-.alpha. In cardiac tissues by extracting the heart of the experimental animal model group divided into the control group and the administration group.
FIG. 7 is a graph showing the expression levels of IL-6, an inflammatory cytokine in cardiac tissue, in the heart of experimental animal model groups divided into the control group and the administration group.

The present invention can be variously modified and can have various embodiments, and the present invention will be described in more detail as follows. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Telomere is a genetic material that is repeatedly present at the end of a chromosome, and is known to prevent damage to the chromosome or its binding to other chromosomes. As the cell divides, the length of the telomere is getting shorter. When there is more than a certain number of cell divisions, the telomere becomes very short, and the cell stops dividing and dies. On the other hand, it is known that lengthening the telomeres prolongs the life of the cells. For example, it is known that the cancer cells secrete an enzyme called telomerase and prevent the shortening of the telomeres. The present inventors confirmed that a peptide derived from telomerase is effective for preventing and treating erectile dysfunction and completed the present invention.

In one aspect of the present invention, the peptide of SEQ ID NO: 1, the peptide of SEQ ID NO: 1, or the peptide having 80% or more of the homology with the peptide sequence is used as a telomerase, specifically in Homo sapiens telomerase Derived peptides.

The peptides disclosed herein may comprise peptides having greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99% sequence homology. Also, the peptide disclosed in the present specification can be prepared by reacting a peptide or a fragment thereof comprising SEQ ID NO: 1 with one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, Or 7 or more amino acid altered peptides.

In one aspect of the invention, the amino acid change is of a property that causes the physicochemical properties of the peptide to change. For example, amino acid changes such as improving the thermal stability of the peptide, altering the substrate specificity, changing the optimum pH, etc. can be performed.

As used herein, the term "amino acid" includes D-isomers and modified amino acids as well as the 22 standard amino acids that are naturally incorporated into the peptide. Accordingly, in one aspect of the present invention, the peptide may be a peptide comprising a D-amino acid. In another aspect of the present invention, the peptide may include post-translationally modified non-standard amino acids and the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, for example, acetylation, myristoylation and palmitoylation), alkylation ), Carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (such as, for example, beta-depleted deamidation , Deamidation) and structural changes (e.g., formation of a disulfide bridge). Also included are amino acid changes such as changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that take place during binding with crosslinkers to form peptide conjugates.

The peptides disclosed herein may be wild type peptides identified and isolated from natural sources. Alternatively, the peptides disclosed herein may be artificial variants, including amino acid sequences in which one or more amino acids are substituted, deleted and / or inserted as compared to peptides that are fragments 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 folding and / or activity of the protein. Examples of conservative substitutions include, but are not limited to, 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) Tryptophan and tyrosine), and small amino acids (glycine, alanine, serine and threonine). In general, amino acid substitutions that do not alter specific activity are known in the art. The most commonly occurring interactions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly, and vice versa. Other examples of conservative substitutions are shown in the following table.

Original amino acid Exemplary residue replacement Preferred residue substitution Ala (A) val; leu; with Val Arg (R) lys; gln; asn Lys Asn (N) gln; feeling; 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; with; val; met; ala; phe Ile Lys (K) arg; gln; asn Arg Met (M) leu; phe; with Leu Phe (F) leu; val; with; 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) with; leu; met; phe; ala; norleucine Leu

Substantial modifications in the biological properties of the peptide include (a) the structure of the polypeptide backbone within the substitution region, e.g., their effect in maintaining the sheet or helical conformation, (b) the charge of the molecule at the target site Or their effect in maintaining hydrophobicity, or (c) their effect in maintaining the bulk of the side chain is significantly different. The natural residues are grouped into the following groups based on their usual side chain properties:

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

 (2) Neutral hydrophilic: cys, ser, thr;

 (3) Acid: asp, glu;

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

 (5) Residues affecting chain orientation: gly, pro; And

 (6) Aromatic: trp, tyr, phe.

Non-conservative substitutions will be made by exchanging one member of these members for another. Any cysteine residue not associated with maintaining the proper stereostructure of the peptide can generally be replaced with a serine to enhance the oxidative stability of the molecule and prevent strange cross-linking. Conversely, cysteine bond (s) can be added to the peptide to improve its stability

Other types of amino acid variants of peptides are those in which the glycosylation pattern of the antibody is altered. The term 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 a carbohydrate moiety attached to the side chain of an 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 moieties to asparagine side chains. 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 May be used.

Addition of the glycosylation site to the peptide is conveniently accomplished by varying the amino acid sequence to contain one or more of the above-mentioned tripeptide sequences (in the case of N-linked glycosylation sites). Such changes may be made by adding one or more serine or threonine residues to the sequence of the original antibody or by substituting these residues (for O-linked glycosylation sites).

Also, the peptide having the sequence of SEQ ID NO: 1, the peptide of the sequence of SEQ ID NO: 1 or the peptide having the sequence homology of 80% or more with the peptide sequence according to one aspect of the present invention has low intracellular toxicity, Is high. SEQ ID NO: 1 in the present invention is a telomerase-derived peptide, which is a peptide consisting of 16 amino acids as described below.

The peptides shown in SEQ ID NO: 1 are shown in Table 2 below. The "name" in Table 2 below is what the peptides are named for. In one aspect of the invention, the peptide of SEQ ID NO: 1 represents the entire peptide of human telomerase. In another aspect of the present invention, a peptide having a sequence of SEQ ID NO: 1, a peptide of a sequence of SEQ ID NO: 1, or a peptide having a sequence homology of 80% or more with the above peptide sequence, And "synthetic peptide" synthesized by selecting peptides at positions. SEQ ID NO: 2 shows the amino acid sequence of the whole telomerase.

SEQ ID NO: name Telomerase phase location order Length One pep1 [611-626] EARPALLTSRLRFIPK 16 aa 2 [1-1132] MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRR
GAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGT TLTALEAAANPALPSDFKTILD 1132 aa

In one aspect of the present invention, there is provided a method for preventing or treating heart disease caused by reperfusion injury after cardiac ischemia, comprising 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, A pharmaceutical composition comprising a peptide having an activity as an active ingredient.

A pharmaceutical composition for prevention and treatment of heart disease caused by reperfusion injury after cardiac ischemia according to one aspect of the present invention comprises, in one aspect, a peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having a sequence homology of 80% Or peptide fragments thereof in an amount of 0.01 mg / mL to 0.1 mg / mL, 1 mg / mL, 10 mg / mL, and 100 mg / mL, . When it is contained in the above-mentioned range or below, it is not only suitable for exhibiting the intended effect of the present invention but also can satisfy both the stability and safety of the composition and may be suitably included in the above range in terms of cost effectiveness .

The composition according to one aspect of the present invention can be applied to all animals including humans, dogs, chickens, pigs, cattle, 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 fragment thereof, which has an effect on erectile dysfunction prevention and treatment ≪ / RTI > The pharmaceutical composition according to one aspect of the present invention can be administered orally, rectally, transdermally, intravenously, intramuscularly, intraperitoneally, intramuscularly, intradermally or subcutaneously.

Formulations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions or emulsions. Formulations for parenteral administration may be, but are not limited to, injections, drops, lozenges, ointments, gels, creams, suspensions, emulsions, suppositories, patches or spraying agents.

The pharmaceutical composition according to one aspect of the present invention may contain additives such as a diluent, an excipient, a lubricant, a binder, a disintegrant, a buffer, a dispersant, a surfactant, a colorant, a fragrance or a sweetener as necessary. The pharmaceutical composition according to one aspect of the present invention can be prepared by a conventional method in the art.

The effective ingredients of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathological condition and severity of the subject to be administered, route of administration, or judgment of the prescriber. Determination of the amount of application based on these factors is within the level of ordinary skill in the art and its daily dose is, for example, from 0.1 to 100 g / kg / day, for example from 10 to 10 g / kg Kg / day, more specifically from 100 μg / kg / day to 1 g / kg / day, more specifically from 500 μg / kg / day to 100 mg / kg / day, It can be adjusted appropriately. The pharmaceutical composition according to one aspect of the present invention may be administered once to three times a day, but is not limited thereto.

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 peptide as a fragment thereof as an active ingredient, A composition for prevention and treatment of heart disease caused by damage is provided.

The formulation of the composition according to one aspect of the present invention is not particularly limited, but may be formulated into, for example, tablets, granules, powders, liquids, solid preparations and the like. Each formulation may be blended without difficulty by a person skilled in the art according to the purpose of formulation or use, in addition to the active ingredient, and the synergistic effect may occur when the composition is applied simultaneously with other ingredients.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The abbreviated term in front of a noun is not intended to limit the quantity but to indicate that there is more than one noun item mentioned. The terms " comprising ", "having ", and" containing "are to be construed as open (i.e., meaning including but not limited to).

To refer to a range of values is an easy way to substitute for referring individually to each distinct value falling within that range and each separate value that is not explicitly stated is referred to individually in the specification Are incorporated herein by reference. All range end values are contained within that range and can be combined independently.

All methods mentioned herein may be performed in any suitable order unless otherwise indicated or clearly contradicted by context. It is to be understood that the use of any embodiment and all of the embodiments or example language (e.g., "such as ") is for the purpose of describing the present invention only, It is not intended to be limiting. No language in the specification should be construed as obliging any non-claimed components to practice the present invention. Unless defined otherwise, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

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

Hereinafter, the constitution 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 for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

Example 1 Synthesis of Peptides

1. Synthesis of peptides

The peptide of SEQ ID NO: 1 (hereinafter referred to as "PEP1") was prepared according to a conventional solid phase peptide synthesis method. Specifically, the peptides were synthesized by coupling one amino acid from the C-terminal through Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). The first amino acid at the C-terminus of the peptides attached to the resin was used as follows. 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

Boc, t-Bu (t-butylester), Pbf (2, 2, 4, 6, 6), which are all amino acid sources used for peptide synthesis, are protected by N-term and Fmoc, 7-pentamethyl dihydro-benzofuran-5-sulfonyl). For example:

Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Arg- 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.

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

Each of the peptides was synthesized by repeating the steps of reacting corresponding amino acids with a starting amino acid having an amino acid protecting group bonded thereto on a solid support, washing with a solvent, followed by deprotection. The synthesized peptide was cleaved from the resin and purified by HPLC. The synthesis was confirmed by MS and lyophilized.

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

A specific procedure for preparing the peptide PEP 1 will be described as follows.

1) Coupling

The protected amino acid (8 eq) and the coupling reagent HBTU (8 eq) / HOBt (8 eq) / NMM (16 eq) were dissolved in DMF and added to NH ₂ -Lys (Boc) -2-chloro-Trityl Resin , And reacted at room temperature for 2 hours and washed with DMF, MeOH and DMF in that order.

2) Fmoc deprotection

Piperidine in DMF in 20% DMF was added, and the reaction was carried out at room temperature for 5 minutes twice, followed by washing with DMF, MeOH and DMF.

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

4) Cleavage: Cleavage cocktail was added to the synthesized peptide Resin to separate the peptide from Resin.

5) Add Cooling diethyl ether to the obtained mixture and centrifuge to precipitate the obtained peptide.

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

Example 2: Therapeutic effect of PEP1 on cardiac ischemic injury

A cardiac ischemic reperfusion animal model was constructed to confirm the efficacy of PEP1 for cardiac ischemic injury. Tissue from an animal model was used to measure the degree of inhibition of inflammation according to injury, and the effect of treating ischemic injury was confirmed.

Cardiac ischemic animal model manufacture

An ischemia / reperfusion animal model for cardiac ischemic injury was prepared by the following method.

One) Subject animal

SD rats (Sprague-Dawley rats) weighing 300-400 g 6-8 weeks old were used. All experiments were carried out in accordance with the protocol of Seoul National University Experimental Animal Committee and were applied to 12 hour arm / 12 hour cycling, and feed and living conditions were kept constant for 1 week and then used for modeling experiments.

2) Cardiac ischemia / perfusion surgery

Procedures for cardiac ischemia reperfusion induction were as follows.

a. After an intraperitoneal anesthesia with SD rats, the animals were anesthetized with a 14-gauge intravenous catheter, and the animals were anesthetized with a 4-0 suture. The tube was connected to a ventilator (Harvard Rodent Ventilator Model; Harvard Apparatus CO, Holliston, MA) (condition: Vt = 10 ml / kg, PEEP = 2 cm H ₂ O, 60 breaths per minute) To ventilate the machine.

b. A midline sternal incision is made to expose the heart and the left anterior descending coronary artery is removed to prepare a snare (snaring with a lasso-type surgical instrument).

c. In the experimental group, PEP1 (0.001, 0.01, 0.1, 1, 5, and 10 mg / kg) prepared by the method described in Example 1 was administered to a 1 ml syringe and a 30 gauge needle through the anterior wall of the right ventricle immediately before inducing myocardial ischemia / ml) is injected at a dose of 1 ml / kg (animal body weight standard dose).

d. Using a 6-0 polypropylene suture and a snare tourniquet, ischemic injury is induced by snaring the left anterior descending artery for approximately 40 minutes.

3) Observation of cardiac ischemic animal model

After the operation of 2) above, the tourniquet is re-opened and reperfused for 10 minutes, and the myocardial changes in the area below the snare are observed. Then, for the analysis of the tissue, the heart is removed immediately after reperfusion for 10 minutes and the sub-snare is immersed in formalin solution.

Observation of cardiac ischemic injury

One) Histology

Hemorrhage and inflammation were analyzed by H & E staining, and dead cells were quantitatively analyzed by TUNEL staining.

2) MPO analysis

MPO is an enzyme that exhibits inflammatory activity (Myeloperoxidase), which is analyzed by quantitative analysis of inflammation.

3) Cytokine analysis

The degree of inflammation was measured by quantitative analysis of the expression levels of TNF-α and IL-6, which are increased when inflammation is activated.

Statistical processing and analysis

All results were expressed as mean standard deviation (SD), and comparisons between the two groups were made by Student's t-test. P <0.05 was considered statistically significant.

Experimental Results and Analysis

One) Histological analysis

a. Hemorrhage

We compared the severity of cardiac bleeding in the groups administered with saline (control), PEP1 at doses of 0.001, 0.01, 0.1, 1, 5, and 10 mg / kg. As a result, bleeding was similar to that of the control group at a concentration of 0.01 mg / kg or less, but bleeding was decreased as the concentration was increased above 0.1 mg / kg, and bleeding was hardly observed in the group administered with 10 mg / kg.

b. Inflammation

The degree of inflammation of the heart was compared with the neutrophile expression level in the control and administration groups. Each group was divided into two groups: the left ventricle (LV) and the right ventricle (RV). Similar to the bleeding severity results, the degree of inflammation decreased with increasing drug concentration, and the degree of inflammation was similar to that of the control group at concentrations below 0.01 mg / kg (see FIG. 2).

c. Apoptosis

Apoptotic cells were analyzed by TUNEL staining. Brown-stained cells (shown in black in the figure) caused apoptotic cells. The higher the concentration of the drug, the less the apoptotic cells tended to decrease (see FIG. 3). At a concentration of 0.01 mg / kg or more, killed cells were significantly reduced compared to the control group (see FIG. 4).

2) MPO analysis result

The degree of inflammation was quantified by MPO analysis. As the concentration of the drug increased, the degree of inflammation decreased. At 1 mg / kg or higher, the MPO activity was significantly decreased as compared with the control group (see FIG. 5).

3) Cytokine analysis results

The expression levels of TNF-α and IL-6 in the heart were quantitatively analyzed. Similar to the previous results, the degree of inflammation was weaker as the drug concentration was increased, and the number of cytokines was significantly lower than that of the control group at 0.1 mg / kg (see FIGS. 6 and 7).

In Example 2, the inventors successfully established a stable cardiac ischemia / reperfusion animal model to produce a reproducible experimental model. As a result of the histological analysis, MPO analysis and cytokine analysis, the degree of inflammation of the cardiac ischemia / reperfusion injury model was evaluated by PEP1 administration in all analyzes And decreased in the results. This shows that PEP1 is effective in preventing and treating cardiac ischemia / reperfusion injury, and PEP1 and a composition containing it can be used for prevention and treatment of cardiac ischemia / reperfusion injury using the same, and cardiac ischemia / reperfusion injury It will be appreciated that preventative and therapeutic methods may be applicable.

As a result, it can be seen that the peptide PEP1 according to the present invention has an effect on cardiac ischemia / reperfusion injury through the above Examples, and a therapeutic agent for preventing and treating cardiac ischemia / reperfusion injury using the peptide, It is possible to develop a method.

<110> KIM, sangjae          Gemvax & KAEL Co., LTD. <120> COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING BY INJURY          REPERFUSION AFTER ISCHEMIA IN HEART &Lt; 130 > <160> 2 <170> Kopatentin 2.0 <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 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 Ser 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 Valle 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 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 (9)

A peptide comprising the amino acid sequence of SEQ ID NO: 1, a peptide having at least 80% sequence homology with the amino acid sequence, or a peptide thereof, or a fragment thereof, to prevent heart disease caused by reperfusion injury after cardiac ischemia &Lt; / RTI &gt;
The composition according to claim 1, wherein the fragment is a fragment consisting of three or more amino acids, wherein the composition is for preventing and treating heart disease caused by reperfusion injury after cardiac ischemia.
The method according to claim 1, wherein the reperfusion injury after cardiac ischemia is a cardiac surgery including cardiac surgery, heart attack, cardiac arrest, cardiac arrest, heart disease, or heart disease due to reperfusion injury after cardiac ischemia &Lt; / RTI &gt;
The method of claim 1, wherein the cardiac disease caused by reperfusion after ischemia is selected from the group consisting of cardiac insufficiency or myocardial damage, tachyarrhythmia, arrhythmia, cardiac events, A composition for preventing and treating heart disease.
5. The composition according to any one of claims 1 to 4, wherein the composition is a pharmaceutical composition, for the prevention and treatment of reperfusion injury after cardiac ischemia.
A method for preventing and treating heart disease caused by reperfusion injury after cardiac ischemia, comprising administering a pharmaceutically effective amount of the composition of claim 1 to a subject suffering from heart disease caused by reperfusion injury after ischemia
7. The method according to claim 6, wherein the composition is administered at a concentration of 0.1 mg / kg or more based on the weight of the individual.
8. The method according to claim 7, wherein the composition is administered at a concentration of 0.1 mg / kg or more and 10 mg / kg or more based on the weight of the individual.
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 peptide thereof, for use in the preparation of a pharmaceutical composition for prevention and treatment of heart disease caused by reperfusion injury after cardiac ischemia Usage.

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