WO2015170790A1

WO2015170790A1 - Composition for treating and preventing ischemic damage

Info

Publication number: WO2015170790A1
Application number: PCT/KR2014/004194
Authority: WO
Inventors: 김상재; 양재석; 김관민; 전상훈; 장지은
Original assignee: 주식회사 카엘젬백스; 김상재
Priority date: 2014-05-09
Filing date: 2014-05-09
Publication date: 2015-11-12

Abstract

The present invention relates to a composition for treating and preventing ischemic damage. More specifically, the present invention relates to a composition that includes a peptide derived from telomerase and that is effective in treating and preventing ischemic damage. A peptide having a sequence of a sequence ID number according to the present invention, or a peptide having 80% homology with said sequence, or a fragment of same is very effective in treating and preventing ischemic damage. Thus, the composition including the peptide according to the present invention may be effectively applied to ischemic damage, particularly, to ischemic reperfusion damage.

Description

Compositions for the treatment and prevention of ischemic injury

The present invention relates to a composition for treating and preventing ischemic injury. More specifically, the present invention relates to a composition comprising a peptide derived from telomerase, which is effective for treating and preventing ischemic injury.

Ischemic damage occurs when blood circulation is blocked in organs that require blood flow, including the heart, brain, kidneys, and lungs (acute infarction, cerebral infarction, and nerve infarction). Height can be called tissue damage. Ischemic injury causes fatal complications in the heart, brain, kidneys and lungs, and delays the transplantation recovery, increases acute rejection and reduces long-term survival.

Substantial reduction in oxygen delivery due to ischemia leads to a condition known as hypoxia. Continued ischemia and hypoxia can lead to tissue loss and even cell death. There are a number of conditions that are both natural and clinical, causing ischemia and hypoxia, including but not limited to obstructive vascular disease, coronary thrombosis, cerebrovascular thrombosis, aneurysm rupture, systemic bleeding, crush injury, sepsis, severe skin burns, Vascular occlusion surgery methods (eg, spinal cord ischemia in thoracoabdominal aneurysm surgery), cardiopulmonary bypass methods, organ transplantation, cardiopulmonary collapse (acute heart death), and asphyxia.

Conventional treatment for ischemia and consequent hypoxia is to restore blood flow and oxygen delivery to normal levels by increasing systemic oxygenation or eliminating the cause of vascular blockage. Compared to situations where ischemia or hypoxia persists for a long time, restoring blood flow may yield improved results. However, there is a problem that, as blood flow and oxygen delivery are restored, additional cell death or loss of function may be caused irrespective of the damage caused by ischemia or hypoxia.

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

Ischemic tissue damage caused by ischemia-reperfusion (IR) during organ transplantation results in delayed recovery of organ function after organ transplantation, which is a poor prognosis for long-term functioning of organs transplanted with inflammatory tissue reactions. Often act as a factor. Early ischemic reperfusion injury that occurs incidentally during transplantation of organs, particularly kidneys or lungs, can lead to subsequent organ deterioration and graft failure.

Recently, renal ischemia-reperfusion injury (IRI) has been newly identified as an acute inflammatory response involving both inflammatory cells of the innate and acquired immune systems.

On the other hand, the flap (Flap) refers to the skin or tissue that is moved from one part of the body to another by attaching a vessel and a corresponding tissue that can be viable to the tissue to be moved. Flap is used for soft tissue defects or chronic wounds that cannot be solved by skin grafts, and is the most widely used surgical procedure in the plastic surgery field.It is useful for restoring not only appearance but also loss of function. Due to the complex transplantation of several tissues, such as muscles and nerves, the primary reconstruction is possible, which allows for rapid recovery. In these flaps, the survival rate of the flaps is closely related to the treatment of ischemic reperfusion injury. The ischemia reperfusion injury treatment method, if there is a method that can stably improve the survival rate of the flap is expected to be very useful.

As described above, ischemia reperfusion injury is an important disease with a high incidence, but there is not enough effective treatment. Therefore, if the effective prevention and treatment for this come out, the ripple effect is likely to be very large.

[Preceding technical literature]

[Non-Patent Documents]

Granger et al. Ann. Rev. Physiol., 57, 311-332, (1995)

Accordingly, the present inventors have made efforts to develop a composition for treating and preventing ischemic injury, in particular, ischemia reperfusion injury, which has excellent effects, and thus, the present invention has been completed.

The inventors have found that peptides derived from telomerase can have an excellent effect on the treatment and prevention of ischemic damage, in particular ischemic reperfusion injury.

It is an object of the present invention to provide a composition having an effect on treating ischemic injury, treating and preventing ischemic reperfusion injury.

According to one aspect of the present invention, there is provided a composition for treating and preventing ischemic injury, comprising 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. .

In the composition according to one aspect of the invention, the fragment may be a fragment consisting of three or more amino acids.

In the composition according to one aspect of the present invention, the ischemic injury is vascular disease, coronary thrombosis, cerebrovascular thrombosis, aneurysm rupture, systemic bleeding, crush injury, sepsis, skin burn, vascular occlusion surgery, cardiopulmonary bypass, organ One or more causes selected from the group consisting of transplantation, cardiopulmonary collapse (acute cardiac death) and asphyxia, and ischemia reperfusion injury.

In the composition according to one aspect of the invention, the ischemic injury may be due to ischemia reperfusion injury.

In the composition according to one aspect of the invention, the ischemia reperfusion injury is cerebrovascular ischemia reperfusion injury, renal ischemia reperfusion injury, hepatic ischemia reperfusion injury, ischemia reperfusion cardiomyopathy, skin ischemia reperfusion injury, intestinal ischemia reperfusion injury, intestinal ischemia reperfusion injury Injury, gastric ischemia reperfusion injury, pulmonary ischemia reperfusion injury, pancreatic ischemia reperfusion injury, skeletal muscle ischemia reperfusion injury, abdominal muscle ischemia reperfusion injury, limb ischemia reperfusion injury, ischemia reperfusion colitis, mesenteric ischemia reperfusion injury, and asymptomatic ischemia reperfusion injury It may be selected.

In the composition according to one aspect of the invention, the ischemia reperfusion injury may be due to organ transplantation.

In a composition according to one aspect of the invention, the ischemia reperfusion injury may be due to lung transplantation.

In the composition according to one aspect of the invention, the ischemia reperfusion injury may be due to kidney transplant.

In the composition according to one aspect of the present invention, the ischemia reperfusion injury may occur in the kidney or lung.

In the composition according to one aspect of the present invention, the ischemia reperfusion injury may occur in the flap.

In a composition according to one aspect of the invention, the peptide may be derived from human telomerase.

In the composition according to one aspect of the invention, the composition may be a pharmaceutical composition.

In the composition according to one aspect of the invention, the composition may be a food composition.

According to another aspect of the present invention there is provided a method of treating and preventing ischemic injury, characterized in that the above-mentioned composition is administered to a subject in need thereof.

Peptides having a sequence of SEQ ID NO: or a peptide having a sequence having at least 80% homology with the sequence or a fragment thereof according to the present invention have an excellent effect in the treatment and prevention of ischemic injury. Thus compositions comprising the peptides of the present invention can be effectively applied to ischemic damage, in particular ischemic reperfusion injury.

1 is a graph measuring the levels of urea nitrogen (BUN) and creatine in blood taken 24 hours after ischemia reperfusion.

Figure 2 is a photograph showing the result of PAS staining kidney tissue 24 hours after ischemia reperfusion.

3 is a graph showing the results of renal tissue injury scoring on renal tissue after 24 hours of ischemia reperfusion.

Figure 4 is a photograph of the result of evaluating the renal tissue TUNNEL staining 24 hours after ischemia reperfusion.

FIG. 5 shows TUNNEL positive cell measurement results of renal tissue evaluated 24 hours after ischemia reperfusion.

FIG. 6 shows the results of evaluation of congenital immune cell infiltration by immunohistostaining of F4 / 80 (macrophage maker) and Gr-1 (neutrophil maker) in renal tissue after 24 hours of ischemia reperfusion.

7 is a graph showing the results of positive cell measurement of F4 / 80 (macrophage maker) and Gr-1 (neutrophil maker) in renal tissue after 24 hours of ischemia reperfusion.

8 to 10 are graphs showing the effect of inhibiting the secretion of inflammatory cytokines in kidney tissues 24 hours after ischemia reperfusion.

11 is a diagram showing the process of inducing ischemia reperfusion injury to evaluate flap survival.

FIG. 12 is a graph showing flap survival measurement results of the PEP1 treated group and the saline treated group 7 days after ischemia reperfusion induction. FIG. 13 is a digital photograph through the ImageJ program.

14 is a graph showing the result of weighing the lower lobe of the transplanted lung immediately after the experiment after rat lung transplantation, and weighing again after drying in a dryer at 60 ° C. for 24 hours.

FIG. 15 is a photograph showing the results of analysis of neutrophil content after instillation and re-extraction of 5 mL of saline into the trachea after rat lung transplantation.

The present invention may be variously modified and have various embodiments. Hereinafter, the present invention will be described in more detail based on examples. However, these examples are not intended to limit the present invention to the specific embodiments, the present invention is capable of various embodiments and applications based on the description of the claims, and all included within the spirit and scope of the present invention It is to be understood to include the transformations, equivalents, or substitutes. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Telomere is a genetic material repeatedly present at the end of a chromosome and is known to prevent damage to the chromosome or binding to another chromosome. Each time a cell divides, the telomeres become slightly shorter. After a certain number of cell divisions, the telomeres become very short, and the cells stop dividing and die. On the other hand, elongation of telomeres is known to prolong cell life. For example, cancer cells secrete an enzyme called telomerase, which prevents telomeres from shortening, so that cancer cells can continue to proliferate without dying. The inventors have found that peptides derived from telomerase are effective in the treatment and prevention of ischemic reperfusion injury and have completed the present invention.

In one aspect of the invention, a peptide of SEQ ID NO: 1, a peptide that is a fragment of SEQ ID NO: 1, or a peptide having a sequence homology of at least 80% with the peptide sequence is selected from telomerase, specifically human (Homo sapiens) telomerase. Peptides derived.

Peptides disclosed herein can include peptides having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. In addition, the peptides disclosed herein, peptides or fragments thereof comprising SEQ ID NO: 1 and 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, six or more amino acids Or peptides with seven or more amino acids changed.

In one aspect of the invention, amino acid changes belong to a property that allows the physicochemical properties of the peptide to be altered. For example, amino acid changes can be made, such as improving the thermal stability of the peptide, altering substrate specificity, changing the optimal pH, and the like.

As used herein, "amino acid" includes not only the 22 standard amino acids that are naturally incorporated into the peptide, but also D-isomers and modified amino acids. Accordingly, in one aspect of the invention the peptide may be a peptide comprising D-amino acids. Meanwhile, in another aspect of the present invention, the peptide may include a non-standard amino acid or the like which has been post-translational modified. 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 (e.g., beta-elimination deimidization) , Deamidation) and structural changes (eg, formation of disulfide bridges). It also includes changes in amino acids, such as changes in amino groups, carboxy groups or side chains, caused by chemical reactions that occur during the linkage with crosslinkers to form peptide conjugates.

Peptides disclosed herein can be wild-type peptides identified and isolated from a natural source. On the other hand, the peptides disclosed herein may be artificial variants, comprising an amino acid sequence in which one or more amino acids are substituted, deleted and / or inserted compared to peptides that are fragments of SEQ ID NO: 1. Amino acid changes in the wild type polypeptide 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). Amino acid substitutions that generally do not alter 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 substitutions are shown in the following table.

Table 1

Original amino acid	Exemplary residue substitutions	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 the peptide include (a) their effect on maintaining the structure of the polypeptide backbone, eg, a sheet or helical conformation, within the substitution region, (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 chains, is carried out by selecting significantly different substitutions. Natural residues are divided into the following groups based on common side chain properties:

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

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

(3) acidic: asp, glu;

(4) basic: 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 a member of one of these classes for another class. Any cysteine residue that is not involved in maintaining the proper conformation of the peptide can generally be substituted with serine to improve the oxidative stability of the molecule and to 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. By change is meant the deletion of one or more carbohydrate residues 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 either N-linked or O-linked. N-linked refers to a carbohydrate moiety attached to the side chain of an asparagine moiety. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrate moieties to asparagine side chains. Thus, the presence of one of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation means attaching one of the sugars N-acetylgalactosamine, galactose or xylose to hydroxyamino acids, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine You can also use

The addition of glycosylation sites to the peptide is conveniently performed by changing the amino acid sequence to contain one or more of the above mentioned tripeptide sequences (for N-linked glycosylation sites). Such changes may also be made by adding or replacing one or more serine or threonine residues with the sequence of the original antibody (for O-linked glycosylation sites).

In the present invention, "ischemic injury" refers to damage caused by reduced oxygen transfer while blocking blood circulation in organs that require blood supply such as heart, brain, kidney, and lung, and impaired tissue function and Fatal damage that can result in cell death. Possible causes of ischemic injury include vascular disease, coronary thrombosis, cerebrovascular thrombosis, aneurysm rupture, systemic bleeding, crush injury, sepsis, severe skin burns, vascular occlusion surgery methods (e.g. spinal cord during thoracoabdominal aneurysm surgery) Ischemia), cardiopulmonary bypass method, organ transplantation, cardiopulmonary collapse (acute cardiac death), and asphyxia.

In addition, "ischemic injury" according to the present invention encompasses ischemic reperfusion injury that may occur by organ transplantation, etc., in addition to ischemic injury that may normally occur. The ischemia reperfusion injury may include cerebrovascular ischemia reperfusion injury, renal ischemia reperfusion injury, hepatic ischemia reperfusion injury, ischemia reperfusion cardiomyopathy, skin ischemia reperfusion injury, intestinal ischemia reperfusion injury, intestinal ischemia reperfusion injury, gastric ischemia reperfusion injury, lung ischemia reperfusion injury , Pancreatic ischemia reperfusion injury, skeletal muscle ischemia reperfusion injury, abdominal muscle ischemia reperfusion injury, limb ischemia reperfusion injury, ischemia reperfusion colitis, mesenteric ischemia reperfusion injury, and asymptomatic ischemia reperfusion injury, and the like.

Ischemic reperfusion injury can occur frequently during organ transplantation. For example, renal transplantation is associated with progressive loss of function and renal impairment of ischemic renal impairment, resulting in activation of the innate immune system. Is known to be one of the important pathogenesis mechanisms.

The peptide described in SEQ ID NO: 1 is shown in Table 2 below. "Name" in Table 2 below is named to distinguish peptides. 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 a sequence of SEQ ID NO: 1, a peptide that is a fragment of SEQ ID NO: 1, or a peptide having at least 80% sequence homology with the peptide sequence corresponds to a peptide included in telomerase. And "synthetic peptides" selected and synthesized at the positional peptides. SEQ ID 2 shows the amino acid sequence of the entire telomerase.

TABLE 2

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

In one aspect of the present invention for the treatment and prevention of ischemic damage comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 (comprising), a peptide having a sequence homology of 80% or more with the amino acid sequence or a fragment thereof as a active ingredient To provide a composition.

In one aspect, the composition for treating and preventing ischemic injury is a peptide comprising an 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. Can be included in an amount of 0.1 μg / mg to 1 mg / mg, specifically 1 μg / mg to 0.5 mg / mg, more specifically 10 μg / mg to 0.1 mg / mg. When included in the above range, not only is the present invention suitable for exhibiting the intended effect, but also satisfies both the stability and safety of the composition, it may be appropriate to include 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 invention, the composition is an ischemic reperfusion injury treatment comprising 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 as a active ingredient And it provides a preventive pharmaceutical composition. The pharmaceutical composition according to one aspect of the present invention may be administered orally, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intramedullary, intradural or subcutaneous.

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, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches or sprays.

Pharmaceutical compositions according to one aspect of the invention may include additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavoring or sweetening agents as needed. Pharmaceutical compositions according to one aspect of the invention may be prepared by conventional methods in the art.

The active ingredient of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. Dosage determination based on these factors is within the level of ordinary skill in the art, and its daily dose is, for example, 0.1 μg / kg / day to 1 g / kg / day, specifically 1 μg / kg / day to 10 mg / kg Per day, more specifically 10 μg / kg / day to 1 mg / kg / day, and more specifically 50 μg / kg / day to 100 μg / kg / day, but is not limited thereto. 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 invention, the composition is an ischemic reperfusion injury treatment comprising 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 as a active ingredient And prophylactic food compositions.

The formulation of the food composition according to one aspect of the present invention is not particularly limited, but may be, for example, formulated into tablets, granules, powders, solutions, solid preparations, and the like. Each formulation may be appropriately selected and formulated by those skilled in the art according to the formulation or purpose of use, in addition to the active ingredient, and may be synergistic when applied simultaneously with other raw materials.

Determination of the dosage of the active ingredient is within the level of those skilled in the art, the daily dosage of which is for example specifically 1 μg / kg / day to 10 mg / kg / day, more specifically 10 μg / kg / day To 1 mg / kg / day, and 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 may vary.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term without the number before the noun is not intended to limit the quantity but rather to the presence of one or more of the mentioned noun articles. The terms "comprising", "having", and "comprising" are interpreted as open terms (ie, meaning "including but not limited to").

Reference to a range of values is merely a substitute for the individual reference to each individual value within the range, unless otherwise indicated, each value applies to the specification as if it were individually mentioned in the specification. do. All range limits are included in the range and can be combined independently.

All methods mentioned herein may be performed in the proper order unless otherwise specified or clearly contradicted by context. Use of one embodiment and all embodiments or exemplary language (eg, "such as") is intended to facilitate the description of the present invention only, unless it is included in the claims, and the scope of the present invention. It is not intended to limit. No language in the specification should be construed as essential to the practice of the invention as to any unclaimed elements. Unless defined otherwise, 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 invention include the most optimal mode known to the inventors for carrying out the 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 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 invention includes all modifications and equivalents of the subject matter referred to in the appended claims, as permitted by patent law. Moreover, any combination of the abovementioned elements within all possible variations is included in the invention unless expressly stated to the contrary or apparently contradictory in context. While the invention has been particularly shown and described with reference to exemplary embodiments, those skilled in the art will understand 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.

In the following Examples, the peptide (PEP 1) having the sequence shown in SEQ ID NO: 1 is administered to the ischemic reperfusion injury of the kidney, lung, and abdominal muscle skin flap, thereby preventing renal damage and improving flap survival, thereby preventing and treating ischemic injury. To check the effect.

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 the purpose of illustration to help understanding of the present invention, but the scope and scope of the present invention is not limited thereto.

실시예 1: 펩티드의 합성Example 1 Synthesis of Peptides

The peptide of SEQ ID NO: 1 (hereinafter referred to as "PEP 1") was prepared according to the solid phase peptide synthesis known in the art. Specifically, peptides were synthesized by coupling amino acids from the C-terminus one by one through Fmoc solid phase peptide synthesis (SPPS) using ASP48S (Peptron, Inc., Daejeon, Korea). As follows, the first amino acid at 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 feedstocks used for peptide synthesis were protected with N-term Fmoc and all residues removed from acid Trt, Boc, t-Bu (t-butylester), Pbf (2,2,4,6, 7-pentamethyl dihydro-benzofuran-5-sulfonyl) and the like were 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.

Coupling reagent is HBTU [2- (1H-Benzotriazole-1-yl) -1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt [N-Hydroxxybenzotriazole] / NMM [4-Methylmorpholine] It was. Fmoc removal was performed using piperidine in DMF in 20% of DMF. The cleavage cocktail (TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / EDT (ethanedithiol) / H ₂ O = 92.5 / 2.5 / 2.5 / 2.5] was used to isolate the synthesized peptide from Resin and remove the protecting group of residues. It was.

Each peptide was synthesized by repeating a process of reacting the amino acids with each other, washing with a solvent, and then deprotecting the amino acid using the state in which the amino acid protecting group was bound to the solid support. The synthesized peptide was separated from the resin and then purified by HPLC, and confirmed by MS and lyophilized.

High performance liquid chromatography on the peptides used in this example showed that all peptides had a purity of at least 95%.

Referring to the specific process of preparing a peptide PEP 1 according to the present invention.

1) coupling

NH ₂ -Lys (Boc) -2-chloro-Trityl Resin protected amino acid (8 equivalents) and coupling reagent HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) dissolved in DMF and added Reaction was performed at room temperature for 2 hours, and washed with DMF, MeOH, and DMF in that order.

2) Fmoc deprotection

Piperidine in DMF at 20% was added thereto, reacted twice at room temperature for 5 minutes, and washed in the order of DMF, MeOH, and DMF.

3) Repeated reaction of 1 and 2, 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: A peptide cocktail was added to the synthesized peptide Resin to remove the peptide from Resin.

5) Cooling diethyl ether is added to the mixture, followed by centrifugation to precipitate the peptide.

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

실시예 2: 신장 허혈 재관류 손상에 대한 억제 효과Example 2: Inhibitory Effect on Renal Ischemia Reperfusion Injury

Ischemia reperfusion induction proceeded as follows.

A mouse model with renal ischemia reperfusion injury was obtained by inducing ischemia reperfusion by bilateral clamping for 30 minutes in the kidney pedicle and restoring blood flow by removing the clamp after 30 minutes. The experimental group was divided into three groups: administration group (PEP 1), control group (PBS: no PEP 1 administration), and Sham (no bilateral clamping). PEP 1 was subcutaneously injected at 1000 nmol / kg 30 minutes before and 12 hours after induction of ischemia reperfusion.

The experiment was performed by inducing ischemia reperfusion kidney injury using C57BL / 6 mice (8 weeks old) (Charles River Laboratories (Walminton, Mass.). The ischemia reperfusion kidney injury model was used to force the pedicle of the kidney into vascular forceps. ) To stop blood flow, induce ischemia for 28 minutes, and then reperfusion.

Peptide PEP 1 was diluted in PBS at a concentration of 1000 nmol / kg and administered intraperitoneally (i.p injection) twice 30 minutes before and 12 hours after ischemia reperfusion. The experimental group was divided into administration group (PEP 1), control group (PBS) and Sham group (group without causing ischemia reperfusion injury and without kidney damage).

시험예 1: 허혈 재관류 손상 유도된 신장기능손상 보호 효과Test Example 1 Protective Effect of Kidney Reperfusion Injury Induced Renal Impairment

Blood was collected 24 hours after ischemia reperfusion and measured BUN (blood urea nitrogen) and creatine, which are renal toxicity indicators, and kidney tissues were extracted to produce paraffin blocks to produce immunohistochemistry and Histologic studies (immunohistochemical and histological studies) were conducted, and protein was extracted to measure cytokine (cytokine) levels. Creatine concentration and BUN were measured using an automatic analyzer (Technicon RA-1000; Bayer, Tarrytown, NY).

As a result, the PEP 1 administration group significantly decreased BUN and creatine values compared to the PBS control group (see FIG. 1).

시험예 2: 신장 조직 손상 보호효과Test Example 2: Protective effect of kidney tissue damage

Twenty four hours after ischemia reperfusion, kidney tissues were subjected to peridodic acid-Schiff (PAS) staining (PAS staining with a PAS kit according to the manufacturer's Polysciences, Inc. (Warington, PA, USA) protocol). After staining, renal tissue injury scoring was evaluated by renal tissue injury scoring. The PEP 1 administration group showed a remarkable alleviation of renal tissue damage compared to the PBS control group (see FIGS. 2 and 3).

시험예 4: 신장 세포사멸(renal apoptosis) 억제효과 Test Example 4: Inhibitory effect of renal apoptosis

Twenty four hours after ischemia reperfusion, renal tissues were evaluated for apoptosis by TUNEL staining. TUNEL staining was performed on paraffin kidney sections via a TUNEL staining kit made by Roche Applied Sciences (Indianapolis, IN, USA).

As a result, it was confirmed that the administration of PEP 1 inhibited renal tissue apoptosis because TUNEL positive cells were significantly reduced compared to the PBS control group (see FIGS. 4 and 5).

시험예 5: 신장 조직에서의 선천면역 세포 침윤 억제 효과Test Example 5: Inhibitory effect of innate immune cell infiltration on renal tissue

Twenty four hours after ischemia reperfusion, kidney tissues were assessed for infiltration of congenital immune cells by F4 / 80 (macrophage maker) and Gr-1 (neutrophil maker) immunohistostaining. Specifically, macrophages and neutrophil cell penetration staining were performed using an antibody specific for macrophages (F4 / 80, abcam., Cambridge, Mass.) By an immunochemical method on paraffin-containing sections.

Compared to the PBS control group, the PEP 1-administered group was found to significantly reduce macrophage and neutrophil infiltration into renal tissues (see FIGS. 6 and 7).

시험예 6: 염증성 사이토카인 분비 억제효과Test Example 6 Inhibitory Effect on Inflammatory Cytokine Secretion

Twenty four hours after ischemia reperfusion, proteins were extracted from renal tissue, and IL-6, MCP-1, and TNF-α levels were measured by a cytometric bead array method. Mouse IL6, MCP-1, TNFa ElISA kits were purchased from R & D Systems and performed according to the manufacturer's protocol.

As a result, the PEP 1 administration group significantly reduced IL-6, MCP-1, and levels compared to the PBS control group, but no significant change was observed in TNF-α (see FIGS. 8 to 10).

As described above, the protective effect of PEP 1 against ischemic reperfusion kidney damage is renal function (BUN, creatine), renal tissue damage (renal apoptosis), renal tissue infiltration (immune cell infiltration) And renal tissue cytokine (cytokine) secretion inhibition.

In the ischemia-reperfusion PBS control group, serum BUN and creatine levels were increased and kidney tissue damage was increased as compared with Sham group. However, in the PEP 1 group, BUN and creatine values were significantly decreased, and renal cell death as well as renal tubular injury were reduced. In addition, the PEP 1 group inhibited the infiltration of inflammatory cells (neutrophils and macrophages) by ischemia reperfusion in the kidney compared to the PBS control group, and significantly inhibited the secretion of inflammatory cytokines (interleukin-6, monocyte chemotactic protein-1).

실시예 3: 복직근 피부판에서의 허혈 재관류 손상에 대한 피판 생존률 향상 효과Example 3 Effect of Enhancing Flap Survival on Ischemic Reperfusion Injury in Abdominal Muscle Skin Flap

Ischemic reperfusion induction of abdominal muscle skin flap proceeded as follows.

Rat models with ischemic reperfusion injury were harvested at 5 cm x 5 cm from the skin of a white rat (Sprague-Dawley Rat, 180 g to 230 g), administered with PEP1 or Saline, and clamped. Ischemia was induced, and after 7 hours, the clamp was removed to induce ischemia reperfusion injury by restoring blood flow (see FIG. 11).

The experimental group was divided into three groups: the administration group (PEP 1 treatment group), the control group (saline treatment group: no PEP 1 administration group), and the Sham group (group without causing ischemic reperfusion injury). PEP 1 was injected at a concentration of 10 mg / 500 μl, and 500 μl of saline was injected intramuscularly 30 minutes before ischemia reperfusion induction and 1, 2, 3, 4, 5, 7 days after each.

Flap Survivability was measured 7 days after induction of ischemia reperfusion. The measurement of flap survival was performed through digital photo analysis through the imageJ program.

After 7 days, the flap survival rate was 34.69% ± 16.44% in the saline treatment group, and the flap survival rate in the PEP1 treated group was 58.88% ± 11.44%. (See Figure 12). Statistical significance (p <0.05) was found between groups.

실시예 4:Example 4: 폐 이식 모델에서의 허혈 재관류 손상 예방 효과 Prevention of Ischemic Reperfusion Injury in Lung Transplantation Model

The lung transplantation model for measuring the preventive effect of PEP1 ischemia reperfusion injury was set as follows.

The experiment was conducted on two rats with the same gene. The left lung was removed from one rat, and the original left lung was removed from the left thoracic cavity of the other rat and lung transplanted. Rats with the same genes were used to fundamentally block the rejection response. Rats were targeted to Sprague-Dawley species with weights of 300 g to 350 g for both donors and recipients. The lungs were then removed from the donor rats and implanted into the recipient rats.

Lung transplanted rats were divided into four groups as follows. In order to determine the appropriate dose level of PEP1, PEP1 was added at low concentration (5 mg) and high concentration (50 mg), respectively.

① 50 mL administration group of normal saline

② Perfadex 50 mL administration group

③ Perfadex 50 mL + PEP1 5 mg administration group

④ Perfadex 50 mL + PEP1 50 mg administration group

The following experiments were performed to evaluate the function of transplanted lung after lung transplantation.

시험예 1. 습건 중량비 (wet/dry weight ratio) 측정Test Example 1. Measurement of wet / dry weight ratio

The lower lobe of the transplanted lung was cut out and weighed immediately, and then weighed again after drying in a dryer at 60 ° C. for 24 hours.

As a result, reperfusion edema in the order of physiological saline administration group (① group), perfadex and PEP1 50 mg group (④ group), Perfadex administration group (② group), and Perfadex and PEP1 5 mg group (③ group) (reperfusion edema) appeared a lot.

Reperfusion edema was significantly decreased (p <0.05) in the Perfadex group (group ②) and in the group administered Perfadex and PEP1 5 mg (group ③), compared to the physiological saline group (group ①). In the group administered with 5 mg of PEP1 (③), the level was the lowest. (See Figure 14)

시험예 2. 기관지 폐포세척액 (BAL, Bronchoalveolar lavage fluid) 분석Experimental Example 2 Bronchoalveolar lavage fluid (BAL) analysis

Neutrophil content was analyzed by instillation of 5 mL of normal saline into the trachea and re-extracted.

As a result, bronchial alveoli in the order of physiological saline administration group (group ①), Perfadex group (group ②), group administered with Perfadex and PEP1 50 mg (group ④), and group administered with Perfadex and PEP1 5 mg (group ③). The percentage of inflammatory cells in the wash solution was high. Arrow indicates inflammatory cells. Perfadex and PEP1 5 mg group (③ group) was confirmed that the least expression of inflammatory cells. (See Figure 15)

As described above, the therapeutic and prophylactic effects of PEP1 on the ischemic injury that may occur in various parts of the human body (kidney, abdominal muscle skin flap, lung) were confirmed.

Claims

A composition for treating and preventing ischemic injury, comprising a peptide comprising an 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 composition for treating and preventing ischemic injury of claim 1, wherein the fragment is a fragment consisting of three or more amino acids.
The method of claim 1, wherein the ischemic injury is ischemia reperfusion injury, vascular disease, coronary thrombosis, cerebrovascular thrombosis, aneurysm rupture, systemic bleeding, crush injury, sepsis, skin burn, vascular occlusion surgery, cardiopulmonary bypass, organ A composition for treating and preventing ischemic injury, which is caused by one or more causes selected from the group consisting of transplantation, cardiopulmonary collapse (acute heart death), and asphyxia.
The method of claim 3, wherein the ischemic injury is due to ischemia reperfusion injury.
The ischemic reperfusion injury, renal ischemia reperfusion injury, hepatic ischemia reperfusion injury, ischemia reperfusion cardiomyopathy, skin ischemia reperfusion injury, intestinal ischemia reperfusion injury, intestinal ischemia reperfusion injury, intestinal ischemia reperfusion injury Injury, pulmonary ischemia reperfusion injury, pancreatic ischemia reperfusion injury, skeletal muscle ischemia reperfusion injury, abs muscle ischemia reperfusion injury, limb ischemia reperfusion injury, ischemia reperfusion colitis, mesenteric ischemia reperfusion injury and asymptomatic ischemia reperfusion injury And prophylactic compositions.
The method of claim 4. Ischemic reperfusion injury is due to organ transplant ischemic damage treatment and prevention composition.
The method of claim 4. Ischemic reperfusion injury is to occur in the kidney ischemic damage treatment and prevention composition.
The method of claim 4. Ischemic reperfusion injury is a composition for the treatment and prevention of ischemic damage that occurs in the flap.
The method of claim 6. Ischemic reperfusion injury is due to lung transplant ischemic damage treatment and prevention composition.
The method of claim 6. The ischemic reperfusion injury is due to kidney transplant ischemic damage treatment and prevention composition.
The method of claim 1. The peptide is derived from human telomerase composition for treating and preventing ischemic damage.
The composition for treating and preventing ischemic injury of claim 1, wherein the composition is a pharmaceutical composition.
The composition for treating and preventing ischemic injury of claim 1, wherein the composition is a food composition.
A method for treating and preventing ischemic injury, comprising administering a composition according to any one of claims 1 to 13 to a subject in need thereof.