Peptides and uses thereof

Abstract

[Problem] To provide a peptide and a use thereof. [Solution] On the basis of the results of the inventive studies performed so far, the present inventors have designed an artificial sequence peptide having the amino acid sequence of SEQ ID NO: 1, and have found that the artificial sequence peptide exhibits a therapeutic effect on traumatic brain injury. Based on these findings, the present application provides peptides comprising artificial sequence peptides having the amino acid sequence of SEQ ID NO: 1 and uses thereof.

Description

Technical Field

[0001] The present application relates to a peptide and its use.

[0002] This application claims the benefit of Japanese Patent Application No. 2022-209604, filed on December 27, 2022, which is hereby incorporated by reference herein in its entirety. Background Art

[0003] Traumatic brain injury (TBI) refers to brain damage caused by external mechanical forces and is recognized as one of the leading causes of morbidity and mortality worldwide. However, currently, treatment options for TBI are limited and mainly consist of medical support and rehabilitation, with no effective pharmacological approaches. Therefore, the development of effective drugs for the treatment of TBI is desirable. Summary of the Invention Problems to be solved by the present invention The purpose of this application is to provide a peptide and its use.

[0004] Means of solving the problem Based on the results of their original research conducted so far, the present inventors have designed an artificial sequence peptide consisting of the amino acid sequence of SEQ ID NO: 1 and found that the artificial sequence peptide exhibits a therapeutic effect on traumatic brain injury. Based on these findings, the present application provides a peptide, such as an artificial sequence peptide consisting of the amino acid sequence of SEQ ID NO: 1, and its use.

[0005] That is, the present disclosure provides the following embodiments.

[0006] [1] A pharmaceutical composition for preventing or treating brain damage, comprising a peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (c) A peptide comprising an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0007] [2] The pharmaceutical composition according to [1], wherein the brain injury is focal or diffuse brain injury.

[0008] [3] The pharmaceutical composition according to [1], wherein the brain injury is selected from cerebral contusion, epidural hematoma, subdural hematoma, intracerebral hematoma, concussion, diffuse axonal injury and subarachnoid hemorrhage.

[0009] [4] The pharmaceutical composition according to [1], wherein the brain injury is traumatic brain injury.

[0010] [5] A peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (c) A peptide comprising an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0011] [6] A composition comprising the peptide according to [5].

[0012] [7] A pharmaceutical composition comprising the peptide according to item 5.

[0013] [8] A nucleic acid encoding the peptide according to [5].

[0014] [9] A vector comprising the nucleic acid according to [8].

[0015]

[10] A host cell comprising the nucleic acid according to [8] or the vector according to [9].

[0016]

[11] A composition comprising the nucleic acid according to [8] or the vector according to [9].

[0017]

[12] A pharmaceutical composition comprising the nucleic acid according to [8] or the vector according to [9].

[0018] [A1] A method for preventing or treating brain damage, comprising the step of administering to a subject an effective amount of a peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (c) A peptide comprising an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0019] [A2] The method according to [A1], wherein the brain injury is focal or diffuse brain injury.

[0020] [A3] The method according to [A1], wherein the brain injury is selected from cerebral contusion, epidural hematoma, subdural hematoma, intracerebral hematoma, concussion, diffuse axonal injury and subarachnoid hemorrhage.

[0021] [A4] The method according to [A1], wherein the brain injury is traumatic brain injury.

[0022] [A5] A method for preventing or treating a disease or pathological condition, comprising the step of administering an effective amount of the peptide described in [A1] to a subject.

[0023] [A6] A method for preventing or treating a disease or pathological condition, comprising the step of administering to a subject a nucleic acid encoding the peptide described in [A1] or a vector comprising the nucleic acid.

[0024] [B1] A peptide for preventing or treating brain damage, selected from the following: (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (c) A peptide comprising an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0025] [B2] The peptide for use according to [B1], wherein the brain injury is focal or diffuse brain injury.

[0026] [B3] The peptide for use according to [B1], wherein the brain injury is selected from the group consisting of cerebral contusion, epidural hematoma, subdural hematoma, intracerebral hematoma, concussion, diffuse axonal injury and subarachnoid hemorrhage.

[0027] [B4] The peptide for use according to [B1], wherein the brain injury is traumatic brain injury.

[0028] [B5] The peptide according to [B1], wherein the peptide is used for preventing or treating a disease or pathological condition.

[0029] [B6] A nucleic acid encoding the peptide described in [B1], for use in preventing or treating a disease or pathological condition.

[0030] [B7] A vector comprising a nucleic acid encoding the peptide described in [B1], for use in preventing or treating a disease or pathological condition.

[0031] [C1] Use of a peptide in the preparation of a medicament for preventing or treating brain damage, wherein the peptide is selected from the following: (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (c) A peptide comprising an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0032] [C2] The use according to [C1], wherein the brain injury is focal or diffuse brain injury.

[0033] [C3] The use according to [C1], wherein the brain injury is selected from cerebral contusion, epidural hematoma, subdural hematoma, intracerebral hematoma, concussion, diffuse axonal injury and subarachnoid hemorrhage.

[0034] [C4] The use according to [C1], wherein the brain injury is traumatic brain injury.

[0035] [C5] Use of the peptide described in [C1] in the preparation of a medicament for preventing or treating a disease or pathological condition.

[0036] [C6] Use of a nucleic acid encoding the peptide described in [C1] or a vector comprising the nucleic acid in the preparation of a medicament for preventing or treating a disease or pathological condition.

[0037] [C7] Use of a vector comprising a nucleic acid encoding the peptide described in [C1] in the preparation of a medicament for preventing or treating a disease or pathological condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] [ Figure 1-1 ] (Acute phase) Graph showing the change in the number of slips over time in a beam balance test in a mouse model of traumatic brain injury. *p<0.05.

[0039] [ Figure 1-2 ] (Acute phase) Graph showing changes in latency to traverse the beam over time in a balance beam test in a mouse model of traumatic brain injury. *p<0.05, **p<0.01.

[0040] [ Figure 2-1 ] (Subacute phase) Graph showing the change in the number of slips over time in a balance beam test in a mouse model of traumatic brain injury. **p<0.01.

[0041] [ Figure 2-2 ] (Subacute phase) Graph showing changes in execution time for crossing a beam in a balance beam test over time in a mouse model of traumatic brain injury. *p<0.05, **p<0.01.

[0042] [ Figure 3-1] (Chronic phase) Graph showing changes in the number of slips over time in a balance beam test in a mouse model of traumatic brain injury. ***p<0.001.

[0043] [ Figure 3-2 ] (Chronic phase) Graph showing changes over time in the execution time of crossing a beam in a balance beam test in a mouse model of traumatic brain injury. **p<0.01, ***p<0.001.

[0044] [ Figure 4 ] (Chronic phase) Graph showing changes in the time spent on the rotor during the rotarod test over time in a mouse model of traumatic brain injury. **p<0.01, ***p<0.001.

[0045] [ Figure 5 Photograph of the apparatus used for the Y-maze test. Three arms (A, B, C) of equal size are connected at 120° to form a Y-shape.

[0046] [ Figure 6 ](Severe) A graph showing changes over time in alternation (%) (referred to as "Y-maze accuracy") in a Y-maze test in a mouse model of traumatic brain injury.

[0047] [ Figure 7-1 ] (Subacute phase) Graph showing changes in the number of slips over time in a balance beam test in a mouse model of traumatic brain injury. *p<0.05, **p<0.01.

[0048] [ Figure 7-2 ] (Subacute phase) Graph showing changes in execution time for crossing a beam in a balance beam test over time in a mouse model of traumatic brain injury. *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0049] The present application provides a certain peptide, a composition comprising the peptide, a pharmaceutical composition comprising the peptide, a nucleic acid encoding the peptide, a composition comprising the nucleic acid, and a pharmaceutical composition comprising the nucleic acid.

[0050] As used herein, "peptide" includes "artificial sequence peptide". As used herein, "artificial sequence peptide" refers to a peptide having an amino acid sequence that does not occur naturally. As used herein, "artificial sequence peptide" is also referred to as "artificial peptide".

[0051] The peptides of the present application include, for example, peptides selected from the following: (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (c) A peptide comprising an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 1.

[0052] The peptides listed in (a) to (c) above include peptides selected from the group consisting of: (1) a peptide consisting of the amino acid sequence described in SEQ ID NO: 1; (2) a peptide consisting of an amino acid sequence in which one or more amino acids in the amino acid sequence described in SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (3) a peptide consisting of an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO: 1; (4) a peptide consisting of a portion of the amino acid sequence described in SEQ ID NO: 1; (5) a peptide encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of the nucleotide sequence described in SEQ ID NO: 2; and (6) A peptide encoded by a DNA consisting of the nucleotide sequence described in SEQ ID NO: 2.

[0053] The peptides described in (a) to (c) and (2) to (6) above may be peptides having an effect of improving motor function or restoring memory function in brain injury. The peptides described in (a) to (c) and (2) to (6) above may be peptides that are functionally equivalent to the peptide comprising the amino acid sequence described in SEQ ID NO: 1 ("functionally equivalent"), and examples of the peptides include peptides comprising the amino acid sequences described in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27, respectively. As used herein, "functionally equivalent" means that the effect of improving motor function or restoring memory function in brain injury is equivalent. For example, the effect can be evaluated in the experimental system described in the working examples of the present application.

[0054] As used herein, "plurality" includes, for example, two to six, two to five, two to five, two to four, two to three, or two.

[0055] As used herein, "about 80% or more" includes, for example, about 80% or more, about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more.

[0056] As used herein, a "peptide comprising the amino acid sequence of SEQ ID NO: 1" includes a "peptide consisting of the amino acid sequence of SEQ ID NO: 1". As used herein, a "peptide comprising the amino acid sequence of SEQ ID NO: 1" has a length of 30 to X (i.e., at least 30 and at most X) or 31 to X (i.e., at least 31 and at most X) amino acids. For example, X is selected from, but not limited to, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, and 31.

[0057] As used herein, "a peptide comprising the amino acid sequence of SEQ ID NO: 1" includes a peptide consisting of an amino acid sequence having one or more amino acids added to the N-terminus and / or C-terminus of the amino acid sequence of SEQ ID NO: 1. As used herein, "and / or" can be used interchangeably with "both or either."

[0058] As used herein, a "peptide consisting of a portion of the amino acid sequence set forth in SEQ ID NO: 1" is from Y to 29 (i.e., at least Y and at most 29) amino acids in length. For example, Y is selected from, but not limited to, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, and 28.

[0059] As used herein, "stringent conditions" may refer to conditions such as hybridization with 6×SSC and 40% formamide at 25° C. and washing with 1×SSC at 55° C. Stringency depends on conditions such as salt concentration, formamide concentration, or temperature, but those skilled in the art can set these conditions to provide the desired stringency.

[0060] When hybridization is performed under stringent conditions, DNAs with highly homologous nucleotide sequences are selected. Thus, the isolated peptides may contain peptides that are functionally equivalent to the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. Highly homologous nucleotide sequences can have, for example, about 60% or greater, about 70% or greater, or about 80% or greater identity.

[0061] The nucleotide sequences described in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 are examples of nucleotide sequences of DNAs encoding the peptides described in SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27, respectively. Other DNA sequences encoding the peptides of the present application can be generated by methods (reverse translation) in which the amino acid residues of the peptides are converted into their corresponding codons using codon tables known to those skilled in the art. Reverse translation can optionally be performed using various software (including programs, algorithms, etc.) developed for amino acid and nucleic acid sequence analysis.

[0062] The peptides of the present application can be obtained by incorporating the DNA encoding them into a suitable expression system as a recombinant or by artificially synthesizing them. Therefore, the peptides of the present application include peptides produced using cells, and those artificially synthesized (ie, synthetic peptides).

[0063] In order to obtain the peptide of the present application by genetic engineering, DNA encoding the peptide can be incorporated into an appropriate expression system and expressed.

[0064] Suitable hosts for the present application include, but are not limited to, prokaryotic cells and eukaryotic cells. The above-mentioned hosts also include, but are not limited to, bacteria (e.g., Escherichia coli (E. coli)), yeast, animal cells (e.g., mammalian cells such as HEK293 cells and CHO cells, and insect cells such as silkworm cells) and plant cells.

[0065] For example, the host / vector system that is suitable for the application can include pGEX expression vector and Escherichia coli.Because pGEX can be used as the fusion protein expression (Gene, 67:31-40,1988) with glutathione S-transferase (GST), so the pGEX containing the DNA encoding the application peptide is introduced into Escherichia coli strains such as BL21 by heat shock, and after the appropriate incubation period, isopropylthio-β-D-galactoside (IPTG) is added to induce the expression of GST fusion peptide.Because the GST of the application is combined with glutathione agarose (Sepharose) 4B, the expression product can be easily separated and purified by affinity chromatography.

[0066] Other suitable host / vector systems can also be used to obtain the recombinant peptides of the present application. For example, when the host is bacteria, expression vectors for fusion proteins using tags and the like are commercially available. The recombinant peptides of the present application also include those to which a tag or a portion of a tag is attached.

[0067] There is no particular limitation on the tag to be attached to the peptide of the present application unless it affects the activity of the peptide. Examples of tags include histidine tags (e.g., 6x His, 10x His), HA tags, FLAG tags, GST tags, T7 tags, HSV tags, E tags, lck tags, and B-tags.

[0068] In yeast, it is known that Pichia species (Pichia sp.) yeast is effective in expressing proteins with polysaccharides. In order to add polysaccharides, it is also useful (Bio / Technology, 6:47-55,1988) to use insect cells as the baculovirus vector expression system of the host. In addition, mammalian cells have been used as hosts for transfection with vectors containing CMV, RSV or SV40 promoters, and any one of these host / vector systems can serve as the expression system of the peptide of the present application. Genes can also be introduced using vectors including but not limited to: plasmid vectors or viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, Sendai virus vectors, Sendai virus envelope vectors and papillomavirus vectors. Vectors may contain promoter DNA sequences that effectively induce gene expression, factors that control gene expression and molecules necessary for maintaining DNA stability.

[0069] The resulting peptide can be isolated from the inside or outside of the host cell (e.g., from the culture medium) and purified to be substantially pure and homogeneous. The separation and purification of the peptide can be carried out by any method commonly used in peptide purification and is not limited to a specific method. For example, techniques such as chromatography columns, filters, ultrafiltration, salt precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization can be appropriately selected and combined to separate and purify the peptide.

[0070] Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Marshak et al., Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Cold Spring Harbor Lab Press, 1996). Chromatography can be performed using liquid chromatography such as HPLC or FPLC.

[0071] Preferably, the peptide of the present application is a substantially purified peptide. As used herein, "substantially purified" means that the degree of purification of the peptide of the present application (percentage of peptide in the total protein fraction) is 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% or higher, or 95% or higher, 100% or close to 100%. The upper limit of "close to 100%" depends on the purification and analytical techniques of those skilled in the art, and can be, for example, 99.999%, 99.99%, 99.9% or 99%.

[0072] A substantially purified peptide can be any peptide purified by any purification method if the peptide is purified to the above extent. Examples of substantially purified peptides include peptides purified by any one or a combination of the methods listed above, including, but not limited to, chromatography columns, filters, ultrafiltration, salt precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization.

[0073] On the other hand, the peptide of the present application can also be artificially synthesized. The peptide synthesis method of the present application allows the chemical synthesis of peptides, for example, using liquid phase or solid phase peptide synthesis. Solid phase peptide synthesis is a common method for peptide chemical synthesis. The method uses polystyrene polymer gel beads with a diameter of about 0.1mm as a solid phase, and its surface is modified with amino groups. Then, the amino acid chain is sequentially extended via a dehydration reaction. Once the desired peptide sequence has been synthesized, it is cut from the solid phase surface to produce a product. Solid phase synthesis also allows the synthesis of ribosomal peptides that are difficult to synthesize in bacteria, the introduction of non-natural amino acids such as D-amino acids, or the use of stable isotopes (for example, 2 H. 13 C or 15 In some embodiments, the present invention relates to a peptide or protein backbone comprising a selenoamino acid, a selenoamino acid, a selenomethionine, a selenothioic ...

[0074] The nucleic acids of the present application include DNA and RNA. RNA includes but is not limited to mRNA. Preferably, the nucleic acids of the present application are substantially purified nucleic acids. The nucleic acids of the present application include both unmodified nucleic acids and modified nucleic acids.

[0075] In the present application, the composition may include one or more peptides or one or more nucleic acids. The peptides, nucleic acids, or vectors containing the peptides of the present application may be dissolved or suspended in a solvent in the composition. Solvents include, but are not limited to, water, saline, or a buffer. Buffers include, but are not limited to, citrate buffer, acetate buffer, or phosphate buffer.

[0076] The compositions of the present application can be used as pharmaceutical compositions or reagent compositions. As used herein, the term "pharmaceutical composition" can be used interchangeably with "drug", "pharmaceutical preparation" or "pharmaceutical composition", and the term "reagent composition" can be used interchangeably with "reagent".

[0077] As used herein, the term "pharmaceutical composition comprising (agent)" is used interchangeably with "pharmaceutical composition comprising (agent) as an active ingredient."

[0078] The peptide or nucleic acid of the present application can be used to prevent or treat traumatic brain injury.In traumatic brain injury, physical damage to the head causes the rupture of brain parenchyma tissue destruction, the rupture of blood vessels in the brain parenchyma and the rupture of blood vessels on the brain surface.In addition, hemorrhage in the peri-brain region (for example, dura mater and arachnoid), brain parenchyma and brain surface causes hematoma and causes damage by compressing the brain. Considering etiology and / or pathogenesis as above, the peptide of the present application can not only be used for treating traumatic brain injury, but also can be used for treating or preventing brain injury.

[0079] In the present application, brain injury includes, but is not limited to, focal brain injury, diffuse brain injury, traumatic brain injury, cerebral contusion, epidural hematoma, subdural hematoma, intracerebral hematoma, concussion, diffuse axonal injury and subarachnoid hemorrhage. The diseases listed above include traumatic diseases or non-traumatic (e.g., intrinsic) diseases. The diseases listed above include diseases in the acute, subacute or chronic stages.

[0080] The diagnostic criteria for these diseases are well known and can be used to assess the prevention and treatment of the disease.

[0081] The peptides or nucleic acids of the present application can also be used in, for example, basic and clinical research. The present application also provides the use of the peptides of the present application in preparing drugs or reagents for basic or clinical research.

[0082] The subject of the present application is not particularly limited and includes, for example, mammals, birds, and fish. Mammals include humans or non-human animals, including but not limited to humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters, guinea pigs, horses, sheep, and whales. As used herein, the term "subject" can be used interchangeably with "patient," "individual," or "animal."

[0083] An effective amount of the peptide or pharmaceutical composition comprising the same, the nucleic acid or pharmaceutical composition comprising the same, or the vector or pharmaceutical composition comprising the same (hereinafter referred to as "pharmaceutical composition, etc.") of the present application is administered to a subject to treat the diseases or symptoms described herein.

[0084] An effective amount as used herein refers to an amount sufficient to prevent or treat a disease or pathological condition described herein. Treatment includes, but is not limited to, alleviation, delay, inhibition, amelioration, alleviation, cure, and complete cure. Prevention includes, but is not limited to alleviation, delay, and inhibition.

[0085] In the present application, the site of administration of the pharmaceutical composition, etc. is not limited. The effect of the pharmaceutical composition, etc. can be achieved when it is administered to a site, such as a site where symptoms of a disease or pathological condition appear or a site near the site, a site different from the site where symptoms of a disease or pathological condition appear or a site near the site (a site other than the site), a site distant from the site where symptoms of a disease or pathological condition appear, a site distal to the site where symptoms of a disease or pathological condition appear, a site distal to and heterologous to the site where symptoms of a disease or pathological condition appear, or any other site.

[0086] In the present application, the effect of a pharmaceutical composition, etc. can be achieved when it is administered to a tissue, such as a tissue different from the tissue where symptoms of a disease or pathological condition appear, a tissue away from the tissue where symptoms of a disease or pathological condition appear, a tissue distal to the tissue where symptoms of a disease or pathological condition appear, or a tissue distal to and heterologous to the tissue where symptoms of a disease or pathological condition appear, or any other tissue.

[0087] In the present application, the application method of pharmaceutical composition etc. comprises oral or parenteral administration.The method for parenteral administration includes but is not limited to intravascular (for example, intraarterial or intravenous), intramuscular, subcutaneous, intradermal, intraperitoneal, intranasal, through lung (transpulmonary) and transdermal administration.Pharmaceutical composition etc. can be by injection, for example intravenous, intramuscular, intraperitoneal or subcutaneous injection, carry out whole body or local application (for example, subcutaneous, intradermal, on skin surface, on eye or eyelid conjunctiva, nasal mucosa, oral cavity and digestive tract mucosa, vagina and uterine mucosa, or at injury site).In other words, the application method of pharmaceutical composition etc. comprises systemic administration and local application.

[0088] The peptide of the present application may also be in the form of a nucleic acid encoding the peptide, a vector comprising the nucleic acid, a cell secreting the peptide, or a gene therapy vector containing a DNA encoding the peptide.

[0089] The peptides of the present application may be in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts include, but are not limited to, hydrochlorides, acetates, and trifluoroacetates. The peptides of the present application may be in the form of solvates of the peptides or solvates of pharmaceutically acceptable salts of the peptides. Solvates are substances in which any number of solvent molecules are coordinated with solute molecules, including but not limited to hydrates. The peptides of the present application may also be in any optical or structural isomer.

[0090] The nucleic acid of the present application may be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, but are not limited to, alkali metal salts, alkaline earth metal salts, metal salts, inorganic salts, inorganic acid salts, amine salts, lower alkane sulfonates, aryl sulfonates, organic salts, organic acid salts, and amino acid salts.

[0091] The nucleic acids of the present application can be administered to a subject using, for example, liposomes, retroviruses, adenoviruses, adeno-associated viruses, and vaccinia vectors.

[0092] Can depend on the age of the patient and the symptom selection method of administration.When using the pharmaceutical composition of the application etc., can for example, at each time use every kg body weight 0.0000001mg to 1000mg scope select dosage.On the other hand, can for example, at each patient 0.00001 to 100000mg / scope select dosage.When using the cell that secretes the peptide of the application or the gene therapy vector containing the DNA encoding the peptide, can so use so that the amount of peptide falls within the above-mentioned scope.But the possible dosage of the pharmaceutical composition of the application is not limited to those dosages mentioned above.

[0093] The pharmaceutical composition of the present application can be prepared using conventional methods, such as those described in the latest edition of Remington's Pharmaceutical Science (Mark Publishing Company, Easton, the U.S.). The pharmaceutical composition may further contain a pharmaceutically acceptable carrier or additive. The example of a carrier or additive includes a surfactant, an excipient, a colorant, a fragrance, a preservative, a stabilizer, a buffer, a suspending agent, an isotonic agent, a binder, a disintegrant, a lubricant, a flow promoter, a flavor modifier, but is not limited to these, and other conventionally used carriers and additives. Its specific example includes light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carboxymethylcellulose calcium, sodium carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinyl pyrrolidone, gelatin, medium chain triglyceride fatty acid esters, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, glucose etc.

[0094] It should be noted that all prior art documents cited herein are incorporated herein by reference.

[0095] The present invention is further illustrated by, but not limited to, the following examples.

[0096] Example Example 1 Evaluation of the efficacy of artificial sequence peptides in acute traumatic brain injury (1) Materials and methods i) Peptide production An artificial sequence peptide consisting of the amino acid sequence described in SEQ ID NO: 1 was chemically synthesized (the obtained peptide was in the form of a trifluoroacetic acid or hydrochloric acid (TFA or HCl) salt).

[0097] ii) Preparation of a mouse model of traumatic brain injury (TBI) Male C57BL / 6J Jcl mice (8 weeks old) are purchased from CLEA, Japan, and then acclimated for five days or longer. Before surgery, mice were anesthetized with isoflurane inhalation anesthesia, the hair on the top of the mouse head was shaved, and the mouse head was then fixed in a stereotaxic head frame. After the parietal skin was disinfected with 70% ethanol, a midline incision was made in the parietal region with a scalpel. Fascia was removed with cotton swabs to expose the skull, the bregma was determined, and a mark was made on the skull 2mm from the right side and the caudal side 2mm from the bregma. A circle of 3mm in diameter was drawn with the mark as the center, bone was cut along the circle with a dental drill, and once the dura mater was reached, skull fragments were slowly removed with tweezers. If there was bleeding, cotton swabs were used to wipe and stop the bleeding. Then use impactor (Leica) to produce traumatic contusion perpendicular to the mouse cortical surface with 2mm diameter, 1.0mm depth, 4.0 meters per second speed, 0.2 second duration and 10 degree slope. The procedure is widely used by researchers studying traumatic brain injury and is the most consistent and reproducible form of injury. After injury, mice are taken out from the stereotaxic head frame and allowed to breathe. The incision is only sutured with a small clip, gentamicin is applied, and then mice are returned to their home cage (home cage). Except taking out skull fragments followed by using impactor to produce traumatic contusion, sham-operated (false) mice experience the same surgical procedure as above. Those with ruptured or damaged dura mater are excluded during preparation and are not used for any further experiment.

[0098] iii) Administration of peptides Traumatic brain injury model mice prepared as described above were divided into a group treated with artificial sequence peptides (n=6) and a control group (n=6). Peptide (hydrochloride) (1.39 mg / kg, or about 0.3 μmol / kg) (10 mL / kg dose volume) was administered once three hours after TBI surgery. The control group was injected with the same volume of saline via the tail vein on the same schedule as the peptide-treated group. Sham-operated (sham) mice (n=6) were also injected with saline via the tail vein in the same manner as the control group.

[0099] iv) Evaluation of motor function Motor function was evaluated by a balance beam test. Specifically, a stainless steel beam 1 m long and 1 cm wide was placed from the front (as the starting point) to the back at a 10 ° slope, and the mice were allowed to walk across the beam spontaneously. The time taken for the mice to cross from the starting point (execution time to cross the beam) and the number of slips during the time were counted. The experiment was performed before the administration of the peptide, basically once a day and once a week. Each mouse was trained twice on two different days before TBI surgery.

[0100] v) Statistical processing methods Each measurement is expressed as mean ± standard error of the mean (mean ± SEM). Comparisons between the control group and the peptide-treated group were performed using the Mann-Whitney U test for the number of slips and the Student's t-test for the execution time across the beam. P values < 0.05, < 0.01 or < 0.001 were considered statistically significant.

[0101] (2) Results In the balance beam test, a significant reduction in the number of slips (*p<0.05) and execution time to cross the beam (*p<0.05, **p<0.01) was observed in the peptide-treated group compared with the control group ( Figure 1-1 and 1-2 ). These results demonstrate the efficacy of artificial sequence peptides in improving motor function in acute traumatic brain injury.

[0102] Example 2 Evaluation of the efficacy of artificial sequence peptides in subacute traumatic brain injury (1) Materials and methods i) Preparation of a mouse model of traumatic brain injury Traumatic brain injury model mice and sham-operated (sham) mice were prepared in the same manner as described in Example 1.

[0103] ii) Administration of peptide The traumatic brain injury model mice prepared as described above were divided into a peptide-treated group (n=6) and a control group (n=6). Peptide (trifluoroacetate) (1.77 mg / kg, or approximately 0.3 μmol / kg) was administered via the tail vein as a single dose (10 mL / kg dose volume) on day 14 after TBI surgery. The control group was injected with the same volume of saline via the tail vein on the same schedule as the peptide-treated group. As in the control group, sham-operated (fake) mice (n=6) were also injected with saline via the tail vein.

[0104] iii) Evaluation of motor function Motor function was assessed by the balance beam test as described in Example 1.

[0105] (2) Results In the balance beam test, a significant reduction in the number of slips (**p<0.01) and execution time to cross the beam (*p<0.05, **p<0.01) was observed in the peptide-treated group compared with the control group ( Figure 2-1 and 2-2 ). These results demonstrate the efficacy of artificial sequence peptides in improving motor function in subacute traumatic brain injury.

[0106] Example 3 Efficacy of artificial sequence peptides in chronic traumatic brain injury (1) Materials and methods i) Preparation of a mouse model of traumatic brain injury (TBI) Traumatic brain injury model mice and sham-operated (sham) mice were prepared by the same method as described in Example 1.

[0107] ii) Administration of peptide The traumatic brain injury model mice prepared as described above were divided into a peptide-treated group (n=10) and a control group (n=10). Peptide (trifluoroacetate) (3 mg / kg, or about 0.5 μmol / kg) was administered via the tail vein (10 mL / kg dose volume) for a total of 16 doses: once a day for five consecutive days starting on day 48 after TBI surgery and then twice a week starting the following week. The control group was injected with the same volume of saline via the tail vein on the same schedule as the peptide-treated group. As in the control group, sham-operated (sham) mice (n=9) were also injected with saline via the tail vein.

[0108] iii) Evaluation of motor function Motor function was evaluated by the balance beam experiment as described in Example 1. In addition, a rotating rod experiment was performed. Each mouse was trained for 2 minutes at 10 rpm, rested for 30 minutes, and then subjected to the actual experiment. During the experiment, the rotating rod (Rotarod) was set to accelerate from 0 rpm to 40 rpm in 5 minutes. The mouse was placed on the rotor, and the time (in seconds) it took for each mouse to no longer keep up with the rotor speed was measured. Starting three days before surgery, the mouse was pre-trained for 2 minutes at 10 rpm once a day for two days.

[0109] iv) Statistical processing methods Each measurement result is expressed as mean ± standard error of the mean (mean ± SEM). Comparisons between the control group and the peptide-treated group were performed using the Mann-Whitney U test for the number of slips and the Student's t-test for the execution time of crossing the beam and the time spent on the rotor. P values < 0.05, < 0.01 or < 0.001 were considered statistically significant (2) Results In the balance beam test, a significant reduction in the number of slips (***p<0.001) and execution time to cross the beam (**p<0.01, ***p<0.001) was observed in the peptide-treated group compared with the control group ( Figure 3-1 and 3-2 In the rotarod test, the peptide-treated group showed a significant increase in the time spent on the rotor compared to the control group (**p<0.01, ***p<0.001) ( Figure 4 ). These results demonstrate the efficacy of artificial sequence peptides in improving motor function in chronic traumatic brain injury.

[0110] Example 4 Evaluation of the efficacy of artificial sequence peptides in severe traumatic brain injury (1) Materials and methods i) Preparation of a mouse model of severe traumatic brain injury (TBI) Traumatic brain injury model mice and sham-operated (sham) mice were prepared by the same method as described in Example 1, except that the contusion conditions were changed to 2.0 mm with the impactor. A deeper impactor depth allowed the production of more severe traumatic brain injury model mice.

[0111] ii) Administration of peptide The traumatic brain injury model mice prepared as described above were divided into a peptide-treated group (n=9) and a control group (n=9). Peptide (hydrochloride) (1.39 mg / kg, or approximately 0.3 μmol / kg) was administered via the tail vein (10 mL / kg dose volume) for a total of 16 doses: once a day for five consecutive days starting on day 32 after TBI surgery and then twice a week starting the following week. The control group was injected with the same volume of saline via the tail vein on the same schedule as the peptide-treated group. As in the control group, saline was also injected into sham-operated (sham) mice (n=7) via the tail vein.

[0112] iii) Evaluation of motor function Motor function was assessed by the balance beam test and the rotarod test as described in Example 3.

[0113] iii) Evaluation of memory function Memory function was evaluated by Y-maze test. Figure 5The device shown in is carried out, it has three arms of the same size (hereinafter referred to as A, B and C) connected with Y-shape. The entrance plate of C arm is blocked to allow only to walk in two directions (A, B), and every mouse training 3 minutes. In actual Y maze experiment, record the direction (from arm A to arm B, from arm A to arm C etc.) that every mouse enters each arm for 8 minutes. When whole body is inside arm, record and enter. Count and observe the number of times (for example, count A→B→C, but do not count A→B→A) that enter different arms for three consecutive times, and the total number of times that arm enters. Use the following formula to calculate alternation, it is an index of spatial working memory (spatial working memory): iv) Statistical processing methods Each measurement result is expressed as mean ± standard error of the mean (mean ± SEM). Comparisons between the control group and the peptide-treated group were performed using the Mann-Whitney U test for the number of slips and the Student's t-test for the execution time of crossing the beam and the time spent on the rotor. P values < 0.05, < 0.01 or < 0.001 were considered statistically significant (2) Results In the balance beam test, the peptide-treated group showed a significant decrease in the number of slips and the execution time to cross the beam compared to the control group, and in the rotarod test, the peptide-treated group showed a significant increase in the time spent on the rotor compared to the control group (figure not shown). In addition, in the Y-maze test, the peptide-treated group showed a trend toward recovery of memory function compared to the control group ( Figure 6 ). These results demonstrate the efficacy of artificial sequence peptides in the treatment of severe traumatic brain injury.

[0114] Example 5 Evaluation of the efficacy of artificial sequence peptides in subacute traumatic brain injury (1) Materials and methods i) Peptide production An artificial sequence peptide consisting of the amino acid sequence described in SEQ ID NO: 3 was chemically synthesized (the obtained peptide was in the form of a hydrochloric acid (HCl) salt).

[0115] ii) Preparation of a mouse model of traumatic brain injury Traumatic brain injury model mice were prepared by the same method as described in Example 1.

[0116] iii) Administration of peptides Traumatic brain injury model mice prepared as described above were divided into a peptide-treated group (n=6) and a control group (n=6). Peptide (hydrochloride) (1.28 mg / kg, or approximately 0.3 μmol / kg) was administered as a single dose (10 mL / kg dose volume) via the tail vein on day 14 after TBI surgery. The control group was injected with the same volume of saline via the tail vein on the same schedule as the peptide-treated group.

[0117] iv) Evaluation of motor function Motor function was assessed by the balance beam test as described in Example 1.

[0118] (2) Results In the balance beam test, the peptide-treated group showed a significant reduction in the number of slips (*p<0.05, **p<0.01) and execution time to cross the beam (*p<0.05, **p<0.01) compared with the control group ( Figure 7-1 and 7-2 ). These results demonstrate that the peptide consisting of the amino acid sequence described in SEQ ID NO: 3 is effective in improving motor function in subacute traumatic brain injury, similarly to the peptide consisting of the amino acid sequence described in SEQ ID NO: 1.

[0119] In addition, peptides (in the form of hydrochloride) consisting of the amino acid sequences described in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 were synthesized and prepared as described above. When these peptides were tested in the same manner as in Example 5, they showed a trend toward improvement in motor function after traumatic brain injury.

[0120] Industrial Applicability The peptides and nucleic acids of the present application can be used to prevent or treat brain damage.

Claims

1. A pharmaceutical composition for preventing or treating brain damage, comprising a peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (c) A peptide comprising an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO:

1.

2. The pharmaceutical composition according to claim 1, wherein the brain injury is focal or diffuse brain injury.

3. The pharmaceutical composition according to claim 1, wherein the brain injury is selected from the group consisting of cerebral contusion, epidural hematoma, subdural hematoma, intracerebral hematoma, concussion, diffuse axonal injury and subarachnoid hemorrhage. The pharmaceutical composition according to claim 1 , wherein the brain injury is traumatic brain injury.

5. A peptide selected from the group consisting of: (a) a peptide comprising the amino acid sequence described in SEQ ID NO: 1; (b) a peptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, deleted, inserted or added; and (c) A peptide comprising an amino acid sequence having about 80% or greater sequence identity with the amino acid sequence set forth in SEQ ID NO:

1. A composition comprising the peptide according to claim 5 .

7. A pharmaceutical composition comprising the peptide according to claim 5.

8. A nucleic acid encoding the peptide according to claim 5.

9. A vector comprising the nucleic acid according to claim 8.

10. A host cell comprising the nucleic acid according to claim 8 or the vector according to claim 9.

11. A composition comprising the nucleic acid according to claim 8 or the vector according to claim 9.

12. A pharmaceutical composition comprising the nucleic acid according to claim 8 or the vector according to claim 9.

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