Pharmaceutical composition for injection comprising MK145 protein and saline for prevention or treatment of ischemic stroke

Abstract

The present invention relates to a protein for the prevention or treatment of neurological diseases and a pharmaceutical composition containing the same. According to one aspect, the protein or polynucleotide exhibits excellent therapeutic effects in animal models of stroke, cerebral palsy, dementia, Parkinson's disease, and autism, and thus has the effect of being useful for the prevention or treatment of neurological diseases that are difficult to regenerate.

Description

Technology Field

[0001] The present invention relates to a protein for the prevention or treatment of neurological diseases and a pharmaceutical composition containing the same. Background Technology

[0002] The neurological system refers to a body control system composed of the brain, spinal cord, cranial nerves, spinal nerves, and the autonomic nervous system. Diseases of the neurological system are diverse, including cerebral palsy, traumatic brain injury, hypoxic brain injury, ischemic brain injury, stroke, cerebral infarction, cerebral hemorrhage, autism, schizophrenia, intellectual disability, Parkinson's disease, Alzheimer's disease, Huntington's disease, dementia, ALS (Lou Gehrig's disease), Pick's disease, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, ataxia, multiple sclerosis, neurological dysfunction, memory loss, epilepsy, encephalitis, prion disease, and neuropathy. Brain injury refers to a condition in which abnormalities in the brain's neural tissue occur due to various internal or external causes, resulting in behavioral or functional abnormalities. Brain injury can be caused by open head injury, closed head injury, deceleration injury, exposure to toxic substances, oxygen deficiency, tumors, infections, or cerebrovascular diseases such as stroke.

[0003] Stroke is caused by ischemia or hemorrhage and is a disease with a high mortality rate, along with cancer and heart disease. In the case of neurological diseases such as stroke, there is a limitation in that brain damage requires periodic examinations and rehabilitation treatment to address neuromotor and musculoskeletal disorders that develop over time.

[0004] Cerebral palsy is not a single disease but a collective term for syndromes with similar clinical characteristics, referring to a clinical syndrome characterized by motor and postural disorders resulting from non-progressive lesions or damage to the immature brain. Cerebral palsy is a condition caused by damage to the developing brain, which leads to problems with muscle control or maintaining posture while walking. While brain damage primarily occurs around birth or during birth, it can occur at any time during pregnancy and even develop in childhood.

[0005] Although brain damage is non-progressive, the patterns of neuromotor disorders and musculoskeletal disorders change over time; therefore, rehabilitation treatment most suitable for the changed clinical manifestations must be provided through periodic examinations. While there are other treatment methods, there is currently no groundbreaking and fundamental cure.

[0006] Neurodegenerative diseases are conditions characterized by degenerative changes in neurons of the central nervous system, leading to various symptoms such as impairment of motor and sensory functions, and inhibition of higher-order causative functions like memory, learning, and computational reasoning. Representative diseases include Alzheimer's disease, Parkinson's disease, and memory impairment. Neurodegenerative diseases involve the death of neurons due to necrosis or apoptosis, which can progress rapidly or slowly. Therefore, understanding the mechanisms of neuronal death is essential for the prevention, control, and development of treatments for neurodegenerative diseases.

[0007] Currently, as the rate of late pregnancies increases, the risk of neurological disorders such as cerebral palsy in children is rising, and as modern society ages rapidly, degenerative neurological diseases such as Alzheimer's disease are also surging.

[0008] Neurological diseases such as stroke and dementia commonly involve the death of brain or nerve cells, resulting in irreversible neurological dysfunction. Currently, various treatments using compounds and stem cells are being attempted in animal models worldwide, but they face many challenges when applied to clinical trials. Therefore, there is a need for the development of therapeutic agents capable of fundamentally treating these neurological diseases. Prior art literature

[0009] Korean Registered Patent No. 10-2079225 The problem to be solved

[0010] One aspect is to provide a protein containing the amino acid sequence of SEQ ID NO. 1.

[0011] Another aspect is to provide a polynucleotide encoding the above protein.

[0012] Another aspect is to provide a recombinant vector containing the above-mentioned polynucleotide.

[0013] Another aspect is to provide cells that have been transformed with the aforementioned vector.

[0015] Another aspect is to provide a composition for cell culture comprising any one selected from the group consisting of the above protein and the above polynucleotide.

[0016] Another aspect is to provide a pharmaceutical composition for the prevention or treatment of brain and nervous system diseases comprising, as an active ingredient, any one selected from the group consisting of the protein and the polynucleotide.

[0017] Another aspect provides a method for preventing or treating neurological diseases, comprising the step of administering the protein, the polynucleotide, or the composition to an individual in need thereof.

[0018] Another aspect is to provide the use of the protein, the polynucleotide, or the composition for use in the manufacture of a pharmaceutical composition for the prevention or treatment of neurological diseases.

[0019] Another aspect is to provide the use of the protein, the polynucleotide, or the composition for the prevention or treatment of neurological diseases. means of solving the problem

[0020] One aspect provides a protein comprising the amino acid sequence of SEQ ID NO. 1.

[0021] In the present invention, SEQ ID NO. 1 represents the sequence of amino acid numbers 255-399 (145 amino acids) among the 3,229 amino acids of Human pericentrin-isoform X5 (PCNT-isoform X5; SEQ ID NO. 3).

[0022] The above Human pericentrin-isoform X5 (PCNT-isoform X5) is a different protein with a different amino acid sequence from the previously known PCNT, and is an undisclosed and unsearched protein. Specifically, compared to the existing PCNT protein sequence, PCNT-isoform X5 is a new protein in which the sequences 1288 to 1298 and 2732 to 2810 are added as new sequences. While the previously known PCNT is known to exist in the cytoplasm and play a role in the nucleus during cell division, PCNT-isoform X5 is different as it is a protein found in plasma.

[0023] In the present invention, the protein containing the amino acid sequence of SEQ ID NO. 1 can be regarded as a new substance different from the 3,229 amino acids of Human pericentrin (PCNT) and Human pericentrin-isoform X5. This is because the protein P145 (MK145) having the amino acid sequence of SEQ ID NO. 1 of the present invention accounts for approximately 4.5% of the total size of the PCNT or PCNT-isoform X5 protein, and thus the protein structures are different, and accordingly, they cannot be considered to perform the same role. Therefore, the protein containing the amino acid sequence of SEQ ID NO. 1 of the present invention is a substance that is newly discovered and defined, although it is composed of an amino acid sequence that matches a part of the PCNT or PCNT-isoform X5 sequence.

[0025] In the present invention, a protein having the amino acid sequence of SEQ ID NO. 1 is defined as "P145 (MK145)".

[0026] Another aspect provides a protein in which a cell-permeable peptide is additionally fused to the above protein.

[0027] In one embodiment, the protein may additionally include a cell-permeable peptide to increase the cell permeability of the protein.

[0028] The cell penetrating peptides (CPPs) mentioned above may be any peptide capable of penetrating a cell membrane, and may be one or more selected from the group consisting of, for example, membrane-translocation sequences (MTS) or fragments thereof (including five or more consecutive amino acids of the membrane-translocation sequence), macromolecule intracellular transduction domains (MTDs), TAT peptides, hydrophobic peptides, and cell-penetrating fusion peptides including basic peptides.

[0029] The above membrane transfer sequence may have an amino acid sequence of, for example, AAVALLPAVLLALLAP (Sequence No. 4), and the fragment thereof may have a peptide fragment consisting of 7 to 16 consecutive amino acids among the above amino acid sequences, for example, AAVALLP (Sequence No. 5) or AVLLALLAP (Sequence No. 6), but is not limited thereto.

[0030] The fusion peptide of the hydrophobic peptide and the basic peptide may comprise: a hydrophobic peptide comprising 5 to 100, 5 to 50, 5 to 40, or 6 to 30 amino acids, comprising hydrophobic amino acids in a ratio of 60% or more, 70% or more, 80% or more, or 90% or more based on the total number of amino acids, e.g., 60 to 100%, 70 to 100%, 80 to 100%, or 90 to 100%; and a basic peptide comprising a peptide unit comprising 1 to 6 amino acids, or a repeating unit comprising 2 to 6 repetitions of the peptide unit, and composed of basic amino acids.

[0031] In one embodiment, the protein may additionally include a sequence for expression and / or purification. For example, for expression and / or purification, it may additionally include 1 to 30 amino acids at the N-terminus, C-terminus, or both ends of SEQ ID NO. 1, but is not limited thereto. The protein to which the sequence for expression and / or purification has been added may include the amino acid sequence of SEQ ID NO. 7, but is not limited thereto.

[0032] The sequence for purification above may be a His-tag sequence, but is not limited thereto.

[0033] Another aspect provides a protein comprising the amino acid sequence of SEQ ID NO. 1, wherein 1 to 30 amino acids are added to the N-terminus of SEQ ID NO. 1 in the direction of the N-terminus starting from the 254th amino acid of SEQ ID NO. 3, or 1 to 30 amino acids are added to the C-terminus of SEQ ID NO. 1 in the direction of the C-terminus starting from the 400th amino acid of SEQ ID NO. 3.

[0034] In the present invention, SEQ ID NO 3 represents the amino acid sequence of Human pericentrin-isoform X5 (PCNT-isoform X5).

[0035] The amino acid added to the above protein may be added to only one side of the N-terminus or C-terminus of the protein, or may be added to both sides.

[0036] The amino acids added to the N-terminus or C-terminus of the above protein may be at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, or 30.

[0037] In this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to polymers of amino acid residues, for example, as commonly found in proteins in their natural state.

[0038] The single (or three) letters of amino acids used in this specification refer to the following amino acids in accordance with standard abbreviation rules in the field of biochemistry: A(Ala): Alanine; C(Cys): Cysteine; D(Asp): Aspartic acid; E(Glu): Glutamic acid; F(Phe): Phenylalanine; G(Gly): Glycine; H(His): Histidine; I(IIe): Isoleucine; K(Lys): Lysine; L(Leu): Leucine; M(Met): Methionine; N(Asn): Asparagine; O(Ply): Pyrrolysine; P(Pro): Proline; Q(Gln): Glutamine; R(Arg): Arginine; S(Ser): Serine; T(Thr): Threonine; U(Sec): Selenocysteine, V(Val): Valine; W(Trp): Tryptophan; Y(Tyr): Tyrosine.

[0039] The peptide of the present invention, i.e., the peptide represented by SEQ ID NO. 1, includes a functional equivalent thereof. A functional equivalent refers to a peptide having at least 70%, preferably 80%, and more preferably 90% sequence homology (or identity) with the amino acid sequence of the peptide of the present invention. For example, it refers to a peptide that exhibits substantially the same physiological activity as the polypeptide of the present invention, comprising a polypeptide having sequence homology of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Here, the term "substantially" means a state of exhibiting the entirety of a specific property or a property of nearly the same degree.

[0040] In this document, "homology or identity" refers to the overall relatedness between polymer molecules, such as peptide molecules. For example, the calculation of homology / identity (%) between two polypeptide sequences may be performed by arranging the two sequences for optimal comparison. Preferably, the length of the sequence arranged for comparison purposes may be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or substantially 100% of the length of the reference sequence. Then, the amino acids at corresponding amino acid sites are compared with each other. If the amino acid located at a site of the first sequence is identical to the amino acid at a corresponding site of the second sequence, the two sequences have identity at that site. The identity (%) of two sequences is a function of the number of regions containing common amino acids in the two sequences, taking into account the number and length of gaps that must be introduced for optimal alignment between the two sequences. The comparison of two sequences and the determination of identity (%) can be performed using mathematical algorithms. For example, sequence identity can be measured using ClustalW (Thompson et al., 1994) with the following parameters: Pair alignment parameter - Method: Accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10; Multi-array parameter - Matrix: PAM, Gap open penalty: 10.00, delay equality (%): 30, penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, residue-specific gap penalties: on, hydrophilic gap penalty: on, hydrophilic residue: GPSNDQEKR. Sequence identity at a specific residue simply involves the same residue that has been derivatized.

[0041] In this document, the term "substantially" means a state exhibiting the entirety or nearly identical degree of a particular property. In this document, the term "substantially identical" is used in relation to the comparison between amino acid or nucleic acid sequences. To those skilled in the art to which the present invention pertains, two sequences will be understood as "substantially identical" if they have identical residues at corresponding sites. As is well known in the art, amino acid or nucleic acid sequences can be compared using various types of algorithms; for example, computer programs such as BLASTN can be used for nucleic acid sequence comparison, and BLASTP, gapped BLAST, and PSI-BLAST can be used for amino acid sequence comparison. Examples of the aforementioned computer programs are described in the following literature: Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to searching for identical sequences, the computer programs mentioned above typically provide the degree of identity of the sequence.Two sequences are considered to be "substantially identical" sequences if, over a certain length of residue, at least 70%, preferably at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% of the residues in the corresponding region are identical. Preferably, the "residue of a certain length" mentioned above may be at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, or more residues.

[0042] In this document, the term “corresponding” is often used to determine the position / identity of amino acid residues of a given polypeptide. To a person skilled in the art, residues within a polypeptide are often designated using a canonical numbering system based on a reference-related polypeptide. Thus, for example, the amino acid “corresponding” to the residue at the 190th position does not necessarily have to be at the 190th position in a specific amino acid chain, and a person skilled in the art can easily understand how to identify the “corresponding” amino acid.

[0043] The "functional equivalents" mentioned above may be produced as a result of addition, substitution, or deletion of a portion of the amino acid sequence of the polypeptide of the present invention. In the above, the substitution of amino acids is preferably a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), and sulfur-containing amino acids (Cys, Met). Additionally, the functional equivalents include variants in which a portion of amino acids is deleted from the amino acid sequence of the polypeptide of the present invention. The deletion or substitution of amino acids is preferably located in a region that is not directly related to the physiological activity of the polypeptide of the present invention. Furthermore, the deletion of amino acids is preferably located in a portion that is not directly involved in the physiological activity of the polypeptide of the present invention. In addition, variants in which several amino acids are added to both ends or within the amino acid sequence of the polypeptide of the present invention are also included. Furthermore, the scope of functional equivalents of the present invention also includes proteins or polypeptide derivatives in which a portion of the chemical structure of the protein is modified while maintaining the basic framework of the protein according to the present invention and its physiological activity. For example, structural modifications to alter the stability, storage properties, volatility, or solubility of the protein of the present invention are included therein.

[0044] In this specification, sequence homology and homology are defined as the percentage of amino acid residues of the candidate sequence relative to the amino acid sequence of the polypeptide of the present invention after aligning the amino acid sequence of the polypeptide of the present invention with the candidate sequence and introducing a gap. If necessary, conservative substitutions are not considered as part of the sequence homology in order to obtain the maximum percentage sequence homology. Furthermore, N-terminal, C-terminal, or internal elongation, deletion, or insertion of the amino acid sequence of the polypeptide of the present invention is not interpreted as a sequence affecting sequence homology or homology. Additionally, said sequence homology can be determined by general standard methods used to compare similar portions of the amino acid sequences of two proteins or polypeptides. BLAST or such computer programs align two proteins or polypeptides so that their respective amino acids are optimally matched (along the full length of one or two sequences or along a predicted portion of one or two sequences). The above program provides a default opening penalty and a default gap penalty, and provides a scoring matrix such as PAM250 (standard scoring matrix; Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp 3, 1978) that can be used in conjunction with a computer program. For example, percentage homogeneity can be calculated as follows: multiply the total number of identical matches by 100 and then divide by the sum of the length of the longer sequence within the corresponding matched span and the number of gaps introduced into the longer sequence to align the two sequences.

[0045] The polypeptide of the present invention can be constructed by genetic engineering methods. First, a polynucleotide sequence encoding the polypeptide of the present invention is constructed according to conventional methods. The polynucleotide sequence can be constructed, for example, by PCR amplifying a polynucleotide encoding the human PCNT or PCNT-isoform X5 gene using appropriate primers as a template. Alternatively, the DNA base sequence may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (sold by Biosearch or Applied Biosystems). The constructed polynucleotide sequence is inserted into a vector containing one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operatively linked to the polynucleotide and regulate the expression of the polynucleotide base sequence, and host cells are transformed with the recombinant expression vector formed therefrom. The generated transformant is cultured under appropriate medium and conditions so that the DNA sequence is expressed, and a substantially pure protein encoded by the DNA sequence is recovered from the culture. The recovery can be performed using methods known in the art (e.g., chromatography).

[0046] In the foregoing, the term "substantially pure polypeptide or protein" means that it substantially does not contain any other protein derived from the protein host cell according to the present invention. For the genetic engineering method for protein synthesis of the present invention, reference may be made to the following literature: Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrook et al., supra; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; and Hitzeman et al., J. Biol. Chem., 255:12073-12080, 1990.

[0047] In addition, the polypeptide of the present invention can be chemically synthesized using techniques known in the art (Creighton, Proteins: Structures and Molecular Principles, WH Freeman and Co., NY (1983)). That is, the polypeptide of the present invention can be prepared using conventional stepwise liquid or solid-phase synthesis, fragment condensation, F-MOC or T-BOC chemistry methods (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, (1997); A Practical Approach, Atherton & Sheppard, Eds., IRL Press, Oxford, England, (1989)). A preferred method of preparation is to use a solid-phase synthesis method. The polypeptide of the present invention can be synthesized by a conventional solid-phase method through a condensation reaction between protected amino acids, starting from the C-terminus and proceeding sequentially according to the identified amino acid sequence. After the condensation reaction, the carrier to which the protecting group and the C-terminal amino acid are linked can be removed by known methods such as acid decomposition or aminolysis. The aforementioned peptide synthesis method is described in detail in a relevant book (Gross and Meienhofer's, The Peptides, vol 2., Academic Press, 1980).

[0048] Proteins produced by genetic engineering methods or chemically synthesized proteins can be separated and purified by methods known in the art, such as extraction, recrystallization, various chromatography (gel filtration, ion exchange, precipitation, adsorption, reverse phase), electrophoresis, and reflux partitioning.

[0049] Another aspect provides a polynucleotide encoding a protein containing the amino acid sequence of SEQ ID NO. 1.

[0050] In one embodiment, the polynucleotide may be a polynucleotide comprising the nucleotide sequence of SEQ ID NO. 2.

[0051] In the present invention, SEQ ID NO 2 represents a DNA sequence that codes (encodes) P145.

[0052] In this specification, “polynucleotide” and “nucleic acid” refer to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in a single-stranded or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.

[0053] The above polynucleotides may be used without limitation as long as they encode the polypeptide of the present invention, and include DNA, cDNA, and RNA sequences. The above polynucleotides may be isolated from nature or prepared by genetic engineering methods known in the art.

[0054] Another aspect provides a recombinant vector containing the above-mentioned polynucleotide.

[0055] In the present invention, "vector" refers to a DNA product containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA within a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. When transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vectors currently, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention. For the purposes of the present invention, it is preferable to use a plasmid vector. A typical plasmid vector that can be used for this purpose has a structure comprising (a) a replication initiation site that enables efficient replication to contain hundreds of plasmid vectors per host cell, (b) an antibiotic resistance gene that enables the selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which an alien DNA fragment can be inserted. Even if a suitable restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adapter or linker according to conventional methods.

[0056] The vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Suitable vectors are expression vectors and may include expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and can be prepared in various ways depending on the purpose. The vectors of the present invention are any means used to deliver nucleic acids encoding the peptides of the present invention to host cells, and preferred vectors are viral vectors such as retroviruses, herpes viruses, adenoviruses, and adeno-associated viruses. Accordingly, the gene encoding the peptides of the present invention is introduced in vivo, in vitro, or in vitro using viral vectors or through direct DNA introduction. Expression in target tissues can be carried out by targeting specific cells with mutant vectors using viral vectors or receptor ligands, using tissue-specific promoters, or using both methods.

[0057] Meanwhile, the standard recombinant DNA and molecular cloning techniques used in the present invention are widely known in the field and are described in the following literature (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989); by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987)).

[0058] Furthermore, in the present invention, "codon optimization" refers to changing the codons of a polynucleotide encoding a protein to be preferentially used in a specific organism so that the encoded protein is expressed more efficiently in the organism. Although the genetic code is degenerate in that most amino acids are represented by a few codons referred to as "synonymous" or "synonymous" codons, the codon usage by a specific organism is not random but biased toward specific codon triplets. This bias in codon usage may be higher in relation to specific genes, genes of common function or ancestral origin, highly expressed proteins versus low-copy number proteins, and collective protein-coding regions of the organism's genome.

[0059] Another aspect provides cells transformed with the above vector.

[0060] The cells of the present invention may be stem cells, progenitor cells, microorganisms, plant cells, or animal cells, but are not limited thereto. Furthermore, the stem cells may specifically be embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPS), mesenchymal stem cells, or reprogrammed stem cells, but are not limited thereto, and may include all stem cells differentiated from the stem cells, such as mesenchymal stem cells derived from embryonic stem cells, mesenchymal stem cells derived from induced pluripotent stem cells, neural stem cells derived from induced pluripotent stem cells, etc. Additionally, the cells may be autologous, allogeneic, allogenic, or xenogenic cells.

[0061] Transformation may be performed using known methods of introducing nucleic acids into an organism, cell, tissue, or organ, and may be carried out by selecting an available technique suitable for the host cell within the scope understood by those skilled in the art. Such methods include, but are not limited to, electroporation, protoplasmic fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring with silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine, etc.

[0062] The protein of the present invention can be easily expressed and mass-produced by culturing the transformed cells in a suitable medium, or by introducing the transformed cells into any animal and culturing them in vivo.

[0063] One embodiment provides a composition for cell culture comprising any one selected from the group consisting of the protein and the polynucleotide.

[0064] The above cell culture composition may include the cell.

[0065] Cells cultured in the composition of the present invention may have their cell functions enhanced by introducing the protein and the polynucleotide into the cell or acting extracellularly, but are not limited thereto.

[0066] The present invention also provides a pharmaceutical composition for the prevention or treatment of neurological diseases, comprising the protein or the polynucleotide as an active ingredient.

[0067] The present invention also provides a method for preventing or treating neurological diseases, comprising the step of administering the protein, the polynucleotide, or the composition to an individual in need thereof.

[0068] The present invention also provides the use of the protein, the polynucleotide, or the composition for use in the manufacture of medicines for the prevention or treatment of neurological diseases.

[0069] The present invention also provides the use of the protein, the polynucleotide, or the composition for the prevention or treatment of neurological diseases.

[0070] In one embodiment, the brain and nervous system disease may include all diseases in which an abnormality or disorder occurs in some or all of the brain, spinal cord, cranial nerves, spinal nerves, autonomic nervous system, etc., constituting the brain and nervous system.

[0071] In one embodiment, the neurological disease may be one or more selected from the group consisting of ischemic stroke, hemorrhagic stroke, hypoxic brain injury, traumatic brain injury, cerebral palsy, mental illness, and degenerative brain disease.

[0072] In another embodiment, the mental disorder may be one or more selected from the group consisting of autism spectrum, schizophrenia, intellectual disability, and Down syndrome.

[0073] In another embodiment, the degenerative brain disease comprises dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, amyloid stroke, systemic amyloid disease, Dutch-type amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's Syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, and frontotemporal dementia. It may be one or more selected from the military, but is not limited thereto.

[0074] The term "prevention" in this invention refers to any act of administering the composition of this invention to an individual to suppress or delay neurological diseases.

[0075] The term "treatment" as used in the present invention refers to any act of administering the composition of the present invention to an individual to improve or benefit from the symptoms of a neurological disease.

[0076] Proteins or polynucleotides included in the pharmaceutical composition of the present invention are not limited as long as they have a therapeutic or preventive effect on brain and nervous system diseases.

[0077] In one embodiment, when the active ingredient is a protein or a polynucleotide, it may be included in an amount of 0.0001 to 99.9% by weight, more specifically 0.01 to 80% by weight, based on the total weight of the final composition. In one embodiment, the protein or polynucleotide included in the pharmaceutical composition of the present invention may be included at a concentration greater than 0 and less than or equal to 10 μM, for example, at a concentration of 0.9 μM to 4.5 μM or 1.8 μM to 3.6 μM. In another embodiment, the protein or polynucleotide included in the pharmaceutical composition of the present invention may be included at a concentration greater than 0 and less than or equal to 500 nM, for example, at a concentration greater than 0 and less than or equal to 100 nM, greater than 0 and less than or equal to 50 nM, or greater than 0 and less than or equal to 40 nM. However, it is not limited thereto, and an effective dose may be appropriately selected depending on the target of administration.

[0078] The pharmaceutical composition of the present invention may further comprise a suitable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions. As used in the present invention, the term "pharmaceuticalally acceptable carrier" means a carrier or diluent that does not stimulate living organisms and does not impair the biological activity and properties of the administered compound.

[0079] The types of carriers usable in the present invention are not particularly limited, and any carrier that is commonly used and pharmaceutically acceptable in the relevant technical field may be used. Non-limiting examples of carriers include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, etc. These may be used alone or in a mixture of two or more types.

[0080] In addition, if necessary, other conventional additives such as antioxidants, buffers and / or bacteriostatic agents may be added and used, and diluents, dispersants, surfactants, binders and / or lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. The pharmaceutical composition of the present invention can be prepared in various formulations depending on whether the intended method of administration is oral or parenteral.

[0081] Non-limiting examples of formulations for oral administration include troches, lozenges, tablets, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs.

[0082] In order to formulate into an oral dosage form such as the above-mentioned tablet or capsule, the formulation may include a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; and a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. Furthermore, in the case of a capsule formulation, in addition to the aforementioned substances, a liquid carrier such as a fatty oil may be additionally contained.

[0083] Formulations for parenteral administration may include, for example, injectable forms such as subcutaneous injection, intravenous injection, or intramuscular injection; suppository administration methods; or formulations for sprays such as aerosols that allow for inhalation through the respiratory tract, but are not limited thereto. To formulate the above-mentioned injectable formulations, the composition of the present invention may be mixed in water with a stabilizer or a buffer to prepare a solution or suspension, and the same may be formulated for unit administration in ampoules or vials. When formulating for sprays such as aerosols, propellants or the like may be combined with additives to disperse the water-dispersed concentrate or wet powder.

[0084] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, wherein the term "pharmaceutically effective amount" in the present invention means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level may be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and elimination rate, the duration of treatment, drugs combined or used concurrently with the composition of the present invention used, and other factors well known in the medical field.

[0085] The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered as a single or multiple doses. Taking all of the above factors into consideration, an amount that obtains maximum effect with a minimum amount without side effects may be administered.

[0086] The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once or several times. The above dosage does not limit the scope of the present invention in any way. Effects of the invention

[0087] Proteins or polynucleotides according to one aspect exhibit excellent therapeutic effects on stroke, cerebral palsy, dementia, Parkinson's disease, and autism spectrum disorders in animal models of stroke, cerebral palsy, dementia, Parkinson's disease, and autism, and thus have the potential to be usefully employed in the prevention or treatment of neurological diseases that are difficult to regenerate. Brief explanation of the drawing

[0088] Figure 1 shows the results of staining with Coomassie brilliant blue of the P145(MK145) recombinant protein obtained by protein expression and purification after transforming pET28a-P145(MK145) plasmid DNA into E. coli. Figure 2 shows the results of confirming the expression level of P145 (MK145) by RT-PCR after transfecting P145 into ES-MSCs. Figure 3 shows the treatment schedule of ES-MSC and P145 (MK145) in MCAO, a stroke model. Figure 4 shows the mNSS test results for days 1, 3, 7, 14, 21, and 28 for groups administered ES-MSC and ES-MSC+P145 (MK145) to an animal model of stroke. Figure 5 shows the scores of improved behavioral ability from day 7 to day 28, the days of stem cell administration. Figure 6 shows the results of the modified Neurological Severity Score (mNSS) test on days 1, 3, 7, 14, 21, and 28 for groups administered P145 (MK145) protein at different concentrations in an animal model of ischemic stroke. Figures 7 and 8 show the results of confirming neuronal markers (NeuN) by immunohistochemistry using brain tissue from an animal model of ischemic stroke administered with P145 (MK145) at 100 μg / kg. Figures 9 to 12 show the results of identifying inflammation markers and apoptosis markers by Western blotting using brain tissue from a stroke animal model administered with P145 (MK145) at 10 μg / kg. Figure 13 shows the treatment schedule of P145 (MK145) in an intracerebral hemorrhage (ICH) model. Figure 14 shows the mNSS test results for days 1, 3, 7, 14, 21, and 28 for groups administered P145 (MK145) at different concentrations to ICH. Figures 15 to 17 show the scores of improved behavioral ability in ICH from day 1 to days 14, 21, and 28, which are the days of P145 (MK145) administration. Figures 18 to 22 show the results of identifying inflammatory markers by RT-PCR using brain tissue from an animal model of cerebral hemorrhage administered with P145 (MK145). Figure 23 shows the treatment schedule of P145 (MK145) for neonatal hypoxic-ischemic brain injury (HI), a cerebral palsy model. Figure 24 shows the results of mNSS and cylinder test behavioral assessment scores 35 days after treatment with P145 (MK145) in a cerebral palsy model. Figures 25 and 26 show the results of identifying inflammation markers and oxidative stress (reactive oxygen stress, ROS) markers using RT-PCR with brain tissue from a cerebral palsy model administered P145 (MK145). Figure 27 shows the treatment schedule of P145 (MK145) in amyloid beta-injected animals, a dementia-inducing model. Figure 28 shows a graph of the Y-Maze and NORT test behavioral assessment scores after treatment with P145 (MK145) in a dementia model. Figures 29 and 30 show images and score graphs of nest-building behavior after treatment with P145 (MK145) in a gene expression dementia model. Figures 31 and 32 show images of stained amyloid plaques and graphs of the number of plaques after treatment with P145 (MK145) in a gene expression dementia model. Figure 33 shows the score graph of the Forced swimming and Tail suspension test behavioral evaluations after treatment with P145 (MK145) in an animal model of Parkinson's disease. Figures 34 and 35 show the results of identifying dopamine-related genes and proteins by ELISA and RT-PCR after treatment with P145 (MK145) in a Parkinson's animal model. Figure 36 shows the treatment schedule of P145 (MK145) in an autistic animal model. Figure 37 shows a graph of the sociality test scores after treatment with P145 (MK145) in an autistic animal model. Figures 38 and 39 show images and graphs of scores from the marble burying test behavior evaluation, an abnormal behavior of covering marbles after treatment with P145 (MK145) in an autistic animal model. Specific details for implementing the invention

[0089] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0090] Terms or words used in the specification and claims of the present invention shall not be interpreted as being limited to their ordinary or dictionary meanings, and shall be interpreted in a meaning and concept consistent with the technical spirit of the present invention, based on the principle that the inventor may appropriately define the concept of the terms to best describe his invention.

[0091] Throughout the specification of the present invention, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0092] Throughout the specification of the present invention, "A and / or B" means A or B, or A and B.

[0094] Example 1. P145 (MK145) Gene cloning

[0096] The P145 (MK145) protein (SEQ No. 1) used in this invention was prepared by recombinant protein expression and purification using the pET-28a expression vector. Using Human pericentrin-isoform X5 (PCNT-isoform X5, SEQ No. 3) as a template, upstream and downstream primers were designed (Cosmogenetech, South Korea) for the DNA sequence (SEQ No. 2) corresponding to amino acids 255–399 (145 amino acids) of the entire PCNT-isoform X5 protein sequence, and polymerase chain reaction (PCR) was performed. The sequences of each primer used are as follows.

[0097] Up primer: 5'-CGCCTA GCTAGC ATGGAGGATTTACAAAAC-3' (underlined is restriction enzyme) Nhe I Recognition sequence)(Sequence No. 8).

[0098] Down primer: 5'-AATATA GGATCC TTACTCCAGTTCGGACTCATG-3' (underlined is restriction enzyme) Bam HI recognition sequence)(Sequence No. 9).

[0099] DNA fragments obtained through PCR with restriction enzymes Nhe I BampET28a-P145 recombinant DNA was produced by introducing it into a pET28a plasmid vector (Novagen, Germany) obtained by cutting with HI and treating with the same restriction enzyme.

[0101] Example 2. P145 (MK145) Protein Expression and Purification

[0102] The constructed pET28a-P145 clone was transduced into E. coli strain BL21(DE3) to express the recombinant protein. After culturing the E. coli in LB broth medium for a certain period of time, isopropyl-β-D-thio-galactoside (IPTG) was added and the culture was further incubated at 30°C for 4 hours. The cultured E. coli was recovered by centrifugation, pulverized with an ultrasonic grinder, and the P145 protein was isolated by Ni-NTA affinity column chromatography.

[0103] The purified protein was verified by staining with Coomassie brilliant blue. The results of staining the P145 recombinant protein with Coomassie brilliant blue are shown in Figure 1.

[0105] Example 3. Transfection of stem cells for overexpression of P145

[0106] The synthesized pET28a-P145 recombinant DNA was transfected into embryonic stem cell-derived mesenchymal stem cells (ES-MSCs) using lipofectamine. Cells were harvested 24 hours after transfection, and the expression level was checked by RT-PCR. The results are shown in Figure 2. It was confirmed that the P145 protein was overexpressed compared to control cells.

[0108] Example 4. Evaluation of the therapeutic efficacy of P145 in a stroke animal model

[0109] 4.1. Establishment of an Animal Model of Ischemic Stroke

[0110] By inserting a monofilament suture through the internal carotid artery (ICA) to occlude the middle carotid artery, thereby blocking blood flow to the middle carotid artery (MCA), maintaining the suture results in a permanent model, while removing the suture to induce reperfusion results in a transient occlusion.

[0111] Male Sprague-Dawley (SD) rats (200–250 g) aged 6–8 weeks were kept at 21°C under 12 hours of light-dark conditions and provided with food and water.

[0112] For the stroke animal model, a transient middle cerebral artery occlusion (MCAO) model in which reperfusion occurred following MCA occlusion was constructed. SD rats were anesthetized by inhalation with 3% isoflurane, and 1.5% isoflurane was maintained during surgery using a respiratory anesthesia device. The midline of the neck was incised, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed while ensuring that the vagus nerve was not damaged. First, the CCA and ECA were sutured together with black silk sutures, and a small hole was made in the wall of the ECA using snips. A fabricated 4-0 nylon filament was inserted from the small hole in the ECA through the right internal carotid artery to the middle cerebral artery (1.5–1.8 mm) to occlude it. The skin of the neck was closed, and after 90 minutes, the inserted filament was removed to reperfuse blood into the middle cerebral artery. During the surgery, the rectal temperature was maintained at 37°C using a rectal thermometer.

[0114] 4.2. Administration of each ES-MSC and ES-MSC+P145 to an ischemic stroke animal model

[0115] ES-MSCs used p12 (passage 12) cells that had undergone 12 passages, and each cell was 5 x 10⁶ per vial. 6 Frozen with dog cells, 3x10 per rat when used 6 Dog cells were dissolved in 1 ml of physiological saline and injected via tail vein. ES-MSC+P145 was harvested 24 hours after P145 transfection, dissolved in physiological saline, and injected via tail vein. The control group was injected with the same amount of physiological saline.

[0116] The therapeutic agent was administered on the 7th day after the MCAO model was created, and all animal model experiments were performed blindly.

[0118] 4.3. Analysis of Behavioral Assessment in Ischemic Stroke Animal Models

[0119] The treatment schedule in the stroke model is shown in Figure 3.

[0120] Behavioral assessments were performed a total of 6 times on days 1, 3, 7, 14, 21, and 28 after surgery.

[0121] Modified Neurological Severity Score (mNSS): An analysis method with a maximum score of 14 points, where 0 points indicate normal behavior and the total score approaches 14 points as neurological deficits become more severe. Limb tone (total 3 points), gait (total 3 points), balance (total 6 points), and sensory function (total 2 points) were comprehensively evaluated and summed. The evaluation results are shown in Figures 4 and 5.

[0122] As a result, at 28 days post-surgery, the mNSS score in the ES-MSC+P145 group was 5.88, which was lower than that of the ES-MSC group (6.25), indicating the best therapeutic effect for stroke. In addition, the difference in behavioral ability scores between 7 days post-surgery and 28 days post-surgery was 4.13 in the ES-MSC+P145 group, which was greater than that of the ES-MSC group (2.63), indicating the best therapeutic effect for stroke.

[0123] Through this, it was found that overexpressing P145 has a superior therapeutic effect on stroke compared to simply treating MSCs. This implies that P145 can be used as a treatment for stroke.

[0125] 4.4. Administration of P145 (MK145) to an animal model of ischemic stroke

[0126] The newly prepared P145 (MK145) protein was diluted with physiological saline at concentrations of 1 μg / kg, 10 μg / kg, and 100 μg / kg. P145 protein was administered intraperitoneally to each rat of an ischemic stroke animal model for 5 days starting from day 2 after stroke model induction. The control group was administered the same amount of physiological saline.

[0128] 4.5. Confirmation of Therapeutic Efficacy Following Administration of P145 (MK145) in an Animal Model of Ischemic Stroke

[0129] As described above, the mNSS test was performed after administering P145 (MK145) protein at different concentrations to an animal model of ischemic stroke. The evaluation results are shown in Figure 6.

[0131] As a result, the higher the dose of P145 (MK145) protein administered, the lower the mNSS score according to the elapsed days. This indicates that neuronal recovery occurs in a dose-dependent manner as the dose of P145 (MK145) protein administered increases.

[0133] Immunostaining was performed using 20 μM thick samples of brain tissue from rats in an ischemic stroke animal model (control group and 100 μg / kg P145 (MK145) protein administration group) to identify NeuN, a neurodifferentiation marker. The results are shown in Figures 7 and 8.

[0134] As a result, it was confirmed that in brain tissue treated with the P145 (MK145) protein, neurons increased in both the cortex and striatum regions due to a neuroprotective effect near the damaged area. This implies that P145 (MK145) can be used as a therapeutic agent for stroke.

[0136] Western blotting was performed using brain tissues from normal rats and ischemic stroke animal model rats (control group and group administered 10 μg / kg P145 (MK145) protein). The results are shown in Figures 9 to 12.

[0137] In the group administered P145 (MK145), the expression levels of TNF-α protein, NLRP3 protein, Bax protein, Bcl-2 protein, ATF6 protein, and GRP78 protein were reduced compared to the control group.

[0138] Through this, it is possible to understand the therapeutic effects of P145 (MK145) protein administration on inflammasome inflammation, apoptosis, and ER stress caused by brain damage, and to understand that P145 (MK145) can be used as a treatment for stroke.

[0140] 4.6. Administration of P145 (MK145) to an animal model of hemorrhagic stroke

[0141] The treatment schedule in a hemorrhagic stroke model is shown in Fig. 13.

[0142] The newly prepared P145 (MK145) protein was diluted with physiological saline at concentrations of 1 μg / kg, 10 μg / kg, and 100 μg / kg. P145 protein was administered intraperitoneally to each rat of the hemorrhagic stroke animal model for 5 days starting from day 1 after induction of the hemorrhagic stroke model. The control group was administered the same amount of physiological saline.

[0144] 4.7. Confirmation of Therapeutic Efficacy of P145 (MK145) Administration in an Animal Model of Hemorrhagic Stroke

[0145] As described above, the mNSS test was performed after administering P145 (MK145) protein at different concentrations to an animal model of hemorrhagic stroke. The evaluation results are shown in Figures 14 and 15 to 17.

[0146] As a result, the higher the administered dose of P145 (MK145) protein, the lower the mNSS score according to the elapsed days. In addition, as the elapsed days increased, the improved scores increased, and the improved scores were higher with higher doses of P145 (MK145) protein. Through this, it can be seen that neurological recovery occurs better in a dose-dependent manner with higher doses of P145 (MK145) protein.

[0147] RT-PCR was performed and analyzed using brain tissues from normal rats and hemorrhagic stroke animal model rats (control group and P145 (MK145) protein administration group). The results are shown in Figures 18 to 22.

[0148] As a result, depending on the cerebral hemorrhage, the mRNA expression levels of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 increased, leading to increased inflammation, but in the group administered P145 (MK145), the expression levels decreased compared to the control group.

[0149] Through this, the therapeutic effect of administering P145 (MK145) protein on inflammasome inflammation caused by brain damage can be understood, and it can be seen that P145 (MK145) can be used as a treatment for stroke.

[0151] Example 5. Evaluation of the therapeutic efficacy of P145 in an animal model of cerebral palsy

[0152] 5.1. Establishment of an Animal Model of Cerebral Palsy

[0153] An animal model of cerebral palsy was constructed using 7-day-old ICR mice.

[0154] The right common carotid artery (CCA) of 7-day-old ICR mice was ligated with 5-0 blue nylon, and then placed in a sealed special container maintained at 37°C with O28% and N292%, after which hypoxic-cerebral ischemia was induced for 1 hour.

[0155] Six days after model creation, the degree of brain tissue damage was visually checked, and models with more than 50% damage were excluded.

[0157] 5.2. Administration of P145 Protein to a Cerebral Palsy Animal Model

[0158] 100 ng of purified and extracted P145 protein was dissolved in 100 μl of physiological saline and administered via intraperitoneal administration.

[0159] The therapeutic agent was administered on the 7th day after the creation of the cerebral palsy model, and all animal model experiments were performed blindly.

[0161] 5.3. Analysis of Behavioral Assessment in Cerebral Palsy Animal Models

[0162] The treatment schedule in the cerebral palsy model is shown in Fig. 23.

[0163] Behavioral evaluation was performed once, 35 days after surgery and 28 days after the administration of the treatment.

[0164] Modified Neurological Severity Score (mNSS): An analysis method with a maximum score of 14 points, where 0 points indicate normal behavior and the total score approaches 14 points as neurological deficits become more severe. Limb tone (total 3 points), gait (total 3 points), balance (total 6 points), and sensory function (total 2 points) were evaluated and summed.

[0165] Cylinder test: As a method to measure exploration ability based on spatial sense and motor function, experimental animals were placed in a glass cylinder with a diameter of 15 cm and a height of 40 cm, and the number of times the animals stood up and touched the wall with their forepaws was observed a total of 20 times. For the analysis, the percentage of times the left forepaw, which showed impairment, touched the wall out of the total 20 times was calculated. The evaluation results are shown in Fig. 24.

[0166] As a result, at 35 days post-surgery, the mNSS score was found to be significantly reduced in the group administered P145, indicating a therapeutic effect for cerebral palsy. In addition, regarding the cylinder evaluation, the frequency of use of the impaired forelimb increased in the group administered P145 (MK145), indicating a therapeutic effect for cerebral palsy. Through this, it was found that P145 (MK145) can be used as a treatment for cerebral palsy.

[0168] 5.4. Confirmation of the therapeutic mechanism of P145 (MK145) in an animal model of cerebral palsy and stroke

[0169] RT-PCR was performed and analyzed using brain tissues from Siamese rats and cerebral palsy stroke animal model rats (control group and P145 (MK145) protein administration group). The results are shown in Figures 25 and 26.

[0170] As a result, depending on brain damage, the mRNA expression levels of inflammatory markers NLRP3, ASC, and Caspase-1, and ROS markers NOX2 and NOX4 increased, but in the group administered P145 (MK145), the expression levels decreased compared to the control group.

[0171] Through this, the therapeutic effect of P145 (MK145) protein administration on inflammation and increased ROS caused by brain damage can be understood, and it can be seen that P145 (MK145) can be used as a treatment for cerebral palsy.

[0173] Example 6. Evaluation of the therapeutic efficacy of P145 (MK145) in an animal model of dementia

[0174] 6.1. Establishment of an amyloid beta injection animal model

[0175] Male B6 mice weighing approximately 23–25 g at 6 weeks of age were used. Five mice were housed in each cage, and conditions were maintained at 21°C with a 12-hour light-dark cycle, and food and water were provided.

[0176] Amyloid beta 1-42, a substance that causes memory deficits, was dissolved in 10% DMSO (dimethyl sulfoxide) saline solution, cultured at 37°C for one week, and prepared at a concentration of 10 μM.

[0177] The experimental animals were anesthetized by inhalation with 3% isoflurane, and 1.5% isoflurane was maintained during surgery using a respiratory anesthesia device. After preparing the stereotaxic frame, the head of the experimental animal was fixed, and 5 μl of amyloid beta-1-42 was injected over 10 minutes using a 10 μl Hamilton microsyringe equipped with a 26-gauge needle at -0.9 mm posteriorly, 1.7 mm laterally, and 2.2 mm deep from the bregma.

[0179] 6.2. Administration of P145 (MK145) protein to amyloid beta injection animal model

[0180] 100 ng of purified and extracted P145 (MK145) protein was dissolved in 100 μl of physiological saline and administered via intraperitoneal (IP) and intravenous (IV) administration.

[0181] The therapeutic agent was administered on the 7th day after the creation of the dementia model, and all animal model experiments were performed blindly.

[0183] 6.3. Behavioral Assessment Analysis of Amyloid Beta-Injected Animal Models

[0184] The treatment schedule in an amyloid beta injection animal model is shown in Fig. 27.

[0185] Prior to the procedure, cognitive ability assessments using the Y-maze and NORT were trained and scored, after which a behavioral assessment was conducted. On the 7th day after the procedure, lesion models were selected to conduct the experiment. After administering the treatment 7 days after the procedure, cognitive function improvement was measured on the 7th and 14th days after treatment.

[0186] Y-maze Test: The device is shaped like the letter Y with three arms extending outwards. Each arm measures 35 cm in length, 15 cm in height, and 5 cm in width, and is positioned at the same angle. The movement of the experimental animals is measured by the number of alterations, which is defined as one alteration occurring when the animals pass through the three arms sequentially in succession (spontaneous alteration). Each animal was instructed to start in one maze and proceed to the other, and the number of successful correct alterations was scored and expressed as a percentage. Measurements were taken for 5 minutes after allowing the animals to move freely for 2 minutes.

[0187] Novel Object Recognition Test (NORT): This is a cognitive experimental technique designed to stimulate the curiosity of rodents. Memory is measured by inducing instinctive curiosity through the replacement of two identical objects with different objects of different colors at the start of measurement, after which the subjects were allowed to move freely with the familiar object for 2 minutes and then switched to the novel object. Measurements were taken for 5 minutes, and the time spent on the novel object was divided by the sum of the times spent on each object and expressed as a percentage. The results are shown in Fig. 28.

[0188] As a result, on days 7 and 14 after administration of P145 (MK145), the group administered P145 intraperitoneally and intravenously recorded significantly higher scores in both the Y-maze evaluation and the new object recognition test compared to the group that was not administered, indicating a therapeutic effect on dementia. Through this, it was found that P145 (MK145) can be used as a treatment for dementia.

[0190] 6.4. Administration of P145 (MK145) Protein to APP / PS1 TG Dementia Animal Model

[0191] After administering P145 intraperitoneally to 12-month-old APP / PS1 TG animal models for 5 days, a nest-building behavior evaluation (Nest test) was performed. The results of the Nest test evaluation for normal rats and APP / PS1 TG animal model rats (control group and P145 (MK145) protein administration group) are shown in Figures 29 and 30.

[0192] As a result, although nest-building behavior is abnormal due to the induction of dementia, the group administered the P145 (MK145) protein showed improved nest-building behavior and scores compared to the control group, indicating that P145 (MK145) can be used as a treatment for dementia.

[0193] Amyloid beta plaques were stained and analyzed using brain tissues from normal rats and APP / PS1 TG animal model rats (control group and P145 (MK145) protein administration group). The results are shown in Figures 31 and 32.

[0194] As a result, it was confirmed that the number of plaques significantly decreased in the group administered P145. This indicates that P145 (MK145) can be used as a treatment for dementia.

[0195] Example 7. Evaluation of the therapeutic efficacy of P145 (MK145) in a Parkinson's disease animal model

[0196] 7.1. Establishment of the MTPT Injection Animal Model

[0197] Male B6 mice weighing approximately 23–25 g at 10 weeks of age were used. Five mice were housed in each cage, and conditions were maintained at 21°C with a 12-hour light-dark cycle, and food and water were provided.

[0198] A Parkinson's model was induced by administering 25 mg / kg of MTPT dissolved in 0.9% NaCl via intraperitoneal administration for 7 days.

[0200] 7.2. Administration of P145 (MK145) Protein to a Parkinson's Disease Animal Model

[0201] 10 μg / kg of purified and extracted P145 protein was dissolved in 100 μl of physiological saline and administered intraperitoneally (IP) a total of 5 times.

[0202] The administration of the therapeutic agent was performed starting from the 7th day after the creation of the dementia model, and all animal model experiments were performed blindly.

[0204] 7.3. Confirmation of Therapeutic Efficacy Following Administration of P145 (MK145) Protein to a Parkinson's Disease Animal Model

[0205] Neurobehavioral evaluation was performed in a Parkinson's animal model after administration of P145 (MK145). The results of the neurobehavioral evaluation of normal rats and Parkinson's animal model rats (control group and P145 (MK145) protein administration group) are shown in Figure 33.

[0206] As a result, although neurobehavioral abnormalities occur due to the induction of Parkinson's disease, the group administered the P145 (MK145) protein showed improved neurobehavior compared to the control group, indicating that P145 (MK145) can be used as a treatment for Parkinson's disease.

[0208] The dopamine expression levels of normal rats and Parkinson's disease animal model rats (control group and P145 (MK145) protein administration group) were analyzed by ELISA. The results are shown in Figure 34.

[0209] As a result, it was confirmed that in the group administered P145 (MK145), the amount of dopamine increased compared to the control group, and the amount of dopamine in the hippocampus increased significantly compared to the normal group and the control group. This indicates that P145 (MK145) can be used as a treatment for Parkinson's disease.

[0211] The expression levels of dopamine-related receptors in brain tissues of normal rats and Parkinson's disease animal model rats (control group and P145 (MK145) protein administration group) were analyzed by RT-PCR. The results are shown in Figure 35.

[0212] As a result, the expression of dopamine type 1 receptors and brain-derived neurotrophic factor (BDNF), which are known to be highly related to neurological function in Parkinson's disease, decreased with the induction of Parkinson's disease. However, in the group administered P145 (MK145), expression improved compared to the control group. Through this, it can be seen that P145 (MK145) can be used as a treatment for Parkinson's disease.

[0214] Example 8. Evaluation of the therapeutic efficacy of P145 (MK145) in an animal model of autism

[0215] 8.1. Establishment of a VPA-Injected Autism Animal Model

[0216] Mother rats on the 12th day of pregnancy were administered VPA subcutaneously and raised until 40 days of age, maintained at 21°C with 12 hours of light and dark, and provided with food and water.

[0218] 8.2. Administration of P145 (MK145) Protein to an Autism Animal Model

[0219] 100 μg / kg of purified and extracted P145 (MK145) protein was administered intraperitoneally (IP) to the patient five times starting from day 47 of age, dissolved in 100 μl of physiological saline.

[0221] 8.3. Confirmation of Therapeutic Efficacy Following Administration of P145 (MK145) Protein to an Autism Animal Model

[0222] The treatment schedule in the autism model is shown in Fig. 36.

[0223] Behavioral evaluation of the autism model was performed for 3 days starting the day after administration of P145 (MK145).

[0225] The socialization responses of normal rats and autism model rats (control group and P145 (MK145) protein administration group) when meeting unfamiliar peers were analyzed. The results are shown in Figure 37.

[0226] As a result, social responses when meeting unfamiliar peers were reduced due to the induction of autism; however, in the group administered the P145 (MK145) protein, improved social responses were observed compared to the control group, indicating that P145 (MK145) can be used as a treatment for autism.

[0228] The behavior of burying beads as a symptom of autism in normal rats and autism model rats (control group and P145 (MK145) protein administration group) was analyzed. The results are shown in Figures 38 and 39.

[0229] As a result, the behavior of burying beads increased with the induction of autism; however, in the group administered the P145 (MK145) protein, the behavior of burying beads decreased compared to the control group, indicating that P145 (MK145) can be used as a treatment for autism.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of ischemic stroke comprising a protein having the amino acid sequence of SEQ ID NO. 1 and saline solution, wherein the composition is formulated into an injectable formulation.

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