WO2020213982A1 - Composition for prevention or treatment of neurodegenerative diseases using cd9 and method for screening therapeutic agent for neurodegenerative diseases - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of neurodegenerative diseases using CD9, and a method for screening a therapeutic agent for neurodegenerative diseases and, more specifically, to a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising, as an active ingredient, an inhibitor against the expression or activity of CD9 protein, and a method for screening a therapeutic agent for neurodegenerative diseases. A material for inhibiting the expression or activity of CD9 exhibits the effect of improving the memory of patients with neurodegenerative diseases, and thus can be effectively used in the development of a therapeutic agent for neurodegenerative diseases, and through assay of the expression level of CD9, candidate materials for the therapeutic agent for neurodegenerative diseases can be screened.

Description

Composition for the prevention or treatment of neurodegenerative diseases and screening method for the treatment of neurodegenerative diseases using CD9

This application claims priority to Korean Patent Application No. 10-2019-0045489 filed on April 18, 2019, and the entire specification is a reference of this application.

The present invention relates to a composition for preventing or treating neurodegenerative diseases using CD9 and a screening method for a therapeutic agent for neurodegenerative diseases, and more particularly, to a prevention or treatment of neurodegenerative diseases comprising an inhibitor of CD9 protein expression or activity as an active ingredient A pharmaceutical composition for use, and (a) treating a test substance on cells expressing CD9 protein or mRNA; (b) measuring the expression level of CD9 protein or mRNA in the cells and control cells not treated with the test substance; And (c) selecting a test substance that reduces the expression level of CD9 protein or mRNA compared to the control cells as a candidate substance for treating neurodegenerative diseases.

Neurodegenerative diseases are associated with symptoms when nerve cells degenerate, lose function, and often die. Patients with neurodegenerative diseases may experience extreme degeneration in cognitive or motor abilities, and because these diseases are primarily progressive, their quality of life and expectations for life may be significantly reduced as a result. .

These diseases include Parkinson's Disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Frontotemporal Dementia, and cortical-basal ganglia. Degeneration (Cortico Basal Degeneration), progressive supranuclear palsy (PSP) and other diseases.

Among various neurodegenerative diseases, dementia is a disease that causes the most extensive cell damage and accompanies degenerative mental disorders. In particular, major symptoms such as memory impairment and loss of judgment are well known. Dementia can be broadly divided into vascular dementia caused by stenosis or blockage of cerebrovascular blood vessels, Alzheimer's dementia, which is known to be caused by accumulation of beta-amyloid protein in the brain, and mixed dementia caused by a combination of these two causes.

Alzheimer's dementia accounts for the largest proportion of dementia patients, and a recent study reports that 1 in 85 people will have the disease by 2050, of which 43% need special care. (Prabhulkar S, Piatyszek R, et al. J Neurochem., 2012, 122, 374-381). Among neurodegenerative diseases, Alzheimer's disease is largely classified into hereditary Alzheimer's disease and sporadic Alzheimer's disease (SAD). Hereditary Alzheimer's disease accounts for 5-10% of all Alzheimer's patients.

The hyperphosphorylated, aggregated tau is a central neuropathological feature of Alzheimer's disease and many human neurodegenerative diseases. Following the discovery of mutations in the MAPT gene, significant reports have been published on the role of tau in recent years. The development of various tau transgenic animal models has made significant progress in tau dependent pathology, including the propagation of tau pathology from neurons to other cells and interactions between Aβ and tau. According to this pathology and tau-related biomarkers, new treatments have been obtained. Nevertheless, the etiological factors leading to neurodegeneration and the exact mechanism of tau-mediated neurotoxicity still need to be elucidated, and the exact cause of the onset is not known to date (Ann Transl Med. 2018 May; 6(10). ): 175.)

Pathological features of Alzheimer's disease include senile plaques that accumulate outside of nerve cells, neurofibrilary tangles that look like tangled bundles of threads in the cell body of nerve cells, and neuroal loss. And the like. These pathological features appear in both hereditary Alzheimer's disease and sporadic Alzheimer's disease. Among them, a toxic protein called amyloid β peptide (Aβ) has been revealed as a major constituent of senile spots. Amyloid beta peptides are insoluble peptides consisting of 40 to 42 amino acids resulting from abnormal cleavage of amyloid proproteins. In addition, it has been reported that excessive accumulation of amyloid beta peptide appears as a phenomenon common to both hereditary Alzheimer's disease and sporadic Alzheimer's disease. Therefore, amyloid beta peptide is regarded as a major pathogenic material for Alzheimer's disease. The overall pathogenesis of Alzheimer's disease is that when mutations in the presenilin 1 and 2 genes (PS 1 and 2) occur, the amyloid proprotein is abnormally cleaved by β-secretase and amyloid beta peptide is produced. do. It is known that necrosis of cranial nerve cells occurs due to the produced amyloid beta peptide, which causes Alzheimer's disease.

Until now, extensive and diverse studies on the cause, etiology and treatment of dementia have been conducted, but the cause is insufficient and the development of effective treatments is also insufficient. Although the FDA approved tacrine, rivastigmine, galantamine, donepezil, and memantine as treatments for Alzheimer's dementia, most dementia treatments currently used are degenerative. It is only a psychodegenerative substance that relieves the symptoms of dementia. Most of these drugs are mainly anti-inflammatory drugs, and have considerable side effects such as hepatotoxicity and damage to the mucous membrane of the digestive tract, and are suitable for symptomatic therapy rather than ultimate cause treatment. There is a limit to being limited.

Meanwhile, CD9 is a tetraspanin-based cell membrane glycoprotein having a molecular weight of 24 to 27 kDa, and there are 34 tetraspanins in mammals, and 33 of them have been identified in humans. Each tetraspanin contains cell adhesion and migration, platelet activity and aggregation, fusion of egg and sperm during fertilization in mammals, development and metastasis of cancer, humoral immune response and allergic response, human immunodeficiency virus-1 (HIV It is known as an antigen that plays an important role in -1) and influenza virus replication. However, studies on the regulation of cellular senescence or aging control of the CD9 antigen in humans have only reported that the expression is increased in aging human vascular endothelial cells, and the effect of the expression and activity of CD9 on neurodegenerative diseases has been reported. There is no bar.

Accordingly, the present inventors have repeatedly conducted extensive research to develop a new treatment for neurodegenerative diseases, and found that inhibition of the expression or activity of the CD9 protein can improve symptoms such as memory loss caused by degenerative brain lesions. The invention was completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an inhibitor of expression or activity of CD9 protein as an active ingredient.

In addition, it is to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases consisting of an inhibitor of the expression or activity of the CD9 protein as an active ingredient.

In addition, it is to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases consisting essentially of an inhibitor of expression or activity of a CD9 protein as an active ingredient.

Another object of the present invention is the steps of (a) treating a test substance on cells expressing CD9 protein or mRNA; (b) measuring the expression level of CD9 protein or mRNA in the cells and control cells not treated with the test substance; And (c) selecting a test substance that reduces the expression level of CD9 protein or mRNA compared to the control cells as a candidate substance for treating a neurodegenerative disease.

Another object of the present invention is to provide a use of an inhibitor of expression or activity of a CD9 protein for preparing a preparation for preventing or treating neurodegenerative diseases.

Another object of the present invention is to provide a method for treating neurodegenerative diseases comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of CD9 protein expression or activity as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an inhibitor of expression or activity of CD9 protein as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an inhibitor of CD9 protein expression or activity as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases essentially consisting of an inhibitor of CD9 protein expression or activity as an active ingredient.

In order to achieve another object of the present invention, the present invention comprises the steps of: (a) treating cells expressing CD9 protein or mRNA with a test substance; (b) measuring the expression level of CD9 protein or mRNA in the cells and control cells not treated with the test substance; And (c) selecting a test substance that reduces the expression level of CD9 protein or mRNA compared to the control cells as a candidate substance for treating a neurodegenerative disease.

In order to achieve another object of the present invention, the present invention provides a use of an inhibitor of expression or activity of a CD9 protein to prepare a preparation for preventing or treating neurodegenerative diseases.

In order to achieve another object of the present invention, the present invention provides a method for treating neurodegenerative diseases comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of CD9 protein expression or activity as an active ingredient. to provide.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an inhibitor of expression or activity of CD9 protein as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising an inhibitor of CD9 protein expression or activity as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating neurodegenerative diseases essentially consisting of an inhibitor of CD9 protein expression or activity as an active ingredient.

In the present specification,'treatment' refers to suppression of occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, reduction of disease progression rate, improvement of disease state, improvement, alleviation or improved prognosis. do. The term'prevention' as used in the present invention means any action that suppresses the onset or delays the progression of a disease.

'Protein' is used interchangeably with'polypeptide' or'peptide', for example, refers to a polymer of amino acid residues as commonly found in proteins in nature. For example, a'fragment' of the CD9 protein refers to a peptide of a portion of the CD9 protein.

In the present invention,'polynucleotide' or'nucleic acid' refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of single or double strands. Unless otherwise limited, also include known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to those of naturally occurring nucleotides. In general, DNA is composed of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), and RNA is composed of uracil (U) instead of thymine. Have. In the double-stranded nucleic acid, A forms a hydrogen bond with T or U, and C forms a hydrogen bond with the G base, and the relationship between these bases is called'complementary'.

On the other hand,'mRNA (messenger RNA or messenger RNA)' is an RNA that serves as a blueprint for polypeptide synthesis (protein translation, translation) by transferring the genetic information of the nucleotide sequence of a specific gene to the ribosome during protein synthesis. Using the gene as a template, single-stranded mRNA is synthesized through a transcription process.

In the present invention, the neurodegenerative disease refers to a disease related to symptoms that appear when nerve cells degenerate, lose function, and die, and Alzheimer's disease, dementia, mild cognitive impairment, Parkinson's disease, progressive nuclear paralysis, multi-system Atrophy, Olive Nucleus-Peripheral-Cerebral Atrophy (OPCA), Shy-Drager Syndrome, Stroke-Black Matter Degeneration, Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS), Essential Tremors, Cortical-basal Nucleus Degeneration, Diffuse Lewy Body Disease , Parkin's-ALS-dementia complex, Niemann-Pick disease, Pick's disease, cerebral ischemia, and may be selected from the group consisting of cerebral infarction, but is not limited thereto, and most preferably Alzheimer's disease or dementia. have.

Meanwhile, CD9 is a tetraspanin-based cell membrane glycoprotein having a molecular weight of 24 to 27 kDa, and there are 34 tetraspanins in mammals, and 33 of them have been identified in humans. Each tetraspanin contains cell adhesion and migration, platelet activity and aggregation, fusion of egg and sperm during fertilization in mammals, development and metastasis of cancer, humoral immune response and allergic response, human immunodeficiency virus-1 (HIV It is known as an antigen that plays an important role in -1) and influenza virus replication.

The term'CD9' as used herein is a cell surface glycoprotein receptor of about 24 to 27 kDa belonging to a member of the tetraspanin family, and a signal that plays an important role in regulating cell development, activity, growth and motility. It is known to regulate signal transduction events. In addition, it can regulate cell adhesion and cell migration, and induce platelet activation and aggregation, which are involved in platelet-induced endothelial cell proliferation. In addition, it is involved in various phenomena within cells, such as promoting muscle cell fusion and contributing to myotube maintenance.

The CD9 protein in the present invention may be derived from mammals, preferably derived from humans. Most preferably, the CD9 protein in the present invention comprises the amino acid sequence of human CD9 isoform 1 (NP_001760.1) represented by SEQ ID NO: 1 or human CD9 isoform 2 (NP_001317241.1) represented by SEQ ID NO: 2 Characterized in that (NCBI Genbank accession number in parentheses).

The CD9 protein expression inhibitor is any one selected from the group consisting of an antisense oligonucleotide complementary to CD9 mRNA, siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme, and protein nucleic acid (PNA). It can be. In addition, the CD9 mRNA most preferably comprises a nucleotide sequence of human CD9 mRNA transcript variant 1 (NM_001769.3) represented by SEQ ID NO: 3 or human CD9 mRNA transcript variant 2 (NM_001330312.1) represented by SEQ ID NO: 4 (NCBI Genbank accession number in parentheses). Features such as the coding region (exon) of the human CD9 mRNA transcript variants are identified in the sequence information displayed by searching the NCBI database with the Genbank accession number in parentheses.

Furthermore, the CD9 protein expression inhibitor according to the present invention may preferably be a shRNA that binds complementarily to CD9 mRNA and causes degradation of the mRNA.

In the present invention, the'siRNA (small interfering RNA or short interfering RNA or silencing RNA)' is artificially introduced into the cell to induce degradation of the mRNA of a specific gene, thereby preventing protein translation, thereby inhibiting gene expression. It is a short double-stranded RNA that causes RNA interference, and is composed of 20 to 25 nucleotides complementary to a specific site of the target mRNA. Among the double strands of siRNA in cells, an antisense strand complementary to the target mRNA binds to the RNA-induced silencing complex (RISC) protein complex and binds to the target mRNA, and the argonaute protein in the RISC complex cleaves and degrades the target mRNA. It suppresses the expression of specific genes through mechanisms such as suppressing the binding of proteins and ribosomes important for protein translation with mRNA.

In the present invention, the shRNA is a substance that induces RNA interference, and is a molecule having a double-stranded structure in the molecule and a hairpin-like structure by partially containing a palindromic nucleotide sequence in a single-stranded RNA. In addition, when a shRNA expression plasmid is introduced into a cell and expressed, a 21-23 base pair siRNA is generated by RNase III (ribonuclease III) enzyme called dicer in the cell to induce RNAi.

In the present invention, the siRNA or shRNA may be obtained by applying various modifications to improve the stability of the oligonucleotide in vivo, impart resistance to nuclease enzymes, and reduce non-specific immune responses. The modification of the oligonucleotide is the OH group at the 2′ carbon position of the sugar structure in one or more nucleotides -CH ₃ (methyl), -OCH ₃ (methoxy), -NH ₂ , -F, -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylami Modification by substitution with dooxyethyl; Modification in which oxygen in the sugar structure in the nucleotide is substituted with sulfur; Alternatively, one or more modifications selected from modifications of nucleotide bonds to phosphorothioate, boranophosphate, and methyl phosphonate bonds may be used in combination, and PNA (peptide nucleic acid), Modification into the form of LNA (locked nucleic acid) or UNA (unlocked nucleic acid) can also be used.

In addition, the CD9 protein activity inhibitor may be any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, natural extracts, and synthetic compounds that specifically bind to CD9 protein.

The pharmaceutical composition according to the present invention can be variously formulated according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier for the treatment of neurodegenerative diseases. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes. Preferably, it is formulated using a transfection reagent, and can be delivered to cells using subcutaneous, blood, bone marrow, or abdominal cavity.

The pharmaceutical composition according to the present invention may be administered to a patient in a pharmaceutically effective amount, that is, in an amount sufficient to prevent or relieve symptoms and treat neurodegenerative diseases. For example, a typical daily dosage may be administered in the range of about 0.01 to 1000 mg/kg, and preferably, it may be administered in the range of about 1 to 100 mg/kg. The pharmaceutical composition of the present invention can be administered in one or several divided doses within a preferred dosage range. In addition, the dosage of the pharmaceutical composition according to the present invention may be appropriately selected by a person skilled in the art according to the route of administration, the subject of administration, age, sex, weight, individual differences, and disease conditions.

The route of administration may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, rectal, or pancreatic It may be intravenous administration, but is not limited thereto.

In the case of oral administration of the pharmaceutical composition of the present invention, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier for oral administration. It can be formulated in the form of such as. Examples of suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.

In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated according to a method known in the art in the form of injections, transdermal administrations and nasal inhalants together with a suitable parenteral carrier.

In addition, the pharmaceutical composition can be administered by any device capable of moving the active substance to the target cell. Preferred modes of administration and formulations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections or instillations. Injectables include aqueous solvents such as physiological saline or ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid ethyl, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin), and other non-aqueous solvents. Stabilizers for preventing deterioration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, and for inhibiting the growth of microorganisms Pharmaceutical carriers such as preservatives (eg, mercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

In the case of such injections, they must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof and/or a solvent or dispersion medium containing vegetable oil. I can. More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be additionally included. In addition, the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.

In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, air rolls, and the like are included. In the above,'transdermal administration' means that the active ingredient in an effective amount contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin. For example, the pharmaceutical composition of the present invention may be prepared in an injectable formulation and administered by lightly pricking the skin with a 30 gauge thin injection needle or applying it directly to the skin. These formulations are described in prescriptions generally known in pharmaceutical chemistry.

In the case of inhalation administration, the compounds used according to the invention can be used in pressurized packs or with suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered from a nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

Other pharmaceutically acceptable carriers may refer to those known in the art.

Pharmaceutical compositions according to the present invention include one or more buffers (e.g., saline or PBS), carbohythrate (e.g., glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and/or preservatives may further be included.

In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

Further, the pharmaceutical composition of the present invention may be administered alone or may be administered in combination with a known compound having an effect of treating neurodegenerative diseases.

In addition, the present invention

(a) treating cells expressing CD9 protein or mRNA with a test substance;

(b) measuring the expression level of CD9 protein or mRNA in the cells and control cells not treated with the test substance; And

(c) It provides a screening method for a neurodegenerative disease treatment comprising the step of selecting a test substance that reduces the expression level of CD9 protein or mRNA compared to the control cell as a candidate substance for treatment of neurodegenerative disease.

The step (a) is a step of contacting cells expressing CD9 protein or mRNA in order to determine whether the test substance to be analyzed has an activity to inhibit the expression of CD9 protein or mRNA.

In the method of the present invention,'contacting' or'treatment' is a general meaning, and two or more agents (e.g., two polypeptides) are combined, or an agent and a cell (e.g., Protein and cell). Contact can occur in vitro. For example, combining two or more agents in a test tube or other container, or combining a test agent with cells or cell lysates and a test agent. Contact can also occur in cells or in situ. For example, two polypeptides are brought into contact in a cell or cell lysate by coexpressing a recombinant polynucleotide encoding two polypeptides in a cell. In addition, a protein chip or a protein array in which the protein to be tested is arranged on the surface of a stationary bed may be used.

In addition, in the method of the present invention, the'test substance' can be used interchangeably with a test agent or agent, and any substance, molecule, element, compound ( Includes a compound, entity, or combination thereof. Examples include proteins, polypeptides, small organic molecules, polysaccharides, and polynucleotides. It may also be a natural product, a synthetic compound or a chemical compound, or a combination of two or more substances.

More specifically, test agents that can be screened by the method of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines. ), oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines or derivatives thereof, structural analogues, or combinations thereof. Test formulations can be synthetic or natural. The test formulations can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Combinatorial libraries can be produced from several types of compounds that can be synthesized in a step-by-step manner. Compounds of multiple combinatorial libraries can be prepared by the encoded synthetic libraries (ESL) method (WO 95/12608, WO93/06121, WO 94/08051, WO95/395503 and WO 95/30642). Peptide libraries can be prepared by the phage display method (WO91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field. Known pharmacological agents can be directed or applied to a random chemical modification, such as acylation, alkylation, esterification, amidification, to prepare structural analogues.

The test agent may be a naturally occurring protein or fragment thereof. Such test formulations can be obtained from natural sources, such as cell or tissue lysates. Libraries of polypeptide preparations can be produced, for example, by conventional methods or obtained from commercially available cDNA libraries. The test agent may be a peptide, such as a peptide having about 5 to 30 amino acids, preferably about 5 to 20, more preferably about 7 to 15 amino acids. The peptide may be a naturally occurring protein, a random peptide, or a cut product of a'biased' random peptide. In addition, the test agent may be a nucleic acid. The nucleic acid test agent can be a naturally occurring nucleic acid, a random nucleic acid, or a'biased' random nucleic acid. For example, cuts of prokaryotic or eukaryotic genomes can be used analogously to those described above.

Further, the test agent may be a small molecule (eg, a molecule having a molecular weight of about 1,000 Da or less). A high throughput assay may be preferably applied to a method for screening a small molecule modulating agent. Many assays are useful for this screening.

In the present specification, "expression" means that a protein or nucleic acid is produced in a cell. The CD9-expressing cell may be a cell that internally expresses CD9, or may be a cell that overexpresses CD9 by being transformed with a recombinant expression vector containing a polynucleotide encoding CD9. Preferably, the cells expressing the CD9 gene may be fibroblasts.

Step (b) is a step of measuring the gene expression level of CD9 in cells expressing CD9 contacted with the test substance and cells expressing CD9 without contact with the test substance.

The measurement of CD9 mRNA expression can be performed without limitation, using conventional methods for determining the expression level in the art, and examples of analysis methods include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR (competitive RT- PCR), real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA sequencing (RNA) sequencing) and the like, but are not limited thereto.

In addition, methods for measuring the expression level of CD9 protein can be used without limitation, methods known in the art, such as western blotting, dot blotting, and enzyme-linked immunosorbent assay. ), Radioactive Immunoassay (RIA), Radioimmune Diffusion Method, Ouchteroni Immunity Diffusion Method, Rocket Immunoelectrophoresis, Immunohistochemical Staining, Immunoprecipitation, Complement Fixation Assay, Flow Cytometry (FACS) or Protein Chip Method And the like, but are not limited thereto.

The step (c) is a step of selecting a test substance that reduces the expression level of CD9 protein or mRNA compared to the control cells as a candidate substance for treating neurodegenerative diseases.

As identified by the present inventors, as inhibiting the activity of CD9 improves memory in an animal model of neurodegenerative disease, substances that inhibit the expression of protein or mRNA of CD9 also exhibit the effect of inhibiting the activity of CD9. It can be clearly understood that it has the potential to be used as a therapeutic agent for neurodegenerative diseases.

The present invention provides a use of a CD9 protein expression or activity inhibitor for preparing a preparation for preventing or treating neurodegenerative diseases.

The present invention provides a method for treating neurodegenerative diseases comprising administering an effective amount of a composition comprising an inhibitor of expression or activity of a CD9 protein as an active ingredient to an individual in need thereof.

The'effective amount' of the present invention refers to an amount showing an effect of improving, treating, preventing, detecting, diagnosing, or inhibiting or reducing neurodegenerative diseases when administered to an individual, and the'individual' is an animal, preferably For example, it may be an animal including a mammal, especially a human, and may be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect.

The'treatment' of the present invention generally refers to improving the symptoms of neurodegenerative diseases or neurodegenerative diseases, which may include curing, substantially preventing, or improving the condition, It includes, but is not limited to, alleviating, curing, or preventing one symptom or most of the symptoms resulting from neurodegenerative disease.

The term'comprising' in the present invention is used in the same way as'containing' or'as a feature', and does not exclude additional component elements or method steps that are not mentioned in the composition or method. . The term “consisting of” means excluding additional elements, steps, or ingredients that are not separately described. The term “essentially consisting of” means including, in the scope of a composition or method, the component elements or steps described, as well as the component elements or steps that do not substantially affect its basic properties.

Substances that inhibit the expression or activity of CD9 have the effect of improving the memory of patients with neurodegenerative diseases and can be usefully used in the development of treatments for neurodegenerative diseases, and candidates for treatment of neurodegenerative diseases through analysis of the expression level of CD9 Can be screened.

1 is a result of confirming the expression of CD9 in AD fibroblasts not subjected to permeabilization.

2 is a result of confirming the expression of CD9 in AD fibroblasts in the case of permeabilization.

Figure 3 shows tau phosphorylation according to treatment with anti-CD9 antibody in AD fibroblasts.

4A to 4F show the effect of improving memory by administering anti-CD9Ab. Each general motor activity (Fig. 4A). The number of alternating repetitions of mice administered with the drug (Fig. 4b), contextual recall (Fig. 4c), freezing time (Fig. 4d), the number of arrivals to the target area in the Morris water maze (Fig. 4e) and the target area The time spent in (Fig. 4f) was measured.

Hereinafter, the present invention will be described in detail.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Experiment method

1. Preparation of cells, antibodies and experimental animals

Female 3xTg mice (JAX 004807) were used as experimental animals. Experimental animals were provided with sterile feed and water, and were reared with less than 10 animals each. The day and night cycle was set to 12 hours. Animal experiments were conducted in an animal breeding facility at Korea University under the guidance and supervision of the Animal Experimental Ethics Committee, and animal experiments were performed according to the protocol approved by Korea University IACUC.

Test antibody inoculation (CD9) was prepared by mixing with PBS (phosphate buffer solution), and 100ul was injected subcutaneously in 8-12 week old mice. Mice (n=4 per group) were divided into groups for each experiment, and PBS (control) was used as a control group. The experiment was carried out by injecting the antibody a total of 4 times once a week.

In the experiment, two types of AD fibroblasts purchased from Coriell (Camden, NJ), (AG07374(AD fibroblast 1), AG05770(AD fibroblast 2)) age-control fibroblasts (AG09857), and normal human tonsils extracted in this laboratory. Fibroblasts were used.

In addition, Mouse control IgG1, anti-CD9 ALB6 (Immunotech) was used as the primary antibody, and goat anti-mouse IgG (Jackson Immunoresearch) labeled with FITC was used as the secondary antibody.

2. FACS staining

Cells were prepared by adding trypsin-EDTA to fibroblasts in culture. Half of the cells were treated with FACS permeabilizing solution 2 purchased from Becton Dickinson Bioscience for 15 minutes, and the other half was left at 4°C. The cells were washed with PBS containing 1% Fetal bovine serum (FBS) and 0.05% sodium azide, transferred to a FACS tube, and reacted with the primary antibody at 4°C for 15 minutes. After washing with FACS wash buffer, secondary antibody was added and reacted at 4° C. for 15 minutes. After washing with FACS buffer, CD9 expression was analyzed with BD FACSCalibur.

3. Animal behavior experiment

3-1. Y-maze

The Y-maze device consisted of three arms 32 cm (length) x 10 cm (width) with 26 cm walls (San Diego Instruments, San Diego, CA). Each mouse was placed in the center of the Y-maze and allowed to move freely through the maze for 5 minutes. It was considered effective if all four paws of the mouse entered the arm of the maze. Alternation was defined as entering 3 different arms (i.e. 1, 2, 3 or 2, 3, 1, etc.) in 3 succession. The percentage shift score was calculated using the following formula.

spontaneous alternation% = (number of spontaneous)/(total number of arm entries -2) x 100

In addition, the total number of arms was used as a measure of the general activity of the animal. The maze was cleaned with 70% ethanol to minimize the odor signal.

3-2. Morris Water Maze

The device used was a white round plastic tank (122 cm in diameter) with a wall of 76 cm, filled with water held at 22u +/- 22uC, made opaque by adding non-toxic white paint, and a detachable square inside (side 10 cm long) Plexiglas platform. The tank was in a test room containing a number of visible visual cues. Mice were placed on the platform for 10 seconds before the first test of the first session. Mice were trained to swim on a platform submerged 1.5 cm below the surface of the water. The platform was in a fixed position equidistant from the center and wall of the tank. Mice were trained three times a day. During each training period, mice were placed in tanks in random order at one of the four designated starting points. The mouse was allowed to find and escape from the submerged platform. If the platform was not found within 60 seconds, it was manually guided to the platform and left there for 10 seconds. Mice were trained to reach a training criterion of 20 seconds (escape waiting time). Mice were evaluated in the probe test for 24 hours after the last training session and consisted of 60 seconds of free swimming in a platformless pool. The performance of each animal acquisition parameters provide data on (waiting to find the platform and distance tracking time), and probe-trial parameters (the number of entries for the target platform area) Any-Maze ^TM video tracking system (Stoelting Co .) was used.

4. Western Blot Analysis

Protein concentration was quantified using Bradford Assay (Bio-Rad Protein Assay 500-0006, Germany, Munchen), and total cell proteins, age-control fibroblasts, and AD-fibroblasts were isolated with 8-10% SDS polyacrylamide gel. . Polyvinylidene difluoride (PVDF) membrane was blocked in Tris buffered saline (5% no fat formula, 0.1% Tween20). As the primary antibody, anti PHF-tau clone AT8 (MN1020, Thermo Scientific, Rockford, USA), p Tau pSer422 (T7944, Sigma), anti-tau clone tau-5 (MAB361, Millipore Billerica, Ma), CD9 were used. I did. All smears were standardized with actin (MAB1501, Chemicon-Millipore, Billerica, Ma).

Experiment result

1. Confirmation of CD9 expression in AD fibroblasts

In the case of cells not subjected to permeabilization, age-control fibroblasts hardly expressed CD9, but AD fibroblasts expressed CD9 at high levels in both types (FIG. 1). This result is consistent with previous data indicating the location of the CD9 molecule in the cell membrane, as well as the difference between the two patient groups was estimated to be related to the progression of Alzheimer's disease. In addition, it was confirmed that CD9 expression was also high in Tonsil fibroblasts in normal humans.

In addition, as shown in Figure 2, after permeabilization, CD9 still did not express age-control fibroblast. In addition, AD fibroblast 1, which showed relatively lower expression of CD9 than AD fibroblast 2 in cell membranes, was hardly expressed even after permeabilization. It is believed that the CD9 molecules on the cell surface and inside the cell were lost in the permeabilization step. Meanwhile, it was found that CD9, a cell membrane protein, was expressed at a high level in AD fibroblast 2 after permeabilization. It was interpreted that the CD9 protein, which normally exists only in the cell membrane, is also present in the nucleoplasm or the nucleus, depending on the progression of alzheimer's disease.

2. Confirmation of Tau hyperphosphorylation in AD fibroblasts

Neurodegeneration through tau hyperphosphorylation Depleted β-amyloid may stimulate tauopathy, and tau hyperphosphorylation is associated with changes in microtubule assembly leading to neurofibrillary tangles (NFTs) and neurodegeneration in Alzheimer's disease. have. In addition, it has been linked to many other neurological disorders, including Alzheimer's and Parkinson's. In the present invention, as a result of confirming the phosphorylation of tau by administration of anti-CD9 antibody, the level (control lane) of phosphorylated tau (p-Ser422) in AD fibroblasts was significantly increased compared to age control fibroblasts (not shown). , When the antibody was administered, it was confirmed that it was significantly reduced (anti-CD9-Ab panel), but the total amount of tau did not show any significant change (FIG. 3 ).

3. The effect of improving memory in Tg mice

Four Tg mice (3xTg+Ab) treated with anti-CD9 Ab in Y-maze and the group treated with placebo (3xTg) had no significant difference in total cancer entry and exit, so there was no difference in general exercise activity. It was found (Fig. 4A). However, the number of alternating repetitions of the mice administered with the drug was higher than that of the control group and the percentage value was higher (Fig. 4B, ^# p<0.05).

In the fear conditioning test, there was no difference between the two groups at the training stage (data not shown). However, 3xTg mice receiving anti-CD9Ab did not show a significant difference in freezing time in contextual recall than in the animal group receiving placebo (FIG. 4C). The 3×Tg mice administered with anti-CD9Ab showed significantly higher freezing time even in the cued recall paradigm than the family administered with placebo (Fig. 4D, ^# p<0.05).

Mice were tested at baseline spatial memory function using the Morris water maze. In this study, we performed a visible platform training, followed by a hidden platform test with four probe attempts per day. All mice in each group were able to reach the training criterion within 4 days and similarly displayed skilled swimming ability. However, in the probe test, 3xTg mice treated with anti-CD9Ab showed a significant increase in the number of arrivals to the target area and time spent in the target area when compared to the control group (Fig. 4E, 4F, ^# p<0.05).

Substances that inhibit the expression or activity of CD9 have the effect of improving the memory of patients with neurodegenerative diseases and can be usefully used in the development of treatments for neurodegenerative diseases, and candidates for treatment of neurodegenerative diseases through analysis of the expression level of CD9 It has high industrial applicability because it can screen.

Claims (16)

1. A pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising an inhibitor of CD9 protein expression or activity as an active ingredient.
2. The method of claim 1, wherein the neurodegenerative disease is Alzheimer's disease, dementia, mild cognitive impairment, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olive nucleus-poni-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatum- Black matter degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremors, cortical-basal ganglia degeneration, diffuse Lewy body disease, Parkin's-ALS-dementia complex, Niemann-Pick disease, Pick's disease , A pharmaceutical composition, characterized in that at least one selected from the group consisting of cerebral ischemia and cerebral infarction.
3. The composition of claim 1, wherein the CD9 protein comprises an amino line sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
4. The method of claim 1, wherein the CD9 protein expression inhibitor is composed of an antisense oligonucleotide complementary to CD9 mRNA, siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme, and protein nucleic acid (PNA). Composition, characterized in that any one selected from the group.
5. The composition of claim 4, wherein the CD9 mRNA comprises a nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
6. The method of claim 1, wherein the CD9 protein activity inhibitor is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, natural extracts, and synthetic compounds that specifically bind to CD9 protein. Composition made by.
7. The composition of claim 1, wherein the inhibitor of CD9 protein activity is an antibody that specifically binds to a CD9 protein.
8. (a) treating cells expressing CD9 protein or mRNA with a test substance;

   (b) measuring the expression level of CD9 protein or mRNA in the cells and control cells not treated with the test substance; And

   (c) A method for screening a therapeutic agent for neurodegenerative diseases comprising the step of selecting a test substance that decreases the expression level of CD9 protein or mRNA compared to a control cell as a candidate substance for treating a neurodegenerative disease.
9. The method of claim 8, wherein the mRNA expression level of CD9 is RT-PCR, quantitative or semi-quantitative RT-PCR (Quantitative or semi-Quantitative RT-PCR), quantitative or semi-quantitative real-time RT-PCR (Quantitative or semi-Quantitative Real-time RT-PCR), Northern blot (northern blot), a screening method characterized in that it is measured using any one method selected from the group consisting of a DNA chip (chip) and an RNA chip.
10. The method of claim 8, wherein the protein expression level of CD9 is Western blot, ELISA, radioimmunoassay, radioimmuno diffusion method, Ouchterlony immunodiffusion method, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation analysis, and fixation of complement. A screening method, characterized in that it is measured using any one method selected from the group consisting of analysis, FACS, and protein chips.
11. Use of an inhibitor of expression or activity of a CD9 protein for preparing a preparation for the prevention or treatment of neurodegenerative diseases.
12. The method of claim 11, wherein the neurodegenerative disease is Alzheimer's disease, dementia, mild cognitive impairment, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olive nucleus-poni-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatum- Black matter degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremors, cortical-basal ganglia degeneration, diffuse Lewy body disease, Parkin's-ALS-dementia complex, Niemann-Pick disease, Pick's disease , Use, characterized in that at least one selected from the group consisting of cerebral ischemia and cerebral infarction.
13. The use according to claim 11, wherein the inhibitor of CD9 protein activity is an antibody that specifically binds to the CD9 protein.
14. A method for treating neurodegenerative diseases comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of CD9 protein expression or activity as an active ingredient.
15. The method of claim 14, wherein the neurodegenerative disease is Alzheimer's disease, dementia, mild cognitive impairment, Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, olive nucleus-poni-cerebellar atrophy (OPCA), Shy-Drager syndrome, striatum- Black matter degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremors, cortical-basal ganglia degeneration, diffuse Lewy body disease, Parkin's-ALS-dementia complex, Niemann-Pick disease, Pick's disease , Treatment method, characterized in that at least one selected from the group consisting of cerebral ischemia and cerebral infarction.
16. The method of claim 14, wherein the inhibitor of CD9 protein activity is an antibody that specifically binds to the CD9 protein.

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