WO2018097628A2 - 신경줄기세포의 분화 촉진 및 보호용 조성물 및 이를 이용하여 신경재생을 유도하는 방법 - Google Patents
신경줄기세포의 분화 촉진 및 보호용 조성물 및 이를 이용하여 신경재생을 유도하는 방법 Download PDFInfo
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Definitions
- the present invention relates to a method of inducing neuronal regeneration comprising administering a MEK 1/2 inhibitor to a patient in need thereof, and a composition comprising a MEK 1/2 inhibitor for use in such a method.
- MEK 1/2 inhibitors differentiate neuronal stem cells into neurons, protect neurons and neural stem cells from cytotoxicity by beta amyloid, or induce nerve regeneration by both.
- the present invention also relates to methods of protecting neurons from loss or damage of neurons, comprising administering MEK 1/2 inhibitors, and compositions comprising MEK 1/2 inhibitors for use in such methods.
- One aspect of the present invention relates to a composition for promoting differentiation of neural stem cells comprising the compound represented by the above [Formula 1].
- the composition does not involve cancerous growth of neural stem cells.
- composition for promoting differentiation of neural stem cells induces differentiation from neural stem cells to neurons even in the presence of beta amyloid.
- Promoting differentiation of neurons by the composition for promoting differentiation of neural stem cells may be due to simultaneously inhibiting MEK1 and MEK2.
- the composition may include certain MEK 1/2 inhibitors in place of the compound of Formula 1.
- the composition induces differentiation of neural stem cells into neurons even in the presence of beta amyloid.
- Another aspect of the present invention relates to a method for differentiating neural stem cells into neurons using the composition for promoting differentiation of the neural stem cells.
- the differentiation method may be one day to 7 days until the differentiation is completed, after processing the composition for promoting differentiation of the neural stem cells to neural stem cells.
- Another aspect of the present invention is to provide a method for promoting the differentiation of neural stem cells, in particular in the presence of beta amyloid to protect neural stem cells and nerve cells and to use to differentiate neural stem cells into neurons It relates to a compound.
- Another aspect of the present invention is also directed to specific MEK 1/2 inhibitors for use in promoting the differentiation of neural stem cells, in particular for differentiating neural stem cells into neurons while protecting the neural stem cells and neurons even in the presence of beta amyloid. It is about.
- Another aspect of the invention relates to a method of inducing neuronal regeneration comprising administering a specific MEK 1/2 inhibitor to a patient in need thereof.
- MEK 1/2 inhibitors differentiate neural stem cells into neural cells, protect neural stem cells and neurons from cytotoxicity by beta amyloid, or induce nerve regeneration by both.
- Most preferred MEK 1/2 inhibitors are compounds of Formula 1.
- a further aspect of the invention relates to certain MEK 1/2 inhibitors, in particular compounds of Formula 1, for use in inducing neuronal regeneration.
- a further aspect of the invention relates to compounds of formula 1 for use in the prevention or treatment of neurodegenerative diseases. It also relates to certain MEK 1/2 inhibitors for use in the prevention or treatment of neurodegenerative diseases.
- Beta-amyloid especially oligomeric beta-amyloid
- MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
- rotenone and oxidopamine treating nerve cell damage causing substances such as oxidopamine) glutamate, lipopolysaccharide (LPS), and S100 calcium-binding protein B (S100B);
- the term “differentiation” means that a cell develops into a specific cell, and specifically, a structure or function is specialized during division and proliferation of cells. Changes in form or function to accomplish a given task. "Differentiation" of neural stem cells is preceded by an asymmetric division in which the parent cell divides into two cells with different characteristics, some of which remain the same stem cells as the parent cell and some of which Differentiation "Differentiation of neural stem cells” includes “proliferation” in that neural stem cells are involved in this asymmetric division process.
- the term "protection” is to take care of the external stimulus, such as danger or destruction, when given, to take good care of it, and to prevent the proliferation or differentiation of neuronal stem cells without killing nerve stem cells, particularly beta amyloid. It means maintaining the state so that the differentiated neurons can survive.
- the term “protection” in the present invention encompasses lowering the A ⁇ (1-42) / A ⁇ (1-40) ratio in the brain to protect nerve stem cells and nerve cells from damage caused by beta amyloid ( Majid et al.
- prevention means any action that inhibits or delays the onset of a neurodegenerative disease by the administration of the pharmaceutical composition according to the present invention
- treatment means the treatment of a neurodegenerative disease by the administration of the pharmaceutical composition. Any action that improves or beneficially alters the symptoms of a suspicious and onset individual.
- Japan Tabacco Inc. Applicant discloses the compound of Example 4-1 in WO2005 / 121142.
- the compound of Formula 1 inhibits both MEK1 and MEK2, which are upstream molecules of ERK, in the mitogen-activated protein kinase / extracellular regulated kinase (MAPK) signaling pathway.
- MEKINIST® is used as an anticancer agent for melanoma and non-small cell cancer.
- the compound of Formula 1 may be used in the form of a free base, or a pharmaceutically acceptable salt or solvate.
- Solvates can be, for example, hydrates or solvates such as dimethylsulfoxide, acetic acid, ethanol, nitromethane, chlorobenzene, 1-pentanol, isopropyl alcohol, ethylene glycol and 3-methyl-1-butanol.
- the inventors have attempted to establish a composition that induces differentiation into neurons using neural stem cells of a mouse, wherein the compound of [Formula 1] is effectively separated from the brain of a mouse embryo or adult One neural stem cell was found to induce differentiation into neurons.
- MEK 1/2 inhibitors that can be used in the present invention are substances that induce neural stem cells to differentiate into neurons and at the same time protect neural stem cells and neurons from toxic substances such as beta amyloid.
- MEK 1/2 inhibitors that can be used in the present invention are as follows: Trametinib, Pimasertib (AS703026), AZD8330, Binimetinib (Binimetinib, MEK162, ARRY-162, ARRY- 438162), Refametinib (RDEA119, Bay 86-9766), PD318088, PD0325901, RO5126766.
- an aspect of the present invention comprises the MEK 1/2 inhibitor, and for protecting neural stem cells and neurons from beta amyloid, in particular oligomeric form A ⁇ (1-42) while at the same time differentiation of nerve stem cells into neurons To a composition.
- trametinib, pimasultip (AS703026), AZD8330, binimetinib, refametinib, PD318088, PD0325901, and RO5126766 induce differentiation of neuronal stem cells into neurons and neural stem cells It was confirmed that there is a protective effect of neurons.
- the compound represented by [Formula 1] has never been used to induce differentiation of neural stem cells into neurons, and MEK1 inhibits activity against both MEK1 and MEK2 through ATP non-competitive binding.
- / 2 inhibitor but only known as a treatment for melanoma and non-small cell cancer.
- Another aspect of the present invention relates to a method for differentiating neural stem cells into neurons by using the compound represented by Formula 1 or another specific MEK 1/2 inhibitor.
- the neural stem cells can be used separately from embryonic and adult brains of animals according to known methods, and can be used by purchasing a commercially available product or cultured according to a conventional culture method, which It is not specifically limited.
- the neuronal stem cells were isolated from the frontal lobe of mouse embryos at 14.5 days and those isolated from the subventricular zone of 8-week-old mice.
- the neural stem cells may be inoculated into the culture medium before differentiation and cultured at 37 ° C.
- the medium for culturing the neural stem cells is not limited as long as it is a serum-free medium composition component to which growth factors are added.
- the medium is, for example, 90-110 ⁇ M putrescine, 20-40 nM selenite, 10-30 in Dulbecco's Modified Eagle's Medium / Nutrient Mixture F12 (DMEM / F12) (1: 1).
- the neural stem cells may be, for example, N2 medium (Dulbecco's modified Eagle's medium (DMEM) / F12 (1: 1), 100 ⁇ M putrescine, 30 nM selenite, 20 nM progesterone, 1.55 mg / ml glucose (d-(+)-glucose), 25 ⁇ g / ml insulin, 0.1 mg / ml apo-transferrin, 0.5 mM Glutamax, 100 IU / ml penicillin, Undifferentiated neural stem cells can be obtained by adding a growth factor to 100 [mu] g / ml streptomycin as a component.
- DMEM Dulbecco's modified Eagle's medium
- F12 1: 1
- 100 ⁇ M putrescine 100 ⁇ M putrescine
- 30 nM selenite 20 nM progesterone
- Differentiating the cultured neural stem cells into neural cells refers to treating the neural stem cells with a composition for inducing differentiation comprising the compound represented by the above [Formula 1], and differentiating according to methods known to those skilled in the art. For example, it is possible to induce differentiation at 37 ° C by adding a composition for promoting differentiation of the neural stem cells of the present invention to the culture medium in which the neural stem cells are cultured.
- the neural stem cells before the addition of the composition for promoting the differentiation of the neural stem cells comprising the compound represented by the formula [1] sufficiently secures the cell concentration, or changes the cells such as proliferation, differentiation or death of neural stem cells
- the culture period of the neural stem cells is the cell concentration It is more preferable that the culture period of at least 1 day and up to 7 days in order to secure enough.
- the compound represented by [Formula 1] is preferably treated to neural stem cells at a concentration of 1 nM to 20 ⁇ M, when the compound represented by the above [Formula 1] is less than 1 nM, the performance of differentiation induction of neural stem cells is reduced. Problem occurs, and if it exceeds 20 ⁇ M, a problem indicating cytotoxicity occurs.
- neural stem cells are typically seeded such that each well of the plate is about 70-80% full. For example, 1 ⁇ 10 5 cells per well for 12-well plates and 5 ⁇ 10 5 cells per well for 6-well plates are seeded.
- the compound represented by [Formula 1] is preferably treated at a concentration of 10 nM to 10 ⁇ M, more preferably 10 nM to 100 nM, if less than 10 nM concentration Since the differentiation rate of the neural stem cells is slowed and the differentiation induction period is long, there is an uneconomic problem, and when it exceeds 10 ⁇ M, the compound represented by the above [Formula 1] is added as an active ingredient in an excessive amount, so that when administered later in vivo Simultaneous inhibition of MEK1 and MEK2, which affect multiple signaling pathways in cells, increases, leading to unwanted reactions that can affect normal cells.
- Trametinib (10 nM), Trametinib (100 nM), Memantine (5 ⁇ M), Memantine (10 ⁇ M), and AS703026 (10 ⁇ M) were each prepared in the undifferentiated mouse adult neural stem cells obtained in step 1A of Example 2. This is the result when processed at the concentration of.
- FIG. 8B shows relative mRNA expression of Tuj1 and TH in mouse embryonic neural stem cells which inhibited expression of either MEK1 or MEK2, or both MEK1 and MEK2 using shMEK1 and shMEK2 as part of the results of Experimental Example 5-1 The amount was confirmed, and the result of confirming the expression of each protein through western blotting.
- FIG. 10 shows the results of Experimental Example 6, FIGS. 10A and 10B show 0.1 ⁇ M, 1.0 ⁇ M, 10 ⁇ M of the MEK 1/2 inhibitors trametinib, AZD8330, PD184352, repametinib, PD318088, binimetinib, and AS703026.
- Figure 10c is PD0325901, RO5126766 , BI847325, and U0126 were treated with adult neural stem cells, and then observed in the morphology of the cells under a phase contrast microscope (FIG. 10C).
- FIG. 11 shows the results of Experiment 7, wherein MEK 1/2 inhibitors, trametinib (0.1 ⁇ M), AS703026 (10 ⁇ M), AZD8330 (1 ⁇ M), PD318088, were treated with or without beta amyloid in mouse adult neural stem cells.
- (1 ⁇ M) vinimetinib (10 ⁇ M), repametinib (1 ⁇ M), PD0325901 (10 ⁇ M), RO5126766 (10 ⁇ M), and cobimetinib (10 known as selective inhibitors for MEK2 over MEK2) It is the result of observing the morphology of a cell by phase contrast microscope after administering [mu] M). Lines marked with “-” are not treated with beta amyloid, while lines marked with “A ⁇ 1-42 ” are treated with 10 ⁇ M of beta amyloid.
- FIG. 14 shows the results of microscopic observation of the sections of the hippocampus (subiculum of hippocampus) of 5XFAD mice to which tramethinib was administered as a result of Experimental Example 8 by staining NeuN by immunohistochemical staining method.
- Figure 16 shows the results of Experiments 9-2 and 9-3, Nissl, NeuN, Dcx and BrdU stained in the section of the dentate gyrus of the hippocampus of 5XFAD mice administered trametinib observed under a microscope Photographs (FIG. 16A) showing one result and the number of cells stained with BrdU are counted (FIG. 16B).
- parts indicated by arrows ( ⁇ ) indicate cells stained with Dcx
- heads ( ⁇ ) of arrows indicate cells stained with BrdU.
- FIG. 18 is a photograph showing the results of experimental example 11 in which the sections of the cerebellum of 5XFAD mice to which trametinib was administered were stained with Tuj1 and calbindin to observe Purkinje neurons of the cerebellum under a microscope. Photographs of two slides (Tuj1 staining) or three (calbindin staining) for each treatment group were attached.
- the inventors of the present invention when the compound of the present invention [Formula 1] to the neural stem cells, the expression of the neuronal marker Tuj1 is increased, dopamine neuronal marker TH, GABAergic (GABAergic) neuronal marker Gad1, exercise ( motor) It was confirmed that the expression of the neuronal marker Isl1 and the cholinergic (Cholinergic) neuronal marker ChAT were all increased (Experimental Example 4). This can be induced by differentiation of neural stem cells into various types of neurons such as dopamine neurons, GABAergic neurons, cholinergic neurons, and motor neurons by the compounds of [Formula 1]. It means that there is.
- the compound of Formula 1 of the present invention can be used for the treatment of various neurodegenerative diseases causing loss or damage of various neurons.
- Parkinson's disease is primarily associated with the loss of dopamine neurons and is associated with atrophic lateral sclerosis (Lou Gehrig's disease, ALS), progressive bulbar palsy (PBP), and progressive muscular atrophy (PMA).
- Motor neuron disease such as primary lateral sclerosis (PLS), pseudobulbar palsy and hereditary spastic paraplegia (HSP), is associated with motor neuron.
- Loss including Alzheimer's disease, vascular dementia, and senile dementia, is primarily associated with the loss of cholinergic neurons.
- Huntington's disease is also mainly associated with the loss of GABA-medium-sized spiny neurons in the striatum of the basal ganglia.
- the compound of [Formula 1] can be used as a therapeutic agent for Alzheimer's disease because it protects the neural stem cells from beta amyloid, which is a major pathological feature of Alzheimer's disease, and differentiates them into neurons.
- the present inventors confirmed that the compound of [Formula 1] increases the number of neurons in the layer 5 of the hippocampus and cerebral cortex of Alzheimer's mouse (5xFAD) with human dementia gene (Experimental Example 8).
- 5xFAD mice have mutations in amyloid precursor protein (APP) and presenilin (PSEN1) genes that are known to cause human familial Alzheimer's disease, and have high amyloid deposits in the hippocampus and cortical layer 5 Of neurons are lost.
- the compound of [Formula 1] of the present invention increased the number of neurons, which means that the compound of [Formula 1] differentiates neural stem cells into neurons in vivo to increase the number of neurons and / or beta. To protect neurons from amyloid.
- the compound of [Formula 1] increased the number of neurons in various parts of the 5xFAD mouse cerebral cortex, especially in the motor cortex, somatosensory cortex cortex compared to the control (Experimental Example 8, Figure 12, 13).
- the compound of Formula 1 can be used for the treatment of diseases caused by neuronal loss of the part, for example motor neuron disease such as amyotrophic lateral sclerosis.
- the compound of [Formula 1] in the cerebellum of the cerebellum (cerebellum) of the 5xFAD mouse increased the arborization of the axon of Purkinje neuron (Purkinje neuron) or preserved the axon structure (Experimental Example 11) , FIG. 18). This means that the compound of Formula 1 can be used for the treatment of cerebellar ataxia caused by the loss or damage of Purkinje cells of the cerebellum.
- the compound of Formula 1 has an effect of inducing neuronal neurogenesis, protecting neurons, or both, thereby inducing neural regeneration or neuro-regeneration.
- the compound of [Formula 1] increases Tuj1 expression, a marker of neurons during neuronal neoplasia in the cerebral somatosensory cortex of Alzheimer's disease model mouse (5xFAD), and hippocampus teeth.
- SGZ subgranular zone
- type 2 or type 3 cell-like cells characteristic during neuronal neurogenesis (as a result of Nissl and NeuN staining in Figure 16)
- immature to express DCX Neurons and BrdU staining increased the number of dividing cells (FIG. 16). This supports that trametinib induced neuronal neurogenesis in the cerebral cortex and hippocampus teeth of mice.
- the compound of [Formula 1] reduced the number of apoptosis cells that can be confirmed by the TUNEL assay in 5xFAD mice (Experimental Example 10, Figure 17), increase axon branching or axon structure of cerebellar Purkinje cells
- the effect of preservation (experimental example 11, FIG. 18) and the ratio of A ⁇ (1-42) / A ⁇ (1-40) of brain tissue (experimental example 13, FIG. 20) was shown. This indicates that the compound of [Formula 1] may contribute to nerve regeneration by protecting nerve cells in an environment where loss or damage of nerve cells occurs and improving the state or activity of nerve cells.
- neurodegenerative disease refers to a degenerative disorder of mental and physical function caused by the progressive structural and functional loss of neurons (neurons), specifically, dementia, Alzheimer's disease, vascular dementia, Geriatric dementia, frontal temporal lobe dementia, Lewy body dementia, Parkinson's disease, multiple system atrophy, corticobasal degeneration, progressive nucleus palsy, Huntington's disease, amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, spinal muscular atrophy Disease, progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pseudobulbar palsy, hereditary spastic paraplegia (HSP), cerebellar ataxia, Creutzfeldt Diseases selected from Jakob disease, multiple sclerosis, Guillain-Barré syndrome and the like.
- neurons specifically, dementia, Alzheimer's disease, vascular dementia, Geriatric dementia, frontal temporal lobe dementia, Lewy body dementia, Parkinson's disease, multiple system atrophy, corticobasal degeneration, progressive
- the term 'comprising as an active ingredient' herein means containing an amount sufficient to inhibit the neurodegenerative diseases of the present invention.
- the prophylactic or therapeutic compositions of the present invention can be prepared in parenteral dosage forms such as formulations common in the art such as oral administration or injection.
- compositions according to the invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of a medicament, each according to a conventional method of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols Oral dosage forms, external preparations, suppositories, patches, and sterile injectable solutions.
- Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, croscarmellose sodium, hydroxypropylmethylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, sodium lauryl sulfate , Colloidal silicon dioxide, croscarmellose sodium and mineral oil.
- Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, shoe, etc. in the pharmaceutical composition of the present invention. Prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
- Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
- Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, patches and the like.
- the non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.
- As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used.
- compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
- the composition of the present invention may be administered at 0.0001 to 10 g / kg, preferably at 0.001 to 8 mg / kg. Administration may be administered once a day or may be divided several times.
- the compound represented by [Formula 1] is 0.1mg to 10mg, 0.1mg to 5mg, 0.1mg to 2mg, 0.1mg to 1mg, 0.1mg to 0.5mg, 0.25mg to 2mg, 0.25mg to 1 day It may be administered in the range of 1 mg, 0.25 mg to 0.5 mg, 0.5 mg to 2 mg, 0.5 mg to 1 mg.
- the compound of Formula 1 may be administered at a dose of 0.1 mg, 0.125 mg, 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg per day.
- the present invention relates to a method for screening a substance capable of protecting neurons while simultaneously inducing neural stem cells into neurons in an environment simulating neurodegenerative diseases including Alzheimer's disease.
- the screening method of the present invention is beta amyloid, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), rotenone, oxidopamine, glutamate, LPS (lipopolysaccharide) ), Using neuron damage causing substances such as S100 calcium-binding protein B (S100B) and mouse-derived neural stem cells, comprising the following steps.
- the neural stem cells are preferably used that derived from the mouse.
- Using neural stem cells of an animal such as a mouse is easier to cultivate cells and has ethical advantages than using human neural stem cells.
- the neural stem cells need to be frequently replaced with a medium, and an expensive growth factor is required.
- the neural stem cells of the mouse can be used to avoid these disadvantages.
- human neural stem cells take more than 7 days to expand and differentiate cells, while mouse neural stem cells take 3 to 4 days to amplify cells and have a short differentiation period. Do.
- the neural stem cells can be isolated and cultured from the adult brain of the mouse.
- Mouse adult neural stem cells can be used isolated from the subventricular zone of 8 to 12 week old mice, for example 8 week old mice.
- neural stem cells of the mouse are often cultured by separating from the frontal lobe of the mouse embryo (embryo) 12 to 16 days after fertilization.
- Mouse embryonic neural stem cells are expected to be more tolerant to toxic environments because they have stronger stem cell abilities compared to neural stem cells derived from mouse adults.
- the present inventors treated beta amyloid with mouse embryonic neural stem cells, all of the cells were killed and differentiation into neurons could not be observed regardless of the treatment of the test substance (see Comparative Example 1 and FIG. 3).
- neural stem cells isolated from mouse adult brains were found to be more resistant to beta amyloid cells, making them suitable for screening of neural stem cell differentiation-inducing substances in an Alzheimer's disease-like environment. Therefore, the neural stem cells of an adult mouse are used in the method of this invention.
- Neural stem cells isolated from adult mice can be seeded in culture medium prior to differentiation and cultured at 37 ° C.
- a medium for culturing the neural stem cells it is preferable to use a serum-free medium composition containing a growth factor.
- an IPM medium containing a growth factor is used.
- N2 medium can be used when cultured and cultured after separation into single cells after neurospheres are formed.
- the IPM medium may contain 1-4% B27 supplement, 0.5-2% Glutamax, 100 IU / ml penicillin, 100 ⁇ g / ml streptomycin in Neurobasal medium, and N2 medium may contain Dulbecco's.
- DMEM / F12 Modified Eagle's Medium / Nutrient Mixture F12 (1: 1) 2.0 mg / ml glucose (d-(+)-glucose), 20-30 ⁇ g / ml insulin, 0.05-0.2 mg / ml apo-transferrin, 0.3-0.6 mMGlutamax, 50-150 IU / Ml penicillin and 50-150 ⁇ g / ml streptomycin added at least one component selected from the group consisting of 10-30 ng / ml bFGF, 10-30 ng / ml EGF and mixtures thereof It may be added to any one or more growth factors selected from the group consisting of.
- the growth factor plays a role of maintaining neural stem cells in undifferentiated state.
- the growth factor is included in the culture medium and cultured neural stem cells to treat test material in the state of differentiation inhibiting the effect of inducing differentiation of neural stem cells by test material. You can judge accurately.
- beta amyloid can be commercially available, and can be obtained, for example, from Gibco (Waltham, Mass.). It is especially preferable to use the thing derived from a human.
- Beta-amyloid is the most common form of A ⁇ (1-40) consisting of 40 amino acids and A ⁇ (1-42) consisting of 42 amino acids, of which A ⁇ (1-42) is more aggregated than A ⁇ (1-40) It is thought to be more associated with the disease state of Alzheimer's disease due to its stronger tendency to form, particularly toxic trimers or tetramers (Dahlgren, et al. (2002) Oligomeric and Fibrillar Species of Amyloid- ⁇ ) Peptides Differentially Affect Neuronal Viability.J. Biol.
- a ⁇ (1-42) is dissolved in hexafluoroisopropanol (HFIP), dried under vacuum, and the dried peptide is resuspended in DMSO to 5 mM. Add DMEM / F12 (without phenol red) to bring the peptide to a concentration of 100 ⁇ M and incubate at 4 ° C. for 24 hours.
- HFIP hexafluoroisopropanol
- MPTP is a prodrug for the neurotoxin MPP + (1-methyl-4-phenylpyridinium), and MPP + permanently induces symptoms of Parkinson's disease by destroying dopaminergic neurons in the black matter of the brain. MPTP is used to induce symptoms of Parkinson's disease in Parkinson's disease animal models.
- Rotenone is a substance that induces the degeneration of cerebral melanoma dopamine neurons by inhibiting the activity of mitochondrial complex I in cells, and is known to cause pathological characteristics of Parkinson's disease.
- Oxidopamine is also called 6-hydroxydopamine (6-OHDA) or 2,4,5-trihydroxyphenethylamine, which selectively destroys dopamine and noradrenaline neurons in the brain. It is a neurotoxic compound used by researchers. Oxidopamine is thought to enter neurons via dopamine and noradrenaline reuptake transporters. Oxidopamine is also used in conjunction with selective noradrenaline reuptake inhibitors (eg desipramine) to selectively damage dopaminergic neurons.
- 6-OHDA 6-hydroxydopamine
- 2,4,5-trihydroxyphenethylamine 2,4,5-trihydroxyphenethylamine
- S100B is a calcium binding protein that is secreted and expressed by astrocytes. S100B has neurotrophic activity in neuronal cell development and maintenance and affects the cognitive function of normal brain. However, abnormal elevations of S100B levels are harmful to neurons because they activate glial cells and cause neuroinflammatory responses.
- test substance After treatment with nerve cell damage causing substances, the test substance is treated with neural stem cells and further cultured with a growth factor treated daily. 12 hours after treatment and as early as 48-72 hours later, morphology analysis can be used to determine whether the test substance is inducing neural stem cell differentiation.
- Step 1 Mouse Embryonic Neural Stem Cell Culture
- Step 1A Culture of Mouse Embryonic Neural Stem Cells in Undifferentiated State
- Neural stem cells were isolated from the brain of mouse embryos at 14.5 days, and 10 ng / ml basic fibroblast growth factor (bFGF) (Peprotech, Princeton, NJ, cat #. 100-) in N2 culture medium. 18B) and 20 ng / ml human epidermal growth factor (EGF) (Peprotech, cat # .AF-100-15) to suspension for 4 days in a 25 cm 2 flask (Nunc, Pittsburgh, PA). Cultured in a) state. After 2 days, the formation of neurospheres was observed.
- bFGF basic fibroblast growth factor
- EGF human epidermal growth factor
- the formed neuro sphere of the single cells counted 4 ⁇ 5X10 5 cells is 200 ⁇ 300 ⁇ l of the number of cells was separated into (single cell) Prepared to be included in the culture (N2 culture medium containing 10 ng / ml bFGF and 20 ng / ml EGF). The coating solution was suctioned just before seeding the cells and evenly spread before the plate dries. Allow the cells to adhere to the plate for about 1 minute, then observe the cell attachment to some extent, then add 1.5 ml of culture medium (N2 culture medium containing 10 ng / ml bFGF and 20 ng / ml EGF). And incubated in a 37 °C incubator.
- the components of the N2 culture medium are as follows.
- Dulbecco's modified Eagle's medium (DMEM) / F12 (1: 1) (Gibco, cat #. 11320033), 100 ⁇ M putrescine (Sigma, cat #. 51799), 30 nM selenite (Sigma, cat # .S5261), 20 nM progesterone (Sigma, cat # .P0130), 1.55 mg / ml glucose (d-(+)-glucose) (Sigma, cat # .G8270), 25 ⁇ g / ml insulin ( insulin) (Gibco, cat #.
- Step 1B Culture of Neural Stem Cells Without Inhibiting Differentiation
- mouse embryonic neural stem cells When isolated and sprayed into single cells, mouse embryonic neural stem cells were cultured in the same manner as in 1A, except that bFGF and EGF were not treated.
- UD undifferentiated: undifferentiated
- D Differentiated (differentiated)
- Neural stem cells Undifferentiated neural stem cells of the UD group has a wide cell body, it is difficult to identify the shape of the neurites, and the cells continue to divide, so the total number of cells is larger than that of the D group.
- Differentiated cells of group D have a small and round cell body, and the neurites are elongated and elongated, clearly distinguishing their shape from UD group cells.
- bFGF basic fibroblast growth factor
- EGF epidermal growth factor
- the formed neurospheres are treated with 0.025% Trypsin-EDTA, separated into single cells, and the number of cells is counted so that 4-5X10 5 cells are included in 200-300 ⁇ l culture medium.
- Cells were prepared.
- N2 medium including 20 ng / ml bFGF and 20 ng / ml EGF
- the coating solution was suctioned just before seeding the cells and evenly spread before the plate dries. Allow the cells to adhere to the plate for about 1 minute, then observe the cell attachment to some extent, then add 1.5 ml of culture medium (N2 culture medium containing 20 ng / ml bFGF and 20 ng / ml EGF). And incubated in a 37 ° C. incubator for 24 hours.
- Step 1B Culture of Neural Stem Cells Without Inhibiting Differentiation
- Beta amyloid was purchased from human A ⁇ (1-42) of Gibco (Waltham, MA) and used for the beta amyloid oligomer formation.
- DMSO Sigma, cat # .D2650
- DMEM / F12 without phenol red
- trametinib was added to 10 nM and 100 nM, memantine (Sigma, cat # .M9292) to 5 ⁇ M and 10 ⁇ M, and AS703026 (pimasertib) to 10 ⁇ M, respectively. EGF and bFGF, beta amyloid and test materials were incubated for 4 days with daily treatment.
- memantine which is currently used as a symptom relieving Alzheimer's disease
- differentiation from neural stem cells to neural cells could not be confirmed, but rather neural stem cells were observed to die.
- Memantine is an NMDA receptor antagonist that is involved in glutamate signaling and helps normal neuronal signaling in Alzheimer's patients, and this function cannot be expected to treat Alzheimer's disease by repairing damaged neurons.
- the results of this experiment which indicate that memantine does not induce differentiation of neural stem cells but rather kill cells, confirm that memantine cannot be a fundamental treatment for dementia.
- Step 1 Mouse Embryonic Neural Stem Cell Culture
- beta amyloid was treated to mouse embryonic neural stem cells cultured in Step 1 above.
- Step 3 Administer Test Substance
- beta amyloid treatment 100 nM of trametinib, 10 ⁇ M of memantine and 10 ⁇ M of AS703026 (pimasertib) were added. EGF and bFGF, beta amyloid and test materials were incubated for 4 days with daily treatment.
- FIG. 3 On the 4th day of culture, the shape of the cells was observed under a phase contrast microscope, and the results are shown in FIG. 3.
- the upper part is a group not treated with beta amyloid in mouse embryonic neural stem cells
- the lower part is a group treated with beta amyloid at a concentration of 10 ⁇ M.
- UD Undifferentiated
- D Differentiated
- a ⁇ treatment group all the cells were killed regardless of the treatment of the test substance, and thus the effect of the test substance could not be confirmed.
- Tuj1 Neuron-specific class III beta-tubulin
- DAPI binds to the DNA of the cell and binds the nucleus to blue fluorescence. It is a dye labeled with.
- Embryonic nerve stem cells cultured by the method of Experimental Example 1 was sprayed on a 24-well plate containing a coverslip and treated with trametinib and AS703026 by concentration for 4 days. The medium was removed and washed with PBS. After fixing for 10 minutes at room temperature with 10% formaldehyde (Sigma, cat #. HT501128) and washed again with PBS. Permeabilization at room temperature for 15 minutes with 0.2% Triton X-100 (Sigma, cat # .93443) and 10% BSA (Sigma, cat # .A2153) + 1% normal goat serum (Vector lab, Burlingame) after PBS washing , CA, cat # .S1000) was incubated at room temperature for 1 hour.
- Tuj1 antibody Cell signaling, Danvers, MA, cat # 4466
- 1% BSA + 1% normal goat serum 1% normal goat serum at a ratio of 1: 200 and incubated overnight at 4 ° C.
- the solution was removed and washed with PBS after incubating the solution containing the secondary antibody (rhodamine attached antibody) in a ratio of 1: 200 in 1% BSA + 1% normal goat serum for 1 hour at room temperature, and washed with 5 ⁇ g
- Incubated with / ml DAPI Sigma, cat # .D9542
- the group treated with trametinib was found to actively increase the differentiation into neurons from the group treated with very low 10 nM concentration.
- the cell morphology was also found to increase the number of neural clumps in the neural outgrowth in the group treated with trametinib 10 nM.
- RNA concentration was measured to calculate 2 ⁇ g of RNA in each group, and the experiment was performed using a reverse transcription kit (Invitrogen, cat # .28025013). After adding 3 sterile water, 1 pM oligo dT, 1 mM dNTP and incubated at 65 ° C for 5 minutes, 5X First-strand buffer, 10mM DTT, M-MLV reverse transcriptase was added, and then 1 hour at 42 ° C and 72 ° C. CDNA made after 15 minutes incubation at 4 ° C. for 30 minutes was stored at ⁇ 20 ° C.
- a reverse transcription kit Invitrogen, cat # .28025013
- results are shown in FIG. 5A and 5B are mean values of relative mRNA expression levels of each group obtained by performing three times of qRT-PCR analysis.
- the relative mRNA expression level is divided by the amount of expression of the mRNA corresponding to each differentiation marker by the amount of GAPDH expression, which is generally not significantly different in order to normalize the total amount of RNA in each group.
- the neural stem cells were changed to N2 culture medium containing no EGF and bFGF, and further cultured for 4 days, RNA and protein were extracted, quantitative RT-PCR (qRT-PCR) and Western blotting was performed to confirm the effect of the activation of MEK1 and MEK2 on neural stem cell differentiation ability, as shown in FIG. 8C.
- Results of FIG. 8C show that differentiation is inhibited when MEK1 and MEK2 are activated in an environment in which differentiation is induced, in contrast to FIG. 8B.
- results of FIG. 8C show that differentiation is inhibited when MEK1 and MEK2 are activated in an environment in which differentiation is induced, in contrast to FIG. 8B.
- Tuj1 and TH When confirmed by qRT-PCR and western blotting, it was confirmed that the expression of the mRNA and protein of the neuronal marker Tuj1 and the dopamine neuronal marker TH was inhibited, especially when both MEK1 and MEK2 were activated. It was confirmed that the expression of Tuj1 and TH is significantly reduced as compared to when one is activated.
- mice mouse adult neural stem cells cultured as well in the plate (seeding) as in step 1A, and after incubation for 24 hours, to the cells 1 ⁇ g / ml of shRNA-MEK1 (CCGGGCCATCCAACATTCTAGTGAACTCGAGTTCACTAGAATGTTGGATGGCTTTTT) or shRNA-MEK2 (CCGGCCTCTCTCGAGATCTCTCTCGAGATC ) Was transfected with Lipofectamin (Invitrogen) to prepare neural stem cells with suppressed expression of MEK1 or MEK2, or both.
- shRNA-MEK1 CCGGGCCATCCAACATTCTAGTGAACTCGAGTTCACTAGAATGTTGGATGGCTTTTT
- shRNA-MEK2 CCGGCCTCTCTCGAGATCTCTCTCTCGAGATC
- Lipofectamin Invitrogen
- the neural stem cells thus prepared were changed to N2 culture medium again after 4 hours, or to medium in which 10 ⁇ M of beta amyloid (A ⁇ ) was treated in N2 medium, and cultured for 2 more days, followed by a microscope. The shape of the cells was observed, qRT-PCR was performed by RNA extraction by the method of Experimental Example 4, and the differentiation capacity into neurons was analyzed.
- a ⁇ beta amyloid
- differentiation was better induced in the case of neural stem cells which inhibited the expression of both MEK1 and MEK2 by simultaneously treating shMEK1 and shMEK2 than when either shMEK1 and shMEK2 were treated.
- beta amyloid was treated, cells were killed in the group not inhibited by MEK1 and MEK2, but when either or both of MEK1 and MEK2 were inhibited, cell death did not occur and differentiation was induced.
- AZD8330, Refametinib, PD318088, Binimetinib, and AS703026 confirmed that each compound induces differentiation only at the concentration at which differentiation is initiated.
- AZD8330, Refametinib, and PD318088 showed differentiation morphology from 1.0 ⁇ M to Binimetinib and AS703026 from 10 ⁇ M.
- Tuj1 a differentiation marker
- PD184352 showed only a slight differentiation induction effect at 0.1 ⁇ M, and both cells died at 1.0 ⁇ M and 10 ⁇ M.
- MEK 1/2 inhibitors PD0325901 (Selleckchem, cat # .S1036), RO5126766 (Selleckchem, cat # .S7170), BI847325 (Selleckchem, cat # .S7843) and U0126 (AGScientific, San Diego, CA, cat # .U-102) was administered to mouse adult neural stem cells cultured as in step 1A of Experimental Example 2 at various concentrations to observe whether there is an effect of inducing differentiation of neural stem cells. On the second day of the culture after the test material treatment, the shape of the cells was observed under a phase contrast microscope, and the results are shown in FIG. 10C. As shown in FIG.
- Step 1A Adult neural stem cells cultured as in Experimental Example 2, Step 1A were treated with beta amyloid 10 ⁇ M as in Step 2 of Experimental Example 2 to induce a cytotoxic environment, and AS703026 (10 ⁇ M), AZD8330, a MEK1 / 2 inhibitor. (1 ⁇ M), PD318088 (1 ⁇ M), Binimetinib (10 ⁇ M), Refametinib (1 ⁇ M), PD0325901 (10 ⁇ M), and RO5126766 (10 ⁇ M), respectively, were treated at the concentrations that best induced differentiation in Experiment 6. Incubated.
- a trametinib 0.1 ⁇ M treatment group and a cobimetinib 10 ⁇ M treatment group known as a selective inhibitor for MEK1 over MEK2, were added, as well as a group in which each compound was incubated without beta amyloid treatment. .
- the shape of the cells was observed under a phase contrast microscope, and the results are shown in FIG. 11.
- Half of the brain tissue (hemisphere) extracted from three mice per group were stored in a deep freezer immediately after extraction for western blotting and ELISA experiments. Brains from the other hemisphere of the other brain and from three mice per other group were placed in 10% formalin solution for one day at 4 ° C and 70%, 80%, 95% 100 Dehydration was performed for 1 hour in% alcohol order, soaked in xylene three times for 1 hour for clearing, and then soaked twice for 1 hour in paraffin in solution. After that, it was fixed well in the frame and made into sections with a thickness of 5 ⁇ m and stored at room temperature in the form of a slide.
- FIG. 13 shows the results of analyzing coronal sections of the motor cortex of the mouse cerebral cortex.
- the group administered only vehicle to 5XFAD mice showed damage of neurons of layer 5 of the motor cortex, but the number of neurons in the group of 5XFAD mice treated with trametinib. It was once again confirmed that it was significantly increased.
- FIG. 13B shows the number of cells stained with NeuN in the motor cortex layer 5, as a percentage of cells per unit area.
- FIG. Six parts of cotton per mouse were analyzed in three mice per group to count the total number of 18 cells and the ratio of NeuN stained cells in tramethinib-treated mice compared to vehicle-only groups. It was shown.
- trametinib has an effect of increasing the number of neurons in the cerebral cortex layer 5 and the hippocampus in 5XFAD mice.
- Fluorescent staining was performed by incubating Tuj1 antibody at 4 ° C for one day on brain tissue slides of 5XFAD mice of Experimental Example 1 and incubating Fluorescein isothiocyanate (FITC) -secondary antibody (Invitrogen, cat # .a21121) for 1 hour at room temperature. Stained with.
- the fluorescence micrographs of the somatosensory cortex of the cerebral cortex are shown in FIG. 15.
- the part indicated by the arrow ( ⁇ ) indicates the cells stained with Tuj1
- the head indicated by the arrow ( ⁇ ) indicates the plaque due to beta amyloid aggregation.
- Nissl staining is a staining method for staining the Nissl body of neurons and confirming the distribution of neurons and the state of neurons in the brain.
- the tissue on the slide of the mouse brain of Experimental Example 8 was rehydrated by soaking for 5 minutes in the order of xylene, alcohol 100%, 90%, 80%, 70%, 50%, water, and then 0.1% cresyl violet The solution was immersed in (Sigma, cat # .C5042) at room temperature for 15 minutes and dehydrated again with alcohol 80%, 90%, 100%, xylene. After covering well with a cover slip, neurons in the brain were observed.
- FIG. 16A The result is shown to FIG. 16A.
- SGZ subgranular zone
- ⁇ immature neurons stained by DCX staining (indicated by arrows ( ⁇ ) in the photo of the column labeled “Dcx” in FIG. 16) were identified, and in the dentate gyrus of the hippocampus of mice treated with trametinib It was confirmed that nerve regeneration is occurring.
- FIG. 16A The result is shown in the photograph of the column shown by "BrdU" of FIG. 16A.
- the cells marked with arrowheads ( ⁇ ) in the photograph are cells stained with BrdU.
- the number of BrdU-stained cells in the dentate gyrus of trametinib-treated 5XFAD mice was increased compared to the vehicle-administered group.
- Figure 16B shows the result of counting the number of BrdU stained cells, which also shows an increase in the number of BrdU stained cells in the trametinib administered group.
- Figure 16b is a graph showing the average value of the number of cells of a total of nine sides by counting the three parts per side in three mice per group.
Abstract
Description
명칭 | 화학구조 | MEK1, MEK2에 대한 IC50(IC50값의 측정방법에 대한 참고문헌) |
트라메티닙 | 0.92 ~ 3.4 nM(International Journal of Oncology 2011;39:23-31) | |
피마설팁(AS703026) | ≤1 μM(US 2009/0093462, Table I, Example 115) | |
AZD8330 | 7 nM (AACR Annual Meeting, 2009, Abst 3696) | |
비니메티닙(ARRY-162, ARRY-438162) | 12 nM(American College of Rheumatology, 2006 Annual Scientific Meeting, Abst 794) | |
레파메티닙(RDEA119, Bay 86-9766) | MEK1: 19 nM, MEK2: 47 nM(Cancer Res. 2009;69(17):6839-47) | |
PD318088 | 1.4 nM(WO 02/06213, Example 40) | |
PD0325901 | 3.6 ~24 nM(Oncotarget 2012;3:1533-1545) | |
RO5126766(CH5126766) | 160 nM(Cancer Res. 2013;73(13): 4050-4060) |
Gene | Forward Primer | Reverse Primer |
GAPDH | 5'- CGTGCCGCCTGGAGAAACC-3' | 5'- TGGAAGAGTGGGAGTTGCTGTTG-3' |
Tuj1 | 5'- GGTCTGGCGCCTTTGGA-3' | 5'- CACCACTCTGACCAAAGATAAAGTTG-3' |
TH | 5'-AGGTATACGCCACGCTGAAG-3' | 5'-CTCGGGTGAGTGCATAGGTG-3' |
Gene | Forward Primer | Reverse Primer |
GAPDH | 5'- CGTGCCGCCTGGAGAAACC-3' | 5'- TGGAAGAGTGGGAGTTGCTGTTG-3' |
Tuj1 | 5'- GGTCTGGCGCCTTTGGA-3' | 5'- CACCACTCTGACCAAAGATAAAGTTG-3' |
TH | 5'-AGGTATACGCCACGCTGAAG-3' | 5'-CTCGGGTGAGTGCATAGGTG-3' |
ChAT | 5'-CCTGCCAGTCAACTCTAGCC-3' | 5'-TACAGAGAGGCTGCCCTGAG-3' |
Gad1 | 5'-TCATGTTATGGAAATCTTGCTTCAG-3' | 5'-CGAGTCACAGAGATTGGTCATATACTACT-3' |
Isl1 | 5'-CGGAGAGACATGATGGTGGT-3' | 5'-GGCTGATCTATGTCGCTTTGC-3' |
Gene | Forward Primer | Reverse Primer |
GAPDH | 5'- CGTGCCGCCTGGAGAAACC-3' | 5'- TGGAAGAGTGGGAGTTGCTGTTG-3' |
Tuj1 | 5'- GGTCTGGCGCCTTTGGA-3' | 5'- CACCACTCTGACCAAAGATAAAGTTG-3' |
TH | 5'-AGGTATACGCCACGCTGAAG-3' | 5'-CTCGGGTGAGTGCATAGGTG-3' |
MEK1 | 5'-CGGCGGTTAACGGGACCA-34' | 5'-GGATTGCGGGTTTGATCTCCA-3' |
MEK2 | 5'-CCTGGATGAGCAGCAAAGGA-3' | 5'-CAGTGAGCCACCATCCATGT-3' |
Claims (20)
- 신경재생이 필요한 환자에게 MEK (mitogen-activated protein kinase kinase) 1 및 MEK 2를 모두 억제하는 화합물(MEK 1/2 억제제)을 투여하는 것을 포함하는 신경재생을 유도하는 방법으로서, 상기 MEK 1/2 억제제는 트라메티닙(trametinib), 피마설팁(pimasertib; AS703026), 비니메티닙(binimetinib), 레파메티닙(refametinib),로 이루어진 군에서 선택되는 하나 이상의 화합물인 방법.
- 제1항에 있어서, MEK 1/2 억제제가 신경줄기세포를 신경세포로 분화시키거나, 베타아밀로이드에 의한 세포 독성으로부터 신경줄기세포 및 신경세포를 보호하거나, 둘 다에 의해 신경재생을 유도하는 것인 방법.
- 제1항에 있어서, MEK 1/2 억제제가 트라메티닙인 방법.
- MEK (mitogen-activated protein kinase kinase) 1 및 MEK 2를 모두 억제하는 화합물(MEK 1/2 억제제)을 투여하는 것을 포함하는, 신경세포의 손실 또는 손상으로부터 신경세포를 보호하는 방법으로서, 상기 MEK 1/2 억제제는 트라메티닙(trametinib), 피마설팁(pimasertib; AS703026), 비니메티닙(binimetinib), 레파메티닙(refametinib),로 이루어진 군에서 선택되는 하나 이상의 화합물인 방법.
- 제4항에 있어서, MEK 1/2 억제제가 트라메티닙인 방법.
- 제6항에 있어서, 신경퇴행성 질환이 치매, 알츠하이머병, 혈관성치매, 노인성치매, 전두측두엽치매, 루이소체치매, 파킨슨병, 다계통위축증, 피질기저핵변성(corticobasal degeneration), 진행성핵상마비, 헌팅턴병, 근위축성 측삭 경화증(루게릭병, ALS), 원발성측삭경화증, 척수근육위축병, 진행성 연수마비(progressive bulbar palsy; PBP), 진행성 근위축증(progressive muscular atrophy; PMA), 가성연수마비(pseudobulbar palsy), 유전성 강직성 하반신마비(hereditary spastic paraplegia; HSP), 소뇌성 운동실조증, 크로이츠펠트-야콥병, 다발성경화증, 및 길랑-바레 증후군으로 이루어진 군에서 선택된 것인 방법.
- 제6항에 있어서, MEK 1/2 억제제가 트라메티닙인 방법.
- 제8항에 있어서, 신경퇴행성 질환이 알츠하이머병인 방법.
- 제8항에 있어서, 신경퇴행성 질환이 혈관성치매인 방법.
- 제8항에 있어서, 신경퇴행성 질환이 근위축성 측삭 경화증(루게릭병, ALS)인 방법.
- 제8항에 있어서, 신경퇴행성 질환이 헌팅턴병인 방법.
- 제8항에 있어서, 트라메티닙이 1일 0.1 mg 내지 2 mg의 용량으로 투여되는 것인 방법.
- 제13항에 있어서, 트라메티닙이 1일 0.1 내지 1 mg의 용량으로 투여되는 것인 방법.
- 제14항에 있어서, 트라메티닙이 1일 0.1 내지 0.5 mg의 용량으로 투여되는 것인 방법.
- 1) 성체 마우스 유래의 신경줄기세포에 베타아밀로이드, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), 로테논(Rotenone), 옥시도파민(oxidopamine), 글루타메이트, LPS (lipopolysaccharide), 및 S100B (S100 calcium-binding protein B)로 이루어진 군에서 선택된 하나 이상의 신경세포 손상 유발 물질을 처리하는 단계;2) 상기 신경세포 손상 유발 물질이 처리된 신경줄기세포에 시험 물질을 투여하는 단계; 및3) 세포의 형태소(morphology) 분석을 통해 신경줄기세포의 분화 및 세포 사멸 여부를 확인하는 단계;를 포함하는, 신경퇴행성 질환 치료제 후보 물질의 스크리닝 방법.
- 제16항에 있어서, 신경세포 손상 유발 물질이 베타아밀로이드인 방법.
- 제17항에 있어서, 베타아밀로이드가 올리고머 형태의 베타아밀로이드인 방법.
- 제18항에 있어서, 올리고머 형태의 베타아밀로이드가 베타아밀로이드의 트라이머(trimer)와 테트라머(tetramer)를 포함하는 것인 방법.
- 제19항에 있어서, 베타아밀로이드가 42개의 아미노산으로 이루어진 Aβ(1-42)인 방법.
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