EA038404B1 - Use of trametinib in treating neurodegenerative disease due to neuronal loss or damage - Google Patents

Abstract

The invention relates to the use of trametinib in treating neurodegenerative disease due to neuronal loss or damage. In particular, according to the invention, the neurodegenerative disease in treated by differentiating neural stem cells into neurons, by protecting neural stem cells and neurons against cytotoxicity of amyloid-betas, or by both of the above.

Description

Technology area

This invention relates to a method for inducing neuroregeneration, comprising administering a MEK 1/2 inhibitor to a patient in need thereof, and to a composition comprising a MEK 1/2 inhibitor for use in the above method. In the present invention, the MEK 1/2 inhibitor induces neuroregeneration by differentiating neural stem cells into neurons, protecting neural stem cells and neurons from the cytotoxic effect of amyloid beta, or both. This invention also relates to a method for protecting neurons from loss or damage, comprising administering a MEK 1/2 inhibitor, and to a composition comprising a MEK1 / 2 inhibitor for use in the above method. In addition, the present invention relates to a method for preventing or treating a neurodegenerative disease due to loss or damage of neurons in a patient in need thereof, comprising administering a MEK 1/2 inhibitor, and to a composition comprising a MEK 1/2 inhibitor for use in the above method.

Prior art

Neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) are common among the elderly, and the number of patients increases exponentially with the aging population. In addition, reports of early-onset neurodegenerative diseases occurring at a young age are not uncommon. Thus, there is great interest in the development of a treatment that will help stop disease progression or repair damaged brain tissue.

The exact causes of such neurodegenerative diseases have not yet been established. At the moment, it is known that damage to nerve cells in certain areas of the brain (for example, the hippocampus or substantia nigra) leads to a decrease in the number of nerve cells, the appearance of defects in the neural network, as a result of which various symptoms of neurodegenerative diseases develop.

Research aimed at finding a cure is being conducted in various directions. Currently, drugs designed to relieve symptoms include memantine (an NMDA receptor antagonist), L-DOPA (a drug analogue of dopamine), etc. they have a therapeutic effect other than temporarily relieving symptoms. Thus, there is a great need for a basic treatment directed at the cause of the neurodegenerative disease.

Nerve stem cells (NSCs) and neural progenitor cells (NPCs), cells that can differentiate into nerve cells, are present in the adult brain. Nerve stem cells are present in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus, and it is in this region that neurogenesis proceeds throughout the animal's life through differentiation and proliferation of neural stem cells (Zhao et al. (2008), Mechanisms and Functional Implications of Adult Neurogenesis. Cell 132: 645-660).

Since neurodegenerative diseases cause damage and loss of brain nerve cells, replacement of damaged or lost neurons with normally functioning neurons due to stimulation of NSC and NPC may be the main treatment for neurodegenerative diseases. This method of treatment includes a method of stem cell treatment in which NSCs and NPCs are isolated from the patient's body, stimulated to differentiate into neurons in vitro, and then transplanted back into the patients. However, there are difficulties in isolating NSC and NPC in patients and their reverse transplantation to patients. In addition, transplanted NSCs and NPCs quickly lose their activity in the brain, which requires repeated transplants. As an alternative to transplanting neural stem cells into patients, a method has recently been proposed for the formation of neurons in the patient's brain by stimulating NSCs and NPCs to differentiate using drugs (Davies et al. (2015), Stemistry: The Control of Stem Cells in Situ Using Chemistry. J. Med Chem 58: 28632894).

Beta-amyloid (Ae) is a 36-43 amino acid peptide that results from the cleavage of amyloid precursor protein (APP), an integral type 1 membrane protein, by β-secretase and γ-secretase. Aggregates of beta-amyloid (Ae) in the form of soluble oligomers of beta-amyloid (Ae), and then protofibrils form insoluble Ae fibrils, which subsequently form clusters in the brain in the form of amyloid plaques. Ae deposition in the brain is associated with synapse damage, neuronal damage and brain atrophy and ultimately leads to impaired memory and cognitive function, two very characteristic symptoms of Alzheimer's disease (AD). Among the various Ae forms, soluble Ae oligomers, in particular trimers and tetramers, are considered the most toxic Ae forms associated with neuronal dysfunction and synapse damage (Murakami, (2014), Conformation-specific antibodies to target amyloid e oligomers and their application to immunotherapy for Alzheimer's disease. Biosci. Biotechnol. Biochem. 78 (8): 1293-1305; Jana et al. (2016), Membrane-bound tetramer and trimer Ae oligomeric species correlate with toxicity towards

- 1 038404 cultured neurons. J. Neurochem. 136 (3): 594-608).

Therefore, the protection of neurons from Ae, especially from oligomeric forms of Ae, is considered a possible target for AD treatment. However, AD patients already have serious neuronal damage, therefore, for the main treatment of AD, in addition to protecting neurons, neuronal regeneration is necessary due to the differentiation of endogenous nerve stem cells.

MEK (mitogen-activated protein kinase kinase; also known as MAP2K or MAPKK) is a member of the MAP kinase (mitogen-activated protein kinase; MAPK) signaling pathway (referred to herein as the MAPK / ERK pathway), which is a chain of reactions catalyzed by Ras- Raf-MEK-ERK. When various signaling molecules, such as growth factors, hormones, cytokines, etc., bind to the receptors of the cell membrane and activate the receptor tyrosine kinase, the Ras GTPase is activated, as a result of which the cytoplasmic protein Raf is recruited to the cell membrane. Activated Raf sequentially phosphorylates and activates MEK and ERK, and activated ERK kinase, in turn, is translocated into the nucleus and activates various transcription factors. These transcription factors then bind to promoters of various genes, regulating cell proliferation, differentiation, and survival. Since the MAPK / ERK signaling pathway is hyperactivated in tumor cells, kinases are considered important targets for suppressing progressed cancer and other proliferative diseases.

There are 7 proteins known in the MEK family (MEK1-MEK7), of which only MEK1 and MEK2 are involved in the Ras-Raf-MEK-ERK signaling pathway. Although MEK1 and MEK2 are encoded by different genes, they have high homology (80%) both in the region of the C-terminal catalytic kinase domain and in most of the N-terminal regulatory region. Despite the fact that oncogenic forms of MEK1 and MEK2 have not been found in human cancers, it is known that constitutive activation of MEK leads to cell transformation. In addition, MEK can also be activated by other oncogenes. Therefore, inhibition of MEK1 and MEK2 has been investigated as a target in the development of anticancer drugs. However, it is unclear what role MEK1 and MEK2 and the MAPK / ERK pathway play in the proliferation and differentiation of adult neural stem cells.

In addition, there are research results that indicate a link between the MAPK / ERK pathway and Ae or tau proteins in the brains of Alzheimer's disease (AD) patients. Unfortunately, it is unclear whether treatment of AD requires activation or inhibition of this signaling pathway, whether the regulation of this signaling pathway can be associated with AD treatment.

Increased expression of MAPK / ERK pathway proteins has been reported in patients with very early stages of Alzheimer's disease (Arendt et al. (1995), Increased Expression and Subcellular Translocation of the Mitogen-Activated Protein Kinase Kinase and Mitogen-Activated Protein Kinase in Alzheimer's Disease . Neuroscience 68 (1): 5-18; Gartner et al. (1999), Elevated Expression of p21ras is an Early Event in Alzheimer's Disease and Precedes Neurofibrillary Degeneration. Neuroscience 91 (1); 1-5), or associations between ERK1 / 2 and MEK1 / 2 with hyperphosphorylation of tau protein in the brain of a patient with Alzheimer's disease (Pei et al. (2002), Up-Regulation of Mitogen-Activated Protein Kinases ERK1 / 2 and MEK1 / 2 is Associated with the Progression of Neurofibrillary Degeneration in Alzheimer's Disease. Brain Res Mol Brain Res. 109 (1-2): 45-55) and increased Ras expression and ERK1 / 2 activation in B103 cells (mouse neuroblastoma cells) expressing amyloid precursor protein (APP) (Chaput et al. (20 12), SILAC-based Proteomic Analysis to Investigate the Impact of Amyloid Precursor Protein Expression in Neuronal-LikeB103 Cells. Electrophoresis 33 (24): 3728-3737).

However, other studies have shown that activation of ERK1 / 2 suppresses apoptosis induced by Ae and decreases accumulation of Ae (Guerra et al. (2004), Plasma Membrane Oestrogen Receptor Mediates Neuroprotection Against β-Amyloid Toxicity Through Activation of Raf-1 / MEK / ERK Cascade in Septal-Derived Cholinergic SN56 Cells. J. Neurochem. 91: 99-109; Watson et al. (2005) Macrophage Inflammatory Protein 2 Inhibits β-Amyloid Peptide (1-42) -Mediated Hippocampal Neuronal Apoptosis through Activation of Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase Signaling Pathways. Molecular Pharmacology 67 (3): 757-765; Mills et al. (1997) Regulation of Amyloid Precursor Protein Catabolism Involves the Mitogen-Activated Protein Kinase Signal Transduction Pathway. J. Neurosci. 17: 9415-9422). In addition, data have been published that ERK1 / 2 reduces the activity of γ-secretase, which induces the formation of Ae from APP and decreases the expression and activity of BACE1 (β-secretase 1) under conditions of oxidative stress (Tamagno et al. (2009), JNK and ERK1 / 2 Pathways Have a Dual Opposite Effect on the Expression of BACE 1. Neurobiology of Aging, 30: 1563-1573).

Thus, it is necessary to clarify the role of regulation of the MAPK / ERK pathway and inhibition of MEK in neurodegenerative diseases such as Alzheimer's disease, as there are conflicting opinions on this.

The essence of the invention

Technical challenge.

The present inventors need to provide a method for inducing neuroregeneration by differentiating neural stem cells into neurons and protecting neural stem cells and neurons from β with a compound that inhibits MEK1 and MEK2. In addition, the inventors need to provide a method for protecting neurons from loss or damage and a method for preventing or treating a neurodegenerative disease due to loss or damage of neurons using a compound that inhibits MEK 1 and MEK2.

The solution of the problem.

The present inventors have found that a compound that inhibits MEK 1 and MEK2 (hereinafter referred to as a MEK 1/2 inhibitor), in particular the compound represented by formula 1, can effectively induce the differentiation of neural stem cells into neurons; more specifically, it protects neural stem cells and neurons from amyloid beta and at the same time induces neural stem cell differentiation into neurons.

I F

Formula 1

One aspect of the present invention relates to a composition for inducing differentiation of neural stem cells comprising a compound of formula 1. The composition does not induce tumor-like growth in neural stem cells.

The composition that induces the differentiation of neural stem cells induces the differentiation of neural stem cells into neurons, even in the presence of beta-amyloid.

The induction of differentiation into neurons under the influence of a composition that induces the differentiation of neural stem cells may result from inhibition of MEK 1 and MEK2.

This composition may include a specific MEK 1/2 inhibitor other than the compound of Formula 1. The composition induces neural stem cell differentiation 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 a composition for inducing neural stem cell differentiation.

The differentiation method may be treating neural stem cells with a composition that induces neural stem cell differentiation and passing 1 to 7 days to complete differentiation.

Another aspect of this invention relates to a compound of formula 1 for use in inducing differentiation of neural stem cells, in particular for use in protecting neural stem cells and neurons and differentiating neural stem cells into neurons even in the presence of amyloid beta. Another aspect of the present invention relates to a particular MEK 1/2 inhibitor for use in inducing differentiation of neural stem cells, in particular for use in protecting neural stem cells and neurons and differentiating neural stem cells into neurons even in the presence of amyloid beta.

Another aspect of the present invention relates to a kit for use in inducing differentiation of neural stem cells into neurons in vitro. The kit may include a composition for inducing differentiation of neural stem cells, culture medium, plates, coating solutions, and additives required for cell culture such as growth factors, etc.

Another aspect of the present invention relates to a method for inducing neuroregeneration comprising administering a particular MEK 1/2 inhibitor to a patient in need thereof. In the present invention, the MEK 1/2 inhibitor induces neuroregeneration by differentiating neural stem cells into neurons, protecting neural stem cells and neurons from the cytotoxic effect of amyloid beta, or both. The most preferred MEK 1/2 inhibitor for this method is a compound of Formula 1. A further aspect of the present invention relates to a particular MEK 1/2 inhibitor, in particular a compound of Formula 1, for use in neuroregeneration.

Another aspect of the present invention relates to a method for protecting neurons from loss or damage, comprising administering a particular MEK 1/2 inhibitor to a patient in need thereof. The most preferred MEK 1/2 inhibitor for this method is a compound of Formula 1. An additional aspect of the present invention relates to a particular MEK 1/2 inhibitor, in particular a compound of Formula 1, for use in protecting neurons from loss or damage.

Another aspect of the present invention relates to a pharmaceutical composition containing a compound of Formula 1 as an active ingredient for the prevention or treatment of neurodegenerative diseases, or a method for preventing or treating neurodegenerative diseases using a compound of Formula 1.

Neurodegenerative diseases refer to functional impairments in various systems, such as the regulation of motor skills, cognitive function, perceptual function, sensory function, and the autonomic nervous system, due to loss or decrease in neuronal function. Examples of neurodegenerative diseases include dementia, Alzheimer's disease (AD), vascular dementia, senile dementia, frontotemporal dementia (FTD), Lewy body dementia (LBD), Parkinson's disease (PD), multiple system atrophy (MSA), corticobasal degeneration ( CBD), progressive supranuclear palsy (PSP), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), primary lateral sclerosis (PLS), spinal muscular atrophy, progressive bulbar palsy (PBP), progressive muscle atrophy (PMA) ), pseudobulbar paralysis, hereditary spastic paraplegia (HSP), cerebellar ataxia, Creutzfeldt-Jakob disease (CJD), multiple sclerosis (MS), Guillain-Barré syndrome (GBS), etc.

Another particular MEK1 / 2 inhibitor other than the compound of Formula 1 may be included in a pharmaceutical composition, may or may be used in a method.

An additional aspect of the present invention relates to a compound of Formula 1 for use in the prevention and treatment of a neurodegenerative disease. In addition, it relates to a particular MEK 1/2 inhibitor for use in the prevention and treatment of neurodegenerative disease.

Another aspect of the present invention relates to a method of screening for compounds that induce the differentiation of neural stem cells into neurons and protect them under conditions that mimic those found in a neurodegenerative disease such as Alzheimer's disease.

The screening method according to the present invention comprises the steps of:

1) treatment of neural stem cells isolated from an adult mouse with substances that cause neuronal damage, such as beta-amyloid (especially in its oligomeric form), MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ), rotenone, oxidopamine, glutamate, LPS (lipopolysaccharide), S100B (S100 calcium-binding protein B);

2) adding a test substance to nerve stem cells treated with the above substances that cause neuronal damage; and

3) study of differentiation or death of nerve stem cells by analyzing morphology.

Various aspects and embodiments of the present invention will now be described in detail.

As used herein, the term neural stem cells refers to the ability to continuously proliferate in an undifferentiated state (self-renewal) and the ability to differentiate into different neurons and glia from a single stem cell (multipotency). The neural stem cell is of animal origin.

The term animal not only refers to humans and primates, but also encompasses animals such as cows, pigs, sheep, horses, mice, rats, cats, etc., and preferably humans. Where the term neural stem cell is used, it encompasses a neural progenitor cell.

In this invention, the term differentiation refers to the development of a cell into a specialized cell. More specifically, it refers to the process by which the structure or function of a cell becomes specialized as a result of cell division, cell proliferation and growth, and to changes in the structure (morphology) or function of cells and tissues to perform their inherent functions. Differentiation of neural stem cells is preceded by an asymmetric division of the mother cell into two cells that have different properties. One of the daughter cells is the same as the parent cell, remaining a stem cell, while the other differentiates into a specialized cell. The fact that the process of such asymmetric division accompanies neural stem cell differentiation means that neural stem cell differentiation involves proliferation.

In this invention, the term proliferation refers to a phenomenon in which a cell divides and proliferates. This, in particular, refers to an increase in the number of cells of the same type due to cell division, an increase in the number of cells due to the reproduction of the same cell shape.

In this invention, the term protection refers to the prevention of cell damage by damaging external stimuli, so that neural stem cells can proliferate or differentiate without causing cell death, and differentiated neurons can survive in the presence of cytotoxic factors, in particular beta-amyloid. In relation to AD, the term protection in this invention includes the protection of neural stem cells and neurons from damage caused by beta-amyloid by decreasing the Av ( _1-42 ) / Av ( _1-40 ) ratio in the brain (Majid et al. (2015 ), Pharmacologic Treatment with Histone Deacetylase 6 Inhibitor (ACY-738) Recovers Alzheimer's Disease Phenotype in Amyloid Precursor Protein / Presenilin 1 (APP / PS1) Mice. Alzheimers Dement. 170-181; Borchelt et al. (1996), Familial Alzheimer's -Linked Presenilin 1 Variants

- 4 038404

Elevate Αβ _1-42 / 1-40 Ratio In Vitro and In Vivo. Neuron. 17: 1005-1013).

As used herein, the term prevention refers to all actions that suppress or slow down the progression of neurodegenerative diseases by administering a pharmaceutical composition of this invention. Treatment refers to any action that relieves symptoms or ameliorates disease in patients who are suspected or diagnosed with neurodegenerative disease.

One aspect of the present invention relates to a composition for inducing differentiation of neural stem cells, comprising a compound represented by formula 1

Formula 1

The common name of the compound represented by the above formula 1 is trametinib, and its chemical name is N- (3- {3-cyclopropyl-5 - [(2-fluoro-4-iodophenyl) amino] 6,8-dimethyl-2,4 , 7-trioxo-3,4,6,7-tetrahydropyrido [4,3-d] pyrimidin-1 (2H) -yl} phenyl) acetamide. It is disclosed in example 4-1 of WO2005 / 121142, where the applicant is Japan Tobacco Inc. The compound of formula 1 inhibits MEK1 and MEK2, components of the MAPK / ERK (mitogen-activated protein kinase / kinase regulated by extracellular signaling) signaling pathway, pre-ERK. This compound is used as an antineoplastic agent in melanoma and non-small cell cancer. In the present invention, it is used in the form of a free base or a pharmaceutically acceptable salt or solvate. Examples of possible solvates are hydrates, dimethyl sulfoxide, acetic acid, ethanol, nitromethane, chlorobenzene, 1-pentanol, isopropyl alcohol, ethylene glycol, 3-methyl-1-butanol, etc.

In particular, one aspect of the present invention relates to a composition comprising a compound of Formula 1 that both protects neural stem cells and neurons and induces neural stem cell differentiation into neurons. Deposits of beta-amyloid in the brain as amyloid plaques are associated with synapse damage, neuronal damage and brain atrophy and are known to ultimately cause the characteristic symptoms of Alzheimer's disease, memory and cognitive impairment. Thus, the protection of neural stem cells and neurons from beta-amyloid during neuronal regeneration as a result of differentiation of endogenous neural stem cells may become the main treatment for Alzheimer's disease.

Λβ ( _1-42 ), consisting of 42 amino acids, has a higher tendency to form aggregates than Λβ ( _1-40 ) and a higher tendency to form more toxic trimers or tetramers, and therefore is considered closely associated with pathological changes in Alzheimer's disease. Thus, a composition is preferred that can protect neural stem cells and induce their differentiation even in the presence of Λβ ( _1-42 ), especially oligomers ^Λβ (1-42).

With the aim of finding a composition that induces differentiation of mouse neural stem cells into neurons, the inventors experimentally confirmed, as shown in the Examples, that the compound of formula 1 is very effective in inducing neuronal differentiation of neural stem cells isolated from the brains of mouse embryos or adult mice.

Nerve stem cells have the ability to differentiate into various neurons or glial cells such as oligodendrocytes, astrocytes and microglia. The compound of formula 1 induces differentiation of neural stem cells predominantly into neurons and restricts differentiation into glial cells. Thus, the compound of formula 1 can effectively induce the formation of neurons (neurogenesis), thereby allowing these new neurons to replace damaged neurons in the brain of patients suffering from neurodegenerative disease accompanied by neuronal damage, and can be used as a drug that promotes neural regeneration. or neuroregeneration.

As shown in specific examples in this invention, the inventors have established in in vitro experiments using oligomeric Αβ, mimicking conditions in the brain of patients suffering from Alzheimer's disease, that the compound of formula 1 protects neural stem cells or neurons from death and induces differentiation of neural stem cells into neurons (Fig. 2).

In addition, one aspect of the present invention relates to a composition for inducing differentiation of neural stem cells, comprising a particular compound that inhibits MEK1 and MEK2.

- 5 038404

In the present invention, a compound that inhibits MEK1 and MEK2 is also referred to as a MEK 1/2 inhibitor. MEK 1/2 inhibitor preferably has an IC50 value in the nanomolar range, and IC ₅₀ values for MEK1 and MEK2, differ less than 10 times, preferably less than 5 times. IC ₅₀ values for MEK1 and 2 can be measured by methods described in publications such as [Yamaguchi et al. (2011) International Journal of Oncology 39: 23-31]. The MEK 1/2 inhibitor that can be used in the present invention is a compound that induces the differentiation of neural stem cells into neurons, while protecting neural stem cells and neurons from toxic substances such as Ab. Examples of MEK 1/2 inhibitors that can be used in this invention are as follows: trametinib, pimacertib (AS703026), AZD8330, binimetinib (MEK162, ARRY-162, ARRY-438162), refametinib (RDEA119, Bay 86-9766), PD318088 , PD0325901, RO5126766.

The chemical structure of the preferred MEK 1/2 inhibitors and their IC ₅₀ values, as well as publications describing methods for measuring IC ₅₀ values, are shown in table. 1.

Table 1

Name Chemical structure IC50 value for MEK 1, MEK2 (reference to how IC50 values are determined) Trametinib Н у / ^ζΝ Λ ^Ν ν ^ΝΗ О ^ν Χκ, 0.92 ~ 3.4 nM (International Journal of Oncology 2011; 39: 23-31) Pimacertib (AS703026) O O O) = / iz i <1 μM (US 2009/0093462, Table I, Example 115) AZD8330 T O O / = z / = \ O rz \% 7nM (AACR Annual Meeting, 2009, Abst 3696) Binimetinib (ARRY162, ARRY-438162) Bg A A ^ ΝΗ (ΪΎ n ¹ O 12 hM (American College of Rheumatology, 2006 Annual Scientific Meeting, Abst 794) Refametinib (RDEA119, Wow 869766) f F MEK1: 19 nM, MEK2: 47 hM (Cancer Res. 2009: 69 (17): 6839-47)

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In addition, one aspect of the present invention relates to a composition containing a MEK 1/2 inhibitor for the differentiation of neural stem cells into neurons while protecting neural stem cells and neurons from amyloid beta, especially the oligomeric form Λβ ( _1-42 ).

The use of a selective inhibitor of either MEK1 or MEK2 rather than a MEK 1/2 inhibitor that inhibits MEK1 and MEK2 is undesirable because its differentiation-inducing activity in neural stem cells is weak. For example, kobimetinib represents MEK1selektivny inhibitor selectivity in respect of MEK1 is 100 or more times higher than for MEK2 (MEK1 IC50 = 0.95 nM, MEK2 IC ₅₀ = 199 nM; Molecules 2017; 22: 1551). Unlike the MEK 1/2 inhibitor, cobimetinib does not induce differentiation of adult mouse neural stem cells even at a high concentration of 10 μM, regardless of the action of beta-amyloid (example 7).

The fact that inhibition of both MEK1 and MEK2 is involved in the induction of NSC differentiation into neurons and the protection of NSCs and neurons was also confirmed experimentally in Example 5. In these experiments, shRNA (short hairpin RNA) was used to inhibit the expression of MEK1 and MEK2 and plasmids with constitutively active MEK1 (CAMEK1) and constitutively active MEK2 (CAMEK2) to activate the expression of MEK1 and MEK2 (example 5).

However, not all MEK 1/2 inhibitors show the same effect. For example, the MEK 1/2 inhibitors U0126, PD184352, and BI847325 exhibited a weak differentiation-inducing effect of neural stem cells (NSCs) into neurons, or caused cytotoxicity, making them unsuitable for use as a differentiation-inducing neural stem cell composition according to this invention (Fig. 10a and 10c). Thus, the differentiation-inducing NSC activity or the NSC- and neuron-protective activity may differ depending on the unique properties that the compound possesses in addition to its MEK 1/2 inhibitory activity. In the present invention, MEK 1/2 inhibitors that have shown differentiation-inducing activity of NSCs into neurons or protecting NSCs and neurons are trametinib, pimacertib (AS7O3026), AZD8330, binimetinib, refametinib, PD318088, PD0325901, and RO5126766.

In particular, the compound of formula 1 (trametinib) showed significantly superior differentiation and protection against β even when compared to other MEK 1/2 inhibitors that can be used in this invention. For example, the compound of Formula 1 exhibited greater differentiation and protection against β compared to AS703026 at a concentration that was 100-fold or more lower. This effect of the compound of Formula 1 is substantially superior to that predicted solely by its MEK1 / 2 inhibitory activity.

The compound of formula 1 has never been used for the induction of neural stem cell differentiation into neurons, and it was only known as an inhibitor of MEK 1/2, suppressing the activity of MEK1 and MEK2 by non-competitive inhibition of ATP binding, and as a drug for the treatment of melanoma and non-small cell carcinoma.

Another aspect of the present invention relates to a method for inducing NSC differentiation into neurons using a compound of Formula 1 or other MEK1 / 2 inhibitors.

With regard to the method disclosed in this invention, neural stem cells can be isolated from the brain of embryos or adults by known methods. Alternatively, neural stem cells can be purchased from commercial sources or can be cultured by any standard method known in the art. There are no specific restrictions on this. In the Examples section below, neural stem cells isolated from

- 7,038404 frontal lobes of 14.5-day-old mouse embryos and subventricular zone of mice at the age of 8 weeks.

Before differentiation, neural stem cells can be introduced into the culture medium and cultured at 37 ° C. The culture medium can be any serum-free medium supplemented with growth factors, without any particular limitation. For example, the medium can be a mixture of Dulbecco's modified Eagle's medium and F12 growth medium (DMEM / F12) (1: 1) with the addition of one or more components selected from the group consisting of 90-110 μM putrescine, 2040 nM selenite, 10 -30 nM progesterone, 1.0-2.0 mg / ml d - (+) glucose, 20-30 μg / ml insulin, 0.05-0.2 mg / ml apo-transferrin, 0.3-0, 6 mM glutamax, 50-150 IU / ml penicillin and 50-150 μg / ml streptomycin and additionally with the addition of growth factors selected from the group consisting of 1030 ng / ml bFGF, 10-30 ng / ml EGF or mixtures thereof.

To obtain undifferentiated neural stem cells, neural stem cells can be cultured in N2 medium supplemented with growth factors [Eagle's medium modified by Dulbecco (DMEM) / F12 (1: 1), supplemented with 100 μM putrescine, 30 nM selenite, 20 nM progesterone, 1 , 55 mg / ml d - (+) - glucose, 25 μg / ml insulin, 0.1 mg / ml apo-transferrin, 0.5 mM glutamax, 100 IU / ml penicillin and 100 μg / ml streptomycin].

Differentiation of cultured neural stem cells into neurons involves exposing the neural stem cells to a differentiation-inducing composition containing a compound of Formula 1 and then allowing differentiation in accordance with a method known in the art. For example, the differentiation-inducing composition of neural stem cells of the present invention is added to a medium containing cultured neural stem cells and differentiation is induced at 37 ° C.

The process of differentiation of neural stem cells into neurons proceeds under various cultivation conditions indicated above (see, for example, the content and amount of medium components and the cultivation time). The cultivation conditions are not limited to the specific conditions mentioned above. Preferably, the culture temperature at which the differentiation of neural stem cells can be induced is 35 to 40 ° C. If the culture temperature is below 35 ° C or above 40 ° C, neural stem cells die before differentiating into neurons.

Before adding the composition for inducing differentiation of neural stem cells containing the compound of formula 1, it is preferable to provide a sufficient concentration of cells in the culture of neural stem cells. In addition, to observe changes in culture, such as cell proliferation, differentiation or death, it is preferable to act on the neural stem cells with a composition that induces the differentiation of neural stem cells containing the compound of formula 1 according to the method described above for a cultivation period of 7 days. or less. For this, the cultivation period of the neural stem cells is preferably at least 1 day to a maximum of 7 days to ensure a sufficient cell concentration.

With regard to the concentration of the compound of Formula 1, it is preferable to add it to the neural stem cells at a concentration of 1 nM to 20 μM. If the concentration is less than 1 nM, the ability to induce the differentiation of neural stem cells decreases, if it exceeds 20 μM, problems with cytotoxicity begin to arise. For compound addition, neural stem cells are usually plated into the wells of a plate to cover up to 70-80% of the well area. For example, 12-well plates are seeded with 1 x 10 ⁵ cells / well, 6-well plates are seeded with ⁵ x 10 5 cells / well.

In addition, as detailed in the Examples section, it is preferable to use the compound of Formula 1 at a concentration of 10 nM to 10 μM, more preferably 10 to 100 nM. If the concentration is less than 10 nM, the differentiation induction time increases, which is uneconomical. However, if it exceeds 10 μM, the compound of formula 1 is in excess as an active ingredient and upon subsequent in vivo administration, inhibition of MEK1 and MEK2 can be very strong, can affect many intracellular signaling pathways and, as a result, can cause many undesirable reactions.

From the moment of addition to the culture of neural stem cells of the composition inducing the differentiation of neural stem cells containing the compound of formula 1, which is an inhibitor of MEK1 and MEK2, until the completion of the differentiation process takes from 1 to 7 days, preferably from 3 to 5 days.

Advantages of the invention

When the composition for inducing neural stem cell differentiation of the present invention is used in patients with a neurodegenerative disease, it induces the differentiation of neural stem cells in the patient's brain into neurons and makes it possible to replace damaged or lost neurons with newly formed neurons. This means that the composition according to the invention induces neural regeneration or neuroregeneration due to neurogenesis from neural stem cells. Thus, the composition can be used to prevent or treat neurodegenerative diseases. In the present invention, neurogenesis means the formation of neurons from nerve stem cells, and neural regeneration or neuroregeneration means the restoration by neurogenesis of the organization and functions of the nervous system, the degeneration of which has occurred due to the death of nerve cells. In addition, the composition of this invention can have a therapeutic or prophylactic effect by protecting neural stem cells or neurons from beta-amyloid oligomers.

In addition, the present invention can be widely used to study the effects of drugs or in various studies in the development of new drugs for neurodegenerative diseases.

Brief description of graphic materials

FIG. 1 depicts a morphology analysis in Example 1, wherein the differentiation of mouse embryonic neural stem cells into neurons upon exposure to trametinib and pimacertib (AS703026) at various concentrations was assessed using a phase contrast microscope. ND and D denote undifferentiated mouse embryonic neural stem cells obtained in Example 1 at stage 1A and differentiated embryonic mouse neural stem cells obtained in stage 1B, respectively, not exposed to any test substance.

FIG. 2 shows an analysis of the morphology in Example 2, where the bottom row shows the results obtained when exposed to 10 uM of oligomeric amyloid-beta (Ar _{1. 42)} on adult neural stem mouse cells, and the top row shows the neural stem cells were not exposed to beta -amyloid. ND and D denote undifferentiated adult mouse neural stem cells obtained in example 2 at stage 1A, and differentiated adult mouse neural stem cells obtained in stage 1B, respectively, not exposed to any test substance. The results obtained on undifferentiated adult mouse stem cells obtained in example 2 at stage 1A, when exposed to the corresponding test substance at the appropriate concentrations: trametinib (10 nM), trametinib (100 nM), memantine (5 μM), memantine (10 μM ) and AS703026 (10 μM).

FIG. 3 compares the cellular morphology of neural stem cells from 14.5-day-old mouse embryos that were exposed (bottom row) or not (top row) to oligomeric beta-amyloid (10 μM) as described in Comparative Example 1. ND ( undifferentiated) and D (differentiated) denote the undifferentiated and differentiated mouse embryonic neural stem cells obtained in Comparative Example 1 in step 1, respectively, not exposed to any test substance. Shown are the results obtained on undifferentiated mouse embryonic stem cells obtained in the comparative example 1 at stage 1, when exposed to the corresponding test substance at the appropriate concentrations: trametinib (100 nM), memantine (10 μM) and AS703026 (10 μM).

FIG. 4 shows the images obtained in example 3 using a fluorescence microscope. In the first row, ND and D indicate undifferentiated and differentiated embryonic nerve stem cells, respectively, not exposed to any test substance. The second and third rows show images of cells not exposed to the respective concentrations of trametinib and AS703026. Blue dots correspond to cell nuclei stained with DAPI, and cells with thin elongated red branches correspond to neurons stained for Tuj 1 with rhodamine conjugate.

FIG. 5 shows the results obtained in Example 4-1, showing the relative mRNA expression levels of the neuron-specific marker Tuj 1 (FIG. 5a) and the dopaminergic neuron marker TH 5b) in mouse embryonic neural stem cells when exposed to various concentrations of trametinib and AS703026.

FIG. 6 shows part of the results obtained in Example 4-2, showing the relative levels of mRNA expression of the marker of dopaminergic neurons TH (Fig.6a), the marker of cholinergic neurons ChAT (Fig.6b), the marker of motor neurons Isl1 (Fig.6c) and the marker of GABAergic neurons Gad1 (FIG. 6d) in mouse embryonic neural stem cells exposed to various concentrations of trametinib.

FIG. 7 shows part of the results of Example 4-2, which shows the relative levels of mRNA expression of the marker of neurons Tuj 1 (Fig.7a), the marker of cholinergic neurons ChAT (Fig.7b) and the marker of dopaminergic neurons TH (Fig.7c) in adult mouse neural stem cells. when exposed to 10 nM trametinib (Tra) and 10 μM AS703026 (AS).

FIG. 8a shows the results of Example 5-1, confirming the presence or absence of expression of the dopaminergic neuron marker TH, as determined by RT-PCR to analyze the ability to differentiate into neurons in mouse embryonic neural stem cells in which the expression of MEK1 or MEK1 and MEK2 was suppressed.

FIG. 8b presents a portion of the results obtained in Example 5-1, showing the relative

- 9,038404 expression levels of Tuj 1 and TH in mouse embryonic neural stem cells in which the expression of MEK1 or MEK2 or MEK1 and MEK2 was suppressed by shMEK1 and shMEK2, and the presence or absence of Tuj 1 and TH proteins was confirmed by Western blotting.

FIG. 8c presents a portion of the results obtained in Example 5-2, which shows the relative expression levels of Tuj 1 and TH mRNA in mouse embryonic neural stem cells in which MEK1 or MEK2 or MEK1 and MEK2 expression is activated by CAMEK1 and CAMEK2, and the presence of or absence of Tuj 1 and TH proteins by Western blotting.

FIG. 9 shows the results obtained in Example 5-3, which shows the cell morphology as observed with a phase contrast microscope (FIG. 9a) and an analysis of the relative levels of mRNA expression of the neuronal marker Tuj 1 (FIG. 9b) in adult mouse neural stem cells, exposed or not exposed to Λβ, where the expression of MEK1 or MEK2 or MEK1 and MEK2 was suppressed by shMEK1 and shMEK2.

FIG. 10 shows the results obtained in Example 6, which shows the morphology of the cells as observed with a phase contrast microscope (Fig.10a) and the analysis of the relative expression level of Tuj1 mRNA (Fig.10b) in mouse embryonic neural stem cells when exposed to trametinib, AZD8330, PD184352, refametinib, PD318088, binimetinib and AS703026 at concentrations of 0.1, 1.0 and 10 μM. FIG. 10c shows the results of phase contrast microscope observations of adult mouse neural stem cells exposed to PD0325901, RO5126766, BI847325 and U0126 (FIG. 10c).

FIG. 11 shows the results obtained using phase contrast microscopy in example 7, which shows the morphology of adult mouse neural stem cells with or without Λβ, followed by exposure to the MEK1 / 2 inhibitors trametinib (0.1 μM), AS703026 (10 μM), AZD8330 (1 μM), PD318088 (1 μM), binimetinib (10 μM), refametinib (1 μM), PD0325901 (10 μM), RO5126766 (10 μM) and cobimetinib (10 μM), known as an inhibitor with selectivity for to MEK1 versus MEK2. Rows marked with - correspond to cells not exposed to amyloid beta, and rows marked Λβ _1-42 correspond to cells exposed to 10 μM amyloid beta.

FIG. 12 shows part of the results obtained in example 8, which shows immunohistochemical staining for NeuN of sections of the somatosensory cortex in 5XFAD mice treated with trametinib (Fig. 12a). FIG. 12b shows the ratio of the number of cells stained for NeuN in mice treated with trametinib versus controls that received only placebo (FIG. 12b).

FIG. 13 shows part of the results obtained in example 8, which shows immunohistochemical staining for NeuN sections of the motor cortex in 5XFAD mice treated with trametinib (Fig. 13a). FIG. 13b shows the ratio of the number of cells stained for NeuN in mice treated with trametinib compared to controls that received only placebo (FIG. 13b).

FIG. 14 shows a portion of the results obtained in Example 8, which shows immunohistochemical staining for NeuN of sections of the hippocampus base in 5XFAD mice treated with trametinib (Fig. 14a). FIG. 14b shows the ratio of the number of cells stained for NeuN in mice treated with trametinib compared to controls that received only placebo (FIG. 14b).

FIG. 15 shows the results obtained in Example 9-1, which shows immunohistochemical staining for Tuj 1 of sections of the somatosensory cortex in 5XFAD mice treated with trametinib. The areas of the images marked with arrows (^ -) show cells stained for Tuj1, and the areas marked with triangles (▼)> show plaques formed by amyloid beta aggregates.

FIG. 16 shows the results obtained in Examples 9-2 and 9-3, showing Nissl staining, NeuN, Dcx staining and BrdU staining of sections of the dentate gyrus of the hippocampus in 5XFAD mice treated with trametinib (FIG.16a) and quantification of cells labeled with BrdU (Fig. 16b). FIG. 16a, the areas marked with arrows (^ -) show cells stained for Dcx, and the areas marked with triangles (▼ / show cells labeled with BrdU.

FIG. 17 shows the results obtained in example 10, which shows staining using the TUNEL method (labeling of dUTP DNA ends with terminal deoxynucleotidyl transferase), an indicator of apoptosis, sections of the hippocampus base and somatosensory cortex in 5XFAD mice treated with trametinib. Cells at the base of the hippocampus that have undergone apoptosis and stained with green fluorescent dye are indicated by arrows (^).

FIG. 18 shows the results obtained in Example 11, which shows staining for Tuj1 and calbindin of Purkinje cells in cerebellar sections of 5XFAD mice treated with trametinib. For each group, images of two (staining for Tuj 1) or three (staining for calbindin) sections are shown.

FIG. 19 shows the results obtained in Example 12, confirming the presence or absence of pERK protein in the cerebral hemispheres of 5XFAD mice treated with trametinib, when analyzed by Western blotting (Fig. 19a); in fig. 19b shows the results of the quantitative determination of the pERK protein (Fig. 19b).

FIG. 20 shows the results obtained in example 13, which shows the quantitative determination of Ab40 and Ab42 and the ratio (Ab42 / Ab40) according to the results of ELISA (enzyme-linked immunosorbent assay) using the cerebral hemispheres of 5XFAD mice treated with trametinib.

An asterisk (*) on the images marks the following results of statistical analysis obtained using the t-test: *: P <0.05, **: P <0.01, ***: P <0.005.

Preferred Embodiments of the Invention

When neural stem cells were treated with a compound of formula 1 according to this invention, the authors observed an increase in the expression level of the Tuj1 neuron marker, as well as an increase in the expression levels of all of the following: the TH dopaminergic neuron marker, the Gad1 marker of GABAergic neurons, the Isl1 motor neuron marker and the ChAT ( example 4). This means that the compound of Formula 1 can differentiate neural stem cells into various types of neurons such as dopaminergic neurons, GABAergic neurons, cholinergic neurons, and motor neurons. Accordingly, the compound of Formula 1 can be used in the treatment of various neurodegenerative diseases that are caused by the loss or damage of various neurons. For example, Parkinson's disease is commonly associated with loss of dopaminergic neurons, motor neuron diseases such as Lou Gehrig's disease / ALS, progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), pseudobulbar palsy (PBP), and hereditary spastic paraplegia (HSP) is associated with loss of motor neurons, and dementia, as in Alzheimer's disease, vascular dementia and senile dementia are commonly associated with loss of cholinergic neurons. In addition, Huntington's disease is usually associated with the loss of G AMK-ergic middle spiny neurons in the striatum of the basal ganglia.

In particular, since the compound of Formula 1 allows neural stem cells to differentiate into neurons while protecting them from Ae, a major neuropathological characteristic of Alzheimer's disease, it can be used in the treatment of Alzheimer's disease.

The present inventors confirmed that the compound of formula 1 increases the number of neurons in the region of the base of the hippocampus and the 5th layer of the cerebral cortex in a model of Alzheimer's disease in mice (5xFAD) expressing genes found in humans with Alzheimer's disease (Example 8). 5xFAD mice carry mutations in the genes encoding APP (amyloid precursor protein) and presenilin (PSEN1), which are known to cause hereditary Alzheimer's disease in humans, and they deposit large amounts of amyloid at the base of the hippocampus and 5th layer of the cerebral cortex with simultaneous loss of neurons in these areas. The observation that the compound of formula 1 increased the number of neurons in the 5xFAD mouse model supports the fact that the compound of formula 1 induces the differentiation of neural stem cells into neurons in vivo with an increase in the number of neurons and / or protects neurons from amyloid beta.

The compound of formula 1 also increased the number of neurons in various parts of the cerebral cortex of 5xFAD mice, especially in the motor cortex and somatosensory cortex, when compared with controls (Example 8, Figs. 12, 13). This indicates that the compound of Formula 1 can be used to treat motor neuron disease such as ALS caused by neuronal loss in this area.

The compound of Formula 1 also increased axon branching or retained the axon structure of Purkinje cells in the cerebellum of 5xFAD mice when compared to controls (Example 11, FIG. 18). This indicates that the compound of Formula 1 can be used to treat cerebellar ataxia caused by loss or damage to the Purkinje cells of the cerebellum.

The compound of formula 1 induces the formation of new neurons (neurogenesis), protects neurons, or both, and thereby induces neural regeneration or neuroregeneration. As the results obtained in example 9 show, the compound of formula 1 increased the expression of the marker of neurons Tuj 1 (a marker of neurons in the process of neurogenesis) in the somatosensory area of the cortex in a model of Alzheimer's disease in mice (5xFAD), increased the number of cells having a type 2 or 3 shape ( the appearance of which is specific for neurogenesis), in the subgranular zone (SGZ) of the dentate gyrus (results of Nissl staining and staining for NeuN in Fig. 16) and increased the number of immature nerve cells expressing DCX and dividing cells stained with BrdU (Fig. 16) ... These results confirm that trametinib induces neurogenesis in the cerebral cortex and dentate gyrus of the mouse hippocampus.

In addition, the compound of Formula 1 reduced the number of dying cells, as evidenced by the results of TUNEL analysis in 5xFAD mice (example 10, Fig. 17, increased axon branching or preserved the axon structure of Purkinje cells of the cerebellum (example 11, Fig. 18) and decreased the ratio Ab (1 _-4 2) / Ab (1 _-4 o) in 5xFAD mouse brain tissue (Example 13, Fig. 20) This shows that the compound of formula 1 can play a role in neuroregeneration, protecting neurons and improving their condition

- 11 038404 or activity under conditions in which damage and loss of neurons occurs.

Accordingly, another aspect of this invention relates to a pharmaceutical composition containing a compound of Formula 1 as an active ingredient for the prevention and treatment of a neurodegenerative disease, to a method for preventing and treating a neurodegenerative disease using a compound of Formula 1, and to a compound of Formula 1 for use in preventing and treating neurodegenerative disease. Instead of a compound of formula 1, other MEK1 / 2 inhibitors can be used in this invention, which cause differentiation of NSCs into neurons, while protecting them from toxic substances such as Ae, but most preferably a compound of formula 1 is used.

As mentioned above, neurodegenerative disease refers to the degeneration of mental and physical function caused by the gradual loss of the structure and function of neurons. In particular, it includes diseases selected from dementia, Alzheimer's disease, vascular dementia, senile dementia, frontotemporal dementia, Lewy body dementia, Parkinson's disease, multiple systemic atrophy, corticobasal degeneration, progressive supranuclear palsy, Huntington's disease, Lou G's disease. eriga / ALS, primary lateral sclerosis, spinal muscular atrophy, progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), pre-bulbar paralysis, hereditary spastic paraplegia (NSP), cerebellar ataxia, Creutzfeldt-sclerosis syndrome, Barre, etc.

In particular, since the compound of Formula 1 exhibits a significant differentiation-inducing effect and neuroprotective effect, even at lower concentrations than the concentrations at which it was previously used due to its antitumor activity, it can be safely administered at a lower dose compared to doses at which it is used to treat cancer. When using a compound of formula 1 as an antineoplastic agent, its recommended dose is 2 mg once a day, and if a dose reduction is required due to side effects, the dose is reduced to 1.5 mg once a day and then to 1 mg once a day. day. In the present invention, in experiments on 5xFAD mice, it was confirmed that the compound of formula 1 demonstrates stem cell differentiation inducing activity already at a dose of 0.1 mg / kg / day. If this dose is transferred to a 60 kg person, it would correspond to 0.48 mg / day (Journal of Basic and Clinical Pharmacy, 7 (2), 27-31.2016).

In this document, the term containing as an active ingredient means the inclusion of the ingredient in an amount sufficient to suppress the neurodegenerative disease associated with this invention.

The prophylactic or therapeutic composition of this invention can be formulated in dosage forms commonly used in the art, for example oral preparations or parenteral preparations such as injectables.

The pharmaceutical composition of this invention may include suitable carriers, excipients and diluents commonly used in pharmaceutical preparations, and may be formulated for oral use such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. etc., or preparations for topical use, suppositories, patches and sterile solutions for injection.

Carriers, excipients and diluents that can be included in the composition of this invention are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, croscarmellose sodium, hydroxypropyl methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, sodium lauryl sulfate, collodionic sodium dioxide, croscarmellose oil, etc.

In pharmaceutical compositions, standard diluents and excipients are used, such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid compositions for oral administration include tablets, pills, powders, granules, capsules, etc. A solid composition is prepared by adding to the composition of this invention at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid compositions for oral administration include suspensions, oral liquids, emulsions, syrups, etc. and in addition to standard simple diluents such as water and liquid paraffin, they can include various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. Compositions for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized compositions, suppositories, and patches. In non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. As a basis for suppositories, you can use witepsol, macrogol, tween 61, cocoa butter, lauric oil, glycero gelatin, etc.

- 12 038404

The composition of this invention can be administered either orally or parenterally, and the administration can be either systemic or topical.

The recommended dose of the therapeutic composition according to this invention may vary depending on the patient's condition, the patient's body weight, the severity of the disease, the dosage form, the route of administration, the duration of treatment, etc. and can be installed appropriately by a person skilled in the art. For example, the composition of this invention can be administered daily at a dose of 0.0001 to 10 g / kg, preferably 0.001 to 8 mg / kg. The dose can be administered once a day or divided into several doses during the day. Preferably, the compound of formula 1 can be administered in a daily dose ranging from 0.1 to 10 mg, 0.1 to 5 mg, 0.1 to 2 mg, 0.1 to 1 mg, 0.1 to 0, 5 mg, 0.25 to 2 mg, 0.25 to 1 mg, 0.25 to 0.5 mg, 0.5 to 2 mg, 0.5 to 1 mg. For example, a compound of Formula 1 can be administered at a daily dose of 0.1, 0.125, 0.25, 0.5, 0.75, 1, 1.5, or 2 mg.

In another aspect of the present invention, a method is disclosed for screening substances that induce the differentiation of neural stem cells into neurons while protecting NSCs and neurons under conditions mimicking a neurodegenerative disease such as Alzheimer's disease. The screening method uses substances that cause neuronal damage such as beta-amyloid, MPTP (1-methyl-4phenyl-1,2,3,6-tetrahydropyridine), rotenone, oxidopamine, glutamate, LPS (lipopolysaccharide) and S100B (S100 calcium -binding protein B) and neural stem cells obtained from the mouse, the method includes the stages:

1) treatment of neural stem cells isolated from an adult mouse with substances that cause neuronal damage, such as beta-amyloid, especially in its oligomeric form, MPTP (1methyl-4-phenyl-1,2,3,6-tetrahydropyridine), rotenone , oxidopamine, glutamate, LPS (lipopolysaccharide) and S100B (S100 calcium binding protein B);

2) adding the test substance to neural stem cells treated with the above substances that cause neuronal damage, and

3) study of differentiation or death of nerve stem cells by analyzing morphology.

In the screening method, it is preferable to use neural stem cells of murine origin. The use of neural stem cells derived from animals such as mice has advantages over human neural stem cells because the methods for their cultivation are simpler and ethical issues are less controversial. Cultivation of human neural stem cells requires more frequent replacement of the culture medium and expensive growth factors, whereas cultures of murine neural stem cells do not. In the cultivation of human nerve stem cells, the processes of reproduction and differentiation of cells take 7 days, while in the cultivation of mouse nerve stem cells, reproduction takes 3-4 days, and differentiation takes only a short period of time, which allows screening to be accelerated.

Nerve stem cells can be isolated from the brains of adult mice and cultured for use. Adult mouse neural stem cells are isolated from mice 8 to 12 weeks old, for example from the subventricular zone of mice 8 weeks old. Isolation of mouse nerve stem cells from the frontal lobes of mouse embryos on 12-16 days of gestation is standard. Compared to adult mouse neural stem cells, mouse embryonic neural stem cells have greater stem cells, which should make them more resistant to toxic conditions. However, the inventors noticed that when mouse embryonic neural stem cells were exposed to amyloid beta, regardless of the addition of the test substance, all cells died and neuronal differentiation could not be observed (Comparative Example 1, Fig. 3). In contrast, adult neural stem cells isolated from the brains of adult mice showed greater resistance to beta-amyloid and were therefore considered more suitable for screening differentiation-inducing substances under conditions mimicking AD. Thus, adult mouse neural stem cells have been used in the methods of the invention.

Nerve stem cells isolated from an adult mouse can be introduced into the culture medium in an undifferentiated state and cultured at 37 ° C. The culture medium for neural stem cells can be any serum-free medium supplemented with growth factors, but is preferably an IPM medium supplemented with growth factors upon initial isolation and culture of adult mouse neural stem cells, and then N2 medium upon isolation and culturing of single cells after neurospheres are formed. ... The IPM medium is a neurobasal medium that can include 1-4% B27 supplementation, 0.5-2% glutamax, 100 IU / ml penicillin, and 100 μg / ml streptomycin. Medium N2 is a mixture of Dulbecco's modified Eagle's medium and F12 growth medium (DMEM / F12) (1: 1), which may additionally contain one or more components selected from the group consisting of 90-110 μM putrescine, 20- 40 nM selenite, 10-30 nM progesterone, 1.0-2.0 mg / ml d - (+) glucose, 20-30 μg / ml insulin, 0.05-0.2 mg / ml apotransferrin, 0.3 -0.6 mM glutamax, 50-150 IU / ml of penicillin and 50-150 μg / ml of streptomycin. V

- 13 038404 medium N2, you can add growth factors selected from the group consisting of 10-30 ng / ml bFGF, 10-30 ng / ml EGF and mixtures thereof. Growth factors are important for keeping neural stem cells in an undifferentiated state. To accurately determine the differentiation-inducing activity of the test substance, the test substance should be added to the neural stem cells, which are maintained in an undifferentiated state by the addition of growth factors that inhibit differentiation.

A commercially available beta-amyloid such as provided by Gibco (Waltham, MA) can be used in the screening method of the present invention. It is preferable to use beta-amyloid of human origin. The most common forms of beta-amyloid are Av (1 _-40 ), consisting of 40 amino acids, and Ap ^ (r), consisting of 42 amino acids. Of the two, Ab (1 _-42 ) has a stronger tendency to form aggregates, especially highly toxic trimers and tetramers, and is therefore considered more closely associated with pathological changes in Alzheimer's disease (Dahlgren et al. (2002), Oligomeric and Fibrillar Species of Amyloid -β Peptides Differentially Affect Neuronal Viability, J. Biol. Chem. 277 (35): 32046-32053; K. Murakami (2014), Conformation-Specific Antibodies to Target Amyloid-β Oligomers and Their Application to Immunotherapy for Alzheimer's Disease, Biosci Biotechnol Biochem 78 (8): 1293-1305). Thus, in the screening method according to the present invention, it is preferable to use Ap ( _{1 - 42} ), in particular the oligomeric form of Ap ( _{1 - 42} ).

In the screening method according to the present invention, the oligomeric form of beta-amyloid should be oligomers of less than 24, preferably 12 or less monomers of beta-amyloid, more preferably a mixture mainly consisting of tetramers and trimers of beta-amyloid, essentially in the absence of protofibrils or amyloid beta fibrils. The oligomeric form of Ap ( _{1 - 42)} in the present invention can be obtained by the method described in the document [Dahlgren et al. (2002) Oligomeric and Fibrillar Species of Amyloid-β Peptides Differentially Affect Neuronal Viability, J. Biol. Chem. 277 (35): 32046-32053]. In particular, Ap _(1-42 ) is dissolved in hexafluoroisopropanol (HFIP), dried under vacuum and then the dried peptide is resuspended in DMSO at a concentration of 5 mM. Add DMEM / F12 (without phenol red) and adjust the peptide concentration to 100 μM, and then incubate it at 4 ° C for 24 h.

In the screening method of the present invention, MPTP is a prodrug for the neurotoxin MPP + (1-methyl-4-phenylpyridine). MPP + causes permanent symptoms of Parkinson's disease (PD) due to the destruction of dopaminergic neurons in the substantia nigra.

It is known that rotenone, a substance that causes degeneration of dopaminergic neurons in the substantia nigra by inhibiting the activity of mitochondrial complex 1 in cells, causes pathological signs of PD.

Oxydopamine, also referred to as 6-hydroxydopamine (6-OHDA) or 2,4,5-trihydroxyphenethylamine, is a neurotoxic compound that we have used to selectively destroy dopaminergic and noradrenergic neurons in the brain. It is believed that oxydopamine enters the neuron through transporters that re-uptake dopamine and norepinephrine. Selective norepinephrine reuptake inhibitors such as desipramine have also been used to selectively damage dopaminergic neurons.

Glutamate acts as a major excitatory neurotransmitter in the central nervous system (CNS), but at high concentrations it is known to damage neurons and cause cell death. Glutamate excitotoxicity is associated not only with acute CNS damage such as ischemic or traumatic brain injury, but also with chronic neurodegenerative disease such as ALS, multiple sclerosis, PD, etc.

LPS (lipopolysaccharide) is a component of the cell surface of gram-negative bacteria, and, for example, LPS isolated from Salmonella typmmurium (Sigma, St. Louis, MO) can be used. LPS induces a pronounced immune response in animals, and also induces an inflammatory response by activating microglia in the nervous system. The secretion of proinflammatory substances due to overactivation of microglia can disrupt homeostasis in the immune system and cause neurodegenerative CNS disease associated with autoimmune disease, such as multiple sclerosis, AD, and PD.

S100B is a calcium-binding protein expressed and secreted by astrocytes. It has neurotrophic activity in the development and maintenance of neurons and affects the normal cognitive functions of the brain. However, abnormally high levels of S100B will activate glia and trigger neuroinflammatory responses that damage neurons.

In the screening method of the present invention, neural stem cells are exposed to substances that damage neurons, such as beta-amyloid, prior to addition of a test substance. In previously published experiments, in order to record the neuroprotective effect of the test substance, neural stem cells were first exposed to the test substances for some time, and then beta-amyloid was added (J. Korean Neural. Assoc. 21 (2): 174-182, 2003) ... This method does not reflect the actual treatment situation when the drug is administered to patients with Alzheimer's disease in whom beta-amyloid is already present in the brain. To more closely simulate the conditions in the brain of patients with Alzheimer's disease at the time of initiation of treatment, the neural stem cells should first be exposed to beta-amyloid, and then add the test substance, as was done in this invention.

The test substance is added to neural stem cells after exposure to beta-amyloid and then cultured by adding growth factors daily. After exposure of the neural stem cells to the test substance, it can be determined whether the test substance has differentiation-inducing activity of neural stem cells by analyzing the morphology at least 12 hours and at most 48-72 hours after exposure.

When analyzing morphology, a phase contrast microscope is used to observe and determine cell morphology. As can be seen at the top of FIG. 2, differentiated (D) neural stem cells clearly differ from undifferentiated (ND) cells. In undifferentiated cells, the cell body is large, the shape of processes such as axons and dendrites is difficult to distinguish, and the total number of cells is high as cells are constantly dividing. In differentiated cells, the cell body is small and round, while the processes are thin and elongated.

The neural stem cells used in the screening method of this invention are relatively fragile compared to normal or tumor cell lines and are susceptible to the toxic effects of the test substance. If the test substance demonstrates cellular toxicity, dead cells can be seen under the microscope, which allows the detection of cellular toxicity simultaneously with the determination of differentiation-inducing activity.

According to the screening method of the present invention, it is possible to screen for substances having a differentiation-inducing activity of neural stem cells under conditions that mimic those in the brain of a patient with Alzheimer's disease, in particular under conditions where oligomeric forms of amyloid beta are present, which are known to closely correlate with neuronal loss. This makes the screening method of the present invention suitable for screening candidate substances that can be used for the main treatment of Alzheimer's disease. In addition, the screening method is convenient in that it allows you to determine by cell morphology whether cells have differentiated without requiring additional experiments. In addition, when neural stem cells are allowed to differentiate naturally in an environment without growth factors, it takes at least 48 hours, after which a change in cellular morphology is observed. In the case of exposure of cells to substances that have a high differentiation-inducing activity, their effect can be observed already 12 hours after exposure and at the latest, 48-72 hours after exposure, despite the presence of growth factors in the cell culture medium to suppress differentiation. Thus, the screening method is fast and efficient, since the toxicity of the test substance can be detected simultaneously with the determination of the differentiation-inducing effect.

In addition, the present invention relates to a kit that can be used to carry out the above screening method. The kit may include neural stem cells obtained from an adult mouse, substances that cause neuronal damage such as amyloid beta, MPTP, rotenone, oxidopamine, glutamate, LPS or S100B, growth factors, culture medium, cell culture supplements, and plates for culturing cells (coated or with separate solutions for immobilization on the surface of the plates).

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples should not be considered as limiting the scope of the invention and its presentation. It should be understood that based on the knowledge set forth in this invention, including examples, a person skilled in the art may make other embodiments of this invention, the practical results of which are not explicitly presented. Such modifications and variations are within the scope of the appended claims.

Examples of

Example 1. Ability of a compound of formula 1 to induce NSC differentiation in mouse embryonic neural stem cells.

Stage 1. Cultivation of mouse embryonic neural stem cells.

Stage 1. Culturing mouse embryonic neural stem cells in an undifferentiated state.

Nerve stem cells were isolated from the brain of a 14.5-day-old mouse embryo and treated with 10 ng / ml human basic fibroblast growth factor (bFGF) (Peprotech, Princeton, NG, cat # 100-18B) and 20 ng / ml human epidermal growth factor (EGF) (Peprotech, cat. Number AF-100-15) in culture medium N2 and cultured in suspension in 25 cm ² flasks (Nunc, Pittsburgh, PA) for 4 days. The formation of neurospheres was observed after 2 days.

To isolate single cells, 6-well plates were prepared the day before by treating the wells with a solution of poly-L-ornithine (Sigma, St. Louis, MO, cat.no. P2533) at a concentration of 15 μg / ml and incubating overnight at 37 ° C for immobilization. On the day of single cell isolation, the poly-L-ornithine solution was removed, the plates were washed three times with PBS and 10 μg / ml fibronectin solution (Gibco, Waltham, MA, cat. No. 33016015) was added and incubated at 37 ° C for 2 hours for immobilization. After the completion of the preparation of the plates, the neurospheres were treated with TryPLE (Gibco cat. No. 12604013), divided into single cells, counted and diluted to a concentration of 4 ~ 5x10 ⁵ cells per 200-300 μl culture solution (N2 culture medium supplemented with 10 ng / ml bFGF and 20 ng / ml EGF). Immediately before inoculation, the immobilizing solution was aspirated and, without allowing the plate to dry, single cells were inoculated, evenly distributing them in the wells of the plate. The cells were allowed to adhere to the plate surface for approximately 1 min and, after the cells had sufficiently adhered, an additional 1.5 ml of culture medium (N2 culture medium supplemented with 10 ng / ml bFGF and 20 ng / ml EGF) was added to the cells and cultured in an incubator with a temperature of 37 ° C.

The composition of the culture medium N2 was as follows.

Dulbecco's Modified Eagle's Medium (DMEM) / F12 (1: 1) (Gibco Cat # 11320033), 100 μM Putrescine (Sigma Cat # 51799), 30 nM Selenite (Sigma Cat # S5261), 20 nM Progesterone (Sigma Cat # P0130), 1.55 mg / ml d - (+) - glucose (Sigma Cat # G8270), 25 μg / ml Insulin (Gibco Cat # 12585014), 0.1 mg / ml apo-transferrin (Sigma cat # T1147), 0.5 mM glutamax (Gibco, cat # A1286001), 100 IU / ml penicillin (Gibco, cat # 15140122), 100 μg / ml streptomycin (Gibco, cat # 15140122).

Stage 1B. Cultivation of neural stem cells without inhibiting differentiation.

Mouse embryonic neural stem cells were cultured as described for step 1A, except for the addition of bFGF and EGF in the separation of neural stem cells into single cells and plating.

Stage 2. Addition of the test substance.

A compound of Formula 1 (hereinafter referred to as trametinib) (Medchem express, Monmouth Junction, NJ, cat.number in various concentrations HY-10999A) and AS703026 (pimacertib) (Selleckchem, Houston, TX, cat.number S1475) were added daily to embryonic nerve cells mice cultured as described in step 1A and cultured for 4 days.

Stage 3. Analysis of morphology.

Cell morphology was examined under a phase contrast microscope after four days of cultivation, the results are shown in FIG. 1.

FIG. 1, the abbreviation ND (undifferentiated) denotes undifferentiated embryonic mouse NSCs obtained in stage 1A, where the cells were not exposed to any test substance. D (differentiated) denotes differentiated embryonic mouse NSCs obtained in stage 1B, where the cells were not exposed to any test substance. Undifferentiated NSCs in the ND group have a large body, the shape of the processes is difficult to distinguish, and the total number of cells is higher than in the D group due to continuous cell division. Differentiated cells of group D have smaller rounded bodies with long thin processes, which makes them easy to distinguish from cells of the ND group.

As shown in FIG. 1, the compound of formula 1 (trametinib) at low concentrations of 10, 25 and 100 nM (0.1 μM) readily induced the differentiation of NSCs into neurons. The concentration of AS703026 required for the onset of neuronal differentiation was 1.0 μM and was 100 times higher than that of trametinib.

Taken together, these results indicate that the compound of formula 1 (trametinib) induces the differentiation of NSCs into neurons and this effect is observed already at very low concentrations (10 nM and higher). This suggests that the compound of Formula 1 can be used in compositions and methods of differentiation to induce NSC differentiation into neurons and to treat neurodegenerative diseases.

Example 2. The ability of a compound of formula 1 to induce NSC differentiation in adult NSC mice (a method for screening substances capable of inducing NSC differentiation into neurons while protecting them simultaneously).

Stage 1. Cultivation of adult NSC mice.

Stage 1A. Cultivation of adult NSC mice in an undifferentiated state.

NSCs were isolated from the subventricular region of the brain of mice at the age of 8 weeks, exposed to 20 ng / ml human basic fibroblast growth factor (bFGF) and 20 ng / ml human epidermal growth factor (EGF) in IPM medium and cultured in suspension in 24-well tablets for 7 days. The formation of neurospheres was observed after 4 days.

2 days before the isolation of single cells, 6-well plates were prepared by treating the wells with a solution of poly-L-ornithine at a concentration of 10 μg / ml and incubating overnight at room temperature for immobilization. On the next day, the poly-L-ornithine solution was removed and the plates were washed 3 times with sterile triple distilled water. Then a 0.5 mg / ml laminin solution (Roche, Upper Bavaria, Germany, cat. No. 11243217001) was added and incubated at

- 16 038404

37 ° C overnight for immobilization. After the completion of the preparation of the plates, the neurospheres were treated with 0.025% Trypsin-EDTA solution, divided into single cells, counted and diluted to a concentration of 4-5x 10 ⁵ cells per 200 ~ 300 μl of culture solution. The culture solution was N2 culture medium (supplemented with 20 ng / ml bFGF and 20 ng / ml EGF). Immediately before inoculation, the immobilizing solution was aspirated and, without allowing the plate to dry, single cells were inoculated, evenly distributing them in the wells of the plate. The cells were allowed to adhere to the plate for approximately 1 min and, after the cells were adhered, an additional 1.5 ml of culture medium (culture medium N2 supplemented with 20 ng / ml bFGF and 20 ng / ml EGF) was added to the cells and cultured in an incubator with a temperature of 37 ° C for 24 hours.

The composition of the IPM and N2 culture medium was as follows.

IPM Medium: Neurobasal Medium (Gibco, Cat # 21103049), Supplement B27 (Gibco, Cat # A3582801), Glutamax, 100 IU / ml penicillin, 100 μg / ml streptomycin.

Medium N2: Eagle's medium modified by Dulbecco (DMEM) / F12 (1: 1), 100 μM putrescine, 30 nM selenite, 20 nM progesterone, 1.55 mg / ml d - (+) - glucose, 25 μg / ml insulin , 0.1 mg / ml apotransferrin, 0.5 mM glutamax, 100 IU / ml penicillin, 100 μg / ml streptomycin.

Stage 1B. Cultivation of neural stem cells without inhibiting differentiation.

Adult mouse neural stem cells were cultured as described for step 1A, except for the addition of bFGF and EGF when the neural stem cells were separated into single cells and plated.

Stage 2. Introduction of beta-amyloid.

After changing the medium in the NSC culture in step 1, 10 μM amyloid beta (Ae) (Gibco, cat # 03112) was added to each well. Wells that did not receive amyloid beta were used as negative controls.

Human Ae ( _1-42 ) was purchased from Gibco's (Waltham, MA) for use as beta-amyloid (Ae) and the following method was used to form oligomers. First, the betaamyloid was dissolved in 100% HFIP (1,1,1,3,3,3-hexafluoro-2-propanol) (Sigma, cat.no.105228) to a concentration of 1 mg / ml and vortexed at room temperature for 1 h. It was then dried for 10 min using a speed Vac, followed by the addition of DMSO (Sigma cat # D2650) to adjust the concentration to 5 mM and then vortex gently at room temperature for 10 min. DMEM / F12 (no phenol red) (Gibco Cat # 21041025) was added to a final concentration of 100 μM. The solution was incubated at 4 ° C for 24 h and then added to the cultured cells.

Stage 3. Addition of the test substance.

Immediately after the addition of beta-amyloid, 10, 100 nM trametinib, 5, 10 μM memantine (Sigma, cat. No. M9292) and 10 μM AS703026 (pimasertiba) were added to the cell cultures. NSCs were cultured for 4 days with daily addition of EGF, bFGF, beta-amyloid, and test substances.

Stage 4. Analysis of morphology.

After four days of cultivation, the morphology of the cells was studied using a phase contrast microscope, the results are shown in FIG. 2.

FIG. 2, the top row shows adult mouse NSCs that were not exposed to beta-amyloid, and the bottom row shows NSCs that were exposed to beta-amyloid at a concentration of 10 μM. ND (undifferentiated) refers to undifferentiated adult mouse NSCs obtained in stage 1A, where cells were not exposed to any test substance. D (differentiated) denotes differentiated adult mouse NSCs obtained in stage 1B, where the cells were not exposed to any test substance. Undifferentiated NSCs in the upper row ND group, not exposed to Ae, have a large body, the shape of the processes is difficult to distinguish, and the total number of cells is higher than in group D due to continuous cell division. Differentiated cells of group D have smaller rounded bodies with elongated thin processes, which makes them easy to distinguish from cells of the ND group.

Trametinib at low concentrations of 10 and 100 nM easily induced the differentiation of NSCs into neurons, regardless of the effect of amyloid beta. For neuronal differentiation, AS703026 was required at a much higher concentration than trametinib, 10 μM, regardless of amyloid-beta exposure. Both substances at the indicated concentrations did not have a toxic effect on cells, which confirmed that they are suitable candidates for Alzheimer's disease (AD).

In addition, in the group where memantine, a drug currently used to relieve AD symptoms, was used, NSC differentiation into neurons was not observed; rather, on the contrary, death of neural stem cells was observed. Memantine is an antagonist of NMDA receptors involved in signal transduction triggered by glutamate and contributes to the normalization of neuronal signal transduction in AD patients. This mechanism of action does not provide the main treatment for AD due to the restoration of damaged neurons. The fact that memantine does not induce NSC differentiation, but rather causes cell death, confirms that it cannot be used for the main treatment of dementia.

Comparative example 1. Effect of beta-amyloid on mouse embryonic neural stem cells.

Stage 1. Culturing mouse embryonic NSCs.

The cells were cultured as described in example 1, in steps 1A and 1B.

Stage 2. Introduction of beta-amyloid.

Embryonic mouse NSCs were cultured as described above in step 1, exposed to beta-amyloid in the same way as described in example 2 in step 2.

Stage 3. Addition of the test substance.

Immediately after the addition of beta-amyloid, 100 nM trametinib, 10 μM memantine, and 10 μM AS703026 (pimacertiba) were added to the cell culture. NSCs were cultured for 4 days with daily addition of EGF, bFGF, beta-amyloid, and test substances.

Stage 4. Analysis of morphology.

After 4 days of cultivation, the morphology of the cells was studied using a phase contrast microscope, the results are shown in Fig. 3.

FIG. 3, the upper row shows embryonic mouse NSCs not treated with beta-amyloid, and the bottom row shows NSCs treated with beta-amyloid. ND (undifferentiated) refers to undifferentiated mouse embryonic NSCs (not treated with any test substance), and D (differentiated) refers to differentiated embryonic mouse NSCs (not treated with any test substance). In the group where Ae was added, all cells died, regardless of the addition of the test substance, and, therefore, it was impossible to assess the effect of the test substances.

Example 3. Immunocytochemical analysis.

To confirm the differentiation of NSCs into neurons in example 1, immunocytochemical staining was performed using markers Tuj1 and DAPI (4 ', 6-diamidino-2-phenylindole). Tuj1 (neuron-specific beta-tubulin class III) is a neuron-specific marker protein that has been conjugated with rhodamine and has red fluorescence, and DAPI is a fluorescent dye that binds to cell DNA and stains the cell nucleus blue.

Embryonic NSCs, which were cultured as described in Example 1, were plated in 24-well coverslip plates and added with trametinib and AS703026 at appropriate concentrations and incubated for 4 days. The medium was removed and the cells were washed once with PBS. After fixation with 10% formaldehyde (Sigma cat. No. HT501128) for 10 min at room temperature, they were washed again with PBS. Permeabilization was performed with 0.2% Triton X-100 solution (Sigma, cat.no.93443) at room temperature for 15 min, washed with PBS and incubated with 10% BSA (Sigma, cat.no. A2153) + 1% normal goat serum (Vector lab, Burlingame, CA, cat. no. S1000) at room temperature for 1 hour. Anti-Tuj 1 antibody (Cell signaling, Danvers, MA, cat. no. 4466) was diluted in 1% BSA + 1% normal goat serum in a ratio of 1: 200 and added to the cells and incubated overnight at 4 ° C. The solution was removed, washed with PBS, and incubated for 1 h at room temperature with a secondary antibody (conjugated with rhodamine) diluted in 1% BSA + 1% normal goat serum in a 1: 200 ratio. The cells were then washed with PBS, incubated with 5 µg / ml DAPI (Sigma, cat # D9542) for 5 min, washed again with PBS and placed on a glass slide for analysis with a fluorescence microscope. The results are shown in FIG. 4. As shown in FIG. 4, in the 1.0 μM AS703026 group, images of thin elongated processes stained red are visible, indicating that neuronal differentiation occurs at this concentration, albeit to a marginal extent. At lower concentrations (0.1 μM), no differentiation was observed.

In contrast, in the group where trametinib was added, differentiation was observed even at a very low concentration of 10 nM, the differentiating activity increased along with the concentration. With regard to cell morphology, an increasing number of neurons with elongated processes were observed when cells were exposed to trametinib at a concentration of 10 nM.

Example 4. Analysis of relative levels of mRNA expression.

Example 4-1. Analysis of Tuj1 and TH mRNA expression induced by the compound of formula 1 in mouse embryonic NSCs.

To determine which cell type NSCs differentiated into in Example 1, the expression of mRNA of the neuron-specific marker Tuj 1 and the marker of dopaminergic neurons TH (tyrosine hydroxylase) was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR).

Stage 1. Isolation of RNA.

After completion of the morphology analysis in Example 1, medium was removed from the cells of each treatment group and added to a TRIzol® plate (Invitrogen, Waltham, MA) and incubated for 5 min at

- 18 038404 room temperature for cell lysis. The cell lysates, together with the TRIzol reagent, were transferred to a tube, mixed thoroughly with chloroform (Sigma, cat. No. 366919), centrifuged, and the supernatant was transferred to a new tube. Isopropanol (Ducsan, GyunggiDo, Korea, cat # 67-63-0) was added and mixed to isolate RNA, then centrifuged again to remove the supernatant and obtain a precipitate. The RNA pellet was resuspended in 75% ethanol (Ducsan, cat. No. 64-17-15), centrifuged, and the supernatant was removed. Then the pellet was resuspended in sterile three times distilled water to obtain mRNA, incubated for 10 min at 55 ° C and stored at -80 ° C.

Stage 2. Reverse transcription.

After measuring the RNA concentration, the amount of RNA in each group was adjusted to 2 μg and the experiment was performed using a reverse transcription kit (Invitrogen, cat. No. 28025013). Sterile triple distilled water, 1 pM oligonucleotide deoxythymidine (dT), and 1 mM dNTP were added to the RNA and incubated for 5 min at 65 ° C. Thereafter, 5X buffer for the synthesis of the first strand, 10 mM DTT and reverse transcriptase M-MLV were added and incubated at 42 ° C for 1 h, then at 72 ° C for 15 min and at 4 ° C for 30 min. The synthesized DNA was stored at -20 ° C.

Stage 3.qRT-PCR.

PCR was performed by mixing 1 μl cDNA (synthesized as described above), 1 pM primers, triple distilled water, and Rotor-Gene SYBR® Green reagent (Qiagen, Venlo, Netherlands, cat # 204074) and used a Rotor-Gene Q ( Qiagen). Primers (Bioneer, Daejeon, Korea) used in the experiment are shown in table. 2.

___________________________________________________________Table 2

Gene Direct primer Reverse primer GAPDH 5'- CGTGCCGCCTGGAGAAACC-3 ' 5'- TGGAAGAGTGGGAGTTGCTGTTG-3 ' Tujl 5'- GGTCTGGCGCCTTTGGA-3 ' 5 CACCACTCTGACCAAAGATAAAGTTG-3 ' TH 5'-AGGTATACGCCACGCTGAAG-3 ' 5'-CTCGGGTGAGTGCATAGGTG-3 '

The results are shown in FIG. 5. In FIG. 5a, 5b show the average values of the relative levels of mRNA expression in each group after 3 rounds of experiments. The term relative mRNA expression level means that to normalize the total RNA level in each group, the mRNA expression level of each differentiation marker is divided by the GAPDH expression level, the expression level of which in cells is sufficiently constant, and then the values for each test substance are compared with those for controls (undifferentiated NSK) in each group.

As shown in FIG. 5, under the influence of trametinib at concentrations of 10, 25 and 100 nM, high levels of expression of the neuron-specific marker Tuj 1 and the marker of dopaminergic neurons TH were observed. These results indicate that trametinib effectively induces NSC differentiation into neurons, in particular into dopaminergic neurons. In addition, trametinib had a differentiation-inducing effect at a very low concentration of 1 nM. This suggests that the composition of this invention can be used to treat a neurodegenerative disease.

AS703026 at 10 μM (10,000 nM) gave results comparable to those of trametinib at 25 nM, indicating that trametinib induces NSC differentiation into neurons at a concentration 400 times (or more) lower than AS703026.

Example 4-2. Analysis of TH, ChAT, Isl1 and Gad1 mRNA expression induced by the compound of formula 1 in embryonic and adult mouse NSCs.

To determine whether the compound of formula 1 induces differentiation of NSCs into cells other than dopaminergic neurons, the expression of mRNA of the marker of cholinergic neurons ChAT (choline acetyltransferase), the marker of motor neurons Isl1 (Islet1) and the marker of GABAergic neurons Gad1 (glutamate 1) was determined. using qRT-PCR.

Nerve stem cells were cultured as described in example 1, and 2 days after the addition of 1, 10, 25 and 100 nM trametinib, RNA was isolated and differentiation markers were examined. The expression of mRNA was determined in the same way as in example 4-1, the primers used are shown in table. 3.

The results are shown in FIG. 6.

Table 3

Gene Direct primer Reverse primer GAPDH 5'- CGTGCCGCCTGGAGAAACC-3 ' 5 '- TGGAAGAGTGGGAGTTGCTGTTG-3' Tujl 5'- GGTCTGGCGCCTTTGGA-3 ' 5'- CACCACTCTGACCAAAGATAAAGTTG3 ' TH 5 '- AGGTATACGCC ACGCTGAAG-3' 5'-CTCGGGTG AGTGC ATAGGTG-3 ' ChAT 5'-CCTGCC AGTC AACTCTAGCC-3 ' 5 '-TAC AG AG AGGCTGCCCTG AG-3' Gadi 5'- TCATGTTATGGAAATCTTGCTTCAG3 ' 5'- CGAGTCACAGAGATTGGTCATATACTACT3 ' Isll 5 '-CGG AG AG AC ATGATGGTGGT-3' 5'-GGCTGATCTATGTCGCTTTGC-3 '

As shown in FIG. 6, the expression of all studied neuronal markers, the marker of dopaminergic neurons TH, the marker of cholinergic neurons ChAT, the marker of motor neurons Isll and the marker of GABAergic neurons Gad1, increased with increasing concentration of added trametinib. This suggests that trametinib can be used to treat neurodegenerative disease caused by damage to various types of neurons.

To establish whether this effect occurs in adult mouse NSCs, 10 nM trametinib and 10 μM AS703026 were separately added for 2 days to adult mouse NSCs, which were cultured as described in Example 2, in step 1A, and the expression levels of neuronal marker mRNA were analyzed Tuj1, marker of dopaminergic neurons TH and marker of cholinergic neurons ChAT by qRT-PCR.

The results are shown in FIG. 7. Trametinib increased the expression of Tuj1, TH and ChAT in adult mouse NSCs, as well as in embryonic NSCs. It should be noted that trametinib caused more pronounced differentiation of NSCs into neurons at concentrations 1000 times lower than AS703026.

Example 5. Study of the ability to induce NSC differentiation by regulating the expression of MEK1 and MEK2 in neural stem cells.

Example 5-1. Inhibition of MEK1 and MEK2 expression in mouse embryonic neural stem cells.

The ability to induce NSC differentiation into neurons was investigated depending on the expression levels of MEK1 and MEK2. To obtain cells where the expression of MEK1 and MEK2 is regulated, the following method was used.

First, to obtain NSCs in which the expression of MEK1 and MEK2 is suppressed, the corresponding shRNAs were used to suppress the expression of MEK1 and MEK2. In particular, embryonic NSC mice were cultured as described in Example 1, in Step 1A, were seeded in the plates, and cultured for 24 hours. The cells were transfected Then 1 ug / ml shRNA-MEK1 (CCGGGCCATCCAACATTCTAGTGAACTCGAGTTCACTAGAATGTTGGATG GCTTTTT) or shRNA-MEK2 (CCGGCCTCCGAGAGAAGCACCAGATCTCGAGATCTGGTGCTTCTCTCGGA GGTTTTTG) using Lipofectamine (Invitrogen, cat # 18324010) to produce NSCs in which MEK1 or MEK2 expression is suppressed, or both.

After 4 hours, the medium in which these transfected cells were located was replaced with culture medium N2, cultured for another 4 days, and then RNA was isolated by the method described in example 4-1. The ability to induce differentiation of NSCs into neurons was analyzed using reverse transcription PCR (RT-PCR). Specifically, RNA was converted to cDNA by reverse transcription, and then EX-Taq DNA polymerase (SG Bio, Kyunggi Do, Korea) and a primer for PCR in a T100 ™ thermal cycler (Bio-rad, Hercules, Calif.) Were added. PCR was carried out by incubation at 95 ° C for 5 min, then 25-35 cycles of the reaction were repeated, in which 1 cycle consisted of incubation at 95 ° C for 30 s, at 55 ~ 62 ° C for 30 s and at 72 ° С for 30 s. The PCR results were examined by electrophoresis in 2% agarose gel, which was then analyzed using an LAS-3000 image analyzer (Fujifilm, Tokyo, Japan).

The primers used for RT-PCR and qRT-PCR in this example, as well as in examples 5-2 and 5-3, are shown in table. 4.

Table 4

Gene Direct primer Reverse primer GAPDH 5'- CGTGCCGCCTGGAGAAACC-3 ' 5 '- TGGAAGAGTGGGAGTTGCTGTTG-3' Tujl 5'- GGTCTGGCGCCTTTGGA-3 ' 5 '- C ACC ACTCTG ACC A A AG ATA A AGTTG-3' TH 5 '- AGGTATACGCC ACGCTGAAG-3' 5'-CTCGGGTG AGTGC ATAGGTG-3 ' MEK1 5 '-CGGCGGTTAACGGG ACC A-3 4' 5'-GGATTGCGGGTTTGATCTCCA-3 ' MEK2 5'-CCTGGATGAGC AGC AAAGGA-3 ' 5 '-C AGTGAGCC ACC ATCC ATGT-3'

The results are shown in FIG. 8a. As shown in FIG. 8a, in NSCs in which the expression of both MEK1 and MEK2 was suppressed as a result of transfection with shMEK1 and shMEK2 (shMEK1 +, shMEK2 +), the expression of the marker of dopamine neurons TH was significantly increased compared to NSCs in which only MEK2 expression was suppressed (shMEK1- , shMEK2 +).

To test again how suppression of MEK1 or MEK2 alone or suppression of both MEK1 and MEK2 affects NSC differentiation into neurons, qRT-PCR and Western blotting were performed. NSCs in which only MEK1 or MEK2 was suppressed, and NSCs in which both MEK1 and MEK2 were suppressed were obtained by the above methods. After 4 hours, the medium containing the transfected cells was replaced with N2 culture medium, cultured for another 4 days, and then qRT-PCR was performed as described in Example 4-1. The results are plotted in FIG. 8b. As shown in the graph in FIG. 8b, the expression of neuron markers Tuj1 and TH significantly increased upon suppression of the expression of MEK1 and MEK2 compared to suppression of the expression of MEK1 alone or MEK2 alone.

To establish whether the induction of NSC differentiation into neurons is observed when the expression of MEK1 and MEK2 is suppressed not only at the mRNA level, but also at the protein level, Western blotting was performed.

Western blotting in each group of cells was performed as follows. After cultivation of NSCs, in which the expression of MEK1 and MEK2 was suppressed, the medium was removed for 4 days and into the plates,

- 20 038404 on ice, added RIPA buffer (0.05 M Tris HCl pH 7.4 (Sigma, cat.no. T3253), 0.15 M NaCl (Ducsan, cat. No. 7647-14-5), 0, 25% deoxycholic acid (Sigma, Cat # D6750), 1% NP-40 (USB, Waltham, MA, Cat # 19628), 1 mM EDTA (Sigma, Cat # EDS), 1 mM PMSF (Acros organics, Geel, Belgium, Cat # 215740050), 1 mM Sodium Orthovanadate (Bio labs, Ipswich, MA, Cat # P0758L), 1mM Sodium Fluoride (Sigma, Cat # HOMepS7920), Protease Inhibitors (Sigma, Cat # P83430) ) and the cells were harvested with a scraper. After incubation on ice for 10 min, the supernatant was collected by centrifugation at 13000 rpm at 4 ° C. Protein concentration was determined, sample loading buffer (0.25 M Tris-HCl pH6.8, 0.05% SDS (Amersco, Solon, OH, cat. No. 227), 50% glycerin (Ducsan, cat. No. 56- 81-5), 0.25 M DTT (Invitrogen, cat # 0861), 0.5 mg / ml BPB (Bio-rad, Hercules, CA, cat # 161-0404) and boiled the sample at 100 ° C for for 10 min and then stored at -20 ° C.

Samples containing 10-20 μg of protein were loaded onto an 8-12% SDS-PAGE gel, separated, transferred onto a nitrocellulose membrane, which was then blocked with 5% skim milk at room temperature for 1 hour. Antibodies to Tuj1, TH (Cell signaling # 2792), MEK1 / 2 (Santa Cruz, Dallas, TX, # Sc-292838) and ERK (Santa Cruz, # Sc-135900) were prepared in TRIS buffered saline (TBS) supplemented with 0, 1% Tween 20 (Sigma, cat.no. P1379) and incubated with the membrane at room temperature for 2 h or at 4 ° C overnight, after which it was incubated with a secondary antibody conjugated with horseradish peroxidase at room temperature for 1 hour. Protein expression was determined using photosensitive equipment, the results are shown in the lower part of FIG. 8b. As shown in FIG. 8b, the expression levels of the neuronal marker Tuj1 and the marker of dopamine neurons TH at the mRNA level and at the protein level were significantly increased in NSCs, in which the expression of both MEK1 and MEK2 was suppressed, due to the use of shMEK1 and shMEK2 (shMEKK, shMEK2 +).

In contrast, in NSCs in which the expression of only MEK1 or only MEK2 was suppressed, a lower level of expression of Tuj1 and TH was observed compared to NSCs in which both MEK1 and MEK2 were suppressed.

Example 5-2. Induction of CAMEK1 and CAMEK2 Expression in Mouse Embryonic Nerve Stem Cells

To study the induction of NSC differentiation into neurons, depending on the activation of MEK1 and MEK2, a plasmid with constitutively active MEK1 (CAMEK1) and a plasmid with constitutively active MEK2 (CAMEK2), having mutations for constitutive expression of MEK1 and MEK2, were used in the experiments. In particular, mouse embryonic NSCs, which were cultured as described in Example 1, in step 1A, were plated and incubated for 24 hours. The cells were then transfected with 1 μg / ml CAMEK1 and CAMEK2 using lipofectamine to obtain NSCs expressing CAMEK1 or CAMEK2 or both. After 4 hours, the medium containing these transfected cells was replaced with N2 culture medium containing no EGF or bFGF, cultured for another 4 days, and then RNA and protein were isolated and quantitative RT-PCR (qRT-PCR) and Western blotting were performed. In these experiments, the effect of activation of MEK1 and MEK2 on the induction of NSC differentiation into neurons was studied, the results are shown in FIG. 8c.

In contrast to FIG. 8b, FIG. 8c shows that upon activation of MEK1 and MEK2 under conditions inducing differentiation, NSC differentiation is inhibited. As shown by qRT-PCR and Western blotting, the expression of the neuronal marker Tuj 1 and the marker of dopaminergic neurons TH at the mRNA and protein levels was suppressed. Expression of Tuj1 and TH was significantly lower in the case of activation of both MEK1 and MEK2 compared with activation of only one of them.

Example 5-3. Inhibition of MEK1 and MEK2 in adult mouse neural stem cells.

To establish whether the inhibition of MEK1 and MEK2 induces NSC differentiation in adult mouse NSC and whether the effect is the same in the presence of amyloid beta, the following experiments were performed.

Adults NSC mice were cultured as described in Example 2, in Step 1A, were seeded in the plates, and cultured for 24 hours. The cells were then transfected with 1 microgram / ml shRNA-MEK1 (CCGGGCCATCCAACATTCTAGTGAACTCGAGTTCACTAGAATGTTGGATG GCTTTTT) or shRNAMEK2 (CCGGCCTCCGAGAGAAGCACCAGATCTCGAGATCTGGTGCTTCTCTCGGA GGTTTTTG) using Lipofectamine (Invitrogen ) to obtain NSCs in which either MEK1 expression or MEK2 expression is suppressed, or both.

After 4 h, the medium containing these transfected cells was replaced with culture medium N2 or culture medium N2 with 10 μM amyloid beta, cultured for another 2 days, and then examined the morphology of the cells using a microscope. RNA was isolated in the same manner as in example 4, qRT-PCR was performed and the induction of NSC differentiation into neurons was analyzed.

The results are shown in FIG. nine.

As shown in FIG. 9a, differentiation was more easily induced in NSCs in which the expression of both MEK1 and MEK2 was suppressed due to transfection with shMEK1 and shMEK2, compared with NSCs,

- 21 038404 transfected with only one of shMEKl and shMEK2.

In the case when the cells were exposed to β, the cells died in the group where neither MEK1 nor MEK2 inhibited, however, in the group where either MEK1, or MEK2, or MEK1 and MEK2 were inhibited, cell death was not observed, but differentiation was induced. ...

In addition, as shown in FIG. 9b, expression of the neuronal marker Tuj 1 was significantly increased in NSCs, in which the expression of both MEK1 and MEK2 was suppressed by shMEK1 and shMEK2 (shMEK1 +, shMEK2 +). In contrast, in NSCs in which either MEK1 or MEK2 (shMEK1, shMEK2) expression was suppressed, expression of the neuronal marker Tuj1 was low, as was the induction of NSC differentiation into neurons, compared to inhibition of both MEK1 and MEK2. In the case of exposure to Ae cells, some protective and differentiation-inducing effect was observed against NSCs in which either MEK1 or MEK2 were suppressed, but in the case of NSCs in which both MEK1 and MEK2 expression were suppressed, the protective and differentiation-inducing effect was significantly higher.

These results indicate that inhibition of MEK1 and MEK2 in adult NSCs not only induces NSC differentiation, but also has a protective effect against amyloid beta.

Example 6. NSC differentiation-inducing effect of other MEK1 / 2 inhibitors

To investigate whether other MEK 1/2 inhibitors besides trametinib demonstrate NSC differentiation inducing and protective effects, mouse embryonic NSCs were exposed to each of the test substances. In particular, mouse embryonic NSCs were cultured as described in Example 1, at stage 1A, with the appropriate MEK1 / 2 inhibitors: AZD8330 (Selleckchem, cat. No. 2134), PD184352 (Selleckchem, cat. No. S1020), refametinib (Selleckchem, cat. S1089), PD318088 (Selleckchem, Cat S1568), binimetinib (Selleckchem, Cat S7007) and AS703026 at concentrations of 0.1, 1.0 and 10 μM and incubated. For comparison, NSCs were also exposed to trametinib at the same concentrations. On the 2nd day of cultivation, the morphology of the cells was studied under a phase contrast microscope, the results are shown in Fig. 10a. FIG. 10b shows an analysis of Tuj 1 mRNA expression in each cell group.

As shown in FIG. 10a and 10b, AZD8330, refametinib, PD318088, binimetinib, and AS703026 induced differentiation, albeit starting at different concentrations. Morphologically, differentiation began with 1 μM AZD8330, refametinib and PD318088, and with 10 μM binimetinib and AS703026. The study of the expression of the marker of neuronal differentiation Tuj 1 by RT-PCR also showed that they induce differentiation at the same concentrations. Compared to other compounds, trametinib showed an excellent differentiation-inducing NSC effect even at a low concentration of 0.1 μM. In contrast, PD184352 showed only a weak differentiation-inducing NSC effect at a concentration of 0.1 μM, while at concentrations of 1.0 and 10 μM, all cells died.

In addition, adult NSC mice, which were cultured as described in Example 2, in step 1A, were exposed to other MEK1 / 2 inhibitors: PD0325901 (Selleckchem, cat number S1036), RO5126766 (Selleckchem, cat number S7170), BI847325 ( Selleckchem, Cat. No. S7843) and U0126 (AG Scientific, San Diego, CA, Cat. No. U-102) at various concentrations, and their differentiation-inducing NSC effects were investigated. After exposure to the test substances, the morphology of the cells was studied under a phase-contrast microscope on the 2nd day of cultivation. The results are shown in FIG. 10s. As shown in FIG. 10c, the differentiation-inducing effect of PD0325901 was manifested starting from 0.1 μM, and RO5126766 - from 1.0 μM. In particular, PD0325901 showed an excellent inducing effect as it induced differentiation over a wide concentration range from 0.1 to 10 μM. In contrast, BI847325 showed only a weak differentiation-inducing effect at a concentration of 0.1 μM, while at concentrations of 1.0 and 10 μM, all cells died. When exposed to BI847325 at lower concentrations (1.0 and 10 nM), cell proliferation was somewhat inhibited, and no differentiation-inducing effect was observed. U0126 inhibited cell proliferation at concentrations of 0.1 and 1.0 μM, and at a concentration of 10 μM had a cytotoxic effect on cells. In addition, U0126 did not show any distinct differentiation-inducing effect at a concentration of 2.5 μM and therefore was not able to induce NSC differentiation at any of the tested concentrations.

These results indicate that not all known MEK1 / 2 inhibitors effectively induce NSC differentiation. Obviously, inhibition of MEK1 and MEK2 is involved in inducing differentiation of NSCs, as evidenced by the results of example 5. However, it is believed that the individual properties of each MEK 1/2 inhibitor affect its ability to induce differentiation of NSCs, resulting in the differences observed in example 6.

Example 7. NSC differentiation-inducing effect of other MEK1 / 2 inhibitors in the presence of cytotoxic factors.

Adult NSCs cultured as described in example 2 in step 1A were exposed to 10 μM amyloid beta as described in example 2 in step 2 to create cytotoxic conditions,

- 22 038404 after which MEK1 / 2 inhibitors were added: AS703026 (10 μM), AZD8330 (1 μM), PD318088 (1 μM), binimetinib (10 μM), refametinib (1 μM), PD0325901 (10 μM) and RO5126766 (10 μM) at concentrations that caused the most pronounced differentiation in example 6, and cultured. For comparison, the group that was exposed to 0.1 μM trametinib and the group that was exposed to 10 μM cobimetinib (known as a selective inhibitor of MEK1 versus MEK2), as well as groups with each of the test compounds, but without exposure to beta-amyloid were included. ... Cell morphology was studied under a phase contrast microscope on the second day of cultivation, the results are shown in Fig. eleven.

FIG. Row 11, marked with - corresponds to the group of adult mouse NSCs without _{beta-amyloid exposure, and the row marked with Ap 1 - 42} , represents the group with 10 μM beta-amyloid exposure. Under conditions when cytotoxicity developed under the influence of 10 μM amyloid beta, it was shown that the addition of AS703026, AZD8330, PD318088, binimetinib, refametinib, PD0325901 and RO5126766, each at an optimal concentration, inducing the most pronounced differentiation, provided cell protection and induced HSC differentiation. In contrast, the addition of cobimetinib, an inhibitor with greater selectivity for MEK1 than MEK2, did not induce differentiation of adult NSCs even at a high concentration of 10 μM, regardless of the addition of amyloid beta.

Example 8. Detection of an increase in the number of neurons in a model of Alzheimer's disease in mice.

Previous experiments have shown that among compounds that inhibit both MEK1 and MEK2, some compounds induce differentiation of NSCs into neurons and protect NSCs and neurons from beta-amyloid, in particular, trametinib showed a pronounced effect. To confirm these results also in a mouse neurodegenerative disease model, the neuroregenerative and therapeutic effects of trametinib were investigated using 5XFAD mice, an animal model showing symptoms of AD, the most common form of neurodegenerative disease. In 5XFAD mice, neuronal pathology, degeneration and cell death of neurons are observed, in particular, in the 5th layer of the somatosensory cortex and the base of the hippocampus (Oakley et al. (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation. J Neurosci. 26 (40): 10129-10140).

Trametinib was administered orally to mice

5XFAD (B6SJL-Tg (APPSwFlLon, PSENl * M146L * L286V) 6799Vas / Mmjax) (BA model) at the age of 12 months daily for 28 days at a dose of 0.1 and 1.0 mg / kg, and mice of the control group were injected with placebo (4% DMSO + corn oil) in the same way (7 mice / group). For subsequent staining with BrdU, 50 mg / kg BrdU (Sigma, cat. No. B5002) was added in a daily dose during the last 5 days. To isolate the brain, the mice were anesthetized and perfused with PBS. Half of the brain tissue (hemisphere) isolated from three mice of each group was immediately placed in a low temperature freezer for subsequent Western blotting and ELISA studies. The other half of the hemispheres and the brain, isolated from 3 other mice from each group, were placed in a 10% formalin solution at 4 ° C for 1 day, after which the following steps were performed to ensure good penetration of paraffin into the brain tissue. For dehydration, brain tissue was sequentially immersed for 1 h in each of the solutions of 70, 80, 95, 100% alcohol, then immersed in xylene 3 times, each time for 1 h, for clarification and embedded in paraffin, twice placed in liquid paraffin, each once every 1 hour. After these procedures, sections of brain tissue embedded in paraffin (paraffin block) with a thickness of 5 μM were prepared, placed on glass, and stored at room temperature. Before immunohistochemical staining, the tissues on the slides were rehydrated by sequentially immersing them in xylene, 100, 90, 80, 70, 50% alcohol and then in water, each time for 5 min. After placing in sodium citrate buffer (10 mM, pH 6) (Sigma, S4641), the antigen was unmasked at 120 ° C for 15 min, blocked with 10% BSA (bovine serum albumin) and incubated with antibodies to the NeuN neuron marker ( Cell signaling, cat.number 24307) at 4 ° C for 1 day. The next day, the slides were incubated with antibodies to rabbit immunoglobulins (Vector lab, cat. Number PI-1000) at room temperature for 1 hour and then stained with diaminobenzidine (DAB) to analyze the distribution of neurons in the brain.

Since 5XFAD mice have extensive neuronal damage in the 5th layer of the cerebral cortex and the base of the hippocampus, these parts of the brain were investigated first.

FIG. 12 shows the results of a study of sagittal sections of the somatosensory area of the cerebral cortex. As shown in FIG. 12a, in 5XFAD mice treated with placebo (4% DMSO + corn oil) (control, first image on the left), the number of cells stained for NeuN (positive) decreased due to significant damage to neurons in the 5th layer of the cerebral cortex. In comparison, 5XFAD mice treated with 0.1 and 1.0 mg / kg trametinib showed a significant increase in the number of neurons stained for NeuN. FIG. 12b shows the number of counted cells stained for NeuN, expressed as a percentage of the number of cells per unit area in the 5th layer of the somatosensory cortex. Samples were taken from each mouse in 3 areas, from 3 mice on

- 23 038404 group, thus, the counting of cells was carried out in a total of 9 areas. The percentage of cells stained for NeuN was calculated in the trametinib group versus the placebo group.

FIG. 13 shows the results of a study of frontal sections of the motor area of the mouse cerebral cortex. As shown in FIG. 13a, 5XFAD mice treated with placebo (control) showed extensive neuronal damage in the 5th layer of the motor cortex and a reduced number of neurons stained for NeuN, while in the group receiving trametinib, a significant increase in the number of neurons was again observed, as in the somatosensory bark. FIG. 13b shows the number of counted cells stained for NeuN, expressed as a percentage of the number of cells per unit area in the 5th layer of the motor cortex. Each mouse was sampled in 6 areas, 3 mice per group, so the cell was counted in a total of 18 areas.

The percentage of cells stained for NeuN was calculated in the trametinib group versus the placebo group.

FIG. 14 shows the results of a study of sagittal sections of the base of the mouse hippocampus. As shown in FIG. 14a, in 5XFAD mice treated with placebo (control), a small number of neurons stained for the NeuN marker of neurons were observed at the base of the hippocampus, while in the group receiving trametinib, a significant increase in the number of neurons was again observed, as in the cerebral cortex. FIG. 14b shows the number of cells counted stained for NeuN expressed as a percentage of the number of cells per unit area in the dashed portion of the hippocampus shown in FIG. 14a. Each mouse was sampled in 3 areas, 3 mice per group, so the cell was counted in a total of 9 areas. The percentage of cells stained for NeuN was calculated in the trametinib group versus the placebo group. The above experiments showed that trametinib causes an increase in the number of neurons in the 5th layer of the cerebral cortex and the base of the hippocampus of 5XFAD mice.

Example 9. Detection of neurogenesis in a mouse model of Alzheimer's disease.

To establish whether the increase in the number of neurons in the 5th layer of the cerebral cortex and the base of the hippocampus in 5XFAD mice treated with trametinib, shown in example 8, is a consequence of neurogenesis under the influence of trametinib, immunohistochemical analysis was performed with various cellular markers that arise during the differentiation of NSCs into neuron.

DCX (doublecortin), a protein expressed in neuronal progenitor cells, is expressed predominantly in migrating or differentiating neurons and indicates that the cell is an immature neuron during neurogenesis. Tuj 1 (neuron-specific class III beta-tubulin), a protein expressed in actively dividing neuronal precursor cells or newly formed immature postmitotic neurons, indicates that the cell is a neuron in the process of neurogenesis. Thus, by determining the presence of DCX or Tuj 1 using immunohistochemical staining, it can be established that neurogenesis occurs as a result of NSC differentiation into neurons.

BrdU (5-bromodeoxyuridine), an analogue of the thymidine nucleotide base, replaces thymidine during DNA synthesis in dividing cells, allowing the detection of dividing cells during neurogenesis by immunohistochemical staining.

Example 9-1. Determination of Tuj1 expression in the somatosensory cortex.

Antibodies to Tuj 1 were added to the 5XFAD mouse brain tissue preparations obtained in Example 8 and incubated at 4 ° C for 1 day, after which the tissue on the slides was stained with a fluorescent dye by incubation with secondary antibodies labeled with fluorescein isothiocyanate (FITC) (Invitrogen A21121) at room temperature for 1 hour. Fluorescence microscope images of the somatosensory cortex are shown in FIG. 15. In FIG. 15, the areas marked with arrows (A) show cells stained for Tuj1, and the areas marked with triangles / by show the plaques formed by amyloid beta aggregates. Plaques are present in both placebo and trametinib groups. However, the 0.1 mg / kg trametinib group in particular showed a significant decrease in the number of cells stained for Tuj1 compared to the placebo group, despite the same amount of amyloid beta plaques present in the samples.

Example 9-2. Nissl staining and determination of NeuN and DCX expression in the dentate gyrus of the hippocampus.

When neurogenesis is activated in the subgranular zone (SGZ) of the dentate gyrus, asymmetric cell division occurs, and as a result, type 2 cells are formed at the very beginning. Type 2 cells have a small body with an atypical nucleus, are short, horizontally oriented, and express nestin or Dcx. Type 3 cells are cells that have undergone further differentiation from type 2 cells and are also referred to as neuroblasts. Type 3 cells are cells at an early stage of differentiation in the neuronal but not glial direction. Type 3 cells are located closer to the granular layer from the subgranular zone of the dentate gyrus, and, unlike type 2 cells, they are not oriented horizontally, but vertically. The presence of type 2 and type 3 cells in the subgranular

- 24 038404 in the dentate gyrus zone indicates that neurogenesis is taking place.

Nissl staining, the staining of the Nissl bodies of a neuron, is a technique that demonstrates the distribution and state of neurons in the brain. The brain tissue on slides obtained in example 8 was rehydrated by sequential immersion in xylene, 100, 90, 80, 70, 50% alcohol and then in water, each time for 5 minutes. After being placed in a 0.1% cresyl violet solution (Sigma, cat. Number C5042) at room temperature for 15 minutes, they were dehydrated by successive immersion in 80, 90, 100% alcohol and xylene. Cover slips were placed on the slides and neurons were examined.

Staining for NeuN was performed as described in example 8, staining for DCX was performed in the same way, but instead of antibodies to NeuN, antibodies to DCX were used (Santa Cruz, cat.no. sc271390)

The results are shown in FIG. 16a. Nissl staining and NeuN staining in 5XFAD mice treated with trametinib showed that the number of cells with type 2 or type 3 cell morphology, the appearance of which is specific for the neurogenesis process, significantly increased in the subgranular zone (SGZ) and in the area located closer to to the granular zone, compared to SGZ in the dentate gyrus. In addition, the presence of immature neurons (indicated by arrows (^) in the Dcx column in FIG. 16) also stained for DCX was noted, confirming that neurogenesis was occurring in the dentate gyrus of the hippocampus in mice receiving trametinib.

Example 9-3. BrdU staining of the dentate gyrus of the hippocampus.

For neurogenesis to occur, asymmetric division of neural stem cells must occur, and this was investigated in the next experiment.

Sections of brain tissue from 5XFAD mice, which were injected with BrdU (administration of 50 mg / kg BrdU daily for 5 consecutive days before euthanasia, as described in example 8), were rehydrated, placed in 1.5 M HCl (hydrochloric acid) and incubated at 37 ° C for 30 min. Sections were blocked with a solution containing 0.5% BSA (bovine serum albumin), 0.3% TritonX-100, and 10% normal goat serum and incubated with anti-BrdU antibody (Cell signaling, ref. 5292) at 4 ° C for 1 day. Immunofluorescence staining was performed the next day by incubating the sections with a secondary antibody conjugated with fluorescein isothiocyanate (FITC) at room temperature for 1 h, after which the staining of the dentate gyrus of the hippocampus was examined using a fluorescence microscope.

The results are shown in FIG. 16a in the column labeled BrdU. Cells labeled with triangles ( ^t ) are cells labeled with BrdU. When compared with the placebo group, in the 5XFAD mice treated with trametinib, an increase in the number of cells stained with BrdU was observed in the dentate gyrus. FIG. 16b shows the number of cells labeled with BrdU, which again indicates that an increase in the number of cells stained with BrdU was observed in the group receiving trametinib. FIG. 16b shows the average number of cells, which were counted in 9 areas (3 mice per group and 3 areas per mouse).

These results indicate that trametinib induces neurogenesis by differentiating NSCs in a mouse model of AD. This suggests that trametinib can be used not only as the main treatment for AD, but also for the treatment and prevention of diseases caused by damage or loss of neurons in the cerebral cortex, in particular in the motor cortex.

Example 10. Neuroprotective activity in a mouse model of AD.

To determine whether trametinib has a neuroprotective effect on the cerebral cortex and base of the hippocampus of 5XFAD mice, immunohistochemical analysis was performed using the TUNEL method (labeling of dUTP DNA ends using terminal deoxynucleotidyl transferase) to detect cell apoptosis. TUNEL assay stains the 3'-hydroxy-terminus of fragmented DNA, which allows the detection of cells undergoing apoptosis in tissue.

Sagittal brain tissue sections prepared in Example 8 were rehydrated and stained using a TUNEL assay kit (Promega, Wisconsin, USA, cat. Number G3250). In particular, brain tissue sections were permeabilized with 20 μg / ml proteinase K solution for 10 min, incubated with equilibration buffer for 10 min, and then incubated with TdT solution at 37 ° C for 1 h. hippocampus, regions where significant cell death is known to occur in 5XFAD mice. TUNEL analysis was also performed on brain tissue sections of normal adult mice (control) to confirm that the staining was performed properly.

The results are shown in FIG. 17. Staining with green fluorescent dye in normal mice (labeled normal mice in FIG. 17) was not observed because apoptosis was absent. However, green fluorescence was observed in many cells in the somatosensory cortex (row indicated by the cortex in FIG. 17) and the base of the hippocampus (row indicated by the base of the hippocampus in FIG. 17) in 5XFAD mice. FIG. 17 stained cells at the base of the hippocampus are indicated by arrows (^); no special marks were made in the somatosensory cortex, since many cells were stained. When

- 25,038404 5XFAD mice treated with trametinib, the number of cells stained by the TUNEL method in the somatosensory cortex and base of the hippocampus was significantly lower compared to the placebo group.

These results demonstrate that trametinib has a protective effect, preventing neuronal death in a mouse model of AD.

Example 11. Protection of cerebellar Purkinje cells in a mouse model of AD.

Sections of brain tissue (5XFAD mice), described in example 8, were incubated with antibodies to Tuj 1 and to calbindin (Cell signaling, cat. No. 13176) at 4 ° C for 1 day and stained for immunofluorescence analysis with secondary antibodies conjugated to fluorescein isothiocyanate (FITC) or rhodamine at room temperature for 1 hour. Immunofluorescence images of the Purkinje cell layer are shown in FIG. 18. For each treatment group, two sections stained for Tuj 1 and three sections stained for calbindin are shown.

As shown in FIG. 18, Purkinje cells in the placebo group had thin protruding axons that looked cut in the middle. In contrast, the trametinib group, in particular the 0.1 mg / kg trametinib group, exhibited Purkinje cells with enhanced axon branching or at least preservation of axonal structure when compared with the placebo control group.

It is known that the cerebellum plays an important role in the integration of proprioceptive sensations with the regulation and coordination of movements. The most amazing cerebellar cells, Purkinje cells, are some of the largest cells in the brain, their complexly branched axons synapse with each other and penetrate deep into the center of the cerebellum to regulate and control motor muscles. The above experimental results indicate that trametinib protects Purkinje cells and increases branching of axons in the cerebellum, possibly allowing the treatment of diseases that are caused by the loss or damage of cerebellar Purkinje cells, such as cerebellar ataxia.

Example 12. Trametinib inhibition of MEK activity in a mouse AD model.

Since drugs for the treatment of neurodegenerative diseases affect the central nervous system, their ability to cross the blood-brain barrier is important. Among MEK 1/2 inhibitors, compounds such as AS703026 are known for their ability to effectively cross the BBB and inhibit MEK and reduce the expression of phosphorylated ERK in the mouse brain (Shaw et al. (2012), Evaluation of brain pharmacokinetics as a potential differentiation factor for the MEK inhibitors , MSC2015103 and pimasertib. Abstract LB-456, American Association for Cancer Research Annual Meeting, Chicago, IL). To confirm that the MEK1 / 2 inhibitor trametinib enters the brain and to obtain the above experimental results when orally administered to 5XFAD mice, the level of pERK expression in the brain tissue was measured.

The cerebral hemispheres of the 5XFAD mice (3 mice per group) from Example 8 were placed in a low temperature freezer, placed in liquid nitrogen and ground in a mortar with liquid nitrogen. The powder was divided into 6 parts, placed in new tubes and placed back in the freezer for low temperature freezing. One of the tubes was removed and used for Western blotting as described in Example 5-2. The powder was carefully suspended with a pipette in RIPA buffer, incubated on ice for 10 min, centrifuged at 13000 rpm at 4 ° C, and the supernatant was collected. After measuring the protein concentration, a buffer was added for applying samples (0.25 M Tris-HCl pH 6.8, 0.05% SDS, 50% glycerol, 0.25 M DTT, 0.5 mg / ml BPB), boiled at 100 ° C for 10 min and stored at -20 ° C. A 10% polyacrylamide gel was loaded with a sample containing 10 μg of protein, electrophoresis was performed in the presence of sodium dodecyl sulfate, transferred to a nitrocellulose membrane and blocked with 5% skim milk at room temperature for 1 hour. Antibodies to pERK (Cell signaling, No. 4370) and ERK in Tris-buffered saline (TBS) with 0.1% Tween 20, incubated at room temperature for 1 h, followed by incubation with secondary antibodies conjugated with horseradish peroxidase at room temperature for 1 h

Protein bands were detected using a photosensitive imaging system (Fig. 19a). To quantify the change in pERK expression, pERK and ERK expression levels were determined using densitometry and the pERK expression level was normalized relative to the ERK expression level. The normalized level of pERK expression in the trametinib group compared to the placebo group is shown in FIG. 19b.

As shown in FIG. 19, the level of pERK protein in the brain in mice treated with 0.1 and 1.0 mg / kg of trametinib was significantly lower than in the group receiving placebo.

This indicates that when trametinib is administered orally, trametinib is delivered to the brain, followed by inhibition of MEK activity and a decrease in pERK expression, resulting in neurogenesis and neuroprotective effect.

Example 13. Decrease in beta-amyloid deposition in a mouse model of AD.

- 26 038404

Since the main pathological characteristic of AD is β deposition, the ability of trametinib to reduce the deposition of AB was investigated. Using ground powder of the cerebral hemispheres of mice 5XFAD, obtained in example 12, the quantitative content _of Ab (1 -4о> and β ₀ - ₄₂ ) was determined using ELISA (enzyme-linked immunosorbent assay). The study was carried out using an ELISA kit (Invitrogen, MA, cat.no. KHB3442) as follows: First, the mouse brain powder was carefully suspended using a pipette in 5 M guanidine-HCl solution, centrifuged at 16000 g (rpm) at 4 ° C and collected the supernatant. After measuring the protein concentration, dilution buffer was added to obtain a solution containing 30-50 μg of protein in 100 μl. In 96-well plates was added 100 ul of each antibody Άβ _{(1. 40)} and Άβ _(1-42), incubated at room temperature for 2 hours, then removed and washed 4 times with wash buffer. 100 μl of each of the secondary antibodies conjugated to HRP was added and incubated at room temperature for 30 min. The solution was then removed and washed again 4 times with wash buffer. 100 μl of protein obtained from mouse brain powder was added to the wells, incubated in the dark for 30 min, and the reaction was stopped by adding 100 μl of a stop solution. Chemiluminescence was then detected at 450 nm using an ELISA plate reader. Absorbance units were converted to concentration using a standard and the concentration of each protein was determined. The amount of β ( _1-40 ) and β ( _1-42 ) was calculated relative to the total amount of protein (30 ~ 50 μg), and then the amount of β ( _1-42 ) was calculated relative to the amount of β ( _1-40 ). The resulting mean values for 3 mice in each group are shown in FIG. 20. As shown in FIG. 20, the amount of β ( _1-42 ) / Άβ ( _1-40 ) decreased in the trametinib group compared to the placebo group.

This indicates that in the BV model in mice, trametinib causes a decrease in the amount of Aβ, in particular, the Άβ form ( _1-42 ), which has a pronounced tendency to form toxic oligomers or aggregates, and thus can protect neurons.

Claims (11)

FORRULE OF INVENTION 1. The use of trametinib for the treatment of neurodegenerative disease caused by loss or damage of neurons, where the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), dementia, vascular dementia, frontotemporal dementia, dementia with Lewy bodies, multiple systemic atrophy, corticobasal degeneration, progressive supranuclear palsy, Huntington's disease, primary lateral sclerosis, spinal muscular atrophy, progressive bulbar palsy (PBP), progressive muscle atrophy (PMΆ), pseudobulbar palsy (hereditary paralysis) ataxia. 2. Use according to claim 1, wherein the neurodegenerative disease is treated by inducing neuroregeneration through differentiation of neural stem cells into neurons. 3. Use according to claim 2, wherein the neurodegenerative disease is further treated by protecting the neural stem cells and neurons from the cytotoxic effects of amyloid beta. 4. Use according to claim 2, wherein the neurons differentiated from neural stem cells are at least a neuron selected from a dopaminergic neuron, a G -ergic neuron, a cholinergic neuron, and a motor neuron. 5. Use according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease. 6. Use according to claim 1, wherein the neurodegenerative disease is vascular dementia. 7. The use according to claim 1, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease). 8. Use according to claim 1, wherein the neurodegenerative disease is Huntington's disease. 9. Use according to any one of claims 1 to 8, wherein trametinib is administered in an amount of 0.1 to 2 mg once a day. 10. Use according to claim 9, wherein trametinib is administered in an amount of 0.1 to 1 mg once a day. 11. Use according to claim 10, wherein trametinib is administered in an amount of 0.1 to 0.5 mg once a day.