Agent for extending axons

Abstract

To provide an agent or a composition useful for, for example, extending axons of nerve cells.SOLUTION: An agent or a composition comprises a component (A) expressing SPARC protein or promoting the expression of SPARC protein.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to an agent or composition capable of stretching axons of nerve cells, etc.

Background Art

[0002] In neurodegenerative diseases such as Alzheimer's disease, it is said that axons of nerve cells (hereinafter also simply referred to as "axons") are atrophied. When axons atrophy, it becomes difficult to send neurotransmitters to other nerve cells through the axons.

[0003] Current approved drugs for Alzheimer's disease all have the effect of enhancing the function of neurotransmitters and do not re-stretch atrophied axons. Therefore, although the progression of the disease state can be delayed, the disease state cannot be improved.

[0004] Non-Patent Document 1 and Non-Patent Document 2 report that by intraperitoneally administering diosgenin to mice, an improvement in memory was observed in normal mice or Alzheimer's disease model mice.

[0005] On the other hand, Patent Document 1 describes that when a SPARC (Secreted Protein Acidic and Rich in Cysteine) protein is added to a medium containing nerve cells, neurite outgrowth is observed, etc.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide an agent or composition capable of extending the axons of nerve cells.

[0009] Another object of the present invention is to provide an agent or composition useful for the treatment and / or improvement of diseases involving axonal dysfunction of nerve cells and / or the improvement and / or maintenance of memory.

Means for Solving the Problems

[0010] The present inventors surprisingly found that in atrophied axons, a specific protein (SPARC protein) is decreased. And the present inventors found that by expressing (increasing, augmenting) the SPARC protein in nerve cells (in nerve cells), the atrophied axons are re-extended.

[0011] In addition, the present inventors focused on the direction in which the axons of nerve cells extend, and found that according to a specific component, the axons of nerve cells can extend (reach) toward the projection target region (for example, in the case of the axons of hippocampal nerve cells, toward the prefrontal area which is their projection target), and through further intensive research, the present invention has been completed.

[0012] Although Non-Patent Documents 1 and 2 describe axonal extension, it was considered that axons extend in various directions rather than in a specific direction, so it was not even anticipated that axons could extend in a specific direction.

[0013] In Patent Document 1 mentioned above, nerve cells are added to a culture medium containing the SPARC protein. Considering the size of the SPARC protein, it is utterly impossible to think that the SPARC protein is incorporated into the nerve cells in this addition. Therefore, what can be recognized from Patent Document 1 is that the SPARC protein stimulates some signal for neurite outgrowth by exerting some influence from outside the nerve cells. The idea of expressing the SPARC protein itself within the nerve cells cannot be conceived at all from Patent Document 1.

[0014] That is, the present invention relates to the following inventions and the like. [1] An agent or composition for axonal outgrowth of nerve cells, comprising a component (A) that expresses the SPARC protein or promotes the expression of the SPARC protein (however, component (A) excludes diosgenins). [2] An agent or composition for extending the axon of a nerve cell to a projection target region (the region that the target axon reaches when normally projected), comprising a component (A) that expresses the SPARC protein or promotes the expression of the SPARC protein. [3] The agent or composition according to [2], wherein the axon is an axon of a hippocampal nerve cell and the projection target region is the prefrontal cortex. [4] An agent or composition for use in the following (1) and / or (2), comprising a component (A) that expresses the SPARC protein or promotes the expression of the SPARC protein (however, component (A) excludes diosgenins). (1) Treatment and / or improvement of diseases involving axonal dysfunction of nerve cells (2) Improvement and / or maintenance of memory [5] The agent or composition according to [4], wherein the disease involving axonal dysfunction of nerve cells is one or more selected from Alzheimer's disease, spinal cord injury, brain contusion, Parkinson's disease, and dementia. [6] The agent or composition according to any one of [1] to [5], wherein component (A) contains one or more components selected from the following (A-1) to (A-4). (A-1) A gene encoding SPARC protein (A-2) One or more enzymes selected from PI3 kinase, MEK1, protein kinase A, and protein kinase C (A-3) One or more enzyme activation components selected from proteins or peptides that activate one or more of the enzymes in (A-2) in nerve cells, vitamins that activate one or more of the enzymes in (A-2) in nerve cells, growth factors or proliferation factors that activate one or more of the enzymes in (A-2) in nerve cells, and steroids that activate one or more of the enzymes in (A-2) in nerve cells (A-4) One or more transcription activation components selected from components that activate transcription via transcription factor c-Jun, and components that activate transcription via transcription factors c-Jun and Fra1 [7] The agent or composition according to any one of [2] to [3] and [6], wherein component (A) contains diosgenins. [8] A screening method for an axon extender of nerve cells, which includes selecting a component that promotes the expression of SPARC protein. [9] A screening method for an agent for extending the axon of nerve cells to the region of the projection destination, which includes selecting a component that promotes the expression of SPARC protein.

[10] A screening method for an agent for use in the following (1) and / or (2), which includes selecting a component that promotes the expression of SPARC protein. (1) Treatment and / or improvement of diseases involving axon dysfunction of nerve cells (2) Improvement and / or maintenance of memory

[0015] In addition, the present invention also includes the following inventions.

[11] A method for axonal outgrowth of neurons by expressing (increasing the expression of) SPARC protein in neurons (in nerve cells).

[12] A method for projecting axons of neurons to the target region by expressing (increasing the expression of) SPARC protein in neurons (in nerve cells).

[13] A method for axonal outgrowth of neurons by administering a component (A) that expresses SPARC protein or promotes the expression of SPARC protein to an animal including a human (provided that the component (A) excludes diosgenins).

[14] A method for extending axons of neurons to the target region by administering a component (A) that expresses SPARC protein or promotes the expression of SPARC protein to an animal including a human.

[15] A method according to the following (1) and / or (2) by administering a component (A) that expresses SPARC protein or promotes the expression of SPARC protein to an animal including a human. (1) Treatment and / or improvement of diseases involving axonal dysfunction of neurons (2) Improvement and / or maintenance of memory

[16] Use of a component (A) that expresses SPARC protein or promotes the expression of SPARC protein for axonal outgrowth of neurons (provided that the component (A) excludes diosgenins).

[17] Use of a component (A) that expresses SPARC protein or promotes the expression of SPARC protein for extending axons of neurons to the target region.

[18] Use of a component (A) that expresses SPARC protein or promotes the expression of SPARC protein for the following uses according to (1) and / or (2). (1) Treatment and / or improvement of diseases involving axonal dysfunction of neurons (2) Improvement and / or maintenance of memory

[19] Component (A) that expresses the SPARC protein or promotes the expression of the SPARC protein for axonal outgrowth of nerve cells (provided that component (A) excludes diosgenins).

[20] Component (A) that expresses the SPARC protein or promotes the expression of the SPARC protein for extending the axons of nerve cells to the target projection area.

[21] Component (A) that expresses the SPARC protein or promotes the expression of the SPARC protein for use in the following use (1) and / or (2) (provided that component (A) excludes diosgenins). (1) Treatment and / or improvement of diseases involving axonal dysfunction of nerve cells (2) Improvement and / or maintenance of memory

Effects of the Invention

[0016] According to the present invention, the axons of nerve cells can be extended. In particular, according to the present invention, the axons of nerve cells can be extended to the target projection area. Moreover, according to the present invention, it is possible to provide an agent or composition useful for the treatment and / or improvement of diseases involving axonal dysfunction of nerve cells and for the improvement and / or maintenance of memory.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] [Agent or Composition] The agent or composition of the present invention contains component (A) that expresses or promotes the expression of SPARC protein and can be used in various applications. The agent or composition of the present invention may contain one or more of component (A).

[0019] [Use] The agent or composition of the first aspect is used for axonal outgrowth of nerve cells. In the agent or composition of the first aspect, component (A) usually does not contain diosgenins (for example, diosgenin, diosgenin derivatives, pharmaceutically acceptable salts thereof, etc.).

[0020] The agent or composition of the second aspect is used to extend the axons of nerve cells to the region of the projection target. That is, the agent or composition of the second aspect is used for a more limited use of extending the axons of nerve cells toward (with directionality to reach) the region of the projection target among the uses (axonal outgrowth use of nerve cells) of the agent or composition of the first aspect. Note that for the agent or composition of the second aspect, at least a part of the axons may be extended toward the region of the projection target, and it does not exclude the case where the axons extend in a direction other than the region of the projection target (regardless of directionality). For example, in the agent or composition of the second aspect, among the atrophied axons, 70% or more (for example, 80% or more, 90% or more, 95% or more, etc.) of the axons may be extended toward the region of the projection target. Note that the ratio of the axons that have extended toward the region of the projection target among the atrophied axons may be determined, for example, using image analysis with a tracer such as the method described in the examples. For example, nerve cells (a) not labeled with a tracer before administration of the agent or composition of the second aspect indicate nerve cells with atrophied axons, and nerve cells (b) labeled with the tracer injected after administration of the agent or composition of the second aspect indicate nerve cells with axons extended to the region of the projection target. By comparing the number of cells (a) and the number of cells (b), the ratio of the axons that have extended toward the region of the projection target among the atrophied axons can be confirmed.

[0021] The agent or composition of the second aspect may be used, for example, for the purpose of extending the axons of hippocampal neurons toward the prefrontal cortex (or the prefrontal cortex region). In normal neurons, in the circuit related to cognitive function, the axons of hippocampal neurons extend toward the prefrontal cortex. Therefore, the agent or composition of the second aspect used for such a purpose can further contribute to the construction of a normal neural circuit related to cognitive function.

[0022] Since the agents or compositions of the first and second aspects can extend axons (further extending toward the target projection region), they can be applied to uses such as the treatment and improvement of symptoms or diseases that are suggested to be related to axonal atrophy (for example, (1) the treatment and / or improvement of diseases involving the dysfunction of axons of nerve cells, as described later). In the agents or compositions of the first and second aspects, the type of nerve cells is not particularly limited, and the axons may be the axons of nerve cells corresponding to the diseases or symptoms to be treated or improved. Also, in the agent or composition of the second aspect, the target projection region may be the region that the axons of the corresponding nerve cells reach when normally projected. For example, in the case of diseases or symptoms involving hippocampal neurons such as Alzheimer's disease, the axons of hippocampal neurons may be extended (further extended toward the prefrontal cortex). Also, when the agents or compositions of the first and second aspects extend the axons of hippocampal neurons, they can be applied to (2) the improvement and / or maintenance of memory.

[0023] The agent or composition of the third aspect is useful for the following uses (1) and / or (2). In the agent or composition of the third aspect, component (A) usually does not contain diosgenins (for example, diosgenin, diosgenin derivatives, pharmaceutically acceptable salts thereof, etc.). (1) Treatment and / or improvement of diseases involving the dysfunction of axons of nerve cells (2) Improvement and / or maintenance of memory Note that such an agent or composition of the third aspect may be accompanied by the extension of nerve axons (the first aspect) or the extension of nerve axons toward the target projection region (the second aspect).

[0024] Diseases involving axonal dysfunction of nerve cells include, but are not particularly limited to, for example, spinal cord injury, brain contusion, Alzheimer's disease (AD), Parkinson's disease, dementia, etc. In the present invention, the dementia does not include Alzheimer's disease (Alzheimer's type dementia), but includes vascular dementia, Lewy body dementia, frontotemporal dementia, Pick's disease, etc. Among these diseases, particularly, AD, spinal cord injury, brain contusion, Parkinson's disease, dementia, etc. are preferred.

[0025] The agent or composition of the present invention (the agent or composition of the first, second, and third aspects) can improve and / or maintain memory regardless of whether a subject has reached any disease or symptom.

[0026] For example, the agent or composition of the present invention may be used (I) to improve and / or maintain the memory of a subject (human) with specific diseases or symptoms as described in (1) above, (II) to improve and / or maintain the memory of a subject (human) with diseases or symptoms other than those described in (1) above, or (III) to improve and / or maintain the memory of a subject (human) without any diseases or symptoms (for example, to improve or enhance the memory decline associated with aging, etc.).

[0027] As described above, the agent or composition of the present invention can improve various symptoms or diseases, and such symptoms or diseases may be those in the chronic stage (chronic diseases, chronic symptoms). The chronic stage is not particularly limited, and usually refers to the state after passing through the fluctuation period (acute stage, the period when symptoms are severe), depending on the symptoms or diseases.

[0028] Usually, it is difficult to treat or improve the symptoms in the chronic stage, but according to the agent or composition of the present invention, even chronic diseases or symptoms can be efficiently treated and / or improved.

[0029] In addition, the agent or composition of the present invention can extend not only atrophied axons but also non-atrophied axons, so it can prevent specific diseases and symptoms as described in (1) above.

[0030] [Component (A)] As component (A), components that can express (increase) SPARC protein in nerve cells or contribute to the expression (increase) can be mentioned. The expression of SPARC protein may be in nerve cells (in nerve cells), may be in the axons of nerve cells, or may be in sites other than axons. Also, when expressed in axons, it may be in atrophied axons or non-atrophied axons. Typically, component (A) may be a component that expresses (or produces SPARC protein or expresses the SPARC gene) or promotes (or stimulates) the expression of SPARC protein.

[0031] The nucleotide sequence of the SPARC gene and the amino acid sequence of the SPARC protein can be easily obtained from known databases (such as DDBJ / GenBank / EMBL, etc.). For example, the accession numbers of the nucleotide sequences of the SPARC genes and the amino acid sequences of the SPARC proteins of humans, mice, and rats are as follows. Human: (BC072457, etc.) (nucleotide sequence), (CAG33080) (amino acid sequence) Mouse: (BC004638, etc.) (nucleotide sequence), (CAJ18514) (amino acid sequence) Rat: (BC061777, etc.) (nucleotide sequence), (XP_032770322) (amino acid sequence)

[0032] Examples of components that express SPARC protein include, for example, genes encoding SPARC protein. Such genes are typically the SPARC gene. Also, such genes may be genes having a sequence homology of 70% or more (for example, 80% or more, etc.) with the SPARC gene.

[0033] Examples of components that promote the expression of SPARC protein include, for example, (A-2) enzymes such as PI3 kinase (PI3K, Phosphoinositide 3-kinase), MEK1 (mitogen-activated protein kinase 1), protein kinase A (PKA), and protein kinase C (PKC); (A-3) in nerve cells, components that activate one or more of the enzymes selected from PI3 kinase, MEK1, protein kinase A, and protein kinase C (enzyme activation components) [for example, proteins or peptides that activate one or more of the above enzymes (for example, peptide hormones such as insulin and leptin, proteins such as osteogenic protein 1, etc.), vitamins that activate one or more of the above enzymes (for example, retinoic acid, etc.), growth factors or proliferation factors that activate one or more of the above enzymes (for example, transforming growth factor β1, platelet-derived growth factor, insulin-like growth factor 1, etc.), steroids that activate one or more of the above enzymes [for example, diosgenins (for example, diosgenin, diosgenin derivatives, pharmaceutically acceptable salts thereof, etc.)], (A-4) transcription activation components such as components that activate transcription via transcription factor c-Jun, and components that activate transcription via transcription factor c-Jun and transcription factor Fra1, etc. When the transcription factor described in (A-4) binds to the promoter region of the SPARC gene, it can promote the transcription of the SPARC gene into mRNA, and thus can promote the expression of SPARC protein.

[0034] Diosgenin has the following chemical formula (I)

Chemical formula

[0035] In the present invention, the diosgenin derivative generally refers to a compound that can be an equivalent of diosgenin. The diosgenin and diosgenin derivatives are not particularly limited, and may be commercially available products, may be those produced according to known methods, methods known per se, or methods equivalent thereto, or may be extracts from natural products. For example, the diosgenin derivative may be an equivalent achievable by chemical modification such as introducing a substituent into diosgenin or converting a substituent, or may be a diosgenin glycoside (such as diosgenin) extracted from a natural product.

[0036] The diosgenin derivative is not particularly limited. For example, specific examples include a compound in which the hydroxyl group at the C3 position of diosgenin is substituted, a compound having a substituent at the C2 position (or a compound in which the hydrogen atom at the C2 position is substituted), a compound having a substituent at the C4 position (or a compound in which the hydrogen atom at the C4 position is substituted), a compound having a substituent at the C6 position (or a compound in which the hydrogen atom at the C6 position is substituted), or salts thereof. For example, ester derivatives of the hydroxyl group at the C3 position (such as amino acid derivatives, aminosulfonic acid derivatives, carbamate derivatives), or halogenated derivatives of the hydroxyl group at the C3 position, etc. can be mentioned.

[0037] Examples of the diosgenin derivative (and its salt) include a compound represented by the following formula (I-1) and its salt (pharmaceutically acceptable salt).

Chemical formula

[0038] R 1In this case, examples of the substituent include hydrocarbon groups {for example, alkyl groups [for example, linear or branched alkyl groups such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, pentyl group (for example, C 1-12 alkyl group, preferably C 1-8 alkyl group)], cycloalkyl groups (for example, C 4-10 cycloalkyl groups such as cyclopentyl group, cyclohexyl group, cycloheptyl group, cyclooctyl group, preferably C 5-8 cycloalkyl group), aralkyl groups (for example, C 6-10 aryl C 1-4 alkyl groups such as benzyl group, phenethyl group), polycyclic aliphatic hydrocarbon groups (for example, decalinyl group, norbornyl group, adamantyl group, dimethyladamantyl group, etc.) and other saturated or unsaturated aliphatic hydrocarbon groups; aryl groups (for example, C 6-10 aryl groups such as phenyl group, tolyl group, xylyl group, etc.) and other aromatic hydrocarbon groups}, heteroatom (nitrogen atom, oxygen atom, sulfur atom, phosphorus atom, etc.)-containing groups {for example, oxygen atom-containing groups [for example, hydroxyl group, oxo group (=O), group -OR a , group -O-CO-R a , group -O-CO-N(R b ) 2 , group -O-CO-O-R a , group -O-CO-S-R a , group -OR c , group -O-SO 2 -OH, group -O-PO 2 -OH, group -(OR d ) k -R e , carboxyl group, group -CO-O-R a , group -CO-N(R b ) 2 , etc.], nitrogen atom-containing groups [for example, amino group, group -NR a R b , group -NR b -CO-O-R a , group -NR b -CO-N(R b ) 2, a nitrogen-containing ring group (for example, a group corresponding to pyridine, pyrroline, pyrrole, indole, etc.), a sulfur atom-containing group [for example, a mercapto group, group -SR a , group -S-S-R a , a sulfo group (-SO 3 H), group -SO 2 -R b , group -S-CO-N(R b ) 2 , etc.], a phosphorus atom-containing group [for example, a phosphoric acid group (H 2 PO 4 -), group -PO 3 H, etc.], an amino acid group [or a residue of an amino acid, for example, a group formed by an ester bond between the hydroxyl group at the 3-position (corresponding to the substitution position of R 1 in the above formula) constituting diosgenin and the carboxyl group of an amino acid (such as glycine, alanine, etc.)], etc.}, a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc.), etc. are mentioned. In the above formula, R a is a hydrocarbon group (for example, the hydrocarbon groups exemplified above such as an alkyl group, etc.), R b is a hydrogen atom or a hydrocarbon group (for example, the hydrocarbon groups exemplified above such as an alkyl group), R c is a sugar (or a sugar chain or a residue of a sugar), R d is an alkylene group (for example, a C 2-4 alkylene group such as an ethylene group, a propylene group, a trimethylene group, etc.), R e is a hydrogen atom, a hydroxyl group or a hydrocarbon group (for example, the hydrocarbon groups exemplified above such as an alkyl group (such as a methyl group, etc.)), k is an integer of 2 or more (for example, 2 to 10), respectively, and R a and R b may be the same or different groups, and when R b is plural, they may be the same or different.

[0039] R a and R bIn this case, the hydrocarbon group (such as an alkyl group) may further have a substituent. The substituent is not particularly limited, but examples include the substituents exemplified above, for example, an oxygen atom-containing group (such as a hydroxyl group, a carboxyl group, group -OR a ), group -O-CO-R a and the like), a nitrogen atom-containing group (such as an amino group, group -NR a R b ), a sulfur atom-containing group (such as a mercapto group, group -SR a ), a sulfo group, group -SO 2 -R b and the like). The hydrocarbon group may have these substituents alone or in combination of two or more. When the hydrocarbon group has a substituent, the number of substituents may be 1 or more, for example, 1 to 10 (for example, 1 to 8), preferably 1 to 6 (for example, 1 to 4), and more preferably about 1 to 3.

[0040] R 1 When it is a substituent, typical R 1 includes, for example, a hydrocarbon group [such as an alkyl group (such as group -(CH 2 ) n -CH 3 ), a cycloalkyl group, an aralkyl group, etc.], a heteroatom-containing group {for example, an oxygen atom-containing group [such as a hydroxyl group, group -O-(CH 2 ) n -CH 3 , group -O-(CH 2 ) m -NH 2 , group -O-(CH 2 ) m -COOH, group -O-(CH 2 ) m -SO 3 H, group -O-CO-(CH 2 ) n -CH 3 , group -O-CO-NH-(CH 2 ) n -CH 3 , group -O-CO-NR-(CH 2 ) n -CH 3, group -O-CO-NH-CH(R b )-COOH, group -O-(CH 2 ) n -CO-NH-AD (wherein, AD represents an adamantyl group (such as 1 - adamantyl group, 2,6 - dimethyladamantan - 1 - yl group, etc.)), group -O-CO-NH-(CH 2 ) m -SO 3 H, group -O-CO-NH-(CH 2 ) m -COOH, group -O-CO-O-(CH 2 ) n -CH 3 , group -O-CO-S-(CH 2 ) n -CH 3 , group -O-SU (wherein, SU represents a sugar chain), group -O-SO 2 -OH, group -O-PO 2 -OH, group -(OCH 2 CH 2 ) m -CH 3 , group -(OCH 2 CH 2 CH 2 ) m -CH 3 , carboxyl group, group -COO(CH 2 ) n CH 3 , group -CO-NH-(CH 2 ) n -CH 3 , group -SO 3 H, group -SO 2 -(CH 2 ) n -CH 3 , group -SO 2 -Ph (wherein, Ph represents a phenyl group.), group -CO-NH-CH(R b )-COOH, group -CO-NH-(CH 2 ) n -SO 3 H, etc.], nitrogen atom - containing groups [for example, amino group, group -NH-(CH 2 ) n -CH 3 , group -NH-(CH 2 ) n -NH2、 group -NH-CH(R b )-COOH, group -NH-(CH 2 ) m -SO 3 H, group -NH-(CH 2 ) m -SO 2 H, group -NH-CO-O-(CH 2 ) n -CH 3 , group -NH-CO-NH 2 , group -NH-CO-NH-AD (wherein, AD represents an adamantyl group (such as 1 - adamantyl group, 2,6 - dimethyladamantan - 1 - yl group, etc.)), group -NH-CO-NH-CH(R b )-COOH, group -NH-CO-NH-(CH 2 ) m -SO 3 H, group -NH-CO-NH-(CH 2 ) m -COOH, etc.], sulfur atom - containing groups [for example, mercapto group, group -S-(CH 2 ) n -CH 3 , group -S-(CH 2 ) m -COOH, group -S-(CH 2 ) m -CH(NH 2 )-COOH, group -S-CO-NH-AD (wherein, AD represents an adamantyl group (such as 1 - adamantyl group, 2,6 - dimethyladamantan - 1 - yl group, etc.)), group -S-S-(CH 2 ) m -CH(NH 2 )-COOH, group -SO 3 H, etc.], phosphorus atom - containing groups [for example, group -PO 3 H, etc.], amino acid groups (for example, group -O-CO-CH 2 -NH 2 , etc.), etc.}, halogen atoms (for example, fluorine atom, chlorine atom, bromine atom, iodine atom, etc.), etc. In the above formula, m represents an integer of 1 or more (for example, 1 to 10, preferably 1 to 4, more preferably 1 or 2), n represents an integer of 0 or more (for example, 0 to 10, preferably 0 to 7), and R bis the same as above [i.e., a hydrogen atom or a hydrocarbon group (e.g., an alkyl group, etc.)].

[0041] R 2 , R 3 and R 4 In the above, the substituents include R 1 Examples of the substituents include those exemplified in the section 1. R 2 and / or R 4 When is a substituent, representative examples of the substituent include an oxygen atom-containing group, a nitrogen atom-containing group, a sulfur atom-containing group, an amino acid group, a halogen atom, and the like. Also, R 3 When is a substituent, representative examples of the substituent include a halogen atom.

[0042] In formula (I-1), R 1 ~R 4 The combination of R is not limited and includes all combinations. 1 ~R 4 Examples of the combination include the following combinations: (1)R 1 is a substituent other than a hydroxyl group, R 2 ~R 4 is a hydrogen atom (2)R 1 is a substituent other than a hydroxyl group, R 2 is a substituent, R 3 and R 4 is a hydrogen atom (3)R 1 is a substituent other than a hydroxyl group, R 3 is a substituent, R 2 and R 4 is a hydrogen atom (4)R 1 is a substituent other than a hydroxyl group, R 2 and R 3 is a substituent, R 4 is a hydrogen atom (5)R 1 is a substituent other than a hydroxyl group, R 2 , R3 and R 4 is a combination where the substituents (6)R 1 is a substituent other than a hydroxyl group, R 2 and R 3 is a hydrogen atom, R 4 is a combination where the substituents (7)R 1 is a substituent other than a hydroxyl group, R 3 is a hydrogen atom, R 2 and R 4 is a combination where the substituents (8)R 1 is a substituent other than a hydroxyl group, R 2 is a hydrogen atom, R 3 and R 4 is a combination where the substituents (9)R 1 is a hydroxyl group, R 2 is a substituent, R 3 and R 4 is a combination where the substituents are hydrogen atoms (10)R 1 is a hydroxyl group, R 3 is a substituent, R 2 and R 4 is a combination where the substituents are hydrogen atoms (11)R 1 is a hydroxyl group, R 2 and R 3 is a substituent, R 4 is a combination where the substituents are hydrogen atoms (12)R 1 is a hydroxyl group, R 2 ~R 4 is a combination where the substituents (13)R 1 is a hydroxyl group, R 2 and R 3 is a hydrogen atom, R 4 is a combination where the substituents (14)R 1 is a hydroxyl group, R 2 is a hydrogen atom, R 3 and R 4 is a combination where the substituents (15)R 1 is a hydroxyl group, R3 is a hydrogen atom, R 2 and R 4 is a combination of substituents

[0043] The diosgenin derivatives are not particularly limited, but specifically, for example, the following formula (II)

Chemical formula

Chemical formula

[0044] In addition, as the diosgenin derivative (or a salt thereof), a commercially available product may be used, or one synthesized by a known method may be used. For example, R 1 When introducing a substituent into, various substituents can be introduced via the hydroxyl group (the hydroxyl group at the 3-position) originally possessed by diosgenin. Also, R 2 and R 3 When introducing a halogen atom into, R 1 is substituted (oxidized) with an oxo group, and the carbon adjacent to the resulting ketone is halogenated to make R 2 and R 3 halogens (and further, if necessary, the oxo group is returned to a hydroxyl group (reduced)), and methods such as this can be adopted. Further, R4 When introducing a halogen atom, it can be introduced via electrophilic halogenation or the like with respect to an unsaturated bond. Furthermore, by using a nucleophile containing a heteroatom (such as an oxygen atom, a nitrogen atom, a sulfur atom, etc.), it is also possible to introduce various substituents.

[0045] In the present invention, the "pharmaceutically acceptable salt" (or "salt") includes those that form a pharmaceutically acceptable salt with diosgenin or the like, and is not particularly limited. Specifically, for example, hydrohalic acid salts (such as hydrofluoric acid salt, hydrochloride, hydrobromic acid salt, hydroiodic acid salt, etc.), inorganic acid salts (such as sulfate, nitrate, perchlorate, phosphate, carbonate, bicarbonate, etc.), organic carboxylates (such as acetate, oxalate, maleate, tartrate, fumarate, citrate, etc.), organic sulfonates (such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, camphorsulfonate, etc.), amino acid salts (such as aspartate, glutamate, etc.), organic amine salts (such as salts with organic bases such as trimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, arginine and lysine), quaternary ammonium salts, alkali metal salts (such as sodium salt, potassium salt, etc.), alkaline earth metal salts (such as magnesium salt, calcium salt, etc.) and the like can be mentioned.

[0046] [Form and Administration] The form of the agent or composition of the present invention is not particularly limited. Component (A) may be used as an agent (for example, powder) or composition as it is, or may be formulated together with other components (for example, carrier, excipient, etc.).

[0047] Examples of other components include components generally used as raw materials for pharmaceuticals, and are not particularly limited. For example, carriers, excipients, binders, disintegrants, lubricants, coating agents, coloring agents, flavoring and deodorizing agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, etc. can be mentioned. These other components may be used alone or in combination of two or more.

[0048] The carrier is not particularly limited. For example, animal and vegetable oils such as soybean oil, beef tallow, and synthetic glycerides; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; ester oils such as octyldodecyl myristate and isopropyl myristate; higher alcohols such as cetostearyl alcohol and behenyl alcohol; silicone resins; silicone oils; surfactants such as polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, and polyoxyethylene polyoxypropylene block copolymers; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethylene glycol, polyvinyl pyrrolidone, and methyl cellulose; lower alcohols such as ethanol and isopropanol; polyhydric alcohols such as glycerin, propylene glycol, dipropylene glycol, and sorbitol; sugars such as glucose and sucrose; inorganic powders such as anhydrous silicic acid, magnesium aluminum silicate, and aluminum silicate; purified water, etc.

[0049] Examples of excipients include lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, silicon dioxide, etc.

[0050] Examples of binders include polyvinyl alcohol, gelatin, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, polyvinyl acetal diethylaminoacetate, corn starch, etc.

[0051] Examples of disintegrants include corn starch, low-substituted hydroxypropyl cellulose, crospovidone, crystalline cellulose, precipitated calcium carbonate, croscarmellose sodium, calcium citrate, dextrin, pectin, calcium carboxymethyl cellulose, etc.

[0052] Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, light anhydrous silicic acid, sucrose fatty acid ester, etc., and examples of the flavoring and odor-correcting agent include cocoa powder, menthol, aromatic powder, peppermint oil, borneol, cinnamon powder, etc.

[0053] The form (dosage form) of the agent or composition is not particularly limited, and examples include tablets, powders, fine granules, granules, dry syrups, coated tablets, orally disintegrating tablets, chewable tablets, capsule agents, soft capsule agents, syrups, oral liquids, troches, jelly agents, inhalants, suppositories, injections, ointments, eye drops, eye ointments, nasal drops, ear drops, poultices, lotions, external liquids, sprays, external aerosol agents, creams, gels, tapes, buccal tablets, sublingual tablets, vaginal suppositories, vaginal tablets, rectal soft capsule agents, etc.

[0054] The administration (taking) form of the agent or composition is not particularly limited, and it may be oral administration or parenteral administration. Examples of parenteral administration include rectal administration, nasal administration, pulmonary administration, injection administration (for example, intravenous administration, intrathecal administration, epidural administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intra-arterial administration, intra-articular administration, intracardiac administration, intracapsular administration, intradermal administration, intralesional administration, intraocular administration, intrathoracic administration, subarachnoid administration, intrauterine administration, intraventricular administration, intracerebral administration, intramedullary administration), etc.

[0055] Although it depends on the type of component (A), the agent or composition of the present invention may particularly preferably be used for oral administration, intravenous administration, intramuscular administration, intramedullary administration, etc.

[0056] When component (A) is a gene, component (A) may be contained in the agent or composition in the form of a vector into which the gene has been introduced (or on which the gene has been mounted). The method for introducing the gene into the vector is not particularly limited, and a known method may be used.

[0057] When administering an agent or composition containing a vector, as the administration form, for example, injection administration (for example, intracerebral administration, intrathecal administration, intraventricular administration, intravenous administration, intraarterial administration, etc.), intramuscular administration, subcutaneous administration, etc. may be preferably used.

[0058] Also, when component (A) is a gene or the like, it may be contained in the agent or composition in a form in which component (A) has been introduced (including). For example, target cells are taken out from the body of an administration subject or non-administration subject by blood collection, biopsy, etc., cultured in vitro, then a gene is introduced into the cells by a vector, and the obtained gene-introduced cells may be administered as an agent or composition (or returned to the body of the administration subject).

[0059] The vector may be a viral vector or a non-viral vector (for example, liposome, plasmid, polymer, etc.). As the vector, a viral vector or the like may be preferably used from the viewpoint of gene introduction efficiency and the like.

[0060] Examples of the virus of the viral vector include DNA viruses or RNA viruses such as retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, vaccinia virus, poxvirus, poliovirus, sindbis virus, Sendai virus, SV40, etc.

[0061] The promoter of the viral vector may be one that promotes specific expression in nerve cells, and examples include synapsin I promoter, neuron-specific enolase promoter, etc.

[0062] In the agent or composition of the present invention, the amount of component (A) is not particularly limited and can be appropriately selected according to the dosage form, dosage, etc.

[0063] The dosage (intake amount, administration amount) of the agent or composition of the present invention varies depending on the type of component (A), the degree of the disease / symptom, age, sex, body weight, administration form, the specific type of the disease, etc. For example, as the amount of component (A) per day, it may be about 0.01 to 50 mg / kg, preferably 0.05 to 10 mg / kg, more preferably about 0.1 to 1 mg / kg.

[0064] When component (A) is a gene, the dosage of the gene is, for example, in the case of intracerebral administration to humans, etc., 1×10 10 ~5×10 12 vg / human, preferably 5×10 10 ~1×10 12 vg / human, more preferably about 1×10 11 ~5×10 11 vg / human.

[0065] The administration may be divided into one or multiple times.

[0066] The agent or composition of the present invention may be a pharmaceutical or a pharmaceutical composition, or may be a quasi-drug.

[0067] The agent or composition of the present invention can also be provided in the form of a kit, if desired. The kit may be a kit provided with a container such as a pack or a dispenser device. Further, the kit may be composed of a container containing component (A) and a container containing other components.

[0068] Depending on the type of component (A), the agent or composition of the present invention may be in the form of a food or drink. Therefore, the present invention also includes foods or drinks containing component (A) (or the agent or composition). Also, from the perspective of component (A) (or the agent or composition), component (A) may be an additive for foods or drinks.

[0069] In such foods or drinks, the preferred embodiments of component (A) etc. may be the same as those described above.

[0070] Food and drink products include, in addition to general foods including so-called health foods, health functional foods such as foods for specified health use and foods with nutrient function claims defined in the health functional food system of the Ministry of Health, Labour and Welfare. Furthermore, supplements (nutritional supplements), feeds, food additives, etc. are also included in the food and drink products of the present invention.

[0071] Also, the food and drink product may be a food and drink product for a specific target group [for example, for the elderly, for patients or sick people (for example, for patients having (the diseases or symptoms such as those exemplified above) such as Alzheimer's disease, etc.)].

[0072] To use component (A) in food and drink products, it can be used as it is or mixed with raw materials of food and drink products such as processed meat and soft drinks together with various nutritional components, etc. to produce food and drink products.

[0073] Also, when using component (A) as a health food, nutritional supplement, etc., for example, it can be prepared into forms such as tablets, capsules (soft capsules, hard capsules, etc.), powders, granules, liquid preparations (suspensions, syrups, etc.), emulsions, jellies, stick shapes, etc. using conventional means. Also, tablets include disintegrating tablets (orally disintegrating tablets).

[0074] The food or drink may contain food additives (additives for food). The food additives are not particularly limited, and examples thereof include excipients (for example, wheat starch, corn starch, cellulose, lactose, sucrose, mannitol, sorbitol, xylitol, pregelatinized starch, casein, magnesium aluminosilicate, calcium silicate, etc.), binders (for example, pregelatinized starch, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.), disintegrants (for example, cellulose, hydroxypropylcellulose, corn starch, etc.), fluidizing agents (for example, light anhydrous silicic acid, sucrose fatty acid ester, etc.), oils (for example, vegetable oils such as soybean oil, sesame oil, olive oil, linseed oil, perilla oil, rapeseed oil, coconut oil, corn oil, etc. or oils derived from animals / fish), nutrients (for example, various minerals, various vitamins, amino acids), flavors, sweeteners, flavor correctives, coloring agents, solvents (ethanol), salts, surfactants, pH adjusters, buffers, antioxidants, stabilizers, gelling agents, thickeners, lubricants, encapsulating agents, suspending agents, coating agents, preservatives, etc.

[0075] The food additives may be used alone or in combination of two or more.

[0076] When component (A) is used as a food or drink additive, the food or drink is not particularly limited, and examples thereof include foods [for example, noodles (buckwheat noodles, udon noodles, Chinese noodles, instant noodles, etc.), confectioneries (candies, candies, gums, chocolates, snack foods (potato chips, etc.), biscuits, cookies, gummies, jelly, jam, butter, cream (such as custard cream), cakes, etc.), breads, processed fishery or livestock products (kamaboko, ham, sausage, etc.), dairy products (processed milk, fermented milk, etc.), oils and fat-processed foods (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing, etc.), seasonings (sauces, gravies, etc.), retort foods (curry, stew, donburi, rice porridge, mixed rice, etc.), frozen desserts (ice cream, sherbet, shaved ice, etc.), fried foods (croquettes, French fries, fried chicken, etc.), etc.], beverages (tea beverages, soft drinks, carbonated beverages, nutritional beverages, fruit beverages, lactic acid beverages, etc.), etc.

[0077] The blending amount of component (A) in the above food and drink products varies depending on the addition form and administration form and can be selected from a wide range. For example, it may be 0.0001% by weight or more (for example, 0.001 to 50% by weight), 0.003% by weight or more (for example, 0.005 to 30% by weight), 0.01% by weight or more (for example, 0.05 to 10% by weight), etc.

[0078] In food and drink products, the intake amount (or administration amount or dosage amount) of component (A) can be selected from the same range as described above.

[0079] [Screening method] The present invention also includes screening methods for various agents, including selecting a component that promotes the expression of SPARC protein. The method may be (i) a screening method for an axon outgrowth agent of nerve cells, (ii) a screening method for an agent for extending the axons of nerve cells to the region of the projection target, (iii) a screening method for an agent for use in the treatment and / or improvement of diseases involving axon dysfunction of nerve cells and / or (2) the improvement and / or maintenance of memory.

[0080] In the screening method of the present invention, the test substance is not particularly limited and may be, for example, nucleic acid, peptide, protein, non-peptidic compound, synthetic compound, fermentation product, cell extract, cell culture supernatant, plant extract, tissue extract of a mammal, plasma, etc. Further, the test substance may be a novel substance or a known substance. The test substance may form a salt. The salt of the test substance may be a salt with a physiologically acceptable acid or base.

[0081] The screening method of the present invention may include, for example, a step of contacting a test substance with cells, a step of measuring the expression level of the SPARC gene of the cells, and a step of comparing the expression level with the expression level of the SPARC gene in cells not contacted with the test substance and selecting a test substance that improves the expression level of the SPARC gene.

[0082] The cells used in the screening method may be in vivo cells or cultured cells. The cultured cells may be primary cultured cells or established cell lines. Furthermore, the cells may be cells having the SPARC gene, or cells into which the SPARC gene has been introduced into cells not having the SPARC gene. Examples of cells having the SPARC gene include nerve cells, fibroblasts, endothelial cells, and the like. Examples of cells not having the SPARC gene include blood cells and the like.

[0083] The method of contacting the test substance with the cells is not particularly limited. When using cultured cells, for example, a method of adding the test substance to the culture medium can be mentioned. In addition, when contacting the test substance with cells in the living body of a non-human animal, systemic administration such as oral administration, intravenous administration, intramuscular administration, intraperitoneal administration, etc., local administration to the target organ or target tissue, intrathecal administration, etc. can be mentioned. It is also preferable to set up a control group that does not contact the test substance.

[0084] The measurement of the expression level of the SPARC gene may measure the amount of SPARC protein expressed or measure the amount of mRNA of the SPARC gene. When measuring the amount of protein expressed, the protein can be extracted from the cells by a known method and quantified using a known method for measuring the amount of protein expressed. Examples of methods for measuring the amount of protein expressed include Western blotting, EIA, ELISA, RIA, and methods using protein measurement reagents. In addition, the measurement of the amount of protein expressed may also be performed by image analysis of immunostaining as described in the examples below. When measuring the amount of mRNA, RNA can be extracted from the cells by a known method and quantified using a known method for measuring the amount of mRNA. Examples of methods for measuring the amount of mRNA include Northern blotting, RT-PCR, quantitative RT-PCR, and RNase protection assay.

[0085] If the amount of SPARC protein expression or the amount of mRNA of the SPARC gene increases when the test substance is contacted, compared with the amount of SPARC protein expression or the amount of mRNA of the SPARC gene in the control group that does not contact the test substance, the test substance can be selected as the target substance. The degree to which the test substance increases the amount of SPARC protein expression or the amount of mRNA of the SPARC gene is not particularly limited. For example, compared with the amount of SPARC protein expression or the amount of mRNA of the SPARC gene in cells not contacted with the test substance, it may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, etc.

Example

[0086] Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

[0087] <Test animals> ddY mice were obtained from Japan SLC (Shizuoka, Japan). 5XFAD mice, which are transgenic mice, were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). 5XFAD mice (male, + / −) were mated with wild-type mice (C57BL / 6*SJL, female, − / −), and genotyping was performed at 4 - 8 weeks after birth. The mice were allowed to freely ingest solid feed and water and were bred under constant temperature and humidity (25 ± 2°C, 50 ± 5%) and a 12-hour light-dark cycle (light period 7:00 - 19:00). Note that 5XFAD mice are Alzheimer's model mice.

[0088] <Axotomy> The operation was started using a stereotaxic apparatus (manufactured by Narishige (Tokyo, Japan)). A small blade was slowly inserted into the brain of a ddY mouse (female, 8 weeks old) at (+0.0 mm anterior-posterior (A-P), +1.5 mm medial-lateral (M-L), -1.6 mm dorsal-ventral (D-V) relative to the previous item) and moved parallelly to +1.5 mm A-P. During and after the procedure, the mouse was placed on a hot plate to maintain its body temperature. Subsequently, the mouse was anesthetized for 1 hour.

[0089] <Drug Administration> Diosgenin was obtained from Tokyo Chemical Industry Co., Ltd. Diosgenin was dissolved in Japanese Pharmacopoeia quality standard olive oil (manufactured by Maruishi Pharmaceutical Co., Ltd.) or a solvent (olive oil). Diosgenin was orally administered to wild-type mice, 5XFAD mice, and axotomized ddY mice at 0.1 μmol / kg / day once a day.

[0090] <Retrograde Labeling> 0.5 μl of Dextran 3000 MW Texas Red (50 mg / ml in aCSF, Thermo Scientific, catalog No. D3328) (hereinafter also simply referred to as "tracer 1"), a retrograde tracer, was injected into the prefrontal cortex (+1.9 mm A-P, +0.3 mm M-L, -2.6 mm D-V) of 5XFAD mice and wild-type mice at a rate of 0.5 ml / min. From 7 days after the injection of tracer 1, drug administration or AAV9 introduction was performed. Subsequently, 0.5 μl of Dextran 3000 MW FITC (50 mg / ml in aCSF, Thermo Scientific, catalog No. D3306) (hereinafter also simply referred to as "tracer 2") was injected into the prefrontal cortex at the same site as tracer 1 over 7 days. In the hippocampal CA1 and / or CA3, neurons that were only positive for tracer 1 (red) indicate those with progressive axonal atrophy. Neurons that were only positive for tracer 2 (green) indicate those with axons extending from the hippocampus to the prefrontal cortex. Neurons that were positive for both tracer 1 and 2 indicate those with neither axonal atrophy nor extension.

[0091] <Laser Capture Microdissection (LCM) and DNA Microarray> Mice were deeply anesthetized and immersed in 20 mL of ice-cold physiological saline. Then, the brain was carefully removed from the skull, rapidly frozen using dry ice, and stored at -30°C. This brain was sectioned at 16 μm in the coronal plane using a cryostat (Leica), mounted on RNase-free glass slides, and stored at -80°C until LCM was performed. Using a PALM MicroBeam (Carl Zeiss, Oberkochen, Germany), 660 neurons with no axonal outgrowth were obtained from three 5XFAD mice administered with vehicle, and 720 neurons with axonal outgrowth were obtained from three 5XFAD mice administered with diosgenin from one slide at room temperature within 1 hour. RNA was extracted from the collected neurons using NucleoSpin RNA (MACHEREYNAGEL GmbH & Co. KG), and T7 RNA polymerase amplification was performed (GeneChip® 3’ IVT Pico Kit, Thermo Scientific). The amplified RNA was hybridized to a mouse Clariom S Array (GeneChip® Hybridization, Wash and Stain Kit, Thermo Scientific). The data were analyzed using analysis software Transcriptome analysis console (Thermo Scientific).

[0092] <Western Blot> For mouse hippocampal neurons cultured initially on 35-mm dishes, the medium was removed in its entirety 3 days after the start of the culture, and the cells were treated with a solvent or diosgenin. Four days later, the cells were rinsed once with PBS and then lysed by adding M-PER (Thermo Fisher Scientific) containing Protease-Phosphatase inhibitor cocktail (Thermo Fisher Scientific), a protease / phosphatase mixed inhibitor. Mercaptoethanol (Wako Pure Chemical Industries) and NuPAGE LDS sample buffer (Thermo Fisher Scientific) were added to the lysate (5 μg / lane), and the mixture was heat-treated at 95°C for 10 minutes, followed by SDS-PAGE. The gel after SDS-PAGE was electrophoresed onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) for 90 minutes to transfer the proteins on the gel to the membrane. Subsequently, the transferred membrane was immersed in a 5% skim milk (Wako Pure Chemical Industries) and 0.1% Tween 20-Tris Buffered Saline (T-TBS) solution and shaken at room temperature for 1 hour for blocking. After washing the membrane three times with T-TBS, Can Get Signal Solution 1 (Toyobo) containing the primary antibody was placed in a plastic bag (ToShin Sangyo), and the reaction was carried out at 4°C overnight while rotating the bag on a rotator. Then, the membrane was washed with T-TBS, Can Get Signal Solution 2 (Toyobo) containing the secondary antibody was placed in the plastic bag, and the reaction was carried out at room temperature for 2 hours under a light-shielded environment while rotating on a rotator. Then, the membrane was washed with T-TBS, and chemiluminescence was detected using the ECL Prime Western blotting detection reagent (GE healthcare) with a LAS4000 (GE healthcare), and an image was acquired. The band intensity was quantified using a CS analyzer (ATTO). The primary antibodies used were goat anti-mouse Sparc antibody (1:1000, R&D systems, catalog No. AF942) and mouse anti-GAPDH antibody (1:1000, Applied biological materials, catalog No. G041), and the secondary antibodies were HRP-conjugated donkey anti-goat IgG (1:2000, Santa Cruz) and HRP-conjugated goat anti-mouse IgG (1:2000, Santa Cruz).

[0093] <AAV9 (Adeno-associated virus) introduction> The AAV9 vector was prepared or purchased from VectorBuilder. AAV9-Syn1-Cerulean-WPRE (Syn1: synapsin I promoter, Cerulean: cyan fluorescence), or AAV9-Syn1-mSparc-IRES-Cerulean-WPRE, was introduced into the hippocampal CA1 (-1.9 mm A-P, +1.7 mm M-L, -1.7 mm D-V) of 5XFAD mice and wild-type mice (female, 7 - 9 months old) at 0.5 ml / min, 1 μl / site.

[0094] <Object recognition memory test> In this example, the object recognition memory test was conducted as follows: Twenty-one days after AAV9 introduction, as the training stage, two identical objects that the mice had never seen before were placed in a square box, and the mice were allowed to explore for 10 minutes. After a 1-hour time interval, the test stage was conducted. In the test stage, one of the objects used in the training stage was replaced with a new object (an object of a different shape and color). In each stage, the number of times the mice contacted the two objects in 10 minutes was recorded. That is, in the test stage, it is a test to confirm whether the mice remember the objects seen in the training stage. In this example, the ratio (%) of the number of explorations of the new object to the total exploration time was calculated as the preference index.

[0095] <Spatial recognition memory test> In this example, the spatial recognition memory test was conducted as follows: The test was conducted in a room with relatively low illumination (about 76 lux). Twenty-three days after AAV9 introduction, as a training stage, two identical objects that the mice had never seen before were placed in a square box, and the mice were allowed to explore for 10 minutes. Among the four inner walls of the box, wallpaper with characteristic patterns (polka dot pattern and vertical stripe pattern) that served as landmarks was pasted on two opposite walls. After a one-hour time interval, the test stage was conducted. In the test stage, one of the objects used in the training stage was placed in a location different from that in the training stage. In each stage, the number of times the mice contacted the two objects in 10 minutes was recorded. That is, in the test stage, it is a test to confirm whether the mice remember the objects seen in the training stage. In this example, the ratio (%) of the number of explorations of the new object to the total exploration time was calculated as the preference index.

[0096] In addition, in the object recognition memory test and the spatial recognition memory test, if the exploration preference index exceeds 50%, it indicates that the memory impairment of the mice has been improved.

[0097] <Anterograde labeling using biotinylated dextran amine (BDA)> After drug administration to the mice, 10% BDA (in PBS, Thermo Scientific, catalog No. D1956) was injected into the hippocampal CA1 (-1.9 mm A-P, +1.7 mm ML, -1.7 mm D-V) of 5XFAD mice and wild-type mice (female, 6 months old) at a rate of 0.5 ml / min. Seven days later, immunohistological analysis was performed.

[0098] <Immunohistological analysis> For mice, under deep anesthesia, after perfusion of the whole brain with ice-cold physiological saline through the heart, the whole brain was removed from the skull and cryopreserved. The brain frozen at -30°C was used to prepare 20-μm thick serial coronal sections using a cryostat (CM3050S, Leica, Heidelberg, Germany). After the sections were returned to room temperature, they were washed with PBS, immersed in a 4% paraformaldehyde (PFA, Wako Pure Chemical Industries, Ltd.) solution for fixation, then the primary antibody solution was added and reacted overnight at 4°C. The next day, after the sections were washed three times with PBS, the secondary antibody solution was added and reacted for 2 hours at room temperature in the dark. After washing three times with PBS, they were mounted with Aqua Poly Mount. Fluorescent images were obtained using a fluorescence microscope (BZ-X700, Keyence). The primary antibodies used were rabbit anti-NeuN (1:500, abcam, catalog No. ab104225), goat anti-mouse Sparc (1:100, R&D systems, catalog No. AF942), and mouse anti-neurofilaments, hypophosphorylated (pNF-H; 1:250, Covance, catalog No. SMI35). The secondary antibodies were Alexa 488-, 568-, 594-, or 647-conjugated affinify-purified secondary anti-mouse IgG, anti-rabbit IgG, anti-goat IgG, and anti-chicken IgY (all at a dilution ratio of 1:400). In addition, nuclear counterstaining was performed using 4',6-diamidino-2-phenylindole (DAPI, 1 μg / ml, Sigma-Aldrich).

[0099] <Primary neuronal cell culture and immunostaining> Primary culture of neurons was performed as follows: fetuses were removed from 14-day-old ddY mice (Japan SLC, Shizuoka), washed with phosphate buffered saline (PBS), and the hippocampus was dissected, minced, and suspended in the primary culture medium [Neurobasal media (Life Technologies, Carlsbad) containing 12% horse serum (Life Technologies), 2 mM L-glutamine, and 0.6% glucose], and then seeded as dispersed cultured cells. The culture was carried out at 10% CO 2 , 37 °C, under saturated water vapor. For immunostaining of cells, the medium was removed from the neurons treated with each treatment, washed with PBS, and then the neurons were fixed with a 4% PFA-PBS solution for 90 minutes. The solution was removed, and the cells were washed twice with a 0.3% TritonX-100 (Wako Pure Chemical Industries)-PBS solution for 5 minutes each time. The primary antibody solution [0.3% TritonX-100-PBS solution, normal goat serum (Wako Pure Chemical Industries), mouse anti-pNF-H monoclonal antibody (1:250, SMI-35, Covance), rabbit anti-MAP2 polyclonal antibody (1:2000, Abcam), goat anti-SPARC monoclonal antibody (1:100, R&D systems)] was added and reacted at 4°C for 24 hours. The solution was removed, and 100 μL of the secondary antibody solution [0.3% TritonX-100-PBS solution, Alexa Fluor 488-, 568-, 594-, 647-labeled goat anti-mouse IgG antibody (1:300, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488-labeled goat anti-rabbit IgG (1:300, Molecular Probes)] was added and reacted at room temperature for 2 hours in the dark. At the same time, nuclear counterstaining was performed using 4’,-6-diamidino-2-phenylindole (DAPI, 1 μg / ml, Sigma-Aldrich). After washing with PBS for 5 minutes, the samples were mounted with Aqua Poly Mount (Polyscience, Warrington). Fluorescent images were acquired using a fluorescence microscope [BZ-X700 (Keyence), or Cell Observer (Carl Zeiss)]. The length of pNF-H positive axons was automatically traced, and the axon length per neuron was measured in the region by dividing the axon length by the number of neurons counted by MAP2 positivity.

[0100] <Image analysis> Image analysis of axonal outgrowth in the brains of 5XFAD mice was performed by counting the number of tracer-positive, DAPI-positive, and NeuN-positive neurons in the CA1 and CA3 regions of the hippocampus in mice injected with a retrograde tracer into the prefrontal cortex using MetaMorph (Molecular Devices, Sunnyvale). The length of pNF-H-positive axons and the quantification of SPARC protein expressed in neurons in primary cultured neurons were quantified using MetaMorph (Molecular Devices, Sunnyvale) or Neurocyte (Kurabo). The amount of SPARC protein expression limited to axons was quantified using Image J. The number of anterograde tracer-positive axons was quantified using Image J.

[0101] <Statistical analysis> Statistical comparisons were performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) by one-way analysis of variance (ANOVA) with post hoc Dunnett’s test and Bonferroni test, two-way ANOVA with multiple comparison Bonferroni test, and unpaired t-test. p < 0.05 was considered significant. Data were presented as the mean ± standard error of the mean (SEM).

[0102] (Example 1) As described above, tracer 1 was injected into the prefrontal cortex of 5XFAD mice (female, 5 - 6 months old). Starting 7 days later, a solvent (olive oil) or diosgenin was orally administered continuously for 14 days. Thereafter, tracer 2 was injected into the prefrontal cortex at the same site as tracer 1 as described above. Thereafter, the brain was removed, and the number of neurons positive only for tracer 2 in the CA1 and CA3 of the hippocampus was quantified. The results are shown in Fig. 1 (CA1) and Fig. 2 (CA3) (**p < 0.01, ***p < 0.001, two-sided test, solvent administration: n = 3 mice, diosgenin administration: n = 4 mice). As shown in FIGS. 1 and 2, in the diosgenin administration group, there were many nerve cells that were positive only for tracer 2 (that is, axons extended from the hippocampus toward the prefrontal cortex).

[0103] From the brains of 5XFAD mice administered with the solvent (n = 3) obtained above, LCM was performed as described above, and nerve cells in which axons did not extend were collected. Also, from the brains of 5XFAD mice administered with diosgenin (n = 3) obtained above, LCM was performed as described above, and nerve cells in which axons extended were collected. From the collected nerve cells, RNA was extracted as described above, and gene changes in the nerve cells were detected using a DNA microarray. The gene changes in the nerve cells detected by the DNA microarray were compared using Hierarchical clustering and Scatter plot. Since the mRNA expression level of the SPARC gene was most increased in the nerve cells in which axons extended, the most increased gene was identified as the SPARC gene (not shown).

[0104] (Test Example 1) Three days after culturing hippocampal neurons primary-cultured from ddY mice, 1 μM diosgenin or a solvent (0.1% ethanol) was added to the medium, and the cells were cultured in a 35-mm dish for 4 days. Thereafter, lysates were prepared from these nerve cells, and the expression level of SPARC protein in each nerve cell was compared with the expression level of GAPDH protein by the Western blot described above. The results are shown in FIG. 3 (**p < 0.01, two-sided test, solvent administration: n number of lysates = 7, diosgenin administration: n number of lysates = 7). As shown in FIG. 3, in the diosgenin administration group, the expression level of SPARC protein was high.

[0105] (Example 2) (Expression of SPARC gene) For hippocampal neurons primary-cultured from ddY mice, 5 × 10 5 , 5 × 10 6 or 5 × 10 7Treat with AAV9-Syn1-Cerulean-WPRE (hereinafter also simply referred to as "AAV-Control") or the viral vector introduced with the Sparc gene, AAV9-Syn1-mSparc-IRES-Cerulean-WPRE (hereinafter also simply referred to as "AAV-Sparc") at a concentration of The expression level of SPARC protein in MAP2-positive neurons was quantified by the above immunostaining and image analysis. The results are shown in Figure 4. In Figure 4, noAAV indicates the group without any treatment (****p < 0.0001 vs. AAV-Control at the same concentration, one-way ANOVA post hoc Bonferroni test, n number of neurons = 337 - 558). As shown in Figure 4, it was confirmed that the Sparc gene was overexpressed in the cells treated with AAV-Sparc.

[0106] (Axonal outgrowth) In addition, for the above AAV-Control treatment, AAV-Sparc treatment (5×10 6 GC / μl), or cells without any treatment, images of neurons were obtained by the above image analysis (Figure 5), and the length of pNF-H-positive axons was quantified. The results are shown in Figure 6 (*p < 0.05, one-way ANOVA post hoc Bonferroni test, n number of images = 10 - 16). As shown in Figure 6, in the mice treated with AAV-Sparc, the axons had the most outgrowth.

[0107] (Example 3) AAV-Control or AAV-Sparc 10 10 ​GC was injected into the hippocampal CA1 of wild-type mice and 5XFAD mice (female, 7 - 9 months old). An object recognition memory test was conducted 21 days after injection, and a spatial recognition memory test was conducted 23 days after injection. The results are shown in Figures 7 and 8 (**p < 0.01, ***p < 0.001, one-way ANOVA post hoc Dunnett’s test. Two-way repeated measures ANOVA showed a significant interaction between gene treatment and memory test. [F(2,23) = 13.77, p = 0.0018 (Figure 7), [F(2,23) = 16.33, p = 0.0010 (Figure 8). p < 0.001, #p < 0.0001, post hoc Bonferroni test, n = 4 mice). In Figures 7 and 8, Cont indicates the AAV-Control treatment group, and Sparc indicates the AAV-Sparc treatment group. As shown in Figures 7 and 8, it was confirmed that memory impairment was improved by AAV-Sparc treatment.

[0108] After the memory test, the brains of the mice were removed and brain sections were prepared. In the hippocampal CA1, the amount of Sparc protein (red) in the injected AAV9-derived Cerulean (blue) fluorescence and NeuN-positive neurons (green) was detected by fluorescence immunostaining. It was confirmed that compared with the AAV-Control treatment, there was more red detection in the AAV-Sparc treatment, indicating that the expression of the Sparc gene was more induced (not shown in the figure).

[0109] (Example 4) As described above, Tracer 1 was injected into the prefrontal cortex of wild-type mice and 5XFAD mice (female, 7 - 9 months old). Seven days later, AAV-Control or AAV-Sparc 10 10GC was continuously administered to the hippocampal CA1 for 21 days. Subsequently, after injecting tracer 2 into the prefrontal cortex at the same site as tracer 1 for 7 days, the brain was removed. In the hippocampal CA1 of the removed brain, the number of neurons positive only for tracer 2, the number of neurons positive only for tracer 1, and the number of neurons positive for both tracers 1 and 2 were quantified. The results are shown in Figs. 9 - 11 (*p < 0.05, ***p < 0.001, ****p < 0.0001, one-way ANOVA post hoc Bonferroni test, n number of mice = 4). In Figs. 9 - 11, Cont indicates the AAV-Control treatment group and Sparc indicates the AAV-Sparc treatment group. As is clear from Figs. 9 - 11, in the AAV-Sparc treatment group, there were many neurons positive only for tracer 2 (i.e., axons extended from the hippocampus to the prefrontal cortex).

[0110] (Example 5) The solvent (olive oil) or diosgenin was orally administered to wild-type mice and 5XFAD mice continuously for 21 days at 0.1 μmol / kg / day. On the 14th day of drug administration, the anterograde tracer BDA was injected into the hippocampal CA1. Seven days after the injection of BDA, brain sections of the mice were prepared, and BDA-positive axons (red), SPARC protein (green), and DAPI (blue) in the prefrontal cortex were detected by fluorescence immunostaining. Since BDA spreads anterogradely to the prefrontal cortex, the axons projected from the hippocampus to the prefrontal cortex are labeled with BDA. The results of quantifying the number of BDA-positive axons are shown in Fig. 12 (*p < 0.05, ***p < 0.001 compared with the solvent administration group of 5XFAD mice, one-way ANOVA post hoc Bonferroni test, n number of mice = 5). In Fig. 12, Veh indicates the solvent administration group and Dios indicates the diosgenin administration group. As shown in Fig. 12, in the diosgenin administration group, there were many neurons with axons extending from the hippocampus to the prefrontal cortex.

[0111] (Test Example 2) Primary cultured neurons isolated from ddY mice (14 days postnatal) were cultured for 3 days and then treated with Aβ25-35 (amyloid β-protein fragment 25-35) (2.5 μM), a causative agent of Alzheimer's disease, for 3 days. Thereafter, the medium containing Aβ25-35 was removed, and the cells were treated with a solvent or diosgenin (0.1 μM or 1 μM) for 4 days, followed by fluorescence immunostaining. Image analysis confirmed that in the diosgenin-administered group, the expression level of SPARC protein increased, particularly on axons and at axon terminals (not shown). Also, the expression level of SPARC protein per axon was quantified by the above immunostaining and image analysis (*p < 0.05, ***p < 0.001, one-way ANOVA post hoc Dunnett’s test compared with the Aβ / Veh group, n = 92 - 427 axons). The results are shown in Fig. 13. In the axons of Aβ25-35-treated neurons, the expression level of SPARC protein increased in the diosgenin-administered group. In Fig. 13, since the Aβ / Veh group had a lower expression level of SPARC protein in axons than the control group (Cont), it can be seen that SPARC protein decreased in atrophied axons.

[0112] (Example 6) Tracer 1 (Dextran Texas Red) was injected into the prefrontal cortex of wild-type and 5XFAD mice, and 7 days later, 10 10 GC of AAV-Control or AAV-Sparc was injected into the hippocampal CA1. Twenty-one days after AAV injection, Tracer 2 (Dextran FITC) was injected into the prefrontal cortex, and 7 days later, the brain was removed. At the stage before AAV injection, the quantitative value of the number of neurons (number of Texas Red-positive neurons) with axonal projections from the hippocampal CA1 to the prefrontal cortex is shown in Fig. 14A, and after AAV injection, the quantitative value of the number of neurons (number of FITC-positive neurons) with axonal projections from the hippocampal CA1 to the prefrontal cortex is shown in Fig. 14B (***p < 0.001, ****p < 0.0001, one-way ANOVA post hoc Bonferroni test, n = 4 for mice). Fig. 14A shows the number of hippocampal CA1 neurons extending axons to the prefrontal cortex before overexpressing the SPARC gene. It is similarly low in the two groups of 5XFAD mice compared to wild-type mice. That is, before SPARC gene expression, about 40% of the axons were similarly atrophied. Fig. 14B shows the situation 28 days after overexpressing the SPARC gene. In the mice expressing only the control vector of 5XFAD, the number of cells extending axons to the prefrontal cortex remains less compared to wild-type mice, but in the SPARC gene expression group, the number of projecting neurons increased beyond that of the wild type. This result indicates that among the axons that were atrophied in the hippocampal CA1, all the axons extended towards the prefrontal cortex.

[0113] (Example 7) Tracer 1 (Dextran Texas Red) was injected into the prefrontal cortex of wild-type and 5XFAD mice, and 7 days later, 10 μl each of AAV-Control or AAV-Sparc was injected into the 10 GC and hippocampal CA3. 21 days after AAV injection, Tracer 2 (Dextran FITC) was injected into the prefrontal cortex, and 7 days later, the brain was removed. At the stage before AAV injection, the quantitative value of the number of neurons (number of Texas Red-positive neurons) with axonal projections from the hippocampal CA3 to the prefrontal cortex is shown in Fig. 15A, and after AAV injection, the quantitative value of the number of neurons (number of FITC-positive neurons) with axonal projections from the hippocampal CA3 to the prefrontal cortex is shown in Fig. 15B (**p < 0.01, ***p < 0.001, one-way ANOVA post hoc Bonferroni test, n = 4 for mice).

[0114] Figure 15A shows the number of hippocampal CA3 neurons extending axons into the prefrontal cortex before overexpressing the SPARC gene. Compared with wild-type mice, the two groups of 5XFAD mice had a similarly small number. That is, before SPARC gene expression, about 40% of the axons were similarly atrophied. Figure 15B shows the situation 28 days after overexpressing the SPARC gene. In the mice expressing only the control vector of 5XFAD, the number of cells extending axons into the prefrontal cortex remained less than that of wild-type mice, but in the SPARC gene expression group, the number of projecting neurons increased beyond that of the wild type. This result indicates that all of the axons that were atrophied in hippocampal CA3 extended toward the prefrontal cortex.

Industrial Applicability

[0115] The agent or composition of the present invention is extremely useful because it can treat or improve diseases involving dysfunction of axons of nerve cells.

Claims

1. An agent or composition for axonal extension of nerve cells, comprising a component (A) that expresses SPARC protein or promotes the expression of SPARC protein (with the proviso that component (A) does not include diosgenins).

2. An agent or composition for extending an axon of a nerve cell to a projection region, comprising a component (A) that expresses a SPARC protein or promotes the expression of a SPARC protein.

3. The agent or composition according to claim 2, wherein the axon is an axon of a hippocampal neuron and the region to which it projects is the prefrontal cortex.

4. An agent or composition for use in the applications (1) and / or (2) below, comprising component (A) that expresses SPARC protein or promotes the expression of SPARC protein (provided that component (A) does not include diosgenins). (1) Treatment and / or improvement of diseases involving dysfunction of nerve cell axons (2) Improving and / or maintaining memory

5. The agent or composition according to claim 4, wherein the disease involving dysfunction of axons of nerve cells is one or more selected from Alzheimer's disease, spinal cord injury, cerebral contusion, Parkinson's disease and dementia.

6. The agent or composition according to any one of claims 1 to 5, wherein the component (A) comprises one or more components selected from the following (A-1) to (A-4): (A-1) Gene encoding SPARC protein (A-2) One or more enzymes selected from PI3 kinase, MEK1, protein kinase A, and protein kinase C. (A-3) One or more enzyme-activating components selected from proteins or peptides that activate one or more of the enzymes of (A-2) in nerve cells, vitamins that activate one or more of the enzymes of (A-2) in nerve cells, growth factors that activate one or more of the enzymes of (A-2) in nerve cells, and steroids that activate one or more of the enzymes of (A-2) in nerve cells. (A-4) One or more transcription activation components selected from a component that activates transcription mediated by the transcription factor c-Jun, and a component that activates transcription mediated by the transcription factor c-Jun and the transcription factor Fra1.

7. The agent or composition according to any one of claims 2 to 3 and 6, wherein component (A) comprises a diosgenin.

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