JP2016210737A - Central nervous system cell protectant, and agent for prevention or treatment of central nervous system degenerative disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel central nervous system cell protectant comprising galacto-N-biose, lacto-N-biose I, or their derivatives as an active ingredient, and an agent for the prevention or treatment of central nervous system degenerative disease.SOLUTION: The present invention provides a compound represented by formula (1) or (2) or a pharmacologically acceptable salt thereof, or their prodrugs (Rs independently represent a halogen atom, an alkyl group, an alkenyl group, an aryl group, a cyano group, -N(R), -NHCOR, or -OR; Rs independently represent H or an alkyl group).SELECTED DRAWING: Figure 2

Description

  The present invention relates to a central nerve cell protective agent and a preventive or therapeutic agent for central neurodegenerative diseases.

  Glutamate is one of excitatory neurotransmitters in the central nervous system and is involved in phenomena such as excitatory post-synaptic potential and long-term potentiation. On the other hand, glutamate also acts as an endogenous excitotoxin, and it is known that excessively released glutamate induces neuronal cell death. Glutamate excitotoxicity is also attracting attention as one of risk factors for central neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). 6).

  Cell death of cultured neurons by glutamate is reduced by the addition of a glutamate receptor antagonist. This suggests that glutamate excitotoxicity is caused by activation of glutamate receptors and downstream mechanisms. Therefore, glutamate receptor antagonists can be candidate compounds for therapeutic agents for central neurodegenerative diseases involving glutamate excitotoxicity.

  However, there are reports that glutamate receptor antagonists cause mental abnormalities in humans and reports that they cause abnormal locomotor activity in rodents, and clinical applications are not so advanced. For this reason, the development of a new central nervous protective agent having a protective action against glutamate excitotoxicity has been desired.

On the other hand, galacto-N-biose is a disaccharide (Galβ1-3GalNAc) in which galactose and N-acetylgalactosamine are β1-3 linked, and is known as the core structure of mucin which is a glycoprotein of the intestinal mucosa. Further, lacto-N-biose I is a disaccharide in which galactose and N-acetylglucosamine are β1-3 linked (Galβ1-3GlcNAc) and constitutes an oligosaccharide (milk oligosaccharide) contained in human breast milk. Known as.
So far, as the pharmacological action of lacto-N-biose I, an anti-obesity action and an action of improving the intestinal barrier function are known (for example, see Patent Documents 1 and 2). However, it has not been known that galacto-N-biose, lacto-N-biose I, or derivatives thereof show a protective effect on central nerve cells.

JP 2011-116725 A JP, 2014-210718, A

Van Den Bosch et al., Biochimica et biophysica acta, 1762, 1068-1082 (2006) Shaw, C.E. et al., Current neurology and neuroscience reports, 1, 69-76 (2001) Schubert, D. et al., The Journal of neuroscience: the official journal of the Society for Neuroscience, 21, 7455-7462 (2001) Dong, X.X. et al., Acta pharmacologica Sinica, 30, 379-387 (2009) Blandini, F. et al., Molecular neurobiology, 12, 73-94 (1996) Estrada Sanchez, A.M. et al., Archives of medical research, 39, 265-276 (2008)

  Therefore, the present invention provides a novel central nerve cell protective agent containing galacto-N-biose, lacto-N-biose I, or a derivative thereof as an active ingredient, and a preventive or therapeutic agent for central neurodegenerative diseases. Is an issue.

Specific means for solving the above problems include the following embodiments.
<1> A central nerve cell protective agent comprising a compound represented by the following formula (1) or (2) or a pharmacologically acceptable salt thereof, or a prodrug thereof as an active ingredient.

(In the formula, ^{R 1} are each independently a halogen atom, an alkyl group, an alkenyl group, an aryl group, a cyano _{^{group, -N (R 2) 2,}} -NHCOR 3, or independently .R ² is showing the -OR ⁴ , if .R ² represents a hydrogen atom or an alkyl group is either an alkyl group, which may form a ring structure by bonding R ² together .R ³ is an alkyl group or an aryl group. R ⁴ represents a hydrogen atom, an alkyl group, an aralkyl group, an alkylcarbonyl group, an arylcarbonyl group, an alkylsulfonyl group, an arylsulfonyl group, or —Si (R ⁵ ) _3. Each R ⁵ independently represents an alkyl group or Represents an aryl group.)

<2> A preventive or therapeutic agent for central neurodegenerative diseases, comprising as an active ingredient a compound represented by the following formula (1) or (2) or a pharmacologically acceptable salt thereof, or a prodrug thereof.

(In the formula, ^{R 1} are each independently a halogen atom, an alkyl group, an alkenyl group, an aryl group, a cyano _{^{group, -N (R 2) 2,}} -NHCOR 3, or independently .R ² is showing the -OR ⁴ , if .R ² represents a hydrogen atom or an alkyl group is either an alkyl group, which may form a ring structure by bonding R ² together .R ³ is an alkyl group or an aryl group. R ⁴ represents a hydrogen atom, an alkyl group, an aralkyl group, an alkylcarbonyl group, an arylcarbonyl group, an alkylsulfonyl group, an arylsulfonyl group, or —Si (R ⁵ ) _3. Each R ⁵ independently represents an alkyl group or Represents an aryl group.)

<3> The agent for preventing or treating central nervous degenerative disease according to <2>, wherein the central neurodegenerative disease is a disease involving glutamate excitotoxicity.

  According to the present invention, there are provided a novel central nerve cell protective agent comprising galacto-N-biose, lacto-N-biose I, or a derivative thereof as an active ingredient, and a preventive or therapeutic agent for central neurodegenerative diseases. Can do.

It is a figure which shows the protective effect with respect to the central nerve cell of galacto-N-biose in Example 1 (MTT assay). It is a figure which shows the protective effect with respect to the central nerve cell of galacto-N-biose in Example 2 (nuclear staining assay). It is a figure which shows the long-term toxicity with respect to the central nerve cell of galacto-N-biose in Example 3 (MTT assay). It is a figure which shows the influence with respect to the number of excitatory synapses (FIG. (A)) and the number of inhibitory synapses (FIG. (B)) of galacto-N-biose in Example 4. It is a figure which shows the protective effect with respect to the central nerve cell of galacto-N-biose and lacto-N-biose I in Example 5 (MTT assay).

Hereinafter, an example of an embodiment of a central nerve cell protecting agent to which the present invention is applied and a preventive or therapeutic agent for central neurodegenerative diseases will be described in detail. However, the present invention is not limited to the following embodiments.
In the present specification, a numerical range indicated using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.

<Central nerve cell protective agent>
The central nerve cell protective agent of the present embodiment is a compound represented by the following formula (1) or (2) or a pharmacologically acceptable salt thereof, or a prodrug thereof (hereinafter collectively referred to as “specific”). Abbreviated as “compound”.) As an active ingredient.

In the above formulas (1) and (2), R ¹ independently represents a halogen atom, an alkyl group, an alkenyl group, an aryl group, a cyano group, —N (R ² ) ₂ , —NHCOR ³ , or —OR ⁴ . Show.

When R ¹ is a halogen atom, examples of the halogen atom include a chlorine atom, a bromine atom, and an iodine atom.

When R ¹ is an alkyl group, the alkyl group preferably has 1 to 20 carbon atoms. The alkyl group may be linear, branched or cyclic. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, n-pentyl group, isopentyl group, cyclopentyl group, n-hexyl group, isohexyl group, cyclohexyl group. Groups and the like.

When R ¹ is an alkenyl group, the alkenyl group preferably has 2 to 20 carbon atoms. The alkenyl group may be linear or branched. Specific examples of the alkenyl group include vinyl group, allyl group, 3-butenyl group, isobutenyl group, 4-pentenyl group, isopentenyl group, 5-hexenyl group, isohexenyl group and the like.

When R ¹ is an aryl group, specific examples include a phenyl group and a naphthyl group.

When R ¹ is —N (R ² ) ₂ , each R ² independently represents a hydrogen atom or an alkyl group. When R ² is an alkyl group, the carbon number of the alkyl group is 1 to 18 is preferred. The alkyl group may be linear, branched or cyclic. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, n-pentyl group, isopentyl group, cyclopentyl group, and n-hexyl group. , Isohexyl group, cyclohexyl group and the like. When both R ² is an alkyl group, R ² may be bonded to each other to form a ring structure. Examples of the group formed by bonding R ² together include a piperidino group.

When R ¹ is —NHCOR ³ , R ³ represents an alkyl group or an aryl group. When R ³ is an alkyl group, the alkyl group preferably has 1 to 15 carbon atoms. The alkyl group may be linear or branched. Examples of the aryl group for R ³ include a phenyl group and a naphthyl group.

When R ¹ is —OR ⁴ , R ⁴ represents a hydrogen atom, an alkyl group, an aralkyl group, an alkylcarbonyl group, an arylcarbonyl group, an alkylsulfonyl group, an arylsulfonyl group, or —Si (R ⁵ ) ₃ .

When R ⁴ is an alkyl group, the alkyl group preferably has 1 to 20 carbon atoms. The alkyl group may be linear or branched.

When R ⁴ is an aralkyl group, examples of the aryl portion of the aralkyl group include a phenyl group and a naphthyl group. Moreover, as for carbon number of the alkyl part of an aralkyl group, 1-10 are preferable. The alkyl moiety may be linear or branched. Specific examples of the aralkyl group include a benzyl group, a diphenylmethyl group, a trityl group, a phenethyl group, and a naphthylmethyl group.

When R ⁴ is an alkylcarbonyl group, the alkyl moiety of the alkylcarbonyl group preferably has 1 to 15 carbon atoms. The alkyl moiety may be linear or branched. Specific examples of the alkylcarbonyl group include acetyl group, propanoyl group, butanoyl group, 2-methylpropanoyl group, pivaloyl group and the like.

When R ⁴ is an arylcarbonyl group, specific examples include a benzoyl group and a naphthoyl group.

When R ⁴ is an alkylsulfonyl group, the alkyl moiety of the alkylsulfonyl group preferably has 1 to 10 carbon atoms. The alkyl moiety may be linear or branched. Specific examples of the alkylsulfonyl group include a methylsulfonyl group, an ethylsulfonyl group, an n-propylsulfonyl group, and an isopropylsulfonyl group.

When R ⁴ is an arylsulfonyl group, specific examples include a benzenesulfonyl group and a naphthalenesulfonyl group.

When R ⁴ is a group represented by —Si (R ⁵ ) ₃ , each R ⁵ independently represents an alkyl group or an aryl group. The number of carbon atoms of the alkyl group in R ⁵ is 1-6 are preferable. The alkyl group may be linear or branched. In addition, examples of the aryl group for R ⁵ include a phenyl group and a naphthyl group. Specific examples of the group represented by —Si (R ⁵ ) ₃ include trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, triisopropylsilyl group, tert-butyldiphenylsilyl group and the like.

The alkyl part or aryl part contained in R ¹ may have a substituent.
For example, the alkyl moiety contained in R ¹ may have a halogen atom as a substituent.
Moreover, the aryl part of the aralkyl group in R ⁴ includes, as a substituent, an alkyl group having 1 to 4 carbon atoms, an aryl group (such as a phenyl group or a naphthyl group), a halogen atom, a nitro group, a cyano group, —COOR ⁶ , — It may have OR ⁶ , —NR ⁷ R ⁸ , or —NR ⁹ COR ¹⁰ . R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ each independently represent an alkyl group having 1 to 10 carbon atoms or an aryl group (phenyl group, naphthyl group, etc.)
Further, arylcarbonyl group or arylsulfonyl group in R ⁴ are as substituents, which may have an alkyl group, a nitro group, or a trifluoromethyl group having 1 to 5 carbon atoms.

The compound represented by the above formula (1) or (2) may be in the form of a pharmacologically acceptable salt. For example, when the compound represented by the above formula (1) or (2) has a basic functional group, it is in the form of a salt with an inorganic acid such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid or phosphoric acid. In the form of a salt with an organic acid such as acetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid There may be.
The “pharmacologically acceptable salt” includes solvates with pharmacologically acceptable solvents such as water and ethanol.

The compound represented by the above formula (1) or (2) or a pharmacologically acceptable salt thereof may be in the form of a prodrug. As used herein, “prodrug” means a compound that is converted into a compound represented by the above formula (1) or (2) or a pharmacologically acceptable salt thereof in vivo.
For example, prodrugs include compounds in which the hydroxy group is acylated, alkylated, phosphorylated, or borated.
When the compound represented by the above formula (1) or (2) has an amino group, examples of the prodrug include compounds in which the amino group is acylated, alkylated, or phosphorylated.

Among the compounds represented by the above formula (1) or (2), R ¹ is a hydroxy group (galacto-N-biose or lacto-N-biose I) as a commercially available product, It can be produced by the method described in JP-A-2008-154495. In addition, the specific compounds other than galacto-N-biose and lacto-N-biose I use galacto-N-biose or lacto-N-biose I as starting materials, and carry out steps such as etherification and esterification according to conventional methods. It can be manufactured by going through.

  The compound represented by the above formula (1) or (2) or a pharmacologically acceptable salt thereof exhibits a protective action against central nerve cells, and particularly suppresses nerve cell death based on glutamate excitotoxicity. it can. For this reason, a central nerve cell protective agent can be manufactured by using the said specific compound. The central nerve cell protecting agent may be used for pharmaceutical use or may be used for uses other than medicine (research use etc.).

  The central nerve cell protective agent may contain components other than the specific compound according to the use mode. For example, the central nerve cell protective agent may contain an organic or inorganic carrier commonly used as a pharmaceutical material. This carrier is used as an excipient, lubricant, binder, disintegrant, etc. in solid preparations, and as a solvent, solubilizer, suspending agent, isotonic agent, buffering agent, etc. in liquid preparations. Blended. The central nerve cell protective agent may contain formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like. In addition, in order to improve the permeability of the blood brain barrier, the central nerve cell protective agent may be one in which the specific compound is encapsulated in a liposome or the like.

  When the central nerve cell protective agent is used for pharmaceutical use, the dosage form of the central nerve cell protective agent is not particularly limited. The dosage form of the central nerve cell protective agent includes tablets, capsules, granules, powders, troches, syrups, emulsions, suspensions, films, etc .; injections, external preparations, suppositories, pellets Nasal preparations, pulmonary preparations (inhalation preparations), parenteral preparations such as eye drops, and the like.

  When the central nerve cell protective agent is used for pharmaceutical use, the dose of the central nerve cell protective agent is appropriately determined according to the administration subject, administration route, target disease, symptom, and the like.

<Preventive or therapeutic agent for central neurodegenerative diseases>
The preventive or therapeutic agent for central nervous degenerative disease of this embodiment contains the specific compound as an active ingredient. Since the specific compound is the same as that of the central nerve cell protective agent, detailed description thereof is omitted.

As described above, the compound represented by the above formula (1) or (2) or a pharmacologically acceptable salt thereof exhibits a protective action on central nerve cells, and in particular, nerve cell death based on glutamate excitotoxicity. Can be suppressed. For this reason, the preventive or therapeutic agent of a central nervous degenerative disease can be manufactured by using the said specific compound.
“Prevention” includes not only the onset of central neurodegenerative diseases but also the delay of onset. “Treatment” includes not only eliminating or reducing symptoms caused by central nervous degenerative diseases, but also suppressing the degree of progression of symptoms.

  The central neurodegenerative disease is preferably a disease involving glutamate excitotoxicity, and specifically, ischemic nerves such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and cerebrovascular disorder. Disorders, cerebellar ataxia, AIDS encephalopathy and the like.

  The preventive or therapeutic agent for central neurodegenerative diseases may contain components other than the specific compound. For example, the preventive or therapeutic agent for central neurodegenerative diseases may contain an organic or inorganic carrier commonly used as a pharmaceutical material. This carrier is used as an excipient, lubricant, binder, disintegrant, etc. in solid preparations, and as a solvent, solubilizer, suspending agent, isotonic agent, buffering agent, etc. in liquid preparations. Blended. In addition, the preventive or therapeutic agent for central neurodegenerative diseases may contain formulation additives such as preservatives, antioxidants, coloring agents, and sweeteners. In order to improve the permeability of the blood brain barrier, the preventive or therapeutic agent for central neurodegenerative diseases may be one in which the specific compound is encapsulated in liposomes or the like.

  The dosage form of the preventive or therapeutic agent for central neurodegenerative diseases is not particularly limited. As a dosage form of a preventive or therapeutic agent for central neurodegenerative diseases, oral preparations such as tablets, capsules, granules, powders, troches, syrups, emulsions, suspensions, films, etc .; injections, external preparations, Suppositories, pellets, nasal preparations, pulmonary preparations (inhalants), parenteral preparations such as eye drops, and the like.

  The dosage of the preventive or therapeutic agent for central neurodegenerative diseases is appropriately determined according to the administration subject, administration route, target disease, symptoms and the like.

  A method for preventing or treating a central neurodegenerative disease is provided by administering a prophylactic or therapeutic agent for a central neurodegenerative disease to a subject. That is, a method for preventing or treating a central neurodegenerative disease comprising administering a pharmaceutical composition containing the specific compound as an active ingredient is provided.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to an Example.

[Example 1: Confirmation of protective effect on central nerve cells of galacto-N-biose (GNB) (MTT assay)]
After anesthetizing embryonic day 18 Wistar rats, the brain was removed by cutting the head, and the removed brain was ice-cooled HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] It was immersed in an ethanesulfonic acid) buffered salt solution (manufactured by Life Technologies Japan). The cortical tissue was treated with papain (Sigma-Aldrich Japan), the tissue was subdivided, and the cells were seeded in a 96-well plate coated with polyethyleneimine (Sigma-Aldrich Japan). Then, 37 ° C., under 5 vol% CO _2, were cultured in B-27 in supplement (manufactured by Life Technologies Japan) and GlutaMAX (Life Technologies Japan Ltd.) Neurobasal medium supplemented with (manufactured by Life Technologies Japan).

  On the 15th day after the start of the culture, a GNB solution in which GNB was dissolved in dimethyl sulfoxide (DMSO) was added to the medium so that the final concentration of GNB was 0.1 μM, and cultured for 21 hours. Thereafter, an aqueous glutamic acid solution was added to the medium so that the final concentration of glutamic acid was 30 μM, and further cultured for 3 hours. The cells after culturing are referred to as “GNB administration group”. In addition, a control group to which the same amount of DMSO as the GNB administration group was added instead of the GNB solution, and a control group to which the same amount of DMSO and water as the GNB administration group were added instead of the GNB solution and glutamic acid aqueous solution were prepared.

Then, MTT assay was performed about the cell of the GNB administration group and the control group using the MTT cell proliferation assay kit (made by Millipore). Specifically, a solution in which 5 mg / mL MTT (3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide) was dissolved in PBS (phosphate buffered saline). , MTT was added to the medium so that the final concentration was 50 μg / mL, and cultured under conditions of 37 ° C. and 5% by volume CO ₂ for 1 hour. Thereafter, the medium was removed and 100 μL of DMSO was added to dissolve the formazan. Then, using a microplate reader (manufactured by Molecular Devices), the absorbance at a wavelength of 570 nm was measured as a control wavelength of 630 nm, and the viability of the cells was determined.

The cell viability measured in the MTT assay is shown in FIG. In FIG. 1, the survival rate is indicated by “average value ± standard error”. However, the average value of each group is normalized so that the average value in the control group to which DMSO and water are added instead of the GNB solution and the glutamic acid aqueous solution is 1.0. The number n of samples in the control group is n = 18, and the number n of samples in the GNB administration group is n = 12.
As can be seen from FIG. 1, when glutamic acid (30 μM) was added, the cell viability was significantly reduced compared to the case where glutamic acid was not added (p <0.01). However, when GNB (0.1 μM) was added in addition to glutamic acid (30 μM), the cell viability increased significantly compared to the case where GNB was not added (p <0.01). From this result, it can be seen that GNB exhibits a protective effect on central nerve cells.

[Example 2: Confirmation of protective effect on central nerve cells of galacto-N-biose (GNB) (nuclear staining assay)]
In the same manner as in Example 1, cells were collected from the brains of 18-day-old Wistar rats, and Neurobasal medium (Life Technologies) supplemented with B-27 supplement (Life Technologies Japan) and GlutaMAX (Life Technologies Japan) was added. In Japan).

  On the 15th day after the start of the culture, a GNB solution in which GNB was dissolved in DMSO was added to the medium so that the final concentration of GNB was 0.1 μM and cultured for 21 hours. After removing the medium and treating the cells with a PBS solution containing 0.01% by mass of DAPI (4 ′, 6-diamidino-2-phenylindole) (manufactured by Dojindo Laboratories), the cells were treated with Washed with PBS. The removed medium was returned, and an aqueous glutamic acid solution was added to the medium so that the final concentration of glutamic acid was 30 μM, followed by further culturing for 3 hours. The cells after culturing are referred to as “GNB administration group”. In addition, a control group to which the same amount of DMSO as the GNB administration group was added instead of the GNB solution, and a control group to which the same amount of DMSO and water as the GNB administration group were added instead of the GNB solution and glutamic acid aqueous solution were prepared.

  Thereafter, 0.05% by mass of PI (propidium iodide) (AnaSpec) was added to the cells and treated for 10 minutes. The cells after PI treatment were washed with PBS and fixed with 0.1 M phosphate buffer containing 4% by mass of paraformaldehyde. Mounted with water-based mounting medium for fluorescent tissue staining (Fluoromount / Plus, manufactured by Diagnostic BioSystems) containing anti-fading agent on fixed cells, and equipped with a digitally cooled CCD (Charge Coupled Device) camera (CoolSNAP HQ2, manufactured by Photometrics) Fluorescence images were obtained using a fluorescent microscope (Eclipse TE2000-E, manufactured by Nikon Corporation). Cells that were nuclear-stained with DAPI (DAPI-positive cells) were dead cells before treatment with glutamic acid, and thus were excluded when measuring the number of cells. Dead cells were confirmed not only by the cell shape but also by the standard that nuclei were stained with PI and not DAPI. Viable cells were confirmed based on the fact that nuclei were not stained by either PI or DAPI. Then, the survival rate was determined by dividing the number of living cells by the total number of cells (excluding DAPI positive cells).

The cell viability measured in the nuclear staining assay is shown in FIG. In FIG. 2, the survival rate is indicated by “average value ± standard error”. The sample number n of the control group to which DMSO and water were added instead of the GNB solution and the glutamic acid aqueous solution was n = 20, and the sample number n of the control group to which DMSO was added instead of the GNB solution was n = 21. The sample number n in the administration group is n = 12.
As can be seen from FIG. 2, when glutamic acid (30 μM) was added, the cell viability decreased significantly compared to the case where glutamic acid was not added (p <0.01). However, when GNB (0.1 μM) was added in addition to glutamic acid (30 μM), the cell viability increased significantly compared to the case where GNB was not added (p <0.01). From this result, it can be seen that GNB exhibits a protective effect on central nerve cells.

[Example 3: Confirmation of toxicity to central nerve cells by long-term exposure to galacto-N-biose (GNB) (MTT assay)]
In the same manner as in Example 1, cells were collected from the brains of 18-day-old Wistar rats, and Neurobasal medium (Life Technologies) supplemented with B-27 supplement (Life Technologies Japan) and GlutaMAX (Life Technologies Japan) was added. In Japan).

  On the 15th day after the start of the culture, a GNB solution in which GNB was dissolved in DMSO was added to the medium so that the final concentration of GNB was 0.1 μM, 1.0 μM, 10 μM, or 100 μM, and cultured for 14 days. After the addition of the GNB solution, the MTT assay was performed in the same manner as in Example 1 using the MTT cell proliferation assay kit (manufactured by Millipore) at the 1st, 2nd, 5th, and 14th day. went.

The cell viability measured in the MTT assay is shown in FIG. In FIG. 3, the survival rate is indicated by “average value ± standard error”. However, the average value of each group is normalized so that the average value in the control group to which the same amount of DMSO as the GNB administration group is added instead of the GNB solution is 1.0. The number n of samples in the control group and the GNB administration group is n = 9.
As can be seen from FIG. 3, when the concentration of GNB was 0.1 μM to 100 μM, the survival rate did not show a significant decrease. From this result, it can be seen that when the concentration of GNB is in the range of 0.1 μM to 100 μM, it exhibits no toxicity to central nerve cells for at least 14 days.

[Example 4: Confirmation of influence on synaptic binding by galacto-N-biose (GNB)]
In the same manner as in Example 1, cells were collected from the brains of 18-day-old Wistar rats, and Neurobasal medium (Life Technologies) supplemented with B-27 supplement (Life Technologies Japan) and GlutaMAX (Life Technologies Japan) was added. In Japan).

On the 15th day after the start of the culture, a GNB solution in which GNB was dissolved in DMSO was added to the medium so that the final concentration of GNB was 0.1 μM, 1.0 μM, 10 μM, or 100 μM, and cultured for 2 days. Thereafter, the cells were fixed with 0.1 M phosphate buffer containing 4% by mass of paraformaldehyde, washed with PBS, and then blocked with 2 × PBS solution containing 5% by mass of normal donkey serum. After blocking, each sample was reacted with the primary antibody at 4 ° C. overnight, washed with PBS, and then reacted with the secondary antibody at room temperature (25 ° C.) for 1 hour. After washing each sample with PBS, cells were mounted with a fluorescent tissue staining aqueous mounting medium (Fluoromount / Plus, manufactured by Diagnostic BioSystems) containing a fading inhibitor, and equipped with a digitally cooled CCD camera (SPOT, manufactured by Diagnostic Instruments) A fluorescence image was obtained using a fluorescence microscope (Eclipse E800, manufactured by Nikon Corporation).
As the primary antibody, mouse anti-vGluT1 antibody (Synaptic Systems), rabbit anti-vGAT antibody (Synaptic Systems), or chicken anti-MAP2 antibody (Merck Millipore) was used. vGluT1 is a marker for excitatory synapses, vGAT is a marker for inhibitory synapses, and MAP2 is a marker for identifying dendrites. As a secondary antibody, a secondary antibody (manufactured by Life Technologies Japan) labeled with Alexa dye was used.

The number of excitatory synapses and the number of inhibitory synapses per 20 μm of dendrites are shown in FIGS. 4A and 4B, respectively. In FIG. 4A, the number of samples n in the group in which the concentration of GNB is 0 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM is n = 29, n = 16, n = 15, n = 8, n = 9. In FIG. 4B, the number of samples n in the group in which the concentration of GNB is 0 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM is n = 11, n = 10, n = 11, and n = 13, n = 12.
As can be seen from FIGS. 4A and 4B, when the concentration of GNB was 0.1 μM to 100 μM, the number of excitatory synapses and the number of inhibitory synapses showed no significant change. From this result, it can be seen that when the concentration of GNB is in the range of 0.1 μM to 100 μM, the synaptic binding is not affected.

[Example 5: Confirmation of protective effect on central nerve cells of galacto-N-biose (GNB) and lacto-N-biose I (LNB) (MTT assay)]
In the same manner as in Example 1, cells were collected from the brains of 18-day-old Wistar rats, and Neurobasal medium (Life Technologies) supplemented with B-27 supplement (Life Technologies Japan) and GlutaMAX (Life Technologies Japan) was added. In Japan).

  On the 15th day after the start of the culture, a GNB solution or LNB solution in which GNB or LNB was dissolved in DMSO was added to the medium so that the final concentration of GNB or LNB was 10 μM, and cultured for 21 hours. The medium was removed, and the cells were treated with a PBS solution containing 0.01% by mass of DAPI (manufactured by Dojindo Laboratories) for 1 minute, and then the cells were washed with PBS. The removed medium was returned, and an aqueous glutamic acid solution was added to the medium so that the final concentration of glutamic acid was 30 μM, followed by further culturing for 3 hours. The cell group to which the GNB solution is added is referred to as “GNB administration group”, and the cell group to which the LNB solution is added is referred to as “LNB administration group”. Also, a control group to which the same amount of DMSO as the GNB administration group or LNB administration group is added instead of the GNB solution or LNB solution, and a GNB administration group or LNB administration group instead of the GNB solution, LNB solution, and glutamic acid aqueous solution. A control group was prepared with the amount of DMSO and water added.

  Thereafter, the MTT assay was performed in the same manner as in Example 1 using the MTT cell proliferation assay kit (manufactured by Millipore) for the cells in the GNB administration group, the LNB administration group, and the control group.

The cell viability measured by the MTT assay is shown in FIG. In FIG. 5, the survival rate is indicated by “mean value ± standard error”. However, the average value of each group is normalized so that the average value in the control group to which DMSO and water are added instead of the GNB solution, the LNB solution, and the glutamic acid aqueous solution is 1.0. The number n of samples in the control group is n = 30, the number n of samples in the GNB administration group is n = 20, and the number of samples n in the LNB administration group is n = 20.
As can be seen from FIG. 5, when glutamic acid (30 μM) was added, the cell viability decreased compared to the case where glutamic acid was not added. However, when GNB (10 μM) or LNB (10 μM) was added in addition to glutamic acid (30 μM), the cell viability increased compared to the case where GNB or LNB was not added. From this result, it can be seen that, like GNB, LNB also exhibits a protective effect on central nerve cells.

Claims (3)

A central nerve cell protecting agent comprising a compound represented by the following formula (1) or (2) or a pharmacologically acceptable salt thereof, or a prodrug thereof as an active ingredient.

(In the formula, ^{R 1} are each independently a halogen atom, an alkyl group, an alkenyl group, an aryl group, a cyano _{^{group, -N (R 2) 2,}} -NHCOR 3, or independently .R ² is showing the -OR ⁴ , if .R ² represents a hydrogen atom or an alkyl group is either an alkyl group, which may form a ring structure by bonding R ² together .R ³ is an alkyl group or an aryl group. R ⁴ represents a hydrogen atom, an alkyl group, an aralkyl group, an alkylcarbonyl group, an arylcarbonyl group, an alkylsulfonyl group, an arylsulfonyl group, or —Si (R ⁵ ) _3. Each R ⁵ independently represents an alkyl group or Represents an aryl group.) A prophylactic or therapeutic agent for central neurodegenerative diseases comprising a compound represented by the following formula (1) or (2) or a pharmacologically acceptable salt thereof, or a prodrug thereof as an active ingredient.

(In the formula, ^{R 1} are each independently a halogen atom, an alkyl group, an alkenyl group, an aryl group, a cyano _{^{group, -N (R 2) 2,}} -NHCOR 3, or independently .R ² is showing the -OR ⁴ , if .R ² represents a hydrogen atom or an alkyl group is either an alkyl group, which may form a ring structure by bonding R ² together .R ³ is an alkyl group or an aryl group. R ⁴ represents a hydrogen atom, an alkyl group, an aralkyl group, an alkylcarbonyl group, an arylcarbonyl group, an alkylsulfonyl group, an arylsulfonyl group, or —Si (R ⁵ ) _3. Each R ⁵ independently represents an alkyl group or Represents an aryl group.)   The preventive or therapeutic agent for central neurodegenerative disease according to claim 2, wherein the central neurodegenerative disease is a disease involving glutamate excitotoxicity.