JP2005126337A - Cranial nerve cell activator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new cranial nerve cell activator which reduces side effects. <P>SOLUTION: The cranial nerve cell activator contains a sugar compound having GalNAc (N-acetyl-D-galactosamine) which exists in a mammal living body and is in a free state at the nonreducing end as an active ingredient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel cerebral nerve cell activator.

  Cell surface sugar chains are thought to activate intracellular information transmission systems and mediate intercellular information transmission (see Non-Patent Document 1). The glycolipid GM2 ganglioside (hereinafter sometimes abbreviated as “GM2”) is also one of the cell surface sugar chains having such biological activity. GM2 is characteristically expressed in normal dendritic extension sites in various animals and is associated with pathological dendritic growth in GM2 gangliosidosis, a disease in which GM2 accumulates pathologically (See Non-Patent Documents 2 and 3). A number of such reports suggest that GM2 is involved in normal or pathological dendritic growth.

However, the molecular mechanism of how GM2 causes dendritic growth was unknown. Therefore, the clarification of the function of GM2 in this mechanism and the mechanism of dendritic extension has been strongly demanded. If this mechanism was elucidated and it became possible to control the extension of dendrites, it was strongly expected to provide useful information for the treatment of neurological diseases, neurological disorders and the like.
On the other hand, regarding GM1 ganglioside, which is the same glycolipid as GM2 (hereinafter sometimes abbreviated as “GM1”), many administration experiments to brain disorder models and Parkinson's disease models have been reported (non-patent literature). 4). For example, when GM1 is administered to a model that has mechanically damaged the cerebral gray matter or a model that has artificially caused ischemic damage, reduction of neuronal atrophy and improvement of the amount of neurotransmitters are observed. It has been reported. In addition, it has been reported that when GM1 is administered to a Parkinson's disease model, recovery of the striatal dopamine amount is observed. However, in these reports, the function of GM1 in nerve cells and the mechanism thereof have not yet been elucidated, and it is not clear whether these functions are the same as the function of GM2 or the mechanism thereof. It was.

  The present inventors have previously conducted an experiment in which GM2 is added to cultured neurons in order to elucidate the function of GM2. As a result, it has been reported that thread-like projections are formed within a very short time of several minutes (see Non-Patent Document 5). In general, however, the formation of dendrites does not occur just because the formation of filamentous protrusions, and the mechanism of dendritic extension by GM2 has not been elucidated.

Therefore, the present inventors further have a synthetic sugar chain GalNAc ₁₀ (hereinafter, similarly, a saccharide) in which ten N-acetylgalactosamines (hereinafter sometimes abbreviated as “GalNAc”) are linked to cultured neurons. An experiment was conducted in which the number of chain linkages (represented by a subscript number) was added to cultured neurons. As a result, it was reported that dendritic elongation was observed within 2 to several days (see Non-Patent Document 6). However, elucidation of the mechanism of dendritic extension by this synthetic sugar chain has been insufficient. In addition, it has not been studied whether or not these effects can be obtained by using glycolipids other than GM2 and other sugar compounds, in particular, sugar compounds that are originally present in the human body. Analysis was desired.
Hakomori and Igarashi, J. Biochem., 118, 1091-1103 (1995) Purpura and Suzuki, Brain Res., 116, 1-21 (1976) Goodman and Walkley, Dev.Brain Res., 93, 162-171 (1996) Susumu Ando, “Brain function and gangliosides – New active substances in neurons”, published by Kyodo Publishing Co., Ltd., pages 142-155 (1997) Hidehide Higashi et al., 21st Annual Meeting of the Japanese Society of Carbohydrates, 73 pages (2000) Higashi Hideyoshi et al., 22nd Annual Meeting of the Japanese Society of Carbohydrates, 10 pages (2001)

  The present invention has been made to provide a novel brain nerve cell activator with few side effects.

As a result of diligent studies to achieve the above-mentioned problems, the present inventors have found that if a saccharide compound having GalNAc that is present in a mammal and is in a free state at the non-reducing end is used, the nerve cell has a dendritic shape. It has been found that extension and / or branching of the protrusion can be promoted. The present invention has been accomplished based on these findings.
That is, according to the present invention,
(1) a cranial nerve cell activator comprising, as an active ingredient, a sugar compound that is present in a mammal and has GalNAc in a free state at a non-reducing end,
(2) The cerebral nerve cell activator according to (1), wherein the sugar compound is a sugar chain part of GM2 ganglioside or asialo GM2 ganglioside,
(3) The cerebral nerve cell activator according to (1) or (2) above, wherein the activation of cerebral nerve cells causes elongation and / or branching of dendrites of the cerebral nerve cells,
(4) Dendritic extension and / or branching is based on the activation of protein kinase A, the brain nerve cell activator according to (3) above, and
(5) The cranial nerve cell activator according to any one of (1) to (4), wherein the cranial nerve cell activator is a therapeutic agent for cranial nerve disorders.

  ADVANTAGE OF THE INVENTION According to this invention, the brain nerve cell activator which is novel and has few side effects is provided. The cranial nerve cell activator is useful for treating cranial nerve disorders and the like in mammals such as humans.

Hereinafter, the present invention will be described in more detail.
In this specification, some sugar compounds may be abbreviated, and their chemical structural formulas are as follows. In the present specification, `` Gal '' is galactose, `` Glc '' is glucose, `` Man '' is mannose, `` Fru '' is fructose, `` GalNAc '' is N-acetylgalactosamine, `` GlcNAc '' is N-acetylglucosamine, “Neu5Ac” represents sialic acid (5-acetylneuraminic acid), respectively. If these sugars are present in the compound, they indicate the residue of those sugars. “Cer” represents ceramide.

Abbreviation Chemical structural formula
(1) GalNAc _n (GalNAc (α1-4)) _n-1 GalNAc (n is an integer from 1 to 100)
(2) Gb ₄ GalNAc (β1-3) Gal (α1-4) Gal (β1-4) GlcCer
(3) Gb ₅ GalNAc (α1-3) GalNAc (β1-3) Gal (α1-4) Gal (β1-4) GlcCer
(4) GD1a Neu5Ac (α2-3) Gal (β1-3) GalNAc (β1-4) (Neu5Ac (α2-3)) Gal (β1-4) GlcCer
(5) GD1b Gal (β1-3) GalNAc (β1-4) (Neu5Ac (α2-8) Neu5Ac (α2-3)) Gal (β1-4) GlcCer
(6) GD2 GalNAc (β1-4) (Neu5Ac (α2-8) Neu5Ac (α2-3)) Gal (β1-4) GlcCer
(7) GD3 Neu5Ac (α2-8) Neu5Ac (α2-3) Gal (β1-4) GlcCer
(8) Gg ₃ GalNAc (β1-4) Gal (β1-4) GlcCer
(9) GM1 Gal (β1-3) GalNAc (β1-4) (Neu5Ac (α2-3)) Gal (β1-4) GlcCer
(10) GM2 GalNAc (β1-4) (Neu5Ac (α2-3)) Gal (β1-4) GlcCer
(11) GM3 Neu5Ac (α2-3) Gal (β1-4) GlcCer
(12) GQ1b Neu5Ac (α2-8) Neu5Ac (α2-3) Gal (β1-3) GalNAc (β1-4) (Neu5Ac (α2-8) Neu5Ac (α2-3)) Gal (β1-4) GlcCer
(13) GT1b Neu5Ac (α2-3) Gal (β1-3) GalNAc (β1-4) (Neu5Ac (α2-8) Neu5Ac (α2-3)) Gal (β1-4) GlcCer

1. Sugar compound contained as an active ingredient in the brain nerve cell activator of the present invention The brain nerve cell activator of the present invention comprises GalNAc (N-acetylgalactosamine) which is present in a mammal and in a free state at the non-reducing end. It is characterized by containing a sugar compound having an active ingredient.
Mammals include, for example, humans, monkeys, cows, pigs, dogs, cats, rabbits, rats, mice and the like, with humans being particularly preferred. The cerebral nerve cell activator of the present invention uses such a saccharide compound that can be present in the living body of mammals as an active ingredient, so that the side effects when the drug is used in humans or other mammals are small. It has an excellent effect.
Examples of the sugar compound include sugar chains, glycolipids, sugar amino acids, glycoproteins, sugar nucleotides, proteoglycans and the like. In the present invention, among these, those having GalNAc existing in a mammal and having a free state at the non-reducing end are used.

  Here, the expression “in a free state” of GalNAc means that it can participate in a biological reaction without various obstacles and has a physiological activity. Specifically, for example, it means a state where GalNAc is not chemically or physically modified, a state where it is not sterically hindered, etc. You may be affected. More specifically, it is preferable that the acetylamino (—NHAc) group at position 2 and the hydroxyl (—OH) group at positions 3, 4, and 6 of GalNAc are in a free state.

  Here, the state having physiological activity is an activity that causes some reaction in the living body, and means a state having, for example, a reaction activity with a receptor. The state that is free to the extent that it has a reaction activity with the receptor is, for example, that the non-reducing terminal GalNAc has not been modified so as to impair the water solubility, and that it is involved in binding to the receptor. This means a spatially free state. For example, even if a small functional group such as a sulfate group is bonded to GalNAc at the non-reducing end, the water solubility of GalNAc is maintained, and there is no significant steric hindrance and physiological activity is not impaired. This can be used. However, for example, when a sugar such as Gal (galactose) or other large functional group is bound to GalNAc at the non-reducing end, the water solubility of GalNAc is reduced or the steric hindrance is caused. The reactivity may be reduced, and the physiological activity may be impaired. Such a sugar compound cannot be used as the sugar compound of the present invention.

  The steric hindrance as described above may be caused when a molecule having a large molecular weight is bound as a side chain to a molecule (sugar, amino acid, lipid, nucleotide, etc.) bound to the reducing end of GalNAc, or a charged molecule is It can also occur when they are connected. The molecule having a large molecular weight includes, for example, a molecule having a molecular weight of 500 or more. When two or more molecules are bonded, it can be determined by the total molecular weight. Generally, however, the bond distance is longer and the degree of freedom of the molecule is higher than that of a single molecule, and steric hindrance is likely to occur. Tend to be. Examples of the molecule having a charge include sialic acid, uronic acid, and sulfated sugar as sugars.

  The sugar compound contained in the cerebral nerve cell activator of the present invention having such properties activates cerebral nerve cells and promotes dendrite extension and / or branching. This action is caused by activation of calcium-independent adenylate cyclase by GalNAc in the free state of the non-reducing end of the sugar compound, followed by intracellular protein kinase A (hereinafter referred to as “PKA”). Is based on being activated. Intracellular PKA further activates cdc42, which is a GTPase with a low molecular weight, and this cdc42 promotes the formation of filamentous processes, and subsequently promotes the extension and / or branching of dendrites. That is, the saccharide compound used in the present invention is a saccharide compound having the ability to activate such a signal cascade among saccharide compounds existing in the living body of mammals.

  Therefore, for use in the brain nerve cell activator of the present invention, whether or not a certain sugar compound selected as having the above-mentioned characteristics can be used as the sugar compound of the present invention by the following method is more determined. It can be confirmed reliably. As a method for this, for example, using cultured nerve cells, the target sugar compound is added and cultured, and the physiological activity of the sugar compound is confirmed by analyzing reactions and changes of the cultured nerve cells. Methods and the like. More specifically, for example, methods using PKA activation, elongation and / or branching of filamentous and dendritic processes, and Gdcation of cdc42 as an index can be mentioned.

  For example, when PKA activation is used as an index, cultured nerve cells are treated by adding a sugar compound to the culture solution, and changes in cells stimulated by the sugar compound are analyzed. Examples of cultured neuronal cells used here include primary cultured neuronal cells, neuroblastoma and glioma hybridomas, neuroblastoma, and the like. Examples of primary cultured neurons include primary cultured neurons such as hippocampus, cerebellum, and cerebral cortex. As a hybridoma of neuroblastoma and glioma, for example, mouse neuroblastoma N18TG2 and rat glioma C6-BU-1 hybridoma NG108-15 cells (Hamprecht B. et al., Methods Enzymol. 109, 316-341 (1985); Amano T. et al., Exp. Cell Res., 85, 399-408 (1974)). As an analysis method, for example, as a method for directly analyzing PKA, an imaging method (Higashi et al., FEBS Lett., 414, 55-60 (1997)) and the like can be mentioned. Examples thereof include a method for measuring cAMP. Examples of cAMP measurement methods include imaging methods, enzyme immunoassays, radioimmunoassays, etc., all of which are commercially available kits (for example, The Biotrak cAMP EIA System (Amersham) in the case of enzyme immunoassays), etc. Can be used. Among these, a method of directly analyzing PKA by an imaging method is preferably used. As a result of the analysis, when activation of PKA is observed, it can be confirmed that the sugar compound can be used as the sugar compound of the present invention.

  In addition, when confirming the extension and / or branching of filamentous processes and dendrites as an index, as in the case of using the above-mentioned PKA activation as an index, the cultured nerve cells are stimulated with a target sugar compound. The resulting protrusion extension and / or branching may be observed. In general, the elongation of filamentous protrusions occurs in a very short time after stimulation, but it is preferable to further culture and observe the elongation and / or branching of dendrites. Further, dendrites may be observed to be elongated, but it is particularly preferable that both elongation and branching are observed. Examples of cultured neuronal cells used here include primary cultured neuronal cells, neuroblastoma and glioma hybridomas, neuroblastoma, and the like, as in the case of analysis of PKA. However, when observing dendritic extension and / or branching, primary cultured neurons are particularly preferably used. The observation of the filamentous protrusion can be performed by, for example, a staining method using a fluorescently labeled phalloidin. The phalloidin staining method is a suitable method for staining an actin polymer, and a fluorescent material used as a label may be selected from rhodamine, fluorescein, oregon green, and the like according to the purpose. The observation of dendrites is preferably performed after culturing for 3 days or more, more preferably 5 days or more. The observation can be performed by immunostaining with a fluorescently labeled phalloidin that stains an actin polymer, an anti-MAP2 antibody that specifically stains nerve cells, and the like. In the case of cerebellar Purkinje cells, methods such as immunostaining with an anti-calbindin antibody can also be used. What is necessary is just to select suitably from these methods according to the cultured nerve cell used.

  Furthermore, the GTP (guanosine 5′-triphosphate) conversion of cdc42 activated by PKA can also be used as an index. Since cdc42 in an active state in cells is bound to GTP (GTP binding type), it is determined whether or not it is activated by analyzing whether intracellular cdc42 is bound to GTP. can do. Preferably, the amount of GTP-linked cdc42 is analyzed for both the cells to which the target sugar compound is added and the cells to which the sugar compound is not added, and the results are compared. The Gdc42 can be analyzed for GTP using, for example, a commercially available kit (cdc42 Activation Assay Kit: manufactured by Upstate Biotechnology).

Hereinafter, the sugar compound thus selected will be described in more detail.
Examples of the sugar chain used as the sugar compound contained in the cerebral nerve cell activator of the present invention include those in which two or more sugars are bonded, and preferably three or more. The upper limit may be any length as long as the sugar chain has water solubility, but is preferably 100 or less, more preferably 50 or less, and particularly preferably 10 or less. Moreover, as long as it has water solubility and does not cause the steric hindrance, the structure may have a branch, and the constituent sugars may be modified.

  Examples of sugars contained in the living body include galactose (Gal), glucose (Glc), mannose (Man), fructose (Fru), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), sialic acid ( Neu5Ac) and the like. The sugar chains used in the present invention may be those in which these sugars are linked in any order as long as they are present in a mammal and have GalNAc in a free state at the non-reducing end.

  Specifically, for example, Gal, GalNAc, GlcNAc, or Man linked to the non-reducing terminal GalNAc (on the reducing terminal side) is preferred, and among them, Gal or GalNAc linked is more preferable. preferable. Here, for example, when Gal is bound as the second sugar, Glc or GlcNAc is preferred as the third sugar. The second Gal may be bound with sialic acid as a side chain, but when sialic acid becomes two molecules, steric hindrance as described above is likely to occur. Further, for example, when GalNAc is bound as the second sugar, Gal or GalNAc is preferable as the third sugar.

Specific examples of such a sugar chain include, for example, a sugar chain portion of GM2 ganglioside (hereinafter sometimes abbreviated as “GM2”), or asialo GM2 ganglioside (hereinafter referred to as “Asialo GM2” or “ The sugar chain part of Gg ₃ ”(sometimes abbreviated as“ Gg ₃ ”) is preferably used. As used herein, “asialo GM2 ganglioside” means sialic acid eliminated from GM2 ganglioside.

The glycolipid used as the sugar compound contained in the cerebral nerve cell activator of the present invention is any glycolipid that exists in the living body of mammals and has GalNAc in a free state at the non-reducing end. Even things can be used. The glycolipid is an amphiphilic substance having a sugar and a lipid in the molecule, and examples of the sugar or sugar chain contained therein include those described above. Examples of lipids include ceramide, acylglycerol, and alkylglycerol. Among the glycolipids composed of these combinations, for example, GM2, Asialo GM2 (Gg ₃ ) and the like are preferably used in the present invention.

  Any sugar amino acid may be used as long as it is a sugar amino acid present in a mammal and has GalNAc in a free state at the non-reducing end. For example, serine, threonine, or asparagine. For example, those in which a sugar or a sugar chain is bonded by an o-glycoside bond are preferably used. Examples of the sugar or sugar chain used here include those described above.

As the glycoprotein, any glycoprotein that exists in the living body of mammals and has a GalNAc in a free state at the non-reducing end can be used. Specifically, for example, as a constituent amino acid, Any protein or peptide containing a sugar amino acid as described above may be mentioned.
Examples of the sugar nucleotide include UDP-GalNAc (UDP: uridine 5′-diphosphate).

  As the proteoglycan, a proteoglycan that exists in a mammalian body and has GalNAc in a free state at a non-reducing end is used. Proteoglycan is a general term for a covalently bonded compound of glycosaminoglycan and protein, and is a biopolymer having a unique repeating structure. Since proteoglycan has a repeating structure, the type of sugar exposed at the non-reducing end may differ depending on the state in which synthesis stops. In the present invention, among proteoglycans such as chondroitin sulfate and dermatan sulfate, those having GalNAc at the non-reducing end are preferably used.

  Among such sugar compounds, the sugar compound used in the brain nerve cell activator of the present invention is preferably a sugar chain or a glycolipid, and particularly preferably a sugar chain. Sugar chains have advantages such as low immunogenicity compared to lipids, proteins, etc., high water solubility, difficulty in aggregation, and ease of handling, and administration to humans or other mammals. In particular, it can be expected to have particularly high effects and small side effects. Moreover, when this is removed from the reaction site, the reaction is reversible, and the reaction rate is fast, so that it has an advantage that it is easy to control in drug treatment.

  Furthermore, various sugar compounds as described above have local effects in the vicinity of nerve cell membranes. This is based on the fact that the sugar compound does not activate intracellular free PKA, but only activates PKA bound in the vicinity of the membrane. Since the effect of the cerebral nerve cell activator of the present invention is based on such a local action, the effect is exerted at a very low concentration and the influence on other signal cascades in the cell is small. It has the advantage that side effects are small. Specifically, the effective concentration of the sugar compound used in the present invention is, for example, about 0.5 to 200 nM, preferably about 1 to 100 nM when the sugar chain portion of GM2 or Asialo GM2 is used. The same applies to GM2.

  Any of the above sugar compounds can be obtained by means of synthesis or extraction / purification from mammals. As a synthesis method, it can be arbitrarily selected from commonly used methods known per se. Extraction and purification from mammals can also be carried out by arbitrarily selecting from commonly used methods known per se. For example, in the case of GM2, cows, pigs, horses, sheep, rabbits, etc. It can be obtained by extracting and purifying a mixture of gangliosides mainly from the brain and digesting it with sialidase, β-galactosidase or the like. By using a specific sialidase, asialo GM2 can be obtained in the same manner. Further, for example, the sugar chain portion of GM2 or asialo GM2 can be prepared by enzymatic digestion of GM2 or asialo GM2 thus obtained using endoglycoceramidase or the like.

  These sugar compounds may be prepared as derivatives as long as their physiological activity is maintained. The method for preparing the derivative can be carried out by arbitrarily selecting from commonly used methods known per se. The derivatization is preferably performed for the purpose of stabilizing the sugar compound used, improving water solubility, and the like. In addition, derivatization for improving the effectiveness when the sugar compound is administered to humans or other mammals or reducing side effects is also preferably performed. For example, derivatization for allowing the sugar compound administered by a method such as oral administration, intravenous injection, intramuscular injection, and intraperitoneal administration to reach the affected area through the blood-brain barrier after being taken into the blood Is mentioned. Specific examples of such derivatization include esterification and alkylation.

2. Utilization of the Brain Nerve Cell Activator of the Present Invention The brain nerve cell activator of the present invention containing the saccharide compound thus prepared is administered to the human or other mammal when the human or other mammal is administered. It has a prominent effect of activating cranial neurons in the brain and promoting dendritic extension and / or branching. As a result, differentiation and maturation of nerve cells can be promoted.

  That is, the cranial nerve cell activator of the present invention can be used as a therapeutic agent for cranial nerve disorders. Examples of cranial nerve disorders include damage due to accidents, cerebral infarction, ischemic damage due to cerebrovascular dementia, and the like, but can also be used for various diseases that cause degeneration of cranial nerve cells. Examples of such diseases include Parkinson's disease, Alzheimer's disease, epilepsy, depression, schizophrenia and the like. In the present specification, the term “brain neuropathy” includes such neuronal degeneration.

  When the cranial nerve cell activator of the present invention is used as a therapeutic agent for cranial nerve disorders, for example, a method of administering a relatively low volume over a long period of time is preferably used. By using such a technique, it is expected to steadily promote dendrite extension and / or branching, promote reconfiguration of the neural network, and improve cranial nerve damage. Of the various sugar compounds described above, it is particularly suitable for such long-term administration if a compound that exhibits a high effect at a very low concentration, has small side effects, and has a reversible reaction is selected.

3. Method for preparing brain nerve cell activator of the present invention The brain nerve cell activator of the present invention containing a sugar compound as described in detail in 1 above as an active ingredient can be prepared by a commonly used method known per se. .
The sugar compound contained in the brain nerve cell activator of the present invention may be physiologically acceptable salts, hydrates, solvates and the like. Examples of the salt of the sugar compound include pharmaceutically acceptable salts such as salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids. Examples include salt. Preferable examples of the salt with an inorganic base include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt and ammonium salt. Preferable examples of the salt with an organic base include, for example, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N′-dibenzylethylenediamine. And salt. Preferable examples of the salt with inorganic acid include salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Preferable examples of the salt with organic acid include formic acid, acetic acid, propionic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid. And salt. Preferable examples of the salt with basic amino acid include salts with arginine, lysine, ortinine and the like, and preferable examples with acidic amino acid include salts with aspartic acid, glutamic acid and the like.

  The sugar compound can be administered by itself to patients with cranial nerve disorders or the like, but can also contain one or more of these active ingredients. Moreover, it is preferable to prepare and administer a pharmaceutical composition using a pharmacologically acceptable additive for pharmaceutical preparation. For example, tablets, capsules, granules, fine granules, powders, pills, microcapsules, liposome preparations, troches, sublinguals, liquids, elixirs, emulsions, suspensions, etc. As an injection, a suppository, an ointment, a patch or the like manufactured orally as a sterile aqueous liquid or oily liquid. These include, for example, the compound in admixture in a unit dosage form required for accepted pharmaceutical practice, along with physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, and the like, It can be produced using methods well known in the art such as filling or tableting. The amount of active ingredient in these pharmaceutical compositions is such that an appropriate volume within the indicated range is obtained.

  For example, additives that can be mixed into tablets, capsules and the like include binders such as gelatin, corn starch, tragacanth and gum arabic, excipients such as crystalline cellulose, fillers such as cellulose, mannitol, and lactose. , Starch, polyvinylpolypyrrolidone, starch derivatives, disintegrating agents such as sodium starch glycolate, lubricants such as magnesium stearate, wetting agents such as sodium lauryl sulfate, sucrose, lactose Alternatively, a sweetening agent such as saccharin, a flavoring agent such as peppermint, red mono oil or cherry is used. In addition, tablets can be coated with an enteric coating agent or the like as necessary. As for the capsule, a liquid carrier such as fats and oils can be further contained in the additive.

  In addition, for example, sterile pharmaceutical compositions used as injections should be formulated in accordance with normal pharmaceutical practice such as dissolving or suspending active substances in vehicles such as water for injection, naturally produced vegetable oils such as sesame oil and coconut oil, etc. Can do. As an aqueous solution for injection, for example, an isotonic solution (D-sorbitol, D-mannitol, sodium chloride, etc.) containing physiological saline, glucose and other adjuvants, etc. are used. , Alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.), nonionic surfactant (polysorbate 80TM, HCO-50 etc.) etc. can be used together. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and can be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. The above pharmaceutical composition includes, for example, a buffer (phosphate buffer, sodium acetate buffer, etc.), a soothing agent (benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (human serum albumin, polyethylene, etc.). Glycol, etc.), preservatives (benzyl alcohol, phenol, etc.), antioxidants and the like may be incorporated. The aqueous liquid for injection thus prepared is usually produced by dissolving the compound in a medium as described above and sterilizing by filtration, and then filling it into a suitable sterilized vial or ampoule. To enhance stability, the water in the vial may be removed under vacuum after the pharmaceutical composition has been frozen. An oily liquid can be produced in substantially the same manner as in the case of an aqueous liquid, but can be preferably produced by suspending the compound in the medium and then sterilizing with ethylene oxide or the like.

Since the pharmaceutical composition thus obtained is safe and has low toxicity, it can be administered, for example, as a preventive or therapeutic agent for cranial nerve disorders and the like to humans and mammals other than humans. The dose of the compound contained in the composition varies depending on the target disease, symptom, target organ, administration target, administration method, etc., for example, when the compound is orally administered for the purpose of treating cranial nerve disorders, In general, for an adult (with a body weight of 60 kg), the dose is about 0.01 to 10 mg, preferably about 0.1 to 5 mg, more preferably about 0.1 to 2 mg per day. In addition, when administered parenterally, for example, when administered to an ordinary adult (as 60 kg) in the form of an injection, about 0.01 to 3 mg per day, preferably about 0.01 to 2 mg, more preferably about It is preferable to administer about 0.01 to 1 mg by intravenous injection. Moreover, it is desirable to administer the daily dose in one to several times. In the case of other animals, an amount converted per 60 kg can be similarly administered.
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited at all by these Examples.

In order to elucidate the phenomenon caused by the addition of certain sugar compounds to cultured cells, we first analyzed changes in intracellular PKA.
(1) Cultured cells Cultured cells were prepared as follows.
Rat hippocampal primary cultured neurons were cultured and prepared as described in Kudo and Ogura, Br. J. Pharmacol., 89, 191-198 (1986). NG108-15 cells, which are hybridomas of mouse neuroblastoma N18TG2 and rat glioma C6-BU-1, were cultured according to the method described in Higashi et al., FEBS Lett., 414, 55-60 (1997). Prepared.

(2) Sugar compound The sugar compound was prepared as follows.
i) Sugars and carbohydrates
GM2 manufactured by Yatron (GSL-102A,> 99% pure) or Sigma (G8397, approx. 95% pure) was purchased and used. Asialo GM2 (Gg ₃ ) was provided by Dr. Yoshio Hirabayashi of RIKEN. Other glycolipids (GM1, GD1a, GD1b, GT1b) were obtained and prepared in the same manner as previously reported (Higashi et al., FEBS Lett., 414, 55-60 (1997), Higashi et al., Biochem. Biophys. Acta, 963, 333-339 (1988)).

The sugar chain part of GM2 was prepared using endoglycoceramidase (Higashi et al., Anal. Biochem., 186, 355-362 (1990)). GalNAc (α1-4) oligomers (GalNAc (α1-4) GalNAc, GalNAc ₅ , or GalNAc ₁₀ ) were purchased from Funakoshi, which was prepared from the polysaccharide produced by the mold paecilomyces sp.I-1 (manufactured by Higeta Shoyu Co., Ltd.) : Takagi H. et al., Agric. Biol. Chem., 49, 3159-3164 (1985)). GlcNAc (β1-4) pentamer (GlcNAc ₅ ) in which five GlcNAc were linked by β1-4 bonds was purchased from Wako Pure Chemical Industries.

Gb ₅ (Forssman antigen) was prepared using sheep erythrocytes according to the method described in Naiki M. et al., Jpn. J. Exp. Med., 42, 205-19 (1972). Gb ₄ was prepared in the same manner as Gb ₅ using human erythrocytes. GD2 was prepared from bovine brain according to the method described in Hirabayashi Y. et al., J. Chromatogr., 445, 377-84 (1988).
In addition, since the ceramide part is not uniform in glycolipid, the nM display of each concentration is an approximate number.

ii) Neoglycoprotein As an oligosaccharide-containing neoglycoprotein, a polyglycoglycoprotein having about 200 molecules of GalNAc ₅ , a neoglycoprotein prepared with maltopentaose or isomaltopentaose, and a neoglycoprotein synthesized with sialylneolactotetraose Using. Maltopentaose and isomaltopentaose were manufactured by Seikagaku Corporation. The sialyl neolactotetraose manufactured by Sigma (A4814) was used. These were prepared according to previous reports (Hashimoto et al., J. Biochem., 123, 468-478 (1998)).

(3) Other reagents
DRII manufactured by Dojindo Laboratories Co., Ltd. was used.
P32-B was prepared by synthesizing a peptide (SEQ ID NO: 1) at the PKA phosphorylation site of DAPP32 and fluorescently labeling it with BODIPY FL C1-IA (Molecular Probes) (Japanese Patent Laid-Open No. 2001-19700). Publication).

(4) PKA activity analysis method
Analysis of PKA activity was performed using the imaging method detailed in Higashi et al., FEBS Lett., 414, 55-60 (1997). Various saccharide compounds prepared in (2) above were added to NG108-15 cells prepared in (1) and rat hippocampal primary cultured neurons, and changes in PKA activity were examined.
The imaging method was performed using two types of fluorescent peptide substrates, DRII and P32-B. These are all peptides having a PKA-specific phosphorylation site in the amino acid sequence. DRII is a fluorescent peptide substrate in which DACM (dimethylaminocoumarin maleimide) is bound to a peptide (SEQ ID NO: 2) whose lipid solubility is enhanced by modifying a part of the sequence (RII) in the regulatory domain of PKA. P32-B is a fluorescent peptide substrate having a sequence of the PKA phosphorylation site of a known protein DAPP32 that is phosphorylated by PKA.

The various sugar compounds prepared in (1) above were prepared from a stock solution of an aqueous solution having a 1000-fold concentration. Addition concentration of each sugar compounds, GM2 is 72 nM, the carbohydrate moiety GM2 is 24 nM, Gg ₃ is 9 nM, GM1 is 65 nM, GD1a is 54 nM, GD1b is 54 nM, GT1b is 47 nM, Gb ₅ is 6.5 nM, GalNAc ₁₀ is 49 nM and GalNAc ₅ were 97 nM. The neoglycoprotein produced with Maltopentaose or isomaltopentaose was 200 pM, the neoglycoprotein produced with GalNAc ₅ was 20 or 200 pM, and the neoglycoprotein produced with sialylneolactotetraose was 200 pM. Gb ₄ was 75 nM, GD2 was 60 nM, GalNAc (α1-4) GalNAc was 240 nM, and GlcNAc ₅ was 97 nM.

First, each cell was mixed with an isotonic buffer solution containing 25 μg / ml DRII or 6 μg / ml P32-B (BSS: pH 7.3, 130 mM NaCl, 5.4 mM KCl, 20 mM HEPES, 5.5 mM glucose, 0.8 mM MgSO ₄ , The substrate was incorporated by holding at 25 ° C. for 30 minutes in 1.8 mM CaCl ₂ ). The cells were stabilized in BSS at 30 ° C. (circulated at 1.4 ml / min), and each sugar compound was added to BSS to stimulate each cell. The fluorescence of cells was excited at 400 nm for DRII and 480 nm for P32-B, and image analysis (Argus100 or 50: manufactured by Hamamatsu Photonics) was performed. After stimulation for 2 minutes, BSS containing no sugar compound was returned. The liquid volume of the petri dish for measurement was kept at 1 ml.

  Furthermore, in order to confirm whether the substrate taken up by the cells was phosphorylated, a 50% acetic acid extract of each cell was prepared and analyzed by HPLC (Higashi et al., FEBS Lett., 414, 55-60 (1997)). For the 50% acetic acid extract of each cell, after removing the extracellular fluid, 0.4 ml of 50% acetic acid was added, the insoluble matter in the extract was centrifuged, and the precipitate was removed. 100 μl of this was collected and subjected to HPLC. The peak obtained as a result of the analysis was compared between cells with and without the addition of a sugar compound, and when the peak showed a phosphorylation-specific shift (migration), it was taken up by the cell It was determined that the fluorescent peptide substrate was phosphorylated.

(5) Analysis results First, as a result of analysis using two different types of fluorescent peptide substrates having PKA-specific phosphorylation sites, the same results were observed regardless of which was stimulated by various sugar compounds. The activated kinase was confirmed to be PKA. Moreover, it was confirmed by the analysis by HPLC that the peptide taken up into the cell was phosphorylated in any substrate. Furthermore, it was found that there was no difference in response to various sugar compounds between NG108-15 cells and rat hippocampal primary cultured neurons. PKA activation occurred in the vicinity of the cell membrane, and PKA activation was confirmed within a few seconds after the addition of the sugar compound, and it returned to the original level by removing the sugar chain.

Among the various sugar compounds used in the addition experiment, it was found that some activated intracellular PKA and some did not. First, GM2 having β-GalNAc at the non-reducing end, the sugar chain portion of GM2, and asialo GM2 (Gg ₃ ) all significantly activated PKA. These were effective even at very low concentrations of 7-70 nM. On the other hand, gangliosides of GM1, GD1a, GD1b, and GT1b that do not have GalNAc at the non-reducing end had no effect of activating PKA. From these results, it was confirmed that the sugar compound having GalNAc at the non-reducing end has a remarkable effect. Further, since no effect of activating PKA was found in GM1, it was found that the activity was inhibited when the -OH group at the 3-position of GalNAc at the non-reducing end was substituted with Gal.

Furthermore, Gb ₅ having α-GalNAc at the non-reducing end, GalNAc α1-4 decamer (GalNAc ₁₀ ) and GalNAc α1-4 pentamer (GalNAc ₁₀ ) which are synthetic sugar chains in which 10 or 5 GalNAc are linked by α1-4 bond ₅ ) also activated PKA at the same concentration as GM2 and the like. From these, it was confirmed that the difference in the structure of α or β could not be distinguished as long as GalNAc was present at the non-reducing end.

On the other hand, Gb ₄ and on the GD2, the non-reducing end despite having a GalNAc, showed no effect of activating PKA. In these sugar chains, even though GalNAc is present at the non-reducing end, it is thought that the reaction is inhibited by spatial concealment by nearby sugars. That is, in the Gb _4, because the GalNAc is attached to Gal by coupling [beta] 1-3, susceptible to steric hindrance by Gal, the non-reducing terminal GalNAc is considered to be in a state not Azukare freely reaction. In addition, in the case of GD2, the same inhibition occurs because two molecules of sialic acid are continuously bound to Gal to which GalNAc is bound, and are affected by the influence of their size and charge. It is thought that. From these, it can be seen that even if GalNAc is present at the non-reducing end, the effect cannot be obtained unless it is in a free state.

In addition, GalNAc (α1-4) GalNAc consisting of disaccharides did not have an effect of activating PKA. Such a disaccharide sugar compound showed no effect, but a saccharide compound consisting of 3 or 4 sugars such as GM2 or Asialo GM2 sugar chain showed a remarkable effect, thus activating PKA In order to do this, it was found that in the case of sugar chains, it is preferable that the number of sugar chains be 3 or more.
Multivalent neoglycoprotein with about 200 molecules of GalNAc ₅ also activated PKA. On the other hand, neoglycoprotein produced by maltopentaose and isomaltopentaose, which are Glc pentamers, neoglycoprotein synthesized by sialylneolactotetraose, GlcNAcβ1-4 pentamer (GlcNAc ₅ ) in which five GlcNAc are linked by β1-4 bonds Did not activate PKA. From the above results, it was shown that, even in the case of glycoproteins, only those having GalNAc at the non-reducing end can activate PKA.

  From the above, it was shown that a sugar compound having free GalNAc at the non-reducing end effectively activates PKA. In particular, since the sugar chain part of GM2 showed the same effect as GM2, it was found that the lipid part, which had been regarded as important in the past, is unnecessary for this reaction, confirming the importance of the signal cascade involving sugar. It was done.

In Example 1 above, it was confirmed that intracellular PKA was activated by certain sugar compounds. Next, in order to analyze a signal cascade having PKA as a member, an experiment using an inhibitor was performed. Went.
(1) Experiment using inhibitor First, in order to confirm that it was PKA that was activated, an experiment using a PKA selective inhibitor was performed. The experiment was carried out in the same manner as in Example 1 above, and was an addition experiment using a cultured cell system and analyzed by the imaging method. As the sugar compound, 72 nM GM2 and 49 nM GalNAc ₁₀ confirmed to activate PKA in Example 1 above were used, and as inhibitors, two different PKA-specific inhibitors H-89 (1 μM: raw Chemical Industry) and RpcAMPS (100 μM: Research Biochemicals International) were used.

Next, an addition experiment using MDL-12,330A (1 mM, manufactured by Calbiochem), which is an adenylate cyclase inhibitor, was performed in the same manner.
Furthermore, PKA was analyzed by the same imaging method with calcium removed from the extracellular fluid. That is, analysis was performed in the same manner as in Example 1 except that calcium was not added to BSS used as the extracellular fluid.

Although GalNAc ₁₀ is a synthetic sugar chain that does not exist in the mammalian body, the same effects as those of other sugar compounds used in the present invention were confirmed in Example 1 above. Using.

(2) Results The results of the experiment, PKA activation by GM2 and GalNAc ₁₀ is inhibited by the two PKA inhibitor, was shown to GM2 and GalNAc ₁₀ is indeed activate PKA.

  PKA activation was also inhibited by MDL-12,330A, an adenylate cyclase inhibitor. This suggests that adenylate cyclase is activated and the cAMP concentration is increased, thereby activating cAMP-dependent PKA. This can also be confirmed from the fact that RpcAMPS, which is a derivative of cAMP, inhibits PKA activation as described above. That is, it was confirmed that PKA is located downstream of adenylate cyclase in this signal cascade.

  Furthermore, it was found that the intracellular calcium ion concentration did not change in nerve cells in which PKA activation was caused by the sugar compound, and the reaction was not inhibited even when calcium ions were removed from the extracellular fluid. . That is, calcium movement accompanying PKA activation was not observed inside or outside the cell. Adenylate cyclase has subtypes such as G protein-dependent adenylate cyclase that functions in a calcium-independent manner and CaM-dependent adenylate cyclase that functions in a calcium-dependent manner. In the signal cascade, it was shown that an adenylate cyclase independent of calcium ions is involved.

In Examples 1 and 2 above, it was confirmed that a sugar compound having free GalNAc at the non-reducing end activates adenylate cyclase and intracellular PKA. Next, we investigated whether this signal cascade of PKA activation is related to actin polymerization and neuronal filamentous process elongation.
(1) Detection of actin polymer in nerve cell In order to analyze the morphological change of actin existing in the cytoskeleton, phalloidin staining method was performed using NG108-15 cells to analyze the elongation of filamentous processes. In addition, double staining with phalloidin and anti-MAP2 antibody was performed using hippocampal primary cultured neurons in order to confirm whether the detected change was a phenomenon occurring only in neurons. Furthermore, C6 glioma (glioma C6-BU-1: Amano T. et al., Exp. Cell Res., 85, 399-408 (1974); Hamprecht B. et al., Methods Enzymol., 109, 316-341 (1985)), actin polymers were similarly detected by phalloidin staining.

NG108-15 cells prepared in the same manner as in Example 1 above and hippocampal primary cultured neurons on culture days 1, 2, 3, and 6 were washed 3 times with BSS and then contained a sugar compound heated to 37 ° C. BSS was added and stimulated for 2 minutes. As the sugar compound, GM2, GM2 sugar chain portion, and GalNAc (α1-4) oligomer (GalNAc ₅ or GalNAc ₁₀ ), GM1, GM3, and GlcNAc ₅ were used. The addition concentration of each sugar compound is shown in Table 1 below. C6 glioma was prepared according to the method described in the above paper and treated with 49 nM GalNAc ₁₀ .

Furthermore, in order to examine the inhibitory effect of the PKA inhibitor H-89 used in Example 2 and MDL-12,330A, which is an adenylate cyclase inhibitor, these addition experiments were performed. All experiments were performed in the same manner as described above, and GalNAc ₁₀ was used as the sugar compound. For comparison, the same experiment was performed using KN-93 (Seikagaku Corporation), which is an inhibitor of CaM-K. Furthermore, an experiment was also conducted in which the peptide St-Ht31 (manufactured by Promega) having the action of inhibiting the binding of PKA regulatory subunit II (RII) and A-kinase anchor protein (AKAP) was added simultaneously with GalNAc ₁₀ (Vijayaraghavan). et al., J. Biol. Chem., 272, 4747-4752 (1997)). As a control experiment for confirming the effect of St-Ht31, an addition experiment of the control peptide St-Ht31P (manufactured by Promega) having no inhibitory action was also conducted. At the same time, forskolin (manufactured by Wako Pure Chemical Industries, Ltd.) and dibutyl cAMP (manufactured by Seikagaku Corporation) having an action of increasing intracellular cAMP were also conducted. The addition concentrations of these drugs are shown in Table 1 below. Incidentally, KN-93 not described in the table was used at 10 μM.

After treatment with each sugar compound or drug, the cells were immediately treated with 4% paraformaldehyde in 100 mM phosphate buffer at 25 ° C. for 15 minutes and fixed. Fixed cells were treated with PBS containing 0.1% Triton X-100 for 5 minutes, 3 times at room temperature, and then with PBS containing 5% goat serum and 0.1% Triton X-100 for 20 minutes at room temperature. (Ono et al., Cell Struct. Func., 21, 491-499 (1996)).
Subsequently, for primary cultured neurons of the hippocampus, anti-MAP2 antibody (mouse monoclonal, MAB364: manufactured by Chemicon International Inc. (Temecula, CA, USA)) was diluted with PBS and added to the cells as a primary antibody, and 4 ° C. For 16 hours. After the reaction, it was washed 3 times with PBS for 5 minutes. Next, as a secondary antibody, fluorescence-labeled anti-mouse IgG: Cappel (Durham, NC, USA) was diluted in PBS and added to the cells. At the same time, Rhodamine-phalloidin (Molecular Probes Inc.) In addition, double staining with phalloidin and anti-MAP2 antibody was performed by reacting at room temperature for 60 minutes. For NG108-15 cells, only phalloidin staining was performed.

  Finally, the cells were washed 3 times with PBS for 5 minutes and observed with a confocal fluorescence microscope (LSM5 Pascal: manufactured by Carl Zeiss). Microspikes (needle-like structures) formed by actin polymers formed at the edge of the cell are fibrillar as defined by Albrecht-Buehler (Albrecht-Buehler, J. Cell Biol ., 69, 275-286 (1976)). Then, cells having 5 or more filamentous processes were counted as cells having filamentous processes.

On the other hand, in order to analyze changes in the total cAMP concentration in cells treated with GalNAc ₁₀ , cAMP concentration was measured using The Biotrak cAMP EIA System (Amersham). GalNAc ₁₀ was added to NG108-15 cells prepared as described above, and a cell extract was prepared according to the method listed in the kit, and this was used as a sample. Measurements were similarly performed on cells not treated with GalNAc ₁₀ , and the results were compared to analyze changes due to sugar compounds.
(2) Results The results are shown in Table 1.

As a result of analysis by immunostaining, when NG108-15 cells were stimulated with GM2 having GalNAc at the non-reducing end, GM2 sugar chain part, and GalNAc oligomer (GalNAc ₅ or GalNAc ₁₀ ), actin polymerization was clearly observed within 2 minutes. Microspike formation occurred. The micro-spikes at the cell edge coincided with the size, morphology, and dynamics described by Albrecht-Buehler as filamentous protrusions. The number of cells with multiple filamentous processes was also significantly increased by stimulation. On the other hand, GM1, GM3, and GlcNAc ₅ , which are sugar compounds having no GalNAc at the non-reducing end, showed no effect. From these facts, it was confirmed that the sugar compound that activates PKA caused the formation of filamentous protrusions.

  The hippocampal primary cultured neurons on the third day of culture also formed filamentous processes in the same manner. In mature cells on day 6 of culture, actin fibers were formed in the cell body of MAP2-positive neurons and almost all of their processes, but such effects were observed in MAP2-negative non-neuronal cells. There wasn't. From this, it was confirmed that this reaction is specific to nerve cells. Actin polymerization is also promoted in the cell bodies and processes of MAP2-positive cells in the primary cultured hippocampal neurons on the first and second day of culture, and even these young neurons have sugars with GalNAc at the non-reducing end. It was shown to have reactivity to the compound. All these reactions occurred within 2 minutes of stimulation.

On the other hand, actin polymerization as described above was not observed in C6 glioma, which is one of the parent cells of NG108-15 cells, indicating that the signal cascade by this sugar compound is specific to neurons.
In addition, as a result of inhibition experiments using the PKA inhibitor H-89 and the adenylate cyclase inhibitor MDL-12, 330A, NG108-15 cells and hippocampal primary cultured neuronal cells formed fibrillar processes by GalNAc ₁₀ . It was confirmed to be inhibited by an inhibitor. On the other hand, KN-93, an inhibitor of CaM-K, did not inhibit the formation of filamentous processes. This means that, similar to the result in Example 2 above, CaM-K is not involved in this signal cascade, and activation of PKA by cAMP generation occurs.

From these results, it was confirmed that actin polymerization and formation of filamentous protrusions by a sugar compound having GalNAc at the non-reducing end are phenomena downstream of the signal cascade of PKA activation.
On the other hand, when treatment with forskolin or dibutyl cAMP (DbcAMP) that significantly increases the total intracellular cAMP concentration regardless of PKA, it was confirmed that the formation of filamentous processes was caused in the cells. However, when cAMP measurement was performed, it was found that it was difficult to detect changes in cAMP at the whole cell level, and cAMP production by sugar compounds was very small. From this, it was predicted that this reaction occurred at a limited site such as near the cell membrane.

  Therefore, the effect of adding peptide St-Ht31 that inhibits the binding of regulatory subunit II (RII) and A-kinase anchor protein (AKAP) was examined. Filamentous formation was inhibited. Further, the control peptide St-Ht31P did not inhibit the formation of filamentous protrusions. This means that the PKA activated by the sugar compound is not a free PKA widely present in cells but a PKA fixed in the vicinity of the cell membrane by AKAP.

  From these results, a sugar compound having free GalNAc at the non-reducing end has an action of activating adenylate cyclase at a very limited site near the cell membrane and activating PKA at the limited site. I understood it. This is consistent with the fact that PKA activation was observed in the vicinity of the cell membrane in the PKA imaging method described in detail in Example 1 above.

In the above examples, it was confirmed that actin polymerization and formation of filamentous protrusions by a sugar compound having GalNAc at the non-reducing end are phenomena downstream of the signal cascade of PKA activation.
PKA is known to activate cdc42 in mast cells, CHO-K1 cells, COS-7 cells and the like (Feoktistov et al., Mol. Pharmacol., 58, 903-910 (2000)). Rho family GTPases cdc42, Rho, and Rac are expressed in neurons including NG108-15 cells, each of which is reported to be involved in characteristic actin reconstitution (Wilk-Blaszczak et al. al., J. Neurosci., 17, 4094-4100 (1997)). Furthermore, cdc42 is said to be involved in the formation of filamentous protrusions by actin polymerization (Nobes and Hall, Cell, 81, 53-62 (1995)).

  Based on these findings, whether cdc42 is involved in the signal cascade of PKA activation by the sugar compound was analyzed by Western blotting and cell immunostaining using a dominant negative mutant. GTP means guanosine 5'-triphosphate and GDP means guanosine 5'-diphosphate.

(1) Analysis by Western blotting In the same manner as in Example 3, NG108-15 cells were cultured, and 49 nM GalNAc ₁₀ was added thereto and stimulated for 2 minutes. Using the cdc42 Activation Assay Kit (manufactured by Upstate Biotechnology), a solubilized fraction of the cells is prepared according to the method included in the kit, and the GTP-binding cdc42 (Cdc42-GTP) and GTP-binding type in the fraction are prepared. rac1 (Rac1-GTP) was precipitated using PAK-1 agarose, which is only reactive with the GTP-bound form. GTP-bound cdc42 is an active form. The precipitate obtained here was used as a sample for separation and analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In Western blotting, the expressed protein was detected using the anti-cdc42 antibody and anti-Rac1 antibody included in the kit.

At the same time, for the cells to which GalNAc ₁₀ was not added, a sample was similarly prepared using a kit as a control. Furthermore, the cells added with GDP (binding to cdc42 or rac1 to form an inactive GDP binding type: negative control), and GTPγS (cdc42 or rac1 binding irreversibly to GTP binding) A sample was prepared in the same manner as a sample added with a mold (positive control). Similarly, cells treated with 1 mM dibutyl cAMP (increasing intracellular total cAMP concentration in a PKA-independent manner) for 2 minutes were similarly prepared as samples. Furthermore, St-Ht31 (10 μM) and its control peptide St-Ht31P (50 μM) used in Example 3 were added simultaneously with GalNAc ₁₀ (49 nM) to treat the cells, and samples were prepared.

Here, for SDS-PAGE, Novex NuPAGE 10% Bis-Tris pre-cast gels using MOPS SDS running buffer (Invitrogen Corporation) was used. For Western blot, PVDF membrane was used, and protein transfer from gel to membrane was performed at 160 mA / 80 cm ² for 30 minutes in transfer buffer (48 mM Tris, 39 mM glycine, 0.037% SDS and 20% methanol). The semi-dry method was used. Detection of the band after the antibody reaction was performed with ECL Plus (Amersham Biosciences Corporation).

(2) Analysis by cellular immunostaining method using dominant negative mutants In the experiments using dominant negative mutants, the function of proteins originally expressed in the cells by the expression of the transfected mutant genes. Can be confirmed and its function is lost.

  Dominant negative mutants used in the experiment (HA-RhoAN19 / pEF-BOS, HA-Rac1N17 / pEF-BOS, and HA-cdc42N17 / pEF-BOS (Katoh et al., Mol. Cell Biol., 20, 7378-7387 (2000)) was provided by Dr. Manabu Negishi of Kyoto University, which mutated cdc42 and the three genes of rhoA and rac1 belonging to the same Rho family, thereby losing GTP binding. Furthermore, it is designed to express HA as a fusion protein as a tag protein, and 1 μg of each plasmid containing each mutant gene was added to Lipofectamine 2000 (Invitrogen Corporation (Carlsbad, CA, USA). ) Was used to transfect NG108-15 cells.

After transfection, GalNAc ₁₀ was added to the cells and stimulated for 2 minutes, and phalloidin staining was performed in the same manner as in Example 3 above. In order to confirm the expression of dominant negative mutants, immunostaining with anti-HA antibody against mouse HA (mouse monoclonal, 12CA5: Roche Diagnostics (Basel, Switzerland)) was also performed. It was. As the secondary antibody, fluorescence-labeled anti-mouse IgG (Cappel (Durham, NC, USA)) was used.

(3) Results The results of analysis by Western blotting are shown in FIG.

As a result of the analysis, an increase in GTP-bound cdc42 was observed in NG108-15 cells treated with GalNAc ₁₀ for 2 minutes, confirming the activation of cdc42. On the other hand, activation of Rac1 and RhoA was not observed. Furthermore, cdc42 activation was inhibited by St-Ht31. From these, it was confirmed that the activation of PKA in the cells caused the activation of cdc42.
Furthermore, in cells expressing a dominant negative mutant of cdc42, the formation of filamentous processes by GalNAc ₁₀ was inhibited. On the other hand, no inhibition occurred in cells expressing dominant negative mutants of Rac1 or RhoA. This means that the formation of filamentous protrusions is specifically caused by cdc42.

These results indicate that PKA-dependent cdc42 activation is involved in the formation of NG108-15 cell filaments by GalNAc ₁₀ .

In Examples 1 to 4 above, it was found that a sugar compound having GalNAc at the non-reducing end activates a signal cascade including adenylate cyclase, PKA, and cdc42. It was also confirmed that this mechanism is involved in actin polymerization and the formation of filamentous protrusions.
Next, it was analyzed whether dendrite elongation and / or branching occurred following the formation of filamentous protrusions when treatment with these sugar compounds was performed for a long period of time.

(1) Observation of dendritic elongation and / or branching by immunostaining To the primary hippocampal neurons prepared in the same manner as in Example 1 above, a sugar chain portion of 100 nM GM2 was added and cultured for 3 days.
The cells were immunized with anti-MAP2B antibody (mouse monoclonal, M41420: manufactured by Transduction Laboratories (Lexington, KY)) as the primary antibody and Alexa Fluor 488 anti-mouse IgG (produced by Molecular Probes Inc.) as the secondary antibody. Stained. Immunostaining was performed in the same manner as in Example 3 above. MAP2B is a subtype of MAP2, and anti-MAP2B antibody is known to be particularly suitable for immunostaining dendrites.

(2) Digitization of neurite branching and length Using a rat cerebellar primary cultured cell containing cerebellar Purkinje cells, dendritic branching and length were digitized.
The cells were prepared according to the method described in Furuya et al., Brain Res. Protoc., 3, 192-198 (1998). To the cells cultured for 10 days, 1, ₁₀ , 30, and 100 nM GalNAc ₁₀ were added, respectively, and further cultured for 4 days.
Similarly, GalNAc ₁₀ was added to cells cultured for 15 days, and cells cultured for 6 days were also prepared. Furthermore, cells added with 1 μM of PKA inhibitor H-89 simultaneously with GalNAc ₁₀ were also prepared in the same manner.

Each of the three types of cultured cells was fixed in the same manner as in Example 3, and Purkinje cells were stained with an anti-calbindin D-28K antibody (Calbiochem-Novabiochem. Corp.). Anti-calbindin antibody is an antibody suitable for specifically staining cerebellar Purkinje cells. The antibody staining method was the same as the method described in Example 3 and the above paper.
Next, a digital photograph of the stained Purkinje cells was taken using a digital camera attached to a microscope. This was subjected to a Macintosh computer program “ColorIt! Plus R” (manufactured by MicroFrontier Inc), the dendrite was traced, and the number of branches and the length thereof were measured using the Macintosh computer program “NIH Image”.

(3) Results In the hippocampal primary cultured neurons, the growth of MAP2B-positive dendrites rich in actin fibers was promoted by stimulation with the sugar chain part of GM2. The absence of a hydrophobic ceramide moiety indicates that this reaction is not due to the sugar chain being inserted into the cell membrane.
Furthermore, as a result of numerical analysis of dendritic elongation and branching using primary rat cerebellar cultured cells including cerebellar Purkinje cells, GalNAc ₁₀ was found to increase both dendritic elongation and branching number ( Figure 2). The effect of the sugar compound was dependent on the concentration, but a clear effect was observed even at 1 nM. This effect of promoting dendritic elongation was also observed in the case of adding GalNAc ₁₀ for 6 days from the 15th day of culture. This means that cerebellar Purkinje cells can respond to GalNAc ₁₀ in the same way, whether they are young or relatively mature.

  The effect of promoting dendritic elongation was inhibited by adding the PKA inhibitor H-89. From this, it was confirmed that the signal cascade by PKA activation is involved in this dendrite growth.

  The cerebral nerve cell activator of the present invention is characterized in that it contains a saccharide compound having GalNAc in a free state at a non-reducing end as an active ingredient, present in a mammal. The sugar compound has, for example, a remarkable effect even with a sugar chain alone, and has advantages such as low antigenicity and easy preparation as a drug. In addition, the action of the sugar compound occurs even at very low concentrations, and is based on a reaction that occurs in a limited site near the cell membrane, so it is very unlikely to cause significant changes in the entire brain nerve cells or other sites and cause side effects It also has the advantage.

  Such a cranial nerve cell activator of the present invention has an effect of activating cranial nerve cells and promoting the extension and / or branching of dendrites. Therefore, it is useful as a therapeutic agent for cranial nerve disorders for various diseases such as injury caused by accidents, ischemic damage caused by cerebral infarction, cerebrovascular dementia, etc., and degeneration of cranial nerve cells.

It is a photograph which shows the result of having analyzed the effect with respect to cdc42 or rac1 of the sugar compound which has GalNAc in a non-reducing terminal by Western blotting. In the figure, Photo A is a photograph of a Western blot immunostained with anti-cdc42 antibody, and B is a photo immunostained with anti-rac1 antibody and anti-cdc42 antibody. 4 is a graph showing dendritic extension and branching promotion effects by a sugar compound having GalNAc at a non-reducing end. In the figure, graph A shows the change in the number of branches depending on the concentration of GalNAc ₁₀ added. The vertical axis represents the number of branches, and the horizontal axis represents the addition concentration. Graph B shows the change of outgrowth by addition concentration of GalNAc _10. The vertical axis represents the length of protrusion extension (mm), and the horizontal axis represents the added concentration. Graph C shows a comparison of the number of branches among the three cells of the cells cells (Non) · stimuli without stimulation by GalNAc ₁₀ (GalNAc ₁₀₎ · inhibitors were added cells (GalNAc ₁₀ + H89). The vertical axis is the cell type, and the horizontal axis is the number of branches. Graph D shows a comparison of process extension between the same three types of cells as Graph C. The vertical axis is the cell type, and the horizontal axis is the length of projection extension (mm). The t test results are shown in the figure, where “**” means P <0.01 and “***” means P <0.001.

Claims (5)

A brain nerve cell activator comprising, as an active ingredient, a sugar compound having GalNAc present in a mammal in vivo and in a free state at a non-reducing end. The cerebral nerve cell activator according to claim 1, wherein the sugar compound is a sugar chain part of GM2 ganglioside or asialo GM2 ganglioside. The cerebral nerve cell activator according to claim 1 or 2, wherein the activation of cerebral nerve cells causes elongation and / or branching of dendrites of the cerebral nerve cells. 4. The brain nerve cell activator according to claim 3, wherein dendritic elongation and / or branching is based on activation of protein kinase A. The cranial nerve cell activator according to any one of claims 1 to 4, wherein the cranial nerve cell activator is a therapeutic agent for cranial nerve disorders.