JPH0930979A - Therapeutic agent for neuropathy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject therapeutic agent comprising a lipid-bonded glycosaminoglycan (lipid bonded GAG) or its salt as an active ingredient, having stretching promoting effect on neurite and recovery promoting action on neurological function, being expected to have reproducing effect on nerve fiber, etc., having extremely low toxicity. SOLUTION: This therapeutic agent for neuropathy comprises a lipid-bonded glycosaminoglycan (lipid bonded GAG) or its salt as an active ingredient, The lipid bonded GAG, for example, is obtained by specifically cleaving a pyranose ring at the reduced end of GAG by oxidizing the saccharide reside at the reduced end of GAG to form carboxyl at the reduced end, subjecting the resultant substance to a lactone formation reaction, then reacting the lactone with a primary amine of a lipid to bond GAG to the lipid by covalent bond. Chondroitin sulfate, hyaluronic acid, and their salts are preferable as GAG of raw material. A primary amino-containing phospholipid is preferable as the lipid. Dipalmitoyl- L-(α-phosphatidyl)ethanolamine-bonded hyaluronic acid, etc., are preferable as the lipid-bonded GAG. palmitoyl. phosphatidyl.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for neurological diseases containing a lipid-bound glycosaminoglycan or a salt thereof as an active ingredient.

[0002]

2. Description of the Related Art Various proteoglycans are present on the surface of cell membranes or in the matrix between cells, and are involved in cell adhesion, spreading, signal transduction, etc. through binding with cytokines and interactions between cells or between cells and substrates. It is thought to change the responsiveness of cells and be involved in development, proliferation, differentiation, growth, aging, canceration, immune response, responsiveness to inflammatory response and the like.

Recently, it has been revealed that many kinds of proteoglycans are present in nerve tissue. It has been considered that these proteoglycans are closely related to the morphogenesis and functional expression of neural tissues such as the brain (RK Margolis and RU Margolis, Experientia., 4
9, 429-446 (1993), A. Oohira et al., Neurosci. Re
s., 20, 195-207 (1994)).

It is considered that the morphology of nerve tissue is formed by phenomena such as differentiation and migration of nerve cells, extension of neurites, recognition of target cells, synapse formation / maintenance and rearrangement with target cells. .

Proteoglycans are composed of a protein part called a core protein and a sugar chain part having a unique structure called a glycosaminoglycan. Addition of chondroitin sulfate proteoglycans in the brain to the culture system of nerve cells promotes neurite outgrowth (N. Iijima et a
l., J. Neurochem., 56, 706-708 (1991)) and sometimes suppress (A. Oohira et al., J. Neurosci., 11, 82).
2-827 (1991)). However, in many cases, the activity is in the core protein portion, and there are many reports that the effect is not observed when glycosaminoglycan is administered alone. On the other hand, neurites cannot invade regions where high concentrations of chondroitin sulfate and keratan sulfate are present (DM Snow et al., J. Neurobiol.
23, 322-336 (1992)), suggesting that glycosaminoglycans may function as guide molecules that direct neurite outgrowth.

[0006] As described above, proteoglycans may act on the nervous system to be effective in the recovery of peripheral nerve damage and central nerve damage, but clinical studies have not been conducted yet. This is probably because proteoglycan is a macromolecule and it is difficult to apply it to the nervous system as it is, and there are differences in animal species, especially in the core protein part, which causes problems such as antibody production.

On the other hand, the present inventors previously synthesized lipid-bound glycosaminoglycans in which lipids and glycosaminoglycans were covalently bonded, and found that they have biological activity such as cell adhesion inhibitory action. (J. Biol. Chem., 26
8, 15779-15787 (1993), JP-A-4-80201, JP-A-4-80202, JP-A-4-82836, and JP-A-5-
236951, JP-A-6-72893).

[0008]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a new drug for improving the symptoms of neurological diseases. In particular, it is an object of the present invention to provide a therapeutic agent for neurological diseases which does not have side effects such as toxicity.

[0009]

Means for Solving the Problems The present invention provides: 1) a therapeutic agent for a neurological disease containing a lipid-bound glycosaminoglycan or a salt thereof as an active ingredient, particularly a neurite outgrowth promoting agent 2) a glycosaminoglycan is chondroitin sulfate Alternatively, the above-mentioned drug, which is hyaluronic acid, is provided.

The therapeutic agent for neurological diseases of the present invention is characterized by promoting neurite outgrowth of peripheral and central nerve cells and recovery of nerve function damaged by nerve damage or nerve defect.

Lipid-bound glycosaminoglycan, which is an active ingredient of the therapeutic agent for neurological diseases of the present invention, is a known substance. For example, Japanese Unexamined Patent Publication No. 80201/1992 or Japanese Unexamined Patent Publication No. 4-802
02, and J. Biol. Chem., 268, 15779-15787.
The compounds described in (1993) are exemplified. However, the present invention is not limited to this, and any lipid-bound glycosaminoglycan in which glycosaminoglycan (hereinafter referred to as GAG) and a lipid are covalently bonded can be used as an active ingredient of the drug of the present invention.

In particular, the lipid-bound glycosaminoglycan is G
Carboxyl group of AG (including lactone), formyl group (including hemiacetal group), hydroxyl group or primary amino group, or the group separately introduced to GAG, and carboxyl group, formyl group or primary amino group of lipid Or a group covalently bonded by an acid amide bond (—CO—NH—), an ester bond or an aminoalkyl bond (—CH ₂ —NH—) formed with the group separately introduced into the lipid. Especially, those having the following bonds are preferable.

[0013] The pyranose ring at the reducing end of GAG is opened, and the acid amide bond (formed by the reaction between the carboxyl group (including lactone) of GAG formed by chemical treatment and the primary amino group of lipid ( -CO-NH
-), The acid amide bond (-CO-NH-) formed by the reaction between the carboxyl group of the uronic acid moiety of GAG and the primary amino group of the lipid, or the pyranose ring at the reducing end of GAG is opened, GA formed by chemical treatment
And formyl group G, aminoalkyl bond formed by reducing the Schiff base formed by the reaction of the primary amino group of the lipid (-CH ₂ -NH-).

An amino group involved in the above covalent bond,
Carboxyl group, formyl group (including hemiacetal group) and hydroxyl group are originally present in GAG or lipid, those formed by chemical treatment of these,
Alternatively, the spacer compound having the functional group at the terminal may be any of those separately introduced by previously reacting with GAG or lipid.

The relationship between the lipid-bound glycosaminoglycan and its raw material compound is schematically shown as follows.

[0016]

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[0017]

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[0018]

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The lipid-bound glycosaminoglycan used in the present invention may be a salt thereof, preferably an alkali metal salt such as sodium or potassium, an alkaline earth metal salt such as calcium or magnesium, or a trialkylamine. Or an organic salt such as pyridine.

As the raw material GAG, any of those extracted from natural products such as animals, those obtained by culturing microorganisms, and those chemically or enzymatically synthesized can be used. Specific examples include hyaluronic acid, chondroitin, chondroitin sulfate (A, C, D, E or K), chondroitin polysulfate, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, keratan polysulfate and the like. These GAGs may be commonly used salts (eg sodium salts). However, chondroitin sulfate, hyaluronic acid and salts thereof are preferable in order to obtain a more effective neurological disease therapeutic action and neurite outgrowth promoting action.

As the raw material lipid, a complex lipid or simple lipid derived from a natural product such as an animal, a plant, a microorganism, or chemically or enzymatically synthesized or partially decomposed can be used. Particularly preferred are phospholipids such as phosphatidylethanolamine, phosphatidylserine, phosphatidylthreonine, ethanolamine plasmalogen, serine plasmalogen, lysophosphatidylcholine, lysophosphatidylinositol, and neutral lipids such as monoacylglycerol and diacylglycerol. . Especially, a phospholipid having a primary amino group is preferable. Further, long-chain fatty acids, long-chain aliphatic amines, cholesterols, sphingosine, and ceramide may be used. The chain length and the degree of unsaturation of the acyl group in the lipid are not particularly limited, but those having 6 or more carbon atoms are preferable. For example, palmitoyl (hexadecanoyl) or stearoyl (octadecanoyl) is exemplified. In addition, these lipids may be commonly used salts.

The method for producing the lipid-bound glycosaminoglycan is not particularly limited, and a known production method (for example, JP-A-4-
No. 80201 and JP-A No. 4-80202) can be adopted. A typical manufacturing method will be described below.

Limited-end limited oxidation method This method involves reducing and limiting oxidation (partial oxidation) of sugar residues at the reducing end of GAG such as xylose residue, galactose residue, uronic acid residue or hexosamine residue. Thereby specifically opening (cleaving) the pyranose ring at the reducing end, and forming a formyl group at the reducing end of the GAG to form an aldehyde compound. The formyl group of the aldehyde compound and the primary amino group of the lipid are combined with each other. Reacting to form a Schiff base, then reducing the Schiff base to form an aminoalkyl bond (—CH ₂ —NH—) to give GAG
And a lipid are covalently bonded. Reduction of the sugar residue at the reducing end of GAG is carried out in an amount of about 5 to 50 equivalents, preferably 25 to 30 equivalents of reducing agent (sodium borohydride, sodium cyanoborohydride, etc., alkali borohydride, etc.). ) In an appropriate aqueous solvent (eg, water, borate buffer, etc.), usually 10 to 30 ° C.,
It can be preferably carried out at 15 to 25 ° C. After the above reduction, limited oxidation is performed to produce an aldehyde compound having a formyl group specifically at the reducing end of GAG. The limited oxidation is performed using 1 to 10 equivalents, preferably 3 to 6 equivalents of an oxidizing agent (such as alkali periodate salts such as sodium periodate and potassium periodate) with respect to the GAG after the reduction, and usually 0 It can be carried out at -10 ° C, preferably 0-4 ° C. The reaction of reacting the obtained aldehyde compound with a lipid having a primary amino group (phospholipid such as phosphatidylethanolamine) to form a Schiff base is an aqueous solvent (water, phosphate buffer, etc.) or a suitable A solution prepared by dissolving the above aldehyde compound in a different organic solvent (dimethylformamide, dimethylsulfoxide, etc.) and a solution prepared by dissolving lipids in a suitable organic solvent (chloroform, methanol, etc.) are mixed, and usually at a temperature of 15 to 60 ° C. Can be reacted. At the time of this reaction or after the completion of the reaction, an appropriate reducing agent (sodium borohydride, alkali borohydride such as sodium cyanoborohydride, etc.) is allowed to act to reduce the Schiff base. When producing a lipid-bound glycosaminoglycan by this reaction method,
Instead of a lipid having a primary amino group, a divalent functional spacer compound having a primary amino group (for example, alkylenediamine such as ethylenediamine, or an amino acid such as lysine) is reacted with the aldehyde compound, A functional group (eg, carboxyl group) capable of forming an aminoalkyl bond (—CH ₂ —NH—) and then reacting with the other functional group (eg, amino group) of the spacer compound.
May be reacted with a lipid (eg, monoacylglycerol dicarboxylic acid ester such as monoacylglycerol succinic acid ester).

Reductive end lactonization method In this method, the pyranose ring at the reducing end is oxidized by oxidizing the sugar residue at the reducing end of GAG such as xylose residue, galactose residue, uronic acid residue or hexosamine residue. A ring group is specifically opened (cleaved) to form a carboxyl group at the reducing end of the GAG, and then subjected to a lactone forming reaction to form a lactone structure at the reducing end of the GAG. And GAG are reacted with each other to form an acid amide bond (-CO-NH-), whereby GAG and a lipid are covalently bonded.
Oxidation of sugar residues at the reducing end of GAG is 2 for GAG.
To about 20 equivalents, preferably about 5 to 15 equivalents of an oxidant (iodine, bromine, etc.), and in an appropriate aqueous solvent (eg, water, phosphate buffer, etc.), usually 0-40 ° C., preferably Can be performed at 15 to 30 ° C. After the above oxidation reaction, a strongly acidic cation exchange resin such as Dowex 50
(Trade name; manufactured by Dow Chemical Co.), Amberlite IR-
120 (trade name; manufactured by Organo) and / or acid (hydrochloric acid,
A lactone compound in which the reducing end of GAG is specifically lactonized can be produced by treatment with an inorganic acid such as sulfuric acid or an acid anhydride of an organic acid such as acetic acid, citric acid, or succinic acid. The reaction between the obtained lactone compound and a lipid having a primary amino group (phospholipid such as phosphatidylethanolamine) is carried out in a suitable aqueous solvent (water, phosphate buffer, etc.) or a suitable organic solvent (dimethylformamide, A solution in which a lactone compound is dissolved in dimethyl sulfoxide) is mixed with a solution in which a lipid is dissolved in an appropriate organic solvent (chloroform, methanol, etc.),
The reaction may be performed at a temperature of 30 ° C, preferably 30 to 60 ° C. As in the case of the reducing end limited oxidation method, instead of the lipid having a primary amino group, a divalent functional spacer compound having a primary amino group is reacted with the lactone compound to form an acid amide bond ( -CO-NH-) may be formed, and the other functional group of the spacer compound may be reacted with the functional group of the lipid (for example, carboxyl group).

Other methods As a method other than the above, for example, a method of reacting the carboxyl group of the uronic acid moiety of GAG with the primary amino group of the lipid to form an acid amide bond (-CO-NH-) is mentioned. Can be mentioned. In the above reaction, a condensing agent (for example, 1-
Ethyl-3- (3-dimethylaminopropyl) carbodiimide, dicyclohexylcarbodiimide, etc.) is used to form an acid amide bond (-CO-NH-), or the carboxyl group of the uronic acid moiety is added in the presence of the condensing agent, After reacting with an activator (for example, N-hydroxysuccinimide, p-nitrophenol, N-hydroxybenzotriazole, etc.) to form an active ester, it is reacted with the lipid to form an acid amide bond (—CO—NH—). Can be formed. In the above reaction, the uronic acid moiety of GAG is made into a salt (amine salt such as triethylamine or tributylamine) which can be dissolved in an organic solvent, and the reaction is carried out in an organic solvent (dimethylformamide, dimethylsulfoxide, pyridine, etc.). Is preferred.

Representative Compounds Suitable compounds of the lipid-bound glycosaminoglycan, which is the active ingredient of the drug of the present invention, are exemplified below. (1) Dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded hyaluronic acid Raw material GAG: Hyaluronic acid (derived from chicken cob, molecular weight 10,000) Lipid: Dipalmitoyl-L- (α-phosphatidyl)
Ethanolamine synthesis method Reduction terminal lactonization method (Japanese Patent Laid-Open No. 80201/1992,
See Example 1- (2) -1).

[0027]

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(2) Dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded chondroitin sulfate Raw material GAG: Chondroitin sulfate (derived from shark cartilage, molecular weight 3)
10,000) Lipid: Dipalmitoyl-L- (α-phosphatidyl)
Ethanolamine synthesis method Reduction terminal lactonization method (Japanese Patent Laid-Open No. 80201/1992,
See Example 1- (2) -2).

[0029]

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(3) Stearoyl palmitoylphosphatidylserine-bonded chondroitin sulfate raw material GAG: Chondroitin sulfate (derived from shark cartilage, molecular weight 3)
10,000) Lipid: Stearoyl palmitoylphosphatidylserine Synthetic method Reduction terminal lactonization method (Japanese Patent Laid-Open No. 80201/1992,
(See Example 1- (3))

[0031]

[Chemical 6]

(4) Monostearoyl glycerol / succinate-bonded chondroitin sulfate Raw material GAG: Chondroitin sulfate (derived from shark cartilage, molecular weight 3)
10,000) Lipid: monostearoyl glycerol / succinate synthetic method reducing terminal amine method (see JP-A-4-80201, Example 2- (3))

[0033]

[Chemical 7]

(5) Dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded hyaluronic acid Raw material GAG: Hyaluronic acid (origin from chicken cob, molecular weight 10,000) Lipid: Dipalmitoyl-L- (α-phosphatidyl)
Ethanolamine synthesis method Reduction end limited oxidation method (see JP-A-4-80202, Example 1- (2) -1)

[0035]

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In the therapeutic agent for neurological diseases of the present invention, an effective amount of a lipid-bound glycosaminoglycan or a salt thereof (hereinafter sometimes abbreviated as lipid-bound GAG), which is the active ingredient, is administered to the peripheral nervous system or the central nervous system. It is a drug capable of treating a mammal, including a human, who is suffering from a neurological disease caused by the above disorder, by treating the animal. Typical diseases include, for example, neuropathy caused by traumatic nerve damage or nerve deficiency (particularly somatic neuropathy, more particularly sensory neuropathy), metabolic disorder polyneuropathy, mechanical neuropathy, and toxic nerve. Various peripheral nerve diseases such as disorders; and therapeutic effects are expected by regenerating nerve fibers such as stroke, cerebral infarction, cerebral hemorrhage, cerebral trauma, memory impairment, senile dementia, Alzheimer's disease and Parkinson's disease Various central nervous system diseases.

The therapeutic agent for neurological diseases of the present invention is a lipid-bound GA
Administer G orally or parenterally (intravenously, intramuscularly,
Intracutaneous administration (injection), enteral administration, transdermal administration, etc.)
As a pharmaceutical agent for administration, it can be formulated into any dosage form such as a liquid preparation, a solid preparation, a semisolid preparation, and is administered to a patient by any administration method.

The above-mentioned preparation is prepared by adding a pharmaceutically acceptable auxiliary agent to the lipid-bound GAG according to a conventional method. Further, it is also possible to prepare a sustained-release preparation by a known technique.

When the agent of the present invention is used for treating a neurological disease caused by disorders of the central nervous system, intramuscular injection, intravenous injection,
Subcutaneous or intraperitoneal injections are preferred.

Lipid-bound GAGs are mostly water soluble,
A liquid formulation can be easily manufactured. In order to manufacture liquid preparations such as injections, lipid-bound GAG may be added as necessary.
pH adjusters (hydrochloric acid, sodium hydroxide, lactic acid, sodium lactate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), isotonic agents (sodium chloride, glucose, etc.)
Alternatively, it may be dissolved in distilled water for injection, aseptically filtered and filled in an ampoule, or mannitol, dextrin, cyclodextrin, gelatin and the like may be further added thereto and freeze-dried under vacuum to give a solution-in-use injection. In addition, for lipid-bound GAG, emulsifier (lecithin, polysorbate 8
0, polyoxyethylene hydrogenated castor oil, etc.) and emulsified in water to give an emulsion for injection.

Further, lipid-bound GAGs and excipients (lactose, starch, crystalline cellulose, etc.), binders (sucrose, hydroxypropylcellulose, etc.), disintegrants (carboxymethylcellulose, etc.), lubricants (magnesium stearate, talc) Formulation aids such as
Solid preparations for oral administration such as granules, capsules, tablets,
Alternatively, a sweetener (sucrose, sorbitol, etc.), water, essential oil, ethanol, etc. may be added to the lipid-bound GAG to prepare a liquid preparation for oral administration such as a syrup.

For rectal administration, a suppository base such as mono-, di- or triglyceride of cocoa fatty acid, polyethylene glycol or the like is added to lipid-bound GAG, and then heated and melted,
Pour this into a mold to cool, or to bind lipid-bonded G
It can be obtained by dissolving AG in polyethylene glycol, soybean oil or the like and then coating it with a gelatin film.

Further, the lipid-bound GAG, white petrolatum,
In addition to beeswax, liquid paraffin, polyethylene glycol, etc., it can be made into an ointment or, if necessary, heated and kneaded to give an external preparation for skin. The tape preparation can be obtained by kneading a lipid-bonded GAG with an adhesive such as rosin or an acrylic ester polymer and spreading the mixture on a non-woven cloth or the like.

The inhalant can be obtained, for example, by dissolving or dispersing the lipid-bound GAG in a pharmaceutically acceptable propellant such as an inert gas, and filling this in a pressure resistant container.

As an injection, the target organ, namely,
For example, liposome preparations and lipid emulsion preparations that can improve the rate of transfer to the central nervous system such as the brain and peripheral nerve bundles are included. The lipid-bound GAG itself forms an aggregate in which 20 to 30 molecules are aggregated in an aqueous solution via a lipid moiety (J. Biol. Chem., 268, 15779-15787).
(1993)), a more effective liposome preparation can be produced by further using an amphipathic substance that forms a liposome. In particular, nanosphere liposomes (ultralipid ultrafine particles) can increase blood concentration without being taken up by reticuloendothelial tissues, reduce the minimum effective dose required for the onset of drug efficacy, and cross the brain blood barrier. Because it ’s easy
It is suitable for use in the treatment of neurological disorders of the brain. The liposome preparation is a known liposome production method (Liposomes: Phy
susial Struvture to Therapeutic Applications, pp.5
1-82, Elsevier, Amsterdam (1981); Proc. Natl. Aca
d.Sci. USA, 75, 4194-4198 (1978)).

The liposome can be produced, for example, by the following method. The above amphipathic substances and additives and the lipid-bound GAG are dissolved or mixed in an organic solvent (single or mixed solvent such as chloroform, dichloromethane, ethanol, methanol, hexane, etc.), and an inert gas (nitrogen) in a container such as a flask. Gas, argon gas, etc.)
The organic solvent is removed in the presence and deposited as a thin film on the vessel wall. Then, a suitable aqueous medium (saline solution,
Buffer solution, phosphate buffered saline, etc.) and stir with a stirrer. In order to obtain a liposome having a small particle size, the liposome is further dispersed by using an ultrasonic emulsifier, a pressure emulsifier, a French press cell crusher or the like. Furthermore, by subjecting this to a membrane filter, nanosphere liposomes (ultrafine particles of lipid; particle size 25
˜50 nm) can be obtained. Alternatively, the liposome may be subjected to fractionation treatment such as ultrafiltration, centrifugation, gel filtration, etc. to remove the lipid-bound GAG separated from the liposome.

As the amphipathic substance forming the liposome, natural phospholipids (egg yolk lecithin, soybean lecithin, sphingomyelin, phosphatidylserine, phosphatidylinositol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine,
A phospholipid such as cardiolipin) or a synthetic phospholipid (such as distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine) is used. In addition, in order to improve the stability of the membrane, fluidity, and membrane permeability of the active ingredient, cholesterols (cholesterol, ergosterol,
Phytosterol, sitosterol, stigmasterol, etc.), substances known to impart a negative charge to liposomes (phosphatidic acid, dicetyl phosphate, etc.), substances known to impart a positive charge (stearylamine, Stearylamine acetate, etc.),
Various known additives such as antioxidants (tocopherol, etc.), oily substances (soybean oil, cottonseed oil, sesame oil, liver oil, etc.) may be used.

The content of lipid-bound GAG in the above-mentioned preparation varies depending on the dosage form, administration method, number of administrations, etc.
0.1 to 10% by weight for injections, 1 to 80% by weight for oral preparations, 0.1 for external preparations
It is about 10% by weight.

The dose of lipid-bound GAG depends on the age of the patient,
It varies depending on symptoms, body weight, and administration method, and cannot be specified in a general way, but in case of intra-organ administration (injection), the daily dose is 1-1, 1.
It is preferable to administer 000 mg once or in several divided doses.

Lipid-bound GAGs are low toxicity compounds,
The safety as a medicine is extremely high. The acute toxicity of lipid-bound GAG was investigated by the following method.

One 4-week-old Sic-ddy strain male and female mice
After preliminarily breeding for a week, when the body weight became 23 to 30 g for males and 20 to 25 g for females, the above-mentioned dipalmitoyl-L- (α
-Phosphatidyl) ethanolamine-bonded chondroitin sulfate or dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded hyaluronic acid was dissolved in local saline to a concentration of 5%, and the solution was intraperitoneally administered. The LD ₅₀ value was calculated using 10 males and 10 females per group. As a result, all compounds had LD ₅₀ values of 2,
Since it is 000 mg / kg or more, there is no problem in safety as a medicine.

[0052]

【Example】

Test Example 1 Effect of PC12 cells on neurite outgrowth A rat adrenal medulla-derived PC12 cell line (provided by Professor Hatanaka, Osaka University) with laminin (1, 5, 10 μg) capable of differentiating into peripheral nerve-like cells was used. / ml) (mouse EH
S-derived; Nitta gelatin Co., Ltd.) coated 24-well culture dish (MS-8024R; Sumitomo Bakelite Co., Ltd.) 1 × 1
Scatter at a density of 0 ⁴ cells / cm ² and 10% fetal bovine serum (FC
S) -containing Dulbecco's modified Eagle medium (DMEM) was cultured at 37 ° C. for 5 hours. Then, the above culture solution was removed, and a DMEM-Ham's F12 medium (Ham's F12) (1: 1) mixed medium containing or not containing phosphatidylethanolamine-bound chondroitin sulfate (CS-PE) was added. After culturing at 37 ° C. for 24 hours, the cells were fixed with 2% glutaraldehyde-containing phosphate buffered saline (PBS), and the state of neurites extending from PC12 cells was observed with a phase contrast microscope. The results are shown in FIG. In addition, FCS
From Flow Labotatories, DMEM and Ham's F12 medium were purchased from Nissui Pharmaceutical. CS-PE is dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bound chondroitin sulfate (as a raw material, GAG is chondroitin sulfate (shark cartilage-derived, molecular weight 30,000), and lipid is dipalmitoyl-L- (α-phosphatidyl). ) Ethanolamine was used, and the reducing terminal lactonization method (Japanese Patent Laid-Open No. 4-80201, Example 1- (2) -2)) was used as a synthetic method. The number of neurites of PC12 cells increased as the concentration of laminin coat increased, and the neurites were elongated, but CS-
When the medium was replaced with PE-containing medium, longer neurite outgrowth was observed.

Test Example 2 Protrusion extension promoting effect of rat fetal brain-derived primary cultured neurons Brains (hippocampus) of 18-day-old embryonic rats were collected, treated with papain (9 units / ml, Worthington), and treated with nerve cells. Was isolated and prepared (Neurosci. Res., 8, 69-82 (199
0)). Add 20 μg / ml polylysine (Sigma) to add 3
Various glycosaminoglycans (chondroitin sulfate (CS), hyaluronic acid (H) were added to a 12-well culture dish (manufactured by Falcon) that had been pretreated overnight at 7 ° C. and washed three times with water.
A)] 50 μg / ml and various lipid-bound glycosaminoglycans [phosphatidylethanolamine-bound chondroitin sulfate (CS-PE), phosphatidylethanolamine-bound hyaluronic acid (HA-PE)] 0.5 μg / ml were added, and the mixture was added at 37 ° C. It was treated for 24 hours. The nerve cells were inoculated to this culture dish at a density of 10 × 10 ⁴ cells / cm ² , and 5% newborn bovine serum (manufactured by Mitsubishi Chemical), 5% heat-treated horse serum (manufactured by Gibco) and 90% Dulbecco. Modified Eagle Medium (DM
EM) -Ham's F12 medium (Ham's F12) (1: 1) mixed medium (containing 1.5 mM HEPES),
The cells were cultured at 37 ° C for 24 hours. Half of the culture solution was exchanged with 4% paraformaldehyde to fix the nerve cells, and the neurite extending from each nerve cell was observed with a phase contrast microscope. The results are shown in FIG. CS is derived from shark cartilage, molecular weight is 30,000, HA is derived from chicken cob, molecular weight is 10,000, CS-PE is the same as that used in Test Example 1, and HA-PE is dipalmitoyl-L- (α-phosphatidyl). Ethanolamine-bonded hyaluronic acid (GAG as a raw material is hyaluronic acid (origin, molecular weight 10,000), lipid is dipalmitoyl-
L- (α-phosphatidyl) ethanolamine and a reducing terminal lactonization method (Japanese Patent Laid-Open No. 4-80201, Example 1- (2) -1) were used as synthetic methods. Treatment with glycosaminoglycan alone showed little effect on neurite formation even at 50 μg / ml. However, for lipid-bound glycosaminoglycans, a clear effect of promoting neurite outgrowth was observed by treatment with CS-PE and HA-PE of 0.5 μg / ml each.

Test Example 3 Protrusion extension promoting effect of rat fetal brain-derived primary cultured nerve cells In the same manner as in Test Example 2, nerve cells were prepared from 18-day-old embryonic rat brain (hippocampus). In the same manner as in Test Example 2, a 96-well culture dish (manufactured by Coaster) pretreated with polylysine and washed three times with water was used to prepare various lipid-bound glycosaminoglycans [phosphatidylethanolamine-bound hyaluronic acid (HA-PE). ) 0.1 and 0.5 μg / ml, phosphatidylethanolamine conjugated chondroitin sulfate (CS-PE) (0.004, 0.02, 0.1, 0.
5 and 1.0 μg / ml)] and treated at 37 ° C. for 24 hours to prepare a culture dish coated with lipid-bound glycosaminoglycan. 10 x 10 of the nerve cells were placed in this culture dish.
Inoculated at a density of ⁴ cells / cm ² , 5% newborn bovine serum (manufactured by Mitsubishi Chemical), 5% heat-treated horse serum (manufactured by Gibco) and 90
% Dulbecco's Modified Eagle Medium (DMEM) -Ham F1
2 medium (Ham's F12) (1: 1) mixed medium (1.5 mM
The cells were cultured at 37 ° C. for 24 hours in a medium containing HEPES. After fixing the nerve cells, the neurites were immunostained by the enzyme antibody method by the method of P. Doherty et al. (J. Neurochem., 42, 1116-1122 (1984)) using an anti-neurofilament antibody (RT97). . That is, half of the culture solution was exchanged with 4% paraformaldehyde and stored for 15 minutes to fix nerve cells. It was treated with methanol cooled to -20 ° C for 30 minutes, and washed 3 times with CMF-PBS (calcium / magnesium-free phosphate buffered saline). Anti-neurofilament antibody (RT9
7) (Boehringer Mannheim) 5% goat serum-containing CMF-PBS solution (10 μg / ml) was added, and the mixture was treated at 4 ° C. overnight. After washing with CMF-PBS, 1: 1,000
A 0-fold diluted peroxidase-conjugated anti-mouse immunoglobulin was added and incubated at room temperature for 60 minutes. CM
After washing 3 times with F-PBS and once with distilled water, 50 μl of 1
A solution of 00 μg / ml tetramethylbenzidine (TMBZ) (manufactured by Dojindo Co., Ltd.) and 0.001% H ₂ O ₂ in 0.1M sodium acetate (pH 6.0) was added and incubated. The reaction was stopped by adding 50 μl of 1N sulfuric acid, and the absorbance at 510 nm was measured. The amount of spread neurites was quantified by calculating the absorbance per cell number. The results are shown in FIG. The same HA-PE and CS-PE as those used in Test Example 2 were used. HA-PE is 0.1 to 0.
At the concentration of 5 μg / ml, CS-PE was 0.02-1.
With the treatment at a concentration of 0 μg / ml, the neurite outgrowth promoting action was observed. Particularly, HA-PE was about 160% coated with 0.5 μg / ml and about 150% coated with CS-PE at 150 μg / ml, which was higher than the control, and thus increased neurofilament production, that is, increased neurite outgrowth. Was given.

Test Example 4 Effect on Rat Nerve Injury Model Under the anesthesia of Nembutal, the left thigh of an SD male rat aged 11 weeks was incised to expose the sciatic nerve, and the sciatic nerve was crushed with a 12 cm Pean forceps for 10 seconds. The right leg is the untreated group,
It was used as a control foot. Immediately after crushing, test liquid (CS-PE 1
0 mg / ml PBS solution) or control solution (PBS) 0.05 ml
Was dripped around the damaged site. After that, the damage to the opening and closing of the toes and the load on the soles of the feet is regarded as a motor dysfunction, and the damage to the sole of the foot, the damage to the toes and the poor extension and loss of the nails are regarded as trauma due to the paralysis or damage of the sensory nerves. Table 1
The score table was used to determine and measure. The pain reaction was measured by pinching with a caliper. This exam is Pain. 47
(1991) According to the method described in p31-39. The same CS-PE as that used in Test Example 1 was used.

[0056]

[Table 1]

Observations were made once a week after the nerve injury regarding motor dysfunction and trauma, and the degree of both was measured as a total disability degree score. The results are shown in FIG.

In the control group to which only PBS was dropped, the number of cases of self-injury (tear off) of the toes increased with time from the second week onward after nerve injury, and it was observed in all 5/5 cases at the fifth week. CS-P
In the E-administered group, almost no rat paw trauma was observed, and an effect significantly different from that of the control group was observed.

It is considered that the injury of the foot is caused by self-injury such as biting off the toe because the sensory nerve function of the lower limb is impaired by the damage of the peripheral nerve. Since almost no trauma was observed by the administration of CS-PE, CS-PE
PE has been shown to promote recovery of peripheral nerve function and be effective in neurological diseases caused by disorders of peripheral nerves including sensory nerves.

Formulation Example Aseptically prepared stearoyl palmitoyl phosphatidyl serine-bonded chondroitin sulfate (CS-PS) and dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded hyaluronic acid (HA-PE), respectively 5
0 mg was dissolved in phosphate buffered saline (PBS) at room temperature to make 50 ml. Aseptic filtration (pore size O.22μm)
The membrane filter was used), and the solution was dispensed in an amount of 2.5 ml and then sealed. Thus, an injectable drug containing 2.5 mg of each of the above active ingredients per ampoule was produced. In addition, CS-P
S is stearoylpalmitoylphosphatidylserine-bonded chondroitin sulfate (GAG is chondroitin sulfate (shark cartilage-derived, molecular weight 30,000) as a raw material, lipid is stearoylpalmitoylphosphatidylserine, and synthetic method is a reducing terminal lactonization method (Japanese Patent Laid-Open No. 4-80201). , Example 1- (3)), HA-PE is dipalmitoyl-L-
(Α-phosphatidyl) ethanolamine-bonded hyaluronic acid (as a raw material, GAG is hyaluronic acid (derived from chicken cob,
The molecular weight is 10,000), the lipid is dipalmitoyl-L- (α-phosphatidyl) ethanolamine, and the synthetic method is a reducing end-limited oxidation method (JP-A-4-80202, Example 1-).
(2) -1)) was used.

[0061]

INDUSTRIAL APPLICABILITY The therapeutic agent for neurological diseases containing the lipid-bound GAG of the present invention as an active ingredient is effective in promoting neurite outgrowth of peripheral and central nerve cells and restoration of nerve function damaged by nerve damage or nerve defect. It is effective for treating various diseases of the peripheral nervous system and the central nervous system because it has an accelerating effect, and is expected to have an effect of preventing degeneration and loss of nerve cells and an effect of regenerating nerve fibers. For example, various peripheral disorders such as neuropathy caused by traumatic nerve injury or nerve deficiency (particularly somatic neuropathy, more particularly sensory neuropathy), metabolic disorder polyneuropathy, mechanical neuropathy, toxic neuropathy, etc. Nervous system diseases; and various central nerves that are expected to have therapeutic effects by regenerating nerve fibers such as stroke, cerebral infarction, cerebral hemorrhage, cerebral trauma, memory impairment, senile dementia, Alzheimer's disease and Parkinson's disease Effective against disease.

Since the lipid-bound GAG, which is the active ingredient of the drug of the present invention, is a compound with extremely low toxicity, it is considered that the drug of the present invention has almost no side effects.

[Brief description of drawings]

FIG. 1 is a phase-contrast photomicrograph of the morphology of an organism showing the neurite outgrowth promoting effect of CS-PE on PC12 cells. A: Laminin 10 μg / ml without CS-PE B: Laminin 10 μg / ml CS-PE 100 μg / ml C: Laminin 5 μg / ml Without CS-PE D: Laminin 5 μg / ml CS-PE 100 μg / ml E: Laminin 1 μg / ml Without CS-PE F: Laminin 1 μg / ml CS-PE 100 μg / ml

FIG. 2 is a phase-contrast photomicrograph of the morphology of an organism showing the effect of GAG and lipid-bound GAG on neurite outgrowth of rat fetal brain-derived primary cultured neurons. A: Control B: CS (chondroitin sulfate) 50 μg / ml C: CS-PE 0.5 μg / ml D: HA (hyaluronic acid) 50 μg / ml E: HA-PE 0.5 μg / ml

[Fig. 3] Primary culture neurons derived from rat fetal brain.
It is a graph which shows the neurite outgrowth promotion effect of HA-PE and CS-PE.

FIG. 4 is a graph showing the degree of motor dysfunction and the degree of trauma in a rat sciatic nerve injury model as a total disability degree score by a score determination method over time.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C08B 37/08 C08B 37/08 Z (72) Inventor Koji Kizen Uedayama, Tenpaku-ku, Nagoya-shi, Aichi 1 chome 1404 (72) Inventor Sachiko Goto 1-39-34 Sakaemachi, Higashimurayama, Tokyo

Claims (3)

[Claims] 1. A therapeutic agent for neurological diseases comprising a lipid-bound glycosaminoglycan or a salt thereof as an active ingredient. 2. The therapeutic agent according to claim 1, wherein the therapeutic agent for neurological diseases is a neurite outgrowth promoting agent. 3. The drug according to claim 1, wherein the glycosaminoglycan is chondroitin sulfate or hyaluronic acid.