JP2003160498A - Inhibitor of distension of epithelial cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new medicament having high safety to a living body, and effectively inhibiting the distension of an epithelial cell. <P>SOLUTION: A lipid-bonded glycosaminoglycan obtained by bonding glycosaminoglycan with a lipid, or a pharmacologically acceptable salt thereof is used as an active ingredient in the inhibitor of the distension of the epithelial cell. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an epithelial cell spreading inhibitor containing a lipid-bound glycosaminoglycan as an active ingredient.

[0002]

2. Description of the Related Art Conventionally, as lipid-bound glycosaminoglycans, for example, those described in Japanese Patent No. 2997018, Japanese Patent No. 2986519, Japanese Patent No. 2987518 and Japanese Patent Laid-Open No. 9-30979 are known. It is known that these substances have cancer metastasis inhibitory activity and cell adhesion inhibitory activity.

Further, it is known that the lipid-bound glycosaminoglycan has an action of suppressing the adhesion of hepatocytes and the like to a substrate and the like and causing spherical agglomeration (Japanese Patent No. 3199405 etc.). However, it is not known that lipid-bound glycosaminoglycans suppress the spreading of epithelial cells.

On the other hand, a drug that suppresses the spread of epithelial cells is considered to be useful as a drug that suppresses the hyperplasia of epithelium. For example, the suppression of the spread of the epithelium of the cornea or conjunctiva is suppressed by the keratoconus, the progression of astigmatism, It is considered to be effective in suppressing the formation of keratoconjunctival scars due to congenital keratoconjunctival epithelial disease and secondary trauma, and suppressing the extension of skin epithelium is effective in suppressing postoperative keloidization. However, a useful and highly safe drug that can be used for these uses has not been obtained so far.

[0005] Therefore, expectations for a drug useful for suppressing the spreading of epithelial cells are increasing.

[0006]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a new drug that is highly safe for living organisms and effectively suppresses the spread of epithelial cells.

[0007]

Means for Solving the Problems The inventors of the present invention, as a result of intensive studies in view of the above problems, surprisingly found that a lipid-bound glycosaminoglycan having a cancer metastasis-suppressing effect has been The present invention was found to be useful as a new drug that effectively suppresses the spread of epithelial cells, and completed the present invention.

That is, the present invention comprises an epithelial cell spreading inhibitor characterized by containing a lipid-bound glycosaminoglycan in which a lipid is bound to glycosaminoglycan or a pharmacologically acceptable salt thereof as an active ingredient, and The gist of the invention is a pharmaceutical composition containing the same.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below with reference to embodiments of the invention. <1> Epithelial Cell Spreading Inhibitor of the Present Invention The epithelial cell spreading inhibitor of the present invention comprises a lipid-bound glycosaminoglycan in which a lipid is bound to glycosaminoglycan or a pharmacologically acceptable salt thereof as an active ingredient. It is characterized by containing.

Glycosaminoglycan, which is a component of lipid-bound glycosaminoglycan, which is an active ingredient of an epithelial cell spreading inhibitor, comprises D-glucosamine or D-galactosamine, D-glucuronic acid, L-iduronic acid and / or Or a polysaccharide or oligosaccharide composed of a repeating unit of disaccharide of D-galactose as a basic skeleton, extracted from natural products such as animals, obtained by culturing microorganisms, chemical or enzymatic Any of those synthesized in 1 can be used. Specifically, for example, hyaluronic acid, chondroitin, chondroitin sulfate (chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate E, chondroitin sulfate K, chondroitin polysulfate, etc.), dermatan sulfate, heparin, heparan sulfate,
Examples thereof include keratan sulfate (including keratan polysulfate), chondroitin sulfate, heparin, and hyaluronic acid are preferable, and chondroitin sulfate is particularly preferable, but not limited thereto.

The molecular weight of chondroitin sulfate is generally about
It is about 1,000 to 100,000, preferably about 2,000 to 80,000, particularly preferably about 3,000 to 70,000. The molecular weight of heparin and heparan sulfate is generally about 1,000 to 60,000, but preferably about 2,000 to 18,000, and particularly about
2,500 to 17,000 is preferable. The molecular weight of hyaluronic acid is generally about 1,000 to 15,000,000, but about 5,000.
~ 10,000,000 is preferable, especially about 10,000-1,000,00
0 is preferable. The molecular weight of glycosaminoglycan usually means the average molecular weight, and generally refers to the weight average molecular weight calculated from the intrinsic viscosity.

As the lipid bound to the above glycosaminoglycan, natural lipids derived from animals, plants, microorganisms, etc., or complex lipids or simple lipids chemically or enzymatically synthesized or partially decomposed are used. Any of glycerolipids such as phospholipids, long-chain fatty acids, long-chain aliphatic amines, cholesterols, sphingolipids and ceramides can be used. Especially phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylthreonine, ethanolamine plasmalogen, serine plasmalogen, phospholipids such as lysophosphatidylcholine, lysophosphatidylinositol, and neutral lipids such as monoacylglycerol and diacylglycerol. Is preferred. Of these, phospholipids are particularly preferred.

The chain length and the degree of unsaturation of the acyl group in the lipid having an acyl group are not particularly limited, but those having 6 or more carbon atoms are preferable. Examples of the acyl group include palmitoyl (hexadecanoyl) and stearoyl (octadecanoyl). In addition, these lipids may be commonly used pharmacologically acceptable salts.

The binding position of the glycosaminoglycan with the lipid is not particularly limited, but the terminal portion of the glycosaminoglycan is preferable, and the binding to the reducing end is particularly preferable. The mode of bonding is not particularly limited, but chemical bonding is particularly preferable, and covalent bonding is most preferable.

In the case of a lipid-bound glycosaminoglycan in which glycosaminoglycan and a lipid are covalently bonded, a carboxyl group (including lactone), formyl group (including hemiacetal group), hydroxyl group or 1 of glycosaminoglycan
A functional group such as a primary amino group, or the functional group separately introduced to glycosaminoglycan, a carboxyl group of a lipid,
An acid amide bond (-CO-NH-), an ester bond or an aminoalkyl bond (-CH ₂ ) formed with a functional group such as a formyl group or a primary amino group, or the functional group separately introduced into a lipid. Those which are covalently bonded by -NH-) are preferred.

Particularly, the reaction of the carboxyl group (including lactone) of glycosaminoglycan formed by the chemical treatment by opening the pyranose ring at the reducing end of glycosaminoglycan and the primary amino group of lipid. Acid-amide bond (-CO-NH-) formed by the reaction, the acid-amide bond (-CO-NH-) formed by the reaction of the carboxyl group of the uronic acid part of glycosaminoglycan and the primary amino group of the lipid
-), Or the Schiff base formed by the reaction of the formyl group of glycosaminoglycan formed by chemical treatment by opening the pyranose ring at the reducing end of glycosaminoglycan and the primary amino group of lipid Those linked by an aminoalkyl bond (—CH ₂ —NH—) formed by reduction of is preferable.

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

[0018] As a method for producing a lipid-bound glycosaminoglycan, which is a particularly preferred lipid-bound glycosaminoglycan in which a lipid is covalently bonded to the reducing end of glycosaminoglycan, for example, Japanese Patent Nos. 2997018 and 2986519 are disclosed. The methods disclosed in Japanese Patent Laid-Open No. 2986518 and Japanese Patent Laid-Open No. 9-30979 can be mentioned. For example, the following methods can be used.

(Reducing end limited oxidation method) In this method, a galactose residue, a uronic acid residue or a hexosamine residue, which is a sugar residue at the reducing end of glycosaminoglycan, is reduced and limited oxidation (partial oxidation) is performed. As a result, the pyranose ring at the reducing end is specifically opened (cleaved),
Forming a formyl group at the reducing terminal of the glycosaminoglycan to give an aldehyde compound, reacting the formyl group of the aldehyde compound with the primary amino group of the lipid to form a Schiff base, and then reducing the Schiff base, It is a method of forming an aminoalkyl bond (-CH ₂ -NH-) to covalently bond a glycosaminoglycan and a lipid.

The sugar residue at the reducing end of glycosaminoglycan is reduced by about 5 to 50 equivalents, preferably 25 to 30 equivalents of reducing agent (sodium borohydride, cyanoborohydride) with respect to glycosaminoglycan. Alkali borohydride salt such as sodium) and a suitable aqueous solvent (for example,
Water, borate buffer, etc.), usually 10 to 30 ° C., preferably
It can be performed at 15 to 25 ° C.

After the above reduction, limited oxidation is carried out to produce an aldehyde compound having a formyl group at the reducing end of glycosaminoglycan. The limited oxidation uses 1 to 10 equivalents, preferably 3 to 6 equivalents of an oxidizing agent (sodium periodate, alkaline periodate such as potassium periodate, etc.) with respect to the reduced glycosaminoglycan. , Usually 0 ~ 10 ℃,
It can be preferably carried out at 0 to 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 carried out in an aqueous solvent (water, phosphate buffer, etc.). ) Or a solution of the above aldehyde compound in a suitable organic solvent (dimethylformamide, dimethylsulfoxide, etc.) and a solution of lipids in a suitable organic solvent (chloroform, methanol, etc.) are mixed, and usually at 15-60 ° C. It is possible to react at the temperature of. During or after this reaction, a suitable reducing agent (alkali borohydride such as sodium borohydride, sodium cyanoborohydride, etc.) can be applied to reduce the Schiff base.

When producing a lipid-bound glycosaminoglycan by this reaction method, a divalent functional spacer compound having a primary amino group (for example, ethylenediamine, etc.) is used instead of the lipid having a primary amino group. Alkylenediamine, or an amino acid such as lysine) with the above aldehyde compound to give an aminoalkyl bond (—CH ₂ —N
H-) and then having a functional group (eg, carboxyl group) capable of reacting with the other functional group (eg, amino group) of the spacer compound (eg, monoacylglycerol such as monoacylglycerol succinate) It may be reacted with a dicarboxylic acid ester).

(Reducing end lactonization method) This method comprises oxidizing a galactose residue, a uronic acid residue or a hexosamine residue which is a sugar residue at the reducing end of glycosaminoglycan to give a pyranose ring at the reducing end. Is specifically opened to form a carboxyl group at the reducing end of the glycosaminoglycan, and then subjected to a lactone formation reaction to form the reducing end of the glycosaminoglycan into a lactone structure. It is a method of forming a acid amide bond (-CO-NH-) by reacting a primary amino group of a lipid with a glycosaminoglycan to form a covalent bond with the lipid.

For the oxidation of the sugar residue at the reducing end of glycosaminoglycan, an oxidizing agent (iodine, bromine, etc.) is used in an amount of about 2 to 20 equivalents, preferably about 5 to 15 equivalents, relative to the glycosaminoglycan. In a suitable aqueous solvent (for example, water, phosphate buffer, etc.), it can be carried out usually at 0-40 ° C, preferably 15-30 ° 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 Co.)
And / or acid (inorganic acid such as hydrochloric acid, sulfuric acid, or acetic acid,
It is possible to produce a lactone compound in which the reducing end of glycosaminoglycan is specifically lactonized by treatment with an acid anhydride of an organic acid such as citric acid or succinic acid.

The reaction of the obtained lactone compound with a lipid having a primary amino group (phospholipid such as phosphatidylethanolamine) can be carried out in a suitable aqueous solvent (water, phosphate buffer, etc.) or a suitable organic solvent ( Dimethylformamide,
A solution of a lactone compound in dimethylsulfoxide, etc.) and a solution of a lipid in an appropriate organic solvent (chloroform, methanol, etc.) are mixed and reacted at a temperature of 5 to 80 ° C, preferably 30 to 60 ° C. Just do it.

As in the case of the reducing end-limited oxidation method, a divalent functional spacer compound having a primary amino group is reacted with the above lactone compound instead of the lipid having a primary amino group to give an acid. An 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 (eg, carboxyl group).

However, the method for producing a lipid-bound glycosaminoglycan in which a lipid is covalently bonded to the reducing end of glycosaminoglycan is not limited to these methods, and a lipid is bound to the reducing end of glycosaminoglycan. It can be produced by any other method as long as it is possible.

As a method for introducing a lipid other than the terminal of glycosaminoglycan, for example, the carboxyl group of the uronic acid moiety of glycosaminoglycan is reacted with the primary amino group of the lipid to form an acid amide bond (--CO -NH-) is formed.

In the above reaction, a condensing agent (eg, 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 reacted with an activator (eg, N-hydroxysuccinimide, p-nitrophenol, N-hydroxybenzotriazole, etc.) in the presence of the condensing agent to form an active ester, It can be reacted to form an acid amide bond (-CO-NH-). By such a method, a plurality of lipids can be bound to one molecule of glycosaminoglycan.

In the above reaction, the uronic acid moiety of glycosaminoglycan is formed into a salt (such as an 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, It is preferably carried out in pyridine).

Examples of the lipid-bound glycosaminoglycan salt include alkali metal salts such as sodium and potassium;
Examples thereof include alkaline earth metal salts such as calcium and magnesium; amine salts such as trialkylamine; salts with organic bases such as pyridine, and the like, but are not particularly limited, and pharmacologically acceptable alkali metal salts are particularly preferable. The sodium salt is preferred and the most preferred.

Specific examples of the above-mentioned lipid-bound glycosaminoglycan include dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bound hyaluronic acid, dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bound chondroitin sulfate. , Stearoyl palmitoyl phosphatidylserine-bonded chondroitin sulfate, monostearoyl glycerol-succinate ester-bonded chondroitin sulfate, dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded hyaluronic acid, dipalmitoyl-L- (α-phosphatidyl) ethanolamine-bonded heparin Etc.

The epithelial cell spread inhibitor of the present invention is a drug that suppresses the spread of epithelial cells. The agents that suppress cell spreading and growth include agents that suppress cell proliferation by damaging cells and consequently suppress cell spreading, and agents that have no cytotoxic effect and suppress cell spreading itself. To be
The epithelial cell spreading inhibitor of the present invention is the latter drug. The fact that the epithelial cell spreading inhibitor of the present invention does not have a cytotoxic effect can be confirmed by, for example, observing a hematoxylin-eosin (HE) stained image histologically. The epithelial cells are skin, cornea, conjunctiva,
Examples include epithelial cells of the digestive tract. For such cells, it is useful as a reagent to be used by adding it to a medium in vitro in order to suppress spreading, and can be applied to a living body as the pharmaceutical composition of the present invention described later.

<2> Pharmaceutical Composition of the Present Invention The pharmaceutical composition of the present invention is a pharmaceutical composition containing the epithelial cell spreading inhibitor of the present invention.

The tissue to which the pharmaceutical composition of the present invention is applied is
Examples include, but are not limited to, skin, cornea, conjunctiva, epithelium of digestive tract, and the like, in particular, treatment and prevention of astigmatism caused by healing of corneal surface damage, treatment and prevention of corneal epithelial hyperplasia such as keratoconus. , Treatment, prevention, surgery of corneal epithelial diseases such as corneal conjunctival scar caused by invasion of conjunctival epithelium into cornea, eyelid adhesion, treatment, prevention of keloid formation on skin caused by injury, treatment of post-cataract, Prevention (JP
9-235239, JP-A-9-291040, etc.) is preferable.

In using the epithelial cell spreading inhibitor of the present invention as a pharmaceutical composition, various dosage forms can be prepared, and various commonly used excipients and additives can be added.

Examples of the dosage form of the pharmaceutical composition of the present invention include eye drops, ointments such as eye ointments, liquids and the like. Examples of the additive include a mixture or combination of salts, preservatives, antibiotics, anti-inflammatory agents and growth factors. Examples of salts include sodium sulfate, sodium chloride, sodium hydrogen phosphate, etc., examples of preservatives include methyl paraoxybenzoate, propyl paraoxybenzoate, benzalkonium chloride, etc., examples of antibiotics include oxytetracycline, anti-inflammatory agents, etc. Examples thereof include diclofenac.

Since the lipid-bound glycosaminoglycan, which is an active ingredient of the pharmaceutical composition of the present invention, is a substance having a high stability, the excipient used in the preparation of ordinary pharmaceutical compositions. Preparation aids such as agents and stabilizers can also be used without any trouble.

The active ingredient of the pharmaceutical composition of the present invention is
Lipid-bound glycosaminoglycans are known to be low-toxic compounds (Japanese Patent Laid-Open No. 6-728936).

[0042]

EXAMPLES The present invention will be described in detail below with reference to examples. <1> Effect of Lipid-Bound Glycosaminoglycan on Rabbit Corneal Epithelial Cell Spreading L- (α-phosphatidyl) ethanolamine dipalmitoyl produced by the method described in Example 1 of Japanese Patent No. 2997018 was used. 1 piece of chondroitin sulfate C (manufactured by Seikagaku Corporation) derived from shark cartilage having a weight average molecular weight of 20,000 attached to the reducing end (hereinafter abbreviated as “CS-PE”)
800 in 199 medium supplemented with 0 mM buffer HEPES (2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid)
It was added at a concentration of μg / ml (this was used as the test medium and CS-PE
-Free medium, and weight average molecular weight of 2 instead of CS-PE
199 medium containing 800 µg / ml of shark cartilage-derived chondroitin sulfate C (manufactured by Seikagaku Corporation) was used as a control medium and a comparative medium, respectively).

Japanese White Native Rabbit (Female 2.7-3.2 kg)
Six birds were euthanized, and a piece of the strong cornea was separated from the excised eyeball and divided into two at the central part of the cornea. One was used as a control medium and the other was used as a test medium, and the cells were cultured at 37 ° C under 5% CO ₂ / air for 15 hours.

After culturing, the scleral corneal pieces were thoroughly washed with phosphate buffered saline (PBS) and fixed with 10% buffered formaldehyde. After 24 hours, the pieces of sclera were further divided into two and fixed for several days. Then, according to the conventional method, embedding in paraffin, slicing, HE
It was dyed. A micrograph was taken at 100 times and the length of the corneal epithelium extended on the cut surface of the corneal stroma was measured (Fig. 1). A photomicrograph after culturing in the control medium is shown in FIG. 2, and a photomicrograph after culturing in the comparative medium is shown in FIG.

When CS-PE was added, the extent of extension was lower than that of the control medium to which nothing was added,
The comparison medium containing only chondroitin sulfate C showed the same degree of spreading as the control medium. From this, it became clear that cell spreading is suppressed by the binding of lipid to glycosaminoglycan. In addition, no cytotoxic effect was observed in any of the HE-stained specimens.

Six eyes in each group were used to perform a test (t-test) of the difference between two corresponding samples with respect to the extended length of the scleral corneal piece cultured in the control medium (FIG. 4).

As a result, no significant difference was found between the control medium and the comparative medium, but a significant difference was found between the control medium and the test medium.

<2> Safety test Four weeks old Sic-ddy male and female mice were preliminarily bred for 1 week, and then male 2
At the time of reaching a body weight of 3 to 30 g and a female weight of 20 to 25 g, CS-PE prepared in <1> and hyaluronic acid having a weight average molecular weight of 10,000 according to the method of Example 1 of Japanese Patent No. 2997018 were prepared. The L- (α-phosphatidyl) ethanolamine dipalmitoyl-bonded hyaluronic acid (HA-PE) 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, it was revealed that each compound had an LD ₅₀ value of 2000 mg / kg or more, and had no problem as a medicine.

<3> Formulation Example (1) Eye drops Sodium chloride 900 mg, Methyl paraoxybenzoate 26 m
g and propyl paraoxybenzoate 14 mg, and sterilized purified water 80 m
Add l, dissolve by heating, return the solution to room temperature, and then
S-PE (3,000 mg) was added and dissolved, and sterile purified water was added to make the total amount 100 ml. 10 ml of this was dispensed into a container to produce an eye drop. An eye drop was produced with the CS-PE amount of the above eye drop being 500 mg. An eye drop was produced with the CS-PE amount of the above eye drop being 100 mg. 100 mg of L- (α-phosphatidyl) ethanolamine dipalmitoyl-bonded hyaluronic acid prepared by using the hyaluronic acid having a weight average molecular weight of 10,000 by the method of Example 1 of Japanese Patent No. 2997018 was added to CS-PE of the above eye drop. Instead, an eye drop was produced. An eye drop was prepared by using 300 mg of glycerol monostearate-bound chondroitin sulfate C prepared by the method of Example 3 of Japanese Patent No. 2986518 in place of the above CS-PE.

(2) Eye ointment Purified lanolin (10 g) and yellow petrolatum (80 g) were heated and melted together, liquid paraffin droplet amount was added to 100 g, and the heat mixture was filtered with a filter paper using a heat-retaining funnel, and at 150 ° C. Sterilized for 1 hour. After returning the mixture to room temperature, 3,000 mg of CS-PE and 100 mg of methyl paraoxybenzoate as a preservative were added and mixed to be uniform. An ointment was manufactured by filling the container with 10 g of the mixture. An ointment was produced with the CS-PE amount of the above ointment being 500 mg. An ointment was produced with the CS-PE amount of the above ointment being 200 mg. An ointment in which 100 mg of L- (α-phosphatidyl) ethanolamine dipalmitoyl-bonded hyaluronic acid prepared by using the hyaluronic acid having a weight average molecular weight of 10,000 by the method of Example 1 of Japanese Patent No. 2997018 was replaced with the above CS-PE. The agent was manufactured.

[0051]

INDUSTRIAL APPLICABILITY The epithelial cell spreading inhibitor of the present invention can effectively and safely promote the prevention and cure of diseases caused by epithelial hyperplasia.

[0052]

[Brief description of drawings]

FIG. 1 is a diagram of tissue staining showing the effect of CS-PE on the spreading of corneal cells on rabbit corneas.

FIG. 2 is a diagram of tissue staining showing the effect of control medium on rabbit corneas.

FIG. 3 is a diagram of tissue staining showing the effect of a comparative medium on rabbit corneas.

FIG. 4 is a view showing the results of t-test on the effect of CS-PE on the spreading of corneal cells on rabbit corneas.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 17/02 A61P 17/02 27/10 27/10 43/00 105 43/00 105 C08B 37/00 C08B 37/00 G (72) Inventor Katsuyoshi Sakurai 2-527-6 Kurashiki, Higashiyamato-shi, Tokyo F-term (reference) 4C086 AA01 AA02 EA20 EA24 EA26 EA27 MA01 MA04 NA14 ZA33 ZA89 ZB21 4C090 AA09 BA62 BA64 BA66 BA67 BA68 DA09 DA23

Claims (9)

[Reference number] J200101400 [Claims] 1. An epithelial cell spreading inhibitor comprising a lipid-bound glycosaminoglycan in which a lipid is bound to glycosaminoglycan or a pharmacologically acceptable salt thereof as an active ingredient. 2. The glycosaminoglycan is a substance selected from the group consisting of hyaluronic acid, chondroitin, chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, and keratan sulfate. Epidermal cell spreading inhibitor. 3. The epithelial cell spreading inhibitor according to claim 1, wherein the lipid is glycerolipid. 4. The epithelial cell spreading inhibitor according to claim 3, wherein the glycerolipid is a phospholipid. 5. The epithelial cell spreading inhibitor according to claim 4, wherein the phospholipid is phosphatidylethanolamine. 6. The epithelial cell spreading inhibitor according to claim 1, wherein the glycosaminoglycan is chondroitin sulfate and the lipid is phosphatidylethanolamine. 7. The epithelial cell spreading inhibitor according to any one of claims 1 to 6, wherein a lipid is covalently bonded to the reducing end of glycosaminoglycan. 8. A pharmaceutical composition comprising the epithelial cell spreading inhibitor according to any one of claims 1 to 7. 9. The pharmaceutical composition according to claim 8, which is a corneal epithelial cell spreading inhibitor.