AU2012201314B2 - Agent for applying to mucosa and method for production thereof - Google Patents

Abstract

An agent for applying to mucosa capable of persistently exerting a therapeutic effect on disorders such as inflammation and lesions in the mucosa even by a lower frequency of 5 administration because the agent can stay at a diseased site for a long period of time by exhibiting a high staying property in a mucosal epithelial layer is provided, said agent for application to mucosal epithelial layer is provided, said agent for application to mucosa containing glycosaminoglycan (GAG) 10 into which a hydrophobic group is introduced via a binding chain, as an active ingredient. C VOf rd%;PEC-229946cx

Description

AUSTRALIA Patents Act COMPLETE SPECIFICATION (ORIGINAL) Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Seikagaku Corporation Actual Inventor(s): Katsuya Takahashi, Yuuji Shimojima, Kenji Miyamoto Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: AGENT FOR APPLYING TO MUCOSA AND METHOD FOR PRODUCTION THEREOF Our Ref : 936081 POF Code: 96621/256659 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- 2 DESCRIPTION AGENT FOR APPLYING TO MUCOSA AND METHOD FOR PRODUCTION THEREOF The present application is a divisional from Australian 5 patent application no 2006300207, which claims the benefit of priority from Japanese provisional application number 2005-297170, filed on 12 October 2005, and Japanese provisional application number 2006-160478, filed on 9 June 2006. The contents of both applications are hereby incorporated by 10 reference in their entirety. (0001] Technical Field The present invention relates to an agent for applying to 15 mucosa containing a hydrophobic group binding type glycosaminoglycan as an active ingredient, and a method for production thereof. [0002] Background Art 20 Conventionally, as substances having healing effects on mucosal disorders such as inflammation and lesions, hyaluronic acid which is a representative glycosaminoglycan (hereinafter described as "GAG") has been known (e.g., Patent Document 1) . However, in mucosa in the cornea, oral cavity and nasal cavity, 25 and conjunctiva which contacts with the external world, and mucosa in the urinary bladder, the mucosal surfaces are washed with secretions and excretions such as tear fluid, salivary fluid and urine, and foreign substances are removed. Thus derivatives of GAG which keep medicinal effects inherent in GAG 30 and exert a high staying property in these mucosal tissues have been demanded. [0003] Meanwhile, photoreactive hyaluronic acid whose water solubility has been increased by binding a photocrosslinking C:\pohod6PEC-826200.docX 3 group such as cinnamic acid to hyaluronic acid and further giving an alkali treatment thereto has been known (e.g., Patent Document 2). This photoreactive hyaluronic acid has been provided by binding a photocrosslinking group such as cinnamic 5 acid to hyaluronic acid in order to provide medical materials such as anti-adhesion materials by giving the photocrosslinking, and does not aim at enhancing the staying property in the mucosal tissue. (0004] 10 (Patent Document 1] Japanese Published Unexamined Patent Publication No. Hei-1-238530 (Patent Document 2] Japanese Published Unexamined Patent Publication No. 2002-24950 [0004a] 15 Throughout the description and claims of the specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps. [0004b] 20 A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims. 25 (0005] Disclosure of the Invention Problem to be Solved by the Invention The present invention aims at providing an agent for applying to mucosa, which exerts an excellent staying property 30 and pharmacological effects in mucosa. [0006] Means for Solving the Problem As a result of an extensive study for solving the above problem, the present inventors have found that "hydrophobic Cl4ctod\SPEC426299 docx 4 group binding type GAG" obtained by binding a hydrophobic group to GAG via a binding chain can be used as an extremely excellent active ingredient in an agent for applying to mucosa because this GAG keeps healing effects inherent in GAG on 5 mucosal disorders such as inflammation and lesions and exhibits a high staying property when applied thereto, and have completed the present invention. [0007] The present invention relates in one aspect to an agent 10 for applying to mucosa which contains glycosaminoglycan (GAG) into which the hydrophobic group is introduced via the binding chain. [0007a] In one aspect the invention relates to an agent when 15 used for the treatment of mucosal disorders of eyes or oral cavity, comprising, as an active ingredient, hyaluronic acid or a salt thereof into which a hydrophobic group selected from the groups as shown below is introduced via -CO-NH- as a binding chain: 20 groups: an alkyl group having 2 to 18 carbon atoms and an arylalkenyl group. In another aspect the invention relates to a method of treating mucosal disorders of eyes or oral cavity, which method comprises applying to a mucosa requiring treatment an 25 agent according to the invention in an amount which effectively treats said mucosal disorder. [0008] Effects of the Invention 30 The agent for applying to mucosa of the present invention can exert the persistent healing effect on the mucosal disorder such as inflammation and lesions even by low frequent administration because this can stay at a SPEC-826299.doCX -5 diseased site for a long period of time by exhibiting high staying property in the mucosa. [0009) Brief Description of the Drawings FIG. 1 is a view showing a spectrum of light transmittance; FIG. 2 is a view showing healed area percentages; FTG. 3 is a view showing healed areas; FIG. 4 is a view showing healing rate; FIG. 5 is a view showing healed areas; FIG. 6 is a view showing healing rate; FIG. 7 is a view showing healed areas; FIG. 8 is a view showing.healing rate; FIG. 9 is a view showing healed areas; FIG. 1.0 is a view showing healing rate; FIG. 11 is a view showing staying property at peeled sites in rabbit corneal epithelia; FIG. 12 is a view showing staying property at peeled sites in rabbit corneal epithelia; FIG. 13 is a view showing photographs of an eyeball after irradiation with ultraviolet rays; FIG. 14 is a view showing amounts of water evaporation in a removed cornea; FIG. 15 is a view showing amounts of water evaporation in a removed cornea; and FIG. 16 is a view showing changes in water evaporation amount ratio in model hamsters for xerostomia. FIG. 17 is a view showing healed areas; FIG. 18 is a view showing healing rate; FIG. 19 is a view showing healed areas; FIG. 20 is a view showing healing rate. [0010] Best Modes for Carrying Out the Invention The present invention will be described in more detail below by the best modes for carrying out the invention.

-6 Herein, an alkyl group refers to a straight or branched aliphatic hydrocarbon group having a described number of carbon atoms. An alkenyl group refers to a straight or branched aliphatic hydrocarbon group having a described number of carbon atoms, having at least one double bond. An alkynyl group refers to a straight or branched aliphatic hydrocarbon group having a described number of carbon atoms, having at least one triple bond. [0011] An aryl group refers to a monocyclic or polycyclic aromatic hydrocarbon group having 6 to 20 carbon atoms as ring-constituting atoms. A heteroaryl group refers to a monocyclic or polycyclic aromatic hydrocarbon group having 3 to 20 carbon atoms and one or more heteroatoms selected from nitrogen, sulfur and oxygen atoms as the ring constituting atoms. [00121 An arylalkyl group refers to the alkyl group defined above substituted with the aryl group defined above. An arylalkenyl group refers to the alkenyl group defined above substituted with the aryl defined above. An arylalkynyl group i-q the alkynyl group defi.nci above substituted with the aryl group defined above. (0013] An amino acid group refers to a group derived by losing a carboxyl group, an amino group or a hydroxyl group by a chemical bond from a natural or synthetic amino acid. [001.4] Herein, the term "treatment" includes prevention, control of progression (prevention of deterioration), improvement (reduction) and cure of the mucosal disorder. The "mucosal disorder" means a condition where morphology, properties and functions to be inherent in the mucosa are disordered in some form. For example, the mucosal disorder can include the conditions such as lesions, -7 defects, erosion, inflammation, ulcers and dryness. GAG into which the hydrophobic group is introduced via the binding chain, which is contained as the active ingredient in the agent for applying to mucosa of the present invention can be any GAG as long as the GAG binds the group having hydrophobicity derived from a hydrophobic compound having a water insoluble and oil soluble nature. This hydrophobic group is bound to GAG via the binding chain. As described later, it is not necessary that all constitutive units of GAG bind the hydrophobic groups. 1) GAG GAG into which the hydrophobic group is introduced via the binding chain contained in the agent for applying to mucosa of the present invention is an acidic polysaccharide having a repeated long chain structure of disaccharide composed of amino sugar and uronic acid (or galactose). Examples of such GAG include hyaluronic acid, chondroitin, chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate and keratan sulfate, and among them, hyaluronic acid is preferable. These GAG may be pharmaceutically acceptable salts thereof. Examples of such salts include sodium salts, potassium salts, magnesium salts and calcium salts, and among them, the sodium salt is preferable. Therefore, it is the most preferable that GAG in the agent for applying to mucosa of the present invention is sodium hyaluronate. An origin of GAG is not particularly limited, and GAG may be derived from an animal or a microorganism or chemically synthesized. For example, when using sodium hyaluronate, those derived from cock's comb can be exemplified. The molecular weight of GAG is not particularly limited, but its weight average molecular weight is preferably 200,000 to 3,000,000, more preferably 500,000 to 2,000,000 and most preferably 600,000 to 1,200,000. When hyaluronic acid or the pharmaceutically acceptable salt thereof is used, its weight average molecular weight is preferably 200,000 to 3,000,000, -8 more preferably 500,000 to 2,000,000 and most preferably 600,000 to 1,200,000. 2) Hydrophobic group The hydrophobic group in GAG into which the hydrophobic group is introduced via the binding chain contained in the agent ior applying to mucosa of the present invention is any group as long as the hydrophobic group is derived from the compound having the water insoluble and oil soluble nature. Examples of such a group can include alkyl groups having 2 to 18 carbon atoms, a)kenyl groups having 2 to 18 carbon atoms, alkynyl groups having 2 to 18 carbon atoms, aryl groups, heteroaryl groups, arylalkyl groups, arylalkenyl groups, arylalkynyl groups and amino acid groups. The alkyl groups having 2 to 18 carbon atoms can include methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t butyl, n--pent.Iyl, t-pentyl, isopentyi, neopentyl, n--heptyl, 5-methylhexyl, 4,4-dimethyl-pentyl, 1,1- dimethyl-pentyl and n-octyl. Among them, the alkyl. groups having 2 to 6 carbon atoms such as n-butyl can be preferably included. The alkenyl groups having 2 to 18 carbon atoms can include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2 methyl-l-propenyl, 1-methyl-1-propenyl, 1-pentenyl, 3 methyl-2-butenyl, 1-heptene-1-yl and 2-heptene-1-yl. Among them, the alkenyl groups having 2 to 6 carbon atoms such as 1-butenyl can be preferably included. The alkynyl groups having 2 to 18 carbon atoms can include ethynyl, 1-propinyl, 2 -propinyl, 1-butynyl, 2- butynyl, 3 butynyl, 1-heptynyl, 2-heptynyl and 3-heptynyl. Among them, the alkynyl groups having 2 to 6 carbon atoms such as l butynyl can be preferably included. The ary. groups can include groups such as phenyl, naphthyl, anthryl and phenanthryl. rhe heteroary). groups can include groups such as furyl, thionyl, thiazolyl, oxazolyl, pyrrolyl, pyridyl, ~9 pyrimidinyl and indolyl. The arylalkyl groups can include groups such as benzyl, phenethyl, naphthylmethyl and naphthylethyl. The arylalkenyl groups can include groups such as 2 phenyl- ethenyl and p- aminophenylethenyl. The arylalkynyl groups can include 'groups such as 2 phenyl- ethynyl and p- aminophenylethynyl. rhe amino acid groups can include groups derived from aliphatic amino acids such as glycine, alanine and p-alanine; branched aliphatic amino acids such as leucine, isoleucine and valine; aromatic amino acids such as phenylalanine and tyrosine; and heterocyclic amino acids such as tryptophan and histidine. These hydrophobic groups may be monosubstituted or polysubstituted with groups such as hydroxyl, carboxyl, cyano, amino (which may be monosubstituted or disubstituted with the above alkyl), nitro, oxo arid al.kylcarbonyloxy. Among the above hydrophohic groups, Aryl groups, arylalkyl groups, arylal.kenyl groups and arylalkynyl groups which are the hydrophobic groups containing the aryl group can be preferably included, and the arylalkenyl group and the aryl group substituted with the alkylcarbonyloxy group can be particularly preferably -10 included. As such an arylalkenyl group, it is possible to specifically use phenylethenyl and p-aminophenylethenyl. As the aryl group, it is possible to preferably use the groups such as CH 3

-(CH

2 ),-COO-phenyl (wherein 1 represents 0 or an integer of 1 to 18). [0025) These hydrophobic groups may also have a function such as ultraviolet ray absorption ability due to having a double bond in the hydrophobic group as shown by a functional group such as phenyl-ethenyl exemplified above contained in the hydrophobic group. For example, when the agent for applying to mucosa of the present invention is used as eye drops described later, it is possible to make the eye drops having the function effectively absorbing the harmful ultraviolet rays by the use of the group having the ultraviolet ray absorption ability as the hydrophobic group, Furthermore, for example, when the agent for applying to mucosa of the present invention is used for the treatment of corneal epithelial layer disorders such as corneal xerosis (dry eye), keratoconjunctivitis, superficial punctate keratitis (SPK), corneal epithelial erosion, cornl.. ep.ithelia1 loss and corneal tumor, it is possible to make the agent for applying to mucosa which has pharmacological effects on the above disorders in combination with the function effectively absorbing the harmful ultraviolet rays by the use of the group having the ultraviolet ray absorption ability as the hydrophobic group. As the group having the ultraviolet ray absorption ability, for example, arylalkenyl group having the conjugation double bond which is exemplified by 2-phenyl-ethenyl and p aminophenylethenyl described above is preferable. When the agent for applying to mucosa of the present invention is used for the corneal disorder, it is preferable that "GAG into which the hydrophobic group is introduced" which is the active ingredie.nt of the agent for applying -11 to miosa of the present invention is made into an aqueous solution of 0.1% by weight, which blocks 70 to 100% transmission of the ultraviolet rays at a wavelength of 200 to 300 nm when an ultraviolet ray transmittance is measured by the method described in the Example described later. Such a hydrophobic group having the ultraviolet ray absorption ability can preferably include the arylalkanyl groups such as 2-phenyl-ethenyl and p aminophenylethenyl. [0026] 3) Binding chain In GAG into which the hydrophobic group is introduced via the binding chain contained in the agent for applying to mucosa of the present invention, the above GAG'. is hound to the above hydrophohic group via the binding chain. GAG has the functional group which is a carboxyl, hydroxyl or sulfonate (-SO3H) group as the side chain. Thus, the hydrophobic group can be bound to GAG via the binding chain obtained by forming an ether bond, carboxylate ester bond, sulfate ester bond, carboxylic acid amide bond or sulfonate amide bond together with these functional groups. Auch a hinding chain can specifically include -CONH-, -COO-, -0-, -SO 3 - and -SO 2 NH-. Among them, the carboxylic acid amide bond of -CONH- and the carboxylate ester bond of -Coo- can be preferably used, and the carboxylic acid amide bond of -CONH- can be particularly preferably used. [0027] Q..Spacercha in In GAG into which the hydrophobic group is introduced via the binding chain contained in the agent for applying to mucosa of the present invention, the hydrophobic group is bound to GAG via the above binding chain, and a spacer chain may further exist between the binding chain and the hydrophobic group. As such a spacer chain, any chain group can be used as long as the spacer -12 group does not completely lose the pharmacological effects which GAG has. Specifically, -(CH2)m- and -(CH 2

)-(OCH

2 )n (wherein m and n are integers of 1 to 18, respectively) can be included. These spacer chains can further have the binding chalis such as -CONH-, -COO-, -0-, -S03- and -SO2NH- which are the same as above at the hydrophobic group side. Such a spacer chain having the binding chain at the hydrophobic group side can specifically include -C00-(CH 2 )m-, -C00 (CH2)-(OCH2)n-, -CONH-(CH 2 )m- and -CONH-(CH 2 )-(OCH2)n- (0028) 5) Ratio having hydrophobic group In GAG into which the hydrophobic yroup is introduced via the binding chain contained in the agent for applying to mucosa of the present invention, it is not necessary that all of GAG constitutive units respectively have the hydrophobic groups. A ratio of the bound hydrophobic group in molar equivalent relative to a disaccharide repeat unit in molar equivalent of GAG (hereinafter, referred to as an "introduction ratio") can be optionally determined depending on the type of the hydrophobic group, the degree of required hydrophobicity, the type of the mucosal disorder administered with the agent for applying to mucosa and the administration site, etc. For example, when using a phenylethenyl group which may be substituted as the hydrophobic group, preferably 5 to 30% dnd more preferably 10 to 20%o of a hydrophobic group in molar equivalent is introduced relative to the disaccharide repeat unit in molar equivalent of GAG (in the case where the crosslinking bond described later is not formed). (00291 6) Crosslink forming group In GAG into which the hydrophobic group is introduced via the binding chain contained in the agent for applying to mucosa of the present invention, the -13 hydrophobic group may form a crosslinking bond between GAG molecules by the functional group contained in the group. As the hydrophobic group capable of forming the crosslinking bond, any group can be used as long as the hydrophobic group produces a photodimerization reaction or a photopolymerization reaction by irradiation of ultraviolet rays and is the same as defined above. The hydrophobic group capable of forming the crosslinking bond includes, for example, phenylethenyl, p aminophenylethenyl, ethenyl, 2-carboxyethenyl and pentane-1,3 dienyl. It is desirable that these groups are bound to GAG via the binding chain which contains the carbonyl group. Among these hydrophobic groups, phenylethenyl or p aminophenylethenyl which is bound to GAG via the binding chain which contains the carbonyl group can be particularly preferably used. In such GAG into which the hydrophobic group is introduced capable of forming the crosslinking bond, GAG molecules can be crosslinked with one another by being subjected to the photodirnerization reaction or the photopolymerization reaction by standard methods. For example, according to the methods described in Japanese Published Unexamined Patent Publication No. 2002-249501, the photodimerization reaction or the photopolymerization reaction can be given. [00303 7) Preferable GAG constitutive unit Representatives of GAG into which the hydrophobic group is introduced via the binding chain contained in the agent for applying to mucosa of the present invention can specifically include the agents for applying to mucosa containing GAG into which Ph-CH=CH-COO- (CH 2 ) tm-NH-; Ph-CH=CH-COO-CH2- (OCH 2 )na-NH-; Ph CH=CH-COtN1H-(CH 2 )m-N1H-; Ph-CH=CH-CONH-CH 2

-(OCH

2 )n-NH-; Ph-CH=CH

COO-(CH

2 )m-O-; Ph-CH=CH-COO-CH 2

-(OCH

2 )n-O-; Ph-CH=CH-CONH-(CH2), o ; Ph -CH.CH- CONH--C11-- (OCH2) n-O-; CH 3 - (CH 2 ) I-COO-Ph-CONH- (CI 2

),,

NH- or CH-(CH 2 ) -COO-Ph-CONl-CH 2 -(OCHi 2 ),-NH- (wherein Ph -14represents phenyl group, m and n represent integers of 1 to 18, respectively, and 1 represents U or an integer of 1 to 18) is introduced, as the active ingredient. The following GAG can be included as the representative. GAG having the repeat unit of the structural unit represented by the Chemical formula 1, as a basic skeleton: [Chemical formula 1) AH OH 0 ' i NHRc wherein, R represents R 1 or R2; Ac represente an acetyl group; R, represents ONa or OH; R, represents (1) Ph-CIH=CIH-COO- (CH).-NH-; (2) Ph-CH=CH -COO-CH 2 - (OCH 2 ) n-NH--; (3) Ph-CH=CH-CONH- (CH 2 ),,-NH; (4) Ph-CH=CH-CONH-CH2-- (0CH2),-NH-; (5) Ph-CH=CH-COO- (CH) 1 bO; (6) Ph-CH=CH-COO-CH 2 - (OCH 2 ) n-O-; (7) Ph-CH=CH-CONH-

(CH

2

)

1 ,-O-; (8) Ph-CH=CH-CONH-CH 2 - (OCH 2 )n-O-; (9) CH 3 - (CH 2

)

1 -COO-Ph-CONH- (CH 2 ) ,-NH ; or (10) CH -(C,) 1 -COO-Ph-CONH-CH2- (OCH2)n-NH--; wherein, Ph represents phenyl group, m and n represent integers of 1 to 10, respectively, and 1 represents 0 or an integer of 1 to :1.8, as a basic skeleton, wherein the ratio of the above structural unit wherein R repr-esents R3 is 5 to 30% in molar equivalent relative to the -15 di-saccharide repeat unit in molar equivalent of the GAG. [00311 8) Method for producing GAG into which hydrophobic group is introduced via binding chain To obtain GAG into which the hydrophobic group is introduced via the binding chain, GAG is reacted with a hydrophobic compound in which the above hydrophobic group has been bound to t.he functional. group such as hydroxyl, carboxyl, amino or sulfonate group which can form an ether bond, carboxylate ester bond, sulfate ester bond, carboxylic acid amide bond or sulfonate amide bond together with the carboxyl, hydroxyl or sulfonate (-SO 3 H) group in GAG. Specifically, when the bond is the carboxylic acid amide bond, GAG having the carboxyl group is reacted with the hydrophobic compound having an amino group to bind the carboxyl group in GAG to the amino group in the hydrophobic compound. In the case of the carboxylate ester bond, GAG is reacted with the hydrophobic compound having hydroxyl or carboxyl group to bind the carboxyl group in GAG to the hydroxyl group in the hydrophobic compound or bind the hydroxyl group in GAG to the carboxyl. group in the hydrophobic compound. Tn the case of the ether bond, GAG having the hydroxyl group is reacted with the hydrophobic compound having the hydroxyl group to react the hydroxyl group in GAG with the hydroxyl group in the hydrophobic compound. In the case of the sulfonate ester bond, GAG is reacted with the hydrophobic compound having the hydroxyl group or sulfonate group to bind the hydroxyl group in GAG to the sulfonate group in the hydrophobic compound or bind the sulfonate group in GAG to the hydroxyl group in the hydrophobic compound. These reactions can be performed by common standard methods, and reaction conditions can be optionally selected by those skilled in the art. [00321 When the spacer chain is present between the binding -16 chain and the hydrophobic group, the order in introducingthe spacer chain and the hydrophobic group to GAG is not particularly limited. For example, either the method in which a spacer compound having the functional group such as the hydroxyl, carboxyl, amino or sulfonate group which can form the ether bond, carboxylate ester bond, sulfate ester bond, carboxylic acid amide bond or sulfonate amide bond together with the functional group in GAG at one end of the above spacer chain is reacted with GAG, and subsequently, the other end of the spacer compound is reacted with the hydrophobic compound which is bound to the functional group such as the hydroxyl, carboxyl, amino or sulfonate group, or the method in which the spacer compound having the functional group such as the hydroxyl., carboxyl, amino or sulfonate group which can form an ether bond, carboxylate ester bond, sulfate ester bond, carboxylic acid amide bond or sulfonate amide bond together with the functional group in the hydrophobic compound at one end is reacted with the hydrophobic compound in which the hydrophobic group has been bound to the functional group such as the hydroxyl, carboxyl, amino or sulfonate group, and subsequently the other end of the spacer compound is reacted with GAG may be used. In particular, the method in which the spacer compound is reacted with the hydrophobic compound followed by being reacted with GAG can be preferably used. [0033) The above-described method can be appropriately carried out by publicly known methods, and preferably performed in the presence of a condensing agent. Such a condensing agent can preferably include water soluble carbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI.HCl), condensing agents such as dicyclohexylcarbodiimide (DCC) and N hydroxysuccinate imide (HOSu). For example, when hyaluronic acid is used as GAG and the cinnamate -17 derivative such as Ph-CH=CH-COO-(CrH 2

),-NH

2 or Ph-CH=CH-C0O

CH

2

-(OCH

2

),-NH

2 (wherein m and n are integers of 1 to 18, respectively) is used as the hydrophobic compound which is bound to the spacer compound, the condensation method using water soluble carbodiimide such as 1-ethyl-3-(3 dimethylaminopropyl)carbodiimide hydrochloride (EDCI.HCl) and N-hydroxysuccinate imide can be preferably used. The ro.action can be accomplished using a mixed solvent of water and a water soluble organic solvent such as dioxane, dimethylformamide or ethanol. The hyaluronate derivative which is highly soluble in an aqueous vehicle can be obtained by treating with a base such as sodium hydrogen carbonate after the completion of the reaction. [0034] When the thou prodrceri GAG into which the hydrophobic group is introduced via the binding chain is subjected to the photodimerization reaction or the photopolymerization reaction to cross link the GAG molecules with one another, for example, the method described in Japanese Published Unexamined Fatent Publication No. 2002-249501 can be used. Specifically, in the case of the compound in which the phenylethenyl group as the hydrophobic group is bound to GAG via -COO-(CH 2 )m-NHCO-, the crosslink can be formed by irradiating light to the solution containing them using an ultraviolet lamp. [0035) 9)Agent for applyinq to mucosa of the present invention The agent for applying to mucosa of the present invention contains one or more GAG into which the hydrophobic group is introduced via the binding chain as the active ingredient, and may also further include other medically, pharmaceutically or biologically acceptable substances other than the GAG into which the hydrophobic group is introduced via the binding chain. Such substances include but are not limited to, salts such as sodium chloride, potassium chloride, disodium hydrogen -18 phosphate, sodium dihydrogen phosphate and monopotassium hydrogen phosphate, and preservatives such as paraoxybenzoate esters, benzalkonium chloride, chlorobutanol and chlorhexidine gluconate, and other pharmacologically active ingredients. The agent for applying to mucosa ot the present invention can be made into any publicly known formulation forms (e.g., solid preparations such as granules and powder, liquid preparations such as aqueous solutions, suspension and emulsion, and gel preparations) as the pharmaceutical for applying to the mucosa. In the agent for applying to mucosa of the present invention, the form thereof upon formulating and distributing and the form thereof upon applying to the mucosa may be the same or different. For example, the agent for applying to mucnosa of the present invention may be formulated in the form of solution and may be applied directly to the mucosa as it is. Also, the agent for applying to mucosa of the present invention may be formulated and distributed in the solid torm, and may be made into solution or gel when being applied to the mucosa. Thus, the agent for applying to mucosa of the present invention can be made into the formulation form for being prepared when used. When being made into the liquid agent by dissolving in water, the amount of GAG into which the hydrophobic group is introduced via the binding chain is preferably 0.02 to 5% by weight, more preferably 0.1 to 3% by weight, extremely preferably 0.1 to 1% by weight and most preferably 0.1 to 0.6% by weight. [0036] <Applied subjects> The agent for applying to mucosa of the present invention aims at applying to the mucosa. Animals to which the agent for applying to mucosa of the present invention is applied are not particularly limited as long as they have the mucosa, and mammalian animals are -19 preferable. The mammali.an animals include, but are not limited to, humans, horses, cattle, dogs, cats, rabbits, hamsters, guinea pigs and mice. The agent for applying to mucosa of the present invention may be of course made into the pharmaceuticals for humans, and can also be made into the pharmaceuticals for the animals. Among them, it is preferable to be made into the pharmaceuticals for humans. The mucosa to which the agent for applying to mucosa of the present invention can be applied is not particularly limited as long as the mucosa is the mucosa present in the animal. Such mucosa include mucosal tissues present in organs and tissues exemplified by the gastrointestinal system such as the stomach and intestines, the cardiovascular system, the respiratory system, the excretion system such as the urinary bladder, rectum and anus, the genital system such as the vagina, and organs such as eyes, nose and oral cavity which contact with the external world. Among them, the agent for applying to mucosa of the present invention can be preferably applied to the cornea, conjunctiva, oral cavity mucosa and urinary bladder mucosa. [0032] <Applied diseases> The agent for applying to mucosa of the present invention can be widely applied to such mucosa. The purpose of the application is not particularly limited, and for example, the purposes such as protection of the mucosal tissue (e.g., prevention of snow blindness by ultraviolet rays, pterygium and cataract), prevention of mucosal dryness and the treatment of mucosal disorder can be exemplified. Thus, the agent for applying to mucosa of the present invention can be applied to not only the mucosa in the abnormal state (e.g., mucosa where the disorder has occurred) but also the mucosa in the normal state. However, since the agent for applying to mucosa of the present invention exerts excellent pharmacological -20 effects in the mucosa where the disorder has occurred, it is possible to preferably use for the treatment of the mucosal disorders, e.g., disorders in the cornea, conjunctiva, oral cavity mucosa and urinary bladder mucosa. [0038] Since the agent for applying to mucosa of the present invention exerts excellent pharmacological effects particularly on disorders in the mucosal epithelia among the mucosal disorders, it is possible to be preferably used for the treatment of the disorders in the mucosal epithelia. [003.91 Examples of such disorders in the mucosal epithelia include corneal epithelial layer disorders such as corneal xerosis (dry eye); keratoconjunctivitis, superficial punctate keratitis (SPK), corneal epithelial erosion, corneal epithelial loss and corneal tumor; oral cavity mucosal disorders such as xerostomia (dry mouth), aphthous ulcer, stomatitis and glossitis; dryness and pruritus of nasal mucosa; urinary bladder mucosal disorders such as interstitial cystitis; ulcerative proctitis, and dryness of the rectum or vagina. Also dryness and lesions of organ mucosa upon surgical operation can be exemplified. Among them, it is possible to be preferably used for the treatment of the corneal epithelial layer disorders, the oral cavity mucosal epithelial disorders and the urinary bladder mucosa epithelial layer disorders. Application method and amount [00401 The agent for applying to mucosa of the present invention can be applied to the mucosal tissues exemplified above, and its application method and application formulation can be appropriately determined by those skilled in the art depending on the position, morphology, property and function of the mucosa to be applied, and the purpose of the application. However, it -21 is preferable that the agent for applying to mucosa of the present invention is applied to the mucosa in the liquid form such as solution in use. In that case, upon producing (formulating) or applying the agent for applying to mucosa of the present invention, the liquid can be obtained by dissolving GAG into which the hydrophobic group is introduced via the binding chain in the'solvent. The solvent is not particularly limited as long as the solvent can dissolve the GAG into which the hydrophobic group is introduced via the binding chain and is the pharmaceutically acceptable solvent. For example, a buffer such as a phosphate buffer or saline can be used, but the solvent is not limited thereto. In this case, the concentration of the GAG into which the hydrophobic group is introduced via the hinting chain in the liquid agent is not particularly limited, and can be appropriately determined depending on the type of the mucosa to be applied and the degree of the mucosal disorder. When the agent for applying to mucosa of the present invention is the eye drops, when the agent for applying to mucosa of the present invention is applied to the oral cavity mucosa or the nrinary bladdimr mucosa, for Poxample, the concentration is preferably 0.02 to 5% by weight, more preferably 0.1 to 3% by weight, still more preferably 0.1 to 1% by weight, still more preferably 0.1 to 0.6% by weight, extremely preferably 0.1 to 0.5% by weight and most preferably 0.1 to 0.3% by weight. [0041] When the agent for applying to mucosa of the present invention is applied to the mucosa in the stomach as the liquid as above, an oral administration or the administration using a catheter can be selected, When applied to the mucosa in the eye, the nose or the oral cavity, for example, the administration method such as instillation of drop, nasal instillation or oral inclusion can be selected. For example, when the agent for applying -22 to mucosa of the present invention is applied to the mucosa in the urinary bladder, rectum or vagina, or the mucosa of organs where the dryness is concerned upon surgical operation, the method of administering by injecting, spraying or applying the agent for applying to mucosa of the present invention to a lumen or a surface of these organs or tissues can be selected, but the methods are not limited thereto. [00423 The amount, the number of times and the frequency of the application (administration of) of the agent for applying to mucosa of the present invention is not particularly limited, and should be determined depending on the mucosa subjected to the application, the purpose of the application, the type, age, hody weight, gender, and degree of mucosal disorder in the animal to be applied. (0043] Specifically, when the agent for applying to mucosa of the present invention is used for the purpose of treating the human corneal epithelial layer disorder, the agent for applying to mucosa of the present invention at the above-described concentration as the liquid formulation for the instillation of drops (eye drops) containing GAG into which the hydrophobic group is introduced via the binding chain can be administered by instilling 1 to 3 drops per administration 1 to 5 times per day, and may be administered by instilling 1 to 3 drops per administration 1 to 3 times per day. [0044) When the agent for applying to mucosa of the present invention is used for the purpose of treating the human oral cavity mucosal disorder, the agent for applying to mucosa of the present invention at the above-described concentration as the liquid containing GAG into which the hydrophobic group is introduced via the binding chain can be administered by putting the agent for applying to -23 mucosa of the present invention in the oral. ravity 1. to 5 times per day and rinsing for approximately several tens of seconds (preferably approximately 20 to 30 seconds) followed by spitting it out. [0045] When the agent for applying to mucosa of the present invention is applied to the urinary bladder mucosal disorder, this is preferably used for the treatment of the urinary bladder mucosal disorders exemplified by non bacterial refractory cystitis exemplified by interstitial cystitis, eosinophilic cystitis and hemorrhagic cystitis which do not respond to anti-bacterial agents although symptoms similar to those ot acute bacterial cystitis are exhibited. In this case, the agent for applying to mucosa of the present invention at the above-dc.rihd concentration as the liquid containing GAG into which the hydrophobic group is introduced via the binding chain can be administered by administering the agent for applying to mucosa of the present invention directly to the urinary bladder at the amount of 50 mL per administration 1 to 7 times per week or administering with a catheter in the or.nary bladder. [0046] The agent for applying to mucosa of the present invention can stay at the diseased site for a longer period of time because the active ingredient contained in the agent exhibits the high staying property in the mucosa, compared with the conventional drugs containing hyaluronic acid ;is the active ingredient in which no hydrophohir group has been bound. Therefore, the agent for applying to mucosa of the present invention can also exert the treating effect persistently even at the low administration frequency on the disorders such as inflammation and lesions in the mucosa. However, the agent for applying to mucosa of the present invention is not limited hy its administration freqnenry.

-24 t004?3 The present invention will be described below by Examples. CExample 1> (1-1) Preparation of cinnamate derivative-introduced sodium hyaluronate A 172 mg / 5 mL aqueous solution of N hydroxysuccinimide (HOSu: Watanabe Chemi cal. Industries, Ltd.), a 143 mg / 5 mL aqueous solution of 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDCI.KCl) (Watanabe Chemical Industries, Ltd.), and a 181 mg / 5 mL aqueous solution of 3-aminopropyl cinnamate hydrochloride (Tokyo Chemical industry Co., Ltd.) were added to a solution of sodium hyaluronate (1.06 g, 2.7 mmol/disaccharide unit, weight-average moleinilar weight 900,000; derived from cock's comb, Seikagaku Corporation) in water (11.5 mL) / dioxane (144 mL) . The mixture was stirred for 3 hours and a 750 mg / 10 mL aqueous solution of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added. Atter stirring further 2 h J min., the reaction was quenched with acetic acid (214mg) and sodium chloride (1.0 g). Ethanol (300 mL) was added and the resulting precipitation was filtered off and washed twice successively by 80% ethanol, 95% ethanol. The solid was dried in vacuo at 40 *C over night to afford white solid (1.06 g) (hereinafter in Examples, "cinnamate derivative introduced sodium hyaluronate" is abbreviated as "cinnamate derivative-introduced HA") . The introduction ratio of the cinnamate derivative was 16 %. The introduction ratio of the cinnamate derivative was calculated based on the amount of cinnamate by an absorbance measurement method (wavelength: 269 nm) and the amount of hyaluronate by a carbazole sulfate method. (1-2) Preparation of cinnamate derivative-introduced HA solution Saline was added to 96 mg of cinnamate derivative- -25 introduced HA obtained in the above (1-1) to give a total amount of 15.45 ml, then the solution was shaken over night with a shaker until uniformly dissolved. The 0.5 % by weight solution of cinnamate derivative-introduced HA in drying loss 10%) was obtained, Likewise, the 0.3 % by weight and 0.1 % by weight solutions of cinnamat.e derivative-introduced HA were obtained. (00481 <Example 2> (2-1) Preparation of cinnamate derivative-introduced HA A 75 mg / 5 mL aqueous solution of HOSu, a 62 mg / 5 mL WFI solution of EDCI.HCl, and a 92 mg / 5 mL WFI solution of 6-aminohexyl cinnamate hydrochloride (Tokyo Chemical Industry Co., Ltd.) were added to a solution of sodium hyaluronate (1.0 g, 2.5 mmol/disaccharide unit, weight-average molecular weight 1,500,000; derived from cock's comb, Seikagaku Corporation) in water for injection (hereinafter referred to as WFI) (150 mL) / dioxane (75 mL). The mixture was stirred for 4 hours and sodium chloride (1.0 g) was added. Etnanol (500 ML) was added and the resulting precipitation was filtered off and washed twice successively by 80% ethanol, ethano.. The solid was dried in vacuo at 40 *C to afford white solid (1.1 g). The introduction ratio of the cinnamate derivative was 2.7 %. (2-2) Preparation of crosslinked cinnamate derivative introduced HA The above cinnamate derivative-introduced HA (12.5 g) was dissolved in phosphate buffered saline (concentration of phosphate: 1.5 mM, hereinafter abbreviated as "PRA") to prepare 2.5% solution of cinnamate derivative-introduced HA (500 mL ). The 2.5% solution of cinnamate derivative introduced HA was irradiated by 800W high pressure mercury lamp and performed by a heat treatment in an autoclave at 121*C for 7.5 min. to yield crosslinked cinnamate derivative-introduced HA. Further, 1 g of the above crosslinked cinnamate -26 derivative-introduced 4A was dissolved in 11.5 ml of WFI to prepare the 0.2 % by weight of crosslinked cinnamate derivative-introduced HA. [0049] <Example 3> (3-1) Preparation of cinnamate derivative-introduced HA A 172 mg / 5 mL aqueous solution of HOSu, a 143 mg / 5 ml, aqueous solution of EDCI.HCrl, and a 181 mg / 5 mL aqueous solution of 3-aminopropyl cinnamate hydrochloride (Tokyo Chemical Industry Co., Ltd.) were added to a solution of sodium hyaluronate (1.0 g, 2.5 mmol/disaccharide unit, weight-average molecular weight 900,000; derived from cock's comb, Seikagaku Corporation) in water (150 mL) / dioxane (75 mL) . The mixture was stirred for 3 hours and a 250 mg / 10 ml, aqueous solution of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added. After stirring further 2 h 30 min., the reaction was quenched with acetic acid (214mg) and sodium chloride (1.0 g) . Ethanol (300 mL,) was added and the resulting precipitation was filtered off and washed twice successively by 80% ethanol, 95% ethanol. The solid was dried in vacuo at 40 *C. to afford white solid (1.0 g) as cinnamate derivative-introduced HA. The introduction ratio of the cinnamate derivative was 10.1 %. (3-2) Preparation of fluorochrome-labeled cinnamate derivative-introduced HA A 3.0 mmol / mL aqueous solution of HOSu, a 1.5 mmol / mL aqueous solution of EDCI.HCl and a 1.5 mmol / mL aqueous solution of 4-aminofluorescein (Tokyo Chemi.cal Industry Co., Ltd.) were added to a solution of cinnamate derivative-introduced HA obtained in the above (3-1) (1.00 g, 2.5 mmol/disaccharide unit) in water (150 mL) / dioxane (75 mL) . The mixture was stirred one day and a 500 mg / 10 mL aqueous solution of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added. After stirring further 4 h 30 min., the reaction was quenched with acetic -27 acid (2 mL) and sodium chloride (6.0 g). Ethanol (500 mL) was added and the resulting precipitation was filtered off and washed four times by 80% ethanol, twice by ethanol. The solid was dried in vacuo over night to afford fluorochrome-labeled solid (782 mg) The introduction ratio of the fluorescence was 0.60%. [0050] <Comparative Example 1> Preparation of fluorochrome-labeled HA A 3.0 mmol / mL aqueous solution of HOSu, a 1.5 mmol / mL aqueous solution of EDCI.HCl and a 1.5 mmol / mL aqueous solution of 4-aminofluorescein (Tokyo Chemical Industry Co., Ltd.) were added to a solution of sodium hyaluronate(l.00 g, 2.5 mmol/disaccharide unit, weight average molecular weight 900,000; derived from cock's comb, Seikagaku Corporation) in water (150 mL) / dioxane (75 mL). The mixture was stirred one day and a 500 mg / 10 mL aqueous solution of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added. After stirring further 4 h 30 min., the reaction was quenched with acetic acid (2 mL) and sodium chloride (6.0 g). Ethanol (500 mL) was added and the resulting precipitation was filtered off and washed four times by 80% ethanol, twice by ethanol. The solid was dried in vacuo over night to afford fluorochrome-labeled solid (830 mg) The introduction ratio of the fluorescence was 0.32%. [0051] <Example 4> Measurement of ultraviolet ray transmittance of cinnamate derivative-introduced HA solution The 0.1 % by weight aqueous solution of cinnamate derivative-introduced HA obtained in the above (1-1) was prepared, and the ultraviolet ray transmittance was measured by an spectrometer (UV-1600, Shimadzu Corporation). A spectrum which indicated the transmittance is shown -28 in FIG. 1, and the transmittance (%) at various wavelengths is shown in Table 1. As a result, 100% of the transmittance was shown at the wavelengths of 340 nm or more, but the LransmiLLance dL the weivelengths of approximately 320 nm or less was 20% or less which was extremely low, and it was demonstrated that this solution effectively blocks the transmission of the ultraviolet ray. In FIG. 1, scales are shown with 65 nm intervals on a horizontal axis and with 20% intervals on a vertical axis. Table 1 _ _ _T(%) 234 19.9 340 106.2 380 101.8 450 98.5 In the table, X and T represent the wavelength and the transmittance (%), respectively. <Example 5> The effect of the cinnamate derivative-introduced HA on the healing of rabbit corneal epithelium. The effect of the cinnamate derivative-introduced HA prepared in Example 1 on the healing of rabbit corneal epithelium with surgical removal (The surgical Model). (5-1) Methods 1) Surgical removal of corneal epithelium (The surgical model) The corneal epithelium of the central region was removed by using a trephine (8 mm I.D), a 23G needle and microscissors after anesthesia with an intravenous injection of 5 mg/kg of ketamine and 2mg/kg of xylazine and topical administration of 0.4% oxybuprocaine hydrochloride. 2) Topical administration One hour and 4 hours after the corneal epithelia were peeled, 150 pal of saline as the control substance was administered in the left eye, and 150 pl of 0.5% by weight -29 cinnamate derivative-introduced RA solution prepared in the above Example (1-2) as the subject substance was administered in the right eye. On one day and 2 days after the peeling, a total of 4 times with 3 hour intervals, and at 3 days after the peeling, with 3 hours interval, the same administration as above was performed. In the administration, 1 ml injection syringes were used. Six model rabbits for the corneal epithelial layer disorder described in the above 1) were used as administration subjects. 3) Photographing of corneal epithelial defective region The rabbit was given general anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the corneal epithelial loss site was stained with 0.2% sodium fluorescein dissolved in PBS, and photographed under ultra-violet light. The photographing was performed just before the administration of the subject substance one hour after the cornea was peeled and 3 hours after the final administration at 3 days after the peeling. When photographed, a focal length was made constant to make a magnification of photographs constant. 4) Measurement of corneal epithelial defective region The area of the corneal epithelial defective region stained with sodium fluorescein was measured on the printed photograph using an image analyzer. A value obtained by subtracting the area of the peeled site 3 hours after the final administration at 3 days after the peeling from the area (peeled area) of the peeled site just before the administration of the subject substance one hour after peeling the corneal epithelia was performed was rendered as "healed area." [0053] (5-2) Study results The results of a healed area percentage in each individual are ohown in PIC. 2, and the reoults of the -30 healed area percentage and a healed area percentage ratio in each individual are shown in Table 2. The healed area percentage and the healed area percentage ratio were calculated as follows. Healed area percentage (%) (Healed area/Peeled area) x 100 Healed area percentage ratio = (Healed area percentage in right eye/Healed area percentage in left eye) x 100 [0054) Table 2 Healed area percentage (%) Specimen right eye Healed area number (administration left eye percentage of cinnamate (administration ratio derivative- of saline) introduced HA) 2. 76.90 66.59 3.3.5.47 2 69.44 85.90 80.85 3 75.23 61.71 121.91 4 75.54 56.79 133.01 5 68.41 64.53 106.01 6 83.76 66.92 125.16 Mean 74.88 67.07 113.73 Standard 5.57 9.95 deviation [0055] For each individual of individual numbers 1 to 6, the left column shows the healed area percentage in the right eye (administration of cinnamate derivative-introduced HA), and the right column shows the healed area percentage in the left eye (administration of saline) . In FIG. 2 and Table 2, the apparent effect to facilitate the healing of the corneal epithelial layer disorder was observed in 5 of the administered 6 individuals. E00563 -31 <Example 6> The effect of the 0.5 % cinnamate derivative-introduced HA on the healing of rabbit corneal epithelium. The effect of the cinnamate derivative-introduced HA prepared in Example 1 on the healing of rabbit corneal epithelium with surgical removal (Th surgicall Model). (6-1) Methods 1) .iirgical removal of corneal epithelium (The surgical model) The corneal epithelium of the central region was removed by using a trephine (8 mm I.D), a 23G needle and microscissors after anesthesia with an intravenous injection of 5 mg/kg of ketamine and 2mg/kg of xylazine and topical administration of 0.4% oxbuprocaine hydrochloride. 2) Topical administration One hour and 4 hours after the corneal. epithelia were peeled, 150 pl of saline as the control substance was administered in the left eye, and 150 pl ot 0.56 by weight cinnamate derivative-introduced HA solution prepared in the above Example (1-2) ast Lhe subject substance was administered in the right eye. On one day and 2 days after the peeling, a total of 4 times with 3 hour intervals, and at 3 days after the peeling, with 3 hour intervals, the same administration as above was performed. In the administration, the 1 ml injection syringes were used. Fourteen model rabbits for the corneal epithelial layer disorder described in the above 1) were used as the administration subjects. 3) Photographing of corneal epithelial defective region The rabbit was given general anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the corneal epithelial loss site was stained with 0.2% sodium fluorescein dissolved in PBS, and photographed under ultra-violet light, The photographing was performed just before the administration -32 of the subject substance one hour after the cornea was peeled and 3 hours after the second administration at I to 3 days after the peeling. When photographed, the focal length was made constant to make the magnification of photographs constant. 4) Measurement of corneal epithelial defective region The area of the corneal epithelial defective region stained with sodium fluorescein was measured on the printed photograph using the image analyzer. The value obtained by subtracting the area of the peeled site 3 hours after the finial administration at 3 days after the peeling from the area (peeled area) of the peeled site just befuC the admUiiniLrtdLiun of the subject substance one hour after the cornea. epithelia were peeled was rendered as "healed area." (6-2) Study results The results of the healed area in each individual are shown in FIG. 3, and the results of a healing rate in each individual are shown in FIG. 4. The healed area and the healing rate were calculated as follows. Healed area = Area after peeling* - Area at each time point (after 1 to 3 days) * Herein after, "area after peeling" means 'the area of the peeled site just before the administration of the subject substance one hour after the cornea was peeled' in calculation of healed area. Healing rate - Mean of healed areas at respective time points (after 1 to 3 days) In FIGS. 3 and 4, it was observed that the healed area of the cornea). epithelia was significantly increased in the eyes administered with 0.5% by weight cinnamate derivative-introduced HA solution, compared with the healed area of the corneal epithelia in the control eyes. And it was also observed that the healing rate was significantly enhanced in the eyes administered with 0.5% -33 by weight cinnamate derivative-introduced HA solution. (0058] <Example 7> The effect of the 0.3 % cinnamate derivative-introduced HA on Lhe healing of rabbit corneal epithelium. The effect of the cinnamate derivative-introduced HA prepared in Example 1 on the healing of rabbit corneal epithelium with surgical removal (The surgical. Model). (7-1) Methods 1) Surgical removal of corneal epithelium (The surgical model) The corneal epithelium of the central region was removed by using a trephine (8 mm I.D), a 23G needle and microscissors after anesthesia with an intravenous injection of 5 mg/kg of ketamine and 2mg/kg of xylazine and topical administration of 0.4% oxbuprocaine hydrochloride. 2) Topical administration One hour and 4 hours atter peeJ.ing the corneal epithelia, 150 1 of saline as the control substance was administered in the left eye, and 150 pl of 0.3% by weight cinnamate derivative-introduced HA solution prepared in the above Example (1-2) as the subject substance was administered in the right eye, At one day and 2 days after the peeling, a total of 4 times with 3 hour intervals, and at 3 days after the peeling, with 3 hour intervals, the same administration as above was performed. In the administration, the 1 ml. injection syringes were used. Fourteen model. rabbits for the corneal epithelial layer disorder described in the above 1) were used as the administration subjects. 3) Photographing of corneal epithelial defective region The rabbit was given general anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the corneal epithelia). loss site was stained with 0.2% sodium fluorescein dissolved in PBS, -34 and photographed under iltra-violet light. The photographing was performed just before the administration of the subject substance one hour after the cornea was peeled and 3 hours after the second administration at 1 to 3 days after the peeling. When photographed, the focal length was made constant to make the magnification of photographs constant. 4) MeAsurement of corneal. epithelial defectivA region The area of the corneal epithelial defective region stained with sodium fluorescein was measured on the printed photograph using the image analyzer. The value obtained by subtracting the area of the peeled site 3 hours after the final administration at 3 days after the peeling from the area (peeled area) of the peeled site just before the administration of the subject substance one hour after the corneal epithelia were peeled was rendered as "healed area." [0059) (7-2) Study results The results of the healed area in each individual are shown in FIG. 5, and the results of a healing rate in each individual are shown in FIG. 6. The healed area and the healing rate were calculated as follows. Healed area = Area after peeling - Area at each time point (after 1. to 3 days) Healing rate - Mean of healed areas at respective time points (after 1 to 3 days) In FIGS. 5 and 6, it was observed that the healed area of the corneal cpithelia woo oignificantly increased in the eyes administered with 0.3% by weight cinnamate derivative-introduced HA solution at all time points of days 1 to 3, compared with the healed area of the corneal epithelia in the control eyes. And it was also observed that the healing rate was significantly enhanced in the eyes administered with 0.3% by weight cinnamate derivative introduced 11A solution.

-35 (0060) <Example 8> The effect of the 0.1 % cinnamate derivative-introduced HA on the healing of rabbit corneal epithelium (0.1% by weight cinnamate derivative-introduced HA aqueous solution and 0.1% by weight HA aqueous solution, 4 times of eye drops per day) The effect of the cinnamate derivative-introduced HA prepared in Example 1 on the healing of rabbit corneal epithelium with surgical removal (The surgical Model). (8-1) Methods 1) Surgical removal of corneal epithelium (The surgical model) The corneal epithelium of the central region was removed by using a trephine (8 mm I.D), a 23G needle and microscissors after anesthesia with an intravenous injection of 5 mg/kg of ketamine and 2mg/kg of xylazine and topical administration of 0 .4% oxbuprocaine hydrochloride. 2) Topical administration One hour and 4 hours after peeling the corneal epithelia, 150 l of an aqueouc solution of 0.1% by weight HA with a weight average molecular weight 600,000 to 1,200,000 as the control substance was administered in the left eye, and 150 ul of 0.1% by weight cinnamate derivative-introduced HA solution prepared in the above Example (1-2) as the subject substance was administered in the right eye. At one day and 2 days after the peeling, a total of 4 times with 3 hour intervals, and at 3 days after the peeling, with 3 hour intervals, the same administration as above was performed. In the administration, the 1 ml injection syringes were used. Eight model rabbits for the corneal epithelial layer disorder described in the above 1) were used as the administration subjects. 3) Photographing of corneal epithelial defective region -36 The rabbit was given general anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the corneal epithelial loss site was stained with 0.2% sdiuom fluorescein dissolved in PBS, and photographed under ultra-violet light. The photographing was performed just before the administration of the subject substance one hour after the cornea was peeled and 3 hours after the second administration at 1 to 3 days after the peeling. When photographed, the focal length was made constant to make the magnification of photographs constant. ') Measurement of corneal epithel.ial defective region The area of the corneal epithelial defective region stained with sodium fluorescein was measured on the printed photograph using the image analyzer. The value obtained by subtracting the area of the peeled site 3 hours after the final administration at 3 days after the peeling from the area (peeled area) of the peeled site just before the administration o the subject substance one hour atter the corneal epithelia were peeled was rendered as "healed area." (8-2) Study results The results of the healed area in each individual are shown in FIG. 7, and the results of the healing rate in each individual are shown in FIG. 8, The healed area and the healing rate were calculated as follows. Healed area - Area after peeling - Area at each time point (after 1 to 3 days) Healing rate " Mean of healed areas at respective time points (after 1 to 3 days) In FIGS. 7 and 8, it was observed that the healed area of the corneal epithelia was significantly increased in the eyes administered with 0.1% by weight cinnamate derivative introduced HA solution, compared with the -37 healed area of the corneal. epithelia in the control eyes administered with the 0.1% by weight HA aqueous solution. And it was also observed that the healing rate was significantly enhanced in the eyes administered with 0.1% by weight cinnamate derivative-introduced HA solution. [0062] <Example 9> The effect of the 0.1 % cinnamate derivative-introduced HA on the healing of rabbit corneal epithelium (0.1% by weight cinnamate derivative-introduced HA aqueous solution and 0.1% by weight HA aqueous solution, one time of eye drops per day) The effect of the cinnamate derivative-introduced HA prepared in Example 1 on the healing of rabbit corneal epithelium with surgical removal (The surgical Model). (9-1) Methods .) Surgical removal. of corneal epithelium (The surgical model) The corneal epithelium ot the central region was removed by using a trephine (8 mm I.D), a 23G needle and microscissors after anesthesia with an intravenous injection of 5 mg/kg of ketamine and 2mg/kg of xylazine and topical administration of 0.4% oxbuprocaine hydrochloride. 2) Topical administration One hour after the corneal epithelia were peeled, 150 pl of the aqueous solution of 0.1% by weight HA with a weight average molecular weight 600,000 to 1,200,000 as the control substance was administered in the left eye, and 150 il of 0.1% by weight cinnamate derivative-introduced HA solution prepared in the above Example (1-2) as the subject substance was administered in the right eye. Furthermore, at one to 3 days after the peeling, once a day, the same administration as above was performed. In the administration, the 1 ml injection syringes were used. Eight model rabbits for the corneal epithelial layer -38disorder described in the above 1) were used as the administration subjects. 3) Photographing of corneal epithelial defective region The rabbit was given general anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the corneal epithelial loss site was stained with 0.2% sodium fluorescein dissolved in PBS, and photographed under ultra-violet light. The photographing was performed just before the administration of the subject substance one hour after the cornea was peeled and 6 hours after the administration at 1 to 3 days after the peeling. When photographed, the focal length was made constant to make the magnification of photographs constant. 4) Measurement of corneal. epi.thelial defective region The area of the corneal epithelial defective region stained with sodium fluorescein was measured on the printed photograph using the image analyzer. The value obtained by subtracting the area of the peeled site 3 hours after the final administration at 3 days after the peeling from the area (peeled area) of the peeled site just before the administration of the subject substance one hour after the corneal epithelia were peeled was rendered as the "healed area." [00633 (9-2) Results rhe results of the healed area in each individual are shown in FIG. 9, and the results of the healing rate in each individual are shown in FIG. 10. The healed area and the healing rate were calculated as follows. Healed area = Area after peeling - Area at each time point (after I to 3 days) Healing rate = Mean of healed areas at respective time points (after I to 3 days) In FIGS. 9 and 10, it was observed that the healed .area of the corneal epithelia was significantly increased -39 in the oyes administered with 0.1% by weight n-innamAt.e derivative-introduced HA solution at all time points of the days 1 to 3, compared with the healed area of the corneal epithelia in the control eyes administered with the 0.1% by weight HA aqueous solution. And it was also observed that the healing rate was significantly enhanced in the eyes administered with 0.1% by weight cinnamate derivative-introduced HA solution. [00641 <Example 10> Staying property at rabbit corneal epithelial peeling site using fluorescence labeled cinnamate derivative-introduced HA The effect of the fluorescence labeled cinnamate derivative-introduced HA prepared in Example 3 and the fluorescence labeled HA prepared in Comparative Example 1 on the residual property of rabbit corneal epithelium with surgical removal. (The surgical Model). (10-1) Methods 1) surgical removal of corneal epithelium (The surgical model) The corneal epithelium of the central region was removed by using a trephine (8 mm I.D), a 23G needle and microscissors after anesthesia with an intravenous injection of 5 mg/kg of ketamine and 2mg/kg of xylazine and topical administration of 0.4% oxbuprocaine hydrochloride. 2) Topical administration One hour after the corneal epithelia were peeled, 150 pl of the aqueous solution of 0.3% by weight fluorescence labeled HA prepared in the above Comparative Example 1 as the control substance was administered in the left eye, and 150 pil of the aqueous solution of 0.3% by weight fluorescence labeled cinnamate derivative-introduced HA prepared in the above Example 3 as the subject substance was administered in the right eye. In the administration, -40 the 1 ml injection syringes were used. Eight model rabbits for the corneal epithelial layer disorder described in the above 1) were used as the administration subjects. 3) Removal of corneal epithelia and production of fro2en blocks Two rabbits were given general anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine per rabbit, and eyeballs were removed 30 minutes, one hour, one and a half hours and 2 hours and 30 minutes after the administration of the subject substance and the control substance. A pore was opened between the cornea and sclera in the removed eyeball using a surgical knife, and only the cornea was taken out using the microscissors. The removed cornea was placed on a biological sample slicing plate (supplied from Nisshin EM Corporation, Cat No. 428), and the portion to be observed was cut out using a single-edged stainless steel razor blade (GEM(R) STAINLESS STEEL UNCOATED, Nisshin EM Corporation, Cat No. 429). The cut out portion was immersed in O.C.T. compound (Tissue-Tek (R) 4583, 1ot. 11/U), then embedded in a cryostat tray (supplied by Murazumi Co., Ltd., Cat. No. 31) filled with the O.C.T. compound so that the portion to be observed was at a bottom, and rapidly frozen using liquid nitrogen in foam polystyrene to make an unfixed frozen block. 4) Production of frozen sections Subsequently, the frozen block was removed from the cryostat tray, and attached on a sample table using the 0.C.T. compound. The sample table and a disposable microtome blade (supplied by Leica Micosytems Japai, Model 818, Lot. No. 913212) were set in a high performance frozen microtome for research, and the block was sliced under the condition of a frozen chamber temperature (CT) at -20 0 C and sample side temperature (OT) at -16 0 C to make sections with a thickness of 5 pm using silane coating -41 slide glasses (supplied by Muto Pure Chemicals Co., Ltd., Star Frost Slide Glass, Cat. No. 5116). 5) Methods of observation and photographing The frozen section was set in an incident-light fluorescence microscope (Olympus Corporation, BH2-RFC), FA images and autofluorescent images were observed at IB cube (BH2-DMIB, excitation wavelength: 495 nm, absorption wavelength: 460 nim) and U cube (BH2-DMU, broad band U excitation, absorption wavelength: 435 nm), respectively. The FA image and the autofluorescent image were photographed using a cooled high sensitivity CCD camera (Keyence Corporation, VB 6010) under the condition of exposure time for one second and ISO sensitivity of 200. [0065) (10-2) Study results Photographs of the sampled cornea were shown in FIGS. 11 and 12, From FIGS. 11 and 12, it was identitied by color development of the fluorescence label that the aqueous solution of 0.3% by weight fluorescence labeled HA which was the control substance stayed until 30 minutes after the administration but did not stay after one hour. MeanwhilG, although the fluorescent color development of the aqueous solution of 0.3% by weight fluorescence labeled cinnamate derivative-introduced HA which was the subject substance was weakened with the elapse of time, the color development was observed at all time points from 30 minutes to 2 hours and 30 minutes after the administration, thereby the high staying performance was confirmed. [0066) <Example 11> Effect of the 0.3 % cinnamate derivative-introduced HA on rabbit eyes after exposure to ultraviolet light The protective effect on the cinnamate derivative introduced HA prepared in Example 1. on the rabbit with corneal superficial disorder.

-42 (11-1) Study procedure 1) Anesthesia and eyelid opening in rabbit Introduced anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine and maintained anesthesia by inhalation of isoflurane were given to the rabbit. Subsequently, the eyelid was always opened using an eyelid retractor for kids. 2) Administration of subject substance and control substance In the condition where the eye was opened, 150 p1 of an aqueous solution of 0.3% by weight HA with a weight average molecular weight 600,000 to 1,200,000 as the control substance was administered in the right eye, and 150 pl of 0.3% by weight cinnamate derivative-introduced HA prepared in the above Example (1-2) as the subject substance was administered in the left eye. In the administration, the 1 ml injection syringes were used. One rabbit described in the above 1) was used as the administration subject. 3) Irradiation of ultraviolet rays to rabbit's cornea Ultraviolet rays were irradiated to both eyes from a distance of approximately 10 cm apart from the rabbit eyeball using a 15 kW germicidal lamp. The irradiation was performed for 3 hours. 4) Photographing of ultraviolet rays irradiated site The eyeball was stained with 0.2% sodium fluorescein under the continuous anesthesia of the rabbit, and photographed under violet light. When photographed, the focal length was made constant to make the magnification of photographs constant. [0067] (11-2) Study results Photographs after the irradiation of the ultraviolet rays were shown in FIG. 13. From FIG. 13, in the eyeball irradiated with the ultraviolet rays after the administration of the control substance, the disordered -43 site stained with 0.2% sodium fluorescein was apparent. Meanwhile, in the eyeball irradiated with the ultraviolet rays after the administration of the subject substance, the disordered site stained with 0.2% sodium fluorescein was clearly smaller than that of the control substance, and the corneal disorder caused by the ultraviolet ray was prevented. [0068] <Example 12> Moisturizing effect using removed cornea Using the removed cornea of the rabbit, the moisturizing performance of the cinnamate derivative introduced HA prepared in Example 1 was validated. (12-1) methods 1) Removal of cornea The rabbit was given general anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine, and the eyeball was removed. A pore was opened between the cornea and the sclera in the removed eyeball using a surgical knite, and only the cornea was taken out using the microscissors. 2) Drying treatment of cornea The drying treatment was performed by placing the removed cornea on a paraffin block with a False-tooth stability material and providing cold air from the distance of approximately 1 m from the cornea using a dryer for 5 minutes. 3) Administration of test substances (subject substance, control substance and negative control substance) After the completion of the drying treatment, 100 il of saline as the negative control substance, the aqueous solution of 0.3% by weight HA with a weight average molecular weight of 600,000 to 1,200,000 as the control substance or the aqueous solution of 0.5% by weight cinnamate derivative-introduced HA prepared in the above Example (1-2) as the subject substance was administered to -44 two corneas. In the administration, the 1 ml injection syringes wei used. Three rabbiLs (6 c. neds) Oescribed in the above 1) were used as the administration subjects. 4) Measurement of water evaporation amount The water evaporation amount was measured using a water evaporation amount measurement apparatus (AS-TW2, ASAHIBIOMED) before the administration of the test substance, after the drying treatment, after the administration of the test substance and until 40 minutes with 10 minute intervals after the administration of the test substance. (12-2) Study re3ultD The results of measuring the water evaporation amount were shown in FIG. 14. From FIG. 14, the water evaporation amount was slightly higher in the HA aqueous solution which was the control substance than in the saline which was the negative control whereas the saline became the value close to 0 after 40 minutes, On the other hand, the water evaporation amount after the administration of the subject substance kept the high value even when 10 minutes passed over, thereby the clear moisturizing performance of the suhjerf.t suhst.ance was confirmed. <Example 13> (13-1) methods 1) Removal of corneal epithelia The rabbit was euthanized, and after removing the eyeball, an entire corneal layer was removed by incising along the 3clera. The removed cornea was preserved in saline, and the corneal epithelia was fixed by placing it on the paraffin block with the False-tooth stability material just before the measurement (hereinafter described as "the cornea to be measured"), 2) Water evaporation in corneal epithelia -45 The cornea to be measured was given cold air hy the dryer from the distance of 30 cm for 5 minutes, and left standing at a room temperature for one hour. 3) Administration of subject substance After the water is evaporated, two drops (approximately 100 14l) of saline as the negative contro. substance, the aqueous solution of 0.3% by weight HA with a weight average molecular weight of 600,000 to 1,200,000 as the control substance or the aqueous solution of 0.5% by weight cinnamate derivative-introduced HA prepared in the above Example (1-2) as the subject substance was administered by the 1 ml syringe. 4) Measurement of water evaporation amounts The amount perceived as an unperceived evaporation amount (released water amount per m 2 per hour) was directly measured as the water evaporation amount from the cornea to be measured using the water evaporation amount measurement apparatus (AS-TW2). [0071] (13-Z) Study results The results of measuring the water evaporation amount were shown in FIG. 15. From FIG. 3.5, the water evaporation amount was slightly higher in the HA aqueous solution which was the control substance than in the saline which was the negative control whereas the saline exhibited the value close to 0 after 40 minutes. On the other hand, the water evaporation amount of the administered subject substance kept the high value even when 40 minutes had passed over, thereby it was confirmed that the subject substance has a more persistent water retention property on the cornea compared with saline and the HA aqueous solution. [0072] <;xample 14> Validation of healing effect of crosslinked cinnamate derivai- ve-introduced HA -46 Using a model hamster for xerostomia, the healing effect of the crosslinked cinnamate derivative-introduced HA prepared in Example 2 on the xerostomia was validated. (14-1) Test method 1) Production of model hamster for xerostomia An inside of the oral cavity of a male Syrian hamster was exposed by inserting a test tube with a diameter of approximately 10 mm in the oral cavity close to a buccal side and reversing it under the anesthesia with Nembutal. By giving hot air for approximately 20 seconds, and subsequently giving cold air for 2 minutes and 40 seconds to the exposed inside of the oral cavity using a dryer, the model hamster for the xerostomia was obtained. The inside of the oral cavity was continuously exposed until the measurement was completed. 2) Administration of subject substance and control substance Immediately after making the xerostomia model, 100 pl of (A) PBS, (B) 0.2% by weight HA solution (supplied by Seikagaku Corporation, weight average molecular weighL: 1,500,000) or (C) 0.2% by weight crosslinked cinnamate derivative-introduced HA solution was administered by applying to the inside of the oral cavity using a microsyringe. Hereinafter, the group administered with (A), the group administered with (B) and the group administered with (C) are referred to as PBS group, hA group and crosslinked cinnamate derivative-introduced HA group, respectively. For the administration rlassification (administration group composition), seven hamsters were used for each of the PBS group, the HA group and the crosslinked cinnamate derivative-introduced HA group. 3) Calculation of water evaporation amount ratio The water evaporation amount in the inside of the oral cavity in the xerostomia model hamster was measured using the water evaporation system (Asahihiomed), and the -47 water evaporation amount ratio was calculated when the measurement value immediately after making the xerostomia model hamster was 1. The higher this water evaporation amount ratio is, the more moisturized condition is maintained (dtgreP of oral cavity dryness is low). The measurement was performed immediately after making the xerostomia model hamster, immediately after the administration, 10 minutes and 20 minutes after the administration. (14-2) Study results The results of measuring the water evaporation amount ratio are shown in FIG. 16. in the figure, circled numbers 1, 2, 3 and 4 on the horizontal axis represent data immediately after making the xerostomia model hamster. immediately after the administration, 10 minutes and 20 minutes after the administration, respectively, P represents a significance level. Immediately after the administration, all of administration groups exhibited the water evaporation amount ratio of 3,3 to 4.5. The water evaporation amount ratio 10 minutes atter the administration was 0.9 on average in the PBS group and 2.3 on average in the HA group. On the other hand, it was 3.2 on average in the crosslinked cinnamate derivative-introduced HA group, thus the higher water evaporation amount ratio than the PBS group and the HA group was shown, Further, the water evaporation amount ratio 20 minutes after the administration was 0.9 on average in the PBS group and 1.3 on average in the HA group. On the other hand, it was 3.2 on average in the crosslinked cinnamate derivative-inLrUduced HA group, thus the exLremely higher watel evaporation amount ratio compared with the PBS group and the HA group was indicated. Furthermore, as is apparent from FIG, 16, the water evaporation amount ratio in the crosslinked cinnamate derivative-introduced HA group was very stable regardless of the elapse of time.

-48 This indicated that the crosslinked cinnamate derivative introduced HA stayed at the administered site for a long period of time and exerted a highly persistent effect. From the above results, GAG into which the hydrophobic group is introduced via the binding chain including the cinnamate derivative-introduced HA and the crosslinked cinnamate derivative-introduced HA has been shown to be suitable for the application to the mucosa and capable of effectively treating the disorder in the mucosal epithelial layer by being applied to the mucosa. It has been also shown that the effect of the treatment is highly persistent. Since no adverse effect due to the administration of the cinnamate derivative-introduced HA and the crosslinked cinnamate derivative-introduced HA was observed in any of the above animal studies, the safety of the agent for applying to mucosa of the present invention can be sufficiently estimated. [0074] <(Ixample 15> (15-1) Preparation of octylamine-introduced sodium hyaluronate A 25.8 mg / 2 mL solution (ethanol:0.lMHCl=1:1) of octylamine, 2 mL of 0.1 M solution (ethanol:water=1:l) of DMT-MM (Wako Pure Chemical Industries, Ltd.) were added to a solution of sodium hyaluronate (502 mg, 1.25 mnol/disaccharide unit, weignt-average molecular weight 900,000) in water (50 mL) / ethanol (50 mL). The mixture was stirred over night and a 376 mg / 5 mL aqueons solution of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added. After stirring further 5 hours, the reaction was quenched with acetic acid (107mg) and sodium chloride (522 mg) . Ethanol (250 mL) was added and the resulting precipitation was filtered off and washed twice successively by 80% ethanol, ethanol. The solid was -49 dried in vacuo to afford white solid (475 mg). The introduction ratio of octylamine was 12.6 % by HPLC. (15-2) Preparation of hexadecylamine-introduced sodium hyaluronate A 30 mg / 3 mL solution (ethanol: 0.1MHCI1=l:1) of hexadecylamine, 1,25 ML of 0.1 M solution ethanoll; water-1:1) of DMT-MM (Wako Pure Chemical Industries, Ltd.) were added to a solution of sodium hyaluronate (501 ma, 1.25 mmol/disaccharide unit, weight-average molecular weight 900,000) in water (50 mL) / ethanol (50 mL). The mixture was stirred over night and a 381 mg / 5 mL aqueous solution of sodium hydrogen carbonate (Japanese Pharmacopoeia) was added. After stirring further 5 hours, the reaction was quenched with acetic acid (107mg) and sodium chloride (497 mg). Ethanol (250 mL) was added and the resulting precipitation was filtered off and washed twice successively by 80% ethanol, ethanol. The solid was dried in vacuo to afford white solid (497 mg). The introduction ratio ot hexadecylamine was 12 % by HPLC. (15-3) Preparation of simple soluLion 64 mg of compound obtained in the above (15-1) was added to 5 mM phosphate buffer saline to give a total amount of 59 ml, then the solution was shaken over night with a shaker. The 0.1 % by weight solution of compound prepared in the above (15-1) was obtained. Likewise, 0.1 % by weight solution of compound prepared in the above (15-2) was obtained. <Example 16> The effect of the 0.1 % cinnamate derivative-introduced HA on the healing of rabbit corneal epithelium. The effect of the cinnamate derivative-introduced HA prepared in Example 1 on the healing of rabbit corneal epithelium with surgical removal (The surgical Model).

-50 (16-1) Methods 1) Surgical removal of corneal epithelium (The surgical model) The corneal epithelium of the central region was removed by using a trephine (8 mm I.D), a 23G needle and microscissors after anesthesia with an intravenous injection of 5 mg/kg of ketamine and 2mg/kg of xylazine and topical administration of 0.4% oxbuprocaine hydrochloride. 2) Topical administration One hour and 4 hours after peeling the corneal epithelia, 150 pil of saline as the control substance was administered in the left eye, and 150 l of 0.1% by weight cinnamate derivative-introduced HA solution prepared in the above Example (1-2) as the subject substance was administered in the right eye. At one day and 2 days after the peeling, a total of 4 times with 3 hour intervals, and at 3 days after the peeling, with 3 hour intervals, the same administration as above was performed. In the administration, the 1 ml injection syringes were used. Fourteen model rabbits for the corneal epithelial layer disorder described in the above 1) were used as the administration subjects. 3) Photographing of corneal epithelial defective region The rabbit was given general anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg of xylazine, subsequently, the corneal epithelial loss site was stained with 0.2% sodium fluorescein dissolved in PBS, and photographed under ultra-violet light. The photographing was performed just before the administration of the subject substance one hour after the cornea was peeled and 3 hours after the second administration at 1 to 3 days after the peeling. When photographed, the focal length was made constant to make the magnification of photographs constant. 4) Measurement of corneal epithclial defective region -51 The area of the corneal epithelial defective region stained with sodium fluorescein was measured on the printed photograph using the image analyzer. The value obtained by subtracting the area of the peeled site 3 hours after the final administration at 3 days after the peeling from the area (peeled area) of the peeled site just before the administration of the subject substance one hour after the corneal epithelia were peeled was rendered as "healed area.," [0078) (16-2) Study results The results of the healed area in each individual are shown in FIG. 17, and the results of a healing rate in each individual are shown in FIG. 18. The healed area and the healing rate were calculated as follows. Healed area = Area after peeling - Area at each time point (after 1 to 3 days) Healing rate = Mean of healed areas at respective time points (after 1 to 3 days) In FIGS. 17 and 18, it was observed that the healed area of the corneal epithelia was significantly increased in the eyes administered with 0.1% by weight cinnamate derivative-introduced hA soluLion aL all time points of days 1 to 3, compared with the healed area of the corneal epithelia in the control. eyes. And it was also observed that the healing rate was significantly enhanced in the eyes administered with 0.1% by weight cinnamate derivative introduced HA solution. (0079) <Example 17> The effect of the 0.1 % octylamine-introduced HA and hexadecylamine-introduced HA on the healing of rabbit corneal epithelium. The effect of the octylamine-introduced HA and hexadecylamine-introduced HA prepared in Example 15 on the healing of rabbit corneal epithelium with surgical.

-52 removal (The surgical Model). (17-1) Methods 1) Surgical removal of corneal epithelium (The surgical model) The corneal epithelium of the central region was removed by using a trephine (8 mm I.D), a 23G needle and microscissors after anesthesia with an intravenous injection of 5 mg/kg of ketamine and 2mg/kg of xylazinA and topical administration of 0.4% oxbuprocaine hydrochloride. 2) Topical administration One hour and 4 hours after peeling the corneal epithelia, 150 pl of saline as the control substance was administered in the left eye, and 150 ptl of 0.1 % octylamine-introduced RA and hexadecylamine-introduced pHA solution prepared in the above Example (15-2) as the subject substance was administered in the right eye. At one day and 2 days after the peeling, a total of 4 times with 3 hour intervals, and at 3 days after the peeling, with 3 hour intervals, the same administration as above was performed. In the administration, the 1 ml injection syringes were used. Eight model each rabbits for the cornea). epithelial layer disorder described in the above 1) were used as the administration subjects. 3) Photographing of corneal epithelial defective region The rabbit was given general. anesthesia by intravenously injecting 5 mg/kg of ketamine and 2 mg/kg ot xylazine, subsequently, the corneal epithelial loss site was stained with 0.2% sodium fluorescein dissolved in PBS, and photographed under ultra-violet light. The photographing was performed just before the administration of the subject substance one hour after the cornea was peeled and 3 hours after the second administration at 1 to 3 days after the peeling. When photographed, the focal length was made constant to make the magnification of photographs constant.

-53 4) Measurement of corneal epithelial defective region The area of the corneal epithelial defective region stained with sodium fluorescein was measured on the printed photograph using the image. analyzer, The value obtained by subtracting the area of the peeled site 3 hours after the final administration at 3 days after the peeling from the area (peeled area) of the peeled site just. before the adininistration of the subject substance one hour after the corneal epithelia were peeled was rendered as "healed area." [00801 (17-2) Study results The results of the healed area in each individual are shown in FIG. 19, and the results of a healing rate in each J'ndividual Arp. shown in FIG. 20. Tn PTG. 19, CB-L(a: control), v-R(b), C16-L(c: control), C16-R(d) represents left eye to which saline was administrated as a control for C8-R, right eye to which 0.1 % octylamine-introduced HA solution was administrated, left eye to which saline was administrated as a control for C16-R, right eye to which 0.1 % hexadecylamine-introduced HA solution was administrated, respectively. In FTG. 20, CA represents results of the above study using octylamine, and C16 represents results of the above study using hexadecylamine. The healed area and the healing rate were calculated as follows. Healed area = Area atter peeling - Area at each time point (after . to 3 days) Healing rate = Mean of healed areas at respective time points (after 1 to 3 days) In FIGS. 19 and 20, it was observed that the healed area of the corneal epithelia was significantly increased in the eyes administered with 0.1 % octylamine-introduced HA and hexadecylamine-introduced HA solution at all time points of days 1 to 3, compared with the healed area of t-he corneal epithelia in the control eyes. And it was also 54 observed that the healing rate was significantly enhanced in the eyes administered with 0.1 % octylamine-introduced HA or hexadecylamine-introduced HA solution.

Claims (10)

1. An agent when used for the treatment of mucosal disorders of eyes or oral cavity, comprising, as an active 5 ingredient, hyaluronic acid or a salt thereof into which a hydrophobic group selected from the groups as shown below is introduced via -CO-NH- as a binding chain: groups: an alkyl group having 2 to 18 carbon atoms and an arylalkenyl group. 10

2. The agent when used for the treatment of mucosal disorders of eyes or oral cavity according to claim 1, wherein the binding chain is -CONH-, and said carboxylic acid amide bond is a binding chain in a carboxyl group of the hyaluronic 15 acid.

3. The agent when used for the treatment of mucosal disorders of eyes or oral cavity according to claim 1 or 2 wherein the hydrophobic group is an alkyl group having 2 to 18 20 carbon atoms.

4. The agent when used for the treatment of mucosal disorders of eyes or oral cavity according to claim 1 or 2 having a spacer chain between the binding chain and the 25 hydrophobic group said spacer chain having a binding chain at a hydrophobic group side: group: -- COO- (CH 2 )m, (wherein m represents an integer of 1 to 18). 30

5. The agent when used for the treatment of mucosal disorders according to claim 4 which is applied to oral cavity mucosa, comprising, as an active ingredient, hyaluronic acid or a salt thereof wherein the binding chain is -CONH-, which is a carboxylic acid amide bond in a carboxyl group of hyaluronic SPEC-826299.docx 56 acid and an arylalkenyl group, said hydrophobic group forming a crosslinking bond.

6. The agent when used for the treatment of mucosal 5 disorders according to claim 5 wherein the hydrophobic group is a phenylethenyl group, and the spacer chain having a binding chain at a hydrophobic group side, is -COO-(CH 2 )m- (wherein m represents integers of 1 to 18). 10

7. The agent when used for the treatment of mucosal disorders according to claim 6 wherein the spacer chain having a binding chain at a hydrophobic group side, is -COO-(CH 2 ) 6 -

8. The agent when used for the treatment of mucosal 15 disorders according to claim 3 which is used for the treatment of corneal epithelial layer disorders.

9. The agent when used according to any one of claims 1 to 8, substantially as hereinbefore described with reference to 20 any of the examples and or figures.

10. A method of treating mucosal disorders of eyes or oral cavity, which method comprises applying to a mucosa requiring treatment an agent according to any one of claims 'l 25 to 9 in an amount which effectively treats said mucosal disorder. SPEC-826299.docx