MX2008006656A - Pharmaceutical comprising ppar agonist - Google Patents

Abstract

Disclosed is a proliferation-promoting agent for a meibomian gland epithelial cell or a corneal epithelial cell. Also disclosed is a therapeutic agent for an ophthalmic disease such as meibomian gland dysfunction and tear-evaporative dry eye. The proliferation promoting agent comprises a PPAR-αor -γagonist as an active ingredient. The therapeutic agent for an ophthalmic disease comprises a PPAR-αor -γagonist as an active ingredient.

Description

RECEPTOR ACTIVATED BY THE PROLOYER OF PEROXISOMA FIELD OF THE INVENTION The present invention relates to an agent for promoting the proliferation of an epithelial cell of the meibomian gland or a corneal epithelial cell comprising an a or d agonist of PPAR (Receptor Activated by the Peroxisome Proliferator) as an active ingredient.

BACKGROUND OF THE INVENTION A meibomian gland is a lipid-producing gland included in the upper and lower eyelids, and secretes a lipid through an opening located on a conjunctival side of the eyelashes of the eyelids. A lipid layer that constitutes a tear fluid contains a lipid supplied from the meibomian gland as a component, and prevents tear fluid from evaporating from the surface of an eye. It is known that, in a dysfunction of the meibomian gland or patient with meibomitis, the function of the meibomian gland is impaired, and the amount of lipid secretion is decreased, causing dry, hyper-evaporative eye, keratoconjunctival epithelial unstable, corneal epithelial erosion and corneal ulcer, which are associated with dry eye, and the like. In addition, a cornea consists of an epithelium REF.:192117 ectodermal and a mesodermal anterior limiting layer (Bowman's membrane) / stroma / posterior limiting layer (Descemet's membrane) / endothelium. Since the cornea is the foremost part of the eyeball, it easily undergoes the influence of the external environment and, as a result, various disorders are generated. Examples of a disease associated with damage or a defect of a corneal epithelial cell include dry eye syndrome, corneal ulcer, superficial punctate keratitis, corneal epithelial erosion, ocular allergic diseases associated with a corneal lesion such as fluid catarrh and keratoconjunctivitis. atopic and similar. On the other hand, PPAR is one of the intranuclear receptors, which is expressed in almost all vertebrates, and is said to be a group of transcription factors closely involved in the intracellular metabolism of saccharide-lipids and the differentiation of a cell. As a subtype, they are known to, d and?. A PPARd is referred to as PPARβ in some cases (non-patent literature 1). As the distribution of PPAR in an ocular tissue, it is known that PPARa and ß are expressed in a corneal epithelial cell of a rabbit (literature 2 not patent). Previously, it has been reported that 5- [4- (6-methoxy-l-methyl-lH-benzimidazol-2-ylmethoxy) benzyl] thiazolidin-2, 4- dione that has an activating action of PPAR can be used in an agent to treat a keratoconjunctival disorder (literatures 1 and 2 patent), and a PPARa agonist, d or? it is administered in the treatment of an ocular disease (conjunctivitis, dry eye syndrome, corneitis or the like) (patent literature 3). Furthermore, it is known that PPARa is distributed in the liver, kidney and the like, and acts on the metabolism / transport of lipids and, furthermore, it has been reported that an agonist thereof can be used as an agent to treat a corneal disease ( patent literature 4). With respect to a PPARa or PPARd agonist, it has been previously reported that the agonist that promotes the proliferation and differentiation of an epithelial cell of rat sebaceous gland (literature 3 not patent), and promotes the healing of skin damage (Literature 4 not of patent). In addition, a method for stimulating the proliferation of a β cell by administration of a non-thiazolidinedione PPAR ligand and a GLP-1 derivative (patent literature 5), and the inhibition of the proliferation of a leukemia cell and a cell of prostate cancer by pioglitazone which is a PPAR agonist? (patent literature 6) are known. However, the expression and function of PPARa, d and? in each animal species and in each tissue or cell they contain many questions to be answered and whose PPAR agonist which is useful for human eye diseases is not known. [Patent Literature 1] WO 2005/039574 [Patent Literature 2] Japanese Patent Application Open to the Public (JP-A) No. 2001-39976 [Patent Literature 3] WO 2002/076177 [Patent Literature 4] JP -Year. 2005-008570 [Patent Literature 5] WO 2002/69994 [Patent Literature 6] WO 1998/25598 [Non-Patent Literature 1] J Med Chem 2000, 43: 527-550 [Non-Patent Literature 2] J Biol Chem 2000, 275: 2837 [Non-Patent Literature 3] Molecular Genetic and Metabolism 2001, 74: 362-369 [Non-Patent Literature 4] Am J Clin Dermatol 2003, 4 (8): 523-530 BRIEF DESCRIPTION OF THE INVENTION An object of the present invention is to provide an agent capable of promoting the proliferation of an epithelial cell of the meibomian glands and a corneal epithelial cell, which can be a fundamental treatment of eye diseases such as dry eye and similar, as well as a therapeutic agent for eye diseases such as dysfunction of the meibomian glands, dry eye, hyper-evaporative and the like, containing said promoter agent.

In view of the aforementioned problems, the present inventors conducted various investigations and as a result, found that a PPARa agonist or od has an activity promoting the proliferation of an epithelial cell of the meibomian glands and a corneal epithelial cell, resulting in the termination of the present invention. Accordingly, the present invention includes at least the following contents. (1) An agent for promoting the proliferation of a meibomian gland epithelial cell, comprising a PPARa agonist or d as an active ingredient. (2) An agent for promoting the proliferation of a corneal epithelial cell, comprising a PPARa agonist or d as an active ingredient. (3) An agent for treating dysfunction of the meibomian gland, comprising a PPARa agonist or d as an active ingredient. (4) An agent for treating a corneal epithelial disorder, comprising a PPARa agonist or d as an active ingredient. (5) An agent for treating the hyper-evaporative dry eye, comprising a PPARa agonist or d as an active ingredient. (6) An agent for promoting the proliferation of an epithelial cell of the meibomian gland, comprising a PPARd agonist as an active ingredient. (7) An agent for promoting the proliferation of a corneal epithelial cell, comprising a PPARd agonist as an active ingredient. (8) An agent for treating dysfunction of the meibomian gland, comprising a PPARd agonist as an active ingredient. (9) An agent for treating a corneal epithelial disorder, comprising a PPARd agonist as an active ingredient. (10) An agent for treating the hyper-ovarian dry eye, comprising a PPARd agonist as an active ingredient. (11) The agent in accordance with any of (1) to (5), wherein the PPARa agonist or d is a compound represented by the formula (I): [Chemical formula 1] (I) wherein: A is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, B is a linker selected from the group consisting of -CO-, -NH-, - (CH2) nS-, - (CH2) n-0 and -0- (CH2) n-0-, where n is an integer from 1 to 3, X and Y are the same or different, and each is a carbon atom or an atom of nitrogen, Z is an oxygen atom, a sulfur atom or -CH2-, Ari is a 5-6 membered aromatic cyclic group which optionally has 1 to 3 substituents, Ri and R2 are the same or different and each is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, and R3 is hydrogen, a halogen atom or an alkyl group having a carbon number of from 1 to 6.0, a pharmacologically acceptable salt thereof . (12) The agent according to any of (6) to (10) characterized in that the PPARd agonist is a compound characterized by the formula (II): [Chemical formula 2] (p) wherein A is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, B is a linker selected from the group consisting of - (CH2) nS- and -0- (CH2) n-0- where n is an integer from 1 to 3, X and Y are the same or different, and each is a carbon atom or a nitrogen atom, Z is an oxygen atom, a sulfur atom or -CH2- , Ari is a 5-6 membered aromatic cyclic group optionally having 1 to 3 substituents, Ri and R2 are the same or different and each is a hydrogen atom or an alkyl group having a carbon number of 1 to 6. , and R3 is hydrogen, a halogen atom or an alkyl group having a carbon number of 1 to 6, or a pharmacologically acceptable salt thereof. (13) The agent according to (11) or (12), characterized in that the PPARd agonist is: (4- (3- (4-acetyl-3-hydroxy-2-propyl) phenoxy) propoxyphenoxy) acetic acid, acid (2-met4- (((4-met2- (4- (trifluoromet phenyl) -5-thiazolyl) met thio) phenoxy) acetic acid or (4 - (((2- (3- fluoro- (4- (trifluoromet phenyl) -4-met5-thiazolyl) met thio) -2-methenoxy) acetic, or a pharmacologically acceptable salt thereof. (14) The agent according to (11), characterized in that the PPARa agonist is the 2- (4- (4-chlorobenzoyl) phenoxy) -2-metropionate of 1-mettor the acid ((4-chloro-6) - ((2,3-dimethenyl) amino) -2-pyrimidinyl) thio) acetic acid, or a pharmacologically acceptable salt thereof. (15) The use of a PPARa agonist or d for the production of an agent to promote the proliferation of an epithelial cell of the meibomian gland. (16) The use of a PPARa agonist or d for the production of an agent to promote the proliferation of a corneal epithelial cell. (17) The use of a PPARa agonist or d for the production of an agent to treat dysfunction of the meibomian gland. (18) The use of a PPARa or d agonist for the production of an agent to treat a corneal epithelial disorder. (19) The use of a PPARa agonist or d for the production of an agent for treating the hyper-evaporative dry eye. (20) The use of a PPARd agonist for the production of an agent to promote the proliferation of a epithelial cell of the meibomian gland. (21) The use of a PPARd agonist for the production of an agent to promote the proliferation of a corneal epithelial cell. (22) The use of a PPARd agonist for the production of an agent to treat dysfunction of the meibomian gland. (23) The use of a PPARd agonist for the production of an agent to treat a corneal epithelial disorder. (24) The use of a PPARd agonist for the production of an agent to treat dry, hyper-evaporative eye. (25) A method for promoting the proliferation of an epithelial cell of the meibomian gland, characterized in that it comprises supplying an effective amount of a PPARa agonist or d to a subject in need of promotion of the proliferation of an epithelial cell of the meibomian gland. (26) A method for promoting the proliferation of a corneal epithelial cell, characterized in that it comprises administering an effective amount of a PPARα agonist or d to a subject in need of promotion of the proliferation of a corneal epithelial cell. (27) The method according to (25), characterized in that it is performed to treat dysfunction of the meibomian gland. (28) The method according to (26), characterized in that it is performed to treat a corneal epithelial disorder. (29) A method for treating the hyper-ovarian dry eye, which comprises administering an effective amount of a PPARa agonist or d to a patient suffering from the hyper-evaporative dry eye. (30) A method for promoting the proliferation of an epithelial cell of the meibomian gland, which comprises administering an effective amount of a PPARd agonist to a subject in need of promoting the proliferation of an epithelial cell of the meib.omiana gland. (31) A method to promote the proliferation of a corneal epithelial cell, which comprises administering an effective amount of a PPARd agonist to a subject in need of promotion of the proliferation of a corneal epithelial cell. (32) The method of compliance with (30), which is performed to treat the dysfunction of the meibomian gland. (33) The method of compliance with (31), which is performed to treat a corneal epithelial disorder. (34) A method for treating the hyper-ovarian dry eye, which comprises administering an effective amount of a PPARd agonist to a patient suffering from the hyper-evaporative dry eye. According to the present invention, a novel agent for promoting proliferation of an epithelial cell and meibomian gland or a novel agent for promoting proliferation of a corneal epithelial cell is provided, and the promoting agent promotes proliferation of an epithelial cell of the meibomian gland and a corneal epithelial cell. In addition, the therapeutic agent of the present invention can be effectively used to treat or ameliorate diseases such as dysfunction of the meibomian gland, corneal epithelial disorder, and the hyper-evaporative dry eye.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the expression of the mRNA of PPARa, d o? in a cultured human corneal epithelial cell (upper column), a cultivated rabbit epithelial corneal cell (intermediate column) and an epithelial cell of the cultured monkey meibomian gland (lower column).

DETAILED DECRIPTION OF THE INVENTION The present invention provides an agent for promoting the proliferation of an epithelial cell of the meibomian gland, comprising a PPARa agonist or d as an active ingredient. The promoter agent promotes the proliferation of an epithelial cell of the meibomian gland. Also, the present invention provides an agent for promoting the proliferation of a corneal epithelial cell comprising a PPARa agonist or d as an active ingredient. The promoter agent promotes the proliferation of a corneal epithelial cell. In the present invention, the agent for promoting cell proliferation means an agent having an action of promoting cell division to increase the number of cells, and an agent having an action of inhibiting cell death to increase the number of cells. The promoter agent of the present invention comprises a PPARa agonist or d as an active ingredient. The PPARa agonist refers to a substance that has an action of binding to and activating PPARa. In addition, the PPARd agonist refers to a substance that has an action of binding to and activating PPARd. As the PPARα or d agonist, various known PPARα or d agonists can be mentioned. The Specific examples of PPARa agonists od include the substances described in, for example, WO2001 / 00603, W099 / 62872, WO2002 / 100813, W097 / 28149, WO2004 / 022551, WO2001 / 79197, WO2003 / 099793, WO2005 / 105736, WO2005 / 105726, WO2005 / 085235, WO2005 / 113600, WO2005 / 105754, WO2005 / 049606, W02004 / 111020, WO2006 / 055187, WO2006 / 084176, WO2005 / 060958, WO2005 / 097786, WO2004 / 092117, WO2005 / 037763, WO2005 / 030694, WO2005 / 016335, WO2003 / 074495, WO2004 / 058174, JP2003-1712753, JP2005-179281A, WO2006 / 031969, WO2005 / 097763, WO2004 / 063165, WO2002 / 050048, WO2005 / 090966, WO2003 / 099793, WO2002 / 076959, WO2002 / 053547, WO2001 / 00603, W097 / 28149, WO2004 / 063184, WO2006 / 090920, WO2006 / 059744, WO2006 / 041197, WO2003 / 033493, WO2003 / 016291, WO2002 / 076957, WO2002 / 046176, WO2002 / 046154, WO2002 / 014291, WO2001 / 079197, etc., and the like. The specific examples of the agonist compound of PPARa or include 2- (4- (4-chlorobenzoyl) phenoxy) -2-methylpropionate 1-methylethyl (fenofibrate; CAS No. 49562-28-9), acid ((4-chloro-6- ((2 , 3-dimethylphenyl) amino) -2-pyrimidinyl) thio) acetic acid (WY-14643; CAS-No .: 50892-23-4), 4- (3- (4-acetyl-3-hydroxy-2-propyl) ) phenoxy) propoxyphenoxy) acetic acid (L-165041; CAS-No .: 79558-09-1), acid (2-methyl-4- (((4-methyl-2- (4- (trifluoromethyl) phenyl) -5- thiazolyl) methyl) thio) phenoxy) acetic acid (GW-501516; CAS No. 317318-70-0), acid (4 - (((2- (3-fluoro- (4- (trifluoromethyl) phenyl) -4- methyl-5- thiazolyl) methyl) thio) -2-methylphenoxy) acetic acid (GW-0742; CAS No. 317318-84-6), 2-methyl-4- ((2R) -2- (3-methyl-5- ( 4- (trifluoromethyl) phenyl) -2-thienyl) propoxy) -benzene propionic acid (CAS-No. 728038-95-7), 2-ethyl-2- (4- (4- (- (4-methoxyphenyl) -piperazine) -1-yl) -2- (4-trifluoromethyl-phenyl) -thiazol-5-ylmethylsulfanyl) -phenoxy) butyric acid (GSK-677954; CAS No. 884324-15-6), (4- (3- (3-phenyl-7-propyl-benzofuran-6-yloxy) -propylsulfañyl) -phenyl) -acetic acid (L-796449; CAS-No .: 194608 -80-5), and 2- (4- (3- (1- (2- (2-chloro-6-fluoro-phenyl) -ethyl) -3- (2,3-dichloro-phenyl) -ureidoic acid ) -propyl) -phenoxy) -2-methylpropionic acid (GW-2433; CAS-No .: 227941-61-9). In the present invention, additional substances (compounds) that activate PPARa or can be obtained by selection from a group of substances that are not known to be PPARα agonists, and such a substance (compound) can be used as an ingredient active of the present invention. The PPARa or d agonist is known to bind to a ligand binding domain (LBD) of PPARa or d, respectively, to activate the receptor, and regulate the transcription of a PPAR target gene. To use this property and, at the same time, to exclude the influence of other nuclear receptors present in a mammalian cell, a new PPARα agonist or d can be selected by a selection method using a yeast LBD and GAL4 chimeric receptor, and a reporter gene. As the selection method, the PPAR GAL4 assay is exemplified (reference literature, TM Willson et al., Journal of Medicinal Chemistry, 2000, vol.33, no.4, p.528-550 and JM Lehmann et al. , The Journal of Biological Chemistry, 1995, vol.270, No. 22, pp. 12953-12956). Specifically, the examples include a method comprising: (a) a step of introducing, within a cell, a vector to express a fused protein of the DNA binding domain (DBD) of a yeast transcription factor GAL4 and LBD of PPARa od, and a reporter plasmid that contains a GAL4 DNA binding element, and a reporter gene. (b) a step of contacting the cell and a test substance, measuring an amount of expression of the reporter gene in the cell, and comparing the amount of expression with an amount of expression in a control cell that is not brought into contact with the test substances; and (c) one step select a substance that activates PPARa od based on the comparison result of (b). In step (a) in the above-mentioned method, a cell used is a cell that does not internally express GAL4, preferably, a mammalian cell. The reporter gene can be a gene that codes for a protein or a detectable enzyme, and examples thereof include a LUC (luciferase) gene, an SPAP (secreted alkaline phosphatase placenteria) gene, a CAT gene (chloramphenicol acetyltransferase), and the like. The preparation of the fused protein expression vector and the reporter plasmid, as well as the introduction of the vector and the plasmid into a cell, can be carried out by the aforementioned reference literature method, or the method known per se. In step (b) of the aforementioned method, the test substance can be any known compound or a novel compound, and examples thereof include a nucleic acid, a saccharide, a lipid, a protein, a peptide, an organic compound of low molecular weight, a library of compounds prepared using the combinatorial chemistry technique, a random peptide library prepared by a solid phase sesis method or a phage display method, and natural components derived from microorganisms, animals and plants, marine organisms or similar. In addition, in step (b), the transcription activity of PPARa or d due to the presence or absence of the test substance is investigated by a so-called reporter assay. The reporter's essay can be done using the method known per se depending on the reporter gene used. In step (c) of the aforementioned method, when the activity of the reporter in the presence of the test substance is significantly higher than the reporter activity in the absence of the same, the test substance can be determined as a compound having the PPARa agonist activity or d. The activity of the PPARa agonist used in the present invention to a human PPARa is not greater than 50 μM, preferably not greater than 10 μM, additionally preferably not greater than 5 μM as expressed by an EC50 value. The activity of the PPARd agonist used in the present invention at a human PPARd is not greater than 10 μM, preferably not greater than 1 μM, additionally preferably not greater than 0.1 μM, as expressed by an EC50 value. The EC50 value is a value measured by a PPAR-GAL4 assay using the human PPARa or d (see T.M. Willson et al., Journal of Medicinal Chemistry, 2000, vol.43, No. 4, p.527-550). Preferable examples of PPARa or d agonists include a compound having a structure represented by the following formula (I): [Chemical formula 3] wherein: A is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, B is a linker selected from the group consisting of -CO-, -NH-, - (CH2) nS-, - (CH2) n-0 and -O- (CH2) n-0-, wherein n is a integer from 1 to 3, X and Y are the same or different, and each is a carbon atom or a nitrogen atom, Z is an oxygen atom, a sulfur atom or -CH2-, Arx is a group 5-6 membered aromatic cyclic having optionally 1 to 3 substituents, Ri and R2 are the same or different and each is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, and R3 is hydrogen , a halogen atom or an alkyl group having a carbon number of from 1 to 6.0, a pharmacologically acceptable salt thereof. In the aforementioned formula (I), examples of "alkyl group having a carbon number of 1 to 6" for A include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3- methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, l-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl and the like. The alkyl group having a carbon number of 1-6 is preferably isopropyl. In the aforementioned formula (I), the linker for B is preferably -CO-, -NH-, -CH2-S- or -0- (CH2) 3-0-, more preferably -CH2-S- or -O - (CH2) 3-0-. Examples of the 5 or 6 membered aromatic ring group for Ari in the aforementioned formula (I) include phenyl, furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1,2,3-oxadiazolyl , 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, furazanyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl , 1,2,4-triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like. The aromatic ring group is preferably phenyl or thiazolyl. Examples of the substituent Ari may have in the aforementioned formula (I), include a halogen atom, a hydroxyl group, an alkyl group having an carbon number of 1-6, a haloalkyl group having a carbon number of 1-6, an acyl group having a carbon number of 2-7, and a phenyl group optionally having an alkyl group having a number of carbons of 1-6 or a haloalkyl group having a carbon number of 1-6. Preferred substituents are a chlorine atom, a hydroxyl group, a methyl group, an acetyl group, a 4-trifluoromethylphenyl group and a 3-fluoro-4-methylphenyl group. Examples of "alkyl group having a carbon number of 1 to 6" for A include the methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, ter- pentyl, 1-methylbutyl, 2-methylbutyl, 1, 2-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1, 3- dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, l-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl and the like. The alkyl group having a carbon number of 1-6 is preferably methyl. Examples of the "halogen atom" for R3 in the aforementioned formula (I) include a fluoro atom, a chlorine atom, a bromine atom and the like. The halogen atom is preferably a chlorine atom. Examples of "alkyl group having a carbon number of 1 to 6" for R3 in formula (I) above mentioned include the groups methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3-3- dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, l-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl and the like. The alkyl group having a carbon number of 1-6 is preferably methyl. Examples of the pharmaceutically acceptable salt of a compound represented by the formula (I) include the salts by addition of acid with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and the like; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, methanesulfonic acid, ethanesulfonic acid, and the like; acidic amino acids such as aspartic acid, glutamic acid, etc., and the like. These salts also include solvates thereof. A compound represented by the formula (I) may have an asymmetric carbon atom or a double bond, and such a compound may contain an optical isomer or a geometric isomer. Such isomers are also encompassed within the scope of the present invention and can be isolated and purified according to a method known per se. Any mixture and isolated forms thereof can be used in the present invention. In the present invention, preferred PPARα agonists from among the compounds represented by formula (I) include 2- (4- (4-chlorobenzoyl) phenoxy) -2-methylpropionate 1-methylethyl (fenofibrate, CAS-No .: 49562-28-9), ((4-chloro-6 - ((2,3-dimethylphenyl) amino) -2-pyrimidinyl) thio) acetic acid (WY-14643; CAS No. 50892-23-4), and the like. Preferred PPARd agonists include 2- (4- (4-chlorobenzoyl) phenoxy) -2-methylpropionate 1-methylethyl (fenofibrate; CAS No. 49562-28-9), ((4-chloro-6- ((2,3-dimethylphenyl) amino) -2-pyrimidinyl) thio) acetic acid (WY-14643; CAS No. 50892-23-4), (4- (3- (4-acetyl-3-hydroxy-2-propyl) phenoxy) propoxyphenoxy) acetic acid (L- 165041; 79558-09-1), acid (2-methyl-4- (((4-methyl-2- (4- (trifluoromethyl) phenyl) -5-thiazolyl) methyl) thio) phenoxy) acetic acid (GW-501516; CAS registered 317318-70-0), acid (- (((2- (3-fluoro- (4- (trifluoromethyl) phenyl) -4-methyl-5-thiazolyl) methyl) thio) -2-methylphenoxy) acetic acid (GW-0742; CAS No. 317318-84-6), and the like. Preferred d agonists include a compound represented by the following formula (II): [Chemical formula 4] wherein A is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, B is a linker selected from the group consisting of - (CH2) nS- and -0- (CH2) n-0- where n is an integer from 1 to 3, X and Y are the same or different, and each is a carbon atom or an atom of nitrogen, Z is an oxygen atom, a sulfur atom or -CH2-, Ari is a 5-6 membered aromatic cyclic group optionally having 1 to 3 substituents, Ri and R2 are the same or different and each is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, and R3 is hydrogen, a halogen atom or an alkyl group having a carbon number of 1 to 6, and a pharmacologically acceptable salt thereof. Examples of "alkyl group that has a number of carbons of 1 to 6"for A in the aforementioned formula (II) include the groups methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, ter-pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, l-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl and the like The binder for B in the The aforementioned formula (II) is preferably -CH2-S- or -O- (CH2) 3-0- The examples of the 5 or 6-membered aromatic ring group for Ari in the above-mentioned formula (I) include phenyl, furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, 1, 2, 3-oxadiazolyl, 1, 2,4-oxadiazolyl, 1,3-oxadiazolyl, furazanyl, 1,2,3-thiadiazolyl , 1, 2 , 4-thiadiazolyl, 1,3,4-thiadiazolyl, 1, 2, 3-triazolyl, 1, 2,4-triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and the like. The aromatic ring group is preferably phenyl or thiazolyl. Examples of the substituent Ari may have in the aforementioned formula (I), include a halogen atom, a hydroxyl group, an alkyl group having an carbon number of 1-6, a haloalkyl group having a carbon number of 1-6, an acyl group having a carbon number of 2-7, and a phenyl group optionally having an alkyl group having a number of carbons of 1-6 or a haloalkyl group having a carbon number of 1-6. Preferred substituents are a chlorine atom, a hydroxyl group, a methyl group, an acetyl group, a 4-trifluoromethylphenyl group and a 3-fluoro-4-methylphenyl group. Examples of "alkyl group having a carbon number of 1 to 6" for Ri or R2 in the aforementioned formula (II) include the methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertbutyl, pentyl, isopentyl, neopentyl, tert-pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1, 2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, l-ethyl-2-methylpropyl, 1,1,2- trimethylpropyl and the like. The alkyl group having a carbon number of 1-6 is preferably methyl. Examples of the "halogen atom" for R3 in the aforementioned formula (II) include a fluoro atom, a chlorine atom, a bromine atom and the like. The halogen atom is preferably a chlorine atom. Examples of "alkyl group that has a number of carbons of 1 to 6"for R3 in the above-mentioned formula (II) include the groups methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertbutyl, pentyl, isopentyl, neopentyl, ter-pentyl, 1- methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2, 2-dimethylbutyl, 2,3-dimethylbutyl, 3, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, l-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl and the like The alkyl group having a number of carbons of 1-6 is preferably methyl Examples of the pharmaceutically acceptable salt of a compound represented by formula (I) include the salts by the addition of acid with mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid and the like; organic acids such as formic acid, ac Alphabet, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, malic acid, tartaric acid, methanesulfonic acid, ethanesulfonic acid and the like; acidic amino acids such as aspartic acid, glutamic acid, etc., and the like. These salts also include solvates thereof. A compound represented by the formula (I) can have an asymmetric carbon atom or a double bond, and such a compound can contain an optical isomer or a geometric isomer. Such isomers are also encompassed within the scope of the present invention and can be isolated and purified according to a method known per se. Any mixture and isolated forms thereof can be used in the present invention. In the present invention, preferred PPARα agonists of the compounds represented by formula (II) include 4- (3- (2-propyl-3-hydroxy-4-acetyl) phenoxy) propoxyphenoxyacetic acid (L-165041; CAS No. 79558-09-1), acid (2-methyl-4- (((4-methyl-2- (4- (trifluoromethyl) phenyl) -5-thiazolyl) methyl) thio) phenoxy) acetic acid (GW -501516; CAS No. 317318-70-0), acid (4 - (((2- (3-fluoro- (4- (trifluoromethyl) phenyl) -4-methyl-5-thiazolyl) methyl) thio) - 2-methylphenoxy) acetic acid (GW-0742; Registered CAS No. 317318-84-6), and the like In the promoter agent of the present invention, the PPARα agonist content od is generally 0.000001-1% by weight , preferably 0.00001-1% by weight, most preferably 0.0001-0.1% by weight The promoter agent of the present invention can contain any carrier in addition to the above-mentioned active ingredient Examples of the carrier include solvents (eg, water, alcohol , etc.), love Tiguadores (for example, phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, tris buffer, glutamic acid, epsilon-aminocaproic acid, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, benzyl alcohol, sodium dehydroacetate, paraoxybenzoates, edetate sodium, boric acid, etc.), isotonicity agents (eg, sodium chloride, potassium chloride, glycerol, mannitol, sorbitol, boric acid, glucose, propylene glycol, etc.) and the like. The promoter agent of the present invention can be used in vivo or in vitro as a medicament or a test reagent. When the promoter agent of the present invention is used as a test reagent, it can be used in various aspects as test reagents in the field of physiology and biochemistry. When used as a medicament, since the promoter agent of the present invention promotes the proliferation of an epithelial cell of the meibomian gland, it is useful as an agent to treat a disease associated with damage or atrophy of an epithelial cell of the meibomian gland, and a disease generated by the deterioration in the function of an epithelial cell of the meibomian gland. Examples of the disease include dysfunction of the meibomian gland.

In addition, since the epithelial cell of the meibomian gland secretes a lipid component in a tear fluid, and this lipid prevents the tear fluid from evaporating and stabilizes the lacrimal fluid layer, the therapeutic agent of the present invention is useful for a disease accompanied with abnormality of the lipids (reduction in the amount of secretion, change of components) in the tear fluid. Examples of disease include hiperevaporative dry eye. In addition, the promoter agent of the present invention is also useful in the treatment of dry eye, corneal ulcer, superficial punctate keratitis, corneal epithelial erosion and the like. In addition, since the promoter agent of the present invention promotes the proliferation of a corneal epithelial cell, it is also useful as an agent to treat a disease associated with damage (eg, a wound or a defect) of a corneal epithelial cell. . The promoter agent of the present invention is useful as an agent for treating a corneal epithelial disorder, specifically, a corneal epithelial disorder associated with an endogenous disease such as Sjogren's syndrome, Stevens-Johnson syndrome or dry keratoconjunctivitis (dry eye); corneal epithelial disorder accompanied by exogenous disease in the case of post-operation, use of drugs, trauma or use of contact lenses; or a corneal epithelial disorder accompanied with corneal ulcer or with an ocular allergic disease associated with a corneal lesion, intense flow catarrh, or atopic keratoconjunctivitis. The promoter agent of the present invention is also useful in the treatment of superficial punctate keratitis and corneal epithelial erosion. In addition, the promoter agent of the present invention is also useful as an agent to promote healing of corneal wounds. In addition, since the promoter agent of the present invention has a proliferative activity of corneal epithelial cells and an activity of proliferation of epithelial cells of the meibomian gland, this shows an effect of acting directly on a corneal tissue and an effect of improving the function of a lacrimal fluid by functioning on a cell of the meibomian gland. As a result, the agent is highly useful particularly as a therapeutic agent for the hyper-evaporative dry eye. In the therapeutic agent of the present invention, the content of the PPARa or d agonist is usually 0.000001 to 1% by weight, preferably 0.00001 to 1% by weight, most preferably 0.0001 to 0.1% by weight. Examples of a subject to which the promoter agent of the therapeutic agent of the present invention is administered include a mammal (e.g., human, mouse, rat, hamster, rabbit, cat, dog, beef, sheep, monkey or similar). The therapeutic agent of the present invention may be used in a dosage form such as ophthalmic drops, patches, skin adhesives, ointments, lotions, creams, oral preparations, or the like, and may contain arbitrary carriers such as pharmaceutically acceptable carriers as well. of the aforementioned active ingredients. In the therapeutic agent of the present invention, the route of administration thereof is not particularly limited as long as the aforementioned therapeutic effect is exercised, but the therapeutic agent is preferably locally administered to the eyes. Examples of a dosage form for ocular local administration include ophthalmic drops and eye ointments. For example, when the therapeutic agent of the present invention is used as an ophthalmic drop or ocular ointment, it can be added as an additive, a stabilizer (eg, sodium acid sulfate, sodium thiosulfate, sodium edetate, citrate sodium, ascorbic acid, dibutylhydroxytoluene and the like), a solubilizing agent (for example, glycerin, propylene glycol, macrogol, polyoxyethylene hardened castor oil, and the like), a suspending agent (e.g. polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, and the like), an emulsifier (e.g., polyvinylpyrrolidone, soy lecithin, egg yolk lecithin, polyoxyethylene hardened castor oil, polysorbate 80, and the like), a buffer (e.g. a phosphate buffer, an acetate buffer, a borate buffer, a carbonate buffer, - a citrate buffer, a Tris buffer, glutamic acid, e-aminocaproic acid, and the like), a thickener (e.g. water-soluble cellulose derivative such as methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose or carboxymethylcellulose, sodium sulfate-chondroitin, sodium hyaluronate, carboxyvinyl polymer, polyvinyl alcohol, polyvinylpyrrolidone, macrogol and the like), a preservative (e.g., benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, chlorobutanol, benzyl alcohol, dehydrated sodium acetate, esters of para-oxybenzoic acid, sodium edetate, boric acid, and the like), an isotonic agent (eg, sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, glucose, propylene glycol, and the like > a pH adjusting agent (eg, hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid and the like), a cooling agent (for example, 1-menthol, d-camphor, d-borneol, peppermint oil and the like), an ointment base (white petrolatum, purified lanolin, liquid paraffin, vegetable oil (olive oil, camellia oil, oil peanuts and the like) and the like). While the amount of these additives varies depending on the type, utility and the like of the additive to be added, the additive can be added at a sufficient concentration to achieve the purpose thereof. When the therapeutic agent of the present invention is formulated in eye drops or eye ointments, these can be produced according to the method that is usually used in the field of pharmacy and, for example, can be produced based on the method described in the Japanese Pharmacopoeia 14th edition, General Rules for the Preparation, item of ophthalmic drops and item of ocular ointments. The present invention provides a method for promoting the proliferation of an epithelial cell of the meibomian gland, which comprises administering an effective amount of a PPARα agonist or d to a subject in need of promoting the proliferation of epithelial cells of the meibomian glands. It is desirable that the method be conducted to treat dysfunction of the meibomian glands. Also, the present invention provides a method for promoting the proliferation of corneal epithelial cells, comprising administering an effective amount of a PPARa agonist or d to a subject in need of promotion of the proliferation of corneal epithelial cells. It is desirable that the method be conducted to treat a corneal epithelial disorder. Also, the present invention provides a method for treating the hyper-ovarian dry eye, including administering an effective amount of a PPARa agonist or d to a patient suffering from hyper-evaporative dry eye. The effective amount of the PPARa agonist or d can not be unconditionally defined, and varies depending on the type of compound, the age, weight and condition of the subject of administration, the purpose of the treatment and the like. When the promoter agent or therapeutic agent of the present invention is administered to a human, for example, a solution containing the PPARa agonist or a concentration usually of 0.000001 to 1% by weight, preferably 0.00001 to 1% by weight, most preferably from 0.0001 to 0.1% by weight, it is administered by 1 or 2 droplets for one eye per administration, eg, approximately 50 to 200 μL per administration, once to 8 times per day. The amount of the PPARa agonist or d contained in the solution having a concentration and a volume within the above ranges, It can be administered as an example of the effective amount.

EXAMPLES In the following, the present invention is described with reference to the following test examples, but the present invention is not entirely limited by them.

[Test example 1] Effect of the PPAR agonist on the increase in the number of cells using the corneal epithelial cell. 1. Animal used A male rabbit was used (Japanese white, KITAYAMA LABES Co., Ltd.). The experimental animal used according to the principles of International Guide for Biomedical Research Involving Animals (International Guiding Principles for Biomedical Research Involing Animáis). 2. Preparation of the corneal epithelial cell A corneal epithelial cell was prepared from rabbit eyeballs. A cornea was removed from isolated eyeballs, and stored in Dulbecco's phosphate-buffered saline (D-PBS; Invitrogen), and transferred to a clean table. The following cell preparation procedures were all performed in a sterile way The isolated corneal segment was washed with D-PBS with 1% penicillin-streptomycin (Invitrogen) added to it three times, and transferred to a minimal essential medium (MEM, Invitrogen). A corneal endothelial cell and a Descemet membrane of the corneal segment immersed in MEM were detached with a razor (Alcon) for ocular operation, and the corneal segment (corneal stroma and corneal epithelium) after detachment, was transferred to MEM which had was added dispasa II (Roche Diagnostics) at 2.4 U / ml. This was incubated at 37 ° C for 1 hour and, after this, the corneal segment treated with dispase II was transferred to MEM. The corneal epithelium of the corneal segment submerged in MEM was detached with the razor for ocular operation and the residual corneal segment (corneal stroma) was removed from MEM. The detached corneal epithelial cell and the MEM containing it were recovered in a 50 ml centrifuge tube, subjected to centrifugation at 1,500 rpm for 5 minutes at room temperature. The supernatant was discarded to give a layer of corneal epithelial cells. The corneal epithelial cell layer was added with 1 ml of trypsin-EDTA (Invitrogen) and, after all of the mixing, the cells were incubated at 37 ° C for 5 minutes to dissociate the addition between the cells. To this 9 were added mi of MEM containing 10% fetal bovine serum (FBS; Invitrogen) to stop the enzymatic reaction, and this was again subjected to centrifugation at room temperature and 1,500 rpm for 5 minutes to obtain the corneal epithelial cell layer. To the resultant corneal epithelial cell layer was added 1 mM of a serum-free liquid medium for proliferation of normal rabbit corneal epithelial cells (RCGM2, Kurabo Industries LTD.) To suspend the cells, and these were seeded in a control box. culture (IWAKI) for cell culture, which had a diameter of 10 cm, to which 9 ml of RCGM2 had been added. The seeded cells were cultured in an incubator (SANYO) adjusted to 37 ° C, 5% C02, 95% air, and 100% humidity. The culture medium was exchanged with a fresh culture medium every 48 hours until the day of the test. 3. Test substance and method of preparation As the test substance, the following compounds were used. The PPARa agonist: WY-14643 (Calbiochem) and fenofibrate (Sigma-Aldrich). PPARd agonist: L-165041 (Sigma-Aldrich). Each test substance was dissociated in ethanol (Wako Puré Chemical Industries, Ltd.) at a concentration of 200 times of a final concentration in the culture medium and stored at -80 ° C until immediately before use. To study the effect of the promotion of cell proliferation for each test substance, a culture medium prepared by removing a mouse-derived epithelial growth factor (mEGF) coupled to RCGM2, from RCGM2, was used as a medium. basal, whereas as a positive control to confirm the effect of the promotion of cell proliferation, a medium with a mEGF added to it (basal medium + 10 ng / ml of mEGF) was used according to the instructions of a protocol of preparation of RCGM2. 4. Test method 1) Collagen treatment of the culture box As a culture box for a cell proliferation promotion test, a 96-well tissue culture box (Corning) was used. On the day before the test, each 50 μL of 0.01% type I collagen (Nitta Gelatin Inc.) was dispensed into each well on the culture box, and the coating was performed at 4 ° C until immediately before the test. proof. On the day of the test, after a type I collagen solution was removed, the bottom of the culture box was washed using D-PBS, and this was used in the test as a culture box treated with collagen. 2) Cell culture and addition of the test substance In the test, rabbit corneal epithelial cells that had been cultured until subconfluence in a culture box having a diameter of 10 cm were used. After the culture medium was removed, the bottom of the culture box was washed twice using D-PBS, to this was added 1 ml of trypsin-EDTA, this was incubated at 37 ° C for 5 minutes and after this the cells were detached from the bottom of the culture box. After incubation, 9 ml of MEM containing 10% FBS was added to the culture box to stop the enzymatic reaction, and this was subjected to centrifugation at room temperature at 1,500 rpm for 5 minutes to obtain a layer of epithelial cells corneal An appropriate amount of RCGM2 was added to the resulting cell at a cell concentration of 2 x 10 5 cells / ml to suspend the cells. Each 64 μL of this cell suspension was supplied in each well to the number of cells per background area (0.32 cm2) of the 96-well culture box treated with collagen, for tissue culture of 4 x 10 4 cells / cm 2. After the end of planting of the cells, the culture box was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity, and this was cultured for 24 hours. After 24 hours of cell seeding, the culture medium was discarded, and every 100 μL of the The next culture medium was mixed fresh in each well of the culture box. [1] Basal medium only (no addition group) [2] Basal medium + mEGF (final concentration: 10 ng / ml, positive control group) [3] Basal medium + WY-14643 (final concentration: 0.1 μM and 1 μM) [4] basal medium + fenofibrate (final concentration: 0.1 μM and 1 μM) [5] basal medium + L-165041 (final concentration: 0.1 μM and 1 μM). To equalize ethanol concentrations in all culture media up to 0.5%, 5 μL / 1 ml of ethanol was added to culture media [1] and [2], respectively. 3) Measurement of cell number After 48 hours of exchange of the culture medium, the culture supernatant from each well was removed and then, each 100 μL of the basal medium with 10% Cell Counting Kit-8 (DOJINDO) added to this one, they were filled inside each well. After the assortment, the culture box was transferred to an incubator set at 37 ° C, 5% C0, 95% air, and 100% humidity, and this was incubated for 2 hours. After incubation for 2 hours, the absorbance at 450 nm was measured using a microplate vector (Dainippon Pharmaceutical Co., Ltd.), and this was used as an index of an increase in the number of cells.

. Statistical analysis Assuming that an average of the absorbance of the group without addition was 100%, the value of each group of the group without addition, each group of addition of test substance and the positive control group were calculated, and the comparison between the group no addition with the addition group of the test substance and the positive control group, was performed using a Dunnet multiple comparison test method (two tails). As a result of the test, less than a level of significance of 5% was determined as significant. 6. Test results The effect of increasing the number of cells of each group is shown in Table 1. Assuming that the cell proliferation of the group without addition was 100%, the cell proliferation in all the addition groups of the test substance and the positive control group was significantly higher than in the group without addition (p <; 0.01) and it was shown that cell proliferation was improved. From this test result, it became clear that the PPARα agonist and the PPARd agonist increase the number of corneal epithelial cells.

[Table 1] Changes in cell number when the PPARα agonist or the PPARd agonist or a mEGF (positive control) was d to a cultured rabbit corneal epithelial cell, are shown as values when an average of the no-tion group was 100% (average ± standard deviation, N = 5). ** in the table indicates a significant difference (p <0.01) compared to the group without tion.

[Test example 2] Effect of the PPARd agonist on the increase in the number of cells using corneal epithelial cells. 1. Animal used A male rabbit (Japanese white, weight: approximately 1.5 kg, KITAYAMA LABES Co., Ltd.) was used. The experimental animal was used according to the International Guiding Principles for Biomedical Research Involving Animals (International Guiding Principles for Biomedical Research Involving). 2. Preparation of the corneal epithelial cell A rabbit corneal epithelial cell was prepared using the same process as that of Test Example 1. 3. Test substance and method of preparation As the test substance, the following compounds were used. The PPARd agonist: L-165041 (Sigma-Aldrich) and GW-501516 (Alexis). Each test substance was dissolved in ethanol (Wako Puré Chemical Industries, Ltd.) at a concentration of 200 times of a final concentration in the culture medium, and this was stored at -80 ° C until immediately before use. To study the effect of promoting cell proliferation for each test substance, a culture medium prepared by removing the factor of Epithelial growth derived from mice (mEGF) linked to RCGM2, from RCGM2, was used as a basal medium, while a medium with mEGF d to it was used as a positive control to confirm the effect of promoting cell proliferation. (basal medium +10 ng / ml of mEGF) according to the instructions of a preparation protocol of RCGM2. 4. Test method 1) Collagen treatment of the culture box The collagen treatment of the culture box was performed using the same process as that of Test Example 1. 2) Cell culture and tion of the test substance In the test , rabbit corneal epithelial cells that had been cultured until subconfluence in a culture box having a diameter of 10 cm, were used. After the culture medium was removed, the bottom of the culture box was washed twice using D-PBS ', to this was d 1 ml of trypsin-EDTA, this was incubated at 37 ° C for 5 minutes and then from this the cells were detached from the bottom of the culture box. After the incubation, 9 ml of MEM containing 10% FBS was d to the culture box to stop the enzymatic reaction, and the latter was subjected to centrifugation. room temperature at 1,500 rpm for 5 minutes to obtain a layer of corneal epithelial cells. An appropriate amount of RCGM2 at a cell concentration of 1 x 10 5 cells / ml was d to the resulting cell to suspend the cells. Each 64 μL of this cell suspension was supplied in each well to the number of cells per background area (0.32 cm2) of the 96-well culture box treated with collagen, for tissue culture of 2 x 10 4 cells / cm 2. After the end of planting of the cells, the culture box was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity, and this was cultured for 24 hours. After 24 hours of cell seeding, the culture medium was discarded, and each 100 μL of the following culture medium was assorted fresh into each well of the culture box. [1] Basal medium only (no tion group) [2] Basal medium + mEGF (final concentration: 10 ng / ml, positive control group) [3] Basal medium + L-165041 (final concentration: 0.1 μM and 1 μM) [4] basal medium + GW-501516 (final concentration: 0.1 μM and 1 μM) To equalize the concentrations of ethanol in all culture media up to 0.5%, 5 μL / 1 ml of ethanol was d to the media. culture [1] and [2], respectively. 3) Measurement of cell number The culture supernatant from each well was removed every 48 hours for the exchange of the culture medium containing a test substance, each 100 μl of the freshly prepared culture medium were assorted into each well. After 120 hours of initial exchange in a culture medium containing a test substance, the culture medium, the culture supernatant from each well was removed and then, every 100 μL of the basal medium with 10% Cell Counting Kit- 8 (DOJINDO) added to it, were filled into each well. After the assortment, the culture box was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity, and this was incubated for 2 hours. After incubation for 2 hours, the absorbance at 450 nm was measured using a microplate vector (Dainippon Pharmaceutical Co., Ltd.), and this was used as an index of an increase in the number of cells.

. Statistical analysis Assuming that an average of the absorbance of the group without addition was 100%, the value of each group of the group without addition, each group of addition of test substance and the positive control group were calculated, and the comparison between the group no addition with group of addition of the test substance and the positive control group, was performed using a Dunnet multiple comparison test method (two tails). As a result of the test, less than a level of significance of 5% was determined as significant. 6. Test results The effect of increasing the number of cells of each group is shown in Table 1. Assuming that the cell proliferation of the group without addition was 100%, the cell proliferation in all the addition groups of the test substance and the positive control group was significantly higher than in the group without addition (p <0.01) and it was shown that cell proliferation was improved. From this test result, it became clear that the compound having a PPARd agonist activity increases the number of corneal epithelial cells.

Table 2 Changes in the number of cells when the PPARd agonist or a mEGF (positive control) was added to cultured rabbit corneal epithelial cells are shown as values relative to the group average without addition as 100% (mean ± standard deviation, N = 5). ** in the table indicates a significant difference (p <0.01) compared to the group without addition.

[Test Example 3] Effect of the PPAR agonist on the increase in the number of cells using epithelial cells of meibomian glands. 1. Animal used A female cynomolgus monkey (Environmental Biological Life Science Research Center). The experimental animal was used according to the International Guiding Principles for Biomedical Research Involving Animals. 2. Preparation of Meibomian Gland Epithelial Cells Meibomian gland epithelial cells were prepared from a monkey eyelid. An eyelid was isolated and stored in Dulbecco's phosphate-buffered saline (D-PBS, Invitrogen), and transferred to a clean table. The following cell preparation procedures were all performed in a sterile manner. After the isolated eyelid was immersed in 80% ethanol for 30 seconds, the eyelid was washed with D-PBS with 1% penicillin-streptomycin (Invitrogen) added to it three times, and transferred to a minimal essential medium (MEM Invitrogen). A fatty tissue and muscle tissue surrounding a tissue of the meibomian gland were removed under a stereomicroscope, and this was transferred to MEM containing 0.475 U / ml of collagenase A (Roche Diagnostics) and 2.4 U / ml of dispase II (Roche Diagnostics), and incubated at 37 ° C overnight. After the completion of the incubation, the enzyme-treated tissue was again placed under the stereomicroscope, the eyelashes and the connective tissue of the eyelid were removed, and the tissue of the meibomian gland was isolated. To the tissue of the isolated gland was added 1 ml of trypsin-EDTA (Invitrogen), and this was incubated at 37 ° C for 5 minutes. After incubation, 9 ml of MEM containing 10% FBS was added to stop the enzymatic reaction, then the suction and exhaust were repeated five times using a 10 ml pipette, and then five times using a pipette. injection syringe equipped with a 21G injection needle to disperse cells that make up the tissue. The cell dispersion was subjected to a 100 μm nylon filter after 40 μm (Cell Strainer; Falcon), to eliminate the cell masses contained in the dispersion that had not been treated with enzyme. The cell suspension that had passed through the filter was recovered in a 50 μl centrifuge tube, and subjected to centrifugation at room temperature and at 1,500 rpm for 5 minutes. To a cell layer containing a target cell obtained by centrifugation, 80 μl of D-PBS containing 0.5% bovine serum albumin (BSA, Sigma-Aldrich) was added to sufficiently suspend the cells, to which was added 20 μl of Anti-fibroblast microspheres (Militenyi Biotec), and this was allowed to stand at room temperature for 30 minutes. After the completion of the reaction with a antibody, to this was added 2 ml of D-PBS containing 0.5% BSA, and this was again subjected to centrifugation at room temperature and 1,500 rpm for 5 minutes. To a cell layer containing the target cells obtained by centrifugation, 1 ml of D-PBS containing 0.5% BSA was added to sufficiently suspend the cells, and these were added dropwise to a column of balanced LD (Militenyi Biotec) in advance using a column wash solution (D-PBS containing 2 mM EDTA (Doj indo Laboratories) and 0.5% BSA). Then, column LD, 2 ml of the column wash solution was added dropwise. Immediately after the dropwise addition of the cell suspension until the completion of the dropwise addition of the wash solution of the column, non-labeled target cells (non-fibroblasts) were recovered with an antibody that had not been adsorbed on the column, in a 50 ml centrifuge tube. The cell recovered in the centrifuge tube was again subjected to centrifugation at room temperature and at 1,500 rpm for 5 minutes to remove the supernatant. To this precipitation was added 1 ml of Defined Keratinocyte Serum Free Medium (DK-SFM; Invitrogen) to suspend the cells and thereby a suspension of meibomian gland epithelial cells was prepared. The prepared cell was seeded on a culture box (IWAKI) for cell culture, which has a diameter of 3.5 cm that had been coated with a type I collagen solution (Nitta Gelatin Inc.) in advance, and to which 3 ml of DK-SFM had been added. The seeded cell was cultivated in an incubator (SANYO) at 37 ° C, 5% C02, 95% air, and 100% humidity, and the culture medium was exchanged with a fresh culture medium every 48 hours until the day of the test. 3. Test substance and method of preparation As a test substance, the following compounds were used. PPARa agonist: fenofibrate (Sigma-Aldrich) 'PPARd agonist: L-165041 (Sigma-Aldrich) PPAR agonist: troglitazone (Calbiochem) Each test substance was dissolved in ethanol (Wako Puré Chemical Industries, Ltd.) to a concentration of 200 times of a final concentration in the culture medium, and this was stored at -80 ° C immediately before use. To study the effect of the promotion of cell proliferation by each test substance, a culture medium obtained by removing the supplement coupled to DK-SFM, from DK-SFM, was used as a basal medium, while as a positive control to confirm the effect of proliferation promotion cell, a medium was used with the supplement added to it (basal medium + supplement) according to the instructions of a DK-SFM preparation protocol. 4. Test Method 1) Collagen treatment of the culture box As a culture box for a cell proliferation promotion test, a 96-well culture plate was used for tissue culture (Corning). On the day before the test, each 50 μl of 0.01% type I collagen (Nitta Gelatin Inc.) was filled into each well of the culture box, and the coating was performed at 4 ° C until immediately before the test. proof. On the day of the test, after a type I collagen solution was removed, the bottom of the culture plate was washed three times using D-PBS, and this was used in the test as a collagen-treated culture plate. . 2) Cell culture and addition of the test substance In the test, monkey meibomian gland epithelial cells, which had been cultured to subconfluence in a culture dish having a diameter of 3.5 cm, were used and cryopreserved in nitrogen liquid. Cells that had been suspended in a cell bank (Nipoon Zenyaku Kogyo Co., Ltd.), and had been cryopreserved, thawed, and transferred to a 50 ml centrifuge tube and a 10-fold amount of DK-SFM was added. Later, these were subjected to centrifugation at room temperature and at 1,500 rpm for 5 minutes to recover a cell layer, an appropriate amount of DK-SFM was added to suspend the cells, so that the resulting cell concentration became 3 x 10 6 cells / ml. Each 64 μl of this cell suspension was dispensed into each well, so that the number of cells per background area (0.32 cm 2) of the 96-well culture plate, treated with collagen for tissue culture, became 6 x 104 cells / cm2. After the termination of the cell seeding, the culture plate was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity, and this was cultured for 24 hours. After 24 hours from the seeding of the cells, the culture medium was discarded, and each 100 μl of the following culture media was freshly added into each well of the culture dish. [1] Basal medium only (no addition group) [2] Basal medium + supplement (positive control group) [3] Basal medium + fenofibrate (final concentration: 1 μM and μM) [4] Basal medium + L-165041 (final concentration 0.1 μM and 1 μm) [5] Basal medium + troglitazone (final concentration: 0.1 μm and 1 μM) To equalize the concentrations of ethanol in all culture media at 0.5%, 5 μl / ml 1 ml of ethanol was added to the culture media [1] ] and [2], respectively. 3) Measurement of cell number After 48 hours from the initial exchange of the culture medium, the culture medium was exchanged with the fresh culture medium of each [1] to [4] mentioned above. In addition, after 48 hours from this, the culture supernatant from each well was removed, then each 100 μl of DK-SFM with 10% Cell Counting Kit-8 (DOJINDO) added to these were filled into each well . After the assortment, the culture box was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity, and this was incubated for 2 hours. After incubation for 2 hours, the absorbance at 450 nm was measured using a microplate vector (Dainippon Pharmaceutical Co., Ltd.), and this was used as an Index of an increase in the number of cells.

. Statistical analysis Assuming that an average of the absorbance of the group without addition was 100%, the value of each group of the group without addition, each addition group of test substance and the positive control group were calculated, and the comparison between the group without addition with addition group of the test substance and the positive control group, was performed using a test method of Dunnet's multiple comparison (two tails). As a result of the test, less than a level of significance of 5% was determined as significant. 6. Test results The effect of increasing the number of cells in each group is shown in Table 1. Assuming that the cell proliferation of the group without addition was 100%, cell proliferation in an addition group of fenofibrate which is an agonist of PPARa and an addition group of L-165041 which is a PPARd agonist, and the positive control group was significantly higher than in the no-addition group (p < 0.01) and cell proliferation was shown to be increased. On the other hand, troglitazone, which is a PPARα agonist, did not show promoter activity of cell proliferation. From this test result, it became clear that the PPARα agonist and the PPARd agonist increase the number of epithelial cells of the meibomian gland.

Table 3 Changes in cell numbers when the PPARα agonist, the PPARd agonist or the PPARα agonist or the supplement (positive control) was added to the epithelial cells of meibomian monkey gland, cultured, they are shown as values when an average of the group without addition was 100% (mean ± standard deviation, N = 5). ** in the table indicates a significant difference (p <0.001) compared to the group without addition.

[Test Example 4] Expression of PPARs in corneal epithelial cells and meibomian gland epithelial cells. 1. Cells used As the rabbit corneal epithelial cells, the cells prepared and cultured were used by the same process as that of Test Example 1. As the epithelial cells of monkey meibomian gland, cells prepared and cultured by the same process were used. than that of (Test Example 3). As the human corneal epithelial cells (Kurabo Industries LTD.), The cultured cells were used in an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity using the serum-free basal medium ( EpiLife; Kurabo Industries LTD.) For the proliferation of normal human corneal epithelial cells. 2. Test Method 1) Total RNA extraction from the cell Total RNA was extracted from each cell according to a conventional Trizol Reagent method (Invitrogen). 2) Preparation of cDNA from extracted RNA Treatment with DNase from extracted total RNA according to a conventional DNA-free method (Ambion) was performed at 37 ° C for 30 minutes, to remove the genomic DNA. The preparation of the cDNA from the extracted RNA was carried out according to a conventional method of the Superscript II Reverse Transcriptase (Invitrogen). That is, using random primers (Invitrogen), a cDNA complementary to this was prepared from 1 μg of a total RNA treated with DNase. 3) Amplification of the PPAR genes (Polymerase Chain Reaction, PCR) The PCR of the PPAR genes was carried out according to a conventional method of Platinum PCR SuperMix (Invitrogen). Primers of PPARs were designed so that the PCR product became approximately 200 base pairs, with reference to the known sequences of a human, a chimpanzee, a cynomolgus monkey, cattle and mice. PPARa: GTAGAATCTGCGGGGACAAG (sense) (SEQ ID NO: 1): GTTGTGTGACATCCCGACAG (antisense) (SEQ ID NO: 2) PPARd: TTCCTTCCAGCAGCTACACA (sense) (SEQ ID NO: 3): GATCGTACGACGGAAGAAGC (antisense) (SEQ ID NO: 4) PPAR ?: CTCCGTGGATCTCTCCGTAA (sense) (SEQ ID NO: 5): GATGCAGGCTCCACTTTGAT (antisense (SEQ ID NO: 6) The PCR reaction was completed by repeating a three-step reaction at 94 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 30 minutes thirty five times, after a reaction at 94 ° C for 2 minutes and 15 seconds. A sample after the PCR reaction was subjected to electrophoresis using a 2% agarose gel and then, the DNA separated in the gel was stained using SYBR Gold (Molecular Probes). The dyed DNA was made to emit light on a UV transilluminator, and this image was stored as digital data. 3. Test results A DNA band stained after electrophoresis is shown in Figure 1. As a result of this test, it was confirmed that all of PPARa, PPARd and PPAR? they are expressed in human corneal epithelial cells and epithelial cells of the meibomian gland, of monkey. In addition, in the rabbit corneal epithelial cells, the expression of PPARd alone was confirmed. Bonazzi et al. report that, among the PPARs, PPARa and PPARβ (= d) are expressed in rabbit corneal epithelial cells (Bonazzi A. et al., J. Biol. Chem. (2000); 275 (4): 2837-2844) and in their report, they use a special method to detect PPARa. As shown in Test Example 1, it is clear that the PPARa agonist promotes the proliferation of epithelial cells of rabbit, and since in the report by Bonazzi et al., PPARa is detected using the special detection method, it is suggested that an amount of expression of a PPARa in a rabbit corneal epithelial cell is very small.

[Test Example 5] Effect of the PPARd agonist on the increase in the number of cells using normal human corneal epithelial cells. 1. Cells used Normal human corneal epithelial cells (KURABO) were used. 2. Test Substance and Method of Preparation As the test substance, the following compounds were used. PPARd agonist: L-165041 (Sigma-Aldrich), GW 501516 (Alexis). Each test substance was dissolved in ethanol (Wako Puré Chemical Industries, Ltd.) at a concentration of 200 times of a final concentration in the culture medium, and stored at -80 ° C until immediately before use. To study the effect of the promotion of cell proliferation by each test substance, as a cell culture medium, the medium (basal medium) was used. in which insulin, hydrocortisone and transirherrin, contained in an HCGS proliferation additive (KURABO) were added to EpiLife (KURABO). In addition, as a positive control to confirm the promoter effect of cell proliferation, a medium was used in which a mouse-derived epithelial growth factor (mEGF) contained in an HCGS proliferation additive kit (KURABO) was added to a basal medium (basal medium + 1 ng / ml of mEGF). 3. Test Method 1) Cell culture and addition of the test substance. Normal human corneal epithelial cells that had been cryopreserved in liquid nitrogen were thawed, the number of cells counted, and a total amount thereof was transferred to 4 ml of EpiLife (complete medium) with all of a proliferation additive kit HCGS (insulin, mouse-derived epithelial growth factor, hydrocortisone, trans-errine, bovine pituitary gland extract) were added to it to suspend them sufficiently. This cell suspension was seeded on a multi well plate coated with fibronectin (24 wells, BECTON DICKINSON) so that the number of cells became 2 x 10 4 cells / 500 μl / well (since the background area is 2 cm 2). , the number is 1 x 104 cells / cm2). After the termination of the cell seeding, the culture plate was cultured in an incubator adjusted to 37 ° C, 5% C02, 95% air, and 100% humidity for 24 hours and then, the culture medium was exchanged with each 400 μl of the basal medium. In addition, 24 hours after this, the culture medium was exchanged with each 400 μl of the following culture media. [1] Basal medium only (no addition group) [2] Basal medium + mEGF (final concentration: 1 ng / ml, positive control group) [3] Basal medium + L-165041 (final concentration: 0.01 μM and 0. 1 μM). [4] Basal medium + GW-501516 (final concentration: 0.001 μM and 0.01 μM). To equalize all ethanol concentrations in all culture media at 0.5%, each 5 μl / 1 ml ethanol was added to culture media [1] and [2], respectively. 2) Measurement of cell number After 24 hours from the start of stimulation with a test substance, the culture supernatant from each well was removed and then, every 200 μl of a basal medium with 10% Cell Counting Kit- 8 (DOJINDO) added to it, were filled into each well. After stocking, the culture plate was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity, this was incubated for 2 hours, and every 100 μl was taken from the supernatant after completion of the reaction in a 96-well culture plate for tissue culture (Corning) The absorbance at 450 nm of the reaction solution that had been transferred to the 96-well culture plate was measured using a microplate reader (Dainippon Sumitomo Pharmaceutical Co., Ltd.), and this was used as an index of an increase in the number of cells. 4. Statistical analysis Assuming that an average of the absorbance of the group without addition was 100%, the value of each group of the group without addition, each addition group of the test substance and the positive control group were calculated, and the comparison between the No addition group with each addition group to the test substance and the positive control group was performed using a Dunnett multiple comparison test method (one tail). As a result of the test, less than a level of significance of 5% was determined to be significant.

. Test results The effect of increasing the number of cells in each group is shown in Table 4. Assuming that cell proliferation of the group without addition was 100%, cell proliferation in the group with addition of test substance (< 0.01) and positive control group (p < 0.05 ) was significantly higher than in the no-addition group, and it was shown that cell proliferation was improved. From this test result, it became clear that the PPARd agonist increases the number of normal human corneal epithelial cells.

Table 4 Changes in cell number when the agonist of PPARd or mEGF (positive control) was added to normal cultured human corneal epithelial cells, shown as values relative to the group average without addition, such as 100% (mean ± standard deviation, N = 3 to 4). * in the table indicates a significant difference compared to the group without addition (<0.05), and ** in the table indicates a significant difference compared to the group without addition (p <0.01).

[Test Example 6] Effect of the PPARd agonist on the increase in the number of cells using normal human corneal epithelial cells. 1. Cells used Normal human corneal epithelial cells (KURABO) were used. 2. Test substance and method of preparation As the test substance, the following compounds were used. PPARd agonist: GW-501516 (Alexis), GW-0742 (Sigma-Aldrich). Each test substance was dissolved in ethanol (Wako Puré Chemical Industries, Ltd.) at a concentration of 200 times of a final concentration in the culture medium, and stored at -80 ° C until immediately before use. To study the effect of the promotion of cell proliferation by each test substance, as a cell culture medium, a medium (basal medium) was used in which insulin, hydrocortisone and transferrin contained in a proliferation additive kit HCGS (KURABO) were added to EpiLife (KURABO). 3. Test Method 1) Cell culture and addition of the test substance. Normal human corneal epithelial cells that had been cryopreserved in liquid nitrogen were thawed, the number of cells was counted, and a total amount of them was transferred to 4 ml of EpiLife (complete medium) with all of a proliferation additive kit HCGS (insulin, mouse-derived epithelial growth factor, hydrocortisone, transferrin, bovine pituitary gland extract) were added to it to suspend them sufficiently. This cell suspension was seeded in a 96-well culture plate for tissue culture (Corning) so that the number of cells became 1.28 x 10 4 cells / 100 μl / well (since the background area is 0.32 cm 2, the number is 4 x 104 cells / cm2). After the termination of the cell seeding, the culture plate was cultured in an incubator adjusted to 37 ° C, 5% C02, 95% air, and 100% humidity for 24 hours and then, the culture medium was exchanged for 100 μl of a basal medium (EpiLife to which insulin, hydrocortisone and transferrin were added). an HCGS proliferation additive). In addition, 24 hours after this, the culture medium was exchanged with each 100 μl of the following culture media. [1] Basal medium only (no addition group) [2] Basal medium + GW-501516 (final concentration: 0.01 μM) [3] Basal medium + GW-0742 (final concentration: 0.01 μM) To equalize all ethanol concentrations in all culture media at 0.5%, 5 μl / 1 ml of ethanol was added to the culture medium [1]. 2) Measurement of cell number After 48 hours from the start of stimulation with a test substance, the culture supernatant from each well was removed and then, every 100 μl of the basal medium with 10% Cell Counting Kit-8 ( DOJINDO) added to these, they were supplied inside each well. After the assortment, the culture plate was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity and this was incubated for 2 hours. The absorbance at 450 nm of each well was measured using a microplate reader (Dainippon Sumitomo Pharmaceutical Co., Ltd.), and this was used as an index of an increase in the number of cells. 4. Statistical analysis Assuming that an average of the absorbance of the group without addition was 100%, the value of the group without addition, and each addition group with test substance was calculated, and the comparison was made between the group without addition with each group of addition of test substance and the positive control group, using a Dunnett multiple comparison test method (one tail). As a result of the test, less than a level of significance of 5% was determined as significant.

. Test results The effect of increasing the number of cells in each group is shown in Table 5. Assuming that the proliferation of the group without addition was 100%, the cell proliferation in both groups of test substance addition, was significantly more higher than in the group without addition, and it was shown that cell proliferation was increased (p <0.01). From this test result, it became clear that the PPARd agonist increases the number of normal human corneal epithelial cells.

Table 5 Changes in the number of cells when the PPARd agonist was added to a cultured human corneal epithelial cell are shown as values relative to the group average without addition as 100% (mean ± standard deviation, N = 4 to 5). ** in the table indicates a significant difference compared to the group without addition (p <0.01.

[Test Example 7] Effect of the PPAR agonist? on the increase in the number of cells using normal human corneal epithelial cells. 1 Cell used A normal human corneal epithelial cell (KURABO) was used. 2. Test substance and method of preparation As the test substance, the following compound was used. PPAR agonist ?: troglitazone (Calbiochem). The test substance was dissolved in ethanol (Wako Puré Chemical Industries, Ltd.) at a concentration of 200 times of a final concentration in the culture medium, and stored at -80 ° C until immediately before use. To study the influence of each test substance on normal human corneal epithelial cells, as a cell culture medium, a medium (EpiLife basal medium) was used, in which insulin, hydrocortisone and transferrin, except for one Mouse-derived epithelial growth factor (mEGF) of additives contained in a group of HCGS proliferation additives (KURABO) were added to EpiLife (KURABO). 3. Test method 1) Cell culture and addition of the test substance Normal human corneal epithelial cells Normal human corneal epithelial cells that had been cryopreserved in liquid nitrogen were thawed, the cell number was counted, and the total number of cells was counted was transferred to EpiLife (complete medium) with all HCGS proliferation additive equipment that contained a mEGF added to it, to suspend them sufficiently. This cell suspension was seeded on a multi well plate which had been coated with collagen as in Test Example 1 (96 wells, Costar), so that the number of cells became 1.28 x 10 4 cells / 64 μl / well ( since the background area is 0.32 cm2, the number is 4 x 104 cells / cm '). After the termination of the cell seeding, the culture plate was cultured in an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity for 24 hours and then, the culture medium was exchanged with each 100 μl of the culture medium containing the next test substance, and the culture was continued. [1] Basal medium only EpiLife (group without addition) [2] Basal medium EpiLife + troglitazone (final concentration: 0.1 μM). To equalize all ethanol concentrations in all culture media at 0.5%, 5 μl / 1 ml of ethanol was added to the culture medium [1]. Between the day of the start of the addition of the test substance and the day of the cell number measurement, the culture medium was exchanged with a culture medium containing the aforementioned test substance every 2 days. 2) Measurement of cell number After 6 days from the start of stimulation with a test substance, the culture supernatant from each well was removed and, subsequently, every 100 μl of a basal EpiLife medium with 10% Cell Counting Kit -8 (DOJINDO) added to it were filled into each well. After the assortment, the culture plate was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity, this was incubated for 2 hours, and the absorbance at 450 nm of each well of the culture plate after completion of the reaction, was measured using a microplate reader (Dainippon Sumitomo Pharmaceuticals Co., Ltd.), and this was used as an index of an increase in the number of cells.

Statistical analysis Assuming that an average of the absorbance of the group without addition was 100%, the value of the group without addition, and each group with addition of the test substance was calculated, and the comparison between the group without addition with each group of Addition of test substance was performed using a Dunnett multiple comparison test method (one tail). As a result of the test, less than a level of significance of 5% was determined as significant.

. Test results The influence of the PPAR agonist? on normal human corneal epithelial cells, is shown in Table 6. From this test result, the effect of increasing the number of corneal epithelial cells in the PPAR? agonist was not recognized.

Table 6 Changes in the number of cells when the PPAR agonist? was added to normal, cultured human corneal epithelial cells are shown as value relative to the group mean without addition as 100% (mean ± standard deviation, N = 3 to 4). ** in the table indicates a significant difference compared to the group without addition (p <0.01).

[Test Example 8] Effect of the PPARd agonist on the increase in the number of epithelial cells of the meibomian gland 1. Preparation of epithelial cells of monkey meibomian gland The preparation of meibomian gland epithelial cells was performed from a monkey eyelid as in Test Example 3. The cells were cultured in an incubator (SANYO) adjusted to 37 ° C, 5% CO 2, 95% air, and 100% humidity using a Defined Keratinocyte Serum Free Medium (DK-SFM; Invitrogen, the coupled supplement was added according to the preparation protocol), and the culture medium was exchanged with a fresh one every 48 hours until the cells were almost subconfluent. 2. Test substance and method of preparation As the test substance, the following compounds were used. PPARd agonist: L-165041 (Sigma-Aldrich) and GW-501516 (Alexis) Each test substance was dissolved in ethanol (Nacalai Tasque, Inc.) at a concentration of 200 times of a final concentration in the culture medium, and stored at -80 ° C until immediately before use. To study the effect of the promotion of cell proliferation by each test substance, a culture medium prepared by the withdrawal of the supplement coupled to DK-SFM, from DK-SFM, was used as a basal medium, while as a positive control to confirm the promoter effect of cell proliferation, a culture medium was used with the supplement added to it (basal medium + supplement ) according to the instructions of a DK-SFM preparation protocol. 3. Test Method 1) Collagen Treatment of the Culture Plate A culture plate for a cell proliferation promotion test was coated using type I collagen (Nitta Gelatin Inc.) as in Test Example 3. 2) Cell culture and addition of the test substance In the test, meibomian monkey gland epithelial cells that had been cultured to subconfluence, suspended in a cell bank (Nippon Zenyaku Kogyo Co., Ltd.), and cryopreserved in liquid nitrogen, were used. The cells that had been cryopreserved were thawed, and transferred to a 50 ml centrifuge tube and a 10-fold amount of DK-SFM was added thereto. Then, it was subjected to centrifugation at room temperature and at 1,500 rpm for 5 minutes to recover a cell layer, an appropriate amount of DK-SFM was added to suspend the cells, so that the resulting cell concentration became 2 x 106 cells / ml. Each 64 μl of this cell suspension was dispensed into each well, so that the number of cells per background area (0.32 cm 2) of the 96-well culture plate, treated with collagen for tissue culture, became 4 x 104 cells / cm2. After the termination of the cell seeding, the culture plate was transferred to an incubator set at 37 ° C, 5% C02, 95% air, and 100% humidity, and this was cultured for 24 hours. After 24 hours from cell seeding, the culture medium was discarded, and each 100 μl of the following culture media was freshly added into each well of the culture plate. [1] Basal medium only (no addition group) [2] Basal medium + supplement (positive control group) [3] Basal medium + L-165041 (final concentration: 0.1 μM and 1 μM) [4] Basal medium + GW- 501516 (final concentration: 0.01 μM and 0. 1 μM) To equalize the concentrations of ethanol in all culture media at 0.5%, 5 μl / 1 ml of ethanol was added to culture media [1] and [2], respectively. 3) Cell number measurement After 48 hours from the initial exchange of the culture medium, the culture medium was exchanged with fresh culture medium from each of [1] to [4]. In addition, after 48 hours of this, the culture medium was exchanged again with fresh culture medium from each of [1] to [4]. In addition, after 48 hours from this, the culture supernatant from each well was removed, then each 100 μl of a basal medium with 10% Cell Counting Kit-8 (DOJINDO) added to it, were dispensed into each water well. After the assortment, the culture plate was transferred to an incubator set at 37 ° C, 5% C02, 95% air and 100% humidity, and this was incubated for 2 hours. After incubation for 2 hours, the absorbance at 450 nM was measured using a microplate reader (Dainippon Pharmaceutical Co., Ltd.), and this was used as an index of an increase in the number of cells. 4. Statistical analysis Assuming that an average of the absorbance of the group without addition was 100%, the value of each group of the group without addition, each group of addition of test substance and the positive control group was calculated, and the comparison between the group without addition with each addition group of test substance and the positive control group was performed using a comparison test method Multiple of Dunnett (one tail). As a result of the test, less than a level of significance of 5% was determined as significant.

. Test results The effect of the promotion of cell proliferation of each group is shown in Table 7. Assuming that the cell proliferation of the group without addition was 100%, cell proliferation in an addition group of L-165041 (10"6 M) which is a PPARd agonist and an addition group of GW-501516 (10" 7 M) which is also a PPARd agonist, and the positive control group, was significantly higher than in the group without addition , and it was shown that cell proliferation was improved. From this test result, it became clear that the PPARd agonist increases the number of epithelial cells of the meibomian gland.

Table 7 Changes in the number of cells when the PPARd / β agonist, or the supplement (positive control) was added to cultured monkey meibomian epithelial cells are shown as values relative to the group average without addition as 100% ( average (standard deviation, N = 5). * in the table indicates a significant difference compared to the group without addition (p <0.05), and ** in the table indicates a significant difference compared to the group without addition (p < 0.01).

[Test Example 9] Study of the promoter activity of the healed epithelial corneal wound by the PPAR agonist ( 1. Animal used A male rabbit (Japanese white, KITAYAMA LABES Co., Ltd.) was used. The experimental animal was used according to the International Guiding Principles for Biomedical Research Involving Animals. 2. Test substance and method for the preparation of ophthalmic eye drops As the test substance, a PPARd agonist (GW-501516 (Alexis Biochemicals) was used, a solution in which GW-501516 was suspended in the next vehicle until 0.05%, it was used as a solution of ophthalmic drops.

Dehydrated sodium diacid phosphate 0.05 g Sodium chloride 0.45 g Ultrapure water c.b.p. Polysorbate 80 0.05 ml NaOH c.b.p. Total amount 50 ml (pH 7.0) As a control for the administration group of the test substance, the aforementioned vehicle administration group that did not contain the drug was established. 3. Experimental method 1) Coating epithelial scraping After the animal was subjected to systemic anesthesia by intramuscular injection (0.9 ml / kg) with a selactar (2% xylazine: Bayer) ketalar (5% ketamine: Sankyo) = 0.5: 1 mixed solution, a solution of ophthalmic drops of oxybuprocaine hydrochloride (solution of 0.4% Benoxil ophthalmic drops; Santen Pharmaceutical Co., Ltd.) was administered, and the eyeballs were exposed. A mark having a diameter of 6 mm was stamped on a corneal epithelium in a central corneal part using a trephine having a diameter of 6 mm, and an entire corneal epithelial layer within a stamped circumference was scraped using a manual spreader under a stereomicroscope. After scraping, a corneal surface was washed using physiological saline solution (Otsuka Pharmaceutical Factory Inc.), and the eyeballs were returned inside the orbit to complete the corneal epithelial scraping treatment. 2) Administration A solution of ophthalmic drops of the test substance or a solution of ophthalmic drops from the vehicle was administered to the treated eyes at 50 μl at a time using a micropipette twice a day on the day of shaving. epithelium, on the next day and after it, four times a day until the end of the test. 3) Evaluation At the time point in which scraping of the corneal epithelium of both eyes of all individuals was completed, this was defined as the time of start of the test (hour 0) and when quantifying the area of corneal epithelial defect at 24, 38 and 48 hours after this, the restoration of the corneal epithelium was evaluated. That is, at each time point, 10 μl of a 0.1% sodium fluorescein solution (Wako Puré Chemical Industries, Ltd.) was administered to the treated eyes, a photograph of an anterior segment of the animal was immediately taken using a slit lamp equipped with a cobalt filter and with this a region of epithelial, dye-stained defect was recorded. A developed photograph was stored as a digital image on a computer, and an area of the part of the stained corneal epithelial defect was measured using the image analysis software (Image-Pro Plus). 4. Statistical analysis Regarding the area of the corneal epithelial defect measured at each time point, a value was calculated for each individual, assuming that its initial value was 100%, and this was adopted as a proportion of the remnant corneal epithelial defect. Regarding the proportion of the remaining corneal epithelial defect at each time point, the comparison between the vehicle administration group and the administration group of the test substance was carried out by means of a T test, and it was determined that a lower level of significance of 5% is significant.

. Test results The proportion of the remaining corneal epithelial defect at each time point of the measurement of the vehicle administration group and the administration group of GW-501516 at 0.05% is shown in Table 8. It was shown that the proportion of the Corneal epithelial defect was significantly decreased in the administration group of GW-501516 at 0.05% 24 hours and 38 hours after scraping the corneal epithelium. After 48 hours, the corneal epithelial defect disappeared in all individuals. From this test result, it became clear that the restoration of the defective corneal epithelium is promoted by the administration of the PPAR agonist to the eyes.

Table 8 A value obtained is shown when calculating a proportion (%) of a corneal epithelial defect remaining after the scraping treatment of the corneal epithelium of rabbit eyes, for each individual, assuming that an initial value was 100% (mean ± standard deviation, N = 4). * in the table indicates a significant difference compared to the vehicle administration group (p <0.05) and indicates a significant difference compared to the vehicle administration group (p <0.01 | INDUSTRIAL APPLICATION According to the present invention, a novel agent is provided to promote the proliferation of meibomian gland epithelial cells, or a new agent to promote the proliferation of corneal epithelial cells, and the promoter agent promotes the proliferation of meibomian gland epithelial cells and corneal epithelial cells. In addition, the therapeutic agent of the present invention can be effectively used in the treatment / amelioration of a disease such as dysfunction of the meibomian gland, corneal epithelial disorder or dry, hyper-evaporative eye. The present invention is based on Japanese Patent Application No. 2005-342025 filed on November 28, 2005, the contents of which are incorporated by reference herein in their entirety.

It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (24)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An agent for promoting the proliferation of a Meibomian gland epithelial cell, characterized in that it comprises a PPARa agonist or d as an active ingredient.
2. 2. An agent for promoting the proliferation of a corneal epithelial cell, characterized in that it comprises a PPARa agonist or d as an active ingredient.
3. 3. An agent for treating dysfunction of the meibomian gland, characterized in that it comprises a PPARa agonist or d as an active ingredient.
4. 4. An agent for treating a corneal epithelial disorder, characterized in that it comprises a PPARa agonist or d as an active ingredient.
5. 5. An agent for treating hyper-evaporative dry eye, characterized in that it comprises a PPARa agonist or d as an active ingredient.
6. 6. An agent for promoting the proliferation of an epithelial cell of the meibomian gland, characterized in that it comprises a PPARd agonist as an active ingredient.
7. 7. An agent for promoting the proliferation of a corneal epithelial cell, characterized in that it comprises a PPARd agonist as an active ingredient.
8. 8. An agent for treating dysfunction of the meibomian gland, characterized in that it comprises a PPARd agonist as an active ingredient.
9. 9. An agent for treating a corneal epithelial disorder, characterized in that it comprises a PPARd agonist as an active ingredient.
10. 10. An agent for treating the hyper-ovarian dry eye, characterized in that it comprises a PPARd agonist as an active ingredient.
11. 11. The agent according to any of claims 1 to 5, characterized in that the agonist of PPARa or d is a compound represented by the formula (I): wherein: A is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, B is a linker selected from the group consisting of -CO-, -NH-, - (CH2) nS-, - (CH2) n-0 and -O- (CH2) n-0-, where n is an integer from 1 to 3, X and Y are the same or different , and each is a carbon atom or a nitrogen atom, Z is an oxygen atom, a sulfur atom or -CH2-, Ari is a 5-6 membered aromatic cyclic group optionally having 1 to 3 substituents, Ri and R2 are the same or different and each is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, and R3 is hydrogen, a halogen atom or an alkyl group having a carbon number from 1 to 6, or a pharmacologically acceptable salt thereof.
12. 12. The agent according to any of claims 6 to 10, characterized in that the agonist of PPARd is a compound represented by the formula (II): wherein A is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, B is a linker selected from the group consisting of - (CH2) nS- and -0- (CH2) n-0- where n is an integer from 1 to 3, X and Y are the same or different, and each is a carbon atom or an atom of nitrogen, Z is an oxygen atom, a sulfur atom or -CH2-, Ari is a 5-6 membered aromatic cyclic group optionally having 1 to 3 substituents, Ri and R2 are the same or different and each is a hydrogen atom or an alkyl group having a carbon number of 1 to 6, and R3 is hydrogen, a halogen atom or an alkyl group having a carbon number of 1 to 6, or a pharmacologically acceptable salt thereof. The agent according to claim 11 or 12, characterized in that the PPARd agonist is: (4- (3- (4-acetyl-3-hydroxy-2-propyl) phenoxy) propoxyphenoxy) acetic acid, (2) -methyl-4- (((4-methyl-2- (4- (trifluoromethyl) phenyl) -5-thiazolyl) methyl) thio) phenoxy) acetic acid or (4- (((2- (3-fluoro- ( 4- (trifluoromethyl) phenyl) -4-methyl-5-thiazolyl) methyl) thio) -2-methylphenoxy) acetic acid, or a pharmacologically acceptable salt thereof 14. The agent according to claim 11, characterized in that the The PPARa agonist is 2- (4- (4-chlorobenzoyl) phenoxy) -2-methylpropionate 1-methylethyl or ((4-chloro-6- ((2,3-dimethylphenyl) amino) -2-pyrimidinyl) thio) acetic acid, or a pharmacologically acceptable salt thereof. 15. The use of a PPARa or d agonist for the production of an agent to promote the proliferation of an epithelial cell of the meibomian gland. 16. The use of a PPARa or d agonist for the production of an agent to promote the proliferation of a corneal epithelial cell. 17. The use of a PPARa agonist or d for the production of an agent to treat dysfunction of the meibomian gland. 18. The use of a PPARa or d agonist for the production of an agent to treat a corneal epithelial disorder. 19. The use of a PPARa agonist or d for the production of an agent for treating the hyper-evaporative dry eye. 20. The use of a PPARd agonist for the production of an agent to promote the proliferation of an epithelial cell of the meibomian gland. 21. The use of a PPARd agonist for the production of an agent to promote the proliferation of a corneal epithelial cell. 22. The use of a PPARd agonist for production of an agent to treat dysfunction of the meibomian gland. 23. The use of a PPARd agonist for the production of an agent to treat a corneal epithelial disorder. 24. The use of a PPARd agonist for the production of an agent for treating hyper-evaporative dry eye.