Composition for preventing or treating xerophthalmia comprising HAPLN1

Abstract

The present invention discloses a pharmaceutical composition for preventing or treating xerophthalmia, which contains hyaluronic acid proteoglycan linking protein 1 (HAPLN1) as an active ingredient, and a method for preventing or treating xerophthalmia using the same. According to the present invention, xerophthalmia can be effectively and stably prevented and treated, and side effects are reduced compared to conventional products.

Description

[Technical field]

[0001] The present invention relates to the field of medical approaches for treating dry eye syndrome (DED), and further to pharmaceutical compositions for the prevention and treatment of dry eye syndrome, and methods for treating dry eye syndrome. [Background technology]

[0002] The eye is a complex organ with different specialized cells and tissues that are regulated to maintain optimal visual function. This function is achieved by the presence of firm barriers at the front and back of the eye, which play a key role by selectively controlling the crossing of fluids and solutes in and / or out. Local and systemic diseases can affect different regions of the eye, but the anatomy of the eye and the complex physiology of the retina and optic nerve make the development of effective drugs difficult. The eye is broadly divided into two compartments: the anterior (front of the eye) and the posterior (back of the eye). The anterior part of the eye refers to the cornea, conjunctiva, anterior sclera (the part of the sclera that is transferred anteriorly to become the cornea), iris, ciliary body, aqueous humor, and lens. The back of the eye (also called the posterior segment) includes the anterior hyaloid membrane and the structures behind this membrane, the posterior sclera (the portion of the sclera that is transferred posteriorly to the optic nerve dura), the vitreous body (containing the vitreous humor and membranes), the retina, macula, choroid, and optic nerve.

[0003] Dry eye disease (DED) is a disease accompanied by symptoms such as stinging, itchy eyes, dry eyes, rough eye surface, eye congestion, abnormal tear secretion due to tear duct blockage, hypersensitivity to light, eye fatigue, discomfort when wearing contact lenses, blurred vision, and foreign body sensation, and the number of patients has been increasing worldwide recently. For reference, according to US statistics, it is expected that 29 million dry eye patients will be born in 2022, and $6.6 billion will be needed for dry eye treatment in 2027. In Korea, there are more than 2.4 million dry eye patients every year. Such an increasing trend of dry eye is believed to be contributed to by digitalization, the incorporation of many electronic devices including smartphones into daily life, and the influence of a worsening surrounding environment.

[0004] Although new products have been developed to alleviate or treat xerophthalmia, it is not easy to completely alleviate or treat the symptoms. The direct cause of xerophthalmia can be said to be insufficient tear production and increased tear evaporation. Although xerophthalmia itself is not life-threatening, patients experience serious discomfort, impaired visual function, and greatly reduced quality of life and productivity.

[0005] As a result of extensive research into HAPLN1 (hyaluronan and proteoglycan link protein 1) protein, the inventors discovered that a composition containing the protein, when administered externally, is effective in preventing and treating xerophthalmia, thereby completing the present invention.

[0006] In relation to HAPLN1, for example, Patent Document 1 discloses a configuration in which, when an expression vector encoding one or more therapeutic polynucleotides is delivered to a joint, the cells differentiate into chondrocytes or cartilage-type cells, and / or such delivery stimulates the chondrocytes in the joint to produce more cells. In the detailed description of the invention, HAPLN1 is mentioned as a gene to be used, among more than 200 protein examples. However, there is no mention of xerophthalmia at all.

[0007] Furthermore, Patent Document 2 relates to metallic nanoparticles coated with glycosaminoglycan and their uses, and relates to a composition for treating symptoms of xerophthalmia, which comprises a plurality of nanoparticles coated with a plurality of linker molecules, at least some of which are attached to at least one glycosaminoglycan chain, wherein the linker is HAPLN1 protein, and the glycosaminoglycan chain comprises hyaluronic acid and / or its derivatives, and discloses that the disclosed composition can be formulated for local treatment. That is, Patent Document 2 discloses a configuration in which HAPLN1 protein is used as a linker to introduce hydrated glycosaminoglycan chains into the skin or eyeball. However, in that the nanoparticle / HAPLN1 conjugate constitutes the invention, the HAPLN1 protein itself is not the main component of the invention, and it is unrelated to the function of the HAPLN1 protein acting independently to increase the expression of the mucin gene and promote tear secretion, thereby alleviating dry eye syndrome.

[0008] In addition, Non-Patent Document 1 is a paper on "Genetic Rescue of Chondrodysplasia and the Perinatal Lethal Effect of Cartilage Link Protein Deficiency," which discloses that mice in which the cartilage link protein (Crtl1) gene has been genetically disrupted show chondrogenesis disorders and die before and after birth, but that if a cartilage-specific link protein is introduced into a Crtl1 null mouse and overexpressed, abnormal cartilage formation is prevented and the mortality rate before and after birth is reduced, and that Crtl1 transcripts and their proteins have been found in all organs from fetuses to adults. However, even here, there is no mention of xerophthalmia at all.

[0009] In addition, existing pharmaceutical compositions for alleviating dry eye syndrome utilize immunosuppression and mechanisms that inhibit T cell migration, and are commercially available under the trade names Restasis (2003, Allergan), Cequa (2018, Sun), Xiidra (2016, Novartis), and Eysuvis (2020, Kala). However, due to insufficient efficacy and side effects, approximately 30% to 70% of dry eye syndrome patients discontinue treatment midway. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US Published Patent US2016 / 0220699 [Patent Document 2] International Patent Publication WO2016 / 0156788 [Non-patent literature]

[0011] [Non-Patent Document 1] Journal of Biological Chemistry. 2003. Vol. 278, No40. Pp 39214-29223 Summary of the Invention [Problem to be solved by the invention]

[0012] As described above, the efficacy of conventional products as preventive and therapeutic agents for dry eye syndrome, including xerophthalmia, is still insufficient and they have many side effects. The present invention overcomes the limitations of such conventional techniques and provides a pharmaceutical composition for effectively and stably treating xerophthalmia and a method for treating xerophthalmia. [Means for solving the problem]

[0013] One aspect of the present invention relates to a pharmaceutical composition for preventing or treating xerophthalmia, comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding same as an active ingredient.

[0014] In one embodiment of the present invention, the protein is a composition having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:1.

[0015] According to one embodiment of the invention, the composition is such that the nucleic acid relating to the gene is contained within an expression vector.

[0016] According to one embodiment of the present invention, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating xerophthalmia, which induces the wound healing ability of cells in the eye.

[0017] According to one embodiment of the present invention, the pharmaceutical composition of the present invention relates to a pharmaceutical composition for preventing or ameliorating xerophthalmia, which reduces the expression of inflammatory factors in corneal epithelial cells.

[0018] According to one embodiment of the present invention, the pharmaceutical composition of the present invention relates to a pharmaceutical composition for preventing or treating xerophthalmia, which increases the expression level of mucin.

[0019] According to one embodiment of the present invention, the pharmaceutical composition of the present invention is for use in preventing or treating dry eye, the single topical dose of the pharmaceutical composition of the present invention being 1 ng / ml to 100 μg / ml.

[0020] According to one embodiment of the present invention, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating xerophthalmia, the pharmaceutical composition being in a dosage form selected from the group consisting of eye drops, artificial tears, eye ointments, tablets, pills, capsules, lozenges, inhalants, injections, patches, and suppositories.

[0021] According to one embodiment of the present invention, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating xerophthalmia, which is intended for parenteral administration.

[0022] According to one embodiment of the present invention, the pharmaceutical composition of the present invention is a pharmaceutical composition for preventing or treating xerophthalmia, further comprising a pharmaceutical additive selected from the group consisting of a pharma- ceutical acceptable carrier, a diluent, a binder, a disintegrant, a lubricant, and any combination thereof.

[0023] According to one embodiment of the present invention, the pharmaceutical composition of the present invention relates to a composition that is contained as a main or auxiliary active ingredient in a composition for preventing or suppressing xerophthalmia.

[0024] Another aspect of the present invention relates to a kit comprising the composition of the present invention and an administration guide.

[0025] Another aspect of the present invention relates to a reagent composition for experiments related to the prevention or suppression of xerophthalmia, which comprises the composition of the present invention.

[0026] Another aspect of the present invention relates to the use of hyaluronic acid proteoglycan link protein 1 (HAPLN1) or a gene encoding same for the preparation of a pharmaceutical composition for preventing or suppressing xerophthalmia.

[0027] Another aspect of the present invention relates to a method for preventing or treating xerophthalmia, which comprises administering hyaluronic acid proteoglycan linking protein 1 (HAPLN1) or a gene encoding it as an active ingredient to an individual in need thereof.

[0028] Furthermore, one aspect of the present invention relates to the use of a composition containing hyaluronic acid proteoglycan link protein 1 (HAPLN1) or the gene encoding it as an active ingredient for the prevention or treatment of xerophthalmia. Effect of the Invention

[0029] As described above, the pharmaceutical composition and treatment method of the present invention can effectively and stably prevent and treat dry eye syndrome, and show reduced side effects compared to conventional products. It is presumed that this is possible because the active ingredient of the composition of the present invention, HAPLN1 protein, is a biological molecule, not a chemically synthesized molecule. [Brief description of the drawings]

[0030] [Figure 1A] FIG. 1 is a diagram showing an experimental design according to Example 1 of the present invention. [Figure 1B] This is the result of an experiment conducted according to the experimental plan of Figure 1A of the present invention, and is a graph showing the experimental results in which the cell viability, part of the xerophthalmia-alleviating effect of HAPLN1 protein, was confirmed in human corneal epithelial cells (HCECs) under hypertonic conditions, 24 hours after administration of the composition of the present invention. [Figure 1C]This is a graph showing the results of an experiment conducted according to the experimental plan of Figure 1A of the present invention, in which the cell viability, part of the xerophthalmia-alleviating effect of HAPLN1 protein, was confirmed in human corneal epithelial cells (HCECs) under hypertonic conditions, 48 ​​hours after administration of the composition of the present invention. [Figure 2A] FIG. 1 shows an experimental design according to Example 2 of the present invention. [Figure 2B] This is the result of an experiment conducted according to the experimental design of Figure 2A of the present invention, and is a microscopic image related to the experimental result confirming the effect of HAPLN1 protein in alleviating xerophthalmia in human corneal epithelial cells (HCEC) under hyperosmotic conditions, including wound recovery via cell proliferation and cell migration. [Figure 2C] FIG. 2C is a graph showing the numerical ratio of the area covered by migratory cells shown on the photograph of FIG. 2B. [Figure 3A] This relates to Example 3 of the present invention, and shows images of human corneal epithelial cells (HCECs) treated with various concentrations of HAPLN1 protein under hypertonic conditions, and then the expression levels of various inflammatory factor markers were confirmed using a Human XL cytokine array (ARY022B (R&D Systems.)). [Figure 3B] This is a graph showing the relative expression levels of various inflammatory factor markers using the Human XL cytokine array (ARY022B (R&D Systems.)) in Figure 3A. [Figure 4A] This relates to Example 4 of the present invention, and is a graph showing the amount of CCL-20 secreted by human corneal epithelial cells (HCECs) after treatment with HAPLN1 protein at further subdivided concentrations under hypertonic conditions, as determined by the dosage of the composition of the present invention (HAPLN1). [Figure 4B]In Example 4 of the present invention, human corneal epithelial cells (HCECs) were treated with various concentrations of HAPLN1 protein under hypertonic conditions, and the activity of NFκB, a transcription factor important in the expression of inflammatory factors, was confirmed by Western blot (upper figure), and the related relative expression level was quantified (lower figure). [Figure 5A] This figure shows the experimental design for Example 5 of the present invention, for investigating the level of mucin gene expression after treating human corneal epithelial cells (HCECs) with HAPLN1 protein at different concentrations under hypertonic conditions in Example 5. [Figure 5B] This is an experiment carried out according to the experimental design of Figure 5A, and is a graph showing the relative quantification of MUC4 mRNA expression level to GAPDH mRNA expression level after treating human corneal epithelial cells (HCECs) with various concentrations of HAPLN1 protein under hypertonic conditions. [Figure 5C] This is an experiment carried out according to the experimental design of Figure 5A, and is a graph showing the relative expression level of MUC16 mRNA to the expression level of GAPDH mRNA after treating human corneal epithelial cells (HCECs) with various concentrations of HAPLN1 protein under hypertonic conditions. [Figure 6A] 13 is a photograph showing the expression of MUC5Ac, a representative mucin protein, in human conjunctival epithelial cells after treatment with various concentrations of HAPLN1 protein under hypertonic conditions in Example 6. [Figure 6B] FIG. 6B is a graph showing the quantification of the ratio of cells secreting MUC5Ac identified in FIG. 6A. [Figure 7A] Experimental design to evaluate the efficacy of HAPLN1 in a mouse dry eye model. [Figure 7B] FIG. 1 is a diagram illustrating sections for recording corneal fluorescein scores. [Figure 7C] FIG. 1 shows the method for tear volume assay. [Figure 8A]1 shows eye photographs showing the degree of corneal damage and the degree of improvement through fluorescent staining in Example 8 of the present invention (0 days, 6 days, 11 days). [Figure 8B] FIG. 8B is a graph showing the numerical score results of the fluorescent score assay in FIG. 8A (0 days, 6 days, and 11 days). [Figure 8C] FIG. 1 is a graph showing the results of a tear volume assay in a mouse dry eye model, i.e., the numerical value of the amount of tears absorbed by Schirmer's test strip (0 days, 6 days, and 11 days). [Figure 9A] 1 is a photograph showing the state of detached corneal epithelial cells on the 16th day after treatment with the composition of the present invention at different concentrations. [Figure 9B] FIG. 9B is a graph showing the number of detached corneal epithelial cells counted and quantified in the photograph of FIG. 9A. [Figure 10A] 1 is a photograph showing an image of goblet cells present in the conjunctiva on the 16th day after treatment with the composition of the present invention at different concentrations. [Figure 10B] FIG. 10B is a graph quantifying the number of goblet cells shown in the photograph of FIG. 10A. [Figure 11A] 1 shows a photograph of the expression level of IL-1β, a representative cytokine of inflammatory responses, in the lacrimal gland, confirmed by immunohistochemistry on the 16th day after treatment with the composition of the present invention at different concentrations. [Figure 11B] FIG. 11B is a graph showing the numerical values ​​of the expression level of IL-1β shown in the photograph of FIG. 11A. [Figure 12A] 1 shows photographs of the expression level of CD45, an inflammatory cell marker, in the lacrimal gland, confirmed by immunofluorescence staining (IF) on the 16th day after treatment with the composition of the present invention at different concentrations. [Figure 12B] FIG. 12B is a graph showing the expression level of CD45 shown in the photograph of FIG. 12A in numerical form. [Figure 13A]1 shows photographs of the expression level of CD11b, an inflammatory cell marker, in the lacrimal gland, confirmed by immunofluorescence staining (IF) on the 16th day after treatment with the composition of the present invention at different concentrations. [Figure 13B] 13B is a graph showing the expression level of CD11b shown in the photograph of FIG. 13A in a numerical value. [Figure 14] FIG. 1 shows the experimental design for inducing a rabbit dry eye model using benzalkonium chloride and evaluating the efficacy of HAPLN1 in the model. [Figure 15A] This is an image of the rabbit's eye before the CFS (corneal fluorescein scores) value was calculated to indicate the degree of damage to the cornea. [Figure 15B] 1 is a graph showing the tear film breakup time (TFBUT) measured for each concentration of the composition of the present invention in a rabbit dry eye model induced using benzalkonium chloride. [Figure 15C] FIG. 15A is a graph showing the corneal fluorescein scores (CFS) values, which indicate the degree of damage to the cornea, measured for each concentration of the composition of the present invention and quantified on days 0, 5, 10, and 15. [Figure 15D] FIG. 15A shows the results of measuring the amount of tears absorbed by a Schirmer's test strip. The tear volume was measured for each concentration of the composition of the present invention, and is shown as a graph quantified on days 0, 5, 10, and 15. [Figure 16A] 1 is a set of photographs showing the degree of corneal epithelial cell detachment on the 16th day after treatment with various concentrations of the composition of the present invention in a rabbit dry eye model. [Figure 16B] FIG. 16A is a graph showing the number of detached corneal epithelial cells counted and quantified for each case. [Figure 17A]1 shows photographs showing the state of goblet cells present in the conjunctiva on the 16th day after treatment with various concentrations of the composition of the present invention in a rabbit dry eye model. [Figure 17B] FIG. 17A is a graph quantifying the number of goblet cells shown in the photographs for each case. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] One aspect of the present invention relates to a pharmaceutical composition for preventing or treating xerophthalmia, which comprises hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding it as an active ingredient.

[0032] Furthermore, according to one embodiment of the present invention, the pharmaceutical composition of the present invention can act to induce wound healing ability of cells in the eye, since the composition of the present invention promotes cell proliferation and migration to the wound.

[0033] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may act to reduce the expression of inflammatory factors in corneal epithelial cells, such as interleukins, CCL / CXCL family, chitinase 3-like 1, and pNFkB.

[0034] According to one embodiment of the present invention, the pharmaceutical composition of the present invention may act to increase the expression level of mucins, since the composition of the present invention promotes the expression of mRNA of various mucin families and galectin-3.

[0035] In the present specification, "rhHAPLN1" is an abbreviation for "recombinant human HAPLN1" and refers to recombinant human HAPLN1.

[0036] In certain embodiments, the pharmaceutical composition of the present invention generally also comprises the molecule and a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, etc., compatible with pharmaceutical administration. Supplementary active compounds may also be included in the composition. In other words, the pharmaceutical composition of the present invention further comprises a pharmaceutical excipient selected from the group consisting of pharma- ceutical acceptable carriers, diluents, binders, disintegrants, lubricants, and any combination thereof.

[0037] The pharmaceutical composition may be formulated to be compatible with the intended route of administration. Desirably, the composition of the present invention is in a dosage form selected from the group consisting of eye drops, artificial tears, eye ointments, tablets, pills, capsules, lozenges, inhalants, injections, patches, and suppositories.

[0038] Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Parenteral administration is preferred.

[0039] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may also contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparabens; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and a tonicity adjusting agent such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, single-use syringes, or multiple dose vials made of glass or plastic.

[0040] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be sufficiently fluid for easy syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be achieved by including in the composition agents which delay absorption, for example, aluminum monostearate and gelatin.

[0041] Sterile injectable solution can be prepared by mixing the active compound in the required amount in a suitable solvent with one or a combination of the above-listed components, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other required components listed above.For the preparation of sterile powder for the preparation of said sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which produces the powder of active ingredient and any additional ingredient desired from the solution that has been previously sterile-filtered.

[0042] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound may be mixed with an excipient and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically suitable binding and / or adjuvant materials may also be included as part of the composition. The tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid primogel or corn starch; a lubricant such as magnesium stearate or sterote; a lubricant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

[0043] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0044] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, transmucosal preparations, bile salts and fusidic acid derivatives. Transmucosal administration can be achieved using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels or creams as generally known in the art.

[0045] The compounds can also be prepared in the form of suppositories (eg, with common suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0046] The data obtained from cell culture assays and animal studies can be used in formulating various dosages for use in humans. The dosage of such compounds is desirably within the range of ED100 with little or no toxicity. 50 The dosage may vary within that range depending upon the dosage form employed and the route of administration utilized.

[0047] The therapeutically effective amount (i.e., effective dosage) of the compositions of the invention will vary depending on the circumstances of the selected patient. For example, a single dose ranging from about 1 pg to 1,000 mg may be administered, and in some embodiments, 10, 30, 100 or 1,000 pg, 10, 30, 100 or 1,000 ng, 10, 30, 100 or 1,000 μg, or 10, 30, 100 or 1,000 mg may be administered.

[0048] In some embodiments, the compositions may be administered at 1 ng / ml to 100 μg / ml, preferably 3 ng / ml to 50 μg / ml, more preferably 5 ng / ml to 500 ng / ml, and most preferably 10 ng / ml to 200 ng / ml. The compositions may be administered from once or more times per day to once or more times per week, including once every other day. A skilled artisan will recognize that certain factors may influence the dosage and timing required to effectively treat an individual, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the individual, and other pre-existing conditions. Furthermore, treating an individual with a therapeutically effective amount of a molecule of the invention may include a single treatment or, preferably, a series of treatments.

[0049] The composition of the present invention may be administered at a dosage of 5 mg / kg / week to 500 mg / kg / week, for example, 5 mg / kg / week, 10 mg / kg / week, 15 mg / kg / week, 20 mg / kg / week, 25 mg / kg / week, 30 mg / kg / week, 35 mg / kg / week, 40 mg / kg / week, 45 mg / kg / week, 50 mg / kg / week, 55 mg / kg / week, 60 mg / kg / week, 65 mg / kg / week, 70 mg / kg / week, 80 mg / kg / week, 85 mg / kg / week, 90 mg / kg / week, 100 mg / kg / week, 150 mg / kg / week, 20 mg / kg / week, 25 mg / kg / week, 30 mg / kg / week, 35 mg / kg / week, 40 mg / kg / week, 45 mg / kg / week, 50 mg / kg / week, 55 mg / kg / week, 60 mg / kg / week, 65 mg / kg / week, 70 mg / kg / week, 85 mg / kg / week, 90 mg / kg / week, 100 mg / kg / week, 120 mg / kg / week, 140 mg / kg / week, 150 mg / kg / week, 160 mg / kg / week, 180 mg / kg / week, 190 mg / kg / week, 200 mg / kg / week, 210 mg / kg / week, 220 mg / kg / week, 230 mg / kg / week, 240 mg / kg / week, 250 mg / kg / week, 260 mg / kg / week, 270 mg / kg / week, 280 mg / kg / week, 290 mg / kg / week, 300 mg / kg / week, 310 mg / kg / g / kg / week, 70 mg / kg / week, 75 mg / kg / week, 80 mg / kg / week, 85 mg / kg / week, 90 mg / kg / week, 95 mg / kg / week, 100 mg / kg / week, 150 mg / kg / week, 200 mg / kg / week, 250 mg / kg / week, 300 mg / kg / week, 350 mg / kg / week, 400 mg / kg / week, 450 mg / kg / week, and 500 mg / kg / week. In certain embodiments, the dosage of a dual acting molecule according to the present invention is in the range of 10 mg / kg / week to 200 mg / kg / week, 20 mg / kg / week to 150 mg / kg / week, or 25 mg / kg / week to 100 mg / kg / week. In certain embodiments, the compositions of the invention are administered 1x per week for 2 weeks to 6 months, e.g., 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 26 weeks, 6 months, 8 months, 10 months, or a year or more. In certain embodiments, the compositions of the invention are administered 2x per week. In other embodiments, the compositions of the invention are administered every other week.

[0050] The composition of the present invention may be formulated into a pharmaceutical composition comprising a pharmacologically effective amount of a HAPLN1 protein molecule and a pharma- ceutically acceptable carrier. A pharmacologically or therapeutically effective amount refers to a content effective to produce an intended pharmacological, therapeutic or preventive result. The phrases "pharmacologically effective amount" and "therapeutically effective amount", or simply "effective amount", refer to an amount of a dual acting molecule effective to produce an intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by 20% or more, then a therapeutically effective amount of a drug for treating that disease or disorder is the amount required to reduce that parameter by at least 20%.

[0051] Properly formulated pharmaceutical compositions of the present invention may be administered by any means known in the art, such as parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical compositions are administered by intravenous or parenteral infusion or injection.

[0052] In general, suitable dosage units of the molecule are in the range of 0.001 to 0.25 mg per kg of recipient body weight per day, 0.01 to 20 mg per kg of recipient body weight per day, or 0.01 to 10 mg per kg of recipient body weight per day, 0.10 to 5 mg per kg of recipient body weight per day, or 0.1 to 2.5 mg per kg of recipient body weight per day. Pharmaceutical compositions containing the molecule may be administered once a day. However, the therapeutic agent may also be administered in dosage units that contain 2, 3, 4, 5, 6, or more subdoses administered at appropriate intervals throughout the day. The dosage units may also be formulated into a single dose over several days, for example, using conventional sustained release dosage forms that provide a sustained and consistent release of the molecule over a period of several days. Sustained release dosage forms are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dosage.

[0053] The pharmaceutical compositions may also be included in a kit, container, pack, or dispenser together with instructions for administration.

[0054] Another aspect of the present invention relates to a method for preventing or treating xerophthalmia, which comprises administering hyaluronic acid proteoglycan linking protein 1 (HAPLN1) as an active ingredient to an individual in need thereof.

[0055] The present invention provides both preventative and therapeutic methods of treating individuals suffering from (or susceptible to) xerophthalmia.

[0056] As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., a molecule of the invention) to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line, having a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease or disorder, for the purpose of curing, healing, mitigating, alleviating, altering, relieving, ameliorating, improving or affecting the condition or condition.

[0057] In one aspect, the invention provides a method of preventing a disease or disorder in an individual, such as those described above, by administering to the individual a composition of the invention. Individuals at risk for a disease can be identified, for example, by any one or combination of the diagnostic or prognostic assays described herein.

[0058] Another aspect of the present invention relates to a method for treating or inhibiting xerophthalmia, i.e., modifying the pathogenesis of xerophthalmia symptoms, by administering to an individual in need thereof a composition comprising hyaluronic acid proteoglycan linking protein 1 (HAPLN1) or a gene encoding same as an active ingredient. These methods can be performed in vitro or, alternatively, in vivo (e.g., by administering a composition of the present invention to an individual).

[0059] Another aspect of the present invention relates to the use of hyaluronan proteoglycan link protein 1 (HAPLN1), or a gene encoding it, in the preparation of a composition for preventing or treating xerophthalmia.

[0060] Furthermore, one aspect of the present invention relates to the use of a composition containing hyaluronic acid proteoglycan linking protein 1 (HAPLN1) as an active ingredient in the prevention or treatment of xerophthalmia. EXAMPLES

[0061] The present invention will be described in more detail below through examples. However, the following examples are merely for the purpose of illustration and are not intended to limit the scope of the present invention.

[0062] [Production Example 1] Production of hyaluronic acid proteoglycan linking protein 1 (HAPLN1) A vector containing a polynucleotide encoding the human HAPLN1 protein of amino acid sequence SEQ ID NO: 1 was inserted into CHO-K1 cells to generate a CHO-K1 cell line producing recombinant human HAPLN1 protein.

[0063] The amino acid sequence of SEQ ID NO:1 is as follows: MKSLLLLVLISICWADHLSDNYTLDHDRAIHIQAENGPHLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEA QQACLDQDAVIASFDQLYDAWRGGLDWCNAGWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYN

[0064] A cell line with excellent protein production and quality was selected as MCB (master cell bank). The MCB was subcultured to obtain 0.40±0.05×10 6 The cells were inoculated into a Thermo Hyperforma SUB 250 L bioreactor at a concentration of 1.0001 cells / mL and fed-batch culture was performed. TMThe medium was mixed with 0.5846 g glutamine, 10.00 g HT Supplement (Thermo Fisher Scientific), 4.29 g 10N NaOH, and 1.80 g NaHCO3. The culture temperature was 36.5°C, the dissolved oxygen (DO) was 40.0%, and the pH was set to 7.00 ± 0.20. 1 M sodium carbonate monohydrate was used as a pH adjusting solution. 181.04 g HyClone was used as a feeding medium (FM). TM Cell Boost 7a + 12.28g FM020a in 10N NaOH, and 94.60g HyClone TM Cell Boost 7b + 105.93g FM020b of 10N NaOH was used.

[0065] From the cells cultured as described above, recombinant human HAPLN1 (rhHAPLN1) protein was separated and purified through processes such as harvest and clarification, ultrafiltration / diafiltration 1 (UF / DF1), anion exchange chromatography, S / D (solvent / detergent) virus inactivation, cation exchange chromatography, mixed-mode chromatography (MMC), hydrophobic interaction chromatography, ultrafiltration / diafiltration 2 (UF / DF2), and intermediate depth filtration (Int.1 DF). In the following examples, such a composition of the present invention was used.

[0066] [Production Example 2] Production of the composition of the present invention containing HAPLN1 protein The HAPLN1 protein purified in Preparation Example 1 was stabilized in 20 mM acetate buffer, 8% (w / v) sucrose, and 0.04% (w / v) PS80 (pH 5.0) to produce a composition of the present invention containing the HAPLN1 protein itself as the main active ingredient.

[0067] [Production Example 3] Preparation of the composition of the invention comprising an expression vector containing a gene encoding the HAPLN1 protein A composition containing an expression vector carrying a gene encoding HAPLN1 protein was prepared using the jetPRIME Transfection Reagent Kit (114-15 product (PolyPlus)). More specifically, a plasmid vector designed to express human HAPLN1 was prepared using RC209274 product (OriGene) (hereinafter referred to as "human HAPLN1 ORF Clone"), which can express the gene it transports in the nucleus of the host cell.

[0068] For reference, the jetPRIME Transfection Reagent Kit is composed of jetPRIME buffer and jetPRIME Transfection reagent, the jetPRIME buffer is used to dilute the plasmid vector to be delivered to the host cell, and the jetPRIME Transfection reagent is used to capture the plasmid vector in the form of a vesicle (liposome) so that it can be penetrated into the host cell.

[0069] The human HAPLN1 ORF clone was diluted in jetPRIME buffer to prepare the composition of the present invention comprising an expression vector carrying a gene encoding the HAPLN1 protein.

[0070] JetPRIME Transfection Reagent was added to the cells, and the mixture was left for 10 minutes to allow the plasmid vector to be captured in the form of vesicles (liposomes), which were then dropped onto the cells in culture. After culturing the cells for 48 hours or more, the increase in human HAPLN1 gene expression was confirmed by real-time qPCR, and the increase in human HAPLN1 protein expression was confirmed by Western blot.

[0071] [Example 1] Confirmation of the effect of the composition of the present invention (HAPLN1 protein) in alleviating xerophthalmia (cell viability) in human corneal epithelial cells (HCEC) under hyperosmotic conditions In most cases of xerophthalmia, the secretion of tears is low or the evaporation of tears is high, resulting in a decrease in the aqueous layer of the eye. At that time, the concentration of proteins, lipids, carbohydrates, etc. in tears remains constant, but the water content decreases, leading to an increase in osmotic pressure. Based on this principle, in cell-level research, NaCl is added to cell culture medium to adjust the osmotic pressure, a xerophthalmia model is established, and the efficacy of drugs is evaluated. In this example, 314 mOsM was set as a normal control group, and 450 mOsM, which is a relatively high osmotic pressure, was set as a xerophthalmia model, and the effect of HAPLN1 protein on human corneal epithelial cells (HCEC) under high osmotic pressure conditions was examined. This is related to the cell viability of human corneal epithelial cells (HCEC).

[0072] In this experiment, CCK-8 (Cell Counting Kit-8) was used, which is a sensitive colorimetric method used to measure cell viability and cytotoxicity. That is, WST-8, a highly water-soluble tetrazolium salt, is added to the cells to reduce the intracellular dehydrogenase activity, allowing the formazan dye to turn orange inside the cells. The amount of the dye correlates with the number of viable cells, so the more dye that is visible, the more viable cells there are.

[0073] That is, the human corneal epithelial cells (HCEC) were seeded in a Petri dish and incubated under hypotonic (314 mOsM) conditions for 24 hours. Then, after incubation under hypertonic (450 mOsM) conditions for 48 hours, the cells were washed with PBS buffer and treated with the composition of the present invention containing HAPLN1 protein at different concentrations. Next, after further incubation for 24 hours and 48 hours, the cells were assayed with a CCK-8 kit (FIG. 1A). FIG. 1B and FIG. 1C are graphs showing the results of an experiment conducted according to the experimental design of FIG. 1A, in which the cell viability of the HAPLN1 protein in the xerophthalmia alleviating effect in human corneal epithelial cells (HCEC) under hypertonic conditions was confirmed 24 hours (FIG. 1B) and 48 hours (FIG. 1C), respectively, after administration of the composition of the present invention. According to these results, when the HAPLN1 protein of the composition of the present invention was treated at concentrations from 5 ng / ml to 100 ng / ml and cultured for 24 or 48 hours, the cells showed CCK-8 absorbance (450 nm) that was greater than or similar to that of normal cells (hyposmolarity control group) at all concentrations.

[0074] For reference, in the graph, CsA refers to cyclosporine A, the main ingredient in the control drug Restasis, and the composition of the present invention surprisingly showed higher values ​​than 0.1 to 0.5 μM cyclosporine A, regardless of the concentration.

[0075] These results confirmed that cell viability decreased with 450 mOsM treatment, but increased in the group treated with the composition containing the HAPLN1 protein of the present invention, proving that the HAPLN1 protein contributes to increased cell viability. In other words, it was found that the composition containing the HAPLN1 protein of the present invention can alleviate xerophthalmia in human corneal epithelial cells (HCEC).

[0076] [Example 2] Confirmation of the effect of the composition of the present invention (HAPLN1 protein) on alleviating xerophthalmia (cell proliferation and cell migration) in human corneal epithelial cells (HCEC) under hyperosmotic conditions In relation to xerophthalmia, one of the effects of the composition containing the HAPLN1 protein of the present invention in alleviating xerophthalmia is that it induces cellular wound healing, which was verified through cell proliferation and cell migration phenomena.

[0077] The human corneal epithelial cells (HCEC) were seeded in 24 wells and incubated under low osmolarity (314 mOsM) conditions for 24 hours. After that, the medium was replaced with a high osmolarity (450 mOsM) medium and incubated under that condition for 48 hours. After that, the surface of the Petri dish was scratched vertically to make a scratch, and after washing twice, the high osmolarity medium was replaced again and treated with the composition of the present invention containing the HAPLN1 protein at different concentrations (Figure 2A). At the time of scratching (0 h), a photograph was taken using a microscope, and another photograph was taken 30 hours later to confirm the reduction in the scratched area (Figure 2B). The area was imaged and the area ratio filled with cells was confirmed compared to the initial area using ImageJ (Figure 2C).

[0078] Figure 2B shows the results of an experiment conducted according to the experimental plan of Figure 2A of the present invention, and is an image confirming the cell proliferation and cell migration of the HAPLN1 protein's effect of alleviating xerophthalmia in human corneal epithelial cells (HCECs) under hyperosmotic conditions. Figure 2C is a graph showing the numerical value of the area covered by the migrated cells shown on the photograph of Figure 2B.

[0079] According to Figures 2B and 2C, when HAPLN1 protein was treated with 5ng / ml to 100ng / ml, cell proliferation and migration phenomena covering the scratch surface were observed in all cases, and in particular, when HAPLN1 was treated with 5 and 10ng / ml, cell proliferation and migration covering about 60% of the scratch surface were observed, which shows a very good efficacy when compared with the result of about 82% for normal cells. Furthermore, it was confirmed that significant efficacy was also observed at other concentrations. For reference, CsA in the graph means cyclosporine A, which is the main component of the control drug Restasis, and the composition of the present invention surprisingly showed a higher value than 0.1μM cyclosporine A regardless of its concentration.

[0080] [Example 3] Confirmation of the inflammation-reducing effect of the composition of the present invention (HAPLN1 protein) in human corneal epithelial cells (HCEC) under hypertonic conditions 1 The fact that a composition containing the HAPLN1 protein of the present invention alleviates inflammation as one of its effects in alleviating dry eye syndrome was verified by examining the expression patterns of inflammatory factors (interleukins, CCL / CXCL family, chitinase 2-like 1) using a Human XL cytokine array (ARY022B (R&D Systems.)).

[0081] First, the human corneal epithelial cells (HCECs) after the fourth passage were seeded in 6 wells and incubated under hypotonic (314 mOsM) conditions for 24 hours. After 48 hours of incubation under hypertonic (450 mOsM) conditions, the cells were treated with the composition of the present invention containing HAPLN1 protein at different concentrations (i.e., 1 μl of 10 μg / ml HAPLN in 1 ml medium → working concentration 10 ng / ml). After further incubation for 24 hours, the culture medium of the cells was collected and assayed for the expression level of each marker using Human XL cytokine array (ARY022B (R&D Systems.)), and the results are shown in Figure 3A. According to the graphs in FIG. 3B showing the relative expression levels of inflammatory factor markers according to the concentration of HAPLN1 protein, it was confirmed that the secretion of inflammation-related factors IL-6, IL-17A, CCL20, CXCL10, CCL5, CD54, and CD176 was increased under high osmolarity conditions of 450 mOsM. However, it can be seen that the expression levels of inflammatory factors are lower in the composition of the present invention at HAPLN1 protein contents of 10 to 100 ng / ml compared to the non-treated group, that is, the inflammation of cells is alleviated. In other words, when the expression levels of the above factors in normal cells are set to 1.0, the expression levels of the abnormal group are all increased to 1 or more, but when treated with the composition containing the rhHAPLN1 recombinant protein of the present invention, the expression levels of various inflammatory factors are reduced to the normal group level or below. In particular, the expression levels of MIP-3α / CCL20 and IP-10 / CXCL10 are significantly higher under high osmolarity conditions (450 mOsM).However, it was confirmed that treatment with a composition containing the HAPLN1 protein of the present invention can lower various inflammatory factors, IL-6, IL-17A, MIP-3α / CCL20 and IP-10 / CXCL10, RANTES5 / CCL5, ICAM-1 / CD54, and Fas ligands / CD178, and that in the case of IL-17A, Fas ligand / CD178, FGF-7, FGF-19, Angiogenin, RBP-4, and chitinase 3-like 1, they were reduced to normal cell levels.

[0082] Based on these results, it was verified that a composition containing the HAPLN1 protein of the present invention alleviates cellular inflammation in human corneal epithelial cells (HCECs) as part of the alleviation of dry eye syndrome.

[0083] [Example 4] Confirmation of the inflammation-reducing effect of the composition of the present invention (HAPLN1 protein) in human corneal epithelial cells (HCEC) under hypertonic conditions 2 In Example 3, the expression of CCL-20 showed the largest change, so in order to reconfirm the effect of the composition of the present invention containing HAPLN1 protein, the concentration was further subdivided and an ELISA experiment was carried out (Figure 4A). In addition, the activity of NFκB, which is an important transcription factor in the expression of inflammatory factors, was confirmed by Western blot (Figure 4B). The experimental preparation method was carried out under the same conditions as the cytokine array carried out above, and the ELISA experimental method was according to the manufacturer's method.

[0084] Similar to the results of the previously conducted cytokine array, the results of the ELISA experiment confirmed that the secretion of the inflammatory factor CCL20 increased by about four-fold under the hyperosmotic condition of 450 mOsM (Figure 4A). However, in the composition containing the HAPLN1 protein of the present invention, the expression level of the inflammatory factor was relatively low at 5, 10, 20, 50 and 100 ng / ml compared to the group not treated with HAPLN1 protein, indicating that cellular inflammation was significantly alleviated.

[0085] In addition, it was confirmed that the phosphorylation of NFκB, a transcription factor that plays an important role in regulating the expression of inflammatory factors, was reduced by the composition of the present invention containing the HAPLN1 protein (Figure 4B).

[0086] Based on these results, it was further verified that the composition of the present invention containing HAPLN1 protein alleviates cellular inflammation in human corneal epithelial cells (HCECs) through the inhibition of NFκB activity as part of the alleviation of dry eye syndrome.

[0087] [Example 5] Confirmation of the effect of the composition of the present invention (HAPLN1 protein) on alleviating xerophthalmia (increasing mucin) in human corneal epithelial cells (HCEC) under hyperosmotic conditions in an in vitro model 1 Mucin, also called mucin, is a sticky liquid that is mainly composed of mucus polysaccharides and glycoproteins. Xerophthalmia induces a decrease in goblet cells, which is closely related to the decrease in secretion of gel-forming mucins, and is therefore an important marker of xerophthalmia. There are two types of mucins: the first group is membrane associated mucins (MAM), which mostly include MUC3A / B, MUC4, MUC11 / 12, MUC16, and MUC17, and the second group is secreted mucins, which include MUC2, MUC5AC, MUC5B, MUC6, and MUC19. Mucins belonging to the first group of MAMs form mucus gel, but their contribution is relatively low, while secreted mucins of the second group play an important role in forming the gel-forming form. The second group of secreted mucins are polymers, macromolecules, synthesized and secreted mainly in specialized cells such as goblet cells, and MUC5AC was confirmed to be expressed in conjunctival cells. The expression of glycocalyx mucins is known to be closely related to xerophthalmia (doi:https: / / doi.org / 10.1167 / iovs.17-23756).

[0088] This example is intended to verify whether the composition of the present invention containing the HAPLN1 protein increases the expression of both MAM and secreted mucin, thereby verifying that the composition of the present invention exhibits an effect of alleviating dry eye syndrome.

[0089] First, human corneal epithelial cells (HCECs) (passage 5) were cultured in corneal epithelial cell basal medium at a concentration of 3.7x10 5 Cells / well were seeded into 6 wells. After 24 hours, human corneal epithelial cells (HCECs) were pretreated with media of different osmolarity (314, 450 mOsM). After 48 hours of incubation, the compositions of the present invention were treated with rhHAPLN1 (0, 5, 10, 20, 50, 100, 200 ng / ml) at different concentrations, and the control drug cyclosporine A (0.1 μM) was treated for 24 hours. After that, the cells were washed and sampled, and RT-qPCR was performed (FIG. 5A).

[0090] Total mRNA was purified from human corneal epithelial cells (HCECs) using the Qiagen kit. cDNA was synthesized using the iscript cDNA synthesis kit. RT-qPCR was then performed, and the resulting data were expressed as relative expression levels (fold) (Figure 5B, Figure 5C).

[0091] The results show that in the xerophthalmia-induced model, the expression levels of MUC4 and MUC16 were reduced compared to the normal group, but in the case of MUC4, the expression level was significantly increased when treated with the composition of the present invention containing 10 ng / ml to 200 ng / ml, particularly 50 ng / ml to 200 ng / ml of HAPLN1 protein compared to the untreated group (Figure 5B).In addition, in the case of MUC16, the expression level was significantly increased when treated with the composition of the present invention containing 20 ng / ml to 200 ng / ml, particularly 50 ng / ml to 200 ng / ml of HAPLN1 protein compared to the untreated group (Figure 5C).

[0092] These results demonstrate that the composition of the present invention containing HAPLN1 protein increases the expression of various mucins in human corneal epithelial cells (HCECs) as part of the alleviation of dry eye syndrome.

[0093] [Example 6] Confirmation of the effect of the composition of the present invention (HAPLN1 protein) in alleviating xerophthalmia (increased mucin) in human corneal epithelial cells (HCEC) under hyperosmotic conditions in an in vitro model 2 In relation to mucin expression, MUC5AC secretion in goblet cells is an important condition for maintaining the tear film. In other words, goblet cells are known to secrete mucin and glycocalyx, which are essential for maintaining the tear film, and their number is known to decrease in dry eyes. MUC5AC is also known as a marker of goblet cells.

[0094] First, 4th passage cultured HConEpiC (human conjunctiva epithelial cells) were seeded in 24-well plates and cultured for 24 hours, and then treated with 450 mOsM hyperosmolarity, the composition of the present invention containing various concentrations of HAPLN1 protein (0, 5, 10, 20, 50, 100, 200 ng / ml), or cyclosporine A (0.1 μM). After 72 hours of culture, the cells were fixed with 4% paraformaldehyde for 30 minutes and washed with buffer. Then, fluorescent staining was performed using anti-MUC5AC (DAPI shows nuclei).

[0095] As experimental results, Figure 6A is a fluorescent photograph showing the protein expression and secretion of MUC5AC, and Figure 6B is a graph showing the quantification of the number of cells secreting MUC5AC as in Figure 6A. According to the graph, when the composition of the present invention was treated with different concentrations of HAPLN1 (10, 20, 50, 100, 200 ng / ml), the number of cells secreting MUC5AC was definitely higher than that of the normal control group in all cases, and depending on the concentration, it was even higher than that of treatment with cyclosporine A (0.1 μM, i.e., 120.261 ng / ml).

[0096] [Example 7] Manufacture of eye drop preparation containing HAPLN1 protein of the present invention In addition to the HAPLN1 protein, which is the main component of the present invention, eye drop preparations were prepared using the components in Table 1 below by a conventional eye drop preparation manufacturing method, and whether or not the functions of the composition of the present invention were correctly demonstrated was confirmed through various tests, including structural analysis (UPLC), functional analysis (linking ability assay), accelerated stress conditions, and stability tests. Based on the results, pharmaceutical compositions containing the HAPLN1 protein of the present invention were prepared in three types of eye drop formulations in Table 2 below. Here, sodium carboxymethylcellulose (CMC-Na) was selected as the viscosity regulator, Tris HCl osmotic regulator was selected as the buffer, and NaCl was selected as the isotonicity agent to establish the formulation.

[0097] In each of the compositions of the present invention, the concentrations of HAPLN1 protein were as shown in Preparation Examples 1, 2 and 3, namely low concentration (0.5 μg / ml), medium concentration (5 μg / ml) and high concentration (50 μg / ml), and all were transparent solutions.

[0098] [Table 1]

[0099] [Table 2]

[0100] *CMC-Na (sodium carboxymethyl cellulose): A component that attracts moisture, thickens the tear layer, and coats the surface of the eye. *The remaining ingredients in the additive composition are made with sterilized distilled water.

[0101] [Example 8] Efficacy evaluation of the eye drop formulation containing the HAPLN1 protein of the present invention in an in vivo dry eye model 1 For the compositions of Preparation Examples 1, 2 and 3 prepared in Example 7, corneal fluorescein scores and tear volume assays were performed on mouse eyes using a low humidity + scopolamine-induced xerophthalmia (DED) model.

[0102] 1. Low Humidity + Scopolamine-Induced Xerophthalmia Model Mice: C57BL / 6, 96 females, 8 weeks old Number of animals at the start of the xerophthalmia test, age in weeks: 58 mice (C57BL / 6, female), 10 weeks old When obtaining animals, the external appearance was observed and inspection and quarantine were performed while referring to the microbial monitoring report of the tester provided by the supplier. Animals that did not have any abnormal appearance were brought into the breeding area and acclimatized for 2 weeks in the animal room where the test was performed. After the 2-week quarantine and acclimatization period, healthy animals were selected by checking whether they were healthy and fit for the experiment. Only animals that were normal during the quarantine and acclimatization periods were used, and xerophthalmia was induced and the degree of corneal damage was evaluated through corneal fluorescent staining, and test groups were separated according to clinical score using the Z-arrangement method. The xerophthalmia test group consisted of eyes with a corneal fluorescent stain score of 7 to 15 points, and in the case of the sham (normal) group, eyes with a corneal fluorescent stain score of 5 points or more were excluded. As a reference, 5 μL of Restasis 0.05%, an immunosuppressant that suppresses T cell production, was administered as a control substance at a concentration of 500 μg / mL.

[0103] [Table 3]

[0104] 2. Induction of xerophthalmia The test animals were kept in a desiccation stress chamber with a relative humidity of 30% or less, and scopolamine hydrobromide (S0929 (Sigma-Aldrich)) prepared at a concentration of 2.5 mg / mL in saline (0.9% normal saline (Chugai Pharmaceutical)) was subcutaneously injected at a dose of 10 mL / kg four times a day (QID (quarter in die)) (09:30 a.m., 00:00 p.m., 15:00 p.m., 17:30 p.m.) for 10 days to induce xerophthalmia. The subcutaneous injection was performed by shifting the injection site to the back in order to minimize damage caused by repeated injection. After the start of administration of the test substance, the mice were kept under conditions of relative humidity of 30% or less, and scopolamine was injected subcutaneously around the neck once a day (QD (quaque die)) (9:30 a.m.) to maintain xerophthalmia.

[0105] 3. Method and frequency of administration From the day after group separation, one drop (5 μl) of each test substance and control substance was administered to both eyes twice a day (BID) (9:30 a.m. and 5:30 p.m.) using a micropipette (FIG. 7A).

[0106] 4. Corneal fluorescence punctum assay (Figure 7B) (1) One drop of mydriatic agent (tropherine) was instilled into the eye to dilate the pupil, and any remaining mydriatic agent was removed by applying a cotton swab to the edge of the canthus. (2) 10 μl of 0.2% sodium fluorescein was instilled into each eye, and the damaged area of ​​the cornea was fluorescently stained. (3) After lightly washing the eyeball with saline, an image of the cornea was taken under blue light or less. According to the NIE (National Institute of Eye) guidelines, the corneal area was divided into 5 regions and each region was scored from 0 to 3 points. The values ​​were then added up to calculate the fluorescein score.

[0107] 5. Tear volume secretion assay (Figure 7C) (1) General anesthesia was induced in mice (10 mg / kg xanthine, 100 mg / kg ketamine (intraperitoneal injection)). (2) In the morning (9:30 a.m.), scopolamine hydrobromide was administered, and the following measurements were performed approximately 3 hours later. (3) Tear volume was measured using a Schirmer's test strip cut to 1 / 2 thickness, which was placed in contact with the inside of the lower eyelid and held for 5 minutes to allow tear absorption, after which a drop (5 μl) of saline was placed on the eyeball. (4) The length of the wetted portion of the test paper was measured with a ruler.

[0108] 6. Details of the experimental process a.Weight measurement The body weight of all animals was measured before evaluation at the time of group separation (day 0) and at each evaluation time point (days 6, 11, and 16 after the start of administration).

[0109] b. Observation of general symptoms All animals were subjected to cage-side observations, including observation of the condition of the eyes, at least once daily from the time of administration of the test substance until the end of the study.

[0110] c. Tear volume measurement (Schirmer's test) and clinical corneal damage evaluation (corneal fluorescein score) After inducing xerophthalmia by desiccating stress and administration of scopolamine hydrobromide, mice were given 10 mg / kg xalazine (Rompun® (Bayer)) and 100 mg / kg ketamine (Ketamine® (Yuhan Co., Ltd.)) by intraperitoneal injection to induce general anesthesia at the time of group separation (day 0) and on days 6, 11, and 16 after administration of the test substance, and then evaluation was performed. If anesthesia was not sufficiently induced, additional anesthesia was performed by intramuscularly injecting 1 / 3 the dose of the anesthetic used to induce the initial anesthesia into the thigh of the mouse (due to the characteristics of the injectable anesthetic, some animals may die from anesthesia during the anesthesia induction process). Evaluation was performed about 3 hours after administration of scopolamine hydrobromide in the morning (09:30). The tear volume was measured using a Schirmer's test strip (Bio Color Tear Test® (Bio Optics)) cut into 1 / 2 thickness. The Schirmer's test strip was placed in contact with the inside of the lower eyelid and left for 5 minutes to allow tears to be absorbed. After the evaluation, one drop (5uL) of saline was dropped onto the eyeball. The length of the wet area (stained area) of the Schirmer's test strip was measured using a ruler. Then, one drop of mydriatic agent (Tropelon (Alcon, Korea)) was instilled to induce mydriasis, and the mydriatic agent remaining in the eyeball was removed by attaching a cotton swab to the edge of the canthus and sucking it up. Next, 10μL of 0.2% sodium fluorescein (Fluorescein® (Alcon, Korea)) was instilled into both eyes, and the damaged area of ​​the cornea was fluorescently stained. Then, after lightly washing the eyeball with saline, an image of the cornea was taken (Micron-IV (Phoenix)) under blue light or less. According to the NIE (National Institute of Eye) guidelines, the cornea was divided into 5 areas and each area was scored with a score of 0 to 3 points. The values ​​were then added up to calculate the fluorescein score.Measurement of tear volume and CFS evaluation from anesthesia were completed within 15 minutes, and once the evaluation was completed, one drop of antibiotic eye drops (Toberan (registered trademark) eye drops (Daewoo Pharmaceutical)) was instilled into the mouse eyeball.

[0111] D. Eye removal On the 18th day after the start of administration, samples were taken from the lacrimal glands, cornea, conjunctiva, and meibomian glands (lower eyelids containing the meibomian glands) of both eyes, and then provided to the client in a frozen state. However, if requested by the client, instead of the above method, both eyes including the upper and lower eyelids can be enucleated, fixed in Davidson's solution and formalin, and then prepared into paraffin tissue slides.

[0112] e. Statistical processing of data All experimental results were expressed as mean ± standard deviation and were tested using GraphPad Prism 6.0 (GraphPad Software (CA)). The Shapiro-Wilk normality test was performed to test for normality of all data. In cases of normal distribution, one-way analysis of variance (ANOVA) was performed, and if significance was observed, Tukey's multiple comparison test was performed to identify test groups with significant differences from the control group (vehicle group). In cases where the distribution was not normal, Kruskal-Wallis test was performed, followed by post-hoc testing using Dunn's multiple comparisons test (significance levels: 5% and 1% on both sides).

[0113] 7. Experimental Results (1) Evaluation of the degree of corneal damage using corneal fluorescence score (CFS) in a mouse dry eye model The image results of the corneal fluorescence score assay are shown in FIG. 8A. Here, Sham corresponds to a normal control group model in which xerophthalmia was not induced, DED control corresponds to a non-treated control group model in which xerophthalmia was induced and treated with a drug-free vehicle, and Cyclosporine A indicates an example of treatment with a conventional therapeutic agent. rhHAPLN1 (0.5 μg / ml) indicates an example of treatment with Preparation Example 1, which is a composition of the present invention, rhHAPLN1 (5 μg / ml) indicates an example of treatment with Preparation Example 2, which is a composition of the present invention, and rhHAPLN1 (50 μg / ml) indicates an example of treatment with Preparation Example 3, which is a composition of the present invention.

[0114] As in 3. above, starting from the day after group separation, one drop (5 μl) of each of the test substance and control substance was administered to both eyes twice a day (BI) (9:30 a.m. and 5:30 p.m.) using a micropipette.

[0115] The results of the corneal fluorescence score assay were quantified and presented graphically in FIG. 8B.

[0116] According to the administration results, the increased corneal fluorescence score (CFS) value in the dry eye model was consistently reduced when treated with the composition of the present invention containing HAPLN1 protein, regardless of the concentration, and in particular, from day 11 onwards, the composition of the present invention containing HAPLN1 protein reduced the corneal fluorescence score (CFS) value more than cyclosporine A (the main ingredient in the control drug Restasis).

[0117] (2) Evaluation of tear volume in a mouse dry eye model The results of the tear volume assay were quantified and shown in Figure 8C.

[0118] According to the administration results, the reduced tear volume in the dry eye model was consistently increased by the composition of the present invention containing HAPLN1 protein, regardless of the concentration, more than by cyclosporine A (the main component of the control drug, Restasis), and in particular, from day 11 onwards, the composition of the present invention containing HAPLN1 protein increased tear volume to a much greater extent than cyclosporine A.

[0119] (3) Evaluation of corneal epithelial cell detachment On the second day after 16 days of drug treatment, the eyeballs were enucleated, and the tissue was then subjected to paraffin block and H&E (hematoxylin and eosin) staining on the prepared slides (Figure 9A). The number of detached corneal epithelial cells was measured and quantified (Figure 9B).

[0120] As a result, in the xerophthalmia model, the number of damaged corneal epithelial cells was increased, but it was confirmed that the number of damaged cells was reduced when treated with the composition of the present invention containing the HAPLN1 protein, regardless of the concentration.

[0121] (4) Evaluation of increase or decrease in goblet cells On the 16th day after drug treatment, the eyeballs were enucleated, blocked in paraffin, and the tissues were stained by PAS staining on the prepared slides (Figure 10A). PAS staining specifically stains goblet cells, which contain a large amount of mucopolysaccharides and mucoproteins. The α-glycol groups of these goblet cells are oxidized by periodic acid to generate aldehydes, which react with Schiff reagent to stain the cells reddish purple.

[0122] Here, the number of goblet cells present in the conjunctiva was measured and quantified (Figure 10B). As a result, the number of damaged goblet cells was reduced in the xerophthalmia model. However, when treated with the composition of the present invention containing HAPLN1 protein, the number of goblet cells was increased more than in the dry eye model, regardless of its concentration, and was confirmed to be at a level equivalent to that of the normal sham control group.

[0123] (5) Anti-inflammatory effect in lacrimal gland 1 When xerophthalmia is induced, an inflammatory response is induced in the eyeball and lacrimal gland, leading to infiltration of immune cells along with an increase in IL-1β, a cytokine representative of inflammatory responses.

[0124] In this example, in order to confirm the anti-inflammatory effect of the composition of the present invention containing the HAPLN1 protein in vivo, the expression of IL-1β in the lacrimal gland was confirmed through immunohistochemistry. For this purpose, the eyeball was enucleated on the second day after 16 days of drug treatment, and paraffin blocking was carried out. Using the prepared slide, immunohistochemistry was carried out, and the expression of IL-1β (brown) was confirmed using an anti-IL1β antibody (FIG. 11A). FIG. 11B is a graph showing the quantification of the expression pattern of IL-1β confirmed in FIG. 11A.

[0125] As a result, in the dry eye syndrome model, the expression of IL-1β was increased in the lacrimal gland. However, when treated with the composition of the present invention containing HAPLN1 protein, the expression of IL-1β was reduced to the level of the sham control group corresponding to normal cells at a concentration of 5 μg / ml or more of HAPLN1, demonstrating the anti-inflammatory effect of the composition of the present invention.

[0126] (6) Anti-inflammatory effect in lacrimal gland 2 Induction of xerophthalmia induces an inflammatory response in the eye and lacrimal gland, and increased expression of the inflammatory marker CD45 on the cell surface of immune cells indicates immune cell infiltration.

[0127] In this example, to confirm the anti-inflammatory effect of the composition of the present invention containing HAPLN1 protein in vivo, the expression of CD45 in the lacrimal gland was confirmed. That is, the eyeball was enucleated on the second day after 16 days of drug treatment, and paraffin blocking was performed. Immunohistochemistry and immunofluorescence were performed on the section slide, and the expression of CD45 (green) was confirmed using an anti-CD45 antibody. Figure 12B is a graph showing the quantification of the expression level in Figure 12A.

[0128] As a result, in the dry eye model, the expression of CD45 was increased in the lacrimal gland. However, when treated with the composition of the present invention containing HAPLN1 protein, the expression of CD45 was reduced, and in particular, the composition of the present invention containing HAPLN1 protein further reduced the expression of CD45 than cyclosporine A (the main component of the control drug Restasis). This proves the anti-inflammatory effect of the composition of the present invention.

[0129] (7) Anti-inflammatory effect in lacrimal gland 3 Induction of xerophthalmia induces an inflammatory response in the eye and lacrimal gland, and increased expression of CD11b, an inflammatory marker, in immune cells indicates immune cell infiltration.

[0130] In this example, to confirm the anti-inflammatory effect of the composition of the present invention containing HAPLN1 protein in vivo, the expression of CD11b in the lacrimal gland was confirmed. That is, the eyeball was enucleated on the second day after 16 days of drug treatment, and paraffin blocking was performed. Immunohistochemistry and immunofluorescence were performed on the section slide, and the expression of CD11b (green) was confirmed using an anti-CD11b antibody. Figure 13B is a graph showing the quantification of the expression level in Figure 13A (Figure 13B).

[0131] As a result, in the dry eye syndrome model, the expression of CD11b was increased in the lacrimal gland. However, when treated with the composition of the present invention containing HAPLN1 protein, the expression of CD11b was reduced, and in particular, the composition of the present invention containing HAPLN1 protein reduced the expression of CD11b much more than cyclosporine A (the main component of the control drug Restasis). This proves the anti-inflammatory effect of the composition of the present invention.

[0132] [Example 9] Efficacy evaluation of the eye drop formulation containing the HAPLN1 protein of the present invention in an in vivo dry eye model 2 For the compositions of Preparation Examples 1, 2 and 3 prepared in Example 7 above, corneal fluorescence scores and tear volume assays were performed on rabbit eyes using a 0.1% benzalkonium chloride (BAC)-induced xerophthalmia (DED) model.

[0133] 1. Establishment of a rabbit model of xerophthalmia According to the following experimental procedure, a model of xerophthalmia was established in rabbits to verify the efficacy of the composition of the present invention (FIG. 14).

[0134] (1) Induction of dry eye One drop (0.05 ml) of 0.1% benzalkonium chloride (BAC) was instilled into both eyes of the rabbits twice a day (9:00 and 17:00). After 14 days of instillation, the eyes were examined (on the 14th day) to confirm that dry eye had been induced, and the rabbits were separated into groups.

[0135] (2) Instillation of the test substance The test substance was instilled into the eyes of the model rabbits in groups twice a day (8:30, 16:30) for 15 to 30 days (total of 16 days).

[0136] [Table 4]

[0137] Experimental animals: 42 New Zealand white rabbits (male, individually housed) Group separation: G1. No DED with vehicle (non-induced dry eye group) G2.DED with vehicle (dry eye group) G3.DED with Restasis (cyclosporine) G4.DED with test substance (0.5μg / ml HAPLN1 dose-1) G5.DED with test substance (5μg / ml HAPLN1 dose-2) G6.DED with test substance (50μg / ml HAPLN1 dose-3) *All groups were euthanized on the second day after 16 days of treatment. *All groups were administered eye drops twice daily according to group. A total of 42 rabbits were used (7 per group).

[0138] (3) Eye examination Anesthesia was induced in rabbits by intramuscular injection of xylazine and ketamine, and one drop of alcaine was instilled into the eye. Excess tears and eye drops were then removed with a Weck cel cellulose spear. STT paper was placed on the lower eyelid, and the length of the wet area for 5 minutes was measured (STT-2). TFBUT (tear film breakup time) was also measured to check the stability of the tear film. Furthermore, a slit test was performed to check the corneal condition and the degree of fluorescent staining.

[0139] (4) Collection and analysis of samples The eyeballs of the rabbits mentioned above, including the conjunctiva, were removed, fixed in a special fixative, embedded in paraffin, sliced, and then stained and analyzed according to the purpose. The eyeballs were stained with H&E (hematoxylin & eosin) to analyze the detachment of the corneal epithelium. After PAS staining, the distribution of goblet cells in the conjunctiva was measured. After impression cytology of the conjunctival epithelium, PAS staining, and goblet cell distribution were also measured.

[0140] 2. Experimental Results (1) Corneal damage assessment using corneal fluorescence score (CFS) The image results of the corneal fluorescence score assay are shown in Figure 15A, and the graph in which it was quantified is shown in Figure 15C. According to the administration results, the corneal fluorescence score (CFS) value, which had increased in the xerophthalmia model, was consistently reduced by treatment with the composition of the present invention containing HAPLN1 protein, regardless of its concentration, and in particular, after 10 days, the composition of the present invention containing HAPLN1 protein reduced the corneal fluorescence score (CFS) value to a greater extent than Restasis.

[0141] (2) Evaluation of the efficacy of the composition of the present invention in improving the stability of the tear film In this embodiment, a method was used to measure the time until a black spot appeared at the fluorescent staining site after dropping one drop of sodium fluorescein on the eyeball, and the black spot appeared within several seconds after blinking, which appears very quickly in dry eye conditions. That is, it measures the TFBUT (tear film breakup time) (measurement of the time it takes for the tear film to break up) and verifies the stability of the tear film.

[0142] The time required for tear film breakdown was measured for each sample, and the experimental results were shown in a graph (FIG. 15B). Here, Sham corresponds to a normal control group model in which xerophthalmia was not induced, DED control corresponds to a non-treated control group model in which xerophthalmia was induced and treated with a drug-free vehicle, and Restasis (active ingredient is cyclosporine A) indicates an example of treatment with a conventional therapeutic agent. HAPLN1 (0.5 μg / ml) indicates an example of treatment with Preparation Example 1, which is a composition of the present invention, HAPLN1 (5 μg / ml) indicates an example of treatment with Preparation Example 2, which is a composition of the present invention, and HAPLN1 (50 μg / ml) indicates an example of treatment with Preparation Example 3, which is a composition of the present invention. In FIG. 15A, HS-601 indicates the experimental name of the HAPLN1 protein-containing composition of the present invention.

[0143] As can be seen from Figure 15B, in the dry eye model, the time until tear film breakdown was very short at 10 seconds or less, but when the composition of the present invention was administered, the time was continuously increased, and in particular, after 10 days, the composition of the present invention containing HAPLN1 protein increased the time until tear film breakdown more than Restasis, regardless of the HAPLN1 concentration. In particular, it can be confirmed that the time was increased to 30 seconds or more at a high concentration of HAPLN1 protein (50 μg / ml).

[0144] (3) Evaluation of tear volume in a rabbit dry eye model The results of the tear volume secretion assay were quantified and shown in FIG. 15D.

[0145] According to the administration results, the tear volume, which was reduced in the dry eye model, was increased by the composition of the present invention containing HAPLN1 protein from the 5th day in a concentration-dependent manner and more consistently than the control drug Restasis, and in particular, from the 10th day onwards, the composition of the present invention containing HAPLN1 protein increased tear volume to a much greater extent than Restasis.

[0146] (4) Evaluation of corneal epithelial cell detachment After the eye examination on the 15th day, the eyeballs were enucleated on the 1st day after the 16th day of drug treatment, and histopathological analysis was performed and paraffin blocks were made. The tissues were stained via H&E staining in the section slides (Figure 16A). 2 The number of corneal epithelial cells in which detachment had occurred was measured and quantified within this range (FIG. 16B).

[0147] As a result, the number of detached, i.e., damaged, corneal epithelial cells increased in the xerophthalmia model. However, it was confirmed that the number of detached cells decreased in a concentration-dependent manner when the composition of the present invention containing HAPLN1 protein was treated. Thus, the degree of detachment was the lowest when the composition of the present invention contained a high concentration (50 μg / ml) of HAPLN1 protein.

[0148] (5) Evaluation of goblet cell proliferation and proliferation in a rabbit dry eye model After the eye examination on the 15th day, the eyeballs were enucleated on the 1st day after the 16th day of drug treatment, and histopathological analysis was performed and paraffin blocks were made. In the section slides, the tissues were stained via PAS staining (Figure 17A). 2 The number of goblet cells was measured and quantified in the range of 0.1 to 1.0 mm (Fig. 17B).

[0149] As a result, the number of damaged goblet cells was drastically reduced in the xerophthalmia model. However, it was confirmed that the number of goblet cells was reliably increased in proportion to the concentration of the composition of the present invention containing HAPLN1 protein, compared to the xerophthalmia model. However, it was confirmed that when the composition of the present invention contained a high concentration of HAPLN1 protein (50 μg / ml), the number of goblet cells was increased much more than the control drug Restasis. [Industrial Applicability]

[0150] As described above, the pharmaceutical composition and treatment method of the present invention can effectively and stably prevent and treat xerophthalmia, and exhibit reduced side effects compared to conventional products. [Sequence List Free Text]

[0151] SEQ ID NO:1: MKSLLLLVLISICWADHLSDNYTLDHDRAIHIQAENGPHLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEA QQACLDQDAVIASFDQLYDAWRGGLDWCNAGWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYN

Claims

1. A pharmaceutical composition for preventing or treating xerophthalmia, comprising hyaluronic acid proteoglycan linkage protein 1 (HAPLN1) or a gene encoding the same as an active ingredient.

2. 2. The pharmaceutical composition for preventing or treating dry eye according to claim 1, wherein the protein has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:

1.

3. 2. The pharmaceutical composition for preventing or treating dry eye according to claim 1, wherein the nucleic acid associated with said gene is contained within an expression vector.

4. The pharmaceutical composition for preventing or treating xerophthalmia according to claim 1 , wherein the composition induces wound healing ability of cells in the eye.

5. The pharmaceutical composition for preventing or treating dry eye according to claim 1 , wherein the composition reduces the expression of inflammatory factors in corneal epithelial cells.

6. The pharmaceutical composition for preventing or treating dry eye according to claim 1 , wherein the composition increases the expression level of mucin.

7. 2. The pharmaceutical composition for preventing or treating dry eye according to claim 1, wherein the composition is administered in a single topical dose of 1 ng / ml to 100 μg / ml.

8. 2. The pharmaceutical composition for preventing or treating dry eye syndrome according to claim 1, wherein the composition is in a dosage form selected from the group consisting of eye drops, artificial tears, eye ointments, tablets, pills, capsules, lozenges, inhalants, injections, patches, and suppositories.

9. The pharmaceutical composition for preventing or treating dry eye according to claim 1 , wherein the pharmaceutical composition is for parenteral administration.

10. The pharmaceutical composition for preventing or treating dry eye according to claim 1, further comprising a pharmaceutical additive selected from the group consisting of a pharma- ceutical acceptable carrier, a diluent, a binder, a disintegrant, a lubricant, and any combination thereof.

11. 2. The pharmaceutical composition for preventing or treating xerophthalmia according to claim 1, wherein the composition is included as a main or auxiliary active ingredient of a composition for preventing or suppressing xerophthalmia.

12. 12. A kit comprising a composition according to any one of claims 1 to 11 and instructions for administration.

13. An experimental reagent composition related to the prevention or suppression of xerophthalmia, comprising hyaluronic acid proteoglycan linkage protein 1 (HAPLN1) or a gene encoding the same, for use in the manufacture of a pharmaceutical composition for preventing or suppressing xerophthalmia.

14. Use of hyaluronic acid proteoglycan linkage protein 1 (HAPLN1) or a gene encoding same for the manufacture of a pharmaceutical composition for preventing or suppressing xerophthalmia.

15. A method for preventing or suppressing xerophthalmia, comprising administering to an individual in need thereof hyaluronic acid proteoglycan linking protein 1 (HAPLN1) or a gene encoding same as an active ingredient.

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