TW202425990A - Eye drop composition comprising cftr modulator compounds - Google Patents

Abstract

The present invention provides an eye drop composition for treatment, prevention or improvement of dry eye disease or dry eye -related diseases, which comprises a compound represented by the following Chemical formula 1 at a concentration of 0.01 to 0.3 %(w/v), or a pharmaceutically acceptable salt, solvate, hydrate, prodrug or stereoisomer thereof as an active ingredient, and is instilled into the eye 1-7 times a day, with 1-3 drops per one eye at one time.

Description

Ophthalmic composition containing CFTR modulator compound

This application claims priority based on Korean Patent Application No. 10-2022-0144645 filed on November 2, 2022, and all the contents disclosed in the specification and drawings of the corresponding application are incorporated into this application. The present invention relates to an eye drop composition, and more specifically, to an eye drop composition comprising a cystic fibrosis transmembrane conductance regulator (CFTR) regulator.

Dry eye disease (DED) is a tear membrane disease that causes irritation to the eye due to a lack of tears, and refers to an eye disease in which the components are imbalanced and the ocular surface is damaged, with symptoms of blurred vision and sensory irritation such as irritation, foreign body sensation, and dryness. The causes of dry eye disease are varied, including insufficient tear secretion, excessive tear evaporation, inflammation of the tear-producing organs, cases with associated systemic diseases such as Sjogren syndrome, Steven Johnson syndrome, and acne vulgaris and the like, and it is one of the most widespread eye diseases, but treatment options are limited. The most common treatment is artificial tear drops, which may occlude tear points to reduce tear volume, reduce ocular surface inflammation, or enhance tear/mucin secretion. Artificial tears are widely used to lubricate the ocular surface in the short term, but the limitation of artificial tears is that it only provides temporary relief rather than a fundamental treatment. As a next step, strategies targeting ocular surface inflammation, tear secretion, and leptomeningeal gland dysfunction are being studied. However, to date, the only drugs that inhibit T cell activation and cytokine production are cyclosporine and lifitegrast. Recently, the P2Y2 receptor agonist diquafosol has been discussed as a next-generation DED therapeutic agent. Ion channels expressed in the ocular epithelium and involved in mucin secretion are becoming new targets for the development of DED therapeutic agents.

On the other hand, cystic fibrosis transmembrane conductance regulator (CFTR), as the main chloride ion channel that stimulates ocular surface fluid secretion, can open up a new treatment strategy for DED. CFTR refers to the membrane protein and chloride ion (Cl ^- ) channel encoded by the CFTR gene. CFTR is a protein located on the cell membrane that acts as a chloride ion channel and induces ocular surface fluid secretion through chloride ion transport. When CFTR present in the cell membrane is activated, CFTR can treat many diseases caused by loss or deterioration of CFTR function. For example, substances that activate the CFTR can correct abnormal tear membranes shown in dry eye disease by activating chloride ion (Cl ^- ) channels and increasing tear volume.

The inventors of the present invention studied methods for treating dry eye disease using CFTR modulators, thereby completing the present invention.

Technical issues

The problem to be solved by the present invention is to provide an eye drop composition, which contains a cystic fibrosis transmembrane conductance regulator (CFTR) regulator, preferably a compound of formula 1 that can act as a CFTR activator. In addition, another problem to be solved by the present invention is to provide a preferred dosage, method of use, prescription dosage and the like of the eye drop of the present invention.

One embodiment of the present invention has the excellent effects of reducing tear secretion caused by dry eye disease, significantly inhibiting keratoconjunctivitis and changes in corneal type, restoring damaged cornea or increasing tear secretion, and thus, it provides a preferred dosage, method of use, prescription dosage and the like of the eye drop composition, which can be used to develop a safe and effective therapeutic agent for dry eye disease or dry eye-related diseases. The scope of the present invention is not limited by these effects.

The problems to be solved by the present invention are not limited to those mentioned above. Those familiar with the art to which the present invention belongs will clearly understand other technical problems not mentioned from the following description. Technical Solution

In order to solve the above problems, the following contents are provided through various aspects of the present invention.

One aspect of the present invention provides an eye drop composition for treating, preventing or improving dry eye disease or dry eye disease-related diseases, the composition comprises a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug or stereoisomer thereof at a concentration of 0.01% (w/v) to 0.3% (w/v) as an active ingredient, and is dropped into the eye 1-7 times a day, 1-3 drops per eye each time. [Chemical formula 1]

Another aspect of the present invention is a method for treating or improving dry eye disease or dry eye-related diseases in an individual, the method comprising the following steps: administering a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt, solvent complex, hydrate, prodrug or stereoisomer as an active ingredient to an individual in need thereof at a concentration of 0.01% (w/v) to 0.3% (w/v), wherein the administration is intraocular instillation 1-7 times a day, 1-3 drops per eye each time. [Chemical formula 1]

Another aspect of the present invention provides a use of a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug or stereoisomer thereof at a concentration of 0.01% (w/v) to 0.3% (w/v), wherein the use is to use a therapeutic agent to treat, prevent or improve dry eye disease or dry eye disease-related diseases, and in the therapeutic agent, the active ingredient is instilled into the eye 1-7 times a day, 1-3 drops per eye each time. [Chemical formula 1]

This will be described in more detail below.

One aspect of the present invention provides an eye drop composition, which comprises a compound of the following chemical formula 1 that can act as a CFTR activator.

The IUPAC name of Chemical Formula 1 is (S)-(4-benzoyl-3-methylpiperazin-1-yl)(7-(3,4-dimethoxyphenyl)pyrazolo[1,5-a]pyrimidin-2-yl)methanone. The compound can be obtained by the method disclosed in International Patent Publication No. WO 2022/084741 A1. The compound of Formula 1 is a pyrazolo[1,5-α]pyrimidine derivative, and the pyrazolo[1,5-α]pyrimidine derivative compound contains a pyrazolo[1,5-α]pyrimidine core and can be obtained by: substitution at position 2 of the core structure with any substituted aryl, any substituted heteroaryl and any substituted heterocyclic substituent, substitution at position 5 of the core structure with a halogen, substitution at position 6 of the core structure with a halogen, any substituted aryl, any substituted (C1-C10)alkyl and any substituted (C1-C10)alkoxy substituent, and substitution at position 7 of the core structure with any substituted aryl, any substituted heteroaryl and any substituted heterocyclic substituent. The compounds described in the present specification may exist not only in crystalline form, powder form and amorphous form, such as polymorphs, pseudopolymorphs, solvates, hydrates, non-solvated polymorphs (including acid anhydrides), morphological polymorphs and amorphous forms of these compounds, but also in various forms including pharmaceutically acceptable salts (including mixtures thereof). The compounds described in the present specification may exist as solvates, especially hydrates, and unless otherwise mentioned, all such solvates and hydrates are intended to be included. Hydrates may be formed during the preparation of the compound or a composition containing the compound, or hydrates may be formed over time due to the hygroscopicity of the compound. The compounds of the present specification may exist as organic solvents, especially including DMF, ether and alcohol solvents. The identification and preparation of any particular solvent complex is within the skill of one familiar with the field of synthetic organic chemistry or medicinal chemistry. In some embodiments, the compounds described in this specification exist in the form of a solvent complex. In some embodiments, when the solvent component of the solvent complex is water, the compounds described in this specification exist in the form of a hydrate. The compounds of the present invention may contain asymmetric or chiral centers, and therefore the compounds may exist in different stereoisomer forms. All stereoisomer forms of the compounds of the present invention, such as non-mirror isomers, mirror isomers, and racemic mixtures, are considered to constitute some part of the present invention. In this specification, "pharmaceutically acceptable salts" refer to salts commonly used in the pharmaceutical industry. Examples include inorganic ion salts prepared from calcium, potassium, sodium, magnesium and the like; inorganic acid salts prepared from hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid, sulfuric acid and the like; inorganic acid salts prepared from acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, glucose Organic acid salts prepared from sugar acid, galacturonic acid, glutaric acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid and the like, sulfonic acid salts prepared from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and naphthalenesulfonic acid and the like, and amino acid salts prepared from glycine, arginine, lysine and the like; and amine salts prepared from trimethylamine, triethylamine, ammonia, pyridine, picoline and the like, but not limited thereto.

In the discovery of ophthalmic drugs including ophthalmic compositions, water solubility is one of the main factors affecting the bioavailability of the eye and ophthalmic preparations. According to the rule of thumb (ROx) of ophthalmic drugs developed by Gukasyan and the like, the calculated optimal solubility value of an effective ophthalmic drug should be 1 μM or higher. The inventors of the present invention conducted research to provide an excellent ophthalmic solution by improving solubility while maintaining the efficacy of CFTR, and thus, they completed the present invention.

Specifically, the inventors of the present invention have conducted research to provide an optimal eye drop method that can provide effects such as increasing tear volume, improving corneal erosion, inhibiting inflammation, and the like, and can be well distributed in target tissues including the cornea and conjunctiva. In addition, they have identified a preferred eye drop method, an eye drop dosage, and the like, thereby completing the present invention.

For the compound of Formula 1, in the tear volume reduction model, the effect of improving tear volume was confirmed, and the excellent solubility, the effect of improving corneal erosion, the effect of reducing the expression of various pro-inflammatory cytokines present in the cornea and conjunctiva, and the like were confirmed. The compound of Formula 1 can be used to treat, prevent or improve dry eye disease or dry eye disease-related diseases. Dry eye disease-related diseases may include diseases caused or aggravated by reduced tear volume, or eye diseases that are prevented or improved by increasing tear volume. For example, the eye disease may include conjunctivitis, leptomeningeal gland dysfunction, ocular surface damage caused by dry eye disease (preferably corneal damage), burning sensation, itching, foreign body sensation or roughness, or the like. There are no specific restrictions on the causes of dry eye disease.

The composition of the present invention may contain an active ingredient at a concentration of 0.01% (w/v) to 0.3% (w/v) to achieve the desired effect or to show stable solubility in the formulation. In addition, in order to provide the advantages of each aspect provided herein, a method of administration suitable for administering the active ingredient at this concentration is 1-7 times a day, 1-3 drops per eye each time.

In a specific embodiment, one aspect of the present invention provides an eye drop composition for treating, preventing or improving dry eye or dry eye-related diseases, the composition comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug or stereoisomer thereof at a concentration of 0.01% (w/v) to 0.3% (w/v) as an active ingredient, and is instilled into the eye 1-7 times a day, 1-3 drops per eye.

On the other hand, in the present invention, "% (w/v)" means the mass (g) of the target component contained in 100 mL of the eye drops or eye drops composition of the present invention. In the present invention, when a salt of the compound of Chemical Formula 1 is contained, the value is the content of the salt of the compound. In addition, in the present invention, when the compound of Chemical Formula 1 or its salt is mixed with a hydrate or a solvate, the value is the content of the hydrate or solvate of the compound of Chemical Formula 1 or its salt.

The pharmaceutical composition of the present invention according to one embodiment may contain 0.5% (w/v) or less of the CFTR activator as an active ingredient based on the total composition. In one embodiment, it may contain 0.01% (w/v) to 0.3% (w/v), 0.04% (w/v) to 0.25% (w/v), 0.05% (w/v) to 0.2% (w/v), 0.01% (w/v) to 0.25% (w/v), 0.01% (w/v) to 0.2% (w/v), 0.03 ...% (w/v), 0.03% (w/v) to 0.3% (w/v), 0.04% (w/v) to 0.25% (w/v), 0.05% (w/v) to 0.2% (w/v), 0.01% (w/v) to 0.2% (w/v), 0.03% (w/v) to 0.3% (w/v), 0.04% (w/v) to 0.25% (w/v), 0.05% (w/v) to 0 % (w/v), 0.03% (w/v) to 0.07% (w/v), 0.04% (w/v) to 0.06% (w/v), 0.05% (w/v), 0.08% (w/v) to 0.12% (w/v), 0.09% (w/v) to 0.11% (w/v), 0.1% (w/v), 0.018% (w/v) to 0.22% (w/v), 0.19% (w/v) to 0.21% (w/v), 0.1% (w/v) of the compound of formula 1.

In one embodiment, the pharmaceutical composition may contain an active ingredient at a concentration of 0.4-3 mg/ml. As a more specific example, the composition of the present invention may contain an active ingredient at a concentration of 0.45-2.5 mg/ml, 0.43-2.3 mg/ml.

When about 50 uL is administered during one instillation in each eye, the amount of active ingredient dropped into the eye during instillation in each eye can be about 3 mg or less, about 2.5 mg or less, about 2.2 mg or less, about 2 mg or less, about 0.01 to about 3 mg, about 0.015 to about 2.8 mg, about 0.018 to about 2.5 mg, about 0.02 to about 2.4 mg, about 0.05 to about 2.3 mg, about 0.08 to about 2.28 mg, about 0.09 to about 2.25 mg, about 0.095 to about 2.2 mg, about 0.01 to about 2 mg, about 0.015 to about 1.8 mg, about 0.018 to about 1.5 mg, about 0.02 to about 1.4 mg, about 0.05 To about 1.3 mg, about 0.08 to about 1.28 mg, about 0.09 to about 1.25 mg, about 0.095 to about 1.2 mg, about 0.095 to about 1.1 mg, about 0.01 to about 1 mg, about 0.015 to about 0.8 mg, about 0.018 to about 0.5 mg, about 0.02 to about 0.4 mg, about 0.05 to about 0.3 mg, about 0.08 to about 0.28 mg, about 0.09 to about 0.25 mg, about 0.095 to about 0.2 mg, about 0.095 to about 0.15 mg, and for example, 0.025 mg, 0.05 mg, 0.075 mg, 0.1 mg, 0.125 mg can be instilled into the eye. , 0.15 mg, 0.175 mg, 0.2 mg, 0.225 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.375 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.525 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.05 mg, 1.2 mg, 1.4 mg, 1.5 mg, 1.8 mg, 2.0 mg or 2.1 mg of active ingredient.

The eye drops of the present invention can be instilled into the eye 1 to 7 times a day, 1 to 3 drops per eye at a time. The interval between instillations of the eye drops of the present invention can be at least one hour or longer. One drop can usually be about 10 to 100 μl, 20 to 80 μl, 30 to 70 μl, 40 to 60 μl, and can be, for example, 50 μl.

The active ingredient of the content can be administered by instilling 1 to 5 drops, 1 to 4 drops, or 1 to 3 drops each time, so as to achieve the purpose or effect of the present invention, such as the stability of the formulation, the distribution on the ocular surface, the effect of reducing corneal erosion, and the like. In addition, the use of the composition can be applied once or multiple times a day, and as an example, the composition can be applied 1 to 7 times a day, 1 to 6 times a day, 1 to 5 times a day, 1 to 4 times a day, 1 to 3 times a day, 1 to 2 times a day, once a day, or frequently or irregularly as needed.

In a specific embodiment, the treatment or improvement includes daily administration of the therapeutic agent during at least one continuous 2-week period. In the treatment regimen, the eye drop composition can be administered once a day, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times a day. In a specific embodiment, the treatment regimen can include daily administration of the therapeutic agent during 3 consecutive weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks or longer.

The content, concentration or amount of the active ingredient contained in the eye drops of the present invention surprisingly shows stable pharmacological performance because the active ingredient is attached to the corneal surface. In addition, the active ingredient can achieve the effect on the ocular surface (target site). The active ingredient having the content range or concentration range allows the provision of an eye drop composition with excellent solubility.

Surprisingly, it has been demonstrated that when the composition according to the present invention is used to treat dry eye or dry eye-related diseases by limiting the dosage and/or administration method, various effects such as distribution degree, reduction of corneal erosion, reduction of ocular inflammation and the like can be obtained.

The composition of the present invention may further include a pharmaceutically acceptable formulation. In one embodiment, the eye drop composition may be a liquid composition or a semisolid composition. In one embodiment, the eye drop composition may be in the form of an eye drop, an ointment, a gel, a cream, a lotion, an emulsion, a suspension or a spray. The eye drop composition of the present invention may be formulated to be compatible with the eye and/or other tissues treated by the composition. For topical application to the eye, the composition of the present invention can be formulated as a sterile aqueous composition (e.g., suspension, solution, emulsion, etc.), and may contain purified water that can be generally contained, such as at least 70 w/v%, more commonly 80 w/v%, and more commonly at least 90 or 95 w/v% purified water. The eye drop composition may be intended for direct application to the corneal surface of the eye, and is generally formulated to have a pH and isotonicity compatible with the eye. In the composition, generally, the pH may have a range of 4 to 9, preferably 5.5 to 8.5, and optimally 5.5 to 8.0. For example, the pH range may be 6.0 to 7.8, and more specifically 6.4 to 7.6. The eye drop composition may be, for example, in the form of an eye drop or an eye ointment. A major advantage of the eye drop composition according to one embodiment is that its use is easy and convenient for the subject. The eye drop can be a solution-type eye drop or a suspension-type eye drop. In addition, the composition can be provided in the form of artificial tears or eye cleaning solution. Since the composition can penetrate the cornea, conjunctiva or inner area of the eye, it can be delivered in a pharmaceutically acceptable ophthalmic medium so as to contact the ocular surface for a sufficient time and maintain. The eye drop composition according to one embodiment can be prepared according to methods well known in this technology. Those familiar with this technology can easily determine appropriate excipients and/or carriers and their amounts according to the type of formulation.

The eye drop composition can be stored by being contained in a container made of various materials. For example, the container can be a container of polyethylene, polypropylene and the like including low-density polyethylene (LDPE) and the like, or a glass container, and when the composition of the present invention is an eye drop, it can be contained in an eye drop container, and more specifically, it can be contained in a multi-dose eye drop container or a unit-dose eye drop container.

A multi-dose eye drop container is an eye drop container equipped with a container body and a cap that can be installed on the container body. It is an eye drop container with a cap that can be opened and resealed freely. In a multi-dose eye drop container, eye drops are usually contained multiple times for use over a certain period of time. A unit-dose eye drop container is an eye drop container with a cap fused at the bottle mouth to form a bag-like shape. The purpose is to break and open the fusion portion between the cap and the bottle body during use. The unit-dose eye drop container contains eye drops for one or more uses. The ophthalmic composition of the present invention may include a lubricant (e.g., hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose (CMC), liquid polyols such as polyvinyl alcohol, propylene glycol, and polyethylene glycol), and may contain an additive for promoting healing (e.g., hyaluronic acid) or an additive for promoting the simulated electrolyte composition of the natural tear film or the retention of the composition on the ocular surface (e.g., a gelling agent such as carbomer).

One aspect of the present invention provides a method for treating or improving dry eye disease or dry eye disease-related diseases in an individual, and specifically, provides a method for treating or improving dry eye disease or dry eye disease-related diseases, the method comprising the following steps: administering to an individual in need thereof a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug or stereoisomer thereof at a concentration of 0.01% (w/v) to 0.3% (w/v) as an active ingredient, wherein the administration is by intraocular instillation 1-7 times a day, 1-3 drops per eye each time [Chemical Formula 1] .

The method for treating or improving can be used as a treatment for dry eye disease or dry eye disease-related diseases. By this treatment method, the individual who drops the composition of the present invention into the individual's eye can show an increase in the amount of tear fluid, an effect of improving corneal erosion, and a decrease in the expression of various inflammatory cytokines present in the cornea and conjunctiva. The compound of Chemical Formula 1 can be used for a treatment method for treating, preventing or improving dry eye disease or dry eye disease-related diseases. Dry eye disease-related diseases may include diseases caused or aggravated by a decrease in the amount of tear fluid, or eye diseases that can be prevented or improved by increasing the amount of tear fluid. For example, the eye disease may include conjunctivitis, leptomeningeal gland dysfunction, ocular surface damage (preferably corneal damage) due to dry eye, burning sensation, itching, foreign body sensation or roughness or the like. The cause of dry eye is not particularly limited.

In the therapeutic method of the present invention, in order to achieve the desired effect of the present invention, the active ingredient can be administered to the individual at a concentration of 0.01% (w/v) to 0.3% (w/v) during one administration. In addition, in order to provide the advantages of each aspect provided herein, the administration method suitable for administering the active ingredient at the above concentration can be provided 1-7 times a day, 1-3 drops per eye each time.

In one specific embodiment, one aspect of the present invention provides a method for treating or improving dry eye or dry eye-related diseases and a method for treating or improving the condition, wherein the method comprises administering to a subject in need thereof a compound represented by the following chemical formula 1 at a concentration of 0.01% (w/v) to 0.3% (w/v) or a pharmaceutically acceptable salt, solvate, hydrate, prodrug or stereoisomer thereof as an active ingredient, and instilling the compound into the eye 1-7 times a day, 1-3 drops per eye each time.

On the other hand, in the present invention, "% (w/v)" is the same as described in the composition.

According to one embodiment, the method may include 0.5% (w/v) or less of the CFTR activator based on the total composition as an active ingredient during one eye drop in each eye in an individual. As an embodiment, it may include 0.01% (w/v) to 0.3% (w/v), 0.04% (w/v) to 0.25% (w/v), 0.05% (w/v) to 0.2% (w/v), 0.01% (w/v) to 0.25% (w/v), 0.01% (w/v) to 0.2% (w/v), 0.03 ...5% (w/v), 0.01% (w/v) to 0.2% (w/v), 0.03% (w/v) to 0.3% (w/v), 0.04% ( % (w/v), 0.03% (w/v) to 0.07% (w/v), 0.04% (w/v) to 0.06% (w/v), 0.05% (w/v), 0.08% (w/v) to 0.12% (w/v), 0.09% (w/v) to 0.11% (w/v), 0.1% (w/v), 0.018% (w/v) to 0.22% (w/v), 0.19% (w/v) to 0.21% (w/v), 0.1% (w/v) of the compound of formula 1.

According to one embodiment, the method may contain an active ingredient at a concentration of 0.4-3 mg/ml during one eye drop in each eye in an individual. As a more specific example, the method of the present invention may contain an active ingredient at a concentration of 0.45-2.5 mg/ml or 0.43-2.3 mg/ml during one eye drop in each eye.

When about 50 uL is administered during one instillation in each eye, the amount of active ingredient dropped into the eye during the instillation in each eye may be about 3 mg or less, about 2.5 mg or less, about 2.2 mg or less, and may be about 0.01 to about 3 mg, about 0.015 to about 2.8 mg, about 0.018 to about 2.5 mg, about 0.02 to about 2.4 mg, about 0.05 to about 2.3 mg, about 0.08 to about 2.28 mg, about 0.09 to about 2.25 mg, about 0.095 to about 2.2 mg, about 0.01 to about 2 mg, about 0.015 to about 1.8 mg, about 0.018 to about 1.5 mg, about 0.02 to about 1.4 mg, about 0.05 to about 1 .3mg, about 0.08 to about 1.28mg, about 0.09 to about 1.25mg, about 0.095 to about 1.2mg, about 0.095 to about 1.1mg, about 0.01 to about 1mg, about 0.015 to about 0.8mg, about 0.018 to about 0.5mg, about 0.02 to about 0.4mg, about 0.05 to about 0.3mg, about 0.08 to about 0.28mg, about 0.09 to about 0.25mg, about 0.095 to about 0.2mg, about 0.095 to about 0.15mg, and for example, 0.025mg, 0.05mg, 0.075mg, 0.1mg, 0.125 , 0.15 mg, 0.175 mg, 0.2 mg, 0.225 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.375 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.525 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.05 mg, 1.2 mg, 1.4 mg, 1.5 mg, 1.8 mg, 2.0 mg or 2.1 mg of active ingredient.

The eye drops of the present invention can be instilled into the eye 1 to 7 times a day, 1 to 3 drops per eye at a time. The interval between instillations of the eye drops of the present invention can be at least one hour or longer. One drop can usually be about 10 to 100 μl, 20 to 80 μl, 30 to 70 μl, 40 to 60 μl, for example 50 μl.

The active ingredient of the content can be administered as an eye drop by instilling 1 to 5 drops, 1 to 4 drops, or 1 to 3 drops each time to achieve the purpose or effect of the present invention, such as the stability of the formulation, the distribution on the ocular surface, the effect of reducing corneal erosion, and the like. In addition, the composition can be used once or multiple times a day, and as an example, the composition can be applied 1 to 7 times a day, 1 to 6 times a day, 1 to 5 times a day, 1 to 4 times a day, 1 to 3 times a day, 1 to 2 times a day, once a day, or frequently or irregularly as needed.

In a specific embodiment, the treatment or improvement includes daily administration of the therapeutic agent for at least 2 consecutive weeks. In the treatment regimen, the eye drop composition can be administered once a day, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times a day. In a specific embodiment, the treatment regimen may include daily administration of the therapeutic agent for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or longer.

"Treatment" and "improvement" as defined in this specification means reducing the severity of symptoms to a certain extent. In the case of treatment or improvement of dry eye or dry eye-related diseases of the eye diseases described in this specification, it may mean increased tear fluid, tear retention, reduced tear evaporation, increased tear production, reduced ocular surface inflammation, and the like. The terms "treatment" and "improvement" used herein are not intended to be absolute terms. Treatment may refer to delayed onset, improved symptoms, improved patient quality of life, and the like. The treatment effect can be compared with untreated individuals or groups of individuals or the same patient at different time points before or during treatment.

The term "treating" or "treatment" refers to treating a disease or medical condition or its treatment in a patient, such as a mammal, particularly a human or an animal, and includes ameliorating the disease or medical condition, i.e., eliminating or stopping the disease or medical condition in a patient; inhibiting the disease or medical condition, i.e., delaying or stopping the disease or medical condition in a patient; or alleviating at least one symptom of the disease or medical condition in a patient. The term encompasses prophylactic treatment of a disease or condition to prevent or reduce the risk of developing or progressing a particular disease or condition, or to prevent or reduce the risk of recurrence.

The "individual" or "patient" used in the present specification may include not only humans, but also other animals, such as other primates, rodents, dogs, cats, horses, sheep, pigs and the like, and preferably, it may include humans.

Unless otherwise specified in this manual, the dosage is based on one application per eye.

The eye drop composition of the present invention has the excellent effect of significantly inhibiting the reduction of tear volume, keratoconjunctivitis and corneal morphological changes caused by dry eye disease, restoring damaged cornea, or increasing tear volume. Therefore, the composition can be used to develop safe and effective therapeutic agents for dry eye disease or dry eye-related diseases. Of course, the scope of the present invention is not limited by such effects. Beneficial effects

The eye drops, eye drop composition and eye drop method according to the present invention not only provide the effect of increasing tear secretion, but also provide the effects of recovering corneal damage and alleviating eye inflammation.

The ophthalmic composition of the present invention can be well distributed to the target tissues of the eye, including the cornea and conjunctiva.

The eye drops of the present invention have sufficient solubility to be used as eye drops preparations.

Best Mode

Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples. Abbreviations used

If abbreviations are not defined below, they have generally accepted meanings. aq. = aqueous solution; AUC = area under the curve; CFTR = cystic fibrous transmembrane conductance regulator; CHO = Chinese hamster ovary; DCM = dichloromethane; DIPEA = N,N-diisopropylethylamine; DMF = dimethylformamide; EA = ethyl acetate; HBT = 3-[bis(dimethylamino)methylinium]-3H-benzotriazole-1-oxide hexafluorophosphate; HRMS = high resolution mass spectrometer; MeOH = methanol; MPLC = medium pressure liquid chromatography; NMR = nuclear magnetic resonance; PBS = phosphate buffered saline; PCR = polymerase chain reaction; PK = pharmacokinetics; rt = room temperature; SAR = structure activity relationship; SPR = structure-property relationship; TBAF =tetra-n-butylammonium fluoride; TBDM = tert-butyldimethylsilyl chloride; TEA = triethylamine; TFA = trifluoroacetic acid; THF = tetrahydrofuran; UPLC = ultra-performance liquid chromatography; YFP = yellow fluorescent protein. 1. Methods

(1) YFP fluorescence quenching assay. CHO-K1 cells expressing wild-type human CFTR with the halogenated sensor YFP-H148Q/I152L were plated at a density of 2 × 10 ⁴ cells/well in a 96-well microplate. CHO-CFTR-YFP cells were incubated at 37°C for 48 h. The assay was performed using a FLUOstar Omega microplate reader (BMG labtech, Allmendgrün, Ortenberg, Germany) and MARS data analysis software (BMG labtech). Briefly, each well of the 96-well plate was washed three times in PBS (200 µL/wash). Then, 100 µL of PBS was added to each well. Test compounds (1 µL) were added to each well at a final concentration of 25 µM. After 10 min, the 96-well plate was transferred to a microplate reader preheated to 37°C for fluorescence assay. Each well was individually assayed for CFTR-mediated I ^- influx by continuously recording fluorescence (400 ms per point) for 2 s (baseline). Then, 100 µL of 140 mM I ^- solution was added at 2 s, and YFP fluorescence was recorded for 14 s. The initial iodine influx rate was determined by nonlinear regression from the slope of the initial fluorescence decrease after iodine infusion.

(2) Solubility test protocol. Phosphate buffered saline (PBS, pH 7.5) was prepared by mixing 81% _{0.0667M Na2HPO4} and 19% _{0.0667M NaH2PO4 ,} and NaCl was added to adjust the isotonicity. Then, the test compound was dissolved in PBS (pH 7.5) at 0.5 mg/mL and vortexed for 90 minutes. The compound solution dissolved in PBS was then filtered through 0.45, 1.2, and 5.0 μm syringe filters (Minisart NML, CA) in sequence. The samples were appropriately diluted using an Agilent 1290 Infinity UPLC coupled to a Sciex triple quadrupole 5500 system, and the concentration of the filtered test compound was measured by LC-MS/MS. To quantify the concentration of the test compound, all calibration curves consist of at least six calibrant concentrations, blank samples (with internal standard) and double blank samples (without internal standard). The calibration curves are constructed by weighted linear or quadratic regression methods (1/x) of the peak area ratio of the analyte to the internal standard versus the actual concentration. The solubility of the test compound is back-calculated by substituting the peak area ratio of the filtered test compound to the internal standard into the calibration curve.

(3) Ussing chamber experiment. Snapwell (Corning Inc., NY, USA) inserts containing FRT cells expressing CFTR and primary cultured human conjunctival epithelial cells were mounted in an Ussing chamber. To measure the apical current of FRT-CFTR cells, the apical bath was filled with half ^Cl- solution and the basolateral bath was filled with HCO ₃ ^-buffered solution to generate a transepithelial ^Cl- gradient (apical, 64 mM; basolateral, 129 mM), and the basolateral membrane was permeabilized with 250 µg/mL amphotericin B. For short-circuit current measurements of primary cultured human conjunctival epithelial cells, the apical and basolateral baths were filled with HCO ₃ ^-buffered solution. The cells were immersed for a 20-min stabilization period and aerated with 95% O ₂ /5% CO ₂ at 37°C. Forskolin, the compound of Formula 1, and CFTR _inh -172 were added sequentially to the apical and basolateral bath solutions. Apical and short-circuit currents were measured using an EVC4000 multichannel V/I clamp (World Precision Instruments, Sarasota, FL) and recorded using a PowerLab 4/35 (AD Instruments, Colorado Springs, CO, USA). Data were collected and analyzed using Labchart Pro 7 software (AD Instruments). The sampling rate was 4 Hz.

(4) Pharmacokinetics and Ocular Tissue Distribution Studies. The purpose of this study was to determine the plasma pharmacokinetics and ocular tissue distribution of the compound of Formula 1 after topical instillation of 50 μL/eye (0.1 mg/eye) into the right eye of naive male New Zealand white rabbits. The compound of Formula 1 was monitored in plasma and ocular tissue (from one eye) for up to 72 hours. The compound of Formula 1 used in this experiment was dissolved in 5% polyethylene glycol 35 castor oil in sodium phosphate buffer. The eye drops containing the compound of Formula 1 were administered to the animals by a single topical instillation of 0.1 mg/eye.

Plasma, tears, cornea, conjunctiva, and retina samples were collected at 0.5, 1, 4, 8, 12, 24, 48, and 72 hours after administration. The concentration of the compound of Formula 1 in plasma, tears, cornea homogenate, conjunctiva homogenate, and retina homogenate samples was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Non-compartmental pharmacokinetic analysis of the plasma, tears, cornea, conjunctiva, and retina concentrations of the compound of Formula 1 in the study animals was performed using Phoenix WinNonlin software (version 6.3 or above, Pharsight). The linear/logarithmic trapezoidal rule was applied to obtain PK parameters. Tear, corneal, conjunctival, retinal, aqueous humor, and lacrimal gland concentration values below the lower limit of quantification (LLOQ) were excluded from the calculation of PK parameters.

(5) Corneal and conjunctival epithelial cell toxicity test. Immortalized human corneal epithelial cells and conjunctival epithelial cells (Innoprot, Bizkaia, Spain) were plated on 96-well microplates. After 24 h of incubation, the cells were treated with 30 μM candidate compounds or 0.01% Triton X-100 (Sigma-Aldrich, St Louis, MO, USA), and then they were incubated for two days. An equal amount of DMSO was added to the control group. The culture medium and compounds were replaced every 12 h. In order to evaluate cell proliferation after 48 h of incubation with the compounds, the cells were incubated with MTS for another 1 h. Soluble formazan produced by MTS cell reduction was quantified by measuring absorbance at 490 nm using an infinite M200 microplate reader (Infinite M200 Pro, Tecan Group Ltd., Grödig, Austria). MTS assays were performed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Madison, WI, USA).

(6) Whole-cell patch clamp. Whole-cell patch clamp recordings were performed on CHO-K1 cells expressing CFTR. The bath solution contained (in mM) 140 NMDG-Cl, 1 CaCl ₂ , 1 MgCl ₂ , 10 Tris-ATP, and 10 HEPES (pH 7.4). The pipette solution contained (in mM) 130 CsCl, 0.5 EGTA, 1 MgCl ₂ , 1 Tris-ATP, and 10 HEPES (pH 7.2). The pipette was drawn from borosilicate glass and had a resistance of 3-5 MΩ after fire polishing. The seal resistance was between 3 and 10 GΩ. After establishing the whole-cell configuration, CFTR was activated by forskolin and/or the compound of Formula 1. Whole-cell currents were elicited by applying voltage pulses hyperpolarizing and depolarizing from a 0 mV holding potential to potentials between -80 mV and +80 mV (20 mV steps). Recordings were performed at room temperature using an Axopatch-200B (Axon instruments, Foster City, CA, USA). Currents were digitized using a Digidata 1440A converter (Molecular Devices Co., Union City, CA USA), filtered at 5 kHz, and sampled at 1 kHz.

(7) ANO1 activity measurement. Snapwell inserts containing FRT cells expressing human ANO1 were mounted in a Ussing chamber. The apical bath was filled with half ^Cl- solution and the basolateral bath was filled with _HCO3 buffer solution to generate a transepithelial Cl gradient (apical, 64 mM; basolateral, 129 mM), and the basolateral membrane was permeabilized with 250 µg/mL amphotericin B. The cells were soaked for a 20-minute stabilization period and aerated with 95% _O2 /5% _CO2 at 37°C. ATP was applied to the apical bath solution to induce an increase in intracellular calcium. The compound of Formula 1 or Ani9 was added to the apical and basolateral bath solutions. Apical membrane current was measured 20 min before ANO1 activation using an EVC4000 multi-channel V/I clamp and a PowerLab 4/35. Data were analyzed using Labchart Pro 7. The sampling rate was 4 Hz.

(8) VRAC activity measurement. HeLa cells were stably transfected with YFP-F46L/H148Q/I152L (halide sensor YFP). After incubation of the cells in a 96-well microplate for 48 hours, each well of the 96-well plate was washed three times in PBS (200 μL/wash) and filled with 50 μL/well of the following isotonic solution (in mM): 140 NaCl, 5 KCl, 20 HEPES (310 mOsm; pH 7.4 using NaOH). In each well, the volume-regulated anion channel (VRAC) expressed on the cells was stimulated by adding 50 μL of the following hypotonic solution (in mM): 5 KCl, 20 HEPES, 90 mannitol (120 mOsm/kg). Test compounds (1 μL) were added to each well in a dose-dependent manner. After 5 minutes, the 96-well plate was transferred to a plate reader for fluorescence assay. VRAC-mediated ^{I- influx} was assayed individually for each well by continuously recording fluorescence (400 ms per point) for 7.6 s. 0.4 s YFP fluorescence was recorded as a baseline, then 100 μL of 140 mM I ^- solution was added at 0.4 s and the fluorescence change was observed. The initial iodine influx rate was determined by nonlinear regression from the slope of the initial fluorescence decrease after the infusion of iodine ions.

(9) Intracellular cAMP measurement. CHO-K1 cells grown on 12-well culture plates were washed three times with PBS and then incubated in PBS containing 100 µM 3-isobutyl-1-methylxanthine (IBMX) at 37°C for 5 min. Cells were treated with the compound of Formula 1 or forskolin and incubated at 37°C for 10 min. After incubation for 10 min, cells were washed with cold PBS, and cytoplasmic cAMP was measured using a cAMP immunoassay kit (parameter cAMP immunoassay kit; R&D Systems, Minneapolis, MN) according to the manufacturer's protocol.

(10) Animals. All animals were used and cared for in strict accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. This study was approved and reviewed by the Institutional Review Board of Severance Hospital, Yonsei College of Medicine (Seoul, South Korea) (IRB No. 2019-0166).

(11) Scopolamine-induced dry eye mouse model and administration of eye drops. Eight-week-old female C57BL/6J mice obtained from Orientbio (Gyeonggi-do, South Korea) were used. The dry eye mouse model was evaluated. The experimental period lasted for 24 days, and the mice were raised in a dry room (temperature: 22°C, humidity: 12%) during the experimental period. To induce the dry eye model, 0.5 mg/0.1 ml of scopolamine hydrobromide was injected subcutaneously 3 times a day (11:00 am, 2:00 pm, 5:00 pm) for 14 days. After inducing the dry eye mouse model, 0.3% diquas sodium (Diquas, ophthalmic solution Santen Pharmaceutical Co. Ltd., Osaka, Japan), vehicle (5% polyethylene glycol 35 castor oil in sodium phosphate buffer) or the compound of Formula 1 (2060 µM, 5% polyethylene glycol 35 castor oil in sodium phosphate buffer) was instilled into each eye 3 times a day (11:00 am, 2:00 pm, 5:00 pm) for 10 days, and 0.5 mg/0.1 ml scopolamine hydrobromide was subcutaneously injected 8 days before the treatment period. The eye drops were applied in both eyes, 5 ul per eye, and maintained for 30 seconds.

(12) Administration of eye drops in wild-type mice. Eight-week-old female C57BL/6J mice obtained from Orient Bio (Seongnam, South Korea) were used. After 7 days of acclimation, each eye drop containing the compound of Chemical Formula 1 was administered once. The eye drops were administered in both eyes, 2.5 μl per eye, and maintained for 30 seconds. This experiment was conducted using different concentrations of the compound of Chemical Formula 1 (1 μM, 10 μM, 100 μM, 1000 μM) and the vehicle.

(13) Tear volume measurement. Tear volume was measured using phenol red thread (Showa Yakuhin Kako Co. Ltd, Tokyo, Japan) by applying the phenol red thread to the lateral canthus area of normal or scopolamine hydrobromide-treated mice with dry eye model for 15 seconds using tweezers, and the length of the wet thread was examined under a microscope by measurement using a vernier caliper. Three measurements were performed in dry eye model mice; before induction of dry eye, after induction of dry eye, and 10 days after application of the eye drops. In experiments using normal mice, tear volume was measured four times over time using phenol red thread, i.e., before application of the eye drops (0 hours, baseline) and 1 hour, 3 hours, and 6 hours after application of the eye drops.

(14) Corneal erosion grading. To evaluate corneal epithelial erosion, 5 μL of 1% fluorescein dye and 0.5% proparacaine were applied to the ocular surface of mice 10 days after each treatment. Anterior segment images were taken using a built-in digital camera under a cobalt blue filter. Each corneal erosion was scored from 0 to 5 according to the Oxford scheme.

(15) Quantitative PCR analysis. Total RNA was isolated using Tri-RNA reagent (FAVORGEN, Ping-Tung, Taiwan), and 1 μg of total RNA was used to synthesize cDNA using RNA to cDNA EcoDry™ premix (TaKaRa, Shiga, Japan) according to the manufacturer's protocol. Relative mRNA levels were assessed using SYBR Green PCR Master Mix (Applied Biosystems) in ViiA7 (Applied Biosystems, Foster City, CA, USA). Target gene expression was normalized to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences used are as follows: GAPDH, sense (5-AACGACCCCTTCATTGACCT-3, Seq. ID NO.1) and antisense (5-ATGTTAGTGGGGTCTCGCTC-3, Seq. ID NO.2), PCR product size is 155 base pairs; IL-1β, sense (5-ACTCATTGTGGCTGTGGAGA-3, Seq. ID NO.3) and antisense (5-TTGTTCATCTCGGAGCCTGT-3, Seq. ID NO.4), PCR product size is 199 base pairs; IL-6, sense (5-CTGCAAGAGACTTCCATCCAG-3, Seq. ID NO.5) and antisense (5-AGTGGTATAGACAGGTCTGTTGG-3, Seq. ID ID NO.6), the PCR product size was 131 base pairs; IL-17, sense (5-GCTGACCCCTAAGAAACCCC-3, Seq. ID NO.7) and antisense (5-GAAGCAGTTTGGGACCCCTT-3, Seq. ID NO.8), the PCR product size was 162 base pairs; TNF-α, sense (5-AGCACAGAAAGCATGATCCG-3, Seq. ID NO.9) and antisense (5-CGATCACCCCGAAGTTCAGT-3, Seq. ID NO.10), the PCR product size was 166 base pairs; MMP-2, sense (5-CGATGTCGCCCCTAAAACAG-3, Seq. ID NO.11) and antisense (5-GCATGGTCTCGATGGTGTTC-3, Seq. ID No. 12), the PCR product size was 176 base pairs; and MMP-9, sense (5-AAAACCTCCAACCTCACGGA-3, Seq. ID No. 13) and antisense (5-GTGGTGTTCGAATGGCCTTT-3, Seq. ID No. 14), the PCR product size was 190 base pairs.

Statistical analysis. The Student's t test was used to assess the significance of the differences and p < 0.05 was considered significant. 2. Results (1) YFP fluorescence and solubility analysis results

A comparison was made between Formula A, which is known to have CFTR activation effect, and the compound of Chemical Formula 1 of the present invention (herein 16d). There was no significant difference in EC ₅₀ , but the solubility value of the compound of Chemical Formula 1 was confirmed to be 134,489 nM, confirming that it has excellent solubility. In this article, solubility refers to solubility in PBS (pH 7.5). Table 1 Compound R EC ₅₀ (nM) ^a Solubility (nM) ^b Solubility/YFP ratio ^cLogD _7.4c A ( Cact-3 ) 18 59 3 2.61 16d twenty three 134,489 5,847 1.73 ^a CFTR channel activity was measured by YFP quench assay in CHO-K1 cells expressing human wild-type CFTR. Results are presented as the mean of three replicates. ^b Compound concentrations after 90 min vortexing in PBS. ^c cLogD _7.4 values were calculated by ACD/Percepta software (ACD/Labs, Toronto, Canada).

The compound of Formula 1 has particularly excellent solubility and high efficacy in CFTR activators. It has been confirmed that the compound of Formula 1 of the present invention is suitable for use as an eye drop formulation. (2) Effect of the compound of Formula 1 on the activity of CFTR chloride ion channel

To investigate the effect of the compound of Formula 1 on the activity of CFTR chloride channel, apical membrane currents were measured in FRT cells expressing human CFTR. The compound of Formula 1 effectively activated CFTR chloride channel in a dose-dependent manner with an IC ₅₀ of 342 nM, and the CFTR chloride current induced by the compound of Formula 1 was completely blocked by the potent and selective CFTR inhibitor CFTR _inh -172 (Fig. 1A, 1B). To further characterize the activation of CFTR by the compound of Formula 1 , whole-cell patch clamp analysis was performed on CHO-K1 cells expressing human CFTR. The CFTR current strongly activated by the compound of Formula 1 at 30 μM showed a linear current/voltage relationship, similar to the CFTR activation induced by forskolin, and the CFTR current induced by the compound of Formula 1 was completely inhibited by CFTR _inh -172 ( Figures 1C -1E ). (3) In vitro manifestations of the compound of Formula 1

To investigate the effects of the compound of Formula 1 on other chloride channels, the effects of the compound of Formula 1 on the calcium-activated chloride channel TMEM16A/Anoctamin 1 (ANO1) and the volume-regulated anion channel (VRAC) were observed. ANO1 apical membrane current was measured in FRT cells expressing human ANO1, and VRAC activity was measured using a YFP fluorescence quenching assay in LN215 cells expressing the halide sensor YFP-F46L/H148Q/I152L. High concentrations (30 μM) of the compound of Formula 1 did not affect the channel activities of ANO1 and VRAC, but ANO1 and VRAC were completely blocked by Ani9 and VI-116 , respectively ( Figures 2A and 2B ). CFTR is activated by the cAMP signaling pathway.

Therefore, the effect of the compound of Chemical Formula 1 on the intracellular cAMP concentration was observed. Compared with the control group, the compound of Chemical Formula 1 slightly increased the cAMP level, but did not increase the cAMP level as strongly as forskolin (Figure 2).

To observe the cytotoxicity of the compound of Chemical Formula 1, the effect of the compound of Chemical Formula 1 on cell viability was evaluated in corneal epithelial (CorE) and conjunctival epithelial (ConjE) cells. The compound of Chemical Formula 1 did not affect the cell viability of CorE and ConjE at 30 μM (Figure 2D).

To investigate whether the compound of Formula 1 can activate endogenous CFTR channels in human ocular epithelium, short-circuit currents were measured in primary cultured human conjunctival epithelial cells. Interestingly, the compound of Formula 1 effectively increased CFTR-dependent chloride ion currents in a dose-dependent manner, and the CFTR current induced by the compound of Formula 1 was completely inhibited by 10 μM CFTR _inh -172. These results indicate that the compound of Formula 1 can effectively and selectively activate human CFTR without causing cytotoxicity to the ocular epithelium. (4) Ocular distribution and plasma pharmacokinetics of the compound of Formula 1 .

Prior to studying the in vivo efficacy of the compound of Formula 1, ocular tissue distribution and plasma pharmacokinetic (PK) were evaluated after topical ocular administration of the compound of Formula 1 in male New Zealand white rabbits. No side effects of the compound of Formula 1 were observed during the PK study. As shown in Figure 3 and Table 2, the concentrations of the compound of Formula 1 in tears, cornea, and conjunctiva maintained an EC ₅₀ value of more than 342 nM (166 ng/mL) for 8 hours. For the mean plasma concentrations of the compound of Formula 1, PK parameters were not determined because they were below the lower limit of quantitation for up to 72 hours, except for 0.5 hours after administration. These results indicate that the compound of Formula 1 is well distributed in the target tissues expressing CFTR (cornea and conjunctiva), maintained for a long period of time, and has negligible systemic exposure. Table 2

Pharmacokinetic parameters of the compound of Formula 1 in rabbits after a single topical instillation of 0.1 mg/eye of the compound of Formula 1 (n=3) PK parameters Mean plasma Mean conjunctival Average cornea Average retinal Average tear C _max (ng/mL or ng/g) ^ND 793.0 7840.0 ^ND 749.0 _Tmax (h) ^ND 0.5 0.5 ^ND 8.0 T _1/2 (h) ^{ND *} 40.6 ^* 23.1 ^{ND *} 29.8 T _last (h) ^ND 72.0 72.0 ^ND 72.0 AUC _0-last (ng·h/mL or ng·h/g) ^ND 9819.0 28349.0 ^ND 16573.0 AUC _0-24 (ng·h/mL or ng·h/g) ^ND 4734.0 19409.0 ^ND 7952.0 AUC _0-inf (ng·h/mL or ng·h/g) ^ND 13741.0 35802.0 ^ND 18750.0 ^a ND: Not determined (parameter not determined due to inadequate definition of the terminal elimination phase) ^* : The adjusted linear regression coefficient of concentration values during the terminal phase is less than 0.9, and T1 _/2 may not accurately estimate the composite mean concentration used in the calculation of PK parameters. (5) Among CFTR activators, it shows particularly rapid action and higher maximum efficacy.

In order to study the pharmacological advantages of the compound (16d) of Chemical Formula 1 compared with Compound A (Cact-3), the effects of 16d and Cact-3 on the tear volume of wild-type (normal) mice were observed. The maximum solubility of Cact-3 is 72 μM, so the administration of Cact-3 is up to 72 μM. In the case of 16d, the maximum solubility is greater than 2 mM, and the administration of 16d is up to 1 mM. As shown in Figure 4, the treatment of both 16d and Cact-3 showed a dose-dependent increase in tear volume, and the E _max of 16d was higher than that of Cact-3. 16d reached the maximum tear volume 1 hour after administration, while Cact-3 showed the maximum tear volume 3 hours after administration. Therefore, compared with Cact-3, 16d has the advantages of higher solubility, higher maximum efficacy and faster action. (6) Increased tear volume and reduced corneal erosion in DED mice.

To investigate the effect of the compound of Formula 1 on tear volume in DED mice, scopolamine-induced tear volume was assessed by phenol red line test in a dry eye mouse model. As shown in FIG5A , subcutaneous injection of scopolamine significantly reduced the length of line wetting in the untreated group and the vehicle-treated group compared with the control group. However, treatment with the compound of Formula 1 significantly and almost completely restored the reduction in tear volume induced by scopolamine, and difos ... To investigate whether the compound of Formula 1 improves ocular surface damage in DED mice, corneal erosion changes were observed in a scopolamine-induced dry eye mouse model treated with vehicle, the compound of Formula 1 , or difosfosol. Each drop of the eye drops was applied to each eye three times a day for 10 days. Compared with the control group, the compound of Formula 1 significantly reduced corneal erosion ( Figures 5B and 5C ) . These results indicate that the compound of Formula 1 enhances tear secretion and reduces corneal erosion in DED mice, and its efficacy is equal to or greater than difosfosol. (7) Reduction in the mRNA expression of proinflammatory cytokines and MMP2 in the ocular epithelium of DED mice.

It is known that the ocular surface of DED mice expresses high levels of infectious interleukins, such as IL-1β, IL-6, IL-17, and TNF-α, as well as matrix metalloproteinases (MMPs)-2 and MMP-9. The mRNA expression levels of MMP-2, MMP-9, and proinflammatory interleukins (including IL-1β, IL-6, IL-17, and TNF-α) were studied by real-time PCR in the cornea and conjunctiva of normal or DED mice in the presence or absence of vehicle, the compound of Formula 1, and defosfoline. When treated with the compound of Formula 1, the mRNA expression levels of IL-1β, IL-17, TNF-α, and MMP-2 in the cornea and conjunctiva were significantly reduced (Figure 6). Among them, defosfoline also significantly reduced the mRNA expression levels of IL-17, TNF-α, and MMP-2. These results indicate that the compound of Formula 1 reduces the mRNA expression levels of IL-1β, IL-17, TNF-α, and MMP-2 in the cornea and conjunctiva of DED mice, and its efficacy is equal to or greater than that of difosfosol. 3. Conclusion

The purpose of this study is to provide an eye drop with excellent solubility to achieve high ocular bioavailability and reduce ocular surface damage.

Electrophysiological studies have shown that the compound of Chemical Formula 1 effectively and selectively activates the CFTR chloride ion channel, and has no cytotoxicity to corneal and conjunctival epithelial cells. In addition, the compound of Chemical Formula 1 is well distributed in the cornea and conjunctiva of rabbits and maintained for a long period of time (>8 hours), and the systemic exposure is negligible. In the following in vivo experiments, the compound of Chemical Formula 1 is equivalent to difosfosol, significantly enhancing the recovery of tear volume and improving corneal erosion in scopolamine-induced dry eye mice. The compound of Chemical Formula 1 also significantly reduces the mRNA expression levels of MMP2 and proinflammatory interleukins (including IL-1β, IL-17 and TNF-α) in the cornea and conjunctiva of scopolamine-induced dry eye mice. 4. Administration of eye drops

The ophthalmic composition was administered to mammals (mice, rabbits and dogs) according to the following ophthalmic administration scheme. The ophthalmic composition is prepared by blending the compound of Formula 1 with a surfactant (polysorbate 80), PEG 40-stearate and polyethylene glycol-35 castor oil, and the composition can be obtained by the method disclosed in Korean Patent Application No. 10-2022-0049160.

The compound of formula 1 contained in the composition is administered at different concentrations and administration times, and when dropped into the eye at the administration concentration and administration frequency of the present invention, the intended effects of the present invention are achieved, such as increasing the amount of tears and inhibiting inflammation. Table 3 Concentration of the compound of formula 1 (mg/ml) Investment schedule 0.5 1.0 2.0 1 drop once 0.025 mg 0.05 mg 0.1 mg 2 drops once 0.05 mg 0.1 mg 0.2 mg 3 drops once 0.075 mg 0.15 mg 0.3 mg 1 drop 2 times 0.05 mg 0.1 mg 0.2 mg 2 drops 2 times 0.1 mg 0.2 mg 0.4 mg 3 drops 2 times 0.15 mg 0.3 mg 0.6 mg 1 drop 3 times 0.075 mg 0.15 mg 0.3 mg 2 drops 3 times 0.15 mg 0.3 mg 0.6 mg 3 drops 3 times 0.225 mg 0.45 mg 0.9 mg 1 drop 4 times 0.1 mg 0.2 mg 0.4 mg 2 drops 4 times 0.2 mg 0.4 mg 0.8 mg 3 drops 4 times 0.3 mg 0.6 mg 1.2 mg 1 drop 5 times 0.125 mg 0.25 mg 0.5 mg 2 drops 5 times 0.25 mg 0.5 mg 1 mg 3 drops 5 times 0.375 mg 0.75 mg 1.5 mg 1 drop 6 times 0.15 mg 0.3 mg 0.6 mg 2 drops 6 times 0.3 mg 0.6 mg 1.2 mg 3 drops 6 times 0.45 mg 0.9 mg 1.8 mg 1 drop 7 times 0.175 mg 0.35 mg 0.525 mg 2 drops 7 times 0.35 mg 0.7 mg 1.05 mg 3 drops 7 times 0.7 mg 1.4 mg 2.1 mg

The ophthalmic composition of the present invention having the above-mentioned administration (eye drops) schedule is excellent in the treatment and improvement of dry eye syndrome or dry eye-related diseases.

The above description of the present invention is for illustrative purposes, and those familiar with the art will understand that the present invention can be easily modified into other specific forms without changing its technical scope or basic characteristics. Therefore, the above embodiments should be understood in all aspects as illustrative rather than restrictive. For example, the scope of each component of the present invention described in a single form is indicated by the following invention patent scope, and all changes or modifications derived from the meaning and scope of the invention patent scope and its equivalent concepts should be interpreted as included in the scope of the present invention. Industrial Applications

The present invention provides an eye drop, which can be used to treat and/or improve dry eye disease.

without

Figure 1 shows the activation of CFTR chloride channel by the compound of Formula 1 (i.e., 16d). (A) Shows representative traces of apical membrane currents in FRT cells expressing human CFTR. CFTR was activated by the indicated concentrations of the compound of Formula 1 in the presence of 50 nM forskolin (FSK) and inhibited by 10 μM CFTR _inh -172. (B) Summary of CFTR activation (mean ± SE, n=3). (C) Whole cell currents were recorded at a holding potential of 0 mV and pulsed with voltages between ±80 mV (20 mV units) in CHO-K1 cells expressing human CFTR. CFTR was activated by 20 μM forskolin or 30 μM of the compound of Formula 1 and inhibited by 20 μM CFTR _inh -172. (D) Current/voltage plot showing the average current in the middle of each voltage pulse. (E) Shows the sum of current density at +80 mV (mean ± SE, n = 3).

FIG. 2 shows the properties of the compound of Formula 1 and the effects on CFTR activity in primary cultured human conjunctival epithelial cells. (A) Apical currents were measured in FRT cells expressing ANO1. ANO1 was activated by 100 μM ATP and inhibited by 10 μM ANO1 inhibitor Ani9. Cells were pretreated with the compound of Formula 1 (30 μM) and Ani9 for 10 minutes. (B) The effects of the compound of Formula 1 on VRAC chloride channel activity were observed in HeLa cells expressing YFP-F46L/H148Q/I152L. Cells were treated with the compound of Formula 1 (30 μM) for 5 minutes in hypotonic solution. VRAC was inhibited by 10 μM VRAC inhibitor VI-116. (C) CHO-K1 cells were treated with the compound of Formula 1 (30 μM) and forskolin (10 μM) in the presence of IBMX (100 μM) for 10 minutes, and then cAMP levels were measured (mean ± SE, n = 3). (D) Corneal epithelial (CorE) and conjunctival epithelial (ConjE) cells were treated with the compound of Formula 1 for 48 hours, and cell viability was measured by MTS assay (mean ± SE, n = 3). (E) Representative traces of short-circuit current in primary cultured human conjunctival epithelial cells are shown. CFTR was activated by the compound of Formula 1 at the indicated concentrations and blocked by 10 μM CFTR _inh -172.

FIG3 shows the mean concentration-time curve of the compound of Formula 1 after a single topical instillation of the compound of Formula 1 eye drops at 0.1 mg/eye in rabbits.

FIG. 4 shows the effects of the compound of Formula 1 and Cact-3 on tear volume in wild-type mice. (A) Tear volume was measured by phenol red line test in each group treated with different concentrations of the compound of Formula 1 (mean ± S.E., n = 6). (B) Tear volume was measured by phenol red line test in each group treated with different concentrations of Cact-3 (mean ± S.E., n = 6). Mice were treated with 2.5 μl of vehicle (5% polyethylene glycol 35 castor oil in sodium phosphate buffer) eye drops, the compound of Formula 1 and Cact-3. * p < 0.05, ** p < 0.01, *** p < 0.001.

FIG. 5 shows the effects of the compound of Formula 1 on tear volume and ocular surface damage in a scopolamine-induced dry eye mouse model. (A) Tear volume of each group was measured by phenol red line test (mean ± S.E., n = 5). (B) Corneal erosion grade of each group was measured by fluorescein staining on a five-point scale (mean ± S.E., n = 5). (C) Representative images of mouse eyes with cornea stained with fluorescein. Mice were treated with 5 μl of vehicle (5% polyethylene glycol 35 castor oil in sodium phosphate buffer), compound of formula 1 (2,060 μM, 5% polyethylene glycol 35 castor oil in sodium phosphate buffer), and diquas three times daily for 10 days while maintaining dry eye. NT: no treatment; Diquas: diquas; ns: not significant. * p < 0.05, ** p < 0.01.

Figure 6 shows the effect of the compound of Formula 1 on the mRNA expression levels of inflammatory interleukins and MMPs in the cornea and conjunctiva. (A-F) shows the mRNA expression levels of IL-1β, IL-6, IL-17, TNF-α, MMP2, and MMP9 in the cornea and conjunctiva. Mice were treated with 5 μl of vehicle (5% polyethylene glycol 35 castor oil in sodium phosphate buffer), the compound of Formula 1 (2,060 μM, 5% polyethylene glycol 35 castor oil in sodium phosphate buffer), and difosfosol three times a day for 10 days while maintaining dry eye (mean ± S.E., n = 5). NT: no treatment; Diquas: difosfosol; ns: not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Domestic storage information (please note in the order of storage institution, date, and number) None Foreign storage information (please note in the order of storage country, institution, date, and number) None

TW202425990A_112142207_SEQL.xml

Claims (17)

An ophthalmic composition for treating, preventing or improving dry eye disease or dry eye disease-related diseases, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug or stereoisomer thereof at a concentration of 0.01% (w/v) to 0.3% (w/v) as an active ingredient, and is dropped into the eye 1-7 times a day, 1-3 drops per eye each time [Chemical Formula 1] . The eye drop composition as described in claim 1, comprising the active ingredient at a concentration of 0.04% (w/v) to 0.25% (w/v). The eye drop composition as described in claim 1, comprising the active ingredient at a concentration of 0.05% (w/v) to 0.2% (w/v). The eye drop composition as described in claim 1, comprising the active ingredient at a concentration of 0.05% (w/v), 0.1% (w/v), or 0.2% (w/v). The eye drop composition as described in claim 1, wherein the composition contains the active ingredient at a concentration of 0.4-3 mg/ml. The ophthalmic composition as described in claim 1, wherein the composition is characterized in that during the intraocular instillation, 0.025 mg, 0.05 mg, 0.075 mg, 0.1 mg, 0.125 mg, 0.15 mg, 0.175 mg, 0.2 mg, 0.225 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.375 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.525 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.05 mg, 1.2 mg, 1.4 mg, 1.5 mg, 1.8 mg, 2.0 mg or 2.1 mg of the active ingredient based on 50 uL is instilled into the eye once per eye. The ophthalmic composition as described in claim 1, wherein the active ingredient is dropped into the eye 1, 2, 3, 4, 5, 6 or 7 times a day. The ophthalmic composition as described in any one of claims 1 to 7, wherein the composition further comprises a pharmaceutically acceptable excipient. The ophthalmic composition as described in claim 8, wherein the excipient comprises a surfactant, an isoosmotic agent or a buffer. The eye drop composition as described in any one of claims 1 to 7, wherein the composition is in the form of an eye drop solution, an ointment, a gel, a cream, a lotion, an emulsion, a suspension, or a spray. A method for treating or improving dry eye disease or dry eye-related diseases in an individual, comprising the following steps: administering a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt, solvate, hydrate, prodrug or stereoisomer as an active ingredient to an individual in need thereof at a concentration of 0.01% (w/v) to 0.3% (w/v), wherein the administration is intraocularly instilled 1-7 times a day, 1-3 drops per eye each time [Chemical formula 1] . The method of claim 11, wherein the active ingredient is dropped into the eye at a concentration of 0.04% (w/v) to 0.25% (w/v). The method of claim 11, wherein the active ingredient is dropped into the eye at a concentration of 0.05% (w/v) to 0.2% (w/v). The method of claim 11, wherein the active ingredient is dropped into the eye at a concentration of 0.05% (w/v), 0.1% (w/v), or 0.2% (w/v). The method of claim 11, wherein the active ingredient is dropped into the eye at a concentration of 0.4-3 mg/ml. The method as described in claim 11, wherein the method is characterized in that during the intraocular instillation, 0.025 mg, 0.05 mg, 0.075 mg, 0.1 mg, 0.125 mg, 0.15 mg, 0.175 mg, 0.2 mg, 0.225 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.375 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.525 mg, 0.6 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.05 mg, 1.2 mg, 1.4 mg, 1.5 mg, 1.8 mg, 2.0 mg or 2.1 mg of the active ingredient is instilled into the eye once per eye. The method of claim 11, wherein the active ingredient is instilled into the eye 1, 2, 3, 4, 5, 6 or 7 times a day.