HK1219224B - Therapeutic agent for keratoconjunctive disorders - Google Patents

Description

Therapeutic agents for keratoconjunctival disorders

Technical Field

The present invention relates to a therapeutic agent for corneal and conjunctival disorders containing an RARγ agonist as an active ingredient.

Background Art

The cornea is a transparent, avascular tissue with a diameter of about 1 cm that covers the front surface of the eyeball. The conjunctiva is a mucous membrane that covers the back of the eyelid and the surface of the eyeball, and is located behind the corneal limbus. The cornea and conjunctiva play an important role in vision. It is known that when a disease occurs in them, visual function is seriously affected. Keratoconjunctival diseases caused by various diseases such as corneal ulcers, keratitis and dry eye are diseases caused by the following: delayed recovery from a disease caused by a certain reason such as external damage, or a disease that has become chronic. Because the cornea is a tissue connected to the conjunctiva, such diseases negatively affect each other in the normal structure of the epithelium, and in some cases can cause damage to the structure or function of the corneal stroma or endothelium.

Collagen (particularly type I collagen) is known to be one of the representative stromal components of corneal parenchymal tissue. Functional disorders caused by stromal degradation occur in diseases caused by corneal and conjunctival disorders. Therefore, inhibiting the degradation of collagen (particularly type I collagen) is believed to be effective for diseases caused by corneal and conjunctival disorders.

In corneal and conjunctival disorders, scar tissue, which typically forms after inflammation has subsided, can interfere with visual function. Therefore, as with the aforementioned inhibition of collagen degradation, inhibition of collagen contraction, if possible, is believed to be effective for scar contraction and formation (hereinafter collectively referred to as "scarring").

Patent Document 1 describes that all-trans retinoic acid (hereinafter also referred to as ATRA) promotes corneal regeneration. However, its effect is weak and its detailed mechanism has not yet been elucidated.

Furthermore, ATRA is an agonist of the retinoic acid receptor (hereinafter also referred to as RAR). However, because ATRA does not have selectivity for the RAR subtypes RARα, RARβ, and RARγ, the contribution of each RAR subtype to corneal regeneration is unknown.

Meanwhile, RAR is involved in various roles such as growth, morphogenesis and differentiation in many cells such as inflammatory cells, immune cells and structural cells.Further, it has been confirmed that there are differences in the distribution of RAR subtypes depending on the tissue or organ of a mammal.

Some effects of RARs are undesirable, such as the increase in triglycerides caused by RARα. Therefore, specificity or selectivity with respect to the subtype in compounds having RAR agonist activity is expected to achieve a reduced risk of side effects.

For the reasons described above, there is a need for RAR agonists that have a potent inhibitory effect on corneal and conjunctival disorders and a high safety profile based on subtype selectivity.

Patent Documents 2 and 3 disclose RAR agonists (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl}vinyl)benzoic acid and its derivatives. Furthermore, Patent Document 2 describes that (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl}vinyl)benzoic acid is useful for emphysema, cancer, and skin diseases. Patent Document 3 describes that the above agonists are useful for neuropathic pain.

In addition, Non-Patent Document 1 describes that the RAR agonist 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthoic acid induces apoptosis in lung cancer cells.

Furthermore, Patent Document 4 describes that the RAR agonist 3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)acetylamino]benzoic acid is useful in muscle repair or regeneration.

However, no pharmacological effects of any RAR agonist on keratoconjunctival disorders or scarring caused by keratoconjunctival disorders have been discussed or reported. In addition, there is no literature suggesting such effects.

Reference List

Patent Literature

Patent Document 1: Japanese Patent Application Laid-Open No. 2009-235031

Patent Document 2: International Publication No. WO 2002/028810

Patent Document 3: International Publication No. WO 2008/057930

Patent Document 4: Japanese Patent Application Laid-Open No. 2013-536855

Non-patent literature

Non-patent literature 1: Sun SY et al., Cancer Research 62(8): 2430-2436 (2002).

Summary of the Invention

SUMMARY OF THE INVENTION

Technical issues

Finding drugs that are effective for ophthalmic diseases, especially corneal and conjunctival disorders, is an important and interesting goal. The object of the present invention is to provide a therapeutic agent that has an effect of inhibiting corneal and conjunctival disorders and has a high safety profile based on subtype selectivity.

Solution to the problem

In diligent research to find drugs effective for corneal and conjunctival disorders, the present inventors discovered, through pharmacological tests using rabbit corneal cells and subconjunctival fibroblasts, that the RARγ agonist (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl}vinyl)benzoic acid (R667: hereinafter also referred to as "RARγ agonist A") exerts an excellent effect of improving corneal and conjunctival disorders and scar formation associated with corneal and conjunctival disorders, among which a potent effect of inhibiting collagen degradation and a significant effect of inhibiting collagen contraction were confirmed. In addition, it was discovered that other RARγ agonists: 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthoic acid (CD437: hereinafter also referred to as "RARγ agonist B") and 3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)acetylamino]benzoic acid (BMS961: hereinafter also referred to as "RARγ agonist C") also showed a significant effect of inhibiting collagen degradation in pharmacological tests using rabbit corneal cells, thereby completing the present invention.

Specifically, the present invention relates to: [1] a therapeutic agent for corneal and conjunctival disorders, comprising an RARγ agonist as an active ingredient; [2] the therapeutic agent of [1], wherein the RARγ agonist is (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl}vinyl)benzoic acid, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene acid, 3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)acetylamino]benzoic acid, an ester thereof, or a salt thereof; and [3] the therapeutic agent of [1] or [2], wherein the corneal and conjunctival disorders are selected from corneal ulcer, corneal epithelial abrasion, and corneal ulcer. abrasion), keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorder, superficial punctate keratopathy, corneal epithelial defect, conjunctival epithelial defect, keratoconjunctivitis sicca, superior limbal keratoconjunctivitis, filamentous keratitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, non-infectious conjunctivitis, corneal scar and conjunctival scar, [4] the therapeutic agent according to any one of the above [1]-[3], wherein the administration form is instillative administration or oral administration, and [5] the therapeutic agent according to any one of the above [1]-[4], wherein the dosage form is instillative, eye ointment, injection, tablet, granule, fine granule, powder or capsule.

Advantageous effects of the invention

The RARγ agonist, which is an active ingredient of the therapeutic agent for corneal and conjunctival disorders of the present invention, can be used as a therapeutic agent for corneal and conjunctival disorders such as corneal ulcer, corneal epithelial abrasion, keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorder, superficial punctate keratopathy, corneal epithelial defect, conjunctival epithelial defect, keratoconjunctivitis sicca, superior limbal keratoconjunctivitis, filamentous keratitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, or non-infectious conjunctivitis by strongly inhibiting corneal and conjunctival collagen degradation.

Furthermore, the RARγ agonist, which is an active ingredient of the therapeutic agent for corneal and conjunctival disorders of the present invention, can also be used as a therapeutic agent for corneal scars or conjunctival scars associated with corneal and conjunctival disorders by strongly inhibiting corneal and conjunctival collagen degradation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the relationship between the concentration (nM) of RARγ agonist A (R667) and collagen degradation (hydroxyproline amount (µg) per well). The vertical axis represents the value (%) when the control hydroxyproline amount is set to 100.

Figure 2 is a graph showing the relationship between the concentration (nM) of RARγ agonist A (R667) and collagen contraction (diameter of collagen gel in the dish (mm)) using subconjunctival fibroblasts. "★" indicates a statistically significant difference (p < 0.05).

Figure 3 is a graph showing the relationship between the concentration (μM) of RARγ agonist A (R667) and collagen contraction (diameter of collagen gel in the dish (mm)) using corneal cells. "★" indicates a statistically significant difference (p < 0.05).

Figure 4 is a graph showing the relationship between the concentration (nM) of RARγ agonist A (R667), RARγ agonist B (CD437), and RARγ agonist C (BMS961) and collagen degradation using corneal cells. The vertical axis represents the ratio (Ratio) of the amount of hydroxyproline (µg) per well in the absence of an RARγ agonist or stimulator, relative to the amount in the presence of an RARγ agonist or stimulator, with the amount in the well being 1. "★" indicates a statistically significant difference (p < 0.05).

Figure 5 shows the relationship between the concentration (μM) of RARγ agonist A (R667) and the expression and activation of matrix metalloproteinases (MMPs) in corneal cells. The top row of Figure 5A shows the expression and activation of MMP-1, the bottom row of Figure 5A shows the expression and activation of MMP-3, and Figure 5B shows the expression and activation of MMP-2 and MMP-9.

Figure 6 shows the results of observations when lipopolysaccharide (LPS) and RARγ agonist A (R667) were administered intraperitoneally into the corneal stroma of one eye of a male Japanese white rabbit. The top row (vehicle) shows the results of administering only a solution without the present invention's RARγ agonist A (R667), while the bottom row (0.1% R667) shows the results of administering a solution containing RARγ agonist A (R667).

DETAILED DESCRIPTION

The keratoconjunctival disorder therapeutic agent of the present invention is not particularly limited and may be any therapeutic agent having an RARγ agonist as an active ingredient. The RARγ agonist of the present invention refers to a compound that can significantly promote the activation of the RARγ receptor by binding to the RARγ receptor compared to the RARα receptor or RARβ receptor.

Examples of such RARγ agonists include (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl}vinyl)benzoic acid (R667) represented by the following formula (I), 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthoic acid (CD437) represented by the following formula (II), 3-fluoro-4-(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl}vinyl)benzoic acid (R667) represented by the following formula (II), -[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)acetylamino]benzoic acid (BMS961), (2E)-3-(4-carboxyphenyl)-1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2-propen-1-one oxime represented by the following formula (IV) (NRX204647: hereinafter also referred to as “RARγ agonist D”), 4-[7-(1-adamantyl)-6-hydroxynaphthalen-2-yl]benzoic acid represented by the following formula (V) (CD1530: hereinafter also referred to as “RARγ agonist E”), esters of such compounds (RARγ agonists A, B, C, D, and E), and salts of such compounds (RARγ agonists A, B, C, D, and E). Preferred examples of the RARγ agonist include RARγ agonist A, RARγ agonist B, RARγ agonist C, esters of such compounds (RARγ agonists A, B and C) and salts of such compounds (RARγ agonists A, B and C).

[Chemical Formula 1]

[Chemical Formula 2]

[Chemical Formula 3]

[Chemical Formula 4]

[Chemical Formula 5]

.

Furthermore, other embodiments of the present invention include a method for treating corneal and conjunctival disorders characterized by administering the RARγ agonist of the present invention to a subject, the RARγ agonist of the present invention for use as a therapeutic agent for corneal and conjunctival disorders, and use of the RARγ agonist of the present invention in the preparation of a therapeutic agent for corneal and conjunctival disorders.

RARγ agonist A, its ester, and its salt, which is one of the active ingredients of the therapeutic agent for corneal and conjunctival disorders of the present invention, are known compounds described in Patent Document 2. They can be produced according to the method described in Patent Document 2 or purchased as commercially available products. Examples of commercially available products include the following product name: Parovatine, manufactured by Shanghai Haoyuan Chemexpress.

Among the active ingredients of the therapeutic agent for corneal and conjunctival disorders of the present invention, RARγ agonist B is a known compound described in Non-Patent Document 1 and Patent Document 4, RARγ agonist C is a known compound described in Patent Document 4, and RARγ agonists D and E are known compounds described in the following document (Shimono K. et al., Nat Med. 17(4): 454-460 (2011)). The compounds, esters or salts thereof can be produced according to conventional methods or purchased as commercially available products. Examples of commercially available products include the following product names: for RARγ agonist B, CD437 (ab141305) manufactured by Abcam and CD437 manufactured by Tocris Bioscience; for RARγ agonist C, BMS961 manufactured by Tocris Bioscience and CD1530 manufactured by Santa Cruz Biotechnology; for RARγ agonist E, CD1530 manufactured by Tocris Bioscience.

Among the esters of the above-mentioned RARγ agonists A, B, C, D, and E, which are active ingredients of the therapeutic agent for corneal and conjunctival disorders of the present invention, there are no particular limitations and may be any ester that is converted into the RARγ agonist A, B, C, D, or E in a reaction utilizing an enzyme or the like under physiological conditions in vivo. Such esters include esters formed by reaction with primary alcohols such as methanol, ethanol, propanol, hexanol, or dodecanol; esters formed by reaction with secondary alcohols such as isopropanol, sec-butanol, or 1-ethylpropanol; esters formed by reaction with tertiary alcohols such as tert-butanol or 1-methyl-1-ethylpropanol; and esters formed by reaction with amino alcohols such as 2-aminoethanol.

The above-mentioned esters can be produced from the above-mentioned RARγ agonists A, B, C, D, E or their synthetic intermediates by known methods.

The salts of the above-mentioned RARγ agonists A, B, C, D and E, which are active ingredients of the therapeutic agent for corneal and conjunctival disorders of the present invention, are not particularly limited and may be any pharmaceutically acceptable salts. Such salts include (1) inorganic acid salts such as hydrochlorides, hydrobromides, hydroiodides, nitrates, sulfates and phosphates as acid addition salts; and organic acid salts such as acetates, trifluoroacetates, benzoates, oxalates, malonates, succinates, maleates, fumarates, tartrates, citrates, methanesulfonates, ethanesulfonates, trifluoromethanesulfonates, benzenesulfonates, p-toluenesulfonates, glutamates and aspartates, and (2) metal salts such as sodium salts, potassium salts, calcium salts and magnesium salts as basic salts; inorganic salts such as ammonium salts; and organic amine salts such as triethylamine salts and guanidine salts.

In the present invention, a corneal or conjunctival disorder refers to a condition in which the cornea or conjunctiva is damaged due to various factors, such as abnormal tear production, metabolic abnormalities, or external injury. Examples of corneal or conjunctival disorders include corneal ulcers, corneal epithelial abrasions, keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosions, persistent corneal disorders, superficial punctate keratopathy, corneal epithelial defects, conjunctival epithelial defects, keratoconjunctivitis sicca, superior limbal keratoconjunctivitis, filamentous keratitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, and non-infectious conjunctivitis. The inhibitory effect on collagen degradation exerts an excellent ameliorative effect. Furthermore, in the present invention, corneal scarring (scarring on the cornea) and conjunctival scarring (scarring on the conjunctiva) associated with corneal or conjunctival disorders are also examples of corneal or conjunctival disorders. The inhibitory effect on collagen contraction exerts an excellent ameliorative effect.

The therapeutic agent for keratoconjunctival disorders of the present invention can be administered orally or parenterally (intravenously, intramuscularly, intraperitoneally, transdermally, intratracheally, intradermally or subcutaneously) in the following forms manufactured by mixing with appropriate pharmacologically acceptable additives: tablets, capsules, powders, syrups, granules, fine granules, pills, liquid preparations, suspensions, emulsions, transdermal absorption agents, suppositories, ointments (preferably eye ointments), lotions, inhalants or injections.

These preparations are produced by well-known methods by using additives such as excipients, lubricants, binders, disintegrants, emulsifiers, stabilizers, flavorings or diluents.

Examples of excipients include organic excipients and inorganic excipients. Examples of organic excipients include: sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; and pullulan. Examples of inorganic excipients include: light anhydrous silicic acid; and sulfates such as calcium sulfate.

Examples of lubricants include: stearic acid; metal salts of stearic acid such as calcium stearate and magnesium stearate; talc; colloidal silicon dioxide; waxes such as beeswax and spermaceti; boric acid; adipic acid; sulfates such as sodium sulfate; ethylene glycol; fumaric acid; sodium benzoate; D,L-leucine, sodium lauryl sulfate; silicic acid such as silicon dioxide and silicic acid hydrate; and starch derivatives among the above excipients.

Examples of the binder include hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyethylene glycol, and the compounds listed above as the excipients.

Examples of disintegrants include: cellulose derivatives such as hydroxypropylcellulose having a low degree of substitution, carboxymethylcellulose, carboxymethylcellulose calcium, and internally cross-linked carboxymethylcellulose calcium; cross-linked polyvinyl pyrrolidone; and chemically modified starch or cellulose derivatives such as carboxymethyl starch and sodium carboxymethyl starch.

Examples of emulsifiers include colloidal clays such as bentonite and magnesium aluminum silicate; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters and sucrose fatty acid esters.

Examples of the stabilizer include: parabens such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; acetic anhydride; and sorbic acid.

Examples of flavoring agents include: sweeteners such as saccharin sodium and aspartame; acidulants such as citric acid, malic acid, and tartaric acid; and flavorings such as lemon extract and orange extract.

The diluent is a compound generally used as a diluent. Examples of diluents include lactose, mannitol, glucose, sucrose, calcium sulfate, hydroxypropylcellulose, microcrystalline cellulose, water, ethanol, polyethylene glycol, propylene glycol, glycerol, starch, polyvinyl pyrrolidone, and mixtures thereof.

The therapeutic agents for keratoconjunctival disorders of the present invention include those in the form of instillations in addition to the above-mentioned dosage forms. These agents can be formulated using well-known methods by appropriately incorporating additives such as isotonicity agents, buffers, pH adjusters, solubilizers, thickeners, stabilizers, and preservatives (antimicrobial agents). Furthermore, a stable instillation can be obtained by adding a pH adjuster, thickener, dispersant, or the like to prepare a drug suspension.

Examples of isotonic agents include glycerol, propylene glycol, sodium chloride, potassium chloride, sorbitol, and mannitol.

Examples of buffering agents include phosphoric acid, phosphates, citric acid, acetic acid, and ε-aminocaproic acid.

Examples of the pH adjuster include hydrochloric acid, citric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, boric acid, borax, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, and sodium bicarbonate.

Examples of solubilizers include polysorbate 80, polyoxyethylene hydrogenated castor oil 60, and polyethylene glycol 4000.

Examples of thickeners and dispersants include: cellulose polymers such as hydroxypropylmethylcellulose and hydroxypropylcellulose; polyvinyl alcohol; and polyvinyl pyrrolidone. Further, examples of stabilizers include edetic acid and edetate sodium.

Examples of preservatives (antimicrobial agents) include commonly used sorbic acid, potassium sorbate, benzalkonium chloride, benzethonium chloride, methylparaben, propylparaben, and chlorobutanol. These preservatives can also be used in combination.

The instillation solution may have any pH within the acceptable range for ophthalmic preparations, but the pH is desirably set to 4.0-8.5.

For ointments (preferably eye ointments), common bases such as white petrolatum or liquid paraffin can be used for preparation.

The dosage of the keratoconjunctival disorder therapeutic agent of the present invention can be appropriately varied depending on the dosage form, the severity of symptoms, age, weight, physician's judgment, and the like of the patient to whom the agent is administered. For oral agents, 0.01-5000 mg, preferably 0.1-2500 mg, and more preferably 0.5-1000 mg, can generally be administered once or several times daily for adults. For instillations, these can be administered once or several times daily at an active ingredient concentration of 0.000001-10% (W/V), preferably 0.00001-3% (W/V), and more preferably 0.0001-1% (W/V). For ophthalmic ointments, these can be administered once or several times daily at an active ingredient concentration of 0.00001-10% (W/W), preferably 0.0001-3% (W/W), and more preferably 0.001-1% (W/W).

Hereinafter, the present invention is described in more detail while providing Examples (Test Examples and Formulation Examples). However, the scope of the present invention is not limited thereto.

(Example 1)

(Test Example) Test on Inhibition of Three-Dimensional Collagen Gel Degradation in Normal Rabbit Primary Corneal Cells by RARγ Agonist A

According to the method of Nishida et al. (Investigative Ophthalmology & Visual Science 42: 1247-1253 (2001)), normal rabbit corneal cells were used to evaluate the inhibitory effect of the test compound on the degradation of three-dimensional collagen gel.

Primary keratocytes collected from normal rabbit eyes were grown to confluence and detached from culture slides using 0.05% trypsin-EDTA. After washing in serum-free medium (Cat. No. 11095; Gibco), the cells were counted. The obtained primary keratocytes were mixed with Cellmatrix Type IA (Cat. No. 637-00653; Nitta Gelatin Inc.) and reconstitution buffer (Cat. No. 635-00791; Nitta Gelatin Inc.) and plated into 24-well plates at a final concentration of 1 × 10 ⁵ cells/well to prepare collagen gels.

After gel preparation, a dimethyl sulfoxide solution containing RARγ agonist A (R667) (R667 concentration: 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM) or a dimethyl sulfoxide solution without RARγ agonist as a control, as well as 10 ng (final concentration 10 ng/ml) IL-1β (Product No. 201-LB-005; R&D Systems) and 60 μg (final concentration 60 μg/ml) plasminogen (Product No. P9156; Sigma) as stimulators were added to MEM culture medium, and the culture medium was overlaid on the previously prepared collagen gel, and culture was initiated (at 37°C and 5% _CO2 ).

After 48 hours of incubation, the supernatant was ultrafiltered, 100 μl of concentrated hydrochloric acid was added and heated, and the collagen was hydrolyzed. The reaction solution after hydrolysis was dried under a nitrogen atmosphere using a Dry Thermo Uni (DTU-2C, Taitec Co. Ltd.) and an evaporating head (E1-20 Taitec Co. Ltd.), and then dissolved in 500 μl of ultrapure water. The amount of hydroxyproline in the solution, a collagen degradation product, was measured according to the method of Bergman et al. (Analytical Chemistry 35 (12): 1961-1965 (1963)) to evaluate the effect of RARγ agonist A in inhibiting collagen degradation. The results are shown in Figure 1.

In this test, RARγ agonist A showed a dose-dependent effect in inhibiting corneal and conjunctival collagen degradation.

(Preparation Example)

(Pharmaceutical Preparation Example 1) Infusion

In 100 ml

RARγ agonist A 100 mg

Sodium chloride 800 mg

Polysorbate 80 appropriate amount

Disodium hydrogen phosphate (appropriate amount)

Sodium dihydrogen phosphate appropriate amount

Sterile purified water in appropriate amount.

Add RARγ agonist A and the other components described above to sterile purified water. Mix the solution thoroughly to prepare an infusion. By varying the amount of RARγ agonist A and other components added, infusions with concentrations of 0.05% (W/V), 0.3% (W/V), 0.5% (W/V), or 1% (W/V) can be prepared.

(Pharmaceutical Preparation Example 2) Eye Ointment

In 100 g

RARγ agonist A 0.3 g

Liquid paraffin 10.0 g

White Vaseline in appropriate amount.

RARγ agonist A is added to uniformly melted white petrolatum and liquid paraffin. The mixture is thoroughly mixed and then gradually cooled to prepare an eye ointment. By varying the amount of added RARγ agonist A, etc., an eye ointment with a concentration of 0.05% (W/W), 0.1% (W/W), 0.5% (W/W), or 1% (W/W) can be prepared.

(Pharmaceutical Preparation Example 3) Tablet

In 100 mg

RARγ agonist A 1 mg

Lactose 66.4 mg

Corn starch 20 mg

Carboxymethylcellulose calcium 6 mg

Hydroxypropylcellulose 6 mg

Magnesium stearate 0.6 mg.

RARγ agonist A, corn starch, and lactose are mixed in a mixer. Carboxymethylcellulose calcium and hydroxypropylcellulose are added to the mixture for granulation. After drying, the particle size of the resulting granules is adjusted. Magnesium stearate is added and mixed with the adjusted granules, and the mixture is prepared into tablets using a tablet press. Furthermore, by varying the amount of RARγ agonist A added, tablets containing 0.1 mg, 10 mg, or 50 mg per 100 mg can be prepared.

Example 2

(Test Example) Test for Inhibition of Three-Dimensional Collagen Gel Contraction in Various Primary Cells Derived from Normal Rabbits by RARγ Agonist A

According to the method of Nishida et al., primary subconjunctival fibroblasts and primary keratocytes were used to evaluate the inhibitory effect of test compounds on the contraction of three-dimensional collagen gels.

As in Example 1, primary subconjunctival fibroblasts were grown and detached from culture slides. After washing, a cell suspension was prepared. The resulting suspension (1.1 × ¹⁰ cells/well in MEM), type I collagen solution (5 mg/ml), 10× MEM reconstitution buffer, and water were mixed on ice at a volume ratio of 0.2:7:1:1:1.8. 0.5 ml of this mixture was inoculated onto a culture dish coated with 1% BSA, and the culture was incubated at 37°C for one hour to prepare a collagen gel.

Subsequently, 0.5 ml of serum-free medium was added to each of the gels. TGF-β1 (R&D Systems) at a specific amount (1 ng/ml) and RARγ agonists at concentrations of 1 nM, 10 nM, and 100 nM were also added to the serum-free medium. The gels were incubated at 37°C, along with the gels to which the serum-free medium was added without reagent. Gel diameters were measured starting 24 hours later. The results of gel diameter measurements after 48 hours are shown in Figure 2.

The gel diameter was measured for primary keratocytes in a similar manner to that for primary subconjunctival fibroblasts. The results are shown in FIG3 .

As shown in Figures 2 and 3, using subconjunctival fibroblasts or keratocytes, RARγ agonist A not only inhibited collagen degradation but also suppressed TGF-induced collagen gel contraction. This demonstrates that RARγ agonists promote collagen turnover and have the potential to inhibit tissue remodeling, namely fibrosis or scarring, that occurs in ocular tissue following inflammation, hemorrhage, infection, surgery, or injury.

Example 3

(Test Example) Test for inhibition of three-dimensional collagen gel degradation in primary corneal cells derived from normal rabbits by RARγ agonist A, B or C

According to the method of Nishida et al., primary corneal cells were used to evaluate the inhibitory effect of the test compound on the degradation of three-dimensional collagen gel by the same method as in Example 1.

As in Example 1, primary corneal cells were grown and detached from culture slides. After washing, a cell suspension was prepared. The resulting suspension (1.1 × ¹⁰ cells/well in MEM), type I collagen solution (5 mg/ml), 10× MEM reconstitution buffer, and water were mixed on ice at a volume ratio of 0.2:7:1:1:1.8. 0.5 ml of this mixture was inoculated into a culture dish coated with 1% BSA, and the culture was incubated at 37°C for one hour to prepare a collagen gel.

After gel preparation, a dimethyl sulfoxide solution (1 nM) containing RARγ agonist A (R667), a dimethyl sulfoxide solution (1 nM, 10 nM) containing RARγ agonist B (CD437), a dimethyl sulfoxide solution (10 nM) containing RARγ agonist C (BMS961), or a dimethyl sulfoxide solution without RARγ agonist as a control, as well as 10 ng (final concentration 10 ng/ml) IL-1β (Product No. 201-LB-005; R&D Systems) and 60 µg (final concentration 60 µg/ml) plasminogen (Product No. P9156, Sigma Aldrich) as stimulators were added to MEM culture medium, and the culture medium was overlaid on the previously prepared collagen gel, and culture was initiated (at 37°C and ₅ % CO2).

After culturing for 48 hours, collagen was hydrolyzed, and the amount of hydroxyproline, a collagen degradation product, was measured by the same method as in Example 1 to evaluate the effect of each RARγ agonist on inhibiting collagen degradation. The results are shown in FIG4 .

In this test, not only RARγ agonist A, but also RARγ agonists B and C showed an effect of inhibiting corneal conjunctival collagen degradation.

Example 4

(Test Example) Test for Inhibition of Expression and Activation of MMP-1, 2, 3, and 9 by RARγ Agonist

The secretion or expression of proteases, matrix metalloproteinases (MMPs), is believed to be involved in the degradation of collagen type I. In this regard, inhibition of the expression and activation of MMP-1, 2, 3, and 9 by RARγ agonists was investigated.

Primary corneal cells collected from normal rabbit eyeballs were cultured in serum-free MEM medium for 24 hours. The RARγ agonist (R667) (R667 concentrations: 1 × ^10-6 µM, 1 × ^10-5 µM, 1 × ^10-4 µM, 1 × ^10-3 µM, 1 × ^10-2 µM) was added to the resulting culture solution, and pretreatment was performed for 12 hours. As a positive control, 10 nM dexamethasone (Dex), a synthetic steroid, was added and pretreated similarly. Subsequently, IL-1β (0.1 ng/ml) was added for stimulation, and the supernatant was collected 24 hours later. The collected culture solution was subjected to Western blot analysis and gelatin zymography analysis as described below.

(Western blot analysis)

After developing the collected culture supernatant on a 10% polyacrylamide gel using SDS-PAGE, the separated proteins were transferred to a nitrocellulose filter. Subsequently, nonspecific sites on the nitrocellulose filter were blocked and incubated with an anti-human MMP-1 antibody (R&D Systems) or an anti-rabbit MMP-3 antibody (Daiichi Fine Chemical Co., Ltd.) at 4°C for 24 hours. Detection was performed using ECL® reagent (GE Healthcare).

The results are shown in Figure 5A . The bands of inactive PRO-MMP-1, PRO-MMP-3, and active MMP-1 and MMP-3 were all weakened, and the amount thereof depended on the concentration of RARγ agonist A. Thus, it was revealed that RARγ agonist A inhibited the expression and activation of MMP-1 and MMP-3 in a concentration-dependent manner.

(Gelatin zymography)

The collected culture supernatant was developed on SDS-PAGE using a 10% polyacrylamide gel containing 0.1% gelatin and then incubated with a TBS solution containing 2.5% Triton X-100 at room temperature for one hour. The incubated gel was stained with Coomassie Brilliant Blue (Wako Pure Chemical Industries, Ltd.) and destained with a 5% methanol-7.5% acetic acid solution (Nacalai Tesque, Inc.).

The results are shown in Figure 5B . The bands of inactive PRO-MMP-2, PRO-MMP-9, and active MMP-2 and MMP-9 were all weakened, and the amount of each was dependent on the concentration of RARγ agonist A. Therefore, it was revealed that RARγ agonist A inhibited the expression and activation of MMP-2 and MMP-9 in a concentration-dependent manner.

Example 5

(Test Example) Test for Inhibition of Corneal Opacity and Ulcers Using RARγ Agonist A

Male Japanese white rabbits (weighing 2.5-3.5 kg; 27 rabbits) were given general anesthesia via intramuscular administration of a ketamine and xylazine mixture solution. Subsequently, local anesthesia was administered via instillation of 0.4% oxybuprocaine hydrochloride. Additionally, 30 µl of 1% LPS (Sigma Aldrich) was injected into the corneal stroma of one eye, while the other eye remained uninjected.

Using a micropipette, 50 µl of a 0.1% PBS/0.1% polysorbate 80 solution containing RARγ agonist A (R667: 0.1%, 24 mM) was administered instilled to rabbits injected with LPS twice after LPS injection on the day of injection, and then four times daily until day 10. As a control, a 0.1% PBS/0.1% polysorbate 80 solution (vehicle) containing no RARγ agonist A was similarly administered. Figure 6 shows representative examples of controls and cases in which RARγ agonist A was administered.

In Figure 6, corneal opacity and ulceration were observed in the control (vehicle) shown in the top row. However, in the case of administration of RARγ agonist A (0.1% R667) shown in the bottom row, corneal opacity and ulceration were not observed. Therefore, it was revealed that RARγ agonist A inhibited corneal opacity and ulceration.

Industrial Applicability

The RARγ agonist, which is an active ingredient in the therapeutic agent for corneal and conjunctival disorders of the present invention, can be used to prevent corneal and conjunctival disorders or as a therapeutic agent for corneal and conjunctival disorders by strongly inhibiting collagen degradation. The corneal and conjunctival disorders include, for example, corneal ulcers, corneal epithelial abrasions, keratitis, dry eye, conjunctivitis, chronic superficial keratitis, corneal erosion, persistent corneal disorders, superficial punctate keratopathy, corneal epithelial defects, conjunctival epithelial defects, keratoconjunctivitis sicca, superior limbal keratoconjunctivitis, filamentous keratitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, and non-infectious conjunctivitis. Furthermore, the RARγ agonist can also be used as a therapeutic agent for corneal scars or conjunctival scars associated with corneal and conjunctival disorders by strongly inhibiting collagen contraction.

Claims (37)

1. Use of (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, or an ester thereof or a salt thereof, in the preparation of a medicament for the prevention or reduction of scarring in the eyes of subjects with dry eye disease. 2. The use of claim 1, wherein the drug is in the form of a liquid formulation. 3. The use of claim 1, wherein the drug is in the form of a powder, syrup, granules, suspension, emulsion, ointment, or infusion. 4. The use of claim 3, wherein the ointment is an ophthalmic ointment. 5. The use of claim 4, wherein the concentration of the (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, or its ester or salt thereof, in the ophthalmic ointment is from 0.0001% to 3% (w/v). 6. The use of claim 5, wherein the concentration of the (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, or its ester or salt thereof, in the ophthalmic ointment is from 0.001% to 1% (w/v). 7. The use of claim 3, wherein the drug is in the form of an infusion. 8. The use of claim 7, wherein the concentration of the (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, or its ester or salt thereof, in the infusion is from 0.00001% to 3% (w/v). 9. The use of claim 8, wherein the concentration of the (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, or its ester or salt thereof, in the infusion is from 0.0001% to 1% (w/v). 10. The use of claim 9, wherein the concentration of the (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, or its ester or salt thereof, in the infusion is 0.3% (w/v), 0.5% (w/v), or 1% (w/v). 11. The use according to any one of claims 1-10, wherein the drug is formulated as a dose taken once or more daily. 12. Use of a RARγ agonist, or an ester or salt thereof, in the preparation of a medicament for the prevention or reduction of scarring in the eye of a subject, wherein said RARγ agonist is (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthyl... Acids, 3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl)acetylamino]benzoic acid, (2E)-3-(4-carboxyphenyl)-1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl)-2-propen-1-one oxime, 4-[7-(1-adamantyl)-6-hydroxynaphthyl-2-yl]benzoic acid, their esters or salts thereof. 13. The use of claim 12, wherein the scar formation in the eye is on the conjunctiva and/or cornea. 14. The use of claim 12, wherein the scarring in the eye is caused by external injury. 15. The use of claim 14, wherein the external injury is surgery. 16. Use of a RARγ agonist, or an ester thereof, or a salt thereof, in the preparation of a medicament for inhibiting collagen degradation and/or collagen contraction in the eye of a subject, wherein said RARγ agonist is (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2- Naphthoic acid, 3-fluoro-4-[2-hydroxy-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphtho-2-yl)acetylamino]benzoic acid, (2E)-3-(4-carboxyphenyl)-1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphtho-2-yl)-2-propen-1-one oxime, 4-[7-(1-adamantyl)-6-hydroxynaphtho-2-yl]benzoic acid, their esters or salts thereof. 17. Use according to any one of claims 12-16, wherein the subject suffers from keratoconjunctival disease. 18. The use of claim 17, wherein the corneal and conjunctival disease is selected from keratitis and conjunctivitis. 19. The use of claim 17, wherein the corneal and conjunctival disease is selected from corneal ulcer, corneal epithelial abrasion, dry eye syndrome, chronic superficial keratitis, corneal erosion, persistent corneal disease, superficial punctate keratitis, corneal epithelial defect, conjunctival epithelial defect, keratoconjunctivitis sicca, superior limbal keratoconjunctivitis, filamentous keratoconjunctivitis, infectious keratitis, non-infectious keratitis, infectious conjunctivitis, non-infectious conjunctivitis, corneal scar, and conjunctival scar. 20. The use of claim 19, wherein the corneal and conjunctival condition is dry eye syndrome. 21. The use of claim 19, wherein the corneal and conjunctival condition is keratoconjunctivitis sicca. 22. The use of claim 19, wherein the corneal and conjunctival condition is corneal scarring. 23. The use of claim 19, wherein the corneal and conjunctival lesion is a conjunctival scar. 24. The use of claim 12 or 16, wherein the RARγ agonist is (E)-4-(2-{3-[(1H-pyrazol-1-yl)methyl]-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthyl-2-yl}vinyl)benzoic acid, its ester, or a salt thereof. 25. The use of claim 24, wherein the drug is in the form of a liquid formulation. 26. The use of claim 24, wherein the drug is in the form of a powder, syrup, granules, suspension, emulsion, ointment or infusion. 27. The use of claim 26, wherein the ointment is an ophthalmic ointment. 28. The use of claim 27, wherein the concentration of the RARγ agonist, or its ester or salt, in the ophthalmic ointment is from 0.0001% to 3% (w/v). 29. The use of claim 28, wherein the concentration of the RARγ agonist, or its ester or salt, in the ophthalmic ointment is from 0.001% to 1% (w/v). 30. The use of claim 26, wherein the drug is in the form of an infusion. 31. The use of claim 30, wherein the concentration of the RARγ agonist, or its ester or salt, in the infusion is from 0.00001% to 3% (w/v). 32. The use of claim 31, wherein the concentration of the RARγ agonist, or its ester or salt, in the infusion is 0.0001% to 1% (w/v). 33. The use of claim 32, wherein the concentration of the RARγ agonist, or its ester or salt thereof, in the infusion is 0.3% (w/v), 0.5% (w/v), or 1% (w/v). 34. Use according to any one of claims 1, 12 or 16, wherein the drug is formulated as a dose taken once or more daily. 35. Use according to any one of claims 1, 12 or 16, wherein the drug comprises a solubilizer. 36. The use of claim 35, wherein the solubilizer is selected from polysorbate 80, polyoxyethylene hydrogenated castor oil 60, and polyethylene glycol 4000. 37. The use of claim 36, wherein the solubilizer is polysorbate 80.