Methods for treating corneal damage

Abstract

This specification discloses a method for treating corneal damage in a subject. Specifically, the method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt thereof to the subject, thereby improving symptoms associated with corneal damage. The compound is selected from the group consisting of the compound represented by formula (I), villavanol A, encainide, fenbutrazate, levonordefrine, permethrin, and 2,2,4,4-tetrahydroxybenzophenone (THBP). JPEG2026511459000028.jpg51102 Here, R1 is H, -SO3H, or The file is JPEG2026511459000029.jpg4639, R2 is H or The file is JPEG2026511459000030.jpg3651.

Description

Technical Field

[0001] The present disclosure relates generally to novel treatments for corneal injuries.

Background Art

[0002] The cornea is one of the transparent layer tissues that make up the eyeball. In humans, the cornea has a diameter of about 12 mm, a central thickness of about 0.5 mm, and a peripheral thickness of about 0.7 mm, and is composed of five layers in order from the surface: the corneal epithelium, Bowman's membrane, corneal stroma, Descemet's membrane, and corneal endothelium. Among the five layers that make up the cornea, corneal endothelial cells play a role in maintaining the water content and thickness of the cornea constant, thereby maintaining the transparency of the cornea. When the corneal endothelial cells are insufficient, corneal opacity occurs and vision is significantly reduced.

[0003] The cornea can be damaged physically (e.g., external force, ophthalmic surgery, laser, etc.), chemically (e.g., exposure to harmful chemicals), or biologically (e.g., microbial infection or certain corneal diseases). When a corneal injury deteriorates to a certain extent, conventional corneal transplantation therapy becomes inevitably necessary. However, although about 185,000 corneal transplants are performed annually in 116 countries and a total of 284,000 corneas are procured from 742 eye banks, it is far from sufficient to supply the estimated 12.7 million patients worldwide who need corneal transplants. Thus, factors such as the shortage of donors who can provide corneas for transplantation and the possible rejection reactions against donor-derived corneas limit the scope of application of corneal transplantation therapy.

[0004] On the other hand, common indications for which corneal transplantation therapy is required include keratoconus and corneal endothelial dysfunction, which account for more than 85% of cases. Keratoconus and corneal endothelial dysfunction usually occur after intraocular surgery, and that intraocular surgery causes corneal injury (i.e., a rapid loss of corneal endothelial cell density), which in turn worsens the conditions of keratoconus and corneal endothelial dysfunction. However, the response to damage of corneal endothelial cells is unclear, and preventing postoperative corneal dysfunction has become a major issue. [Overview of the project] [Problems that the invention aims to solve]

[0005] In light of the above situation, there is a need in related technological fields for novel treatment methods to treat corneal damage that improve corneal wound healing (e.g., after intraocular surgery) and prevent corneal dysfunction, thereby reducing the need for corneal transplant surgery. [Means for solving the problem]

[0006] One aspect of this disclosure aims to provide a novel treatment for corneal injury. Accordingly, as embodied and broadly described herein, this disclosure relates to a method for treating corneal injury in a subject. The method comprises administering an effective amount of a compound or a pharmaceutically acceptable salt thereof to the subject, the compound being selected from the group consisting of the compound represented by formula (I), villavanol A, encainide, fenbutrazate, levonordefrine, permethrin, and 2,2,4,4-tetrahydroxybenzophenone (THBP). [ka] Here, R1 is H, -SO3H, or [ka] And, R2 is H, or [ka] That is the case.

[0007] According to embodiments of this disclosure, in a compound represented by formula (I), R1 and R2 are independently H; R1 is -SO3H and R2 is H; R1 is H and R2 is [ka] is; or R1 is [ka] And R2 is H.

[0008] According to embodiments of this disclosure, the compound represented by formula (I) is in the form of a salt. In some embodiments, the compound represented by formula (I) is in the form of a fumarate. In other embodiments, the compound represented by formula (I) is in the form of a tartrate.

[0009] According to preferred embodiments of this disclosure, the corneal injury is corneal endothelial injury. In some embodiments, the corneal injury is caused by intraocular surgery. In other embodiments, the corneal injury is due to apoptosis during wound healing.

[0010] According to embodiments of the present disclosure, the compounds of the present disclosure may be administered in amounts ranging from about 0.1 ng / kg to about 1 μg / kg.

[0011] To help you understand, ophthalmic anti-infective agents can be used in combination with the compounds of the Disclosure in the manner of the Disclosure to improve corneal wound healing. The ophthalmic anti-infective agents can be administered to the subject before, concurrently with, or after the application of the compounds of the Disclosure. Non-limiting examples of ophthalmic anti-infective agents include chloramphenicol, azidamphenicol, tetracycline, chlortetracycline, oxytetracycline, dihydrostreptomycin, neomycin, flamycin, kanamycin, amikacin, tobramycin, gentamicin, netylmycin, micronomycin, erythromycin, natamycin, ampicillin, benzylpenicillin, polymyxin B, tyrotricin, rifamycin, fusidic acid, sulfamethizol, sulfafurazole, sulfadiclamide, sulfase Examples of compounds include, but are not limited to, tamide, sulfafenazole, idoxuridine, trifluridine, acyclovir, interferon, vidarabine, famciclovir, homivirsen, ganciclovir, nitrofural, vibrocator, resorcinol, sodium borate, hexamidine, chlorhexidine, sodium propionate, ofloxacin, norfloxacin, ciprofloxacin, dibromopropamidine, propamidine, picroxidine, lomefloxacin, povidone-iodine, levofloxacin, and gatifloxacin. Furthermore, to achieve better therapeutic effects, the compounds of this disclosure are administered to the subject intracavitaryally.

[0012] In all embodiments of this disclosure, the subjects suitable to receive treatment of the methods of this disclosure are mammals, preferably humans.

[0013] Many of the accompanying features and benefits of this disclosure will be better understood by referring to the following detailed description, which will be considered in conjunction with the attached drawings.

[0014] These and other features, aspects, and advantages of this disclosure will be better understood by referring to the following description, the appended claims, and the appended drawings. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows the response of corneal endothelial cells after laser ablation treatment according to an embodiment of the present disclosure. [Figure 2] Figure 2 shows the wound healing status of laser-damaged corneal endothelial cells after treatment with the compound of the present disclosure according to an embodiment of the present disclosure. [Figure 3] Figure 3 shows the density of laser-damaged corneal endothelial cells on the second day after treatment with the compound of the present disclosure according to another embodiment of the present disclosure, where * indicates P < 0.05.

Mode for Carrying Out the Invention

[0016] The detailed description provided below in connection with the accompanying drawings is intended to describe embodiments of the present disclosure and is not intended to represent the only form in which the embodiments of the present disclosure can be constructed or utilized. The foregoing description sets forth the functions of the embodiments and the order of steps for constructing and operating the embodiments. However, the same or equivalent functions and order can also be achieved by different embodiments.

[0017] I. Definitions For convenience, specific terms used in the specification, examples, and appended claims are summarized herein. Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have the meanings generally understood and used by those skilled in the art. Also, unless otherwise required by context, the singular form shall include the plural of the same thing, and the plural form shall be understood to include the singular form. Specifically, as used in this specification and the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Also, as used in this specification and the claims, the terms "at least one" and "one or more" have the same meaning and include 1, 2, 3, or more. The implementation of the present invention uses conventional techniques of cell biology and pharmacology within the technical scope of those skilled in the art unless otherwise indicated. Such techniques are well described in the literature.

[0018] Although the numerical ranges and parameters representing the broad scope of the present invention are approximate, the numerical values ​​shown in specific examples are reported as accurately as possible. However, any numerical value inherently contains some degree of error, which is inevitable from the standard deviation present in each test measurement. Furthermore, the term “about” as used herein generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” should be understood, as considered by those skilled in the art, to be modified by the term “about” in all examples, except in examples where it means within the acceptable standard error of the mean, or unless otherwise explicitly specified. This applies to all numerical ranges, quantities, values, and percentages such as quantities of material, durations of time, temperatures, operating conditions, and ratios of quantities disclosed herein. Accordingly, unless otherwise indicated, the numerical parameters shown in this disclosure and the appended claims are approximate values ​​that may vary as desired. At a minimum, each numerical parameter should be interpreted in light of the number of significant figures reported and by applying the usual rounding method.

[0019] As used herein, the term "cornea" refers to the transparent membrane on the surface of the central part of the eyeball, which has the structure to protect the eye from the outside, and to allow light to pass through and refract, thereby enabling vision.

[0020] As used herein, the term “corneal injury” refers to any part of the cornea that is damaged and may be treatable by the methods described herein. Parts of the cornea include, but are not limited to, the corneal epithelium, Bowman’s membrane, corneal stroma, Descemet’s membrane, or corneal endothelium. Damage can be caused by physical (e.g., trauma, intraocular surgery, contact lens use, ultraviolet (UV) radiation), chemical (e.g., exposure to chemicals), or biological (e.g., infection).

[0021] As used herein, the term “corneal disease” refers to all diseases or injuries that cause damage to the cornea, thereby resulting in loss of clear vision. Specifically, corneal disease may be any of the following, but is not limited to: dry eye syndrome, conjunctival hyperemia, neovascularization, and keratitis. It may also include any disease that may be accompanied by conjunctival hyperemia or that may result from neovascularization.

[0022] As used herein, the terms “treatment” and “treating” may refer to curative or palliative measures. In particular, as used herein, “treating” refers to applying the methods disclosed herein (or administration of any compound described in the methods disclosed herein) to a subject having corneal damage or symptoms associated with corneal damage, for the purpose of partially or completely alleviating, improving, reducing, delaying the onset, inhibiting the progression, reducing the severity, and / or reducing the incidence of one or more symptoms or characteristics of corneal damage.

[0023] The terms “administered,” “administering,” and “administration” are interchangeable herein and mean directly administering at least one compound described in the methods of this disclosure to a subject, or administering a pharmaceutical composition containing the same.

[0024] As used herein, the term “an effective amount” refers to the amount effective in a given dose and duration to achieve the desired therapeutic outcome in the treatment of corneal damage or conditions associated with corneal damage. For therapeutic purposes, an effective amount is one in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the component. A particular effective or sufficient amount will vary depending on the specific condition being treated, the patient’s physical condition (e.g., size, age, or sex), the type of mammal or animal being treated, the duration of treatment, the nature of any concurrent therapies, the specific formulation employed, and the specific route of administration, as well as similar factors within the scope of the healthcare professional’s knowledge and expertise. An effective amount may be expressed, for example, in grams, milligrams, or micrograms, or in milligrams per kilogram of body weight (mg / kg). Alternatively, an effective amount may be expressed in terms of the concentration of the active component (e.g., the compound described in the method of this disclosure), such as molar concentration, mass concentration, volume concentration, molality, mole fraction, mass fraction, and mixing ratio. A person skilled in the art can calculate the human equivalent dose (HED) of a pharmaceutical (compound described in the method of this disclosure) based on doses determined from animal models. For example, when estimating the maximum safe dose for use in human subjects, one can follow the industry guidance published by the U.S. Food and Drug Administration (FDA) entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers."

[0025] The term “pharmaceutically acceptable salt” as used herein refers to a salt formed by the interaction of a pharmaceutically acceptable acid (e.g., a compound represented by formula (I) of this disclosure) with a base, including organic or inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, methylsulfonic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, carbonic acid, cinnamic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, salicylic acid, p-aminosalicylic acid, 2-phenoxybenzoic acid, and 2-acetoxybenzoic acid. In one preferred example, the compound represented by formula (I) is in the form of a fumarate (e.g., formoterol fumarate). In another example, the compound represented by formula (I) is in the form of a tartrate (e.g., formoterol tartrate or alformoterol tartrate).

[0026] The terms “subject” or “patient” refer to animals, including human species, that can be treated by the methods of this disclosure. Unless the context specifically indicates one sex, the terms “subject” or “patient” are intended to refer to both males and females. Thus, the terms “subject” or “patient” include any mammal that could benefit from the treatment of corneal damage or conditions associated with corneal damage. Examples of “subject” or “patient” include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cattle, horses, dogs, cats, birds, and poultry. In one exemplary embodiment, the patient is human.

[0027] As used herein, the phrase “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material, that is involved in transporting or delivering the pharmaceutical product in question from one organ (or part of the body) to another organ (or part of the body). Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation. The pharmaceutical formulation comprises at least one compound described in the methods of this disclosure in combination with one or more pharmaceutically acceptable components. For clinical use of the methods of the present invention, the pharmaceutical compositions of the present invention are formulated into formulations suitable for the intended route of administration.

[0028] II. Detailed Description of the Invention The inventors of this disclosure unexpectedly discovered that a series of compounds individually possess the ability to suppress apoptosis of corneal endothelial cells. Therefore, these compounds are potential candidates for developing pharmaceuticals to treat (manage) corneal damage or corneal disease.

[0029] 1. Compounds of the present disclosure and pharmaceutical compositions containing the same Accordingly, a first aspect of the present disclosure relates to compounds or pharmaceutically acceptable salts thereof that have the ability to suppress corneal decompensation, and these compounds or pharmaceutically acceptable salts thereof may be suitable for treating corneal damage. The compounds may be any one of the compounds represented by formula (I), villavanol A, encainide, fenbutrazate, levonordefrine, permethrin, or 2,2,4,4-tetrahydroxybenzophenone (THBP), [ka] Here, in the compound represented by formula (I), R1 is H, -SO3H, or [ka] And, R2 is H, or [ka] That is the case.

[0030] According to some embodiments, in the compound represented by formula (I), R1 and R2 are independently H. Therefore, the compound has the structure of formula (I)-1 (formoterol). [ka]

[0031] In other embodiments, in the compound represented by formula (I), R1 is -SO3H and R2 is H. Therefore, the compound has the structure of formula (I)-2 (formoterol sulfate). [ka]

[0032] In further embodiments, in a compound represented by formula (I), R1 is H and R2 is [ka] Therefore, the compound has the structure of formula (I)-3 (formoterol benzyl glucuronide, formoterol BG). [ka]

[0033] In yet another embodiment, in a compound represented by formula (I), R1 is [ka] And R2 is H, and therefore the compound has the structure of formula (I)-4 (formoterol phenol glucuronide, formoterol PG). [ka]

[0034] Preferably, the compound represented by formula (I) is in the form of a salt, such as a fumarate or tartrate. In some embodiments, the compound represented by formula (I) is in the form of a fumarate (e.g., formoterol fumarate). In other embodiments, the compound represented by formula (I) is in the form of a tartrate (e.g., formoterol tartrate).

[0035] To your understanding, while the compounds described above may be administered as single compounds to treat corneal damage, they are generally administered in the form of pharmaceutical compositions comprising one or more of the above compounds with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition may comprise two of the above compounds (e.g., formoterol and vilavanol A; formoterol and encainide; vilavanol A and encainide; etc.). In some embodiments, the pharmaceutical composition may comprise three of the above compounds (e.g., formoterol, vilavanol A, and encainide; formoterol, levonordefrine, and THBP; levonordefrine, THBP, and vilavanol A; levonordefrine, THBP, and encainide; etc.).

[0036] Optionally, a pharmaceutical composition comprising one or more compounds of the present disclosure may be administered alone or in combination with other therapeutic agents (e.g., ophthalmic anti-infective agents). Suitable ophthalmic anti-infective agents that may be used with the pharmaceutical composition include antibiotics (e.g., chloramphenicol, azidamphenicol, tetracycline, chlortetracycline, oxytetracycline, dihydrostreptomycin, neomycin, flamycetin, kanamycin, amikacin, tobramycin, gentamicin, netylmycin, micronomycin, erythromycin, natamycin, ampicillin, benzylpenicillin, polymyxin B, tyrotricin, rifamycin, or fusidic acid); sulfonamides (e.g., sulfamethizol, sulfafurazole, sulfadiclamide, sulf) This includes, but is not limited to, acetamide or sulfafenazole; antiviral agents (e.g., idoxuridine, trifluridine, acyclovir, interferon, vidarabine, famciclovir, fomivirsen, or ganciclovir); or other pharmaceuticals (e.g., nitrofural, vibrocator, resorcinol, sodium borate, hexamidine, chlorhexidine, sodium propionate, ofloxacin, norfloxacin, ciprofloxacin, dibromopropamidine, propamidine, picroxidine, lomefloxacin, povidone-iodine, levofloxacin, or gatifloxacin).

[0037] Pharmaceutical compositions comprising one or more compounds of the Disclosure may be in any dosage form at the time of provision or storage, depending on their intended use and a suitable pharmaceutically acceptable carrier selected to achieve such use. Pharmaceutical compositions comprising one or more compounds of the Disclosure may be in solid, semi-solid, or liquid dosage forms, gels, creams, etc. Pharmaceutical compositions comprising one or more compounds of the Disclosure may generally be administered as oral preparations such as tablets, capsules, powders, granules, pills, syrups, eye drops, eye ointments, transdermal preparations, ophthalmic injections, or sprays. Of these, topical preparations for the eye, including eye drops, eye ointments, ophthalmic injections, and sprays, are preferred, with eye drops and sprays being more preferred because they can be easily administered topically to the eye with minimal invasiveness.

[0038] Conventional bases such as white petrolatum and liquid paraffin can be prepared as ophthalmic ointments. Eye drops can be prepared using, as needed, isotonic agents (e.g., sodium chloride, potassium chloride, glycerol, propylene glycol, etc.); buffering agents (e.g., sodium phosphate, sodium acetate, sodium borate, sodium carbonate, etc.); surfactants (e.g., polyoxyethylene sorbitan fatty acid ester, polyoxyl stearate 40, polyoxyethylene polyoxypropylene glycol, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, etc.); stabilizers (e.g., disodium edetate, sodium citrate, etc.); preservatives (e.g., benzalkonium chloride, sorbic acid, methyl parahydroxybenzoate, etc.); thickeners (e.g., methylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, etc.); antioxidants (e.g., ascorbic acid, tocopherol, etc.). The pH may be within an acceptable range for ophthalmic formulations, preferably in the range of 4 to 8. To adjust the pH, hydrochloric acid, phosphoric acid, citric acid, sodium hydroxide, potassium hydroxide, sodium carbonate, etc., can be used.

[0039] Generally, one or more compounds of the present disclosure are present in a pharmaceutical composition at a level of about 0.01% to 99% by weight, based on the total weight of the pharmaceutical composition. In some embodiments, one or more compounds of the present disclosure are present at a level of at least 0.1% by weight, based on the total weight of the pharmaceutical composition. In certain embodiments, one or more compounds of the present disclosure are present at a level of at least 1% by weight, based on the total weight of the pharmaceutical composition. In other embodiments, one or more compounds of the present disclosure are present at a level of at least 5% by weight, based on the total weight of the pharmaceutical composition. In yet another embodiment, one or more compounds of the present disclosure are present at a level of at least 10% by weight, based on the total weight of the pharmaceutical composition. In yet another embodiment, one or more compounds are present at a level of at least 25% by weight, based on the total weight of the pharmaceutical composition.

[0040] 2.Treatment method Therefore, the Disclosure also includes methods for treating corneal damage in subjects requiring such treatment. These methods include administering an effective amount of the pharmaceutical composition of the Disclosure to the subject.

[0041] The corneal injuries treatable by the compounds, pharmaceutical compositions, and / or methods of this disclosure are caused by intraocular surgery, but are not limited to those caused by intraocular surgery. Because the compounds of this disclosure inhibit cellular apoptosis and promote wound healing, the pharmaceutical compounds of this disclosure are useful for treating any corneal injuries involving the processes described above, regardless of their location or cause.

[0042] According to the method of this disclosure, the pharmaceutical composition of this disclosure is administered to the subject in an amount of approximately 0.1 pg to 100 mg (0.1 pg / kg to 100 mg / kg) per kg of body weight of the subject, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 ,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,150,200,250,300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000pg / kg (=1ng / kg), 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 5 3, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000ng / kg(=1μg / kg)、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1,000μg / kg(=1mg / kg),2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,Alternatively, it is administered in an amount of 100 mg / kg. Preferably, it is about 10 pg to 1 mg per kg of body weight of the subject (10 pg / kg to 1 mg / kg), more preferably about 0.1 ng to 1 μg per kg of body weight of the subject (0.1 ng / kg to 1 μg / kg), and even more preferably about 2 ng to 30 ng per kg of body weight of the subject (2 ng / kg to 30 ng / kg). In some examples, the pharmaceutical compositions of this disclosure are administered to subjects in amounts of approximately 2.748 ng / kg (for levonordefrine), approximately 3.75375 ng / kg (for THBP), approximately 5.166 ng / kg (for formoterol), approximately 6.3675 ng / kg (for formoterol sulfate), approximately 7.8075 ng / kg (for formoterol PG), approximately 7.8075 ng / kg (for formoterol BG), approximately 23.8875 ng / kg (for vilavanol A), approximately 5.5125 ng / kg (for fenbutrazate), approximately 5.8695 ng / kg (for permethrin), and approximately 5.2875 ng / kg (for encainide). The dose may be administered as a single dose or alternatively as multiple doses. Those skilled in the art or clinicians may adjust the dose or regimen according to the patient's physical condition or the severity of the disease. ,

[0043] Those skilled in the art or healthcare professionals can adjust the administration regimen of the pharmaceutical compositions of this disclosure in accordance with various factors such as age, sex, weight, and other treatments (if any). For example, the pharmaceutical compositions of this disclosure may be administered to a subject 1 to 7 times per week (e.g., 1, 2, 3, 4, 5, 6, or 7 times per week) over 1, 2, 3, 4, or more consecutive weeks. Alternatively, the pharmaceutical compositions of this disclosure may be administered to a subject 1 to 10 times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) every 2 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks, or every month, every 2 months, every 3 months, or longer intervals. Preferably, the pharmaceutical composition of the Disclosure is administered to a subject daily for at least one day, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 days or longer. More preferably, the pharmaceutical composition of the Disclosure is administered to a subject daily for at least 28 days (i.e., 4 weeks). More preferably, the pharmaceutical composition of the Disclosure is administered to the subject daily for at least 14 days (i.e., 2 weeks). According to one embodiment of the Disclosure, in order to produce a therapeutic effect, the pharmaceutical composition of the Disclosure is administered to the subject only once a day for the entire duration of treatment (i.e., 2 days).

[0044] Optionally, the methods of the Disclosure include further administration of an ophthalmic anti-infective agent to a subject in combination with the administration of the pharmaceutical composition of the Disclosure. Examples of ophthalmic anti-infective agents are as described above and may be formulated in pharmaceutical compositions or pharmaceutical kits together with the compounds used in the methods of the Disclosure. The ophthalmic anti-infective agent may be administered to the subject before, simultaneously with, or after the application of the pharmaceutical composition of the Disclosure, at different doses, at time intervals, and via the same or different routes. The dose and time interval may vary due to the factors described above and depend on the professional judgment of the healthcare professional. The routes may be oral, enteral, buccal, nasal, percutaneous, transmucosal, intravenous, intraperitoneal, intra-arterial, intradermal, subcutaneous, intramuscular, and anterior chamber routes. Preferably, the route of administration suitable for use in the methods of the Disclosure is the anterior chamber route, and the active compound of the Disclosure is administered anteriorly in the form of eye drops or by direct anterior chamber injection.

[0045] In general, the subjects treatable by the methods of this disclosure are mammals, and preferably, humans.

[0046] The following examples are provided to illustrate specific aspects of the present invention and to assist those skilled in the art in carrying it out. These examples should not be considered to limit the scope of the present invention in any way. Without further detail, those skilled in the art will be able to make the most of the present invention based on the description herein. All publications cited herein are incorporated herein by reference in their entirety. [Examples]

[0047] Materials and methods 1. Pharmaceutical preparation Levonordefrine (Sigma), formoterol (Santa Cruz), formoterol sulfate (Synzil), formoterol BG (Synzil), formoterol PG (Synzil), 2,2,4,4-tetrahydroxybenzophenone (THBP) (Sigma), permethrin (Sigma), villavanol A (Sigma), fenbutrazate (Santa Cruz), or encainide (Sigma) were dissolved in DMSO (Sigma) to obtain a stock solution with a final concentration of 10 mM, which was then stored at -20°C for later use. To prepare the working solution, each stock solution was diluted with equilibrium salt solution (BSS) (Alcon) before anterior chamber injection to obtain the desired working concentration.

[0048] 2. Mouse strain nT / nG transgenic mice (Jax #023035; Jackson Laboratory) possess a Cre reporter allele containing nuclear-targeted tandem-dimer tomato (tdTomato; orange fluorescent protein) and enhanced green fluorescent protein (EGFP) (Prigge, Wiley et al., 2013; “Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei.”). These mice were used in this study to investigate cell density and healing processes in the corneal endothelium. The mice were characterized by broad fluorescence expression of tdTomato (localized to the cell nucleus) before Cre recombination, and detection of nuclear-localized green fluorescence after Cre recombination. All procedures involving the mice were approved by and followed the guidelines of the Animal Ethics Committee of National Taiwan University.

[0049] 3. Animal bioimaging For in vivo imaging, mice were anesthetized by intramuscular injection of 50-80 mg / kg of zoletil (Virbac). The corneal surface was anesthetized with 0.4% oxybuprocaine hydrochloride (Sigma) dissolved in physiological saline. Following a previous report (Wu, Wang et al., 2019; “Intravital multiphoton microscopic imaging platform for ocular surface imaging.”), an ophthalmic holder was used to minimize artifacts during in vivo imaging. In vivo imaging was performed using a laser wavelength of 880 nm to excite tdTomato at a laser output of approximately 35 mW on the sample surface to optimize image quality without causing photobleaching. After the laser light was reflected by a main dichroic mirror (Semloc), the laser light was focused onto the eye using a water-immersion 20x / NA1.0 objective lens (Olympus). The tdTomato signal was spectrally separated using a 585 / 40 nm bandpass filter (Semloc). To prevent drying of the ocular surface during imaging, an ophthalmic gel with a refractive index of 1.338 (Vidisic gel, Dr. Gerhardmann Chem-Firm) was used as an immersion medium on the cornea. Image stacks were acquired along the z-axis with a step size of 1 μm, and the dimensions of the images acquired in this study were 512 × 512 pixels.

[0050] 4. Corneal endothelial cell ablation To remove corneal endothelial cells, cell ablation was induced using a multiphoton femtosecond laser. This was due to its ability to precisely control cell ablation temporally and spatially while minimizing damage to adjacent tissues (Rompolas, Deschene et al., 2012; “Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration.”). After identifying the corneal endothelial cells to be removed, laser ablation was performed on a selected area (50 μm × 50 μm) in a single scan at a laser wavelength of 880 nm and a laser power of 500 mW, with repeated laser adjustments along the z-axis to achieve optimal results.

[0051] 5. Immunofluorescence assay After laser ablation, the excised eyeballs were fixed overnight at 4°C in 4% paraformaldehyde solution (PFA) (Santa Cruz), and then washed with PBS (Protec). Next, the corneas were excised from the eyeballs under a dissecting microscope and blocked overnight at 4°C with 5% BSA (Biobasic) / PBS containing 1% Triton-X 100 (VWR). The corneas were incubated overnight at 4°C in blocking buffer with N-cadherin antibody (1:200, Cell Signaling), and then stained overnight at 4°C with conjugated secondary antibody AlexaFluor 647 (1:500, Jackson ImmunoResearch). Finally, apoptotic cells were detected using the TUNEL kit (Promega), and the stained corneas were mounted using VECTASHIELD anti-fade mounting medium (Vector). All confocal images were acquired using an SP8 confocal microscope (Leica).

[0052] 6. Intracavitary drug delivery Following laser ablation, the aforementioned drug was immediately injected into the anterior chamber of the mouse eye using a customized needle attached to a 5 μl syringe (Hamilton). To avoid movement, a cotton swab was used to support the eyeball during the anterior chamber injection in the peripheral corneal region. A single dose of 0.5 μl of a different drug was slowly applied to each eye. A non-toxic fluorescein dye was used concurrently to ensure that the drug remained in the aqueous humor without leakage.

[0053] 7. Cell density quantification Two days after laser ablation, mice were anesthetized and euthanized after receiving the indicated treatment (as shown in Figures 1-3). Their eyeballs were extracted and fixed overnight in 4% paraformaldehyde (PFA) at 4°C. After dissecting the cornea from the eyeball, the cornea was cut into four lobes, placed on a glass slide, mounted with mounting medium (vector), and fixed with a coverslip. All fluorescence images were acquired using a Leica SP8 confocal microscope with a 20× objective lens set to 1 μm steps per z section. Cell nuclei were manually segmented using an Imaris (Oxford Instruments) spot plug-in. For each sample, to quantify cell density under different pharmaceutical treatments, a 300 μm diameter circle area was selected and the number of cells was counted.

[0054] 8.Statistical analysis All experimental data were processed using Prism and expressed as mean ± SEM. Statistical significance of differences between experimental groups was assessed by Student's t-test. A p-value < 0.05 was considered statistically significant.

[0055] Example 1. The compounds of the present disclosure improved the healing of laser-damaged corneas.

[0056] 1.1 Establishment of an animal model of laser-damaged cornea This study investigated the effects of the compounds disclosed herein on damaged corneas. For this purpose, an animal model of corneal injury using a laser to simulate injury caused by external forces such as ophthalmic surgery was first established in this study following the procedure described in the "Materials and Methods" section. Laser ablation treatment caused loss of corneal endothelial cells in the treated area, as evidenced by the absence of N-cadherin staining (data not shown). Furthermore, TUNEL staining of damaged cells showed a significant increase, suggesting that corneal endothelial cells underwent apoptosis after laser treatment (Figure 1).

[0057] 1.2 Intracavitary injection of pharmaceuticals The test compounds were administered intrachorally to mice after corneal laser ablation, and corneal opacity was initially evaluated via macroscopic corneal photography. The cornea was transparent before and after laser ablation, but holes approximately 0.1–0.2 mm in diameter were observed immediately after intrachorally administered injection (data not shown). On day 2, only the BSS group showed mild corneal inflammation and irritation. In contrast, all test drugs, including levonordefrine, formoterol, formoterol sulfate, formoterol BG, formoterol PG, THBP, permethrin, vilavanol A, fenbutrazate, and encainide, maintained a moist and transparent ocular surface. The fenbutrazate group, however, showed the formation of slightly hazy spots. All of the above observations are summarized in Table 1. Overall, all test drugs were found to be suitable for intrachorally administered injection without causing significant adverse effects on the ocular surface.

[0058] [Table 1]

[0059] 1.3 Promotion of corneal wound healing The efficacy of the investigational drugs that promote corneal wound healing was investigated by multiphoton microscopy analysis, which tracked changes at the wound site after drug injection. On day 1 after laser ablation, corneal endothelial cells in the BSS group were unable to close the wound defect, and the impaired integrity of the corneal endothelium persisted until day 2 (Figure 2). In contrast, intrachorally administered 6 μM (27.48 ng / kg) levonordefrine and 6 μM (37.5375 ng / kg) THBP promoted wound healing and closed the defect on day 1. On the other hand, intrachorporeal injections of 6 μM (51.66 ng / kg) formoterol, 6 μM (63.675 ng / kg) formoterol sulfate, 6 μM (78.075 ng / kg) formoterol prostaglandin, 6 μM (78.075 ng / kg) formoterol butylene glycol, 30 μM (238.875 ng / kg) vilavanol A, 6 μM (55.125 ng / kg) fenbutrazate, 6 μM (58.695 ng / kg) permethrin, and 6 μM (52.875 ng / kg) encainide promoted wound healing on day 2. The aforementioned medications promoted wound healing to varying degrees. All of them promoted corneal endothelial wound healing within 2 days.

[0060] 1.4 Protection from damage to corneal endothelial cells The protective effect of the compounds of this disclosure on corneal endothelial cells against injury was evaluated by counting the number and / or density of corneal endothelial cells, rather than corneal cells in the corneal stroma or other cell populations in the trabecular meshwork. The results are shown in Figure 3 and Table 2. The pharmaceuticals were found to protect corneal endothelial cells in the following order: formoterol sulfate > formoterol PG > formoterol BG > formoterol > THBP > permethrin > fenbutrazate > levonordefrine > encainide > vilavanol A. Overall, the pharmaceuticals described herein were demonstrated to have the potential to promote primary wound healing and protect corneal endothelial cells from injury.

[0061] [Table 2]

[0062] In summary, this disclosure provides a novel method for treating corneal injury by promoting corneal wound healing and preventing corneal dysfunction through the use of one or more of the pharmaceuticals described above, thereby reducing the need for corneal transplant surgery.

[0063] The above description of embodiments is given for illustrative purposes only, and it will be understood that various modifications can be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of exemplary embodiments of the present invention. Various embodiments of the present invention are described above with some specificity or by reference to one or more individual embodiments, but those skilled in the art can make numerous modifications to the disclosed embodiments without departing from the spirit or scope of the present invention.

Claims

1. The use of a compound or a pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical for the treatment of corneal damage, wherein the compound is selected from the group consisting of the compound represented by formula (I), vilavanol A, encainide, fenbutrazate, levonordefrine, permethrin, and 2,2,4,4-tetrahydroxybenzophenone (THBP), 【Chemistry 1】 Here, R １ H, -SO ３ H, or 【Chemistry 2】 And, R ２ is H, or 【Transformation 3】 It is used.

2. In the compound represented by formula (I), R １ and R ２ H is independent of H; R １ Ha-SO ３ H and R ２ is H; R １ is H, and R ２ is 【Chemistry 4】 is; or R １ teeth 【Transformation 5】 And R ２ The use described in claim 1, wherein is H.

3. The use according to claim 2, wherein the compound represented by formula (I) is in the form of a tartrate or fumarate.

4. The use according to claim 1, wherein the corneal injury is corneal endothelial injury.

5. The use according to claim 1, wherein the corneal damage is caused by intraocular surgery or by apoptosis during wound healing.

6. The use according to claim 1, wherein the pharmaceutical product is for anterior chamber administration.

7. The use according to claim 5, wherein the pharmaceutical product is in the form of eye drops or an injectable solution.

8. The use according to claim 1, wherein the pharmaceutical product is to be administered before, simultaneously with, or after an ophthalmic anti-infective agent.

9. The aforementioned ophthalmic anti-infective drugs include chloramphenicol, azidamphenicol, tetracycline, chlortetracycline, oxytetracycline, dihydrostreptomycin, neomycin, flamycetin, kanamycin, amikacin, tobramycin, gentamicin, netylmycin, micronomycin, erythromycin, natamycin, ampicillin, benzylpenicillin, polymyxin B, tyrotricin, rifamycin, fusidic acid, sulfamethizol, sulfafurazole, sulfaziclamide, sulfacetamide, and sulf The use according to claim 8, selected from the group consisting of afenazole, idoxuridine, trifluridine, acyclovir, interferon, vidarabine, famciclovir, homivirsen, ganciclovir, nitrofural, vibrocator, resorcinol, sodium borate, hexamidine, chlorhexidine, sodium propionate, ofloxacin, norfloxacin, ciprofloxacin, dibromopropamidine, propamidine, picroxidine, lomefloxacin, povidone-iodine, levofloxacin, and gatifloxacin.

10. A method for treating corneal damage in a subject, comprising administering an effective amount of a compound or a pharmaceutically acceptable salt thereof to the subject, wherein the compound is selected from the group consisting of a compound represented by formula (I), vilavanol A, encainide, fenbutrazate, levonordefrine, permethrin, and 2,2,4,4-tetrahydroxybenzophenone (THBP), 【Transformation 6】 Here, R １ H, -SO ３ H, or 【Transformation 7】 And, R ２ is H, or 【Transformation 8】 The method.

11. In the compound represented by formula (I), R １ and R ２ H is independent of H; R １ Ha-SO ３ H and R ２ is H; R １ is H and R ２ teeth 【Chemistry 9】 is; or R １ teeth 【Chemistry 10】 And R ２ The method according to claim 10, wherein is H.

12. The method according to claim 11, wherein the compound represented by formula (I) is in the form of a tartrate or fumarate.

13. The method according to claim 10, wherein the corneal injury is corneal endothelial injury.

14. The method according to claim 10, wherein the corneal injury is caused by intraocular surgery.

15. The method according to claim 10, wherein the corneal damage is caused by apoptosis during wound healing.

16. The method according to claim 10, wherein the compound is administered to the subject in an amount of about 0.1 ng / kg to 1 μg / kg.

17. The method according to claim 10, wherein the compound is administered to the subject intracavitaryally.

18. The method according to claim 17, wherein the compound is administered in the form of eye drops or by direct injection.

19. The method according to claim 10, wherein the subject is a human.

20. The method according to claim 10, further comprising administering an ophthalmic anti-infective agent to the subject before, simultaneously with, or after the administration of the compound.

21. The aforementioned ophthalmic anti-infective drugs include chloramphenicol, azidamphenicol, tetracycline, chlortetracycline, oxytetracycline, dihydrostreptomycin, neomycin, flamycetin, kanamycin, amikacin, tobramycin, gentamicin, netylmycin, micronomycin, erythromycin, natamycin, ampicillin, benzylpenicillin, polymyxin B, tyrotricin, rifamycin, fusidic acid, sulfamethizol, sulfafurazole, sulfaziclamide, sulfacetamide, and sulf The method according to claim 20, wherein a substance selected from the group consisting of afenazole, idoxuridine, trifluridine, acyclovir, interferon, vidarabine, famciclovir, homivirsen, ganciclovir, nitrofural, vibrocator, resorcinol, sodium borate, hexamidine, chlorhexidine, sodium propionate, ofloxacin, norfloxacin, ciprofloxacin, dibromopropamidine, propamidine, picroxidine, lomefloxacin, povidone-iodine, levofloxacin, and gatifloxacin.

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