Topical medications to prevent or reduce corneal scarring

Abstract

The present invention relates to compositions, systems, and methods for treating a subject with corneal injury and / or existing corneal scarring using a composition comprising an ACE-2 receptor antagonist (e.g., losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, or the losartan metabolite EXP3174. In certain embodiments, the ACE-2 receptor antagonist is present in the composition at a concentration of about 0.2 mg / ml to 0.9 mg / ml or about 0.1 mg / ml to 2.0 mg / ml.

Description

[Technical field]

[0001] Federal Funding Statement This invention was made with Government support under W81XWH-19-1-0846 awarded by the Department of Defense (CDMRP). The U.S. Government has certain rights in this invention.

[0002] Sequence Listing The text of the computer readable sequence listing entitled "39765-601_SEQUENCE_LISTING", generated on August 30, 2022, and having a file size of 1,929 bytes, is submitted herewith and is incorporated by reference in its entirety herein.

[0003] The present invention relates to compositions, systems, and methods for treating a subject with corneal injury and / or existing corneal scarring using a composition comprising an ACE-2 receptor antagonist (e.g., losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, or the losartan metabolite EXP3174. In certain embodiments, the ACE-2 receptor antagonist is present in the composition at a concentration of about 0.2 mg / ml to 0.9 mg / ml or about 0.1 mg / ml to 2.0 mg / ml. [Background technology]

[0004] Corneal cicatricial fibrosis mediated by myofibroblast development after trauma, microbial infection, scarring disease, and some corneal surgery is one of the most important causes of vision loss in the United States and worldwide (Witcher et al., 2001). According to WHO statistics, 5.1% of bilateral blindness is corneal blindness, with stromal scarring being the largest subcategory (Witcher et al., 2001). Corneal opacity due to bacterial keratitis or trauma is also a common reason for corneal transplantation in the United States (Ghosheh et al., 2008). Persistent corneal scarring due to trauma, infection, disease, or surgery occurs in humans through the same myofibroblast-related mechanism (Cockerham and Hidayat, 1999; Lee et al., 2001) as in rabbits, mice, rats, chickens, and other species (Mohan et al., 2003; Netto et al., 2006; Martinez-Garcia, et al., 2006; Mohan et al., 2008; Hindman et al., 2019; Joung et al., 2020; de Oliveira et al., 2021). Summary of the Invention

[0005] The present invention relates to compositions, systems, and methods for treating a subject with corneal injury and / or existing corneal scarring using an ACE-2 receptor antagonist (e.g., losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, or the losartan metabolite EXP3174), or a composition comprising another ACE-2 receptor antagonist. In certain embodiments, the ACE-2 receptor antagonist is present in the composition at a concentration of about 0.2 mg / ml to 0.9 mg / ml or about 0.1 mg / ml to 3.0 mg / ml (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0..., 2.5..., or 3.0 mg / ml).

[0006] In some embodiments, provided herein are compositions that include: a) a pharmaceutical agent, including an ACE-2 receptor antagonist; b) water; and c) at least one of: i) one or more salts present at levels such that the composition, when in aqueous form, has an approximately physiological concentration of the one or more salts and an approximately physiological pH; ii) one or more gelling agents present at levels such that the composition is in the form of a gel; and iii) one or more ointment forming agents present at levels such that the composition is in the form of an ointment; d) an optional preservative; and e) an optional soothing agent.

[0007] In certain embodiments, provided herein is a method comprising delivering a system to a subject, the subject having an eye comprising a corneal injury or an existing corneal scar, the system comprising: a) an eye dropper or contact lens, and b) a composition comprising: i) a pharmaceutical agent comprising an ACE-2 receptor antagonist; ii) water; and iii) at least one of: A) one or more salts present at levels such that the composition, when in aqueous form, has an approximately physiological concentration of one or more salts and an approximately physiological pH, B) one or more gelling agents present at levels such that the composition is in the form of a gel; and C) one or more ointment forming agents present at levels such that the composition is in the form of an ointment; iv) an optional preservative; and v) an optional soothing agent.

[0008] In further embodiments, a system is provided herein that includes: a) a composition comprising i) a drug comprising an ACE-2 receptor antagonist; ii) water; and iii) at least one of: A) one or more salts present at a level such that the composition has an approximately physiological concentration of one or more salts and an approximately physiological pH when in aqueous form; B) one or more gelling agents present at a level such that the composition is in the form of a gel; and C) one or more ointment forming agents present at a level such that the composition is in the form of an ointment; iv) an optional preservative; and v) an optional soothing agent; and b) an eye dropper or contact lens.In certain embodiments, the system comprises an eye dropper, and the composition is present inside the eye dropper.In further embodiments, the system comprises a contact lens, and the composition is present inside or on the inner surface of the contact lens.

[0009] In some embodiments, provided herein is a method of treating a subject for corneal damage and / or existing corneal scarring comprising administering a composition to the subject's cornea or providing a composition to a subject such that the subject administers the composition to the cornea, wherein the subject's cornea comprises corneal damage and / or existing corneal scarring, and the composition comprises: a) an agent comprising an ACE-2 receptor antagonist; b) water; and c) at least one of: i) one or more salts present at levels such that the composition, when in aqueous form, has an approximately physiological concentration of one or more salts and an approximately physiological pH; ii) one or more gelling agents present at levels such that the composition is in the form of a gel; and iii) one or more ointment forming agents present at levels such that the composition is in the form of an ointment; d) an optional preservative; and e) an optional soothing agent.

[0010] In certain embodiments, the agent is present in the composition at a concentration of 0.1 mg / ml to 2.0 mg / ml. In other embodiments, the administering or administration is accomplished at least daily for at least one week, or at least two weeks, or at least one month, and the subject has a best corrected visual acuity (BSCVA) that is 20 / X immediately before the administering or administration, and then 20 / Y at the end of at least one week, at least two weeks, or at least one month, where Y is at least 5 points (or 10, 15, or 20, or 25, or 30 points) lower than X.

[0011] In certain embodiments, the composition is free of or detectably free of any additional reagents other than the drug, water, and one or more salts. In other embodiments, the composition includes a soothing agent, and the composition is free of or detectably free of any additional reagents other than the drug, water, one or more salts, and the soothing agent. In some embodiments, the composition further includes a preservative, and the composition is free of or detectably free of any additional reagents other than the drug, water, one or more salts, and the preservative. In certain embodiments, the composition further includes a preservative and a soothing agent, and the composition is free of or detectably free of any additional reagents other than the drug, water, one or more salts, the preservative, and the soothing agent.

[0012] In certain embodiments, the antiseptic is selected from the group consisting of benzalkonium chloride, sodium chlorite, sodium perborate, purite, benzododecinium bromide, ethylenediaminetetraacetic acid (EDTA), chlorobutanol, thiomersal, disodium edetate, and oxychloro complex (SOC). In certain embodiments, a soothing agent is present in the composition, and the soothing agent is optionally selected from the group consisting of carboxymethylcellulose, polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, and hyaluronic acid.

[0013] In some embodiments, the composition is present in an eye dropper container, optionally the eye dropper container is a disposable container. In further embodiments, the composition comprises one or more salts, is in liquid form, and is free or detectably free of one or more gelling agents and one or more ointment formers. In additional embodiments, the composition comprises one or more gelling agents and / or one or more ointment formers, and is in the form of a gel or ointment, optionally the gelling agent is selected from the group consisting of hypromellose (e.g., about 0.3%), carbomer homopolymer (e.g., about 0.5%), and carboxymethylcellulose (e.g., about 1%), and optionally the ointment former is mineral oil (e.g., about 40-50%) and / or petrolatum (e.g., 40-60%).

[0014] In some embodiments, the administering or administration is at least 4 or 6 or 8 times per day for at least one week. In further embodiments, the administering or administration is performed about every 30 minutes for at least 8 hours. In additional embodiments, the subject's cornea contains a corneal injury, and the administering or administration is performed at least daily for at least one week, beginning within 1-5 days from the appearance of the corneal injury, such that one month after the appearance of the corneal injury, the cornea has a Fantes slit lamp corneal haze score of 0, 0.5, 1, or 2, and the corneal injury, if left untreated, would result in a Fantes slit lamp corneal haze score of 3 or 4 after one month. In other embodiments, the subject's cornea contains a corneal injury, and the administering or administration is accomplished at least daily for at least one week beginning within one to five days from the appearance of the corneal injury, and one month after the appearance of the corneal injury, the cornea has a Fantes slit lamp corneal haze score of 0, 0.5, or 1, and the corneal injury, if left untreated, would result in a Fantes slit lamp corneal haze score of 2, 3, or 4 after one month.

[0015] In certain embodiments, the agent is selected from the group consisting of losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, and the losartan metabolite EXP3174. In other embodiments, the one or more salts include one or more or all of the following: i) about 0.64% or 0.60%-0.070% sodium chloride, ii) about 0.075% or 0.070-0.080% potassium chloride, iii) about 0.048% or 0.040-0.055% calcium chloride dihydrate, iv) about 0.03% or 0.01-0.05% magnesium chloride hexahydrate, v) about 0.39% or 0.30-0.50% sodium acetate trihydrate, and / or vi) about 0.17% or about 0.10-0.30% sodium citrate dihydrate.

[0016] In certain embodiments, the method further comprises administering a corticosteroid to the subject's cornea or providing a corticosteroid to the subject such that the subject administers the corticosteroid to the cornea, where the corticosteroid is present in the composition or in a separate composition. In other embodiments, the composition is present in a conjunctival reservoir or other continuous delivery device that slowly releases the composition into the subject's tear fluid over time. In certain embodiments, the composition is present in a porous collagen therapeutic contact lens that releases the composition over time.

[0017] In some embodiments, the composition comprises an antiseptic (e.g., an antibiotic). In further embodiments, the administering or administration is accomplished at least daily for at least one week, or at least two weeks, or at least one month, and the subject has a myopia score of X diopters immediately prior to the administering or administration and Y diopters at the end of at least one week, at least two weeks, or at least one month, where Y is at least one diopter lower than X. In certain embodiments, the subject is a human, a cat, a dog, a horse, a cow, or a pig.

[0018] In certain embodiments, the subject's cornea comprises a corneal injury, and the corneal injury occurs 1, 3, 6, 12, 24, or 48 hours before administering or administering. In other embodiments, administering the composition to the subject's cornea comprises the subject administering the composition to his or her cornea. In other embodiments, the subject's cornea comprises a corneal injury, and the injury is caused by trauma, chemical burn, microbial infection, or surgery. In certain embodiments, the subject's cornea comprises a corneal injury, and the injury is caused by photorefractive keratectomy (PRK) or therapeutic laser keratectomy (PTK).

[0019] In certain embodiments, the corneal damage occurs within 5 days of administration or administration. In further embodiments, the corneal damage occurs within 24 hours of administration or administration. In additional embodiments, the agent comprises losartan. In certain embodiments, the composition is present in an eye dropper. In other embodiments, the composition is present in a contact lens.

[0020] In some embodiments, a method of treating a subject with corneal injury (e.g., recent occurrence) and / or existing corneal scarring (e.g., that has been present for 3, 4, 5, 6 months or more) comprising administering (e.g., topically) a first composition to the cornea of ​​a subject or providing the composition to the subject such that the subject administers the composition to the cornea, wherein the cornea of ​​the subject comprises corneal injury and / or existing corneal scarring, the composition comprises an agent, and the agent comprises an ACE-2 receptor antagonist; and optionally, Provided herein is a method, wherein the agent is selected from the group consisting of losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, and the losartan metabolite EXP3174, and the method optionally further comprises administering a corticosteroid to the subject's cornea or providing a corticosteroid to the subject such that the subject administers the corticosteroid to the cornea, wherein the corticosteroid is present in a first composition or a second composition. In some embodiments, the first composition and / or the second composition is in the form of an oil-in-water emulsion or micelles. In certain embodiments, the ACE-2 receptor antagonist is present in the first composition and / or the second composition (e.g. if present in an oil-in-water emulsion or micelles) at 1 to 30 mg / ml (such as 1.0..., 5.0..., 10.0..., 15.0..., 20.0..., 25.0.., or 30.0 mg / ml).

[0021] In certain embodiments, provided herein is a method comprising delivering a system to a subject, the subject having an eye with a corneal injury or existing corneal scar, the system comprising: a) an eye dropper or contact lens; and b) a first composition comprising a drug, the drug comprising an ACE-2 receptor antagonist, the drug being optionally selected from the group consisting of losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, and the losartan metabolite EXP3174; and c) an optional corticosteroid, the corticosteroid being present in the first composition or the second composition. In certain embodiments, the drug is present in the composition at a concentration of about 0.2 mg / ml to 0.9 mg / ml or about 0.1 mg / ml to 3.0 mg / ml. In certain embodiments, a corticosteroid is used in the method and is present in the first composition. In other embodiments, a corticosteroid is used in the method and is present in the second composition. In some embodiments, the first composition and / or the second composition are in the form of an oil-in-water emulsion or micelles. In certain embodiments, the ACE-2 receptor antagonist is present in the first composition and / or the second composition (e.g., if present in an oil-in-water emulsion or micelles) at 1-30 mg / ml (such as 1.0..., 5.0..., 10.0..., 15.0..., 20.0..., 25.0..., or 30.0 mg / ml).

[0022] In some embodiments, the delivery or administration (e.g., of the first composition and / or the second composition) is accomplished by a pharmacy personnel, a physician, a nurse, or other medical personnel. In certain embodiments, the corneal damage occurs within 5 days (e.g., 5, 4, 3, 2, or 1 day) of delivery or administration of the first composition and / or the second composition. In other embodiments, the corneal damage occurs within 24 hours (e.g., 24..., 12..., 6..., 3..., 2..., or 1 hour) of delivery or administration of the first composition and / or the second composition.

[0023] In some embodiments, provided herein is a composition comprising or consisting essentially of a drug and saline, and an optional corticosteroid, wherein the drug comprises an ACE-2 receptor antagonist, and the drug is optionally selected from the group consisting of losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, and the losartan metabolite EXP3174.

[0024] In certain embodiments, provided herein is a system comprising: a) a first composition comprising an ACE-2 receptor antagonist, the drug being optionally selected from the group consisting of losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, and the losartan metabolite EXP3174; and b) an eye dropper or contact lens; and c) the optional corticosteroid, the corticosteroid being present in the first composition or the second composition. In certain embodiments, the system comprises an eye dropper, and the first composition and / or the second composition are present within the eye dropper. In further embodiments, the system comprises a contact lens, and the first composition and / or the second composition are present within or on the inner surface of the contact lens.

[0025] In certain embodiments, the compositions herein are sterile. In some embodiments, the agent is present in the first composition at a concentration of about 0.2-0.9 mg / ml or about 0.1 mg / ml-2.0 mg / ml. In further embodiments, the concentration of the agent in the first composition of 0.2-0.9 mg / ml is about 0.8 mg / ml, or about 0.7 mg / ml, or about 0.6 mg / ml, or about 0.5 mg / ml, or about 0.4 mg / ml, or about 0.3 mg / ml, or about 0.2 mg / ml. In certain embodiments, the first composition and / or the second composition is in the form of a liquid or gel and further comprises saline. In additional embodiments, the first composition consists or consists essentially of the agent and saline; and / or the composition has a pH of 7.0-7.2. In some embodiments, the first composition and / or the second composition is in the form of an oil-in-water emulsion or micelles. In certain embodiments, the ACE-2 receptor antagonist is present in the first composition and / or the second composition (e.g. if present in an oil-in-water emulsion or micelles) at 1 to 30 mg / ml (such as 1.0..., 5.0..., 10.0..., 15.0..., 20.0..., 25.0.., or 30.0 mg / ml).

[0026] In certain embodiments, the first composition and / or the second composition further comprises an antiseptic substance. In some embodiments, the antiseptic substance is selected from the group consisting of benzalkonium chloride, sodium chlorite, sodium perborate, purite, or benzododecinium bromide. In additional embodiments, the first composition and / or the second composition does not comprise an antiseptic substance.

[0027] In some embodiments, the first composition and / or the second composition are present in an eye dropper container, hi other embodiments, the eye dropper container is a disposable container.

[0028] In certain embodiments, the administering or dosing (e.g., of an ACE-2 receptor antagonist and / or a corticosteroid) is at least 4 times per day (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 times per day) for at least one week (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 weeks). In other embodiments, the administering or dosing (e.g., of an ACE-2 receptor antagonist and / or a corticosteroid) is at least 8 times per day for at least one week. In certain embodiments, the administering or dosing (e.g., of an ACE-2 receptor antagonist and / or a corticosteroid) is accomplished about every 30 minutes for at least 8 hours (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 hours..., or 72 hours, or up to 3 days).

[0029] In some embodiments, the subject's cornea contains a corneal injury, and administering or administration (e.g., of an ACE-2 receptor antagonist and / or a corticosteroid) is accomplished at least daily for at least one week beginning within one to five days from the appearance of the corneal injury, and one month after the appearance of the corneal injury, the cornea has a Fantes slit lamp corneal haze score of 0, 0.5, 1, or 2, and the corneal injury, if left untreated, would result in a Fantes slit lamp corneal haze score of 3 or 4 after one month. In other embodiments, the subject's cornea contains a corneal injury, and administering or administration (e.g., of an ACE-2 receptor antagonist and / or a corticosteroid) is accomplished at least daily for at least one week beginning within one to five days from the appearance of the corneal injury, and one month after the appearance of the corneal injury, the cornea has a Fantes slit lamp corneal haze score of 0, 0.5, or 1, and the corneal injury, if left untreated, would result in a Fantes slit lamp corneal haze score of 2, 3, or 4 after one month. In additional embodiments, the subject's cornea contains a corneal injury, and administering or administration (e.g., of an ACE-2 receptor antagonist and / or a corticosteroid) is accomplished at least daily for at least one week beginning within one to five days from the appearance of the corneal injury, and one month after the appearance of the corneal injury, the cornea has a Fantes slit lamp corneal haze score of 0, 0.5, or 1, and the corneal injury, if left untreated, would result in a Fantes slit lamp corneal haze score of 1, 2, 3, or 4 after one month. In some embodiments, the subject's cornea contains a corneal injury, and administering or dosing (e.g., of an ACE-2 receptor antagonist and / or a corticosteroid) is accomplished at least daily for at least one week beginning within one to five days from the appearance of the corneal injury, and one month after the appearance of the corneal injury, the cornea has a Fantes slit lamp corneal haze score of 0, 0.5, 2, or 3, and the corneal injury, if left untreated, would result in a Fantes slit lamp corneal haze score of 4 after one month.

[0030] In certain embodiments, the first composition and / or the second composition are in the form of an ointment, liquid, or gel. In other embodiments, the first composition and / or the second composition are present in a conjunctival reservoir or other continuous delivery device that slowly releases the ACE-2 receptor antagonist (e.g., losartan) and / or corticosteroid into the subject's tear fluid over time. In other embodiments, the first composition and / or the second composition are present in a porous collagen therapeutic contact lens that releases the ACE-2 receptor antagonist (e.g., losartan) or corticosteroid over time. In additional embodiments, the first composition and / or the second composition further comprises an antibiotic. In certain embodiments, the antibiotic is selected from the group consisting of ciprofloxacin, ofloxacin, gatifloxacin, levofloxacin, moxifloxacin, besifloxacin, gentamicin, tobramycin, amikacin, neomycin, amphotericin B, natamycin, chlorhexidine digluconate, polyhexamethyline biguanide, or broline. In certain embodiments, the composition containing the Ace-2 receptor antagonist comprises at least one antiviral agent (e.g., for herpes simplex virus, such as acyclovir, valcyclovir, famciclovir, ganciclovir, and trifluridine) and / or an anti-acanthamoeba drug (such as propamidine isethionate, hexamidine, and pentamidine).

[0031] In certain embodiments, the subject is a human subject. In additional embodiments, the subject's cornea comprises a corneal injury, and the corneal injury occurred 1, 3, 6, 12, 24, or 48 hours prior to administering or administering the first composition and / or the second composition. In some embodiments, the subject's cornea comprises a corneal scar, and the administering or administration (of the first composition and / or the second composition) is performed at least daily for at least one week such that the Fantes slit lamp corneal haze score of the cornea is reduced by at least 0.5 or at least 1 from the initial Fantes slit lamp corneal haze score of the cornea. In some embodiments, the subject's cornea comprises a corneal injury, and the injury was caused by trauma, chemical burn, microbial infection, or surgery. In other embodiments, the subject's cornea comprises a corneal injury, and the injury was caused by laser refractive keratectomy or therapeutic laser keratectomy. In further embodiments, the corneal injury occurred within 5 days of administering or administering the first composition and / or the second composition. In other embodiments, the corneal injury occurs within 24 hours of administering or administering the first composition and / or the second composition. In some embodiments, the agent comprises losartan. In certain embodiments, the agent comprises the losartan metabolite EXP3174.

[0032] In certain embodiments, the compositions herein may contain any of the following reagents: methylcellulose (e.g., 0.5%, 1%, 1.5%, or 2%, or any concentration between 0.5% and 2%); hydroxypropyl methylcellulose (e.g., 0.5%, 1%, 1.5%, or 2%, or any concentration between 0.5% and 2%); dextran (e.g., 0.1% or 0.2%); glycerin (e.g., 0.1% to 2%); carbomer (e.g., 0.1% to 0.5%); hyaluronic acid (e.g., 0.03%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, or any concentration between 0.03% and 1%; and further comprising one or more of higher viscosity lipids such as phospholipids, saturated and unsaturated fatty acids, or triglycerides (e.g., 0.5% to 5%), which may increase corneal permeability and improve patient comfort.

[0033] In some embodiments, the compositions herein further comprise one or more of the following agents: components that increase corneal epithelial permeability to increase losartan or other agents herein penetration into the stroma for greater TGFβ blockade. Such agents may be used, for example, typically for about 1-5 days after infection or injury for greater blockade of TGFβ in the early phase of the scarring response. Components added to accomplish this include, for example, benzalkonium chloride (e.g., 0.02%), and sodium ethylenediaminetetraacetate (e.g., 0.01%).

[0034] In other embodiments, the compositions herein further comprise an anti-inflammatory agent, such as a corticosteroid (e.g., for use 1 day to 2 weeks post-injury). In certain embodiments, the anti-inflammatory agent is selected from prednisolone acetate (e.g., 0.1%, 0.2%, 0.5%, 1%, or any concentration between 0.1% and 1%), fluromethalone (e.g., 0.1%, 0.25%, 0.5%, or any concentration between 0.1% and 0.5%), dexamethasone sodium phosphate (e.g., 0.1% to 0.2%), loteprednol (e.g., 0.1% to 1%), and difluprednate (e.g., 0.01% to 0.1%). [Brief description of the drawings]

[0035] [Figure 1]Standardized slit lamp photographs of representative intact sham-operated corneas and corneas one month after an 8 mm central Descemet's membrane stripping procedure treated with topical and / or oral losartan solutions or corresponding vehicle solutions. Sham-operated intact control (Con) corneas treated with oral losartan (A) or topical losartan (B). C. Corneas after Descemet's membrane stripping procedure and treatment with oral vehicle and topical vehicle for one month. Arrowheads indicate neovascularization. D. Corneas after Descemet's membrane stripping procedure and treatment with oral losartan 5 mg / kg three times daily. Arrowheads indicate neovascularization. E. Corneas after Descemet's membrane stripping procedure and treatment with topical losartan 0.4 mg / kg six times daily for one month. Note peripheral corneal clarity compared to C and D, as well as reduced central opacity and areas of clearing (arrows), and reduced corneal neovascularization compared to C or D. F. The cornea treated with Descemet's membrane stripping surgery and both topical losartan 0.4 mg / ml 6 times a day and oral losartan 5 mg / kg 3 times a day also had greater peripheral corneal clarity and reduced central opacity compared to C and D. Areas of peripheral opacity clearing were also present in this cornea (arrows). G. Graph showing opacity intensity measured by ImageJ in a 2.5 mm diameter circle for each cornea. Mean and standard error for each group are shown. * and ** indicate that the opacity intensity for DMR+topical losartan and DMR+topical losartan and oral losartan, respectively, was significantly different from the other groups but not from each other. DMR, Descemet's membrane stripping surgery. Un, intact sham surgery. OR, oral. T or TOP, topical. L or Los, losartan. [Figure 2A]Dual IHC of α-smooth muscle actin (SMA) marker for myofibroblasts and keratocan marker for keratocytes in corneas after one month of treatment with losartan or vehicle. Representative central sections from two corneas in each group are shown. Note that the thickness of the fibrotic SMA-positive (red) layer was thicker in corneas in the DMR oral vehicle and topical vehicle groups (DMR vehicle, panels A, B) and DMR oral losartan groups (panels E, F) compared to corneas in the DMR topical losartan group (panels C, D) and DMR topical losartan and oral losartan groups (G, H). SMA-positive cells noted in the limbus of some corneas are pericytes associated with limbal blood vessels. The DMR topical losartan cornea shown in panel D had the least SMA-positive fibrosis in the posterior cornea in that group. Note that posterior corneal fibrosis was absent in intact sham-operated corneas treated with oral (panels I, J) or topical (panels K, L) losartan. Blue is DAPI staining of cell nuclei. [Figure 2B] Graphical results of the individual SMA-positive fibrotic areas in each cornea in each group are provided. Individual measurements and the mean ± standard error for each group are shown. For statistical comparisons between groups using Kruskal-Wallis test followed by post-hoc Dunn's-Bonferroni test, see Table 4. [Figure 3A]Double IHC for type IV collagen and TGFβ-1. Column A. In intact sham-operated corneas treated with topical losartan for 1 month, type IV collagen was detected in Descemet's membrane (arrow) but little, if any, was detected in the stroma or keratocytes (which were primarily keratocytes). TGFβ-1 was produced in corneal endothelial cells (arrowhead) but only in a few scattered keratocytes. TGFβ-1 penetration into the stroma from corneal endothelial cells or aqueous humor is inhibited by intact Descemet's membrane and its components, including type IV collagen and perlecan. The results were identical in intact sham-operated groups that were not treated (not shown) or treated with oral vehicle alone (not shown). Column B. One month after Descemet's membrane stripping surgery and one month after treatment with topical and oral vehicle, type IV collagen was present at high levels in the posterior stroma (delineated by the square brackets), which is dominated by myofibroblasts and corneal fibroblasts, but was absent in the more anterior stroma, which is dominated by keratocytes. TGFβ-1 was detected at high levels throughout the fibrotic posterior stroma in both keratocytes and stromal tissue. Column C. One month after Descemet's membrane stripping surgery and one month after treatment with topical losartan alone, type IV collagen was significantly decreased in the posterior stroma, except adjacent to the posterior corneal surface (arrows), which lacked Descemet's membrane and endothelium. TGFβ-1 was present in abundance at the posterior stromal surface and throughout the posterior stroma. Column D. One month after Descemet's membrane stripping surgery and one month after treatment with oral losartan alone, type IV collagen was present at high levels in the posterior stroma, which is mainly occupied by myofibroblasts and corneal fibroblasts, but was absent in the more anterior stroma, which is mainly occupied by keratocytes. TGFβ-1 was detected in large amounts at the posterior stromal surface and in some keratocytes. Column E. One month after Descemet's membrane stripping surgery and one month after treatment with both topical and oral losartan, type IV collagen was significantly reduced in the posterior stroma. TGFβ-1 was detected at high levels at the posterior stromal surface and in some keratocytes. Blue is DAPI in all composite images.The yellow dashed rectangle shows an example of the area quantified for collagen IV by ImageJ, and the average of three non-overlapping rectangles is taken as the value for that cornea. [Figure 3B] Graphical results of individual collagen type IV staining intensity in the posterior stroma analyzed by ImageJ in a 0.75 width x 0.5 height rectangle tangent to the posterior corneal surface for each cornea in each group. Individual measurements and the mean ± standard error for each group are shown. The double diagonal line at the top of the topical and oral vehicle column indicates that the highest value in this group actually exceeded the units of the Y axis (the value for this cornea was an average of 6.586 x 103 intensity). See Table 5 for Kruskal-Wallis test followed by post-hoc Dunn's-Bonferroni statistical comparison between groups. [Figure 4A] 1 shows the chemical structure of losartan. [Figure 4B] The chemical structure of the losartan metabolite EXP3174 is shown. [Figure 4C] 1 shows the chemical structure of telmisartan. [Figure 4D] 1 shows the chemical structure of valsartan. [Figure 4E] 1 shows the chemical structure of olmesartan. [Figure 4F] 1 shows the chemical structure of candesartan. [Figure 5A] Standardized slit-lamp photographs of rabbit corneas one month after a 1-minute exposure to 1N NaOH on 5-mm diameter filter paper, and continuous treatment six times per day for one month with topical vehicle (VEH), 0.8 mg / ml losartan, 1% prednisolone acetate, or 0.8 mg / ml losartan + 1% prednisolone acetate. Note that the opacity in each cornea is composed of a central darker zone (*) and a peripheral lighter zone (**). Arrows indicate central corneal neovascularization. Dotted circles show examples of ImageJ analysis of total opacity for individual corneas, including a combination of the central darker zone and the peripheral lighter zone. Magnification 15x. [Figure 5B]Graph of total opacity area measured by ImageJ in individual corneas. Mean ± standard error of the mean is shown for each group. * indicates opacity was significantly different from the vehicle BSS control group. Table 8 shows post-hoc Dunn's-Bonferroni test p-values ​​for comparison between groups following Kruskal-Wallace. [Figure 5C] Graph of total opacity in pixel intensity measured by ImageJ in individual corneas. Mean ± standard error of the mean is shown for each group. * indicates opacity was significantly different from the vehicle BSS control group. Table 9 shows post-hoc Dunn's-Bonferroni test p-values ​​for comparison between groups following Kruskal-Wallace. [Figure 6A] Dual immunohistochemistry for the keratocyte-specific marker keratocan (green) and myofibroblast-specific marker α-SMA (red) in intact control corneas and one month after alkali-burn injury and topical treatment. Fragile peripheral epithelium and persistent epithelial defects were noted in all alkali-burned corneas. In corneas treated with losartan alone or losartan plus prednisolone acetate, α-SMA-positive myofibroblasts tended to be localized in the posterior cornea, while the anterior cornea was repopulated with keratocan-positive keratocytes. Two examples of corneas treated with prednisolone acetate alone are shown to demonstrate the variability observed in this group. In #1, α-SMA-positive myofibroblasts occupied the entire thickness of this cornea and there was a persistent epithelial defect. In #2, despite the presence of persistent epithelial defects, α-SMA-positive myofibroblasts were present only in the posterior stroma. All alkali-burned corneas were devoid of corneal endothelium over an 8-10 mm diameter area of ​​the posterior cornea. Arrows indicate areas with posterior α-SMA positive myofibroblasts. LOS, topical losartan; Acetate Pred, 1% prednisolone acetate; BSS Veh, balanced salt solution vehicle; e, epithelium; S, stroma. [Figure 6B]Graph of total α-SMA positive stromal area determined using ImageJ in each central corneal section in each group. * indicates that the mean was significantly different from the BSS vehicle control group. # indicates that the mean was significantly different from the prednisolone acetate alone group. Table 10 shows the post-hoc Dunn's-Bonferroni test p-values ​​for statistical comparisons between groups following Kruskal-Wallace. [Figure 6C] Graph of total α-SMA positive intensity per corneal section in pixels as determined using ImageJ in each central corneal section in each group. * indicates that the mean was significantly different from the BSS vehicle control group. # indicates that the mean was significantly different from the prednisolone acetate alone group. Note that the mean and standard error of the mean are lower in the losartan + prednisolone acetate combination group and higher in the prednisolone acetate only group. Table 11 shows the post-hoc Dunn's-Bonferroni test p-values ​​for statistical comparisons between groups following Kruskal-Wallace. [Figure 7A] Dual immunohistochemistry for TGFβ-1 and collagen type IV in both the anterior and posterior stroma. Representative IHC for each group is shown. Arrows indicate Descemet's membrane or remnants of Descemet's membrane in each cornea. Representative ImageJ quantification rectangles (100×50 units) for both the anterior stroma (long side of rectangle at anterior stromal surface) and posterior stroma (long side of rectangle at posterior stromal surface, just anterior to Descemet's membrane or its remnants). No differences in TGFβ-1 were noted between groups. [Figure 7B] ImageJ quantification of IHC intensity units of collagen IV in the anterior stroma in the groups. * and ** indicate that the mean was significantly different from the vehicle group. Table 12 shows the post-hoc Dunn's-Bonferroni test p-values ​​for statistical comparisons between groups following Kruskal-Wallace. [Figure 7C]ImageJ quantification of IHC intensity units of type IV collagen in the posterior stroma in the 16- and 17-treated groups. * indicates that the mean was significantly different from the vehicle group. Table 12 shows the post-hoc Dunn's-Bonferroni test p-values ​​for statistical comparisons between groups following Kruskal-Wallace. [Figure 8] Corneal angiogram images of initial intravenous fluorescein filling in corneal neovascularization (CNV). The yellow line outlines the approximate area of ​​each cornea free of corneal neovascularization. In some eyes, the nictitating membrane or eyelid covers a portion of the peripheral cornea, and the peripheral area free of CNV was estimated as noted from direct slit lamp examination. Magnification 20x. [Figure 9] Immunohistochemistry for α-SMA on representative central sections from each cornea was analyzed by ImageJ to determine total α-SMA positive stromal area and total α-SMA intensity in pixels. Arrows indicate localized areas of α-SMA staining that were present only in the posterior-most stroma of corneas treated with the losartan + prednisolone acetate combination. [Figure 10A] Slit lamp opacification after -9D PRK and one month of topical treatment with vehicle or losartan. Standardized slit lamp photographs of each cornea. Note that the light reflections are all located in similar locations. The yellow dotted circle indicates the central 3.5 mm diameter area within the excimer laser ablation zone of each corneal image that was analyzed by ImageJ to determine the average total pixels of opacification. [Figure 10B] Graph of mean central opacity pixels in corneas analyzed by ImageJ. Note the higher mean and greater variability (larger standard error) in vehicle-treated corneas compared to losartan-treated corneas. [Figure 11A]Double immunohistochemistry for myofibroblast marker α-SMA and keratocyte marker keratocan (same labels as in Fig. 10A-B) in the central 0.5 mm of each cornea. Composites of IHC for α-SMA and keratocan with DAPI staining of all cell nuclei for each cornea are shown along with the corresponding panel showing α-SMA alone analyzed for total pixels of α-SMA staining by ImageJ. The yellow dashed box indicates the area of ​​analysis by ImageJ that captured all α-SMA staining in the central 0.5 mm of each cornea. * indicates an example of artifactual displacement of epithelium from stroma that occurred in most corneas during sectioning. This was likely related to the incomplete regeneration of EBM present in nearly all rabbit corneas with -9D PRK one month after surgery. 6, 7 Blue is DAPI staining of cell nuclei in all panels. In many panels in both groups, cells in the stroma with DAPI-positive nuclei but SMA- and keratocan-negative are probably primarily corneal fibroblasts, although some immune cells may persist even one month after PRK. [Figure 11B] Graph of total α-SMA pixels determined by ImageJ analysis of the squares shown in A. Note the higher mean and also the greater variability (larger standard error) in the vehicle-treated group compared to the losartan-treated group. [Figure 12A]Representative double-stranded immunohistochemistry for collagen IV (green) and TGFβ-1 (magenta) in the central cornea. In intact corneas, collagen IV (COL IV) was primarily localized to the epithelial basement membrane (arrow) and Descemet's membrane (arrowhead), with little detection in the stroma. In the vehicle-treated group 1 month after -9D PRK, COL IV was present in a broad band posterior to the epithelial basement membrane (indicated by arrow) and in intact Descemet's membrane. In the losartan-treated group 1 month after -9D PRK, COL IV was localized to the epithelial basement membrane (arrow) with only relatively small amounts detected in the underlying stroma. Descemet's membrane (not shown for this losartan-treated cornea) also had abundant COL IV. In the COL IV only panels for each -9D PRK cornea, the dotted rectangle is a 900 pixel x 235 pixel analysis rectangle in ImageJ that encloses all stromal type IV collagen in each cornea in both treatment groups. e is epithelium. Blue is DAPI in all panels. [Figure 12B] Graph of total COL IV pixels determined by ImageJ analysis of the squares (as shown in A). Note the significant downregulation of total COL IV stained pixels in the losartan-treated group compared to the vehicle-treated group (p=0.004).

[0036] definition As used herein, the terms "host," "subject," and "patient" refer to any animal, including but not limited to humans and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, etc.), that is being treated, studied, analyzed, tested, or diagnosed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention relates to compositions, systems, and methods for treating a subject with corneal injury and / or existing corneal scarring using a composition comprising an ACE-2 receptor antagonist (e.g., losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, or the losartan metabolite EXP3174. In certain embodiments, the ACE-2 receptor antagonist is present in the composition at a concentration of about 0.2 mg / ml to 0.9 mg / ml or about 0.1 mg / ml to 2.0 mg / ml.

[0038] In studies conducted during the development of embodiments of the present disclosure, topical losartan was shown to be effective in the limited myofibroblast development and fibrosis induced by more anterior to mid-corneal injury (such as trauma, chemical burns, microbial infections, and surgery such as photorefractive or therapeutic laser keratectomy) or more posterior corneal injury (such as endothelial trauma during cataract or other surgery, Descemet's membrane peeling surgery, or endotheliitis). Trauma, chemical burns, microbial infections, surgery, or disease may involve any layer of the cornea. In other studies conducted during the development of embodiments of the present disclosure, an ACE-2 receptor antagonist and corticosteroid combination was shown to be effective in treating ocular trauma.

[0039] In certain embodiments, the ACE-2 receptor antagonist and / or corticosteroid is present in one or more compositions that include water (e.g., about 90% water) and an oil vehicle (e.g., to benefit the ocular surface by restoring the lipid layer of the tear film and protecting the aqueous layer from drying). In some embodiments, the composition is in the form of an oil-in-water emulsion (e.g., Restasis®, Lacrinmune®, and Ikervis®, but containing an ACE-2 receptor inhibitor and / or corticosteroid instead of CsA) or a micelle-based solution (e.g., Papilock Mini®, Modusik-A Ofteno®, and Taejoon [TJ] Cyporin®, but containing an ACE-2 receptor inhibitor and / or corticosteroid instead of CsA). In certain embodiments, the composition contains one or more of the following: i) solubilizers / enhancing substances (e.g., castor oil, medium chain triglycerides, polyoxly-40 stearate ethanol, polysorbate 80 ethanol, or corn oil); ii) surfactants (e.g., polysorbate 80, tyloxapol, poloxamer 188, or cetalkonium chloride); iii) preservatives (e.g., boric acid or sorbic acid); iv) stabilizing substances (e.g., carbomer copolymer type A, or sodium EDTA); v) viscosity adjusting substances (e.g., hypromellose or sodium hyaluronate); vi) pH adjusting substances (e.g., NaOH, NaH2PO4, or sodium bisulfite); vii) osmotic agents (e.g., glycerol or NaCl); and / or vii) diluents (e.g., water, petrolatum, lanolin, or alcohol).

[0040] In certain embodiments, improvement in corneal damage (compared to leaving untreated) or treated existing corneal scars is measured by the Fantes slit lamp corneal haze score as described in Fantes et al., Arch. Opthalmol., 1990, 108(5):665-675 (hereby incorporated by reference) and is shown in Table 6 below. [Table 1]

[0041] Although the present disclosure is not limited to any particular mechanism, it is believed that oral losartan described in the following examples was not effective in reducing corneal fibrosis after DMR because it did not reach sufficient concentration in the corneal stroma compared to the concentration achieved by local delivery.This is not surprising, since oral losartan has been used by millions of patients for hypertension or other diseases since it was approved by the FDA in 1995, and no beneficial effect on corneal fibrosis has been reported.

[0042] Damage to the corneal epithelium, such as by abrasion or other trauma, is quickly repaired (usually within 24-48 hours) by the proliferation of rapidly dividing epithelial cells. However, this rapid proliferation of corneal epithelial cells is often accompanied by the development of scar tissue. The presence of scar tissue in the cornea results in "corneal haze" (a clouding of the cornea in which vision is dramatically reduced due to the inability of light to pass through the cornea). Treatment of corneal haze varies depending on the extent of scar tissue formation. In cases where scarring remains mild and affects only the surface of the cornea, surgical or laser removal is used as treatment. In situations where scar tissue extends deeper into the cornea, removal of the entire tissue and transplantation of a new cornea is often used. Thus, prevention of scarring in this tissue after injury is an important step in preserving vision.

[0043] Many corneal injuries are known to typically result in scarring of the cornea. These fall into three broad categories: trauma, infection, and disease conditions, all of which are intended to be treated by the agents herein. Natural trauma (such as abrasion or chemical burns) and trauma associated with medical correction of vision (such as photoablation or contact lens-induced damage) cause the destruction of the normal corneal epithelium, leading to the rapid proliferation of these cells and often the formation of scar tissue. Damage to the cornea resulting from surgery (such as transplantation) also usually leads to scarring of this tissue.

[0044] Ocular infections with bacteria, viruses, fungi, acanthamoeba, and other organisms can also lead to scarring. For example, ocular infections with strains of Herpes Simplex Virus type I, Streptococcus pneumoniae, Staphylococcus spp., Escherichia coli, Proteus spp., Klebsiella spp., and Pseudomonas spp. are known to cause ulceration on the surface of the cornea. Such ulcers not only destroy the surrounding epithelial layer, but also penetrate and damage the corneal stroma, further aided by acute inflammatory cells and collagenase released by the damaged epithelial cells themselves. Such deep and widespread damage to the cornea and surrounding tissues leads to extensive scarring. Other non-ulcerative pathogens are also known to lead to corneal scarring. One such organism is the varicella zoster virus (shingles), and infection with this organism often leads to scarring.

[0045] Many disease conditions not immediately caused by pathogens or trauma also contribute to corneal opacification due to scarring. Two such conditions are cicatricial pemphigoid and Stevens-Johnson syndrome (SJS). Cicatricial pemphigoid is an autoimmune blistering disease affecting the oral mucosa and ocular conjunctiva, where inflammation of the corneal epithelium leads to scarring. SJS is a severe form of erythema multiforme (immune complex-mediated hypersensitivity reaction). The ocular manifestation of this disease is epithelial ulceration followed by severe scarring.

[0046] The majority of patients develop varying degrees of corneal haze following excimer laser photorefractive keratectomy (PRK). Corneal haze typically peaks at 2-4 months and has been noted to increase with the degree of myopic correction. Such haze can lead to loss of best-corrected visual acuity after PRK of one or more lines. Corneal stromal remodeling influences the degree of corneal haze after PRK, and corneal haze is thought to be responsible for the reduction in best-corrected visual acuity possible, regression of refractive correction, and poor predictability of attempted correction. The formation of corneal haze after PRK is a result of laser corneal ablation and stromal wound healing. Despite significant advances made in understanding PRK technology (e.g., laser-tissue interactions, optical profiling of the laser beam, multizone multipass approaches, and edge smoothing techniques), characterization of the biological aspects associated with PRK (e.g., wound healing) still presents important limitations associated with PRK technology.

[0047] In certain embodiments, the compositions herein containing an ACE-2 receptor antagonist (e.g., losartan) are used to treat endothelial replacement surgery (e.g., DSAEK, DMEK, etc.). 31、32 , especially when the implant partially displaces from its intended base, leaving the posterior stroma area uncovered by Descemet's membrane and endothelium. 19、22 In corneal diseases associated with severe inflammation (such as alkali burns), the compositions herein containing ACE-2 receptor antagonists are used to modulate posterior corneal fibrosis that may develop. 20 Severe inflammation damages the fibers and matrix of the corneal stroma, which is also the cause of corneal opacity. 22Since it is a highly effective treatment, it can be a very effective treatment. Some other exemplary corneal diseases in which the compositions herein (containing ACE-2 receptor antagonists) may be effective in both prophylactically inhibiting the development of stromal fibrosis and therapeutically treating fibrosis once it has developed include viral, bacterial, fungal, and acanthamoeba keratitis, corneal trauma, chemical burns, and other corneal surgery-induced fibrosis. Myofibroblasts are involved in pathophysiology (conjunctival scarring disorders such as trachoma), and in the pathogenesis of corneal fibrosis ... 33 and fibrosis of conjunctival blebs after glaucoma filtration surgery 34 The compositions herein may also be used to inhibit fibrosis in other areas of the anterior segment of the eye that have a role in ocular function (such as retinal fibrosis, ... and ocular hypertrophy). EXAMPLES

[0048] Example 1 Topical losartan inhibits corneal cicatricial fibrosis after injury This example examines and describes the effect of topical and / or oral TGF-β blocker losartan on corneal stromal fibrosis developed in rabbit corneas after Descemet's membrane stripping surgery (removal of central Descemet's membrane and corneal endothelium). Twenty-eight New Zealand White rabbits were included, with one eye either undergoing an 8 mm central Descemet's membrane stripping surgery or a sham control surgery without Descemet's membrane stripping surgery. Groups of four eyes without Descemet's membrane stripping surgery were treated with no medication, topical losartan, or oral losartan for one month. Groups of four eyes with Descemet's membrane stripping surgery were treated with topical and oral vehicle, topical losartan, oral losartan, or both topical and oral losartan for one month. Standardized slit lamp photographs were obtained with central opacity intensity measured by ImageJ. Corneal posterior fibrotic zones were measured for α-smooth muscle actin (SMA) and keratocan immunohistochemistry using QuPath analysis. Type IV collagen expression in the posterior cornea was quantified by ImageJ and double immunohistochemistry for type IV collagen and TGFβ-1. After Descemet's membrane stripping surgery, topical but not oral losartan reduced the intensity of central stromal opacity and reduced peripheral corneal scarring compared to corneas treated with Descemet's membrane stripping and vehicle alone. Topical losartan also reduced corneal neovascularization that developed after Descemet's membrane stripping surgery. Topical losartan reduced type IV collagen production by cells in the posterior stroma compared to vehicle treatment 1 month after Descemet's membrane stripping surgery.

[0049] Myofibroblast development and scarring stromal fibrosis are mediated by transforming growth factor (TGF) beta and possibly other growth factors such as platelet-derived growth factor (PDGF) after injury to the anterior and posterior cornea (de Oliveira et al., 2021; Wilson, 2019). A recently characterized model for posterior corneal fibrosis is the surgical removal of a portion of the Descemet's membrane-endothelial complex in rabbits (Sampaio et al., 2021). Aqueous humor is likely an important source of TGF beta-1 and TGF beta-2 that subsequently drives the development of myofibroblasts from corneal fibroblasts and fibrocytes in the absence of growth factor-regulatory functions of the Descemet's basement membrane (Medeiros et al., 2020; Sampaio et al., 2021).

[0050] Losartan is an oral medication used to treat hypertension, diabetic kidney disease, heart failure, and left ventricular dilation (Simpson and McClellan, 2000). It is an angiotensin II receptor antagonist (Michel et al., 2013), but has also been shown to be an inhibitor of TGFβ (Lim et al., 2001; Lavoie et al., 2005; Cohn et al., 2007; Wylie-Sears et al., 2014; Geirsson et al., 2012; Park et al., 2012). The aim of this study was to determine whether losartan inhibited cicatricial fibrosis after corneal injury using a previously shown rabbit Descemet's membrane stripping surgical model to induce posterior corneal myofibroblast development and fibrosis. This study shows that topical, but not oral, losartan significantly reduces stromal opacification, stromal fibrosis, and stromal type IV collagen production after Descemet's membrane stripping surgical injury in rabbits.

[0051] method animal All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic Foundation (Cleveland, OH, USA) and the Animal Care and Use Review Office of the Department of the Army (Fort Detrick, MD). All animals were treated in accordance with the principles of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The study included 28 female New Zealand White rabbits (4 in each group), 10-15 weeks of age, each weighing 2.5-3.0 kg.

[0052] Resection of the central Descemet's membrane (DM)-endothelium complex (Descemet's membrane stripping surgery) Rabbits were anesthetized with an intramuscular injection of 30 mg / kg ketamine hydrochloride and 5 mg / kg xylazine. In addition, topical proparacaine hydrochloride 1% (Alcon, Ft Worth, TX, USA) was applied to each eye prior to surgery. One randomly selected cornea in each group underwent either a sham operation without Descemet's membrane stripping or an 8 mm diameter central Descemet's membrane-endothelial resection. The 1 month postoperative time point was selected for the study because myofibroblasts and fibrosis in the posterior cornea were maximal at this time point in a previous time-course Descemet's membrane stripping surgery study (Sampaio et al., 2021). Descemet's membrane stripping surgery was performed as described in a previous study (Sampaio et al., 2021). Briefly, a wire-lid specula was placed into one eye of each rabbit, and an 8 mm diameter gentian violet circle was marked on the epithelial surface for reference. A limbal incision was made with a 1.6 mm blade (Bausch and Lomb, Rochester, NY, USA) and 0.3 ml of Healon OVD (Abbott Medical Optics Inc. Santa Ana, CA, USA) was injected into the anterior chamber. An inverted Sinskey hook (Katena, Denville, New Jersey, USA) was placed into the anterior chamber and an 8 mm diameter disk of Descemet's membrane and endothelium underlying the 8 mm area previously marked on the anterior corneal surface was excised without subsequent endothelial keratoplasty. Removal of Descemet's membrane or endothelium was not performed in intact sham-operated eyes. Remaining Healon OVD was removed with a Simcoe irrigation and aspiration cannula (Bausch & Lomb, Storz, Rochester, NY, USA) and the anterior chamber was filled with balanced salt solution (BSS). A single 10-0 nylon suture was placed at the incision site to prevent leakage from the anterior chamber. One drop of ciprofloxacin (Alcon, Ft. Worth, TX, USA) was applied to the cornea four times a day immediately after surgery and until the epithelial penetration wound healed, approximately 2 days later.

[0053] Beginning immediately after surgery and depending on group (Table 1), Descemet's membrane stripping and intact control eyes were treated with 0.4 mg / ml losartan (Merck and Co, Kenilworth, NJ) in 30 μl of pH 7.0 saline or 30 μl of vehicle saline, pH 7.0, six times per day (every 2 h between 8 am and 6 pm). [Table 2]

[0054] Animals receiving oral losartan were administered 5 mg / kg in oral solution or oral vehicle solution alone three times per day at 8 am, 1 pm, and 6 pm using an oral applicator equipped with a syringe. The oral solution was prepared with 5 ml distilled water + 50 ml ora-plus (suspension vehicle - Perrigo, Dublin, Ireland) + 45 ml Ora-sweet (flavored syrup vehicle - Perrigo).

[0055] Standardized slit lamp photographs and ImageJ analysis of scar intensity One month after treatment, the study eyes were dilated with 2 drops of 1% tropicamide (Akorn Co., Lake Forest, IL) for 30 minutes. After general anesthesia, standardized slit-lamp photographs were taken at 20x magnification with a Haag Streit (Mason, OH, USA) BX900 slit-lamp photography system using identical illumination position and intensity in the study eyes of each rabbit. For each study eye, the intensity of a central circle with a diameter of 2.5 mm (not including light reflection, example shown in Figure 1) was determined using ImageJ 1.53a analysis software. Statistical comparison of opacity intensity was performed using the Kruskal-Wallis test followed by a post-hoc Dunn's-Bonferroni test, with p<0.05 considered statistically significant.

[0056] Corneal fixation, immunohistochemistry, and fibrosis area analysis One month after surgery and treatment, an intravenous injection of 100 mg / kg Beuthanasia (Shering-Plough, Kenilworth, NJ) was given for euthanasia of the animals under general anesthesia. Sharp Westcott scissors (Fairfield, CT, USA) and 0.12 forceps (Storz, St. Louis, MO) were used to remove the corneal-scleral rims of sham-operated intact and DMR eyes without touching the cornea. Each cornea was placed in the center of a 24×24×5 mm mold (Fisher Scientific, Pittsburgh, PA, USA) and the mold was filled with liquid OCT compound (Sakura Finetek, Torrance, CA, USA). The molds and corneal-scleral rims were snap frozen on dry ice and stored at −80°C until sectioning.

[0057] Each OCT block was bisected exactly in the center of the cornea, and 8-μm-thick transverse sections were cut from the central cornea within the previous DMR lesion or the intact sham-operated cornea with a cryostat (HM 505M; Micron GmbH, Walldorf, Germany), and three sections per slide were placed on 25 mm × 75 mm × 1 mm Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA, USA). Slides were kept at −20°C until immunohistochemistry.

[0058] Double immunohistochemistry (IHC) was performed after masking of slides by an investigator not involved in the analysis using methods described previously (de Oliveira et al., 2021) and used primary antibodies confirmed by Western blot and IHC to recognize rabbit antigens (Table 2) or isotype control antibodies (ThermoFisher Scientific, Waltham, MA), as well as secondary fluorescent tag antibodies (Table 3). [Table 3] [Table 4]

[0059] The AB769 (Millipore, Temecula CA) collagen type IV antibody used in this study was raised against purified human and bovine collagen type IV, affinity purified by cross-linking human and bovine collagen type IV to agarose, and cross-absorbed by the manufacturer with human and bovine collagen types I, II, III, V, and VI to remove cross-reactivity. This antibody was previously shown to bind rabbit collagen type IV in IHC (Sampaio et al., in press). The keratocan antibody was raised against the peptide H2N-LRLDGNEIKPPIPIDLVAC-OH (SEQ ID NO: 1). Antibody to TGFβ-1 (GeneTex, Irvine, CA) binds rabbit TGFβ-1 in IHC and shows no reactivity to TGFβ-2 or TGFβ-3 (de Oliveria et al., 2021).

[0060] The area of ​​SMA-positive posterior stromal fibrosis was quantified in the original group masked to the analyst by standardized photography with a 10x objective on a Leica DM6B upright microscope equipped with an automated stage and a Leica 7000T camera using LAS X software (Leica Microsystems, GmbH, Wetzlar, Germany). The area of ​​SMA-positive posterior stromal fibrosis was measured by manually outlining the area on the corneal sections with calibrated QuPath v0.2.3 software using a method previously described (Bankhead et al., 2017). Statistical analysis for SMA-positive area was performed using the Kruskal-Wallis test followed by a post-hoc Dunn's-Bonferroni test, with p<0.05 considered statistically significant.

[0061] Collagen type IV intensity in immunohistochemistry images was quantified in a 0.75 wide x 0.5 high posterior stromal rectangle tangent to the posterior corneal surface using ImageJ on 7.62 cm wide x 5.69 cm high and 300 DPI images generated using standardized microscope illumination settings applied to all sections. If Descemet's membrane was present in the analyzed specimen, the quantification rectangle was positioned so that the posterior side of the rectangle tangent to the posterior corneal surface just anterior to Descemet's membrane. The mean collagen type IV intensity of three non-overlapping rectangles in the corresponding area of ​​the intact cornea of ​​Descemet's membrane stripping or sham surgery was used as the value for that cornea. p values ​​for all statistical comparisons between groups were performed by Kruskal-Wallis test followed by post-hoc Dunn's-Bonferroni test.

[0062] result Slit lamp photograph and central corneal opacity Representative standardized slit lamp photographs from the different groups after one month of treatment are shown in Figure 1. Neither oral losartan at 5 mg / kg three times per day (A) nor topical losartan at 0.4 mg / ml six times per day (B) had any effect on the transparency of sham-operated intact corneas. One month after Descemet's membrane stripping surgery and treatment with oral vehicle solution three times per day and topical vehicle solution six times per day (C), all corneas had severe cicatricial fibrosis extending to the limbus with prominent neovascularization. There was no difference in cicatricial fibrosis and neovascularization compared to control corneas that underwent Descemet's membrane stripping surgery and vehicle treatment one month after Descemet's membrane stripping surgery and treatment with oral losartan at 5 mg / kg three times per day for one month (D). One month after Descemet's membrane stripping surgery and upon treatment with topical losartan at 0.4 mg / kg six times per day for one month (E), there was an overall reduction in cicatricial fibrosis, a decrease in the intensity of central opacity, clarity in the peripheral cornea allowing iris details to be clearly observed, and areas of clearing of intermediate stromal opacity.One month after Descemet's membrane stripping surgery and upon treatment with topical losartan at 0.4 mg / kg six times per day and oral losartan at 5 mg / kg three times per day for one month (F), the changes in corneal opacity and neovascularization were not different from those shown by treatment with topical losartan alone.

[0063] When the intensity of opacity in a 2.5 mm circle of the central cornea was quantified by ImageJ (FIG. 1G), there was a significant decrease (P<0.05, Kruskal-Wallis test followed by post hoc Dunn's-Bonferroni test) in the group that underwent Descemet's membrane stripping with topical losartan for 1 month or with topical and oral losartan for 1 month compared with the group that underwent Descemet's membrane stripping with topical vehicle and oral vehicle solution for 1 month or with oral losartan alone for 1 month. There was no significant difference in the intensity of opacity in the central cornea between the group that underwent Descemet's membrane stripping with topical losartan for 1 month and the group that underwent Descemet's membrane stripping with topical and oral losartan for 1 month.

[0064] Immunohistochemistry for α-smooth muscle actin (SMA), a myofibroblast marker, and keratocan, a keratocyte marker Representative results for immunohistochemistry for SMA and keratocan are shown in Figure 2. Corneas that underwent Descemet's membrane stripping surgery and treatment with topical and oral vehicle for one month (A, B) had significant posterior corneal fibrosis as outlined by IHC for SMA myofibroblast markers. Corneas that underwent Descemet's membrane stripping surgery and treatment with topical losartan 0.4 mg / ml six times per day for one month (C, D) all had a significant reduction in the SMA-positive fibrotic zone in the posterior cornea. The cornea shown in Figure 2D had the smallest area of ​​posterior fibrosis of any cornea that underwent Descemet's membrane stripping surgery in any group. Corneas that underwent Descemet's membrane stripping surgery and treatment with oral losartan 5 mg / kg three times per day for one month (E, F) had posterior fibrosis similar to Descemet's membrane stripping surgery corneas treated with topical and oral vehicle solution for one month. Corneas treated with Descemet's membrane stripping surgery and topical losartan 6 times per day and oral losartan 3 times per day for both months (G,H) also had reduced SMA-positive posterior corneal fibrosis, but this was not significantly different from corneas treated with Descemet's membrane stripping surgery and topical losartan alone. Intact corneas treated with either oral losartan 3 times per day (I,J) or topical losartan 6 times per day (K,L) did not develop SMA-positive posterior corneal fibrosis.

[0065] The graph in FIG. 2M shows the results of quantification of the area of ​​the posterior fibrotic zone of each cornea in each group. There was no SMA-positive fibrosis in any of the intact corneas, whether they were untreated, treated with topical losartan 6 times per day, or with oral losartan 3 times per day. In the group that underwent Descemet's membrane stripping surgery, those treated with topical losartan 0.4 mg / ml 6 times per day had a significantly lower mean SMA-positive posterior fibrotic area (Table 4) compared to the other groups that underwent Descemet's membrane stripping surgery (whether they were treated with oral losartan alone for 1 month, oral and topical losartan for 1 month, or oral and topical vehicle solution for 1 month). The mean area of ​​the SMA-positive posterior fibrotic area was highest in the group that was treated with oral losartan alone for 1 month, and the variability of the area of ​​SMA-positive posterior fibrosis was also greater in that group. However, the mean SMA-positive posterior fibrosis area of ​​Descemet's membrane stripping corneas treated with oral losartan alone was not statistically significantly different from the Descemet's membrane stripping groups treated with topical and oral vehicle for one month or topical and oral losartan for one month. Table 4 provides p values ​​for all statistical comparisons between groups performed by Kruskal-Wallis test followed by post hoc Dunn's-Bonferroni test. [Table 5]

[0066] Immunohistochemistry for type IV collagen and TGFβ-1, and quantification of type IV collagen staining intensity in the posterior stroma Representative results of immunohistochemistry for type IV collagen in combination with TGFβ-1 are shown in Figure 3. In sham-operated intact corneas treated with topical (A) or oral (B) losartan (not shown) or not treated (not shown), type IV collagen was localized mainly in the Descemet's basement membrane and epithelial basement membrane (not shown), and only rare keratinocytes contained type IV collagen. TGFβ-1 was localized in the endothelium and rarely in keratinocytes and in the corneal epithelium (not shown).

[0067] All Descemet's membrane stripping corneas one month after surgery were devoid of central Descemet's membrane and corneal endothelium. In corneas that had undergone Descemet's membrane stripping surgery and treatment with both topical and oral vehicles for one month (Figure 3B), the posterior stroma contained a thick posterior fibrotic zone filled with SMA-positive myofibroblasts and an overlying layer of SMA-negative keratocan-negative cells that were primarily corneal fibroblasts (this can be seen in Figure 2). These cells occupied the posterior fibrotic zone indicated by the square bracket in Figure 3B, and this entire zone had high levels of type IV collagen. Many of the cells in the fibrotic zone of these corneas had TGFβ-1 associated with them. In corneas that had undergone Descemet's membrane stripping surgery and treatment with topical losartan alone for one month (Figure 3C), type IV collagen was detected primarily adjacent to the posterior surface. Many cells in the posterior stroma of these corneas had TGFβ-1 associated with them. As shown in the cornea shown in Figure 3C, some but not all of these corneas also had a densa that accumulated TGFβ-1 at the posterior corneal surface. Corneas that underwent Descemet's membrane stripping and were treated with oral losartan alone (Figure 3D) were not significantly different from corneas that underwent Descemet's membrane stripping and were treated with topical vehicle and oral vehicle for one month (Figure 3B). Corneas that underwent Descemet's membrane stripping and were treated with both topical and oral losartan for one month (Figure 3E) did not differ as a group with respect to type IV collagen from corneas that underwent Descemet's membrane stripping and were treated with topical losartan alone. Thus, oral losartan alone had no significant effect on type IV collagen production in the posterior stroma, and oral losartan treatment did not have an additive effect to topical losartan treatment alone.

[0068] Figure 3F is a graph showing type IV collagen intensity quantified in a 0.75 width x 0.5 height ImageJ rectangle of the posterior stroma abutting the posterior corneal surface, not including Descemet's membrane, of intact corneas as shown in Figures 3A-3E. Treatment with topical losartan, or topical losartan and oral losartan, significantly reduced type IV collagen production in the posterior stroma 1 month after Descemet's membrane stripping surgery compared to topical vehicle and oral vehicle treated corneas.

[0069] Table 5 provides p values ​​for statistical comparisons between all groups of type IV collagen levels in the posterior stroma, performed by Kruskal-Wallis test followed by post-hoc Dunn's-Bonferroni test. [Table 6]

[0070] The lower mean collagen type IV intensity in the Descemet's membrane stripping surgery and oral losartan treatment alone group compared to the Descemet's membrane stripping surgery and topical and oral vehicle treatment groups was likely due to one cornea in the latter group having higher stromal levels of collagen type IV, but the difference between the groups was not statistically significant, as shown in the graph in Figure 3F. No effect of topical and / or oral losartan treatment on the levels of TGFβ-1 in the posterior stroma or posterior stromal surface compared to vehicle-treated corneas was demonstrated, with considerable variation in TGFβ-1 protein levels detected in all posterior stroma of the Descemet's membrane stripping surgery group.

[0071] Although the present disclosure is not limited to any particular mechanism, and understanding of the mechanism is not necessary to practice the present invention, the main importance of the results shown in FIG. 3 is believed to show that losartan penetrates into the cornea and blocks TGFβ, the main growth factor that stimulates fibrosis by driving the development of myofibroblasts. TGFβ also stimulates the production of many collagens that are unregulated in the cornea when myofibroblasts are present underneath. Type IV collagen is one of the collagens that can be traced well because it is found only in the epithelial basement membrane and Descemet's basement membrane, not in the stroma, in the normal intact cornea. It can be observed in FIG. 3. Others, such as type 1 collagen, are also produced by myofibroblasts. The myofibroblasts themselves and the unregulated collagens they produce are the opacity in scar fibrosis. Therefore, topical losartan is believed to inhibit the development of myofibroblasts in the stroma and, if some myofibroblasts develop, reduce the production of scar collagen.

[0072] The Descemet's membrane removal (DMR) model induces severe posterior stromal myofibroblast development and fibrosis in rabbits without overtly affecting the anatomy or function of the corneal epithelium (Figure 1; Figure 2; Medeiros et al., 2019; Sampaio et al., in press). The observation that topical losartan influenced myofibroblast and fibrosis development in the posterior stroma of DMR-treated corneas demonstrates the possibility that topical losartan penetrated into the posterior corneal stroma in this study.

[0073] Type IV collagen is composed of six distinct α chains (α1-α6) that assemble into a heterotrimer (Pozzi et al., 2017). Virtually all basement membranes contain type IV collagen, with the most common trimer composed of two α1 and one α2 chain. In the intact cornea, type IV collagen is prominent in the epithelial basement membrane (de Oliveria et al., in press) and Descemet's membrane (Figure 3 and Sampaio et al., in press), but type IV collagen is barely detectable in keratocytes of the intact cornea (Figure 3). Recent studies have shown that type IV collagen protein is produced by corneal fibroblasts and myofibroblasts in the posterior cornea after central Descemet's membrane peel surgery, and that TGFβ-1 upregulates the production of type IV collagen mRNA in corneal fibroblasts and myofibroblasts (Sampaio et al., in press). An important property of type IV collagen is that it directly binds to TGFβ-1 and TGFβ-2 (Paralkar et al., 1991; Shibuya et al., 2006) and PDGF (Paralkar et al., 1991), thereby reducing their binding to their cognate receptors. Although the present invention is not limited to any particular mechanism, it is believed that type IV collagen produced by corneal fibroblasts and myofibroblasts in the posterior cornea after Descemet's membrane peeling surgery functions to downregulate the effects of TGFβ-1 and TGFβ-2 on keratocytes in the area of ​​production and reduce the penetration of TGFβ-1 and TGFβ-2 into the more anterior stroma. Thus, type IV collagen produced by corneal fibroblasts and myofibroblasts in the cornea may provide a negative feedback regulatory mechanism to regulate TGFβ effects on keratocytes.

[0074] In this example, topical losartan treatment for one month after Descemet's membrane stripping surgery significantly downregulated type IV collagen levels in the posterior stroma compared to vehicle except at the posterior corneal surface (Figure 3). This indicated that topical losartan penetrated into the posterior corneal stroma and inhibited type IV collagen production by keratinocytes in response to TGFβ-1, except at the posterior corneal surface, which lacks Descemet's basement membrane and corneal endothelium at this time point one month after Descemet's membrane stripping surgery, and the localized stromal TGFβ-1 concentration was most likely due to access to the TGFβ-1 reservoir in the aqueous humor. This observation provided direct evidence that the TGFβ regulatory effect in the injured cornea is downregulated by topical losartan, regardless of whether TGFβ is derived from the aqueous humor and / or locally produced by keratinocytes after injury (Figure 3). The results in Figure 3F indicate a trend towards oral losartan alone possibly having some effect on posterior stroma type IV collagen levels 1 month after Descemet's membrane stripping surgery compared to topical vehicle and oral vehicle treated corneas, but the two groups were not statistically significantly different and small differences, if they do exist, are unlikely to be of clinical relevance. A consequence of this example is that IHC and other assays that detect type IV collagen expression will enable monitoring of the anti-TGFβ effects of losartan and other modulators.

[0075] In conclusion, this study suggests that topical losartan may be effective in the prophylactic prevention and treatment of corneal cicatricial fibrosis caused by trauma, infection, disease, and surgery. Because myofibroblasts depend on TGFβ for survival (Wilson, 2020), the TGFβ inhibitory effects of losartan may also be useful even in established corneal scars.

[0076] References for Example 1 Bankhead, et al., 2017. QuPath: Open source software for digital pathology image analysis. Sci Rep. 7, 16878. Cockerham and Hidayat, 1999. Retrocorneal membrane with myofibroblasts after perforating injury: an immunohistochemical and ultrastructural study of 11 cases. Cornea. 18, 700-6. Cohn, et al., 2007. Angiotensin II type 1 receptor blockade attenuates TGF-[beta]-induced failure of muscle regeneration in multiple myopathic states. Nat. Med. 13, 204-10. de Oliveira et al., (in press). Epithelial basement membrane regeneration after PRK-induced epithelial-stromal injury in rabbits: Fibrotic vs. non-fibrotic corneal healing. J. Ref. Surg., in press. de Oliveira, et al., Exp. Eye Res. 202, 108325. de Oliveira and Wilson, S.E., 2020. Biological effects of mitomycin C on late corneal haze stromal fibrosis following PRK. Exp Eye Res. 200, 108218. Geirsson et al., 2012, 126, S189-97. Ghosheh, et al., 2008., Eye Contact Lens. 34, 211-4. Hindman, et al., 2019, Exp. Eye Res. 181, 49-60. Jester, et al., 1997, Cornea. 16, 177-87. Joung, et al., 2020, Int. J. Mol. Sci. 21, 2990. Lavoie, et al., 2005, J. Hypertens. 23, 1895-1903 Lee, et al., 2001, J. Refract. Surg. 17, 334-41. Lim, et al., 2001, Circulation. 103, 789-91. Martinez-Garcia, et al., 2006, Exp. Eye Res. 83, 728-35. Medeiros, et al. 2019, Vis. Sci. 60:1010-20. Michel, et al., 2013, Pharmacol. Rev. 65, 809-48. Mohan, et al., 2003, Exp. Eye Res. 76, 71-87. Mohan, et al., 2008, Exp. Eye Res. 86, 235-40. Moller-Pedersen, et al., 1998, Curr. Eye Res. 17, 736-47. Netto et al., 2006, Exp. Eye Res. 82, 788-97. Paralkar, et al., 1991, Dev. Biol. 143, 303-308. Park, et al., 2012, Cell Transplant. 21, 2407-24. Pozzi et al., 2017, Matrix Biol. 57, 58:1-11. Sampaio,. Exp. Eye Res., 2021 Dec;213:108803. Shibuya, et al., 2006,. J. Dermatol. Sci. 41, 187-195. Simpson, et al., 2000, Drugs Aging. 16, 227-50. Srinivasan, 2012, Arch. Ophthalmol. 130, 143-50. Wilson, 2019, J. Refract. Surg. 35, 506-16. Wilson, 2020, Exp. Eye Res. 201, 108272. Wilson, 2021, Invest. Ophth. Vis. Sci. 62, 8. Wilson, 2021b, Exp. Eye Res. 207:108594. Witcher, et al., 2001, Bull. World Health Organ. 79, 214-21. Wylie-Sears, et al., 2014, et al., Biochem. Biophys. Res. Commun. 446, 870-5. Yao et al., 2008, J. Huazhong Univ. Sci. Technolog. Med. Sci. 28, 543-8.

[0077] Example 2 Topical losartan and corticosteroids additively inhibit corneal stromal myofibroblastogenesis and cicatricial fibrosis after alkali burn injury This example evaluates and describes the efficacy of losartan and prednisolone acetate in inhibiting corneal fibrosis after alkali burn injury in rabbits. Briefly, the study included 16 New Zealand White rabbits. Alkaline burn injury was produced using 1N NaOH on a Whatman #1 filter paper with a diameter of 5 mm. Four corneas in each group were treated with 50 μl of 1) 0.8 mg / ml losartan in BSS, 2) 1% prednisolone acetate, 3) a combination of 0.8 mg / ml losartan and 1% prednisolone acetate, or 4) BSS, six times per day for one month. The area of ​​opacity and total opacity were analyzed by ImageJ on standardized slit lamp photographs. Corneas in both groups were cryofixed in OCT 1 month after surgery and immunohistochemistry (IHC) was performed for α-SMA and keratocan or TGFβ-1 and type IV collagen. Treatment with the combination of topical losartan and prednisolone acetate significantly reduced the area and intensity of slit-lamp opacification. The combination also significantly reduced the area and intensity of stromal myofibroblast α-SMA staining per section, confining myofibroblasts to the posterior stroma only and repopulating the anterior and middle stroma with keratocan-positive keratocytes 1 month after treatment. Corneal fibroblasts produce type IV collagen that is not associated with the basement membrane (especially in the anterior and posterior corneal stroma), and this production was reduced by topical losartan. In conclusion, the combination of topical losartan and prednisolone acetate was effective in reducing myofibroblast-associated fibrosis after corneal alkali burn producing full-thickness injury (including damage to the corneal endothelium).

[0078] Materials and Methods animal Animal treatment and care were approved by the Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic Foundation (Cleveland, OH, USA) and the Animal Care and Use Review Office of the Department of the Army (Fort Detrick, MD). All rabbits were treated in accordance with the principles of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. This example included 16 female New Zealand White rabbits, 10-15 weeks old, each weighing 2.5-3.0 kg.

[0079] Corneal alkali burn method Beginning 24 hours prior to alkali exposure and continuing for 5-7 days after treatment, rabbits were given 60 ml of pediatric liquid acetaminophen (Johnson and Johnson, Ft. Washington, PA) per liter of drinking water. Prior to all alkali exposures and tests, rabbits were anesthetized with an intramuscular injection of 30 mg / kg ketamine hydrochloride and 5 mg / kg xylazine. In addition, topical proparacaine hydrochloride 1% (Alcon, Ft Worth, TX, USA) was applied to each eye. If necessary, rabbits were also given 0.05 mg / kg buprenorphine by subcutaneous injection twice daily upon signs of pain.

[0080] Alkaline injury was performed using 5 mm diameter circular Whatman number 1 filter paper (catalog no. 1001-6508, Fisher Scientific) moistened with 1 N sodium hydroxide (Sigma, St. Louis, MO) and 100 μl of 1 N NaOH solution in balanced salt solution (BSS, 0.64% sodium chloride, 0.075% potassium chloride, 0.048% calcium chloride dihydrate, 0.03% magnesium chloride hexahydrate, 0.39% sodium acetate trihydrate, 0.17% sodium citrate dihydrate, pH 7.5, Alcon, Ft. Worth, TX) according to a previously published method. 10 Alkaline burn injury was produced in one rabbit eye by IV irrigation. Following alkali burn injury, the cornea was liberally irrigated with BSS. Each injured cornea also received one drop of ciprofloxacin (Alcon, Ft. Worth, TX, USA) 10 min after surgery and four times a day for one week, at least 15 min apart from the study medication.

[0081] Treatment with topical losartan and / or prednisolone acetate Beginning immediately after the alkali burn injury, four corneas in each group were treated six times per day (approximately 8:00 AM, 10:00 AM, 12:00 PM, 2:00 PM, 4:00 PM, and 6:00 PM) for one month with 1) 0.8 mg / ml losartan (Merck & Co., Inc., Kenilworth, NJ) in 50 μl BSS, 2) 50 μl 1% prednisolone acetate (Alcon, Ft. Worth, TX), 3) 0.8 mg / ml losartan and 50 μl 1% prednisolone acetate (Alcon, Ft. Worth, TX) in 50 μl BSS at least 5 minutes apart, or 4) 50 μl BSS.

[0082] Fluorescein staining of epithelial defects 2 weeks after injury Two weeks after the alkali burn injury, topical 0.5% fluorescein in BSS was applied to each eye and the presence or absence of epithelial defect(s) was recorded.

[0083] Standardized slit lamp photographs, corneal angiograms, and ImageJ analysis of corneal opacities One month after sodium hydroxide exposure and treatment, the eyes of rabbits under general anesthesia with ketamine-xylazine were dilated with 2 drops of 1% tropicamide (Akorn Co., Lake Forest, IL) for 30 minutes. Slit-lamp photographs were taken of the study eyes of each rabbit at 20x magnification with a Topcon (Oakland, NJ, USA) SL-D7 slit-lamp photography system with standardized illumination levels and angles. For each study eye, the total area of ​​opacity was measured in mm 2 Opacities were determined by outlining them with a hand-drawn selection tool using ImageJ 1.53a analysis software calibrated to . The "raw internal density" in pixels for the opacified area in each cornea was also determined using ImageJ.

[0084] All corneas were treated with 100 μg / kg / day of 11 Fluorescence angiography was performed at the peak of dye passage in the cornea immediately after injection of 1.5 ml of 10% sodium fluorescein (McKesson, Irvine, TX) into the central ear vein using a slit lamp system and a digital camera system that broadly illuminated the cornea and used a "barrier filter" (transmitting only the peak of fluorescein emission, 520 to 530 nm).

[0085] Corneal fixation and sectioning One month after exposure and topical treatment, rabbits were euthanized with 100 mg / kg Beuthanasia (Shering-Plough, Kenilworth, NJ) intravenously and bilateral pneumothorax after general anesthesia with ketamine-xylazine. The corneal-scleral rim of the eye was removed with sharp Westcott scissors (Fairfield, CT, USA) and 0.12 forceps (Storz, St. Louis, MO). The cornea was placed in the center of a 24×24×5 mm mold (Fisher Scientific, Pittsburgh, PA, USA), which was filled with OCT compound (Sakura Finetek, Torrance, CA, USA) and flash frozen on dry ice. The blocks were stored at −80°C until sectioning.

[0086] The OCT blocks were bisected at the center of the cornea, and 8 μm thick transverse sections were cut from the central cornea with a cryostat (HM 505M; Micron GmbH, Walldorf, Germany). Sections from each cornea were placed on 25 mm × 75 mm × 1 mm Superfrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA, USA). Slides with sections were kept at −20°C prior to immunohistochemistry.

[0087] Immunohistochemistry and fibrotic area opacity intensity analysis Multiplex immunohistochemistry (IHC) was performed as previously described. 8 This was accomplished using primary antibodies that were confirmed by Western blot and IHC to recognize rabbit antigens (Table 7) or isotype control antibodies (Thermo Fisher Scientific, Waltham, MA), as well as secondary fluorescent tag antibodies (Table 7). [Table 7]

[0088] The type IV collagen antibody (catalog no. AB769, Millipore, Temecula, CA) was raised against purified human and bovine type IV collagen, which was affinity purified by cross-linking human and bovine type IV collagen to agarose and cross-absorbed by the manufacturer with human and bovine collagen types I, II, III, V, and VI to remove cross-reactivity. This type IV collagen antibody binds to rabbit type IV collagen in IHC. 8、9 It has previously been shown to recognize the alpha-1 / alpha-2 chains but not the alpha-3 through alpha-6 chains. The keratocan antibody is raised against the peptide H2N-LRLDGNEIKPPIPIDLVAC-OH (SEQ ID NO: 1). This marker was used to identify keratocytes in situ. The TGFβ-1 antibody (GeneTex, Irvine, CA) binds to rabbit TGFβ-1 in IHC and shows no reactivity to TGFβ-2 or TGFβ-3. 5

[0089] mm 2 The α-SMA positive stromal area in mm and total α-SMA opacity in pixels were quantified using standardized images acquired with a 10x objective on a Leica DM6B upright microscope equipped with an automated stage and a Leica 7000T camera using LAS X software (Leica Microsystems, GmbH, Wetzlar, Germany). An average of three central corneas was analyzed from each cornea to provide the α-SMA positive stromal area and total α-SMA opacity in mm for each cornea. 2The α-SMA positive stromal area in was determined from the complete diameter and thickness of the central corneal images (300 DPI, 885 width × 500 height for all images, all converted to identical +50% brightness increase files in Photoshop) by ImageJ 1.53a analysis software using a hand-drawn selection tool to outline the α-SMA positive staining area(s). In some corneas, two or more distinct areas were present and the sum of these areas was used as the value for that cornea. In these same sections for each cornea, the total α-SMA positive intensity in pixels was also determined by ImageJ by using the sum if distinct α-SMA positive areas were present.

[0090] All corneas in each group underwent IHC for collagen type IV. Images from each cornea were converted to a uniform 300 DPI, 875 x 568 pixel file. Each cornea had three measurements of signal intensity in three randomly located 100 x 50 ImageJ analysis squares in both the anterior cornea (anterior side of a square at the anterior stromal surface posterior to the EBM, if present) and posterior cornea (posterior side of a square at the posterior stromal surface anterior to Descemet's membrane, if present). The staining intensity within each box was determined by the analysis histogram function, and the average of the three boxes was the intensity value in the anterior or posterior stroma for that cornea.

[0091] statistics Statistical analysis was performed using the Kruskal-Wallis test followed by a post-hoc Dunn's-Bonferroni test, with p<0.05 considered statistically significant.

[0092] result Persistent epithelial defects following alkali burns At 2 and 4 weeks after alkali burn, all corneas in all groups were found to have epithelial defects of at least 1 mm in diameter, with no differences between treatment groups.

[0093] Slit-lamp stromal opacity and central corneal neovascularization (CNV) after alkali burn Using the method of 100 μl 1N NaOH on a 5 mm filter paper circle for 1 min, followed by treatment with topical 0.8 mg / ml losartan, 1% prednisolone acetate, 0.8 mg / ml losartan and 1% prednisolone acetate, or BSS vehicle 6 times per day for 1 month, the central stromal opacification of each of the corneas was as shown in FIG. 5A. An example of the ImageJ visualization used to measure the total stromal opacification area is shown in one cornea for each treatment. Note that these quantification areas for each imaged cornea contain a very dense center (e.g., * in LOS-2) and a less dense periphery (e.g., ** in LOS-2). The mm 2 The total area of ​​opacity in the 100-mL naphthalene stenosis was shown in FIG. 5B. Statistical comparisons between groups are shown in Table 8. [Table 8]

[0094] The losartan alone group, the prednisolone alone group, and the losartan and prednisolone combined group were significantly different from the BSS vehicle group, but were not significantly different from each other.ImageJ was also used to determine the total opacity intensity in pixels for the combination of high density central area and lower density peripheral area in each cornea (as shown by the dashed line in one cornea from each group), and the data is shown in Figure 5C.The statistical comparison between groups is shown in Table 9. [Table 9]

[0095] The losartan alone group and the losartan and prednisolone acetate combination group were significantly different from the BSS vehicle group, but not from each other. The difference between the 1% prednisolone acetate group and the BSS vehicle group did not reach statistical significance. However, the losartan and prednisolone acetate combination group had the lowest standard error of the mean for both parenchymal opacity area and total opacity intensity.

[0096] Central corneal neovascularization (Figure 8) developed in all four BSS vehicle-treated corneas, in two corneas treated with topical 0.8 mg / ml losartan alone, in one of the corneas treated with 1% prednisolone acetate alone, and in none of the corneas treated with both 0.8 mg / ml losartan and 1% prednisolone acetate.

[0097] IHC for keratocan-positive keratocytes and α-SMA-positive myofibroblasts Figure 6A shows representative central sections from corneas in each group. All alkali-injured corneas in this study had no corneal endothelium within the central 6-10 mm at 1 month after injury. No α-SMA-positive myofibroblasts were found in intact corneas. All corneas treated with BSS vehicle had full or near full thickness α-SMA-positive myofibroblasts, as shown in Figure 6A, although some patches of keratocan-positive keratocytes were present. In corneas treated with losartan alone, α-SMA-positive myofibroblasts were restricted primarily to the posterior half of the stroma in all corneas, as shown by the example in Figure 6A. The localization of α-SMA-positive myofibroblasts tended to be more variable in the prednisolone acetate group. In two corneas in that group, α-SMA-positive myofibroblasts were found throughout the stroma (as shown in Example #1 in FIG. 6A), and in two corneas, α-SMA-positive myofibroblasts were present only in the posterior half of the stroma (as shown in Example #2 in FIG. 6A). In all four corneas in the Losartan + Prednisolone Acetate combination group, α-SMA-positive myofibroblasts were restricted to the very posterior part of the stroma, with repopulation of the more anterior part of the stroma with keratocan-positive keratocytes (as shown in FIG. 6A).

[0098] FIG. 9 shows α-SMA positive staining in representative sections from each cornea used for ImageJ analysis of total area of ​​stromal α-SMA positive staining (and total α-SMA positive opacity). The variability of stromal α-SMA positive staining among the prednisolone acetate groups can be noted in this figure. The ImageJ analysis results for total α-SMA area in each cornea are shown in FIG. 6B. Table 10 shows the statistical comparison between groups. [Table 10]

[0099] The losartan-prednisolone acetate combination group had significantly less α-SMA area than the vehicle control group. The losartan-prednisolone acetate combination group had significantly less α-SMA area than the vehicle control group. It can be noted that the losartan group tended to be significantly different from the vehicle control group, although other differences did not reach statistical significance. The ImageJ analysis results of the total α-SMA opacity intensity in pixels for each cornea are shown in Figure 6C. Table 11 shows the statistical comparison between the groups. [Table 11]

[0100] Again, the losartan-prednisolone acetate combination group had highly significantly less α-SMA opacity intensity in pixels than the vehicle control group. Additionally, the losartan-prednisolone acetate combination group had significantly less total α-SMA opacity intensity than the prednisolone acetate alone group. Other differences did not reach statistical significance. Importantly, note the low variability for both total α-SMA area (FIG. 6B) and total α-SMA opacity intensity (FIG. 6C) in the losartan + prednisolone acetate combination group.

[0101] Figure 7A shows representative double IHC for collagen IV and TGFβ-1 in the anterior and posterior stroma for a representative cornea in each group, with exemplary ImageJ quantification rectangles. Figure 7B shows quantification for collagen IV staining intensity in the anterior stroma of corneas in each group. Both losartan treatment and losartan + 1% prednisolone acetate significantly reduced the mean intensity units of collagen IV in the anterior stroma compared to vehicle treatment. Prednisolone acetate alone produced a trend toward reduced collagen IV staining intensity in the anterior stroma compared to vehicle treatment, but the difference did not reach statistical significance. Table 12 shows the statistical comparison for collagen IV intensity in the anterior stroma between groups. [Table 12]

[0102] Figure 7C shows quantification of collagen IV staining intensity in the posterior stroma of the cornea in each group. Only topical losartan treatment was significantly different from vehicle treatment, while the losartan + 1% prednisolone acetate combination group trended toward significance. Table 13 shows statistical comparison of collagen IV intensity in the anterior and posterior between groups. [Table 13]

[0103] Chemical injury to the cornea caused by sodium hydroxide (NaOH) ranges from mild self-healing ocular surface damage to devastating burns affecting the corneal epithelium, limbus, stroma, and corneal endothelium. 10、12~17 Severe alkali burn injury is frequently associated with corneal neovascularization (CNV) and persistent epithelial defects. 12~17 Severe NaOH corneal burns can also damage the trabecular meshwork, iris, ciliary body, lens, retina, and optic nerve. 12

[0104] The 1N NaOH corneal burn injury method used in this example has been used in many previous rabbit studies. 10、13、14 A time point of 1 month was chosen for the analysis of the effects of alkali burn injury and the potential effects of topical agents because 1 month is the same as previous studies. 2~6This is because this is the time when the wound healing response to injury to the cornea peaks in corneal keratinocytes. This example shows that a severe chemical injury with 100 microliters of 1N NaOH, applied using a 5 mm filter paper delivery system for 1 minute, penetrated through the stroma and uniformly damaged a large area underlying the corneal endothelium and often Descemet's membrane (approximately 8-10 mm in diameter). Using this method, no evidence of limbal injury was shown. Similarly, no evidence of iris or lens damage was shown in rabbit eyes following this injury. In one particular experiment, even a 15 second exposure to 1N NaOH using this method damaged the corneal endothelium (Sampaio LP and Wilson SE, unpublished data, 2021), so dilutions of NaOH will likely be necessary to create a model of injury restricted to the epithelium and anterior stroma of the cornea.

[0105] The mode of cell death of affected epithelium, keratocytes, and corneal endothelium caused by 1 N NaOH has previously been reported to be necrosis. 15、16 Cell necrosis, possibly together with degeneration of the underlying collagen fibrils, 17 , induced a severe corneal inflammatory response that was noted by slit lamp in all corneas in this study between the first few days and 2 weeks after injury.

[0106] The opacities remaining one month after injury and treatment in all groups were characterized by a central zone of high density surrounded by a ring of lower density (Figure 5A). The central region of high density was thought to represent denatured and disorganized collagen fibers generated by the original NaOH injury, together with developed myofibroblasts and the abundant disorganized extracellular matrix produced by these fibrotic cells. 5、6、18 The lower density ring may be associated with less severely damaged stromal collagen and corneal fibroblasts, together with a lower amount of disorganized extracellular matrix produced by the corneal fibroblasts.

[0107] Losartan is an angiotensin-converting enzyme (ACE) II receptor antagonist that also inhibits TGFβ signaling. 9、19~24 In this example, topical treatment with 0.8 mg / ml losartan, 1% prednisolone acetate, or the combination of losartan and prednisolone acetate in BSS 6 times per day reduced the total corneal opacity area measured by ImageJ on standardized slit-lamp images (Figure 5B). The difference in the total area of ​​opacity was not significantly different between the losartan, prednisolone acetate, or combination of losartan and prednisolone acetate groups (Table 8). The total opacity in pixels in the corneal opacity area measured by ImageJ was significantly lower in the 0.8 mg / ml losartan group or the 0.8 mg / ml losartan + 1% prednisolone acetate group compared to the vehicle BSS group. The 1% prednisolone acetate alone group tended to have reduced total opacity compared to the vehicle BSS group, but the difference did not reach statistical significance (Table 9).

[0108] One of the most interesting findings in this example relates to the development of myofibroblasts and stromal fibrosis in the different treatment groups (Figure 6A). All corneas following alkali burn followed by treatment with vehicle BSS had α-SMA positive myofibroblasts and fibrosis throughout the full thickness of the cornea, but this fibrosis appeared to be greatest adjacent to the anterior and posterior stromal surfaces (Figure 9), likely due to higher concentrations of TGFβ-1 and TGFβ-2 penetrating into the stroma from the tears, epithelium, remaining peripheral corneal endothelium, and aqueous humor at the corneal surface. 5、6 In NaOH-injured corneas treated with 0.8 mg / ml topical losartan, the greatest density of α-SMA-positive myofibroblasts tended to be demonstrated in the posterior half of the stroma, whereas lower amounts of anterior stromal α-SMA-positive myofibroblasts were demonstrated in the two losartan-treated corneas after 1 month of treatment (Figure 9). The persistent corneal epithelial defect itself was associated with the development of anterior stromal myofibroblasts and fibrosis. 25, anterior myofibroblasts shown in the two losartan-treated corneas may have some role. Alkaline-injured corneas treated with 1% prednisolone acetate alone were more variable in stromal myofibroblast development (Figure 9). However, after injury and treatment with the combination of losartan and prednisolone acetate, α-SMA-positive myofibroblasts in all four corneas were restricted to the very posterior part of the stroma (Figure 9). As can be seen in the representative cornea in Figure 6A, it is important to note that by the 1-month time point, the corneal endothelium and Descemet's membrane had not regenerated in any of the corneas in the treatment group.

[0109] When the area of ​​α-SMA positive myofibroblasts in each central cornea was determined using ImageJ (FIG. 6B), the losartan plus prednisolone acetate combination group was significantly different from the vehicle BSS treatment group (p=0.0005, Table 10). This losartan plus prednisolone acetate combination group also had a lower standard error of the mean for the area of ​​α-SMA staining (FIG. 6B). The losartan and prednisolone acetate combination group had significantly less area of ​​α-SMA staining than the prednisolone acetate alone group. Similarly, when ImageJ was used to determine the total α-SMA intensity per corneal section in each of the corneas (FIG. 6C), the combined losartan + prednisolone acetate treatment group was significantly lower than the vehicle BSS treatment group (p=0.002, Table 11), and the combined losartan + prednisolone acetate treatment group was significantly lower than the prednisolone acetate alone group (p=0.01). Although the present invention is not limited to any particular mechanism, and an understanding of the mechanism is not necessary to practice the present invention, the inventors hypothesize that the efficacy of the combined losartan + prednisolone acetate treatment after severe alkali burn was due to corticosteroid modulation of inflammation due to severe tissue necrosis, as well as losartan modulation of profibrotic TGFβ effects on stromal myofibroblast development and thus unregulated collagen production by these cells. In addition to corneal fibroblasts, bone marrow-derived fibrocytes also invade the stroma from the limbus after injury and are precursors to TGFβ-driven myofibroblasts. 26、27Corticosteroids inhibit the proliferation of fibrocytes, which is necessary for the generation of large numbers of myofibroblasts. 28 , and also induces fibrocyte apoptosis. 29 Thus, topical corticosteroids may contribute to the losartan inhibition of myofibroblast development from both corneal fibroblasts and fibrocytes through these mechanisms.

[0110] This example demonstrates that severe sodium hydroxide injury is usually associated with damage to the corneal endothelium and Descemet's membrane that increases the corneal fibrotic response, similar to findings regarding the effects of chemical burns caused by biological warfare agents (such as sulfur mustard), where damage to the corneal endothelium is a major determinant of the long-term outcome of the injury. 30、31 Topical losartan and corticosteroid combinations were also able to reduce myofibroblast production and corneal cicatricial fibrosis that occurs in response to these chemical biowarfare agents.

[0111] References for Example 2 1. Witcher et al., Bull World Health Organ. 2001;79:214-21. 2. Torricelli et al., Invest Ophth Vis Sci. 2013:54:4026-33. 3. Marino et al., J Ref Surg. 2017;33:337-346. 4. Marino et al., Exp Eye Res. 2017;161:101-105. 5. de Oliveira et al., Exp Eye Res. 2021;202:108325 6. de Oliveira et al., J Ref Surg. 2022;38:50-60. 7. Medeiros et al., Invest Ophthalmol Vis Sci. 2018;59:4044-4053. 8. Sampaio et al., Exp Eye Res. 2021;213:108803. 9. Sampaio et al., Exp Eye Res. 216:108940. 10. Ishizaki et al., Invest Ophthalmol Vi Sci. 1993;34:3320-8. 11. Nirankari et al., Ophthalmology 1993;100:111-8. 12. Paschalis et al., J Pathol. 2017;187:1327-1342. 13. Lee et al., Graves Arch Clin Exp Ophthalmol. 2014;252:951-61. 14. Burns et al., Invest Ophthalmol Vi Sci. 1989;30:1569-75. 15. Yi et al., Trans Ophthalmol Soc U K. 1978;98:379-82. 17. Maskati et al., Ind J Ophthalmol. 1987;35:396-400. 18. Jester et al., J Cell Sci. 1999;112:613-22. 19. Wylie-Sears et al., Biochem Biophys Res Commun. 2014;446: 870-875. 20. Geirsson et al., Circulation. 2012;126(11 Suppl 1):S189-197. 21. Park et al., Cell Transplantation. 2012;21:2407-2424. 22. Lim et al., Circulation. 2001;103:789-791. 23. Lavoie et al., J Hypertens.2005;23:1895-1903. 24. Cohn et al., Nat Med.2007;13:204-210. 25. Wilson et al., J Ref Surg. 2018;34:59-64. 26. Lassance et al., Exp Eye Res. 2018;170:177-187. 27. de Oliveira et al., Invest Ophthalmol Vis Sci. 2020;61:28-35. 28. Hayashi et al., Biomed Res Int. 2014;2014:738625. 29. Lo et al., J Allergy Clin Immunol. 2015;135:1186-95.e1-6. 30. McNutt et al., Invest Ophthalmol Vis Sci. 2013;54:6735-44. 31. McNutt et al., Cornea. 2020;39:640-648. 32. Paralkar et al., Dev. Biol. 1991;143:303-308. 33. Shibuya et al., J Dermatol Sci. 2006;41:187-195. 34. Wilson et al., Matrix Bio. 2022;109:162-172

[0112] Example 3 Losartan inhibition of myofibroblast formation and late haze (scarring fibrosis) after photorefractive keratectomy (PRK) in rabbits This example evaluates and describes the effects of topical losartan compared to vehicle on myofibroblast generation and the development of delayed haze cicatricial fibrosis following PRK in rabbits.

[0113] summary Briefly, rabbits (12) underwent -9D PRK in one eye followed by 50 μl of topical losartan 0.8 mg / ml or 50 μl of vehicle six times per day for one month. Standardized slit-lamp photographs were obtained prior to euthanasia. Duplex IHC was performed on cryofixed corneas for myofibroblast marker α-smooth muscle actin (α-SMA) and keratocyte markers keratocan or type IV collagen and transforming growth factor (TGF) β-1. ImageJ was utilized for quantification. Topical losartan significantly reduced corneal opacity (P=0.04) and anterior stromal myofibroblast production (P=0.01) compared to vehicle 1 month after PRK. Topical losartan also reduced anterior stromal non-basement membrane type IV collagen (P=0.004) compared to vehicle 1 month after PRK. The topical ACEII receptor inhibitor losartan, a known inhibitor of TGFβ signaling, reduced delayed haze scar fibrosis and myofibroblast production after -9D PRK in rabbits compared to vehicle. It also reduced TGFβ-regulated corneal fibroblast-produced non-basement membrane stromal type IV collagen.

[0114] background Clinically significant delayed corneal haze (also called stromal cicatricial fibrosis) continues to be reported as a complication after photorefractive keratectomy (PRK). (1) The incidence of delayed haze after PRK has decreased significantly after widespread adoption of a single dose of intraoperative mitomycin C (2, 3), but it continues to be demonstrated in some eyes despite mitomycin C application, and it has been termed "breakthrough haze." (4) Late haze is most commonly seen after correction of moderate to high myopia or hyperopia with PRK, but delayed haze is occasionally seen after PRK correction for low myopia, especially when intraoperative mitomycin C treatment is omitted or when there is a persistent epithelial defect following surgery. (2, 3)

[0115] Several studies in rabbits have demonstrated that defective regeneration of epithelial basement membrane (EBM) and development of subepithelial myofibroblasts after PRK underlie the development of delayed haze corneal stromal fibrosis. (5-7) Myofibroblasts develop in the cornea from both keratocyte-derived corneal fibroblasts and bone marrow-derived fibrocytes through a cellular development program driven primarily by transforming growth factor (TGF) β-1 and TGF β-2. (8, 9) TGF β-1 and TGF β-2 continuously invade the stroma from the corneal epithelium and tear fluid after PRK when there is delayed regeneration of EBM, which, together with apical epithelial barrier function, regulates TGF β passage into the stroma. (6, 10-12) Perlecan and type IV collagen are EBM components that serve as gatekeepers regulating the passage of TGF β-1 and TGF β-2 into the stroma. (10-12) Myofibroblasts are critically dependent on an adequate and continuous source of TGFβ-1 and / or TGFβ-2 for complete development and survival, and upon deprivation of signaling by these growth factors, these fibrotic cells and their progenitors undergo apoptosis (8, 9).

[0116] Materials and Methods Animals and surgery Animal procedures were approved by the Institutional Animal Care and Use Committee at the Cleveland Clinic Foundation, and animals were treated in accordance with the principles of the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Twelve female New Zealand White rabbits, 12–15 weeks of age, each weighing 2.5–3 kg, were included. Rabbits were administered 60 ml pediatric liquid acetaminophen (Johnson & Johnson, Ft. Washington, PA) per liter of drinking water starting 24 h before surgery and continuing for 5 days after PRK. One eye of each rabbit was randomly selected to have −9.0 D (on the myopia scale) PRK and was administered 2 drops of 1% topical proparacaine hydrochloride (Alcon, Fort Worth, TX, USA) prior to surgery. PRK with manual epithelial debridement using a #6400 Beaver blade (MedexSupply, Passaic, NJ) was performed in rabbits under general anesthesia with 30 mg / kg ketamine hydrochloride and 5 mg / kg xylazine by intramuscular (IM) injection and local anesthesia with 1% proparacaine (Alcon, Ft. Worth, TX) using a VISX (Santa Clara, CA) S4 IR excimer laser according to previously published methods. 6、7、10 This was accomplished using 6 The effect of contralateral PRK was previously reported in 5、6、7、10 The contralateral cornea was included as an intact control, as not shown in .

[0117] Medicines Beginning immediately after surgery, six eyes that underwent PRK were treated six times daily (approximately at 8:00 AM, 10:00 AM, 12:00 PM, 2:00 PM, 4:00 PM, and 6:00 PM) with 50 μl of balanced salt solution (BSS, 0.64% sodium chloride, 0.075% potassium chloride, 0.048% calcium chloride dihydrate, 0.03% magnesium chloride hexahydrate, 0.39% sodium acetate trihydrate, 0.17% sodium citrate dihydrate, pH 7.5), and six eyes that underwent PRK were treated six times daily with 0.8 mg / ml losartan (Merck & Co., Inc., Kenilworth, NJ, USA) in 50 μl of BSS. Treatment with vehicle or losartan was continued for one month after PRK surgery. PRKed eyes were treated with one drop of topical ciprofloxacin three times daily, at least 5 minutes away from other topical medications, until the epithelium closed (the epithelium in all eyes was closed by 5 days after surgery). No corticosteroids were administered in either group.

[0118] Standardized slit-lamp photographs and ImageJ measurement of corneal opacity One month after PRK and treatment, each rabbit was anesthetized with ketamine-xylazine and the eye was dilated with 2 drops of 1% tropicamide (Akorn Co., Lake Forest, IL, USA) for 30 minutes. Slit-lamp photographs were taken of the study eye in each rabbit at 20x magnification with a Topcon (Oakland, NJ, USA) SL-D7 slit-lamp photography system with standardized illumination levels and angles. For each study cornea, the mean opacity in pixels in the central 3.5 mm PRK ablated zone was determined by using ImageJ 1.53a analysis software (National Institutes of Health, Bethesda, MD, USA).

[0119] Corneal cryofixation and sectioning Rabbits were euthanized under general anesthesia with ketamine-xylazine with 100 mg / kg Beuthanasia (Shering-Plough, Kenilworth, NJ, USA) intravenously followed by bilateral pneumothorax. The corneal-scleral rim was removed with sharp Westcott scissors (Fairfield, CT, USA) and 0.12 forceps (Storz, St. Louis, MO, USA). The corneal-scleral rim was placed in the center of a 24 mm × 24 mm × 5 mm mold (Fisher Scientific, Pittsburgh, PA, USA), which was filled with optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) and flash frozen on dry ice. Blocks were stored at −80°C until sectioning. The blocks were bisected at the center of the cornea, and 10 μm thick transverse sections were cut from the central cornea with a cryostat (HM 505M; Micron GmbH, Walldorf, Germany). Sections from each cornea were placed on 25 mm × 75 mm × 1 mm Superfrost Plus microscope slides (Fisher Scientific). Slides with sections were kept at −20°C prior to immunohistochemistry (IHC).

[0120] Immunohistochemistry (IHC) and quantification of parenchymal α-smooth muscle actin and type IV collagen Dual IHC for 1) α-smooth muscle actin and keratocan, or 2) type IV collagen and TGFβ-1 was performed using methods previously described and primary antibodies or isotype non-specific control antibodies confirmed by Western blot and IHC to recognize rabbit antigens (ThermoFisher Scientific, Waltham, MA, USA), and secondary fluorescent tag antibodies previously described (Table 14). 20 [Table 14]

[0121] The type IV collagen antibody (catalog AB769, Millipore, Temecula, CA, USA) was raised against purified human and bovine type IV collagen (affinity purified by human and bovine type IV collagen cross-linked to agarose, then cross-absorbed by the manufacturer with human and bovine collagen types I, II, III, V, and VI to remove cross-reactivity). This type IV collagen antibody binds to rabbit type IV collagen in IHC. 6、20 It has previously been shown that it binds to the α-1 / α-2 chains but not the α-3 to α-6 chains.

[0122] A keratocyte-specific keratocan antibody was raised against the peptide H2N-LRLDGNEIKPPIPIDLVAC-OH (SEQ ID NO: 1). The TGFβ-1 antibody used (GeneTex, Irvine, CA, USA) binds to rabbit TGFβ-1 in IHC and shows no reactivity to TGFβ-2 or TGFβ-3. 6、20 Images were acquired at 100x total magnification on a Leica DM6B upright microscope equipped with an automated stage and a Leica 7000T camera using LASX software (Leica Microsystems, GmbH, Wetzlar, Germany).

[0123] All images were converted to 300 DPI, 900 pixel wide x 672 pixel high images with Photoshop 22.1.1 (Adobe, San Jose, CA). The average pixels for stromal α-SMA or stromal collagen type IV were determined in a 900 pixel wide x 235 pixel high rectangle (for quantification of either α-SMA or collagen type IV) with ImageJ for three sections of each cornea using an image panel showing only the color of interest. The average from the three corneal sections was used for each individual cornea as the value for stromal α-SMA or stromal collagen type IV.

[0124] statistics Comparisons between groups were performed using the Kruskal Wallis test followed by post-hoc Dunn's-Bonferroni test. P<0.05 was considered statistically significant.

[0125] result Figure 10A provides standardized slit lamp photographs from each cornea in the vehicle and losartan groups after -9D (on the myopia scale) PRK and one month of topical drug treatment. This figure also shows a uniform central circular 3.5 mm diameter area within the PRK ablation in each cornea analyzed for opacification using ImageJ. Figure 10B shows the average pixels of opacification within the 3.5 mm diameter area analyzed for each cornea in the vehicle and losartan treatment groups. The difference between the vehicle group (mean ± SEM, 93 ± 6 pixels) and the losartan group (mean ± SEM, 77 ± 3) was statistically significant (p = 0.04).

[0126] FIG. 11A shows representative dual immunohistochemistry for α-SMA and keratocan in central corneas in vehicle and losartan groups after −9D PRK and one month of topical drug treatment. Also shown for each cornea is a 900 pixel wide by 235 pixel high area analyzed for total pixels of α-SMA staining intensity by ImageJ on the panel showing staining for α-SMA only. FIG. 11B shows a graph of total pixels of α-SMA staining within the square analyzed for each cornea. The difference between the vehicle-treated group (mean ± SEM, 1630 ± 840 pixels) and the losartan-treated group (mean ± SEM, 120 ± 60 pixels) was statistically significant (p = 0.01). Note the much larger variation (higher SEM) in the vehicle group compared to the losartan group.

[0127] FIG. 12A shows representative exemplary double immunohistochemistry for type IV collagen and TGFβ-1 in a representative intact control central cornea, and in vehicle and losartan-treated corneas after -9D PRK and one month of topical drug treatment. In intact corneas, TGFβ-1 was localized in large amounts in the epithelium and corneal endothelium, with lesser amounts detected in the stroma. In intact corneas, type IV collagen was mainly localized in the epithelial basement membrane and Descemet's membrane, with little detection in the stroma. In corneas with PRK and one month of vehicle or losartan treatment, TGFβ-1 was still localized in the epithelium and corneal endothelium, but was also prominently localized in the epithelial basement membrane in both treatment groups. In vehicle-treated corneas, a prominent line of TGFβ-1 was present in the stroma just below the type I collagen band in each cornea in that group.

[0128] Figure 12A shows that high levels of type IV collagen were present in bands in the subepithelial stroma beneath the epithelial basement membrane even in the vehicle-treated group 1 month after -9D PRK. In the losartan-treated group 1 month after -9D PRK, type IV collagen was primarily localized to the epithelial basement membrane, with much smaller amounts of type IV collagen detected in the underlying anterior stroma.

[0129] Figure 12B is a graph of the total pixels of collagen IV signal within a stromal rectangle of 900 pixels wide by 235 pixels high analyzed for each cornea. The difference between the vehicle-treated group (628700±37100) and the losartan-treated group (191000±9000) was statistically significant (p=0.004).

[0130] Late corneal haze after PRK is a clinical sign of stromal fibrosis resulting from the development and persistence of subepithelial myofibroblasts after surgery. These fibroblasts are themselves impermeable due to downregulation of corneal crystallins compared to keratocytes. 21Myofibroblasts develop from precursor cells, including local corneal fibroblasts (derived from keratocytes) and bone marrow-derived fibrocytes, which invade the cornea from limbal blood vessels in response to corneal injury. 22、23 Once mature myofibroblasts develop in the subepithelial stroma, they produce large amounts of disorganized extracellular matrix components (such as collagen type I and collagen type III), which further impairs corneal transparency. 22 The development of myofibroblast precursors into mature myofibroblasts, and the persistence of myofibroblasts in tissues, depends on a continuous and adequate supply of TGFβ-1 and / or TGFβ-2 and other growth factors, including platelet-derived growth factor. 6、10

[0131] Several studies have demonstrated that delayed or defective regeneration of the epithelial basement membrane (EBM), which is produced through the coordinated efforts of epithelium and keratocytes / corneal fibroblasts, underlies the development of late haze fibrosis. 6、10 Perlecan and type IV collagen are important EBM components that regulate the entry of TGFβ-1 and TGFβ-2 from the epithelium and tear film into the stroma. The development of late haze fibrosis reduces visual acuity after PRK, possibly through a combination of stromal opacification, irregularities transmitted to the corneal surface, and regression of the refractive effects of PRK. 22 Once late haze develops in rabbit or human corneas, it persists until normal EBM is regenerated, a process that typically takes several months in rabbits and months to years in humans after the development of late haze cicatricial fibrosis. In this example, one month was chosen for analysis because many studies have shown that this is the peak of corneal late haze fibrosis after PRK in rabbits. 5~7、10 Regeneration of normal mature EBM re-establishes barrier function for epithelial and tear TGFβ-1 and TGFβ-2, reduces levels of these profibrotic growth factors in the subepithelial stroma, and induces apoptosis of myofibroblasts. 24, or possibly reverting myofibroblasts to a corneal fibroblast phenotype, although the latter mechanism of disappearance has not yet been demonstrated in situ. Once myofibroblasts in the stroma decline, corneal fibroblasts and keratocytes re-enter the affected stromal tissue and resorb / reorganize the disorganized extracellular matrix produced by myofibroblasts, increasing corneal transparency. 22

[0132] Each of the corneas in Figure 10A-9D one month after treatment with PRK and topical losartan had residual central opacity despite a reduction in myofibroblasts, primarily due to disorganized extracellular matrix (ECM) components (such as collagen type I and collagen type III) produced by corneal fibroblasts and the small number of myofibroblasts that developed despite losartan treatment at this early time point after surgery. 21、22 Many corneas with PRK without myofibroblast development develop clinically insignificant haze due to reduced crystallin production by corneal fibroblasts and the production of relatively low levels of unregulated ECM by these cells. 21、22 This transient opacity typically diminishes over several months as corneal fibroblasts that developed in response to surgery are eliminated by a combination of apoptosis and reversion to a keratocyte phenotype. 22 Therefore, the corneas in both groups in this study would probably have become clearer with longer follow-up.

[0133] This example demonstrates that topical losartan reduces non-basement membrane stromal type IV collagen production, which is also upregulated in corneal fibroblasts via TGFβ signaling (FIGS. 12A-B). 19、20、27 Parenchymal type IV collagen appears to function by directly binding TGFβ-1 and TGFβ-2 and regulating the interaction of TGFβ with its receptors. 27One might be concerned that this inhibition of type IV collagen production might have deleterious effects on the overall TGFβ-mediated fibrotic response, however this was not shown in this example.

[0134] References for Example 3 1. Lipshitz et al., Ophthalmology. 1997;104:369-73 2. Raviv et al., J Cataract Refract Surg. 2000;26:1105-6. 3. de Oliveira R et al., Exp Eye Res. 2020;200:108218. 4. Kaiserman et al., Cornea. 2017;36:961-966. 5. Torricelli et al., Invest Ophthalmol Vis Sci. 2013;54:4026-33. 6. de Oliveira et al., J Refract Surg. 2022;38:50-60. 7. Marino et al., J Refract Surg. 2017;33:337-346. 8. Wilson et al., Corneal Invest Ophthalmol Vis Sci. 2020;61:28. 10. de Oliveira et al., Exp Eye Res. 2021;202:108325. 11. Wilson et al., Exp Eye Res. 2021;207:108594. 12. Wilson SE. Cell Mol Life Sci. 2022;79:144. 13. Wylie-Sears et al., Biochem Biophys Res Commun. 2014;446: 870-5. 14. Geirsson et al., Circulation. 2012;126(11 Suppl 1):S189-97. 15. Park et al., Cell Transplant. 2012;21:2407-24. 16. Lim D-S, et al., Circulation. 2001;103:789-91. 17. Lavoie et al., J Hypertens. 2005;23:1895-1903. 18. Cohn et al., Nat Med. 2007;13:204-10. 19. Sampaio et al., Exp Eye Res. 2022;216:108940. 20. Sampaio LP, Hilgert GSL, Shiju TM, Santhiago MR, Wilson SE. Topical losartan and corticosteroid additively inhibit corneal stromal myofibroblast generation and scarring fibrosis after alkali burn injury. Trans Vis Sci Tech, in press. 21. Jester et al., J Cell Sci. 1999;112:613-22. 22. Wilson et al., Invest. Ophth. Vis. Sci. 2022;63:22. 23. Lassance et al., Exp. Eye Res. 2018;170:177-187. 24. Wilson et al., Exp. Eye Res. 2007;85:305-11. 25. Jester et al., Cornea. 1997;16:177-87. 26. Angiotensin II Receptor Antagonists, In: LiverTox: Clinical and research information on drug-induced liver injury [Internet]. Bethesda (MD): National Institute of Diabetes and Digestive and Kidney Diseases;2012-2017. 27. Wilson et al., Matrix Biol. 2022;109:162-172. 28. Majmudar et al., Ophthalmology. 2000;107:89-94. 29. Medeiros et al., Invest Ophthalmol Vis Sci. 2019;60:1010-1020. 30. Sampaio et al., Exp Eye Res. 2021;213:108803. 31. Waldrop et al., Cornea. 2020;39:1227-1234. 32. Kobayashi et al., Ophthalmology. 2013;120:923-927. 33. Abu el-Asrar et al., Eye (Lond). 1998;12 ( Pt 3a):453-60. 34. Schlunck et al., Exp Eye Res. 2016;142:76-82.

[0135] All publications and patents mentioned in the above specification are incorporated herein by reference.Various modifications and variations of the described compositions and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the present invention.

Claims

1. A composition for use in a method for treating corneal injury and / or existing corneal scarring in a subject, comprising: The composition comprises: a) a drug comprising an ACE-2 receptor antagonist; b) water, and c) Below: i) one or more salts present at levels such that the composition, when in aqueous form, has an approximately physiological concentration of said salt or salts and an approximately physiological pH; ii) one or more gelling agents present at levels such that the composition is in the form of a gel; and iii) one or more ointment forming agents, present at levels such that the composition is in the form of an ointment at least one of; d) an optional preservative; and e) an optional soothing agent; A composition comprising:

2. A composition for use according to claim 1 defined by one of the following: i. the agent is present in the composition at a concentration of 0.1 mg / ml to 2.0 mg / ml; ii. the composition is for at least daily administration to the subject for at least one week, or at least two weeks, or at least one month, wherein the subject has a best corrected visual acuity (BSCVA) of 20 / X immediately before administering, and then 20 / Y at the end of the at least one week, the at least two weeks, or the at least one month, wherein Y is at least 5 points lower than X, or wherein Y is at least 15 points lower than X; iii. the composition does not contain or detectably does not contain any additional reagents other than the drug, the water, and the one or more salts; iv. the composition comprises the soothing agent, and the composition is free of or detectably free of any additional reagents other than the drug, the water, the one or more salts, and the soothing agent; vi. a) the composition further comprises the preservative, and the composition is free of or detectably free of any additional reagents other than the agent, the water, the one or more salts, and the preservative; or b) the composition further comprises the preservative and the soothing agent, and the composition is free of or detectably free of any additional reagents other than the drug, the water, the one or more salts, the preservative, and the soothing agent; the preservative is selected from the group consisting of benzalkonium chloride, sodium dichloride, sodium perborate, purite, benzododecinium bromide, ethylenediaminetetraacetic acid (EDTA), chlorobutanol, thiomersal, disodium edetate, and oxychloro complex (SOC); v. the soothing agent is present in the composition, and the soothing agent is optionally selected from the group consisting of carboxymethylcellulose, polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, and hyaluronic acid; vii. the composition is present in an eye dropper container; viii. the composition comprises the one or more salts, is in liquid form, and is free of or detectably free of the one or more gelling agents and the one or more ointment forming agents; ix. the composition is in the form of a gel or ointment comprising the one or more gelling agents and / or the one or more ointment formers, optionally wherein the gelling agent is selected from the group consisting of hypromellose, carbomer homopolymer, and carboxymethylcellulose, and optionally wherein the ointment former is mineral oil and / or petrolatum; x. the composition is for administration at least 4, or 6, or 8 times daily for at least 1 week; xi. the composition is for administration about every 30 minutes for at least 8 hours; xii. the subject's cornea contains the corneal injury, and the composition is for administration at least daily for at least one week starting within one to five days from the appearance of the corneal injury, such that one month after the appearance of the corneal injury, the cornea has a Fantes slit lamp corneal haze score of 0, 0.5, 1, or 2, and the corneal injury, if left untreated, would result in a Fantes slit lamp corneal haze score of 3 or 4 after said one month; xiii. the cornea of ​​the subject contains the corneal injury, and the composition is for administration at least daily for at least one week starting within one to five days from the appearance of the corneal injury, such that one month after the appearance of the corneal injury, the cornea has a Fantes slit lamp corneal haze score of 0, 0.5, or 1, and the corneal injury, if left untreated, would result in a Fantes slit lamp corneal haze score of 2, 3, or 4 after the one month; xiv. the drug is selected from the group consisting of losartan, telmisartan, valsartan, olmesartan, candesartan, irbesartan, eprosartan, azilsartan, and the losartan metabolite EXP3174; xv. the one or more salts are: i) about 0.64% sodium chloride; ii) about 0.075% potassium chloride; iii) about 0.048% calcium chloride dihydrate; iv) about 0.03% magnesium chloride hexahydrate; v) about 0.39% sodium acetate trihydrate, and / or vi) about 0.17% sodium citrate dihydrate including one or more or all of the following: xvi. the composition further comprises a corticosteroid for use in treating the cornea of ​​the subject, wherein the corticosteroid is present in the composition or in a separate composition; xvii. the composition is present in a conjunctival reservoir or other continuous delivery device that slowly releases the composition into the subject's tear fluid over time; xviii. the composition is present in a porous collagen therapeutic contact lens that releases the composition over time; xix. the composition comprises the preservative; xx. the composition is for administration to the subject at least daily for at least one week, or at least two weeks, or at least one month, wherein the subject has a myopia score of X diopters at or immediately before said administering and Y diopters at the end of said at least one week, said at least two weeks, or said at least one month, wherein Y is at least 1 diopter lower than X; xxi. The subject is a human; xxii. the cornea of ​​the subject contains the corneal injury, and the corneal injury occurred 1, 3, 6, 12, 24, or 48 hours prior to the use in the method; xxiii. the composition is for administration by the subject to their own cornea; xxiv. the cornea of ​​the subject contains a corneal injury, and the injury is caused by trauma, a chemical burn, a microbial infection, or surgery; xxv. the cornea of ​​the subject contains the corneal injury, and the injury was caused by photorefractive keratectomy (PRK) or therapeutic laser keratectomy (PTK); xxvi. the corneal injury occurs within 5 days of use; xxvii. the corneal injury occurs within 24 hours of the use; xxviii. The drug comprises losartan.

3. A system for use in delivery to a subject, comprising: the subject has an eye containing a corneal injury or a pre-existing corneal scar; The system is a) an eye dropper or contact lens, and b) i) a drug comprising an ACE-2 receptor antagonist; ii) water, and iii) Below: A) one or more salts present at levels such that the composition, when in aqueous form, has an approximately physiological concentration of said salt or salts and an approximately physiological pH; B) one or more gelling agents present at levels such that the composition is in the form of a gel; and C) one or more ointment forming agents present at levels such that the composition is in the form of an ointment at least one of; iv) an optional preservative; and v) an optional soothing agent; A composition comprising A system including:

4. A system for use according to claim 3 defined by one of the following: i. the agent is present in the composition at a concentration of 0.1 mg / ml to 2.0 mg / ml, 0.2 to 0.9 mg / ml, 0.7 to 0.8 mg / ml, or 0.4 to 0.6 mg / ml; ii. the composition does not contain or detectably contains any additional reagents other than the drug, the water, and the one or more salts; iii. the composition is present in the eye dropper container; iv. the composition comprises the soothing agent, wherein the soothing agent is optionally selected from the group consisting of carboxymethylcellulose, polyvinyl alcohol, hydroxypropylmethylcellulose, hydroxypropylcellulose, and hyaluronic acid, and the composition does not contain or detectably does not contain any additional reagents other than the drug, the water, the one or more salts, and the soothing agent; the composition further comprises the preservative and the soothing agent, and the composition is free of or detectably free of any additional reagents other than the drug, the water, the one or more salts, the preservative, and the soothing agent; v. the composition further comprises the preservative, wherein the composition is free of or detectably free of any additional reagents other than the agent, the water, the one or more salts, and the preservative, and the preservative is selected from the group consisting of benzalkonium chloride, sodium perchlorate, sodium perborate, purite, or benzododecinium bromide; vi. The eye dropper container is a disposable container; vii. the composition comprises the at least one ointment forming agent and is in the form of an ointment; viii. the composition is present in the contact lens; ix. the composition comprises the preservative; x. the composition further comprises a soothing agent, wherein the soothing agent is selected from the group consisting of methylcellulose, hydroxypropylmethylcellulose, dextran, glycerin, carbomer, hyaluronic acid, phospholipids, saturated fatty acids, unsaturated fatty acids, triglycerides, benzalkonium chloride, and sodium ethylenediaminetetraacetate; xi. The subject is a human subject; xii. the cornea of ​​the subject contains the corneal injury, and the corneal injury occurred 1, 3, 6, 12, 24, 48 hours, or 5 days prior to the delivery; xiii. The cornea of ​​the subject contains a corneal injury, and the injury is caused by trauma, a chemical burn, a microbial infection, or surgery; xiv. the cornea of ​​the subject contains the corneal injury, and the injury was caused by photorefractive laser keratectomy or therapeutic laser keratectomy; xv. the composition comprises the one or more salts, is in liquid form, and is free of or detectably free of the one or more gelling agents and the one or more ointment forming agents; xvi. the corneal injury occurs within 24 hours of the delivery; xvii. The drug comprises losartan.

5. a) a drug comprising an ACE-2 receptor antagonist; b) water, and c) Below: i) one or more salts present at levels such that the composition, when in aqueous form, has an approximately physiological concentration of said salt or salts and an approximately physiological pH; ii) one or more gelling agents present at levels such that the composition is in the form of a gel; and iii) one or more ointment forming agents, present at levels such that the composition is in the form of an ointment at least one of; d) an optional preservative; and e) an optional soothing agent; A composition comprising:

6. 6. The composition of claim 5, wherein the agent is present in the composition at a concentration of 0.2 to 0.9 mg / ml.

7. the composition comprises the one or more gelling agents and / or the one or more ointment forming agents and is in the form of a gel or ointment, optionally wherein the gelling agent is selected from the group consisting of hypromellose, carbomer homopolymer, and carboxymethylcellulose, and optionally wherein the ointment forming agent is mineral oil and / or petrolatum; 6. The composition of claim 5, wherein the composition is in liquid form and does not contain or detectably contains any additional reagents other than the agent, the water, and the one or more salts.

8. 6. The composition of claim 5, wherein the composition is in liquid form and comprises the soothing agent, and the composition does not contain or detectably does not contain any additional reagents other than the drug, the water, the one or more salts, and the soothing agent.

9. 6. The composition of claim 5, wherein the composition further comprises the preservative and the soothing agent, and wherein the composition does not contain or detectably does not contain any additional reagents other than the drug, the water, the one or more salts, the preservative, and the soothing agent.

10. the composition further comprises the preservative, and the composition is free of or detectably free of any additional reagents other than the agent, the water, the one or more salts, and the preservative; 6. The composition of claim 5, wherein the antiseptic material is selected from the group consisting of benzalkonium chloride, sodium perchlorate, sodium perborate, purite, or benzododecinium bromide.

11. 6. The composition of claim 5, wherein the agent is present in the composition at a concentration of 0.7 to 0.9 mg / ml.

12. The composition of claim 5 , wherein the composition comprises the one or more gelling agents and is in the form of a gel.

13. the composition comprises the antiseptic, and the antiseptic comprises an antibiotic; or 6. The composition of claim 5, wherein the composition comprises the soothing agent, and the soothing agent is selected from the group consisting of methylcellulose, hydroxypropylmethylcellulose, dextran, glycerin, carbomer, hyaluronic acid, phospholipids, saturated fatty acids, unsaturated fatty acids, triglycerides, benzalkonium chloride, and sodium ethylenediaminetetraacetate.

14. a) i) a drug comprising an ACE-2 receptor antagonist; ii) water, and iii) Below: A) one or more salts present at levels such that the composition, when in aqueous form, has an approximately physiological concentration of said salt or salts and an approximately physiological pH; B) one or more gelling agents present at levels such that the composition is in the form of a gel; and C) one or more ointment forming agents present at levels such that the composition is in the form of an ointment at least one of; iv) an optional preservative; and v) an optional soothing agent; a composition comprising: b) eye dropper or contact lens; A system containing 15. The system of claim 14 defined by one of the following: i) the system comprises the eye dropper and the composition is present within the eye dropper; ii) the system comprises the contact lens and the composition is present within the contact lens or on the inner surface of the contact lens; iii) the agent is present in the composition in a concentration of from 0.1 mg / ml to 2.0 mg / ml; iv) the agent is present in the composition in a concentration of from 0.7 mg / ml to 0.9 mg / ml;

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