Dexamethasone sodium phosphate loaded nanoparticles for prevention and treatment of vesicant agent induced ocular injury

Abstract

Provided herein are methods of preventing and / or treating vesicant agent induced ocular injury. The methods comprise treating the eyes of a subject injured or at risk of being injured by a vesicant agent with dexamethasone sodium phosphate (DSP) loaded nanoparticles. In some aspects, the vesicant agent is mustard gas.

Description

[0001] DEXAMETHASONE SODIUM PHOSPHATE LOADED NANOPARTICLES FOR PREVENTION AND TREATMENT OF VESICANT AGENT INDUCED OCULAR INJURY

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of United States provisional patent application 63 / 756,559 filed February 10, 2025.

[0003] STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support under grant numbers R01EY027827 and R21EY035974 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

[0005] BACKGROUND OF THE INVENTION

[0006] Technical Field

[0007] The invention generally relates to methods of preventing and / or treating vesicant agent induced ocular injury in a subject. In particular, the invention provides methods of preventing and / or treating vesicant agent (e.g. mustard gas) induced ocular injury in the subject by administering dexamethasone sodium phosphate (DSP) loaded nanoparticles to the eyes of the subject.

[0008] Description of Related Art

[0009] Vesicating chemical warfare agents have been widely deployed as a terrorist weapons causing significant threat to human health. Upon exposure, they can cause severe dermal, gastrointestinal, respiratory, and ocular injuries. Mustard gases were first discovered in the 18th century and their simplicity of synthesis, potent toxic manifestations, and the presence of massive stockpiles, make them one of most feared agents of warfare and terrorism even today. Sulfur mustard (SM) and nitrogen mustard (NM) are widely used vesicating agents during warfare, leading to battlefield injuries and casualties. Due to their high reactivity and alkylating characteristics, mostly attributable to the alkylation of DNA at the N-7 position of guanine, both SM and NM are considered to have similar mechanisms of toxicity. NM is a bifunctional analogue of SM, sharing similar structural and toxicological properties, and has been widely used to study ocular injuries. NM possesses crosslinkingproperties similar to SM, is commercially available, and has been widely used in research of vesicant-induced damage in lieu of SM since the latter is a strictly controlled substance with limited access by research facilities. Exposure to NM causes severe injuries to the skin and eyes, inhalation effects to the lungs, and several systemic toxicides depending upon the dose, duration, and route of exposure. Ocular exposure is the most destructive of the several routes of vesicant exposure, making the eyes the most vulnerable organ to NM exposure. Mild exposure causes photophobia, irritation, inflammation, lacrimation, swelling / edema, and blepharospasm. Furthermore, long and severe effects are comeal opacity, comeal ulceration, chronic inflammation, dryness, epithelial abrasion, keratopathy, corneal neovascularization, conjunctival scarring, and blindness.

[0010] In the current clinical setting, prophylactic and therapeutic interventions for mustard-induced ocular injury are limited to the use of protective equipment, ocular irrigation with water, use of local lubricants, antibiotics, or ocular hypotensive medications as required. To overcome these limitations, numerous efforts have been made in exploring effective medical countermeasures including the use of treatments such as colchicine, a naturally occurring alkaloid that affects axoplasmic transport, diltiazem, deferoxamine combined with zinc / gallium, and diphoterine rinsing solutions. However, these interventions have not fully met the required standards for successful treatment due to pitfalls like systemic toxicity, increased intraocular pressure (IOP), and suboptimal efficacy.

[0011] One of the most promising therapy options is the use of corticosteroids, whose antiinflammatory properties lend themselves to be repurposed for management of vesicant-induced corneal inflammation. Dexamethasone sodium phosphate (DSP) is an FDA-approved corticosteroid which is routinely used in clinical settings to treat ocular inflammations ad is typically administered topically as eye drops. However, topical corticosteroids have low ocular bioavailability and require repetitive dosing, which lowers patient compliance, especially when dosing frequency is high. Repetitive dosing of corticosteroid can also have adverse side effects, such as increased IOP, glaucoma, and cataracts. The current standard for DSP eyedrop (0.1%, USP) application is two to three times a day, with severe inflammatory conditions requiring dosing every two to 3 h.

[0012] Thus, for treating vesicant-induced comeal injuries, there exists a pressing clinical need for delivery of therapeutic levels of corticosteroids to the anterior chamber for an extended period of time.SUMMARY OF THE INVENTION

[0013] Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

[0014] The invention generally relates to methods of preventing and / or treating vesicant agent induced ocular injury. In particular, the invention provides methods of preventing and / or treating ocular injury caused by exposure of the eyes of a subject to a vesicant agent. The methods comprise administering to at least one eye of the subject nanoparticles comprising at least one a corticosteroid such as dexamethasone sodium phosphate (DSP). In some aspects, the disclosure provides glucocorticoid phosphate prodrugs, which are chelated within nanoparticles, for example, via a dexamethasone phosphate-zinc chelation. In particular, what is administered is a sodium or potassium salt of e.g. a dexamethasone phosphate salt. The nanoparticles release the corticosteroid slowly over time, decreasing the need for frequent dosing. The administration (e.g. by subconjunctival (SCT) injection) of formulations comprising the nanoparticles advantageously does not cause an appreciable increase in intraocular pressure (IOP)

[0015] It is an object of this invention to provide a method of preventing or treating at least one symptom of vesicant agent induced injury to at least one eye of a subject in need thereof, comprising administering to the at least one eye a composition comprising nanoparticles loaded with at least one corticosteroid. In some aspects, the at least one corticosteroid is dexamethasone sodium phosphate (DSP). In further aspects, the nanoparticles comprise a polymer or copolymer comprising one or more poly(hydroxyacid) polymers, optionally coated with one or more hydrophilic polymers. In additional aspects, the copolymer is comprised of poly(D,L-lactic acid-glycolic acid (LA:GA) in a 50:50 ratio and the one or more hydrophilic polymers is polyethylene glycol (PEG). In yet further aspects, the at least one corticosteroid is present in the nanoparticles at a loading of 5-10% by weight. In some aspects, the vesicant agent is a chemical warfare agent, an arsenical, a halogenated oxime or a pharmaceutical vesicant. In further aspects, the chemical warfare agent is a sulfur mustard or a nitrogen mustard; the arsenical is Lewisite; the halogenatedoxime is phosgene oxime; and the pharmaceutical vesicants is a chemotherapy drug. In other aspects, the chemotherapy drug is doxorubicin or vincristine. In some aspects, the at least one symptom is selected from the group consisting of: upregulation of inflammatory cytokines, angiogenic markers, fibrotic markers, comeal ulceration, corneal neovascularization, corneal opacity, increased comeal thickness, and epithelial degradation. In additional aspects, the step of administering is performed by subconjunctival (SCT) injection. In other aspects, the step of administering forms a depot of nanoparticles that releases the at least one corticosteroid over a period of at least a plurality of days or weeks. In some aspects, the at least one corticosteroid is released over a period of at least two weeks.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Fig. 1A-E. Progression of corneal ulceration following nitrogen mustard (NM) exposure. Both eyes of healthy Sprague Dawley rats were injured with NM at varying concentrations (0.5%, 1%, and 2% w / v) for two exposure durations (1 min and 2 min). At day 7 and 14 post NM injury (PID7 and PID14), corneal ulceration was evaluated using ophthalmic fluorescein staining. (A) Representative slit-lamp images on PID14 showing corneal ulceration following NM exposure. (B) Quantification of corneal ulceration on PID7 and (C) PID14, with comparisons between exposure durations at all tested NM concentrations. (D) Quantification of comeal scarring on PID7 and (E) PID14. Mean ± SEM, n = 6; Two-way ANOVA followed by Tukey’s multiple comparison test: $p < 0.05, $$p < 0.01, $$$ / ? < 0.001 represents comparison of fluorescence intensity between pre-injury (healthy control) and tested NM concentrations for 1 min exposure. #p < 0.05, ##p < 0.01, ###p < 0.001 represents comparison of fluorescence intensities between pre-injury (healthy control) and tested NM concentrations for 2 min exposure. *p < 0.05; **p < 0.01; ***p < 0.001 represents comparison of fluorescence intensities between 1 min and 2 min exposure at tested NM concentrations.

[0018] Fig. 2A-E. Progression of comeal neovascularization and opacity following NM exposure. Both eyes of healthy Sprague Dawley rats were injured with NM at varying concentrations (0.5%, 1%, and 2% w / v) for two exposure durations (1 min and 2 min). On day 7 and 14 post-NM injury (PID7 and PID14), length of new blood vessels invading the cornea was evaluated and corneal opacity was quantified with scoring method. (A) Representative digital microscopic images on PID14 showing growth of blood vessels in the cornea withapparent corneal opacity following NM exposure. (B) Quantification of corneal opacity on PID7 and (C) PID14 with comparisons between exposure durations at all tested NM concentrations. (D) Quantification of corneal neovascularization (NV) length on PID7 and (E) PID14, with comparisons between exposure durations at all tested NM concentrations. Mean ± SEM, n = 6. Two-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001 represents comparison between 1-min and 2-min exposure durations at tested NM concentrations.

[0019] Fig. 3A-C. Histopathologic changes in the cornea following NM injury. Both eyes of healthy Sprague Dawley rats were injured with NM at varying concentrations (0.5%, 1%, and 2% w / v) for two exposure durations (1 min and 2 min). On day 14 post NM injury (PID14), rats were euthanized, and eyeballs were collected followed by histopathologic analysis using H&E staining. (A) Quantification of comeal epithelial thickness and (B) total comeal thickness on PID14 with comparisons between exposure durations at all tested NM concentrations. (C) Representative histopathologic images of rat corneas for all experimental groups on PID14. Surface epithelial cells are represented with asterisk (*), stromal keratocytes are represented with black arrows, and inflammatory cells are represented with white arrows. Mean ± SEM, n = 3. Two-way ANOVA followed by Tukey’s multiple comparison test: $p < 0.05, $$p < 0.01, $$$ / ? < 0.001 represent comparisons between pre-injury (healthy control) and tested NM concentrations for l-min exposure. #p < 0.05, ##p < 0.01, ###p < 0.001 represent comparisons between pre-injury (healthy control) and tested NM concentrations for 2-min exposure. *p < 0.05; **p < 0.01; ***p < 0.001 represent comparisons between 1-min and 2-min NM exposure. Scale bar: 100 pm.

[0020] Fig. 4. NM exposure upregulates the proinflammatory and angiogenic cytokines in injured cornea. Both eyes of healthy SD rats were injured with 1% NM for 1 min. On day 14 post NM injury (PID14), rats were euthanized, and the eyeballs were collected followed by comeal separation. Proinflammatory cytokines (TNFa, IFN-y, NF-kB, IL-6, IL-ip, and NF-kB) and angiogenic cytokines (MMP-2, MMP-9, and VEGF) in rat corneas were evaluated by qPCR. Mean ± SEM, n = 3. Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001 represents comparison between healthy cornea (Ctrl) and NM injured cornea (NM).

[0021] Fig. 5A and B. Physicochemical properties of PLGA-DSP-NP. (A) Representative transmission electron microscope (TEM) image of PLGA-DSP-NP. (B) In vitro drug releaseof DSP from PLGA-DSP-NP under sink conditions at 37 °C. Mean ± SD, n = 3.

[0022] Fig. 6A-C. PLGA-DSP-NP prevents corneal ulcer progression following a single SCT injection after exposure to 1% NM for 1 min. All groups received their respective treatments immediately after the NM exposure and were followed for 14 days. (A) Representative slitlamp images demonstrating corneal ulceration on PID14. (B) Quantitative analysis of comeal ulceration expressed as mean fluorescence intensity (a.u.) on PID14. (C) Quantitative analysis of comeal scarring score on PID14 for different treatment groups. Mean ± SEM, n = 7. Two-way ANOVA followed by Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001.

[0023] Fig. 7A-D. PLGA-DSP-NP prevents comeal neovascularization and opacity following a single SCT injection after injury by 1% NM for 1 min. All groups received their respective treatments immediately after the NM exposure and were followed for 14 days. (A) Representative microscopic images of rat corneas on PID14 representing comeal NV and opacity. (B) Quantitative analysis of comeal NV length, (C) corneal NV score, and (D) comeal opacity scores on PID14 for different treatment groups. Mean ± SEM, n = 7. Two-way ANOVA followed by Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001.

[0024] Fig. 8A-G. A single SCT injection of PLGA-DSP-NP prevents upregulation of inflammatory, angiogenic and profibrotic cytokines following injury by 1% NM for 1 min. All groups received their respective treatments immediately after the NM exposure and were followed for 14 days. qPCR analysis of relative expression of (A) MMP-2, (B) MMP-9, (C) PDGFRa, (D) PDGFRp, (E) VEGF, (F) IFN-y and (G) TGFpi in corneal tissues collected on day 14 post injury (PID14) compared to healthy corneas. Mean ± SEM, n = 3. Two-way ANOVA followed by Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001.

[0025] Fig. 9A-C. A single subconjunctival (SCT) injection of PLGA-DSP-NP preserves corneal health with intact corneal layers (epithelium, stroma, and endothelium) following exposure to 1% NM for 1 min. All groups received their respective treatments immediately following NM exposure and were then followed for 14 days. (A) Representative H&E-stained comeal sections from different treatment groups on PID14 following 1% NM exposure for 1 min.

[0026] (B) Quantification of corneal epithelial thickness and (C) total corneal thickness on PID14 for different treatment groups. Surface epithelial cells are represented with asterisk (*),-1-stromal keratocytes are represented with black arrows, and inflammatory cells are represented with white arrows. Mean ± SEM, n = 3. Two-way ANOVA followed by Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar: 100 pm.

[0027] Fig 10A-F. Quantification of comeal ulceration for (a) 1 min NM exposure and (b) 2 min NM exposure demonstrating comparisons between ulcer progression at PID7 and PID14 at all tested NM concentrations. Quantification of corneal opacity score for (c) 1 min NM exposure and (d) 2 min NM exposure demonstrating comparisons between corneal opacification at PID7 and PID14 at all tested NM concentrations. Quantification of comeal NV length for (e) 1 min NM exposure and (f) 2 min NM exposure with comparisons between comeal NV lengths at PID7 and PID14 at all tested NM concentrations. Data are represented as Mean ± SEM, n=6. Two-way ANOVA followed by Tukey’s multiple comparison test:#p < 0.05,##p < 0.01,###p < 0.001 represents comparison of fluorescence intensities between pre-injury (healthy control) and tested NM concentrations on PID7.$p < 0.05,$$p < 0.01,sss< 0.001 represents comparison of fluorescence intensity between preinjury (healthy control) and tested NM concentrations on PID14. *p < 0.05; **p < 0.01; ***p < 0.001 represents comparison of corneal NV length between PID7 and PID14 at tested NM concentrations in fig 10(e) and 10(f).

[0028] DETAILED DESCRIPTION

[0029] Vesicating agents are used e.g. in warfare due to their easy and low-cost production and effectiveness (high absorption through tissues, acute to chronic effects on biological systems, and the ability to disable military and / or civilians in battlefields). Thus, preparedness and preventive measures against vesicant-induced injuries at different stages of chemical attacks is deemed necessary. Also, some anticancer agents are vesicating agents and can cause deleterious side effects. The formulations comprising glucocorticoid loaded polymeric nanoparticles described herein, when administered to the eye, are effective in both the prevention and treatment of vesicant induced comeal injury. The formulations provide high drug concentrations in the anterior segment of the eye. Further, since the polymeric nanoparticles form a depot and provide slow release of the corticosteroids, the need for repeated administration is advantageously precluded, i.e. the frequency of administration is decreased. In addition, as described in the Examples provided herein, administration of the formulations by subconjunctival (SCT) injection advantageously does not cause anappreciable increase in intraocular pressure (IOP), e.g. not more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% increase per injection, or no increase at all.

[0030] DEFINITIONS

[0031] Intraocular injection refers to an injection administered to an eye, including but not limited to subconjunctival, intravitreal, subvitreal, subretinal and suprachoroidal.

[0032] A subconjunctival (SCT) injection in the eye is a minimally invasive procedure used to deliver medication directly into the subconjunctival space (SCS), which is the space located between the sclera (the white outer layer) and the choroid (the middle vascular layer). This method is designed to provide targeted therapy to the back of the eye, specifically the retina, retinal pigment epithelium (RPE), and choroid, while minimizing contact with the anterior segment, which can reduce side effects like cataracts and high eye pressure. SCT injection usually utilizes a specialized microneedle (approximately 900-1100 pm in length) that penetrates the sclera to reach the suprachoroidal space without entering the vitreous cavity. Injected medication spreads circumferentially and posteriorly, allowing for high, localized bioavailability to the back of the eye.

[0033] A "drug depot" refers to a long-acting medication delivery system, usually an injection, that creates a storage site (a depot) in the body, releasing the drug slowly over weeks or months for sustained effects, improving patient compliance, and reducing dosing needs.

[0034] Corticosteroids are a broad class of steroid hormones from the adrenal cortex, including both glucocorticoids (like cortisol, prednisone, dexamethasone which affect sugar levels and inflammation) and mineralocorticoids (like aldosterone, which affect salt / water balance). While technically distinct, the terms “corticosteroids” and “glucocorticoids” are often used interchangeably in medicine to refer to anti-inflammatory drugs. However, synthetic glucocorticoids are the most common therapeutic type. They suppress immune responses and reduce inflammation for conditions like asthma, allergies, and arthritis. They are primarily synthesized industrially from steroid precursors, often starting with plant sterols like diosgenin or stigmasterol, using complex biochemical conversions involving microbial fermentation and chemical modifications to create potent analogues like prednisone, dexamethasone, or hydrocortisone for medicinal uses.

[0035] GLUCOCORTICOIDS

[0036] Common synthetic glucocorticoid drugs used in the present methods include but arenot limited to: prednisone, dexamethasone, methylprednisolone, prednisolone, hydrocortisone, cortisone, triamcinolone, budesonide, and betamethasone. Other examples include aclometasone, budesonide, clobetasol, clobetasone, cortivazol, desonide, fluocinolone, fluocortolone flunisolide, fluticasone, methylprednisolone, mometasone, paramethasone, rimexolone, and tixocortol pivalate. Glucocorticoids that are loaded into the nanoparticles have different potencies. For example, 1 mg of dexamethasone is as effective as 25 mg of hydrocortisone. Preferred glucocorticoids include dexamethasone, hydrocortisone, prednisone, prednisolone and methylprednisolone.

[0037] The glucocorticoids that are incorporated into the nanoparticles described herein are usually in a salt form to enhance solubility. Water soluble glucocorticoid salts may be obtained commercially or synthesized using conventional chemistry. Preferred salts include phosphates, such as dexamethasone sodium phosphate and hydrocortisone sodium phosphate, and carboxylates such as hydrocortisone sodium succinate and methylprednisolone sodium succinate.

[0038] POLYMERIC NANOPARTICLES

[0039] This disclosure provides polymeric nanoparticles comprising one or more (at least one) glucocorticoid such as those described above. “Polymeric nanoparticles” (NPs) refers to particles having dimensions within the size range from 1 to 1000 nm. They can be loaded with active compounds entrapped within the polymeric core (matrix) of the nanoparticle and / or surface-adsorbed onto the polymeric core.

[0040] The polymers that are utilized to make the nanoparticles used in the present formulations are generally hydrophobic block copolymers and may be homopolymeric or copolymeric. Examples of suitable hydrophobic polymers include polyhydroxyacids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acids); polyhydroxyalkanoates such as poly3-hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones; poly (orthoesters); poly anhydrides; poly(phosphazenes); poly(hydroxyalkanoates); poly(lactide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates); hydrophobic poly ethers; polyurethanes; poly etheresters; poly acetals; polycyanoacrylates; poly acrylates; polymethylmethacrylates; poly siloxanes; poly(oxyethylene) / poly(oxypropylene) copolymers; polyketals; polyphosphates;polyhydroxy valerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), as well as copolymers thereof.

[0041] The polymer can be a polyanhydride. The polyanhydride can be an aliphatic polyanhydride, an unsaturated polyanhydride, or an aromatic polyanhydride. Representative polyanhydrides include polyadipic anhydride, polyfumaric anhydride, polysebacic anhydride, polymaleic anhydride, polymalic anhydride, polyphthalic anhydride, polyisophthalic anhydride, polyaspartic anhydride, polyterephthalic anhydride, polyisophthalic anhydride, poly carboxyphenoxypropane anhydride, polycarboxyphenoxyhexane anhydride, as well as copolymers of these polyanhydrides with other polyanhydrides at different mole ratios. Other suitable polyanhydrides are disclosed in U.S. Pat. Nos. 4,757,128, 4,857,311, 4,888,176, and 4,789,724, the complete contents of which are hereby incorporated by reference in entirety. The polyanhydride can also be a copolymer containing polyanhydride blocks. In certain embodiments, the polymer is polysebacic anhydride. In certain embodiments, the polymer is poly(l,6-bis(p-carboxyphenoxy)hexane-co-sebacic acid) (poly(CPH-SA). In certain embodiments, the polymer is poly(l,3-bis(p-carboxyphenoxy)propane-co-sebacic acid) (poly(CPP-SA).

[0042] The molecular weight of the polymer can be varied to prepare polymeric nanoparticles that form particles having properties, such as drug release rate, optimal for specific applications. The polymers that are utilized generally have a molecular weight of about 150 Da to 1 MDa, inclusive. In certain embodiments, the polymer has a molecular weight of from about 1 kDa to about 100 kDa, more preferably from about 1 kDa to about 50 kDa, most preferably from about 1 kDa to about 25 kDa, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 kDa, including all integers between these values to the nearest 0.5 kDa.

[0043] The NPs typically have an average diameter in the range of from about 100 to about 500 nm, such as about 100, 150, 200, 250, 300, 350, 400, 450 or 500 nm. In some aspects, the average diameter is about 250 + / -10 nm.

[0044] The NPs generally have a surface charge of from about 5-15 mV, such as about 5, 6, 7, 8, 9, 10 15 mV. In some aspects, the surface charge is about -11.

[0045] In some embodiments, the polymer is a polyhydroxy ester such as polylactic acid, poly glycolic acid or a copolymer thereof. The ratio of glycolic acid to lactic acid can be optimized to control the rate of degradation. In a preferred embodiment, the NP is aPoly(D,L-lactic-co-glycolic acid NP having an LA:GA ratio of 50:50.

[0046] In certain optional aspects, the nanoparticles are coated with one or more hydrophilic polymers. The one or more hydrophilic polymers may be described as forming a hydrophilic polymer segment. These polymers are generally hydrophilic, biocompatible (i.e., it does not induce a significant inflammatory or immune response), non-toxic polymers or copolymers. Examples of suitable polymers include but are not limited to poly(alkylene glycols) such as polyethylene glycol (PEG), polypropylene glycol) (PPG), and copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinyl alcohol), and copolymers, terpolymers, and mixtures thereof.

[0047] In preferred embodiments, the one or more hydrophilic polymer segments contain a poly(alkylene glycol) chain. The poly(alkylene glycol) chains typically contain between 8 and 500 repeat units, and more preferably between 40 and 500 repeat units. Suitable poly(alkylene glycols) include polyethylene glycol), polypropylene 1,2-glycol, polypropylene oxide), polypropylene 1,3-glycol, and copolymers thereof. In certain embodiments, the one or more hydrophilic polymers are PEG chains. In such cases, the PEG chains can be linear or branched, such as those described in U.S. Pat. No. 5,932,462, the complete contents of which is hereby incorporated by reference in entirety. In certain embodiments, the PEG chains are linear.

[0048] Each of the one or more hydrophilic polymer segments can independently have a molecular weight of about 300 Da to 1 MDa. In some aspects, the hydrophilic polymer segment has a molecular weight of between about 1 kDa and about 20 kDa, more preferably between about 1 kDa and about 15 kDa, most preferably between about 1 kDa and about 10 kDa. In a preferred embodiment, each of the one or more hydrophilic polymer segments has a molecular weight of about 5 kDa.

[0049] The polymeric nanoparticles contain one or more glucocorticoids, preferably complexed by chelation of metal ions with phosphate or carboxyl groups, most preferably carboxyl end groups at the terminus of the biodegradable polymer such as a polymer containing an ester or other hydrolysable moiety. The glucocorticoid may be derivatized into a water-soluble salt and then incorporated into the polymeric nanoparticle. Generally, drug loading into the NPs is in the range of from about 1-20%, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20%, including all integers and decimal fractions in between these values. In some aspects, the drug loading is from about 5-10% %, such asabout 5, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10 %.

[0050] In some aspects, loading of the glucocorticoid (e.g. DSP) is facilitated by bridging of the carboxyl ends of one or more polymers that make up the NPs with the phosphate group of the glucocorticoid by divalent zinc cations.

[0051] Further description of suitable polymeric nanoparticles, methods of synthesis thereof, and formulations thereof is found in issued United States patent 10 / 195,212, the complete contents of which is hereby incorporated by reference in entirety.

[0052] COMPOSITIONS

[0053] The MPs described herein are generally delivered (administered) in a pharmaceutical composition and the present invention encompasses such formulations / compositions. The compositions generally include purified MPs as described herein (i.e., comprising at least glucocorticoid), and a pharmacologically suitable (physiologically compatible) carrier.

[0054] Generally, such compositions are prepared as liquid solutions or suspensions. However, solid forms suitable for solution in, or suspension in, liquids prior to administration are also contemplated (e.g., lyophilized forms of the MPs), as are emulsified preparations. In some aspects, the formulations are liquid and are aqueous or oil-based suspensions or solutions.

[0055] In some aspects, the drug-loaded MPs are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients, e.g., pharmaceutically acceptable salts. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, preservatives, and the like. The compositions of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration, particularly administration into the eye. The final amount of drug-loaded MPs in the formulations varies but is generally from about 1-99%. Still other suitable formulations for use in the present invention are found, for example, in Remington's Pharmaceutical Sciences, 22nd ed. (2012; eds. Allen, Adejarem Desselle and Felton).

[0056] Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as Tween® 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate. Releasing agents, coating agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0057] "Pharmaceutically acceptable salts" of the compounds refer to the relatively nontoxic, inorganic and organic acid addition salts and base addition salts of the glucocorticoids. In some aspects, these salts are prepared in situ during the final isolation and purification of the glucocorticoids. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-P-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, 1-19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodiumhydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like. In preferred aspects, the disclosure provides phosphate prodrugs, chelated within a nanoparticle, for example, via a (dexamethasone) phosphate-zinc chelation. In this aspect, the salt is a sodium or potassium salt of (dexamethasone) phosphate salt. In these aspects, an HC1 salt does not work.

[0058] The pharmaceutical compositions of the present invention are preferably packaged in a unit dosage form. The term "unit dosage form" refers to a form that is suitable for dosing a patient, i.e., such that each unit produces the desired therapeutic effect, either alone or in combination with one or more additional units. For example, a unit dosage form may be a unit package comprising a needle prefilled with a dose of a pharmaceutical composition suitable for intraocular administration; or a unit dosage form may be a unit package comprising an ampule or other container prefilled with a single dose or multiple doses of a pharmaceutical composition suitable for intraocular administration and ready to be loaded into a syringe. Such units may be available e.g. in a blister pack or other packaging suitable for shipping and storage. All such packaged doses of the pharmaceutical compositions are encompassed herein.

[0059] The polymeric matrix described herein can be formed into implants, microparticles, nanoparticles, or combinations thereof for delivery to the eye. Upon administration, the one or more glucocorticoids are released over an extended period of time, either upon degradation of the polymer matrix, diffusion of the one or more glucocorticoids out of the polymer matrix, or a combination thereof. By employing a polymeric nanoparticle, particles can be formed with more controlled drug loading and drug release profiles.METHODS

[0060] In general, the methods disclosed herein involve administering to at least one eye, usually both eyes, of a subject in need thereof, a composition (formulation) comprising polymeric nanoparticles comprising one or more glucocorticoids. The one or more glucocorticoid(s) is / are dispersed or encapsulated or otherwise comprised within a polymeric matrix of the nanoparticles and is administered by intraocular injection.

[0061] Subjects in need of treatments disclosed herein include but are not limited to subjects whose eyes are at risk of being exposed to or who have been exposed to a vesicating agent. Examples of vesicant agents that can damage the eye include but are not limited to: chemical warfare agents (also known as “blister agents”): mustards which may be gases such as sulfur mustard (mustard gas) and nitrogen mustards; arsenicals such as Lewisite (which contains arsenic); halogenated oximes such as phosgene oxime (CX); certain medical / pharmaceutical vesicants such as the chemotherapy drugs doxorubicin and vincristine; and other medications (e.g., potassium chloride, calcium chloride); and the like. The subjects are generally mammals, such as humans, but veterinary uses are not precluded, e.g. such as canines, horses, etc. that serve in the armed forces or in law enforcement. Humans that are treated may be, for example, members of the military and / or civilians that are likely to be or have been exposed to a chemical warfare agent, including soldiers, advisors, translators, intelligence agents, etc. Exposure may be deliberate (e.g. in warfare) or accidental (e.g. in a hospital during chemotherapy treatments).

[0062] The compositions disclosed herein are generally administered to a subject in need thereof periocularlly, such as by subconjunctival injection, generally by an ophthalmologist. In some aspects, the compositions are administered by subconjunctival injection in one or both eyes.

[0063] Typically, a volume of 1 to 100 pL, e.g. 25 pL, 50 pL, or 100 pL, and usually no more than 100 pL, of the subject composition is delivered to the eye by subconjunctival injection without removing the vitreous. This amount typically delivers from about 50 to about 300 pg DSP, such as about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300 pg DSP. In some aspects, the amount that is delivered ranges from about 150 to 200 pg DSP, such as about 150, 155, 160, 165, 170, 175, 180, 185, 190 or 200 pg DSP. For subconjunctival administration, the NPs can be delivered in the form of a suspension.

[0064] Typically, the process involves numbing the eye, cleaning the surface with an antiseptic(e.g., povidone-iodine) and using a sterile speculum to hold the eyelids open. In other words, a topical anesthetic is applied to the surface of the eye followed by a topical antiseptic solution. The eye is held open, with or without instrumentation, and the composition is injected through the sclera with a short, narrow needle, for example a 30-gauge needle, into the vitreous cavity of the eye of a subject, under direct observation of a medical professional, e.g., an ophthalmologist. The ophthalmologist carefully injects a suitable amount of the composition comprising the drug-loaded MPs through the eye and into the vitreous humor. An antibiotic drop is typically administered after the injection.

[0065] Subconjunctival administration is generally well tolerated. At the conclusion of the procedure, there is sometimes mild redness at the injection site. There is occasional tenderness, but most patients do not report any pain. No eye patch or eye shield is necessary after this procedure, and activities are not restricted. Sometimes, an antibiotic eye drop is prescribed for several days to help prevent infection. Alternatively, an antibiotic can be included in the NPs.

[0066] In some aspects, the deposition of a composition containing the microparticles as described herein is in effect the placement or formation of a “depot” for slow, extended release of the one or more glucocorticoids, The slow or extended release generally occurs and provides preventive protection or treatment for at least 1-4 weeks, such as for about 1, 2, 3 or 4 or more weeks. In some aspects, protection lasts for months, such as for 1-12 months, e.g. about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12 months.

[0067] Since the subject that is treated may be susceptible to exposure to one or move vesicating agents over a long period of time (e.g. several days or weeks), the injections may be repeated for as long as the exposure or chance of damage due to prior exposure persists.

[0068] The compositions disclosed herein may be administered with other medicinal agents and / or treatments. For example: immediate decontamination to remove all contaminated clothing immediately and thoroughly wash the skin with soap and water, or a specific reactive lotion like RSDL (Reactive Skin Decontaminant Lotion) if available, or a dilute bleach solution (0.5% sodium hypochlorite); and flushing / rising the eyes with copious amounts of sterile saline or water. Additional treatments include but are not limited to British Anti-Lewisite (BAL), an antidote for arsenic poisoning, used for lewisite exposure. For e.g. sulfur mustard supportive treatment to manage pain and inflammation, similar to severe bums, may be given, including but not limited to: corticosteroid drops or sprays, e.g.dexamethasone drops or sprays; antibiotics and / or antimicrobials; topical and / or systemic analgesics, etc.

[0069] The methods disclosed herein comprise administering to the subject a therapeutically effective amount of at least one NP formulation as disclosed herein. A “therapeutically effective amount” refers to an amount that entirely prevents or decreases at least one symptom of vesicant injury in the subject, e.g. the symptom is abolished, or at least the level or amount of the symptom (e.g. of neovascularization, upregulation of inflammation, etc.) that occurs is less than would occur in the absence of administration of the formulation. If the symptom is already present, the level is decreased compared to the level that was present before exposure or in the absence of administration of the formulation after exposure. The compositions disclosed herein, when administered to a subject that has been or is at risk of being exposed to a vesicating agent, prevent and / or treat at least one symptom of such exposure. Examples of symptoms that are prevented and / or treated include but are not limited to: upregulation of inflammatory cytokines (e.g. TNFa, IFN-y, NF-kB, IE-6 and IE-1 P), angiogenic markers (e.g. MMP-2, MMP-9, VEGF, PDGFRP and PDGFRa), fibrotic markers; corneal ulceration; corneal neovascularization; corneal opacity; increased corneal thickness; and epithelial degradation.

[0070] It is noted that subconjunctival injection of PLGA-DSP-NPs does not cause a significant increase in intraocular pressure (IOP).

[0071] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0072] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Representative illustrative methods and materials are herein described; methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0074] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual dates of public availability and may need to be independently confirmed.

[0075] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as support for the recitation in the claims of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitations, such as "wherein [a particular feature or element] is absent", or "except for [a particular feature or element]", or "wherein [a particular feature or element] is not present (included, etc.)...".

[0076] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0077] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.EXAMPLE

[0078] Dexamethasone sodium phosphate loaded nanoparticles for prevention of nitrogen mustard (NM) induced comeal injury

[0079] 1. Polymeric nanoparticles (NPs) offer a safe approach to meet the needs of controlled drug release at target tissues, thus improving therapeutic efficacy and minimizing adverse side effects. We have developed DSP-loaded Poly(D,L-lactic-co-glycolic acid (PLGA) NPs through a noncovalent cationic zinc bridging between DSP and carboxyl-terminated PLGA that provided sustained DSP release. This Example describes DSP-loaded PLGA nanoparticles that can release DSP for 2 weeks in an in vitro setting. A single subconjunctival (SCT) injection of PLGA-DSP-NP prevents the progression of NM-induced comeal injury in a mammalian animal model.

[0080] 2. Materials and methods

[0081] 2.1. Animals The animal experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Virginia Commonwealth University (VCU). Animals used in the experiment were taken care of in accordance with the Association for Research in Vision and Ophthalmology (ARVO) concerning the use of animals in ophthalmic research. Male Sprague Dawley (SD) rats (8-weeks old) were obtained from Envigo (Indianapolis, IN). The experimental animals were cared for by the VCU’s Department of Animal Resources. For experimental procedures, animals were anesthetized with an intramuscular injection of 50 mg / kg ketamine hydrochloride mixed with 5 mg / kg xylazine. Topical instillation of 0.5% proparacaine hydrochloride (Bausch & Lomb), and 1% tropicamide (Bausch & Lomb), was used for topical anesthesia and pupil dilation, respectively. At the predetermined experimental time point (post- injury day 14, PID14), animals were sacrificed by CO2 euthanasia followed by chest opening.

[0082] 2.2. Development of NM-induced corneal injury rat model The dose and exposure time dependency of NM-induced comeal injury was evaluated in healthy SD rats. Three doses of NM (0.5%, 1%, and 2% w / v) and two exposure durations (1 min and 2 min) were evaluated. All NM solutions were freshly prepared in sterile saline before the experiment. Briefly, SD rats (8-weeks old) were anesthetized with an intramuscular injection of 50 mg / kg ketamine hydrochloride and 5 mg / kg xylazine. Topical anesthesia was provided by instilling a drop of0.5% proparacaine hydrochloride solution into the eye. A piece of filter paper (5-mm diameter) was soaked in NM solution (0.5%, 1%, or 2% w / v) and applied to the cornea for 1 or 2 min. After application, both eyes were washed with 25 ml of PBS to remove any remaining NM from the ocular surface. The animals were followed daily for signs of pain / discomfort until the end of study on PID14 following NM exposure.

[0083] 2.3. Clinical observations and evaluations Clinical evaluations for corneal neovascularization (NV), opacity, ulceration and scarring were conducted on PID7 and PID14 by slit lamp imaging, microscopic imaging, and digital quantification. Microscopic images were captured via a Carl Zeiss Stemi 305 microscope using a Labcam adapter ilabcam.com). All animals were observed for clinical evaluations and histopathology graded in a masked manner by an ophthalmologist and ophthalmic pathologist, respectively.

[0084] 2.3.1. Corneal ulceration and scarring Comeal ulceration was evaluated via slit lamp examination (Carl Zeiss, Germany) under cobalt blue light at PID7 and PID14. Animals were anesthetized as per the described protocol and fluorescein dye was instilled into both eyes using fluorescein sodium ophthalmic strips (FluoroTouch, Madhu Instruments, India). A drop of balanced salt solution (BSS) was added onto the ophthalmic strip and allowed to drop onto the eye surface, without direct contact of the strip and the eye. Under cobalt blue light, fluorescein- stained regions with depleted corneal epithelium emits a green fluorescence. The mean fluorescence intensity (in arbitrary units a.u.) was analyzed using ImageJ software. Comeal scarring was evaluated using the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) scale for comeal scarring grading using slit lamp images.

[0085] 2.3.2. Corneal neovascularization and opacity The extent of comeal NV was evaluated with ophthalmic microscope images and ImageJ software on PID7 and PID14. Animals were anesthetized and 0.5% tropicamide solution was administered to dilate pupils before imaging. The quantification of corneal NV was represented through blood vessel length and average corneal NV scores. To evaluate the blood vessel length, an arc was drawn along the limbus in the vascularized area and the distance from blood vessel tip to limbus was calculated at 5 different intersection points of the arc. The average of five measured length was reported as comeal NV length for that eye. Average corneal NV scores were assigned following previously established grading criteria, based on the percentage increase in length of blood vessels from all four quadrants of the cornea. (Table 1). Corneal opacity causesclouding of the cornea, reducing its transparency. Corneal opacity was evaluated using a semiquantitative clinical scoring method (Table 2) described previously for laboratory

[0086] species used in ocular research.

[0087] Table 1: Corneal neovascularization scoring criteria

[0088]

[0089] Table 2: Corneal opacity scoring criteria

[0090]

[0091] 2.3.3. Histopathological evaluation On PID14, the enucleation specimens were collected

[0092] and fixed in Davidson’s fixative solution for 24 h and then transferred to 70% ethanol.

[0093] Following embedding in paraffin, axial sections (5 pm) with anteroposterior orientation

[0094] (from cornea to optic nerve) were prepared and stained with hematoxylin and eosin (H&E).

[0095] Three eyeballs per group were used. The corneal sections were examined and imaged using

[0096] a Nikon E800 light microscope equipped with a digital camera (Tokyo, Japan). Comeal

[0097] thickness was assessed using a magnification of 40x, approximately 1.0 mm away from the

[0098] limbus region, by use of ImageJ software. For measurements of corneal and epithelial thicknesses, the corneal sections were segregated into four distinct regions and five random measurements were taken per region. The average comeal thickness and epithelial thickness per cornea were reported as the mean of all such measurements. H&E- stained slides were evaluated in a masked manner by an ophthalmic pathologist.

[0099] 2.4. RNA isolation and quantitative PCR On PID14, the rats were euthanized followed by enucleation. Briefly, corneas were dissected out from the eyeballs, wrapped in aluminum

[0100] foil and flash freezed in liquid nitrogen. The frozen corneas were immediately smashed by a hammer followed by homogenization in 400 pl of TRIzol® using a Bullet Blender Stormhomogenizer (Raymer town, NY). The samples were then incubated at room temperature for 5 min followed by addition of chloroform. Following centrifugation (12000g, 15mins), the supernatant was extracted with isopropanol and mRNA was isolated. Total isolated mRNA in cornea was quantified using a Thermo-fisher Nanodrop 2000 (Waltham, MA). cDNA was synthesized using the High-Capacity cDNA reverse transcription kit following manufacturer’s instruction. The mRNA expression for inflammatory and angiogenic cytokines used in the experiment were quantified using PowerUP SYBR® Green Master Mix using QuantStudio 3 real-time PCR system (Hampton, NH). The primers sequences used for qPCR mRNA expression levels were normalized to glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) and were analyzed using the AACT method.

[0101] 2.5. Preparation and characterization of PLGA-DSP-NP PLGA-DSP-NP were prepared by a modified nanoprecipitation method. PLGA-DSP-NP were characterized for particle size, surface charge, drug loading, morphology, and in vitro drug release profile, as follows. Preparation and characterization of PLGA-DSP-NP

[0102] Poly(D,L-lactic-co-glycolic acid; LA:GA ratio 50:50, acid terminated) (PLGA) with Mw of 7kDa was purchased from Evonik Industries (Birmingham, Al). To prepare the PLGA-DSP-NP, initially, 0.5M zinc acetate was added to an equal volume of DSP aqueous solution containing 20 mg DSP to form the DSP-Zinc complex. Following centrifugation of the DSP-Zinc complex at 20,000g for 5 min, the precipitate was collected and dissolved with 100 mg PLGA in 5 ml of THF. The pH of this solution was adjusted to between 7 and 8 using triethanolamine. Then the solution was slowly injected into a beaker containing 75 ml of 5% w / v Pluronic Fl 27 aqueous solution under stirring (700 rpm) using a Hamilton syringe. After 1 hour, THF was removed from the mixture using a rotary evaporator apparatus (Buchi Co., DE). To solubilize and remove any unencapsulated DSP-Zinc complexes, 1 ml of 0.5 M EDTA was added to the mixture. PLGA-DSP-NPs were centrifuged at 8000g and washed with 5% Pluronic F127. The collected nanoparticles were resuspended in 0.2 ml ultrapure water.

[0103] The particle size (number mean) and surface charge (measured by ^-potential) of PLGA-DSP-NP was measured by Zetasizer Nano ZS90 (Malvern Instruments, MA). The morphology of PLGA-DSP-NP was characterized by transmission electron microscopy (TEM) using a Hitachi H7600 microscope (Hitachi Co. Ltd., Japan). To calculate the drug loading, 15 pl of PLGA-DSP-NP was lyophilized, weighed, and dissolved in a combinationof 500 (il of acetonitrile and 500 pl of 50 mM EDTA. DSP concentration was then measured by high-performance liquid chromatography / ultraviolet (HPLC / UV) on a reverse phase Shimadzu Prominence LC system (Kyoto, Japan) using a Pursuit 5 C18 column. Isocratic separation was performed with mobile phase consisting of acetonitrile / water (60 / 40 v / v) containing 0.1% trifluoro acetic acid, monitored at a UV detection of 241 nm. The drug loading (DL) was calculated as follows:

[0104] DL (wt %) = (amount of DSP in particles) / (weight of particles) To investigate the in vitro drug release of PLGA-DSP-NP, a cellulose membrane dialysis tubing with a molecular weight cutoff of 10 kDa (Sigma- Aldrich, MO) was used.

[0105] 400 pl of PLGA-DSP-NP suspension was sealed inside the dialysis membrane and placed inside 50 ml conical tubes. 12 ml of phosphate-buffered saline (PBS, pH 7.4) was used as the release medium. The setup was incubated at 37 °C on a platform shaker operating at 100 rpm. At predetermined timepoints, the release media was collected and replaced in its entirety with fresh PBS. HPLC / UV analysis was performed to calculate DSP concentration in the collected release media for all timepoints.

[0106] 2.6. Treatment of NM-induced corneal injury in rats After the optimization of NM-induced comeal injury model, the exposure of 1% NM solution for 1 min was used to establish the comeal injury for the following efficacy studies. Following NM exposure, rats were randomly assigned into five groups receiving the following different treatments: I. PLGA-DSP-NP, SCT (180 pg DSP); II. sterile isotonic saline, SCT; III. placebo NP, SCT; IV. DSP solution, SCT (180 pg DSP); and V. DSP eyedrops (0.1% USP, 3x / day). Site and volume (30 pL) of all SCT injections was kept consistent across the groups. All animals received their respective treatments within 5 min post-NM exposure. The clinical observations of comeal NV, ulceration and opacity were conducted on PID7 and PID14. All animals were euthanized on PID14, and eyeballs were collected for further qPCR and histopathologic analysis.

[0107] 2.7. Statistical analysis Two groups were compared for statistical significance using two-tailed Student’s t-test whereas three or more groups were compared using two-way ANOVA followed by Tukey post-hoc test using Graphpad Prism 10. Differences were considered statistically significant if the p value was <0.05. The data are presented as the mean ± standard error of the mean (SEM).

[0108] 3. Results 3.1. NM exposure induces corneal ulceration, scarring, opacity andneovascularization

[0109] 3.1.1. Corneal ulceration and scarring Comeal ulceration was evident in all rats in the tested NM concentrations and exposure durations on PID7 and PID14 (Fig. 1A). The depleted epithelial layer leads to exposed stroma, causing an uptake of fluorescein.

[0110] Fluorescence intensity was evaluated under cobalt blue light using a slit lamp on PID7 and PID14. On PID7, amongst all 1-min exposure groups, significant difference in fluorescence intensity compared to healthy control was observed only in the 1% NM group (2.2-fold higher). We did not observe significant disparity in 0.5% NM and 2% NM groups when compared to healthy control. A plausible explanation for this could be the presence of severe comeal edema and necrosis which hinders fluorescein uptake. Amongst the 2-min exposure groups, fluorescence intensity was significantly higher for both 1% NM group (3-fold higher) and 2% NM group (3-fold higher) as compared to healthy control. However, such statistical difference was not observed in the 0.5% NM group. Observations indicate that ulceration severity increases in an exposure duration-dependent manner, where higher fluorescence intensities were observed for the animals exposed to NM for 2 min. However, statistical significance between the 1 min and 2 min exposure was observed only for the 2% NM groups (Fig. IB). On PID14, amongst the 1-min NM exposure groups, only the 1% and 2% NM groups presented significant ulceration (2-fold) higher compared to the healthy control. For the 2-min NM-exposure, all three NM concentrations demonstrated significant increase in fluorescence intensity compared to the healthy control. The most prominent comeal ulceration was observed in the rats subjected to 2% NM for 2 min with 3- fold higher fluorescent staining than healthy control, along with severe edema, necrosis, and comeal scarring. Consistent with our observations on PID7, severity of ulceration increased with the duration of NM exposure. However, significant difference between the exposure durations was observed only for 2% NM group (p < 0.001) (Fig. 1C). In addition, we observed that the increase in concentration of NM correlates to an increased severity of ulceration, though this trend was only apparent for the 2-min NM exposure groups (2% NM > 1% NM > 0.5% NM). Within the tested concentrations, the reported data suggests that NM-induced comeal ulceration is more dependent on the duration of NM exposure rather than NM concentration. The progression of ulceration severity from PID7 to PID14 was evaluated for both the 1-min and 2-min exposure groups. Amongst the 1-min exposure groups (Fig. 10a), highest fluorescence intensity was observed for the 1% NM on both PID7and PID14 (58 ± 7.6 a.u. and 54 ± 3.6 a.u. respectively). For the 2-min exposure groups, the mean fluorescence intensity surged to a maximum of 78 ± 12.1 a.u. (2% NM) on PID7 and 87 ± 9.3 a.u. (1% NM) on PID14 (Fig. 10b). Comeal scarring analysis post NM exposure showed that the scar formation was obvious with the increasing concentration of NM dose. For 1-min NM exposure, maximum scar was observed for 1% NM group with average scores 2.3 ± 1.8 and 2.0 ± 1.6 at PID7 and PID14 respectively (Fig. ID). For 2-min NM exposure, rise in scar formation was proportional to the dose of NM, with maximum scar observed in 2% NM group at both PID7 (average score 3.6 ± 0.5) and PID14 (average score 3.3 ± 0.8) (Fig. IE).

[0111] 3.1.2. Corneal opacity On PID7 and PID14 (Fig. 2A), comeal opacity was observed in all animals subjected to NM, at all test concentrations and exposure durations, although to varying degrees. The opacity score was higher for all 2-min exposure groups compared to 1-min exposure on PID7. Notably, 2-min exposure of NM caused severe comeal opacity (scores >3), while a moderate opacity (scores 2 to 3) was observed for 1- min exposure. However, significant disparity between 1-min and 2-min exposure was observed only in the 0.5% NM (p < 0.05) and 2% NM (p < 0.05) groups (Fig. 2B). By PID14, the corneal opacity of the 1-min exposure groups progressed noticeably, becoming more severe (scores >3) (Fig. 10c). Since the 2-min exposure groups swiftly developed severe opacity on PID7, a less pronounced increase was noted by PID14 (Fig. lOd). Additionally, statistical significance was not observed between the 1-min and 2-min exposure opacity scores on PID14, regardless of NM concentration. Moreover, no discernible pattern correlating the concentration of NM to the severity of opacity was observed (Fig. 2C).

[0112] 3.1.3. Corneal neovascularization Varying degrees of comeal NV were observed in all the experimental animals on PID7 and PID 14 (Fig. 2A). 1-min exposure caused corneal NV ranging from 1.0 mm to 1.4 mm on PID7. Some variability was observed in the 2% NM group, but the 1% NM group reliably developed the longest comeal NV (1.4 mm) after 1-min exposure. As shown in Fig. 2D, increased corneal NV length on 2-min exposure was observed in 1% NM (1.3-fold) and 2% NM (1.8-fold) groups compared to 1-min exposure. There was no obvious difference in the comeal NV lengths between the corneal NV lengths in the 1-min and 2-min exposure groups using 0.5% NM. A general increase in the comeal NV lengths was observed by PID14. Compared to 1-min exposure, the corneal NV length increased in 0.5% NM (1.1-fold, / ? < 0.05), 1% NM (1.2-fold, / ? > 0.05), and 2% NM (1.1-fold, p > 0.05) groups on 2-min NM exposure (Fig. 2E). However, due to the pathologic progression of NM-induced corneal injury in this study period, prominent opacity was observed in some eyes, especially in the 2% NM groups. This made it challenging to view and quantify corneal NV and thus, reported lengths for this group may not represent the actual corneal NV growth. As the study progressed, an increase in the comeal NV length was observed from PID7 to PID14. On 1-min exposure to NM, a significant increase in the NV length was observed in the 0.5% NM (2.2-fold, p < 0.001) and 1% NM (1.8-fold, p < 0.01) groups on PID14 compared to PID7 (Fig. lOe). Following a similar trend, significant growth of the comeal NV was noted in 0.5% NM (2.9-fold, / ? < 0.001) and 1% NM (1.6-fold, / ? < 0.001) groups on 2-min exposure at PID14 (Fig. lOf). Although the 2% NM group displayed 1.5-fold increase on 1-min exposure, it was not statistically significant. The drop in corneal NV lengths for 2% NM groups, especially on 2-min exposure, can be attributed to the obscuration of blood vessels due to development of severe opacity.

[0113] 3.2. NM exposure promotes epithelial degradation & increases corneal thickness Representative eyeballs with NM-induced comeal injury were sectioned and stained with H&E followed by the quantification of epithelial degradation (epithelial thickness) and total comeal thickness. Sections of the comeal epithelium on PID14 demonstrated histopathologic alterations in all NM-injured corneas. NM exposure caused significant deterioration and thinning of the corneal epithelium when compared to healthy control (Fig.

[0114] 3 A). On l-min exposure, significant corneal epithelial degradation was observed for 1% NM (1.4-fold) and 2% NM (3.8-fold) groups as compared to healthy control, with the exception of the 0.5% NM group. Likewise, epithelial degradation was prominent at all three NM concentrations on 2-min exposure. Compared to healthy control, decrease in epithelial thickness by 1.5-fold, 7.8-fold and over 50-fold was observed for the 0.5%, 1%, and 2% NM groups, respectively. On the contrary, NM-injury caused significant increase in the total comeal thickness across all groups. This increase in thickness was notably observed in the stromal region, indicating alterations induced by NM exposure. For the 1-min exposure duration groups, increase in comeal thickness by 2-fold, 2.2-fold, and 1.7-fold as compared to healthy control was observed on PID14 for the 0.5%, 1%, and 2% groups respectively. For 2-min NM exposure, 0.5% NM had 2-fold, 1% NM had 1.6-fold, and 2% NM had 2.2-fold increase in the total corneal thickness on PID14, as compared to healthy control. There was no significant difference between the 1-min and 2-min exposure durations for 0.5%NM. Significant differences in the total corneal thickness were observed between 1-min and 2-min exposure durations for the 1% (p < 0.001) and 2% (p < 0.001) NM groups. However, based on these histopathologic findings, no correlation could be found between the increase of total corneal thickness and NM concentration / exposure duration (Fig. 3B). Moreover, infiltration of inflammatory cells and an increase in the number of keratocytes in the stromal region were noted in the NM-injured corneas. H&E images revealed aberrant histomorphology in the squamous epithelial cells, in all NM-injured groups. Notably, ulcerations (areas completely devoid of squamous epithelium) were identified all 1% and 2% NM injured groups at both Imin and 2 min exposure duration at PID14 (Fig. 3C).

[0115] 3.3. NM exposure upregulates inflammatory and angiogenic cytokines Comeal inflammation caused by NM injury elevates the expression of inflammatory and angiogenic mediators as shown in Fig. 4. Based on the aforementioned results, NM-induced injury by exposure to 1% NM for 1 min was selected for quantitative polymerase chain reaction (qPCR) analysis of relevant genes. The presence of several inflammatory cytokines like TNFa, IFN-y, IL-ip, IL-6, NF-kB, and angiogenic markers like MMP-2, MMP-9, and VEGF in the cornea was evaluated on PID14. The NM-injured corneas demonstrated significant upregulation of tested inflammatory cytokines as compared to healthy control. We observed significant elevation of TNFa (13-fold), IFN-y (4-fold), IL-ip (90-fold), IL-6 (10-fold), and NF-kB (1.6-fold) as compared to healthy control. Similarly, the angiogenic markers MMP-2, MMP-9, and VEGF were upregulated in a marked manner by 2-fold, 4-fold, and 7-fold, respectively.

[0116] 3.4. Formulation and characterization of PLGA-DSP-NP Efficient loading of DSP into PLGA7kDa was facilitated by bridging of the carboxyl ends of PLGA7kDa with the phosphate group of DSP by divalent zinc cations. The PLGA-DSP-NP exhibited a spherical morphology (Fig. 5 A), with an average diameter of 253 ± 2.5 nm and a poly dispersity index of 0.096, indicating uniform particle size distribution (Fig. 5A) (Table 3).

[0117] The nearly neutral surface charge (- 11 mV) on the PLGA-DSP-NP can be attributed to the dense PEG coating (Fig. 5B). 6.5 wt% DSP was successfully loaded into the PLGA-DSP-NP and a sustained release was observed for more than 2 weeks in vitro (Fig. 5C). The limited initial burst release suggests that the DSP was primarily dispersed inside the nanoparticles rather than localized near, or adsorbed onto, the surface.Table 3. Physicochemical characterization of PLGA-DSP-NP and Placebo NP.

[0118]

[0119] 3.5. PLGA-DSP-NP prevents NM induced corneal ulceration and scarring After exposure to 1% NM for 1 min, eyes were immediately given with various treatments. On PID14, most control groups demonstrated progressive corneal ulceration and edema (Fig. 6A). Animals in the saline, placebo NP, DSP eyedrops, and DSP solution control groups exhibited severe edema and ulceration of the cornea by PID14. No edema was observed in the PLGA-DSP-NP group. Although some ulceration was observed, it remained significantly less than the other control groups (p < 0.01). Only one out of seven rats in PLGA-DSP-NP group presented evidence of ulceration on PID14. Compared to the PLGA-DSP-NP treatment, the saline (2-fold), DSP eyedrop (1.8-fold), placebo NP (2-fold), and DSP solution (2.1-fold) groups showed significant increase in mean fluorescence intensity measurements (Fig. 6B). Similarly, PLGA-DSP-NP significantly prevented corneal scarring when compared to all other control groups. DSP eyedrops was also effective in the prevention of comeal scarring with no significant difference in comeal scarring score when compared to PLGA-DSP-NP. The average comeal scarring scores were 0.38 ± 0.7 for PLGA-DSP-NP, 1.15 ± 0.9 for DSP eyedrops while Saline, DSP solution and Placebo NP had average scores >2 at PID14.

[0120] 3.6. PLGA-DSP-NP prevents NM induced corneal neovascularization Evaluations on PID14 demonstrated that a single SCT injection of PLGA-DSP-NP effectively halted the progression of comeal NV. Conversely, control groups exhibited a rise in corneal neovascularization (Fig. 7A). There was a significant disparity in the average length of blood vessels between PLGA-DSP-NP and other groups. The average length of corneal NV in PLGA-DSP-NP group was 0.3 ± 0.05 mm, which increased to 2.65 ± 0.45 for saline control, 1.26 ± 0.16 for DSP eyedrop, 1.56 ± 0.25 for placebo NP and 1.33 ± 0.08 for DSP solution treated group (Fig. 7B). Similarly, comeal NV score was presented as the percentage of length of the blood vessel growth in the cornea, averaged from four quadrants of the cornea. Following a similar trend, on PID14, a score of over 3 (longest vessel length between 50% and 75% of the radius of the cornea) was observed for the saline group. DSPeyedrop, placebo NP, and DSP solution displayed scores of between 2 and 3 (longest vessel length between 25% and 50% of the radius of the cornea). A single SCT injection of PLGA-DSP-NP effectively halted the progression of corneal NV as observed on PID14 with an overall corneal NV score of less than 1 (longest vessel length less than 25% of the radius of the cornea) (Fig. 7C).

[0121] 3.7. PLGA-DSP-NP prevents NM induced corneal opacity A progressive increase in the corneal opacity was observed following NM injury, with significant increase by PID14. On PID14, the groups treated with saline, DSP eyedrop, and placebo NP had notable corneal opacity, with average opacity scores of 3.0 ± 0.5, 2.8 ± 0.47, and 2.5 ± 0.31 respectively (Fig. 7D). Despite three-times-per-day administration of DSP eyedrops for 14 days, prominent opacity was noted in 6 of 7 animals. However, a single SCT injection of PLGA-DSP-NP was effective in inhibiting the progression of corneal opacity with an average comeal opacity score of less than 1 (0.5 ± 0.2).

[0122] 3.8. PLGA-DSP-NP prevents upregulation of inflammatory, angiogenic and profibrotic cytokines On PID14, there was no significant difference in the mRNA expression of most inflammatory and proangiogenic cytokines between healthy control and PLGA-DSP-NP group, with the exception of MMP-2, where PLGA-DSP-NP showed significantly lower (p < 0.001) mRNA expression (Fig. 8). We observed a significant upregulation of inflammatory cytokines such as IFN-y (p < 0.001) and proangiogenic factors like MMP-2 (p < 0.001), MMP-9 ( < 0.001), PDGFRa (p < 0.001) and PDGFRp (p < 0.05) in the saline-treated group when compared to the PLGA-DSP-NP treated group, with the exception of VEGF (which showed no significant difference). PLGA-DSP-NP were more effective than DSP solution in suppressing mRNA expression of MMP-2 (p < 0.001), MMP-9 (p < 0.001), PDGFRa ( < 0.001), IFN-y (p < 0.001) and VEGF (p < 0.001). Similarly, PLGA-DSP-NP showed significant reduction in the expression of all key pro-angiogenic, and inflammatory cytokines compared to placebo NP group. DSP eyedrops (three times a day) was comparable to treatment with PLGA-DSP-NP in terms of mRNA expression of VEGF, PDGFRa, PDGFRp, and MMP-9; however, there was a significant increase in mRNA expression of MMP-2 (p < 0.05) and IFN- y (p < 0.01). On note, the expression of TGFpi was downregulated by the PLGA-DSP-NP compared to Saline (p < 0.001), Placebo NP (p < 0.01) and DSP solution group (p < 0.05). TGFpi expression for PLGA-DSP-NP, however, was comparable to DSP eyedrop treated group. This study shows that the PLGA-DSP-NPprevents the fibrosis and cornea scar formation associated with NM induced corneal injury.

[0123] 3.9. PLGA-DSP-NP prevents corneal epithelial degradation As observed in Section 3.2, NM-injury results in the degradation of corneal epithelium. The H&E corneal sections collected on PID14 from the saline and placebo NP groups showed corneal thickening and ulceration (absent epithelial layer). Extensive infiltration of inflammatory cells was observed throughout the cornea in the saline group. The corneas of DSP eyedrops and DSP solution groups also presented significant epithelial degradation, loss of structural integrity, and stomal thickening. In comparison, the corneas from the PLGA-DSP-NP treated group had intact epithelial, stromal, and endothelial layers. Slight increase in comeal thickness (p > 0.05) and absence of inflammatory cell infiltration were observed in the PLGA-DSP-NP group as compared to healthy control (Fig. 9A). PLGA-DSP-NP successfully prevented epithelial degradation, with epithelial thickness comparable to that of healthy control.

[0124] However, there was a 11-fold reduction in epithelial thickness for the saline group, a 1.8-fold reduction for the DSP eyedrops group, and a 2.4-fold reduction for the placebo NP. No significant difference in epithelial thickness was detected between the PLGA-DSP-NP and DSP solution groups (Fig. 9B). Apparent augmentation of comeal thickness was observed in the saline, placebo NP, DSP eyedrop, and DSP solution control groups as compared to healthy control and PLGA-DSP-NP. NM-injury induced thickening of the cornea was effectively mitigated by PLGA-DSP-NP treatment, surpassing the efficacy of other treatment groups. Compared to healthy control, PLGA-DSP-NP group had a mere 11% rise in corneal thickness (p > 0.05), whereas, saline, DSP eyedrop, placebo NP, and DSP solution caused a 50%, 33%, 57%, and 39% rise in comeal thickness respectively at PID14 (Fig. 9C).

[0125] 4. Discussion Our current knowledge of the mechanisms of ocular injury from exposure to vesicating agents is limited, presenting a major challenge in devising effective therapeutic strategies and interventions. The ocular injuries resulting from NM exposure have been well documented, since ocular surface is identified as the tissue most vulnerable to such injuries). These studies have revealed that the alkylating effects of NM are associated with significant clinical manifestations such as severe corneal inflammation, ulceration, neovascularization, and corneal thickening. Similarly, increased oxidative stress and elevated cellular expression of inflammatory and angiogenic cytokines are other hallmarks of NM-induced comeal injury. Following exposure to NM, the primary objective of therapy is to mitigate ocularinflammation, facilitate the healing of comeal epithelium, and reinstate corneal transparency during the acute phase. Currently, there is lack of such specific and effective treatments to tackle the mustard-induced comeal injuries. The existing treatment protocols lack specificity and fail to address the fundamental pathophysiology of this type of injury.

[0126] The use of eyedrops is currently the primary method for delivering corticosteroids, owing to ease of application. However, topical eye drops suffer from low efficiency, since most of an applied solution is lost via blinking and drainage, leading to suboptimal treatment. Moreover, there is evidence to suggest that frequent topical administration of dexamethasone can lead to high systemic exposures, which may result in unwanted systemic side effects. While ex-vivo transscleral dmg penetration is higher for water-soluble DSP in comparison to the hydrophobic dexamethasone, the therapeutic effects of free DSP solution are short-lived due to quick clearance from the subconjunctival space. The resulting low ocular bioavailability of DSP calls for frequent injections, which adversely affects patient compliance.

[0127] The need to address current challenges, coupled with the clinical importance of DSP in treating corneal inflammation motivated us to develop a new, sustained-release DSP formulation that is appropriate for SCT injection. We found that PLGA-DSP-NP provides sustained DSP delivery to the anterior segment of eye while minimizing systemic dmg exposure. We investigated the course of comeal injury across three concentrations of NM (0.5%, 1%, and 2% w / v) and two exposure durations (1 min and 2 min) in a rat model. We assessed the key clinical and histopathologic outcomes of NM-induced corneal injury.

[0128] Similarly, there was a significant increase in the expression of inflammatory cytokines like TNFa, IFN-y, NF-kB, IL-6, IL-ip, and angiogenic markers like MMP-2, MMP-9, and VEGF in the NM injured cornea on PID14 compared to healthy control. Compromised integrity of the corneal epithelial barrier leads to impaired hydration of the cornea, resulting in an increase in the thickness of the comeal stroma. This outcome has been consistently noted in our current research. Furthermore, infiltration of inflammatory cells into the stroma possibly exacerbates corneal thickening. We observed that an increase in exposure duration of NM typically enhanced progression of clinical symptoms. The clinical parameters like ulceration, NV, and opacity were significantly upregulated for 2-min NM exposure compared to 1-min exposure on both PID7 and PID14. After a detailed comparative study of the corneal injury by various NM concentrations and exposure durations, we selected 1%NM exposure for 1 min as the condition for evaluation of treatment efficacy. This injury model displayed an apparent corneal ulceration (2-fold high), corneal NV (2.6-fold high), and corneal opacity (3.6-fold high) compared to healthy control after 14 days. Also, significant increase in inflammatory cells, increased comeal thickness and epithelial degradation was observed in the H&E-stained images for the cornea exposed to 1% NM for 1 min. Following treatment with PLGA-DSP-NP, the upregulation of major inflammatory (IFNy), angiogenic (VEGF, MMP-2, MMP-9, PDGFRp, PDGFRa and fibrotic markers (TGFpi) was observed in the NM injured corneas.

[0129] Long-term treatment with topical corticosteroids has been associated with safety concerns like increased IOP. SCT injection of PLGA-DSP-NP does not cause significant an IOP increase, inflammation, change in body weight or other pathological manifestations, unlike DSP eyedrops. Additionally, the PLGA-DSP-NP primarily comprises PLGA and Zinc (in addition to the drug), both of which are extensively utilized in ophthalmic products. Nevertheless, if instances of deleterious effects arise post-injection of PLGA-DSP-NP, the depot of SCT injected nanoparticles can be moved from the subconjunctival space.

[0130] 5. Conclusion Polymeric nanoparticles loaded with water soluble dexamethasone sodium phosphate (PLGA-DSP-NP) provide sustained release of DSP in the anterior segment of the eye after a single subconjunctival injection. Subconjunctival injection of PLGA-DSP-NP successfully prevented progression of NM-induced comeal injury in rats. Sustained release of DSP holds great potential with improved therapeutic efficacy and enhanced patient compliance for the management of comeal injuries by vesicant exposure. Lurthermore, our study offers a comprehensive comparison showing differences of NM-induced corneal injury progression at different NM doses and exposure durations in a rat model. This allows for an extensive study and deeper insight into a variety of pathological issues caused by comeal exposure to NM and the development of novel treatment ventures.

[0131] While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMSWe claim:

1. A method of preventing or treating at least one symptom of vesicant agent induced injury to at least one eye of a subject in need thereof, comprisingadministering to the at least one eye a composition comprising nanoparticles loaded with at least one corticosteroid.

2. The method of claim 1, wherein the at least one corticosteroid is dexamethasone sodium phosphate (DSP).

3. The method of claim 1, wherein the nanoparticles comprise a polymer or copolymer comprising one or more poly (hydroxy acid) polymers, optionally coated with one or more hydrophilic polymers.

4. The method of claim 3, wherein the copolymer is comprised of poly(D,L-lactic acid-glycolic acid (LA:GA) in a 50:50 ratio and the one or more hydrophilic polymers is polyethylene glycol (PEG).

5. The method of claim 1, wherein the at least one corticosteroid is present in the nanoparticles at a loading of 5-10% by weight.

6. The method of claim 1, wherein the vesicant agent is a chemical warfare agent, an arsenical, a halogenated oxime or a pharmaceutical vesicant.

7. The method of claim 6, wherein: the chemical warfare agent is a sulfur mustard or a nitrogen mustard; the arsenical is Lewisite; the halogenated oxime is phosgene oxime; and the pharmaceutical vesicants is a chemotherapy drug.

8. The method of claim 7, wherein the chemotherapy drug is doxorubicin or vincristine.

9. The method of claim 1, wherein the at least one symptom is selected from the group consisting of: upregulation of inflammatory cytokines, angiogenic markers, fibrotic markers, comeal ulceration, corneal neovascularization, corneal opacity, increased comeal thickness, and epithelial degradation.

10. The method of claim 1, wherein the step of administering is performed by subconjunctival (SCT) injection.

11. The method of claim 1, wherein the step of administering forms a depot of nanoparticles that releases the at least one corticosteroid over a period of at least a plurality of days or weeks.

12. The method of claim 13, wherein the at least one corticosteroid is released over a period of at least two weeks.

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