Ppara agonist loaded microemulsion and methods of use thereof

Abstract

An oil-in-water microemulsion is provided. The microemulsion contains 1-5% v / v oil, 10-25% v / v of at least one emulsifier, 1-10% v / v of at least one co-emulsifier, and a peroxisome proliferat or- activated receptor-a (PPARa) agonist. Methods of preparing the microemulsion and for the ocular delivery of the microemulsion are also provided. The microemulsion may be used for the treatment of ocular injury or ocular degeneration.

Description

[0001] PPARa AGONIST LOADED MICROEMULSION AND METHODS OF USE

[0002] THEREOF

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under grant numbers R01 EY033477 and UG3EY036558 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0005] FIELD OF THE INVENTION

[0006] The invention is generally related to oil-in-water emulsions for the ocular delivery of PPARa agonists.

[0007] BACKGROUND OF THE INVENTION

[0008] Despite the prohibition of chemical warfare on battlefields by the 1925 Geneva Protocol, the threat of chemical agents still persists, causing a widespread harm on human beings [1- 3]. Mustard agents, particularly sulfur mustard (SM) and nitrogen mustard (NM), are of significant concern due to their ease of synthesis, potent toxic effects, large unused stockpiles and odorless / colorless nature, making them some of the most feared warfare agents [4—6]. Their ability to dissolve in both polar and non-polar media allows them to easily penetrate exposed tissues, leading to detrimental effects on multiple organs [7-9], Clinical studies on mustard gases exposed victims have highlighted ocular surface as the most vulnerable and destructive tissue to injury upon vesicants exposure [1 ,7,10],

[0009] NM is a bifunctional analog of SM, sharing both structural similarity and toxicological mechanisms -primarily the alkylation of DNA at the N-7 positions of guanine

[0011] , NM has been widely used to study ocular injuries and vesicant-induced comeal damage, serving as a substitute for SM, which is a strictly controlled substance with limited accessibility to most research facilities

[0012] , Mild exposure to NM results in photophobia, irritation, inflammation, lacrimation, edema, and blepharospasm. Whereas, severe exposure can lead to persistent keratopathy, corneal neovascularization (NV), scarring, compromised corneal transparency, and irreversible vision impairment, potentially requiring interventions such as corneal or limbal stem cell transplantation [1,7,13-15], Current understanding of NM-induced comeal injury and its pathogenesis is limited. In the clinical setting, prophylactic and therapeutic interventions for mustard gases-induced ocular injury are limited to the use of protective equipment, irrigating solutions, application of topical lubricants, antibiotics, and ocular hypotensive medications to manage symptoms and prevent complications [16-18], However, these interventions mainly focus on symptomatic relief and do not address the underlying pathophysiology of ocular injuries. Despite efforts to develop effective medical countermeasures for mustard gases-induced ocular injuries, the explored treatment interventions have shown limited success and present their own drawbacks. Colchicine, while demonstrating some anti-inflammatory effects in rabbit models of SM corneal injury, has a narrow therapeutic window and demonstrated severe side effects, such as gastrointestinal distress and bone marrow suppression

[0018] , The use of chelating agents like deferoxamine in combination with zinc or gallium has shown the potential to reduce oxidative stress and inflammation; however, their efficacy in treating mustard gases-induced ocular injuries remains uncertain, and may cause unintended systemic effects [19J. Diphot erine rinsing solutions, although effective in decontaminating the ocular surface, do not address the underlying pathophysiology and may not prevent long-term complications

[0020] ,

[0010] Peroxisome proliferator-activated receptor-a (PPARa) is a ligand-activated transcription factor that is crucial in regulating lipid metabolism

[0021] , Fenofibrate, a synthetic PPARa agonist, has been used clinically to treat dyslipidemia for over three decades

[0022] , After absorption, fenofibrate is rapidly metabolized in tissues and plasma to its active metabolite, fenofibric acid

[0023] , A recent study has revealed that PPARa is highly expressed in the cornea and fenofibrate promotes corneal wound healing

[0024] , PPARa has been shown to ameliorate oxidative stress, inflammation, and angiogenesis by regulating numerous genes involved in lipid metabolism, mitochondrial function, and inflammatory pathways [25-30]. Furthermore, previous studies have demonstrated that PPARa overexpression in the cornea enhances corneal wound healing, while PPARa knockout delays the wound healing process

[0024] ,

[0011] Fenofibrate is an FDA approved oral medication for treating dyslipidemia, but its efficacy in treating corneal disorders via oral administration is limited due to poor ocular distribution [31,32], Eyedrops are the most widely used and accepted topical ophthalmic dosage forms due to their ease of manufacturing and application [33,34], However, this dosage form has several limitations, such as rapid drainage of the drug from the eye surface, either to the outside of the eye or to the systemic circulation through the nasolacrimal duct, and a very short corneal contact time, which may result in the absorption of only 5% of the administered dose into ocular tissues [35,36], To address these limitations, various formulation strategies have been employed, including viscosity enhancers [37,38], penetration enhancers

[0039] , use of bioadhesive polymers

[0040] , ocular implants [41,42], contact lenses [43,44], ocular inserts

[0045] , ocular injections, and colloidal systems, such as liposomes, nanosuspensions, microparticles, nanoparticles, nanoemulsions, and microemulsions (ME) [46-48], According to the biopharmaceutical classification system (BCS), Fenofibrate is a class II drug with low solubility and high permeability. A formulation for this class of drugs should improve its solubility and corneal contact time

[0052] ,

[0012] Therefore, improved formulations for the delivery of PPARa agonists are needed.

[0013] SUMMARY

[0014] Described herein is a PPARa agonist loaded microemulsion prepared using biocompatible, non-toxic, biodegradable, and emulsifying excipients that can provide higher drug loading, longer stability, precorneal retention and permeation through the eyes.

[0015] An aspect of the disclosure provides an oil-in-water microemulsion, comprising 1- 40% v / v at least one oil, such as glyceryl monooleate oil; 5-75% v / v at least one emulsifier; 5-75% v / v at least one co-emulsifier; and a PPARa agonist. In some embodiments, the PPARa agonist is fenofibrate or fenofibric acid. In some embodiments, the at least one emulsifier comprises a mixture of hydrophobic and hydrophilic emulsifiers. In some embodiments, the at least one emulsifier has a hydrophilic-lipophilic balance (IILB) value greater than 10. In some embodiments, the at least one co-emulsifier comprises caprylocaproyl macrogol-8- glycerides. In some embodiments, the microemulsion has an average droplet size of 10-200 nm. In some embodiments, a ratio of emulsifier to co-emulsifier is from 3:1 to 1 :3.

[0016] Another aspect of the disclosure provides a pharmaceutical composition comprising an oil-in-water microemulsion as described herein, wherein the pharmaceutical composition is formulated for topical administration to an eye.

[0017] Another aspect of the disclosure provides a method of preparing an oil-in-water microemulsion as described herein, comprising adding a dispersed phase to a continuous phase to provide a combined sample, wherein the dispersed phase comprises the PPARa agonist, the oil, the emulsifier, and the co-emulsifier, and wherein the continuous phase comprises water; and sonicating the combined sample under conditions sufficient to produce the oil-in-water microemulsion.

[0018] Another aspect of the disclosure provides a method for treating an ocular injury or ocular degeneration in a subject in need thereof, comprising topically administering to an eye of the subject a therapeutically effective amount of an oil-in-water microemulsion as described herein. In some embodiments, the ocular injury or ocular degeneration is caused by a vesicant. In some embodiments, the vesicant is nitrogen mustard or sulfur mustard. In some embodiments, the ocular injury or ocular degeneration is caused by diabetic keratopathy.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1. Pseudo-ternary phase diagram with microemulsion region.

[0021] Figures 2A-F. (A) The dynamic viscosity (Pa s) vs. temperature (°C); (B) flow curves (dynamic viscosity as a function of the shear rate) performed at 35°C; (C) Average droplet size, (D) polydispersity index, (E) zeta potential, and (F) percentage drug content of Feno ME eyedrop after storage for 7 months at room temperature (25±2°C) (mean ± SEM; n = 3). There was no significant difference in any of the studied parameters (p > 0.05) compared to the initial values obtained from the freshly prepared formulation.

[0022] Figures 3A-D. Feno ME and placebo ME eyedrops were dosed to rat eyes 3 times / day for 7 days. (A) Representative slit lamp images captured on day 7 after application of fluorescein; (B) Tear weight (mg); (C) Average number of blinks counted in 3 minutes; and (D) Change in the intraocular pressure. (IOP). Blink rate and tear weight were measured on day 0, day 1, day 3, and day 7 after administration of saline, Placebo ME, and Feno ME. No Significant difference between the groups. (Mean ± SEM, n = 6-8).

[0023] Figure 4. Comeal retention study of Feno ME, Systane® Ultra and Saline in rats. Quantification of fluorescein intensity in the eye at 10 s, 1 min, 5 min, 10 min, and 30 min. Relative mean fluorescence intensity (MFI) is calculated as the MFI at observation time / MFI at 10 s. Results are presented as the mean ± SEM (n = 3).

[0024] Figures 5A-C. Feno ME exhibited sustained drug concentrations in tear fluid up to 12 hours after the last administration. (A) Fenofibrate and fenofibric acid concentrations were measured in tear fluid at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h following a single dose of Feno ME (0.5% w / v) (n = 6-9 independent animals). Drug tissue biodistribution after 7 days of 3x daily dosing (n = 6-9 independent animals) demonstrates the distribution of (B) fenofibrate and (C) fenofibric acid across various ocular tissues, including the cornea, conjunctiva, sclera, and aqueous humor (n=4-6). Results are presented as the mean ± SEM.

[0025] Figures 6A-E. Feno ME alleviates corneal ulceration and suppresses neovascularization after NM-exposure. Topical eyedrops (thrice / day) were dosed Ih after NM-exposure. (A) Representative slit-lamp images demonstrating comeal ulceration and (B) quantification of corneal ulceration (fluorescence intensity) on PID14. (C) Representative microscopic images of rat corneas on PID14 representing corneal NV and opacity. (D) Quantitative analysis of comeal NV length and (E) comeal opacity scores on PID14 for different groups, (mean ± SEM, n = 14 independent eye). Two-way ANOVA followed by Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001.

[0026] Figures 7A-D. (A) Representative H&E-stained comeal sections from different treatment groups at PID14. (B) Quantification of corneal epithelial thickness and (C) total comeal thickness at PID14 for different treatment groups. Surface epithelial cells, necrotic keratinocytes, and inflammatory cells are represented with arrows. Mean ± SEM, n = 6-7. Two-way ANOVA followed by Tukey’s multiple comparison test: * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bar: 100 pm.

[0027] Figures 8A-C. Sprague Dawley rats were subjected to NM injury in the cornea, and treatment was followed for 14 days with Feno ME and Saline. PPARa staining was quantified in (A) comeal epithelium, (B) stroma, and (C) endothelium using ImageJ (mean ± SEM, n = 3). Two-way ANOVA followed by Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001.

[0028] Figures 9A-H. Feno microemulsion preserves IICEC migration and shows no migration-inhibitory effects. (A) Screening of individual emulsifiers / components used in formulation: representative scratch- wound images of primary human corneal epithelial cells (HCECs) acquired at the indicated times to assess any migration-inhibitory effects. (B) Summary of Plutonic® selection versus hydrophilic-lipophilic balance (HLB): filled symbols indicate microemulsions that formed stably; open symbols indicate unstable systems. (C) Characteristics of Pluronics® used for optimization (block composition / HLB). (D) Representative images comparing the optimized Feno microemulsion versus placebo microemulsion in the scratch assay. (E-F) Under high-glucose conditions (25 mM), HCECs treated with the optimized Feno microemulsion (0.5% eyedrop; 20 pM fenofibrate equivalent) showed no inhibition of migration and demonstrated enhanced wound closure versus vehicle / placebo; quantification at 24 h. (G-H) Under HNE-induced oxidative stress, Feno microemulsion enhanced migration relative to vehicle / placebo and approached normal (no-stress) levels; quantification at 24 h shown in (H). Data are mean ± SEM, n = 3 independent experiments; P < 0.01. Vehicle indicates placebo microemulsion. Images were analyzed in ImageJ to calculate acellular (wound) area.

[0029] Figures 10A-G. Rheology, morphology, stability, and release of the Feno microemulsion. (A) Dynamic viscosity (Pa-s) versus temperature (°C) comparing Feno microemulsion and Systane® Ultra; the Feno microemulsion shows a modest temperature- responsive increase in viscosity, consistent with Pluronic®-based thermo-gelling behavior, while remaining readily flowable from a dropper. (B) Flow curves at 35 °C (dynamic viscosity versus shear rate) demonstrating shear-thinning at high shear (-5,000 s ', blinkmimicking) for both Feno microemulsion and Systane® Ultra, indicating lubricating behavior compatible with blink comfort. (C) Ciyo-TEM image of diluted Feno microemulsion showing uniform, nanometer- scale droplets consistent with a microemulsion architecture (scale bar: 20 nm). Long-term storage at room temperature (25 ± 2 °C) for 12 months showed no significant changes in mean (D) droplet size, (E) polydispersity index, (F) zeta potential, or (G) percent drug content relative to freshly prepared Feno microemulsion (mean ± SD; n = 3).

[0030] Figures 11A-J. Safety, precorneal retention, and ocular PK of the Feno microemulsion. Safety in rats dosed three times daily for 7 days with Feno microemulsion or placebo (saline control): representative corneal fluorescein staining on Day 7 (A); intraocular pressure (B), tear secretion (C), and body weight (D) measured on Days 0, 2, 4, and 7 showed no significant differences among groups (mean ± SEM; n = 6-8). Precorneal retention in rats under anesthesia with manual blinking using fluorescein-labeled drops: representative IVIS images (E) and relative mean fluorescence intensity (MFI) over time (F). The Feno microemulsion exhibited significantly longer corneal residence than Systane® Ultra at 30 min (mean ± SEM; n = 3). (G) Corneal retention in New Zealand rabbits under natural blinking (no anesthesia) assessed by OCT: Feno microemulsion remained detectable after five blinks, whereas saline and Systane® Ultra were not detectable. (H) Tear-fluid pharmacokinetics in rats after a single 20 p L dose of Feno microemulsion (0.5% w / v): Feno and fenofibric acid were quantified at 5 min to 8 h, showing detectable levels up to 12 h (mean ± SEM; n = 6-9). Ocular tissue distribution after 7 days of dosing (3x / day): concentrations of Feno (I) and fenofibric acid (J) in cornea, conjunctiva / sclera, and aqueous humor at 1 h and 4 h after the final dose (mean ± SEM; n = 4—6). Conjunctiva / sclera showed the highest levels; plasma and controls were below detection.

[0031] Figures 12A-J. Feno microemulsion normalizes mitochondrial respiration and restores bioenergetic proteins under hyperglycemia. (A) Seahorse XFe96 mitochondrial stress test in ex vivo mouse corneal punches showing OCR traces (sequential oligomycin, FCCP, and rotenone / antimycin A). (B-D) Quantification from (A): basal respiration (B), maximal respiration (C), and ATP-linked / spare respiratory capacity (D). Feno microemulsion increases OCR parameters toward control compared with high glucose / placebo. (E) In vitro Seahorse OCR traces for HCECs cultured under high D- glucose (L-glucose osmotic control), then treated with placebo or Feno microemulsion (20 pM fenofibrate equivalent). (F) Quantification from (E) demonstrating recovery of mitochondrial function with Feno microemulsion versus placebo under high glucose. (G) Representative Western blots of PPARa, TOMM20, and PGC-la ( -actin loading control) across groups (low-glucose control, high-glucose, placebo, Feno microemulsion). Densitometry of PPARa (H), TOMM20 (I), and PGC-la (J) normalized to -actin, showing restoration toward low-glucose control with Feno microemulsion, whereas placebo remains reduced.

[0032] Figures 13A-L. Topical Feno microemulsion accelerates comeal epithelial wound closure in STZ, db / db, and db / m mice. (A-D) STZ-diabetic mice: (A) Representative fluorescein-stained corneas at baseline and follow-up after 2-mm Algerbrush abrasion. (B) Time course of wound healing over 0-48 h shows faster healing with Feno microemulsion versus placebo microemulsion and saline. (C-D) Quantification of residual wound area at 24 h and 48 h; Feno microemulsion reduces the remaining defect compared with controls. (E H) db / db mice: (E) Representative images and (F) time course demonstrate accelerated healing with Feno microemulsion. (G-H) at 24 h and 48 h confirm significantly smaller wounded areas with Feno microemulsion compared to placebo and saline. (I-L) db / m control mice: (I) Representative images and (J) show enhanced healing with Feno microemulsion. (K-L) Quantification at 24 h and 48 h indicates smaller wounded areas with Feno microemulsion compared to saline and placebo. Mice received twice-daily topical dosing (5-10 pL / eye) of Feno microemulsion, placebo microemulsion, or saline. Wounds were imaged after 0.1% fluorescein, and residual wound area was measured in Image!

[0033] Bars / points show mean ± SEM.

[0034] DETAILED DESCRIPTION

[0035] Embodiments of the disclosure provide a PPARa agonist loaded emulsion as a topical drug delivery system comprising an oil, at least one emulsifier, and at least one co-emulsifier. The oil is dispersed in the aqueous phase (water) and stabilized using the emulsifier and coemulsifier. As shown in the Examples, the emulsions described herein have a low polydispersity index (below 0.2), zeta potential near neutral, high drug loading (90-110%), osmolality (171-1711 mOsm / kg), and physical stability (no phase separation, no drug precipitation, no turbidity). The emulsions improve treatment efficacy and reduce the potential side effects caused by high oral dose.

[0036] The term "PPARa agonist" as used herein refers to any agonist of peroxisome proliferator-activated receptor alpha. For example, a PPARa agonist is a compound or composition which when combined with PPARa directly or indirectly (e.g., binding directly to PPARa) stimulates or increases an in vivo or in vitro reaction typical for the receptor, e.g., transcriptional regulation activity as measured by known assays in the art.

[0037] Suitable PPARa agonists include fibrate drugs. Fibrates are a class of amphipathic carboxylic acids and esters, such as, but not limited to, fenofibrate, fenofibric acid, pemafibrate, clofibrate, aluminum clofibrate, simfibrate, ronifibrate, etofibrate, clofibride, clinofibrate, gemfibrozil, ciprofibrate, bezafibrate, binifibrate, etofylline clofibrate, pirifibrate, or a pharmaceutically acceptable salt thereof. In some embodiments, the fibrate drug is a derivative, for example, an acid or ester derivative. In some embodiments, the PPARa agonist is a non-fibrate drug such as A 190.

[0038] The amount of agonist loaded in the emulsion depends upon the compound, and indication for which it is used. Exemplary ranges include 0.001 mg / ml to 50 mg / ml, or 0.01 mg / ml to 50 mg / ml, such as 0.01 mg / ml to 30 mg / ml or about 0.01 to about 5 percent (w / v) of the composition.

[0039] An emulsion contains two immiscible liquids, which are finely dispersed in each other. For oil-in water emulsions, the oily phase (dispersed phase) is evenly mixed into the aqueous phase (continuous phase). The terms “emulsion”, “microemulsion”, and “nanoemulsion” may be used interchangeably throughout. Microemulsions as described herein form spontaneously upon mixing of the aqueous phase and oily phase. The microemulsion is, therefore, a thermodynamically stable system, with particles dispersed in the continuous phase. The droplet size of the dispersed phase in a microemulsion is less than 500 nm, generally in the range between 10 nm and 200 nm, inclusive, e.g. about 10-30 nm.

[0040] The oily phase may include non-polar compounds, e.g., non-polar active ingredients; at least one emulsifier / surfactant; at least one co-emulsifier / surfactant; oils; non-polar solvents; preservatives; and microemulsion stabilizers. Other lipophilic and / or amphiphilic components can be included in the oily phase. In some embodiments, the oily phase contains only oil, emulsifier, co-emulsifier, and active ingredient. The aqueous phase may include components in addition to water such as polar solvents, e.g. polar protic solvents, such alcohols, typically alcohols having more than one hydroxy group such as dihydroxy and trihydroxy alcohols, such as glycerol and propylene glycol; emulsifiers / surf actants; preservatives; and emulsion stabilizers. In some embodiments the aqueous phase contains only water or water and emulsifiers / surf act ants.

[0041] Suitable oils for the oil in water microemulsion include one or more of vegetable oils, animal oils, and synthetic or semisynthetic oils, or mixtures thereof. In some embodiments, the oil phase is of synthetic origin and is selected from caprylic / capric triglycerides (e.g., Miglyol® 810, Captex® 355), medium-chain mono- / di-glycerides (e.g., Capmul® MCM / MEM), long-chain monoglycerides (e.g., glyceryl monooleate / Peceol®), fatty acids (e.g., oleic acid, caprylic acid), and fatty-acid esters (e.g., ethyl oleate, ethyl butyrate, isopropyl myristate, isopropyl palmitate), or a mixture thereof; in certain embodiments the one or more oils comprise Capryol® 90. In other embodiments, the oils are of natural origin and are selected from soybean (soya) oil, cottonseed oil, corn oil, linseed oil, sunflower oil, krill oil, cod-liver oil, fish oil, avocado oil, almond oil, babassu oil, borage oil, carob oil, cashew nut oil, grapeseed oil, coconut oil, Oryza sativa (rice) bran oil, castor oil, hemp seed oil, jojoba oil, peanut oil, poppy seed oil, sesame oil, walnut oil, olive oil, wheat-germ oil, argan oil, blackcurrant seed oil, and oils enriched in polyunsaturated fatty acids (PUFAs) by >10%, or mixtures thereof. In some embodiments, the composition contains about 1-40% v / v oil (e.g., about 1-15% v / v, 1-10% v / v, e.g., about 1-5% v / v).

[0042] The microemulsion also contains at least one emulsifier and at least one co-emulsifier. Emulsifiers (or surfactants) are substances that are amphipathic, which means that they contain both hydrophobic and hydrophilic groups. The hydrophilic-lipophilic balance (HLB) of an emulsifier is measured on an empirical scale developed by Griffin (W. C. Griffin, J. Cosmet. Chem., 1, 31 1, 1949). This scale can range from 0 to 40 or higher, with lower values representing a more lipophilic molecule and higher values representing a more hydrophilic molecule. In some embodiments, the emulsifier and / or co-emulsifier has a HLB value of 10 or higher, e.g. 20 or higher, e.g. about 20-30. In some embodiments, the at least one emulsifier and / or co-emulsifier comprises a mixture of hydrophobic and hydrophilic emulsifiers. In some embodiments, the at least one co-emulsifier has an HLB value of 10-18, e.g. about 12-14.

[0043] The emulsifier or co-emulsifier can be nonionic. For example, nonionic emulsifiers or co-emulsifiers can be selected from among Pluronic® (poloxamers), Labrasol® (caprylocaproyl macrogol-8-glycerides), Cremophor®, Kolliphor®, Polysorbates (Tween™), lauryl dimethyl ammine oxide, polyethoxylated alcohol, polyoxyl lauryl ether, Brij®, polyoxyethylated castor oil, lecithin, polyethylene glycol, and glycerol esters of fatty acids. In some embodiments, the total amount of emulsifier is about 5-75% v / v, e.g. about 5-30% v / v or 10-25% v / v of the total composition. In some embodiments, the total amount of co- emulsifier is about 1-15% v / v, e.g. about 1-10% v / v. In some embodiments, a ratio of emulsifier to co-emulsifier is about 3:1 to 1 :3, e.g. about 2:1.

[0044] In some embodiments, the emulsion remains stable at a temperature from 4-37°C for at least 6-12 months, e.g. at least 1-3 years, such that an average droplet size of the nanoemulsion does not increase more than 1-10 nm.

[0045] The polydispersity index (PDI) is within the range 0-0.4, inclusive, or 0.02 to 0.3, inclusive.

[0046] Another aspect of the disclosure provides a dosage form or pharmaceutical composition comprising an emulsion as described herein. A composition comprising the emulsion may contain one or more pharmaceutically acceptable earners. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0047] Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelate, carbohydrates such as lactose, amylose or starch, magnesium stearate talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. Other suitable excipients include, for example an inert diluent such as lactose; granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate.

[0048] The compositions may include pharmaceutically acceptable salts of the compounds described herein including the acid addition and base salts thereof.

[0049] The compositions of the present disclosure may also contain other components such as, but not limited to, antioxidants, additives, adjuvants, buffers, tonicity agents, bioadhesive polymers, stabilizing agents, thickening agents, and preservatives. It should be appreciated that the compositions of the present disclosure may be buffered by any common buffer system such as phosphate, borate, acetate, citrate, carbonate and borate-polyol complexes, with the pH and osmolality adjusted in accordance with well-known techniques to proper physiological values.

[0050] An additive such as a sugar, a glycerol, and other sugar alcohols, can be included in the compositions of the present disclosure. Pharmaceutical additives can be added to increase the efficacy or potency of other ingredients in the composition. For example, a pharmaceutical additive can be added to a composition of the present disclosure to improve the stability of the bioactive agent, to adjust the osmolality of the composition, to adjust the viscosity of the composition, or for another reason, such as effecting drug delivery. Non-limiting examples of pharmaceutical additives of the present disclosure include sugars, such as trehalose, mannose, D-galactose, and lactose.

[0051] In an embodiment, if a preservative is desired, the compositions may optionally be preserved with any well-known system such as benzyl alcohol with / without EDTA, benzalkonium chloride, chlorhexidine, Cosmocil® CQ, or Dowicil 200.

[0052] In some embodiments, the emulsions are preservative free. To avoid microbial growth during long term storage, the emulsions may be sterilized using sterile filtration, wherein the finished emulsion preparation is passed through a sterilizing-grade membrane during fill of the final product container. Embodiments of the disclosure include methods of preparing compositions as described herein. The method may comprise adding a dispersed phase to a continuous phase to provide a combined sample, wherein the dispersed phase comprises the PPARa agonist, the oil, the emulsifier, and the co-emulsifier and wherein the continuous phase comprises water optionally with an emulisifier; and sonicating the combined sample under conditions sufficient to produce the oil-in-water emulsion. The ultrasonication high-shearing method is applied to generate disruptive energy to disperse the oil into the aqueous phase and to form nano-sized droplets. Other preparation methods known in the art include emulsifying techniques such as high-pressure homogenization, high-shear mixing, and microfluidization.

[0053] The present disclosure also provides a method for the ocular delivery of a PPARa agonist to a subject in need thereof, comprising topically administering an oil-in-water emulsion as described herein to the subject. The compositions of the disclosure may be useful for the treatment of any disease or disorder that the included PPARa agonist is useful for treating. In particular embodiments, the compositions are formulated for ophthalmic administration, and are useful for treatment of ocular injury or ocular degeneration. Such conditions and disorders, include, but are not limited to, injury induced by vesicating chemical agents or other chemical agents (e.g. nitrogen mustard or sulfur mustard) or other corneal bums with corneal neovascularization and corneal opacity and corneal diseases such as diabetic keratopathy, dry eye syndrome, infectious keratitis, comeal ulcer, cornea neovascularization, comeal dystrophies, or keratoconus, macular degeneration, glaucoma, infection, inflammation, allergy, and diabetic retinopathy.

[0054] In some embodiments, the composition contains at least a second active agent for treatment of an ocular injury or degeneration. Such agents include, a prostaglandin inhibitor selected from among latanoprost, travoprost, bimatoprost, unoprostone, and pharmaceutically acceptable derivatives, hydrates, solvates, metabolites and salts thereof, or a polymorphic crystalline form thereof, and / or an anti-angiogenic agent selected from among sorafenib, sorafenib tosylate, regorafenib, regorafenib tosylate, regorafenib isethionate, regorafenib, ethylsulfonate apremilast, radotinib, spironolactone, and pharmaceutically acceptable derivatives, hydrates, solvates, metabolites and salts thereof, or a polymorphic crystalline form thereof; and / or an anti-oxidant selected from among nordihydroguaiaretic acid, mesonordihydroguaiaretic (masoprocol) and pharmaceutically acceptable derivatives, hydrates, solvates, metabolites and salts thereof, or a polymorphic crystalline form thereof. In some embodiments, the composition does not contain a second therapeutic / active agent.

[0055] Administration can be affected by any route, such as topically using eye drops, and by injections, such as intravitreal injection, subconjunctival injection, sub-tenon injection, retrobulbar injection, subretinal injection, or suprachoroidal injection. Topical administration to the eye refers to localized administering to a surface of an eye, for example, to any exterior aspect of the eye normally accessible between the eyelids. The composition may be provided in any form that allows local or direct administration thereof to the eye, including but not limited to, a solution, drops, mist / spray, plasters and pressure sensitive adhesives, ointment, lotion, cream, gel, lyophilized / spray-dried forms, rods, beads, emulsions, lenses, patch, plug, elixir, etc. Topical administration to the eye generally may be achieved, for example, with drops, irrigants, ointments, or sprays.

[0056] In one embodiment, the pharmaceutical composition is formulated as eyedrops. For example, the composition may be presented in a rigid bottle fitted with a combination screw- cap / bulb pipette dropper cap. In one embodiment the composition is presented in a squeeze bottle fitted with a tip constructed and arranged to serve as a dropper and a removable cap to cover the tip. In a typical embodiment the eyedrops are dispensed as 20 microliter to 300 microliter single drops.

[0057] A patient or subject to be treated by any of the compositions or methods of the present disclosure can mean either a human or a non-human animal including, but not limited to mammals, e.g. dogs, horses, cats, rabbits, gerbils, hamsters, rodents, birds, aquatic mammals, cattle, pigs, camelids, and other zoological animals.

[0058] In some embodiments, the active agent is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of active agent to treat the disease or disorder at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific active agent employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels or frequencies lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage or frequency until the desired effect is achieved. However, the daily dosage of the active agent may be varied over a wide range from 0.01 to 1 ,000 mg per adult per day. In particular, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, in particular from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day. In some embodiments, the composition is administered daily or 2, 3, 4 or more times weekly.

[0059] In one embodiment, the administering involves topically administering a single drop of the composition to the eye to be treated. Such single-drop administration can include administration 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, or 12 times a day or more.

[0060] Whilst the beneficial effects of the disclosure are particularly apparent in ocular delivery, the utility of the disclosure is not limited and compositions according to the invention may also used for oral, intranasal, buccal, rectal, vaginal, ocular, intraperitoneal, and parenteral drug delivery.

[0061] Before exemplary embodiments of the present invention are described in greater detail, 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.

[0062] 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 or both of those included limits are also included in the invention.

[0063] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. The term "about" when used in connection with percentages will mean.+-. l%.

[0064] 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. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0065] 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 publication dates which may need to be independently confirmed.

[0066] 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 antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0067] 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.

[0068] 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.

[0069] EXAMPLE 1

[0070] Vesicants are highly toxic chemicals used as warfare agents, primarily absorbed through the skin, inhalation, or ocular surface. Nitrogen mustard (NM), a potent vesicant, often leads to blindness due to the inadequacies of existing therapies. This study explores the role of peroxisome proliferator-activated receptor-a (PPARa) in NM-induced ocular injury and evaluates the efficacy of fenofibrate, a PPARa agonist, in preventing corneal injury progression. Fenofibrate, an FDA-approved drug for treating dyslipidemia, has shown off- label potential in promoting comeal wound healing. However, its limited ocular distribution following oral administration reduces its therapeutic efficacy. We developed fenofibrate - loaded oil-in-water microemulsion (Feno ME) eyedrop formulation to enhance aqueous solubility and ocular bioavailability of fenofibrate, aiming to improve the therapeutic efficacy in treating NM-induced corneal injury. The formulation, characterized by a droplet size of approximately 20 nm, was stable for seven months, showed extended ocular retention, and demonstrated well-accepted rheological properties without ocular irritation or toxicity. A 14- day treatment with 0.5% Feno ME (3X / day) significantly reduced comeal ulceration, neovascularization (NV), and opacity in NM-induced ocular injury in rats. Histopathological analysis confirmed preserved comeal integrity and reduced inflammation.

[0071] Furthermore, Feno ME demonstrated sustained drug levels in ocular tissues with minimal systemic absorption. Immunohistochemistry revealed that NM injury significantly reduced PPARa levels in the comeal epithelium, which was restored with Feno ME treatment, offering a targeted therapeutic approach. Our Feno ME formulation offers a promising localized delivery system for fenofibrate by effectively mitigating NM-induced corneal injury and introducing new treatment interventions for vesicant exposure.

[0072] Materials and methods

[0073] Materials Fenofibrate (Cat. No: F6020) was purchased from Sigma- Aldrich (St. Louis, MO, USA). Fenofibrate-d6 (internal standard, IS) was obtained from Toronto Research Chemicals (Toronto, ON, Canada). Caprylocaproyl macrogol-8-glycerides (Labrasol®) and glyceryl monooleate (Peceol™) were generously provided by Gattefosse (Saint-Priest, France). Pluronic® P61 (EO2-PO31-EO2), P103 (E017-P060-E017), and F127 (E0100-P065- EOIOO) were donated by BASF Corporation (Florham Park, NJ, USA). Ketamine HC1 (100 mg / mL) and xylazine (100 mg / mL) were purchased from Covetrus (Des Moines, IA, USA). Sterile tear flow test strips (Tear Flo) were obtained from HUB Pharmaceuticals (Rancho Cucamonga, CA, USA). Sterile irrigation solution (BSS) was purchased from Alcon (Fort Worth, TX, USA).

[0074] Preparation and optimization of Feno ME

[0075] The saturation solubility of fenofibrate in various oils, including Peceol™, oleic acid, Capryol® 90, and Maisine® oil, was determined. After selection of oil based on solubility, a series of emulsifiers and co-emulsifiers were tested for the miscibility with the oil to form a stable monophasic system. The final selected components included Peceol1Mas oil, mixture of Pluronic®-L61, P103, and F127 as emulsifiers, Labrasol® as the co-emulsifier, and deionized water as the aqueous phase.

[0076] To evaluate the effects of the percent of ME components such as oil, emulsifiers, and co-emulsifier (independent variables) on the physicochemical properties of the Feno ME such as droplet size, poly dispersity index (PDI), zeta potential, and drug content (dependent variables), a design of experiments (DoE) was constructed using JMP Pro 14 software (SAS Institute, Cary, NC, USA). The choice of these significant variables were identified through the screening design and preliminary findings

[0055] . The objective function for the DoE was to maximize drug content and surface charge, while minimize droplet size and PDI to obtain a stable ME. The Feno ME formulation was optimized using central composition design (CCD) with 4-factor, 3-level, and 2 replicates of the center points. In addition, the main effects, interaction effects, and quadratic effects of independent variables were studied. The independent variables were studied at three levels: low (-1), medium (0), and high (+1) as shown in Table 1, resulting in 26 experimental runs.

[0077] The levels of independent variables were selected based on the maximum desirability. A desirability function was computed for each of the observed variables (i.e., droplet size, PDI, zeta potential, and drug content) and for the response to be minimized defined as [56,57] :

[0078] For a response to be maximized, desirability function was defined as: where Ymin, Ymax, and Yi represented the lowest possible value, highest possible value, and the experimental value, respectively. An overall desirability function, DF, was calculated to identify optimum conditions for preparation of final formulation which could be calculated using the formula:

[0079] Where n was the number of responses.

[0080] Feno ME was prepared using high energy emulsification technique with slight modification [56,58], In brief, Feno was first dissolved in the oil at 40°C in a water bath while sonicating for 30 minutes (Branson CPX2800H, USA), followed by addition of emulsifiers and co-emulsifier. After vigorous vortex mixing, deionized water was added to the mixture and immediately sonicated using an ultrasonic Liquid Processor VCX 500 (SONICS & MATERIALS, INC., CT, USA) for 6 minutes and amplitude of 40%. The obtained ME was left overnight at room temperature to stabilize and become clear. During the optimization process, the turbidity and optical clarity of the ME were assessed, and only formulations exhibiting minimal turbidity and maximum transparency were selected for further characterization.

[0081] Table 1: Variables in central composition design (CCD) for Feno ME

[0082]

[0083] Physicochemical characterization of Feno ME

[0084] The droplet size, PDI, and zeta potential (measured by ^-potential) of the Feno ME were evaluated using Zetasizer® Nano ZS90 (Malvern Instruments, Malvern, UK). Each Feno ME formulation was diluted at a 1 :50 v / v ratio in 0.01 M NaCl solution to stabilize the ionic environment and sonicated for 1 minute to minimize multiple scattering effects and measured at 25 °C. The osmolality of Feno ME was determined using Vapro® 5600 vapor pressure osmometer (ELITechGroup, Logan, USA). Mean osmolarity values of three repeated measurements was reported as the result. Rheological properties of the Feno ME was evaluated with Rheometer (MCR 702e, Anton Paar GmbH, Austria) using a system of parallel steel plates and a standard-size concentric cylinder geometry, taking the formulation composition system into account. The dynamic viscosity of the samples was registered as a function of shear rate (1-5000 s -1) at 35 °C, using 21 measuring points and no time setting. From each flow curve, the value of zero shear viscosity (qO, viscosity in the range of Newtonian plateau) was obtained. The pO values reported were the mean values of the three measurements. Feno ME was also characterized for viscosity at 1 shear rate 0 s -1 and G ' at 1 Hz frequency in range of temperatures (25 - 35°C) to con linn the influence of thermogelation behavior at room temperature and application site temperature. As comparison of rheologoical property, two commercialized ocular products: Akom® Artificial Tears Ointment and Systane® Ultra lubricant eyedrop, were used as controls.

[0085] The drug content in the Feno ME was determined by diluting each formulation with acetonitrile, followed by filtration through a 0.2 pm PTFE syringe filter (Thermo scientific, USA). The concentration of Feno in the ME system was quantified using an HPLC system (LC-2030C 3D Plus, Shimadzu, Japan) equipped with a UV detector. Chromatographic separation was performed on an Agilent® Pursuit XRs 5 C18 column (250 x 4.6 mm). The mobile phase consisted of acetonitrile and water, both containing 0.1% trifluoroacetic acid (TFA), in a 25:75 v / v ratio. The flow rate was set at 1 mL / min, and the column temperature was maintained at 25 °C. A sample injection volume of 10 pL was used, and detection was conducted at a wavelength of 284 nm.

[0086] Stability tests of Feno ME

[0087] The selected Feno MF was subjected to the thermodynamic stability tests, including freeze-thaw cycle, centrifugation test, and heating -cooling cycle

[0059] , For freeze-thaw cycle, Feno ME was exposed to 3 cycles of freezing (-20°C) and thawing (25 °C) and was observed for 48 h. The centrifugation test was conducted to study the occurrence of phase separation due to centrifugal force. Feno ME was subjected to centrifugation at 5000 rpm for 30 min in a centrifuge. For heating-cooling cycle test, Feno ME was exposed to six cycles of heating (45°C) and cooling (4°C) for 48h. In each test, Feno ME was assessed for phase separation, creaming or cracking of ME along with physical appearance, including precipitation and turbidity or cloudiness.

[0088] To study the physical stability, Feno ME was stored at room temperature for 7 months. At predetermined time intervals, droplet size, PDI, zeta potential, and percent drug content remaining were measured. In addition, Feno ME was checked visually for any instability (turbidity, transparency, precipitation, and color change). Each experiment was repeated three times, and the results were presented as mean ± SEM.

[0089] Animals

[0090] 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; 200-250 g) were obtained from Envigo (Indianapolis, IN). The experimental animals were cared for by the VCU's Department of Animal Resources (DAR). The animals were housed and maintained on a 12 h light / dark cycles with ad libitum access to a standard laboratory diet and ion-sterilized water, under housing conditions of temperature (23 ± 2 °C) and relative humidity (55 ± 10%). 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, USA) was used for anesthesia during NM injury induction, and 1% tropicamide (Bausch + Lomb, USA) for pupil dilation during clinical evaluations. At the predetermined time point (post-injury day 14, PID14), animals were sacrificed by CO2 euthanasia followed by cervical dislocation.

[0091] Safety and ocular tolerance of Feno ME

[0092] The ocular safety of Feno ME was tested by installing 20 pL of the optimum Feno ME and the placebo ME (without drag) in SD rats’ eyeballs (n = 3), administered 3 times / day for 7 days. Control eyes received sterile saline at the same administration frequency. Eyes were examined for any signs of imitation or toxicities. On day 7, any changes to the ocular surface, including signs of inflammation, imitation, corneal ulceration, or epithelial damage, were assessed after the corneas were stained with fluorescein sodium, using a slit lamp microscope (Carl Zeiss, Germany). Corneal opacity was also monitored on the same day using microscopic imaging (Carl Zeiss Stemi 305) [50,60], Evaluation of possible side effects or imitant effects of Feno ME or vehicles was assessed by Schirmer test. In brief, TearFlo™ paper strip was placed in the eye's lower lid 30 minutes after Feno ME administration. After 1 minute, the paper strips was removed and weighed for total tear content [61,62], Blink-rate measurements were conducted by counting the number of blinks in a 3-minutes period. All the blink- rates measurements were performed 3 h after the Feno ME administration

[0062] , Noninvasive IOP measurements were conducted using a tonometer (Icare, Helsinki, Finland) immediately before application of the ME formulation (baseline) and at predetermined time intervals for 7 days. Six consecutive IOP readings were averaged for each individual eye at each measurement

[0060] ,

[0093] Ocular retention study of Feno ME

[0094] To evaluate the ocular retention of Feno ME in SD rats, noninvasive fluorescence imaging was performed using the IVIS® Lumina X5 (PerkinElmer, USA). Healthy rats were anesthetized as previously described, and IVIS imaging of the head region was performed using an in vivo imaging system equipped with filter sets (excitation / emission, 480 / 520 nm). Feno ME eyedrops, Systane® Ultra eyedrops, and saline were loaded with fluorescein sodium 2% and topically administered (20 |1L) in healthy rats. The fluorescence signal was measured at the predetermined time points (10 s, 1 min, 5 min, 10 min, and 30 min) after the topical administration of each formulation.

[0095] Pharmacokinetic and tissue biodistribution studies of Feno ME

[0096] For pharmacokinetic sampling, tear samples were collected at pre-dose, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h after a single administration of Feno ME. Schirmer test strips were used to absorb tears from the lower conjunctival sac, and the tear volume was calculated by weighing the strips before and after collection. Separate animals were used for each time point, and the strips were stored at -80 °C until analysis.

[0097] The concentration of Feno in the ocular tissues were analyzed using a liquid chromatography-mass spectrometry (LC-MS) method. Rats were administered with 20 pE of Feno ME or Feno suspension for 6 days (3x / day) with a limit of quantitation of 780 pg / mL. At day 7, rats were sacrificed at 1 h and 4 h after Feno ME or Feno suspension administration. The eyes were immediately dissected to separate cornea, aqueous humor, conjunctiva, and sclera, and stored at -80°C until further analysis.

[0098] On the day of analysis, tissues, tear samples, and plasma were thawed at room temperature, mixed with ice-cold methanol and acetonitrile (1:1, v / v). After homogenization using a tissue homogenizer, the sample mixtures were centrifuged at 14,000 rpm for 15 minutes at 4°C, supernatant collected in glass vials, and evaporated to dryness. The resulting residue was reconstituted with 100 pL of the mobile phase, filtered using syringe filters, and injected into the LC-MS system (LCMS-2020, Shimadzu, Japan) for drug quantification. The stationary phase consisted of an Eclipse C18 column (3.0 mm x 100 mm, 1.8 pm) maintained at 40°C. The mobile phase (water with 0.1% formic acid: methanol with 0.1% formic acid, 20:80, v / v) was run at a flow rate of 0.2 mL / min. The MS detector operated in MRM mode, with transitions for fenofibrate and fenofibric acid at m / z 361.0 to 121 (positive ESI) and m / z 319.0 to 232.9 (negative ESI), respectively.

[0099] NM induced corneal injury model and treatment with Feno ME

[0100] NM induced comeal injury in SD rats was developed using a 1 % w / v NM solution with a 2 minute exposure, as previously described

[0012] , Rats were randomly divided into three groups after NM exposure and given respective treatments topically I) Placebo ME, II) 0.5% Feno ME, III) Saline. After 1 h of NM exposure, each rats received respective treatments (20 pL / eye, bilaterally, 3 times a day) for 14 days. To prevent claw scratching, animals were held for at least one minute after applying the eyedrops. All animals were euthanized on PID14, and their eyeballs were collected for further analysis. Clinical assessments for NV, comeal ulceration, and opacity post treatment were carried out on PID14 as described previously

[0012] , Histopathological evaluation

[0101] The enucleation specimens were collected on PID14 and were fixed in Davidson's fixative solution for 24 h, followed by storage in 70% ethanol until paraffin embedding. The embedded specimens were sectioned axially (5 pm thickness) with anteroposterior orientation (from cornea to optic nerve). These sections were then stained with hematoxylin and eosin (H&E). Three eyeballs per group were examined using a Nikon® E800 light microscope with a digital camera. Comeal thickness was assessed approximately 1.0 mm away from the limbus region at 40X magnification using ImageJ software. Five random measurements were taken from corneal sections to determine the average corneal and epithelial thickness.

[0102] Immunohistochemistry

[0103] Ten-micrometer-thick corneal sections were stained with primary antibody against PPARa (dilution, 1 :100; #NB600-636 Novus, Centennial, CO, USA), overnight at 4°C, followed by incubation with the secondary donkey anti-rabbit IgG (H+L) Alexa Fluor® 488 antibody (dilution, 1 :400; #711-545-152, Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. The slides were mounted with Vectashield® mounting buffer containing DAPI (#H-1200 Vector Laboratories, Newark, CA, USA). Immunofluorescent images were obtained using Zeiss® Microscope (Observer Zl, Pleasanton, CA, USA). The fluorescein intensities were calculated using ImageJ software. Briefly, the region of interest, defined as the epithelium, stroma, and endothelium layers, was determined using a freehand selection tool. The mean gray values were used to compare the average fluorescein intensity among the groups

[0063] ,

[0104] Statistical analysis

[0105] Two groups were compared for statistical significance using a two-tailed Student's t-test, whereas three or more groups were compared using two-way ANOVA followed by a 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).

[0106] Results

[0107] Preparation and optimization of Feno ME

[0108] Selection of appropriate oil, emulsifier, and co-emulsifier is crucial for improving drug solubility, achieving high drug loading, and preventing drug precipitation as well as stability issues throughout the shelf life of a ME

[0064] , The oils, emulsifiers, and co-emulsifiers were selected based on the solubility of fenofibrate. In addition, all excipients chosen for the Feno ME formulation were among generally recognized as safe (GRAS) category. Fenofibrate is a lipophilic drug (LogP ~ 5), which indicates its higher solubility in non-aqueous phases, such as the oil phase of ME. We used Peceol™ as oil, which is primarily composed of long-chain fatty acids (Cl 8) and supports uniform mixing in ME. These fatty acids enhance the solubility of hydrophobic drugs and contribute to the stability of emulsified systems. They provide structural integrity by forming a stable interface between the hydrophobic and hydrophilic phases in the ME. The interaction between these long-chain fatty acids and surfactants is crucial, as it influences the overall stability and functionality of the ME

[0065] , Consequently, the longer fatty acid chains in Peceol™ necessitate a carefully balanced surfactant system to ensure consistency and prevent phase separation in the ME formulation [65,66].

[0109] Choosing the optimal emulsifier or emulsifier / co-emulsifier mixture is essential to improve the stability of the ME. As emulsifiers, we selected a combination of pluronics based on their hydrophilic-lipophilic balance (HLB) value. To prepare a stable ME formulation loaded with hydrophobic drug such as fenofibrate, we selected two hydrophobic Pluronics®, namely L61 and P 103, with HLB values of 2 and 9, respectively. These two hydrophobic Pluronics® in ME system help fenofibrate to be stable and prevent precipitation of the drug throughout the shelf life. In addition, we observed that the mixture of L61 and Pl 03 at a 1 :1 ratio (v / v) has higher drug solubility and avoids the turbidity of ME over time. To further increase the stability of the ME formulation, we included Pluronic® Fl 27, a safe hydrophilic emulsifier with HLB value of 22, to increase the stability of the oil phase in an aqueous phase and mask the potential irritation produced by other ingredients. Besides, Labrasol® is used in the ME formulation as co-emulsifier and penetration enhancer. It is reported that Labrasol® as co-emulsifier can maintain the droplet size distribution in the ME.

[0110] A 4-factor, 3 -level central composite design was adopted to determine the impact of percentage of oil, emulsifiers, and co-emulsifier on droplet size, PDI, zeta potential, and drug loading. In addition, stability factors such as transparency, turbidity, and phase separation of the obtained ME were considered. Three levels were selected for percentages of oil (1 %, 2.5%, and 4%), emulsifier 1 (12.5%, 15%, and 17.5%), emulsifier 2 (1.5%, 3%, and 4.5%), and co- emulsifier (2.5%, 5%, and 7.5%). To further assess the reproducibility of ME formulation, the center point was evaluated in duplicated, making the total runs to 26. Studying the effects of aforementioned independent variables on the dependent variables (responses), the 3D response surface plots were obtained (data not shown). We established desirability criteria ranging from 20 to 100 nm for droplet size, 0 to 0.3 for PDI, -10 to 10 mV for zeta potential, and >90% for drug content. After optimization, we found that 2:1 (v / v) is the ideal emulsifier / co-emulsifier ratio to make stable and clear ME. Quadratic polynomial models were determined to offer the greatest match for all the three criteria. The model terms revealed that independent variables directly influenced dependent variables (responses). All the observed responses were compared to discover the ideal experimental parameter. P-values demonstrated the effect of each component on chosen replies (Table 2). It was demonstrated that the percentage of oil (Peceol™) significantly affected droplet size, PDI, and drug content (P < 0.05), with increases in Peceol™ concentration resulting in larger droplet size, higher PDI, and higher drug content. This suggests that the oil concentration plays a crucial role in influencing the physicochemical properties of the microemulsion. Similarly, the percentage of emulsifiers I showed a significant effect on PDI, with higher concentrations leading to a more consistent distribution of droplet sizes.

[0111] Pseudo-ternary phase diagram for Feno ME was constructed using optimized concentrations of the oil, emulsifiers, and co-emulsifiers as obtained from the DOE. The phase diagram was plotted using the Peceol™, mixture of Pluronic® L61, Pluronic® P103, and Plutonic® F127, and Labrasol® as the oil, emulsifiers, and co-emulsifier, respectively. Furthermore, the choice of emulsifier / co-emulsifier and the ratio was based on the need to reduce interfacial tension, maximize the dispersion entropy of the ME system, and hinderance to coalescence, thereby forming a stable Feno ME [65,67], The circle represented on the pseudo-ternary phase diagram confirms that the optimum (maximum desirability) ME obtained from the DOE lies in the ME area as illustrated by the shaded region in Figure 1. In addition, this optimum composition was determined based on the minimum percentage of oil and emulsifier / co-emulsifier used to produce stable and transparent ME with maximum drug loading. Thus, the optimum Feno ME was composed of 3% of Peceol™ as the oil phase, 12.5% of Pluronics L-61 / Pluronics P103 (1:1 v / v) as the emulsifier I, and 2.6% of a 10% (w / v) solution of Pluronics Fl 27 in deionized water as emulsifier II, 7.5% of Labrasol as the coemulsifier, and 74.4% as the aqueous phase (Table 3).

[0112] Table 2: Summary of results of regression analysis for responses Yi, Y2, Y3 and Y4

[0113]

[0114] Table 3: Composition of optimized Feno ME.

[0115] Characterizations of Feno ME eyedrop

[0116] The Feno ME prepared by a conventional high energy emulsification method was transparent and monophasic. Table 4 lists the mean droplet size, PDI, and zeta potential values of both placebo and Feno ME formulations. The freshly prepared Placebo ME and Feno ME exhibited a monomodal size distribution with a low PDI of 0.06 ± 0.02 and 0.07 ± 0.01, and an average hydrodynamic droplet size of 18.4 ± 0.3 nm and 20.4 ± 0.1 nni, respectively. These findings demonstrated that incorporating drug into the oil phase of the ME did not affect the droplet size and PDI of the formulation, suggesting that Feno was fully solubilized in the oil phase. The smaller PDI values (<0.2) indicates that the Feno ME formulations are homogeneous and have a narrow droplet size distribution, which is likely due to the selection of ME composition from the ME region of the pseudo-ternary phase diagram. This resulted in the formation of nano-sized droplets and resistance to physical destabilization, including phase separation and drug precipitation

[0068] , The small droplet size is critical for achieving effective in vivo results, as it significantly enhances the transcomeal penetration of the Feno ME formulation. It is well-documented that droplet sizes below 200 nm are required to facilitate passive drug targeting across biological membranes, improving drug absorption and therapeutic efficacy [69,70]. Additionally, the nanodroplets in ME can remain in the precorneal area for extended periods due to their small size. This nanometer-scale dimension increases the surface area-to- volume ratio, enabling greater interaction with the mucin layer of the tear film. This enhanced interaction allows the nanodroplets to penetrate deeper and maintain prolonged contact with the comeal epithelium, improving adhesion to the ocular surface

[0071] , Furthermore, nanosized particles are more effective at navigating through the mucus layer than larger particles, preventing rapid clearance by tear turnover and blinking

[0072] ,

[0117] The small droplet size of Feno ME was expected because of the presence of Labrasol® as a co-emulsifier. Labrasol® can penetrate the film formed by the mixture of emulsifiers (Pluronics® Fl 27, L61 , and P103) at the oil / water interface. As a result, it lowers the mixture's viscosity, leading to the formation of nanodroplets with a smaller radius curvature and resulting in the production of transparent microemulsion systems [73,74],

[0118] Both the formulations (placebo and Feno ME) showed zeta potential values near zero (-0.9 mV and 0.7 mV, respectively), indicating neutral charges of the ME. The inclusion of non-ionic emulsifiers such as Pluronics® and Labrasol® might have contributed to the neutral charge of the ME [75-77], A neutral zeta potential can be beneficial in minimizing irritation and improving ocular tolerability, as demonstrated in studies where neutral-charged emulsions and nanoparticles exhibited lower irritation and better compatibility with ocular tissues [78— 80], Feno ME and Placebo ME had osmolarity of 457.3 and 398.2 mOsmol / kg, respectively. Studies have shown that ophthalmic products may be tolerated over a fairly wide range of tonicity (0.5%-5% sodium chloride, equivalent to about 171-1711 mOsm / kg), which indicates that Feno ME can be used safely for ocular applications without causing significant discomfort or harm to the eye

[0081] .

[0119] Table 4: Physicochemical characterization of Feno ME and Placebo ME

[0120] Rheological studies have been performed to predict the behavior of Feno ME after administration. We compared the Feno ME rheological behavior with marketed Akom® Artificial Tears Ointment and Systane® Ultra eyedrop because they have optimized drainage based on their zero-shear viscosity; therefore, this clinically accepted viscosity was chosen as the target value for the following studies. Figure 2A shows the viscosity of the Feno ME, Akorn® Artificial Tears Ointment, and Systane® Ultra eyedrop in the temperature range of 25 to 40°C in constant low shear rate (1 shear rate 0 s-1). For Feno ME, slight increase in the viscosity from 119.8 to 214.1mPas was observed as the temperature increased from 25°C (room temperature) to 35 °C (temperature of the corneal surface). On contrary, marketed artificial tear ointment and eyedrop exhibited a significant decrease in viscosity, dropping from 1802 to 214.9 mPas and 18.7 to 15 mPas, respectively. The increase in viscosity for Feno ME at increased temperature is attributed to the presence of Pluronics® and their thermogelation behavior, which has also been reported in earlier studies [62,82-84], This behavior of Feno ME is expected to improve the comeal retention. However, there is a concern that the higher viscosity of Feno ME compared to marketed ocular products might result in discomfort for patients when blinking. Figure 2B illustrates the dynamic viscosity of Feno ME and other ocular products as a function of the shear rate at 35°C. At high shear rates (5000 1 / s), which mimics blinking, both Feno ME and Systane® Ultra eyedrops showed shearthinning (lubricating) behavior, which is necessary for ocular comfort when blinking [62,85], Systane® Ultra eyedrops have clinically accepted blink tolerance, and Feno ME exhibited similar viscosity behavior in high shear rate conditions. Therefore, Feno ME is expected to have good blink tolerability and resistance to rapid clearance during blinking conditions. In addition, the optimized Feno ME formulation exhibited a high drug loading capacity, achieving a drug content of 95.6%, which indicates the efficient solubilization of fenofibrate within the ME system.

[0121] Stability of Feno ME eyedrop

[0122] Thermodynamic stability confers a long shelf life to the ME system compared to ordinary emulsions, which possess only kinetic stability and will eventually undergo phase separation. The thermodynamic stability of ME arises from the optimal combination of emulsifiers and co-emulsifiers, which reduce interfacial tension and create a stable interface between the oil and aqueous phases. This stability is further enhanced by the presence of Pluronics® and Labrasol® in the formulation, which not only stabilize the droplet size but also prevent coalescence and phase separation. Furthermore, ultrasonication during the preparation process significantly contributes to the thermodynamic stability of the ME by uniformly dispersing the droplets and reducing their size, which enhances the stability of the formulation. Studies have shown that ultrasonication can improve the thermodynamic stability of colloidal systems by reducing droplet size and ensuring uniformity across the emulsion [86,87], Feno ME passed all stability tests, including freeze-thaw cycles, centrifugation, and heating -cooling cycles, with no observed turbidity, phase separation, precipitation, or change in drug content (data not shown).

[0123] We evaluated the long-term storage stability of the optimized Feno ME for 7 months at room temperature (25 ± 2°C). No signs of physical instability, including creaming, turbidity, phase separation, or precipitation were observed, demonstrating the physical stability of the Feno ME eye drops (data not shown). Similarly, the Feno ME on long-term storage at room temperature did not show significant change in the mean hydrodynamic droplet size, PDI, zeta potential, or drug loading compared to the freshly Feno ME (Figure 2C). These findings indicate that Feno ME is resistant to droplets coalescence and Ostwald ripening, the major mechanisms responsible for ME destabilization and phase separation [88,89], Furthermore, the impact of gravitational force is significantly minimized due to smaller droplet sizes of Feno ME, allowing Brownian motion to dominate. As a result, creaming, flocculation, and

[0124] Ostwald ripening do not take place

[0090] ,

[0125] Safety and ocular tolerance of Feno ME

[0126] Upon administrations of Feno ME and placebo ME in healthy rats for 7 consecutive days, slit-lamp biomicroscopic examination results showed Feno ME and placebo ME did not cause any obvious changes like redness, swelling, ulceration of corneal surface compared to the saline control. Also, microscopic images showed no signs of irnlalion, such as redness or swelling. There was no aqueous humor flare, and the lens were transparent (Figure 3A). Furthermore, tear secretion, blink rate, and IOP remained close to the baseline, and no significant change was observed for Feno ME and placebo ME eyedrops compared to the control group after 7 days (p > 0.05) (Figure 3B-D). Notably, all the excipients used in the Feno ME formulation are GRAS, further supporting the safety profile of the formulation. The safety and tolerability of these excipients have been confirmed in previous studies, showing no adverse effects when used in ocular formulations [91-93],

[0127] Ocular retention

[0128] To evaluate the retention of fluorescent Feno ME on the corneal surface, IVIS imaging was performed in rats following a single topical administration of Feno ME, Systane® Ultra, and saline, all containing fluorescein sodium dye for this study. Images were captured at different time points. The fluorescence intensity became weaker over time due to tear turnover, blinking, and metabolism. As shown in Figure 4, the fluorescence intensity of saline significantly decreased (PcO.Ol) within 30 min, indicating poor comeal retention. While Systane® Ultra showed better retention compared to saline, its retention was still significantly less than Feno ME (P < 0.05). In contrast, Feno ME demonstrated a longer residence time on the cornea, with higher fluorescence intensity persisting at the 30-minute mark. These results indicate that Feno ME exhibits significantly enhanced corneal retention, providing ample time for the formulation to penetrate the corneal surface effectively. The higher retention of Feno ME can be attributed to its viscosity, which, as shown in our rheology results, remains constant and slightly increases at physiological temperatures. Increase in the viscosity improved comeal retention by minimizing the rate of fluid clearance from the ocular surface. The augmented viscosity prolongs the residence time of the Feno ME on the comeal surface, thereby facilitating extended contact and enhanced dmg permeation. In contrast, both Systane® Ultra and saline are less viscous, leading to faster clearance from the eye and shorter residence times.

[0129] Pharmacokinetic and drug-tissue biodistribution studies

[0130] Figure 5A shows the concentrations of fenofibrate and fenofibric acid in tear fluids following a single administration of Feno ME eyedrops. At 5 minutes post- administration, the concentrations of fenofibrate and fenofibric acid were 787.5 ng / mg and 5.9 ng / mg, respectively, indicating rapid ocular absorption. Fenofibrate is a prodrug that undergoes hydrolysis into fenofibric acid, its pharmacologically active form, primarily through esterases in tear fluid

[0094] , A significant decline in both fenofibrate and fenofibric acid concentrations was observed over time, with levels dropping to 90.1 ng / mg and 2.7 ng / mg, respectively, at 4 hours post-administration. By 12 hours, concentrations further decreased to 56.3 ng / mg for fenofibrate and 1.9 ng / mg for fenofibric acid, suggesting sustained presence of the drug and its metabolite in tear fluid, though at reduced levels. These results align with typical pharmacokinetic profiles observed in ophthalmic drug delivery, where rapid absorption is followed by a gradual decrease in drag concentrations over time. The extended presence of both fenofibrate and fenofibric acid in tear fluid up to 8 hours post-administration suggests that Feno ME eyedrop formulation provides prolonged ocular retention.

[0131] The tissue distribution of fenofibrate and fenofibric acid, in ocular tissues of rats after 7 days of Feno ME eye drop administration is shown in Figures 5B and 5C. Tissue samples were collected 1 and 4 hours following the final topical administration, and the concentrations were measured in four ocular tissues (cornea, conjunctiva, sclera, aqueous humor) and plasma. At 1 hour, fenofibrate and fenofibric acid concentrations in the cornea were 280 ng / g and 1277.3 ng / g, respectively. By 4 hours, both concentrations decreased to 55 ng / g and 248.19 ng / g, demonstrating rapid absorption followed by clearance typical of ocular drag kinetics.

[0132] Higher drag concentrations were observed in the conjunctiva / sclera tissues. After 1 hour, the concentrations of fenofibrate and fenofibric acid were 3463.1 ng / g and 6455.3 ng / g, respectively, suggesting enhanced penetration and retention in these tissues. At 4 hours, the concentrations decreased but remained elevated, with fenofibrate at 200. 1 ng / g and fenofibric acid at 564.0 ng / g. Notably, the highest concentrations of both fenofibrate and fenofibric acid were observed in the conjunctiva / sclera tissue at both the 1 hour and 4 hour time points, highlighting these tissues as the primary site for drag retention. In the aqueous humor, fenofibrate concentrations were much lower, measuring 7.5 ng / g at 1 hour and decreasing to 3.5 ng / g by 4 hours. Similarly, fenofibric acid concentrations were 131.0 ng / g at 1 hour, dropping to 27.5 ng / g by 4 hours, indicating limited penetration into this compartment. Drug levels in plasma and tissues from the control group were below the detection limit, confirming enhanced absorption of Feno ME and minimal systemic absorption.

[0133] Overall, fenofibric acid concentrations were significantly higher than fenofibrate concentrations in all ocular tissues at both time points, suggesting rapid conversion of fenofibrate to its active form and enhanced retention of fenofibric acid in ocular tissues.

[0134] Efficacy of Feno ME in NM-induced corneal injury - Clinical observation

[0135] An earlier study demonstrated that dexamethasone-loaded nanoparticles significantly mitigated corneal ulceration, neovascularization, and opacity in NM-injured rats

[0012] , In line with these findings, we evaluated the efficacy of Feno ME in reducing NM-induced corneal injury. On PID14, animals treated with saline and placebo ME exhibited severe corneal edema and ulceration. In contrast, significantly less edema was observed in the Feno ME group, with only 2 out of 7 animals showing signs of edema (Figure 6A). While some ulceration was present in the Feno ME group, it was notably less severe compared to the saline and placebo ME groups (p < 0.05 and P < 0.01, respectively). Only one out of seven rats in the Feno ME group demonstrated evidence of severe ulceration on PID14. Furthermore, a significant increase in mean fluorescence intensity was observed in the saline (1.2-fold) and placebo ME (1.3-fold) treated groups at PID14 when compared to Feno ME (Figure 6B), indicating a more pronounced inflammatory response in these groups.

[0136] Furthermore, Feno ME administration (3 times / day) prevented NM-induced comeal NV and comeal opacity. Conversely, saline and placebo ME groups exhibited a rise in corneal NV (Figure 6C). There was a significant difference in the average length of blood vessels between Feno ME and other groups. The average length of corneal NV in the Feno ME group was 1.88 ± 0.15 mm, whereas saline and placebo ME demonstrated comeal NV length of 3.13 ± 0.39 mm and 2.61 ± 0.40 mm, respectively (Figure 6D). A progressive increase in the corneal opacity was observed following NM injury, with significant increase by PID14. On PIDI4, the groups treated with saline and placebo ME had notable comeal opacity, with average opacity scores of 3.64 ± 0.5 and 3.42 ± 0.8, respectively (Figure 6E). However, Feno ME was effective in inhibiting the progression of comeal opacity with an average corneal opacity score of 2.28 ± 1.1.

[0137] Histopathological evaluation The H&E-stained corneal sections of the saline and placebo ME treated groups presented significant epithelial degradation, loss of structural integrity, and stromal thickening (Figure 7A). In comparison, the corneas from the Feno ME group had intact epithelial, stromal, and endothelial layers. Extensive infiltration of inflammatory cells and presence of necrotic keratinocytes was observed throughout the cornea in both Placebo-ME and saline treated groups. An increase in corneal thickness and the presence of inflammatory cell infiltration was observed in the Feno ME group compared to the healthy control (p > 0.05). However, it is still significantly lower compared to the saline and placebo ME groups. Feno ME successfully prevented epithelial degradation, with epithelial thickness comparable to that of healthy control. Also, epithelial squamous cells were observed in both healthy and Feno ME groups, indicating that Feno ME successfully maintained the integrity of the epithelium. However, there was an 8.2-fold reduction in epithelial thickness for the saline group and a 2.2-fold reduction for the Placebo ME group compared to healthy group. Similarly, epithelial thickness was significantly higher for Placebo ME compared to saline group (p < 0.001), which may be attributed to the lubrication effect of placebo ME eyedrop and potentially reducing mechanical stress and promoting a more stable tear film in NM injury (Figure 7B). An apparent increase in comeal thickness was observed in the saline and placebo ME groups when compared to the healthy and Feno ME groups. However, when treated with Feno ME, the thickening of the cornea induced by NM injury was effectively mitigated, surpassing the efficacy of other groups. Compared to the healthy control, the Feno ME group had a 33% rise in comeal thickness, whereas saline and Feno ME caused a 54% and 49% rise in comeal thickness, respectively, at PID14 (Figure 7C).

[0138] Immunohistochemistry

[0139] Immunohistochemical analysis was conducted to investigate the correlation between PPARa expression in the corneal epithelium and NM-induced injury and to determine the cellular localization of PPARa before and after treatment with Feno ME. PPARa is predominantly localized in the nuclei of corneal epithelial cells. In NM-wounded corneas (saline-treated group), PPARa expression in the epithelium was markedly reduced compared to healthy controls and the Feno ME-treated group. Notably, 14 days of treatment with Feno ME led to increased PPARa expression in the comeal epithelium, restoring levels closer to those observed in healthy tissue.

[0140] Quantification of PPARa staining using ImageJ revealed a significant decrease (P<0.01) in PPARa levels in the comeal epithelium following NM-induced injury compared to healthy controls (Figure 8A). The Feno ME-treated group showed a slight increase in PPARa levels in the corneal epithelium compared to NM-injured group following a 7-day administration post-NM injury. Quantification of PPARa levels in the stroma and endothelium revealed no significant differences or correlations among the healthy, NM-injured, and Feno ME-treated groups. This suggests that PPARa expression in these corneal layers was not affected by the injury or the treatment (Figure 8B and C). These findings demonstrate that NM injury significantly decreases PPARa levels in epithelium, validating our rationale of using PPARa agonist in NM injury to increase the ligand-receptor interaction and overcome PPARa level downregulation in the corneal epithelium.

[0141] Conclusion

[0142] Current understanding of the mechanisms underlying ocular injury caused by vesicating agents remains limited, posing challenges in the development of effective targeted treatments. Existing therapies primarily manage symptoms but fail to address the root pathophysiology. Our findings, particularly from immunohistochemistry, revealed that PPARa expression is significantly downregulated following NM-induced corneal injury, identifying PPARa as a novel therapeutic target for corneal damage caused by vesicating agents. The administration of fenofibrate, a PPARa agonist, in the form of eyedrops effectively mitigated vesicant- induced complications, demonstrating significant therapeutic use in treating these types of corneal injuries.

[0143] Eyedrops are commonly prescribed for the long-term treatment of eye conditions. However, a significant challenge in their effectiveness is the rapid clearance from the eye surface by the eyelids and tear film, leading to reduced drug retention and therapeutic effect. We designed an o / w ME to enhance fenofibrate’ s solubility and topical ocular delivery. As demonstrated by pharmacokinetic and biodistribution studies, Feno ME eye drop facilitated efficient fenofibrate penetration through ocular barriers, delivering significantly higher amounts of the drug to the anterior segment of the eye compared to free drug suspension. Following topical administration, the drug concentrations showed a gradient: sclera and conjunctiva > cornea > aqueous humor, suggesting that fenofibrate was delivered through a periocular pathway to reach the anterior segment tissues. This gradient also supports the hypothesis that the drug penetrates through the cornea.

[0144] Additionally, the presence of significant amounts of fenofibrate and its active metabolite, fenofibric acid, 12 hours after a single administration confirms the desirable drug retention of our formulation, with mass spectrometry detecting both fenofibrate and fenofibric acid in the ocular tissues and tear, suggesting that these tissues possess the esterases necessary to convert fenofibrate into its active form. Notably, negligible amounts (below LOQ) of fenofibrate and fenofibric acid were observed in plasma, minimizing the risk of systemic side effects. In summary, we successfully developed and optimized a biocompatible, non-toxic Feno ME for topical ophthalmic use, offering a therapeutic approach to address these limitations. Our formulation remains stable for at least seven months, is biocompatible, and provides high drug-loading capacities, offering a long-term solution for vesicant-induced comeal injuries.

[0145] EXAMPLE 2

[0146] Diabetic keratopathy is a common complication of diabetes that causes impaired comeal wound healing, nerve degeneration with decreased corneal sensitivity, epithelial thickness changes, corneal ulceration, and stromal edema. Currently, there is no satisfactory treatment. Diabetic corneas exhibit impaired mitochondrial metabolism, which is believed to play a crucial pathogenic role. Peroxisome proliferator-activated receptor-a (PPARa) is a ligand-activated transcription factor regulating lipid metabolism. Fenofibrate (Feno), a PPARa agonist, has been used clinically for dyslipidemia for over 30 years. PPARa ameliorates oxidative stress, inflammation, and angiogenesis by regulating target genes involved in lipid metabolism, mitochondrial function, and inflammation. Corneal PPARa overexpression enhances wound healing, whereas PPARa knockout delays healing and induces nerve degeneration, reduced sensitivity, and ulceration, recapitulating diabetic keratopathy.

[0147] Our studies show that Feno microemulsion exhibits nano-size (<50 nm), high drug content (>1 %), thermodynamic stability (>12 months), and scalability using low-energy input; promotes corneal epithelial cell migration under diabetic conditions; is safe with repeated dosing; shows long ocular surface retention; and accelerates wound healing in db / db mice. We hypothesize that Feno microemulsion eyedrops are effective for diabetic keratopathy and represent a new therapeutic approach. Table 5: Physicochemical characterization of Feno microemulsion and Placebo microemulsion

[0148] To ensure the safety of our Feno microemulsion eyedrop formulation, only the ingredients that do not compromise cell migration were selected. Scratch assay was performed on primary HCEC to evaluate the potential toxicity or cell migration inhibitory effects of each component in the formulation. After applying a scratch to the cell monolayer, we added components of the formulation (oil, emulsifier, and co-emulsifier) to the cell media and monitored cell migration for 24 h (Fig.9A). We confirmed that Peceol™ as oil and Labrasol® as co-emulsifier were safe and did not show any toxicity or inhibitory effect in HCEC cell migration. We further assessed the generally recognized as safe (GRAS) materials, the poly(ethylene glycol)-poly(propylene oxide)-poly(ethylene glycol) copolymers (known as Pluronics®) with various hydrophilic-lipophilic balance (HLB) values. We found that more hydrophilic Pluronics® with HLB >20 (e.g. F87, Fl 08, Fl 27, F68) did not compromise the cell migration, while the more hydrophobic Pluronics® with HLB <20 (e.g. L61, P103, P105, P65) induced significant inhibition in cell migration (Fig. 9A-C). Among these safe Pluronics®, only F87, F108 and F127 (filled symbols in Fig.9B) can produce Feno microemulsion with high drug content and good stability, and they are used in the development of novel eyedrops.

[0149] Furthermore, our optimized Feno microemulsion eyedrop was shown to be safe without inhibitory effects on HCEC migration under normal condition after 24h (Fig. 9D).

[0150] High D-glucose exposure is a standard in-vitro model that mimics the diabetic corneal microenvironment, inducing osmotic / oxidative stress and delayed epithelial repair, while L- glucose serves as an osmotic control. The optimized Feno microemulsion was evaluated in HCEC migration assay under high-glucose conditions (L-glucose control; D-glucose high glucose). After 24 h, no inhibitory effect on cell migration was observed for the optimized Feno microemulsion in the high-glucose condition; moreover, under the diabetic condition, Feno microemulsion significantly promoted HCEC migration compared with the Placebo microemulsion (Fig. 9E-F), indicating the therapeutic use of Feno microemulsion for diabetic keratopathy by enhancing corneal epithelial cell migration.

[0151] Under 4-hydroxynonenal (HNE) induced oxidative stress, HCEC cells were treated with Placebo or Feno microemulsion. After 24 h, Feno microemulsion enhanced cell migration under HNE stress to levels comparable to the normal (no-stress) condition and significantly greater than the Placebo microemulsion group, indicating preservation of migratory capacity under oxidative stress (Fig. 9G-H).

[0152] Rheology studies were performed to predict the behavior of the Feno microemulsion following ocular instillation and to assess blink tolerance. The rheological profile of the Feno microemulsion was compared with a marketed artificial tear, Systane® Ultra. The viscosity of the Feno microemulsion increased modestly from -148.8 to -210 mPa-s as temperature rose from 25 °C to 35 °C (corneal surface temperature), whereas Systane® Ultra decreased from -16 to -11 mPa s (Fig. 10A). The temperature-responsive increase in viscosity can be attributed, to Pluronics® with thermo-gelling behavior and may enhance comeal residence time. Importantly, at 0.5% drug load, the Feno microemulsion remains readily flow able from a dropper and easy to administer (see inset).

[0153] Dynamic viscosity as a function of shear rate at 35 °C showed that, at high shear (-5000 s ') mimicking blinking, both the Feno microemulsion and Systane® Ultra exhibited shear-thinning (lubricating) behavior necessary for blink comfort (Fig. 10B). The Feno microemulsion demonstrated viscosity behavior comparable to Systane® Ultra under high shear.

[0154] Formation and nanostructure of the Feno microemulsion were confirmed by cryo- TEM, which revealed uniform, nanometer-scale dispersed droplets consistent with a microemulsion structure (Fig. 10C).

[0155] To assess physical stability, the Feno microemulsion was stored at room temperature (25 ± 2 °C) for 12 months. At predetermined intervals, droplet size, PDI, zeta potential, and percent drug content remaining were measured, and samples were visually inspected for turbidity, loss of transparency, precipitation, color change, or phase separation. Each experiment was performed in triplicate and reported as mean ± SD. Over 12 months, no signs of physical instability (e.g., creaming, turbidity, phase separation, precipitation) were observed, and the Feno microemulsion showed no significant change in mean hydrodynamic droplet size, PDI, zeta potential, or drug content compared with freshly prepared Feno microemulsion (Fig. 10D-G). These findings indicate resistance to droplet coalescence and Ostwald ripening; the small droplet size likely minimizes gravitational effects, allowing Brownian motion to dominate and thereby limiting creaming, flocculation, and ripening.

[0156] Topical Feno microemulsion eyedrop is safe. To evaluate the safety profile of our Feno microemulsion eyedrop, rats received Feno microemulsion and placebo eyedrops three times daily for 7 days; control eyes received sterile saline. Feno microemulsion and placebo showed no damage to the corneal surface compared with saline (Fig. 11 A) and showed no significant changes in intraocular pressure, tear secretion, or body weight (Fig. 11B-D), demonstrating no signs of ocular toxicity or irritation from the Feno microemulsion or placebo.

[0157] Feno microemulsion shows prolonged comeal retention. IVIS imaging was used to assess retention on the rat comeal surface following a single topical administration of fluorescein-labeled Feno microemulsion or Systane® Ultra under anesthesia with manual blinking. Feno microemulsion exhibited significantly longer residence time on the cornea than the long-lasting eyedrop Systane® Ultra at 30 min post-dose under same conditions (Fig. 11E- F). Retention was further supported by OCT in rabbits under natural blinking (no anesthesia): after five blinks, Feno microemulsion remained detectable on the ocular surface, whereas saline and Systane® Ultra were no longer detectable (Fig. 11G).

[0158] Given the extended precorneal residence of the Feno microemulsion, we anticipated greater ocular exposure and therefore evaluated pharmacokinetics and tissue distribution. As shown in Fig. 11H, following a single administration of Feno microemulsion eyedrops, Feno and fenotibric acid concentrations in tear fluid at 5 min were -8,348 ng / mg and -2.9 ng / mg, respectively, indicating rapid ocular uptake and distribution. Feno is a prodrug that hydrolyzes to fenofibric acid — its active form — and esterases reported in tear fluid may contribute to this conversion. By 8 h, concentrations declined to -20.9 ng / mg (Feno) and -3.91 ng / mg (fenofibric acid), consistent with sustained, though decreasing, levels over time. In contrast, the Feno suspension group remained below the limit of detection (UOD, 970 pg / mU) at all sampled time points. The detectable presence of both Feno and fenofibric acid in tear fluid up to 8 h supports that the Feno microemulsion provides prolonged ocular retention and exposure.

[0159] Tissue distribution after repeated dosing was evaluated in rats following 7 days of Feno microemulsion eyedrops (Fig. 11I-J). Drug concentrations of Feno and fenofibric acid were quantified in cornea, conjunct! va / sclera, aqueous humor, and plasma at 1 and 4 h after the final administration. At 1 h, corneal concentrations were -472.2 ng / g (Feno) and -1,328.6 ng / g (fenofibric acid); by 4 h they declined to -40.6 ng / g and -205.8 ng / g, respectively, indicating rapid absorption followed by clearance typical of ocular kinetics. Conjunctiva / sclera exhibited the highest concentrations at both time points, suggesting enhanced penetration and retention in these tissues; levels decreased by 4 h but remained elevated relative to other matrices. In the aqueous humor, Feno concentrations were much lower, indicating limited penetration into this compartment. Plasma and control-group samples were below the detection limit, supporting minimal systemic exposure and confirming ocular targeting by the Feno microemulsion. Overall, fenofibric acid exceeded Feno in all ocular tissues at both time points, consistent with rapid in situ conversion of Feno to its active form and enhanced tissue retention of fenofibric acid.

[0160] Mitochondrial respiration was assessed by Seahorse XFe96 mitochondrial stress testing in ex vivo mouse comeal punches and in vitro HCECs. Under hyperglycemic stress (D-glucose; L-glucose osmotic control), HCECs treated with the Feno microemulsion exhibited basal and maximal OCR values comparable to the low-glucose control, indicating normalization of mitochondrial respiratory function; placebo did not restore OCR (Fig. 12A- D). In ex vivo corneal punches, Feno microemulsion increased ATP-linked respiration and spare respiratory capacity relative to hyperglycemic / diabetic controls, consistent with improved mitochondrial efficiency (Fig.l2E-F). Western blot analysis showed increased PPARa protein and recovery of key mitochondrial markers of oxidative phosphorylation in Feno microemulsion-treated samples versus placebo under high-glucose conditions, supporting a PPARa-dependent restoration of corneal bioenergetics (Fig. 12G-J).

[0161] Comeal epithelial wounds were created with a 2-mm Algerbrush burr, stained with 0.1% fluorescein, and imaged daily on Micron IV. STZ-diabetic, db / db, and db / m mice were randomized to receive Feno microemulsion, placebo microemulsion, or sterile saline (5-10 pL / eye, twice daily). Wound area was quantified from fluorescent pixels in ImageJ.

[0162] Across models, Feno microemulsion accelerated epithelial closure versus placebo and saline. In STZ-diabetic mice, Feno microemulsion reduced the 24-h residual wound area to near non-diabetic levels and at 48 h produced significantly smaller wounds than placebo or saline (Fig. 13 A-D). In db / db mice, Feno microemulsion significantly improved healing at 24 h and remained superior at 48 h (Fig. 13E-H). In db / m controls, Feno microemulsion also enhanced 24-h closure versus placebo and saline and maintained the smallest residual area at 48 h (Fig. 13 I-L). Together, these data show that topical Feno microemulsion consistently restores and accelerates comeal epithelial repair in diabetic keratopathy models and confers measurable benefit even in non-diabetic corneas.

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[0263] While the invention has been described in terms of its preferred 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

We claim:

1. A method for treating an ocular injury or ocular degeneration in a subject in need thereof, comprising topically administering to an eye of the subject a therapeutically effective amount of an oil-in-water microemulsion, wherein the oil-in-water microemulsion comprises:1 -40% v / v at least one oil:5-75% v / v at least one emulsifier;5-75% v / v at least one co-emulsifier; and a peroxisome proliferator-activated receptor-a (PPARa) agonist.

2. The method of claim 1, wherein the ocular injury or ocular degeneration is caused by a vesicant.

3. The method of claim 2, wherein the vesicant is nitrogen mustard or sulfur mustard.

4. The method of claim 1, wherein the ocular injury or ocular degeneration is caused by diabetic keratopathy.

5. The method of claim 1, wherein the PPARa agonist is fenofibrate or fenofibric acid.

6. The method of claim 1, wherein the at least one emulsifier comprises a mixture of hydrophobic and hydrophilic emulsifiers.

7. The method of claim 1, wherein the at least one emulsifier has a hydrophilic-lipophilic balance (HLB) value greater than 10.

8. The method of claim 1, wherein the at least one co-emulsifier comprises caprylocaproyl macrogol-8-glycerides.

9. The method of claim 1, wherein the microemulsion has an average droplet size of 10-200nm.

10. The method of claim 1, wherein a ratio of emulsifier to co-emulsifier is from 3:1 to 1:3.11 . An oil-in-water microemulsion, comprising1-40% v / v at least one oil;5-75% v / v at least one emulsifier;5-75% v / v at least one co-emulsifier; and a peroxisome proliferator-activated receptor-a (PPARa) agonist.

12. The oil-in-water microemulsion of claim 11, wherein the PPARa agonist is fenofibrate or fenofibric acid.

13. The oil-in-water microemulsion of claim 11, wherein the at least one emulsifier comprises a mixture of hydrophobic and hydrophilic emulsifiers.

14. The oil-in-water microemulsion of claim 11, wherein the at least one emulsifier has a hydrophilic-lipophilic balance (HLB) value greater than 10.

15. The oil-in-water microemulsion of claim 11, wherein the at least one co-emulsifier comprises caprylocaproyl macrogol-8-glycerides.

16. The oil-in-water microemulsion of claim 11, wherein the microemulsion has an average droplet size of 10-200 nm.

17. The oil-in-water microemulsion of claim 11, wherein a ratio of emulsifier to co- emulsifier is from 3: 1 to 1 :3.

18. A pharmaceutical composition comprising the oil-in-water microemulsion of claim 11, wherein the pharmaceutical composition is formulated for topical administration to an eye.

19. A method of preparing the oil-in-water microemulsion of claim 11, comprisingadding a dispersed phase to a continuous phase to provide a combined sample, wherein the dispersed phase comprises the PPARa agonist, the oil, the emulsifier, and the co-emulsifier, and wherein the continuous phase comprises water; and sonicating the combined sample under conditions sufficient to produce the oil-in- water microemulsion.

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