(T1030-03-14) Novel Microemulsion-Based Fenofibrate Eyedrop for Treating Nitrogen Mustard-Induced Corneal Injury

Purpose: Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, has been used clinically for the treatment of dyslipidemia for over 30 years [1]. The role of PPARα in mitigating oxidation, inflammation, and angiogenesis has previously been established and is attributed to the regulation of various genes involved in lipid metabolism, mitochondrial function, and inflammation processes. Furthermore, PPARα is highly expressed in the cornea [2]. Nitrogen mustard (NM) is a potent vesicating chemical warfare agent that causes severe ocular injuries leading to corneal inflammation, ulceration, and neovascularization. Effective therapies for vesicant-induced ocular injuries are not yet available [3]. This study aims to formulate and evaluate fenofibrate microemulsion (Feno ME) eyedrop for treating NM-induced corneal injuries.

Methods: 0.5% Feno ME eyedrop formulation was prepared using the high-energy emulsification method. The formulation was then characterized for droplet size, polydispersity index (PDI), zeta potential, osmolarity, rheological behavior, and drug loading. The safety profile of the eyedrops was evaluated by investigating the changes in blink rate, intraocular pressure (IOP) and tear secretions following Feno-ME eyedrop administration (3x per day) (Figure 1) [4]. NM-induced corneal injury was established on SD rats through the topical application of 1% NM solution for 2 mins. Then SD Rats with NM-induced corneal injury were treated with Feno ME, Placebo ME and saline eyedrops (3× per day, n=14 eyes each group). Routine clinical observations and evaluations for corneal opacity, corneal ulceration, and corneal neovascularization were performed on day 14 post injury (Figure 2). Histological analysis was conducted to assess corneal integrity following treatment with Feno ME (Figure 3).

Results: The prepared Feno ME eyedrop had a droplet size of 20.4 ± 0.1 d.nm, PDI of 0.07 ± 0.01, a near-neutral zeta potential (-0.7 ± 0.4 mV), an osmolarity of 457 mOsmol/kg, and a drug loading of above 95.6 %. The rheological evaluation demonstrated that when the temperature of Feno ME eyedrop is increased from room temperature to 35°C, which is close to corneal temperature, the viscosity remains high. This property may contribute to better retention on the corneal surface. Additionally, Feno ME eyedrops exhibit high viscosity at low shear rates, which can enhance residence time, while their low viscosity at high shear rates suggests they are unlikely to cause discomfort during blinking. Safety evaluations showed no signs of toxicity or irritation to the eyes, nor changes in blink rate, tear secretion, or IOP after 7 days of eyedrop administration. Efficacy studies revealed that Feno ME eyedrop significantly inhibited corneal ulceration (1.2-fold, P < 0.05), neovascularization (1.7-fold, P < 0.001), and opacity (1.8-fold, P < 0.001) over a duration of two weeks in rats with NM-exposed corneas compared to saline treated group. On the other hand, the group treated with placebo ME eyedrop significantly developed severe corneal ulceration (1.2-fold, P < 0.01), neovascularization (1.4-fold, P < 0.001), and opacity (1.5-fold, P < 0.01) compared to Feno ME eyedrop treated group. In addition, histology evaluations revealed that Feno ME eyedrop significantly inhibits epithelial degradation (P < 0.001) and decreases total corneal thickness (18-20%) compared to saline (P < 0.01) and placebo ME eyedrop (P < 0.05) in NM-injured corneas, in which the corneal epithelia were degraded with extensive inflammation in the corneal stroma.

Conclusion: This novel microemulsion eyedrop (Feno ME) provided a significant drug loading capacity, demonstrated a favorable safety profile, and presented a more desirable option for patients compared to conventional invasive injectable interventions. Feno ME eyedrop could serve as a promising treatment option for the treatment of corneal injuries induced by vesicants.

References: 1. Lee CH, Olson P, Evans RM: Minireview: lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors. Endocrinology 2003, 144:2201-7.
2. Liang, Wentao, et al. "Peroxisome proliferator-activated receptor-α (PPARα) regulates wound healing and mitochondrial metabolism in the cornea." Proceedings of the National Academy of Sciences 120.13 (2023): e2217576120.
3. Poudel, Sagun, et al. "Dexamethasone sodium phosphate loaded nanoparticles for prevention of nitrogen mustard induced corneal injury." Experimental Eye Research (2024): 109902.
4. Zagon, Ian S., et al. "Spontaneous episodic decreased tear secretion in rats is related to opioidergic signaling pathways." Investigative ophthalmology & visual science 53.6 (2012): 3234-3240.


Figure 1: 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) Average number of blinks counted in 3 minutes; (C) Tear weight (mg); 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).


Figure 2: (A) Representative corneal fluorescein staining images. (B) quantification of corneal ulceration (fluorescence intensity) on D14 post NM-exposure. (C) Representative slit-lamp images of rat corneas. (D) Quantification of corneal neovascular length. (E) Quantification of corneal opacity (mean ± SEM, n = 14 independent eye). Topical eyedrop, thrice/day. *P <0.05; **P <0.01; ***P <0.001



Figure 3: Representative H&E-stained corneal sections from different treatment groups at D14 post-NM exposure. (B) Quantification of corneal epithelial thickness and (C) total corneal thickness at D14 post-NM exposure for different treatment groups. Surface epithelial cells are represented with red arrows, necrotic keratinocytes with green arrows, and inflammatory cells are represented with yellow 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 μm.