Elucidating the ocular absorption pathway of fasudil hydrochloride towards developing advanced ophthalmic formulations

Glaucoma is a progressive eye disease that damages the optic nerve and can cause irreversible blindness, often linked to elevated intraocular pressure (IOP). Elevated IOP arises from impaired aqueous humour outflow, primarily involving the trabecular meshwork and Schlemm's canal, necessitating effective treatments to prevent vision loss. Fasudil hydrochloride, a selective ROCK inhibitor, offers a promising approach by improving aqueous outflow through the trabecular meshwork, reducing IOP, enhancing ocular blood flow, and protecting optic nerve cells. However, its hydrophilic nature poses challenges, leading to low bioavailability and necessitating advanced delivery systems to improve its therapeutic potential. This study aims to elucidate the ocular absorption pathway of Fasudil and develop delivery systems to enhance its ocular surface absorption by potentially extending ocular residence time or improving penetration into deeper tissues. Methods: A novel HPLC method, validated according to FDA guidelines, was used to quantify Fasudil permeation across bovine cornea and sclera. Three delivery systems—chitosan nanoparticles, PLGA microparticles, and ocular inserts—were formulated to enhance Fasudil bioavailability. Chitosan nanoparticles, prepared via ionic gelation and optimized using statistical design, were evaluated for particle size, PDI, zeta potential, entrapment efficiency, in vitro release, ex vivo permeation, and ocular tolerability using BCOP and HET-CAM tests. Cytotoxicity on human lens epithelial cells was assessed with the NRU assay. PLGA microparticles, produced by spray drying, were characterized for size, charge, morphology, and release profile. Ex vivo corneal and scleral permeation studies were performed, along with ocular irritation assessments via BCOP and HET-CAM tests and cytotoxicity evaluation on uveal melanoma cells. Ocular inserts, developed using the solvent-casting method with film-forming polymers (PVA, CMC, ALG), were analysed for physicomechanical properties, mucoadhesion, and drug release. Ocular irritation potential was assessed using the HET-CAM test and BCOP assay. Results: The HPLC method demonstrated high accuracy and selectivity with a detection limit of 0.22 µg/ml and quantification limit of 0.65 µg/ml. Permeation studies revealed significantly higher Fasudil penetration through the sclera than the cornea. Optimized chitosan nanoparticles exhibited small size, high zeta potential, low PDI, and high encapsulation efficiency, enabling controlled release and corneal permeation compared to a simple solution. They were no ocular irritation observed, and lens epithelial cells showed good tolerance at low concentrations. PLGA microparticles, with a size of 2.7 ± 0.5 µm and negative charge, provided sustained release over 24 hours, were well-tolerated, and showed no cytotoxicity on the uveal melanoma cell line. Ocular inserts demonstrated uniform thickness (156.9 ± 3.0 µm), consistent weight (5.5 ± 0.40 mg), and compatibility with a surface pH of 6.8 ± 0.2. These inserts exhibited sustained drug release, excellent ocular tolerance, and desirable mechanical properties, including tensile strength of 20.4 ± 4.3 MPa and strain of 76.1% ± 5.2. Conclusion: The ocular absorption pathway of Fasudil was elucidated, and three delivery systems were subsequently developed and evaluated to enhance its ocular absorption and potentially bioavailability. Chitosan nanoparticles were promising, significantly enhancing corneal and scleral permeation. All investigated systems demonstrated good ocular tolerability and biocompatibility, making them viable options for effective Fasudil delivery for glaucoma treatment.