Enhanced in vivo performance of topical ocular acetazolamide nanocrystals: A novel approach for glaucoma treatment
Highlights
- Acetazolamide nanocrystals were successfully obtained by wet bead milling technique.
- Acetazolamide nanocrystals improved drug aqueous solubility.
- Nanocrystal technology allowed a therapeutic alternative for ocular application.
- Acetazolamide nanocrystals eye drop allowed 32% of intraocular pressure reduction.
Introduction
Glaucoma is one of the leading causes of blindness worldwide with a high prevalence among ocular pathologies (Tribble et al., 2023). Glaucoma is clinically divided into primary, secondary, and pediatric glaucoma. The most common primary glaucoma types include primary acute angle-closure glaucoma, primary chronic angle-closure glaucoma, and primary open-angle glaucoma which is more prevalent (Verma et al., 2024). Secondary glaucoma includes a glaucoma condition derived from vascular diseases, inflammation, or a contusion (Mueller et al., 2024).
Glaucoma is an ophthalmic neurodegenerative disorder involving progressive optic nerve damage ending in visual loss (Crish and Calkins, 2011). It primarily arises from the deterioration of retinal ganglion cell axons as they pass through the optic nerve head, leading to a persistent impairment of the retinal nerve fiber layer and triggering biomechanical remodeling of the lamina cribrosa (Wang et al., 2025). The abnormal increase in intraocular pressure (IOP) is the most important identifiable risk factor for the development of glaucoma, which can cause stress on the optic nerve and its subsequent damage, leading to blindness (Chan et al., 2017a, Chan et al., 2017b, Da Silva and Lira, 2022, Lusthaus and Goldberg, 2019, Wang et al., 2025). IOP-mediated stress triggers a retinal ganglion cell axonal injury at the level of the optic nerve head, inducing cell stress signaling pathways in retinal ganglion cell axons and soma, as well as microglia, and astrocytes. This process can eventually lead to the activation of cell death signals (Asrani et al., 2024). Thus, the deterioration of retinal ganglion cells is multifactorial, involving molecular mechanisms such as axonal transport disruptions, mitochondrial dysfunction, a lack of neurotrophic support, excitotoxicity, glial activation and oxidative stress (Basavarajappa et al., 2023, Garcia-Sanchez et al., 2024, Soto and Howell, 2014).
Given the impact of elevated IOP on ocular cell mechanisms that lead to the deterioration of retinal ganglion cells, its control is the main focus of glaucoma treatment. This rise in the IOP is primarily associated with impaired aqueous humor outflow either due to trabecular meshwork dysfunction in open-angle glaucoma or mechanical blockage in closed-angle glaucoma (Kong et al., 2023), excess production of aqueous humor, structural changes in the eye, neovascularization and chronic corticosteroid treatment (Asrani et al., 2024). IOP management may require a multifaceted approach, including both surgical and pharmacological treatments. Topical ocular pharmacological treatments include mainly prostaglandin analogous (Zhou et al., 2022), beta-blockers (Skov et al., 2022), and carbonic anhydrase inhibitors (Popovic et al., 2022, Stoner et al., 2022). However, available ocular hypotensive agents present several limitations in managing elevated IOP. Most of these treatments achieve only a partial reduction in IOP (no more than 33 %), which is often insufficient to control the condition adequately (Liu et al., 2022, Murgatroyd and Bembridge, 2008). Consequently, patients often require combination therapies with multiple medications to achieve the desired therapeutic effect and the application of several eye drops throughout the day, which can decrease patient therapy compliance, increase the risk of side effects, and compromise overall treatment efficacy (Broadway and Cate, 2015, Moore et al., 2023). In addition, oral drugs are often used combined to ensure IOP reduction such as carbonic anhydrase inhibitors (Popovic et al., 2022) and hyperosmotic agents (Casson, 2022). Among them, acetazolamide (AZM) is a drug of choice (Greiner et al., 2022, Sabri and Levin, 2006).
AZM is a sulfonamide derivative with diuretic, antiglaucoma, and anticonvulsant properties. It is a potent carbonic anhydrase inhibitor that regulates fluid secretion and reduces the IOP Kaur et al., 2002). Notably, AZM inhibits carbonic anhydrase II found in the epithelium of the ciliary processes which has a crucial role in aqueous humor secretion by regulating bicarbonate transport into the posterior chamber of the eye. Since AZM presents around 20 times the effect on carbonic anhydrase II relative to the other isoenzymes, this drug has remained as a carbonic anhydrase inhibitor for systemic administration to lower IOP for decades (Gulati and Aref, 2021).
AZM is considered a class IV drug (Kumari et al., 2024) according to the Biopharmaceutics Classification System (BCS) (Ghadi and Dand, 2017), exhibiting low aqueous solubility and low permeability that compromises its intraocular bioavailability and hinders the development of effective eye drops for the clinical management of glaucoma. Thus, AZM is employed in the glaucoma treatment predominantly via oral tablets, encompassing dosages from 125 to 500 mg administered once or twice daily (Gulati and Aref, 2021). Due to AZM’s unfavorable pharmacokinetic properties, high oral doses are required to achieve effective drug concentrations in the eye. However, systemic drug distribution can cause severe adverse effects, including gastrointestinal disturbances, electrolyte imbalances, metabolic acidosis, a propensity for kidney stone formation, potential allergic reactions and sensitivity in sulfonamide-allergic individuals, among others (Arbabi et al., 2022, Gulati and Aref, 2021, Schmickl et al., 2020). Most patients struggle to tolerate the side effects of oral AZM, often leading to treatment discontinuation (Manchanda and Sahoo, 2017).
While its analogs, brinzolamide and dorzolamide, are carbonic anhydrase inhibitors available for topical ocular administration with good water solubility, enough corneal penetration and comparable hypotensive effects, they can be ineffective in lowering IOP in certain types of glaucoma and can cause side effects. Furthermore, several studies demonstrated that the IOP-lowering effect of oral AZM is greater than that of topical dorzolamide in both eyes with or without glaucoma (Hayashi et al., 2017, Liu et al., 2019, Maus, 1997, Portellos et al., 1998). Thus, even though brinzolamide and dorzolamide represent significant progress in topical glaucoma treatment with carbonic anhydrase inhibitors, there remains a need for more effective topical therapies. In this context, developing AZM formulations for topical ocular administration may provide a potent hypotensive effect while minimizing systemic discomfort in patients.
Nonetheless, topical ocular drug administration involves a great challenge associated with overcoming the eye anatomical and physiological barriers that may hinder efficient drug concentration at the target site (Gaudana et al., 2010, Qi et al., 2023). Layers of tissue in the cornea and conjunctiva act as barriers to the passage of molecules, the nasolacrimal drainage system constantly replenishes ocular fluids and blinking washes away topical formulations. Therefore, frequent medication administration and high drug doses are required to achieve pharmacological effects (Qi et al., 2023). In addition, the drug’s physical–chemical characteristics can hinder the availability of adequate formulations for ocular application. Due to all the aforementioned reasons, no efficient topical pharmaceutical formulations containing AZM have been available.
In this regard, nanotechnology is a widely employed tool enabling the development of drug delivery systems that can overcome these challenges. Drug nanocrystals (NC) are crystalline nanoparticles (<1 µm) formed only by the pure drug and stabilized by a surfactant layer of either polymers or surfactants (Formica et al., 2022, Tuomela et al., 2016). NC are often obtained in suspension (nanosuspensions, NS), with further solvent removal leading to NC redispersible powder. NC presents nanometric size typically ranging from 200 to 500 nm (Müller and Keck, 2012), increased saturation solubility, higher dissolution rate and enhanced adhesion (Möschwitzer, 2013, Parmar et al., 2021, Tuomela et al., 2016), leading to enhanced bioavailability and therapeutic effectiveness (Tuomela et al., 2016).
NC showed pharmacological efficiency in several in vitro and in vivo animal models (McGuckin et al., 2022), aiming for multiple administration routes including oral (Tian et al., 2021, Wang et al., 2021), parenteral (Lu et al., 2016, Rossier et al., 2024), pulmonary and intranasal (Lombardo et al., 2021, Mangal et al., 2017, Zingale et al., 2022), transdermal (Mangal et al., 2017, Parveen et al., 2023) and ocular (Geng et al., 2024, Peters et al., 2020), among others. Recently, NC of a corticosteroid drug applied to ocular delivery exhibited high therapeutic effects after subconjunctival injection, using half the dose of those used in clinical practice (Formica et al., 2023).
NC could be considered an effective platform for ocular drug delivery due to their ability to enhance the apparent solubility and dissolution rate of poorly water-soluble drugs, which can lead to improved drug bioavailability (Sood et al., 2024). Their nanometric size can facilitate penetration through ocular barriers (Fathi-Karkan et al., 2024, Peters et al., 2020), and may offer higher adhesion to biological barriers (Kim et al., 2023, Zhang et al., 2024) and reduce irritation. Likewise, NC can be incorporated into various delivery systems, making them versatile and well-suited for treating ocular diseases (McGuckin et al., 2022).
Regarding the advantages of NC, this study aims to develop and characterize acetazolamide nanocrystals (AZM-NC) as a novel topical treatment for IOP-lowering, addressing drug solubility and permeability limitations while minimizing systemic side effects.
Materials and reagents
Hamoudi Ghassan Awde Alfonso, Luis Ignacio Tártara, Alejandro Javier Paredes, Santiago Daniel Palma, María Lina Formica,
Enhanced in vivo performance of topical ocular acetazolamide nanocrystals: A novel approach for glaucoma treatment,
International Journal of Pharmaceutics, 2025, 125440, ISSN 0378-5173,
https://doi.org/10.1016/j.ijpharm.2025.125440.