New trends towards glaucoma treatment: Topical osmoprotective microemulsions loaded with latanoprost.

The chronic use of hypotensive agents eventually leads to ocular surface damage and poor patient compliance during glaucoma management. Thus, new sustained drug delivery systems are needed. This work aimed to develop osmoprotective latanoprost-loaded microemulsion formulations as new potential glaucoma treatments with ocular surface protective properties. The microemulsions were characterized and latanoprost encapsulation efficacy determined. In-vitro tolerance, osmoprotective efficacy, cell internalization as well as cell-microemulsion interactions and distribution were performed. In vivo hypotensive activity was conducted in rabbits to assess intraocular pressure reduction and relative ocular bioavailability. Physicochemical characterization showed nanodroplet sizes within 20-30 nm, being in vitro tolerance within 80 and 100% viability in corneal and conjunctival cells. Besides, microemulsions exhibited higher protection under hypertonic conditions than untreated cells. Cell fluorescence lasted for 11 days after short exposure to coumarin-loaded microemulsions (5 min) showing extensive internalization in different cell compartments by electronic microscopy. In vivo studies exhibited that a single instillation of latanoprost-loaded microemulsions reduced the intraocular pressure for several days (4-6 days without polymer and 9-13 days with polymers). Relative ocular bioavailability was 4.5 and 19 times higher than the marketed formulation. These findings suggest the use of these microemulsions as potential combined strategies for extended surface protection and glaucoma treatment.

Development of an osmoprotective microemulsion as a therapeutic platform for ocular surface protection.

Self-emulsified osmoprotective ophthalmic microemulsions (O/A) were prepared by combining betaine/leucine, clusterin/oleanolic acid, and hyaluronic acid or Dextran. The microemulsions contained an internal oily phase (1.2%), an external aqueous phase (96.3%), cosolvents (1%), and surfactants (1.5%). Physicochemical characterization and in vivo and in vitro tolerance were analyzed. The formulations' osmoprotective in vitro activity was assayed in a hyperosmolar model in human corneal cells. Average internal phase sizes were 16-26 nm for the microemulsions including Dextran. Addition of hyaluronic acid increased the size range (25-39 nm). Addition of osmoprotectants did not change nanodroplet size. The formulations were isotonic (280-290 mOsm/L) with neutral pH (≈7) and zeta potential (-10 to 0 mV), low surface tension (≈35-40mN·m-1), and low viscosity (≈1 mPa·s), except for the microemulsions containing hyaluronic acid (≈4-5 mPa·s). SEM and cryo-TEM showed that all formulations exhibited sphere-shaped morphology with good cell tolerance (≈100%) and were stable at 8 °C for 9 months. Osmoprotective formulations were well tolerated in vitro and in vivo, protecting cells from hypertonic stress. We therefore developed stable microemulsions compatible with the ocular surface that could constitute a novel tool for treatment of ophthalmic diseases.

Combined hyperosmolarity and inflammatory conditions in stressed human corneal epithelial cells and macrophages to evaluate osmoprotective agents as potential DED treatments.

PURPOSE: To develop an easy-to-perform combined model in human corneal epithelial cells (HCECs) and Balb/c mice macrophages J774.A1 (MP) for preliminary screening of potential ophthalmic therapeutic substances. METHODS: HCECs were exposed to different osmolarities (350-500 mOsm/L) and MTT assay was employed for cell survival and flow cytometry to assess apoptosis-necrosis and relative cell size (RCS) distribution. Effectiveness of Betaine, L-Carnitine, Taurine at different concentrations (ranging from 20 mM to 200 mM) was studied. Also, mucoadhesive polymers such as Hyaluronic acid (HA) and Hydroxypropylmethylcellulose (HPMC) (0.4 and 0.8%) were evaluated. Cells were pre-incubated with the compounds (8h) and then exposed to hyperosmotic stress (470 mOsm/L) for 16h. Moreover, anti-inflammatory activity was performed in LPS-stimulated MP. RESULTS: Exposure to hyperosmotic solutions between 450 and 500 mOsm/L promoted the highest cell death after 16h exposures (p < 0.0001) with a drop in viability to 34.96% ± 11.77 for 470 mOsm/L. Pre-incubation with Betaine at 150 mM and 200 mM provided the highest cell survival against hyperosmolarity (66.01% ± 3.65 and 65.90% ± 0.78 respectively) while HA 0.4% was the most effective polymer in preventing cell death (42.2% ± 3.60). Flow cytometry showed that Betaine and Taurine at concentrations between 150-200 mM and 20-80 mM respectively presented the highest anti-apoptotic activity. Also, HA and HPMC polymers reduced apoptotic-induced cell death. All osmoprotectants modified RCS, and polymers increased their value over 100%. L-Carnitine 50 mM, Taurine 40 mM and HA 0.4% presented the highest TNF-α inhibition activity (60%) albeit all of them showed anti-inflammatory inhibition percentages higher than 20% CONCLUSIONS: HCECs hyperosmolar model combined with inflammatory conditions in macrophages allows the screening of osmoprotectants by simulating chronic hyperosmolarity (16h) and inflammation (24h).

Donadores de óxido nítrico como hipotensores en glaucoma / Nitric oxide-donating compounds for IOP lowering in glaucoma

INTRODUCCIÓN: En relación con la progresión del glaucoma, la presión intraocular (PIO) elevada es el principal factor de riesgo sobre el que se puede actuar. Las estrategias farmacológicas destinadas a reducir la PIO tienen como objetivo la reducción de la producción de humor acuoso (HA) y/o el aumento de su drenaje a través de la vía uveoescleral. Sin embargo, en la actualidad no hay ninguna estrategia farmacológica de primera elección que de forma principal esté destinada a facilitar la salida de HA por la vía convencional. La producción de óxido nítrico (NO) a nivel ocular tiene lugar en las rutas de flujo de HA y en el músculo ciliar, modulando la respuesta celular en situaciones de PIO elevada. MÉTODOS: En esta revisión se describe el mecanismo de acción del NO endógeno así como de las nuevas moléculas donadoras de NO que se encuentran en fase de investigación. Además se incluye información acerca de los estudios preclínicos y clínicos realizados hasta la fecha con estos nuevos compuestos, discutiendo su potencial terapéutico en el tratamiento farmacológico de la hipertensión ocular en glaucoma. RESULTADOS: La administración de compuestos donadores de NO por vía tópica oftálmica proporciona un descenso de la PIO marcado y mantenido en modelos experimentales de glaucoma y en sujetos con hipertensión ocular. CONCLUSIONES: El mecanismo de acción de estos compuestos es novedoso y la evidencia científica muestra resultados prometedores. Sin embargo, para poder valorar su uso en terapias crónicas son necesarios más estudios que demuestren su seguridad y la eficacia a largo plazo

INTRODUCTION: An elevated intraocular pressure (IOP) remains the main risk factor for progression of glaucoma upon which we can efficiently act. Pharmacological strategies to reduce IOP are directed towards the reduction of aqueous humour (AH) production and/or the increase in AH drainage through the uveoscleral pathway. However, there are no drugs currently available as first-line treatment to increase AH outflow primarily via the conventional route. Ocular nitric oxide (NO) production takes place in AH outflow pathways and in the ciliary muscle, modulating the cellular response to elevated IOP. METHODS: This review describes the mechanism of action of endogenous NO and NO-donating compounds that are under research. It includes information regarding pre-clinical and clinical studies previously conducted with these compounds, discussing their role and therapeutic potential in the pharmacological treatment of ocular hypertens in in glaucoma. RESULTS: The topical ocular administration of NO-donating compounds significantly lowered IOP and maintained it in animal models of glaucoma and subjects with ocular hypertension. CONCLUSIONS: The mechanism of action of these compounds is novel and scientific evidence that shows promising results. However, there is a need for more comprehensive studies to assess long-term safety and tolerability in order to properly evaluate their use in chronic therapies

Nitric oxide-donating compounds for IOP lowering in glaucoma. / Donadores de óxido nítrico como hipotensores en glaucoma.

INTRODUCTION: An elevated intraocular pressure (IOP) remains the main risk factor for progression of glaucoma upon which we can efficiently act. Pharmacological strategies to reduce IOP are directed towards the reduction of aqueous humour (AH) production and/or the increase in AH drainage through the uveoscleral pathway. However, there are no drugs currently available as first-line treatment to increase AH outflow primarily via the conventional route. Ocular nitric oxide (NO) production takes place in AH outflow pathways and in the ciliary muscle, modulating the cellular response to elevated IOP. METHODS: This review describes the mechanism of action of endogenous NO and NO-donating compounds that are under research. It includes information regarding pre-clinical and clinical studies previously conducted with these compounds, discussing their role and therapeutic potential in the pharmacological treatment of ocular hypertension in glaucoma. RESULTS: The topical ocular administration of NO-donating compounds significantly lowered IOP and maintained it in animal models of glaucoma and subjects with ocular hypertension. CONCLUSIONS: The mechanism of action of these compounds is novel and scientific evidence that shows promising results. However, there is a need for more comprehensive studies to assess long-term safety and tolerability in order to properly evaluate their use in chronic therapies.

Pharmaceutical microscale and nanoscale approaches for efficient treatment of ocular diseases.

Efficient treatment of ocular diseases can be achieved thanks to the proper use of ophthalmic formulations based on emerging pharmaceutical approaches. Among them, microtechnology and nanotechnology strategies are of great interest in the development of novel drug delivery systems to be used for ocular therapy. The location of the target site in the eye as well as the ophthalmic disease will determine the route of administration (topical, intraocular, periocular, and suprachoroidal administration) and the most adequate device. In this review, we discuss the use of colloidal pharmaceutical systems (nanoparticles, liposomes, niosomes, dendrimers, and microemulsions), microparticles (microcapsules and microspheres), and hybrid systems (combination of different strategies) in the treatment of ophthalmic diseases. Emphasis has been placed in the therapeutic significance of each drug delivery system for clinical translation.

Novel Water-Soluble Mucoadhesive Carbosilane Dendrimers for Ocular Administration.

The purpose of this research was to determine the potential use of water-soluble anionic and cationic carbosilane dendrimers (generations 1-3) as mucoadhesive polymers in eyedrop formulations. Cationic carbosilane dendrimers decorated with ammonium -NH3(+) groups were prepared by hydrosylilation of Boc-protected allylamine and followed by deprotection with HCl. Anionic carbosilane dendrimers with terminal carboxylate groups were also employed in this study. In vitro and in vivo tolerance studies were performed in human ocular epithelial cell lines and rabbit eyes respectively. The interaction of dendrimers with transmembrane ocular mucins was evaluated with a surface biosensor. As proof of concept, the hypotensive effect of a carbosilane dendrimer eyedrop formulation containing acetazolamide (ACZ), a poorly water-soluble drug with limited ocular penetration, was tested after instillation in normotensive rabbits. The methodology used to synthesize cationic dendrimers avoids the difficulty of obtaining neutral -NH2 dendrimers that require harsher reaction conditions and also present high aggregation tendency. Tolerance studies demonstrated that both prototypes of water-soluble anionic and cationic carbosilane dendrimers were well tolerated in a range of concentrations between 5 and 10 µM. Permanent interactions between cationic carbosilane dendrimers and ocular mucins were observed using biosensor assays, predominantly for the generation-three (G3) dendrimer. An eyedrop formulation containing G3 cationic carbosilane dendrimers (5 µM) and ACZ (0.07%) (289.4 mOsm; 5.6 pH; 41.7 mN/m) induced a rapid (onset time 1 h) and extended (up to 7 h) hypotensive effect, and led to a significant increment in the efficacy determined by AUC0(8h) and maximal intraocular pressure reduction. This work takes advantage of the high-affinity interaction between cationic carbosilane dendrimers and ocular transmembrane mucins, as well as the tensioactive behavior observed for these polymers. Our results indicate that low amounts of cationic carbosilane dendrimers are well tolerated and able to improve the hypotensive effect of an acetazolamide solution. Our results suggest that carbosilane dendrimers can be used in a safe range of concentrations to enhance the bioavailability of drugs topically administered in the eye.

Absorción de fármacos por vía tópica. Papel de la conjuntiva / Ocular drug absorption by topical route. Role of conjunctiva

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