A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

We assessed the sustained delivery effect of poly (lactic-co-glycolic) acid (PLGA)/vitamin E (VitE) microspheres (MSs) loaded with glial cell-derived neurotrophic factor (GDNF) alone (GDNF-MSs) or combined with brain-derived neurotrophic factor (BDNF; GDNF/BDNF-MSs) on migration of the human adult retinal pigment epithelial cell-line-19 (ARPE-19) cells, primate choroidal endothelial (RF/6A) cells, and the survival of isolated mouse retinal ganglion cells (RGCs). The morphology of the MSs, particle size, and encapsulation efficiencies of the active substances were evaluated. In vitro release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability, terminal deoxynucleotidyl transferase (TdT) deoxyuridine dUTP nick-end labelling (TUNEL) apoptosis, functional wound healing migration (ARPE-19; migration), and (RF/6A; angiogenesis) assays were conducted. The safety of MS intravitreal injection was assessed using hematoxylin and eosin, neuronal nuclei (NeuN) immunolabeling, and TUNEL assays, and RGC in vitro survival was analyzed. MSs delivered GDNF and co-delivered GDNF/BDNF in a sustained manner over 77 days. The BDNF/GDNF combination increased RPE cell migration, whereas no effect was observed on RF/6A. MSs did not alter cell viability, apoptosis was absent in vitro, and RGCs survived in vitro for seven weeks. In mice, retinal toxicity and apoptosis was absent in histologic sections. This delivery strategy could be useful as a potential co-therapy in retinal degenerations and glaucoma, in line with future personalized long-term intravitreal treatment as different amounts (doses) of microparticles can be administered according to patients’ needs

Six month delivery of GDNF from PLGA/vitamin E biodegradable microspheres after intravitreal injection in rabbits

Local long-term delivery of glial cell line derived neurotrophic factor (GDNF) from vitamin E/poly-lactic-co-glycolic acid microspheres (MSs) protects retinal ganglion cells in an animal model of glaucoma for up to 11 weeks. However, the pharmacokinetics of GDNF after intravitreal injection of MSs is not known. We evaluated the GDNF levels after a single intravitreal injection of GDNF/VitE MSs. Biodegradable MSs were prepared by the solid-oil-in-water emulsion-solvent evaporation technique and characterized. Rabbits received a single intravitreal injection (50 μL) of GDNF/VitE MSs (4%w/v; 24 right eyes; 74.85 ng GDNF), blank MSs (4%w/v; 24 left eyes), and balanced salt solution (4 eyes). Two controls eyes received no injections. At 24 hours, 1, 4, 6, 8, 12, 18, and 24 weeks after injection, the eyes were enucleated, and the intravitreal GDNF levels were quantified. Pharmacokinetic data were analysed according to non-compartmental model. Intraocular GDNF levels of 717.1 ± 145.1 pg/mL were observed at 24 hours for GDNF-loaded MSs, followed by a plateau (745.3 ± 25.5 pg/mL) until day 28. After that, a second plateau (17.4 ± 3.7 pg/mL) occurred from 8 to 24 weeks postinjection, significantly higher than the basal levels. Eyes injected with GDNF/vitE and Blank-MSs did not show any abnormalities during the six-months follow up after administration. The single injection of GDNF/VitE MSs provided a sustained controlled release of the neurotrophic factor in a controlled fashion for up to six months

Six month delivery of GDNF from PLGA/vitamin E biodegradable microspheres after intravitreal injection in rabbits

Local long-term delivery of glial cell line derived neurotrophic factor (GDNF) from vitamin E/poly-lactic-co-glycolic acid microspheres (MSs) protects retinal ganglion cells in an animal model of glaucoma for up to 11 weeks. However, the pharmacokinetics of GDNF after intravitreal injection of MSs is not known. We evaluated the GDNF levels after a single intravitreal injection of GDNF/VitE MSs. Biodegradable MSs were prepared by the solid-oil-in-water emulsion-solvent evaporation technique and characterized. Rabbits received a single intravitreal injection (50 μL) of GDNF/VitE MSs (4%w/v; 24 right eyes; 74.85 ng GDNF), blank MSs (4%w/v; 24 left eyes), and balanced salt solution (4 eyes). Two controls eyes received no injections. At 24 hours, 1, 4, 6, 8, 12, 18, and 24 weeks after injection, the eyes were enucleated, and the intravitreal GDNF levels were quantified. Pharmacokinetic data were analysed according to non-compartmental model. Intraocular GDNF levels of 717.1 ± 145.1 pg/mL were observed at 24 hours for GDNF-loaded MSs, followed by a plateau (745.3 ± 25.5 pg/mL) until day 28. After that, a second plateau (17.4 ± 3.7 pg/mL) occurred from 8 to 24 weeks postinjection, significantly higher than the basal levels. Eyes injected with GDNF/vitE and Blank-MSs did not show any abnormalities during the six-months follow up after administration. The single injection of GDNF/VitE MSs provided a sustained controlled release of the neurotrophic factor in a controlled fashion for up to six months

Diseño y evaluación de microesferas biodegradables de agentes neuroprotectores para el tratamiento de patologías degenerativas que afectan al segmento posterior del ojo

Las patologías neurodegenerativas que afectan al segmento posterior del ojo se caracterizan por ser devastadoras y comprometer la visión en un gran número de sujetos. Representan un grupo de enfermedades que cursan con un daño progresivo del nervio óptico y pérdida de fotorreceptores y células ganglionares de la retina (CGR), que sin un tratamiento continuado, pueden conducir a ceguera irreversible. Las nuevas tendencias en el tratamiento de estas enfermedades neurodegenerativas oculares están basadas en la neuroprotección, siendo la administración de factores neurotróficos una de las estrategias más prometedoras. Al tratarse de patologías que presentan una etiología multifactorial, el tratamiento combinado con distintos agentes terapéuticos (agentes neuroprotectores y antioxidantes) que actúen sobre los diversos mecanismos implicados en la patogénesis puede resultar de gran utilidad. Debido a que este tipo de afecciones son generalmente crónicas, se hace patente la necesidad de formas de administración oftálmicas capaces de proporcionar concentraciones terapéuticas de las sustancias activas en su lugar de acción durante periodos de tiempo prolongados que permitan disminuir el número de administraciones. Además, si el sistema desarrollado es biodegradable, desaparecerá una vez ejercido su efecto, evitando así un proceso quirúrgico al ser retirado. El desarrollo de esta Tesis Doctoral se ha basado en el diseño de nuevas formulaciones de terapia combinada, microesferas biodegradables cargadas con agentes neuroprotectores (GDNF, Factor Neurotrófico Derivado de la línea celular Glial y BDNF, Factor Neurotrófico Derivo del Cerebro) y vitamina E como agente antioxidante, para el tratamiento de patologías neurodegenerativas que afectan al segmento posterior del ojo. Para esto, se ha desarrollado un nuevo método de microencapsulación con el objeto de preservar la integridad del producto biotecnológico encapsulado. Posteriormente, se ha procedido a la esterilización de las formulaciones, puesto que están destinadas a ser administradas por vía intravítrea, proponiendo la combinación de distintos recursos tecnológicos que atenuaran la degradación del factor neuroprotector. Finalmente, se ha evaluado el posible efecto antiangiogénico con el empleo de estas nuevas formulaciones. En este caso, se han encapsulado dos factores neurotróficos (GDNF y BDNF) junto con vitamina E. Cada uno de estos trabajos ha sido objeto de una publicación científica