RESUMO
Phosphine-borane complexes are recently developed redox-active drugs that are neuroprotective in models of optic nerve injury and radioprotective in endothelial cells. However, a single dose of these compounds is short-lived, necessitating the development of sustained-release formulations of these novel molecules. We screened a library of biodegradable co- and non-block polyester polymer systems for release of incorporated phosphine-borane complexes to evaluate them as drug delivery systems for use in chronic disease. Bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) was combined with biodegradable polymers based on poly(D,L-lactide) (PDLLA), poly(L-lactide) (PLLA), poly(caprolactone) (PCL), poly(lactide-co-glycide) (PLGA), or poly(dioxanone-co-caprolactone) (PDOCL) to make polymer microdiscs, and release over time quantified. Of 22 polymer-PB1 formulations tested, 17 formed rigid polymers. Rates of release differed significantly based on the chemical structure of the polymer. PB1 released from PLGA microdiscs released most slowly, with the most linear release in polymers of 60:40 LA:GA, acid endcap, Mn 15 000-25 000 and 75:25 LA:GA, acid endcap, Mn 45 000-55 000. Biodegradable polymer systems can, therefore, be used to produce sustained-release formulations for redox-active phosphine-borane complexes, with PLGA-based systems most suitable for very slow release. The sustained release could enable translation to a clinical neuroprotective strategy for chronic diseases such as glaucoma.
Assuntos
Boranos/farmacocinética , Portadores de Fármacos/farmacocinética , Liberação Controlada de Fármacos , Fármacos Neuroprotetores/farmacocinética , Fosfinas/farmacocinética , Poliésteres/farmacocinética , Boranos/química , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Portadores de Fármacos/química , Fármacos Neuroprotetores/química , Fosfinas/química , Poliésteres/químicaRESUMO
Within the field of glaucoma research, neuroprotection is defined as slowing the functional loss in glaucoma by a mechanism independent of lowering of intraocular pressure. There is currently a great potential for research surrounding neuroprotection as it relates to glaucoma. Anatomical targets for neuroprotection should focus on upstream rather than downstream factors, and could include any part of the retinal ganglion cell, the glia, especially astrocytes or Muller cells, and vasculature. The great number of anatomical targets is exceeded only by the number of possible biochemical pathways and potential treatments. Successful treatment may be accomplished through the targeting of one or even a combination of multiple pathways. Once a treatment is shown effective in vitro, it should be evaluated in vivo with carefully chosen animal models and studied in sufficient numbers to detect statistically and clinically significant effects. Such a drug should have few systemic side effects and its delivery should be optimized so as to encourage compliance. There are still a multitude of possible screens available to test the efficacy of a neuroprotective drug and a single gold standard is ideal for the accurate assessment and comparison of new drugs. Future studies in neuroprotection should investigate the genetic component of the disease, novel pharmaceutical agents for new or known pathways, modulations of scleral biomechanics, and relation to research of other complex disorders of the central nervous system.
Assuntos
Astrócitos/fisiologia , Glaucoma/tratamento farmacológico , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Doenças do Nervo Óptico/tratamento farmacológico , Células Ganglionares da Retina/efeitos dos fármacos , Pesquisa Translacional Biomédica , Animais , Axônios/fisiologia , Humanos , Células Ganglionares da Retina/fisiologiaRESUMO
Phosphine-borane complexes are novel cell-permeable drugs that protect neurons from axonal injury in vitro and in vivo. These drugs activate the extracellular signal-regulated kinases 1/2 (ERK1/2) cell survival pathway and are therefore neuroprotective, but do not scavenge superoxide. In order to understand the interaction between superoxide signaling of neuronal death and the action of phosphine-borane complexes, their biochemical activity in cell-free and in vitro assays was studied by electron paramagnetic resonance (EPR) spectrometry and using an intracellular dithiol reporter that becomes fluorescent when its disulfide bond is cleaved. These studies demonstrated that bis(3-propionic acid methyl ester) phenylphosphine-borane complex (PB1) and (3-propionic acid methyl ester) diphenylphosphine-borane complex (PB2) are potent intracellular disulfide reducing agents which are cell permeable. EPR and pharmacological studies demonstrated reducing activity but not scavenging of superoxide. Given that phosphine-borane complexes reduce cell injury from mitochondrial superoxide generation but do not scavenge superoxide, this implies a mechanism where an intracellular superoxide burst induces downstream formation of protein disulfides. The redox-dependent cleavage of the disulfides is therefore a novel mechanism of neuroprotection.
Assuntos
Boranos/metabolismo , Líquido Intracelular/metabolismo , Neuroproteção/fisiologia , Fosfinas/metabolismo , Animais , Boranos/química , Linhagem Celular , Dissulfetos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Líquido Intracelular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Oxirredução , Fosfinas/química , Ratos , SuínosRESUMO
PURPOSE: RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype. METHODS: 661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology. RESULTS: Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested. CONCLUSIONS: 661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Ganglionares da Retina/fisiologia , Animais , Linhagem Celular , Forma Celular/efeitos dos fármacos , Camundongos , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Ratos , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/ultraestrutura , Estaurosporina/farmacologiaRESUMO
Exposure to radiation can damage endothelial cells in the irradiated area via the production of reactive oxygen species. We synthesized phosphine-borane complexes that reduce disulfide bonds and had previously been shown to interfere with redox-mediated signaling of cell death. We hypothesized that this class of drugs could interfere with the downstream effects of oxidative stress after irradiation and rescue endothelial cells from radiation damage. Cultured bovine aortic endothelial cells were plated for clonogenic assay prior to exposure to varying doses of irradiation from a (137)Cs irradiator and treated with various concentrations of bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1) at different time points. The clone-forming ability of the irradiated cells was assessed seven days after irradiation. We compared the radioprotective effects of PB1 with the aminothiol radioprotectant WR1065 and known superoxide scavengers. PB1 significantly protected bovine aortic endothelial cells from radiation damage, particularly when treated both before and after radiation. The radioprotection with 1 µM PB1 corresponded to a dose-reduction factor of 1.24. Radioprotection by PB1 was comparable to the aminothiol WR1065, but was significantly less toxic and required much lower concentrations of drug (1 µM vs. 4 mM, respectively). Superoxide scavengers were not radioprotective in this paradigm, indicating the mechanisms for both loss of clonogenicity and PB1 radioprotection are independent of superoxide signaling. These data demonstrate that PB1 is an effective redox-active radioprotectant for endothelial cells in vitro, and is radioprotective at a concentration approximately 4 orders of magnitude lower than the aminothiol WR1065 with less toxicity.
Assuntos
Boranos/farmacologia , Células Endoteliais/efeitos dos fármacos , Raios gama/efeitos adversos , Fosfinas/farmacologia , Protetores contra Radiação/farmacologia , Superóxidos/antagonistas & inibidores , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/efeitos da radiação , Bovinos , Células Cultivadas , Células Clonais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/efeitos da radiação , Humanos , Mercaptoetilaminas/farmacologia , Metaloporfirinas/farmacologia , Oxirredução , Polietilenoglicóis/farmacologia , Transdução de Sinais , Superóxido Dismutase/farmacologia , Superóxidos/metabolismoRESUMO
PURPOSE: Light-induced oxidative stress is an important risk factor for age-related macular degeneration, but the downstream mediators of photoreceptor and retinal pigment epithelium cell death after photic injury are unknown. Given our previous identification of sulfhydryl/disulfide redox status as a factor in photoreceptor survival, we hypothesized that formation of one or more disulfide-linked homo- or hetero-dimeric proteins might signal photoreceptor death after light-induced injury. METHODS: Two-dimensional (non-reducing/reducing) gel electrophoresis of Wistar rat retinal homogenates after 10 hours of 10,000 lux (4200°K) light in vivo, followed by mass spectrometry identification of differentially oxidized proteins. RESULTS: The redox proteomic screen identified homodimers of visual arrestin (Arr1; S antigen) after toxic levels of light injury. Immunoblot analysis revealed a light duration-dependent formation of Arr1 homodimers, as well as other Arr1 oligomers. Immunoprecipitation studies revealed that the dimerization of Arr1 due to photic injury was distinct from association with its physiological binding partners, rhodopsin and enolase1. Systemic delivery of tris(2-carboxyethyl)phosphine, a specific disulfide reductant, both decreased Arr1 dimer formation and protected photoreceptors from light-induced degeneration in vivo. CONCLUSIONS: These findings suggest a novel arrestin-associated pathway by which oxidative stress could result in cell death, and identify disulfide-dependent dimerization as a potential therapeutic target in retinal degeneration.