Abstract
Purpose:
Our group previously synthesized and characterized phosphine-borane complexes that are novel redox-active drugs. We showed that they are neuroprotective for retinal ganglion cells (RGCs) in vitro and in vivo in rodent optic nerve transection and experimental glaucoma models. We hypothesized that the protective borane group is removed from the phosphine in biological fluids, and that the deprotected form of the molecule then reacts with disulfides to inhibit oxidative signaling. In this study we explored the metabolic pathways associated with one of these compounds, bis (3-propionic acid methyl ester) phenylphosphine borane complex 1 (PB1), in the presence of cultured cells, in order to test this hypothesis and better understand the mechanism of action of these compounds.
Methods:
RGC-5 cells were plated in 150 cm2 tissue culture dishes and allowed to reach approximately 40% confluency before treatment with 200 µM PB1 or vehicle. After 24 hours, a small volume of media was collected to determine extracellular metabolites of PB1, and then the cells were briefly rinsed with warm phosphate-buffered saline and water. After removal of the water rinse, plates of cells were flash frozen with liquid nitrogen, and the cells scraped and collected in a small volume of either acetonitrile or 9:1 methanol:chloroform. Cells were sonicated, Insoluble debris pelleted, and the supernatant frozen at -80 C for subsequent analysis. Liquid chromatography-mass spectroscopy analysis was carried out at the UW School of Pharmacy Analytical Instrumentation center on an Agilent 1100 LC-MSD quadrupole SL after separation on a 150 mm C18 column, and peak differences between the vehicle- and PB1-treated cells were isolated and assigned formulas from their mass.
Results:
PB1 underwent many of the predicted modifications, with a peak for the borane-deprotected species at the expected elution time in the PB1-treated condition. Surprisingly, PB1 was abundant in the media, suggesting that the protective borane group was more stable than expected.
Conclusions:
The presence of PB1 with an intact protective borane raises the possibility that the neuroprotective effects of these compounds could be independent of a reactive phosphine. Further analyses of the intra- and extracellular products of PB1 metabolism will aid in the understanding of phosphine-borane chemistry and cytoprotection.
Keywords: 615 neuroprotection •
634 oxidation/oxidative or free radical damage •
531 ganglion cells