Abstract
Purpose: :
Phosphine-borane (PB) complexes bis (3-propionic acid methyl ester) phenylphosphine borane complex (PB1) and (3-propionic acid methyl ester) diphenylphosphine borane complex (PB2) are novel neuroprotective compounds. We previously demonstrated that they have a positive, dose-dependent effect on the viability of RGCs after axonal injury (Schlieve et al., Exp Eye Res 83:1252, 2006), which we hypothesize results from their ability to chemically reduce critical disulfides formed as a result of oxidation by reactive oxygen species. We performed in vitro testing of the effectiveness of these complexes and their utility as drugs, focusing on their reducing power, solubility, blood-brain barrier permeability, and accelerated stability.
Methods: :
Relative reducing power was tested using Ellman’s reagent (DTNB), which turns yellow when its disulfide linkage is reduced. Reaction of various concentrations of dithiothreitol, tris (carboxyethyl) phosphine, PB1, or PB2 with 30 mM DTNB in methanol was carried out in buffer at pH 5, 7, and 9. Octanol/water partitioning was carried out on PB1 and PB2 to determine their solubility in aqueous solution. A modified PAMPA assay was used to determine blood-brain barrier permeability of PB1. An artificial membrane was created on a filter plate using porcine polar brain lipids dissolved in hexanes. PB1 in PBS was placed in the donor well above the membrane, and PBS placed in the acceptor well below the membrane. Concentration of PB1 in each well after 18 hours was calculated by comparison to a standard curve of PB1 in PBS. Accelerated stability was determined by incubating 4 mg of pure PB1 and PB2 in sand baths at 40°C and 60°C for 37 days, followed by HPLC.
Results: :
There was a linear relation between concentration and reducing power for each compound. PB1 and PB2 had maximal reducing capability at pH 7. log Poct/wat for PB1 was 0.65 (somewhat soluble) and for PB2 was 1.1 (minimally soluble). log Pe for PB1 was -5.86 (Pe = 1.38x10-6), a low BBB permeability similar to that of caffeine and aldosterone. Peak areas for PB1 at 40°C and 60°C were 80% and 72% of control respectively at 37 days. PB2 peak areas were 100% of control for both conditions at 37 days.
Conclusions: :
Both PB complexes are effective reducers of disulfides. PB2, while highly lipophilic, is more stable than its water-soluble counterpart.
Keywords: neuroprotection • oxidation/oxidative or free radical damage • apoptosis/cell death