Methods development for testing aqueous formulations of Met12, a novel inhibitor of photoreceptor cell death, in vitro and in vivo

摘要

Purpose: A significant consequence of retina-RPE separation is death of photoreceptor (PR) cells. We previously reported that Met12, a small molecule antagonistic of the Fas receptor, inhibits Fas-mediated PR death in a rodent retinal detachment (RD) model. Those previous studies all had Met12 dissolved in the organic solvent dimethyl sulfoxide (DMSO). The purpose of this study was to develop an assay to screen new aqueous solvents for Met12 that would measure caspase inhibition in vitro and increase PR survival in vivo, with the eventual goal to develop this drug for therapeutic application in humans. Methods: For this study, Met12 was dissolved in either DMSO or citrate buffer at concentrations ranging between 0.07uM - 175uM. Cultured 661w cells incubated with Fas-ligand and various concentrations of Met12-citrate buffer. Caspase 8 activity was measured using Caspase-Glo 8 Assay (Promega). For in vivo testing, RDs were created in Brown Norway rats as previously described, followed by intravitreal administration of Met12, dissolved in either DMSO or citrate buffer, in a 5 ?μL volume. Eyes were harvested at 24 hours for caspase 8 activity assays, 3 days for TUNEL staining or at 2 months for PR cell counts. Results: Doses of 0.5, 5 and 50 ?μg of Met12-DMSO, administered intravitreally, were equally effective at inhibiting caspase 8 activation, in vitro, with 5 ?μg being the lowest dose that provided maximal suppression of photoreceptor TUNEL staining and preservation of outer nuclear layer cell counts in vivo.br / br / In Met12-citrate assays, 100uM Met12 yielded maximum inhibition of Caspase 8 activity following Fas activation in vitro, with an IC50 of approximately 20uM. In vivo, Met12-citrate inhibited PR apoptosis as measured by TUNEL staining, with a less potent effect than Met12-DMSO. Conclusions: Met12-DMSO inhibits PR apoptosis in a dose-dependent fashion. Compared to Met12-DMSO, Met12-citrate was a less potent inhibitor of caspase activity in vitro, and this translated to a less effective inhibition of PR apoptosis in vivo. As such, we successfully demonstrate the ability of our in vitro assay to predict in vivo efficacy and the usefulness of our in vitro assay to screen aqueous formulations of Met12.