Antibody-based Imaging of Bioreductive Prodrug Release in Hypoxia

Abstract

Regions of hypoxia occur in most tumors and predict for poor patient prognosis. Hypoxia-activated prodrugs provide an ideal strategy to target the aggressive, hypoxic fraction of a tumor while protecting the normal tissue from toxicity. A key challenge associated with the development of novel hypoxia-activated prodrugs, however, is the ability to visualize the delivery of the prodrug to hypoxic regions and determine where it has been activated. Here we report a modified version of the commonly used nitroimidazole bioreductive group that incorporates the fluoroethyl epitope of the antibody-based hypoxia imaging agent, EF5. Attachment of this group to the red fluorescent dye, DCM, enabled us to correlate release of the DCM dye with imaging of the reduced bioreductive group using the EF5 antibody. This study confirmed that the antibody was imaging reduction and fragmentation of the pro-fluorophore. We next employed the modified bioreductive group to synthesize a new prodrug of the KDAC inhibitor Panobinostat, EF5-Pano. Release of EF5-Pano in hypoxic multiple myeloma cells was imaged using the EF5 antibody, and the presence of an imaging signal correlated with apoptosis and a reduction in cell viability. Therefore, EF5-Pano is an imageable hypoxia-activated prodrug with proven cytotoxic effect in multiple myeloma, which could be utilized in future in vivo experiments.