Examination of the impact molecular charge has on NTSR1-targeted agents incorporated with cysteine protease inhibitors.

Our laboratory has previously reported a strategy of employing cysteine cathepsin (CC) inhibitors as adduct forming, trapping agents to extend the tumor residence time of neurotensin receptor subtype 1 (NTSR1)-targeted radiopharmaceuticals. As a follow-up, we herein report a small library of CC trapping agent (CCTA)-incorporated, NTSR1-targeted conjugates with structural modifications that reduce the number of charged functional groups for both the CCTA and the peptide targeting sequence. These modifications were pursued to reduce the renal uptake and increase the translational potential of the CCTA-incorporated, NTSR1-targeted agents as radiotherapeutics. The biological performance of these constructs was examined using a battery of in vitro and in vivo studies employing the NTSR1-positive HT-29 human colon cancer cell line as our model. In vitro studies confirmed the ability of these constructs to target the NTSR1 and efficiently form intracellular adducts with cysteine proteases. Biodistribution studies using an HT-29 xenograft mouse model revealed that truncation (removal of Lys6-Pro7) of the NTSR1-targeted peptide (177Lu-NE2a) had the greatest (3.7-fold) effect at lowering renal recognition/uptake relative to our previously reported construct. Other charge-reducing modifications to the CCTA resulted in unexpected increases in renal uptake. All of the constructs demonstrated similar levels of in vivo NTSR1-positive tumor targeting with the highest tumor residualization resulting from the construct containing the zwitterionic CCTA (177Lu-NE2a). In vivo adduct formation of the conjugates was confirmed using autoradiographic SDS-PAGE analysis.