Abstract
Several studies have focused on the development and application of radiolabeled DOTA-AE105 for targeting the urokinase-type plasminogen activator receptor (uPAR), which is expressed on various cancer types. The aim of this project was to design and evaluate novel uPAR-targeting radiopeptides with improved pharmacokinetic properties in view of their therapeutic application. Five peptides (uPAR-01, uPAR-02, uPAR-03, uPAR-04, and uPAR-05) were synthesized based on the AE105 peptide backbone, a DOTA chelator, and the 4-(p-iodophenyl)butanoate moiety as an albumin binder. The peptides were obtained in 20-29 synthetic steps using solid-phase peptide synthesis with a 6-34% overall yield. In saline, the (177)Lu-labeled peptides (100 MBq/nmol) were stable (>93% intact radiopeptides) in the presence of l-ascorbic acid over 24 h. The new radiopeptides were also stable (>98% intact radiopeptides) in mouse and human blood plasma, while only ∼13% of [(177)Lu]Lu-DOTA-AE105 was intact after a 4 h incubation period. The uPAR-binding affinities (K(D) values) determined with uPAR-transfected human embryonic kidney cells (HEK-uPAR) ranged from 10 to 57 nM and were, thus, similar to that of [(177)Lu]Lu-DOTA-AE105 (K(D): 20 ± 1 nM). Compared to [(177)Lu]Lu-DOTA-AE105, the radiopeptides showed the anticipated increased binding affinity to plasma proteins both in mouse (31- to 104-fold) and human blood plasma (43- to 136-fold). The tissue distribution of the novel radiopeptides in nude mice bearing HEK-uPAR xenografts showed substantial activity retention in the blood (12-16% IA/g and 4.5-13% IA/g at 4 and 24 h p.i., respectively), while [(177)Lu]Lu-DOTA-AE105 was rapidly cleared (<0.1% IA/g at 4 h p.i.). As a result, the accumulation of the new radiopeptides in HEK-uPAR xenografts (3.6-11% and 3.1-10% IA/g at 4 and 24 h p.i., respectively) was increased in comparison to that of [(177)Lu]Lu-DOTA-AE105 (<1% IA/g at 4 h p.i.). Importantly, the metabolic stability of the new radiopeptides in mice was enhanced as compared to that of [(177)Lu]Lu-DOTA-AE105. [(177)Lu]Lu-uPAR-02 showed the most promising tissue distribution profile with over 10-fold higher activity retention in the HEK-uPAR xenograft than observed after injection of [(177)Lu]Lu-DOTA-AE105. As a result, the xenograft-to-kidney ratio of [(177)Lu]Lu-uPAR-02 was >3-fold higher than that of [(177)Lu]Lu-DOTA-AE105.