PEG spacers of different length influence the biological profile of bombesin-based radiolabeled antagonists

The gastrin-releasing peptide receptor (GRPR) was shown to be expressed with high density on several types of cancers. Radiolabeled peptides for imaging and targeted radionuclide therapy have been developed. In this study, we evaluated the potential of statine-based bombesin antagonists, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) through oligoethyleneglycol spacers, labeled with 177Lu and we determined the effect of polyethyleneglycol (PEG) spacer length on in vitro and in vivo properties. The bombesin antagonists were synthesized on solid phase using Fmoc chemistry; the spacers Fmoc-dPEGx-OH (x = 2, 4, 6 and 12) and the DOTA(tBu)3 were coupled using a standard procedure. The peptides were labeled with 177Lu and evaluated in vitro (lipophilicity, serum stability, internalization and binding affinity assays). Biodistribution studies were performed in PC-3 tumor-bearing nude mice. The solid-phase synthesis was straightforward with an overall yield ranging from 30% to 35% based on the first Fmoc cleavage. The hydrophilicity increased with spacer length (logD: − 1.95 vs − 2.22 of PEG2 and PEG12 analogs, respectively). There is a tendency of increased serum stability by increasing the spacer length (T1/2 = 246 ± 4 and 584 ± 20 for PEG2 and PEG6 analogs, respectively) which seems to reverse with the PEG12 analog. The IC50 values are similar with the only significant difference of the PEG12 analog. The 177Lu-labeled PEG4 and PEG6 conjugates showed similar pharmacokinetic with high tumor uptake and excellent tumor-to-kidney ratios (7.8 and 9.7 at 4 h for the PEG4 and PEG6 derivatives, respectively). The pancreas uptake was relatively high at 1 h but it shows fast washout (0.46% ± 0.02% IA/g and 0.29% ± 0.08% IA/g already at 4 h). Among all the studied analogs the PEG4 and PEG6 showed significantly better properties. The very high tumor-to-non-target organ ratios, in particular tumor-to-kidney ratios, already at early time point will be important in regard to safety concerning kidney toxicity.