Design, Synthesis, In Vitro, and Initial In Vivo Evaluation of Heterobivalent Peptidic Ligands Targeting Both NPY(Y1)- and GRP-Receptors—An Improvement for Breast Cancer Imaging?

Abstract

Heterobivalent peptidic ligands (HBPLs), designed to address two different receptors independently, are highly promising tumor imaging agents. For example, breast cancer has been shown to concomitantly and complementarily overexpress the neuropeptide Y receptor subtype 1 (NPY(Y1)R) as well as the gastrin-releasing peptide receptor (GRPR). Thus, radiolabeled HBPLs being able to bind these two receptors should exhibit an improved tumor targeting efficiency compared to monospecific ligands. We developed here such bispecific HBPLs and radiolabeled them with 68Ga, achieving high radiochemical yields, purities, and molar activities. We evaluated the HBPLs and their monospecific reference peptides in vitro regarding stability and uptake into different breast cancer cell lines and found that the 68Ga-HBPLs were efficiently taken up via the GRPR. We also performed in vivo PET/CT imaging and ex vivo biodistribution studies in T-47D tumor-bearing mice for the most promising 68Ga-HBPL and compared the results to those obtained for its scrambled analogs. The tumors could easily be visualized by the newly developed 68Ga-HBPL and considerably higher tumor uptakes and tumor-to-background ratios were obtained compared to the scrambled analogs in and ex vivo. These results demonstrate the general feasibility of the approach to use bispecific radioligands for in vivo imaging of breast cancer.

Current State of Radiolabeled Heterobivalent Peptidic Ligands in Tumor Imaging and Therapy

“…For [ 177 Lu]Lu-6, comparably promising in vitro results were obtained compared to [ 68 Ga]Ga-6. As found for the 68 Ga-labeled analog, the heterodimer [ 177 Lu]Lu-6 showed a favorable pharmacokinetic profile in mice carrying PC3 and LNCaP tumors and slightly higher uptakes in both tumor types when compared with the respective monomers, confirming the positive results obtained for [ 68 Ga]Ga-6. Furthermore, the found tumor uptakes were higher in both tumor types than in any other organ at 3 h post injection (p.i.).…”

“…On the other hand, highly rigid linkers could limit the motility of the molecule, impeding the first peptide to bind although the second binder might be forced to stay near the cell surface, increasing its binding probability. However, an effect of linker rigidity on the peptide-receptor interaction of peptide homo-or heterodimers-although having been investigated in some studies-could not be shown thus far [59,62,66,68].…”

“…The radiolabeled heterobivalent peptides described thus far are discussed in the following paragraphs and summarized in Table 1. Recently, not only the addressed target receptors broadened significantly, but also the potential application of the agents expanded from mainly PET imaging to PET and SPECT imaging as well as tumor therapy, entailing the use of different diagnostic ( 18 F, 64 Cu, 68 Ga, 86 Y, 99m Tc, and 111 In) as well as therapeutic ( 90 Y, 125 I, and 177 Lu) radionuclides ( Table 2). Although approaches to combine GRPR and PSMA binders to radiolabeled heterobivalent ligands emerged only in 2014, there were several agents already described in this group of heterobivalent peptidic agents, some of them showing high clinical potential.…”

“…In another approach, iPSMA (Glu-urea-Lys-Nal, used as PSMA-targeting agent) was connected to a full-length analog of bombesin (Lys 3 -bombesin for GRPR binding) over a very short cysteine building block carrying a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelator to enable radiolabeling not only with the diagnostic radionuclide 68 Ga, but also offering the possibility to introduce a therapeutic radionuclide to obtain a theranostic pair (6) [77,78]. The 68 68 Ga]Ga-DOTA-iPSMA: 1.0 ± 0.3 nM for PSMA) and even slightly higher uptakes in GRPR-positive PC3 and PSMA-positive LNCaP cells compared with the respective monomers. These results could be confirmed by in vivo tumor imaging of mice carrying PC3 and LNCaP tumors where the heterodimer visualized the tumors with higher SUV max (SUV = standardized uptake value) than the corresponding radiolabeled monomers.…”

“…However, no complete biodistribution data were provided in this study, preventing an assessment of achievable tumor-to-background ratios. Interestingly, a non-optimal 68 Ga-radionuclide incorporation and a relatively low radiochemical yield (RCY) of 62 ± 4% were observed during radiolabeling. In contrast, 177 Lu-labeling proceeded with high efficiency, giving [ 177 Lu]Lu-6 in high RCY of 99 ± 1% [78].…”