Linker modifications in radiolabeled RM26-based antagonists to gastrin-releasing peptide receptor (GRPR) improved tracers’ pharmacokinetics

Radionuclide targeting of gastrin-releasing peptide receptor (GRPR) holds significant diagnostic and therapeutic potential, particularly in PSMA-negative/low-grade prostate cancer, estrogen receptor-positive breast cancer, and other malignancies. Recently, our group reported the development and results of the Phase I clinical evaluation of [^99mTc]Tc-maSSS-PEG2-RM26, an antagonist GRPR-targeting SPECT imaging agent. This study focuses on developing the next generation of RM-26-based GRPR antagonists with enhanced metabolic stability and improved pharmacokinetics. Four new RM-26-based agents containing sarcosine (Sar) at position 11 to improve the in vivo stability and with more hydrophilic linkers were designed: Pep1 – maSSS-PEG2-[Sar¹¹]RM26, Pep2 – maSSS-PEG6-[Sar¹¹]RM26, Pep3 – maSSS-PEG2-Pip-[Sar¹¹]RM26, and Pep4 – maSSS-EEE-[Sar¹¹]RM26. These analogs were compared both in vitro and in vivo with [^99mTc]Tc-maSSS-PEG2-RM26 as a reference. In PC-3 cells, [^99mTc]Tc-Pep1, [^99mTc]Tc-Pep2 and [^99mTc]Tc-Pep3, but not [^99mTc]Tc-Pep4, specifically bound to GRPR and exhibited low nanomolar affinity. When compared in vivo, [^99mTc]Tc-Pep1, [^99mTc]Tc-Pep2, and [^99mTc]Tc-Pep3 demonstrated rapid blood clearance with different degrees of hepatobiliary excretion, particularly [^99mTc]Tc-Pep2 and [^99mTc]Tc-Pep3 had significantly lower activity uptake in the liver and gastrointestinal tract compared to [^99mTc]Tc-maSSS-PEG2-RM26. Both [^99mTc]Tc-Pep2 and [^99mTc]Tc-Pep3 showed improved metabolic stability, bound specifically to GRPR in vivo, and demonstrated a tendency (not statistically significant) for higher tumor activity uptake compared to the reference peptide. Biodistribution data were confirmed by SPECT/CT imaging. In conclusion, the analogs with an elongation of the PEG2-linker either up to PEG6 (Pep2) or with the addition of a basic piperidine-containing moiety (Pep3) demonstrated an improvement of the pharmacokinetic properties of these agents and justify further investigations.