Simultaneous targeting of the prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) could improve the diagnostic accuracy in prostate cancer (PCa). The aim of this study was to develop a PSMA/GRPR-targeting bispecific heterodimer for SPECT and positron emission tomography (PET) diagnostic imaging of PCa. The heterodimer NOTA-DUPA-RM26 was produced by manual solid-phase peptide synthesis. NOTA-DUPA-RM26 was labeled with ¹¹¹In and ⁶⁸Ga, with yields >98%, and demonstrated a high stability and binding specificity to PSMA and GRPR. IC₅₀ values for ^natIn-NOTA-DUPA-RM26 were 4 ± 1 nM towards GRPR and 824 ± 230 nM towards PSMA. An in vivo binding specificity 1 h pi of ¹¹¹In-NOTA-DUPA-RM26 in PC3-PIP-xenografted mice demonstrated partially blockable tumor uptake when co-injected with an excess of PSMA- or GRPR-targeting agents. Simultaneous co-injection of both agents induced pronounced blocking. The biodistribution of ¹¹¹In-NOTA-DUPA-RM26 and ⁶⁸Ga-NOTA-DUPA-RM26 revealed fast activity clearance from the blood and normal organs via the kidneys. Tumor uptake exceeded normal organ uptake for both analogs 1 h pi. ⁶⁸Ga-NOTA-DUPA-RM26 had a significantly lower tumor uptake (8 ± 2%ID/g) compared to ¹¹¹In-NOTA-DUPA-RM26 (12 ± 2%ID/g) 1 h pi. Tumor-to-organ ratios increased 3 h pi, but decreased 24 h pi, for ¹¹¹In-NOTA-DUPA-RM26. MicroPET/CT and microSPECT/CT scans confirmed biodistribution data, suggesting that ⁶⁸Ga-NOTA-DUPA-RM26 and ¹¹¹In-NOTA-DUPA-RM26 are suitable candidates for the imaging of GRPR and PSMA expression in PCa shortly after administration.

Prostate-specific membrane antigen (PSMA) is overexpressed in the majority of prostate cancer cells and is considered to be an important target for the molecular imaging and therapy of prostate cancer. Herein, we present the design, synthesis, and evaluation of 11 PSMA-binding radioligands with modified linker structures, focusing on the relationship between molecular structure and targeting properties. The linker design was based on 2-naphthyl-L-alanine-tranexamic acid, the linker structure of PSMA-617. X-ray crystal-structure analysis of PSMA and structure-based design were used to generate the linker modifications, suggesting that substitution of tranexamic acid could lead to interactions with Phe546, Trp541, and Arg43 within the binding cavity. After synthesis through SPPS, analogues were labelled with indium-111 and evaluated in vitro for their specific binding, affinity, and cellular retention. Selected compounds were further evaluated in vivo in PSMA-expressing tumour-bearing mice. Based on the results, 2-naphthyl-L-alanine appears to be crucial for good targeting properties, whereas tranexamic acid could be replaced by other substituents. [¹¹¹In]In-BQ7859, consisting of a 2-naphthyl-L-alanine-L-tyrosine linker, demonstrated favourable targeting properties. The substitution of tranexamic acid for L-tyrosine in the linker led to an improved tumour-to-blood ratio, highlighting [¹¹¹In]In-BQ7859 as a promising PSMA-targeting radioligand.

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are promising targets for molecular imaging of prostate cancer (PCa) lesions. Due to the heterogenic overexpression of PSMA and GRPR in PCa, a heterodimeric radiotracer with the ability to bind to both targets could be beneficial. Recently, our group reported the novel heterodimer BQ7800 consisting of a urea-based PSMA inhibitor, the peptide-based GRPR antagonist RM26 and NOTA chelator. The study reported herein, aimed to improve the affinity of BQ7800 towards PSMA by changing the composition of the two linkers connecting the PSMA- and GRPR-targeting motifs. Three novel heterodimeric analogues were synthesized by incorporation of phenylalanine in the functional linker of the PSMA-binding motif and/or shortening the PEG-linker coupled to RM26. The heterodimers were labeled with indium-111 and evaluated in vitro. In the competitive binding assay, BQ7812, featuring phenylalanine and shorter PEG-linker, demonstrated a nine-fold improved affinity towards PSMA. In the in vivo biodistribution study of [¹¹¹In]In-BQ7812 in PC3-pip tumor-bearing mice (PSMA and GRPR positive), the activity uptake was two-fold higher in the tumor and three-fold higher in kidneys than for [¹¹¹In]In-BQ7800. Herein, we showed that the affinity of a bispecific PSMA/GRPR heterodimer towards PSMA could be improved by linker modification.

Gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA) are overexpressed in most prostate cancers. GRPR expression is higher in early stages while PSMA expression increases with progression. The possibility of targeting both markers with a single theranostics radiotracer could improve patient management. Three GRPR/PSMA-targeting bispecific heterodimers (urea derivative PSMA-617 and bombesin-based antagonist RM26 linked via X-triazolyl-Tyr-PEG2, X = PEG2 (BO530), (CH₂)₈ (BO535), none (BO536)) were synthesized by solid-phase peptide synthesis. Peptides were radio-iodinated and evaluated in vitro for binding specificity, cellular retention, and affinity. In vivo specificity for all heterodimers was studied in PC-3 (GRPR-positive) and LNCaP (PSMA-positive) xenografts. [¹²⁵I]I-BO530 was evaluated in PC-3pip (GRPR/PSMA-positive) xenografts. Micro single-photon emission computed tomography/computed tomography (microSPECT/CT) scans were acquired. The heterodimers were radiolabeled with high radiochemical yields, bound specifically to both targets, and demonstrated high degree of activity retention in PC-3pip cells. Only [¹²⁵I]I-BO530 demonstrated in vivo specificity to both targets. A biodistribution study of [¹²⁵I]I-BO530 in PC-3pip xenografted mice showed high tumor activity uptake (30%–35%ID/g at 3 h post injection (pi)). Activity uptake in tumors was stable and exceeded all other organs 24 h pi. Activity uptake decreased only two-fold 72 h pi. The GRPR/PSMA-targeting heterodimer [¹²⁵I]I-BO530 is a promising agent for theranostics application in prostate cancer.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in prostate cancer (PCa) and in hormone-driven breast cancer (BCa). The aim of this phase I clinical trial was to evaluate safety, biodistribution, and dosimetry after the administration of the recently developed GRPR-targeting antagonistic bombesin analogue [^99mTc]Tc-maSSS-PEG₂-RM26 in PCa and BCa patients. Planar and whole-body SPECT/CT imaging was performed in six PCa patients and seven BCa patients 2, 4, 6, and 24 h post the intravenous administration of 40 µg of [^99mTc]Tc-maSSS-PEG₂-RM26 (600–700 MBq). No adverse events or pathological changes were observed. The rapid blood clearance of [^99mTc]Tc-maSSS-PEG₂-RM26 was observed with predominantly hepatobiliary excretion. The effective doses were 0.0053 ± 0.0007 for male patients and 0.008 ± 0.003 mSv/MBq for female patients. The accumulation of [^99mTc]Tc-maSSS-PEG₂-RM26 in tumors was observed in four out of six PCa and in seven out of seven BCa patients. In four BCa patients, a high uptake of the agent into the axillary lymph nodes was detected. Immunohistochemistry revealed positive GRPR expression in 60% of primary PCa, 71.4% of BCa tumors, and 50% of examined BCa lymph nodes. In conclusion, a single administration of [^99mTc]Tc-maSSS-PEG₂-RM26 was safe and well tolerated. [^99mTc]Tc-maSSS-PEG₂-RM26 SPECT may be useful for tumor detection in PCa and BCa patients, pending further studies.

Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies. In this study, we evaluated a scaffold protein, designed ankyrin repeat protein (DARPin) Ec1, for imaging of EpCAM in TNBC. DARPin Ec1 was labeled with a non-residualizing [¹²⁵I]I-para-iodobenzoate (PIB) label and a residualizing [^99mTc]Tc(CO)₃ label. Both imaging probes retained high binding specificity and affinity to EpCAM-expressing MDA-MB-468 TNBC cells after labeling. Internalization studies showed that Ec1 was retained on the surface of MDA-MB-468 cells to a high degree up to 24 h. Biodistribution in Balb/c nu/nu mice bearing MDA-MB-468 xenografts demonstrated specific uptake of both [¹²⁵I]I-PIB-Ec1 and [^99mTc]Tc(CO)₃-Ec1 in TNBC tumors. [¹²⁵I]I-PIB-Ec1 had appreciably lower uptake in normal organs compared with [^99mTc]Tc(CO)₃-Ec1, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by micro-Single-Photon Emission Computed Tomography/Computed Tomography (microSPECT/CT) imaging. In conclusion, an indirectly radioiodinated Ec1 is the preferable probe for imaging of EpCAM in TNBC.