We have recently introduced the potent GRPR-antagonist [⁶⁸Ga]SB3 ([⁶⁸Ga-DOTA-p-aminomethylaniline-diglycolic acid-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt), showing excellent tumor localizing efficacy in animal models and in patients. By replacement of the C-terminal the novel GRPR-antagonist NeoBOMB1 was generated and labeled with different radiometals for theranostic use. We herein report on the biological profile of resulting [^67/68Ga/¹¹¹In/¹⁷⁷Lu]NeoBOMB1 radioligands in GRPR-expressing cells and mouse models. First evidence of prostate cancer lesion visualization in man with [⁶⁸Ga]NeoBOMB1 and PET/CT is also presented. Methods: NeoBOMB1 was radiolabeled with ^67/68Ga, ¹¹¹In and ¹⁷⁷Lu according to published protocols. The respective metalated species [natGa/natIn/natLu]NeoBOMB1 were also synthesized and used in competition binding experiments against [¹²⁵I-Tyr4]BBN in GRPR-positive PC-3 cell membranes. Internalization of [⁶⁷Ga/¹¹¹In/¹⁷⁷Lu]NeoBOMB1 radioligands was studied in PC-3 cells at 370C and their metabolic stability in peripheral mouse blood was determined by HPLC-analysis of blood samples. Biodistribution was performed by injecting a [⁶⁷Ga/¹¹¹In/¹⁷⁷Lu]NeoBOMB1 bolus (74 kBq/74 kBq /370 kBq, respectively, 100 µL, 10 pmol total peptide ±40 nmol [Tyr4]BBN: for in vivo GRPR-blockade) in SCID mice bearing PC-3 xenografts. PET/CT images with [⁶⁸Ga]NeoBOMB1 were acquired in prostate cancer patients. Results: NeoBOMB1 and [natGa/natIn/natLu]NeoBOMB1 bound to GRPR with high affinity (IC50: 1-2 nM). [⁶⁷Ga/¹¹¹In/¹⁷⁷Lu]NeoBOMB1 specifically and strongly bound on the cell-membrane of PC-3 cells displaying low internalization, as expected for receptor antagonists. They showed excellent metabolic stability in peripheral mouse blood (>95% intact at 5 min postinjection (pi)). After injection in mice, all three [⁶⁷Ga/¹¹¹In/¹⁷⁷Lu]NeoBOMB1 showed comparably high and GRPR-specific uptake in the PC-3 xenografts (e.g. 30.6±3.9%ID/g, 28.6±6.0%ID/g and >35%ID/g at 4 h pi, respectively), clearing from background predominantly via the kidneys. During a translational study in prostate cancer patients [⁶⁸Ga]NeoBOMB1 rapidly localized in pathological lesions achieving high contrast imaging. Conclusion: The GRPR-antagonist radioligands [⁶⁷Ga/¹¹¹In/¹⁷⁷Lu]NeoBOMB1, independent of the radiometal applied, have shown comparable behavior in prostate cancer models, in favor of future theranostic use in GRPR-positive cancer patients. Such translational prospects were further supported by the successful visualization of prostate cancer lesions in man applying [⁶⁸Ga]NeoBOMB1 and PET/CT.

The gastrin-releasing peptide receptor (GRPR) is expressed in high numbers in a variety of human tumors, including the frequently occurring prostate and breast cancers, and therefore provides the rationale for directing diagnostic or therapeutic radionuclides on cancer lesions after administration of anti-GRPR peptide analogs. This concept has been initially explored with analogs of the frog 14-peptide bombesin, suitably modified at the N-terminus with a number of radiometal chelates. Radiotracers that were selected for clinical testing revealed inherent problems associated with these GRPR agonists, related to low metabolic stability, unfavorable abdominal accumulation, and adverse effects. A shift toward GRPR antagonists soon followed, with safer analogs becoming available, whereby, metabolic stability and background clearance issues were gradually improved. Clinical testing of three main major antagonist types led to promising outcomes, but at the same time brought to light several limitations of this concept, partly related to the variation of GRPR expression levels across cancer types, stages, previous treatments, and other factors. Currently, these parameters are being rigorously addressed by cell biologists, chemists, nuclear medicine physicians, and other discipline practitioners in a common effort to make available more effective and safe state-of-the-art molecular tools to combat GRPR-positive tumors. In the present review, we present the background, current status, and future perspectives of this endeavor.

The clinical success of radiolabeled somatostatin analogs in the diagnosis and therapy—“theranostics”—of tumors expressing the somatostatin subtype 2 receptor (SST₂R) has paved the way for the development of a broader panel of peptide radioligands targeting different human tumors. This approach relies on the overexpression of other receptor-targets in different cancer types. In recent years, a shift in paradigm from internalizing agonists to antagonists has occurred. Thus, SST₂R-antagonist radioligands were first shown to accumulate more efficiently in tumor lesions and clear faster from the background in animal models and patients. The switch to receptor antagonists was soon adopted in the field of radiolabeled bombesin (BBN). Unlike the stable cyclic octapeptides used in the case of somatostatin, BBN-like peptides are linear, fast to biodegradable and elicit adverse effects in the body. Thus, the advent of BBN-like antagonists provided an elegant way to obtain effective and safe radiotheranostics. Likewise, the pursuit of gastrin and exendin antagonist-based radioligands is advancing with exciting new outcomes on the horizon. In the present review, we discuss these developments with a focus on clinical results, commenting on challenges and opportunities for personalized treatment of cancer patients by means of state-of-the-art antagonist-based radiopharmaceuticals.

Diagnostic imaging and radionuclide therapy of prostate (PC) and breast cancer (BC) using radiolabeled gastrin-releasing peptide receptor (GRPR)-antagonists represents a promising approach. We herein propose the GRPR-antagonist based radiotracer [^99mTc]Tc-DB15 ([^99mTc]Tc-N₄-AMA-DGA-_DPhe⁶,Sar¹¹,LeuNHEt¹³]BBN(6-13); N₄: 6-carboxy-1,4,8,11-tetraazaundecane, AMA: aminomethyl-aniline, DGA: diglycolic acid) as a new diagnostic tool for GRPR-positive tumors applying SPECT/CT. The uptake of [^99mTc]Tc-DB15 was tested in vitro in mammary (T-47D) and prostate cancer (PC-3) cells and in vivo in T-47D or PC-3 xenograft-bearing mice as well as in BC patients. DB15 showed high GRPR-affinity (IC₅₀ = 0.37 ± 0.03 nM) and [^99mTc]Tc-DB15 strongly bound to the cell-membrane of T-47D and PC-3 cells, according to a radiolabeled antagonist profile. In mice, the radiotracer showed high and prolonged GRPR-specific uptake in PC-3 (e.g., 25.56 ± 2.78 %IA/g vs. 0.72 ± 0.12 %IA/g in block; 4 h pi) and T-47D (e.g., 15.82 ± 3.20 %IA/g vs. 3.82 ± 0.30 %IA/g in block; 4 h pi) tumors, while rapidly clearing from background. In patients with advanced BC, the tracer could reveal several bone and soft tissue metastases on SPECT/CT. The attractive pharmacokinetic profile of [^99mTc]DB15 in mice and its capability to target GRPR-positive BC lesions in patients highlight its prospects for a broader clinical use, an option currently being explored by ongoing clinical studies.

Background: The GRPR-antagonist-based radioligands [^67/68Ga/¹¹¹In/¹⁷⁷Lu]NeoBOMB1 have shown excellent theragnostic profiles in preclinical prostate cancer models, while [⁶⁸Ga]NeoBOMB1 effectively visualized prostate cancer lesions in patients. We were further interested to explore the theragnostic potential of NeoBOMB1 in GRPR-positive mammary carcinoma, by first studying [⁶⁷Ga]NeoBOMB1 in breast cancer models; Methods: We investigated the profile of [⁶⁷Ga]NeoBOMB1, a [⁶⁸Ga]NeoBOMB1 surrogate, in GRPR-expressing T-47D cells and animal models; Results: NeoBOMB1 (IC₅₀s of 2.2 ± 0.2 nM) and [^natGa]NeoBOMB1 (IC₅₀s of 2.5 ± 0.2 nM) exhibited high affinity for the GRPR. At 37 °C [⁶⁷Ga]NeoBOMB1 strongly bound to the T-47D cell-membrane (45.8 ± 0.4% at 2 h), internalizing poorly, as was expected for a radioantagonist. [⁶⁷Ga]NeoBOMB1 was detected >90% intact in peripheral mouse blood at 30 min pi. In mice bearing T-47D xenografts, [⁶⁷Ga]NeoBOMB1 specifically localized in the tumor (8.68 ± 2.9% ID/g vs. 0.6 ± 0.1% ID/g during GRPR-blockade at 4 h pi). The unfavorably high pancreatic uptake could be considerably reduced (206.29 ± 17.35% ID/g to 42.46 ± 1.31% ID/g at 4 h pi) by increasing the NeoBOMB1 dose from 10 pmol to 200 pmol, whereas tumor uptake remained unaffected. Notably, tumor values did not decline from 1 to 24 h pi; Conclusions: [⁶⁷Ga]NeoBOMB1 can successfully target GRPR-positive breast cancer in animals with excellent prospects for clinical translation.

We have recently shown that treatment of mice with the neutral endopeptidase (NEP) inhibitor phosphoramidon (PA) improves the bioavailability and tumor uptake of biodegradable radiopeptides. For the truncated gastrin radiotracer [111In-DOTA]MG11 ([(DOTA)DGlu10]gastrin(10–17)), this method led to impressively high tumor-to-kidney ratios. Translation of this concept in the clinic requires the use of certified NEP inhibitors, such as thiorphan (TO) and its orally administered prodrug racecadotril (Race). Besides NEP, angiotensin-converting enzyme (ACE) has also been implicated in the catabolism of gastrin analogs. In the present study, we first compared the effects induced by NEP inhibition (using PA, TO, or Race) and/or by ACE inhibition (using lisinopril, Lis) on the biodistribution profile of [111In-DOTA]MG11 in mice. In addition, we compared the efficacy of PA and TO at different administered doses to enhance tumor uptake. [111In-DOTA]MG11 was coinjected with (a) vehicle, (b) PA (300 μg), (c) TO (150 μg), (d) Lis (100 μg), (e) PA (300 μg) plus Lis (100 μg), or (f) 30–40 min after intraperitoneal (ip) injection of Race (3 mg) in SCID mice bearing AR42J xenografts. In addition, [111In-DOTA]MG11 was coinjected with vehicle, or with progressively increasing amounts of PA (3, 30, or 300 μg) or TO (1.5, 15, and 150 μg) in SCID mice bearing twin A431-CCK2R(+/−) tumors. In all above cases, biodistribution was conducted at 4 h postinjection (pi). During NEP inhibition, the uptake of [111In-DOTA]MG11 in the AR42J tumors impressively increased from 1.8 ± 1.0 % ID/g (controls) to 15.3 ± 4.7 % ID/g (PA) and 12.3 ± 3.6 % ID/g (TO), while with Race tumor values reached 6.8 ± 2.8 % ID/g. Conversely, Lis had no effect on tumor uptake and no additive effect when coinjected with PA. During the dose dependence study in mice, PA turned out to be more efficacious in enhancing tumor uptake of [111In-DOTA]MG11 in the CCK2R-positive tumors compared to equimolar amounts of TO. In all cases, renal accumulation remained low, resulting in notable increases of tumor-to-kidney ratios. This study has confirmed NEP as the predominant degrading enzyme of [111In-DOTA]MG11 and ruled out the involvement of ACE in the in vivo catabolism of the radiotracer. NEP inhibition with the clinically tested NEP inhibitors TO and Race resulted in significant enhancement of tumor-to-kidney ratios vs. controls. However, compared with PA, TO and its prodrug Race induced less potent increases of tumor uptake, highlighting the significance of inhibitor type, administration route, and dose for implementing a first proof-of-principle study in human.