The gastrin-releasing peptide receptor (GRPR) plays a critical role in numerous physiological and pathological processes including the progression of various cancers. GRPR is significantly overexpressed in several tumor types, including prostate, breast, pancreatic, and small-cell lung cancers, where it promotes cell proliferation, angiogenesis, and metastasis through activation of signaling pathways such as MAPK and PI3K/AKT. (1,2) This elevated GRPR expression in tumors, has positioned it as a compelling target for both cancer imaging and therapy, particularly in the context of nuclear medicine. A broad spectrum of GRPR-targeted peptidic analogues, encompassing both agonistic and antagonistic profiles, have been developed and extensively evaluated, demonstrating significant potential. However, early clinical attempts utilizing GRPR-targeting agonists were unsuccessful, primarily due to significant gastrointestinal side effects observed at therapeutic peptide doses. (3) To address these limitations, GRPR antagonists were promptly introduced and evaluated for their potential application for both imaging and radionuclide therapy. (4,5) The transition from agonists to antagonists was further supported by findings from somatostatin receptor research, which revealed that radiolabeled antagonists can bind multiple receptor sites and exhibit superior pharmacokinetic profiles compared to agonists. (6) These insights have further supported the clinical interest in GRPR-targeting antagonists as a safer and potentially more effective strategy for theranostic purposes.
In the evolving landscape of prostate cancer imaging, the complementary use of GRPR and prostate-specific membrane antigen (PSMA) radioligands marks a significant step forward. By combining these two approaches, clinicians can achieve more accurate diagnosis, better staging, and improved therapeutic guidance, particularly for challenging cases such as PSMA-negative or heterogeneous tumors. (7) Beyond diagnostic applications, the emergence of new-generation GRPR antagonist-based radiopeptides is paving the way for realistic theranostic approaches, broadening the scope of precision medicine in prostate cancer management.
The application of radiolabeled GRPR-targeting agents such as RM2, AMTG, SAR-BBN (NCT05633160) and NeoB (NCT03872778) as theranostic tools has demonstrated significant efficacy in the diagnosis and treatment of various cancers that exhibit high expression of the GRPR. These malignancies include prostate cancer, breast cancer, gastrointestinal tumors, lung cancer, and glioblastomas, as supported by multiple clinical studies and trials. (4,8−13) By combining diagnostic imaging and targeted radionuclide therapy, they have shown promising results in enhancing tumor detection and delivering selective cytotoxic radiation to cancerous tissues.
Despite these encouraging developments, the clinical translation of GRPR-targeting radioligands faces several key challenges. One of the primary limitations is their susceptibility to enzymatic degradation in vivo, which significantly shortens their biological half-life and limits their therapeutic efficacy. (13,14) This rapid metabolic breakdown necessitates ongoing efforts to improve the chemical stability, receptor-binding affinity, and pharmacokinetic profiles of these peptide-based radiopharmaceuticals. (13−19)
This study provides a detailed preclinical assessment of LF1 (AAZTA 5-Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH 2), a GRPR-targeting statine based antagonist developed by our group. (20) LF1 is functionalized with the chelator AAZTA 5 (6-[Bis(carboxymethyl)amino]-1,4-bis(carboxymethyl)-6-methyl-1,4-diazepane) via the positely charged Pip spacer (4-amino-1-carboxymethylpiperidine) and was previously radiolabeled with a variety of radionuclides and characterized in vitro by us. (20) Here, we further assess [177 Lu]Lu-LF1 through in vitro and in vivo studies, including GRPR-binding assays, pharmacokinetics, metabolic stability, biodistribution, and SPECT/CT imaging. Its therapeutic efficacy was tested in prostate cancer models using two regimens: [177 Lu]Lu-LF1 alone and combined with the mTOR inhibitor everolimus, aiming to enhance treatment response through the mTOR pathway inhibition. (21)
The favorable properties of AAZTA 5 enabled fast and efficient synthesis of [177 Lu]Lu-LF1 under mild conditions. The molar activities ranged from 10 to 44 GBq/μmol, depending on the experiment. The radiochemical yield and purity exceeded 99%, confirmed by radio-TLC and radio-HPLC. The tracer remained highly stable (99 ± 0.01%, n = 10) up to 72 h postlabeling. After 6 days, slight tailing was observed, and the radiochemical stability was assessed as 88% after integration of the signal.
The mesocyclic structure of AAZTA 5, when conjugated to a GRPR antagonist, enables efficient radiolabeling under mild conditions with high molar activities, up to 44 GBq/μmol without further purification, suggesting even higher values are possible. However, previous work from our group (20) indicated that AAZTA 5 is suboptimal for gallium-68, limiting its use in [177 Lu]Lu-LF1/[68 Ga]Ga-LF1 theranostic pairs. This challenge can be overcome by employing RM2, a GRPR-targeted tracer effectively labeled with gallium-68. (22) Despite differences in chelators (DOTA vs AAZTA 5), RM2 and LF1 share similar peptide and spacer structure, supporting their potential as a complementary theranostic pair. Moreover, AAZTA 5 shows promise for other radionuclide pairs such as scandium-44/47 and copper-64/67, (23) warranting further investigation. The longer half-lives of scandium-44 (3.97 h) and copper-64 (12.7 h) compared to gallium-68 (67.7 min) allow for delayed imaging, which may enhance GRPR-targeted imaging due to improved background clearance, as shown in both preclinical (24,25) and clinical studies. (26)
[177 Lu]Lu-LF1 exhibited a high binding affinity with a K d value of 0.12 ± 0.01 nM, indicating strong and specific receptor interaction. The kinetic analysis revealed a moderate association rate constant (K on) of 2.5 × 10 5 ± 0.2 × 10 5 M–1 s–1 and a very slow dissociation rate constant (K off) of 3.2 × 10–5 ± 0.1 × 10–5 s–1. [177 Lu]Lu-LF1 showed an improved kinetic profile compared to the existing GRPR antagonist [177 Lu]Lu-RM2 (K d = 5.4 ± 0.8 nM and IC 50 = 7.7 ± 3.3 nM). (20,27) The slow off-rate in [177 Lu]Lu-LF1 is particularly favorable for therapeutic applications, as it suggests prolonged receptor occupancy and sustained radiation delivery to GRPR-expressing tumors. These results highlight the strong potential of [177 Lu]Lu-LF1 as a GRPR-targeted radiopharmaceutical, combining both high receptor affinity and favorable kinetic for effective tumor targeting and retention. The much lower K d obtained with LigandTracer compared to previously reported IC 50 values for RM2 (18,27) and K d value of LF1 (20) might appear to indicate a major increase in affinity, but this discrepancy should be interpreted with caution. The values were generated using different experimental strategies, cell-based competition assays versus real-time kinetic binding, which are influenced by factors such as tracer concentration, incubation conditions, receptor density, and data-fitting models. As such, the LigandTracer-derived K d of 0.1 nM should not be directly compared with IC 50 values from earlier assays, but rather considered a complementary measure that provides higher-resolution insight into binding kinetics.
Figure 1. Sensogram of the kinetic profile of [177 Lu]Lu-LF1 in PC-3 cells using LigantTracer Gray. For the association phase measurements, two increasing concentration were used, 1 and 3 nM of [177 Lu]Lu-LF1 in complete medium.
The biodistribution and tumor-to-tissue ratios of [177 Lu]Lu-LF1 are summarized in Figure 2 and Table 1, with detailed ex vivo data provided in Table S1 (supplementary). [177 Lu]Lu-LF1 showed rapid blood clearance, with ∼ 1% IA/g at 1 h p.i., further decreasing over time. Tumor uptake was high and rapid (>40% IA/g at 1 and 4 h p.i.), with slow clearance, retaining 3.9 ± 1.1% IA/g at 144 h p.i. High uptake was initially observed in GRPR-rich organs such as the pancreas (71 ± 8.1% IA/g at 1 h p.i.), however, decreased quickly, dropping by a factor of 4.7 at 4 h and to <1% IA/g at later time points. This finding is in agreement with the behavior previously observed with other radiolabeled GRPR antagonists. (18,24,25,28) Tumor-to-pancreas ratios rose from 0.5 at 1 h to >20 at later time points. Renal excretion was the primary elimination route, with low liver uptake (<1% IA/g) across the investigated time points. Kidney uptake peaked at 7.1 ± 1.1% IA/g at 1 h p.i., dropping below 2% IA/g by 24 h. GRPR-specific targeting was confirmed by a 70% and 95% reduction in tumor and pancreas uptake, respectively, following coinjection with the blocking agent.
Figure 2. Biodistribution data of [177 Lu]Lu-LF1 in PC-3-mice at 1, 4, 24, 48, 72, 96, and 144 h p.i along with blocking studies data at 4 h p.i. Data have been calculated as %IA/g of tissue and are presented as mean ± SD (n = 3–4).
Table 1. Tumor to Organ (Blood, Liver, Kidney, Muscle, and Pancreas) Ratios of [177 Lu]Lu-LF1 in PC-3-mice at 1, 4, 24, 48, 72, 96, and 144 h p.i
Among the evaluated GRPR-based radioligands (RM2, RM26, AMTG, NeoB, RGD-Glu-[DO3A]-6-Ahx-RM2) (Table S2), [177 Lu]Lu-LF1 exhibited the highest tumor uptake (42%IA/g at 1 h p.i. and 18%IA/g at 24 h p.i.) demonstrating strong and sustained tumor targeting. (18,24,28,29) When comparing these results, it is important to account for differences in experimental conditions between the studies, particularly with respect to the mice strain and gender. [177 Lu]Lu-LF1 showed significantly high pancreatic uptake at 1 h p.i. (70%IA/g), which declined sharply to 0.9%IA/g by 24 h p.i., indicating rapid clearance from GRPR-rich healthy tissues. Additionally, kidney and liver uptake remained consistently low, reflecting a favorable pharmacokinetic profile. In comparison, [177 Lu]Lu-NeoB displayed substantially lower tumor uptake (9%IA/g at 1 h p.i. and 8%IA/g at 24 h p.i.), but with a longer biological half-life (t 1/2 = 50 h), potentially advantageous for delayed imaging or therapeutic applications. (24) However, its pancreatic retention at 24 h p.i. (4%IA/g) suggests slower clearance from nontarget tissues, which may raise concerns regarding off-target exposure. (24) The AMTG analogs labeled with lutetium-177 and terbium-161, particularly [161 Tb]Tb-AMTG, achieved moderate tumor uptake (15%IA/g at 1 h p.i., 10%IA/g at 24 h p.i.), along with manageable pancreatic retention and very low hepatic accumulation. (18) These features, combined with the favorable emission characteristics of terbium-161, make it a promising candidate for both imaging and therapy. [177 Lu]Lu-RM2 showed relatively lower tumor uptake (12%IA/g at 1 h p.i. and 8%IA/g at 24 h p.i.), but with excellent clearance from the pancreas and liver. (18) The heterodimer 86 Y/90 Y-[RGD-Glu-DO3A]-6-Ahx-RM2 demonstrated the lowest tumor uptake (9%IA/g at 1 h p.i. and 5%IA/g at 24 h p.i.), with minimal accumulation in nontarget tissues. (28) The tumor uptake for RM26 analogs; [111 In]In-X-PEG 2-RM2 (X = NOTA, NOTAGA, DOTA, DOTAGA) was around 3%IA/g at 4 h p.i. with a further decrease to approximately 1.5%IA/g at 24 h p.i. (29) While this may limit its therapeutic impact, it could be advantageous for diagnostic purposes due to its favorable biodistribution profile. In summary, [177 Lu]Lu-LF1 emerges as one of the most promising radioligands, offering superior tumor targeting and favorable clearance kinetics. Nonetheless, agents such as [161 Tb]Tb-AMTG and [177 Lu]Lu-RM2 also present balanced biodistribution profiles that may be better suited for specific therapeutic or safety-related applications.
At 5 and 15 min after radioligand injection, 3.2% and 2.5% of the activity, respectively, were bound to murine plasma proteins. These data very well support the biodistribution data which showed very low activity concentration in blood pool. The low degree of protein binding may be attributed to the hydrophilic nature of [177 Lu]Lu-LF1, as indicated by its LogD value of 2.9 ± 0.04, determined in our previous studies. (20) HPLC analysis revealed proteolytic degradation, with three main metabolites increasing over time (Figure 3). The intact radioligand decreased from 67.6 ± 2.2% at 5 min to 38.8 ± 0.9% at 15 min p.i..
Figure 3. (A) Chemical structure of [177 Lu]Lu-LF1 and (B) representative HPLC chromatograms of blood samples collected at 5 min (blue) and 15 min (red) p.i., revealing three metabolites (M1, M2, and M3). Additionally, the reference chromatogram of [177 Lu]Lu-LF1 prior to its injection in PC-3 tumor-bearing mice is shown in black.
The GRPR-based antagonists are prone to proteolytic degradation, particularly by neutral endopeptidase (NEP), which can impair their in vivo targeting and theranostic efficacy. (15−17,19) To improve their stability and performance in diagnostic imaging and radionuclide therapy, two main strategies are employed: structural modi
