The gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of primary prostate cancer lesions, with persistent expression in lymph nodes and bone metastases, making it a legitimate molecular target for diagnostic imaging and staging. This study presents the synthesis and preclinical evaluation of [18 F]MeTz-PEG 2-RM26, a GRPR antagonist which utilises the Inverse Electron Demand Diels-Alder (IEDDA) reaction for 18 F-labelling. This click-chemistry approach allows for site-specific incorporation of fluorine-18 under mild conditions, preserving the peptide’s structural integrity and biological activity. Receptor specificity and affinity of [18 F]MeTz-PEG 2-RM26 were evaluated in vitro using GRPR-expressing PC-3 cells. Furthermore, the biodistribution profile of [18 F]MeTz-PEG 2-RM26 was assessed in NMRI mice and its tumour-targeting capability was investigated in mice bearing PC-3 xenografts.
The labelling of TCO-PEG 2-RM26 precursor involved three steps: (1) synthesis of an 18 F-labelled activated ester on a quaternary methyl ammonium (QMA) cartridge, (2) conjugation of the labelled ester to a tetrazine amine, and (3) attachment to TCO-PEG 2-RM26 via an IEDDA click reaction. This production method of [18 F]MeTz-PEG 2-RM26 afforded a high apparent molar activity of 3.5–4.3 GBq/µmol and radiochemical purity exceeding 98%, with 43–70 MBq activity incorporation, while the entire synthesis was completed within 75 min. Both in vitro and in vivo studies confirmed the specific binding of [18 F]MeTz-PEG 2-RM26 to GRPR, with a significant reduction in activity uptake observed upon receptor saturation. The radioligand exhibited rapid blood clearance and minimal bone uptake, confirming the stability of the fluorine-carbon bond. However, high hepatic uptake (12–13% IA/g at 1 h post-injection) indicated predominant hepatobiliary excretion. Receptor-mediated uptake was observed in the tumours and pancreatic tissue, although the overall activity uptake in tumours was low, likely due to the rapid hepatobiliary clearance of [18 F]MeTz-PEG 2-RM26.
These findings demonstrate the effectiveness of the IEDDA click reaction for fluorine-18 labelling of GRPR-targeting PET tracers. Future studies should focus on increasing the hydrophilicity of the imaging probe to improve the targeting properties and biodistribution profile of the radioligand.
Positron Emission Tomography (PET) is a powerful medical imaging modality that visualises the distribution of tracers labelled with positron-emitting radioisotopes. Widely employed in both biomedical research and clinical practice, PET is used to study biological processes in vivo and diagnose conditions such as cancer, neurological disorders, and cardiovascular diseases. The most commonly used PET tracer is fluorodeoxyglucose ([18 F]FDG), a glucose analogue labelled with the positron-emitting radioisotope fluorine-18, which has a physical half-life of 110 min. Beyond small molecules, PET imaging also extends to larger biomolecules like peptides and proteins. These biomolecules often exhibit high affinity and selectivity for specific receptors or enzymes along with rapid blood clearance, leading to high-contrast images shortly after administration. In addition to fluorine-18, another common PET radioisotope is gallium-68 with a physical half-life of 68 min. Gallium-68 labelled peptides have proven valuable for precise tumour imaging and receptor characterisation. However, their utility is somewhat constrained by the shorter half-life and the limitations of generator-based production. Therefore, fluorine-18 labelling could be preferred due to its high-yielding radionuclide production and lower positron energy. The half-life of fluorine-18 is also well-aligned with the biological half-life and pharmacokinetic properties of peptides (Zhang et al. 2004). However, the incorporation of fluorine-18 into targeting ligands and biomolecules can pose significant challenges. Traditional radiolabelling methods, such as nucleophilic substitution with [18 F]fluoride, often require harsh reaction conditions that are unsuitable for peptides and proteins (Jacobson et al. 2015).
To address these challenges, alternative approaches have been developed, including the use of prosthetic groups like [18 F]fluorobenzaldehyde and N-succinimidyl-4-[18 F]fluorobenzoate ([18 F]SFB), which provide more controlled labelling processes and have been extensively used to form stable oxime and amide bonds with peptides (Li et al. 2021). Chelation strategies have recently gained traction for fluorine-18 labelling due to the development of chelates allowing aluminium [18 F]fluoride to be incorporated at low temperatures (Cleeren et al. 2018; Mcbride et al. 2009; Wegrzyniak et al. 2024). Despite this progress, concerns about complex stability remain in certain applications (Archibald and Allott 2021). In addition to these methods, click chemistry has emerged as an increasingly important strategy in PET radiochemistry and fluorine-18 labelling. The copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) has been successfully employed for the high-yield labelling of peptides (Gill and Marik 2011; Li et al. 2017). Another promising click chemistry approach is the Inverse Electron-Demand Diels-Alder (IEDDA) reaction, which offers distinct advantages over copper-catalysed reactions for bioconjugation and labelling of biologically active peptides. The IEDDA click reaction can, for instance, take place between an 18 F-labelled tetrazine ([18 F]Tz) and a trans-cyclooctene (TCO) functionalised moiety, providing a versatile and efficient method for fluorine-18 labelling. This bioorthogonal chemistry enables the site-specific incorporation of fluorine-18 into various molecules under mild conditions, including physiological media, without the need for a metal-catalyst (Cheung et al. 2023; Schlein et al. 2024; Syvänen et al. 2020; Wegrzyniak et al. 2023). This approach holds great promise for advancing the development of next-generation PET imaging agents. The diagnostic accuracy could be improved due to combination of the favourable imaging properties of fluorine-18 and the specific biological targeting capabilities of proteins and peptides. Various techniques and methods have been applied to label gastrin releasing peptide receptor (GRPR)-targeting bombesin (BBN) analogues with fluorine-18 (Baratto et al. 2017). Besides conventional nucleophilic substitution with [18 F]fluoride, which is feasible for compounds with suitable leaving groups in activated positions, such as 4-(N, N,N-trimethylammonium)benzoate triflate (AlJammaz et al. 2014) or aromatic rings activated with electron-withdrawing groups (Becaud et al. 2009; Höner et al. 2011), alternative strategies include isotopic exchange reactions. This can be performed on di-tert-butylfluorosilanes or ammoniomethyl-trifluoroborates, allowing labelling under milder conditions (Dialer et al. 2013). However, labelling of BBN analogues using this method has only been achieved with di-tert-butylfluorosilane (Dialer et al. 2013; Pourghiasian et al. 2015).
