A variety of radiolabelled minigastrin analogues targeting the cholecystokinin 2 (CCK2) receptor were developed and compared in a concerted preclinical testing to select the most promising radiotracer for diagnosis and treatment of medullary thyroid carcinoma (MTC). DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04) after labelling with (111)In displayed excellent characteristics, such as high stability, receptor affinity, specific and persistent tumour uptake and low kidney retention in animal models. Therefore, it was selected for further clinical evaluation within the ERA-NET project GRAN-T-MTC. Here we report on the development of a pharmaceutical freeze-dried formulation of the precursor CP04 for a first multi-centre clinical trial with (111)In-CP04 in MTC patients. The kit formulation was optimised by adjustment of buffer, additives and radiolabelling conditions. Three clinical grade batches of a final kit formulation with two different amounts of peptide (10 or 50 μg) were prepared and radiolabelled with (111)In. Quality control and stability assays of both the kits and the resulting radiolabelled compound were performed by HPLC analysis. Use of ascorbic acid buffer (pH4.5) allowed freeze-drying of the kit formulation with satisfactory pellet-formation. Addition of methionine and gentisic acid as well as careful selection of radiolabelling temperature was required to avoid extensive oxidation of the Met(11)-residue. Trace metal contamination, in particular Zn, was found to be a major challenge during the pharmaceutical filling process in particular for the 10 μg formulation. The final formulations contained 10 or 50 μg CP04, 25mg ascorbic acid, 0.5mg gentisic acid and 5mg L-methionine. The radiolabelling performed by incubation of 200-250 MBq (111)InCl3 at 90 °C for 15 min resulted in reproducible radiochemical purity (RCP) >94%. Kit-stability was proven for >6 months at +5 °C and at +25 °C. The radiolabelled product was stable for >4h at +25 °C. A kit formulation to prepare (111)In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product.

The α-fetoprotein derived growth inhibitory peptide (GIP) is a 34-amino acid peptide composed of three biologically active subfragments. GIP-34 and its three constituent segments have been synthesized, purified, and studied for biological activity. The GIP-34 and GIP-8 have been characterized as anticancer therapeutic peptides. An multicenter study was initiated to elucidate the means by which these peptide drugs could be targeted to tumor cells. The study first established which cancer types were specifically targeted by the GIP peptides in both in vitro and in vivo investigations. It was next demonstrated that radiolabeled peptide (¹²⁵I GIP-34) is specifically localized to rodent breast tumors at 24 h post-injection. The radionuclide studies also provided evidence for a proposed cell surface receptor; this was confirmed in a further study using fluorescent-labeled GIP-nanobeads which localized at the plasma membrane of MCF-7 breast cancer cells. Finally, it was readily demonstrated that GIP conjugated to either fluorescein or doxorubicin (DOX) underwent tumor cell uptake; subsequently, DOX–GIP conjugates induced cytotoxic cell destruction indicating the utility of GIP segments as cancer therapeutic agents. Following a discussion of the preceding results, a candidate cell surface receptor family was proposed which correlated with previous published reports for a putative AFP/GIP receptor.

Despite significant advances in nuclear medicine for diagnosing and treating prostate cancer (PCa), research into new ligands with increasingly better biological properties is still ongoing. Prostate-specific membrane antigen (PSMA) ligands show great potential as radioisotope carriers for the diagnosis and therapy of patients with metastatic PCa. PSMA is expressed in most types of prostate cancer, and its expression is increased in poorly differentiated, metastatic, and hormone-refractory cancers; therefore, it may be a valuable target for the development of radiopharmaceuticals and radioligands, such as urea PSMA inhibitors, for the precise diagnosis, staging, and treatment of prostate cancer. Four developed PSMA-HYNIC inhibitors for technetium-99m labeling and subsequent diagnosis were subjected to preclinical in vitro and in vivo studies to evaluate and compare their diagnostic properties. Among the studied compounds, the PSMA-T4 (Glu-CO-Lys-L-Trp-4-Amc-HYNIC) inhibitor showed the best biological properties for the diagnosis of PCa metastases. [^99mTc]Tc-PSMA-T4 also showed effectiveness in single-photon emission computed tomography (SPECT) studies in humans, and soon, its usefulness will be extensively evaluated in phase 2/3 clinical trials.

A new PSMA ligand (PSMA-D4) containing the Glu-CO-Lys pharmacophore connected with a new linker system (L-Trp-4-Amc) and chelator DOTA was developed for radiolabeling with therapeutic radionuclides. Herein we describe the synthesis, radiolabeling, and preliminary biological evaluation of the novel PSMA-D4 ligand. Synthesized PSMA-D4 was characterized using TOF-ESI-MS, NMR, and HPLC methods. The novel compound was subject to molecular modeling with GCP-II to compare its binding mode to analogous reference compounds. The radiolabeling efficiency of PSMA-D4 with ¹⁷⁷Lu, ⁹⁰Y, ⁴⁷Sc, and ²²⁵Ac was chromatographically tested. In vitro studies were carried out in PSMA-positive LNCaP tumor cells membranes. The ex vivo tissue distribution profile of the radioligands and Cerenkov luminescence imaging (CLI) was studied in LNCaP tumor-bearing mice. PSMA-D4 was synthesized in 24% yield and purity >97%. The radio complexes were obtained with high yields (>97%) and molar activity ranging from 0.11 to 17.2 GBq mcmol⁻¹, depending on the radionuclide. In vitro assays confirmed high specific binding and affinity for all radiocomplexes. Biodistribution and imaging studies revealed high accumulation in LNCaP tumor xenografts and rapid clearance of radiocomplexes from blood and non-target tissues. These render PSMA-D4 a promising ligand for targeted therapy of prostate cancer (PCa) metastases.