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Radiolabelled somatostatin-based antagonists show a higher uptake in tumour-bearing mouse models than agonists of similar or even distinctly higher receptor affinity. Very similar results were obtained with another family of G protein-coupled receptor ligands, the bombesin family. We describe a new conjugate, RM2, with the chelator DOTA coupled to D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as (111)In and (68)Ga.RM2 was synthesized on a solid support and evaluated in vitro in PC-3 cells. IC(50) and K(d) values were determined. The antagonist potency was evaluated by immunofluorescence-based internalization and Ca(2+) mobilization assays. Biodistribution studies were performed in PC-3 and LNCaP tumour-bearing mice with (111)In-RM2 and (68)Ga-RM2, respectively. PET/CT studies were performed on PC-3 and LNCaP tumour-bearing nude mice with (68)Ga-RM2.RM2 and (111)In-RM2 are high-affinity and selective ligands for the GRP receptor (7.7 ± 3.3 nmol/l for RM2; 9.3 ± 3.3 nmol/l for (nat)In-RM2). The potent antagonistic properties were confirmed by an immunofluorescence-based internalization and Ca(2+) mobilization assays. (68)Ga- and (111)In-RM2 showed high and specific uptake in both the tumour and the pancreas. Uptake in the tumour remained high (15.2 ± 4.8%IA/g at 1 h; 11.7 ± 2.4%IA/g at 4 h), whereas a relatively fast washout from the pancreas and the other abdominal organs was observed. Uptake in the pancreas decreased rapidly from 22.6 ± 4.7%IA/g at 1 h to 1.5 ± 0.5%IA/g at 4 h.RM2 was shown to be a potent GRPr antagonist. Pharmacokinetics and imaging studies indicate that (111)In-RM2 and (68)Ga-RM2 are ideal candidates for clinical SPECT and PET studies.

PURPOSE: Diethylenetriamine-pentaacetic acid (DTPA)-coupled minigastrins are unsuitable for therapeutic application with the available beta-emitting radiometals due to low complex stability. Low tumour-to-kidney ratio of the known radiopharmaceuticals is further limiting their potency. We used macrocyclic chelators for coupling to increase complex stability, modified the peptide sequence to enhance radiolytic stability and studied tumour-to-kidney ratio and metabolic stability using (111)In-labelled derivatives. METHODS: Gastrin derivatives with decreasing numbers of glutamic acids were synthesised using (111)In as surrogate for therapeutic radiometals for in vitro and in vivo studies. Gastrin receptor affinities of the (nat)In-metallated compounds were determined by receptor autoradiography using (125)I-CCK as radioligand. Internalisation was evaluated in AR4-2J cells. Enzymatic stability was determined by incubating the (111)In-labelled peptides in human serum. Biodistribution was performed in AR4-2J-bearing Lewis rats. RESULTS: IC(50) values of the (nat)In-metallated gastrin derivatives vary between 1.2 and 4.8 nmol/L for all methionine-containing derivatives. Replacement of methionine by norleucine, isoleucine, methionine-sulfoxide and methionine-sulfone resulted in significant decrease of receptor affinity (IC(50) between 9.9 and 1,195 nmol/L). All cholecystokinin receptor affinities were >100 nmol/L. All (111)In-labelled radiopeptides showed receptor-specific internalisation. Serum mean-life times varied between 2.0 and 72.6 h, positively correlating with the number of Glu residues. All (111)In-labelled macrocyclic chelator conjugates showed higher tumour-to-kidney ratios after 24 h (0.37-0.99) compared to (111)In-DTPA-minigastrin 0 (0.05). Tumour wash out between 4 and 24 h was low. Imaging studies confirmed receptor-specific blocking of the tumour uptake. CONCLUSIONS: Reducing the number of glutamates increased tumour-to-kidney ratio but resulted in lower metabolic stability. The properties of the macrocyclic chelator-bearing derivatives make them potentially suitable for clinical purposes.

Purpose: Targeting of tumours positive for somatostatin receptors (sst) with radiolabelled peptides is of interest for tumour localization, staging, therapy follow-up and targeted radionuclide therapy. The peptides used clinically are exclusively agonists, but recently we have shown that the radiolabelled somatostatin-based antagonist 111In-DOTA-sst2-ANT may be preferable to agonists. However, a comprehensive study of this radiolabelled antagonist to determine its significance was lacking. The present report describes the evaluation of this novel antagonist labelled with 111In and 177Lu in three different tumour models. Methods: Radiopeptide binding, internalization and dissociation studies were performed using cells expressing HEK293-rsst2. Biodistribution studies were performed in HEK293-rsst2, HEK293-hsst2 and HEK293-rsst3 xenografted mice. Results: Saturation binding analysis confirmed earlier IC50 data for 111/natIn-DOTA-sst2-ANT and showed similar affinity of 177/natLu-DOTA-sst2-ANT for the sst2. Only low internalization was found in cell culture (6.68 ± 0.06% at 4h), which was not unexpected for an antagonist, and this could be further reduced by the addition of sucrose. No internalization was observed in HEK293 cells not expressing sst. Both results indicate that the internalization was specific. 111In-DOTA-sst2-ANT and 177Lu-DOTA-sst2-ANT were shown to target tumour xenografts expressing the rat and the human sst2 receptor with no differences in their uptake or pharmacokinetics. The uptake in rsst2 and hsst2 was high (about 30 %IA/g 4h after injection) and surprisingly long-lasting (about 20-23 %IA/g 24h after injection). Kidney uptake was blocked by approximately 50% by lysine or Gelofusine. Conclusion: These results indicate that radiolabelled somatostatin-based antagonists may be superior to corresponding agonists. The long tumour retention time of 177Lu-DOTA-sst2-ANT indicates that this new class of compounds is of relevance not only in diagnostic imaging but also in targeted radionuclide therapy of sst-positive tumours

A number of (111)In- and (99m)Tc-folate-based tracers have been evaluated as diagnostic agents for imaging folate receptor (FR)-positive tumours. A (68)Ga-folate-based radiopharmaceutical would be of great interest, combining the advantages of PET technology and the availability of (68)Ga from a generator. The aim of the study was to develop a new (68)Ga-folate-based PET radiotracer.Two new DOTA-folate conjugates, named P3026 and P1254, were synthesized using the 1,2-diaminoethane and 3-{2-[2-(3-amino-propoxy)-ethoxy]-ethoxy}-propylamine as a spacer, respectively. Both conjugates were labelled with (67/68)Ga. Binding affinity, internalization and externalization studies were performed using the FR-positive KB cell line. Biodistribution and PET/CT imaging studies were performed in nude mice, on a folate-deficient diet, bearing KB and HT1080 (FR-negative) tumours, concurrently. The new radiotracers were evaluated comparatively to the reference molecule (111)In-DTPA-folate ((111)In-P3139).The K(d) values of (67/68)Ga-P3026 (4.65 ± 0.82 nM) and (67/68)Ga-P1254 (4.27 ± 0.42 nM) showed high affinity for the FR. The internalization rate followed the order (67/68)Ga-P3026 (67/68)Ga-P1254 (111)In-P3139, while almost double cellular retention was found for (67/68)Ga-P3026 and (67/68)Ga-P1254, compared to (111)In-P3139. The biodistribution data of (67/68)Ga-DOTA-folates showed high and receptor-mediated uptake on the FR-positive tumours and kidneys, with no significant differences compared to (111)In-P3139. PET/CT images, performed with (68)Ga-P3026, showed high uptake in the kidneys and clear visualization of the FR-positive tumours.The DOTA-folate conjugates can be efficiently labelled with (68)Ga in labelling yields and specific activities which allow clinical application. The characteristics of the (67/68)Ga-DOTA-folates are comparable to (111)In-DTPA-folate, which has already been used in clinical trials, showing that the new conjugates are promising candidates as PET radiotracers for FR-positive tumours.