Radiopharmaceuticals for somatostatin receptor imaging

The aim of this review is to summarize the developments and briefly characterize the somatostatin analogs which are currently used for somatostatin receptor imaging in clinical routine or in early phase clinical trials. Somatostatin (sst) receptor targeting with radiolabeled peptides has become an integral part in nuclear oncology during the last 20 years. This integration process has been initiated in Europe with the introduction to the market of 111In-DTPA-DPhe1-octreotide [111In-pentetreotide]. Introducing 99mTc in somatostatin receptor targeting radiopeptides resulted in much better image quality, higher sensitivity of tumor detection and lower mean effective dose for the examined patient. The next generation are 68Ga labeled somatostatin analogs. Due to the spatial resolution of PET technique and increasing number of PET scanners, the PET or PET/CT technique became very important in somatostatin receptor imaging. Until up to a couple of years ago the analogs of somatostatin were constructed aiming at their agonistic behavior, expecting that their internalization with the receptor acti­vated by the radiolabeled ligand and its retention within the tumor cell are crucial for efficient imaging and therapy. Recently it has been shown that the antagonists recognize more binding sites at the tumor cell membrane and hence offer an improved diagnostic efficacy, especially when the density of sst receptors is low. This approach may in future improve diagnostic value of somatostatin receptor imaging techniques. The developments in tracer design are followed by the improvements in imaging techniques. The new SPECT scanners offer resolution close to that of PET, which might open a new era for 99mTc and other SPECT radiotracers

68Ga-PET: a powerful generator-based alternative to cyclotron-based PET radiopharmaceuticals

PET (positron emission tomography) is a powerful diagnostic and imaging technique which requires short-lived positron emitting isotopes. The most commonly used are accelerator-produced 11C and 18F. An alternative is the use of metallic positron emitters. Among them 68Ga deserves special attention because of its availability from long-lived 68Ge/68Ga generator systems which render 68Ga radiopharmacy independent of an onsite cyclotron. The coordination chemistry of Ga3+ is dominated by its hard acid character. A variety of mono- and bifunctional chelators have been developed which allow the formation of stable 68 Ga3+ complexes and convenient coupling to biomolecules. 68Ga coupling to small biomolecules is potentially an alternative to 18F- and 11C-based radiopharmacy. In particular, peptides targeting G-protein coupled receptors overexpressed on human tumour cells have shown preclinically and clinically high and specific tumour uptake. Kit-formulated precursors along with the generator may be provided, similar to the 99Mo/99mTc-based radiopharmacy, still the mainstay of nuclear medicine

Comprehensive evaluation of a somatostatin-based radiolabelled antagonist for diagnostic imaging and radionuclide therapy

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 tumour

Development of new folate-based PET radiotracers: preclinical evaluation of 68Ga-DOTA-folate conjugates

Purpose: A number of 111In- and 99mTc-folate-based tracers have been evaluated as diagnostic agents for imaging folate receptor (FR)-positive tumours. A 68Ga-folate-based radiopharmaceutical would be of great interest, combining the advantages of PET technology and the availability of 68Ga from a generator. The aim of the study was to develop a new 68Ga-folate-based PET radiotracer. Methods: 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 67/68Ga. 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 111In-DTPA-folate (111In-P3139). Results: The Kd values of 67/68Ga-P3026 (4.65 ± 0.82 nM) and 67/68Ga-P1254 (4.27 ± 0.42 nM) showed high affinity for the FR. The internalization rate followed the order 67/68Ga-P3026 > 67/68Ga-P1254 > 111In-P3139, while almost double cellular retention was found for 67/68Ga-P3026 and 67/68Ga-P1254, compared to 111In-P3139. The biodistribution data of 67/68Ga-DOTA-folates showed high and receptor-mediated uptake on the FR-positive tumours and kidneys, with no significant differences compared to 111In-P3139. PET/CT images, performed with 68Ga-P3026, showed high uptake in the kidneys and clear visualization of the FR-positive tumours. Conclusion: The DOTA-folate conjugates can be efficiently labelled with 68Ga in labelling yields and specific activities which allow clinical application. The characteristics of the 67/68Ga-DOTA-folates are comparable to 111In-DTPA-folate, which has already been used in clinical trials, showing that the new conjugates are promising candidates as PET radiotracers for FR-positive tumour

DOTA-PESIN, a DOTA-conjugated bombesin derivative designed for the imaging and targeted radionuclide treatment of bombesin receptor-positive tumours

Purpose: We aimed at designing and developing a novel bombesin analogue, DOTA-PEG4-BN(7-14) (DOTA-PESIN), with the goal of labelling it with 67/68Ga and 177Lu for diagnosis and radionuclide therapy of prostate and other human cancers overexpressing bombesin receptors. Methods: The 8-amino acid peptide bombesin (7-14) was coupled to the macrocyclic chelator DOTA via the spacer 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG4). The conjugate was complexed with Ga(III) and Lu(III) salts. The GRP receptor affinity and the bombesin receptor subtype profile were determined in human tumour specimens expressing the three bombesin receptor subtypes. Internalisation and efflux studies were performed with the human GRP receptor cell line PC-3. Xenografted nude mice were used for biodistribution. Results: [GaIII/LuIII]-DOTA-PESIN showed good affinity to GRP and neuromedin B receptors but no affinity to BB3. [67Ga/177Lu]-DOTA-PESIN internalised rapidly into PC-3 cells whereas the efflux from PC-3 cells was relatively slow. In vivo experiments showed a high and specific tumour uptake and good retention of [67Ga/177Lu]-DOTA-PESIN. [67Ga/177Lu]-DOTA-PESIN highly accumulated in GRP receptor-expressing mouse pancreas. The uptake specificity was demonstrated by blocking tumour uptake and pancreas uptake. Fast clearance was found from blood and all non-target organs except the kidneys. High tumour-to-normal tissue ratios were achieved, which increased with time. PET imaging with [68Ga]-DOTA-PESIN was successful in visualising the tumour at 1h post injection. Planar scintigraphic imaging showed that the 177Lu-labelled peptide remained in the tumour even 3days post injection. Conclusion: The newly designed ligands have high potential with regard to PET and SPECT imaging with 68/67Ga and targeted radionuclide therapy with 177L

New AMD3100 derivatives for CXCR4 chemokine receptor targeted molecular imaging studies: synthesis, anti-HIV-1 evaluation and binding affinities

CXCR4 is a target of growing interest for the development of new therapeutic drugs and imaging agents as its role in multiple disease states has been demonstrated. AMD3100, a CXCR4 chemokine receptor antagonist that is in current clinical use as a haematopoietic stem cell mobilising drug, has been widely studied for its anti-HIV properties, potential to inhibit metastatic spread of certain cancers and, more recently, its ability to chelate radiometals for nuclear imaging. In this study, AMD3100 is functionalised on the phenyl moiety to investigate the influence of the structural modification on the anti-HIV-1 properties and receptor affinity in competition with anti-CXCR4 monoclonal antibodies and the natural ligand for CXCR4, CXCL12. The effect of complexation of nickel(II) in the cyclam cavities has been investigated. Two amino derivatives were obtained and are suitable intermediates for conjugation reactions to obtain CXCR4 molecular imaging agents. A fluorescent probe (BODIPY) and a precursor for 18F (positron emitting isotope) radiolabelling were conjugated to validate this route to new CXCR4 imaging agents

In vitro characterization of 177Lu-radiolabelled chimeric anti-CD20 monoclonal antibody and a preliminary dosimetry study

Purpose: 131I- and 90Y-labelled anti-CD20 antibodies have been shown to be effective in the treatment of low-grade, B-cell non-Hodgkin's lymphoma (NHL). However, the most appropriate radionuclide in terms of high efficiency and low toxicity has not yet been established. In this study we evaluated an immunoconjugate formed by the anti-CD20 antibody rituximab and the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). DOTA-rituximab was prepared as a kit formulation and can be labelled in a short time (<20min) with either 177Lu or 90Y. Materials and methods: Immunoconjugates with different numbers of DOTA molecules per rituximab were prepared using p-SCN-Bz-DOTA. In vitro immunoreactivity and stability were tested and preliminary dosimetric results were acquired in two patients. Results: The immunological binding properties of DOTA-rituximab to the CD20 antigen were found to be retained after conjugation with up to four chelators. The labelled product was stable against a 105 times excess of diethylenetriaminepentaacetic acid (DTPA, 37°C, 7days). Two patients with relapsed NHL were treated with 740MBq/m2 body surface 177Lu-DOTA-rituximab. Scintigraphic images showed specific uptake at tumour sites and acceptable dosimetric results. The mean whole-body dose was found to be 314mGy. The administration of 177Lu-DOTA-rituximab was tolerated well. Conclusion: Our results show that DOTA-rituximab (4:1) can be labelled with 177Lu with sufficient stability while the immunoconjugate retains its immunoreactivity. 177Lu-DOTA-rituximab is an interesting, well-tolerated radiolabelled antibody with clinical activity in a low dose range, and provides an approach to the efficient treatment with few side effects for patients with relapsed NH

Development of a potent DOTA-conjugated bombesin antagonist for targeting GRPr-positive tumours

Purpose: 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-NH2 via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as 111In and 68Ga. Methods: RM2 was synthesized on a solid support and evaluated in vitro in PC-3 cells. IC50 and Kd values were determined. The antagonist potency was evaluated by immunofluorescence-based internalization and Ca2+ mobilization assays. Biodistribution studies were performed in PC-3 and LNCaP tumour-bearing mice with 111In-RM2 and 68Ga-RM2, respectively. PET/CT studies were performed on PC-3 and LNCaP tumour-bearing nude mice with 68Ga-RM2. Results: RM2 and 111In-RM2 are high-affinity and selective ligands for the GRP receptor (7.7±3.3nmol/l for RM2; 9.3±3.3nmol/l for natIn-RM2). The potent antagonistic properties were confirmed by an immunofluorescence-based internalization and Ca2+ mobilization assays. 68Ga- and 111In-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 1h; 11.7±2.4%IA/g at 4h), 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 1h to 1.5±0.5%IA/g at 4h. Conclusion: RM2 was shown to be a potent GRPr antagonist. Pharmacokinetics and imaging studies indicate that 111In-RM2 and 68Ga-RM2 are ideal candidates for clinical SPECT and PET studie

Macrocyclic chelator-coupled gastrin-based radiopharmaceuticals for targeting of gastrin receptor-expressing tumours

Purpose: Diethylenetriamine-pentaacetic acid (DTPA)-coupled minigastrins are unsuitable for therapeutic application with the available β-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 111In-labelled derivatives. Methods: Gastrin derivatives with decreasing numbers of glutamic acids were synthesised using 111In as surrogate for therapeutic radiometals for in vitro and in vivo studies. Gastrin receptor affinities of the natIn-metallated compounds were determined by receptor autoradiography using 125I-CCK as radioligand. Internalisation was evaluated in AR4-2J cells. Enzymatic stability was determined by incubating the 111In-labelled peptides in human serum. Biodistribution was performed in AR4-2J-bearing Lewis rats. Results: IC50 values of the natIn-metallated gastrin derivatives vary between 1.2 and 4.8nmol/L for all methionine-containing derivatives. Replacement of methionine by norleucine, isoleucine, methionine-sulfoxide and methionine-sulfone resulted in significant decrease of receptor affinity (IC50 between 9.9 and 1,195nmol/L). All cholecystokinin receptor affinities were >100nmol/L. All 111In-labelled radiopeptides showed receptor-specific internalisation. Serum mean-life times varied between 2.0 and 72.6h, positively correlating with the number of Glu residues. All 111In-labelled macrocyclic chelator conjugates showed higher tumour-to-kidney ratios after 24h (0.37-0.99) compared to 111In-DTPA-minigastrin 0 (0.05). Tumour wash out between 4 and 24h 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 purpose