Long-Term Tumor Control Following Targeted Alpha Therapy (TAT) of Low-Grade Gliomas (LGGs): A New Treatment Paradigm?

The median survival time has been reported to vary between 5 and 8 years in low-grade (WHO grade 2) astrocytoma, and between 10 and 15 years for grade 2 oligodendroglioma. Targeted alpha therapy (TAT), using the modified peptide vector [213Bi]Bi/[225Ac]Ac-DOTA-substance P, has been developed to treat glioblastoma (GBM), a prevalent malignant brain tumor. In order to assess the risk of late neurotoxicity, assuming that reduced tumor cell proliferation and invasion should directly translate into good responses in low-grade gliomas (LGGs), a limited number of patients with diffuse invasive astrocytoma (n = 8) and oligodendroglioma (n = 3) were offered TAT. In two oligodendroglioma patients, TAT was applied as a second-line treatment for tumor progression, 10 years after targeted beta therapy using [90Y]Y-DOTA-substance P. The radiopharmaceutical was locally injected directly into the tumor via a stereotactic insertion of a capsule-catheter system. The activity used for radiolabeling was 2-2.5 GBq of Bismuth-213 and 17 to 35 MBq of Actinium-225, mostly applied in a single fraction. The recurrence-free survival times were in the range of 2 to 16 years (median 11 years) in low-grade astrocytoma (n = 8), in which TAT was administered following a biopsy or tumor debulking. Regarding oligodendroglioma, the recurrence-free survival time was 24 years in the first case treated, and 4 and 5 years in the two second-line cases. In conclusion, TAT leads to long-term tumor control in the majority of patients with LGG, and recurrence has so far not manifested in patients with low-grade (grade 2) astrocytomas who received TAT as a first-line therapy. We conclude that targeted alpha therapy has the potential to become a new treatment paradigm in LGG.

Radiochemistry and Preclinical PET Imaging of 68Ga-Desferrioxamine Radiotracers Targeting Prostate-Specific Membrane Antigen.

Radiotracers incorporating the urea-based Glu-NH-C(O)-NH-Lys group have gained prominence due to their role in targeting prostate-specific membrane antigen (PSMA)-a clinical biomarker of prostate cancer. Here, the synthesis, radiolabeling, and in vitro and in vivo characterization of two 68Ga-radiolabeled Glu-NH-C(O)-NH-Lys radiotracers conjugated to the desferrioxamine B (DFO) chelate were evaluated. Two linker groups based on amide bond and thiourea coupling chemistries were employed to develop 68Ga-DFO-Nsucc-PSMA (68Ga-4) and 68Ga-DFO- pNCS-Bn-PSMA (68Ga-7), respectively. Radiosynthesis proceeded quantitatively at room temperature with high radiochemical yields, chemical/radiochemical purities, and specific activities. Pharmacokinetic profiles of 68Ga-4 and 68Ga-7 were assessed using positron-emission tomography (PET) in mice bearing subcutaneous LNCaP tumors. Data were compared to the current clinical benchmark radiotracer 68Ga-HBED-CC-PSMA (68Ga-1) (HBED = N,N'-Bis(2-hydroxy-5-(ethylene-beta-carboxy)benzyl)ethylenediamine N,N'-diacetic acid). Results indicated that the target binding affinity, protein association, blood pool and background organ clearance properties, and uptake in PSMA-positive lesions are strongly dependent on the nature of the chelate, the linker, and the spacer groups. Protein dissociation constants ( Kd values) were found to be predictive of pharmacokinetics in vivo. Compared to 68Ga-1, 68Ga-4 and 68Ga-7 resulted in decreased tumor uptake but enhanced blood pool clearance and reduced residence time in the kidney. The study highlights the importance of maximizing protein binding affinity during radiotracer optimization.

The prognostic and predictive value of sstr2-immunohistochemistry and sstr2-targeted imaging in neuroendocrine tumors.

PURPOSE: Our aim was to assess the prognostic and predictive value of somatostatin receptor 2 (sstr2) in neuroendocrine tumors (NETs). METHODS: We established a tissue microarray and imaging database from NET patients that received sstr2-targeted radiopeptide therapy with yttrium-90-DOTATOC, lutetium-177-DOTATOC or alternative treatment. We used univariate and multivariate analyses to identify prognostic and predictive markers for overall survival, including sstr2-imaging and sstr2-immunohistochemistry. RESULTS: We included a total of 279 patients. In these patients, sstr2-immunohistochemistry was an independent prognostic marker for overall survival (HR: 0.82, 95 % CI: 0.67 - 0.99, n = 279, p = 0.037). In DOTATOC patients, sstr2-expression on immunohistochemistry correlated with tumor uptake on sstr2-imaging (n = 170, p < 0.001); however, sstr2-imaging showed a higher prognostic accuracy (positive predictive value: +27 %, 95 % CI: 3 - 56 %, p = 0.025). Sstr2-expression did not predict a benefit of DOTATOC over alternative treatment (p = 0.93). CONCLUSIONS: Our results suggest sstr2 as an independent prognostic marker in NETs. Sstr2-immunohistochemistry correlates with sstr2-imaging; however, sstr2-imaging is more accurate for determining the individual prognosis.

AMD3100: A Versatile Platform for CXCR4 Targeting (68)Ga-Based Radiopharmaceuticals.

CXCR4 is a G protein-coupled receptor (GPCR), which is overexpressed in numerous diseases, particularly in multiple cancers. Therefore, this receptor represents a valuable target for imaging and therapeutic purposes. Among the different approaches, which were developed for CXCR4 imaging, a CXCR4 antagonist biscyclam system (AMD3100, also called Mozobil), currently used in the clinic for the mobilization of hematopoietic stem cells, was radiolabeled with different radiometals such as (62)Zn, (64)Cu, (67)Ga, or (99m)Tc. However, cyclam is not an ideal chelator for most of these radiometals, and could lead to the release of the radionuclide in vivo. In the current study, a new family of CXCR4 imaging agents is presented, in which AMD3100 is used as a carrier for specific delivery of an imaging reporter, i.e., a (68)Ga complex for PET imaging. AMD3100 was functionalized on the phenyl moiety with different linkers, either ethylenediamine or diamino-polyethylene glycol 3 (PEG3). The resulting AMD3100 analogues were further coupled with two different chelators, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-acetic acid (NODAGA). Five potential CXCR4 targeting agents were obtained. The derived AMD3100-based ligands were labeled with (68)Ga, highlighting the influence of the spacer nature on the (68)Ga-labeling yield. The lipophilic character of the different systems was also investigated, as well as their affinity for the CXCR4 receptor. The most promising compound was further evaluated in vivo in H69 tumor xenografts by biodistribution and PET imaging studies, validating the proof of principle of our concept.

Stereochemistry of amino acid spacers determines the pharmacokinetics of (111)In-DOTA-minigastrin analogues for targeting the CCK2/gastrin receptor.

The metabolic instability and high kidney retention of minigastrin (MG) analogues hamper their suitability for use in peptide-receptor radionuclide therapy of CCK2/gastrin receptor-expressing tumors. High kidney retention has been related to N-terminal glutamic acids and can be substantially reduced by coinjection of polyglutamic acids or gelofusine. The aim of the present study was to investigate the influence of the stereochemistry of the N-terminal amino acid spacer on the enzymatic stability and pharmacokinetics of (111)In-DOTA-(d-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-D) and (111)In-DOTA-(l-Glu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 ((111)In-PP11-L). Using circular dichroism measurements, we demonstrate the important role of secondary structure on the pharmacokinetics of the two MG analogues. The higher in vitro serum stability together with the improved tumor-to-kidney ratio of the (d-Glu)6 congener indicates that this MG analogue might be a good candidate for further clinical study.

Copper-64 Labeled Macrobicyclic Sarcophagine Coupled to a GRP Receptor Antagonist Shows Great Promise for PET Imaging of Prostate Cancer.

The gastrin-releasing peptide receptor (GRPr) is an important molecular target for the visualization and therapy of tumors and can be targeted with radiolabeled bombesin derivatives. The present study aims to develop statine-based bombesin receptor antagonists suitable for labeling with 64Cu for imaging by positron emission tomography (PET). The potent GRPr antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 was conjugated to the sarcophagine (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane=Sar) derivative 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar) via PEG4 (LE1) and PEG2 (LE2) spacers and radiolabeled with 64Cu2+ with >95% yield and specific activities of about 100 MBq/nmol. Both Cu(II) conjugates have high affinity for GRPr (IC50: natCu-LE1, 1.4±0.1 nM; natCu-LE2, 3.8±0.6 nM). The antagonistic properties of both conjugates were confirmed by Ca2+-flux measurements. Biodistribution studies of Cu-64-LE1 exhibited specific targeting of the tumor (19.6±4.7% IA/g at 1 h p.i.) and GRPr-positive organs. Biodistribution and PET images at 4 and 24 h postinjection showed increasing tumor-to-background ratios with time. This was illustrated by the acquisition of PET images showing high tumor-to-normal tissue contrast. This study demonstrates the high affinity of the MeCOSar-PEGx-bombesin conjugates to GRPr. The stability of 64Cu complexes of MeCOSar, the long half-life of 64Cu, and the suitable biodistribution profile of the 64Cu-labeled peptides lead to PET images of high contrast suitable for potential translation into the clinic.

Hybrid bombesin analogues: combining an agonist and an antagonist in defined distances for optimized tumor targeting.

Radiolabeled hybrid ligands with defined distances between an agonist and an antagonist for the gastrin-releasing peptide receptor were found to have excellent tumor-targeting properties. Oligoprolines served as rigid scaffolds that allowed for tailoring distances of 10, 20, and 30 Å between the recognition elements. In vitro and in vivo studies revealed that the hybrid ligand with a distance of 20 Å between the recognition elements exhibits the highest yet observed tumor cell uptake and retention time in prostate cancer cells.

Somatostatin-based radiotherapy with [90Y-DOTA]-TOC in neuroendocrine tumors: long-term outcome of a phase I dose escalation study.

BACKGROUND: We describe the long-term outcome after clinical introduction and dose escalation of somatostatin receptor targeted therapy with [90Y-DOTA]-TOC in patients with metastasized neuroendocrine tumors. METHODS: In a clinical phase I dose escalation study we treated patients with increasing [90Y-DOTA]-TOC activities. Multivariable Cox regression and competing risk regression were used to compare efficacy and toxicities of the different dosage protocols. RESULTS: Overall, 359 patients were recruited; 60 patients were enrolled for low dose (median: 2.4 GBq/cycle, range 0.9-7.8 GBq/cycle), 77 patients were enrolled for intermediate dose (median: 3.3 GBq/cycle, range: 2.0-7.4 GBq/cycle) and 222 patients were enrolled for high dose (median: 6.7 GBq/cycle, range: 3.7-8.1 GBq/cycle) [90Y-DOTA]-TOC treatment. The incidences of hematotoxicities grade 1-4 were 65.0%, 64.9% and 74.8%; the incidences of grade 4/5 kidney toxicities were 8.4%, 6.5% and 14.0%, and the median survival was 39 (range: 1-158) months, 34 (range: 1-118) months and 29 (range: 1-113) months. The high dose protocol was associated with an increased risk of kidney toxicity (Hazard Ratio: 3.12 (1.13-8.59) vs. intermediate dose, p = 0.03) and a shorter overall survival (Hazard Ratio: 2.50 (1.08-5.79) vs. low dose, p = 0.03). CONCLUSIONS: Increasing [90Y-DOTA]-TOC activities may be associated with increasing hematological toxicities. The dose related hematotoxicity profile of [90Y-DOTA]-TOC could facilitate tailoring [90Y-DOTA]-TOC in patients with preexisting hematotoxicities. The results of the long-term outcome suggest that fractionated [90Y-DOTA]-TOC treatment might allow to reduce renal toxicity and to improve overall survival. (ClinicalTrials.gov number NCT00978211).

Radiopharmaceutical development of radiolabelled peptides.

Receptor targeting with radiolabelled peptides has become very important in nuclear medicine and oncology in the past few years. The overexpression of many peptide receptors in numerous cancers, compared to their relatively low density in physiological organs, represents the molecular basis for in vivo imaging and targeted radionuclide therapy with radiolabelled peptide-based probes. The prototypes are analogs of somatostatin which are routinely used in the clinic. More recent developments include somatostatin analogs with a broader receptor subtype profile or with antagonistic properties. Many other peptide families such as bombesin, cholecystokinin/gastrin, glucagon-like peptide-1 (GLP-1)/exendin, arginine-glycine-aspartic acid (RGD) etc. have been explored during the last few years and quite a number of potential radiolabelled probes have been derived from them. On the other hand, a variety of strategies and optimized protocols for efficient labelling of peptides with clinically relevant radionuclides such as (99m)Tc, M(3+) radiometals ((111)In, (86/90)Y, (177)Lu, (67/68)Ga), (64/67)Cu, (18)F or radioisotopes of iodine have been developed. The labelling approaches include direct labelling, the use of bifunctional chelators or prosthetic groups. The choice of the labelling approach is driven by the nature and the chemical properties of the radionuclide. Additionally, chemical strategies, including modification of the amino acid sequence and introduction of linkers/spacers with different characteristics, have been explored for the improvement of the overall performance of the radiopeptides, e.g. metabolic stability and pharmacokinetics. Herein, we discuss the development of peptides as radiopharmaceuticals starting from the choice of the labelling method and the conditions to the design and optimization of the peptide probe, as well as some recent developments, focusing on a selected list of peptide families, including somatostatin, bombesin, cholecystokinin/gastrin, GLP-1/exendin and RGD.

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 (111)In-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 (111)In and (177)Lu in three different tumour models. METHODS: Radiopeptide binding, internalization and dissociation studies were performed using cells expressing HEK293-rsst(2). Biodistribution studies were performed in HEK293-rsst(2), HEK293-hsst(2) and HEK293-rsst(3) xenografted mice. RESULTS: Saturation binding analysis confirmed earlier IC(50) data for (111/nat)In-DOTA-sst2-ANT and showed similar affinity of (177/nat)Lu-DOTA-sst2-ANT for the sst(2). Only low internalization was found in cell culture (6.68 ± 0.06 % at 4 h), 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. (111)In-DOTA-sst2-ANT and (177)Lu-DOTA-sst2-ANT were shown to target tumour xenografts expressing the rat and the human sst(2) receptor with no differences in their uptake or pharmacokinetics. The uptake in rsst(2) and hsst(2) was high (about 30 %IA/g 4 h after injection) and surprisingly long-lasting (about 20-23 %IA/g 24 h 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 (177)Lu-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.

Biokinetics and dosimetry of 111In-DOTA-NOC-ATE compared with 111In-DTPA-octreotide.

PURPOSE: The biokinetics and dosimetry of (111)In-DOTA-NOC-ATE (NOCATE), a high-affinity ligand of SSTR-2 and SSTR-5, and (111)In-DTPA-octreotide (Octreoscan™, OCTREO) were compared in the same patients. METHODS: Seventeen patients (10 men, 7 women; mean age 60 years), referred for an OCTREO scan for imaging of a neuroendocrine tumour (15), thymoma (1) or medullary thyroid carcinoma (1), agreed to undergo a second study with NOCATE. Whole-body anterior-posterior scans were recorded 0.5 (100 % reference scan), 4, 24 and 48 h (17 patients) and 120 h (5 patients) after injection. In 16 patients the OCTREO scan (178 ± 15 MBq) was performed 16 ± 5 days before the NOCATE scan (108 ± 14 MBq) with identical timing; 1 patient had the NOCATE scan before the OCTREO scan. Blood samples were obtained from 14 patients 5 min to 48 h after injection. Activities expressed as percent of the initial (reference) activity in the whole body, lung, kidney, liver, spleen and blood were fitted to biexponential or single exponential functions. Dosimetry was performed using OLINDA/EXM. RESULTS: Initial whole-body, lung and kidney activities were similar, but retention of NOCATE was higher than that of OCTREO. Liver and spleen uptakes of NOCATE were higher from the start (p < 0.001) and remained so over time. Whole-body activity showed similar α and ß half-lives, but the ß fraction of NOCATE was double that of OCTREO. Blood T (1/2)ß for NOCATE was longer (19 vs. 6 h). As a result, the effective dose of NOCATE (105 µSv/MBq) exceeded that of OCTREO (52 µSv/MBq), and the latter result was similar to the ICRP 106 value of 54 µSv/MBq. Differential activity measurement in blood cells and plasma showed an average of <5 % of NOCATE and OCTREO attached to globular blood components. CONCLUSION: NOCATE showed a slower clearance from normal tissues and its effective dose was roughly double that of OCTREO.

In vivo imaging of folate receptor positive tumor xenografts using novel 68Ga-NODAGA-folate conjugates.

The overexpression of the folate receptor (FR) in a variety of malignant tumors, along with its limited expression in healthy tissues, makes it an attractive tumor-specific molecular target. Noninvasive imaging of FR using radiolabeled folate derivatives is therefore highly desirable. Given the advantages of positron emission tomography (PET) and the convenience of (68)Ga production, the aim of our study was to develop a new (68)Ga-folate-based radiotracer for clinical application. The chelator 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA) was conjugated to folic acid and to 5,8-dideazafolic acid using 1,2-diaminoethane as a spacer, resulting in two novel conjugates, namely, P3246 and P3238, respectively. Both conjugates were labeled with (68/67)Ga. In vitro internalization, efflux, and saturation binding studies were performed using the FR-positive KB cell line. Biodistribution and small-animal PET imaging studies were performed in nude mice bearing subcutaneous KB xenografts. Both conjugates were labeled with (68)Ga at room temperature within 10 min in labeling yields >95% and specific activity ~30 GBq/µmol. The K(d) values of (68/67)Ga-P3246 (5.61 ± 0.96 nM) and (68/67)Ga-P3238 (7.21 ± 2.46 nM) showed high affinity for the FR. (68/67)Ga-P3246 showed higher cell-associated uptake in vitro than (68/67)Ga-P3238 (approximately 72 and 60% at 4 h, respectively, P < 0.01), while both radiotracers exhibited similar cellular retention up to 4 h (approximately 76 and 71%, respectively). Their biodistribution profile is characterized by high tumor uptake, fast blood clearance, low hepatobiliary excretion, and almost negligible background. Tumor uptake was already high at 1 h for both (68)Ga-P3246 and (68)Ga-P3238 (16.56 ± 3.67 and 10.95 ± 2.12% IA/g, respectively, P > 0.05) and remained at about the same level up to 4 h. Radioactivity also accumulated in the FR-positive organs, such as kidneys (91.52 ± 21.05 and 62.26 ± 14.32% IA/g, respectively, 1 h pi) and salivary glands (9.05 ± 2.03 and 10.39 ± 1.19% IA/g, respectively, 1 h pi). The specificity of the radiotracers for the FR was confirmed by blocking experiments where tumor uptake was reduced by more than 85%, while the uptake in the kidneys and the salivary glands was reduced by more than 90%. Reduction of the kidney uptake was achieved by administration of the antifolate pemetrexed 1 h prior to the injection of the radiotracers, which resulted in an improvement of tumor-to-kidney ratios by more than a factor of 3. In line with the biodistribution results, small-animal PET images showed high uptake in the kidneys, clear visualization of the tumor, accumulation of radioactivity in the salivary glands, and no uptake in the gastrointestinal tract. (68)Ga-P3246 and (68)Ga-P3238 showed very high tumor-to-background contrast in PET images; however, the tumor-to-kidney ratio remained low. The new radiotracers, especially (68)Ga-P3246, are promising as PET imaging probes for clinical application due to their facile preparation and improved in vivo profile as compared to the other folate-based PET radiotracers.

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

PURPOSE: 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. 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 (67/68)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). RESULTS: 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. CONCLUSION: 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.

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-NH(2) via the cationic spacer 4-amino-1-carboxymethyl-piperidine for labelling with radiometals such as (111)In and (68)Ga. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Tetraamine-derived bifunctional chelators for technetium-99m labelling: synthesis, bioconjugation and evaluation as targeted SPECT imaging probes for GRP-receptor-positive tumours.

Owing to its optimal nuclear properties, ready availability, low cost and favourable dosimetry, (99m)Tc continues to be the ideal radioisotope for medical-imaging applications. Bifunctional chelators based on a tetraamine framework exhibit facile complexation with Tc(V)O(2) to form monocationic species with high in vivo stability and significant hydrophilicity, which leads to favourable pharmacokinetics. The synthesis of a series of 1,4,8,11-tetraazaundecane derivatives (01-06) containing different functional groups at the 6-position for the conjugation of biomolecules and subsequent labelling with (99m)Tc is described herein. The chelator 01 was used as a starting material for the facile synthesis of chelators functionalised with OH (02), N(3) (04) and O-succinyl ester (05) groups. A straightforward and easy synthesis of carboxyl-functionalised tetraamine-based chelator 06 was achieved by using inexpensive and commercially available starting materials. Conjugation of 06 to a potent bombesin-antagonist peptide and subsequent labelling with (99m)Tc afforded the radiotracer (99m)Tc-N4-BB-ANT, with radiolabelling yields of >97% at a specific activity of 37 GBq micromol(-1). An IC(50) value of (3.7+/-1.3) nM was obtained, which confirmed the high affinity of the conjugate to the gastrin-releasing-peptide receptor (GRPr). Immunofluorescence and calcium mobilisation assays confirmed the strong antagonist properties of the conjugate. In vivo pharmacokinetic studies of (99m)Tc-N4-BB-ANT showed high and specific uptake in PC3 xenografts and in other GRPr-positive organs. The tumour uptake was (22.5+/-2.6)% injected activity per gram (% IA g(-1)) at 1 h post injection (p.i.). and increased to (29.9+/-4.0)% IA g(-1) at 4 h p.i. The SPECT/computed tomography (CT) images showed high tumour uptake, clear background and negligible radioactivity in the abdomen. The promising preclinical results of (99m)Tc-N4-BB-ANT warrant its potential candidature for clinical translation.

Detection of Insulinomas Using Dual-Time-Point 68Ga-DOTA-Exendin 4 PET/CT.

PURPOSE: Insulinomas are predominantly benign neuroendocrine tumors originating from beta cells within the islets of Langerhans of the endocrine pancreas. Because surgical resection represents the only curative therapy option, exact tumor localization and discrimination of insulinomas from focal or diffuse manifestations of congenital hyperinsulinism are crucial for optimal treatment strategies. We investigated the diagnostic value of glucagon-like peptide 1 receptor PET/CT using Ga-DOTA-exendin 4 for detecting insulinomas and compared the diagnostic value of PET scans performed at 2 time points. METHODS: In 10 patients with clinically and biochemically suspected insulinoma, PET/CT was performed at 1 hour (PET1) and 2 hours (PET2) after injection of Ga-DOTA-exendin 4. In this retrospective analysis, tracer uptake was visually assessed in both scans by 2 independent readers. SUVmax and tumor-to-background ratio (TBR) of focal lesions were assessed. Imaging results were compared with histopathologic findings, if patients underwent resection. RESULTS: Increased focal Ga-DOTA-exendin 4 uptake was observed in 8 of 10 patients concordantly by both readers. Seven patients with focal uptake underwent surgery with tumor enucleation and histopathologic proof of insulinoma (7/8). Two of 10 patients without focal uptake were considered to suffer from diffuse form of congenital hyperinsulinism and consequently received medical treatment. A significant increase of tumoral SUVmax on PET2 (PET1: SUVmax 20.2 ± 8.2 g/mL; PET2: SUVmax 24.7 ± 7.9 g/mL; P = 0.0018) did not result in a significant improvement in TBR (PET1: TBR 4.9 ± 1.7; PET2: TBR 4.3 ± 1.2; P = 0.2892). CONCLUSIONS: Focal uptake of Ga-DOTA-exendin 4 reliably indicated insulinomas as histopathologically confirmed in all patients undergoing consecutive surgery. The diagnostic value of PET2 was not found to be superior to PET1, indicating that a single 1-hour Ga-DOTA-exendin 4 PET/CT scan is a sufficient and convenient approach for patient care.

Highly efficient in vivo agonist-induced internalization of sst2 receptors in somatostatin target tissues.

UNLABELLED: The successful peptide receptor imaging of tumors, as exemplified for somatostatin receptors, is based on the overexpression of peptide receptors in selected tumors and the high-affinity binding to these tumors of agonist radioligands that are subsequently internalized into the tumor cells in which they accumulate. Although in vitro studies have shown ample evidence that the ligand-receptor complex is internalized, in vivo evidence of agonist-induced internalization of peptide receptors, such as somatostatin receptors, is missing. METHODS: Rats subcutaneously transplanted with the somatostatin receptor subtype 2 (sst(2))-expressing AR42J tumor cells were treated with intravenous injections of various doses of the sst(2) agonist [Tyr(3), Thr(8)]-octreotide (TATE) or of the sst(2) antagonist 1,4,7,10-tetraazacyclododecane-N,N',N'',N''',-tetraacetic acid (DOTA)-Bass and were sacrificed at various times ranging from 2.5 min to 24 h after injection. The tumors and pancreas were then removed from each animal. All tissue samples were processed for sst(2) immunohistochemistry using sst(2)-specific antibodies. RESULTS: Compared with the sst(2) receptors in untreated animals, which localized at the plasma membrane in pancreatic and AR42J tumor cells, the sst(2) receptors in treated animals are detected intracellularly after an intravenous injection of the agonist TATE. Internalization is fast, as the receptors are already internalizing 2.5 min after TATE injection. The process is extremely efficient, as most of the cell surface receptors internalize into the cell and are found in endosomelike structures after TATE injection. The internalization is most likely reversible, because 24 h after injection the receptors are again found at the cell surface. The process is also agonist-dependent, because internalization is seen with high-affinity sst(2) agonists but not with high-affinity sst(2) antagonists. The same internalization properties are seen in pancreatic and AR42J tumor cells. They can further be confirmed in vitro in human embryonic kidney-sst(2) cells, with an immunofluorescence microscopy-based sst(2) internalization assay. CONCLUSION: These animal data strongly indicate that the process of in vivo sst(2) internalization after agonist stimulation is fast, extremely efficient, and fully functional under in vivo conditions in neoplastic and physiologic sst(2) target tissues. This molecular process is, therefore, likely to be responsible for the high and long-lasting uptake of sst(2) radioligands seen in vivo in sst(2)-expressing tumors.

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

PURPOSE: (131)I- and (90)Y-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 (<20 min) with either (177)Lu or (90)Y. 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 10(5) times excess of diethylenetriaminepentaacetic acid (DTPA, 37 degrees C, 7 days). Two patients with relapsed NHL were treated with 740 MBq/m(2) body surface (177)Lu-DOTA-rituximab. Scintigraphic images showed specific uptake at tumour sites and acceptable dosimetric results. The mean whole-body dose was found to be 314 mGy. The administration of (177)Lu-DOTA-rituximab was tolerated well. CONCLUSION: Our results show that DOTA-rituximab (4:1) can be labelled with (177)Lu with sufficient stability while the immunoconjugate retains its immunoreactivity. (177)Lu-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 NHL.

New pansomatostatin ligands and their chelated versions: affinity profile, agonist activity, internalization, and tumor targeting.

PURPOSE: Somatostatin receptor (sst) targeting is an established method to image and treat sst-positive tumors. Particularly, neuroendocrine tumors express the receptor subtype 2 in high density, but sst1, sst3, sst4, and sst5 are also expressed to some extent in different human tumors. Currently used targeting peptides mainly have sst2 affinity. We aimed at developing (radio)peptides that bind with high affinity to all receptor subtypes. EXPERIMENTAL DESIGN: Carbocyclic octapeptides were coupled with macrocyclic chelators for radiometal labeling. Affinity, internalization, and agonist potencies were determined on sst1- to sst5-expressing cell lines. Biodistribution was determined on nude mice bearing HEK-sst2 or AR4-2J and HEK-sst3 tumors. RESULTS: High affinity to all receptor subtypes was found. Y(III)-KE88 showed agonistic properties at all five sst receptor subtypes as it inhibits forskolin-stimulated cyclic AMP production. Surprisingly, very low or even absent sst2 receptor internalization was found compared with currently clinically established octapeptides, whereas the sst3 internalization was very efficient. Biodistribution studies of [(111)In]KE88 and [(67)Ga]KE88/[(68)Ga]KE88 reflected the in vitro data. In nude mice with s.c. implanted sst2 (HEK-sst2, AR4-2J)-expressing and sst3 (HEK-sst3)-expressing tumors, high and persistent uptake was found in sst3-expressing tumors, whereas the uptake in the sst2-expressing tumors was lower and showed fast washout. The kidney uptake was high but blockable by coinjection of lysine. CONCLUSION: This peptide family shows pansomatostatin potency. As radiopeptides, they are the first to show a full pansomatostatin profile. Despite some drawback, they should be useful for imaging sst2-expressing tumors with short-lived radiometals, such as (68)Ga, at early time points and for sst3-expressing tumors at later time points with longer-lived radiometals, such as (64)Cu or (86)Y.

Accuracy of [68Ga]Ga-RM2-PET/CT for diagnosis of primary prostate cancer compared to histopathology.

INTRODUCTION: Prostate cancer (PCa) often shows an overexpression of the gastrin-releasing peptide receptor (GRPr). Therefore, GRPr is a possible theragnostic target. An interesting antagonist GRPr-ligand is RM2 or BAY86-7548. This study examines the accuracy of positron emission tomography (PET) with [68Ga]Ga-RM2 for diagnostic imaging of primary PCa (pPCa) compared to histopathology in patients undergoing radical prostatectomy (RP). METHODS: [68Ga]Ga-RM2-PET examinations were performed in 15 patients before RP. All prostate specimens were histopathologically examined based on predefined spatial octants. Each prostate volume on PET was subdivided into octants, which were correlated to histopathology and evaluated according to presence of tumor by two experienced examiners. Additionally, PET data was evaluated by volume of interest (VOI) analyses in terms of maximum standardized uptake value (SUVmax) and normalized SUVmax relative to background activity (rSUVmax). Receiver operating characteristic (ROC) curves for SUVmax and rSUVmax were calculated. RESULTS: At least one focus of increased [68Ga]Ga-RM2 uptake corresponding to a tumor manifestation on histology was found in 14 of 15 patients (93%). Spatial concordance of visual PET readings with histopathology was very variable. Intraindividual agreement reached from ≤2 octants in three, 3-5 octants in six to ≥6 octants in six patients, resulting in a relatively low correlation of visual PET readings with histopathology (accuracy = 0.63; p = 0.0018). Lesion-based analysis found a sensitivity of 69% and a positive predictive value of 73%. Concordantly, the octant-based ROC curves for SUVmax and rSUVmax indicated a relatively low diagnostic performance (area under the curve of 0.59 and 0.61, respectively). CONCLUSIONS: [68Ga]Ga-RM2-PET shows only a relatively low diagnostic accuracy for pPCa compared to histopathology on an octant basis, which may be explained to some extent by methodological weaknesses. Further studies need to explore, whether the observed high interindividual variability of agreement between [68Ga]Ga-RM2-PET and histopathology can be explained by different tumor biologies or other coincident prostatic pathologies.