No abstract

The chelators do influence the affinity, antagonistic potency, and pharmacokinetics of the conjugates. The promising preclinical results warrant clinical translation of these probes for SPECT and PET.

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,N9,N$,N9$,-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 kidneysst 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.

The gastrin-releasing peptide receptor (GRPr) is overexpressed in prostate cancer and is an attractive target for radionuclide therapy. In addition, inhibition of the protein kinase mammalian target of rapamycin (mTOR) has been shown to sensitize various cancer cells to the effects of radiotherapy. Methods: To determine the effect of treatment with rapamycin and radiotherapy with a novel 177 Lu-labeled GRPr antagonist ( 177 Lu-RM2, BAY 1017858) alone and in combination, in vitro and in vivo studies were performed using the human PC-3 prostate cancer cell line. PC-3 cell proliferation and 177 Lu-RM2 uptake after treatment with rapamycin were assessed in vitro. To determine the influence of rapamycin on 177 Lu-RM2 tumor uptake, in vivo small-animal PET studies with 68 Ga-RM2 were performed after treatment with rapamycin. To study the efficacy of 177 Lu-RM2 in vivo, mice with subcutaneous PC-3 tumors were treated with 177 Lu-RM2 alone or after pretreatment with rapamycin. Results: Stable expression of GRPr was maintained after rapamycin treatment with doses up to 4 mg/kg in vivo. Monotherapy with 177 Lu-RM2 at higher doses (72 and 144 MBq) was effective in inducing complete tumor remission in 60% of treated mice. Treatment with 37 MBq of 177 Lu-RM2 and rapamycin in combination led to significantly longer survival than with either agent alone. No treatment-related toxicity was observed. Conclusion: Radiotherapy using a 177 Lu-labeled GRPr antagonist alone or in combination with rapamycin was efficacious in inhibiting in vivo tumor growth and may be a promising strategy for treatment of prostate cancer.

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...