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New macrobicyclic cage amine or “sarcophagine” (sar) bifunctional chelators have been synthesised that form copper complexes of exceptional in vivo stability and incorporate isothiocyanate (-NCS) functional groups for conjugation to an antibody. The chelators were synthesised from the methyl-capped complex [MgII(CH3)(NH2)sar]2+. Coordination of MgII within the cavity of the cage amine ligand protects the secondary amine atoms from reacting with the -NCS functional groups. Two different [MgII(NCS-sar)]2+ derivatives were conjugated to the HER2/neu-targeting antibody trastuzumab and the progress of the reaction monitored by electrospray mass spectrometry. The MgII ion was removed from the immunoconjugates under mild conditions (0.1 M citrate buffer, pH 6). Labelling of the (CH3)(p-NCS-Ph)sar-trastuzumab conjugate with 64CuII, a radioisotope suitable for positron emission tomography (PET), was fast (∼5 min) and easily performed at room temperature with high radiochemical purity (＞95%). Biodistribution and PET imaging studies in vivo showed that 64Cu-labelled (CH3)(p-NCS-Ph)sar-trastuzumab maintained high stability under physiological conditions with high and selective uptake in a HER2-positive cancer cell line. The stability of the copper complex and the 12.7 h half-life of the radioisotope allows clear visualisation of tumours out to 48 h. [ABSTRACT FROM AUTHOR]

The use of copper radioisotopes in cancer diagnosis and radionuclide therapy is possible using chelators that are capable of binding CuII with sufficient stability in vivo to provide high tumour-to-background contrast. Here we report the design and synthesis of a new bifunctional chelator, 5-(8-methyl- 3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar), that forms copper complexes of exceptional stability by virtue of a cage amine (sarcophagine) ligand and a new conjugate referred to as SarTATE, obtained by the conjugation of MeCOSar to the tumour-targeting peptide Tyr3-octreotate. Radiolabeling of SarTATE with 6CuII, a radioisotope suitable for positron emission tomography (PET), was fast (∼20 min), easily performed at room temperature and consistently resulted in high radiochemical purity (>99%). In vitro and in vivo evaluation of 64CuSarTATE demonstrated its high selectivity for tumour cells expressing somatostatin receptor 2 (sstr2). Biodistribution and PET imaging comparisons were made between 64CuSarTATE and 64Cu-labeled DOTA-Tyr3-octreotate (64CuDOTATATE). Both radiopharmaceuticals showed excellent uptake in sstr2-positive tumours at 2 h post-injection. While tumour uptake of 64CuDOTATATE decreased significantly at 24 h, 64CuSarTATE activity was retained, improving contrast at later time points. 64CuSarTATE accumulated less than 64CuDOTATATE in the nontarget organs, liver and lungs. The uptake of 64CuSarTATE in the kidneys was high at 2 h but showed significant clearance by 24 h. The new chemistry and pre-clinical evaluation presented here demonstrates that MeCOSar is a promising bifunctional chelator for Tyr3-octreotate that could be applied to a combined imaging and therapeutic regimen using a combination of 64Cu- and 67CuSarTATE complexes, owing to improved tumour-to-non-target organ ratios compared to 64CuDOTATATE at longer time points. [ABSTRACT FROM AUTHOR]

Peptide receptor radionuclide therapy (PRRT) using radiolabeled octreotate is an effective treatment for somatostatin receptor 2-expressing neuroendocrine tumors. The diagnostic and therapeutic potential of 64 Cu and 67 Cu, respectively, offers the possibility of using a single somatostatin receptor-targeted peptide conjugate as a theranostic agent. A sarcophagine cage amine ligand, MeCOSar (5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid), conjugated to (Tyr 3 )-octreotate, called 64 Cu-CuSarTATE, was demonstrated to be an imaging agent and potential prospective dosimetry tool in 10 patients with neuroendocrine tumors. This study aimed to explore the antitumor efficacy of 67 Cu-CuSarTATE in a preclinical model of neuroendocrine tumors and compare it with the standard PRRT agent, 177 Lu-LuDOTA-Tyr 3 -octreotate ( 177 Lu-LuTATE). Methods: The antitumor efficacy of various doses of 67 Cu-CuSarTATE in AR42J (rat pancreatic exocrine) tumor-bearing mice was compared with 177 Lu-LuTATE. Results: Seven days after a single administration of 67 Cu-CuSarTATE (5 MBq), tumor growth was inhibited by 75% compared with vehicle control. Administration of 177 Lu-LuTATE (5 MBq) inhibited tumor growth by 89%. Survival was extended from 12 d in the control group to 21 d after treatment with both 67 Cu-CuSarTATE and 177 Lu-LuTATE. In a second study, the efficacy of fractionated delivery of PRRT was assessed, comparing the efficacy of 30 MBq of 67 Cu-CuSarTATE or 177 Lu-LuTATE, either as a single intravenous injection or as two 15-MBq fractions 2 wk apart. Treatment of tumors with 2 fractions significantly improved survival over delivery as a single fraction ( 67 Cu-CuSarTATE: 47 vs. 36 d [ P = 0.036]; 177 Lu-LuTATE: 46 vs. 29 d [ P = 0.040]). Conclusion: This study demonstrates that 67 Cu-CuSarTATE is well tolerated in BALB/c nude mice and highly efficacious against AR42J tumors in vivo. Administration of 67 Cu-CuSarTATE and 177 Lu-LuTATE divided into 2 fractions over 2 wk was more efficacious than administration of a single fraction. The antitumor activity of 67 Cu-CuSarTATE in the AR42J tumor model demonstrated the suitability of this novel agent for clinical assessment in the treatment of somatostatin receptor 2-expressing neuroendocrine tumors., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Imaging of somatostatin receptor expression is an established technique for staging of neuroendocrine neoplasia and determining the suitability of patients for peptide receptor radionuclide therapy. PET/CT using 68 Ga-labeled somatostatin analogs is superior to earlier agents, but the rapid physical decay of the radionuclide poses logistic and regulatory challenges. 64 Cu has attractive physical characteristics for imaging and provides a diagnostic partner for the therapeutic radionuclide 67 Cu. Based on promising preclinical studies, we have performed a first-time-in-humans trial of 64 Cu-MeCOSar-Tyr 3 -octreotate ( 64 Cu-SARTATE) to assess its safety and ability to localize disease at early and late imaging time-points. Methods: In a prospective trial, 10 patients with known neuroendocrine neoplasia and positive for uptake on 68 Ga-DOTA-octreotate ( 68 Ga-DOTATATE) PET/CT underwent serial PET/CT imaging at 30 min, 1 h, 4 h, and 24 h after injection of 64 Cu-SARTATE. Adverse reactions were recorded, and laboratory testing was performed during infusion and at 1 and 7 d after imaging. Images were analyzed for lesion and normal-organ uptake and clearance to assess lesion contrast and perform dosimetry estimates. Results: 64 Cu-SARTATE was well tolerated during infusion and throughout the study, with 3 patients experiencing mild infusion-related events. High lesion uptake and retention were observed at all imaging time-points. There was progressive hepatic clearance over time, providing the highest lesion-to-liver contrast at 24 h. Image quality remained high at this time. Comparison of 64 Cu-SARTATE PET/CT obtained at 4 h to 68 Ga-DOTATATE PET/CT obtained at 1 h indicated comparable or superior lesion detection in all patients, especially in the liver. As expected, the highest early physiologic organ uptake was in the kidneys, liver, and spleen. Conclusion: 64 Cu-SARTATE is safe and has excellent imaging characteristics. High late-retention in tumor and clearance from the liver suggest suitability for diagnostic studies and for prospective dosimetry for 67 Cu-SARTATE peptide receptor radionuclide therapy, and the half-life of 64 Cu would also facilitate good-manufacturing-practice production and distribution to sites without access to 68 Ga., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

The use of copper radioisotopes in cancer diagnosis and radionuclide therapy is possible using chelators that are capable of binding Cu(II) with sufficient stability in vivo to provide high tumour-to-background contrast. Here we report the design and synthesis of a new bifunctional chelator, 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar), that forms copper complexes of exceptional stability by virtue of a cage amine (sarcophagine) ligand and a new conjugate referred to as SarTATE, obtained by the conjugation of MeCOSar to the tumour-targeting peptide Tyr(3)-octreotate. Radiolabeling of SarTATE with (64)Cu(II), a radioisotope suitable for positron emission tomography (PET), was fast (~20 min), easily performed at room temperature and consistently resulted in high radiochemical purity (>99%). In vitro and in vivo evaluation of (64)CuSarTATE demonstrated its high selectivity for tumour cells expressing somatostatin receptor 2 (sstr2). Biodistribution and PET imaging comparisons were made between (64)CuSarTATE and (64)Cu-labeled DOTA-Tyr(3)-octreotate ((64)CuDOTATATE). Both radiopharmaceuticals showed excellent uptake in sstr2-positive tumours at 2 h post-injection. While tumour uptake of (64)CuDOTATATE decreased significantly at 24 h, (64)CuSarTATE activity was retained, improving contrast at later time points. (64)CuSarTATE accumulated less than (64)CuDOTATATE in the non-target organs, liver and lungs. The uptake of (64)CuSarTATE in the kidneys was high at 2 h but showed significant clearance by 24 h. The new chemistry and pre-clinical evaluation presented here demonstrates that MeCOSar is a promising bifunctional chelator for Tyr(3)-octreotate that could be applied to a combined imaging and therapeutic regimen using a combination of (64)Cu- and (67)CuSarTATE complexes, owing to improved tumour-to-non-target organ ratios compared to (64)CuDOTATATE at longer time points.