No abstract

A new hexaazamacrobicyclic cage ligand, 1-N-(4-aminobenzyl)- 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine (SarAr) has been designed for conjugation to proteins. SarAr was synthesised and characterised by microanalyses, 1 H NMR and electrospray mass spectrometry. The complexation of selected transition metal ions (Cu(), Ni() and Co() at 10 Ϫ6 M) by SarAr was complete within 30 min over pH 6 to 8. The [ 64 Cu(SarAr)] 2ϩ complex was investigated with a view to applications in radioimaging. The [ 64 Cu(sar)] 2ϩ complex was found to be stable in human plasma for at least 174 h and biodistribution studies in mice, showed that the [ 64 Cu(SarAr)] 2ϩ complex was rapidly excreted through the renal system unlike the free 64 Cu 2ϩ . Overall, the simple synthesis, ready complexation behaviour of SarAr, the kinetic inertness of the [Cu(SarAr)] 2ϩ complex to dissociation of 64 Cu and its facile elimination from mice make it an attractive prospect for use in nuclear medicine.

The advancement of positron emission tomography (PET) depends on the development of new radiotracers that will complement 18 F-FDG. Copper-64 ( 64 Cu) is a promising PET radionuclide, particularly for antibody-targeted imaging, but the high in vivo lability of conventional chelates has limited its clinical application. The objective of this work was to evaluate the novel chelating agent SarAr (1-N-(4-aminobenzyl)-3, 6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine) for use in developing a new class of tumorspecific 64 Cu radiopharmaceuticals for imaging neuroblastoma and melanoma. The anti-GD2 monoclonal antibody (mAb) 14.G2a, and its chimeric derivative, ch14.18, target disialogangliosides that are overexpressed on neuroblastoma and melanoma. Both mAbs were conjugated to SarAr using carbodiimide coupling. Radiolabeling with 64 Cu resulted in >95% of the 64 Cu being chelated by the immunoconjugate. Specific activities of at least 10 Ci/ g (1 Ci ‫؍‬ 37 GBq) were routinely achieved, and no additional purification was required after 64 Cu labeling. Solid-phase radioimmunoassays and intact cell-binding assays confirmed retention of bioactivity. Biodistribution studies in athymic nude mice bearing s.c. neuroblastoma (IMR-6, NMB-7) and melanoma (M21) xenografts showed that 15-20% of the injected dose per gram accumulated in the tumor at 24 hours after injection, and only 5-10% of the injected dose accumulated in the liver, a lower value than typically seen with other chelators. Uptake by a GD2-negative tumor xenograft was significantly lower (<5% injected dose per gram). MicroPET imaging confirmed significant uptake of the tracer in GD-2-positive tumors, with minimal uptake in GD-2-negative tumors and nontarget tissues such as liver. The 64 Cu-SarAr-mAb system described here is potentially applicable to 64 Cu-PET imaging with a broad range of antibody or peptide-based imaging agents.

The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metalbinding sites, GMTCXXC, at the N terminus. The metalbinding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent K m and V max values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease.

Introduction Use of copper radioisotopes in antibody radiolabeling is challenged by reported loss of the radionuclide from the bifunctional chelator used to label the protein. The objective of this study was to investigate the relationship between the thermodynamic stability of the 64Cu-complexes of five commonly used bifunctional chelators (BFCs) and the biodistribution of an antibody labeled with 64Cu using these chelators in tumor-bearing mice. Methods The chelators (p-NH2-Bn-NOTA, BAT-6, p-NH2-Bn-DOTA, DOTA, and SarAr) were conjugated to the anti-GD2 antibody ch14.18, and the conjugated antibody was labeled with 64Cu and injected into mice bearing subcutaneous human melanoma tumors (M21) (n = 3-5 for each study). Biodistribution data were obtained from PET images acquired at 1, 24 and 48 hours post-injection, and at 48 hours post-injection a full ex vivo biodistribution study was carried out. Results The biodistribution, including tumor targeting, was similar for all the radioimmunoconjugates. At 48 h post-injection, the only statistically significant differences in radionuclide uptake (p < 0.05) were between blood, liver, spleen and kidney. For example, liver uptake of [64Cu]ch14.18-p-NH2-Bn-NOTA was 4.74 ± 0.77 per cent of the injected dose per gram of tissue (%ID/g), and for [64Cu]ch14.18-SarAr was 8.06 ± 0.77 %ID/g. Differences in tumor targeting correlated with variations in tumor size rather than which BFC was used. Conclusions The results of this study indicate that differences in the thermodynamic stability of these chelator-Cu(II) complexes were not associated with significant differences in uptake of the tracer by the tumor. However, there were significant differences in tracer concentration in other tissues, including those involved in clearance of the radioimmunoconjugate (e.g., liver and spleen).