The success of positron emission tomography (PET) in personalised medicine and drug development requires radioisotopes that provide high quality images and flexible chemistry for a broad application. 64Cu is arguably one of the most suitable PET isotopes for imaging with the evolving target agents, but there are not many appropriate chelating agents for 64Cu and this has limited its wider application. The bi-functional chelator, SarAr is known to bind 64Cu2+ quantitatively (i.e. one metal per ligand present) and rapidly (<2 min) at 10(-6) M over a range of pH (4-9). In this paper the conjugation of SarAr to the whole and fragmented antibody is described. Conjugation of the SarAr to the protein does not impair its coordination of the 64Cu. It complexes the 64Cu2+ rapidly, quantitatively and essentially irreversibly at pH 5. Animal studies show that the 64Cu-SarAr-immunoconjugates maintain their specificity for the target and are stable in vivo. Also, SarAr is a platform technology, is easy to use in a kit formulation and is readily adaptable for the wider application in 64Cu PET imaging.

M Cu / Anion exchange / Radionuclide / 61 Ga waste / Cyclotron / Radionuclide separation SummaryLow acid concentration aqueous/organic mixtures with an anion exchange resin (AG1-X8) have been used to separate carrierfree '"Cu from the by-products of cyclotron produced 67 Ga. The specific activity of the M Cu (5 X10 14 Bq/g) was found to be higher than that reported previously. Trace levels (<1%) of 67 Cu were also present. The new separation method is easier and faster (2 hours) than those currently employed in routine purification of both 67 Cu and "Cu production. We believe that the new method is the first reported use of organic solvents in the separation and purification of these radioisotopes and represents the first effort towards full characterisation of by-products for appropriate waste management.

A novel hexa aza cage, N(1)-(4-isothiocyanatobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1,8-diamine (SarAr-NCS) was synthesized in good yield and characterized by (1)H NMR and electrospray mass spectrometry. A new method for the synthesis of the related N(1)-(4-carboxybenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane-1,8-diamine (AmBaSar) using the p-carboxybenzaldehyde is reported. The complexation of Cu(2+), Co(2+) and Zn(2+) by the two ligands over a range of pHs was found to be similar to the parent derivative SarAr. SarAr-NCS was conjugated to both silica particles (≈90 nm diam.) and the model B72.3 murine antibody. The SarAr-NCSN-silica particles were radiolabeled with Cu(2+) doped (64)Cu and the number of ligands conjugated was calculated to be an average of 7020 ligands per particle. Conjugation of SarAr-NCS to the B72.3 antibody was optimized over a range of conditions. The SarAr-NCSN-B72.3 conjugate was stored in buffer and as a lyophilized powder at 4 °C over 38 days. Its radiolabeling efficiency, stability and immunoreactivity were maintained. The development of a high yielding synthesis of SarAr-NCS should provide an entry point for a wide range of Cu and Zn radiometal PET imaging agents and potentially radiotherapeutic agents with (67)Cu.

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The B72.3 monoclonal antibody was radiolabelled with 123I, and with 111In and 64Cu, using DTPA and SarAr, respectively. Their biodistribution in tumour-bearing nude mice was used to calculate the dosimetry of their respective therapeutic analogue, using 131I, 90Y, 67Cu, and 64Cu. Two dosimetry models were used: one using the classical approach and a second model that takes into consideration the chemical stability of the radiolabelling methods employed and the biological clearance of each radioimmunoconjugate. Results clearly show that the 64Cu-SarAr-B72.3 could be used as a therapeutic agent and, theoretically, be at least as effective as any of the other therapeutic radionuclides currently studied, such as 131I, 90Y, and 67Cu.

A diaminodihydroxyaryl derivative of ethylenediaminetetraacetic acid (DAHA-EDTA) was synthesised in two steps and evaluated for Cu-64 radiolabelling of the B72.3 antibody. The ligand complexes Cu-64 rapidly in a pH range 4 to 7. The Cu-64 complex of the parent species N,N′-bis(carboxymethyl)-N,N′-bis(2-hydroxyacetanilido)-1,2-diaminoethane (DHA-EDTA) shows good stability in serum at 37°C for up to 72 h. Conjugation of the Cu-64-DAHA-EDTA to the B72.3 antibody was achieved using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the activating agent. The reaction conditions were optimized for protein concentration and molar ratio of Cu-64-DAHA-EDTA and EDC to antibody. The specific activity of the final [Cu-64-DAHA-EDTA]-B72.3 product was 49 MBq mg−1 at the end of synthesis. The biodistribution of [Cu-64-DAHA-EDTA]-B72.3 in LS174t tumour-bearing nude mice was monitored over a 24 h period. Maximum tumour uptake (25.8 ± 7.5 % ID g−1) was achieved at 16 h and maintained at 24 h (21.6 ± 1.8 % ID g−1). Rapid clearance of the [Cu-64-DAHA-EDTA]-B72.3 from the blood resulted in good tumour-to-blood ratios (≈ 3.3) within a shorter period (6 h) than previously reported with B72.3 whole antibody and the LS174t tumour bearing nude mouse model.

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