High radiolabeling efficiency, preferably to high specific activity, and good stability of the radioimmunoconjugate are essential features for a successful immunoconjugate for imaging or therapy. In this study, the radiolabeling efficiency, in vitro stability and biodistribution of immunoconjugates with eight different bifunctional chelators labeled with 64 Cu were compared. The anti-CD20 antibody, rituximab, was conjugated to four macrocyclic bifunctional chelators (p-SCN-Bn-DOTA, p- , three DTPA derivatives (p-SCN-Bn-DTPA, p-SCN-CHX-A"-DTPA and ITC-2B3M-DTPA) and a macrobicyclic hexamine ("sarcophagine") chelator (sar-CO 2 H) = (1-NH 2 -8-NHCO(CH 2 ) 3 CO 2 H)sar where sar = sarcophagine = 3, 6,10,13,16,19-hexaazabicyclo[6.6.6]icosane). Radiolabeling efficiency under various conditions, in vitro stability in serum at 37°C and in vivo biodistribution and imaging in normal mice over 48 h were studied. All chelators except sar-CO 2 H were conjugated to rituximab by thiourea bond formation with an average of 4.9 +/− 0.9 chelators per antibody molecule. Sar-CO 2 H was conjugated to rituximab by amide bond formation with 0.5 chelators per antibody molecule. Efficiencies of 64 Cu radiolabeling were dependent on the concentration of immunoconjugate. Notably, the 64 Cu-NOTA-rituximab conjugate demonstrated highest radiochemical yield (95%) under very dilute conditions (31 nM NOTA-rituximab conjugate). Similarly, sar-CO-rituximab, containing 1/10 th the number of chelators per antibody compared to other conjugates retained high labeling efficiency (98 %) at an antibody concentration of 250 nM. In contrast to the radioimmunoconjugates containing DTPA derivatives, which demonstrated poor serum stability, all macrocyclic radioimmunoconjugates were very stable in serum with <6 % dissociation of 64 Cu over 48 h. In vivo biodistribution profiles in normal female Balb/C mice were similar for all the macrocyclic radioimmunoconjugates with most of the activity remaining in the blood pool up to 48 h. Whilst all the macrocyclic bifunctional chelators are suitable for molecular imaging using 64 Cu-labeled antibody conjugates, NOTA and sar-CO 2 H show significant advantages over the others in that they can be radiolabeled rapidly at room temperature, under dilute conditions resulting in high specific activity. Europe PMC Funders Group

Myocardial hypoxia is an attractive target for diagnostic and prognostic imaging, but current approaches are insufficiently sensitive for clinical use. The PET tracer copper(II)-diacetyl-bis (N4-methylthiosemicarbazone) ( 64 Cu-ATSM) has promise, but its selectivity and sensitivity could be improved by structural modification. We have therefore evaluated a range of 64 Cu-ATSM analogs for imaging hypoxic myocardium. Methods: Isolated rat hearts (n 5 5/group) were perfused with normoxic buffer for 30 min and then hypoxic buffer for 45 min within a custom-built triple-g-detector system to quantify radiotracer infusion, hypoxiadependent cardiac uptake, and washout. A 1-MBq bolus of each candidate tracer (and 18 F-fluoromisonidazole for comparative purposes) was injected into the arterial line during normoxia, and during early and late hypoxia, and their hypoxia selectivity and pharmacokinetics were evaluated. The in vivo pharmacokinetics of promising candidates in healthy rats were then assessed by PET imaging and biodistribution. Results: All tested analogs exhibited hypoxia sensitivity within 5 min. Complexes less lipophilic than 64 Cu-ATSM provided significant gains in hypoxic-to-normoxic contrast (14:1 for 64 Cu-2,3-butanedione bis(thiosemicarbazone) (ATS), 17:1 for 64 Cu-2,3-pentanedione bis(thiosemicarbazone) (CTS), 8:1 for 64 Cu-ATSM, P , 0.05). Hypoxic first-pass uptake was 78.2% 6 7.2% for 64 Cu-ATS and 70.7% 6 14.5% for 64 Cu-CTS, compared with 63.9% 6 11.7% for 64 Cu-ATSM. Cardiac retention of 18 F-fluoromisonidazole increased from 0.44% 6 0.17% during normoxia to 2.24% 6 0.08% during hypoxia. In vivo, normoxic cardiac retention of 64 Cu-CTS was significantly lower than that of 64 Cu-ATSM and 64 Cu-ATS (0.13% 6 0.02% vs. 0.25% 6 0.04% and 0.24% 6 0.03% injected dose, P , 0.05), with retention of all 3 tracers falling to less than 0.7% injected dose within 6 min. 64 Cu-CTS also exhibited lower uptake in liver and lung. Conclusion: 64 Cu-ATS and 64 Cu-CTS exhibit better cardiac hypoxia selectivity and imaging characteristics than the current lead hypoxia tracers, 64 Cu-ATSM and 18 F-fluoromisonidazole.

Background[18F]BF4−, the first 18F-labelled PET imaging agent for the sodium/iodide symporter (NIS), was produced by isotopic exchange yielding a product with limited specific activity (SA, ca. 1 GBq/μmol) posing a risk of sub-optimal target-to-background ratios (TBR) in PET images due to saturation of NIS in vivo. We sought to quantify this risk and to develop a method of production of [18F]BF4− with higher SA.MethodsA new radiosynthesis of [18F]BF4− was developed, involving reaction of [18F]F− with boron trifluoride diethyl etherate under anhydrous conditions, guided by 11B and 19F NMR studies of equilibria involving BF4− and BF3. The SA of the product was determined by ion chromatography. The IC50 of [19F]BF4− as an inhibitor of [18F]BF4− uptake was determined in vitro using HCT116-C19 human colon cancer cells expressing the human form of NIS (hNIS). The influence of [19F]BF4− dose on biodistribution in vivo was evaluated in normal mice by nanoPET imaging and ex vivo tissue counting.ResultsAn IC50 of 4.8 μΜ was found in vitro indicating a significant risk of in vivo NIS saturation at SA achieved by the isotopic exchange labelling method. In vivo thyroid and salivary gland uptake decreased significantly with [19F]BF4− doses above ca. 10 μg/kg. The new radiosynthesis gave high radiochemical purity (>99 %) and moderate yield (15 %) and improved SA (>5 GBq/μmol) from a starting activity of only 1.5 GBq.Conclusions[18F]BF4− produced at previously reported levels of SA (1 GBq/μmol) can lead to reduced uptake in NIS-expressing tissues in mice. This is much less likely in humans. The synthetic approach described provides an alternative for production of [18F]BF4− at higher SA with sufficient yield and without need for unusually high starting activity of [18F]fluoride, removing the risk of NIS saturation in vivo even in mice.Trial registrationISRCTN75827286.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0188-5) contains supplementary material, which is available to authorized users.

A series of metal-chelating lipid conjugates has been designed and synthesized. Each member of the series bears a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) macrocycle attached to the lipid head group, using short n-ethylene glycol (n-EG) spacers of varying length. Liposomes incorporating these lipids, chelated to Gd3+, 64Cu2+, or 111In3+, and also incorporating fluorescent lipids, have been prepared, and their application in optical, magnetic resonance (MR) and single-photon emission tomography (SPECT) imaging of cellular uptake and distribution investigated in vitro and in vivo. We have shown that these multimodal liposomes can be used as functional MR contrast agents as well as radionuclide tracers for SPECT, and that they can be optimized for each application. When shielded liposomes were formulated incorporating 50% of a lipid with a short n-EG spacer, to give nanoparticles with a shallow but even coverage of n-EG, they showed good cellular internalization in a range of tumour cells, compared to the limited cellular uptake of conventional shielded liposomes formulated with 7% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)2000] (DSPE-PEG2000). Moreover, by matching the depth of n-EG coverage to the length of the n-EG spacers of the DOTA lipids, we have shown that similar distributions and blood half lives to DSPE-PEG2000-stabilized liposomes can be achieved. The ability to tune the imaging properties and distribution of these liposomes allows for the future development of a flexible tri-modal imaging agent.