Kavitha Sunassee

Comparison of 64Cu-Complexing Bifunctional Chelators for Radioimmunoconjugation: Labeling Efficiency, Specific Activity, and in Vitro/in Vivo Stability

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-SCN-Bn-Oxo-DO3A, p-SCN-NOTA, and p-SCN-PCTA), three DTPA derivatives (p-SCN-Bn-DTPA, p-SCN-CHX-A″-DTPA, and ITC-2B3M-DTPA), and a macrobicyclic hexamine (sarcophagine) chelator (sar-CO2H) = (1-NH2-8-NHCO(CH2)3CO2H)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-CO2H were conjugated to rituximab by thiourea bond formation with an average of 4.9 ± 0.9 chelators per antibody molecule. Sar-CO2H 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 the highest radiochemical yield (95%) under very dilute conditions (31 nM NOTA-rituximab conjugate). Similarly, sar-CO-rituximab, containing 1/10th the number of chelators per antibody compared to that of 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. While all the macrocyclic bifunctional chelators are suitable for molecular imaging using (64)Cu-labeled antibody conjugates, NOTA and sar-CO2H show significant advantages over the others in that they can be radiolabeled rapidly at room temperature, under dilute conditions, resulting in high specific activity.

Efficient bifunctional gallium-68 chelators for positron emission tomography: tris(hydroxypyridinone) ligands

A new tripodal tris(hydroxypyridinone) bifunctional chelator for gallium allows easy production of (68)Ga-labelled proteins rapidly under mild conditions in high yields at exceptionally high specific activity and low concentration.

National Electrical Manufacturers Association NU-4 Performance Evaluation of the PET Component of the NanoPET/CT Preclinical PET/CT Scanner

The NanoPET/CT represents the latest generation of commercial preclinical PET/CT systems. This article presents a performance evaluation of the PET component of the system according to the National Electrical Manufacturers Association (NEMA) NU-4 2008 standard. Methods: The NanoPET/CT consists of 12 lutetium yttrium orthosilicate:cerium modular detectors forming 1 ring, with 9.5-cm axial coverage and a 16-cm animal port. Each detector crystal is 1.12 × 1.12 × 13 mm, and 1 module contains 81 × 39 of these crystals. An optical light guide transmits the scintillation light to the flat-panel multianode position-sensitive photomultiplier tubes. Analog-to-digital converter cards and a field-programmable gate array–based data-collecting card provide the readout. Spatial resolution, sensitivity, counting rate capabilities, and image quality were evaluated in accordance with the NEMA NU-4 standard. Energy and temporal resolution measurements and a mouse imaging study were performed in addition to the standard. Results: Energy resolution was 19% at 511 keV. The spatial resolution, measured as full width at half maximum on single-slice rebinning/filtered backprojection–reconstructed images, approached 1 mm on the axis and remained below 2.5 mm in the central 5-cm transaxial region both in the axial center and at one-quarter field of view. The maximum absolute sensitivity for a point source at the center of the field of view was 7.7%. The maximum noise equivalent counting rates were 430 kcps at 36 MBq and 130 kcps at 27 MBq for the mouse- and rat-sized phantoms, respectively. The uniformity and recovery coefficients were measured with the image-quality phantom, giving good-quality images. In a mouse study with an 18F-labeled thyroid-specific tracer, the 2 lobes of the thyroid were clearly distinguishable, despite the small size of this organ. The flexible readout system allowed experiments to be performed in an efficient manner, and the system remained stable throughout. Conclusion: The large number of detector crystals, arranged with a fine pitch, results in excellent spatial resolution, which is the best reported for currently available commercial systems. The absolute sensitivity is high over the field of view. Combined with the excellent image quality, these features make the NanoPET/CT a powerful tool for preclinical research.

Incorporation of paramagnetic, fluorescent and PET/SPECT contrast agents into liposomes for multimodal imaging

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.

Aluminium hydroxide stabilised MnFe2O4 and Fe3O4 nanoparticles as dual-modality contrasts agent for MRI and PET imaging

Magnetic nanoparticles (NPs) MnFe2O4 and Fe3O4 were stabilised by depositing an Al(OH)3 layer via a hydrolysis process. The particles displayed excellent colloidal stability in water and a high affinity to [18F]-fluoride and bisphosphonate groups. A high radiolabeling efficiency, 97% for 18F-fluoride and 100% for 64Cu-bisphosphonate conjugate, was achieved by simply incubating NPs with radioactivity solution at room temperature for 5 min. The properties of particles were strongly dependant on the thickness and hardness of the Al(OH)3 layer which could in turn be controlled by the hydrolysis method. The application of these Al(OH)3 coated magnetic NPs in molecular imaging has been further explored. The results demonstrated that these NPs are potential candidates as dual modal probes for MR and PET. In vivo PET imaging showed a slow release of 18F from NPs, but no sign of efflux of 64Cu.

Performance Evaluation of an MRI-Compatible Pre-Clinical PET System Using Long Optical Fibers

We have designed and constructed an MR-compatible PET system for fully simultaneous PET/MR studies of small animals. The scanner uses long optical fibers to distance the magnetic field sensitive PET PMTs from the high magnetic field at the center of an MR scanner. It is a single slice system with an inner diameter of 7 cm. A full evaluation of the performance of the PET system and the results of an MR compatibility assessment in a Philips Achieva whole body 3 T MRI scanner are presented. The reconstructed resolution of the PET scanner is 1.5 mm at the center falling to 2.5 mm at the edge of the field of view; the system sensitivity is 0.95%; the count rate is linear up to an activity of 6 MBq (~4 kcps) and the scatter fraction is 42% which can be reduced to 26% using MR-compatible gamma shields. Simultaneous PET/MR images of phantoms and a mouse have been acquired. The system is highly MR compatible, as demonstrated here, showing no degradation in performance of either the MR or PET system in the presence of the other modality. The system will be used to demonstrate novel pre-clinical applications of simultaneous PET/MR.

Biocompatible inorganic nanoparticles for [18F]-fluoride binding with applications in PET imaging.

A wide selection of insoluble nanoparticulate metal salts was screened for avid binding of [(18)F]-fluoride. Hydroxyapatite and aluminium hydroxide nanoparticles showed particularly avid and stable binding of [(18)F]-fluoride in various biological media. The in vivo behaviour of the [(18)F]-labelled hydroxyapatite and aluminium hydroxide particles was determined by PET-CT imaging in mice. [(18)F]-labelled hydroxyapatite was stable in circulation and when trapped in various tissues (lung embolisation, Subcutaneous and intramuscular), but accumulation in liver via reticuloendothelial clearance was followed by gradual degradation and release of [(18)F]-fluoride (over a period of 4 h) which accumulated in bone. [(18)F]-labelled aluminium hydroxide was also cleared to liver and spleen but degraded slightly even without liver uptake (Subcutaneous and intramuscular). Both materials have properties that are an attractive basis for the design of molecular targeted PET imaging agents labelled with (18)F.

In vivo biocompatibility, clearance, and biodistribution of albumin vehicles for pulmonary drug delivery.

The development of clinically acceptable albumin-based nanoparticle formulations for use in pulmonary drug delivery has been hindered by concerns about the toxicity of nanomaterials in the lungs combined with a lack of information on albumin nanoparticle clearance kinetics and biodistribution. In this study, the in vivo biocompatibility of albumin nanoparticles was investigated following a single administration of 2, 20, and 390 μg/mouse, showing no inflammatory response (TNF-α and IL-6, cellular infiltration and protein concentration) compared to vehicle controls at the two lower doses, but elevated mononucleocytes and a mild inflammatory effect at the highest dose tested. The biodistribution and clearance of 111In labelled albumin solution and nanoparticles over 48 h following a single pulmonary administration to mice was investigated by single photon emission computed tomography and X-ray computed tomography imaging and terminal biodistribution studies. 111In labelled albumin nanoparticles were cleared more slowly from the mouse lung than 111In albumin solution (64.1 ± 8.5% vs 40.6 ± 3.3% at t = 48 h, respectively), with significantly higher (P < 0.001) levels of albumin nanoparticle-associated radioactivity located within the lung tissue (23.3 ± 4.7%) compared to the lung fluid (16.1 ± 4.4%). Low amounts of 111In activity were detected in the liver, kidneys, and intestine at time points > 24 h indicating that small amounts of activity were cleared from the lungs both by translocation across the lung mucosal barrier, as well as mucociliary clearance. This study provides important information on the fate of albumin vehicles in the lungs, which may be used to direct future formulation design of inhaled nanomedicines.

PET performance evaluation of a pre-clinical SiPM based MR-compatible PET scanner

We have carried out a PET performance evaluation of an SiPM based scanner designed for fully simultaneous preclinical PET/MR studies. The PET scanner has an inner diameter of 20 cm with a crystal size of 1.3 by 1.3 by 10 mm. The crystals are read out using MR-compatible SiPMs to allow the PET scanner to be located within a Philips 3T Achieva MRI scanner. The spatial resolution of the system, measured using SSRB and 2D FBP is just under 2.4mm in the trans-axial and axial directions. The system sensitivity is 0.6% for a point source at the centre of the field of view. The true coincidence count rate shows no sign of saturating at 30 MBq, at which point the randoms fraction is 9%, and the scatter fraction for a rat sized object is approximately 23%. Artefact-free images of phantoms have been obtained using SSRB/FBP and iterative reconstructions. The current performance is limited because only one of three axial ring positions is currently populated with detectors, and limitations of the first-generation detector readout ASIC used in the system. The performance of the system as described is sufficient for imaging rat-sized animals and large organs within the mouse. We have demonstrated here the feasibility of using the system to investigate dynamic processes simultaneously in a mouse using PET and MR with a dual labeled PET/MR probe. Extrapolating from the current performance results we anticipate that population of all three detector rings and upgrading of the readout ASIC will result in an MR-compatible PET scanner with performance similar to that of state-of-the-art non-MR-compatible pre-clinical systems.

PET Imaging of Copper Trafficking in a Mouse Model of Alzheimer Disease

Alzheimer disease (AD) is a fatal neurodegenerative disorder characterized by progressive neuronal loss and cognitive decline. The lack of reliable and objective diagnostic markers for AD hampers early disease detection and treatment. Growing evidence supports the existence of a dysregulation in brain copper trafficking in AD. The aim of this study was to investigate brain copper trafficking in a transgenic mouse model of AD by PET imaging with 64Cu, to determine its potential as a diagnostic biomarker of the disorder. Methods: Brain copper trafficking was evaluated in 6- to 8-mo-old TASTPM transgenic mice and age-matched wild-type controls using the 64Cu bis(thiosemicarbazone) complex 64Cu-GTSM (glyoxalbis(N4-methyl-3-thiosemicarbazonato) copper(II)), which crosses the blood–brain barrier and releases 64Cu bioreductively into cells. Animals were intravenously injected with 64Cu-GTSM and imaged at 0–30 min and 24–25 h after injection. The images were analyzed by atlas-based quantification and texture analysis. Regional distribution of 64Cu in the brain 24 h after injection was also evaluated via ex vivo autoradiography and compared with amyloid-β plaque deposition in TASTPM mice. Results: Compared with controls, in TASTPM mice PET image analysis demonstrated significantly increased (by a factor of ∼1.3) brain concentration of 64Cu at 30 min (P < 0.01) and 24 h (P < 0.05) after injection of the tracer and faster (by a factor of ∼5) 64Cu clearance from the brain (P < 0.01). Atlas-based quantification and texture analysis revealed significant differences in regional brain uptake of 64Cu and PET image heterogeneity between the 2 groups of mice. Ex vivo autoradiography showed that regional brain distribution of 64Cu at 24 h after injection did not correlate with amyloid-β plaque distribution in TASTPM mice. Conclusion: The trafficking of 64Cu in the brain after administration of 64Cu-GTSM is significantly altered by AD-like pathology in the TASTPM mouse model, suggesting that 64Cu-GTSM PET imaging warrants clinical evaluation as a diagnostic tool for AD and possibly other neurodegenerative disorders.