In vitro and preclinical systematic dose-effect studies of Auger electron- and beta particle-emitting radionuclides and external beam radiation for cancer treatment.

PURPOSE: Despite a rise in clinical use of radiopharmaceutical therapies, the biological effects of radionuclides and their relationship with absorbed radiation dose are poorly understood. Here, we set out to define this relationship for Auger electron-emitters [99mTc]TcO4 and [123I]I, and ß--particle-emitter [188Re]ReO4. Studies were carried out using genetically-modified cells that permitted direct radionuclide comparisons. METHODS AND MATERIALS: Triple-negative MDA-MB-231 breast cancer cells, expressing the human sodium/iodide symporter (hNIS) and green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used. In vitro radiotoxicity of [99mTc]TcO4, [123I]I and [188Re]ReO4 was determined using clonogenic assays. Radionuclide uptake, efflux, and subcellular location were used to calculate nuclear-absorbed doses using the Medical Internal Radiation Dose formalism. In vivo studies were performed using female NSG mice bearing orthotopic MDA-MB-231.hNIS-GFP tumors and compared to X-ray-treated (12.6-15 Gy) and untreated cohorts. Absorbed dose per unit activity in tumors and NIS-expressing organs were extrapolated to reference human adult models using OLINDA/EXM®. RESULTS: [99mTc]TcO4- and [123I]I reduced the survival fraction only in hNIS-expressing cells, whereas [188Re]ReO4 reduced survival fraction in hNIS-expressing and parental cells. [123I]I required 2.4-fold and 1.5-fold lower decays/cell to achieve 37% survival compared to [99mTc]TcO4- and [188Re]ReO4, respectively, following 72 hours incubation. Additionally, [99mTc]TcO4-, [123I]I and [188Re]ReO4 had superior cell killing effectiveness in vitro compared to X-rays. In vivo, X-ray led to a greater median survival compared to [188Re]ReO4 and [123I]I (54 days versus 45 and 43 days, respectively). Unlike the X-ray cohort, no metastases were visualized in the radionuclide-treated cohorts. Extrapolated human absorbed doses of [188Re]ReO4 to a 1 g tumor were 13.8-fold and 11.2-fold greater than for [123I]I in female and male models, respectively. CONCLUSIONS: This work reports reference dose-effect data using cell and tumor models for [99mTc]TcO4, [123I]I, and [188Re]ReO4, for the first time. We further demonstrate the tumor controlling effects of [123I]I, and [188Re]ReO4 in comparison to EBRT.

In vivo real-time positron emission particle tracking (PEPT) and single particle PET.

Positron emission particle tracking (PEPT) enables 3D localization and tracking of single positron-emitting radiolabelled particles with high spatiotemporal resolution. The translation of PEPT to the biomedical imaging field has been limited due to the lack of methods to radiolabel biocompatible particles with sufficient specific activity and protocols to isolate a single particle in the sub-micrometre size range, below the threshold for capillary embolization. Here we report two key developments: the synthesis and 68Ga-radiolabelling of homogeneous silica particles of 950 nm diameter with unprecedented specific activities (2.1 ± 1.4 kBq per particle), and the isolation and manipulation of a single particle. We have combined these developments to perform in vivo PEPT and dynamic positron emission tomography (PET) imaging of a single radiolabelled sub-micrometre size particle using a pre-clinical positron emission tomography/computed tomography scanner. This work opens possibilities for quantitative assessment of haemodynamics in vivo in real time, at the whole-body level using minimal amounts of injected radioactive dose and material.

Combined Vorinostat and Chloroquine Inhibit Sodium-Iodide Symporter Endocytosis and Enhance Radionuclide Uptake In Vivo.

PURPOSE: Patients with aggressive thyroid cancer are frequently failed by the central therapy of ablative radioiodide (RAI) uptake, due to reduced plasma membrane (PM) localization of the sodium/iodide symporter (NIS). We aimed to understand how NIS is endocytosed away from the PM of human thyroid cancer cells, and whether this was druggable in vivo. EXPERIMENTAL DESIGN: Informed by analysis of endocytic gene expression in patients with aggressive thyroid cancer, we used mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake, and NanoBRET to understand the mechanisms of NIS endocytosis in transformed cell lines and patient-derived human primary thyroid cells. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and gene expression in BALB/c mice. RESULTS: We identified an acidic dipeptide within the NIS C-terminus that mediates binding to the σ2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine (CQ) modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, CQ treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor vorinostat/SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. CONCLUSIONS: NIS internalization is specifically druggable in vivo. Our data, therefore, provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. See related commentary by Lechner and Brent, p. 1220.

An indium-111-labelled membrane-targeted peptide for cell tracking with radionuclide imaging.

Cell labelling agents that enable longitudinal in vivo tracking of administered cells will support the clinical development of cell-based therapies. Radionuclide imaging with gamma and positron-emitting radioisotopes can provide quantitative and longitudinal mapping of cells in vivo. To make this widely accessible and adaptable to a range of cell types, new, versatile and simple methods for directly radiolabelling cells are required. We have developed [111In]In-DTPA-CTP, the first example of a radiolabelled peptide that binds to the extracellular membrane of cells, for tracking cell distribution in vivo using Single Photon Emission Computed Tomography (SPECT). [111In]In-DTPA-CTP consists of (i) myristoyl groups for insertion into the phospholipid bilayer, (ii) positively charged lysine residues for electrostatic association with negatively charged phospholipid groups at the cell surface and (iii) a diethylenetriamine pentaacetate derivative that coordinates the γ-emitting radiometal, [111In]In3+. [111In]In-DTPA-CTP binds to 5T33 murine myeloma cells, enabling qualitative SPECT tracking of myeloma cells' accumulation in lungs immediately after intravenous administration. This is the first report of a radiolabelled cell-membrane binding peptide for use in cell tracking.

Imaging zinc trafficking in vivo by positron emission tomography with zinc-62.

Non-invasive imaging techniques to dynamically map whole-body trafficking of essential metals in vivo in health and diseases are needed. Despite 62Zn having appropriate physical properties for positron emission tomography (PET) imaging (half-life, 9.3 h; positron emission, 8.2%), its complex decay via 62Cu (half-life, 10 min; positron emission, 97%) has limited its use. We aimed to develop a method to extract 62Zn from a 62Zn/62Cu generator, and to investigate its use for in vivo imaging of zinc trafficking despite its complex decay. 62Zn prepared by proton irradiation of natural copper foil was used to construct a conventional 62Zn/62Cu generator. 62Zn was eluted using trisodium citrate and used for biological experiments, compared with 64Cu in similar buffer. PET/CT imaging and ex vivo tissue radioactivity measurements were performed following intravenous injection in healthy mice. [62Zn]Zn-citrate was readily eluted from the generator with citrate buffer. PET imaging with the eluate demonstrated biodistribution similar to previous observations with the shorter-lived 63Zn (half-life 38.5 min), with significant differences compared to [64Cu]Cu-citrate, notably in pancreas (>10-fold higher at 1 h post-injection). Between 4 and 24 h, 62Zn retention in liver, pancreas, and kidney declined over time, while brain uptake increased. Like 64Cu, 62Zn showed hepatobiliary excretion from liver to intestines, unaffected by fasting. Although it offers limited reliability of scanning before 1 h post-injection, 62Zn-PET allows investigation of zinc trafficking in vivo for >24 h and hence provides a useful new tool to investigate diseases where zinc homeostasis is disrupted in preclinical models and humans.

New Bifunctional Chelators Incorporating Dibromomaleimide Groups for Radiolabeling of Antibodies with Positron Emission Tomography Imaging Radioisotopes.

Positron Emission Tomography (PET) imaging with antibody-based contrast agents frequently uses the radioisotopes [64Cu]Cu2+ and [89Zr]Zr4+. The macrobicyclic chelator commonly known as sarcophagine (sar) is ideal for labeling receptor-targeted biomolecules with [64Cu]Cu2+. The siderophore chelator, desferrioxamine-B (dfo), has been widely used to incorporate [89Zr]Zr4+ into antibodies. Here, we describe new bifunctional chelators of sar and dfo: these chelators have been functionalized with dibromomaleimides (dbm), that enable site-specific and highly stable attachment of molecular cargoes to reduced, solvent-accessible, interstrand native disulfide groups. The new sar-dbm and dfo-dbm derivatives can be easily conjugated with the IgG antibody trastuzumab via reaction with reduced interstrand disulfide groups to give site-specifically modified dithiomaleamic acid (dtm) conjugates, sar-dtm-trastuzumab and dfo-dtm-trastuzumab, in which interstrand disulfides are rebridged covalently with a small molecule linker. Both sar- and dfo-dtm-trastuzumab conjugates have been radiolabeled with [64Cu]Cu2+ and [89Zr]Zr4+, respectively, in near quantitative radiochemical yield (>99%). Serum stability studies, in vivo PET imaging, and biodistribution analyses using these radiolabeled immunoconjugates demonstrate that both [64Cu]Cu-sar-dtm-trastuzumab and [89Zr]Zr-dfo-dtm-trastuzumab possess high stability in biological milieu. Dibromomaleimide technology can be easily applied to enable stable, site-specific attachment of radiolabeled chelators, such as sar and dfo, to native interstrand disulfide regions of antibodies, enabling tracking of antibodies with PET imaging.

Imaging niacin trafficking with positron emission tomography reveals in vivo monocarboxylate transporter distribution.

INTRODUCTION: A sufficient dietary intake of the vitamin niacin is essential for normal cellular function. Niacin is transported into the cells by the monocarboxylate transporters: sodium-dependent monocarboxylate transporter (SMCT1 and SMCT2) and monocarboxylate transporter (MCT1). Despite the importance of niacin in biological systems, surprisingly, its in vivo biodistribution and trafficking in living organisms has not been reported. The availability of niacin radiolabelled with the short-lived positron emitting radionuclide carbon-11 ([11C]niacin) would enable the quantitative in vivo study of this endogenous micronutrient trafficking using in vivo PET molecular imaging. METHODS: [11C]Niacin was synthesised via a simple one-step, one-pot reaction in a fully automated system using cyclotron-produced carbon dioxide ([11C]CO2) and 3-pyridineboronic acid ester via a copper-mediated reaction. [11C]Niacin was administered intravenously in healthy anaesthetised mice placed in a high-resolution nanoScan PET/CT scanner. To further characterize in vivo [11C]niacin distribution in vivo, mice were challenged with either niacin or AZD3965, a potent and selective MCT1 inhibitor. To examine niacin gastrointestinal absorption and body distribution in vivo, no-carrier-added (NCA) and carrier-added (CA) [11C]niacin formulations were administered orally. RESULTS: Total synthesis time including HPLC purification was 25 ± 1 min from end of [11C]CO2 delivery. [11C]Niacin was obtained with a decay corrected radiochemical yield of 17 ± 2%. We report a rapid radioactivity accumulation in the kidney, heart, eyes and liver of intravenously administered [11C]niacin which is consistent with the known in vivo SMCTs and MCT1 transporter tissue expression. Pre-administration of non-radioactive niacin decreased kidney-, heart-, ocular- and liver-uptake and increased urinary excretion of [11C]niacin. Pre-administration of AZD3965 selectively decreased [11C]niacin uptake in MCT1-expressing organs such as heart and retina. Following oral administration of NCA [11C]niacin, a high level of radioactivity accumulated in the intestines. CA abolished the intestinal accumulation of [11C]niacin resulting in a preferential distribution to all tissues expressing niacin transporters and the excretory organs. CONCLUSIONS: Here, we describe the efficient preparation of [11C]niacin as PET imaging agent for probing the trafficking of nutrient demand in healthy rodents by intravenous and oral administration, providing a translatable technique to enable the future exploration of niacin trafficking in humans and to assess its application as a research tool for metabolic disorders (dyslipidaemia) and cancer.

Imaging Biotin Trafficking In Vivo with Positron Emission Tomography.

The water-soluble vitamin biotin is essential for cellular growth, development, and well-being, but its absorption, distribution, metabolism, and excretion are poorly understood. This paper describes the radiolabeling of biotin with the positron emission tomography (PET) radionuclide carbon-11 ([11C]biotin) to enable the quantitative study of biotin trafficking in vivo. We show that intravenously administered [11C]biotin is quickly distributed to the liver, kidneys, retina, heart, and brain in rodents-consistent with the known expression of the biotin transporter-and there is a surprising accumulation in the brown adipose tissue (BAT). Orally administered [11C]biotin was rapidly absorbed in the small intestine and swiftly distributed to the same organs. Preadministration of nonradioactive biotin inhibited organ uptake and increased excretion. [11C]Biotin PET imaging therefore provides a dynamic in vivo map of transporter-mediated biotin trafficking in healthy rodents. This technique will enable the exploration of biotin trafficking in humans and its use as a research tool for diagnostic imaging of obesity/diabetes, bacterial infection, and cancer.

Protocols for Dual Tracer PET/SPECT Preclinical Imaging.

BACKGROUND: Multi-tracer PET/SPECT imaging enables different modality tracers to be present simultaneously, allowing multiple physiological processes to be imaged in the same subject, within a short time-frame. Fluorine-18 and technetium-99m, two commonly used PET and SPECT radionuclides, respectively, possess different emission profiles, offering the potential for imaging one in the presence of the other. However, the impact of the presence of each radionuclide on scanning the other could be significant and lead to confounding results. Here we use combinations of 18F and 99mTc to explore the challenges posed by dual tracer PET/SPECT imaging, and investigate potential practical ways to overcome them. METHODS: Mixed-radionuclide 18F/99mTc phantom PET and SPECT imaging experiments were carried out to determine the crossover effects of each radionuclide on the scans using Mediso nanoScan PET/CT and SPECT/CT small animal scanners. RESULTS: PET scan image quality and quantification were adversely affected by 99mTc activities higher than 100 MBq due to a high singles rate increasing dead-time of the detectors. Below 100 MBq 99mTc, PET scanner quantification accuracy was preserved. SPECT scan image quality and quantification were adversely affected by the presence of 18F due to Compton scattering of 511 keV photons leading to over-estimation of 99mTc activity and increased noise. However, 99mTc:18F activity ratios of > 70:1 were found to mitigate this effect completely on the SPECT. A method for correcting for Compton scatter was also explored. CONCLUSION: Suitable combinations of injection sequence and imaging sequence can be devised to meet specific experimental multi-tracer imaging needs, with only minor or insignificant effects of each radionuclide on the scan of the other.

Imaging of changes in copper trafficking and redistribution in a mouse model of Niemann-Pick C disease using positron emission tomography.

Niemann-Pick C disease (NPC) is an autosomal recessive lysosomal storage disorder resulting from mutations in the NPC1 (95% of cases) or NPC2 genes. Disturbance of copper homeostasis has been reported in NPC1 disease. In this study we have used whole-body positron emission tomography (PET) and brain electronic autoradiography with copper-64 (64Cu), in the form of the copper(II) bis(thiosemicarbazonato) complex 64Cu-GTSM, to image short-term changes in copper trafficking after intravenous injection in a transgenic mouse model of NPC1 disease. 64Cu-GTSM is taken up in all tissues and dissociates rapidly inside cells, allowing monitoring of the subsequent efflux and redistribution of 64Cu from all tissues. Significantly enhanced retention of 64Cu radioactivity was observed in brain, lungs and blood at 15 h post-injection in symptomatic Npc1-/- transgenic mice compared to wildtype controls. The enhanced retention of 64Cu in brain was confirmed by electronic autoradiography, particularly in the midbrain, thalamus, medulla and pons regions. Positron emission tomography imaging with 64Cu in selected chemical forms could be a useful diagnostic and research tool for the management and understanding of NPC1 disease.

Studies of copper trafficking in a mouse model of Alzheimer's disease by positron emission tomography: comparison of 64Cu acetate and 64CuGTSM.

Alzheimer's disease can involve brain copper dyshomeostasis. We aimed to determine the effect of AD-like pathology on 64Cu trafficking in mice, using positron emission tomography (PET imaging), during 24 hours after intravenous administration of ionic 64Cu (Cu(ii) acetate) and 64Cu-GTSM (GTSMH2 = glyoxalbis(thiosemicarbazone)). Copper trafficking was evaluated in 6-8-month-old and 13-15 month-old TASTPM transgenic and wild-type mice, by imaging 0-30 min and 24-25 h after intravenous administration of 64Cu tracer. Regional 64Cu distribution in brains was compared by ex vivo autoradiography to that of amyloid-ß plaque. 64Cu-acetate showed uptake in, and excretion through, liver and kidneys. There was minimal uptake in other tissues by 30 minutes, and little further change after 24 h. Radioactivity within brain was focussed in and around the ventricles and was significantly greater in younger mice. 64CuGTSM was taken up in all tissues by 30 min, remaining high in brain but clearing substantially from other tissues by 24 h. Distribution in brain was not localised to specific regions. TASTPM mice showed no major changes in global or regional 64Cu brain uptake compared to wildtype after administration of 64Cu acetate (unlike 64Cu-GTSM) but efflux of 64Cu from brain by 24 h was slightly greater in 6-8 month-old TASTPM mice than in wildtype controls. Changes in copper trafficking associated with Alzheimer's-like pathology after administration of ionic 64Cu are minor compared to those observed after administration of 64Cu-GTSM. PET imaging with 64Cu could help understand changes in brain copper dynamics in AD and underpin new clinical diagnostic imaging methods.

Simple, mild, one-step labelling of proteins with gallium-68 using a tris(hydroxypyridinone) bifunctional chelator: a 68Ga-THP-scFv targeting the prostate-specific membrane antigen.

BACKGROUND: Labelling proteins with gallium-68 using bifunctional chelators is often problematic because of unsuitably harsh labelling conditions such as low pH or high temperature and may entail post-labelling purification. To determine whether tris(hydroxypyridinone) (THP) bifunctional chelators offer a potential solution to this problem, we have evaluated the labelling and biodistribution of a THP conjugate with a new single-chain antibody against the prostate-specific membrane antigen (PSMA), an attractive target for staging prostate cancer (PCa). A single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, was prepared in order to achieve biokinetics matched to the half-life of gallium-68. The scFv, J591c-scFv, was engineered with a C-terminal cysteine. RESULTS: J591c-scFv was produced in HEK293T cells and purified by size-exclusion chromatography. A maleimide THP derivative (THP-mal) was coupled site-specifically to the C-terminal cysteine residue. The THP-mal-J591c-scFv conjugate was labelled with ammonium acetate-buffered gallium-68 from a 68Ge/68Ga generator at room temperature and neutral pH. The labelled conjugate was evaluated in the PCa cell line DU145 and its PSMA-overexpressing variant in vitro and xenografted in SCID mice. J591c-scFv was produced in yields of 4-6 mg/l culture supernatant and efficiently coupled with the THP-mal bifunctional chelator. Labelling yields > 95% were achieved at room temperature following incubation of 5 µg conjugate with gallium-68 for 5 min without post-labelling purification. 68Ga-THP-mal-J591c-scFv was stable in serum and showed selective binding to the DU145-PSMA cell line, allowing an IC50 value of 31.5 nM to be determined for unmodified J591c-scFv. Serial PET/CT imaging showed rapid, specific tumour uptake and clearance via renal elimination. Accumulation in DU145-PSMA xenografts at 90 min post-injection was 5.4 ± 0.5%ID/g compared with 0.5 ± 0.2%ID/g in DU145 tumours (n = 4). CONCLUSIONS: The bifunctional chelator THP-mal enabled simple, rapid, quantitative, one-step room temperature radiolabelling of a protein with gallium-68 at neutral pH without a need for post-labelling purification. The resultant gallium-68 complex shows high affinity for PSMA and favourable in vivo targeting properties in a xenograft model of PCa.

Complement inhibition by hydroxychloroquine prevents placental and fetal brain abnormalities in antiphospholipid syndrome.

Placental ischemic disease and adverse pregnancy outcomes are frequently observed in patients with antiphospholipid syndrome (APS). Despite the administration of conventional antithrombotic treatment a significant number of women continue to experience adverse pregnancy outcomes, with uncertain prevention and management. Efforts to develop effective pharmacological strategies for refractory obstetric APS cases will be of significant clinical benefit for both mothers and fetuses. Although the antimalarial drug, hydroxychloroquine (HCQ) is increasingly used to treat pregnant women with APS, little is known about its efficacy and mechanism of action of HCQ. Because complement activation plays a crucial and causative role in placental ischemia and abnormal fetal brain development in APS we hypothesised that HCQ prevents these pregnancy complications through inhibition of complement activation. Using a mouse model of obstetric APS that closely resembles the clinical condition, we found that HCQ prevented fetal death and the placental metabolic changes -measured by proton magnetic resonance spectroscopy in APS-mice. Using 111In labelled antiphospholipid antibodies (aPL) we identified the placenta and the fetal brain as the main organ targets in APS-mice. Using this same method, we found that HCQ does not inhibit aPL binding to tissues as was previously suggested from in vitro studies. While HCQ did not affect aPL binding to fetal brain it prevented fetal brain abnormal cortical development. HCQ prevented complement activation in vivo and in vitro. Complement C5a levels in serum samples from APS patients and APS-mice were lower after treatment with HCQ while the antibodies titres remained unchanged. HCQ prevented not only placental insufficiency but also abnormal fetal brain development in APS. By inhibiting complement activation, HCQ might also be an effective antithrombotic therapy.

Lung inflammation does not affect the clearance kinetics of lipid nanocapsules following pulmonary administration.

Lipid nanocapsules (LNCs) are semi-rigid spherical capsules with a triglyceride core that present a promising formulation option for the pulmonary delivery of drugs with poor aqueous solubility. Whilst the biodistribution of LNCs of different size has been studied following intravenous administration, the fate of LNCs following pulmonary delivery has not been reported. We investigated quantitatively whether lung inflammation affects the clearance of 50nm lipid nanocapsules, or is exacerbated by their pulmonary administration. Studies were conducted in mice with lipopolysaccharide-induced lung inflammation compared to healthy controls. Particle deposition and nanocapsule clearance kinetics were measured by single photon emission computed tomography/computed tomography (SPECT/CT) imaging over 48 h. A significantly lower lung dose of (111)In-LNC50 was achieved in the lipopolysaccharide (LPS)-treated animals compared with healthy controls (p<0.001). When normalised to the delivered lung dose, the clearance kinetics of (111)In-LNC50 from the lungs fit a first order model with an elimination half-life of 10.5±0.9h (R(2)=0.995) and 10.6±0.3h (R(2)=1.000) for healthy and inflamed lungs respectively (n=3). In contrast, (111)In-diethylene triamine pentaacetic acid (DTPA), a small hydrophilic molecule, was cleared rapidly from the lungs with the majority of the dose absorbed within 20min of administration. Biodistribution to lungs, stomach-intestine, liver, trachea-throat and blood at the end of the imaging period was unaltered by lung inflammation. This study demonstrated that lung clearance and whole body distribution of lipid nanocapsules were unaffected by the presence of acute lung inflammation.

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

UNLABELLED: 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 (64)Cu, 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 (64)Cu bis(thiosemicarbazone) complex (64)Cu-GTSM (glyoxalbis(N(4)-methyl-3-thiosemicarbazonato) copper(II)), which crosses the blood-brain barrier and releases (64)Cu bioreductively into cells. Animals were intravenously injected with (64)Cu-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 (64)Cu in the brain 24 h after injection was also evaluated via ex vivo autoradiography and compared with amyloid-ß 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 (64)Cu at 30 min (P < 0.01) and 24 h (P < 0.05) after injection of the tracer and faster (by a factor of ~5) (64)Cu clearance from the brain (P < 0.01). Atlas-based quantification and texture analysis revealed significant differences in regional brain uptake of (64)Cu and PET image heterogeneity between the 2 groups of mice. Ex vivo autoradiography showed that regional brain distribution of (64)Cu at 24 h after injection did not correlate with amyloid-ß plaque distribution in TASTPM mice. CONCLUSION: The trafficking of (64)Cu in the brain after administration of (64)Cu-GTSM is significantly altered by AD-like pathology in the TASTPM mouse model, suggesting that (64)Cu-GTSM PET imaging warrants clinical evaluation as a diagnostic tool for AD and possibly other neurodegenerative disorders.

Quantification of experimental venous thrombus resolution by longitudinal nanogold-enhanced micro-computed tomography.

INTRODUCTION: The assessment of thrombus size following treatments directed at preventing thrombosis or enhancing its resolution has generally relied on physical or histological methods. This cross-sectional design imposes the need for increased numbers of animals for experiments. Micro-computed tomography (microCT) has been used to detect the presence of venous thrombus in experimental models but has yet to be used in a quantitative manner. In this study, we investigate the use of contrast-enhanced microCT for the longitudinal assessment of experimental venous thrombus resolution. MATERIALS AND METHODS: Thrombi induced by stenosis of the inferior vena cava in mice were imaged by contrast-enhanced microCT at 1, 7 and 14 days post-induction (n=18). Thrombus volumes were determined longitudinally by segmentation and 3D volume reconstruction of microCT scans and by standard end-point histological analysis at day 14. An additional group of thrombi were analysed solely by histology at 1, 7 and 14 days post-induction (n=15). RESULTS: IVC resident thrombus was readily detectable by contrast-enhanced microCT. MicroCT-derived measurements of thrombus volume correlated well with time-matched histological analyses (ICC=0.75, P<0.01). Thrombus volumes measured by microCT were significantly greater than those derived from histological analysis (P<0.001). Intra- and inter-observer analyses were highly correlated (ICC=0.99 and 0.91 respectively, P<0.0001). Further histological analysis revealed noticeable levels of contrast agent extravasation into the thrombus that was associated with the presence of neovascular channels, macrophages and intracellular iron deposits. CONCLUSION: Contrast-enhanced microCT represents a reliable and reproducible method for the longitudinal assessment of venous thrombus resolution providing powerful paired data.

Technetium-99m and rhenium-188 complexes with one and two pendant bisphosphonate groups for imaging arterial calcification.

The first (99m)Tc and (188)Re complexes containing two pendant bisphosphonate groups have been synthesised, based on the mononuclear M(v) nitride core with two dithiocarbamate ligands each with a pendant bisphosphonate. The structural identity of the (99)Tc and stable rhenium analogues as uncharged, mononuclear nitridobis(dithiocarbamate) complexes was determined by electrospray mass spectrometry. The (99m)Tc complex showed greater affinity for synthetic and biological hydroxyapatite, and greater stability in biological media, than the well-known but poorly-characterised and inhomogeneous bone imaging agent (99m)Tc-MDP. It gave excellent SPECT images of both bone calcification (mice and rats) and vascular calcification (rat model), but the improved stability and the availability of two pendant bisphosphonate groups conferred no dramatic advantage in imaging over the conventional (99m)Tc-MDP agent in which the bisphosphonate group is bound directly to Tc. The (188)Re complex also showed preferential uptake in bone. These tracers and the biological model of vascular calcification offer the opportunity to study the biological interpretation and clinical potential of radionuclide imaging of vascular calcification and to deliver radionuclide therapy to bone metastases.

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 [(18)F]-fluoride and bisphosphonate groups. A high radiolabeling efficiency, 97% for (18)F-fluoride and 100% for (64)Cu-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 (18)F from NPs, but no sign of efflux of (64)Cu.

Monitoring the efficacy of dendritic cell vaccination by early detection of (99m) Tc-HMPAO-labelled CD4(+) T cells.

DC vaccines have been used to induce tumour-specific cytotoxic T cells . However, this approach to cancer immunotherapy has had limited success. To be successful, injected DCs need to migrate to the LNs where they can stimulate effector T cells . We and others have previously demonstrated by MRI that tumour antigen-pulsed-DCs labelled ex vivo with superparamagnetic iron oxide nanoparticles migrated to the draining LNs and are capable of activating antigen-specific T cells . The results from our study demonstrated that ex vivo superparamagnetic iron oxide nanoparticles-labelled and OVA-pulsed DCs prime cytotoxic CD8(+) T-cell responses to protect against a B16-OVA tumour challenge. In the clinic, a possible noninvasive surrogate marker for efficacy of DC vaccination is to image the specific migration and accumulation of T cells following DC vaccination.

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 Gd(3+), (64)Cu(2+), or (111)In(3+), 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.