In Vivo Trafficking of the Anticancer Drug Tris(8-Quinolinolato) Gallium (III) (KP46) by Gallium-68/67 PET/SPECT Imaging.

KP46 (tris(hydroxyquinolinato)gallium(III)) is an experimental, orally administered anticancer drug. Its absorption, delivery to tumours, and mode of action are poorly understood. We aimed to gain insight into these issues using gallium-67 and gallium-68 as radiotracers with SPECT and PET imaging in mice. [67Ga]KP46 and [68Ga]KP46, compared with [68Ga]gallium acetate, were used for logP measurements, in vitro cell uptake studies in A375 melanoma cells, and in vivo imaging in mice bearing A375 tumour xenografts up to 48 h after intravenous (tracer level) and oral (tracer and bulk) administration. 68Ga was more efficiently accumulated in A375 cells in vitro when presented as [68Ga]KP46 than as [68Ga]gallium acetate, but the reverse was observed when intravenously administered in vivo. After oral administration of [68/67Ga]KP46, absorption of 68Ga and 67Ga from the GI tract and delivery to tumours were poor, with the majority excreted in faeces. By 48 h, low but measurable amounts were accumulated in tumours. The distribution in tissues of absorbed radiogallium and octanol extraction of tissues suggested trafficking as free gallium rather than as KP46. We conclude that KP46 likely acts as a slow releaser of gallium ions which are inefficiently absorbed from the GI tract and trafficked to tissues, including tumour and bone.

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.

Non-invasive radionuclide imaging of trace metal trafficking in health and disease: "PET metallomics".

Several specific metallic elements must be present in the human body to maintain health and function. Maintaining the correct quantity (from trace to bulk) and location at the cell and tissue level is essential. The study of the biological role of metals has become known as metallomics. While quantities of metals in cells and tissues can be readily measured in biopsy and autopsy samples by destructive analytical techniques, their trafficking and its role in health and disease are poorly understood. Molecular imaging with radionuclides - positron emission tomography (PET) and single photon emission computed tomography (SPECT) - is emerging as a means to non-invasively study the acute trafficking of essential metals between organs, non-invasively and in real time, in health and disease. PET scanners are increasingly widely available in hospitals, and methods for producing radionuclides of some of the key essential metals are developing fast. This review summarises recent developments in radionuclide imaging technology that permit such investigations, describes the radiological and physicochemical properties of key radioisotopes of essential trace metals and useful analogues, and introduces current and potential future applications in preclinical and clinical investigations to study the biology of essential trace metals in health and disease.

In vivo trafficking of a tumor-targeting IgE antibody: molecular imaging demonstrates rapid hepatobiliary clearance compared to IgG counterpart.

IgE antibodies elicit powerful immune responses, recruiting effector cells to tumors more efficiently and with greater cytotoxicity than IgG antibodies. Consequently, IgE antibodies are a promising alternative to conventional IgG-based therapies in oncology (AllergoOncology). As the pharmacokinetics of IgE antibodies are less well understood, we used molecular imaging in mice to compare the distribution and elimination of IgE and IgG antibodies targeting the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4). Anti-CSPG4 IgE and IgG1 antibodies with human Fc domains were radiolabeled with 111In. CSPG4-expressing A375 human melanoma xenografts implanted in NOD-scid IL2rg-/- mice were also engrafted with human immune cells by intravenous administration. 111In-anti-CSPG4 antibodies were administered intravenously. Their distribution was determined by single-photon emission computed tomography (SPECT) and ex vivo gamma-counting over 120 h. SPECT imaging was conducted from 0 to 60 min after antibody administration to precisely measure the early phase of IgE distribution. 111In-labeled anti-CSPG4 IgG and IgE showed serum stability in vitro of >92% after 5 days. In A375 xenograft-bearing mice, anti-CSPG4 IgE showed much faster blood clearance and higher accumulation in the liver compared to anti-CSPG4 IgG. However, tumor-to-blood and tumor-to-muscle ratios were similar between the antibody isotypes and higher compared with a non-tumor-targeting isotype control IgE. IgE excretion was much faster than IgG. In non-tumor-bearing animals, early SPECT imaging revealed a blood clearance half-life of 10 min for IgE. Using image-based quantification, we demonstrated that the blood clearance of IgE is much faster than that of IgG while the two isotypes showed comparable tumor-to-blood ratios.

Targeted Auger electron-emitter therapy: Radiochemical approaches for thallium-201 radiopharmaceuticals.

INTRODUCTION: Thallium-201 is a radionuclide that has previously been used clinically for myocardial perfusion scintigraphy. Although in this role it has now been largely replaced by technetium-99 m radiopharmaceuticals, thallium-201 remains attractive in the context of molecular radionuclide therapy for cancer micrometastases or single circulating tumour cells. This is due to its Auger electron (AE) emissions, which are amongst the highest in total energy and number per decay for AE-emitters. Currently, chemical platforms to achieve this potential through developing thallium-201-labelled targeted radiopharmaceuticals are not available. Here, we describe convenient methods to oxidise [201Tl]Tl(I) to chelatable [201Tl]Tl(III) and identify challenges in stable chelation of thallium to support future synthesis of effective [201Tl]-labelled radiopharmaceuticals. METHODS: A plasmid pBR322 assay was carried out to determine the DNA damaging properties of [201Tl]Tl(III). A range of oxidising agents (ozone, oxygen, hydrogen peroxide, chloramine-T, iodogen, iodobeads, trichloroisocyanuric acid) and conditions (acidity, temperature) were assessed using thin layer chromatography. Chelators EDTA, DTPA and DOTA were investigated for their [201Tl]Tl(III) radiolabelling efficacy and complex stability. RESULTS: Isolated plasmid studies demonstrated that [201Tl]Tl(III) can induce single and double-stranded DNA breaks. Iodo-beads, iodogen and trichloroisocyanuric acid enabled more than 95% conversion from [201Tl]Tl(I) to [201Tl]Tl(III) under conditions compatible with future biomolecule radiolabelling (mild pH, room temperature and post-oxidation removal of oxidising agent). Although chelation of [201Tl]Tl(III) was possible with EDTA, DTPA and DOTA, only radiolabeled DOTA showed good stability in serum. CONCLUSIONS: Decay of [201Tl]Tl(III) in proximity to DNA causes DNA damage. Iodobeads provide a simple, mild method to convert thallium-201 from a 1+ to 3+ oxidation state and [201Tl]Tl(III) can be chelated by DOTA with moderate stability. Of the well-established chelators evaluated, DOTA is most promising for future molecular radionuclide therapy using thallium-201; nevertheless, a new generation of chelating agents offering resistance to reduction and dissociation of [201Tl]Tl(III) complexes is required.

The Hantzsch reaction for nitrogen-13 PET: preparation of [13N]nifedipine and derivatives.

Nitrogen-13 is an attractive but under-used PET radionuclide for labelling molecules of biological and pharmaceutical interest, complementing other PET radionuclides. Its short half-life (t1/2 = 9.97 min) imposes synthetic challenges, but we have expanded the hitherto limited pool of 13N labelling strategies and tracers by adapting the multicomponent Hantzsch condensation reaction to prepare a library of 13N-labelled 1,4-dihydropyridines from [13N]ammonia, including the widely-used drug nifedipine. This represents a key advance in 13N PET radiochemistry, and will serve to underpin the renewed interest in clinical opportunities offered by short-lived PET tracers.

Manipulating the In Vivo Behaviour of 68Ga with Tris(Hydroxypyridinone) Chelators: Pretargeting and Blood Clearance.

Pretargeting is widely explored in immunoPET as a strategy to reduce radiation exposure of non-target organs and allow the use of short-lived radionuclides that would not otherwise be compatible with the slow pharmacokinetic profiles of antibodies. Here we investigate a pretargeting strategy based on gallium-68 and the chelator THPMe as a high-affinity pair capable of combining in vivo. After confirming the ability of THPMe to bind 68Ga in vivo at low concentrations, the bifunctional THPMe-NCS was conjugated to a humanised huA33 antibody targeting the A33 glycoprotein. Imaging experiments performed in nude mice bearing A33-positive SW1222 colorectal cancer xenografts compared pretargeting (100 µg of THPMe-NCS-huA33, followed after 24 h by 8-10 MBq of 68Ga3+) with both a directly labelled radioimmunoconjugate (89Zr-DFO-NCS-huA33, 88 µg, 7 MBq) and a 68Ga-only negative control (8-10 MBq of 68Ga3+). Imaging was performed 25 h after antibody administration (1 h after 68Ga3+ administration for negative control). No difference between pretargeting and the negative control was observed, suggesting that pretargeting via metal chelation is not feasible using this model. However, significant accumulation of "unchelated" 68Ga3+ in the tumour was found (12.9 %ID/g) even without prior administration of THPMe-NCS-huA33, though tumour-to-background contrast was impaired by residual activity in the blood. Therefore, the 68Ga-only experiment was repeated using THPMe (20 µg, 1 h after 68Ga3+ administration) to clear circulating 68Ga3+, producing a three-fold improvement of the tumour-to-blood activity concentration ratio. Although preliminary, these results highlight the potential of THPMe as a 68Ga clearing agent in imaging applications with gallium citrate.

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.

Convergent synthesis of 13N-labelled Peptidic structures using aqueous [13N]NH3.

BACKGROUND: Nitrogen-13 has a 10-min half-life which places time constraints on the complexity of viable synthetic methods for its incorporation into PET imaging agents. In exploring ways to overcome this limitation, we have used the Ugi reaction to develop a rapid one-pot method for radiolabelling peptidic molecules using [13N]NH3 as a synthetic precursor. METHODS: Carrier-added [13N]NH3 (50 µL) was added to a solution of carboxylic acid, aldehyde, and isocyanide in 2,2,2-TFE (200 µL). The mixture was heated in a microwave synthesiser at 120 °C for 10 min. Reactions were analysed by radio-HPLC and radio-LCMS. Isolation of the target 13N-labelled peptidic Ugi compound was achieved via semi-preparative radio-HPLC as demonstrated for Ugi1. RESULTS: Radio-HPLC analysis of each reaction confirmed the formation of radioactive products co-eluting with their respective reference standards with radiochemical yields of the crude products ranging from 11% to 23%. Two cyclic γ-lactam structures were also achieved via intra-molecular reactions. Additional radioactive by-products observed in the radio-chromatogram were identified as 13N-labelled di-imines formed from the reaction of [13N]NH3 with two isocyanide molecules. The desired 13N-labelled Ugi product was isolated using semi-preparative HPLC. CONCLUSION: We have developed a one-pot method that opens up new routes to radiolabel complex, peptidic molecules with 13N using aqueous [13N]NH3 as a synthetic precursor.

Evaluation of [18F]-tetrafluoroborate as a potential PET imaging agent for the human sodium/iodide symporter in a new colon carcinoma cell line, HCT116, expressing hNIS.

PURPOSE: Accumulation of iodide and other substrates via the human sodium/iodide symporter (hNIS) is fundamental to imaging and therapy of thyroid disease, hNIS reporter gene imaging and hNIS-mediated gene therapy. There is no readily available positron emission tomography (PET) tracer for hNIS. Our aim was to develop a colon carcinoma cell line stably expressing hNIS, and use it to evaluate a novel hNIS PET tracer, [18F]-tetrafluoroborate. METHODS: Colon carcinoma cell line, HCT116, was stably transfected with hNIS, thus producing a cell line, HCT116-C19, with high hNIS expression. A Fisher rat thyroid cell line, FRTL5, which expresses rat sodium/iodide symporter when stimulated with thyroid-stimulating hormone, was used for comparison. Accumulation of [188Re]-perrhenate, [99mTc]-pertechnetate and [18F]-tetrafluoroborate was evaluated with and without perchlorate inhibition using an automated radioimmune assay system, LigandTracer. The affinity of [18F]-tetrafluoroborate for hNIS, and its half-maximal inhibitory concentration (IC50) for the inhibition of [99mTc]-pertechnetate transport were determined from the plateau accumulation of [18F]-tetrafluoroborate and [99mTc]-pertechnetate, respectively, as a function of tetrafluoroborate concentration. RESULTS: [18F]-tetrafluoroborate accumulated effectively in both FRTL5 and HCT116-C19 cells. The accumulation in HCT116-C19 cells (plateau accumulation 31%) was comparable to that of [188Re]-perrhenate (41%) and [99mTc]-pertechnetate (46%). Its affinity for hNIS and half-maximal inhibitory concentration (IC50) for the inhibition of pertechnetate uptake was approximately micromolar. CONCLUSION: We have produced a human colon cell line with a stable constitutive expression of functional hNIS (HCT116-hNIS-C19). [18F]-tetrafluoroborate accumulates in cells expressing hNIS or rat sodium/iodide symporter and is a potential PET imaging agent in thyroid disease and hNIS reporter gene imaging.