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.

Oxorhenium(V) and Oxotechnetium(V) Complexes of N3S Tetradentate Ligands with a Styrylpyridyl Functional Group: Toward Imaging Agents to Assist in the Diagnosis of Alzheimer's Disease.

Alzheimer's disease (AD) is associated with the presence of amyloid plaques in the brain mainly comprised of aggregated forms of amyloid-ß (Aß). Molecules radiolabeled with technetium-99m that cross the blood-brain barrier (BBB) and selectively bind to Aß plaques have the potential to assist in the diagnosis of AD using single-photon emission computed tomography imaging. In this work, three new tetradentate ligands of pyridyl, amide, amine and thiol donors, featuring a styrylpyridyl group that is known to interact with amyloid plaques, were prepared. The new ligands formed charge-neutral and lipophilic complexes with the [Tc═O]3+ and [Re═O]3+ motifs, and two rhenium complexes were characterized by X-ray crystallography. The rhenium(V) complexes interact with synthetic Aß1-40 and amyloid plaques on human brain tissue. Two of the new ligands were radiolabeled with 99mTc using a kit-based approach, and their biodistribution in wild-type mice was evaluated. The presence of amide donors in the tetradentate ligand increased the stability of the respective [Tc═O]3+ complexes but reduced brain uptake. While the complexes were able to cross the BBB, the degree of uptake in the brain was not sufficient to justify further investigation of these complexes.

Resolving the cellular specificity of TSPO imaging in a rat model of peripherally-induced neuroinflammation.

The increased expression of 18 kDa Translocator protein (TSPO) is one of the few available biomarkers of neuroinflammation that can be assessed in humans in vivo by positron emission tomography (PET). TSPO PET imaging of the central nervous system (CNS) has been widely undertaken, but to date no clear consensus has been reached about its utility in brain disorders. One reason for this could be because the interpretation of TSPO PET signal remains challenging, given the cellular heterogeneity and ubiquity of TSPO in the brain. The aim of the current study was to ascertain if TSPO PET imaging can be used to detect neuroinflammation induced by a peripheral treatment with a low dose of the endotoxin, lipopolysaccharide (LPS), in a rat model (ip LPS), and investigate the origin of TSPO signal changes in terms of their cellular sources and regional distribution. An initial pilot study utilising both [18F]DPA-714 and [11C]PK11195 TSPO radiotracers demonstrated [18F]DPA-714 to exhibit a significantly higher lesion-related signal in the intracerebral LPS rat model (ic LPS) than [11C]PK11195. Subsequently, [18F]DPA-714 was selected for use in the ip LPS study. Twenty-four hours after ip LPS, there was an increased uptake of [18F]DPA-714 across the whole brain. Further analyses of regions of interest, using immunohistochemistry and RNAscope Multiplex fluorescence V2 in situ hybridization technology, showed TSPO expression in microglia, monocyte derived-macrophages, astrocytes, neurons and endothelial cells. The expression of TSPO was significantly increased after ip LPS in a region-dependent manner: with increased microglia, monocyte-derived macrophages and astrocytes in the substantia nigra, in contrast to the hippocampus where TSPO was mostly confined to microglia and astrocytes. In summary, our data demonstrate the robust detection of peripherally-induced neuroinflammation in the CNS utilising the TSPO PET radiotracer, [18F]DPA-714, and importantly, confirm that the resultant increase in TSPO signal increase arises mostly from a combination of microglia, astrocytes and monocyte-derived macrophages.

Endoplasmic Reticulum Stress Mediates Vascular Smooth Muscle Cell Calcification via Increased Release of Grp78 (Glucose-Regulated Protein, 78 kDa)-Loaded Extracellular Vesicles.

OBJECTIVE: Vascular calcification is common among aging populations and mediated by vascular smooth muscle cells (VSMCs). The endoplasmic reticulum (ER) is involved in protein folding and ER stress has been implicated in bone mineralization. The role of ER stress in VSMC-mediated calcification is less clear. Approach and Results: mRNA expression of the ER stress markers PERK (PKR (protein kinase RNA)-like ER kinase), ATF (activating transcription factor) 4, ATF6, and Grp78 (glucose-regulated protein, 78 kDa) was detectable in human vessels with levels of PERK decreased in calcified plaques compared to healthy vessels. Protein deposition of Grp78/Grp94 was increased in the matrix of calcified arteries. Induction of ER stress accelerated human primary VSMC-mediated calcification, elevated expression of some osteogenic markers (Runx2 [RUNX family transcription factor 2], OSX [Osterix], ALP [alkaline phosphatse], BSP [bone sialoprotein], and OPG [osteoprotegerin]), and decreased expression of SMC markers. ER stress potentiated extracellular vesicle (EV) release via SMPD3 (sphingomyelin phosphodiesterase 3). EVs from ER stress-treated VSMCs showed increased Grp78 levels and calcification. Electron microscopy confirmed the presence of Grp78/Grp94 in EVs. siRNA (short interfering RNA) knock-down of Grp78 decreased calcification. Warfarin-induced Grp78 and ATF4 expression in rat aortas and VSMCs and increased calcification in an ER stress-dependent manner via increased EV release. CONCLUSIONS: ER stress induces vascular calcification by increasing release of Grp78-loaded EVs. Our results reveal a novel mechanism of action of warfarin, involving increased EV release via the PERK-ATF4 pathway, contributing to calcification. This study is the first to show that warfarin induces ER stress and to link ER stress to cargo loading of EVs.

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.

Investigating the effects of ebselen, a potential new lithium mimetic, on glutamate transmission.

Ebselen, a potential new lithium-like drug, treatment reduced brain glutamate, as measured by PET imaging using the mGluR5 radioligand [18 F]FPEB in rats.

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.

Poly(ADP-Ribose) Links the DNA Damage Response and Biomineralization.

Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.

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.

Development of [18F]FAMTO: A novel fluorine-18 labelled positron emission tomography (PET) radiotracer for imaging CYP11B1 and CYP11B2 enzymes in adrenal glands.

INTRODUCTION: Primary aldosteronism accounts for 6-15% of hypertension cases, the single biggest contributor to global morbidity and mortality. Whilst ~50% of these patients have unilateral aldosterone-producing adenomas, only a minority of these have curative surgery as the current diagnosis of unilateral disease is poor. Carbon-11 radiolabelled metomidate ([11C]MTO) is a positron emission tomography (PET) radiotracer able to selectively identify CYP11B1/2 expressing adrenocortical lesions of the adrenal gland. However, the use of [11C]MTO is limited to PET centres equipped with on-site cyclotrons due to its short half-life of 20.4 min. Radiolabelling a fluorometomidate derivative with fluorine-18 (radioactive half life 109.8 min) in the para-aromatic position ([18F]FAMTO) has the potential to overcome this disadvantage and allow it to be transported to non-cyclotron-based imaging centres. METHODS: Two strategies for the one-step radio-synthesis of [18F]FAMTO were developed. [18F]FAMTO was obtained via radiofluorination via use of sulfonium salt (1) and boronic ester (2) precursors. [18F]FAMTO was evaluated in vitro by autoradiography of pig adrenal tissues and in vivo by determining its biodistribution in rodents. Rat plasma and urine were analysed to determine [18F]FAMTO metabolites. RESULTS: [18F]FAMTO is obtained from sulfonium salt (1) and boronic ester (2) precursors in 7% and 32% non-isolated radiochemical yield (RCY), respectively. Formulated [18F]FAMTO was obtained with >99% radiochemical and enantiomeric purity with a synthesis time of 140 min from the trapping of [18F]fluoride ion on an anion-exchange resin (QMA cartridge). In vitro autoradiography of [18F]FAMTO demonstrated exquisite specific binding in CYP11B-rich pig adrenal glands. In vivo [18F]FAMTO rapidly accumulates in adrenal glands. Liver uptake was about 34% of that in the adrenals and all other organs were <12% of the adrenal uptake at 60 min post-injection. Metabolite analysis showed 13% unchanged [18F]FAMTO in blood at 10 min post-administration and rapid urinary excretion. In vitro assays in human blood showed a free fraction of 37.5%. CONCLUSIONS: [18F]FAMTO, a new 18F-labelled analogue of metomidate, was successfully synthesised. In vitro and in vivo characterization demonstrated high selectivity towards aldosterone-producing enzymes (CYP11B1 and CYP11B2), supporting the potential of this radiotracer for human investigation.

Manganese-52: applications in cell radiolabelling and liposomal nanomedicine PET imaging using oxine (8-hydroxyquinoline) as an ionophore.

The ionophore 8-hydroxyquinoline (oxine) has been used to radiolabel cells and liposomal medicines with 111In and, more recently, 89Zr, for medical nuclear imaging applications. Oxine has also shown promising ionophore activity for the positron-emitting radionuclide 52Mn that should allow imaging of labelled cells and nanomedicines for long periods of time (>14 days). However, to date, the radiometal complex formed and its full labelling capabilities have not been fully characterised. Here, we provide supporting evidence of the formation of [52Mn]Mn(oxinate)2 as the metastable complex responsible for its ionophore activity. The cell labelling properties of [52Mn]Mn(oxinate)2 were investigated with various cell lines. The liposomal nanomedicine, DOXIL® (Caelyx) was also labelled with [52Mn]Mn(oxinate)2 and imaged in vivo using PET imaging. [52Mn]Mn(oxinate)2 was able to label various cell lines with moderate efficiency (15-53%), however low cellular retention of 52Mn (21-25% after 24 h) was observed which was shown not to be due to cell death. PET imaging of [52Mn]Mn-DOXIL at 1 h and 24 h post-injection showed the expected pharmacokinetics and biodistribution of this stealth liposome, but at 72 h post-injection showed a profile matching that of free 52Mn, consistent with drug release. We conclude that oxine is an effective ionophore for 52Mn, but high cellular efflux of the isotope limits its use for prolonged cell tracking. [52Mn]Mn(oxinate)2 is effective for labelling and tracking DOXIL in vivo. The release of free radionuclide after liposome extravasation could provide a non-invasive method to monitor drug release in vivo.

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.

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.

Successful removal of intrathyroidal parathyroid adenoma diagnosed and accurately located preoperatively by parathyroid scintigraphy (SPECT-CT).

We describe the case of a large intrathyroidal parathyroid adenoma in a 46-year-old woman who had a history of recently diagnosed hypercalcaemia and a 2-year history of an asymptomatic enlargement of the right lobe of the thyroid. This rare case highlights the potential difficulties that can arise in the evaluation of hyperparathyroidism, especially in cases of multinodular goiter. In some cases, including this one, even a thorough preoperative evaluation that includes radiological studies (ultrasonography and computed tomography [CT]) may not allow for a definitive preoperative diagnosis due to limited sensitivity, especially in multinodular goiter. The overlapping histological features between thyroid and parathyroid lesions can also be problematic at the time of the intraoperative frozen-section evaluation. We present a case in which, with parathyroid scintigraphy and combination of structural and functional imaging (SPECT-CT), we could accurately locate the intrathyroidal parathyroid adenoma in a patient with multinodular goiter.