In vivo [64Cu]CuCl2 PET imaging reveals activity of Dextran-Catechin on tumor copper homeostasis.

Given the strong clinical evidence that copper levels are significantly elevated in a wide spectrum of tumors, copper homeostasis is considered as an emerging target for anticancer drug design. Monitoring copper levels in vivo is therefore of paramount importance when assessing the efficacy of copper-targeting drugs. Herein, we investigated the activity of the copper-targeting compound Dextran-Catechin by developing a [64Cu]CuCl2 PET imaging protocol to monitor its effect on copper homeostasis in tumors. Methods: Protein expression of copper transporter 1 (CTR1) in tissue microarrays representing 90 neuroblastoma patient tumors was assessed by immunohistochemistry. Western blotting analysis was used to study the effect of Dextran-Catechin on the expression of CTR1 in neuroblastoma cell lines and in tumors. A preclinical human neuroblastoma xenograft model was used to study anticancer activity of Dextran-Catechin in vivo and its effect on tumor copper homeostasis. PET imaging with [64Cu]CuCl2 was performed in such preclinical neuroblastoma model to monitor alteration of copper levels in tumors during treatment. Results: CTR1 protein was found to be highly expressed in patient neuroblastoma tumors by immunohistochemistry. Treatment of neuroblastoma cell lines with Dextran-Catechin resulted in decreased levels of glutathione and in downregulation of CTR1 expression, which caused a significant decrease of intracellular copper. No changes in CTR1 expression was observed in normal human astrocytes after Dextran-Catechin treatment. In vivo studies and PET imaging analysis using the neuroblastoma preclinical model revealed elevated [64Cu]CuCl2 retention in the tumor mass. Following treatment with Dextran-Catechin, there was a significant reduction in radioactive uptake, as well as reduced tumor growth. Ex vivo analysis of tumors collected from Dextran-Catechin treated mice confirmed the reduced levels of CTR1. Interestingly, copper levels in blood were not affected by treatment, demonstrating potential tumor specificity of Dextran-Catechin activity. Conclusion: Dextran-Catechin mediates its activity by lowering CTR1 and intracellular copper levels in tumors. This finding further reveals a potential therapeutic strategy for targeting copper-dependent cancers and presents a novel PET imaging method to assess patient response to copper-targeting anticancer treatments.

Synthesis and biological characterisation of 18F-SIG343 and 18F-SIG353, novel and high selectivity σ2 radiotracers, for tumour imaging properties.

BACKGROUND: Sigma2 (σ2) receptors are highly expressed in cancer cell lines and in tumours. Two novel selective 18F-phthalimido σ2 ligands, 18F-SIG343 and 18F-SIG353, were prepared and characterised for their potential tumour imaging properties. METHODS: Preparation of 18F-SIG343 and 18F-SIG353 was achieved via nucleophilic substitution of their respective nitro precursors. In vitro studies including radioreceptor binding assays in the rat brain membrane and cell uptake studies in the A375 cell line were performed. In vivo studies were carried out in mice bearing A375 tumours including positron emission tomography (PET) imaging, biodistribution, blocking and metabolite studies. RESULTS: In vitro studies showed that SIG343 and SIG353 displayed excellent affinity and selectivity for σ2 receptors (Ki(σ2) = 8 and 3 nM, σ2:σ1 = 200- and 110-fold, respectively). The σ2 selectivity of 18F-SIG343 was further confirmed by blocking studies in A375 cells, however, not noted for 18F-SIG353. Biodistribution studies showed that both radiotracers had similar characteristics including moderately high tumour uptake (4%ID/g to 5%ID/g); low bone uptake (3%ID/g to 4%ID/g); and high tumour-to-muscle uptake ratios (four- to sevenfold) up to 120 min. Although radiotracer uptake in organs known to express σ receptors was significantly blocked by pre-injection of competing σ ligands, the blocking effect was not observed in the tumour. PET imaging studies indicated major radioactive localisation in the chest cavity for both ligands, with approximately 1%ID/g uptake in the tumour at 120 min. Metabolite studies showed that the original radiotracers remained unchanged 65% to 80% in the tumour up to 120 min. CONCLUSIONS: The lead ligands showed promising in vitro and in vivo characteristics. However, PET imaging indicated low tumour-to-background ratios. Furthermore, we were unable to demonstrate that uptake in the A375 tumour was σ2-specific. 18F-SIG343 and 18F-SIG343 do not display ideal properties for imaging the σ2 receptor in the A375 tumour model. However, since the radiotracers show promising in vitro and in vivo characteristics, longer scans using appropriate half-life isotopes and alternative tumour models will be carried out in future studies to fully validate the imaging characteristics of these radiotracers.

Central nervous system expression and PET imaging of the translocator protein in relapsing-remitting experimental autoimmune encephalomyelitis.

UNLABELLED: Glial neuroinflammation is associated with the development and progression of multiple sclerosis. PET imaging offers a unique opportunity to evaluate neuroinflammatory processes longitudinally in a noninvasive and clinically translational manner. (18)F-PBR111 is a newly developed PET radiopharmaceutical with high affinity and selectivity for the translocator protein (TSPO), expressed on activated glia. This study aimed to investigate neuroinflammation at different phases of relapsing-remitting (RR) experimental autoimmune encephalomyelitis (EAE) in the brains of SJL/J mice by postmortem histologic analysis and in vivo by PET imaging with (18)F-PBR111. METHODS: RR EAE was induced by immunization with PLP(139-151) peptide in complete Freund's adjuvant. Naive female SJL/J mice and mice immunized with saline-complete Freund's adjuvant were used as controls. The biodistribution of (18)F-PBR111 was measured in 13 areas of the central nervous system and compared with PET imaging results during different phases of RR EAE. The extents of TSPO expression and glial activation were assessed with immunohistochemistry, immunofluorescence, and a real-time polymerase chain reaction. RESULTS: There was significant TSPO expression in all of the central nervous system areas studied at the peak of the first clinical episode and, importantly, at the preclinical stage. In contrast, only a few TSPO-positive cells were observed at the second episode. At the third episode, there was again an increase in TSPO expression. TSPO expression was associated with microglial cells or macrophages without obvious astrocyte labeling. The dynamics of (18)F-PBR111 uptake in the brain, as measured by in vivo PET imaging and biodistribution, followed the pattern of TSPO expression during RR EAE. CONCLUSION: PET imaging with the TSPO ligand (18)F-PBR111 clearly reflected the dynamics of microglial activation in the SJL/J mouse model of RR EAE. The results are the first to highlight the discrepancy between the clinical symptoms of EAE and TSPO expression in the brain, as measured by PET imaging at the peaks of various EAE episodes. The results suggest a significant role for PET imaging investigations of neuroinflammation in multiple sclerosis and allow for in vivo follow-up of antiinflammatory treatment strategies.

PET imaging of brain inflammation during early epileptogenesis in a rat model of temporal lobe epilepsy.

BACKGROUND: Recently, inflammatory cascades have been suggested as a target for epilepsy therapy. Positron emission tomography (PET) imaging offers the unique possibility to evaluate brain inflammation longitudinally in a non-invasive translational manner. This study investigated brain inflammation during early epileptogenesis in the post-kainic acid-induced status epilepticus (KASE) model with post-mortem histology and in vivo with [18F]-PBR111 PET. METHODS: Status epilepticus (SE) was induced (N = 13) by low-dose injections of KA, while controls (N = 9) received saline. Translocator protein (TSPO) expression and microglia activation were assessed with [125I]-CLINDE autoradiography and OX-42 immunohistochemistry, respectively, 7 days post-SE. In a subgroup of rats, [18F]-PBR111 PET imaging with metabolite-corrected input function was performed before post-mortem evaluation. [18F]-PBR111 volume of distribution (Vt) in volume of interests (VOIs) was quantified by means of kinetic modelling and a VOI/metabolite-corrected plasma activity ratio. RESULTS: Animals with substantial SE showed huge overexpression of TSPO in vitro in relevant brain regions such as the hippocampus and amygdala (P < 0.001), while animals with mild symptoms displayed a smaller increase in TSPO in amygdala only (P < 0.001). TSPO expression was associated with OX-42 signal but without obvious cell loss. Similar in vivo [18F]-PBR111 increases in Vt and the simplified ratio were found in key regions such as the hippocampus (P < 0.05) and amygdala (P < 0.01). CONCLUSION: Both post-mortem and in vivo methods substantiate that the brain regions important in seizure generation display significant brain inflammation during epileptogenesis in the KASE model. This work enables future longitudinal investigation of the role of brain inflammation during epileptogenesis and evaluation of anti-inflammatory treatments.

Decreased plasma peptide YY accompanied by elevated peptide YY and Y2 receptor binding densities in the medulla oblongata of diet-induced obese mice.

It is well known that the peripheral peptide YY (PYY)-central neuropeptide Y (NPY) Y2 receptor axis plays an important role in promoting negative energy balance regulation. Both the hypothalamus and medulla oblongata express a high level of Y2 receptors; however, the functional role of this receptor in chronic high-fat diet-induced obesity has not been fully examined. Using quantitative autoradiography, this study measured binding densities of total [(125)I]PYY and Y2 receptors in the hypothalamus and medulla of chronic high-fat diet-induced obese (DIO), obese-resistant, and low-fat-fed mice. Plasma PYY was also measured using RIA after 22 wk of dietary intervention. The results revealed that body weight gain was significantly higher in the obese mice, compared with the lean mice. Furthermore, PYY and NPY Y2 receptor binding densities in the medulla of the obese mice were significantly higher, compared with the lean mice, whereas the level of plasma PYY was significantly lower in the DIO mice than the low-fat-fed mice. In conclusion, this study showed that the DIO mice had low plasma PYY, which may have caused a compensatory up-regulation of PYY and Y2 receptor densities in the medulla. A low-level response of PYY-medullary regulation to positive energy balance may have contributed to the development of high-fat diet-induced obesity in DIO mice; conversely, a normal response of this regulatory axis in the obese-resistant mice may have contributed to the maintenance of body weight while on a high-fat diet.