Improved PET Imaging of Tumors in Mice Using a Novel 18 F-Folate Conjugate with an Albumin-Binding Entity

Purpose: The folate receptor (FR) is a promising target for nuclear imaging due to its overexpression in many different cancer types. A drawback of using folate radioconjugates is the high accumulation of radioactivity in the kidneys. Therefore, the aim of this study was to develop a 18 F-labeled folate conjugate with an albumin-binding entity to enhance the blood circulation time and hence improve the tumor-to-kidney ratio. Procedures: The novel 18 F-folate was prepared by conjugation of a 18 F-labeled glucose azide to an alkyne-functionalized folate precursor containing an albumin-binding entity via Cu(I)-catalyzed 1,3-dipolar cycloaddition. The radioconjugate was tested in vitro on FR-positive KB tumor cells and by biodistribution and positron emission tomography (PET) imaging studies using KB tumor-bearing mice. Results: The radiosynthesis of the albumin-binding [18 F]fluorodeoxyglucose-folate ([18 F]3) resulted in a radiochemical yield of 1-2% decay corrected (d.c.) and a radiochemical purity of ≥95%. The specific activity of [18 F]3 ranged from 20 to 50GBq/μmol. In vitro experiments revealed FR-specific binding of [18 F]3 to KB tumor cells. In vivo we found an increasing uptake of [18 F]3 into tumor xenografts over time reaching a value of ∼ 15% injected dose (ID)/g at 4h post-injection (p.i.). Uptake in the kidneys (∼ 13% ID/g; 1h p.i.) was approximately fourfold reduced compared to previously published 18 F-labeled folic acid derivatives. An excellent visualization of tumor xenografts with an unprecedentedly high tumor-to-kidney ratio (∼ 1) was obtained by PET imaging. Conclusions: [18 F]3 showed a favorable accumulation in tumor xenografts compared to the same folate conjugate without albumin-binding properties. Moreover, the increased tumor-to-kidney ratios improved the PET imaging quality significantly, in spite of a somewhat higher background radioactivity which was a consequence of the slower blood clearance of [18 F]

Direct in vitro and in vivo comparison of 161Tb and 177Lu using a tumour-targeting folate conjugate

Purpose: The radiolanthanide 161Tb (T 1/2 = 6.90days, Eβ− av = 154keV) was recently proposed as a potential alternative to 177Lu (T 1/2 = 6.71days, Eβ− av = 134keV) due to similar physical decay characteristics but additional conversion and Auger electrons that may enhance the therapeutic efficacy. The goal of this study was to compare 161Tb and 177Lu in vitro and in vivo using a tumour-targeted DOTA-folate conjugate (cm09). Methods: 161Tb-cm09 and 177Lu-cm09 were tested in vitro on folate receptor (FR)-positive KB and IGROV-1 cancer cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. In vivo 161Tb-cm09 and 177Lu-cm09 (10MBq, 0.5nmol) were investigated in two different tumour mouse models with regard to the biodistribution, the possibility for single photon emission computed tomography (SPECT) imaging and the antitumour efficacy. Potentially undesired side effects were monitored over 6months by determination of plasma parameters and examination of kidney function with quantitative SPECT using 99mTc-dimercaptosuccinic acid (DMSA). Results: To obtain half-maximal inhibition of tumour cell viability a 4.5-fold (KB) and 1.7-fold (IGROV-1) lower radioactivity concentration was required for 161Tb-cm09 (IC50 ~0.014MBq/ml and ~2.53MBq/ml) compared to 177Lu-cm09 (IC50 ~0.063MBq/ml and ~4.52MBq/ml). SPECT imaging visualized tumours of mice with both radioconjugates. However, in therapy studies 161Tb-cm09 reduced tumour growth more efficiently than 177Lu-cm09. These findings were in line with the higher absorbed tumour dose for 161Tb-cm09 (3.3Gy/MBq) compared to 177Lu-cm09 (2.4Gy/MBq). None of the monitored parameters indicated signs of impaired kidney function over the whole time period of investigation after injection of the radiofolates. Conclusion: Compared to 177Lu-cm09 we demonstrated equal imaging features for 161Tb-cm09 but an increased therapeutic efficacy for 161Tb-cm09 in both tumour cell lines in vitro and in vivo. Further preclinical studies using other tumour-targeting radioconjugates are clearly necessary to draw final conclusions about the future clinical perspectives of 161Tb

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic β-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic β-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand

Development and optimization of targeted radionuclide tumor therapy using folate based radiopharmaceuticals

The folate receptor (FR) has been used for a quarter of a century as a tumor-associated target for selective delivery of drugs and imaging agents to cancer cells. While several folic acid radioconjugates have been successfully employed for imaging purposes in (pre)clinical studies, a therapeutic application of folic acid radioconjugates has not yet reached the critical stage which would allow a clinical translation. Due to a substantial expression of the FR in the proximal tubule cells, radiofolates accumulate in the kidneys which are at risk of damage by particle-radiation. To improve this situation, we aimed to develop and evaluate strategies for the performance of FR-targeted radionuclide therapy by decreasing the renal uptake of radiofolates and thereby reducing potential nephrotoxic effects. Two different strategies were investigated. First, the combination of radiofolates with chemotherapeutic agents such as pemetrexed (PMX) and 5-fluorouracil (5-FU) and secondly, an approach based on radioiodinated folates and their partial in vivo deiodination. It was known from the previous work in our group that PMX reduces the kidney uptake of radiofolates up to ~ 4-fold. Thus, we evaluated the combined application of a therapeutic radiofolate with the antifolate PMX as an option to perform FR-targeted radionuclide therapy. A first therapy study was performed in nude mice bearing FR-positive cervical KB tumors. Injections of subtherapeutic amounts of PMX and the $^{177}$Lu-labeled DOTA-folate ( $^{177}$Lu-EC0800, 20 MBq) showed a 50% prolonged survival time when compared to control animals. To evaluate potential nephrotoxic effects of such a therapy, an additional experiment was performed in mice without tumors. Therapeutic amounts of $^{177}$Lu-EC0800 were injected into the animals either with or without pre-injection of subtherapeutic amounts of PMX. The mice which received only $^{177}$Lu-EC0800 showed significantly increased levels of blood plasma parameters such as blood urea nitrogen (5-fold increased) and creatinine (10-fold increased) when compared to control animals after 6 months. In the same time range SPECT/CT studies using $^{99m}$Tc-DMSA showed an 85% lower uptake in the kidneys compared to controls which confirmed an impaired kidney function. In contrast, analysis of plasma parameters and the results of the $^{99m}$Tc-DMSA studies showed consistently intact kidney function in animals which received PMX in addition to $^{177}$Lu-EC0800. PMX is also known as a radiosensitizing agent. Therefore, we investigated the combined application of $^{177}$Lu-EC0800 and PMX in vitro which showed a pronounced synergistic effect in FR-positive KB and ovarian cancer cell lines, IGROV-1 and SKOV-3. It could also be shown in vivo that the therapeutic efficacy of $^{177}$Lu-EC0800 was enhanced by therapeutic amounts of PMX using two different tumor mouse models. A novel DOTA-folate radioconjugate ( $^{177}$Lu-cm09) was also investigated in vitro in combination with the radiosensitizing agent 5-FU in two different cancer cell lines. The simultaneous application of $^{177}$Lu-cm09 and 5-FU resulted in a synergistic anticancer effect in KB and IGROV-1 tumor cells similar to the results obtained with $^{177}$Lu-EC0800 and PMX. To reduce the cell viability in the combined treatment to 75%, a 5-fold lower quantity of 5-FU was needed compared to the single treatment in both cell lines. The amount of $^{177}$Lu-cm09 was reduced 6-fold in KB and 3-fold in IGROV-1cells. These findings demonstrated the radio sensitizing potential of 5-FU, which is routinely used in combination with external radiotherapy in the clinic. In the second approach we studied three different radioiodinated folate conjugates. Biodistribution studies performed in tumor-bearing nude mice showed specific accumulation of all $^{125}$I-radiofolates in KB tumor xenografts (~ 3 % ID/g; 4 h p.i.). The tumor-to-kidney ratio of $^{125}$I-tyrosine-click-folate was increasing over time (from 0.12; 1 h p.i. to 0.27; 24 h p.i). With these findings the hypothesis was confirmed that partial deiodination of radioiodinated folate conjugates would improve tumor-to- background contrast over time. An 11-fold increase in the tumor-to-background contrast was achieved with $^{125}$I-tyrosine-click-folate if the mice were pre-injected with PMX. The introduction of an albumin-binding entity in the structure improved the tumor-to-background ratio further and resulted in an unprecedented high tumor-to-kidney ratio of approximately 2 (24 h p.i.). The SPECT/CT image showed uptake of radioactivity in FR-positive tumors and in the thyroid gland. Based on these findings the more promising approach that may allow a therapeutic application of folate radioconjugates in the future is the combination of therapeutic radiofolates ( $^{177}$Lu-EC0800, $^{177}$Lu-cm09) with chemotherapeutics (PMX, 5-FU) that can also act as radiosensitizing agents. If the kidney uptake reducing effect of PMX could be confirmed in patients, this therapy concept would be promising for the treatment of non-small cell lung cancer because it shows frequent FR- overexpression and because PMX is already approved for the therapy

First Insights into Infants’ and Children's Aha-Experiences: A Parent Report Study

Previous research has revealed that aha-experiences improve learning and enhance motivation. Hence, knowledge about aha-experiences in children may enable educators to enhance learning experiences. The current study aims to provide a basic knowledge about aha-experiences in children, for example, at what age they appear and on which topics. To answer these questions, we collected parental reports from two different populations (a Norwegian sample and an international sample of English-speaking parents). A content analysis of more than 600 stories of children's aha-experiences yielded three main findings: (1) Parents reported that children have aha-experiences already as infants; (2) Children have aha-experiences on various topics related to action and cognition; (3) The focus of the aha-stories shifts from action to cognition with age. In addition to the main findings, there are indications that children's aha-experiences share qualities with aha-experiences in adults. Our findings provide first insights into aha-experiences in infancy and childhood and may have implications for understanding what motivates children's learning and cognitive development

First Insights into Infants’ and Children's Aha-Experiences: A Parent Report Study

This study present the first study of children's aha-experiences. Using parent reports, we have documented stories of children having aha-experiences already in infancy and that the focus of children's aha-experiences shifts from action to cognition by age

Folate receptor-targeted radionuclide therapy: preclinical investigation of anti-tumor effects and potential radionephropathy

INTRODUCTION Application of therapeutic folate radioconjugates is a promising option for the treatment of folate receptor (FR)-positive tumors, although high uptake of radiofolates in the kidneys remains a critical issue. Recently, it was shown that enhancing the blood circulation of radiofolates results in increased tumor uptake and reduced retention of radioactivity in the kidneys. In this study, we investigated and compared the anti-tumor effects and potential long-term damage to the kidneys after application of an albumin-binding ((177)Lu-cm09), and a conventional ((177)Lu-EC0800) folate radioconjugate. METHODS In vivo studies were performed with KB tumor-bearing nude mice. (177)Lu-EC0800 and (177)Lu-cm09 were applied at variable quantities (10-30MBq/mouse), and the tumor growth was monitored over time. Mice without tumors were injected with the same radiofolates and investigated over eight months by determination of creatinine and blood urea nitrogen plasma levels and by measuring renal uptake of (99m)Tc-DMSA using SPECT. At the study end, the morphological changes were examined on renal tissue sections using variable staining methods. RESULTS Compared to untreated controls, dose-dependent tumor growth inhibition and prolonged survival was observed in all treated mice. In line with the resulting absorbed dose, the treatment was more effective with (177)Lu-cm09 than with (177)Lu-EC0800, enabling complete tumor remission after application of ≥20MBq (≥28Gy). Application of radiofolates with an absorbed renal dose ≥23Gy showed increased levels of renal plasma parameters and reduced renal uptake of (99m)Tc-DSMA. Morphological changes observed on tissue sections confirmed radionephropathy of variable stages. CONCLUSIONS (177)Lu-cm09 showed more favorable anti-tumor effects and significantly less damage to the kidneys compared to (177)Lu-EC0800 as was expected based on improved tumor-to-kidney ratios. It was demonstrated that enhancing the blood circulation time of radiofolates was favorable regarding the risk-benefit profile of a therapeutic application. These results hold promise for future translation of the albumin-binder concept to the clinics, potentially enabling FR-targeted radionuclide therapy in patients