[68Ga]ABY-028: an albumin-binding domain (ABD) protein-based imaging tracer for positron emission tomography (PET) studies of altered vascular permeability and predictions of albumin-drug conjugate transport.

BACKGROUND: Albumin is commonly used as a carrier platform for drugs to extend their circulatory half-lives and influence their uptake into tissues that have altered permeability to the plasma protein. The albumin-binding domain (ABD) protein, which binds in vivo to serum albumin with high affinity, has proven to be a versatile scaffold for engineering biopharmaceuticals with a range of binding capabilities. In this study, the ABD protein equipped with a mal-DOTA chelator (denoted ABY-028) was radiolabeled with gallium-68 (68Ga). This novel radiotracer was then used together with positron emission tomography (PET) imaging to examine variations in the uptake of the ABD-albumin conjugate with variations in endothelial permeability. RESULTS: ABY-028, produced by peptide synthesis in excellent purity and stored at - 20 °C, was stable for 24 months (end of study). [68Ga]ABY-028 could be obtained with labeling yields of > 80% and approximately 95% radiochemical purity. [68Ga]ABY-028 distributed in vivo with the plasma pool, with highest radioactivity in the heart ventricles and major vessels of the body, a gradual transport over time from the circulatory system into tissues and elimination via the kidneys. Early [68Ga]ABY-028 uptake differed in xenografts with different vascular properties: mean standard uptake values (SUVmean) were initially 5 times larger in FaDu than in A431 xenografts, but the difference decreased to 3 after 1 h. Cutaneously administered, vasoactive nitroglycerin increased radioactivity in the A431 xenografts. Heterogeneity in the levels and rates of increases of radioactivity uptake was observed in sub-regions of individual MMTV-PyMT mammary tumors and in FaDu xenografts. Higher uptake early after tracer administration could be observed in lower metabolic regions. Fluctuations in the increased permeability for the tracer across the blood-brain-barrier (BBB) direct after experimentally induced stroke were monitored by PET and the increased uptake was confirmed by ex vivo phosphorimaging. CONCLUSIONS: [68Ga]ABY-028 is a promising new tracer for visualization of changes in albumin uptake due to disease- and pharmacologically altered vascular permeability and their potential effects on the passive uptake of targeting therapeutics based on the ABD protein technology.

Irreversible inhibition of cytosolic thioredoxin reductase 1 as a mechanistic basis for anticancer therapy.

Cancer cells adapt to their inherently increased oxidative stress through activation of the glutathione (GSH) and thioredoxin (TXN) systems. Inhibition of both of these systems effectively kills cancer cells, but such broad inhibition of antioxidant activity also kills normal cells, which is highly unwanted in a clinical setting. We therefore evaluated targeting of the TXN pathway alone and, more specifically, selective inhibition of the cytosolic selenocysteine-containing enzyme TXN reductase 1 (TXNRD1). TXNRD1 inhibitors were discovered in a large screening effort and displayed increased specificity compared to pan-TXNRD inhibitors, such as auranofin, that also inhibit the mitochondrial enzyme TXNRD2 and additional targets. For our lead compounds, TXNRD1 inhibition correlated with cancer cell cytotoxicity, and inhibitor-triggered conversion of TXNRD1 from an antioxidant to a pro-oxidant enzyme correlated with corresponding increases in cellular production of H2O2 In mice, the most specific TXNRD1 inhibitor, here described as TXNRD1 inhibitor 1 (TRi-1), impaired growth and viability of human tumor xenografts and syngeneic mouse tumors while having little mitochondrial toxicity and being better tolerated than auranofin. These results display the therapeutic anticancer potential of irreversibly targeting cytosolic TXNRD1 using small molecules and present potent and selective TXNRD1 inhibitors. Given the pronounced up-regulation of TXNRD1 in several metastatic malignancies, it seems worthwhile to further explore the potential benefit of specific irreversible TXNRD1 inhibitors for anticancer therapy.

ß3-Adrenergically induced glucose uptake in brown adipose tissue is independent of UCP1 presence or activity: Mediation through the mTOR pathway.

OBJECTIVE: Today, the presence and activity of brown adipose tissue (BAT) in adult humans is generally equated with the induced accumulation of [2-18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) in adipose tissues, as investigated by positron emission tomography (PET) scanning. In reality, PET-FDG is currently the only method available for in vivo quantification of BAT activity in adult humans. The underlying assumption is that the glucose uptake reflects the thermogenic activity of the tissue. METHODS: To examine this basic assumption, we here followed [18F]FDG uptake by PET and by tissue [3H]-2-deoxy-d-glucose uptake in wildtype and UCP1(-/-) mice, i.e. in mice that do or do not possess the unique thermogenic and calorie-consuming ability of BAT. RESULTS: Unexpectedly, we found that ß3-adrenergically induced (by CL-316,243) glucose uptake was UCP1-independent. Thus, whereas PET-FDG scans adequately reflect glucose uptake, this acute glucose uptake is not secondary to thermogenesis but is governed by an independent cellular signalling, here demonstrated to be mediated via the previously described KU-0063794-sensitive mTOR pathway. CONCLUSIONS: Thus, PET-FDG scans do not exclusively reveal active BAT deposits but rather any tissue possessing an adrenergically-mediated glucose uptake pathway. In contrast, we found that the marked glucose uptake-ameliorating effect of prolonged ß3-adrenergic treatment was UCP1 dependent. Thus, therapeutically, UCP1 activity is required for any anti-diabetic effect of BAT activation.

Preclinical PET imaging of EGFR levels: pairing a targeting with a non-targeting Sel-tagged Affibody-based tracer to estimate the specific uptake.

BACKGROUND: Though overexpression of epidermal growth factor receptor (EGFR) in several forms of cancer is considered to be an important prognostic biomarker related to poor prognosis, clear correlations between biomarker assays and patient management have been difficult to establish. Here, we utilize a targeting directly followed by a non-targeting tracer-based positron emission tomography (PET) method to examine some of the aspects of determining specific EGFR binding in tumors. METHODS: The EGFR-binding Affibody molecule ZEGFR:2377 and its size-matched non-binding control ZTaq:3638 were recombinantly fused with a C-terminal selenocysteine-containing Sel-tag (ZEGFR:2377-ST and ZTaq:3638-ST). The proteins were site-specifically labeled with DyLight488 for flow cytometry and ex vivo tissue analyses or with (11)C for in vivo PET studies. Kinetic scans with the (11)C-labeled proteins were performed in healthy mice and in mice bearing xenografts from human FaDu (squamous cell carcinoma) and A431 (epidermoid carcinoma) cell lines. Changes in tracer uptake in A431 xenografts over time were also monitored, followed by ex vivo proximity ligation assays (PLA) of EGFR expressions. RESULTS: Flow cytometry and ex vivo tissue analyses confirmed EGFR targeting by ZEGFR:2377-ST-DyLight488. [Methyl-(11)C]-labeled ZEGFR:2377-ST-CH3 and ZTaq:3638-ST-CH3 showed similar distributions in vivo, except for notably higher concentrations of the former in particularly the liver and the blood. [Methyl-(11)C]-ZEGFR:2377-ST-CH3 successfully visualized FaDu and A431 xenografts with moderate and high EGFR expression levels, respectively. However, in FaDu tumors, the non-specific uptake was large and sometimes equally large, illustrating the importance of proper controls. In the A431 group observed longitudinally, non-specific uptake remained at same level over the observation period. Specific uptake increased with tumor size, but changes varied widely over time in individual tumors. Total (membranous and cytoplasmic) EGFR in excised sections increased with tumor growth. There was no positive correlation between total EGFR and specific tracer uptake, which, since ZEGFR:2377 binds extracellularly and is slowly internalized, indicates a discordance between available membranous and total EGFR expression levels. CONCLUSIONS: Same-day in vivo dual tracer imaging enabled by the Sel-tag technology and (11)C-labeling provides a method to non-invasively monitor membrane-localized EGFR as well as factors affecting non-specific uptake of the PET ligand.

A method for comparing intra-tumoural radioactivity uptake heterogeneity in preclinical positron emission tomography studies.

BACKGROUND: Non-uniformity influences the interpretation of nuclear medicine based images and consequently their use in treatment planning and monitoring. However, no standardised method for evaluating and ranking heterogeneity exists. Here, we have developed a general algorithm that provides a ranking and a visualisation of the heterogeneity in small animal positron emission tomography (PET) images. METHODS: The code of the algorithm was written using the Matrix Laboratory software (MATLAB). Parameters known to influence the heterogeneity (distances between deviating peaks, gradients and size compensations) were incorporated into the algorithm. All data matrices were mathematically constructed in the same format with the aim of maintaining overview and control. Histograms visualising the spread and frequency of contributions to the heterogeneity were also generated. The construction of the algorithm was tested using mathematically generated matrices and by varying post-processing parameters. It was subsequently applied in comparisons of radiotracer uptake in preclinical images in human head and neck carcinoma and endothelial and ovarian carcinoma xenografts. RESULTS: Using the developed algorithm, entire tissue volumes could be assessed and gradients could be handled in an indirect manner. Similar-sized volumes could be compared without modifying the algorithm. Analyses of the distribution of different tracers gave results that were generally in accordance with single plane preclinical images, indicating that it could appropriately handle comparisons of targeting vs. non-targeting tracers and also for different target levels. Altering the reconstruction algorithm, pixel size, tumour ROI volumes and lower cut-off limits affected the calculated heterogeneity factors in expected directions but did not reverse conclusions about which tumour was more or less heterogeneous. CONCLUSIONS: The algorithm constructed is an objective and potentially user-friendly tool for one-to-one comparisons of heterogeneity in whole similar-sized tumour volumes in PET imaging.

[11C]MADAM Used as a Model for Understanding the Radiometabolism of Diphenyl Sulfide Radioligands for Positron Emission Tomography (PET).

In quantitative PET measurements, the analysis of radiometabolites in plasma is essential for determining the exact arterial input function. Diphenyl sulfide compounds are promising PET and SPECT radioligands for in vivo quantification of the serotonin transporter (SERT) and it is therefore important to investigate their radiometabolism. We have chosen to explore the radiometabolic profile of [11C]MADAM, one of these radioligands widely used for in vivo PET-SERT studies. The metabolism of [11C]MADAM/MADAM was investigated using rat and human liver microsomes (RLM and HLM) in combination with radio-HPLC or UHPLC/Q-ToF-MS for their identification. The effect of carrier on the radiometabolic rate of the radioligand [11C]MADAM in vitro and in vivo was examined by radio-HPLC. RLM and HLM incubations were carried out at two different carrier concentrations of 1 and 10 µM. Urine samples after perfusion of [11C]MADAM/MADAM in rats were also analysed by radio-HPLC. Analysis by UHPLC/Q-ToF-MS identified the metabolites produced in vitro to be results of N-demethylation, S-oxidation and benzylic hydroxylation. The presence of carrier greatly affected the radiometabolism rate of [11C]MADAM in both RLM/HLM experiments and in vivo rat studies. The good concordance between the results predicted by RLM and HLM experiments and the in vivo data obtained in rat studies indicate that the kinetics of the radiometabolism of the radioligand [11C]MADAM is dose-dependent. This issue needs to be addressed when the diarylsulfide class of compounds are used in PET quantifications of SERT.

Imaging of a clinically relevant stroke model: glucose hypermetabolism revisited.

BACKGROUND AND PURPOSE: Ischemic stroke has been shown to cause hypermetabolism of glucose in the ischemic penumbra. Experimental and clinical data indicate that infarct-related systemic hyperglycemia is a potential therapeutic target in acute stroke. However, clinical studies aiming for glucose control in acute stroke have neither improved functional outcome nor reduced mortality. Thus, further studies on glucose metabolism in the ischemic brain are warranted. METHODS: We used a rat model of stroke that preserves collateral flow. The animals were analyzed by [2-(18)F]-2-fluoro-2-deoxy-d-glucose positron emission tomography or magnetic resonance imaging during 90-minute occlusion of the middle cerebral artery and during 60 minutes after reperfusion. Results were correlated to magnetic resonance imaging of cerebral blood flow, diffusion of water, lactate formation, and histological data on cell death and blood-brain barrier breakdown. RESULTS: We detected an increased [2-(18)F]-2-fluoro-2-deoxy-d-glucose uptake within ischemic regions succumbing to infarction and in the peri-infarct region. Magnetic resonance imaging revealed impairment of blood flow to ischemic levels in the infarct and a reduction of cerebral blood flow in the peri-infarct region. Magnetic resonance spectroscopy revealed lactate in the ischemic region and absence of lactate in the peri-infarct region. Immunohistochemical analyses revealed apoptosis and blood-brain barrier breakdown within the infarct. CONCLUSIONS: The increased uptake of [2-(18)F]-2-fluoro-2-deoxy-d-glucose in cerebral ischemia most likely reflects hypermetabolism of glucose meeting increased energy needs of ischemic and hypoperfused brain tissue, and it occurs under both anaerobic and aerobic conditions measured by local lactate production. Infarct-related systemic hyperglycemia could serve to facilitate glucose supply to the ischemic brain. Glycemic control by insulin treatment could negatively influence this mechanism.

Comparison of methods for evaluating radiolabelled Annexin A5 uptake in pre-clinical PET oncological studies.

PURPOSE: The uptakes of radiolabel led AnnexinA5 (AnxA5) and a size-matched control protein in experimental tumours were evaluated by kinetic analyses and compared with standard uptake values (SUVs) to investigate whether the method of analysis may impact on the conclusions that can be drawn. PROCEDURES: PET scans of the (11)C-labelled proteins performed in untreated and doxorubicin-treated mice with head and neck carcinoma xenografts were retrospectively analysed. The appropriateness of using the Logan graphical analyses for reversibly binding radiotracers in these models was evaluated and confirmed. Distribution volume ratios (DVRs) of the regions of interest to reference muscle tissue were compared to those based on the image-derived input function from arterial blood. SUVs were calculated in the same individuals. RESULTS: DVRs based on reference muscle tissue gave results similar to those based on the arterial blood and may be preferred since they are simpler to calculate. In the inter-group comparisons of baseline versus chemotherapy treatment or AnxA5 versus control protein, differences in DVR quantifications had a 20- to 40-fold higher statistical significance than differences in SUVs. As quantified using the control protein, the amount of free ligand in the vascular space of tumours may be large due to enhanced permeability and retention (EPR) contributions at baseline and affected during treatment, which has implications for quantifications of the specifically bound radioligand. CONCLUSIONS: These results demonstrate that the quantification method as well as the controls used can be important for interpreting the uptake in tumours of the medium-sized protein ligand AnxA5 and its use in monitoring the effects of therapy on cell death in the tumours. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: These results provide additional support for the recognition that more detailed investigations on the effects of the tumour microenvironment on the targeting capability of imaging radiopharmaceuticals are needed.

Visualization of angiogenesis during cancer development in the polyoma middle T breast cancer model: molecular imaging with (R)-[11C]PAQ.

BACKGROUND: Vascular endothelial growth factor receptor 2 (VEGFR2) is a crucial mediator of tumour angiogenesis. High expression levels of the receptor have been correlated to poor prognosis in cancer patients. Reliable imaging biomarkers for stratifying patients for anti-angiogenic therapy could therefore be valuable for increasing treatment success rates. The aim of this study was to investigate the pharmacokinetics and angiogenesis imaging abilities of the VEGFR2-targeting positron emission tomography (PET) tracer (R)-[11C]PAQ. METHODS: (R)-[11C]PAQ was evaluated in the mouse mammary tumour virus-polyoma middle T (MMTV-PyMT) model of metastatic breast cancer. Mice at different stages of disease progression were imaged with (R)-[11C]PAQ PET, and results were compared to those obtained with [18 F]FDG PET and magnetic resonance imaging. (R)-[11C]PAQ uptake levels were also compared to ex vivo immunofluorescence analysis of tumour- and angiogenesis-specific biomarkers. Additional pharmacokinetic studies were performed in rat and mouse. RESULTS: A heterogeneous uptake of (R)-[11C]PAQ was observed in the tumorous mammary glands. Ex vivo analysis confirmed the co-localization of areas with high radioactivity uptake and areas with elevated levels of VEGFR2. In some animals, a high focal uptake was observed in the lungs. The lung uptake correlated to metastatic and angiogenic activity, but not to uptake of [18 F]FDG PET. The pharmacokinetic studies revealed a limited metabolism and excretion during the 1-h scan and a distribution of radioactivity mainly to the liver, kidneys and lungs. In rat, a high uptake was additionally observed in adrenal and parathyroid glands. CONCLUSION: The results indicate that (R)-[11C]PAQ is a promising imaging biomarker for visualization of angiogenesis, based on VEGFR2 expression, in primary tumours and during metastasis development.

Selective intra-arterial administration of ¹8F-FDG to the rat brain -- effects on hemispheric uptake.

INTRODUCTION: The purpose of this study was to investigate the radioligand uptake and iodine contrast distribution in the intra- and extracranial circulation of the rat, after intra-arterial injections to the common carotid artery and different parts of the internal carotid artery. METHODS: All animal experiments were carried out in accordance with Karolinska Institutet's guidelines and were approved by the local laboratory animal ethics committee. We used clinical neurointerventional systems to place microcatheters in the extra- or intracranial carotid artery of 15 Sprague-Dawley rats. Here, injection dynamics of iodine contrast was assessed using digital subtraction angiography. Maintaining the catheter position, the animals were placed in a micro PET and small-animal positron emission tomography (PET) was used to analyze injections [2-(18)F]-2-fluoro-2-deoxy-D-glucose ((18)F-FDG). RESULTS: Microcatheters had to be placed in the intracranial carotid artery (iICA) for the infusate to distribute to the brain. Selective injection via the iICA resulted in a 9-fold higher uptake of (18)F-FDG in the injected hemisphere (p < 0.005) compared to both intravenous and more proximal carotid artery injections. Furthermore, selective injection gave a dramatically improved contrast between the brain and extracranial tissue. CONCLUSION: Intra-arterial injection increases the cerebral uptake of a radiotracer dramatically compared to systemic injection. This technique has potential applications for endovascular treatment of malignancies allowing intra-interventional modifications of injection strategy, based on information on tumor perfusion and risk to surrounding normal parenchyma. Furthermore the technique may increase diagnostic sensitivity and avoid problems due to peripheral pharmacological barriers and first passage metabolism of labile tracers.

Evaluation of efficacy of a new MEK inhibitor, RO4987655, in human tumor xenografts by [(18)F] FDG-PET imaging combined with proteomic approaches.

BACKGROUND: Inhibition of mitogen-activated protein kinase (MEK, also known as MAPK2, MAPKK), a key molecule of the Ras/MAPK (mitogen-activated protein kinase) pathway, has shown promising effects on B-raf-mutated and some RAS (rat sarcoma)-activated tumors in clinical trials. The objective of this study is to examine the efficacy of a novel allosteric MEK inhibitor RO4987655 in K-ras-mutated human tumor xenograft models using [(18)F] FDG-PET imaging and proteomics technology. METHODS: [(18)F] FDG uptake was studied in human lung carcinoma xenografts from day 0 to day 9 of RO4987655 therapy using microPET Focus 120 (CTI Concorde Microsystems, Knoxville, TN, USA). The expression levels of GLUT1 and hexokinase 1 were examined using semi-quantitative fluorescent immunohistochemistry (fIHC). The in vivo effects of RO4987655 on MAPK/PI3K pathway components were assessed by reverse phase protein arrays (RPPA). RESULTS: We have observed modest metabolic decreases in tumor [(18)F] FDG uptake after MEK inhibition by RO4987655 as early as 2 h post-treatment. The greatest [(18)F] FDG decreases were found on day 1, followed by a rebound in [(18)F] FDG uptake on day 3 in parallel with decreasing tumor volumes. Molecular analysis of the tumors by fIHC did not reveal statistically significant correlations of GLUT1 and hexokinase 1 expressions with the [(18)F] FDG changes. RPPA signaling response profiling revealed not only down-regulation of pERK1/2, pMKK4, and pmTOR on day 1 after RO4987655 treatment but also significant up-regulation of pMEK1/2, pMEK2, pC-RAF, and pAKT on day 3. The up-regulation of these markers is interpreted to be indicative of a reactivation of the MAPK and activation of the compensatory PI3K pathway, which can also explain the rebound in [(18)F] FDG uptake following MEK inhibition with RO4987655 in the K-ras-mutated human tumor xenografts. CONCLUSIONS: We have performed the first preclinical evaluation of a new MEK inhibitor, RO4987655, using a combination of [(18)F] FDG-PET imaging and molecular proteomics. These results provide support for using preclinical [(18)F] FDG-PET imaging in early, non-invasive monitoring of the effects of MEK and perhaps other Ras/MAPK signaling pathway inhibitors, which should facilitate a wider implementation of clinical [(18)F] FDG-PET to optimize their clinical use.

[18 F]FDG-PET imaging is an early non-invasive pharmacodynamic biomarker for a first-in-class dual MEK/Raf inhibitor, RO5126766 (CH5126766), in preclinical xenograft models.

BACKGROUND: Positron emission tomography (PET) with [2-18 F]-2-fluoro-2-deoxy-D-glucose ([18 F]FDG-PET) was acquired at multiple time-points a) to monitor the early response to RO5126766 (CH5126766) in xenograft models b) to evaluate non-invasive small animal [18 F]FDG-PET imaging as a biomarker for MEK inhibitors for translation into dose-finding studies in cancer patients and c) to explore the underlying mechanism related to FDG uptake in tumors treated with RO5126766. METHODS: [18 F]FDG uptake was studied in HCT116 (K-ras), COLO205 (B-raf) mutants and COLO320DM (wild type) xenografts from day 0 to 3 of RO5126766 treatment using a microPET Focus 120 and complemented with in vitro incubations, ex-vivo phosphor imaging and immunohistochemical (IHC) analyses. RESULTS: In the HCT116 (K-ras) and COLO205 (B-raf) mutant xenografts, significant decreases in [18 F]FDG uptake were detected in vivo on day 1 with 0.3 mg/kg and ex vivo on day 3 with 0.1 mg/kg RO5126766. [18 F]FDG changes correlated with decreases in tumor cells proliferation (Ki-67) and with changes in expression levels of GLUT1. No effects were observed in drug resistant COLO320DM cells. The cellular fractionation and Western blotting analyses suggested that the change of [18 F]FDG uptake associated with RO5126766 is due to translocation of GLUT1 from membrane to cytosol, similar to the results reported in the literature with EGFR tyrosine kinase inhibitors, which also target the MAPK pathway. CONCLUSIONS: RO5126766 inhibition resulted in a rapid time - and dose - dependent decline in [18 F]FDG uptake in both mutant xenografts. These results strongly resemble the clinical observations obtained with MEK/Raf inhibitors support the use of preclinical [18 F]FDG-PET as a translational tool for decision support in preclinical and early clinical development of MEK inhibitors.

Metabolism of epidermal growth factor receptor targeting probe [11C]PD153035: impact on biodistribution and tumor uptake in rats.

UNLABELLED: Several tracers have been evaluated as probes for noninvasive epidermal growth factor receptor (EGFR) quantification with PET. One of the most promising candidates is the (11)C-labeled analog of the EGFR tyrosine kinase inhibitor PD153035. However, previous in vitro studies indicated extensive metabolism of the tracer, which could be disadvantageous for the assessment of receptor density in vivo. The aim of this study was to investigate the in vivo metabolism of [(11)C]PD153035 to determine whether alterations in metabolite formation are accompanied by changes in biodistribution and tumor uptake. METHODS: EGFR-overexpressing human epidermoid carcinoma xenografts in rats were used in all examinations of tumor uptake. Cytochrome P450 enzymes of subfamilies CYP2D and CYP3A were inhibited before intravenous injection of [(11)C]PD153035 into healthy and tumor-bearing male rats. Samples were taken from arterial blood and urine, and the occurrence of radioactive metabolites was assessed with radio-high-performance liquid chromatography. Dynamic PET examinations of healthy and tumor-bearing animals were performed. In 1 rat, the effect of local intraarterial administration was examined. RESULTS: [(11)C]PD153035 labeled at position 6 was metabolized extensively in vivo in male rats, resulting in very low levels of the intact tracer in plasma only minutes after injection. The major identified radiolabeled metabolites found were the N-oxide and metabolites arising from O demethylation at position 7. They were reduced by inhibition of CYP2D and CYP3A enzymes. PET revealed enzyme activity-dependent changes in the radioactivity distribution in the liver and tumors. Local administration of [(11)C]PD153035 greatly increased radioactivity levels in the adjacent tumor compared with levels typically found after intravenous administration. The highest tumor-to-muscle ratio at 60 min after intravenous injection was found in the untreated animals, whereas the overall highest ratio was found in the tumor near the intraarterial administration site. CONCLUSION: We suggest that the metabolism of [(11)C]PD153035 should be taken into consideration when this tracer is used to quantify EGFR expression, as our results indicated that the distribution of radioactivity to EGFR-overexpressing tumors was affected by the rate of metabolism and the route of administration.

Site-specifically 11C-labeled Sel-tagged annexin A5 and a size-matched control for dynamic in vivo PET imaging of protein distribution in tissues prior to and after induced cell death.

BACKGROUND: Radiolabeled annexin A5 (AnxA5) is widely used for detecting phosphatidylserine exposed on cell surfaces during apoptosis. We describe here a new method for labeling AnxA5 and a size-matched control protein with short-lived carbon-11, for probing the specificity of in vivo cell death monitoring using positron emission tomography (PET) imaging. METHODS: AnxA5 and the control protein were recombinantly expressed with a C-terminal "Sel-tag", the tetrapeptide -Gly-Cys-Sec-Gly-COOH. The proteins were then labeled either fluorescently for in vitro corroborations of binding behaviors or with 11C for dynamic in vivo PET studies. RESULTS: AnxA5 demonstrated retained calcium-dependent binding to apoptotic cells after the C-terminus modification. The control protein showed no functional binding. The 11C-ligands demonstrated similar in vivo pharmacokinetic behavior in healthy mice except for higher uptake in kidney and higher intact elimination to urine of AnxA5. After inducing hepatic apoptosis, however, the uptake of labeled AnxA5 in the targeted tissue increased compared to baseline levels while that of the control protein tended to decrease. CONCLUSIONS: These data suggest that the combined use of these two tracers can facilitate differentiating specific AnxA5 binding and its changes caused by induced cell death from uptake due to non-specific permeability and retention effects at baseline or after therapy. GENERAL SIGNIFICANCE: The Sel-tag enables rapid and mild reactions with electrophilic agents giving site-specifically labeled proteins for multi-probe analyses. The combined use of 11C-labeled AnxA5 and a size-matched control protein with dynamic PET can be useful for evaluating drug effects on target as well as off-target tissues.

Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes.

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved ß-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

Combining [11C]-AnxA5 PET imaging with serum biomarkers for improved detection in live mice of modest cell death in human solid tumor xenografts.

BACKGROUND: In vivo imaging using Annexin A5-based radioligands is a powerful technique for visualizing massive cell death, but has been less successful in monitoring the modest cell death typically seen in solid tumors after chemotherapy. Here we combined dynamic positron emission tomography (PET) imaging using Annexin A5 with a serum-based apoptosis marker, for improved sensitivity and specificity in assessment of chemotherapy-induced cell death in a solid tumor model. METHODOLOGY/PRINCIPAL FINDINGS: Modest cell death was induced by doxorubicin in a mouse xenograft model with human FaDu head and neck cancer cells. PET imaging was based on (11)C-labeled Sel-tagged Annexin A5 ([(11)C]-AnxA5-ST) and a size-matched control. 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]-FDG) was utilized as a tracer of tissue metabolism. Serum biomarkers for cell death were ccK18 and K18 (M30 Apoptosense® and M65). Apoptosis in tissue sections was verified ex vivo for validation. Both PET imaging using [(11)C]-AnxA5-ST and serum ccK18/K18 levels revealed treatment-induced cell death, with ccK18 displaying the highest detection sensitivity. [(18)F]-FDG uptake was not affected by this treatment in this tumor model. [(11)C]-AnxA5-ST gave robust imaging readouts at one hour and its short half-life made it possible to perform paired scans in the same animal in one imaging session. CONCLUSIONS/SIGNIFICANCE: The combined use of dynamic PET with [(11)C]-AnxA5-ST, showing specific increases in tumor binding potential upon therapy, with ccK18/K18 serum measurements, as highly sensitive markers for cell death, enabled effective assessment of modest therapy-induced cell death in this mouse xenograft model of solid human tumors.

HER2-positive tumors imaged within 1 hour using a site-specifically 11C-labeled Sel-tagged affibody molecule.

UNLABELLED: A rapid, reliable method for distinguishing tumors or metastases that overexpress human epidermal growth factor receptor 2 (HER2) from those that do not is highly desired for individualizing therapy and predicting prognoses. In vivo imaging methods are available but not yet in clinical practice; new methodologies improving speed, sensitivity, and specificity are required. METHODS: A HER2-binding Affibody molecule, Z(HER2:342), was recombinantly fused with a C-terminal selenocysteine-containing tetrapeptide Sel-tag, allowing site-specific labeling with either (11)C or (68)Ga, followed by biodistribution studies with small-animal PET. Dosimetry data for the 2 radiotracers were compared. Imaging of HER2-expressing human tumor xenografts was performed using the (11)C-labeled Affibody molecule. RESULTS: Both the (11)C- and (68)Ga-labeled tracers initially cleared rapidly from the blood, followed by a slower decrease to 4-5 percentage injected dose per gram of tissue at 1 h. Final retention in the kidneys was much lower (>5-fold) for the (11)C-labeled protein, and its overall absorbed dose was considerably lower. (11)C-Z(HER2:342) showed excellent tumor-targeting capability, with almost 10 percentage injected dose per gram of tissue in HER2-expressing tumors within 1 h. Specificity was demonstrated by preblocking binding sites with excess ligand, yielding significantly reduced radiotracer uptake (P = 0.002), comparable to uptake in tumors with low HER2 expression. CONCLUSION: To our knowledge, the Sel-tagging technique is the first that enables site-specific (11)C-radiolabeling of proteins. Here we present the finding that, in a favorable combination between radionuclide half-life and in vivo pharmacokinetics of the Affibody molecules, (11)C-labeled Sel-tagged Z(HER2:342) can successfully be used for rapid and repeated PET studies of HER2 expression in tumors.

Brief Report: alterations in cerebral blood flow as assessed by PET/CT in adults with autism spectrum disorder with normal IQ.

Specific biological markers for autism spectrum disorder (ASD) have not yet been established. Functional studies have shown abnormalities in the anatomo-functional connectivity of the limbic-striatal "social" brain. This study aimed to investigate regional cerebral blood flow (rCBF) at rest. Thirteen patients with ASD of normal intelligence and ten IQ-, sex- and age-matched healthy controls (HC) underwent PET/CT using [1-(11)C]butanol, a perfusion tracer. As compared to HC, ASD showed significant CBF increases in the right parahippocampal, posterior cingulate, primary visual and temporal cortex, putamen, caudatus, substantia nigra and cerebellum. No statistically significant correlation between CBF and IQ was found. The limbic, posterior associative and cerebellar cortices showed increased blood flow in ASD, confirming previous findings about the neurobiology of ASD.

Radiolabelled receptor-tyrosine-kinase targeting drugs for patient stratification and monitoring of therapy response: prospects and pitfalls.

Transmembrane receptor tyrosine kinases (RTKs) are overexpressed in many malignancies. RTK signalling triggers cell proliferation, suppression of apoptosis, increased motility, and recruitment of neovasculature. Overexpressed RTKs are the molecular targets for an increasing number of anticancer drugs. Monoclonal antibodies block the ligands or their binding sites and prevent receptor dimerisation, thereby hindering RTK signalling. The antibody-dependent cellular cytotoxicity can boost the therapeutic effect. Small-molecule tyrosine kinase inhibitors (TKIs) hamper downstream RTK signalling by targeting the intracellular kinase domain. These drugs have significantly increased survival in several patient groups. Improved patient stratification and therapy monitoring might further enhance the efficacy of anti-RTK therapy. Radionuclide-based molecular imaging can provide methods for localising and estimating the expression of RTKs. It can potentially identify patients who have tumours that overexpress RTK and would, therefore, most likely benefit from a targeted treatment. Monitoring changes in RTK expression during therapy could help avoid overtreatment and undertreatment. Radionuclide-based methods are less invasive and less sensitive to expression heterogeneity than more conventional sampling methods. The biochemical information is also obtained in an anatomical context. The development of radiolabelled anti-RTK drugs and their analogues is the subject of intensive preclinical and translational research. In this review, we present current approaches to developing imaging probes for in-vivo RTK visualisation and discuss their advantages and disadvantages.

Vascular endothelial growth factor B controls endothelial fatty acid uptake.

The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb(-/-) mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.