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INTRODUCTION: Vitamin K antagonists (VKA) and non-vitamin K oral antagonist anticoagulants (NOAC) are used in the clinic to reduce risk of thrombosis. However, they also exhibit vascular off-target effects. The aim of this study is to compare VKA and NOAC on atherosclerosis progression and calcification in an experimental setup.MATERIAL AND METHODS: Female Apoe-/- mice (age 12 weeks) were fed Western-type diet as control or supplemented with dabigatran etexilate or warfarin for 6 or 18 weeks. Vascular calcification was measured in whole aortic arches using µCT and [18 F]-NaF. Atherosclerotic burden was assessed by (immuno)histochemistry. Additionally, in vitro effects of warfarin, thrombin, and dabigatran on primary vascular smooth muscle cells (VSMC) were assessed.RESULTS: Short-term treatment with warfarin promoted formation of atherosclerotic lesions with a pro-inflammatory phenotype, and more rapid plaque progression compared with control and dabigatran. In contrast, dabigatran significantly reduced plaque progression compared with control. Long-term warfarin treatment significantly increased both presence and activity of plaque calcification compared with control and dabigatran. Calcification induced by warfarin treatment was accompanied by increased presence of uncarboxylated matrix Gla protein. In vitro, both warfarin and thrombin significantly increased VSMC oxidative stress and extracellular vesicle release, which was prevented by dabigatran.CONCLUSION: Warfarin aggravates atherosclerotic disease activity, increasing plaque inflammation, active calcification, and plaque progression. Dabigatran lacks undesired vascular side effects and reveals beneficial effects on atherosclerosis progression and calcification. The choice of anticoagulation impacts atherosclerotic disease by differential off target effect. Future clinical studies should test whether this beneficial effect also applies to patients.

Although anatomical imaging modalities (X-ray, computed tomography (CT), magnetic resonance imaging (MRI)) still have a higher spatial resolution (0.1–1 mm) than molecular imaging modalities (single-photon emission computed tomography (SPECT), positron emission tomography (PET), optical imaging (OI)), the advantage of molecular imaging is that it can detect molecular and cellular changes at the onset of a disease before it leads to morphological tissue changes, which can be detected by anatomical imaging. During the last decades, noninvasive diagnostic imaging has encountered a rapid growth due to the development of dedicated imaging equipment for preclinical animal studies. In addition, the introduction of multimodality imaging (PET/CT, SPECT/CT, PET/MRI) which combines high-resolution conventional anatomical imaging with high sensitivity of tracer-based molecular imaging techniques has led to successful accomplishments in this exciting field. In this book chapter, we will focus on chemical synthesis techniques for site-specific incorporation of radionuclide chelators. Subsequently, radiolabeling based on complexation of a radionuclide with a chelator will be discussed, with focus on: diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-triacetic acid (NOTA), hexa-histidine (His-tag), and 6-hydrazinonicotinic acid (HYNIC) that allow the production of peptides labeled with 18F, 68Ga, 99mTc, and 111In – the currently most widely used isotopes.

The authors wish to replace the ‘Author Contributions’ statement and the affiliation for Jochen Maurer of this article [...]

Cancers 14(1), 230 (2022). doi:10.3390/cancers14010230 special issue: "Special Issue "Radiopharmaceuticals for Oncological Diseases" / Special Issue Editor: Prof. Dr. Melpomeni Fani, Guest Editor", Published by MDPI, Basel

Purpose: Reliable quantification of radioactivity in nuclear medicine is becoming increasingly important in various therapeutic applications requiring a high accuracy of nuclear medicine measuring equipment, such as radionuclide calibrators. In this study the accuracy of four different radionuclide calibrators was assessed for Tc-99m,In-111,Ga-68 and F-18 for measurement geometries clinically used.Methods: Syringes and vials were prepared with a reference activity using a stock solution of which the activity concentration was determined using gamma-ray spectroscopy. The accuracy of four different radionuclide calibrator systems, ISOMED 2000, ISOMED 2010, VIK-202 and Capintec CRC-25R, was assessed by comparing the measured activity to the reference activity.Results: Deviations in measured activity from reference values were found up to 12.5%, 32.0%, 29.0% and 12.6% for Tc-99m,In-111,Ga-68 and F-18, respectively. For Ga-68 all radionuclide calibrators systematically overestimated the activity by 10-20%. For(111)In, large differences in activity measurements were observed between different source geometries, in particular between syringes and vials. Deviations between radionuclide calibrator systems were found up to 11.8%, 44.4%, 14.4% and 8.7% for( 99m)Tc,In-111,Ga-68 and F-18, respectively. When comparing similar syringe types of different brands filled with identical stock solution volume, deviations up to 1.8%, 5.8%, 10.2% and 3.2% were found for Tc-99m,In-111,Ga-68 and F-18.Conclusion: Substantial deviations in measured activity were found for all radionuclides and radionuclide calibrators, which may result in erroneous activity dosing and image quantification. This underlines the importance of thorough validation of radionuclide calibrators for all measurement geometries and radionuclides clinically used.

International journal of molecular sciences 22(11), 5544 (2021). doi:10.3390/ijms22115544 special issue: "Special Issue "Cancer Molecular Imaging 2.0" / Special Issue Editor: Prof. Dr. Christophe V. van de Wiele, Guest Editor", Published by Molecular Diversity Preservation International, Basel

The metabolism of ceramides is deregulated in the brain of Alzheimer’s disease (AD) patients and is associated with apolipoprotein (APO) APOE4 and amyloid-β pathology. However, how the ceramide metabolism changes over time in AD, in vivo, remains unknown. Distribution and metabolism of [18F]F-HPA-12, a radio-fluorinated version of the ceramide analog N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide, was investigated in the brain of AD transgenic mouse models (FAD) on an APOE4 or APOE3 genetic background, by positron emission tomography and by gamma counter. We found that FAD mice displayed a higher uptake of [18F]F-HPA-12 in the brain, independently from the APOE4 or APOE3 genetic background. FAD mice could be distinguished from littermate control animals with a sensitivity of 85.7% and a specificity of 87.5%, by gamma counter measurements. Metabolic analysis of [18F]F-HPA-12 in the brain suggested that the tracer is degraded less efficiently in the FAD mice. Furthermore, the radioactive signal registered in the hippocampus correlated with an increase of Cer d18:1/20:2 levels measured in the same brain region by mass spectrometry. Our data gives additional proof that ceramide metabolism is different in FAD mice compared to controls. Ceramide analogs like HPA-12 may function as metabolic probes to study ceramide disbalance in the brain.

Background In vivo imaging of glucose analogue 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) via positron emission tomography (PET) is the current gold standard to visualize and assess brown adipose tissue (BAT) activity. However, glucose metabolism is only a part of the metabolic activity of BAT. [18F]FDG-PET has been shown in clinical trials to often fail to visualize BAT under insulin-resistant conditions associated with aging and weight gain. We employed a novel developed triglyceride-based tracer to visualize BATs metabolic activity under different temperature conditions as well as under diabetic and obese conditions in preclinical models. Results [18F]BDP-TG-chylomicron-like particles visualized BAT in control, streptozocin-induced diabetes and obese mice. Increased BAT tracer uptake was found in control mice acutely exposed to cold but not in cold-acclimated animals. Diabetes did not remove BAT tracer uptake, but did limit BAT tracer uptake to levels of control mice housed at 21 °C. In obese animals, BAT tracer uptake was significantly reduced, although the stimulating effect of cold exposure could still be noted. Conclusion BAT was visualized in control, diabetic and obese conditions. Streptozocin-induced diabetes, but not obesity, inhibited the stimulatory effect of cold exposure.

Purpose: Brown adipose tissue (BAT) research has evolved from an underestimated to a fast developing field. Its assumed curing properties for the world wide epidemic obesity, and its related diseases, makes this tissue an interesting target for a broad amount of non-invasive molecular BAT tracers. Apart from F-18-FDG PET/CT there are several methods to detect BAT and measure its metabolism in a more appropriate way. Especially interesting is the measure of lipid turnover, because fatty acids comprise the main fuel for active BAT. This review outlines different imaging modalities suitable for BAT imaging with the overall goal to explain the yet not completely understood mechanism in BAT and its quantitative contribution to whole body lipid and energy metabolism.Methods: Publications with focus on brown adipose tissue and lipid metabolism imaging are analyzed, different imaging approaches are introduced and promising BAT tracers are presented.Results: Radiolabelled and fluorescent fatty acids, labelled particles, H-3-Triolein and ADIFAB staining can give information about the inflow and therefore about the utilization of fatty acids which represents the activation state in vivo/in vitro. Non-invasive scanning with CT or MRI is a useful addition to those techniques.Conclusion: Lipid metabolism imaging offers the opportunity to visualize and quantify yet undiscovered aspects of BAT metabolic activities and is key to completely clarify its role in whole body lipid and energy metabolism. (C) 2017 Elsevier Inc. All rights reserved.

Background: Lumbar disc herniation is often associated with an inflammatory process. In this context, inflammation has been considered a key factor in the modulation of pain. Here, we present a case of inflammatory activity directly documented in a patient with a lumbar disc herniation. Case Description: A 49-year-old male presented with progressive low back pain and left-sided S1 radiculopathy, without a focal neurological deficit. The lumbar MR revealed a prominent herniated disc at the L5-S1 level, with compression of the left S1 root. The patient underwent a L5-S1 discectomy using a standard interlaminar approach. Although initially he was pain free, he required three additional operations to address recurrent pain complaints. As research indicates that local inflammation contributes to neuropathic pain, we had the patient undergoes single-photon emission computed tomography (SPECT) imaging using technetium-99m-labeled-infliximab (an anti-tumor necrosis factor [TNF]-alpha monoclonal antibody) before a proposed fourth operation. The SPECT study documented a strong signal at the site of the herniated disc, thus confirming the diagnosis of a pro-inflammatory process involving the S1 nerve root. Nine months after the fourth operation, the patient was pain free. Of interest, the second SPECT study in the now asymptomatic patient demonstrated no detectable/ residual signal at the operative/disc site. Conclusion: Absence of a SPECT TNF-alpha signal in a pain-free patient following a lumbar discectomy correlates with the reduction/resolution of the local preoperative inflammatory response.

Background Personalized molecular radiotherapy based on theragnostics requires accurate quantification of the amount of radiopharmaceutical activity administered to patients both in diagnostic and therapeutic applications. This international multi-center study aims to investigate the clinical measurement accuracy of radionuclide calibrators for 7 radionuclides used in theragnostics: 99mTc, 111In, 123I, 124I, 131I, 177Lu, and 90Y. Methods In total, 32 radionuclide calibrators from 8 hospitals located in the Netherlands, Belgium, and Germany were tested. For each radionuclide, a set of four samples comprising two clinical containers (10-mL glass vial and 3-mL syringe) with two filling volumes were measured. The reference value of each sample was determined by two certified radioactivity calibration centers (SCK CEN and JRC) using two secondary standard ionization chambers. The deviation in measured activity with respect to the reference value was determined for each radionuclide and each measurement geometry. In addition, the combined systematic deviation of activity measurements in a theragnostic setting was evaluated for 5 clinically relevant theragnostic pairs: 131I/123I, 131I/124I, 177Lu/111In, 90Y/99mTc, and 90Y/111In. Results For 99mTc, 131I, and 177Lu, a small minority of measurements were not within ± 5% range from the reference activity (percentage of measurements not within range: 99mTc, 6%; 131I, 14%; 177Lu, 24%) and almost none were outside ± 10% range. However, for 111In, 123I, 124I, and 90Y, more than half of all measurements were not accurate within ± 5% range (111In, 51%; 123I, 83%; 124I, 63%; 90Y, 61%) and not all were within ± 10% margin (111In, 22%; 123I, 35%; 124I, 15%; 90Y, 25%). A large variability in measurement accuracy was observed between radionuclide calibrator systems, type of sample container (vial vs syringe), and source-geometry calibration/correction settings used. Consequently, we observed large combined deviations (percentage deviation > ± 10%) for the investigated theragnostic pairs, in particular for 90Y/111In, 131I/123I, and 90Y/99mTc. Conclusions Our study shows that substantial over- or underestimation of therapeutic patient doses is likely to occur in a theragnostic setting due to errors in the assessment of radioactivity with radionuclide calibrators. These findings underline the importance of thorough validation of radionuclide calibrator systems for each clinically relevant radionuclide and sample geometry.

Radiolabeled amino acids as metabolic tracers are increasingly important for imaging and therapy applications. There are only a few reports on 99mTc-labeled amino acids that have been evaluated in vivo. In this work we coupled a small and highly potent chelator for the fac-[99mTc(CO)3]+ core to l-tyrosine through a poly(ethylene glycol) linker and investigated the in vivo biodistribution of the 99mTc-labeled l-tyrosine derivative. In vivo evaluation and biodistribution in mice bearing C6-tumor xenografts revealed little tumor and pancreas uptake. The microSPECT images showed rapid clearance from the blood pool. Fast uptake into the gallbladder and an extended residence time makes this labeled tyrosine suitable for gallbladder imaging and associated dysfunctions.

Ceramide levels are increased in blood and brain tissue of Alzheimer's disease (AD) patients. Since the ceramide transporter protein (CERT) is the only known protein able to mediate non-vesicular transfer of ceramide between organelle membranes, the modulation of CERT function may impact on ceramide accumulation. The competitive CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl) dodecanamide (HPA-12) interferes with ceramide trafficking. To understand the role of ceramide/CERT in AD, HPA-12 can be a useful tool to modulate ceramide trafficking. Here we first report the synthesis and in vitro properties of HPA-12 radiolabeled with fluorine-18 and present preliminary in vitro and in vivo positron emission tomography (PET) imaging and biodistribution data. In vitro results demonstrated that the fluorination did not alter the biological properties of HPA-12 since the [fluorine-19] HPA-12, interferes with 5-DMB-ceramide trafficking in HeLa cells. Radiolabeled HPA-12, [fluorine-18] HPA-12, was obtained with a radiochemical yield of 90% and a specific activity of 73 MBq/mu mol. PET imaging on wild-type mice showed hepatobiliary clearance and a brain uptake on the order of 0.3 standard uptake value (SUV) one hour post injection. Furthermore, the biodistribution data showed that after removal of the blood by intracardial perfusion, radioactivity was still measurable in the brain demonstrating that the [fluorine-18] HPA-12 crosses the blood brain barrier and is retained in the brain.

European journal of nuclear medicine and molecular imaging 43(13), 2433-2447 (2016). doi:10.1007/s00259-016-3480-8, Published by Springer-Verl., Heidelberg [u.a.]

Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [(68)Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing (68)Ga for preclinical CA IX imaging are scarce, and this is one of the first effective (68)Ga compounds reported for PET imaging of CA IX.

Brown adipose tissue (BAT) is present in human adults and the current gold standard to visualize and quantify BAT is [18F]FDG PET-CT. However, this method fails to detect BAT under insulin-resistant conditions associated with ageing and weight gain, such as type 2 diabetes. The aim of this study was to develop a novel triglyceride-based tracer for BAT. For this purpose we designed a dual-modal fluorescent/PET fatty acid tracer based on commercially available BODIPY-FL-C16, which can be esterified to its correspondent triglyceride, radiolabeled and incorporated into pre-synthesized chylomicron-like particles. BODIPY-FL-C16 was coupled to 1,2-diolein with a subsequent radiolabeling step resulting in [18F]BODIPY-C16-triglyceride that was incorporated into chylomicron-like particles. Various quality control steps using fluorescent and radioactive methods were conducted before BAT visualization was tested in mice. Triglyceride synthesis, radiolabeling and subsequent incorporation into chylomicron-like particles was carried out in decent yields. This radiotracer appeared able to visualize BAT in vivo, and the uptake of the radiotracer was stimulated by cold exposure. The here reported method can be used to incorporate radiolabeled triglycerides into pre-synthesized chylomicron-like particles. Our approach is feasible to visualize and quantify the uptake of triglyceride-derived fatty acids by BAT.

Noninvasive molecular imaging, using positron emission tomography (pet), is an important technique to visualize metabolic processes in vivo. It also allows to visualize the process of apoptosis, by using radiolabeled compounds such as annexin v, that bind to extracellular phosphatidylserine (ps). This chapter describes the radiosynthesis of 68ga-labeled annexin v and how to noninvasively image apoptosis in vivo.key wordsannexin v68gapetnoninvasive imaging.

We present the combination of the clinically well-proven chemotherapeutic agent, Doxorubicin, and (99m)Tc, an Auger and internal conversion electron emitter, into a dual-action agent for therapy. Chemical conjugation of Doxorubicin to (99m)Tc afforded a construct which autonomously ferries a radioactive payload into the cell nucleus. At this site, damage is exerted by dose deposition from Auger radiation. The (99m)Tc-conjugate exhibited a dose-dependent inhibition of survival in a selected panel of cancer cells and an in vivo study in healthy mice evidenced a biodistribution which is comparable to that of the parent drug. The homologous Rhenium conjugate was found to effectively bind to DNA, inhibited human Topoisomerase II, and exhibited cytotoxicity in vitro. The collective in vitro and in vivo data demonstrate that the presented metallo-conjugates closely mimic native Doxorubicin.

Atherosclerotic events are usually acute and often strike otherwise asymptomatic patients. Although multiple clinical risk factors have been associated with atherosclerosis, as of yet no further individual prediction can be made as to who will suffer from its consequences based on biomarker analysis or traditional imaging methods like CT, MRI or angiography.Previously, non-invasive imaging with F-18-fluorodeoxyglucose (F-18-FDG) PET was shown to potentially fill this niche as it offers high sensitive detection of metabolic processes associated with inflammatory changes in atherosclerotic plaques. However, F-18-FDG PET imaging of arterial vessels suffers from non specificity and has still to be proven to reliably identify vulnerable plaques, carrying a high risk of rupture. Therefore, it may be regarded only as a secondary marker for monitoring treatment effects and it does not offer alternative treatment options or direct insight in treatment mechanisms.In this review, an overview is given of the current status and the potential of PET imaging of inflammation and angiogenesis in atherosclerosis in general and special emphasis is given to imaging of alpha 7 nicotinic acetylcholine receptors (alpha 7 nAChRs). Due to the gaps that still exist in our understanding of atherogenesis and the limitations of the available PET tracers, the search continues for a more specific radioligand, able to differentiate between stable atherosclerosis and plaques prone to rupture. The potential role of the alpha 7 nAChR as imaging marker for plaque vulnerability is explored. Today, strong evidence exists that nAChRs are involved in the atherosclerotic disease process. They are suggested to mediate the deleterious effects of the major tobacco component, nicotine, a nAChR agonist.Mainly based on in vitro data, alpha 7 nAChR stimulation might increase plaque burden via increased neovascularization. However, in animal studies, alpha 7 nAChR manipulation appears to reduce plaque size due to its inhibitory effects on inflammatory cells. Thus, reliable identification of alpha 7 nAChRs by in vivo imaging is crucial to investigate the exact role of alpha 7 nAChR in atherosclerosis before any therapeutic approach in the human setting can be justified.In this review, we discuss the first experience with alpha 7 nAChR PET tracers and developmental considerations regarding the "optimal" PET tracer to image vascular nAChRs. (C) 2017 Published by Elsevier Inc.

PURPOSE: Brown adipose tissue (BAT) has transformed from an interfering tissue in oncological 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) to an independent imaging research field. This review takes the perspective from the imaging methodology on which human BAT research has come to rely on heavily. METHODS: This review analyses relevant PubMed-indexed publications that discuss molecular imaging methods of BAT. In addition, reported links between BAT and human diseases such as obesity are discussed, and the possibilities for imaging in these fields are highlighted. Radiopharmaceuticals aiming at several different biological mechanisms of BAT are discussed and evaluated. RESULTS: Prospective, dedicated studies allow visualization of BAT function in a high percentage of human subjects. BAT dysfunction has been implicated in obesity, linked with diabetes and associated with cachexia and atherosclerosis. Presently, 18F-FDG PET/CT is the most useful tool for evaluating therapies aiming at BAT activity. In addition to 18F-FDG, other radiopharmaceuticals such as 99mTc-sestamibi, 123I-metaiodobenzylguanidine (MIBG), 18F-fluorodopa and 18F-14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid (FTHA) may have a potential for visualizing other aspects of BAT activity. MRI methods are under continuous development and provide the prospect of functional imaging without ionizing radiation. CONCLUSION: Molecular imaging of BAT can be used to quantitatively assess different aspects of BAT metabolic activity.

PURPOSE: The purpose of this paper is to study molecular imaging of apoptosis and necrosis, two key players in atherosclerosis instability, using a multimodal imaging approach combining single photon emission computed tomography (SPECT), positron emission tomography (PET), and computed tomography (CT). PROCEDURES: Collar-induced carotid atherosclerosis ApoE knockout mice were imaged with 99mTc-AnxAF568 SPECT-CT to study apoptosis and sequentially with PET-CT following 124I-Hypericin (124I-Hyp) injection to visualize necrosis. RESULTS: SPECT depicted increased 99mTc-AnxAF568 uptake in both atherosclerotic carotid arteries, whereas our data suggest that this uptake is not merely apoptosis related. Although PET of 124I-Hyp was hampered by the slow blood clearance in atherosclerotic mice, 124I-Hyp was able to target necrosis in the atherosclerotic plaque. CONCLUSION: Both 99mTc-AnxAF568 and 124I-Hyp uptake are increased in atherosclerotic carotid vasculature compared to control arteries. While apoptosis imaging remains challenging, necrosis imaging can be feasible after improving the biodistribution characteristics of the probe.

To study whether formulation influences biodistribution, necrosis avidity and tumoricidal effects of the radioiodinated hypericin, a necrosis avid agent for a dual-targeting anticancer radiotherapy.Iodine-123- and 131-labeled hypericin ((123)I-Hyp and (131)I-Hyp) were prepared with Iodogen as oxidant, and formulated in dimethyl sulfoxide (DMSO)/PEG400 (polyethylene glycol 400)/water (25/60/15, v/v/v) or DMSO/saline (20:80, v/v). The formulations with excessive Hyp were optically characterized. Biodistribution, necrosis avidity and tumoricidal effects were studied in rats (n = 42) without and with reperfused liver infarction and implanted rhabdomyosarcomas (R1). To induce tumor necrosis, R1-rats were pre-treated with a vascular disrupting agent. Magnetic resonance imaging, tissue-gamma counting, autoradiography and histology were used.The two formulations differed significantly in fluorescence and precipitation. (123)I-Hyp/Hyp in DMSO/PEG400/water exhibited high uptake in necrosis but lower concentration in the lung, spleen and liver (p 0.01). Tumor volumes of 0.9 ± 0.3 cm(3) with high radioactivity (3.1 ± 0.3% ID/g) were detected 6 days post-treatment. By contrast, (131)I-Hyp/Hypin DMSO/saline showed low uptake in necrosis but high retention in the spleen and liver (p 0.01). Tumor volumes reached 2.6 ± 0.7 cm(3) with low tracer accumulation (0.1 ± 0.04%ID/g).The formulation of radioiodinated hypericin/hypericin appears crucial for its physical property, biodistribution, necrosis avidity and tumoricidal effects.

As CD13 is selectively expressed in angiogenesis, it can serve as a target for molecular imaging tracers to noninvasively visualize angiogenic processes in vivo. The CD13-targeting moiety NGR was synthesized and cyclized by native chemical ligation (NCL) instead of disulfide bridging, leading to a cyclic peptide backbone: cyclo(Cys-Asn-Gly-Arg-Gly) (coNGR). Beside this new monomeric coNGR, a tetrameric NGR peptide co(NGR)4 was designed and synthesized. After radiolabeling, their in vitro and in vivo characteristics were determined. Both coNGR-based imaging agents displayed considerably higher standardized uptake values (SUVs) at infarcted areas compared to the previously reported disulfide-cyclized cNGR imaging agent. Uptake patterns of 111In-coNGR and 111In-co(NGR)4 coincided with CD13 immunohistochemistry on excised hearts. Blood stability tests indicated better stability for both novel imaging agents after 50 min blood incubation compared to the disulfide-cyclized cNGR imaging agent. In mice, both coNGR peptides cleared rapidly from the blood mainly via the kidneys. In addition, co(NGR)4 showed a significantly higher specific uptake in infarcted myocardium compared to coNGR and thus is a promising sensitive imaging agent for detection of angiogenesis in infarcted myocardium.

PLoS one 12(8), e0182297 (2017). doi:10.1371/journal.pone.0182297, Published by PLoS, Lawrence, Kan.

Background: Motor impairments are among the major complications that develop after cortical damage caused by either stroke or traumatic brain injury. Motor cortex stimulation (MCS) can improve motor functions in animal models of stroke by inducing neuroplasticity. Objective: In the current study, the therapeutic effect of chronic MCS was assessed in a rat model of severe cortical damage. Methods: A controlled cortical impact (CCI) was applied to the forelimb area of the motor cortex followed by implantation of a flat electrode covering the lesioned area. Forelimb function was assessed using the Montoya staircase test and the cylinder test before and after a period of chronic MCS. Furthermore, the effect of MCS on tissue metabolism and lesion size was measured using [F-18]-fluorodesoxyglucose (FDG) mu PET scanning. Results: CCI caused a considerable lesion at the level of the motor cortex and dorsal striatum together with a long-lasting behavioral phenotype of forelimb impairment. However, MCS applied to the CCI lesion did not lead to any improvement in limb functioning when compared to non-stimulated control rats. Also, MCS neither changed lesion size nor distribution of FDG. Conclusion: The use of MCS as a standalone treatment did not improve motor impairments in a rat model of severe cortical damage using our specific treatment modalities. This work was supported in part by a grant from Hasselt University (BOF11603BOFMO) to SH and a grant from the Hersenstichting Nederland and Medtronic Europe (A1196117) to YT and AJ.

EJNMMI Research 6(1), 44 (2016). doi:10.1186/s13550-016-0199-2, Published by Springer, Berlin ; Heidelberg

Background— 18 F-fluorocholine ( 18 F-FCH) uptake is associated with cell proliferation and activity in tumor patients. We hypothesized that 18 F-FCH could similarly be a valuable imaging tool to identify vulnerable plaques and associated intraplaque inflammation and atheroma cell proliferation. Methods and Results— Ten consecutive stroke patients (90% men, median age 66.5 years, range, 59.4–69.7) with ipsilateral >70% carotid artery stenosis and who underwent carotid endarterectomy were included in the study. Before carotid endarterectomy, all patients underwent positron emission tomography to assess maximum 18 F-FCH uptake in ipsilateral symptomatic carotid plaques and contralateral asymptomatic carotid arteries, which was corrected for background activity, resulting in a maximum target-to-background ratio (TBRmax). Macrophage content was assessed in all carotid endarterectomy specimens as a percentage of CD68 + -staining per whole plaque area (plaqueCD68 + ) and as a maximum CD68 + percentage (maxCD68 + ) in the most inflamed section/plaque. Dynamic positron emission tomography imaging demonstrated that an interval of 10 minutes between 18 F-FCH injection and positron emission tomography acquisition is appropriate for carotid plaque imaging. TBRmax in ipsilateral symptomatic carotid plaques correlated significantly with plaqueCD68 + (Spearman’s ρ=0.648, P =0.043) and maxCD68 + (ρ=0.721, P =0.019) in the 10 corresponding carotid endarterectomy specimens. TBRmax was significantly higher ( P =0.047) in ipsilateral symptomatic carotid plaques (median: 2.0; interquartile range [Q1–Q3], 1.5–2.5) compared with the contralateral asymptomatic carotid arteries (median: 1.4; Q1–Q3, 1.3–1.6). TBRmax was not significantly correlated to carotid artery stenosis (ρ=0.506, P =0.135). Conclusions— In vivo uptake of 18 F-FCH in human carotid atherosclerotic plaques correlated strongly with degree of macrophage infiltration and recent symptoms, thus 18 F-FCH positron emission tomography is a promising tool for the evaluation of vulnerable plaques. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT01899014.

Noninvasive molecular imaging, using positron emission tomography (PET), is an important technique to visualize metabolic processes in vivo. It also allows to visualize the process of apoptosis, by using radiolabeled compounds such as Annexin V, that bind to extracellular phosphatidylserine (PS). This chapter describes the radiosynthesis of (68)Ga-labeled Annexin V and how to noninvasively image apoptosis in vivo.

Due to an enhanced demand for amino acids, the L-type amino acid transporter 1 (LAT1) is overexpressed in many tumor cell lines. LAT1 represents therefore an attractive target for cancer therapy and diagnosis. On the basis of our reported aqueous synthesis of (Cp-R)Tc-99m(CO)(3)]-type complexes,(1-S) we describe the preparation of unnatural amino acid analogues (Cp-CH2CH(NH2)COOH)Mn(CO)(3)] and (Cp-CH(NH2)COOH)M(CO)(3)] (M = Mn, Re, Tc-99m). Starting from fully protected HC5H5-aa (aa = amino acid), (Cp-aa)Tc-99m(CO)(3)] complexes are accessible in quantitative yields and in a one-step synthesis from (TcO4)-Tc-99m](-). The rhenium and manganese analogues were prepared and structurally characterized to confirm the authenticity of the Tc-99m complex. The inhibition constant of natural phenylalanine (phe) for LAT1 is in the range 70 +/- 10 mu M. The K-i value of (Cp-CH(NH2)COOH)Mn(CO)(3)] (1a) is 53 +/- 11 mu M, whereas K-i for the ``true'' phe analogue (Cp-CH2CH(NH2)COOH)Mn(CO)(3)] (2) was surprisingly high at 277 +/- 37 mu M. Complex la caused efflux when exposed to cells, underlining its active transport by LAT1 into the cell. Tc-99m analogues of small biological lead structures such as amino acids are generally not recognized anymore by their targets, in particular by trans-membrane transporters. The bioorganometallic analogues presented here are, however, actively transported and corroborate the importance of organometallic complexes as mimics of organic lead structures in life sciences.

Molecular imaging of biological processes may allow detection of therapy effects before the tumor is reduced in size. The most frequently used PET tracer in oncology, 2-[F-18]fluoro-2-deoxyglucose (FDG), suffers from low specificity due to uptake in inflammatory cells. The proliferation marker, 3'-[F-18]fluoro-3'-deoxy-L-thymidine (FLT), is less influenced by the inflammatory response following therapy but here disease-and drug-specific effects need to be considered. Since cancer therapy mainly intends to eliminate cancer cells, imaging of cell death offers a direct way to image therapy response. This review gives an overview of the radiopharmaceutical development and in vivo evaluation of radio-ligands that have emerged so far for detection and assessment of apoptosis and necrosis. Two radiopharmaceuticals that can image cell death have made it to clinical trials for follow up of tumor treatment: i) Tc-99m-and I-123-labelled AnxA5 for the response to treatment of for example lymphoma and lung cancer and ii) F-18-ML10 for the evaluation of brain tumors post-radiation. Other agents need further optimization.

As an alternative to directly targeting of necrotic tissue using hypericin, we synthesized a conjugate of hypericin to biotin for use in a pretargeting approach. With this conjugate, we explored the possibility of a two-step pretargeting strategy using (123)I-labeled avidin as effector molecule directed against necrotic RIF-1 tumors. Hypericin was conjugated to biotin-ethylenediamine in a straightforward coupling method using n-hydroxysuccinimide and dicyclohexylcarbodiimide. The necrosis avidity of the conjugate was first confirmed in necrotic liver tissue by means of fluorescence microscopy. Using autoradiography imaging and whole body-biodistribution, the accumulation of (123)I-avidin in necrotic tumor tissue was evaluated 24 h after administration and 48 h after pretargeting with hypericin-biotin. Analysis of autoradiography images show a higher accumulation of (123)I-avidin in pretargeted compared to nontargeted tissue. However, absolute accumulation of (123)I-avidin in necrotic tumors was low as shown by biodistribution experiments. Direct injection of hypericin-biotin or biotin-fluorescein did not substantially improve (123)I-avidin accumulation after pretargeting, pointing towards a poor penetration of avidin in necrotic tissue. Our results show the feasibility of a pretargeting technique using a small molecule as targeting agent. However, for a more efficient accumulation of the effector molecule in necrotic tissue, other pretargeting strategies need to be investigated.

Purpose Two variants of Annexin A5 (Cys2-AnxA5 and Cys165-AnxA5) were labelled with Gallium-68 in order to evaluate their biological properties. Procedures Biodistribution and pharmacokinetics of the radiotracers were studied with μPET in healthy mice and in a mouse model of hepatic apoptosis. μPET imaging after IV injection of the tracers in combination with μMRI was performed in Daudi tumor bearing mice before and after treatment with a combination of chemotherapy and radiotherapy. Results The biodistribution data indicated a fast urinary clearance with only minor hepatobilliary clearance, although a high retention in the kidneys was observed. Animals treated with anti-Fas showed a 3 to 8 times higher liver uptake as compared to healthy animals. Tumor uptake of 68 Ga-Cys2-AnxA5 and 68 Ga-Cys165-AnxA5 was low but significantly increased after therapy. Conclusion Both 68 Ga-Cys2-AnxA5 and 68 Ga-Cys165-AnxA5 show a clear binding to apoptotic cells and are promising tracers for rapid evaluation of cancer therapy.

Objectives (68)Ga-Dotatoc has become the PET radiopharmaceutical of choice for the diagnosis and treatment follow-up of neuroendocrine tumours. (68)Ga-Dotatoc is prepared on-site through a so-called magisterial preparation. The use of an appropriate buffer during the radiolabelling step is essential to maximize the labelling yield and the specific activity. Such a buffer should be nontoxic, able to buffer in the pH range of 3.5-5.0, not compete with gallium ions and preferentially have a weak metal complexing capacity to avoid the formation of colloidal gallium. In addition, the buffer should be allowed for human use. In view of the high radiation dose to the operator when manually handling (68)Ga, especially to the extremities, we also tested the buffers in a semi-automated system. Methods HEPES, acetate, succinate, Tris, glutamate, lactate, oxalate and tartrate were tested as potential buffers in the manual radiosynthesis of (68)Ga-Dotatoc. Temperature, heating time and substrate concentration were optimized. Results Buffers based on HEPES, acetate and succinate were found to be the most appropriate. Optimal labelling yields were achieved with a 5-min heating time for the manual synthesis and 8 min for a semi-automated system, whereas the optimal amount of Dotatoc was 30 and 40 microg, respectively. Conclusion Although the use of HEPES, acetate and succinate as buffering substances yielded comparable results, only acetate is currently recognized as a substance for pharmaceutical use and also for human use. Therefore, acetate buffer should be used for (68)Ga-Dotatoc synthesis. The semi-automated system allowed for a shorter radiosynthesis time, thereby increasing the overall yield.

SummaryAim: To assess the accuracy of ECG-gated micro (µ)-SPECT in a mouse myocardial infarction (MI) model in comparison to 3D-echocardiography. Animals, methods: In a mouse (Swiss mice) MI model we compared the accuracy of technetium-99m sestamibi (99mTc-sestamibi) myocardial perfusion, electrocardiogram (ECG) gated µSPECT to 3D-echocardiography in determining left ventricular function. 3D-echocardiography and myocardial perfusion ECG-gated µSPECT data were acquired in the same animal at baseline (n = 11) and 7 (n = 8) and 35 (n = 9) days post ligation of the left anterior descending coronary artery (LAD). Sham operated mice were used as a control (8, 6 and 7 mice respectively). Additionally, after day 35 µSPECT scans, hearts were harvested and 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining and autoradiography was performed to determine infarct size. Results: In both infarcted and sham-operated mice we consistently found comparable values for the end-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (EF) obtained by 3D-echocardiography and ECG-gated µSPECT. Excellent correlations between measurements from 3D-echocardiography and ECG-gated µSPECT were found for EDV, ESV and EF (r = 0.9532, r = 0.9693 respectively and r = 0.9581) in infarcted mice. Furthermore, comparable infarct size values were found at day 35 post MI by TTC staining and autoradiography (27.71 ± 1.80% and 29.20 ± 1.18% with p = 0.43). Conclusion: We have demonstrated that ECG-gated µSPECT imaging provides reliable left ventricular function measurements in a mouse MI model. Obtained results were comparable to the highly accurate 3D-echocardiography. This, in addition to the opportunity to simultaneously image multiple biological processes during a single acquisition makes µSPECT imaging a serious option for studying cardiovascular disease in small animals.

The Na+-independent l -type LAT1 amino acid transport system for large and neutral amino acids has been shown to be expressed higher in tumour tissue relative to normal tissue and has been regarded as a key point for the development of new amino acid based tumour tracers for molecular imaging. We developed a new fluorinated phenylalanine analogue, 2-[18F]fluoromethyl- l -phenylalanine, considering that the spatial volume of FCH3 is comparable with that of the iodine atom in 2-I- l -phenylalanine, of which we have proven that it is taken up excellently in tumours by the LAT1 system. The substrate molecule for radiolabeling, Boc-2-bromomethyl- l -phenylalanine-tButylester, was prepared by radical bromination of Boc-2-methyl- l -phenylalanine-tButylester. [18F−] for bromine exchange is performed within 3 min in conditions comparable to the [18F]FDG synthesis with a radiochemical yield of at least 85%. After deprotection and semi-preparative HPLC purification, the 2-[18F]fluoromethyl- l -phenylalanine is recovered n.c.a. (57%) with a high purity and 3.7 MBq were injected into R1M rhabdomyosarcoma tumour-bearing rats. Imaging was performed with a human PET camera from 5 to 45 min p.i. The tumour/background and tumour/blood ratios obtained from PET acquisition were at least 2.5. DUR values for the tumours were at least about 5. Furthermore, a small tumour implanted near a kidney could be well visualized completely separated from this kidney. Moreover in all tumours the “active” tumour tissue can clearly be differentiated from less active tumour tissue. This proves that 2-[18F]fluoromethyl- l -phenylalanine has a great potential as a new tracer for specific tumour diagnosis with PET.

The aim of this study was the comparison of the tumour uptake and the long-term retention of [123I]-2-I-L-phenylalanine and [123I]-2-I-D-phenylalanine with those of [123I]-2-I-L-tyrosine and [123I]-2-I-D-tyrosine in R1M rhabdomyosarcoma tumour-bearing rats. The biodistribution of the radioactivity as a function of time in R1M tumour-bearing rats was measured by planar gamma camera imaging (dynamic and static). If dissection was applied, the activity in the tumours and tissues of interest was measured by gamma counting. [123I]-2-iodo-L-phenylalanine, [123I]-2-iodo-D-phenylalaine, [123I]-2-I-L-tyrosine showed a considerable tumour uptake reaching a maximum between 10 and 30 min. At 30 min p.i. the differential uptake ratio values of this uptake were, respectively, 2.1, 2.3, 2.5 and 1.7. The activity in the tumour was shown to be related to a tumour cell uptake and not to an increased blood pool activity. All the tracers showed a clearance from the blood to the bladder without renal retention. At longer times both L- and D- [123I]-2-I-tyrosine were cleared for a large part from the tumours and the body. [123I]-2-I-L-Phe and [123I]-2-I-D-Phe showed a considerable and equal retention in the tumours: as compared with 0.5 h, 91% at 24 h and 80% at 48 h. This was related to the longer retention of activity in the blood pool noticed for these compounds (81% at 24 h and 65% at 48 h). The tumour-to-background ratio increased with 25% at those longer times. At short times all the tracers were taken up to a considerable extent in the tumours. In the R1M-bearing Wag/Rij rat model only [123I]-2-I-L-phenylalanine and [123I]-2-I-D-phenylalanine showed an especially high retention at long times without any significant difference between the enantiomers. Copyright © 2007 John Wiley & Sons, Ltd.

OBJECTIVE: To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice. METHODS: Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry. RESULTS: Hr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were

Medicine 94(20), e725 (2015). doi:10.1097/MD.0000000000000725, Published by Lippincott Williams & Wilkins, Baltimore, Md.

Radiolabeling allows noninvasive imaging by single photon emission computed tomography (SPECT) or positron emission tomography (PET) for assessing the biodistribution of nanostructures. Herein, the synthesis of a new coating ligand for gold nanoparticles (AuNPs) and quantum dots (QDs) is reported. This ligand is multifunctional; it combines the metal chelate with conjugating functions to biological vectors. The concept allows the coupling of any targeting function to the chelator; an example for the prostate specific membrane antigen is given. Derivatized NPs can directly be labeled in one step with [(99m) Tc(OH2 )3 (CO)3 ](+) . AuNPs in particular are highly stable, a prerequisite for in vivo studies excluding misinterpretation of the biodistribution data. AuNPs with differing sizes (7 and 14 nm core diameter) were administered intravenously into nude NMRI mice bearing LNCaP xenografts. MicroSPECT images show for both probes rapid clearance from the blood pool through the hepatobiliary pathway. The 7 nm AuNPs revealed a significantly higher bone uptake than the 14 nm AuNPs. The high affinity towards bone mineral is further confirmed in vitro with hydroxyapatite.