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The catecholamine analogue [123 I]mIBG has been used for scintigraphic imaging of neuroblastoma since 1984. It is taken up by the noradrenaline transporter (NAT), which is present in most neuroblastoma cells. An alternative imaging method could be PET with 6-[18 F]fluorodopamine, which is also taken up by NAT, but-in contrast to mIBG-also by dopamine transporter (DAT), present in neuroblastoma cells (NAT > DAT). An enzymatic method was established allowing a rapid, quantitative transformation of FDOPA to FDA by DOPA decarboxylase within 25 minutes. This strategy was applied to [18 F]FDOPA, which was produced via nucleophilic synthesis (RCY 15%, 10 GBq, 50 GBq/μmol) and subsequently converted to [18 F]FDA (RCY 35%-50%, n = 5). Uptake and metabolism of FDOPA and FDA were analyzed in human Kelly and SK-N-SH neuroblastoma cell lines and in human Caki-1 kidney cells that can take up catecholamines and mIBG via an organic cation transporter (OCT). FDOPA and FDA were taken up by all three cells, but FDOPA could only be converted to FDA in neuroblastoma cells. As today, [18 F]FDOPA is well available in high yields, efficient enzymatic conversion to [18 F]FDA to be used for NAT/DAT PET imaging in neuroendocrine tumors is an attractive, alternative synthesis route.

Mitogen-activated protein kinase (MAPK) signaling plays an important role in inflammatory diseases such as rheumatoid arthritis (RA).The aim of our study was to elucidate the therapeutic potential of the highly selective p38 MAPK inhibitor Skepinone-L and the dual inhibitor LN 950 (p38 MAPK and JNK 3) in the K/BxN serum transfer model of RA. Additionally, we aimed to monitor MAPK treatment non-invasively in vivo using the hypoxia tracer [18F]fluoromisonidazole ([18F]FMISO) and positron emission tomography (PET). To induce experimental arthritis, we injected glucose-6-phosphate isomerase autoantibody-containing serum in BALB/c mice. MAPK inhibitor or Sham treatment was administered per os once daily. On days 3 and 6 after arthritis induction, we conducted PET imaging with [18F]FMISO. At the end of the experiment, ankles were harvested for histopathological analysis. Skepinone-L and LN 950 were applicable to suppress the severity of experimental arthritis confirmed by reduced ankle swelling and histopathological analysis. Skepinone-L (3.18 ± 0.19 mm) and LN 950 (3.40 ± 0.13 mm) treatment yielded a significantly reduced ankle thickness compared to Sham-treated mice (3.62 ± 0.11 mm) on day 5 after autoantibody transfer, a time-point characterized by severe arthritis. Hypoxia imaging with [18F]FMISO revealed non-conclusive results and might not be an appropriate tool to monitor MAPK therapy in experimental RA. Both the selective p38 MAPK inhibitor Skepinone-L and the dual (p38 MAPK and JNK 3) inhibitor LN 950 exhibited significant therapeutic effects during experimental arthritis. Thus, our study contributes to the ongoing discussion on the use of p38 MAPK as a potential target in RA.

Although preclinical studies clearly indicate an effect of 5-HTTLPR genotype on 5-HT transporter (5-HTT) expression, studies in humans provided inconclusive results, hypothetically due to environmental factors and differences in individual behavior. For example, nicotine and other constituents of tobacco smoke elevate serotonin (5-HT) levels in the brain and may thereby cause homeostatic adaptations in 5-HTT availability that moderate effects of 5-HTTLPR genotype. To test whether 5-HTT availability in the midbrain is affected by smoking status and 5-HTTLPR genotype, we pooled data from prior studies on in vivo 5-HTT availability (BPND) measured with positron emission tomography (PET) and [11C]DASB. In total, we reanalyzed 5-HTT availability in 116 subjects using ANCOVA statistics. ROI analysis revealed that current smokers and non-smokers do not differ in midbrain BPND. Interestingly, smoking status significantly interacted with 5-HTTLPR genotype: active smoking was associated with reduced 5-HTT availability only in LL subjects but not in carriers of the S-allele. From the perspective of genotype effects, non-smokers showed the expected association with 5-HTTLPR, i.e. higher 5-HTT availability in LL subjects compared to carriers of the S-allele, whereas this pattern was actually reversed for active smokers. Our study indicates that smoking status moderates the association of 5-HTTLPR genotype and 5-HTT expression, which may help to explain inconsistent findings in previous studies. Regarding the mechanism, we suggest that smoking may induce epigenetic processes such as methylation of SLC6A4, which can differ depending on its genetic constitution.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells of the myeloid compartment and major players in the tumor microenvironment (TME). With increasing numbers of studies describing MDSC involvement in cancer immune escape, cancer metastasis and the dampening of immunotherapy responses, MDSCs are of high interest in current cancer therapy research. Although heavily investigated in the last decades, the in vivo migration dynamics of MDSC subpopulations in tumor- or metastases-bearing mice have not yet been studied extensively. Therefore, we have modified our previously reported intracellular cell labeling method and applied it to in vitro generated MDSCs for the quantitative in vivo monitoring of MDSC migration in primary and metastatic cancer. MDSC migration to primary cancers was further correlated to the frequency of endogenous MDSCs. Methods: Utilizing a 64Cu-labeled 1,4,7-triazacyclononane-triacetic acid (NOTA)-modified CD11b-specific monoclonal antibody (mAb) (clone M1/70), we were able to label in vitro generated polymorphonuclear (PMN-) and monocytic (M-) MDSCs for positron emission tomography (PET) imaging. Radiolabeled PMN- and M-MDSCs ([64Cu]PMN-MDSCs and [64Cu]M-MDSCs, respectively) were then adoptively transferred into primary and metastatic MMTV-PyMT-derived (PyMT-) breast cancer- and B16F10 melanoma-bearing experimental animals, and static PET and anatomical magnetic resonance (MR) images were acquired 3, 24 and 48 h post cell injection. Results: The internalization of the [64Cu]NOTA-mAb-CD11b-complex was completed within 3 h, providing moderately stable radiolabeling with little detrimental effect on cell viability and function as determined by Annexin-V staining and T cell suppression in flow cytometric assays. Further, we could non-invasively and quantitatively monitor the migration and tumor homing of both [64Cu]NOTA-αCD11b-mAb-labeled PMN- and M-MDSCs in mouse models of primary and metastatic breast cancer and melanoma by PET. We were able to visualize and quantify an increased migration of adoptively transferred [64Cu]M-MDSCs than [64Cu]PMN-MDSCs to primary breast cancer lesions. The frequency of endogenous MDSCs in the PyMT breast cancer and B16F10 melanoma model correlated to the uptake values of adoptively transferred MDSCs with higher frequencies of PMN- and M-MDSCs in the more aggressive B16F10 melanoma tumors. Moreover, aggressively growing melanomas and melanoma-metastatic lesions recruited higher percentages of both [64Cu]PMN- and [64Cu]M-MDSCs than primary and metastatic breast cancer lesions as early as 24 h post adoptive MDSC transfer, indicating an overall stronger recruitment of cancer-promoting immunosuppressive MDSCs. Conclusion: Targeting of the cell surface integrin CD11b with a radioactive mAb is feasible for labeling of murine MDSCs for PET imaging. Fast internalization of the [64Cu]NOTA-αCD11b-mAb provides presumably enhanced stability while cell viability and functionality was not significantly affected. Moreover, utilization of the CD11b-specific mAb allows for straightforward adaptation of the labeling approach for in vivo molecular imaging of other myeloid cells of interest in cancer therapy, including monocytes, macrophages or neutrophils.

Hypoxia is essential for the development of autoimmune diseases such as rheumatoid arthritis (RA) and is associated with the expression of reactive oxygen species (ROS), because of the enhanced infiltration of immune cells. The aim of this study was to demonstrate the feasibility of measuring hypoxia noninvasively in vivo in arthritic ankles with PET/MRI using the hypoxia tracers 18F-fluoromisonidazole (18F-FMISO) and 18F-fluoroazomycinarabinoside (18F-FAZA). Additionally, we quantified the temporal dynamics of hypoxia and ROS stress using L-012, an ROS-sensitive chemiluminescence optical imaging probe, and analyzed the expression of hypoxia-inducible factors (HIFs). Methods: Mice underwent noninvasive in vivo PET/MRI to measure hypoxia or optical imaging to analyze ROS expression. Additionally, we performed ex vivo pimonidazole-/HIF-1α immunohistochemistry and HIF-1α/2α Western blot/messenger RNA analysis of inflamed and healthy ankles to confirm our in vivo results. Results: Mice diseased from experimental RA exhibited a 3-fold enhancement in hypoxia tracer uptake, even in the early disease stages, and a 45-fold elevation in ROS expression in inflamed ankles compared with the ankles of healthy controls. We further found strong correlations of our noninvasive in vivo hypoxia PET data with pimonidazole and expression of HIF-1α in arthritic ankles. The strongest hypoxia tracer uptake was observed as soon as day 3, whereas the most pronounced ROS stress was evident on day 6 after the onset of experimental RA, indicating that tissue hypoxia can precede ROS stress in RA. Conclusion: Collectively, for the first time to our knowledge, we have demonstrated that the noninvasive measurement of hypoxia in inflammation using 18F-FAZA and 18F-FMISO PET imaging represents a promising new tool for uncovering and monitoring rheumatic inflammation in vivo. Further, because hypoxic inflamed tissues are associated with the overexpression of HIFs, specific inhibition of HIFs might represent a new powerful treatment strategy.

Introduction Radiolabeled 2-nitroimidazoles (azomycins) are a prominent class of biomarkers for PET imaging of hypoxia. [ 18 F]Fluoro-azomycin- α -arabinoside ([ 18 F]FAZA) – already in clinical use – may be seen as α -configuration nucleoside, but enters cells only via diffusion and is not transported by cellular nucleoside transporters. To enhance image contrast in comparison to [ 18 F]FAZA our objective was to 18 F-radiolabel an azomycin-2´-deoxyriboside with β -configuration ([ 18 F]FAZDR, [ 18 F]- β - 8 ) to mimic nucleosides more closely and comparatively evaluate it versus [ 18 F]FAZA. Methods Precursor and cold standards for [ 18 F]FAZDR were synthesized from methyl 2-deoxy-d-ribofuranosides α - and β - 1 in 6 steps yielding precursors α - and β - 5 . β - 5 was radiolabeled in a GE TRACERlab FX F-N synthesizer in DMSO and deprotected with NH 4 OH to give [ 18 F]FAZDR ([ 18 F]- β - 8 ). [ 18 F]FAZA or [ 18 F]FAZDR was injected in BALB/c mice bearing CT26 colon carcinoma xenografts, PET scans (10min) were performed after 1, 2 and 3h post injection (p.i.). On a subset of mice injected with [ 18 F]FAZDR, we analyzed biodistribution. Results [ 18 F]FAZDR was obtained in non-corrected yields of 10.9±2.4% (9.1±2.2GBq, n=4) 60min EOB, with radiochemical purity >98% and specific activity >50GBq/μmol. Small animal PET imaging showed a decrease in uptake over time for both [ 18 F]FAZDR (1h p.i.: 0.56±0.22% ID/cc, 3h: 0.17±0.08% ID/cc, n=9) and [ 18 F]FAZA (1h: 1.95±0.59% ID/cc, 3h: 0.87±0.55% ID/cc), whereas T/M ratios were significantly higher for [ 18 F]FAZDR at 1h (2.76) compared to [ 18 F]FAZA (1.69, P 18 F]FAZDR showed an inverse correlation between tracer uptake in carcinomas and oxygen breathing, while muscle tissue uptake was not affected by switching from air to oxygen. Conclusions First PET imaging results with [ 18 F]FAZDR showed advantages over [ 18 F]FAZA regarding higher tumor contrast at earlier time points p.i. Availability of precursor and cold fluoro standard together with high output radiosynthesis will allow for a more detailed quantitative evaluation of [ 18 F]FAZDR, especially with regard to mechanistic studies whether active transport processes are involved, compared to passive diffusion as observed for [ 18 F]FAZA.

The benefits of PET imaging of tumor hypoxia in patient management has been demonstrated in many examples and with various tracers over the last years. Although, the optimal hypoxia imaging agent has yet to be found, 2-nitroimidazole (azomycin) sugar derivatives&mdash, mimicking nucleosides&mdash, have proven their potential with [18F]FAZA ([18F]fluoro-azomycin-&alpha, arabinoside) as a prominent representative in clinical use. Still, for all of these tracers, cellular uptake by passive diffusion is postulated with the disadvantage of slow kinetics and low tumor-to-background ratios. We recently evaluated [18F]fluoro-azomycin-&beta, deoxyriboside (&beta, [18F]FAZDR), with a structure more similar to nucleosides than [18F]FAZA and possible interaction with nucleoside transporters. For a deeper insight, we comparatively studied the interaction of FAZA, &beta, FAZA, &alpha, FAZDR and &beta, FAZDR with nucleoside transporters (SLC29A1/2 and SLC28A1/2/3) in vitro, showing variable interactions of the compounds. The highest interactions being for &beta, FAZDR (IC50 124 &plusmn, 33 &micro, M for SLC28A3), but also for FAZA with the non-nucleosidic &alpha, configuration, the interactions were remarkable (290 &plusmn, 44 &micro, M {SLC28A1}, 640 &plusmn, 10 &micro, M {SLC28A2}). An improved synthesis was developed for &beta, FAZA. For a PET study in tumor-bearing mice, &alpha, [18F]FAZDR was synthesized (radiochemical yield: 15.9 &plusmn, 9.0% (n = 3), max. 10.3 GBq, molar activity &gt, 50 GBq/&micro, mol) and compared to &beta, [18F]FAZDR and [18F]FMISO, the hypoxia imaging gold standard. We observed highest tumor-to-muscle ratios (TMR) for &beta, [18F]FAZDR already at 1 h p.i. (2.52 &plusmn, 0.94, n = 4) in comparison to [18F]FMISO (1.37 &plusmn, 0.11, n = 5) and &alpha, [18F]FAZDR (1.93 &plusmn, 0.39, n = 4), with possible mediation by the involvement of nucleoside transporters. After 3 h p.i., TMR were not significantly different for all 3 tracers (2.5&ndash, 3.0). Highest clearance from tumor tissue was observed for &beta, [18F]FAZDR (56.6 &plusmn, 6.8%, 2 h p.i.), followed by &alpha, [18F]FAZDR (34.2 &plusmn, 7.5%) and [18F]FMISO (11.8 &plusmn, 6.5%). In conclusion, both isomers of [18F]FAZDR showed their potential as PET hypoxia tracers. Differences in uptake behavior may be attributed to a potential variable involvement of transport mechanisms.

In a variety of diseases, from benign to life-threatening ones, inflammation plays a major role. Monitoring the intensity and extent of a multifaceted inflammatory process has become a cornerstone in diagnostics and therapy monitoring. However, the current tools lack the ability to provide insight into one of its most crucial aspects, namely, the alteration of the extracellular matrix (ECM). Using a radiolabeled platelet glycoprotein VI-based ECM-targeting fusion protein (GPVI-Fc), we investigated how binding of GPVI-Fc on fibrous tissue could uncover the progression of several inflammatory disease models at different stages (rheumatoid arthritis, cutaneous delayed-type hypersensitivity, lung inflammation and experimental autoimmune encephalomyelitis). Methods: The fusion protein GPVI-Fc was covalently linked to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and subsequently labeled with 64Cu. We analyzed noninvasively in vivo 64Cu-GPVI-Fc accumulation in murine cutaneous delayed-type hypersensitivity, anti-glucose-6-phosphate isomerase serum-induced rheumatoid arthritis, lipopolysaccharide-induced lung inflammation and an experimental autoimmune encephalomyelitis model. Static and dynamic Positron Emission Tomography (PET) of the radiotracer distribution was performed in vivo, with ex vivo autoradiography confirmation, yielding quantitative accumulation and a distribution map of 64Cu-GPVI-Fc. Ex vivo tissue histological staining was performed on harvested samples to highlight the fusion protein binding to collagen I, II and III, fibronectin and fibrinogen as well as the morphology of excised tissue. Results: 64Cu-GPVI-Fc showed a several-fold increased uptake in inflamed tissue compared to control tissue, particularly in the RA model, with a peak 24 h after radiotracer injection of up to half the injected dose. Blocking and isotype control experiments indicated a target-driven accumulation of the radiotracer in the case of chronic inflammation. Histological analysis confirmed a prolonged accumulation at the inflammation site, with a pronounced colocalization with the different components of the ECM (collagen III and fibronectin notably). Binding of the fusion protein appeared to be specific to the ECM but unspecific to particular components. Conclusion: Imaging of 64Cu-GPVI-Fc accumulation in the ECM matrix appears to be a promising candidate for monitoring chronic inflammation. By binding to exposed fibrous tissue (collagen, fibronectin, etc.) after extravasation, a new insight is provided into the fibrotic events resulting from a prolonged inflammatory state.

2-Deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) has been used as a standard clinical positron emission tomography (PET) tracer for the follow-up of the rare but life-threatening parasitic disease alveolar echinococcosis (AE). Given that the disease is endemic in many countries in the northern hemisphere and the diagnosis is still challenging, the aim of our study was to evaluate further clinically relevant PET tracers as possible diagnostic tools for AE in vitro and in vivo. Various clinically used PET tracers were evaluated in vitro and assessed in an in vivo AE animal model based on PET/magnetic resonance (MR) measurements. In vitro binding assays displayed high uptake of [18F]FDG in a cell suspension of E. multilocularis tissue, whereas 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT) and [11C]choline were found to be taken up strongly by E. multilocularis vesicles. [18F]FDG and [18F]FLT displayed an elevated uptake in vivo, which appeared as several foci throughout the parasite tissue as opposed to [18F]fluoro-azomycinarabinofuranoside ([18F]FAZA) and [11C]choline. Our data clearly demonstrate that the clinically applied PET tracer [18F]FDG is useful for the diagnosis and disease staging of AE but also has drawbacks in the assessment of currently inactive or metabolically weak parasitic lesions. The different tested PET tracers do not show the potential for the replacement or supplementation of current diagnostic strategies. Hence, there is still the need for novel diagnostic tools.

Background. 1-α-D-(5-Deoxy-5-[18F]fluoroarabinofuranosyl)-2-nitroimidazole ([18F]FAZA) is a PET radiotracer that demonstrates excellent potential in imaging regional hypoxia, and is clinically used in diagnosing a wide range of solid tumors in cancer patients. [18F]FAZA, however, is radiofluorinated in only moderate recovered radiochemical yield (rRCY, ~12%). It is postulated that the relative stability of the C1’ β-anomeric bond at C5’ will make 1-β-D-(5-fluoro-5-deoxyarabinofuranosyl)-2-nitroimidazole (β-FAZA), the β-conformer of FAZA, an attractive candidate for clinical hypoxia imaging. Objectives. The principle goals were to synthesize β-FAZA and β-Ac2TsAZA, the radiofluorination precursor, to establish the radiofluorination chemistry leading to β-[18F]FAZA, and to investigate the biodistribution of β-[18F]FAZA in an animal tumor-bearing model using PET imaging. Methods. The appropriately-protected furanose sugar was coupled with 2-nitroimidazole to afford 1-β-D-(2,3-di-O-acetylarabinofuranosyl)-2-nitroimidazole (β-Ac2AZA). Fluorination of β-Ac2AZA with DAST, followed by alkaline hydrolysis, afforded β-FAZA (21%). The radiolabeling synthon, 1-β-D-(5-O-toluenesulfonyl-2,3-di-O-acetylarabinofuranosyl)-2-nitroimidazole (β-Ac2TsAZA), on radiofluorination using the 18F/K222 complex under various reaction conditions, followed by base-catalyzed deacetylation, afforded β-[18F]FAZA. β-[18F]FAZA was radiochemically stable for at least 8 h when stored in aqueous ethanol (8%) at 22 °C. A preliminary PET imaging-based biodistribution study of β-[18F]FAZA was performed in A431 tumor-bearing nude mice. Results. β-FAZA and β-Ac2TsAZA were synthesized in satisfactory yield. Radiochemistry of [18F]FAZA was established. PET images showed strong uptake in hypoxic regions of the tumor. Conclusions. The synthesis of β-FAZA and β-[18F]FAZA are reported. Radiofluorination of β-Ac2TsAZA and the deprotection of β-Ac2[18F]FAZA were facile, but led to a more complex mixture of radiofluorinated by-products than observed with the corresponding precursor of α-[18F]FAZA. PET images were indicative of hypoxia-selective accumulation of β-[18F]FAZA in tumor.

Blockade of the μ-opioid receptor (MOR) by naltrexone reduces relapse risk in a subpopulation of alcohol-dependent patients. Previous positron-emission-tomography (PET) studies using the MOR ligand [11C]carfentanil have found increased MOR availability in abstinent alcoholics, which may reflect either increased MOR expression or lower endogenous ligand concentration. To differentiate between both effects, we investigated two cohorts of alcoholic subjects using either post-mortem or clinical PET analysis. Post-mortem brain tissue of alcohol-dependent subjects and controls (N=43/group) was quantitatively analyzed for MOR ([3H]DAMGO) binding sites and OPRM1 mRNA in striatal regions. [11C]carfentanil PET was performed in detoxified, medication free alcohol-dependent patients (N=38), followed by a randomized controlled study of naltrexone versus placebo and follow up for one year (clinical trial number: NCT00317031). Because the functional OPRM1 variant rs1799971:A>G affects the ligand binding, allele carrier status was considered in the analyses. MOR binding sites were reduced by 23–51 in post-mortem striatal tissue of alcoholics. In the PET study, a significant interaction of OPRM1 genotype, binding potential (BPND) for [11C]carfentanil in the ventral striatum, and relapse risk was found. Particularly in G-allele carriers, lower striatal BPND was associated with a higher relapse risk. Interestingly, this effect was more pronounced in the naltrexone treatment group. Reduced MOR is interpreted as a neuroadaptation to an alcohol-induced release of endogenous ligands in patients with severe alcoholism. Low MOR availability may explain the ineffectiveness of naltrexone treatment in this subpopulation. Finally, low MOR binding sites are proposed as a molecular marker for a negative disease course.

Although T cells can be labeled for noninvasive in vivo imaging, little is known about the impact of such labeling on T-cell function, and most imaging methods do not provide holistic information about trafficking kinetics, homing sites, or quantification. Methods We developed protocols that minimize the inhibitory effects of (64)Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) ((64)Cu-PTSM) labeling on T-cell function and permit the homing patterns of T cells to be followed by PET. Thus, we labeled ovalbumin (OVA) T-cell receptor transgenic interferon (IFN)-γ-producing CD4(+) T (Th1) cells with 0.7-2.2 MBq of (64)Cu-PTSM and analyzed cell viability, IFN-γ production, proliferation, apoptosis, and DNA double-strand breaks and identified intracellular (64)Cu accumulation sites by energy dispersive x-ray analysis. To elucidate the fate of Th1 cell homing by PET, 10(7 64)Cu-OVA-Th1 cells were injected intraperitoneally or intravenously into healthy mice. To test the functional capacities of (64)Cu-OVA-Th1 cells during experimental OVA-induced airway hyperreactivity, we injected 10(7 64)Cu-OVA-Th1 cells intraperitoneally into OVA-immunized or nonimmunized healthy mice, which were challenged with OVA peptide or phosphate-buffered saline or remained untreated. In vivo PET investigations were followed by biodistribution, autoradiography, and fluorescence-activated cell sorting analysis. Results PET revealed unexpected homing patterns depending on the mode of T-cell administration. Within 20 min after intraperitoneal administration, (64)Cu-OVA-Th1 cells homed to the perithymic lymph nodes (LNs) of naive mice. Interestingly, intravenously administered (64)Cu-OVA-Th1 cells homed predominantly into the lung and spleen but not into the perithymic LNs. The accumulation of (64)Cu-OVA-Th1 cells in the pulmonary LNs (6.8 ± 1.1 percentage injected dose per cubic centimeter [%ID/cm(3)]) 24 h after injection was highest in the OVA-immunized and OVA-challenged OVA airway hyperreactivity-diseased littermates 24 h after intraperitoneal administration and lowest in the untreated littermates (3.7 ± 0.4 %ID/cm(3)). As expected, (64)Cu-OVA-Th1 cells also accumulated significantly in the pulmonary LNs of nonimmunized OVA-challenged animals (6.1 ± 0.5 %ID/cm(3)) when compared with phosphate-buffered saline-challenged animals (4.6 ± 0.5 %ID/cm(3)). Conclusion Our protocol permits the detection of Th1 cells in single LNs and enables temporal in vivo monitoring of T-cell homing over 48 h. This work enables future applications for (64)Cu-PTSM-labeled T cells in clinical trials and novel therapy concepts focusing on T-cell-based immunotherapies of autoimmune diseases or cancer.

Purpose Tumor hypoxia is a major cause of radiation resistance, often present in various solid tumors. Dynamic [18 F]-fluoromisonidazole (FMISO) PET imaging is able to reliably assess tumor hypoxia. Comprehensive characterization of tumor microenvironment through FMISO-PET and dynamic contrast enhanced (DCE) MR multimodality imaging might be a valuable alternative to the dynamic FMISO-PET acquisition. The aim of this work was to explore the correlation between the FMISO-PET and DCE-MRI kinetic parameters. Methods This study was done on head and neck cancer patients (N = 6), who were imaged dynamically with FMISO-PET and DCE-MRI on the same day. Images were registered and analyzed for kinetics on a voxel basis. FMISO-PET images were analyzed with the two-tissue compartment three rate-constant model. Additionally, tumor-to-muscle ratio (TMR) maps were evaluated. DCE-MRI was analyzed with the extended Tofts model. Voxel-wise Pearson's coefficients were calculated for each patient to assess pairwise parameter correlations. Results Median correlations between FMISO uptake parameters and DCE-MRI kinetic parameters varied across the parameter pairs in the range from -0.05 to 0.71. The highest median correlation of r = 0.71 was observed for the pair Vb -vp , while the K1 -Ktrans median correlation was r = 0.45. Median correlation coefficients for the K1 -vp and the Ki -Ktrans pairs were r = 0.42 and r = 0.32, respectively. Correlations between FMISO uptake rate parameter Ki and DCE-MRI kinetic parameters varied substantially across the patients, whereas correlations between the FMISO and DCE-MRI vascular parameters were consistently high. Median TMR-K1 and TMR-Ktrans correlations were r = 0.52 and r = 0.46, respectively, but varied substantially across the patients. Conclusions Based on this clinical evidence, we can conclude that the vascular fraction parameters obtained through DCE-MRI kinetic analysis or FMISO kinetic analysis measure the same biological property, while other kinetic parameters are unrelated. These results might be useful in the design of future clinical trials involving FMISO-PET/DCE-MR multimodality imaging for the assessment of tumor microenvironment.

Most frequently, gram-negative bacterial infections in humans are caused by Enterobacteriaceae and remain a major challenge in medical diagnostics. We non-invasively imaged moderate and severe systemic Yersinia enterocolitica infections in mice using the positron emission tomography (PET) tracer 3’-deoxy-3’-[18F]fluorothymidine ([18F]FLT), which is a marker of proliferation, and compared the in vivo results to the ex vivo biodistributions, bacterial loads, and histologies of the corresponding organs. Y. enterocolitica infection is detectable with histology using H&E staining and immunohistochemistry for Ki 67. [18F]FLT revealed only background uptake in the spleen, which is the main manifestation site of systemic Y. enterocolitica-infected mice. The uptake was independent of the infection dose. Antibody-based thymidine kinase 1 (Tk-1) staining confirmed the negative [18F]FLT-PET data. Histological alterations of spleen tissue, observed via Ki 67-antibody-based staining, can not be detected by [18F]FLT-PET in this model. Thus, the proliferation marker [18F]FLT is not a suitable tracer for the diagnosis of systemic Y. enterocolitica infection in the C57BL/6 animal model of yersiniosis.

Here, we describe the efficacy of the novel small molecule c-Met inhibitor BAY 853474 in reducing tumor growth in the Hs746T gastric cancer xenograft model and tested the suitability of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) versus 3'-deoxy-3'-18F-fluorothymidine ([(18)F]FLT) for response monitoring in a gastric cancer xenograft mouse model using small animal PET.The c-Met inhibitor or vehicle control was administered orally at various doses in tumor-bearing mice. Glucose uptake and proliferation was measured using PET before, 48 and 96 h after the first treatment. The PET data were compared to data from tumor growth curves, autoradiography, Glut-1 and Ki-67 staining of tumor sections, and biochemical analysis of tissue probes, i.e., c-Met and ERK phosphorylation and cyclin D1 levels.BAY 853474 significantly reduces tumor growth. [(18)F]FDG uptake in Hs746T tumors was significantly reduced in the groups receiving the drug, compared with the control group. The [(18)F]FLT uptake in the tumor tissue was completely absent 96 h after treatment. Autoradiographic, immunohistochemical, and biochemical analyses confirmed the PET findings. Treatment with the c-Met inhibitor did not affect body weight or glucose levels, and no adverse effects were observed in the animals.These preclinical findings suggest that clinical PET imaging is a useful tool for early response monitoring in clinical studies.

Autoradiolysis describes the degradation of radioactively labeled compounds due to the activity of the labeled compounds themselves. It scales with activity concentration and is of importance for high activity and microfluidic PET tracer synthesis. This study shows that microfluidic devices can be shaped to reduce autoradiolysis by geometric exclusion of positron interaction. A model is developed and confirmed by demonstrating in-capillary storage of non-stabilized [18F]FDG (2-[18F]Fluoro-2-deoxy- d -glucose) at max. 23 GBq/ml while maintaining > 90 % radiochemical purity over 14 h.

Noninvasive in vivo imaging of biologic processes using PET is an important tool in preclinical studies. We observed significant differences in 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) uptake in arthritic ankles and carcinomas between dynamic and static PET measurements when mice breathed oxygen. Thus, we suspected that air or oxygen breathing and the anesthesia protocol might influence 18F-FLT tracer uptake. Methods: We injected arthritic, healthy, and CT26 colon carcinoma–bearing mice with 18F-FLT before static or dynamic small-animal PET measurements. The spontaneously oxygen- or air-breathing mice were kept conscious or anesthetized with ketamine and xylazine during 18F-FLT uptake before the 10-min static PET measurements. For dynamic PET scans, mice were anesthetized during the entire measurement. 18F-FLT uptake was reported in percentage injected dose per cubed centimeter by drawing regions of interest around ankles, carcinomas, and muscle tissue. Additionally, venous blood samples were collected before 18F-FLT injection and after PET measurement to analyze pH, carbon dioxide partial pressure (pCO2), and lactate values. Results: A significantly reduced 18F-FLT uptake was measured in arthritic ankles and in CT26 colon carcinomas when the mice breathed oxygen and were conscious during tracer uptake, compared with mice that were anesthetized during 18F-FLT uptake. Breathing air completely abolished this phenomenon. Analysis of blood samples that were obtained from the mice before 18F-FLT injection and after the PET scan implicated respiratory acidosis that was induced by oxygen breathing and consciousness during tracer uptake. Acidosis was found to be the primary factor responsible for the reduced 18F-FLT uptake, as reflected by increased pCO2 and reduced pH and lactate values. Conclusion: Oxygen-breathing conscious mice sustained respiratory acidosis and, consequently, reduced cell proliferation and 18F-FLT uptake in arthritic ankles and CT26 colon carcinomas. Thus, we suggest the use of air instead of oxygen breathing for 18F-FLT PET measurements.

For detection of hypoxic tumor tissue, all radiotracers synthesized until now, are based on the concept that cellular uptake is being controlled by diffusion. As a new approach, we chose the concept to have the tracer hypothetically transported into the cells by well known carrier systems like the amino acid transporters. For this purpose, radiosynthesis of O-[2-[18F]fluoro-3-(2-nitro-1H-imidazole-1yl)propyl]tyrosine ([18F]FNT]) was carried out from methyl 2-(benzyloxycarbonyl)-3-(4-3-(2-nitro-1H-imidazol-1-yl)-2-(tosyloxy)propoxy) phenyl)propanoate via no-carrier-added nucleophilic aliphatic substitution. After labelling, 81 ± 0.9% of labelled intermediate i.e. methyl 2-(benzyloxycarbonyl)-3-(4-(2-[18F]fluoro-3-(2-nitro-1H-imidazole-1-yl)propoxy) phenyl)propanoate was obtained at 140 °C. At the end of radiosynthesis, [18F]FNT was obtained in an overall radiochemical yield of 40 ± 0.9% (not decay corrected) within 90 min in a radiochemical purity of >98% in a formulation ready for application in the clinical studies for PET imaging of hypoxia.

// Valerie S. Honndorf 1 , Stefan Wiehr 1 , Anna-Maria Rolle 1 , Julia Schmitt 1 , Luisa Kreft 1 , Letitia Quintanilla-Martinez 2 , Ursula Kohlhofer 2 , Gerald Reischl 1 , Andreas Maurer 1 , Karsten Boldt 3 , Michael Schwarz 4 , Holger Schmidt 5 , Bernd J. Pichler 1 1 Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University, Tuebingen, Germany 2 Institute of Pathology, University Hospital, Eberhard Karls University, Tuebingen, Germany 3 Medical Proteome Center, Institute for Ophthalmic Research, Eberhard Karls University, Tuebingen, Germany 4 Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Toxicology, Eberhard Karls University, Tuebingen, Germany 5 Department of Radiology, Diagnostic and Interventional Radiology, Eberhard Karls University, Tuebingen, Germany Correspondence to: Valerie S. Honndorf, email: Valerie.Schmidt-Honndorf@med.uni-tuebingen.de Keywords: PET, genistein and cetuximab, A431 and Colo205 xenografts, molecular tumor imaging Received: December 21, 2015 Accepted: February 28, 2016 Published: April 07, 2016 ABSTRACT The natural phytoestrogen genistein is known as protein kinase inhibitor and tumor suppressor in various types of cancers. We studied its antitumor effect in two different xenograft models using positron emission tomography (PET) in vivo combined with ex vivo histology and nuclear magnetic resonance (NMR) metabolic fingerprinting. Procedures: A431 and Colo205 tumor-bearing mice were treated with vehicle or genistein (500 mg/kg/d) over a period of 12 days. Imaging was performed with 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG) and 3′-deoxy-3′-[ 18 F]fluorothymidine ([ 18 F] FLT). In a second study A431 tumor-bearing mice were treated with vehicle, genistein (500 mg/kg/d), cetuximab (1 mg/3d) or a combination of the compounds and imaged using [ 18 F]FDG, [ 18 F]FLT and [ 64 Cu]NODAGA-cetuximab. Data were compared to histology and principal components analysis (PCA) of NMR fingerprinting data. Results: Genistein reduced tumor growth significantly in both xenografts. [ 18 F] FLT uptake was consistent in both models and corresponded to histological findings and also PCA whereas [ 18 F]FDG and [ 64 Cu]NODAGA-cetuximab were not suitable for therapy monitoring. Conclusions: As mono-therapy the natural isoflavone genistein has a powerful therapeutic effect in vivo on A431 and Colo205 tumors. [ 18 F]FLT has superior consistency compared to the other tested tracers in therapy monitoring, while the treatment effect could be shown on the molecular level by histology and metabolic fingerprinting.

UNLABELLED The pharmacokinetics of (18)F-fluorodeoxythymidine (FLT), (18)F-FDG, (11)C-choline, and (18)F-fluoroethylcholine (FEC) in 2 hormone-independent (PC-3, DU145) and 2 hormone-dependent (CWR22, PAC120) prostate cancer xenograft mouse models were evaluated by PET and compared by immunohistochemistry. Further investigation was performed to determine whether PET can detect early changes in tumor metabolism after androgen ablation therapy through surgical castration. METHODS PET was performed on 4 consecutive days. In addition, the CWR22 and PAC120 tumor models were surgically castrated after the baseline measurement and imaged again after castration. The tracer uptake was analyzed using time-activity curves, percentage injected dose per volume (%ID/cm(3)), and tumor-to-muscle ratio (T/M). RESULTS Regarding the hormone-independent prostate tumor models, (18)F-FLT showed the best T/M and highest %ID/cm(3) in PC-3 (2.97 ± 0.63 %ID/cm(3)) and DU145 (2.06 ± 0.75 %ID/cm(3)) tumors. (18)F-FDG seemed to be the tracer of choice for delineation of the PC-3 tumors but not for the DU145 tumors. Using (11)C-choline (PC-3: 1.33 ± 0.29 %ID/cm(3), DU145: 1.60 ± 0.27 %ID/cm(3)) and (18)F-FEC, we did not find any significant uptake in the tumors, compared with muscle tissue. Regarding the hormone-dependent prostate tumor models, the CWR22 model showed a highly significant (P < 0.01) decrease in tumor (18)F-FDG uptake from 4.11 ± 1.29 %ID/cm(3) to 2.19 ± 1.45 %ID/cm(3) after androgen ablation therapy. However, the (18)F-FLT, (11)C-choline, or (18)F-FEC tracers did not provide sufficient uptake or reliable information about therapy response in CWR22 tumors. The PAC120 model showed a significant increase in (18)F-FLT tumor uptake (P = 0.015) after androgen ablation therapy. The accumulation of (18)F-FEC (before: 2.32 ± 1.01 %ID/cm(3), after: 1.36 ± 0.39 %ID/cm(3)) was found to be the next highest after (18)F-FDG (before: 2.45 ± 0.93 %ID/cm(3), after: 2.18 ± 0.65 %ID/cm(3)) in PAC120 tumors before castration and is better suited for monitoring therapy response. CONCLUSION This comprehensive study in 2 hormone-dependent and 2 hormone-independent prostate tumor mouse models shows that (18)F-FLT and (18)F-FDG are the most appropriate tracers for delineation of PC-3, DU145 (except (18)F-FDG), and CWR22 tumors, but not for PAC120 tumors. (18)F-FEC and (11)C-choline, in particular, revealed insufficient T/M ratio in the prostate tumor models. The results may indicate that radiolabeled choline and choline derivatives compete with a high concentration of the precursor dimethylaminoethanol, resulting in reduced uptake in small-rodent tumor models, a hypothesis that is currently under investigation in our laboratory.

As model reactions for the introduction of 18F into protected aromatic amino acids, the replacement of NO2, Cl, Br and F by [18F]fluoride in 2-isophthalaldehyde and 2-terephthalaldehyde derivatives which model 18F-DOPA and 18F-tyrosine was investigated by comparing labelling yields and reaction rates with those of corresponding mono-aldehyde compounds. All isophthalaldehydes showed maximum radiochemical yields (79 to 86%) at 140°C and in comparison with the corresponding mono-aldehydes the reaction proceeded faster. At lower temperature the reaction already resulted in high yields, e.g. 2-nitroisophthalaldehyde was labelled with a yield of 78% at 25°C after 7 min, whereas 2-nitrobenzaldehyde only reached a yield of 1.7% under the same reaction conditions. The 18F/NO2 exchange in nitroterephthalaldehydes proceeded more slowly and with lower radiochemical yields when compared with corresponding isophthalaldehydes and monoaldehydes. The decarbonylation of 18F-labelled aromatic dialdehydic compounds with 4 eq. of Wilkinson's catalyst at 150°C in benzonitrile resulted in high yields, e.g. 2-[18F]fluoro-5-methoxyisophthalaldehyde and 4-[18F]fluoro-2-methoxy-5-methylisophthalaldehyde were decarbonylated efficiently with yields of 67±3% and 72±2%, respectively. Copyright © 2010 John Wiley & Sons, Ltd.

An improved, automated synthesis of [(18)F]FDOPA including four synthetic steps (fluorination, reductive iodination, alkylation and hydrolysis) is reported with each step optimized individually. In a home-made automatic synthesizer, 9064+/-3076 MBq of [(18)F]FDOPA were produced within 120 min from EOB (n=5). Radiochemical purity and enantiomeric excess were bothor= 95%. Specific activity was ca. 50 GBq/micromol at EOS. This automatically operable synthesis is well suited for the multi-patient-dose routine production of n.c.a. [(18)F]FDOPA.

As model reactions for the introduction of [ 18 F]fluorine into aromatic amino acids, the replacement of NO 2 by [ 18 F]fluoride ion in mono- to tetra-methoxy-substituted ortho -nitrobenzaldehydes was systematically investigated. Unexpectedly, the highly methoxylated precursors 2,3,4-trimethoxy-6-nitrobenzaldehyde and 2,3,4,5-tetramethoxy-6-nitrobenzaldehyde showed high maximum radiochemical yields (82% and 48% respectively). When the electrophilicity of the leaving group substituted carbon atom is expressed by its 13 C NMR chemical shift a good correlation with the reaction rate at the beginning of the reaction (first min) was found ( R 2 = 0.89), whereas the maximum radiochemical yields correlated much poorer with this electrophilicity parameter. This may be caused by side reactions becoming influencial in the further reaction course. As possible side reactions the demethylation of methoxy groups and intramolecular redox reactions could be detected by HPLC/MS.

Radiolabeled arginine-glycine-aspartate (RGD) peptides are increasingly used in preclinical and clinical studies to assess the expression and function of the αvβ3 integrin, a cellular adhesion molecule involved in angiogenesis and tumor metastasis formation. To better understand the PET signal obtained with radiolabeled RGD peptides, we have constructed a compartmental model that can describe the time–activity curves in tumors after an intravenous injection. Methods: We analyzed 60-min dynamic PET scans obtained with 64Cu-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA)-RGD in 20 tumor-bearing severe combined immunodeficient (SCID) mice after a bolus dose (18,500 kBq [500 μCi]), using variations of the standard 2-compartment (4k) tissue model augmented with a compartment for irreversible tracer internalization. αvβ3 binding sites were blocked in 5 studies with a coinjection of cold peptide. In addition, 20 h after injection, static PET was performed on 9 of 20 mice. We fitted 2k (k3 = k4 = 0), 3k (k4 = 0), 4k, and 4kc (k4 = constant) models to the PET data and used several criteria to determine the best model structure for describing 64Cu-DOTA-RGD kinetics in mice. Akaike information criteria (AIC), calculated from model fits and the ability of each model to predict tumor concentration 20 h after tracer injection, were considered. Results: The 4kc model has the best profile in terms of AIC values and predictive ability, and a constant k4 is further supported by Logan–Patlak analysis and results from iterative Bayesian parameter estimation. The internalization compartment allows quantification of the putative tracer internalization rate for each study, which is estimated here to be approximately an order of magnitude less than k3 and thus does not confound the apparent specific binding of the tracer to the tumor integrin during the first 60 min of the scan. Analysis of specific (S) and nonspecific or nondisplaceable (ND) binding using fitted parameter values showed that the 4kc model provided expected results when comparing αvβ3 blocked and nonblocked studies. That is, specific volume of distribution, [VS = (K1k3)/(k2k4)], is much higher than is nondisplaceable volume of distribution, [VND = (K1/k2)], in nonblocking studies (2.2 ± 0.6 vs. 0.85 ± 0.14); VS and VND are about the same in the blocking studies (0.46 ± 1.6 vs. 0.56 ± 0.09). Also, the ratio of static tumor and plasma measurements at 60 and 10 min [CT(60)/CP(10)] is highly correlated (RS = 0.92) to tumor VS. Conclusion: We have developed and tested a compartmental model for use with the 64Cu-DOTA-RGD PET tracer and demonstrated its potential as a tool for analysis and design of preclinical and clinical imaging studies.

Iodine-124 was produced via the (124)Te(p,n)(124)I reaction by 15 MeV proton irradiation of an in-house solid mass tellurium dioxide target, using the Tübingen PETtrace (General Electric Medical Systems) cyclotron. 1-(2-Deoxy-beta-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene (dRFIB), a stable, non-polar thymidine mimic nucleoside, was synthesized in 5 steps following a literature method, for radioiodination with [(124)I] iodide via isotope exchange in the presence of copper sulphate and ammonium sulphate in methanol-water. The radiolabelling procedure was optimized with respect to temperature, amount of dRFIB, amount of sodium hydroxide and reaction time, to produce radiochemical yields of up to 85% with a 1-h reaction at 140 degrees C. With routine I-124 production of 30 MBq/run, relatively high specific activities, approaching 100 MBq/mmol, can be expected. The activation energy for dRFIB radioiodination was calculated from temperature-time RCY data to be approximately 100 kJ/mol using no-carrier-added [(124)I]iodide.

The hypoxia-inducible factor-1 alpha (HIF-1 alpha) activates angiogenesis in response to cellular hypoxia, suggesting a spatial correlation between angiogenesis and tissue hypoxia. Methods: Using digital autoradiography of coinjected F-18-labeled azomycin arabinoside (F-8-FAZA) (assessing regional hypoxia) and a glycosylated RGD-containing peptide (I-125-3-iodo-DTyr(4)-cyclo(-Arg-Gly-Asp-DTyr-Lys(SAA)-), or I-125-Gluco-RGD) (assessing angiogenesis via binding to alpha v beta 3 integrin receptors on endothelial cells) performed on 22 EMT6 tumor xenografts, we investigated the intratumoral spatial distribution of these tracers. We applied a Bayesian bivariate image analysis using the mean tumor-to-muscle ratio as a discriminator, resulting in 4 groups: FAZA high/RGD high (Q1), FAZA low/RGD high (Q2), FAZA low/RGD low (Q3), and FAZA high/RGD low (Q4). In an additional 18 xenografts, the immunohistochemically derived HIF-1 alpha protein distribution was compared with F-18-FAZA autoradiography. Animals were divided into groups breathing either room air or carbogen (95% oxygen, 5% CO2) for 4 In until sacrifice. Results: Under room air conditions, roughly 60% of the tumor surface displayed a spatial coupling of F-18-FAZA and I-125-Gluco-RGD uptake: either high (Q1) or low (Q3) uptake for both tracers, with Q1 indicating spatial association of hypoxia and angiogenesis and Q3 indicating adequate oxygenation without active angiogenesis. However, the remaining approximately 40% of the tumor surface showed discordant F-18-FAZA and I-125-Gluco-RGD uptake, indicating that hypoxia and angiogenesis are not necessarily spatially linked to each other and highlighting substantial intratumoral heterogeneity of the F-18-FAZA and I-125-Gluco-RGD uptake. Although carbogen breathing conditions significantly decreased the mean F-18-FAZA tumor-to-muscle ratio, no significant changes were observed for I-125 -Gluco-RGD, indicating that an acute increase in tumor oxygenation did not influence alpha v beta 3 integrin receptor expression. The HIF-1 alpha-positive (HIFpos tumor cell fraction was not significantly influenced by breathing conditions and covered between 0% and 35% of the total tumor section surface. However, the HIFpos tumor section surface was much smaller than the tumor section surface of increased F-18-FAZA uptake, suggesting that both markers are identifying distinctly different biologic processes associated with hypoxia. Conclusion: The study revealed a substantial spatial discordance of the F-18-FAZA and I-125-Gluco-RGD tumor distribution suggesting that hypoxia and angiogenesis are not necessarily spatially linked in malignancies. These results may prove essential in developing advanced targeted systemic chemotherapeutic approaches (such as combinations of hypoxia-activated cytotoxins and antiangiogenic drugs) for hypoxic tumors.

Serotonergic dysfunction may contribute to negative mood states in affective disorders. Some in vivo imaging studies showed reduced availability of serotonin transporters (5-HTT) in the brainstem and thalamus of patients with major depression. We tested the hypothesis that 5-HTT availability is reduced in unmedicated unipolar patients with major depression compared to healthy control subjects matched for gender, age, genotype and smoking status. Availability of 5-HTT was measured in vivo with positron emission tomography and [(11)C]-3-amino-4-(2-dimethylaminomethyl-phenylsulfanyl)-benzonitrile (DASB) in the midbrain, thalamus and amygdala. DASB binding was correlated with the severity of depression (Beck's Depression Inventory), anxiety (Spielberger's State-Trait Anxiety Inventory) and personality traits (Temperament and Character Inventory). Patients with major depression displayed reduced 5-HTT availability in the thalamus (P=0.005). In patients, low serotonin transporter availability correlated with high anxiety (thalamus: r=-0.78, P=0.004; midbrain: r=-0.78, P=0.004; amygdala: r=-0.80, P=0.003). Correlations with severity of depression were weaker and did not survive correction for multiple testing. These results support the hypothesis that central serotonergic dysfunction is associated with negative mood states in affective disorders. In the thalamus, a low serotonin reuptake capacity may interfere with thalamic control of cortical excitability and contribute to anxiety rather than depression per se in major depression.

For a series of benzaldehydes only with a leaving group or with both a leaving group and a single methoxy substituent 18F-fluorination via nucleophilic aromatic substitution (SNAr) was studied in DMF and Me2SO. In general, the radiochemical yields were clearly higher in DMF than in Me2SO. In the fluorodehalogenation reaction (leaving group: halogen = Br, Cl), extremely low radiochemical yields were observed in Me2SO (

For 18F-labelling of aromatic amino acids such as tyrosine and DOPA simple and efficient procedures have been under development for quite a while. The direct introduction of 18F using [18F]fluoride can principally be realized by nucleophilic aromatic substitution (SNAr). However, this requires the presence of an appropriate leaving group and an auxiliary substituent, which is able to reduce the electron density of the benzene ring. Furthermore, this auxiliary substituent should be removable easily after the introduction of 18F. The electron-withdrawing formyl substituent meets both requirements. It facilitates nucleophilic attack in the ortho and/or para position and is easily removed in a decarbonylation reaction mediated by Wilkinson's catalyst (RhCl(PPh3)3). In order to evaluate the reaction conditions for a possible synthesis of 2-[18F]fluoro-5-hydroxyphenylalanine ([18F]-m-tyrosine), 2-[18F]fluoro-4-hydroxyphenylalanine ([18F]-p-tyrosine) or 2-[18F]fluoro-4,5-hydroxyphenylalanine ([18F]FDOPA), the dependence of the decarbonylation reaction on solvent, temperature, reaction time and catalyst concentration was studied using appropriate model compounds. Optimum yields of 81%, 89% and 88% could be achieved using benzonitrile as solvent and 2M equivalents of RhCl(PPh3)3 (based on labelling precursor) at 150 degrees C reaction temperature within 20 min reaction time for compounds modelling [18F]-m-tyrosine, [18F]-p-tyrosine and [18F]FDOPA, respectively.

Reduced availability of brainstem serotonin transporters (5-HTT) has been observed in vivo in obsessive-compulsive disorder (OCD). However, results vary and may be influenced by competition with endogenous serotonin. Using positron emission tomography (PET) and [11C]DASB, a specific 5-HTT ligand that showed no competition with serotonin for 5-HTT binding in vitro, we tested the hypothesis that 5-HTT availability is reduced in OCD patients and correlated with OCD severity. METHODS. 5-HTT availability in the thalamus and the midbrain was measured in nine drug-free OCD patients and compared with 19 healthy controls, matched for the individual combination of 5-HTT genotype, gender and smoking status. OCD severity was assessed with the Yale-Brown obsessive compulsive scale (Y-BOCS). RESULTS. 5-HTT availability was significantly reduced in the thalamus and midbrain of OCD patients. Age and 5-HTT in the thalamus explained 83% of OCD severity in patients that were drug-free for at least 1 year. CONCLUSION. This PET study confirms a central role of the serotonergic system, particularly the thalamus in the pathogenesis of obsessive compulsive disorder.

Homozygote carriers of two long (L) alleles of the serotonin transporter (5-HTT) regulatory region displayed in vitro a twofold increase in 5-HTT expression compared with carriers of one or two short (S) alleles. However, in vivo imaging studies yielded contradictory results. Recently, an AG exchange leading to differential transcriptional activation of 5-HTT mRNA in lymphobalstoid cell lines was discovered in the 5-HTT regulatory region. In vitro and in vivo evidence suggests that [(11)C]DASB, a new 5-HTT ligand offers some advantages over the ligands used in previous studies in measuring 5-HTT density independent of synaptic levels of serotonin.We assessed 5-HTT binding potential (BP (2)) in the midbrain of 19 healthy subjects with positron emission tomography and [(11)C]DASB. Accounting for the hypothesized functional similarity of L (G) and S in driving 5-HTT transcription, we assessed whether L (A) L (A) homozygotes display increased midbrain BP (2) compared with carriers of at least one S allele.BP (2) in the midbrain was significantly increased in L (A) L (A) homozygotes compared with carriers of at least one S allele. Interestingly, the genotype effect on the midbrain was significantly different from that on the thalamus and the amygdala where no group differences were detected.This in vivo study provides further evidence that subjects homozygous for the L (A) allele display increased expression of 5-HTT in the midbrain, the origin of central serotonergic projections.

Summary3′[18F]Fluoro-3′-deoxy-thymidine ([18F]FLT) (III) has been discussed to be a promising tracer for assessing tumor proliferation. In order to perform clinical studies for evaluating [18F]FLT a simplified labeling procedure was developed using 2,3′-anhydrothymidine with benzoyl as a protecting group in the 5′-position (I). In DMSO the labeling yield was 46% at 160 °C in 10 min. Hydrolysis was efficiently performed with 0.25% NaOH at room temperature within 10 min. The labeling procedure was transferred to a remote controlled synthesis module allowing the production of [18F]FLT in high activities. The overall radiochemical yield was 18.1 ± 5.4% (n= 55) with absolute yields of 9.2 ± 2.6 GBq of [18F]FLT at EOS ready for injection (60 min after EOB; irradiation parameters: 35 μA, 60 min) and specific activities of 100–220 GBq/μmol. A convenient cartridge method for metabolite analysis was developed and validatedversusHPLC showing that after 90 min 69.0 ± 7.0% of the radioactivity in plasma (less than 20% of initial radioactivity) was unchanged [18F]FLT (26 patients with various tumors).

2,6-Di-tert-butyl-4-iodophenol (1) does not react to the keto carbene 4 under strongly basic conditions and exclusion of oxygen, in contrast to a report in the literature. In the presence of oxygen, however, oxidation takes place. Reaction times and product pattern strongly depend on the concentration of O2. Besides biphenyl 2, diphenoquinone 3, and quinone 5, a new (oxocyclohexadienylidene)bisphenol 6 was isolated, and its molecular structure was determined by X-ray crystallography. A radical mechanism is suggested for the formation of the above products from 1. The bisphenol 6 was also synthesized by thermolysis of the quinone diazide 11 in the presence of 3.

As an example of applying the electrochemical method in radiofluorination a new procedure was developed for preparing [18F]fluorophenylalanine by direct use of [18F]fluoride. Several protecting groups for amino and carboxylic function of phenylalanine were investigated. Best results were obtained for N-trifluoroacetylphenylalanine methyl ester (1) (10.5±2.5%) with a relative isomeric distribution of 50.5±1.5% ortho isomer, 11.5±1.5% meta isomer and 38.8±2.1% para isomer. Optimized reaction conditions were with Et3N·3HF as supporting electrolyte and temperatures between 0 and –10°C. By variation of working potential (1.5–2.0 V) and chloride concentration (added as supporting electrolyte, 0.0–0.36 M) it was demonstrated that an indirect electrolysis as reaction mechanism or a halogen exchange (halex) reaction with primary formation of a chlorinated product followed by halogen exchange are highly improbable. Copyright © 2005 John Wiley & Sons, Ltd.

The synthesis of [11C]DASB is known to be performed by reacting the demethyl-DASB precursor with [11C]methyl iodide but the necessary reaction parameters had hitherto not been determined. In this work the dependence of the labelling yield on reaction temperature, reaction time, precursor concentration and type of solvent was investigated. Best results were found when methylation was carried out at 100 °C within 2 min using 4 mg/ml demethyl precursor in DMSO. With respect to [11C]methyl iodide a radiochemical yield of 74%±3% [11C]DASB was achieved. In addition, the synthesis was transferred to a remotely controlled module. A carbon-11 production for 1 h at 50 μA resulted in 3800-4300 MBq [11C]DASB with a specific activity of 152-211 GBq/μmol (4.1-5.7 Ci/μmol) (EOS) and a radiochemical purity of >95% in a sterile solution ready for injection. The total synthesis time was 45 min after EOB.

Electrochemical fluorination of various monosubstituted aromatic compounds was performed with [18F]fluoride using potentiostatic anodic oxidation on platinum electrodes in an undivided cell in acetonitrile with a mixture of Et3N·3HF/Et3N·HCl as electrolyte. Maximum radiochemical yields were obtained after a charge of 50 C passed the solution. The results showed a clear dependence of the radiochemical yields on the oxidation potentials E1/2 of the substrates as a consequence of different substituents namely CH3CO-, F-, Cl-, Br- and tert.-Butyl. With increasing E1/2, the fluorination yields decreased from 7.9% (tert.-butylbenzene) to 1.5% (acetophenone). The ratio between F-for-X substitution and F-for-H substitution correlated with the bond energies of the C–X bond. With higher bond energies, less X-substitution was observed.

The aim of this study was to evaluate the use of 124I positron emission tomography (PET) to determine the dosimetry of radioiodine therapy in hyperthyroidism and thyroid cancer. Phantom studies to assess the accuracy of PET were performed using an EEC phantom with spheres of different diameters filled with 3-30 MBq of 124I. Patient dosimetry was derived from PET data obtained 1-13 days after simultaneous oral administration of a therapeutic dose of 131I and a diagnostic dose of 124I. The obtained data were compared with findings from intratherapeutic probe measurements and clinical outcome. The phantom studies confirmed that 124I can be quantitated by PET (imprecisionor =10%), and volumetry is feasible for nodules13 mm (imprecisionor =20%). Any influence of contamination with 123I or the simultaneous administration of 131I on the accuracy of the PET quantification and the probe measurements was ruled out by phantom measurements with solutions of 131I, 124I and 123I in various ratios. In autonomous nodular goitres, radioiodine uptake measured by PET varied from 25.4% to 64.3% and was not significantly different from that obtained by a scintillation probe (24.1%-73.1%, correlation coefficient r=0.91). Comparison of uptake and effective half-life in normal tissue versus autonomous nodules revealed significant differences in uptake but not in effective half-life [uptake 2.0-8.3 kBq/(ml x MBq) in normal tissue vs 12.6-29.3 kBq/(ml x MBq) in nodules; half-life 97.8-156.7 h in normal tissue vs 73.3-192.3 h in nodules]. Calculated radiation doses ranged between 177 and 633 Gy for autonomous nodules and between 47 and 126 Gy for normal tissue. In thyroid cancer patients, doses between 350 and 1,420 Gy were achieved in thyroid remnants and between 70 and 170 Gy in tumour metastases. It is concluded that 124I and PET are suitable for evaluation of the dosimetry of radioiodine therapy in benign and malignant thyroid diseases. The applied technique might be particularly useful for quantitative dose-response studies in radioiodine treatment and further investigations of stunning phenomena.

Tumor hypoxia can be identified by [18F]FAZA positron emission tomography, or invasively using oxygen probes. The impact of anesthetics on tumor hypoxia remains controversial. The aim of this comprehensive study was to investigate the impact of isoflurane and ketamine/xylazine anesthesia on [18F]FAZA uptake and partial oxygen pressure (pO2) in carcinoma and muscle tissue of air- and oxygen-breathing mice. Methods CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O2). We performed 10 min static PET scans 1 h, 2 h and 3 h after [18F]FAZA injection and calculated the [18F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO2 probe in the tumor as well as in the gastrocnemius muscle to measure the pO2 and perfusion. Results Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [18F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O2: 4.4 vs. IF-O2: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [18F]FAZA uptake in the carcinomas. Invasive pO2 probe measurements yielded enhanced intra-tumoral pO2 values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O2 9.73 mmHg, IF-O2: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003). Conclusion [18F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [18F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO2-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.

Bivalent oxidation of 3,5-di-tert-butyl-hydroquinone monoesters leads to phenoxenium ions, which can transfer an acyl group to nucleophiles. Based on this principle, dipeptides, glyco-amino acids and N-sulfonyl-amino acids were synthesized from hydroquinone esters of amino acids and p-toluenesulfonic acid. For this reaction, direct anodic and indirect mediated oxidation, as well as chemical oxidation with NBS or trisarylammoniumyl salts, was used. The mechanism of the acyl transfer is discussed in terms of a direct and/or a mediated process. A spirocyclic key intermediate was isolated and its molecular structure determined by X-ray crystallography.

18F-labeling of aromatic compounds normally is achieved by electrophilic substitution. In that case [18F]fluoride cannot be applied although it is produced very efficiently at medical cyclotrons. By the use of electrochemical methods, however, benzene can be oxidized and thus, the electron density is reduced in a way that nucleophilic attack of [18F]fluoride occurs. For the first time benzene was shown to be labeled with [18F]fluoride after being electrochemically oxidized in a 2 ml electrolysis cell with 0.033M Et3N.3HF and 0.066M Et3N.HCl in CH3CN and benzene in various concentrations. After 50 Coulombs (60-90 min) maximum of labeling was reached. With the highest concentration of aromatic compound (1.0M) the radiochemical yields were 16±9% with specific activities up to 27 GBq/mmol.

For labeling reactions [18F]fluoride has to be separated from [18O]water and transferred into an organic solvent suitable for nucleophilic substitutions. An electrolytical method is described for depositing [18F]fluoride on a vitreous carbon electrode and releasing it directly into CH3CN or DMSO. In the presence of Et3N×3HF, [18F]fluoride is almost quantitatively released into acetonitrile. When using n.c.a conditions, i.e., Et3N.HCl, desorption of the 18F activity is almost 70% and 60% in acetonitrile and DMSO, respectively, already within 5 minutes.

Quantification of the binding of [ 11 C]methylphenidate to the dopamine transporter (DAT) using positron emission tomography (PET) is often used to evaluate the integrity of dopaminergic neurons in the striatal regions of the brain. Over the past decade, many genetically engineered mouse models of human disease have been developed and have become particularly useful for the study of disease onset and progression over time. Quantitative imaging of small structures such as the mouse brain is especially challenging. Thus, the aims of this study were (1) to evaluate the accuracy of quantifying DAT binding using in vivo PET and (2) to examine the impact of different methodologies. Methods Eight mice were scanned with [ 11 C]methylphenidate under true or transient equilibrium conditions using a bolus and constant infusion protocol or a bolus injection protocol to evaluate the accuracy of the Logan graphical approach for [ 11 C]methylphenidate imaging in mice. Displacement with unlabeled methylphenidate (0.1, 3 and 10 mg/kg) was used to verify specific binding. In a second experiment, 30 mice were lesioned by injection of 6-hydroxydopamine (6-OHDA) at doses of 0, 2 or 4 μg (n = 10) into the right striatum to assess the dose-dependent correlation between the PET signal and dopaminergic degeneration. In addition, we performed test–retest experiments and used ex vivo autoradiography (AR) to validate the effect of partial volume on the accuracy of the [ 11 C]methylphenidate PET quantification in the mouse striatum. Results The binding potentials ( BP ND ) calculated from the Logan graphical analysis under transient equilibrium conditions (1.03 ± 0.1) were in excellent agreement with those calculated at true equilibrium (1.07 ± 0.1). Displacement of specific binding with 0.1, 3 and 10 mg/kg methylphenidate resulted in 38%, 77% and 81% transporter occupancy in the striatum. Intra-striatal injections of 6-OHDA caused a dose-dependent decrease in the specific binding of [ 11 C]methylphenidate to the DAT in the striatum. The BP ND was reduced by 49% and 61% after injection with 2 and 4 μg of 6-OHDA, respectively. The test–retest reproducibility was 6% in the healthy striatum and 27% in the lesioned striatum. In addition, only a small (15%) difference was found between the [ 11 C]methylphenidate DVR-1 values determined by PET and AR on the healthy side, and no differences were observed on the lesioned side. Conclusion The present work demonstrates for the first time that [ 11 C]methylphenidate PET is useful for the quantification of striatal dopamine transporters at the dopaminergic nerve terminals in the mouse striatum; therefore, this marker may be used as a biomarker in genetically engineered mouse models of neurodegenerative disorders. However, only changes resulting in greater than 10% differences in BP ND values can reliably be detected in vivo .

BACKGROUND The prostate specific membrane antigen (PSMA) is expressed by virtually all prostate cancers and represents an ideal target for diagnostic and therapeutic strategies. This article compares the in vivo behavior and tumor uptake of three different radiolabeled anti-PSMA monoclonal antibodies (mAbs) and corresponding F(ab)2 and Fab fragments thereof. METHODS The mAbs 3/A12, 3/F11, and 3/E7 and fragments of 3/A12 were conjugated with the chelating agent DOTA and radiolabeled with 64Cu. For the microPET imaging studies, SCID mice bearing PSMA-positive C4-2 and PSMA-negative DU 145 prostate cancer xenografts were used. Each animal received 20–30 µg radiolabeled mAb or fragment corresponding to an activity of 8–14 MBq. Imaging was performed 3, 24, and 48 hr post-injection. After the last scan, mice were sacrificed and tracer in vivo biodistribution was measured by gamma-counting. RESULTS Static microPET images of mice with PSMA-positive tumors revealed a high uptake of the mAbs in the C4-2 tumors at 24 and 48 hr after tracer injection and only a minimal distribution in the DU 145 tumors and other organs. In contrast, the F(ab)2 and Fab fragments of 3/A12 were detected at a high extend in the kidney but not in the C4-2 tumors. These results were confirmed by gamma counting of dissected organs after the final imaging. CONCLUSIONS Due to the high and specific uptake of the 64Cu-labeled mAbs in PSMA-positive tumors, these antibodies represent excellent tools for prostate cancer imaging. Prostate 70: 1413–1421, 2010. © 2010 Wiley-Liss, Inc.

Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein, is highly expressed by virtually all prostate cancers and is currently the focus of several diagnostic and therapeutic strategies. We have previously reported on the generation of several monoclonal antibodies (mAb) and antibody fragments that recognize and bind with high affinity to the extracellular domain of cell-adherent PSMA. This article reports the in vivo behavior and tumor uptake of the radiolabeled anti-PSMA mAb 3/A12 and its potential as a tracer for PET. Methods: The mAb 3/A12 was conjugated with the chelating agent 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and radiolabeled with 64Cu. Severe combined immunodeficient mice bearing PSMA-positive C4-2 prostate carcinoma xenografts were used for small-animal PET imaging. Mice with PSMA-negative DU 145 tumors served as controls. For PET studies, each animal received 20–30 μg of radiolabeled mAb corresponding to an activity of 7.6–11.5 MBq. Imaging was performed 3, 24, and 48 h after injection. After the last scan, the mice were sacrificed and tracer in vivo biodistribution was measured by γ-counting. Results: Binding of the mAb 3/A12 on PSMA-expressing C4-2 cells was only minimally influenced by DOTA conjugation. The labeling efficiency using 64Cu and DOTA-3/A12 was 95.3% ± 0.3%. The specific activity after 64Cu labeling was between 327 and 567 MBq/mg. After tracer injection, static small-animal PET images of mice with PSMA-positive tumors revealed a tumor-to-background ratio of 3.3 ± 1.3 at 3 h, 7.8 ± 1.4 at 24 h, and 9.6 ± 2.7 at 48 h. In contrast, no significant tracer uptake occurred in the PSMA-negative DU 145 tumors. These results were confirmed by direct counting of tissues after the final imaging. Conclusion: Because of the high and specific uptake of 64Cu-labeled mAb 3/A12 in PSMA-positive tumors, this ligand represents an excellent candidate for prostate cancer imaging and potentially for radioimmunotherapy.

18F-labeling of the nitroimidazole nucleoside analogue 1-(5-fluoro-5-deoxy-alpha-D-arabinofuranosyl)-2-nitroimidazole (FAZA) was developed to use this tracer in PET for detection of hypoxia. Parameters for labeling and hydrolysis were optimized with regard to amount of precursor, temperature and time. Labeling yields reached a maximum of 62+/-4% at 100 degrees C within 5 min using 5 mg of precursor. Hydrolysis was best performed with 1 mL of 0.1 N NaOH at 20 degrees C for 2 min. Transfer of these conditions to an automated synthesizer resulted in an overall radiochemical yield of 20.7+/-3.5%. Absolute yields at EOS were 9.8+/-2.3 GBq of [18F]FAZA ready for injection (n=21; 50 min after EOB; irradiation parameters: 35 microA, 60 min). Thus, a convenient approach suitable for large-scale production of [18F]FAZA was developed by an automated process.

The efficiency of serotonergic signal transduction is controlled by the density of serotonegic synapses and by the activity of the serotonin transporter (SERT), which selectively clears the synaptic cleft of the neurotransmitter. SERT is located in axons, where it is concentrated in varicosities and terminal boutons and thus is an exquisite marker for serotonergic synapses. This finding has been taken advantage of for neuroimaging serotonergic synaptic contact sites. Previous positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies were often carried out using radioligands that bind with high affinity to SERTs in the brainstem but also exhibit high affinity for dopamine and norepinephrine transporters and therefore did not allow quantification of serotonergic innervations in brain regions also containing dopaminergic or noradrenergic terminals. In order to visualize SERT availability more selectively, in recent years new tracers have been developed, one of which is [11C]DASB (N,N-dimethyl-2-2-amino-4-cyanophenylthiobenzylamine). Here, we have performed a detailed pharmacological characterization of unlabelled as well as radioactive DASB on recombinant human monoamine transporter proteins. Our results show that DASB selectively binds to SERT with high affinity (KD = 3.5 nm) to a site distinct from the serotonin (5-HT) recognition/translocation site. 5-HT inhibits DASB binding to SERT with more than one order of magnitude lower affinity than that of DASB binding (IC50 = 82.4 nm). These findings suggest DASB to be a highly selective PET tracer to visualize the density of serotonergic synapses in human brain.

The synthesis of carbon-11 amino function labelled uncharged Thioflavin T derivatives is known to be performed by reaction of the demethyl-precursors with [11C]methyl iodide but the labelling yields are only mediocre. The use of [11C]methyl triflate improved the radiochemical yield of three potential beta-amyloid imaging PET-radiotracers significantly. Performance of the labelling reaction by reacting the corresponding precursor molecules with [11C]methyl triflate for 1 min at 80 degrees C led to radiochemical yields of 44+/-10% (n=5) for [11C]6-Me-BTA-1, 68+/-4% (n=10) for [11C]BTA-1 and 58+/-2% (n=5) for [11C]6-OH-BTA-1 with respect to [11C]methyl triflate. In production runs (60 min, 50 microA) up to 6500 MBq (mean: 4000+/-1900 MBq) of [11C]6-Me-BTA-1, 7900 MBq (mean: 6000+/-1000 MBq) of [11C]BTA-1 and 7100 MBq (mean: 6300+/-600 MBq) of [11C]6-OH-BTA-1 could be obtained ready for intravenous injection. The radiochemical purity was >95% with specific activities in the range of 80-120 GBq/micromol (EOS) within a total synthesis time of less than 40 min after EOB.