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Aim/Introduction: The question of whether the presence of the APOE ε4 allele impacts α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) availability in Alzheimer’s dementia (AD) was so far mainly studied post-mortem, and is subject of a controversial debate. We aimed to answering this question in vivo using the recently developed α4β2-nAChR-specific radioligand (-)-[18F] Flubatine and PET. Materials and Methods: Non-smoking, drug-naïve AD-APOE ε4+ (n=7; 76±6ys; 6 females; MMSE 24±3) and AD-APOE ε4- (n=9; 75±7ys; 7 females; MMSE 24±2, n. sign. vs. AD-APOE ε4+) were investigated using (-)-[18F]Flubatine (370 MBq, ECAT Exact HR+, 0-90min p.i.) and compared with non-smoking healthy controls (HC; n=13; 72±4ys; 7 females). For quantification of the α4β2-nAChR availability, kinetic modeling (1TCM, Logan) was performed and the distribution volume (VT) was calculated. VOI analyses of a-priori selected brain regions and exploratory SPM analyses were carried out (ANCOVA, significance at P3.0; P

Both (+)-[18F]flubatine and its enantiomer (−)-[18F]flubatine are radioligands for the neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). In a clinical study in patients with early Alzheimer’s disease, (+)-[18F]flubatine ((+)-[18F]1) was examined regarding its metabolic fate, in particular by identification of degradation products detected in plasma and urine. The investigations included an in vivo study of (+)-flubatine ((+)-1) in pigs and structural elucidation of formed metabolites by LC-MS/MS. Incubations of (+)-1 and (+)-[18F]1 with human liver microsomes were performed to generate in vitro metabolites, as well as radiometabolites, which enabled an assignment of their structures by comparison of LC-MS/MS and radio-HPLC data. Plasma and urine samples taken after administration of (+)-[18F]1 in humans were examined by radio-HPLC and, on the basis of results obtained in vitro and in vivo, formed radiometabolites were identified. In pigs, (+)-1 was monohydroxylated at different sites of the azabicyclic ring system of the molecule. Additionally, one intermediate metabolite underwent glucuronidation, as also demonstrated in vitro. In humans, a fraction of 95.9 ± 1.9% (n = 10) of unchanged tracer remained in plasma, 30 min after injection. However, despite the low metabolic degradation, both radiometabolites formed in humans could be characterized as (i) a product of C-hydroxylation at the azabicyclic ring system, and (ii) a glucuronide conjugate of the precedingly-formed N8-hydroxylated (+)-[18F]1.

This paper presents a new approach for integrated vehicle motion control, coordinating multiple vehicle subsystems of a passenger car including friction brake system, near-wheel drive electric motors, wheel steer actuators, camber angle actuators, dynamic tire pressure system and actuators generating additional normal forces. The proposed algorithms are based on restriction weights into the cost function of optimization-based control allocation. Hardware-in-the-loop investigation using a test rig with hardware components of friction brake system and dynamic tire pressure system showed that the proposed approach allows to achieve lower energy consumption and energy losses without significant impairment of motion stability and vehicle handling as compared to conventional control allocation.

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Ziel: Bei der Translation neuartiger Radiotracer in die klinische Phase ist eine Abschätzung der Strahlenexposition vor Erstanwendung am Menschen notwendig. Hierbei werden die Organdosen (OD) sowie die effektive Dosis (ED) am Tiermodell abgeschätzt, welche nach i.v. Injektion eines Radiotracers entstehen. Erstmalig wurde Kleintier-PET/MRT zur rein bildgebungsbasierten Inkorporationsdosimetrie mit CD-1 Mäusen eingesetzt. Um den Einfluss von Speziesunterschieden zu untersuchen wurden PET/CT-Studien an Ferkeln durchgeführt und die Ergebnisse mit am Menschen erhobenen Ergebnissen (PET/CT) verglichen. Methodik: Nach i.v. Injektion von (-)- oder (+)-[18F]Flubatine (a, b) bzw. (S)- oder (R)-[18F]Fluspidine (c,d) wurden (i) In-vivo-PET/MRT- (MEDISO nanoScan, Budapest) und PET/CT-Scans (SIEMENS Biograph 16) bis zu 7 h p.i. durchgeführt, die List-Mode Daten unter Nutzung der Standardkorrekturen rekonstruiert und die Organaktivitäten (OA) mit ROVER (ABX, Radeberg) bestimmt; (ii) (a, c, d) Ex-vivo-Organentnahme an Mäusen und Messung der OA in einem Gammacounter durchgeführt (Goldstandard). Nach Extrapolation der Tierdaten auf menschliche Verhältnisse, wurden die OD und die ED mit OLINDA für 3 Spezies berechnet. Ergebnisse: Die Dosimetrie für a/b ergab eine ED (µSv/MBq) von 12,5/12,1 (30 Mäuse), 13,4/14,3 (8 Ferkel), 22,3/23,0 (n=6 Menschen) und für (c/d) 12,9/14,0 (6 Mäuse), 21,0/n.a. (4 Menschen). Während a und b eine vergleichbare Biokinetik sowie ED zeigen, ist die ED von c und d signifikant (p=0,025) verschieden basierend auf Enantiomeren Unterschieden. Weiterhin zeigt sich eine Unterschätzung der ED erhoben mit Tierdaten im Vergleich zum Menschen von 38% (Ferkel) bis 47% (Mäuse). Schlussfolgerung: Die Strahlenexposition nach i.v. Applikation von a,b,c,d liegt im Bereich der durch andere F-18-markierter Radiotracer. Die Abschätzung der ED unter Nutzung von Tiermodellen mit Hilfe eines Kleintier-PET/MRT ist unter Berücksichtigung der genannten Limitationen möglich und liefert mit dem Ex-vivo-Goldstandard vergleichbare Ergebnisse.

Both (+)-[18F]flubatine and its enantiomer (-)-[18F]flubatine are radioligands for the neuroimaging of a4ß2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). Within a clinical study in patients with early Alzheimer’s disease, (+)-[18F]flubatine ((+)-[18F]1) was examined regarding its metabolic fate, in particular by identification of degradation products detected in plasma and urine. The investigations included an in vivo study of (+)-flubatine ((+)-1) in pig and structural elucidation of formed metabolites by LC-MS/MS. Incubations of (+)-1 and (+)-[18F]1 with human liver microsomes were performed to generate in vitro metabolites as well as radiometabolites, which enabled an assignment of their structures by comparison of LC-MS/MS and radio-HPLC data. Plasma and urine samples taken after administration of (+)-[18F]1 into human were examined by radio-HPLC and, on the basis of results obtained in vitro and in vivo, formed radiometabolites were identified. In pig, (+)-1 was monohydroxylated at different sites of the azabicyclic ring system of the molecule. Additionally, one intermediate metabolite underwent glucuronidation, as also demonstrated in vitro. In human, 95.9 ? 1.9% (N = 10) of unchanged tracer remained in plasma, 30 min after injection. However, despite the low metabolic degradation, both radiometabolites formed could be characterized as i.) a product of C-hydroxylation at the azabicyclic ring system and ii.) a glucuronide conjugate of previously N8-hydroxylated (+)-[18F]1.

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Radiolabeled tracers targeting the prostate-specific membrane antigen (PSMA) have become important radiopharmaceuticals for the PET-imaging of prostate cancer. In this connection, we recently developed the fluorine-18-labelled PSMA-ligand [18F]PSMA-1007 as the next generation radiofluorinated Glu-ureido PSMA inhibitor after [18F]DCFPyL and [18F]DCFBC. Since radiosynthesis so far has been suffering from rather poor yields, novel procedures for the automated radiosyntheses of [18F]PSMA-1007 have been developed. We herein report on both the two-step and the novel one-step procedures, which have been performed on different commonly-used radiosynthesisers. Using the novel one-step procedure, the [18F]PSMA-1007 was produced in good radiochemical yields ranging from 25 to 80% and synthesis times of less than 55 min. Furthermore, upscaling to product activities up to 50 GBq per batch was successfully conducted. All batches passed quality control according to European Pharmacopoeia standards. Therefore, we were able to disclose a new, simple and, at the same time, high yielding production pathway for the next generation PSMA radioligand [18F]PSMA-1007. Actually, it turned out that the radiosynthesis is as easily realised as the well-known [18F]FDG synthesis and, thus, transferable to all currently-available radiosynthesisers. Using the new procedures, the clinical daily routine can be sustainably supported in-house even in larger hospitals by a single production batch.

Radiolabelled tracers targeting the prostate-specific membrane antigen (PSMA) have become important radiopharmaceuticals for the PET-imaging of prostate cancer. In this connection we recently developed the fluorine-18 labelled PSMA-ligand [18F]PSMA-1007 as next generation radiofluorinated Glu-ureido PSMA inhibitor after [18F]DCFPyL and [18F]DCFBC. Since radiosynthesis so far was suffering for rather poor yields novel procedures for the automated radiosyntheses of [18F]PSMA-1007 have been developed. We herein report on both the two-step and the novel one-step procedures, which have been performed on different commonly used radiosynthesisers. Using the novel one-step procedure the [18F]PSMA-1007 was produced in good radiochemical yields ranging from 30-70 % and synthesis times less than 55 minutes. Furthermore, upscaling to product activities up to 50 GBq per batch was successfully conducted. All batches passed quality control according to European Pharmacopoeia standards. Therefore, we were able to disclose a new, simple and at the same time high yielding production pathway for the next generation PSMA radioligand [18F]PSMA-1007. Actually it turned out that the radiosynthesis is as easily realised as the well-known [18F]FDG synthesis and thus transferable onto all currently available radiosynthesisers. Using the new procedures, clinical daily routine can be sustainably supported in-house even in larger hospitals by a single production batch.

Erythrina poeppigiana is a medicinal plant which is widely used in Asia, Latin America, and Africa in traditional remedies for gynecological complications and maladies. In continuation of studies for the discovery of novel phytoestrogens, four erythroidine alkaloids, namely α-erythroidine, β-erythroidine, and their oxo-derivatives 8-oxo-α-erythroidine and 8-oxo-β-erythroidine, were isolated and structurally characterized from the methanolic extract of the stem bark of E. poeppigiana. Due to the high amounts of erythroidines in the extract and considering the widespread utilization of Erythrina preparations in traditional medicine, the exploration of their estrogenic properties was performed. The estrogenicity of the isolated erythroidines was assayed in various estrogen receptor-(ER)-dependent test systems, including receptor binding affinity, cell culture based ER-dependent reporter gene assays, and gene expression studies in cultured cells using reverse transcription polymerase chain reaction techniques. α-Erythroidine and β-erythroidine showed binding affinity values for ERα of 0.015 ± 0.010 % and 0.005 ± 0.010 %, respectively, whereas only β-erythroidine bound to ERβ (0.006 ± 0.010 %). In reporter gene assays, both erythroidines exhibited a significant dose-dependent estrogenic stimulation of ER-dependent reporter gene activity in osteosarcoma cells detectable already at 10 nM. Results were confirmed in the MVLN cells, a bioluminescent variant of MCF-7 breast cancer cells. Further, α-erythroidine and β-erythroidine both induced the enhanced expression of the specific ERα-dependent genes trefoil factor-1 and serum/glucocorticoid regulated kinase 3 in MCF-7 cells, confirming estrogenicity. Additionally, using molecular docking simulations, a potential mode of binding on ERα, is proposed, supporting the experimental evidences. This is the first time that an estrogenic profile is reported for erythroidine alkaloids, potentially a new class of phytoestrogens.

Introduction (−)-[ 18 F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α 4 β 2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimer's disease (AD) patients and 20 healthy controls (HCs), the following parameters were determined: plasma protein binding, metabolism and activity distribution between plasma and whole blood. Methods Plasma protein binding and fraction of unchanged parent compound were assessed by ultracentrifugation and HPLC, respectively. The distribution of radioactivity (parent compound+metabolites) between plasma and whole blood was determined ex vivo at different time-points after injection by gamma counting after separation of whole blood by centrifugation into the cellular and non-cellular components. In additional experiments in vitro , tracer distribution between these blood components was assessed for up to 90min. Results A fraction of 15%±2% of (−)-[ 18 F]Flubatine was found to be bound to plasma proteins. Metabolic degradation of (−)-[ 18 F]Flubatine was very low, resulting in almost 90% unchanged parent compound at 90min p.i. with no significant difference between AD and HC. The radioactivity distribution between plasma and whole blood changed in vivo only slightly over time from 0.82±0.03 at 3min p.i. to 0.87±0.03 at 270min p.i. indicating the contribution of only a small amount of metabolites. In vitro studies revealed that (−)-[ 18 F]Flubatine was instantaneously distributed between cellular and non-cellular blood parts. Discussion (−)-[ 18 F]Flubatine exhibits very favourable characteristics for a PET radiotracer such as slow metabolic degradation and moderate plasma protein binding. Equilibrium of radioactivity distribution between plasma and whole blood is reached instantaneously and remains almost constant over time allowing both convenient sample handling and facilitated fractional blood volume contribution assessment.

Both enantiomers of the epibatidine analogue flubatine display high affinity towards the α4β2 nicotinic acetylcholine receptor (nAChR) in vitro, accompanied by negligible interactions with diverse off-target proteins. Extended single dose toxicity studies in rodent indicated a NOEL (No Observed Effect Level) of 6.2μg/kg for (-)-flubatine and 1.55μg/kg for (+)-flubatine. We developed syntheses for both flubatine enantiomers and their corresponding precursors for radiolabeling. The newly synthesized trimethylammonium precursors allowed for highly efficient (18)F-radiolabelling in radiochemical yields >60% and specific activities >750GBq/μmol, thus making the radioligands practical for clinical investigation.

(−)-[18F]flubatine is a promising agent for visualization by PET of cerebral α4β2 nicotinic acetylcholine receptors (nAChRs), which are implicated in psychiatric and neurodegenerative disorders. Here, we describe a substantially improved two-step radiosynthesis strategy for (−)-[18F]flubatine, based on the nucleophilic radiofluorination of an enantiomerically pure precursor followed by deprotection of the intermediate. An extensive leaving group/protecting group library of precursors was tested. Application of a trimethylammonium-iodide precursor with a Boc-protecting group provided the best results: labeling efficiencies of 80–95%, RCY of 60±5%, radiochemical purity of >98%, and a specific activity of >350 GBq/μmol. The radiosynthesis is easily transferable to an automated synthesis module.

Objectives: Radiometabolites can affect PET imaging dramatically due to their expected different properties. Therefore identification of radiometabolites is an important step to understand the metabolic fate of a radioligand. The approach presented demonstrates how LC-MS supports in vitro experiments and contributes to explore the metabolic profile of two tracers recently studied in human brain [1, 2]. Methods: (+)-[18F]Flubatine ([18F]1) and (S)-[18F]Fluspidine ([18F]2) (Figure 1), as well as nonradioactive references were incubated with human liver microsomes (HLM) in presence of NADPH and/or activated glucuronic acid (UDPGA) at 37°C. Radiometabolite patterns were monitored by radio-HPLC and structures were identified by LC-MS of non-radioactive incubations using different MS-methods (EPI, MS3). Plasma (30 min p.i.) and urine (90 min p.i.) from human subjects receiving [18F]1 or [18F]2 during clinical studies were investigated and compared with results von microsomal incubations. Results: During HLM incubations in presence of NADPH, mono-hydroxylation was predominant for both, 1 and 2, beside debenzylation of 2. In presence of UDPGA 1 and 2 underwent glucuronidation, but only after previous hydroxylation. Corresponding in vitro radiometabolites were detected by radio-HPLC and assigned regarding their structure. Samples obtained from humans showed high stability of both tracers, whereby [18F]1 (97.0% in plasma 30 min p.i, n=6) proved to be more stable than [18F]2 (85.3% in plasma 30 min p.i, n=3). However, hydroxylation and subsequent glucuronidation was found to be the major metabolic pathway of both tracers. Conclusions: Using in vitro studies and LC-MS, in vivo radiometabolites could be identified. Beside high metabolic stability, [18F]1 and [18F]2 show similar major pathways, namely glucuronidation after previous hydroxylation. Acknowledgements: Supported by the Helmholtz Validation Fund (HVF) and the German Research Foundation (DFG). References: [1] Sattler et al. (2015), J Nucl Med, 56, suppl. 3, 1020; [2] Sattler et al. (2016), J Nucl Med, 57, suppl. 2, 1022.

Objectives: In an ongoing clinical study (–)-(S)-[18F]fluspidine (1, 2) is being investigated for imaging of sigma-1 receptors in patients with major depression by PET. Beside estimation of the fraction of unchanged radiotracer in plasma and urine, radiometabolites formed should be detected and their structures elucidated. Methods: (–)-(S)-[18F]Fluspidine or unlabelled reference were incubated with human liver microsomes (HLM) in presence of NADPH and/or activated glucuronic acid (UDPGA) at 37°C. Metabolites were detected by radio-HPLC or LC-MS and characterized by aid of reference compounds or structural elucidation using different MS methods (EPI, MS3). Plasma (10, 20, 30 min p.i) and urine (90 min p.i.) samples of human subjects receiving 268 (245-290) MBq (–)-(S)-[18F]fluspidine i.v. were investigated and compared to results from HLM incubations. Results: By incubation with HLM mainly 3 hydroxylation products and one debenzylation product were formed. The product hydroxylated at the alkyl side chain was glucuronidated subsequently. Plasma samples (10, 20, 30 min p.i.) obtained from human showed 97.7%, 93.8%, and 88.6% (SD=2.6-7.9, n=6-8) of unchanged tracer. In urine (90 min p.i.) the fraction of tracer was 0–7.8% (average of 2.3%, n=10) and up to 3 main radiometabolites were detected (Figure 1). The one with the highest intensity, also found in plasma, matched the glucuronide formed in vitro. Debenzylation of the parent tracer was detected with a small proportion. Conclusions: The results demonstrate an appropriate metabolic stability of (–)-(S)-[18F]fluspidine for the application as PET ligand. The structure of the major radiometabolite found in plasma and urine could be assigned by means of in vitro studies. Routes of metabolism are currently investigated in more detail. 302 References: [1] German Clinical Trial Register, DRKS-ID: DRKS00008321 [2] Fischer et al. Eur. J. Nucl. Med. Mol. Imaging 2011, 38, 540–551

Background Both enantiomers of [18F]flubatine are new radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors with positron emission tomography (PET) exhibiting promising pharmacokinetics which makes them attractive for different clinical questions. In a previous preclinical study, the main advantage of (+)-[18F]flubatine compared to (−)-[18F]flubatine was its higher binding affinity suggesting that (+)-[18F]flubatine might be able to detect also slight reductions of α4β2 nAChRs and could be more sensitive than (−)-[18F]flubatine in early stages of Alzheimer’s disease. To support the clinical translation, we investigated a fully image-based internal dosimetry approach for (+)-[18F]flubatine, comparing mouse data collected on a preclinical PET/MRI system to piglet and first-in-human data acquired on a clinical PET/CT system. Time-activity curves (TACs) were obtained from the three species, the animal data extrapolated to human scale, exponentially fitted and the organ doses (OD), and effective dose (ED) calculated with OLINDA. Results The excreting organs (urinary bladder, kidneys, and liver) receive the highest organ doses in all species. Hence, a renal/hepatobiliary excretion pathway can be assumed. In addition, the ED conversion factors of 12.1 μSv/MBq (mice), 14.3 μSv/MBq (piglets), and 23.0 μSv/MBq (humans) were calculated which are well within the order of magnitude as known from other 18F-labeled radiotracers. Conclusions Although both enantiomers of [18F]flubatine exhibit different binding kinetics in the brain due to the respective affinities, the effective dose revealed no enantiomer-specific differences among the investigated species. The preclinical dosimetry and biodistribution of (+)-[18F]flubatine was shown and the feasibility of a dose assessment based on image data acquired on a small animal PET/MR and a clinical PET/CT was demonstrated. Additionally, the first-in-human study confirmed the tolerability of the radiation risk of (+)-[18F]flubatine imaging which is well within the range as caused by other 18F-labeled tracers. However, as shown in previous studies, the ED in humans is underestimated by up to 50 % using preclinical imaging for internal dosimetry. This fact needs to be considered when applying for first-in-human studies based on preclinical biokinetic data scaled to human anatomy. Electronic supplementary material The online version of this article (doi:10.1186/s40658-016-0160-5) contains supplementary material, which is available to authorized users.

Both enantiomers of [18F]flubatine are promising radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). To support clinical studies in patients with early Alzheimer’s disease, a detailed examination of the metabolism in vitro and in vivo has been performed. (+)- and (−)-flubatine, respectively, were incubated with liver microsomes from mouse and human in the presence of NADPH (β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt). Phase I in vitro metabolites were detected and their structures elucidated by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Selected metabolite candidates were synthesized and investigated for structural confirmation. Besides a high level of in vitro stability, the microsomal incubations revealed some species differences as well as enantiomer discrimination with regard to the formation of monohydroxylated products, which was identified as the main metabolic pathway in this assay. Furthermore, after injection of 250 MBq (+)-[18F]flubatine (specific activity > 350 GBq/μmol) into mouse, samples were prepared from brain, liver, plasma, and urine after 30 min and investigated by radio-HPLC (high performance liquid chromatography with radioactivity detection). For structure elucidation of the radiometabolites of (+)-[18F]flubatine formed in vivo, identical chromatographic conditions were applied to LC-MS/MS and radio-HPLC to compare samples obtained in vitro and in vivo. By this correlation approach, we assigned three of four main in vivo radiometabolites to products that are exclusively C- or N-hydroxylated at the azabicyclic ring system of the parent molecule.

Both enantiomers of [18F]flubatine are promising radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). To support clinical studies in patients with early Alzheimer’s disease, a detailed examination of the metabolism in vitro and in vivo has been performed. (+)- and (−)-flubatine, respectively, were incubated with liver microsomes from mouse and human in the presence of NADPH (β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt). Phase I in vitro metabolites were detected and their structures elucidated by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Selected metabolite candidates were synthesized and investigated for structural confirmation. Besides a high level of in vitro stability, the microsomal incubations revealed some species differences as well as enantiomer discrimination with regard to the formation of monohydroxylated products, which was identified as the main metabolic pathway in this assay. Furthermore, after injection of 250 MBq (+)-[18F]flubatine (specific activity > 350 GBq/μmol) into mouse, samples were prepared from brain, liver, plasma, and urine after 30 min and investigated by radio-HPLC (high performance liquid chromatography with radioactivity detection). For structure elucidation of the radiometabolites of (+)-[18F]flubatine formed in vivo, identical chromatographic conditions were applied to LC-MS/MS and radio-HPLC to compare samples obtained in vitro and in vivo. By this correlation approach, we assigned three of four main in vivo radiometabolites to products that are exclusively C- or N-hydroxylated at the azabicyclic ring system of the parent molecule.

Both enantiomers of [(18)F]flubatine are promising radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET). To support clinical studies in patients with early Alzheimer's disease, a detailed examination of the metabolism in vitro and in vivo has been performed. (+)- and (-)-flubatine, respectively, were incubated with liver microsomes from mouse and human in the presence of NADPH (β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt). Phase I in vitro metabolites were detected and their structures elucidated by LC-MS/MS (liquid chromatography-tandem mass spectrometry). Selected metabolite candidates were synthesized and investigated for structural confirmation. Besides a high level of in vitro stability, the microsomal incubations revealed some species differences as well as enantiomer discrimination with regard to the formation of monohydroxylated products, which was identified as the main metabolic pathway in this assay. Furthermore, after injection of 250 MBq (+)-[(18)F]flubatine (specific activity350 GBq/μmol) into mouse, samples were prepared from brain, liver, plasma, and urine after 30 min and investigated by radio-HPLC (high performance liquid chromatography with radioactivity detection). For structure elucidation of the radiometabolites of (+)-[(18)F]flubatine formed in vivo, identical chromatographic conditions were applied to LC-MS/MS and radio-HPLC to compare samples obtained in vitro and in vivo. By this correlation approach, we assigned three of four main in vivo radiometabolites to products that are exclusively C- or N-hydroxylated at the azabicyclic ring system of the parent molecule.

CH-acidic methyl groups can easily be converted into dithioesters by use of sulfur transfer reagents possessing a S2-unit and two leaving groups. A possible mechanism is discussed.

The design of radioligands selective for particular GABAA receptor isoforms is challenging because of their great potential for the diagnostic imaging of neuropsychiatric diseases such as epilepsy and sleep disorders. A subtype specificity higher than that of the classical benzodiazepines makes the novel pyrazolopyrimidine indiplon an attractive candidate structure. In the present work, N-(3-[18F]fluoro-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine- 7-yl]-phenyl}-acetamide ([18F]fluoro-propyl-indiplon; [18F]FPI) was characterized and validated as a candidate tracer for positron emission tomography imaging of α1-GABAA receptors. In vitro, receptor affinity and autoradiography of [18F]FPI were assessed in cerebellar homogenates and sagittal brain slices of rats, respectively. The regional brain and organ uptake was investigated in NMRI mice at various times after i.v. application of [18F]FPI by ex vivo autoradiography and dissection, respectively. Radiometabolites of [18F]FPI were determined in blood, brain, and urine samples by highperformance liquid chromatography. [18F]FPI binds to cerebellar receptors with high affinity (KD 2.93 nM), and the in vitro labelling pattern appears to be more selective for α1-containing GABAA receptors than [3H]flunitrazepam. Intravenous application of [18F]FPI revealed (i) a strong biotransformation, (ii) low brain-to-plasma ratios of 18F ( < 0.4 at all times after tracer injection), (iii) homogenous and non-specific 18F distribution in the rat brain, and (iv) high radioactivity uptake in liver and femur. Thus, despite the promising results obtained by in vitro evaluation, [18F]FPI is excluded from further development as PET tracer.

The function of gamma-aminobutyric acid type A receptors (GABA(A) receptors) is enhanced by various clinically important drugs including benzodiazepines that act on an allosteric site formed at the interface between the alpha and gamma subunits. In contrast to classical benzodiazepines, the novel pyrazolopyrimidine indiplon (N-methyl-N-{3-[7-(thiophene-2-carbonyl)-1,5,9-triazabicyclo[4.3.0]nona-2,4,6,8-tetraen-2-yl]phenyl}acetamide; N-methyl-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide) demonstrates relative binding selectivity for the alpha1 subunit containing receptor subtypes, which are the most frequently expressed in the mammalian central nervous system. To investigate the pharmacological properties at GABA(A) receptors and to promote the development of alpha1 subunit selective radiotracers for positron emission tomography imaging, we have started with the evaluation of various fluorinated indiplon derivatives. Binding affinities were determined in homogenates from newborn and adult rats suggesting an alpha1 preference of the reference compounds indiplon, zaleplon as well as for all newly synthesized indiplon derivatives. In homogenated cerebellar tissue obtained from adult rat brain, known to primarily express alpha1 containing GABA(A) receptors, the high affinity of the basic indiplon structure was only slightly affected by an elongation of the alkyl substituent of the amide N from methyl (indiplon; K(i) 3.1 nM) via ethyl (2a, N-(2-fluoro-ethyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 5.4 nM) to propyl (2b, N-(3-fluoro-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidine-7-yl]phenyl}-acetamide; K(i) 2.4 nM). Whole cell patch-clamp recordings at neuronal and recombinant GABA(A) receptors indicated that the fluorinated derivatives 2a and 2b have a high potency at alpha1beta3gamma2L isoforms comparable to indiplon (EC(50): 105, 158, and 81 nM, respectively), with 2b displaying the most pronounced efficacy at alpha3beta3gamma2L subtypes. In conclusion, the affinity profiles and functional properties of the newly synthesised fluorinated indiplon derivatives make compounds 2a and 2b suitable for the development of [(18)F]-labelled ligands at GABA(A) receptors containing the alpha1 subunit.

The involvement of gamma amino butyric acid (GABA) receptors in a variety of neurological and psychiatric diseases has promoted the development and use of radiolabelled benzodiazepines (BZ) for brain imaging by PET. However, these radioligands are unable to distinguish between the various subtypes of GABAA receptors. Novel non-BZ such as the pyrazolo-pyrimidine indiplon proved to be selective for the α1-subunit of the GABAA receptor. Here, we describe the syntheses of four novel 18F-labelled indiplon derivatives. Radiosyntheses were performed via n.c.a. 18F-nucleophilic substitution starting from the tosyl, bromo, and 4-nitrobenzoyl precursors to obtain fluorine substituted N-alkylamide side chain derivatives of indiplon, followed by multistep purification using semi-preparative high-performance liquid chromatography and solid phase extraction. Tosyl and bromo precursors were converted into 18F-labelled indiplon derivatives with good and reproducible radiochemical yield (RCY) (35–70%, decay corrected), high radiochemical purity (≥98.5%), and high specific activity ( > 150 GBq/µmol). By contrast, a low RCY (5–10%) and specific activity (10–15 GBq/µmol) were achieved for the 4-nitrobenzoyl precursor. Copyright © 2008 John Wiley & Sons, Ltd.

With the aim of imaging and quantification of oxytocin receptors (OTRs) in living brain using positron emission tomography (PET), we developed a (18)F-labeled small molecule radiotracer and investigated its in vivo pharmacokinetics in mice and pig. [(18)F]6b (KD = 12.3 nM) was radiolabeled by a two-step procedure using a microwave system with radiochemical yields of 26.9 ± 4.7%. Both organ distribution and small animal PET studies revealed limited brain uptake of [(18)F]6b in mouse (mean SUV of 0.04 at 30 min pi). Besides, significant radioactivity uptake in the pituitary gland was observed (SUV of 0.7 at 30 min pi). In a dynamic PET study in one piglet, we detected a higher uptake of [(18)F]6b in the olfactory bulb (SUV of 0.34 at 30 min pi) accompanied by a low uptake in the whole brain. In vitro autoradiographic studies on porcine brain sections indicated interaction of [(18)F]6b with several off-target receptors.

International audience; [18F]FMTEB, [18F]FPEB, [18F]flumazenil, [18F]DAA1106, [18F]MFBG, [18F]FDOPA, [18F]FMT and [18F]FDA are prepared from the corresponding arylboronic esters and [18F]KF/K222 in the presence of Cu(OTf)2py4. The method was successfully applied using three radiosynthetic platforms, and up to 26 GBq of non-carrier added starting activity of 18F-fluoride.

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Background: Both enantiomers of [18F]flubatine are promising radioligands for neuroimaging of α4β2 nicotinic acetylcholine receptors with positron emission tomography (PET) exhibiting promising pharmacokinetics which makes them attractive for different clinical questions. Ina previous preclinical study, the main advantage of (+)-[18F]flubatine compared to (-)-[18F]flubatine was its higher binding affinity suggesting that (+)-[18F]flubatine might be able to detect also slight reductions of α4β2 nAChRs and could be more sensitive than (-)-[18F]flubatine in early stages of Alzheimer's disease. To support the clinical translation, we investigated a fully image-based internal dosimetry approach for (+)-[18F]flubatine, coparing mouse data collected on a preclinical PET/MR system to piglet and first-in-human data acquired on a clinical PET/CT system. Time-activity curves (TACs) were obtained from the three species, the animal data extrapolated to human scale, exponentially fitted and the organ doses (OD), and the effective dose (ED) calculated with OLINDA. Results: The excreting organs (urinary bladder, kidneys, and liver) receive the highest organ doses in all species. Hence, a renal/hepatobiliary excretion pathway can be assumed. In addition, the ED conversion factors of 12.1 µSv/MBq (mice), 14.3 µSv/MBq (piglets) and 23.0 µSv/MBq (humans) were calculated which are well within the order of magnitude as known from other 18F-labeled radiotracers. Conclusion: Although both enantiomers of [18F]flubatine] exhibit different binding kinetics in the brain due to the respective affinities, the effective dose revealed no enantiomer-specific differences among the investigated species. The preclinical dosimetry and biodistribution of (+)-[18F]flubatine was shown and the feasibility of a dose assessment based on image data acquired on a small animal PET/MR and a clinical PET/CT was demonstrated. Additionally, the first-in-human study confirmed the tolerability of the radiation risk of (+)-[18F]flubatine imaging which is well within the range as caused by other 18F-labeled tracers. However, as shown in previous studies, the ED in humans is underestimated by up to 50 % using preclinical imaging for internal dosimetry. This fact needs to be considered when applying for first-in-human studies based on preclinical biokinetic data scaled to human anatomy.

Ziel: In einer jüngst begonnenen klinischen Studie (1) wird die Veränderung der Verfügbarkeit von Sigma-1-Rezeptoren bei Patienten mit Depression mittels (–)-(S)-[18F]Fluspidine (2) und PET untersucht. Neben der Bestimmung des Anteils an Radiotracer in Plasma und Urin, war die Strukturaufklärung nachgewiesener Radiometaboliten Gegenstand unserer Arbeiten. Methodik: Nach Injektion von 255±9 MBq (n=3) (–)-(S)-[18F]Fluspidine, wurden Plasma (15, 30 min p.i.) und Urin (120 min p.i.) der Versuchsteilnehmer per Radio-HPLC untersucht. Für In-vitro-Studien wurden der Radiotracer bzw. unmarkiertes (–)-(S)-Fluspidine jeweils mit Lebermikrosomen des Menschen (HLM) sowie NADPH und /oder aktivierter Glucuronsäure (UDPGA) bei 37°C in PBS-Puffer inkubiert und die Ansätze mittels Radio-HPLC bzw. LC-MS untersucht. Mit Hilfe verschiedener MS-Methoden (z. B. EPI, MS3) wurden Strukturen von Metaboliten identifiziert und im Menschen gebildeten Radiometaboliten zugeordnet. Ergebnisse: Bei ersten Messungen an Probanden lag der Anteil an (–)-(S)-[18F]Fluspidine im Plasma nach 30 min bei 85%. Die Extraktionsausbeute der Plasmafällung betrug 96-98%. Im Urin wurde ein Radiotracer-Anteil von 0 - 4% (120 min, p.i., n=3) bestimmt. Es wurden 3 Hauptmetaboliten detektiert. Durch Inkubation mit HLM wurden unter oxidativen Bedingungen, Metaboliten in vitro gebildet. Mit UDPGA wurde zudem die Bildung von Glucuroniden beobachtet, von denen eines chromatographisch mit einem im Menschen gefundenen Haupt-Radiometaboliten übereinstimmt. LC-MS-Untersuchungen zeigten, dass dieser durch Glucuronidierung infolge einer Oxidation am nicht-benzylischen Molekülteil entstand. Schlussfolgerungen: Erste klinische Untersuchungen zeigten eine für die Anwendung geeignete metabolische Stabilität, wobei einer der Hauptmetaboliten bereits identifiziert werden konnte. Über weitere Daten im Studienverlauf sowie über Strukturaufklärung wird berichtet. Literatur: (1) DRKS-ID:DRKS00008321 (2) Fischer et al. Eur J Nucl Med Mol Imaging (2011) 38: 540-551

kein Abstract verfügbar

Expression of system L amino acid transporters (LAT) is strongly increased in many types of tumor cells. The purpose of this study was to demonstrate that 18F-labeled amino acids, for example, 3-O-methyl-6- 18 F-fluoro-L-dopa ( 18 F-OMFD), that accumulate in tumors via LAT represent an important class of imaging agents for visualization of tumors in vivo by PET. Methods: 18 F-OMFD uptake kinetics, transport inhibition, and system L messenger RNA expression were studied in vitro in human adenocarcinoma (HT-29), squamous cell carcinoma (FaDu), macrophages (THP1), and primary aortic endothelial cells (HAEC) and in vivo in the corresponding mouse tumor xenograft models. Results: Uptake of 18F-OMFD in all cell lines tested was mediated mainly by the sodium-independent high-capacity LAT. We found higher uptake inFaDu cells (Vmax,10.6 61.1nmol/min ·mg of cellprotein) and in the corresponding FaDu tumor xenografts than in the other cells and corresponding xenograft models studied. Quantitative messenger RNA analysis revealed that tumor cells and xenografts have a higher expression of LAT1 than do HAEC and THP-1 macrophages. However, only in the FaDu tumor model did an increased 18 F-OMFD uptake seem to be explained by increased LAT expression. Furthermore, we demonstrated a high expression of LAT4, a recently identified LAT. Conclusion: Ourfindingssupport thehypothesis that 18F-OMFD is atracer for visualization of tumor cells. 18 F-OMFD particularly seems to be a suitable tracer for diagnostic imaging of amino acid transport in poorly differentiated squamous cell head and neck carcinoma with increased LAT1 and LAT4 expression.

Background The norepinephrine transporter (NET) has been demonstrated to be relevant to a multitude of neurological, psychiatric and cardiovascular pathologies. Due to the wide range of possible applications for PET imaging of the NET together with the limitations of currently available radioligands, novel PET tracers for imaging of the cerebral NET with improved pharmacological and pharmacodynamic properties are needed. Methods The present study addresses the radiosynthesis and first preclinical evaluation of the novel NET PET tracer [11C]Me@HAPTHI by describing its affinity, selectivity, metabolic stability, plasma free fraction, blood–brain barrier (BBB) penetration and binding behaviour in in vitro autoradiography. Results [11C]Me@HAPTHI was prepared and displayed outstanding affinity and selectivity as well as excellent in vitro metabolic stability, and it is likely to penetrate the BBB. Moreover, selective NET binding in in vitro autoradiography was observed in human brain and rat heart tissue samples. Conclusions All preclinical results and radiosynthetic key-parameters indicate that the novel benzothiadiazole dioxide-based PET tracer [11C]Me@HAPTHI is a feasible and improved NET radioligand and might prospectively facilitate clinical NET imaging.

For the electrophilic preparation of 6-[18F]fluoro-L-m-tyrosine ([18F]FMT), a PET tracer for measuring changes in dopaminergic function in movement disorders, a novel precursor, N-(tert-butoxycarbonyl)-3-(tert-butoxycarbonyloxy)-6-trimethylstannnyl-L-phenylalanine ethyl ester, was synthesized in four steps and 26% yield starting from L-m-tyrosine. [18F]FMT produced by two methods at two institutions was comparable in both radiochemical yield, 25-26%, and quality (chemical, enantiomeric, and radiochemical purity and specific activity) as that obtained with the original N-trifluoroacetyl-3-acetyl-6-trimethylstannyl-L-m-tyrosine ethyl ester [18F]FMT precursor.

In the last decade radiolabeled tropane analogs based on β-CIT have proven indispensable for the imaging of the dopamine transporter. However, further improvements in their pharmacodynamic and pharmacokinetic features are desirable. An important improvement, yielding in higher affinity to the dopamine transporter (DAT) vs. serotonin transporter (SERT), can be achieved by a simple replacement of the carboxylic methyl ester group in β-CIT by a fluoroethyl ester. The preparation and ex vivo evaluation of this new β-CIT-analog ([18F]FE@CIT) is presented here. Precursor and standard were prepared from β-CIT and analyzed by spectroscopic methods. Yields of precursor and standard preparation were 61% and 42%, respectively. [18F]FE@CIT was prepared by distillation of [18F]bromofluoroethane ([18F]BFE) and reaction with (1R-2-exo-3-exo)8-methyl-3-(4-iodo-phenyl)-8-azabicyclo[3.2.1] octane-2-carboxylic acid. After 10 min at 150°C the product was purified using a C-18 SepPak. The radiosynthesis evinced radiochemical yields of >90% (based on [18F]BFE), the specific radioactivity was >416 GBq/μmol. An average 30 μAh cyclotron irradiation yielded more than 2.5 GBq [18F]FE@CIT. For the ex vivo bioevaluation, 20 male Sprague-Dawley rats were sacrificed at 5, 15, 30, 60, and 120 min after injection. Organs were removed, weighed, and counted. For autoradiographic experiments, transverse brain slices of about 100 μm were prepared. The ex vivo evaluation showed highest brain uptake in striatal regions, followed by thalamus and cerebellum. The highest striatum to cerebellum ratio was 3.73 and the highest thalamus to cerebellum ratio was 1.65. Autoradiographic images showed a good and differentiated uptake in striatal regions with a good target-to-background ratio. Synapse 55:73–79, 2005. © 2004 Wiley-Liss, Inc.

1. Ziel Oxytocin ist ein zyklisches Nonapeptid, welches im Hypothalamus synthetisiert und in der Hypophyse gespeichert wird. Sein Rezeptor (OTR) ist in spezifischen Hirnarealen exprimiert und wird mit psychiatrischen Erkrankungen, wie Schizophrenie, Autismus und Depression in Zusammenhang gebracht. Ziel unserer Arbeiten ist die Entwicklung eines nicht-peptidischen, F-18-markierten Radiotracers zur Untersuchung der Expressionsdichte des OTR im gesunden bzw. erkrankten Gehirn. 2. Methodik Die Bindungsaffinitäten neuer Derivate zum humanen OTR wurden mittels Radioligand-Verdrängungsstudien bestimmt. Die Radiomarkierung von [18F]ABX163 erfolgte sowohl thermisch als auch mikrowellenunterstützt in einer Zwei-Schritt-Synthese ausgehend von einem MOM-geschützen Tosylatpräkursor. Metabolismus und Organverteilung wurden in Mäusen untersucht. Dynamische PET-Scans erfolgten in Mäusen und in einem Ferkel. 3. Ergebnisse Gegenüber der thermischen Reaktionsführung führte die mikrowellenunterstützte F-18-Markierung und Entschützung von [18F]ABX163 (Ki=13,3 nM ) zu höheren radiochemischen Ausbeuten in kürzerer Zeit (RCA: 25,4 ± 3,1% (n = 5); SA: 35-160 GBq/µmol). Sowohl die Organverteilung als auch PET-Scans von [18F]ABX163 in der Maus zeigten eine Anreicherung in der Hypophyse (SUV60=0,85), jedoch wurde eine geringe Hirnaufnahme (SUV60=0,04) beobachtet. Im Ferkel lagen die SUV-Werte für das Gesamthirn bei 0,43 (120 Min.), mit einer höheren Aufnahme im Bulbus olfactorius (SUV120=0,73; OTR-reiche Hirnregion). In-vitro-Autoradiographien am Rattenhirn zeigten eine Anreicherung von [18F]ABX163 in OTR-typischen Regionen, die jedoch nur partiell durch Oxytocin blockiert werden konnte. Selektivitätsstudien deuten auf eine Bindung von ABX163 an den ebenfalls im Gehirn exprimierten Vasopressin-Rezeptor V2 hin. 4. Schlussfolgerung Auf Grund der geringen Hirnaufnahme und der vermuteten unzureichenden Selektivität ist eine Weiterentwicklung dieses Radioliganden nicht vorgesehen.

Objectives: The peptide oxytocin is synthesized in the hypothalamus and acts as neurotransmitter to regulate a diverse range of CNS functions. Its receptor (OTR) is expressed in specific brain areas related to psychiatric diseases. So far, a non-invasive investigation of the OTR in brain is hampered by a lack of suitable radiotracers. To develope a PET ligand with high affinity toward OTR, we synthesized a series of fluorinated non-peptidic small molecules and performed radiofluorination of a selected candidate to investigate its in vivo properties in mice and pigs. Methods: Binding affinities of the compounds to the human OTR were determined by radioligand displacement studies. The radiosynthesis of [18F]ABX163 (KD = 12.3 nM) was performed via thermal and microwave heating (see scheme 1). Metabolism and organ distribution of the radiotracer were studied in female CD-1 mice. Dynamic PET scans were performed in mice (animal PET/MR; 60 min) and in one female piglet (PET; 120 min). Results: Using microwave heating for the synthesis of [18F]ABX163 provided higher RCY in shorter reaction time compared to thermal heating (RCY 25.4 ± 3.1% (n=5); SA 35-160 GBq/µmol; RP > 97%). Both organ distribution and dynamic PET imaging studies revealed limited uptake of the radiotracer in mouse brain (SUVmean = 0.04). Besides, significant uptake in the pituitary gland was observed (SUV55 min p.i. = 0.85), which indicates target-specific binding of [18F]ABX163. By a dynamic PET study in pig, a mean SUV of 0.43 was estimated for the whole brain at 120 min p.i. A two fold higher uptake was observed in the olfactory bulb (SUV120 min p.i. = 0.73), a region with high expression of OTR. In vitro autoradiography studies on rat and pig brain slices revealed an interaction of [18F]ABX163 with several off target receptors. Conclusion: Due to the low brain uptake and the insufficient selectivity, [18F]ABX163 is not suitable for imaging of OTR in living brain. Acknowledgment: The authors thank the SAB (Sächsische AufbauBank) for funding this project.