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The cerebral cholinergic system is involved in several cognitive processes and neuropsychiatric 2 diseases. For research purposes and later on in routine clinical settings new PET radioligands with more favorable characteristics than the established 3-pyridylether derivatives with their slow kinetics are necessary. Here we present the first in-human brain PET imaging data of the new α4β2 nicotinic acetylcholine receptor (nAChR)-targeting radioligand (+)-[18F]Flubatine. Primary aim of this study was to develop a kinetic modeling-based approach to quantify the α4β2 nAChR availability in the human brain and to compare respective data of healthy controls (HCs) with those of patients with Alzheimer’s disease (AD). Secondary aims were to investigate whether (+)-[18F]Flubatine binding was correlated to cognitive test data or β-amyloid radiotracer accumulation. Furthermore, the partial volume effect (PVE) on regional (+)-[18F]Flubatine binding was investigated. We examined 11 non-smoking HCs and 9 non-smoking patients with mild AD without anti-dementive drugs. Prior to (+)-[18F]Flubatine PET, all subjects underwent an extensive neuropsychological testing and a β-amyloid [11C]PiB PET/MRI examination. To evaluate the (+)-[18F]Flubatine PET data, we used full kinetic modeling (one and two tissue compartment 16 modeling (1TCM and 2TCM)) and regional as well as voxel-based analyses. 270 min p.i., the unchanged parent compound in arterial blood amounted to 97±2%. As revealed by regional analysis, (+)-[18F]Flubatine distribution volume (binding) was significantly reduced in the bilateral mesial temporal cortex in AD patients compared to HCs (right: AD: 10.6±1.1 vs HC: 11.6±1.4, p=0.049; left: AD: 11.0±1.1 vs HCs:12.2±1.8, p=0.046). Voxel-based analysis detected further clusters of reduced (+)-[18F]Flubatine in left precuneus/posterior cingulate cortex, right superior temporal and left middle temporal cortex (k>30, p0.5, p

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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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Introduction (+)-[18F]Flubatine ((+)[18F]NCFHEB), the enantiomer of the recently introduced radioligand for quantificati(-)-[18F]Flubatine, was investigated in a clinical trial with patients suffering from Alzheimer’s disease compared to healthy controls. In order to be able to apply full kinetic modelling biological parameters such as plasma protein binding, amount of parent compound over time and distribution between cellular and noncellular blood components, were determined. In addition the kinetics of the tracer distribution between plasma and whole blood was assessed. Methods Plasma protein binding was evaluated in vitro by means of ultracentrifugation using a blood sample from each subject prior to injection. The amount of unchanged tracer over time was determined at 11 time points ranging from 3 to 270 min p.i. by radio-HPLC analysis of protein free plasma obtained by centrifugation as described previously. The distribution of radioactivity between cellular and non-cellular blood components was determined at 11 time points p.i. after separation of the blood components by centrifugation. Results/Discussion Plasma protein binding of (-)-[18F]Flubatine was found to be 0.140.02 (meansd) without significant difference between AD and HC groups. Metabolic degradation of (+)-[18F]Flubatine was very low: the amount of parent compound was found to be 100 and 972 % at 90 and 270 min p.i., respectively. The activity distribution between plasma and whole blood was found to be 0.820.05 and did not change with time. Kinetics for the distribution of the tracer between plasma and whole blood was determined over a time period of 1.5 h and equilibrium was found to be reached instantaneously. Conclusions Biological parameters such as plasma protein binding, metabolism and tracer/activity distribution between plasma and whole blood were investigated within a clinical trial using (+)-[1842 subtype of nAChRs. From the biological data obtained within this study we, conclude that (+)-[18F]Flubatine is a very suitable radiotracer for the determination of the 42 nAChRs by kinetic modelling since plasma protein binding is moderate and the equilibrium between whole blood and plasma is reached instantaneously. Furthermore, metabolic degradation of the radiotracer is negligible.

Objectives: Nicotinic α4β2* acetylcholine receptors (nAChR) are an important target for diagnostic neuroimaging because of their involvement e.g. in Alzheimer's and Parkinson's diseases, and nicotine addiction. The development of new PET-tracers for these receptors is an active field of research. Here we present the PET quantification of the new nAChR-ligand (+)-[18F]Flubatine, an enantiomer of (-)-[18F]Flubatine, also known as (-)-[18F]NCFHEB, which is already used for clinical PET imaging of nAChRs. Methods: After intravenous administration of 284.1±13.2 MBq (+)-[18F]Flubatine PET brain recordings were performed in 11 healthy non-smoking subjects (age 66.6±4.4 years) using an ECAT EXACT HR+ system in 3D-acquisition mode. 41 frames were acquired from 0-270 min post injection and motion corrected with SPM2. Kinetic modeling using 1- and 2-tissue compartment models (1TCM, 2TCM) with arterial input-function was applied to the volume of interest (VOI) based tissue time-activity curves (TACs) generated for 36 brain regions (anatomically defined via MRI co-registration). Time ranges from 0 to 90 and 0 to 270 min were investigated. Model-based receptor parameter was the total distribution volume VT (ml/cm-3). Metabolites in plasma were measured by radioactivity HPLC and the free fraction of (+)-[18F]Flubatine in plasma was determined by ultracentrifugation. Results: The tracer showed high stability in vivo with more than 97% remaining as untransformed parent compound at 90 and 270 min. The free fraction in plasma was high and showed only small interindividual variations (0.86±0.02, n=11). Given the negligible amounts of metabolites present in plasma the arterial input function was not corrected for metabolites. TACs of all 36 regions could be described with the 1- or 2TCM. VT in all cortical regions could be reliably estimated from 90 min PET data. VT increased with receptor density as expected. Using the 2TCM and 270 min PET-data: CC (VT: 7.9±1.5, n=11), frontal cortex (10.8±1.2), pons (16.3±3.1), thalamus (41.3±8.7). For 90 min PET data the distribution volumes were very similar: CC (VT: 7.9±1.3), frontal cortex (10.3±1.4), pons (15.4±3.0), thalamus (41.0±9.0). The distribution volumes computed with the 1TCM were comparable to the results of the 2TCM: Frontal cortex (10.9±1.3, 270 min) and (10.2±1.4, 90 min). The previously investigated enantiomer (-)-[18F]Flubatine showed very similar VT in cortical structures e.g., frontal (10.4±1.3, n=12) but lower VT in pons (12.4±1.9) and especially thalamus (27.6±4.2) (2TCM, 90 min; Sabri et al., 2015). Conclusions: (+)-[18F]Flubatine has an optimal metabolic profile. The amount of metabolites is very low and no metabolite correction has to be applied to the arterial input function. The high VT values in subcortical structures are favorable. Receptor parameters can be estimated with a 1- or 2TCM from 90 min PET-data. If a model derived receptor parameter is used in a classification problem, e.g., distinguishing patients with Alzheimer’s disease from healthy controls the bias-variance tradeoff problem associated with the simpler 1TCM (higher bias) and the more complex 2TCM (higher variance) has to be solved. The final decision which model should be used will be made on the basis of the PET-data of both groups. Reference: Sabri O et al. (2015) Neuroimage 118:199-208 Figure Legend: 2TCM fits (270 min) in 8 brain regions. Abscissa: Time in min, ordinate: Activity (kBq/cm3). Computed total tracer amount in tissue is presented but also the tracer amount in the non-displaceable and specific tissue compartment, the total distribution volume V and influx rate constant K1.

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.

α4β2* nicotinic receptors (α4β2* nAChRs) could provide a biomarker in neuropsychiatric disorders (e.g., Alzheimer's and Parkinson's diseases, depressive disorders, and nicotine addiction). However, there is a lack of α4β2* nAChR specific PET radioligands with kinetics fast enough to enable quantification of nAChR within a reasonable time frame. Following on from promising preclinical results, the aim of the present study was to evaluate for the first time in humans the novel PET radioligand (−)-[18F]Flubatine, formerly known as (−)-[18F]NCFHEB, as a tool for α4β2* nAChR imaging and in vivo quantification. Dynamic PET emission recordings lasting 270 min were acquired on an ECAT EXACT HR+ scanner in 12 healthy male non-smoking subjects (71.0 ± 5.0 years) following the intravenous injection of 353.7 ± 9.4 MBq of (−)-[18F]Flubatine. Individual magnetic resonance imaging (MRI) was performed for co-registration. PET frames were motion-corrected, before the kinetics in 29 brain regions were characterized using 1- and 2-tissue compartment models (1TCM, 2TCM). Given the low amounts of metabolite present in plasma, we tested arterial input functions with and without metabolite corrections. In addition, pixel-based graphical analysis (Logan plot) was used. The model's goodness of fit, with and without metabolite correction was assessed by Akaike's information criterion. Model parameters of interest were the total distribution volume VT (mL/cm3), and the binding potential BPND relative to the corpus callosum, which served as a reference region. The tracer proved to have high stability in vivo,with 90% of the plasma radioactivity remaining as untransformed parent compound at 90 min, fast brain kinetics with rapid uptake and equilibration between free and receptor bound tracer. Adequate fits of brain TACs were obtained with the 1TCM. VT could be reliably estimated within 90 min for all regions investigated, and within 30 min for low-binding regions such as the cerebral cortex. The rank order of VT by region corresponded well with the known distribution of α4β2* receptors (VT [thalamus] 27.4±3.8, VT [putamen] 12.7±0.9, VT [frontal cortex] 10.0±0.8, and VT [corpus callosum] 6.3±0.8). The BPND, which is a parameter of α4β2* nAChR availability, was 3.41±0.79 for the thalamus, 1.04±0.25 for the putamen and 0.61 ± 0.23 for the frontal cortex, indicating high specific tracer binding. Use of the arterial input function without metabolite correction resulted in a 10% underestimation in VT, and was without important biasing effects on BPND.

Ziel (+)[18F]Flubatine ist ein neuer vielversprechender Radiotracer zur Bildgebung nikotinischer Acetylcholin-Rezeptoren (α4β2) mit PET. Zur Abschätzung des Strahlenrisikos am Menschen wurde die Biodistribution erhoben und inkorporationsdosimetrische Messungen an 3 gesunden Probanden vorgenommen. Die Organdosis (OD), sowie die Effektive Dosis (ED) wurden berechnet und mit dem (-)-Enantiomer des Liganden verglichen. Methodik 3 gesunde Probanden (Alter 58,3 ± 5,8 a, Gewicht: 80,7 ± 5,5 kg) wurden nach i.v. Injektion von 28 6 ± 13MBq (+)[18F]Flubatine sequentiell bis zu 7 h an einem PET/CT (SIEMENS Biograph16) gemessen. Das Untersuchungsprotokoll umfasste 9 Bettpositionen (BP), 1,5 – 6 min/BP, CT-Schwächungskorrektur, und iterative Rekonstruktion (OSEM, 4 Iterationen, 8 Subsets). In den Scanpausen wurde der Urin gesammelt, in einem Gammacounter gemessen und in der Dosisberechnung berücksichtigt. Mit Hilfe von ROVER (ABX, Radeberg, Germany) wurden 13 den Tracer anreichernde Organe identifiziert und per VOI-Analyse die entsprechenden Aktivitäten/Organ ermittelt. Die Zeit-Aktivitäts Daten wurden durch exponentielle Kurven approximiert, die Anzahl der Zerfälle/Organ berechnet und die OD mit OLINDA (V.1.0) abgeschätzt. Die ED wurde unter Hinzuziehung der Gewebewichtungsfaktoren aus ICRP 103 ermittelt. Ergebnisse Die Harnblase erhält die höchste OD (µSv/MBq) mit 102,4, gefolgt von der Leber (53,1), Nieren (38,1), Colon transversum (32,4), Dünndarm (29,4) und Gehirn (28,6). Den größten Beitrag zur ED (µSv/MBq) leistet die Harnblase (4,1), gefolgt von den Lungen (3,24), Magen (2,4), Leber (2,1), Colon transversum (1,9) und rotem Knochenmark (1,8). Resultierend ergibt sich die ED am Menschen nach i.v. Injektion von (+)[18F]Flubatine zu 23,0 µSv/MBq. Schlussfolgerung Die ED, als Maß für das Gesamtstrahlenrisiko, nach Injektion von 300 MBq (+)[18F]Flubatine ergibt sich zu 6,9 mSv. Sie liegt in der Größenordnung von anderen 18F-markierten Tracern (z.B. FDG = 5,7 mSv) und ist mit der ED des (-) Enantiomers (6,7 mSv) nahezu identisch.