Stephan Wilke

First-in-human PET quantification study of cerebral α4β2* nicotinic acetylcholine receptors using the novel specific radioligand (−)-( 18 F)Flubatine☆

α4β2* nicotinic receptors (α4β2* nAChRs) could provide a biomarker in neuropsychiatric disorders (e.g., Alzheimers and Parkinsons 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 (-)-[(18)F]Flubatine, formerly known as (-)-[(18)F]NCFHEB, as a tool for α4β2* nAChR imaging and in vivo quantification. Dynamic PET emission recordings lasting 270min were acquired on an ECAT EXACT HR+ scanner in 12 healthy male non-smoking subjects (71.0±5.0years) following the intravenous injection of 353.7±9.4MBq of (-)-[(18)F]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 models goodness of fit, with and without metabolite correction was assessed by Akaikes information criterion. Model parameters of interest were the total distribution volume VT (mL/cm(3)), 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 90min, 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 90min for all regions investigated, and within 30min 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. Altogether, kinetics and imaging properties of (-)-[(18)F]Flubatine appear favorable and suggest that (-)-[(18)F]Flubatine is a very suitable and clinically applicable PET tracer for in vivo imaging of α4β2* nAChRs in neuropsychiatric disorders.

Fully automated radiosynthesis of both enantiomers of [18F]Flubatine under GMP conditions for human application

A fully automatized radiosynthesis of (+)- and (-)-[(18)F]Flubatine ((+)- and (-)NCFHEB) by means of a commercially available synthesis module (TRACERlab FX FN) under GMP conditions is reported. Radiochemical yields of 30% within an overall synthesis time of 40 min were achieved in more than 70 individual syntheses. Specific activities were approximately 3000 GBq/μmol and radiochemical purity was determined to be at least 97%.

Evaluation of metabolism, plasma protein binding and other biological parameters after administration of (−)-( 18 F)Flubatine in humans

INTRODUCTION (-)-[(18)F]Flubatine is a PET tracer with high affinity and selectivity for the nicotinic acetylcholine α4β2 receptor subtype. A clinical trial assessing the availability of this subtype of nAChRs was performed. From a total participant number of 21 Alzheimers 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.

Internal Dose Assessment of (–)-18F-Flubatine, Comparing Animal Model Datasets of Mice and Piglets with First-in-Human Results

(−)-18F-flubatine is a promising tracer for neuroimaging of nicotinic acetylcholine receptors (nAChRs), subtype α4β2, using PET. Radiation doses after intravenous administration of the tracer in mice and piglets were assessed to determine the organ doses (ODs) and the effective dose (ED) to humans. The results were compared with subsequent clinical investigations in human volunteers. Methods: Twenty-seven female CD1 mice (weight ± SD, 28.2 ± 2.1 g) received intravenous injection of 0.75 ± 0.33 MBq of (−)-18F-flubatine. Up to 240 min after injection, 3 animals per time point were sacrificed and the organs harvested, weighed, and counted in a γ counter to determine mass and activity, respectively. Furthermore, whole-body PET scans of 5 female piglets (age ± SD, 44 ± 3 d; weight ± SD, 13.7 ± 1.7 kg) and 3 humans (2 men and 1 woman; age ± SD, 59.6 ± 3.9 y; weight ± SD, 74.3 ± 3.1 kg) were obtained up to 236 min (piglets) and 355 min (humans) after injection of 186.6 ± 7.4 and 353.7 ± 10.2 MBq of (−)-18F-flubatine, respectively, using a PET/CT scanner. The CT was used for delineation of the organs. Exponential curves were fitted to the time–activity-data, and time and mass scales were adapted to the human anatomy. The ODs were calculated using OLINDA/EXM (version 1.0); EDs were calculated with the tissue-weighting factors of ICRP103. Results: After the injection of (−)-18F-flubatine, there were no adverse or clinically detectable pharmacologic effects in any of the subjects. The highest activities after injection were found in the kidneys, urinary bladder, and liver. The urinary bladder receives the highest OD in all investigated species, followed by the kidneys and the liver for animals and humans, respectively. On the basis of mouse, piglet, and human kinetic data, the projected human ED of (−)-18F-flubatine was estimated to be 12.5 μSv/MBq in mice, 14.7 ± 0.7 μSv/MBq in piglets, and 23.4 ± 0.4 μSv/MBq in humans. Conclusion: As has been demonstrated for other PET radiotracers, preclinical (i.e., animal-derived) dosimetry underestimates the ED to humans, in the current case of (−)-18F-flubatine by 34%–44%.

First in man study to image alpha4beta2 cerebral nicotinic acetylcholine receptors (nAChRs) in early Alzheimer's disease (AD) with the new radioligand (-)-[18F]-norchloro-fluoro-homoepibatidine (NCFHEB) and PET

Osama Sabri, StephanWilke, Susanne Graef, Ulrike Lengler, HermanJosef Gertz, Peter Sch€onknecht, Bernd Habermann, Georg Becker, Julia Luthardt, Marianne Patt, Kai Kendziorra, Philipp Meyer, Swen Hesse, Henryk Barthel, Gudrun Wagenknecht, Alexander Hoepping, Ulrich Hegerl, Peter Brust, University of Leipzig, Leipzig, Germany; Research Center Juelich, Juelich, Germany; Advanced Biochemical Compounds (ABX), Radeberg, Germany; Helmholtz-Zentrum Dresden-Rossendorf, Leipzig, Germany.

Cognitive correlates of α4β2 nicotinic acetylcholine receptors in mild Alzheimer's dementia

Whether α4β2 nicotinic acetylcholine receptor (α4β2-nAChR) expression is reduced in early Alzheimer’s disease is controversial. Using (-)-[18F]Flubatine PET, Sabri, Meyer et al. report α4β2-nAChR deficiency in mild Alzheimer’s dementia, especially within the basal forebrain-cortical and septohippocampal cholinergic projections. Reduced α4β2-nAChR availability correlates with impaired episodic memory and executive function/working memory.

PET Imaging of Cerebral Nicotinic Acetylcholine Receptors (nAChRs) in Early Alzheimer’s Disease (AD) Assessed with the New Radioligand (–)-[18F]Norchloro-Fluoro-Homoepibatidine ((–)-[18F]Flubatine)

Background: Alzheimer’s disease (AD) with early onset often presents with a distinct cognitive profile, potentially reflecting a different distribution of underlying neuropathology. The purpose of this study was to examine the relationships between age and both in vivo fibrillary amyloid deposition and glucose metabolism in AD. Methods: Dynamic [C]Pittsburgh compound-B (PIB) (90 minutes) and static [F]fluorodeoxyglucose (FDG) (15 minutes) scans were obtained in 100 AD patients and 20 healthy controls. Parametric nondisplaceable binding potential images of [C]PIB and standardized uptake value ratio images of [F]FDG were generated using cerebellar grey matter as reference tissue. Nine [C]PIB negative patients were excluded. The remaining patients were categorized into younger (n = 45, age: 56±4) and older (n = 46, age: 69±5) groups, based on the median age (62) at time-of-diagnosis. Results: Younger patients showed more severe impairment on visuo-spatial function, attention and executive function composite scores (p < 0.05), while we found a trend towards poorer memory performance for older patients (p = 0.11). Repeated measures ANOVA showed no main effect of age for [C]PIB or [F]FDG, suggesting that overall, the extent of amyloid deposition or glucose hypometabolism did not differ between groups. Regional distributions of [C]PIB and [F]FDG (both p for interaction < 0.05) differed between groups, however, largely due to increased [C]PIB binding and decreased [F]FDG uptake in the parietal cortex of younger patients (both p < 0.05). Linear regression analyses showed negative associations between visuo-spatial functioning and parietal [C]Pittsburgh compound-B binding for younger patients (standardized b: 0.37) and between visuo-spatial functioning and occipital binding for older patients (standardized b: 0.39). For [F]fluorodeoxyglucose, associations were found between parietal uptake with visuo-spatial (standardized b: 0.55), attention (standardized b: 0.39) and executive functioning (standardized b: 0.37) in younger patients, and between posterior cingulate uptake and memory in older patients (standardized b: 0.41, all p < 0.05). Conclusions: These in vivo findings suggest that clinical differences between younger and older AD patients are not restricted to topographical differ-