Purpose: The adenosine A_2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A_2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A_2A receptor radiotracer and report herein the preclinical evaluation of [¹⁸F]FLUDA, a deuterated isotopologue of [¹⁸F]FESCH. Methods: [¹⁸F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. Results: [¹⁸F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72–180 GBq/μmol. Autoradiography proved A_2A receptor–specific accumulation of [¹⁸F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [¹⁸F]FLUDA compared to that of [¹⁸F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [¹⁸F]FLUDA towards striatal A_2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [¹⁸F]FLUDA. Conclusions: The new radiotracer [¹⁸F]FLUDA is suitable to detect the availability of the A_2A receptor in the brain with high target specificity. It is regarded ready for human application.

Background: The level of expression of cannabinoid receptor type 2 (CB₂R) in healthy and diseased brain has not been fully elucidated. Therefore, there is a growing interest to assess the regional expression of CB₂R in the brain. Positron emission tomography (PET) is an imaging technique, which allows quantitative monitoring of very low amounts of radiolabelled compounds in living organisms at high temporal and spatial resolution and, thus, has been widely used as a diagnostic tool in nuclear medicine. Here, we report on the radiofluorination ofN-aryl-oxadiazolyl-propionamides at two different positions in the lead structure and on the biological evaluation of the potential of the two tracers [¹⁸F]1and [¹⁸F]2as CB₂receptor PET imaging agents.Results: High binding affinity and specificity towards CB₂receptors of the lead structure remained unaffected by the structural changes such as the insertion of the aliphatic and aromatic fluorine in the selected labelling sites of1and2. Aliphatic and aromatic radiofluorinations were optimized, and [¹⁸F]1and [¹⁸F]2were achieved in radiochemical yields of ≥30% with radiochemical purities of ≥98% and specific activities of 250 to 450 GBq/μmol. Organ distribution studies in female CD1 mice revealed that both radiotracers cross the blood–brain barrier (BBB) but undergo strong peripheral metabolism. At 30 min after injection, unmetabolized [¹⁸F]1and [¹⁸F]2accounted for 60% and 2% as well as 68% and 88% of the total activity in the plasma and brain, respectively. The main radiometabolite of [¹⁸F]2could be identified as the free acid [¹⁸F]10, which has no affinity towards the CB₁and CB₂receptors but can cross the BBB.Conclusions: N-aryl-oxadiazolyl-propionamides can successfully be radiolabelled with¹⁸F at different positions. Fluorine substitution at these positions did not affect affinity and specificity towards CB₂R. Despite a promisingin vitrobehavior, a rather rapid peripheral metabolism of [¹⁸F]1and [¹⁸F]2in mice and the generation of brain permeable radiometabolites hamper the application of these radiotracersin vivo. However, it is expected that future synthetic modification aiming at a replacement of metabolically susceptible structural elements of [¹⁸F]1and [¹⁸F]2will help to elucidate the potential of this class of compounds for CB₂R PET studies.

Purposes: We present the first in-human brain PET imaging data of the new α4β2 nicotinic acetylcholine receptor (nAChR)–targeting radioligand (+)-[¹⁸F]Flubatine. Aims were to develop a kinetic modeling-based approach to quantify (+)-[¹⁸F]Flubatine and compare the data of healthy controls (HCs) and patients with Alzheimer’s disease (AD); to investigate the partial volume effect (PVE) on regional (+)-[¹⁸F]Flubatine binding; and whether (+)-[¹⁸F]Flubatine binding and cognitive test data respective β-amyloid radiotracer accumulation were correlated. Methods: We examined 11 HCs and 9 mild AD patients. All subjects underwent neuropsychological testing and [¹¹C]PiB PET/MRI examination. (+)-[¹⁸F]Flubatine PET data were evaluated using full kinetic modeling and regional as well as voxel-based analyses. Results: With 270-min p.i., the unchanged parent compound amounted to 97 ± 2%. Adequate fits of the time-activity curves were obtained with the 1 tissue compartment model (1TCM). (+)-[¹⁸F]Flubatine distribution volume (binding) was significantly reduced in bilateral mesial temporal cortex in AD patients compared with HCs (right 10.6 ± 1.1 vs 11.6 ± 1.4, p = 0.049; left 11.0 ± 1.1 vs 12.2 ± 1.8, p = 0.046; one-sided t tests each). PVE correction increased not only (+)-[¹⁸F]Flubatine binding of approximately 15% but also standard deviation of 0.4–70%. Cognitive test data and (+)-[¹⁸F]Flubatine binding were significantly correlated in the left anterior cingulate, right posterior cingulate, and right parietal cortex (r > 0.5, p < 0.05 each). In AD patients, (+)-[¹⁸F]Flubatine binding and [¹¹C]PiB standardized uptake value ratios were negatively correlated in several regions; whereas in HCs, a positive correlation between cortical (+)-[¹⁸F]Flubatine binding and [¹¹C]PiB accumulation in the white matter was found. No adverse event related to (+)-[¹⁸F]Flubatine occurred. Conclusion: (+)-[¹⁸F]Flubatine is a safe and stable PET ligand. Full kinetic modeling can be realized by 1TCM without metabolite correction. (+)-[¹⁸F]Flubatine binding affinity was high enough to detect group differences. Of interest, correlation between white matter β-amyloid PET uptake and (+)-[¹⁸F]Flubatine binding indicated an association between white matter integrity and availability of α4β2 nAChRs. Overall, (+)-[¹⁸F]Flubatine showed favorable characteristics and has therefore the potential to serve as α4β2 nAChR–targeting PET ligand in further clinical trials.

Phosphodiesterases (PDEs) are enzymes that play a major role in cell signalling by hydrolysing the secondary messengers cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP) throughout the body and brain. Altered cyclic nucleotide-mediated signalling has been associated with a wide array of disorders, including neurodegenerative disorders. Recently, PDE5 has been shown to be involved in neurodegenerative disorders such as Alzheimer’s disease, but its precise role has not been elucidated yet. To visualize and quantify the expression of this enzyme in brain, we developed a radiotracer for specific PET imaging of PDE5. A quinoline-based lead compound has been structurally modified resulting in the fluoroethoxymethyl derivative ICF24027 with high inhibitory activity towards PDE5 (IC₅₀ = 1.86 nM). Radiolabelling with fluorine-18 was performed by a one-step nucleophilic substitution reaction using a tosylate precursor (RCY_(EOB) = 12.9% ± 1.8%; RCP > 99%; SA_(EOS) = 70–126 GBq/μmol). In vitro autoradiographic studies of [¹⁸F]ICF24027 on different mouse tissue as well as on porcine brain slices demonstrated a moderate specific binding to PDE5. In vivo studies in mice revealed that [¹⁸F]ICF24027 was metabolized under formation of brain penetrable radiometabolites making the radiotracer unsuitable for PET imaging of PDE5 in brain.

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.

(S)-[¹⁸F]fluspidine ((S)-[¹⁸F]1) has recently been explored for positron emission tomography (PET) imaging of sigma-1 receptors in humans. In the current report, we have used plasma samples of healthy volunteers to investigate the radiometabolites of (S)-[¹⁸F]1 and elucidate their structures with LC-MS/MS. For the latter purpose additional in vitro studies were conducted by incubation of (S)-[¹⁸F]1 and (S)-1 with human liver microsomes (HLM). In vitro metabolites were characterized by interpretation of MS/MS fragmentation patterns from collision-induced dissociation or by use of reference compounds. Thereby, structures of corresponding radio-HPLC-detected radiometabolites, both in vitro and in vivo (human), could be identified. By incubation with HLM, mainly debenzylation and hydroxylation occurred, beside further mono- and di-oxygenations. The product hydroxylated at the fluoroethyl side chain was glucuronidated. Plasma samples (10, 20, 30 min p.i., n = 5-6), obtained from human subjects receiving 250–300 MBq (S)-[¹⁸F]1 showed 97.2, 95.4, and 91.0% of unchanged radioligand, respectively. In urine samples (90 min p.i.) the fraction of unchanged radioligand was only 2.6% and three major radiometabolites were detected. The one with the highest percentage, also found in plasma, matched the glucuronide formed in vitro. Only a small amount of debenzylated metabolite was detected. In conclusion, our metabolic study, in particular the high fractions of unchanged radioligand in plasma, confirms the suitability of (S)-[¹⁸F]1 as PET radioligand for sigma-1 receptor imaging.