Radiosynthesis and biological evaluation of [18F]AG-120 for PET imaging of the mutant isocitrate dehydrogenase 1 in glioma.

Glioma are clinically challenging tumors due to their location and invasiveness nature, which often hinder complete surgical resection. The evaluation of the isocitrate dehydrogenase mutation status has become crucial for effective patient stratification. Through a transdisciplinary approach, we have developed an 18F-labeled ligand for non-invasive assessment of the IDH1R132H variant by using positron emission tomography (PET) imaging. In this study, we have successfully prepared diastereomerically pure [18F]AG-120 by copper-mediated radiofluorination of the stannyl precursor 6 on a TRACERlab FX2 N radiosynthesis module. In vitro internalization studies demonstrated significantly higher uptake of [18F]AG-120 in U251 human high-grade glioma cells with stable overexpression of mutant IDH1 (IDH1R132H) compared to their wild-type IDH1 counterpart (0.4 vs. 0.013% applied dose/µg protein at 120 min). In vivo studies conducted in mice, exhibited the excellent metabolic stability of [18F]AG-120, with parent fractions of 85% and 91% in plasma and brain at 30 min p.i., respectively. Dynamic PET studies with [18F]AG-120 in naïve mice and orthotopic glioma rat model reveal limited blood-brain barrier permeation along with a low uptake in the brain tumor. Interestingly, there was no significant difference in uptake between mutant IDH1R132H and wild-type IDH1 tumors (tumor-to-blood ratio[40-60 min]: ~1.7 vs. ~1.3). In conclusion, our preclinical evaluation demonstrated a target-specific internalization of [18F]AG-120 in vitro, a high metabolic stability in vivo in mice, and a slightly higher accumulation of activity in IDH1R132H-glioma compared to IDH1-glioma. Overall, our findings contribute to advancing the field of molecular imaging and encourage the evaluation of [18F]AG-120 to improve diagnosis and management of glioma and other IDH1R132H-related tumors.

Development of a Highly Specific 18F-Labeled Radioligand for Imaging of the Sigma-2 Receptor in Brain Tumors.

Novel ligands with the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline or 5,6-dimethoxyisoindoline pharmacophore were designed and synthesized for evaluation of their structure-activity relationship to the sigma-2 (σ2) receptor and developed as suitable PET radioligands. Compound 1 was found to possess nanomolar affinity (Ki(σ1) = 2.57 nM) for the σ2 receptor, high subtype selectivity (>2000-fold), and high selectivity over 40 other receptors and transporters. Radioligand [18F]1 was prepared with radiochemical yield of 37-54%, > 99% radiochemical purity, and molar activity of 107-189 GBq/µmol. Biodistribution and blocking studies in mice and micro-PET/CT imaging of [18F]1 in rats indicated excellent binding specificity to the σ2 receptors in vivo. Micro-PET/CT imaging of [18F]1 in the U87MG glioma xenograft model demonstrated clear tumor visualization with high tumor uptake and tumor-to-background ratio. Co-injection with CM398 (5 µmol/kg) led to a remarkable reduction of tumor uptake (80%, 60-70 min), indicating high specific binding of [18F]1 in U87MG glioma xenografts.

Specific and safe targeting of glioblastoma using switchable and logic-gated RevCAR T cells.

Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo.

Radiosynthesis and Preclinical Evaluation of an 18F-Labeled Triazolopyridopyrazine-Based Inhibitor for Neuroimaging of the Phosphodiesterase 2A (PDE2A).

The cyclic nucleotide phosphodiesterase 2A is an intracellular enzyme which hydrolyzes the secondary messengers cAMP and cGMP and therefore plays an important role in signaling cascades. A high expression in distinct brain areas as well as in cancer cells makes PDE2A an interesting therapeutic and diagnostic target for neurodegenerative and neuropsychiatric diseases as well as for cancer. Aiming at specific imaging of this enzyme in the brain with positron emission tomography (PET), a new triazolopyridopyrazine-based derivative (11) was identified as a potent PDE2A inhibitor (IC50, PDE2A = 1.99 nM; IC50, PDE10A ~2000 nM) and has been radiofluorinated for biological evaluation. In vitro autoradiographic studies revealed that [18F]11 binds with high affinity and excellent specificity towards PDE2A in the rat brain. For the PDE2A-rich region nucleus caudate and putamen an apparent KD value of 0.24 nM and an apparent Bmax value of 16 pmol/mg protein were estimated. In vivo PET-MR studies in rats showed a moderate brain uptake of [18F]11 with a highest standardized uptake value (SUV) of 0.97. However, no considerable enrichment in PDE2A-specific regions in comparison to a reference region was detectable (SUVcaudate putamen = 0.51 vs. SUVcerebellum = 0.40 at 15 min p.i.). Furthermore, metabolism studies revealed a considerable uptake of radiometabolites of [18F]11 in the brain (66% parent fraction at 30 min p.i.). Altogether, despite the low specificity and the blood−brain barrier crossing of radiometabolites observed in vivo, [18F]11 is a valuable imaging probe for the in vitro investigation of PDE2A in the brain and has potential as a lead compound for further development of a PDE2A-specific PET ligand for neuroimaging.

Novel Radioiodinated and Radiofluorinated Analogues of FT-2102 for SPECT or PET Imaging of mIDH1 Mutant Tumours.

Isocitrate dehydrogenases (IDHs) are metabolic enzymes commonly mutated in human cancers (glioma, acute myeloid leukaemia, chondrosarcoma, and intrahepatic cholangiocarcinoma). These mutated variants of IDH (mIDH) acquire a neomorphic activity, namely, conversion of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate involved in tumourigenesis. Thus, mIDHs have emerged as highly promising therapeutic targets, and several mIDH specific inhibitors have been developed. However, the evaluation of mIDH status, currently performed by biopsy, is essential for patient stratification and thus treatment and follow-up. We report herein the development of new radioiodinated and radiofluorinated analogues of olutasidenib (FT-2102) as tools for noninvasive single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging of mIDH1 up- and dysregulation in tumours. Nonradiolabelled derivatives 2 and 3 halogenated at position 6 of the quinolinone scaffold were synthesised and tested in vitro for their inhibitory potencies and selectivities in comparison with the lead compound FT-2102. Using a common organotin precursor, (S)-[125I]2 and (S)-[18F]3 were efficiently synthesised by radio-iododemetallation and copper-mediated radiofluorination, respectively. Both radiotracers were stable at room temperature in saline or DPBS solution and at 37 °C in mouse serum, allowing future planning of their in vitro and in vivo evaluations in glioma and chondrosarcoma models.

Structure-Based Design, Optimization, and Development of [18F]LU13: A Novel Radioligand for Cannabinoid Receptor Type 2 Imaging in the Brain with PET.

The cannabinoid receptor type 2 (CB2R) is an attractive target for the diagnosis and therapy of neurodegenerative diseases and cancer. In this study, we aimed at the development of a novel 18F-labeled radioligand starting from the structure of the known naphthyrid-2-one CB2R ligands. Compound 28 (LU13) was identified with the highest binding affinity and selectivity versus CB1R (CB2RKi = 0.6 nM; CB1RKi/CB2RKi > 1000) and was selected for radiolabeling with fluorine-18 and biological characterization. The new radioligand [18F]LU13 showed high CB2R affinity in vitro as well as high metabolic stability in vivo. PET imaging with [18F]LU13 in a rat model of vector-based/-related hCB2R overexpression in the striatum revealed a high signal-to-background ratio. Thus, [18F]LU13 is a novel and highly promising PET radioligand for the imaging of upregulated CB2R expression under pathological conditions in the brain.

Quantitation of the A2A Adenosine Receptor Density in the Striatum of Mice and Pigs with [18F]FLUDA by Positron Emission Tomography.

The cerebral expression of the A2A adenosine receptor (A2AAR) is altered in neurodegenerative diseases such as Parkinson's (PD) and Huntington's (HD) diseases, making these receptors an attractive diagnostic and therapeutic target. We aimed to further investigate the pharmacokinetic properties in the brain of our recently developed A2AAR-specific antagonist radiotracer [18F]FLUDA. For this purpose, we retrospectively analysed dynamic PET studies of healthy mice and rotenone-treated mice, and conducted dynamic PET studies with healthy pigs. We performed analysis of mouse brain time-activity curves to calculate the mean residence time (MRT) by non-compartmental analysis, and the binding potential (BPND) of [18F]FLUDA using the simplified reference tissue model (SRTM). For the pig studies, we performed a Logan graphical analysis to calculate the radiotracer distribution volume (VT) at baseline and under blocking conditions with tozadenant. The MRT of [18F]FLUDA in the striatum of mice was decreased by 30% after treatment with the A2AAR antagonist istradefylline. Mouse results showed the highest BPND (3.9 to 5.9) in the striatum. SRTM analysis showed a 20% lower A2AAR availability in the rotenone-treated mice compared to the control-aged group. Tozadenant treatment significantly decreased the VT (14.6 vs. 8.5 mL · g-1) and BPND values (1.3 vs. 0.3) in pig striatum. This study confirms the target specificity and a high BPND of [18F]FLUDA in the striatum. We conclude that [18F]FLUDA is a suitable tool for the non-invasive quantitation of altered A2AAR expression in neurodegenerative diseases such as PD and HD, by PET.

Development and Biological Evaluation of the First Highly Potent and Specific Benzamide-Based Radiotracer [18F]BA3 for Imaging of Histone Deacetylases 1 and 2 in Brain.

The degree of acetylation of lysine residues on histones influences the accessibility of DNA and, furthermore, the gene expression. Histone deacetylases (HDACs) are overexpressed in various tumour diseases, resulting in the interest in HDAC inhibitors for cancer therapy. The aim of this work is the development of a novel 18F-labelled HDAC1/2-specific inhibitor with a benzamide-based zinc-binding group to visualize these enzymes in brain tumours by positron emission tomography (PET). BA3, exhibiting high inhibitory potency for HDAC1 (IC50 = 4.8 nM) and HDAC2 (IC50 = 39.9 nM), and specificity towards HDAC3 and HDAC6 (specificity ratios >230 and >2080, respectively), was selected for radiofluorination. The two-step one-pot radiosynthesis of [18F]BA3 was performed in a TRACERlab FX2 N radiosynthesizer by a nucleophilic aliphatic substitution reaction. The automated radiosynthesis of [18F]BA3 resulted in a radiochemical yield of 1%, a radiochemical purity of >96% and a molar activity between 21 and 51 GBq/µmol (n = 5, EOS). For the characterization of BA3, in vitro and in vivo experiments were carried out. The results of these pharmacological and pharmacokinetic studies indicate a suitable inhibitory potency of BA3, whereas the applicability for non-invasive imaging of HDAC1/2 by PET requires further optimization of the properties of this compound.

Amphiphilic Anionic Oligomer-Stabilized Calcium Phosphate Nanoparticles with Prospects in siRNA Delivery via Convection-Enhanced Delivery.

Convection-enhanced delivery (CED) has been introduced as a concept in cancer treatment to generate high local concentrations of anticancer therapeutics and overcome the limited diffusional distribution, e.g., in the brain. RNA interference provides interesting therapeutic options to fight cancer cells but requires nanoparticulate (NP) carriers with a size below 100 nm as well as a low zeta potential for CED application. In this study, we investigated calcium phosphate NPs (CaP-NPs) as siRNA carriers for CED application. Since CaP-NPs tend to aggregate, we introduced a new terpolymer (o14PEGMA(1:1:2.5) NH3) for stabilization of CaP-NPs intended for delivery of siRNA to brain cancer cells. This small terpolymer provides PEG chains for steric stabilization, and a fat alcohol to improve interfacial activity, as well as maleic anhydrides that allow for both labeling and high affinity to Ca(II) in the hydrolyzed state. In a systematic approach, we varied the Ca/P ratio as well as the terpolymer concentration and successfully stabilized NPs with the desired properties. Labeling of the terpolymer with the fluorescent dye Cy5 revealed the terpolymer's high affinity to CaP. Importantly, we also determined a high efficiency of siRNA binding to the NPs that caused very effective survivin siRNA silencing in F98 rat brain cancer cells. Cytotoxicity investigations with a standard cell line resulted in minor and transient effects; no adverse effects were observed in organotypic brain slice cultures. However, more specific cytotoxicity investigations are required. This study provides a systematic and mechanistic analysis characterizing the effects of the first oligomer of a new class of stabilizers for siRNA-loaded CaP-NPs.

Non-Invasive Assessment of Locally Overexpressed Human Adenosine 2A Receptors in the Heart of Transgenic Mice.

A2A adenosine receptors (A2A-AR) have a cardio-protective function upon ischemia and reperfusion, but on the other hand, their stimulation could lead to arrhythmias. Our aim was to investigate the potential use of the PET radiotracer [18F]FLUDA to non-invasively determine the A2A-AR availability for diagnosis of the A2AR status. Therefore, we compared mice with cardiomyocyte-specific overexpression of the human A2A-AR (A2A-AR TG) with the respective wild type (WT). We determined: (1) the functional impact of the selective A2AR ligand FLUDA on the contractile function of atrial mouse samples, (2) the binding parameters (Bmax and KD) of [18F]FLUDA on mouse and human atrial tissue samples by autoradiographic studies, and (3) investigated the in vivo uptake of the radiotracer by dynamic PET imaging in A2A-AR TG and WT. After A2A-AR stimulation by the A2A-AR agonist CGS 21680 in isolated atrial preparations, antagonistic effects of FLUDA were found in A2A-AR-TG animals but not in WT. Radiolabelled [18F]FLUDA exhibited a KD of 5.9 ± 1.6 nM and a Bmax of 455 ± 78 fmol/mg protein in cardiac samples of A2A-AR TG, whereas in WT, as well as in human atrial preparations, only low specific binding was found. Dynamic PET studies revealed a significantly higher initial uptake of [18F]FLUDA into the myocardium of A2A-AR TG compared to WT. The hA2A-AR-specific binding of [18F]FLUDA in vivo was verified by pre-administration of the highly affine A2AAR-specific antagonist istradefylline. Conclusion: [18F]FLUDA is a promising PET probe for the non-invasive assessment of the A2A-AR as a marker for pathologies linked to an increased A2A-AR density in the heart, as shown in patients with heart failure.

Imaging of Gαq Proteins in Mouse and Human Organs and Tissues.

G protein-coupled receptors (GPCRs) transfer extracellular signals across cell membranes by activating intracellular heterotrimeric G proteins. Several studies suggested G proteins as novel drug targets for the treatment of complex diseases, e.g., asthma and cancer. Recently, we developed specific radiotracers, [³H]PSB-15900-FR and [³H]PSB-16254-YM, for the Gαq family of G proteins by tritiation of the macrocyclic natural products FR900359 (FR) and YM-254890 (YM). In the present study, we utilized these potent radioligands to perform autoradiography studies in tissues of healthy mice, mouse models of disease, and human tissues. Specific binding was high, while non-specific binding was extraordinarily low, giving nearly identical results for both radioligands. High expression levels of Gαq proteins were detected in healthy mouse organs showing the following rank order of potency: kidney > liver > brain > pancreas > lung > spleen, while expression in the heart was low. Organ sub-structures, e.g., of mouse brain and lung, were clearly distinguishable. Whereas an acute asthma model in mice did not result in altered Gαq protein expressions as compared to control animals, a cutaneous melanoma model displayed significantly increased expression in comparison to healthy skin. These results suggest the future development of Gαq-protein-binding radio-tracers as novel diagnostics.

Development of [18F]LU14 for PET Imaging of Cannabinoid Receptor Type 2 in the Brain.

Cannabinoid receptors type 2 (CB2R) represent an attractive therapeutic target for neurodegenerative diseases and cancer. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor receptor density and/or occupancy during a CB2R-tailored therapy, we herein describe the radiosynthesis of cis-[18F]1-(4-fluorobutyl-N-((1s,4s)-4-methylcyclohexyl)-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamide ([18F]LU14) starting from the corresponding mesylate precursor. The first biological evaluation revealed that [18F]LU14 is a highly affine CB2R radioligand with >80% intact tracer in the brain at 30 min p.i. Its further evaluation by PET in a well-established rat model of CB2R overexpression demonstrated its ability to selectively image the CB2R in the brain and its potential as a tracer to further investigate disease-related changes in CB2R expression.

Development of fluorinated and methoxylated benzothiazole derivatives as highly potent and selective cannabinoid CB2 receptor ligands.

The upregulation of the CB2 receptors in neuroinflammation and cancer and their potential visualization with PET (positron emission tomography) could provide a valuable diagnostic and therapy-monitoring tool in such disorders. However, the availability of reliable CB2-selective imaging probes is still lacking in clinical practice. We have recently identified a benzothiazole-2-ylidine amide hit (6a) as a highly potent CB2 ligand. With the aim of enhancing its CB2 over CB1 selectivity and introducing structural sites suitable for radiolabeling, we herein describe the development of fluorinated and methoxylated benzothiazole derivatives endowed with extremely high CB2 binding affinity and an exclusive selectivity to the CB2 receptor. Compounds 14, 15, 18, 19, 21, 24 and 25 displayed subnanomolar CB2Ki values (ranging from 0.16 nM to 0.68 nM) and interestingly, all of the synthesized compounds completely lacked affinity at the CB1 receptor (Ki > 10,000 nM for all compounds), indicating their remarkably high CB2 over CB1 selectivity factors. The fluorinated analogs, 15 and 21, were evaluated for their in vitro metabolic stability in mouse and human liver microsomes (MLM and HLM). Both 15 and 21 displayed an exceptionally high stability (98% and 91% intact compounds, respectively) after 60 min incubation with MLM. Contrastingly, a 5- and 2.8-fold lower stability was demonstrated for compounds 15 and 21, respectively, upon incubation with HLM for 60 min. Taken together, our data present extremely potent and selective CB2 ligands as credible leads that can be further exploited for 18F- or 11C-radiolabeling and utilization as PET tracers.

In vitro and in vivo evaluation of fluorinated indanone derivatives as potential positron emission tomography agents for the imaging of monoamine oxidase B in the brain.

Monoamine oxidases (MAOs) play a key role in the metabolism of major monoamine neurotransmitters. In particular, the upregulation of MAO-B in Parkinson's disease, Alzheimer's disease and cancer augmented the development of selective MAO-B inhibitors for diagnostic and therapeutic purposes, such as the anti-parkinsonian MAO-B irreversible binder l-deprenyl (Selegiline®). Herein we report on the synthesis of novel fluorinated indanone derivatives for PET imaging of MAO-B in the brain. Out of our series, the derivatives 6, 8, 9 and 13 are amongst the most affine and selective ligands for MAO-B reported so far. For the derivative 6-((3-fluorobenzyl)oxy)-2,3-dihydro-1H-inden-1-one (6) exhibiting an outstanding affinity (KiMAO-B = 6 nM), an automated copper-mediated radiofluorination starting from the pinacol boronic ester 17 is described. An in vitro screening in different species revealed a MAO-B region-specific accumulation of [18F]6 in rats and piglets in comparison to L-[3H]deprenyl. The pre-clinical in vivo assessment of [18F]6 in mice demonstrated the potential of indanones to readily cross the blood-brain barrier. Nonetheless, parallel in vivo metabolism studies indicated the presence of blood-brain barrier metabolites, thus arguing for further structural modifications. With the matching analytical profiles of the radiometabolite analysis from the in vitro liver microsome studies and the in vivo evaluation, the structure's elucidation of the blood-brain barrier penetrant radiometabolites is possible and will serve as basis for the development of new indanone derivatives suitable for the PET imaging of MAO-B.

Synthesis of Novel Fluorinated Xanthine Derivatives with High Adenosine A2B Receptor Binding Affinity.

The G protein-coupled adenosine A2B receptor is suggested to be involved in various pathological processes accompanied by increased levels of adenosine as found in inflammation, hypoxia, and cancer. Therefore, the adenosine A2B receptor is currently in focus as a novel target for cancer therapy as well as for noninvasive molecular imaging via positron emission tomography (PET). Aiming at the development of a radiotracer labeled with the PET radionuclide fluorine-18 for imaging the adenosine A2B receptor in brain tumors, one of the most potent and selective antagonists, the xanthine derivative PSB-603, was selected as a lead compound. As initial biodistribution studies in mice revealed a negligible brain uptake of [3H]PSB-603 (SUV3min: 0.2), structural modifications were performed to optimize the physicochemical properties regarding blood-brain barrier penetration. Two novel fluorinated derivatives bearing a 2-fluoropyridine (5) moiety and a 4-fluoro-piperidine (6) moiety were synthesized, and their affinity towards the four adenosine receptor subtypes was determined in competition binding assays. Both compounds showed high affinity towards the adenosine A2B receptor (Ki (5) = 9.97 ± 0.86 nM; Ki (6) = 12.3 ± 3.6 nM) with moderate selectivity versus the other adenosine receptor subtypes.

Design, Radiosynthesis and Preliminary Biological Evaluation in Mice of a Brain-Penetrant 18F-Labelled σ2 Receptor Ligand.

The σ2 receptor (transmembrane protein 97), which is involved in cholesterol homeostasis, is of high relevance for neoplastic processes. The upregulated expression of σ2 receptors in cancer cells and tissue in combination with the antiproliferative potency of σ2 receptor ligands motivates the research in the field of σ2 receptors for the diagnosis and therapy of different types of cancer. Starting from the well described 2-(4-(1H-indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline class of compounds, we synthesized a novel series of fluorinated derivatives bearing the F-atom at the aromatic indole/azaindole subunit. RM273 (2-[4-(6-fluoro-1H-pyrrolo[2,3-b]pyridin-1-yl)butyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline) was selected for labelling with 18F and evaluation regarding detection of σ2 receptors in the brain by positron emission tomography. Initial metabolism and biodistribution studies of [18F]RM273 in healthy mice revealed promising penetration of the radioligand into the brain. Preliminary in vitro autoradiography on brain cryosections of an orthotopic rat glioblastoma model proved the potential of the radioligand to detect the upregulation of σ2 receptors in glioblastoma cells compared to healthy brain tissue. The results indicate that the herein developed σ2 receptor ligand [18F]RM273 has potential to assess by non-invasive molecular imaging the correlation between the availability of σ2 receptors and properties of brain tumors such as tumor proliferation or resistance towards particular therapies.

Development of 18F-Labeled Radiotracers for PET Imaging of the Adenosine A2A Receptor: Synthesis, Radiolabeling and Preliminary Biological Evaluation.

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

Improved in vivo PET imaging of the adenosine A2A receptor in the brain using [18F]FLUDA, a deuterated radiotracer with high metabolic stability.

PURPOSE: The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A 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 A2A 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 A2A 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 A2A receptor in the brain with high target specificity. It is regarded ready for human application.