The ghrelin receptor (GhrR) is a widely investigated target in several diseases. However, the current knowledge of its role and distribution in the brain is limited. Recently, the small and non-peptidic compound (S)-6-(4-bromo-2-fluorophenoxy)-3-((1-isopropylpiperidin-3-yl)methyl)-2-methylpyrido[3,2-d]pyrimidin-4(3H)-one ((S)-9) has been described as a GhrR ligand with high binding affinity. Here, we describe the synthesis of fluorinated derivatives, the in vitro evaluation of their potency as partial agonists and selectivity at GhrRs, and their physicochemical properties. These results identified compounds (S)-9, (R)-9, and (S)-16 as suitable parent molecules for 18F-labeled positron emission tomography (PET) radiotracers to enable future investigation of GhrR in the brain.

Monocarboxylate transporters 1 and 4 (MCT1 and MCT4) are involved in tumour development and progression. Their level of expression is particularly upregulated in glycolytic cancer cells and accordingly MCTs are considered as promising drug targets for treatment of a variety of human cancers. The non-invasive imaging of these transporters in cancer patients via positron emission tomography (PET) is regarded to be valuable for the monitoring of therapeutic effects of MCT inhibitors. Recently, we developed the first 18F-radiolabelled MCT1/MCT4 inhibitor [18F]FACH and reported on a two-step one-pot radiosynthesis procedure. We herein describe now a unique one-step radiosynthesis of this radiotracer which is based on the approach of using a methylsulfonate (mesylate) precursor bearing an unprotected carboxylic acid function. With the new procedure unexpected high radiochemical yields of 43 ± 8% at the end of the radiosynthesis could be obtained in a strongly reduced total synthesis time. Moreover, the radiosynthesis was successfully transferred to a TRACERlab FX2 N synthesis module ready for future preclinical applications of [18F]FACH.

The adenosine A2B receptor has been proposed as a novel therapeutic target in cancer, as its expression is drastically elevated in several tumors and cancer cells. Noninvasive molecular imaging via positron emission tomography (PET) would allow the in vivo quantification of this receptor in pathological processes and most likely enable the identification and clinical monitoring of respective cancer therapies. On the basis of a bicyclic pyridopyrimidine-2,4-dione core structure, the new adenosine A2B receptor ligand 9 was synthesized, containing a 2-fluoropyridine moiety suitable for labeling with the short-lived PET radionuclide fluorine-18. Compound 9 showed a high binding affinity for the human A2B receptor (Ki(A2B) = 2.51 nM), along with high selectivities versus the A1, A2A, and A3 receptor subtypes. Therefore, it was radiofluorinated via nucleophilic aromatic substitution of the corresponding nitro precursor using [18F]F-/K2.2.2./K2CO3 in DMSO at 120 °C. Metabolic studies of [18F]9 in mice revealed about 60% of radiotracer intact in plasma at 30 minutes p.i. A preliminary PET study in healthy mice showed an overall biodistribution of [18F]9, corresponding to the known ubiquitous but low expression of the A2B receptor. Consequently, [18F]9 represents a novel PET radiotracer with high affinity and selectivity toward the adenosine A2B receptor and a suitable in vivo profile. Subsequent studies are envisaged to investigate the applicability of [18F]9 to detect alterations in the receptor density in certain cancer-related disease models.

Monocarboxylate transporters 1-4 (MCT1-4) are involved in several metabolism-related diseases, especially cancer, providing the chance to be considered as relevant targets for diagnosis and therapy. [18F]FACH was recently developed and showed very promising preclinical results as a potential positron emission tomography (PET) radiotracer for imaging of MCTs. Given that [18F]FACH did not show high blood-brain barrier permeability, the current work is aimed to investigate whether more lipophilic analogs of FACH could improve brain uptake for imaging of gliomas, while retaining binding to MCTs. The 2-fluoropyridinyl-substituted analogs 1 and 2 were synthesized and their MCT1 inhibition was estimated by [14C]lactate uptake assay on rat brain endothelial-4 (RBE4) cells. While compounds 1 and 2 showed lower MCT1 inhibitory potencies than FACH (IC50 = 11 nM) by factors of 11 and 25, respectively, 1 (IC50 = 118 nM) could still be a suitable PET candidate. Therefore, 1 was selected for radiosynthesis of [18F]1 and subsequent biological evaluation for imaging of the MCT expression in mouse brain. Regarding lipophilicity, the experimental log D7.4 result for [18F]1 agrees pretty well with its predicted value. In vivo and in vitro studies revealed high uptake of the new radiotracer in kidney and other peripheral MCT-expressing organs together with significant reduction by using specific MCT1 inhibitor α-cyano-4-hydroxycinnamic acid. Despite a higher lipophilicity of [18F]1 compared to [18F]FACH, the in vivo brain uptake of [18F]1 was in a similar range, which is reflected by calculated BBB permeabilities as well through similar transport rates by MCTs on RBE4 cells. Further investigation is needed to clarify the MCT-mediated transport mechanism of these radiotracers in brain.

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.

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.

This study describes the synthesis, radiofluorination and purification of an anionic amphiphilic teroligomer developed as a stabilizer for siRNA-loaded calcium phosphate nanoparticles (CaP-NPs). As the stabilizing amphiphile accumulates on nanoparticle surfaces, the fluorine-18-labeled polymer should enable to track the distribution of the CaP-NPs in brain tumors by positron emission tomography after application by convection-enhanced delivery. At first, an unmodified teroligomer was synthesized with a number average molecular weight of 4550 ± 20 Da by free radical polymerization of a defined composition of methoxy-PEG-monomethacrylate, tetradecyl acrylate and maleic anhydride. Subsequent derivatization of anhydrides with azido-TEG-amine provided an azido-functionalized polymer precursor (o14PEGMA-N3) for radiofluorination. The 18F-labeling was accomplished through the copper-catalyzed cycloaddition of o14PEGMA-N3 with diethylene glycol-alkyne-substituted heteroaromatic prosthetic group [18F]2, which was synthesized with a radiochemical yield (RCY) of about 38% within 60 min using a radiosynthesis module. The 18F-labeled polymer [18F]fluoro-o14PEGMA was obtained after a short reaction time of 2-3 min by using CuSO4/sodium ascorbate at 90 °C. Purification was performed by solid-phase extraction on an anion-exchange cartridge followed by size-exclusion chromatography to obtain [18F]fluoro-o14PEGMA with a high radiochemical purity and an RCY of about 15%.

The adenosine A2A receptor (A2AR) has emerged as a potential non-dopaminergic target for the treatment of Parkinson's disease and, thus, the non-invasive imaging with positron emission tomography (PET) is of utmost importance to monitor the receptor expression and occupancy during an A2AR-tailored therapy. Aiming at the development of a PET radiotracer, we herein report the design of a series of novel fluorinated analogs (TOZ1-TOZ7) based on the structure of the A2AR antagonist tozadenant, and the preclinical evaluation of [18F]TOZ1. Autoradiography proved A2AR-specific in vitro binding of [18F]TOZ1 to striatum of mouse and pig brain. Investigations of the metabolic stability in mice revealed parent fractions of more than 76% and 92% of total activity in plasma and brain samples, respectively. Dynamic PET/magnetic resonance imaging (MRI) studies in mice revealed a brain uptake but no A2AR-specific in vivo binding.

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.

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.

The development of cannabinoid receptor type 2 (CB2R) PET radioligands has been intensively explored due to the pronounced CB2R upregulation under various pathological conditions. Herein, we report on the synthesis of a series of CB2R affine fluorinated indole-2-carboxamide ligands. Compound RM365 was selected for PET radiotracer development due to its high CB2R affinity (Ki = 2.1 nM) and selectivity over CB1R (factor > 300). Preliminary in vitro evaluation of [18F]RM365 indicated species differences in the binding to CB2R (KD of 2.32 nM for the hCB2R vs KD > 10,000 nM for the rCB2R). Metabolism studies in mice revealed a high in vivo stability of [18F]RM365. PET imaging in a rat model of local hCB2R(D80N) overexpression in the brain demonstrates the ability of [18F]RM365 to reach and selectively label the hCB2R(D80N) with a high signal-to-background ratio. Thus, [18F]RM365 is a very promising PET radioligand for the imaging of upregulated hCB2R expression under pathological conditions.

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.

The expression of monocarboxylate transporters (MCTs) is linked to pathophysiological changes in diseases, including cancer, such that MCTs could potentially serve as diagnostic markers or therapeutic targets. We recently developed [18F]FACH as a radiotracer for non-invasive molecular imaging of MCTs by positron emission tomography (PET). The aim of this study was to evaluate further the specificity, metabolic stability, and pharmacokinetics of [18F]FACH in healthy mice and piglets. We measured the [18F]FACH plasma protein binding fractions in mice and piglets and the specific binding in cryosections of murine kidney and lung. The biodistribution of [18F]FACH was evaluated by tissue sampling ex vivo and by dynamic PET/MRI in vivo, with and without pre-treatment by the MCT inhibitor α-CCA-Na or the reference compound, FACH-Na. Additionally, we performed compartmental modelling of the PET signal in kidney cortex and liver. Saturation binding studies in kidney cortex cryosections indicated a KD of 118 ± 12 nM and Bmax of 6.0 pmol/mg wet weight. The specificity of [18F]FACH uptake in the kidney cortex was confirmed in vivo by reductions in AUC0-60min after pre-treatment with α-CCA-Na in mice (-47%) and in piglets (-66%). [18F]FACH was metabolically stable in mouse, but polar radio-metabolites were present in plasma and tissues of piglets. The [18F]FACH binding potential (BPND) in the kidney cortex was approximately 1.3 in mice. The MCT1 specificity of [18F]FACH uptake was confirmed by displacement studies in 4T1 cells. [18F]FACH has suitable properties for the detection of the MCTs in kidney, and thus has potential as a molecular imaging tool for MCT-related pathologies, which should next be assessed in relevant disease models.

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.

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.

The adenosine A2A receptor (A2AR) is regarded as a particularly appropriate target for non-dopaminergic treatment of Parkinson's disease (PD). An increased A2AR availability has been found in the human striatum at early stages of PD and in patients with PD and dyskinesias. The aim of this small animal positron emission tomography/magnetic resonance (PET/MR) imaging study was to investigate whether rotenone-treated mice reflect the aspect of striatal A2AR upregulation in PD. For that purpose, we selected the known A2AR-specific radiotracer [18F]FESCH and developed a simplified two-step one-pot radiosynthesis. PET images showed a high uptake of [18F]FESCH in the mouse striatum. Concomitantly, metabolism studies with [18F]FESCH revealed the presence of a brain-penetrant radiometabolite. In rotenone-treated mice, a slightly higher striatal A2AR binding of [18F]FESCH was found. Nonetheless, the correlation between the increased A2AR levels within the proposed PD animal model remains to be further investigated.

The development of cannabinoid receptor type 2 (CB2R) radioligands for positron emission tomography (PET) imaging was intensively explored. To overcome the low metabolic stability and simultaneously increase the binding affinity of known CB2R radioligands, a carborane moiety was used as a bioisostere. Here we report the synthesis and characterization of carborane-based 1,8-naphthyridinones and thiazoles as novel CB2R ligands. All tested compounds showed low nanomolar CB2R affinity, with (Z)-N-[3-(4-fluorobutyl)-4,5-dimethylthiazole-2(3H)-ylidene]-(1,7-dicarba-closo-dodecaboranyl)-carboxamide (LUZ5) exhibiting the highest affinity (0.8 nM). Compound [18F]LUZ5-d8 was obtained with an automated radiosynthesizer in high radiochemical yield and purity. In vivo evaluation revealed the improved metabolic stability of [18F]LUZ5-d8 compared to that of [18F]JHU94620. PET experiments in rats revealed high uptake in spleen and low uptake in brain. Thus, the introduction of a carborane moiety is an appropriate tool for modifying literature-known CB2R ligands and gaining access to a new class of high-affinity CB2R ligands, while the in vivo pharmacology still needs to be addressed.