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.

Enhanced Survival of High-Risk Medulloblastoma-Bearing Mice after Multimodal Treatment with Radiotherapy, Decitabine, and Abacavir.

Children with high-risk SHH/TP53-mut and Group 3 medulloblastoma (MB) have a 5-year overall survival of only 40%. Innovative approaches to enhance survival while preventing adverse effects are urgently needed. We investigated an innovative therapy approach combining irradiation (RT), decitabine (DEC), and abacavir (ABC) in a patient-derived orthotopic SHH/TP53-mut and Group 3 MB mouse model. MB-bearing mice were treated with DEC, ABC and RT. Mouse survival, tumor growth (BLI, MRT) tumor histology (H/E), proliferation (Ki-67), and endothelial (CD31) staining were analyzed. Gene expression was examined by microarray and RT-PCR (Ki-67, VEGF, CD31, CD15, CD133, nestin, CD68, IBA). The RT/DEC/ABC therapy inhibited tumor growth and enhanced mouse survival. Ki-67 decreased in SHH/TP53-mut MBs after RT, DEC, RT/ABC, and RT/DEC/ABC therapy. CD31 was higher in SHH/TP53-mut compared to Group 3 MBs and decreased after RT/DEC/ABC. Microarray analyses showed a therapy-induced downregulation of cell cycle genes. By RT-PCR, no therapy-induced effect on stem cell fraction or immune cell invasion/activation could be shown. We showed for the first time that RT/DEC/ABC therapy improves survival of orthotopic SHH/TP53-mut and Group 3 MB-bearing mice without inducing adverse effects suggesting the potential for an adjuvant application of this multimodal therapy approach in the human clinic.

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.

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.

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.

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.

Synthesis and Biological Evaluation of a Novel 18F-Labeled Radiotracer for PET Imaging of the Adenosine A2A Receptor.

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.

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.

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.

Preclinical Evaluation of [18F]FACH in Healthy Mice and Piglets: An 18F-Labeled Ligand for Imaging of Monocarboxylate Transporters with PET.

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.

Development of a Radiofluorinated Adenosine A2B Receptor Antagonist as Potential Ligand for PET Imaging.

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.

Preclinical Incorporation Dosimetry of [18F]FACH-A Novel 18F-Labeled MCT1/MCT4 Lactate Transporter Inhibitor for Imaging Cancer Metabolism with PET.

Overexpression of monocarboxylate transporters (MCTs) has been shown for a variety of human cancers (e.g., colon, brain, breast, and kidney) and inhibition resulted in intracellular lactate accumulation, acidosis, and cell death. Thus, MCTs are promising targets to investigate tumor cancer metabolism with positron emission tomography (PET). Here, the organ doses (ODs) and the effective dose (ED) of the first 18F-labeled MCT1/MCT4 inhibitor were estimated in juvenile pigs. Whole-body dosimetry was performed in three piglets (age: ~6 weeks, weight: ~13-15 kg). The animals were anesthetized and subjected to sequential hybrid Positron Emission Tomography and Computed Tomography (PET/CT) up to 5 h after an intravenous (iv) injection of 156 ± 54 MBq [18F]FACH. All relevant organs were defined by volumes of interest. Exponential curves were fitted to the time-activity data. Time and mass scales were adapted to the human order of magnitude and the ODs calculated using the ICRP 89 adult male phantom with OLINDA 2.1. The ED was calculated using tissue weighting factors as published in Publication 103 of the International Commission of Radiation Protection (ICRP103). The highest organ dose was received by the urinary bladder (62.6 ± 28.9 µSv/MBq), followed by the gall bladder (50.4 ± 37.5 µSv/MBq) and the pancreas (30.5 ± 27.3 µSv/MBq). The highest contribution to the ED was by the urinary bladder (2.5 ± 1.1 µSv/MBq), followed by the red marrow (1.7 ± 0.3 µSv/MBq) and the stomach (1.3 ± 0.4 µSv/MBq). According to this preclinical analysis, the ED to humans is 12.4 µSv/MBq when applying the ICRP103 tissue weighting factors. Taking into account that preclinical dosimetry underestimates the dose to humans by up to 40%, the conversion factor applied for estimation of the ED to humans would rise to 20.6 µSv/MBq. In this case, the ED to humans upon an iv application of ~300 MBq [18F]FACH would be about 6.2 mSv. This risk assessment encourages the translation of [18F]FACH into clinical study phases and the further investigation of its potential as a clinical tool for cancer imaging with PET.

Sigma-1 Receptor Positron Emission Tomography: A New Molecular Imaging Approach Using (S)-(-)-[18F]Fluspidine in Glioblastoma.

Glioblastoma multiforme (GBM) is the most devastating primary brain tumour characterised by infiltrative growth and resistance to therapies. According to recent research, the sigma-1 receptor (sig1R), an endoplasmic reticulum chaperone protein, is involved in signaling pathways assumed to control the proliferation of cancer cells and thus could serve as candidate for molecular characterisation of GBM. To test this hypothesis, we used the clinically applied sig1R-ligand (S)-(-)-[18F]fluspidine in imaging studies in an orthotopic mouse model of GBM (U87-MG) as well as in human GBM tissue. A tumour-specific overexpression of sig1R in the U87-MG model was revealed in vitro by autoradiography. The binding parameters demonstrated target-selective binding according to identical KD values in the tumour area and the contralateral side, but a higher density of sig1R in the tumour. Different kinetic profiles were observed in both areas, with a slower washout in the tumour tissue compared to the contralateral side. The translational relevance of sig1R imaging in oncology is reflected by the autoradiographic detection of tumour-specific expression of sig1R in samples obtained from patients with glioblastoma. Thus, the herein presented data support further research on sig1R in neuro-oncology.

PET Imaging of the Adenosine A2A Receptor in the Rotenone-Based Mouse Model of Parkinson's Disease with [18F]FESCH Synthesized by a Simplified Two-Step One-Pot Radiolabeling Strategy.

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.

Development of Novel Analogs of the Monocarboxylate Transporter Ligand FACH and Biological Validation of One Potential Radiotracer for Positron Emission Tomography (PET) Imaging.

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.

Radiosynthesis and Biological Investigation of a Novel Fluorine-18 Labeled Benzoimidazotriazine- Based Radioligand for the Imaging of Phosphodiesterase 2A with Positron Emission Tomography.

A specific radioligand for the imaging of cyclic nucleotide phosphodiesterase 2A (PDE2A) via positron emission tomography (PET) would be helpful for research on the physiology and disease-related changes in the expression of this enzyme in the brain. In this report, the radiosynthesis of a novel PDE2A radioligand and the subsequent biological evaluation were described. Our prospective compound 1-(2-chloro-5-methoxy phenyl)-8-(2-fluoropyridin-4-yl)-3- methylbenzo[e]imidazo[5,1-c][1,2,4]triazine, benzoimidazotriazine (BIT1) (IC50 PDE2A = 3.33 nM; 16-fold selectivity over PDE10A) was fluorine-18 labeled via aromatic nucleophilic substitution of the corresponding nitro precursor using the K[18F]F-K2.2.2-carbonate complex system. The new radioligand [18F]BIT1 was obtained with a high radiochemical yield (54 ± 2%, n = 3), a high radiochemical purity (≥99%), and high molar activities (155-175 GBq/µmol, n = 3). In vitro autoradiography on pig brain cryosections exhibited a heterogeneous spatial distribution of [18F]BIT1 corresponding to the known pattern of expression of PDE2A. The investigation of in vivo metabolism of [18F]BIT1 in a mouse revealed sufficient metabolic stability. PET studies in mouse exhibited a moderate brain uptake of [18F]BIT1 with a maximum standardized uptake value of ~0.7 at 5 minutes p.i. However, in vivo blocking studies revealed a non-target specific binding of [18F]BIT1. Therefore, further structural modifications are needed to improve target selectivity.

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor.

On the basis of a pyrazine core structure, three new adenosine A2B receptor ligands (7a-c) were synthesized containing a 2-fluoropyridine moiety suitable for 18F-labeling. Compound 7a was docked into a homology model of the A2B receptor based on X-ray structures of the related A2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A1 receptor, 7a was radiolabeled as the most suitable candidate (Ki(A2B) = 4.24 nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [18F]7a with a standardized uptake value (SUV) of ≈1 at 5 min post injection followed by a fast wash out. Metabolism studies of [18F]7a in mice revealed the formation of a blood-brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.

Synthesis, Structure-Activity Relationships, Radiofluorination, and Biological Evaluation of [18F]RM365, a Novel Radioligand for Imaging the Human Cannabinoid Receptor Type 2 (CB2R) in the Brain with PET.

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.

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.