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Upregulation of the cannabinoid type 2 receptors (CB2R) unveils inflammation processes of pathological disorders, such as cancer, pain, or neurodegenerative diseases. Among others, CB2R agonist A-836339 has been labeled with carbon-11 for PET imaging of the CB2R and displayed promising results in a mouse model of Alzheimer's disease. The aim of the present work was to develop fluorinated analogs of A-836339 for labeling with fluorine-18 to design a new PET tracer for CB2R imaging. Seven fluorinated analogs of A-836339 were synthesized in two to three steps and their binding affinities and selectivities for both the human and the mouse CB2R were measured as well as their early ADME profiles. Among them, compound 2f (KihCB2R = 0.1 nM, KihCB1R/KihCB2R = 300) displayed high affinity and selectivity for CB2R but also promising lipophilicity, kinetic solubility, and membrane permeation properties and was further selected for in vitro metabolism studies. Incubation of 2f with human or rat liver microsomes followed by LC/MS analysis revealed the presence of six different metabolites mainly resulting from oxidation reactions. A tosylated precursor of 2f was synthesized in two steps and radiolabeled with fluorine-18 to afford [18F]2f in 15 ± 5% radiochemical yield and a molar activity of 110 ± 30 GBq/μmol. Autoradiographies of rat spleen and biodistribution studies in healthy rats including pretreatments with either CB2R or CB1R-specific compounds suggested that [18F]2f is a specific tracer for the CB2R in vivo. We have therefore demonstrated here that [18F]2f is a promising novel tracer for imaging CB2R in vivo using PET. Further investigation in animal models of inflammation will follow.

The translocator protein 18kDa (TSPO) is today a validated target for a number of therapeutic applications, but also a well-recognized diagnostic/imaging biomarker for the evaluation of inflammatory related-disease state and progression, prompting the development of specific and dedicated TSPO ligands worldwide. For this purpose, pyrazolo[1,5-a]pyrimidine acetamides constitute a unique class of high affinity and selectivity TSPO ligands; it includes DPA-714, a fluorine-containing derivative that has also been labelled with the positron-emitter fluorine-18, and is nowadays widely used as a Positron Emission Tomography imaging probe. Recently, to prevent defluorination issues encountered invivo with this tracer, a first series of analogues was reported where the oxygen atom bridging the phenyl ring of the core structure and the fluorinated moiety was replaced with a more robust linkage. Among this new series, CfO-DPA-714 was discovered as a highly promising TSPO ligand. Herein, a novel series of fluorinated analogues of the latter molecule were synthesized and invitro characterized, where the pharmacomodulation at the amide position of the molecule was explored. Thirteen compounds were thus prepared from a common key-ester intermediate (synthesized in 7 steps from 4-iodobenzoate - 11% overall yield) and a set of commercially available amines and obtained with moderate to good yields (23-81%) and high purities (>95%). With one exception, all derivatives displayed nanomolar to subnanomolar affinity for the TSPO and also high selectivity versus the CBR (Ki (CBR)/Ki (TSPO)>103). Within this series, three compounds showed better Ki values (0.25, 0.26 and 0.30nM) than that of DPA-714 (0.91nM) and CfO-DPA-714 (0.37nM), and favorable lipophilicity for brain penetration (3.67.4&nbsp

Parkinson’s disease (PD) is a slowly progressing neurodegenerative disorder caused by loss of dopaminergic neurons in the substantia nigra (SN), leading to severe impairment in motor and non-motor functions. Endogenous subventricular zone (SVZ) neural stem cells constantly give birth to new cells which might serve as a possible source for regeneration in the adult brain. However, neurodegeneration is accompanied by neuroinflammation and dopamine depletion, potentially compromising regeneration. We therefore employed in vivo imaging methods to study striatal deafferentation ([123I]Ioflupane SPECT, DaTscanTM) and neuroinflammation in SN and striatum ([18F]DPA-714 PET) in the intranigral 6-hydroxydopamine (6-OHDA) PD mouse model. Additionally, we transduced cells in the SVZ with a lentivirus encoding firefly luciferase and followed migration of progenitor cells in the SVZ – olfactory bulb (OB) axis via bioluminescence imaging (BLI) under disease and control conditions. We found that activation of microglia in the SN is an acute process coming along with the degeneration of dopaminergic cell bodies in the SN. Dopaminergic deafferentation of the striatum does not influence the generation of Dcx+ neuroblasts in the SVZ, but generates chronic astrogliosis in the nigrostriatal system.

The effects of metoclopramide on the central nervous system (CNS) in patients suggest substantial brain distribution. Previous data suggest that metoclopramide brain kinetics may nonetheless be controlled by ATP-binding cassette (ABC) transporters expressed at the blood–brain barrier. We used 11C-metoclopramide PET imaging to elucidate the kinetic impact of transporter function on metoclopramide exposure to the brain. Methods:11C-metoclopramide transport by P-glycoprotein (P-gp; ABCB1) and the breast cancer resistance protein (BCRP; ABCG2) was tested using uptake assays in cells overexpressing P-gp and BCRP. 11C-metoclopramide brain kinetics were compared using PET in rats (n = 4–5) in the absence and presence of a pharmacologic dose of metoclopramide (3 mg/kg), with or without P-gp inhibition using intravenous tariquidar (8 mg/kg). The 11C-metoclopramide brain distribution (VT based on Logan plot analysis) and brain kinetics (2-tissue-compartment model) were characterized with either a measured or an imaged-derived input function. Plasma and brain radiometabolites were studied using radio–high-performance liquid chromatography analysis. Results:11C-metoclopramide transport was selective for P-gp over BCRP. Pharmacologic dose did not affect baseline 11C-metoclopramide brain kinetics (VT = 2.28 ± 0.32 and 2.04 ± 0.19 mL⋅cm−3 using microdose and pharmacologic dose, respectively). Tariquidar significantly enhanced microdose 11C-metoclopramide VT (7.80 ± 1.43 mL⋅cm−3) with a 4.4-fold increase in K1 (influx rate constant) and a 2.3-fold increase in binding potential (k3/k4) in the 2-tissue-compartment model. In the pharmacologic situation, P-gp inhibition significantly increased metoclopramide brain distribution (VT = 6.28 ± 0.48 mL⋅cm−3) with a 2.0-fold increase in K1 and a 2.2-fold decrease in k2 (efflux rate), with no significant impact on binding potential. In this situation, only parent 11C-metoclopramide could be detected in the brains of P-gp–inhibited rats. Conclusion:11C-metoclopramide benefits from favorable pharmacokinetic properties that offer reliable quantification of P-gp function at the blood–brain barrier in a pharmacologic situation. Using metoclopramide as a model of CNS drug, we demonstrated that P-gp function not only reduces influx but also mediates the efflux from the brain back to the blood compartment, with additional impact on brain distribution. This PET-based strategy of P-gp function investigation may provide new insight on the contribution of P-gp to the variability of response to CNS drugs between patients.

International audience; Translocator protein 18 kDa (TSPO) expression at the mitochondrial membrane of glial cells is related to glial activation. TSPO radioligands such as [$^{18}$F]DPA‐714 are useful for the non‐invasive study of neuroimmune processes using positron emission tomography (PET). Anesthetic agents were shown to impact mitochondrial function and may influence [$^{18}$F]DPA‐714 binding parameters and PET kinetics. [$^{18}$F]DPA‐714 PET imaging was performed in $Papio\ anubis$ baboons anesthetized using either intravenous propofol ($n$ = 3) or inhaled isoflurane ($n$ = 3). Brain kinetics and metabolite‐corrected input function were measured to estimate [$^{18}$F]DPA‐714 brain distribution ($V_T$). Displacement experiments were performed using PK11195 (1.5 mg/kg). In vitro [$^{18}$F]DPA‐714 binding experiments were performed using baboon brain tissue in the absence and presence of tested anesthetics. Brain radioactivity peaked higher in isoflurane‐anesthetized animals compared with propofol (SUVmax = 2.7 ± 0.5 vs. 1.3 ± 0.2, respectively) but was not different after 30 min. Brain $V_T$ was not different under propofol and isoflurane. Displacement resulted in a 35.8 ± 8.4% decrease of brain radioactivity under propofol but not under isoflurane (0.1 ± 7.0%). In vitro, the presence of propofol increased TSPO density and dramatically reduced its affinity for [$^{18}$F]DPA‐714 compared with control. This in vitro effect was not significant with isoflurane. Exposure to propofol and isoflurane differentially influences TSPO interaction with its specific radioligand [$^{18}F$]DPA‐714 with subsequent impact on its tissue kinetics and specific binding estimated in vivo using PET. Therefore, the choice of anesthetics and their potential influence on PET data should be considered for the design of imaging studies using TSPO radioligands, especially in a translational research context.

DPA-714 (N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide) is a recentlydiscovered fluorinated ligand of the translocator protein 18 kDa (TSPO). Labelled with the short-lived positron emitterfluorine-18, this structure is today the radioligand of reference for in vivo imaging of microglia activation andneuroinflammatory processes with positron emission tomography. In the present work, an isotopically tritium-labelledversion was developed ([3H]DPA-714), in order to access high resolution in vitro and ex vivo microscopic autoradiographystudies, repeated and long-lasting receptor binding studies and in vivo pharmacokinetic determination at late time points.Briefly, DPA-714 as reference, and its 3,5-dibrominated derivative as precursor for labelling, were both prepared from DPA-713 in nonoptimized 32% (two steps) and 10% (three steps) yields, respectively. Reductive debromination using deuteriumgas and Pd/C as catalyst in methanol, performed at the micromolar scale, confirmed the regioselective introduction of twodeuterium atoms at the meta positions of the phenyl ring. Tritiodebromination was analogously performed using no-carriertritium gas. HPLC purification provided >96% radiochemically pure [3H]DPA-714 (7 GBq) with a 2.1 TBq/mmol specificradioactivity. Interestingly, additional hydrogen-for-tritium exchanges were also observed at the 5-methyl and 7-methylpositions of the pyrazolo[1,5-a]pyrimidine, opening novel perspectives in the labelling of compounds featuring thisheterocyclic core.

Purpose: The purpose of the study was to validate [18F]DPA-714, a translocator protein (TSPO)18 kDa radioligand, as a probe to non-invasively quantify the inflammatory state in inflammatorybowel disease (IBD) animal models. Procedures: Quantitative positron emission tomography (PET) imaging of intestinal inflammationwas conducted with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) a glucose metabolismsurrogate marker and [18F]DPA-714 a ligand of the 18 kDa TSPO, on two IBD models. The firstmodel was induced using dextran sodium sulfate (DSS), creating global inflammation in thecolon. The second model was induced by rectally administering trinitrobenzenesulfonic acid(TNBS), creating local and acute inflammation. Results: The level of inflammation was analyzed using PET imaging on days 7 and 8. The analysisobtained with [18F]DPA-714, yielded a significant difference between the DSS treated (0.50±0.17%ID/cc) and non-treated rats (0.35±0.15%ID/cc). [18F]FDG on the other hand did not yield asignificant difference.We did observe a mean glucose consumption in the colon increase from 0.40±0.11%ID/cc to 0.54±0.17%ID/cc. In the TNBSmodel, the uptake level of [18F]DPA-714 increasedsignificantly from 0.46±0.23%ID/cc for the non-treated group, to 1.30±0.62%ID/cc for those treated.PET signal was correlated with increased TSPO expression at cellular level. Conclusions: Results indicate that [18F]DPA-714 is suitable for studying inflammation in IBDmodels. [18F]DPA-714 could be a good molecular probe to non-invasively evaluate the level andlocalization of inflammation. Moreover, in vivo imaging using this TSPO ligand is potentially apowerful tool to stage and certainly to follow the evolution and therapeutic efficiency atmolecular level within this disease family.

DPA-C5yne, the lead compound of a novel series of DPA-714 derivatives in which the fluoroethoxy chain linked to thephenylpyrazolopyrimidine scaffold has been replaced by a fluoroalkyn-1-yl moiety, is a high affinity (Ki: 0.35 nM) and selectiveligand targeting the translocator protein 18 kDa. In the present work, DPA-C5yne was labelled with no-carrier-added [18F]fluoride based on a one-step tosyloxy-for-fluorine nucleophilic substitution reaction, purified by cartridge and HPLC, andformulated as an i.v. injectable solution using a TRACERLab FX N Pro synthesizer. Typically, 4.3–5.2GBq of [18F]DPA-C5yne,ready-to-use, chemically and radiochemically pure (> 95%), was obtained with specific radioactivities ranging from 55 to110GBq/μmol within 50–60 min, starting from a 30GBq [18F]fluoride batch (14–17%). LogP and LogD of [18F]DPA-C5yne weremeasured using the shake-flask method and values of 2.39 and 2.51 were found, respectively. Autoradiography studiesperformed on slices of ((R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique (AMPA)-lesioned rat brains showed a hightarget-to-background ratio (1.9 ± 0.3). Selectivity and specificity of the binding for the translocator protein was demonstratedusing DPA-C5yne (unlabelled), PK11195 and Flumazenil (central benzodiazepine receptor ligand) as competitors. Furthermore,DPA-C5yne proved to be stable in plasma at 37°C for at least 90min.

The TSPO (translocator protein), also known as the peripheral benzodiazepine receptor, is upregulated in the brain of subjects suffering from neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's disease. Moreover, this overexpression has been proved to be linked to microglia activation making thus the TSPO a marker of choice of neuroinflammatory processes and therefore a potential target for the development of radioligands for positron emission tomography imaging. The discovery of selective TSPO ligands and their labelling with the short-lived positron-emitter isotopes carbon-11 and fluorine-18 emerged in the mid-1980s with the preparation of the 3-isoquinolinecarboxamide [(11) C]PK11195. To date, an impressive number of promising compounds-[(11) C]PK11195-challengers-have been developed; some radioligands-for example, [(11) C]PBR28, [(11) C]DPA-713, [(18) F]FEDAA1106 and [(18) F]DPA-714-are currently used in clinical trials. As illustrated in this review, the methodologies applied for the preparation of these compounds remain mainly [(11) C]methylations using [(11) C]MeI or [(11) C]MeOTf and SN 2-type nucleophilic aliphatic [(18) F]fluorinations-two processes illustrating the state-of-the-art arsenal of reactions that involves these two short-lived radioisotopes-but alternative processes, such as [(11) C]carbonylations using [(11) C]CO and [(11) C]COCl2 as well as SN Ar-type nucleophilic [(18) F]fluorinations, have also been reported and as such, reviewed herein.

International audience; The development of dyskinesias following chronic L-DOPA replacement therapy remains a major problem in the long-term treatment of Parkinson's disease. This study aimed at evaluating the effect of IRC-082451 (base of BN82451), a novel multitargeting hybrid molecule, on L-DOPA-induced dyskinesias (LIDs) and hypolocomotor activity in a non-human primate model of PD. IRC-082451 displays multiple properties: it inhibits neuronal excitotoxicity (sodium channel blocker), oxidative stress (antioxidant) and neuroinflammation (cyclooxygenase inhibitor) and is endowed with mitochondrial protective properties. Animals received daily MPTP injections until stably parkinsonian. A daily treatment with increasing doses of L-DOPA was administered to parkinsonian primates until the appearance of dyskinesias. Then, different treatment regimens and doses of IRC-082451 were tested and compared to the benchmark molecule amantadine. Primates were regularly filmed and videos were analyzed with specialized software. A novel approach combining the analysis of dyskinesias and locomotor activity was used to determine efficacy. This analysis yielded the quantification of the total distance travelled and the incidence of dyskinesias in 7 different body parts. A dose-dependent efficacy of IRC-082451 against dyskinesias was observed. The 5 mg/kg dose was best at attenuating the severity of fully established LIDs. Its effect was significantly different from that of amantadine since it increased spontaneous locomotor activity while reducing LIDs. This dose was effective both acutely and in a 5-day sub-chronic treatment. Moreover, positron emission tomography scans using radiolabelled dopamine demonstrated that there was no direct interference between treatment with IRC-082451 and dopamine metabolism in the brain. Finally, post-mortem analysis indicated that this reduction in dyskinesias was associated with changes in cFOS, FosB and ARC mRNA expression levels in the putamen. The data demonstrates the antidyskinetic efficacy of IRC-082451 in a primate model of PD with motor complications and opens the way to the clinical application of this treatment for the management of LIDs.