The translocator protein (18 kDa) (TSPO), formerly known as the peripheral benzodiazepine receptor, is dramatically upregulated under pathologic conditions. Activated microglia are the main cell type expressing the TSPO at sites of central nervous system pathology. Radioligands for the TSPO can therefore measure active disease in the brain. This article details the synthesis, radiofluorination, and pharmacologic evaluation of a new TSPOspecific pyrazolopyrimidine, DPA-714. Methods: The affinity of DPA-714 for the TSPO was measured in rat kidney membranes with 3 H-PK11195. The in vitro functional activity of DPA-714 was measured in a steroidogenic assay in which the ability of DPA-714 to increase pregnenolone synthesis was measured with rat C6 glioma cells. The radiofluorination of DPA-714 was achieved by nucleophilic 18 F-fluoride displacement of the tosylate precursor. 18 F-DPA-714 was assessed in rats harboring unilateral quinolinic acid (QA) lesions. In addition, pretreatment experiments were performed with PK11195 (5 mg/kg), DPA-714 (1 mg/kg), and DPA-713 (1 mg/kg). The in vivo binding and biodistribution of 18 F-DPA-714 were determined in a baboon with PET. Experiments involving presaturation with PK11195 (1.5 mg/kg) and displacement with DPA-714 (1 mg/kg) were conducted to evaluate the specificity of radioligand binding. Results: In vitro binding studies revealed that DPA-714 displayed a high affinity for the TSPO (dissociation constant, 7.0 nM). DPA-714 stimulated pregnenolone synthesis at levels 80% above the baseline. 18 F-DPA-714 was prepared at a 16% radiochemical yield and a specific activity of 270 GBq/mmol. In rats harboring unilateral QA lesions, an 8-fold-higher level of uptake of 18 F-DPA-714 was observed in the ipsilateral striatum than in the contralateral striatum. Uptake in the ipsilateral striatum was shown to be selective because it was inhibited to the level in the contralateral striatum in the presence of PK11195, nonlabeled . PET studies demonstrated rapid penetration and good retention of 18 F-DPA-714 in the baboon brain. Pretreatment with PK11195 effectively inhibited the uptake of 18 F-DPA-714 in the whole brain, indicating its selective binding to the TSPO. The injection of nonlabeled DPA-714 20 min after the injection of 18 F-DPA-714 resulted in radioligand washout, demonstrating the reversibility of 18 F-DPA-714 binding. Conclusion: 18 F-DPA-714 is a specific radioligand for the TSPO, displaying promising in vivo properties and thus warranting further investigation.

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Two novel series of iodinated N-substituted analogs of 2beta-carbomethoxy-3beta-(4'-iodophenyl)tropane (beta-CIT) and N-(3-iodoprop-(2E)-enyl)-2beta-carbomethoxy-3beta-(3',4'-dis ubstituted phenyl)nortropane were synthesized. They were evaluated for their inhibitory properties on dopamine (DA(T)), serotonin (5-HT(T)), and norepinephrine (NE(T)) transporters in rat brain homogenates using [3H]GBR-12935, [3H]paroxetine, and [3H]nisoxetine as specific ligands. All new N-substituted analogs of beta-CIT exhibited higher DAT selectivity over both 5-HT(T) and NE(T) than beta-CIT. Moreover compounds with the N-substituents propynyl (6), crotyl (4), 2-bromoprop-(2E)-enyl (5), and 3-iodoprop-(2E)-enyl (3d) showed similar to higher DA(T) affinities than beta-CIT (respectively 14, 15, 30, and 30 nM vs 27 nM). Compound 3d was found to be the most selective DA(T) agent of this series (5-HTT/DA(T) = 32.0 vs 0.1 for beta-CIT). The N-(3-iodoprop-(2E)-enyl) chain linked to the tropane nitrogen was therefore maintained on the tropane structure, and phenyl substitution was carried out in order to improve DA(T) affinity. K(i) values of N-(3-iodoprop-(2E)-enyl)-2beta-carbomethoxy-3beta-(3',4'-dis ubstituted phenyl)nortropanes revealed that phenyl, 4'-isopropyl, and 4'-n-propyl derivatives weakly inhibited specific binding to DA(T), whereas phenyl substitution with 4'-methyl (3c), 3',4'-dichloro (3b), and 4'-iodo (3d) yielded high-DA(T) reuptake agents with increased DA(T) selectivity compared to beta-CIT. These results demonstrate that the combination of a nitrogen and a phenyl substitution yields compounds with high affinity and selectivity for the dopamine transporter which are usable as SPECT markers for DA neurons.

We studied concomitantly the level of neuroinflammation and β-amyloid (Aβ) load in the APPswePS1dE9 transgenic mouse model of Alzheimer's disease using positron emission tomography. The translocator protein 18 kDa (TSPO) tracer [(18)F]DPA-714 was used to measure neuroinflammation and [(18)F]AV-45 for Aβ load in mice at 6, 9, 12, 15, and 19 months of age. At 19 months, we also analyzed the neuroinflammatory and neuroanatomic status of mice brains. The main affected brain areas were the cortex and hippocampus, with a concomitant progression of neuroinflammation with increased amyloid burden. At 19 months, no increase in TSPO binding was observed in the cerebellum; immunostaining revealed W0-2-positive plaques, indicating that the amyloid deposits seemed not stimulate inflammation. This finding was in agreement with the observed level of microglia and astrocytes staining. Our findings provide a better understanding of the relationships between neuroinflammation and plaque accumulation in the course of the disease in this mouse model. The monitoring of both processes should be of value to validate potential therapeutic approaches.

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LBT‐999 (8‐((E)‐4‐fluoro‐but‐2‐enyl)‐3‐beta‐p‐tolyl‐8‐aza‐bicyclo[3.2.1]octane‐2‐beta‐carboxylicacid methyl ester) is a recently developed cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands (KD: 9 nM for the DAT and IC50 > 1000 nM for the serotonin and norepinephrine transporter). Initial fluorine‐18‐labelling of LBT‐999 was based on the robust and reliable two‐step radiochemical pathway often reported for such tropane derivatives, involving first the preparation of (E)‐1‐[18F]fluoro‐4‐tosyloxybut‐2‐ene followed by a N‐alkylation reaction with the appropriate nor‐tropane moiety. In the present work, a simple one‐step fluorine‐18‐labelling of LBT‐999 is reported, based on a chlorine‐for‐fluorine nucleophilic aliphatic substitution, facilitating as expected both automation and final high‐performance liquid chromatography (HPLC) purification. The process involves: (A) reaction of K[18F]F–Kryptofix®222 with the chlorinated precursor (3.5–4.5 mg) at 165°C for 10 min in DMSO (0.6 mL) followed by (B) C‐18 PrepSep cartridge pre‐purification and finally (C) semi‐preparative HPLC purification on a Waters Symmetry® C‐18. Typically, 3.70–5.92 GBq of [18F]LBT‐999 (> 95% chemically and radiochemically pure) could be obtained with specific radioactivities ranging from 37 to 111 GBq/µmol within 85–90 min (HPLC purification and Sep‐Pak‐based formulation included), starting from a 37.0 GBq [18F]fluoride batch (overall radiochemical yields: 10–16%, non‐decay‐corrected). Copyright © 2007 John Wiley & Sons, Ltd.

These results demonstrate that CLINDE is suitable for TSPO in vivo SPECT imaging to explore their involvement in neurodegenerative disorders associated with microglial activation.