title: DPA-714, a new translocator protein-specific ligand: synthesis, radiofluorination, and pharmacologic characterization.
abstract: 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 TSPO-specific 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/mumol. 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 DPA-714, or DPA-713. 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.
  
  
  CONCLUSIONS
  (18)F-DPA-714 is a specific radioligand for the TSPO, displaying promising in vivo properties and thus warranting further investigation.
authors.affiliation: Department of Pharmacology, University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: James
authors.forename: Roger R
authors.initials: RR
authors.lastname: Fulton
authors.forename: Johnny
authors.initials: J
authors.lastname: Vercoullie
authors.forename: David J
authors.initials: DJ
authors.lastname: Henderson
authors.forename: Lucette
authors.initials: L
authors.lastname: Garreau
authors.forename: Sylvie
authors.initials: S
authors.lastname: Chalon
authors.forename: Frederic
authors.initials: F
authors.lastname: Dolle
authors.forename: Barbara
authors.initials: B
authors.lastname: Costa
authors.forename: Silvia
authors.initials: S
authors.lastname: Selleri
authors.forename: Denis
authors.initials: D
authors.lastname: Guilloteau
authors.forename: Michael
authors.initials: M
authors.lastname: Kassiou
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D005462:Fluorine Radioisotopes; D054879:Halogenation; D008024:Ligands; D048529:Papio hamadryas / Q000378:metabolism; D011284:Pregnenolone / Q000096:biosynthesis; D011720:Pyrazoles / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D011743:Pyrimidines / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D011854:Radiochemistry; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D013379:Substrate Specificity; D014018:Tissue Distribution
chemical_list: D002352:Carrier Proteins; D005462:Fluorine Radioisotopes; D008024:Ligands; C530129:N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo(1,5-a)pyrimidin-3-yl)acetamide; D011720:Pyrazoles; D011743:Pyrimidines; D011963:Receptors, GABA-A; C069997:Tspo protein, rat; D011284:Pregnenolone