No 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 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.

Indole and indazole synthetic cannabinoids (SCs) featuring l-valinate or l-tert-leucinate pendant group have recently emerged as prevalent recreational drugs, and their use has been associated with serious adverse health effects. Due to the limited pharmacological data available for these compounds, 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and their analogues were synthesized and assessed for cannabimimetic activity in vitro and in vivo. All SCs acted as potent, highly efficacious agonists at CB1 (EC50 = 0.45-36 nM) and CB2 (EC50 = 4.6-128 nM) receptors in a fluorometric assay of membrane potential, with a general preference for CB1 activation. The cannabimimetic properties of two prevalent compounds with confirmed toxicity in humans, 5F-AMB and MDMB-FUBINACA, were demonstrated in vivo using biotelemetry in rats. Bradycardia and hypothermia were induced by 5F-AMB and MDMB-FUBINACA doses of 0.1-1 mg/kg (and 3 mg/kg for 5F-AMB), with MDMB-FUBINACA showing the most dramatic hypothermic response recorded in our laboratory for any SC (>3 °C at 0.3 mg/kg). Reversal of hypothermia by pretreatment with a CB1, but not CB2, antagonist was demonstrated for 5F-AMB and MDMB-FUBINACA, consistent with CB1-mediated effects in vivo. The in vitro and in vivo data indicate that these SCs act as highly efficacious CB receptor agonists with greater potency than Δ(9)-THC and earlier generations of SCs.

Focal cerebral ischemia leads to an inflammatory reaction involving an overexpression of the peripheral benzodiazepine receptor (PBR)/18-kDa translocator protein (TSPO) in the cerebral monocytic lineage (microglia and monocyte) and in astrocytes. Imaging of PBR/TSPO by positron emission tomography (PET) using radiolabeled ligands can document inflammatory processes induced by cerebral ischemia. We performed in vivo PET imaging with [(18)F]DPA-714 to determine the time course of PBR/TSPO expression over several days after induction of cerebral ischemia in rats. In vivo PET imaging showed significant increase in DPA (N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide) uptake on the injured side compared with that in the contralateral area on days 7, 11, 15, and 21 after ischemia; the maximal binding value was reached 11 days after ischemia. In vitro autoradiography confirmed these in vivo results. In vivo and in vitro [(18)F]DPA-714 binding was displaced from the lesion by PK11195 and DPA-714. Immunohistochemistry showed increased PBR/TSPO expression, peaking at day 11 in cells expressing microglia/macrophage antigens in the ischemic area. At later times, a centripetal migration of astrocytes toward the lesion was observed, promoting the formation of an astrocytic scar. These results show that [(18)F]DPA-714 provides accurate quantitative information of the time course of PBR/TSPO expression in experimental stroke.

No abstract

There are growing concerns about potential delayed, neuropsychiatric consequences (e.g, cognitive decline, mood or anxiety disorders) of sports-related traumatic brain injury (TBI). Autopsy studies of brains from a limited number of former athletes have described characteristic, pathologic changes of chronic traumatic encephalopathy (CTE) leading to questions about the relationship between these pathologic and the neuropsychiatric disturbances seen in former athletes. Research in this area will depend on in vivo methods that characterize molecular changes in the brain, linking CTE and other sports-related pathologies with delayed emergence of neuropsychiatric symptoms. In this pilot project we studied former National Football League (NFL) players using new neuroimaging techniques and clinical measures of cognitive functioning. We hypothesized that former NFL players would show molecular and structural changes in medial temporal and parietal lobe structures as well as specific cognitive deficits, namely those of verbal learning and memory. We observed a significant increase in binding of [11C]DPA-713 to the translocator protein (TSPO), a marker of brain injury and repair, in several brain regions, such as the supramarginal gyrus and right amygdala, in 9 former NFL players compared to 9 age-matched, healthy controls. We also observed significant atrophy of the right hippocampus. Finally, we report that these same former players had varied performance on a test of verbal learning and memory, suggesting that these molecular and pathologic changes may play a role in cognitive decline. These results suggest that localized brain injury and repair, indicated by increased [11C]DPA-713 binding to TSPO, may be linked to history of NFL play. [11C]DPA-713 PET is a promising new tool that can be used in future study design to examine further the relationship between TSPO expression in brain injury and repair, selective regional brain atrophy, and the potential link to deficits in verbal learning and memory after NFL play.

Molecular imaging refers to the use of non-invasive imaging techniques to detect signals that originate from molecules, often in the form of an injected tracer, and observe their interaction with a specific cellular target in vivo. Differences in the underlying physical principles of these measurement techniques determine the sensitivity, specificity and length of possible observation of the signal, characteristics that have to be traded off according to the biological question under study. Here, we describe the specific characteristics of single photon emission computed tomography (SPECT) relative to other molecular imaging technologies. SPECT is based on the tracer principle and external radiation detection. It is capable of measuring the biodistribution of minute (<10(-10) molar) concentrations of radio-labelled biomolecules in vivo with sub-millimetre resolution and quantifying the molecular kinetic processes in which they participate. Like some other imaging techniques, SPECT was originally developed for human use and was subsequently adapted for imaging small laboratory animals at high spatial resolution for basic and translational research. Its unique capabilities include (i) the ability to image endogenous ligands such as peptides and antibodies due to the relative ease of labelling these molecules with technetium or iodine, (ii) the ability to measure relatively slow kinetic processes (compared with positron emission tomography, for example) due to the long half-life of the commonly used isotopes and (iii) the ability to probe two or more molecular pathways simultaneously by detecting isotopes with different emission energies. In this paper, we review the technology developments and design tradeoffs that led to the current state-of-the-art in SPECT small animal scanning and describe the position SPECT occupies within the matrix of molecular imaging technologies.

Synthetic cannabinoid (SC) designer drugs featuring bioisosteric fluorine substitution are identified by forensic chemists and toxicologists with increasing frequency. Although terminal fluorination of N-pentyl indole SCs is sometimes known to improve cannabinoid type 1 (CB1) receptor binding affinity, little is known of the effects of fluorination on functional activity of SCs. This study explores the in vitro functional activities of SC designer drugs JWH-018, UR-144, PB-22, and APICA, and their respective terminally fluorinated analogues AM-2201, XLR-11, 5F-PB-22, and STS-135 at human CB1 and CB2 receptors using a FLIPR membrane potential assay. All compounds demonstrated agonist activity at CB1 (EC50 = 2.8-1959 nM) and CB2 (EC50 = 6.5-206 nM) receptors, with the fluorinated analogues generally showing increased CB1 receptor potency (∼2-5 times). Additionally, the cannabimimetic activities and relative potencies of JWH-018, AM-2201, UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-135 in vivo were evaluated in rats using biotelemetry. All SCs dose-dependently induced hypothermia and reduced heart rate at doses of 0.3-10 mg/kg. There was no consistent trend for increased potency of fluorinated SCs over the corresponding des-fluoro SCs in vivo. Based on magnitude and duration of hypothermia, the SCs were ranked for potency (PB-22 > 5F-PB-22 = JWH-018 > AM-2201 > APICA = STS-135 = XLR-11 > UR-144).