Aaron Reynolds

Radiolabelled Molecules for Imaging the Translocator Protein (18 kDa) Using Positron Emission Tomography

The translocator protein (18 kDa) (TSPO), formerly known as the peripheral benzodiazepine receptor (PBR), was originally identified as an alternate binding site for the central benzodiazepine receptor (CBR) ligand, diazepam, in the periphery, but has now been distinguished as a novel site. The TSPO is ubiquitously expressed in peripheral tissues but only minimally in the healthy brain and increased levels of TSPO expression have been noted in neuroinflammatory conditions such as Alzheimers disease, Parkinsons disease and stroke. This increase in TSPO expression has been reported to coincide with the process of microglial activation, whereby the brains intrinsic immune system becomes active. Therefore, by using recently developed high affinity, selective TSPO ligands in conjunction with functional imaging modalities such as positron emission tomography (PET), it becomes possible to study the process of microglial activation in the living brain. A number of high affinity ligands, the majority of which are C,N-substituted acetamide derivatives, have been successfully radiolabelled and used in in vivo studies of the TSPO and the process of microglial activation. This review highlights recent achievements (up to December 2008) in the field of functional imaging of the TSPO as well as the radiosyntheses involved in such studies.

Initial Evaluation of 11C-DPA-713, a Novel TSPO PET Ligand, in Humans

Translocator protein (TSPO) is upregulated in activated microglia and thus can serve as a marker of neuroinflammation. Recently, a novel radioligand, 11C-N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide (11C-DPA-713), has been described that binds to TSPO with high affinity. Here, we report the first examination of 11C-DPA-713 in human subjects using PET. Methods: Five healthy controls were studied with PET for 90 min after a bolus injection of high-specific-activity 11C-DPA-713. For comparison, 2 additional healthy controls were studied with 11C-R-PK11195. Arterial blood sampling and metabolite analysis were performed to allow the accurate quantification of tracer kinetics. Tracer uptake was evaluated for several brain regions. Tissue time–activity curves were fitted using 1- and 2-tissue-compartment models, with goodness-of-fit tests showing a preference for the 2-tissue model. Results: In the healthy brain, the average plasma-to-tissue clearance and the total volume of distribution were an order of magnitude larger than measured for 11C-R-PK11195. Accordingly, dose-normalized time–activity curves showed that 11C-DPA-713 gives a larger brain signal. Conclusion: Studies in patient populations will help determine whether 11C-DPA-713 provides better sensitivity for evaluating increased TSPO expression. This initial study in humans shows that 11C-DPA-713 is a promising ligand for evaluating TSPO binding with PET.

Initial evaluation in healthy humans of [18F]DPA-714, a potential PET biomarker for neuroinflammation

INTRODUCTION The translocator protein 18 kDa (TSPO), although minimally expressed in healthy brain, is up-regulated in pathological conditions, coinciding with microglial activation. It is thereby a suitable in vivo biomarker of neuroinflammation for detection, evaluation and therapeutic monitoring of brain diseases. We aimed to estimate the radiation dosimetry of the positron emission tomography (PET) TSPO radioligand [(18)F]DPA-714, and we evaluated in healthy volunteers its whole-body uptake and cerebral kinetics. METHODS Biodistribution data from mice were used for the prediction of radiation dosimetry. In human studies, a 90-min dynamic PET scan was performed in seven healthy volunteers after injection of [(18)F]DPA-714 (245±45 MBq). Arterial and venous samples were collected from two subjects, and two additional subjects were submitted to whole-body acquisition. Regions of interest were defined over cerebral structures to obtain mean time-activity curves and to estimate the distribution volume ratios by Logan graphical analysis, and over peripheral organs to obtain standard uptake values. RESULTS The effective dose estimated from biodistribution in mice was 17.2 μSv/MBq. Modeling of regional brain and plasma data showed good in vivo stability of [(18)F]DPA-714 in humans, with only 20% of blood metabolites 20 min postinjection (p.i.). Maximum cerebral uptake was observed 5 min p.i., followed by two decreasing phases: a rapid washout (5-30 min) followed by a slower phase for the remainder of PET acquisition. Whole-body images demonstrate high activity in the gallbladder, heart, spleen and kidneys. CONCLUSIONS This initial study in humans shows that [(18)F]DPA-714 is a promising PET radioligand with excellent in vivo stability and biodistribution, and acceptable effective dose estimation. Therefore, [(18)F]DPA-714 could provide a sensitive measure of neuroinflammatory changes in subsequent clinical investigations.

Selective recognition of pyrophosphate in water using a backbone modified cyclic peptide receptor

A cyclic peptide based receptor, bearing two dipicolylamino arms complexed to zinc(II) ions, binds pyrophosphate ions with high affinity and selectivity in aqueous solution as determined using an indicator displacement assay.

Effects of translocator protein (18 kDa) ligands on microglial activation and neuronal death in the quinolinic-acid-injected rat striatum.

There is evidence that excitotoxicity and prolonged microglial activation are involved in neuronal death in neurodegenerative disorders. Activated microglia express various molecules, including the translocator protein 18 kDa (TSPO; formerly known as the peripheral benzodiazepine receptor) on the outer mitochondrial membrane. The TSPO is a novel target for neuroprotective treatments which aim to reduce microglial activation. The effect of PK 11195 and three other TSPO ligands on the level of microglial activation and neuronal survival was evaluated in a quinolinic acid (QUIN) rat model of excitotoxic neurodegeneration. All three ligands were neuroprotective at a level comparable to PK 11195. All of the ligands decreased microglial activation following the injection of QUIN but had no effect on astrogliosis. Interestingly, we also observed neuroprotective effects from the vehicle, dimethyl sulfoxide (DMSO).

Pyrazolo[1,5-a]pyrimidine acetamides: 4-Phenyl alkyl ether derivatives as potent ligands for the 18 kDa translocator protein (TSPO).

Herein, we report the synthesis of four new phenyl alkyl ether derivatives (7, 9-11) of the pyrazolo[1,5-a]pyrimidine acetamide class, all of which showed high binding affinity and selectivity for the TSPO and, in the case of the propyl, propargyl, and butyl ether derivatives, the ability to increase pregnenolone biosynthesis by 80-175% over baseline in rat C6 glioma cells. While these compounds fit our in silico generated pharmacophore for TSPO binding the current model does not account for the observed functional activity.

Extracellular Loops 2 and 4 of GLYT2 Are Required for N-Arachidonylglycine Inhibition of Glycine Transport

Concentrations of extracellular glycine in the central nervous system are regulated by Na+/Cl−-dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonylglycine (NAGly) is an endogenous inhibitor of GLYT2 with little or no effect on GLYT1 and is analgesic in rat models of neuropathic and inflammatory pain. Understanding the molecular basis of NAGly interactions with GLYT2 may allow for the development of novel therapeutics. In this study, chimeric transporters were used to determine the structural basis for differences in NAGly sensitivity between GLYT1 and GLYT2 and also the actions of a series of related N-arachidonyl amino acids. Extracellular loops 2 and 4 of GLYT2 are important in the selective inhibition of GLYT2 by NAGly and by the related compounds N-arachidonyl-γ-aminobutyric acid and N-arachidonyl-d-alanine, whereas only the extracellular loop 4 of GLYT2 is required for N-arachidonyl-l-alanine inhibition of transport. These observations suggest that the structure of the head group of these compounds is important in determining how they interact with extracellular loops 2 and 4 of GLYT2. Site-directed mutagenesis of GLYT2 EL4 residues was used to identify the key residues Arg531, Lys532, and Ile545 that contribute to the differences in NAGly sensitivity.

Insights into structure-activity relationships and CNS therapeutic applications of NR2B selective antagonists.

Excessive stimulation of NMDA receptors is involved in various CNS pathologies such as Parkinsons disease, acute and chronic pain and cerebral ischaemia. The use of NMDA antagonists as therapeutic agents has been restricted as a result of unwanted side effects including hallucinations and loss of co-ordination. NR2B subtype selective antagonists have previously shown a therapeutic effect without causing the side effects of broad spectrum NMDA antagonists. Considerable research has since been devoted to the development of orally bioavailable, selective NR2B antagonists and their applications in various neurological diseases. The improved therapeutic index of these compounds is expected to be the result of the subtype selectivity and cellular location of the NR2B receptors within the CNS. This review describes recent advances in the development of NR2B antagonists as well as their therapeutic applications.

Inhibition of human recombinant T‐type calcium channels by N‐arachidonoyl 5‐HT

BACKGROUND AND PURPOSE N‐arachidonoyl 5‐HT (NA‐5HT) has anti‐nociceptive effects reported to be mediated by inhibitory actions at the transient receptor potential vanilloid receptor 1 (TRPV1) and fatty acid amide hydrolase (FAAH). Anandamide and N‐arachidonoyl dopamine (NA‐DA), endocannabinoids that activate TRPV1 or are metabolized by FAAH, also inhibit T‐type calcium channels (ICa). T‐type ICa are expressed by many excitable cells, including neurons involved in pain detection and processing. We sought to determine whether NA‐5HT also modulates T‐type ICa.

Trishomocubane as a scaffold for the development of selective dopamine transporter (DAT) ligands

In our continued exploration of trishomocubane derivatives with central nervous system (CNS) activity, N-arylalkyl-8-aminopentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecanes (10-13) displaying affinity for the sigma (σ) receptor were also found, in several cases, to interact with the dopamine transporter (DAT). Compound 12 was identified as the first trishomocubane-derived high affinity DAT ligand (K(i) = 1.2 nM), with greater than 8300-fold selectivity over the monoamine transporters NET and SERT, and only low to moderate affinity for σ(1) and σ(2) receptors.