Soumen Paul

Adenosine A1 Receptors in the Central Nervous System: Their Functions in Health and Disease, and Possible Elucidation by PET Imaging

Adenosine is a neuromodulator with several functions in the central nervous system (CNS), such as inhibition of neuronal activity in many signaling pathways. Most of the sedating, anxiolytic, seizure-inhibiting and protective actions of adenosine are mediated by adenosine A(1) receptors (A(1)R) on the surface of neurons and glia. Positron Emission Tomography (PET) is a powerful in vivo imaging tool which can be applied to investigate the physiologic and pathologic roles of A(1)R in the human brain, and to elucidate the mechanism of action of therapeutic drugs targeting adenosine receptors, nucleoside transporters and adenosine-degrading enzymes. In this review article, we discuss (i) functions of adenosine and its receptors in cerebral metabolism; (ii) radioligands for A(1)R imaging: xanthine antagonists, non-xanthine antagonists, and agonists; (iii) roles of A(1)R in health and disease, viz. sleep-wake regulation, modulation of memory retention and retrieval, mediating the effects of alcohol consumption, protecting neurons during ischemia and reperfusion, suppression of seizures, modulating neuroinflammation and limiting brain damage in neurodegenerative disorders. The application of PET imaging could lead to novel insights in these areas. Finally (iv), we discuss the application of PET in pharmacodynamic studies and we examine therapeutic applications of adenosine kinase inhibitors, e.g. in the treatment of pain, inflammation, and epilepsy.

Small-Animal PET Study of Adenosine A 1 Receptors in Rat Brain: Blocking Receptors and Raising Extracellular Adenosine

Activation of adenosine A1 receptors (A1R) in the brain causes sedation, reduces anxiety, inhibits seizures, and promotes neuroprotection. Cerebral A1R can be visualized using 8-dicyclopropylmethyl-1-11C-methyl-3-propyl-xanthine (11C-MPDX) and PET. This study aims to test whether 11C-MPDX can be used for quantitative studies of cerebral A1R in rodents. Methods: 11C-MPDX was injected (intravenously) into isoflurane-anesthetized male Wistar rats (300 g). A dynamic scan of the central nervous system was obtained, using a small-animal PET camera. A cannula in a femoral artery was used for blood sampling. Three groups of animals were studied: group 1, controls (saline-treated); group 2, animals pretreated with the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 mg, intraperitoneally); and group 3, animals pretreated (intraperitoneally) with a 20% solution of ethanol in saline (2 mL) plus the adenosine kinase inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d] pyrimidine dihydrochloride (ABT-702) (1 mg). DPCPX is known to occupy cerebral A1R, whereas ethanol and ABT-702 increase extracellular adenosine. Results: In groups 1 and 3, the brain was clearly visualized. High uptake of 11C-MPDX was noted in striatum, hippocampus, and cerebellum. In group 2, tracer uptake was strongly suppressed and regional differences were abolished. The treatment of group 3 resulted in an unexpected 40%–45% increase of the cerebral uptake of radioactivity as indicated by increases of PET standardized uptake value, distribution volume from Logan plot, nondisplaceable binding potential from 2-tissue-compartment model fit, and standardized uptake value from a biodistribution study performed after the PET scan. The partition coefficient of the tracer (K1/k2 from the model fit) was not altered under the study conditions. Conclusion: 11C-MPDX shows a regional distribution in rat brain consistent with binding to A1R. Tracer binding is blocked by the selective A1R antagonist DPCPX. Pretreatment of animals with ethanol and adenosine kinase inhibitor increases 11C-MPDX uptake. This increase may reflect an increased availability of A1R after acute exposure to ethanol.

Development of [18F]-labeled pyrazolo[4,3-e]-1,2,4- triazolo[1,5-c]pyrimidine (SCH442416) analogs for the imaging of cerebral adenosine A2A receptors with positron emission tomography

Cerebral adenosine A2A receptors (A2ARs) are attractive therapeutic targets for the treatment of neurodegenerative and psychiatric disorders. We developed high affinity and selective compound 8 (SCH442416) analogs as in vivo probes for A2ARs using PET. We observed the A2AR-mediated accumulation of [18F]fluoropropyl ([18F]-10b) and [18F]fluoroethyl ([18F]-10a) derivatives of 8 in the brain. The striatum was clearly visualized in PET and in vitro autoradiography images of control animals and was no longer visible after pretreatment with the A2AR subtype-selective antagonist KW6002. In vitro and in vivo metabolite analyses indicated the presence of hydrophilic (radio)metabolite(s), which are not expected to cross the blood-brain-barrier. [18F]-10b and [18F]-10a showed comparable striatum-to- cerebellum ratios (4.6 at 25 and 37 min post injection, respectively) and reversible binding in rat brains. We concluded that these compounds performed equally well, but their kinetics were slightly different. These molecules are potential tools for mapping cerebral A2ARs with PET.

Cerebral adenosine A1 receptors are upregulated in rodent encephalitis

Adenosine A1 receptors (A1Rs) are implied in the modulation of neuroinflammation. Activation of cerebral A1Rs acts as a brake on the microglial response after traumatic brain injury and has neuroprotective properties in animal models of Parkinsons disease and multiple sclerosis. Neuroinflammatory processes in turn may affect the expression of A1Rs, but the available data is limited and inconsistent. Here, we applied an animal model of encephalitis to assess how neuroinflammation affects the expression of A1Rs. Two groups of animals were studied: Infected rats (n=7) were intranasally inoculated with herpes simplex virus-1 (HSV-1, 1 × 10(7) plaque forming units), sham-infected rats (n=6) received only phosphate-buffered saline. Six or seven days later, microPET scans (60 min with arterial blood sampling) were made using the tracer 8-dicyclopropyl-1-(11)C-methyl-3-propyl-xanthine ((11)C-MPDX). Tracer clearance from plasma and partition coefficient (K₁/k₂ estimated from a 2-tissue compartment model fit) were not significantly altered after virus infection. PET tracer distribution volume calculated from a Logan plot was significantly increased in the hippocampus (+37%) and medulla (+27%) of virus infected rats. Tracer binding potential (k₃/k₄ estimated from the model fit) was significantly increased in the cerebellum (+87%) and the medulla (+148%) which may indicate increased A1R expression. This was confirmed by immunohistochemical analysis showing a strong increase of A1R immunoreactivity in the cerebellum of HSV-1-infected rats. Both the quantitative PET data and immunohistochemical analysis indicate that A1Rs are upregulated in brain areas where active virus is present.

Use of 11C-MPDX and PET to Study Adenosine A1 Receptor Occupancy by Nonradioactive Agonists and Antagonists

Adenosine A1 receptors (A1Rs) in human and rodent brains can be visualized with the radioligand 8-dicyclopropylmethyl-1-11C-methyl-3-propylxanthine (11C-MPDX) and PET. Here we investigated whether A1R occupancy by nonradioactive agonists and antagonists can be assessed with this technique. Methods: Small-animal PET scans with arterial blood sampling were obtained for 4 groups of isoflurane-anesthetized Wistar rats: controls (n = 7); pretreated with a centrally active A1R agonist, N6-cyclopentyladenosine (CPA; 0.25 mg/kg intraperitoneally; dissociation constant, 0.48 nM; n = 7); pretreated with a moderate dose of caffeine (antagonist for A1Rs and adenosine A2A receptors; 4 mg/kg intraperitoneally; dissociation constant, 11 μM; n = 6); and pretreated with a high dose of caffeine (40 mg/kg intraperitoneally; n = 6). Results: The administration of CPA resulted in a strong reduction (>50%) in the heart rate, and caffeine administration resulted in a small increase (10%–15%). A caffeine dose of 4 mg/kg (n = 6) resulted in 65.9% A1R occupancy, and a dose of 40 mg/kg (n = 6) resulted in 98.5% occupancy (calculated from a modified Lassen plot). However, the administration of CPA resulted in an increase in 11C-MPDX binding in the brain. Conclusion: Small-animal PET with 11C-MPDX can be used to assess antagonist but not agonist binding at A1Rs. Changes in tracer uptake after the administration of CPA resembled previously reported changes induced by treatment of rats with ethanol and an adenosine kinase inhibitor (ABT702). Thus, the administration of an exogenous agonist or increasing the level of an endogenous agonist have similar effects. Agonists and antagonists may bind to different sites on the A1R protein having allosteric interactions.

PET Imaging of Adenosine A1 Receptor Occupancy

TO THE EDITOR: We have recently published 2 articles about the use of 11C-MPDX (8-dicyclopropylmethyl-1-11C-methyl-3propylxanthine) and PET for measurement of adenosine A1 receptor occupancy by nonradioactive agonists and antagonists (1,2). Dose-dependent occupancy of A1 receptors in the rodent brain by antagonists (caffeine, DPCPX [1,3-dipropyl-8-cyclopentyl xanthine]) could be assessed, but administration of an exogenous agonist (CPA [N6-cyclopentyladenosine]) or raising the levels of endogenous adenosine by treating animals with ethanol and the adenosine kinase inhibitor ABT-702 (4-amino-5-(3-bromophenyl)-7(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine) did not result in measurable competition of agonist and tracer. A paradoxic increase of tracer uptake was observed under these conditions. Kinetic modeling of the PET data suggested that there was an increase in tracer binding potential rather than in tracer delivery to the brain or passage of the tracer across the blood–brain barrier. This phenomenon was unexpected and could not be explained. At the recent Purines 2014 meeting in Bonn, a possible explanation was offered by Drs. Andreas Bauer (Düsseldorf, Germany) and Renata Ciccarelli (Chieti, Italy). These experts pointed out that many ligands for adenosine A1 receptors that initially were considered antagonists are in fact inverse agonists. Inverse agonism has been proven for WRC-0571 (8-(N-methylisopropyl) amino-N6-(59-endohydroxy-endonorbornyl)-9-methyladenine) (3), CGS-15943 (9-chloro-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]quinazolin-5amine) (3), DPCPX (3), and CPFPX (8-cyclopentyl-(3-(3-fluoropropyl)1-propylxanthine) (4). The last 2 ligands are structurally similar to our tracer, MPDX. Inverse agonists such as DPCPX display a high affinity for the uncoupled, or inactive, state of the A1 receptor and a lower affinity for the G-protein–coupled state (5). Paradoxic increases in 11CMPDX binding in the rodent brain on administration of CPA or treatment of rats with ethanol and adenosine kinase inhibitor may therefore be explained in the following way: agonists (such as CPA or adenosine) increase the fraction of A1 receptors in the uncoupled state, because the ternary complex consisting of agonist, activated receptor, and G-protein is not stable in living tissue. Guanosine triphosphate binding results in decoupling of the G-protein from the complex and relaxation of the receptor to the inactive conformation, possessing low affinity for the agonist but high affinity for an inverse agonist such as 11C-MPDX. Thus, the binding potential for 11C-MPDX is increased after administration of a pharmacologic dose of an agonist. An increase (23%–55%) similar to that we observed for 11C-MPDX binding in the rodent brain after agonist treatment was seen for 3H-DPCPX binding in human brain slices on the addition of guanosine triphosphate (6). This hypothesized explanation of our PET findings could be tested by radiolabeling a neutral antagonist and repeating the experiments with that tracer. Binding of an antagonist tracer should not be increased in the presence of an excess of agonist, in contrast to binding of an inverse agonist such as 11C-MPDX or 18F-CPFPX. To achieve this goal, a neutral antagonist with nanomolar affinity should be developed that is amenable to labeling (7). It may also be possible to use the nonxanthine PET tracer 11C-FR194921 (11C2-(1-methyl-4-piperidinyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)3(2H)-pyridazinone (8).

Evaluation of a novel PET radioligand to image O-GlcNAcase in brain and periphery of rhesus monkey and knock-out mouse.

Accumulation of hyperphosphorylated tau, a microtubule-associated protein, plays an important role in the progression of Alzheimer disease. Animal studies suggest that one strategy for treating Alzheimer disease and related tauopathies may be inhibition of O-GlcNAcase (OGA), which may subsequently decrease pathologic tau phosphorylation. Here, we report the pharmacokinetics of a novel PET radioligand, 18F-LSN3316612, which binds with high affinity and selectivity to OGA. Methods: PET imaging was performed on rhesus monkeys at baseline and after administration of either thiamet-G, a potent OGA inhibitor, or nonradioactive LSN3316612. The density of the enzyme was calculated as distribution volume using a 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. The radiation burden for future studies was based on whole-body imaging of monkeys. Oga∆Br, a mouse brain-specific knockout of Oga, was also scanned to assess the specificity of the radioligand for its target enzyme. Results: Uptake of radioactivity in monkey brain was high (∼5 SUV) and followed by slow washout. The highest uptake was in the amygdala, followed by striatum and hippocampus. Pretreatment with thiamet-G or nonradioactive LSN3316612 reduced brain uptake to a low and uniform concentration in all regions, corresponding to an approximately 90% decrease in distribution volume. Whole-body imaging of rhesus monkeys showed high uptake in kidney, spleen, liver, and testes. In Oga∆Br mice, brain uptake of 18F-LSN3316612 was reduced by 82% compared with control mice. Peripheral organs were unaffected in Oga∆Br mice, consistent with loss of OGA expression exclusively in the brain. The effective dose of 18F-LSN3316612 in humans was calculated to be 22 μSv/MBq, which is typical for 18F-labeled radioligands. Conclusion: These results show that 18F-LSN3316612 is an excellent radioligand for imaging and quantifying OGA in rhesus monkeys and mice. On the basis of these data, 18F-LSN3316612 merits evaluation in humans.