Shivashankar Khanapur

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.

Adenosine A 2A Receptor Antagonists as Positron Emission Tomography (PET) Tracers

The adenosine A(2A) receptor (A(2A)R) is highly concentrated in the striatum, and a therapeutic target for Parkinsons disorder (PD) and Huntingtons disease. High affinity and selective radiolabeled A(2A)R antagonists can be important research and diagnostic tools for PD. Positron Emission Tomography (PET) can play an important role by measuring radiolabeled A(2A) antagonists non-invasively in the brain. However, till date no complete review on A(2A)R PET ligands is available. The present article has been therefore focused on available PET tracers for A(2A)R and their detailed biological evaluation in rodents, nonhuman primates and humans. Drug design and development by molecular modeling including new lead structures that are potential candidates for radiolabeling and mapping of cerebral A(2A)Rs is discussed in the present article. A brief overview of functions of adenosine in health and disease, including the relevance of A(2A)R for PD has also been presented.

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.

Synthesis and preclinical evaluation of 2-(2-furanyl)-7-[2-[4-[4-(2-[11C]methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-5-amine ([11C]Preladenant) as a PET tracer for the imaging of cerebral adenosine A2A receptors

2-(2-Furanyl)-7-[2-[4-[4-(2-[(11)C]methoxyethoxy)phenyl]-1-piperazinyl]ethyl]7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-5-amine [(11)C]-3 ([(11)C]Preladenant) was developed for mapping cerebral adenosine A2A receptors (A2ARs) with PET. The tracer was synthesized in high specific activity and purity. Tissue distribution was studied by PET imaging, ex vivo biodistribution (BD), and in vitro autoradiography (ARG) experiments. Regional brain uptake of [(11)C]-3 was consistent with known A2ARs distribution, with highest uptake in striatum. The results indicate that [(11)C]-3 has favorable brain kinetics and exhibits suitable characteristics as an A2AR PET tracer.

In vivo evaluation of [11C]preladenant positron emission tomography for quantification of adenosine A2A receptors in the rat brain

[11C]Preladenant was developed as a novel adenosine A2A receptor positron emission tomography radioligand. The present study aims to evaluate the suitability of [11C]preladenant positron emission tomography for the quantification of striatal A2A receptor density and the assessment of striatal A2A receptor occupancy by KW-6002. Sixty- or ninety-minute dynamic positron emission tomography imaging was performed on rats. Tracer kinetics was quantified by the two-tissue compartment model, Logan graphical analysis and several reference tissue-based models. Test–retest reproducibility was assessed by repeated imaging on two consecutive days. Two-tissue compartment model and Logan plot estimated comparable distribution volume (VT) values of ∼10 in the A2A receptor-rich striatum and substantially lower values in all extra-striatal regions (∼1.5–2.5). The simplified reference tissue model with midbrain or occipital cortex as the reference region proved to be the best non-invasive model for quantification of A2A receptor, showing a striatal binding potential (BPND) value of ∼5.5, and a test–retest variability of ∼5.5%. The brain metabolite analysis showed that at 60-min post injection, 17% of the radioactivity in the brain was due to radioactive metabolites. The ED50 of KW-6002 in rat striatum for i.p. injection was 0.044–0.062 mg/kg. The study demonstrates that [11C]preladenant is a suitable tracer to quantify striatal A2A receptor density and assess A2A receptor occupancy by A2A receptor-targeting molecules.

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.

Potential therapeutic applications of adenosine A2A receptor ligands and opportunities for A2A receptor imaging

Adenosine A2A receptors (A2ARs) are highly expressed in the human striatum, and at lower densities in the cerebral cortex, the hippocampus, and cells of the immune system. Antagonists of these receptors are potentially useful for the treatment of motor fluctuations, epilepsy, postischemic brain damage, or cognitive impairment, and for the control of an immune checkpoint during immunotherapy of cancer. A2AR agonists may suppress transplant rejection and graft‐versus‐host disease; be used to treat inflammatory disorders such as asthma, inflammatory bowel disease, and rheumatoid arthritis; be locally applied to promote wound healing and be employed in a strategy for transient opening of the blood–brain barrier (BBB) so that therapeutic drugs and monoclonal antibodies can enter the brain. Increasing A2AR signaling in adipose tissue is also a potential strategy to combat obesity. Several radioligands for positron emission tomography (PET) imaging of A2ARs have been developed in recent years. This review article presents a critical overview of the potential therapeutic applications of A2AR ligands, the use of A2AR imaging in drug development, and opportunities and limitations of PET imaging in future research.

Preclinical Evaluation and Quantification of 18F-Fluoroethyl and 18F-Fluoropropyl analogs of SCH442416 as Radioligands for PET Imaging of the Adenosine A2A Receptors in Rat Brain

The cerebral adenosine A2A receptor is an attractive therapeutic target for neuropsychiatric disorders. 18F-fluoroethyl and 18F-fluoropropyl analogs of 18F-labeled pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH442416) (18F-FESCH and 18F-FPSCH, respectively) were developed as A2A receptor–specific PET ligands. Our aim was to determine an appropriate compartmental model for tracer kinetics, evaluate a reference tissue approach, and select the most suitable PET ligand. Methods: A 90-min dynamic PET scan with arterial blood sampling and metabolite analysis was acquired for 22 healthy male Wistar rats starting at the time of 18F-FESCH (n = 12) and 18F-FPSCH (n = 10) injection. For each tracer, half the animals were vehicle-treated whereas the other half were pretreated with the A2A receptor–selective antagonist KW-6002, inducing full blocking. Regional tissue total volume of distribution (VT) was estimated by 1- and 2-tissue-compartment modeling (1TCM and 2TCM, respectively) and Logan graphical analysis. Midbrain, cerebellum, and hippocampus were evaluated as the reference region by comparing baseline VT with VT under full blocking conditions and comparing striatal nondisplaceable binding potential (BPND) using a simplified reference tissue model (SRTM) with distribution volume ratio minus 1 (DVR − 1) for 60- and 90-min scans. Results: On the basis of the Akaike information criterion, 1TCM and 2TCM were the most appropriate models for 18F-FPSCH (baseline striatal VT, 3.7 ± 1.1) and 18F-FESCH (baseline striatal VT, 5.0 ± 2.0), respectively. Baseline striatal VT did not significantly differ between tracers. After pretreatment, striatal VT was reduced significantly, with no significant decrease in hippocampus, midbrain, or cerebellum VT. Baseline striatal SRTM BPND did not differ significantly from DVR − 1 except for 18F-FPSCH when using a 60-min scan and midbrain as the reference region, whereas Bland–Altman analysis found a smaller bias for 18F-FESCH and a 60-min scan. After pretreatment, striatal SRTM BPND did not significantly differ from zero except for 18F-FPSCH when using hippocampus as the reference region. Striatal SRTM BPND using midbrain or cerebellum as the reference region was significantly lower for 18F-FPSCH (range, 1.41–2.62) than for 18F-FESCH (range, 1.64–3.36). Conclusion: Dynamic PET imaging under baseline and blocking conditions determined 18F-FESCH to be the most suitable PET ligand for quantifying A2A receptor expression in the rat brain. Accurate quantification is achieved by a 60-min dynamic PET scan and the use of either cerebellum or midbrain as the reference region.

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).