In Vivo Positron Emission Tomography Imaging of Adenosine A2A Receptors

As an invasive nuclear medical imaging technology, positron emission tomography (PET) possess the possibility to imaging the distribution as well as the density of selective receptors via specific PET tracers. Inspired by PET, the development of radio-chemistry has greatly promoted the progress of innovative imaging PET tracers for adenosine receptors, in particular adenosine A2A receptors (A2ARs). PET imaging of A2A receptors play import roles in the research of adenosine related disorders. Several radio-tracers for A2A receptors imaging have been evaluated in human studies. This paper reviews the recent research progress of PET tracers for A2A receptors imaging, and their applications in the diagnosis and treatment of related disease, such as cardiovascular diseases, autoimmune diseases, neurodegenerative and psychiatric disease. The future development of A2A PET tracers were also discussed.


INTRODUCTION
As an extracellular endogenous messenger, adenosine play important roles in biochemical processes, signal transduction and neurotransmission (Estrela and Abraham, 2011). In physiological and pathological conditions, it acts as a cytoprotectant and a neuromodulator in response to organ and tissue stress (Khanapur et al., 2013). It also holds the capability to reduce energy demand or increase energy supply to organs or tissues which are damaged or disturbed. It is known that cytoprotective and neuromodulatory function in the brain are mediated by four adenosine receptors (ARs), namely A 1 , A 2A , A 2B , and A 3 (Jacobson and Gao, 2006;Khanapur et al., 2013). A2ARs are ubiquitously distributed in brain, heart, lungs and spleen, and A2ARs mainly facilitates neurotransmissions and other physiological functions. A 2A Rs are involved in multiple physiological processes (Tang et al., 2019;Chen and Cunha, 2020), as well as in various pathological conditions (Illes et al., 2016;Burnstock, 2017). The dysfunction of A 2A Rs are related to many diseases such as cardiovascular diseases, autoimmune Diseases, neurodegenerative and psychiatric disease. However, many of their functions in pathophysiological processes remain unknown, partly due to the lack of available techniques for spatial and temporal control of purinergic signaling. Positron emission tomography (PET) is a nuclear medical technology that allows in vivo imaging and quantification of specific targets, as well as molecular and cellular processes in the living body. For example, with specific brain-targeted radio-tracers, PET therefore enables the in vivo imaging of local brain function, including receptor-binding ability, cerebral blood flow, and molecular metabolism (Mishina and Ishiwata, 2014). At present, PET imaging studies on adenosine receptors are mainly focused on A1 and A2A receptors, and for the diagnose of related diseases (Figure 1). In this paper, we will discuss the recent progress of lead compounds and related radio tracers for PET imaging for A 2A Rs. In addition, this review also outlines PET imaging for adenosine A 2A receptors in health and diseases subjects. Furthermore, the direction of future development of A 2A PET tracers were also discussed.

DEVELOPMENT OF ADENOSINE A 2A BASED POSITRON EMISSION TOMOGRAPHY TRACERS
In 1988, 3,7-dimethyl-1-propylxanthine (DMPX) was identified as an A 2A R-targeted selective antagonists (Seale et al., 1988), several xanthine based radio-tracers were also successfully developed thereafter. In addition, shortly after the discovery and report of a novel pyrazolol-pyrimidine based compound as a potent and selective A 2A R antagonist (Poucher et al., 1995;Baraldi et al., 1996;Zocchi et al., 1996;Baraldi et al., 1998), these compounds with a fused heterocycles were also regarded as lead compounds for A 2A R PET tracers ( Figure 2). Therefore, current PET tracers for A 2A receptors can be subdivided into the following two categories ( Figure 2)

Triazolopyrimidine Based A2 Adenosine Receptor Positron Emission Tomography Tracers
Based on the findings of antagonism for A 2A R from triazolopyrimidine based compounds such as ZM241385 (Poucher et al., 1995) and SCH58261 , another class of A 2A R PET tracers were developed based on the novel triazolopyrimidine structure. Todde et al. prepared [11C]SCH442416 by O-methylation (Todde et al., 2000), and this radio-ligand exhibits the highest selectivity and affinity among all A 2A PET ligands reported as present. In addition, several nonxanthine heterocycles have also been synthesized and radiolabeled for   . Preliminary investigations of these tracers revealed a similar distribution pattern with the known expressions of A 2A R in rat brain (Khanapur et al., 2017 Among all xanthine and non-xanthine based ligands, the most potent affinity for A 2A Rs was observed in SCH442416. IS-DMPX, KF21213 and SCH442416 exhibited higher A 2A R selectivity. The selective uptake in striatum was observed in validation studies in rodents for all radio-labeled compounds, which is correspond to A 2A Rs expressions. However, most of the radioligands also showed a considerable degree of specific binding in the cerebral cortex and cerebrum, which is not observed with [11C]KF21213. Therefore, based on the uptake on the receptor poor cerebellum to receptorrich striatum,  (Leussis et al., 2008;Bar-Yehuda et al., 2009;Ishiwata et al., 2010). Theophylline stimulation confirmed the specific binding of [11C] TMSX to adenosine A 2A receptor (Ishiwata et al., 2005). Thus, the good reproducibility of [11C]TMSX PET in striatum was confirmed. The highest A 2A receptor density was observed in nucleus putamen in [11C]TMSX PET images, followed by caudate head and thalamus. And the relative low density of A 2A receptor was observed in cerebral cortex and frontal lobe. (Ishiwata et al., 2005;Leussis et al., 2008). Autopsy and nonhuman studies have found that [11C]TMSX PET shows great binding potential in the adenosine A 2A receptor-rich striatum, but [11C]TMSX binds more strongly in the human thalamus than in other mammals (Mishina and Ishiwata, 2014). Moreover, as the first non-xanthine A 2A R PET tracer, [11C]SCH442416 showed highest binding in putamen and the lowest binding in cerebellar was observed in unaffected people (Brooks et al., 2010). The specific binding of [11C]SCH442416 was also calculated with cerebellum as the reference region to study the different binding potentials in the putamen by Ramlackhansingh et al., (2011).
With a good maximal striatal to cerebellar ratio in rodents but low in primates, [11C]SCH442416 was not suitable for the receptor occupancy quantification studies. Barret et al. used (Barret et al., 2015). PET imaging with [18F]MNI-444 showed a rapid brain distribution, and the uptake pattern was consistent with known A 2A R densities in the human brain. The favorable kinetic properties of [18F]MNI-444 may promote the PET imaging of A 2A R in research related to neurodegenerative and psychiatric diseases.
What's more, the clinical study of [11C]preladenant showed the individual organ and total-body administration of [11C] preladenant were comparable with other 11C-labeled tracers.
As is known to all, the highest signal level of A 2A Rs was observed in the basal ganglia, followed by cerebral cortex and thalamus.
[11C]preladenant's regional distribution in healthy human brain is consistent with A 2A R density. [11C] preladenant provides a feasible approach for imaging of adenosine A 2A R in the brain. Therefore, A 2A R density can be quantified using the cerebellum as a reference tissue model for the reference region. Further inhibition studies in the human brain may be needed to fully verify the existence of reference regions.
In  (Lahesmaa et al., 2019). [11C] TMSX binding with BAT decreases when BAT is exposed to cold, which indicates that endogenous adenosine and irradiated oligosaccharide competition receptors show high binding (Sousa and Diniz, 2017). Interestingly, the reduction of [11C]TMSX binding is related to increased perfusion in BAT, further indicating that endogenous adenosine release in BAT is accompanied by the increased oxidative metabolism. This implies that adenosine and A 2A R are significant in the BAT activation induced by cold, which provides a new therapeutic direction for the fight against obesity and diabetes. [11C]SCH442416 In order to avoid photoisomerization generated by xanthine analogues, Todde et al. labelled the first non-xanthine A 2A antagonist, [11C]SCH442416, whose kinetic behavior in rodents suggests that it may be used for in vivo imaging of the A 2A adenosine receptor in future (Todde et al., 2000).
[11C] SCH442416, as an in vivo marker of A2A effectiveness, can selectively and reversibly bind to striatum A2A receptor with nanoscale affinity. PET imaging with [11C]SCH442416 was used to observe the expression of levodopa-induced dyskinesias (LIDs) in patients with Parkinson's disease (Ramlackhansingh et al., 2011). This implies that A2A antagonists may have value in levodopa-induced dyskinesias intervention while reducing levodopa dose.
[11C]SCH442416 PET provides an efficient and robust approach for in vivo studies of the effectiveness of A2A.
[11C]SCH442416 PET also can be used to determine the dose occupation of other A2A antagonists. In addition, [18F] FESCH and [18F]FPSCH are prepared as the analogs of SCH442416 Khanapur et al., 2017).

[11C]Preladenant
[11C]SCH442416 and [11C]TMSX are the most favorable tracers for imaging A 2A Rs in brain. However, low target-to-nontarget ratios, high nonspecific binding and low binding potentials are the disadvantages of these tracers. Thus, the newly improved radioactive ligand [11C]preladenant was developed for imaging A 2A Rs in the living brain, including human brain, rat brain and monkey brain (Sakata et al., 2017;Zhou et al., 2017a;Zhou et al., 2017b;Zhou et al., 2017c). It is a non-xanthine heterocyclic compound with high selectivity, sufficient affinity for image receptors without affecting the quantification of receptors, and this compound also showed good pharmacokinetic properties (Zhou et al., 2014). With superior target-to-nontarget ratios and excellent pharmacokinetic properties, this tracer was advanced into human studies. Recently, studies have shown that [11C] preladenant is applied to healthy human brains in a manner consistent with A 2A R density. Thus, it indicated that [11C] preladenant is suitable for imaging of A 2A Rs in the living brain (Sakata et al., 2017). In addition, compared with other ARs, [11C]preladenant showed high affinity and significant selectivity for A 2A R (Neustadt et al., 2007;Zhou et al., 2014). Recently, Ishibashi et al. reported [11C]preladenant PET can be used to calculate the occupancy rate of Istradefylline to A 2A R (Ishibashi et al., 2018). These results demonstrated that [11C] preladenant is a suitable tracer to evaluate A2A receptor occupancy and quantify striatal A2A receptor density by A2A receptor-targeting molecules (Sakata et al., 2017;Zhou et al., 2017a;Zhou et al., 2017c In healthy rat, although [11C]KW-6002 shows some potential as a PET ligand, it also showed low cerebral cortex and cerebellar retention, and it may proved to be insufficiently selective to be a useful in vivo radio-tracer, at least in rodents; however, it also binds to the outer fissure region, so its potential as a PET tracer needs further studies (Hirani et al., 2001 Although adenosine can also be tested by in situ hybridization and immunochemistry probes in recent years, PET imaging of A 2A R can further be used to capture changes in A 2A Rs distribution and density as the disease progresses, as well as to monitor treatment responses to these changes. In addition, PET can also determine the A 2A R occupancy in the brain can be measured by PET, and hence providing a useful method for drug discovery (Tavares et al., 2013). The PET radio-tracers provided valuable information for the diagnosis and treatment of diseases associated with altered ARs expression, following of the summary picture.
Molecular imaging plays a crucial role in improving accuracy by quantifying, characterizing and visualizing biological processes at the molecular and cellular levels in living body, which provides an achievable basis for precision medicine. Therefore, how to realize the personalized diagnosis and treatment of A2A-related diseases with PET imaging technology will become an important research direction in the future. In addition, the application of PET molecular imaging technology in assessing A2A disease risk and understanding disease mechanisms would also make a significant contribution to the medical profession.

AUTHOR CONTRIBUTIONS
MS and YZ prepared the manuscript, review and editing by XW and FL.

FUNDING
This work is supported by Sichuan Science and Technology Program (No. 2017JY0324).