Preclinical evaluation of the first adenosine A₁receptor agonist radioligand for positron emission tomography (PET) imaging

Objectives: The adenosine A₁ receptor (A₁R) is implicated in sleep modulation and highly expressed in the brain. When activated by its endogenous agonist, adenosine, A₁R blocks neurotransmitter release, reduces neuron firing rate, and has a negative chronotropic effect in the heart. Further examination of the role of the A₁R in the sleep-wake cycle would require a PET radiotracer capable of indicating subtle changes in the levels of endogenous adenosine. Although A₁R PET radioligands have been heavily studied, the current well-established ones are all A₁R antagonists based on a xanthine core structure. These antagonists, however, fail to compete with adenosine, most likely due to having different binding sites. Meanwhile, no successful A₁R agonist tracer has been developed. Since most agonists are derived from a purine nucleoside, the hydrophilic ribose makes these compounds difficult to cross the blood-brain barrier (BBB) at low concentrations. Here we report the synthesis of the first non-xanthine, non-nucleoside A₁R agonist radiotracer, as well as its use in preclinical PET studies.

Methods: 4-Phenylpyridine and 4-phenlypyrimidine were chosen as templates and sixteen compounds were generated through parallel synthesis. In vitro structure-activity relationship (SAR) studies were performed to screen their A₁R/A_2AR binding and functional activities. Preliminarily, four selected compounds were radiolabeled to test their BBB permeability and biodistribution in the rat brain (Wistar), followed by PET scans conducted on a Focus-220 scanner to assess their pharmacokinetics, in vivo binding affinities, specificity (self-blocking study), and selectivity over A_2AR (blocking study with Preladenant, a selective A_2AR antagonist).

Results: Compound 8 appears to be a potent and selective partial A₁R agonist based on in vitro data (hA₁R K_i=0.8 nM, hA₁R % activation at 10 μM=11.8 %, hA_2AR % replacement at 10 μM= 74.5%). [¹¹C]8 was synthesized with its desmethyl precursor and [¹¹C]CH₃I in moderate radiochemical yield (RCY=18±4%, n=6), high radiochemical purity (>99%) and superior specific activity (18.5±7.0 Ci/µmol @EOB, n=25). [¹¹C]8 showed great BBB permeability (SUV, ~1 g/mL at 10 min after iv administration, logD_7.4=3.26) and test-retest reproducibility in the rat brain (n=3). Brain uptake of [¹¹C]8 was blocked significantly by non-radioactive compound 8 pretreatment (iv, 1 mg/kg, n=3), but not Preladenant (selective A_2AR antagonist) pretreatment, which reflects high A₁R selectivity (Figure 1). Conclusion: We developed the first adenosine A₁R agonist radioligand, [¹¹C]8, for CNS applications. Rodent PET studies demonstrated that [¹¹C]8 has excellent in vivo properties and further brain studies on competitive inhibition by endogenous adenosine are under way. This radiotracer might not only be a useful tool to investigate the roles of A₁R and endogenous adenosine in sleep mechanisms, but may also be a good drug template for adenosinergic abnormalities. Figure 1. Structure of [¹¹C]8 and representative rat brain PET scan

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