Role Investigation of Adenosine Receptor Ligands on Neuroinflammation and Neurodegenerative Diseases

Neuroinflammation is a complex and chronic response of the nervous system (NS) against infections, oxidative stress, neurodegenerative diseases or injuries. This pathological condition is produced by an aberrant activation of microglia and astrocytes, which represent the neuroimmune brain cells. Nowadays adenosine receptors (ARs) are emerging as potential and attractive targets to treat several diseases like cardiovascular problems, sleep disorders, ischemia, cancer, immune pathologies, neuroinflammation and neurodegenerative diseases. ARs belong to G-protein coupled receptors and are divided into A1, A2A, A2B, and A3, among them, A1AR and A2AAR play the major roles in neuroinflammation modulation. In fact, it has been discovered that the activation of A1AR and the blockade of A2AAR could conferee beneficial effects to neuroinflammatory pathologies like Alzheimer’s or Parkinson’s disease. For these reasons, the synthesis and development of new ligands for these receptors are of great interest for the discovery of new potential drugs. The scope of this work was to study the pharmacological profile of a series of A1AR agonists (CPA derivatives) present in the chemical library and new potential A2AAR antagonists substituted at 2, N6, 8, or 9 positions. The investigation has been centred in their role in protection and/or recovery from neuroinflammation both in in vitro and in in vivo models of neuroinflammation experiments. After their biological characterization through binding and cAMP assay, all the A1AR agonists and the three most potent and selective A2AAR antagonists (Compound 6, 7 and 16) were selected for further in vitro assays. These compounds were tested on mixed glial cell cultures in presence or absence of proinflammatory cytokine cocktail (TNF-α, IL-1β and IFN-γ) through viability assay, Griess technique and Hoechst staining. The two best neuroprotective ligands, the A1AR partial agonist 2’- dCCPA, (endowed with submicromolar affinity versus A1AR) and the A2AAR antagonist compound 7 (possessing subnanomolar affinity versus A2AAR), were subsequently tested in other cell lines to ensure and provide solider results of their positive effects. 2’-dCCPA and compound 7 were then studied on N13 (mouse microglial cells) and primary neuronal cell culture, resulting in the reaffirmation of their protective and restoring effects against cytokine inflammation. Since these compounds showed good biological effects at cell level the study continued on in vivo rat neuroinflammation model in order to test their in vivo potential. Animals were treated with lipopolysaccharides (LPS) in presence or absence of the partial agonist (2’-dCCPA) or antagonist (compound 7). Both of the ligands demonstrated not to alter the velocity, distance and permanence time of the animals in Open Field. On the other hand, both ligands ameliorate the animal’s memory in Y Maze after the treatment with LPS, allowing the rat to remember which arm was closed and which one was opened. Lastly, despite not showing significant differences in Object Displacement, animals treated with 2’-dCCPA or compound 7 were able to elucidate which object was moved, while LPS treated animals did not. Collectively, these data provide new evidence that the use of selective and potent A1AR partial agonists and A2AAR antagonists result as a promising therapeutic approach to improve the functional recovery of patients with neuroinflammation or associated pathologies.