TRR469, a potent A1 adenosine receptor allosteric modulator, exhibits anti-nociceptive properties in acute and neuropathic pain models in mice

• The A1 adenosine receptor enhancer TRR469 has anti-nociceptive effects in mice.
• TRR469 improves the binding and the effect of adenosine agonists.
• TRR469 has a better side effect profile than the agonist CCPA.
• The inhibitory effect of A1 activation on aspartate release is enhanced by TRR469.

A1 adenosine receptors (ARs) have been identified as a potential target for the development of anti-nociceptive compounds. The present study explores the analgesic effects of a novel A1AR positive allosteric modulator, TRR469, in different models of acute and chronic pain in mice. To evaluate the allosteric enhancement, in vitro binding experiments were performed. The anti-nociceptive properties were investigated in formalin and writhing tests, and in the streptozotocin-induced diabetic neuropathic pain model. Rotarod and catalepsy tests were used to identify potential side effects, while the functional effect of TRR469 was studied using [3H]-d-aspartate release from synaptosomes. TRR469 effectively inhibited nociceptive responses in the formalin and writhing tests, with effects comparable to those of the reference analgesic morphine. Isobolographic analysis of the combination of TRR469 and morphine revealed an additive interaction. TRR469 was anti-allodynic in the neuropathic pain model and did not display locomotor or cataleptic side effects. TRR469 enhanced the binding of the agonist radioligand [3H]-CCPA and induced a 33-fold increase of adenosine affinity in spinal cord membranes. In mouse spinal cord synaptosomes, TRR469 enhanced the inhibitory effect of A1AR activation on [3H]-d-aspartate release, a non-metabolizable analogue of glutamate. In conclusion, this research demonstrates the anti-nociceptive effect of the novel compound TRR469, one of the most potent and effective A1AR positive allosteric modulators so far synthesized. The use of TRR469 allows for the possibility of exploiting analgesic properties of endogenous adenosine, with a minor potential to develop the various side effects often associated with the use of direct receptor agonists.