Characterization of a selective and potent antagonist of human P2X7 receptors, AZ11645373

Background and purpose: The ATP-gated P2X7 receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X7 receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized. Experimental approach: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1β release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP). Key results: AZ11645373 up to 10 μM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X1, rat P2X2, human P2X3, rat P2X2/3, human P2X4, or human P2X5 receptors expressed in HEK cells. AZ11645373 inhibited human P2X7 receptor responses in HEK cells in a non-surmountable manner with KB values ranging from 5 - 20 nM, with mean values not significantly different between assays. KB values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1β release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC50 = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X7 receptor-mediated currents with less than 50% inhibition occurring at 10 μM. Conclusions and implications: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X7 receptors and will have much practical value in studies of human cells. British Journal of Pharmacology (2006) 149, 880–887. doi:10.1038/sj.bjp.0706933