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

Background and purpose: The ATP-gated P2X(7) 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 P2X(7) 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-1 beta release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP ( BzATP). Key results: AZ11645373 up to 10 mu M, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X(1), rat P2X(2), human P2X(3), rat P2X(2/3), human P2X(4), or human P2X(5) receptors expressed in HEK cells. AZ11645373 inhibited human P2X(7) receptor responses in HEK cells in a non-surmountable manner with K-B values ranging from 5 - 20 nM, with mean values not significantly different between assays. K-B values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1 beta release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC50 = 90 nM. AZ11645373 was > 500-fold less effective at inhibiting rat P2X(7) receptor-mediated currents with less than 50% inhibition occurring at 10 mu M. Conclusions and implications: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X(7) receptors and will have much practical value in studies of human cells.