Fig 3. In vitro Characterization of CCX168 Using C5aR-Expressing U937 Cells.

(A) CCX168 inhibition of [¹²⁵I]-C5a binding to U937 cells with a potency (IC₅₀ value) of 0.1 nM; each data point is the mean of 4 replicates ± standard error; study repeated 5 separate times, representative experiment shown. (B) Effects of vehicle control (■) and 1 nM (○) or 10 nM (●) CCX168 on C5a-mediated chemotaxis of U937 cells in buffer; each data point is the mean of 8 replicates ± standard error; study repeated 5 separate times, representative experiment shown. (C) C5a-mediated chemotaxis of U937 cells in the absence (●) or presence (■) of 1 μM CCX168, as well as following 1 μM CCX168 / 3x wash (□) and 1 μM CCX168 / 6x wash (Δ) treatments; each data point is the mean of 8 replicates ± standard error; study repeated 2 separate times, representative experiment shown. (D) C5a-induced intracellular calcium release in U937 cells, as measured by FLIPR, in the presence of vehicle control (●) and various concentrations of CCX168, 1 nM (○), 10 nM (■), or 100 nM (▲); each data point is the mean of 4 replicates ± standard error; study repeated 2 separate times, representative experiment shown. (E) C5a-mediated chemotaxis of U937 cells in 100% human plasma in the presence of vehicle control (■) and 1 nM (○) or 10 nM (●) CCX168; each data point is the mean of 8 replicates ± standard error; study repeated 2 separate times, representative experiment shown. (F) Inhibition by CCX168 of U937 cell chemotaxis towards 0.1 nM C5a in the presence (□) or absence (●) of α1-acid glycoprotein (AGP, 5 mg/mL) in buffer containing 5% human serum albumin (HSA); each data point is the mean of 8 replicates ± standard error; study repeated 2 separate times, representative experiment shown.