A preclinical pharmacodynamic analysis of CU201, a novel bradykinin antagonist, using a calcium flux assay in lung cancer cell lines

A105

Background: Neuroendocrine features, including paracrine and autocrine growth stimulation by various neuropeptides, are characteristics of all small cell lung cancers (SCLC) and many non-small cell lung cancers (NSCLC). CU201, a novel bradykinin antagonist, acts as a biased agonist for neuropeptides by blocking G_αq signaling and activating G_α12,13 signaling (Chan DC, et al. Clin Cancer Res 2002;8:1280-1287). A pharmacodynamic test is sought to incorporate into future phase I/II studies with this compound, at feasible plasma concentrations in clinical trial participants.Methods: Cell growth was assessed using a modified tetrazolium salt (MTT) assay. Gene expression profiling was performed using Affymetrix U133A plus 2.0 microarray on SCLC cell lines (SHP77 and H69) and Affymetrix U133A microarray on NSCLC cell lines (H2126 and H1703). Cell necrosis and apoptosis was determined by flow cytometry after cells were treated with CU201 (range 0.1-10 uM) over 48 hours followed by loading with YO-PRO-1 and propidium iodide. Intracellular calcium flux was determined by flow cytometry after cells were loaded with calcium marker Indo-1-AM and were exposed to bradykinin (range 0.1-10 uM) or CU201 (range 0.1-100 uM) followed by bradykinin. Plasma was obtained from 3 healthy volunteers. Calcium flux was then determined using fresh, thawed, and heat-inactivated plasma, respectively. Plasma was spiked with CU201 to a final concentration of 1 uM and then heat-inactivated and frozen at -70 C for 10 days, then thawed and exposed to cell lines followed by the addition of 0.1 uM bradykinin.Results: IC₅₀ (uM) values for growth inhibition were 4.0, 1.9, 3.0, and 1.2 uM and BKR2 expression signal intensities were 1460, 1267, 4.76, 10.73 in H2126, SHP77, H1703, and H69; respectively. Necrosis₅₀ values by the apoptosis assay was seen with 8 uM and 4.5 uM CU201 in H2126 and SHP77, respectively. Bradykinin at 0.1uM caused maximum flux (>95%) within seconds in SHP77 and H2126. No calcium flux was seen in H1703 and H69 even at 20 uM bradykinin. CU201 alone at concentrations ranging from 0.1-10 uM did not cause calcium flux. However, at concentrations ranging from 20-100 uM, there was a rapid and persistent calcium flux >99%, as a result of rapid cell death. CU201 at 1 uM effectively blocked calcium flux after 0.1 uM bradykinin was added to SHP77 and H2126 with over 3 minutes observation. Fresh plasma caused persistent calcium flux in seconds, while it took 2 minutes for thawed plasma to cause a persistent calcium flux. These persistent fluxes may be due to the presence of lipopolysaccharides (LPS) in plasma. Interestingly, heat-inactivated plasma alone only caused a transient calcium flux that resolved after 80 seconds. Heat-inactivated plasma with 1 uM CU201 (added at least 10 days prior) caused a transient calcium flux resolving in 15-30 seconds. After the consecutive addition of 0.1 uM bradykinin to heat-inactivated plasma with 1 uM CU201, progressively smaller transient calcium fluxes were seen, each lasting less than 15 seconds. This suggests the antagonistic activity of CU201 persisted after pretreatment in heat-inactivated plasma.Conclusions: CU201 inhibits calcium flux in cell lines with high BKR2 expression signal by microarray at doses less than IC₅₀ and 1/4 of necrosis₅₀ in SHP77 and H2126. CU201 alters the calcium flux pattern of heat-inactivated plasma. These results on plasma may be due to CU201 suppression of lipopolysaccharides in plasma and further testing is ongoing. Effective calcium flux blockade by CU201 is maintained in heat-inactivated plasma. Patient samples can be heat-inactivated and frozen for testing (for at least 10 days) for calcium flux inhibition in SHP77 and H2126 cell lines. Our study provides a method for analyzing the pharmacodynamic properties of CU201 in treated patients for a clinical phase I/II study.