Pharmacodynamic Evaluation of novel Catechol-O-methyltransferase Inhibitors

TThe incubation of liver MB-COMT and S-COMT and brain MB-COMT samples with increasing concentrations of adrenaline resulted in the concentration-dependent formation of metanephrine (Figure 1). The kinetic parameters for liver S-COMT and MB-COMT and brain MB-COMT samples are given in Table 1. The novel COMT tight-binding inhibitors were then tested against liver MB- and S-COMT and brain MB-COMT samples using a fixed amount of protein (2 mg/ml), in the presence of a saturating concentration of adrenaline (5 times the corresponding Km; 1000 µM for liver S-COMT and 10 µM for liver and brain MB-COMT) (Figure 2). The IC50 values for inhibition of MB- and S-COMT activity in liver and brain are given in Tables 2 and 3. Exposure of SK-HEP-1 and SK-N-SH cells to 1, 10 and 50 µM tolcapone or CNCAPE for 24 h reduced cell viability in a concentration-dependent manner (Figures 3 and 4). This pattern was apparent in both cell lines during their linear growth phase (at 48 h post seeding) and when reaching monolayer confluence (72 h post seeding). In general, the cytotoxicity was greater for SK-N-SH cells, in comparison to SK-HEP-1 cells. SK-N-SH cells also showed a greater reduction in cell viability with different concentrations of tolcapone and CNCAPE, in comparison to SK-HEP-1 cells. Cells in the linear growth phase were generally more susceptible to the cytotoxic effects of tolcapone and CNCAPE, in comparison to cells in monolayer. The cytotoxicity of tolcapone and CNCAPE was quite similar for each concentration in liver SK-HEP-1 cells during their linear growth phase (Figure 3). Only the highest concentration (50 µM) produced a statistically significant decrease in cell viability. In SK-HEP-1 cells when reaching monolayer confluence, as shown in Figure 3, only the highest concentration of CNCAPE produced a statistically significant decrease in cell viability, although there was a concentration-dependent decrease in cell viability for both COMT inhibitors. As shown in Figure 4, all concentrations of tolcapone and CNCAPE used on SK-N-SH cells in growth phase produced a statistically significant decrease in cell viability. In SK-N-SH cells when reaching monolayer confluence, only 50 µM of tolcapone and 10 and 50 µM of CNCAPE produced a statistically significant decreases in cell viability (Figure 4).