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).将肝脏MB-COMT（膜结合型儿茶酚-O-甲基转移酶）、S-COMT（可溶性儿茶酚-O-甲基转移酶）及脑组织MB-COMT样本与梯度浓度的肾上腺素共同孵育，可生成浓度依赖性的变肾上腺素（图1）。肝脏S-COMT、MB-COMT及脑组织MB-COMT样本的动力学参数详见表1。随后，我们采用固定蛋白量（2 mg/ml），在饱和浓度肾上腺素（为对应Km值的5倍：肝脏S-COMT为1000 µM，肝脏及脑组织MB-COMT为10 µM）存在的条件下，针对新型COMT紧密结合抑制剂，开展了针对肝脏MB-COMT、S-COMT及脑组织MB-COMT样本的活性测试（图2）。肝脏及脑组织中MB-COMT与S-COMT活性抑制的半数抑制浓度（IC50）详见表2与表3。将SK-HEP-1与SK-N-SH细胞暴露于1、10及50 µM的托卡朋（tolcapone）或CNCAPE中孵育24小时，可引发浓度依赖性的细胞活力降低（图3与图4）。该浓度依赖性效应在两种细胞系的线性生长阶段（接种后48小时）及单层汇合状态（接种后72小时）下均显著可见。总体而言，SK-N-SH细胞的细胞毒性效应强于SK-HEP-1细胞；相较于SK-HEP-1细胞，SK-N-SH细胞在不同浓度托卡朋与CNCAPE处理下的细胞活力降幅也更为显著。相较于处于单层汇合状态的细胞，处于线性生长阶段的细胞通常对托卡朋与CNCAPE的细胞毒性更为敏感。在处于线性生长阶段的肝脏SK-HEP-1细胞中，各浓度下托卡朋与CNCAPE的细胞毒性水平基本相当（图3）；仅最高浓度（50 µM）可引发具有统计学显著性的细胞活力降低。如图3所示，在达到单层汇合状态的SK-HEP-1细胞中，尽管两种COMT抑制剂均呈现出浓度依赖性的细胞活力降低趋势，但仅最高浓度的CNCAPE可引发具有统计学显著性的细胞活力下降。如图4所示，对处于生长阶段的SK-N-SH细胞施加各浓度的托卡朋与CNCAPE，均可引发具有统计学显著性的细胞活力降低。在达到单层汇合状态的SK-N-SH细胞中，仅50 µM的托卡朋以及10 µM、50 µM的CNCAPE可引发具有统计学显著性的细胞活力降低（图4）。