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).

对肝脏MB-COMT、S-COMT和脑部MB-COMT样本进行肾上腺素浓度梯度的培养，结果呈现浓度依赖性的甲氧基去甲肾上腺素（MN）的形成（见图1）。肝脏S-COMT、MB-COMT及脑部MB-COMT样本的动力学参数列于表1中。随后，通过固定蛋白量（2 mg/ml）及饱和浓度的肾上腺素（Km值的5倍；肝脏S-COMT为1000 µM，肝脏和脑部MB-COMT为10 µM）条件下，对新型COMT紧结合型抑制剂进行了肝MB-和S-COMT以及脑MB-COMT样本的活性测试（见图2）。抑制肝脏和脑部MB-和S-COMT活性的IC50值分别列于表2和表3中。将SK-HEP-1和SK-N-SH细胞暴露于1、10和50 µM的托卡朋或CNCAPE中，持续24小时，可导致细胞活力以浓度依赖性方式下降（见图3和图4）。此效应在细胞线性生长阶段（播种后48小时）及达到单层汇合时（播种后72小时）均十分明显。总体而言，与SK-HEP-1细胞相比，SK-N-SH细胞的细胞毒性更强。与SK-HEP-1细胞相比，SK-N-SH细胞对托卡朋和CNCAPE的不同浓度表现出更显著的细胞活力下降。在细胞线性生长阶段，细胞对托卡朋和CNCAPE的细胞毒性比对单层细胞的细胞毒性更为敏感。在细胞线性生长阶段，托卡朋和CNCAPE对肝脏SK-HEP-1细胞的细胞毒性在各浓度下均较为相似（见图3）。仅最高浓度（50 µM）导致了细胞活力的统计学上显著的下降。当SK-HEP-1细胞达到单层汇合时，如图3所示，仅最高浓度的CNCAPE导致了细胞活力的统计学上显著的下降，尽管两种COMT抑制剂均表现出浓度依赖性的细胞活力下降。如图4所示，在生长阶段的所有浓度下，托卡朋和CNCAPE对SK-N-SH细胞的细胞活力均产生了统计学上显著的下降。当SK-N-SH细胞达到单层汇合时，仅50 µM的托卡朋和10及50 µM的CNCAPE导致了细胞活力的统计学上显著的下降（见图4）。