Department of Ion Channels, Merck Research Laboratories, Rahway, New Jersey, USA.
Mol Pharmacol. 2012 Mar;81(3):488-97. doi: 10.1124/mol.111.075226. Epub 2011 Dec 21.
Biological, genetic, and clinical evidence provide validation for N-type calcium channels (Ca(V)2.2) as therapeutic targets for chronic pain. A state-dependent Ca(V)2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl Ca(V)2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent Ca(V)2 inhibitor (3R)-5-(3-chloro-4-fluorophenyl)-3-methyl-3-(pyrimidin-5-ylmethyl)-1-(1H-1,2,4-triazol-3-yl)-1,3-dihydro-2H-indol-2-one (TROX-1) has an improved therapeutic window compared with ziconotide. Here we characterize TROX-1 inhibition of Cav2.2 channels in more detail. When channels are biased toward open/inactivated states by depolarizing the membrane potential under voltage-clamp electrophysiology, TROX-1 inhibits Ca(V)2.2 channels with an IC(50) of 0.11 μM. The voltage dependence of Ca(V)2.2 inhibition was examined using automated electrophysiology. TROX-1 IC(50) values were 4.2, 0.90, and 0.36 μM at -110, -90, and -70 mV, respectively. TROX-1 displayed use-dependent inhibition of Ca(V)2.2 with a 10-fold IC(50) separation between first (27 μM) and last (2.7 μM) pulses in a train. In a fluorescence-based calcium influx assay, TROX-1 inhibited Ca(V)2.2 channels with an IC(50) of 9.5 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the Ca(V)2 subfamily. Depolarized IC(50) values were 0.29, 0.19, and 0.28 μM by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 μM by calcium influx for Ca(V)2.1, Ca(V)2.2, and Ca(V)2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non-subtype-selective Ca(V)2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent Ca(V)2.2-selective inhibitor ziconotide in preclinical models of chronic pain.
生物、遗传和临床证据为 N 型钙通道（Ca(V)2.2）作为慢性疼痛治疗靶点提供了验证。与临床上使用的肽基 Ca(V)2.2 抑制剂（如 ziconotide）相比，状态依赖性 Ca(V)2.2 抑制剂可能提供更好的治疗窗口。支持这一观点，我们最近报道称，在临床前模型中，状态依赖性钙通道抑制剂（3R）-5-（3-氯-4-氟苯基）-3-甲基-3-（嘧啶-5-基甲基）-1-（1H-1,2,4-三唑-3-基）-1,3-二氢-2H-吲哚-2-酮（TROX-1）与 ziconotide 相比具有更好的治疗窗口。在这里，我们更详细地描述了 TROX-1 对 Ca(V)2.2 通道的抑制作用。当膜电位在电压钳电生理学中被去极化而使通道偏向开放/失活状态时，TROX-1 以 0.11 μM 的 IC50 抑制 Ca(V)2.2 通道。使用自动电生理学研究了 Ca(V)2.2 抑制的电压依赖性。TROX-1 的 IC50 值分别为-110 mV 时为 4.2、-90 mV 时为 0.90 和-70 mV 时为 0.36 μM。TROX-1 对 Ca(V)2.2 表现出使用依赖性抑制，在一串中的第一个（27 μM）和最后一个（2.7 μM）脉冲之间，10 倍 IC50 分离。在基于荧光的钙内流测定中，TROX-1 在超极化条件下以 9.5 μM 的 IC50 抑制 Ca(V)2.2 通道，在去极化条件下以 0.69 μM 的 IC50 抑制 Ca(V)2.2 通道。最后，研究了 TROX-1 在 Ca(V)2 亚家族中的效力。使用匹配条件的手动电生理学测量的去极化 IC50 值分别为 0.29、0.19 和 0.28 μM，而钙离子内流的 IC50 值分别为 0.69、1.8 和 1.1 μM，用于 Ca(V)2.1、Ca(V)2.2 和 Ca(V)2.3。这些体外数据共同支持这样一种观点，即与相对状态独立的 Ca(V)2.2 选择性抑制剂（如 ziconotide）相比，状态依赖性、非亚型选择性 Ca(V)2 通道抑制剂可以在慢性疼痛的临床前模型中实现更好的治疗窗口。
