Abstract

Biological, genetic, and clinical evidence provide validation for N-type calcium channels (Ca_V2.2) as therapeutic targets for chronic pain. A state-dependent Ca_V2.2 inhibitor may provide an improved therapeutic window over ziconotide, the peptidyl Ca_V2.2 inhibitor used clinically. Supporting this notion, we recently reported that in preclinical models, the state-dependent Ca_V2 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_V2.2 channels with an IC₅₀ of 0.11 μM. The voltage dependence of Ca_V2.2 inhibition was examined using automated electrophysiology. TROX-1 IC₅₀ 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_V2.2 with a 10-fold IC₅₀ 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_V2.2 channels with an IC₅₀ of 9.5 μM under hyperpolarized conditions and 0.69 μM under depolarized conditions. Finally, TROX-1 potency was examined across the Ca_V2 subfamily. Depolarized IC₅₀ 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_V2.1, Ca_V2.2, and Ca_V2.3, respectively. Together, these in vitro data support the idea that a state-dependent, non–subtype-selective Ca_V2 channel inhibitor can achieve an improved therapeutic window over the relatively state-independent Ca_V2.2-selective inhibitor ziconotide in preclinical models of chronic pain.