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.