Absence seizures are a common seizure type in children with genetic generalized epilepsy and are characterized by a temporary loss of awareness, arrest of physical activity, and accompanying spike-and-wave discharges on an electroencephalogram. They arise from abnormal, hypersynchronous neuronal firing in brain thalamocortical circuits. Currently available therapeutic agents are only partially effective and act on multiple molecular targets, including γ-aminobutyric acid (GABA) transaminase, sodium channels, and calcium (Ca(2+)) channels. We sought to develop high-affinity T-type specific Ca(2+) channel antagonists and to assess their efficacy against absence seizures in the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Using a rational drug design strategy that used knowledge from a previous N-type Ca(2+) channel pharmacophore and a high-throughput fluorometric Ca(2+) influx assay, we identified the T-type Ca(2+) channel blockers Z941 and Z944 as candidate agents and showed in thalamic slices that they attenuated burst firing of thalamic reticular nucleus neurons in GAERS. Upon administration to GAERS animals, Z941 and Z944 potently suppressed absence seizures by 85 to 90% via a mechanism distinct from the effects of ethosuximide and valproate, two first-line clinical drugs for absence seizures. The ability of the T-type Ca(2+) channel antagonists to inhibit absence seizures and to reduce the duration and cycle frequency of spike-and-wave discharges suggests that these agents have a unique mechanism of action on pathological thalamocortical oscillatory activity distinct from current drugs used in clinical practice.

Using human Kv1.5 channels expressed in HEK293 cells we assessed the ability of H+o to mimic the previously reported action of Zn2+ to inhibit macroscopic hKv1.5 currents, and using site‐directed mutagenesis, we addressed the mechanistic basis for the inhibitory effects of H+o and Zn2+. As with Zn2+, H+o caused a concentration‐dependent, K+o‐sensitive and reversible reduction of the maximum conductance (gmax). With zero, 5 and 140 mm K+o the pKH for this decrease of gmax was 6.8, 6.2 and 6.0, respectively. The concentration dependence of the block relief caused by increasing [K+]o was well fitted by a non‐competitive interaction between H+o and K+o, for which the KD for the K+ binding site was 0.5‐1.0 mm. Additionally, gating current analysis in the non‐conducting mutant hKv1.5 W472F showed that changing from pH 7.4 to pH 5.4 did not affect Qmax and that charge immobilization, presumed to be due to C‐type inactivation, was preserved at pH 5.4. Inhibition of hKv1.5 currents by H+o or Zn2+ was substantially reduced by a mutation either in the channel turret (H463Q) or near the pore mouth (R487V). In light of the requirement for R487, the homologue of Shaker T449, as well as the block‐relieving action of K+o, we propose that H+ or Zn2+ binding to histidine residues in the pore turret stabilizes a channel conformation that is most likely an inactivated state.

Voltage-gated calcium channel (Ca v )2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Ca v 2.2 channels, is efficacious in treating refractory pain but exhibits a narrow therapeutic window and must be administered intrathecally. We have discovered an N-triazole oxindole, (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), as a small-molecule, state-dependent blocker of Ca v 2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Ca v 2.2 channels under depolarized conditions (IC 50 ϭ 0.27 M) compared with hyperpolarized conditions (IC 50 Ͼ 20 M). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited -conotoxin GVIA-sensitive calcium currents (Ca v 2.2 channel currents), with greater potency under depolarized conditions (IC 50 ϭ 0.4 M) than under hyperpolarized conditions (IC 50 ϭ 2.6 M), indicating state-dependent Ca v 2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Ca v 2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Ca v 2 family channels. TROX-1 reversed inflammatory-induced hyperalgesia with maximal effects equivalent to nonsteroidal anti-inflammatory drugs, and it reversed nerve injury-induced allodynia to the same extent as pregabalin and duloxetine. In contrast, no significant reversal of hyperalgesia was observed in Ca v 2.2 gene-deleted mice. Mild impairment of motor function in the Rotarod test and cardiovascular functions were observed at 20-to 40-fold higher plasma concentrations than required for analgesic activities. TROX-1 demonstrates that an orally available state-dependent Ca v 2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.Inflammatory diseases and neuropathic insults are frequently accompanied by severe debilitating pain, which can become chronic and unresponsive to conventional analgesic treatments. Intrathecal administration of conventional agents, including morphine, may be required in more severe C.A. and O.B.M. contributed equally to this work. Article, publication date, and citation information can be found at

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.

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 M. 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 M at Ϫ110, Ϫ90, and Ϫ70 mV, respectively. TROX-1 displayed usedependent inhibition of Ca V 2.2 with a 10-fold IC 50 separation between first (27 M) and last (2.7 M) 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 M under hyperpolarized conditions and 0.69 M 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 M by manual electrophysiology using matched conditions and 1.8, 0.69, and 1.1 M 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.