Analgesic Effects of a Substituted N-Triazole Oxindole (TROX-1), a State-Dependent, Voltage-Gated Calcium Channel 2 Blocker

Abstract

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

Pharmacologic Inhibition of the Renal Outer Medullary Potassium Channel Causes Diuresis and Natriuresis in the Absence of Kaliuresis

“…35 S]MK-499 binding (Schmalhofer et al, 2010), the human voltage-gated sodium channel Nav1.5 (HEK-hNav1.5) using a fluorescence resonance energy transfer (FRET)-based membrane potential assay (Felix et al, 2004), and the L-type calcium channel Cav1.2 (HEK-hCav1.2) in a fluorescence calcium influx assay (Abbadie et al, 2010) have been previously described elsewhere.…”

Characterization of the SubstitutedN-Triazole Oxindole TROX-1, a Small-Molecule, State-Dependent Inhibitor of Cav2 Calcium Channels

“…TROX-1, a substituted N-triazole oxindole, is a Ca V 2.2 inhibitor which exhibits efficacy in a number of animal pain models with a therapeutic window for both cardiovascular and central nervous system side effects (Abbadie et al, 2010). Here we show electrophysiologically that TROX-1 inhibits Ca V 2.2 channels in both a state-dependent and use-dependent manner.…”

“…Blocking these Ca V 2.2 channels with conopeptides attenuates nociception in behavioral models of neuropathic and inflammatory pain (Malmberg and Yaksh, 1995;Scott et al, 2002). Furthermore, Ca V 2.2 knockout mice display reduced pain sensitivity in a number of pain models (Hatakeyama et al, 2001;Kim et al, 2001;Saegusa et al, 2001;Abbadie et al, 2010). Perhaps most convincing are the clinical data from ziconotide, a selective peptide blocker of Ca V 2.2 channels, which is efficacious in the treatment of chronic pain (Miljanich, 2004).…”

“…Ca V 2.2 channels are highly ex-pressed in laminae I and II of the spinal cord (Gohil et al, 1994;Westenbroek et al, 1998) and are up-regulated in behavioral pain models (Cizkova et al, 2002;Abbadie et al, 2010). Laminae I and II serve as critical relay points in the transmission of pain information into the central nervous system, where primary nociceptors make synaptic connections with dorsal horn neurons of the spinal cord.…”

Selective, Direct Activation of High-Conductance, Calcium-Activated Potassium Channels Causes Smooth Muscle Relaxation

“…The activity of test compounds on the voltage-gated sodium channel Nav1.5, and L-type calcium channel Cav1.2 was determined in functional assays, as described previously (Felix et al, 2004;Abbadie et al, 2010). The interaction of compounds with the hERG channel was evaluated in a [ 35 S]MK-499 binding assay to membranes prepared from HEK293 cells expressing hERG as described previously (Schmalhofer et al, 2010).…”