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

Voltage-gated calcium channel (Cav)2.2 (N-type calcium channels) are key components in nociceptive transmission pathways. Ziconotide, a state-independent peptide inhibitor of Cav2.2 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 Cav2 channels, and we investigated the therapeutic advantages of this compound for analgesia. TROX-1 preferentially inhibited potassium-triggered calcium influx through recombinant Cav2.2 channels under depolarized conditions (IC50 = 0.27 μM) compared with hyperpolarized conditions (IC50 > 20 μM). In rat dorsal root ganglion (DRG) neurons, TROX-1 inhibited ω-conotoxin GVIA-sensitive calcium currents (Cav2.2 channel currents), with greater potency under depolarized conditions (IC50 = 0.4 μM) than under hyperpolarized conditions (IC50 = 2.6 μM), indicating state-dependent Cav2.2 channel block of native as well as recombinant channels. TROX-1 fully blocked calcium influx mediated by a mixture of Cav2 channels in calcium imaging experiments in rat DRG neurons, indicating additional block of all Cav2 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 Cav2.2 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 Cav2 channel blocker may achieve a therapeutic window suitable for the treatment of chronic pain.

[1]  Miljanich Gp,et al.  Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. , 2004 .

[2]  Sheryl E. Koch,et al.  Functional disorders of the sympathetic nervous system in mice lacking the α1B subunit (Cav 2.2) of N-type calcium channels , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[3]  T. Yaksh,et al.  Quantitative assessment of tactile allodynia in the rat paw , 1994, Journal of Neuroscience Methods.

[4]  D. Kullmann,et al.  Dysfunction of the brain calcium channel CaV2.1 in absence epilepsy and episodic ataxia. , 2005, Brain : a journal of neurology.

[5]  G. Zamponi,et al.  Molecular Pharmacology of High Voltage-Activated Calcium Channels , 2003, Journal of bioenergetics and biomembranes.

[6]  J. Sandkühler,et al.  Pre‐ and postsynaptic contributions of voltage‐dependent Ca2+ channels to nociceptive transmission in rat spinal lamina I neurons , 2004, The European journal of neuroscience.

[7]  T. Aoki,et al.  Spinal mechanism of standard analgesics: evaluation using mouse models of allodynia. , 2010, European journal of pharmacology.

[8]  R. Tsien,et al.  Ablation of P/Q-type Ca(2+) channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the alpha(1A)-subunit. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  K. Valentino,et al.  Cardiovascular effects of omega-conopeptides in conscious rats: mechanisms of action. , 1992, Journal of cardiovascular pharmacology.

[10]  E. Mathews,et al.  Splicing of alpha 1A subunit gene generates phenotypic variants of P- and Q-type calcium channels. , 1999, Nature Neuroscience.

[11]  H. Schaible,et al.  Effects of antagonists to high-threshold calcium channels upon spinal mechanisms of pain, hyperalgesia and allodynia , 2000, Pain.

[12]  Yongxiang Wang,et al.  Interactions of intrathecally administered ziconotide, a selective blocker of neuronal N-type voltage-sensitive calcium channels, with morphine on nociception in rats , 2000, PAIN®.

[13]  Primary Afferent Depolarization Evoked by a Painful Stimulus , 1969, Science.

[14]  M. Williams,et al.  Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. , 1992, Science.

[15]  F. Colpaert,et al.  Evidence that adjuvant arthritis in the rat is associated with chronic pain , 1987, Pain.

[16]  Miljanich Gp,et al.  Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. , 2004, Current medicinal chemistry.

[17]  David John Adams,et al.  ω-Conotoxin inhibition of excitatory synaptic transmission evoked by dorsal root stimulation in rat superficial dorsal horn , 2008, Neuropharmacology.

[18]  J. Hell,et al.  Molecular cloning of the alpha-1 subunit of an omega-conotoxin-sensitive calcium channel. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Vasko,et al.  Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons , 1996, Brain Research.

[20]  G. Wang,et al.  Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. , 1998, Biochemistry.

[21]  D. Pruneau,et al.  Haemodynamic and humoral effects of omega-conotoxin GVIA in normotensive and spontaneously hypertensive rats. , 1992, European journal of pharmacology.

[22]  Dong Kwan Kim,et al.  Altered Nociceptive Response in Mice Deficient in the α1B Subunit of the Voltage-Dependent Calcium Channel , 2001, Molecular and Cellular Neuroscience.

[23]  M. Nishida,et al.  Ca(2+) channel alpha(1B) subunit (Ca(V) 2.2) knockout mouse reveals a predominant role of N-type channels in the sympathetic regulation of the circulatory system. , 2002, Trends in cardiovascular medicine.

[24]  William B. Dobyns,et al.  Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel , 1997, Nature Genetics.

[25]  J. Angus,et al.  Actions of intrathecal omega-conotoxins CVID, GVIA, MVIIA, and morphine in acute and neuropathic pain in the rat. , 2002, European journal of pharmacology.

[26]  W. Jänig.  Hyperalgesia and Allodynia , 1993 .

[27]  R. Luther,et al.  Selective N-type neuronal voltage-sensitive calcium channel blocker, SNX-111, produces spinal antinociception in rat models of acute, persistent and neuropathic pain. , 1996, The Journal of pharmacology and experimental therapeutics.

[28]  J. Angus,et al.  Postural hypotension following N‐type Ca2+ channel blockade is amplified in experimental hypertension , 2000, Journal of hypertension.

[29]  O. McManus,et al.  A high-throughput assay for evaluating state dependence and subtype selectivity of Cav2 calcium channel inhibitors. , 2008, Assay and drug development technologies.

[30]  M. Adams,et al.  CALCIUM CHANNEL DIVERSITY AND NEUROTRANSMITTER RELEASE : THE OMEGA -CONOTOXINS AND OMEGA -AGATOXINS , 1994 .

[31]  T. Soong,et al.  Splicing of α1A subunit gene generates phenotypic variants of P- and Q-type calcium channels , 1999, Nature Neuroscience.

[32]  Dennis E Bulman,et al.  Familial Hemiplegic Migraine and Episodic Ataxia Type-2 Are Caused by Mutations in the Ca2+ Channel Gene CACNL1A4 , 1996, Cell.

[33]  Á. Díaz,et al.  Blockade of spinal N- and P-type, but not L-type, calcium channels inhibits the excitability of rat dorsal horn neurones produced by subcutaneous formalin inflammation , 1997, Pain.

[34]  Jin Mo Chung,et al.  An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat , 1992, PAIN.

[35]  J. Ludbrook,et al.  A method for the analysis of sigmoid stimulus-response curves. , 1979, The Australian journal of experimental biology and medical science.

[36]  Yu-Qing Cao,et al.  Voltage-gated calcium channels and pain , 2006, Pain.

[37]  A. Takahara,et al.  Recent updates of N-type calcium channel blockers with therapeutic potential for neuropathic pain and stroke. , 2009, Current topics in medicinal chemistry.

[38]  D. Kullmann,et al.  Dysfunction of the brain calcium channel CaV2.1 in absence epilepsy and episodic ataxia , 2004 .

[39]  T. Kanazawa,et al.  Purification of cardiac sarcolemmal vesicles: high sodium pump content and ATP-dependent, calmodulin-activated calcium uptake. , 1982, Journal of biochemistry.

[40]  T. Yaksh,et al.  Localization of N-type Ca2+ channels in the rat spinal cord following chronic constrictive nerve injury , 2002, Experimental Brain Research.

[41]  V. Gribkoff,et al.  Use-dependent blockade of Cav2.2 voltage-gated calcium channels for neuropathic pain. , 2005, Biochemical pharmacology.

[42]  A. Malmberg,et al.  Effect of continuous intrathecal infusion of ω-conopeptides, N-type calcium-channel blockers, on behavior and antinociception in the formalin and hot-plate tests in rats , 1995, Pain.

[43]  W. Catterall,et al.  Localization of Ca2+ Channel Subtypes on Rat Spinal Motor Neurons, Interneurons, and Nerve Terminals , 1998, The Journal of Neuroscience.

[44]  G. Kaczorowski,et al.  Evidence for distinct sites coupled to high affinity omega-conotoxin receptors in rat brain synaptic plasma membrane vesicles. , 1988, Biochemical and biophysical research communications.

[45]  Michael E. Adams,et al.  P-type calcium channels in rat central and peripheral neurons , 1992, Neuron.