Pharmaceutical Research

, Volume 30, Issue 5, pp 1409–1422 | Cite as

In Vivo and Ex Vivo Inhibition of Spinal Nerve Ligation-Induced Ectopic Activity by Sodium Channel Blockers Correlate to In Vitro Inhibition of NaV1.7 and Clinical Efficacy: A Pharmacokinetic-Pharmacodynamic Translational Approach

  • Ivana Kalezic
  • Lei Luo
  • Per-Eric Lund
  • Anders B Eriksson
  • Tjerk Bueters
  • Sandra A. G. Visser
Research Paper



In vivo and ex vivo inhibition of ectopic activity of clinically used and newly developed sodium channel (NaV) blockers were quantified in the rat spinal nerve ligation (SNL) model using a pharmacokinetic-pharmacodynamic (PKPD) approach and correlated to in vitro NaV1.7 channel inhibition and clinical effective concentrations.


In vivo, drug exposure and inhibition of ectopic activity were assessed in anaesthetized SNL rats at two dose levels. Ex vivo, compounds were applied at increasing concentrations to dorsal root ganglias isolated from SNL rats. The inhibitory potency (IC 50 ) was estimated using PKPD analysis. In vitro IC 50 was estimated using an electrophysiology-based assay using recombinant rat and human NaV1.7 expressing HEK293 cells.


In vivo and ex vivo inhibition of ectopic activity correlated well with the in vitro inhibition on the rat NaV1.7 channel. The estimated IC 50s for inhibition of ectopic activity in the SNL model occurred at similar unbound concentrations as clinical effective concentrations in humans.


Inhibition of ectopic activity in the SNL model could be useful in predicting clinical effective concentrations for novel sodium channel blockers. In addition, in vitro potency could be used for screening, characterization and selection of compounds, thereby reducing the need for in vivo testing.


ectopic activity NaV1.7 pharmacokinetics and pharmacodynamics sodium channels spinal nerve ligation rat model 



artificial cerebral spinal fluid


concentration in the biophase


congenital insensitivity to pain


concentration in plasma


dorsal root ganglion


effect at a certain concentration


baseline of effect


maximal attainable effect




concentration at which 50% inhibition is achieved


rate constant between plasma and biophase concentration


liquid chromatography with mass spectrometry detection


hill slope factor


voltage-gated sodium channel




spinal nerve ligation


Acknowledgments and Disclosures

The authors would like to thank Vibeke Täpp for technical help regarding the preparation of the animal model and Sveinn Briem and Yvonne Jaksch for their help with the bioanalysis of plasma and protein binding samples.

Supplementary material

11095_2013_979_MOESM1_ESM.jpg (709 kb)
Figure S1 In vivo electrophysiology observations for all non-selective sodium channel blockers tested. Left panels: Observed and fitted plasma concentrations in a simultaneous analysis with additional intravenous PK data (data not shown). Middle panels: observed and simultaneously fitted time-course of the inhibition of ectopic activity at two dose levels. Right panels: the relationship between the biophase concentration and the observed and predicted inhibition of ectopic activity. (JPEG 708 kb)
11095_2013_979_MOESM2_ESM.jpg (647 kb)
Figure S2 In vivo electrophysiology observations for newly developed sodium channel blockers tested. Left panels: Observed and fitted plasma concentrations in a simultaneous analysis with additional intravenous PK data (data not shown). Middle panels: observed and simultaneously fitted time-course of the inhibition of ectopic activity at two dose levels. Right panels: the relationship between the biophase concentration and the observed and predicted inhibition of ectopic activity. (JPEG 647 kb)


  1. 1.
    Dray A. Neuropathic pain: emerging treatments. Br J Anaesth. 2008;101(1):48–58.PubMedCrossRefGoogle Scholar
  2. 2.
    Priest BT, Kaczorowski GJ. Blocking sodium channels to treat neuropathic pain. Expert Opin Ther Targets. 2007;11(3):291–306.PubMedCrossRefGoogle Scholar
  3. 3.
    Bhattacharya A, Wickenden AD, Chaplan SR. Sodium channel blockers for the treatment of neuropathic pain. Neurotherapeutics. 2009;6(4):663–78.PubMedCrossRefGoogle Scholar
  4. 4.
    Krafte DS, Bannon AW. Sodium channels and nociception: recent concepts and therapeutic opportunities. Curr Opin Pharmacol. 2008;8(1):50–6.PubMedCrossRefGoogle Scholar
  5. 5.
    Cox JJ, Reimann F, Nicholas AK, Thornton G, Roberts E, Springell K, et al. An SCN9A channelopathy causes congenital inability to experience pain. Nature. 2006;444(7121):894–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Ahmad S, Dahllund L, Eriksson AB, Hellgren D, Karlsson U, Lund PE, et al. A stop codon mutation in SCN9A causes lack of pain sensation. Hum Mol Genet. 2007;16(17):2114–21.PubMedCrossRefGoogle Scholar
  7. 7.
    Cummins TR, Dib-Hajj SD, Waxman SG. Electrophysiological properties of mutant Nav1.7 sodium channels in a painful inherited neuropathy. J Neurosci. 2004;24(38):8232–6.PubMedCrossRefGoogle Scholar
  8. 8.
    Fertleman CR, Baker MD, Parker KA, Moffatt S, Elmslie FV, Abrahamsen B, et al. SCN9A mutations in paroxysmal extreme pain disorder: allelic variants underlie distinct channel defects and phenotypes. Neuron. 2006;52(5):767–74.PubMedCrossRefGoogle Scholar
  9. 9.
    Attal N, Bouhassira D. Translating basic research on sodium channels in human neuropathic pain. J Pain. 2006;7(1 Suppl 1):S31–7.PubMedGoogle Scholar
  10. 10.
    Dib-Hajj SD, Black JA, Waxman SG. Voltage-gated sodium channels: therapeutic targets for pain. Pain Med. 2009;10(7):1260–9.PubMedCrossRefGoogle Scholar
  11. 11.
    Gabrielsson J, Dolgos H, Gillberg PG, Bredberg U, Benthem B, Duker G. Early integration of pharmacokinetic and dynamic reasoning is essential for optimal development of lead compounds: strategic considerations. Drug Discov Today. 2009;14(7–8):358–72.PubMedCrossRefGoogle Scholar
  12. 12.
    Krekels EH, Angesjo M, Sjogren I, Moller KA, Berge OG, Visser SA. Pharmacokinetic-pharmacodynamic modeling of the inhibitory effects of naproxen on the time-courses of inflammatory pain, fever, and the ex vivo synthesis of TXB2 and PGE2 in rats. Pharm Res. 2011;28(7):1561–76.PubMedCrossRefGoogle Scholar
  13. 13.
    Van Der Graaf PH, Gabrielsson J. Pharmacokinetic-pharmacodynamic reasoning in drug discovery and early development. Future Med Chem. 2009;1(8):1371–4.CrossRefGoogle Scholar
  14. 14.
    Nakamura S, Atsuta Y. Effect of sodium channel blocker (mexiletine) on pathological ectopic firing pattern in a rat chronic constriction nerve injury model. J Orthop Sci. 2005;10(3):315–20.PubMedCrossRefGoogle Scholar
  15. 15.
    Yates JM, Smith KG, Robinson PP. The effect of carbamazepine on injury-induced ectopic discharge in the lingual nerve. Brain Res. 2005;1051(1–2):1–7.PubMedCrossRefGoogle Scholar
  16. 16.
    Ritter AM, Ritchie C, Martin WJ. Relationship between the firing frequency of injured peripheral neurons and inhibition of firing by sodium channel blockers. J Pain. 2007;8(4):287–95.PubMedCrossRefGoogle Scholar
  17. 17.
    Su X, Liang AH, Urban MO. The effect of amitriptyline on ectopic discharge of primary afferent fibers in the L5 dorsal root in a rat model of neuropathic pain. Anesth Analg. 2009;108(5):1671–9.PubMedCrossRefGoogle Scholar
  18. 18.
    Kirillova I, Teliban A, Gorodetskaya N, Grossmann L, Bartsch F, Rausch VH, et al. Effect of local and intravenous lidocaine on ongoing activity in injured afferent nerve fibers. Pain. 2011;152(7):1562–71.PubMedCrossRefGoogle Scholar
  19. 19.
    Macsari I, Sandberg L, Besidski Y, Gravenfors Y, Ginman T, Bylund J, et al. Phenyl isoxazole voltage-gated sodium channel blockers: structure and activity relationship. Bioorg Med Chem Lett. 2011;21(13):3871–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Kers I, Macsari I, Csjernyik G, Nylof M, Skogholm K, Sandberg L, et al. Phenethyl nicotinamides, a novel class of Na(V)1.7 channel blockers: structure and activity relationship. Bioorg Med Chem Lett 2012;22(19):6108–15.Google Scholar
  21. 21.
    Kers I, Csjernyik G, Macsari I, Nylof M, Sandberg L, Skogholm K, et al. Structure and activity relationship in the (S)-N-chroman-3-ylcarboxamide series of voltage-gated sodium channel blockers. Bioorg Med Chem Lett. 2012;22(17):5618–24.PubMedCrossRefGoogle Scholar
  22. 22.
    Macsari I, Besidski Y, Csjernyik G, Nilsson LI, Sandberg L, Yngve U, et al. 3-Oxoisoindoline-1-carboxamides: Potent, State-Dependent Blockers of Voltage-Gated Sodium Channel Na(V)1.7 with Efficacy in Rat Pain Models. J Med Chem. 2012;55(15):6866–80.PubMedCrossRefGoogle Scholar
  23. 23.
    Beaudoin S, Laufer-Sweiler MC, Markworth CJ, Marron BE, Millan DS, Rawson DJ. Sulfonamide derivatives. 2010. [Patent].Google Scholar
  24. 24.
    Liu X, Zhou JL, Chung K, Chung JM. Ion channels associated with the ectopic discharges generated after segmental spinal nerve injury in the rat. Brain Res. 2001;900(1):119–27.PubMedCrossRefGoogle Scholar
  25. 25.
    Schroeder K, Neagle B, Trezise DJ, Worley J. Ionworks HT: a new high-throughput electrophysiology measurement platform. J Biomol Screen. 2003;8(1):50–64.PubMedCrossRefGoogle Scholar
  26. 26.
    Bueters T, Dahlstrom J, Kvalvagnaes K, Betner I, Briem S. High-throughput analysis of standardized pharmacokinetic studies in the rat using sample pooling and UPLC-MS/MS. J Pharm Biomed Anal. 2011;55(5):1120–6.PubMedCrossRefGoogle Scholar
  27. 27.
    Briem S, Martinsson S, Bueters T, Skoglund E. Combined approach for high-throughput preparation and analysis of plasma samples from exposure studies. Rapid Commun Mass Spectrom. 2007;21(13):1965–72.PubMedCrossRefGoogle Scholar
  28. 28.
    Borgegård T, Minidis A, Jureus A, Malmborg J, Rosqvist S, Gruber S, et al. In vivo analysis using a presenilin-1-specific inhibitor: presenilin1-containing g-secretase complexes mediate the majority of CNS Ab production in the mouse. Alzheimer’s Dis Res J. 2011;3(1):30–45.Google Scholar
  29. 29.
    Sheiner LB, Stanski DR, Vozeh S, Miller RD, Ham J. Simultaneous modeling of pharmacokinetics and pharmacodynamics: application to d-tubocurarine. Clin Pharmacol Ther. 1979;25(3):358–71.PubMedGoogle Scholar
  30. 30.
    Liu X, Eschenfelder S, Blenk KH, Janig W, Habler H. Spontaneous activity of axotomized afferent neurons after L5 spinal nerve injury in rats. Pain. 2000;84(2–3):309–18.PubMedCrossRefGoogle Scholar
  31. 31.
    Devor M. Ectopic discharge in Abeta afferents as a source of neuropathic pain. Exp Brain Res. 2009;196(1):115–28.PubMedCrossRefGoogle Scholar
  32. 32.
    Gold MS. Spinal nerve ligation: what to blame for the pain and why. Pain. 2000;84(2–3):117–20.PubMedCrossRefGoogle Scholar
  33. 33.
    Pan HL, Eisenach JC, Chen SR. Gabapentin suppresses ectopic nerve discharges and reverses allodynia in neuropathic rats. J Pharmacol Exp Ther. 1999;288(3):1026–30.PubMedGoogle Scholar
  34. 34.
    Abdi S, Lee DH, Chung JM. The anti-allodynic effects of amitriptyline, gabapentin, and lidocaine in a rat model of neuropathic pain. Anesth Analg. 1998;87(6):1360–6.PubMedGoogle Scholar
  35. 35.
    Burchiel KJ. Carbamazepine inhibits spontaneous activity in experimental neuromas. Exp Neurol. 1988;102(2):249–53.PubMedCrossRefGoogle Scholar
  36. 36.
    Dick IE, Brochu RM, Purohit Y, Kaczorowski GJ, Martin WJ, Priest BT. Sodium channel blockade may contribute to the analgesic efficacy of antidepressants. J Pain. 2007;8(4):315–24.PubMedCrossRefGoogle Scholar
  37. 37.
    Tao H, Guia A, Xie B, Santaana D, Manalo G, Xu J, et al. Efficient characterization of use-dependent ion channel blockers by real-time monitoring of channel state. Assay Drug Dev Technol. 2006;4(1):57–64.PubMedCrossRefGoogle Scholar
  38. 38.
    Liu CN, Wall PD, Ben-Dor E, Michaelis M, Amir R, Devor M. Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury. Pain. 2000;85(3):503–21.PubMedCrossRefGoogle Scholar
  39. 39.
    Liu X, Chung K, Chung JM. Ectopic discharges and adrenergic sensitivity of sensory neurons after spinal nerve injury. Brain Res. 1999;849(1–2):244–7.PubMedCrossRefGoogle Scholar
  40. 40.
    Dayneka NL, Garg V, Jusko WJ. Comparison of four basic models of indirect pharmacodynamic responses. J Pharmacokinet Biopharm. 1993;21(4):457–78.PubMedCrossRefGoogle Scholar
  41. 41.
    Shimada S, Nakajima Y, Yamamoto K, Sawada Y, Iga T. Comparative pharmacodynamics of eight calcium channel blocking agents in Japanese essential hypertensive patients. Biol Pharm Bull. 1996;19(3):430–7.PubMedCrossRefGoogle Scholar
  42. 42.
    Visser SAG, Kalezic I, Luo L, Bueters T, Besidski Y, Nyström JE, et al. Pharmacokinetic-pharmacodynamic validation of in vivo and ex vivo electrophysiology markers for NaV1.7 inhibition in spinal nerve injured rats. In: Danhof M, Van der Graaf PH, Holford N, editors. Advances in simultaneous pharmacokinetic-pharmacodynamic modelling; 2010. p. 111–113. [Contribution to a Book].Google Scholar
  43. 43.
    Goldberg YP, Price N, Namdari R, Cohen CJ, Lamers MH, Winters C, et al. Treatment of Na(v)1.7-mediated pain in inherited erythromelalgia using a novel sodium channel blocker. Pain. 2012;153(1):80–5.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Ivana Kalezic
    • 1
  • Lei Luo
    • 1
  • Per-Eric Lund
    • 1
  • Anders B Eriksson
    • 1
  • Tjerk Bueters
    • 2
  • Sandra A. G. Visser
    • 3
  1. 1.Neuroscience, CNSP Innovative MedicinesAstraZeneca R&DSödertäljeSweden
  2. 2.DMPK, CNSP Innovative MedicinesAstraZeneca R&DSödertäljeSweden
  3. 3.Global DMPK, Centre of Excellence Innovative MedicinesAstraZeneca R&DSödertäljeSweden

Personalised recommendations