A-803467, a Potent and Selective Nav1.8 Sodium Channel Blocker, Attenuates Neuropathic and Inflammatory Pain in the Rat

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A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat Michael F. Jarvis*†, Prisca Honore*, Char-Chang Shieh*, Mark Chapman‡, Shailen Joshi*, Xu-Feng Zhang*, Michael Kort*, William Carroll*, Brian Marron‡, Robert Atkinson‡, James Thomas‡, Dong Liu‡, Michael Krambis‡, Yi Liu‡, Steve McGaraughty*, Katharine Chu*, Rosemarie Roeloffs‡, Chengmin Zhong*, Joseph P. Mikusa*, Gricelda Hernandez*, Donna Gauvin*, Carrie Wade*, Chang Zhu*, Madhavi Pai*, Marc Scanio*, Lei Shi*, Irene Drizin*, Robert Gregg*, Mark Matulenko*, Ahmed Hakeem*, Michael Gross‡, Matthew Johnson‡, Kennan Marsh*, P. Kay Wagoner‡, James P. Sullivan*, Connie R. Faltynek*, and Douglas S. Krafte‡§ *Neuroscience Research, Abbott Laboratories, Abbott Park, IL 60064; and ‡Icagen, Inc., Durham, NC 27703 Edited by William A. Catterall, University of Washington School of Medicine, Seattle, WA, and approved March 25, 2007 (received for review December 20, 2006) Activation of tetrodotoxin-resistant sodium channels contributes pain or chronic local hyperalgesia also show increased Nav1.8 to action potential electrogenesis in neurons. Antisense oligonu- channel expression proximal to a peripheral injury site (12–14). cleotide studies directed against Nav1.8 have shown that this In the absence of subtype selective sodium channel blockers, channel contributes to experimental inflammatory and neuro- Nav1.8 knockout mice have provided insights for the specific role pathic pain. We report here the discovery of A-803467, a sodium of Nav1.8 channels in pain states (15–18). These mice do not express channel blocker that potently blocks tetrodotoxin-resistant cur- Nav1.8-mediated TTX-R currents in DRG neurons and show a -nM) and the generation of spontaneous and decreased sensitivity to mechanical stimuli and delayed develop 140 ؍ rents (IC50 electrically evoked action potentials in vitro in rat dorsal root ment of thermal hyperalgesia. However, interpretation of the ganglion neurons. In recombinant cell lines, A-803467 potently Nav1.8 knockout phenotype has been complicated by a compen- .(nM) and was >100-fold selective satory increased expression of other sodium channel subtypes (19 8 ؍ blocked human Nav1.8 (IC50 > ␮ In addition, nociceptor-specific knockout of Nav1.8 and another vs. human Nav1.2, Nav1.3, Nav1.5, and Nav1.7 (IC50 values 1 M). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of sodium channel did not prevent the development of neuropathic wide dynamic range neurons in the rat spinal dorsal horn. pain in the mouse (20). A-803467 also dose-dependently reduced mechanical allodynia in Additional evidence for Nav1.8 involvement in transduction of nociceptive information was derived from studies using antisense ؍ a variety of rat pain models including: spinal nerve ligation (ED50 (AS) oligodeoxynucleotides (ODNs) (11, 21–23). Selective knock- -mg/kg, i.p.), capsa 85 ؍ mg/kg, i.p.), sciatic nerve injury (ED50 47 down of Na 1.8 protein after Na 1.8 AS ODN treatment reversed icin-induced secondary mechanical allodynia (ED50 Ϸ 100 mg/kg, v v i.p.), and thermal hyperalgesia after intraplantar complete mechanical allodynia and thermal hyperalgesia after peripheral inflammation and nerve injury (22, 24). This Nav1.8 knockdown mg/kg, i.p.). A-803467 was 41 ؍ Freund’s adjuvant injection (ED50 inactive against formalin-induced nociception and acute thermal strategy has also demonstrated Nav1.8 involvement in afferent and postoperative pain. These data demonstrate that acute and nerve sensitization after chemical irritation of the bladder and visceral pain (23). Collectively, these findings strongly support a key selective pharmacological blockade of Nav1.8 sodium channels in vivo produces significant antinociception in animal models of role for Nav1.8 in regulating the properties of nociceptive neurons and, consequently, nociceptive behaviors. neuropathic and inflammatory pain. Although it is widely appreciated that blockade of sodium allodynia ͉ electrophysiology ͉ hyperalgesia ͉ sensory neurons channels contributes to the analgesic activity of a large number of nonopioid analgesics like lamotrigine and amitriptyline (19, 25, 26), the identification of specific blockers of individual sodium channels he excitability of peripheral nerves is governed by the expres- Tsion of voltage-gated sodium channels (1). Certain tetrodotox- in-resistant (TTX-R) sodium channels have high activation thresh- Author contributions: M.F.J., P.H., C.-C.S., M. Kort, W.C., B.M., R.K.W., J.P.S., C.R.F., and olds, slow inactivation kinetics, and channel opening leads directly D.S.K. designed research; P.H., C.-C.S., M.C., S.J., X.-F.Z., R.A., J.T., D.L., M. Krambis, Y.L., S.M., K.C., R.R., C. Zhong, J.P.M., G.H., D.G., C.W., C. Zhu, M.P., M.S., L.S., I.D., R.G., M.M., to the generation of action potentials (2, 3). Nav1.8 is a TTX-R A.H., M.G., M.J., and K.M. performed research; M. Kort, W.C., B.M., R.A., J.T., M.S., I.D., R.G., sodium channel with these properties and is highly expressed in L.S., M.M., A.H., M.G., and M.J. contributed new reagents/analytic tools; M.F.J., P.H., C.-C.S., small-diameter sensory neurons (4, 5). The expression and bio- M.C., S.J., S.M., K.M., P.K.W., J.P.S., C.R.F., and D.S.K. analyzed data; and M.F.J., P.H., and D.S.K. wrote the paper. physical properties of Nav1.8 channels can be modulated by ongoing Conflict of interest statement: All authors are employees of either Abbott Laboratories or nociceptive input. For example, there is increased Nav1.8 channel Icagen, Inc. expression in the rat digital nerve and dorsal root ganglion (DRG) after intraplantar administration of complete Freund’s adjuvant This article is a PNAS Direct Submission. (CFA) or carrageenan (CARR), respectively (6, 7). Similarly, local Abbreviations: CARR, carrageenan; CFA, complete Freund’s adjuvant; DRG, dorsal root ganglion; TTX, tetrodotoxin; TTX-R, TTX-resistant; WDR, wide dynamic range. administration of prostaglandin-E2, adenosine, or serotonin pro- See Commentary on page 8205. duces an increase in TTX-R sodium current magnitude in periph- †To whom correspondence may be addressed at: Abbott Laboratories, R-4PM, AP9A/3,100 eral nerves through modulation of the voltage-dependent proper- Abbott Park Road, Abbott Park, IL 60064. E-mail: [email protected]. tiesofNav1.8 and other sodium channels (8–10). Direct nerve §To whom correspondence may be addressed at: Icagen, Inc., P.O. Box 14487, Research injury (e.g., L5/L6 spinal nerve ligation) also leads to immunocy- Triangle Park, NC 27709. E-mail: [email protected]. tochemical and electrophysiological changes in Nav1.8 channels This article contains supporting information online at www.pnas.org/cgi/content/full/ suggesting a potential role for this channel in the sensation of 0611364104/DC1. neuropathic pain (11). Human patients with chronic neurogenic © 2007 by The National Academy of Sciences of the USA 8520–8525 ͉ PNAS ͉ May 15, 2007 ͉ vol. 104 ͉ no. 20 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611364104 Downloaded by guest on September 30, 2021 Table 1. A-803467 selectively blocks human Nav1.8 sodium channels SEE COMMENTARY IC50, ␮M † V0*V1/2 hNaV1.8 0.079 Ϯ 0.01 0.008 Ϯ 0.002 hNaV1.2 9.49 Ϯ 0.23 7.38 Ϯ 0.17 hNaV1.3 11.76 Ϯ 0.91 2.45 Ϯ 0.05 hNaV1.5 32.82 Ϯ 4.89 7.34 Ϯ 0.21 hNaV1.7 35.34 Ϯ 0.30 6.74 Ϯ 0.03 *Resting state protocol: the prepulse voltage at which 100% of channels are available to be activated. (V0 ϭϪ100 mV for hNaV1.8; V0 ϭϪ120 mV for Fig. 1. A-803467 is a selective blocker of Nav1.8 channels. (A) Chemical hNaV1.2, hNaV1.3, hNaV1.7; V0 ϭϪ150 mV for hNaV1.5.) structure of A-803467. (B) Block of recombinant and native Nav1.8 currents by †Inactivated state protocol: the prepulse voltage at which 50% of channels are 100 nM A-803467. Current records were generated during voltage steps to 0 inactivated. (V1/2 ϭϪ40 mV for hNaV1.8; V1/2 ϭϪ60 mV for hNaV1.2, hNaV1.3, mV in control and after application of A-803467 (100 nM) for human Nav1.8 or hNa 1.7; V ϭϪ90 mV for hNa 1.5.) native DRG TTX-R currents. Holding potentials were set at Ϫ100 mV, and each V 1/2 V test pulse was preceded by an 8-s prepulse to Ϫ40 mV, followed by 20 ms at Ϫ100 mV before a 20-ms depolarization to 0 mV. [Scale bars, 0.3 nA and 5 ms TTX-R currents with IC values of Ͼ30 ␮M (data not shown). (Nav1.8) and 1.5 nA and 5 ms (TTX-R).] (C) Representative concentration– 50 response curves were generated after application of different concentrations Small-diameter DRG neurons express at least two subtypes of of A-803467 and assessment of current block for human Nav1.8 (IC50 ϭ 8 Ϯ 1 TTX-R currents, NaV1.8 and NaV1.9 (27, 28). Under the present ϭ Ϯ nM), and TTX-R currents (IC50 140 14 nM) in rat DRG cells and hNav1.2, experimental conditions, the contribution of NaV1.9 to the peak Ϯ hNav1.3, hNav1.5, and hNav1.7. Data represent means SEM from at least current evoked at 0 mV was minimal (27) (M.C., unpublished three separate experiments. Percent inhibition was calculated as 1 Ϫ (test observations). At a resting state, A-803467 also potently blocked current/(control current ϩ wash current/2)) and plotted vs. test concentration. human Nav1.8 channels (IC50 ϭ 79 nM, Table 1).
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  • The Nav1.7 Sodium Channel: from Molecule to Man

    The Nav1.7 Sodium Channel: from Molecule to Man

    REVIEWS The NaV1.7 sodium channel: from molecule to man Sulayman D. Dib-Hajj1,2, Yang Yang1,2, Joel A. Black1,2 and Stephen G. Waxman1,2 Abstract | The voltage-gated sodium channel NaV1.7 is preferentially expressed in peripheral somatic and visceral sensory neurons, olfactory sensory neurons and sympathetic ganglion neurons. NaV1.7 accumulates at nerve fibre endings and amplifies small subthreshold depolarizations, poising it to act as a threshold channel that regulates excitability. Genetic and functional studies have added to the evidence that NaV1.7 is a major contributor to pain signalling in humans, and homology modelling based on crystal structures of ion channels suggests an atomic-level structural basis for the altered gating of mutant NaV1.7 that causes pain. Neuropathic pain Voltage-gated sodium channels are essential for electro- that are not seen when these channels are expressed in 11 Pain resulting from lesions or genesis in excitable cells. Nine pore-forming α‑subunits HEK 293 cells . Methods are now available that allow the diseases of the somatosensory of such channels (referred to as channels hereinafter), expression and functional profiling of sodium channels in system. 1 NaV1.1–NaV1.9, have been identified in mammals . peripheral neurons, which more closely mimic the in vivo These isoforms share a common overall structural environment of such channels12. motif (FIG. 1). They are each composed of a long poly- In humans, gain‑of‑function mutations in SCN9A, neuropathic pain peptide (1,700–2,000 amino acids) that folds into four which encodes NaV1.7, lead to severe , homologous domains (DI–DIV) that are linked by three whereas loss‑of‑function mutations in this gene lead to loops (L1–L3), with each domain having six transmem- an indifference to pain13.