WO2012162732A1

WO2012162732A1 - Assays for sodium ion channel modulators and uses thereof

Info

Publication number: WO2012162732A1
Application number: PCT/AU2012/000597
Authority: WO
Inventors: Richard James Lewis; Irina VETTER
Original assignee: University of Queensland UQ
Current assignee: University of Queensland UQ
Priority date: 2011-06-02
Filing date: 2012-05-29
Publication date: 2012-12-06
Anticipated expiration: 2013-12-02

Abstract

This invention relates generally to novel screening assays for modulating sodium channels, particularly voltage-gated sodium channels. The assays employ mammalian cells that endogenously express a voltage-gated sodium channel in the context of one or more endogenously co-expressed β subunits and accessory β subunits. The mammalian cells are useful in high throughput assays for identifying drugs with therapeutic value against diseases or conditions associated with sodium channel activity including pain, inflammation, cancer, neurodegeneration, neuroendocrine disorders and cardiovascular disease.

Description

TITLE OF THE INVENTION

ASSAYS FOR SODIUM ION CHANNEL MODULATORS AND USES THEREOF

FIELD OF THE INVENTION

[0001] This invention relates generally to novel screening assays for modulating sodium channels, particularly voltage-gated sodium channels. The assays employ mammalian cells that endogenously express a voltage-gated sodium channel in the context of one or more endogenously co-expressed a subunits and accessory β subunits. The mammalian cells are useful in high throughput assays for identifying drugs with therapeutic value against diseases or conditions associated with sodium channel activity including pain, inflammation, cancer, neurodegeneration,

neuroendocrine disorders and cardiovascular disease.

BACKGROUND OF THE INVENTION

[0002] Voltage-gated sodium channels (Na_v) are complex transmembrane proteins comprised of a pore-forming a subunit and accessory β subunits that play an essential role in the initiation and propagation of action potentials in excitable cells. Na_v channels open to permit influx of sodium ions when the membrane potential is depolarized and close on repolarization. They also close on continuous depolarization by a process termed inactivation, which leaves the channel refractory (i.e., unable to open again for a period of time).

[0003] To date, apart from the related Na_x, which has been suggested to function as a sodium sensor [Shimizu, H., et al. , Neuron, 2007. 54(1): p. 59-72;

Hiyama, T.Y., et al. , Nat Neurosci, 2002. 5(6): p. 511 -2], nine isoforms termed Na_v 1.1 - Na_v 1.9 have been functionally defined as sodium-selective ion channels [Yu, F.H. and W.A. Catterall, Genome Biol, 2003. 4(3): p. 207]. Their distinct tissue distribution as well as amenability to modulation by toxins and drugs has led to significant interest in Na_v channels as therapeutic targets in a number of poorly treated conditions, ranging from epilepsy to cardiac arrhythmias and pain [Clare, J. J., et al., Drug Discov Today, 2000. 5(11): p. 506-520].

[0004] In particular, in recent years Na_vl .7 has emerged as what appears to be a near perfect pharmaceutical target - its expression is restricted to a subset of nociceptive neurons, which can be expected to limit on-target side effects of pharmacological modulators of Na_v1.7 [Dib-Hajj, S.D., et al., Annu Rev Neurosci. 33: p. 325-47], In addition, loss-of-function mutations in humans have provided the promise that Na_v1.7 blockage could produce complete loss of pain sensations without dose- limiting side effects [Cox, J.J;, et ai, Nature, 2006. 444(7121): p. 894-8]. However, it remains unclear to date if such an effect can be translated to the clinic since there is a lack of subtype-specific Na_vl .7 blockers in later stage clinical development, in part reflecting a lack of functionally relevant assays amenable to high throughput screening.

[0005] Several approaches have been taken to identify novel, subtype- selective modulators of Na_v channels. While electrophysiology can provide high quality data that offers mechanistic insights into the state-dependence of inhibition, despite the development of novel fully automated patch-clamp technology this approach can currently achieve low to medium throughput, is expensive, and requires highly skilled personnel [Gonzalez, J.E., et al. , Drug Discov Today, 1 99. 4(9): p. 431-439; Dunlop, J., et i, Comb Chem High Throughput Screen, 2008. 11(7): p. 514-22; Xu, J., et al , Drug Discov Today, 2001. 6(24): p. 1278-1287] . In contrast, fluorescence-based assays measuring changes in membrane potential or intracellular sodium concentration are industry standard approaches for high throughput compound screening despite at times being prone to artifacts and sensitivity problems [Gonzalez, J.E., et al, 1999. 'supra; Xu, J., et al, 2001. supra]. Similarly, radioligand binding assays are also prone to high false negative rate due to the large number of allosteric sites on Na_v that cannot be simultaneously assayed with a single radioligand. Also, difficulties associated with the cloning and heterologous expression of the Na_v channel complexes have further restricted the development of high throughput assays for specific sodium channel subtypes. In fact, some studies have reported an inability to detect modulation of heterologously expressed Na_vl .7 by state-dependent gating modifiers such as ProTxI using membrane potential dyes [Bhattacharya, A., et al. , FASEB J. , 2009. 23 ((Meeting Abstract Supplement)): p. 998.31]. Moreover, questions have been raised about the physiological relevance of assays assessing the function of Na_v over-expressed in the absence of auxiliary subunits [Gonzalez, J.E., et al, 1999. supra; Dunlop, J., et al , 2008. supra; Xu, J., et al, 2001. supra]. SUMMARY OF THE INVENTION

(0006] In contrast to prior art assays that were unable to detect modulation of heterologously expressed Na_v channels by state-dependent gating modifiers, various Na_v channel modulators including state-dependent gating modifiers (e.g., ProTxII), pore blockers (e.g., tetrodotoxin, μ-conotoxin Till A, and clinically used anesthetic compounds such as amitriptyline and tetracaine) have been shown by the present inventors to be surprisingly effective in modulating Na_v channels that are endogenously expressed by mammalian cells in the context of endogenously co-expressed a subunits and accessory β subunits. Without wishing to be bound by any one theory or mode of operation, it is believed that this discrepancy results from (1) endogenous expression of Na_v channels giving rise to a more physiological membrane potential than the membrane potential achieved using commonly used over-expression systems; and/or (2) endogenous co-expression of Na_v channels with functionally relevant β-subunits, which may be missing in the prior art heterologous expression systems. These discoveries have led the present inventors to develop novel screening assays for modulators of Na_v isoforms, which use a mammalian cell that endogenously expresses a Na_v isoform of interest in the context of one or more endogenously co-expressed a subunits and accessory β subunits. In specific embodiments, the mammalian cell is a neuroblastoma cell (e.g., a human neuroblastoma cell line, such as SH-SY5Y), which endogenously expresses at least one Na_v channel (e.g. , 1 , 2 or all) a subunits selected from Na_vl .2, Na_v1.3 and Na_v1.7. Suitably, the neuroblastoma cell (e.g., a human neuroblastoma cell line, such as SH-SY5Y) endogenously co-expresses at least one accessory β subunit (e.g., 1 or both) selected from β2 and β3 subunits.

[0007] The present inventors have also determined that activation of these Na_v isoforms (e.g. , using sodium channel openers/activators, illustrative examples of which include veratridine, grayanotoxin, aconitine, batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion toxins (e.g., OD-1), sea anemone toxins, β scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, δ- conotoxins) leads to sodium influx, which results in membrane depolarization and subsequent Ca²⁺ influx through endogenously or heterologously expressed voltage- gated calcium channels (VGCC). In specific embodiments, this permits the use of voltage sensors including ion transport-indicating agents (e.g., sodium-indicating agents and calcium-indicating agents) and membrane potential-indicating agents for determining the activity of the Na_v isoform of interest, including the influence of a candidate agent on modulating that activity.

[0008] Thus, in one aspect, the present invention provides methods for identifying an agent that modulates the activity of a voltage-gated sodium channel (Na_v) isoform of interest that is endogenously expressed by a neuroblastoma cell. In some embodiments, these methods comprise: (a) contacting the neuroblastoma cell with a candidate agent under conditions permitting, promoting or supporting ion transport across the membrane of the cell; and (b) detecting a change in the intracellular level of the ion, which results from contacting the cell with the candidate agent, wherein the change indicates that the candidate agent modulates the activity of the Na_v isoform of interest. Desirably, the candidate agent blocks, abrogates, inhibits or otherwise reduces the activity of the Na_v isoform of interest.

[0009] In some embodiments, the methods employ at least one Na_v isoform- inhibiting agent (e.g., conotoxin TIIIA, ProTxII, an antagonist antigen-binding molecule that is specifically immuno-interactive with an individual Na_v isoform, or a nucleic acid molecule [e.g., siRNA, shR A, antisense etc.] that inhibits expression of an individual Na_v isoform) to inhibit the level or activity of one or more Na_v isoforms other than an Na_v isoform of interest that is the subject of investigation, under conditions supporting ion transport across the membrane of the cell, to thereby permit determination of the activity of the Na_v isoform of interest. Accordingly, in a related aspect, the present invention provides methods for identifying an agent that modulates the activity of a voltage-gated sodium channel (Na_v) isoform of interest that is endogenously expressed by a mammalian cell (e.g., a neuroblastoma cell line such as SH-SY5Y), wherein the mammalian cell further expresses at least one other Na_v isoform. These methods generally comprise (a) contacting the mammalian cell, in the presence and absence of a candidate agent, with a Na_v isoform-inhibiting agent that inhibits the level or activity of the at least one other Na_v isoform under conditions permitting, promoting or supporting ion transport across the membrane of the cell; and (b) detecting a change in the intracellular level of the ion, which results from the presence of the candidate agent, wherein the change indicates that the candidate agent modulates the activity of the Na_v isoform of interest. In some advantageous embodiments, the method comprise detecting a change in the intracellular level of calcium ions. [0010] Candidate agents testing positive in the methods/assays of the present invention (e.g. , agents that block, abrogate, inhibit or otherwise reduce the activity of the Na_v isoform of interest) find utility in drug discovery, including drugs with therapeutic value against a disease or condition associated with sodium channel activity such as, but not limited to, pain, inflammation, neurodegeneration, neuroendocrine disorders and cardiovascular disease. Thus, in yet another aspect, the present invention provides methods of producing an agent that is useful for treating or preventing a disease or condition associated with sodium channel activity. These methods generally comprise: identifying an agent that modulates the activity of a voltage-gated sodium channel (Na_v) isoform of interest, as broadly described above; and synthesizing the agent on the basis that it tests positive for the modulation. Suitably, the methods further comprise derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing the disease or condition associated with sodium channel activity.

[0011] In a related aspect, the present invention provides methods for treating or preventing a disease or condition associated with sodium channel activity (e.g. , aberrant activity or hyperactivity) in a subject. These methods generally comprise administering an effective amount of an agent that modulates (e.g., blocks or reduces) the level or activity of a voltage-gated sodium channel (Na_v) isoform of interest, wherein the agent is identified by the methods/assays of the present invention.

[0012] Still another aspect of the present invention provides kits for assessing or assaying the potential of an agent to modulate the activity of a voltage-gated sodium channel (Na_v) isoform of interest. These kits generally contain (1) a mammalian cell (e.g., a neuroblastoma cell that is suitably of human origin), which enodgenously expresses the Na_v isoform of interest and suitably at least one other Na_v isoform, (2) at least one Na_v isoform-inhibiting agent (e.g., conotoxin TIIIA, ProTxII, an antagonist antigen-binding molecule that is specifically immuno-interactive with an individual Na_v isoform, or a nucleic acid molecule [e.g., siRNA, shRNA, antisense etc.] that inhibits expression of an individual Na„ isoform) to inhibit the level or activity of one or more of the other Na_v isoforms that are not the subject of investigation; and (3) a sodium channel opener/activator (e.g., veratridine, grayanotoxin, aconitine, batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion toxins (e.g., OD-1), sea anemone toxins, β scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, δ- conotoxins) that opens/activates the Na_v isoform of interest and optionally the other Na_v isoform, or if more than one, at least one of the other Na_v isoforms. In some

embodiments, the kits further comprise a voltage sensor, illustrative examples of which are selected from ion transport-indicating agents (e.g., sodium-indicating agents and calcium-indicating agents) and membrane potential-indicating agents. In certain embodiments, the kits may further contain instructions for conducting the assessment or assay.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is a graphical and photographic representation showing that SH-SY5Y cells endogenously express Na_v and accessory β subunits. Expression of Na_v a and accessory β subunits was assessed by semi-quantitative PCR. (A) Amplification of endogenously expressed human Na_vl .2, Na_vl .3, Na_v 1.4, Na_v 1.5 and Na_vl .7 was detected in SH-SY5Y cells, with Na_vl .7 being the most abundantly expressed Na_v isoforms. (B) SH-SY5Y cells endogenously expressed human β2 and β3, but not βΐ or β4 subunits. (C) Representative gel of Na_v 1.1- Na_v 1.9 subunits endogenously expressed in SH-SY5Y cells. Far left and right lanes; size marker (bp) (D)

Representative gel of βΐ - β4 subunits endogenously expressed in SH-SY5 Y cells. Far left lane; size marker (bp). Data are presented as mean ± SEM of n = 3 independent experiments.

[001 ] Figure 2 is a photographic representation showing that endogenously expressed Na_v channels in SH-SY5Y cells are located at the plasma membrane. SH- SY5Y cells stained with an anti-Na_vl .7 antibody ((A) and (B)) and anti-Na_vl .3 antibody ((C) and (D)) showed fluorescence located predominately in the plasma membrane, indicative of functional Na_v expression. Scale bar; 10 μπι.

[0015] Figure 3 is a graphical representation showing that activation of endogenously expressed Na_v in SH-SY5Y by veratridine causes membrane

depolarization. (A) and (B) The Na_v activator veratridine caused a concentration- dependent change in membrane potential with an EC50 of 28.5 μΜ (pICso 4.54 ± 0.06) that is mediated through activation of TTX-sensitive Na_v as TTX (300 nM) completely blocked responses. (B) Addition of 50 μΜ veratridine causes a transient change in membrane potential in SH-SY5Y cells mediated through endogenously expressed TTX- sensitive Na_v. Data are presented as mean ± SEM of n = 3-4 wells and are representative of at least 3 independent experiments.

[0016] Figure 4 is a graphical representation showing that activation of endogenously expressed TTX-sensitive Na_v by veratridine and P-CTX-1 elicits Ca responses in SH-SY5Y cells. (A) Veratridine elicited concentration-dependent increases in intracellular Ca ⁺in SH-SY5Y cells with an EC₅₀ of 21.9 μΜ (pIC₅₀ 4.66 ± 0.04). The Ca²⁺ responses elicited by veratridine were completely blocked in the presence of 300 nM TTX, providing evidence that the responses were mediated solely through TTX- sensitive Na_v isoforms endogenously expressed in SH-SY5 Y cells. (B) Tetrodotoxin concentration-dependently blocked Ca responses elicited by addition of 50 μΜ veratridine with an IC₅o of 8.6 nM; consistent with the inhibition of TTX-sensitive Na_v activated by veratridine. (C) P-CTX-1 caused concentration-dependent Ca²⁺ responses with an EC₅₀ of 3.7 nM (pEC₅o 8.42 ± 0.32), respectively. The Ca²⁺ responses elicited by P-CTX-1 were completely blocked in the presence of 300 nM TTX, providing evidence that the responses were mediated solely through TTX-sensitive Na_v isoforms endogenously expressed in SH-SY5Y cells. (D) Tetrodotoxin concentration- dependently blocked Ca²⁺ responses elicited by addition of 30 nM P-CTX-1 with an IC50 of 2.4 nM; consistent with the inhibition of TTX-sensitive Na_v activated by P-CTX- 1. Data are presented as mean ± SEM of n = 3-6 wells and are representative of at least 3 independent experiments.

[0017] Figure 5 is a graphical representation showing that L-type and N-type VGCC contribute to the veratridine- and P-CTX-1 -induced Ca²⁺response in SH-SY5Y cells. (A) The L-type VGCC blocker nifedipine concentration-dependently inhibited veratridine-induced responses by 68-88 % (76 ± 4.4 %) with an IC50 of 10.7 nM (pICjo 7.97 ± 0.2). In contrast, the P/Q-type VGCC blocker agatoxin did not significantly inhibit veratridine-elicited Ca²⁺ responses, (B) The N-type VGCC blocker CVID concentration-dependently inhibited the veratridine-induced Ca responses by 23-33 % (31.8 ± 1.1 %) with an IC₅₀ of 19.7 nM (pIC₅₀ 7.7 ± 0.5). Block by CVID together with nifedipine was additive, with responses to veratridine completely abolished in the presence of 10^'μΜ nifedipine and 1 μΜ CVID. (C) Nifedipine concentration- dependently inhibited P-CTX- 1 -induced Ca²⁺ responses by 65.9 - 85.1% (75.5 ± 3.9 %) with an IC₅₀ of 19.8 nM (pIC₅₀ 7.7 ± 0.4), while agatoxin TK did not inhibit P-CTX-1 responses. (D) CVID concentration-dependently inhibited P-CTX-1 responses with an IC50 of 7.9 nM (pIC₅₀ 8.12 ± 0.53). Block of P-CTX-1 responses by CVID was partial with maximum inhibition of 28.7 ± 5.8 % (20.2 - 42.8%) and was additive with inhibition by nifedipine as responses were completely abolished in the presence of 10 μΜ nifedipine and 1 μΜ CVID. Data are presented as mean ± SEM of n = 3-4 wells and are representative of at least 3 independent experiments.

[0018] Figure 6 is a graphical representation showing that veratridine-and P-

CTX-1 -induced Ca²⁺ responses are partially mediated by activation of endogenously expressed Na_vl .2. (A) The Na_v1.2 Na_v 1.4-selective blocker TIIIA reduced veratridine- induced responses with an ICsoof 290 nM (pICso 6.54 ± 0.09). This effect was mediated by Na_vl .2, as the Na_v 1.4-selective blocker GIIIA did not affect veratridine responses at concentrations up to 10 μΜ. (B) In the presence of 1 μΜ TIIIA, the magnitude of the veratridine-induced responses was decreased to 58.7 + 2.8 % of maximum and there was a small but significant (p< 0.05) rightward shift of the veratridine concentration- response curve to an EC₅₀ of 45.8 μΜ (pEC₅₀ 4.33 ± 0.16). (C) The Na_v 1.4 inhibitor GIIIA did not significantly affect P-CTX-1 responses, however, the Na_vl .2 Na_v 1.4 inhibitor TIIIA decreased P-CTX-1 -induced responses by 9 - 32.7 % (22.3 ± 4.4 %) with an IC₅₀ of 79.5 nM (pIC₅₀ 7.10 ± 0.14). (D) The EC₅₀ of the P-CTX-1

concentration-response curve was not significantly affected in the presence of 1 μΜ TIIIA, although the magnitude of the P-CTX-1 response was decreased by 22.4% (p = 0.08). Data are presented as mean ± SEM of n = 3-4 wells and are representative of 3-4 independent experiments.

[0019] Figure 7 is a graphical representation showing that activation of Na_vl .7 contributes to the veratridine-induced Ca²⁺ response in SH-SY5 Y cells. (A) In the presence of 1 μΜ TIIIA, the Na_vl .7 -selective blocker ProTxII concentration- dependently inhibited veratridine-induced responses with an IC50 of 206.9 pM (pICso 9.68 ± 0.15), consistent with inhibition of Na_v1.7.In the absence of TIIIA, ProTxII concentration-deperidently inhibited veratridine-induced responses with a two-site fit with IC50S of 151.7 pM and 56 nM (pICso 9.82 ± 0.23 and pICso 7.25 ± 0.26), respectively. (B) Activation of endogenously expressed Na_vl .2 and Na_vl .7 by veratridine results in an assay with excellent performance, with Z' scores of 0.7 ± 0.05. (C) ProTxII inhibited P-CTX-1 responses with an IC₅₀ of 1.9 μΜ (pIC₅₀5.71 ± 0.05), consistent with activation of Na_v isoforms other than Na_v1.7 by P-CTX-1. (D)

Activation of endogenously expressed Na_vl .2 and Na_vl .3 by P-CTX-1 produces an assay with Z' scores of 0.68 ± 0.04. Data are presented as mean ± SEM of n = 3-4 wells and are representative of 3-4 independent experiments.

[0020] Figure 8 is a graphical representation showing that Na_vl .3 contributes to P-CTX-1 responses in SH-SY5Y cells. SH-SY5Y cells were transfected with shRNA targeting Na_vl .3 and Ca²⁺ responses to 1 nM P-CTX-1 or 60 mM KC1 measured in Fura-2-loaded cells using the high content imaging platform BD Pathway 855. (A) Responses to 1 nM P-CTX-1 were decreased in SH-SY5Y cells expressing Na_v1.3 - targeting shRNA, compared to non-expressing, GFP-negative cells. (B) Responses to depolarization with 60 mM KCl were not significantly affected by expression of Na„l .3 -targeting shRNA. Arrows indicate addition of P-CTX-1 and KCl.

[0021] Some figures contain colour representations or entities. Colour illustrations are available from the Applicant upon request or from an appropriate Patent Office. A fee may be imposed if obtained from the Patent Office.

DETAILED DESCRIPTION OF THE INVENTION

/. Definitions

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0023] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0024] The term "activation," as used herein, refers to the transition from a resting (non-conducting) state of an ion channel to the activated (conducting) state.

[0025] By "activation threshold" is meant the lowest potential above which measurable opening of a channel occurs.

[0026] The term "agent" includes a compound that induces a desired pharmacological and/or physiological effect. The term "agent" is not to be construed narrowly but can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide.a nucleic acid, a carbohydrate, a lipid, or a combination thereof. The term "agenf'extends to small molecules, macromolecules as well as cellular agents.

[0027] As used herein, the terms "antagonist," "inhibitor" and "blocker" are used interchangeably to refer to agents that reduce, inhibit, impair or prevent ion transfer across a cell membrane.

[0028] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to

immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.

[0029] The term "candidate agent," as used herein, refers to a chemical to be tested by one or more screening method(s) of the invention as a putative modulator. A candidate agent Usually, various predetermined concentrations of candidate agents are used for screening, such as 0.01 μΜ, 0.1 μΜ, 1 μΜ and 10 μΜ. Candidate agent controls can include the measurement of a signal in the absence of the candidate agent or comparison to an agent known to modulate the target.

[0030] The term "depolarization," as used herein, refers to a change in the electrical potential difference across the membrane of a cell (between the inside of the cell and the outside of the cell, with outside taken as ground potential), where that electrical potential difference is reduced, eliminated, or reversed in polarity. Activation of a Na_v channel will typically increase in the permeability of the cell membrane to sodium and other ions (e.g., Ca²⁺) effective to reduce the magnitude, and may nearly or completely eliminate, the electrical potential difference across a cell membrane.

[0031] As used herein, the terms "disease" and "condition" may be used interchangeably or may be different in that the particular malady or condition may not have a known causative agent (so that etiology has not yet been determined) and it is . therefore not yet recognized as a disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms has been identified by clinicians.

[0032] By "effective amount," is meant the administration of an amount of active agent to a subject, either in a single dose or as part of a series or slow release system, which is effective for prevention or treatment. The effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors.

[0033] The term "endogenously expressed" refers to a molecule such as a nucleic acid or polypeptide (e.g., a Na_v channel, an accessory subunit etc. or their encoding genes), which is naturally or natively produced by a host cell without external manipulation or the insertion of a new genetic sequence.

[0034] The term "heterologously expressed" refers to a molecule such as a nucleic acid or polypeptide (e.g. , a Na_v channel, an accessory subunit etc. or their encoding genes), which is not naturally or natively produced by a host cell and which results from external manipulation or the insertion of a new genetic sequence.

[0035] The term "hit" refers to a candidate agent that shows desired properties in an assay. [0036] The term "inactivation" means that an ion channel moves into the inactivated state.

[0037] The term "inactivated" refers to a voltage-dependent ion channel in a particular non-conducting conformational state. Transitions into and out of the inactivated state are generally slow relative to transitions between other conformational states. The inactivated state is usually the preferred state at elevated transmembrane potentials. At low transmembrane potentials, the inactivated state is unstable and relaxes to the resting state.

[0038] As used herein, the term "library" means a collection of molecules.

[0039] The term "multi well plate" refers to a two dimensional array of addressable wells located on a substantially flat surface. Multiwell plates may comprise any number of discrete addressable wells, and comprise addressable wells of any width or depth. Common examples of multiwell plates include 96 well plates, 384 well plates and 3456 well Nanoplates™.

[0040] The term "naturally occurring" refers to a component produced by cells in the absence of artificial genetic or other modifications of those cells.

[0041] As used herein, the term "pain" refers to all categories of pain and is recognized to include, but is not limited to, neuropathic pain, inflammatory pain, nociceptive pain, idiopathic pain, neuralgic pain, orofacial pain, burn pain, burning mouth syndrome, somatic pain, visceral pain, myofacial pain, dental pain, cancer pain, chemotherapy pain, trauma pain, surgical pain, post-surgical pain, childbirth pain, labor pain, reflex sympathetic dystrophy, brachial plexus avulsion, neurogenic bladder, acute pain (e.g. , musculoskeletal and post-operative pain), chronic pain, persistent pain, peripherally mediated pain, centrally mediated pain, chronic headache, migraine headache, familial hemiplegic migraine, conditions associated with cephalic pain, sinus headache, tension headache* phantom limb pain, peripheral nerve injury, pain following stroke, thalamic lesions, radiculopathy, HIV pain, post-herpetic pain, non-cardiac chest pain, irritable bowel syndrome and pain associated with bowel disorders and dyspepsia, and combinations thereof.

[0042] The terms "patient," "subject," "host" or "individual" used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents (e.g. , mice rats, guinea pigs), lagomorphs (e.g. , rabbits, hares), bovines (e.g. , cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs) and felines (e.g., cats). In specific embodiments, the subject is a primate (e.g., a human, monkey, chimpanzee) in need of treatment or prophylaxis for a condition or disease associated with sodium channel activity (e.g., aberrant activity or hyperactivity). However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0043] As used herein, the terms "prevent," "prevented," or "preventing," refers to a prophylactic treatment which increases the resistance of a subject to developing a disease or condition that associates with sodium channel activity (e.g., aberrant activity or hyperactivity) or, in other words, decreases the likelihood that the subject will develop that disease or condition as well as a treatment after the disease or condition has begun in order to reduce or eliminate it altogether or prevent it from becoming worse.

[0044] The term "resting" or "resting state" refers to a voltage-dependent ion channel that is closed, but free from inactivation.

[0045] The term "selective" refers to agents that modulate (e.g. , inhibit or display antagonism towards) a Na_v channel of interest without displaying substantial modulation of (e.g., inhibition or antagonism towards) one or more other Na_v channels. Accordingly, an agents that is selective for Na_vl .7 exhibits Na_vl .7 selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to modulation of (e.g., inhibition or antagonism towards) one or more other Na_v channels (i.e., a Na_v channel other than Na_v1.7 such as Na_v1.2 and Na_v1.3 ). In some embodiments, selective agents display at least 50-fold, at least 100-fold, at least 500- fold, at least 1000-fold greater modulation of (e.g. , inhibition or antagonism towards) Na_v1.7 than of Na_v1.2. In other embodiments, selective agents display at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold greater modulation of (e.g., inhibition or antagonism towards) Na_vl 7 than of Na_vl .3. In still other embodiments, selective agents display at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold greater modulation of (e.g., inhibition or antagonism towards) Na_v1.3 than of Na_v1.2. In still other embodiments, selective agents display at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold greater modulation of (e.g., inhibition or antagonism towards) Na_v1.2 than of Na_v1.3. In still other embodiments, selective agents display at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold greater modulation of (e.g., inhibition or antagonism towards) Na_v1.2 than of Na_v1.7.

[0046] As used herein a "small molecule" refers to a composition that has a molecular weight of less than 3 kilodaltons (kDa), and typically less than 1.5 kilodaltons, and more preferably less than about 1 kilodalton. Small molecules may be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. As those skilled in the art will appreciate, based on the present description, extensive libraries of chemical and/or biological mixtures, often marine, reptilian, insect, fungal, bacterial, or algal extracts, may be screened with any of the assays of the invention to identify compounds that modulate a bioactivity. A "small organic molecule" is an organic compound (or organic compound complexed with an inorganic compound (e.g., metal)) that has a molecular weight of less than 3 kilodaltons, less than 1.5 kilodaltons, or even less than about 1 kDa. .

[0047] The terms "treating" or "treatment" as used herein cover the treatment of a disease or condition of interest in a mammal (e.g., a human) having the disease or condition of interest, and includes: (a) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition, i.e., arresting its development; (c) relieving the disease or condition, i.e., causing regression of the disease or condition; or (d) relieving the symptoms resulting from the disease or condition, /. e. , relieving pain without addressing the underlying disease or condition.

[0048] The term "voltage sensor" includes FRET based voltage sensors, electrochromic transmembrane potential dyes, transmembrane potential redistribution dyes, extracellular electrodes, field effect transistors, radioactive ions, ion sensitive fluorescent or luminescent dyes, and ion sensitive fluorescent or luminescent proteins, that are capable of providing an indication of the transmembrane potential. 2. Abbreviations

[0049] The following abbreviations are used throughout the application:

d = days

h = hours

s = seconds

g = grams

kg = kilograms

Na_v = voltage-gated sodium channel

BTX = batrachotoxin

TTX = tetrodotoxin

STX = saxitoxin

FRET = fluorescence resonance energy transfer

FLIPR = fluorescent imaging plate reader

VIPR = voltage/ion probe reader

PSS = physiological salt solution

HTS = high throughput screening

MTS = medium throughput screening

3. Screening for modulators of endogenously expressed Na channels

[0050] The present invention provides novel assays that are useful in screening of agents for their ability to modulate (increase or decrease activity) an Na_v channel (also referred to interchangeably herein as "sodium channel") that is endogenously expressed by a mammalian cell, including a primate cell {e.g. , a human, monkey or ape). In specific embodiments, the cell is a neuronal cell such as a neuroblastoma cell, including a neuroblastoma cell line such as SH-S Y5 Y, which endogenously expresses at least one Na_v channel a subunit {e.g. , 1 , 2 or all) selected from Na_vl .2, Na_vl .3 and Na_vl .7. Desirably, the mammalian cell endogenously co- expresses accessory β subunits, which co-assemble with the Na_v channel a subunit(s). In some embodiments, the mammalian cell endogenously co-expresses β subunits selected from one or both of β2 and β3 subunits. Suitably, the mammalian cell endogenously co- expresses at least one Na_v channel a subunit {e.g. , 1 , 2 or all) selected from Na_vl .2, Na_v1.3 and Na_v1.7 as well as at least one β subunit {e.g., 1 or both) selected from β2 and β3 subunits.

[0051] These assays are useful, therefore, for identifying Na_v channel modulators, particularly Na_v channel blockers, that are useful as therapeutic agents. The Na_v channel-modulating agents so identified are then tested in a variety of in vivo models so as to determine if they alleviate the symptoms of diseases or conditions associated with sodium channel activity such as, but not limited to, pain, inflammation, cancer, neurodegeneration, neuroendocrine disorders and cardiovascular disease. In specific embodiments, the Na_v channel modulator modulates the activity of the Na_v channel of interest downwards, inhibits the activity of the Na_v channel of interest, and/or reduces or prevents sodium ion flux across a cell membrane by preventing an activity of the Na_v channel of interest such as ion flux. Any such modulation, whether it be partial or complete inhibition or prevention of ion flux, is sometimes referred to herein as "blocking," "inhibiting" or "antagonizing" and corresponding agents as "blockers," "inhibitors" or "antagonists," respectively.

[0052] Assays for the identification of these agents may make use of these mammalian cells in a variety of different formats as described for example below. Animal models can also be used for determining the in vivo effects of such agents. In specific embodiments, the cells or animals also may be contacted with additional sodium channel blockers in combination with a putative modulator of Na_v channel function in order to determine- whether the effect of such sodium channel blockers is increased or decreased as a result of the presence of the candidate agent. An alteration in Na_v channel activity, expression or processing in the presence of the candidate agent will indicate that the candidate agent is a modulator of the activity.

[0053] In specific embodiments, the assays of the present invention identify a candidate agent as being capable of inhibiting Na_v channel activity, by measuring or determining the activity of the Na_v channel in the absence of the added candidate agent. The candidate agent suspected of blocking the activity of the Na_v channel is contacted with the cell and the activity of the Na_v channel in the presence of the candidate agent is determined. A candidate agent which is inhibitory or blocking would decrease the sodium channel activity.

[0054] Identification of modulators of sodium channels can be assessed using a variety of in vitro and in vivo assays, e.g., measuring current, measuring membrane potential, measuring ion flux, (e.g., sodium, calcium or guanidinium), measuring sodium concentration, measuring second messengers and transcription levels, and using e.g., voltage-sensitive dyes, and radioactive tracers. These assays can be carried out in cells, or cell or tissue extracts endogenously expressing the Na_v channel(s) of interest (/. e. , expressing the sodium channel(s) in a natural endogenous setting).

[0055] In accordance with the present invention, in vitro assays will involve mammalian cells that endogenously express one or more Na_v channels of interest. Illustrative mammalian cells include without limitation primary mammalian cells e.g. , neurons as well as neuronal (e.g. , neuroblastoma) cell lines such as SH-SY5Y, which naturally express the Na_v channel(s) of interest. The cells are plated in an appropriate support e.g., in multi-well poly-D-lysine-coated black wall-clear bottom culture plates, at a suitable concentration (e. g. , 1 -2x 10^s cells/well). The cells are typically maintained at about 37° C in an atmosphere containing about 5% C0₂.

[0056] In some embodiments, ion flux assays can be used to assess sodium channel activity. In representative examples of these assays, sodium channels are stimulated to open by contacting the cell with an ion transport-activating agent, including sodium channel openers/activators (e.g. , veratridine, grayanotoxin, aconitine, batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion toxins (e.g., OD-1), sea anemone toxins, β scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, δ-conotoxins). If desired, a channel-stabilizing (e.g., a positive Na_v modulator) may also be employed, which stabilizes the channel in an open state. Channel blockers are suitably identified by their ability to prevent ion influx. In some embodiments, the assays use radioactive ²²[Na] and ^l4[a] guanidinium ions as tracers. FlashPlate & Cytostar-T plates in living cells avoids separation steps and are suitable for high throughput screening (HTS). Scintillation plate technology has also advanced this method to HTS suitability.

[0057] Advantageous assays, including HTS assays may involve the use of optical readouts of transmembrane potential, or ion channel conductance. Such assays include the use of transmembrane potential or ion sensitive dyes, or molecules, that typically exhibit a change in their fluorescent or luminescent characteristics as a result of changes in ion channel conductance or transmembrane potential. In some

embodiments of such assays, a Fluorescent Imaging Plate Reader (FLIPR™) system membrane potential kit available from Molecular Dynamics (a division of Amersham Biosciences, Piscataway, N.J.) is used to measure redistribution of membrane potential. The FLIPR™ system is particularly suited to ion flux assays and may be used to monitor, for example, sodium channel opener-evoked increases in intracellular Ca²⁺. In illustrative examples of this type, a calcium-indicating agent, such as Fluo-4-AM, is loaded into the cells and the cells are monitored, in real-time, using the FLIPR™.

Briefly, cells are incubated with 4 μΜ Fluo-4-AM in physiological salt solution (PSS) for 30 min at 37° C. They are then washed with PSS to remove extracellular calcium- indicating agent and plates containing the cells are transferred to the FLIPR™. The cells are incubated for 5 min in FLIPR™ buffer, in the absence (control) or presence of the candidate agent, prior to addition of veratridine (40 μ ). Cell fluorescence

495 nm;

nm) is monitored both before and after the addition of a sodium channel opener (e.g., veratridine, grayanotoxin, aconitine, a batrachotoxin, BTG502, an antillatoxin, hoiamide A, an a scorpion toxin (e.g. , OD-1), a sea anemone toxin, a β scorpion toxin, pumiliotoxin B, brevetoxins, a ciguatoxin such as pacific ciguatoxin-1 (P-CTX-1), a versutoxin, a pyrethroid insecticide, a δ-conotoxin). Peak fluorescence intensity, after sodium channel opener addition, is determined using the FLIPR™ software. Curve fitting and parameter estimation (pIC₅o) can be performed using any suitable software, illustrative examples of which include Screenworks™ (Molecular Devices).

[0058] It will be understood, however, that the use of calcium-indicating agents is not limited to the FLIPR™ assay and encompasses any assay that measures Ca²⁺ influx as a surrogate marker of sodium channel activity. Several different types of calcium indicating agent are known, representative examples of which include Fura-2, Fluo-3, Fluo-4, Mag-Fluo-4, Fluo-5, Oregon green, calcium green, calcium orange, BAPTA-1 , BAPTA-2, BAPTA-5, BAPTA-6, Rhod-1 , Rhod-2, and Rhod-3.

[0059] Alternatively, sodium-indicating agents can be used to measure the rate or amount of sodium ion influx through the sodium channel. This type of assay measures Na⁺ influx directly. CoroNa Red, SBFI and/or sodium green (Molecular Probes, Inc. Eugene Oreg.) can be used to measure Na influx; all are Na responsive dyes. If desired, they can be used in combination with the FLIPR instrument.

[0060] In another HTS assay format, FRET based voltage sensors are used to measure the ability of a candidate agent to directly block Na⁺ influx. Commercially available HTS systems include the VIPR™ II FRET system (Aurora Biosciences Corporation, San Diego, Calif., a division of Vertex Pharmaceuticals, Inc.) which may be used in conjunction with FRET dyes, also available from Aurora Biosciences. A VIPR™ II FRET system is equipped with instrumentation capable of electrical stimulation of cells, which allows manipulation of the membrane potential and modulates the Na_v channel conductance. Sodium channels have brief (~l-3 ms) open times, so a train of electric field pulses is used to cycle the channel through open and closed conformations repeatedly. Membrane potential changes caused by the sodium influx through the channels is converted to optical signals using the Aurora FRET voltage sensitive dyes, described above. Cells stained with CC2-DMPE and

DiSBAC6(3) are excited at 405 nm. The instrument is able to continually monitor the fluorescent output at two wavelengths for FRET measurement. Fluorescence responses are obtained at two wavelengths, 460 nm for CC2-DMPE and 580 nm for DiSBAC₆(3), VIPR™ II FRET assays measure sub-second responses to voltage changes. There is no requirement for a modifier of channel function. The assays measure depolarization and hyperpolarizations, and provides ratiometric outputs for quantification. A somewhat less expensive medium throughput screening (MTS) version of these assays employs the FLEXstation™ (Molecular Devices Corporation) in conjunction with FRET dyes from Aurora Biosciences. In a non-limiting example of a VIPR™ II FRET assay, cells endogenously expressing a Na_v channel of interest are cultured on multi-well plates (e.g., Costar tissue culture treated 96-well flat bottom plates, Corning). To prevent detachment of cells during plate washing, these plates are pre-coated with 0.5% Growth Factor Reduced matrigel matrix in DMEM for 1 hour at room temperature before use for cell culture. About 40,000 cells are seeded to each well and incubated at 38° C for 24 hours before assay. Assay is performed at room temperature. The cell plates are first washed three times with bath solution using automatic plate washer (ELx405, Biotek), leaving a residual volume of 50 μΙ,ΛνβΙΙ. Subsequently, cells are incubated with Aurora FRET voltage sensitive dyes in the form of a mixed dye solution for 30 min in the dark at room temperature. The mixed dye solution is prepared with external solution and consists of 10 μΜ CC2-DMPE (chlorocoumarin-2-dimyristoyl

phosphatidylethanolamine), 2.4 μΜ DISBAC6(3) (bis-(l,3-dihexyl-thiobarbituric acid) trimethine oxonol), 0.5% β-cyclodextrin, 20 μg/mL pluronic F-127 and ESS Acid Yellow 17 (ESS AY- 17). Thereafter, the cells are washed three times again with bath solution and then incubated with bath solution containing 0.5 raM ESS AY- 17 in the absence (control) or presence of the candidate agent (at desired concentrations) for 10 min before assay.

[0061] Other assays can be selected which allow the investigator to identify candidate agents that block specific states of the sodium channel of interest, such as the open state, closed state or the resting state, or which block transition from open to closed, closed to resting or resting to open. Those skilled in the art are generally familiar with such assays. In specific embodiments, candidate agents are screened for their ability to inhibit the ion flux through an endogenously expressed Na_v isoform, wherein the agent is a state or frequency dependent modifier of the isoform, having a low affinity for the rested/closed state and a high affinity for the inactivated state. These compounds are likely to interact with overlapping sites located in the inner cavity of the sodium conducting pore or a subunit of the channel similar to that described for other state-dependent sodium channel blockers (Cestele, S., et al, Biochimie 2000. 82: 883- 892). These compounds may also be likely to interact with sites outside of the inner cavity and have allosteric effects on sodium ion conduction through the channel pore.

[0062] In some embodiments of the present in which the mammalian cell comprises a Na_v channel isoform of interest as well as one or more other Na_v channel isoforms, the assays will generally employ one or more Na_v channel isoform-inhibiting agents to block or inhibit ion transport across the other Na_v channel isoform(s) so as to direct or focus ion transport across the Na_v channel isoform of interest. Thus, for example, when the mammalian cell comprises a Na_v channel isoform of interest and a single other Na_v channel isoform, the cell is contacted with a Na_v channel isoform- inhibiting agent to block or inhibit ion transport across the other Na_v channel isoform before measuring or determining ion transport across the Na_v channel isoform of interest. Similarly, when the mammalian cell comprises a Na_v channel isoform of interest as well as a first other Na_v channel isoform and a second other Na_v channel isoform, the cell is contacted with a first Na_v channel isoform-inhibiting agent to block or inhibit ion transport across the first other Na_v channel isoform and with a second Na_v channel isoform-inhibiting agent to block or inhibit ion transport across the second other Na_v channel isoform before measuring or determining ion transport across the Na_v channel isoform of interest. Suitably, the first and second Na_v channel isoform- inhibiting agents may be the same or different. Non-limiting examples of Na_v isoform- inhibiting agents include: Na_v channel pore blockers (e.g. , conotoxin TIIIA); Na_v channel gating modifiers (e.g., ProTxI and ProTxII); antagonist antigen-binding molecules (e.g., antagonist antibodies and antibody fragments) that are specifically immuno-interactive with an individual other Na_v channel isoform and which reduce, inhibit, impair or prevent ion transfer across that isoform; and nucleic acid molecules (e.g., siRNA, shRNA, antisense etc.) that inhibits expression of a gene encoding an individual other Na_v channel isoform.

[0063] In specific embodiments, the mammalian cell (e.g. , a human cell or a cell of human origin), which is suitably a neuronal cell such as a neuroblastoma cell (e.g., SH-SY5Y), endogenously expresses two or more Na_v channels selected from Na_vL2, Na_vl .3 and Na_vl .7. In illustrative examples of this type, the cell expresses Na_vl .2 and Na_vl .7 and the Na_v isoform of interest is Na_vl .7. In some of these examples, a Na_vl .2-inhibiting agent (e.g., conotoxin TIIIA, an antagonist antigen-binding molecule that is specifically immuno-interactive with Na_v1.2 and a nucleic acid molecule (e.g., siRNA, shRNA, antisense etc.) that inhibits expression of Na_v1.2) is used to selectively reduce ion flux activity of Na_vl .2 as compared to flux activity of Na_vl .7 so as to selectivity or preferentially direct ion transport_. across Na_vl .7 under conditions permitting or supporting ion transport across the membrane of the cell (e.g., veratridine-evoked ion influx). The cell is then exposed to a candidate agent and a Na_vl .7 blocker is identified by its ability to further prevent ion influx into the cell.

[0064] In other embodiments, OD- 1 , which is an a-like toxin from the venom of the Iranian yellow scorpion, Odonthob thus doriae, is used to selectively activate Na_vl .7 so as to preferentially direct ion transport across that channel. In these embodiments, it is not necessary to use Na_v-inhibiting agents to selectively reduce ion flux activity of the other Na_v channels (i.e., Na_vl .2 or Na_v1.3) as these channels would either not activate in the presence of OD-1 or activate to a much lower degree than Na_vl .7. If desired, OD-1 may be used in combination with veratridine to synergistically activate Na„1.7. In these embodiments, veratridine is generally used at a concentration that does not lead to activation of the other Na_v channels ( . e. , Na_v 1.2 or Na_v 1.3) or that leads to less activation of those channels, as compared to the activation of Na_v1.7 (e.g., the ion transport across an individual other Na_v channel is less than 30%, 25%, 20%, 15%, 10%, 5%, 1% of the ion transport across Na_vl .7).

[0065] In other illustrative examples, the cell expresses Na_vl .2 and Na_vl .7 and the Na_v isoform of interest is Na_vl .2. In these examples, a Na_vl .7-inhibiting agent (e.g., ProTxII, an antagonist antigen-binding molecule that is specifically immuno- interactive with Na_vl .7 and a nucleic acid molecule (e.g. , siRNA, shRNA, antisense etc.) that inhibits expression of Na_vl.7) is used to selectively reduce ion flux activity of Na_vl .7 as compared to flux activity of Na_vl .2 so as to selectivity or preferentially direct ion transport across Na_vl .2 under conditions permitting or supporting ion transport across the membrane of the cell (e.g., veratridine-evoked ion influx). The cell is then exposed to a candidate agent and a Na_v1.2 blocker is identified by its ability to further prevent ion influx into the cell. [0066] In still other illustrative examples, the cell expresses Na_vl .3 and Na_vl .7 and the Na_v isoform of interest is Na_vl .3. In these examples, a Na_vl .7-inhibiting agent (e.g., ProTxII, an antagonist antigen-binding molecule that is specifically immuno-interactive with Na_vl .7 and a nucleic acid molecule (e.g. , siRNA, shRNA, antisense etc.) that inhibits expression of Na_vl.7) is used to selectively reduce ion flux activity of Na_vl .7 as compared to flux activity of Na_vl .3 so as to selectivity or preferentially direct ion transport across Na_v1.3 under conditions permitting or supporting ion transport across the membrane of the cell (e.g., ciguatoxin- or brevetoxin-evoked ion influx). The cell is then exposed to a candidate agent and a Na_vl .3 blocker is identified by its ability to further prevent ion influx into the cell.

[0067] In specific embodiments, the mammalian cell comprises voltage-gated calcium channels (VGCC) and activation of the Na_v isoform(s), which leads to sodium

7

influx, results in membrane depolarization and subsequent Ca influx through theVGCC. In these embodiments, Ca²⁺ influx acts as a surrogate marker of sodium influx, which permits the use of calcium-indicating agents for determining the activity of the Na_v isoform of interest, including the influence of a candidate agent on^' modulating that activity. The VGCC may be endogenously or heterologously expressed by the cell. In this regard, VGCC are generally found in many cells where, among other functions, they play important roles in signal transduction. In these instances, it is possible to measure Ca²⁺ influx through the endogenously expressed VGCC.

Alternatively, in instances in which the mammalian cell lacks endogenous VGCC or does not express VGCC at levels permitting detection of Ca²⁺ influx, one or more heterologous VGCC may be introduced into the cell, for example, by recombinant means.

[0068] Multiple types of VGCC have been identified in mammalian cells from various tissues, including skeletal muscle, cardiac muscle, lung, smooth muscle and brain, [see, e.g., Bean, B. P. Ann. Rev. Physiol. 1989. 51: 367-384 and Hess, P. Ann. Rev. Neurosci. 1990. 56: 337]. The different types of VGCC have been broadly categorized into five classes, L-, P/Q-, N-, R- and T-, distinguished by current kinetics, holding potential sensitivity and sensitivity to calcium channel agonists and antagonists (see, e.g., Swandulla, D. et al, Trends in Neuroscience 1991. 14: 46; Catterall W.A., Annu Rev Cell Dev Biol. 2000. 16: 52 1-555; Benarroch, E.E., Neurology 2010. 74(16): 1310-1315). The cDNA and corresponding amino acid sequences of the al , cc2, β, γ and δ subunits of the different VGCC are available (e.g. , GenBank), which facilitates the construction of chimeric contracts from which these subunits are expressible and their introduction into appropriate host cells. Illustrative host cells for introduction of VGGC-encoding nucleic acid molecules will endogenously express the Na_v isoform of interest and optionally one or more other Na_v isoforms. In some embodiments, the host cell is a primary, germ, or stem cell, including an embryonic stem cell. In other embodimentss, the host cell is an immortalized cell. The host cell may be derived from a primary or immortalized cell from mesoderm, ectoderm, or endoderm layers, illustrative examples of which include endothelial, epidermal, mesenchymal, neural, renal, hepatic, hematopoietic, or immune host cells. One of ordinary skill in the art will understand that different known or unknown accessory factors may interact with or alter the function or expression of the recombinantly or heterologously expressed VGCC depending on the choice of host cell type.

[0069] As will be appreciated by those of skill in the art, any vector that is suitable for use with the host cell may be used to introduce a nucleic acid encoding a VGCC subunit into the host cell. In instances where a plurality of vectors is used to express a plurality of different VGCC subunits, they may be the same type or may be of different types. Examples of vectors that may be used to introduce the VGCC subunit- encoding nucleic acids into host cells include but are not limited to plasmids, viruses, including retroviruses and lentiviruses, cosmids, artificial chromosomes and may include for example, pCMV-Script, pcDNA3.1 Hygro, pcDNA3.1neo, pcDNA3.1puro, pSV2neo, pIRES puro, pSV2 zeo.

4. Candidate Agents

[0070] A potential modulator assayed using the methods of the present invention comprises a candidate agent. As used herein, the terms "candidate agent,"

"test agent" "test substance" and "test compounds" are used interchangeably herein, and each refers to a substance or agent that is suspected of interacting with a Na_v isoform of interest, including any synthetic, recombinant, or natural product or composition. A test substance suspected of interacting with a Na_v isoform of interest can be subsequently evaluated for such an interaction. A test substance can comprise a peptide, an oligomer, a nucleic acid (e.g., an aptamer), a small molecule (e.g., a chemical compound), an antibody or fragment thereof, a nucleic acid-protein fusion, a peptidomimetic, a carbohydrate, a lipid or other organic (carbon containing) or inorganic molecules, a carbohydrate, any other affinity agent, and combinations thereof. Alternatively, or in addition, a test substance can comprise a carbohydrate, a vitamin or derivative thereof, a hormone, a neurotransmitter, a virus or receptor binding domain thereof, an opsin or rhodopsin, an odorant, a pheromone, a toxin, a growth factor, a platelet activation factor, a neuroactive peptide, or a neurohormone. A candidate substance to be tested can be a purified molecule, a homogenous sample, or a mixture of molecules or compounds.

[0071] Small (non-peptide) molecule modulators are particularly

advantageous as they are more readily absorbed after oral administration, have fewer potential antigenic determinants, or are more likely to cross the cell membrane than larger, protein-based pharmaceuticals. Small organic molecules may also have the ability to gain entry into an appropriate cell and affect the expression of a gene (e.g., by interacting with the regulatory region or transcription factors involved in gene expression); or affect the activity of a gene by inhibiting or enhancing the binding of accessory molecules. Small molecules generally .have a molecular weight of less than about 3,000 daltoms, usually less than 1 ,000 daltons, less than about 750 daltons, less than about 600 daltons, less than about 500 daltons. A small molecule also suitably has a computed log octanol-water partition coefficient in the range of about -4 to about +14, more suitably in the range of about -2 to about +7.5.

[0072] The present invention also extends to the screening of known modulators of sodium channels as well as compounds that are structurally related to known modulators of sodium channels. The active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. However, prior to testing of such compounds in humans or animal models, it will be necessary to test a variety of candidates to determine which have potential.

[0073] Accordingly, the active compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. Accordingly, the present invention provides screening assays to identify agents which inhibit or otherwise treat a disease or condition associated with sodium channel activity (e.g., aberrant activity or

hyperactivity). It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine animals, plants and other samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents.

[0074] It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate agents identified by the present invention may be polypeptide, polynucleotide, small molecule inhibitors or any other inorganic or organic chemical compounds that may be designed through rational drug design starting from known agents that are used in the intervention of a disease or condition associated with sodium channel activity. (e.g., aberrant activity or

hyperactivity).

[0075] Test substances can be obtained or prepared as a library. A library can contain a few or a large number of different molecules, varying from about ten molecules to several billion molecules or more. A molecule can comprise a naturally occurring molecule, a recombinant molecule, or a synthetic molecule. A plurality of test substances in a library can be assayed simultaneously. Optionally, test substances derived from different libraries can be pooled for simultaneous evaluation. A library can comprise a random collection- of molecules. Alternatively, a library can comprise a collection of molecules having a bias for a particular sequence, structure, or

conformation. See e.g., U.S. Pat. Nos. 5,264,563 and 5,824,483.

[0076] There are a number of different libraries used for the identification of small molecule modulators including chemical libraries, natural product libraries and combinatorial libraries comprised or random or designed peptides, oligonucleotides or organic molecules. Generally, libraries of test substances will consist of structural analogs of known compounds or compounds that are identified as hits or leads via natural product screening or from screening against a potential therapeutic target.

Natural product libraries are collections of products from microorganisms, animals, plants, insects or marine organisms which are used to create mixtures of screening by, e.g., fermentation and extractions of broths from soil, plant or marine organisms.

Natural product libraries include polypeptides, non-ribosomal peptides and non- naturally occurring variants thereof. For a review see Science 282:63 68 (1998).

Combinatorial libraries are composed of large numbers of peptides oligonucleotides or organic compounds as a mixture. They are relatively simple to prepare by traditional automated synthesis methods, PCR cloning or other synthetic methods. Of particular interest will be libraries that include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial and polypeptide libraries. A review of

combinatorial libraries and libraries created therefrom, see Myers Curr. Opin.

Biotechnol. 8: 701 707 (1997). A candidate modulator identified by the use of various libraries described may then be optimized to modulate the level or activity of a Na_v isoform of interest through, for example, rational drug design.

[0077] Representative libraries include but are not limited to a peptide library (U.S. Pat. Nos. 6,156,511, 6,107,059, 5,922,545, and 5,223,409), an oligomer library (U.S. Pat. Nos. 5,650,489 and 5,858,670), an aptamer library (U.S. Pat. No. 6,180,348 and 5,756,291), a small molecule library (U.S. Pat. Nos. 6,168,912 and 5,738,996), a library of antibodies or antibody fragments (U.S. Pat. Nos. 6,174,708, 6,057,098, 5,922,254, 5,840,479, 5,780,225, 5,702,892, and 5,667988), a library of nucleic acid- protein fusions (U.S. Pat. No. 6,214,553), and a library of any other affinity agent that can potentially bind to a sodium channel (e.g., U.S. Pat. Nos. 5,948,635, 5,747,334, and 5,498,538). Methods for preparing libraries containing diverse populations of various types of molecules are known in the art, for example as described in U.S. patents cited herein above. Numerous libraries are also commercially available.

5. Selectivity and T oxicology of Candidate Modulators

[0078] Once identified, candidate Na_v modulators can be evaluated for selectivity and toxicological effects using known methods (see, e.g., Lu, Basic

Toxicology, Fundamentals, Target Organs, and Risk Assessment, Hemisphere

Publishing Corp., Washington (1985); U.S. Pat. No. 5,196,313 to Culbreth and U.S. Pat. No. 5,567,952 to Benet.

[0079] In specific embodiments, the modulatory agents identified using the assays of the present invention modulate are selective for a Na_v isoform of interest as opposed to other sodium channel alpha subunits. Such selectivity of modulation is suitably at least 10%, 50%, 100%, 10 times, 20 times, 100 times, 1000 times, 10,000 times or higher for the Na_v isoform of interest over any other sodium channel alpha subunit. Alternatively, an agent that is selective for the Na_v isoform of interest may not demonstrate an absolute preference for that isoform, the agent may show a preference for modulating the Na_v isoform of interest as compared to any other sodium channel. In illustrative examples of this type, the methods disclosed herein for identifying an agent that modulates, suitably blocks, a Na_v isoform of interest, comprise first identifying such agent and then testing such agent for effects on expression or activity of at least one other sodium channel gene or polypeptide, as the case may be, suitably at least two other such genes, or polypeptides, with little or no effect.

[0080] In other embodiments, an agent identified as having inhibitory activity against an Na_v isoform of interest by an assay of the invention is further tested to identify whether it also blocks activity of other sodium channels, other ion channels and/or other proteins. Such testing may be performed by a wide variety of methods, including systematic in vitro evaluations.

[0081] In some embodiments, exemplary modulatory agent are those that inhibit the Na_v isoform of interest at a lower concentration than any other ion channel protein. In particular, for a modulatory agent of the invention, the IC-50 of the Na_v isoform of interest is lower than the IC-50 of the next closest ion channel by a multiple of at least 1.1 , 1.2, 1.5, 1.7, 2, 3, 4, 5, 10, 20, 25, 50, 75, 100, 200, 500, 1000, 2000, 5000, 10000 or more. Thus, in a specific embodiments, the ratio of IC-50 of said next closest ion channel to the IC-50 of the Na_v isoform of interest is at least 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 10, 20, 25, 50, 75, 100, 200, 500, 1000, 2000, 5000, 10000 or more.

[0082] In certain embodiments, a modulatory agent identified by the assays of the present invention has a ratio of IC-50 of a sodium channel selected from among Na_vl.l, Na_v1.2, Na_v1.3, Na_v1.4, Na_v1.5, Na_v1.6, Na_v1.8, and Na_v1.9 to the IC-50 for Na_vl .7 that is at least 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 10, 20, 25, 50, 75, 100, 200, 500, 1000, 2000, 5000, or 10000. In other embodiments, a modulatory agent identified by the assays of the present invention has a ratio of IC-50 of a sodium channel selected from among Na_vl .1 , Na_vl .3, Na_vl .4, Na_vl .5, Na_vl .6, Na_vl .7, Na_vl .8, and Na_vl .9 to the IC-50 for Na_v1.2 that is at least 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 10, 20, 25, 50, 75, 100, 200, 500, 1000, 2000, 5000, or 10000. In still other embodiments, a modulatory agent identified by the assays of the present invention has a ratio of IC-50 of a sodium channel selected from among Na_vl .1 , N .2, Na_vl .4, Na_vl .5, Na_vl .6, Na_vl .7, Na_vl .8, and Na_vl .9 to the IC-50 for Na_v1.3 that is at least 1.1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 10, 20, 25, 50, 75, 100, 200, 500, 1000, 2000, 5000, or 10000.

[0083] The cells used for assessing selectivity desirably express at least 2, 3,

4, 5 or more Na_v channels in a natural setting. In specific embodiments, the cells employed a neuronal cells, including neuroblastoma cells and cell lines (e.g., SH- 5YSY). [0084] The toxicological effects of the Na_v modulators identified by the instant assays can be evaluated, for example, using primary cell lines or tissue slices in order to screen for the effect of the candidate modulator on the response of the ion channel of interest in its native physiological context. For example, to screen for drugs that exhibit specific and/or selective effects on heart cells it may be desirable to use myocytes or other in vitro cell culture model cell lines. In this case, a primary screen could be completed in a myocyte derived cell line to identify compounds that either shorten, prolong or block electrically-induced action potentials.

[0085] The secondary screen would then be designed to identify compounds that exhibit potentially adverse effects on the body. For example, this can be accomplished by screening for the effects of the candidate drug on electrically excitable tissues such as heart or neuronal tissues, or immortalized cell cultures derived from these tissues. These tissues play critical roles within an organism and any undesired effect of the candidate drug on the ability of these tissues to be electrically stimulated would be predicted to create potential serious side effects when administered. As a consequence, active compounds that also impaired the ability of these tissues to function could be eliminated from consideration as a drug candidate at an early stage, or have medicinal chemistry performed to reduce the side effects.

[0086] Additional toxicological analysis of candidate modulators can be established by determining in vitro toxicity towards a cell line, such as a mammalian (preferably human) cell line. Candidate modulators can be treated with, for example, tissue extracts, such as preparations of liver, including microsomal preparations, to determine increased or decreased toxicological properties of the chemical after being metabolized by a whole organism, or via their ability to be degraded via Cytochrome P450 systems. The results of these types of studies are often predictive of toxicological properties of chemicals in animals, such as mammals, including humans.

[0087] The toxicological activity can be measured using reporter genes that are activated during toxicological activity or by cell lysis (see WO 98/13353, published Apr. 2, 1998) or by using human models of drug metabolism, illustrative examples of which are disclosed in WO 2002/083897, published Oct. 24, 2002.

[0088] Alternatively, or in addition to these in vitro studies, the

bioavailability and toxicological properties of a candidate modulator in an animal model, such as mice, rats, rabbits, or monkeys, can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford (1979), Osweiler,

Toxicology, Williams and Wilkins, Baltimore, Md. (1995), Yang, Toxicology of Chemical Mixtures; Case Studies, Mechanisms, and Novel Approaches, Academic Press, Inc., San Diego, Calif. (1994), Burrell et al., Toxicology of the Immune System; A Human Approach, Van Nostrand Reinhld, Co. (1997), Niesink et al., Toxicology, Principles and Applications, CRC Press, Boca Raton, Fla. (1996)). Depending on the toxicity, target organ, tissue, locus, and presumptive mechanism of the candidate modulator, the skilled artisan would not be burdened to determine appropriate doses, LD50 values, routes of administration, and regimes that would be appropriate to determine the toxicological properties of the candidate modulator. In addition to animal models, human clinical trials can be performed following established procedures, such as those set forth by national or regional regulatory bodies, including the United States Food and Drug Administration (USFDA). These toxicity studies provide the basis for determining the therapeutic utility of a candidate modulator in vivo.

[0089J Candidate agent testing positive in the assays of the present invention, and suitably showing desirable therapeutic activities, may be derivatised to increase half-life, improve stability, reduce immunogenicity, and/or control solubility and hence bioavailability and pharmaco-kinetic properties, or to enhance solubility of actives or viscosity of solutions containing the derivatised agent.

[0090] A successful therapeutic agent of the present invention will typically meet some or all of the following criteria. Oral availability should be at or above 20%. Animal model efficacy is less than about 0.1 μg to about 100 mg/kg body weight and the target human dose is between 0.1 μg to about 100 mg/kg body weight, although doses outside of this range may be acceptable ("mg/kg" means milligrams of compound per kilogram of body mass of the subject to whom it is being administered). The therapeutic index (or ratio of toxic dose to therapeutic dose) should be greater than 100. The potency (as expressed by IC50 value) should be less than 10 μΜ, preferably below 1 μΜ and more preferably below 50 riM. The IC50 ("Inhibitory Concentration-50%") is a measure of the amount of compound required to achieve 50% inhibition of ion flux through a sodium channel, over a specific time period, in an assay of the invention. 6. Phamaceutical compositions and methods of treatment or prevention

[0091] The present invention also contemplates using the Na_v-modulating agents identified by the assays of the presen invention or their derivatives in methods for treating, preventing or ameliorating a disease or a condition in a mammal, suitably a human, wherein the disease or condition is associated with sodium channel activity (also referred to herein as a "sodium channel -mediated disease or condition"). Thus, where clinical applications are contemplated, it will be necessary to prepare the Nav- modulating agents a identified by the present invention as pharmaceutical compositions, i.e. , in a form appropriate for in vivo applications. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[0092] Administration of the Na_v-modulating agents of the invention, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of the invention can be prepared by combining a Na_v-modulating agent of the invention with an appropriate pharmaceutically acceptable carrier, including any suitable diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. Pharmaceutical

compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain an effective amount of a compound of the invention for treatment of a disease or condition of interest in accordance with the teachings of this invention.

[0093] The pharmaceutical compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable carriers include, but are not limited to, liquids, such as water, saline, glycerol and ethanol, and the like. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).Thus, the present invention provides compositions for treating, preventing and/or relieving the symptoms of a sodium channel-mediated disease or condition, comprising an effective amount of a Na_v-modulating agent and a pharmaceutically acceptable carrier, diluent or excipient. Pharmaceutically acceptable carriers include without limitation any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

Supplementary active ingredients also can be incorporated into the compositions.

[0094) Generally, the Na_v-modulating agents of the present invention are formulated in a neutral or salt form. Pharmaceutically-compatible salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0095] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the pharmaceutically active compounds are contained in an effective amount to achieve their intended purpose. The dose of active compounds administered to a patient should be sufficient to achieve a beneficial response in the patient over time such as reducing or ameliorating at least one symptom associated with a sodium channel-mediated disease or condition, preventing the disease or condition condition from occurring, i.e., prophylactic treatment of a patient; ameliorating the disease or condition, i.e., eliminating or causing regression of the disease or condition in a patient; suppressing the disease or condition, i.e., slowing or arresting the

development of the disease or condition in a patient; or alleviating the symptoms of the disease or condition in a patient. Thus, in terms of treatment, an effective amount of the given therapeutic agent is an amount sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of a disease or condition associated with sodium channel activity or otherwise reduce the pathological consequences of such a disease or condition. The effective amount is generally determined by the physician on a case-by- case basis and is within the skill of one in the art. Several factors are typically taken into account when determining, an appropriate dosage. These factors include age, sex and weight of the patient, the condition being treated, the severity of the condition and the form of the Na_v-modulating agent being administered. An effective amount can be administered in one or more doses. In any event, those of skill in the art may readily determine suitable dosages of the Na_v-modulating agents of the invention.

[0096] Generally, an effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g).

[0097] The ranges of effective doses provided herein are not intended to be limiting and represent preferred dose ranges. However, the most preferred dosage will be tailored to the individual subject, as is understood and determinable by one skilled in the relevant arts, (see, e.g., Berkow et al., eds., The Merck Manual, 16^th edition, Merck and Co., Rahway, N.J., 1992; Goodmanetna., eds., Goodman and Cilman's The

Pharmacological Basis of Therapeutics, 10^th edition, Pergamon Press, Inc., Elmsford, N.Y., (2001); Avery's Drug Treatment: Principles and Practice of Clinical

Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987), Ebadi, Pharmacology, Little, Brown and Co., Boston, (1985); Osolci al., eds., Remington's Pharmaceutical Sciences, 18^th edition, Mack Publishing Co., Easton, Pa. (1990); Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk, Conn. (1992)). [0098] The total dose required for each treatment can be administered by multiple doses or in a single dose over the course of the day, if desired. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. The diagnostic pharmaceutical compound or composition can be administered alone or in conjunction with other diagnostics and/or pharmaceuticals directed to the pathology, or directed to other symptoms of the pathology. The recipients of administration of compounds and/or compositions of the invention can be any vertebrate animal, such as mammals. Among mammals, the preferred recipients are mammals of the Orders Primate (including humans, apes and monkeys), Arteriodactyla (including horses, goats, cows, sheep, pigs), Rodenta

(including mice, rats, rabbits, and hamsters), and Carnivora (including cats, and dogs). Among birds, the preferred recipients are turkeys, chickens and other members of the same order. The most preferred recipients are humans.

[0099] The active compositions of the present invention include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. The pharmaceutical compositions may be introduced into the subject by any conventional method, e.g., by intravenous, intradermal, intramusclar,

intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term release); by oral, sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical implantation at a particular site, e.g., embedded under the splenic capsule, brain, or in the cornea. The treatment may consist of a single dose or a plurality of doses over a period of time.

[0100] The Na_v-modulating agents may be prepared for administration as solutions of free base or pharmacologically acceptable salts in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0101] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0102] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0103] A typical composition for intramuscular or intrathecal administration will consist of a suspension or solution of active ingredient in an oil, for example arachis oil or sesame oil. A typical composition for intravenous or intrathecal administration will consist of a sterile isotonic aqueous solution containing, for example active ingredient and dextrose or sodium chloride, or a mixture of dextrose and sodium chloride. Other examples are lactated Ringer's injection, lactated Ringer's plus dextrose injection, Normosol-M and dextrose, Isolyte E, acylated-Ringer's injection, and the like. Optionally, a co-solvent, for example, polyethylene glycol; a chelating agent, for example, ethylenediamine tetracetic acid; a solubilizing agent, for example, a cyclodextrin; and an anti-oxidant, for example, sodium metabisulphite, may be included in the formulation. Alternatively, the solution can be freeze dried and then reconstituted with a suitable solvent just prior to administration.

[0104] For oral administration the Na_v-modulating agents of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

[0105] The present invention also provides methods for treating or preventing a disease or condition associated with sodium channel activity (e.g. , aberrant activity or hyperactivity), wherein the methods comprise administering to a subject in need thereof an effective amount of a Na_v-modulating agent. Representative diseases or conditions associated with sodium channel activity generally include all disease states and/or conditions that are acknowledged now, or that are found in the future, to be associated with the activity of sodium channels. Such disease states and/or conditions include, but are not limited to, pathophysiological disorders, including hypertension, cardiac arrhythmogenesis, angina, insulin-dependent diabetes, non-insulin dependent diabetes mellitus, diabetic neuropathy, seizures, tachycardia, ischemic heart disease, cardiac failure, myocardial infarction, transplant rejection, autoimmune disease, sickle cell ^' anemia, respiratory diseases, muscular dystrophy, gastrointestinal disease, mental disorder, sleep disorder, anxiety disorder, eating disorder, neurosis, alcoholism, inflammation, multiple sclerosis, cerebrovascular ischemia, CNS diseases, epilepsy, stroke, Parkinson's disease, asthma, incontinence, urinary dysfunction, micturition disorder, irritable bowel syndrome, restenosis, subarachnoid hemorrhage, Alzheimers disease, drug dependence/addiction, schizophrenia, Huntington's chorea, pain and depression.

[0106] A sodium channel-mediated disease or condition broadly includes pain associated with HIV, HIV treatment induced neuropathy, trigeminal neuralgia, glossopharyngeal neuralgia, neuropathy secondary to metastatic infiltration, adiposis dolorosa, thalamic lesions, hypertension, autoimmune disease, asthma, drug addiction (e.g., opiate, benzodiazepine, amphetamine, cocaine, alcohol, butane inhalation), Alzheimer, dementia, age-related memory impairment, Korsakoff syndrome, restenosis, urinary dysfunction, incontinence, Parkinson's disease, cerebrovascular ischemia, neurosis, gastrointestinal disease, sickle cell anemia, transplant rejection, heart failure, myocardial infarction, reperfusion injury, intermittant claudication, angina, convulsion, respiratory disorders, cerebral or myocardial ischemias, long-QT syndrome,

Catecholeminergic polymorphic ventricular tachycardia, ophthalmic diseases, spasticity, spastic paraplegia, myopathies, myasthenia gravis, paramyotonia congenita,

hyperkalemic periodic paralysis, hypokalemic periodic paralysis, alopecia, anxiety disorders, psychotic disorders, mania, paranoia, seasonal affective disorder, panic disorder, obsessive compulsive disorder (OCD), phobias, autism, Aspergers Syndrome, Retts syndrome, disintegrative disorder, attention deficit disorder, aggressivity, impulse control disorders, thrombosis, pre clampsia, congestive cardiac failure, cardiac arrest, Freidrich's ataxia, Spinocerebellear ataxia, myelopathy, radiculopathy, systemic lupus erythamatosis, granulomatous disease, olivo-ponto-cerebellar atrophy, spinocerebellar ataxia, episodic ataxia, myokymia, progressive pallidal atrophy, progressive

supranuclear palsy and spasticity, traumatic brain injury, cerebral oedema,

hydrocephalus injury, spinal cord injury, anorexia nervosa, bulimia, Prader-Willi syndrome, obesity, optic neuritis, cataract, retinal haemorrhage, ischaemic retinopathy, retinitis pigmentosa, acute and chronic glaucoma, macular degeneration, retinal artery occlusion, Chorea, Huntington's chorea, cerebral edema, proctitis, post-herpetic neuralgia, eudynia, heat sensitivity, sarcoidosis, irritable bowel syndrome, Tourette syndrome, Lesch-Nyhan Syndrome, Brugado syndrome, Liddle syndrome, Crohns disease, multiple sclerosis and the pain associated with multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), disseminated sclerosis, diabetic neuropathy, peripheral neuropathy, Charcot marie tooth syndrome, arthritic, rheumatoid arthritis, osteoarthritis, chondrocalcinosis, atherosclerosis, paroxysmal dystonia, myasthenia syndromes, myotonia, myotonic dystrophy, muscular dystrophy, malignant

hyperthermia, cystic fibrosis, pseudoaldosteronism, rhabdomyolysis, mental handicap, hypothyroidism, bipolar depression, anxiety, schizophrenia, sodium channel toxin related illnesses, familial erythermalgia, primary erythermalgia, rectal pain, cancer, epilepsy, partial and general tonic seizures, febrile seizures, absence seizures (petit mal), myoclonic seizures, atonic seizures, clonic seizures, Lennox Gastaut, West Syndome (infantile spasms), multiresistant seizures, seizure prophylaxis (anti-epileptogenic), familial Mediterranean fever syndrome, gout, restless leg syndrome, arrhythmias, fibromyalgia, neuroprotection under ischaemic conditions caused by stroke or neural trauma, tachy-arrhythmias, atrial fibrillation and ventricular fibrillation and as a general or local anaesthetic.

[0107] In specific embodiments, the disease or condition is selected from the group consisting of neuropathic pain, inflammatory pain, visceral pain, cancer pain, chemotherapy pain, trauma pain, surgical pain, post-surgical pain, childbirth pain, labor pain, neurogenic bladder, ulcerative colitis, chronic pain, persistent pain, peripherally mediated pain, centrally mediated pain, chronic headache, migraine headache, sinus headache, tension headache, trigeminal neuralgia, cluster headache, phantom limb pain, peripheral nerve injury, and combinations thereof.

[01 8] In certain embodiments, the disease or condition is selected from the group consisting of pain associated with HIV, HIV treatment induced neuropathy, trigeminal neuralgia, post-herpetic neuralgia, eudynia, heat sensitivity, tosarcoidosis, irritable bowel syndrome, Crohns disease, pain associated with multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), diabetic neuropathy, peripheral neuropathy, arthritic, rheumatoid arthritis, osteoarthritis, atherosclerosis, paroxysmal dystonia, myasthenia syndromes, myotonia, malignant hyperthermia, cystic fibrosis,

pseudoaldosteronism, rhabdomyolysis, hypothyroidism, bipolar depression, anxiety, schizophrenia, sodium channel toxin related illnesses, familial erythermalgia, primary erythermalgia, familial rectal pain, cancer, epilepsy, partial and general tonic seizures, restless leg syndrome, arrhythmias, fibromyalgia, neuroprotection under ischaemic conditions caused by stroke or neural trauma, tachy-arrhythmias, atrial fibrillation and ventricular fibrillation.

7. Combination Therapy

[0109] A Na_v-modulating agent of the present invention may be usefully combined with one or more other Na_v-modulating agents of the invention or one or more other therapeutic agents or in any combination thereof, in the treatment of sodium channel-mediated diseases and conditions. For example, a Na_v-modulating agent of the invention may be administered simultaneously, sequentially or separately in

combination with other therapeutic agents, including, but not limited to: opiates analgesics, e.g., morphine, heroin, cocaine, oxymorphine, levorphanol, levallorphan, oxycodone, codeine, dihydrocodeine, propoxyphene, nalmefene, fentanyl, hydrocodone, hydromorphone, meripidine, methadone, nalorphine, naloxone, naltrexone,

buprenorphine, butorphanol, nalbuphine and pentazocine; non-opiate analgesics, e.g., acetomeniphen, salicylates (e.g. , aspirin); nonsteroidal antiinflammatory drugs

(NSAIDs), e.g., ibuprofen, naproxen, fenoprofen, ketoprofen, celecoxib, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetin and zomepirac;

anticonvulsants, e.g., carbamazepine, oxcarbazepine, lamotrigine, valproate, topiramate, gabapentin and pregabalin; antidepressants such as tricyclic antidepressants, e.g., amitriptyline, clomipramine, despramine, imipramine and nortriptyline; COX-2 selective inhibitors, e.g., celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, and lumiracoxib; alpha-adrenergics, e.g., doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, and 4-amino-6,7-dimethoxy-2-(5-methane sulfonamido- 1 ,2,3 ,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl) quinazoline; barbiturate sedatives, e.g., amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobartital, secobarbital, talbutal, theamylal and thiopental; tachykinin ( K) antagonist, particularly an NK-3, N -2 or NK-1 antagonist, e.g., ( R,9R)-7-[3,5-bis(trifluoromethyl)benzyl)]-8,9,10,l l-tetrahydro-9- methyl-5-(4-methylphenyl)-7H-[l,4]diazocino[2,l-g][l,7]-naphthyridine-6-13- -dione (TAK-637), 5-[[2R,3S)-2-[(lR)-l-[3,5-bis(trifluoromethylphenyl]ethoxy-3-(4- fluorophe- nyl)-4-morpholinyl]-methyl]-l ,2-dihydro-3H-l ,2,4-triazol-3-one (MK-869), aprepitant, lanepitant, dapitant or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]- methylamino]-2-phenylpiperidine (2S,3S); coal-tar analgesics, in particular

paracetamol; serotonin reuptake inhibitors, e.g., paroxetine, sertraline, norfluoxetine (fluoxetine desmethyl metabolite), metabolite demethylsertraline, '3 fluvoxamine, paroxetine, citalopram, citalopram metabolite desmethylcitalopram, escitalopram, d,I- fenfluramine, femoxetine, ifoxetine, cyanodothiepin, litoxetine, dapoxetine, nefazodone, cericlamine, trazodone and fluoxetine; noradrenaline (norepinephrine) reuptake inhibitors, e.g., maprotiline, lofepramine, mirtazepine, oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion, buproprion metabolite hydroxybuproprion, nomifensine and viloxazine (Vivalan™)), especially a selective noradrenaline reuptake inhibitor such as reboxetine, in particular (S,S)-reboxetine, and venlafaxine duloxetine neuroleptics sedative/anxiolytics; dual serotonin-noradrenaline reuptake inhibitors, such as venlafaxine, venlafaxine metabolite O-desmethylvenlafaxine, clomipramine, clomipramine metabolite desmethylclomipramine, duloxetine, milnacipran and imipramine; acetylcholinesterase inhibitors such as donepezil; 5-HT₃ antagonists such as ondansetron; metabotropic glutamate receptor (mGluR) antagonists; local anaesthetic such as mexiletine and lidocaine; corticosteroid such as dexamethasone;

antiarrhythimics, e.g., mexiletine and phenyloin; muscarinic antagonists, e.g., tolterodine, propiverine, tropsium t chloride, darifenacin, solifenacin, temiverine and ipratropium; cannabinoids; vanilloid receptor agonists {e.g., resinferatoxin) or antagonists (e.g., capsazepine); sedatives, e.g. , glutethimide, meprobamate,

methaqualone, and dichloralphenazone; anxiolytics such as benzodiazepines, antidepressants such as mirtazapine, topical agents (e.g., lidocaine, capsacin and resiniferotoxin); muscle relaxants such as benzodiazepines, baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol and orphrenadine; anti-histamines or HI antagonists; NMDA receptor antagonists; 5-HT receptor agonists/antagonists;

PDEV inhibitors; Tramadol™; cholinergic (nicotine) analgesics; alpha-2-delta ligands; prostaglandin E2 subtype antagonists; leukotriene B4 antagonists; 5-lipoxygenase inhibitors; and 5-HT₃ antagonists; and AT₂ receptor antagonists as described for example in WO 2006/066361 published Jun 29, 2006 and WO 2007/106938 published September 27, 2007.

[0110] Sodium channel-mediated diseases and conditions that may be treated and/or prevented using such combinations include but not limited to, pain, central and peripherally mediated, acute, chronic, inflammatory, neuropathic pain as well as other diseases with associated pain and other central nervous disorders such as epilepsy, anxiety, depression and bipolar disease; or cardiovascular disorders such as

arrhythmias, atrial fibrillation and ventricular fibrillation; neuromuscular disorders such as restless leg syndrome and muscle paralysis or tetanus; neuroprotection against stroke, neural trauma and multiple sclerosis; and channelopathies such as erythromyalgia and familial rectal pain syndrome.

8. Kits

[0111] Also provided by the present invention are kits for practicing the methods and screening assays described herein. These kits will generally contain (1) the mammalian cell (e.g. , a neuroblastoma cell that is suitably of human origin), which enodgenously expresses an Na_v isoform of interest and suitably at least one other Na_v isoform, (2) at least one Na_v isoform-inhibiting agent (e.g., conotoxin Till A, ProTxII, an antagonist antigen-binding molecule that is specifically immuno-interactive with an individual Na_v isoform, or a nucleic acid molecule [e.g. , siRNA, shRNA, antisense etc.] that inhibits expression of an individual Na_v isoform) to inhibit the level or activity of one or more of the other Na_v isoforms that are not the subject of investigation; and (3) a sodium channel opener/activator (e.g., veratridine, grayanotoxin, aconitine,

batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion toxins, sea anemone toxins, β scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, δ-conotoxins) that opens/activates the Na_v isoform of interest and optionally the other Na_v isoform, or if more than one, at least one of the other Na_v isoforms. In some embodiments, the kits further comprise a voltage sensor, illustrative examples of which are selected from ion transport-indicating agents (e.g., sodium- indicating agents and calcium-indicating agents) and membrane potential-indicating agents. In certain embodiments, the kits may further contain instructions for conducting the assesment or assay. Suitably, the kits may comprise one or more containers (e.g. , multiwell plates) for conducting the assessment or assay.

[0112] In some embodiments, the kits of the invention include at least one candidate agent screening apparatus, where the apparatus comprises the mammalian cell. In certain embodiments, the kits further include a positive or negative control, e.g. , a positive control, such as a known agonist or antagonist of the Na_v isoform of interest. Other optional components of the kits include: reagents for detection ion transport (e.g., chemical reagents to facilitate detection of sodium or calcium influx or changes in membrane potential, buffers; etc. The various components of the kits may be present in separate containers or certain compatible components may be precombined into a single container, as desired.

[0113] In addition to above-mentioned components, the subject kits may further include instructions for using the components of the kit to practice the methods and assays of the present invention. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. , via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0114] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLES

EXAMPLE 1

ASSAY FOR NA_V1.7 BLOCKERS USING VERATRIDINE-EVOKED CALCIUM INFLUX AND Μ-CONOTOXIN TIIIA TO BLOCK ΙΘΝ TRANSPORT THROUGH A YL .2

[0115] SH-S Y5 Y cells are loaded with Fluo-4 (Invitrogen) by incubating the cells in PSS containing 0.3% bovine serum albumin and 4 μΜ Fluo-4-AM (Invitrogen) for 30 min at 37° C. To remove extracellular dye and facilitate dye hydrolysis, cells are washed with PSS for 5-15 min prior to loading of plates into the FLIPR^TCTRA+

(Molecular Devices, Sunnyvale, CA) fluorescent plate reader. Fluorescence (excitation 470-495 nm; emission 515-575 nm) is measured using a cooled CCD camera with camera gain and excitation intensity adjusted for each plate to yield an average baseline fluorescence value of 1000 AFU. After 10 baseline reads, μ-conotoxin TIIIA is added to the cells at a final concentration 1 μΜ to block Na_v1.2 and the fluorescence response is measured every 10 seconds for 150 reads. Buffer or putative Na_v 1.7 blocker (/^'. e. , candidate agent) is then added to the cells and the fluorescence response is measured every second for 300 reads. Veratridine is then added to a final concentration of 50 μΜ, which preferentially activates both Na_vl .2 and Na_vl .7 as compared to Na_vl .3.

Fluorescence measurements are then taken every second for a further 300 seconds. Na_v1.7 blockers are identified by their ability to further prevent ion influx into the cell. EXAMPLE 2

ASSAY FOR NA 1.7 BLOCKERS USING OD-1-EVOKED CALCIUM INFLUX

[0116] SH-S Y5Y cells are loaded with Fluo-4 (Invitrogen) by incubating the cells in PSS containing 0.3% bovine serum albumin and 4 μΜ Fluo-4-AM (Invitrogen) for 30 min at 37° C. To remove extracellular dye and facilitate dye hydrolysis, cells are washed with PSS for 5-15 min prior to loading of plates into the FLIPR^TCTRA+

(Molecular Devices, Sunnyvale, CA) fluorescent plate reader. Fluorescence (excitation 470-495 nm; emission 515-575 nm) is measured using a cooled CCD.camera with camera gain and excitation intensity adjusted for each plate to yield an average baseline fluorescence value of 1000 AFU. Buffer or putative Na_vl .7 blocker (i.e. , candidate agent) is then added to the cells and the fluorescence response is measured every second for 300 reads. OD-1 (Jalali et al., FEBS Letters 2005. 579: 4181-4186; Maertens et al,

J Pharmacol. 2006. 70(1): 405-414) is added to a final concentration of 50 μΜ, which preferentially activates Na_vl .7. Alternatively, a combination of OD-1 (10 nM) and veratridine (3 μΜ) may be used in which veratridine synergises with OD-1 to improve the signal to noise ratio of the assay. As with Example 1, fluorescence measurements are taken every second for a further 300 seconds and Na_v1.7 blockers are identified by their ability to further prevent ion influx into the cell.

EXAMPLE 3

ASSAY FOR NA_K1.7 BLOCKERS USING VERATRIDINE-EVOKED CALCIUM INFLUX AND

SHRNA OR SIRNA TO KNOCK DOWN EXPRESSION OF NA_KL2

[0117] SH-S Y5 Y cells are transfected with siRNA (ON-TARGETplus Set of 4 Scn2al, Thermo Scientific Dharmacon RNAi Technologies, Boulder, CO, USA) or shRNA-expressing vector (Set of 3 Human Lentiviral shRNA Constructs SCN2A, Thermo Scientific Dharmacon RNAi Technologies, Boulder, CO, USA) according to the manufacturer's instructions. The cells are subsequently incubated in PSS containing 0.3% bovine serum albumin and 4 μΜ Fluo-4-AM (Invitrogen) for 30 min at 37° C as in Example 1. Buffer or putative Na_v 1.7 blocker (/^*. e. , candidate agent) is then added to the cells and the fluorescence response is measured every second for 300 reads. Veratridine is then added to a final concentration of 30-50 μΜ, which preferentially activates both Na_vl .2 and Na_vl .7 as compared to Na_vl .3, and fluorescence measurements are taken every second for a further 300 seconds. Fluorescence measurements are then taken every second for a further 300 seconds. Na_vl .7 blockers are identified by their ability to further prevent ion influx into the cell.

EXAMPLE 4

ASSAY FOR NA 1.2 BLOCKERS USING VERATRIDINE-EVOKED CALCIUM INFLUX AND

PROTXII TO BLOCK ION TRANSPORT THROUGH NA 1.7

[0118] SH-SY5Y cells are loaded with Fluo-4 (Invitrogen) by incubating the cells in PSS containing 0.3% bovine serum albumin and 4 μΜ Fluo-4- AM (Invitrogen) for 30 min at 37° C. To remove extracellular dye and facilitate dye hydrolysis, cells are washed with PSS for 5-15 min prior to loading of plates into the FLIPR^TETRA+

(Molecular Devices, Sunnyvale, CA) fluorescent plate reader. Fluorescence (excitation 470-495 nm; emission 515-575 nm) is measured using a cooled CCD camera with camera gain and excitation intensity adjusted for each plate to yield an average baseline fluorescence value of 1000 AFU. After 10 baseline reads, ProTxII is added to the cells at a final concentration 30 nM to block Na_v1.7 and the fluorescence response is measured every 10 seconds for 150 reads. Buffer or putative Na_v1.2 blocker (i.e., candidate agent) is then added to the cells and the fluorescence response is measured every second for 300 reads. Veratridine is then added to a final concentration of 30-50 μΜ, which preferentially activates both Na_vl .2 and Na_vl .7 as compared to Na_vl .3, and fluorescence measurements are taken every second for a further 300 seconds. Na_v1.2 blockers are identified by their ability to further prevent ion influx into the cell.

EXAMPLE 5

ASSAY FOR NA_K1.2 BLOCKERS USING VERATRIDINE-EVOKED CALCIUM INFLUX AND

SHRNA OR SIRNA TO KNOCK DOWN EXPRESSION OF NA 1.7

[0119] SH-S Y5 Y cells are transfected with siRNA (ON-TARGETplus Set of 4 SCN7A, Thermo Scientific Dharmacon RNAi Technologies, Boulder, CO, USA) or shRNA-expressing vector (Set of 3 Human Lentiviral shRNA Constructs SCN7A, Thermo Scientific Dharmacon RNAi Technologies, Boulder, CO, USA) according to the manufacturer's instructions. The cells are subsequently incubated in PSS containing 0.3% bovine serum albumin and 4 μΜ Fluo-4-AM (Invitrogen) for 30 min at 37° C as in Example 3. Buffer or putative Na_v1.2 blocker (i.e., candidate agent) is then added to the cells and the fluorescence response is measured every second for 300 reads. Veratridine is then added to a final concentration of 30-50 μΜ, which activates both Na_vl .2 and Na_vl .7 but not Na_vl .3, and fluorescence measurements are taken every second for a further 300 seconds. Na_vl .2 blockers are identified by their ability to further prevent ion influx into the cell.

EXAMPLE 6

ASSAY FOR NA_K1.3 BLOCKERS USING CIGUATOXIN BREVETOXIN-EVOKED CALCIUM INFLUX AND PROTXII TO BLOCK ION TRANSPORT THROUGH NA _v1.7

[0120] SH-SY5Y cells are loaded with Fluo-4 (Invitrogen) by incubating the cells in PSS containing 0.3% bovine serum albumin and 4 μΜ Fluo-4- AM (Invitrogen) for 30 min at 37° C. To remove extracellular dye and facilitate dye hydrolysis, cells are washed with PSS for 5- 15 min prior to loading of plates into the FLIPR^"* *

(Molecular Devices, Sunnyvale, CA) fluorescent plate reader. Fluorescence (excitation 470-495 nm; emission 515-575 nm) is measured using a cooled CCD camera with camera gain and excitation intensity adjusted for each plate to yield an average baseline fluorescence value of 1000 AFU. After 10 baseline reads, ProTxII is added to the cells at a final concentration 30 nM to block Na_y1.7 and the fluorescence response is measured every 10 seconds for 150 reads. Buffer or putative Na_vl .3 blocker (i.e. , candidate agent) is then added to the cells and the fluorescence response is measured every second for 300 reads. Ciguatoxin (e.g., P-CTX-1) or brevetoxin is then added to a final concentration of 10 - 100 nM, which activates Na_v1.3 but not Na_v1.7, and fluorescence measurements are taken every second for a further 300 seconds. Na_vl .3 blockers are identified by their ability to further prevent ion influx into the cell.

EXAMPLE 7 ASSAY FOR N A _v\ .3 BLOCKERS USING CIGUATOXIN/BREVETOXIN-EVOKED CALCIUM INFLUX AND SHRNA OR SIRNA TO KNOCK DOWN EXPRESSION OF NA_K1.7

[0121] SH-S Y5 Y cells are transfected with siRNA (ON-T ARGETplus Set of 4 SCN7A, Thermo Scientific Dharmacon RNAi Technologies, Boulder, CO, USA) or shRNA-expressing vector (Set of 3 Human Lentiviral shRNA Constructs SCN7A, Thermo Scientific Dharmacon RNAi Technologies, Boulder, CO, USA) according to the manufacturer's instructions. The cells are subsequently incubated in PSS containing 0.3% bovine serum albumin and 4 μΜ Fluo-4-AM (Invitrogen) for 30 min at 37° C as in Example 5. Buffer or putative Na_vl .3 blocker (i.e., candidate agent) is then added to the cells and the fluorescence response is measured every second for 300 reads. Ciguatoxin (e.g. , P-CTX- 1 ) or brevetoxin is then added to a final concentration ofl 0- 100 nM, which activates Na_v1.3 but not Na_v1.7, and fluorescence measurements are taken every second for a further 300 seconds. Na_v1.3 blockers are identified by their ability to further prevent ion influx into the cell.

EXPERIMENTAL RESULTS

SH-SYSY Cells Endoeenouslv Express TTX-sensitive Na_v Channels

[0122] Expression of human TTX-sensitive and TTX-resistant Na_v isoforms and the accessory β subunits present in SH-SY5Y cells was assessed by semiquantitative PCR. As previously reported, SH-SY5Y cells expressed mainly the TTX- sensitive isoforms Na_v1.3 and Na_vl .7 as well as Na_v1.2 (Figure 1 A) [Blum, R., et al, Nature, 2002. 419(6908): 687-93]. In addition, some amplification of Na_vl .4 and Na_vl .5 was detected, although in contrast to previous reports the present inventors were unable to detect Na_vl .9 [Blum, R., et al. , 2002, supra], while Na_vl .7 was consistently found to be the most highly expressed Na_v isoform in SH-SY5Y cells. In addition, accessory subunits β2 and β3, but not βΐ or β4, were amplified, with the β3 subunit being most abundant (Figure 1 B). The present inventors also confirmed that Na_vl .7 and Na_vl .3 protein trafficked correctly by immunofluorescence (Figures 2 A and B). SH-SY5Y cells stained with Na_vl .7 and Na_vl .3 antibodies showed fluorescence localized to the plasma membrane (Figures 2 A and B), indicative of functional Na_v expression.

Endozenously Expressed TTX-sensitive Na_v in SH-SYSY cells are Activated by

Veratridine and Cieuatoxin

[0123] To establish that Na_v isoforms expressed in SH-SY5Y cells are functional, in membrane potentials were assessed in response to the Na_v specific activator, veratridine. Addition of veratridine caused a concentration-dependent membrane depolarization with an EC50 of 28.5 μΜ (pIC₅o 4.54 ± 0.06), confirming that Na_v channels endogenously expressed in SH-SY5 Y cells are indeed functional (Figure 3 A). The veratridine-induced membrane depolarization was mediated only through activation of TTX-sensitive Na_v, as pre-treatment with 300 nM TTX completely abolished these responses (Figure 3 A and B). However, as membrane potential assays are prone to artifacts and are costly, the present inventors sought to establish a Na_v assay based on the more robust and cost-effective fluorescent calcium (Ca ) responses.

Addition of veratridine also caused a concentration-dependent increase in intracellular calcium with an EC50 of 21.9 μΜ (pICso 4.66 ± 0.04) that was not significantly different to the potency obtained using the membrane potential assay (Figure 4 A). The Hill slope of the veratridine-induced response was surprisingly steep (3.25 ± 0.9 in the membrane potential assay and 4.2 ± 0.6 in the Ca²⁺ response assay) suggesting a mode of activation of the endogenously expressed Na_v and subsequent cell depolarization involving positive cooperativity. The Ca²⁺ responses elicited by veratridine were completely blocked in the presence of 300 nM TTX, providing further evidence that the responses were mediated solely through TTX-sensitive Na_v isoforms endogenously expressed in SH-SY5Y cells (Figure 4 A and B). The IC50 of TTX-mediated inhibition of veratridine responses was 8.6 nM (pIC₅o 8.06 ± 0.08) (Figure 4 B), consistent with the inhibition of TTX-sensitive Na_v channels. [0124] Addition of P-CTX-1 caused a concentration-dependent increase in intracellular Ca²⁺ (Figure 4 C) with an EC_S0 of 3.7 nM (pEC₅₀ 8.43 ± 0.32), consistent with low nanomolar activation of Na_v expressed in DRG neurons [Birinyi-Strachan, L.C., et al., Toxicol Appl Pharmacol, 2005. 204(2): 175-86]. The Ca²⁺ responses elicited by P-CTX-1 were both mediated through TTX-sensitive Na_v isoforms, with an IC50 of TTX-mediated inhibition (Figure 4 D) of 2.4 nM (pIC₅₀ 8.74 ± 0.07). The Ca²⁺ responses elicited by P-CTX-1 were completely blocked in the presence of 300 nM TTX, indicating that the responses were mediated solely by TTX-sensitive Na_v isoforms endogenously expressed in SH-SY5Y cells. L-type and N-type Calcium Channels Contribute to the Veratridine- and P-CTX-

1 -Induced Responses

[0125] To assess which voltage-gated calcium channels contribute to the depolarization-induced Ca²⁺ influx after addition of veratridine, the present inventors assessed the effects of nifedipine to block L-type voltage-gated calcium channels (VGCC), co-conotoxin CVID to block N-type VGCC, and ω-agatoxin TK to block P/Q- type VGCC, on the veratridine-induced responses (Figure 5 A and B). Pre-treatment with nifedipine concentration-dependently inhibited veratridine-induced responses with an IC50 of 10.7 nM (pICso 7.97 ± 0.2) (Fig 5 A). However, nifedipine did not completely abolish veratridine-induced responses, with 23.9 ± 4.4 % of the response remaining in the presence of saturating concentrations of nifedipine. The veratridine-induced response was also mediated by N-type VGCC, as CVID also caused a partial (31.8 ± 1.1%) concentration-dependent block (ρ ¼ο 7.7 ± 0.5) of the veratridine-induced response (Figure 5 B). Co-addition of nifedipine (10 μΜ) and CVID (1 μΜ ) completely abolished veratridine-mediated responses (Figure 5 B). In contrast, the P/Q- type antagonist agatoxin TK did not inhibit veratridine responses (Figure 5A), supporting a major role of L-type VGCC and a smaller role for N-type VGCC in the depolarization-induced calcium influx elicited by activation of endogenously expressed Na_v.

[0126] Nifedipine also concentration-dependently inhibited P-CTX- 1 -induced Ca²⁺ responses by 75.5 ± 3.9 % with an IC50 of 19.8 nM (pIC50 7.7 ± 0.4), while agatoxin TK did not inhibit P-CTX-1 responses (Figure 5 C), supporting a role for L- type but not P/Q-type VGCC contributing to the P-CTX-1 response. In addition, N-type VGCC contributed to the Ca ⁺ influx elicited by P-CTX-1 , as CVID concentration's - dependently inhibited P-CTX-1 responses (Figure 5 D) with an IC50 of 7.9 nM (pICso 8.12 ± 0.53). Similar to inhibition of veratridine-induced responses, block of P-CTX-1 responses by CVID was partial (maximum inhibition of 28.7 ± 5.8 %) and additive with nifedipine, with P-CTX-1 responses completely abolished in the presence of nifedipine (10 μΜ) and CVID (1 μΜ).

Nay Subtypes Contributing to the Veratridine- and P-CTX-1 -Induced Responses

[0127] Na_v subtype-specific inhibitors were used to elucidate the contribution of various Na_v subtypes to the veratridine- and P-CTX- 1 -induced Ca²⁺ response.

Although Na_vl .2 was not the most abundantly expressed Na_v isoform in SH-SY5Y cells, the Na_vl .2 1\ .4-selective blocker TIIIA [Lewis, R.J., et al. , Mol Pharmacol, 2007. 71(3): 676-85] reduced veratridine-induced responses by 42.6 ± 6.8 % (Figure 6 A; n= 4 independent experiments) with an ICso of 290 nM (pICso 6.54 ± 0.09). This component was mediated exclusively by Na_v1.2, as the Na_vl .4-selective blocker GIIIA [Lopez- Vera, E., et al, Biochemistry, 2008. 47(6): 1741-51] did not affect veratridine responses (Figure 6 A) at concentrations up to 10 μΜ. A small (p < 0.05) rightward shift of the veratridine concentration-response curve was observed in the presence of 1 μΜ TIIIA (EC50 45.8 μΜ; pEC₅o 4.33 ± 0.16, n = 3 independent experiments) and TIIIA again decreased the magnitude of the response by 41.3 ± 2.8% (Figure 6 B).

[0128} While the Na„l .4 inhibitor GIIIA did not significantly affect P-CTX- 1 responses (Figure 6 C), the Na_vl .2 /Na_vl .4 inhibitor TIIIA decreased P-CTX- 1 -induced responses by 22.3 ± 4.4 %. (Figure 6 C; n= 7 independent experiments) with an IC50 of 79.5 nM (pICso 7.10 ± 0.14). However, in the presence of 1 μΜ TIIIA, the EC50 of the P-CTX-1 concentration-response curve was not significantly affected, although the magnitude of the P-CTX-1 response was decreased by 22.4% (p - 0.08; Figure 6 D), consistent with previous reports of activation of Na_vl .2 by ciguatoxins [Yamaoka, K., et al, Br J Pharmacol, 2004. 142(5): 879-89; Yamaoka, K., et al., JBiol Chem, 2009. 284(12): 7597-605],

[0129] The component of the veratridine-induced response observed in the presence of TIIIA was completely and concentration-dependently blocked by the Na_vl .7-selective blocker ProTxII [Schmalhofer, W.A., et al. , Mol Pharmacol, 2008. 74(5): 1476-84] (IC_S0 of 206.9 pM; pIC₅₀ 9.68 ± 0.15, n = 4 independent experiments) consistent with inhibition of Na_v1.7 (Figure 7 A). In the absence of TIIIA, ProTxII blocked veratridine-induced responses with a two-site fit with IC50S of 151.7 pM and 56 nM (pICso 9.82 ± 0.23 and pIC₅₀ 7.25 ± 0.26; n = 3 independent experiments)(Figure 7 A). In contrast, ProTxII inhibited P-CTX-1 responses only with low affinity (Figure 7C), providing evidence that P-CTX-1 responses do not involve activation of Na_v1.7 endogenously expressed in SH-SY5Y cells. The inhibition of P-CTX-1 responses by ProTxII preferentially fit a one-site model, and with an IC50 of 1.9 μΜ, was consistent with activation of Na_v isoforms other than Na_v1.7 by P-CTX-1 [Schmalhofer, W.A., et al, Mol Pharmacol, 2008, supra]. In light of minor contribution of Na_v1.2 and no contribution of Na_vl .7 to the P-CTX-1 -induced Ca²⁺ responses in SH-SY5Y cells, Na_v1.3 remained as the only endogenously expressed TTX-sensitive Na_v isoform that could contribute to P-CTX-1 -induced responses in SH-SY5Y cells. Indeed, P-CTX-1 - induced responses were significantly decreased in cells expressing Na_vl .3 shRNA, while KCl-induced Ca²⁺ responses were not significantly decreased in Na_v1.3 shRNA- expressing SH-SY5Y cells (Figure 8 A and B), confirming a major role of Na_v1.3 and a minor role of Na_vl .2 in P-CTX-1 -induced responses in SH-SY5Y cells.

(0130] The assays presented herein display excellent robustness and reproducibility with a Z' factor [Zhang, J.H., et al, J Biomol Screen, 1999. 4(2): 67-73] of 0.70 ± 0.05 (Figure 7 B) for veratridine-induced responses and 0.68 ± 0.04 for P- CTX-1 induced responses and can be adapted to detect endogenously expressed human Na_vl .2, Na_vl .7 or Na_vl .3 specifically in the presence of appropriate inhibitors. DISCUSSION

[0131] Despite rapidly growing interest in Na_v channels as putative therapeutic targets, the discovery of subtype-specific inhibitors has been hampered by a lack of functionally relevant assays amenable to high throughput screening, limiting especially the early phases of the drug discovery process. Assays for hit and lead compounds identification should ideally display high sensitivity and specificity, yield high throughput and high content information, display high robustness and flexibility, and be physiologically relevant, and cost effective. To simultaneously address at least some of these criteria the present inventors developed certain embodiments of the assays broadly described herein, which are based on fluorescent imaging assays for detecting inhibitors of pain-specific Na_vl .3 and 1.7 channels.

[0132] While it is possible to use membrane potential dyes in the assays of the invention, Ca²⁺ imaging, including fluorescent Ca²⁺ imaging, is arguably the high throughput method of choice for a range of pharmaceutical targets including voltage- or Hgand-gated ion channels permeable to Ca and G-protein coupled receptors coupled to intracellular Ca²⁺ stores [Hansen, K.B., et al, Methods Mol Biol, 2009. 552: 269-78; Belardetti, F., et al, Assay Drug Dev Technol, 2009. 7(3): 266-80]. Across cell membranes, concentration difference of approximately 10,000 fold between the extracellular and intracellular side provide a large Ca²⁺ gradient which essentially drives the extremely high sensitivity and low noise inherent to measurement of Ca²⁺. These properties also allow excellent temporal resolution of Ca²⁺ signals. In addition, the availability of several platforms including the high throughput plate reader FLIPR (Molecular Devices) makes fluorescent Ca ⁺ measurement one of the highest throughput options for the identification of putative drug leads using functional assays.

[0133] In specific embodiments of the assays described herein, the present inventors have developed three novel FLIPR Ca²⁺ assays to detect toxin activation of Na_v channels endogenously expressed in human neuroblastoma cells, including the SH- SY5Y neuroblastoma cell line. The alkaloid veratridine preferentially activated endogenously expressed Na_vl .2 and Na_vl .7, while P-CTX- 1 preferentially activated Na_vl .2 and Na_vl .3. Activation of endogenously expressed Na_v results in influx of Na* ions and subsequent membrane depolarization. This membrane depolarization triggers a Ca²⁺ influx through endogenously expressed voltage-gated L- and N-type calcium channels which can be detected by fluorescent Ca ⁺ dyes such as Fluo-4 or Fura-2 in high throughput or high content format. For veratridine-induced responses, block of Na_v1.2 by the conotoxin TIIIA produces a Na_v1.7 -specific assay, and conversely block of Na_vl .7 by low concentrations of ProTxII isolates Na_v responses mediated exclusively by Na_vl .2. Similarly, for P-CTX-1 responses, block of Na_vl .2 by the conotoxin TIIIA produced a Na_vl .3 -specific assay.

[0134] The present inventors also confirmed expression of Na_vl .2, Na_vl .3 and

Na_vl .7 in SH-SY5Y cells at the mRNA level, consistent with previous reports [Blum, R., et al, 2002, supra], although they were unable to detect Na_vl .9 previously reported to be also expressed in these cells. Consistent with a lack of TTX-resistant Na_v isoforms in SH-SY5Y cells, both Ca²⁺ and membrane potential responses elicited by veratridine and P-CTX-1 were completely abolished by low concentrations of TTX. Although low levels of Na_v1.4 mRNA transcripts were also detected, veratridine has been reported to block rather than activate the skeletal muscle Na_v isoform Na_vl .4 [Wang, G.K. et al. , J Physiol, 2003. 548(Pt 3): 667-75], further supporting the lack of effect of the Na_vl .4 inhibitor GI1IA on the veratridine-elicited Ca²⁺ responses. In addition, Na_vl .4 did not contribute to the P-CTX- 1 -induced responses.

[0135] The present inventors were also able to confirm for the first time the expression of Na_vl .7 and Na_vl .3 in SH-SY5Y cells at the protein level, with Na_vl .7 and Na_vl .3 immunofluorescence shown to be localized predominantly at the plasma membrane. The endogenous expression of human Na_vl .7 and Na„l .3 with functionally relevant β subunits make SH-SY5Y cells well suited to the study of native human Na_v pharmacology.

[0136] Pharmacological characterization of the veratridine-induced responses in SH-SY5Y cells shows that despite relatively high expression of Na_vl .3, this isoform does not appear to contribute significantly to the veratridine-induced responses. While veratridine has been reported to affect gating of most Na_v isoforms albeit with reduced efficacy at Na_v1.8 [Vickery, R.G., et al, Receptors Channels, 2004. 10(1): 11-23] by binding to site 2 in the S6 segment of the pore-forming a subunit, the relative sensitivity of Na_v isoforms to veratridine, as well as its relative efficacy across different Na_v isoforms has not been established [Ulbricht, W., Rev Physiol Biochem Pharmacol, 1998. 133: 1-54]. In addition, veratridine has been reported to be a partial agonist in fetal mouse brain cells and rat heart cells [Catterall, W.A., et al., Mol Pharmacol, 1981. 20(3): 533-42; Couraud, F., et al., JNeurosci, 1986. 6(1): 192-8]. Thus, differences in the sensitivity or efficacy of activation of Na_v subtypes by veratridine could contribute to preferential activation of endogenously expressed Na_vl .2 and Na_vl .7 in SH-SY5 Y cells. It is also unclear if expression of accessory subunits could alter activation of Na_v isoforms by veratridine. Specifically, the altered inactivation kinetics of Na_vl .3 in the presence of β3 subunits J Biol Chem. 285(43): 33404-12] could contribute to the absence of Na_vl .3 -mediated responses to veratridine.

[0137] However, Na_vl.3 endogenously expressed in SH-SY5Y cells is functional, as P-CTX- 1 elicited responses predominantly mediated through Na_v1.3 and to a lesser degree through Na_vl .2. While activation of Na_vl .2 by ciguatoxins has been reported previously [Yamaoka, K., et al, 2009, supra] and the results presented here are consistent with a contribution of Na_v1.2 to P-CTX- 1 -induced responses, this is the first time that Na_v 1.3 has been shown to be activated by P-CTX- 1. Surprisingly, while activation of Na_vl .7 by ciguatoxins has previously been proposed to be mechanistically involved in the painful symptoms of ciguatera [Yamaoka, K., et al, 2009, supra], the present inventors present evidence herein that Na_vl .7 is not activated by low

concentrations of P-CTX-1 and is thus unlikely to contribute to the symptomatology of ciguatera.

[0138] Compounds affecting function ofVGCC can also produce positive responses in this system. However, such potential artifacts can provide valuable information on putative off-target effects of the test compounds. For example, compounds that cause an increase in intracellular Ca²⁺ upon addition are likely to possess undesirable off-target effects and can be excluded immediately from further study. The effect of compounds on VGCC can easily be verified using a K+

depolarization assay and fluorescent Ca²⁺ imaging in SH-SY5Y cells.

[0139J Some studies have reported an inability to detect modulation of heterologously expressed Na_v1.7 by state-dependent gating modifiers such as ProTxI using membrane potential dyes [Bhattacharya, A., et ai, FASEB J., 2009. 23 ((Meeting Abstract Supplement)): 998.31]. However, the present inventors were able to detect inhibition by ProTxII as well as pore blockers such as tetrodotoxin, μ-conotoxin Till A and clinically used compounds including amitriptyline and tetracaine (data not shown). This discrepancy may reflect expression of endogenous sodium channels in SH-SY5Y cells at a more physiological membrane potential compared to commonly used over- expression systems such as HEK293 cells Biosens Bioelectron, 2006. 21(8): 1483-92]. In addition, the human Na_v channels in SH-SY5Y cells are co-expressed with functionally relevant β-subunits, which could affect inhibition of Na_v activity by state- dependent blockers such as ProTxII.

[0140] While certain embodiments of the assays described herein are readily available and able to measure changes in Na_v function using any platform that is capable of detecting changes in fluorescence at suitable wavelengths, the assays are particularly amenable to high throughput screening using platforms such as provided by the FLIPR platform in 96-, 384 or 1536-well format. The exceptional signal-to-noise ratio, exemplified by the high Z' score of 0.7, make these assays particularly suitable to the identification of novel Na_v blockers early in the drug discovery process.

[0141] In summary, novel assays are described herein for detecting activity of

Na_v, channels, including Na_vl .2, Na_vl .3 and Na_vl .7, which are endogenously expressed by mammalian cells, especially neuroblastoma cells such as the human neuroblastoma cell line SH-SY5Y. This is the first time that functional human Na_vl .7 and Na_vl .3 expression in SH-SY5Y has been reported at the protein level. The assays of the present invention provide a flexible, low cost alternative for the identification of both Na_v pore blockers as well as gating modifier modulators that are amenable to high throughput screening. MATERIALS AND METHODS

Materials

[0142] Veratridine was obtained from Ascent Scientific (Bristol, UK), tetrodotoxin (TTX) was from Enzo Life Sciences (Farmingdale, NY, USA) and ProTxII and agatoxin TK were from Peptides International (Louisville, KY, USA). Pacific ciguatoxin-1 (P-CTX-1) was isolated as previously reported. Briefly, ciguatoxins including P-CTX-1 were isolated through a series of HPLC chromatography steps from the viscera of Moray eel obtained from the Republic of Kiribati. CVID, TIIIA and GIIIA were kind gifts from Prof Paul Alewood, The University of Queensland, Australia. P-CTX-1 was prepared as a 10 μΜ stock in 50% methanol/HaO and stored at -20° C. AU dilutions of P-CTX- 1 were made with buffer containing 0.3% BSA to avoid loss to plastic. All other reagents, unless otherwise stated, were obtained from Sigma Aldrich (Castle Hill, NSW, Australia).

Cell Culture

[0143] SH-SY5Y human neuroblastoma cells were a kind gift from Victor Diaz (Max Planck Institute for Experimental Medicine, Goettingen, Germany). Cells were routinely maintained in RPMI medium (Invitrogen) supplemented with 15% foetal bovine serum and L-glutamine and passaged every 3-5 days using 0.25% trypsin/EDTA (Invitrogen). Cells were plated at a density of 120, 000-150, 000 cells/well on 96- well or 30, 000-50, 000 cells/well on 384-well black-walled imaging plates (Corning) 48 h prior to the assay.

Measurement of Membrane Potential Chanees

[0144] To assess changes in membrane potential, SH-SY5Y cells were loaded with the red membrane potential dye kit (Molecular Devices, Sunnyvale, CA) according to the manufacturer's instructions. In brief, membrane potential dye was reconstituted with a volume of physiological salt solution (PSS; composition in mM: NaCl 140, glucose 11.5, KC1 5.9, MgCl₂ 1.4, NaH₂P0 1.2, NaHC0₃ 5, CaCl₂ 1.8, HEPES 10) as specified in the manufacturer's instructions and after a wash with PSS, cells were incubated with 100 of the membrane potential solution at 37° C for 30 min. The cells were then transferred to the FLiPR^TETRA+ fluorescent plate reader and changes in fluorescence (excitation 510-545 nm; emission 565-625 nm) in response to addition of agonists was measured every second for 300 seconds.

Measurement of Calcium Responses

[0145] SH-SY5V cells were loaded with the fluorescent calcium dye Fluo-4 (Invitrogen) by incubating the cells in physiological salt solution (PSS; composition in mM: NaCl 140, glucose 1 1.5, KC1 5.9, MgCl₂ 1.4, NaH₂P0₄1.2, NaHC0₃ 5, CaCl₂ 1.8, HEPES 10) containing 0.3% bovine serum albumin and 4 μΜ Fluo-4- AM (Invitrogen) for 30 min at 37° C. To remove extracellular dye and facilitate dye hydrolysis, cells were washed with PSS for 5-15 min prior to loading of plates into the FL_PR^TETRA+ (Molecular Devices, Sunnyvale, CA) fluorescent plate reader. Fluorescence (excitation 470-495 nm; emission 515-575 nm) was measured using a cooled CCD camera with camera gain and excitation intensity adjusted for each plate to yield an average baseline fluorescence value of 1000 AFU. After 10 baseline reads; buffer or antagonists were added and the fluorescence response was measured every second for 300 reads, followed by addition of agonists and fluorescence measurements every second for a further 300 seconds. For ProTxII, an additional read interval of 150 reads every 10 seconds was incorporated prior to addition of agonists to extend the total incubation time to 30 min. Raw fluorescence readings were converted to response over baseline using the analysis tool of Screenworks™ 3.1.1.4 (Molecular Devices) and were expressed relative to the maximum increase in fluorescence of control responses.

Transfection with shRNA and Wen Content Ca²* Imagine

[0146] pGIPZ-shRNA targeting Na_v1.3 (Oligomer ID V2LHS_203470) was obtained from Open Biosystems and transfected into SH-SY5Y cells using Arrest-In

(Open Biosystems) as described by the manufacturer. Briefly, 0.1 μg plasmid DNA was mixed with 0.5 μg Arrest-In transfection reagent, incubated at room temperature for 20 min and added to SH-SY5Y cells plated at a density of 70, 000 cells/well on 96-well plates 24 hours prior to transfection. After 6 h, an equal volume growth medium containing 30% FBS was added and cells cultured for a further 48 h. SH-SY5Y cells were then loaded with Fura-2 by incubating for 30 min at 37° C in PSS containing 0.3%

BSA and 5 μΜ Fura-2-AM (Invitrogen), and after 2 washes with PSS, were transferred to the recording chamber of the high content imaging platform BD Pathway 855. SH- SY5Y cells were stimulated with 1 nM P-CTX- 1 or 60 mM KC1 and responses of shRNA-expressing GFP-positive and non-transfected GFP-negative cells were plotted as Δ F/F values, by subtracting baseline fluorescence values from all subsequent time points and dividing these values by the baseline fluorescence.

Immunofluorescence

[0147] SH-SY5Y cells were plated on PDL-coated glass coverslips at a density of 1 x 10⁵ cells/well in 12 well plates and grown for 48-72 h. After a wash with PBS (phosphate buffered saline; Invitrogen) cells were fixed for 30 min at room temperature with Histochoice^® MB fixative (Solon, OH, USA), permeabilized for 10 min with 0.1% Triton-X and blocked with 3% BSA for 30 min at room temperature. After a 1 h incubation with rabbit anti-Na_vl .7 and rabbit anti Na_vl .3 primary antibodies (Alomone Labs, Jerusalem), cells were washed several times with PBS and stained with anti-rabbit Alexa-488 (Na_v1.7 ) and anti-rabbit Alexa-555 (Na_v1.3 )(Invitrogen) and DAPI to visualize nuclei. Cells were imaged with a Zeiss Axiovert 200 Inverted Laser Scanning Confocal microscope using a Plan Apochromat lOOx/1.4 oil immersion lens.

Semi-Quantitative PCR

(0148] SH-SY5Y cells were grown on 10 cm dishes, washed twice with ice- cold PBS and total RNA isolated using the Qiagen R easy™ Plus Mini Kit (Qiagen) according to the manufacturer's instructions with on-column DNA digestion. The

Omniscript Reverse Transcription Kit (Qiagen) was used to reverse transcribe 1 μg of RNA, as determined by spectrophotometric absorbance at 260 nm, and 20 ng of the resulting cDNA was amplified using the Platinum™ Pfx kit (Invitrogen). PCR reactions additionally contained final concentrations of 2 x amplification buffer, 0.3 mM dNTP, 1 mM MgCl₂, 0.4 μΜ primers and 1 U Pfx polymerase in a volume of 50 and were amplified under the following conditions: 94° C for 5 min, 30 cycles of 94° C for 15 sec, 60-64° C for 30 sec, 68° C for 1 min and a final extension at 68° C for 10 min. Human Na_v primers were designed using Primer BLAST, and human β subunit primers were as previously described in the literature [Diss, J.K., et al, Prostate Cancer Prostatic Dis, 2008. 11(4): 325-33] (see Table 1). Plasmids encoding for Na_vl .1-1.8 and β1-β3 subunits verified amplification of the correct products for each subtype (data not shown). All reaction products were analyzed on 2% agarose gels and band density was determined using BioRad Quantity One V4.5.2 build 70 with background correction. Z' Factor Determination

[0149] The Z' factor was determined as previously described [Zhang, J.H., et al., JBiomol Screen, 1999. 4(2): 67-73], with 48 replicates of a negative control (PSS) and 48 replicates of positive controls (50 μΜ veratridine or 10 nM P-CTX-1) per plate. Mean and standard deviation for positive and negative controls were determined using GraphPad Prism™ (Version 4.00, San Diego, California) and the Z' factor for each plate determined according to the following equation:

[0150] Z' = 1 - (QSOposMve + 3SD negative)/ (^meanpositive ~™e „_egative))

Data Analysis

[0151] Unless otherwise stated, all data are expressed as the mean ± standard error of the mean (SEM) determined from at least n = 3 replicates and are representative of at least three independent experiments. To establish concentration-response curves, responses after addition of compounds were plotted against agonist concentration and a 4-parameter Hill equation with variable Hill slope or a two-site model was fitted to the data using GraphPad Prism (Version 4.00, San Diego, California). Potency of agonists and antagonists are reported as the mean ± SEM of 3-4 separate experiments. Statistical significance was determined using an unpaired student's t-test with statistical significance defined as p < 0.05 unless otherwise stated.

TABLES

TABLE 1

PRIMER PAIRS USED FOR PCR OF NA_V SUBTYPES AND B SUBUNITS.

[0152] Human Na_v primers were designed using Primer BLAST, and human β subunit primers were as previously described in the literature [Diss, J.K., et al, Prostate Cancer Prostatic Dis, 2008. 11(4): 325-33]. For each Na_v subtype and β subunit, GenBank accession numbers, forward and reverse primer sequences, expected PCR product size and locations are listed.

βΐ ΝΜ 001037.4 5'-AGAAGGGCACTGAGGAGTTT-3' 379 315-693 5'-GCAGCGATCTTCTTGTAGCA-3'

β2 ΝΜ 004588.4 5'-GAGATGTTCCTCCAGTTCCG-3' 310 423-732

5'-TGACCACCATCAGCACCAAG-3'

Ρ3 ΝΜ 018400.3 5'-CTGGCTTCTCTCGTGCTTAT-3' 353 834-1186 ΝΜ 001040151.1 5'-TCAAACTCCCGGGACACATT-3' 435-787

Μ ΝΜ 174934.3 5'-TAACCCTGTCGCTGGAGGTG-3' 459 322-780 ΝΜ 001142349.1 5 -TGAGGATGAGGAGCCCGATG-3' 259-717

{0153] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0154] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0155] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features..Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for identifying an agent that modulates the activity of a voltage- gated sodium channel ( a_v) isoform of interest that is endogenously expressed by a neuroblastoma cell, the method comprising:

- contacting the neuroblastoma cell with a candidate agent under conditions permitting, promoting or otherwise supporting ion transport across the membrane of the cell; and

- detecting a change in the intracellular level of the ion, which results from contacting the cell with the candidate agent, wherein the change indicates that the candidate agent modulates the activity of the Na_v isoform of interest.

2. A method according to claim 2, wherein the neuroblastoma cell is SH-

SY5Y.

3. A method according to claim 1 or claim 2, wherein the Na_v isoform is selected from Na_vl .7, Na_vl .3 and Na_vl .2.

4. A method according to any one of claims 1 to 3, wherein the conditions are achieved by a process comprising contacting the cell with an ion transport-activating agent that activates the Na_v isoform to thereby permit, promote or otherwise support ion transport therethrough.

5. A method according to claim 4, wherein the ion transport-activating agent is selected from veratridine, grayanotoxin, aconitine, batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion toxins, sea anemone toxins, β scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, δ-conotoxins.

6. A method according to claim 5, wherein the ion transport-activating agent is veratridine, which activates a Na_v isoform selected from Na_vl .2 and Na_vl .7.

7. A method according to claim 6, wherein the neuroblastoma cell expresses

Na_v1.2 and Na_v1.7 and the method further comprises contacting the cell with a Na_v isoform-inhibiting agent that inhibits the level or activity of Na_v1.2 to thereby determine whether the candidate agent modulates the activity of Na_v 1.7.

8. A method according to claim 7, wherein the Na_v isoform-inhibiting agent is selected from conotoxin TIIIA, an antagonist antigen-binding molecule that is specifically immuno-interactive with Na_v1.2 and a nucleic acid molecule (e.g., siRNA, shRNA, antisense etc.) that inhibits expression of Na_vl.2.

9. A method according to claim 5, wherein the neuroblastoma cell expresses

Na_v1.2 and Na_v1.7 and the method further comprises contacting the cell with a Na_v isoform-inhibiting agent that inhibits the level or activity of Na_vl .7 to thereby determine whether the candidate agent modulates the activity of Na_vl .2.

10. A method according to claim 9, wherein the Na_v isoform-inhibiting agent is selected from ProTxII, an antagonist antigen-binding molecule (e.g., antibody, antibody fragment etc.) that is specifically immuno-interactive with Na_v1.7 and a nucleic acid molecule (e.g., siRNA, shRNA, antisense etc.) that inhibits expression of Na_vl.7.

11. A method according to claim 5, wherein the ion transport-activating agent is pacific ciguatoxin-1 (P-CTX-1), which activates a Na_v isoform selected from Na_v1.2 and Na_v1.3.

12. A method according to claim 1 1, wherein the neuroblastoma cell expresses

Na_v1.2 and Na_v1.3 and the method further comprises contacting the cell with a Na_v isoform-inhibiting agent that inhibits the level or activity of Na_v1.2 to thereby determine whether the candidate agent modulates the activity of Na_vl .3.

13. A method according to claim 12, wherein the Na_v isoform-inhibiting agent is selected from conotoxin Till A, an antagonist antigen-binding molecule (e.g., antibody, antibody fragment etc.) that is specifically immuno-interactive with Na_v1.2 and a nucleic acid molecule (e.g. , siRNA, shRNA, antisense etc.) that inhibits expression of Na_vl.2.

14. A method according to any one of claims 1 to 13, wherein the ion transported across the membrane of the cell is Ca²⁺.

15. A method according to claim 14, wherein the change in the intracellular level of the ion is measured using a calcium-indicating agent.

16. A method according to claim 15, wherein the calcium-indicating agent is detectably labeled (e.g. , with a fluorophore).

17. A method according to any one of claims 1 to 13, wherein the ion transported across the membrane of the cell is Na⁺.

18. A method according to claim 17, wherein the change in the intracellular level of the ion is measured using a sodium-indicating agent.

19. A method according to claim 18, wherein the sodium-indicating agent is detectably labeled (e.g., with a fluorophore).

20. A method according to any one of claims 1 to 13, wherein the change in the intracellular level of the ion is measured using a membrane potential-indicating agent.

21. A method according to claim 20, wherein the membrane potential-indicating agent is detectably labeled (e.g. , with a fluorophore).

22. A method for identifying an agent that modulates the activity of a voltage- gated sodium channel (Na_v) isoform of interest that is endogenously expressed by a mammalian cell, wherein the mammalian cell further expresses at least one other Na_v isoform, the method comprising:

- contacting the mammalian cell, in the presence and absence of a candidate agent, with a Na_v isoform-inhibiting agent that inhibits the level or activity of the at least one other Na_v isoform under conditions permitting, promoting or otherwise supporting ion transport across the membrane of the cell; and

- detecting a change in the intracellular level of the ion, which results from the presence of the candidate agent, wherein the change indicates that the candidate • agent modulates the activity of the Na_v isoform of interest.

23. A method according to claim 22, wherein the mammalian cell is a neuroblastoma cell.

24. A method according to claim 23, wherein the neuroblastoma cell is SH- SY5Y.

25. A method according to claim 22 or claim 23, wherein the conditions are achieved by a process comprising contacting the cell with an ion transport-activating agent that activates the Na_v isoform of interest and one or more of the other Na_v isoforms to thereby permit, promote or otherwise support ion transport therethrough.

26. A method according to claim 25, wherein the ion transport-activating agent is selected from veratridine, grayanotoxin, aconitine, batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion toxins, sea anemone toxins, β scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, δ- conotoxins.

27, A method according to claim 26, wherein the cell expresses Na_v 1.2 and

Na_v1.7.

28. A method according to claim 27, wherein the ion transport-activating agent is veratridine, which activates Na_vl .2 and Na_vl .7.

29. A method according to claim 27 or claim 28, wherein the Na_v isoform of interest is Na_vl .7 and the at least one other Na_v isoform is Na_vl.2.

30. A method according to claim 29, wherein the Na_v isoform-inhibiting agent is selected from conotoxin TIIIA, an antagonist antigen-binding molecule that is specifically immuno-interactive with Na_v1.2 and a nucleic acid molecule {e.g., siRNA, shR A, antisense etc.) that inhibits expression of Na_v1.2.

31. A method according to claim 27 or claim 28, wherein the Na_v isoform of interest is Na_vl .2 and the at least one other Na_v isoform is Na_vl .7.

32. A method according to claim 31, wherein the Na_v isoform-inhibiting agent is selected from ProTxII, an antagonist antigen-binding molecule {e.g., antibody, antibody fragment etc.) that is specifically immuno-interactive with Na_v1.7 and a nucleic acid molecule (e.g., siRNA, shRNA, antisense etc.) that inhibits expression of Na .7. \

33. A method according to claim 26, wherein the cell expresses Na_v1.2 and

Na_v1.3.

34. A method according to claim 33, wherein the ion transport-activating agent is pacific ciguatoxin-l (P-CTX-1), which activates Na_vl .2 and Na_vl .3.

35. A method according to claim 33 or claim 34, wherein the Na_v isoform of interest is Na_vl .3 and the at least one other Na_v isoform is Na_vl .2.

36. A method according to claim 35, wherein the Na_v isoform-inhibiting agent is selected from conotoxin TI1IA, an antagonist antigen-binding molecule that is specifically immuno-interactive with Na_v1.2 and a nucleic acid molecule (e.g., siRNA, shRNA, antisense etc.) that inhibits expression of Na_v1.2.

37. A method according to any one of claims 22 to 36, wherein the ion transported across the membrane of the cell is Ca²⁺.

38. A method according to claim 37, wherein the change in the intracellular level of the ion is measured using a calcium-indicating agent.

39. A method according to claim 38, wherein the calcium-indicating agent is detectably labeled (e.g. , with a fluorophore).

40. A method according to any one of claims 22 to 36, wherein the ion transported across the membrane of the cell is Na⁺.

41. A method according to claim 40, wherein the change in the intracellular level of the ion is measured using a sodium-indicating agent.

42. A method according to claim 41, wherein the sodium-indicating agent is detectably labeled (e.g., with a fluorophore).

43. A method according to any one of claims 22 to 36, wherein the change in the intracellular level of the ion is measured using a membrane potential-indicating agent.

44. A method according to claim 43, wherein the membrane potential-indicating agent is detectably labeled (e.g. , with a fluorophore).

45. A method of producing an agent that is useful for treating or preventing a disease or condition associated with sodium channel activity, the method comprising: identifying an agent that modulates the activity of a voltage-gated sodium channel (Na_v) isoform of interest, according to the method of any one of claims 1 to 44; and synthesizing the agent on the basis that it tests positive for the modulation.

46. A method according to claim 45, further comprising derivatising the agent, and optionally formulating the derivatised agent with a pharmaceutically acceptable carrier or diluent, to improve the efficacy of the agent for treating or preventing the disease or condition associated with sodium channel activity.

47. A method for treating or preventing a disease or condition associated with sodium channel activity (e.g., aberrant activity or hyperactivity) in a subject, the method comprising: adminsitering an effective amount of an agent that modulates (e.g., blocks or reduces) the level or activity of a voltage-gated sodium channel (Na_v) isoform of interest, wherein the agent is identified or produced by the method of any one of claims 1 to 46.

48. A method according to any one of claims 45 to 47, wherein the disease or condition associated with sodium channe/ activity, pain, cancer, inflammation, neurodegeneration, neuroendocrine disorders and cardiovascular disease.

49. A kit for assessing or assaying the potential of an agent to modulate the activity of a voltage-gated sodium channel (Na_v) isoform of interest, the kit comprising

(1) a mammalian cell (e.g., a neuroblastoma cell that is suitably of human origin), which enodgenously expresses the Na_v isoform of interest and suitably at least one other Na_v isoform, (2) at least one Na_v isoform-inhibiting agent (e.g., conotoxin TULA, ProTxII, an antagonist antigen-binding molecule that is specifically immuno-interactive with an individual Na_v isoform, or a nucleic acid molecule [e.g., siRNA, shRNA, antisense etc.] that inhibits expression of an individual Na_v isoform) to inhibit the level or activity of one or more of the other Na_v isoforms that are not the subject of investigation; and (3) a sodium channel opener/activator (e.g., veratridine, grayanotoxin, aconitine, batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion toxins, sea anemone toxins, β scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, δ-conotoxins) that opens/activates the Na_v isoform of interest and optionally the other Na_v isoform, or if more than one, at least one of the other Na_v isoforms.

50. A kit according to claim 49, further comprising a voltage sensor.

51. A kit according to claim 50, wherein the voltage sensor is selected from ion transport-indicating agents (e.g., sodium-indicating agents, calcium-indicating agents etc) and membrane potential-indicating agents.

52. A kit according to claim 49 or claim 50, further comprising instructions for conducting the assesment or assay.