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WO2012162732A1 - Essais pour modulateurs des canaux de l'ion sodium et leurs utilisations - Google Patents

Essais pour modulateurs des canaux de l'ion sodium et leurs utilisations Download PDF

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WO2012162732A1
WO2012162732A1 PCT/AU2012/000597 AU2012000597W WO2012162732A1 WO 2012162732 A1 WO2012162732 A1 WO 2012162732A1 AU 2012000597 W AU2012000597 W AU 2012000597W WO 2012162732 A1 WO2012162732 A1 WO 2012162732A1
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isoform
agent
cell
activity
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Richard James Lewis
Irina VETTER
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University of Queensland UQ
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University of Queensland UQ
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • 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 ⁇ 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,
  • Voltage-gated sodium channels are complex transmembrane proteins comprised of a pore-forming a subunit and accessory ⁇ 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).
  • 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].
  • 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.
  • 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.
  • Na v channel modulators including state-dependent gating modifiers (e.g., ProTxII), pore blockers (e.g., tetrodotoxin, ⁇ -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 ⁇ subunits.
  • state-dependent gating modifiers e.g., ProTxII
  • pore blockers e.g., tetrodotoxin, ⁇ -conotoxin Till A
  • clinically used anesthetic compounds such as amitriptyline and tetracaine
  • 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 v l .2, Na v 1.3 and Na v 1.7.
  • the neuroblastoma cell e.g., a human neuroblastoma cell line, such as SH-SY5Y
  • 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, ⁇ scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, ⁇ - conotoxins) leads to sodium influx, which results in membrane depolarization and subsequent Ca 2+ influx through endogenously or heterologously expressed voltage- gated calcium channels (VGCC).
  • sodium channel openers/activators illustrative examples of which include veratridine, grayanotoxin, aconitine, batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion
  • 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.
  • ion transport-indicating agents e.g., sodium-indicating agents and calcium-indicating agents
  • 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.
  • 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.
  • 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.
  • the candidate agent blocks, abrogates, inhibits or otherwise reduces the activity of the Na v isoform of interest.
  • 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.
  • a 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,
  • 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.
  • a mammalian cell e.g., a neuroblastoma cell line such as SH-SY5Y
  • 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.
  • the method comprise detecting a change in the intracellular level of calcium ions.
  • 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.
  • 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.
  • 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.
  • 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.
  • a disease or condition associated with sodium channel activity e.g. , aberrant activity or hyperactivity
  • 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.
  • 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 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 Naont 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
  • 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.
  • ion transport-indicating agents e.g., sodium-indicating agents and calcium-indicating agents
  • membrane potential-indicating agents e.g., membrane potential-indicating agents.
  • the kits may further contain instructions for conducting the assessment or assay.
  • Figure 1 is a graphical and photographic representation showing that SH-SY5Y cells endogenously express Na v and accessory ⁇ subunits. Expression of Na v a and accessory ⁇ subunits was assessed by semi-quantitative PCR.
  • A Amplification of endogenously expressed human Na v l .2, Na v l .3, Na v 1.4, Na v 1.5 and Na v l .7 was detected in SH-SY5Y cells, with Na v l .7 being the most abundantly expressed Na v isoforms.
  • B SH-SY5Y cells endogenously expressed human ⁇ 2 and ⁇ 3, but not ⁇ or ⁇ 4 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 D)
  • FIG. 1 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 v l .7 antibody ((A) and (B)) and anti-Na v l .3 antibody ((C) and (D)) showed fluorescence located predominately in the plasma membrane, indicative of functional Na v expression.
  • Scale bar 10 ⁇ .
  • Figure 3 is a graphical representation showing that activation of endogenously expressed Na v in SH-SY5Y by veratridine causes membrane
  • FIG. 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.
  • Veratridine elicited concentration-dependent increases in intracellular Ca + in SH-SY5Y cells with an EC 50 of 21.9 ⁇ (pIC 50 4.66 ⁇ 0.04).
  • the Ca 2+ 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.
  • FIG. 5 is a graphical representation showing that L-type and N-type VGCC contribute to the veratridine- and P-CTX-1 -induced Ca 2+ 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).
  • 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 ⁇ nifedipine and 1 ⁇ CVID.
  • Figure 6 is a graphical representation showing that veratridine-and P-
  • CTX-1 -induced Ca 2+ responses are partially mediated by activation of endogenously expressed Na v l .2.
  • the Na v 1.2 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 v l .2, as the Na v 1.4-selective blocker GIIIA did not affect veratridine responses at concentrations up to 10 ⁇ .
  • Figure 7 is a graphical representation showing that activation of Na v l .7 contributes to the veratridine-induced Ca 2+ response in SH-SY5 Y cells.
  • A In the presence of 1 ⁇ TIIIA, the Na v l .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 v 1.7.
  • pICso 9.68 ⁇ 0.15 the Na v l .7 -selective blocker
  • FIG. 8 is a graphical representation showing that Na v l .3 contributes to P-CTX-1 responses in SH-SY5Y cells.
  • SH-SY5Y cells were transfected with shRNA targeting Na v l .3 and Ca 2+ 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 v 1.3 - targeting shRNA, compared to non-expressing, GFP-negative cells.
  • B Responses to depolarization with 60 mM KCl were not significantly affected by expression of Naforcel .3 -targeting shRNA. Arrows indicate addition of P-CTX-1 and KCl.
  • activation refers to the transition from a resting (non-conducting) state of an ion channel to the activated (conducting) state.
  • activation threshold is meant the lowest potential above which measurable opening of a channel occurs.
  • agent includes a compound that induces a desired pharmacological and/or physiological effect.
  • 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 terms "antagonist,” “inhibitor” and “blocker” are used interchangeably to refer to agents that reduce, inhibit, impair or prevent ion transfer across a cell membrane.
  • antigen-binding molecule a molecule that has binding affinity for a target antigen. It will be understood that this term extends to
  • immunoglobulins immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
  • candidate agent 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 ⁇ , 0.1 ⁇ , 1 ⁇ and 10 ⁇ .
  • 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.
  • depolarization 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., Ca 2+ ) effective to reduce the magnitude, and may nearly or completely eliminate, the electrical potential difference across a cell membrane.
  • 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.
  • an 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.
  • 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.
  • a nucleic acid or polypeptide e.g., a Na v channel, an accessory subunit etc. or their encoding genes
  • 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.
  • a nucleic acid or polypeptide e.g. , a Na v channel, an accessory subunit etc. or their encoding genes
  • hit refers to a candidate agent that shows desired properties in an assay.
  • activation means that an ion channel moves into the inactivated state.
  • 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.
  • library means a collection of molecules.
  • 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 NanoplatesTM.
  • Naturally occurring refers to a component produced by cells in the absence of artificial genetic or other modifications of those cells.
  • 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.
  • patient refers 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.
  • 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).
  • a primate e.g., a human, monkey, chimpanzee
  • a condition or disease associated with sodium channel activity e.g., aberrant activity or hyperactivity.
  • 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.
  • 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.
  • resting or “resting state” refers to a voltage-dependent ion channel that is closed, but free from inactivation.
  • 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 v l .7 exhibits Na v l .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 v 1.7 such as Na v 1.2 and Na v 1.3 ).
  • 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 v 1.7 than of Na v 1.2. 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 v l 7 than of Na v l .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 v 1.3 than of Na v 1.2.
  • 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 v 1.2 than of Na v 1.3. 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 v 1.2 than of Na v 1.7.
  • 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.
  • 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. .
  • treating 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.
  • 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.
  • Na v voltage-gated sodium channel
  • TTX tetrodotoxin
  • FRET fluorescence resonance energy transfer
  • MTS medium throughput screening
  • 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).
  • 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.
  • the mammalian cell endogenously co- expresses accessory ⁇ subunits, which co-assemble with the Na v channel a subunit(s).
  • the mammalian cell endogenously co- expresses at least one Na v channel a subunit ⁇ e.g.
  • Na v channel modulators particularly Na v channel blockers
  • 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.
  • 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.
  • blocking 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • in vitro assays will involve mammalian cells that endogenously express one or more Na v channels of interest.
  • 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% C0 2 .
  • ion flux assays can be used to assess sodium channel activity.
  • 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, ⁇ scorpion toxins, pumiliotoxin B, brevetoxins, ciguatoxins, versutoxin, pyrethroid insecticides, ⁇ -conotoxins).
  • sodium channel openers/activators e.g. , veratridine, grayanotoxin, aconitine, batrachotoxin, BTG502, antillatoxin, hoiamide A, a scorpion toxins (e.g., OD-1), sea anemone toxins, ⁇ scorpion toxins, pumili
  • a channel-stabilizing e.g., a positive Na v modulator
  • Channel blockers are suitably identified by their ability to prevent ion influx.
  • 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.
  • Advantageous 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.
  • a Fluorescent Imaging Plate Reader (FLIPRTM) system membrane potential kit available from Molecular Dynamics (a division of Amersham Biosciences, Piscataway, N.J.) is used to measure redistribution of membrane potential.
  • FLIPRTM Fluorescent Imaging Plate Reader
  • the FLIPRTM system is particularly suited to ion flux assays and may be used to monitor, for example, sodium channel opener-evoked increases in intracellular Ca 2+ .
  • a calcium-indicating agent such as Fluo-4-AM, is loaded into the cells and the cells are monitored, in real-time, using the FLIPRTM.
  • cells are incubated with 4 ⁇ 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 FLIPRTM. The cells are incubated for 5 min in FLIPRTM buffer, in the absence (control) or presence of the candidate agent, prior to addition of veratridine (40 ⁇ ).
  • PSS physiological salt solution
  • 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 ⁇ scorpion toxin, pumiliotoxin B, brevetoxins, a ciguatoxin such as pacific ciguatoxin-1 (P-CTX-1), a versutoxin, a pyrethroid insecticide, a ⁇ -conotoxin).
  • Peak fluorescence intensity, after sodium channel opener addition is determined using the FLIPRTM software. Curve fitting and parameter estimation (pIC 5 o) can be performed using any suitable software, illustrative examples of which include ScreenworksTM (Molecular Devices).
  • calcium-indicating agents is not limited to the FLIPRTM assay and encompasses any assay that measures Ca 2+ 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.
  • 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.
  • 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 VIPRTM 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 VIPRTM 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 DiSBAC 6 (3), VIPRTM 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 FLEXstationTM (Molecular Devices Corporation) in conjunction with FRET dyes from Aurora Biosciences.
  • a VIPRTM 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 ⁇ , ⁇ .
  • ELx405, Biotek automatic plate washer
  • the mixed dye solution is prepared with external solution and consists of 10 ⁇ CC2-DMPE (chlorocoumarin-2-dimyristoyl
  • phosphatidylethanolamine 2.4 ⁇ DISBAC6(3) (bis-(l,3-dihexyl-thiobarbituric acid) trimethine oxonol), 0.5% ⁇ -cyclodextrin, 20 ⁇ g/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.
  • DISBAC6(3) bis-(l,3-dihexyl-thiobarbituric acid) trimethine oxonol
  • 0.5% ⁇ -cyclodextrin 20 ⁇ g/mL pluronic F-127 and ESS Acid Yellow 17 (ESS AY- 17).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • nucleic acid molecules e.g.
  • the mammalian cell e.g. , a human cell or a cell of human origin
  • a neuronal cell such as a neuroblastoma cell (e.g., SH-SY5Y)
  • endogenously expresses two or more Na v channels selected from Na v L2, Na v l .3 and Na v l .7.
  • the cell expresses Na v l .2 and Na v l .7 and the Na v isoform of interest is Na v l .7.
  • a Na v l .2-inhibiting agent e.g., conotoxin TIIIA, an antagonist antigen-binding molecule that is specifically immuno-interactive with Na v 1.2 and a nucleic acid molecule (e.g., siRNA, shRNA, antisense etc.) that inhibits expression of Na v 1.2
  • a Na v l .2-inhibiting agent e.g., conotoxin TIIIA, an antagonist antigen-binding molecule that is specifically immuno-interactive with Na v 1.2 and a nucleic acid molecule (e.g., siRNA, shRNA, antisense etc.) that inhibits expression of Na v 1.2)
  • a nucleic acid molecule e.g., siRNA, shRNA, antisense etc.
  • the cell is then exposed to a candidate agent and a Na v l .7 blocker is identified by its ability to further prevent ion influx into the cell.
  • OD- 1 which is an a-like toxin from the venom of the Egyptian yellow scorpion, Odonthob thus doriae, is used to selectively activate Na v l .7 so as to preferentially direct ion transport across that channel.
  • Na v -inhibiting agents 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 v l .2 or Na v 1.3) as these channels would either not activate in the presence of OD-1 or activate to a much lower degree than Na v l .7.
  • OD-1 may be used in combination with veratridine to synergistically activate Na opinion1.7.
  • 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 v 1.7 (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 v l .7).
  • the cell expresses Na v l .2 and Na v l .7 and the Na v isoform of interest is Na v l .2.
  • a Na v l .7-inhibiting agent e.g., ProTxII, an antagonist antigen-binding molecule that is specifically immuno- interactive with Na v l .7 and a nucleic acid molecule (e.g.
  • siRNA, shRNA, antisense etc. that inhibits expression of Na v l.7) is used to selectively reduce ion flux activity of Na v l .7 as compared to flux activity of Na v l .2 so as to selectivity or preferentially direct ion transport across Na v l .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 v 1.2 blocker is identified by its ability to further prevent ion influx into the cell.
  • the cell expresses Na v l .3 and Na v l .7 and the Na v isoform of interest is Na v l .3.
  • a Na v l .7-inhibiting agent e.g., ProTxII, an antagonist antigen-binding molecule that is specifically immuno-interactive with Na v l .7 and a nucleic acid molecule (e.g.
  • siRNA, shRNA, antisense etc. that inhibits expression of Na v l.7) is used to selectively reduce ion flux activity of Na v l .7 as compared to flux activity of Na v l .3 so as to selectivity or preferentially direct ion transport across Na v 1.3 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 v l .3 blocker is identified by its ability to further prevent ion influx into the cell.
  • the mammalian cell comprises voltage-gated calcium channels (VGCC) and activation of the Na v isoform(s), which leads to sodium
  • Ca 2+ 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 Ca 2+ influx through the endogenously expressed VGCC.
  • one or more heterologous VGCC may be introduced into the cell, for example, by recombinant means.
  • VGCC Multiple types 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.
  • cDNA and corresponding amino acid sequences of the al , cc2, ⁇ , ⁇ and ⁇ 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.
  • the host cell is a primary, germ, or stem cell, including an embryonic stem cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • a potential modulator assayed using the methods of the present invention comprises a candidate agent.
  • 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.
  • a nucleic acid e.g., an aptamer
  • a small molecule e.g., a chemical compound
  • an antibody or fragment thereof e.g., a nucleic acid-protein fusion
  • a peptidomimetic e.g., a carbohydrate, a lipid or other organic (carbon containing) or inorganic molecules, a carbohydrate, any other affinity agent, and combinations thereof.
  • 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.
  • 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.
  • 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.
  • 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
  • the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds.
  • 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
  • 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.
  • test substances derived from different libraries can be pooled for simultaneous evaluation.
  • a library can comprise a random collection- of molecules.
  • a library can comprise a collection of molecules having a bias for a particular sequence, structure, or
  • 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.
  • 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 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.
  • 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.
  • candidate Na v modulators can be evaluated for selectivity and toxicological effects using known methods (see, e.g., Lu, Basic
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • exemplary modulatory agent are those that inhibit the Na v isoform of interest at a lower concentration than any other ion channel protein.
  • 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.
  • 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.
  • 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 v l.l, Na v 1.2, Na v 1.3, Na v 1.4, Na v 1.5, Na v 1.6, Na v 1.8, and Na v 1.9 to the IC-50 for Na v l .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.
  • 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 v l .1 , Na v l .3, Na v l .4, Na v l .5, Na v l .6, Na v l .7, Na v l .8, and Na v l .9 to the IC-50 for Na v 1.2 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.
  • 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 v l .1 , N .2, Na v l .4, Na v l .5, Na v l .6, Na v l .7, Na v l .8, and Na v l .9 to the IC-50 for Na v 1.3 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.
  • the cells used for assessing selectivity desirably express at least 2, 3,
  • the cells employed a neuronal cells, including neuroblastoma cells and cell lines (e.g., SH- 5YSY).
  • 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.
  • myocytes or other in vitro cell culture model cell lines it may be desirable to use myocytes or other in vitro cell culture model cell lines.
  • 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.
  • 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.
  • candidate modulators can be established by determining in vitro toxicity towards a cell line, such as a mammalian (preferably human) cell line.
  • 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.
  • tissue extracts such as preparations of liver, including microsomal preparations
  • 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.
  • bioavailability and toxicological properties of a candidate modulator in an animal model 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,
  • Candidate agent testing positive in the assays of the present invention 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.
  • 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 ⁇ g to about 100 mg/kg body weight and the target human dose is between 0.1 ⁇ g 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 ⁇ , preferably below 1 ⁇ and more preferably below 50 riM.
  • the IC50 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
  • 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").
  • sodium channel activity also referred to herein as a "sodium channel -mediated disease or condition”
  • 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.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • 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
  • Carnivora including cats, and dogs
  • the preferred recipients are turkeys, chickens and other members of the same order.
  • the most preferred recipients are humans.
  • 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,
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • 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.
  • 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.
  • 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.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a co-solvent for example, polyethylene glycol
  • a chelating agent for example, ethylenediamine tetracetic acid
  • a solubilizing agent for example, a cyclodextrin
  • an anti-oxidant for example, sodium metabisulphite
  • the solution can be freeze dried and then reconstituted with a suitable solvent just prior to 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).
  • 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.
  • 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.
  • a disease or condition associated with sodium channel activity e.g. , aberrant activity or hyperactivity
  • 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.
  • pathophysiological disorders including hypertension, cardiac arrhythmogenesis, angina, insulin-dependent diabetes, non-insulin dependent diabetes mellitus
  • 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,
  • drug addiction e.g., opiate, benzodiazepine, amphetamine, cocaine, alcohol, but
  • 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
  • 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.
  • 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.
  • 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,
  • MS multiple sclerosis
  • ALS amyotrophic lateral sclerosis
  • diabetic neuropathy peripheral neuropathy
  • arthritic rheumatoid arthritis
  • osteoarthritis atherosclerosis
  • paroxysmal dystonia myasthenia syndromes
  • myotonia malignant hyperthermia
  • 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.
  • 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.
  • a Na v -modulating agent of the invention may be administered simultaneously, sequentially or separately in
  • opiates analgesics e.g., morphine, heroin, cocaine, oxymorphine, levorphanol, levallorphan, oxycodone, codeine, dihydrocodeine, propoxyphene, nalmefene, fentanyl, hydrocodone, hydromorphone, meripidine, methadone, nalorphine, naloxone, naltrexone,
  • opiates analgesics e.g., morphine, heroin, cocaine, oxymorphine, levorphanol, levallorphan, oxycodone, codeine, dihydrocodeine, propoxyphene, nalmefene, fentanyl, hydrocodone, hydromorphone, meripidine, methadone, nalorphine, naloxone, naltrexone,
  • 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;
  • NSAIDs e.g., ibuprofen, naproxen, fenoprofen, ketoprofen, celecoxi
  • 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-tetrahydr
  • 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 hydroxybuprop
  • 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,
  • 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 PDEV inhibitors; TramadolTM; cholinergic (nicotine) analgesics; alpha-2-delta ligands; prostaglandin E2 subtype antagonists; leukotriene B4 antagonists; 5-lipoxygenase inhibitors; and 5-HT 3 antagonists; and AT 2 receptor antagonists as described for example in WO 2006/066361 published Jun 29, 2006 and WO 2007/106938 published September 27, 2007.
  • 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 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.
  • kits for practicing the methods and screening assays described herein 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., 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., conotoxin Till A, ProTxII, an antagonist antigen-binding molecule that is specifically immuno-interactive with an individual Na v isoform, or a nucleic acid
  • a sodium channel opener/activator e.g., veratridine, grayanotoxin, aconitine,
  • 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.
  • the kits may further contain instructions for conducting the assesment or assay.
  • the kits may comprise one or more containers (e.g. , multiwell plates) for conducting the assessment or assay.
  • kits of the invention include at least one candidate agent screening apparatus, where the apparatus comprises the mammalian cell.
  • 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.
  • 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.
  • 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.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • 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.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., CD-ROM, diskette, etc.
  • 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.
  • 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 ⁇ 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+
  • 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.
  • ⁇ -conotoxin TIIIA is added to the cells at a final concentration 1 ⁇ to block Na v 1.2 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 ⁇ , which preferentially activates both Na v l .2 and Na v l .7 as compared to Na v l .3.
  • SH-S Y5Y cells are loaded with Fluo-4 (Invitrogen) by incubating the cells in PSS containing 0.3% bovine serum albumin and 4 ⁇ 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+
  • 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 v l .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,
  • 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 ⁇ 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 ⁇ , which preferentially activates both Na v l .2 and Na v l .7 as compared to Na v l .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 v l .7 blockers are identified by their ability to further prevent ion influx into the cell.
  • SH-SY5Y cells are loaded with Fluo-4 (Invitrogen) by incubating the cells in PSS containing 0.3% bovine serum albumin and 4 ⁇ 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+
  • Fluorescence excitation 470-495 nm; emission 515-575 nm
  • Fluorescence 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.
  • ProTxII is added to the cells at a final concentration 30 nM to block Na v 1.7 and the fluorescence response is measured every 10 seconds for 150 reads.
  • Buffer or putative Na v 1.2 blocker i.e., candidate agent
  • Veratridine is then added to a final concentration of 30-50 ⁇ , which preferentially activates both Na v l .2 and Na v l .7 as compared to Na v l .3, and fluorescence measurements are taken every second for a further 300 seconds.
  • Na v 1.2 blockers are identified by their ability to further prevent ion influx into the cell.
  • 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 ⁇ Fluo-4-AM (Invitrogen) for 30 min at 37° C as in Example 3.
  • Buffer or putative Na v 1.2 blocker i.e., candidate agent
  • Veratridine is then added to a final concentration of 30-50 ⁇ , which activates both Na v l .2 and Na v l .7 but not Na v l .3, and fluorescence measurements are taken every second for a further 300 seconds.
  • Na v l .2 blockers are identified by their ability to further prevent ion influx into the cell.
  • SH-SY5Y cells are loaded with Fluo-4 (Invitrogen) by incubating the cells in PSS containing 0.3% bovine serum albumin and 4 ⁇ 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 ⁇ "* *
  • Fluorescence excitation 470-495 nm; emission 515-575 nm
  • Fluorescence 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.
  • ProTxII is added to the cells at a final concentration 30 nM to block Na y 1.7 and the fluorescence response is measured every 10 seconds for 150 reads.
  • Buffer or putative Na v l .3 blocker i.e. , candidate agent
  • 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 v 1.3 but not Na v 1.7, and fluorescence measurements are taken every second for a further 300 seconds.
  • Na v l .3 blockers are identified by their ability to further prevent ion influx into the cell.
  • 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 ⁇ Fluo-4-AM (Invitrogen) for 30 min at 37° C as in Example 5.
  • Buffer or putative Na v l .3 blocker i.e., candidate agent
  • Buffer or putative Na v l .3 blocker is then added to the cells and the fluorescence response is measured every second for 300 reads.
  • Ciguatoxin e.g. , P-CTX- 1
  • brevetoxin is then added to a final concentration ofl 0- 100 nM, which activates Na v 1.3 but not Na v 1.7, and fluorescence measurements are taken every second for a further 300 seconds.
  • Na v 1.3 blockers are identified by their ability to further prevent ion influx into the cell.
  • VGCC L-type voltage-gated calcium channels
  • CVID co-conotoxin CVID
  • ⁇ -agatoxin TK to block P/Q- type VGCC
  • 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 ( ⁇ 1 ⁇ 4 ⁇ 7.7 ⁇ 0.5) of the veratridine-induced response ( Figure 5 B).
  • Co-addition of nifedipine (10 ⁇ ) and CVID (1 ⁇ ) completely abolished veratridine-mediated responses (Figure 5 B).
  • Nifedipine also concentration-dependently inhibited P-CTX- 1 -induced Ca 2+ 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.
  • 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).
  • Na v subtype-specific inhibitors were used to elucidate the contribution of various Na v subtypes to the veratridine- and P-CTX- 1 -induced Ca 2+ response.
  • Ca 2+ imaging including fluorescent Ca 2+ imaging
  • Ca 2+ imaging is ideally 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 Ca 2+ 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].
  • the present inventors have developed three novel FLIPR Ca 2+ 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 v l .2 and Na v l .7, while P-CTX- 1 preferentially activated Na v l .2 and Na v l .3.
  • Activation of endogenously expressed Na v results in influx of Na* ions and subsequent membrane depolarization.
  • This membrane depolarization triggers a Ca 2+ 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.
  • fluorescent Ca + dyes such as Fluo-4 or Fura-2 in high throughput or high content format.
  • block of Na v 1.2 by the conotoxin TIIIA produces a Na v 1.7 -specific assay, and conversely block of Na v l .7 by low concentrations of ProTxII isolates Na v responses mediated exclusively by Na v l .2.
  • block of Na v l .2 by the conotoxin TIIIA produced a Na v l .3 -specific assay.
  • the present inventors also confirmed expression of Na v l .2, Na v l .3 and
  • the present inventors were also able to confirm for the first time the expression of Na v l .7 and Na v l .3 in SH-SY5Y cells at the protein level, with Na v l .7 and Na v l .3 immunofluorescence shown to be localized predominantly at the plasma membrane.
  • the endogenous expression of human Na v l .7 and Na gripl .3 with functionally relevant ⁇ subunits make SH-SY5Y cells well suited to the study of native human Na v pharmacology.
  • 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].
  • Na v l.3 endogenously expressed in SH-SY5Y cells is functional, as P-CTX- 1 elicited responses predominantly mediated through Na v 1.3 and to a lesser degree through Na v l .2. While activation of Na v l .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 v 1.2 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.
  • 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].
  • the human Na v channels in SH-SY5Y cells are co-expressed with functionally relevant ⁇ -subunits, which could affect inhibition of Na v activity by state- dependent blockers such as ProTxII.
  • 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.
  • Na v , channels including Na v l .2, Na v l .3 and Na v l .7, which are endogenously expressed by mammalian cells, especially neuroblastoma cells such as the human neuroblastoma cell line SH-SY5Y.
  • 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.
  • 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 ⁇ 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).
  • 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.
  • RPMI medium Invitrogen
  • trypsin/EDTA Invitrogen
  • membrane potential dye was reconstituted with a volume of physiological salt solution (PSS; composition in mM: NaCl 140, glucose 11.5, KC1 5.9, MgCl 2 1.4, NaH 2 P0 1.2, NaHC0 3 5, CaCl 2 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.
  • PSS physiological salt solution
  • 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, MgCl 2 1.4, NaH 2 P0 4 1.2, NaHC0 3 5, CaCl 2 1.8, HEPES 10) containing 0.3% bovine serum albumin and 4 ⁇ 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.
  • PSS physiological salt solution
  • FL_PR TETRA+ Molecular Devices, Sunnyvale, CA
  • 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 ScreenworksTM 3.1.1.4 (Molecular Devices) and were expressed relative to the maximum increase in fluorescence of control responses.
  • pGIPZ-shRNA targeting Na v 1.3 (Oligomer ID V2LHS_203470) was obtained from Open Biosystems and transfected into SH-SY5Y cells using Arrest-In
  • plasmid DNA was mixed with 0.5 ⁇ g 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%
  • 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 ⁇ F/F values, by subtracting baseline fluorescence values from all subsequent time points and dividing these values by the baseline fluorescence.
  • SH-SY5Y cells were plated on PDL-coated glass coverslips at a density of 1 x 10 5 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.
  • PBS phosphate buffered saline
  • Histochoice ® MB fixative Solon, OH, USA
  • SH-SY5Y cells were grown on 10 cm dishes, washed twice with ice- cold PBS and total RNA isolated using the Qiagen R easyTM Plus Mini Kit (Qiagen) according to the manufacturer's instructions with on-column DNA digestion.
  • Qiagen R easyTM Plus Mini Kit Qiagen
  • Omniscript Reverse Transcription Kit (Qiagen) was used to reverse transcribe 1 ⁇ g of RNA, as determined by spectrophotometric absorbance at 260 nm, and 20 ng of the resulting cDNA was amplified using the PlatinumTM Pfx kit (Invitrogen).
  • PCR reactions additionally contained final concentrations of 2 x amplification buffer, 0.3 mM dNTP, 1 mM MgCl 2 , 0.4 ⁇ 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 ⁇ 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 v l .1-1.8 and ⁇ 1- ⁇ 3 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
  • 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 ⁇ veratridine or 10 nM P-CTX-1) per plate. Mean and standard deviation for positive and negative controls were determined using GraphPad PrismTM (Version 4.00, San Diego, California) and the Z' factor for each plate determined according to the following equation:

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Abstract

L'invention concerne de manière générale de nouveaux essais de criblage pour la modulation des canaux sodiques, notamment des canaux sodiques tensiodépendants. Les essais utilisent des cellules mammaliennes qui expriment de manière endogène un canal sodique tensiodépendant dans le contexte d'une ou de plusieurs sous-unités β et sous-unités β accessoires co-exprimées de manière endogène. Les cellules mammaliennes sont utiles dans les essais à rendement élevé pour l'identification de médicaments ayant une valeur thérapeutique contre les maladies ou troubles associés avec l'activité des canaux sodiques, y compris la douleur, l'inflammation, le cancer, la neurodégénération, les troubles endocriniens et les maladies cardiovasculaires.
PCT/AU2012/000597 2011-06-02 2012-05-29 Essais pour modulateurs des canaux de l'ion sodium et leurs utilisations Ceased WO2012162732A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105853417A (zh) * 2016-05-20 2016-08-17 中国中医科学院中药研究所 乌头碱在治疗红斑狼疮中的应用
WO2021123086A1 (fr) 2019-12-20 2021-06-24 F. Hoffmann-La Roche Ag Oligonucléotides améliorés pour inhiber l'expression de scn9a
CN113607707A (zh) * 2021-08-05 2021-11-05 国家食品安全风险评估中心 一种基于钠离子通道Nav1.1的海洋神经毒素荧光快速筛查方法
US12116576B2 (en) 2018-06-22 2024-10-15 Hoffmann-La Roche Inc. Oligonucleotides for modulating SCN9A expression

Citations (1)

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WO2007109324A2 (fr) * 2006-03-21 2007-09-27 Xenon Pharmaceuticals, Inc. Bloqueurs puissants et sélectifs du canal sodique nav1.7

Patent Citations (1)

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WO2007109324A2 (fr) * 2006-03-21 2007-09-27 Xenon Pharmaceuticals, Inc. Bloqueurs puissants et sélectifs du canal sodique nav1.7

Non-Patent Citations (2)

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PARK, J. ET AL.: "High expression of large-conductance Ca2+-activated K+ channel in the CD 133(+) subpopulation of SH-SY5Y neuroblastoma cells", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 396, 2010, pages 637 - 642 *
VETTER, I. ET AL.: "Characterisation of NaV types endogenously expressed in human SH-SY5Y neuroblastoma cells", BIOCHEMICAL PHARMACOLOGY, vol. 83, 2012, pages 1562 - 1571 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105853417A (zh) * 2016-05-20 2016-08-17 中国中医科学院中药研究所 乌头碱在治疗红斑狼疮中的应用
US12116576B2 (en) 2018-06-22 2024-10-15 Hoffmann-La Roche Inc. Oligonucleotides for modulating SCN9A expression
WO2021123086A1 (fr) 2019-12-20 2021-06-24 F. Hoffmann-La Roche Ag Oligonucléotides améliorés pour inhiber l'expression de scn9a
CN113607707A (zh) * 2021-08-05 2021-11-05 国家食品安全风险评估中心 一种基于钠离子通道Nav1.1的海洋神经毒素荧光快速筛查方法
CN113607707B (zh) * 2021-08-05 2024-03-12 国家食品安全风险评估中心 一种基于钠离子通道Nav1.1的海洋神经毒素荧光快速筛查方法

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