AU2008207631A1 - Using selective antagonists of persistent sodium current to treat neurological disorders and pain - Google Patents

Description

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00 Australian Patents Act 1990 Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Using selective antagonists of persistent sodium current to treat neurological disorders and pain The following statement is a full description of this invention, including the best method of performing it known to me:- P/00/011 5951 P NVYPD0S\CRN\RWSpec\02330 div spec.doc-281O8QO0 8 TITLE OF THE INVENTION (Ni [01] Using Selective Antagonists of Persistent Sodium Current to Treat Neurological Disorders and Pain (Ni CROSS REFERENCES TO RELATED APPLICATIONS [02] This application is a divisional application of Australian Patent Application No.

0O 10 2004268633, the entire disclosure which is incorporated herein by reference.

BACKGROUND OF THE INVENTION [03] Field of the Invention [04] This invention relates generally to the fields of neurobiology, physiology, biochemistry and medicine and can be directed toward the treatment of pain and, in particular, to the therapeutic use of compounds that selectively reduce persistent sodium currents to treat neurological disorders and to the therapeutic use of compounds that selectively reduce persistent sodium currents to treat chronic pain.

Background Information [06] The lipid bilayer membrane of all cells forms a barrier that is largely impermeable to the flux of ions and water. Residing within the membrane are a superfamily of proteins called ion channels, which provide selective pathways for ion flux. Precisely regulated conductances produced by ion channels are required for intercellular signaling and neuronal excitability. In particular, a group of ion channels that open upon depolarization of excitable cells are classified as voltage-gated and are responsible for electrical activity in nerve, muscle and cardiac tissue. In neurons, ion currents flowing through voltage-gated sodium channels are responsible for rapid spike-like action potentials. During action potentials the majority of sodium channels WO 2005/020982 PCT/US2004/02077 00 open very briefly. These brief openings result in transient sodium currents.

Z However, a subset of voltage-gated sodium channels does not close rapidly, but remain open for relatively long intervals. These channels therefore generate sustained or persistent sodium currents. The balance between transient and persistent sodium current is crucial for maintaining normal physiological function and Selectrical signaling throughout the entire nervous system.

r"- O [07] In conditions characterized by aberrant levels of persistent sodium current, 00 normal function is disrupted when neurons discharge signals inappropriately and include, neuropathies; hypoxias and ischemias; behavioral disorders and dementia; and movement and neurodegenerative diseases. For example, in the case of the neuropathies embraced by epilepsy, there can be a brief electrical "storm" arising from neurons that are inherently unstable because of a genetic defect as in various types of inherited epilepsy, or from neurons made unstable by metabolic abnormalities such as low blood glucose, or alcohol. In other cases, the abnormal discharge can come from a localized area of the brain, such as in patients with epilepsy caused by head injury or brain tumor. In the case of ischemic injuries, such as, cerebral ischemia and myocardial ischemia, there can be prolonged electrical activity arising from neurons in which persistent sodium channel expression or activity is increased. Such aberrant electrical activity can cause or contribute to neuronal death, which can lead to debilitating injury or death of an individual.

Aberrant electrical activity also can contribute to neurodegenerative disorders such as, without limitation, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and multiple sclerosis.

[08] Clinical pain encompasses nociceptive and neuropathic pain. Each type of pain is characterized by hypersensitivity at the site of damage and in adjacent normal tissue. While nociceptive pain usually is limited in duration and responds well to available opioid therapy, neuropathic pain can persist long after the initiating event has healed, as is evident, for example, in phantom pain that often follows amputation.

Chronic pain syndromes such as neuropathic pain can be triggered by a variety of causes, including, without limitation, a traumatic insult, such as, a compression injury, a spinal cord injury, a limb amputation, an inflammation or a surgical procedure; an ischemic event, such as, a stroke; an infectious agent; a toxin exposure, such as, a drug or alcohol; or a disease such as, an WO 2005/020982 PCT/US2004/028077 00

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inflammatory disorder, a neoplastic tumor, acquired immune deficiency syndrome S(AIDS) or a metabolic disease.

c [09] At present, treatments for many diseases characterized by aberrant levels of persistent sodium channel current are inadequate or non-existent. Current therapies, Ssuch as, Berger et al., Treatment of Neuropathic Pain, U.S. Patent No. 5688830 NO (Nov. 18, 1997); Marquess et al., Sodium Channel Drugs and Uses, U. S. Patent No.

O 6479498 (Nov. 12, 2002); Choi et al., Sodium Channel Modulators, U.S. Patent No.

00 6646012 (Nov. 11, 2003); and Chinn et al., Sodium Channel Modulators, U.S. Patent No. 6756400 (Jun 29, 2004), encompass general sodium channel modulators that systemically effect transient currents. As such, the usefulness of available sodium channel blocking drugs is severely limited by potentially adverse side effects, such as, paralysis and cardiac arrest.

[010] Unfortunately, chronic pain such as chronic neuropathic pain is generally resistant to available opioid and nonsteroidal antiinflammatory drug therapies.

Available drug treatments for chronic neuropathic pain, such as tricyclic antidepressants; anti-convulsants/anti-epileptic, such as, carbamazepine, phenytoin and lamotrigine; and local anesthetics/antiarrythmics, such as, e.g., lidocaine, mexiletine, tocainide and flecainide, only temporarily alleviate symptoms and to varying degrees. In addition, current therapies have serious side effects that can include cognitive changes, sedation, nausea, emesis, dizziness, ataxia, tinnitus and, in the case of narcotic drugs, addiction. Further, many patients suffering from neuropathic and other chronic pain are elderly or have medical conditions that limit their tolerance to the side effects associated with available analgesic therapy, such as, cardiotoxicity, hepatic dysfunction and leukopenia. The inadequacy of current therapy in relieving chronic pain without producing intolerable side effects is reflected in the high rate of depression and suicide in chronic pain sufferers.

[011] Recent evidence suggests that increased persistent sodium current may be an underlying basis for chronic pain, such as, inflammatory and neuropathic pain, see Fernando Cervero Jennifer M. A. Laird, Role of Ion Channels in Mechanisms Controlling Gastrointestinal Pain Pathways, 3(6) CURR. OPIN.

PHARMACOL. 608-612 (2003); Joel A. Black et al., Changes in the Expression of WO 2005/020982 PCT/US2004/028077 00 0 STetrodotoxin-Sensitive Sodium Channels Within Dorsal Root Ganglia Neurons in Inflammatory Pain, 108(3) PAIN 237-247 (2004) and Li Yunru et al., Role of Persistent Sodium and Calcium Currents in Motoneuron Firing and Spasticity in Chronic Spinal Rats, 91(2) J. NEUROPHYSIOL. 767-783 (2004), which are hereby incorporated by reference in their entirety. However, at present, treatments for C chronic pain characterized by aberrant levels of sodium channel current, such as,

NO

Berger et al., Treatment of Neuropathic Pain, U.S. Patent No. 5688830 (Nov.

18, 1997); Marquess et al., Sodium Channel Drugs and Uses, U. S. Patent No.

0 0 6479498 (Nov. 12, 2002); Choi et al., Sodium Channel Modulators, U.S. Patent No.

6646012 (Nov. 11, 2003); and Chinn et al., Sodium Channel Modulators, U.S. Patent No. 6756400 (Jun 29, 2004), encompass general sodium channel modulators that effect transient currents. As such, the usefulness of available sodium channel blocking drugs is severely limited by potentially adverse side effects, such as, e.g., paralysis and cardiac arrest.

[012] Thus, there exists a need to identify new uses of compounds for therapeutic methods that can selectively treat conditions characterized by aberrant levels of persistent sodium current, such as, neuropathies; hypoxias and ischemias; behavioral disorders and dementia; movement and neurodegenerative diseases; and pain, and to protect the brain from the damaging effects of persistent sodium current.

The present invention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION [013] The present invention provides uses of compounds for treating neurological disorders and chronic pain in a mammal, including a human. In one embodiment, the method involves administering to the mammal an effective amount of a selective persistent sodium current antagonist that has at least 20-fold selectivity for a persistent sodium current relative to transient sodium current. In further embodiments, the antagonist has at least 50-fold selectivity for a persistent sodium current, at least 200-fold selectivity for a persistent sodium current, at least 400-fold selectivity for a persistent sodium current, at least 600-fold selectively for a persistent sodium current, or at least 1000-fold selectively for a persistent sodium current, WO 2005/020982 PCT/US2004/028077 00

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bt relative to a transient sodium current. A variety of mammals can be treated by the methods of the invention including, without limitation, humans.

[014] The present invention provides uses of compounds for treating a variety of conditions characterized by aberrant levels of persistent sodium current. In certain Sembodiments, the methods are directed to treating neuropathies, including, without IN limitation, amyloidosis, autoimmune disorders, palsies, connective tissue disorders, epilepsies, and conditions associated with neuropathies like alcoholism, cancers, 00 infectious diseases, organ disorders and vitamin deficiencies. In other embodiments, the methods are directed to treating hypoxic and ischemic conditions, such as, e.g., cerebral ischemia, myocardial ischemia, ischemia retinae, diabetes ischemia and postural ischemia. In still other embodiments, the methods are directed to treating behavioral disorders, dementia, movement disorders, and neurodegenerative diseases, such as, without limitation, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and multiple sclerosis. In further embodiments, the methods are directed to treating diabetic retinopathy. In yet other embodiments, the methods are directed to treating conditions characterized by aberrant levels of intracellular nitric oxide. In additional embodiments, the methods provide for reducing neuronal death associated with aberrant levels of persistent sodium current. In other certain embodiments, the methods are directed to treating neuropathic pain, inflammatory pain such as arthritic pain, visceral pain, postoperative pain, pain resulting from cancer or cancer treatment, headache pain, irritable bowel syndrome pain, fibromyalgia pain, and pain resulting from diabetic neuropathy.

[015] A variety of selective persistent sodium current antagonists can be useful in the methods of the invention. In one embodiment, a method of the invention is practiced by administering an effective amount of a selective antagonist that has at least 20-fold selectivity for a persistent sodium current relative to a transient sodium current. In further embodiments, the antagonist has at least 50-fold selectivity for a persistent sodium current; at least 200-fold selectivity for a persistent sodium current; at least 400-fold selectivity for a persistent sodium current; at least 600-fold selectively for a persistent sodium current; or at least 1000-fold selectively for a persistent sodium current, relative to transient sodium current.

WO 2005/020982 PCT/US2004/028077 00 0

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S[016] A variety of selective persistent sodium current antagonists can be useful in the methods of the invention. In one embodiment, a method of the invention is practiced by administering an effective amount of a selective Nay1.3 antagonist that has at least 20-fold selectivity for Nay1.3 persistent sodium current relative to transient sodium current. In further embodiments, the antagonist has at least c selectivity for the Nay1.3 persistent sodium current; at least 200-fold selectivity for the IN Nav1.3 persistent sodium current; at least 400-fold selectivity for the Nav1.3 persistent sodium current; at least 600-fold selectively for the Na,1.3 persistent sodium current; 00 or at least 1000-fold selectively for the Nay1.3 persistent sodium current, relative to transient sodium current.

[017] In further embodiments, the methods of the invention involve administering an effective amount of a selective persistent sodium current antagonist belonging to one of the disclosed structural classes of selective persistent sodium current antagonists.

Such a selective persistent sodium channel antagonist can be, without limitation, a compound represented by a formula selected from Formula 1: S Y-Ar 1 Ar 2

C)

n

-N

3

R

1

R

[018] wherein, [019] Ar 1 is an aryl group; [020] Ar 2 is an aryl group; [021] Y is absent or is selected from: 0 0 0

R

4

O

[022] R 1 is selected from hydrogen, C1-C8 alkyl, aryl, arylalkyl; WO 2005/020982 WO 205100982PCTIUS2004/028077 00 00 [023] R 2 and R' are independently selected from hydrogen, 01-08 alkyl, aryl, arylalkyl, hydroxy, fluoro, CI-G8 carbocyclic ring, or CI-C8 heterocyclic ring; [024] R 4 is selected from hydrogen, CI-C8 alkyl, aryl, arylalkyl; [025] RWand R 6 are selected from hydrogen, fluoro, C, to 08 alkyl, hydroxy; [026] R 7 is selected from hydrogen, C, to C8 alkyl, aryl, arylalkyl; and [027] n is an integer of from 1 to 6; [028] Formula 2 Ar -Y 1

-C-X

1 -Ar 3

-NR

8

R'

[029] [030] [031] [032] wherein, Ar 3 is an aryl group; Ar 4 is an aryl group;

X

1 and Y' are independently selected from 0 0 0 0 12 0 11 0 0 [033] R 5 and R 6 are independently selected from hydrogen, fluoro, C, to 08 alkyl, hydroxy; [034] R 7 is selected from hydrogen, C, to 08 alkyl, aryl, arylalkyl; WO 2005/020982 PCT/US2004/028077 00 [035] R 8 and R 9 are selected from hydrogen, Cj-C 8 alkyl, aryl, arylalkyl, C0R 12 ;Z

COCF

3 [036] R 10 and R" 1 are selected from hydrogen, halogen, hydroxyl, CI-C8 alkyl, aryl, arylalkyl, and [037] R 12 is selected from hydrogen, Cj-C8 alkyl, aryl, arylalkyl; 00 [038] Formula 3 Ar 5-j

A

6 y 2

Z

2 [039] wherein, [040] Ar 5 is an aryl group; [041] Arr'is anaryl group; [042] X 2 is 0, S, or N 4 [043] y 2 is N or CR 15 [044] Z 2 is N or CR 1 6 [045] R 5 and R 6 are selected from hydrogen, fluoro, C, to C 8 alkyl, hydroxy; [046] R 7 is selected from hydrogen, C 1 to C 8 alkyl, aryl, arylalkyl; [047] R 13 is selected from halogen Cl-C8 alkyl, arylalkyl, and (CR 5

R

6 )cN(R 7 2 [048] R 14 is selected from hydrogen, halogen, C1 to C 8 alkyl, CE 3

OCH

3

NO

2

(CR

5

R

6 )cN(R 7 2 [0491 R 1 5 is selected from hydrogen, halogen, C, to C 8 alkyl, CF 3

OCH

3

NO

2 (CR 5 R 6 7 2 WO 2005/020982 PCT/US2004/028077

O

00 0 00 [050] R 1 6 is selected from hydrogen, halogen, C, to Ce alkyl, CF 3

OCH

3

NO

2

(CR

5

R

6 )cN(R 7 2 and [051] c is 0 or an integer from 1 to 5; and [052] Formula 4 R19 R 17 R X

R

18

X

217 Ra [053] wherein, [054] Ar 7 is an aryl group; [055] R is selected from halogen, Cl-C8 alkyl, NR2R 23

OR

2 2 [056] R 5 and R 6 are selected from hydrogen, fluoro, C, to C8 alkyl, hydroxy; [057] R 7 is selected from hydrogen, CI to C8 alkyl, aryl, arylalkyl; [058] R 1 7 and R 1 8 are independently selected hydrogen, C1-C8 alkyl, aryl, arylalkyl, hydroxy; [059] R 1 9 and R 20 are independently selected from hydrogen, halogen, C1-C, alkyl, hydroxy, amino, CF 3 [060] R 21

R

2 2 and R 23 are independently selected from hydrogen, aryl or C1-C8 alkyl; [061] a is 0 or an integer from 1 to 5; and [062] m is 0 or and integer from 1 to 3.

P \WPD0CS\CRN\RMHSpcc\2423MI3 dv spa dw.28A)nI2X)8 00 [063] A compound corresponding to any of the above formulas also can be a pharmaceutical acceptable salt, ester, amide, or geometric, steroisomer, or racemic mixture.

[064] Any of the variety of routes of administration can be useful for treating chemical Opain according to a method of the invention. In particular embodiments, administration is performed peripherally, systemically or orally.

00 0 Thus according to an aspect of the present invention as claimed there is provided 1 10 a method of treating chronic pain in a mammal, comprising administering to said mammal an effective amount of a selective persistent sodium channel antagonist, wherein said antagonist has at least 20-fold selectivity for a persistent sodium current relative to a transient sodium current, and wherein said agonist is a compound included in formula or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemate mixture thereof.

According to another aspect of the invention as claimed there is provided use of a selective persistent sodium channel antagonist for the manufacture of a medicament for the treatment of chronic pain in a mammal, wherein said antagonist has at least selectivity for a persistent sodium current relative to a transient sodium current and wherein said agonist is a compound included in formula or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemate mixture thereof.

These and other aspects of the invention are described below and in the claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS [065] Fig. 1 shows four compounds that are selective persistent sodium current antagonists.

00 O [066] Fig. 2 shows inhibition of persistent current-dependent depolarization by sodium channel blockers. In the this assay, cells are resting in wells containing 80 pi of TEA-MeSO 3 (sodium-free box) to which is added 240 pl of NaMeSO 3 buffer containing 13 mM KMeS03 for a final K' concentration of 10 mM and a final Na concentration of 110 mM (sodium/potassium-addition). This elicits a robust depolarizing response. Following the resolution of the sodium-dependent Sdepolarization, a second aliquot of KMeSO 3 is added to the well bringing the final K concentration to 80 mM (High potassium-addition). This addition results in a second depolarizing response. Compounds that reduce the sodium-dependent, but not the potassium-dependent depolarizations are selected as persistent sodium channel C blockers. Circles indicate the control response with 0.1% DMSO added, triangles show the effects of the sodium channel inhibitor tetracaine (10 pM) and the diamonds show the response during the application of a non-specific channel blocker.

[067] Fig. 3 shows data from assays in which the screening window for the persistent current assay is determined. To evaluate the size of the "screening window," data was examined from assays in which responses to sodium-dependent depolarization were measured in the presence of 10 pM Tetracaine to completely block the sodium-dependent depolarization or in the presence of a 0.1% DMSO WO 2005/020982 PCT/US200/02077 00 0 control to obtain a maximum depolarization. Data were binned into histograms and a Sscreening window was calculated from the mean and standard deviation for the 0 maximal and minimum values according to the equation: Z 1 (3 x STDax 3 x STDMin)/(MeanMax MeanMin). Histograms A, B and C represent data obtained from three different assay plates. The screening window for a run was considered T adequate 1.0 Z O [068] Fig. 4 shows sodium current traces before and after the addition of 3 CiM 00 Compound 1 or 500 nM TTX. HEK cells expressing Nay1.3 channels were patch clamped in the perforated -patch mode. Currents were elicited by 200 msec test pulses to 0 mV from a holding potential of -90 mV.

[069] Fig. 5 shows a dose-response curve for Compound 1. The peak amplitudes of transient Na current (It) and the steady state amplitude of the persistent current (1p) were measured at various Compound 1 concentrations, normalized to amplitude of the control currents. The percent block was then plotted against drug concentration. Solid lines represent fits to the data with the Hill equation. The calculated EC50 values and Hill coefficients are as follows: Hillslope, It is 0.354 and Ip is 0.733; EC 50 It is 0.167 M and Ip is 3.71 x 10" M.

[070] Fig. 6 shows the effects of intraperitoneally administered Compound 1 on paw withdrawal threshold (mean±SEM) in a test of mechanical allodynia in the spinal nerve ligation model of neuropathic pain. Paw withdrawal threshold (gram force) was determined using von Frey filament stimulation and the Dixon's up-down method.

Allodynic response was measured at baseline (0 min) and at 15, 30, 60 and 120 min after of 10 mg/kg IP injection of Compound 1 or vehicle control. Percent reversal of allodynia compared with non-injected rats was calculated. Six rats were used at each dose. Data were analyzed by analysis of variance and Dunnett's test reversal of allodynia was considered significant if DETAILED DESCRIPTION OF THE INVENTION [071] I. Voltage-gated Sodium Channels WO 2005/020982 PCT/US200/028077 00 ;[072] In the normal functioning of the nervous system, neurons are capable of receiving a stimulus, and in response, propagating an electrical signal away from Stheir neuron cell bodies (soma) along processes (axons). From the axon, the signal is delivered to the synaptic terminal, where it is transferred to an adjacent neuron or t other cell. Voltage-sensitive sodium channels have an important role in nervous IND system function because they mediate propagation of electrical signals along axons.

00 [073] Voltage-gated sodium channels are members of a large mammalian gene family encoding at least 9 alpha- and 3 beta- subunits. While all members of this family conduct Na ions through the cell membrane, they differ in tissue localization, regulation and, at least in part, in kinetics of activation and inactivation, see, e.g., William A. Catterall, From Ionic Currents to Molecular Mechanism: The Structure and Function of Voltage-gated Sodium Channels, 26(1) NEURON 13-25 (2000); and Sanja D. Novakovic et al., Regulation of Na Channel Distribution in the Nervous System, 24(8) TRENDS NEUROSCI. 473-478 (2001), which are hereby incorporated by reference in their entirety.

[074] Generally, under resting conditions, sodium channels are closed until a stimulus depolarizes the cell to a threshold level. At this threshold, sodium channels begin to open and then rapidly generate the upstroke of the action potential.

Normally during an action potential, sodium channels open briefly (one millisecond) and then close (inactivate) until the excitable cell returns to its resting potential and the sodium channels re-enter the resting state.

[075] Without wishing to be bound by the following, this behavior of voltage-gated sodium channels can be understood as follows. Sodium channels can reside in three major conformations or states. The resting or "closed" state predominates at negative membrane potentials -60 mV). Upon depolarization, channels open and allow current to flow. Transition from the resting state to the open state occurs within a millisecond after depolarization to positive membrane potentials. Finally, during sustained depolarization 1-2 ms), channels enter a second closed or inactive state. Subsequent re-opening of channels requires recycling of channels from an inactive state to a resting state, which occurs when the membrane potential returns WO 2005/020982 PCIUS2004/28077 00 0 Oto a negative value (repolarization). Therefore, membrane depolarization not only causes sodium channels to open, but also causes them to close, during sustained depolarization.

[076] A small fraction of the sodium channels can fail to inactivate even with c sustained depolarization. This non-inactivating sodium current is called a "persistent" IND sodium current. Four sodium channels, Nav1.3, Nav1.5, Nav1.6 and Nav1.9, have Shistorically been known to generate a persistent current. Recent evidence, however, 0 0 suggests that all voltage-gated sodium channels are capable of producing a 010 persistent current, see, Abraha Taddese Bruce P. Bean, Subthreshold Sodium Current from Rapidly Inactivating Sodium Channels Drives Spontaneous Firing of Tubermammillary Neurons, 33(4) NEURON 587-600 (2002); Michael Tri H.

Do Bruce P. Bean, Subthreshold Sodium Currents and Pacemaking of Subthalamic Neurons: Modulation by Slow Inactivation, 39(1) NEURON 109-120 (2003), which are hereby incorporated by reference in their entirety. The mechanism that produces a persistent current is poorly understood. Two hypothesis are that different sodium channels produce transient and persistent currents, and that a sodium channel capable of producing transient sodium current enters a different gating mode to produce a persistent current. Persistent sodium channels can open at more negative membrane potentials relative to normal sodium channels and inactivate at more positive potentials, see, Jacopo Magistretti Angel Alonso, Biophysical Properties and Slow-voltage Dependent Inactivation of a Sustained Sodium Current in Entorhinal Cortex Layer-11 Principal Neurons: A Whole-Cell and Single-Channel Study 114(4) J. GEN. PHYSIOL. 491-509 (1999). Persistent sodium current have been observed at membrane potentials as negative as -80 mV, see, Peter K. Stys, Anoxic and Ischemic Injury of Myelinated Axons in CNS White Matter: From Mechanistic Concepts to Therapeutics, 18(1) J. CEREB. BLOOD FLOW METAB. 2-25 (1998) and have been shown to persist for seconds following depolarization at potentials as positive as 0 mV, see, Magistretti Alonso, supra, (1999). Thus, persistent sodium current is distinct from, and can be readily distinguished from, transient sodium current.

[077] Although the physiological role of persistent sodium current is not fully understood, such current can function in generating rhythmic oscillations; integrating synaptic input; modulating the firing pattern of neurons; and regulating neuronal WO 2005/020982 PCT/US200/028077 00 0 J excitability and firing frequency, see, Wayne E. Crill, Persistent Sodium Current in Mammalian Central Neurons 58 ANNU. REV. PHYSIOL. 349-362 (1996); and David S. Ragsdale Massimo Avoli, Sodium Channels as Molecular Targets for Antiepileptic Drugs, 26(1) BRAIN RES. BRAIN RES. REV. 16-28 (1998). Aberrant persistent sodium current can contribute to the development or progression of many cpathological conditions. For example, persistent sodium current are thought to S u induce deleterious phenomena, including, neuropathies, cardiac arrhythmia, O epileptic seizure, neurodegeneration, and neuronal cell death under hypoxic and 0 0 ischemic conditions, see, Christoph Lossin et al., Molecular Basis of an 010 Inherited Epilepsy 34(6) NEURON 877-84 (2002); Peter K. Stys et al., Ionic Mechanisms of Anoxic Injury in Mammalian CNS White Matter: Role of Na Channels and Na(+)-Ca2+ Exchanger, 12(2) J. NEUROSCI. 430-439 (1992); Peter K. Stys et al., Noninactivating, Tetrodotoxin-Sensitive Na* Conductance in Rat Optic Nerve Axons, 90(15) PROC. NATL. ACAD. SCI. USA, 6976-6980 (1993); and Giti Garthwaite et al., Mechanisms of Ischaemic Damage to Central White Matter Axons: A Quantitative Histological Analysis Using Rat Optic Nerve, 94(4) NEUROSCIENCE 1219-1230 (1999).

Thus, aberrant persistent sodium current can contribute to development or progression of pathological conditions by collapsing the normal cell transmembrane gradient for sodium, leading to reverse operation of the sodium-calcium exchanger, and resulting in an influx of intracellular calcium, which injures the axon, see, e.g., Stys et al., supra, (1992). As disclosed herein, pain conditions associated with aberrant persistent sodium current can be treated by selectively inhibiting or reducing persistent sodium current without compromising normal transient sodium current function.

[078] While abnormal activity of a persistent current can underlie a wide array of neurological disorders, the underlying mechanisms appears to be similar. It is generally understood that abnormally increased persistent sodium current can depolarize the resting membrane potential or reduce the rate of repolarization during an action potential. Either effect may produce a state of hyper-excitability in which aberrant neuronal behavior is manifested. This aberrant neuronal behavior can result in a neuron with increased firing rates, enhanced sensitivity to synaptic input or abnormal repetitive or rhythmic firing patterns. It is also generally understood that abnormally high levels of persistent current generate sustained membrane depolarization and a concomitant increase of Na* within the cell. This high Na* influx, WO 2005/020982 PC1T/US2004/028077 00 0 bt in turn, drives the sodium/calcium exchanger, which in turn, results in detrimental levels of Ca 2 to accumulate inside affected cells. Abnormally high levels of Ca 2 result in neuronal cell dysfunction and neuronal cell death. Thus, by collapsing the normal cell transmembrane gradient for sodium, a persistent current can reverse the operation-of the sodium-calcium exchanger, and the resulting an influx of intracellular calcium would cause injures or death to a nerve. As disclosed herein, conditions N associated with aberrant persistent sodium current can be treated by selectively inhibiting or reducing persistent sodium current without compromising normal 00 transient sodium current function, thereby allowing normal neuronal function (excitability).

[079] II. Neurological disorders and persistent sodium current [080] The methods of the invention can be used to reduce or eliminate aberrant levels of persistent sodium current in a mammal, and thus can be used, for example, to treat any of a variety of neurological conditions that involve aberrant levels of persistent sodium current. Neuronal disturbance, including neuronal dysfunction and neuronal death, associated with unwanted persistent neuronal firing can contribute to, or cause, a variety of disorders of the central and peripheral nervous systems.

Therefore, a compound that decreases persistent sodium current without a similar decrease in non-pathological transient sodium current can effectively treat such conditions, without harmful side effects that generally accompany non-selective sodium channel blockers currently in use. Because all sodium channels seem capable of generating a persistent current, and since any condition whose underlying cause includes an aberrant persistent sodium current, a very wide range of neurological abnormalities can be treated using a persistent sodium channel antagonist. Conditions that can be treated according to a method of the invention include, without limitation, neuropathies such as, epilepsies, palsies, connective tissue disorders and conditions associated with neuropathies, like, alcoholism, cancers, infectious diseases, organ disorders and vitamin deficiencies; hypoxia and ischemia, such as, cerebral hypoxia/ischemia, myocardial hypoxia/ischemia, myoischemia, diabetes ischemia and hypoxia/ischemic retinopathy; and behavioral disorders, dementia, movement disorders and neurodegenerative conditions such as.

Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and multiple sclerosis.

I.

WO 2005/020982 PCT/US2004/028077 00 ;[081] Based on the identification of selective persistent sodium current antagonists Sthat have at least 20-fold selectivity for persistent sodium current relative to transient sodium current, the present invention provides therapeutic methods that involve selectively antagonizing persistent sodium channel. Whereas certain conditions Mc, have been treated using non-selective sodium channel blockers, albeit with ID significant side effects, the methods of the present invention involve administering to a mammal an effective amount of a selective persistent sodium current antagonist 00 that has at least 20-fold selectivity for persistent sodium current relative to transient sodium current.

[082] By preventing or reducing aberrant levels of persistent sodium current, the progression of various conditions associated with unwanted persistent neuronal firing can be stopped or slowed, and improvement in the pathophysiology or symptoms appreciated. As used herein, the term "conditions associated with unwanted persistent neuronal firing" means a disorder in which persistent membrane sodium conductance causes or contributes to functional changes resulting from disease or injury. Such functional changes, or pathophysiology, can involve either neuronal damage, including neuronal death; unwanted persistent neuronal firing; or both. As used herein, the term "reducing," when used in reference to neuronal death means preventing, decreasing or eliminating unwanted persistent neuronal firing or aberrant levels of persistent sodium current. Reducing aberrant levels of persistent sodium current by administering a selective persistent sodium current antagonist can be an effective method for treating conditions involving neuronal dysfunction or neuronal death, for example, for treating conditions characterized by aberrant levels of persistent sodium current or aberrant levels of intracellular nitric oxide.

[083] III. Treatment of neuropathies using a selective persistent sodium current antagonist [084] The present invention provides methods of treating a neuropathy by administering an effective amount of a selective persistent sodium current antagonist having at least 20-fold selectivity for persistent sodium current relative to transient sodium current. Aberrant levels of sodium current are associated with a variety of neuropathic conditions that led to neuronal dysfunction or neuronal death. As used WO 2005/020982 PCT/US004/0281077 00 0

(N

herein, the term "neuropathic condition" means any condition resulting in nerve damage, including, motor nerve damage, sensory nerve damage, autonomic nerve damage. Neuropathic conditions include a heterogeneous group of conditions of the central or peripheral nervous system that include, without limitation, headache, pain, inflammatory diseases, movement disorders, tumors, birth injuries, Sdevelopmental abnormalities, neurocutaneous disorders, autonomic disorders, and Sparoxysmal disorders. As such, a neuropathic condition have a wide range of O different etiologies, including, hereditary or sporatic, secondary to a toxic or 00 metabolic process, and can result from an injury, trauma, disease, or infection. Such conditions can be characterized by abnormalities of relatively specific regions of the brain or specific populations of neurons. The particular cell groups affected in different neuropathic conditions typically determine the clinical phenotype of the condition.

[085] Exemplary examples of neuropathies include, without limitation, amyloidosis; autoimmune disorders such as, Guillain-Barr6 syndrome, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease; movement disorders like palsies involving injury or damage to nerves such as, cerebral palsy, Bell's palsy, diver's palsy, extrapyramidial cerebral palsy, lead palsy, Ramsey Hunt syndrome, obstetrical palsy, like Erb palsy and Klumpke palsy, posticus palsy, Scrivener's palsy, tardy median palsy, tardy ulnar palsy, and progressive supranuclear palsy; arthritis/connective tissue disorders such as, e.g., osteoarthritis, rheumatoid arthritis, juvenile arthritis, gouty arthritis; spondyloarthritis, scleroderma, fibromyalgia, osteoporosis, noise sensitivity, multiple chemical sensitivity and asthma; conditions associated with neuropathies like alcoholism, cancers, infectious diseases, organ disorders and vitamin deficiencies; and epilepsies, seizures and paroxysmal conditions. The skilled person understands that these and other mild, moderate or severe neuropathic conditions can be treated according to a method of the invention.

[086] As a non-limiting example, epilepsies are conditions that can be characterized by aberrant levels of persistent sodium current. Epilepsies and other seizure disorders, are a group of neuronal dysfunction disorders of the central nervous WO 2005/020982 PCT/US2004/028)077 00 0

(N

Ssystem and are generally characterized by sudden seizures, muscle contractions, Sand partial or total loss of consciousness. Epilepsy is a disorder characterized by the occurrence of at least 2 unprovoked seizures. Seizures are the manifestation of abnormal hypersynchronous discharges of cortical neurons. The clinical signs or symptoms of seizures depend upon the location and extent of the propagation of the c discharging cortical neurons. That seizures are a common nonspecific manifestation N of neurologic injury and disease should not be surprising, because the main function O of the brain is the transmission of electrical impulses. The lifetime likelihood of 0 0 experiencing at least one epileptic seizure is about and the lifetime likelihood of being diagnosed as having epilepsy is almost However, the prevalence of active epilepsy is only 0.8%.

[087] Epilepsies can be divided into two major categories. Partial-onset seizures begin in one focal area of the cerebral cortex, while generalized-onset seizures have an onset recorded simultaneously in both cerebral hemispheres. Some seizures are difficult to fit into one particular class, and they are considered as unclassified seizures. Partial-onset seizures include, simple partial seizures, complex partial seizures and secondarily generalized tonic-clonic seizures. Generalized-onset seizures include, absence seizures, tonic seizures, clonic seizures, myoclonic seizures, primary generalized tonic-clonic seizures, and atonic seizures. Likewise, epileptic syndromes can be classified into two major groups, localization-related syndromes and generalized-onset syndromes.

[088] Voltage-gated sodium channels, which play an important role in initiation and transmission of action potentials, are involved in the etiology of epilepsy, and appear to include Nav1.1, Na,1.2, Nay1.3, Na,1.5, and Nav1.6, see, Rudiger Kohling, Voltage-gated Sodium Channels in Epilepsy, 43(11) Epilepsia 1278-1295 (2002); Michael M. Segal Sodium Channels and Epilepsy Electrophysiology, 241 NOVARTIS FOUND. SYMP. 173-180 (2002), which are hereby incorporated by reference in their entirety. Examination of individuals suffering from hereditary forms of epilepsy has revealed these individuals carried deleterious mutations in Nav1.1 or Nay1.2, see, Lossin et al., supra, (2002); Miriam H. Meisler et al., Mutations of Voltage-gated Sodium Channels in Movement Disorders and Epilepsy, 241 NOVARTIS FOUND. SYMP.

72-81 (2002); J. Spampanato et al., Generalized Epilepsy with Febrile Seizures Plus Type 2 Mutation W1204R Alters Voltage-Dependent Gating of Na(V)1.1 Sodium WO 2005/020982 PCT/US2004/028077 00 0 J) Channels, 116(1) NEUROSCIENCE 37-48 (2003); Paolo Bonanni et al., Generalized Epilepsy with Febrile Seizures Plus (GEFS+): Clinical Spectrum in Seven Italian Families Unrelated to SCN1A, SCN1B, And GABRG2 Gene Mutations, 45(2) EPILEPSIA 149-158 (2004); Berten P. G. M. Ceulemans et al., Clinical Correlations Of Mutations in the SCN1A Gene: from Febrile Seizures to Severe Myoclonic Epilepsy Sin Infancy, 30(4) PEDIATR. NEUROL. 236-243 (2004); Goryu Fukuma et al., Mutations F" of Neuronal Voltage-Gated Na+ Channel Alpha 1 Subunit Gene SCN1A in Core 0 Severe Myoclonic Epilepsy in Infancy (SMEI) and in Borderline SMEI (SMEB), 45(2) 00 EPILEPSIA 140-148 (2004); and Kazusaku Kamiya et al., A Nonsense Mutation of the Sodium Channel Gene SCN2A in a Patient with Intractable Epilepsy and Mental Decline, 24(11) J. NEUROSCI. 2690-2698 (2004), which are hereby incorporated by reference in their entirety. In addition, epilepsy appears to be caused by abnormal nerve discharges in the brain that result in aberrant levels of persistent sodium current, see, Newton Agrawal et al., Increased Persistent Sodium Currents in Rat Entorhinal Cortex Layer V Neurons in a Post-Status Epilepticus Model of Temporal Lobe Epilepsy, 44(12) EPILEPSIA 1601-1604 (2003); and Martin Vreugdenhil et al., Persistent Sodium Current in Subicular Neurons Isolated from Patients with Temporal Lobe Epilepsy, 19(10) EUR. J. NEUROSCI. 2769-2778 (2004), which are hereby incorporated by reference in their entirety. Both Nay1.1 and Na,1.6 are thought to be capable of producing a persistent current, see, Joshua P.

Klein et al., Dysregulation of Sodium Channel Expression in Cortical Neurons in a Rodent Model of Absence Epilepsy, 1000(1-2) BRAIN RES. 102-109 (2004), which is hereby incorporated by reference in its entirety. In view of the role of persistent sodium currents in epilepsy, a selective persistent sodium current antagonist can be advantageously used to treat epilepsy without deleterious side effects associated with non-selective sodium channel blockers.

[089] IV. Treatment of hypoxias and ischemias using a selective persistent sodium current antagonist [090] The present invention also provides methods of treating a hypoxia or ischemia by administering an effective amount of a selective persistent sodium current antagonist having at least 20-fold selectivity for persistent sodium current relative to transient sodium current. Neuronal damage or death occurring as a result of changes induced by hypoxia or ischemia appears to be associated with increased WO 2005/020982 PCT/US2(104/08077 00 j persistent sodium current, see, Anna K. M. Hammarstrom Peter W. Gage, Hypoxia and Persistent Current, 31 EUR. BIOPHYS. J. 323-330 (2002), which is hereby incorporated by reference in its entirety. As used herein, the term "hypoxia" means an incident during which the oxygen supply to a tissue is diminished or eliminated. A hypoxia can include, cerebral hypoxia, diffusion hypoxia, hypoxic Shypoxia, cell hypoxia, ischemic hypoxia, or any other accidental or purposeful Sreduction or elimination of oxygen supply to a tissue. As used herein, the term O "ischemia" means an incident during which the blood supply to a tissue is reduced or 00 completely obstructed. An ischemia can include, cerebral ischemia, myocardiac ischemia, myoischemia, diabetes ischemia, ischemia retinae, postural ischemia, or any other accidental or purposeful reduction or complete obstruction of blood supply to a tissue. That a reduction or complete obstruction of blood to a tissue necessarily means a reduction or elimination of oxygen supply to that tissue, ischemias and hypoxias are usually related. The skilled person understands that these and other mild, moderate or severe hypoxic and ischemic conditions can be treated according to a method of the invention.

[091] As a non-limiting example, cerebral ischemia occurs when a blood vessel bringing oxygen and nutrients to the brain bursts or is clogged by a blood clot or other material. Because of this rupture or blockage, part of the brain is deprived of its normal blood flow and the oxygen it contains. In the absence of oxygen, nerve cells in the affected area of the brain undergo deleterious changes and die. This neuronal cell death can lead to a stroke, resulting in loss of control of the body part normally controlled by these nerve cells. The devastating effects of stroke are often permanent because damaged nerve cells are not replaced.

[092] Cerebral hypoxia or ischemia can result, without limitation, from CNS surgery, open heart surgery or any procedure during which the function of the cardiovascular system is compromised; trauma that results in reduction of blood flow to the brain; disease that causes reduction of blood flow to the brain, including cerebrovascular disease, such as chronic subdural hematoma, cavernous angioma, arteriovenous malformation, vascular dementia, carotid or circle of Willis hypertensive encephalopathy, multiple embolic infarctions, hypertensive encephalopathy and cerebral hemorrhage; infectious diseases that can cause cranial swelling that reduces blood flow to neurons, such as meningitis, Lyme encephalopathy, Herpes WO 2005/020982 MA1~S2004/028077 00 0 jI) encephalitis, Creutzfeld-Jakob disease, cerebral toxoplasmosis and the like; trauma, such as head trauma and traumatic brain injury that cause a reduction in blood flow to neurons; and proliferative disorders that cause a reduction in blood flow to neurons, including diseases associated with the overgrowth of connective tissues, such as various fibrotic diseases, vascular proliferative disorders, and benign tumors.

SProliferative disorders of the central nervous system include, for example, cerebellar astrocytomas and medulloblastomas, ependymomas, gliomas, germinomas, and O metastatic adenocarcinoma, metastatic bronchogenic carcinoma, meningioma, 00 sarcoma and neuroblastoma.

[093] Sodium channel inhibitors, such as, tetrotoxin (TTX) and lidocaine, and extracelluar Na' ions protect neurons from hypoxic and ischemic damage, suggesting that voltage-gated sodium channel activity is an early and important step in sensing oxygen levels and cell damage in neurons. It was subsequently shown that these oxygen sensing channels generated a persistent current, and hypoxic/ischemic conditions increased the activity of these persistent current channels that result in an abnormally high intake of see, Anna H. K.

Hammarstr6m Peter W. Gage, Oxygen-sensing Persistent Sodium Channels in Rat Hippocampus, 529(1) J. PHYSIOL. 107-118 (2000), which is hereby incorporated by reference in its entirety. The influx of Na would drive the sodium/calcium exchanger, which in turn, would result in detrimental levels of Ca2+ accumulate inside affected cells and cell death, see, Peter Lipton, Ischemic Cell Death in Brain Neurons, 79(4) PHYSIOL. REV. 1431-1568 (1999), which is hereby incorporated by reference in its entirety. Therefore, application of a selective persistent sodium current antagonist can serve as a neuroprotectant against cerebral hypoxia or ischemia, without the deleterious side effects associated with non-selective sodium channel blockers.

[094] As another non-limiting example, myocardial ischemia is a disorder of cardiac function caused by insufficient blood flow to the muscle tissue of the heart. The decreased blood flow may be due to narrowing of the coronary arteries (coronary arteriosclerosis), to obstruction by a thrombus (coronary thrombosis), or less commonly, to diffuse narrowing of arterioles and other small vessels within the heart.

Severe interruption of the blood supply to the myocardial tissue results in a concomitant interruption in oxygen which may lead to necrosis of cardiac muscle (myocardial infarction).

WO 2005/020982 PCT/US200/028077 00 0 S[095] Abnormal levels of a persistent sodium current, which become prominent following cardiac hypoxia or ischemia, are associated with arrhythmias, which can C trigger a heart attack, see, Hammarstr6m Gage, supra, (2002). Cardiac cells, such as, Purkinje fibers and ventricular myocytes, generate a persistent sodium Scurrent. Examination of ventricular myocytes in the presence or absence of oxygen Sindicates that persistent sodium current increases during hypoxia, and that this O aberrant current could trigger early after depolarization, arrhythmia, and heart failure, 00 see, Y. K. Ju et al., Hypoxia Increases Persistent Sodium Current in Rat Ventricular Myocytes, 497(2) J. PHYSIOL. 337-347 (1996), which is hereby c incorporated by reference in its entirety. Additionally, application of tetrotoxin (TTX), a voltage-gated sodium channel inhibitor, reduces the action potential duration of a persistent current in human ventricular myocytes, as well as abolishes the early after depolarization in myocytes isolated from heart failure patients, see, Victor A.

Maltsev et al., Novel, Ultraslow Inactivating Sodium Current in Human Ventricular Cardiomyocytes, 98(23) CIRCULATION 2545-2552 (1998), which is hereby incorporated by reference in its entirety.

[096] Several voltage-gated sodium channels are localized in specific regions of the heart where they are believed to regulate distinct activities. The persistent sodium channel Na,1.5 is found in the intercalated disks/AV node and seems to be involved primarily in initiation and propagation of the action potential from cell to cell. On the other hand, Nay1.1 and Na,1.3 appear to generate a persistent current in the transverse tubules/SA node and may function in coordinating and synchronizing the action potential from the cell surface into the interior, see, Sebastian K. G. Maier et al., An Unexpected Requirement for Brain-Type Sodium Channels for Control of Heart Rate in the Mouse Sinoatral Node, 100(6) PROC. NATL. ACAD. Sci. U. S. A.

3507-3512 (2003); and Sebastian K. G. Maier et al., Distinct Subcellular Localization of Different Sodium Channel Alpha and Beta Subunits in Single Ventricular Myocytes from Mouse Heart, 109(11) CIRCULATION 1421-1427 (2004), which are hereby incorporated by reference in their entirety. Furthermore, a missense mutation in that accelerates channel activation is associated with individuals diagnosed with cardiac arrhythmia, see, Igor Splawski et al., Variant of SCN5A Sodium Channel Implicated in Risk of Cardiac Arrhythmia, 297 SCIENCE 1333-1336 (2002), which is hereby incorporated by reference in its entirety. Thus, as seen in cerebral WO 2005/020982 PCT/US2004/028077 00

O

O

jhypoxia/ischemia, as described above, elevated Na* levels due to an increased persistent current, will cause the sodium/calcium exchanger to import abnormally high levels Ca2+, thereby triggering myocardial cell death. Thus, a selective persistent sodium current antagonist can be used beneficially to prevent cardiac hypoxia or ischemia without the harmful side effects associated with current non- Sselective sodium channel blockers.

NO

C[097] In a third non-limiting example, ischemia retinae is a diminished blood supply Sin the retina due to diminished or failed blood circulation that can result in bilateral transitory or permanent blindness. Ischemia of the neuroretina and optic nerve can arise during an embolism, such as, retinal branch vein occlusion, retinal branch artery occlusion, central retinal artery occlusion, central retinal vein occlusion; as a result of a disease, such, diabetic retinopathy; during intravitreal surgery; by poisoning, such as, quinine; in retinal degenerations such as, retinitis pigmentosa, and in age-related macular degeneration; during an inflammation; during an infection; or exsanguination from recurring profuse haemorrhages in parturition, gastric and duodenal ulcers, and pulmonary tuberculosis). The skilled person understands that the methods of the invention can be used to treat these and other types of ischemia known in the art.

[098] The earliest ophthalmolscopic indication of an ischemic retinopathy is the appearance of microaneruysms, which correspond with areas of focal ischemia, see, Thomas W. Gardner et al., Diabetic Retinopathy: More than Meets the Eye, 47(Suppl. 2) SURV. OPHTHALMOL. S253-S262 (2002); and Alistair J. Barder, A New View of Diabetic Retinopathy: A Neurodegenerative Disease of the Eye, 27(2) PROG.

NEUROPSYCHOPHARMACOL. BIOL. PSYCHIATRY. 283-290 (2003), which are hereby incorporated by reference in their entirety. Coincident with or preceding these clinical findings, significant electrophysiological changes can be observed, including reduction in oscillatory potentials, delays in visual evoked potentials and changes in pattern and multi-focal electroretinograms, see, Erich Lieth et al., Retinal Neurodegeneration: Early Pathology in Diabetes, 28(1) CLIN. EXPERIMENT.

OPHTHALMOL. 3-8 (2000), which is hereby incorporated by reference in its entirety.

Alterations in the normal ionic conductances of the neural retinia, including retinal ganglion cells and their axons, have been associated with ischemic retinopathy, see, Quasthoff, (1998), which is hereby incorporated by reference in its entirety. Key WO 2005/020982 PCT/US2004021077 00 J) observations include a decrease in conduction velocity, a dysfunction of nodal sodium channels and an increase in intracellular Na concentration, see, Tom Brismar, Abnormal Na-Currents in Diabetic Rat Nerve Nodal Membrane, 10(Suppl. 2) DIABET. MED. 110S-112S (1993), which is hereby incorporated by reference in its entirety. The increased influx of Na would drives the sodium/calcium exchanger to cimport abnormally high levels of intracellular calcium, a major cause of neuronal cell death, see, Lipton, supra, (1999). The gradual loss of neurons in the retina O indicates that progress of the disease is ultimately irreversible, since these cells 00 cannot usually be replaced. Selectively reducing persistent sodium current can provide an effective means for reducing symptoms or pathophysiology of retinal ischemia. Thus, analogous to the neuroprotective effects of selective persistent sodium current blockers during cerebral hypoxia/ischemia, the present invention discloses a method that prevents retinal ischemias.

[099] V. Treatment of neurodegenerative conditions using a selective persistent sodium current antagonist [0100] The present invention further provides methods of treating a neurodegenerative condition by administering an effective amount of a selective persistent sodium current antagonist having at least 20-fold selectivity for persistent sodium current relative to transient sodium current. Aberrant levels of sodium current are associated with a variety of neurodegenerative conditions. As used herein, the term "neurodegenerative condition or disorder" means a condition characterized by progressive loss of neural tissue. Neurodegenerative conditions include a heterogeneous group of aberrant conditions of the central or peripheral nervous system that include, without limitation, behavioral disorders, dementia, neuromuscular disorders, movement disorders, inflammatory disorders and demyelinating diseases. Such conditions have many different etiologies such as, without limitation, sporatic or hereditary, secondary to toxic or metabolic processes, and can result from an injury, a trauma, a disease, or an infection.

Neurodegenerative conditions are progressive conditions that can be age associated or chronic. Such conditions can be characterized by abnormalities of relatively specific regions of the brain or specific populations of neurons. The particular cell groups affected in different neurodegenerative conditions typically determine the clinical phenotype of the condition. In particular, neurodegenerative conditions can WO 2005/020982 PCT/US2004/028077 00

O

O

tJb be associated with atrophy of a particular affected central or peripheral nervous system structure, and aberrant levels of sodium current and subsequent elevation of intracellular sodium can be a cause or contributing factor to this atrophy.

[0101] Exemplary neurodegenerative conditions include, but are not limited to, Motor SNeuron Disease (ALS), Parkinsonian Syndromes, diffuse sclerosis, amyotrophic lateral sclerosis, multiple sclerosis, diffuse cerebral cortical atrophy, Lewy-body O dementia, Pick disease, mesolimbocortical dementia, thalamic degeneration, bulbar 0 0 palsy, Huntington chorea, cortical-striatal-spinal degeneration, cortical-basal ganglionic degeneration, cerebrocerebellar degeneration, familial dementia with spastic paraparesis, polyglucosan body disease, Shy-Drager syndrome, olivopontocerebellar atrophy, progressive supranuclear palsy, dystonia musculorum deformans, Hallervorden-Spatz disease, Meige syndrome, familial tremors, Gilles de la Tourette syndrome, acanthocytic chorea, Friedreich ataxia, Holmes familial cortical cerebellar atrophy, AIDS related dementia, Gerstmann-Straussler-Scheinker disease, progressive spinal muscular atrophy, progressive balbar palsy, primary lateral sclerosis, hereditary muscular atrophy, spastic paraplegia, peroneal muscular atrophy, hypertrophic interstitial polyneuropathy, heredopathia atactica polyneuritiformis, optic neuropathy, diabetic retinopathy, Alzheimer's disease and ophthalmoplegia. The skilled person understands that these and other mild, moderate or severe neurodegenerative conditions can be treated according to a method of the invention.

[0102] As a non-limiting example, multiple sclerosis is a condition that can be characterized by aberrant levels of persistent sodium current. Multiple Sclerosis (MS) an chronic inflammatory disease of the central nervous system affecting white matter tissue impacts more than 350,000 persons in the United States and worldwide may affect as many as 30 cases per 100,000 population. MS can therefore be considered a nerve fiber, or axonal disease. MS can cause damage in a random manner within the CNS causing lesions or plaques to appear in CNS axons. A lesion is characterized by a loss of myelin (demyelination), the material that insulates axons. Demyelination profoundly effects the electrical properties of the axon, slowing or blocking nervous impulses from occurring. A variety of bodily functions are affected as a result of the adverse effects on axon physiology. During the course of the disease axons are destroyed classifying MS as a neurodegenerative disease.

WO 2005/020982 PCT/US200/028077 00 0

(N

Many people with the disorder are affected during what normally would be the most Sproductive years of their lives since the age of onset is often between 28 and Drug therapies currently available at best may slow down the disease or lessen the 1 symptoms. It is obvious that there is an unmet need for therapies to treat this form of neurological disorder.

N [0103] This neurodegenerative condition is typically marked by lack of muscle O coordination, muscle weakness, speech problems, paresthesia, and visual 00 impairments. In human patients with multiple sclerosis as well as animal models of this condition, there is evidence that onset of multiple sclerosis produces changes in cN the expression pattern of sodium channels within Purkinje cells. Dysregulated sodium channel expression can contribute to symptoms of multiple sclerosis. For example, a persistent sodium current can trigger calcium-mediated axonal injury via reverse sodium-calcium exchange, see, Stephen G. Waxman, Sodium Channels as Molecular Targets in Multiple Sclerosis, 39(2) J. REHABIL. RES. DEV.

233-242 (2002); and Stephen G. Waxman, Ion Channels and Neuronal Dysfunction in Multiple Sclerosis, 59(9) ARCH. NEUROL. 1377-1380 (2002), which are hereby incorporated by reference in their entirety. In myelinated axons, voltage-gated sodium channels Nay1.2 and Nay1.6 specifically cluster at the nodes of Ranvier.

However, both exhibit altered expression along demyelinated axons derived from patients suffering with multiple sclerosis. In addition, Nay1.6 and the sodium/calcium exchanger co-localize within axons expressing P-APP, a maker of axonal injury in multiple sclerosis. Thus in patients suffering from multiple sclerosis, altered distribution of Na,1.6 is thought to produce a persistent current that results in aberrantly high influx of Na which drives a sodium/calcium exchanger to import abnormally high levels of intraaxonal calcium, which triggers the neuronal damage seen in these individuals, see, Matthew J Craner et al., Molecular Changes in Neurons In Multiple Sclerosis: Altered Axonal Expression of Na,1.2 and Nal,.6 Sodium Channels And Na+/Ca2+ Exchanger, 101(21) PROC. NATL. ACAD. SCI. U. S.

A. 8168-8173 (2004); and Matthew J Craner et al., Co-Localization of Sodium Channel Na,1.6 and the Sodium-Calcium Exchanger at Sites of Axonal Injury in the Spinal Cord in EAE, 127(2) BRAIN 294-303 (2004), which are hereby incorporated by reference in their entirety. Thus, selectively reducing this abnormally high persistent sodium current can provide an effective means for treating an individual having multiple sclerosis.

WO 2005/020982 PCT/US2004/0281077 00 O0

O

[0104] As another non-limiting example, amyotrophic lateral sclerosis (ALS) or \"Motor Neuron Disease" is a neurodegenerative disorder of both the upper and lower motor neurons. The mean age of onset is approximately 55 years and the incidence of ALS is about two per 100,000. The prevalence of ALS in the USA is about 11 per c 100,000 affecting approximately 30,000 people. There are about 5,000 new cases per year, or 15 per day. ALS is characterized by progressive weakness of the lower Sand upper extremities as well as stiffness, muscle twitching and shaking and muscle 0 0 atrophy. ALS is a fatal disease with only 20% of those inflicted surviving 5 years. At present Riluzole is only FDA-approved drug that appears to slow down progression of the disease.

[0105] The etiology of ALS is unknown but one hypothesis proposes that glutamate excitotoxicity causes neuronal cell death associated with the disease. Interestingly, in an in vitro model of neuronal excitotoxicity, voltage-gated sodium channels, NMDA receptors and glutamate release were shown to mediate delayed neurodegeneration via nitric oxide formation, see, Paul J. Strijbos et al, Vicious Cycle Involving Na Channels, Glutamate Release, and NMDA Receptors Mediates Delayed Neurodegeneration Through Nitric Oxide Formation, 16(16) J. NEUROSCI. 5004-5013 (1996), which is hereby incorporated by reference in its entirety. It is thought that glutamate release requires the activation of voltage-gated Na' channels and therefore blocking these channels can prevent cytotoxic effects from excess spillover of glutamate. In a transgenic mouse model of ALS it was found that the morphological changes associated with neurogenenration in the peripheral axons of these mice were accompanied by changes in membrane conductance and excitability Jasna Kriz et al, Altered Ionic Conductances in Axons Of Transgenic Mouse Expressing the Human Neurofilament Heavy Gene: A Mouse Model of Amyotrophic Lateral Sclerosis, 163(2) EXP. NEUROL. 414-421 (2000). These authors suggested that the inactivation rate of the sodium channels from the axons of the transgenic mice were significantly slowed compared to controls. Moreover, of the many drugs tested for ALS the only drug shown to slow the progression of the disease (Riluzole) was found to block voltage-gated Na' channels and subsequent glutamate release, see, A Stefani et al., Differential Inhibition by Riluzole, Lamotrigine, and Phenytoin of Sodium and Calcium Currents in Cortical Neurons: Implications for Neuroprotective Strategies, 147(1) EXP. NEUROL. 115-122 (1997); WO 2005/020982 PCT/US200/028077 00 0

(N

j) and Thomas Anger et al., Medicinal Chemistry of Neuronal Voltage-Gated Sodium SChannel Blockers, 44(2) J. MED. CHEM. 115-137 (2001), which are hereby Sincorporated by reference in their entirety. It was subsequently shown that Riluzole targets persistent sodium currents, see, Andrea Urbani Ottorino Belluzzi, Riluzole Inhibits the Persistent Sodium Current in Mammalian CNS Neurons, 12(10) c EUR. J. NEUROSCI. 3567-3574 (2000); and Francesca Spadoni et al., Lamotrigine Derivatives and Riluzole Inhibit INa,P in Cortical Neurons, 13(9) NEUROREPORT.

O 1167-1170 (2002), which are hereby incorporated by reference in their entirety. Thus 00 persistent sodium currents appear to play a role in the progression of ALS, and selectively reducing this aberrantly high persistent sodium current can provide an effective means for treating an individual having ALS.

[0106] VI. Treatment of ocular conditions using a selective persistent sodium current antagonist [0107] The present invention also provides a method for treating an ocular condition by administering an effective amount of a selective persistent sodium current antagonist having at least 20-fold selectivity for persistent sodium current relative to transient sodium current. Unwanted neuronal firing and neuronal death induced by aberrant levels of persistent sodium channels can be a cause or contributing factor in ocular conditions.

[0108] An ocular condition can include a disease, aliment or condition which affects or involves the eye or one of the parts or regions of the eye. Broadly speaking the eye includes the eyeball and the tissues and fluids which constitute the eyeball, the periocular muscles (such as the oblique and rectus muscles) and the portion of the optic nerve which is within or adjacent to the eyeball. An anterior ocular condition is a disease, ailment or condition which affects or which involves an anterior front of the eye) ocular region or site, such as a periocular muscle, an eye lid or an eye ball tissue or fluid which is located anterior to the posterior wall of the lens capsule or ciliary muscles. Thus, an anterior ocular condition primarily affects or involves, the conjunctiva, the cornea, the conjunctiva, the anterior chamber, the iris, the posterior chamber (behind the retina but in front of the posterior wall of the lens capsule), the lens or the lens capsule and blood vessels and nerve which vascularize or innervate an anterior ocular region or site. A posterior ocular condition is a disease, ailment or WO 2005/020982 PCT/US2004/028077 00 0 condition which primarily affects or involves a posterior ocular region or site such as choroid or sclera (in a position posterior to a plane through the posterior wall of the lens capsule), vitreous, vitreous chamber, retina, optic nerve the optic disc), and blood vessels and nerves which vascularize or innervate a posterior ocular region or site.

S[0109] Thus, a posterior ocular condition can include a disease, ailment or O condition, such as for example, macular degeneration (such as non-exudative age 00 related macular degeneration and exudative age related macular degeneration); choroidal neovascularization; acute macular neuroretinopathy; macular edema (such as cystoid macular edema and diabetic macular edema); Behcet's disease, retinal disorders, diabetic retinopathy (including proliferative diabetic retinopathy); retinal arterial occlusive disease; central retinal vein occlusion; uveitic retinal disease; retinal detachment; ocular trauma which affects a posterior ocular site or location; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy; photocoagulation; radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; non-retinopathy diabetic retinal dysfunction, retinitis pigmentosa and glaucoma. Glaucoma can be considered a posterior ocular condition because the therapeutic goal is to prevent the loss of or reduce the occurrence of loss of vision due to damage to or loss of retinal cells or optic nerve cells neuroprotection).

[0110] An anterior ocular condition can include a disease, ailment or condition, such as for example, aphakia; pseudophakia; astigmatism; blepharospasm; cataract; conjunctival diseases; conjunctivitis; corneal diseases;, corneal ulcer; dry eye syndromes; eyelid diseases; lacrimal apparatus diseases; lacrimal duct obstruction; myopia; presbyopia; pupil disorders; refractive disorders and strabismus. Glaucoma can also be considered to be an anterior ocular condition because a clinical goal of glaucoma treatment can be to reduce a hypertension of aqueous fluid in the anterior chamber of the eye reduce intraocular pressure).

[0111] Examples of ocular conditions that can be treated using a method of the invention include, but are not limited to, maculopathies and retinal degeneration, such as Non-Exudative Age Related Macular Degeneration (ARMD), Exudative Age WO 2005/020982 PCT/US2004/028077 00

O

O

W Related Macular Degeneration (ARMD), Choroidal Neovascularization, Diabetic Retinopathy, Central Serous Chorioretinopathy, Cystoid Macular Edema, Diabetic 0 Macular Edema, Myopic Retinal Degeneration; inflammatory diseases, such as Acute Multifocal Placoid Pigment Epitheliopathy, Behcet's Disease, Birdshot Retinochoroidopathy, Infectious (Syphilis, Lyme, Tuberculosis, Toxoplasmosis), cIntermediate Uveitis (Pars Planitis), Multifocal Choroiditis, Multiple Evanescent White Dot Syndrome (MEWDS), Ocular Sarcoidosis, Posterior Scleritis, Serpiginous O Choroiditis, Subretinal Fibrosis and Uveitis Syndrome, Vogt-Koyanagi-Harada 0 0 Syndrome, Punctate Inner Choroidopathy, Acute Posterior Multifocal Placoid Pigment Epitheliopathy, Acute Retinal Pigement Epitheliitis, Acute Macular Neuroretinopathy; vascular and exudative diseases, such as Diabetic retinopathy, Central Retinal Arterial Occlusive Disease, Central Retinal Vein Occlusion, Disseminated Intravascular Coagulopathy, Branch Retinal Vein Occlusion, Hypertensive Fundus Changes, Ocular Ischemic Syndrome, Retinal Arterial Microaneurysms, Coat's Disease, Parafoveal Telangiectasis, Hemi-Retinal Vein Occlusion, Papillophlebitis, Central Retinal Artery Occlusion, Branch Retinal Artery Occlusion, Carotid Artery Disease (CAD), Frosted Branch Angiitis, Sickle Cell Retinopathy and other Hemoglobinopathies, Angioid Streaks, Familial Exudative Vitreoretinopathy; Eales Disease; traumatic, surgical and environmental disorders, such as Sympathetic Ophthalmia, Uveitic Retinal Disease, Retinal Detachment, Trauma, Retinal Laser, Photodynamic therapy, Photocoagulation, Hypoperfusion During Surgery, Radiation Retinopathy, Bone Marrow Transplant Retinopathy; proliferative disorders, such as Proliferative Vitreal Retinopathy and Epiretinal Membranes; infectious disorders, such as Ocular Histoplasmosis, Ocular Toxocariasis, Presumed Ocular Histoplasmosis Syndrome (POHS), Endophthalmitis, Toxoplasmosis, Retinal Diseases Associated with HIV Infection, Choroidal Disease Associate with HIV Infection, Uveitic Disease Associate with HIV Infection, Viral Retinitis, Acute Retinal Necrosis, Progressive Outer Retinal Necrosis, Fungal Retinal Diseases, Ocular Syphilis, Ocular Tuberculosis, Diffuse Unilateral Subacute Neuroretinitis, Myiasis; genetic disorders, such as Retinitis Pigmentosa, Systemic Disorders with Accosiated Retinal Dystrophies, Congenital Stationary Night Blindness, Cone Dystrophies, Stargardt's Disease And Fundus Flavimaculatus, Best's Disease, Pattern Dystrophy of the Retinal Pigmented Epithelium, X-Linked Retinoschisis, Sorsby's Fundus Dystrophy, Benign Concentric Maculopathy, Bietti's Crystalline Dystrophy, pseudoxanthoma elasticum; retinal injuries, such as Macular WO 2005/020982 PCT/US00/02077 00 SHole, Giant Retinal Tear; retinal tumors, such as Retinal Disease Associated With Tumors, Congenital Hypertrophy Of The RPE, Posterior Uveal Melanoma, Choroidal Hemangioma, Choroidal Osteoma, Choroidal Metastasis, Combined Hamartoma of the Retina and Retinal Pigmented Epithelium, Retinoblastoma, Vasoproliferative Tumors of the Ocular Fundus, Retinal Astrocytoma, and Intraocular Lymphoid CTumors.

17- [0112] VII. Neurological conditions and intracellular nitric oxide 00 O0 [0113] The present invention further provides a method for treating a neurological condition associated with abnormal levels of nitric oxide by administering an effective amount of a selective persistent sodium current antagonist having at least selectivity for persistent sodium current relative to transient sodium current. As disclosed herein, selective persistent sodium current antagonists are useful for selectively reducing persistent sodium current, thereby providing a neuroprotective benefit for acute and chronic neuronal insults. Such antagonists also are useful for reducing deleterious cellular effects resulting from inappropriately high levels of intracellular nitric oxide, and therefore, can effectively treat conditions characterized by aberrant levels of intracellular nitric oxide. As used herein, the term "condition characterized by aberrant levels of intracellular nitric oxide" means a disorder characterized by amounts of nitric oxide in the cells of an individual, that are increased compared to normal amounts of nitric oxide. Such excessive amounts of nitric oxide can result, for example, from excess or unregulated synthesis of nitric oxide.

[0114] Nitric oxide is a free radical gas that functions as a signaling molecule in at least three systems: white blood cells, where nitric oxide mediates tumoricidal and bactericidal effects; blood vessels, where it represents endothelium-derived relaxing factor activity, and in neurons, where it functions much like a neurotransmitter. In addition to its normal role in neurons, nitric oxide can also function as a neurotoxic mediator under pathophysiological conditions. For example, mice having a deletion of the nitric oxide synthase gene were found to be resistant to focal and transient global ischemia, see, N. Panahian et al., Attenuated Hippocampal Damage After Global Cerebral Ischemia in Mice Mutant in Neuronal Nitric Oxide Synthase, 72(2) NEUROSCIENCE 343-354 (1996), which is hereby incorporated by reference in its WO 2005/020982 PCT/US2004/028077 00

O

O

tJb entirety. Therefore, without wishing to be bound by the following, nitric oxide can cause neuronal death by activating persistent sodium channels and causing intracellular calcium overload. As non-limiting examples, conditions characterized by aberrant levels of intracellular nitric oxide include vascular shock, stroke, diabetes, neurodegeneration, asthma, arthritis and chronic inflammation, see, Nobuyuki cMiyasaka Yukio Hirata, Nitric Oxide and Inflammatory Arthritides, 61(21) LIFE SCl.

FN- 2073-2081 (1997); Juan P. Bolanos Angeles Almeida, Roles of Nitric Oxide in Brain Hypoxia-lschemia, 1411(2-3) BIOCHIM. BIOPHYS. ACTA. 415-436 (1999); Joel E.

00 Barbato Edith Tzeng Nitric Oxide and Arterial Disease 40(1) J. VASC. SURG. 187- 193 (2004); Kevin J. Barnham at al., Neurogegenerative diseases and Oxidative Stress, 3(3) NAT. REV. DRUG. DIS. 205-214 (2004); Hossein A. Ghofrani et al., Nitric Oxide Pathway and Phosphodiesterase Inhibitors in Pulmonary Arterial Hypertension, 43(12 Suppl. S) J. AM. COLL. CARDIOL. 68S-72S (2004); Maria A. Moro et al., Role of Nitric Oxide after Brain Ischaemia, 36(3-4) CELL CALCIUM 265-275 (2004); S A. Mulrennan A. E. Redington, Nitric Oxide Synthase Inhibition: Therapeutic Potential in Asthma, 3(2) TREAT. RESPIR. MED. 79-88 (2004); Fabio L. M.

Ricciardolo et al., Nitric Oxide in Health and Disease of the Respiratory System, 84(3) PHYSIOL. REV. 731-765 (2004); and Sharma S. Prabhakar, Role of Nitric Oxide in Diabetic Nephropathy, 24(4) SEMIN. NEPHROL. 333-344 (2004), which are hereby incorporated by reference in their entirety.

[0115] Nitric oxide can cause neurodegeneration and neurotoxicity via voltage-gated sodium channels, see, Garthwaite et al, supra, (1999). For example in the optic nerve, the neurodestructive effects of nitric oxide donors were shown to be ameliorated by compounds that block voltage-gated Na channels such as TTX, see, Gita Garthwaite et al, Nitric Oxide Toxicity in CNS White Matter: An in Vitro Study Using Rat Optic Nerve, 109(1) NEUROSCIENCE 145-155 (2000a); and Gita Garthwaite et al., Soluble Guanylyl Cyclase Activator YC-1 Protects White Matter Axons From Nitric Oxide Toxicity and Metabolic Stress, Probably Through Na(+) Channel Inhibition, 61(1) MOL. PHARMACOL. 97-104 (2000b), which are hereby incorporated by reference in their entirety. Thus blocking voltage-gated Na channels would appear to be a protective strategy against the injurious effects of nitric oxide toxicity. However, the normal rapidly inactivating Na channels do not appear to be the targets for this strategy. For example, increases in either endogenous or exogenous levels of intracellular nitric oxide generate aberrant WO 2005/020982 PCT/US2004/028077 00

O

O

persistent sodium currents in central neurons and cardiac cells, see, Anna K. M.

Hammarstr6m Peter W. Gage, Nitric Oxide Increases Persistent Sodium Current in Rat Hippocampal Neurons, 520(2) J. PHYSIOL. 451-461 (1999); and Gerard P. Ahern et al., Induction of Persistent Sodium Current by Exogenous and Endogenous Nitric Oxide, 275(37) J. BIOL. CHEM. 28810-28815 (2000), which are hereby incorporated Sby reference in their entirety. This up-regulation of persistent sodium current by nitric I oxide is independent of guanylate cyclase and thus independent of cGMP formation, O see, Ahern et al., supra, (2000). As such, nitric oxide may contribute to 00 neurodestruction or neurodegeneration via activating or increasing persistent sodium current. Blocking persistent sodium current upregulated by nitric oxide should therefore prevent cellular Na and subsequent Ca 2 4 overload associated with neuronal cell death under pathophysiological conditions where this current plays a role. Thus blocking the persistent sodium current in neurons may afford a neuroprotective benefit in the treatment acute and chronic neuronal insults including neurodegenerative diseases where nitric oxide is thought to play a neurodestructive role.

[0116] An additional advantage of targeting the persistent sodium channel/current is that it appears to be the final common effector in the neurodestructive pathway caused by nitric oxide. For example, activation of NMDA receptors under excitotoxic conditions results in excess nitric oxide production, see, Strijbos et al, supra, (1996) that then up-regulates persistent sodium currents, see, Garthwaite et al, supra, (2000b). Activation of persistent sodium channels would then lead to further membrane depolarization (leading to further glutamate release), elevated intracellular Ca2+ (via Ca 2 influx through NMDA receptor channels) and reversal of the sodium/calcium exchanger. Elevation of Ca 2 through the NMDA receptor and reverse sodium/calcium exchange would further exacerbate the situation since additional Ca 2 entry would activate more nitric oxide synthase causing a pernicious cycle of neurodestruction.

[0117] It is understood that conditions characterized by aberrant levels of persistent sodium current or aberrant levels of intracellular nitric oxide can be identified or confirmed using routine methods, including methods described herein. It is also understood that one or more transient sodium currents also can be increased.

Similarly, a level of intracellular nitric oxide in a cell from a subject having a disease WO 2005/020982 PCT/US2004/028077 00

O

O

Sor pathological condition can be compared to a level of intracellular nitric oxide in a cell from a normal or non-diseased subject. An increased level of intracellular nitric oxide can typically be observed in at least one cell type of a subject having a condition characterized by aberrant levels intracellular nitric oxide. Human conditions characterized by aberrant levels of persistent sodium current or aberrant levels of M intracellular nitric oxide in addition to these described herein above can be identified F" by those skilled in the art.

00 [0118] VIII. Chronic pain and Persistent Sodium Current 010 [0119] There is strong evidence that altered voltage-gated sodium channel activity plays a critical role in chronic pain, such as, inflammatory and neuropathic pain, see, Mark D. Baker John N. Wood, Involvement of Na' Channels in Pain Pathways, 22(1) TRENDS PHARMACOL. SCI. 27-31 (2001); John N. Wood et al., Sodium Channels in Primary Sensory Neurons: Relationship to Pain States, 241 NOVARTIS FOUND. SYMP. 159-168 (2002); Josephine Lai et al., The Role of Voltagegated Sodium Channels in Neuropathic Pain, 13(3) CURR. OPIN. NEUROBIOL. 291-297 (2003); Philip LoGrasso Jeffrey McKelvy, Advances in Pain Therapeutics, 7(4) Curr. Opin. Chem. Biol. 452-456 (2003); Phillip J. Birch et al., Strategies to Identify Ion Channel Modulators: Current and Novel Approaches to Target Neuropathic Pain, 9(9) DRUG DISCOV. TODAY 410-418 (2004); and Josephine Lai et al., Voltage-gated sodium channels and hyperalgesia, 44 ANNU. REV. PHARMACOL. TOXICOL. 371-397 (2004), which are hereby incorporated by reference in their entirety. Alterations in sodium channel expression and/or function has a profound effect on the firing pattern of neurons in both the peripheral and central nervous systems. For example, injury to sensory primary afferent neurons often results in rapid redistribution of voltagegated sodium channels along the axon and dendrites and in abnormal, repetitive discharges or exaggerated responses to subsequent sensory stimuli. Such an exaggerated response is considered to be crucial for the incidence of spontaneous pain in the absence of external stimuli that is characteristic of chronic pain. In addition, inflammatory pain is associated with lowered thresholds of activation of nociceptors in the periphery and altered sodium channel function is thought to underlie aspects of this phenomenon. Likewise, neuropathic pain states resulting from peripheral nerve damage is associated with altered sodium channel activity and ectopic action potential propagation.

WO 2005/020982 PCT/US2004/028077 00

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O

[0120] Importantly, sodium channel inhibitors are clinically effective in the treatment of many types of chronic pain. For example, local anesthetics (such as, e.g., lidocaine, mexiletine, tocainide and flecainide) have been reported to provide effective relief in painful diabetic neuropathy, neuralgic pain, lumbar radiculopathies, Scomplex regional pain syndrome Type I and Type II and traumatic peripheral injuries.

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Anticonvulsants (such as, carbamazepine and phenytoin) used as analgesics to Streat chronic pain associated with neuralgic pain, trigeminal neuralgia, diabetic 0 0 neuropathy. Anti-epileptic agents (such as, lamotrigine) are used with trigeminal neuralgia, diabetic neuropathy, postherpetic neuralgia, complex regional pain syndrome Type II and phantom pain. However, the usefulness of available sodium channel blocking drugs is severely limited by their failure to discriminate adequately between sodium channel a subunits. Highly systemic concentration would be associated with devastating side-effects, such as, periodic paralyses in muscle, cardiac arrest due to ventricular fibrillation and delayed cardiac repolarization in the heart, and epilepsy in the central nervous system, see, Baker Wood, supra, (2001); and Lai et al., supra, (2004).

[0121] Recent evidence has revealed that increased activity from a persistent sodium current may be responsible for the underlying basis of chronic pain, see e.g., Cervero Laird, supra, (2003); Black et al., supra, (2004); and Yunru et al., supra, (2004), which are hereby incorporated by reference in their entirety. An example of a sodium channel capable of mediating persistent current is the type III sodium channel Nav1.3. Under pathological pain circumstances, Nav1.3 expression can become upregulated while other sodium channels are concomitantly downregulated. For example, in adult rodents, damage to sensory neurons results in upregulation of Nav1.3 and downregulation of Nay1.8 and Nay1.9, see, Birch et al., supra, (2004), which is hereby incorporated by reference in its entirety. Furthermore, this Nav1.3 upregulation after nerve injury is associated with increased membrane potential oscillations that appear to underlie spontaneous activity, see, Bryan C.

Hains et al., Upregulation of Sodium Channel Na,1.3 and Functional Involvement in Neuronal Hyperexcitability Associated With Central Neuropathic Pain After Spinal Cord Injury, 23(26) J. NEUROSCI. 8881-8892 (2003); and Bryan C. Hains et al., Altered Sodium Channel Expression in Second-Order Spinal Sensory Neurons Contributes to Pain after Peripheral Nerve Injury, 24(20) J. NEUROSCI. 4832-4839 WO 2005/020982 PCT/US2004/02077 00 P (2004), which are hereby incorporated by reference in their entirety. Selective reduction in the expression or activity of sodium channels capable of mediating persistent current relative to any reduction in normal voltage-gated (transient) sodium current can be useful for treating conditions associated with increased persistent sodium current.

IND

[0122] Therefore, chronic pain is an example of a condition associated with Sincreased persistent sodium current. As described herein, a compound that 00 00decreases persistent sodium current without a similar decrease in normal transient sodium current can effectively treat chronic pain without harmful side effects that generally accompany non-selective sodium channel blockers. As disclosed in Example 5, a selective persistent sodium current antagonist can effectively reverse allodynia in an animal model of neuropathic pain. Therefore, based on the identification of selective persistent sodium channel antagonists that have at least fold selectivity for persistent sodium channel relative to transient sodium current, and the demonstration of the effectiveness of treating pain by selectively antagonizing persistent sodium current, the present invention provides a method of treating chronic pain in a mammal by selectively antagonizing persistent sodium current. The method involves administering to the mammal an effective amount of a selective persistent sodium channel antagonist that has at least 20-fold selectivity for persistent sodium current relative to transient sodium current.

[0123] The methods of the invention are useful for treating any of a variety of types of chronic pain, and, as non-limiting examples, pain that is neuropathic, visceral or inflammatory in origin. In particular embodiments, the methods of the invention are used to treat neuropathic pain; visceral pain; post-operative pain; pain resulting from cancer or cancer treatment; fibromyalgia pain, and inflammatory pain.

[0124] As used herein, the term "pain" encompasses both acute and chronic pain.

As used herein, the term "acute pain" means immediate, generally high threshold, pain brought about by injury such as a cut, crush, burn, or by chemical stimulation such as that experienced upon exposure to capsaicin, the active ingredient in chili peppers. The term "chronic pain," as used herein, means pain other than acute pain and includes, without limitation, neuropathic pain, visceral pain, inflammatory pain, headache pain, muscle pain and referred pain. It is understood that chronic pain WO 2005/020982 PCT/US2004/028077 00 0 Soften is of relatively long duration, for example, months or years and can be continuous or intermittent.

(N

[0125] In one embodiment, the methods of the invention are used to treat "neuropathic pain," which, as used herein, means abnormal sensory input by either C the peripheral nervous system, central nervous systems, or both resulting in discomfort. Neuropathic pain typically is long-lasting or chronic and can develop Sdays or months following an initial acute tissue injury. Symptoms of neuropathic pain 0 can involve persistent, spontaneous pain, as well as allodynia, which is a painful response to a stimulus that normally is not painful, hyperalgesia, an accentuated response to a painful stimulus that usually a mild discomfort, such as a pin prick, or hyperpathia, a short discomfort becomes a prolonged severe pain. Neuropathic pain generally is resistant to opioid therapy. Neuropathic pain can be distinguished from nociceptive pain, which is pain caused by the normal processing of stimuli resulting from acute tissue injury. In contrast to neuropathic pain, nociceptive pain usually is limited in duration to the period of tissue repair and usually can be alleviated by available opioid and non-opioid analgesics.

[0126] The methods of the invention are useful for treating both centrally-generated and peripherially-generated neuropathic pain resulting from, without limitation, a trauma or disease of peripheral nerve, dorsal root ganglia, spinal cord, brainstem, thalamus or cortex. Examples of neuropathic pain that can be treated by the methods of the invention include neuralgia, such as, trigeminal neuralgia, postherpetic neuralgia, glossopharyngeal neuralgia, sciatica and atypical facial pain; deafferentation pain syndromes, such as, injury to the brain or spinal cord, poststroke pain, phantom pain, paraplegia, peripheral nerve injuries, brachial plexus avulsion injuries, lumbar radiculopathies and postherpetic neuralgia; complex regional pain syndromes (CRPSs) such as, reflex sympathetic dystrophy (CRPS Type I) and causalgia (CRPS Type II); and polyneuropathic pain, such as, diabetic neuropathy, chemotherapy-induced pain, treatment-induced pain, and postmastectomy syndrome. It is understood that the methods of the invention are useful in treating neuropathic pain regardless of the etiology of the pain. As nonlimiting examples, the methods of the invention can be used to treat neuropathic pain resulting from a peripheral nerve disorder such as neuroma; from nerve compression; from nerve crush or stretch, nerve entrapment or incomplete nerve WO 2005/020982 PCT/US2004/028077 00

O

O

transsection; or from a mononeuropathy or a polyneuropathy. As further non-limiting Sexamples, the methods of the invention are useful in treating neuropathic pain resulting from a disorder such as dorsal root ganglion compression; inflammation of C the spinal cord; contusion, tumor or hemisection of the spinal cord; and tumors or trauma of the brainstem, thalamus or cortex.

O [0127] As indicated above, the methods of the invention can be useful for treating O neuropathic pain resulting from a mononeuropathy, polyneuropathy, complex 00 regional pain syndromes or deafferentation. A neuropathy is a functional disturbance or pathological change in the peripheral nervous system and is characterized c clinically by sensory or motor neuron abnormalities. The term mononeuropathy indicates that a single peripheral nerve is affected, while the term polyneuropathy indicates that several peripheral nerves are affected. Deafferentation indicates a loss of the sensory input from a portion of the body, and can be caused by interruption of either peripheral sensory fibres or nerves from the central nervous system. The etiology of a neuropathy can be known or unknown. Known etiologies include complications of a disease or toxic state such as diabetes, which is the most common metabolic disorder causing neuropathy, or irradiation, ischemia or vasculitis.

Polyneuropathies that can be treated by a method of the invention can result, without limitation, from post-polio syndrome, diabetic neuropathy, alcohol neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia, vitamin deficiencies, chemotherapy, 2',3'-didexoycytidine (ddC) treatment, Guillain-Barr6 syndrome or Fabry's disease. It is understood that the methods of the invention can be used to treat chronic pain of these or other chronic neuropathies of known or unknown etiology.

[0128] The methods of the invention also can used for treating chronic pain resulting from excessive muscle or nerve tension, such as certain types of back pain, such as that resulting from a herniated disc; a bone spur, sciatica, sprains, strains and joint pain. The methods of the invention can further be used for treating chronic pain resulting from activity, such as, as non-limiting examples, long hours of work at a computer, work with heavy objects or heavy machinery, or spending long hours on one's feet, and repetitive motion disorders (RMDs). RMDs are a variety of muscular conditions that can cause chronic pain. RMDs can be caused by overexertion, incorrect posture, muscle fatigue, compression of nerves or tissue, too many uninterrupted repetitions of an activity or motion, or friction caused by an unnatural or WO 2005/020982 PCT/US200/028077 00 awkward motion such as twisting the arm or wrist. Common RMDs occur in the hands, wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, and ankles, however, the hands and arms are most often affected. The methods of the invention can be used to treat chronic pain arising from any type of RMD. The methods of the invention further can be used to treat chronic muscle pain, chronic pain associated with substance abuse or withdrawal, and other types of chronic pain of known or Iunknown etiology.

00 [0129] Similarly, the methods of the invention can be used to treat chronic pain resulting from an inflammatory disorder, for example, from arthritis/connective tissue disorders such as, osteoarthritis, rheumatoid arthritis, juvenile arthritis, gouty arthritis; spondyloarthritis, scleroderma and fibromyalgia; autoimmune diseases such as, Guillain-Barr6 syndrome, myasthenia gravis and lupus erythematosus; inflammation caused by injury, such as a crush, puncture, stretch of a tissue or joint; inflammation caused by infection, such as tuberculosis; or neurogenic inflammation.

[0130] The methods of the invention can also be used to treat visceral pain, such as, functional visceral pain including chronic gastrointestinal inflammations like Crohn's disease, ulcerative colitis, gastritis, irritable bowel syndrome; orangic visceral pain including pain resulting from a traumatic, inflammatory or degenerative lesion of the gut or produced by a tumor impinging on sensory innervation; and treatmentinduced visceral pain, for example, attendant to chemotherapy or radiation therapy.

[0131] The methods of the invention can be used for treating chronic pain resulting from headache, including, without limitation, tension-type headache, migraine headache, cluster headache, hormone headache, rebound headache, sinus headache, and organic headache. The methods of the invention can be used for treating chronic pain resulting infections, such as, Lymes disease, HIV/AIDS and leprosy.

[0132] IX. Selective persistent sodium current blockers [0133] The methods of the invention involve administering a compound that selectively reduces persistent sodium current relative to transient sodium current. As used herein, the term "selective," when used herein in reference to a compound, WO 2005/020982 PCT/US2004/028077 00 2 such as an antagonist, means a compound that, at least one particular dose reduces Z persistent sodium current at least 20-fold more than transient sodium current is reduced. Therefore, a compound that selectively reduces persistent sodium current has at least 20-fold selectively for persistent sodium current relative to transient sodium current, and can have, for example, at least 50-fold selectively for persistent Ssodium current relative to transient sodium current, at least 100-fold, at least 200- ID fold, at least 400-fold, at least 600-fold, or at least 1000-fold selectively for persistent O sodium current relative to transient sodium current.

00 [0134] As used herein, the term "persistent sodium current" means a sodium channel mediated current that is non-transient; that can remain active during prolonged depolarization or that activates at voltage more negative than -60 mV and thus can contribute to hyperexcitability of the neural membrane. Prolonged depolarization refers to depolarization that occurs over a time period greater than the time period during which a transient current typically inactivates. As a non-limiting example, prolonged depolarization can occur within a time period greater than the time period during which the transient current of a sodium channel, such as Navl.2, typically inactivates. Therefore, prolonged depolarization refers to depolarization that persists for at least 0.002 second, such as at least 0.01 second, at least 0.1 second and at least 1 second.

[0135] A compound that selectively reduces persistent sodium current can be, for example, a persistent sodium channel antagonist. As used herein, the term "persistent sodium channel antagonist," means a compound that inhibits or decreases persistent current mediated through a sodium channel by binding to the sodium channel. It is understood that a persistent sodium channel antagonist can act by any antagonistic mechanism, such as by directly binding a persistent sodium channel at the pore entrance, thereby inhibiting movement of ions through the channel, or by binding a channel at another site to alter channel conformation and inhibit movement of ions through the channel. Exemplary selective persistent sodium channel antagonists that represent four structural classes of organic molecules are disclosed herein as Formulas 1, 2, 3 and 4.

[0136] It further is understood that a compound that selectively reduces persistent sodium current can act indirectly, for example, by reducing or down-regulating WO 2005/020982 PCT/US200/028)077 00 expression of a persistent sodium channel, for example, by inactivating a positive ;regulator of transcription or activating a negative regulator of transcription, without a corresponding reduction transient sodium channel; by increasing the expression or ri activity of a molecule that inactivates or reduces persistent sodium channel function, such as a protease, modifying enzyme or other molecule, without a corresponding reduction in transient sodium current; or by decreasing the expression or activity of a INO molecule that transmits a downstream signal from a persistent sodium current without a corresponding reduction in transient sodium current, for example, without 00 substantially altering the downstream signal from a transient sodium channel.

00 [0137] As disclosed herein, structurally unrelated molecules can have at least selectivity for reducing persistent sodium current relative to transient sodium current and, therefore, can be useful in the methods of the invention. For example, such a compound can be a naturally or non-naturally occurring macromolecule, such as a peptide, peptidomimetic, nucleic acid, carbohydrate or lipid. The compound further can be an antibody, or antigen-binding fragment thereof such as a monoclonal antibody, humanized antibody, chimeric antibody, minibody, bifunctional antibody, single chain antibody (scFv), variable region fragment (Fv or Fd), Fab or F(ab) 2 The compound also can be a partially or completely synthetic derivative, analog or mimetic of a naturally occurring macromolecule, or a small organic or inorganic molecule.

[0138] A selective persistent sodium current antagonist that is a nucleic acid can be, for example, an anti-sense nucleotide sequence, an RNA molecule, or an aptamer sequence. An anti-sense nucleotide sequence can bind to a nucleotide sequence within a cell and modulate the level of expression of a persistent sodium channel gene, or modulate expression of another gene that controls the expression or activity of a persistent sodium channel. Similarly, an RNA molecule, such as a catalytic ribozyme, can bind to and alter the expression of a persistent sodium channel gene, or other gene that controls the expression or activity of a persistent sodium channel.

An aptamer is a nucleic acid sequence that has a three dimensional structure capable of binding to a molecular target, see, Sumedha D. Jayasena, Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics, 45(9) OLIN.

CHEM. 1628-1650 (1999), which is hereby incorporated by reference in its entirety.

As such, an aptamer can serve as a persistent sodium current selective compound.

WO 2005/020982 PCT/US2004/028077 00

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O

[0139] A selective persistent sodium current antagonist that is a nucleic acid also 0can be a double-stranded RNA molecule for use in RNA interference methods. RNA interference (RNAi) is a process of sequence-specific gene silencing by posttranscriptional RNA degradation, which is initiated by double-stranded RNA (dsRNA) homologous in sequence to the silenced gene. A suitable double-stranded RNA S(dsRNA) for RNAi contains sense and antisense strands of about 21 contiguous r"- O nucleotides corresponding to the gene to be targeted that form 19 RNA base pairs, 00 leaving overhangs of two nucleotides at each 3' end (Sayda M. Elbashir et al., Duplexes of 21-nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells, 411(6836) NATURE 494-498 (2001); B. L. Bass, RNA Interference. The Short Answer, 411(6836) NATURE 428-429 (2001); Phillip D. Zamore, RNA Interference: Listening to the Sound of Silence, 8(9) NAT. STRUCT. BIOL. 746-750 (2001), which are hereby incorporated by reference in their entirety. dsRNAs of about 25-30 nucleotides have also been used successfully for RNAi (Anton Karabinos et al., Essential Roles for Four Cytoplasmic Intermediate Filament Proteins in Caenorhabditis elegans Development, 98(14) PROC. NATL. ACAD. Scl. USA 7863- 7868 (2001), which is hereby incorporated by reference in its entirety. dsRNA can be synthesized in vitro and introduced into a cell by methods known in the art.

[0140] A persistent sodium channel selective compound that is an antibody can be, for example, an antibody that binds to a persistent sodium channel and inhibits movement of ions through the channel, or alters the activity of a molecule that regulates persistent sodium current expression or activity, such that sodium current is decreased. It is understood that such a compound binds selectively such that a corresponding reduction in transient sodium current is not affected.

[0141] A persistent sodium channel selective compound that is a small molecule can have a variety of structures. In several embodiments, a compound that selectively reduces persistent sodium current that has at least 20-fold selectivity for reducing persistent sodium current to non-persistent sodium current is an organic molecule represented by a formula shown herein below, or a pharmaceutically acceptable salt, ester, amide, steroisomer or racemic mixture thereof. As disclosed herein in Fig. 1, several identified compounds are selective for persistent sodium current relative to transient sodium current, with selectivities of 32-fold, 38-fold, 110-fold and 453-fold.

WO 2005/020982 PCT/US2004/028077 00

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It is understood that these and other compounds with at least 20-fold selectivity for persistent sodium current relative to transient sodium current, for example, identified C by the methods disclosed herein in Examples 1, 2, 3, 4 and 5 can be useful for treating chronic pain according to a method of the invention.

C [0142] In one embodiment, a compound useful in a method of the invention, or a

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pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture i thereof, has a structure from Formula 1: 00 N /Y-Ar 1 Ar 2 (C)n-N R3 R 1 R [0143] wherein, [0144] Ar 1 is an aryl group; [0145] Ar is an aryl group; [0146] Y is absent or is selected from: N-I [0147] R 1 is selected from hydrogen, Ci-Ce alkyl, aryl, or arylalkyl; [0148] R 2 and R 3 are independently selected from hydrogen, C1-Ce alkyl, aryl, arylalkyl, hydroxy, fluoro, C1-C carbocyclic ring, or C1-C8 heterocyclic ring; [0149] R 4 is selected from hydrogen, C1-C8 alkyl, aryl, or arylalkyl; [0150] R 5 and R 6 are selected from hydrogen, fluoro, C, to Ce alkyl, or hydroxy; [0151] R 7 is selected from hydrogen, C, to C8 alkyl, aryl, or arylalkyl, and [0152] n is an integer of from 1 to 6.

[0153] In one aspect of this embodiment, Ar' is thienyl, or substituted thienyl. For example, the thienyl can be substituted with one or more of halogen, C,-C8 alkyl,

NO

2

CF

3

OCF

3

OCF

2 H, CN, (CR 5 R')cN(R) 2 wherein c is 0 or an integer from 1 to and WO 2005/020982 PCT/US2004/028077

F--

O

00

O

(N

0"0 [0154] In another aspect of this embodiment, Ar 2 is phenyl or substituted phenyl. For example, the phenyl can be substituted with halogen, C1-Ca alkyl, arylalkyl, NO 2

CF

3

OCF

3

OCF

2 H, CN and (CR 5

R

6

N(R

7 2 wherein c is 0 or an integer from 1 to [0155] In another embodiment, a compound useful in a method of the invention, or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture thereof, has a structure from Formula 2:

I

Ar 4 Y _C-X 1 -Ar 3

-NR

8

R

9 11 [0156] wherein, [0157] Ar 3 is an aryl group; [0158] Ar 4 is an aryl group; [0159] X' and Y 1 are independently selected from: 0~o- 0 J-o-L- 0 12 0 ,12

R

0 o 1 0 0 [0160] R 5 and R 6 are independently selected from: hydrogen, fluoro, C1 to Cg alkyl, hydroxy; [0161] R 7 is selected from hydrogen, C1 to Ce alkyl, aryl, arylalkyl; [0162] R 8 and R 9 are selected from hydrogen, C1-C8 alkyl, aryl, arylalkyl, COR 1 2

COCF

3 WO 2005/020982 PCT/US200/028077 00 0 [0163] R'O and R" are selected from hydrogen, halogen, hydroxyl, C-C8 alkyl, aryl, Z arylalkyl; and [0164] R 12 is selected from hydrogen, C1-C8 alkyl, aryl, arylalkyl.

S[0165] In one aspect of this embodiment, Ar 3 can be phenyl or substituted phenyl.

SFor example, the phenyl can be substituted with one or more of halogen, C1-Ca alkyl, O NO 2

CF

3

OCF

3

OCF

2 H, CN, (CRsR 6

)N(R

7 2 wherein c is 0 or an integer from 1 to 00 [0166] In another aspect of this embodiment, Ar 4 is substituted with one or more of halogen, Cl-C8 alkyl, arylalkyl, NO 2

CF

3

OCF

3

OCF

2 H, CN or (CRR 6 )cN(R 7 2 wherein c is 0 or an integer from 1 to [0167] In yet another embodiment, a compound useful in a method of the invention, or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture thereof, has a structure from Formula 3: Ar 6 y 2

Z

2 [0168] wherein, [0169] Ar 5 is an aryl group; [0170] Ar 6 is an aryl group; [0171] X 2 is O, S, or NR1 4 [0172] Y 2 is N or CR 15 [0173] Z 2 is N or CR1 6 [0174] R 5 and R 6 are selected from hydrogen, fluoro, C, to Ca alkyl, hydroxy; WO 2005/020982 PCT/US2004/028077 t1o

;Z

[0175] R 7 is selected from hydrogen, C, to C8 alkyl, aryl, arylalkyl; [0176] R 13 is selected from halogen C 1

-C

8 alkyl, arylalkyl, and (CR'R 6 )cN(R 7 2 [0177] R 14 is selected from hydrogen, halogen,

(CR

5

R

6 )cN(R 7 2 [0178] R' 5 is selected from hydrogen, halogen,

(CR

5

R

6

)N(R

7 2 [0179] R 1 6 is selected from hydrogen, halogen,

(CR

5

R

6 )cN(R 7 2 and [0180] wherein c is 0 or an integer from 1 to C1 to Cs alkyl, CF 3

OCH

3

NO

2 C, to C8 alkyl, CF 3 OCHa, NO 2 C1 to C8 alkyl, CF 3

OCH

3

NO

2 [0181] In one aspect of this embodiment, Ar s is phenyl or substituted phenyl. For example, the phenyl can be substituted with one or more of halogen, C1-C8 alkyl,

NO

2

CF

3

OCF

3

OCF

2 H, CN, or (CR 5

R

6 )cN(R 7 2 wherein c is 0 or an integer from 1 to [0182] In another aspect of this embodiment, Ar 6 is substituted with halogen, CI-CB alkyl, arylalkyl, NO 2

CF

3

OCF

3

OCF

2 H, CN or (CR 5

R

6 )cN(R 7 2 wherein c is 0 or an integer from 1 to [0183] In yet another aspect of this embodiment, Ar 6 is selected from:

N-R

1 3 R

NR

13 [0184] In yet another embodiment, a compound useful in a method of the invention, or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture thereof, has a structure from Formula 4: WO 2005/020982 PCT/US2004/02077 00

O

O

R19

R

18 17

R

21 Ar 7 N Ra r"- 0 [0185] wherein,

(N

00 S[0186] Ar 7 is an aryl group; Ni [0187] Ra is selected from halogen, C1-C8 alkyl, NR2R 23

OR

22 [0188] R and R 6 are selected from hydrogen, fluoro, C, to Ce alkyl, hydroxy; [0189] R 7 is selected from hydrogen, C, to C8 alkyl, aryl, arylalkyl; [0190] R 1 7 and R 18 are independently selected hydrogen, C1-C8 alkyl, aryl, arylalkyl, and hydroxy; [0191] R 1 9 and R 20 are independently selected from hydrogen, halogen, Ci-C8 alkyl, hydroxy, amino, CF 3 [0192] R 21

R

22 and R 2 3 are independently selected from hydrogen, aryl or CI-Cs alkyl; [0193] a is 0 or an integer from 1 to 5, and [0194] m is 0 or and integer from 1 to 3.

[0195] In one aspect of this embodiment, Ar 7 is phenyl or substituted phenyl. For example the phenyl can be substituted with one or more of halogen C,-Ce alkyl,

NO

2 CF3, OCF 3

OCF

2 H, CN, (CR 5

R

6

),N(R

7 2 wherein c is 0 or an integer from 1 to WO 2005/020982 PCT/US2004/0280(77 00 0 [0196] In another aspect of this embodiment, R is amino or

N

H

[0197] In yet another aspect of this embodiment, R 17 is isopropyl; in one c embodiment, R 18 is methyl.

0O CN [0198] Exemplary compounds that are persistent sodium channel antagonists useful in a method of the invention are shown as Formulas 1, 2, 3 and 4. In addition, the compounds shown in Fig. 1 have selectivities for persistent sodium current of 32-fold, 38-fold, 110-fold, and 453-fold, relative to transient sodium current.

[0199] As used herein, the term "alkyl" means a straight-chain, branched or cyclic saturated aliphatic hydrocarbon. For example, an alkyl group can have 1 to 12 carbons, such as from 1 to 7 carbons, or from 1 to 4 carbons. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like. An alkyl group may be optionally substituted with one or more substituents are selected from the group consisting of hydroxyl, cyano, alkoxy, =0,

NO

2 halogen, dimethyl amino, and SH.

[0200] As used herein, the term "alkenyl" means a straight-chain, branched or cyclic unsaturated hydrocarbon group containing at least one carbon-carbon double bond.

For example, an alkenyl group can have 1 to 12 carbons, such as from 1 to 7 carbons, or from 1 to 4 carbons. An alkenyl group can optionally be substituted with one or more substituents. Exemplary substituents include hydroxyl, cyano, alkoxy,

NO

2 halogen, dimethyl amino, and SH.

[0201] As used herein, the term "alkynyl" means a straight-chain, branched or cyclic unsaturated hydrocarbon containing at least one carbon-carbon triple bond. For example, an alkynyl group can have 1 to 12 carbons, such as from 1 to 7 carbons, or from 1 to 4 carbons. An alkynyl group can optionally be substituted with one or more WO 2005/020982 PCT/US2004/028077 00

O

O

i) substituents. Exemplary substituents include hydroxyl, cyano, alkoxy, NO 2 halogen, dimethyl amino, and SH.

[0202] As used herein, the term "alkoxyl" means an "O-alkyl" group.

C [0203] As used herein, the term "aryl" means an aromatic group which has at least

IO

one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups. An aryl group can optionally be substituted with 00 one or more subtituents. Exemplary substituents include halogen, trihalomethyl, O 10 hydroxyl, SH, OH, NO 2 amine, thioether, cyano, alkoxy, alkyl, and amino.

[0204] As used herein, the term "alkaryl" means an alkyl that is covalently joined to an aryl group. The alkyl can be, for example, a lower alkyl.

[0205] As used herein, the term "carbocyclic aryl" means an aryl group wherein the ring atoms are carbon.

[0206] As used herein, the term "heterocyclic aryl" means an aryl group having from 1 to 3 heteroatoms as ring atoms, the remainder of the ring atoms being carbon.

Heteroatoms Include oxygen, sulfur, and nitrogen. Thus, heterocyclic aryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like.

[0207] As used herein, the term "hydrocarbyl" means a hydrocarbon radical having only carbon and hydrogen atoms. For example, an hydrocarbyl radical can have from 1 to 20 carbon atoms, such as from 1 to 12 carbon atoms or from 1 to 7 carbon atoms.

[0208] As used herein, the term "substituted hydrocarbyl" means a hydrocarbyl radical wherein one or more, but not all, of the hydrogen and/or the carbon atoms are replaced by a halogen, nitrogen, oxygen, sulfur or phosphorus atom or a radical including a halogen, nitrogen, oxygen, sulfur or phosphorus atom, e.g. fluoro, chloro, cyano, nitro, hydroxyl, phosphate, thiol, etc.

WO 2005/020982 PCT/US2004/028077 00

O

O

(N

[0209] As used herein, the term "amide" means wherein R' is alkyl, aryl, alkylaryl or hydrogen.As used herein, the term "thioamide" means wherein R' is alkyl, aryl, alkylaryl or hydrogen. As used herein, the term "amine" means a group, wherein R" and are independently selected from the group consisting of alkyl, aryl, and alkylaryl. As used herein, the term "thioether" Smeans wherein R" is alkyl, aryl, or alkylaryl. As used herein, the term "sulfonyl" refers to -S(O) 2 where is aryl, C(CN)=C-aryl, CH 2 CN, alkyaryl, O sulfonamide, NH-alkyl, NH-alkylaryl, or NH-aryl.

00 [0210] X. Screening assays [0211] The ability of a compound to selectively reduce persistent sodium current relative to transient sodium current can be determined using a variety of assays.

Such assays can be performed, for example, in a cell or tissue that expresses an endogenous or recombinantly expressed persistent sodium current, and generally involve determining persistent and transient sodium current prior to and following application of a test compound.

[0212] Methods for measuring sodium current are well known to those skilled in the art, and are described, see, Joseph S. Adorante, Inhibition of Noninactivating Na Channels of Mammalian Optic Nerve as a Means of Preventing Optic Nerve Degeneration Associated with Glaucoma, U.S. Patent No. 5,922,746 (Jul 13, 1999); Bert Sakmann Erwin Neher, SINGLE CHANNEL RECORDING (Plenum Press, 2 nd ed.

1995); and Tsung-Ming Shih et al., High-level Expression and Detection of Ion Channels in Xenopus Oocytes, 529-556 (METHODS IN ENZYMOLOGY: ION CHANNELS PART B, Vol. 293, P. Michael Conn ed., Academic Press 1998), which are hereby incorporated by reference in their entirety. These protocols are routine procedures well within the scope of one skilled in the art and from the teaching herein (see, e.g., Examples 1, 2, 3, 4 and Since the rate at which sodium currents open and close is rapid and the speed at which ions flow through the channel is high, channel function can be studied using an electrophysiological approach, which is capable of measuring the ion flux at the rate of one million ions per second with a millisecond time resolution. In addition, as shown in Examples 1, 2 and 3, a method for identifying a selective persistent sodium channel antagonist or other persistent sodium current antagonist can involve using a fluorescent dye that is sensitive to WO 2005/020982 PCT/US200/028077 00 Schange in cell membrane potential in order to enable optical measurement of cell membrane potential. As disclosed herein below, a compound to be tested is added to a well containing cells that express a sodium channel capable of mediating a persistent sodium current, and express a potassium channel or a sodium/potassium ATPase or both.

Cc

NO

[0213] Methods for measudng membrane potential with voltage-sensitive dyes are (Ni well known to those skilled in the art, and are described, see, lain D. Johnson, 00 0Fluorescent Probes for Living Cells 30(3) HISTOCHEM. J. 123-140 (1998); and IMAGING NEURONS: A LABORATORY MANUAL (Rafael Yuste, et al., eds., Cold Spring Harbor Laboratory Press, 2000). In particular, the example listed below takes advantage of the high temporal and spatial resolution that derives from utilization of fluorescence resonance energy transfer (FRET) in the measurement of membrane potential by voltage-sensitive dyes as described, see, Jesus E. Gonzalez Roger Y. Tsien, Improved Indicators of Cell Membrane Potential That Use Fluorescence Resonance Energy Transfer 4(4) CHEM. BIOL. 269-277 (1997); Roger Y. Tsien Jesus E. Gonzalez, Voltage Sensing by Fluorescence Resonance Energy Transfer, U.S. Patent No. 5661035 (Aug. 26, 1997); Roger Y. Tsien Jesus E.

Gonzalez, Detection of Transmembrane Potentials by Optical Methods, U.S. Patent No. 6342379 (Jan. 29, 2002); Jesus E. Gonzalez Michael P. Maher, Cellular Fluorescent Indicators and Voltage/Ion Probe Reader (VIPR) Tools for Ion Channel and Receptor Drug Discovery, 8(5-6) RECEPTORS CHANNELS 283-295, (2002); and Michael P. Maher Jesus E. Gonzalez, High Throughput Method and System for Screening Candidate Compounds for Activity Against Target Ion Channels, U.S.

Patent No. 6686193 (Feb. 3, 2004), which are hereby incorporated by reference in their entirety.

[0214] In addition, the selectivity of a compound for persistent sodium current versus transient sodium current can be confirmed, as shown in the teaching herein (see, Examples 2 and 3).

[0215] A variety of cell types, including naturally occurring cells and genetically engineered cells can be used in an in vitro assay to detect persistent sodium current.

Naturally occurring cells having non-inactivating sodium current include, for example, several types of neurons, such as squid axon, cerebellar Purkinje cells, neocortical WO 2005/020982 PCT/US2004/028077 00 pyramidal cells, thalamic neurons, CA1 hipppocampal pyramidal cells, striatal neurons and mammalian CNS axons. Other naturally occurring cells having persistent sodium current can be identified by those skilled in the art using methods disclosed herein below and other well known methods. Cells for use in testing a compound for its ability to alter persistent sodium current can be obtained from a Smammal, such as a mouse, rat, pig, goat, monkey or human, or a non-mammal containing a cell expressing a sodium channel capable of mediating persistent O sodium current.

00 [0216] Genetically engineered cells having persistent sodium current can contain, for example, a cDNA encoding a sodium channel capable of mediating a persistent current such as Na,1.3; or can be a cell engineered to have increased expression of a sodium channel capable of mediating a persistent current, decreased expression of a sodium channel mediating a transient current, or both. Recombinant expression is advantageous in providing a higher level of expression of a sodium channel capable of mediating a persistent sodium current than is found endogenously and also allows expression in cells or extracts in which the channel is not normally found. One or more recombinant nucleic acid expression constructs generally contain a constitutive or inducible promoter of RNA transcription appropriate for the host cell or transcription-translation system, operatively linked to a nucleotide sequence that encodes one or more polypeptides of the channel of interest. The expression construct can be DNA or RNA, and optionally can be contained in a vector, such as a plasmid or viral vector. Based on well-known and publicly available knowledge of nucleic acid sequences encoding subunits of many sodium channels, including several sodium channels capable of mediating a persistent sodium current, one skilled in the art can express desired levels of a biologically active persistent or transient sodium channels using routine laboratory methods as described, see, e.g., Molecular Cloning A Laboratory Manual (Joseph Sambrook David W. Russell eds., Cold Spring Harbor Laboratory Press, 3 rd ed. 2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al., eds., John Wiley Sons, 2004), which are hereby incorporated by reference in their entirety. cDNAs for several families of sodium channels have been cloned and sequenced, and are described, see, e.g, Alan L. Goldin, Diversity of Mammalian Voltage-gated Sodium Channels, 868 ANN. N.Y. ACAD. SCI. 38-50 (1999), William A. Catterall, From Ionic Currents to Molecular Mechanisms: The Structure and Function of Voltage-gated Sodium WO 2005/020982 PCT/US2004/028077 00

O

O

Channels, 26(1) NEURON 13-25 (2000); John N. Wood Mark D. Baker, Voltagegated Sodium Channels, 1(1) CURR. OPIN. PHARMACOL. 17-21 (2001); and Frank H.

Yu William A. Catterall, Overview of the Voltage-Gated Sodium Channel Family, 4(3) GENOME BIOL. 207 (2003), which are hereby incorporated by reference in their entirety. In addition, both nucleotide and protein sequences all currently described Ssodium channels are publicly available from the GenBank database (National N Institutes of Health, National Library of Medicine, http://www.ncbi.nlm.nih.gov/), which O is hereby incorporated by reference in its entirety.

00 [0217] Exemplary host cells that can be used to express recombinant sodium channels include isolated mammalian primary cells; established mammalian cell lines, such as COS, CHO, HeLa, NIH3T3, HEK 293-T and PC12; amphibian cells, such as Xenopus embryos and oocytes; and other vertebrate cells. Exemplary host cells also include insect cells such as Drosophila, yeast cells such as S. cerevisiae, S. pombe, or Pichia pastoris and prokaryotic cells (such as E. coli,) engineered to recombinantly express sodium channels.

[0218] XI. Reaction schemes [0219] A compound used in a method of the invention can be synthesized by general synthetic methodology, such as by the specific synthetic reaction schemes and methodologies described below and in Examples 6, 7, 8 and 9. Modifications of these synthetic methodologies will become readily apparent to the practicing synthetic organic chemist in view of the following disclosure and general knowledge available in the art.

[0220] The reaction schemes disclosed below are directed to the synthesis of exemplary compounds used in a method of the invention. The synthetic processes described herein are adaptable within the skill of the practicing organic chemist and can be used with such adaptation for the synthesis of compounds useful in a method of the invention that are not specifically described. Reaction schemes 1, 2, 3 and 4 disclose synthetic routes to compounds having Formulas 1, 2, 3 and 4, respectively.

Examples 6, 7, 8 and 9 describe methodology useful for synthesizing exemplary compounds representative of Formulas 1, 2, 3 and 4, respectively.

WO 2005/020982 PCT/US2004/028077 [0221] The specific reaction conditions described in Examples 6, 7, 8 and 9 are directed to the synthesis of exemplary compounds useful in a method of the invention. Whereas each of the specific and exemplary synthetic methods shown in Examples 6, 7, 8 and 9 describe specific compounds within the scope of general Formulas 1 through 4, the synthetic processes and methods used therein are adaptable within the skill of the practicing organic chemist and can be used with such adaptation for the synthesis of compounds useful in a method of the invention that are not specifically described herein as examples.

[0222] Reaction scheme 1 R2 O Ar 2 -()n-N-S-Ar 1 R3O 0 CI- S-Ar' 0

CH

2

CI

2 Et a

N

02 I o O=C=N-Arl

R

3

CH

2

CI

2

R

2

-NH

1 3 1 0 CI Ar'

CH

2

CI

2 Et 3

N

R2 0 Ar2-(C),-N 11 A, 13 R'l

R

0 R 4

CH

2

CI

2 Et 3

N

R2 A-(C)nNJLN 11 Arl I3Al A4 WO 20051020982 WO 205100982PCT/US2004/028077 [0223] Reaction Scheme 2 0 Ar 4 -1CI H04-A3-NRWR

R"

R12N-} A3-NR9 0 RIO C1 1 r-RR

R'

1

CH

2

CI

2 Et 3

N

0 Ar4 C1

CH

2

CI

2 Et 3

N

Ar4-OH

CH

2

CI

2 Et 3

N

0

RI

Ar4k 0+Ar-NRBR9 0

RI

0 Ar4A N+ r-RR

R'

2

R

1 1 0 RI 0 Ar- -L A3N1R

R"

[0224] Reaction Scheme 3 0 Ar 5 QkEt

NH

2

NH

2

EIOH

0 Ar5- 'NHNH 2

NH

H

2 N)'Ar 6

H

Ar 5 -r

N-N

0 SArk cl Et 3 N, CH 2

CI

2 POC1 3 r>OAr6

N-N

jThiourea, THF p 2 s 5 pyridine As-( s v-Ar6

N-N

WO 2005/020982 PCT/US2004/028077 00

O

O

a'\ [0225] Reaction Scheme 4

R

19

R

1 7

R

20

R

R 0 0

R

2 Ar 7 KOH, DMSO KOH, DMSO

OHC

5O- CH3 R= NH 2 a=1

CH

2

CI

2 Na 2

SO

4 [0226] XII. Animal models [0227] The efficacy of a compound that selectively reduces persistent sodium current, such as a selective persistent sodium current antagonist, in treating a condition characterized by aberrant levels of sodium current or aberrant levels of intracellular nitric oxide in a mammal can be confirmed using a variety of well-known methods. Well-known animal models can be useful for determining the ability of a compound, such as a selective persistent sodium current antagonist, to reduce neuronal death or treat a condition characterized by aberrant levels of sodium current or condition characterized by aberrant levels of intracellular nitric oxide. Ischemia can be induced in several animal species using any of several surgical procedures, which can employ, for example, any of intralumenal occlusions, extralumenal WO 2005/020982 PCT/US2004/028077 00

O

O

occlusions, vascular clips, miniature hydraulic occluders or Ameroid occluders.

SSpecific animal models of ischemia are well known to those skilled in the art, and exemplary ischemia models including rodent, monkey, baboon, dog, gerbil and rabbit models are described, see, Douglas E. McBean Paul A. T. Kelly, Rodent Models of Global Cerebral Ischemia: A Comparison of Two-Vessel Occlusion and SFour-Vessel Occlusion 30(4) GEN. PHARMACOL. 431-434 (1998); E. M. Nemoto, SMonkey Model of Complete Global Ischemia, 24(2) STROKE 328-329 (1993); R. F.

O Spetzler et al., Chronic Reversible Cerebral Ischemia: Evaluation of a New Baboon 00 Model, 7(3) NEUROSURGERY 257-261 (1980); A. Mitro et al., Method of the Development of Irreversible, Complete Cerebral Ischemia in Dog, 21(2) NEUROPATOL. POL. 315-321 (1983); T. Yoshimine T Yanagihara, Regional Cerebral Ischemia by Occlusion of the Posterior Communicating Artery and the Middle Cerebral Artery in Gerbils, 58(3) J. NEUROSURG. 362-367 (1983); R. Pluta, Experimental Treatment with Prostacyclin of Global Cerebral Ischemia in Rabbit-New Data, 28(3-4) NEUROPATOL. POL. 205-215 (1990); J. Guo Y. D. Chao, Modification of a Model for Cerebral Ischemia in the Cat: A New Method to Occlude the Middle Cerebral Artery, 25(1) NEUROSURGERY 49-53 (1989).

[0228] Animal models of neurodegenerative disorders are well known in the art, and include, for example, include Alzheimer's disease models, such as transgenic mice over-expressing mutant forms of amyloid precursor protein and presenilin-1, and models in which animals treated with amyloid 1-peptide or excitotoxins. An exemplary model of Parkinson's disease involves administration of the toxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to animals, such as monkeys and mice, which results in selective loss of substantia nigra dopaminergic neurons and associated motor dysfunction. Neurodegenerative disease models can employ a variety of animals including, but not limited to, mice, gerbils, rats, rabbits, pigs, cats, dogs, sheep and primates, see, Senile Dementia of Alzheimer Type: Early Diagnosis, Neuropathology and Animal Models (J Traber W. H. Gispen, eds., Springer Verlag, 1985); CENTRAL NERVOUS SYSTEM DISEASES: INNOVATIVE ANIMAL MODELS FROM LAB TO CLINIC (Dwaine F. Emerich et al., eds., Humana Press, 1999).

[0229] End-points useful for assessing the effect of a compound that selectively reduces persistent sodium current, such as a selective persistent sodium current WO 2005/020982 PCT/US200/02r077 00 0 )antagonist, on the extent of neuronal death or dysfunction in comparison to a control animal that has not received the compound depend, in part, on the condition to be treated and are well known to those skilled in the art. Such end points included, for example, reduction in lesion size, improved physiological function, and improved behavior.

S[0230] The activity of a compound that selectively reduces persistent sodium current, O such as a selective persistent sodium current antagonist, also can be confirmed in a 00 cell-based model of neuronal damage. Such a cell-based model can provide another read-out for the activity of an antagonist prior to its use in an animal model, and can also be used to identify antagonists or other compounds useful for reducing death of cultured neurons. Exemplary cell-based assays include cell models of ischemiainduced neuronal damage in which neurons demonstrate one or more indicia of apoptosis in response to a substance or condition, such as hypoxia, glucose deprivation, oxidative or excitotoxic insult. Exemplary cell-based models of ischemiainduced neuronal damage are known to those of skill in the art and are described, for example, in Lalitha Tenneti et al., Role of Caspases in N-Methyl-D-Aspartate-lnduced Apoptosis in Cerebrocortical Neurons, 71(3) J. NEUROCHEM. 946-959 (1998); and R.

James White lan J. Reynolds, Mitochondrial Depolarization in Glutamate- Stimulated Neurons: An Early Signal Specific to Excitotoxin Exposure, 16(18) J.

NEUROSCI. 5688-5697 (1996).

[0231] The ability of a compound that selectively reduces persistent sodium current, such as a selective persistent sodium current antagonist, to reduce neuronal death or dysfunction can be assessed by analyzing an observable sign or symptom of nerve cell destruction in the presence and absence of treatment with the compound.

Initiation of apoptotic death of neurons can have observable effects on cell function and morphology, as well as observable effects on tissues, organs and animals that contain dysfunctional or apoptotic neurons. Therefore, an indicator of neuronal damage can include observable parameters of molecular changes, such as increased expression of apoptosis-induced genes; cell function changes, such as reduced mitochondrial functions; cell morphological changes, such as cell shrinkage and blebbing; organ and tissue functional and morphological changes, such as the presence of an infarct or other lesion, the severity of which can be assessed by parameters including lesion volume and lesion size; physiological changes in animal WO 2005/020982 PCT/US2004/028077 00 I models, including functional changes, such as loss of motor function, increased Z mortality and decreased survival, and behavioral changes, such as onset of dementia or loss of memory.

[0232] A reduction in an indicator of neuronal damage can be assessed in a cell, M tissue, organ or animal by comparing an indicator of neuronal damage in at least two states of a cell, tissue, organ or animal. Thus, a reduction in an indicator of neuronal damage can be expressed relative to a control condition. A control condition can be, 00 for example, a cell, tissue, organ or animal prior to treatment, in the absence of treatment, in the presence of a different treatment, in a normal animal or another condition determined to be appropriate by one skilled in the art.

[0233] XIII. Pain models [0234] The ability of a compound that selectively reduces persistent sodium current relative to transient sodium current to treat chronic pain in a mammal can be confirmed using a variety of well-known assays. Such essays include, but are not limited to, the Mouse Writhing Assay, the Tail Flick Assay, the Sciatic Nerve Ligation assay, the Formalin Test and the Dorsal Root Ganglia Ligation assay.

[0235] An accepted standard for detecting and comparing the analgesic activity of different classes of analgesic compounds for which there is a correlation with human analgesic activity is the prevention of acetic acid induced writhing in mice, see, e.g., R. Koster et al., Acetic Acid for Analgesic Screening, 18 FED. PROC. 412-416 (1959).

In the Mouse Writhing Assay, mice are treated with various doses of a test compound or vehicle, followed by intraperitoneal injection with a standard challenge dose of acetic acid 5 minutes prior to a designated observation period. The acetic acid can be prepared as a 0.55% solution and injected at a volume of 0.1 ,grams of body weight. For scoring purposes a "writhe" is indicated by whole body stretching or contracting of the abdomen during an observation period beginning about five minutes after the administration of acetic acid.

[0236] Another model that has been used to define or monitor analgesic levels following exposure to a variety of compounds is the Tail Flick Assay, see, e.g., William L. Dewey et al., The Effect of Narcotics and Narcotic Antagonists on the Tail- WO 2005/020982 PCT/US2004/028077 00

O

O

Flick Response in Spinal Mice, 21(8) J. PHARM. PHARMACOL. 548-550 (1969). In this assay, an apparatus can be used to test mice, rats or monkeys by focusing a beam of light on the tail and evaluating latency to tail-flick. This test has proven useful for screening weak and strong analgesics. In the Tail flick Assay, mice are treated with various doses of a test compound or vehicle. At a selected time point after Sadministration, mice are placed in a holding tube and the time required for each Smouse to react (tail flick) to the heat from a beam of light focused on the tail is O recorded on a Tail Flick Apparatus (Columbus Instruments, Columbus, OH).

00 [0237] An accepted model for assessment of neuropathic pain analgesia is the Chung model of peripheral neuropathic pain, see, Sun H. Kim Jin M. Chung, An Experimental Model for Peripheral Neuropathy Produced by Segmental Spinal Nerve Ligation in the Rat, 50(3) PAIN 355-363 (1992), which is hereby incorporated by reference in its entirety. The Chung model is a selective spinal neurectomy model that involves introducing partial nerve injury by performing a spinal nerve ligation procedure. These protocols for this procedure are routine and well within the scope of one skilled in the art and from the teaching herein (see, Example [0238] Another accepted model for assessment of neuropathic pain analgesia is the Sciatic Nerve Ligation model, see, Gary J. Bennett and Yi-Kuan Xie, A Peripheral Mononeuropathy in Rat That Produces Disorders of Pain Sensation Like Those Seen in Man, 33(1) PAIN 87-107 (1988); and Youn-Woo Lee et al., Systemic and Supraspinal, but not Spinal, Opiates Suppress Allodynia in a Rat Neuropathic Pain Model, 199(2) NEUROSCI. LETT. 186:111-114 (1995), which are hereby incorporated by reference in their entirety. In the Sciatic Nerve Ligation model, rats are anesthetized and a nerve ligation procedure performed. The common sciatic nerve is exposed and 4 ligatures are tied loosely around it with about 1 mm spacing.

One day to 10 weeks after surgery, nociceptive testing is performed. Responses to noxious heat are determined by placing the rats in a chamber with a clear glass floor and aiming at the plantar surface of the affected foot a radiant heat source from beneath the floor. Increased latency to withdraw the hindpaw is demonstrative of analgesic activity. Responses to normally innocuous mechanical stimuli are determined by placing the rats in a chamber with a screen floor and stimulating the plantar surface of the hind paw with graduated von Frey hairs which are calibrated by the grams of force required to bend them. Rats with sciatic nerve ligation respond to WO 2005/020982 PC3T/US200/0281077 00 0 lower grams of mechanical stimulation by reflexive withdrawal of the foot than unoperated rats, demonstrating allodynia. An increase in the grams of mechanical 0 force required to produce foot withdrawal is demonstrative of anti-allodynic activity.

[0239] The Formalin Test is a well accepted model of inflammatory pain, see, e.g., SAnnika B. Malmberg Tony L. Yaksh, Antinociceptive Actions of Spinal Nonsteroidal ND Anti-Inflammatory Agents on the Formalin Test in the Rat, 263(1) J. PHARMACOL.

O EXP. THER. 136-146 (1992). Rats are anesthetized, and, following a loss of 00 spontaneous movement, they are injected subcutaneously in the dorsal surface of the hindpaw with 50 microliters of 5% formalin solution using a 30 gauge needle.

Rats are then individually placed in an open Plexiglas chamber for observation, and within a maximum interval of 1 to 2 minutes, the animals display recovery from anesthesia with spontaneous activity and normal motor function. Pain behavior is quantified by periodically counting the incidents of spontaneous flinching/shaking of the injected paw. The flinches are counted for 1-minute periods at 1- to 5- to 6and 5 minute intervals during the interval from 10 to 60 minutes. Inhibition of the flinching/shaking of the injected paw is demonstrative of an analgesic activity.

[0240] Using any of these assays, those skilled in the art recognize that EDso values and their standard errors of the mean can be determined using accepted numerical methods, see, Roger E. Kirk, EXPERIMENTAL DESIGN: PROCEDURES FOR THE BEHAVIORAL SCIENCES, (Wadsworth Publishing, 3 rd ed. 1994), which is hereby incorporated by reference in its entirety. One skilled in the art understands that any of the above or other well known models of pain can be useful for corroborating that a selective persistent sodium current antagonist, including a selective persistent sodium channel antagonist, is useful for treating chronic pain.

[0241] XIV. Pharmaceutical compositions [0242] As disclosed herein a selective persistent sodium current antagonist is administered to a mammal to treat a condition characterized by aberrant levels of sodium current or aberrant levels of intracellular nitric oxide. As used herein, the term "treating," when used in reference to administering to a mammal an effective amount of a selective persistent sodium current antagonist, means reducing a symptom of a condition characterized by aberrant levels of sodium current or WO 2005/020982 PCT/US2004/028077 00 aberrant levels of intracellular nitric oxide, or delaying or preventing onset of a ;symptom of a condition characterized by aberrant levels of sodium current or aberrant levels of intracellular nitric oxide in the mammal. For example, the term "treating" can mean reducing a symptom of a condition characterized by aberrant levels of sodium current or aberrant levels of intracellular nitric oxide by at least 60%, 70%, 80%, 90% or 100%. The effectiveness of a selective persistent Isodium current antagonist in treating a condition characterized by aberrant levels of r"- O sodium current or aberrant levels of intracellular nitric oxide can be determined by 00 observing one or more clinical symptorfis or physiological indicators associated with the condition. An improvement in a condition characterized by aberrant levels of sodium current or aberrant levels of intracellular nitric oxide also can be indicated by a reduced need for a concurrent therapy. Those of skill in the art will know the appropriate symptoms or indicators associated with specific conditions and will know how to determine if an individual is a candidate for treatment with a selective persistent sodium current antagonist. In particular, it is understood that those skilled in the art will be able to determine if a condition if characterized by aberrant persistent sodium current, for example, by comparison of levels of persistent sodium channel expression or activity in cells from the individual with a normal control cells.

[0243] As disclosed herein, a selective persistent sodium current antagonist is administered to a mammal to treat chronic pain. As used herein, the term "treating chronic pain," when used in reference to administering to a mammal an effective amount of a selective persistent sodium current antagonist, means reducing a symptom of chronic pain, or delaying or preventing onset of a symptom of chronic pain in the mammal. For example, the term "treating chronic pain" can mean reducing a symptom of chronic pain by at least 30%, 40%, 60%, 70%, 80%, 90% or 100%. The effectiveness of a selective persistent sodium current antagonist in treating chronic pain can be determined by observing one or more clinical symptoms or physiological indicators associated with pain. For example, a reduction in chronic pain can include an arrest or a decrease in clinical symptoms of chronic pain or physiological indicators associated with chronic pain. A reduction in chronic pain also can be indicated by a reduced need for a concurrent therapy for chronic pain, such as reduced need for analgesic therapy, TENS, counterirritation, trigger point injection, spray and stretch, or physical therapy. Those of skill in the art will know the appropriate symptoms or indicators associated with specific types of chronic pain and WO 2005/020982 PCT/US004/028077 00 0

(N

0 will know how to determine if an individual is a candidate for treatment with a selective persistent sodium current antagonist.

[0244] The appropriate effective amount to be administered for a particular application of the methods can be determined by those skilled in the art, using the guidance provided herein. For example, an effective amount can be extrapolated N from in vitro and in vivo assays as described herein above. One skilled in the art will 0 recognize that the condition of the patient can be monitored throughout the course of 00 therapy and that the effective amount of a selective persistent sodium current 010 antagonist that is administered can be adjusted accordingly.

[0245] The invention also can be practiced by administering an effective amount of persistent sodium current antagonist together with one or more other agents including, but not limited to, one or more analgesic agents. In such "combination" therapy, it is understood that the antagonist can be delivered independently or simultaneously, in the same or different pharmaceutical compositions, and by the same or different routes of administration as the one or more other agents.

[0246] Exemplary compounds that have at least 20-fold selectivity for reducing persistent sodium current relative to non-persistent sodium current include those shown in Formulas 1, 2, 3 and 4. Also encompassed by the invention are pharmaceutically acceptable salts, esters and amides derived from Formulas 1, 2, 3 or 4. Suitable pharmaceutically acceptable salts of the antagonists useful in the invention include, without limitation, acid addition salts, which can be formed, for example, by mixing a solution of the antagonist with a solution of an appropriate acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

Where an antagonist carries an acidic moiety, suitable pharmaceutically acceptable salts thereof can include alkali salts such as sodium or potassium salts; alkaline earth salts such as calcium or magnesium salts; and salts formed with suitable organic ligands, for example, quaternary ammonium salts. Representative pharmaceutically acceptable salts include, yet are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, WO 2005/020982 PCT/US2004/028077 00

O

O

hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, Nmethylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, Ssulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide and I valerate.

00 [0247] Thus, it is understood that the functional groups of antagonists useful in the invention can be modified to enhance the pharmacological utility of the compound.

Such modifications are well within the knowledge of the skilled chemist and include, without limitation, esters, amides, ethers, N-oxides, and pro-drugs of the indicated antagonist. Examples of modifications that can enhance the activity of an antagonist include, for example, esterification such as the formation of C1 to C6 alkyl esters, such as C1 to C4 alkyl esters, wherein the alkyl group is a straight or branched chain.

Other acceptable esters include, for example, C5 to C7 cycloalkyl esters and arylalkyl esters such as benzyl esters. Such esters can be prepared from the compounds described herein using conventional methods well known in the art of organic chemistry.

[0248] Other pharmaceutically acceptable modifications include the formation of amides. Useful amide modifications include, for example, those derived from ammonia; primary C1 to C6 dialkyl amines, where the alkyl groups are straight or branched chain; and arylamines having various substitutions. In the case of secondary amines, the amine also can be in the form of a 5- or 6-member ring.

Methods for preparing these and other amides are well known in the art.

[0249] It is understood that, where an antagonist useful in the invention has at least one chiral center, the antagonist can exist as chemically distinct enantiomers. In addition, where an antagonist has two or more chiral centers, the compound exists as diastereomers. All such isomers and mixtures thereof are encompassed within the scope of the indicated antagonist. Similarly, where an antagonist possesses a structural arrangement that permits the structure to exist as tautomers, such tautomers are encompassed within the scope of the indicated antagonist.

Furthermore, in crystalline form, an antagonist can exist as polymorphs; in the WO 2005/020982 PCT/US2004028077 00 presence of a solvent, an antagonist can form a solvate, for example, with water or a Z common organic solvent. Such polymorphs, hydrates and other solvates also are encompassed within the scope of the indicated antagonist as defined herein.

[0250] A selective persistent sodium current antagonist or other compound useful in Sthe invention generally is administered in a pharmaceutical acceptable composition.

IAs used herein, the term "pharmaceutically acceptable" refer to any molecular entity O or composition that does not produce an adverse, allergic or other untoward or 00 unwanted reaction when administered to a human or other mammal. As used herein, the term "pharmaceutically acceptable composition" refers to a therapeutically effective concentration of an active ingredient. A pharmaceutical composition may be administered to a patient alone, or in combination with other supplementary active ingredients, agents, drugs or hormones. The pharmaceutical compositions may be manufactured using any of a variety of processes, including, without limitation, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, and lyophilizing. The pharmaceutical composition can take any of a variety of forms including, without limitation, a sterile solution, suspension, emulsion, lyophilizate, tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosage form suitable for administration.

[0251] It is also envisioned that a pharmaceutical composition disclosed in the present specification can optionally include a pharmaceutically acceptable carriers that facilitate processing of an active ingredient into pharmaceutically acceptable compositions. As used herein, the term "pharmacologically acceptable carrier" refers to any carrier that has substantially no long term or permanent detrimental effect when administered and encompasses terms such as "pharmacologically acceptable vehicle, stabilizer, diluent, auxiliary or excipient." Such a carrier generally is mixed with an active compound, or permitted to dilute or enclose the active compound and can be a solid, semi-solid, or liquid agent. It is understood that the active ingredients can be soluble or can be delivered as a suspension in the desired carrier or diluent.

Any of a variety of pharmaceutically acceptable carriers can be used including, without limitation, aqueous media such as, distilled, deionized water, saline; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic and absorption delaying agents; or any other inactive ingredient. Selection of a pharmacologically acceptable carrier can depend on the mode of administration.

WO 2005/020982 PCTUS004028077 00 0 j) Except insofar as any pharmacologically acceptable carrier is incompatible with the Sactive ingredient, its use in pharmaceutically acceptable compositions is Scontemplated. Non-limiting examples of specific uses of such pharmaceutical carriers can be found in PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (Howard C. Ansel et al., eds., Lippincott Williams Wilkins Publishers, 7' t Sed. 1999); REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R.

N Gennaro ed., Lippincott, Williams Wilkins, 20 h ed. 2000); GOODMAN GILMAN'S O THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Joel G. Hardman et al., eds., 00 McGraw-Hill Professional, 10 t ed. 2001); and HANDBOOK OF PHARMACEUTICAL EXCIPIENTS (Raymond C. Rowe et al., APhA Publications, 4 th edition 2003) which are hereby incorporated by reference in their entirety. These protocols are routine procedures and any modifications are well within the scope of one skilled in the art and from the teaching herein.

[0252] It is further envisioned that a pharmaceutical composition disclosed in the present specification can optionally include, without limitation, other pharmaceutically acceptable components, including, without limitation, buffers, preservatives, tonicity adjusters, salts, antioxidants, physiological substances, pharmacological substances, bulking agents, emulsifying agents, wetting agents, sweetening or flavoring agents, and the like. Various buffers and means for adjusting pH can be used to prepare a pharmaceutical composition disclosed in the present specification, provided that the resulting preparation is pharmaceutically acceptable. Such buffers include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline and borate buffers. It is understood that acids or bases can be used to adjust the pH of a composition as needed. Pharmaceutically acceptable antioxidants include, without limitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

Useful preservatives include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate and a stabilized oxy chloro composition, for example, PURITE. Tonicity adjustors useful in a pharmaceutical composition include, without limitation, salts such as, sodium chloride, potassium chloride, mannitol or glycerin and other pharmaceutically acceptable tonicity adjustor. The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in WO 2005/020982 PCTIUS2004/028077 00 aqueous or other protonic solvents than are the corresponding free base forms. It is ;understood that these and other substances known in the art of pharmacology can be included in a pharmaceutical composition useful in the invention.

[0253] An antagonist useful in a method of the invention is administered to a Smammal in an effective amount. Such an effective amount generally is the minimum IND dose necessary to achieve the desired therapeutic effect, which can be, for example, r'that amount roughly necessary to reduce the discomfort caused by the pain to 00 tolerable levels or to achieve a significant reduction in pain, or, as another example, that amount roughly necessary to reduce the symptoms associated with a neurological disorder, such as, epilepsy, cerebral hypoxia, cardiac ischemia, multiple sclerosis and amyotrophic lateral sclerosis. For example, the term "effective amount' when used with respect to treating a neurological disorder or chronic pain can be a dose sufficient to reduce pain, for example, by at least 30%, 40%, 60%, 70%, 80%, 90% or 100%. Such a dose generally is in the range of 0.1-1000 mg/day and can be, for example, in the range of 0.1-500 mg/day, 0.5-500 mg/day, 0.5-100 mg/day, 0.5-50 mg/day, 0.5-20 mg/day, 0.5-10 mg/day or 0.5-5 mg/day, with the actual amount to be administered determined by a physician taking into account the relevant circumstances including the severity of the neurological disorder or chronic pain, the age and weight of the patient, the patient's general physical condition, the cause of the neurological disorder or chronic pain and the route of administration. Where repeated administration is used, the frequency of administration depends, in part, on the half-life of the antagonist. Suppositories and extended release formulations can be useful in the invention and include, for example, dermal patches, formulations for deposit on or under the skin and formulations for intramuscular injection. It is understood that slow-release formulations also can be useful in the methods of the invention. The subject receiving the selective persistent sodium channel antagonist can be any mammal or other vertebrate capable of experiencing chronic pain, for example, a human, primate, horse, cow, dog, cat or bird.

[0254] Various routes of administration can be useful for treating a neurological disorder or chronic pain according to a method of the invention. A pharmaceutical composition useful in the methods of the invention can be administered to a mammal by any of a variety of means depending, for example, on the type and location of the WO 2005/020982 PCT/US2004/028077 00 neurological disorder or chronic pain to be treated, the antagonist or other compound Z to be included in the composition, and the history, risk factors and symptoms of the subject. Routes of administration suitable for the methods of the invention include both systemic and local administration. As non-limiting examples, a pharmaceutical composition useful for treating a neurological disorder or chronic pain can be Sadministered orally or by subcutaneous pump; by dermal patch; by intravenous, IN subcutaneous or intramuscular injection; by topical drops, creams, gels or ointments; as an implanted or injected extended release formulation; as a bioerodible or non- 00 bioerodible delivery system; by subcutaneous minipump or other implanted device; by intrathecal pump or injection; or by epidural injection. An exemplary list of biodegradable polymers and methods of use are described in, Heller, Biodegradable Polymers in Controlled Drug Delivery (CRC CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS, Vol. 1. CRC Press, 1987); Vernon G. Wong, Method for Reducing or Preventing Transplant Rejection in the Eye and Intraocular Implants for Use Therefor, U.S. Patent No. 6,699,493 (Mar. 2, 2004); Vernon G.

Wong Mae W. L. Hu, Methods for Treating Inflammation-mediated Conditions of the Eye, U.S. Patent No. 6,726,918 (Apr. 27, 2004); David A. Weber et al., Methods and Apparatus for Delivery of Ocular Implants, U.S. Patent Publication No. US2004/ 20040054374 (Mar. 18, 2004); Thierry Nivaggioli et al., Biodegradable Ocular Implant, U.S. Patent Publication No. US2004/ 0137059 (Jul. 15, 2004), which are hereby incorporated by reference in their entirety. It is understood that the frequency and duration of dosing will be dependent, in part, on the relief desired and the half-life of the selective persistent sodium current antagonist.

[0255] In particular embodiments, a method of the invention is practiced by peripheral administration of a selective persistent sodium current antagonist. As used herein, the term "peripheral administration" or "administered peripherally" means introducing an agent into a subject outside of the central nervous system.

Peripheral administration encompasses any route of administration other than direct administration to the spine or brain. As such, it is clear that intrathecal and epidural administration as well as cranial injection or implantation are not within the scope of the term "peripheral administration" or "administered peripherally." It further is clear that some selective persistent sodium current antagonists can cross the blood-brain barrier and, thus, become distributed throughout the central and peripheral nervous systems following peripheral administration.

WO 2005/02)982 PCT/US2004/0280077 00 ;Z [0256] Peripheral administration can be local or systemic. Local administration results in significantly more of a pharmaceutical composition being delivered to and about the site of local administration than to regions distal to the site of administration. Systemic administration results in delivery of a pharmaceutical Scomposition to essentially the entire peripheral nervous system of the subject and ID may also result in delivery to the central nervous system depending on the properties O of the composition.

00 [0257] Routes of peripheral administration useful in the methods of the invention encompass, without limitation, oral administration, topical administration, intravenous or other injection, and implanted minipumps or other extended release devices or formulations. A pharmaceutical composition useful in the invention can be peripherally administered, for example, orally in any acceptable form such as in a tablet, liquid, capsule, powder, or the like; by intravenous, intraperitoneal, intramuscular, subcutaneous or parenteral injection; by transdermal diffusion or electrophoresis; topically in any acceptable form such as in drops, creams, gels or ointments; and by minipump or other implanted extended release device or formulation.

[0258] It is understood that modifications which do not substantially affect the activity of the various embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention.

EXAMPLES

Example 1 High-throughput Screening Assay for Identification of Inhibitors of Persistent Sodium Current [0259] To identify compounds that inhibit persistent sodium current, a primary highthroughput screen was employed, see, Joseph S. Adorante et al., High- WO 2005/020982 PCT/US2004/028077 00

O

O

throughput Screen for Identifying Channel Blockers that Selectively Distinguish STransient from Persistent Sodium Channels, U.S. Patent Publication No.

2002/0077297 (Jun. 20, 2002), which is hereby incorporated by reference in its C entirety.

S[0260] I. Compound Identification Assay Overview

NO

O [0261] To examine the ability of test compounds to alter persistent sodium current, 00 human embryonic kidney (HEK) cells were transfected with Nay1.3 sodium channel to obtain cells that express sodium current capable of mediating persistent sodium current. HEK cells expressing Nay1.3 (HEK-Nal1.3) were added to assay plate wells containing a Na -free media and physiologic concentrations of K (4.5 mM) and preincubated for 20 minutes with ion-sensitive FRET dyes and either 5 pM of a test compound or a DMSO control. The assay plates were then transferred to a voltage/ion probe reader (VIPR) (Aurora Biosciences, San Diego, CA) and the VIPR was adjusted so that the fluorescent emission ratio from the donor ands acceptor FRET dyes equaled 1.0. To elicit persistent sodium current, a double addition protocol was performed by first adding an isotonic solution to adjust the concentration of sodium and potassium ions in the well to 110 mM and 10 mM, respectively, and measuring the resulting sodium-dependent depolarization and second by adding K to a final concentration of 80 mM, and measuring K -dependent depolarization. Test compounds that block the Na dependent signal, but not the K* dependent signal were selected for further analysis. The Na -dependent depolarization resulting from the persistent Na* was measured as shown in Fig. 2. The labeled boxes indicate the application of Na or Circles indicate the control response with 0.1% DMSO added, triangles show the effects of the Na' channel inhibitor tetracaine (10 pM), and the diamonds show the response during the application of a non-specific channel blocker.

[0262] In this high-throughput assay, non-specific agents that inhibit membrane depolarizations induced by any effector must be distinguished from true persistent Na 4 current antagonists, which block only Na-dependent depolarizations. Therefore, a counter-screen to determine the ability of compounds to alter K*-dependent depolarization was performed. As shown in Fig. 2, following pre-incubation with vehicle alone (DMSO) both Na and K additions produced a robust depolarization as WO 2005/020982 PCT/US2004/028077 00

O

O

Oindicated by the increase in Rf/Ri. Tetracaine, a Na channel blocker, inhibited the Na'-dependent, but not the K-dependent change in Rf/Ri. In contrast, a non-specific \inhibitor of Na and K-dependent depolarization blocked the change in Rf/Ri following either addition. This data demonstrates that selective antagonists of the persistent sodium current can be identified using the described method.

IND

[0263] To eliminate compounds that non-specifically inhibited the Na+-dependent Sdepolarization, data obtained using the above procedure was analyzed with respect 0 0 to a counter-screen that used K+-dependent depolarization as a readout. To select 010 hits from the primary screen, the data were plotted as histograms. Inhibition of the Na'-dependent depolarization was plotted against inhibition of the K -dependent depolarization. Based on these data, the criteria for selection as a hit, was a greater or equal to 90% inhibition of the Na'-dependent depolarization and a less than or equal to 20% inhibition of the K-dependent depolarization. This protocol provided a distinction between compounds that were inert or non-specific in their effects and compounds that specifically block the persistent sodium current.

[0264] II. Solutions [0265] Solution compositions and volumes used in the assay are described below.

Functions of some components of the solutions using the assay are as follows: (1) CC2-DMPE: a stationary coumarin-tagged phospholipid resonance energy donor.

This dye is excited at 405 nm wavelength light and in the absence FRET emits fluorescence at 460 nm. DiSBAC2 or DiSBAC6(3): mobile resonance energy acceptors that partition across the membrane as a function of the electric field. The excitation spectra for these dyes overlap the emission of the coumarin donor and, thus, they act as FRET acceptors. They have an emission spectrum in the range of 570 nm. ESS-AY17: reduces the background fluorescence that complicates the assay. CdC12 (400 pM) was included in the pre-incubation solutions to stabilize the membrane potential of the cells at negative resting potential, resulting in the maximum number of Na channels being available for activation. Extracellular Clwas replaced with MeSO 3 during preincubation and throughout the assay. This eliminates a complicating Cl- current during the assay and results in an amplified and more stable voltage-change induced by the persistent Na current. 1st K addition: functions to depolarize the test cells to a voltage that activates substantial WO 2005/020982 PCT/US2004/02077 00 0 numbers of Na channels. 2nd K* addition: this addition produces a K*dependent depolarization, which is used as a counterscreen to eliminated nonspecific blockers.

[0266] III. Cell Culture [0267] HEK-293 cells were grown in Minimum Essential Medium (Invitrogen, Inc., 0 N' Carlsbad, CA) supplemented with 10% Fetal Bovine Serum (Invitrogen, Inc., 0 Carlsbad, CA) and 1% Pennicillin-Streptomycin (Invitrogen, Inc., Carlsbad, CA).

Medium for HEK-Navl.3 cells also contained 500 mg/ml G418 Geneticin (Invitrogen, Inc., Carlsbad, CA) and 2 pM TTX (Calbiochem, Inc., San Diego, CA) for maintaining selective pressure. Cells were grown in vented cap flasks, in 90% humidity and C02, to about 80% confluence and generally split by trypsinization 1:5 or 1:10.

[0268] HEK-Nav1.3 cells were seeded in 96-well plates (Becton-Dickinson, San Diego, CA) coated with Matrigel (Becton-Dickinson,. San Diego, CA) at 40,000 cells (in 100 pl culture medium) per well, and assayed the following day (16-20 hours).

Cells were sometimes incubated in 96-well plates at somewhat lower densities (20,000 per well), and incubated for up to 40-48 hours.

[0269] IV. HEK-Nal1.3 Handling and Dye Loading [0270] Approximately 16 to 24 hours before the assay, HEK-Nal1.3 cells were seeded in 96-well poly-lysine coated plates at 40,000 per well. On the day of the assay, medium was aspirated and cells were washed 3 times with 150 uL of Bath Solution #1 (BS#1) using CellWash (Thermo LabSystems, Franklin, MA).

[0271] A 20 pM CC2-DMPE solution was prepared by mixing coumarin stock solution with 10% Pluronic 127 1:1 and then dissolving the mix in the appropriate volume of BS#1. After the last wash, 50 ml of 20 pM CC2-DMPE solution was added to 50 mL of residual bath in each well to make 10 pM coumarin staining buffer. Plates were incubated in the dark for 30-60 minutes at room temperature.

[0272] While the cells were being stained with coumarin, a 10 pM DiSBAC2(3) solution in TEA-MeSO3 bath was prepared. In addition to oxonol, this solution WO 2005/020982 PCT/US2004/028077 00

O

O

contained any drug(s) being tested, at 4 times the desired final concentration ;pM for 5 pM final), 1.0 mM ESS-AY17, and 400 pM CdCl 2 C [0273] After 30-60 minutes of CC2-DMPE staining, the cells were washed 3 times with 150 pL of TEA-MeSO 3 buffer. Upon removing the bath, the cells were loaded with 80 pL of the DiSBAC2(3) solution and incubated for 20-30 minutes as before.

0Typically, wells in one column on each plate column 11) were free of test 0 drug(s) and served as positive and negative controls.

00 [0274] Once the incubation was complete, the cells were ready to be assayed on c VIPR for sodium addback. 240 pL of NaMeSO3 buffer was added to stimulate the cells, resulting in a 1:4 dilution of the drugs; 240 pL of TEA-MeSO 3 buffer or 1 pM TTX was used as a positive control.

[0275] V. VIPR Instrumentation and Data Process [0276] Optical experiments in microtiter plates were performed on the Voltage/Ion Probe Reader (VIPR) using two 400 nm excitation filters and filter sticks with 460 nm and 570 nm filters on the emission side for the blue and red sensitive PMTs, respectively. The instrument was run in column acquisition mode with 2 or 5 Hz sampling and 30 seconds of recording per column. Starting volumes in each well were 80 ml; usually 240 mL was added to each well during the course of the experiment. The lamp was allowed to warm up for about 20 minutes, and power to the PMTs was turned on for about 10 minutes prior to each experiment.

[0277] Ratiometric measurements of changes in fluorescent emissions at 460- and 570 nm on the VIPR platform (Aurora Bioscience, San Diego, CA) demonstrated that this assay format produces a robust and reproducible fluorescent signal upon depolarization of HEK-Nav1.3 cells with a Na K addition. From a normalized ratio of 1.0 in Na'-free media, Na'-dependent depolarization resulted in an increase in the 460/570 ratio to over 2.2 (Fig. Inter-well analysis of the ratios indicated that the amplitude of signal was large enough and consistent enough to be used in highthroughput screening.

WO 2005/020982 PCT/US2004/028077 00

O

O

tb [0278] Data were analyzed and reported as normalized ratios of intensities measured in the 460 nm and 580 nm channels. The VIPR sampling rate varied between 2 and 5 Hz in different experiments, with 5 Hz used for higher resolution of the peak sodium responses. The process of calculating these ratios was performed as follows. On all plates, column 12 contained TEA-MeSO 3 buffer with the same -DiSBAC2(3) and ESS-AY17 concentrations as used in the cell plates; however no cells were included in column 12. Intensity values at each wavelength were averaged for the duration of the scan. These average values were subtracted from 0 0 intensity values in all assay wells. The initial ratio obtained from samples 5-10 (Ri) was defined as: Intensity 460 nm, samples 5-10 background46 nm Ri Intensity 580 nm, samples 5-10 background 58 o nm [0279] and the ratio obtained from sample f (Rf) was defined as: Intensity 46 nm, sample f background 4 6 0 nm Rf- Intensity 580 nm, sample f backgrounds 0 nm [0280] Final data were normalized to the starting ratio of each well and reported as Rf/Ri. The fluorescent response in the Na,1.3 persistent current assay reached a peak approximately 10 seconds following the start of the run, therefore, the maximum ratio was selected as the readout for the assay (Fig. 3).

[0281] VI. Assay reproducibility and resolution [0282] The assay format described above allows for quality assurance by measuring both negative (DMSO and positive (tetracaine 10 pM) controls. Every plate in an assay run was a control plate. The data from these plates were used to verify that the assay conditions were optimal and to normalize the data from the test compounds. Fig. 3 shows results from control plates from multiple assays.

[0283] In Fig. 3, control plates having wells containing either 0.1% DMSO or 10 pM tetracaine were run after every ninth assay plate. The response to Na-dependent WO 2005/020982 PCT/US2004/028077 00

O

O

Sdepolarization was measured and the data were binned into histograms as shown.

The mean maximum response (Max) obtained in the presence of DMSO) and 0the mean minimum response (Min) obtained in the presence of 10 pM tetracaine were determined. For quality control, data variance was compared to the difference between the maximum and minimum signals. This was accomplished by calculating C a screening window for each control plate. Data for the run was accepted if 1.0

NO

Z 2

O

o0 3xSTD max +3x STD min SZ=1 Meanmax -Meanmin Example 2 Moderate -throughput Screening Assay for Selectivity of Inhibitors of Persistent Sodium Current [0284] Compounds obtained by the high-throughput screening described in Example 1 were tested for selectivity of blockade of persistent sodium current with respect to blockade of transient sodium current using a moderate-throughput screen. The selectivity assay utilizes Estim technology (Aurora Bioscience, San Diego, CA) to induce channel activation. This assay has an inherently greater time resolution than the high-throughput assay, and thus allows the measurement of both the transient and persistent components of the Na currents within a single experiment.

[0285] I. Compound Selectivity Assay Overview [0286] The Estim technology involves instrumenting 96-well plates with electrodes so that application of an appropriate voltage gradient across the well (electric field stimulation, EFS) can be used for activation of the ion channels in the target cells.

EFS of HEK-293 cells expressing Nay1.3 channels resulted in a rapid depolarization followed by a delayed repolarization. The transient Na current drives the rapid depolarization while the persistent Na' current sustains the delayed repolarization.

When similar experiments were performed in cells expressing channels that do not exhibit persistent currents, only rapid depolarization was seen. For quantification of WO 2005/020982 PCT/US2004/028077 00

O

O

(N

Sthe block of transient current, the amplitude of peak response was averaged for seven stimuli. The average response was converted to activity by normalizing O\ against the difference between the responses in Ringer's solution with DMSO and Ringer's solution containing 10 pM tetracaine. Persistent current activity was calculated by integrating under the curve. The area obtained for each compound C was normalized against the responses obtained with the DMSO control and in the presence of 10 pM tetracaine.

O

00 [0287] II. Cell Culture [0288] Approximately 16 to 24 hours before the assay, HEK-Navl.3 cells were seeded in 96-well poly-lysine coated plates at 60,000 per well. On the day of the assay, medium was aspirated were cells were washed 3 times with 150 pL of HBSS using CellWash (Thermo LabSystems, Franklin, MA).

[0289] III. HEK-Na,1.3 Handling and Dye Loading [0290] A 20 pM CC2-DMPE solution was prepared by mixing coumarin stock solution with 10% Pluronic 127 1:1 and then dissolving the mix in the appropriate volume of HBSS. After the last wash, 50 pL of 20 pM CC2-DMPE solution was added to 50 pL of residual bath in each well to make 10 pM coumarin staining buffer.

Plates were incubated in the dark for 30 minutes at room temperature.

[0291] While the cells were being stained with CC2-DMPE, a 0.2 pM DiSBAC6(3) solution in HBSS was prepared.

[0292] After 30 minutes of CC2-DMPE staining, the cells were washed 3 times with 150 pL of HBSS. After the last wash, 50 pL of 0.2 pM DiSBAC6(3) solution was added to 50 pL of residual bath in each well to make 0.1 pM oxonol staining buffer.

Plates were then incubated in the dark for 15 minutes.

[0293] After 15 minutes of DiSBAC6(3) staining, the cells were washed again 3 times with 150 pL of HBSS. After the last wash, 50 pL of 1.0 pM ESS-AY17 solution was added to 50 pL of residual bath in each well to make 0.5 pM ESS. This solution also contained any drug(s) being tested, at twice the desired final concentrations. Plates WO 2005/020982 PCT/US2004/028077 00 0

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were incubated in the dark again for 15 minutes. Once the incubation was complete, the cells were assayed on EFSNSP reader.

[0294] III. Fast FRET Reader Instrumentation and Data Process C[0295] Optical experiments in microtiter plates were performed on the fast FRET

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Reader using two 400 nm excitation filters and filter sticks with 460 nm and 580 nm filters on the emission side for the blue and red sensitive PMTs, respectively. The 0 instrument was run in column acquisition mode with 100 Hz sampling and 12 0 10 seconds of recording per column. Seven pulses were applied at 1 Hz, starting at 2 seconds. The lamp was allowed to warm up for about 20 minutes, and power to the PMTs was turned on for about 10 minutes prior to each experiment.

[0296] Data were analyzed and reported as normalized ratios of intensities measured in the 460 nm and 580 nm channels. The process of calculating these ratios was performed as follows. On all plates, column 12 contained HBSS with the same ESS-AY17 concentration as used in the cell plates; however no cells were included in column 12. Intensity values at each wavelength were averaged for the duration of the scan. These average values were subtracted from intensity values in all assay wells. The initial ratio obtained from samples 50-100 (Ri) was defined as: Intensity 46 nm, samples 50-100 background46 nm Intensity 580 nm, samples 50-100 background5o nm [0297] and the ratio obtained from sample f (Rf) was defined as: Intensity 460 nm, sample f background 460 nm Rf= Intensity 5 8 nm, sample background nm [0298] Data were normalized to the starting ratio of each well and reported.as Rf/Ri.

The transient Na+-current signal was calculated as average of the peaks resulting from the seven electric pulses applied in the course of recording. The persistent Na*current signal was calculated integrating the area under the total response during the seven electric pulses applied in the course of recording. Selectivity was determined WO 2005/020982 PCT/US200/028)077 00 by comparison of concentrations of agent required to block 50% of the persistent current (ICso) vs. the ICso for the transient current.

Example 3 CElectrophysiological Assay for

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Selectivity of Inhibitors of Persistent Sodium Current 0 00 [0299] To confirm the blocking selectivity of test compounds for persistent sodium 0 10 current, individual compounds were examined using a whole-cell patch clamp method.

[0300] HEK cells transfected with Nay1.3 sodium channels that express transient and persistent sodium currents were plated onto glass coverslips and cultured in MEM cell culture media with Earle's salts and GlutaMAX (Invitrogen, Inc., Carlsbad, CA) supplemented with:10% Fetal bovine serum, heat inactivated (Invitrogen, Inc., Carlsbad, CA), 0.1 mM MEM non-essential amino acids (Invitrogen, Inc., Carlsbad, CA), 10 mM HEPES (Invitrogen, Inc., Carlsbad, CA), 1% Penicillin/Streptomycin (Invitrogen, Inc., Carlsbad, CA).

[0301] After an incubation period of from 24 to 48 hours the culture medium was removed and replaced with external recording solution (see below). Whole cell patch clamp experiments were performed using an EPC10 amplifier (HEKA Instruments, Lambrecht, Germany.) linked to an IBM compatible personal computer equipped with PULSE software. Borosilicate glass patch pipettes were pulled to a fine tip on a pipette puller (Sutter Instrument Co., Novato, CA) and were polished (Microforge, Narishige, Japan) to a resistance of about 1.5 Mohm when filled with intracellular recording solution (Table 1).

WO 2005/020982 00

OT

Table 1. Patch Clamp Solutions PCT/US2004/028077 0

GO

0 0

O

O 0 External Recording Solution Internal Recording Solution Compound Concentration Compound Concentration NaCI 127 mM CsMeSO 3 125 mM HEPES (free acid) 10 mM CsCI 25 mM KCI 5 mM NaHEPES 10 mM CsCI 5 mM Amphotericin 240 pg/ml Glucose 10 mM MgCI 2 0.6 mM CaCI 2 1.2 mM CdCI 2 200 pM pH to 7.4 with NaOH room temp. 290 pH 7.20 with CsOH300 mOsm mOsm.

[0302] Persistent and transient currents in HEK cells expressing Nay1.3 channels were measured by applying 200-msec depolarizations from a holding potential of mV to 0 mV. Background currents that remained in the presence of 500 nM TTX were subtracted from all traces. Drugs were perfused directly into the vicinity of the cells using a microperfusion system.

[0303] Under control conditions, depolarizing pulses elicited a large transient inward current that declined to a smaller persistent current, which remained stable during the remainder of the pulse (Fig. 4, control). Addition of 500 nM TTX completely blocked both the transient and persistent currents (Fig. 4, TTX).

Application of 3 pM of exemplar compound Compound 1, produced a much different effect. Inspection of Fig. 4 reveals that the Compound 1 blocked 99% of the persistent current while only reducing the transient current by 16%. Dose-response analysis for Compound 1 demonstrates its significant selectivity for blocking the persistent sodium current relative to the transient sodium current over a four order of magnitude range (Fig. Example 4 Administering a Selective Persistent Sodium Current Antagonist in a Rodent Model Results in Reduced Epileptic Seizures WO 2005/020982 PCT/US2004/028077 00 [0304] This study examined the anti-seizure efficacy of Compound 1 against to Sreference compounds (Diazepam and Sipatrigine) using the audiogenic mouse model as the test platform.

C- [0305] DBA mice are well established in the literature as a model for audiogenic Nseizures (AGS). This genetically based model is attractive for testing potential O therapeutics in that no treatment protocol is required to create the condition. It is 00 classified as inherited idiopathic epilepsy with no known associated organic disease.

[0306] The AGS response in these mice consists of a progressive sequence of behaviors. The latency to onset may vary from 2 to 15 seconds, and is age variant.

The initial wild running phase may be divided into an early running phase that varies from 5 to 20 seconds, followed by a wild running phase that continues for 10 seconds. Wild running may progress to tonic/clonic seizures, with loss of righting ability, respiratory suppression and death.

[0307] We used a scoring system to quantify the effects of the test compounds on the induction of seizures.. The behavioral sequence was assigned ascending numerical values (Table and the highest numerical value reached by an animal was that animal's score. Results are reported as an average score for ten animals in a treatment group.

Table 2. Seizure Scale 1 Staring 2 Head/body tremors/jerks 3 Tonic contraction/Straub tail 4 Wild running Wild running/Jumping 6 Tonic Clonic Seizure 7 Convulsion 8 Death [0308] Animals. Male DBA/2 mice were obtained from Jackson Laboratories at 21 day post partum. The animals were acclimated for 1 or 2 days prior to use. Individual WO 2005/020982 PCT/US2004/028077 00 animal weights were recorded immediately before treatment. Surviving animals were euthanized within three days of completion of testing.

[0309] Seizure Induction. Mice were placed in a test chamber (25 cm and exposed to pure tone sound of 11 kHz at a minimum of 116dB for approximately 60 s Suntil a sequential seizure response, consisting of an early wild running phase,

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followed by generalized myoclonus and tonic flexion and extension, was obtained.

Non-responders were challenged up to five times. Ten animals were obtained by this 00 method for each intended treatment group, with a standard deviation in their maximum score of less than 25%. The acoustic stimulus signal was produced using a signal generator and projected via four high-frequency ceramic speakers mounted on the roof of the chamber. Chamber calibration was performed daily to ensure consistent sound pressure. AGS behaviors were videotaped for later analysis.

[0310] injections: The 10 animals in each treatment group were injected intraperitoneally with the test drug in a volume of 10 mL/kg of body weight. Injections were 60 min prior to sound challenge.

[0311] Data analyses: Data were tested for normal distribution, and (when normal) were analyzed using ANOVA followed by Student's t test. When the data were not normally distributed, analysis was with the Mann-Whitney rank sum test.

Significance was set at p [0312] Results: Mean AGS scores for each group (with standard deviations) are shown for each treatment in Table 3. No control animals experienced full- blown convulsive seizures. Control values near five indicate that these animals entered the wild running phase and either did not progress to convulsions or exhibited infrequent or mild tonic-clonic seizures. Control animals exhibited tonic body contraction (back arching, hind leg rigidity), running and some body clonus (jerks, tremors). The response was quite variable with approximately 20% of the animals exhibiting very mild (if any) symptoms. No control animals developed powerful TCS symptoms that progressed to death.

[0313] Although the lack of a full-blown seizure response in the control animals limited the power of this study, compounds (Diazepam and Sipatrigine) that are WO 2005/020982 PCT/US2004/028077 00

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O

b known to reduce the behavioral response to sound-induced seizures in DBA mice were effective at the predicted doses. Compound 1 (1 mg/kg) also significantly 0 reduced seizure response at 60 minutes after dosing. The estimated plasma concentration for Compound 1 at this time would be on approximately 5 pM, a concentration that should reduce the persistent sodium current by more than Swhile having only a limited effect on the transient current. The fact that statistically significant effects on seizure response were detected at this concentration in spite of both the high variability and the reduced therapeutic window in this assay indicates 0 0 the efficacy of Compound 1 derives from its effect on the persistent sodium current.

Table 3. Reduction of seizure score in DBA mice following treatment with potential anti-epileptic compounds.

Dose Treatment (mg/kg) Mean s.d. p Control 4.8+ 1.9 Diazepam 0.15 3.1 1.1 0.02 Sipatrigine 3 3.3 1.7 0.04 Compound 1 1 3.2 2.3 0.04 Example Administering a Selective Persistent Sodium Current Antagonist in a Rodent Model Results in Reduced Pain [0314] This example describes reversal of allodynia in an animal model of neuropathic pain by administering a selective persistent sodium channel antagonist.

[0315] Compound 1 was tested in a rodent model of neuropathic pain known to be predictive of clinical activity, see, Kim Chung, supra, (1992). Following ligation of two spinal nerves, the animals developed sensitivity to normally nonpainful stimuli such as touch. The ability of Compound 1 to reverse this sensitivity, called allodynia, was tested 30 minutes after dosing by intraperitoneal administration.

As shown in Fig. 6, Compound 1 produced an 80% reduction in allodynia with respect to a vehicle control.

WO 2005/020982 PCT/US2004/028077 00 O[0316] The animal model used involved the surgical ligation of the L5 (and optionally Z the L6) spinal nerves on one side in experimental animals. Rats recovering from the surgery gained weight and displayed a level of general activity similar to that of normal rats. However, these rats developed abnormalities of the foot in which the hindpaw was moderately everted and the toes were held together. More importantly, Sthe hindpaw on the side affected by the surgery became sensitive to pain from low- IDthreshold mechanical stimuli, such as that producing a faint sensation of touch in a r"- O human, within about 1 week following surgery. This sensitivity to normally non- 00 painful touch is called "tactile allodynia" and lasts for at least two months. The response includes lifting the affected hindpaw to escape from the stimulus, licking the paw and holding it in the air for many seconds. None of these responses is normally seen in the control group.

[0317] Rats were anesthetized before surgery. The surgical site was shaved and prepared either with betadine or Novacaine. Incision was made from the thoracic vertebra XIII down toward the sacrum. Muscle tissue was separated from the spinal vertebra (left side) at the L4-S2 levels. The L6 vertebra was located and the transverse process was carefully removed with a small rongeur to expose the L4-L6 spinal nerves. The L5 and L6 spinal nerves were isolated and tightly ligated with silk thread. The same procedure was performed on the right side as a control, except that no ligation of the spinal nerves was performed.

[0318] A complete hemostasis was confirmed, then the wounds were sutured. A small amount of antibiotic ointment was applied to the incised area, and the rat was transferred to the recovery plastic cage under a regulated heat-temperature lamp.

On the day of the experiment, at least seven days after the surgery, six rats per test group were administered the test drugs by intraperitoneal injection. For i.p.

injection, Compound #1 was formulated in approximately 50% DMSO and given in a volume of 1 ml/kg body weight. Compound #1 was tested 10 mg/kg.

[0319] Tactile allodynia was measured prior to and 30 minutes after drug administration using von Frey hairs, which are a series of fine hairs with incremental differences in stiffness. Rats were placed in a plastic cage with a wire mesh bottom and allowed to acclimate for approximately 30 minutes. The von Frey hairs were applied perpendicularly through the mesh to the mid-plantar region of the rats' WO 2005/020982 PCT/US2004/028077 00 0

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Shindpaw with sufficient force to cause slight buckling and held for 6-8 seconds. The applied force has been calculated to range from 0.41 to 15.1 grams. If the paw is sharply withdrawn, it is considered a positive response. A normal animal will not respond to stimuli in this range, but a surgically ligated paw will be withdrawn in response to a 1-2 gram hair.

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[0320] In summary, results shown in this example indicate that a selective persistent

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Csodium current antagonist can be used to effectively reduce pain in a mammal.

00 Example 6 Synthesis of Exemplary Compounds Representative of Formula 1 [0321] A compound having general Formula 1, exemplified by thiophene-2carboxylic acid (4-phenyl-butyl)-amide (Compound 1; Fig. 1) can be prepared as follows. A solution of thiophene-2-carbonyl chloride (147 mg, 1.0 mmol), triethylamine (101 mg, 1.0 mmol) in dichloromethane is treated with 4phenylbutylamine (149 mg, 1.0 mmol). The reaction mixture is stirred until no further reaction occurs and is quenched by the addition of aqueous NaHCO 3 solution. The organic phase is collected and concentrated to give the title compound.

Example 7 Synthesis of Exemplary Compounds Representative of Formula 2 [0322] A compound having general Formula 2, exemplified by 1-Benzyl-4-(5-phenyl- [1,3,4]oxadiazol-2-yl)-pyridine (Compound 2; Fig. 1) can be prepared as follows. A solution of 4-(5-phenyl-[1,3,4]oxadiazol-2-yl)-pyridine (223 mg, 1.0 mmol) is prepared by the method of H. Smith Broadbent, et al., Quinoxalines. I. Preparation and Stereochemistry of Decahydroquinoxalines, 82(1) J. AMER. CHEM. Soc. 189-193 (1960) in chloroform is treated with benzylbromide (171 mg, 1.0 mmol). The reaction is stirred until no further reaction occurs. The reaction mixture is concentrated to give the title compound.

Example 8 WO 2005/020982 PC'FUS2004028077 00 0 Synthesis of Exemplary Compounds Representative of Formula 3 [0323] A compound having general Formula 3, exemplified by 6-lsopropyl-3-methyl- 2-{4-[(4-propoxy-benzylidene)-amino]-benzylidene}-cyclohexanone (Compound 3; C- Fig. 1) can be prepared as follows. A solution of menthone (154 mg, 1.0 mmol) and S4-aminobenzaldehyde (121 mg, 1.0 mmol) in dimethylsulfoxide is treated with O potassium hydroxide (56 mg, 1.0 mmol). The reaction is stirred until no futher 00 reaction occurs. The reaction mixture is poured into ethyl acetate and water. The organic phase is collected, dried and concentrated to give 2-(4-Amino-benzylidene)- 6-isopropyl-3-methyl-cyclohexanone. The 2-(4-Amino-benzylidene)-6-isopropyl-3methyl-cyclohexanone is dissolved in dichloromethane and treated with 4propoxybenzaldehyde (164 mg, 1.0 mmol) and anhydrous Na 2

SO

4 The reaction mixture is stirred until no further reaction occurs. The reaction mixture is filtered and concentrated to give the title compound.

Example 9 Synthesis of Exemplary Compounds Representative of Formula 4 [0324] A compound having general Formula 4, exemplified by 3-(2,2,2-Trifluoroacetylamino)-benzoic acid 2-oxo-2-phenyl-ethyl ester (Compound 4; Fig. 1) can be prepared as follows. A solution of 3-aminobenzoic acid (137 mg, 1.0 mmol) in dichloromethane is treated with trifluoroacetic anhydride (420 mg, 2.0 mmol). The reaction mixture is stirred until no further reaction occurs. The reaction mixture is concentrated to give 3-(2,2,2-Trifluoro-acetylamino)-benzoic acid. A solution of 3- (2,2,2-Trifluoro-acetylamino)-benzoic acid (233 mg, 1.0 mmol) and 2hydroxyacetophenone (136 mg, 1.0 mmol) in dimethylformamide and diisopropylethylamine (260 mg, 2.0 mmol) is treated with HBTU (379 mg, 1.0 mmol).

The reaction mixture is stirred until no further reaction occurs. The reaction is poured into ethyl acetate and water. The organic phase is collected, dried and concentrated to give the title compound.

Example WO 2005/020982 PCT/US2004/028077 00

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)Oral Administration of a Persistent Sodium Current Blocker to Treat Epilepsy [0325] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB Scompound to treat a neuropathy. While the example illustrates the use of a PSCB to treat eplilepsy, any neuropathic condition resulting from aberrant activity of a 0 persistent current, such as, headache, pain, inflammatory diseases, movement 00 disorders, tumors, birth injuries, developmental abnormalities, neurocutaneous disorders, autonomic disorders, and paroxysmal disorders, can also be treated using this method.

[0326] A patient presents neuropathic symptoms that are diagnosed as an epilepsy.

The patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB over a period of several months. The patent is reassessed after this treatment and it is found that the patient's epileptic seizures have subside. Repeated administration of the PSCB composition maintains this sustained relief from epileptic seizures.

Example 11 Oral Administration of a Persistent Sodium Current Blocker to Treat Cerebral Hypoxia [0327] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat a hypoxia. While the example illustrates the use of a PSCB to treat cerebral hypoxia, any hypoxia resulting from a loss of oxygen to a portion of the body, such as, diffusion hypoxia, hypoxic hypoxia, cell hypoxia, ischemic hypoxia, or any other accidental or purposeful reduction or elimination of oxygen supply to a tissue, can also be treated using this method.

[0328] A patient presents symptoms that are diagnosed as cerebral hypoxia. The patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB over a period of several months. The WO 2005/020982 PCT/US2004/028077 00

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Spatent is reassessed after this treatment and it is found that the patient's symptoms have subside. Continued administration of the PSCB composition maintains O alleviation of these symptoms.

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Example 12

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Oral Administration of a Persistent Sodium Current Blocker to Treat Cardiac Ischemia 00 [0329] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat an ischemia. While the example illustrates the use of a PSCB to treat myocardiac ischemia, any ischemia resulting from a loss of blood to a portion of the body, such as, cerebral ischemia, myoischemia, diabetes ischemia, ischemia retinae, postural ischemia, or any other accidental or purposeful reduction or complete obstruction of blood supply to a tissue, can also be treated using this method.

[0330] A patient presents symptoms that are diagnosed as myocardiac ischemia.

The patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB over a period of several months. The patent is reassessed after this treatment and it is found that the patient's symptoms have subside. Continued administration of the PSCB composition maintains alleviation of these symptoms.

Example 13 Oral Administration of a Persistent Sodium Current Blocker to Treat Multiple Sclerosis [0331] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat multiple sclerosis.

WO 2005/020982 PCT/US200/028077 00 0 [0332] A patient presents symptoms that are diagnosed as multiple sclerosis. The Spatient is treated orally with a therapeutically-effective amount of a pharmaceutically Sacceptable composition comprising a PSCB over a period of several months. The patent is reassessed after this treatment and it is found that the patient's symptoms have stablize. Continued administration of the PSCB composition maintains prevents continued progression of the disease.

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0 Example 14 00

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Oral Administration of a Persistent Sodium Current Blocker to Treat Amyotrophic Lateral Sclerosis [0333] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat amyotrophic lateral sclerosis.

[0334] A patient presents symptoms that are diagnosed as amyotrophic lateral sclerosis. The patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB over a period of several months. The patent is reassessed after this treatment and it is found that the patient's symptoms have stablize. Continued administration of the PSCB composition maintains prevents continued progression of the disease.

Example Oral Administration of a Persistent Sodium Current Blocker to Treat Aberrant Nitric Oxide Levels [0335] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat aberrant nitric oxide levels.

A patient presents symptoms that are diagnosed as aberrant nitric oxide levels. The patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB over a period of several months. The WO 2005/020982 PCT/US200402077 00 0

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Spatent is reassessed after this treatment and it is found that the patient's symptoms have subside. Continued administration of the PSCB composition maintains 0 alleviation of these symptoms.

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Example 16

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Oral Administration of a Persistent Sodium Current Blocker Sto Treat Neuropathic Pain from Trigeminal Neuralgia 00 [0336] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat a neuralgic pain. While the example illustrates the use of a PSCB to treat trigeminal neuralgia, any acute spasmodic pain that travels along one or more nerves, such as, post-herpetic neuralgia, glossopharyngeal neuralgia, sciatica and atypical facial pain, can also be treated using this method.

[0337] A patient presents pain symptoms that are diagnosed as trigeminal neuralgia.

She describes the pain as a sudden sharp stabbing pain on the right side of her face, eyes and lips. The pain is triggered when she tries to chew her food while eating and each episode lasts for several seconds and may repeat many times over the course of the day. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated.

Repeated administration of the PSCB composition maintains this pain relief.

Example 17 Oral Administration of a Persistent Sodium Current Blocker to Treat Neuropathic Pain from Phantom Pain [0338] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat a deafferentation pain syndrome. While the example illustrates the use of a PSCB to treat phantom pain, any pain resulting from a loss of the sensory input from a portion of the body, such as, an injury to the brain, spinal WO 2005/020982 PCT/US2004/028077 00 0 Scord, or a peripheral nerve, post-stroke pain, phantom pain, paraplegia, brachial plexus avulsion and postherpetic neuralgia, can also be treated using this method.

[0339] A patient with an amputated right arm presents symptoms that are diagnosed as phantom pain. That patient is treated orally with a therapeutically-effective Camount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Repeated administration of the PSCB composition maintains this pain 0 0 relief.

010 Example 18 Oral Administration of a Persistent Sodium Current Blocker to Treat Neuropathic Pain from Chemotherapy Treatment [0340] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat a polyneuropathic pain. While the example illustrates the use of a PSCB to treat pain induced from chemotherapy treatment, any pain involving two or more peripherial nerves, such as, diabetic neuropathy, treatment-induced pain, postmastectomy syndrome, post-polio syndrome, diabetes, alcohol, amyloid, toxins, HIV, hypothyroidism, uremia, vitamin deficiencies, 2',3'-didexoycytidine (ddC) treatment and Fabry's disease, can also be treated using this method.

[0341] A cancer patient undergoing chemotherapy presents symptoms that are diagnosed as chemotherapy-induced pain. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Repeated administration of the PSCB composition maintains this pain relief.

Example 19 Oral Administration of a Persistent Sodium Current Blocker to Treat Allodynia WO 2005/020982 PCT/US00/02077 00 0 [0342] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat allodynia.

S[0343] A patient presents pain symptoms that are diagnosed as allodynia. She indicates that whenever something gently touches her lest forearm, she feels an 0 (Ni intense pain like a sudden burning sensation. That patient is treated orally with a Stherapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Repeated administration of the PSCB composition maintains this pain relief.

Example Oral Administration of a Persistent Sodium Current Blocker to Treat Hyperalgesia [0344] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat hyperalgesia.

[0345] A patient presents pain symptoms that are diagnosed as hyperalgesia. He indicates that whenever he mildly bumps his right thigh against a hard object, like a table corner, a great shooting pain occurs to such an extent that he needs to sit down. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated.

Repeated administration of the PSCB composition maintains this pain relief.

Example 21 Oral Administration of a Persistent Sodium Current Blocker to Treat Hyperpathia WO 2005/020982 PCT/US200/028077 00 0 [0346] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat hyperpathia.

[0347] A patient presents pain symptoms that are diagnosed as hyperpathia. That M patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration C- of a PSCB therapy, the patient's pain is substantially alleviated. Repeated 00 0administration of the PSCB composition maintains this pain relief.

Example 22 Oral Administration of a Persistent Sodium Current Blocker to Treat Chronic Pain from a Migraine Headache Pain [0348] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat pain from a headache. While the example illustrates the use of a PSCB to treat pain resulting from a migraine Headache, any headache pain, such as, tension-type headache, cluster headache, hormone headache, rebound headache, sinus headache and organic headache, can also be treated using this method.

[0349] A patient presents pain symptoms that are diagnosed as resulting from a migraine headache. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Repeated administration of the PSCB composition maintains this pain relief.

Example 23 Oral Administration of a Persistent Sodium Current Blocker to Treat Pain Associated with Rheumatoid Arthritis WO 2005/020982 PCT/US2004/028077 00 [0350] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat chronic pain resulting from inflammatory disorder. While the example illustrates the use of a PSCB to treat chronic pain resulting from a rheumatoid arthritis, any inflammatory disorder-induced pain, such as, e.g., Cc osteoarthritis, gouty arthritis, spondylitis or autoimmune diseases such as lupus erythematosus, can also be treated using this method.

00 [0351] A patient presents pain symptoms that are diagnosed as resulting from rheumatoid arthritis. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Repeated administration of the PSCB composition maintains this pain relief.

Example 24 Oral Administration of a Persistent Sodium Current Blocker to Treat Chronic Back Pain [0352] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat chronic pain resulting from excessive muscle tension. While the example illustrates the use of a PSCB to treat chronic lower back pain, any excessive muscle tension-induced pain can also be treated using this method.

[0353] A patient presents with a non-spasmodic muscle pain localized at the lumbar region of the back due to a herniated disc. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Repeated administration of the PSCB composition maintains this pain relief.

Example WO 2005/020982 PCT/US2004/028077 00 Oral Administration of a Persistent Sodium Current Blocker to Pain Associated with Treat Irritable Bowel Syndrome [0354] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB Scompound to treat pain resulting from chronic gastrointestinal inflammations. While the example illustrates the use of a PSCB to treat the pain associated with irritable bowel syndrome, any gastrointestinal inflammation-induced pain, such as, e.g., 00 Crohn's disease, ulcerative colitis and gastritis, can also be treated using this method.

[0355] A patient presents pain symptoms that are diagnosed as resulting from irritable bowel syndrome. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Repeated administration of the PSCB composition maintains this pain relief.

Example 26 Oral Administration of a Persistent Sodium Current Blocker to Treat Post-Operative Pain [0356] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat post-operative pain.

[0357] A patient presents pain symptoms resulting from a surgical operation. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Administration of the PSCB composition continues for about 1 to about 4 weeks to maintain this pain relief.

Example 27 WO 2005/020982 PCT/US2004/028077 00

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SOral Administration of a Persistent Sodium Current Blocker to Treat Pain Associated with Fibromyalgia [0358] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB c compound to treat pain associated with fibromyalgia.

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[0359] A patient presents pain symptoms that are diagnosed as resulting from 00 fibromyalgia. That patient is treated orally with a therapeufically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated.

Repeated administration of the PSCB composition maintains this pain relief.

Example 28 Oral Administration of a Persistent Sodium Current Blocker to Treat Pain Associated with Repetitive Motion Disorder of the Wrist [0360] This example shows a method of persistent sodium current blocker (PSCB) therapy using a pharmaceutically acceptable composition comprising a PSCB compound to treat pain resulting from repetitive motion disorders (RMDs). While the example illustrates the use of a PSCB to treat the pain associated with RMDs of the wrist, any RMD-induced pain occurring in, hands, elbows, shoulders, neck, back, hips, knees, feet, legs, and ankles, can also be treated using this method.

[0361] A patient presents pain symptoms that are diagnosed as resulting from an RMD of the wrist. That patient is treated orally with a therapeutically-effective amount of a pharmaceutically acceptable composition comprising a PSCB. Within one day after the administration of a PSCB therapy, the patient's pain is substantially alleviated. Repeated administration of the PSCB composition maintains this pain relief.

[0362] Although the present invention has been described with reference to the disclosed embodiments, one skilled in the art will readily appreciate that the specific P NPDOCSCRN\RMSpc=2OG4Z383 dv p= doc.2M8A/2008 00 Sexperiments disclosed are only illustrative of the present invention. Various modifications can be made without departing from the spirit of the present invention.

Throughout this specification and the claims which follow, unless the context 5 requires otherwise, the word "comprise", and variations such as "comprises" or t' ."comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers c or steps.

00 The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (37)

1. A method of treating chronic pain in a mammal, comprising administering to said mammal an effective amount of a selective persistent sodium channel antagonist, wherein said antagonist has at least 20-fold selectivity for a persistent sodium current relative to a transient sodium current, and wherein said antagonist is a compound included in formula 1, or a pharmaceutically acceptable salt, ester, amide, stereoisomer or racemic mixture thereof: ,Y-Ar 1 2 Ar -N R 3 R 1 R wherein Ar 1 is an aryl group; Ar 2 is an aryl group; Y is absent or is selected from the group consisting of 0 NJlrj 0 1_u 1 0 andl -S-I 0 wherein, R 4 is selected from hydrogen, a C 1 -C 8 alkyl, an aryl or an arylalkyl; R 1 is selected from hydrogen, a C, to Ca alkyl, an aryl, and an arylalkyl; R 2 and R 3 are independently selected from hydrogen, hydroxy, fluoro, a C, to C8 an alkyl, an aryl, an arylalkyl, a Cj-C8 carbocyclic ring and a Ci-C 8 heterocyclic ring; and n is 1, 2, 3, 4, 5, or 6. P \WPDOCS\CRN\RMH\Spec\20423830 div claims doc 00 O CN 2. The method of claim 1, wherein said persistent sodium current is a Nay1.1 persistent Scurrent, a Na,1.2 persistent current, a Na,1.3 persistent current, a Nav1.4 persistent current, a Nav1.5 persistent current, a Na,1.6 persistent current, a Na,1.7 persistent current, a Nav1.8 persistent current, or a Nav1.9 persistent current.

3. The method of claim 1 or claim 2, wherein said mammal is a human. CD

4. The method of any one of claims 1 to 3, wherein said antagonist has at least 00 selectivity for said persistent sodium current relative to said transient sodium current. 00 CN 5. The method of claim 4, wherein said antagonist has at least 200-fold selectivity for said persistent sodium current relative to said transient sodium current.

6. The method of claim 5, wherein said antagonist has at least 400-fold selectivity for said persistent sodium current relative to said transient sodium current.

7. The method of any one of claims 1 to 6, wherein said Ar 1 is a thienyl or substituted thienyl.

8. The method of claim 7, wherein said substituted thienyl is substituted with one or more of halogen, a C1-C 8 alkyl, NO 2 CF 3 OCF 3 OCF 2 H, CN, or a (CRsR 6 )cN(R 7 2 wherein, R 5 and R 6 are independently selected from hydrogen, hydroxy, fluoro, and a C1 to Ca alkyl; and R 7 is selected from hydrogen, a C, to C8 alkyl, an aryl and an arylalkyl; and c is 1, 2, 3, 4, or

9. The method of any one of claims 1 to 8, wherein said Ar 2 is a phenyl or a substituted phenyl. P:\WPDOCS\CRN\RMH\Spec\20423830 div claims doc 00 O O C 10. The method of claim 9, wherein said substituted phenyl is substituted with one or more of halogen, a C 1 -C 8 an alkyl, an arylalkyl, NO 2 CF 3 OCF 3 OCF 2 H, CN or a (CRSR 6 )cN(R 7 2 wherein, R 5 and R 6 are independently selected from hydrogen, hydroxy, fluoro, and a C1 to Ca alkyl; and R 7 is selected from hydrogen, a C, to Ca alkyl, an aryl and an arylalkyl; and 00 Sc is 0, 1, 2, 3, 4, or

11. The method of claim 7, wherein said Ar 1 is thienyl.

12. The method of claim 9, wherein said Ar 2 is phenyl.

13. The method of any one of claims 1 to 12, wherein said R 1 is hydrogen, methyl, ethyl, propyl, or isopropyl.

14. The method of any one of claims 1 to 13, wherein said R 2 is hydrogen, methyl, ethyl, propyl, or isopropyl. The method of any one of claims 1 to 14, wherein said R 3 is hydrogen, methyl, ethyl, propyl, or isopropyl.

16. The method of any one of claims 1 to 15, wherein said n is 3, 4 or

17. The method of claim 16, wherein said n is 4.

18. The method of claim 11, wherein said antagonist is thiophene-2-carboxylic acid (4-phenyl-butyl)-amide.

19. The method of claim 12, wherein said antagonist is P:\WPDOCS\CRN\RMH\Spec\20423830 div clims doc 00 O \O 0 (N 00 20. The method of any one of claims 1 to 19, wherein said chronic pain is a Smononeuropathy or a polyneuropathy.

21. The method of any one of claims 1 to 19, wherein said chronic pain results from cancer or cancer treatment.

22. The method of any one of claims 1 to 19, wherein said chronic pain results from diabetic neuropathy or fibromyalgia.

23. The method of any one of claims 1 to 19, wherein said chronic pain is associated with substance withdrawal or a headache.

24. The method of any one of claims 1 to 19, wherein said chronic pain is associated with a repetitive motion disorder pain or excessive muscle tension. The method of any one of claims 1 to 19, wherein said chronic pain is associated with inflammation.

26. The method of claim 25, wherein said inflammation results from an inflammatory disorder, an injury, an infection, or a chronic visceral pain.

27. The method of any one of claims 1 to 19, wherein said chronic pain is a neuropathic pain.

28. The method of claim 27, wherein said neuropathic pain is a neuralgia. P:\WPDOCS\CRN\RMH\Spec\20423830 div claims.doc 00 O NC 29. The method of claim 28, wherein said neuralgia is selected from the group consisting of a trigeminal neuralgia, a post-herpetic neuralgia, a glossopharyngeal neuralgia, a sciatica and an atypical facial pain. The method of claim 28, wherein said neuropathic pain is a deafferentation pain syndrome. CD

31. The method of claim 30, wherein said deafferentation pain syndrome is selected from NC the group consisting of an injury to the brain or spinal cord, a post-stroke pain, a 00 Sphantom pain, a paraplegia, a peripheral nerve injury, a brachial plexus avulsion injury NC and a lumbar radiculopathy.

32. The method of claim 28, wherein said neuropathic pain is a complex regional pain syndrome (CRPS).

33. The method of claim 32, wherein said complex regional pain syndrome is selected from the group consisting of a reflex sympathetic dystrophy (CRPS Type I) and a causalgia (CRPS Type II).

34. The method of claim 33, wherein said complex regional pain syndrome is selected from the group consisting of a diabetic neuropathy, a chemotherapy-induced pain, a treatment-induced pain, and a postmastectomy syndrome. The method of claim 28, wherein said neuropathic pain is a polyneuropathic pain.

36. The method of claim 28, wherein said neuropathic pain is a centrally-generated neuropathic pain.

37. The method of claim 36, wherein said centrally-generated neuropathic pain is selected from the group consisting of a dorsal root ganglion compression, an inflammation of the spinal cord, a contusion, a tumor of the spinal cord, a hemisection of the spinal cord, a tumor of the brainstem, a tumor of the thalamus, a tumor of the cortex, a trauma of the brainstem, a trauma of the thalamus and a trauma of the cortex. P:\WPDOCS\CRN\RMH\Spec\20423830 div claims doc 00 NC 38. The method of claim 28, wherein said neuropathic pain is a peripherially-generated neuropathic pain.

39. The method of claim 38, wherein said peripherially-generated neuropathic pain is selected from the group consisting of a neuroma, a nerve compression, a nerve crush, a _nerve stretch, a nerve entrapment and an incomplete nerve transsection. IND The method of claim 28, wherein said neuropathic pain is an allodynia, a hyperalgesia or C1 a hyperpathia. 00

41. The method of any one of claims 1 to 40, wherein said effective amount reduces the symptoms of said chronic pain by at least

42. The method of claim 41, wherein said effective amount reduces the symptoms of said chronic pain by at least

43. The method of claim 42, wherein said effective amount reduces the symptoms of said chronic pain by at least

44. The method of claim 43, wherein said effective amount reduces the symptoms of said chronic pain by at least The method of any one of claims 1 to 44, wherein said antagonist is administered peripherally or systemically.

46. The method of any one of claims 1 to 44, wherein said antagonist is administered orally.

47. The method of any one of claims 1 to 44, wherein said antagonist is administered in a sustained release formula.

48. The method of any one of claims 1 to 44, wherein said antagonist is administered in an bioerodible delivery system or a non-bioerodible delivery system.

49. Use of a selective persistent sodium channel antagonist as defined in any one of claims 1 to 19 for the manufacture of a medicament for the treating chronic pain in a mammal. P:\WPDOCS\CRN\RMH\Spec\20423830 div claims doc 00 O Use of claim 49, wherein said chronic pain is a neuropathic pain or wherein said chronic pain results from cancer or cancer treatment, diabetic neuropathy or fibromyalgia or wherein said chronic pain is associated with substance withdrawal or a headache, repetitive motion disorder pain, excessive muscle tension or inflammation. IN 00 0O