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  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/72—Nitrogen atoms
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  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
  • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
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  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• R 1 is a member selected from the group consisting of substituted or unsubstituted branched (C 3 -C 8 )alkyl, substituted or unsubstituted (C 3 -C 8 )cycloalkyl, substituted or unsubstituted (C 3 - C 8 )heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
• Ion channels of the KCNQ family have only recently been cloned and expressed. These voltage-gated potassium channels underlie the M-current, which functions to control resting membrane potential and excitability in a variety of neuronal cells (see, e.g., Wang et al, Science 282:1890-1893 (1998)). This application shows that KCNQ channels and M-currents are expressed in neurons of the dorsal root ganglion, which are an important part of the sensory neuronal pathway involved in pain.
• KCNQ channels e.g., KCNQ2/3 heteromeric channels
• the mechanism for treating pain by opening KCNQ channels was previously unknown.
• the present application provides a mechanism for treating pain disorders, and an assay for identifying compounds that open KCNQ potassium channels and reduce pain.
• Modulation of KCNQ-type channels therefore represents a novel approach to the treatment of pain, including both somatic and neuropathic pain.
• modulation of KCNQ channels is used for pain syndromes not treatable by opiates or non-steroidal anti-inflammatory drugs.
• Such drugs that open KCNQ channels should also be free of the side-effects associated with opiates (e.g., addiction and respiratory depression) or non-steroidal anti-inflammatory drugs (gastrointestinal ulceration).
• the number of punished lever presses on test days is compared to the mean on baseline days.
• the positive control chlordiazepoxide, increases punished lever pressing by > 50%.
• a compound that produces an increase of approximately 40% or greater is generally considered to be of interest as a rapid-onset anxiolytic.
• a selective KCNQ2/3 channel opener increased punished responding in a dose dependent, statistically significant manner (Figure 6).
• KCNQ modulator i.e., a selective KCNQ2/3 channel opener
• KCNQ channel openers can be used to treat anxiety disorders.
• modulators are identified using the in vitro and in vivo assays described herein.
• KCNQ polypeptide or “KCNQ subunit” refers to a polypeptide that is a subunit or monomer of a voltage-gated, KCNQ potassium channel, a member of the KCNQ gene family, and a member of the Kv superfamily of potassium channel monomers.
• KCNQ polypeptide e.g., KCNQ 1, 2, 3, or 4
• KCNQ 1, 2, 3, or 4 is part of a KCNQ potassium channel, either a homomeric or heteromeric potassium channel, the channel has voltage-gated activity.
• KCNQ polypeptide therefore refers to polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have a sequence that has greater than about 60% amino acid sequence identity, preferably about 65, 70, 75, 80, 85, 90, or 95% amino acid sequence identity, to a KCNQ gene family member such as those described in Biervert et al, Science 279:403-406, Kubisch et al, Cell 96:437-446 (1999), Yang et al, J. Biol. Chem. 273: 19419-19423 (1998); Wang et al, Nature Genet. 12:17 (1996); Wei et al, Neuropharmacol. 35:805 (1996); Singh et al, Nature Genet.
• KCNQ potassium channels, KCNQ polynucleotides, and KCNQ nucleic acids are identified, isolated, expressed, purified, and expressed in recombinant cells according to methods well known to those of skill in the art.
• Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5x SSC and 1% SDS incubated at 42° C or 5x SSC and 1% SDS incubated at 65° C, with a wash in 0.2x SSC and 0.1% SDS at 65° C.
• Algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al, J. Mol Biol. 215:403-410 (1990), respectively.
• W wordlength
• E expectation
• Inhibitors refer to inhibitory or activating molecules identified using in vitro and in vivo assays for KCNQ channel function.
• inhibitors, activators, and modulators refer to compounds that increase KCNQ channel function, thereby reducing pain in a subject, as assayed using a formalin algesia test or a hotplate test in vivo, or thereby reducing anxiety in a subject, as assayed using a Geller conflict test (see Example section).
• Inhibitors are compounds that decrease, block, prevent, delay activation, inactivate, desensitize, or down regulate the channel, or speed or enhance deactivation.
• Beta subunit is a polypeptide monomer that is an auxiliary subunit of a potassium channel composed of alpha subunits; however, beta subunits alone cannot form a channel (see, e.g., U.S. Patent No. 5,776,734).
• Beta subunits are known, for example, to increase the number of channels by helping the alpha subunits reach the cell surface, change activation kinetics, and change the sensitivity of natural ligands binding to the channels. Beta subunits can be outside of the pore region and associated with alpha subunits comprising the pore region. They can also contribute to the external mouth of the pore region.
• the membrane potential of cells depends primarily on their potassium channels and is typically between -60 and -100 mV for mammalian cells. This value is also known as the "reversal potential" or the "Nemst” potential for potassium. Some voltage-gated potassium channels undergo inactivation, which can reduce potassium efflux at higher membrane potentials. Potassium channels can also allow potassium influx in certain instances when they remain open at membrane potentials negative to E (see, e.g., Adams & Nonner, in Potassium Channels, pp. 40-60 (Cook, ed., 1990)).
• the characteristic of voltage gating can be measured by a variety of techniques for measuring changes in current flow and ion flux through a channel, e.g., by changing the [K + ] of the external solution and measuring the activation potential of the channel current (see, e.g., U.S. Patent No. 5,670,335), by measuring current with patch clamp techniques or voltage clamp under different conditions, and by measuring ion flux with radiolabeled tracers or voltage-sensitive dyes under different conditions.
• saturated hydrocarbon radicals include groups such as methyl, ethyl, n- propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n- octyl, and the like.
• An unsaturated alkyl group is one having one or more double bonds or triple bonds.
• alkylene by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by -CH 2 CH CH 2 CH 2 -, and further includes those groups described below as “heteroalkylene.”
• an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
• a “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
• alkoxy alkylamino and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.
• heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
• the heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group.
• the heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule.
• heteroalkylene by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by -CH - CH 2 -S-CH 2 CH 2 - and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 -.
• heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).
• cycloalkyl and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively.
• heterocycloalkyl a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include eye lopentyl, cyclohexyl, 1-cyclohexenyl, 3- cyclohexenyl, cycloheptyl, and the like.
• arylalkyl is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (l-naphthyloxy)propyl, and the like).
• alkyl group e.g., benzyl, phenethyl, pyridylmethyl and the like
• an oxygen atom e.g., phenoxymethyl, 2-pyridyloxymethyl, 3- (l-naphthyloxy)propyl, and the like.
• R', R" and R'" each independently refer to hydrogen, unsubstituted (C ⁇ -C 8 )alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C ⁇ -C 4 )alkyl groups.
• R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7- membered ring.
• -NR'R is meant to include l-pyrrolidinyl and 4- morpholinyl.
• Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CH 2 ) q -U-, wherein T and U are independently -NH-, -O-, -CH 2 - or a single bond, and q is an integer of from 0 to 2.
• pharmaceutically acceptable salts or “pharmaceutically acceptable carrier” is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
• base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
• pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
• acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
• Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
• inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and
• the neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
• the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
• Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
• Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.
• Samples or assays that are treated with a potential potassium channel inhibitor or activator are compared to control samples without the test compound, to examine the extent of modulation.
• Control samples (untreated with activators or inhibitors) are assigned a relative potassium channel activity value of 100.
• Inhibition of KCNQ channels is achieved when the potassium channel activity value relative to the control is about 90%, preferably 50%, more preferably 25-0%.
• Activation of KCNQ channels is achieved when the potassium channel activity value relative to the control is 110%, more preferably 150%, more preferably 200-500% higher, preferably 1000% or higher.
• the effects of the test compounds upon the function of the channels can be measured by changes in the electrical currents or ionic flux or by the consequences of changes in currents and flux.
• Changes in electrical current or ionic flux are measured by either increases or decreases in flux of ions such as potassium or rubidium ions.
• the cations can be measured in a variety of standard ways. They can be measured directly by concentration changes of the ions or indirectly by membrane potential or by radio- labeling of the ions. Consequences of the test compound on ion flux can be quite varied. Accordingly, any suitable physiological change can be used to assess the influence of a test compound on the channels of this invention.
• the effects of a test compound can be measured by a toxin binding assay.
• the compounds tested as modulators of KCNQ channels can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid.
• test compounds will be small chemical molecules and peptides.
• any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
• the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St.
• combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al, Nature 354:84-88 (1991)).
• Other chemistries for generating chemical diversity libraries can also be used.
• Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No.
• N-aryl benzamide compounds of the present invention can be prepared using standard procedures as outlined in Scheme la below.
• an N-phenyl benzamide i, wherein Y 1 and Y 2 represent substituents, including multiple substituents on the aryl groups
• reagents such as Lawessons's reagent to provide the thioamides, ii.
• Alkylation of ii, with, for example, methyl iodide produces iii which can be converted to target structures iv, v and vi.
• treatment of iii with sodium hydride (or another suitable base) and sulfamide provides the sulfamoylimino derivative, iv.
• treatment of iii with sodium hydride or another base, followed by cyanamide provides v. Conversion of v to vi can be accomplished with HC1.
• an aromatic amine of the invention is converted to the corresponding isothiocyanate by the action of thiophosgene.
• the resulting isothiocyanate is coupled to an amine of the invention, thereby forming either a homo- or heterodimeric species.
• the isothiocyanate is coupled with an amine-containing backbone, such as polylysine, thereby forming a conjugate between a polyvalent framework and a compound of the invention.
• the polylysine is underlabeled with the first isothiocyanate and subsequently labeled with one or more different isothiocyanates.
• a mixture of isothiocyanates is added to the backbone. Purification proceeds by, for example, size exclusion chromatography, dialysis, nanofiltration and the like.
• compositions of the present invention are determined in part by the particular composition being administered (e.g., nucleic acid, protein, modulatory compounds or transduced cell), as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 17 ed., 1989).
• Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
• liquid solutions such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or PEG 400
• capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
• suspensions in an appropriate liquid such as water, saline or PEG 400
• Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
• Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
• a flavor e.g., sucrose
• an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
• Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base.
• Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
• gelatin rectal capsules which consist of a combination of the compound of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
• Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
• compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
• Parenteral administration, oral administration, and intravenous administration are the prefe ⁇ ed methods of administration.
• the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
• Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.
• the pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component.
• the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
• the composition can, if desired, also contain other compatible therapeutic agents.
• the compounds utilized in the pharmaceutical method of the invention are administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily.
• the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed.
• the dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time.
• the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
• Example 1 Expression of KCNO2 and KCNO3 mRNA in human dorsal root ganglion
• KCNQ2 and KCNQ3 mRNA were detected by PCR amplification of human dorsal root ganglion (DRG) cDNA.
• cDNA was prepared by reverse transcription of total RNA from human DRG using standard procedures.
• a second mock reverse-transcription reaction was also performed, which was identical to the first, except for the omission of the reverse transcriptase.
• 35 cycles of amplification were performed on a single microliter of human DRG cDNA, using oligonucleotide primers designed to amplify either KCNQ2 or KCNQ3.
• the oligonucleotides used for the amplification were: 5'-
• the oligonucleotides were: 5'- GCCCACGGTCCTGCCTATCTT-3' (sense) and 5'-CATTGGTGTCCCCGCTGGTAA- 3' (antisense).
• PCR amplified DNA fragments were separated by agarose gel electrophoresis, visualized using ethidium bromide staining and sized by comparison to DNA fragments of known size (see Figure 1; M, lane 1).
• PCR failed to amplify KCNQ2 or KCNQ3 fragments from reverse-transcription samples generated in the absence of reverse transcriptase (-), indicating that the DRG RNA samples were not contaminated with genomic DNA.
• PCR did however, amplify both KCNQ2 (lane 3) and KCNQ3 (lane 5) from reverse transcribed cDNA samples, indicating that KCNQ2 and KCNQ3 mRNA are expressed in human dorsal root ganglion.
• Example 2 Expression of recombinant KCNO2/3 channel CHO cells
• a cloned KCNQ2/3 channel was expressed in Chinese hamster ovary cells (CHO-K1 cells) according to standard methodology.
• CHO-K1 cells were transfected with human KCNQ2/3 nucleic acid using lipofectamine reagent according to the manufacturer's instructions.
• Cells stably expressing KCNQ2/3 were identified by their resistance to G418 (400 ⁇ g/ml).
• CHO-K1 cells stably transfected with the KCNQ2/3 tandem construct were maintained in Ham's F-12 supplemented with 10% heat- inactivated fetal bovine serum and 400 ⁇ g/ml G418 in an incubator at 37°C with a humidified atmosphere of 5% CO 2 .
• a benzanilide KCNQ channel opener was applied to the cells. The compound increased holding current at —40 mV (see Figure 2) and hyperpolarized the membrane potential.
• Example 3 Expression of endogenous KCNO2/3 channel in DRGs
• DRGs were isolated from 1 day old Sprague-Dawley rats. DRGs were dissociated using trypsin (0.25%) and protease type XXIII (2 mg/ml) and neurons were maintained in culture in 90% Eagles MEM (without L-glutamate), 10% FCS, 100 U/m; penicillin, 100 g/ml streptomycin, in an incubator at 37°C with a humidified atmosphere of 5% CO 2 .
• a benzanilide KCNQ channel opener was applied to the cells. The opener increased holding current at -30 mV and hyperpolarized the membrane potential (see Figure 3).
• Example 4 In vivo formalin algesia test The analgesic effect of a KCNQ modulator was assessed in vivo, using the formalin algesia test. All animal experiments were conducted in accordance with the Declaration of Helsinki and with the guide for the care and use of laboratory animals.
• Example 5 In vivo Hotplate test for pain
• Example 6 In vivo Geller conflict test for anxiolytics In the Geller conflict test (see, e.g., Geller & Seifter,
• a compound with selective KCNQ2/3 channel opening activity increased punished responding in a dose-dependent manner (see Figure 6).
• the increase in punished responding was statistically significant(paired t-test p ⁇ 0.05) at 10, 17, 30, and 56 mg/kg PO with increases of 40% or greater at 30 and 56 mg/kg. Responding in the unpunished phase was not disrupted, indicating that the animals were not impaired at the doses tested.

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