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WO 2009/038722 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (43) International Publication Date PCT (10) International Publication Number 26 March 2009 (26.03.2009) WO 2009/038722 Al (51) International Patent Classification: (74) Agents: SHEA, Timothy, J., Jr. et al; Sterne, Kessler, AOlN 43/90 (2006.01) Goldstein & Fox P.L.L.C, 1100 New York Avenue, N.W., (21) International Application Number: Washington, DC 20005-3934 (US). PCT/US2008/0 10823 (81) Designated States (unless otherwise indicated, for every (22) International Filing Date: kind of national protection available): AE, AG, AL, AM, 17 September 2008 (17.09.2008) AO, AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, (25) Filing Language: English CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, (26) Publication Language: English EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, (30) Priority Data: LR, LS, LT, LU, LY,MA, MD, ME, MG, MK, MN, MW, 60/973,080 17 September 2007 (17.09.2007) US MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, (71) Applicant (for all designated States except US): RED- RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TJ, POINT BIO CORPORATION [US/US]; 7 Graphics TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, Drive, Ewing, NJ 08628 (US). ZW (71) Applicants and (72) Inventors: LEE, S., Paul [US/US]; 5 Aspen Court, New (84) Designated States (unless otherwise indicated, for every ton, PA 18940 (US). BUBER, TuIu [US/US]; 32 Redwood kind of regional protection available): ARIPO (BW, GH, Drive, Newton, PA 18940 (US). CERNE, Rok [SI/US]; GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, 1518 White Pine Circle, Lawrenceville, NJ 08648 (US). ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), BRYANT, Robert, W. [US/US]; 133 Sayre Drive, Prince European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, ton, NJ 08540 (US). FR, GB, GR, HR, HU, IE, IS, IT, LT,LU, LV,MC, MT, NL, [Continued on next page] (54) Title: MODULATION OF THE COOPERATIVITY BETWEEN THE ION CHANNELS TRPM5 AND TRPAl (57) Abstract: The present invention is related to modulating TRPAl ion channel activity by targeting the ion channel TRPM5. The cooperativity between the ion channels can be used to modulate pain, mechanosensation and taste responses triggered through TRPAl by modulating the activity of TRPM5. FIG.1 NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, Published: MODULATION OF THE COOPERATIVITY BETWEEN THE ION CHANNELS TRPM5 AND TRPAl BACKGROUND OF THE INVENTION Field of the Invention [0001] The present invention is related to modulating TRPAl ion channel activity by targeting the ion channel TRPM5 and vice versa through the cooperativity mechanism identified herein. More specifically, the present invention relates to methods of modulating pain, mechanosensation and taste responses triggered through the ion channels. Background [0002] Ion channels are transmembrane proteins that form pores in a membrane and allow ions to pass from one side to the other (reviewed in B. Hille (Ed), 1992, Ionic Channels of Excitable Membranes 2nd ed., Sinauer, Sunderland, Mass.). Several ion channels have been shown to be essential for taste transduction (Perez et al, Nature Neuroscience 5:1 169-1176 (2002); Zhang et al, Cell 772:293-301 (2003)). The effects that well known taste compounds have on ion channel activity have also begun to be analyzed. For example, menthol has been shown to activate the transient receptor potential (TRP) channel M8 (TRPM8) (Behrendt, H.-J., et al, Brit. J. Pharm. 141:131- 745 (2004)). [0003] The TRP channel Al (TRPAl) is also a member of the superfamily of TRP channels. TRPAl was initially described as a cold sensitive, nonselective cation channel (Story, G.M. et al, Cell 772:819-829 (2003)), but it also functions as a ligand-gated channel in heterologous expression systems and sensory neurons. (Ramsey, LS. et al, Ann. Rev. Physiol. 65:619-647 (2006)). Noxious stimuli, including natural compounds such as cinnamaldehyde and the ingredients in mustard (allyl isothiocyanate, AITC), cold temperatures and environmental irritants all activate TRPAl (Jordt, S.E., et al, Nature 427:260-265 (2004); Macpherson, L.J., et al, Curr. Biol. 75:929-934 (2005); Macpherson, L.J., et al, Nature 445:541-545 (2007); Bautista, D.M. et al, Proc. Natl. Acad. ScL USA 702:12248-12252 (2005); Bandell, M., et al, Neuron 47:849-857 (2004); Kwan, K.Y., et al, Neuron 50:277-289 (2006)). TRPAl has also been shown to be important in responses to pain. (Bautista, D.M. et al, Cell 724:1269-1282 (2006); Trevisani et al. Proc. Natl Acad. Sd. USA 704:13519-13524 (2007)). [0004] Recent studies have shown noxious stimuli activate TRPAl through an unusual mechanism involving covalent modification of cysteine and lysine residues within the N-terminal cytoplasmic domain of the channel protein (Hinman, A., et al, Proc. Natl. Acad. Sd. USA 705:19564-19568 (2006); Macpherson, LJ. et al (2007)). In addition, one model suggests TRPAl activation by bradykinin, a potent algogenic (pain related) substance released in response to tissue injury and inflammation, occurs through two possible mechanisms: (1) through PLC-mediated increases in intracellular Ca2+ or other metabolites; or (2) via Ca2+ influx through TRPVl (Dorener, J.F. et al, J. Biol. Chem. 282: 13180-13189 (2007); Bautista, D.M. et al, (2006); Akopain et al. J. Physiol. 553:175-193 (2007)). [0005] Pain is a sensory experience distinct from sensations of touch, pressure, heat and cold. It is often described by sufferers by such terms as bright, dull, aching, pricking, cutting or burning and is generally considered to include both the original sensation and the reaction to that sensation. This range of sensations, as well as the variation in perception of pain by different individuals, renders a precise definition of pain difficult, however, many individuals suffer with severe and continuous pain. [0006] TRPM5 is another member of the TRP superfamily. TRPM5 is believed to be activated by stimulation of a receptor pathway coupled to 2+ phospholipase C and by IP3-mediated Ca release. The opening of this channel is dependent on a rise in Ca2+ levels (Hofmann et al, Current Biol. 73:1 153-1 158 (2003)). TRPM5 is also a necessary part of the taste-perception machinery and has been shown to play a role in bitter, sweet and umami taste (Talavera, K. et al, Nature 435:1022-1025 (2005)). [0007] An earlier study that analyzed TRP channel distribution in mice demonstrated that TRPM5 expression is quite limited (Kunert-Keil et al. BMC Genomics, 7:159 (2006)). This earlier study did not identify TRPM5 expression in nerve tissue or its association with pain. [0008] Therefore, there exists a need in the art to provide a method to modulate the activity of these ion channels. The present invention identifies a cooperativity mechanism between TRPAl and TRPM5. Identification of this mechanism allows for the specific modulation of the cognate channels through their common pathway. The common pathway also provides the basis for modulating their activity, especially with respect to modulating taste, mechanosensation and decreasing pain responses. BRIEF SUMMARY OF THE INVENTION [0009] A new cooperativity between the ion channels TRPAl and TRPM5 has been identified. The common pathway provides the basis for modulating their activity, especially with respect to modulating taste, mechanosensation and decreasing pain responses. [0010] An embodiment of the invention is a method for modulating TRPAl- mediated processes comprising administering a modulator of TRPM5 activity. In one embodiment, the TRPAl and TRPM5 are human hi another embodiment, the administration is done in vivo. In yet another embodiment, the TRPAl is present in a TRPM5-expressing cell or cultured neuron. In a further embodiment, the modulated processes are selected from the group consisting of pain, mechanosensation and taste. The TPRM5 activities may be either increased or decreased. [0011] In another embodiment, the invention relates to inhibiting TRPAl- mediated pain signaling by inhibiting TRPAl activity, comprising administering to a subject in need thereof an inhibitor of TRPM5 expression. In one embodiment, the TRPAl is present in a TRPM5-expressing cell or cultured neuron. In another embodiment, the TRPAl and TRPM5 are human. In a further embodiment, TRPM5 expression is inhibited using RNA interference, antisense oligonucleotides, ribozymes, aptamers or antibodies. In yet another embodiment, the TRPAl activity is measured by measuring calcium influx in said TRPAl -expressing cell or by measuring the enzymatic activity of the phospholipase C polypeptide. The enzymatic activity can be the breakdown of phosphatidylinositol-4,5-bisphospate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). In another embodiment, the type of pain is selected from the group consisting of acute, chronic, neuropathic and nociceptive. [0012] In another embodiment, the invention relates to a method of inhibiting TRPAl -mediated signaling comprising administering an inhibitor of TRPM5. [0013] In another embodiment, the invention relates to a method of increasing TRPAl expression in a cell comprising expressing TRPM5 in said cell. In one embodiment, TRPM5 expression is at a greater level than expressed in wild- type cells. In another embodiment, the TRPM5 is exogenously added to said TRPAl expressing cell. [0014] In another embodiment, the invention relates to a method of amplifying TRPM5 activation comprising administering an activator of TRPAl activity. In one embodiment, the activator of TRPAl is selected from the group consisting of cinnamaldehyde, eugenol, gingerol, methyl salicylate, AITC and allicin.
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