(19) TZZ ZZ ¥_T (11) EP 2 504 023 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 38/16 (2006.01) A61K 38/00 (2006.01) 15.07.2015 Bulletin 2015/29 C07K 14/435 (2006.01) (21) Application number: 10816761.0 (86) International application number: PCT/IB2010/002275 (22) Date of filing: 13.09.2010 (87) International publication number: WO 2011/033358 (24.03.2011 Gazette 2011/12) (54) NOVEL PEPTIDES ISOLATED FROM SPIDER VENOM, AND USES THEREOF NEUARTIGE AUS SPINNENGIFT ISOLIERTE PEPTIDE SOWIE VERWENDUNGEN DAVON NOUVEAUX PEPTIDES ISOLÉS DE VENIN D’ARAIGNÉE ET LEURS UTILISATIONS (84) Designated Contracting States: • F. BOSMANS: "Four Novel Tarantula Toxins as AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Selective Modulators of Voltage-Gated Sodium GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Channel Subtypes", MOLECULAR PL PT RO SE SI SK SM TR PHARMACOLOGY, vol. 69, no. 2, 1 January 2005 (2005-01-01), pages419-429, XP055031555, ISSN: (30) Priority: 15.09.2009 US 272336 P 0026-895X, DOI: 10.1124/mol.105.015941 • FRANK BOSMANS ET AL: "Deconstructing (43) Date of publication of application: voltage sensor function and pharmacology in 03.10.2012 Bulletin 2012/40 sodium channels", NATURE, vol. 456, no. 7219, 13 November 2008 (2008-11-13), pages 202-208, (73) Proprietor: Alomone Preclinical Ltd. XP055054589, ISSN: 0028-0836, DOI: 91042 Jerusalem (IL) 10.1038/nature07473 • PARK S P ET AL: "A tarantula spider toxin, (72) Inventors: GsMTx4, reduces mechanical and neuropathic • MEIR, Alon pain", PAIN, ELSEVIER SCIENCE PUBLISHERS, Jerusalem (IL) AMSTERDAM, NL, vol. 137, no. 1, 30 June 2008 • CHERKI, Ronit, Simcha (2008-06-30) , pages 208-217, XP025574189, Jerusalem (IL) ISSN: 0304-3959, DOI: 10.1016/J.PAIN. •KOLB,Ela 2008.02.013 [retrieved on 2008-03-24] Kiryat Moshe • Y. XIAO ETAL: "Tarantula Huwentoxin- IV Inhibits Jerusalem (IL) Neuronal Sodium Channels by Binding to • LANGUT, Yael Receptor Site 4 and Trapping the Domain II Jerusalem (IL) Voltage Sensor in the Closed Configuration", • BAJAYO, Nissim JOURNAL OF BIOLOGICAL CHEMISTRY, vol. Beit Hakerem 283, no. 40, 1 January 2008 (2008-01-01), pages Jerusalem (IL) 27300-27313, XP055054686, ISSN: 0021-9258, DOI: 10.1074/jbc.M708447200 (74) Representative: Becker Kurig Straus • DATABASE SWISS-PROT [Online] 16 June 2009 Patentanwälte XP008159659 Database accession no. P60975 Bavariastrasse 7 • CABRELE ET AL.: ’Redox-Active Cyclic Bis 80336 München (DE) (cysteinnyl)peptides as Catalysts for In Vitro Oxidative Protein Folding.’ CHEMISTRY & (56) References cited: BIOLOGY vol. 9, no. 6, June 2002, pages 731 - 740, US-A- 5 877 026 US-A1- 2004 192 886 XP026924884 US-A1- 2006 270 832 Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 504 023 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 504 023 B1 • OSTROW ET AL.: ’cDNA sequence and in vitro • DATABASE SWISS-PROT [Online] 16 June 2009 folding of GsMTx4, a specific peptide inhibitor of XP008159431 Database accession no. POC2P5 mechanosensitive channels.’ TOXICON. vol. 42, no. 3, 2003, pages 263 - 274, XP002375376 • SHCHERBATKO ET AL.: ’Voltage-Dependent Sodium Channel Function Is Regulated Through Membrane Mechanics.’ BIOPHYS J. vol. 77, no. 4, October 1999, pages 1945 - 1959, XP008159429 2 EP 2 504 023 B1 Description FIELD 5 [0001] The presently described subject matter relates to isolated spider venom peptides, which are used as a potent andselective ionchannel blockers and toa composition and methods for useof thepeptides inthe treatment of neuropathic pain. BACKGROUND 10 [0002] Voltage dependent sodium (Na v1) and in some cases low voltage activated calcium (Ca v3) channels are mem- brane proteins that in all excitable cells including neurons, lay in the basis of action potential generation and propagation. Inhibition of Na v1 channels causes a neuron to be incapable of firing action potentials and therefore incapable of encoding and transmitting information. 15 [0003] Neuropathic pain is a compound neuronal process involving both peripheral hyperexcitability and central sen- sitization. Peripheral hyperexcitability could be due to ectopic spontaneous firing of damaged dorsal root ganglion (DRG) neurons which is transmitted to the central nervous system (CNS) and sensed as pain. Tackling this phenomenon by specifically inhibiting Na v1 (or Ca v3) channels in damaged sensory nociceptive neurons in the periphery may be effective in reducing or eliminating neuropathic pain. This could be achieved by either local application of non-specific Na v blockers 20 (such as local anesthetics) or by systemic application of Nav1 blockers, which specifically recognize Nav1 channels in DRG neuron membranes. One generic approach to increase this specificity is by avoiding the CNS (in which most Na v1 isoforms are crucial for neuronal activity). The other approach is to find selective blockers to vNa1 isoforms, which expression is restricted to hyperexcitable DRG neurons (Devor, 2006; Cummins et al. 2007). One of the main issues regarding Nav1 blockers specificity is avoidance of cardiac blockade activity. 25 [0004] The Tetrodotoxin (TTX)-sensitive, Na v1.3, channel is normally expressed in the CNS and the peripheral nervous system (PNS) during the embryonic stage and its expression is heavily down regulated with maturation. However, up- regulation of the channel expression is reported following neuronal injury. These observations, suggest that specifically targeting Nav1.3 isoforms, could block exclusively damaged-hyperexcitable DRG neurons (Devor, 2006; Cummins et al. 2007). 30 [0005] The TTX-resistant, Na v1.8 channel is expressed almost exclusively in the PNS and has been shown to mediate most of the well documented TTX resistant component of DRG neurons action potentials. Recently, a paper has been published, emphasizing the importance of Nav1.8 channels as a target in pain control (Jarvis, M. F., et al. 2007; Zim- mermann, K., et al. 2007). [0006] The importance of treating pain, by targeting a protein which is expressed specifically in the periphery is high- 35 lighted by the case of ASIC1 channels which are expressed in the spinal cord and their inhibition induces analgesia in a rat model of neuropathic pain. However, administration of the peptide modulator, should be direct as in systemic administration the peptides do not cross the blood brain barrier to reach their target protein (Mazzuca, M., et al. 2007, Nat. Neurosci. 10, 943). [0007] Bosmans F. ("Four novel Tarantula toxins as selective modulators of voltage-gated sodium channel subtypes"; 40 Molecular Phramacology, Vol: 69, No. 2, Jan 1, 2005, p. 419-429) discloses four tarantula toxins as selective modulators of voltage-gated sodium channel subtypes. [0008] Park S. et al. ("A Tarantula spider toxin, GsMTx4, reduces mechanical and neuropathic pain"; Pain, Elsevier Science Publishers, Vol. 137, No. 1, Jun 30, 2008, p. 208-217) study the peptide GsMTx4 concluding that it reduces mechanical and neuropathic pain. 45 [0009] Xiao Y. et al. ("Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain II voltage sensor in the closed configuration"; Journal of Biological Chemistry, Vol. 283, No. 40, Jan 1, 2008, p. 27300-27313) analyze huwentoxin activity on VGSCs and disclose that it interacts with Nav1.7 suggesting the peptide can reduce pain behavior in rats. [0010] US 5,877,026 relates to analgesia-inducing peptides obtainable from venom of Grammostola spatulata. 50 SUMMARY [0011] Provided is a peptide isolated from spider venom, comprising an amino acid sequence as identified by SEQ ID NO: 3. Also provided is a C-terminally amidated form of the peptide. 55 [0012] Also, the present invention provides an isolated peptide consisting of an amino acid sequence as identified by SEQ ID NO: 3, and a C-terminally amidated form thereof. [0013] According to one embodiment, the isolated peptide is for treating pain. Preferably, the isolated peptide is for treating neurogenic and/or neuropathic pain; for treating cancer pain, post-surgical pain, oral or dental pain, pain from 3 EP 2 504 023 B1 referred trigeminal neuralgia, pain from post-herpetic neuralgia, or pain due to reflex sympathetic dystrophy, or for treating pain associated with an inflammatory condition, or for treating pain associated with one or more conditions selected from the group consisting of acute pain, migraine, headache pain, migraine headache, traumatic nerve injury, nerve compression, nerve entrapment, postherpetic neuralgia, trigeminal neuralgia, diabetic neuropathy, chronic lower back 5 pain, phantom limb pain, chronic pelvic pain, neuroma pain, complex regional pain syndrome, chronic arthritic pain, and pain associated with cancer, chemotherapy, HIV and HIV treatment-induced neuropathy, irritable bowel syndrome and related disorders and Crohns disease. [0014] According to another aspect, the isolated peptide is used as a medicament. [0015] According to another embodiment, a pharmaceutical composition is provided comprising the isolated peptide 10 and a pharmaceutically acceptable carrier or diluent. [0016] According to a further embodiment, the pharmaceutical