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Vm23 and Vm24, Two Scorpion Peptides That Block Human T-Lymphocyte Potassium Channels (Sub-Type Kv1.3) with High Selectivity

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Vm23 and Vm24, Two Scorpion Peptides That Block Human T-Lymphocyte Potassium Channels (Sub-Type Kv1.3) with High Selectivity (19) & (11) EP 2 158 213 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 14/435 (2006.01) A61K 38/04 (2006.01) 14.12.2011 Bulletin 2011/50 G01N 33/68 (2006.01) G01N 33/94 (2006.01) A61P 37/06 (2006.01) (21) Application number: 07734817.5 (86) International application number: (22) Date of filing: 14.05.2007 PCT/IB2007/001544 (87) International publication number: WO 2008/139243 (20.11.2008 Gazette 2008/47) (54) VM23 AND VM24, TWO SCORPION PEPTIDES THAT BLOCK HUMAN T-LYMPHOCYTE POTASSIUM CHANNELS (SUB-TYPE KV1.3) WITH HIGH SELECTIVITY VM23 UND VM24, ZWEI SKORPIONPEPTIDE, DIE HUMANE T-LYMPHOZYTEN-KALIUMKANÄLE (UNTERTYP KV1.3) MIT HOHER SELEKTIVITÄT BLOCKIEREN VM23 ET VM24, DEUX PEPTIDES DE SCORPION QUI BLOQUENT LES CANAUX POTASSIQUES DES LYMPHOCYTES T HUMAINS (SOUS-TYPE KV1.3) AVEC UNE SÉLECTIVITÉ ÉLEVÉE (84) Designated Contracting States: (74) Representative: Daniels, Jeffrey Nicholas AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Page White & Farrer HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE Bedford House SI SK TR John Street London WC1N 2BF (GB) (43) Date of publication of application: 03.03.2010 Bulletin 2010/09 (56) References cited: EP-A- 0 916 681 WO-A-2006/116156 (73) Proprietor: UNIVERSIDAD NACIONAL AUTONOMA DE MEXICO • BATISTA CESAR V F ET AL: "Two novel toxins C.P. 04510 Mexico from the Amazonian scorpion Tityus cambridgei D.F. Mexico (MX) that block Kv1.3 and Shaker B K+- channels with distinctly different affinities." BIOCHIMICA ET (72) Inventors: BIOPHYSICAACTA, vol.1601, no. 2, 16 December • POSSANI POSTAY, Lourival, Domingos 2002 (2002-12-16), pages 123-131, XP002464263 CEP-62210 Morelos (MX) ISSN: 0006-3002 • GURROLA-BRIONES, Georgina • OLAMENDI-PORTUGALET AL: "Novel alpha- KTx CEP-62210 Morelos (MX) peptides from the venom of the scorpion • SALAS-CASTILLO, Saida Patricia Centruroides elegans selectively blockade Kv1.3 CEP-92880 Veracruz (MX) over IKCa1 K<+> channels of T cells" TOXICON, • FERREIRA BATISTA, Cesar Vicente ELMSFORD, NY, US, vol. 46, no. 4, 15 September CEP-62210 Morelos (MX) 2005 (2005-09-15), pages 418-429, XP005045659 • VARGA, Zoltán, S. ISSN: 0041-0101 H-Debrecen 4028 (HU) • DE LA VEGA RICARDO C RODRIGUEZ ET AL: • PANYI, György "Current views on scorpion toxins specific for H-40314 Debrecen (HU) K+-channels" TOXICON, vol. 43, no. 8, 15 June • GÁSPÁR, Rezsö 2004 (2004-06-15), pages 865-875, XP002464264 H-Debrecen 4034 (HU) ISSN: 0041-0101 cited in the application 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 2 158 213 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 158 213 B1 • GIANGIACOMO KATHLEEN M ET AL: "Molecular • PANYI GYRGY ET AL: "Ion channels and basis of alpha-KTx specificity" TOXICON, vol. 43, lymphocyte activation" IMMUNOLOGY no. 8, 15 June 2004 (2004-06-15), pages 877-886, LETTERS, vol. 92, no. 1-2, 29 March 2004 XP002464265 ISSN: 0041-0101 cited in the (2004-03-29), pages 55-66, XP002464267 & 12TH application SYMPOSIUM ON SIGNALS AND SIGNAL • TYTGAT J ET AL: "A unified nomenclature for PROCESSINGIN THE IMMUNE SYSTEM - AN EFIS short-chain peptides isolated from scorpion (EUROPEAN FEDERATION; SOPRON, venoms: alpha-KTx molecular subfamilies" HUNGARY; SEPTEMBER 03-07, 2003 ISSN: TRENDS IN PHARMACOLOGICAL SCIENCES, 0165-2478 cited in the application ELSEVIER, HAYWARTH, GB, vol. 20, no. 11, 1 • VARGA Z ET AL: "Involvement of membrane November 1999 (1999-11-01), pages 444-447, channels in autoimmune disorders" CURRENT XP004182937 ISSN: 0165-6147 cited in the PHARMACEUTICAL DESIGN 2007 application NETHERLANDS, vol. 13, no. 24, 2007, pages • PANYI G ET AL: "K+ channel blockers: Novel 2456-2468, XP002464268 ISSN: 1381-6128 tools to inhibit T cell activation leading to specific immunosuppression" CURRENT PHARMACEUTICAL DESIGN, vol. 12, no. 18, 2006, pages 2199-2220, XP002464266 ISSN: 1381-6128 cited in the application 2 EP 2 158 213 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention relates generally to the fields of biochemistry, molecular biology, immunology and elec- trophysiology. Disclosed are peptides, their pharmaceutical compositions, and methods for their use to block Kv1.3 potassium channels, their use for the treatment of various immunological conditions and for diagnostic applications, and methods for the chemical synthesis and correct folding procedures for peptides corresponding to two protein components isolated from the venom of the Mexican scorpion Vaejovis mexicanus smithi (here abbreviated thereafter: V. mexicanus), 10 which constitute a new sub-family of potassium channel specific ligands, capable of blocking with high affinity and specificity a sub-type of potassium channels (hKv1.3) shown to be implicated in immunological diseases and graft rejections. Methods and techniques used for their chemical and functional characterization are disclosed, as well as the results of in vivo experiments on the DTH-response of sensitized rats when treated with Vm24. This peptide (Vm24), its homologous Vm23 and their functional equivalent analogs are lead compounds, candidates for the treatment of various 15 immunological conditions and diagnostic applications. BACKGROUND OF THE INVENTION General considerations 20 [0002] Several aspects should be taken into consideration concerning the subject of the invention reported here, among which are the important advances in the fields of biochemistry, molecular biology, immunology and electrophys- iology related to the knowledge generated about: 25 1)- the presence of integral proteins of biological membranes termed "ion-channels" playing a fundamental role in cellular communication, signal transduction pathways and general homeostasis of tissues and various organ func- tions; 2)- different levels of expression of these channels in cells of the immune system, mainly in T-lymphocytes, shown 30 to play a clear role in events related to the onset of autoimmune diseases; 3)- the possible control of the channel function, hence possible treatment of disorders, through the addition of various chemicals, natural ligands and synthetically prepared substances, either reproducing the ligand as found in nature or preparing similar derivatives (peptidomimetics). 35 [0003] A multitude of potassium (K) channels have been discovered and reported to exist in the last fifteen years permitting their isolation, individual expression and functional analysis. [0004] They are multimeric proteins implicated in the determination of cell membrane potential, thereby controlling smooth muscle tone, synaptic excitability, neurotransmitter release, and other processes. In this invention we want to 40 emphasize the importance of a K channel species, sub-type Kv1.3, and its role in lymphocyte proliferation and in the control of autoimmune diseases by means of inhibiting this channel. This is a delayed-rectifier channel predominantly expressed in T lymphocytes [Grissmer et al., 1990; Lewis and Cahalan, 1995], different from the sub- types Kv1.1, Kv1.2 widely distributed in brain or Kv1.5 in heart tissue, to mention just a few of the sub-types of K channels. [0005] The mechanisms by which modulation of Kv1.3 channels activity affects lymphocyte proliferation are being 45 investigated in several laboratories and were the object of many recent publications (reviewed in [Beeton and Chandy, 2005; Judge and Bever, 2006, Panyi et al., 2006], including some patents (v.gr. US 5,397,702 by Cahalan et al. 1995 and US 6,077,680 by Kem et al. 2000). [0006] Autoimmune diseases are known for their considerable worldwide morbidity. Among these diseases are: type- 1 diabetes mellitus (insulin dependent), multiple sclerosis (MS), rheumatoid arthritis, Sjogren’s syndrome, mixed con- 50 nective tissue disease, systemic lupus erythematosus (SLE), myasthenia gravis, to mention just some of them. A relevant experimental model for autoimmune diseases is the experimental autoimmune encephalomyelitis (EAE). It is generally accepted that these autoimmune diseases result from the response of the immune system destroying specific tissues, either by a direct attack to the cells, or by producing auto-antibodies. The over-expression of Kv1.3 channels is a characteristic feature of autoreactive T cells thereby providing an excellent opportunity for the modification of their 55 proliferation by blockers of Kv 1.3. [0007] In these lines of research and experimentation, several substances were described and even patented. One such example is the toxin ShK from the sea anemone Stichodactyla helianthus and several derivatives of it, claimed to have a protective effect against several autoimmune diseases (v.gr. US 6,077,680 by Kem et al. 2000). 3 EP 2 158 213 B1 [0008] Among other natural ligands that are capable of affecting the function of ion- channels are toxic peptides isolated from scorpion venoms. K channel specific peptides isolated from these venoms are short-chain peptides containing 22 to 42 amino acids compacted by either three or four disulfide bridges. They are blockers of many different sub- types of channels, with a huge variability in selectivity and affinity (reviewed in [Giangiacomo et al., 2004; Rodriguez de la Vega 5 and Possani, 2004]). For example, charybdotoxin is a potent blocker of Kv1.1, Kv1.2 and 1.3Shaker type delayed rectifier channels but also blocks maxi-type K(Ca) channels [Rauer et al., 2000]. Margatoxin, another scorpion venom peptide, lacks K(Ca) channel blocking activity, but maintains a high affinity block of Kv1.3 channels. [Garcia-Calvo et al., 1993]. Agitoxin, noxiustoxin, kaliotoxin are examples of scorpion toxins that affect different types of K channels with distinct affinities and selectivities, but usually modify more than one sub-types of channels (recent reviewed in [Panyi et 10 al., 2006]).
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