(19) TZZ_¥¥ _T (11) EP 1 866 332 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C07K 14/46 (2006.01) G01N 33/68 (2006.01) 17.09.2014 Bulletin 2014/38 A61K 47/42 (2006.01) (21) Application number: 06721611.9 (86) International application number: PCT/BR2006/000052 (22) Date of filing: 17.03.2006 (87) International publication number: WO 2006/096953 (21.09.2006 Gazette 2006/38) (54) Use of Crotamine and Composition Verwendung von Crotamin und Zusammensetzung Utilisation de la crotamine et composition (84) Designated Contracting States: (74) Representative: Denjean, Eric et al AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Cabinet Laurent & Charras HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI "Le Contemporain" SK TR 50, Chemin de la Bruyère 69574 Dardilly Cedex (FR) (30) Priority: 18.03.2005 BR PI0501037 (56) References cited: (43) Date of publication of application: • KERKIS A ET AL: "Crotamine is a novel cell- 19.12.2007 Bulletin 2007/51 penetratingprotein from the venom of rattlesnake crotalus durissus terrificus" FASEB,, vol. 18, 1 (73) Proprietor: Fundacao de Amparo a Pesquisa do September 2004 (2004-09-01), pages 1407-1409, Estado de Sao XP002478903 Paulo - FAPESP • MORRIS M C ET AL: "TRANSLOCATING CEP-05468-901 Sao Paulo, SP (BR) PEPTIDES AND PROTEINS AND THEIR USE FOR GENE DELIVERY" CURRENT OPINION IN (72) Inventors: BIOTECHNOLOGY, LONDON, GB, vol. 11, no. 5, • YAMANE, Tetsuo 1 October 2000 (2000-10-01), pages 461-466, Butantã XP001022944 ISSN: 0958-1669 Brazil 05579-010 São Paulo, Sp (BR) • FUTAKI SHIROH ET AL: "Arginine- rich peptides: • KERKIS, Irina An abundant source of membrane-permeable 01421-001 São Paulo, SP (BR) peptides having potential as carriers for • KERKIS, Alexandre intracellular protein delivery" JOURNAL OF 01421-001 São Paulo, SP (BR) BIOLOGICAL CHEMISTRY, AL, vol. 276, no. 8, 17 • BAPTISTA, Gandhi, Rádis November 2000 (2000-11-17), pages 5836-5840, 04084-006 São Paulo, Sp (BR) XP002210171 ISSN: 0021-9258 • HAYASHI, Mirian, Akemi, Furuie • OGUIURA ET AL: "New view on crotamine, a 01403-020 São Paulo, SP (BR) small basic polypeptide myotoxin from South • KONNO, Katsuhiro American rattlesnake venom" TOXICON, 05503-900 São Paulo, SP (BR) ELMSFORD, NY, US, vol. 46, no. 4, 15 September • DA SILVA, Alvaro, Rossan, B., P. 2005 (2005-09-15), pages 363-370, XP005045652 01324-001 São Paulo, SP (BR) ISSN: 0041-0101 • PEREIRA, Lygia, da Veiga 05508-900 São Paulo, SP (BR) • DE OLIVEIRA, Eduardo, Brandt 14049-900 Ribeirao Preto, SP (BR) 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 1 866 332 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 1 866 332 B1 • KERKIS A. ET AL.: ’Crotamine is a novel cell- • SANTOS M C DOS ET AL: ’Purification and penetratingprotein from the venom of rattlesnake properties of a crotamine analog from Crotalus Crotalus durissus terrificus’ FASEB J. vol. 18, no. durissus ruruima venom’ TOXICON vol. 31, no. 2, 12, September 2004, pages 1407 - 1409, 1993, & IVTH PAN AMERICAN SYMPOSIUM ON XP002478903 ANIMAL, PLANT AND MICROBIAL TOXINS (IST); • NICASTRO G. ET AL.: ’Solution structure of CAMPINAS, BRAZIL; JULY 27-31, 1992, page 166, crotamine, a Na+ channel affecting toxin from XP008160555 ISSN: 0041-0101 -& DATABASE Crotalus durissus terrificus venome’ EUR. J. Uniprot, [Online] 11 October 2004 (2004-10-11), BIOCHEM. vol. 270, no. 9, May 2003, pages 1969 DOS SANTOS M C ET AL: "Purification and - 1979, XP003022428 properties of a crotamine analog from Crotalus • LUNDBERG P. ET AL.: ’A brief introduction to durissus ruruima venom", XP007921611, cell-penetrating peptides’ J. MOL. RECOGNIT. retrieved from Uniprot Database accession no. vol. 16, no. 5, September 2003 - October 2003, P63327 pages 227 - 233, XP003022429 • FRANC BENJAMIN L ET AL: "Breaching •G.Rádis- Baptista ET AL: "Nucleotide sequence biological barriers: protein translocation of crotamine isoform precursors from a single domains as tools for molecular imaging and South American rattlesnake (Crotalus durissus therapy", MOLECULAR IMAGING, MIT PRESS, terrificus)", Toxicon, vol. 37, no. 7, 1 July 1999 US, vol. 2, no. 4, 1 October 2003 (2003-10-01) , (1999-07-01), pages973-984, XP055055102, ISSN: pages 313-323, XP009118803, ISSN: 1535-3508, 0041-0101, DOI: 10.1016/S0041-0101(98)00226-8 DOI: 10.1162/153535003322750655 -& DATABASE Uniprot, [Online] 27 September 2004 (2004-09-27), BATISTIC R F ET AL: "Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus)", XP007921613, retrieved from Uniprot Database accession no. O57540 -& DATABASE Uniprot, [Online] 27 September 2004 (2004-09-27), BATISTIC R F ET AL: "Structure and chromosomal localization of the gene for crotamine, a toxin from the South American rattlesnake, Crotalus durissus terrificus", XP007921618, retrieved from Uniprot Database accession no. Q9PWF3 -& DATABASE Uniprot, [Online] 27 September 2004 2 EP 1 866 332 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention refers, in one aspect, to the uses of crotamine and compositions containing it, based on its novel characteristics of internalization into mammalian cells cytoplasm and nucleus, and of interaction with genetic material. [0002] In one of its aspects, the invention refers to a series of applications of crotamine in submicromolar amounts, in which the concentration range of this polypeptide is no longer toxic, besides still presenting its characteristics of cell 10 penetration, molecule transport to the surface, cytoplasm or cell nucleus and a particularly selective cell penetration into actively proliferating cells. [0003] In another aspect, the invention refers to compositions comprising a pharmaceutically effective concentration of crotamine and said compositions for use in the treatment of cancers, based on its characteristics of interaction with genetic material, such as DNA and RNA, and its cell selectivity. 15 BACKGROUND OF THE INVENTION TOXICITY 20 [0004] To the eyes of an expert in the art, and bearing in mind the knowledge in the state of the art, toxic characteristics of crotamine could not be incentive, and they may even inhibit, the research of its use for any beneficial purpose for human beings and other living organisms. [0005] Crotamine is a toxin, more specifically a myotoxin, isolated from the venom of South American rattlesnake, Crotalus durissus terrificus (Gonçalves, J. M. (1956), Estudos sobre Venenos de Serpentes Brasileiras, II - Crotalus 25 durissus crotaminicus, Subspécie Biológica, An. Acad. Brasileira Ciências 28, 365-367). Crotamine is one of the most abundant components in the venom of rattlesnake, corresponding to approximately 10% of dry weight of the venom. Crotamine is a basic polypeptide with low molecular weight of about 4,800 Daltons and with isoelectric point above 9.5. It is constituted by 42 amino acid residues (YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG, residues 23 to 64 of SEQ ID NO: 5), presenting six cysteines forming three disulphide bridges, and it is rich in basic amino acids, 30 such as lysine and arginine. DOS SANTOS et al. ((1993), Purification and properties of a crotamine analog from Crotalus durissus ruruima venom, Toxicon 31(2), 166) and RADIS-BAPTISTA et al. ((1999), Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus), Toxicon 37(7), 973-984) reported a crotamine analog where the leucine residue at position 19 is replaced by isoleucine (SEQ ID NO: 4). [0006] When injected intraperitoneally, crotamine causes a quick paralysis, in less than 15 minutes, of the hind legs 35 of mice, which is the typical physiological effect of this toxin. Furthermore, difficulty in breathing and rigidity are also observed, suggesting veratrine-simile action (Gonçalves, J. M. (1956), as mentioned above. At cellular and molecular levels, crotamine induces an increase in the voltage-dependent sodium current (as mediated by sodium channels) by causing high depolarization (reduction of rest potential) of the membrane of myocites of muscle fibers next to motor plates - a mechanism which is prevented by tetradotoxin (TTX) (Hong, S. J., Chang, C. (1985), Electrophysiological 40 Studies of Myotoxin as Isolated from Prairie Rattlesnake (Crotalus viridis viridis) Venom on Murine Skeletal Muscle, Toxicon 23, 927-937). Consequently, the massive inflow of sodium ions causes dilatation of the sarcoplasmatic reticulum of myocites and the slow induction of myonecrosis restricted to the cells of skeletal muscles (Ownby, C. L., Cameron, M. S., Tu, A. T. (1976), Isolation of Myotoxin Component from Rattlesnake (Crotalus viridis viridis) Venom, Am. J. Pathol. 85,149-166). 45 [0007] KERKIS et al. ((2004), Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus, FASEB Journal 18, 1407-1409) reported the cellular uptake of Cy3-crotamine, as well as the fact that crotamine is a marker of actively proliferating living cells. [0008] This mechanism, through which crotamine exerts its toxicity, is similar to myotoxin-a from Crotalus viridis viridis, a more extensively studied toxin. 50 [0009] Crotamine is able to form dimers through linkages between disulphide chains, acquiring a form close to a sphere, which covalently links extra subunits. [0010] Therefore, there are no publications in the state of the art suggesting or indicating, in any form, benefits incurred from the effects of crotamine on living organisms. 55 CELL PENETRATION [0011] Cell-penetrating peptides (CPP, or "cell-translocating peptides") are known in the state of the art. A few char- acteristics of this type of peptide were already published, such as its cationic character, low molecular weight and high 3 EP 1 866 332 B1 content of basic amino acids.