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(12) Patent Application Publication (10) Pub. No.: US 2009/0005340 A1 Kristiansen (43) Pub US 20090005340A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0005340 A1 Kristiansen (43) Pub. Date: Jan. 1, 2009 54) BOACTIVE AGENTS PRODUCED BY 3O Foreigngn AppApplication PrioritVty Data SUBMERGED CULTIVATION OFA BASDOMYCETE CELL Jun. 15, 2005 (DK) ........................... PA 2005 OO881 Jan. 25, 2006 (DK)........................... PA 2006 OO115 (75) Inventor: Bjorn Kristiansen, Frederikstad Publication Classification (NO)NO (51) Int. Cl. Correspondence Address: A 6LX 3L/75 (2006.01) BROWDY AND NEIMARK, P.L.L.C. CI2P I/02 (2006.01) 624 NINTH STREET, NW A6IP37/00 (2006.01) SUTE 300 CI2P 19/04 (2006.01) WASHINGTON, DC 20001-5303 (US) (52) U.S. Cl. ............................ 514/54:435/171; 435/101 (57) ABSTRACT (73) Assignee: MediMush A/S, Horsholm (DK) - The invention in one aspect is directed to a method for culti (21) Appl. No.: 11/917,516 Vating a Basidiomycete cell in liquid culture medium, said method comprising the steps of providing a Basidiomycete (22) PCT Filed: Jun. 14, 2006 cell capable of being cultivated in a liquid growth medium, e - rs and cultivating the Basidiomycete cell under conditions (86). PCT No.: PCT/DK2OO6/OOO340 resulting in the production intracellularly or extracellularly of one or more bioactive agent(s) selected from the group con S371 (c)(1) sisting of oligosaccharides, polysaccharides, optionally gly (2), (4) Date: Ul. 31, 2008 cosylated peptides or polypeptides, oligonucleotides, poly s e a v-9 nucleotides, lipids, fatty acids, fatty acid esters, secondary O O metabolites Such as polyketides, terpenes, steroids, shikimic Related U.S. Application Data acids, alkaloids and benzodiazepine, wherein said bioactive (60) Provisional application No. 60/690,477, filed on Jun. agent comprises one or more desirable activities, such as 15, 2005, provisional application No. 60/761,745, anti-tumour activity, immune stimulating activity, and filed on Jan. 25, 2006. enhancement of survival of an individual. E. co 12 O 5 2O culture time (h) Patent Application Publication Jan. 1, 2009 Sheet 1 of 2 US 2009/0005340 A1 Fig. 1 E. coli K12 O 5 culture time (h) Patent Application Publication Jan. 1, 2009 Sheet 2 of 2 US 2009/0005340 A1 Fig. 2 Bioactive agent 50 --MRC-5 --Kelly OO -v- MT mem C-26 gO RS MRC-5 -- FEMX s 50 S s Kelly MT-1 FEMX O C-26 O.O. O. 0.2 0.3 0.4 0.5 mg ml US 2009/0005340 A1 Jan. 1, 2009 1 BOACTIVE AGENTS PRODUCED BY Japanese Journal of Pharmacology 1994, Vol. 66, Iss 2. SUBMERGED CULTIVATION OFA p265-271. Ito et al., Anticancer Research 1997, Vol. 17, BASDOMYCETE CELL Iss 1A, p 277-284; Fujimiya et al., Cancer Immunology Immunotherapy 1998, Vol. 46, Iss 3, p 147-159; TECHNICAL FIELD OF THE INVENTION Fujimiya et al., Journal of the Japanese Society for Food Science and Technology—Nippon Shokuhin Kagaku 0001. The present invention relates to a method for sub- Kogaku Kaishi 1998. Vol 45, Iss 4. p 246-252: Ebina et merged cultivation of Basidiomycete cells, bioactive agents al., Biotherapy 1998, Vol. 11, Iss 4. p 259-265; Fujimiya obtained from Such a cultivation, and methods for using said et al., Anticancer Research 1999, Vol. 19, Iss 1A, p bioactive agents. The bioactive agent is preferably a polysac- 113-118; Mizuno et al., Biochemistry and Molecular charide comprising an immune stimulating activity and/oran Biology International 1999, Vol. 47, Iss 4, p 707-717; anti-cancer activity. Takaku et al., Journal of Nutrition 2001, Vol. 131, Iss 5, p 1409-1413: Ohno et al., Biological and Pharmaceuti BACKGROUND OF THE INVENTION cal Bulletin 2001, Vol. 24, Iss 7, p. 820-828; Delmanto et 0002. Submerged cultivation of Basidiomycete cells is al., Mutation Research Genetic Toxicology and Envi well known in the art and various beneficial effects have been ronmental Mutagenesis 2001, Vol. 496, Iss 1-2, p 15-21) attributed to compounds obtained from Basidiomycete cells 0005 Anti-angiogenic: (Kimura, Y: In Vivo 2005, Vol e.g. of the Agaricus family: 19, Iss 1, p.37-60; Kimura, Yet al., Cancer Science 2004, Vol 95, Iss9, p 758-764; Takaku et al., Journal of Nutri 0003 Stimulation of immune system (Kimura, Y. In tion 2001, Vol. 131, Iss 5, p 1409-1413) Vivo 2005, Vol 19, Iss 1, p 37-60; Kawamura et al., 0006 Protection against X-rays (Kubo et al., Interna Immunology 2005, Vol. 114, Iss3, p.397-409: (killer cell tional journal of Molecular Medicine 2005, Vol 15, Iss. activity increased): Ahn et al., International Journal of 3, p. 401–406) Gynecological Cancer; (killer cell activity—increased): 0007 Hepatoprotective effect (Barbisan et al., Journal Kaneno R et al., Food and Chemical Toxicology 2004, of Ethnopharmacology 2002, Vol 83, Iss 1-2, p 25-32) Vol 42, Iss 6. p909-916; Chenet al., International Immu 0008 Inhibiting growth of Candida albicans (Paccola nopharmacology 2004, Vo. 4, Iss 3. p 403-409; (killer et al., Brazilian Journal of Microbiology 2001, Vol. 32. cell activity increased): Takimoto et al., Biological and Iss 3, p 176-178) Pharmaceutical Bulletin 2004, Vol.27, Iss 3, p. 404–406; 0009 Anti-hypertensive (Wantanabe et al., Journal of (activates complement system) Shimizu et al., Phy the Japanese Society for food Science and technology tomedicine 2002, Vol. 9, Iss 6, p 536-545; Kuo et al., Nippon Shokuhin Kagaku Kogaku Kaishi 2002, Vol. 49, Journal of Laboratory and clinical medicine 2002, Vol. Iss 3, p 166-173) 140, Iss 3, p 176-187; Nakajima et al., International 0.010 Increasing NO production (Nagata et al., Journal Immunopharmacology 2002, Vol. 2, Iss 8, p 1205-1211; of the Japanese Society for Food Science and Technol Mizuno et al., Bioscience Technology and Biochemistry ogy—Nippon Shokuhin Kagaku Kogaku Kaishi 2001, 1998, Vol. 62, Iss 3, p. 434–437: Fujimiya et al., Antican Vol. 48, Iss 12, p. 939-942) cer Research 1999, Vol. 19, Iss 1A, p 113-118; Ooi et al., 0.011 Stimulating hair growth (Towatari et al., Journal Current Medicinal Chemistry 2000, Vol. 7, Iss 7, p 715 of Wood Science 2001, Vol. 47, Iss 5, p. 410-413) 729; Sorimachi et al., Cell structure and function 2001, 0012 Antibacterial effects (Bernardshaw, S et al., Vol. 26, Iss 2, p 103-108.) Immunology 2004: Genomic Issues, Immune System 0004 Anticancer/antitumour/antimutagenic: (Kimura, Activation and Allergy, 473–477, 2004; Osaki et al., Y: In Vivo 2005, Vol 19, Iss 1, p 37-60; Kim et al., Food Yakugaku Zasshi Journal of the Pharmaceutical Soci Science and Biotechnology 2004, Vol 13, Iss 6, p. 852; ety of Japan 1994, Vol. 114, Iss 5. p 342-350) Kim et al., Food Science and Biotechnology 2004, vol 0013 Inhibition of cytopathic effect induced by West 13, Iss 3. p 347-352; Guterrez et al., Texicology in Vitro ern Equine encephalitis virus (Sorimachi K. et al., Bio 2004, Vol 18, Iss 3, p 301-309; Ribeiro et al., Mutation science Biotechnology and Biochemistry 2001, Vol. 65, Research Reviews in Mutation Research 2003, Vol 54, Iss. 7, p 1645-1647) Iss 2-3, p 195.201; Pinheiro et al., Food and Chemical 0.014 Skin barrier against harmful effects of environ Toxicology 2003, Vol. 41, Iss 11, p 1543-1550; (Inhib mental toxins, pollution, chemicals, and radiation iting tumour growth) Lee et al., Experimental Animals (Uchiyama, Shoji; et al.: US200201 19164) 2003, Vol. 52, p 371-375; Luiz et al., Mutation 0.015 Autoimmune disorders (Uchiyama, Shoji; et al.: research Fundamental and Molecular Mechanisms of US200201 19164) Mutagenesis 2003, Vol. 528, Iss 1-2, p 75-79; Bellini et 0016 anti-inflammatory or antiallergic (Maekawa, al., Texicology in Vitro 2003, Vol 17, Iss 4, p. 465-469; Ashida et al., Food Factors in Health Promotion and Takaaki; et al.: US20030104.006) Disease Prevention 2003, Vol. 851, p. 235-248; (anti 0017 Anti-diabetes (Itokawa, Mark: US200201 10564: genotoxic effect) de Oliveira et al., Food and Chemical Ha et al., KR 2004062814) Toxicology 2002, Vol. 40, Iss 12, p 1775-1780; Kuo et 0.018 Anti-AIDS (U.S. Pat. No. 6,120,772) al., Journal of Laboratory and clinical medicine 2002, SUMMARY OF THE INVENTION Vol. 140, Iss3, p 176-187: Oshiman et al., Planta Medica 2002, Vol. 68, Iss 7, p 610-614; Meloniet al., Mutation 0019. The present invention relates to novel bioactive Research Genetic Toxicology and Environmental agents such as polysaccharides as well as to novel methods Mutagenesis 2001, Vol. 496, Iss 1-2, p 5-13: Osaki et al., for liquid cultivation of microbial cells of the class Basidi Yakugaku Zasshi Journal of the Pharmaceutical Soci omycete, for example microbial cells of the genera Agaricus, ety of Japan 1994, Vol. 114, Iss 5. p 342-350; Itoh et al., Schizophyllum, Lentinus, Trametes, Ganoderma and Grifola, US 2009/0005340 A1 Jan. 1, 2009 in particular Agaricus Blazei, Schizophyllum commune, group consisting of oligosaccharides, polysaccharides, Lentinus edodes, Trametes versicolor; Ganoderma applana optionally glycosylated peptides or polypeptides, oligonucle tum and Grifola frondosa. otides, polynucleotides, lipids, fatty acids, fafty acid esters, 0020. The polysaccharides can in principle be obtained secondary metabolites such as polyketides, terpenes, Ste from liquid cultivation of any Basidiomycete cell. Basidi roids, shikimic acids, alkaloids and benzodiazepins. omycetes of the genera Agaricus, Schizophyllum, Lentinus, 0028. The above-mentioned Basidiomycete cells are pref Trametes, Ganoderma and Grifola represent examples in this erably selected from the groups consisting of the Agaricus sp. respect. A. blazei and A. bisporus, the Schizophyllum sp. Schizophyl 0021. In one embodiment, the polysaccharide according lum commune, the Lentinus sp. Lentinus edodes, the Tram to the present invention has a molar ratio of galactose:man etes sp.
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