• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention relates to pharmacologically active polypeptide-sugar conjugates that exhibit physicochemical stability with identified consensus sequences and have in vivo pharmacological and clinical activity related to immunocontrolling activity that cannot be represented by isolated components. Such conjugates can be used in the pharmaceuticals of humans and animals.
    公开号:KR20010112290A
    申请号:KR1020017010906
    申请日:1999-10-21
    公开日:2001-12-20
    发明作者:브리에바델가도오로라；가르시아빌라루비아비센테；구에레로고메즈파모안토니오；피벨라니에리주안파블로；길레르모 기메네즈갈레고；마트지투드리조세안토니오
    申请人:인더스트리얼 파르머서티카 칸타브리아 에스에이；
    IPC主号:
    专利说明:

    Pharmacologically Active Polypeptide Glycoconjugates
    [2] Recently, as knowledge of the function and control mechanisms of the immune system have been accumulated, the possibility of therapeutic changes in their function is increasing.
    [3] The vast variety of natural and synthetic substances of different origins, which can alter some immune systems, is to some extent due to their own origins and the possibility of recognizing different substances. GH Werner, P Folles "Immunostimulating agents: what next . A review of their present and potential medical applications" Eur. J. Biochem 242, 1-19 (1996), describes some recently developed products with their therapeutic relationships.
    [4] Among the various endogenous mediators are tumor necrosis factor (TNF) causing significant therapeutic changes. This molecule exhibits several specialized unique features (R Ksontini, SLD MacKay, LL Moldawer "Revisiting the role of tumor necrosisfactor α and the response to surgical injury and inflammation" Arch. Surg. 133, 558-567 (1998) , JL Alonso "La compleja fisiologia del factor de necrosis tumoral." Inmunologia 8, (3) 73-94 (1989)], T. Calandra "Importance des cytokines dans les syndromes septiques." Med. Hyg. 49, 609-614 (1991), A Eigler, B. Sinha, G Hartman, S Endres "Taming TNF: strategies to restrain this pro inflmmatory cytokines." Immunology Today 18, 487-492 (1997)], [R Gonzalez- amaro, C Garcia-Monzon, L. Garcia-Buey, R Moreno-Otero, JL Alonso, E Yague, JP Pivel, M Lopez-Cabrera, E Fernandez-Ruiz, F Sanchez-Madrid "Induction of Tumor Necrosis Factor, a production by Human Hepatocytes in Chronic Viral Hepatitis. "J. Exp. Med 179, 841-848 (1994)).
    [5] Tumor necrosis factor (TNF) is a diversity expressing cytokine with a large number of cells responding to it. Normally produced by mononuclear cells and bound to the membrane of secretory cells, and free form induced during the preparation of the tumor necrosis factor or bound to the secretory cell membrane by metalloproteinases, TACE converting enzymes. There are two active forms.
    [6] T lymphocytes in addition to mononuclear cells (AG Santis, MR Campanero, JL Alonso, F Sanchez-Madrid "Regulation of tumor necrosis factor (TNF) -α synthesis and TNF receptors expression in T lymphocytes through the CD2 activation pathway." Eur. J. Immunol. 22, 3155-3160 (1992), AG Santis, MR Campanero, JL Alonso, A Tugores, MA Alonso, E Yague, JP Pivel, F Sanchez-Madrid "Tumor necrosisfactor-α production induced in T lymphocytes through the AIM / CD69 activation pathway. "Eur. J. Immunol 22, 1253-1259 (1992)) and NK cells (Imelero, MA Balboa, JL Alonso, E Yague, JP Pivel, F Sanchez-Madrid, M). Other cells such as Lopez-botet "Singnaling through the LFA-1 Leucyte integrin actively regulates intercellular adhesion and tumor necrosis factor α production in natural killer cells." See Eur. J. Immunol. 23, 1859-1865 (1993). It has been reported to participate in the synthesis of TNFα.
    [7] Several molecules have been found to induce TNF production, including bacterial endotoxins or lipopolysaccharides (LPS), superantigens derived from bacteria, viruses, or parent cells, and other cytokines.
    [8] Like other cytokines, TNFs also exhibit nano-femtomol levels on neighboring tissues (endocrine activity) or on distant cells (endocrine activity) as well as on secretory cells themselves (self-endocrine activity) and on adjacent cells (proximal activity). It acts in a reactive nitrogen way to its concentration. This means that the activity of this molecule, like other cytokines, is heavily influenced by the "state" of the receptor cell and in particular its interaction with the extracellular matrix.
    [9] Thus, it has been found that circulating TNF is not always a basic parameter because the cytokines may be present in high concentrations locally, even if the TNF is normal in various situations.
    [10] Two receptors of different cell types were found, TNF receptor p55 (TNFR p55) and 75 (TNFR p75).
    [11] When these receptors interact with free or bound TNF, they exhibit a variety of responses that can be classified into three broad classes. First, it activates inflammatory chain amplification in the light of the fact that TNF belongs to a group of related proteins including IL1, IL6, GM-CSF and the like. On the other hand, it activates cell mediated responses, particularly to lesion attack by intracellular pathogens. Thirdly, it results in cell death or planned cell death, especially in tumorous cells. However, TNF may have two responses in the cell death response, which, on the one hand, activates the NFkB transcription factor to prevent some cell populations from dying during acute infection, but nevertheless overproduction of TNF This can be caused by cell death. TNF is implicated in many pathologies in that it can prevent cell death. The relationship between their local and / or systemic overproduction and the onset and exacerbation of many pathological processes has been extensively studied (R Ksontini, SLD MacKay, LL Moldawer “Revisiting the role of tumor necrosis factor α and the response to surgical injury and inflammation "Arch. Surg. 133, 558-567 (1998)], JL Alonso" La compleja fisiologia del factor de necrosis tumoral. "Inmunologia 8, (3) 73-94 (1989)], [ T. Calandra "Importance des cytokines dans les syndromes septiques." Med. Hyg. 49, 609-614 (1991)], A Eigler, B. Sinha, G Hartman, S Endres "Taming TNF: strategies to restrain this pro inflmmatory cytokines. "Immunology Today 18, 487-492 (1997)], [R Gonzalez-amaro, C Garcia-Monzon, L. Garcia-Buey, R Moreno-Otero, JL Alonso, E Yague, JP Pivel, M Lopez-Cabrera , E Fernandez-Ruiz, F Sanchez-Madrid "Induction of Tumor Necrosis Factor, a production by Human Hepatocytes in Chronic Viral Hepatitis." J. Exp. Med 179, 841-848 (1994)]. Because TNF is produced by various cell types, for example, skin and digestive tract disorders associated with host graft reactions (PF Piguet, GE Grau, B Allet, P Vassalli, "Tumor Necrosis Factor / Cachectin is an effector of skin and gut lesions of the acute phase of GRAFT-VS-HOST disease. "J. Exp. Med. 166, 1280-1289 (1987)), pneumonia (CE Reed." Hypersensitivity pneumonitis and occupational lung disease from inhaled) endotoxin. "Immunology and Allergy Clinics of North America. 12 N ° 4 (1992)) or neurological lesions (SW Barger" Tumor Necrosis Factor. The Good, the Bad and the Umbr. "Neuroprotective Signal Transduction. Edited by MP Mattson Humana Press Inc. Totowa NJ.], lung lesions, chronic lesions (intestinal inflammatory diseases and rheumatoid arthritis, etc.), and sepsis. Cytokines also play an important role in two high-incidence lesions: asthma and chronic obstructive pulmonary disease (see P. Norman "Pulmonary diseases. Disease trends and market opportunities" Financial Times Pharmaceuticals management Reports (1999)).
    [12] Due to the above background, it is difficult to devise an effective treatment based on the control of TNF in various lesion situations.
    [13] New drug designs should be able to construct and explain experimental pharmacology models that reproduce the most important aspects of the lesion in question. One of the most used models for drug investigations that can regulate the production of TNF is a rat model induced by TNF systemically by bacterial endotoxin (LPS). Another widely used model is one that is investigating in vitro stimulation of cells belonging to the granulocyte macrophage lineage for the production of the cytokines.
    [14] The most surprising from a scientific point of view is that the chemically diverse products with the ability to control TNF excess production in various in vivo and in vitro experimental models. For example, antioxidants (N Satomi, A Sakurai, R haranaka, K Haranaka "Preventive Effects of Several Chemicals Against Lethality of Recombinant Human Tumor Necrosis Factor." Journal of Biological Response Modifiers. 7, 54-64 (1988) ), Cannabinoids (R Gallily, A Yamin, Y Waksmann, H Ovadia, J Weidenfeld, A Bar-Joseph, A Biegon, R Mechoulanm, E Shohami. "Protection against Septic Shock and Suppression of Tumor Necrosis factor α and Nitric Oxide Production by Dexanabinol (HU-211), a Nonpshychotropic Cannabinoid. "The Journal of Pharmacol. And Experimental Therapeut. 283, 918-924 (1997)), IL10 (SR Smith, C Terminelli, G Denhardt, S Narula, G Jeanette Thorbecke "Administration of Interleukin-10 and the Time of Priming Protects Corynebacterium parvum-Primed Mice Against LPS- and TNF-α-induced Lethality." Cellular Immunology 173, 207-214 (1996)), thalidomide (AL Moreira, J Wang, EN Sarno, G Kaplan. "Thalidomide protects mice against LPS-induced shock. "Brazilian Journal of Medical and Biological Research 30: 1199-1207 (1997)], SM McHugh, TL Rowland" Thalidomide and derivatives: immunologycal investigations of tumour necrosis factor-alpha (TNF-α) inhibition suggest drugs capable of selective gene regulation. "Clin Exp. Immunol 110: 151-154 (1997)], JD Klausner, VH Freedman, G Kaplan "Thalidomide as an Anti-TNF-α Inhibitor: Implications for Clinical Use." Clinical Immunology and Immunopathology. 81, 219-223 (1996)), chloropromacin (M. Gadina, R. Bertini, M. Mengozzi, M. Zandalasini, A. Mantovani and P. Ghezzi "Protective Effect of Chlorpromazine on Endotoxin Toxicity and TNF Production in Glucocorticoid-Sensitive and Glucocorticoid-Resistant Models of Endotoxic Shock. "J. Exp. Med. 273, 1305-1310 (1991)), benzidamine (A. Gluglielmotti, L. Aquilini, MT Rosignoli, C, Landolfi, L. Soldo, I. Coletta and M. Pinza "Benzydamine protection in a mouse model of endotoxemia." Inflamm. Resp. 46, 332-335 (1997)), hydrazine sulfate (R. silverstein, BR Turley, CA Christoffersen, DC Johnson and DC Morrison "Hydrazine Sulfate Protects D-Galactosamine-sensitized Mice against Endotoxin and Tumor necrosis factor / Canchectin Lethality: Evidence of a Role for the Pituitary." J. Exp. Med. 173, 357-365 (1991)) and natural extracts (H. Ueda and M. Yanazaki "inhibition of Tumor Necrosis Factor a Production by Orally Adm" inistering a Perilla leaf Extract. "Biosci. Biotech. Biochem. 61, 1292-1295 (1997).
    [15] In the study of the clinical situation of patients with lesions known to involve TNF in the progression of the lesion, the effects of various active ingredients on the control of in vitro TNF production in place of isolated mononuclear cells of peripheral blood were also studied. . For example, ciflofloxacin (S Bailly, M Fay, B Ferrua, MA Gougerot-Pocidalo "Ciprofloxacin treatment in vivo increases the ex vivo capacity of lipopolysaccharide-stimulated human monocytes to produce IL-1, IL-6 and tumour necrosis factor-alpha. "Clin. Exp. Immunol 85, 331-334 (1991)], and rolipram (J Semmler, H Wachtel, S Endres" The specific type IV phosphodiesterase inhibitor rolipram suppresses Tumor Necrosis Factor -α production by human mononuclear cells. "Int. J. Immunopharmac. 15, 409-413 (1993)), Besnarinone (T Kambayashi, N Mazurek, Cho Jacob, N Wei, M Fong and G Strassmann). "Vesnarinone as a selective inhibitor of Macrophage TNF-α release." Int J. Immunnopharmac, 18, 371-378 (1996)), prostacycline analogs (A Jorres, H Dinter, N Topley, GM Gahl, U Frei, P Scholz "Inhibition of Tumor Necrosis Factor production in endotoxin-stimulated human mononuclear leukocytes by the prostacyclin analogue iloprost: Cellular mechanisms. "Cytokine 9, 119-125 (1997)), pentoxifylline (BJ Dezube, ML Sherman, JL Fridovich-keil, J Allen-T Ryan, A B Pardee. "Down-regulation of tumor necrosis factor expresion by pentoxifylline in cancer patients: apilot study." Cancer Immunol Immunogher 36: 57-60 (1993)]. In particular cases, corticoids are known to be involved in the inhibition of the TNF gene (S Abe, T Yamamoto, S Iihara, M Yamazaki, D Minuzo. "A possible role of glucocorticoids: an intrinsic inhibitor of the cytotoxic activity of Tumor necrosis Jpn. J, Cancer Res. (Gann) 79: 305-308 (1988). J Han, P Thompson, B Beutler "Dexamethasone and Pentoxifylline Inhibit Endotoxin-induced Cachectin / Tumor necrosis Factor Synthesis at Separate Points in the Signaling pathway. "J. Exp. Med. 172, 391-394 (1990). IMH Debets, TJM Ruers, MPMH Van Der Linden, CJ Van Den Linder, WA Buurman." Inhibitory effect of corticosteroids on the secretion of tumor necrosis factor ( TNF) by monocytes is dependent of the stimulus inducing TNF synthesis. "Clin. Exp. Immunol 78: 224-229 (1989)). It is significant that regulation of cytokine levels has already been mentioned as a specific "target" in new drug design (K Cooper, H Masamune "Cytokine Modulation as a Medicinal Chemistry Target." Annual Reports in Medicinal Chemistry-27, Chapter 22]. Another attempt to modulate the effects of cytokines in the condition of sepsis, as well as ulcerative colitis and rheumatoid arthritis, has been associated with the development of monoclonal anti-TNF antibodies (A Trilla, P Aloso "Anticuerpos monoclonales en el tratamiento del shock septico. "Med. Clin. 99: 778-780 (1992)], JG Sinkovics" Monoclonal antibodies in the treatment of endotoxin shock "Acta Microbiologica Hungarica 37: (1990)], SB Porter" Current Status of Clinical Trials With Anti-TNF "Chest 112: 6 (1997)], JR O'Dell" Anticytokine therapy. A new era in the treatment of rheumatoid arthritis "New Eng. J. Med. 340, 310-312 (1999)], RA van Hogenzand, HW Verspaget "The future role of anti-tumor necrosis factor a products in the treatment of Crohn's disease" Drugs 56, 299-305 (1998), F Mackay, JL Browning, P Lawton, SA Shah, M Comiskey, AK Bhan, E Mizoguchi, C Terhorst, SJ Simpson "Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis "Gaestroenteroloty 115, 1464-1475 (1998)"). However, despite extensive knowledge of cytokines, including molecular biology, no safe and effective therapeutics have been developed to control their overproduction.
    [16] A rigorous analysis of all of these possible treatments has shown recent successes in cases of rheumatoid arthritis and ulcerative colitis for monoclonal anti-TNF, but has not been effective for acute lesions and has been shown to affect cytokines. It also showed diversity in terms of affinity (JR O'Dell "Anticytokine therapy. A new era in the treatment of rheumatoid arthritis" New Eng. J. Med. 340, 310-312 (1999)), RA van Hogenzand , HW Verspaget "The future role of anti-tumor necrosis factor a products in the treatment of Crohn's disease" Drugs 56, 299-305 (1998)]. Other products showed very strong toxicity as in the case of thalidomide, or activity that was difficult to handle, such as ciprofloxacin or rolipram. In other cases, the chemical reliability was low, resulting in insufficient regeneration of the extract from batch to batch. Finally, corticosteroids, inhibitors of TNF gene expression, showed a significant group of contraindications.
    [17] As knowledge of the functional mechanisms of the immune system has accumulated over the years, the development of a substance known as an immunomodulator has been developed. Lesions in which the supply of immune control agents is of particular importance, such as counter imbalances of the immune system, sensitizing immunosuppression (e.g., implants, anti-neoplastic therapies or especially traumatic surgery) or environmentally-immune autoimmune diseases (caused by stress or pollution) There is a situation. Meanwhile, many treatment protocols today include immunomodulators as adjuvant to specific anti-tumor genes or anti-infective therapies (E Garaci, F Pica, G Rasi, AT Palamara, C Favalli "Combination therapy with BRMs in cancer and infectious diseases "Mechanisms of Ageing and development 96, 103-116 (1997)).
    [18] Important among immunomodulators are those designed to promote natural immune mechanisms, in particular NK (natural killing) activity and phagocytic and bactericidal activity of mononuclear phagocytes. Examples include bacterial extracts, BCG, Corynebacterium pathogens, muramyldipeptide derivatives and polysaccharides, especially glucans extracted from yeast (A Aszalos "Immunstimulators of microbial origin" in "Antitumor compounds of natural origin" CRC Press (1982). However, although these molecules have been shown to be effective as activators of monocyte-macrophage systems, they involve two unwanted side effects. One is that they block the liver metabolic system, which is also present in other immunomodulatory substances, which interferes with the administration of adjuvants for other treatments such as antibiotics or cytostatic agents. Another very special case is that these molecules are susceptible to bacterial endotoxins, so that endotoxins released by antibiotic action may become larger toxins in patients receiving immunosuppressive agents (M Trautmann, R Zick, T Rukavina). , AS Cross, R Marre "Antibiotic-induced release of endotoxin: In vitro comparison of meropenem and other antibiotics" J. Antimicr. Chemother. 41, 163-169 (1998).
    [19] Thus, as described above, efforts have been made to find products that can selectively inhibit some TNF action without blocking or further promoting natural immune responses.
    [20] One controversial molecular type for its use is peptide-type immunomodulators. The problem is that although these types of molecules exhibit very promising activities, such as specific interactions with receptors, specific inhibition of other proteins (proteinase inhibitors), etc., bioavailability, in particular oral availability, Problems such as susceptibility to proteases, short half-lives and allergic or anaphylactic reactions. Recent reviews of peptides and proteins as immunomodulators highlight these features (see JE Talmadge "Pharmacodynamic aspects of peptide administration biological response modifiers" Advanced Drug Delivery Reviews 33, 241-252 (1998)). The therapeutic activity of proteins is distinguished in various paradigms when compared to traditional low molecular weight drugs. This difference is mainly related to the pharmacokinetic properties of the protein. Thus, it is important not only to optimize their therapeutic activity or more generally to identify the activity, but also to understand the pharmacology of these drugs. The paradigm includes the need for subcutaneous or continuous intravenous administration to achieve short half-life and maximum activity of the protein, a pronounced bell-shaped response, the need for long-term administration associated with the cognitive mechanism of molecular activity, chemotherapy and / or radiotherapy. It is a fact that the optimal and maximum adjuvant immunotherapeutic activity of the drug as adjuvant in combination is found in patients with minimal residual disease.
    [1] The present invention relates to substantially pure specific polypeptides, conjugate formation of specific polysaccharides and their pharmacological properties for the preparation of therapeutic compositions for the treatment of immunological abnormalities, infections and / or tumors.
    [63] 1 is a diagram showing an infrared spectrum of the sugar binder of the present invention.
    [21] An object of the present invention is to be able to form bonds with specific peptides or proteins having unique physicochemical properties defined by strict structural requirements, and these bindings result in activity by oral administration in controlling human or animal immune responses. It allows one to form non-covalent bonds with specific polysaccharide molecules defined by visible structural features. The control means stimulating the mononuclear phagocyte system and expanding the granulocyte macrophage zone but down-regulating TNF production induced under specific experimental conditions without inhibiting the liver metabolic system.
    [22] Two points of particular importance can be emphasized for the purposes of the present invention.
    [23] The first is that the non-covalent conjugates formed are activated by oral administration, which is novel in the field of biologically active peptides by the oral route and overcomes the disadvantages of the inherent mode of route administration. These disadvantages are described in detail in the following documents (BL Ferraiolo, LZ Benet "Peptides and proteins as drugs" Pharmaceutical Research 4, 151-194 (1985)), FM Rollwagen, S Baqar "Oral cytokine administration" Immunol. Today 17, 548-550 (1996)], Solis-Pereyra, N Aattouri, D Lemonnier "Role of food in the stimulation of cytokine production" Am. J. Clin. Nutr. 66, 521S-525S (1997) ], [A Fasano "Innovative strtegies for the oral delivery of drugs and peptides" Trends in Biotech. 16, 152-157 (1998)], GM Pauletti, S Gangwar, TJ Siahaan, J Aube, RT Borchardt "Improvement of oral peptide bioavailability: Peptidomimetics and prodrug strategies "Adv. Drug Deliv. Rev. 27, 235-256 (1997)], JJ Hols, C Deacon, MB Toft-Nielsen, L Bjerre-Knudsen" On the treatment of diabetes mellitus with Glucagon -like peptide-1 "Ann. New York Acad. Sci. 865, 336-343 (1998)). In this respect, it should be noted that this is not the only example of protein activity by the oral route (Y Nagao, K Yamashiro, N hara, Y Horisawa, K Kato, A Uemera "Oral administration of IFN-α potentiates immune response in mice "J Interferon and Cytokine Res. 18, 661-666 (1998)], S Kaminogawa" Food Allergy, oral tolerance and immunomodulation. Their molecular and cellular mechnisms "Biosci. Biotec, Biochem. 60, 1749-1756 ( 1996), [H Uwata, TT Yip, K Yamauchi, S Teraguchi, H Hayasawa, M Tomita, TW Hutchens "The survival of ingested lactoferrin in the gastrointestinal tract of adult mice" Biochem. J. 334, 321-223 (1998 ), [J Xu-amano, WK Aicher, T Taguchi, H Kiyono, JR McGhee "Selective induction of Th2 cells in murine Peyer's patches by oral immunization: internat. Immunol. 4, 433-445 (1992)].
    [24] The second important point describes the covalently bound polysaccharide-protein complexes as well as the non-covalent bonds with biological activity, but these are contrary to the object of the present invention by adding certain peptides to T-dependent and low reactivity polysaccharide antigens only. It is generally associated with increased antigenic response of weak immunogen polysaccharides to achieve dependence (HK Guttormsen, LM Wetzler, RW Finberg, DL Kasper "Immunologic memory induced by a glycoconjugate vaccine in a murine adoptive lymphocyte transfer model"). Infection and immunity 66, 2026-2032 (1998)], MA Avanzini, AM Carre, R Macario, M. Zecca, G Zecca, A Pession, P Comoli, M Bozzola, A Prete, R Esposito, F Bonetti, F Locatelli "Immunization with Haemophilus influenzae type b conjugate vaccine in children given bone marrow transplantation: Comparison with healthy age-matched controls" J. Clin. Immunol. 18, 193-301 (1998)], EFE B abiker, A Hiroyuki, N Matsudomi, H Iwata, T Ogawa, N Bando, A Kato "Effect of polysaccharide conjugation or transglutaminase treatment on the allergenecity and functional properties of soy protein" J. Agric. Food Chem. 46, 866-871 ( 1998)]. It should be noted in these documents, which describe polysaccharide-protein binding with immunomodulatory activity, that, unlike the present invention, the binding is covalent and derived from the same source (K Noda, N Ohno, K Tanaka, M Okuda). , T Yadomae, K Nomoto, T Shoyama "A new type of biological response modifier from chlorella vulgaris which nees protein moiety to show antitumor activity" Phytotherapy Res. 12, 309-319 (1998)], DSabolobvic, L Galoppin "Effect of a protein bound polysaccharide (PS-K on tumor development and infections in splenectomized rats and mice "Int. J. Immunopharmac. 8, 41-46 (1986)).
    [25] It will be described in detail below to better understand the features of the present invention.
    [26] The present invention relates to the formation of conjugates of substantially pure specific polypeptides and specific polysaccharides and their pharmacological properties for the preparation of therapeutic compositions for the treatment of immunological abnormalities, infections and / or tumors. The binder is pharmacologically active but its components (polypeptides or polysaccharides) do not show the pharmacological activity of the binder. The conjugates also show different stereochemistry in the polysaccharide-polypeptide relationship, and the pharmacological activity depends on this stereochemistry.
    [27] The technique of the present invention is described according to the following configuration: a) conditions to be followed by the polysaccharide molecules which are the raw materials of the present invention; b) conditions to be followed by a polypeptide which is a raw material of the present invention; c) result of the above conditions: formation of a polysaccharide-polypeptide conjugate; d) biological activity of the polysaccharide-polypeptide conjugate.
    [28] a) Conditions to be followed by polysaccharide molecule which is raw material of this invention
    [29] The conditions which the polysaccharide molecule which is a raw material of this invention should follow are as follows.
    [30] The polysaccharide molecules of the invention must be derived from bacteria, not viruses, in particular from yeast walls. It should have an average molecular weight of 50 to 250 KDa, be soluble in water or saline media with ionic strength similar to that obtained by sodium chloride solutions at concentrations from 0 to 250 mM, and dissolve at least 0.1 mg / ml under these conditions. The polysaccharide molecules of the present invention should exhibit a charge in a solution of neutral medium, mainly due to phosphates that impart specific reactivity to aa T cells (A Salerno, F Dieli "Role of aa T Lymphocytes in immune response in humans"). and mice "Critical Rev. Immunol 18, 327-357 (1998)], the ratio of phosphate residues to monosaccharides should be from 1/5 to 1/25. No sulfate or carboxylate groups should be shown. In the composition of monosaccharides, mannose should be the main component (40% or more) and the remainder should consist of glucose and / or galactose. The nitrogen monosaccharide content cannot exceed 5% of the total. The main backbone preferably has 1 to 2 branches and the monosaccharides in the branches should be formed by 1 to 6 bonds in a manner not exceeding 60%. There should be no linked lipid groups.
    [31] With regard to physicochemical behavior, the polysaccharide molecules of the present invention should have domains that can interact with octadecylsilane in aqueous media and should not gel in aqueous or saline media, especially when calcium is present at concentrations of 2 mM or less. The polysaccharide molecule of the present invention must be able to form a conjugate with a polypeptide or peptide having the characteristics described in the next section, which conjugate should be stable under physiological conditions.
    [32] Polysaccharide molecules of the invention should not exhibit anticoagulant activity. It must be able to withstand the physicochemical and enzymatic conditions of the gastrointestinal tract, thus ensuring the activity of the conjugates by the oral route. This activity originates in the interaction of the conjugate with gastrointestinal lymphoid tissue and the generation of a systematic response via aa T cell bridges (AK Abbas, AHLichtman, JS Pober "Cellular and molecular immunology" WB Saunders Co. Philadelphia, pp 232- 236 (1994)], TW Mak, DA Ferrick "The aa T-cell bridge: Linkage innate and adquired immunity" Nature Med. 4, 764-765 (1998)), which bridges are particularly reduced with age. (See G Pawelec, R Solana, E Remarque, E Mariani "Impact of aging on innate immunity" J. Leuk. Biol. 64, 703-712 (1998)).
    [33] b) Conditions to be followed by a polypeptide molecule as a raw material of the present invention:
    [34] The raw polypeptide molecules of the present invention must satisfy the following paradigm.
    [35] Polypeptide molecules of the invention must be able to withstand the physicochemical and enzymatic conditions of the gastrointestinal tract, and therefore the activity of the conjugates by the oral route must be ensured.
    [36] Polypeptide molecules of the present invention should be able to form conjugates with polysaccharides having the features described in the previous section, which should be stable under physiological conditions.
    [37] It is contemplated that polypeptides that are stabilized through disulfide bridges or stabilized through chemical manipulations to form dimethylene bridges will be particularly advantageous.
    [38] This type of structure simultaneously shows the chemical stability of the low molecular weight drug and the stereospecificity of the polypeptide.
    [39] Sources that can provide this type of molecule include vegetable rind polypeptides, plant defense polypeptides, plant sweetener polypeptides, and the like.
    [40] In order to satisfy the above properties, the polypeptide molecule must satisfy the following conditions.
    [41] Molecular Weight: 4 to 30 KDa
    [42] Solubility: Must be dissolved in a concentration of at least 0.1 mg / ml in water or saline medium having ionic strength similar to that produced by 0 to 0.25 M sodium chloride solution.
    [43] In its natural state, the polypeptide molecules of the present invention must be able to fight trypsin-type proteases, chemotrypsin and / or pepsin (under optimal activity conditions of the enzyme) and, in nature, acidic for at least one hour. It must be able to withstand pH (similar to gastrointestinal tract).
    [44] Polypeptide molecules of the invention must be able to withstand the physicochemical and enzymatic conditions of the gastrointestinal tract, and therefore the activity of the conjugates by the oral route should be ensured. The activity originates in the interaction of the conjugate with gastrointestinal lymphoid tissue and the generation of a systematic response via aa T cell bridges (AK Abbas, AH Lichtman, JS Pober "Cellular and molecular immunology" WB Saunders Co. Philadelphia, pp. 232-236 (1994)], TW Mak, DA Ferrick "The aa T-cell bridge: Linkage innate and adquired immunity" Nature Med. 4, 764-765 (1998)). Reduced (see G Pawelec, R Solana, E Remarque, E Mariani "Impact of aging on innate immunity" J. Leuk. Biol. 64, 703-712 (1998)).
    [45] Polypeptides of the present invention are restored to their natural state simply by diluting the sensitive drug when sensitized by the addition of a drug such as 8M guanidine chloride or 6M urea in the presence of a disulfide bridge reducing agent such as dithiothreitol or mercaptoethanol at a concentration of 6.4 mM (Validated by a circular bicolor spectrum in the range of 280 to 200 nm).
    [46] It is preferable that the polypeptide of the present invention does not contain glycosylate.
    [47] Polypeptides of the invention may be oligomers, in particular dimers, stabilized by disulfide or dimethylene bridges, with at least two disulfide or dimethylene intrachain bridges.
    [48] In order to satisfy the above conditions, polypeptides of the present invention should include the following consensus sequences in their sequences.
    [49] Z 3-48 CZ 9-13 C (Q, E, R, K) Z (Z Hydrophobic ) (LIVM) Z 15-39 CC (Z Hydrophilic ) (Q, E, H) (L, V) Z 6 CZC Z 2 (L, I) Z 13-56 GZ 15-26 CZ (V, I, L, M) Z 1-8 CZ 1-12
    [50] In the above, () represents one amino acid, and the sequences in parentheses are possible preferred sequences. Zn represents n amino acids whatever it is. This sequence has a CZnC domain (see Tamaki et al "Folding motifs induced and stabilized by distinct cystine frameworks" Protein engineering 11, 649-659 (1988)).
    [51] In the case of dimeric polypeptides, the consensus sequence can be placed between two subunit sequences, meaning that there is a hydrolysis point that must be present in one of the regions indicated by Zn of this sequence.
    [52] Polypeptides of the invention should show a significant proliferative effect in a model of murine splenocytes (threefold multiplier compared to the control). The effect of treating polypeptides on spleen cells of rat Balb / c in vitro was evaluated. Assays were performed on microplates and proliferation was quantified by chromatographic methods (T Mosmann "Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytoxicity assays" J. Immunol. Methods 65, 55-63 (1983) ] Reference).
    [53] c) formation of polysaccharide-polypeptide conjugates
    [54] Formation of polysaccharide-polypeptide conjugates occurs spontaneously starting from a solution of the two components in water or saline solution where the ionic strength does not exceed the sodium chloride solution level of 0.15 M concentration at room temperature. The molar ratio of the polysaccharide-polypeptide conjugate may be in the range of 1/1 to 1/19 (mol / mol). The polypeptide and polysaccharide solution, which are retained in an appropriate amount (to satisfy the molar ratio) in each other in the medium, are mixed at a temperature of 15 to 40 ° C. and gently shaken at 1 to 100 rpm to form a conjugate. The solution mixture is left to shake for 5 to 60 minutes. Once the conjugate has been formed, it can be administered as it is or in any suitable formulation prior to sterile filtration when used via the parenteral, intramuscular or subcutaneous route.
    [55] One polysaccharide and two polypeptides may form a conjugate as long as the polysaccharide / total polypeptide ratio described above is maintained, and the conjugate must satisfy the following conditions in addition to the conditions described above.
    [56] a) the molar ratio between the two polypeptides is from 1/3 to 3/1
    [57] b) Both polypeptides have the same biological origin.
    [58] c) both polypeptides have at least 25% sequence homology (absolute sum of at least 50%).
    [59] d) form of preparation
    [60] Injectable Pharmaceutical Forms: The conjugates were sterilized by dialysis or filtration against apirogen sterile saline solution and filtered to 0.22i under apirogen sterile conditions.
    [61] Oral Form: The solution in which the binder is obtained, or an instant solution in which the binder is lyophilized in water, in any conventional pharmaceutical formulation such as tablets, pills or capsules using the necessary prosthesis, or syrup Or in any oral liquid pharmaceutical form.
    [62] Pharmaceutical Forms for Topical Administration: The conjugates of the present invention may be formulated in conventional forms of topical formulations such as gels, creams and ointments having a concentration of 1 to 5% (w / w) using conventional pharmaceutical prostheses. Can be.
    [64] (Example 1)
    [65] 1. Preparation of Polysaccharides
    [66] G Kogan, J Sandula, V Simkovicova "Glucomannan from Candida utilis. Structural investigation" Folia Microbiol (Praha) 38, 219-224 (1993), KH Rademacher, Y Koch "(Structure of the cell wall mannans of synchronously multiplying candida utilis cells) "Z All. Microbio 19, 65-67 (1979)] was prepared as follows.
    [67] In this example, the polysaccharide, which is an essential component of the conjugate of the present invention, was prepared from a commercial dry candida utility which can be used in humans according to the following method.
    [68] About 100 g of 1.1 soybeans were weighed and soaked in water for 24 hours.
    [69] 1.2 The soybeans were washed several times.
    [70] 1.3 These were ground with a mortar and pestle.
    [71] 2 L of an aqueous solution containing 6.25 g / L 1.4 MnSO 4 .H 2 O and 3.33 g / L CoCl 2 H 2 O was prepared. The temperature was kept at 37 ° C. Stir with a magnetic stirrer, add 0.21 g / l MnO 2 , 62.5 g / l dry Candida utility, and 12.5 g / l soy flour.
    [72] Incubate for 48 hours at 37 ° C. in an orbital stirrer stirred at 1.5 200 rpm.
    [73] 1.6 left to separate the supernatant and centrifuged at 2300 x g for 10 minutes at room temperature. The centrifuge supernatant was vacuumed with paper and filtered through a 0.45i filter.
    [74] Dialysis was carried out three to five times of water in MilliRQ water (4-8 ° C.) five times the magnetic volume for 1.7 days.
    [75] When the 1.8 precipitate appeared, it was removed by centrifugation at 2300 × g for 10 minutes at room temperature.
    [76] 1.9 If necessary, the dialysate or centrifuged dialysate was lyophilized.
    [77] Purification by conventional methods such as 1.10 molecular permeation chromatography (permeation in gels such as Sephacryl S-200 or S-400), ultrafiltration (pass 50.000 molecular cleavage membranes such as Amicon).
    [78] 1.11 It can be lyophilized if desired.
    [79] 1.12 This method yielded 0.2 to 6.4 g of polysaccharide per 100 g of yeast as a pure product. The yield is at a level that enables industrial scale.
    [80] The average molecular weight of the polysaccharide thus obtained was 150 ± 30 when 10 mM phosphate buffer, 0.3 M NaCl (pH 7.4) was used as the eluent, and molecular exclusion was performed on a TSK40 column and the refractive index was detected by comparison with the Fluka Dextra standard. Measured by KDa. The phosphate content determined according to the method of Hethwa Deere showed one phosphate residue per 15 monosaccharide residues (HH Hess, JE Deer "Assay of inorganic and organic phosphrous in the 0.1-5 nanomolrange." Anal Biochem 63: 607-613 (1975). The composition of monosaccharides is described in A Novotny "Basic exercises in immunochemistry" S. Verlag Ed. Berlin, Heildelberg, New York pp 127-131 (1979)], G Keleti, WH Leaderer "Handbook of Micromethods for the Biological Science" Ed. Van Nostrand Reinhold. New York. pp 55-57 and HP Burchfield, EE Storrs "Biochemical Applications of Gas Chromatography" Academic Press New York (1962), as determined by gas chromatography of hydrolysis, reduction, acetylation and alditol acetylated derivatives. Mannose 84 ± 6%, glucose 7 ± 3% and galactose 1 ± 1%. According to the structural analysis determined by the decomposition method of Smith (see F Smith, R Montgomery Meth Biochem Anal 3: 153 (1956)), the polysaccharide shows a linear structure 1-6 (45 ± 5% monosaccharide found) ), One to two branches (45 ± 5% of monosaccharides may be found). There is no positive reaction to carboxylate or sulphate. The polysaccharide thus obtained was interacted with octadecylsilane when injected into the column of this character in an aqueous medium (column C18 Vydac, at least 25% acetonitrile for elution). The polysaccharide thus obtained was subjected to chromatographic behavior in the aforementioned TSK 40 column or phosphate content after incubation for 1 hour with shaking at 50 to 100 rpm in incomplete gastric fluid (2 g / L NaCl, 7 mL / L concentrated hydrochloric acid) at 37 ° C. There was no change. The polysaccharide thus obtained does not gel when calcium chloride is present at a concentration of less than 10 mM. The polysaccharides thus obtained showed no anticoagulant activity in vitro (see TA Harper "Laboratory guide to disordered haemostasis" pp 76-77 Butterworths (1970)).
    [81] 2. Preparation of Polypeptides
    [82] FS Sharief, SSl Li "Aminoacid sequence of small and large subunits protein from Ricinus communis" J. Biol. Chem. 257, 14753-14759 (1982)], J Godinho da Silva Jr, OLT Machado, C Izumi, JC Padovan, BT Chait, UA Mirzaa, LJ Geene "Aminoacid sequence of a new 2S albumin which is part of a 29 kDa precursor protein "Arch. Biochem. Biophys. 336, 10-18 (1996)], GM Neumann, R Condron, GM Polya "Purification and sequencing of napin-like protein small and large subunits from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca 2+-dependent protein kinase "Biochem. Biophys. Acta 1298, 223-240 (1996)], MEH Bashir, I Hubatsch, HP Leinenbach, M Zeppezauer, RC Panzani, IH Hussein "Ric c1 and Ric c3, the allergenic 2S albumin storage proteins of Ricinus communis: Complete primary structures and phylogenetic relationships "Int. Arch. Allergy Immunol. 115, 73-82 (1998), according to the method described in the following manner.
    [83] In this example, the polypeptide, which is an essential component of the conjugate of the present invention, was prepared from non-germinated seeds of Ricinus communis by the method described below.
    [84] 2.1 First, 100 g of seeds washed with water were ground until a fine paste was obtained.
    [85] 2.2 The extract was obtained by magnetic stirring of the paste with 500 ml of water at 4 ° C. for 18 hours.
    [86] 2.3 Next, through a lightweight Hiflo hypercell 0.2 mm coated stainless steel filter mesh and with a polypropylene prefilter and a nitro cellulose or analogous filter having a diameter of 40 mm, 80im, 8im, 5im, 0.45im and 0.22im Seed residue was removed.
    [87] 2.4 The filtrate was acidified to pH 1.5 with phosphoric acid diluted to 50% (volume / volume) using MilliQ water.
    [88] Heated to 56 ° C. for 120 minutes with moderate magnetic stirring in a water bath with a 2.5 thermometer.
    [89] 2.6 Centrifuged at 2300 x g for 15 minutes at room temperature. The supernatant was carefully separated to avoid contamination with precipitate.
    [90] 2.7 The supernatant was neutralized to pH 7.0-7.5 using 20% (weight / volume) NaOH solution.
    [91] Centrifuged at 2300 x g for 15 minutes at room temperature. The supernatant was carefully separated to avoid contamination with precipitate.
    [92] The supernatant was ultrafiltered with a 5000 Da molecular cut membrane until the 2.9 volume was approximately 1/2. Milliq water was added to make an initial volume, which was again filtered to 1/2 volume. This process was repeated four times.
    [93] 2.10 The concentrated and washed supernatant resulting from the previous step was examined by reverse phase column (Vydac C4) chromatography, and the polypeptide was purified by eluting with acetonitrile concentration adjusted to 18-22%.
    [94] 2.11 Solvents were evaporated by freeze drying and excess salts were removed by filtration or chromatography in BioGel P10 or the like.
    [95] 2.12 If desired, it can be lyophilized.
    [96] 2.13 This procedure yielded 0.2-1.0 g of polypeptide per 100 g of Lysinus seed as a pure product. The yield is at a level that enables industrial scale.
    [97] The average molecular weight of the polypeptide thus obtained was 12 ± 0.5 KDa as determined by mass spectroscopy, and was dimer as determined by polyacrylamide gel electrophoresis under sensitizing and reducing conditions (H Schagger, G von Jagow "). tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa: Anal.Biochem. 166, 368-379 (1987)). The dimers are bound by disulfide bridges. It can be inferred from the fact that reducing conditions are required for interpretation by electrophoresis The polypeptides produced are also 24 at 37 ° C. in trypsin (0.1m Tris-HCl, pH 8.5, at a polypeptide protease ratio of 30: 1). Incubated for hours), pepsin (incubated for 24 hours at 37 ° C. in 0.01M HCl at a polypeptide protease ratio of 25: 1) The it met the rest of the conditions described in the detailed description. Sequence determined by the Edman decomposition method was as follows.
    [98] Small Units: ESKGEREGSSSQQ C RQEVQRKDLSS CE RY L RQSSSRR
    [99] Major subunits:
    [100] QQQESQQLQQ CC N QV KQVRDE C Q C EA I KYIAEDQIQQGQLH G EESERVAQRAGEIVSS C G V R C MRQTR
    [101] (Amino acids specified in common sequences are underlined)
    [102] The polypeptide thus obtained induces proliferation of splenocytes with a maximum proliferation index of 5 at a concentration of 3 μg / ml per se.
    [103] 3. Formation of Conjugates
    [104] The polysaccharide prepared as described in Example 1 of this Example was dissolved in water so as to have a total volume of 50 ml at a concentration of 1 mg / ml, and the polypeptide prepared as described in 2 of this Example at a concentration of 1 mg / ml. It was dissolved in water so that the total volume was 10 ml. 34 ml of the polysaccharide solution and 6.5 ml of the polypeptide solution were poured into a glass container at room temperature, water was added so that the final volume was 300 ml, and the mixture was shaken at 50 rpm for 30 minutes. Thereafter, 1 ml aliquots were taken and stored frozen until administration to experimental animals.
    [105] 4. Biological Activity: Inhibition of Tumor Necrosis Factor (TNF) Production Induced by Bacterial Endotoxin (LPS) in BalB / C Rat Serum
    [106] The polysaccharide-polypeptide conjugate of the present invention was administered to Balb / c mice for 6 consecutive days by oral route in 0.5 ml of the solution prepared as described above, followed by 25 μg of E coli endotoxin serotype 055: B5 per animal. Injection. This treatment inhibited TNF serum levels by 65% 90 minutes after LPS administration.
    [107] When administered separately at doses similar to those in the conjugate, neither of the polysaccharide and polypeptide components showed activity.
    [108] TNF was determined by bioassay to measure serum cytotoxicity against L929 cell line (T Mosmann "Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytoxicity assays" J. Immunol. Methods 65, 55- 63 (1983).
    [109] (Example 2)
    [110] 1. Preparation of Polysaccharides
    [111] Prepared from Candida utility as described in Example 1-1.
    [112] 2. Preparation of Polypeptides
    [113] FS Sharief, SSl Li "Aminoacid sequence of small and large subunits protein from Ricinus communis" J. Biol. Chem. 257, 14753-14759 (1982)], J Godinho da Silva Jr, OLT Machado, C Izumi, JC Padovan, BT Chait, UA Mirzaa, LJ Geene "Aminoacid sequence of a new 2S albumin which is part of a 29 kDa precursor protein "Arch. Biochem. Biophys. 336, 10-18 (1996)], GM Neumann, R Condron, GM Polya "Purification and sequencing of napin-like protein small and large subunits from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca 2+ - dependent protein kinase "Biochem. Biophys. Acta 1298, 223-240 (1996)], MEH Bashir, I Hubatsch, HP Leinenbach, M Zeppezauer, RC Panzani, IH Hussein "Ric c1 and Ric c3, the allergenic 2S albumin storage proteins of Ricinus communis: Complete primary structures and phylogenetic relationships "Int. Arch. Allergy Immunol. 115, 73-82 (1998), according to the method described in the following manner.
    [114] In this example, the polypeptide, which is an essential component of the conjugate of the present invention, was prepared from non-germinated seeds of Ricinus communis by the method described below.
    [115] 2.1 First, 100 g of seeds washed with water were ground until a fine paste was obtained.
    [116] 2.2 The extract was obtained by magnetic stirring of the paste with 500 ml of water at 4 ° C. for 18 hours.
    [117] 2.3 Next, through a lightweight Hiflo hypercell 0.2 mm coated stainless steel filter mesh and with a polypropylene prefilter and a nitro cellulose or analogous filter having a diameter of 40 mm, 80im, 8im, 5im, 0.45im and 0.22im Seed residue was removed.
    [118] 2.4 The filtrate was acidified to pH 1.5 with phosphoric acid diluted to 50% (volume / volume) using MilliQ water.
    [119] Heated to 56 ° C. for 120 minutes with moderate magnetic stirring in a water bath with a 2.5 thermometer.
    [120] 2.6 Centrifuged at 2300 x g for 15 minutes at room temperature. The supernatant was carefully separated to avoid contamination with precipitate.
    [121] 2.7 The supernatant was neutralized to pH 7.0-7.5 using 20% (weight / volume) NaOH solution.
    [122] Centrifuged at 2300 x g for 15 minutes at room temperature. The supernatant was carefully separated to avoid contamination with precipitate.
    [123] The supernatant was ultrafiltered with a 5000 Da molecular cleavage membrane until the 2.9 volume was approximately 1/2. Milliq water was added to make an initial volume, which was again filtered to 1/2 volume. This process was repeated four times.
    [124] 2.10 The concentrated and washed supernatant resulting from the previous step was examined by reversed phase column (Vydac C4) chromatography, and the polypeptide was purified by eluting with acetonitrile concentration adjusted to 22-24%.
    [125] 2.11 Solvents were evaporated by freeze drying and excess salts were removed by filtration or chromatography in BioGel P10 or the like.
    [126] 2.12 If desired, it can be lyophilized.
    [127] 2.13 This procedure yielded 0.2-1.0 g of polypeptide per 100 g of Lysinus seed as a pure product. The yield is at a level that enables industrial scale.
    [128] The average molecular weight of the polypeptide thus obtained was 11 KDa ± 0.5 KDa as determined by mass spectroscopy, and was dimer as determined by polyacrylamide gel electrophoresis under sensitization and reducing conditions (H Schagger, G von Jagow). "tricine-sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa: Anal. Biochem. 166, 368-379 (1987)]. The dimers are bound by disulfide bridges. This can be inferred from the fact that reducing conditions are required for interpretation by electrophoresis The prepared polypeptide is also 37 ° C. in trypsin (30: 1 polypeptide protease ratio at 0.1 m Tris-HCl, pH 8.5). Incubated for 24 hours at), pepsin (incubated for 24 hours at 37 ° C. in 0.01M HCl at a polypeptide protease ratio of 25: 1), It met the rest of the conditions described in the detailed description of the invention. Sequence determined by the Edman decomposition method was as follows.
    [129] Small Units: PSQQG C RGQIQEQQNLRQ CQ EY I KQQVSGQGPRR
    [130] Major subunits:
    [131] QERSLRG CC D HL KQMQSQ C R C EG L RQAIEQQQSQGQLQ G QDVFEAFRTAANLPSM C G V SPTE C RF
    [132] (Amino acids specified in common sequences are underlined)
    [133] The polypeptide thus obtained in itself induced proliferation of splenocytes with a maximum growth index of 4 at a concentration of 6 μg / ml.
    [134] 3. Formation of the conjugate
    [135] The polysaccharide prepared as described in 1 of Example 1 was dissolved in water at a concentration of 1 mg / ml so as to have a total volume of 50 ml, and the polypeptide prepared as described in 2 of this Example was 1 mg / ml. It was dissolved in water so that the total volume was 10 ml at the concentration. 34 ml of the polysaccharide solution and 6.5 ml of the polypeptide solution were poured into a glass container at room temperature, water was added so that the final volume was 300 ml, and the mixture was shaken at 50 rpm for 30 minutes.
    [136] 4. Biological Activity: Inhibition of Tumor Necrosis Factor (TNF) Production Induced by Bacterial Endotoxin (LPS) in BalB / C Rat Serum
    [137] The polysaccharide-polypeptide conjugate of the present invention was administered to Balb / c mice for 6 consecutive days via the oral route in 0.5 ml of the solution prepared as described above, followed by 25 μg per E coli endotoxin serotype 055: B5 per animal. Intravenously injected. This treatment resulted in a 55% inhibition of TNF serum levels 90 minutes after LPS administration.
    [138] When administered separately at doses similar to those in the conjugate, neither of the polysaccharide and polypeptide components showed activity.
    [139] TNF was determined by bioassay to measure serum cytotoxicity against L929 cell line (T Mosmann "Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytoxicity assays" J. Immunol. Methods 65, 55- 63 (1983).
    [140] (Example 3)
    [141] 1. Preparation of Polysaccharides
    [142] Prepared from Candida utility as described in Example 1-1.
    [143] 2. Preparation of Polypeptides
    [144] FS Sharief, SSl Li "Aminoacid sequence of small and large subunits protein from Ricinus communis" J. Biol. Chem. 257, 14753-14759 (1982)], J Godinho da Silva Jr, OLT Machado, C Izumi, JC Padovan, BT Chait, UA Mirzaa, LJ Geene "Aminoacid sequence of a new 2S albumin which is part of a 29 kDa precursor protein "Arch. Biochem. Biophys. 336, 10-18 (1996)], GM Neumann, R Condron, GM Polya "Purification and sequencing of napin-like protein small and large subunits from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca 2+ - dependent protein kinase "Biochem. Biophys. Acta 1298, 223-240 (1996)], MEH Bashir, I Hubatsch, HP Leinenbach, M Zeppezauer, RC Panzani, IH Hussein "Ric c1 and Ric c3, the allergenic 2S albumin storage proteins of Ricinuscommunis: Complete primary structures and phylogenetic relationships "Int. Arch. Allergy Immunol. 115, 73-82 (1998), according to the method described in the following manner.
    [145] In this example, the polypeptide, which is an essential component of the conjugate of the present invention, was prepared from non-germinated seeds of Ricinus communis by the method described below.
    [146] 2.1 First, 100 g of seeds washed with water were ground until a fine paste was obtained.
    [147] 2.2 The extract was obtained by magnetic stirring of the paste with 500 ml of water at 4 ° C. for 18 hours.
    [148] 2.3 Next, through a lightweight Hiflo hypercell 0.2 mm coated stainless steel filter mesh and with a polypropylene prefilter and a nitro cellulose or analogous filter having a diameter of 40 mm, 80im, 8im, 5im, 0.45im and 0.22im Seed residue was removed.
    [149] 2.4 The filtrate was acidified to pH 1.5 with phosphoric acid diluted to 50% (volume / volume) using MilliQ water.
    [150] Heated to 56 ° C. for 120 minutes with moderate magnetic stirring in a water bath with a 2.5 thermometer.
    [151] 2.6 Centrifuged at 2300 x g for 15 minutes at room temperature. The supernatant was carefully separated to avoid contamination with precipitate.
    [152] 2.7 The supernatant was neutralized to pH 7.0-7.5 using 20% (weight / volume) NaOH solution.
    [153] Centrifuged at 2300 x g for 15 minutes at room temperature. The supernatant was carefully separated to avoid contamination with precipitate.
    [154] The supernatant was ultrafiltered with a 5000 Da molecular cleavage membrane until the 2.9 volume was approximately 1/2. Milliq water was added to make an initial volume, which was again filtered to 1/2 volume. This process was repeated four times.
    [155] 2.10 Total up to elution positive for Lowry reaction (OH Lowry, HJ Rosenbrough, AL Farr, RJ Randall "Protein measurement with the Folin phenol reagent." J. Biol. Chem. 193,265-275 (1951)) The volume was purified by Molecular Permeation Chromatography in BioGel P10 (area through area) and the volume of eluate equal to or greater than the entire layer was discarded.
    [156] 2.11 If desired, it can be lyophilized.
    [157] 2.12 This procedure yielded polypeptides in the range of 0.4 to 1.2 g per 100 g of Lysinus seed. The polypeptide was a mixture of the two polypeptides described above (Examples 1 and 2), with a mixing ratio of polypeptide 1 / polypeptide 2 ranging from 35/75 to 75/35. The yield is at a level that enables industrial scale.
    [158] When the polypeptide thus obtained was administered together at the obtained ratio (12 kDa polypeptide / 11 kDa polypeptide = 1/2), the splenocytes were induced with a maximum growth index of 6 at a concentration of 3 µg / ml per se.
    [159] 3. Formation of the conjugate
    [160] The polysaccharide prepared as described in Example 1 1 was dissolved in water at a concentration of 3.75 mg / ml so that the total volume was 150 ml, and the polypeptide prepared as described in Example 2 2 had a concentration of 0.75 mg / ml. It was dissolved in water so that the total volume of the furnace was 150 ml. 34 ml of the polysaccharide solution and 6.5 ml of the polypeptide solution were poured into a glass container at room temperature, put in a magnet, and shaken at 50 rpm for 30 minutes. It was then frozen, lyophilized and kept frozen until administration to experimental animals and dissolved in distilled water at a suitable concentration appropriate for the dosage required at the time of administration.
    [161] The infrared spectrum of the sugar binder thus obtained is shown in FIG. 1. This spectrum uses various slots for 6 minutes on an infrared spectrophotometer Perkin-Elmer model 881, passing through 4000 to 600 cm -1 with a resolution of 1000 cm -1 and spectral noise of 0.5% T and reducing the sugar binder concentration to 0.2 It was carried out in potassium bromide pellets in%, electronically adjusted by the Savitzky / Golay method, and the absorbance was automatically expanded.
    [162] 4. Biological Activity: Inhibition of Tumor Necrosis Factor (TNF) Production Induced by Bacterial Endotoxin (LPS) in BalB / C Rat Serum
    [163] E coli endotoxin per animal after administration of the polysaccharide-polypeptide conjugates of the invention to Balb / c mice for 6 consecutive days via the oral route in a 0.5 ml solution prepared as described above at a dose of 3 mg / kg. Intravenous injection of 25 μg of serotype 055: B5. This treatment inhibited TNF serum levels by 65% 90 minutes after LPS administration.
    [164] TNF was determined by bioassay to measure serum cytotoxicity against L929 cell line (T Mosmann "Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytoxicity assays" J. Immunol. Methods 65, 55- 63 (1983).
    [165] Effect of Multiple Dose of Polysaccharide-Polypeptide on the Production of TNF Induced by Serum in Mice
    [166] Multiple doses of the conjugates of the present invention were administered to mice according to the same treatment schedule as previously described. The results showed a dose effect relationship between the inhibition of TNF and the dose of the conjugate. Dose effect curves showed a bell type with a maximum inhibition of 90% at a dose of 48 mg / kg.
    [167] Increased hematopoietic activity assessed by increased progenitor cell number in granulocyte-macrophage (CFU-GM)
    [168] Intravenous administration of the polysaccharide-polypeptide conjugate to rat C57BI / 6 at a single dose of 2 mg / kg in 0.25 ml resulted in the formation of progenitor cells in granulocyte-macrophage lines measured 5 days post-dose.
    [169] Intravenous administration of the polypeptide at a dose similar to that in the conjugate increased the number of precursor cell CFU-GM by 227%. Administration of the polysaccharide separately in the intravenous route at the same dose as in the conjugate showed no effect in this assay. When the conjugate was administered at the previously described doses, the activity increased by 3763%.
    [170] Increased Survival in Mice Infected and Immunized with Monoclonal Listeria
    [171] Infection after administration of the conjugates of the present invention to Swiss rats by oral route at 3 mg / kg in a volume of 0.5 ml for 6 consecutive days protected mice that were immunosuppressed with silica and infected with monocytic Listeria. Immunosuppression was induced by intraperitoneal administration of 120 mg / kg of silica one day prior to infection. The preventive capacity of the conjugates of the present invention was confirmed to increase the lethal dose 50 in animals treated with the conjugates of the present invention to a level similar to that of non-immunosuppressed animals.
    [172] Restoration of Anti-tumor Cytotoxin Activity of NK Cells in Immunocompromised Animals
    [173] Treatment of the conjugates of the present invention at 3 mg / kg in a volume of 0.5 ml for 4 consecutive days increased NK activity in splenocytes of normal rats and reduced cyclone to a single dose of 180 mg / kg with aging rats, even when reduced. Normalized to the extent occurring in mice immunosuppressed with phosphamide.
    [174] Effect on Macrophage Function in Rats
    [175] The oral route of administration of the conjugate of the present invention to Balb / c rats at a dose of 0.9 mg / kg in a volume of 0.2 ml for 6 consecutive days increased the phagocytic bactericidal ability of peritoneal macrophages against Staphylococcus aureus. A clear relationship was observed between the response levels.
    [176] When macrophages fight intracellular phagocytic Candida guillermondy, the phagocytic value as well as the bactericidal activity was observed to increase.
    [177] Activity against pulmonary edema induced by intranasal endotoxin
    [178] Pulmonary edema was induced by dropping 400ig of E. coli 0.55: B5 (Sigma) LPS, and pulmonary edema was evaluated by visual inspection of the lung surface on day 3 after administration. The intraperitoneal administration of the conjugates of the present invention in various doses (0.9 to 4.5 mg / kg) in 0.5 ml of apirogen sterile water from the day of LPS administration to the sacrifice of the animals clearly reduced edema. The effective amount 50 was 1.67 mg / kg.
    [179] Acute Toxicity Analysis in Rats
    [180] When the oral administration of the polysaccharide-polypeptide conjugate of the present invention to the CD1 line mice at 200 mg / kg dose in 1 ml, there is no reading of no change in body weight or macroscopic or microscopic weight and appearance of major life support organs. This was confirmed.
    [181] Activity on hepatic metabolism
    [182] When the oral route was administered to Sprague-Daurie rats at a dose of 3 mg / kg in 0.5 ml of the conjugate of the present invention, it did not inhibit the antipyrine removal.
    [183] When the conjugate of the present invention was administered by oral route to rats of the same strain for 6 consecutive days in a single dose of 3 times or more in 0.5 ml, the contents of cytochrome P450, cytochrome b5 and NADPH cytochrome c reductase were unchanged and cytochrome P450 ( There was no change in the biotransformation enzyme activity associated with phase II binding enzyme in phase I) or in hepatic microsomes of rats.
    [184] As described above, according to the present invention there is provided a polypeptide-polysaccharide noncovalent conjugate that is active by oral administration in controlling a human or animal immune response. The polypeptide-sugar conjugate can stimulate the mononuclear phagocyte system and expand the granulocyte macrophage zone, but can down-regulate TNF production induced under certain experimental conditions without inhibiting the liver metabolic system.
    权利要求:
    Claims (6)
    [1" claim-type="Currently amended] In the sugar conjugate formed by the non-covalent linkage of the polysaccharide and the polypeptide, the polysaccharide fraction has a molecular weight of 50 to 250 KDa, and accommodates the phosphate functional group in the ratio of one phosphate group to 5 to 25 monosaccharide residues, and 40% of mannose Wherein the remainder may be glucose and / or galactose, consisting of a main backbone bound by 1 to 6 bonds, having no more than 60% 1 to 2 branches, wherein the polypeptide fraction is Z 3-48 CZ 9-13 C (Q, E, R, K) Z (Z Hydrophobic ) (LIVM) Z 15-39 CC (Z Hydrophilic ) (Q, E, H) (L, V) Z 6 CZC Z 2 (L, I) Z 13-56 GZ 15-26 CZ (V, I, L, M) Z 1-8 CZ 1-12 (wherein the symbols indicate amino acids, the parentheses indicate the preferred sequence, Zn Is a n-amino acid, whatever it is.).
    [2" claim-type="Currently amended] The polypeptide-glycol conjugate of claim 1, wherein the polypeptide fraction is comprised of one or two polypeptides, wherein the mole / molar ratio of the two polypeptides is 1/3 to 3/1.
    [3" claim-type="Currently amended] The polypeptide-sugar binder according to claim 1, wherein the polypeptide fraction is a dimer having a molecular weight of 12 ± 0.5 KDa and has an amino acid sequence selected from the following.
    Small Units: ESKGEREGSSSQQ C RQEVQRKDLSS CE RY L RQSSSRR
    PSQQG C RGQIQEQQNLRQ CQ EY I KQQVSGQGPRR
    Major subunits:
    QQQESQQLQQ CC N QV KQVRDE C Q C EA I KYIAEDQIQQGQLH G EESERVAQRAGEIVSS C G V R C MRQTR
    QERSLRG CC D HL KQMQSQ C R C EG L RQAIEQQQSQGQLQ G QDVFEAFRTAANLPSM C G V SPTE C RF
    (Underlined are amino acids specified in consensus sequence.)
    [4" claim-type="Currently amended] The polypeptide-sugar conjugate of claim 1, wherein the structure of the polypeptide fraction is stabilized by disulfide or dimethylene bridges, may be oligomers or dimers, and has two or more disulfide or dimethylene intrachain bridges. .
    [5" claim-type="Currently amended] The polypeptide-sugar conjugate of claim 1, which has pharmacological activity and can be used in a drug for treating immune system abnormalities.
    [6" claim-type="Currently amended] Use of the polypeptide-sugar conjugate of claim 1 in a pharmaceutical for use in a formulation of a conventional herbal form.
    类似技术:
    公开号 | 公开日 | 专利标题
    JP5491485B2|2014-05-14|Transmucosal agent for use in transmucosal administration of a conjugate in which a pharmacologically active substance and a mucoadhesive polymer are covalently bonded
    US20190169231A1|2019-06-06|Compositions containing hc-ha/ptx3 complexes and methods of use thereof
    TWI601744B|2017-10-11|A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
    EP0335423B2|2003-03-05|Modified human G-CSF
    EP0640622B1|2000-08-09|Polysaccharide derivative and drug carrier
    EP0491829B1|1997-06-04|Composition for immune system activation
    KR102011924B1|2019-08-21|Improved peptide pharmaceuticals for insulin resistance
    US20180360922A1|2018-12-20|Medicament for treatment of tumors and the use thereof
    CA2006596C|2000-09-05|Chemically-modified g-csf
    EP0862455B1|2007-12-26|Interferon-polymer conjugates and process for preparing the same
    CN102757505B|2015-10-07|There is the physiologically active polypeptide conjugate of the Half-life in vivo of prolongation
    US6838450B2|2005-01-04|Drug complex
    US7803757B2|2010-09-28|Peptides based on the sequence of human lactoferrin and their use
    US6166183A|2000-12-26|Chemically-modified G-CSF
    Siemion et al.1999|Tuftsin: on the 30-year anniversary of Victor Najjar’s discovery
    JP4633361B2|2011-02-23|Therapeutic treatment
    ES2371865T3|2012-01-10|Conjugates of hydroxialquilalmidón-active principle.
    JP4011106B2|2007-11-21|Platelet growth promoter
    ES2568899T3|2016-05-05|Procedure to control the activity of an immunofunctional molecule
    Fan et al.2012|Partial characterization and anti-tumor activity of an acidic polysaccharide from Gracilaria lemaneiformis
    Kimura et al.2006|Antitumor and antimetastatic activity of a novel water-soluble low molecular weight β-1, 3-D-glucan | isolated from Aureobasidium pullulans 1A1 strain black yeast
    Di Luzio1985|Update on the immunomodulating activities of glucans
    Daba et al.2003|Anti-cancer effect of polysaccharides isolated from higher basidiomycetes mushrooms
    US20010041189A1|2001-11-15|Poly| as a drug carrier
    JP4067058B2|2008-03-26|PEGylated HGF
    同族专利:
    公开号 | 公开日
    AU770046B2|2004-02-12|
    CN1344169A|2002-04-10|
    BR9917150A|2002-01-22|
    CA2363095A1|2000-08-31|
    US20050203004A1|2005-09-15|
    JP2002537361A|2002-11-05|
    ES2163966B2|2003-07-01|
    AU6477799A|2000-09-14|
    CN1265839C|2006-07-26|
    KR100599176B1|2006-07-12|
    WO2000050087A1|2000-08-31|
    US7241735B2|2007-07-10|
    ES2163966A1|2002-02-01|
    EP1163911A1|2001-12-19|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1999-02-26|Priority to ES009900408A
    1999-02-26|Priority to ESP9900408
    1999-10-21|Application filed by 인더스트리얼 파르머서티카 칸타브리아 에스에이
    2001-12-20|Publication of KR20010112290A
    2006-07-12|Application granted
    2006-07-12|Publication of KR100599176B1
    优先权:
    申请号 | 申请日 | 专利标题
    ES009900408A|ES2163966B2|1999-02-26|1999-02-26|Glicoconjugados constituted by the non-covalent association of polisacarids with polypeptides.|
    ESP9900408|1999-02-26|
    [返回顶部]