• 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:
    For the Contracting States : BE, CH, DE, FR, GB, IT, LI, NL, SE 1. Oligopeptide isolable from sound erythrocytes or granulocytes, respectively, selectively inhibiting the growth or proliferation, respectively, of normal and leukemic myeloid cells, characterized in that it has the gross amino acid composition Tau1 , Asx1 , Ser2 , Thr1 , Glx3 , Gly2 , Ala1 , (PO4 )1 in which Tau represents a radical of taurine, Asx means a radical of L-asparagin and/or L-asparaginic acid, Ser represents a radical of L-serine, Thr means a radical of L-threonine, Glx represents a radical of L-glutamine and/or L-glutamic acid, Gly means a radical of glycine and Ala represents a radical of L-alanine and exhibits at a pH-value of 6.5 a negative charge and a relative electrophoretic mobility of from -0.55 to -0.65 referred to asparaginic acid or at a pH-value of 1.9 no charge and a relative mobility of about 0.26 referred to epsilon-(dinitrophenyl)-lysine and has a positive reaction to chlorotolidine. For the Contracting State : AT 1. A process for preparing an oligopeptide isolable from sound erythrocytes or granulocytes, respectively, selectively inhibiting the growth or proliferation, respectively, of normal and leukemic myeloid cells having the gross amino acid composition Tau1 , Asx1 , Ser2 , Thr1 , Glx3 , Gly2 , Ala1 , (PO4 )1 in which Tau represents a radical of taurine, Asx means a radical of L-asparagin and/or L-asparaginic acid, Ser represents a radical of L-serine, Thr means a radical of L-threonine, Glx represents a radical of L-glutamine and/or L-glutamic acid, Gly means a radical of glycine and Ala represents a radical of L-alanine and exhibits at a pH-value of 6.5 a negative charge and a relative electrophoretic mobility of from -0.55 to -0.65 referred to asparaginic acid or at a pH-value of 1.9 no charge and a relative mobility of about 0.26 referred to epsilon-(dinitrophenyl)-lysine and having a positive reaction to chlorotolidine, characterized in that one a) homogenises sound erythrocytes gained from animal or human blood, particularly horse blood, in a buffer solution having a pH-value of from 7 to 8, one centrifuges the liquid homogenisate, one separates from the supernantant liquid the dissolved components having molecular weights below 10 000, one subjects this fraction to a further fractionation, suitably by gel chromatography, and separates the fraction precipitating at volume values ve /vo of from 1.15 to 1.45 and optionally lyophilises it or b) homogenises animal organs containing granulocytes, particularly calf spleen, in an organic solvent miscible with water, one separates from the obtained liquid suspension the solids and extracts them with an organic solvent dissolving fats, one extracts the thus obtained solids with water, one separates from the liquid suspension obtained the insoluble part and one separates from the liquid phase the dissolved components having molecular weights below 10 000, one subjects this fractions to a further fractionation, suitably by gel chromatography, and one separates the fraction precipitating at volume values ve /vo of from 1.3 to 2.5 and optionally lyophilises and one purifies the concentrate of active principle obtained according to one of the above variants a) and b) by chromatographing fractionating on account of the ionic charge and particle size and/or by paper-electrophoresis and one separates the oligopeptide having at a pH-value of 6.5 a negative charge and a relative mobility of from -0.55 to -0.65 referred to asparaginic acid or exhibiting at a pH-value of 1.9 no charge and a relative mobility of about 0.26 referred to epsilon-(dinitrophenyl)-lysine, respectively and positively reacting to chlorotolidine and optionally lyophilises it.
    公开号:SU1367837A3
    申请号:SU813230703
    申请日:1981-01-14
    公开日:1988-01-15
    发明作者:Балаж Андраш；Шайго Михай；Кишфалуди Лайош；Клупп Тибор；Барабаш Миклошне
    申请人:Рихтер Гедеон Ведьесети Дьяр Рт (Инопредприятие)；
    IPC主号:
    专利说明:


    ace with
     cm
    This invention relates to medicine, in particular to methods for producing effective substances from healthy blood cells.
    The aim of the invention is to increase the activity of the target product.
    The method is carried out as follows.
    Healthy white blood cells, obtained from the blood of animals or humans, preferably from horse blood, are homogenized in a buffer solution of pH 7-8, the liquid homogenisate is centrifuged, and soluble components with a molecular weight less than 10,000 are separated by molecular filtration. and then this fraction is subjected to further fractionation by gel chromatography. A fraction, precipitated between the volume values of Vg / VQ 1.15 and 1.45, was isolated and lyophilized.
    In addition, the method can be carried out as follows.
    Animal organs, preferably the spleen, containing granulocytes, are homogenized in a water-miscible solvent, preferably in acetone. A solid is separated out from the liquid suspension obtained by centrifuging, extracted with a fat-dissolving organic solvent, preferably a chlorine-containing hydrocarbon, the solid residue is extracted with water (it is advisable to separate the insoluble part by centrifugation) and the constituent parts with a molecular weight less than 10,000 are separated. by molecular filtration. This fraction is further fractionated, predominantly by gel chromatography. The fraction that precipitates between Vg / v 1.3 and 2.5 is separated, and the latter is lyophilized.
    Then, the product obtained according to one of the above variants of the method (fraction G1-3) is purified by chromatography or paper electrophoresis, and the peptide (protein) positively reacting to chlorotolidine, showing a negative charge at pH 6.5 and relative mobility between - 0.55 and -0.65, based on aspartic acid, and the absence of charge at pH 1.9 and mobility
    five
    0,26, counting on-DNP-lysine, then lyophilized.
    Example 1. Obtaining a fraction of G1-3 and-z blood of a horse.
    10 liters of horse blood are treated with 3500 units / l of heparin, then they are kept at room temperature for 30 minutes for sedimentation. The upper leukocyte-rich layer is separated and centrifuged at 800 g. The cell population obtained as a precipitate containing 80% granulocytes is then suspended in 200 ml of a 0.06 M phosphate buffer solution with a pH of 7.4
    (composition: 9,500 g of dibasic acid sodium orthophosphate, and 1,815 g of dibasic acid potassium orthophosphate in 1 liter of distilled water) and again
    0 is centrifuged at 800 g. The granulocyte pellet is suspended in the same phosphate buffer solution at a concentration of 410 cells / ml and homogenized in a Potter homogenizer.
    5 at 10 rpm
    The obtained homogenisate is centrifuged at 1550 g and the separated liquid is subjected to ultrafiltration through an Amikon PM 10 membrane under a pressure of 3 atm, the separation fraction is with a molecular mass less than 10,000. The lycified filtrate is lyophilized, the lyophilized is dissolved in ammonium carbonate buffer solution (pH 7.9) and subjected to gel chromatography on a Sephadex-G 10 column. The fraction precipitated between Vg / v 1.15 and 1.45 values is separated and lyophilized. Thus, 65 mg of the concentrate of the effective substance (fraction G1-3) are obtained in the form of a white powder.
    PRI mme R 2. Obtaining fraction G1-3 from the calf spleen.
    1000 g of the cleaned and crushed calf spleen is mixed with acetone cooled to 4 ° C to obtain a total volume of 4 liters, the mixture is homogenized at this temperature for 60 minutes, incubated and then centrifuged. The separated solid is washed and dried under vacuum at 20 ° C.
    The dry solid is extracted with 2 liters of chloroform for 30 minutes, then the solid is separated, dried at room temperature for 16 hours, then stirred with 3 liters of double-distilled water and centrifuged at 20,000 g.
    0
    five
    0
    five
    The separated liquid is lyophilized, the lyophilisate is dissolved in a 0.05 M buffer solution of ammonium hydrogen carbonate (pH 7.9) and subjected to gel chromatography on a Sephadex G-15 column, the fraction precipitated between 1.3 and 2 Vg / Vjj, 5, are separated and lyophilized. In this way, 3.54 concentrates of the effective substance G1-3 are obtained in the form of a white powder.
    PRI me R 3. Obtaining a pure effective substance GP.
    300 mg of the concentrate of the effective substance G1-3 (obtained in Example 1 or 2) are applied on Whatman chromatographic paper 3 mm (the starting point is located in the middle of the sheet, and the total amount of the applied sample is distributed over a length of 30 cm). The electrophoresis is carried out in a buffer mixture of pyridine, acetic acid and water in a volume ratio of 90: 4: 900 (pH 6.5) in an electrophoresis apparatus with a horizontal arrangement with a voltage gradient of 50 V / cm for 2 hours. The position of the acid is positive the chlorine reactant and the ninhydrin negative component are determined by the color of chlorotolidine. The active component thus obtained is eluted from paper with a buffer mixture of acetic and formic acids and water in a volume ratio of 8: 2: 90 (pI 1.9) and in the same buffer mixture is subjected to additional electrophoresis with a voltage gradient of 80 V / cm min The position of the active component is determined with chlorotolidine. The active component is eluted with the same buffer mixture (pH 1.91).
    Selective action of 100 μg / ml GP. on the introduction of 3 H-thymidine in acid-insoluble deoxyribonucleic acid in cultures of bone marrow and Thjraius
    12 14
    53.2 59.9
    67837
    and the eluate is lyophilized. In this way, 8 μg of the pure effective substance GP is obtained in the form of a white V-glove.
    PRI me R 4. Obtaining active material GP from the blood of the calf.
    Of the 10 liter defibrinated blood of the calf, a fraction containing 86% granulocytes and 14% single nuclei is selected by spontaneous precipitation. Then hemolyzed for 5 min with a 0.85% chloride solution
    ammonium, centrifuged, wash the precipitate (with physiological saline solution. Cells obtained in this way are homogenized in a buffer solution and treated as in Example 1.
    PRI me R 5. Obtaining active material GP from human blood. 1 l of blood from healthy donors, belonging to one group, is subjected to sedimentation at 4 ° C for 4 hours. The obtained white blood cells contain approximately 75% of granulocytes and 25% of lymphocytes. The cells are then homogenized in a buffer solution and treated as in Example 1.
    In this case, in examples 4 and 5, respectively, 60 and 5.6 mg of the active substance concentrate, treated as in Example 3, were obtained with the same characteristics.
    The results of experiments carried out in vitro to determine the effectiveness of the GP substance isolated by ion-exchange chromatography or paper electrophoresis of GP B — compared with fraction G1–3, are listed in Table. 1 and 2.
    Table 1
    0.001
    14.6 7.3
    0.2
    Table 2

    100 430
    8 90
    GP
    2.5
    7,8
    0.2 1.6
    IEC - minimal effective concentration.
    Therefore, the effective substance GP obtained in accordance with the invention can be used as a biological effective substance for inhibiting the proliferation of normal or leukemic human blood cells or bone marrow cells in vitro in concentrations from 0.2 to 10.0 pmol / ml.
    A new substance isolated from granulocytes as described above in a lyophilized form (effective substance GP) has a more effective action: it inhibits the specific reproduction of myeloid cells, but is ineffective with respect to normal thymocytes, PHA-stimulating lymphocytes, subacute lymphoid leukemic synthesis.
    mocites, human lymphoid leukemic blood cells and Helia human tumor cells.
    The partially purified fraction G1-3, obtained as an intermediate product in accordance with the proposed method, also contains an effective substance GP, but not in pure
    Q form, and together with various active ingredients and, mainly, with a significant amount of inactive impurities, the same fraction is obtained by the method prototype. Dunn
    Fraction 15 also has an inhibitory effect against the reproduction of myeloid cells; In vitro, the minimum effective dose of G1-3 fractions is when inhibition of H-thymidine intake into
    2Q acid-insoluble DNA 110 µg / ml, in the case of colonies - 8 µg / ml.
    权利要求:
    Claims (1)
    [1]
    Invention Formula
    Gnoco6 for producing an agent that selectively inhibits the growth or reproduction of normal and leukemic myeloid cells by isolating from human blood or calves granulocyton or lymphocytes, homogenizing, centrifuging the homogenizate in an isotonic aqueous solution, followed by separating the liquid phase, characterized in increase the activity of the target product; horse blood is used as a raw material, which is homogenized in a buffer solution with a pH of 7.4, then, after centrifugation, the liquid phase is subjected to molecular In filtering, a fraction fraction with a molar mass of at least 10,000, after which the final fraction is applied to a chromatographic column and separated
    a fraction precipitating between VB / VO values of 1.15 and 1.45, which is lyophilized and then purified by chromatography and / or paper electrophoresis, followed by repeated lyophilization.
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    同族专利:
    公开号 | 公开日
    DK155712C|1989-09-25|
    DK15281A|1981-07-16|
    DK155712B|1989-05-08|
    DE3161195D1|1983-11-24|
    AU542044B2|1985-02-07|
    US4384991A|1983-05-24|
    EP0035102B1|1983-10-19|
    AU6620081A|1982-04-22|
    HU182087B|1983-12-28|
    SU1228776A3|1986-04-30|
    JPS56138118A|1981-10-28|
    AT5071T|1983-11-15|
    EP0035102A1|1981-09-09|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO1995011659A2|1993-10-18|1995-05-04|Tsentr Embrionalnych Tkanei 'emcell'|Pharmaceutical preparation with immuno-correcting properties based on a cell suspension, and a method of treating sugar diabetes using said preparation|US3973001A|1954-04-27|1976-08-03|Solco Basel Ag|Tissue cell stimulating blood extracts|
    US3937816A|1970-07-21|1976-02-10|Solco Basel Ag|Growth regulating compositions extracted from spleen|
    GB1579120A|1975-08-05|1980-11-12|Union International Co Ltd|Extracts of the haemopoietic system|EP0085254A1|1981-12-28|1983-08-10|Yoshio Sakagami|Proteins and their extraction|
    US4572834A|1984-04-10|1986-02-25|Clinical Reference Laboratory, Inc.|Biologic and method of preparing same|
    US5149544A|1989-11-13|1992-09-22|Research Corporation Technologies, Inc.|Method of inhibiting progenitor cell proliferation|
    WO2006032269A2|2004-09-24|2006-03-30|Salama Zoser B|Pharmaceutical product containing blood constituents and or kda, and the use of the same for the prophylaxis and treatment of defects of the immune system|
    EP1640012A1|2004-09-24|2006-03-29|Salama, Zoser B. nat.rer.Dr.|Pharmaceuticals agents comprising blood components10 kDa and their use for prophylaxis and treatment of defects of the immune system|
    DE102004047262A1|2004-09-24|2006-04-06|Ipss International Pharmaceutical Strategies & Solutions|A pharmaceutical composition comprising blood products and their use as injection or infusion agents for the prophylaxis and treatment of immune system defects in humans|
    US20060067942A1|2004-09-24|2006-03-30|Salama Zoser B|Pharmaceutical agent comprising amino acids, peptides, proteins and/or fractions and fragments thereof and the use of same in the prophylaxis and treatment of immune system deficiency in humans and animals|
    CA2585698A1|2004-11-04|2006-05-18|Peter Xianqi Wang|Opiate intermediates and methods of synthesis|
    US7511060B2|2005-10-21|2009-03-31|Mallinckrodt Inc.|Opiate intermediates and methods of synthesis|
    US7622586B2|2005-10-21|2009-11-24|Mallinckrodt Inc.|Opiate intermediates and methods of synthesis|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    HU8068A|HU182087B|1980-01-15|1980-01-15|Process for preparing an active substance for the selective inhibition of the multiplication of normal cells and of cells in myeloide leukemia|
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