WO2008020743A2 - Procédé permettant d'extraire un inhibiteur de l'agrégation plaquettaire du venin d'agkistrodon blomhoffii ussuriensis - Google Patents

Procédé permettant d'extraire un inhibiteur de l'agrégation plaquettaire du venin d'agkistrodon blomhoffii ussuriensis Download PDF

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Publication number
WO2008020743A2
WO2008020743A2 PCT/MN2006/000005 MN2006000005W WO2008020743A2 WO 2008020743 A2 WO2008020743 A2 WO 2008020743A2 MN 2006000005 W MN2006000005 W MN 2006000005W WO 2008020743 A2 WO2008020743 A2 WO 2008020743A2
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WO
WIPO (PCT)
Prior art keywords
buffer
tris hcl
column
poison
hcl buffer
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Application number
PCT/MN2006/000005
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English (en)
Russian (ru)
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WO2008020743A3 (fr
Inventor
Georgiy Volkov
Alexiy Savchuk
Vitaly Karbovskyy
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Wisdom Asset Company
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Application filed by Wisdom Asset Company filed Critical Wisdom Asset Company
Publication of WO2008020743A2 publication Critical patent/WO2008020743A2/fr
Publication of WO2008020743A3 publication Critical patent/WO2008020743A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/58—Reptiles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors

Definitions

  • the invention relates to the industry of bioactive substances and can be used in biotechnological industries, medicine and the pharmaceutical industry, namely the production of medicines and diagnostic preparations from snake venom.
  • a platelet aggregation inhibitor is a protein that has an inhibitory effect on the platelet activation process by various activation agents.
  • platelet activation inhibitors There are a significant number of platelet activation inhibitors from various sources. All of them differ in the strength of inhibition and the mechanisms by which this inhibition is carried out.
  • One of the effective sources of platelet activation inhibitors is snake venoms, which contain only protein components and have a stable composition.
  • Some methods for producing active enzymes from snake venom are known.
  • a method is known for producing active enzymes from the snake venom of Triteresir okppaepsis by fractionation of poisoned with buffer on Sephadex G-100, chromatography on SM-Touorerl 650M in two stages and final purification on Mono Q gel.
  • Purificapofepofitofibf -Race spake (Agistrodop asites) venom., Toxicon.-1999.-V.37, N7.-P.999-1013); from Yard Agcistrodop Asitis, in which the poison dissolved in the buffer is subjected to separation of the DEAE Sephadex A-50 anion exchanger, then chromatographed twice on Sephadex G-200 (Quaupg C, Hong J. et al. Aspirates Vpom apd its comraritiop with bovipe tlombip., Thromb.Diath.haymorrth. -1971.
  • a method for isolating snake venom of platelet aggregation PAl is proposed, the essence of which includes a method for determining the ability to inhibit platelet aggregation in snake venom, a method for purifying an inhibitor of platelet activity from various samples of snake venom, its characterization and a truncated form containing a modified lysine residue that specifically inhibits fibrin binding or von Willebrand factor with GPPb-Sha. (WO 90/15620, 06/16/1989, Platellet agregatiphibitors, COR ⁇ S, INC).
  • the disadvantage of the prototype is both a low level of profitability of the production process, a low level of purity and a large investment of time.
  • the basis of the invention is the task to develop a method for isolating a platelet activation inhibitor from Agkistrodop Yothqffii poison syriepsis poison, which allows by means of affinity chromatography followed by ion exchange chromatography and the use of carriers with higher chromatographic properties and physicochemical stability to increase the profitability of the production process and reduce the isolation time / fibrinoliptic protein and increase its purity.
  • the problem is solved in a method of an inhibitor of platelet activation from the Agkistrodop Yothoffii venereus poison in that affinity chromatography is carried out on a column with a carrier of Clin Serharose FF using 1OmM Tris HCl with pH 7.4 as a buffer solution.
  • the fraction of a material unrelated to Blue Serum FF was diluted 8 times with 5OmM Tris HCl buffer with pH 8.2 and applied to a column filled with DEAE Serophoros FF anion exchanger, previously equilibrated with 5OmM Tris HCl buffer with pH 8.2.
  • the platelet activation inhibitor was eluted with 5OmM Tris HCl buffer at pH 8.2 with a content of 0.2M NaCl.
  • the protein peak containing the platelet activation inhibitor is collected, concentrated, and applied to replace the buffer with a Serhadex G25 column equilibrated with 5OmM Tris HCl buffer with a pH of 7.4 containing 0.1 ZM NaCl.
  • the problem is solved by the proposed method, when the parameters of the process of isolation of platelet activation inhibitorg are in the ranges indicated in the claims. When the parameters are changed in the direction of increasing or decreasing, the characteristics of the resulting preparation worsen.
  • the solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate 0.5 ml / min ) After application, 1OmM Tris HCl buffer with a pH of 7.4 is passed through the column at a speed of 2 ml / min and all unbound material (60 ml) is collected.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • the fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger.
  • the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength 0.2M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • a protein peak containing a platelet activation inhibitor is collected (6 ml) and dried by lyophilization. Characteristic Yield, mg 2.0 mg (4.2%)
  • the solution of the poison is applied to a column (C 10/10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate 0.5 ml / min )
  • 1OmM Tris HCl buffer with a pH of 7.4 is passed through the column at a speed of 2 ml / min and all unbound material (53 ml) is collected.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • the fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger.
  • the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength of 0.2 M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • the protein peak containing the platelet activation inhibitor is collected (5.2 ml) and dried by lyophilization. Characteristic Yield, mg 1.7 mg (3.9%) Biological activity IC 5O 232nM
  • the solution of the poison is applied to a column (CU / 10, volume 7 ml) with an affinity sorbent Vlie Serharose FF, previously equilibrated with 50 ml of 1OmM Tris HCl buffer with a pH of 7.4 (column equilibration rate of 2 ml / min, poison application rate of 0.5 ml / min).
  • 1OmM Tris HCl buffer with pH 7.4 is passed through the column at a speed of 2 ml / min and all material unbound with the sorbent (75 ml) is collected.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • the fraction of material unrelated to Vlhe Serofaros FF was diluted 8 times with 5OmM Tris HCl with a pH 8.2 buffer and applied to a column filled with DEAE Serocos FF anion exchanger (column size Ix8cm, volume 7ml), pre-equilibrated with 5OmM Tris HCl 2 buffered with a pH of 2 buffered with 2 buffered with a pH of 2 buffered with 8.2 buffer min (Sample application speed lml / min). The bulk of the proteins (85%) are collected on the anion exchanger.
  • the platelet activation inhibitor is eluted with a stepwise gradient of ionic strength 0.2M NaCl in 5OmM Tris HCl buffer with pH 8.2 at a rate of 2 ml / min.
  • the optical density was recorded continuously using a flow monitor REC 102 (Parmacia biotech).
  • a protein peak containing a platelet activation inhibitor is collected (7.1 ml) and dried by lyophilization.

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  • Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne la production de substances bioactives, et peut être utilisée dans des processus de production biotechnologiques, en médecine et dans l'industrie pharmaceutique, en particulier pour produire des préparations médicinales et diagnostiques à partir de venin de serpent. Le procédé selon l'invention est caractérisé en ce qu'il consiste : à effectuer une chromatographie d'affinité sur une colonne contenant un support Blue Sepharose FF, à l'aide d'une solution tampon se présentant sous la forme de Tris HCl présentant un pH de 7,4, puis à diluer huit fois la fraction du matériau non conjugué à Blue Sepharose FF à l'aide d'une solution tampon de Tris HCl présentant un pH de 8,2, et à appliquer sur une colonne remplie d'un échangeur d'anions de DEAE Sepharose FF et préalablement équilibrée par la solution tampon de Tris HCl; puis à laver le sorbant pour éliminer le matériau non conjugué, à éluer un inhibiteur de l'activation plaquettaire dans la solution tampon de Tris HCl contenant du NaCl, à recueillir et à concentrer le pic protéique contenant l'inhibiteur de l'activation plaquettaire, à l'appliquer, afin de remplacer la solution tampon, sur une colonne Sephadex G25 équilibrée par la solution tampon de HCl contenant du NaCl, età procéder enfin à un séchage par lyophilisation.
PCT/MN2006/000005 2006-08-16 2006-08-30 Procédé permettant d'extraire un inhibiteur de l'agrégation plaquettaire du venin d'agkistrodon blomhoffii ussuriensis WO2008020743A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
MN381206 2006-08-16
MN3812 2006-08-16

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WO2008020743A2 true WO2008020743A2 (fr) 2008-02-21
WO2008020743A3 WO2008020743A3 (fr) 2008-07-10

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1293795A (en) * 1969-07-04 1972-10-25 Twyford Lab Ltd Improvements relating to anticoagulants
WO1990015620A1 (fr) * 1989-06-16 1990-12-27 Cor Therapeutics, Inc. Inhibiteurs de l'agregation des plaquettes
CN1504569A (zh) * 2002-12-05 2004-06-16 合肥兆峰科大药业有限公司 蝰蛇蛇毒血凝酶的层析纯化方法
RU2271219C1 (ru) * 2004-07-28 2006-03-10 Государственное образовательное учреждение высшего профессионального образования "Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова Министерства здравоохранения Российской Федерации" Способ получения тромбиноподобных ферментов из яда змеи

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1293795A (en) * 1969-07-04 1972-10-25 Twyford Lab Ltd Improvements relating to anticoagulants
WO1990015620A1 (fr) * 1989-06-16 1990-12-27 Cor Therapeutics, Inc. Inhibiteurs de l'agregation des plaquettes
CN1504569A (zh) * 2002-12-05 2004-06-16 合肥兆峰科大药业有限公司 蝰蛇蛇毒血凝酶的层析纯化方法
RU2271219C1 (ru) * 2004-07-28 2006-03-10 Государственное образовательное учреждение высшего профессионального образования "Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова Министерства здравоохранения Российской Федерации" Способ получения тромбиноподобных ферментов из яда змеи

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