WO1992006118A1 - Proteine extraite d'hirudo medicinalis - Google Patents

Proteine extraite d'hirudo medicinalis Download PDF

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Publication number
WO1992006118A1
WO1992006118A1 PCT/EP1991/001856 EP9101856W WO9206118A1 WO 1992006118 A1 WO1992006118 A1 WO 1992006118A1 EP 9101856 W EP9101856 W EP 9101856W WO 9206118 A1 WO9206118 A1 WO 9206118A1
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WO
WIPO (PCT)
Prior art keywords
protein
new protein
new
hirudin
activity
Prior art date
Application number
PCT/EP1991/001856
Other languages
German (de)
English (en)
Inventor
Karl-Hermann Strube
Siegfried Bialojan
Original Assignee
Basf Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Publication of WO1992006118A1 publication Critical patent/WO1992006118A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a new protein from the leech Hirudo medicinalis, its muteins and their production and use.
  • fibrinolytics that break down fibrin or fibrinogen directly, and plasinogen activators.
  • the fibrin or fibrinogen-degrading substances include hementin (from He enteria ghilianii) and destabilase (from Hirudo medicinalis; I.P. Baskova et al. Folia Haatol., Leipzig, 115, 1988, p. 166).
  • a plasminogen activator has been described in secretions from Hementeria depressa.
  • Inhibitors of blood coagulation e.g. the thrombin inhibitor hirudin (from Hirudo medicinalis), a factor Xa inhibitor (from Hirudo medicinalis), trypsin and plasmin or chymotrypsin and elastase inhibitors such as e.g. the Bdellin or Egline (from Hirudo medicinalis) (Leech Biology and Behavior, Vol. II, Oxford, Science Publication / R.T. Sawyer, 1986).
  • hirudin from Hirudo medicinalis
  • factor Xa inhibitor from Hirudo medicinalis
  • trypsin and plasmin or chymotrypsin and elastase inhibitors such as e.g. the Bdellin or Egline (from Hirudo medicinalis) (Leech Biology and Behavior, Vol. II, Oxford, Science Publication / R.T. Sawyer, 1986).
  • the invention relates to a new protein from Hirudo medi ⁇ cinalis, which extends the clotting time of the blood, is relatively heat-stable and has a molecular weight of 25 to 34 KDa, and its muteins.
  • the new protein significantly increases the intrinsic clotting time (APTT) and the extrinsic clotting time (PT) of the blood. It differs in its anticoagulant properties from hirudin and the factor Xa inhibitor. In fractions in which the APTT prolonging activity is present, neither appropriate amounts of hirudin (by ELISA) nor factor
  • Muteins of the new protein are to be understood as those proteins which are derived from the new protein by exchange, deletion and / or addition of amino acids or peptides without their properties being very different from the new protein.
  • the new protein can be obtained from leeches (Hirudo medicinalis). For this, the leeches are placed in water. After stimulation with arginine, the leeches release the factor into the aqueous solution, from which the new protein is isolated in a known manner by ion exchange, affinity, chelate and gel permeation chromatography or by hydrophobic chromatography.
  • the expression systems include prokaryotes such as E. coli or Bacillus subtilis on the one hand, but also eukaryotes such as yeasts and mammalian cells (e.g. Cl27 mouse fibroblasts, hamster and insect cells).
  • the new protein and its muteins are suitable for the treatment of diseases, in particular for the treatment and prophylaxis of thrombotic conditions.
  • diseases in particular for the treatment and prophylaxis of thrombotic conditions.
  • Extracts from Hirudo medicinalis were obtained using the following methods:
  • the liquids obtained according to a) were heated individually or together for 10 minutes at 90 ° C. and then immediately cooled on ice. After centrifugation (20 min, 10,000 rpm, Sorvall® SA 600 rotor), the supernatant was first diluted 1: 2 to 1: 5 with PBS. The pH was adjusted to 7.4. The solution was then equilibrated to PBS
  • Fractions containing activity were pooled, diluted 1: 2 with 20 mM phosphate buffer, 250 mM NaCl pH 7.0 and applied to a Cu chelate column (1 ml). After rinsing with 10 to 15 column volumes of equilibration buffer (20 mM Na phosphate, 250 mM NaCl pH 7.0), the elution was carried out by applying a linear gradient from 0 to 100% buffer B (20 mM succinic acid, 1 M NaCl pH 4.0). . The column was then post-eluted either with 20 mM phosphate, 2 M NaCl, 10 mM EDTA, pH 7 or with 20 mM succinic acid 2 M NaCl, 100 M histidine pH 4.
  • the concentrates were diluted to 20 mM succinic acid pH 2.5 (final concentration) and applied to an S-Sepharose® column equilibrated with 20 mM succinic acid pH 2.5. Any hirudin present was separated off by elution with 30 mM succinic acid pH 4.5. Elution of the new factor? was carried out by applying a buffer gradient according to PE pH 7.5. A pH value of 5.5-7 was measured in the value fractions of S-Sepharose. Fractions containing the activity (thrombin inhibition) were further tested in a hirudin Elisa (see Example 6a).
  • the fractions of S-Sepharose® chromatography exhibiting thrombin inhibition were further purified by reversed phase HPLC on a Bio-RAD rp304® column with a 20 M ammonium acetate, pH 6, 20 M ammonium acetate, 50% acetonitrile 1 system .
  • the value fractions were concentrated to a volume of 200 to 500 ⁇ l and eluted from the rHPLC by applying an acetonitrile gradient.
  • the new factor elutes at an acetonitrile concentration of 7.5% + _ 3%.
  • the activity of the new thrombin inhibitor is retained and can be demonstrated by tricin-SDS-polyacrylamide gel electrophoresis or APTT determination.
  • the new protein could be concentrated by Centricon® 10, but not by Centricon® 30. This means that the new protein must have a molecular weight between 10 and 30 kDA.
  • the gel electrophoresis was carried out in accordance with the instructions (Schägger, H. and von Jagow, G .; Analytical Biochemistry, 166, 368-379 (1987) at 20 A and 1400 V, 30 W, with a power supplier from LKB and a running chamber of the " mighty s all system ". After completion of the electrophoresis, the gel was first incubated for 30 min in a 2.5% solution of Triton X100 and then on an agarose plate containing thrombin and fibrinogen twice
  • the thrombin concentration in the agarose was adjusted so that the fibrinogen was polymerized under the action of the thrombin within 2 to 4 hours.
  • the presence of the new thrombin inhibitor was confirmed by unpolymerized sites in the
  • the molecular weight marker (calibration proteins: intact myoglobin 17.2 kDA, cyanogen bromide cyanide myoglobin I + Uli 14.6 kDa, cyanogen bromide cyanogen myoglobin I 8.2 kDa, bromocyanopeptide myoglobin II 6.4 kDa, bromocyanopeptide myoglobin III 2.6 kDa, myoglobin 1-14), the molecular weight of the new thrombin inhibitor was determined to be 14.6 + _ 3 kDa.
  • the new factor was tested in various assay systems to determine how and which factors in the coagulation as ( ade it inhibits.
  • APTT *** activated partial thromboplastin time
  • Samples to be tested were diluted in 50 ⁇ l human plasma (eg Preciclot®, Boehringer / Ma, No. 654 370).
  • 50 ⁇ l of PTT a reagent (Boehringer / Ma, No. 886 050) were pipetted in and incubated at 37 ° C. for 5 min.
  • the coagulation cascade was started by adding 50 ⁇ l of 25 mM CaCl. The time until the occurrence of plasma clots was measured.
  • the samples showed a significant increase in the plasma clotting time (intrinsically).
  • PT prothrombin time, quick test
  • ⁇ OD C ⁇ D 60m in-OD5 min ] + FXa - [OD 6 0min- OD 5minJ'-FXa
  • a sample of the new protein was diluted in 100 ⁇ l plasma.
  • the coagulation was started by adding 50 ⁇ l of thrombin reagent (Boehringer / Ma, No. 126 594). The time until the occurrence of plasma clots was determined turbid etrically.
  • the evaluation was carried out using a calibration curve with known thrombin inhibitors (eg hirudin). The results obtained show that the new protein is a thrombin inhibitor. 6. Attempts to differentiate the new protein from hirudin
  • Microtiter plates (Flow 77-173-05) - streptavidin-peroxidase complex (Boehringer / Ma 1089153) Tetramethylbenzidin (Serva 35926)
  • Peroxidase substrate 0.1 ml TBM solution (42 mM TBM in DMSO) and 10 ml substrate buffer (0, Mix 1 M Na acetate pH 4.9) slowly; then add 14.7 ⁇ l 3% H2O2
  • Standard dilutions were applied and gave a linear dependence in the range of approx. 60 to 2 ng / ml.
  • Samples with unknown hirudin concentrations were used in parallel to the calibration sample in different dilutions in the test. An amount of hirudin corresponding to an activity of at most 5 ATU / ml could be detected in the measured samples.
  • the measurement of the TZ showed an antithrombin activity of 200 ATU / ml. This value was a factor> 40 above the value determined for hirudin by ELISA. This proves that the new protein is not hirudin.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Hematology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Diabetes (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une nouvelle protéine extraite d'hirudo medicinalis qui prolonge le temps de coagulation du sang, qui est relativement stable à la chaleur et qui a un poids moléculaire compris entre 25 et 34 KDa, ainsi que ses mutéines, utiles pour traiter des maladies.
PCT/EP1991/001856 1990-10-06 1991-09-27 Proteine extraite d'hirudo medicinalis WO1992006118A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4031731.5 1990-10-06
DE4031731A DE4031731A1 (de) 1990-10-06 1990-10-06 Protein aus hirudo medicinalis

Publications (1)

Publication Number Publication Date
WO1992006118A1 true WO1992006118A1 (fr) 1992-04-16

Family

ID=6415763

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1991/001856 WO1992006118A1 (fr) 1990-10-06 1991-09-27 Proteine extraite d'hirudo medicinalis

Country Status (5)

Country Link
EP (1) EP0551320A1 (fr)
JP (1) JPH06501255A (fr)
CA (1) CA2093432A1 (fr)
DE (1) DE4031731A1 (fr)
WO (1) WO1992006118A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0209061A2 (fr) * 1985-07-17 1987-01-21 Hoechst Aktiengesellschaft Peptides à activité anticoagulante, leur procédé de préparation, d'obtention, leur application et les agents les contenant

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0209061A2 (fr) * 1985-07-17 1987-01-21 Hoechst Aktiengesellschaft Peptides à activité anticoagulante, leur procédé de préparation, d'obtention, leur application et les agents les contenant

Also Published As

Publication number Publication date
CA2093432A1 (fr) 1992-04-07
DE4031731A1 (de) 1992-04-09
EP0551320A1 (fr) 1993-07-21
JPH06501255A (ja) 1994-02-10

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