WO1994009039A1 - Nouvelle proteine extraite de la sangsue sylvestre haemadipsa sylvestris, qui augmente le temps de coagulation - Google Patents

Nouvelle proteine extraite de la sangsue sylvestre haemadipsa sylvestris, qui augmente le temps de coagulation Download PDF

Info

Publication number
WO1994009039A1
WO1994009039A1 PCT/EP1993/002793 EP9302793W WO9409039A1 WO 1994009039 A1 WO1994009039 A1 WO 1994009039A1 EP 9302793 W EP9302793 W EP 9302793W WO 9409039 A1 WO9409039 A1 WO 9409039A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
factor
leu
amino acid
new protein
Prior art date
Application number
PCT/EP1993/002793
Other languages
German (de)
English (en)
Inventor
Karl-Hermann Strube
Thomas Meyer
Siegfried Bialojan
Burkhard Kroeger
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 WO1994009039A1 publication Critical patent/WO1994009039A1/fr

Links

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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the new protein (PT factor) has the following physicochemical properties.
  • the protein shows two bands, which are assigned a molecular mass of 8200 ⁇ 2000 Da and 18600 ⁇ 2000 Da (FIG. 2).
  • Example 8a In the APTT test (Example 8a) and in the PT test (Example 8b), the protein in each case shows significant increases in the plasma coagulation time. Furthermore, it shows a specific factor Xa inhibition in a factor Xa inhibition test (example 8d). In an analog factor VII-He test (FIG. 3b) an inhibitory effect is also found; however, this can also occur indirectly through the inhibition of factor Xa.
  • the proteins determined the following N-terminal amino acid sequence:
  • the new proteins can be obtained from the country's blood leeches
  • the proteins can be further purified from the solution obtained in this way by chromatographic methods, preferably ion exchange chromatography and / or reversed phase HPLC.
  • the purification of the proteins can be followed using the activity tests described in Example 8.
  • a cDNA library is created from the leech in a manner known per se.
  • the gene coding for the protein according to the invention can be isolated from this gene bank by, for example, producing a DNA sample whose sequence is obtained from the N-terminal amino acid sequence described above by back-translation according to the genetic code. The corresponding gene can be found and isolated by hybridization with this DNA sample.
  • a primer the sequence of which was obtained by back-translation from the N-terminal amino acid sequence described above
  • a second primer the sequence of which is complementary to the 3 'end of the cDNA gene fragment, preferably with the sequence poly (dT)
  • the corresponding gene can also be isolated by creating an expression gene bank of leeches and screening them with an antibody which is directed against the protein according to the invention.
  • the corresponding gene After the corresponding gene has been isolated, it can be genetically engineered in organisms, e.g. in bacteria, yeasts or higher eukaryotic cells can be expressed in a manner known per se with the aid of an expression vector.
  • the protein can be isolated from these recombinant host systems on the basis of the physicochemical properties described above.
  • the protein according to the invention is preferably used in the form of its pharmaceutically acceptable salts.
  • the new protein has anticoagulant properties. It can be used, for example, for the prophylaxis of thromboses or arterial reocclusions, for the treatment of thromboses, for the preservation of blood or for extracorporeal circulation.
  • the new protein is an effective anticoagulant. It can be used alone or together with known coagulation factors, for example thrombin inhibitors such as hirudin, as a medicament.
  • 150 g of live landing gel (Haemadipsa sylvestris) were homogenized in 400 ml of 20 mM sodium phosphate buffer (pH 7.4) with a mixer at 4 ° C. for 10 minutes. The homogenate was centrifuged for 15 minutes at 2000 rpm (Sorvall RC-5B, rotor GS-3) and then after removing the precipitate for 30 minutes at 8000 rpm. The precipitate was discarded and the supernatant was diluted to a volume of about 600 ml with 50 mM tris (hydroxymethyl) amino methane / HCl buffer pH 8.5 (Tris / HCl buffer).
  • the protein solution had a volume of 580 ml, the protein concentration was 4.49 mg / ml and the thrombin-inhibiting activity was 22.2 U / ml.
  • the coagulation time (APTT, see Example 8) of the blood was prolonged by about 200 sec in a 1:10 dilution and the partial thromboplastin time (PT) was prolonged by about 60 sec in a 1:10 dilution.
  • the new protein can be purified from homogenates of leech heads.
  • the leeches were first decapitated and the head preparations obtained in this way were homogenized in an analogous manner.
  • Purification from leech heads has the advantage that the homogenate contains a smaller amount of protein with approximately the same amount of activity.
  • the new protein is already here in a higher purity.
  • a significantly reduced proteolytic activity was determined in the head homogenate.
  • the disadvantage of isolation from leech heads is the limited amount of substance.
  • the protein solution obtained from the Egelhomogenaten was (50-100 ml, 2.5 cm diameter) on a 8.5 equilibrated with 50 mM Tris / HCl buffer pH Q-Sepharose ® column applied. After washing away unbound material with the equilibration buffer, the bound proteins were washed with a
  • the new protein elutes from the column at a salt concentration of 300 to 500 mM NaCl.
  • the value fraction of the Q-Sepharose chromatography was concentrated using a 3000 D membrane (Filtron Omega Alpha Cat. No. AM003062) and buffered to 20 mM succinic acid buffer pH 4.0 or simply with the succinic acid buffer pH 4.0 diluted and the pH adjusted if necessary. This fraction was applied to an S-Sepharose FF column (diameter 1.5-2.5 cm, volume 10-30 ml) equilibrated with 20 mM succinic acid at a flow of 40-80 ml / h. After washing away unbound material with equilibration buffer, the bound proteins were eluted from the column using a linear gradient from 0 to 1 M NaCl in 20 mM succinic acid pH 4.0. The elution was monitored at 280 nm in the UV and
  • the value fraction of S-Sepharose chromatography was dissolved in the smallest possible volume of 0.1% by weight trifluoroacetic acid and on a reversed phase (r) HPLC column (BioRad rp304) after 5 minutes of rinsing with 0.1% by weight.
  • Trifluoroacetic acid using a linear gradient (0.1 wt .-% TFA in water / 0.1 wt .-% TFA in acetonitrile in 65 min to 0.1 wt .-% TFA in 50% acetonitrile) .
  • the one from the HPLC column Eluting proteins were detected by UV detection at 210 nm and fractionated by hand.
  • the PT-prolonging activity contained in the individual fractions was determined after removing the solvent and resuspending in water.
  • the PT factor eluted between 20 and 28% acetonitrile from the column under these conditions.
  • Electrophoresis was carried out as described by Schgger and Jagow (Analytical Biochemistry, 166, 368-379 (1987)). An aliquot (20 ⁇ g) was separated on a 16% Tris / Tricine gel (BAI GmbH, Bensheim). Fig. 3 shows the result after staining the gel with Coomassie Brillant Blau (A) or Silber (B). The new protein (A: lane 2; B: lane 2) shows two bands with molecular masses of 8200 ⁇ 1000 and 18600 ⁇ 2000 Da.
  • the new factor was tested in various assay systems to determine how and which factors in the coagulation cascade are inhibited by it.
  • APTT activated partial thromboplastin time
  • the samples were diluted as described above, with the modification that the corresponding chromogenic substrate (25 ⁇ l) was pipetted in.
  • the new protein very effectively inhibits both factor Xa and the formation of factor VIIa.
  • Thrombin (Boehringer / Mannheim) became a final concentration of (25 mU / ml) in phosphate buffered saline (PBS) (0.8 g / 1 NaCl; 0.2 g / 1 HCl; 0.144 g / 1 sodium phosphate; 0 , 2 g / 1 potassium phosphate, pH 7.5).
  • PBS phosphate buffered saline
  • Chromozym TH (Boehringer / Mannheim) was dissolved in 20 ml H 2 0 / bottle.
  • chromozyme 50 ⁇ l of chromozyme as well as 25 ⁇ l of sample or buffer were added to the wells of a microtiter plate. Immediately afterwards, the absorption at 405 nm was measured at time 0 and after 30 min at 37 ° C. If the sample had a strong intrinsic color, a further control without thrombin was treated as above.
  • FIG. 2 Tris / Tricine - SDS polyacrylamide gel electrophoresis
  • the molar mass of the value fraction of the (r) HPLC separation was determined on a 16% Tris / Tricine gel.
  • Lanes 1.3 Molar mass calibration substance, intact myoglobin 17.2 kDa, cyanogen bromide peptide myoglobin I + III 14.6 kDa, cyanogen bromide cyanide 8.2 kDa, cyanogen bromide cyanide 6.4 kDa, cyanogen bromide cyanide 2.6 kDa,
  • FIG. 3 Measurement of the influence of PT factor on extrinsic coagulation.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une protéine extraite de sangsues sylvestres du genre Haemadipsa dont la séquence aminoacide N-terminale est la suivante : A-Val-Lys-Phe-Cys-Gly-His-Pro-Leu-Ser-Leu-Pro-Thr-Ala-Leu-Cys-Ala, dans laquelle A désigne un aminoacide n'ayant pas à être déterminé de manière univalente. La protéine est appropriée pour lutter contre des maladies.
PCT/EP1993/002793 1992-10-16 1993-10-12 Nouvelle proteine extraite de la sangsue sylvestre haemadipsa sylvestris, qui augmente le temps de coagulation WO1994009039A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19924234921 DE4234921A1 (de) 1992-10-16 1992-10-16 Neues, die Gerinnungszeit des Blutes verlängerndes Protein aus dem Landblutegel Haemadipsa sylestris
DEP4234921.4 1992-10-16

Publications (1)

Publication Number Publication Date
WO1994009039A1 true WO1994009039A1 (fr) 1994-04-28

Family

ID=6470630

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1993/002793 WO1994009039A1 (fr) 1992-10-16 1993-10-12 Nouvelle proteine extraite de la sangsue sylvestre haemadipsa sylvestris, qui augmente le temps de coagulation

Country Status (2)

Country Link
DE (1) DE4234921A1 (fr)
WO (1) WO1994009039A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8501241B1 (en) 2012-09-17 2013-08-06 Biopep Solutions, Inc. Treating cancer with a whole, leech saliva extract
CN108101975A (zh) * 2017-12-14 2018-06-01 中国科学院昆明动物研究所 一种森林山蛭抗血栓多肽Sylvestin及其体外表达制备方法和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0346894A2 (fr) * 1988-06-16 1989-12-20 Merrell Dow Pharmaceuticals Inc. Extraction et purification d'une substance anticoagulante de la sangsue d'amérique du sud, haementeria ghilianii
EP0352903A2 (fr) * 1988-06-24 1990-01-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Facteur inhibant le facteur Xa bovin et compositions pharmaceutiques le contenant
WO1993011239A1 (fr) * 1991-11-26 1993-06-10 Basf Aktiengesellschaft Nouvelles proteines inhibitrices de thrombine provenant de la sangsue des campagnes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0346894A2 (fr) * 1988-06-16 1989-12-20 Merrell Dow Pharmaceuticals Inc. Extraction et purification d'une substance anticoagulante de la sangsue d'amérique du sud, haementeria ghilianii
EP0352903A2 (fr) * 1988-06-24 1990-01-31 Yissum Research Development Company Of The Hebrew University Of Jerusalem Facteur inhibant le facteur Xa bovin et compositions pharmaceutiques le contenant
WO1993011239A1 (fr) * 1991-11-26 1993-06-10 Basf Aktiengesellschaft Nouvelles proteines inhibitrices de thrombine provenant de la sangsue des campagnes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
M. SCHARF ET AL.: "Primary structures of new 'iso-hirudins'", FEBS LETTERS, vol. 255, no. 1, September 1989 (1989-09-01), AMSTERDAM NL, pages 105 - 110 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9149498B2 (en) 2012-09-17 2015-10-06 Biopep Solutions, Inc. Treating a parasitic disease with a whole, leech saliva extract
US9265802B2 (en) 2012-09-17 2016-02-23 Biopep Solutions, Inc. Treating colorectal cancer with a whole, leech saliva extract
US8784896B2 (en) 2012-09-17 2014-07-22 Biopep Solutions, Inc Antioxidant therapy with a whole, leech saliva extract
US8790711B2 (en) 2012-09-17 2014-07-29 Biopep Solutions, Inc. Treating diabetes with a whole, leech saliva extract
US8962034B2 (en) 2012-09-17 2015-02-24 Biopep Solutions, Inc. Antiviral therapy with a whole, leech saliva extract
US9017732B1 (en) 2012-09-17 2015-04-28 Biopep Solutions, Inc. Antibacterial therapy with a whole, leech saliva extract
US8685462B1 (en) 2012-09-17 2014-04-01 Biopep Solutions, Inc. Whole, leech saliva product and applications thereof
US9265803B2 (en) 2012-09-17 2016-02-23 Biopep Solutions, Inc. Treating a melanoma with a whole, leech saliva extract
US8501241B1 (en) 2012-09-17 2013-08-06 Biopep Solutions, Inc. Treating cancer with a whole, leech saliva extract
US9433648B2 (en) 2012-09-17 2016-09-06 Biopep Solutions, Inc. Treating renal cancer with a whole, leech saliva extract
US9433649B2 (en) 2012-09-17 2016-09-06 Biopep Solutions, Inc. Treating a lymphoma with a whole, leech saliva extract
US9480720B1 (en) 2012-09-17 2016-11-01 Biopep Solutions, Inc. Treating damaged dermal or mucosal tissue with a whole, leech saliva extract
US9597361B2 (en) 2012-09-17 2017-03-21 Biopep Solutions, Inc. Treating a bacterial skin infection with a whole, leech saliva extract
US10064897B2 (en) 2012-09-17 2018-09-04 Biopep Solutions, Inc. Treating a bacterial skin infection with a whole, leech saliva extract
CN108101975A (zh) * 2017-12-14 2018-06-01 中国科学院昆明动物研究所 一种森林山蛭抗血栓多肽Sylvestin及其体外表达制备方法和应用

Also Published As

Publication number Publication date
DE4234921A1 (de) 1994-04-21

Similar Documents

Publication Publication Date Title
DE68920915T2 (de) Extraktion und Reinigung einer Antigerinnungssubstanz aus den südamerikanischen Blutegeln Haementeria ghilianii.
EP0142860A2 (fr) Désulfatohirudine, son procédé de préparation et agent pharmaceutique
DE69823218T2 (de) Aminoterminal verkürztes mcp-2 als chemokin antagonist
DE68918246T2 (de) Verbindungen zur Verhinderung von Thrombose.
DD297977A5 (de) Verfahren zur reinigung von annexinen
Chopin et al. Amino acid sequence determination and biological activity of therin, a naturally occuring specific trypsin inhibitor from the leech Theromyzon tessulatum
EP0616642B1 (fr) Nouvelles proteines inhibitrices de thrombine provenant de la sangsue des campagnes
EP0318703A1 (fr) Préparation par génie génétique de la protéine anticoagulante PP4
WO1994009039A1 (fr) Nouvelle proteine extraite de la sangsue sylvestre haemadipsa sylvestris, qui augmente le temps de coagulation
WO2002040512A2 (fr) Beta-defensine-3 humaine
DE4140381A1 (de) Neue synthetische isohirudine mit verbesserter stabilitaet
EP0677107B1 (fr) Inhibiteur de thrombine provenant de salive de protostomiens
EP0612349B1 (fr) Nouvelle proteine inhibitrice de la trombine isolee dans les reduves
EP0610246B1 (fr) Nouvelle proteine inhibitrice de la trombine isolee dans les tiques
EP0493494A1 (fr) Nouvelles proteines servant a combattre des maladies et a proteger le betail contre les tiques
DE3850407T2 (de) Protein mit inhibierender wirkung auf die entzündungserregende phospholipase a2.
DE4427531A1 (de) Humanes zirkulierendes beta-Defensin hBD-1
WO2002066513A2 (fr) Fragments humains de lekti circulants hf7072, hf7638 et hf14448 ainsi que leur utilisation
DE4134814A1 (de) Neues thrombininhibitorisches protein aus zecken
Brandt et al. Variants of wheat histone H1 with N-and C-terminal extensions
EP0364942A2 (fr) Isohirudine
DE4031731A1 (de) Protein aus hirudo medicinalis
EP0502876B1 (fr) Nouvelles proteines dissolvant les caillots sanguins, et leur preparation a partir de la sangsue hirudo medicinalis
DE60126026T2 (de) PROTEIN MIT HäMOLYTISCHER AKTIVITäT UND DIESES PROTEIN KODIERENDE GEN
DE19724791B4 (de) Thrombin hemmende Peptide, deren Verwendung und diese enthaltende pharmazeutische Zusammensetzungen

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WA Withdrawal of international application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA