GB2247239A - Hirudin conjugates with polyoxyethylene - Google Patents

Hirudin conjugates with polyoxyethylene Download PDF

Info

Publication number
GB2247239A
GB2247239A GB9018464A GB9018464A GB2247239A GB 2247239 A GB2247239 A GB 2247239A GB 9018464 A GB9018464 A GB 9018464A GB 9018464 A GB9018464 A GB 9018464A GB 2247239 A GB2247239 A GB 2247239A
Authority
GB
United Kingdom
Prior art keywords
hirudin
desulphatohirudin
gly
gln
asn
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
GB9018464A
Other versions
GB9018464D0 (en
Inventor
Peter Goddard
Lyndon Nigel Brown
Karel Petrak
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis AG
Original Assignee
Ciba Geigy AG
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 Ciba Geigy AG filed Critical Ciba Geigy AG
Priority to GB9018464A priority Critical patent/GB2247239A/en
Publication of GB9018464D0 publication Critical patent/GB9018464D0/en
Publication of GB2247239A publication Critical patent/GB2247239A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A61K38/58—Protease inhibitors from animals; from humans from leeches, e.g. hirudin, eglin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

A conjugate of hirudin and polyoxyethylene (which has a molecular weight of from 20,000 to 50,000 Da) may be used as a blood anticoagulant.

Description

Hirudin Derivatives The present invention relates to derivatives of hinidin, and especially to conjugates of hirudin.
Hirudin which has been known for a long time and which occurs naturally in leeches (Hirudo medicinalis) (Walsman, P. and Markwardt, F. (1981) Pharmazie 36, 653) is the strongest thrombin inhibitor of all naturally occurring and synthetic anticoagulants known with a complex dissociation constant of 2 x 10-14 M, thus preventing the formation of fibrin from its precursor fibrinogen. Other enzymes of the blood coagulation cascade are not inhibited by hirudin. In contrast to heparin which is the preferred anticoagulant in conventional anticoagulation therapy, hirudin exerts its inhibiting action directly on thrombin and, unlike the former, does not act through antithrombin III. No effect on heart rate, respiration, blood pressure, thrombocyte count, fibrinogen and haemoglobin could be observed after intravenous administration of hirudin to dogs, even in high doses.In tests on rats, pigs and dogs, hirudin has proved effective in experimental thrombosis (induced either by stasis, vascular damage or by the injection of thrombin), in endotoxin shock, and also in DIC (disseminated intravascular coagulation).
Hirudin is not a single polypeptide species but a class of equally acting polypeptides consisting of at least four representatives designated hirudin variant 1 (HV1), hirudin variant 2 (HV2; EP Application 0 158 564), hirudin variant PA (PCT Application WO 88/03493), and "des-(Val)2-hirudin" (EP Application 0 158 986). The variants differ from each other by a number of amino acids, for example at the N-terminal sequence which is Val-Val-Tyr for HV 1, lle-Thr-Tyr for HV2 and PA and Thr-Tyr for "des-(Val)2-hirudin".
Based on NMR studies, HV1 is composed of an N-terminal core domain with a protruding "finger" (residues 31-36), and an acidic terminal loop (Clore et al., EMBO Journal 6, 529, 1987). All above-mentioned hirudin variants have an accumulation of hydrophobic amino acids at the N-terminus and an accumulation of polar amino acids at the C-terminus, a tyrosine residue (Tyr 63) present as sulphate monoester, three disuiphide bridges and the anticoagulant activity in common.
Recently, cDNAs and synthetic genes coding for hirudin variants have been cloned and expressed in microbial hosts. Although the expression products lack the sulphate monoester group at Tyr 63 - and were, therefore, designated "desulphato hirudins1 - they turned out to exhibit approximately the same biological activity as the natural sulphated hirudins. Desulphatohirudin variant HV1 has been expressed in Escherichia coli (European Patent Applications No. 158 564 and 168 342) and in Saccharomvces cerevisiae (European Patent Applications No. 168 342,200 655, 225 633 and 252 854).
Similarly, desulphatohirudin HV2 has been expressed in Escherichia coli (European Patent Application No.200655, PCT-Application No. 86/01224) and des-(Val)2-desulphatohirudin has been expressed in Escherichia coli (European Patent Application No. 158 986).
In the present application, the term hirudin, when not otherwise stated, is intended to embrace (1) all naturally occurring or synthetic hirudin variants and hirudin derivatives, such as hirudin fragments which retain the anticoagulant activity, and (2) all desulphatohirudin variants and desuiphatohirudin derivatives, such as C-terminally shortened sulphatohirudins, which are described in the literature or are obtainable by methods of recombinant DNA technology.
Examples of such hirudins are: (1) hirudin or a hirudin variant of type HV1 with the formula 10 H-4-Zl-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly- 20 -Gln-A sn-lLeu-Cys-Leu-Cys -Glu-Gly-Ser-Asn- 30 -Val-Cys-Gly-Gln-Gly-Asn-Z2-Cys-2e-Leu- (I) 40 -Gly-Ser-Asp-Gly-Glu-5-Asn-Gln-Cys-Val- 50 -Thr-Gly-Glu-Gly-Thr-Pro-Z4--Z6-S er- 60 -Z7-Z8-Asp-GIy-Asp-Phe-Glu-Glu-ne-Pro- -Glu-Glu-Tyr(R)-Leu-Gln-OH, wherein - (R)is the phenolic hydroxygroup of Tyr (desulphatohirudin) or a -0-S03H group, and - the whole molecule can be shortened by the C-terminal amino acid Gln, the C-terminal dipeptide Leu-Gln, the C-terminal tripeptide Tyr-Leu-Gln or the C-terminal tetrapeptide Glu-Tyr-Leu-Gln, and wherein Zo is a direct bond or represents Val, Ile or Gly or the dipeptidyl radicals Gly-Val or Met-Val, Z1 is Val, ne or Thr, Z2 is Lys, Gln, Asn, Leu, Arg or Val, Z3 represents Lys, Arg, Asn, Val, Leu, Gln, Z4 represents Lys, Arg, Asn, Val or Leu, 5 represents Pro or Gly, Z6 and Z8 independently from each other represent Gln, Asn or Met, and Z7 represents His, Gln or Asn, (2) desulphatohirudin variants of type HV1 with the formula 10 H-Val-Val-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly 20 -Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn- 30 -Val-Cys-Gly-Gln-Gly-Asn-Lys-Cys-Ile-Leu- (11) 40 -Gly-Ser-Asp-Gly-Glu-Lys-Asn-Gln-Cys-Val 50 -Thr-Gly-Glu- Gly-Thr-Pro-Lys -Pro-Gln-S er- 60 His-Asn-Y1-Gly-Asp-Phe-Y2-Y3-lle-Pro- -Y4-Y5-Y6-Leu-Y7-OH, wherein Y1 represents Asp or the radical of a neutral genetically encoded amino acid, Y2 and Y3 independently from each other represent Giu, Gln, Asn or the radical of a lipophilic genetically encoded amino acid, Y4 and Y5 independently from each other represent Glu, Gln or the radical of a neutral genetically encoded amino acid, Y6 represents Tyr or the radical of an acidic genetically encoded amino acid and Y7 represents Gln or the dipeptidyl radical Gln-Pro, (3) a hirudin variant of type HV2 with the formula 10 H-Ile-Thr-Tyr-Thr-Asp-Cys-Thr-Glu-Ser-Gly 20 -Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn- 30 -Val-Cys-Gly-Lys-Gly-Asn-Lys-CysJie-Leu- (III) 40 -Gly-Ser-Asn-Gly-Lys-Gly-Asn-Gln-Cys-Val- 50 -Thr-Gly-Glu-Gly-Thr-Pro-Asn-Pr > Glu-Ser- 60 -His-Asn-Asn-Gly-Asp-Phe-Glu-Glu-lle-Pro- -Glu-Glu-Tyr (R)-Leu-Gln-OH, wherein - (R)is the phenolic hydroxygroup of Tyr (desulphatohirudin) or a -0-S03H group, and - Ile 1 can be replaced by Val and Thr 2 by Val (HV2 modified) or - Asn 47 can be replaced by Lys or Arg or His or - Tyr 63 can be replaced by Glu or Asp, (4) a hirudin variant of type PA (HV3) with the formula 10 H-Ile-Thr-Tyr-Thr-A sp- Cys -Thr-Glu- S er-Gly- 20 -Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-Ser-Asn 30 -Val-Cys-Gly-Lys-Gly-Asn-Lys-Cys-Ile-Leu- (IV) 40 -Gly-Ser-Gln-Gly-Lys-Asp-Asn-Gln-Cys-Val- 50 -Thr-Gly-Glu-Gly-Thr-Pro-Lys -Pro-Gln-Ser- 60 -His-Asn-Gln-Gly-Asp-Phe-Glu-Pro-Ile-Pro- -Glu-Asp-Ala-Tyr (R)-Asp-Glu-OH, wherein - (R)is the phenolic hydroxygroup of Tyr (desulphatohirudin) or a -0-S03H group, and - the polypeptide chain can be shortened at the C-terminius by 18, 10, 9, 6,4 or 2 amino acids, or - the polypeptide chain can be shortened at the N-terminus by 1 or 2 amino acids.
Examples of hirudins of the formula (I) are desulphatohirudin HV1 in which (R) is the phenolic hydroxygroup of Tyr, Z0 represents Val, Z1 is Val, Z2, Z3 and Z4 are each Lys, Z5 is Pro, Z6 is Gln, Z7 is His and Z5 represents Asn, or des-(Val)2-desulphatohirudin, in which 4 represents a direct bond, Z1 is Thr and Z2-z8 and (R) are as defined for HV1.
Further examples are variants of HV1 like [Asn27]-desulphatohirudin, [Asn36]-desulphatohirudin, [Val36] -desuiphatohirudin, [Gly48]-desulphatohirudin, [Met49]desulphatohirudin, [Me2] -desuiphatohirudin, [Asn51]-desulphatohirudin, [Gin27, Arg47]-desulphatohirudin, [Gly27, Gin36, Arg47]-desulphatohirudin, [Arg36, Arg47]-desulphatohirudin, [Arg27, Arg47]-desulphatohirudin, Glycyl-[Gln27,Gln36, Arg47]-desulphatohirudin, Methionyl-[Gln27Arg47]-desulphatohirudin, [Ilel, Ile2]-desulphatohirudin and [Glyl]-desulphatohirudin.
Neutral genetically encoded amino acids are the following L-amino acids: Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Met, Phe, Try and Pro, furthermore the amino acid Gly.
Lipophilic genetically encoded amino acids are the following L-amino acids: Ala, Val, Leu, Ile, Phe and Gly.
Acidic genetically encoded amino acids are Asp and Glu.
Examples of desulphatohirudin variants of the formula (II) are [Gly61.62] -desulphatohirudin, [Leu61,62]-desulphatohirudin, [Asn61,62]-desulphatohirudin, [1-eu57,58,6 61,62]-desulphatohirudin, [Asn57,58,61,65 -desulphatohirudin, [Ala53]-desulphatohirudin, [Asp63] -desuiphatohirudin, [Glu63]-desulphatohirudin, [Pro66]-desulphatohirudin, rGln57,58,61,623 -desulphatohirudin.
Examples of hirudin variants of type HV2 of the formula (I) are desulphatohirudin HV2 or desulphatohirudin HV2 (Lys47).
An example of a hirudin variant of type PA of the formula aV) is desulphatohirudin PA.
The desulphatohirudin variants of the formulae ('), (11), (m) and JV) can be prepared by conventional recombinant DNA technology well known in the art. Following the isolation and cloning of the hirudin gene mutation of defined codons (like e.g. base exchanges, base deletions or base extensions) within the cloned DNA is achieved in vitro by the method of site-directed mutagenesis using suitable mutagenic primers. The resulting mutant gene is integrated in an appropriate expression vector and transformed in a microbial host like e.g.
Escherichia coli, Bacillus subtilis or Saccharomvces cerevisiae. Transformants carrying the hybrid vector which preferably comprises a signal sequence linked in the proper reading frame to the DNA-sequence encoding the mutant gene are cultivated by employing conventional techniques. The desulphatohirudin variants are isolated from the culture broth and purified by means well known to anybody of ordinary skill in the art.
Protein or polypeptide drugs are generally by necessity administered parentally in vivo.
Intravenously administered peptide drugs are, in general rapidly eliminated from the bloodstream via a process which is a combination of metabolism and of excretion.
Large proteins having sizes above the glomerulan filtration limit (i.e. diameter 745 AO) are eliminated from the body by metabolism. Small proteins/peptides, of a size below the glomerular filtration limit can be either filtered out of the body, metabolised by the body or both. Hirudin is known to be almost fully removed from the blood by the kidney. This removal is relatively rapid (tV2".5h), limiting the duration of biological action of the compound.
Various methods for modifying proteins to increase the time during which they remain in circulation have been proposed. In the case of hirudin, it has been proposed to link it to a carrier such as polysaccharide such as dextran or polyethylene glycol with a molecular weight of 1500-15000 Da - see EP 0 345 616.
In order to limit the rate of filtration of any compound through the kidneys, its effective size needs to be increased close to the size cut-off of the kidney membrane (i.e., 45 A).
Therefore, the size of the polymer to be attached to hirudin needed to be selected accordingly.
We have now found that when hirudin is conjugated with polyoxyethylene (the term to include alkoxy derivatives) having a molecular weight of from 20,000 to 50,000 Da, the circulating half life is much extended.
Accordingly, the present invention provides a conjugate of hirudin and polyoxyethylene which has a molecular weight of from 20,000 to 50,000 Da The conjugate has an extended plasma half-life and can be used as a long-acting anticoagulant for therapeutic purposes.
The polyoxyethylene is conjugated to the hirudin via the available amino groups of the hirudin. Usually between three and four amino groups are substituted with polyoxyethylene, making the molecular weight of the product > 100 k Da.
The conjugate may be prepared by first forming a chloroformate derivative of polyoxyethylene (POE-OCOCl) by reacting POE with phosgene in dry acetonitrile by the method described by Muther et al., Reactive Polymers 6, 99-107 (1987). This is then reacted with hirudin at an alkaline pH e.g. 7.5-10, to give a hirudin -POE conjugate free from cross-linked material.
The therapeutically effective amount of hirudin is normally in the dosage range from about 0.001 to 10 mgkg of body weight, e.g. from 0.01 to 3 mg/kg. Because of the longer life of the product of the invention in the body, the dosage range may be lowered from that used with hirudin itself. Administration is made by intravenous, intramuscular or subcutaneous injection.
The following Examples illustrate the invention. In the Examples the hirudin used is desulphatohirudin HV1 made by the method of Example 1 of EP 0 367 713.
Example Polyoxyethylene (POE) having a molecular weight of 35 k Da is dissolved in tetrahydrofuran and precipitated by the addition of diethyl ether. This is repeated twice and the precipitated material is collected and dried over phosphorus pentoxide in a vacuum desiccator.
The dry POE (33g) is dissolved in dry acetonitrile (200ml) and reacted with (lOg) phosgene. The resulting bifunctional POE chloroformate is isolated by removal of solvent and excess phosgene, and precipitation of the product from dichloromethane into ether.
The bifunctional POE chioroformate (910mg) is reacted with hirudin (9mg) in aqueous phosphate buffer at a pH of 8.5. The resulting conjugate is isolated and purified by ultra filtration through a 100 kDa membrane using a 50 x cell volume wash.
Size exclusion chromatography shows the conjugate to be polydisperse with a peak molecular weight of about 100 kDa. Elemental analysis (C/N ratio) indicates the substitution of between three and four amino groups (C/N =52.13=28.54 mol % Hirudin and 71.46 mol % POE., by weight 7.3% Hirudin and 92.7% POE).
Example 2 A comparative duration of action study is carried out following intravenous administration in rats (4 used per experiment). A dosage of 100 mgRg of conjugate is given to provide an initial anticoagulant activity (measured after 2 minutes) equivalent to 0.1 mg/kg of Hirudin. After 30 minutes the activities of Hirudin and Hirudin-POE are 23% and 91% of the initial value. After 60 min the activities are 15% and 62% respectively. These figures illustrate the significant increase in half life of the conjugate compared to hirudin itself.

Claims (6)

Claims
1. A conjugate of hirudin and polyoxyethylene which has a molecular weight of from 20,000 to 50,000 Da.
2. A conjugate as claimed in claim 1 which has between three and four polyoxyethylene chains per molecule conjugated via the available amino groups on hirudin.
3. A conjugate as claimed in claim 1 or 2 having a molecular weight of > 100 kDa.
4. A conjugate as claimed in any preceding claim in which the hirudin is selected from naturally occurring or synthetic variants, derivatives and fragments as well as desulphatohirudin variants and derivatives.
5. A conjugate as claimed in any preceding claim in which the hirudin is desulphatohirudin HV1.
6. The use of a conjugate as claimed in any preceding claim as a long acting blood anticoagulant.
GB9018464A 1990-08-22 1990-08-22 Hirudin conjugates with polyoxyethylene Withdrawn GB2247239A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
GB9018464A GB2247239A (en) 1990-08-22 1990-08-22 Hirudin conjugates with polyoxyethylene

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
GB9018464A GB2247239A (en) 1990-08-22 1990-08-22 Hirudin conjugates with polyoxyethylene

Publications (2)

Publication Number Publication Date
GB9018464D0 GB9018464D0 (en) 1990-10-03
GB2247239A true GB2247239A (en) 1992-02-26

Family

ID=10681080

Family Applications (1)

Application Number Title Priority Date Filing Date
GB9018464A Withdrawn GB2247239A (en) 1990-08-22 1990-08-22 Hirudin conjugates with polyoxyethylene

Country Status (1)

Country Link
GB (1) GB2247239A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0503829A2 (en) * 1991-03-08 1992-09-16 Ciba-Geigy Ag Hirudin for the inhibition of cancer metastasis
EP0658585A2 (en) * 1993-12-10 1995-06-21 BEHRINGWERKE Aktiengesellschaft Aminophenylalanine derivatives, process for their production, their use and agents containing them as anti-coagulants
WO2000061121A2 (en) * 1999-04-08 2000-10-19 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Use of molecular weight-amplified hirudin as an anticoagulant in extracorporeal renal replacement therapy

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0333356A2 (en) * 1988-03-04 1989-09-20 Biogen, Inc. Hirudin peptides
EP0345616A2 (en) * 1988-06-04 1989-12-13 Hoechst Aktiengesellschaft Hirudin derivatives with delayed action

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0333356A2 (en) * 1988-03-04 1989-09-20 Biogen, Inc. Hirudin peptides
EP0345616A2 (en) * 1988-06-04 1989-12-13 Hoechst Aktiengesellschaft Hirudin derivatives with delayed action

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0503829A2 (en) * 1991-03-08 1992-09-16 Ciba-Geigy Ag Hirudin for the inhibition of cancer metastasis
EP0503829A3 (en) * 1991-03-08 1993-06-02 Ciba-Geigy Ag Hirudin for the inhibition of cancer metastasis
EP0658585A2 (en) * 1993-12-10 1995-06-21 BEHRINGWERKE Aktiengesellschaft Aminophenylalanine derivatives, process for their production, their use and agents containing them as anti-coagulants
EP0658585A3 (en) * 1993-12-10 1995-10-18 Behringwerke Ag Aminophenylalanine derivatives, process for their production, their use and agents containing them as anti-coagulants.
US5571844A (en) * 1993-12-10 1996-11-05 Behringwerke Aktiengesellschaft Amidinophenylalanine derivatives, a process for their preparation, their use and compositions containing these as anticoagulants
WO2000061121A2 (en) * 1999-04-08 2000-10-19 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V., Berlin Use of molecular weight-amplified hirudin as an anticoagulant in extracorporeal renal replacement therapy
WO2000061121A3 (en) * 1999-04-08 2001-04-26 Max Planck Gesellschaft Use of molecular weight-amplified hirudin as an anticoagulant in extracorporeal renal replacement therapy

Also Published As

Publication number Publication date
GB9018464D0 (en) 1990-10-03

Similar Documents

Publication Publication Date Title
US6060451A (en) Thrombin inhibitors based on the amino acid sequence of hirudin
FI102183B (en) A process for the preparation of novel thrombin inhibitors
US5538946A (en) Hirudin derivatives with delayed action
EP0291982B1 (en) Cyclic anticoagulant peptides
EP0725797B1 (en) Bivalent thrombin inhibitors
EP0526192B1 (en) Hexapeptide
WO2001010450A1 (en) Targeted thrombolytic agents
JP2669510B2 (en) Thrombin inhibitors based on the amino acid sequence of hirudin
GB2247239A (en) Hirudin conjugates with polyoxyethylene
AU641215B2 (en) Stabilized sulfonate, sulfate, phosphonate and phosphate derivatives of hirudin
JP2023551050A (en) Polypeptides containing disulfide bonds having serine protease inhibitory activity, hybrid peptides derived therefrom, and applications thereof
HUT63180A (en) Process for producing anticoagulant peptides
US5880258A (en) Anticoagulant hirudin variants and methods for their production
US5681925A (en) Trifunctional antithrombin and antiplatelet peptides
US5837808A (en) Analogs of hirudin
US5858972A (en) Antithrombotic agents and methods of use
JP4031994B2 (en) Cyclic pentapeptides and methods for their preparation
EP0531537B1 (en) Hirudin analogue or salt thereof, production thereof, and anticoagulant containing the same as active ingredient
US5371071A (en) Peptide and pseudopeptide compounds which are therapeutically active in the blood coagulation cascade
JP2719891B2 (en) Pharmaceutical composition for promoting TNF secretion
AU761011B2 (en) Trivalent thrombin inhibitor
WO1996030407A1 (en) Bifunctional thrombin inhibitors bearing highly truncated fibrinogen recognition exosite binding component
WO1995007295A1 (en) Analogs of hirudin
JPS6330499A (en) Opioid peptide-polypeptide complex
NZ503669A (en) Trivalent thrombin inhibitor comprising (S subsite blocking segment)-(S' subsite blocking segment)-(fibrinogen recognition exosite blocking segment)

Legal Events

Date Code Title Description
WAP Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1)