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WO1996017928A1 - Utilisation du domaine protease de l'activateur plasminogene humain dans le traitement de maladies thromboemboliques - Google Patents

Utilisation du domaine protease de l'activateur plasminogene humain dans le traitement de maladies thromboemboliques Download PDF

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Publication number
WO1996017928A1
WO1996017928A1 PCT/EP1995/004717 EP9504717W WO9617928A1 WO 1996017928 A1 WO1996017928 A1 WO 1996017928A1 EP 9504717 W EP9504717 W EP 9504717W WO 9617928 A1 WO9617928 A1 WO 9617928A1
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Prior art keywords
plasminogen activator
amino acids
wild type
active molecule
protein
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PCT/EP1995/004717
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English (en)
Inventor
Ulrich Kohnert
Anne Stern
Ulrich Martin
Stephan Fischer
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Boehringer Mannheim Gmbh
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Publication date
Priority claimed from DE19944443273 external-priority patent/DE4443273A1/de
Priority claimed from US08/456,566 external-priority patent/US5723122A/en
Application filed by Boehringer Mannheim Gmbh filed Critical Boehringer Mannheim Gmbh
Priority to EP95941060A priority Critical patent/EP0796323A1/fr
Priority to AU42594/96A priority patent/AU4259496A/en
Publication of WO1996017928A1 publication Critical patent/WO1996017928A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • 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 invention is concerned with the use of the protease domain of human tissue-type plasminogen activator, medicaments for the treatment of thromboembolic diseases, pharmaceutical compositions which contain, as an active component, said protein, and the uses thereof.
  • Tissue-type plasminogen activator is a serine protease consisting of several domains, which catalyzes the conversion of plasminogen to plasmin and is employed for fibrinolytic therapy.
  • fibrinolysis is regulated partly by the interaction between t-PA and the plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor from the serpine family.
  • PAI-1 plasminogen activator inhibitor 1
  • the binding of PAI-1 to t-PA is essentially accomplished via amino acids 296-302 of t- PA. Mutation of this region causes a reduction of the inhibitory influence of PAI-1 on t-PA (E.L. Madison et al. (1990)).
  • Extensive investigations have been carried out on the mechanism of the interaction between amino acid region 296-302 of t-PA and PAI-1 (cf. E.L. Madison, Nature 339 (1989) 721-723, R.V.
  • Unmodified t-PA in its form as occurs in plasma (i.e., "wild type” t-PA), consists of 527 amino acids, and can be split by plasmin into two chains which are then still held together via a disulfide bridge.
  • the A chain also referred to as the heavy chain
  • the finger domain (amino acids 1-49) displays certain similarities to the finger structures in fibronectin.
  • the growth factor domain (amino acids 50-86), is to a certain extent, homologous to murine and human epidermal growth factors.
  • the two kringle domains (amino acids 87-175 and 176-262) are to a large extent homologous to the fourth and fifth kringle domain of plasminogen.
  • the finger domains and the kringle 2 domains of t-PA are especially involved in fibrin binding and in the stimulation of proteolytic activity by fibrin.
  • the B chain of t-PA (amino acids 276-527, protease domain) is a serine protease and is largely homologous to the B chains of urokinase and plasmin (T. J.R Harris, supra and J. Krause, supra).
  • t-PA variants which exhibit lower bleeding side effects are described in WO 93/24635 and by B.A. Keyt et al., Proc. Natl. Acad. Sci. USA 91: 3670-3674 (1994). These t-PA variants have an additional glycosylation site at amino acid positions 103-105. In addition, these t-PA variants may be modified at amino acids 296-302, whereby fibrin specificity is increased.
  • the object of the present invention is to provide thrombolytically active proteins which have, inter alia, lower bleeding side effects as compared to the known plasminogen activators.
  • the invention comprises a method for treating a thromboembolic condition comprising administering to a subject in need thereof an effective amount of a thrombolytically active molecule which is not stimulated by CNBr fragments of fibrinogen, has plasminogenolytic activity less than about 25 KU/mg, is a protein or glycoprotein and which has less than about 10% of the fibrin binding activity of wild type human tissue type plasminogen activator; as well as the use of said molecules for manufacturing a therapeutic agent for treating a thromboembolic condition.
  • a method for treating a thromboembolic condition comprising administering to a subject in need thereof a thrombolytically active molecule which is a protein or glycoprotein, the protein portion of which consists of an amino acid sequence which
  • a further object of the invention is a method in which the protein portion of the thrombolytically active molecule consists of amino acid 276 through 527 to wild type, human tissue type plasminogen activator and the use of said molecule for manufacturing a therapeutic agent for treating a thromboembolic condition.
  • compositions which is useful in treating a thromboembolic condition comprising said thrombolytically active molecule, and a pharmaceutically acceptable carrier.
  • the object of the invention is accomplished via a thrombolytically active protein and medicaments based upon it which are used for the treatment of thromboembolic diseases, wherein the protein contains, as the only structure effecting thrombolytic activity, the protease domain of human tissue-type plasminogen activator (i.e., amino acids 262-527, as set forth supra).
  • the protein consists of the essential parts of the protease domain.
  • the proteins have a specific plasminogenolytic activity of 25 KU/mg or less, more preferably less than about 10 KU/mg, and most preferably less than about 2.5 KU/mg using CNBr fragments of fibrinogen as a stimulator.
  • amino acids Ile276 to C terminal end are required, at a minimum.
  • the N-terminus may, however, begin with any amino acids beginning with Ser262 of wild type t-PA up to Ile276.
  • the N-terminus starts with Ser262, Cys264, and the C-terminus is 527.
  • thrombolytically active proteins containing alterations within amino acids 296-302, and/or 274-277 of wild type t-PA.
  • Cys264 is desirably included because it forms a disulfide bond with Cys395, thus stabilizing three dimensional structure.
  • the N-terminus may also begin with any of amino acids Gly-3 up through Ile5, preferably any of amino acids Serl, through Gln3, followed immediately by one of the N-terminus options presented supra (for numbering see Harris, Prot. Eng. 1: 449-458 (1987), incorporated herein by reference).
  • amino acids encoded by nucleotides positioned in the region of the human t-PA DNA before the intron/exon junction of the finger domain correspond to amino acids encoded by nucleotides positioned in the region of the human t-PA DNA before the intron/exon junction of the finger domain.
  • Ser262 to Ile276 Ser262 is positioned at an intron/exon junction.
  • minor modifications such as substitutions, deletions, additions, and/or modifications of one or more amino acids are possible, as long as the proteolytic character of the molecule is not changed.
  • Ser262 is especially preferred as the N-terminus of the molecule with or without some further N-terminal amino acids which correspond to the original N-terminus of tPA, especially all or a portion of the amino acids of positions -5 through 3, or more preferably of positions 1 to 3 of wild type tPA.
  • Proteolytic activity can be determined very easily, using any of the well known assays for determining this property, including those set forth herein.
  • Thrombolytically active proteins in accordance with the invention exhibit low fibrin binding and very low plasminogenolytic activity in vitro (determined according to Verheijen, J. et al., Thromb. Haemostas. 48: 266-269 (1982)) and have therefore not been considered as suitable thrombolytic therapeutic agents up to now.
  • the properties of these proteins include plasminogenolytic activity of about 25 KU/mg or less, preferably about 10 KU/mg or less, most preferably about 2.5 KU/mg or less, and less than about 10% of fibrin binding activity of wild type human tissue type plasminogen activator. These molecules are also not stimulated by CNBr fragments of fibrinogen. Surprisingly, a high potency and a distinctly reduced risk of bleeding compared to known plasminogen activators and high activity are found, however, when they are used in vivo.
  • the fibrin binding in accordance with the invention can be determined in a fibrin binding test, wherein thrombin at a final concentration of 2.5 NIH units/ml is added to a solution of the protease and 1.2 mg/ml or increasing concentrations of fibrinogen in buffer solution (e.g. Veronal 15 mmol 1, NaCl 28 mmol 1, CaC12 0.5 mmol/1, MgC12 0.2 mmol 1, Tris HC1 5 mmol 1 and 0.005% (v/v) Tween ® 80, pH 7.75).
  • buffer solution e.g. Veronal 15 mmol 1, NaCl 28 mmol 1, CaC12 0.5 mmol/1, MgC12 0.2 mmol 1, Tris HC1 5 mmol 1 and 0.005% (v/v) Tween ® 80, pH 7.75.
  • the resulting fibrin clots are removed by centrifugation at 12,000xg for 8 minutes and the amount of the thrombolytically active protein retained in the supernatant is determined by ELISA.
  • the method set forth in example 5, infra can also be used to determine fibrin binding. Indeed, this is the preferred method.
  • the proteins in accordance with the invention display at least 50% lower blood loss compared to human tissue-type plasminogen activator, when a radioactively labeled thrombus is formed in the jugular vein, 100 IU/kg of heparin are administered subcutaneously, and a single intravenous bolus injection of 1 mg kg protein is given and the bleeding is determined according to J. Clin. Invest. 71: 368-376 (1993).
  • thrombolytically active proteins of the invention it is thus possible, by using the thrombolytically active proteins of the invention, to retain a therapeutically relevant thrombolytic effect and to reduce the bleeding side effects observed clinically, by more than 50%. This property is not lost even when the dose of protein is increased.
  • the thrombolytically active proteins of the invention are suitable for use as intravenous bolus injections.
  • therapy using protease results in less than 50% of the blood loss found when using wild type t- PA.
  • This property is highly significant with respect to the safety profile of thrombolytically active proteins, since it reduces the side effect of bleeding which otherwise usually occurs in the case of thrombolytic agents, such as wild type t-PA.
  • the proteins of the invention exceedly valuable thrombolytic agents for the treatment of all thromboembolic diseases.
  • thrombolytic agents that have hitherto been mainly used for diseases which pose a very great danger to life, such as cardiac infarction and massive pulmonary embolism
  • thrombolytic agents based on the use of the protease domain as the sole, thrombolytically active domain can now be applied in a much wider range than before, since the prior danger of bleeding complications is no longer an issue.
  • the inventive proteins can also be used advantageously for acute diseases, such as cardiac infarction or pulmonary embolism.
  • the preparation of the thrombolytically active proteins in accordance with the invention can be carried out in eukaryotic or prokaryotic cells according to the methods known to one skilled in the art.
  • the compounds according to the invention are prepared by genetic engineering techniques. Such methods are described, for example, in WO 90/09437, EP-A 0 297 066, EP-A 0 302 456, EP-A 0 245 100 and EP-A 0 400 545 all of which are incorporated herein by reference with regard to such methods of production.
  • Mutations can be inserted into the cDNA of t-PA or a derivative thereof by means of oligonucleotide-directed site-specific mutagenesis.
  • Site-specific mutagenesis is described, for instance, by Zoller and Smith, DNA 3: 479-488 (1984), modified according to T.A. Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985) and Morinaga et al., Biotechnology 21 : 634 (1984).
  • the method of PCR mutagenesis which is described, for example, in Ausubel et al., Current Protocols In Molecular Biology, Vol. 2, Chapter 15 (Greene Publ. Associates & Wiley Interscience 1991) is also suitable. These methods are also incorporated by reference.
  • nucleic acid molecules obtained in this way serves the expression of the t-PA derivative employed according to the invention, especially if it is present on an expression vector suitable for the host cell used.
  • nucleic acid molecules which code for proteins according to the invention can also be modified. Examples of such modifications include:
  • nucleotide sequence - supplementing the nucleotide sequence with additional regulatory and transcription elements so as to optimize expression in the host cell.
  • glycosylated proteins employed according to the invention is carried out in eukaryotic host cells.
  • the preparation of non-glycosylated proteins according to the invention may be carried out in either eukaryotic host cells, wherein the resulting glycosylated product must be deglycosylated by methods known to one skilled in the art, or, preferably, by expression in non-glycosylating host cells, more preferably in prokaryotic host cells.
  • Suitable prokaryotic host organisms include E.coli, Streptomyces spec, or Bacillus subtilis.
  • the prokaryotic cells are cultivated in the usual manner and, after harvest of the bacteria, the protein is isolated in the usual manner. If the protein is obtained in inactive form (e.g. as inclusion bodies), it is solubi ⁇ zed and renatured according to the methods known to one skilled in the art. According to these, it is also possible to secrete the protein from the microorganisms in the form of an active protein.
  • An expression vector suitable for this purpose preferably contains a signal sequence appropriate for the secretion of proteins in the employed host cells, and the nucleotide sequence coding for the protein.
  • the protein expressed by this vector is secreted either into the medium (in the case of gram-positive bacteria) or into the penplasmatic region (in the case of gram-negative bacteria). It is preferred to employ a sequence coding for a cleavage site between the signal sequence and the sequence coding for the proteins of the invention, which allows the splitting off of the protein either during processing or by treatment with protease.
  • the selection of the base vector into which the nucleic acid molecule coding for the protein of the invention is inserted is dependent upon the host cells that are used for expression.
  • the skilled artisan is aware of appropriate plasmids as well as of the minimum requirements to be met by a plasmid of this type (e.g. replication origin, restriction cleavage sites).
  • a plasmid e.g. replication origin, restriction cleavage sites.
  • a cosmid the replicative double-stranded form of phages (M13), or other vectors known to one skilled in the art can be used within the framework of the invention.
  • purification of the proteins according to the invention is carried out in the presence of L-arginine, in particular at an arginine concentration of from about 10 to about 1000 mmol/1.
  • the separation of foreign proteins is performed, preferably, by means of affinity chromatography, and more preferably via an adsorber column on which ETI (Erythrina trypsin inhibitor) is immobilized.
  • ETI Errythrina trypsin inhibitor
  • carrier material CNBr-activated Sepharose , e.g., may be used.
  • Purification via an ETI adsorption column offers the advantage that the ETI adsorption column material can be loaded directly from the concentrated renaturation batch even in the presence of arginine concentrations as high as 0.8 mol 1 arginine.
  • purification of the thrombolytically active proteins according to the invention is carried out via an ETI adsorption column in the presence of 0.6 to 0.8 mol/1 arginine.
  • the solution applied to the column preferably has a pH higher than 7, more preferably between 7.5 and 8.6.
  • Elution from the ETI column of the proteins of the invention is accomplished by lowering the pH both in the presence and in the absence of arginine under conditions at which the proteins of the invention are readily soluble.
  • the pH value is preferably within the acidic range, preferably between about pH 4.0 and pH 6.5, more preferably between pH 4.0 and pH 5.5, most preferably between pH 4.5 and pH 5.5.
  • Another aspect of the invention is a pharmaceutical composition containing a thrombolytically active protein, wherein the protein contains as the only structure effecting thrombolytic activity the protease domain of human tissue-type plasminogen activator.
  • the proteins of the invention can be formulated, in a manner known to one skilled in the art, in therapeutic agents, wherein the compounds according to the invention usually are combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier typically, such compositions contain as a dose an effective amount of 0.1 to 7 mg kg, preferably 0.3 to 7 mg/kg or 0.7 to 5 mg kg, and more preferably 1 to 3 mg kg body weight.
  • the therapeutic compositions usually are present as sterile aqueous solutions or as sterile, soluble dry formulations, such as lyophilisates.
  • the compositions usually contain an appropriate amount of a pharmaceutically acceptable salt with which an isotonic solution is prepared.
  • buffers such as arginine buffer or phosphate buffer, can be applied to stabilize the compositions at an appropriate pH value (preferably 5.5 to 7.5).
  • the dosage level of the compounds according to the invention can be easily determined by a person skilled in the art. Relevant factors include, e.g., the mode of application (infusion or bolus) and the duration of therapy. Due to their extended half-life the compounds (preferably 11 to 19 minutes) according to the invention are particularly useful for bolus application (single bolus, multiple bolus). For instance, an ampoule containing 25 to 1000 mg of a compound according to the invention, arginine, and a buffer may be used in bolus application. Application is preferably performed intravenously, but may also be accomplished subcutaneously, intramuscularly, intra-aortically or intra-arterially. The protein can also be infused or applied locally.
  • the compounds according to the invention can be applied as a multiple bolus (preferably as a double bolus).
  • Appropriate time intervals for multiple boli are between 20 and 180 minutes, an interval between 30 and 90 minutes being more preferred and an interval between 30 and 60 minutes being most preferred. It has been found, however, that, surprisingly, the proteins are sufficiently effective in single bolus application.
  • the dosage preferably is about 1 mg/kg of body weight.
  • the compounds according to the invention are useful, in particular, for the treatment of all thromboembolic diseases such as, e.g., acute cardiac infarction, cerebral infarction, pulmonary embolism, deep vein thrombosis, especially of the leg, acute arterial occlusion, etc. More preferably, the compounds according to the invention are applied for treating subchronic thromboembolic diseases where thrombolysis must be carried out for an extended period of time.
  • thromboembolic diseases such as, e.g., acute cardiac infarction, cerebral infarction, pulmonary embolism, deep vein thrombosis, especially of the leg, acute arterial occlusion, etc.
  • the compounds according to the invention are applied for treating subchronic thromboembolic diseases where thrombolysis must be carried out for an extended period of time.
  • the compounds according to the invention in combination with a substance inhibiting coagulation (anticoagulant) such as heparin or hirudin, and/or a substance inhibiting platelet aggregation, whereby the vessel-opening effect is enhanced with little side effects being involved. It is also preferred to add substances stimulating the blood flow or substances which improve microcirculation.
  • Figure 1 presents a comparison of clot lysis activity of recombinant, wild type human tissue type plasminogen activator ("t-PA"), and a thrombolytically active protein in accordance with the invention, consisting of amino acids 1-3 and 262-527 of wild type t-PA.
  • Figure 2 compares fibrin binding of recombinant human tissue type plasminogen activator to a thrombolytically active protein in accordance with the invention (a protein containing amino acids 1-3 and 262-527 of wild type t-PA), over various concentrations of fibrin.
  • FIG. 3 is a schematic description of the plasma clot penetration and lysis model.
  • the pressure was provided by a buffer compartment (hatched area).
  • the mixture of buffer and the plasma above the clot was avoided by the installation of a bubble trap.
  • 1 buffer reservoir
  • 2 peristaltic pump
  • 3 bubble trap
  • 4 syringe for injection of the fibrinolytic agent
  • 5 pipet tip with clot (netted area)
  • 6 hose clip
  • 7 pressure element.
  • Figure 4 presents a comparison of CHO-t-PA and the protease domain in the plasma clot penetration and lysis model. Increasing concentrations of both plasminogen activators were added to 1 ml plasma on top of a preformed plasma clot. The lysis was determined as described in Example 3b. All values are the mean of five separate experiments.
  • the starting plasmid pA27fd contains the following components: tac promoter, lac operator region with an ATG starting codon, the coding region for a t-PA derivative consisting of the kringle-2 domain and the protease domain (amino acids 1-3 and 176-527), and the fd transcription terminator.
  • Plasmid pKK223-3 is the starting vector.
  • the formation of the heteroduplex was performed as described in EP 0 382 174, incorporated by reference.
  • the heteroduplex preparation together with the plasmid pUBS520 (Brinkmann et al., 1989, Gene 85:109) was transformed in E.coli C600+.
  • the transformants were selected by adding to the nutrient medium ampicillin and kanamycin (50 ⁇ g/ml each).
  • the resulting plasmid was called pA27Protease; it is distinguished from the starting plasmid by the absence of an EcoRI cleavage site.
  • the E.coli strain C600+ was cultured with the plasmids pA27Protease and pUBS520 in LB medium (Sambrook et al., supra) in the presence of ampicillin and kanamycin (50 ⁇ g/ml each) until an OD at 550 nm of 0.4 was attained.
  • the expression was initiated by adding 5 mmol 1 EPTG.
  • the culture was incubated for a further four hours.
  • the E.coli cells were collected by centrifugation and resuspended in buffer (50 mmol/1 Tris HC1 pH 8, 50 mmol/1 EDTA); lysis of the cells was brought about by sonication.
  • the insoluble protein fractions were collected and resuspended in the above-mentioned buffer by sonication.
  • the suspension was mixed with 1/4 volume of loading buffer (250 mmol/1 Tris HC1 pH 6.8, 10 mmol/EDTA, 5% SDS, 5% mercaptoethanol, 50% glycerol and 0.005% bromophenol blue) and analyzed with the aid of a 12.5% SDS polyacrylamide gel.
  • loading buffer 250 mmol/1 Tris HC1 pH 6.8, 10 mmol/EDTA, 5% SDS, 5% mercaptoethanol, 50% glycerol and 0.005% bromophenol blue
  • the same preparation was carried out using a culture of E.coli with the two above-mentioned plasmids which had not been induced with IPTG, and separated in the gel.
  • Alteplase (recombinant, wild type tissue-type plasminogen activator "t-PA", commercially available as Actilyse ® from Thomae, Biberach, Germany), the protein described in Example 1, streptokinase (commercially available as Streptase ® from Behring, Marburg, Germany) or solvent (0.2 M arginine phosphate buffer) were administered to the rabbits intravenously.
  • t-PA tissue-type plasminogen activator
  • the placebo group was given an intravenous single bolus injection of 1 ml/kg solvent.
  • the alteplase group was given, intravenously, a total dose of 1.45 mg/kg, of which 0.2 mg/kg were in the form of a starting bolus injection, 0.75 mg/kg in the form of a 30 minutes' infusion, immediately followed by 0.5 mg/kg as a 60-minutes continuous infusion (total infusion: 90 min.).
  • the streptokinase group was given a 60-minutes intravenous infusion of 64,000 IU/kg.
  • the protease group was given an intravenous single bolus injection of 1 mg/kg or 2 mg/kg. For alteplase and streptokinase, these are accepted, standard regimes.
  • thrombolysis Two hours after the start of therapy, any thrombus remaining was removed, and the extent of the dissolution of the thrombus (thrombolysis) was determined by means of the decrease of radioactivity in the thrombus. Blood samples for obtaining plasma were taken prior to therapy and 2 hours after the start of therapy for use in recovering plasma. Activated partial thromboplastin time was measured, using a standard method. Furthermore, any blood loss caused by the thrombolytic therapy, was quantified. To this end, prior to the administration of the thrombolytic agents, a defined skin incision (4 cm in length and 0.3 cm in depth) was made on the thigh of the animals using a template and a scalpel. The bleeding which occurred as a result stopped as a result of natural coagulation.
  • a sponge was placed on the wound, soaking up the blood from the bleeding which had newly started as a result of the thrombolysis.
  • the amount of blood issuing from the wound was measured and in this way specified the extent of the bleeding side effect.
  • Both alteplase and the protease of the invention are highly active thrombolytic substances and, compared to the solvent control both significantly dissolved the thrombi. However, a dose of 1 mg/kg (invention) instead of 1.45 mg/kg of (alteplase), i.e., a 31% lower dose, was effective.
  • the dose and mode of administration (three-stage infusion) used for alteplase corresponds to the dose and mode of administration customarily applied in the clinic when treating cardiac infarction (see GUSTO study: N. Engl. J. Med., 1993; 329: 673-82).
  • t-PA and the recombinant protease were adjusted with buffer to the concentrations given in Figure 1 and their activity was determined in the clot lysis assay.
  • the sample is adjusted to the protein concentration required in the particular case by adding buffer (0.06 M Na2HPO4, pH 7.4, 5 mg/ml BSA (bovine serum albumin), 0.01% Tween ).
  • buffer 0.06 M Na2HPO4, pH 7.4, 5 mg/ml BSA (bovine serum albumin), 0.01% Tween ).
  • 0.1 ml of the sample were mixed with 1 ml human fibrinogen solution (DMCO) (2 mg/ml 0.006 M Na2HPO4, pH 7.4, 0.5 mg/ml BSA, 0.01% Tween ® 80 ) and incubated at 37°C for 5 minutes.
  • DMCO human fibrinogen solution
  • the substances according to the invention are investigated under conditions which are quite similar to the in vivo conditions.
  • the substances are administered into the plasma at the top of the clot under the influence of a peristaltic pressure which resembles the pressure caused by the heartbeat.
  • 200 ⁇ l citrate plasma were mixed with 20 ⁇ l of a 0.25 mol 1 CaCl2 solution and incubated at 37°C. 0.16 U thrombin were added and the mixture was transferred into a 1 ml-pipet tip (Eppendorff, Hamburg, FRG).
  • the pipet trip was stored vertically for 2 min at 23°C, incubated for 60 min in 0.01 mol 1 Tris/HCl, pH 7.4, 0.15 mol 1 NaCl, 0.025 mol/1 CaCl2, 0.01% Tween® 80 and transferred into the clot lysis apparatus.
  • the clot lysis activity was determined in a circuit system built up from elastic tubes ( Figure 1). The flow is provided by a peristaltic pump and is split into two parallel branches. Branch A contains the 1 ml-pipet tip filled with the plasma clot which seals this branch. Branch B is a blind line parallel to branch A The pressure was adjusted by a hose clip in branch B to 10 mbar. Plasma (1 ml) was loaded on the top of the clot.
  • the pump was switched on and the stability of each clot was checked for 15 min.
  • the fibrinolytic agent (final plasma concentration between 0.5 and 10 and 20 ⁇ g/ml for the protease domain and CHO-t-PA, respectively) was injected carefully into the plasma with a 1 ml-tuberculin syringe and a hypodermic needle for intramuscular injection (Braun, Melsungen, FRG).
  • the clot lysis time was calculated as the time difference between addition of the fibrinolytic enzyme and the decrease of pressure to 50% of the value before the addition of the fibrinolytic agent.
  • the pressure was determined with a piezoelectric pressure registration system calibrated with water and documented by a computer aided documentation program.
  • This example tests the thrombolytically active protein of Example 1 for its ability to bind to fibrin, and also compares it to recombinant, human tissue type plasminogen activator for this property.
  • Actilyse is recombinant human tissue type plasminogen activator produced in Chinese Hamster Ovary (CHO) cells. Samples of Actilyse, and a protein in accordance with the invention, were prepared as solutions of 1.5 ⁇ g protein/ml.
  • the components were mixed, and incubated for one hour at 37°C. Subsequently, supernatant was separated from the fibrin clot by centrifugation (15 minutes, 13,000 RPM, at 4°C), and the amount of plasminogen activator protein present in the supernatant was determined by a standard ELISA.
  • the results are set forth in Figure 2.
  • the amount of recombinant t-PA which bound to the fibrin clot ranged from 15% (10 ⁇ g/ml fibrin), up to 65%, at 300 ⁇ g/ml.
  • the thrombolytically active protein showed insignificant binding at low concentrations, and only rose to about 10% at the top of the range. This low value can be attributed to non-specific inclusion of the protein in the fibrin net, rather than to specific binding between the protein and the clot.
  • thrombolytically active protein in accordance with the invention does not significantly bind to fibrin.
  • the reagents used in this assay include fibrinogen fragments, "Lys-plasminogen”, and aprotinin-Eupergit.
  • the first of these, which acts as a thrombolytically active protein stimulator, was prepared by treating human fibrinogen with cyanogen bromide (1 g human fibrinogen, 1.3 g CNBr in 100 ml water), in 70% v/v formic acid, for 17 hours at room temperature, followed by dialysis against distilled water.
  • Lys-plasminogen is human plasminogen which contains primarily lys-plg, which has been isolated from Cohn fraction HI, via affinity chromatography on lysine sepharose, followed by treatment with aprotinin-Eupergit to remove plasmin.
  • aprotinin-Eupergit 39 mg aprotinin were coupled to 4g of EupergitTMC oxirane acrylic beads, following the manufacturer's instructions.
  • Actilyse was determined to have activity of from about 400 to about 700 KU/mg, as compared to the compound of the invention, with an activity of about 2.5 KU/mg.
  • the invention has 1/160 to 1/300 the plasmmogeneolytic activity of the recombinant t-PA.
  • fibrinogen fragments There was no stimulation of the compound of the invention by the fibrinogen fragments, as compared to the t-PA, which was stimulated by a factor of from 30 to 50.
  • the plasminogenolytic activity was determined in a method similar to that described by Verheijen et al. (1982), Thromb. Haemost. 48, 266-269.
  • 25 ⁇ l of the sample correspondingly pre-diluted with 0.1 mol/1 Tris, pH 7.5, 0.15% Tween ® 80, are pipetted into a well of a microtiter plate. Subsequently, 200 ⁇ l of reagent mixture are added and the extinction is determined at 405 nm over a period of 2 h against the blank value (25 ⁇ l, 0.1 mol/1 Tris, pH 7.5, 0.15% Tween ® 80 with 200 ⁇ l reagent mixture).
  • test buffer 0.1 mol/1 Tris, pH 7.5, 0.15% Tween® 80
  • t-PA stimulator (1 mg/ml bromocyan fragments from human fibrinogen) 1 ml substrate solution (3 mrnol/1 S2251, H-D-Val-Leu-Lys-pNA; Chromogenix, Moelndal,
  • the activity in the presence of the t-PA stimulator is divided by the activity in the absence of the t-PA stimulator.
  • the dilution in each case is to be selected such that approximately the same extinctions are achieved in both preparations.
  • To the reaction mixture without the t-PA stimulator is added 1 ml H2O, instead of 1 ml t-PA stimulator.
  • the measurement of activity is carried out in the same manner both in the absence of stimulator and in the presence of stimulator.
  • the stimulation factor F is calculated as follows:
  • the specific activity is the quotient for the plasminogenolytic activity (KU/ml) and the protein concentration (mg/ml).
  • P Lethal arrhythmias or hypotension were not observed after P. It is concluded that P is a very effective and safe thrombolytic agent in vivo in contrast to in vitro.

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Abstract

L'invention concerne l'utilisation de protéines thromboemboliques, lesquelles contiennent, en tant que seule portion structurelle possédant une activité thrombolytique, le domaine protéase d'un activateur du plasminogène d'un type de tissu humain. Ces dérivés possèdent peu d'effets secondaires et présentent notamment une réduction du saignement, tout en démontrant une remarquable efficacité in vivo. Cette efficacité est surprenante étant donné les propriétés in vitro de ces dérivés.
PCT/EP1995/004717 1994-12-06 1995-11-30 Utilisation du domaine protease de l'activateur plasminogene humain dans le traitement de maladies thromboemboliques WO1996017928A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP95941060A EP0796323A1 (fr) 1994-12-06 1995-11-30 Utilisation du domaine protease de l'activateur plasminogene humain dans le traitement de maladies thromboemboliques
AU42594/96A AU4259496A (en) 1994-12-06 1995-11-30 Use of the protease domain of human plasminogen activator for the treatment of thromboembolic diseases

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19944443273 DE4443273A1 (de) 1994-12-06 1994-12-06 Verwendung der Proteasedomäne von humanem Plasminogenaktivator zur Behandlung von thromboembolischen Erkrankungen
DEP4443273.9 1994-12-06
US08/456,566 US5723122A (en) 1995-06-01 1995-06-01 Use of the protease domain of human plasminogen activator for the treatment of thromboembolic diseases
US08/456,566 1995-06-01

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WO1996017928A1 true WO1996017928A1 (fr) 1996-06-13

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009185A1 (fr) * 1997-08-13 1999-02-25 Roche Diagnostics Gmbh Activateur du plasminogene avec zymogenite amelioree
WO1999009184A1 (fr) * 1997-08-13 1999-02-25 Roche Diagnostics Gmbh Activateur du plasminogene a pouvoir zymogene ameliore et a liaison a la fibrine reduite

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0382174A1 (fr) * 1989-02-07 1990-08-16 Roche Diagnostics GmbH Dérivé de l'activateur tissulaire de plasminogène

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0382174A1 (fr) * 1989-02-07 1990-08-16 Roche Diagnostics GmbH Dérivé de l'activateur tissulaire de plasminogène

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
M-J GETHING ET AL: "Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain", EMBO JOURBAL, vol. 7, no. 9, pages 2731 - 2740 *
P. BURCK ET AL: "Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 265, no. 9, 25 March 1990 (1990-03-25), MD US, pages 5170 - 5177 *
ROBINSON J H ET AL: "REDESIGNING T-PA FOR IMPROVED THROMBOLYTIC THERAPY", TRENDS IN BIOTECHNOLOGY, vol. 9, no. 3, 1 March 1991 (1991-03-01), pages 86 - 90, XP000175392 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009185A1 (fr) * 1997-08-13 1999-02-25 Roche Diagnostics Gmbh Activateur du plasminogene avec zymogenite amelioree
WO1999009184A1 (fr) * 1997-08-13 1999-02-25 Roche Diagnostics Gmbh Activateur du plasminogene a pouvoir zymogene ameliore et a liaison a la fibrine reduite

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AU4259496A (en) 1996-06-26

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