EP0701606A4 - Procede d'amelioration de thrombolyse - Google Patents

Procede d'amelioration de thrombolyse

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Publication number
EP0701606A4
EP0701606A4 EP94915405A EP94915405A EP0701606A4 EP 0701606 A4 EP0701606 A4 EP 0701606A4 EP 94915405 A EP94915405 A EP 94915405A EP 94915405 A EP94915405 A EP 94915405A EP 0701606 A4 EP0701606 A4 EP 0701606A4
Authority
EP
European Patent Office
Prior art keywords
aspirin
binding domain
bolus
vcl
domain polypeptide
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Withdrawn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
EP94915405A
Other languages
German (de)
English (en)
Other versions
EP0701606A1 (fr
Inventor
Leonard Garfinkel
Marian Gorecki
Amos Panet
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.)
Savient Pharmaceuticals Inc
Original Assignee
Savient Pharmaceuticals Inc
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Publication date
Application filed by Savient Pharmaceuticals Inc filed Critical Savient Pharmaceuticals Inc
Publication of EP0701606A1 publication Critical patent/EP0701606A1/fr
Publication of EP0701606A4 publication Critical patent/EP0701606A4/fr
Withdrawn legal-status Critical Current

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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
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/49—Urokinase; Tissue plasminogen activator
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present application is directed towards the use of a fragment of von Willebrand Factor (vWF) comprising the platelet glycoprotein lb (GPIb) binding domain in conjunction with known therapies to obtain enhanced protection against thrombosis, improved thrombolysis, and a decreased likelihood of reocclusion following thrombolytic treatment.
  • vWF von Willebrand Factor
  • GPIb platelet glycoprotein lb
  • Von Willebrand Factor is a large plasma protein which is synthesized in the endothelial cells which form the inner surface lining of the blood vessel wall, and by megakarocytes, the precursor of platelets.
  • Mature vWF is a multivalent molecule comprising domains which constitute binding sites for several proteins.
  • One of the domains constitutes a binding site for the platelet glycoprotein lb (GPIb) . Using proteolytic digests this site has been .localized to the region between amino acid residues 449 and 7?8 of mature vWF.
  • vWF has at least two collagen binding sites, at least two heparin binding sites, a Factor VIII binding site, and a RGD site which binds to the platelet GP Ilb/I Ia receptor.
  • von Willebrand factor is essential for the formation of platelet thrombi, especially under flow conditions characterized by high shear stress, such as occur in stenosed coronary arteries (24) .
  • Von Willebrand factor interacts with glycoprotein lb receptors on the platelet membrane to initiate platelet adhesion (13-15) and to activate platelet release of ADP, thromboxane, and serotonin, which cause platelet aggregation and thrombus formation (16-20) . Therefore, efforts have been made to prevent platelet adhesion and thrombus formation by blocking the platelet glycoprotein lb receptors. For example, aurin tricarboxylic acid blocks the platelet glycoprotein lb recognition site on von Willebrand factor and may be useful in antithrombotic therapy (25) .
  • vWF von Willebrand Disease
  • BSS Bernard-Soulier Syndrome
  • Hemostasis is a dynamic and ongoing process. It includes on one hand formation of clots to prevent bleeding and on the other hand dissolution and lysis of unwanted fibrin deposits and platelet aggregates in order to maintain blood flow throughout the body.
  • thrombolytic agents such as tissue plasminogen activator (t-PA) and streptokinase have been effective in the treatment of thrombosis in patients with acute transmural myocardial infarction ("Q-wave infarcts") (8-10), and are often administered with adjuncts such as anticoagulants, e.g. heparin and aspirin.
  • t-PA tissue plasminogen activator
  • Q-wave infarcts acute transmural myocardial infarction
  • adjuncts such as anticoagulants, e.g. heparin and aspirin.
  • 15-20% of patients do not achieve reperfusion.
  • reocclusion of coronary arteries has limited the efficacy of thrombolytic therapy in some patients (11-12) .
  • vWF von Willebrand factor
  • vWF a multivalent protein
  • vWF a multivalent protein
  • the adhesion of platelets to the subendothelium results in the activation of platelets and the release of adenosine diphosphate (.ADP) , thromboxane, and serotonin. These in turn activate additional platelets.
  • a platelet plug forms (16-20) . Therefore, preventing platelet adhesion may be useful in preventing thrombosis and reocclusion in coronary arteries (21) .
  • VCL One such peptide, designated VCL, is a recombina ⁇ t fragment of von Willebrand factor, Leu 504 - Lys 728 , with a single intrachain disulfide bond linking residues Cy ⁇ 509 and Cys 695 (23) .
  • VCL is disclosed in coassigned International Patent Application Publication No. WO91/13903 which also discloses a method of inhibiting platelet aggregation in a subject prior to or after the subject has undergone angioplasty, thrombolytic treatment or coronary bypass surgery by use of the vWF fragment alone to achieve the desired effect.
  • a vWF polypeptide fragment comprising the GPIb binding domain in conjunction with thrombolytic therapy provides greatly improved therapeutic results.
  • the time required for thrombolysis is considerably lessened as evidenced by the shortened time to reperfusion.
  • vWF GPIb binding domain polypeptide and aspirin synergistically prevent complications following traumatic vascular damage.
  • the present invention provides a method of enhancing thrombolysis comprising administering a vWF GPIb binding domain polypeptide in conjunction with a thrombolytic agent and an anticoagulant which results in the shortening of the time to thrombolysis and reducing the incidence of reocclusion following thrombolysis.
  • the invention provides a method for preventing complications following traumatic vascular damage in a subject comprising administering to the subject an amount of a vWF GPIb binding domain polypeptide in conjunction with an amount of aspirin, which together are effective to prevent the complications.
  • This figure shows plasmid pvWF-VC3 which expresses a vWF GPIb binding domain polypeptide under the control of the deo P t P 2 promoter.
  • This plasmid has been deposited in E. coli S ⁇ 930 under ATCC Accession No. 68241.
  • This figure shows plasmid pvWF-VCL which is under control of the ⁇ P L promoter and the deo ribosomal binding site and expresses the same vWF GPIb binding domain polypeptide as pvWF-VC3.
  • This plasmid has been deposited in E.coli 4300 (F") under ATCC Accession No. 68242.
  • This figure shows the translated sequence of the von Willebrand Factor GPIb binding domain polypeptide expressed by plasmids pvWF-VC3 (ATCC Accession No. 68241) and pvWF-VCL (ATCC Accession No. 68242) .
  • the sequence consists of 226 amino acids including an N-terminal methionine.
  • the nucleotide and amino acid numbering starting from 1 are shown in the margins. Met 1 is the initiator methionine.
  • the sequence Leu 2 - Lys 226 is identical to the sequence Leu 304 - Lys 728 of von Willebrand factor as shown in Figure 12 of International Application Publication No. WO91/13093.
  • ACT Activated clotting times
  • FIG. 11 Platelet Aggregation Following VCL Treatment
  • the present invention is directed to a method of enhancing a thrombolytic treatment in a subject comprising administering to the subject, in conjunction with a thrombolytic agent and an anticoagulant, an amount of a von Willebrand factor glycoprotein lb binding domain polypeptide effective to enhance the thrombolytic treatment.
  • Enhancing a thrombolytic treatment is hereby defined as shortening the time to thrombolysis and reperfusion, and reducing the incidence of reocclusion following thrombolysis.
  • the thrombolytic agent may be any thrombolytic agent known to those skilled in the art.
  • thrombolytic agents include tissue plasminogen activator (tPA) and streptokinase. It is also envisaged that the combination treatments of the invention may also be used in conjunction with other known methods (e.g. angioplasty) of obtaining revascularization of blocked blood vessels and of maintaining blood vessel patency.
  • the thrombolytic treatment includes, in addition to the thrombolytic agent, other pharmaceutical substances e.g. anticoagulants such as heparin and aspirin separately or in conjunction. Other anticoagulants known to the skilled artisan administering the thrombolytic treatment may also be used.
  • anticoagulants such as heparin and aspirin separately or in conjunction.
  • Other anticoagulants known to the skilled artisan administering the thrombolytic treatment may also be used.
  • the polypeptide is administered intravenously.
  • the intravenous administration is a bolus, continuous infusion, or bolus followed by continuous infusion.
  • the polypeptide is administered intravenously as a bolus of 0.4-40 mg/kg body weight.
  • the polypeptide is administered intravenously as a bolus of 1-20 mg/kg body weight.
  • the polypeptide is administered intravenously as a bolus of 2-10 mg/kg body weight.
  • the polypeptide is administered intravenously by continuous infusion at a rate of 0.2-20 mg/kg body weight per hour.
  • the polypeptide is administered intravenously by continuous infusion at a rate of 1-10 mg/kg body weight per hour.
  • the polypeptide is administered intravenously as a bolus of 0.4- 4 mg/kg body weight followed by continuous infusion of 0.2- 20 mg/kg body weight per hour.
  • the invention relates to situations in which occurrence of thrombosis may be foreseen, such as during surgery, for example cardiovascular surgery.
  • pretreatment with the vWF GPIb binding domain polypeptide may reduce the occurrence of thrombosis.
  • the invention provides a method for preventing complications following traumatic vascular damage in a subject comprising administering to the subject an amount of a vWF GPIb binding domain polypeptide in conjunction with an amount of aspirin, which together are effective to prevent the complications.
  • the cause of the traumatic vascular damage is exemplified by but not limited to coronary artery bypass surgery, angioplasty, thrombolysis, or unstable angina. Traumatic vascular damage caused by other therapeutic or clinical occurrences is also encompassed.
  • the complications resulting from the traumatic vascular damage are exemplified by but not limited to myocardial infarction, ischemia, coronary bypass surgery, repeat angioplasty, or death. Other complications resulting from traumatic vascular damage are also encompassed.
  • the effective amount of the vWF GPIb binding domain polypeptide is in the range of about 0.1-20 mg/kg bolus and 0.1-20 mg/kg/hr continuous infusion of and the effective amount of aspirin is in the range of about 0.1-50 mg/kg.
  • the precise dosages will be readily determined by one skilled in the art based on the details of the case in treatment.
  • vWF GPIb binding domain polypeptide may be administered by any clinically appropriate means known to one skilled in the art; intravenous administration is a presently preferred embodiment.
  • the aspirin may be administered by any clinically acceptable means such as orally or parenterally and may be administered in a single dose, in multiple doses or by continuous administration.
  • Parenteral adminisration refers to intravenous, intraperitoneal, subcutaneous or intramuscular administration.
  • the glycoprotein lb (GPIb) binding domain of von Willebrand factor may be obtained from a variety of sources such as from naturally occurring von Willebrand factor or by recombinant protein production, e.g. in bacteria, fungi, plant, insect or mammalian cells.
  • von Willebrand factor glycoprotein lb binding domain polypeptide also encompasses a homolog of the GPIb binding domain which may be used in the methods of the invention with the proviso that the homolog has GPIb binding activity.
  • a homolog of the polypeptide is a polypeptide which has substantially the same amino acid sequence and substantially the same biological activity as such polypeptide.
  • a homolog may differ from the polypeptide of the invention by the addition, deletion, or substitution of one or more non-essential amino acid residues, provided that the resulting polypeptide retains the biological activity of the polypeptide.
  • homologs of the polypeptide of the subject invention are deletion homologs containing less than all the residues specified in the subject polypeptide, substitution homologs wherein one or more residues specified are replaced by other residues, and addition homologs wherein one or more amino acids residues is added to a terminal or medial portion of the polypeptide, all of which homologs share the biological activity of the polypeptide of the subject invention.
  • Substantially the same amino acid sequence is herein defined as encompassing the addition or deletion of fewer than four amino acids at the N-terminus of the amino acid sequence of the polypeptide.
  • substitutions and/or deletions in the sequence which do not eliminate the biological activity of the protein. Examples of substitutions are ser for cys and ala for gly. Other substitutions are known to those skilled in the art. Substitutions may encompass up to 10 residues in accordance with the homologous or equivalence groups described by e.g. Lehninger, Biochemistry. 2nd ed. Worth Pub., N.Y. (1975); Creighton, Protein Structure, a Practical Approach. IRL Press at Oxford Univ. Press, Oxford, England (1989) ; and Dayhoff, Atlas of Protein Sequence and Structure 1972, National Biomedical Research Foundation, Maryland (1972) .
  • Substantially the same biological activity refers to biological activity the same as that of the polypeptide possibly differing slightly in degree or level which would still be known by the skilled artisan to be the same biological activity.
  • von Willebrand factor glycoprotein lb binding domain polypeptide also encompasses various mutants and variants of the vWF GPIb binding domain having different amino acid sequences, but having substantially the same biological activity.
  • the biological activity of the polypeptides encompassed by the invention is the ability to bind to glycoprotein lb of platelets or to prevent platelet adhesion to subendothelial matrix.
  • plasmids and hosts for production of recombinant polypeptides comprising the vWF GPIb binding domain are plasmids pvWF-VC3 in Escherichia coli S ⁇ 930 and pvWF-VCL in Escherichia coli 4300(F) which were deposited on February 26, 1990, pursuant to, and in satisfaction of, the re ⁇ quirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 68241 and 68242 respectively.
  • ATCC American Type Culture Collection
  • Example 1 Production and Purification of Oxidized. Folded and Biologically Active vWF GPIb Binding Domain Polypeptide
  • Plasmid pvWF-VCL ( Figure 2) was constructed as described in coassigned, copending patent application U.S.S.N. 753,815, filed September 3, 1991 and is maintained in E.coli strain A4300(F " ) under ATCC Accession No. 68242.
  • This host/plasmid system was fermented essentially as known in the art for vectors harboring a gene expressed under control of the ⁇ P L promoter, i.e. growth at 30°C until mid-log stage followed by induction at 42°C for about two hours, essentially as described in U.S. Patent No. 5,126,252. Following fermentation and induction, the growth medium was centrifuged and a cell cake obtained which was stored frozen until further processing.
  • the resulting pellet containing the inclusion bodies is dissolved at about 10% w/v in a solution such that the final concentrations are 8M urea, 20mM DTT, 20mM HEPES pH 8, and lOOmM NaCl.
  • the solution may be further purified by ion exchange ch.romatography as described below.
  • the inclusion bodies may be solubilized in a buffer containing 6M guanidine hydrochloride followed by buffer exchange to urea.
  • the inclusion bodies may also be dissolved at different concentrations of urea, guanidine hydrochloride or any other denaturant or in the absence of denaturants, for example, at extremes of pH.
  • This step eliminates most of the contaminants and produces the vWF GPIb binding domain polypeptide at greater than 90% purity.
  • Any cation exchange method e.g. carboxymethyl
  • CM-Sepharose fast flow (Pharmacia) chromatography may be used in this step such as CM-Sepharose fast flow (Pharmacia) chromatography.
  • the functional group may be carboxymethyl, phospho group or sulphonic groups such as sulphopropyl.
  • the matrix may be based on inorganic compounds, synthetic resins, polysaccharides, or organic polymers; possible matrices are agarose, cellulose, trisacryl, dextran, glass beads, oxirane acrylic beads, acrylamide, agarose/polyacrylamide copolymer (Ultrogel) or hydrophilic vinyl polymer (Fractogel) .
  • the polypeptide is loaded onto a CM-Sepharose FF column equilibrated with 8M urea, ImM DTT, 20mM HEPES pH 8, lOO M NaCl. Pure polypeptide elutes in 8M urea, ImM DTT, 20mM HEPES pH 8 and 200mM NaCl. Up to about 30 OD 280 units of solubilized inclusion bodies may be loaded per ml CM-Sepharose FF. At this ratio the eluted polypeptide typically has a concentration of 4-5 OD 280 /ml.
  • the polypeptide solution eluted from the cation exchange step above is treated with 6M guanidine hydrochloride (GuCl) to disrupt any aggregates.
  • the polypeptide is then diluted to 0.05 OD 2 g 0 /ml in 2M GuCl, pH 5-11, preferably 20mM HEPES pH 8, O.lmM GSSG (glutathione, oxidized form) . This mixture is allowed to stand overnight at room temperature.
  • the phproducts are analyzed by gel filtration on fast protein liquid chromatography (FPLC) such as Superose 12 before proceeding.
  • FPLC fast protein liquid chromatography
  • Oxidations may also be performed in urea instead of in GuCl, or in any other denaturant or in the absence of denaturants under appropriate buffer conditions in which, for example, pH, ionic strength, and hydrophobicity are varied.
  • the preferred concentration of urea is in the range 0.5M to 10M, preferably 4M, and the preferred oxidant is GSSG in the range O.OlmM to 5mM preferably O.lmM.
  • Other oxidants such as Cud, may be used or alternatively no oxidant may be added, thereby utilizing air oxidation only.
  • 4M urea is the presently preferred solution for the oxidation step.
  • the filtrate is quite clear as the material is relatively clean and most of the contaminants are large enough not to pass through the 30K membrane. It is thus possible to reuse the filtrate for performing oxidations. No difference in the oxidation products of oxidations performed in reused versus freshly prepared 2M GuCl was detectable by FPLC analysis. E . Dialysis
  • dialysis against 20mM HEPES pH8, lOOmM NaCl is performed in dialysis tubing with 2-3 changes of buffer, or alternatively by diafiltration against the same buffer in a tangential flow ultrafiltration system with a 10K MW cutoff membrane.
  • the yield of correctly oxidized monomer can be greatly increased by further recovery of correctly oxidized monomer from the precipitate.
  • the solution containing the correctly oxidized monomer is clarified by centrifugation.
  • the supernatant containing the correctly oxidized monomer is saved.
  • the pellet containing disulfide - linked dimers and reduced and incorrectly oxidized monomer is treated with DTT to reduce disulfide bonds. Accordingly, the pellet is dissolved in a minimal volume of 6M GuCl, 20ml HEPES pH 8, 150mM NaCl, 20mM DTT.
  • the solution is passed through Sephadex G25 in a buffer similar to the dissolution buffer but containing only ImM DTT (instead of 20mM) .
  • the combined supernatant of the dialysate of steps E and F is concentrated by binding to CM Sepharose in 20mM HEPES pH8, lOO M NaCl. Elution is with 20mM HEPES pH8, 400mM NaCl . The eluate is exclusively monomeric despite the high salt concentration. Concentrations of up to 3 mg/ml have been achieved by this method and that is not the upper limit.
  • This step can alternatively be performed with Heparin-Sepharose which also binds the purified monomer in lOmM Tris pH 7.4, 150mM NaCl. Elution from Heparin- Sepharose is performed using lOmM Tris-HCl pH 7.4, 500mM NaCl.
  • the product of the previous step is dialyzed against 20mM HEPES pH8, 150mM NaCl.
  • the purified vWF GPIb binding domain polypeptide may be lyophilized.
  • the resultant solution contains exclusively monomeric protein showing no traces of dimers or other multimers on FPLC.
  • the protein was loaded onto a CM Sepharose column equilibrated with 8M urea, ImM DTT, 20mM HEPES pH 8, lOOmM NaCl.
  • the protein eluted at 200mM NaCL in 8M urea, 20mM HEPES pH 8, ImM DTT, and was saved.
  • Oxidation was performed overnight at room temperature.
  • VCL purified vWF GPIb binding domain polypeptide
  • VCL VCL migrated under non-reducing conditions at a lower apparent molecular weight than under reducing conditions (?- mercaptoethanol) .
  • This shift from compact to less compact configuration is consistent with the reduction of a disulfide bond.
  • Such an intramolecular bond is formed between the cysteines at positions 509 and 695. (The shift in molecular weight is not large enough to be consistent with the reduction of an intermolecular bond.)
  • Example 2 Use of the vWF GPIb Binding Domain in Conjunction with Thrombolytic Treatment to Prevent Reocclusion
  • the ability of a GPIb binding domain polypeptide fragment of vWF to enhance thrombolysis by shortening the time to reperfusion and to prevent reocclusion following thrombolysis was studied.
  • the GPIb binding domain fragment tested was the VCL polypeptide described in Example 1.
  • a canine model was evaluated for the effect of VCL on (l) the formation of intracoronary thrombosis, (2) the duration of thrombolysis, and (3) the incidence of reocclusion of coronary arteries after thrombolysis with t-PA. The results suggest that VCL delays thrombus formation, shortens the duration of thrombolysis, and reduces the incidence reocculsion of coronary arteries following thrombolysis.
  • thrombus formation is induced in a coronary artery by electrical stimulation. Following thrombus formation, thrombolysis is induced by infusion of tissue plasminogen activator (tPA) .
  • tissue plasminogen activator tPA
  • the effect of the GPIb binding domain polypeptide in preventing thrombus formation and in reducing the incidence of reocclusion following thrombolysis was tested.
  • the GPIb binding domain polypeptide proved effective in delaying onset of electrically stimulated thrombus formation.
  • the GPIb binding domain polypeptide shortened the time to reperfusion during thrombolysis and reduced the incidence of reocclusion when used in the thrombolytic therapy in conjunction with heparin and aspirin.
  • Plastic catheters were placed in a carotid artery for monitoring aortic pressure and in a jugular and a peripheral vein for drug and fluid administration.
  • a left fifth intercostal space thoracotomy was performed, and the heart suspended in a pericardial cradle.
  • a 1- to 2-cm segment of left anterior descending coronary artery was carefully exposed and nearby branches ligated.
  • An ultrasonic Doppler flow probe (Hartley Instruments, Houston, TX) was placed around the proximal portion of the exposed left anterior descending coronary artery to measure the velocity of blood flow.
  • Baseline hemodynamics including heart rate, systolic and diastolic aortic pressures and phasic and mean coronary blood flow velocities were recorded on an eight-channel recorder (Gould, Model 3000, Cleveland, OH) .
  • a needle electrode (the 8-mm tip of a 25-gauge needle crimped on the end of a l0-cm length of 30-gauge Teflon- insulated silver wire) was inserted obliquely approximately 4 mm into the lumen of the exposed left anterior descending coronary artery at a site distal to the Doppler flow probe.
  • the needle was stabilized on the vessel with 6-0 silk suture.
  • heat-shrink tubing was applied to the needle/wire and soldered connection.
  • a ground wire was connected to the subcutaneous tissue to complete the electrical circuit.
  • Thrombus formation was determined by the reduction of coronary blood flow velocity, which was monitored by the externally positioned Doppler flow probe. The electric current was maintained until 30 min after persistent thrombotic occlusion had occurred.
  • the amount of time elapsed from the beginning of electrical stimulation to the total occlusion of the coronary arteries was recorded.
  • This treatment was intended to induce lysis of the thrombi formed in the coronary arteries.
  • a thrombus was considered to be lysed (and the artery reperfused) when the flow velocity of the coronary artery returned to at least 70% of the value that existed before thrombus formation.
  • the amount of time elapsed from t-PA administration to reperfusion was recorded as thrombolysis or reperfusion time. Dogs in whom reperfusion had not occurred after 90 min of t-PA infusion were excluded from further study. Dogs in whom reperfusion did occur were further monitored until the coronary arteries reoccluded or until 180 min had elapsed without reocclusion. The time from reperfusion to reocclusion was recorded as reocclusion time. Dogs in whom coronary arteries had not reoccluded after 180 min of reperfusion were considered not to have reoccluded. Dogs in whom reocclusion did occur were monitored for 30 min to verify persistent reocclusion.
  • Hematocrit was checked before and at the end of t-PA administration in all dogs in protocol 2.
  • Activated whole blood clotting time was measured in these dogs before and 5, 60, 120, and 180 min after the administration of t-PA on an automated blood coagulation timing device (HemoTec 2001370, Englewood, CO) .
  • Ex vivo platelet aggregation was analyzed before and 10 min after the administration of VCL and aspirin in dogs in protocol 1. Blood samples were collected in plastic tubes containing a 3.8% solution of sodium citrate (9 volumes blood:1 volume sodium citrate). Platelet-rich plasma was obtained by centrifuging blood samples at 200 x g for 20 min and platelet-poor plasma was obtained by centrifuging the residual blood at 3000 x g for 10 min.
  • the platelet count in platelet-rich plasma was adjusted to 300,000/mm 3 .
  • a four-channel platelet aggregometer (Bio-Data, Model PAP-4, Horsham, PA) was used for the assay.
  • Agonists and their final concentrations were ADP (Sigma, St. Louis, MO) at 5, 10, and 20 ⁇ M; botrocetin (produced as described by Fujimura et al., Biochemistry !_:1957-1964 (1991)) at 1.1, 2.2, and 4.4 ⁇ g/ml; and arachidonic acid (Sigma, St. Louis, MO) at 12.5, 25, and 50 ⁇ g/ml.
  • Insertion of the electrode needle into the coronary artery caused some stenosis in the arteries of all animals, as determined by a reduction of blood flow velocity to approximately 65% of the baseline level (Tables 1 and 2 ) .
  • all animals developed total occlusion of the affected coronary arteries.
  • protocol 1 the elapsed time from electrical stimulation to total occlusion of the coronary arteries was significantly longer in dogs treated with VCL (p ⁇ 0.001) and aspirin (p ⁇ 0.05) than in dogs treated with saline ( Figure 4) .
  • aortic blood pressure and heart rate changed slightly after the occlusion of coronary arteries.
  • heart rate and mean aortic pressure increased at each time-point (Table 1) .
  • AOM mean aortic pressure
  • HR heart rate
  • MNFLO mean flow velocity in the coronary artery
  • PHFLO phasic flow velocity in the coronary artery
  • t-PA tissue-type plasminogen activator
  • AOM mean aortic pressure
  • HR heart rate
  • MNFLO mean flow velocity in the coronary artery
  • PHFLO phasic flow velocity in the coronary artery
  • t-PA tissue-type plasminogen activator.
  • dogs were not pretreated before their coronary arteries were occluded, and 3 h after the occlusion of coronary arteries, they received thrombolytic treatments: t-PA and heparin induced thrombolysis in 5 of 7 dogs (71%) ; t-PA, heparin, and VCL induced thrombolysis in 6 of 7 dogs (86%) ; t-PA, heparin, and aspirin induced thrombolysis in 7 of 8 dogs (85%) ; and t-PA, heparin,VCL, and aspirin induced thrombolysis in 8 of 8 dogs (100%) .
  • Activated clotting time was significantly prolonged immediately after t-PA and heparin administration ( Figure 10) . It returned to 1.5 times the baseline value 1 h after treatment and to just above the baseline value 3 h after treatment. The addition of aspirin or VCL, or a combination of aspirin and VCL did not affect activated clotting time.
  • Intravenous administration of VCL before electrical injury to the coronary artery significantly increased the time required for formation of an occlusive thrombus in vivo.
  • the ex vivo platelet aggregation induced by botrocetin was completely inhibited by the treatment.
  • Coronary artery reocclusion has limited the efficacy of thrombolytic therapy in patients with acute myocardial infarction (11, 12, 29-31). Most clinical trials have used adjunct treatment with antiplatelet agents (e.g. aspirin) to prevent reocclusion (32-33) .
  • antiplatelet agents e.g. aspirin
  • VCL was more effective than aspirin in the same conditions.
  • Bleeding is a common complication of thrombolytic therapy.
  • treatment with VCL, plus t-PA and heparin caused mild to moderate bleeding around the surgical incisions, and the combination of VCL, aspirin, t-PA, and heparin resulted in severe bleeding in some cases.
  • blocking platelet glycoprotein lb receptors with VCL may be effective in diminishing the formation of thrombi in injured coronary arteries.
  • VCL is comparable or superior to aspirin as an adjunctive treatment with t-PA and heparin. Treatment with VCL and aspirin, in addition to t-PA and heparin, may completely prevent reocclusion.
  • GPIb binding domain polypeptide was investigated using a model of traumatic vascular damage.
  • This model is essentially that described by Kelly et al. (34) .
  • segments of 2 mm collagen coated polytetrafluoroethylene (e-PFTE) tubes are inserted as extension pieces into chronic arteriovenous access shunts in baboons.
  • the baboons are injected with autologous •• 'in- labeled platelets and then administered one of the treatments being studied. At specified times, the amount of deposited platelets was determined with a scintillation camera.
  • the first set of studies was directed to the effect of the GPIb binding domain polypeptide alone in the model.
  • the reference for this study was the lowest dose from a dose response study previously performed, i.e. 1 mg/kg bolus and 2 mg/kg/hr continuous infusion for one hour. As seen in Figure 13, this dose delayed the occlusion of the tubes to about 60 minutes compared to about 20 minutes in controls.
  • the second set of studies was directed to determining the effect of aspirin alone in the model. Three animals were examined. 35 mg/kg aspirin were given orally two hours before the study. All specimens occluded less than 30 minutes into the study. Qualitatively and quantitively, the aspirin curves were found to be similar to the control curves obtained using pure non-anticoagulated blood. Platelet counts were similar in both groups.
  • the final set of experiments included study of the activity of the GPIb binding domain polypeptide in the presence of aspirin. Three animals were studied. Animals were given 35 mg/kg aspirin orally two hours before the study. The GPIb binding domain polypeptide dose used was 1 mg/kg bolus and 2 mg/kg/hr continuous infusion for one hour.
  • the synergistic effect of the GPIb binding domain polypeptide and aspirin on platelet deposition was found to be equivalent to a four-fold higher dose of 4 mg/kg bolus plus 8 mg/kg/hr continuous infusion of the GPIb binding domain polypeptide when administered alone.
  • bleeding times were measured in the experiments described above. They are presented in Table 3. Bleeding times increased in a dose dependent manner, from 6 minutes at the lowest dose of the GPIb binding domain polypeptide to 22.5 minutes at the highest dose of the GPIb binding domain polypeptide. Aspirin alone resulted in a bleeding time of 6 minutes. The combination of aspirin plus the low dose of the GPIb binding domain polypeptide resulted in a slight increase to 7 minutes. However, this combination had the anti-platelet deposition effect of a dose of the GPIb binding domain polypeptide four times higher (4 mg/kg bolus and 8 mg/kg/hr infusion) with a bleeding time of 16.5 minutes. Thus, combination of the GPIb binding domain polypeptide with aspirin resulted in a dramatic increase in efficacy without a concomitant increase in bleeding time.

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Abstract

L'invention se rapporte à un procédé d'utilisation d'un fragment du Facteur von Willebrand (vWF) comprenant le domaine de liaison de la glycoprotéine plaquettaire Ib (GPIb), utilisé en association avec des thérapies connues afin d'obtenir une protection améliorée contre la thrombose, une thrombolyse améliorée et une probabilité réduite de réocclusion succédant à un traitement thrombolytique. En outre, un procédé d'utilisation du fragment de vWF et d'aspirine pour prévenir des complications résultant de lésions traumatiques vasculaires est également décrit.
EP94915405A 1993-04-23 1994-04-22 Procede d'amelioration de thrombolyse Withdrawn EP0701606A4 (fr)

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PCT/US1994/004418 WO1994025582A1 (fr) 1993-04-23 1994-04-22 Procede d'amelioration de thrombolyse

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DE4437544A1 (de) * 1994-10-20 1996-04-25 Behringwerke Ag Einsatz von vWF-enthaltenden Konzentraten als Kombinationstherapie bei Therapie mit Antithrombotika und Fibrinolytika

Citations (3)

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Publication number Priority date Publication date Assignee Title
US4601895A (en) * 1983-12-23 1986-07-22 Bayer Aktiengesellschaft Delayed-action acetylsalicylic acid formulations for oral administration
WO1991013093A1 (fr) * 1990-03-02 1991-09-05 Bio-Technology General Corp. CLONAGE ET PRODUCTION DE POLYPEPTIDES HUMAINS DE DOMAINE DE LIAISON DE GPIb DE FACTEUR DE VON WILLEBRAND ET LEURS PROCEDES D'UTILISATION
WO1993016712A1 (fr) * 1992-02-26 1993-09-02 The Scripps Research Institute FRAGMENTS DE GPIbα MUTANTS ET EXPRESSION RECOMBINEE DE CES FRAGMENTS

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EP0255206B1 (fr) * 1986-05-30 1995-03-22 The Scripps Research Institute Peptides inhibant la liaison du facteur von Willebrand
WO1992006999A1 (fr) * 1990-10-17 1992-04-30 The Scripps Research Institute Fragments therapeutiques du factor de von willebrand
MX9203763A (es) * 1991-06-28 1993-08-01 Rhone Poulenc Rorer Int Polipeptidos terapeuticos a base del factor de von willebrand

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4601895A (en) * 1983-12-23 1986-07-22 Bayer Aktiengesellschaft Delayed-action acetylsalicylic acid formulations for oral administration
WO1991013093A1 (fr) * 1990-03-02 1991-09-05 Bio-Technology General Corp. CLONAGE ET PRODUCTION DE POLYPEPTIDES HUMAINS DE DOMAINE DE LIAISON DE GPIb DE FACTEUR DE VON WILLEBRAND ET LEURS PROCEDES D'UTILISATION
WO1993016712A1 (fr) * 1992-02-26 1993-09-02 The Scripps Research Institute FRAGMENTS DE GPIbα MUTANTS ET EXPRESSION RECOMBINEE DE CES FRAGMENTS

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of WO9425582A1 *
YAO ET AL: "Blockade of platelet glycoprotein Ib receptors offers better protection than cyclooxygenase inhibition in preventing coronary artery reocclusion after thrombolysis", CLINICAL RESEARCH, vol. 41, no. 2, March 1993 (1993-03-01), pages 1993, XP002085729 *

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AU677659B2 (en) 1997-05-01
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IL109383A0 (en) 1994-07-31
WO1994025582A1 (fr) 1994-11-10
ZA942818B (en) 1995-01-30
CA2159508A1 (fr) 1994-11-10
JPH08512291A (ja) 1996-12-24

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