WO1993009807A1 - Procedes d'inhibition de la thrombose par elevation des taux de proteine c activee endogene dans la circulation - Google Patents

Procedes d'inhibition de la thrombose par elevation des taux de proteine c activee endogene dans la circulation Download PDF

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Publication number
WO1993009807A1
WO1993009807A1 PCT/US1992/009978 US9209978W WO9309807A1 WO 1993009807 A1 WO1993009807 A1 WO 1993009807A1 US 9209978 W US9209978 W US 9209978W WO 9309807 A1 WO9309807 A1 WO 9309807A1
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thrombin
agent
apc
protein
composition
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PCT/US1992/009978
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English (en)
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John H. Griffin
Andras Gruber
Stephen R. Hanson
Laurence A. Harker
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The Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4833—Thrombin (3.4.21.5)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
    • A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
    • 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)
    • C12Y304/21005—Thrombin (3.4.21.5)

Definitions

  • the present invention relates to the use of agents such as thrombin that increase circulating level of activated protein C (APC) to inhibit arterial, venous, and microvascular thrombosis and thromboe bolism.
  • agents such as thrombin that increase circulating level of activated protein C (APC) to inhibit arterial, venous, and microvascular thrombosis and thromboe bolism.
  • Adverse effects of the most potent antithrombotic agents at therapeutically efficacious doses limit their use in patients and make these drugs unsuitable for the prevention of thrombosis and embolism in apparently healthy individuals.
  • the use of antithrombotic and/or thrombolytic therapy has undesired side effects, such as bleeding or reocclusion during thrombolytic treatment in myocardial infarction, bleeding or thrombosis following surgery, and thrombosis following surgery that employs grafts or other cardiovascular prosthetic devices.
  • the optimal antithrombotic therapy would have anticoagulant, antiplatelet and fibrinolytic properties simultaneously, without the attendant hazards of hemorrhage.
  • APC activated protein C
  • a thrombus is an aggregate of elements formed in the living heart or vessels from constituents of the blood in response to a thrombogenic stimulus.
  • Thrombosis the process of thrombus formation, can occur through distinct but usually interactive mechanisms.
  • Platelet aggregation occurs as a result of platelets being activated by a thrombogenic stimulus such as a vessel wall lesion.
  • Fibrin formation is the result of activation of the coagulation cascade system whose final step is typically considered the conversion of fibrinogen into fibrin by thrombin, i.e., fibrin formation.
  • the purpose of fibrin formation in thrombi and hemostatic plugs appears, in part, to be stabilization of the aggregated platelets and stabilization of the thrombus or plug.
  • venous thrombosis occurs under low flow rate conditions and has been shown to involve a combined and equivalent consumption of platelets and elements of the coagulation cascade system. Harker et al., N. Encr. J. Med. 287: 999-1005 (1972).
  • venous thrombi are typically relatively unstructured masses composed of aggregated platelets, interspersed fibrin and red blood cells; hence the name "red thrombus”. See Freiman, in He ostasis and Thrombosis: Basic Principles and Clinical Practice. 2d ed. , Coleman, et al.
  • fibrin formation is not dominant in arterial thrombosis because procoagulant material, such as thrombin, fibrin monomers, or small fibrin aggregates, is swept away from the thrombogenic focus by the rapid arterial flow before coagulation becomes fully activated and before fibrin polymerizes.
  • procoagulant material such as thrombin, fibrin monomers, or small fibrin aggregates
  • arterial thrombi are typically either composed predominantly of platelets (a "white thrombus") or are a complex structure composed of a basal or primary mass of platelets and a secondary mass of platelets and fibrin that overlays, and extends downstream from, the primary mass. See Freiman, supra .
  • arterial thrombosis at least in its early stages, can be characterized as being platelet dependent. Harker et al., in Vascular Diseases: Current Research and Clinical Application, Strandess et al., (eds.) Orlando, Grune & Stratton, pp. 271-283 (1987) .
  • agents that affect either platelet aggregation or fibrin formation has been found to depend on the type of thrombosis being treated.
  • agents such as aspirin or ticlopidine that inhibit platelet function i.e., inhibit the ability of platelets to aggregate
  • agents such as heparin that inhibit the ability of thrombin to form fibrin have been shown to be therapeutically effective against stasis-type venous thrombosis but not arterial thrombosis. Harker et al., Thro b. Diat .
  • thrombin a potent inducer of thrombosis via its clotting of fibrinogen and its activation of platelets, could be administered at low doses over a prolonged period of time in the presence of a thrombus or a thrombogenic stimulus to achieve a significant antithrombotic effect.
  • thrombin functions as an effective antithrombotic agent and a potent stimulator of endogenous APC levels, particularly in primates, including man.
  • agents that are capable of upregulating the plasma level or generation of APC can be used for the treatment or prevention of thrombosis and thromboembolism.
  • agents include thrombin, active site acylated-thrombin, or thrombin-like enzymes; soluble thrombin:thrombomodulin complex or its analogs; agents that would prevent clearance or decay of thrombin:thrombomodulin complexes; peptides that selectively interfere with inhibition of APC; factor Xa or its analogs; plasmin or trypsin analogs; venoms (e.g., protac, Russel Viper venom [RW]); recombinant or plasma-derived PC or its analogs, agents that enhance PC synthesis or thrombomodulin concentration; specific antibodies against PC inhibitors; and agents that delay clearance of PC zymogen, to name a few examples.
  • thrombin active site acylated-thrombin, or thrombin-like enzymes
  • the present invention contemplates various methods for treating and inhibiting thrombosis and/or thromboembolism formation in a patient.
  • One such method comprises administering to the patient an effective amount of an agent capable of increasing the blood activated protein C level in the patient.
  • the agent comprises a serine protease capable of activating protein C via enzymatic cleavage.
  • An example of such a protease is thrombin, including human thrombin.
  • the thrombin is selected from a group comprising plasma- derived or recombinant ⁇ -thrombin, thrombin E192Q, thrombin K52E, other naturally-occurring or mutated thrombins, and active site acylated-thrombin.
  • Other appropriate agents capable of increasing the activated protein C level include thrombin analogs, Protac (a snake venom PC activator) , factor Xa, venoms, or any enzyme — natural or synthetic — that is capable of activating protein C.
  • the effective amount of agent administered is that sufficient to inhibit arterial and/or venous thromboembolic events.
  • the agent is administered intravenously, via bolus injection or via continuous infusion, for as long a period as is efficacious.
  • the present invention further contemplates that an effective amount of thrombin, and more particularly ⁇ -thrombin, is administered in the dosage range of about 0.05U/kg/min (approximately 17 ng/kg/min) to about 2U/kg/min (approximately 0.666 ⁇ g/kg/min) .
  • the contemplated effective amount of thrombin is sufficient to achieve an APC concentration in the blood 3-5 times the normal level of APC, or more.
  • the effective amount of thrombin is sufficient to achieve an APC concentration in the blood of at least about 10 ng/ml.
  • the effective amount of thrombin is sufficient to maintain the activated protein C concentration for a time period of at least about 30 minutes.
  • an effective amount of thrombin is sufficient to achieve an activated protein C concentration in the blood of from 10 to 760 ng/ml.
  • the amount of thrombin is administered over a period of at least 20 minutes.
  • the effective amount of thrombin is sufficient to achieve an activated protein C concentration in the blood three to five times the normal level of activated protein C, or higher.
  • Yet another variation contemplates the administration of an antithrombotic agent in addition to an agent capable of increasing the activated protein C level in the blood.
  • the antithrombotic agent may be selected from the group comprising fibrinolytic agents, oral anticoagulants, dextrans, pentoxifilline, snake venom, soluble thrombomodulin, antiplatelet drugs, cyclooxygenase inhibitors, cAMP modulators, ticlopidine, thrombolytic agents, streptokinase, Eminase, urokinase, tissue plasminogen activator, anticoagulant peptides, protein C, and activated protein C.
  • the present invention also contemplates a therapeutic method in which blood of a patient circulates through a prosthetic device, which method comprises administering an effective amount of an agent to an individual that elevates the endogenous APC level substantially concurrently with exposing the blood to a prosthetic surface on the device.
  • substantially concurrently is defined as occurring within about 48 hours of performing a medical or surgical procedure on a patient.
  • the therapeutic method further comprises a method of inhibiting thrombosis or thrombus formation in a prosthetic device, which method comprises administering an effective amount of an agent to the patient substantially concurrently with exposing the blood to a prosthetic surface on the device, wherein the agent is capable of increasing the blood activated protein C level in the patient.
  • the prosthetic device may be selected from the following group, for example: (a) cardiopulmonary assist devices, (b) hemodialysis devices, (c) an arterial prostheses, (d) venous prostheses, (e) arteriovenous shunts, (f) metallic or polymeric endoprostheses; (g)heart valve implants or artificial heart valves, and (h) an artificial heart.
  • the present invention contemplates a method of treating surgically induced thrombosis, which method comprises administering an effective amount of an agent to a patient substantially concurrently with performing an invasive surgical procedure on the patient, wherein the agent is capable of increasing the blood activated protein C level in the patient.
  • the invention further discloses a method of performing invasive surgery on a patient, which method comprises administering an effective amount of an agent to the patient, the administration being performed during a time period of up to one hour prior to exposing the circulating blood of the patient to a prosthetic surface on a cardiopulmonary assist device, wherein the agent is capable of increasing the blood activated protein C level in the patient.
  • the agent is administered during or subsequent to the invasive surgical procedure, or both.
  • the invention contemplates a method of performing a medical procedure on a patient, which method comprises administering an effective amount of an agent to the patient, the administration being performed during a time period of up to one hour prior to initiating the medical procedure, wherein the agent is capable of increasing the blood activated protein C level in the patient.
  • the invention further comprises continuing to administer the effective amount of agent during or subsequent to the performance of the medical procedure, or both.
  • contemplated medical procedures include angioplasty, hemodialysis, treatment of heparin-induced thrombosis, use of endoprosthetic devices (e.g. stents) , use of intravascular access devices or indwelling endovascular devices (e.g. cannulas, catheters, and devices used with injections and infusions) , or thrombolytic therapy.
  • the present invention contemplates methods for determining the presence of thrombomodulin, particularly that which is active or functional, in an individual, comprising the steps of (a) obtaining a first plasma sample from the individual (which sample may be collected in one or more vials, tubes, or other appropriate vessels, or which may be divided into separate portions for testing) ; (b) determining the protein C level in a first portion of the sample; (c) determining the activated protein C level in a second portion of the sample; (d) intravenously injecting a thrombomodulin- dependent protein C activating agent into a vein of the individual for 5 minutes; (e) obtaining a second plasma sample from the individual (i.e., a sample taken subsequently to the intravenous injection of protein C activating agent; (f) determining the activated protein C level in the second sample; and (g) comparing the activated protein C levels determined for the first and second samples to determine the presence of functional thrombomodulin in the individual.
  • the protein C activating agent comprises thrombin, which may be administered at a rate of O.lU/kg/ in or more.
  • the present invention also contemplates therapeutic compositions suitable for administration via injection, comprising an effective amount of an agent capable of increasing the blood activated protein C level via enzymatic cleavage of protein C in a pharmaceutically acceptable carrier or excipient, wherein the effective amount of the agent is administered at the dosage of 0.05U/kg/min to 2U/kg/min.
  • the agent is present in a solution of up to 0.5U agent per ml of carrier; it may also be infused over a period of at least 20 minutes.
  • the agent is thrombin; in other embodiments, the agent may comprise plasma- derived or recombinant ⁇ -thrombin, thrombin E192Q, thrombin K52E, other naturally-occurring or mutated thrombins, active site acylated-thrombin, thrombin analogs, Protac (a snake venom PC activator) , factor Xa, venoms, or any enzyme —natural or synthetic — that is capable of activating protein C. It is further contemplated that active site acylated thrombin may be administered in conjunction with an anticoagulant specific for thrombin.
  • acyl-thrombin may be co-administered with one or more of such anticoagulants, or in a "cocktail” form with one/or more such anticoagulants including peptides such as polypeptides derived from prothrombin, or "PT polypeptides".
  • the present invention further contemplates various diagnostic compositions comprising an effective amount of a thrombomodulin-dependent protein C activating agent in a pharmaceutically acceptable carrier or excipient, wherein the agent is administered at a suggested dosage of 0.lU/kg/min to 2U/kg/min.
  • the agent may be present in a solution of up to 0.5U agent per ml of carrier and may be infused over a period of at least 5 minutes.
  • the agent is thrombin.
  • the present invention also contemplates the ex vivo activation of protein C via use of an external shunt apparatus in conjunction with an apparatus containing a thrombin:thrombomodulin complex, followed by reintroduction of APC into the individual.
  • the present invention also contemplates a method of decreasing the rate of clearance of endogenous APC in order to elevate the concentration of circulating APC.
  • administration of inhibitors of PCI, PAI- I, ⁇ -1-antitrypsin, and ⁇ -2-antiplasmin is suggested.
  • Yet another embodiment contemplates the inhibition of thrombosis or thro boembolism via increasing endogenous zymogen levels and/or APC levels, via administration of anabolic steroids (e.g. danazolol) , for example.
  • anabolic steroids e.g. danazolol
  • Another aspect of the present invention contemplates the administration of thrombin to an individual as a means of assaying for the presence of thrombomodulin in vivo .
  • Figure 1 illustrates a thrombogenic device in an AV shunt such as that used in the present study of arterial and venous thrombosis. It illustrates the direction (from the Dacron graft end through the Teflon chamber portion) and speed (100 ml/min) of blood flow, as well as the approximate dimensions of the graft portions (20mm in length and 4 mm i.d. for the Dacron portion; 20 mm in length and 9.3 mm i.d. for the Teflon portion) . Examples of construction materials and gamma camera range are also indicated.
  • Figure 2 illustrates the generation of circulating activated protein C during infusion of thrombin.
  • Activated protein C (APC) in nanograms (ng) /ml is plotted against time, in minutes. Mean data from the control animals (open triangles) is compared against that from animals that are receiving thrombin infusions in lU/kg/min (open squares) and 2U/kg/min (open circles) doses. In all animals, a thrombogenic device is present.
  • Figure 3 illustrates platelet deposition in the dacron graft segment in control animals and in experimental animals, during infusion of thrombin.
  • Platelets deposited are plotted against time (in minutes) . In all instances, a thrombogenic device is present. Experimental animals are receiving thrombin infusions in lU/kg/min (open squares) and 2U/kg/min (open circles) amounts. Control data is illustrated using open triangles.
  • Figure 4 illustrates platelet deposition in the teflon chamber portion of the grafts in control animals (open triangles) and in experimental animals, during infusion of thrombin. Platelets deposited (in billions) are plotted against time (in minutes) . In all instances, a thrombogenic device is present. Experimental animals are receiving thrombin infusions in lU/kg/min (open squares) and 2U/kg/min (open circles) amounts.
  • Figure 5 illustrates the deposition of fibrin in thrombogenic devices during infusion of thrombin.
  • Fibrin deposition in milligrams, mg
  • T thrombin
  • Deposition of fibrin in the dacron portion of the graft and in the teflon chamber portion is compared to the controls.
  • Figure 6 illustrates the prolongation of clotting time during infusion of thrombin.
  • Activated partial thromboplastin time (APTT, measured in seconds) is plotted against time (in minutes) for control (open triangles) and test animals.
  • APTT Activated partial thromboplastin time
  • a thrombogenic device is present.
  • Experimental animals are receiving thrombin infusions in lU/kg/min (open squares) and 2U/kg/min (open circles) amounts.
  • Figure 7 illustrates the inhibition of thrombus formation during in vivo protein C activation by thrombin.
  • Inhibition of platelet (PLT) and fibrin (FN) deposition is plotted against thrombin infusion, in U/kg/min.
  • Inhibition in the dacron and teflon chamber graft segments is illustrated.
  • Data for platelet deposition in the dacron portion is illustrated using open triangles; that for platelets in the teflon chamber portion is shown using an X-shaped mark.
  • Fibrin deposition in the dacron portion is illustrated with open circles; that for fibrin in the teflon chamber is shown using open squares.
  • Figure 8 illustrates the inhibition of thrombus formation during in vivo protein C activation by thrombin. Percent inhibition of platelet (PLT) and fibrin (FN) deposition is plotted against percent increase in activated protein C (APC) , in thousands. Inhibition in the dacron and teflon chamber graft segments is illustrated. Data for platelet deposition in the dacron portion is illustrated using open triangles; that for platelets in the teflon chamber portion is shown using an X-shaped mark. Fibrin deposition in the dacron portion is illustrated with open circles; that for fibrin in the teflon chamber is shown using open squares.
  • Figure 9 illustrates the correlation between APC levels and PC activity in 67 blood donors (each donor is indicated by a square) .
  • APC activity in percent (%) is shown and is plotted against PC activity in percent (%) , which is diagrammed on the horizontal axis.
  • r has a value of 0.61
  • p has a value of ⁇ 0.001.
  • Activated protein C refers to a member of the serine protease family subgroup involved in the blood coagulation pathway.
  • Protein C (PC) is a zymogen, that is, it is inactive until converted into APC through interaction with thrombin, another serine protease active in the blood coagulation pathway or through interaction with other proteases.
  • PC and APC differ in primary structure only in a dodecapeptide which is present at the amino-terminal end of PC and absent in APC. The 12 amino acid peptide is removed by proteolytic cleavage. The role of APC is to inactivate coagulation cofactors Va and Villa.
  • Anticoagulant refers to an agent that interrupts coagulation and thereby inhibits fibrin formation.
  • Coagulation refers to the sequential process in which the multiple coagulation factors of the blood interact resulting in the formation of fibrin.
  • Enzyme refers to a protein or polypeptide capable of accelerating or producing by catalytic action some change in a substrate for which it is often specific.
  • Prosthetic device refers to a biologic or synthetic vascular prosthesis that is inserted into the vasculature so as to receive and/or transport blood.
  • Protein as used herein refers to a protein that catalyzes the cleavage of peptide bonds in other proteins.
  • Serine protease refers to a member of a family of proteases that share an active site functional domain defined by amino acid residues Asp 102 , Ser 195 , and His 57 of chymotrypsin.
  • serine proteases include those in the complement convertase family (e.g., factors Clr, Cls, D, C3 convertase) ; those in the plasminogen activator family (e.g., plasmin, tissue plasminogen activator (tPA) , urinary plasminogen activator (uPA) ) ; those in the blood coagulation pathway family (e.g., factors Xlla, XIa, Xa, IXa, Vila, thrombin, plasma kallikrein, APC) , those in the digestive enzyme family (e.g., trypsin, chymotrypsin, pancreatic elastase, enterokinase) , those in the hormone processing family (e.g., tissue kallikreins, post proline cleaving enzyme) , and the like.
  • Serine protease is further intended to encompass all substantially homologous molecules.
  • substantially homologous means that a particular subject sequence or molecule, for example, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between reference and subject sequences.
  • amino acid sequences having greater than 90 percent similarity, equivalent biological activity, and equivalent expression characteristics are considered substantially homologous and are included within the scope of proteins defined by the terms "serine protease” and "thrombin”. Amino acid sequences having greater than 40 percent similarity are considered substantially similar.
  • Thrombin is a multifunctional serine protease. As a procoagulant enzyme, thrombin clots fibrinogen, activates clotting factors V, VIII, and XIII, and activates platelets. It is also capable of cleaving the dodecapeptide which is present at the amino- terminal end of PC and absent in APC; the 12 amino acid peptide is removed by proteolytic cleavage.
  • Thrombin is further intended to encompass all substantially homologous molecules, including the mutated thrombins known as E192Q and K52E. [See Le Bonniec, PNAS USA 88: 7371-7375 (1991) , and Wu, et al., PNAS USA 88: 6775-6779 (1991).]
  • One unit (U) of thrombin, as used herein, is generally known in the art, and means equivalent fibrinogen clotting activity to 1 N.I.H. unit of reference enzyme using the same assay (see Fenton, et al. , Thromb. Res. 4.: 809-817 (1974) .
  • Thrombin may be administered according to the present application in a pharmaceutically acceptable form. . B. Introduction
  • Activated protein C is an anticoagulant enzyme that exhibits potent antithrombotic effects when given at pharmacologic doses in venous, microvascular and arterial thrombosis models.
  • APC inhibits the blood coagulation pathways and the formation of thrombin at least in part by proteolytic cleavage of coagulation cofactors Va and Villa, and APC also enhances fibrinolysis, at least in vitro.
  • APC can be generated from its vitamin K-dependent zymogen, protein C (PC) , upon enzymatic activation by thrombin, thrombomodulin-bound thrombin, factor Xa bound to thrombomodulin, trypsin or various venoms, including snake venoms, in vitro .
  • PC protein C
  • APC is unique among known anticoagulants, since it circulates in blood, and it has now been shown to inhibit many kinds of thrombosis (arterial, microvascular, and venous) at pharmacologic doses that do not compromise primary hemostasis.
  • thrombosis arterial, microvascular, and venous
  • antithrombotic therapy with purified plasma-derived APC or recombinant APC requires infusion of considerable quantities (>0.07 mg/kg/h) of enzyme to reach therapeutically efficacious plasma levels of the active enzyme in arterial-type thrombosis.
  • the parenteral route of administration requires close supervision by medical personnel.
  • APC is a member of the serine protease family subgroup involved in the blood coagulation pathway.
  • Protein C is a zymogen, that is, it is inactive until converted into APC through interaction with thrombin, another serine protease active in the blood coagulation pathway.
  • PC and APC are structurally different only in a dodecapeptide which is present in PC and absent in APC.
  • the 12 amino acid peptide is removed by proteolytic cleavage, and such proteolytic cleavage permits the acquisition of enzymatic activity by APC.
  • One major role of APC is to inactivate coagulation cofactors Va and Villa. Therefore, APC can down-regulate thrombosis through its activity on factors Va and Villa.
  • APC is inactivated by the protease inhibitors, ⁇ -1-anti-trypsin, plasminogen activator inhibitor-1, ⁇ -2-antiplasmin, ⁇ -2- macroglobulin, and possibly by other nonspecific prote
  • the level of APC and/or PC in a body fluid sample has medical relevance.
  • the incidence of hereditary PC and protein S deficiency among thrombophilic patients [Gladson et al., Thromb. Haemost. 59: 18-22 (1988)] is higher than in the normal population [Miletich et al., N. Engl. J. Med. 317: 991-996 (1987) ] and many patients have been described with heterozygous PC deficiency and familial thrombophilia [Griffin et al. , J. Clin. Invest. 68: 1370-1373 (1981); Horellou et al. , Br. Med. J.
  • Thrombotic complications of PC deficiency can be controlled with PC or APC replacement therapy (Seligsohn et al., Taylor et al., and Snow et al., supra) or liver transplantation [Casella et al., Lancet 1: 435-437 (1988)].
  • PC or APC replacement therapy Steligsohn et al., Taylor et al., and Snow et al., supra
  • liver transplantation Thrombotic complications of PC deficiency
  • the presence of measurable quantities of APC-inhibitor complexes in plasma samples from patients with intravascular coagulation indicates that APC is generated in vivo [Heeb et al., Blood 73: 455-461 (1989); Tabernero et al., Thromb. Haemost. 63: 380-382 (1990)].
  • thrombin a natural activator of the PC zymogen, reasoning that thrombin, by combining with its endothelial cofactor, thrombomodulin (TM) , would generate active circulating APC enzyme in non-human primates with experimental thrombosis. We then hoped to observe whether induced APC at levels significantly higher than normal concentrations in plasma would potently inhibit both venous-type and arterial-type thrombus formation in thrombogenic devices and the vascular tree.
  • TM endothelial cofactor
  • the method of inhibiting thrombosis disclosed herein, which utilizes the body's own, endogenous protein C pathway, is an entirely new therapeutic modality. Its clinical significance is comparable to the introduction of plasminogen activators (streptokinase, urokinase, tissue-type plasminogen activator, streptokinase-plasminogen complexes, etc.) in the treatment of thrombotic vascular occlusions, since those approaches also utilize an endogenous antithrombotic pathway, the fibrinolytic pathway of the body.
  • plasminogen activators streptokinase, urokinase, tissue-type plasminogen activator, streptokinase-plasminogen complexes, etc.
  • Arterial prosthetic devices having surfaces exposed to arterial blood when operatively inserted into a patient's circulation are well known in the art. See, Biologic and Synthetic Vascular Prostheses, J. Stanley, ed., Grune and Stratton, N.Y. (1982).
  • Exemplary biological arterial prostheses include, without limitation, autologous arterial grafts, particularly autologous saphenous vein arterial grafts, dialdehyde starch-tanned bovine heterografts, human umbilical vein grafts and the like.
  • Synthetic arterial prostheses are also well known in the art and include Dacron grafts, expanded polytetrafluoroethylene grafts such as those described in U.S. Patent No. 3,962,153, hydrophobic polymer- lined grafts such as those described in the U.S. Patent No. 4,687,482, and the like.
  • Exemplary arterial prosthetic surfaces include arterial stents, A-V shunts, and the like.
  • A-V shunts are typically sections of non-endothelialized tubing, usually constructed of a polymeric material, that are used to transport arterial blood in a vein, either directly or first through and ex vivo therapeutic device.
  • Exemplary ex vivo therapeutic devices include hemodialysis and cardiopulmonary assist devices, and the like. The use of ex vivo therapeutic devices is well known in the art.
  • a thrombo-resistant vascular prosthesis of the present invention is produced by a method comprising removably affixing an endogenous APC-level-increasing agent onto the luminal surface of the prosthesis.
  • removably affixing such an agent to the luminal surface of a vascular prosthetic device is a method for improving the thrombo-resistance of that device.
  • Acute arterial thrombotic occlusion including coronary, cerebral or peripheral arteries.
  • b Acute thrombotic occlusion and restenosis after angioplasty.
  • Angioplasty a commonly performed procedure in cardiology and vascular medicine, disrupts intimal surfaces of diseased arteries and may cause acute occluding thrombosis. It also may frequently produce restenosis several months later, due to platelet-mediated intimal thickening. These complications represent a major unsolved problem in cardiology.
  • Thrombolytic agents such as tissue plasminogen activator (tPA) , streptokinase, or Eminase salvage ischemic tissue when used within hours of acute heart attack or stroke by re-establishing blood flow in the occluded artery.
  • tPA tissue plasminogen activator
  • streptokinase streptokinase
  • Eminase salvage ischemic tissue when used within hours of acute heart attack or stroke by re-establishing blood flow in the occluded artery.
  • Endogenous APC should have greater efficacy than heparin in preventing reocclusion because it inhibits arterial thrombosis.
  • Small caliber vascular graft occlusion vascular graft occlusion.
  • Vascular grafts of small caliber, e.g., 3mm diameter have a high frequency of thrombotic occlusion.
  • Endogenous APC alone or in combination with other antithrombotic agents could be a useful agent to prevent occlusion.
  • Endogenous APC could be used in other instances of arterial thrombosis or thromboembolism where other applied therapeutic measures (e.g. heparin, aspirin, thrombolytic agents, etc.) are either contraindicated or ineffective.
  • exogenous APC could improve the results in the treatment of the following conditions, for example: acute pre- or post-capillary occlusion, including transplantations, retinal thrombosis, heparin-induced thrombosis, microthrombotic necrosis of any organ, complicating infections, or tumors, thus possibly reducing the medical and social costs.
  • heparin is only partially effective, thereby limiting the reuse of dialyzers.
  • heparin has a number of troublesome side effects and complications.
  • Endogenous APC could replace heparin or complement its beneficial effects.
  • Cardiopulmonary bypass surgery To prevent thrombus formation in the oxygenator and pump apparatus, heparin is currently used. However, it fails to protect against platelet activation and the resultant transient platelet dysfunction which predisposes bleeding problems post-operatively.
  • the antithrombotic effects of endogenous APC in the absence of compromised surgical hemostasis is an important aspect of the presently disclosed strategy for the use of APC induction with surgical procedures, h. Left ventricular cardiac assist device.
  • This prosthetic pump is highly thrombogenic and results in life threatening thromboembolic events — complications that are only partially reduced by use of conventional anticoagulants (e.g., heparin or coumarin-type drugs) .
  • Convention anticoagulants e.g., heparin or coumarin-type drugs
  • Heart replacement use of artificial heart.
  • This prosthetic device has four artificial valves and biomer chamber construction, which are all highly thrombogenic. Although heparin is partially effective, it fails to prevent platelet-mediated thromboembolic events. Artificial hearts could have much greater utility if the risk of thromboembolism could be reduced by using the body's own ability to produce circulating APC.
  • Deep vein thrombosis and pulmonary embolism are examples of embolism.
  • Thrombotic occlusion of the major veins and subsequent embolism are commonly occurring conditions with relatively high acute mortality. Although other anticoagulants and fibrinolytic agents are partly effective, they do not substantially reduce the mortality of pulmonary embolism. Postthrombotic syndrome still occurs, even after and during conventional antithrombotic therapy. Induction of the PC pathway proximal to the thrombosis, i.e., on the endothelial microvascular surface, could reduce the damage caused by venous occlusion by releasing the powerful endogenous anticoagulant right at the site it is needed. k. Microvascular thrombosis.
  • Thrombotic occlusion of the precapillary, capillary and postcapillary system results in irreversible ischemic tissue necrosis unless the formation of the microthrombi are prevented or the thrombi are dissolved.
  • Tissue necrosis besides the loss of function of the necrotized tissue, can lead to generalized toxic conditions often leading to fatality.
  • Disseminated intravascular coagulation can occur without complete occlusion of microvessels but with substantial fibrin deposition in the microvessel, resulting in failure of normal gas and electrolyte exchange and metabolism.
  • Fibrin deposition and thrombotic occlusion of microvessels may occur as a result of infections, hemolysis, antigen-antibody reactions, adult respiratory distress syndrome, amniotic fluid embolism during labor and delivery, and several other conditions.
  • Traditional antithrombotic agents e.g. ,heparin, fibrinolytic agents, oral anticoagulants
  • Infused purified APC has been shown to be efficacious and to prevent fatality in toxic microthrombosis followed by experimental E. coli infection.
  • an increase in the circulating levels of endogenous APC could provide antithrombotic protection in microvascular thrombosis.
  • Agents Which May Be Combined to Produce an Increase in Endogenous APC Levels A number of agents may be administered according to the present invention, in order to achieve the desired increase in endogenous blood APC levels.
  • thrombin may be administered in combination with APC, protein C, fibrinolytic agents, and oral anticoagulants, to name a few examples.
  • agents which may be used to increase endogenous APC levels and/other agents useful in a combined therapy with agents that increase circulating APC are set forth in Table 1 below. It should be noted that some enzyme inhibitors including hirudin, PPACK, heparin, heparinoids, SP54, dermatan sulfate, and the like are not recommended for use in combination with thrombin. They may, however, be administered in combination with other agents that increase circulating APC, including protein C and APC itself.
  • Block enzyme Coagulation Enzyme inhibitors specificity factors hirudin, PPACK, heparin,
  • Antiplatelet drugs surface cyclooxygenase inhibitors (aspirin, NSAD) , cAMP modulators (dipyridamole, PGI-, and analogs) , ticlopidme
  • compositions which contain antithrombotic or anticoagulant agents as active ingredients is well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active ingredient is often mixed with inorganic and/or organic excipients which are pharmaceutically acceptable and compatible with the active ingredient (e.g., a protein C-activating or APC-increasing agent) .
  • Preferred excipients are protein carriers, such as those used in insulin formulations.
  • Suitable excipients may include, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents which enhance the effectiveness of the active ingredient.
  • composition is conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic or diagnostic composition used in the present invention refers to physically discrete units suitable as unitary dosages for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic or diagnostic effect in association with the required excipient.
  • the composition is administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's blood antithrombotic system to utilize the active ingredient, the degree of antithrombotic effect desired. Precise amount of active ingredient required to be administered depends on the judgement of the practitioner and are peculiar to each individual.
  • suitable therapeutic dosage ranges for thrombin, and particularly human ⁇ - thrombin are of the order of 0.05U to 2U per kilogram body weight per minute and depend on the route of administration.
  • Suitable diagnostic dosage ranges for thrombin are of the order of 0.05U to 2U per kilogram body weight per minute and depend on the route of administration, as well.
  • thrombin in a solution of about 0.5U/ml or less is suggested.
  • Pharmaceutically acceptable, biodegradable excipients suitable for use in slow release formulations are well known and include polymers (e.g., polyethylene glycol) , polyamino acids (e.g., polyglycolic acid) , and the like.
  • polymers e.g., polyethylene glycol
  • polyamino acids e.g., polyglycolic acid
  • the local concentration of an agent in the blood i.e. , the concentration of the agent that increases the APC level at the blood-luminal surface interface
  • the excipient dissolution rate i.e. , the concentration of the agent that increases the APC level at the blood-luminal surface interface
  • thrombin may be administered in a bolus — i.e., a more concentrated mass — as opposed to steady infusion.
  • Reversible acylation of the active center of serine proteases, e.g. trypsin, plasmin and thrombin has been shown to modify the biochemical properties of the enzymes.
  • acylation of the active center of the streptokinase-plasminogen complex led to the development of a new thrombolytic agent known as APSAC (anisolated plasminogen streptokinase activator complex — which has also been referred to as Eminase or Anistreplase) , that exerts its effect by slow deacylation in the circulation.
  • APSAC anisolated plasminogen streptokinase activator complex
  • Eminase is currently used clinically for thrombolysis.
  • active site acylated-thrombin can be used as a bolus injection rather than a slow, continuous infusion in patients with thrombosis, to activate protein C.
  • a small molecular inhibitor e.g., by diisopropylfluorophosphate
  • TM endothelial receptor
  • small molecular weight active site inhibitors do ablate thrombin's interaction with its substrates, e.g., fibrinogen, coagulation factors and cofactors (for example. Factors V and VIII) , and antithrombins.
  • substrates e.g., fibrinogen, coagulation factors and cofactors (for example. Factors V and VIII)
  • antithrombins for example. Factors V and VIII
  • acyl-thrombin may be used via bolus injection for the purpose of increasing endogenous circulating APC levels with equivalent — or superior — efficacy compared to continuous infusion of thrombin, and will provide a wider therapeutic window.
  • Acylated forms of other enzymes e.g., factor Xa
  • Acyl- thrombin would preferably be administered in a dosage sufficient to achieve essentially a "controlled release" in the 0.05U-2U/kg/min range suggested for infused thrombin.
  • acyl-thrombin does not cleave fibrinogen, activate platelets or activate factors V and VIII, it is ideal not only for solo administration, but can function efficaciously when administered in conjunction with an anticoagulant specific for thrombin.
  • acyl-thrombin may be co- administered with one or more of such anticoagulants, or in a "cocktail" form with one/or more such anticoagulants.
  • anticoagulants specific for thrombin include, but are not limited to, peptides such as polypeptides derived from prothrombin, or "PT polypeptides".
  • a PT polypeptide inhibits the activity of its corresponding serine protease, prothrombin, and has anticoagulant activity.
  • a serine protease binding domain sequence is an amino acid residue sequence that defines a surface-exposed region of a native and naturally folded serine protease protein, a region that participates in an essential protein-protein interaction between the serine protease and another protein during the catalytic process of the serine protease.
  • essential protein-protein interaction is meant that if the interaction is perturbed by a competition reaction using a PT polypeptide, the catalytic activity of the serine protease is diminished or extinguished. Thus, the interaction is essential for the protease to exert its catalytic activity.
  • exosite indicates a region of a serine protease that is defined by amino acid residues which are not located at the region of the protease responsible for the catalytic activity; i.e., an exosite is not located at the catalytic site.
  • Other anticoagulant peptides may also be administered in conjunction with acyl-thrombin.
  • thrombin exerts its PC activating effect via binding to the endothelial thrombin receptor, thrombomodulin (TM) .
  • TM endothelial thrombin receptor
  • TM endothelial thrombin receptor
  • Deficiency in TM i.e., an insufficient number of these cellular receptors for thrombin, either acquired or inherited, could lead to a pathological condition characterized by a deleterious distribution of thrombin molecules between thrombomodulin and procoagulant receptors.
  • thrombin When exogenous thrombin is infused, the newly- formed thrombin:thrombomodulin complexes generate circulating APC from protein C. In cases of TM deficiency, this response to thrombin infusion will be decreased. Thus, assaying the APC increase after thrombin infusion provides a method of detecting TM functional deficiency in individuals.
  • TM active, functioning TM
  • An assay for TM may be formed essentially as follows. First, the functional PC levels of the individual to be tested for TM deficiency should be assayed and found normal; then, if necessary, they should be normalized via exogenous PC infusion. Second, blood should be drawn from the individual and assayed, using the APC ECA or some other appropriate method, to determine the baseline APC level.
  • the present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of protease in a fluid sample.
  • a diagnostic system includes, in an amount sufficient for at least one assay, a subject protease activity-free immobilized anti-protease antibody as a separately packaged immunochemical reagent. Instructions for use of the packaged reagent are also typically included.
  • the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits a protease activity-free anti-protease antibody of the present invention in an immobilized composition.
  • a package can be a microtiter plate well to which microgram quantities of a contemplated antibody have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antigen, and being protease-free.
  • Preferred are anti-APC antibodies, and particularly MabC3 in an immobilized composition.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
  • the packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.
  • the vascular prosthesis of the present invention in its preferred embodiment, is substantially non- compliant.
  • non- compliant means showing less than 10 percent expansion of the inner diameter between systole and diastole under normal arterial pressures (less than 250 m Hg) .
  • the external surface of the vascular prosthesis permits tissue anchoring upon implantation in a human or other mammals, as is common for currently commercially available prostheses.
  • the tubular segment of the vascular prosthesis may be constructed of materials that exhibit the requisite strength, durability and suturability. Commercially available materials suitable for use in fabricating the prosthesis or graft include a polyester such as Dacron (C. R. Bard, Inc., Billerica, MA) and a polyfluorocarbon such as Teflon (Gore-Tex, W. L. Gore, Flagstaff, AZ) .
  • the luminal surface is comprised of polymers that form a relatively smooth, non-polar and hydrophobic surface. Such materials and either use in forming a portion of a luminal surface as described in U.S. Patent No. 4,687,482 to Hanson, whose disclosures are incorporated herein by reference. The following examples are intended to illustrate, but not limit, the present invention.
  • a chronic arteriovenous shunt was surgically implanted between the femoral artery vein (A-V shunt) of a normal, 10-12 kilogram male baboon.
  • the permanent shunt system consisted of two 25 centimeter (cm) lengths of silastic tubing having an inner diameter (i.d.) of 3 millimeters (mm; Dow Corning Corp., Midland, MI) connected to 13- and 15- gauge Teflon vessel tips (Lifemed, Venitron Corp., Compton, CA) .
  • the two Silastic lengths were fixed with Dacron sewing cuffs (E.I. duPont de Nemours and Co., Wilmington, DE) at skin exit sites.
  • Thrombus formation was initiated by incorporation of a thrombogenic device into the shunt.
  • Figure 1 is illustrative of the device described.
  • the thrombogenic segment consisted of a Dacron vascular graft segment (2cm long, 4mm i.d.) exhibiting high- shear undisturbed blood flow, similar to arterial flow.
  • Distal to the Dacron graft was placed a Teflon- coated chamber (2cm long, 9.3mm i.d.) exhibiting low- shear annular disturbed blood flow and stasis, which more closely mimics venous flow.
  • the procedures and the model have been extensively described and tested for reproduceability. (See, e.g., Hanson, et al., Thromb. Haemost.
  • Blood passed into the arterial end of the device at a flow rate of about 100 ml/min. Turbulence generated in the venous portion of the device was observed to simulate that of an actual vein.
  • Both graft- and chamber-induced acute thrombosis was indicated by continuous deposition of platelets and fibrinogen from the circulation onto the thrombogenic surfaces. This device generated two types of thrombi during perfusion. The proximal Dacron segment accumulated platelet-rich arterial-type thrombi, while the distal chamber accumulated erythrocyte and fibrin-rich thrombi. [See also
  • the blood flow in the shunt and device was detected using a Doppler flow meter. Progressive thrombotic occlusion of the grafts occurred after 70 minutes of blood flow through the shunt.
  • the number of deposited platelets and the quantity of fibrin deposited was used as a quantitative marker of thrombus formation. Platelet deposition was measured in real time using radiolabeled autologous platelets and gamma-camera imaging, according to the methods of Hanson, et al.. Arteriosclerosis 5: 595-603 (1985) and Cadroy, et al., PNAS USA 88: 1177-1181 (1991).
  • Fibrin deposition was quantified as described in Gruber, et al., supra (1990) or Cadroy, et al., supra (1991).
  • the thrombogenic device was incorporated into the shunt at 0 minutes, and was removed after 60 minutes of blood perfusion through the shunt; then, the normal flow was restored in the shunt.
  • thrombin In order to increase circulating levels of APC, we increased the concentration of thrombin:thrombomodulin complexes in the vasculature. This was achieved by infusion of thrombin ( ⁇ -thrombin was a gift of Dr. John W. Fenton, Albany, NY) distal to the thrombogenic devices, into the inferior vena cava.
  • the ⁇ -thrombin is preferably purified according to the method described by Fenton, et al. , in Chemistry and Biology of Thrombin. Lundblad, Fenton and Mann (eds.), Ann Arbor Science Publ. Inc., 1977, pp.
  • thrombin was infused at two doses on different subsequent days: 1 U/kg/min and 2 U/kg/min. Calculating with an estimated 1 L/min cardiac output and negligible inhibition of the enzyme within the seconds necessary to reach the heart, these doses resulted in maximal estimated thrombin concentrations of 75 and 150 pM, respectively, in the pulmonary artery. Because of thrombin's prothrombotic properties, we also measured platelet and fibrinogen consumption from the circulation during thrombin infusions.
  • ECA Enzyme Capture Assay
  • Purified IgG was absorbed to immobilized PC in 0.01 M Tris, pH 7.4, 0.14 M NaCl (TBS), 0.02% Na-azide, and subsequently eluted using either 3 M Na-thiocyanate in 0.05 M Tris, pH 7.4, 1.0 M NaCl, 0.02 % Na-azide or 0.1 M glycine, pH 2.5, 0.1 M NaCl.
  • the thiocyanate eluate was dialyzed against 0.05 M Tris, pH 7.4, 0.5 M NaCl; the glycine eluate was dialyzed against TBS. Both eluates were stored at -70°C until use.
  • the two antibody preparation methods gave equivalent results. ii. Activation
  • APC was prepared as follows.
  • the activated form of PC (APC) was prepared by treating the inactive zymogen PC prepared above with ⁇ -thrombin-Sepharose beads.
  • Thrombin was purchased from Enzyme Research Laboratories (South Bend, IN) and prepared from homogeneous human prothrombin by activation with Factor Xa, Factor Va and phospholipid.
  • Human thrombin was homogeneous as judged by 10% SDS- PAGE.
  • the purified thrombin was coupled to CNBr-activated Sepharose as described. Thrombin- Sepharose beads were mixed with purified PC solutions continuously and the activation of PC was monitored.
  • Activation was by the method of Marlar, et al., Blood 59: 1067 (1982) .
  • the amidolytic activity was determined in an assay using the chromogenic substrate, S-2238 (H-D-Phenylalanyl-L- pipecolyl-L-arginine-p-nitroanilide dihydrochloride, Kabi-Vitrum, Franklin, OH) .
  • Other preferred substrates include S-2366 and Spectrozyme PCa
  • APC product was determined to be greater than 95% pure when analyzed by SDS-PAGE as described above.
  • the specific anticoagulant activity of APC was determined to be 250 Units/mg.
  • 1U of APC corresponds to the activity of PC in 1ml of human plasma after full activation.
  • concentration of the APC used in the assay was initially optimized with respect to the sensitivity of the assay towards APC-induced prolongation of clotting time compared to clotting time without APC.
  • Immunoaffinity purified APC preparations were tested for anticoagulant activity and preparations exhibiting maximal specific activity (250 U/mg) were used as standards in the APC ECA.
  • Murine Mabs to PC were prepared by a modification of the method of Kohler and Milstein [Nature 256: 495
  • mice 25 the mice were injected with 35 ⁇ g antigen in incomplete Freund's adjuvant. On day 35, three days prior to cell fusion, 35 ⁇ g of purified PC was injected intravenously (IV) .
  • Spleen cells (8.6 X IO 8 ) from immunized mice were fused with P3X63-Ag8.653 (available from ATCC as CRL15800) murine myeloma cells (1.7 X10 8 ) using 30%
  • Tissue Culture 109 medium, oxaloacetate (l mmol/L) , pyruvate (0.45 mmol/L), glutamine (2 mmol/L), penicillin and streptomycin (5ml each to 720 medium) , Hepes (20 mmol/L) , hypoxanthine (1 x IO “4 mol/L) , and thymidine (3 X 10 "5 mol/L) .
  • the hybridomas were screened for growth. Approximately 15% were growing.
  • each well of a 96-well plastic microtiter plate (Immulon II microtiter plates, Dynatech Laboratories) was coated overnight at 4°C with 6 ⁇ g/ml PC in 0.05 M borate buffer, pH 8.4. The plates were washed with 50 mM Tris/HCl, 0.14 M NaCl, 0.05% NaN 3 , pH 7.2, and 7 mM EDTA (TBS/EDTA) , and then coated with 3% BSA in TBS/EDTA (blocking buffer) for 1 hr at 37°C.
  • RIA radioimmunoassay
  • the plates were washed three times with TBS/EDTA. Fifty ⁇ l of supernatant from each clone were added to each well, along with 5 ul of 70 mM EDTA, and incubated for 1 hr at room temperature. The plate was washed four times with Buffer A/BSA + EDTA (50 mM Tris/HCl, 0.14 M NaCl, 0.05% NaN 3 , 0.1% BSA, 1.5 mM MgCl 2 , 0.05% Tween- 20, pH 7.2, 7 mM EDTA). Iodinated rabbit anti-mouse
  • IgG (diluted in Buffer A/BSA + EDTA) was added to each plate and it was allowed to incubate 2 hr at 37°C. Finally, the plate was washed four times with Buffer A/BSA + EDTA, the plate was dried, and the wells cut out and counted. Wells demonstrating radioactivity higher than that recorded in control plates (not coated with PC or not treated with antibody) were indicative of immunoreaction of the iodinated second antibody with anti-PC bound to immobilized PC, and thus were deemed to have been contacted with monoclonal antibody immunoreactive with PC.
  • hybridomas including hybridoma 22A101CS3B2 (referred to herein as C3) , were determined by these procedures to immunoreact with PC and were then cloned by limiting dilution.
  • Hybridoma C3 has been deposited pursuant to Budapest Treaty requirements with the American Type Culture Collection (ATCC) , Rockville, MD, on July 3, 1991, and was assigned accession number HB 10820.
  • Hybridoma C3 was deposited in a depository affording permanence of the deposit and ready accessibility thereto by the public upon the issuance of a patent, under conditions which assure that access to the hybridoma will be available during the pending of the patent application to those deemed by the Commissioner to be entitled to such access, and that all restrictions on the availability to the public of the hybridoma as deposited will be irrevocably removed upon the granting of the patent.
  • the deposited hybridoma will be maintained by the ATCC for the term of the patent or 30 years from the date of deposit, whichever is longer, and in all events for at least five years after the date of the last request for access.
  • Thymus cells (5 X 10 5 /ml) from BALB/c mice were added to the wells as a feeder layer during cloning.
  • the clones were evaluated in the above-described assay for production of antibodies specific for PC. Clones that were again found positive were recloned by the same procedure to ensure monoclonality and were again screened by one of the assays. Positive cell lines were selected for injection (1 X 10 6 cells per animal) into the peritoneal cavity of BALB/c mice that had been injected with 300-500 ⁇ l pristane about 10 days prior to injection with hybridoma cells. The ascitic fluids obtained from mice injected with clones producing monoclonal antibodies C3, C4, and CIO were screened for their ability to bind radiolabelled PC as follows. The IgG fractions were purified from the fluid using anion exchange chromatography, as described in Geiger et al.
  • 125 I-PC was prepared by iodination of purified PC using the standard chloramine T method [McConahey et al. (1966) Int. Arch. All. Immunol. 29: 185-189]. Fifty ul of 125 I-PC (200,000 CPM/well, specific activity 10 ⁇ Ci/ ⁇ g) in 0.01 M Na-phosphate buffer containing 0.14 M NaCl (PBS), 3.0% BSA, 1 mM EDTA, 0.05% Tween 20, 0.05% Na- azide, pH 7.4, were added to each well and the plate was incubated for 90 min at 37°C. After washing the plates, wells were cut out and counted in a Micromedic 4/600 automatic gamma counter.
  • the wells of a 96 well flat bottom Immulon II microtiter plate (Dynatech Laboratories, Chantilly, USA) were coated with 250 ⁇ l of C3-Mab (50 to 100 ⁇ g/ml, purified as described above) in coating buffer at 4°C.
  • the following coating buffers were used with equivalent results: 0.01 M sodium carbonate, pH 9.2, 0.02% Na-azide; or 0.02 sodium carbonate, pH 8.5, 0.02% Na-azide; or 0.02 Tris-HCl, pH 7.8, 0.02% Na- azide.
  • Coating buffer alone was added to negative control plates. The plates were allowed to incubate for 14 hours at 4°C.
  • the plates were then blocked with 1% casein in coating buffer (300 ⁇ l/well) for at least 1 hour at 37°C or overnight at 4°C, the latter being preferred.
  • the blocking buffer was stored at 4°C with immobilized soybean trypsin inhibitor (1 ml bead to 500 ml buffer; Pierce, Rockford, IL) , and was filtered through 0.2 ⁇ m pore size syringe filter prior to use.
  • the C3-Mab coated plates were washed with washing buffer (0.02 M Tris, pH 7.4, 0.15 M NaCl, 0.02
  • Example 2 were treated with 250 ⁇ l/well of either DFP
  • dilution buffer 0.05 M Hepes, pH 7.24, 0.2 M NaCl, 0.05 M benzamidine, 0.02 M EDTA, 0.4% casein, 0.6% ovalbumin, 2% BSA, 0.04 % Na-azide, and 0.05% Tween-80; filtered with 0.2 ⁇ m pore size filter
  • soybean trypsin inhibitor had been added prior to benzamidine addition.
  • a synthetic oligopeptide chromogenic substrate for APC S-2366 (0.45-1.0 mM in TBS, pH 8.0, 0.05% Na-azide) was added to the wells.
  • S-2366 is ⁇ Glu-Pro-Arg-pNA (Kabi Diagnostica, Uppsala, Sweden) , it was prepared aseptically at 4°C and filtered through a 0.2 ⁇ m filter.
  • the lyophilized substrate was diluted to 4 mM using sterile water. Prior to the assay the substrate was further diluted to 0.4 mM using filtered washing buffer in a disposable sterile reagent tray.
  • NASH Formula A The blood was centrifuged in the bag at 300 x g for 10 minutes.
  • the supernatant platelet- rich plasma (PRP) was then transferred to a second bag and the pH adjusted to 6.5 by the addition of 0.15M citric acid (O.lml/lOml PRP).
  • the red blood cell fraction was returned to the donor mammal.
  • the platelets were formed into a pellet by centrifugation of the PRP at 1300 x g for 15 minutes.
  • the supernatant platelet-poor plasma (PPP) was completely decanted and discarded.
  • the bag containing the platelet pellet was carefully washed once by overlaying with 30 ml of Ringer's citrate dextrose (RCD, pH 6.5) that was then decanted and discarded. The pellet was then gently resuspended in 5.0 ml RCD, and incubated for 30 minutes with 500-700 micro Ci 111 In-oxine (Amersham Corp. , Arlington Heights, IL) . Contaminating red cells were removed by a final slow centrifugation at 200 x g for 5 minutes.
  • RCD Ringer's citrate dextrose
  • Labeling efficiency was determined by diluting 200 microliters of the labeled-platelet concentrate with 5.0 ml RCD, and comparing the activity in 0.5 ml of the diluted platelet suspension with the activity in 0.5 ml of cell-free supernatant following centrifugation at 3000 x g for 30 minutes. A measured volume of labeled platelet suspension containing approximately 13 percent non-platelet bound isotope was then injected directly into the recipient mammals following the preparation of a 100 microliter standard. Additional washing procedures to remove non-platelet bound isotope were deemed undesirable since they may produce in vitro cell damage.
  • Circulating platelet 111 In-activity was determined from 4 ml blood samples drawn prior to and following graft placement, and collected in 2 mg/ml
  • EDTA ethylenedinitrilo-tetraacetic acid
  • EDTA ethylenedinitrilo-tetraacetic acid
  • One ml of each sample was used for platelet counting, and 1.0 ml was counted for whole blood 11l In-activity. The remaining 2 ml were centrifuged at 3000 x g for 30 minutes, and 1.0 ml of the supernatant (PPP) was counted for plasma T11 In-activity. All blood and plasma samples were counted using a gamma spectrometer (Nuclear Chicago, Chicago, IL) . Platelet counts were performed on whole blood using a electronic platelet counter (Clay Adams UF-100, Parsippany, NJ) .
  • the baboons were injected with 15 I-labeled fibrinogen (FGN) (0.005mCi) before each experiment.
  • the grafts were removed if occlusion occurred before 60 minutes from the beginning of the experiment and were washed and stored in 2.5% glutaraldehyde.
  • 125 I- labeled FGN radioactivity was measured in counts per minute (cpm) after allowing the graft 111 In activity (from the labeled autologous platelets) to decay for at least 30 days. At 30 days, the remaining platelet- bound 111 In radioactivity was 0.0006% of the value at 0 minutes, compared with 70% of 125 I radioactivity that remained at 30 days.
  • Clotting time was measured after recalcification using lOO ⁇ l of 25mM CaCl 2 in a fibro eter (BBL Fibrosystem) .
  • the activated partial thromboplastin time (APTT) returned to quasi-normal (41.9 seconds) 3 hours after termination of thrombin infusion (not shown) .
  • APTT activated partial thromboplastin time
  • Figure 5 illustrates the decrease in fibrin deposition. Infusion of 2 U/kg/min thrombin for one hour reduced platelet counts by 30% versus controls without significant increase in antithrombotic effect (see Table 6) . This suggests that lU/kg/min thrombin induced near maximal antithrombotic effect. Protein C zymogen level was not depleted during one hour infusion of 1 U/kg/min thrombin (-3%, from 77.3 % to 75%) . Bleeding times were not prolonged significantly at the 2 U/kg/min thrombin dose (from 3.1 + 0.6 minutes to 4 + 0.7 minutes) . No adverse effects were observed during and after one hour thrombin infusion at either dose.
  • Activated partial thromboplastin time (APTT, SEC)
  • thrombin Thrombogenic device absent; l ⁇ /kg/min. thrombin added
  • Activated partial thromboplastin time (APTT, SEC)
  • Activated partial thromboplastin time (APTT, SEC)
  • Activated protein C (APC, ng/ml)
  • Thrombogenic device present; lU/kg/min. thrombin added
  • Activated protein C (APC, ng/ml)
  • Thrombogenic device present; 2U/kq/min. thrombin added
  • Activated partial thromboplastin time (APTT, SEC)
  • Activated protein C (APC, ng/ml)
  • thrombin itself clots blood and is capable of inducing thrombosis (it is "prothrombotic") via activating platelets and factors V and VIII, and via clotting fibrinogen, reduction or abolition of thrombus formation on thrombogenic surfaces during thrombin infusion was a novel finding that contradicted previous, "common” knowledge about thrombin. (See, e.g., Walz, et al., eds., "Bioregulatory functions of thrombin", in Ann. N.Y. Acad. Sci. 485: 5-413 (1986).) In fact, many current antithrombotic therapies are based on inhibiting thrombin.
  • Another useful method of increasing circulating APC levels comprises the infusion of protein C zymogen. It is reported herein that protein C zymogen levels correlate with APC levels; see, e.g.. Fig. 9. Furthermore, infusion of protein C into homozygous protein C deficient patients A and B with severe thrombophilia increases circulating protein C levels (see Tables 7 and 8 below) . TABLE 7
  • the baboon platelet aggregating activity of K52E was about 20% of ⁇ -thrombin.
  • the following data were gathered previously in six (6) similar size baboons receiving 1200 amidolytic units of ⁇ -thrombin under similar experimental conditions (mean values) , as illustrated in Table 10 below. 120'
  • K52E at a 2400 U dose resulted in less circulating FPA by 60 minutes than ⁇ -thrombin at a 1200 U dose.
  • the baboon receiving K52E proved to be partially deficient in PC zymogen (50% levels) , it produced over 400 ng/ml of circulating APC.
  • the APTT values corresponded to this APC level.
  • This baboon might have produced APC levels reaching 900 ng/ml if the PC zymogen was normal. Fibrinogen and platelet consumption was negligible with K52E.
  • the 3-fold increase in prothrombin fragment Fl-2 suggested efficacious inhibition of prothrombin cleavage.
  • K52E at this dose very efficaciously inhibited both arterial type (Dacron platelets, 78% inhibition) and venous type (Teflon platelets, 96% inhibition) thrombus formation without adverse effects (i.e., bleeding or toxicity at a very high dose) .
  • the above data indicate that increasing the circulating concentration of the zymogen protein C will increase the circulating levels of APC.
  • the data also suggest that any agent that increases the activation of protein C, thus increasing the level of APC, could be used in conjunction with infusion of the purified zymogen for (a) preventing the depletion of the endogenous zymogen pool during the induced anticoagulant therapy, and (b) reaching higher levels of APC.
  • compositions useful in the inhibition of thrombosis formation in an individual.
  • an agent capable of increasing the blood activated protein C level in an individual is admixed with a pharmaceutically acceptable carrier or excipient.
  • the agent is capable of increasing the blood activated protein C level via enzymatic cleavage of protein C.
  • the agent may comprise a serine protease, such as thrombin, and more preferably, human thrombin.
  • the thrombin used may be selected from the group comprising plasma-derived or recombinant ⁇ -thrombin, thrombin E192Q, thrombin K52E, and active site acylated-thrombin, to name a few examples.
  • the effective amount of agent included in the composition may vary according to the therapeutic modality, the needs of the individual, and the recommendations of the physician. In one variation, e.g. when the therapeutic agent comprises thrombin, an effective amount of thrombin is in the range of from 0.05U/kg/min to 2U/kg/min.
  • compositions of the present invention may further be formulated so that the effective amount of the activated protein C level- increasing agent — such as thrombin — may be administered over a period of at least 20 minutes.
  • an effective amount of the therapeutic agent, such as thrombin is sufficient to achieve an activated protein C concentration in the blood of an individual three to five times the normal level of activated protein C.
  • the effective amount of agent e.g., thrombin
  • the effective amount of agent is sufficient to achieve an activated protein C concentration in the blood of the individual of from 10 to 760 ng/ml. It may also be preferred that the effective amount of agent (e.g., thrombin) is sufficient to maintain the activated protein C concentration for a time period of at least 30 minutes.
  • compositions according to the present invention may be formulated for intravenous administration, or for continuous or bolus injection.
  • Another variation includes the admixture of an antithrombotic agent into the composition.
  • the antithrombotic agent is selected from the group comprising fibrinolytic agents, oral anticoagulants, dextrans, pentoxifilline, snake venom, soluble thrombomodulin, antiplatelet drugs, cyclooxygenase inhibitors, cAMP modulators, ticlopidine, thrombolytic agents, streptokinase, Eminase, urokinase, tissue plasminogen activator, anticoagulant peptides, protein C, and activated protein C.
  • the present invention also contemplates methods of preparing compositions formulated for administration to individuals suffering from surgically induced thrombosis, or for administration substantially concurrently with exposing the blood of an individual to a prosthetic surface on a prosthetic device.
  • prosthetic devices include (a) a cardiopulmonary assist device; (b) a hemodialysis device; (c) an arterial prosthesis; (d) a venous prosthesis; (e) an arteriovenous shunt; (f) a cardiopulmonary bypass oxygenator and/or pump apparatus; (g) heart valve implants or artificial heart valves; and (h) an artificial heart.

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Abstract

L'invention concerne différents procédés de prévention et de traitement de la thrombose par élévation des taux de protéine C activée endogène dans la circulation et en régularisant vers le haut le taux de plasma ou la génération de protéine C activée. La présente invention concerne également des compositions appropriées destinées à être utilisées pour inhiber la thrombose. Elle concerne en outre des procédés et des compositions pour la détermination de la quantité de thrombomoduline chez un individu.
PCT/US1992/009978 1991-11-18 1992-11-18 Procedes d'inhibition de la thrombose par elevation des taux de proteine c activee endogene dans la circulation WO1993009807A1 (fr)

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Cited By (9)

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Publication number Priority date Publication date Assignee Title
WO1995007712A1 (fr) * 1993-09-14 1995-03-23 Regents Of The University Of Minnesota Procede d'elevation de la production de thrombomoduline apc a l'aide de proteines cationiques
WO1998040474A2 (fr) * 1997-03-12 1998-09-17 Baxter Aktiengesellschaft Facteur sanguin active dependant de la vitamine k et procede de preparation dudit facteur
EP0882453A2 (fr) * 1997-06-05 1998-12-09 Eli Lilly And Company Méthodes de traitement de problèmes thrombotiques
US6110721A (en) * 1993-11-12 2000-08-29 Gilead Sciences, Inc. Polypeptides and coagulation therapy
US7060484B1 (en) 1993-11-12 2006-06-13 Gilead Sciences, Inc. Polypeptides and coagulation therapy
US7517965B2 (en) 2002-07-22 2009-04-14 Chugai Seiyaku Kabushiki Kaisha Non-neutralizing anti-aPC antibodies
EP2103310A1 (fr) * 2008-03-19 2009-09-23 Universiteit Maastricht Procédé pour la prévention ou le traitement d'une blessure de réperfusion d'ischémie
WO2014005183A1 (fr) 2012-07-04 2014-01-09 The University Of Sydney Traitement de troubles cutanés inflammatoires
US11491214B2 (en) 2014-04-16 2022-11-08 Zz Biotech Llc Treatment of abnormal cutaneous scarring

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Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585095A (en) * 1993-09-14 1996-12-17 Regents Of The University Of Minnesota Method to enhance thrombomodulin APC generation using cationic proteins
US5674489A (en) * 1993-09-14 1997-10-07 Regents Of The University Of Minnesota Method to enhance thrombomodulin APC generation using cationic proteins
WO1995007712A1 (fr) * 1993-09-14 1995-03-23 Regents Of The University Of Minnesota Procede d'elevation de la production de thrombomoduline apc a l'aide de proteines cationiques
US6110721A (en) * 1993-11-12 2000-08-29 Gilead Sciences, Inc. Polypeptides and coagulation therapy
US7060484B1 (en) 1993-11-12 2006-06-13 Gilead Sciences, Inc. Polypeptides and coagulation therapy
WO1998040474A2 (fr) * 1997-03-12 1998-09-17 Baxter Aktiengesellschaft Facteur sanguin active dependant de la vitamine k et procede de preparation dudit facteur
WO1998040474A3 (fr) * 1997-03-12 1999-02-11 Immuno Ag Facteur sanguin active dependant de la vitamine k et procede de preparation dudit facteur
EP0882453A3 (fr) * 1997-06-05 2001-04-04 Eli Lilly And Company Méthodes de traitement de problèmes thrombotiques
WO1998055142A1 (fr) * 1997-06-05 1998-12-10 Eli Lilly And Company Procede de traitement de troubles thrombotiques
EP0882453A2 (fr) * 1997-06-05 1998-12-09 Eli Lilly And Company Méthodes de traitement de problèmes thrombotiques
US7517965B2 (en) 2002-07-22 2009-04-14 Chugai Seiyaku Kabushiki Kaisha Non-neutralizing anti-aPC antibodies
EP2103310A1 (fr) * 2008-03-19 2009-09-23 Universiteit Maastricht Procédé pour la prévention ou le traitement d'une blessure de réperfusion d'ischémie
WO2009115548A2 (fr) * 2008-03-19 2009-09-24 Universiteit Maastricht Méthode de traitement des lésions de l’ischémie-reperfusion
WO2009115548A3 (fr) * 2008-03-19 2009-12-23 Universiteit Maastricht Méthode de traitement des lésions de l’ischémie-reperfusion
WO2014005183A1 (fr) 2012-07-04 2014-01-09 The University Of Sydney Traitement de troubles cutanés inflammatoires
US11617785B2 (en) 2012-07-04 2023-04-04 Zz Biotech Llc Treatment of inflammatory skin disorders
US11491214B2 (en) 2014-04-16 2022-11-08 Zz Biotech Llc Treatment of abnormal cutaneous scarring

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