WO1992018139A1 - Molecules chimeriques ayant une activite d'activation de plasminogene et affinite pour les plaques atherosclerotiques - Google Patents

Molecules chimeriques ayant une activite d'activation de plasminogene et affinite pour les plaques atherosclerotiques Download PDF

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WO1992018139A1
WO1992018139A1 PCT/US1992/003009 US9203009W WO9218139A1 WO 1992018139 A1 WO1992018139 A1 WO 1992018139A1 US 9203009 W US9203009 W US 9203009W WO 9218139 A1 WO9218139 A1 WO 9218139A1
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moiety
plasminogen activator
chimeric molecule
fibrin
plasminogen
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PCT/US1992/003009
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English (en)
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Joseph Loscalzo
Boris Pasche
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Brigham And Women's Hospital
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Publication of WO1992018139A1 publication Critical patent/WO1992018139A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally relates to agents that are useful in treating thrombotic and atherothrombotic disorders and methods of treatment using such agents.
  • the invention specifically relates to compositions and methods of treatment comprising a chimeric molecule containing a plasminogen activator moiety linked to a moiety that recognizes a component in a pathologic thrombus.
  • the fibrinofytic system contains a proenzyme, plasminogen, which can be converted to the active enzyme plasmin by the action of several different types of plasminogen activators.
  • Plasmin is a serine protease that digests fibrin to soluble degradation products. Natural inhibition of the fibrinolytic system occurs both at the level of the plasminogen activator and at the level of plasmin (Cohen, D., Les Cahiers de la
  • Plasminogen is a single chain glycoprotein converted by plasminogen activators to plasmin by cleavage of the Arg560-Val561 peptide bond.
  • Plasminogen activators are serine proteases with a high specificity for plasminogen. The hydrolysis of the Arg560-Val561 peptide bond of plasminogen yields the active enzyme, plasmin.
  • Streptokinase is a protein that is produced by beta.hemolytic streptococci. Streptokinase activates the fibrinofytic system indirectly by forming a complex with plasminogen. The formation of this complex exposes an active site in the plasminogen moiety, whereby the complex becomes a potent plasminogen activator.
  • Anisoylated plasminogen streptokinase activator complex is an inactive derivative of the plasminogen streptokinase activator complex obtained by acylation of its active site serine. Spontaneous deacylation at physiological pH with a t of approximately 35 minutes promotes its reactivation.
  • Urokinase is a serine protease composed of two polypeptide chains, connected by a disulfide bridge. Urokinase activates plasminogen directly to plasmin.
  • Single chain urokinase-type plasminogen activator (scu-PA) or pro-urokinase is a single chain glycoprotein which is converted to high molecular weight two-chain urokinase by hydrolysis of the Lysl58-Ilel59 peptide bond.
  • scu-PA activates fibrin-bound plasminogen much more readily than it activates circulating plasminogen.
  • Tissue-type plasminogen activator is a serine protease. It occurs either as a single chain or as a two chain proteolytic derivative, t-
  • PA is a poor plasminogen activator in the absence of fibrin, but it binds specifically to fibrin and activates plasminogen at the fibrin surface several hundred-fold more efficiently than it activates circulating plasminogen.
  • c ⁇ -antiplasmin is a glycoprotein of the serine protease inhibitor
  • Plasminogen activator inhibitor-1 is a fast acting inhibitor of t-PA and urokinase occurring at very low concentration in the blood, but which may be significantly increased in several disease states including venous thromboembolism and ischemic heart disease.
  • Plasminogen activators are thrombolytic agents that convert plasminogen, the inactive proenzyme of the fibrinofytic system in blood, to plasmin. Plasmin not only dissolves the fibrin of a blood clot, but may also lead to hemorrhage by degrading normal components of the hemostatic system. Several plasminogen activators have been either used clinically or are currently under clinical investigation.
  • streptokinase include streptokinase, urokinase, recombinant tissue-type plasminogen activator, anisoylated plasminogen streptokinase activator complex and single chain urokinase- type plasminogen activator (Collen D. et al, Verh. Acad. Geneeskd (Belgium) 52:191 (1990); Collen et al. Ann. Rev. Med. 39:405 (1990)).
  • Single-chain urokinase-type plasminogen activator has been developed as another relatively clot- selective plasminogen activator, and it has been shown in some trials to produce less depletion of plasma fibrinogen than t-PA (Loscalzo, J., et al, Circulation 79:116 (1989)).
  • the remaining plasminogen activators vary in their degree of fibrin-specificity but similarly are not absolutely selective for fibrin.
  • c ⁇ -antiplasmin may be depleted by the over-production of systemic plasmin that is produced during plasminogen activator therapy using non-fibrin-specific plasminogen activators. This depletion may lead to bleeding complications during treatment.
  • New developments towards further improved efficacy and fibrin- specificity of thrombolytic therapy include the use of combinations of synergistic thrombolytic agents, mutants of t-PA and scu-PA chimeric t- PA/scu-PA molecules, antibody-targeted thrombofytic agents, and/or combinations of fibrin-dissolving agents with anti-platelet strategies (Bode, C et al, Clin. Cardiol 13:315 (1990).
  • Bode C et al, Science 229:165 (1985). These include chemical, immunologic, or recombinant linkage of fibrin-specific antibodies with thrombolytic agents.
  • chemical conjugates between a fibrin- specific and an activator-specific antibody, and biosynthetically produced heteroduplex antibodies that are both fibrin and activator-specific could bind to fibrin and localize endogenous or exogenous activator.
  • These conjugates display significantly enhanced clot-specific lysis in vitro and, in an animal model, in vivo. (Collen, D. et al, Verh. Acad.. Geneeskd (Belgium) 51:191 (1990); Collen et al Ann. Rev. Med. 39:405 (1990) ;
  • the resultant molecule might combine the fibrin affinity of t-PA (which is responsible for its concentration at the clot surface) with both the enzymatic properties of scu-PA (which is responsible for its stability in plasma) and the kinetic fibrin selectivity of scu-PA.
  • t-PA which is responsible for its concentration at the clot surface
  • scu-PA which is responsible for its stability in plasma
  • kinetic fibrin selectivity of scu-PA One such chimera has been studied in detail (Nelles, L. et al, J. Biol Chem. 262:10855 (1987)).
  • this chimera has a higher fibrin affinity than scu-PA, its affinity is not as high as that of intact t-PA.
  • Studies of the chimera in vivo indicate that it has maintained most of the thrombofytic potential of scu- PA, but does not appear to be a superior agent for thrombolysis (Haber, E. et al, Thromb. Haemost. 57:253 (1987)).
  • a principal drawback to the therapeutic value of the available native and modified plasminogen activators is that the currently available activators cannot distinguish a pathologic thrombus from protective hemostatic plugs, the dissolution of which may also be accompanied by hemorrhage.
  • Acute myocardial infarction in particular, is associated with the development of an occlusive thrombus at the site of a fissured (activated) atherosclerotic plaque in a coronary artery as a direct proximate cause of the clinical event.
  • plasminogen activators i.e., tissue-type plasminogen activator, streptokinase, or urokinase-type plasminogen activators
  • Numerous clinical trials have confirmed the benefits of this treatment in terms of the firm endpoints of preservation of ventricular function and improvement in mortality (ISIS-2 Study Group, Lancet 336:65 (1990); Guerin et al, NFJM 327:1613 (1990); Wilcox et al, Lancet 2:525 (1988)).
  • lysis of normal protective hemostatic plugs occurs.
  • This novel strategy comprises the development of a molecule which 1) activates plasminogen efficiently, 2) inhibits thrombin, and 3) is localized to a pathologic thrombus, avoiding protective hemostatic plugs.
  • This molecule is useful in the treatment of a variety of atherothrombotic and thrombotic disorders. These include acute myocardial infarction, unstable angina, deep venous thrombosis, pulmonary embolism, peripheral arterial occlusion, and cerebrovascular accident.
  • This novel strategy also comprises the application of adjunctive therapy wherein said therapy is adjunctive to plasminogen activation therapy and wherein the adjunctive agent inhibits thrombin and is localized to a pathologic thrombus, avoiding protective hemostatic plugs.
  • the adjunctive agent is unlinked to the plasminogen activator in this mode of therapy.
  • An adjunctive therapy with these characteristics is described below.
  • the present invention addresses a need in the field of thrombofytic therapy to provide an effective thrombolytic agent that minimizes hemorrhagic complications.
  • the present invention comprises a novel plasminogen activator molecule that activates plasminogen efficiently and is localized to a pathologic thrombus, avoiding interaction not only with systemic components of the fibrinofytic system but also with normal protective hemostatic plugs.
  • the invention further comprises the use of said novel plasminogen activator molecule in thrombolytic therapy, i.e., in the treatment of atherothrombotic disorders.
  • the invention further comprises the use of a thrombin .inhibiting agent which is localized to a pathologic thrombus as an adjunct to plasminogen activator therapy for the treatment of atherothrombotic disorders.
  • the present invention includes compositions that comprise a chimeric molecule with a plasminogen activator moiety that does not directly bind fibrin and wherein that moiety is linked to a second moiety that targets a component that is found in pathologic thrombi but not in normal protective thrombi.
  • the invention encompasses the linkage of any suitable plasminogen activator with any desired molecule that has affinity for a component found in a pathologic thrombus, but not a normal protective hemostatic plug.
  • the chimeric molecule contains a moiety that has thrombin-inhibitor activity and the linkage between the moieties is hydrolyzable in vivo and is at the active site of the plasminogen activator moiety.
  • low molecular weight single- chain urokinase-type plasminogen activator is linked at its serine-protease active site to heparin cofactor II by means of an anisoyl-based coupling that is hydrolyzable in vivo.
  • the present invention also encompasses methods of treatment of various atherothrombotic disorders using the compositions of the present invention.
  • the present invention also includes compositions wherein a plasminogen activator is used in combination with a second component wherein the second component targets a component that is found in pathologic thrombi but not in normal protective thrombi.
  • This composition encompasses the use of any suitable plasminogen activator with any suitable component that has affinity for a component found in the pathologic thrombus but not in a normal protective hemostatic plug.
  • the composition contains an adjunctive component that is a component added with a plasminogen. activator wherein the component is heparin cofactor II.
  • the desirable plasminogen activator is administered in conjunction with the administration of the heparin cofactor II.
  • the present invention also encompasses compositions and methods of treatment of various atherothrombotic disorders using the compositions of the present invention in adjunctive therapy.
  • the present invention relates to chimeric molecules that contain at least two moieties: a plasminogen activator moiety and a moiety that targets the molecule to a pathologic thrombus by means of the targeting moiety having affinity for a component found in a pathologic thrombus but not in a normal clot.
  • the first and second moieties are chemically linked at the active site of the plasminogen activator moiety by a linkage that is hydrolyzable in vivo.
  • a linkage that is hydrolyzable in vivo By linking these two molecules chemically and using a slowly hydrolyzable active site coupling, the plasminogen activator remains inactive until localization to the thrombus has occurred, after which the plasminogen activator and the targeting moiety dissociate at physiologic pH.
  • a preferred linkage between the first and second moieties is an anisoyl-based coupling that slowly hydrolyzes at physiological pH.
  • An alternative linkage between the first and second moieties is a plasmin-sensitive linker domain comprising an alanine 12-mer which links the first and second moieties and which contains a plasmin sensitive bond within the 12-mer.
  • the 12-mer is a 12 amino acid oligopeptide wherein all of the amino acids are alanine moieties. Plasmin will hydrolyze any peptide bond to lysine or arginine. Therefore, the placement of lysine or arginine within the 12-mer (perhaps as lys-fys or arg-val) will provide a hydrolysis site. This site facilitates the release of the plasminogen activator into the thrombus after the chimeric molecule has bound to the de ⁇ natan sulfate in the vessel wall.
  • the linkage need not be at the active site and need not be degradable in vivo.
  • Covalent linkage, not involving the active site, that is not degradable may be used in the invention.
  • a fusion protein is formed by recombinant DNA methods, as by forming a hybrid DNA that comprises a sequence coding for a plasminogen activator linked in the same transcriptional orientation to a sequence that codes for the targeting moiety. Upon translation of the RNA, a fusion protein, containing both activities, results.
  • the invention encompasses immunological linkage.
  • the second moiety contains anti-thrombin activity so that at the site of the pathologic thrombus, fibrin deposition is inhibited.
  • the first moiety comprises low molecular weight single chain urokinase-type plasminogen activator or a functional derivative thereof.
  • Low molecular weight scu-PA will be used because it does not bind to fibrin directfy, but only exerts its relatively fibrin-selective action kineticalfy, by preferentially converting fibrin-bound glu-plasminogen to plasmin. Because this plasminogen activator does not bind directfy to fibrin, it does not by itself have an affinity for either normal protective clots or pathologic thrombi but is directed to thrombi only by means of the targeting moiety to which it is attached.
  • the plasminogen activator Since the targeting moiety targets pathologic thrombi, the plasminogen activator has access only to those thrombi. Further, because this activator activates only fibrin-bound plasminogen, circulating plasminogen is not activated and thus, systemic degradation of normal components of the fibrinolytic system is avoided.
  • the dermatan sulfate binding domain is isolated and linked to urokinase thereby conferring plaque specificity without thrombin-inhibiting activity.
  • the present invention also relates to compositions and methods of treatment with those compositions wherein the compositions comprise a plasminogen activator and a molecule that is specific for a pathologic thrombus, said molecule having affinity for a component found in a pathologic thrombus but not in a normal clot. This molecule has thrombin inhibiting activity.
  • a desired plasminogen activator is used in conjunction with heparin cofactor II.
  • low molecular weight single chain urokinase type plasminogen activator or a functional derivative thereof is used in conjunction with heparin cofactor II.
  • compositions comprising a desired plasminogen activator and heparin cofactor II, and therapy encompassing the use of these compositions as well as the chimeric molecules discussed above, encompass the use of specific mutants of heparin cofactor II which increase the selectivity of binding for dermatan over heparin.
  • the invention is directed to, for example, the use of lysine 173 mutated to glutamine or leucine (Whinna et al, J. Biol. Chem. 266:8129 (1991)) that increases the selectivity of binding for dermatan over heparin by several hundredfold. Accordingly, the invention is directed to heparin cofactor II, not only in its native form but in its mutant forms as well, used in combination with the plasminogen activators as described above.
  • mutant forms of heparin cofactor II may be linked to any desirable plasminogen activator.
  • mutant forms of heparin cofactor II are linked to low molecular weight single chain urokinase-type plasminogen activator as described above, which linkage may or may not be at the active site.
  • the linkage may be hydrolyzable in vivo, such as by means of the anisoyl-based coupling described above, or by means of a plasmin sensitive linker also as described above.
  • mutant heparin cofactor II may be used in conjunction, where they are not linked to, plasminogen activators such as high molecular two-chain urokinase, low molecular weight two-chain urokinase, single chain t-PA or two chain t-PA
  • mutant heparin cofactor II is used to form compositions for adjunctive therapy with single chain urokinase type plasminogen activator (scu-PA).
  • Glu 1 3 -Leu 14 peptide bond in scu-PA It was shown to have very similar properties to those of intact scu-PA including its conversion to an active two-chain molecule by plasmin, its intrinsic plasminogen activating potential, and its ability to induce relatively fibrin-specific clot lysis in the absence of direct fibrin binding (8). In addition, it has similar thrombofytic properties to those of intact scu-PA in vivo (Stump et al, Blood 69:592 (1987)). - • A recombinant form of this molecule has been obtained (Lijnen et al, J. Biol. Chem. 263:5594 (1988)).
  • the low molecular weight scu-PA is linked to the targeting moiety by chemical methods using a hydrolyzable anisoyl group bound to the serine-protease active site of scu-PA.
  • the low molecular weight form of scu-PA is less readily cleared .from the circulation than is the intact native molecule, thus prolonging somewhat its plasma half-life after dissociation of the linkage.
  • the plasminogen activator moiety comprises a different plasminogen activator.
  • scu-PA itself, high molecular weight two-chain urokinase, low molecular weight two-chain urokinase, single chain t-PA and two-chain t-PA.
  • the targeting moiety comprises heparin cofactor II.
  • Heparin cofactor II is chosen because it binds to dermatan sulfate, a component associated with atherosclerotic plaques but not with normal thrombi. Plaque activation with rupture exposes dermatan sulfate, heparin cofactor II binds to the dermatan sulfate, and plasminogen activation is thus localized to the site of thrombus. At the thrombus site, heparin cofactor II also inhibits thrombin generation in a selective manner that is catalyzed by dermatan sulfate-induced conformational changes in the serine protease inhibitor
  • the targeting moiety recognizes other components in a plaque, such as chondroitin sulfate or collagen type III (in atheromas, a change in the predominant form of collagen occurs from type to type) and athero- endothelial leukocyte adhesion molecule
  • Human plasma contains two heparin-independent inhibitors of thrombin: antithrombin III and heparin cofactor II.
  • Heparin cofactor II inhibits thrombin by formation of stable 1:1 complexes with the protease (Tollefeen et al, J. Biol. Chem. 258:6113 (1983).
  • the present invention is also directed to methods of treatment using the hybrid molecules or compositions comprising a plasminogen activator as described above in conjunction with a thrombin inhibitor that specifically recognizes pathologic thrombi, such as heparin cofactor II and its mutant forms and functional derivatives.
  • a thrombin inhibitor that specifically recognizes pathologic thrombi, such as heparin cofactor II and its mutant forms and functional derivatives.
  • the various embodiments of the chimeric molecule or the composition comprising plasminogen activator not linked to heparin cofactor II (or other thrombus-specific thrombin inhibitor) are administered in doses sufficient to treat thrombotic and atherothrombotic diseases of animals.
  • the diseases include, but are not limited to acute myocardial infarction, unstable angina, deep venous thrombosis, pulmonary embolism, peripheral arterial occlusion, and cerebrovascular accident.
  • Any thrombotic or atherothrombotic disorder is potentially amenable to treatment with the chimeric molecules or with the composition comprising a plasminogen activator not linked to heparin cofactor II (or other thrombus-specific thrombin-inhibitor of the present invention).
  • treating is intended the administration to subjects of the compositions of the invention for purposes which include prophylaxis, amelioration, or cure of disease.
  • administer any method of treating a subject with a substance, such as orally, intranasalfy, parenterally (intravenously, intramuscularly, or subcutaneously), or rectalfy.
  • administer is also intended simultaneous or sequential administration of the individual components of the present invention.
  • the plasminogen activator and the thrombin inhibitor which specifically recognizes dermatan sulfate or other components of atherosclerotic plaques, may be administered at different times. Therefore, administration may be simultaneous, within minutes, or up to around 3 hours of each other. The time frame for sequential administration will depend upon the specific plasminogen activator in the composition.
  • administration would be simultaneous or the thrombin inhibitor is administered just prior to the administration of the plasminogen activator.
  • simultaneous or sequential "co-administration" is intended that there is a temporal overlap of the biological activities of the co-administered components.
  • animal any living creature that contains components of a fibrinofytic system such that specific thrombofysis may be induced by the administration of agents of this invention.
  • animals Foremost among such animals are humans; however, the invention is not intended to be so-limiting, it being within the contemplation of the present invention to apply the compositions of the invention to any and all animals which may experience the benefits of the application.
  • disease is intended any deviation from or interruption of the normal structure or function of any part, organ, or system (or combination thereof) of the body that is manifested by a characteristic set of symptoms and signs.
  • functional derivative of heparin cofactor II is intended, for the purpose of the invention, any protein or peptide fragment based on the sequence of heparin cofactor II and that is similar to heparin cofactor II in that it inhibits thrombin and is activated by dermatan sulfate.
  • the derivatives of low molecular weight scu-PA and heparin cofactor II may be derived from the naturally-occurring molecule or synthesized chemically or by recombinant methods based on the sequence of the naturally occurring-molecule.
  • chimeric molecule is intended, for the purpose for the invention, a hybrid molecule constructed to contain functional moieties from two different proteins or two different genes.
  • chimeric molecule is meant a hybrid protein which possesses a moiety that has plasminogen-activating activity and a moiety that has affinity for a non-fibrin component in an atherosclerotic plaque.
  • chimeric molecule is also meant a hybrid oligonucleotide that possesses a DNA sequence that encodes a protein having plasminogen-activating activity and a DNA sequence that encodes a protein having affinity for a non-fibrin component in an atherosclerotic plaque.
  • mutant is intended, for the purpose of the present invention, a heparin cofactor II peptide sequence containing one or more amino acid substitutions, deletions, or insertions.
  • the effect of said deletion, substitution or insertion is to increase the selectivity of binding for dermatan over heparin relative to th e selectivity of binding of the native heparin cofactor II sequence.
  • Mutations can be generated by any of the routine manipulations taught in the art such as in vitro site-specific mutagenesis.
  • alanine 12-mer is intended, for the purpose of the present invention, a dodecamer of alanine that links the two functional domains, i.e., the plasminogen activator moiety and the second moiety which has affinity for a non-fibrin component found in atherosclerotic plaques.
  • the second moiety may be heparin cofactor II or mutants thereof.
  • the alanine 12-mer is a polyalanine linkage region that has no organized structure and which allows the domain of the second moiety to be separate from the plasminogen activator moiety so that, following dissociation, the two moieties may move independently. Further, this dodecamer is the site of a plasmin sensitive covalent bond.
  • plasmin sensitive covalent bond is intended, for the purpose of the present invention, a dipeptide bond wherein the dipeptide bond is covalentfy linked to and internal to the alanine dodecamer.
  • the dipeptide lysine-fysine or arginine-valine in the middle of the alanine 12-mer would be sensitive to hydrolysis after having been exposed to plasmin. Any lysine or arginine bond is plasmin sensitive.
  • lysine-X or arginine-X where X is any amino acid, would be plasmin sensitive, and placement of such dipeptides within the alanine dodecamer would render the dodecamer susceptible to hydrolysis with plasmin, thus separating any moieties linked by said dodecamer.
  • Arginine-valine is the most common dipeptide.
  • the purpose of including a plasmin sensitive linker domain is to facilitate the release of the plasminogen activator into the thrombus after the chimeric molecule has bound to the dermatan sulfate in the vessel wall.
  • the chimeric protein of the invention is a single peptide wherein the functional moieties described above are chemically linked to each other through a peptide bond or other covalent linkage.
  • the moieties may be directly linked to each other or a coupling or conjugating agent (peptide or non-peptide) may be inserted between the functional moieties.
  • Indirect linkage may be achieved by utilizing any of the several intermolecular cross-linking reagents. (See, for example, Means, G.E. and Feeney, R.E. Chemical Modification of Proteins, Holden-Day, 1974, pp.39-43).
  • N-succinimidyl 3-(2-pyridyldithio) propionate SPDP
  • N-N'- (1,3- phenylene) bismalemide both are highly specific for sulfhydiyls, and form irreversible linkages
  • N-N'-ethylenebis-(iodoacetamide) or other such reagent having 6 and 11 carbon methylene bridges Relatively specific for sulfhydiyl groups
  • l,5-dif_uoro-2,4-dinitrobenzene forms irreversible linkages with amino and tyrosine groups
  • p,p'-difluoro-m-m'- dinitrodiphenylsulfone forms irreversible cross-linkages with amino and phenolic groups
  • dimethyl adipimidate specific for amino groups
  • anisoyl-based coupling is intended the type of bond formed as by using the following procedure. Using a total urokinase concentration of 1 mg/ml in 0.1 M Tris-HCl, 0.9% w/v NaCl, 20% v v glycerol, pH 7.4, acylation is accomplished by incubating the solution with 1 mM p-amidinophenyl p'-anisate-HCl at 0° for 1 hr. The solution is then exhaustively dialyzed prior to storage at 4° or lyophilization (Tanizawa et al, J. Am. Chem. Soc. 99:4495 (1977); Smith et al, Nature 290:505 (1981)).
  • the chimeric protein of the invention is a dipeptide wherein the functional moieties described above are not covalently linked to each other but wherein the moieties associate with each other with sufficient affinity so as to provide the protein of this embodiment to the patient's lesion in the dipeptide form.
  • This association includes, but is not limited to, ionic and hydrogen bonding, immunological affinity as between antigen and cognate antibody, and affinity as between an enzyme and its substrate.
  • the conditions and concentrations useful for obtaining the couples of the invention can be readily adjusted by those of skill in the art by reference to known literature or by no more than routine experimen ⁇ tation.
  • the compounds of the invention can be formulated in various pharmaceutical preparations adapted for administration in manners similar to those used for other plasminogen activator compounds.
  • one aspect of the invention involves pharmaceutical compositions for human beings or animals provided by using a conventional pharmaceutical carrier, diluent, and/or excipient with an effective amount of the chimeric molecule of the invention.
  • compositions of the present invention may be administered by any means that provide thrombolytic activity.
  • the pharmaceutical compositions of the invention can be formulated in dosage forms for oral, parenteral or rectal administration. Sohd dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is admixed with at least one inert diluent such as sucrose, lactose or starch.
  • inert diluent such as sucrose, lactose or starch.
  • Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluent.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with an enteric coating.
  • Liquid dosage forms for oral administration include pharmaceutically accepted emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the pharmaceutical arts. Besides inert diluents, such compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents and sweetening. Compositions for rectal administration are suppositories which may contain in addition to the active substance, excipients such as cocoa butter or suppository wax.
  • Suitable formulations for parenteral administration are, above all, aqueous solutions of the active compounds in water-soluble forms, for example, in the form of water-soluble salts, and also suspensions of the active compounds, such as appropriate oily injection suspensions, for which suitable lipophilic solvents are used or aqueous injection suspensions which contain substances which increase the viscosity and optionally also contain stabilizers.
  • Other methods of administration can be intravenous administration, oral administration, intraperitoneal administration, intramuscular administration, and subcutaneous administration).
  • the dosage of active ingredients in the composition of this invention may be varied; however, it is necessary that the amount of the active ingredient be formulated such that a suitable dosage form is obtained.
  • the dosage form depends upon the desired therapeutic effect, on the route of the administration, and on the duration of the treatment. Treatment can be carried out for any period of time, depending on the severity of the disease.
  • Administration dosage and frequency will depend on the age and general health condition of the patient, taking into consideration the possibility of side-effects.
  • the administration will also be dependent on concurrent treatment with other drugs and patients' tolerance of the administered drug.
  • the preferred percent by weight of the plasminogen activator in compositions formulated according to the present invention is in the range of approximately 0.1 - 3.0%.
  • compositions of the present invention is 0.01 - 2.0 mg/kg body weight.

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Abstract

La présente invention concerne des compositions et de procédés de traitement qui sont utiles en thérapie thrombolytique. Les compositions de la présente invention sont destinées à réduire au minimum les complications dues au saignement et qui sont associées à une thérapie thrombolytique actuellement disponible. En conséquence, les compositions comprennent une molécule chimèrique construite avec une fraction d'activateur plasminogène qui ne peut se lier à la fibrine et une fraction qui cible les thrombus pathologiques. Cette construction a comme résultat une molécule qui possède une affinité uniquement pour les thrombus pathologiques, évitant ainsi les thrombus normaux et les composants de circulation du système fibrinolytique. L'activateur plasminogène peut être couplé à un inhibiteur de thrombine spécifique de la classe serpine qui s'oppose à la production de thrombines lors d'une thrombolyse. La présente invention concerne également des compositions et des procédés de traitement où l'activateur plasminogène n'est pas couplé à la fraction qui cible les thrombus pathologiques, mais où cette fraction est utilisée comme élément d'adjonction séparé pour une thérapie utilisant un activateur plasminogène.
PCT/US1992/003009 1991-04-09 1992-04-09 Molecules chimeriques ayant une activite d'activation de plasminogene et affinite pour les plaques atherosclerotiques WO1992018139A1 (fr)

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Cited By (3)

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EP0669394A1 (fr) * 1993-07-15 1995-08-30 Grünenthal GmbH Dérivés bifonctionels de l'urokinase ayant des propriétés améliorées fibrinolytiques et inhibant la thrombine
EP0714982A2 (fr) * 1994-11-30 1996-06-05 Grünenthal GmbH Protéines hybrides, ayant des propriétés fibrinolytiques et inhibant la thrombine
WO2011036444A1 (fr) * 2009-09-22 2011-03-31 Ximmune Ab Fragments du cofacteur ii de l'héparine ayant une activité anti-inflammatoire et anticoagulante

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0669394A1 (fr) * 1993-07-15 1995-08-30 Grünenthal GmbH Dérivés bifonctionels de l'urokinase ayant des propriétés améliorées fibrinolytiques et inhibant la thrombine
US5681721A (en) * 1993-07-15 1997-10-28 Gruenenthal Gmbh Bifunctional urokinase variants with improved fibrinolytic characteristics and thrombin inhibiting effect
US5747291A (en) * 1993-07-15 1998-05-05 Gruenenthal Gmbh Bifunctional urokinase variants with improved fibrinolytic characteristics and thrombin inhibiting effect
CN1057124C (zh) * 1993-07-15 2000-10-04 格吕伦塔尔有限公司 具改进纤溶特性和凝血酶抑制活性的双功能尿激酶变异体
EP0714982A2 (fr) * 1994-11-30 1996-06-05 Grünenthal GmbH Protéines hybrides, ayant des propriétés fibrinolytiques et inhibant la thrombine
EP0714982A3 (fr) * 1994-11-30 1999-09-08 Grünenthal GmbH Protéines hybrides, ayant des propriétés fibrinolytiques et inhibant la thrombine
US6133011A (en) * 1994-11-30 2000-10-17 Gruenenthal Gmbh Chimeric proteins having fibrinolytic and thrombin-inhibiting properties
WO2011036444A1 (fr) * 2009-09-22 2011-03-31 Ximmune Ab Fragments du cofacteur ii de l'héparine ayant une activité anti-inflammatoire et anticoagulante
JP2013505030A (ja) * 2009-09-22 2013-02-14 エクセルミューネ・アクチエボラーグ 抗炎症及び抗凝固活性を有するヘパリンコファクターｉｉ断片
US9169315B2 (en) 2009-09-22 2015-10-27 Ximmune Ab Heparin cofactor II fragments with anti-inflammatory and anti-coagulant activity

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