WO1992018139A1 - Chimeric molecule with plasminogen activator activity and affinity for atherosclerotic plaques - Google Patents

Abstract

The present invention is directed to compositions and methods of treatment that are useful in thrombolytic therapy. The compositions of the present invention are designed to minimize the bleeding complications that are associated with presently available thrombolytic therapy. Accordingly, the compositions comprise a chimeric molecule constructed with a plasminogen activator moiety that cannot bind to fibrin and a moiety that targets pathologic thrombi. This construction results in a molecule that has affinity only for pathologic thrombi, thus avoiding normal thrombi and circulating components of the fibrinolytic system. The plasminogen activator may be coupled to a specific thrombin inhibitor of the serpin class that will counteract thrombin generation during thrombolysis. The present invention is also directed to compositions and methods of treatment wherein the plasminogen activator is not coupled with the moiety that targets pathologic thrombi, but wherein this moiety is used as a separate adjunct for plasminogen activator therapy.

Description

TITLE OF THE INVENTION

CHE ERIC MOLECULE WITH PLASMINOGEN ACTIVATOR ACΠVΠΎ AND AFFINITY FOR ATHEROSCLEROTIC PLAQUES

Background of the Invention

1. Field of the Invention

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.

2. Description of the Background Art

Components of the Fibrinofytic System (Collen et al, Verh. Acad. Geneeskd (Belgium) 51:191 (1990); Collen D. et al. Ann. Rev. Med. 39:405 (1990)). 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

Foundation Louis Jeantet de Medicine 2:41 (1986); Bachman, F. in Thrombosis and Haemostasis Eds. Verstraete, M. et al, pp. 227 (1987); Collen, D., Thromb. Haemost. 43:11 (1980)).

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 (t-PA) 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. cώ-antiplasmin is a glycoprotein of the serine protease inhibitor

(serpin) superfamily. α2-antiplasmin inactivates plasmin. It forms a reversible but inactive complex which is then slowly converted into an irreversible complex. 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 Activator Therapy

Cardiovascular disease is often complicated by thrombosis. Death or disability are often the result of this complication. Thrombolysis could beneficially affect the outcome of such cardiovascular diseases as myocardial infarction, cerebrovascular thrombosis and venous thromboembolism. 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. These 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)).

Significant shortcomings of plasminogen activator therapy remain, however, that limit its overall effectiveness, among which is a lack of available agents with absolute selectivity for fibrin. Despite the fact that the theoretical basis upon which the biotechnical development of t-PA is grounded is its "fibrin selectivity," in actual practice t-PA is hardly selective for fibrin. Treatment with t-PA leads not only to lysis of the fibrin composing the offending thrombus, but also to depletion of plasma fibrinogen, which itself may be at least in part responsible for the hemorrhagic complications attendant to plasminogen activator therapy (Loscalzo, J., Drugs 37:191 (1989)). 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.

Another significant shortcoming of the available plasminogen activators is the need for large doses of these activators in order to achieve therapeutic efficacy. Large doses are associated with systemic activation of plasminogen and unpredictable bleeding, cώ-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).

Antibody-targeted thrombolytic therapy

Several alternatives to target the action of thrombolytic agents towards the thrombus with the use of fibrin-specific antibodies are presently being investigated (Haber, E. et al, Science 243:51 (1989); Dewerchin, M., Eur. J. Biochem. 285:141 (1989); Bode, C. et al, ibid;

Bode C, et al, Science 229:165 (1985). These include chemical, immunologic, or recombinant linkage of fibrin-specific antibodies with thrombolytic agents. Alternatively, 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) ;

Bode, C. et al, J. Biol Chem. 264:944 (1989); Branscomb, E.E. et al, Clin. Res. 35-.264A (1987)).

Chimers of t-PA and scu-PA

Another approach has been to construct chimeric (hybrid) molecules that contain domains of both the t-PA and scu-PA molecules.

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. One such chimera has been studied in detail (Nelles, L. et al, J. Biol Chem. 262:10855 (1987)).

Although 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)).

Deposition of Fibrin at the Site of the Thrombus

In addition to the problem of fibrin-specificity and the related problem of hemorrhage, another often- noted problem with current plasminogen activator therapy is the delay in time to lysis of the thrombus (Fitzgerald et al, Proc. Nat'l Acad. Sci. 86:1585 (1989)). One reason for this delay is that platelet and thrombin activation accompany plasminogen activator therapy, thereby leading to the simultaneous and competitive processes of fresh platelet and fibrin accrual on a dissolving thrombus leading to slower net lysis rates than would be expected if such prothrombotic processes were inhibited. Recent data support the view that inhibition of platelet function, thrombin action, or both, enhance rates of thrombolysis (Vaughan et al, Blood 73:1213 (1989); Fitzgerald et al, Proc. Nail. Acad. Sci. USA 86:1585 (1989); Am. J. Cardiol 67:1A (1991)). In addition, many investigators believe that thrombin is the primary antagonist in this schema, since it both converts fibrinogen to fibrin and is the primary platelet agonist in vivo (Eisenberg, P.R., Am. J. Cardiol 67:19A (1991)).

Specificity for Pathologic Thrombi

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. Conversion of plasma plasminogen to plasmin by the administration of pharmacologic doses of plasminogen activators (i.e., tissue-type plasminogen activator, streptokinase, or urokinase-type plasminogen activators) leads to prompt lysis of the thrombus with restoration of patency of the involved vessel. 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)). However, in addition to lysis of the pathologic clot, lysis of normal protective hemostatic plugs occurs. There is, thus, a need for a plasminogen activator that is selective for a pathologic thrombus and avoids normal thrombi.

Given the observations and problems discussed above, Applicants have developed a novel strategy to optimize therapy with a thrombolytic agent. 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.

A novel molecule with these characteristics is described below.

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.

Summary of the Invention

The present invention addresses a need in the field of thrombofytic therapy to provide an effective thrombolytic agent that minimizes hemorrhagic complications. Accordingly, 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.

Accordingly, 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.

In preferred embodiments 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. In a specific disclosed embodiment, 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.

Accordingly, 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. In preferred embodiments the composition contains an adjunctive component that is a component added with a plasminogen. activator wherein the component is heparin cofactor II. Thus 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.

Description of the Preferred Embodiments

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.

In a preferred embodiment of the present invention 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. 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 deπnatan 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. For example, in alternative embodiments 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. Alternatively, the invention encompasses immunological linkage.

In a further preferred embodiment of the invention the second moiety contains anti-thrombin activity so that at the site of the pathologic thrombus, fibrin deposition is inhibited.

In a highly preferred embodiment of the present invention, 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. 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.

In an alternative embodiment of the invention 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.

In a preferred embodiment of the invention, a desired plasminogen activator is used in conjunction with heparin cofactor II. In a highly preferred embodiment of the present invention, low molecular weight single chain urokinase type plasminogen activator or a functional derivative thereof is used in conjunction with heparin cofactor II.

In further embodiments of the invention, 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.

Specifically, mutant forms of heparin cofactor II may be linked to any desirable plasminogen activator. In a preferred embodiment, 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.

Similarly, the 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 In a highly preferred embodiment the mutant heparin cofactor II is used to form compositions for adjunctive therapy with single chain urokinase type plasminogen activator (scu-PA).

Low Molecular Weight Single Chain Urokinase-Type Plasminogen Activator

Low molecular weight single-chain urokinase-type plasminogen activator with a molecular weight of 32,000 has been purified from the conditioned medium of a human lung adenocarcinoma cell line (Stump et al, J. Biol Chem. 261:11120 (1986)) and from monkey kidney cells (Wijngaards et al, Thromb. Res. 42:149 (1986)). This low molecular weight derivative of scu-PA was generated by specific cleavage of the

Glu^{1 3}-Leu¹⁴ 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)). This recombinant has biological properties similar to those of the natural molecule. In a preferred molecule, 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.

In alternative embodiments of the invention the plasminogen activator moiety comprises a different plasminogen activator. Examples are scu-PA itself, high molecular weight two-chain urokinase, low molecular weight two-chain urokinase, single chain t-PA and two-chain t-PA.

In a further highly preferred embodiment of the invention, 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

(serpin), while scu-PA activates thrombus-bound plasminogen.

In alternative embodiments, 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

(adhesion molecules that are expressed on the surface of endothelial cells in or adjacent to atheromata).

Heparin Cofactor II

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 anticoagulant activity of dermatan sulfate was first demonstrated when it was shown that this glycosaminoglycan prolonged the partial thromboplastin time in vitro (Teien et al, Thromb. Res. 8:859

(1976)). Recent research work has provided evidence that the anticoagulant activity of dermatan sulfate is mediated through heparin cofactor II (Tollefeen et al, J. Biol Chem. 258:6113 (1983)). This work demonstrated that the rate of inhibition of thrombin by purified heparin cofactor II was increased nearly one thousand-fold by the addition of dermatan sulfate. Furthermore, the addition of dermatan sulfate to plasma containing ¹²⁵I- thrombin resulted in the formation of complexes between thrombin and heparin cofactor II. These observations suggested that heparin cofactor II is activated by dermatan sulfate (Salem et al, Develop. Biol Standard 67:61 (1987)). Dermatan sulfate has been shown to accumulate in natural and diet-induced atherosclerotic plaques

(Robbins et al, Am. J. Pathol 134:615 (1989)).

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. In preferred methods of treatment, 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).

By the term "treating" is intended the administration to subjects of the compositions of the invention for purposes which include prophylaxis, amelioration, or cure of disease.

By the term "administer" is intended any method of treating a subject with a substance, such as orally, intranasalfy, parenterally (intravenously, intramuscularly, or subcutaneously), or rectalfy. By the term "administer" is also intended simultaneous or sequential administration of the individual components of the present invention. For example, 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. Optionally, administration would be simultaneous or the thrombin inhibitor is administered just prior to the administration of the plasminogen activator. By simultaneous or sequential "co-administration" is intended that there is a temporal overlap of the biological activities of the co-administered components.

By the term "animal" is intended 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. 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. By the term "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.

By the term "functional derivative" of low molecular weight scu-PA for the purpose of the present invention is intended, any protein or peptide fragment based on the sequence of low molecular weight scu-PA and that is similar to low molecular weight scu-PA in that it activates plasminogen, does not bind fibrin directfy but binds to fibrin-bound plasminogen, and does not bind to circulating plasminogen. By the term "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.

By "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. As used herein, by "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. As used herein, by "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.

By the term "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. For the purpose of the invention, 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.

By the term "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.

By "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. For example, 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. Therefore, 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.

In one embodiment, 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. In this embodiment, 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). Among these reagents are, for example, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or 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); ρhenyl-2,4-disulfonylchloride (reacts principally with amino groups); hexamethylenedfisocyanate or diisothiocyanate, or azophenyl-p-diiso- cyanate (reacting principally with amino groups); glutaraldehyde (reacting with several different side chains) and bisdiazobenzidine (reacting primarily with tyrosine and histidine). These are onfy a few of several cross-linking agents that can be utilized.

By "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)).

In another embodiment, 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. Thus, 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.

The compositions of the present invention may be administered by any means that provide thrombolytic activity. For example, 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.

In such sohd dosage forms, the active compound is admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluent. In the case of capsule, tablets, and pills, 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%.

The preferred dosage range for compositions of the present invention is 0.01 - 2.0 mg/kg body weight. Having now generally described the invention, the various modifications and uses that are encompassed therein will be evident to the person of ordinary skill in the art.

Claims

What is new and claimed and intended to be covered by a letters patent of the United States is:CLAIMS

1. A chimeric molecule comprising a first moiety and a second moiety, wherein said first moiety has plasminogen-activator activity and does not bind to fibrin and wherein said second moiety has affinity for a non-fibrin component found in atherosclerotic plaques.

2. The chimeric molecule of claim 1 wherein said first and second moieties are dissociable from each other in vivo.

3. The chimeric molecule of claim 2 wherein said dissociation occurs by hydrolysis of a covalent bond linking the two moieties.

4. The chimeric molecule of claim 3 wherein said bond is an anisoyl-based coupling.

5. The chimeric molecule of claim 3 wherein said dissociation occurs by hydrolysis of a plasmin-sensitive covalent bond.

6. The chimeric molecule of claim 5 wherein said covalent bond is in a polyalanine 12-mer oligopeptide and wherein said oligopeptide is between and covalently linked to the first and second moieties.

7. The chimeric molecule of claim 6 wherein the covalent bond is in a lys-lys or arg-val dipeptide and wherein said dipeptide is internal to and covalently joined to said oligopeptide.

8. The chimeric molecule of claim 2 wherein said dissociation occurs at the active site of the plasminogen activator moiety.

9. The chimeric molecule of claim 1 wherein said plasminogen activator moiety is low molecular weight single chain urokinase-type plasminogen activator or a functional derivative thereof.

10. The chimeric molecule of claim 1 wherein said second moiety has thrombin inhibitor activity.

11. The chimeric molecule of claim 10 wherein said second moiety is heparin cofactor II or a functional derivative thereof.

12. A method of treating atherothrombotic disease in an animal comprising administering to said animal an effective amount of a chimeric protein comprising a first moiety and a second moiety, wherein said first moiety has plasminogen-activator activity and does not bind to fibrin and wherein said second moiety has affinity for a non-fibrin component found in atherosclerotic plaques.

13. The method of claim 12 wherein said disease is selected from the group consisting of acute myocardial infarction, unstable angina, deep venous thrombosis, pulmonary embolism, peripheral arterial occlu¬ sion, and cerebrovascular accident.

14. The method of claim 12 wherein said first and second moieties are dissociable from each other in vivo.

15. The method of claim 14 wherein said dissociation occurs by hydrolysis of a covalent bond linking the two moieties.

16. The method of claim 15 wherem said bond is an anisoyl- based coupling.

17. The method of claim 14 wherein said dissociation occurs at the active site of the plasminogen activator moiety.

18. The method of claim 12 wherein said plasminogen activator moiety is low molecular weight single chain urokinase-type plasminogen activator or a functional derivative thereof.

19. The method of claim 12 wherein said second moiety has thrombin inhibitor activity.

20. The method of claim 19 wherein said second moiety is heparin cofactor II or a functional derivative thereof.

21. A pharmaceutical composition useful in the treatment of atherothrombotic disease, said composition comprising an effective amount of the chimeric molecule of claim 1 together with a pharmaceutically acceptable carrier.

22. A composition comprising a first moiety and a second moiety wherein said first moiety has plasminogen activator activity and does not bind to fibrin and wherein said second moiety has affinity for a non-fibrin component found in atherosclerotic plaques.

23. The composition of claim 22 wherein said plasminogen activator moiety is low molecular weight, single chain urokinase-type plasminogen activator or a functional derivative thereof.

24. The composition of claim 22 wherein said second moiety has thrombin inhibitor activity.

25. The composition of claim 24 wherein said second moiety is heparin cofactor II or a mutant or a functional derivative thereof.

26. A method of treating atherothrombotic disease in an animal comprising administering to said animal an effective amount of a composition comprising a first moiety and a second moiety, wherein said first moiety has plasminogen activator activity and does not bind to fibrin and wherein said second moiety has affinity for a non-fibrin component and in atherosclerotic plaques.

27. The method of claim 26 wherein said disease is selected from the group consisting of acute myocardial infarction, unstable angina, deep venous thrombosis, pulmonary embolism, peripheral arterial occlusion, and cerebrovascular accident

28. The method of claim 26 wherein said plasminogen activator moiety is low molecular weight single-chain urokinase-type plasminogen activator or a functional derivative thereof.

29. The method of claim 26 wherein said second moiety has thrombin inhibitor activity.

30. The method of claim 29 wherein said second moiety is heparin cofactor II or a mutant or a functional derivative thereof.

31. A pharmaceutical composition useful in the treatment of atherothrombotic disease, said composition comprising an effective amount of the composition of claim 22 together with a pharmaceutically effective carrier.