10 New thrombolytic agents and strategies

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New thrombolytic agents and strategies DESIRE C O L L E N H. R O G E R LIJNEN

One approach to the treatment of thrombosis consists of the pharmacological dissolution of the blood clot via the intravenous infusion of plasminogen activators, which activate the blood fibrinolytic system. The mechanisms involved in the regulation of the fibrinolytic system are discussed in Chapter 1. The recognition that early administration of thrombolytic therapy to patients with acute myocardial infarction (AMI) results in recanalization of occluded coronary arteries, has provided the basis for large scale clinical application of thrombolysis in patients with AMI. Currently, five thromboylic agents are available for clinical use: streptokinase (SK), anisoylated plasminogen streptokinase activator complex (APSAC), two-chain urokinase-type plasminogen activator (tcuPA, urokinase), recombinant single-chain u-PA (rsu-PA, pro-urokinase) and recombinant tissue-type plasminogen activator (rt-PA). In clinical trials, reduction of infarct size, preservation of ventricular function and reduction in mortality has been demonstrated with SK, rt-PA and APSAC. However, all available thrombolytic agents still suffer significant shortcomings, including the need for a large therapeutic dose, short plasma half-life, limited fibrin-specificity, reocclusion, and bleeding complications (reviewed in Collen and Lijnen, 1991). Recent approaches to improve the thrombolytic properties ofplasminogen activators include the construction and evaluation of mutant plasminogen activators, of chimeric molecules comprising portions of different plasminogen activators, of antibody-targeted plasminogen activators using fibrin-specific or platelet-specific monoclonal antibodies and of plasminogen activators from other animal or bacterial sources (reviewed in Lijnen and Collen, 1991; Madison, 1994). Conjunctive antithrombotic and antiplatelet strategies to improve thrombolytic therapy are also evaluated (reviewed in Gold, 1990). MUTANTS AND VARIANTS OF scu-PA

The stuctural properties and the mechanism of action of scu-PA are discussed in Chapters 1 and 2. Some approaches to improve the thrombolytic profile of rscu-PA are briefly summarized below; mutants or variants Baillidre "s Clinical Haematology425 Vol. 8, No. 2, June 1995 ISBN 0-7020-1957-7

Copyright © 1995, by Bailli~re Tindall All rights of reproduction in any form reserved

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with improved thrombolytic potency have, however, not yet been reported. Because clot lysis with scu-PA in a plasma milieu is associated with a higher degree of fibrin-specificity as compared to tcu-PA several investigators have constructed mutants of scu-PA in which the plasmin cleavage site was destroyed by site-specific mutagenesis (reviewed in Lijnen and Collen, 1991). Such plasmin-resistant mutants of scu-PA (i.e rscu-PA K158A, rscu-PA K158G) have, however, a thrombolytic potency in rabbits with jugular vein thrombosis that is about five-fold lower than that of scuPA (Collen et al, 1989). The thrombolytic potency of such mutants thus appears to be too low to allow their efficient use in man. A low M~ derivative of human scu-PA, lacking the NH2-terminal 143 residues, was purified from cell cultures (Stump et al, 1986) or was obtained by recombinant DNA technology (rscu-PA-32k) (Lijnen et al, 1988). In a rabbit jugular vein thrombosis model, comparable clot lysis with less pronounced systemic fibrinogen breakdown was obtained with rscu-PA-32k as with 33 kDa tcu-PA (Lijnen et al, 1988). rscu-PA-32k may thus represent a useful alternative for large scale production of a singlechain u-PA species by recombinant DNA technology. MUTANTS AND VARIANTS OF t-PA

The structural properties and the mechanism of action of t-PA are discussed in Chapters 1 and 2. rt-PA mutants have been constructed with altered pharmacokinetic or functional properties, including binding to fibrin and stimulation by fibrin, and resistance to plasma protease inhibitors (reviewed in Lijnen and Collen, 1991; Madison, 1994). rt-PA mutants with deletion of the finger (F), epidermal growth factor (E) and/or kringle 1 (K1) domains were shown to have a significantly reduced plasma clearance in several animal models. This was, however, frequently associated with a reduced specific thrombolytic activity, resulting in an unchanged or only marginally improved thrombolytic potency. The pharmacokinetic and thrombolytic properties in animal models of one of such mutants, consisting of the kringle 2 ( K 2 ) and protease (P) domains of t-PA (K2P, BM 06.022, LY 210825), suggested that this molecule, because of its prolonged plasma half-life, may be used for coronary artery thrombolysis by bolus administration (Jackson et al, 1992; Martin et al, 1992; Nicolini et al, 1992). In patients with acute myocardial infarction, BM 06.022 was found to be a relatively fibrin-selective compound which did not cause major bleeding complications (Miiller et al, 1992). Prolonged half-lives have also been obtained by substitution or deletion of one or of a few selected amino acids in the finger or epidermal growth factor domains (reviewed in Lijnen and CoUen, 1991; Madison, 1994). Such mutants may have a better preserved specific thrombolytic activity than domain deletion mutants. One of such t-PA molecules in which cysteine 84 in the E-domain is replaced by serine, has a half-life in man of more than 20 minutes, as compared to 6 minutes for native t-PA. This molecule has been used successfully for bolus administration in a canine femoral artery thrombosis model (Suzuki et al,

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1994) and in a multicentre trial in patients with acute myocardial infarction (Kawai et al, 1992). A rt-PA mutant in which threonine 103 is substituted by asparagine and the sequence Lys 296-His-Arg-Arg is mutagenized to Ala-Ala-Ala-Ala was found to have both a prolonged half-life and resistance to PAI-1. This mutant has an increased potency on platelet-rich plasma clots (rich in PAIl) in animal models (Refino et al, 1993). Additional substitution in this mutant of asparagine 117 by glutamine resulted in a rt-PA variant with eightfold slower clearance and 200-fold enhanced resistance to PAI-1. In in vivo models of thrombolysis in rabbits, this mutant was shown to have an increased potency on platelet-rich clots, to conserve fibrinogen and to be effective upon bolus administration at a lower dose as compared to rt-PA (Keyt et al, 1994). Similar results were obtained in combined arterial and venous thrombosis model in the dog (Collen et al, 1994). Such mutants with prolonged half-life and resistance to PAI-1 may be useful to reduce a potential contribution of high PAI-1 levels to reocclusion. More zymogenic mutants of rt-PA (with a larger difference in enzymatic activity between one-chain and two-chain forms) have been obtained by site-specific mugenesis in the proteinase domain (Bennett et al, 1991; Madison et al, 1992). Thus, it appears to be possible to engineer several properties into human t-PA in order to produce a molecule with a potentially enhanced therapeutic potential. The t-PA of saliva from the vampire bat Desmodus rotundus (bat-PA) was found to constitute a potent and fibrin-specific thrombolytic agent in rabbits and dogs with femoral arterial thrombosis (Gardell et al, 1991; Mellott et al, 1992). From a family of four Desmodus plasminogen activators encoded by four distinct genes, one of the two larger forms, rDSPAoq, was shown to be an efficient and fibrin-specific thrombolyfic agent in rats with experimental pulmonary embolism (Witt et al, 1992) and in dogs with copper coil-induced coronary thrombosis (Witt et al, 1994). It remains to be shown in direct comparative studies whether any of these rt-PA mutants or variants offers advantages over wild-type rt-PA for the treatment of thromboembolic disease. R E C O M B I N A N T C H I M E R I C P L A S M I N O G E N ACTIVATORS Recombinant chimeric plasminogen activators have been constructed primarily using different regions of t-PA and scu-PA, although several altemative combinations have been evaluated to some extent (reviewed in Lijnen and Collen, 1991). In vivo evaluation in animal models of thrombosis indicated that one of these variants (K1K2Pu) had a markedly enhanced thrombolytic potency towards venous and arterial thrombi (Collen et al, 1991). K~K2Puconsists of kringles 1 and 2 of rt-PA (amino acids Ser 1-Gln 3 and Asp 87-Phe 274) and of the serine proteinase part of rscu-PA (amino acids Ser 138-Leu 411). The delayed plasma clearance of K~K2P, with relatively preserved specific thrombolytic activity suggested that a significant reduction of the total amount of material required for

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thrombolytic therapy, and its administration by bolus injection may be possible in patients with thromboembolic disease. A small feasibility study of coronary thrombolysis with K~K2P, was performed in patients with acute myocardial infarction (Van de Weft et al, 1993). Clot lysis within 30 minutes was obtained in two of four patients given two 10mg boluses of K~K2Po with a 15 minute interval. Administration of K~K2P, did not induce an overt systemic lyric state, as shown by virtually unchanged levels of fibrinogen and %-antiplasmin. KlK2P.-related antigen disappeared from plasma with an initial half-life of about 9 minutes and a terminal half-life of 70 minutes, corresponding to a plasma clearance of approximately 50 ml/minute. Although the small number of patients studied precludes valid estimation of the frequency of coronary recanalization with K,K2Po and of the adequacy of the dose used, this preliminary trial suggests that two bolus injections of 10 mg K~K2P, may produce fibrin-specific coronary thrombolysis. Other recombinant chimeric plasminogen activators which are presently evaluated in animal models of thrombosis include FK2tu-PA and K2tu-PA (F and K2 domains or K2 domain only of t-PA linked to the protease domain of scu-PA). In a rabbit jugular vein thrombosis model the thrombolytic activity of K2tu-PA was reported to be significantly higher than that of both t-PA and scu-PA, whereas systemic effects were not different (Agnelli et al, 1992). In contrast, hybrids containing the growth factor domain of u-PA and the K2 and protease domain of t-PA were found to have a prolonged half-life but to be virtually inactive in a rabbit jugular vein thrombosis model (Asselbergs et al, 1993). An acylated recombinant chimera consisting of the fibrin-binding domains of plasminogen covalently linked to the protease domain of t-PA, was more potent and more fibrin-selective than t-PA upon bolus administration in a guinea-pig puhnonary embolism model (Robinson et al, 1992). CONJUGATES OF PLASMINOGEN ACTIVATORS AND ANTIFIBRIN MONOCLONAL ANTIBODIES The fibrin-specificity of thrombolytic agents may be improved by targetting the agent to a fibrin clot by conjugation with monoclonal antibodies which are fibrin-specific and do not cross-react with fibrinogen (Haber et at, 1989). Monoclonal antibodies that have been used for this purpose include antibodies against the BI}-chain of fibrin (MA-59D8) and against crosslinked human fibrin fragment D-dimer (MA-15C5) (reviewed in Dewerchin and Collen, 1991). In a rabbit thrombolysis model the t-PA59D8 conjugate was three to 10 times more potent than t-PA, and at equivalent thrombolytic concentrations it degraded less fibrinogen and consumed less oL2-antiplasmin (Runge et al, 1987). Chemical conjugates of scu-PA with MA-15C5, had a three- to six-fold enhanced thrombolytic potency in venous thrombosis models in rabbits and baboons. A recombinant chimeric plasminogen activator rscu-PA-32k/MA-15C5Hu, which consists of humanized MA-15C5 (MA-15C5Hu) and rscu-PA-32k, had a

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11-fold higher thrombolytic potency than rscu-PA in rabbits with jugular vein thrombosis (Dewerchin et al, 1992). Alternatively, a single-chain chimeric plasminogen activator consisting of a synthetic single-chain variable region (Fv) fragment of MA-15C5 and scu-PA-33k (Ala 132 through Leu 411 of scu-PA), had a 10-fold increased specific thrombolytic activity and a six-fold increased thrombolytic potency as compared to scuPA in a hamster pulmonary embolism model (Holvoet et al, 1993). These smaller fragments may be preferred moieties for the construction of recombinant chimeric scu-PA/antibody proteins for fibrin-targetted thrombolytic therapy. STAPHYLOKINASE Mature staphylokinase consists of 136 amino acids in a single polypeptide chain without disulphide bridges. Like streptokinase, staphylokinase is not an enzyme but it forms a 1 : I stoichiometic complex with plasmin(ogen) that activates other plasminogen molecules. Streptokinase and plasminogen produce a complex which exposes the active site in the plasminogen molecule without proteolytic cleavage, whereas generation of plasmin is required for exposure of the active site in the complex with staphylokinase (reviewed in Collen and Lijnen, 1994). Staphylokinase does not bind to fibrin, and fibrin stimulates the initial rate of plasminogen activation by staphylokinase only four-fold, as compared to two-fold by streptokinase. In purified systems oh-antiplasmin rapidly inhibits the plasmin.staphylokinase complex (second-order inhibition rate constant of approximately 2 × 1 0 6 M" s~), although it does not inhibit the plasmin(ogen).streptokinase complex. Addition of 6-aminohexanoic acid or of fibrin-like substances (e.g. CNBr-digested fibrinogen) induces a more than 100-fold reduction of the inhibition rate of the plasmin.staphylokinase complex by oL2-antiplasmin.Rapid inhibition by a2antiplasmin indeed requires the availability of the lysine-binding sites in the plasminogen moiety of the complex. More detailed studies on the interaction between staphylokinase, plasmin(ogen) and a~-antiplasmin have shown that neutralization of the plasmin.staphylokinase complex by o~2antiplasmin results in dissociation of functionally active staphylokinase from the complex, followed by its recycling to other plasminogen molecules. In plasma, conversion of plasminogen.staphylokinase to plasmin.staphylokinase does not occur at a significant rate because it is prevented by oL2antiplasmin; without plasmin.staphytokinase complex, no significant plasminogen activation occurs. In the presence of fibrin, generation of the ptasmin(ogen).staphylokinase complex is facilitated and inhibition of plasmin.staphylokinase by oh-antiplasmin at the clot surface is delayed. Recycling of staphylokinase to fibrin-bound plasminogen, after neutralization of the plasmin.staphylokinase complex, will result in more efficient generation of the active complex. This mechanism is mediated via the lysine-binding sites of plasminogen and results in a signficantly enhanced

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plasminogen activation at the fibrin surface. These regulatory properties of fibrin and a2-antiplasmin suggest that the fibrin-specificity of staphylokinase is due to rapid inhibition of generated plasmin.staphylokinase complex by oh-antiplasmin, and by a more than 100-fold reduced inhibition rate at the fibrin surface (reviewed in Collen and Lijnen, 1994). Recombinant staphylokinase (STAR) was found to have a potency for venous clot |ysis in hamsters and rabbits comparable to that of streptokinase (Lijnen et al, 1991). Additional studies in hamsters and dogs suggested that STAR may be relatively more potent than streptokinase towards platelet-rich clots, and potentially less immunogenic (Collen et al, 1992a). These findings were subsequently confirmed in baboons, where STAR was shown to have a thrombolytic potency towards jugular vein blood clots comparable to that of streptokinase, but to be less immunogenic and less allergenic. Indeed, repeated administration of STAR, in contrast to streptokinase, did not induce resistance to clot lysis in this model. In addition, STAR was found to be significantly more effective than streptokinase for the dissolution of platelet-rich arterial eversion graft thrombi (Collen et al, 1993). These encouraging results have formed the basis for the evaluation, on a pilot scale, of the phannacokinetic, thrombolytic and immunogenic properties of STAR in patients with acute myocardial infarction (Collen and Van de Weft, 1993). In four of five patients with acute myocardial infarction, 10 mg STAR given intravenously over 30 minutes was found to induce angiographically documented coronary artery recanalization within 40 minutes. Plasma fibrinogen and oh-antiplasmin levels were unaffected and allergic reactions were not observed. In a second series of five patients with acute coronary occlusion, intravenous administration of 10 mg STAR over 30 minutes induced recanalization in all patients within 20 minutes, without associated fibrinogen degradation (Schlott et al, 1994). However, in these patients, neutralizing antibodies were consistently demonstrable in plasma at 14-35 days (Collen and Van de Weft, 1993). Thus, with respect to immunogenicity, the initial observations in man are not as encouraging as the experience in baboons. Definition of the relative therapeutic benefit, or lack thereof of straphylokinase, will require more detailed dose-finding studies, followed by randomized clinical trials against presently available thrombolytic agents. CONJUNCTIVE ANTIPLATELET STRATEGIES The conjunctive use of potent and selective antiplatelet agents with plasminogen activators, might constitute improved thrombolytic strategies (Gold, 1990; Runge, 1990). Several approaches to reduce platetet aggregation via pathways other than cyclooxygenase inhibition have been explored in animal models. These include the use of platelet glycoprotein IIb/IIIa receptor-blocking agents, of thromboxane synthase inhibitors and antagonist of the serotonin or endoperoxide receptors (reviewed in Lijnen and Collen, 1993).

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Glycoprotein IIb/IIIa inhibitors include monoclonal antibodies that bind to the fibrinogen receptor, or small peptides that contain the arginine-glycine-aspartic acid (RGD) sequence. Following detailed evaluation in a number of animal models (reviewed in Coller, 1992), F(ab')2 fragments of monoclonal antibody 7E3 have been administered to humans with unstable angina (Gold et al, 1990) and Fab fragments to patients with AMI treated with rt-PA (Kleiman et al, 1991). Excessive haemorrhage was not observed in these patients and those receiving the Fab fragment of the antibody suffered the fewest thrombotic complications. However, no firm conclusions regarding efficacy could be drawn because of the small number of patients. Several snake venoms containing the RGD sequence have been evaluated in animal models of thrombolysis with rt-PA. These include bitistatin (from Bitis arietans), echistatin and kistrin (from Agkistrodon rhodostroma) (reviewed in Lijnen and Collen, 1993). Taken together, these studies indicate that the RGD-containing proteins yeided results comparable to those obtained with monoclonal antibody 7E3. No studies on humans have, however, been reported. Multiple antiplatelet agents other than glycoprotein IIb/IIIa antagonists have been developed which block different steps in the mechanism of platelet activation. These include cyclooxygenase inhibitors, thromboxane antagonists, serotonergic receptor antagonists and prostacyclin or its analogues (reviewed in Jang et al, 1992). Recently, combined thromboxane A2 synthase inhibitor/prostaglandin endoperoxide receptor antagonists have been developed. Thus, ridogrel was shown to enhance and sustain recanalization of platelet-rich arterial thrombosis with rt-PA in dogs (Collen et al,1992b). CONJUNCTIVE ANTITHROMBOTIC STRATEGIES Recent studies have shown a correlation between the efficiency of heparinization and coronary artery patency after treatment of AMI with rtPA (Amout et al, 1992; de Bono et al, 1992; Hsia et al, 1992). Thus, adequate anticoagulation with intravenous heparin appears to be required in order to sustain coronary artery patency after rt-PA therapy. More selective thrombin inhibition may allow prevention of platelet-rich arterial thrombosis, acceleration of clot lysis and reduction of early and late reocclusion after reflow (Gold, 1990; Runge, 1990). Hirudin, a 65 amino acid polypeptide isolated from the leech Hirudo medicinalis is a very potent and specific thrombin inhibitor. Hirudin-derived COOH-terminal peptides with a minimal length of 12 amino acids were also shown to have anticoaglant activity. Hirugen a synthetic peptide derived from residues 53-64 of hirudin, was shown to enhance rt-PA induced thrombolysis and to delay reocclusion in a canine left anterior descending coronary artery model (Yao et al, 1992). Different types of very selective synthetic thrombin inhibitors have been developed (reviewed in Hauptmann and Markwardt, 1992). In vivo studies in animal models have been performed with PPACK

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(D-Phe-Pro-Arg-CH2Cl) and with argatroban. In a hamster femoral vein platelet-rich thrombosis model, a synergistic effect was obtained with the combination of G-4120, a RGD-containing synthetic peptide, and argatroban (reviewed in Lijnen and Collen, 1993). Several alternatives to thrombin inhibition have recently been evaluated as antithrombotic strategies, including a monoclonal antibody neutralizing tissue factor activity, and selective inhibition of factor Xa with the tick anticoagulant peptide (TAP). A combination of activated protein C and tcuPA was shown to produce substantial and efficient antithrombotic effects without impairing haemostatic function in baboons with femoral arteriovenous shunt (reviewed in Lijnen and Collen, 1993). These different antithrombotic compounds may represent useful conjunctive strategies to accelerate thrombolysis and to prevent reocclusion. SUMMARY

Despite their widespread use in patients with acute myocardial infarction, all currently available thrombolytic agents suffer from a number of significant limitations, including resistance to reperfusion, the occurence of acute coronary reocclusion, and bleeding complications. Several lines of research towards improvement of thrombolytic therapy are being explored, including strategies to enhance the fibrinolytic potency of plasminogen activators and to improve conjunctive antiplatelet or antithrombotic agents. Mutants and variants of plasminogen activators, chimeric plasminogen activators, and conjugates of plasminogen activators with monoclonal antibodies have been constructed, and plasminogen activators from animal or bacterial origin have been evaluated. Some of these new thrombolytic agents have shown promise in animal models of venous or arterial thrombosis and in pilot studies in patients with acute myocardial infarction. Such molecules include mutants of tissue-type plasminogen activator (t-PA) with prolonged half-life and/or resistance to protease inhibitors and staphylokinase. Antiplatelet strategies include the use of platelet glycoprotein IIb/IIIa receptor blocking agents, of thromboxane synthase inhibitors and endoperoxide receptor antagonists. Antithrombotic strategies include the use of selective inhibitors of thrombin, tissue factor or factor Xa. The efficiency and safety of these new agents in man will have to be carefully evaluated.

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activator-induced thrombolysis and delays reocclusion. American Journal of Physiology 262: H374-H379. Van de Werf F, Lijnen HR & Collen D (1993) Coronary thrombolysis with KjK2P~,a chimeric tissuetype and urokinase-type plasminogen activator. A feasibility study in six patients with acute myocardial infarction. Coronary Artery Disease 4: 929-933. Witt W, Baldus B, Bringmann P e t al (1992) Thrombolytic properties of Desmodus rotundus (vampire bat) salivary plasminogen activator in experimental pulmonary embolism in rats. Blood 79: 1213-1217. Witt W, Maass B, Baldus Bet al (1994) Coronary thrombolysis with Desmodus salivary plasminogen activator in dogs. Fast and persistent recanalization by intravenous bolus administration. Circulation 90:421-426.