Reteplase (r-PA): a new plasminogen activator

Drug Evaluation Cardiovascular & Renal

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Reteplase (r-PA): a new plasminogen activator Christoph Bode, Benedikt Kohler, Martin Moser, Mark Schmittner, Richard W Smalling & Ruth H Strasser Reteplase (r-PA) is a genetically engineered deletion mutant of wild-type tissue-type plasminogen activator. The structural differences lead to different functional properties, such as a prolonged half-life. The compound demonstrated good thrombolytic efficacy in in vitro as well as in animal studies. In angiographically controlled patency studies (GRECO, GRECO-2 RAPID-1, RAPID-2), the double-bolus application scheme was established, and a superior patency profile for reteplase in comparison to alteplase was demonstrated. Mortality studies established reteplase as a safe drug with a 30-day mortality at least equivalent to streptokinase (INJECT) and very similar to alteplase (GUSTO-3). A possible advantage may be the doublebolus application without a need for weight adjustment, especially in a prehospital setting. Thus, reteplase can be regarded as an excellent alternative to streptokinase or alteplase for thrombolytic therapy in acute myocardial infarction. Key words: acute myocardial infarction, alteplase, plasminogen activators, reteplase, streptokinase, thrombolysis Exp. Opin. Invest. Drugs (1997) 6(8):1099-1104

1.

Introduction

Thrombolytic therapy has become an accepted form of treatment for acute myocardial infarction. The GUSTO trial showed that mortality reduction correlates with early, complete and sustained patency of the infarct-related coronary artery [1,2]. Current thrombolytic regimens achieve patency 90 min after initiation of treatment in only about 81% of cases, and only about 54% experience complete (TIMI grade 3) reperfusion; earlier patency rates are even more disappointing. Early reocclusion further limits the preservation of left ventricular function. In addition, even though patients are carefully selected, bleeding - especially intracranial bleeding - is a feared side-effect, limiting the applica-

bility of this form of treatment. In order to improve the risk/benefit ratio of thrombolytic therapy for patients, a number of new thrombolytic agents are being developed, primarily with the aim of reducing mortality by establishing more rapid, more complete and more stable coronary patency. This article summarises the molecular characteristics and pharmacological properties of reteplase obtained in vitro and in experimental animals, and reviews clinical results obtained in patency and mortality studies with this new drug. Although most studies of reteplase have expressed doses in megaunits (MU), on the basis of an amidolytic assay, doses in this review are cited in units (U) based on a new clot lysis assay. One MU equals one U in the new system.

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2.

Molecular characteristics of reteplase

Reteplase (recombinant plasminogen activator [r-PA]; BM 06.022; Boehringer Mannheim) is a genetically engineered deletion mutant of human tissue-type plasminogen activator (t-PA). Reteplase is produced by expression of an appropriately constructed plasmid in Escherichia coli, where it is localised in inclusion bodies. As for many proteins expressed in prokaryotic cells, the fully functional, non-glycosylated protein becomes available after an in vitro refolding process. SDS-polyacrylamide electrophoresis and amino acid analysis reveal a single-chain, non-glycosylated protein of 39.6 kDa, which consists of amino acids 1 - 3 and 176 - 527 of human t-PA. Reteplase consists of the kringle-2 and protease domains of human t-PA; the kringle-1, finger and epidermal growth factor domains have been deleted. The one-chain form can be cleaved by plasmin to a two-chain form [3,4]. The structural changes relative to human t-PA result in markedly different properties in vitro and in vivo.

3.

In vitro studies with reteplase

The structure and function of the enzymatic domain of human t-PA are largely retained by reteplase, although reteplase was shown to have only low affinity for fibrin; however, it is fibrin-specific in vivo at appropriate dosages [3]. The plasminogenolytic activity of reteplase in the absence of stimulatory CNBr fibrinogen fragments is similar to that of alteplase; however, in the presence of stimulator, its activity is about 4-fold less when compared on a molar basis [3]. Plasminogen activator inhibitor-1 (PAI-1) inhibition was similar for reteplase and t-PA, indicating that the structures of the kringle 2 and protease domains responsible for this were identical in the two molecules [5]. Presumably due to the lack of glycosylation and/or the lack of finger and growth factor domains, reteplase has been reported to exhibit a lower affinity for endothelial cells than t-PA [3]. Extensive studies on in vitro lysis of fresh, aged, platelet-poor and platelet-rich as well as whole blood clots have been performed [6]. To achieve 50% clot lysis 4 h into the experiment, 6.4 times higher molar concentrations of reteplase (vs. alteplase) had to be used. The data suggest that, on a molar basis in vitro, reteplase is less potent than alteplase, especially in lysing aged clots and platelet-rich clots. In further experiments, clots incubated with reteplase or alteplase were transfered to plasminogen activator free plasma. Clot lysis continued for 3 h with alteplase; in contrast, no further lysis occurred with reteplase. These differences may well be of interest in a clinical situation. © Ashley Publications Ltd. All rights reserved.

4.

Reteplase in experimental animals

The intended prolongation of half-life relative to t-PA could be demonstrated in rats, dogs and non-human primates [8]. In a rabbit model of jugular vein thrombosis, reteplase proved to be 5.3-times more effective than alteplase when both activators were given as a bolus. Plasma clearance was 4.3-fold lower than that of alteplase; thus the apparent higher potency may well be due to the lower clearance rate. At equipotent dosages, residual fibrinogen was similar for both activators; thus no relative loss in specificity was observed [8]. In a canine model of coronary thrombosis, reteplase was compared to alteplase, anistreplase, urokinase and streptokinase. Reperfusion was achieved significantly more rapidly with reteplase than with the other tested plasminogen activators, and, remarkably, bleeding time was least affected [9]. In another study utilising a dog model of coronary thrombosis, the dose of intravenous reteplase required to achieve 50% reperfusion was 11.6-fold lower than that for intravenous alteplase [10]. Further experiments suggest that an antithrombotic adjunct may be useful for preservation of patency after fibrinolysis with reteplase [11,12]. Double-bolus administration of reteplase was shown to be more effective than infusion or single-bolus dose regimens [13]. Reteplase also proved useful in reversing pulmonary hypertension in a dog model of pulmonary embolism. Due to its bolus application, reteplase acted faster than other plasminogen activators in these experiments [14]. Reteplase and alteplase had no effect on platelet count or, when given alone, on ex vivo platelet aggregation. In animals pretreated with aspirin, reteplase significantly reduced platelet aggregation in comparison with alteplase [15]; however, this difference was not observed in patients [16].

5.

Clinical efficacy

Phase I studies evaluating the effects of reteplase in healthy volunteers provided no data preventing Phase II testing [17,18]. The first Phase II trial with reteplase was designed as an open, sequential dose-finding study in patients with acute myocardial infarction [19]. With the first tested dose of 10 units (U) of reteplase, the predefined minimal 90-min patency of 70% was not achieved. A sequential probability ratio test after treatment of 42 patients yielded a 90-min patency rate (TIMI grade 2 or 3) of only 67%. An increased dose of 15 U reteplase, administered as a single bolus, resulted in a 90-min patency rate (TIMI grade 2 or 3) of 76% in the following 100 patients (74% at 60 min). Complete patency (TIMI grade 3) after 90 min was achieved for 69% of patients in the 15 U group and 52% of those treated with 10 U. Exp. Opin. Invest. Drugs (1997) 6(8)

Cardiovascular & Renal - Drug Evaluation 1101 5.1 GRECO

II

In an attempt to increase efficacy further, double-bolus administration of reteplase was investigated in an open, non-controlled, dose-finding study in 50 patients (GRECO 2). The 60- and 90-min patency rates (TIMI grade 2 or 3) after a 10 U + 5 U (30 min apart) double-bolus regimen were 72% and 78%, respectively; TIMI grade 3 patency was seen in 50% after 60 min and 58% after 90 min [20].

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5.2 RAPID-1

The 15 U bolus regimen, the 10 U + 5 U double-bolus regimen and a new 10 U + 10 U regimen of reteplase were compared to conventional alteplase (100 mg over 3 h) in an international, randomised study enrolling 606 patients with acute myocardial infarction (RAPID1) [21]. The 10 U + 10 U reteplase regimen was superior to the other reteplase regimens, and also achieved better 60- and 90-min TIMI grade 3 patency than alteplase (60-min: reteplase 51.0% vs. alteplase 32.7%, p < 0.01; 90-min: reteplase 62.7% vs. alteplase 49.3%, p < 0.05). Similarly, overall patency (TIMI grade 2 and 3) was superior with reteplase 10 U + 10 U, although this did not attain statistical significance (60-min: reteplase 77.6% vs. alteplase 66.3%; 90-min: reteplase 85.2% vs. alteplase 77.8%). Patency was achieved more rapidly with reteplase than with alteplase: 60-min TIMI grade 3 patency for reteplase (51.0%) was similar to 90-min TIMI grade 3 patency for alteplase (49.3%). Superior speed and completeness of reperfusion resulted in better preservation of left ventricular function in the 10 U + 10 U reteplase group [22]. The risk of bleeding was not significantly different for reteplase and conventional alteplase [23], and there was no significant difference between reteplase and alteplase with respect to platelet aggregation or thrombin activity during and up to 12 h after treatment of patients with acute myocardial infarction [16]. 5.3 INJECT

The INJECT study was designed to assess the safety and efficacy of reteplase in a larger study population [24]. Reteplase 10 U + 10 U was compared to streptokinase in 6010 patients with acute myocardial infarction in a double-blind, randomised trial with 35-day mortality as the primary end-point. The primary aim of the inject study was to demonstrate that the 10 U + 10 U double-bolus regimen of reteplase is at least equivalent to the standard regimen of streptokinase in terms of mortality. Equivalence was defined as a 35-day mortality rate for reteplase not more than 1% higher than for streptokinase with a 95% probability. The trial was not powered to demonstrate superiority and had design features (recruitment up to 12 h, trial confined to Europe) that made it difficult to compare to other trials © Ashley Publications Ltd. All rights reserved.

(e.g., GUSTO-1). Death up to 35 days after the index infarction occurred in 9.02% of 2994 patients in the reteplase group and 9.53% of 2992 patients in the streptokinase group. The difference of -0.51% (90% confidence interval [CI] -1.74 to 0.73) in favour of reteplase was non-significant. Thus, reteplase showed efficacy at least equivalent to that of streptokinase according to the above stated definition. Mortality at 6 months was also equivalent, with 11.02% in the reteplase group and 12.05% in the streptokinase group (difference -1.03%; 90% CI -2.65 to 0.59%). Patient follow-up was 99.6% complete. The incidence of cardiac shock (reteplase vs. streptokinase: 4.7% vs. 6.0%), heart failure (23.6% vs. 26.3%), hypotension (15.5% vs. 17.6%) and atrial fibrillation (7.2% vs. 8.8%) was significantly lower in the reteplase group (p < 0.05). The INJECT study also provided comparative data for safety and tolerability. The overall incidence of bleeding (reteplase 15% vs. streptokinase 15.3%) and significant bleeding (4.6% vs. 4.7%) was very similar. Also, there was no significant difference in the total incidence of stroke (reteplase 1.23% vs. streptokinase 1.00%) or haemorrhagic stroke (0.77% vs. 0.37%). Data from the invasive RAPID-1 and RAPID-2 studies support these findings in showing no excess side-effects for reteplase versus alteplase. 5.4

RAPID-2

After the GUSTO-1 study [1,2] had shown that an accelerated dose-regimen for alteplase (100 mg over 90 min) was superior to streptokinase, and possibly also to conventional alteplase therapy with respect to efficacy and safety, the RAPID-2 study was undertaken. The 10 U + 10 U reteplase regimen was compared to the GUSTO regimen of alteplase in an open, randomised trial enrolling 324 patients. The study design differed from RAPID-1 in that there was no age limit (RAPID-1 < 75 years) and patients were included up until 12 h after onset of pain (RAPID-1 < 6 h). Infarct-related 90-min coronary artery patency (TIMI grades 2 and 3) and complete patency (TIMI grade 3) rates were significantly higher in the reteplase-treated patients (TIMI grades 2 and 3: reteplase 83.4% vs. alteplase 73.3%; p = 0.03; TIMI grade 3: 59.9% vs. 45.2%; p = 0.01). At 60 min, the incidence of both patency and complete patency was also significantly higher in reteplase-treated patients (TIMI grades 2 and 3: reteplase 81.8% vs. alteplase 66.1%; p = 0.01; TIMI grade 3: 51.2% vs. 37.4%; p = 0.03). Reteplase-treated patients required fewer additional acute coronary interventions within the first 6 h post-dose (13.6% vs. 26.5%; p < 0.01), and there was no apparent increase in death (reteplase 4.1% vs. alteplase 8.4%), stroke (1.8% vs. 2.6%), haemorrhagic stroke (1.2% vs. 1.9%), Exp. Opin. Invest. Drugs (1997) 6(8)

1102 Reteplase (r-PA): a new plasminogen activator - Bode et al. reocclusion (9.0% vs. 7.0%) or bleeding rate (12.4% vs. 9.7%) [25].

United Kingdom), the United States of America and New Zealand.

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5.5 GUSTO-3

The GUSTO-3 study of 15,000 patients was designed to investigate whether the apparent advantages of reteplase in achieving early and complete patency would translate into a further decrease in mortality. As reported at the 1997 Annual Meeting of the American College of Cardiology, a preliminary analysis indicated that this was not the case. Mortality at 30 days was 7.43% for reteplase and 7.22% for front-loaded alteplase (p = n.s.). Furthermore, there was no significant advatage for any of the prespecified subgroups. Time to randomisation did not significantly influence results, although there was a slight trend towards patients coming in early faring better with reteplase (mortality in patients presenting within 0 - 2 h after onset of pain: reteplase 5.9% vs. alteplase 6.2%, p = n.s.). Infarct location was also not associated with a significant advantage for either drug (mortality in patients with anterior infarcts: reteplase 10.1 % vs. alteplase 9.3%, p= n.s.; mortality in patients with inferior infarcts: reteplase 4.9% vs. alteplase 5.2%, p = n.s.). There was also no difference in the mortality of patients stratified according to age group. The rate of stroke (reteplase 1.67% vs. alteplase 1.83%) and haemorrhagic stroke (0.91 vs. 0.88%) was also no different between the two agents. The GUSTO-3 Steering Committee formulated four conclusions based on the preliminary analysis of the mortality and safety data. • In this study of 15000 patients, r-PA did not significantly reduce mortality compared with accelerated t-PA. • No statistical differences in stroke or net benefit (death or nonfatal, disabling stroke) with r-PA versus accelerated t-PA were observed. • With 95% confidence, it is not possible to exclude the possibility that absolute mortality for accelerated t-PA is 1.1% better than for r-PA, or that mortality for r-PA is 0.7% better than for accelerated t-PA. • There is still some uncertainty as to whether these drugs can be regarded as equivalent. Choice of treatment will depend on further analysis of these data with long-term follow-up, cost of the drugs, ease of administration, and other issues.

6.

Regulatory status

Reteplase has been licensed for clinical use in the European Community (Austria, Belgium, Denmark, Finland, France, Germany, Greece, Ireland, Italy, Luxembourg, Portugal, Spain, Sweden, The Netherlands, © Ashley Publications Ltd. All rights reserved.

7.

Expert opinion

Double-bolus administration (10 U + 10 U) of reteplase results in effective, rapid and complete lysis of coronary thrombi in the majority of patients. With respect to these parameters, the 10 U + 10 U regimen was superior to established alteplase regimens in the RAPID studies. Patency appears to be an appropriate surrogate end-point for mortality, but only when investigated in large, multicentre, randomised trials with blinded, centralised assessment of results. That these requirements appear mandatory may be delineated from two examples. Double-bolus administration of alteplase was first investigated in a relatively small, single-centre, non-randomised study [26], suggesting very high patency rates, superior to those achievable with an accelerated alteplase infusion. In a larger study, the alteplase double-bolus in fact turned out to be slightly less effective than the accelerated alteplase infusion [27], and was associated with a higher mortality in a still unpublished mortality trial. A similar pattern could be noted when the GUSTO-2b investigators showed patency results for acute percutaneous transluminal coronary angioplasty (PTCA) much inferior to those previously reported in smaller trials [28,29]. In fact, the patency rates for reteplase-treated patients recruited within 6 h in the RAPID-2 study (TIMI grades 2 and 3: 86.5%; TIMI grade 3: 65%) are closer to those reported for acute PTCA in the GUSTO-2b trial (TIMI grades 2 and 3: 82%; TIMI grade 3: 75%) than those of other thrombolytic regimens, so that a large trial comparing prehospital thrombolysis with double-bolus reteplase (exploiting this logistic advantage of thrombolysis) to acute PTCA would be an interesting comparison. The GUSTO-3 study, however, provided evidence that the superior patency results for reteplase shown in the appropriately sized RAPID studies did not translate into a decrease in mortality. The reasons for this are under active investigation. One possible explanation may be sought in differences in mechanical revascularisation, especially rescue PTCA in GUSTO-3. Another possibility may be a differene in platelet activation, leading to a higher rate of early reocclusion for reteplase. Until proven otherwise, the higher early patency shown in RAPID-2 must be regarded as an advantage for reteplase; albeit an advantage that we are presently unable to preserve long enough to translate into a reduced 30-day mortality. Thus, the design of GUSTO-4, combining a potent antiplatelet agent (c7E3, Reopro) with reteplase, is a particularly interesting one. Prevention of early reocclusion may well have the advantage of Exp. Opin. Invest. Drugs (1997) 6(8)

Cardiovascular & Renal - Drug Evaluation 1103 tor BM 06.022 in rats, dogs, and nonhuman primates. Fibrinolyis (1992) 6:39-43.

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higher early patency rates, and may lead to reduced mortality in comparison to other thrombolytic agents. 8.

MARTIN U, FISCHER S, KOHNERT U et al.: Thrombolysis with an Escherichia coli-produced recombinant plasminogen activator (BM06.022) in the rabbit model of jugular vein thrombosis. Thromb. Haemost. (1991) 65:560564.

9.

MARTIN U, SPONER G, STREIN K: Evaluation of thrombolytic and systemic effects of the novel recombinant plasminogen activator BM 06.022 compared with alteplase, anistreplase, streptokinase and urokinase in a canine model of coronary artery thrombosis. J. Am. Coll. Cardiol. (1992) 19:433-440.

10.

MARTIN U, FISCHER S, KOHNERT U et al.: Coronary thrombolytic properties of a novel recombinant plasminogen activator (BM 06.022) in a canine model. J. Cardiovasc. Pharmacol. (1991) 18:111-119.

11.

MARTIN U, SPONER G, STREIN K: Hirudin and sulotroban improve coronary blood flow after reperfusion induced by the novel recombinant plasminogen activator BM 06.022 in a canine model of coronary artery thrombosis. Int. J. Hematol. (1992) 56:143-153.

12.

MARTIN U, FISCHER S, SPONER G: Influence of heparin and systemic lysis on coronary blood flow after reperfusion induced by novel recombinant plasminogen activator BM 06.022 in a canine model of coronary thrombosis. J. Am. Coll. Cardiol. (1993) 22:914-920.

13.

MARTIN U, SPONER G, KONIG R et al.: Double-bolus administration of the novel recombinant plasminogen activator (BM 06.022) improves coronary blood flow after reperfusion in a canine model of coronary thrombosis. Blood Coagul. Fibrinolysis (1992) 3:139-147.

14.

THE GUSTO INVESTIGATORS: An international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. New Engl. J. Med. (1993) 329:673-682.

MARTIN U, SPONER G, STREIN K: Rapid reversal of canine thromboembolic pulmonary hypertension by bolus injection of the novel recombinant plasminogen activator BM 06.022. J. Cardiovasc. Pharmacol. (1993) 7:365372.

15.

THE GUSTO ANGIOGRAPHIC INVESTIGATORS: The effects of tissue plasminogen activator, streptokinase, or both on coronary-artery patency, ventricular function and survival after acute myocardial infarction. New Engl. J. Med. (1993) 329:1615-1622.

MARTIN U, DALCHAU H, SPONER G: Effects of the novel recombinant plasminogen activator BM 06.022 on platelets and bleeding time in rabbits. Platelets (1992) 3:247-253.

16.

BODE C, KOHLER B, NORDT T et al.: Thrombin activity during and after thrombolysis with reteplase or alteplase in patients with acute myocardial infarction. Circulation (1994) 90(Suppl.):I-563. Abstract.

17.

MARTIN U, VON ME, AKPAN W et al.: Dose-ranging study of the novel recombinant plasminogen activator BM 06.022 in healthy volunteers. Clin. Pharmacol. Ther. (1991) 50:429-436.

18.

MARTIN U, VON ME, AKPAN W et al.: Pharmacokinetic and hemostatic properties of the recombinant plasminogen activator BM 06.022 in healthy volunteers. Thromb. Haemost. (1991) 66:569-574.

19.

NEUHAUS KL, VON ESSEN R, VOGT A et al.: Dose finding with a novel recombinant plasminogen activator (BM 06.022) in patients with acute myocardial infarction: results of the German Recombinant Plasminogen Activator Study. J. Am. Coll. Cardiol. (1994) 24:55-60.

20.

TEBBE U, VON ESSEN R, SMOLARZ A et al.: Open, noncontrolled dosefinding study with a novel recombinant plasminogen activator (BM 06.022) given as a double-

New thrombolytic agents, such as antibody-targeted plasminogen activators, have been produced as conjugates [30] and recombinant molecules [31], and may improve thrombolytic therapy further. The potency and specificity of these agents compare favourably to conventional plasminogen activators in different animal models [31,32]. More specific thrombolytic agents, which leave the clotting system largely intact, may well require equally specific anticoagulants as adjuncts [33]. While other new approaches are on the horizon, reteplase has proven at present to be an extremely potent and safe thrombolytic agent in patients with acute myocardial infarction, and one that offers the convenience of double-bolus injection. Although reteplase is currently marketed in the US and Germany at the exact same price as alteplase, competition may ultimately lead to a price reduction for both drugs, to the benefit of our health care systems and ultimately our patients. Advancements in mechanical strategies (e.g., newer stent generations) and possible combinations of both thrombolytic and ‘rescue’ mechanical strategies deserve further attention. The addition of ‘platelet-passivating’ agents, e.g., GP IIb/IIIa receptor blockers in the GUSTO-4 study, may well improve the outcome of thrombolytic, mechanical and combined strategies.

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Christoph Bode1, Benedikt Kohler1, Marti n Mos er1, Mark Schmittner 1, Richard W Smalling2 & Ruth H Strasser1 † Author for correspondence 1Medizinische Klinik III (Kardiologie, Angiologie und Pulmologie), Universität Heidelberg, 69115 Heidelberg, Germany 2Division of Cardiology, University of Texas Medical School at Houston, 6431 Fannin, Room 1.246 MSB, Houston, TX 77030, USA Address for correspondence Christoph Bode, M.D., Medizinische Klinik III (Kardiologie), Bergheimerstrasse 58, 69115 Heidelberg, Germany (Tel.: +49 6221 568611; Fax.: +49 6221 565513)

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