What do new lytics add to t-PA?

What do new lytics add to t-PA? Frans Van de Werf, MD, PhD Leuven, Belgium

Current thrombolytic therapy fails to induce early, complete, and sustained reperfusion in ±50% of the patients with ST-segment elevation acute coronary syndromes. There are two complementary approaches to improve thrombolytic therapy: the development of new fibrinolytics with enhanced fibrin specificity and/or reduced plasma clearance and the coadministration of new antithrombotic agents. The results obtained so far suggest that single-bolus fibrinolytic therapy is likely to replace the current infusions in the near future. This may result in a significantly earlier (prehospital) treatment of patients. The concomitant intravenous administration of a glycoprotein IIb/IIIa receptor antagonist (in combination with a reduced dose of a fibrinolytic) appears to be able to further enhance the efficacy for clot lysis without increasing the risk for bleeding complications. (Am Heart J 1999;138:S115-S120.)

Tissue-type plasminogen activator (t-PA), or alteplase, remains the “golden molecule” for fibrinolysis. No agent or thrombolytic regimen has been shown to be superior or even equivalent to an accelerated infusion of t-PA in association with intravenous heparin. Nevertheless, accelerated infusion of t-PA fails to induce early, complete, and sustained coronary artery patency in ±50% of the patients and is associated with an intracranial hemorrhage rate of 0.7% to 0.9%, depending on the proportion of elderly patients treated.1 The efforts for improving thrombolytic therapy have focused on the development of new fibrinolytics with enhanced fibrin specificity and/or prolonged plasma half-life and on the use of new antithrombotic cotherapies. The properties and clinical effects of the following new fibrinolytics will be discussed in comparison with alteplase: reteplase, lanoteplase, TNK-t-PA, saruplase, and staphylokinase. The key properties of these agents are summarized in Table 1.

Alteplase, t-PA Early experiments with wild-type and variants of t-PA have allowed identification of the regions involved in plasma clearance (epidermal growth factor and finger domains, the carbohydrate moieties in the kringle-1 and kringle-2 regions), in the enzymatic activity and interaction with Plasminogen Activator Inhibitor-1 (serine protease domain), and in the enhancement of plasminogen activation by fibrin (finger domain, kringle-2)2-4 (Fig 1). Collen et al5 have proposed several strategies for the genetic modification of the wild-type t-PA molecule.

Reteplase Reteplase (rPA) was the first of the third-generation fibrinolytics. This agent is already on the market in several countries. It is a deletion mutant of t-PA lacking the kringle-1, finger, and epidermal growth factor domains and the carbohydrate side chains. As a result, it has a plasma half-life twice that of alteplase and a decreased fibrin specificity. The clinical efficacy of reteplase was tested in the International Joint Efficacy Comparison of Thrombolytics (INJECT) trial, showing a small, nonsignificant benefit over streptokinase.6 In the Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO)-III trial, reteplase therapy did not improve 30day survival as compared with alteplase.7 From a strictly statistical point of view, the mortality results of GUSTOIII do not support equivalence between the two agents because the lower 95% boundary of the difference is >1%. These results are also in contrast with the improved patency rates observed with reteplase after 60 and 90 minutes in earlier angiographic trials.8,9 When judging these results, one should keep in mind that angiography gives merely a “snapshot” in time of the process of coronary artery recanalization. Therefore, significant differences at other time points may not be evident. For example, in a small subgroup of patients of the RAPID-2 angiographic study, reteplase therapy was associated with higher patency rates than alteplase at 60 or 90 minutes, whereas at 30 minutes higher patency rates were observed with alteplase. This initial advantage of alteplase may be clinically relevant.

Lanoteplase

From the Department of Cardiology, Gasthuisberg University Hospital. Reprint requests: Frans Van de Werf, MD, Department of Cardiology, Gasthuisberg University Hospital, Herestraat 49, B-3000 Leuven, Belgium. Copyright © 1999 by Mosby, Inc. @MS:0002-8703/99/$8.00 + 0 4/0/98545

Lanoteplase (n-PA) is a deletion mutant of alteplase with one amino acid substitution in position 117 of kringle-1 deleting a glycosylation site (Fig 2). Due to these structural changes lanoteplase has a markedly prolonged plasma half-life. In the Intravenous n-PA for Treatment of Infarcting Myocardium Early (InTIME)-I

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Table I. New vs established fibrinolytic agents SK

rt-PA

TNK-t-PA

r-PA

n-PA

rscu-PA

SAK

Mr (Daltons) 47,000 70,000 70,000 39,000 53,500 46,500 16,500 23-29 4-8 ±20 15 23 9 6 Plasma t1⁄2 (min) Fibrin specificity – ++ +++ + + ± ++++ Plasminogen activation indirect direct direct direct direct direct indirect Dose* 1.5 MU/60 min 100 mg/90 min 0.5/mg/kg bolus 2 × 10 MU bolus 120 U/kg bolus 80 mg/60 min 20-30 mg/30 min 30 min apart Antigenic + – – – – – + Hypotension + – – – – – – Patency at 90 min + +++ +++(+?) ++++ ++++ +++ +++(+?) Hemorrhagic stroke + ++ + or ++ ++ ? ++ ? Mortality reduction + ++ ? ++ ? +(+) ? Cost + +++ +++(?) +++ +++(?) ++(?) ++(?) Concomitant heparin† ? + + + + + + SK, Streptokinase; rt-PA, recombinant tissue-type plasminogen activator (alteplase); AMI, acute myocardial infarction; SAK, recombinant staphylokinase; TNK-t-PA, TNK-variant of t-PA; r-PA, reteplase; n-PA, lanoteplase; rscu-PA, recombinant single-chain urokinase type plasminogen activator (saruplase). *Most frequently used/tested. †With the exception of SK and rt-PA, the need for concomitant heparin has not been formally tested. (Reproduced from Circulation, 1998, volume 97, No. 16. With permission from the American Heart Association.)

trial, a single bolus of lanoteplase induced more TIMI grade 3 flow than alteplase (57.1% vs 46.4%, respectively).10 The finding of higher patency rates is in accordance with the lower incidence of adverse events at 30 days: The combined incidence of death, reinfarction, heart failure, and major bleeding was 11% with lanoteplase and 25% with alteplase.10 The large mortality trial, InTIME-2, has compared single-bolus lanoteplase with alteplase in 15,000 patients with acute myocardial infarction. The results will be available in 1999.

TNK-t-PA The t-PA mutant screening program identified a number of mutants with potentially interesting biological properties.11 TNK-t-PA is a triple-combination mutant of t-PA. In comparison with the wild-type t-PA molecule, 6 amino acids are substituted at 3 sites (Fig 2): asparagine for threonine at position 103 (“T”), glutamine for asparagine at position 117 (“N”), and a tetra-alanine sequence for the lysine-hystidine-arginine-arginine sequence at positions 296-299 (“K”).12 These mutations were conceived for the prolongation of the plasma halflife (T and N) and for the increase in fibrin specificity and of the resistance to Plasminogen Activator Inhibitor-1 inhibition (K). This new fibrinolytic has been tested in the phase I, dose-ranging Thrombolysis in Myocardial Infarction (TIMI)-10A trial.13 One hundred thirteen patients between 19 and 69 years of age were enrolled. The inclusion criteria were the presence of ischemic pain lasting for ≥30 minutes associated with ST-segment elevation of ≥0.1 mV in 2 or more contiguous leads or new left bundle-branch block and the ability to treat within 12 hours of symptom onset. A control arm of t-PA was

not included in this dose-escalation study. TIMI grade 3 flow was observed in 55% to 66% of the patients given 30 to 50 mg TNK-t-PA. These promising angiographic results were obtained at a high level of fibrin specificity, as shown by the minimal decrease in plasma levels of fibrinogen and plasminogen 60 minutes after the TNK-t-PA bolus (3% mean reduction of fibrinogen and <13% reduction of plasminogen).13 In contrast, frontloaded alteplase may induce a drop of 50% to 60% in fibrinogen and plasminogen levels.14,15 Administration of a single bolus of TNK-t-PA resulted in a plasma concentration profile similar to that of accelerated t-PA.16 The phase II evaluation program of TNK-t-PA comprised 2 studies: the efficacy trial, Thrombosis in Myocardial Infarction (TIMI)-10B, and the safety trial, ASsessment of the Safety of a New Thrombolytic: TNKt-PA (ASSENT)-I. These trials were conducted in parallel and their ultimate goal was to identify the optimal dose for testing in a large phase III trial. The TIMI-10B trial compared the efficacy of singlebolus TNK-t-PA and that of accelerated alteplase. A total of 886 patients were randomly assigned to receive either a single bolus of TNK-t-PA at 30 or 50 mg doses or front-loaded t-PA. Patients then underwent immediate angiography. During the initial phase of the trial, there were 3 intracranial hemorrhages among the 78 patients treated with the 50 mg TNK-t-PA dose (3.8%). This rate was considered unacceptably high; therefore the 50 mg dose was discontinued and replaced by a 40 mg dose in both the TIMI-10B and ASSENT-I trials. Concomitantly, the heparin dose was decreased. Similar results of TIMI grade 3 flow at 90 minutes after the start of thrombolysis were observed in the 40 mg dose TNKt-PA and the PA arms (62.8% vs 62.7%; P = not significant).17 The 30 mg dose of TNK-t-PA had a significantly

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Figure 1

Function-structure relation of t-PA.

lower rate of TIMI grade 3 flow than t-PA (54.3%), whereas the 50 mg dose achieved 65.8% TIMI grade 3 flow. At 60 minutes there were no significant differences in the rates of TIMI grade 3 flow or patency. Similar results were obtained with a TIMI frame count analysis. The median TIMI frame count at 60 minutes was slightly but not significantly lower for 40 mg TNKt-PA compared with t-PA (34 vs 40 frames, P = .33), suggesting faster and more complete reperfusion.17 A stratified analysis showed that the rate of TIMI grade 3 flow was also related to body weight. Indeed, when “weightcorrected” doses were calculated for patients receiving TNK-t-PA (as the dose per kilogram of body weight), it was shown that patients receiving higher “weight-corrected” doses (≥0.5 mg/kg) more often had TIMI grade 3 flow at the 90 minute angiogram than those receiving <0.5 mg/kg (62% to 63% vs 51% to 54%, respectively). An analysis of the corrected TIMI frame count also showed faster flow in patients who received higher doses per kilogram of body weight. On the basis of these findings, a “weight-corrected” dose regimen was selected for use in the phase III trial. The safety profile of single-bolus TNK-t-PA was tested in the ASSENT-I trial. This trial represents the largest phase II study ever performed with a new thrombolytic agent. A total of 3235 patients was randomized: 1705 received 30 mg, 1457 received 40 mg, and 73 received 50 mg TNK-t-PA.18 The patient outcome was favorable, with high survival rates and net clinical benefit (survival without stroke), especially in patients treated within 6 hours. No striking differences were found between the 30 mg and 40 mg TNK-t-PA groups with the exception of a

higher incidence of reinfarction in the 30 mg TNK-t-PA group. The 30-day total stroke rate was 1.5%, a value similar or slightly lower than those observed in other large trials of thrombolytic therapy. Intracranial hemorrhage occurred in 0.77% of the patients within the first 30 days after treatment. The incidence of bleeding complications and the need for transfusions were low (<3%). The decrease of the heparin doses early in the two trials resulted in a significant reduction of bleeding complications (including intracranial hemorrhage) without loss of thrombolytic efficacy.19 The rationale for performing a large phase II safety study was that phase II efficacy trials may give misleading results with respect to the risk of bleeding complications. This may be due to the extra risk of bleeding at the site of arterial access and, more importantly, the frequent use of additional antithrombotic therapies during the procedures. The latter may explain the higher incidence of intracranial hemorrhage observed in nearly all angiographic trials testing new thrombolytic agents (including the angiographic twin study TIMI-10B) as compared with the mortality trials performed subsequently with the same agent at the same dose. On the basis of the results of TIMI-10B and ASSENT-I, a dose of 0.50 to 0.55 mg TNK-t-PA/kg was selected for testing against accelerated t-PA in the phase III trial: Assessment of the Safety and Efficacy of a New Thrombolytic: TNK-t-PA (ASSENT-II). In the latter study 17,000 patients with ST-segment elevation acute myocardial infarction were randomly assigned, with allcause death at 30 days as the primary end point. The results will be available in the spring of 1999.

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Figure 2

Molecular structure of t-PA (alteplase), n-PA (lanoteplase), r-PA (reteplase), and TNK-t-PA. Reproduced from Brener SJ and Topol EJ. In: Topol EJ, editor. Acute coronary syndromes. Reprinted by courtesy of Marcel Dekker, Inc.

Saruplase Single-chain urokinase-type plasminogen activator (pro-urokinase) is a naturally occurring protein that is rapidly converted to urokinase by plasmin. However, it has intrinsic plasminogen-activating properties. Recombinant pro-urokinase, saruplase, has been studied in several clinical trials. In the Pro-urokinase in Myocardial Infarction (PRIMI) trial, saruplase therapy was associated with higher patency rates and slightly less fibrinogen breakdown than was streptokinase.20 In a comparison with alteplase in the Study in Europe with Saruplase and Alteplase in Myocardial Infarction (SESAM) trial, similar rates of TIMI grade 3 flow were observed after saruplase and after a 180-minute t-PA infusion.21 In the Comparative Trial of Saruplase vs Streptokinase (COMPASS) study, saruplase has been compared with streptokinase in 3089 patients. Thirty-day mortality rates were lower with saruplase than with streptokinase (5.7% vs 6.7%) at the cost, however, of an increased rate of intracranial hemorrhage (0.9% vs 0.3%).22

Staphylokinase Staphylokinase is a protein of bacterial origin that exhibits a unique mechanism of fibrin specificity.23

Two small angiographic studies have shown that recombinant staphylokinase was at least as effective as alteplase and significantly more fibrin-specific.24,25 The major disadvantage is that as a result of its bacterial origin, staphylokinase therapy induces an immune response. However, the immunogenicity may be reduced by site-directed mutagenesis. It is likely that a phase III large-scale mortality trial will be performed with a less immunogenic variant of staphylokinase.

New anticoagulant agents The major limitations of heparin cotherapy are related to its complex pharmacokinetics, with high variability in the individual response and an increased risk of bleeding when used in conjunction with thrombolytic therapy. The need for better anticoagulants resulted in the development of low-molecular-weight heparins and antithrombin III–independent agents. Low-molecular-weight heparins are associated with fewer variations in the anticoagulant response and are as effective as standard heparin in preventing thrombosis at lower activated partial thromboplastin time levels. Low-molecular-weight heparins will be tested as adjunctive therapy to fibrinolytic agents in the near future.

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Recently, the synthetic analogue of the antithrombin III–binding sequence of heparin, a pentasaccharide with pure anti–factor Xa activity, showed promising results in an animal model of thrombolysis.26 This agent is now being compared with standard heparin in conjunction with accelerated t-PA in 320 patients with acute myocardial infarction (PENTALYSE study). Anti–thrombin III–independent agents include hirudin and hirulog, direct factor Xa inhibitors (tick anticoagulant peptide), and tissue factor pathway inhibitor. Hirudin has been studied in the GUSTO-IIb trial and TIMI-9B trials.27,28 No significant, durable benefit over heparin was observed. The effects of hirudin with different fibrinolytics was not equal in GUSTO-IIb. There was little benefit when given in association with alteplase, but a significant improvement in clinical outcomes was observed in combination with streptokinase.29 These results are in accord with the findings of the Hirulog Early Reperfusion Occlusion (HERO)-1 trial.30 The 48% TIMI grade 3 flow rate obtained in this study with a combination of streptokinase, aspirin, and high-dose hirulog was superior to the patency rates obtained with streptokinase alone or with a combination of streptokinase and heparin. The effects of a combination of hirulog and streptokinase will be further tested in 17,000 patients in the HERO-2 mortality trial.

New antiplatelet agents The fact that aspirin brings a substantial benefit to thrombolysis is well known. However, this drug has similar limitations, including the relatively slow onset of action and the inhibition of only one of the several mechanisms responsible for platelet activation. A better approach to platelet inhibition may be to block the glycoprotein IIb/IIIa receptor, a key factor in the final common pathway of platelet aggregation. The first agent of this new class, abciximab, is a humanized monoclonal antibody to the glycoprotein IIb/IIIa receptor. The potential synergism between abciximab and thrombolytic therapy was first tested in the TAMI-8 pilot study.31 Coronary angiography performed after a mean of 121 hours showed TIMI grade 3 flow in 92% of the patients given abciximab versus 50% after conventional (3 hours) alteplase therapy. A combination of a reduced dose of a fibrinolytic and a full dose of a glycoprotein IIb/IIIa antagonist (abciximab) is currently being tested in angiographic trials (TIMI-14 and SPEED).32,33 A large mortality trial with this combination is being planned as well (GUSTO-IV Acute Myocardial Infarction).

Conclusions The new fibrinolytic agents discussed above fail to induce early TIMI grade 3 flow in >70% of the patients. Therefore these results may be considered disappoint-

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ing. Nevertheless, a new fibrinolytic that could induce early TIMI grade 3 flow in ±65% of the patients after a single bolus (as some of the new agents seem to be able to) may greatly facilitate prehospital thrombolysis and therefore may be associated with a significant increase in survival, particularly in patients who seek medical help in the first hours after the onset of symptoms. In parallel to the development of new fibrinolytics, new antithrombotic cotherapies, such as direct antithrombins, Xa-inhibitors, and glycoprotein IIb/IIIa antagonists, are being tested. The very encouraging results of a combination of abciximab and a 50% reduced dose of alteplase and reteplase observed in the TIMI-14 and SPEED trials should be mentioned here. It can be expected that combinations of some of the new fibrinolytics with abciximab or other glycoprotein IIb/IIIa inhibitors will also be tested in the near future.

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