Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

Transcription

1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Meyer G, Vicaut E, Danays T, et al. Fibrinolysis for patients with intermediate-risk pulmonary embolism. N Engl J Med 2014;370: DOI: /NEJMoa

2 Pulmonary Embolism Thrombolysis Study (PEITHO) ClinicalTrials.gov number: NCT New England Journal of Medicine Submission # Supplement This supplement contains the following items: 1. Original protocol, all subsequent amendments to original protocol (including final protocol), summary of changes; 2. Final statistical analysis plan (please note: the original statistical analysis plan was part of the original protocol and not a separate document)

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74 Thrombolysis in Pulmonary Embolism Page TP 1 Assistance Publique des Hôpitaux de Paris Délégation à la Recherche Clinique Clinical Trial Protocol Sponsor trial Number P EudraCT number Investigational product: Tenecteplase (TNK-tPA; TNK-tissue plasminogen activator) Title: PEITHO Pulmonary EmbolIsm THrOmbolysis Study A prospective, randomized, double-blind, placebo-controlled, international, multicentre, parallel-group comparison trial evaluating the efficacy and safety of single i.v. bolus tenecteplase plus standard anticoagulation as compared with standard anticoagulation in normotensive patients with acute pulmonary embolism and with echocardiographic (or spiral CT) and laboratory evidence of right ventricular dysfunction. Thrombolysis in Acute Pulmonary Embolism Clinical Phase: Chairman of Steering Committee : Co-Chairman of the Steering Committee and Principal Investigator: Coordinating Investigators: III Stavros KONSTANTINIDES Guy MEYER Giancarlo AGNELLI Samuel Z. GOLDHABER Nils KUCHER Eric VICAUT Date of Protocol: Second version 15 december 2008 Planned Dates of Trial: Second version December 15, 2008 Nov 2006 (FPI) Dec 2011 (LPO) Page 1 of 71

75 Thrombolysis in Pulmonary Embolism Page TP 2 ABBREVIATIONS AE(s) AMI aptt BI CDS cf. CI(s) CPR e-crf(s) CT scan CRO DSMB ECG(s) e.g. FPI GAMP GCP hr(s) IB ICH ICU IEC IRB ISF IU IV kg L LMWH LPI LPO Adverse Events Acute Myocardial Infarction Activated Partial Thromboplastin Time Boehringer Ingelheim Corporate Drug Safety Confer Confidence Interval(s) Cardiopulmonary Resuscitation electronic -Case Report Form(s) Computed Tomographic scanning Clinical Research Organization Data Safety and Monitoring Board Electrocardiogram(s) For Example First Patient In Good Automated Manufacturing Practice Good Clinical Practice Hour(s) Investigational Brochure 1) International Conference on Harmonization 2) Intracranial Hemorrhage Intensive Care Unit Independent Ethics Committee Institutional Review Board Investigators Site File International Unit(s) Intravenous Kilograms(s) Litre(s) Low Molecular Weight Heparin Last Patient In Last Patient Out Second version December 15, 2008

76 Thrombolysis in Pulmonary Embolism Page TP 3 MAH mg min mm mmhg MRI NS PE PI QA rt-pa SAE SOM SOPs SWFI TNK-tPA UFH VS Market Authorisation Holder (license holder) Milligram(s) Minute(s) Millimeter(s) Millimeter(s) of Mercury Magnetic Resonance Imaging Not Significant Pulmonary embolism Principle Investigator Quality Assurance Recombinant Tissue-type Plasminogen Activator Serious Adverse Event Standard Operating Manual Standard Operation Procedures Sterile Water for Injection TNK Tissue-type Plasminogen Activator Unfractionated heparin Versus Second version December 15, 2008

77 Thrombolysis in Pulmonary Embolism Page TP 4 CLINICAL TRIAL PROTOCOL SUMMARY Name of Sponsor: Assistance Publique des Hôpitaux de Paris Name of finished product: Metalyse Name of active ingredient: Tenecteplase SecondVersion December 15, 2008 Tabulated Study Protocol Trial number: Planned Study P period: Nov 2006 (FPI) Dec 2011 (LPO) A prospective, randomized, double-blind, placebo-controlled, international, multicentre, parallel-group comparison trial evaluating the efficacy and safety of single i.v. bolus tenecteplase plus standard anticoagulation as compared with standard anticoagulation in normotensive patients with acute pulmonary embolism and with echocardiographic (or spiral CT) and laboratory evidence of right ventricular dysfunction. Investigators: Study centre(s): Clinical phase: Objectives: Thrombolysis in Acute Pulmonary Embolism Multicentre Approximately 150 study centres in 10 countries III The primary objective is to demonstrate the clinical benefits of tenecteplase over placebo in normotensive patients with acute pulmonary embolism and right ventricular dysfunction. The secondary objective is to assess the safety after administration of tenecteplase in normotensive patients with acute pulmonary embolism and with echocardiographic (or spiral CT) and laboratory evidence of right ventricular dysfunction. Methodology: No. of subjects total: each treatment: Prospective, randomized, double-blind, multicentre, placebocontrolled Approximately 1,000 patients Approximately 500 patients Second version December 15, 2008

78 Thrombolysis in Pulmonary Embolism Page TP 5 Name of Sponsor: Assistance Publique des Hôpitaux de Paris Name of finished product: Metalyse Name of active ingredient: Tenecteplase Protocol date: Second Version December 15, 2008 Diagnosis and main criteria for inclusion: Test product: dose: mode of admin: Trial number: P Tabulated Study Protocol Planned Study period: Nov 2006 (FPI) December 2011 (LPO) Patients suffering from acute pulmonary embolism (first symptoms occurring within 15 days) confirmed by lung scanning or a positive spiral computed tomogram, or a positive pulmonary angiogram, presenting with right ventricular dysfunction on echocardiography (or spiral CT) and tested troponin I or T positive. Tenecteplase (Group A) Single body-weight (known or estimated) adjusted IV bolus administered over 5-10 seconds not later than 30 minutes after randomization, and not later than 2 hours after the diagnosis of RV dysfunction Weight (kg) Dose in mg Dose in units Dose in ml <60 30 mg 6000 U 6 ml >60 to <70 35 mg 7000 U 7 ml >70 to <80 40 mg 8000 U 8 ml >80 to <90 45 mg 9000 U 9 ml >90 50 mg U 10 ml Concomitant therapy-unfractionated heparin The dose of 80 IUxKg -1 as an intravenous bolus, followed by an infusion of 18 IUxKg -1 xh -1,(with a maximum of 1800 IU xh -1 ) to be administered immediately after randomization in all patients for at least 48 hours following randomization. Beyond this period, intravenous UFH may be substituted with subcutaneous heparin (LMWH) treatment. The bolus will be omitted when heparin was started before randomisation. Second version December 15, 2008

79 Thrombolysis in Pulmonary Embolism Page TP 6 Reference therapy Placebo (Group B) dose: Single body-weight (known or estimated) adjusted IV bolus mode of admin: administered over 5-10 seconds not later than 30 minutes after randomization, and not later than 2 hours after the diagnosis of RV dysfunction. Weight (kg) Dose in ml <60 6 ml >60 to <70 7 ml >70 to <80 8 ml >80 to <90 9 ml >90 10 ml Concomitant therapy-unfractionated heparin The dose of 80 IUxKg -1 as an intravenous bolus, followed by an infusion of 18 IUxKg -1 xh -1, (with a maximum of 1800 IU xh -1 ) to be administered immediately after randomization in all patients for at least 48 hours following randomization. Beyond this period, intravenous UFH may be substituted with subcutaneous heparin (LMWH) treatment. The bolus will be omitted when heparin was started before randomisation Name of Sponsor: Assistance Publique Hopitaux de Paris Name of finished product: Metalyse Name of active ingredient: Tenecteplase Protocol date: Second version 15/12/08 Duration of treatment: Reference therapy: Tabulated Study Protocol Trial number: Planned Study P period: Nov 2006 (FPI) December 2011 (LPO) Tenecteplase, as single IV bolus over 5-10 seconds. Tenecteplase placebo, as single IV bolus over 5-10 seconds Second version December 15, 2008

80 Thrombolysis in Pulmonary Embolism Page TP 7 Criteria for efficacy: Primary end point: Clinical composite endpoint of all-cause mortality or haemodynamic collapse within 7 days Haemodynamic collapse is defined as: need for cardiopulmonary resuscitation; or systolic blood pressure < 90 mm Hg for at least 15 min or drop of systolic blood pressure by at least 40 mm Hg for at least 15 min with signs of end organ hypoperfusion (cold extremities or low urinary output < 30 ml/h or mental confusion); or the need for catecholamines to maintain adequate organ perfusion and a systolic blood pressure of >90 mm Hg (including dopamine at the rate of >5 micrograms / kg per minute). Secondary end points: Death within 7 days Haemodynamic collapse within 7 days Confirmed symptomatic pulmonary embolism recurrence within 7 days Death within 30 days Criteria for safety: Safety secondary end-points: Total strokes (intra cranial haemorrhage or ischaemic stroke) within 7 days Major bleeding (other than ICH) within 7 days defined as: Moderate or severe bleed. Serious adverse events within 30 days Second version December 15, 2008

81 Thrombolysis in Pulmonary Embolism Page TP 8 Statistical methods: Intent-to-treat analysis on superiority of tenecteplase over placebo. Primary criterion: The primary ITT analysis on the primary endpoint will be carried out by a two-sided chi-square test on proportions. In addition the 95% confidence interval on the oddsratio will be presented. Secondary Efficacy Criteria: The secondary ITT analysis will be carried out by the chi-square test on proportions. In addition, the corresponding 95% confidence interval on the odds ratio will be presented. The survival status during 30 days follow-up will be analyzed by showing Kaplan-Meier-curves and treatment differences will be compared by means of log-rank test. Safety Analysis: Safety end-points and (Serious) adverse events will be tabulated per treatment group. All (dichotomized) endpoints will be analyzed by chi-square test on proportions and the 95% confidence interval on the odds-ratio will be presented. In addition, continuous safety monitoring (in a blinded fashion) will be done by the Data and Safety Monitoring Board. Interim analyses Interim analyses will be made by the Data Centre after recruitment of 25%, 50%, and 75% of the total number of patients. These analyses will be made in order to allow sample size reassessment or early stop of the trial for efficacy or futility. For these reasons, the α-spending functions determining the boundary limits for the interim analysis will be chosen to require an adjustment of total P value by less than (P=0.047 for final analysis). Second version December 15, 2008

82 Thrombolysis in Pulmonary Embolism Page TP 9 FLOW CHART Enrolment In-hospital 7-day follow-up 30-day follow-up Medical history X Demographic data X Baseline information Haematology a Troponin I/T c Echocardiography d Inclusion - exclusion criteria Informed consent Randomisation Study drug administration X X b X b X aptt monitoring X X Observation/Recording of: Haemodynamic collapse X X Symptomatic Pulmonary Embolism recurrence Major and minor bleeds X e X e X X X X X X e X e Stroke (haemorrhagic or ischaemic) X e X Serious Adverse Events X X X X Mortality X X X X a b c According to hospital practice, but it is recommended to include at least haemoglobin, haematocrit and platelet count; except for the baseline status and the case of major bleeds, measurements may be performed at any time during the in-hospital stay; values will not be recorded in the CRF. In case of major bleeds (blood transfusions required), haemoglobin, haematocrit and platelets should be measured according to local practice, but need not be recorded in the CRF. Only number of blood units (<2/>2 units) and a drop in Hb levels by >2 g/dl need to be entered in the CRF. Either troponin I or troponin T testing performed according to hospital practice. d RV dysfunction can be assessed using multidetector-row CT if echocardiography is not available. e To be assessed/recorded at any time during hospitalization if there are signs or symptoms suggestive of pulmonary embolism recurrence or of a bleeding complication. Second version December 15, 2008

83 Thrombolysis in Pulmonary Embolism Page TP 10 TABLE OF CONTENTS ABBREVIATIONS...2 CLINICAL TRIAL PROTOCOL SUMMARY...4 FLOW CHART...9 TABLE OF CONTENTS INTRODUCTION SCIENTIFIC BACKGROUND DRUG PROFILE Tenecteplase Unfractionated Heparin (UFH) RATIONALE FOR AND DESCRIPTION OF THE TRIAL Echocardiography Spiral CT Cardiac troponins Benefit/risk assessment STUDY OBJECTIVES GENERAL AIM / PRIMARY OBJECTIVE PRIMARY ENDPOINT SECONDARY ENDPOINTS Efficacy Safety STUDY POPULATION NUMBER OF PATIENTS PLANNED INCLUSION CRITERIA Criteria for right ventricular dysfunction Troponin I or T testing EXCLUSION CRITERIA WRITTEN INFORMED CONSENT TREATMENT TREATMENT REGIMENS TO BE COMPARED Investigational product Comparator drug Dosage and treatment schedule Group A Group B Order and timing of study medication given / procedures Administration of study drug Labelling and packaging Storage conditions CONCOMITANT THERAPY Non rescue concomitant dopamine Rescue medication and additional treatment in case of haemodynamic collapse...33 Second version December 15, 2008

84 Thrombolysis in Pulmonary Embolism Page TP Anticoagulation - Administration of Vitamin K antagonists Other medication Restrictions TREATMENT COMPLIANCE OBSERVATIONS EFFICACY Primary end point Secondary endpoints SAFETY OTHER Medical history and physical examination Lung scanning or spiral computed tomogram or pulmonary angiogram Echocardiography Cardiac troponin T or I aptt monitoring Chest X-ray Haematology Modified Rankin scale INVESTIGATIONAL PLAN STUDY DESIGN AND PLAN STUDY ORGANIZATION Steering Committee Executive Committee STUDY PROCEDURES Enrolment and in-hospital period Concomitant medication Clinical assessment of efficacy and safety Follow-up period day follow-up day follow up assessments ADHERENCE TO PROTOCOL Visit schedule Criteria and rules for stopping subject treatment Criteria for termination of the trial STATISTICS STATISTICAL DESIGN / MODEL NULL AND ALTERNATIVE HYPOTHESES PLANNED ANALYSES Populations to be analysed Patient accountability Baseline characteristics Subgroup analyses Interim analyses EFFICACY ANALYSIS Main Efficacy Criterion Secondary Efficacy Criteria...50 Second version December 15, 2008

85 Thrombolysis in Pulmonary Embolism Page TP SAFETY ANALYSIS EXPLORATORY ANALYSIS HANDLING OF MISSING DATA RANDOMISATION SAMPLE SIZE ISSUES ADMINISTRATIVE ISSUES ETHICS Institutional Review Board or Independent Ethics Committee Informed consent and subject information Unblinding RECORDS Drug accountability Emergency code break (also see Unblinding, above) Case Report Forms Source documents Direct access to source data / documents Processing of data QUALITY ASSURANCE AUDIT Audit Monitoring PROCEDURES Adverse Events Background Expedited safety reporting in the study The Safety Group Data and Safety Monitoring Board RULES FOR AMENDING PROTOCOL DISCONTINUATION OF THE TRIAL BY THE SPONSOR PATIENT CONFIDENTIALITY PUBLICATION POLICY SIGNATURE PAGE REFERENCES APPENDICES APPENDIX 1: HEPARIN NOMOGRAM...70 APPENDIX 2: PULMONARY EMBOLISM-RELATED EVENTS APPENDIX 3: DEFINITIONS...72 Second version December 15, 2008

86 Thrombolysis in Pulmonary Embolism Page TP INTRODUCTION 1.1. SCIENTIFIC BACKGROUND Pulmonary Embolism (PE) afflicts more than 100,000 patients annually in the United States and Europe and has a high mortality rate. For example, in the International Cooperative Pulmonary Embolism Registry (ICOPER) which included 2,454 PE patients from 7 countries, 3-month mortality was as high as 15% [1]. Thrombolysis is standard therapy in patients with massive PE and cardiogenic shock. However, the use of thrombolysis in PE patients with preserved systemic arterial pressure remains controversial. If thrombolysis is harmful or not helpful in these patients, then it should not be administered. A positive study in favor of thrombolysis will expand the use of thrombolysis to patients with right ventricular dysfunction (i.e. echo-positive and troponin-positive patients), with savings of lives and a reduction in other serious adverse events and particularly haemodynamic collapse necessitating cardiopulmonary resuscitation or catecholamine administration. A negative clinical trial will lead to a significant reduction in thrombolysis use, with reduction of major bleeding, including intracranial haemorrhage, reduced length of hospital stay, and cost savings. There are only 11 randomized PE trials of thrombolysis versus heparin to date [2-12], with a total of 748 patients. In a recent meta-analysis, there was a (non-significant) trend towards reduction of mortality or PE recurrence with thrombolysis compared to heparin alone [13]. Table 1 shows the mortality data which are available for 10 of these studies [2-4;6-12] with a total number of 717 randomized patients. Second version December 15, 2008

87 Thrombolysis in Pulmonary Embolism Page TP 14 Table 1. Mortality rates in randomized PE trials. Study Heparin/Thrombolysis N n/n Mortality (n/n) Relative Risk* (95%CI) UPET (1973) /82 7/ ( ) Tibbutt (1974) 30 17/13 1/ ( ) Ly (1978) 25 11/14 2/ ( ) Marini (1988) 30 10/20 0/0 - PIOPED (1990) 13 4/9 0/ ( ) Levine (1990) 58 25/33 0/ ( ) PAIMS 2 (1992) 36 16/20 1/ ( ) Goldhaber (1993) /46 2/ ( ) Jerjes-Sanchez (1995) 8 4/4 4/ ( ) Konstantinides (2002) /118 3/ ( ) Overall / (5.6%) / 15 (4.2%) 0.75 ( ) *for mortality with thrombolysis. The potential benefit of PE thrombolysis must be weighed against the increased risk of major haemorrhage. In an overview of the trials presented in Table 1, thrombolysis appears to have increased the rate of major bleeding (Table 2). Second version December 15, 2008

88 Thrombolysis in Pulmonary Embolism Page TP 15 Table 2. Major Bleeding Episodes in randomized PE Trials. Heparin/Thrombolysis Study n n/n Major bleeding (n/n) Relative Risk (95%CI)* UPET (1973) /82 11/ ( ) Tibbutt (1974) 30 17/13 1/ ( ) Ly (1978) 25 11/14 2/ ( ) Marini (1988) 30 10/20 0/0 - PIOPED (1990) 13 4/9 0/ ( ) Levine (1990) 58 25/33 0/0 - PAIMS 2 (1992) 36 16/20 2/ ( ) Goldhaber (1993) /46 1/ ( ) Jerjes-Sanchez (1995) 8 4/4 0/0 - Konstantinides (2002) /118 5/ ( ) Overall / (5.0%) /38 (10.6%) 2.11 ( ) *for major bleeding after thrombolysis compared to heparin alone. Of note, the difference in major bleedings observed between thrombolytic therapy and heparin is mainly driven by the UPET study, in which all patients were submitted to invasive diagnostic procedures. When the bleeding rate is tabulated from the studies using noninvasive procedures for the diagnosis of PE [8;10;12] the bleeding rate is lower in both treatment groups and does not differ significantly between thrombolytic therapy (3 major bleedings among 201 patients; 1.5%) and heparin (6 major bleedings among 222 patients; 2.7%). MAPPET-3: Results and Limitations The largest randomized double-blind thrombolysis trial of 256 haemodynamically stable patients with submassive PE (MAPPET-3) [12] compared 100 mg tpa over 2 hours versus therapy with heparin alone. This study included patients with evidence of right ventricular dysfunction by echocardiography, ECG parameters, or right heart catheterization. The combined end point, defined as death or escalation of therapy (defined as the need for cardiopulmonary resuscitation, pressors, mechanical ventilation, interventional or surgical Second version December 15, 2008

89 Thrombolysis in Pulmonary Embolism Page TP 16 embolectomy, or secondary rescue thrombolysis) was significantly reduced in patients who received thrombolysis plus heparin compared to heparin alone. On the other hand, there was no difference in mortality, which was low in both treatment groups, and no difference in the individual end points comprising escalation of treatment, except for rescue thrombolysis. Rescue thrombolysis was administered when the treating physician judged that the patient s clinical condition was deteriorating based on the following criteria: 1) worsening of symptoms, particularly dyspnea; 2) worsening respiratory failure; 3) arterial hypotension or shock; 4) persistent or worsening pulmonary hypertension or right ventricular dysfunction. Rescue thrombolysis was administered in 23.2% of patients in the heparin group and to 7.6% of those in the thrombolysis group. However, since the publication of MAPPET-3, rescue thrombolysis (cross-over) has been criticized as a rather soft end-point which drove the composite end point to statistical significance DRUG PROFILE Tenecteplase Tenecteplase is a derivative of the wild-type tissue-type plasminogen activator, tpa (identical to the recombinant marketed agent, rtpa, alteplase) with three amino acid site substitutions: 1) a threonine (T) replaced by an asparagine, which adds a glycosylation site to position 103; 2) an asparagine (N) replaced by a glutamine, thereby removing a glycosylation site from position 117; and 3) four amino acids, lysine (K), histidine (H), arginine (R), and arginine (R), replaced by four alanines at the third site. In pharmacokinetic studies in patients with acute myocardial infarction, tenecteplase was shown to exhibit biphasic disposition kinetics with an initial half-life of min and a terminal half-life of min. The initial half-life accounted for appr. 70 % of AUC (area under the curve) [14]. Second version December 15, 2008

90 Thrombolysis in Pulmonary Embolism Page TP 17 Tenecteplase has a longer half-life, a higher fibrin-selective activity and a lower affinity for PAI-1 than rt-pa [15]. The efficacy of a single bolus of Tenecteplase in restoring coronary artery flow in patients with acute myocardial infarction (AMI) has been clearly demonstrated [16,17]. The results of these studies suggested that a bodyweight-adjusted single bolus of mg/kg Tenecteplase is equivalent to a 90-minute regimen of rt-pa with regard to efficacy and safety in patients with AMI. To definitively show this equivalence, single-bolus Tenecteplase was compared with front-loaded rt-pa in AMI in a double blind randomized trial [16]. The increased fibrin specificity and single bolus administration of Tenecteplase do not increase the risk of intracranial bleeding with respect to rt-pa, but are associated with less non-cerebral bleeding, especially among high-risk patients [17]. Recent observations support the potential use of Tenecteplase in patients affected by PE [18,19]. Tenecteplase is less inactivated by PAI-I and this could make this drug particularly effective in the treatment of PE. PAI-I is mostly contained in platelets. Recent observations supporting the role of platelets in PE could be in favor of the use of Tenecteplase for this indication [20]. Unfractionated Heparin (UFH) Intravenous unfractionated heparin (UFH) remains the mainstay of immediate treatment of pulmonary embolism [21]. For patients receiving thrombolytics, heparin (i.e. antithrombin) therapy is also important, since it inhibits both the thrombin which has already been formed prior to thrombolysis and the thrombin which is generated as a consequence of administration of the thrombolytic agent. Furthermore, thrombin is one of the main stimuli responsible for platelet activation in the setting of thrombolysis. UFH used as adjuvant to thrombolytics can inhibit thrombin activity and, to a lesser extent, thrombin generation [22]. Although combination of thrombolytic agents with low molecular weight heparins (LMWH) is theoretically also possible, there is very limited experience with the use of LMWH in submassive PE (i.e., in patients with known right ventricular dysfunction). Furthermore, administration of LMWH as an adjuvant to thrombolysis has not yet been clinically tested in the setting of acute pulmonary embolism. Second version December 15, 2008

91 Thrombolysis in Pulmonary Embolism Page TP RATIONALE FOR AND DESCRIPTION OF THE TRIAL In haemodynamically unstable patients with acute massive PE, thrombolysis may be lifesaving [11]. However, recent registries and prospective trials also suggested a beneficial effect of thrombolysis in patients with PE who do not present with shock or hypotension [10,12,23]. For example, in one of the largest trials to date [10], thrombolytic treatment resulted both in an improvement of lung perfusion as assessed by scintigraphy (by 14.6%, compared to 1.5% in patients who received heparin alone) and in an increase (in 39% vs. 17% of the patients) of right ventricular wall motion on echocardiography. However, PE has a wide spectrum of severity at presentation and, given the bleeding risk associated with thrombolytic treatment, it is conceivable that the use of more aggressive treatments should be reserved to patients at high risk for adverse outcome. Hence, it is of critical importance to identify predictors of adverse outcome in patients with pulmonary embolism. Risk stratification in the proposed trial will include cardiac troponin testing and echocardiography in all patients. Echocardiography In the large ICOPER registry, right ventricular dysfunction on the baseline echocardiogram was an important predictor of in-hospital death [1]. In the present trial, right ventricular dysfunction will be diagnosed if at least one of the following echocardiographic findings is present [24]: Right ventricular end-diastolic diameter (RVEDD) > 30 mm (parasternal long-axis or short-axis view), Right/left ventricular end-diastolic diameter (RVEDD/LVEDD) >0.9 (apical or subcostal 4-chamber view), Right ventricular free wall hypokinesis from any view, Tricuspid systolic velocity > 2.6 m/s from the apical or subcostal 4-CH view, parasternal short-axis view. Second version December 15, 2008

92 Thrombolysis in Pulmonary Embolism Page TP 19 Spiral CT Several studies have assessed the value of spiral CT for the diagnosis of right ventricular dysfunction in patients with PE. The ratio of the RV to LV short axis diameter was used in most studies to assess right ventricular dysfunction. RV and LV dimensions are measured by identifying the maximal distance between the ventricular endocardium and the interventricular septum perpendicular to the long axis. The RV/LV diameter ratio can be measured on multiplanar reformats of axial CT data or directly on axial CT images [25,26]. Two different cutoff values of RV/LV diameter ratio have been reported: a ratio greater than 0.9 and a ratio greater than 1.0. Both values are significantly associated with an adverse clinical outcome. In one study the Hazard Ratio of RV/LV diameter ratio > 0.9 for predicting 30-day death was 5.17 (95% CI: 1.63 to 16.35; p = 0.005), after adjusting for confounders [25]. In another series, the positive predictive value for PE related mortality with an RV/LV ratio greater than 1.0 was 10% [26]. Cardiac troponins Recently, cardiac troponins have been shown to be highly useful for identifying patients at risk for PE-related death and other serious adverse clinical outcomes [27]. Release of cardiac troponins in patients with acute PE is induced by myocardial shear stress due to severe right ventricular pressure overload rather than by myocardial ischaemia due to coronary artery disease. The prognostic information obtained from troponin testing adds to established prognostic indicators such as clinical severity of PE and evidence of right ventricular dysfunction by echocardiography. In a study of 106 PE patients, the negative predictive value of troponin I and T for adverse clinical outcomes was 92% and 93%, respectively [28]. In another study, all 8 patients with PE who died in-hospital presented with elevated troponin T or I levels [29]. A recent overview confirmed that currently available cardiac troponin I and T assays all have a high negative predictive value for in-hospital mortality [30]. More recently, data from 20 studies (1985 patients) using several different troponin assays, were included in a meta-analysis [31]. Overall, 122 of 618 patients with elevated troponin levels died (19.7%;95% CI, 16.6 to 22.8) compared with 51 of 1367 with normal troponin levels (3.7%; 95% CI,2.7 to 4.7). Elevated troponin levels were significantly associated with short-term mortality (odds ratio, 5.24; 95% CI, 3.28 to 8.38) and with adverse outcome events (odds ratio, 7.03; 95% CI, 2.42 to 20.43). Four studies (252 patients) reported the incidence of Second version December 15, 2008

93 Thrombolysis in Pulmonary Embolism Page TP 20 adverse outcome in patients with normal blood pressure at hospital admission. Adverse outcome events were seen in 38 of 103 patients with elevated troponin levels (36.9%) compared with 32 of 149 patients with normal troponin levels (21.5%). In these patients, elevated troponin levels were significantly associated with adverse outcome events (odds ratio, 4.12; 95% CI, 0.71 to 23.86). Results were consistent for troponin I or T over a wide range of threshold value ranging from 0.06 µg/l to 2 µg/l for troponin I and from 0.01 µg/l to 0.10 µg/l for troponin T. [31]. In the present trial, we will include troponin-positive patients only. A positive troponin test will be defined as follows: Criteria for a positive cardiac Troponin test: Troponin I > 0.06 µg/l or troponin T > 0.01 µg/l using the test of the Department of Clinical Chemistry at the participating site. The predictive value for adverse outcomes increases when results of echocardiography and troponin tests are combined. In a study of 91 patients with acute PE, the positive predictive value of the combination of echocardiography and troponin I for adverse clinical outcomes was higher (75%; 95%CI, 55-88%) compared with each test alone (echocardiography: 41%; 95%CI, 28-56%; troponin I: 64%; 95%CI, 46-79%) [32]. From the available data [27,28,32], it can be expected that approximately 20-25% of all patients with acute PE will present with troponin elevation and echocardiographically confirmed right ventricular dysfunction (Table 4): Study N Troponin + ECHO positive N (%) Konstantinides, (20) Giannitsis, (31) Kucher, (26) Total (24) Second version December 15, 2008

94 Thrombolysis in Pulmonary Embolism Page TP 21 Benefit/risk assessment A more rapid lysis of pulmonary emboli and a more rapid reduction of pulmonary hypertension can be obtained by thrombolytic agents as compared with heparin. However, it is still undefined whether these advantages of thrombolytic treatment result in a clinical benefit, outweighing the increased risk of bleeding complications. For example, the haemodynamic benefits of thrombolysis compared to heparin treatment are confined to the first few weeks or even days [33,34], but it needs to be emphasized that that most in-hospital deaths from acute pulmonary embolism occur very early during hospitalization, i.e. within the first 1-2 hours after admission [35]. Furthermore, randomized trials showed thrombolysis to be associated with a significant risk of major bleeding (Table 2), including a 2% intracranial or fatal bleeding rate, but this needs to be weighed against the 10-20% in-hospital death risk of selected patients with PE and right ventricular dysfunction [36-39]. In addition, and importantly, the (more recent) trials which used non-invasive procedures for the diagnosis of PE [8,10,11,12], showed lower bleeding rates both in the thrombolysis and in the heparin groups. To date, no conclusive clinical outcome studies aimed at comparing thrombolysis and heparin in patients with pulmonary embolism are available. Recent meta-analyses [13] appear to suggest that stable patients with PE may not significantly benefit from thrombolytic treatment in terms of a reduction in in-hospital mortality, but it must be emphasized that most of the trials that were included in these analyses were not (with the exception of MAPPET-3) designed to test clinical end points, and many of them did not have the statistical power to do so due to their small size. MAPPET-3 [12] did suggest that early treatment with alteplase may improve the clinical course of stable patients with acute PE, and particularly that it may reduce the risk of clinical deterioration requiring emergency escalation of treatment, but the differences in the combined end point were driven entirely by a reduction in the rate of secondary thrombolysis. Thus the results of this trial cannot be regarded as definitive proof of the benefits of thrombolysis in stable patients with PE. Second version December 15, 2008

95 Thrombolysis in Pulmonary Embolism Page TP STUDY OBJECTIVES 2.1. GENERAL AIM / PRIMARY OBJECTIVE The aim is to show superiority of tenecteplase over placebo for the primary composite clinical end point of all-cause mortality or haemodynamic collapse within 7 days. Haemodynamic collapse is defined as: need for cardiopulmonary resuscitation; or systolic blood pressure < 90 mm Hg for at least 15 min, or drop of systolic blood pressure by at least 40 mm Hg for at least 15 min with signs of end organ hypoperfusion (cold extremities or low urinary output < 30 ml/h or mental confusion); or need for catecholamine administration to maintain adequate organ perfusion and a systolic blood pressure of > 90 mm Hg (including dopamine at the rate of > 5 micrograms / kg per minute) The primary objective is to demonstrate the clinical benefits of tenecteplase over placebo in normotensive patients with acute pulmonary embolism and with echographic (or spiral computed tomography) and laboratory evidence of right ventricular dysfunction. The secondary objective of the study is to assess the safety after administration of tenecteplase in normotensive patients with acute pulmonary embolism and with echographic (or spiral computed tomography) and laboratory evidence of right ventricular dysfunction PRIMARY ENDPOINT Primary efficacy endpoint: Clinical composite endpoint of all-cause mortality or haemodynamic collapse (as defined above) within 7 days. Second version December 15, 2008

96 Thrombolysis in Pulmonary Embolism Page TP SECONDARY ENDPOINTS Efficacy The secondary endpoints of this study are: Death within 7 days Haemodynamic collapse (as defined above) within 7 days Confirmed symptomatic pulmonary embolism recurrence within 7 days Death within 30 days Criteria for Symptomatic Recurrent Pulmonary Embolism: For diagnosis of symptomatic recurrent pulmonary embolism, clinical symptoms and/or signs suggesting recurrent pulmonary embolism will have to be objectively confirmed by lung scan, or a positive spiral computed tomogram, or a positive pulmonary angiogram. In particular, symptomatic recurrent pulmonary embolism will be diagnosed if at least one of the following findings is present: a new filling defect revealed by pulmonary angiography or spiral computed tomography a new high probability perfusion defect revealed by lung scan There will be no routine surveillance for asymptomatic recurrent venous thromboembolism Safety Safety criteria for this study are: Total strokes (intracranial haemorrhage or ischaemic stroke) within 7 days Major bleeding (excluding ICH) within 7 days defined as moderate or severe bleeding Definition of moderate bleeding: A bleeding episode requiring blood transfusion(s), but which is not deemed life-threatening and does not lead to haemodynamic compromise requiring emergency fluid replacement, inotropic support, or interventional treatment. Definition of severe bleeding: A bleeding episode that leads to haemodynamic compromise requiring emergency intervention (such as replacement of fluid and/or blood products, inotropic support, or surgical treatment), or life-threatening or fatal bleeding. Serious adverse events within 30 days. Second version December 15, 2008

97 Thrombolysis in Pulmonary Embolism Page TP STUDY POPULATION 3.1. NUMBER OF PATIENTS PLANNED Approximately one thousand patients (two groups of 500 patients; tenecteplase or matching placebo) suffering from acute pulmonary embolism (first symptoms occurring 15 days or less before randomisation) confirmed by lung scan, a positive spiral computed tomogram, or a positive pulmonary angiogram, and presenting with right ventricular dysfunction confirmed by echocardiography (or spiral computed tomography) and a positive troponin (I or T) test will be eligible for randomisation subject to inclusion /exclusion criteria. Each investigational site is expected to enrol a minimum of 5-10 patients per year. Sites that fail to randomize at least two patients within six months of study initiation may be closed at the discretion of the Sponsor. Study enrolment will be regularly assessed and, if necessary, additional sites will be initiated. Patient enrolment will be competitive with no restriction on any site. Investigators will be notified when the appropriate number of patients has been enrolled and advised when no further recruitment is allowed and randomization is closed INCLUSION CRITERIA Patients must fulfil the following inclusion criteria: Age 18 years or older; acute PE (first symptoms occurring 15 days or less before randomisation) confirmed by lung scan, or a positive spiral computed tomogram, or a positive pulmonary angiogram; right ventricular dysfunction confirmed by echocardiography or spiral computed tomography of the chest and a positive troponin I or T test. Criteria for right ventricular dysfunction At least one of the following echocardiographic criteria is needed to confirm right ventricular dysfunction : Right ventricular end-diastolic diameter (RVEDD) > 30 mm (parasternal long-axis or short-axis view), Second version December 15, 2008

98 Thrombolysis in Pulmonary Embolism Page TP 25 Right/left ventricular end-diastolic diameter (RVEDD/LVEDD) > 0.9 (apical or subcostal 4-chamber view), Hypokinesis of the right ventricular free wall (any view), Tricuspid systolic velocity > 2.6 m/s from the apical or subcostal 4-chamber view. Echocardiography is strongly recommended for diagnosis of right ventricular dysfunction in all patients. Only if echocardiography is not available, right ventricular dysfunction may be assessed on CT images by measuring the minor axis of the right and left ventricle in the transverse plane and calculating the RVd/LVd ratio (a ratio > 0.9 denotes RVD). Troponin I or T testing Criteria for a positive troponin test in the present trial are the following: Troponin I > 0.06 µg/l or troponin T > 0.01 µg/l using the test of the Department of Clinical Chemistry at the participating site. After diagnosis of RV dysfunction by echocardiography and troponin testing, patients who fulfil the inclusion criteria should be randomised as early as possible. In particular, every effort should be made to administer the study medication (TNK vs. placebo) within 2 hours after the investigator had received the result of the second of the two criteria for RVD, whichever comes the latest EXCLUSION CRITERIA Subjects who meet any of the following criteria will be excluded from randomization into the study: Haemodynamic collapse at presentation as defined above Known significant bleeding risk Administration of thrombolytic agents within the previous 4 days Vena cava filter insertion or pulmonary thrombectomy within the previous 4 days Uncontrolled hypertension defined as systolic BP >180 mm Hg and/or diastolic BP >110 mm Hg at randomisation Second version December 15, 2008

99 Thrombolysis in Pulmonary Embolism Page TP 26 Treatment with an investigational drug under another study protocol in the previous 7 days or greater, according to local requirements Previous enrolment in this study Known hypersensitivity to tenecteplase, alteplase, unfractionated heparin, or to any of the excipients Pregnancy, lactation or parturition within the previous 30 days. Women of childbearing age must have a negative pregnancy test or use a medically accepted method of birth control Known coagulation disorder (including vitamin K antagonists and platelet count < /mm 3 ) Any other condition that the investigator feels would place the patient at increased risk if the investigational therapy is initiated WRITTEN INFORMED CONSENT Patients fulfilling the above criteria may be randomized following receipt of written informed consent. Second version December 15, 2008

100 Thrombolysis in Pulmonary Embolism Page TP TREATMENT 4.1. TREATMENT REGIMENS TO BE COMPARED We intend to use the same therapeutic regimen of Tenecteplase that was validated in patients with AMI. Tenecteplase has been shown to be effective in patients with AMI when given as a weight-adjusted i.v. bolus. The same regimen has been chosen for the treatment of patients with acute PE. We presume that the same dose of Tenecteplase shown to be effective in AMI will be also effective in PE. This is in analogy, with some limitations, with alteplase in AMI and PE, respectively. Group A (experimental; fibrinolytic treatment) Tenecteplase, as a single IV bolus over 5-10 seconds. Group B (reference) Placebo, as a single IV bolus over 5-10 seconds Investigational product Tenecteplase Provided as 50 mg sterile, lyophilized powder in a vial for reconstitution with a pre-filled syringe with 10ml sterile water for IV injection. Comparator drug Placebo Provided as 50 mg sterile, lyophilized powder in a vial for reconstitution with a pre-filled syringe with 10ml sterile water for IV injection. Second version December 15, 2008

101 Thrombolysis in Pulmonary Embolism Page TP 28 Dosage and treatment schedule Group A Tenecteplase: Single body-weight (known or estimated) adjusted IV bolus administered over 5-10 seconds not later than 30 minutes after randomization, and not later than 2 hours after the diagnosis of RV dysfunction. Weight (kg) Dose in mg Dose in units Dose in ml *) <60 30 mg 6000 U 6 ml >60 to <70 35 mg 7000 U 7 ml >70 to <80 40 mg 8000 U 8 ml >80 to <90 45 mg 9000 U 9 ml >90 50 mg U 10 ml Concomitant therapy - Unfractionated heparin The dose of 80 IUxKg -1 as an intravenous bolus, followed by an infusion of 18 IUxKg -1 xh -1 (with a maximum initial infusion rate of 1800 IU xh -1 ), to be administered immediately after randomisation in all patients for at least 48 hours following randomisation. Beyond this period, intravenous UFH may be substituted with subcutaneous heparin (LMWH) or fondaparinux treatment. The bolus will be omitted when heparin was started before randomisation. Heparin infusion will be adjusted in order to achieve and maintain aptt corresponding with therapeutic heparin levels (equivalent to 0.3 to 0.7 IU/mL by factor Xa inhibition) and will last until treatment with vitamin K antagonists results in therapeutic INR values ( ) as determined on two consecutive days, or until the administration of low molecular weight heparin or fondaparinux at a weight-adjusted dosage. In both groups A and B, aptt will be assessed immediately (last available value) before study drug administration, and after study drug administration, approximately 3 hours, approximately 6 hours, approximately 12 hours after start of heparin treatment, and subsequently at least once daily as long as the patient is on UFH infusion. If adjustments of heparin dosage are necessary, the aptt will also be measured approximately 3 hours after each adjustment. Second version December 15, 2008

102 Thrombolysis in Pulmonary Embolism Page TP 29 *) 50 mg reconstituted in 10 ml solvent (sterile water for injection) Group B Placebo: Single body-weight (known or estimated) adjusted IV bolus administered over 5-10 seconds not later than 30 minutes after randomization, and not later than 2 hours after the diagnosis of RV dysfunction. Weight (kg) Dose in ml *) <60 6 ml >60 to <70 7 ml >70 to <80 8 ml >80 to <90 9 ml >90 10 ml Concomitant therapy - Unfractionated heparin. The dose of 80 IUxKg -1 as an intravenous bolus, followed by an infusion of 18 IUxKg -1 xh -1 (with a maximum initial infusion rate of 1800 IU xh -1 ), to be administered immediately after randomisation in all patients for at least 48 hours following randomisation. Beyond this period, intravenous UFH may be substituted with subcutaneous heparin (LMWH) or fondaparinux treatment. The bolus will be omitted if heparin is started before randomisation. Heparin infusion will be adjusted in order to achieve and maintain aptt corresponding with therapeutic heparin levels (equivalent to 0.3 to 0.7 IU/mL by factor Xa inhibition) and will last until treatment with vitamin K antagonists results in therapeutic INR values ( ) as determined on two consecutive days, or until the administration of low molecular weight heparin or fondaparinux at a weight-adjusted dosage. In both groups A and B, aptt will be assessed immediately (last available value) before study drug administration, and, after study drug administration, approximately 3 hours, approximately 6 hours, approximately 12 hours after start of heparin treatment, and subsequently at least once daily as long as the patient is on UFH infusion. If adjustments of heparin dosage are necessary, the aptt will also be measured approximately 3 hours after each adjustment. *) 50 mg reconstituted in 10 ml solvent (sterile water for injection) Second version December 15, 2008

103 Thrombolysis in Pulmonary Embolism Page TP 30 Order and timing of study medication given / procedures For both treatment groups: As soon as possible after the diagnosis of acute pulmonary embolism, confirmation of eligibility criteria and randomisation, an intravenous line will be established. Unfractionated heparin will be administered immediately after randomization in all patients as an intravenous bolus of 80 IUxKg -1,followed by an infusion of 18 IUxkg -1 xh -1 (with a maximum initial infusion rate of 1800 IU xh -1 ), and will be continued for at least 48 hours. Unfractionated heparin is NOT provided in the treatment kits. For patients already receiving unfractionated heparin or any low molecular weight heparin (LMWH) or fondaparinux before randomisation, the initial bolus of UFH will be omitted. Patients already receiving low molecular weight heparin or fondaparinux at therapeutic dosage can be included in the study. In these patients, the start of unfractionated heparin will be delayed until 12 after the last LMWH injection and until 24 hours after the last fondaparinux injection. Heparin infusion will be adjusted in order to achieve and maintain aptt corresponding with therapeutic heparin levels (equivalent to 0.3 to 0.7 IU/mL by factor Xa inhibition) and will overlap with the administration of vitamin K antagonists until therapeutic INR values ( ) are reached as determined on two consecutive days. aptt will be assessed immediately (last available value) before study drug administration, and, after study drug administration, approximately 3 hours, approximately 6 hours, approximately 12 hours after start of heparin infusion, and subsequently at least once daily as long as the patient is on UFH infusion. aptt will also be assessed approximately 3 hours after any dosage adjustment. As an alternative to the early initiation of oral anticoagulation, it will be possible to switch patients from UFH infusion to treatment with subcutaneous injections of LMWH or fondaparinux at a weight adjusted dosage 48 hours or later after randomization. A suggested standardized nomogram for titration of the heparin dose is illustrated in Appendix 1. Second version December 15, 2008

104 Thrombolysis in Pulmonary Embolism Page TP 31 Administration of study drug Study drug (Tenecteplase or placebo) should be administered not later than 30 minutes after randomisation, and not later than 2 hours after the diagnosis of RV dysfunction. Instructions for reconstitution of study drug are given on the leaflet in the treatment kit. The dose of study drug will be adjusted according to body weight (actual or estimated) as shown in the tables above. The dose of study drug must NOT be administered in dextrose (glucose) or Ringer s solution. Study drug should be administered not later than 30 minutes after randomization, and not later than 2 hours after the diagnosis of RV dysfunction, (defined as the time when the investigator receives the result of the second of the two criteria for RVD, whichever comes the latest) over 5 10 seconds. To avoid any untoward drug interactions between the study drug and any other IV treatments ideally the IV line should be flushed with a sufficient amount of saline solution (not glucose or Ringer s solution) prior to and following the intravenous administration. Aseptic procedures should be used during preparation and administration of the study drug. Labelling and packaging Study medication kits will be labelled as follows: Study n :P A description of the contents Medication Number Directions for use Storage Statement Expiry Date Caution Statement Sponsor Name and Address NOTE: The kits labels will be multilingual booklets and according to specific requirements of all countries. Samples of the labels will be kept in Investigator s Site File. Second version December 15, 2008

105 Thrombolysis in Pulmonary Embolism Page TP 32 Emergency code break instructions for the unblinding of study medication will be provided for the Investigator (Please, refer to section 8.1.3). The study medication will be provided by Boehringer Ingelheim and labeled, packed and distributed by a contract research organization (CRO) selected by the Sponsor. The clinical trial supply consists of 2 different treatment kits (boxes), one for each of the treatment groups. Each patient will be randomised to receive one of the 2 treatment kits. The kits will only contain the study drug (tenecteplase or placebo) and a pre-filled syringe of SWFI, sufficient for treating the patient according to protocol. For concomitant heparin and any further treatment during hospitalization is at the discretion of the investigator, with consideration of the restrictions in Section 4.2, Concomitant therapy commercial hospital stocks will be used. Trial medication in a kit may not be used for any patient other than those randomised. A record of opened kits (used and unused) as well as unopened, expired, damaged or unused study drug should be kept. All kits remaining at a site should be returned to the Sponsor with an inventory of clinical material returned, unless alternative local procedures have been agreed upon and approved by the Sponsor. Double-blind treatment kits contain: 1 vial of 50 mg lyophilized powder of teneteplase or placebo for IV injection after reconstitution; 1 x pre-filled syringe with 10 ml of sterile water for injection for the reconstitution of the study drug; multilingual instructions for preparation, handling, administration and dosing of the study drug; Storage conditions Trial kits are to be stored under normal hospital pharmacy conditions in a locked area with access limited to the investigator, study coordinator pharmacist or other authorized study personnel. Kits must not be frozen. Lyophilized tenecteplase vials should be protected from direct sunlight during extended periods. Because the reconstituted tenecteplase solution contains no preservatives, it is Second version December 15, 2008

106 Thrombolysis in Pulmonary Embolism Page TP 33 strongly recommended to be used immediately after reconstitution, and under no circumstances to be kept for longer than 8 hours at 2-8 C (36-46 F) before being used CONCOMITANT THERAPY Any concomitant or additional medication is NOT provided in the treatment kits. Non rescue concomitant dopamine Dopamine can be used in both treatment groups at the discretion of the investigator at a dosage of < 5 micrograms / kg per minute. Rescue medication and additional treatment in case of haemodynamic collapse In case of haemodynamic collapse (which means that the patient has reached the end point of the trial), the following emergency therapeutic measures can be performed at the discretion of the investigator: 1) inotropic or vasopressive drugs at any dosage regarded necessary (including dopamine at a dosage of > 5 micrograms / kg per minute); 2) rescue thrombolytic treatment; 3) surgical embolectomy; or 4) catheter thrombus fragmentation. Reperfusion medical treatment or procedures, i.e. rescue thrombolysis, emergency surgical embolectomy, or emergency catheter thrombus fragmentation, should not be given or performed unless the patient suffers haemodynamic collapse. This policy is in accordance with current guidelines which discourage these forms of treatment in haemodynamically stable patients with pulmonary embolism [40]. Rescue thrombolysis and additional treatment(s) will be recorded in the CRF. Second version December 15, 2008

107 Thrombolysis in Pulmonary Embolism Page TP 34 Anticoagulation - Administration of Vitamin K antagonists Heparin infusion should be maintained within the therapeutic range for at least 48 hours following randomisation. Beyond this period, intravenous UFH may be substituted with subcutaneous heparin (LMWH) or fondaparinux treatment at the discretion of the Investigator. Oral anticoagulation will be initiated between day 2 and day 10 from study drug administration, at the discretion of the attending physician. Heparin anticoagulation must be continued, overlapping with the administration of warfarin until the INR value is within the therapeutic range ( ) for two consecutive days. Oral anticoagulants will be continued for at least 6 months. Other medication Any other medication can be used at the discretion of the investigator. Intramuscular injections should be avoided. In case of severe bleeding complications, administration of an antifibrinolytic drug may be considered. Restrictions Please refer to TREATMENT COMPLIANCE As all study treatments will be given in-hospital no compliance problems are foreseen. The only reasons for deviations from the treatment schedule in the protocol would be for patient safety or due to the rare events of dosing mistakes or damaged treatment kits. Information on dosing will be collected in the CRF. Second version December 15, 2008

108 Thrombolysis in Pulmonary Embolism Page TP OBSERVATIONS For timing of study evaluations, see flow chart (page 11) EFFICACY Efficacy will be evaluated by using clinically relevant outcome variables as a composite endpoint. Primary end point The primary end point of this study is: Death or haemodynamic collapse within 7 days Secondary endpoints The secondary endpoints of this study are: Death within 7 days Haemodynamic collapse within 7 days Confirmed symptomatic pulmonary embolism recurrence within 7 days Death within 30 days SAFETY Safety secondary endpoints: Total strokes (intracranial haemorrhage or ischaemic stroke) within 7 days; Major bleeding (other than intracranial haemorrhage) within 7 days as defined above (definitions also provided in Appendix 3); Serious adverse events within 30 days Except for non-serious bleeds which will be collected up to day 7, only serious adverse events (SAEs) observed (objective or subjective) will be recorded in the e-crf. When collecting information on subjective events, leading questions will be avoided. For definition of AEs/SAEs and reporting procedures, see section Second version December 15, 2008

109 Thrombolysis in Pulmonary Embolism Page TP OTHER Medical history and physical examination Medical history data will be assessed in hospital, which will include information on previous cardiovascular, metabolic or haematologic disease and prior medication. Physical examination of the patient will also be performed in hospital. Lung scanning or spiral computed tomogram or pulmonary angiogram. After clinical assessment, all patients will undergo an imaging diagnostic procedure in order to confirm PE objectively. The diagnosis of PE will be confirmed on at least one of the following criteria: High probability lung scan (either perfusion or ventilation and perfusion lung scan) according to PIOPED or PISAPED criteria [41,42] Spiral CT scan showing at least one segmental filling defect; Positive pulmonary angiography showing at least one segmental filling defect. The use of non-invasive procedures (lung scan or spiral CT) is preferred over conventional pulmonary angiography as first-line diagnostic option. Echocardiography Transthoracic echocardiography will be performed as soon as possible following clinical suspicion of PE. In patients with objectively confirmed PE (see above), at least one of the following echocardiographic criteria is needed to confirm right ventricular dysfunction [24]: Right ventricular end-diastolic diameter (RVEDD) > 30 mm (parasternal long-axis or short-axis view), Right/left ventricular end-diastolic diameter (RVEDD/LVEDD) > 0.9 (apical or subcostal 4-chamber view), Hypokinesis of the right ventricular free wall ( any view), Tricuspid systolic velocity > 2.6 m/s. Echocardiography is strongly recommended for diagnosis of right ventricular dysfunction in all patients. Second version December 15, 2008

110 Thrombolysis in Pulmonary Embolism Page TP 37 Only if echocardiography is not available, right ventricular dysfunction may be assessed on CT images by measuring the minor axis of the right and left ventricle in the transverse plane and calculating the RVd/LVd ratio (> 0.9 denotes RVD). Cardiac troponin T or I Cardiac troponin measurements will be performed as soon as possible following clinical suspicion of PE. Troponin T or I will be measured according to local practice. In patients with objectively confirmed PE (see above), the following cut-off values will be used to define a positive Troponin test [28]: Criteria for a positive troponin test in the present trial are the followings: Troponin I > 0.06 µg/l or troponin T > 0.01 µg/l using the test of the Department of Clinical Chemistry at the participating site. Measured values will not be recorded in the CRF. aptt monitoring Heparin infusion will be adjusted in order to achieve and maintain aptt corresponding with therapeutic heparin levels (equivalent to 0.3 to 0.7 IU/mL by factor Xa inhibition) and will overlap with the administration of vitamin K antagonists until therapeutic INR values ( ) are reached as determined on two consecutive days. aptt will be assessed immediately (last available value) before study drug administration, and, after study drug administration, approximately 3 hours, approximately 6 hours, approximately 12 hours after start of heparin infusion, and subsequently at least once daily as long as the patient is on UFH infusion. aptt will also be assessed approximately 3 hours after any dosage adjustment. As an alternative to the early initiation of oral anticoagulation, it will be possible to switch patients from UFH infusion to treatment with subcutaneous injections of LMWH or fondaparinux at a weight adjusted dosage 48 hours or later after randomization. A suggested standardized nomogram for titration of the heparin dose is illustrated in Appendix 1. Second version December 15, 2008

111 Thrombolysis in Pulmonary Embolism Page TP 38 Chest X-ray It is at the discretion of the investigator to perform chest X-rays as part of the diagnostic workup for suspected pulmonary embolism. Haematology Haematological laboratory tests will be performed according to local hospital practice as part of initial patient management. In addition, these tests should be repeated whenever clinically indicated during a post treatment period of at least 48 hours. The tests should include, as a minimum, hemoglobin, haematocrit and a platelet count. Haematological parameters will be used to classify severity of (suspected) bleeding complications, to guide therapeutic interventions, and to monitor the adequacy of treatment (e.g.blood transfusions), but they will not be recorded in the CRF with the exception of drop in hemoglobin levels by > 2 g/dl. Modified Rankin scale For patients who experienced a non-fatal stroke, the modified Rankin scale will be determined where possible at day 30. Second version December 15, 2008

112 Thrombolysis in Pulmonary Embolism Page TP INVESTIGATIONAL PLAN 6.1. STUDY DESIGN AND PLAN This study is a prospective, randomized, double-blind, placebo-controlled, international, multicentre, parallel-group trial evaluating the efficacy and safety of tenecteplase in normotensive patients with acute pulmonary embolism and with right ventricular dysfunction. The trial design is as follows: Second version December 15, 2008

113 Thrombolysis in Pulmonary Embolism Page TP 40 Haemodynamically stable patient with PE Meets inclusion criteria, no exclusion criteria Chest CT, VQ scan, or pulmonary angiogram: positive Troponin I or T: positive ECHO: RV dysfunction Obtain consent, Randomise Placebo Tenecteplase Primary Endpoint Death or haemodynamic collapse within 7 days Secondary Endpoints Death within 7 days Haemodynamic collapse within 7 days Confirmed symptomatic pulmonary embolism recurrence within 7 days SAEs within 30 days Death within 30 days. Second version December 15, 2008

114 Thrombolysis in Pulmonary Embolism Page TP 41 In case of haemodynamic collapse (which means that the patient has reached the primary end point of the trial), the following emergency therapeutic measures can be performed at the discretion of the investigator: 1) inotropic or vasopressive drugs at any dosage regarded necessary (including dopamine at a dosage of > 5 micrograms / kg per minute); 2) rescue thrombolytic treatment; 3) surgical embolectomy; or 4) catheter thrombus fragmentation. Reperfusion medical treatment or procedures, i.e. rescue thrombolysis, emergency surgical embolectomy, or emergency catheter thrombus fragmentation, should not be given or performed unless the patient suffers haemodynamic collapse. This policy is in accordance with current guidelines which discourage these forms of treatment in haemodynamically stable patients with pulmonary embolism [40]. Reperfusion therapy, particularly thrombolysis within 48 hours of study drug treatment, may be preceded by unblinding. Haemodynamic collapse occurring at least 48 hours after study drug treatment needing rescue therapy, bleedings or death at any time after study drug treatment should not need unblinding. Rescue medication and additional treatment(s) will be recorded in the CRF STUDY ORGANIZATION An independent Data and Safety Monitoring Board (DSMB, Please, refer to section ) will monitor the accruing outcome and safety data. Two other independent committees, the Steering Committee and the Executive Committee, supervise and support the conduct of the study. Steering Committee The Steering Committee is composed of the Study Chairman, Co-chairman, the Coordinating Investigators, and the Principal Investigators acting as representatives /coordinators for each one of the participating countries. Further members include the sponsor representatives and, non-voting members will include representatives of the MAH of tenecteplase. The Steering Committee will meet periodically to assess the progress, provide scientific input, and address policy issues and operational aspects of the protocol and recommendations of the DSMB. Second version December 15, 2008

115 Thrombolysis in Pulmonary Embolism Page TP 42 At the end of the trial, the Steering Committee will meet in a closed session to discuss the trial results. The interpretation of these results will then be presented to the Sponsor in a meeting immediately following the closed session. Executive Committee The Executive Committee is composed of the Study Chairman, the Co chairman and Coordinating Investigators. Further members include the sponsor representatives and, nonvoting members will include representatives of the MAH of tenecteplase. The executive committee gives scientific input on the protocol and possible amendments as well as on the state of the art and any on-going development during the study, which could have consequences for the performance of the study. The Executive Committee is responsible for proposing actions which need to be discussed and approved by the Steering Committee. When the results of the study become available, the Executive Committee will provide a publication policy and provide advice on the interpretation of the results and the eventual impact on current therapy STUDY PROCEDURES The study is divided in two distinct periods: Enrolment and in-hospital period; Follow-up period. The study flow chart shows a summary of different assessments and procedures during the different study periods. Second version December 15, 2008

116 Thrombolysis in Pulmonary Embolism Page TP 43 Enrolment and in-hospital period Start of period: When a given patient is randomised. End of period: 7 days after randomisation Concomitant medication Please refer to section Clinical assessment of efficacy and safety During the in-hospital phase, patients will be monitored for serious adverse events, intracranial haemorrhage (ICH) and non-serious bleeds as described in section Subjects with a clinical suspicion of stroke at any time after randomisation up to 7 days, should be taken for immediate (within 24 hours) computed tomographic scanning (CT) or magnetic resonance imaging (MRI) to determine whether an ICH has occurred. CT is preferred, but MRI is also acceptable. In addition, all such patients should also be seen by a neurologist who could assist with the completion of the necessary documentation. In case of fatal stroke, if no imaging was done, an autopsy should be performed whenever possible. e-stroke FORM In every case of a detected/suspected stroke an e-sae Report Form must be completed in an expedited fashion (within one working day). This will be processed to meet regulatory requirements by the Safety group in Leuven (Leuven Safety Group, LSG; see section 8.4.1). Additional stroke data will also be recorded in the specific e-crf page. e-sae REPORT FORM For all serious adverse events (including clinical suspicion of stroke), requiring expedited reporting, an e-sae form must be completed and will trigger an alert to the central safety group in Leuven (Leuven Safety Group, LSG) for further processing and distribution (see section 8.4.1). Second version December 15, 2008

117 Thrombolysis in Pulmonary Embolism Page TP 44 e-crf ENTRIES Medical history Haemodynamic collapse criteria Stroke, including intracranial haemorrhage Any bleeds, both major and minor (other than ICH) (for definition refer to appendix 4) Transfusions given (red blood cells or whole blood) Serious adverse events Haemoglobin drop by > 2 g/d aptt monitoring Death from any cause Primary cause of death Symptomatic recurrence of pulmonary embolism Hospital discharge At day 7 it will be recorded if the patient was discharged or not. Hospital discharge is defined as end of hospitalisation or if the patient is transferred for long-term rehabilitation. Follow-up period day follow-up Start of period: Day 8 after randomisation Duration: Up to day 30 after randomisation The patient s vital status and need for any first re-hospitalisation will be established 30 day after randomisation. Second version December 15, 2008

118 Thrombolysis in Pulmonary Embolism Page TP 45 This may be done by clinic appointment or by contact (phone or mail) with the patient, a family member or the family physician. This follow-up is ideally completed on day 30, but may be postponed up to 7 days. If done later, than at the actual day the vital status should be given for day 30. It may NOT be given for an earlier date unless the patient died between day 7 and day 30 (even so it will be recorded that the patient is dead at day 30) day follow up assessments In previous acute pulmonary embolism studies thrombolysis has little or no clinical effect as compared to anticoagulant therapy alone after 7 days of administration, therefore only the following events will be reported in the CRF: Death from any cause Primary cause of death Modified Rankin scale for patients who had a non-fatal stroke Serious adverse events Re hospitalisation and primary reason for it (Symptomatic recurrent PE, Deep Venous Thrombosis, Major bleed, Stroke, Other) Note: Primary cause of death and cause of re-hospitalisation are SAEs day follow-up The patient s vital status will be recorded six months after randomisation. This may be done by clinic appointment or by contact (phone or mail) with the patient, a family member or the family physician. This six-month follow-up will not delay the database lock and reporting, which will be done based on the in-hospital and 30-day follow-up data. The follow-up can be done between days, but the status should be given for day 180 after randomisation, not earlier. If done earlier, a repeated contact with the patient must be made on or after day 180. If done later, than at the actual day, the vital status should be given for day 180. The following assessments will be done: Death from any cause Primary cause of death (Note: Primary cause of death is a SAE) Second version December 15, 2008

119 Thrombolysis in Pulmonary Embolism Page TP 46 Severity of dyspnoea on exertion using the NYHA scale. If echocardiography can be performed as part of standard care of the 6-month follow-up at the participating center, the following echocardiographic data will be recorded: Systolic pulmonary artery pressure Persistent right ventricular dysfunction defined as at least one of the following echocardiographic criteria : o Right ventricular end-diastolic diameter (RVEDD) > 30 mm (parasternal long-axis or short-axis view), o Right/left ventricular end-diastolic diameter (RVEDD/LVEDD) > 0.9 (apical or subcostal 4-chamber view), o Hypokinesia of the right ventricular free wall ( any view), o Tricuspid systolic velocity > 2.6 m/s. If indicated by the six-month follow-up, it can be decided to further extend the follow-up period by another six months or longer ADHERENCE TO PROTOCOL The rules set out in the protocol should be well known by all persons involved in the study. This is of particular importance for the site personnel and can be achieved by sufficient training of the staff and a well defined procedure for delegation and authorisation of different tasks to various staff members. This training and authorisation process at any site is the responsibility of the investigator. Guidelines and example logs will also be available on the trial web site Visit schedule The visits are already described in the previous sections on Study procedures, 6.2 and outlined in the flow chart on page 9 and can be summarized as follows: Enrolment period; In-hospital period; 7-day follow-up Second version December 15, 2008

120 Thrombolysis in Pulmonary Embolism Page TP 47 The 30 day follow-up to be performed in the window days, but give the status at 30 days be done by clinic appointment or by contact (phone or mail) with the patient, a family member or the family physician. The 180 day follow-up to be performed in the window days, but give the status at 180 days be done by clinic appointment or by contact (phone or mail) with the patient, a family member or the family physician Criteria and rules for stopping subject treatment Not applicable (single IV bolus injection). Criteria for termination of the trial Premature termination of the trial may happen under the following conditions: Occurrence of unknown or increase of known adverse events that render the risk/benefit ratio unacceptable; Interim analysis indicates reason (see section 7.3.5) An unacceptable high number of SAEs; Ethical justification; Recruitment rate is too low such that it is unrealistic to consider completion of the trial within an acceptable period of time; At the request of the MAH Decision of the authorities. Second version December 15, 2008

121 Thrombolysis in Pulmonary Embolism Page TP STATISTICS 7.1. STATISTICAL DESIGN / MODEL The trial is a prospective, international, multi-centre, randomised (1:1), double-blind, parallel group comparison conducted for investigating the efficacy and safety of tenecteplase compared to placebo in normotensive patients with acute pulmonary embolism and with echographic and laboratory evidence of right ventricular dysfunction NULL AND ALTERNATIVE HYPOTHESES The primary aim of the trial is to demonstrate superiority in the intent-to-treat analysis of tenecteplase over placebo with regard to primary composite endpoint as the incidence of death or haemodynamic collapse at 7 days (= 7daydeahaemcol). The null and alternative hypotheses are as follows: H 0 : 7daydeahaemcol tenecteplase = 7daydeahaemcol placebo versus H 1 : 7daydeahaemcol tenecteplase 7daydeahaemcol placebo 7.3. PLANNED ANALYSES Populations to be analysed The main analysis will be based on the intent-to-treat population (ITT) of all patients randomised (irrespective of which study treatment is given or if any study treatment is received). In addition, an explanatory analysis (PP) of all patients randomized & treated without major protocol violations/deviations will be carried out. Pre-defined major protocol violations/deviations are: (1) missing data for the primary / co-primary efficacy endpoints, (2) no study drug received, (3) violation of inclusion criteria, (4) additional protocol violations will be possibly defined during the blind data review. Second version December 15, 2008

122 Thrombolysis in Pulmonary Embolism Page TP 49 Patient accountability Disposition of patients, patient status and patients excluded from ITT. PP populations will be summarised by treatment group. Descriptive statistics for primary reason for patient s withdrawal will be also presented by treatment group as well as a list of these patients sorted by treatment group. DROP-OUTS Reasons for drop-outs in each treatment group will be displayed. A detailed list of drop-out patients will also be provided. Baseline characteristics Baseline characteristics will be tabulated and comparability / differences between the treatment groups will be examined by means of descriptive statistics. Subgroup analyses The following subgroup analyses will be carried out: gender, age and country. Interim analyses Interim analyses will be made after recruitment of 25%, 50%, and 75% of the total number of patients. These analyses will be made in order to allow sample size reassessment or early stop of the trial for efficacy or futility. For these reasons, the α-spending functions determining the boundary limits for the interim analysis will be chosen to require an adjustment of total P value by less than (P=0.047 for final analysis). Calculations for boundary limits and for sample size reassessments will be made using East Software (Cytel, Cambridge, MA, USA) or equivalent validated software. In case of a non-futile interim analysis, a decision may be made to increase reasonably the sample size if the interim result looks promising [43]. In addition, safety monitoring will be done by the Data and Safety Monitoring Board, according to the charter. Second version December 15, 2008

123 Thrombolysis in Pulmonary Embolism Page TP EFFICACY ANALYSIS Main Efficacy Criterion The primary ITT analysis on the primary endpoint will be carried out by a two-sided chisquare test on proportions. In addition the 95% confidence interval on the odds-ratio will be presented. Secondary Efficacy Criteria The secondary ITT analysis will be carried out by the chi-square test on proportions. In addition, the corresponding 95% confidence interval on the odds ratio will be presented. The survival status during 30 days follow-up will be analyzed by showing Kaplan-Meiercurves and treatment differences will be compared by means of log-rank test SAFETY ANALYSIS (Serious) adverse events will be tabulated per treatment group. All (dichotomized) endpoints will be analyzed by chi-square test on proportions and the 95% confidence interval on the odds-ratio will be presented. In addition, continuous safety monitoring (in a blinded fashion) will be done by the Data and Safety Monitoring Board EXPLORATORY ANALYSIS Potentially important prognostic factors for the main efficacy / safety endpoint will be explored and utilized as covariates in the relative risk analyses by means of logistic regression or Cox-model HANDLING OF MISSING DATA In the intent-to-treat analysis missing data for the primary endpoint will be imputed according to the worst case principle (endpoint reached). In case of large differences between PP and ITT populations, an analysis of sensitivity using different methods for missing data replacement, including multiple imputation technique, will be carried out. Second version December 15, 2008

124 Thrombolysis in Pulmonary Embolism Page TP RANDOMISATION The randomisation will be stratified by centre and, within the centres, performed in blocks to ensure balanced distribution of the treatment groups at any time. Patients are considered to be randomised as soon as the randomisation number has been assigned SAMPLE SIZE ISSUES Based on previous studies in pulmonary embolism and registry data, it is estimated that the primary endpoint (incidence of 7 day death or haemodynamic collapse) in the placebo group will be approximately 7%. A two group χ 2 test (not continuity-recorded) with a two-sided significance level will have 80% power to detect the difference between a group placebo proportion, π 1, of and a group tenecteplase proportion, π 2, of (odds ratio of 0.411) when the sample size in each group is 474. Second version December 15, 2008

125 Thrombolysis in Pulmonary Embolism Page TP ADMINISTRATIVE ISSUES The trial will be carried out: in compliance with the protocol, in compliance with Conventions of the Council of Europe on Human Rights and Biomedicine, UNESCO Declaration,. in accordance with the Declaration of Helsinki, version as of Oct. 1996,the ICH Harmonized Tripartite Guideline for Good Clinical Research Practice (GCRP) and in accordance with applicable regulatory requirements. Insurance Cover The Sponsor will take out no-fault insurance cover for all subjects included in the trial. The conditions of this insurance cover are available to the investigator in the e-isf. The investigator can make this information available to subjects on request ETHICS Institutional Review Board or Independent Ethics Committee The trial will not be initiated before the protocol and informed consent and subject information form have been reviewed and received approval / favourable opinion from an Independent Ethics Committee (IEC) or the local Institutional Review Board (IRB). Should a protocol amendment be made that needs IEC / IRB approval, the changes in the protocol will not be implemented until the amendment and revised informed consent (if appropriate) has been reviewed and an approval / favourable opinion has been received from IEC or the local IRB. A protocol amendment intended to eliminate an apparent immediate hazard to subjects may be implemented immediately providing that the appropriate regulatory authorities and IEC / IRB are notified as soon as possible and an approval is requested. Protocol amendments only for logistical or administrative changes may be implemented immediately; the IEC / IRB needs to be informed only. The constitution of the IEC / IRB must meet the requirements of the participating countries. A list of the IEC / IRB members, with names and qualifications, will be requested. If such a list is unavailable, the investigator must provide the name and address of the IEC/IRB along with a statement from the IEC / IRB that it is organized according to current ICH GCP guidelines and the applicable local laws and regulations. The IEC / IRB must also perform all duties outlined by the requirements of the participating countries. Second version December 15, 2008

126 Thrombolysis in Pulmonary Embolism Page TP 53 In accordance with local national regulations, the principal investigator is responsible for keeping the IEC / IRB informed of the progress of the study. According to current ICH GCP guidelines, this must occur at least once annually and the principal investigator should also keep the IEC / IRB informed of serious adverse drug reaction reports coming to her/his attention (e.g. case category-1 events). Upon completion of the trial the principal investigator shall notify the IEC / IRB, where applicable. Informed consent and subject information Ethical and legal requirements in accordance with international declarations (e.g. Declaration of Helsinki, Conventions of the Council of Europe on Human Rights and Biomedicine, UNESCO Declaration) will be fulfilled in this study. The person who informs the subject should be a physician. An informed consent document that includes both information about the trial and the consent form will be prepared and given to the subject. This document will comply with all local requirements and the requirements set out in ICH GCP. The document must be in a language understandable to the subject and must specify who informed the subject. After reading the informed consent document, the subject is asked to give consent in writing. The subject's consent must be confirmed at the time of consent by the personally dated signature of the subject or the subject's legally authorized representative (means an individual or judicial or other body authorized under applicable law to consent on behalf of a prospective subject to the subject's participation in the procedures involved in research). A copy of the signed consent document must be given to the subject or the subject's legally authorized representative. The original signed consent document will be retained by the investigator.(a copy may be requested by the sponsor) The subject must be informed that his / her medical records may be examined by Clinical Quality Assurance auditors appointed by the Sponsor, or by appropriate IEC / IRB members or by inspectors from regulatory authorities. Should a protocol amendment be made, the subject consent form and subject information form may need to be revised to reflect the changes to the protocol. It is the responsibility of the investigator to ensure that an amended consent form is reviewed and received approval / favourable opinion from the ICE or IRB, and that it is signed by all subjects subsequently entered in the trial and those currently in the trial, if affected by the amendment. Second version December 15, 2008

127 Thrombolysis in Pulmonary Embolism Page TP Unblinding Unblinding will be available 24 hours a day, 7 days a week to the investigator and/or pharmacist through the e-crf and data management system. Should the e-crf be unavailable, a back-up manual procedure will be used as instructed in the SOM. In case of unblinding the Sponsor will be informed immediately. Unblinding must only be done in emergency situations when the identity of the study drug must be known by the investigator in order to provide appropriate medical treatment. In particular, unblinding may be requested by the investigator in the case of: haemodynamic collapse as defined under 2.1., page 22 (meaning that the patient has reached the primary end point); or any adverse event that requires unblinding for appropriate patient management. After unblinding, the use of inotropic or vasopressive drugs (at any dosage regarded necessary), or the use of rescue thrombolytic treatment, emergency surgical embolectomy, or catheter thrombus fragmentation can be performed at the discretion of the investigator. Rescue medication and additional treatment(s) will be recorded in the CRF RECORDS Drug accountability Drug supplies, which will be provided by the Sponsor, must be kept in a secure, limited access storage area at room temperature as defined in section The investigator / study coordinator and/or pharmacist must maintain records of the delivery of study medication to subjects and the return to the Sponsor or any alternative disposition as agreed of unused study medication. Drug accountability forms to be filled in and signed by the site personnel capturing all necessary information will be provided in the Investigator Site File. Emergency code break (also see Unblinding, above) The reason for any code break must be documented at the site along with the date and the initials of the person who broke the code. Second version December 15, 2008

128 Thrombolysis in Pulmonary Embolism Page TP 55 Case Report Forms The study will be conducted using the Hypernet electronic data capture system, validated according to 21CRF11 and FDA 1678 Guidance, GAMP and GCP guidelines. Data are entered by the principal investigators or sub-/co-investigators in the electronic case report form (e-crf) provided by the CRO, and saved in an Oracle database. Only authorized persons (principal investigator and sub-/co-investigators) can access the e-crf at the study sites by means of personalised username and password. The principal investigators at the study sites who have the right to enter data and sign study documents should be listed on an Staff list/authorisation Log. Data transmission to the web-server is performed by means of an Internet connection and no specific software has to be installed at the study sites. Hypernet system requirements are the following: Software: Windows 98, Windows NT Millennium Edition, Windows 2000, Windows XP Browser: Microsoft Internet Explorer 5.0 or higher Hardware: Internet connection with a 56kbit/sec modem (or higher), ISDN, ADSL, LAN (i.e. Ethernet), Fastweb. Subjects will be identified on the ecrf by a sequential subject number within site. The investigator must make a separate, confidential record of any details (Enrolment Log, see Investigator Site File) to permit identification of any subject enrolled in the trial in case follow-up is required. On-line dictionaries will be available to code adverse events (med DRA). Electronic e-crfs are used to record clinical trial data as defined in other sections of the protocol, and are an integral part of the trial and subsequent reports. It is the responsibility of the Investigator to maintain adequate and accurate e-crfs, by keeping them current to reflect status at each phase during the course of the trial. Data may be corrected until the visit is closed by the investigator. Subsequent data corrections can only be done by the data management centre on the basis of equeries issued to the investigators or sub-/coinvestigators (see 8.2.6). A track of any correction is maintained in an electronic audit trial. A declaration ensuring accuracy of data recorded in the e-crfs must be acknowledged by the principal investigator or, if permitted by local regulations, by his authorized sub-/coinvestigator when the e-crf is complete. Second version December 15, 2008

129 Thrombolysis in Pulmonary Embolism Page TP 56 A print out of the e-crf is possible at any stage of the study. A printed final copy of the ecrf of each patient should be made and signed by the principal investigator. These records should be archived at the study site. The principal investigator will be responsible for retaining/archiving all records pertaining to the trial in accordance with local regulatory guidelines. Source documents Source documents provide evidence for the existence of the subject and substantiate the integrity of the data collected. Source documents are filed at the investigator s site. Data reported on the Case Report Forms must be consistent with the source documents. Worksheets may be used to facilitate the data collection process. Data from these worksheets will be transferred to the official e-crf, such worksheets will be considered source data and should be stored and archived at the site. Direct access to source data / documents The investigator / institution will permit trial-related monitoring, audits, IEC / IRB review and regulatory inspection, providing direct access to all related source data / documents. Signed informed consents and all source documents, including progress notes and copies of laboratory and medical test results, must be available at all times for review by the Sponsor s clinical trial monitor and/or inspection by health authorities. Electronic case report forms (e-crfs) will be accessible and printable at any moment by the Sponsor s clinical trial monitor. The accuracy of the data will be verified by performing site visits / on selected sites to review study procedures, accuracy of data and review of the documents described in section Processing of data Study data will be entered into the electronic case report forms (e-crfs) by the authorized principal investigators or sub-/co-investigators. The data-management of the study database Second version December 15, 2008

130 Thrombolysis in Pulmonary Embolism Page TP 57 maintenance will be performed by the CRO according to its own Standard Operating Procedures. The Hypernet equery tool will be used by to clarify data inconsistencies. The investigators or sub-/co-investigators can also issue equeries to correct data an already closed e-crf visits. All equeries and data corrections will be electronically tracked. Reconciliation of the study database and the safety database will be performed on an ongoing basis. Data evaluated by the DSMB will be extracted periodically from the trial database and the safety database. After data base lock, the database will be transferred to the Sponsor QUALITY ASSURANCE AUDIT Audit A quality assurance (QA) audit of this trial may be conducted by the Sponsor or the Sponsor s designees. The quality assurance auditor will have access to all medical records, the investigator s trial related files and correspondence and all informed consent documentation that are relevant to this clinical trial. Monitoring The sponsor will organise a site monitoring plan with the CRO. It is planned to conduct at least one site visit to each centre, in each participating country The Sponsor may also arrange for cause monitoring as needed PROCEDURES Adverse Events All serious adverse events up to day 30 will be recorded in the e-crf. All bleeds up to day 7 will be recorded in the e-crf. Second version December 15, 2008

131 Thrombolysis in Pulmonary Embolism Page TP Background Definition of an Adverse Event : An adverse event (AE) is any untoward medical occurrence, including an exacerbation of a pre-existing condition in a patient or clinical investigation subject administered a pharmaceutical product, and which does not necessarily have to have a causal relationship with this treatment. Lack of effect is not an adverse event in clinical studies because the purpose of the clinical study is to establish drug effect. Cases of pregnancy are not adverse events but should be reported for tracking purposes. Administered means that the drug has been given during or before the AE occurred. This is true, regardless of how much time has elapsed between exposure and event. It also does not matter whether the reported event occurred during the pre-specified observational period as per protocol, or whether it has been reported later, for whatever reason. An adverse event can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding), symptom or disease temporally associated with the use of a medicinal (investigational) product, whether or not considered related to the medicinal (investigational) product. In case a disease is diagnosed, the adverse event is the disease, rather than the associated sign or symptom observed. However, if the diagnosis is unknown to the investigator, the observed symptom, or clinically significant laboratory abnormality will be recorded as the adverse event. If a subject has an adverse experience, which has begun before informed consent was given, such experience is not an adverse event, but is based upon a pre-existing condition. Adverse event reporting starts when informed consent or presumed informed consent has been given, regardless if study medication has been administered. (this is not a pre- existing condition unless it already existed before signing informed consent. Causal relationship Medical judgment should be used to determine the relationship, considering all relevant factors, including pattern of reaction, temporal relationship, de-challenge or re-challenge, confounding factors such as concomitant medication, concomitant diseases and relevant history. Assessment of causal relationship should be recorded in the case report forms. Second version December 15, 2008

132 Thrombolysis in Pulmonary Embolism Page TP 59 Yes: There is a reasonable causal relationship between the investigational drug administered and the AE. No: There is no reasonable causal relationship between the investigational drug administered and the AE. The causal relationship must be provided by the investigator for both tenecteplase and placebo. Definition of a Serious Adverse Event (SAE): A Serious Adverse Event is an event, which fulfils one or more of the following criteria: fatal; immediately life-threatening; results in persistent or significant disability/incapacity; requires or prolongs in-patient hospitalisation; is a congenital anomaly / birth defect; any other reason representing a significant hazard comparable to the criteria mentioned above. All serious adverse events occurring during the course of the trial (i.e. from signing the informed consent onwards through the observational phase i.e. up to day 30) will be collected, documented and reported to the sponsor by the investigator according to the specific definitions and instructions detailed in the Adverse Event Reporting section of the Investigator Site File. NOTE! Death is an outcome, not an event (except for sudden death and when no cause of death can be defined). Expedited safety reporting in the study Case reports that must be reported by the investigator expeditiously (within 24 hours) using safety forms (e-sae form): All SAE s considered by the investigator to have a reasonable relationship to tenecteplase, to placebo or to the study design/ trial procedure. Second version December 15, 2008

133 Thrombolysis in Pulmonary Embolism Page TP 60 Any SAE considered not related by the investigator to tenecteplase, to placebo or to the study design/ trial procedure and not listed in the list of Disease Related Events (see below). All haemorrhagic strokes are considered drug related SAEs. An additional e-stroke Form will also be completed. All fatal cases Where to report SAE cases expeditiously? All SAE case reports and stroke forms are included in the e-crf and will trigger an alert at the Leuven Safety Group (see section 8.4.3). For other information, contact persons, fax and telephone numbers, see the Investigator Site File. List of Pulmonary Embolism-Related Events (exempted events) These events are known to occur as common complications of the qualifying pulmonary embolism and are exempted from regulatory reporting unless considered causally related to the study medication by the investigator. For list of events, see Appendix, The Safety Group A suitably qualified safety group will act as the central safety processing group for the whole study and will assess the drug relationship, the category classification and listedness and seriousness of reported SAE cases. This group will also co-ordinate handling of the events and prepare the necessary narratives. All required SAE data will be entered into a database provided by the MAH and transmitted to the MAH Regional Centres for Drug Safety in Europe (RCE). The SAEs will also be transmitted to all investigators for transmission to IECs/IRBs as per local regulations. Prompt identification of category-1 and -2 cases is of particular importance from a regulatory reporting perspective. A case is classified as category-1 or -2, if at least one event fulfils the following 3 criteria: is serious according to the previously given criteria in section 8.4.2; Second version December 15, 2008

134 Thrombolysis in Pulmonary Embolism Page TP 61 there is a reasonable causal relationship to tenecteplase (in the opinion of either the investigator or the Sponsor); the event is unlisted for tenecteplase (category-1), or it is listed for tenecteplase (category- 2). An event is considered listed (expected for tenecteplase) if it is referenced in the labelling or the tenecteplase Investigational Brochure (IB). The Safety group will also provide the DSMB regularly with safety/efficacy data. SAEs summaries will also be sent to the Sponsor. Data and Safety Monitoring Board The DSMB will consist of independent members representing cardiology, pulmonology, internal medicine, and biostatistics. At regular intervals the DSMB will review safety and outcome data provided by LSG and report to the chairman of the Steering Committee. The DSMB will also conduct analyses of data at the request of the Steering Committee. In order to assure patient safety, the DSMB will be provided in a timely fashion with early, unclean data on bleeds (both, major and minor), all detected symptomatic strokes and all SAEs reportable expeditiously in periodic cumulative reports RULES FOR AMENDING PROTOCOL All amendments must be documented, dated and signed by all signatories (or their successors) of the original protocol. Rules regarding submission for ethical review of amendments were presented in section DISCONTINUATION OF THE TRIAL BY THE SPONSOR The Sponsor reserves the right to discontinue this trial at any time for failure efficacy safety or any other administrative reasons. Premature termination of the trial must be reported to the regulatory authorities and the IEC/IRB as appropriate PATIENT CONFIDENTIALITY Individual subjects medical information obtained as a result of this study is considered confidential and disclosure to third parties is prohibited with the exceptions noted below. Second version December 15, 2008

135 Thrombolysis in Pulmonary Embolism Page TP 62 Subject confidentiality will be further ensured by utilizing subject identification code numbers to correspond to treatment data in the computer files. Such medical information may be given to the patient's personal physician or to other appropriate medical personnel responsible for the subject's welfare. Data generated as a result of this trial are to be available for inspection on request by the participating physicians, the Sponsor s representatives, by the IEC / IRB and the regulatory health authorities 8.8. PUBLICATION POLICY The Sponsor is dedicated to supporting the process of free exchange of relevant scientific information. Any publication of the results of this trial must be consistent with the publication policy of the Sponsor, Marketing Authorisation Holder and Executive Committee. Some general rules are laid down below. This study represents a joint effort between the Sponsor and the investigators. A publication policy will be presented by the Executive Committee (including Sponsor representative) and approved by the Steering Committee. All investigators and members of these committees agree to abide by the policy. Agreement on authorship of the main paper Conditions for secondary manuscripts It is foreseen that the Study Chairman, Co chairman and the Executive Committee will prepare a manuscript plan to ensure timely and high quality presentation and publication of the study results and provide this to the Sponsor for due consideration. Local publication of part(s) of the results is not allowed before the publication of the main study. The Study Chairman and Co-chairman will submit any manuscript to members of the Steering and Operational Committees for comment and release prior to the actual submission to a learned society or scientific journal. After this, Investigators may submit secondary papers, but these should be provided according to the publication policy for review and approval. Second version December 15, 2008

136 Thrombolysis in Pulmonary Embolism Page TP 63 One week should be allowed for review by the Sponsor and the Marketing Authorisation Holder of abstracts planned to be submitted to a learned society, and 3 weeks should be allowed for review of papers planned to be submitted to a scientific journal. The rights of the investigator and of the Sponsor with regard to publication of the results of this trial are also described in the investigator contract. Second version December 15, 2008

137 Thrombolysis in Pulmonary Embolism Page TP SIGNATURE PAGE Second version December 15, 2008

138 Thrombolysis in Pulmonary Embolism Page TP 65 Local signature page ( to be adapted for local requirements ) I hereby certify that I agree to adhere to the protocol and to all the documents and procedures referenced in the protocol. National Principal Investigator Date Signature Investigator: Name Institution /Department Co-Investigator(if applicable) Name Institution /Department Date Date Signature Signature Second version December 15, 2008

139 Thrombolysis in Pulmonary Embolism Page TP REFERENCES 1. Goldhaber SZ, Visani L, De Rosa M ; Acute pulmonary embolism: clinical outcomes in the international cooperative pulmonary embolism registry Lancet 1999;353: The urokinase pulmonary embolism trial. A national cooperative study. Circulation 1973; 47: II Tibbutt DA, Davies JA, Anderson JA, Fletcher EW, Hamill J, Holt JM, Thomas ML, Lee G, Miller GA, Sharp AA, Sutton GC. Comparison by controlled clinical trial of streptokinase and heparin in treatment of life-threatening pulmonary embolism. Br Med J 1974; 1: Ly B, Arnesen H, Eie H, Hol R. A controlled clinical trial of streptokinase and heparin in the treatment of major pulmonary embolism. Acta Med Scand 1978; 203: Dotter CT, Seamon AJ, Rosch J, et al. Streptokinase and heparin in the treatment of pulmonary embolism: a randomized comparison. Vasc Surg 1979; 13: Marini C, Di Ricco G, Rossi G, Rindi M, Palla R, Giuntini C. Fibrinolytic effects of urokinase and heparin in acute pulmonary embolism: a randomized clinical trial. Respiration 1988; 54: Tissue plasminogen activator for the treatment of acute pulmonary embolism. A collaborative study by the PIOPED Investigators. Chest 1990; 97: Levine M, Hirsh J, Weitz J, Cruickshank M, Neemeh J, Turpie AG, Gent M. A randomized trial of a single bolus dosage regimen of recombinant tissue plasminogen activator in patients with acute pulmonary embolism. Chest 1990; 98: Dalla-Volta S, Palla A, Santolicandro A, Giuntini C, Pengo V, Visioli O, Zonzin P, Zanuttini D, Barbaresi F, Agnelli G. PAIMS 2: alteplase combined with heparin versus heparin in the treatment of acute pulmonary embolism. Plasminogen activator Italian multicenter study 2. J Am Coll Cardiol 1992; 20: Goldhaber SZ, Haire WD, Feldstein ML, Miller M, Toltzis R, Smith JL, Taveira da Silva AM, Come PC, Lee RT, Parker JA. Alteplase versus heparin in acute pulmonary embolism: randomised trial assessing right-ventricular function and pulmonary perfusion. Lancet 1993; 341: Jerjes-Sanchez C, Ramírez-Rivera A, de Lourdes G, Arriaga-Nava R, Valencia S, Rosado-Buzzo A, Pierzo JA, Rosas E. Streptokinase and heparin versus heparin alone in massive pulmonary embolism: a randomized controlled trial. J Thromb Thrombolysis 1995; 2: Konstantinides S, Geibel A, Heusel G, Heinrich F, Kasper W. Heparin plus alteplase compared with heparin alone in patients with submassive pulmonary embolism. N Engl J Med 2002; 347: Wan S, Quinlan DJ, Agnelli G, Eikelboom JW. Thrombolysis compared with heparin for the initial treatment of pulmonary embolism - Circulation 2004;110: Second version December 15, 2008

140 Thrombolysis in Pulmonary Embolism Page TP Tanswell P, Modi N, Danays T, et al. Pharmacokinetics and pharmacodynamics of tenecteplase in fibrinolytic therapy of acute myocardial infarction - Clin. Pharmacokinet 2002; 41(15): Keyt BA, Paoni NF, Refino CJ, et al. Faster-acting and more potent form of tissue plasminogen activator - Proc Natl Acad Sci USA. 1994; 91: ASSENT-II Investigators, Single-bolus tenecteplase compared with front-loaded alteplase in acute myocardial infarction: the ASSENT-2 double-blind randomised trial - Lancet 1999; 354: Van de Werf F, Barron HV, Armstrong PW, Granger CB, Berioli S, Barbash G, Pehrsson K, Verheugt FW, Meyer J, Betriu A, Califf RM, Li X, Fox NL; ASSENT-2 Investigators. Assessment of the Safety and Efficacy of a New Thrombolytic. Incidence and predictors of bleeding events after fibrinolytic therapy with fibrin-specific agents: a comparison of TNK-tPA and rt-pa. - Eur Heart J 2001 Dec;22(24): Sze DY, Carey MBL, Razavi MK Treatment of massive pulmonary embolus with catheter-directed Tenecteplase - J Vasc Interv Radiol 2001; 12: Caldicott D, Parasivam S, Harding J, Edwards N, Bochner F Tenecteplase for massive pulmonary embolus - Resuscitation 2002; Sobieszczyk P, Fishbein MC, Goldhaber SZ Acute pulmonary embolism. Don t ignore the platelet - Circulation 2002; 106: Hyers TM, Agnelli G, Russell HD, et al. Antithrombotic therapy for venous thromboembolic disease - Chest 2001;119: 176S-93S. 22. Granger CB, Becker R, Tracy RP, Califf RM, Topol EJ, Pieper K, Ross AM, Roth S, Lambrew C and Bovill EG for the GUSTO-I Hemostasis Substudy Group. Thrombin generation, inhibition and clinical outcomes in patients with acute myocardial infarction treated with thrombolytic therapy and heparin: results from the GUSTO-I trial - J Am Coll Cardiol 1998;31(3): Konstantinides S, Geibel A, Olschewski M, Heinrich F, Grosser K, Rauber K et al. Association between thrombolytic treatment and the prognosis of hemodynamically stable patients with major pulmonary embolism - Circulation 1997;96: Goldhaber SZ. Echocardiography in the management of pulmonary embolism Ann Intern Med 136: , Schoepf UJ, Kucher N, Kipfmuller 26. van der Meer RW, Pattynama PM, 27. Giannitsis E, Muller-Bardorf M, Kurowsky V, et al. Independent prognostic value of cardiac troponin T in patients with confirmed pulmonary embolism - Circulation 2000;102: Konstantinides S, Geibel A, Olschewski M, et al. Importance of cardiac troponins I and T in risk stratification of patients with acute pulmonary embolism - Circulation 2002; 106: Second version December 15, 2008

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143 Thrombolysis in Pulmonary Embolism Page TP APPENDICES APPENDIX 1: HEPARIN NOMOGRAM Initial dosing : Bolus dose: 80 IUxKg -1 intravenous bolus. Infusion rate: 18 IU UxKg -1 xh IU xh -1 ) (with a maximum initial infusion rate of The IV heparin sodium infusion is adjusted to maintain an aptt (Activated Partial Thromboplastin Time) range between x aptt control. Heparin infusion will be adjusted in order to achieve and maintain aptt (range between x aptt control ) corresponding with therapeutic heparin levels (equivalent to 0.3 to 0.7 IU/mL by factor Xa inhibition) and will overlap with the administration of vitamin K antagonists until therapeutic INR values ( ) are reached as determined on two consecutive days. aptt will be assessed before study drug administration, and after study drug administration, approximately 3 hours, approximately 6 hours, approximately 12 hours after start of the heparin infusion, and subsequently at least once daily as long as the patient is on UFH infusion. aptt will also be assessed approximately 3 hours after any dosage adjustment. The suggested standardized nomogram for titration of the heparin dose is: Table 1a: Goal of therapy = aptt x control Heparin titration nomogram when standard laboratory reagents are used. aptt (sec) Bolus Dose Stop heparin infusion Rate change* ) (IU/Kg/h) Repeat aptt <1.2 x control 80IU/Kg 0 min hrs x 40IU/Kg 0 min hrs control x 0 0 min. 0 (no change) 6-12 hrs. control x 0 0 min hrs control > min hrs Second version December 15, 2008

144 Thrombolysis in Pulmonary Embolism Page TP 71 *) Based on a concentration of 50 IU of heparin sodium in 1 cc (1 ml) of fluid and a control aptt of seconds. APPENDIX 2: PULMONARY EMBOLISM-RELATED EVENTS Dyspnea Chest pain Hypoxemia Syncope Deep vein thrombosis (e.g. leg pain, swelling) Neoplasm, Cancer Arrhythmia Pleural effusion Pulmonary infection, Pneumonia Pulmonary infarction Electrocardiograph abnormalities: atrial arrhythmias complete right bundle branch block peripheral low voltage pseudo infarction pattern (Q waves) in leads III and avf ST segment changes (elevation or depression) over the left precordial leads Acute cor pulmonale (right heart failure) Jugular distension Acute tricuspid regurgitation Congestive heart failure Cardiogenic shock Shock Cardiac arrest Pulseless electric activity (PEA) Haemodynamic collapse Recurrence of PE Second version December 15, 2008

145 Thrombolysis in Pulmonary Embolism Page TP APPENDIX 3: DEFINITIONS 1. Disabling stroke by the modified Rankin Scale Grade 0: Grade 1: Grade 2: Grade 3: Grade 4: Grade 5: no symptoms at all no significant disability despite symptoms: Able to carry out all usual duties and activities slight disability: Unable to carry out all previous activities but able to look after own affairs without assistance moderate disability: Requiring some help but able to walk without assistance moderate severe disability: Unable to walk without assistance and unable to attend to own bodily needs without assistance severe disability: Bedridden, incontinent, and requiring constant nursing care and attention 2. Bleeds 2.1 Major bleeds Severe bleed: bleeding that leads to haemodynamic compromise requiring intervention (e.g. blood or fluid replacement, inotropic support, ventricular assist device, surgical repair) or lifethreatening or fatal bleeds. Moderate bleed: bleeding requiring transfusion of blood but which does not lead to haemodynamic compromise requiring intervention. 2.2 Minor bleeds Mild bleed: bleeding neither requiring blood transfusion nor leading to haemodynamic compromise. 3. Thrombocytopenia 3.1 Mild thrombocytopenia < cells/micro liter 3.2 Moderate thrombocytopenia < cells/micro liter Second version December 15, 2008

146 Thrombolysis in Pulmonary Embolism Page TP Severe thrombocytopenia < cells/micro liter Second version December 15, 2008

147 Thrombolysis in Pulmonary Embolism Assistance Publique des Hôpitaux de Paris Délégation à la Recherche Clinique Clinical Trial Protocol Sponsor trial Number P EudraCT number Investigational product: Tenecteplase (TNK-tPA; TNK-tissue plasminogen activator) Title: PEITHO Pulmonary EmbolIsm THrOmbolysis Study A prospective, randomized, double-blind, placebo-controlled, international, multicentre, parallel-group comparison trial evaluating the efficacy and safety of single i.v. bolus tenecteplase plus standard anticoagulation as compared with standard anticoagulation in normotensive patients with acute pulmonary embolism and with echocardiographic (or spiral CT) and laboratory evidence of right ventricular dysfunction. Thrombolysis in Acute Pulmonary Embolism GLOBAL AMENDMENT n. 1 Version 15 th December 2008 Page 1

148 Thrombolysis in Pulmonary Embolism GLOBAL AMENDMENT n. 1: list of changes 1) Six-month follow-up 2) Criteria for a positive troponin test. 3) Study timelines 4) Spiral CT 5) Platelet count 6) Maximal initial dosage of heparin 7) Delay between the diagnosis of right ventricular dysfunction and randomisation 8) Use of fondaparinux 9) aptt monitoring Page 2

149 Thrombolysis in Pulmonary Embolism 1) Six-month follow-up Previous version: 6-month follow-up was not mentioned. New version: day follow-up The patient s vital status will be recorded six months after randomisation. This may be done by clinic appointment or by contact (phone or mail) with the patient, a family member or the family physician. This six-month follow-up will not delay the database lock and reporting, which will be done based on the in-hospital and 30- day follow-up data. The follow-up can be done between days, but the status should be given for day 180 after randomisation, not earlier. If done earlier, a repeated contact with the patient must be made on or after day 180. If done later, than at the actual day, the vital status should be given for day The following assessments will be done: Death from any cause Primary cause of death (Note: Primary cause of death is a SAE) Severity of dyspnoea on exertion using the NYHA scale. If echocardiography can be performed as part of standard care of the 6-month follow-up at the participating center, the following echocardiographic data will be recorded: Systolic pulmonary artery pressure Persistent right ventricular dysfunction defined as at least one of the following echocardiographic criteria : o Right ventricular end-diastolic diameter (RVEDD) > 30 mm (parasternal long-axis or short-axis view), o Right/left ventricular end-diastolic diameter (RVEDD/LVEDD) > 0.9 (apical or subcostal 4-chamber view), o Hypokinesia of the right ventricular free wall ( any view), o Tricuspid systolic velocity > 2.6 m/s. If indicated by the six-month follow-up, it can be decided to further extend the follow-up period by another six months or longer. 2) Criteria for a positive troponin test Previous version: In the present trial, we will include troponin-positive patients only. A positive troponin test will be defined as follows: Troponin I (ng/ml) Centaur, Bayer: > 0.06 ng/ml Axsym, Abbott: > 0.06 ng/ml TroponinT (ng/ml) Elecsys, Roche: > 0.04 ng/ml Page 3

150 Thrombolysis in Pulmonary Embolism New version: In the present trial, we will include troponin-positive patients only. A positive troponin test will be defined as follows: Criteria for a positive cardiac Troponin test: Troponin I > 0.06 µg/l or troponin T > 0.01 µg/l using the test of the Department of Clinical Chemistry at the participating site. 3) Study timelines Previous version: Nov 2006 (FPI) Oct 2008 (LPO) New version: Nov 2006 (FPI) Dec 2011 (LPO) 4) Spiral CT Previous version: A prospective, randomized, double-blind, placebo-controlled, international, multicentre, parallel-group comparison trial evaluating the efficacy and safety of single i.v. bolus tenecteplase plus standard anticoagulation as compared with standard anticoagulation in normotensive patients with acute pulmonary embolism and with echocardiographic and laboratory evidence of right ventricular dysfunction. New version: A prospective, randomized, double-blind, placebo-controlled, international, multicentre, parallel-group comparison trial evaluating the efficacy and safety of single i.v. bolus tenecteplase plus standard anticoagulation as compared with standard anticoagulation in normotensive patients with acute pulmonary embolism and with echocardiographic (or spiral CT) and laboratory evidence of right ventricular dysfunction. 5) Platelet count Previous version: Known coagulation disorder (including vitamin K antagonists) New version: Known coagulation disorder (including vitamin K antagonists and platelet count < /mm 3 ) 6) Maximal initial dosage of heparin Previous version: The dose of 80 IUxKg -1 as an intravenous bolus, followed by an infusion of 18 IUxKg -1 xh -1, to be administered immediately after randomisation in all patients for at least 48 hours following randomisation. Page 4

151 Thrombolysis in Pulmonary Embolism New version: The dose of 80 IUxKg -1 as an intravenous bolus, followed by an infusion of 18 IU x Kg -1 x h -1 (with a maximum initial infusion rate of 1800 IU x h -1 ), to be administered immediately after randomisation in all patients for at least 48 hours following randomisation. 7) Delay between the diagnosis of right ventricular dysfunction and randomisation Previous version: After diagnosis of RV dysfunction by echocardiography and troponin testing, patients who fulfil the inclusion criteria should be randomised as early as possible. In particular, every effort should be made to administer the study medication (TNK vs. placebo) within 2 hours. New version: After diagnosis of RV dysfunction by echocardiography and troponin testing, patients who fulfil the inclusion criteria should be randomised as early as possible. In particular, every effort should be made to administer the study medication (TNK vs. placebo) within 2 hours after the investigator has received the result of the of the second of the two criteria for RVD, whichever comes the latest. 8) Use of fondaparinux Previous version: The dose of 80 IUxKg -1 as an intravenous bolus, followed by an infusion of 18 IUxKg -1 xh -1, to be administered immediately after randomisation in all patients for at least 48 hours following randomisation. Beyond this period, intravenous UFH may be substituted with subcutaneous heparin (LMWH) treatment. The bolus will be omitted when heparin was started before randomisation. New version: The dose of 80 IUxKg -1 as an intravenous bolus, followed by an infusion of 18 IUxKg -1 xh -1 (with a maximum initial infusion rate of 1800 IU xh -1 ), to be administered immediately after randomisation in all patients for at least 48 hours following randomisation. Beyond this period, intravenous UFH may be substituted with subcutaneous heparin (LMWH) or fondaparinux treatment. The bolus will be omitted when heparin was started before randomisation. 9) aptt monitoring Previous version: In both groups A and B, aptt will be assessed immediately before study drug administration, and approximately 3 hours, approximately 6 hours, approximately 12 hours after administration of the study drug (tenecteplase or placebo), and subsequently at least once daily as long as the patient is on UFH infusion. If adjustments of heparin dosage are necessary, the aptt will also be measured approximately 3 hours after each adjustment. Page 5

152 Thrombolysis in Pulmonary Embolism New version:. In both groups A and B, aptt will be assessed immediately (last available value) before study drug administration, and after study drug administration, approximately 3 hours, approximately 6 hours, approximately 12 hours after start of heparin treatment, and subsequently at least once daily as long as the patient is on UFH infusion. If adjustments of heparin dosage are necessary, the aptt will also be measured approximately 3 hours after each adjustment. Page 6

153 Thrombolysis in Pulmonary Embolism Page TP 1/72 Assistance Publique des Hôpitaux de Paris Délégation à la Recherche Clinique Clinical Trial Protocol Sponsor trial Number P EudraCT number Investigational product: Tenecteplase (TNK-tPA; TNK-tissue plasminogen activator) Title: PEITHO Pulmonary EmbolIsm THrOmbolysis Study A prospective, randomized, double-blind, placebo-controlled, international, multicentre, parallel-group comparison trial evaluating the efficacy and safety of single i.v. bolus tenecteplase plus standard anticoagulation as compared with standard anticoagulation in normotensive patients with acute pulmonary embolism and with echocardiographic (or spiral CT) and laboratory evidence of right ventricular dysfunction. Thrombolysis in Acute Pulmonary Embolism Clinical Phase: Chairman of Steering Committee : Co-Chairman of the Steering Committee and Principal Investigator: Coordinating Investigators: III Stavros KONSTANTINIDES Guy MEYER Giancarlo AGNELLI Samuel Z. GOLDHABER Nils KUCHER Eric VICAUT Date of Protocol: Version 1.0 Aug 31, 2006 Version 2.0 Dec 15, 2008 Version 3.0 Mar 21, 2011 Planned Dates of Trial: Nov 2007 (FPI) Jun 2013 (LPO) version 3.0 March 21, 2011