Clinical trial NCT04419493
Conditions: Healthy
Sponsor: Boehringer Ingelheim
Start date: 2020-06-23; Completion date: 2021-06-21
Enrollment: 30 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
To establish the bioequivalence of alteplase derived from two different manufacturing processes.

Detailed description:

Arms: arm 1: None arm 2: None arm 3: Open-label arm 4: None
Interventions: intervention 1: Alteplase (from modified manufacturing process) intervention 2: Alteplase (from current manufacturing process) intervention 3: Heparin-Natrium-5000-ratiopharm (unfractionated heparin)
Intervention descriptions: intervention 1: Alteplase, TPA-05 intervention 2: Alteplase, TPA-02 intervention 3: Heparin-Natrium-5000-ratiopharm (unfractionated heparin)

Protocol documents:
Prot_000.pdf: 
001-MCS-40-106-RD-13 (6.0) / Saved on: 13 Jun 2018
Clinical Trial Protocol
TITLE PAGE
Document Number:
c30603221-08
EudraCT No.
EU Trial No.
2019-004932-40
NA
BI Trial No.
0135-0340
BI Investigational
Medicinal Product
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 mg/vial)
Title
Bioequivalence of alteplase derived from two different manufacturing 
processes following intravenous administration in healthy male 
volunteers
Lay Title
A study in healthy men to compare 2 different formulations of alteplase
Clinical Phase
I
Clinical Trial Leader
Phone:
 
Fax: 
Investigator
Phone:
 
Fax: 
Status
Final Protocol (Revised Protocol (based on global amendment 7))
Version and Date
Version: 8.0
Date: 14 April 2021
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© 2021 Boehringer Ingelheim International GmbH or one or more of its affiliated companies. All rights reserved.
This document may not - in full or in part - be passed on, reproduced, published or otherwise used without prior written permission


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CLINICAL TRIAL PROTOCOL SYNOPSIS
Company name
Boehringer Ingelheim
Protocol date
17 February 2020
Revision date
14 April 2021
BI trial number
0135-0340
Title of trial
Bioequivalence of alteplase derived from two different manufacturing processes 
following intravenous administration in healthy male volunteers
 Investigator:
Trial site
Clinical phase
I
Trial rationale
To allow a change of manufacturing process of alteplase (new process: TPA-05)
Trial objective
To establish the bioequivalence of alteplase derived from two different 
manufacturing processes (new process, TPA-05, vs. current process, TPA-02)
Trial design
open-label, randomised, 2-way cross-over design with at least 24 h wash-out
Trial endpoints:
Primary endpoints: AUC0-tz and Cmax
Secondary endpoints: AUC0-
Number of subjects 
total entered
72
each treatment
72
The trial part A was performed with 12 subjects and resulted in PK-data that cannot 
be used for the assessment of bioequivalence. Therefore  part B of this trial will be 
performed following a 2-stage design with an adaptive sample size re-estimation. 
Initially 18 subject will be dosed (stage 1, Part B). After an interim analysis a 
maximum of 42 subjects may be dosed in stage 2 (Part B).   
Diagnosis
Not applicable
Main criteria
for inclusion
Healthy male subjects, age of 18 to 45 years (inclusive), body mass index (BMI) of 
18.5 to 29.9 kg/m2 (inclusive)
Test product
alteplase, TPA-05 (50 mg vial)
dose
0.2 mg/kg body weight
mode of admin.
iv-infusion over 30 min


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Reference product
alteplase, TPA-02 (50 mg vial)
dose
0.2 mg/kg body weight
mode of admin.
iv-infusion over 30 min
Auxiliary medicinal 
product
Heparin-Natrium-5000-ratiopharm® (unfractionated heparin)
dose
5000 IU
mode of admin.
iv-bolus (5 min prior to start of each alteplase infusion)
Duration of treatment
single dose for each treatment
Statistical methods
Part A:
Only descriptive analyses are performed since the generated PK-data that cannot be 
used for the confirmatory bioequivalence assessment.
Part B:
The study design is an adaptive two-stage group-sequential design allowing for 
unblinded sample size re-estimation after stage 1. The assessment of bioequivalence 
will be based upon 2-sided repeated confidence intervals (CIs) for the ratios of 
geometric means (test/reference) for the primary endpoints using an acceptance 
range of 80.00 to 125.00%. The alpha level for stage 1 is fixed to 0.03585 and a 
user-defined alpha spending function will be used such that the full level of 0.05 is 
spent at the final analysis. The final analysis will be performed according to the 
inverse normal approach.
The statistical model for each stage of the trial will be an analysis of variance 
(ANOVA) on the logarithmic scale including effects for sequence, subjects nested 
within sequences, period and treatment. p-values will be calculated based on the 
residual error from the ANOVA and the t-distribution.
Descriptive statistics will be calculated for all endpoints.


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FLOW CHART
Trial period
Visit
Day
Planned time (relative to 
start of infusion [h:min]
Approximate clock time  
of actual day [h:min]
Event and comment
Safety laboratory
Alteplase  Infusion
PK blood (x) / ADA (y)
Biomarkers 8
Vital signs (BP, PR)
Questioning for AEs and 
concomitant therapy 6
SCR
1
-21 to -1
Screening (SCR)1
A
x
Treatments periods will be performed on 2 consecutive days
2
-1
-12:00
20:00
Admission to site, check of restrictions
x5
x
1
-1:30
06:30
Allocation to treatment2
x2
x2,8
x2
x2
-0:15
07:45
B
x,y
x8,9
-0:05
07:55
i.v.-bolus of standard heparin
0:00
08:00
Start of alteplase infusion
▲
0:05
08:05
│
x
0:10
08:10
│
x
0:15
08:15
│
x
0:20
08:20
│
x
0:25
08:25
│
x
x8
0:30
08:30
End of infusion
▼
x
x
0:32
08:32
x
0:34
08:34
x
0:36
08:36
x
0:40
08:40
x
0:45
08:45
x
0:50
08:50
x
1:00
09:00
240 mL fluid intake
x
x8,9
x
1:20
09:20
x
1:40
09:40
x
2:00
10:00
Snack , 240 mL fluid intake
x
x8
x
3:00
11:00
Assessment of local tolerability
x
x
4:00
12:00
lunch3
B
x
x
x
6:00
14:00
x
x
7:30
15:30
Snack 
10:45
18:45
Assessment of local tolerability10
(all subjects together)
x7
x10
11:00
19:00
Dinner 
3
1
-1:30
06:30
x2
x2,8
x2
x2
-0:15
07:45
B
x
x8,9
-0:05
07:55
i.v.-bolus of standard heparin
0:00
08:00
Start of alteplase infusion
▲
0:05
08:05
│
x
0:10
08:10
│
x
0:15
08:15
│
x
0:20
08:20
│
x
0:25
08:25
│
x
x8
0:30
08:30
End of infusion
▼
x
x
0:32
08:32
x
0:34
08:34
x
0:36
08:36
x
0:40
08:40
x
0:45
08:45
x
0:50
08:50
x


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1:00
09:00
240 mL fluid intake
x
x8,9
x
1:20
09:20
x
Period
Visit
Day
Planned time (relative to 
start of infusion [h:min]
Approximate clock time  
of actual day [h:min]
Event and comment
Safety laboratory
Alteplase  Infusion
PK blood (x) / ADA (y)
Biomarkers 8
Vital signs (BP, PR)
Questioning for AEs and 
concomitant therapy 6
3
1
1:40
09:40
x
2:00
10:00
Snack, 240 mL fluid intake
x
x8
x
3:00
11:00
Assessment of local tolerability
x
x
4:00
12:00
lunch3
B
x
x
x
6:00
14:00
x
x
7:30
15:30
Snack 
10:45
18:45
Assessment of local tolerability10
(all subjects at that time)
x10
11:00
19:00
Dinner 
2
24:00
08:00
Assessment of local tolerability,
Breakfast (voluntary), 
discharge from trial site
B
x8
x
x
EOT
4
8-22
End of trial (EOT) examination4
C
x
x
1.
Subject must be informed and written informed consent obtained prior to starting any screening procedures. Screening 
procedures include physical examination, check of vital signs, ECG, safety laboratory (including drug screening, fecal 
occult blood test), demographics (including determination of body height and weight, smoking status and alcohol 
history), relevant medical history, concomitant therapy and review of inclusion/exclusion criteria. Pharmacogenetic 
samples will be collected if needed.
2.
The time is approximate; the procedure is to be performed and completed within 3 h prior to drug administration.
3.
If several actions are indicated at the same time point, the intake of meals will be the last action.
4.
End of trial examination includes physical examination, vital signs, ECG, safety laboratory, recording of AEs and 
concomitant therapies. 
5.
Only urine drug screening and alcohol breath test
6.
AEs and concomitant therapies will be recorded throughout the trial, but will be specifically asked for at the time points 
indicated in the Flow Chart above.
7.
Urine stix only
10. tolerance time for these measurements is ± 90 min


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TABLE OF CONTENTS
TITLE PAGE ......................................................................................................1
CLINICAL TRIAL PROTOCOL SYNOPSIS ................................................2
FLOW CHART...................................................................................................4
TABLE OF CONTENTS ...................................................................................6
ABBREVIATIONS.......................................................................................... 11
1.
INTRODUCTION.................................................................................... 14
1.1
MEDICAL BACKGROUND............................................................................14
1.2
DRUG PROFILE OF ALTEPLASE................................................................15
1.2.1
General information .............................................................................15
1.2.2
Paradox effect of alteplase on the coagulation system.......................15
1.2.3
Safety in patients after systemic administration of Actilyse® ..........17
1.2.4
Clinical experience with alteplase in healthy subjects.......................17
1.2.6
Residual Effect Period..........................................................................28
1.3
DRUG PROFILE OF HEPARIN.....................................................................28
1.4
RATIONALE FOR PERFORMING THE TRIAL........................................28
1.4.2
Rationale for bioequivalence trial in humans ....................................29
1.5
BENEFIT - RISK ASSESSMENT ...................................................................29
1.5.1
Procedure-related risks ........................................................................30
1.5.2
Risk resulting from the trial design.....................................................30
1.5.3
Drug-related risks and safety measures..............................................30
1.5.3.1
Risks associated to alteplase administration...........................30
1.5.3.2
Risk associated to heparin administration...............................33
1.5.3.3
Risk of combined administration of alteplase and heparin.....34
1.5.3.4
Drug-induced liver injury (DILI)............................................34
1.5.4
Overall assessment................................................................................35
2
TRIAL OBJECTIVES AND ENDPOINTS........................................... 36
2.1
MAIN OBJECTIVES, PRIMARY AND SECONDARY ENDPOINTS ......36
2.1.1
Main objectives......................................................................................36


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2.1.2
Primary endpoints ................................................................................36
2.1.3
Secondary endpoint ..............................................................................36
2.2.2.3
Safety and tolerability..............................................................37
3
DESCRIPTION OF DESIGN AND TRIAL POPULATION.............. 38
3.1
OVERALL TRIAL DESIGN AND PLAN ......................................................38
3.2
DISCUSSION OF TRIAL DESIGN, INCLUDING THE CHOICE OF 
CONTROL GROUP..........................................................................................41
3.3
SELECTION OF TRIAL POPULATION ......................................................41
3.3.1
Main diagnosis for trial entry..............................................................42
3.3.2
Inclusion criteria...................................................................................42
3.3.3
Exclusion criteria ..................................................................................42
3.3.4
Withdrawal of subjects from treatment or assessments ...................44
3.3.4.1
Discontinuation of trial treatment............................................44
3.3.4.2
Withdrawal of consent to trial participation ............................44
3.3.4.3
Discontinuation of the trial by the sponsor..............................45
3.3.5
Replacement of subjects .......................................................................45
4
TREATMENTS........................................................................................ 46
4.1
INVESTIGATIONAL TREATMENTS ..........................................................46
4.1.1
Identity of the Investigational Medicinal Products ...........................46
4.1.2
Selection of doses in the trial................................................................47
4.1.3
Method of assigning subjects to treatment groups ............................47
4.1.4
Drug assignment and administration of doses for each subject .......47
4.1.5
Blinding and procedures for unblinding ............................................49
4.1.6
Packaging, labelling, and re-supply ....................................................49
4.1.7
Storage conditions.................................................................................49
4.1.8
Drug accountability ..............................................................................49
4.2
OTHER TREATMENTS, EMERGENCY  PROCEDURES,
RESTRICTIONS ...............................................................................................50
4.2.1
Other treatments and emergency procedures....................................50
4.2.2
Restrictions............................................................................................50
4.2.2.1
Restrictions regarding concomitant treatment .........................50
4.2.2.2
Restrictions on diet and life style.............................................50
4.3
TREATMENT COMPLIANCE.......................................................................51
5
ASSESSMENTS....................................................................................... 52
5.1
ASSESSMENT OF EFFICACY.......................................................................52
5.2
ASSESSMENT OF SAFETY............................................................................52


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5.2.1
Physical examination ............................................................................52
5.2.2
Vital signs...............................................................................................52
5.2.3
Safety laboratory parameters..............................................................52
5.2.4
Electrocardiogram................................................................................55
5.2.5
Other safety parameters.......................................................................55
5.2.5.1
Local tolerability......................................................................55
5.2.6
Assessment of adverse events...............................................................55
5.2.6.1
Definitions of adverse events...................................................55
5.2.6.1.1 Adverse event ............................................................55
5.2.6.1.2 Serious adverse event ................................................56
5.2.6.1.3 AEs considered ‘Always Serious’.............................56
5.2.6.1.4 Adverse events of special interest .............................57
5.2.6.1.5 Intensity (severity) of AEs.........................................57
5.2.6.1.6 Causal relationship of AEs ........................................57
5.2.6.2
Adverse event collection and reporting ...................................58
5.2.6.2.1 AE collection.............................................................58
5.2.6.2.2 AE reporting to the sponsor and timelines ................59
5.2.6.2.3 Information required..................................................59
5.3
DRUG CONCENTRATION MEASUREMENTS AND 
PHARMACOKINETICS..................................................................................60
5.3.1
Assessment of pharmacokinetics .........................................................60
5.3.2
Methods of sample collection...............................................................60
5.3.2.1
Blood sampling for pharmacokinetic analysis of alteplase......60
5.4
ASSESSMENT OF BIOMARKER (S) ............................................................64
5.5
BIOBANKING ...................................................................................................64
5.6
OTHER ASSESSMENTS .................................................................................64
5.7
APPROPRIATENESS OF MEASUREMENTS.............................................64
6
INVESTIGATIONAL PLAN.................................................................. 65
6.1
VISIT SCHEDULE............................................................................................65
6.2
DETAILS OF TRIAL PROCEDURES AT SELECTED VISITS ................65
6.2.1
Screening................................................................................................65
6.2.2
Treatment period ..................................................................................65
6.2.3
Follow-up period and trial completion ...............................................66


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7
STATISTICAL METHODS AND DETERMINATION OF
SAMPLE SIZE......................................................................................... 67
7.1
STATISTICAL DESIGN – MODEL ...............................................................67
7.2
NULL AND ALTERNATIVE HYPOTHESES ..............................................67
7.3
PLANNED ANALYSES....................................................................................69
7.3.1
Primary endpoint analyses...................................................................70
7.3.2
Secondary endpoint analyses ...............................................................71
7.3.4
Safety analyses.......................................................................................72
7.4
INTERIM ANALYSES .....................................................................................73
7.5
HANDLING OF MISSING DATA ..................................................................74
7.5.1
Safety......................................................................................................74
7.5.2
Pharmacokinetics..................................................................................74
7.6
RANDOMISATION ..........................................................................................74
7.7
DETERMINATION OF SAMPLE SIZE ........................................................75
8
INFORMED CONSENT, TRIAL RECORDS, DATA 
PROTECTION, PUBLICATION POLICY, AND 
ADMINISTRATIVE STRUCTURE...................................................... 76
8.1
TRIAL APPROVAL, SUBJECT INFORMATION, INFORMED 
CONSENT ..........................................................................................................76
8.2
DATA QUALITY ASSURANCE .....................................................................77
8.3
RECORDS..........................................................................................................77
8.3.1
Source documents .................................................................................77
8.3.2
Direct access to source data and documents.......................................78
8.3.3
Storage period of records.....................................................................78
8.4
EXPEDITED REPORTING OF ADVERSE EVENTS .................................79
8.5
STATEMENT OF CONFIDENTIALITY AND SUBJECT PRIVACY.......79
8.5.1
Collection, storage and future use of biological samples and 
corresponding data ...............................................................................79
8.6
TRIAL MILESTONES .....................................................................................79
8.7
ADMINISTRATIVE STRUCTURE OF THE TRIAL ..................................80
9
REFERENCES......................................................................................... 81
9.1
PUBLISHED REFERENCES...........................................................................81
9.2
UNPUBLISHED REFERENCES.....................................................................86
10
APPENDICES .......................................................................................... 87
10.1
INSTRUCTIONS FOR RECONSTITUTING ALTEPLASE .......................87
11
DESCRIPTION OF GLOBAL AMENDMENT(S).............................. 89
11.1
GLOBAL AMENDMENT 1 .............................................................................89
11.2
GLOBAL AMENDMENT 2 .............................................................................90


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11.3
GLOBAL AMENDMENT 3 .............................................................................91
11.4
GLOBAL AMENDMENT 4 .............................................................................92
11.5
GLOBAL AMENDMENT 5 .............................................................................93
11.6
GLOBAL AMENDMENT 6 .............................................................................96
11.7
GLOBAL AMENDMENT 7 .............................................................................98


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ABBREVIATIONS
ADA
Anti-drug antibody
AE
Adverse event
AESI
Adverse events of special interest
ANOVA
Analysis of variance
AUC0-∞
Area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity
%AUCtz-∞
Percentage of AUC0-∞obtained by extrapolation
AUC0-tz
Area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point
BI
Boehringer Ingelheim
BMI
Body mass index (weight divided by height squared)
BP
Blood pressure
CA
Competent authority
CHO
Chinese hamster ovary
CI
Confidence interval
CL
Total clearance of the analyte in plasma after intravascular administration
Cmax
Maximum measured concentration of the analyte in plasma
Cmin
Minimum measured concentration of the analyte in plasma
CML
Clinical Monitor Local
CRF
Case Report Form, paper or electronic (sometimes referred to as ‘eCRF’)
CT
Computed tomography
CTP
Clinical trial protocol
CTR
Clinical trial report
DILI
Drug induced liver injury

Bioequivalence margin
ECG
Electrocardiogram
eCRF
Electronic case report form
eDC
Electronic data capture
EDTA
Ethylenediaminetetraacetic acid
EoTrial
End of trial
EudraCT
European Clinical Trials Database
F1+2
Prothrombin fragment F1+2
FOB
Faecal occult blood
GCP
Good Clinical Practice
gCV
Geometric coefficient of variation
gMean
Geometric mean


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GMR
geometric mean ratio
IB
Investigator’s brochure
IEC
Independent Ethics Committee
IRB
Institutional Review Board
ISF
Investigator site file
i.v.
Intravenous(ly)
kDa
Kilodalton
kg
Kilogram(s)
l
Liter(s)
z
Terminal rate constant of the analyte in plasma
MDA
Methylenedioxyamphetamine
MDMA
Methylenedioxymethamphetamine
MedDRA
Medical Dictionary for Regulatory Activities
mg
Milligram(s)
PAI-1
Plasminogen activator inhibitor 1
PAP
Plasmin - alpha2-antiplasmin complex
PD
Pharmacodynamic(s)
PK
Pharmacokinetic(s)
PKS
Pharmacokinetic set
PR
Pulse rate
QT
Time between start of the Q-wave and the end of the T-wave in an 
electrocardiogram
QTc
QT interval corrected for heart rate using the method of Fridericia (QTcF) 
or Bazett (QTcB)
R
Reference treatment
REP
Residual effect period
SAE
Serious adverse event
SCR
Screening
SmPC
Summary of Product Characteristics
SOP
Standard operating procedure
SUSAR
Suspected unexpected serious adverse reaction
T
Test product or treatment
TAT
Thrombin-antithrombin complex
t-PA/TPA
Alteplase (tissue plasminogen activator)
t1/2
Terminal half-life of the analyte in plasma
tmax
Time from (last) dosing to the maximum measured concentration of the 
analyte in plasma
TS
Treated set


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tz
Time of last measurable concentration of the analyte in plasma
TSAP
Trial statistical analysis plan
TxA2
Thromboxane A2
TxB2
Thromboxane B2
ULN
Upper limit of normal
Vz
Apparent volume of distribution during the terminal phase after 
intravascular administration
XTC
Ecstasy 


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1.
INTRODUCTION
Alteplase is a tissue plasminogen activator (t-PA) produced by recombinant DNA 
technology. Alteplase is the active pharmaceutical ingredient of medicinal products currently 
approved in the Europe under the brand names Actilyse® or Actilyse® Cathflo® which are 
used for thrombolytic treatment in several indications (see below).
Boehringer Ingelheim intends a post-approval change of the drug substance manufacturing 
process for alteplase concerning both approved medicinal products (Actilyse® and Actilyse®
Cathflo®). Compared to the current registered manufacturing process for alteplase (referred to 
as TPA-02) the proposed manufacturing process TPA-05 contains modifications to upstream 
and downstream processing including modifications to raw and starting materials used, cell 
culturing procedures and purification methodology. This trial aims to establish  
bioequivalence of TPA-02 compared with TPA-05  following intravenous administration of 
0.2 mg/kg body weight.
1.1
MEDICAL BACKGROUND
Alteplase is a tissue plasminogen activator (t-PA) produced by recombinant DNA 
technology. Tissue plasminogen activator binds to fibrin clots and activates plasminogen, 
leading to the generation of plasmin and to the degradation of fibrin clots or blood coagulates. 
Physiologic dissolution of a thrombus depends primarily on the proteolytic action of plasmin 
formed from plasminogen at the site of a clot. Plasminogen, the zymogen precursor of 
plasmin, may be activated by substances found in the plasma milieu, in tissue (tissue-type 
plasminogen activator or t-PA), urine (urokinase), and in bacteria (streptokinase). The 
generated plasmin may be inhibited by plasma proteins such as α2-antiplasmin and α2-
macroglobulin.
Tissue plasminogen activator is an endogenous protein and was first isolated from a human 
melanoma cell line [P86-20349, P84-97880]. Tissue plasminogen activator was described to 
be a serine protease with a molecular mass of about 72 kDa and occurred as either a one- or a 
two-chain molecule, depending on the isolation procedures employed. Both the one- and two-
chain forms appear to have equal catalytic efficiencies for their natural substrate, 
plasminogen, only in the presence of fibrin. Kinetic analyses suggest that plasminogen 
activation in the presence of fibrin occurs after the binding of plasminogen and t-PA at the 
clot site. Plasminogen-t-PA complex results in an increased concentration of fibrin-bound 
plasmin [P84-97878].
Actilyse® (available as powder and solvent for solution for injection and infusion at dose 
strengths of 10, 20 and 50 mg) is currently indicated for the thrombolytic treatment of acute 
myocardial infarction, acute massive pulmonary embolism and acute ischaemic stroke. 
Actilyse® Cathflo® (available as powder and solvent for solution for injection and infusion at 
a dose strength of 2 mg) is indicated for the thrombolytic treatment of occluded central 
venous access devices including those used for haemodialysis.


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1.2
DRUG PROFILE OF ALTEPLASE
1.2.1
General information
Alteplase is a recombinant wild type DNA-derived version of t-PA (rt-PA). It is a 
glycoprotein consisting of 527 amino acids with a molecular mass of 59 kDa without 
consideration of the carbohydrate moieties attached at positions Asn117, Asn142, Asn184 
and Asn448. The total relative molecular mass is approximately 65 kDa. Alteplase is cleaved 
into a two-chain form by plasmin between amino acids 275 and 276 connected by a disulfide 
bridge between Cys264 and Cys395. The single-chain form and the two-chain form show 
comparable fibrinolytic activity in vitro. Alteplase is expressed in Chinese hamster ovary 
(CHO) cells. It is manufactured using standard mammalian production techniques, followed 
by a series of protein purification steps including several chromatography steps.
Mode of action: Alteplase activates plasminogen directly to plasmin. When administered 
intravenously, alteplase remains relatively inactive in the circulatory system. Once bound to 
fibrin, it is activated, inducing the conversion of plasminogen to plasmin leading to the 
dissolution of the fibrin clot.
The pharmacokinetic profile of alteplase after intravenous administration is well documented: 
alteplase is cleared rapidly from the circulating blood and metabolised mainly by the liver 
(plasma clearance 550 - 680 ml/min.). The relevant plasma half-life t1/2α is 4 – 5 minutes. 
This means that after 20 minutes less than 10% of the initial value is present in the plasma. 
For the residual amount remaining in a deep compartment, a beta-half-life of about 40 
minutes was measured [U87-0935, U85-0697, U86-0354, U87-0368, P89-49823].
For a more detailed description of alteplase profile, please refer to the current Investigator’s 
Brochure [c29758839-04].
1.2.2
Paradox effect of alteplase on the coagulation system
In the treatment of acute myocardial infarction (AMI) success of thrombolytic therapy was 
limited by failure of clot lysis or reocclusion. As indicators of an unfavorable outcome of 
thrombolytic therapy markers of elevated thrombin activity were identified. So it could be 
demonstrated that initiation of a fibrinolytic therapy caused a rapid increase of plasma 
concentrations of thrombin/antithrombin complex (TAT, marker of thrombin activation), 
prothrombin fragment F1+2 (F1+2, marker of thrombin generation) and fibrinopeptiode A 
(FPA, a marker of thrombin activity) [ P91-62123, P92-68159, P95-86478, R99-0792, P88-
39175, P89-45379, P92-68175]
The fact of thrombin stimulation by a drug that is administered in order to lyse a thrombus is 
called “thrombolytic paradox”. The thrombolytic paradox seems to be related to the extent of 
systemic plasmin activation, which is more pronounced with non-fibrin specific thrombo-
lytics (e.g. streptokinase) compared to more fibrin-specific thrombolytics (e.g. alteplase, 
tenecteplase) [P88-37698, P89-49897, P99-00076, P20-11344].


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Which are the underlying mechanisms of the thrombolytic paradox ? 
Based on the in-vitro work of Ewald and Eisenberg thrombolytic drugs cause a plasmin 
mediated activation of the factor XII-kallikrein system (contact system of coagulation) [P96-
2741]. Based on in-vitro data also other coagulation factors (e.g. V, VII, VIII) seem to be 
involved in plasmin-mediated thrombin activation [R20-4206, P99-01489, R20-4207]. In 
contrast the activation of the factor XII-kallikrein pathway has been proven in several clinical 
trials including alteplase regimen in the treatment of AMI. In these trials a combined increase 
of plasmin-antiplasmin complex (PAP, marker of plasmin activation), kallikrein/factor XIIa 
and TAT (marker of thrombin activation) could be demonstrated [P99-00076, P00-02754, 
P03-10831].
A mechanism probably contributing to the procoagulant effects is the activation of platelets 
which appears to occur immediately after initiation of a fibrinolytic therapy [P94-80579]. On 
the one hand this seems to be a secondary effect to increased thrombin formation, because 
thrombin is potent platelet activator. On the other hand there are direct effects of plasmin on 
platelets. Based on intensive in-vitro investigations plasmin may induce or inhibit platelet 
function (for review see R20-4253). 
Clinical data describing effects of plasminogen activators on platelet function are rare. 
Fitzgerald et al could demonstrate a marked increase of thromboxane A2 (TXA2) formation 
(measured as urinary excretion of its main metabolite 2,3-dinor TXB2) after thrombolytic 
therapy with streptokinase in AMI patients. Considering, that platelets are the major source of 
TXA2 a marked platelet activation can be concluded by these findings [R99-2284]. Similar 
results could be demonstrated during coronary thrombolysis with t-PA. In this study a parallel 
group of patients received a single dose of 325 mg aspirin that could abolish the increase in 
urinary metabolites of TXA2 and prostacyclin [P90-52264].  
In an ex-vivo study tissue-type plasminogen activator added to platelet rich plasma from 12 
healthy subjects increased platelet surface P-selectin (marker of platelet activation). This 
effect could be inhibited by administration of aspirin over 7 days prior to blood sampling 
[R20-4252]. In a canine-model of coronary thrombosis the iv administration of the thrombin 
inhibitor argatroban (starting 30 min prior to t-PA infusion until 2 h after reperfusion) did not 
alter the increase of urinary 2,3-dinor TXB2 (marker of thromboxane biosynthesis) indicating 
that there might be thrombin-independent mechanisms of platelet activation during 
thrombolysis [P90-56499]. 
The activation of platelets may be important, because platelet function is not much inhibited 
by heparin. This might explain why heparin could not completely prevent thrombin activation 
in patients with AMI undergoing a thrombolytic therapy [P91-62123, P95-86478, P98-2656, 
P99-00076, P03-10831]. Activated platelets release procoagulant (coagulation factor V, VIII, 
XIII; TxA2) and antifibrinolytic (PAI-I) substances which may attenuate fibrinolytic efficacy 
and maintain prothrombotic status [P94-80579].


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1.2.3
Safety in patients after systemic administration of Actilyse®
Actilyse® is a well-established product, first authorised in Europe for marketing in June 1987. 
Nowadays it has been authorised worldwide in more than 100 countries. 
The most frequent adverse reaction associated with Actilyse® is bleeding in different forms 
resulting in a fall in haematocrit and/or haemoglobin values. Haemorrhage at any site or body 
cavity can occur and may result in life-threatening situations, permanent disability or death.
The type of bleeds associated with thrombolytic therapy can be divided into two broad 
categories:
-
superficial bleeding, normally from punctures or damaged blood vessels
-
internal bleeding at any site or body cavity.
Compared to other indications patients with acute ischaemic stroke treated with Actilyse®
have a markedly increased risk of intracranial haemorrhage as the bleeding occurs predomi-
nantly into the infarcted area. The incidence of symptomatic intracranial haemorrhage in 
acute myocardial infarction is <1% [P99-02520], whereas it is about 4%-5% in acute 
ischaemic stroke [P16-06898].
With intracranial haemorrhage neurological symptoms such as somnolence, aphasia, 
hemiparesis, convulsion may be associated. Advanced age, lower weight, female gender, 
prior cerebrovascular disease and systolic and diastolic hypertension on admission are 
significant predictors of intracranial haemorrhage.
Immune-mediated hypersensitivity reactions associated with the administration of Actilyse®
can be caused by the active substance alteplase, gentamicin (a trace residue from the 
manufacturing process), any of the excipients or the stopper of the glass vials which contains 
natural rubber (a derivative of latex). Angio-oedema represents the most common
hypersensitivity reaction reported with Actilyse®.
For a more detailed description of drug safety after systemic administration of Actilyse®, 
please refer to the SmPC of Actilyse® [R20-0187].
1.2.4
Clinical experience with alteplase in healthy subjects
In several clinical trials alteplase has been administered to healthy subjects:
Verstraete et al (1986) report a trial in which an in initial dose of 40 mg or 60 mg of 
recombinant tissue plasminogen activator (rt-PA) was administered intravenously over 90 
min to two groups of 6 healthy subjects, which was followed by a maintenance dose of 30 mg 
over 6 h in both groups. This dosing regimen resulted in maximum serum concentrations of 
1080 ng/ml (40 mg group) and 1560 ng/ml (60 mg group) and in steady state levels of 250 
ng/ml during maintenance infusion. At the end of the initial infusion there was a dose 
dependent decrease of fibrinogen to 74% and 57%, of plasminogen to 55% and 48% and of 
α2-antiplasmin to 28% and 18% of initial values in both groups. 
In the 60 mg dose group two minor bleeding events occurred. In one subject oozing from an 
oral blister (not detected prior to start of infusion) was observed. The infusion was stopped 
and oozing seezed within 30 min. For this subject a Cmax-value of 2590 ng/ml is reported. The 
other subject had subcutaneous hematomas at the i.v.-catheter sites and at a site in the 


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forearm where a contusion occurred the day before (also in this subject the infusion was 
stopped). No further side effects have been reported [P86-5232].
Seyfried et al (1988) report a trial in which 8 healthy subjects received 0.25 mg rt-PA per kg 
body weight as i.v.-infusion over 30 min. Maximum rt-PA concentrations were 973 ng/ml or 
813 ng/ml dependent on whether or not the samples had been treated with D-Phe-Pro-Arg-
CH2Cl. In this trial the infusion was well tolerated without adverse reactions [P88-34055].  
Transwell et al (1989) report a trial in which three groups of 6 subjects received either 
placebo, 0.25 mg/kg rt-PA or 0.5 mg/kg rt-PA as i.v.-infusion over 30 min. Maximum rt-PA 
antigen concentrations were 960 ng/ml and 1830 ng/ml in both active groups. While 
plasminogen levels decreased only slightly, α2-antiplasmin after 60 min decreased to 85% 
and 65% of baseline values in the lower and the higher dose group. Fibrinogen levels were 
not affected. The infusions were tolerated with no adverse reactions [P89-49823].
The studies 135.57 and 135.67 are two bioequivalence trials that were performed 1989 and 
1992 in the
in 
 comparing alteplase derived from two 
different manufacturing processes. In both trials two groups of 12 subjects (parallel-group 
design) received an i.v. infusion of 0.25 mg/kg alteplase over 30 min.
In the 135.57 trial mean maximum alteplase concentrations of 774 ng/ml and 739 ng/ml have 
been achieved. At 1 hour after infusion plasminogen and fibrinogen decreased by about 10% 
compared to baseline. After 24 hours there was no change of plasminogen and fibrinogen 
compared to baseline. No adverse events were reported during this trial [U91-0823].
In the 135.67 trial mean maximum alteplase concentrations of 917 ng/ml and 933 ng/ml have 
been achieved. One hour after start of infusion plasminogen decreased by 8% and 9% in both 
treatment groups. The respective decrease of α2-antiplasmin was -16% and -14%. These 
changes were not detectable any more at 24 hours. For fibrinogen no directional change was 
seen. One subject complained about mild nausea at about 2 hours after start of infusion (AE 
recovered within 1 hour). Otherwise both treatments were well tolerated [U94-2138].
De Boer et al (1993) reported a trial that investigated the effect of nifedipine (20 mg p.o.) on 
endogenous and recombinant tissue-type plasminogen activator (t-PA and rt-PA). Following 
a randomized, three-way cross-over design with a wash-out period of one week between the 
treatments nine healthy subjects received an intravenous infusion of 35 mg rt-PA over 120 
min with nifedipine capsules (a), with placebo nifedipine capsules (b) and an i.v. saline 
infusion over 120 min with nifedipine (c). According to Figure 2 in this paper maximum 
plasma concentrations of total rt-PA antigen at the end of infusion were in the range of 700 –
800 ng/ml. With the exception of vasovagal reactions in one subject the authors did not report 
adverse events from this trial. All subjects completed the study [P93-77079].


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1.2.6
Residual Effect Period
The Residual Effect Period (REP) of alteplase is 24 hours in this trial. This is the period after 
the last dose with measurable drug levels and/or pharmacodynamic effects is still likely to be 
present.
1.3
DRUG PROFILE OF HEPARIN
Heparin is an anticoagulant drug, which binds antithrombin III (AT III) and enhance its 
activity by 700-fold. In turn, AT III counteracts coagulation by inhibiting several activated 
clotting factors, including factor IIa, VIIa, Xa, XIa and XIIa. Furthermore, heparin inhibits 
platelets activity and may prevent fibrin formation (in very high doses).
Heparin can only be administered via parenteral route (subcutaneous or intravenous) due to 
lacking gut absorption. After intravenous injection peak plasma concentration and pharmaco-
dynamic effects are observed immediately, while after subcutaneous administration effects 
appear with 20-30 min delay. Administration of 5000 IU twice daily leads to average plasma 
concentration of 2-8 IU/mL.  
Heparin is highly bound to plasma proteins, such as LDL, globulin and fibrinogen with an 
average volume of distribution of 0.07 L/kg. The average elimination half-life is 90-120 min. 
Heparin can be degraded in liver and excreted mainly as inactive form in urine. Drug 
elimination is highly variable and depends on proper liver and kidney function as well as 
comorbidities
Therapeutic doses of heparin are 5000 IU intravenous loading dose (bolus), followed by 
1000 IU / h continuous infusion (by infusion pump), while prophylactic doses are 
approximately 5000-7500 I.E. subcutaneous daily. 
Adverse reactions to heparin include bleeding and prolonged bleeding disorder, heparin-
induced thrombocytopenia type I and II, allergic reactions (including anaphylactic shock) or 
increased liver enzymes (e.g. GOT, GPT). The antidote for heparin is protamine, which can 
be used to counteract heparin overdose or life-threatening heparin-induced bleeding. 
For a complete listing of adverse reactions, including frequency of occurrence, please refer to 
the current SmPC [R20-4172].  
1.4
RATIONALE FOR PERFORMING THE TRIAL
Actilyse®  is used in more than 100 countries for the treatment of acute ischaemic stroke, 
myocardial infarction, and acute massive pulmonary embolism. To cover the rising world-


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wide demand for Actilyse®  the manufacturing process has been modified. Alteplase provided 
by the updated manufacturing process is TPA-05 (alteplase obtained from the current process 
is TPA-02). 
The modified production process resulted in changes of the glycosylation pattern of TPA-05 
compared to TPA-02 [q00263495-01]. Because it could not be ruled out that these differences 
in glycosylation pattern might influence the plasma clearance of TPA-05, a pharmacokinetic 
study in rabbits was done.
1.4.2
Rationale for bioequivalence trial in humans
Although bioequivalence for TPA-05 and TPA-02 could be shown in the animal model there 
are remaining uncertainties if these results are also quantitatively predictive for the human 
situation. To resolve these uncertainties bioequivalence between TPA-02 and TPA-05 should 
be demonstrated in a clinical trial. This trial aims to establish bioequivalence of TPA-05
compared with TPA-02 following intravenous administration of 0.2 mg/kg body weight in 
healthy male subjects.
1.5
BENEFIT - RISK ASSESSMENT
Participation in this clinical trial is without any (therapeutic) benefit for healthy subjects. 
Their participation, however, is of major importance for thrombolytic treatment of myocar-


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dial infarction, acute massive pulmonary embolism and acute ischaemic stroke. Subjects are 
exposed to risks of study procedures and risks related to the exposure to the trial medication.
1.5.1
Procedure-related risks
The use of an indwelling venous catheter or venepuncture for e.g. blood sampling may result 
in mild bruising, subcutaneous hematomas at the site of i.v.-catheter, syncope, feeling of 
lightheadedness, and in rare cases, in transient inflammation of the wall of the vein, or nerve 
injury, potentially resulting in paraesthesia, reduced sensibility, and/or pain for an indefinite 
period.
The total volume of blood withdrawn per subject during the entire study will not exceed the 
volume of a normal blood donation (500 mL). No health-related risk to healthy subjects is 
expected from withdrawal of this volume of blood.
1.5.2
Risk resulting from the trial design
In this cross-over trial two single doses of alteplase (0.2 mg/kg) will be infused on 2 consecu-
tive days keeping a wash-out period of at least 24 hours. This short interval has been chosen 
to prevent any effect of potentially formed anti-drug antibodies on alteplase kinetics. 
Considering the short beta-half-life of alteplase of about 40 min this wash-out period is 
justified.  In the sister trials 135.57 and 135.67 laboratory parameters of fibrinolytic system 
(fibrinogen, plasminogen and α2-antiplasmin) were only minimally affected by a chosen dose 
of 0.25 mg/kg alteplase (infusion in 30 min) and after 24 hours these changes were not 
detectable any more [U91-0823, U94-2138]. Considering that there is neither a PK- nor a PD-
overlap no undue risk to healthy subjects is expected from the chosen wash-out period. 
1.5.3
Drug-related risks and safety measures
1.5.3.1
Risks associated to alteplase administration
Bleeding risk
The fibrinolytic effect of alteplase is relatively clot-specific. This is attributed to the very low 
affinity of t-PA to circulating plasminogen in contrast to its high affinity to fibrin bound 
plasminogen. Thus alteplase binds to fibrin in the thrombus and converts plasminogen to 
plasmin on the surface of the clot, thereby initiating local fibrinolysis without activating 
systemic plasminogen substantially [P84-97878, P88-37492].
The fact that alteplase exerts its fibrinolytic effect predominantly on a local basis (on the clot) 
and shows only limited effects on systemic plasminogen is in line with the tolerability obser-
ved in the healthy subject trials cited in section 1.2.2. Alteplase was overall well tolerated by 
the subjects. Only in one trial two minor bleeding events occurred: oozing from an (pre-
existing) oral blister and subcutaneous hematomas at the i.v.-catheter sites. Both cases can be 
at least partly explained by alteplase induced lysis of hemostatic fibrin in pre-existing 
injuries. The subject with the bleeding blister had a maximum alteplase concentration of 2590 
ng/ml which was the by far highest concentration reported in this trial [P86-5232].


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In the trial reported by Transwell at al. the infusion of 0.5 mg/kg rt-PA in 30 min resulted in 
maximum alteplase concentrations of 1830 mg/kg, which is the highest reported mean value 
of rt-PA concentrations in the cited healthy volunteer trials (see 1.2.2). This exposure was 
tolerated without any adverse events [P89-49823]. Infusion of 0.25 mg/kg rt-PA in 30 min 
were tested in several trials and resulted in maximum alteplase concentrations of 900 -1000 
ng/ml [P89-49823, P88-34055, U94-2138]. 
The alteplase dose to be administered in this trial is even lower. Based on trials 135.67 [U94-
2138] and 135.57 [U91-0823] and the assumption of linear pharmacokinetics, the planned 
infusion of 0.2 mg/kg rt-PA in 30 min is expected to result in maximum alteplase 
concentrations of approximately 620-750 ng/ml, thus providing a safety margin of more than 
2 compared to the concentration reported in the Transwell study and of more than 3 
compared to the concentration reported from the subject with the bleeding blister. 
To mitigate the bleeding risk the following safety measures have been undertaken:

A low dose of 0.2 mg/kg has been chosen

A minimum body weight of 65 kg has been defined (see 3.3.2)

Trial specific exclusion criteria haven been defined to exclude subjects who are 
perceived to have an increased risk of bleeding (see 3.3.3)

Trial specific restrictions have been defined to reduce the risk of injury prior to dosing 
(see 4.2.2)

Inhouse confinement of 24 h after start of infusion (covering the complete elimination 
of alteplase and heparin) to reduce the risk of injury after dosing

Thorough lab testing (including urinestix)

Frequent AE-questioning (focus on bleeding events)
Considering the mode of action of alteplase focusing on fibrin bound plasminogen, taking 
into account the safety measures described above and referring to the good tolerability of rt-
PA infusions in the cited healthy volunteer trials the bleeding risk for healthy subjects partici-
pating in this trial is expected to be low.    
Subcutaneous hematomas at the injection site of i.v.-catheters are typical procedure related 
side effect in clinical trials. They happen more often with anticoagulants and are expected 
also in this trial. Subcutaneous hematomas are no undue risk to healthy subjects. At the time 
points given in the Flow Chart inspections of the injection sites will be performed. If 
necessary cooling bags may be applied.
Risk of thromboembolic complications
In part A of the trial clinical observations have been made indicating a procoagulant status. 
An increase of fibrin/fibrinogen degradation products has been reported also in previous trials 
in healthy subjects [P86-5232, P87-3595]. However, at that time the origin of the fibrin in 
healthy subjects was unclear. 
Meanwhile it is known that fibrinolytic therapy is associated with a paradox thrombin 
stimulation. An increased generation of F1+2 (marker of thrombin generation), TAT (marker 
of thrombin activity) and FPA (marker of thrombin activity) after start of alteplase infusion 
has been demonstrated in several clinical trials (for references see 1.2.2).


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In these trials patients with AMI have been treated with therapeutic doses of up to 100 mg 
alteplase. In the current trial doses of 13-20 mg alteplase (depending on body weight) will be 
administered to healthy subjects. Considering, that the activation of coagulation seems to be 
related to the extent of systemic plasmin activation (see 1.2.2) lower alteplase doses are 
supposed to cause less thrombin stimulation compared to higher doses. Taking into account 
also the hypercoagulatory status in patients with AMI the risk of thromboembolic 
complications following an activation of coagulation after administration of 13 – 20 mg 
alteplase to healthy subjects is substantially lower compared to the cited AMI patient trials. 
Based on its favorable pharmacodynamic profile [R20-4172] including an inhibition of factor 
XIIa, XIa, Xa, VIIa and IIa (very strong effect) heparin has been successfully used to increase 
the patency rate of coronary arteries after fibrinolytic therapy. Nevertheless thrombin 
activation could not completely prevented by heparin in the cited patient trials [P91-62123,
P95-86478, P98-2656, P99-00076, P03-10831].
To mitigate the risks resulting from activated coagulation after alteplase infusion the 
following safety measures are planned for Part B of this trial:
-
Trial specific exclusion criteria have been defined to exclude subjects who are 
perceived to have an increased risk of thromboembolic complications (see 3.3.3)
-
i.v. bolus of 5000 IU heparin prior to each alteplase infusion
-
measurement of an extended set of biomarkers (see 2.2.2.2)
-
safety review meeting after the first two cohorts in Part B (see 3.1)
-
implementation of trial specific stopping criteria (see 3.3.4.3)
The planned administration of an i.v.-bolus of 5000 IU heparin is identical to the clinical use 
of heparin in acute myocardial infarction. Considering the comparably low dose of alteplase 
and the clinical dose of heparin that are planned to be given to healthy subjects without 
additional risk factors the risk of thromboembolic complications is regarded to be low in this 
trial. To cover remaining uncertainties with respect to the efficacy of heparin markers of 
coagulation will be measured. The respective results from the first two cohorts will be 
discussed in a safety review meeting (see 3.1). Dosing of further subjects will be stopped if a 
clinically relevant activation of coagulation is concluded in this meeting.
Immunogenicity risk
Polypeptide pharmaceuticals and therapeutic proteins may cause the formation of anti-drug 
antibodies (ADAs) when administered to humans. After treatment with alteplase a sustained 
antibody formation to the recombinant human t-PA molecule has not been observed. 
However, there is no systematic experience with re-administration of alteplase [c29758839-
04].
According to Reed et al 
 from 1686 patients who received rt-PA during clinical trials 
(total dose of 60 mg – 150 mg, delivered over 1-24 h) were assessed for the presence of anti-
drug- antibodies (ADAs). Only in 3 patients low ADA titers could be found. These positive 
samples were obtained between 7 and 32 days after treatment. Additional samples obtained 
between day 12 and 440 were negative. Clinical sequelae related to the occurrence of antibo-
dies were not observed. The authors conclude that rt-PA can be given repeatedly without the 
risk of immunologic complications seen with streptokinase [P90-58876]. 


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This is confirmed by Schwieder et al who report the repeated administration of 20 mg rt-PA 
to 137 patients with deep vein thrombosis over 4-7 days. In this trial alteplase was admini-
stered either locally (via a dorsal pedal vein) or systemically for 4 hours each day. The 
authors describe several bleeding events but no immunologic complications [P95-4459].
Cugno et al determined anti-rt-PA antibodies in blood samples of 200 healthy subjects and 60 
patiens with acute myocardial infarction, 43 of which had been treated with rt-PA. 15 days 
after t-PA treatment six patients had increased levels of anti rt-PA antibodies, which 
progressively declined during the following 6 months. Interestingly there were three patients 
with a previous rt-PA treatment. While two of them did not develop anti rt-PA antibodies, the 
3rd patient, who was exposed to rt-PA three years ago, showed the highest and long lasting 
antibody titer. Furthermore in vitro experiments demonstrated that binding of rt-PA to 
forming fibrin was not affected by patients rt-PA binding IgG. Considering also the 
favourable outcome of those patients forming these antibodies the authors conclude that anti 
rt-PA antibodies do not interfere with the physiological fibrinolytic activity [P96-4257]. 
In contrast to streptokinase the tissue plasminogen activator (t-PA) is physiologically 
occurring in humans and therefore should not be immunogenic. The low number of patients 
with a positive ADA titer might indicate that rt-PA is very similar to physiological t-PA. 
Considering the lack of interference with fibrinolytic function and the reported disappearance 
of detected ADAs within 1-2 years the immunogenic risk to healthy subjects in this trial is 
assessed to be low.   
1.5.3.2
Risk associated to heparin administration
Heparin has been tested in several clinical trials with healthy subjects.  
Swan et al investigated the safety and anticoagulant effects of argatroban and heparin in two 
randomised, parallel group dose escalation studies. In the first study 18 subjects received 
escalating single doses of up to 240 IU/kg (N=3) / 120 IU/kg (N=15) as iv-bolus. In the 
second study i.v. infusion of heparin at escalating dosage levels (up to 0.30 IU/kg/min) over
4 h with (N=9) and without (N=9) a loading bolus of 125 IU/kg. The most common adverse 
events in the heparin treated subjects were headache (11.1%), pain at the injection/infusion 
site (8.3%) and dizziness (5.6%). These adverse events were of mild to moderate severity and 
resolved spontaneously. Beyond one case of nose bleed (minor) and one case of bruising at 
the injection/ infusion site no further bleeding events were reported for the heparin group 
[R20-4203].
In a drug-drug interaction study, conducted by Teng et al, 28 healthy subjects received a 
single dose of 180 mg ticagrelor alone, unfractionated heparin 100 IU/kg as iv-bolus alone 
and both drugs together in a tree period cross-over design. Study treatments were well 
tolerated by all subjects. No significant bleeding related events occurred. Drug related advese 
events were reported for 2 (ticagrelor alone), 2 (heparin alone) and 3 (ticagrelor + heparin) 
subjects. No clinically relevant changes in 12-lead ECG, vital signs, urinalysis and physical 
findings were observed [R20-4204].
According to Gretler et al the combined administration of the GPIIb-IIIa receptor antagonist 
eptifibatide (180µg/kg bolus followed by 2 µg/kg/min for 48 h) and unfractionated heparin 
(iv bolus of 5000 IU followed by an infusion of 1000 IU/h adjusted to maintain aPTT 


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between 50-70 seconds) was well tolerated by healthy subjects with the exception of one 
subject. In this case study drug administration was discontinued due to treatment related 
thrombocytopenia [R20-4202] 
Pernerstorfer et al describe the administration of 10.000 IU heparin as iv-bolus to healthy 
subjects followed by an initial infusion rate of 1000 IU/h (titrated at 3 h and 12 h to keep an 
aPTT ratio of 2.0 - 2.5 over 24 h. Statements on tolerability have not been made in this 
publication [R20-4200].
In the given examples heparin doses have been administered to healthy subjects, that equal or 
substantially exceed the planned administration of 5000 IU in this trial. Heparin was well 
tolerated in these trials. Minor bleedings did occur (e.g. nose bleed), but these events can be 
handled in the setting of a phase I trial. Major bleedings have not been reported, although in 
some reported cases a further anticoagulant has been administered on top of heparin.
Considering also the short elimination half-life of 90 - 120 minutes no undue risk is expected 
from the planned heparin administration in this trial.
1.5.3.3
Risk of combined administration of alteplase and heparin
The combined administration of heparin and alteplase is a standard procedure in the treatment 
of myocardial infarction and pulmonary embolism. 
Based on the synergistic mode of action (anticoagulation + fibrinolysis) the combined 
administration of heparin and alteplase is associated with an increased bleeding risk in 
general. Bleedings are the most frequent side effects of both drugs when used clinically. 
However, the administration of alteplase up to doses of 0.5 mg/kg did not cause any bleeding 
events in healthy subjects so far. In contrast, in Part A of this trial the administration of 0.2 
mg/kg alteplase was rather associated with procoagulant effects. In Part B of this trial heparin 
should be used to mitigate the risk of thromboembolic complications resulting from these 
effects. Therefore, no additional bleeding risk is expected from combined administration of 
0.2 mg/kg alteplase and heparin to healthy subjects in this trial. 
The use of heparin may cause minor bleeding events. These events are limited by the short 
half-life of heparin and can be handled within the setting of a phase I trial. Thromboembolic 
complications have to be avoided. Therefore, the combined use of heparin and alteplase is 
justified and does not represent an undue risk to trial participants.   
1.5.3.4
Drug-induced liver injury (DILI)
Although rare, a potential for drug-induced liver injury (DILI) is under constant surveillance 
by sponsors and regulators. Therefore, this trial requires timely detection, evaluation, and 
follow-up of laboratory alterations in selected liver laboratory parameters to ensure subjects´ 
safety. See also Section 5.2.6.1.4, adverse events of special interest.


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1.5.4
Overall assessment
Alteplase has been successfully used for the thrombolytic treatment of acute myocardial 
infarction, acute massive pulmonary embolism and acute ischaemic stroke for many years. To 
satisfy the growing world-wide demand for alteplase the manufacturing process has been 
modified. This trial will be performed to establish bioequivalence between alteplase obtained 
from the current process (TPA-02) and alteplase produced with the modified process (TPA-
05). 
The administration of alteplase up to doses of 0.5 mg/kg did not cause any bleeding events in 
healthy subjects so far. However, in Part A of this trial the start of alteplase infusion was 
associated with procoagulant effects that have not been observed before in phase I trials. To 
mitigate the risk of thromboembolic complications in Part B of the trial several safety 
measures will be implemented including trial specific exclusion criteria, the measurement of 
coagulation parameters, a safety review meeting and the administration of a heparin bolus 
prior to start of alteplase infusion. Considering these measurements, and taking into account 
the short half-life of both drugs the risks to healthy subjects participating in this trial are 
assessed to be outweighted by the proven benefit future patients will have from treatment 
with alteplase.


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2
TRIAL OBJECTIVES AND ENDPOINTS
2.1
MAIN OBJECTIVES, PRIMARY AND SECONDARY ENDPOINTS
2.1.1
Main objectives
The main objective of this trial is to establish the bioequivalence of TPA-05 compared with 
TPA-02 following intravenous administration of 0.2 mg/kg body weight.
2.1.2
Primary endpoints
The following pharmacokinetic parameters will be determined for TPA-02 and TPA-05:

AUC0-tz (area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point)

Cmax (maximum measured concentration of the analyte in plasma)
2.1.3
Secondary endpoint
The following pharmacokinetic parameter will be determined for TPA-02 and TPA-05:

AUC0-∞(area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity)


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2.2.2.3
Safety and tolerability
Safety and tolerability of TPA-02 and TPA-05 will be assessed based on:  

Adverse events (including clinically relevant findings from the physical examination)

Safety laboratory tests

TAT, F1+2 and D-dimer values will be assessed in a safety review meeting (see 3.1)

12-lead ECG and vital signs (blood pressure, pulse rate)


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3
DESCRIPTION OF DESIGN AND TRIAL POPULATION
3.1
OVERALL TRIAL DESIGN AND PLAN
Part A of this trial has been terminated after 12 subjects due to procoagulant effects that 
interfered also with alteplase bioanalysis (PK-data from Part A cannot be used for assessment 
of bioequivalence, see 1.2.5.4). After implementation of measures to mitigate the risk of 
thromboembolic complications and to avoid methodological problems with alteplase 
analytics, Part B of the trial will be performed following a 2-stage design as described below:
The resumed study will be performed as a randomised, open-label, two-way crossover trial
with two stages in healthy subjects in order to compare the test treatment (T) to the reference 
treatment (R):
Test treatment (T): 
0.2 mg TPA-05 / kg body weight (iv-infusion over 30 min)
Reference treatment (R): 0.2 mg TPA-02 / kg body weight (iv-infusion over 30 min)
For each stage, the subjects will be randomly allocated to the 2 treatment sequences (T-R or 
R-T). For details, refer to Section 4.1. There will be a washout period of at least 24 hours
between the treatments, i.e. both treatments will be administered on 2 consecutive days.
For a single subject the schedule of trial participation can be displayed as follows:
-
Screening examination: 
up to 21 days
-
Treatment period 1 (incl. follow-up): 
1 day
-
Treatment period 2 (incl. follow-up):
7 days
-
Follow-up examination:
up to 15 days 
Considering the flexible time frame for screening and follow-up examination the expected 
total trial duration for a single subject is about 4-6 weeks.
For the whole trial a maximum cohort size of 4 subjects will apply, i.e. the number of 
subjects that are dosed on one day will not exceed 4. No more than 2 subjects will be infused 
in parallel. The cohort size has been chosen for logistical reasons.  
Stage 1 (Part B) will comprise n1 = 18 subjects. After the first stage, recruitment will be 
stopped and an assessment for bioequivalence will be performed. Based on unblinded data 
from the first stage it will be evaluated whether the trial continues to stage 2 or not. If the 
confidence intervals for the comparison of Test vs. Reference of all primary and secondary 
endpoints are within the pre-specified boundaries 80.00% to 125.00%, the trial will be closed 
after stage 1 due to proven bioequivalence. If bioequivalence is not shown for all endpoints, 
then a decision regarding continuation of the trial will be made. A sample size re-estimation 
based on the unblinded data from the first stage will be performed to calculate the number of 
subjects required for the second stage (Part B) of the trial (n2). For the sample size re-
estimation it is planned to use the observed variability (geometric coefficient of variation, 
gCV) and the observed geometric mean ratio (GMR) from the (unblinded) stage 1 data. For a 
more detailed description, please refer to Section 7.
An overview of all relevant trial activities is provided in the Flow Chart. For visit schedule 
and details of trial procedures at selected visits, refer to Sections 6.1 and 6.2, respectively.
A schematic diagram of the trial design is displayed on the following pages.


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Period 1, Day 1
Period 2, Day 1
Figure 3.1: Schedule of stage 1 of 
this trial
Interim analysis: Performance of stage 2 necessary?
Screening
Follow-up
TPA-02 or TPA-05
TPA-05 or TPA-02


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Period 1, Day 1
Period 2, Day 1
Figure 3.2: Schedule of stage 2 of this trial
Screening
Follow-up
TPA-02 or TPA-05
TPA-05 or TPA-02


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After the first 2 cohorts (planned sample size is N=3 for each cohort) a documented safety 
review meeting will take place to check whether an activation of coagulation occurred after 
start of alteplase infusion. The minimum data set for this safety review meeting consists of 
the following:

AEs (Note: AEs may be ongoing at the time of the meeting and AE information may 
be subject to change prior to Database Lock)

Results from safety lab

Results from TAT, D-dimer and F1+2 

blinded review of PK-data (reliable concentration-time curve?), if possible

Check of criteria for stopping the trial as per Section 3.3.4.3
The decision to continue the trial will be made jointly by the Principal investigator (or an 
authorized deputy) and the Clinical Trial Leader (or an authorised deputy) after in-depth 
analysis of data listed above . Continuation of the trial after the first 2 cohorts will only be 
permitted if no safety concerns exist neither in the opinion of the Principal Investigator (or an 
authorised deputy) nor the Clinical Trial Leader (or an authorised deputy). 
Safety Reviews can be conducted face-to-face or by video/telephone conference. The Clinical 
Trial Leader is responsible for the organisation and minutes of the reviews. Minutes will be 
signed off by the Principal Investigator (or an authorised deputy) and Clinical Trial Leader 
(or an authorised deputy), and will be filed in the ISF and TMF.
3.2
DISCUSSION OF TRIAL DESIGN, INCLUDING THE CHOICE OF 
CONTROL GROUP
For bioequivalence trials, the crossover design is preferred because of its efficiency: since 
each subject serves as his own control, the comparison between treatments is based on an 
intra-subject comparison, thus removing inter-subject variability from the comparison 
between treatments [R94-1529].
The open-label treatment is not expected to bias results, since the study endpoints are derived 
from measurement of plasma concentrations of the analyte, which are provided by a 
bioanalytical laboratory that is blinded to treatment allocation.
3.3
SELECTION OF TRIAL POPULATION
In part A of this trial, 12 subjects have been dosed. For part B of this trial, it is planned that 
18 healthy male subjects will enter stage 1/Part B of the study, with a maximum number of 
42 additional subjects for stage 2/Part B, if required. They will be recruited from the 
volunteers' pool of the trial site.
A log of all subjects enrolled into the trial (i.e. who have signed informed consent) will be 
maintained in the ISF irrespective of whether they have been treated with investigational drug 
or not.


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3.3.1
Main diagnosis for trial entry
The study will be performed in healthy male subjects.
3.3.2
Inclusion criteria
Subjects will only be included in the trial if they meet the following criteria:
1. Healthy male subjects according to the assessment of the investigator, as based on a 
complete medical history including a physical examination, vital signs (BP, PR), 12-lead 
ECG, and clinical laboratory tests 
2. Age of 18 to 45 years (inclusive)
3. BMI of 18.5 to 29.9 kg/m2 (inclusive)
4. Body weight of 65 – 100 kg (inclusive) at screening
5. Signed and dated written informed consent prior to admission to the study, in accordance 
with GCP and local legislation
3.3.3
Exclusion criteria
Subjects will not be allowed to participate, if any of the following general criteria apply:
1. Any finding in the medical examination (including BP, PR or ECG) deviating from 
normal and assessed as clinically relevant by the investigator
2. Repeated measurement of systolic blood pressure outside the range of 90 to 140 mmHg, 
diastolic blood pressure outside the range of 50 to 90 mmHg, or pulse rate outside the 
range of 45 to 90 bpm
3. Any laboratory value outside the reference range that the investigator considers to be of 
clinical relevance, e.g. thrombotic predisposition according to thrombophilic testing
4. Any evidence of a concomitant disease assessed as clinically relevant by the investigator
5. Gastrointestinal, hepatic, renal, respiratory, cardiovascular, metabolic, immunological or 
hormonal disorders
6. Diseases of the central nervous system (including but not limited to any kind of seizures 
or stroke), and other relevant neurological or psychiatric disorders 
7. History of relevant orthostatic hypotension, fainting spells, or blackouts
8. Chronic or relevant acute infections
9. History of relevant allergy or hypersensitivity (including allergy to the trial medication or 
its excipients like gentamicin) 
10. Use of drugs within 30 days of planned administration of trial medication that might 
reasonably influence the results of the trial or that may pose a potential safety risk to a 
subject, e.g. risk of bleeding
11. Intake of an investigational drug in another clinical trial within 60 days of planned  
administration of investigational drug in the current trial, or concurrent participation in 
another clinical trial in which investigational drug is administered
12. Smoker (more than 3 cigarettes or 1 cigar or 1 pipe per day)
13. Inability to refrain from smoking on specified trial days
14. Alcohol abuse (consumption of more 24 g per day)


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15. Drug abuse or positive drug screening
16. Blood donation of more than 100 mL within 30 days of planned administration of trial 
medication or intended blood donation during the trial
17. Intention to perform excessive physical activities (including martial arts, contact sports
such as soccer and handball, high risk sports such as motor cycling and down hill racing) 
within one week prior to the administration of trial medication or during the trial
18. Inability to comply with the dietary regimen of the trial site
19. Subject is assessed as unsuitable for inclusion by the investigator, for instance, because 
the subject is not considered able to understand and comply with study requirements, or 
has a condition that would not allow safe participation in the study 
In addition, the following trial-specific exclusion criteria apply:
20. Hemostatic disorders in the last 6 months 
21. known haemorrhagic diathesis
22. Concomitant treatment with other anticoagulants (e.g. unfractionated heparin, low 
molecular weight heparins, heparin derivatives, oral anticoagulants)
23. manifest or recent severe or dangerous bleeding
24. Any history of central nervous system damage (i.e. neoplasm, aneurysm, intracranial or 
spinal surgery)
25. Bacterial endocarditis or pericarditis
26. Acute pancreatitis
27. Neoplasm with increased bleeding risk
28. Major surgery or significant trauma in past 3 months
29. Subjects who in the investigators judgement are perceived as having an increased risk of 
bleeding, for example because of: recent intracranial hemorrhage, condition after 
subarachnoidal hemorrhage, recent puncture of a non-compressible blood vessel (e.g. 
subclavia or jugular vein puncture), current or recent gastrointestinal ulceration, known or 
suspected oesophageal varices, arteriovenous malformations, vascular aneurysms, major 
intraspinal or intracerebral vascular abnormalities, recent ophthalmic surgery
30. Existing or history of confirmed venous thromboembolism, family history of venous
thromboembolism, and other known factors for venous thromboembolism
31. Existing or history of arterial thrombotic or embolic processes, conditions which 
predispose to them, e.g. disorders of clotting processes, valvular heart disease and atrial 
fibrillation
32. Hypersensitivity to heparin (e.g. known heparin induced thrombocytopenia)
33. During COVID-19 pandemic: laboratory test indicative of an ongoing SARS-CoV-2 
infection
For study restrictions, refer to Section 4.2.2.


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3.3.4
Withdrawal of subjects from treatment or assessments
Subjects may discontinue trial treatment or withdraw consent to trial participation as a whole 
(‘withdrawal of consent’) with very different implications; please see sections 3.3.4.1 and 
3.3.4.2 below.
If a subject is removed from or withdraws from the trial prior to the first administration of 
trial medication, the data of this subject will not be entered in the case report form (CRF) and 
will not be reported in the clinical trial report (CTR). If a subject is removed from or 
withdraws from the trial after the first administration of trial medication, this will be 
documented and the reason for discontinuation must be recorded in the CRF; in addition, the 
data will be included in the CRF and will be reported in the CTR. At the time of 
discontinuation, a complete end of trial examination will be performed, if possible, and the 
information will be recorded in the CRF. If the discontinuation occurs before the end of the 
REP (see Section 1.2.3), the discontinued subject should be questioned for AEs and 
concomitant therapies at or after the end of the REP in order to ensure collection of AEs and 
concomitant therapies throughout the REP, if not contrary to any consent withdrawal of the 
subject.
3.3.4.1
Discontinuation of trial treatment
An individual subject will discontinue trial treatment if:
1. The subject wants to discontinue trial treatment, without the need to justify the decision
2. The subject has repeatedly shown to be non-compliant with important trial procedures 
and, in the opinion of both, the investigator and sponsor representative, is not willing or 
able to adhere to the trial requirements in the future.
3. The subject needs to take concomitant medication that interferes with the investigational 
medicinal product or other trial treatment
4. The subject can no longer receive trial treatment for medical reasons (such as surgery, 
adverse events [AEs], or diseases)
In addition to these criteria, the investigator may discontinue subjects at any time based on 
his or her clinical judgment.
Even if the trial treatment is discontinued, the subject remains in the trial and, given his/her 
agreement, will undergo the procedures for early treatment discontinuation and follow up as 
outlined in the Flow Chart and section 6.2.3.
3.3.4.2
Withdrawal of consent to trial participation
Subjects may withdraw their consent to trial participation at any time without the need to 
justify the decision. If a subject wants to withdraw consent, the investigator should be
involved in the discussion with the subject and explain the difference between trial treatment 
discontinuation and withdrawal of consent to trial participation, as well as explain the options 
for continued follow up after trial treatment discontinuation, please see section 3.3.4.1 above.


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3.3.4.3
Discontinuation of the trial by the sponsor
Boehringer Ingelheim reserves the right to discontinue the trial at any time for any of the 
following reasons:
1. Failure to meet expected enrolment goals overall or at a particular trial site
2. New toxicological findings, serious adverse events, or any safety information invalidating
the earlier positive benefit-risk-assessment. More specifically, the trial will be terminated 
if more than 50% of the subjects have drug-related and clinically relevant adverse events 
of moderate or severe intensity, or if at least 1 drug-related serious adverse event is 
reported
3. Clinically relevant activation of coagulation as assessed at the safety review meeting (see 
3.1)
4.
Preliminary PK-data obtained from a blinded review after the first two cohorts indicate 
non-reliable concentration-time profiles (i.e. data do not reflect systemic alteplase 
concentrations due to methodological problems)  
5. Violation of GCP, or the CTP impairing the appropriate conduct of the trial
6. The sponsor decides to discontinue the further development of the investigational product
7. BI is not allowed to produce alteplase furthermore (withdrawal of production licence)
8. The trial site is assessed to be not suitable by the sponsor, ethics committee or authorities
9. Infeasibility to achieve statistical objectives (e.g. based on the results of the interim 
analysis after stage 1, see section 7.4)
The trial will be regularly terminated after stage 1, if bioequivalence could be demonstrated
after stage 1 based on interim analysis (see chapter 3.1)
3.3.5
Replacement of subjects
In case more than 4 subjects do not complete stage 1 (of Part B), the Clinical Trial Leader
together with the Trial Pharmacokineticist and the Trial Statistician are to decide, if and how 
many subjects will be replaced. A replacement subject will be assigned a unique trial subject 
number, and will be assigned to the same treatment as the subject he or she replaces.
Replacing subjects is only allowed, if the total number of exposed subjects will not exceed 
72.


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4
TREATMENTS
4.1
INVESTIGATIONAL TREATMENTS
4.1.1
Identity of the Investigational Medicinal Products
The characteristics of the test product are given below:
Substance:
alteplase (from modified manufacturing process: TPA-05)
Protein content:
51.5 mg/vial (Reference: Certificate of Analysis)
Pharmaceutical formulation: Powder and Solvent for Solution for Injection/Infusion
Source:
BI Pharma GmbH & Co. KG, Germany
Unit strength:
50 mg powder and 50 ml solvent
Posology:
1-0-0 (0.2 mg/kg body weight)
Route of administration:
i.v.
Duration of use:
infusion over 30 min (single dose)
The characteristics of the reference product are given below:
Substance:
alteplase (from current manufacturing process: TPA-02)
Protein Content:
50.1 mg/vial (Reference: Certificate of Analysis)
Pharmaceutical formulation: Powder and Solvent for Solution for Injection/Infusion
Source:
BI Pharma GmbH & Co. KG, Germany
Unit strength:
50 mg powder and 50 ml solvent
Posology:
1-0-0 (0.2 mg/kg body weight)
Route of administration:
i.v.
Duration of use:
infusion over 30 min (single dose
Sterile water for Injection/Infusion is used as solvent for both alteplase products. Alteplase 
(TPA-02 and TPA-05) solution for injection/infusion will be prepared within 30 min prior to 
its intended use. A detailed reconstitution instruction is given in Appendix 10.1.
Alteplase will be administered as short infusion using a syringe infusion pump (Perfusor fm®
). For intravenous drug administration and PK sampling two 
different intravenous cannulas will be used. They must not be on the same arm during the 
drug administration. 


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The characteristics of the Auxiliary medicinal product are given below:
Drug product:
Heparin-Natrium-5000-ratiopharm® (Ampullen)
Pharmaceutical formulation: vial containing solution for injection
Source:
Unit strength:
0.2 ml (= 5000 IU Heparin-sodium)
Posology:
1-0-0 
Route of administration:
i.v.
Duration of use:
bolus injection (5 min prior to start of each alteplase infusion)
4.1.2
Selection of doses in the trial
The alteplase dose selected for this trial is lower than the standard clinical dose for safety 
reasons. It is also 20% lower compared to the reference trials 135.57 and 135.67 performed in 
the 
(see section 1.2.2) to further improve the safety of participating subjects. There is no 
evidence of non-linearities. Based on assumed dose-linearity, levels are sufficiently high 
above endogenous concentrations, suggesting a relevant exposure (AUC) will be achieved.
The administration of 5000 IU heparin as i.v.bolus is a standard therapeutic dose (see 1.3).
4.1.3
Method of assigning subjects to treatment groups
The randomisation list will be provided to the trial site in advance.
Subjects will be allocated to treatment sequences prior to the first administration of trial 
medication in the morning of Day 1 (Visit 2). For this purpose, numbers of the randomisation 
list will be allocated to the subjects by drawing lots. Subjects are then assigned to a treatment 
sequence according to the randomisation list.
Once a subject number has been assigned, it cannot be reassigned to any other subject.
The randomisation procedure is described in Section 7.6.
4.1.4
Drug assignment and administration of doses for each subject
This trial is a 2-way crossover study. All subjects will receive the 2 treatments in randomised 
order at 2 consecutive days. The treatments to be evaluated are outlined in Table 4.1.4: 1
below. 
Table 4.1.4: 1
Dosage and treatment schedule
Treatment
Substance
Formulation
Unit strength
Dosage
Total  dose
T (Test)
TPA-05
Powder and Solvent 
for Solution for 
Injection/Infusion
50 mg
0.2 mg/kg body weight
Depending on 
body weight
R (Reference)
TPA-02
Powder and Solvent 
for Solution for 
Injection/Infusion
50 mg
0.2 mg/kg body weight
Depending on 
body weight


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The specific alteplase dose to be administered to a given subject will be calculated based on 
the body weight measured at screening examination. The weight will be always rounded to 
the lower value of full kilogram. Considering the inclusion criterion 4 on total body weight 
the following doses may apply in this trial:
Table 4.1.4: 2
Body weight and alteplase dose in 0135-0340 trial
Body weight
65 kg
66 kg
67 kg
68 kg
69 kg
Alteplase dose
13 mg
13.2 mg
13.4 mg
13.6 mg
13.8 mg
Table 4.1.4: 2
Body weight and alteplase dose in 0135-0340 trial (continued)
Body weight
Alteplase dose
Body weight
Alteplase dose
Body weight
Alteplase dose
70 kg
14 mg
80 kg
16 mg
90 kg
18 mg
71 kg
14.2 mg
81 kg
16.2 mg
91 kg
18.2 mg
72 kg
14.4 mg
82 kg
16.4 mg
92 kg
18.4 mg
73 kg
14.6 mg
83 kg
16.6 mg
93 kg
18.6 mg
74 kg
14.8 mg
84 kg
16.8 mg
94 kg
18.8 mg
75 kg
15 mg
85 kg
17 mg
95 kg
19 mg
76 kg
15.2 mg
86 kg
17.2 mg
96 kg
19.2 mg
77 kg
15.4 mg
87 kg
17.4 mg
97 kg
19.4 mg
78 kg
15.6 mg
88 kg
17.6 mg
98 kg
19.6 mg
79 kg
15.8 mg
89 kg
17.8 mg
99 kg
19.8 mg
100 kg
20.0 mg
The respective alteplase dose will be administered as a continuous intravenous infusion over 
30 minutes under supervision of the investigating physician or an authorised designee. Start 
and end time of the infusion will be recorded. For reconstitution and drug administration, the 
so-called four-eye principle (two-person rule) should be applied. For this, one authorised 
employee of the trial site should witness the administration of trial medication, and its 
preparation (reconstitution), if correct dosage cannot be ensured otherwise.


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Both treatments will be separated by a wash-out phase of at least 24 hours. Subjects will be 
kept under close medical surveillance until 24 h after the last study drug administration.
4.1.5
Blinding and procedures for unblinding
This Phase I trial will be handled in an open fashion throughout (that is, during the conduct,
including data cleaning and preparation of the analysis). This is considered acceptable 
because the potential for bias seems to be low and does not outweigh practical considerations.
Emergency envelopes will not be provided, because the dose of trial medication is known to 
investigators and subjects.
PK samples will be labelled in such a way that treatment allocation cannot be derived by the 
analytical site.
4.1.6
Packaging, labelling, and re-supply
The investigational medicinal products will be provided by BI. They will be packaged and 
labelled in accordance with local law and the principles of Good Manufacturing Practice. 
For details of packing and the description of the label, refer to the ISF.
The telephone number of the sponsor and the name, address and telephone number of the trial 
site are provided in the subject information form. The EudraCT number is indicated on the 
title page of this protocol as well as on the subject information and informed consent forms. 
4.1.7
Storage conditions
Drug supplies will be kept in their original packaging and in a secure limited access storage 
area in accordance with the recommended (labelled) storage conditions. If necessary, a 
temperature log must be maintained to make certain that the drug supplies are stored at the 
correct temperature. If the storage conditions are found to be outside the specified range, the 
local clinical monitor (as provided in the list of contacts) is to be contacted immediately.
4.1.8
Drug accountability
The investigator or designee will receive the investigational drugs delivered from the sponsor
following requirements are fulfilled:

Approval of the clinical trial protocol by the IRB / ethics committee

Approval/notification of the regulatory authority,  e.g. competent authority

Availability of the curriculum vitae of the Principal Investigator

Availability of a signed and dated clinical trial protocol  
Only authorised personnel documented in the form 'Trial Staff List' may dispense medication 
to trial subjects. The trial medication must be administered in the manner specified in the 
CTP.
The investigator or designee must maintain records of the product’s delivery to the trial site, 
the inventory at the site, the use by each subject, and the disposal of unused products.These 


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records will include dates, quantities, batch / serial numbers, expiry ('use-by') dates, and the 
unique code numbers assigned to the investigational medicinal product and trial subjects. The 
investigator or designee will maintain records that document adequately that the subjects 
were provided the doses specified by the CTP and reconcile all investigational medicinal 
products received from the sponsor. At the time of disposal of remaining trial medication, the 
investigator or designee must verify that no remaining supplies are in the investigator’s 
possession.
All unused medication will be disposed of locally by the trial site upon written authorisation 
of the clinical trial leader. Receipt, usage and disposal of trial medication must be 
documented on the appropriate forms. Account must be given for any discrepancies.
4.2
OTHER TREATMENTS, EMERGENCY  PROCEDURES,
RESTRICTIONS
4.2.1
Other treatments and emergency procedures
There are no special emergency procedures to be followed. No additional treatment is 
planned. However, if adverse events require treatment, the investigator can authorise 
symptomatic therapy. In those cases, subjects will be treated as necessary and, if required, 
kept under supervision at the trial site or transferred to a hospital until all results of medical 
evaluations are acceptable.
4.2.2
Restrictions
4.2.2.1
Restrictions regarding concomitant treatment
In principle, no concomitant therapy is allowed. All concomitant or rescue therapies will be 
recorded (including time of intake on study days) on the appropriate pages of the CRF.
The intake of analgesics known to inhibit cyclooxygenase (e.g. acetylsalicylic acid, ibuprofen 
and diclofenac) is strongly forbidden starting from 1 week prior to first study drug 
administration until discharge from trial site.  The same refers to the use of anticoagulants 
(heparins, heparin ointment).
4.2.2.2
Restrictions on diet and life style
While admitted to the trial site, the subjects will be instructed not to consume any foods or 
drinks other than those provided by the staff. Standardised meals will be served at the times
indicated in the Flow Chart. No food is allowed for at least 2 h after start of infusion.
Starting from 1 hour before start of infusion until lunch, fluid intake is restricted to 240 mL of 
water served at 1 h and 2 h after start of infusion (mandatory for all subjects). Beyond this 
time interval fluid intake is not restricted.
Green tea, grapefruits, Seville oranges (sour or bitter oranges) and their juices, and dietary 
supplements and products containing St. John's wort (Hypericum perforatum) are not 
permitted from 3 days before the first administration of trial medication until discharge from 
trial site. 


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Poppy-seeds containing products should not be consumed starting 3 days before trial drug 
administration until discharge from trial site.
Alcoholic beverages are not permitted starting 48 h before trial drug administration until 
discharge from trial site. 
Methylxanthine-containing drinks or foods (such as coffee, tea, cola, energy drinks, or 
chocolate) are not allowed during the in-house confinement at the trial site.
Smoking is not allowed during in-house confinement.
Excessive physical activity (including martial arts, contact sports such as soccer and handball, 
high risk sports such as motor cycling and down hill racing) should be avoided from 7 days 
before the first administration of trial medication until the end of trial examination.
4.3
TREATMENT COMPLIANCE
Compliance will be assured by administration of all trial medication in the study centre under 
supervision of the investigating physician or a designee. The measured plasma concentrations 
of trial medication will provide additional confirmation of compliance.
Subjects who are non-compliant (for instance, who do not appear for scheduled visits or 
violate trial restrictions) may be removed from the trial and the CRF will be completed 
accordingly (for further procedures, please see Section 3.3.4.1).


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5
ASSESSMENTS
5.1
ASSESSMENT OF EFFICACY
Not applicable.
5.2
ASSESSMENT OF SAFETY
5.2.1
Physical examination
At screening, the medical examination will include demographics, height and body weight, 
smoking and alcohol history (results not mandatory to be entered into CRF or to be reported),
relevant medical history and concomitant therapy, review of inclusion and exclusion criteria, 
review of vital signs (BP, PR), 12-lead ECG, laboratory tests (including fecal occult blood 
test), and a physical examination. At the end of trial examination, it will include review of 
vital signs, 12-lead ECG, laboratory tests, and a physical examination.
5.2.2
Vital signs
Systolic and diastolic blood pressures (BP) as well as pulse rate (PR) will be measured by a 
blood pressure monitor (Dinamap Pro 100, 
) at the 
times indicated in the Flow Chart, after subjects have rested for at least 5 min in a supine
position. All recordings should be made using the same type of blood pressure recording 
instrument on the same arm, if possible.
5.2.3
Safety laboratory parameters
For the assessment of laboratory parameters, blood and urine samples will be collected by the 
trial site at the times indicated in the Flow Chart.
The parameters that will be determined are listed in Tables 5.2.3: 1 and 5.2.3: 2. Reference 
ranges will be provided in the ISF, Section 10. 
Manual differential white blood cell count or urine sediment examinations will only be 
performed if there is an abnormality in the automatic blood cell count or in the urinalysis, 
respectively.
Fecal occult blood testing, using an immunochemical test kit for hemoglobin (e.g. BioNexia®
FOBplus,
), will be performed at the 
trial site.


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Table 5.2.3: 1
Routine laboratory tests
Functional lab group
BI test name [comment/abbreviation]
A1
B1
C1
Haematology
Haematocrit
Haemoglobin
Red Blood Cell Count/Erythrocytes 
White Blood Cells/Leucocytes
Platelet Count/Thrombocytes (quant)
X
X
X
X
X
X
X
X
X
X
-
-
-
-
-
X
X
X
X
X
Automatic WBC 
differential, relative
Neutrophils/Leukocytes; Eosinophils/Leukocytes; Basophils/ 
Leukocytes; Monocytes/Leukocytes; Lymphocytes/Leukocytes 
X
-
-
X
Automatic WBC 
differential, absolute 
Neutrophil, absol.; Eosinophils, absol.; Basophils, absol.; 
Monocytes, absol.; Lymphocytes, absol.
X
-
-
X
Manual differential 
WBC (if automatic
differential WBC is 
abnormal)
Neut. Poly (segs); Neut. Poly (segs), absol.; Neutrophils Bands;  
Neutrophils Bands, absol.; Eosinophils/Leukocytes; Eosinophils, 
absol.; Basophils/ Leukocytes; Basophils, absol.; Monocytes/ 
Leukocytes; Monocytes, absol.; Lymphocytes/Leukocytes; 
Lymphocytes, absol.
Coagulation
Activated Partial Thromboplastin Time 
Prothrombin time – INR (International Normalization Ratio)
Fibrinogen
Antithrombin III
Protein C 
Protein S
Activated protein C resistance
X
X
X
X
X
X
X
X
X
X
X
X
X
Enzymes
AST [Aspartate transaminase] /GOT, SGOT 
ALT [Alanine transaminase] /GPT, SGPT
Alkaline Phosphatase
Gamma-Glutamyl Transferase 
Amylase
Lipase
X
X
X
X
X
X
-
-
-
-
-
-
-
-
-
-
-
-
X
X
X
X
-
-
Hormones
Thyroid Stimulating Hormon
X
-
-
-
Substrates
Glucose (Plasma)
Creatinine 
Bilirubin, Total 
Bilirubin, Direct
Protein, Total
C-Reactive Protein (Quant)  
X
X
X
X
X
X
-
-
-
-
-
-
-
-
-
-
-
-
X
X
X
X
X
X
Electrolytes
Sodium
Potassium
Calcium
X
X
X
-
-
-
-
-
-
X
X
X
Urinalysis2 (Stix)
Urine Nitrite (qual)
Urine Protein (qual)
Urine Glucose (qual)
Urine Ketone (qual)
Urobilinogen (qual)
Urine Bilirubin (qual)
Urine RBC/Erythrocytes (qual)
Urine WBC/Leucocytes (qual)
Urine pH
X
X
X
X
X
X
X
X
X
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
X
X
X
X
X
X
X
X
X
Urine sediment2
Only positive findings will be reported (for instance, the presence 
of sediment bacteria, casts in sediment, squamous epithelial cells, 
erythrocytes, leukocytes)
1 A, B, and C laboratory profiles to be done at time points specified in the Flow Chart. 
2 Microscopic examination if erythrocytes, leukocytes, or protein are abnormal in urine


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The tests listed in Table 5.2.3: 2 are exclusionary laboratory tests that may be repeated as 
required. The results will not be entered in the CRF/database and will not be reported in the 
CTR. Infectious serology will be performed at screening only. Drug screening will be 
performed at screening and at start of in-house confinement.
Table 5.2.3: 2
Exclusionary laboratory tests
Functional lab group
Test name 
Drug screening (urine)
Amphetamine/MDA
Barbiturates
Benzodiazepine
Cannabis
Cocaine 
Methadone
Methamphetamines/MDMA/XTC
Opiates 
Phencyclidine
Tricyclic antidepressants
Infectious serology (blood)
Hepatitis B surface antigen (qualitative)
Hepatitis B core antibody (qualitative)
Hepatitis C antibodies (qualitative)
HIV-1 and HIV-2 antibody (qualitative)
To encourage compliance with alcoholic restrictions, a breath alcohol test (e.g. Alco True 
M®,
) will be performed at start of in-house 
confinement, and may be repeated at any time during the study at the discretion of an 
investigator or designee. The results will not be included in the CTR
The laboratory tests listed in Tables 5.2.3: 1 and 5.2.3: 2 will be performed at 
 
, with the exception of 
drug screening tests. These tests will be performed at the trial site using the Multidrogen-
Pipettiertest M-10/14-PDT, or comparable test systems.
For fecal occult blood testing the subjects will receive a test tube with an integrated bar for 
stool sampling. On the next occasion they will take three samples from the stool with the 
collection bar and give it into the test tube. The test tubes should be closed carefully to not 
destroy the collection bar. These samples will be analysed for occult blood in faeces by 
immunological reaction according to the manufacturers instruction. As subjects may not be 
able to defecate at the trial site in the morning of the respective visit they may collect the 
specimen at home and bring the test specimen to the trial site.
In the evening of the first treatment day an urine stix will be performed in-house. For this 
purpose a suitable test system (e.g. the Combur-10 test system [
, 
]) will be used. 
Laboratory data will be transmitted electronically from the laboratory to the trial site.


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5.2.4
Electrocardiogram
Twelve-lead ECGs (I, II, III, aVR, aVL, aVF, V1 - V6) will be recorded using a 
computerised electrocardiograph (CardioSoft EKG System, 
, 
) at the times provided in the Flow Chart.
To achieve a stable heart rate at rest and to assure high quality recordings, the site personnel 
will be instructed to assure a relaxed and quiet environment, so that all subjects are at 
complete rest.
All ECGs will be recorded for a 10 sec duration after subjects have rested for at least 5 min in 
a supine position. ECG assessment will always precede all other study procedures scheduled 
for the same time to avoid compromising ECG quality. 
All ECGs will be stored electronically on the Muse CV Cardiology System (
 
). Electrode placement will be performed according to the 
method of Wilson, Goldberger and Einthoven modified by Mason and Likar (hips and 
shoulders instead of ankles and wrists). 
All locally printed ECGs will be evaluated by the investigator or a designee. Abnormal 
findings will be reported as AEs (during the trial) or baseline conditions (at screening) if 
assessed to be clinically relevant by the investigator. Any ECG abnormalities will be 
carefully monitored and, if necessary, the subject will be removed from the trial and will 
receive the appropriate medical treatment. 
ECGs may be repeated for quality reasons (for instance, due to alternating current artefacts, 
muscle movements, or electrode dislocation) and the repeated ECG will be used for analysis. 
Additional (unscheduled) ECGs may be collected by the investigator for safety reasons. 
5.2.5
Other safety parameters
5.2.5.1
Local tolerability
Local tolerability will be assessed by the investigator on the basis of swelling, induration, 
heat, redness, pain, and other findings. Abnormal findings will be recorded as adverse event.
5.2.6
Assessment of adverse events
5.2.6.1
Definitions of adverse events
5.2.6.1.1
Adverse event
An adverse event (AE) is defined as any untoward medical occurrence in a patient or clinical 
investigation subject administered a medicinal product and which does not necessarily have 
to have a causal relationship with this treatment.
An AE can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of a medicinal 
product, whether or not considered related to the medicinal product.
The following should also be recorded as an AE in the CRF and BI SAE form (if applicable):


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
Worsening of the underlying disease or of other pre-existing conditions 

Changes in vital signs, ECG, physical examination, and laboratory test results, if they 
are judged clinically relevant by the investigator
If such abnormalities already pre-exist prior to trial inclusion, they will be considered as 
baseline conditions and should be collected in the eCRF only.
5.2.6.1.2
Serious adverse event
A serious adverse event (SAE) is defined as any AE which fulfils at least one of the 
following criteria:

Results in death 

Is life-threatening, which refers to an event in which the patient was at risk of death 
at the time of the event; it does not refer to an event that hypothetically might have 
caused death if more severe 

Requires inpatient hospitalisation 

Requires prolongation of existing hospitalisation

Results in persistent or significant disability or incapacity 

Is a congenital anomaly/birth defect 

Is deemed serious for any other reason if it is an important medical event when based 
upon appropriate medical judgment which may jeopardise the patient and may 
require medical or surgical intervention to prevent one of the other outcomes listed in 
the above definitions. Examples of such events are intensive treatment in an 
emergency room or at home for allergic bronchospasm, blood dyscrasias or 
convulsions that do not result in hospitalisation or development of dependency or 
abuse
5.2.6.1.3
AEs considered ‘Always Serious’
Cancers of new histology and exacerbations of existing cancer must be classified as a serious 
event regardless of the time since discontinuation of the trial medication and must be reported 
as described in 5.2.6.2, subsections ‘AE Collection’ and ‘AE reporting to sponsor and 
timelines’.
In accordance with the European Medicines Agency initiative on Important Medical Events, 
Boehringer Ingelheim has set up a list of further AEs, which, by their nature, can always be 
considered to be ‘serious’ even though they may not have met the criteria of an SAE as 
defined above.
The latest list of ‘Always Serious AEs’ can be found in the eDC system, an electronic data 
capture system which allows the entry of trial data at the trial site. These events should 
always be reported as SAEs as described above.


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5.2.6.1.4
Adverse events of special interest
The term adverse events of special interest (AESI) relates to any specific AE that has been 
identified at the project level as being of particular concern for prospective safety monitoring 
and safety assessment within this trial, e.g. the potential for AEs based on knowledge from 
other compounds in the same class. AESIs need to be reported to the sponsor’s 
Pharmacovigilance Department within the same timeframe that applies to SAEs, please see 
section 5.2.6.2.2.
The following are considered as AESIs:

Hepatic injury
A hepatic injury is defined by the following alterations of hepatic laboratory 
parameters:
o
An elevation of AST (aspartate transaminase) and/or ALT (alanine 
transaminase) ≥3-fold ULN combined with an elevation of total bilirubin 
≥2-fold ULN measured in the same blood sample, or
o
Aminotransferase (ALT, and/or AST) elevations ≥10 fold ULN
These lab findings constitute a hepatic injury alert and the subjects showing these lab 
abnormalities need to be followed up according to the ‘DILI checklist’ provided in the 
ISF. In case of clinical symptoms of hepatic injury (icterus, unexplained 
encephalopathy, unexplained coagulopathy, right upper quadrant abdominal pain, 
etc.) without lab results (ALT, AST, total bilirubin) available, the Investigator should 
make sure that these parameters are analysed, if necessary in an unscheduled blood 
test. Should the results meet the criteria of hepatic injury alert, the procedures 
described in the DILI checklist should be followed.
5.2.6.1.5
Intensity (severity) of AEs
The intensity (severity) of the AE should be judged based on the following:
Mild:
Awareness of sign(s) or symptom(s) that is/are easily tolerated
Moderate:
Sufficient discomfort to cause interference with usual activity
Severe:
Incapacitating or causing inability to work or to perform usual activities
5.2.6.1.6
Causal relationship of AEs
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.
Arguments that may suggest that there is a reasonable possibility of a causal relationship 
could be:

The event is consistent with the known pharmacology of the drug 

The event is known to be caused by or attributed to the drug class

A plausible time to onset of the event relative to the time of drug exposure


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
Evidence that the event is reproducible when the drug is re-introduced

No medically sound alternative aetiologies that could explain the event (e.g. pre-
existing or concomitant diseases, or co-medications)

The event is typically drug-related and infrequent in the general population not 
exposed to drugs (e.g. Stevens-Johnson syndrome)

An indication of dose-response (i.e. greater effect size if the dose is increased, smaller 
effect size if dose is reduced)
Arguments that may suggest that there is no reasonable possibility of a causal relationship 
could be:

No plausible time to onset of the event relative to the time of drug exposure is evident 
(e.g. pre-treatment cases, diagnosis of cancer or chronic disease within days / weeks 
of drug administration; an allergic reaction weeks after discontinuation of the drug 
concerned)

Continuation of the event despite the withdrawal of the medication, taking into 
account the pharmacological properties of the compound (e.g. after 5 half-lives). Of 
note, this criterion may not be applicable to events whose time course is prolonged 
despite removing the original trigger

Additional arguments amongst those stated before, like alternative explanation (e.g. 
situations where other drugs or underlying diseases appear to provide a more likely 
explanation for the observed event than the drug concerned)

Disappearance of the event even though the trial drug treatment continues or remains 
unchanged
5.2.6.2
Adverse event collection and reporting
5.2.6.2.1
AE collection
Upon enrolment into a trial, the subject’s baseline condition is assessed (for instance, by 
documentation of medical history/concomitant diagnoses), and relevant changes from 
baseline are noted subsequently. 
Subjects will be required to report spontaneously any AEs as well as the time of onset, end
time, and intensity of these events. In addition, each subject will be regularly assessed by the 
medical staff throughout the clinical trial and whenever the investigator deems necessary. As 
a minimum, subjects will be questioned for AEs (and concomitant therapies) at the time 
points indicated in the Flow Chart. Assessment will be made using non-specific questions 
such as ‘How do you feel?’. Specific questions will be asked wherever necessary in order to 
more precisely describe an AE.
A carefully written record of all AEs shall be kept by the investigator in charge of the trial. 
Records of AEs shall include data on the time of onset, end time, intensity of the event, and 
any treatment or action required for the event and its outcome.
The following must be collected and documented on the appropriate CRF(s) by the 
investigator:

From signing the informed consent onwards until an individual subject’s end of trial: 


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o
All AEs (serious and non-serious) and all AESIs 
o
The only exception to this rule are AEs (serious and non-serious) and AESIs 
in Phase I trials in healthy volunteers, when subjects discontinue from the trial 
due to screening failures prior to administration of any trial medication. In 
these cases, the subjects’ data must be collected at trial site but will not be 
entered in the CRF or trial database and will not be reported in the CTR.

After the individual subject’s end of trial: 
o
The investigator does not need to actively monitor the subject for AEs but 
should report any SAE and occurrence of cancer of which the investigator may 
become aware of by any means of communication, e.g. phone call. Those AEs 
should, however, not be reported in the CRF. For possibly related SAEs and 
related AESIs rules for expedited reporting (see 5.2.6.2.2) will apply
5.2.6.2.2
AE reporting to the sponsor and timelines
The Investigator must report SAEs, AESIs, and non-serious AEs which are relevant for the 
reported SAE or AESI, on the BI SAE form via fax immediately (within 24 hours) to the 
sponsor’s unique entry point (country specific contact details will be provided in the ISF). 
The same timeline applies if follow-up information becomes available. In specific occasions 
the Investigator could inform the sponsor upfront via telephone. This does not replace the 
requirement to complete and fax the BI SAE form. 
With receipt of any further information to these events, a follow-up SAE form has to be 
provided. For follow-up information, the same rules and timeline apply as for initial 
information.
5.2.6.2.3
Information required
All (S)AEs, including those persisting after the individual subject’s end of trial, must be 
followed up until they have resolved, have been sufficiently assessed as ‘chronic’ or ‘stable’, 
or no further information can be obtained.


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5.3
DRUG CONCENTRATION MEASUREMENTS AND 
PHARMACOKINETICS
5.3.1
Assessment of pharmacokinetics
For the assessment of pharmacokinetics, blood samples will be collected at the time points 
indicated in the Flow Chart. The actual sampling times will be recorded and used for 
determination of pharmacokinetic parameters. To keep the venous catheters (cannulas) patent 
they will be flushed with 1 ml heparin/saline solution (10 U/ml) after each sampling. Prior to 
the next sampling the first 2 ml blood will be discarded. This procedure refers to each PK-
sampling time point given in the Flow Chart (no flushing after last PK-sampling at 6 h p.a.). 
5.3.2
Methods of sample collection
5.3.2.1
Blood sampling for pharmacokinetic analysis of alteplase
For quantification of analyte concentrations in plasma, 2.7 mL of blood will be drawn from 
an antecubital or forearm vein into a K2-EDTA (dipotassium ethylenediaminetetraacetic
acid)-anticoagulant blood drawing tube at the times indicated in the Flow Chart. Blood will 
be withdrawn by means of either an indwelling venous catheter or by venepuncture with a 
metal needle.  
Within 5 minutes after sampling 54µl 100 µM PPACK-inhibitor solution (D-Phe-Pro-Arg-
CH2-CL) will be added to the blood sample to get a final PPACK concentration of 2 µM.
Thereafter the samples should be gently inverted 10 times while avoiding rigorous shaking. 
These blood samples will be centrifuged for approximately 10 minutes at approximately
2000 g to 4000 g and at 4 to 8 °C. Prior to centrifugation the samples should be incubated for 
at least 15 min in an ice bath or on ice.
Two plasma aliquots will be obtained and stored in polypropylene tubes. The first aliquot 
should contain at least 0.5 mL of plasma. The process from blood collection until transfer of 
plasma aliquots into the freezer should be completed within 60 minutes. The time each 
aliquot was placed in the freezer will be documented. Until transfer on dry ice to the 
analytical laboratory, the aliquots will be stored upright at approximately -20°C or below at 
the trial site. The second aliquot will be transferred to the analytical laboratory after the 
bioanalyst has acknowledged safe arrival of the first aliquot. At the analytical laboratory, the 
plasma samples will be stored at approximately -20°C or below until analysis.
At a minimum, the sample tube labels should list BI trial number, subject number, visit, and 
planned sampling time. 
After completion of the trial, the plasma samples may be used for further methodological 
investigations (e.g. for stability testing). However, only data related to the analyte will be 
generated by these additional investigations. Furthermore the back-up samples may be used 
for determination of any laboratory parameters of plasmin activation, coagulation and 
fibrinolytic cascade and platelet function. The study samples will be discarded after 
completion of the additional investigations but not later than 5 years after the CTR is 
archived.


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5.4
ASSESSMENT OF BIOMARKER (S)
Not applicable.
5.5
BIOBANKING
Not applicable.
5.6
OTHER ASSESSMENTS
5.7
APPROPRIATENESS OF MEASUREMENTS
All measurements performed during this trial are standard measurements and will be 
performed in order to monitor subjects’ safety and to determine pharmacokinetic parameters 
in an appropriate way. The scheduled measurements will allow monitoring of changes in vital 
signs, standard laboratory values, and ECG parameters that might occur as a result of 
administration of trial medication. The safety assessments are standard, are accepted for 
evaluation of safety and tolerability of an intravenously administered drug, and are widely 
used in clinical trials. The pharmacokinetic parameters and measurements outlined in Section 
5.4 are generally used assessments of drug exposure.


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6
INVESTIGATIONAL PLAN
6.1
VISIT SCHEDULE
Exact times of measurements outside the permitted time windows will be documented. The 
acceptable time windows for screening and the end of trial examination are provided in the 
Flow Chart.
Study measurements and assessments scheduled to occur ‘before’ trial medication 
administration on Day 1 are to be performed and completed within a 3 h-period prior to the 
trial drug administration. The acceptable deviation from the scheduled time for all procedures 
on Day -1 will be ± 2 hours.
The acceptable deviation from the scheduled time for vital signs, and laboratory tests will be 
± 30 min, it is ± 2 hours for the urine sticks on Day 1.
If scheduled in the Flow Chart at the same time as a meal, blood sampling and vital signs
have to be done first. Furthermore, if several measurements including venepuncture are 
scheduled for the same time, venepuncture should be the last of the measurements due to its 
inconvenience to the subject and possible influence on physiological parameters. The subjects 
may have the snack in the afternoon and the dinner together. In the morning of Day 2 (Visit 
3) subjects may have their breakfast prior to all other measures. 
For planned blood sampling times, refer to the Flow Chart. While these nominal times should 
be adhered to as closely as possible, the actual sampling times will be recorded and used for 
the determination of pharmacokinetic parameters.
If a subject misses an appointment, it will be rescheduled if possible. The relevance of 
measurements outside the permitted time windows will be assessed no later than at the Report 
Planning Meeting.
6.2
DETAILS OF TRIAL PROCEDURES AT SELECTED VISITS
6.2.1
Screening
After having been informed about the trial, all subjects will provide written informed consent 
in accordance with GCP and local legislation prior to enrolment in the study.
For information regarding laboratory tests (including drug and virus screening and FOB-test), 
ECG, vital signs, and physical examination, refer to Sections 5.2.3 to 5.2.5.
Genotyping will be performed in those volunteers whose genotypes have not been previously 
determined (for details, see Section 5.3).
6.2.2
Treatment period
Each subject is expected to participate in 2 treatment periods, which will be performed on 2 
consecutive days. The start of infusions will be separated by a wash-out phase of at least 24
hours. 
Study participants will be admitted to the trial site in the evening before the planned first drug 
administration. They are kept under close medical surveillance for at least 24 h following the 


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start of last drug administration. The subjects will then be allowed to leave the trial site after 
formal assessment and confirmation of their fitness.
For details on time points and procedures for collection of plasma samples for PK analysis, 
refer to Flow Chart and Section 5.3.2.
The safety measurements performed during the treatment period are specified in Section 5.3
of this protocol and in the Flow Chart. For details on times of all other trial procedures, refer 
to the Flow Chart. AEs and concomitant therapy will be assessed continuously from 
screening until the end of trial examination.
6.2.3
Follow-up period and trial completion
The EOT visit will be performed at least 1 week after the last trial drug administration within 
the time span defined in the Flow Chart.
For AE assessment, laboratory tests, recording of ECG and vital signs, and physical 
examination during the follow-up period, see Sections 5.2.2 to 5.2.5.
Subjects who discontinue treatment before the end of the planned treatment period should 
undergo the EoTrial Visit.
All abnormal values (including laboratory parameters) that are assessed as clinically relevant 
by the investigator will be monitored using the appropriate tests until a return to a medically 
acceptable level is achieved. (S)AEs persisting after a subject’s EoTrial Visit must be 
followed until they have resolved, have been sufficiently characterised, or no further 
information can be obtained.


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7
STATISTICAL METHODS AND DETERMINATION OF
SAMPLE SIZE
7.1
STATISTICAL DESIGN – MODEL
The main objective of this trial is to establish the bioequivalence of TPA-05 compared with 
TPA-02 following intravenous administration of 0.2 mg/kg body weight on the basis of the 
primary and secondary pharmacokinetic endpoints, as listed in Section 2.1.2 and 2.1.3. The 
trial is designed to allow intra-subject comparisons and will be evaluated statistically by use 
of a linear model for logarithmically transformed PK endpoints.
Part A of this trial has been terminated after 12 subjects due to procoagulant effects that 
interfered also with alteplase analytics (see 1.2.5). The PK-data could not be used for 
hypothesis testing and hence the data was only analysed descriptively.
After implementation of measures to mitigate the risk of thromboembolic complications and 
to avoid methodological problems with alteplase analyticsPart B will be performed following 
a 2-stage design.
The establishment of bioequivalence will be only based on data of Part B (stage 1 and 2).
The study flow will in principle follow the adaptive sample size re-estimation (SSR) 
algorithm provided in [R19-3175] based on the standard inverse normal combination test. An 
outline including further details of this algorithm is provided below.
7.2
NULL AND ALTERNATIVE HYPOTHESES
Part A: no formal hypothesis will be tested. The same statistical analyses as for Part B will be 
performed but p-values and confidence intervals will be only considered descriptively.
Part B (stage 1 and stage 2):
The study design is an adaptive two-stage group sequential design according to [R14-1197] 
and [R19-3175].
Primary and secondary endpoints
For each stage, the following hypotheses are considered:
Null hypothesis H0 (Inequivalence):
δ
μ
μ
δ
μ
μ
R
T
R
T





or
where 
T

and 
R

are the means of the log-transformed endpoint for the test and reference 
treatments, respectively, and δ is the bioequivalence limit that defines the acceptance range 
on the logarithmic scale. Alternative hypothesis Ha (Equivalence):
δ
μ
μ
δ
R
T




In this trial, the bioequivalence limit δ is ln(1.25). By back-transforming (exponentiation), 
this translates to an acceptance range of 80.00 to 125.00% for the ratio of the geometric 
means (test/reference) for endpoints on the original scale.


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Although there are multiple primary endpoints, an alpha adjustment with respect to multiple 
endpoints is not needed because it is required that all primary and secondary endpoints are 
significant (either at interim or at the final stage, see Section 7.3). The alpha level for the 
significance testing will be adjusted for considering multiple stages (i.e. looks). The standard 
inverse normal combination test as described in [R19-3175] with pre-specified weights of √
and √1 − for stage 1 and stage 2, respectively, will be used. The alpha spent for the first 
stage is fixed to 0.03585 (one-sided) and a user-defined alpha spending function [R10-2517] 
will be used such that the full level of 0.05 is spent at the final analysis. The information 
fraction w will be calculated as  = 0.8 ⋅
∗/, where 1 = 18 and 1
∗is the actual number 
of subjects contributing at least one evaluable PK parameter for at least one study period (i.e. 
are in the PKS, see Section 7.3) at the time of the interim analysis. Thus, the ratio 1
∗/1
accounts for example for subjects that drop-out early or do not provide evaluable PK 
parameters for any of the periods. It is expected that 1
∗= 1and therefore the planning 
values are w = 0.8 and 1 = 2 = 0.03585; these alpha values correspond to Pocock 
boundaries. For illustration purposes the following table provides examples on how the 
adjusted alpha value 2 and the critical value (see below) will look like depending on 
different values of 
∗.
Table 7.2: 1
Information fraction, adjusted alpha level and critical value for stage 
two depending on different values of 
∗.

∗
w
2
c2
17
0.75556
0.03359
1.83050
16
0.71111
0.03159
1.85797
15
0.66667
0.02980
1.88381
At the interim analysis an assessment for BE will be performed. Thus, at the interim analysis 
the p-values p1j based on stage 1 data, where j = 1, 2 denotes the index of the two one-sided 
tests will be compared to the value 0.03585. This is equivalent to comparing the repeated 
confidence interval with the boundaries 80.00% to 125.00%. If the study continues to the 
second stage, let p2j denote the two p-values of the second stage, j = 1, 2. Note that p2j are 
obtained using only the data from stage 2. BE is concluded (i.e. the null hypothesis is
rejected) after the second stage if the combination of the p-values p1j and p2j (j=1, 2) is greater 
than the critical boundary c2 for the final assessment, cf. [R14-1197] and [R19-3175]:
√−1 1 −1 + 1 −−1 1 −2 ≥2,
where j = 1, 2 and j again refers to the j-th hypothesis of the TOST procedure and  refers to 
the cumulative distribution function of the standard normal distribution. Note that based on 
the elaborations above this results in planning values of c2 = 1.80107, the critical value
possibly being adapted according to the alpha spending function described above.


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7.3
PLANNED ANALYSES
The analyses for endpoint apply for both study parts Part A and Part B. However, since the 
data of Part A could not be used for a formal statistical assessment all analyses in Part A are 
only considered to be descriptive.
The data from Part A will be analyses separately and will not contribute to the bioequivalence 
assessment.
Analysis sets
Statistical analyses will be based on the following analysis sets:
Part A:

Treated set - part A (TS-A): The treated set includes all subjects who were 
randomized and treated with at least one dose of study drug. The treated set will be 
used for safety analyses.
Pharmacokinetic parameter analysis set - part A (PKS-A): This set includes all 
subjects in the treated set - part A (TS-A) who provide at least one PK endpoint that 
was defined as primary or secondary and was not excluded due to a protocol violation 
relevant to the evaluation of PK or due to PK non-evaluability (as specified in the 
following subsection ‘Pharmacokinetics’). Thus, a subject will be included in the 
PKS, even if he/she contributes only one PK parameter value for one period to the 
statistical assessment. Descriptive and model based analyses of PK parameters will 
be based on the PKS.
Part B (stage I and II):

Treated set - part B (TS-B): The treated set includes all subjects who were 
randomized and treated with at least one dose of study drug. The treated set will be 
used for safety analyses.
Pharmacokinetic parameter analysis set - part B(PKS-B): This set includes all subjects 
in the treated set - part B (TS-B) who provide at least one PK endpoint that was 
defined as primary or secondary and was not excluded due to a protocol violation 
relevant to the evaluation of PK or due to PK non-evaluability (as specified in the 
following subsection ‘Pharmacokinetics’). Thus, a subject will be included in the 
PKS, even if he/she contributes only one PK parameter value for one period to the 
statistical assessment. Descriptive and model based analyses of PK parameters will 
be based on the PKS.
Adherence to the protocol will be assessed by the trial team. Important protocol deviation
(IPD) categories will be described in the IQRMP, IPDs will be identified no later than in the 
Report Planning Meeting, and the IPD categories will be updated as needed.
Pharmacokinetics
The pharmacokinetic parameters listed in Section 2 will be calculated according to the 
relevant BI internal procedures. .
Individual endogenous TPA will be determined for each period as a mean of two 
measurements at baseline. Individual baseline levels will then be subtracted from all 


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concentration measurements in that treatment period. In case the resulting concentration 
value is negative, it will be set to zero.
Plasma concentration data and parameters of a subject will be included in the statistical 
pharmacokinetic (PK) analyses if they are not flagged for exclusion due to a protocol 
violation relevant to the evaluation of PK (to be decided no later than in the Report Planning 
Meeting) or due to PK non-evaluability (as revealed during data analysis, based on the 
criteria specified below). Exclusion of a subject’s data will be documented in the CTR.
Relevant protocol violations may be

Incorrect trial medication taken, i.e. the subject received at least one dose of trial
medication the subject was not assigned to

Incorrect dose of trial medication taken

Use of restricted medications
Plasma concentrations and/or parameters of a subject will be considered as non-evaluable, if 
for example

A predose concentration (mean baseline concentration in Visit 3 corrected by the 
mean baseline concentration in Visit 2) is >5% Cmax value of that subject in Visit 3

Missing samples/concentration data at important phases of PK disposition curve
Plasma/concentration data and parameters of a subject which is flagged for exclusion will be 
reported with its individual values but will not be included in the statistical analyses. 
Descriptive and inferential statistics of PK parameters will be based on the PKS.
Only concentration values within the validated concentration range and actual sampling times 
will be used for the calculation of pharmacokinetic parameters. Concentrations used in the 
pharmacokinetic calculations will be in the same format provided in the bioanalytical report, 
(that is, to the same number of decimal places provided in the bioanalytical report). 
7.3.1
Primary endpoint analyses
Primary analysis
The statistical model used for the analysis of the primary endpoints for each stage of the trial 
will be an analysis of variance (ANOVA) model on the logarithmic scale. That is, the PK 
endpoints will be log-transformed (natural logarithm) prior to fitting the ANOVA model. 
This model will include effects accounting for the following sources of variation: sequence, 
subjects within sequences, period and treatment. All effects will be considered as fixed. The 
model is described by the following equation:
yijkm = + i + sim + j + k + eijkm, where
yijkm = logarithm of response measured on subject m in sequence i receiving treatment  
k in period j,
= the overall mean,


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i = the ith sequence effect, i = 1, 2,
sim = the effect associated with the mth subject in the ith sequence,
m = 1, 2, ..., ni
j = the jth period effect, j = 1, 2,
k = the kth treatment effect, k = 1, 2,
eijkm = the random error associated with the mth subject in sequence i who received
treatment k in period j.
where e !"# ~ N(0, σ(
) ) i.i.d. Note that each stage will be analyzed separately based on data 
from only this stage, see Section 7.2.
Point estimates for the ratios of the geometric means (test/reference) for the primary 
endpoints (see Section 2.1) and their two-sided confidence intervals (CIs) will be provided.
At stage 1, the (1 – 21)*100% confidence interval for the gMean ratio will be calculated. If 
the study continues to stage 2, the repeated confidence interval corresponding to the test 
decision will be calculated, in conjunction with the median unbiased point estimate as 
estimate for the final adjusted geometric mean ratio.
Bioequivalence is considered established if the confidence intervals of the geometric means 
for the primary endpoints are contained in the pre-defined acceptance range, see Section 7.2.
7.3.2
Secondary endpoint analyses
Primary analysis
The secondary endpoints (refer to Section 2.1.3) will be calculated according to the relevant 
BI internal procedures and will be assessed statistically using the same methods as described 
for the primary analysis of the primary endpoints (see also Section 7.2).


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7.3.4
Safety analyses 
Safety will be analysed based on the assessments described in Section 2.2.2.2. All treated 
subjects (TS, refer to Section 7.2) will be included in the safety analysis. Safety analyses will 
be descriptive in nature and based on BI standards. No hypothesis testing is planned.
For all analyses, the treatment actually administered (= treatment at onset) to the subject will 
be used (any deviations from the randomised treatment will be discussed in the minutes of the 
Report Planning Meeting).
Treatments will be compared in a descriptive way. Tabulations of frequencies/proportions 
will be used to evaluate categorical (qualitative) data, and tabulations of descriptive statistics 
will be used to analyse continuous (quantitative) data.
Measurements (such as ECG, vital signs, or laboratory parameters) or AEs will be assigned to 
treatments (see Section 4.1) based on the actual treatment at the planned time of the 
measurement or on the recorded time of AE onset (concept of treatment emergent AEs).
Therefore, measurements planned or AEs recorded prior to first intake of trial medication will 
be assigned to the screening period, those between first trial medication intake and end of 
REP (see Section 1.2.3) will be assigned to the treatment period. Events occurring after the 
REP but prior to next intake or end of trial termination date will be assigned to ’follow-up’. 
In case of two or more treatments, the follow-up will be summarized according to the 
previous treatment. These assignments including the corresponding time intervals will be 
defined in detail in the Trial statistical analysis plan (TSAP). Note that AEs occurring after 
the last per protocol contact but entered before final database lock will be reported to 
Pharmacovigilance only and will not be captured in the trial database.
Additionally, further treatment intervals (analysing treatments) may be defined in the TSAP 
in order to provide summary statistics for time intervals, such as combined treatments, on-
treatment totals, or periods without treatment effects (such as screening and follow-up 
intervals).
Adverse events will be coded using the Medical Dictionary for Regulatory Activities 
(MedDRA). Frequency, severity, and causal relationship of AEs will be tabulated by 
treatment, system organ class, and preferred term. SAEs, AESIs (see Section 5.2.6.1), and 
other significant AEs (according to ICH E3) will be listed separately.
Previous and concomitant therapies will be presented per treatment group without 
consideration of time intervals and treatment periods.


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Laboratory data will be compared to their reference ranges. Values outside the reference 
range as well as values defined as possibly clinically significant will be highlighted in the 
listings. Additionally, differences from baseline will be evaluated.
Vital signs or other safety-relevant data will be assessed with regard to possible on-treatment 
changes from baseline. Relevant ECG findings will be reported as AEs.
ADA status and ADA titer will be analysed descriptively if applicable.
7.4
INTERIM ANALYSES
Part A:
The study was stopped for safety reasons and PK-data from Part A cannot be used for 
assessment of bioequivalence. Therefore, all analysis were only considered descriptively.
Part B
Blinded review of PK-data 
After the first 2 cohorts a blinded review of PK-data will be performed. If these data indicate 
non-reliable concentration-time profiles (data do not reflect systemic alteplase concentrations 
due to methodological problems, as it has been observed in Part A), the trial will be 
discontinued (see 3.3.4.3). 
Review of biomarkers
Biomarkers (see 2.2.2.2) can be assessed at any time point during the trial. After the first two 
cohorts a safety review meeting is planned (see 3.1) to check whether an activation of 
coagulation could be observed.
Neither the PK review nor the biomarker review will influence the sample size planning of 
the study. Both, PK and biomarker review are not part of the adaptive design.
Two stage design with formal interim analysis:
After n1 = 18 subjects have completed their crossover (Part B), recruitment will be stopped 
and an assessment for bioequivalence will be performed. Based on unblinded data from the 
first stage it will be evaluated whether the trial continues to stage 2 (Part B) or not. This 
analysis will be performed in an unblinded manner and will be conducted by the trial team. 
As described in Section 7.2, the p-values based on stage 1 (Part B) data are planned to be 
compared to the value 0.03585.
Equivalently, if the corresponding (adjusted) confidence intervals for the comparison of the
primary and secondary endpoints are within the pre-specified boundaries 80.00% to 125.00% 
(original scale), the trial will be closed after stage 1 (Part B) due to proven bioequivalence. If 
bioequivalence is not shown for all primary and secondary endpoints, then a decision 
regarding continuation of the trial will be made by the Principal Investigator, the Trial 
Statistician, and the Clinical Trial Lead or their respective designees.
The power of the TOST procedure based on the first stage data will be calculated (using the 
planned treatment effect, the observed variability and the adjusted alpha level for stage 1). If 
this power is greater or equal to 0.9, the trial is recommended to stop for futility, cf. [R19-
3175]. Note that this futility rule is considered nonbinding. Note also that there will be no 


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futility criterion with respect to the confidence interval of the gMean ratio after stage 1, in 
contrast to the proposed algorithm in [R19-3175]. Otherwise a sample size re-estimation 
(SSR) based on the unblinded data from the first stage will be performed and the trial 
continues to stage 2 (Part B). 
The SSR will be based on the conditional power as described in [R19-3175], i.e. it will use 
the conditional error rates as well as the estimated conditional target power for the second 
stage. For the SSR it is planned to use the observed variability (gCV) and the observed 
geometric mean ratio (GMR) from the (unblinded) stage 1 data. The overall target power to 
declare bioequivalence at the end of the trial is planned to be 90%. Note that a specific power 
adjustment due to multiple endpoints will not be done [R17-0365] and the sample size 
recalculation, if required, will be based on observed results from primary and secondary PK 
parameters. The calculation will be performed for each parameter and the highest sample size 
will be used. Note that the minimum sample size for stage 2 is set to 4, as done and 
recommended in [R19-3175]. Moreover, for this study a maximum overall sample size of 72
will be considered. Therefore, the number of subjects in stage 2 will not exceed 42, even if 
the calculated sample size based on stage 1 data is higher than 42.
If bioequivalence is shown for one endpoint but not the other(s), only the endpoint(s) where 
bioequivalence has not been shown will be tested a second time at the end of the trial [R17-
1639]. Note, however, that for completeness all PK parameters and corresponding confidence 
intervals for the comparison(s) as described in Section 7.3.1 and Section 7.3.2 will be 
calculated.
7.5
HANDLING OF MISSING DATA
7.5.1
Safety
It is not planned to impute missing values for safety parameters.
7.5.2
Pharmacokinetics
Handling of missing PK data will be performed according to the relevant BI internal 
procedures.
PK parameters that cannot be reasonably calculated based on the available drug 
concentration-time data will not be imputed.
7.6
RANDOMISATION
Subjects will be randomised to one of the 2 treatment sequences in a 1:1 ratio. The block size 
will be documented in the CTR.
The sponsor will arrange for the randomisation as well as packaging and labelling of trial 
medication. The randomisation list will be generated using a validated system that uses a 
pseudo-random number generator and a supplied seed number so that the resulting allocation 
is both reproducible and non-predictable.
The randomisation list will contain additional blocks to allow for subject replacement (refer 
to Section 3.3.5).


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7.7
DETERMINATION OF SAMPLE SIZE
There is limited knowledge about the intra-individual variability of the primary endpoints. 
Previous trials in healthy volunteers (135.57 and 135.67) with TPA-02 were performed as a 
parallel design and reported a total variability (gCV) of 20.8% and 12%, respectively (both 
AUC and Cmax had similar variability). Another trial in rabbits (n00267264) with TPA-02 and 
TPA-05 that has been performed as a crossover trial reported an intra-individual variability of 
approximately 15%. This latter trial observed a gMean ratio of about 100% for Cmax and 
109% for AUC. Therefore, in lack of further evidence it is assumed that the gMean ratio 
ranges between 100% and 109%.
All calculations below are based on the specifications given in Section 7.2. Particularly, a 
weight of 0.8 for the inverse normal approach, a sample size n2 for stage 2 between 4 and 42 
and usage of the observed gCV and gMean ratio are assumed. Using a sample size of n1 = 18
subjects for the first stage, the probability to conclude bioequivalence after the first stage is 
79% and the overall power to conclude bioequivalence is at least 95%, assuming an intra-
individual gCV of 15% and a treatment difference of 9% (ratio scale) (Table 7.7: 1). In order 
to investigate the sensitivity of the sample size, a range of gCVs around 15% and a range of 
gMean ratios around 109% are presented.
Table 7.7: 1
Power for concluding bioequivalence overall, power for concluding 
bioequivalence after stage 1 and average sample size (acceptance 
range 80-125%) based on an intra-individual geometric coefficient of 
variation around 15% and for expected ratios of geometric means 
(test/reference) around 109% in a two-stage 2x2 crossover trial (n1 = 
18)
Ratio T/R [%]
gCV [%]
104
109
114
10
>99; >99; 18.0
98.5; 98.2; 18.0
95.3; 79.4; 22.2
15
97.3; 95.4; 18.3
95.2; 79.0; 22.4
83.0; 48.3; 35.8
20
95.5; 77.3; 22.9
89.2; 56.1; 32.1
61.8; 31.6; 43.8
From the above table, even with such variations in assumptions the power is considered to be 
sufficient to meet the study objectives. The calculations were performed as described by 
Maurer et al. [R19-3175] using the function power.tsd.in of the R package Power2Stage 
Version 0.5.2 in R Version 3.6.1.


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8
INFORMED CONSENT, TRIAL RECORDS, DATA 
PROTECTION, PUBLICATION POLICY, AND 
ADMINISTRATIVE STRUCTURE
The trial will be carried out in compliance with the protocol, the ethical principles laid down 
in the Declaration of Helsinki, in accordance with the ICH Harmonized Guideline for Good 
Clinical Practice (GCP), relevant BI Standard Operating Procedures (SOPs), the EU 
regulation 536/2014,  and other relevant regulations. Investigators and site staff must adhere 
to these principles.
Standard medical care (prophylactic, diagnostic, and therapeutic procedures) remains the 
responsibility of the subject’s treating physician.
The investigator will inform the sponsor immediately of any urgent safety measures taken to 
protect the trial subjects against any immediate hazard, as well as of any serious breaches of 
the protocol or of ICH GCP.
Within 1 year after trial termination the Sponsor will submit a summary of the trial report 
covering all relevant results of the trial to the Competent Authorithy and to the Independent 
Ethics Committee.
The Boehringer Ingelheim transparency and publication policy can be found on the following 
web page: trials.boehringer-ingelheim.com. As a general rule, no trial results should be 
published prior to archiving of the CTR.
The terms and conditions of the insurance coverage are made available to the investigator and 
the subjects, and are stored in the ISF.
8.1
TRIAL APPROVAL, SUBJECT INFORMATION, INFORMED CONSENT
This trial will be initiated only after all required legal documentation has been reviewed and 
approved by the respective Institutional Review Board (IRB / Independent Ethics Committee 
(IEC and competent authority (CA) according to national and international regulations. The 
same applies for the implementation of changes introduced by amendments.
Prior to a subject’s participation in the trial, written informed consent must be obtained from 
each subject (or the subject’s legally accepted representative) according to ICH-GCP and to 
the regulatory and legal requirements of the participating country. Each signature must be 
personally dated by each signatory and the informed consent and any additional subject-
information form retained by the investigator as part of the trial records. A signed copy of the 
informed consent and any additional subject information must be given to each subject or the 
subject’s legally accepted representative.
The subject must be given sufficient time to consider participation in the trial. The 
investigator or delegate obtains written consent of the subject’s own free will with the 
informed consent form after confirming that the subject understands the contents. The 
investigator or 
delegate must sign (or place a seal on) and date the informed consent form. 
If a trial collaborator has given a supplementary explanation, the trial collaborator also signs 
(or places a seal on) and dates the informed consent.


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Re-consenting may become necessary when new relevant information becomes available and 
should be conducted according to the sponsor’s instructions.
The consent and re-consenting process should be properly documented in the source 
documentation.
8.2
DATA QUALITY ASSURANCE
A risk-based approach is used for trial quality management. It is initiated by the assessment 
of critical data and processes for trial subject protection and reliability of the results as well as 
identification and assessment of associated risks. An Integrated Quality and Risk 
Management Plan documents the rationale and strategies for risk management during trial 
conduct including monitoring approaches, vendor management and other processes focusing 
on areas of greatest risk. 
Continuous risk review and assessment may lead to adjustments in trial conduct, trial design 
or monitoring approaches. 
A quality assurance audit/inspection of this trial may be conducted by the sponsor, sponsor’s 
designees, or by IRB / IEC or by regulatory authorities. The quality assurance auditor will
have access to all medical records, the investigator’s trial-related files and correspondence, 
and the informed consent documentation of this clinical trial..
8.3
RECORDS
CRFs for individual subjects will be provided by the sponsor. For drug accountability, refer 
to Section 4.1.8.
ClinBaseTM
In the
– the 
validated ClinBaseTM system is used for processing information and controlling data 
collected in clinical studies. In addition to its function as a procedure control system, 
ClinBaseTM serves as data base. Instead of being entered into CRFs, selected data are directly 
entered into the system.
8.3.1
Source documents
In accordance with regulatory requirements, the investigator should prepare and maintain 
adequate and accurate source documents and trial records for each trial subject that include 
all observations and other data pertinent to the investigation. Source data as well as reported 
data should follow the ‘ALCOA principles’ and be attributable, legible, contemporaneous, 
original, and accurate. Changes to the data should be traceable (audit trail).
Data reported on the CRF must be consistent with the source data or the discrepancies must 
be explained.
Before providing any copy of subjects’ source documents to the sponsor, the investigator 
must ensure that all subject identifiers (e.g., subject’s name, initials, address, phone number, 
and social security number) have properly been removed or redacted to ensure subject 
confidentiality.


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If the subject is not compliant with the protocol, any corrective action (e.g. re-training) must 
be documented in the subject file.
For the CRF, data must be derived from source documents, for example:

Subject identification: sex, year of birth (in accordance with local laws and 
regulations)

Subject participation in the trial (substance, trial number, subject number, date subject 
was informed)

Dates of subject’s visits, including dispensing of trial medication

Medical history (including trial indication and concomitant diseases, if applicable)

Medication history

AEs and outcome events (onset date [mandatory], and end date [if available])

SAEs (onset date [mandatory], and end date [if available])

Concomitant therapy (start date, changes)

Originals or copies of laboratory results and other imaging or testing results, with 
proper documented medical evaluation (in validated electronic format, if available)

ECG results (original or copies of printouts)

Completion of subject’s participation in the trial (end date; in case of premature 
discontinuation, document the reason for it, if known)

Prior to allocation of a subject to a treatment into a clinical trial, there must be 
documented evidence in the source data (e.g. medical records) that the trial participant 
meets all inclusion criteria and does not meet any exclusion criteria. The absence of 
records (either medical records, verbal documented feedback of the subject or testing 
conducted specific for a protocol) to support inclusion/exclusion criteria does not 
make the subject eligible for the clinical trial.
Data directly entered into ClinBaseTM (that is, without prior written or electronic record) are 
considered to be source data. The place where data are entered first will be defined in a trial 
specific Source Data Agreement. The data in ClinBaseTM are available for inspection at any 
time.
8.3.2
Direct access to source data and documents
The investigator /institution will allow site trial-related monitoring, audits, IRB / IEC review 
and regulatory inspections. Direct access must be provided to the CRF and all source docu-
ments/data, including progress notes, copies of laboratory and medical test results, which 
must be available at all times for review by the Clinical Research Associate, auditor and regu-
latory inspector (e.g. FDA). They may review all CRFs and informed consents. The accuracy 
of the data will be verified by direct comparison with the source documents described in 
section 8.3.1. The sponsor will also monitor compliance with the protocol and GCP.
8.3.3
Storage period of records
Trial site:
The trial site must retain the source and essential documents (including ISF) according to the 
local requirements valid at the time of the end of the trial.


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Sponsor:
The sponsor must retain the essential documents according to the sponsor’s SOPs.
8.4
EXPEDITED REPORTING OF ADVERSE EVENTS
BI is responsible to fulfil their legal and regulatory reporting obligation in accordance with 
regulatory requirements.
8.5
STATEMENT OF CONFIDENTIALITY AND SUBJECT PRIVACY
Individual subject data obtained as a result of this trial is considered confidential and 
disclosure to third parties is prohibited with the exceptions noted in section 8.7. 
Data protection and data security measures are implemented for the collection, storage and 
processing of patient data in accordance with the principles 6 and 12 of the WHO GCP 
handbook.
Personalised treatment data may be given to the subject’s personal physician or to other 
appropriate medical personnel responsible for the subject’s welfare. Data generated at the site 
as a result of the trial need to be available for inspection on request by the participating 
physicians, the sponsor’s representatives, by the IRB / IEC and the regulatory authorities.
8.5.1
Collection, storage and future use of biological samples and corresponding 
data
Measures are in place to comply with the applicable rules for the collection, biobanking and 
future use of biological samples and clinical data, in particular

Sample and data usage has to be in accordance with the separate biobanking informed 
consent

The BI-internal facilities storing biological samples from clinical trial participants as 
well as the external banking facility are qualified for the storage of biological samples 
collected in clinical trials.

An appropriate sample and data management system, incl. audit trail for clinical data 
and samples to identify and destroy such samples according to ICF is in place

A fit for the purpose documentation (biomarker proposal, analysis plan and report) 
ensures compliant usage

A fit for purpose approach will be used for assay/equipment validation depending on 
the intended use of the biomarker data
Samples and/or data may be transferred to third parties and other countries as specified in the 
biobanking ICF.
8.6
TRIAL MILESTONES
The start of the trial is defined as the date of the enrolment of the first subject in the trial.
The end of the trial is defined as the ‘date of the last visit of the last subject in whole trial’ 
(‘Last Subject Completed’) or ‘end date of the last open AE’ or ‘date of the last follow-up 
test’ or ‘date of an AE has been decided as sufficiently followed-up’, whichever is latest.


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Early termination of the trial is defined as the premature termination of the trial for any 
reason before the end of the trial as specified in this protocol.
Temporary halt of the trial is defined as any unplanned interruption of the trial by the 
sponsor with the intention to resume it.
Suspension of the trial is defined as an interruption of the trial based on a Health Authority 
request.
The EC/competent authority in each participating EU member state will be notified about the 
trial milestones according to the laws of each member state. 
A final report of the clinical trial data will be written only after all subjects have completed 
the trial in all countries (EU or non-EU), so that all data can be incorporated and considered 
in the report.
The sponsor will submit to the EU database a summary of the final trial results within one 
year from the end of a clinical trial as a whole, regardless of the country of the last patient 
(EU or non-EU).
8.7
ADMINISTRATIVE STRUCTURE OF THE TRIAL
The trial is sponsored by Boehringer Ingelheim (BI).
The trial will be conducted at the 
 of 
 
, under the supervision of the Principal Investigator. Relevant 
documentation on the participating (Principal) Investigators (e.g. their curricula vitae) will be 
filed in the ISF.
BI has appointed a Clinical Trial Leader, responsible for coordinating all required trial 
activities, in order to 

Manage the trial in accordance with applicable regulations and internal SOPs

Direct the clinical trial team in the preparation, conduct, and reporting of the trial

ensure appropriate training and information of local clinical monitors (CML), Clinical 
Research Associates, and investigators of participating trial sites
The trial medication will be provided by the 
 
.
Safety laboratory tests will be performed by the local laboratory of the trial site (
 
).
Analyses of alteplase concentrations in plasma and analyses of ADAs will be performed at 
the 
.
Coagulation, fibrinolysis and platelet parameters will be measured at 
, 
and at 
.
On-site monitoring will be performed by BI or a contract research organisation appointed by 
BI.
Data management and statistical evaluation will be done by BI according to BI SOPs.
Tasks and functions assigned in order to organise, manage, and evaluate the trial are defined 
according to BI SOPs. A list of responsible persons and relevant local information can be 
found in the ISF.


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9
REFERENCES 
9.1
PUBLISHED REFERENCES
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Szabo S, Letsch R, Ehlers R, Walter T, Kazmaier S, Helber U, Hoffmeister 
HM. Absence of paradoxical thrombin activation by fibrin-specific 
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P16-06898
Whiteley WN, Emberson J, Lees KR, Blackwell L, Albers G, Bluhmki E, et 
al, Stroke Thrombolysis Trialists' Collaboration. Risk of intracerebral 
haemorrhage with alteplase after acute ischaemic stroke: a secondary 
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Published Online: 08 June 2016, doi: 10.1016/S1474-4422(16)30076-X 
Lancet Neurol 2016. 15(9):825-933.
P20-11344
Hoffmeister HM. The thrombolytic paradox. Thromb Res 2001;103:S51
P84-97878
Hoylaerts M, Rijken DC, Lijnen HR, Collen D. Kinetics of the activation of 
plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol 
Chem 1982;257:2912-9.
P84-97880
Rijken DC, Collen D. Purification and characterization of the plasminogen 
activator secreted by human melanoma cells in culture. J Biol Chem 
1981;256:7035-41.
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Rijken DC, Wijngaards G, Zaal-de Jong M, Welbergen J. Purification and 
partial characterization of plasminogen activator from human uterine tissue. 
Biochim Biophys Acta 1979;580:140-53.
P86-5232
Verstraete M, Su CAPF, Tanswell P, Feuerer W, Collen D. 
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volunteers. Thromb Haemost 1986. 56(1):1-5. 
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Seifried E, Transwell P, Rijken DC, Kluft C, Hoegee E, Nieuwenhuizen W. 
Fibrin degradation products are not specific markers for thrombolysis in 
myocardial infarction. Lancet 1987. 2:333-334
P88-34055
Seifried E, Tanswell P, Rijken DC, Barrett-Bergshoeff MM, Su CAPF, 
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type plasminogen activator after infusion in healthy volunteers. 
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P88-36274
Fry ETA, Sobel BE. Lack of interference by heparin with thrombolysis or   
binding of tissue-type plasminogen activator to thrombi. Blood 1988;71(5):   
1347-1352.
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Sobel BE. Fibrinolysis and activators of plasminogen. Heart Lung 1987. 
16(6, Part 2):775-779.
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Eisenberg PR, Miletich JP, Sobel BE, Jaffe AS. Differential effects of  
activation of prothrombin by streptokinase compared with urokinase and 
tissue-type plasminogen activator (t-PA). Thromb Res 1988;50(5):707-717.
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Owen J, Friedman KD, Grossman BA, Wilkins C, Berke AD, Powers ER. 
Thrombolytic therapy with tissue plasminogen activator or streptokinase 
induces transient thrombin activity. Blood 1988;72(2):616-620.
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Rapold HJ, Kuemmerli H, Weiss M, Baur H, Haeberli A. Monitoring of 
fibrin generation during thrombolytic therapy of acute myocardial 
infarction with recombinant tissue-type plasminogen activator. Circulation 
1989;79:980-989.P89-49823
Tanswell P, Seifried E, Su CAPF, Feuerer 
W, Rijken DC. Pharmacokinetics and systemic effects of tissue-type 
plasminogen activator in normal subjects. Clin Pharmacol Ther 1989. 
46(2):155-162. 
P89-49823
Tanswell P, Seifried E, Su CAPF, Feuerer W, Rijken DC. Pharmacokinetics 
and systemic effects of tissue-type plasminogen activator in normal 
subjects. Clin Pharmacol Ther 1989;46(2):155-162.
P89-49897
Eisenberg PR, Miletich JP. Induction of mearked thrombin activity by 
pharmacologic concentrations of plasminogen activators in 
noanticoagulated whole blood. Thromb Res 1989. 55(5):635-643
P90-52264
Kerins DM, Roy L, Fitzgerald GA, Fitzgerald DJ. Platelet and vascular 
function during coronary thrombolysis with tissue-type plasminogen 
activator. Circulation 1989;80(6):1718-1725.
P90-56499
Fitzgerald DJ, Fitzgerald GA. Role of thrombin and thromboxane A(2) in 
reocclusion following coronary thrombolysis with tissue-type plasminogen  
activator. Proc Natl Acad Sci USA 1989;86(19):7585-7589.
P90-58876
Reed BR, Chen AB, Tanswell P, Prince WS, Wert RM, Glaesle-Schwarz L, 
Grossbard EB. Low incidence of antibodies to recombinant human tissue-
type plasminogen activator in treated patients. Thromb Haemost 1990; 64: 
276-280
P91-62123
Gulba DC, Barthels M, Westhoff-Bleck M, Jost S, Rafflenbeul W, Daniel 
WG, Hecker H, Lichtlen PR. Increased thrombin levels during thrombolytic 
therapy in acute myocardial infarction - Relevance for the success of 
therapy.   Circulation 1991;83(3):937-944.
P92-68159
Eisenberg PR, Sobel BE, Jaffe AS. Activation of prothrombin 
accompanying thrombolysis with recombinant tissue-type plasminogen 
activator. J Am Coll Cardiol 1992;19(5):1065-1069.


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P92-68175
Rapold HJ, Bono D de, Arnold AER, Arnout J, Cock F de, Collen D, 
Verstraete M. Plasma fibrinopeptide A levels in patients with acute 
myocardial infarction treated with alteplase: correlation with concomitant 
heparin, coronary artery patency, and recurrent ischemia. Circulation 
1992;85(3):928-934.
P93-77079
Boer A de, Kluft C, Kasper FJ, Kroon JM, Schoemaker HC, Breimer DD, 
Soons PA, Cohen AF. Interaction study between nifedipine and 
recombinant tissue-type plasminogen activator in healthy subjects. Br J Clin 
Pharmacol 36, 99 - 104 (1993)P95-4459 Schwieder G, Grimm W, Siemens 
HJ, Flor B, Hilden A, Gmelin E, Friedrich HJ, Wagner T. Intermittent 
regional therapy with rt-PA is not superior to systemic thrombolysis in deep 
vein thrombosis (DVT) - a German multicenter trial. Thromb Haemost 
1995. 74(5):1240-1243.
P94-80579
Eisenberg PR. Procoagulant effects of fibrinolytic agents. In: Sobel BE,   
Collen D, editors. Coronary Thrombolysis in Perspective: Principles   
Underlying Conjunctive and Adjunctive Therapy. (Fundamental and 
Clinical Cardiology Series; vol 16). New York: Marcel Dekker; 1993. p. 
77-99.
P95-86478
Merlini PA, Bauer KA, Oltrona L, Ardissino D, Spinola A, Cattaneo M,   
Broccolino M, Mannucci PM, Rosenberg RD. Thrombin generation and 
activity during thrombolysis and concomitant heparin therapy in patients 
with acute myocardial infarction. J Am Coll Cardiol 1995;25(1):203-209.
P96-2741
Ewald GA, Eisenberg PR. Plasmin-mediated activation of contact system in  
response to pharmacological thrombolysis. Circulation 1995;91(1):28-36.
P96-4257
Cugno M, Cicardi M, Colucci M, Bisiani G, Merlini PA, Spinola A, 
Paonessa R, Agostoni A. Non neutralizing antibodies to tissue type 
plaminogen activator in the serum of acute myocardial infarction patients 
treated with the recombinant protein. Thromb Haemost 76 (2), 234 - 238 
(1996).
P98-2656
Granger CB, Becker R, Tracy RP, Califf RM, Topol EJ, Pieper KS, Ross 
AM, Roth S, Lambrew C, Bovill EG. 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):497-505.
P99-01489
Tsujioka H, Suehiro A, Kakishita E. Activation of coagulation factor VII by   
tissue-type plasminogen activator. Am J Hematol 1999;61(1):34-39.
P99-00076
Hoffmeister HM, Szabo S, Kastner C, Beyer ME, Helber U, Kazmaier S, 
Wendel HP, Heller W, Seipel L. Thrombolytic therapy in acute myocardial 
infarction:comparison of procoagulant effects of streptokinase and alteplase 
regimens with focus on the kallikrein system and plasmin. Circulation 
1998;98(23):2527-2533.


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P99-02520
Werf F van de. Single-bolus tenecteplase compared with front-loaded 
alteplase in acute myocardial infarction: the ASSENT-2 double-blind 
randomised trial. Lancet 1999;354(9180):716-722. (P99-02520 / MF-Nr. 
10676)
R10-2517
ADDPLAN Adaptive Designs – Plans and Analyses: user’s guide, release 
4. http://www.addplan.com (2007).
R14-1197
Lehmacher W, Wassmer G. Adaptive sample size calculations in group 
sequential trials. Biometrics 55, 1286 – 1290 (1999).
R17-0365
Hauck WW, Hyslop T, Anderson S, Bois FY, Tozer TN. Statistical and
regulatory considerations for multiple measures in bioequivalence testing.
Clin Res Regul Aff 12 (4), 249 - 265 (1995)
R17-1639
Patterson S, Jones B. Bioequivalence and statistics in clinical 
pharmacology. 2nd ed. Boca Raton: CRC Press (2017)
R19-3175 
Maurer W, Jones B, Chen Y. Controlling the type 1 error rate in two-stage 
sequential designs when testing for average bioequivalence. Stat Med. 
2018; 37(10): 1587--1607.
R20-0187
Actilyse (Boehringer Ingelheim) (Fachinformation, Stand der Information: 
03/2019).
R20-4172
Heparin-Natrium-5000-ratiopharm® (ratiopharm GmbH) (Fachinformation, 
Stand der Information: 07/2017). 
R20-4200
Pernerstorfer T, Eichler HG, Stohlawetz P, Speiser W, Jilma B. Effects of 
heparin and aspirin on circulating P-selectin, E-selectin and von Willebrand
Factor levels in healthy men. Atherosclerosis 2001;155:389-393.
R20-4201
Favresse J, Lippi G, Roy PM, Chatelain B, Jacqmin H, Cate H ten, et al. D-
dimer: preanalytical, analytical, postanalytical variables, and clinical 
applications. Crit Rev Clin Lab Sci 2018;55(8):548-577.
R20-4202
Gretler DD. Pharmacokinetic and pharmacodynamic properties of 
eptifibatide in healthy subjects receiving unfractionated heparin or the low-
molecular-weight heparin enoxaparin. Clin Ther 2003;25(10):2564-2574.
R20-4203
Swan SK, St Peter JV, Lambrecht LJ, Hursting MJ. Comparison of 
anticoagulant effects and safety of argatroban and heparin in healthy 
subjects. Pharmacotherapy 2000;20(7):756-770.
R20-4204
Teng R, Butler K. Lack of clinically significant pharmacological 
interactions between ticagrelor and enoxaparin or unfractionated heparin in 
healthy subjects. J Clin Pharm Ther 2012;37:704-711.
R20-4205
Lijnen HR, Uytterhoeven M, Collen D. Inhibition of trypsin-like serine   
proteinases by tripeptide arginyl and lysyl chloromethylketones. 
ThrombRes 1984;34(5):431-437
R20-4206
Lee CD, Mann KG. Activation/inactivation of human factor V by plasmin. 
Blood 1989;73(1):185-190.


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R20-4207
Ogiwara K, Nogami K, Nishiya K, Shima M. Plasmin-induced 
procoagulant effects in the blood coagulation: a crucial role of coagulation 
factors V and VIII. Blood Coagul Fibrinolysis 2010;21(6):568-576
R20-4253
Vorm LN van der, Remijn JA, Laat B de, Huskens D. Effects of plasmin on 
von Willebrand factor and platelets: a narrative review. TH Open 
2018;2(2):e218-e228.
R20-4252
Kawano K, Aoki I, Aoki N, Homori M, Maki A, Hioki Y, et al. Human 
platelet activation by thrombolytic agents: effects of tissue-type 
plasminogen activator and urokinase on platelet surface P-selectin 
expression. Am Heart J 1998;135(2, Part 1):268-271.
R94- 1529
Chow SC, Liu JP. Design and Analysis of Bioavailability and 
Bioequivalence Studies. New York: Marcel Dekker Inc 1992
R99-0792
Eisenberg PR, Sherman LA, Jaffe AS. Paradoxic elevation of fibrinopeptide 
A after streptokinase: evidence for continued thrombosis despite intense  
fibrinolysis. J Am Coll Cardiol 1987;10(3):527-529.
R99-2284
Fitzgerald DJ, Catella F, Roy L, FitzGerald GA. Marked platelet activation 
in vivo after intravenous streptokinase in patients with acute myocardial   
infarction. Circulation 1988;77(1):142-150.


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9.2
UNPUBLISHED REFERENCES
c29758839-04
Investigators brochure: TPA-05 Alteplase Powder and Solvent for Solution 
for Injection and Infusion 10, 20, 50 mg and 2 mg, 31 Mar 2020
n00267264
. Comparison of the Pharmacokinetics of Alteplase TPA-
05 and TPA-02 After an Intravenous Dose to Rabbits in a Crossover 
Design. May 2019
q00263495-01
 Manufacturing Process Development, Alteplase, 17 Jan 2020
U85-0697
. Report on a pharmacokinetic study of 
recombination human tissue type plasminogen activator (RT-PA) 
(G11021).
U86-0354
. Pharmacokinetics of recombinant tissue type plasminogen 
activator (0.25 mg/kg) after 10 minute intravenous infusion of 2 different 
formulations in healthy volunteers.
U87-0368
. Pharmacokinetics, pharmacodynamics and tolerance of a 30 
minute infusion of 0.25 mg/kg bodyweight and 0.50 mg/kg bodyweight of 
rt-PA in healthy volunteers.
U87-0935
. A pharmacokinetic and safety 
study of recombinant human tissue type plasminogen activator in human 
volunteers.
U91-0823
. Pharmacokinetics, systemic effects and 
tolerance of a 30 minute infusion of Actilyse® derived from two different 
manufacturing processes (current and modified) at a dose of 0.25 mg/kg 
body weight in healthy volunteers. Phase I: Protocol IP 135.57. 08 May 
1991
U94-2138
. Pharmacokinetics, systemic effects 
and tolerability of a 30 minute infusion of rt-PA, derived from two different 
manufacturing processes (current and modified) at a dose of 0.25 mg/kg 
body weight in healthy volunteers. Phase I: Clinical Trial Report IP 135.67. 
02 August 1994


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10
APPENDICES
10.1
INSTRUCTIONS FOR RECONSTITUTING ALTEPLASE
According to this protocol alteplase should be reconstituted within 30 min prior to the 
intended start of infusion. 
Reconstitution should be done according to the following steps taken from the German 
SMPC (Summary of Product Characteristics) [R20-0187]:
1. Entfernen Sie die Schutzkappe der Durchstechflasche mit dem Wasser für 
Injektionszwecke bzw. der Durchstechflasche mit der Alteplase-Trockensubstanz, 
indem Sie sie mit dem Daumen nach oben drücken.
2. Wischen Sie die Gummistopfen der Durchstechflaschen mit einem Alkoholtupfer ab.
3. Nehmen Sie die Überleitungskanüle aus ihrer Verpackung. Die Überleitungskanüle ist 
steril und muss nicht nochmals desinfiziert oder sterilisiert werden. Nehmen Sie 
zunächst nur eine Schutzkappe ab.
4. Stellen Sie die Durchstechflasche mit dem Wasser für Injektionszwecke aufrecht auf 
eine stabile Fläche. Stechen Sie von oben vorsichtig aber fest mit der 
Überleitungskanüle vertikal durch die Mitte des Gummistopfens, ohne die Kanüle zu 
drehen.
5. Halten Sie die Durchstechflasche mit Wasser für Injektionszwecke und die 
Überleitungskanüle mit einer Hand an den beiden seitlichen Griffplatten fest. Nehmen 
Sie die zweite Schutzkappe von der Überleitungskanüle ab.
6. Halten Sie die Durchstechflasche mit dem Wasser für Injektionszwecke und die 
Überleitungskanüle mit einer Hand an den beiden seitlichen Griffplatten fest. Halten 
Sie die Durchstechflasche mit der Alteplase-Trockensubstanz über die 
Überleitungskanüle und platzieren Sie die Spitze der Kanüle direkt in der Mitte des 
Gummistopfens. 
Drücken Sie die Durchstechflasche mit der Trockensubstanz von oben auf die 
Überleitungskanüle und durchstechen Sie vorsichtig aber fest vertikal den 
Gummistopfen, ohne zu drehen.
7. Drehen Sie die beiden Durchstechflaschen um, sodass das gesamte Wasser in die 
Durchstechflasche mit der Trockensubstanz fließen kann.
8. Ziehen Sie die leere Durchstechflasche zusammen mit der Überleitungskanüle ab und 
entsorgen Sie diese.
9. Schwenken Sie die Durchstechflasche mit der gebrauchsfertig zubereiteten Alteplase-
Lösung vorsichtig, bis das restliche Pulver vollständig gelöst ist. Nicht schütteln, um 


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Schaumbildung zu vermeiden. Falls sich Schaum gebildet hat, sollte die Lösung 
einige Minuten lang stehen gelassen werden, damit sich die Blasen auflösen können.
10. Die Lösung enthält 1 mg/ml Alteplase. Sie sollte klar und farblos bis leicht gelblich 
sein und keine Partikel enthalten.
11. Entnehmen Sie die benötigte Menge mit Nadel und Spritze. Um ein Auslaufen zu 
vermeiden, sollte nicht die Einstichstelle der Überleitungskanüle verwendet werden.


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11
DESCRIPTION OF GLOBAL AMENDMENT(S)
11.1
GLOBAL AMENDMENT 1
Date of amendment
02 April 2020
EudraCT number
EU number
2019-004932-40
BI Trial number
0135-0340
BI Investigational Medicinal 
Product(s)
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 
mg/vial)
Title of protocol
Bioequivalence of alteplase derived from two different 
manufacturing processes following intravenous 
administration in healthy male volunteers
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
To be implemented immediately in order to eliminate hazard – IRB / IEC / 
Competent Authority to be notified of change with request for approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
1. Section 1.3
2. Section 3.1
3. Section 3.3.3
4. Section 3.3.4.3 and Section 7.4
5. Section 5.2.6.2.1
6. Section 6.2.3
7. Section 8
Description of change
1.Preclinical data have been added.
2.Precise duration of trial participation for a single 
subject has been added; a schematic diagram of the 
trial design has been added
3. Exclusion criterion 10 was added by drugs that 
may pose a potential safety risk to a subject
4. Criteria for trial discontinuation have been added
5. Reporting rules for AEs after a subject`s end of 
trial have been changed
6. Time frame of EOT visit has been added.
7. A reporting rule (according to §13 GCP-V) has 
been added.
Rationale for change
1.-7. Requested by Competent Authority.


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11.2
GLOBAL AMENDMENT 2
Date of amendment
08 Jun 2020
EudraCT number
EU number
2019-004932-40
BI Trial number
0135-0340
BI Investigational Medicinal 
Product(s)
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 
mg/vial)
Title of protocol
Bioequivalence of alteplase derived from two different 
manufacturing processes following intravenous 
administration in healthy male volunteers
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
To be implemented immediately in order to eliminate hazard – IRB / IEC / 
Competent Authority to be notified of change with request for approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
Section 5.3.2.1 and 5.3.2.1
Description of change
Details of sample processing (e.g. incubation time) 
had to be added
Rationale for change
Advice from bioanalyst


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11.3
GLOBAL AMENDMENT 3
Date of amendment
22 Jul 2020
EudraCT number
EU number
2019-004932-40
BI Trial number
0135-0340
BI Investigational Medicinal 
Product(s)
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 
mg/vial)
Title of protocol
Bioequivalence of alteplase derived from two different 
manufacturing processes following intravenous 
administration in healthy male volunteers
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
To be implemented immediately in order to eliminate possible hazard – IRB / 
IEC / Competent Authority to be notified of change with request for approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
Flow Chart, 5.2.3
Description of change
Lab panel was inserted to check additional 
coagulation parameters (D-Dimer, aPTT, PT/INR)
Rationale for change
In the light of 2 occluded venous catheters 
(occlusion occurred within 60 min after start of 
alteplase infusion) additional coagulation parameters 
should be measured to check, whether there is any 
systemic effect on coagulation. Coagulation
parameters are safety parameters – therefore this 
Amendment is regarded as substantial from a 
regulatory view point.


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11.4
GLOBAL AMENDMENT 4
Date of amendment
10 Aug 2020
EudraCT number
EU number
2019-004932-40
BI Trial number
0135-0340
BI Investigational Medicinal 
Product(s)
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 
mg/vial)
Title of protocol
Bioequivalence of alteplase derived from two differ-
rent manufacturing processes following intravenous 
administration in healthy male volunteers
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
To be implemented immediately in order to eliminate possible hazard – IRB / 
IEC / Competent Authority to be notified of change with request for approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
5.3.2
Description of change
PK- and PD-back-up samples of all subjects may be 
used to determine any lab parameters of coagulation 
(e.g. F1+2) and fibrinolytic (e.g. D-Dimer) cascade.
Rationale for change
To better understand earlier made study observations 
of increased D-dimer in the context of coagulation 
and fibrinolysis pathways.
The trial is kept on-hold. Clinical activities will only 
re-start after a new benefit-risk assessment has been 
approved by authorities.
In the light of this situation it has been decided to 
close stage 1 and to start with interim analysis. 
Statistical hypotheses, models and analyses remain 
unchanged. In particular, the alpha spending 
procedure as planned will be used, i.e. the alpha for 
the first stage is 0.03585.


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11.5
GLOBAL AMENDMENT 5
Date of amendment
18 December 2020
EudraCT number
EU number
2019-004932-40
BI Trial number
0135-0340
BI Investigational Medicinal 
Product(s)
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 
mg/vial)
Title of protocol
Bioequivalence of alteplase derived from two differ-
rent manufacturing processes following intravenous 
administration in healthy male volunteers
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
To be implemented immediately in order to eliminate possible hazard – IRB / 
IEC / Competent Authority to be notified of change with request for approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
1.) Synopsis, 3.1, 7.1, 7.2, 7.3
2.) Flow Chart, 2.2.1, 2.2.2.2, 2.2.2.3
3.) 1.2.2
4.) 1.2.5
5.) 1.3, 1.5.3.2 and 1.5.3.3
6.) 1.5.3.1
7.) 1.4.4
8.) 3.1
9.) 3.3.3, 5.2.3
10.)
3.3.4.3
11.)
4.1.1, 4.1.2
12.)
5.3.1
13.)
5.3.2.3, 5.3.3.3, 5.3.3.4 
14.)
7.3.2 and 7.5.2, 9.2
15.)
7.4
16.)
8.7
Description of change
1.) Trial is splitted in Part A and B. Total 
number of subjects is increased to 72. 
Heparin will be given prior to start of 
alteplase infusion.
2.) More biomarker added
more 
measurement time points for biomarker


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3.) New chapter inserted
4.) New chapter inserted
5.) New chapters inserted
6.) Passage on risk of thromboembolic 
complication added
7.) Update of overall assessment for Part B
8.) Safety review meeting after first 2 cohorts
9.) Adaptation of inclusion criteria and lab 
parameters to be tested at screening
10.)
New stopping criteria have been 
added (clinically relevant activation of 
coagulation, non-reliable PK-data)
11.)
Information to heparin added
12.)
Cannulas will be flushed with heparin 
solution.
13.)
To describe sample handling for 
measurement of biomarkers.
14.)
Reference to SOP deleted.
15.)
A blinded review of PK data will be 
performed after the first 2 cohorts. 
Biomarkers can be assessed at any time.
16.)
New lab for biomarkers added.
Rationale for change
1.) PK-data of Part A (N=12) do not reflect the 
concentration-time profile of an iv-infusion. 
They are not reliable and can`t be used for 
assessment of bioequivalence. Heparin will 
be given to address increased coagulation 
observed in Part A.  
2.) To describe potential effects on plasmin 
formation, coagulation and platelet function; 
to review the safety of the subjects
3.) Background information to explain activation 
of coagulation by thrombolytic drugs
4.) Description and interpretation of results of 
Part A (why the trial was stopped after 12 
subjects). Consequences for part B.
5.) To provide drug profile of heparin and to 
describe the risks associated with the use of 
heparin (+ alteplase) in healthy subjects
6.) Based on the observations in Part A and the 
background information provided in 1.2.2 the 
risk of thromboembolic complications and 
measurements to address it are described. 
This includes the use of i.v. heparin.
7.) To include procoagulatory effects observed 


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in Part A and the planned use of heparin to 
address this into the benefit-risk assessment. 
8.) To evaluate safety of subjects, assess 
potential effects of alteplase on coagulation 
and to decide on trial continuation
9.) To cover thromboembolic risks of alteplase
10.)
Based on procoagulatory effects and 
methodological problems observed in Part A
11.)
Heparin will be given to address 
increased coagulation observed in Part A.
12.)
To prevent clotting in cannulas.
13.)
New biomarkers have been added.
14.)
SOP became invalid.
15.)
In case of non-reliable PK-data 
(observed in Part A) the trial will be stopped. 
Biomarker will be reviewed to assure 
subjects safety.
16.)
Extent of biomarkers was increased.


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11.6
GLOBAL AMENDMENT 6
Date of amendment
11 March 2021
EudraCT number
EU number
2019-004932-40
BI Trial number
0135-0340
BI Investigational Medicinal 
Product(s)
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 
mg/vial)
Title of protocol
Bioequivalence of alteplase derived from two differ-
rent manufacturing processes following intravenous 
administration in healthy male volunteers
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
To be implemented immediately in order to eliminate possible hazard – IRB / 
IEC / Competent Authority to be notified of change with request for approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
1. Flow Chart, Headline of table
2. Flow Chart
3. Flow Chart
4. Table 5.2.3:1
5. Section 5.3.1
6. Section 5.3.2.3
Description of change
1. Footnote 8 removed from vital signs.
2. Safety lab is moved from 1 h p.a. to 4 h p.a. 
3. Time window of ± 90 min was defined for 
tolerability assessment and AE questioning at 
planned time 10:45 after start of infusion.
4. Protein C (photometry) changed to Protein C.
5. After last PK-sampling (6 h p.a.) the cannula 
will not be flushed with heparin/saline.
6. Indomethacin should be added to aliquots for 
measurement of platelet parameters.
Rationale for change
1. Footnote 8 does not refer to vital signs.
2. Safety lab at 4 h p.a. better fits with study 
conditions of Part B (including heparin 
administration). The results are available in 
the evening of the same day (unchanged). 
Thus, the safety of subjects is not affected.
3. To allow that tolerability assessment and AE-
questioning can be performed together for all 
subjects of one cohort.


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Trial Protocol
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001-MCS-40-106-RD-13 (6.0) / Saved on: 13 Jun 2018
4. To be in line with BI naming of lab values.
5.
After last PK-sampling the cannula will be 
removed. Flushing does not make sense.
6.
To assure stability of thromboxane values.


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11.7
GLOBAL AMENDMENT 7
Date of amendment
14 April 2021
EudraCT number
EU number
2019-004932-40
BI Trial number
0135-0340
BI Investigational Medicinal 
Product(s)
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 
mg/vial)
Title of protocol
Bioequivalence of alteplase derived from two differ-
rent manufacturing processes following intravenous 
administration in healthy male volunteers
To be implemented only after approval of the IRB / IEC / Competent 
Authorities
To be implemented immediately in order to eliminate possible hazard – IRB / 
IEC / Competent Authority to be notified of change with request for approval
Can be implemented without IRB / IEC / Competent Authority approval as 
changes involve logistical or administrative aspects only
Section to be changed
1.) 5.2.3
2.) 5.3.2.3
Description of change
1.) A suitable test system may be used for urine 
stix
Rationale for change
1.) To allow more flexibility


 
 
 
 
APPROVAL / SIGNATURE PAGE 
 
Document Number: 
 
Technical Version Number:
 
Document Name: 
 
 
 
                                                                                                                 
 
Title: 
 
 
 
 
                                                                                                                   
 
Signatures (obtained electronically) 
 
Meaning of Signature  
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Date Signed 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
c30603221
8.0
clinical-trial-protocol-version-08
Bioequivalence of alteplase derived from two different manufacturing processes
following intravenous administration in healthy male volunteers
Author-Trial Clinical
Pharmacokineticist
14 Apr 2021 14:37 CEST
Approval-Therapeutic Area 
14 Apr 2021 14:52 CEST
Approval-Team Member Medical
Affairs
14 Apr 2021 14:57 CEST
Author-Clinical Trial Leader
14 Apr 2021 19:10 CEST
Author-Trial Statistician
15 Apr 2021 09:07 CEST
Verification-Paper Signature
Completion
16 Apr 2021 10:56 CEST


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c30603221
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SAP_001.pdf: 
TITLE PAGE
TRIAL STATISTICAL ANALYSIS PLAN
c32989625-02
BI Trial No.:
0135-0340
Title:
Bioequivalence of alteplase derived from two different 
manufacturing processes following intravenous administration in 
healthy male volunteers
Final Protocol (Revised Protocol (based on global amendment 1) 
(c30603221-02) ) 
Investigational
Product(s):
alteplase (TPA-05, 50 mg/vial and TPA-02, 50 mg/vial)
Responsible trial 
statistician(s):
Phone:
Fax:
Phone:
Fax:
Date of statistical 
analysis plan:
19 APR 2021 SIGNED
Version:
1
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reserved.
This document may not - in full or in part - be passed on, reproduced, published or otherwise used without prior written permission.


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1.
TABLE OF CONTENTS
TITLE PAGE ...........................................................................................................................1
1.
TABLE OF CONTENTS......................................................................................2
LIST OF TABLES...................................................................................................................4
2.
LIST OF ABBREVIATIONS ..............................................................................5
3.
INTRODUCTION.................................................................................................6
4.
CHANGES IN THE PLANNED ANALYSIS OF THE STUDY......................7
5.
ENDPOINTS .........................................................................................................8
5.1
PRIMARY ENDPOINT .......................................................................................8
5.2
SECONDARY ENDPOINTS...............................................................................8
6.
GENERAL ANALYSIS DEFINITIONS ..........................................................10
6.1
TREATMENTS...................................................................................................10
6.2
IMPORTANT PROTOCOL DEVIATIONS....................................................11
6.3
SUBJECT SETS ANALYSED...........................................................................12
6.5
POOLING OF CENTRES .................................................................................14
6.6
HANDLING OF MISSING DATA AND OUTLIERS....................................14
6.7
BASELINE, TIME WINDOWS AND CALCULATED VISITS ...................14
7.
PLANNED ANALYSIS......................................................................................16
7.1
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS.........17
7.2
CONCOMITANT DISEASES AND MEDICATION .....................................17
7.3
TREATMENT COMPLIANCE........................................................................18
7.4
PRIMARY ENDPOINT .....................................................................................18
7.4.1
Primary analysis of the primary endpoint........................................................18
7.5
SECONDARY ENDPOINTS.............................................................................21
7.5.1
Key secondary endpoint .....................................................................................21
7.5.2
Secondary endpoints...........................................................................................21
7.7
EXTENT OF EXPOSURE.................................................................................22
7.8
SAFETY ANALYSIS..........................................................................................22
7.8.1
Adverses Events...................................................................................................22
7.8.2
Laboratory data ..................................................................................................23
7.8.3
Vital signs.............................................................................................................24


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7.8.4
ECG......................................................................................................................24
7.8.5
Others...................................................................................................................24
8.
REFERENCES....................................................................................................25
9.
HISTORY TABLE..............................................................................................26


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LIST OF TABLES
Table 6.1: 1
Flow chart of analysis phases for statistical analyses of AEs, safety 
laboratory and vital signs .................................................................................10
Table 6.2: 1
Handling of iPDs..............................................................................................12
Table 10: 1
History table .....................................................................................................26


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2.
LIST OF ABBREVIATIONS
Term
Definition / description
ADME
Absorption, Distribution, Metabolism and Excretion
ALT
Alanine aminotransferase
AST
Aspartate aminotransferase
AUC0-∞
Area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity
AUC0-tz
Area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point
BI
Boehringer Ingelheim
Cmax
Maximum measured concentration of the analyte in plasma
CV
Arithmetic coefficient of variation
gCV
geometric coefficient of variation
gMean
Geometric mean
ICH
International Council for Harmonisation
PKS
Pharmacokinetic parameter analysis set
RAGe
Report appendix generator
SD
Standard Deviation
tmax
Time from (last) dosing to the maximum measured concentration of the 
analyte in plasma
TS
Treated set
ULN
Upper limit of normal range


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3.
INTRODUCTION
As per ICH E9 (1), the purpose of this document is to provide a more technical and detailed 
elaboration of the principal features of the analysis described in the revised CTP and to 
include detailed procedures for executing the statistical analysis of the primary and secondary 
variables and other data.
This TSAP assumes familiarity with the CTP and its amendments. In particular, the TSAP is 
based on the planned analysis specification as written in CTP Section 7 ‘Statistical Methods 
and Determination of Sample Size’. Therefore, TSAP readers may consult the revised CTP 
for more background information on the study, e.g., on study objectives, study design and 
population, treatments, definition of measurements and variables, planning of sample size, 
randomisation.
Study data will be stored in a trial database within the RAVE EDC system. 
The statistical analyses will be performed within the validated working environment CARE, 
including SASTM (current Version 9.4 or higher, by 
), and 
a number of SASTM-based tools (e.g., macros for the analyses of AE data or laboratory data; 
Report Appendix Generator system (RAGe) for compilation/formatting of the CTR 
appendices). 
The trial is kept on-hold. Clinical activities will only re-start after a new benefit-risk 
assessment has been approved by authorities.
Part A of this trial has been terminated after 12 subjects due to procoagulant effects that 
interfered also with alteplase analytics. The PK-data could not be used for hypothesis testing 
and hence the data was only analysed descriptively.
After implementation of measures to mitigate the risk of thromboembolic complications and 
to avoid methodological problems with alteplase analyticsPart B will be performed following 
a 2-stage design.
The establishment of bioequivalence will be only based on data of Part B (stage 1 and 2).


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4.
CHANGES IN THE PLANNED ANALYSIS OF THE STUDY
All analyses described in this TSAP are in accordance with the statistical methods planned in 
the CTP.


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5.
ENDPOINTS
5.1
PRIMARY ENDPOINT
The following pharmacokinetic parameters will be determined for TPA-02 and TPA-05:

AUC0-tz (area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 to the last quantifiable data point)

Cmax (maximum measured concentration of the analyte in plasma)
5.2
SECONDARY ENDPOINTS
The following pharmacokinetic parameter will be determined for TPA-02 and TPA-05:

AUC0-∞(area under the concentration-time curve of the analyte in plasma over the 
time interval from 0 extrapolated to infinity)(AUC0-inf_pred and supportive AUC0-
inf_obs)


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6.
GENERAL ANALYSIS DEFINITIONS
6.1
TREATMENTS
For basic study information on treatments to be administered, assignment of treatment 
groups, and selection of doses, cf. Section 4 of the CTP.
All subjects will receive the 2 treatments (TPA-02 and TPA-05) in randomised order at 2 
consecutive days. The specific alteplase dose to be administered to a given subject will be 
calculated based on the body weight measured at screening examination. Prior to the first 
administration of trial medication, subjects will be assigned to treatment sequences T-R or R-
T.
For statistical analysis of AEs, safety laboratory data, vital signs and ECG, the following 
analysis phases are defined for each subject:
Table 6.1: 1
Flow chart of analysis phases for statistical analyses of AEs, safety laboratory 
and vital signs 
Study analysis 
phase
Label
Start (inclusive)
End (exclusive)
Screening
Screening
Date of informed consent
Date/time of first administration 
of TPA-02/ TPA-05
On-treatment
TPA-02 
0.2 mg/kg body 
weight
Date/time of first administration 
of TPA-02 
Date/time of first administration 
of TPA-05 or 12:00 a.m. on day 
after subject's trial termination 
date, whichever occurs earlier
On-treatment
TPA-05 
0.2 mg/kg body 
weight
Date/time of first administration 
of TPA-05
Date/time of first administration 
of TPA-02 or 12:00 a.m. on day 
after subject's trial termination 
date, whichever occurs earlier
Analysis phases for statistical analysis of AEs are defined for each subject as described in the
table above. 
CTR Section 15, Appendix 16.1.13.1.8.2 and Appendix 16.1.13.1.8.3 AE displays will 
present results for the on-treatment phase and screening phase.
In CTR Section 15 AE tables (but not in Appendix 16.1.13.1.8.2 and Appendix 16.1.13.1.8.3 
AE tables), the following totals will be provided in addition:

"Total on-trt", defined as the total over all on-treatment phases involving BI
Safety laboratory data and vital signs will be analysed by treatment (TPA-02 or TPA-05).


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More details on the technical implementation of these analyses are provided in the ADS Plan 
of this TSAP.
6.2
IMPORTANT PROTOCOL DEVIATIONS
Consistency check listings (for identification of deviations from time windows) and a list of 
protocol deviations (e.g. deviations in drug administration, in blood sampling times, etc.) will 
be provided to be discussed at the Report Planning Meeting for the final analysis. At this 
meeting, it will be decided whether a discrepant data value can be used in analyses or 
whether it must be corrected in the clinical database. Each protocol deviation must be 
assessed to determine whether it is an important protocol deviation (IPD). For definition of 
IPDs, and for the process of identification of these, refer to the BI reference document 
"Identify and Manage Important Protocol Deviations (IPD)" (2).
For the interim analysis a meeting to discuss deviations related to PK will be conducted and 
the decisions will be documented in a decision log. 
If any IPDs are identified, they are to be summarised into categories and will be captured in 
the decision log. Categories which are considered to be IPDs in this trial are defined in the 
integrated quality and risk management plan (IQRMP).
IPDs will be summarized and listed. Table 6.2: 1 below specifies which kind of IPDs could 
potentially lead to exclusion from which analysis set. The decision on exclusion of subjects 
from analysis sets will be made at the latest at the Report Planning Meeting, after discussion 
of exceptional cases and implications for analyses. If the data show other IPDs, this table will 
be supplemented accordingly by the time of the Report Planning Meeting.


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Table 6.2: 1
Handling of iPDs
iPD code
iPD Category & Brief Description
Excluded from which 
analysis set
A1
Inclusion Criteria violated
PKS
A2
Exclusion Criteria violated
PKS
B1
Informed consent not available/not done
TS, PKS
B2
Informed consent too late
None
C1
Incorrect trial medication intake
PKS
C2
Randomisation not followed 
PKS
C3
Non-compliance 
PKS
C4
Medication code broken inappropriately
PKS
C5
Incorrect intake of trial medication
PKS
C6
Improper washout between treatments
PKS
D1
Prohibited medication use
PKS
D2
Mandatory medication not taken
PKS
D3
Improper washout of concomitant medication
PKS
E1
Certain violations of procedures used to measure 
primary or secondary data
PKS
F1
Certain violations of time schedule used to 
measure primary or secondary data
PKS
G1
Incorrect intake of meal
PKS
6.3
SUBJECT SETS ANALYSED
Subject sets will be used as defined in the Section 7.3 of the CTP:
Part A:

Treated set - part A (TS-A): The treated set includes all subjects who were 
randomized and treated with at least one dose of study drug. The treated set will be 
used for safety analyses.
Pharmacokinetic parameter analysis set - part A (PKS-A): This set includes all 
subjects in the treated set - part A (TS-A) who provide at least one PK endpoint that 
was defined as primary or secondary and was not excluded due to a protocol 
violation relevant to the evaluation of PK or due to PK non-evaluability (as specified 
in the following subsection ‘Pharmacokinetics’). Thus, a subject will be included in 
the PKS, even if he/she contributes only one PK parameter value for one period to the 


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statistical assessment. Descriptive and model based analyses of PK parameters will 
be based on the PKS.
Part B (stage I and II):

Treated set - part B (TS-B): The treated set includes all subjects who were 
randomized and treated with at least one dose of study drug. The treated set will be 
used for safety analyses.
Pharmacokinetic parameter analysis set - part B(PKS-B): This set includes all 
subjects in the treated set - part B (TS-B) who provide at least one PK endpoint that 
was defined as primary or secondary and was not excluded due to a protocol 
violation relevant to the evaluation of PK or due to PK non-evaluability (as specified 
in the following subsection ‘Pharmacokinetics’). Thus, a subject will be included in 
the PKS, even if he/she contributes only one PK parameter value for one period to the 
statistical assessment. Descriptive and model based analyses of PK parameters will 
be based on the PKS.
Table 6.3: 1
Subject sets analysed
Part A:
Subject set
Class of endpoint
TS-A
PKS-A
Disposition
X
IPDs
X
Primary endpoints
X
Secondary endpoints
X
Further PK endpoints
X
Safety parameters 
X
Demographic/baseline 
conditions 
X
Exposure
X
Part B:


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Subject set
Class of endpoint
TS-B
PKS-B
Disposition
X
IPDs
X
Primary endpoints
X
Secondary endpoints
X
Further PK endpoints
X
Safety parameters 
X
Demographic/baseline 
conditions 
X
Exposure
X
6.5
POOLING OF CENTRES
This section is not applicable because the trial is performed in only one centre.
6.6
HANDLING OF MISSING DATA AND OUTLIERS
Data of screened subjects who were withdrawn from the trial prior to first administration of 
any study drug will not be reported in the CTR.
Data of subjects who failed to complete all periods of the study (dropouts or withdrawals) 
will be reported in the CTR as far as their data are available. All withdrawals will be 
documented and the reason for withdrawal reported in the CTR.
CTP Section 7.5.1: It is not planned to impute missing values for safety parameters.
One exception where imputation might be necessary for safety evaluation are AE dates. 
Missing or incomplete AE dates are imputed according to BI standards (3).
Missing data and outliers of PK data are handled according to BI standards (4) and (5).
CTP Section 7.5.2: PK parameters that cannot be reasonably calculated based on the 
available drug concentration-time data will not be imputed.
6.7
BASELINE, TIME WINDOWS AND CALCULATED VISITS
For all parameters, the last non-missing value determined prior to first dosing of the trial 
medication of the respective treatment period will be defined as baseline. 


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For plasma concentrations:
Individual endogenous TPA will be determined for each period as a mean of two 
measurements at baseline. Individual baseline levels will then be subtracted from all 
concentration measurements in that treatment period. In case the resulting concentration 
value is negative, it will be set to zero.
Time windows are defined in Section 6.1 of the CTP. Adherence to time windows will be 
checked at the RPM.


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7.
PLANNED ANALYSIS
The analyses for endpoint apply for both study parts Part A and Part B. However, since the 
data of Part A could not be used for a formal statistical assessment all analyses in Part A are 
only considered to be descriptive.
The data from Part A will be analyses separately and will not contribute to the bioequivalence 
assessment.
The format of the listings and tables will follow the BI guideline "Standards for Reporting of 
clinical trials and project summaries" (6).
The individual values of all subjects will be listed. Listings will be sorted by treatment 
group/sequence, subject number and visit (if visit is applicable in the respective listing). AE 
listings will be sorted by assigned treatment (see Section 7.8.1 below for details). The listings 
will be contained in Appendix 16.2 (SDL) of the CTR.
The following standard descriptive statistical parameters will be displayed in summary tables 
of continuous variables:
N 
number of non-missing observations
Mean 
arithmetic mean
SD 
standard deviation
Min 
minimum
Median 
median
Max 
maximum
For plasma concentrations as well as for all PK parameters the following descriptive statistics 
will additionally be calculated:
CV 
arithmetic coefficient of variation
gMean
geometric mean
gCV
geometric coefficient of variation
For PK parameters the following descriptive statistics will additionally be calculated:
P10
10th percentile
Q1
1st quartile
Q3
3rd quartile
P90
90th percentile
The data format for descriptive statistics of plasma concentrations will be identical with the 
data format of the respective concentrations. The descriptive statistics of PK parameters will 
be calculated using the individual values with the number of decimal places as provided by 
the evaluation program. Then the individual values as well as the descriptive statistics will be 
reported with three significant digits in the CTR.
Tabulations of frequencies for categorical data will include all possible categories and will 
display the number of observations in a category as well as the percentage (%) relative to the 


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respective treatment group. Percentages will be rounded to one decimal place. The category 
missing will be displayed if and only if there actually are missing values. Percentages will be 
based on all subjects in the respective subject set whether they have non-missing values or 
not.
Exclusion of PK parameters
The ADS ADPP contains column variables APEXC and APEXCO indicating 
inclusion/exclusion (APEXC) of a PK parameter and an analysis flag comment (APEXCO). 
All analyses based on the PKS are based on PK parameter values which are not flagged for 
exclusion, i.e. with APEXC equal to "Included" (see protocol Section 7.3).
Exclusion of plasma concentrations
The ADS ADPC (PK concentrations per time-point or per time-interval) contains column 
variables ACEXC or ACEXCO indicating inclusion/exclusion (ACEXC) of a concentration 
and an analysis flag comment (ACEXCO). Exclusion of a concentration depends on the 
analysis flag comment ACEXCO. For example, if ACEXCO is set to "ALL CALC", the 
value will be excluded for all types of analyses based on concentrations. If ACEXCO is set to 
"DESC STATS" the value will be excluded from descriptive evaluations per planned time 
point/time interval. If ACEXCO contains the addition "TIME VIOLATION" or "TIME 
DEVIATION", the value can be used for further analyses based on actual times. If ACEXCO 
is set to "HALF LIFE", the value will be excluded from half-life calculation only; the value is 
included for all other analyses. Excluded concentration itself will be listed in the CTR 
associated with an appropriate flag.
Further details are given in "Noncompartmental Pharmacokinetic / Pharmacodynamic 
Analyses of Clinical Studies"(4) and "Description of Analytical Transfer Files and PK/PD 
Data Files" (5)
7.1
DEMOGRAPHIC AND OTHER BASELINE CHARACTERISTICS
Only descriptive statistics are planned for this section of the CTR.
7.2
CONCOMITANT DISEASES AND MEDICATION
Concomitant diseases will be coded according to the most recent version of MedDRA. 
Concomitant medication will be coded according to the most recent version of the World 
Health Organisation – Drug Dictionary. Concomitant non-drug therapies will be coded 
according to the most recent version of MedDRA.
A medication will be considered concomitant, if it

is ongoing at the time of first study drug administration, or

starts within the analysis phase of the respective treatment (see Section 6.1 for a 
definition of treatments and analysis phases).


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The relevance of the concomitant therapies to the evaluation of PK will be decided no later 
than at the RPM.
Only descriptive statistics are planned for this section of the CTR.
7.3
TREATMENT COMPLIANCE
Treatment compliance will not be analyzed as a specific endpoint. Any deviations from 
complete intake will be addressed in the Report Planning Meeting (cf. Section 6.2) and 
described in the CTR.
7.4
PRIMARY ENDPOINT
7.4.1
Primary analysis of the primary endpoint
The statistical model used for the analysis of the primary endpoints for each stage of the trial 
will be an analysis of variance (ANOVA) model on the logarithmic scale as described in the 
CTP, section 7.3.1.
Each stage will be analyzed separately based on data from only this stage, see CTP Section
7.2.
Stage 1 analysis
After stage 1, an assessment for bioequivalence will be performed. Data of stage one will be 
analyzed with the statistical model described in the CTP, section 7.3.1.
CTP section 7.3.1 Point estimates for the ratios of the geometric means (test/reference) for 
the primary endpoints (see Section 2.1) and their two-sided confidence intervals (CIs) will be 
provided. At stage 1, the (1 – 21)*100% confidence interval for the gMean ratio will be 
calculated.
The alpha spent for the first stage is fixed to 0.03585 (one-sided), see CTP section 7.2.
The analysis of stage 1 data will be implemented using SAS PROC MIXED:
PROC MIXED DATA=pks_stage1;
  CLASS subject treatment(ref=”TPA-02”) sequence period;
  MODEL logkp = treatment sequence period subject / DDFM=KR;
  LSMEANS treatment / PDIFF CL ALPHA=0.0717;
RUN;
Bioequivalence is concluded, if the corresponding (adjusted) confidence intervals for the 
comparison of the primary and secondary (same model, see CTP section 7.5.2) endpoints are 
within the pre-specified boundaries 80.00% to 125.00% (original scale).


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1 A) If bioequivalence is shown, the trial will be closed after stage 1.
1 B) If bioequivalence is not shown for all primary and secondary endpoints after stage 1, 
then a decision regarding continuation of the trial will be made by the Principal Investigator, 
the Trial Statistician, and the Clinical Trial Lead or their respective designees.
As stated in the CTP, for all endpoints not meeting the bioequivalence criterion, the 
following calculations will be performed.
The power of the TOST procedure based on the first stage data will be calculated 
(using the planned treatment effect, the observed variability and the adjusted alpha 
level for stage 1). If this power is greater or equal to 0.9, the trial is recommended to 
stop for futility, cf. (11) Note that this futility rule is considered nonbinding. 
The SSR will be based on the conditional power as described in (11), i.e. it will use
the conditional error rates as well as the estimated conditional target power for the 
second stage. For the SSR it is planned to use the observed variability (gCV) and the 
observed geometric mean ratio (GMR) from the (unblinded) stage 1 data. Note that a 
specific power adjustment due to multiple endpoints will not be done and the sample 
size recalculation, if required, will be based on observed results from primary and 
secondary PK parameters. The calculation will be performed for each parameter and 
the highest sample size will be used. Note that the minimum sample size for stage 2 is 
set to 4, as done and recommended in (11). Moreover, for this study a maximum 
overall sample size of 60 will be considered.
This step may be implemented using function interim.tsd.in from the R package 
Power2Stage:
interim.tsd.in(weight = 0.8, max.comb.test = FALSE, targetpower = 
0.9, 
               GMR1 = GMR1, CV1 = CV1, n1 = n1, , 
               GMR = 1.09, usePE = TRUE, max.n = 60, fCrit = "No")
For GMR1 and CV1 it is planned to use the observed results from stage 1, i.e. the 
gMean ratio and gCV as derived from the above proc mixed call. Similarly, df1 and 
SEM1 are the degrees of freedom and standard error of the difference of LSmeans, 
respectively, and are to be taken (on the log-scale) from the same proc mixed call. n1 
is the number of subjects in the PKS from stage 1,
Note that the weight of 0.8 may be adjusted as described in the CTP Section 7.2.
If applicable, Stage 2 analysis
Step 2 A)
Data of stage two will be analyzed with the same statistical model described in the CTP, 
section 7.3.1 using in principle the same SAS dummy code as described above in Section 


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7.4.1 of the TSAP. Note again that the input data for the proc mixed call will be PKS data 
from stage 2 only.
PROC MIXED DATA=pks_stage2;
  CLASS subject treatment(ref=”TPA-02”) sequence period;
  MODEL logkp = treatment sequence period subject / DDFM=KR;
  LSMEANS treatment / PDIFF CL ALPHA=2*alpha2;
RUN;
where alpha2 is planned to be 0.03585 and may be adjusted based in accordance with the 
weight.
Step 2 B)
Study success will then be determined based on the standard inverse normal combination test.
CTP section 7.2 If the study continues to the second stage, let p2j denote the two p-values of 
the second stage, j = 1, 2. Note that p2j are obtained using only the data from stage 2. BE is 
concluded (i.e. the null hypothesis is rejected) after the second stage if the combination of the 
p-values p1j and p2j (j=1, 2) is greater than the critical boundary c2 for the final assessment, 
cf. (10) and (11):
√	1 − + √1 −	1 − ≥,
where j = 1, 2 and j again refers to the j-th hypothesis of the TOST procedure and  refers to 
the cumulative distribution function of the standard normal distribution.
Note that 2  and the critical value , will possibly adapted according the user-defined alpha 
spending function as described in the CTP, section 7.2.
CTP section 7.3.1. If the study continues to stage 2, the repeated confidence interval 
corresponding to the test decision will be calculated, in conjunction with the median 
unbiased point estimate as estimate for the final adjusted geometric mean ratio. For details 
see (12).
Bioequivalence is concluded, if the corresponding (repeated) confidence intervals for the 
comparison of the primary and secondary endpoints (that did not show bioequivalence in the 
first stage) are within the pre-specified boundaries 80.00% to 125.00% (original scale).
This step B) may be implemented using function final.tsd.in from the R package 
Power2Stage:
final.tsd.in(weight = 0.8, max.comb.test = FALSE,
             GMR1 = GMR1, CV1 = CV1, n1 = n1, 
             GMR2 = GMR2, CV2 = CV2, n2 = n2)
where GMR1, CV1, n1 are the same values as described above and GMR2, CV2, n2are the 
corresponding values obtained from stage 2 data only, i.e. from the proc mixed call in Step 2 
A).


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7.5
SECONDARY ENDPOINTS
7.5.1
Key secondary endpoint
Not applicable.
7.5.2
Secondary endpoints
The analysis of secondary endpoints will be based on the PKS.
The secondary endpoints will be assessed statistically using the same methods as described 
for the primary analysis of the primary endpoints.


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7.7
EXTENT OF EXPOSURE
Descriptive statistics are planned for this section of the report.
7.8
SAFETY ANALYSIS
All safety analyses will be performed on the TS.
7.8.1
Adverses Events
AEs will be coded with the most recent version of MedDRA. The Residual Effect Period 
(REP) of alteplase is 24 hours (i.e. 23.5h after end of infusion) in this trial, see CTP Section 
1.2.3.
The analyses of AEs will be descriptive in nature. All analyses of AEs will be based on the 
number of subjects with AEs and not on the number of AEs.
For further details on summarization of AE data, please refer to "Analysis and Presentation of 
Adverse Event Data from Clinical Trials" (7) and "Handling of missing and incomplete AE 
dates" (3). 
The analysis of AEs will be based on the concept of treatment emergent AEs. That means 
that all AEs will be assigned to screening or on-treatment phases as defined in Section 6.1. 
AEs will be analysed based on actual treatments, as defined in Table 6.1: 1.
An overall summary of AEs will be presented. This overall summary will comprise summary 
statistics for the class of other significant AEs according to ICH E3 (8) and for the class of 
AESIs.
CTP Section 5.2.6.1.4: The following are considered as AESIs:

Hepatic injury
A hepatic injury is defined by the following alterations of hepatic laboratory 
parameters:
○
An elevation of AST (aspartate transaminase) and/or ALT (alanine 
transaminase) ≥3-fold ULN combined with an elevation of total bilirubin ≥2-
fold ULN measured in the same blood sample, or 


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○
Aminotransferase (ALT, and/or AST) elevations ≥10 fold ULN
The investigator had to classify on the eCRF whether an observed AE was an AESI or not.
According to ICH E3 (8) AEs classified as "other significant" need to be reported and will 
include those non-serious and non-significant AEs
(i) which are marked haematological or other lab abnormalities, or
(ii) which were reported with "action taken = discontinuation" or "action taken = reduced", or
(iii) which lead to significant concomitant therapy as identified by the Clinical 
Monitor/Investigator at a Medical Quality Review Meeting. 
The frequency of subjects with AEs will be summarised by treatment, primary SOC and 
preferred term. AEs which were considered by the investigator to be drug related will be 
summarised separately. Separate tables will also be provided for subjects with SAEs, subjects 
with AESIs and subjects with other significant AEs (according to ICH E3 (8)). AEs will also 
be summarized by maximum intensity.
The SOCs and preferred terms within SOCs will be sorted by descending frequency over all 
treatment groups.
For disclosure of AE data typically intended for ClinicalTrials.gov, the frequency of subjects 
with non-serious AEs occurring with an incidence of greater than 5 % (in preferred terms) 
will be summarised by treatment, primary SOC and preferred term. The frequency of subjects 
with SAEs will also be summarised.
For disclosure of AE data in the EudraCT register, the frequency of AEs, the frequency of 
non-serious AEs with an incidence of greater than 5 % (in preferred terms) and the frequency 
of SAEs will be summarized.
For support of lay summaries, the frequency of subjects with drug-related SAEs will be 
summarized by treatment, primary SOC and preferred term.
7.8.2
Laboratory data
The analyses of laboratory data will be descriptive in nature and will be based on BI 
standards "Display and Analysis of Laboratory Data" (9).
Analyses will be based on normalised values, which mean transforming to a standard unit and 
a standard reference range. The original values will be analysed if the transformation into 
standard unit is not possible for a parameter.
Descriptive statistics of absolute values and change from baseline from laboratory parameters
over time (see Section 6.7) will be provided. Frequency tables of changes between baseline 
and last value on treatment with respect to the reference range will be presented.
Individual line plots as well as plots on aggregated data presenting median with Q1 and Q3 as 
error bars will be provided for plasminogen and α2-antiplasmin.


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Unscheduled measurements of laboratory data will be assumed to be repeat measurements of 
the most recent scheduled measurement (e.g. for follow-up or confirmation of a particular 
value). Therefore, unscheduled measurements will be assigned to the planned time point of 
the previous scheduled measurement. Descriptive statistics will be calculated by planned time 
point based on the worst value of the subject at that planned time point (or assigned to that 
planned time point).
Possibly clinically significant abnormal laboratory values are only those identified either in 
the Investigator's comments or at the RPM at the latest. It is the Investigator's responsibility 
to decide whether a lab value is clinically significant abnormal or not. Standard or project-
specific rules for flagging clinically significant values in an automated manner will not be 
applied in this study.
Clinically relevant findings in laboratory data will be reported as baseline conditions (prior to 
first administration of study treatment) or as AEs (after first administration of study 
treatment) if judged clinically relevant by the investigator, and will be analyzed as such.
7.8.3
Vital signs
The analyses of vital signs (blood pressure and pulse rate) will be descriptive in nature. 
Descriptive statistics of absolute values and change from baseline from vital signs over time 
(see Section 6.7) will be provided.
Unscheduled measurements of vital signs will be assigned to planned time points in the same 
way as described above for laboratory data. However, for vital signs, descriptive statistics 
will be calculated by planned time point based on the last value of the subject at that planned 
time point (or assigned to that planned time point).
Clinically relevant findings in vital signs data will be reported as baseline conditions (prior to 
first administration of study treatment) or as AEs (after first administration of study 
treatment) if judged clinically relevant by the investigator, and will be analyzed as such.
7.8.4
ECG
Abnormal findings will be reported as baseline conditions (prior to first study drug 
administration) or as AEs (from first study drug administration onwards) if judged clinically 
relevant by the investigator. 
7.8.5
Others
Physical examination findings will be reported as relevant medical history/baseline condition 
(if a condition already exists before first administration of study treatment) or as AE (if 
condition emerges after first administration of study treatment) and will be summarized as 
such. No separate listing or analysis of physical or neurological examination findings will be 
prepared.


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8.
REFERENCES
1
CPMP/ICH/363/96: "Statistical Principles for Clinical Trials", ICH Guideline 
Topic E9; Note For Guidance on Design, Conduct, Analysis and Evaluation of 
Clinical Trials, current version
2
001-MCS-40-413_1.0: "Identify and Manage Important Protocol Deviations 
(iPD)", current version; IDEA for CON
3
KM Asset BI-KMED-BDS-HTG-0035: "Handling of missing and incomplete AE 
dates", current version; KMED
4
001-MCS-36-472_RD-01: "Noncompartmental Pharmacokinetic / 
Pharmacodynamic Analyses of Clinical Studies", current version; IDEA for CON
5
001-MCS-36-472_RD-03: "Description of Analytical Transfer Files and PK/PD 
Data Files", current version; IDEA for CON
6
KM Asset BI-KMED-BDS-HTG-0045: "Standards for Reporting of Clinical Trials 
and Project Summaries", current version; KMED
7
KM Asset BI-KMED-BDS-HTG-0066: "Analysis and Presentation of Adverse 
Event Data from Clinical Trials", current version; KMED
8
CPMP/ICH/137/95: "Structure and Content of Clinical Study Reports", ICH 
Guideline Topic E3; Note For Guidance on Structure and Content of Clinical Study 
Reports, current version
9
KM Asset BI-KMED-BDS-HTG-0042: "Display and Analysis of Laboratory Data", 
current version; KMED
10 Lehmacher W, Wassmer G. Adaptive sample size calculations in group sequential 
trials. Biometrics 55, 1286 – 1290 (1999). [R14-1197]
11 Maurer W, Jones B, Chen Y. Controlling the type 1 error rate in two-stage 
sequential designs when testing for average bioequivalence. Stat Med. 2018; 
37(10): 1587--1607. [R19-3175]
12 Wassmer G, Brannath W. Group Sequential and Confirmatory Adaptive Designs in 
Clinical Trials. Springer 2016. doi: 10.1007/9783319325620. [R20-2316]


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9.
HISTORY TABLE
Table 10: 1
History table
Version
Date
(DD-MMM-
YY)
Author
Sections 
changed
Brief description of change
1
14-AUG-2020
None
This is the final TSAP