Clinical trial NCT02876601
Conditions: Healthy Volunteers Endotoxemia
Keywords: Coagulation Inflammation Defibrotide Fibrinolysis
Sponsor: Bernd Jilma
Start date: 2017-04-18; Completion date: 2018-02-12
Enrollment: 20 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
Defibrotide is an anti-inflammatory and anti-coagulatory agent approved for treatment of veno-occlusive disease. Although it has been in clinical use for almost 30 years, the exact mechanism of action has never been fully elucidated. Thus, the effects of defibrotide will be investigated in the human endotoxemia model in order to gather further information on its actions.

Detailed description:
Defibrotide (DF) is a highly complex polydisperse mixture of single-stranded phosphodiester oligodeoxyribonucleotides derived from the controlled depolymerization of porcine intestinal mucosal DNA. The entire mode of action remains unknown. Its actions may be summarized to pro-fibrinolytic, anti-inflammatory and anti-coagulatory actions. To better define the mechanisms of Defibrotide the effects of the substance will be investigated in the well-established endotoxemia model. Sixteen healthy volunteers will be randomized to receive LPS±defibrotide/placebo and four subjects will be randomized to receive Placebo± defibrotide/placebo in a single center, randomized, double blind, placebo controlled, two-way crossover trial. Immediately after a 2h infusion of 6,25mg/kg bodyweight defibrotide or placebo a LPS bolus of 2ng/kg bodyweight will be infused. Analyses will be performed by blood sampling at pre-defined time-points.

Arms: arm 1: 2ng/kg lipopolysaccharide period I: 6.25mg/kg bodyweight defibrotide or placebo (0.9% sodium chloride) period II: vice versa

16 healthy volunteers will receive 2ng/kg bodyweight LPS plus Defibrotide or Placebo 4 healthy volunteers will receive 0.9% saline (NO LPS) plus Defibrotide or Placebo arm 2: 2ng/kg lipopolysaccharide period I: 6.25mg/kg bodyweight defibrotide or placebo (0.9% sodium chloride) period II: vice versa

16 healthy volunteers will receive 2ng/kg bodyweight LPS plus Defibrotide or Placebo 4 healthy volunteers will receive 0.9% saline (NO LPS) plus Defibrotide or Placebo arm 3: Placebo (0.9% sodium chloride) period I: 6.25mg/kg bodyweight defibrotide or placebo (0.9% sodium chloride) period II: vice versa

16 healthy volunteers will receive 2ng/kg bodyweight LPS plus Defibrotide or Placebo 4 healthy volunteers will receive 0.9% saline (NO LPS) plus Defibrotide or Placebo arm 4: Placebo (0.9% sodium chloride) period I: 6.25mg/kg bodyweight defibrotide or placebo (0.9% sodium chloride) period II: vice versa

16 healthy volunteers will receive 2ng/kg bodyweight LPS plus Defibrotide or Placebo 4 healthy volunteers will receive 0.9% saline (NO LPS) plus Defibrotide or Placebo
Interventions: intervention 1: Defibrotide intervention 2: Placebo (0.9% sodium chloride) intervention 3: Lipopolysaccharide intervention 4: Placebo (0.9% sodium chloride bolus)
Intervention descriptions: intervention 1: 6.25mg/kg bodyweight over 2h infusion intervention 2: (0.9% sodium chloride) infusion over 2h infusion intervention 3: bolus infusion of 2ng/kg bodyweight lps intervention 4: (0.9% sodium chloride) bolus infusion

Protocol documents:
Prot_SAP_000.pdf: 
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Defibrotide in the human endotoxemia model - an exploratory trial investigating 
the effects and the mechanisms of Defibrotide 
A randomized, double blind trial in healthy volunteers 
 
Eudract-Nr.:  
 
2016-001375-77 
Clinical Trials.gov:   
NCT02876601 
 
Principal Investigator:  
Bernd Jilma, Ao.Univ.Prof.Dr. 
 
 
 
 
Medical University of Vienna 
 
 
 
 
Department of Clinical Pharmacology 
 
 
 
 
Währinger Gürtel 18-20, 1090 Vienna 
 
Sponsor: 
 
 
Medical University of Vienna 
 
 
 
 
Department of Clinical Pharmacology 
 
 
 
 
Währinger Gürtel 18-20, 1090 Vienna 
 
 
 
 
 
 
 
 
 
 
 


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Synopsis: 
Title:   
 
Defibrotide in the human endotoxemia model 
Study Phase  
IV 
 
Background: 
 Defibrotide (DF) is approved for the use in sinusoidal obstruction 
syndrome (SOS), which mainly occurs after chemotherapy and after 
hematopoietic stem cell transplantation. Furthermore it seems to 
reduce the incidence of graft versus host disease (GVHD). Although 
effective in clinical trials, the mode of action is still not fully 
understood. The manifold effects of DF may be summarized as anti-
inflammatory, anti-coagulatory and profibrinolytic. However, in 
vivo data are scarce and no comprehensive data on the effects of DF 
in inflammatory states are available. This trial is designed to 
investigate the effects of DF in a human inflammation model using 
Lipopolysaccheride (LPS) as a stimulus.  
Additionally, we will investigate the effects of DF on coagulation 
tests ex-vivo. Eight to 16 healthy volunteers will be included in this 
ex-vivo spiking study and blood sampling will be performed.  
 
Trial hypothesis:  DF reduces the activation of coagulation, the inflammatory 
response and the endothelial activation caused by infusion of LPS in 
healthy volunteers.  
 
Trial objectives:  
The objectives of this trial are: 


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 To investigate the effects of DF on coagulation parameters 
and tests ex-vivo 
 To investigate the anti-coagulant effects of DF 
 To investigate the anti-inflammatory effects of DF 
 To investigate the effects of DF on endothelial activation 
 To investigate the safety of DF infusion in healthy volunteers 
 To investigate the effects of DF on thrombeolastometry ex 
vivo 
 To investigate LPS pharmacokinetics in plasma of healthy 
volunteers 
 
Study design:  
This trial will be conducted as a randomized, double-blind, 
crossover trial in 20 healthy volunteers. Sixteen healthy volunteers 
will be treated with an LPS bolus (2ng/kg bodyweight) and an 
infusion of 6.25mg/kg bodyweight DF or placebo. A washout period 
of 6 weeks will be planned between two study periods. 
Additionally, four healthy volunteers will receive a placebo instead 
of LPS and will be treated with DF or placebo to investigate the 
effects of DF in healthy volunteers without LPS as a stimulus.  
 
Additionally we will investigate the effects of DF on 
thrombelastometry ex vivo.  
 
Trial Products:  
Defibrotide (Defitelio, Jazz Pharmaceuticals), 6.25mg/kg 
bodyweight 
 
LPS: 2ng/kg bodyweight US Standard Reference LPS (E.coli) 


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Paracetamol: 500mg Paracetamol Genericon (Genericon Pharma) 
on demand to alleviate flu-like symptoms 
 
Placebo: 0,9% NaCl solution not distinguishable from the original 
products will be used 
 
Primary variable:  Prothrombin fragments F1+2 (F1+2) as a measure of thrombin 
generation and coagulation 
 
Secondary variables:  
 vWF antigen and activity 
 microparticle associated tissue factor 
 TNF-α levels  
 tPA 
 PAI-1 
 D-Dimer 
 Thrombin-Antithrombin-Complexes (TAT) 
 Circulating endothelial cells 
 E-Selectin 
 Thrombelastometry (clotting time, clot formation time, 
maximum lysis, etc.) 
 Proinflammatory exosome 
 Differential blood counts 
 Safety parameters 
 LPS concentration 
 


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Risks/Benefits: 
There will be no benefits for participating subjects. However, by 
gaining knowledge about DF and better defining its effects in acute 
inflammatory states treatment of patients may be improved. 
Furthermore, our results may generate hypotheses about the use of 
DF in other diseases.   
 
On the other hand use of DF in healthy volunteers may contain 
certain risks for participants. Exclusion criteria comprise 
pregnancy or breastfeeding, as well as intake of other medication 
beside contraceptives. Furthermore all participants will be 
required to perform effective contraception for the duration of the 
trial. However, these measures are precautions and due to a lack of 
information rather than known side effects. In studies, the side 
effect profile of DF was overall good. There were no reports of 
hypersensitivities or anaphylaxis in the marketing trials. However, 
in previously marketed DF formulations such reactions were 
reported and must therefore be considered. The most common side 
effects were hemorrhage, hypotension and coagulopathy. However, 
this may also be due to the selection of patients as they are typically 
severely thrombopenic, refractory to thrombocyte infusion and 
critically ill. Information about DF in healthy volunteers is scarce. 
Two studies are published investigating the effects of intravenous 
DF in healthy volunteers. No serious or severe side effects were 
reported.  
 
In the ex-vivo spiking study 17 ml blood will be drawn which 
should not impose any significant risk on participating subjects.  


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1. Index 
1. Index ................................................................................................................................................. 6 
2. Introduction ................................................................................................................................... 8 
2.1. Defibrotide ............................................................................................................................................ 8 
2.2. The human endotoxemia model .................................................................................................... 9 
2.3. Circulating endothelial cells ........................................................................................................ 10 
2.4. Rationale of the trial ....................................................................................................................... 10 
2.5. Hypotheses and Objectives. .......................................................................................................... 11 
3. Trial Design ................................................................................................................................. 11 
3.1. Flow Chart ........................................................................................................................................... 11 
3.2. Trial design and rationale ............................................................................................................. 12 
3.3. Medication, dose rationale and dosing schedule .................................................................. 12 
3.3.1. Defibrotide .................................................................................................................................................. 12 
3.3.2 LPS ................................................................................................................................................................... 13 
3.3.3 Timing ............................................................................................................................................................ 13 
3.3.4 Sodium Chloride solution ....................................................................................................................... 13 
3.3.5. Blinding of the medication ........................................................................................................ 14 
3.4. Trial Schedule .................................................................................................................................... 14 
4. Trial Population ........................................................................................................................ 14 
4.1. Trial Population................................................................................................................................ 14 
4.2. Withdrawal Criteria ........................................................................................................................ 15 
4.4. Subject Replacement ....................................................................................................................... 16 
5. Methods and Assessments ..................................................................................................... 16 
5.1. Visits and Procedures ..................................................................................................................... 16 
5.1.1.Ex-vivo spiking Trial ................................................................................................................................ 16 
5.1.2. Screening Visit ........................................................................................................................................... 16 
5.1.3. Period 1 Trial Day 1 ................................................................................................................................. 17 
5.1.4. Period 1 Trial Day 2 ................................................................................................................................. 18 
5.1.5. Period 2 Trial Day 1 ................................................................................................................................. 18 
5.1.6. Period 2 Trial Day 2 ................................................................................................................................. 19 
5.1.7. Final Visit ..................................................................................................................................................... 19 
5.2. Efficacy analysis ................................................................................................................................ 19 
5.2.1. Primary endpoint ..................................................................................................................................... 19 
5.2.2. Secondary endpoints ............................................................................................................................... 19 
5.2.3. Rationale of the chosen endpoints .................................................................................................... 20 
5.3. Safety Endpoints ............................................................................................................................... 22 
5.4. Blood Sampling schedule, tubes and volume ......................................................................... 22 
5.5. Preparations of samples ................................................................................................................ 23 
5.5.1. Thrombelastometry ................................................................................................................................ 23 
5.5.2. Coagulation specific and fibrinolytic parameters ....................................................................... 23 
5.5.3. Inflammatory biomarkers ..................................................................................................................... 24 
5.5.4. Endothelial biomarkers ......................................................................................................................... 24 
5.5.5. Differential blood counts ....................................................................................................................... 24 
5.5.6. Blood Chemistry ........................................................................................................................................ 24 
5.5.7 Global coagulation assays ...................................................................................................................... 25 


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5.5.8. Circulating endothelial cells ................................................................................................................. 25 
6. Trial Materials ............................................................................................................................ 26 
6.1. Trial Products .................................................................................................................................... 26 
6.2. Packaging and Labeling of trial products ................................................................................ 27 
6.3. Storage and Drug Accountability of Trial Product(s) ......................................................... 27 
6.4. Randomization and Blinding ....................................................................................................... 28 
6.5. Potential inconveniences and risks for the subjects ........................................................... 29 
6.5.1. Defibrotide .................................................................................................................................................. 29 
6.5.2. LPS .................................................................................................................................................................. 29 
6.5.3. Paracetamol ................................................................................................................................................ 30 
6.5.4. Blood sampling .......................................................................................................................................... 30 
6.5.5. Risk/benefit assessment ....................................................................................................................... 30 
6.6. Safety precautions ....................................................................................................................................... 31 
7. Concomitant Illness and Medication .................................................................................. 32 
7.1. Concomitant Medication during the Trial ............................................................................... 32 
7.2. Precautions/Overdosage .............................................................................................................. 32 
8. Adverse Events ........................................................................................................................... 32 
8.1. Definitions .......................................................................................................................................... 32 
8.1.1. Adverse Events .......................................................................................................................................... 32 
8.1.2. Clinical Laboratory Adverse Events .................................................................................................. 33 
8.1.3. Serious Adverse Events (SAE) ............................................................................................................. 33 
8.1.4. Non-Serious Adverse Event ................................................................................................................. 34 
8.1.5. Severity Assessment Definitions ........................................................................................................ 34 
8.1.6. Relationship to Trial Drug Assessment Definitions: .................................................................. 34 
8.1.7. Outcome Categories and Definitions: ............................................................................................... 34 
8.2. Collection, Recording and Reporting of Adverse Events.................................................... 35 
8.3. Follow-up of Adverse Events ........................................................................................................ 36 
8.4. Case record forms ............................................................................................................................ 36 
8.5. Rules for completing CRFs ............................................................................................................ 36 
8.6. Correction of CRFs ........................................................................................................................... 37 
8.7. CRF Flow .............................................................................................................................................. 37 
9. Monitoring procedures ........................................................................................................... 38 
10. Data Management ................................................................................................................... 38 
11. Statistical Considerations .................................................................................................... 39 
11.1. Sample Size Calculation ............................................................................................................... 39 
11.2. Methods ............................................................................................................................................. 39 
11.2.1 Efficacy Analysis ...................................................................................................................................... 39 
11.3. Safety Analysis ............................................................................................................................................ 40 
11.4. Missing, unused and spurious data ......................................................................................... 40 
12. Ethics ........................................................................................................................................... 41 
12.1. Informed Consent Form of trial subjects (AMG §38) ........................................................ 41 
12.2. Independent Ethics Committees (AMG §§ 40, 41) ............................................................. 41 
12.3. Regulatory Authorities ................................................................................................................ 42 
12.4. Insurance (AMG §32(2)) ............................................................................................................. 43 
12.5. Confidentiality ................................................................................................................................ 43 
13. Audits and Inspections ......................................................................................................... 43 
14. Publication policy ................................................................................................................... 43 
15. Premature Termination of the trial ................................................................................. 43 
16. Retention of Clinical Trial Documentation .................................................................... 44 


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17. References: ............................................................................................................................... 44 
 
2. Introduction 
2.1. Defibrotide 
 
Defibrotide (DF) is a highly complex polydisperse mixture of single-stranded 
phosphodiester oligodeoxyribonucleotides derived from the controlled 
depolymerization of porcine intestinal mucosal DNA1.  
 
The entire mode of action of DF remains unknown and it seems to induce multiple anti-
coagulatory, anti-inflammatory and profibrinolytic effects2. DF increases levels of 
prostaglandins3 and reduced thromboxane levels4. Furthermore, DF significantly and 
dose-dependently increased tissue plasminogen activator (tPA) antigen and activity, and 
dose-dependently decreased PAI-1 antigen in vitro using endothelial cells and 
lipopolysaccheride (LPS) as a stimulus5. Interestingly, only in microvascular endothelial 
cell cultures, DF decreased the LPS induced tissue factor (TF) antigen expression5. 
Infusion of DF in healthy volunteers caused a significant decrease of PAI-1 and 
significantly increased prostacyclin and tissue factor pathway inhibitor6. In healthy 
volunteers, combined infusion of DF and heparin significantly enhanced the effect of 
heparin alone and prolonged the activated partial thromboplastin time and increased 
antithrombin III levels7. Moreover, in a model using endothelial cells and sera from 
patients undergoing autologous stem cell transplantation treatment with DF reduced 
expression of von Willebrand factor8. DF was effective in reducing thrombosis in a 
placebo controlled trial after general surgery 9. In a malaria model using endothelial 


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cells incubated with plasmodium falciparum infected erythrocytes DF reduced factor X 
activation, which may be caused by reduced tissue factor expression of these cells10.  
 
DF is mainly used to treat veno-occlusive disease (VOD) of the liver after stem cell 
transplantation2. The current pathophysiological model of VOD suggests that high-dose 
chemotherapy causes endothelial injury with increased expression of TF and PAI-1 
leading to a prothrombotic, hypofibrinolytic state with extracellular, subendothelial 
deposition of fibrinogen and factor VIII/vWF11. These aggregates may occlude the small 
pores in the liver endothelium leading to obstruction of the venous outflow and portal 
hypertension12. It was demonstrated in multiple trials that DF reduces mortality in VOD 
and that the use of DF significantly reduces the risk of developing a VOD and therefore 
may be used as prophylaxis13.  
 
2.2. The human endotoxemia model 
 
The human endotoxemia model is well established and widely used to mimic systemic 
inflammatory disease states experimentally. The infusion of LPS (usually 1-4ng/kg 
bodyweight) leads to the release of proinflammatory and procoagulant mediators and 
consequently to increased parameters of inflammation, activation of coagulation, 
increased body temperature, hypotension and tachycardia3. LPS infusion leads to a 
systemic activation of inflammation and coagulation by increasing TF-mRNA, thrombin-
anthithrombin complexes, D-Dimer, prothrombin fragments F1+2 (F1+2), TNF-α, IL-6, 
PAI-1, TPA, E-Selectin, P-Selectin, vWF-antigen, ICAM, VCAM and soluble 
thrombomodulin14,15. Thrombelastometry, a point-of-care whole blood assay that 
evaluates the viscoelastic properties of whole blood during clot formation and clot lysis 
is also affected by LPS and shows a significant activation of coagulation, correlating with 


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F1+2 (r=-0,83) and increased clot lysis, which correlated well with tPA levels (r=0,82). 
Infusion of LPS increases levels of circulating endothelial cells several fold in healthy 
volunteers16. This goes along with an increase in other endothelial activation markers 
such as E-Selectin, thrombomodulin and vWF15.  
 
2.3. Circulating endothelial cells 
Circulating endothelial cells are a useful marker to monitor endothelial activation 
and/or damage17. Interestingly, levels of circulating endothelial cells increase after 
engraftment in patients with acute GVHD. On the contrary, in patients, who do not 
develop GVHD, after engraftment levels of circulating endothelial cells seem to 
decrease18. DF protects the endothelium from activation19. Moreover, pediatric patients 
after hematopoietic stem cell transplantation, who were treated with DF, had a lower 
incidence of developing acute GVHD than the control group20. However, the effect of DF 
on circulating endothelial cell levels as a marker of endothelial cell activation is not 
known16.  
 
2.4. Rationale of the trial 
There is a lack of knowledge on the concise mode of action of DF. Additionally, available 
data mainly stems from cell based studies. However, human in vivo data is currently 
lacking. This project will investigate the anti-inflammatory effects, anti-coagulatory 
effects and effects on the endothelial activation of DF in a well-established human model 
of acute, transient systemic inflammation in healthy volunteers. The effects of many 
other anti-coagulatory and anti-inflammatory drugs on LPS infusion have been 
investigated similarly 14,21-25. Thus, comparisons of DF with these substances may 
possibly be drawn and further hypotheses may be generated based on our data.  


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Additionally, the effects of DF on thrombelastography will be investigated and its 
potential use as a DF therapy-monitoring device will be examined. Therefore we will 
spike different doses of DF in whole blood of healthy volunteers ex-vivo.  
 
2.5. Hypotheses and Objectives.  
We hypothesize that DF infusion reduces the LPS induced activation of coagulation and 
the inflammatory response. Furthermore we hypothesize that DF infusion reduces the 
endothelial activation induced by LPS. Moreover we will investigate the influence of DF 
on thrombelastometry and the utility of this device for potential therapy monitoring of 
DF. Finally, the safety of DF infusion in healthy volunteers will be assessed.  
3. Trial Design 
3.1. Flow Chart 
 
 


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3.2. Trial design and rationale 
Before initiation of the trial we will investigate the effects of DF on thrombelastometry 
ex vivo. In this part blood samples will be drawn from a minimum of eight healthy 
volunteers and after ex-vivo spiking of blood samples with increasing doses of DF 
thrombelastometry will be performed. The effects of activators (i.e. recombinant tissue 
factor) on DF in thrombelastometry will be investigated. The number of participants 
may be increased to 16 after analysis of the results of the first eight subjects.  
 
This trial is designed as a randomized, double blind, crossover trial in 20 healthy 
volunteers. Sixteen healthy volunteers will be randomly assigned to receive LPS, 
whereas four healthy volunteers will be randomly assigned to the placebo group. In each 
group two treatment periods will be performed. In one treatment period all subjects will 
be treated with DF. In the other treatment period all subjects will be treated with 
placebo. A washout period of 6 weeks will be planned between both treatment periods 
to reduce the risk of any carry-over effects. Whether a subject starts with the placebo or 
the DF period will also be randomized.  
3.3. Medication, dose rationale and dosing schedule 
3.3.1. Defibrotide 
 
Defibrotide is a highly complex polydisperse mixture of single-stranded phosphodiester 
oligodeoxyribonucleotides that exerts anti-inflammatory and anti-coagulatory effects1. It 
is mainly used for treatment of the sinusoidal obstruction syndrome, also called veno-
occlusive disease (VOD)2. The approved dose of DF is 6.25mg/kg bodyweight every six 
hours. This sums up to daily doses of 25mg/kg bodyweight DF26. This dose is based on 
clinical data. In a trial investigating the effects of DF in patients with VOD treatment with 
DF was started at 10mg/kg bodyweight split into four daily doses and increased every 


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two to four days by 10mg/kg depending on tolerability and response. Most responses 
were observed at daily doses between 20 and 40mg/kg bodyweight. However, doses 
ranged between 10 and 60mg/kg bodyweight27. A phase II trial compared 25mg/kg 
bodyweight daily with 40mg/kg bodyweight daily and found no differences in clinical 
outcomes, except for a slightly higher bleeding rate in the higher dose group28. The FDA   
approved Defibrotide on March 30th 29. 
3.3.2 LPS 
In the human endotoxemia model  doses of 2-4ng/kg bodyweight are regularly used 30. 
WE will use an intravenous bolus of 2ng/kg bodyweight US Standard Reference E.coli 
LPS in this trial. This dose increases F1+2 reliably about eight- to tenfold31,32. Maximum 
effects in coagulatory parameters are usually detected two to six hours after infusion, 
whereas after 24 hours most levels have returned to the baseline. Moreover, in various 
performed trials this dose was demonstrated to be safe and well-tolerated30. In previous 
trials the dose of 2ng/kg bodyweight was better tolerated by subjects compared to 
4ng/kg bodyweight, without eliminating the activation of the coagulation system.  
3.3.3 Timing 
For this trial we chose to infuse 6.25mg/kg bodyweight over a period of two hours, 
which is the approved dose of this drug. The LPS bolus will be infused 1h after the start 
of the defibrotide infusion. We chose this dosing regimen based on the Tmax of DF of two 
hours, the T1/2 of 0.7 hours and the effects of LPS with a maximum approximately four 
hours after infusion of the bolus. 
3.3.4 Sodium Chloride solution 
All subjects will receive an intravenous infusion of 0,9% sodium chloride at a rate of 
100ml/h to maintain adequate hydration for healthy volunteers, as they will be kept in a 


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fasting state during the first four hours. This should prevent any interference of ingested 
food with any of the trial’s endpoints or side effects such as nausea. A light, fat–reduced 
meal may be served after four hours, when nausea is no longer encountered.  
 
3.3.5. Blinding of the medication 
DF, after dissolution, is a colorless fluid not distinguishable from sodium chloride 
solution. A pharmacist not otherwise involved in the conduct of the trial will prepare 
weight adjusted medication of infusion labeled with Defibrotide/Placebo or 
LPS/Placebo.  
 
 
3.4. Trial Schedule 
Ex-vivo spiking study 
 
 
April 2017 
 
Planned inclusion of the first subject  
May 2017 
Planned recruitment period 
 
6 weeks 
Planned completion of the last subject 
November 2017 
Planned completion of the trial report 
January 2018 
4. Trial Population 
4.1. Trial Population 
The trial will be performed in 20 healthy volunteers. Sixteen healthy volunteers will be 
randomly assigned to the LPS group receiving two doses of 2ng/kg LPS once with a 
placebo and once with DF. Four healthy volunteers will be randomized to the control 
group and will not receive any LPS but a placebo, again once with DF and once with a 
placebo. In- and exclusion criteria will comprise: 
Inclusion Criteria:  


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 >18 years of age 
 <90kg body weight 
 Normal findings in medical history and physical examination unless the 
investigator considers the abnormality to be clinically irrelevant 
 Normal laboratory values unless the investigator considers abnormalities to be 
clinically irrelevant   
 Ability to understand the purpose and nature of the study, as well as the 
associated risks 
 
Exclusion Criteria: 
 Intake of any drugs that may interfere with the trial’s endpoints or drugs (i.e. 
platelet inhibitors, anticoagulants, etc.) 
 Positive results of HIV or hepatitis virology 
 Acute illness with systemic inflammatory reactions 
 Known allergies, hypersensitivities or intolerances to any of the used 
substances 
 Acute or recent bleeding episodes, increased risk of bleeding at the discretion 
of the investigator 
 Participation in an LPS trial within 6 weeks of the first study day 
 Pregnancy or breastfeeding 
4.2. Withdrawal Criteria 
The subject may be withdrawn from the trial at the discretion of the investigator if 
judged non-compliant with trial procedures or due to safety concern.  


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4.4. Subject Replacement 
A subject will be considered evaluable if he or she has completed the first six hours of 
the trial’s second period. Otherwise another subject may replace him. Based on previous 
trials, this is expected to occur in less than 10% of the cases.  
5. Methods and Assessments 
5.1. Visits and Procedures 
5.1.1.Ex-vivo spiking Trial 
Healthy volunteers will report to the study ward and before blood sampling is 
performed the following steps will be performed:  
 Informed consent  
 Medical History and physical examination 
 Height and weight measurement 
 Blood pressure and heart rate measurement 
 Check for Ex- and inclusion criteria 
Blood sampling will be performed thereafter. The needed blood volume will be 17ml.  
5.1.2. Screening Visit 
During the screening visit eligibility of the subjects for participation in the trial will be 
checked. This contains obtaining informed consent before any trial related activity is 
performed.  
The following examinations will be performed at least one week before the first trial 
day: 
 In-/Exclusion Criteria 
 Physical examination and medical history 
 Height and weight 


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 Blood pressure, heart rate, ECG evaluation 
 Laboratory analysis incl. blood counts, blood chemistry, global coagulation assays 
and urinalysis 
 Pregnancy test, if applicable 
 Virology incl. HIV and Hepatitis C  
5.1.3. Period 1 Trial Day 1 
The subjects will report to the study ward at 08:00 at the Department of Clinical 
Pharmacology after an overnight fast. In- and exclusion criteria will be checked, a 
physical examination will be performed, intake of medication and other medical 
conditions since the screening visit will be noted. Vital parameters will be measured in a 
supine position (heart rate, blood pressure, oxygen saturation and body temperature) 
and urinalysis will be performed. If applicable, a pregnancy test will be performed. 
Baseline blood sampling will be performed.  
Two indwelling intravenous catheters will be placed in appropriate antecubital veins 
and infusion of the trial drugs will be started. In one of the catheter the LPS bolus will be 
injected. It will then be flushed using 0,9% sodium chloride and continuous intravenous 
infusion of sodium chloride at a rate 100ml/h will be started. The other catheter will be 
used for infusion of DF, which is initiated 1h before LPS bolus infusion.  
Vital signs will be measured and documented every hour until discharge. 
Blood sampling will be performed at the baseline, before any drug is infused, as well as 
one, two, four, six and 24 hours after infusion of the LPS bolus.   
In three out of 20 subjects, 3 additional blood draws will be performed at 5, 10 and 20 
minutes after LPS administration, respectively. These samples will serve for exploratory 
quantification of LPS. Each of the blood draws will be performed by single 


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venipunctures on the arm contralateral to LPS infusion. Participation to this part of the 
trial will be optional for each healthy volunteer.   
Four hours after the LPS bolus subjects will be receiving a standardized fat-reduced 
meal. After six to eight hours subjects will be discharged from the ward depending on 
their medical condition.  
5.1.4. Period 1 Trial Day 2 
24 hours after infusion of the LPS bolus subjects will report to the Department of Clinical 
Pharmacology for further blood sampling, urinalysis and for a physical examination. Any 
signs of bleedings will be documented.  
 
5.1.5. Period 2 Trial Day 1 
Similar to Trial Day 1 of the period 1 the subjects will report to the study ward at 08:00 
at the Department of Clinical Pharmacology after an overnight fast. In- and exclusion 
criteria will be checked, a physical examination will be performed, intake of medication 
and other medical conditions since first period will be noted. Vital parameters will be 
measured in a supine position (heart rate, blood pressure, oxygen saturation and body 
temperature) and urinalysis will be performed. If applicable, a pregnancy test will be 
performed. Baseline blood sampling will be performed.  
Two indwelling intravenous catheters will be placed in appropriate antecubital veins 
and infusion of the trial drugs will be started. In one of the catheter the LPS bolus will be 
injected. It will then be flushed using 0,9% sodium chloride and continuous intravenous 
infusion of sodium chloride at a rate 100ml/h will be started. The other catheter will be 
used for infusion of DF.  
Vital signs will be measured and documented every hour until discharge. 


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Blood sampling will be performed at the baseline, before any drugs are infused, as well 
as one, two, four, six and 24 hours after infusion of the LPS bolus.   
Four hours after the LPS bolus subjects will be receiving a standardized fat-reduced 
meal. After eight hours subjects will be discharged from the ward depending on their 
condition.  
5.1.6. Period 2 Trial Day 2 
24 hours after infusion of the LPS bolus subjects will report to the Department of Clinical 
Pharmacology for further blood sampling, urinalysis and for a physical examination. Any 
signs of bleedings will be documented.  
 
5.1.7. Final Visit 
The final safety visit will take place 7-14 days after the second trial period. A urinalysis, 
physical examination, blood sampling for safety parameters, ECG evaluation and 
documentation of all other relevant information including concomitant medication and 
adverse events, will be performed.  
This visit will be the final visit of the trial and all subjects will be discharged from the 
trial thereafter unless a medical reason exists for another follow-up visit.  
 
5.2. Efficacy analysis 
5.2.1. Primary endpoint 
The primary endpoint of this trial will be levels of F1+2.  
 
5.2.2. Secondary endpoints 
Secondary endpoints will include the following: 
 


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 vWF antigen and activity 
 microparticle associated tissue factor 
 inflammatory biomarkers (TNF-α, etc.) 
 Tissue-type plasminogen activator (t-PA) 
 Plasminogen activator inhibitor-1 (PAI-1) 
 D-Dimer 
 Thrombin-Antithrombin-Complexes (TAT) 
 Circulating endothelial cells 
 E-Selectin 
 Thrombelastometry parameters 
 Proinflammatory exosome 
 Differential blood counts 
 Proinflammatory exosomes 
 LPS concentration 
 
5.2.3. Rationale of the chosen endpoints 
F1+2 is a biomarker of thrombin generation and therefore coagulation activation. It has 
been repeatedly shown that infusion of a LPS bolus reliably increases F1+2 about 6-
10fold30-32. We hypothesize that the tissue-factor driven activation of coagulation and 
therefore thrombin generation is reduced by treatment with DF. Levels of F1+2 will be 
measured by enzyme-linked immunoassay (ELISA) at predefined time-points.  
 
Secondary endpoints include other markers of coagulation activation, such as TAT or 
microparticle associated tissue factor levels by enzyme-linked immunoassay or the 
clotting time or the clot formation time in thrombelastometry, which is a global 


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coagulation assay demonstrated to be sensitive to LPS induced coagulation activation33. 
However, it was demonstrated that DF treatment affects the fibrinolytic system and we 
will pay special interest to biomarkers of this system2. We will measure PAI-1, t-PA and 
D-Dimer levels by enzyme-linked immunoassays. Maximum lysis, a parameter of 
thrombelastometry also offers information on the fibrinolytic state of whole blood. 
Moreover, we will assess biomarkers of endothelial activation. Infusion of LPS is known 
to activate endothelial cells. DF was demonstrated to reduce endothelial activation 2. 
Thus, vWF activity and antigen, E-Selectin (all by enzyme-linked immunoassay) and 
circulating endothelial cells (by flowcytometry analysis) will be measured. Moreover, 
anti-inflammatory effects of DF will be investigated and assessed by measuring 
inflammatory biomarkers such as TNF-a levels by enzyme-linked immunoassay. Finally 
differential blood counts will be performed repeatedly to assess effects of DF and LPS on 
cell counts.   
The increase in tissue factor activity bearing microparticles is well known. However, the 
effects of endotoxemia on the release of proinflammatory exosomes, membrane vesicles 
smaller than microparticles, have not yet been investigated. Furthermore we want to 
assess the impact of DF on proinflammatory exosomes.  
After administration, LPS is cleared very rapidly from the bloodstream. After 
intravenous bolus injection of 2ng/kg bodyweight, studies have reported absence of 
detectable endotoxin levels as soon as 30 minutes post-dose 34. 
 In a recently published clinical trial, colistin was shown to markedly reduce the 
inflammatory response in LPS-treated healthy volunteers 35. This effect is thought to be 
due to binding and neutralization of circulating LPSby colistin. However, due to 
technical reasons this study did not include measurement of LPS plasma levels, and the 
ultimate evidence for the abovementioned hypothesis is still lacking.  


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Now, a collaboration with a research group from Uppsala, Sweden would provide us 
with the opportunity of measuring LPS by means of a chromogenic limulus amoebocyte 
lysate (LAL) assay.  
The quantification of LPS kinetics as attempted in the present study is to be regarded as  
exploratory and will provide crucial information for potential future investigations on 
the topic. 
The availability of a reliable assay for LPS quantification might trigger additional in vitro 
and in vivo experiments aiming at demonstrating the link between increase of colistin 
concentrations and decline of LPS levels in a more stringent way. 
5.3. Safety Endpoints 
Serum CRP, GPT levels and blood cell count at 24h, hemodynamic and febrile responses, 
as well as adverse events are considered safety endpoints.  
We will specifically assess any signs of bleeding and perform a follow-up visit 1-2 weeks 
after the last study day.  
5.4. Blood Sampling schedule, tubes and volume 
Blood samples will be drawn at predefined time-points. Baseline measurements will be 
performed before infusion of any trial drug. Total blood volume will not exceed 240ml 
per period.  
 
Table 1. Parameters, Samples and Schedule  
Parameter 
Sample 
Time points 
Coagulation biomarkers 
Citrated plasma 
Pre, 0h, 1h, 2h, 4h, 6h, 24h 
Fibrinolysis biomarkers 
Citrated plasma 
Pre, 0h, 1h 2h, 4h, 6h, 24h 
Endothelial biomarkers 
EDTA-plasma/ Citrate 
plasma 
Pre, 0h, 1h 2h, 4h, 6h, 24h 
Inflammatory 
biomarkers 
EDTA-plasma, Serum, 
Citrate-plasma  
Pre, 0h, 1h 2h, 4h, 6h, 24h 
FACS analysis 
Citrated blood 
Pre, 0h, 1h 2h, 4h, 6h, 24h,  


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MPTF 
Citrated plasma 
Pre, -0h, 2h, 4h, 6h, 24h 
Chemistry 
Serum 
Pre,  24h 
Differential blood counts 
EDTA 
Pre, 0h, 1h 2h, 4h, 6h, 24h  
Global coagulation assays 
Citrate 
Pre, 0h, 1h 4h, 24h 
LPS quantification 
Citrate 
0h, 5min ,10min, 20min 
Pre= Before Defibrotide/Placebo infusion and before LPS, 0h=Before LPS injection, 
other time-points relate to the LPS injection 
5.5. Preparations of samples 
 
5.5.1. Thrombelastometry 
Thrombelastometry (ROTEM; Pentapharm GmbH, Munich, Germany) will be performed 
using citrate anti-coagulated whole blood as previously described33. In short, 
thrombelastometry measures the viscoelastic properties of whole blood during clot 
formation and clot lysis. Thrombelastometry is sensitive to infusion of 2ng/kg 
bodyweight LPS33. 
 
5.5.2. Coagulation specific and fibrinolytic parameters 
F1+2, TAT, t-PA, PAI-1, D-Dimer and MPTF activity will all be detected in citrate anti-
coagulated plasma samples. Whole blood samples will be centrifuged at 2000g for 15 
minutes at 4 degrees Celsius and supernatant plasma will be transferred to aliquots of 
approximately 500µl each. These aliquots will be frozen and stored until batch analysis. 
MPTF will be centrifuged twice to generate platelet free plasma.  F1+2, TAT, t-PA, PAI-1 
and D-Dimer will be assessed by commercially available enzyme-linked immunoassays. 
MPTF activity will be assessed as previously demonstrated32. 
 


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 5.5.3. Inflammatory biomarkers 
EDTA-anticoagulated blood will be centrifuged at 2000g for 15 minutes at 4 degrees 
Celsius and supernatant plasma will be transferred to aliquots of approximately 500µl 
each. These aliquots will be frozen and stored until batch analysis. TNF-a will be 
measured by commercially available enzyme-linked immunoassays. We will assess 
changes in the proinflammatory exosome and the influence of DF on exosomes. 
Therefore, citrate anticoagulated blood will be centrifuged twice and isolated exosomes 
will be analyzed by mass spectrometry.  
 
5.5.4. Endothelial biomarkers 
vWF antigen levels and activity and E-Selectin levels will be assessed by commercially 
available enzyme linked immunoassays. EDTA- and citrate-anticoagulated blood 
samples will be centrifuged at 2000g for 15 minutes at 4 degrees Celsius and 
supernatant plasma will be transferred to aliquots of approximately 500µl each. These 
aliquots will be frozen and stored until batch analysis. 
 
5.5.5. Differential blood counts 
Differential blood counts will be performed at the central laboratory of the General 
Hospital of Vienna. 
 
5.5.6. Blood Chemistry 
Blood chemistry will be performed at the central laboratory of the General Hospital of 
Vienna before infusion of any trial drugs and 24 hours after infusion of LPS. 
 


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5.5.7 Global coagulation assays 
Global coagulation assays will be performed at the central laboratory of the General 
Hospital of Vienna.  
 
5.5.8. Circulating endothelial cells 
Circulating endothelial cells will be assessed by flowcytometric analysis as previously 
described36. 1ml of freshly drawn EDTA-anticoagulated blood will be first mixed with 
10ml of FACS lysing solution (10x, diluted 1:10) according to the manufacturers 
instructions. The white cells will then be blocked with 20μl of specific Fc-receptor 
antibodies (Octagam) and 200μl of mouse serum (Sigma, Gillingham, UK) for a minimum 
of 20min at room temperature. Next, the cells will be incubated with fluorochrome-
labelled monoclonal anti-human mouse antibodies, namely FITC-CD45, PE-CD146 and 
PE-Cy5-CD34 (Becton Dickinson, Oxford, UK) for 20 min at room temperature, washed 
with cell buffer solution [PBS + bovine serum albumin (BSA) 1% + sodium azide 0.05%] 
and centrifuged at 500g to repellet the cells. The cells will be fixed with 200μl of 2% 
paraformaldehyde for 20min at 4°C and made up to a final volume of 1ml with cell 
buffer solution ready for analysis. Each sample will be analysed in a 3-colour FACScan 
flow cytometer (Becton Dickinson, Oxford, UK). Cells will be plotted according to 
forward scatter and side scatter profiles (a measure of size and granularity of an event, 
respectively) and gated to include only mononuclear cell events and excluding cell 
doublets, platelets, dead cells/debris, microparticles and high side scatter events (e.g. 
dead cells and polynuclear cells have higher side scatter). A second gate will be used to 
include only those cells negative for CD45 (FITC) (CD45 is a pan-leukocyte marker, and a 
‘dump’ channel will be constructed to exclude all CD45 positive cells including CD45dim 
cells as included in these are endothelial progenitor cells) and are low to medium side 


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scatter singlets. A third gate will be used to analyse cells doubly positive for CD146 (PE) 
and CD34 (PE-Cy 5) expression, and only high intensity doubly fluorescent cells (i.e. 
CD146+++/CD34+++) will be defined as CECs. The sample will be analysed for a 
minimum of 500,000 mononuclear cellular events and a pre-set time period, 
corresponding to a known volume of sample (predetermined from previous validation 
studies, data not shown). Fluorochrome-matched isotype controls (FITC-IgG1, PE- IgG1, 
PE-Cy5– IgG1; Becton Dickinson, Oxford, UK) as well as non-stained samples will be 
used to set the appropriate gate parameters and served as negative controls.  
5.5.9. LPS quantification 
Blood for LPS quantification will be collected in citrate tubes and put on ice. Within 30 
minutes from collection, samples will be centrifuged at 4°C and 2000 g for 10 minutes and 
resulting plasma transferred into 3 aliquots of approximately 500 μL each. 
All aliquots will be snap frozen at -20°C and later stored at -80°C until analysis. LPS will 
be measured at the Department of Pharmaceutical Biosciences, Uppsala University, 
Sweden, by means of a chromogenic limulus amoebocyte lysate assay. 
Optionally, a second method of LPS quantification (Haemotox rFC, Haemochrom 
Diagnostica, Germany) might be used and results compared with the LAL assay. 
6. Trial Materials 
6.1. Trial Products 
LPS:  
 US Standard Reference Endotoxin, CC-RE Lot 2, vialed by 
MARP/NCI 
Manufacturer:  
FROC 2ng/ml 
Dose:  
 
2ng/kg administered at 0 min as an i.v. bolus over 1-2 min 
 
Defibrotide:   
Defitelio, 80mg/ml,  Ampullae of 2,5ml 
Manufacturer: 
Jazz Pharmaceuticals 


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Dose:  
 
6.25mg/kg bodyweight as a countinuous infusion over 2 hours 
 
Physiologic Saline: Isotone Kochsalz-Lösung 0,9% Braun 
Manufacturer: 
Braun 
Dose:  
 
100ml/h for six hours 
 
Paracetamol:  
Paracetamol Genericon 
Manufacturer: 
Genericon Pharma  
Dose:   
 
Paracetamol 500mg p.o. on demand 
 
6.2. Packaging and Labeling of trial products 
Not applicable. 
 
6.3. Storage and Drug Accountability of Trial Product(s) 
All trial products should be stored in a secure place.  
Defitelio should be stored at room temperature. After dissolution it should be kept 
refrigerated and used within 24 hours according to the manufacturer.  
LPS should be kept refrigerated at 4-8 °C.  
Saline and paracetamol should be stored at room temperature.  
A record of all supplies dispatched to the investigator will be maintained by the Monitor. 
The investigator will keep accurate records of all used and unused sup-plies. No trial 
products may be dispensed to any person except to the subjects en-rolled in the trial. 
 


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6.4. Randomization and Blinding 
All screened subjects will receive a screening number. This number will be the identifier 
of each subject within the trial. A subject identification list, which links the screening 
numbers with the names and birth dates of each participating subject will be kept in the 
trial master file and in the “study maker” (the computer system of the Department of 
Clinical Pharmacology). In future publications we will only refer to numbers of subjects, 
but not to names or birth dates.  
 
A physician not performing other tasks in the trial will observe the subjects.  
Block randomization will be performed using an open access randomization generator 
(www.randomization.com). 
Three sets of sealed code/label with the randomization number containing information 
about the treatment allocation (LPS or placebo group, DF or placebo period) for the 
particular subject will be prepared for each subject.  
The code for a particular subject can be broken in a medical emergency if knowing the 
identity of the treatment allocation would influence treatment of the subject. Whenever 
a code is broken, the person breaking the code must record the time, date and reason as 
well as their initials in the source documents.  
All codes (whether broken or not) must be kept throughout the trial period.  
Breaking of the code, however, does not necessarily mean that the plasma obtained from 
the subject is not eligible for analyses, which can still be performed in a blinded fashion. 


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6.5. Potential inconveniences and risks for the subjects 
6.5.1. Defibrotide 
Defibrotide is approved by the FDA and the EMA for treatment of the sinosoidal 
obstruction syndrome (also called veno-occlusive disease) after hematopoietic stem cell 
transplantation or chemotherapy. Only few studies exist investigating the effects of DF 
in healthy volunteers7,37. No serious or severe adverse events were reported in these 
trials.  
The summary of product characteristics suggest that the most important adverse events 
of DF are hemorrhage, hypotension and coagulopathy26. Of note, compared to a 
historical control group these side effects did not occur more frequently in the DF group 
indicating that these side effects may be due to the selection of patients, who were all 
thrombopenic critically ill patients38.  
No hypersensitivities or anaphylactic reaction were reported in the marketing trials20,38. 
However, in previously marketed defibrotide products hypersensitivities and 
anaphylactic reactions have been reported 26. Thus, all participant will be made aware of 
that risk and special attention will be paid to potential anaphylactic reactions.   
We expect minor anti-coagulatory effects after infusion of DF. However, studies suggest 
that the effects of DF are only short termed and DF should be eliminated completely 
within 24 hours. All subjects will be made aware of an increased bleeding risk on the 
treatment days.  
 
6.5.2. LPS 
2-4ng/kg bodyweight are safe doses, which have already been used in clinical trials 30 
and at the investigational site in over 1000 subjects. LPS produces flu-like symptoms 
consisting of fever, chills, myalgias, arthralgias, nausea and headache which are most 


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prominent 2-3h following endotoxin, and begin to resolve after 6 hours. Overall, infu-
sion of endotoxin is generally well tolerated and trial subjects are virtually free of 
symptoms after 8 hrs. 
 
6.5.3. Paracetamol 
At therapeutic dosages (0.5 to 1g orally bid or tid) paracetamol is usually well tolerated 
and no common adverse effect has been established. Rarely allergic skin reactions may 
be seen. Chronic abuse or overdose can lead to disturbances in kidney and liver function. 
 
6.5.4. Blood sampling  
A total amount of approx. 500ml of blood will be collected during the duration of 8 
weeks. This is about the volume of one blood donation. Between the two study periods a 
washout period of 6 weeks will apply. Thus, the blood volume should not impose any 
dangers on subjects. Care will be taken to avoid formation of subcutaneous haematomas 
after venipunctures. 
 
6.5.5. Risk/benefit assessment 
Endotoxin infusion in humans is a safe standard model, which induces a systemic 
inflammatory response and which has been safely used in more than 1000 volunteers at 
our institution. This inflammatory response is limited in duration and severity and can 
be used with restrictions as a model for the early phase of human sepsis.  
Defibrotide has been used in clinical trials and clinical practice for over 30 years. 
Although many in vitro and animal studies have been published and effects in patients 
with SOS38, with atherosclerosis obliterans39 or peripheral arterial occlusive disease40, 


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as a prophylaxis against deep venous thrombosis after surgery9,  and other diseases 
have been investigated, the mode of action is still not entirely understood. However, in 
all those studies the use of DF was safe and no serious or severe AEs occurred.   
The aim of this trial is to better define the pharmacodynamic effects of DF in a well 
established model of acute, transient inflammation in healthy volunteers. Furthermore, 
this trial will allow comparison of DF and other drugs that have already been 
investigated in this model and will help to generate further hypotheses about new 
indications of DF. The main risk of DF is a potential allergic reaction or bleeding 
episodes. However, the anti-coagulatory effects of DF will be closely monitored and 
strong anticoagulatory effects are not expected. Allergic reactions are rare, but must be 
considered a potential risk.  
In conclusion we consider the benefits to clearly outweigh the risks in this study.  
6.6. Safety precautions 
Throughout the trial-period the subjects will be observed by a physician. In addition, a 
physician with experience in emergency medicine will be at the unit during the first 
eight hours. The clinical trial center is equipped with monitoring facilities comparable to 
an intensive care unit. 
The subjects will be supine with their chest tilted up throughout the trial period and 
their vital parameters monitored (ECG, heart rate and oxygen saturation continuously; 
blood pressure at 20 min intervals). Oral temperature will be checked every hour during 
the first 6 hours.  
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Although our experience with more than 1000 LPS challenges showed no severe 
untoward effects, all adverse events will be managed by adequate medical measures 
according to standard practice. Evaluation of the safety parameters will be performed 
for each trial subject, which includes checks of C-reactive protein, GPT-levels blood cell 
count 24 hours after the endotoxin bolus. 
7. Concomitant Illness and Medication 
Concomitant illness: any illness that is present at the start of the trial  
Concomitant medication: any medication other than the trial product that is taken 
during the trial, including screening and run-in periods 
7.1. Concomitant Medication during the Trial  
No concomitant medication is planned. 
7.2. Precautions/Overdosage  
As all drugs will be administered at the study site by a physician and controlled by an 
independent staff member according to SOP, no overdose are described in the 
Investigator’s Brochure.  
 
8. Adverse Events 
8.1. Definitions 
8.1.1. Adverse Events  
An AE is any undesirable medical event occurring to a subject in a clinical trial, whether 
or not considered related to the trial product(s). This includes events not seen at 
baseline or worsened if present at baseline. The following should not be re-corded as 
AEs, if recorded at screening (on Screening Form or CRF):  


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Pre-planned procedure, unless the condition for which the procedure was planned has 
worsened since baseline  
Pre-existing conditions found as a result of screening procedures. 
 
8.1.2. Clinical Laboratory Adverse Events 
A clinical laboratory AE is any clinical laboratory abnormality that suggests a dis-ease 
and/or organ toxicity and is of a severity which requires active management (i.e. 
changes of dose, discontinuation of drug, more frequent follow-up or diagnostic 
investigation). 
8.1.3. Serious Adverse Events (SAE) 
An SAE is any adverse drug experience that at any dose results in any of the follow-ing 
outcomes:  
 death  
 a life-threatening* experience  
 in-patient hospitalization or prolongation of existing 
hospitalization  
 a persistent or significant disability/incapacity  
 a congenital anomaly/birth defect.  
 
Important medical events that may not result in death, be life-threatening, or require 
hospitalization may be considered a serious adverse drug experience when, based upon 
appropriate medical judgment, they may jeopardize the health of the subject or patient 
and may require medical or surgical intervention to prevent one of the out-comes listed 


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in this definition.  
*The term life-threatening in the definition of serious adverse event refers to an event in 
which the subject was at risk of death at the time of the event. It does not refer to an 
event, which hypothetically might have caused death if it was more severe. 
8.1.4. Non-Serious Adverse Event 
A non-serious adverse event is any AE, which does not fulfill the definition of an SAE. 
8.1.5. Severity Assessment Definitions 
Mild: transient symptoms, no interference with the subject’s daily activities.  
Moderate: marked symptoms, moderate interference with the subject’s daily activities.  
Severe: considerable interference with the subject’s daily activities unacceptable.  
 
8.1.6. Relationship to Trial Drug Assessment Definitions: 
Probably: Good reasons and sufficient documentation to assume a causal relationship  
Possible: A causal relationship is conceivable and cannot be dismissed  
Unlikely: The event is most likely related to an aetiology other than the trial treatment  
Impossible to assess: Causality is not assessable, e.g. because of insufficient evidence, 
conflicting data or poor documentation. 
 
8.1.7. Outcome Categories and Definitions: 
Recovered  
Stabilized: An AE is stabilized when, according to the Investigator, the subject is in a 
clinically stable condition. This term should only be used for chronic conditions and for a 
given subject only when he/she has completed the protocol  


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Recovered with sequelae: As a result of the SAE, the subject suffered persistent and 
significant disability/incapacity (e.g. became blind, deaf, paralyzed). Any AE re-covered 
with sequelae should be rated as an SAE.  
Not yet recovered  
Died  
Unknown. 
 
8.2. Collection, Recording and Reporting of Adverse Events  
All events meeting the definition of an adverse event must be collected and reported 
from the first trial-related activity after the subject signs the informed consent and until 
the end of the protocol required post-treatment follow-up period.  
At each contact to the site (visit or telephone, excluding safety visits, where the subject 
is not seeing the Investigator or his staff (e.g. visits to the laboratory)), the subject must 
be asked about adverse events. All adverse events, either observed by the Investigator 
or reported by the subject, must be recorded by the Investigator and evaluated. 
The subjects will be asked a general question “How are you?” as well as a specific 
question “Have you experienced any problems since the last contact?”  
The Investigator should record the diagnosis, if available. If no diagnosis is available the 
Investigator should record each sign and symptom as individual Adverse Events.  
All adverse events must be recorded by the Investigator on the standard Adverse Event 
Form. One single Adverse Event Form/line must be used per adverse event from start to 
resolution. For serious adverse events, the Serious Adverse Event Supplementary pages 
1-4 must also be completed.  


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The Investigator must inform the Health Authorities and IRBs/IECs in accordance with 
the local requirements in force and ICH guideline for GCP. The Monitor must be in-
formed accordingly. 
8.3. Follow-up of Adverse Events 
During and following a subject’s participation in a clinical trial, the 
Investigator/institution will ensure that adequate medical care is provided tot he 
subject for any adverse events, including clinically significant laboratory values related 
to the trial. The investigator/institution will inform the subject when medical care is 
needed for adverse event(s) of which the investigator becomes aware. The active post-
treatment follow-up period will end withthe post-trial examination.  
All adverse events classified as serious or severe or possibly/probably related to the 
trial product must be followed until the subject has recovered, stabilized, recovered with 
sequelae or died, and until all queries have been resolved.  
All other adverse events must be followed until the subject has recovered or stabi-lized 
or until the end of the protocol required post-treatment follow-up whichever comes 
first, and until all adverse event related queries for the subject have been re-solved. 
 
8.4. Case record forms 
A case record form will be completed for each subject. The entries will be checked by 
trained personnel and any errors or inconsistencies will be corrected immediately. CRFs 
will be completed by trained personnel at the trial site. 
 
8.5. Rules for completing CRFs 
Print legibly using a ball-point pen. Ensure that all relevant questions are answered and 
that no empty data blocks exist.  


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If a test/assessment is not done and will not be available, indicate this by writing „N/D“ 
(Not done) in the respective answer field in the CRF. If the question is irrelevant (e.g. is 
not applicable) indicate this by writing „N/A“ (Not applicable) in the respective answer 
field.  
The investigator or investigator’s authorized staff must ensure that all information 
derived from source documents should be consistent with the source information. By 
signing the affirmation statement, the investigator confirms that the information is 
complete and correct.  
8.6. Correction of CRFs 
Corrections to the data on the CRFs must only be made by drawing a straight line 
through the incorrect data and by writing the correct value next to data that has been 
crossed out. Each correction must be initialed and dated and explained (if necessary) by 
the Investigator or the Investigator’s authorized staff. If corrections are made by the 
Investigator’s authorized staff after the date of the investigator’s signature on the 
affirmation statement, the statement must be signed and dated again by the investigator.  
Corrections necessary after the CRFs have been removed from the Investigator’s site 
must be documented on a Query Resolution Form (QRF). If the affirmation statement for 
the subject has not been signed, corrections must be approved by the Investigators or 
her/his authorized staff. If the affirmation statement fort he subject has been signed, 
only the investigator can approve the correction.  
8.7. CRF Flow 
Paper CRFs will be used and stored at the trial side. Laboratory report of pre-and post-
trial screening examinations will be attached as copies to the CRFs, in case of clinically 
relevant deviations from normal this will be protocolled in the CRF. The Investigator or 


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another authorized physician will sign analysis results (e.g. laboratory, ECG, etc.) as soon 
as they are available to verify that the results are reviewed. 
9. Monitoring procedures 
During the course of the trial, the Monitor will visit the investigational site 
approximately two times during each part and must be available for discussions by 
telephone. The purpose of these visits is to ensure that the CRFs are completed 
correctly, the protocol is adhered to, to monitor drug accountability, and to collect 
completed pages of the CRFs.  
The Monitor must be given direct access to source documents (original documents, data 
and records). Direct access includes permission to examine, analyze, verify and 
reproduce any record(s) and report(s) that are important to evaluation of the clinical 
trial. Source data will be available for all data in the CRFs, including all laboratory re-
sults.  
The Monitor will ensure that the CRFs are collected. 
10. Data Management 
The subjects will be identified by subject number, initials, site, and trial identification 
number. Subject initials will be retained in the database only during the period of trial 
data processing. Upon completion of the Integrated Clinical Trial Report, subject initials 
will be deleted or encrypted to ensure subject confidentiality.  
Electronic data transfer of any CRF or subject related data must be approved by the 
responsible Data Management Unit(s). In cases where data is transferred via non-secure 
electronic networks, data must be encrypted at source. 


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11. Statistical Considerations 
11.1. Sample Size Calculation 
 
 
The sample size calculation is based on the experience with prior trials using anti-
coagulants in the human endotoxemia model. Ten to 15 subjects in each group were 
sufficient to detect significant differences in the primary outcome parameter (F1+2 
levels) between treatment groups in a parallel-group study14,23,24. Currently, we do not 
have a good estimate for the size of effect of defibrotide in the same model. In a prior 
LPS trial F1+2 levels of approximately 819±173 (Standard deviation) pmol/ml were 
measured 41. This corresponds to an approximate 6fold increase in F1+2 levels and was 
similar in other endotoxemia trials42. The median intraindividual coefficient of variation 
was 15% in a recent study, the mean was 25%32. Thus, a sample size of 16 volunteers 
will be sufficient to detect a 25% decrease in the defibrotide period.  
The placebo group consisting of four healthy volunteers will be an additional control 
group, which will also allow to assess the effects of DF in healthy volunteers without 
LPS. However, this analysis is exploratory and no sample size calculation for this group 
is performed.  
11.2. Methods 
Two-sided tests and a 5% critical value will be used throughout the trial. Test 
procedures to reduce multiple testing will be applied. Refer to the detailed, separate 
statistical analysis plan for further information. 
 
11.2.1 Efficacy Analysis 
The efficacy analysis will compare changes in the primary and secondary efficacy 
endpoints described above between the placebo and the DF periods.  


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The repeated measures analysis of variance (ANOVA) will be used for analysis of 
treatment and period effects (treatment=independent factor, period=independent 
factor, outcome variable=dependent factor). When significant, post hoc comparisons will 
be performed with nonparametric tests for reasons of robustness: The Friedman ANOVA 
and the Wilcoxon test for post hoc comparisons will be used.  
Furthermore, graphs showing the time profiles at the individual subject and group mean 
levels will be presented. 
Multiple parameters will be tested. However, to reduce the number of tests a stepwise 
statistical analysis plan will be applied. Moreover, this study is of exploratory nature and 
correcting for multiple testing is therefore not necessary.  
 
11.3. Safety Analysis 
All subjects who will receive trial drugs (including placebo) will be included in the safety 
analysis, which will compare the safety endpoints specified above between treatment 
periods. Tables of summary statistics will be presented. Detailed analysis specifications 
will be presented in a statistical analysis plan prior to analysis.  
As LPS-infusion is associated with a number of adverse effects, particular interest will be 
focused on potential specific adverse effects of DF such as bleeding events or allergic 
reactions. 
11.4. Missing, unused and spurious data 
Missing or spurious data will not be intrapolated, but set missing. Statistical analyses 
will be performed for the intention to treat and the per-protocol population and 
compared. If results differ significantly it will be mentioned in any reports of trial 
results.  


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12. Ethics 
The trial will be conducted in accordance with the Declaration of Helsinki for biomedical 
research involving human subjects. 
12.1. Informed Consent Form of trial subjects (AMG §38)  
In obtaining and documenting informed consent, the investigator must comply with the 
applicable regulatory requirement(s) and adhere to ICH guideline for GCP and the 
requirements in the Declaration of Helsinki.  
Prior to any trial-related activity, the investigator or an authorized physician must give 
the subject oral and written information about the trial in a form that the subject can 
read and understand.  
A voluntary, signed and dated informed consent form will be obtained from the subject 
prior to any trial-related activity. The subject must have consented to participate after 
the nature, scope and possible consequences of the trial have been explained in a form 
understandable to him or her.  
The written informed consent must be signed by the person who conducted the 
informed consent.  
If information becomes available that may be relevant to the subjects’s willingness to 
continue participating in the trial, the investigator must inform the subject in a timely 
manner, and a revised written informed consent must be obtained.  
 
12.2. Independent Ethics Committees (AMG §§ 40, 41) 
Prior to commencement of the trial, the protocol, any amendments, Subject Informa-


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tion/Informed Consent Form, any other written information to be provided to the sub-
ject, subject recruitment procedures (e.g. advertisements), if any, Investigator’s Bro-
chure 10, package insert (if marketed product), information about payments and 
compensation available to subjects if not mentioned in the subject information, the 
Investigator’s current CV and/or other documentation evidencing qualifications, and 
other documents as required by the local Independent Ethics Committee (IEC) should be 
submitted. The submission letter should clearly identify (by including ver-sion no. 
and/or date of the document) which documents have been submitted to the IRB/IEC. 
Written approval/favorable opinion must be obtained from the IEC prior to 
commencement of the trial.  
During the trial, the Investigator must promptly report the following to the IEC: Up-
dates to IB, unexpected SAEs where a causal relationship cannot be ruled out, 
amendments to the protocol, notes of administrative changes, deviations to the pro-
tocol implemented to eliminate immediate hazards to the trial subjects, new informa-
tion that may affect adversely the safety of the subjects or the conduct of the trial, 
annually written summaries of the trial status, and other documents as required by the 
local IEC.  
Amendments must not be implemented before approval/favorable opinion, unless  
necessary to eliminate immediate hazards to the subjects. 
 
12.3. Regulatory Authorities 
The Regulatory Authorities will receive the protocol, amendments, reports on SAEs, and 
the Final Report according to Austrian regulations. 
 


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12.4. Insurance (AMG §32(2)) 
All subjects participating in this clinical trial will be insured through the Department of 
Clinical Pharmacology, Vienna University Hospital. 
12.5. Confidentiality 
All subject names will be kept secret in the investigator's files. 
13. Audits and Inspections 
Upon request, the Investigator will make all study-related source data and records 
available to a qualified quality assurance auditor mandated by the sponsor or to 
competent authority inspectors. The main purposes of an audit or inspection are to 
confirm that the rights and welfare of the subjects have been adequately protected, and 
that all data relevant for assessment of safety and efficacy of the investigational product 
have appropriately been reported to the sponsor. 
All material used in clinical studies are subject to quality control.  
14. Publication policy 
The findings of this study will be published by the sponsor 43 in a scientific journal and 
presented at scientific meetings. The manuscript will be circulated to all co-Investigators 
before submission. Confidentiality of subjects in reports/publications will be 
guaranteed.  
15. Premature Termination of the trial 
The Investigator may decide to stop the trial or part of the trial at any time, but agree-
ment on procedures to be followed must be obtained.  


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If a trial is prematurely terminated or suspended, the Investigator should promptly 
inform the subjects and assure appropriate therapy and follow-up. Furthermore, the 
Investigator should promptly inform the IRB/IEC and provide a detailed written ex-
planation. The pertinent regulatory authorities should be informed according to na-
tional regulations. 
16. Retention of Clinical Trial Documentation 
Subject notes must be kept for the maximum time period as permitted by the hospital, 
institution or private practice. Other source documents and the Investigator’s trial file 
must be retained for at least 10 years or longer in accordance with local regulation. 
However, the Subject Identification Codes must be kept for at least 15 years.  
The Investigator must agree to archive the documentation pertaining to the trial in an 
archive after completion or discontinuation of the trial, if not otherwise notified. 
 
 
 
 
17. References:  
 
 
 
 
 
 


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