The Prophylactic Effect of Levosimendan in Reducting Acute Kidney Injury Postoperatively in Pediatric Patients Undergoing Corrective Heart Surgery
Phase: PHASE2
Conditions: Congenital Heart Defects
Sponsor: Göteborg University

Clinical Study Protocol 
Medical 
Levosimendan 
Study Code: 
MiLe-1 
EudraCT No: 
2013-003105-25 
Date: 
2013-09-01 version 5 
 
The prophylactic effect of Levosimendan in reducting acute kidney 
injury postoperatively in pediatric patients undergoing corrective 
heart surgery  
 
 
 
Principal 
Investigator: 
Doc. Albert Castellheim 
Sahlgrenska University Hospital 
The Queen Silvia Children’s Hospital 
AnOpIVA 
416 85 Gothenburg 
Tel: 031-3435212 
Mobile: 070 897 98 20 
Albert.castellheim@gu.se 
  
 
 
Co-investigator: 
Elin Thorlacius 
Sahlgrenska University Hospital 
The Queen Silvia Children’s Hospital 
AnOpIVA 
416 85 Gothenburg 
Tel: 031-3436433 
Mobile: 
elin.thorlacius@vgregion.se 
 
 
 
Study Site: 
The Queen Silvia Children’s Hospital 
Department of anesthesia and 
intensive care unit 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The following Amendment(s) and Administrative Changes have been made to this protocol since the date of 
preparation: 
Amendment  No. 
 
Date: 
 
Admin. Change  No. 
 
Date: 
 
 
 
 
 
  
 
 
 
 
 
  
 
 
 
 
 
  

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PROTOCOL SYNOPSIS 
The prophylactic effect of Levosimendan in reducting acute kidney injury 
postoperatively in pediatric patients undergoing corrective heart surgery  
 
Principal Investigator 
Doc. Albert Castellheim 
Sahlgrenska University Hospital 
The Queen Silvia Children’s Hospital, AnOpIVA, Göteborg  
Study centre(s) 
The Queen Silvia Children’s Hospital, Department of anesthesia and intensive care unit 
Study period  
Estimated date of first subject enrolled: June 2014 
Estimated date of last subject completed: June 2016 
Background 
Acute kidney injury (AKI) is a major cause of morbidity and mortality in critically ill 
patients including critically ill children. The etiology of AKI is multifaceted which may 
partly explain the lack of a causative therapy. The treatment of AKI remains supportive 
and consists of, in milder cases urine production enhancing medications, and in more 
severe cases renal replacement therapy. There is evidence that Levosimendan may 
protect the kidney from injury in conjunction with cardiac surgery in adults [1]. In this 
study we will use Levosimendan to intervene with renal hypoperfusion and assess its 
efficacy in reducing creatinine, which is used most frequently as a clinical marker of 
kidney injury. The study is prospective, randomized, and double-blinded. The trial will 
contain two study groups, 35 patients in each, where the one group will receive 
Levosimendan and the second group will receive Milrinone as an inotrope agent 
perioperatively. Milrinone is currently used as the drug of choice for inotropic therapy in 
our department like many other pediatric cardiac surgery centers. It remains to see if 
Levosimendan can exert a kidney protecting function in addition to its inotropic one. 

Clinical Study Protocol Synopsis 
Drug Substance Levosimendan  
Study Code 2013-003105-25 
Date  
3(62) 
The aim of the study is to assess the ability of Levosimendan to reduce the 
postoperative acute kidney injury in pediatric patients undergoing surgery for 
congenital heart disease (CHDs). 
 Primary objective 
The primary objective of this study is to investigate the preventive effect of 
Levosimendan on postoperative acute kidney injury in pediatric patients undergoing 
surgery for their CHDs. Creatinine levels postoperatively will be the primary endpoint. 
Study design 
This is a parallel-group, prospective, randomized, double-blinded controlled study in 
pediatric patients in need for surgery due to their CHD.  
A total of 70 male and female pediatric patients will be included in the study and 
undergo heart surgery according to routine procedures at the hospital. Patients will be 
randomly assigned to receive either Levosimendan (test group) or Milrinone (control 
group). Creatinine, the common marker of kidney injury, will be measured daily 
according to the clinical routines. The length of treatment (Levosimendam or Milrinone) 
is 24 hours. The treatment will be started during the operation at the same time-point at 
which Milrinone infusion routinely is started. Blood samples will be obtained at five 
occasions, during 24 hours perioperatively. Patients will be followed 4 days after 
termination of treatment.  
Target subject population 
Young children, between the age of 1 to 12 months, with congenital heart disease in need 
of elective heart surgery  
Inclusion criteria 
The inclusion criteria will be any of the diagnoses:  
1. Non-restrictive VSD (corrective surgery) 
2. Complete AVSD (biventricular repair) 
3. Tetralogy of Fallot  
Exclusion criteria 
The exclusion criteria will be 

Clinical Study Protocol Synopsis 
Drug Substance Levosimendan  
Study Code 2013-003105-25 
Date  
4(62) 
1. Unbalanced AVSD 
2. Age less than one month and more than one year 
3. Acute operation that is unscheduled operation during the first 24 hours after 
presentation to the department for thoracic surgery 
4. Mild, moderate, or severe kidney dysfunction 
5. Liver impairment or disease 
 
6. Ongoing infection 
7.  Use of nephrotoxic drugs (like ibuprofen, angiotensin-converting-enzyme inhibitors, 
gentamicin, vancomycin) preoperative or postoperative until first post operative day 
8. Use of inhibitors of membrane transport proteins (Cimetedin, cetirizine, trimethoprim, 
probenecid, rifampin and gemfibrosil 
9. Allergy to Levosimendan or substance included in the preparation or previous use of 
Levosimendan 
10. Severe arrhythmias needing pace-maker treatment prior to the operation 
11. Severe cardiac dysfunction needing for treatment with extracorporeal membrane 
oxygenation (ECMO) prior to the operation 
12. Severe uncorrected hypotension where the initiation of anesthesia is postponed for 
its correction 
13. Re-operation 
Investigational product, dosage and mode of administration 
Active: Levosimendan 2.5 mg/mL solution, diluted to 0.05 mg/mL i.v. 
Control: Milrinone i.v. standard of care 
Duration of treatment 
24 hours 
Duration of study 

Clinical Study Protocol Synopsis 
Drug Substance Levosimendan  
Study Code 2013-003105-25 
Date  
5(62) 
30 days (24 hours treatment + 4 days follow up + 30-days-mortality registration) 
Outcome variable(s):  
Creatinine is primary outcome in this study. Inflammatory biomarkers, NGAL, and other 
relevant biomarkers belong to secondary outcome variables.    
Safety 
Adverse Events will be recorded. Serious Adverse Events will be reported. 
Statistical methods 
The number of patients needed for the study will be 70 and is based on a 20% reduction 
in creatinine in the Levosimendan group compared with the Milrinone group 
postoperatively. The power of study is set on 80 %.  
Patients will be randomly assigned to one of two treatment groups: Levosimendan 
(n=35) and Milrinone group (n=35).  
Analysis of data will be performed by a professional statistician. Univariate analyses will 
be used where needed. Multivariable regression analysis will be used to identify 
association between the occurrences of AKI (based on creatinine results) and the 
measured clinical variables. Descriptive statistics will be applied on the secondary 
outcomes including clinical data (fluid balance, length of surgery, length of CPB, length of 
mechanical ventilation postoperatively, length of vasoactive therapy, number of 
vasoactive medications, total amount of infused vasoactive drugs, hemodynamic 
parameters). 
Safety and tolerability data will be summarized descriptively by treatment and 
presented in tabular and/or graphical form. All adverse event data will be listed 
individually and summarized using MedDRA terminology. 

Clinical Study Protocol Synopsis 
Drug Substance Levosimendan  
Study Code 2013-003105-25 
Date  
6(62) 
STUDY STRUCTURE 
Principle 
investigator: 
Dr. Albert Castellheim, MD, PhD, 
Associated professor 
Sahlgrenska University Hospital 
The Queen Silvia Children’s 
Hospital 
AnOpIVA/ Plan 7 
416 85 Göteborg 
 
Tel: 031-3435212 
Mobile: 070 8979820 
Albert.castellheim@gu.se 
Co-investigator: 
Dr. Elin Thorlacius, MD, Specialist 
Sahlgrenska University Hospital 
The Queen Silvia Children’s 
Hospital 
AnOpIVA/ Plan 7 
416 85 Göteborg 
 
Tel: 031-343 6433 
elin.thorlacius@vgregion.se 
Collaborators: 
Dr. Håkan Wåhlander, MD, PhD, 
Consultant Pediatric Cardiologist 
Division of Cardiology 
The Queen Silvia Childrens´s 
Hospital 
 
Dr. Birgitta Romlin, MD, PhD 
Sahlgrenska University Hospital 
The Queen Silvia Children’s 
Hospital 
AnOpIVA/ Plan 7 
416 85 Göteborg 
 
 
 
Prof. Sven-Erik Ricksten 
Institution för kliniska 
vetenskaper 
Avd för Anestesiologi och 
Intensivvård 
Sahlgrenska University Hospital 
413 45 Göteborg 
Tel: 031-342 7433 

Clinical Study Protocol Synopsis 
Drug Substance Levosimendan  
Study Code 2013-003105-25 
Date  
7(62) 
 
Study nurse: 
Anna-Lena Jansson 
The Queen Silvia Children’s 
Hospital 
AnOpIVA/ Plan 6 
 
Tel: 031-343 5689 
 
 
Lena Larsson 
The Queen Silvia Children’s 
Hospital 
AnOpIVA/ Plan 6 
 
Tel: 031-343 5689 
 
Study Monitor: 
Annette Erichsen Andersson 
 
Tel: 031-786 6073 
Study Statistician: 
Hans Wedel, Professor 
Gothia Forum 
Sahlgrenska University Hospital 
 
Tel: 070 829 7288 
Hans.wedel@biostat.se 
 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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TABLE OF CONTENTS 
PAGE 
PROTOCOL SYNOPSIS ............................................................................................................. 2 
TABLE OF CONTENTS ............................................................................................................. 8 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ................................................... 12 
1. 
INTRODUCTION .................................................................................................... 13 
1.1 
The importance of acute kidney injury............................................................... 13 
1.2 
Etiology of AKI ...................................................................................................... 13 
1.3 
Diagnosis of AKI .................................................................................................... 13 
1.4 
Prevention of AKI ................................................................................................. 14 
1.5 
Levosimendan ....................................................................................................... 14 
1.5.1 
Mechanism of action ............................................................................................ 14 
1.5.2 
Effects in adults .................................................................................................... 14 
1.5.3 
Effects and pharmacokinetics in Children .......................................................... 15 
1.5.4 
Dosage and interactions with other medications .............................................. 17 
1.6 
Milrinone ............................................................................................................... 18 
1.7 
Research hypothesis ............................................................................................ 18 
1.8 
Rationale for conducting this study .................................................................... 18 
1.9 
Benefit/risk assessment ...................................................................................... 18 
1.10 
Ethical considerations .......................................................................................... 19 
2. 
STUDY OBJECTIVES AND ENDPOINT(S) ............................................................ 19 
2.1 
Primary objective ................................................................................................. 19 
2.2 
Primary endpoint ................................................................................................. 19 
2.3 
Secondary endpoints ............................................................................................ 20 
3. 
STUDY DESIGN ..................................................................................................... 20 
3.1.1 
Stopping criteria for dose change ....................................................................... 23 
3.2 
Rationale for study design, doses and control groups ...................................... 23 
4. 
SUBJECT POPULATION ........................................................................................ 23 
4.1 
Inclusion criteria .................................................................................................. 24 
4.2 
Exclusion criteria .................................................................................................. 24 
4.3 
Subject enrolment and randomization ............................................................... 25 
4.3.1 
Safety and risk minimization in the scope of randomization ........................... 25 
4.4 
Discontinuation and Withdrawal of subjects ..................................................... 26 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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4.5 
Temporary or Premature termination of the study .......................................... 26 
5. 
TREATMENTS ....................................................................................................... 26 
5.1 
Test and control drugs ......................................................................................... 26 
5.1.1 
Levosimendan ....................................................................................................... 27 
5.1.2 
Milrinone ............................................................................................................... 28 
5.2 
Doses, preparations and treatment regimens.................................................... 28 
5.2.1 
Organization for preparation of the study drugs ............................................... 28 
5.2.2 
Preparing final concentrations ............................................................................ 29 
5.2.3 
Infusion rates ........................................................................................................ 29 
5.2.4 
Dosage scheme in present study ......................................................................... 29 
5.2.5 
Quality control of the medication preparation .................................................. 29 
5.2.6 
Storage conditions ................................................................................................ 30 
5.2.7 
Blinding and unblinding procedures .................................................................. 30 
5.2.8 
Labelling ................................................................................................................ 30 
5.3 
Concomitant and post-study treatment(s) ......................................................... 31 
5.4 
Treatment compliance ......................................................................................... 31 
5.5 
Accountability ....................................................................................................... 31 
6. 
STUDY CONDUCT AND METHODS ...................................................................... 31 
6.1 
Start and termination of study infusion and follow-up ..................................... 31 
6.2 
Efficacy variables .................................................................................................. 32 
6.2.1 
Blood for study-specific analysis......................................................................... 32 
6.2.2 
Blood for pharmacokinetics ................................................................................... 32 
7. 
SAFETY .................................................................................................................. 32 
7.1.1 
Safety procedures ................................................................................................. 32 
7.1.1.1 
During surgery ...................................................................................................... 32 
7.1.1.2 
Post-surgery follow-up ........................................................................................ 32 
7.1.2 
Medical emergencies ............................................................................................ 33 
7.1.3 
Common AEs after CHD surgery ......................................................................... 33 
7.1.4 
Adverse reactions of study medication .............................................................. 33 
7.1.5 
Laboratory safety assessment ............................................................................. 34 
7.1.6 
Definition of adverse events (AE) ....................................................................... 34 
7.1.7 
Definitions of serious adverse event (SAE) ........................................................ 34 
7.1.8 
Recording of adverse events ............................................................................... 35 
7.1.8.1 
Variables ................................................................................................................ 35 
7.1.8.2 
Definitions for intensity rating ............................................................................... 35 
7.1.9 
Adverse Events based on examinations and tests ............................................. 36 
7.1.10 
Follow-up of unresolved adverse events ........................................................... 36 
7.1.11 
Reporting of Suspected unexpected serious adverse reaction (SUSAR) ......... 37 
7.1.12 
Registering safety data in CRF ............................................................................. 37 
7.1.13 
Randomization code breaking ............................................................................. 37 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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7.1.14 
Postoperative course after pediatric cardiac surgery is quite eventful ........... 38 
7.1.15 
Evaluation and reversing of AEs ......................................................................... 38 
8. 
BIOLOGICAL SAMPLING PROCEDURES.............................................................. 39 
8.1 
Volume of blood .................................................................................................... 39 
8.2 
Handling, storage and destruction of biological samples ................................. 40 
9. 
STUDY AND DATA MANAGEMENT ..................................................................... 40 
9.1 
Audits and inspections ......................................................................................... 40 
9.2 
Recording of data.................................................................................................. 41 
9.2.1 
Source data............................................................................................................ 41 
9.3 
Training of study site personnel ......................................................................... 41 
9.4 
Monitoring of the study ....................................................................................... 41 
10. 
STATISTICAL METHODS AND SAMPLE SIZE ..................................................... 42 
10.1 
Sample size calculation ........................................................................................ 42 
10.2 
Statistical analysis ................................................................................................ 42 
11. 
STUDY TIMETABLE AND END OF STUDY .......................................................... 43 
12. 
LIST OF REFERENCES ....................................................................................... 44 
13. 
PREPARATION OF THE STUDY DRUGS .............................................................. 48 
13.1 
Logistics ................................................................................................................. 48 
13.2 
Levosimendan and Milrinone preparations ....................................................... 48 
13.3 
Infusion rate for a normal dose ........................................................................... 48 
13.4 
Bolus dose / half-bolus dose ............................................................................... 49 
13.5 
Doubling the dose continuously .......................................................................... 49 
13.6 
The calculations .................................................................................................... 49 
13.6.1 
Infusion rate and the dose delivered .................................................................. 49 
13.6.2 
Bolus dose ............................................................................................................. 49 
13.6.2.1 Levosimendan ....................................................................................................... 49 
13.6.2.2 Milrinone ............................................................................................................... 50 
 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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LIST OF APPENDICES 
Appendix A 
Signatures  
Appendix B 
The preparation of the study drugs and the doses  
Appendix C  
Additional Safety Information 
Appendix D 
 Ethics and regulatory review 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS  
The following abbreviations and special terms are used in this study protocol. 
Abbreviation or 
special term 
Explanation 
AE 
Adverse event 
AKI 
Acute Kidney Injury 
ARF 
Acute Renal Failure 
AVSD 
Atrioventricular septum defect 
CHDs 
Congenital heart diseases 
CPB 
Cardiopulmonary bypass 
CRF 
Case report form 
DAE 
Discontinuation due to Adverse Event 
EC 
Ethics Committee, synonymous to Institutional Review Board (IRB) 
and Independent Ethics Committee (IEC) 
ECG 
Electrocardiogram 
ECMO 
Extracorporeal membrane oxygenation 
GCP 
Good Clinical Practice 
ICU 
Intensive care unit 
ICH 
International Conference on Harmonisation 
KATP 
Potassium-sensitive adenosine triphosphate channels 
MMP-9 
Matrix metalloproteinase-9 
OAE 
Other Significant Adverse Event (ie, adverse events of particular 
clinical importance, other than SAEs and those AEs leading to 
discontinuation of the subject from study treatment 
NGAL 
Neutrophil gelatinase-associated lipocalin 
PICU 
Pediatric intensive care unit 
SAE 
Serious adverse event 
SIRS 
Systemic inflammatory response syndrome 
SOPs 
Standard Operating Procedures 
SUSARs 
Suspected unexpected severe adverse reactions 
TOF 
Tetralogy of Fallot 
VSD 
Ventricle septum defect 
 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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1. 
INTRODUCTION 
1.1 
The importance of acute kidney injury 
Acute renal failure (ARF), which recently has been refined and defined as acute kidney 
injury (AKI), is a common and serious condition in critically ill pediatric and adult 
patients. Due to lack of standard definition of ARF the reported incidence of AKI has 
varied considerably, namely between 5 and 50% of critically ill patients, both children 
and adults. The morbidity and mortality of AKI is unacceptably high. The reported 
mortality rates of about 40-50% in dialysis-dependent AKI, demonstrates the 
seriousness of the condition.  AKI is an independent factor for mortality in critically ill 
patients, meaning that the patients do not simply die with AKI but also from AKI. The 
treatment is supportive in addition to reversing the underlying causes. The supportive 
measures include optimizing fluid and cardiorespiratory status, avoiding nephrotoxic 
medications and renal replacement therapy [2]. 
1.2 
Etiology of AKI 
It is well known that cardiopulmonary bypass (CPB) is associated with AKI [3-6]. 
Pediatric patients with cyanotic congenital heart disease undergoing CPB are at 
increased risk for developing AKI [7-9]. The pathophysiology of CPB-related AKI include 
renal 
hypoperfusion, 
ischemia-reperfusion 
injury 
and 
CPB-induced 
systemic 
inflammatory response with activation of the blood cascade systems [2]. The 
complement system is one of the initiators of the systemic inflammation. The cause of 
systemic inflammatory response syndrome (SIRS) is the contact between blood 
components and CPB artificial surfaces. This contact is perceived as a danger signal by 
the body [10]. Other danger signals, and hence SIRS contributors, are ischemia-
reperfusion, surgical trauma, and probably endotoxemia. The danger signals activate 
leukocytes and endothelial cells as well as the plasma cascade systems [11].  The blood 
cascade systems are comprised by the coagulation, fibrinolytic, kallikrein-kinin, and 
complement systems. In SIRS there is an unproportional activation of these systems, 
which is harmful to patients especially in those who have other risk factors. These 
cascade systems are mutually correlated and “cross talk” to each other. This means that 
activation of one system may lead to the activation of the other systems [12, 13].  
1.3 
Diagnosis of AKI 
The diagnosis of AKI is based on clinically and laboratory variables like decreased urine 
output and increased serum creatinine (the cornerstone of the AKI) [14]. However, the 
fact that creatinine is a late marker of AKI, has led to a considerable search for other 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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biomarkers enable to early predict the proceeding AKI. Early prediction of AKI is 
important for timely initiation of prophylactic treatment options. One promising 
biomarker is NGAL [15]. 
1.4 
Prevention of AKI 
There is no causal treatment for AKI, highlighting the importance of the preventive 
measures. As mentioned previously, renal hypoperfusion is one of the etiologies to AKI. 
In this study we aim to intervene renal hypoperfusion (by Levosimendan).  
1.5 
Levosimendan 
1.5.1 
Mechanism of action 
Levosimendan belongs to a relatively new group of drugs which is called calcium-
sensitizing. Levosimendan enhances the contraction of heart muscle fibers at the same 
level of cytosolic calcium concentration. It performs this by holding the calcium-binding 
site of troponin C in its active conformation which leads to more binding at lower 
intracardiac calcium concentrations. The advantage of this approach, compared with 
other inotropes, is the needlessness of increased cytosolic calcium. Hence, the negative 
effects of increased cytosolic calcium on oxygen consumption and mitochondrial 
function can be avoided in addition to the avoidance of the activation of various calcium-
dependent proteases and phospholipases which mediate systemic inflammation during 
ischemia-reperfusion. Levosimendan also stimulates membrane potassium-sensitive 
adenosine triphosphate (KATP) channels. This leads to dilation of peripheral and 
coronary vessels. Levosimendan also stimulates the same channels in mitochondria and 
opens them, which may be an important mechanism of pharmacologic preconditioning 
and cytoprotection. Levosimendan differs from other inotropic agents like dopamine, 
amrinone, and Milrinone, with regard to maintaining the positive inotropic properties 
even in the abnormal myocardium.  Levosimendan mechanism of action indicates 
limited, if any, potential to initiation of arrhythmias, which is confirmed by clinical 
experience. At much greater concentrations, it does inhibit phosphodiesterase III (like 
Milrinone), but this effect does not appear to be relevant at clinical concentrations [16].  
1.5.2 
Effects in adults 
Clinical effects include improved cardiac output, reduced ventricular filling pressures, 
and decreased pulmonary vascular resistance during the acute treatment of adult 
patients with either stable or decompensated heart failure. In addition there are 
evidence for improved cardiac performance in the post cardiotomy and post bypass 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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setting. There have also demonstrated beneficial responses in adult patients who 
appeared to be poorly responsive to other inotropes [17].  
 
1.5.3 
Effects and pharmacokinetics in Children 
Levosimendan is commonly used in the pediatric intensive care units (PICUs), despite 
the fact that the drug, like many other drugs in PICUs, is not registered for use in 
children. In two recent reports [18, 19] the authors assessed the current practices at 
European hospitals related to the prevention and treatment of low cardiac output 
syndrome in children undergoing open heart surgery. Results demonstrated that 
Levosimendan belongs to the most common drug used for this indication in European 
countries. The most common drugs were Milrinone, dopamine, epinephrine, dobutamine 
and Levosimendan, which together comprised up to 85.9% of the total drug usage for 
this indication. The dosage of the off-label usage of Levosimendan in PICUs was usually 
similar to that of adult dosage regimen, i.e. 12 μg/kg loading dose followed by an 
infusion of 0.1-0.2 μg/kg/min. This dosage is consistent with current recommendation 
from the pharmacy at the Queen Silvia Children's Hospital for the use of levosimendan in 
the PICU. However, in some centers the initial bolus dose was excluded to avoid an 
eventual iatrogenic hypotension. In some other centers norepinephrine infusion was 
administrated simultaneous with the start of Levosimendan infusion. The exclusion of a 
bolus dose in some hospitals occurred when Levosimendan was first introduced and 
clinicians were more cautious, both in adults and children. Below follows a brief 
literature review on the use of levosimendan in pediatric cardiac surgery. 
The first two case reports on the use of Levosimendan in infants with CHDs were 
published in 2004 [20, 21]. In the first report Levosimendan was used after cardiac 
surgery in a two months old infant with complicated postoperative course; acute left 
ventricle failure and pulmonary vascular hypertension [20]. The infant received 24 µg 
/kg Levosimendan as a bolus dose followed by an infusion of 0.2 µg /kg/minute for the 
next 48 hours. Immediately after administration of bolus dose a significant increase in 
left ventricle function could be demonstrated by echocardiography. In this case 
Levosimendan caused a breakthrough in the treatment and increasing the function of left 
ventricle decreased also the vascular resistance. The other case report [21] (in German) 
was a report on an infant with myocardial infarction due to congenital stenosis of the left 
coronary artery, left ventricular dysfunction, mitral regurgitation and refractory 
pulmonary hypertension The authors proposed the use of Levosimendan as a long-term 
inotropic agent and pulmonary vasodilator in children with depressed cardiac function. 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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In the same year, the first report on the pharmacokinetics of Levosimendan in children 
was published [22]. The children were between three months and seven years old and 
were being evaluated for cardiac surgery in a cardiac catheter laboratory. All children 
received 12 µg/kg Levosimendan as an intravenous infusion over 10 minutes during the 
catheterization. The authors of this small study concluded that the pharmacokinetic 
profile of Levosimendan in children with congenital heart disease was similar to that in 
adult patients with congenital heart diseases. In 2006, in a small retrospective 
observational safety study, it was demonstrated that Levosimendan did not worsen the 
clinical conditions, and the patients did not suffer from adverse events attributable to 
Levosimendan [23]. The number of patients in this study was nineteen; they were 
between seven days and ten years old, suffered from established low cardiac output 
syndrome after congenital heart surgery. The dose of Levosimendan was 12 µg/kg over 
60 minutes followed by 0.1 µg/kg/minute for 24 hour. In another retrospective cohort 
study from the same year, fifteen pediatric patients with ventricular dysfunction were 
included and the hemodynamics after administration of Levosimendan was analyzed 
[24]. The patients (aged seven days to eighteen years) had different diagnosis and 
underwent different procedures. All patients received a bolus dose of Levosimendan (6-
12 µg/kg over 10 minutes) followed by either a continuous infusion of 0.1 µg/kg/minute 
for 24 hours (in thirteen children) or 0.05 µg/kg/minute for 48 hours (in two children). 
The authors concluded that Levosimendan could safely be administered to infants and 
children with severe heart failure. In 2007 the first report on the successful use of 
Levosimendan in a premature infant with congestive heart failure following cardiac 
surgery was published [25]. The patient was a 32 weeks gestational old premature 
neonate, weighted 1525 grams and suffered from transposition of great arteries. The 
postoperative course became complex immediately after surgery. Levosimendan 
infusion was initiated by a dose of 0.05 µg/kg/minute which was increased to 0.1 
µg/kg/minute. The infusion lasted for 24 hours. Within 6 hours after start of 
Levosimendan infusion a marked clinical improvement could be demonstrated. The 
authors concluded that Levosimendan was a potent inotropic agent in this patient. In 
2009 Osthaus and his colleagues published a report on the intraoperative Levosimendan 
use in seven infants with body weight between 2.6 and 6.3 kg [26]. In four children 
Levosimendan infusion was started during weaning from CPB, while in the other three 
patients it was administered after the termination of CPB but before the chest closure. 
All patients received Levosimendan as a loading dose of 12 µg/kg over 10 minutes, 
followed by an infusion of 0.2 µg/kg/minute for 24 hours. The authors demonstrated 
that the intraoperatively administered Levosimendan was well tolerated in these infants 
with severe myocardial dysfunction after complex congenital heart surgery. In 2011, 
Momeni and her colleagues published a paper on comparing the hemodynamical and the 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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biochemical effects of Levosimendan and Milrinone when used as inotropic support after 
congenital heart surgery [27]. The study was prospective, randomized and double 
blinded. Forty-one patients between 0 and 5 years old were enrolled; thirty-six patients 
completed the study. The patients were randomized to a continuous infusion of either 
Levosimendan (0.05 µg/kg/minute) or Milrinone  (0.4 µg/kg/minute). Epinephrine was 
started after aortic cross-clamp release in both groups (0.02 µg/kg/minute). The authors 
concluded that Levosimendan is at least as efficacious as Milrinone after corrective 
congenital heart surgery. In 2012, Lechner and her colleagues reported a study on the 
prophylactic use of Levosimendan versus Milrinone in neonates and infants after 
corrective heart surgery [28]. The study was prospective, randomized and double blind 
and included 40 infants younger than one year of age. At weaning from CPB, either a 24-
hours infusion of 0.1 µg/kg/minute Levosimendan or 0.5 µg/kg/minute Milrinone were 
administered. The authors observed an increase in cardiac output and cardiac index over 
time in the Levosimendan group, whereas cardiac output and cardiac index remained 
stable in the Milrinone group.   
The elimination half-life of Levosimendan is about 1 hour, but there is at least one 
metabolite that has prolonged effects of at least 80 hours. This may account for the 
observations of sustained benefit after discontinuation of the drug. Levosimendan can be 
infused simultaneously with any other medications. However, cautious should be taken 
in case of infusion of arrythmogenic drugs. It should be added that pharmacokinetic data 
on Levosimendan in children, infants, and neonates is scarce. It is preferable to gain a 
better understanding of Levosimendan pharmacokinetics in these groups of patients. In 
the current study we intend to analyze the blood samples in respect to the 
Levosimendan concentrations in addition to other analyses such as neurobiomarkers, 
complement system and NGAL. 
1.5.4 
Dosage and interactions with other medications 
As earlier mentioned, Levosimendan is not registered for use in a paediatric population. 
In the lack of pediatric and neonatal data, the drug has been used with an adult dosage 
regimen with successful results, which has been presented above. The adult dosage of 
Levosimendan is 12 μg/kg loading dose followed by an infusion of 0.1-0.2 μg/kg/min. 
This is the also the recommendation dose of the pharmacy at Queen Silvias Children’s 
Hospital for use of Levosimendan in PICU.  The elimination half-life of Levosimendan is 
about 1 hour, but there is at least one metabolite that has prolonged effects of about 80 
hours. This may account for the observations of sustained benefit after discontinuation 
of the drug. Levosimendan can be infused simultaneously with any other medications. 
However, cautious should be taken in case of infusion of arrythmogenic drugs. 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
18(62) 
1.6 
Milrinone 
Milrinone is a inotrope drug used in infants and children undergoing surgery for 
congenital heart disease and is indicated for the short-term treatment of postoperative 
severe heart failure who do not respond to conventional maintenance therapy. It s also 
indicated for short-term treatment (up to 35 hours) of pediatric patients with acute 
heart failure, including conditions of low cardiac output after cardiac surgery.  
1.7 
Research hypothesis 
Can Levosimendan reduce the incidence of acute renal dysfunction after cardiac surgery 
in paediatric children with congenital heart disease?  
1.8 
Rationale for conducting this study 
In this study we will compare the kidney protective ability of Levosimendan compared 
with Milrinone [29-31]. Levosimendan is an inotrope drug not registered for use in 
pediatric populations for any indication, however it is currently used as an inotrope in 
PICUs. Milrinone is another inotrope drug, which is registered for short-term use in 
pediatric populations . Milrinone is normally used in PICUs in conjunction with pediatric 
cardiac surgery. There is evidence that Levosimendan, in addition to its inotropic effect, 
preserves adequate renal perfusion and protects kidneys from injury. This should lead to 
a less increased creatinine postoperatively. Our aim is to assess the efficacy of 
Levosimendan in reducing creatinine postoperatively compared to Milrinone.  
1.9 
Benefit/risk assessment  
Levosimendan is primarily indicated in adults for short term treatment of acutely de-
compensated severe chronic heart failure in situations where conventional therapy is 
inadequate or insufficient. However, Levosimendan has also been used also in children 
and neonates in both Sweden and Europe in connection with complex congenital heart 
surgery.  
Levosimendan, Milrinone, dopamine, epinephrine, dobutamine are all common drugs 
used in the prevention and treatment of low cardiac output syndrome in European 
children undergoing open heart surgery. Milrinone is used routinely in our clinic. It is a 
registered inotropic drug in the paediatric population. 
Levosimendan and Milrinone has been considered well tolerated and has a potential 
benefit on postoperative hemodynamic and metabolic parameters. An initial 
hemodynamic adverse effect may be a reduction in systolic and diastolic blood pressure.  

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
19(62) 
We estimate that we do not expose patients to any additional significant risk. Infusion of 
Levosimendan and Milrinone is indicated for use only in hospitals where adequate 
supervision and experience with inotropic agents are available. Solid routines are 
followed during surgery, the operation team are well trained in case of emergency and 
the patients are thoroughly monitored during the stay in the intensive care unit. 
Prophylactic measures are always taken, e.g. infusion of vasoactive medication for 
preventing hypotension and insertion of pace-maker leads for treatment of therapy 
resistant arrhythmias. 
1.10 
Ethical considerations 
This study will be conducted in compliance with ICH Good Clinical Practice and 
applicable regulatory requirements and in accordance with the ethical principles in the 
Declaration of Helsinki. For detailed information regarding ethics and regulatory review, 
informed consent, data protection and audits and inspections see Appendix D (Ethics).  
Written approval must be obtained from the regional ethics committees and regulatory 
authorities prior to start of the trial. 
The investigator will ensure that both parents/guardians (legal representative (s) of the 
child) are given full and adequate oral and written information about the nature, 
purpose, possible risk and benefit of the study and are notified that they/their child are 
free to discontinue from the study at any time. The parent/guardian will be given the 
opportunity to ask questions and allowed time to consider the information provided. 
Signed and dated informed consent should be obtained before conducting any study 
specific procedures and stored in the Study Master File. A copy of the signed Informed 
Consent Form is kept by the parents/guardians (legal representative (s) of the child). 
2. 
STUDY OBJECTIVES AND ENDPOINT(S) 
2.1 
Primary objective 
The aim of this study is to assess the efficacy of Levosimendan to reduce the 
postoperative acute kidney injury in pediatric patients undergoing surgery for 
congenital heart disease (CHDs) having creatinine as an endpoint marker. 
2.2 
Primary endpoint 
AKI is usually defined as 50% increase in creatinine within 48 hours. Accordingly, the 
primary outcome in this study is the postoperative creatinine levels which will be 
measured according to table 1.  

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
20(62) 
2.3 
Secondary endpoints 
The secondary endpoints are the hemodynamic parameters, fluid balance, the level of 
inflammation and other plasma kidney biomarkers (like cystatin C and NGAL), and 
markers of neural injury. Other parameters will also be assessed such as length of 
surgery, length of CPB, length of mechanical ventilation postoperatively, length of 
vasoactive therapy, number of vasoactive medications and total amount of infused 
vasoactive drugs.  
3. 
STUDY DESIGN 
This is a prospective, randomized, parallel-group, and double-blinded study in pediatric 
patients in need for surgery due to congenital heart disease. The study will be performed 
at the Department of anesthesia and intensive care unit at The Queen Silvia Children’s 
Hospital, Göteborg, Sweden. 
A total of 70 male and female pediatric patients will be included in the study and 
undergo heart surgery according to routine procedures at the hospital. Patients will be 
randomly assigned to receive either Levosimendan (test group) or Milrinone (control 
group). Creatinine will be measured daily according to the clinical routines. The length of 
treatment (Levosimendam or Milrinone) will be 24 hours. The treatment will be started 
during the operation at the same time-point at which Milrinone infusion routinely starts. 
Study-specific blood samples will be obtained at five occasions, during 24 hours 
perioperatively. Patients will be followed up as by routine as long as they are in PICU. 
Patients will be followed for an additional 4 days after treatment termination regardless 
if they are in ICU or they are being cared at in the ward. Final follow-up will occur 30 
days after surgery. Overall study design and study activities are shown in Figures 1 and 2 
and Table 1. 
Figure 1 
 
Study design 
Levosimendan (active)  
n=35 
Milrinone (control) 
 
n=35 
Time points:  
 
0 
 
2h 
6h 
 
 
24h 
48h 
3d 
4d 
5d        30d 
T0 
T1 
T2 
T3 
 
 
T4 
 
Clinical f-up 
R 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
21(62) 
Table 1  
Study Activities 
Pre-
study 
Surgery 
Post-surgery 
Follow-up 
 
T0 
T1 
T2 
T3 
T4 
 
 
 
At:
 
Baseline 
 
2 hours 6 hours 24 hours 48 hours 4, 5 days 30 days  
Informed consent 
X 
 
 
 
 
 
 
 
 
Demography 
X 
 
 
 
 
 
 
 
 
Weight, height 
X 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
Physical examination 
X 
 
 
 
 
 
 
 
 
Inclusion/exclusion 
criteria 
X 
X 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
 
Surgery 
 
X 
 
 
 
 
 
 
 
Levosimendan/Milrinone 
 
X 
X 
X 
X 
X 
 
 
 
Pulse, Blood pressure 
 
X 
X 
X 
X 
X 
X 
X 
 
Routine laboratory 
screening (minimum 
Creatinine, S-K) 
 
X 
X 
X 
X 
X 
X 
X 
 
 
Pharmacokinetics 
 
 
 
 
X 
X 
X 
X 
 
Study specific blood 
 
X 
X 
X 
X 
X 
 
 
 
Continuous ECG 
monitoring 
 
X 
X 
X 
X 
X 
X 
 
 
Continuous 
hemodynamic and 
respiratory monitoring 
 
X 
X 
X 
X 
X 
 
 
 
Serious Adverse event 
recording 
 
X 
X 
X 
X 
X 
 
 
 
Urine production 
 
X 
X 
X 
X 
X 
 
X 
 
Adverse Events registr* 
 
X 
X 
X 
X 
 X 
X 
X 
X 
Registration of mortality 
 
 
 
 
 
 
 
 
X 
Registration of length of 
stay (bed-days) 
 
 
 
 
 
 
 
 
X 
Routine echocardiography (preoperative, after CPB termination, first postoperative day, and at discharge)  
* Registration of adverse events will also be performed at day 30 (e.g. telephone contact with the parents if 
the patient is at home)  
 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
22(62) 
 
 
 
Figure 2 
Study flow chart 
 
 
The infusion of Levosimendan/Milrinone will start perioperatively when the patient is 
on cardiopulmonary bypass. The infusion will thereafter continue in the intensive care 
unit (ICU) and will be terminated after 24 hours. Patients in both groups will be followed 
up according the normal clinical routines. Routine echocardiography will be performed 
at pre-defined time-points as a clinical routine (pre-operatively, after termination of CPB 
intra-operatively, at first postoperative day, and at discharge). Extra study specific blood 
samples will be taken from the included patients. Patients who develop AKI will be 
identified by increase in s-Creatinine >50% from baseline. The usual clinical and 
laboratory data will be routinely obtained and recorded in the hospital electronic 
records. Study-specific parameters will be registered in a CRF. 
Follow-up of the patients will continue 4 days after termination of Levosimendan 
/Milrinone infusion. The follow-up parameters, included in case report form (CRF), 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
23(62) 
include among others treatment-needing hypotension, treatment-needing arrhythmias, 
s-Creatinine, s-Potassium and albumin.  
3.1.1 
Stopping criteria for dose change 
General stopping criteria are hypersensitivity reactions to the active substance or 
any recipients.  
The children are thoroughly monitored during surgery and postoperatively as per 
routine at the clinic including continuous blood pressure, pulse, and ECG monitoring.  It 
should be noted that the initiation of Levosimendan/Milrinone occurs during the 
cardiopulmonary bypass time where cardiopulmonary function parameters (like blood 
pressure) are optimized before the termination of cardiopulmonary bypass. 
3.2 
Rationale for study design, doses and control groups 
Our department in Sahlgrenska University Hospital, the Department of anesthesiology 
and intensive care, includes both the adults and children sections. The adult section in 
our department has been proactive in producing evidence that Levosimendan may 
protect the kidney from injury in conjunction with cardiac surgery in adults [1]. This 
study was published in 2013. The overall rationale for the planned children study is the 
same as we reported in the adult study from 2013. The aim is to investigate whether the 
findings in adults are applicable in children, given the facts that the impact of cardiac 
surgery is the same in adults and children and the pharmacokinetics of the 
Levosimendan, despite scarce of data, are principally the same. In practice, the adult 
doses of Levosimendan are used in infants and children. Levosimendan in children is 
used as an inotropic drug, and to our knowledge it has not been used as a kidney 
protective agent in children yet.  
Control group: Milrinone is the inotrope standard of care in this setting. According to 
Good Clinical Practice if there is a standard of care, it should be used instead for placebo. 
To our knowledge the kidney-related effects of Milrinone per se has not been 
investigated yet.  
4. 
SUBJECT POPULATION 
A total of 70 children between 1 and 12 months of age will be selected from the pediatric 
patients who undergo corrective cardiac surgery at the Queen Silvia´s children hospital 
in Göteborg, Sweden. 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
24(62) 
Subject population should be selected without bias. Investigator(s) must keep a record of 
all subjects even those who entered pre-trial screening but were never enrolled. Use a 
subject screening log. Each subject must meet all of the inclusion criteria and none of the 
exclusion criteria for this study.  
CHDs include some twenty major diagnoses, with many anatomical combinations and 
physiologic variations as well as prognoses. The birth prevalence of CHD is about 9 per 
1000 live births. With a couple of exceptions, the prevalence of any single diagnosis is 
also very low which influence the research in this group of patients due to limited 
number of patients treated in each center. In this study we have chosen a study 
population made of three diagnoses. 
4.1 
Inclusion criteria 
For inclusion in the study subjects must fulfill the following criteria.  
1. Provision of informed consent prior to any study specific procedures 
2. Female and male children between 1 and 12 months of age 
3. Non-restrictive VSD (corrective surgery) 
4. Complete AVSD (biventricular repair) 
5. Tetralogy of Fallot  
4.2 
Exclusion criteria 
Subjects must not enter the study if any of the following exclusion criteria are fulfilled  
1. Unbalanced AVSD 
2. Age less than one month and more than one year 
3. Acute operation that is unscheduled operation during the first 24 hours after 
presentation to the department for thoracic surgery 
4. Mild, moderate, or severe kidney dysfunction 
5. Liver impairment or disease 
 
6. Ongoing infection 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
25(62) 
7.  Use of nephrotoxic drugs (like ibuprofen, angiotensin-converting-enzyme inhibitors, 
gentamicin, vancomycin) preoperative or postoperative until first post operative day 
8. Use of inhibitors of membrane transport proteins (Cimetedin, cetirizine, trimethoprim, 
probenecid, rifampin and gemfibrosil 
9. Allergy to Levosimendan or substance included in the preparation or previous use of 
Levosimendan 
10. Severe arrhythmias needing pace-maker treatment prior to the operation 
11. Severe cardiac dysfunction needing for treatment with extracorporeal membrane 
oxygenation (ECMO) prior to the operation 
12. Severe uncorrected hypotension where the initiation of anesthesia is postponed for 
its correction 
13. Re-operation 
4.3 
Subject enrolment and randomization 
The principal investigator will obtain signed informed consent from the potential subject 
i.e. parent/guardian (legal representative (s) of the child) before any study specific 
procedures are performed and determine subject eligibility. 
Randomization codes will be assigned strictly sequentially as subjects are eligible for 
randomization according to a pre-defined treatment schedule. If a subject discontinues 
participation in the study, his/her enrolment/subject number cannot be reused. 
Discontinued subjects are not allowed to re-enter the study.  
4.3.1 
Safety and risk minimization in the scope of randomization  
A nurse who is not directly involved in the patient care will perform the randomization 
(by a net-based randomization program) prior to the operation (“randomizer”). Two or 
three nurses will be trained in working with the randomization soft ware to cover the 
risk of the absence of randomizer. The randomizers will follow the rule of confidentiality 
regarding the result of randomization. The randomization result will be kept in a 
predefined locked safe at our PICU.  
Children undergoing surgery for congenital heart diseases belong to different prognostic 
groups, and their outcomes vary depending on the specific prognostic group they belong 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
26(62) 
to [32]. The statistical methods of either stratified randomization or minimization 
should be used in these cases to achieve approximate balance of important 
characteristics in each group.  
 
In clinical trials with larger sample size and need for several stratifying variables, 
stratified randomization would be appropriate while in the smaller studies needing 
several stratifying variables the statistical method called minimization is more 
appropriate [33, 34].  However, in smaller studies with only one or two stratification 
variables stratified randomization may be used. In the current study, the diagnosis and 
the age of the patients constitute known prognostic factors, which should be used as 
stratifying variables. Hence, we will use stratified randomization with the forehead 
mentioned stratifying factors for randomization of the patients.   
4.4 
Discontinuation and Withdrawal of subjects 
The parent(s) are at any time free to discontinue his/her child’s participation in the 
study, without prejudice to further treatment. The subjects may be withdrawn from the 
study at the discretion of the investigator due to safety concerns, adverse events or if 
judged non-compliant with study procedures. Other reasons for discontinuing a subject 
are incorrect enrolment and subjects lost to follow-up. Other reasons for discontinuing a 
subject are incorrect enrolment subjects lost to follow-up, new diagnosis, significant 
complications. In either case serious adverse events will be followed up. 
4.5 
Temporary or Premature termination of the study 
The Investigator may decide to temporary or permanently stop the trial or part of the 
trial at any time in case of a substantial amendment or as a part of an urgent safety 
measure. Urgent safety measures may be taken without prior notification. If a trial is 
prematurely terminated or suspended, the Investigator should promptly inform the 
parent/guardian and ensure appropriate therapy and follow-up. Furthermore, the 
Investigator should promptly inform the Ethics committee and provide a detailed 
written explanation. The regulatory authority should be informed according to national 
regulations. 
5. 
TREATMENTS 
5.1 
Test and control drugs  
Levosimendan (test) and Milrinone (control) will be used in the present study. 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
27(62) 
 
 
 
Table 2 
 
Identity of investigational products  
Investigational product 
Dosage form and strength 
Manufacturer 
Levosimendan (Simdax®) 
2.5 mg/mL solution. Diluted to 
0.05 mg/mL 
Orion Corporation,   
Orionintie 1, FI-02200 Espoo, 
Finland 
Milrinone Abcur 
Solution for Injection 1 mg / ml Abcur AB. Box 1452,            
251 14 Helsingborg 
 
5.1.1 
 Levosimendan  
Levosimendan, a calcium-sensitizer (ATC-kod: C01CX08), has been developed for short-
time treatment of de-compensated heart failure and is used intravenously when patients 
with heart failure require immediate initiation of drug therapy and when conventional 
therapy is insufficient. It increases cardiac contractility and induces vasodilatation.  
The pharmacokinetics of Levosimendan is linear at the therapeutic dose range of 0.05-
0.2 µg /kg/minute. The short half-life (about 1 hour) of the parent drug, Levosimendan, 
enables fast onset of drug action, although the effects are long-lasting. The maximum 
concentrations of metabolites (OR-1855 and OR-1896) are seen on average 2 days after 
stopping a 24-hour infusion. It is completely metabolised and negligible amounts of 
unchanged parent compound are excreted in urine and faeces. 
Limited data suggest that the pharmacokinetics of levosimendan in children (3 months - 
6 years of age) after a single dose is similar to that in adults. 
There are no particular risks with short-term use regarding general toxicity and gene 
toxicity. 
Limited data suggest that the pharmacokinetics of Levosimendan in children (3 months-
6 years of age) after a single dose is similar to that in adults. The pharmacokinetics of the 
active metabolite has not been studied in children. 
No pharmacokinetic interactions have been observed. However, cautious should be 
taken in case of all arrythmogenic drugs or those which decrease blood pressure like 
beta blockades. 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
28(62) 
5.1.2 
Milrinone 
Milrinone is a phosphodiesterase inhibitor (ATC-kod: C01CE02).  It is a positive 
inotropic and vasodilating substance. Milrinone is indicated for short term treatment (up 
to 35 hours ) of pediatric patients with acute heart failure, including permits low output 
after cardiac surgery.   
In published studies, selected doses for infants and children were i.v. loading dose of 50-
75 g/kg during 30 to 60 minutes or continuous i.v. infusion initiated on the basis of the 
hemodynamic response and the potential occurrence of adverse events between 0.25-
0.75 micrograms/kg/min for a period of up to 35 hours. 
Milrinone eliminats faster in children than in adults and neonates have significantly 
lower clearance than children. It has a mean terminal half-life of 2 to 4 hours in infants 
and children 
Milrinone is slowly injected intravenously into a large vein to avoid local irritation. 
5.2 
Doses, preparations and treatment regimens 
For further details see Appendix B. 
5.2.1 
Organization for preparation of the study drugs 
An experienced specialized nurse in anesthesia or PICU will have the responsibility for 
preparation of the medications for the day. The candidate nurses will receive extra 
training regarding the course of the trial. The randomizer delivers the result of the 
randomization confidentially to the nurse responsible for the drug preparation.  The 
drug preparation responsible nurse will not be involved in the care of that particular 
patient and will follow the rule of confidentiality regarding the result of randomization.  
In our department, the department of anesthesiology and intensive care, there are 
“medication cards” for drugs used regularly. Each drug has its own card which is 
approved by the hospital pharmacy. These cards include an exact presentation of the 
drug as well as the exact process of preparing them. These cards are available in drug 
preparing rooms and used every day by nurses preparing the drugs. The cards are 
prepared for pediatric populations where the final concentration of them usually differs 
from that for adults. The potent drugs, like Milrinon and Levosimendan are prepared 
only in one standard concentration described in the medication card.   

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
29(62) 
The study drugs will be prepared according to the existing medication cards and in 
accordance with usual clinical work. There will be neither a change in the final 
concentration neither of the drugs nor in the process of preparing them.    
5.2.2 
Preparing final concentrations 
Both Levosimendan and Milrinone are diluted in glucose 50 mg/mL and prepared in 50 
mL infusion syringes according to the respective medication card. The final 
concentration of Levosimendan will be 0.05 mg/mL and final concentration of Milrinone 
will be 0.2 mg/mL.  
5.2.3 
Infusion rates  
With the final concentrations described above and for delivering a normal dose, 
Levosimendan is infused in a rate of 0.12 mL/kg/h (which delivers 0.1 μg/kg/min) and 
Milrinone is infused in a rate of 0.15 mL/kg/h (which delivers 0.5 μg/kg/min).  
The recommended dose of Levosimendan is 0.1-0.2 μg/kg/min and the recommended 
dose of Milrinone is 0.3 – 1.0 μg/kg/min. 
5.2.4 
Dosage scheme in present study 
In a double blind study like the present study, it is obvious that the drugs must be 
infused in one single infusion rate to protect the blindness of the study. In the present 
study, the infusion rate will be 0.12 mL/kg/h. With this infusion rate the dose delivered 
will thus be 0.1 μg/kg/min for Levosimendan and 0.4 μg/kg/min for Milrinone.  
For delivering a bolus dose we will infuse the study drug at a rate of 1.44 mL/kg/h in 10 
minutes (the pump will also be programmed for a maximal infusion volume of 0.24 
mL/kg). This will deliver a bolus dose 12 μg/kg of Levosimendan, which is the 
recommended bolus dose for Levosimendan and 48 μg/kg of Milrinon (recomended 
bolus dose for Milrinone is 50 μg/kg).  
5.2.5 
Quality control of the medication preparation 
According to the routines of our department drug preparations should always be 
performed according to the medication cards. The cards should be put on the 
preparation table and the described steps should be followed step by step. In addition 
there should be one additional nurse who checks the correctness of the drug preparation 
for each drug.  In this study we will follow the routines of our department in all aspects. 
The second nurse, who quality controls the preparation of the medication, will not be 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
30(62) 
involved in the care of the patient and will follow the rule of confidentiality regarding the 
result of randomization. 
5.2.6 
Storage conditions 
The product should be used immediate after solving but can be stored at 2-8°C for a 
maximum time of 24 hours. 
All study drugs should be kept in a secure place under appropriate storage conditions as 
specified on the pack label and handled according to the manufacturer’s instructions. 
Handling of Levosimendan/Mirinone will follow recommendations in FASS and the 
medication cards at the department. 
After preparation of the study medication for 24 hours (the length of infusion), the 
syringes will be coupled to the infusion sets. The infusion sets will be filled with the 
content of the syringes so that they are ready to be coupled to the patient.  A label with 
the word “Study” and “the inclusion number of the patient” will be paced on each 
syringe. The syringes and the sets are placed in a drug paper tray and stored in the drug 
refrigerator in PICU. The first syringe will be used for start of infusion during operation 
and cardiopulmonary bypass.   
5.2.7 
Blinding and unblinding procedures 
Levosimendan and Milrinone will be prepared in brown syringes originally made for 
medications needing to be protected from daylight. The reason for this is that the color 
of prepared Levosimendan is yellow. From the scope of blindness, it is important that 
Levosimendan and Milrinone have the same appearance. The brown syringes and 
infusion sets will act well in this regard. For the same reason of blindness, that lumen of 
the central venous catheter, which will be used for infusion of the study medication, will 
be covered by surgical soft tape.   
The treatment code must not be broken except in medical emergencies when the 
appropriate management of the subject necessitates knowledge of the treatment 
randomization. 
5.2.8 
Labelling 
The syringes and study medication will be kept separately from other medications in the 
surgery ward. The study medication, syringes and related materials will be stored in a 
box labelled with: study code, i.v. Levosimendan/Milrinone, dosage form, strength, 
quantity, dosage instructions, medical cards with dosage instructions, name of 

Clinical Study Protocol 
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Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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investigator (to be filled in at study site), expiry date, storage instruction, “for clinical 
study use only”, address and telephone number of sponsor/investigator. 
5.3 
Concomitant and post-study treatment(s) 
Medication, which is used as standard therapy in cardiac surgery as well as other 
medications considered necessary for the subject’s safety and well-being, may be given 
at the discretion of the investigator. 
5.4 
Treatment compliance 
As treatment will be administered in the hospital, at the intensive care unit, under the 
supervision of medical staff, compliance is expected to be 100%.  
5.5 
Accountability 
The study drug provided for this study will be used only as directed in the study 
protocol. 
An experienced nurse at the anesthesia and intensive care unit will be appointed as 
"study medication preparer” for the day and will be responsible for preparing the study 
medication according to the routines for medicine preparation at the department.  
6. 
STUDY CONDUCT AND METHODS 
6.1 
Start and termination of study infusion and follow-up 
The infusion of Levosimendan/Milrinone starts during CPB and will be continued in our 
PICU for 24 hours. After 24 hours the infusion rate will be decreased and set to the half 
infusion rate for 2 hours. If the patient does not tolerate this dose reduction, we will shift 
from test medication to Milrinone and start a Milrinone infusion. If the patient tolerates 
the dose reduction for 2 hours, then we will stop completely the infusion of the test 
medication. Two 2 hours after reducing the infusion rate by 50%, we will stop the 
infusion completely.  
Both groups are followed up according to the normal clinical routines as long as they 
stay at PICU. Extra blood samples, in addition to those needed for usual clinical care, will 
be taken from the patients according the specified scheme presented in this document.  
The trial follow-up of the patients will continue 4 days after termination of 
Levosimendan/Milirone infusion. CRF includes the clinical and laboratory follow-up 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
32(62) 
parameters as well as adverse events (like treatment needing hypotension and 
arrhythmias), adverse reactions and SUSARS (se section 7). 
6.2 
Efficacy variables 
The usual clinical and laboratory data connected to this kind of heart surgery will be 
routinely obtained prior to surgery, during surgery, and during the care in PICU (see 
table 4).   
6.2.1 
Blood for study-specific analysis 
Blood samples for the analyses of pharmacokintetics, inflammatory biomarkers like 
complement components, neurodegenerative biomarkers, NGAL, and other biological 
markers relevant for AKI will be obtained. Samples will be analyzed after termination of 
the study. 
7. 
SAFETY 
Detailed information regarding safety is seen in Appendix C. 
7.1.1 
Safety procedures 
7.1.1.1 During surgery 
Solid routines will be followed during surgery including ECG-monitoring, invasive 
monitoring of hemodynamics (blood pressure and pulse), respiratory monitoring, and 
monitoring of cardiopulmonary bypass parameters including blood gases. Hemostasis is 
monitored with advanced in-site monitoring devices, and blood and blood products as 
well as coagulation factors are administered if needed.  The pediatric cardiac surgery 
operation theatre is a place for most advanced physiologic monitoring procedures. The 
cardiac surgery staff is among the most trained health care personnel in dealing with 
acute cardiac complications.       
7.1.1.2 Post-surgery follow-up 
The doctors, nurses, and other caring staff are well trained in emergencies related to 
post cardiac pediatric surgery. The patients are thoroughly monitored routinely during 
the stay in the PICU. Prophylactic measures are always taken, e.g. infusion of vasoactive 
medication for preventing hypotension and insertion of pace-maker leads for treatment 
of therapy resistant arrhythmias. Children will be carefully monitored during the 
treatment with Levosimendan/Milrinone (24 hours). They will be followed up by an 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
33(62) 
additional 48 hours after the termination of the treatment. Hypotension, arrhythmias, s-
creatinine and albumin will be checked and corrected if needed.   
7.1.2 
Medical emergencies  
The principal investigator(s) is responsible for ensuring that procedures and expertise 
are available to handle medical emergencies during the study. A medical emergency 
usually constitutes an SAE and is to be reported as such. 
It is of the utmost importance that all staff involved in the study is familiar with the 
content of this section. The principal investigator is responsible for ensuring this. 
7.1.3 
Common AEs after CHD surgery 
Cardiac adverse effects occur in a usual basis in intensive care units after cardiac 
surgery. Some prophylactic measures are always taken, i.e. infusion of vasoactive 
medication for preventing hypotension and insertion of pace-maker leads for treatment 
of therapy resistant arrhythmias (Table 3).  
Table 3  
Adverse events dependent on the disease or surgical treatment postoperatively 
 
Occurrence 
Treatable 
Usual 
Length of 
episodes 
Prophylactic 
Measures 
Watchfulness 
and 
Preparedness 
for initiation 
of treatment 
Careful 
monitoring 
Hypotension 
episodes 
Usual 
Yes 
Minutes 
Taken 
routinely 
Present 
Present 
Arrhythmias 
episodes 
Usual  
Yes 
Minutes to 
hours 
Taken 
routinely 
Present 
Present 
 
7.1.4 
Adverse reactions of study medication 
Data regarding adverse effects of Levosimendanmendan and Milrinone in a pediatric 
population are scare. The reported adverse reactions of Levosimendanmendan in adults 
are hypotension and arrhythmias (both ventricular and supra ventricular). Reported 
adverse reactons of Milrinone are headache, ventricular tachycardia, supraventricular 
arrhythmias and hypotension. The reactions are probable to occur in the present study 
and are treatable. Careful and adequate monitoring will be present 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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7.1.5 
Laboratory safety assessment 
Routine blood sample for laboratory safety screen will be taken at baseline and every 
morning in PICU as long as the patient is in PICU according to our clinical guidelines.  
Table 4 
Laboratory screen 
Clinical Chemistry 
Haematology 
S/P-Creatinine 
B-Haemoglobin (Hb) 
S/P-Cystatin C 
B-Platelet count (PPC) 
S/P -Bilirubin, total, 
conjugated/unconjugated 
B-Leukocytes, total count (LPC) 
S/P -Glucose 
B-Leukocytes diff.absolute count: 
S/P -Potassium (K) 
          B-Basofila 
S/P -Albumin 
          B-Eosinof 
S/P -Sodium (Na) 
          B-Lymfocyter 
S/P Reactive protein (CRP) 
          B-Monocyter 
 
          B Neutrofila 
 
B-Hematocrit (Hct) 
 
The blood samples will be analysed according to routine standards at the Department of 
Clinical Chemistry at the hospital. Valid reference values of all routine analyses will be 
obtained. Laboratory values outside the reference limit suspected to be of any clinical 
significance will be repeated.  
7.1.6 
Definition of adverse events (AE) 
An adverse event is the development of an undesirable medical condition or the 
deterioration of a pre-existing medical condition following or during exposure to a 
pharmaceutical product, whether or not considered causally related to the product. An 
undesirable medical condition can be symptoms (eg, nausea, chest pain), signs (eg, 
tachycardia, enlarged liver) or the abnormal results of an investigation (eg, laboratory 
findings, electrocardiogram). In clinical studies, an AE can include an undesirable 
medical condition occurring at any time, including run-in or washout periods, even if no 
study treatment has been administered. 
The term AE is used to include both serious and non-serious AEs 
7.1.7 
Definitions of serious adverse event (SAE) 
A serious adverse event is an AE occurring during any study phase (i.e, run-in, treatment, 
wash-out, follow-up), that fulfils one or more of the following criteria: 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
35(62) 
• 
Results in death 
• 
Is immediately life-threatening 
• 
Requires in-patient hospitalisation or prolongation of existing hospitalisation  
• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital abnormality or birth defect 
• 
Is an important medical event that may jeopardise the subject or may require 
medical intervention to prevent one of the outcomes listed above. 
For reporting purposes, any suspected transmission via a medicinal product of an 
infectious agent is also considered an SAE and is reported in an expedited manner. Any 
organism, virus or infectious particle (for example prion protein transmitting 
Transmissible 
Spongiform 
Encephalopathy), 
pathogenic 
or 
non-pathogenic, is 
considered an infectious agent. 
For further guidance on the definition of a SAE, see Appendix B to the Clinical Study 
Protocol. 
7.1.8 
Recording of adverse events 
Adverse Events will be collected from, randomization throughout the treatment period 
including the follow-up period that is five days after start of infusion of 
Milrinone/Levosimendan. In addition the adverse events will also be registered at day 
30 (by telephone conversation with the parents if the patient is at home).  
7.1.8.1 
Variables 
The following variables will be recorded in the CRF for each AE; description of the AE, 
the date and time when the AE started and stopped, maximum intensity, whether the AE 
is serious or not, causality rating (yes or no), action taken with regard to investigational 
product, AE caused subject to discontinue study and outcome. 
7.1.8.2 
Definitions for intensity rating 
• 
mild (awareness of sign or symptom, but easily tolerated) 
• 
moderate (discomfort sufficient to cause interference with normal activities) 
• 
severe (incapacitating, with inability to perform normal activities) 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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It is important to distinguish between serious and severe AEs. Severity is a measure of 
intensity whereas seriousness is defined by the criteria in Section 7.1.7. An AE of severe 
intensity need not necessarily be considered serious. For example, nausea that persists 
for several hours may be considered severe nausea, but not a SAE. On the other hand, a 
stroke that results in only a limited degree of disability may be considered a mild stroke 
but would be a SAE. 
The Investigator will assess causal relationship between Study medication and Adverse 
Events, and answer “yes” or “no” to the question “Do you consider that there is a 
reasonable possibility that the event may have been caused by the investigational 
product. 
Causal relationship in cases where the disease under study has deteriorated due to lack 
of effect will be classified as no, reasonable or possibility.  
For SAEs causal relationship will also be assessed for other medication and/or study 
procedure. Note that for SAEs that could be associated with any study procedure the 
causal relationship is implied as “yes”. 
7.1.9 
Adverse Events based on examinations and tests 
Deterioration as compared to baseline in protocol-mandated laboratory values, vital 
signs and other safety variables will only be reported as AEs if they fulfil any of the SAE 
criteria or are the reason for discontinuation of treatment with the investigational 
product. If deterioration in a laboratory value/vital sign is associated with clinical signs 
and symptoms, the sign/symptom will be reported as an AE and the associated 
laboratory result/vital sign will be considered as additional information. Any new or 
aggravated clinically relevant abnormal medical finding at a physical examination as 
compared with the baseline assessment will be reported as an AE. 
Clinically relevant deterioration in non-protocol-mandated measurements will be 
reported as AE(s). 
Wherever possible the reporting investigator uses the clinical, rather than the 
laboratory term (eg, anaemia versus low haemoglobin value). 
7.1.10 
Follow-up of unresolved adverse events 
Any AEs that are unresolved at the patient’s last AE assessment in the study are followed 
up by the investigator for as long as medically indicated, but without further recording in 
the CRF.  

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
37(62) 
7.1.11 
Reporting of Suspected unexpected serious adverse reaction (SUSAR) 
Any serious adverse reaction that is suspected and unexpected will immediately be 
reported by sponsor to the EudraVigilance database and to the EC. 
Fatal and life-threatening events must be reported within 7 calendar days after they 
have come to the investigator's knowledge. A detailed written report must be submitted 
after another 8 days. For other SUSARs 15 days apply. 
For the current study, the responsible investigator will delegate registration of SUSARs 
into the EudraVigilance database to the MPA. The CIOMS form will be used and send 
electronically to registrator@mpa.se. 
All SAEs have to be reported, whether or not considered causally related to the 
investigational product, or to the study procedure(s). All SAEs will be recorded in the 
CRF.  
The investigators are responsible for informing the Ethics Committee and the Regulatory 
Authority of the SAE as per local requirements. 
An annual safety statement including a summary of all SAEs and SUSARs must be 
submitted to the MPA and the EPN as long as the study is ongoing. The current 
reference safety information contained in the SPC in FASS applies. 
7.1.12 
Registering safety data in CRF 
Adverse events will be registered from the time of patient randomization until the end of 
the follow up period. Registrations will be made in CRFs and in hospital medical journals 
according to information in this section regarding duration, severity and relationship. 
7.1.13 
Randomization code breaking 
If an AE is assessed to be a SAE or SUSAR, the study code will be broken, the infusion of 
the study medicine will be terminated and prompt measures for reversing the course of 
the events will be taken until stability is reached. The patient will be followed up as long as 
medically indicated. The patient will be included in the intention-to-treat-analysis and 
described in the Clinical Study Report 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
38(62) 
7.1.14 
Postoperative course after pediatric cardiac surgery is quite eventful 
In the 1970s only a small fraction of children with congenital heart diseases could 
survive. The mortality rates have decreased markedly during the last two three decades, 
thanks to the continuous development of operation techniques and perioperative care 
measures. However, the nature of caring for these patients remains quite challenging. A 
quite many of these children experience an eventful and unstable postoperative course. 
This partly depends on the patient’s diagnoses and partly on the undertaken corrective 
surgery. The diagnosis alone quite often affects the function of the heart, lungs, and other 
remote organs profoundly. The corrective surgery in many cases is extensive and 
demanding. The result is that the postoperative period in these patients is adherent to 
quite many adverse events. This demanding period craves a highly skilled staff to 
promptly reverse the AEs, to correctly recognize ARs , and to mange adequately SAEs, 
and SUSARs. 
7.1.15 
Evaluation and reversing of AEs  
The evaluation of the AEs will be made on the clinical and scientific basis. All caring 
physicians and nurses are familiar with the measures usually taken in case of adverse 
events. The adverse events of Levosimendan do not differ from those usually 
encountered in our PICU. In case of hypotension fluid therapy will be instituted initially. 
If the patient does not respond to the fluid therapy and hypotension is evaluated as fluid-
resistant then therapy with vasoactive medications (noradrenalin, adrenalin) will be 
instituted. A quite common scenario is that the patient is already receiving vasoactive 
medication or medications as a continuous infusion or infusions, in this case a dose 
adjustment of the vasoactive medication or medications will be undertaken. In case of 
arrhythmia fluid therapy and/or anti-arrhythmic therapy, depending on the clinical 
situation, will be started. There are established routines in our department for dealing 
with post cardiac surgery arrhythmia (e.g. fluid therapy, cooling down the patient, and 
treatment with Amiodarone or Metoprolol). If the arrhythmia is therapy resistant pacing 
of the heart with routinely pre-inserted pacemaker leads will be instituted. It is 
noteworthy that the heart is paced routinely postoperatively in many pediatric cardiac 
surgery patients. In summary, the occurrence of AEs is quite usual in the setting of a post 
thoracic surgery intensive care unit. In these units, like our PICU, there are clinical 
routines for prompt recognition, evaluation, and treatment of these AEs.  The probable 
adverse effects of Levosimendan to be encountered in this trial will not differ from the 
other adverse effects usually encountered in our department in connection with the use 
of other potent cardiovascular medications and in conjunction with cardiac surgery. A 
list of AEs is included in the CRF with existing appropriate fields (not related, not likely, 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
39(62) 
possible, probable, and certain) for evaluating of each AE  in respect to a probable causal 
relationship with Levosimendan. 
8. 
BIOLOGICAL SAMPLING PROCEDURES  
8.1 
Volume of blood 
The patients usually stay in the intensive care unit for 2-4 days depending on the 
postoperative course. Each day during the stay at the ICU, 10-15 mL blood for routine 
laboratory screen will be drawn. The amount of blood may increase to 25-30 mL in case 
of complicated postoperative conditions (disease/operation/post operative course).  
In the present study extra blood will be drawn for the analysis of study-specific 
biomarkers e.g. inflammatory markers and the complement system, NGAL and markers 
of neuronal injuries.   
The total volume of extra blood that will be drawn from each subject in this study is 
shown in Table 5.  
Table 5           Volume of blood to be drawn from each subject  
 
Time point 
mL blood 
T0 
Prior to CPB 
2 mL 
T1 
At the end of CPB 
2 mL 
T2 
2 hours after termination of CPB 
2 mL 
T3 
6 hours after termination of CPB 
2 mL 
T4 
24 hours after termination of CBP 
2 mL 
At day 5 or at discharge from hospital 
2 mL 
Total 
 
12 mL 
 
The total amount of blood taken extra from the included patients will be 10 mL in the 
operation day and the first postoperative day. The following table shows the proportion of the 
extra blood taken from an included patient compared with that amount of the blood needed for 
normal routine clinical use. The patient in this example has a weight of 3.5 kg and a blood 
volume of 90 mL/kg (total blood volume 3.5 x 90 = 315 mL). It should be noted that the 
majority of pediatric cardiac surgery patients receive either blood products or blood (or both), 
and the length of stay may be considerably longer. In addition to blood drawn according to 
table 1, at day 5 or at the discharge from hospital 2 mL blood will be drawn for among others 
the analysis of Levodsimendan metabolites (pharmacokinetics). 
 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
40(62) 
Table 6 
Blood taken for lab screen and for study-specific analyses 
Days in PICU 
Routine blood samples in 
uncomplicated cases       
mL (% of total blood vol.) 
Routine blood samples in 
complicated cases 
 mL (% of total blood vol.) 
Blood taken for               
this study 
 mL (% of total blood vol.) 
Day 1 (operation day) 
15 (4.8 %) 
30  (9.5%) 
10  (3.2%) 
Day 2 
15  (4.8 %) 
30  (9.5%) 
- 
Day 3 
15 (4.8 %) 
30  (9.5%) 
- 
Total after 3 PICU days 
45  (14.4 %) 
90  (28.5 %) 
10  (3.2%) 
 
 
8.2 
Handling, storage and destruction of biological samples 
The samples for routine analysis will be used up or disposed after analyses and are not 
applicable for Biobank. 
The blood samples will be analysed after finalized study. A minor amount of the 
biological samples from each patient will be stored in Biobank at the hospital for a 
maximum of 15 years for future research. The results from future analysis will not be 
reported in the Clinical Study Report but separately in a Clinical Study Report 
Amendment and/or Scientific Report or Scientific Publication. 
A full chain of custody is maintained for all samples throughout their lifecycle. 
The principal investigator keeps full tractability of collected biological samples from the 
subjects while in storage at the centre. 
The sample receiver keeps full tractability of the samples while in storage and during use 
until used or disposed. 
9. 
STUDY AND DATA MANAGEMENT  
9.1 
Audits and inspections 
A regulatory authority or the Ethics Committee may perform audits or inspections at the 
centre, including source data verification. The purpose of an audit or inspection is to 
systematically and independently examine all study-related activities and documents, to 
determine whether these activities were conducted, and data were recorded, analysed, 
and accurately reported according to the protocol, Good Clinical Practice (GCP), 
guidelines of the International Conference on Harmonisation (ICH), and any applicable 
regulatory requirements.  

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
41(62) 
9.2 
Recording of data 
Paper case record forms (CRF), will be used to record all data. Data entry, editing and 
analyses will be done by the responsible investigators and study nurse(s). The 
investigators will ensure that all data collected in the study will be provided. He/she 
ensures the accuracy, completeness, legibility and timeliness of the data recorded in the 
appropriate sections of the CRF and according to any instructions provided.  
Clean File/database lock must be documented.  The reason for any excluded data or 
protocol deviations will be described in the study report. Any changes in the database 
after Clean File/database lock must be documented. 
Data will thereafter be entered into an Excel datasheet and analysed with S.P.S.S.19 
statistical software. 
CRF documentation and other source data will be retained for at least 10 years after 
finalization of the clinical study. 
9.2.1 
Source data 
All patient source data such as analysis results from the hospital laboratories and other 
measurements made e.g. ECG readings will be stored at the hospital according to 
routines.  
The CRFs serves as the source for demographic data, medical history and physical 
examination and measurements as well as blood analysis.  
9.3 
Training of study site personnel 
The principal investigator will ensure that appropriate training relevant to the study is 
given to all of these staff, and that any new information relevant to the performance of 
this study is forwarded to the staff involved.  
The principal investigator will maintain a record of all individuals involved in the study 
(medical, nursing and other staff). 
9.4 
Monitoring of the study 
An independent study nurse not directly involved in the study will be appointed for 
monitoring the study. The monitor will be appropriately trained and informed about the 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
42(62) 
nature of the study, patient written information, GCP and applicable regulatory 
requirements. Monitor’s qualifications will be documented. 
The monitor will verify informed consents of participating subjects, confirm that 
facilities remain acceptable, that the investigational team is adhering to the protocol that 
data are being accurately recorded in the Case Report Forms (CRF) and that therapy 
accountability is being carried out. The monitor will also ensure source data verification 
(comparison of the data in the CRF with the hospital/practice and other records at the 
investigational site).  
10. 
STATISTICAL METHODS AND SAMPLE SIZE  
10.1 
Sample size calculation 
The number of patients needed for the study is based on a 20% reduction in creatinine 
in the Levosimendan group compared with the Milrinone group in the day after stopping 
the infusion of Levosimendan/Milrinone (second postoperative day). The power of study 
is set on 80 %.  
The study will contain two groups; Levosimendan (n=35) and Milrinone n=35). Patients 
in the Levosimendan group will receive Levosimendan as inotrope agent and patients in 
the Milrinone group will receive Milrinone as inotrope agent. Patients will randomly be 
assigned to either group. The caregivers (physicians, nurses and nurse-assistants) will 
be blinded for the study. The same infusion rate and total volume of Levosimendan and 
Milrinone will be applied to both groups. 
10.2 
Statistical analysis 
Analysis of data will be performed by a professional statistician. T-test for independent 
groups will be used for primary outcome. Descriptive statistics will be applied on the 
secondary outcomes including 30-days mortality, length of hospital stay and kidney 
function 5 days after start of infusion of Milrinone/Levosimendan. Additionally, clinical 
data (fluid balance, length of surgery, length of CPB, length of mechanical ventilation 
postoperatively, length of vasoactive therapy, number of vasoactive medications, total 
amount of infused vasoactive drugs, hemodynamic parameters) will be registered and 
analyzed. Univariate analyses will be used where needed. Multivariable regression 
analysis will be used to identify association between the occurrences of AKI (based on 
creatinine results) and the measured clinical variables.  

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
43(62) 
Other statistical methods might be used in an exploratory fashion but no formal 
inference will be made. 
Safety and tolerability data will be summarized descriptively by treatment and 
presented in tabular and/or graphical form. All adverse event data will be listed 
individually and summarized using MedDRA terminology. 
11. 
STUDY TIMETABLE AND END OF STUDY 
The study will be terminated when the follow-up of the last patient included is 
completed. The follow-up period for each patient included is 4 days after termination of 
Levosimendan/Milrinone infusion. 
First Subject in: June 2014. Last Subject Last Visit: June 2016 
Final Study Report:  within 12 months from end of study. 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
44(62) 
 
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EudraCT No: 013-003105-25  
Date  
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Braun JP, Schneider M, Kastrup M, Liu J: Treatment of acute heart failure in an 
infant after cardiac surgery using levosimendan. Eur J Cardiothorac Surg 2004, 
26(1):228-230. 
21. 
Luther YC, Schulze-Neick I, Stiller B, Ewert P, Redlin M, Nasseri B, Muhler EG, 
Hetzer R, Lange PE: [Levosimendan-long-term inodilation in an infant with 
myocardial infarction]. Z Kardiol 2004, 93(3):234-239. 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
46(62) 
22. 
Turanlahti M, Boldt T, Palkama T, Antila S, Lehtonen L, Pesonen E: 
Pharmacokinetics of levosimendan in pediatric patients evaluated for cardiac 
surgery. Pediatr Crit Care Med 2004, 5(5):457-462. 
23. 
Egan JR, Clarke AJ, Williams S, Cole AD, Ayer J, Jacobe S, Chard RB, Winlaw DS: 
Levosimendan for low cardiac output: a pediatric experience. J Intensive Care 
Med 2006, 21(3):183-187. 
24. 
Namachivayam P, Crossland DS, Butt WW, Shekerdemian LS: Early experience 
with Levosimendan in children with ventricular dysfunction. Pediatr Crit Care 
Med 2006, 7(5):445-448. 
25. 
Lechner E, Moosbauer W, Pinter M, Mair R, Tulzer G: Use of levosimendan, a new 
inodilator, for postoperative myocardial stunning in a premature neonate. Pediatr 
Crit Care Med 2007, 8(1):61-63. 
26. 
Osthaus WA, Boethig D, Winterhalter M, Huber D, Goerler H, Sasse M, Sumpelmann 
R: First experiences with intraoperative Levosimendan in pediatric cardiac 
surgery. Eur J Pediatr 2009, 168(6):735-740. 
27. 
Momeni M, Rubay J, Matta A, Rennotte MT, Veyckemans F, Poncelet AJ, Clement de 
Clety S, Anslot C, Joomye R, Detaille T: Levosimendan in congenital cardiac 
surgery: a randomized, double-blind clinical trial. J Cardiothorac Vasc Anesth 
2011, 25(3):419-424. 
28. 
Lechner E, Hofer A, Leitner-Peneder G, Freynschlag R, Mair R, Weinzettel R, Rehak 
P, Gombotz H: Levosimendan versus milrinone in neonates and infants after 
corrective open-heart surgery: a pilot study. Pediatr Crit Care Med 2012, 
13(5):542-548. 
29. 
Hoffman TM, Wernovsky G, Atz AM, Bailey JM, Akbary A, Kocsis JF, Nelson DP, 
Chang AC, Kulik TJ, Spray TL et al: Prophylactic intravenous use of milrinone 
after cardiac operation in pediatrics (PRIMACORP) study. Prophylactic 
Intravenous Use of Milrinone After Cardiac Operation in Pediatrics. Am Heart J 
2002, 143(1):15-21. 
30. 
Hoffman TM, Wernovsky G, Atz AM, Kulik TJ, Nelson DP, Chang AC, Bailey JM, 
Akbary A, Kocsis JF, Kaczmarek R et al: Efficacy and safety of milrinone in 
preventing low cardiac output syndrome in infants and children after corrective 
surgery for congenital heart disease. Circulation 2003, 107(7):996-1002. 
31. 
McNamara PJ, Shivananda SP, Sahni M, Freeman D, Taddio A: Pharmacology of 
milrinone in neonates with persistent pulmonary hypertension of the newborn 
and suboptimal response to inhaled nitric oxide. Pediatr Crit Care Med 2013, 
14(1):74-84. 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
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32. 
Jenkins KJ, Gauvreau K, Newburger JW, Spray TL, Moller JH, Iezzoni LI: 
Consensus-based method for risk adjustment for surgery for congenital heart 
disease. J Thorac Cardiovasc Surg 2002, 123(1):110-118. 
33. 
Altman DG, Bland JM: Treatment allocation by minimisation. BMJ 2005, 
330(7495):843. 
34. 
Scott NW, McPherson GC, Ramsay CR, Campbell MK: The method of minimization 
for allocation to clinical trials. a review. Control Clin Trials 2002, 23(6):662-674. 
 

Clinical Study Protocol 
Study Code: MiLe-1 
Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
Date  
48(62) 
 
Appendix B - The preparation of the study drugs and the doses   
 
13. 
PREPARATION OF THE STUDY DRUGS 
13.1 
Logistics 
Logistically, it is not possible to use the hospital pharmacy for preparation dilution of the 
study medications. The hospital pharmacy does not offer such a services. Hence, we 
should do this by ourselves, using “Drug Cards” currently used in our department; 
Department of Pediatric Anesthesia and Intensive Care. The Drug Cards has been 
produced as a result of cooperation between the hospital pharmacy and the clinicians. In 
our department, the nurses prepare all the drugs we currently use.  The nurses, who are 
either specialist in anesthesia or specialist in intensive care do this according to our 
strictly regulated routines. 
13.2 
Levosimendan and Milrinone preparations 
Levosimendan and Milrinone, prepared according to the Drug Cards, have different final 
Concentrations; Levosimendan 0.05 mg/mL and Milrinone 0.2 mg/mL. Despite this, 
Levosimendan and Milrinone have fairly similar infusion rates to deliver a normal dose 
of the drugs to the patients; Levosimendan 0.12 mL / kg / hr and Milrinone 0,15 mL / kg 
/ hr. These infusion rates supply the patient with 0.1 μg / kg / min of Levosimendan and 
0.5 μg / kg / min of Milrinone 
13.3 
Infusion rate for a normal dose  
Because the study is blinded, we must have the same infusion rate in each included 
patient. If we have an infusion rate of 0.12 mL / kg / hr, then we deliver 0.1 μg / kg / min 
of Levosimendan (normal dose) and 0.4 μg / kg / min of Milrinone. The normal dose of 
Milrinone is 0.3 - 1.0 μg / kg / min. In the clinic, we begin normally infuse Milrinone at a 
rate of 0.15 mL / kg / hr which corresponds a dose 0.5 μg / kg / min. Hence, the 
infusion rate of the drugs in this study is 0.12 mL / kg / hr. If the patient needs more 
inotropic support there are two options; delivering a bolus doses (or half-bolus dose) or 
doubling the dose continuously.   

Clinical Study Protocol 
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13.4 
Bolus dose / half-bolus dose 
For administration of bolus doses, we should use the same principle that is the volume of 
the bolus dose should be constant and this volume should deliver the actual bolus dose 
for both drugs. The bolus dose for Levosimendan is 12 μg/kg given in ten minutes. An 
infusion rate of 1.44 ml / kg / hr in ten minutes (a maximum volume 0.24 ml / kg) 
delivers this volume dose. In case of Milrinone, 1.44 ml / kg / hr in ten minutes (a 
maximum volume 0.24 ml / kg) delivers 48 μg / kg of the drug. The standard loading 
dose of Milrinone is 50 μg / kg. Hence, the bolus dose in this study is administered as 
1.44 ml / kg / hr in ten minutes (a maximum volume 0.24 ml / kg). To deliver a half-
bolus dose we infuse 0. 72 mL / kg / hr for 10 minutes (a maximum volume 0.12 ml / 
kg). 
13.5 
Doubling the dose continuously 
If we double the infusion rate to 0.24 ml / kg / hr, this delivers 0.2 μg / kg / min of 
Levosimendan (maximum dose of Levosimendan) and 0.8 μg  / kg / min of Milrinone. 
Maximum dose of Milrinone is 1.0 μg / kg / min. 
13.6 
The calculations 
13.6.1 
Infusion rate and the dose delivered 
What dose do we deliver to the patient with a infusion rate of 0.12 mL/kg/hr? 
Infusion rate 
Delivers 
0.15 mL/kg/hr 
0.5 μg/kg/min 
0.12 mL/kg/hr 
X = 0.12 x 0.5 / 0.15 = 0.4 μg/kg/min 
 
13.6.2 
Bolus dose 
13.6.2.1 Levosimendan 
Is it correct that bolus infusion rate of 1.44 mL/kg/hr in ten minutes delivers 12 μg/kg of 
Levosimendan? The concentration of Levosimendan is 0.05 mg/mL. 
mL 
minutes 
 

Clinical Study Protocol 
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1.44 
60 
 
x 
10 
X = 1.44 x 10 / 60 = 0.24 mL 
 
This means that in 10 minutes we deliver 0.24 mL of Levosimendan. How much active 
drug is in this volume? 
mL 
mg 
 
1 
0.05 
 
0.24 
X 
X = 0.05 x 0.24 / 1 = 0.012 mg = 12 μg 
 
Hence, it is correct, 1.44 mL/kg/hr in ten minutes delivers 12 μg/kg of Levosimendan. 
13.6.2.2 Milrinone 
How much active drug is delivered with the bolus infusion rate of 1.44 mL/kg/hr in ten 
minutes? The concentration of Milrinone is 0.2 mg/mL. 
mL 
min 
 
1.44 
60 
 
X 
10 
X = 1.44 x 10 / 60 = 0.24 mL 
 
 
 
mL 
mg 
 
1 
0.2 
 
0.24 
X 
X = 0.2 x 0.24 / 1 = 0.048 mg = 48 μg 
 
 
 

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Drug Substance: Levosimendan 
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Appendix C - Definitions of Adverse Events  
Definitions: 
Adverse Events 
Serious Adverse Events 
A medical emergency usually constitutes an SAE and is to be 
reported as such 
Intensity rating 
Causal relationship 
Action taken 
Reporting in CRF 
Adverse Events based on signs and symptoms 
Final outcome assessment 
Reporting of serious adverse events 
Further guidance on Serious Adverse Events: 
Life threatening 
Hospitalisation 
Important medical event or medical intervention 
A guide to interpreting the causality QUESTION 
Other significant Adverse Events 
Overdose 
 

Clinical Study Protocol 
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DEFINITIONS OF ADVERSE EVENTS AND PROCEDURES IN CASE OF 
PREGNANCY 
It is of the utmost importance that all staff involved in the study is familiar with the content of 
this section. The Principal Investigator is responsible for ensuring this.  
Adverse Events 
An adverse event is the development of an undesirable medical condition or the deterioration 
of a pre-existing medical condition following or during exposure to a pharmaceutical product, 
whether or not considered causally related to the product. An undesirable medical condition 
can be symptoms (eg, nausea, chest pain), signs (eg, tachycardia, enlarged liver) or the 
abnormal results of an investigation (eg, laboratory findings, electrocardiogram). In clinical 
studies, an AE can include an undesirable medical condition occurring at any time, including 
run-in or washout periods, even if no study treatment has been administered. 
The term AE is used to include both serious and non-serious AEs  
Serious Adverse Events and suspected unexpected serious adverse reactions (SUSAR) 
It is important to distinguish between Serious Adverse Events (SAEs) and severe adverse 
events (AEs). Severity is a measure of intensity whereas seriousness is defined by the criteria 
listed below. An AE of severe intensity need not necessarily be considered serious. For 
example, nausea which persists for several hours may be considered severe nausea, but not an 
SAE. On the other hand, a stroke which results in only a limited degree of disability may be 
considered a mild stroke but would be a SAE. 
A medical emergency usually constitutes an SAE and is to be reported as such 
A serious adverse event is an AE occurring during any study phase (i.e. run-in, pre-entry, 
screening, treatment, wash-out, follow-up), and at any dose of the investigational product, 
comparator or placebo, that fulfils one or more of the following criteria: 
• results in death 
• is immediately life-threatening 
• requires in-patient hospitalisation or prolongation of existing hospitalisation  
• results in persistent or significant disability or incapacity 
• is a congenital abnormality or birth defect 

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• is an important medical event that may jeopardise the subject or may require medical 
intervention to prevent one of the outcomes listed above.  
For reporting purposes, any suspected transmission via a medicinal product of an infectious 
agent is also considered an SAE and is reported in an expedited manner. Any organism, virus 
or infectious particle (for example prion protein transmitting Transmissible Spongiform 
Encephalopathy), pathogenic or non-pathogenic, is considered an infectious agent. 
Definitions for severity rating 
0. None – No symptoms 
1. Mild - Transient symptoms; awareness of sign or symptom, but easily tolerated. No 
interference with the subject’s daily activities 
2. Moderate - Marked symptoms; discomfort sufficient to cause interference with normal 
activities. Moderate interference with the subject’s daily activities 
3. Severe - Considerable interference with the subject’s daily activities; unacceptable, 
incapacitating. Inability to perform normal activities 
N/A Not Applicable 
Causal relationship 
The causality of (S)AEs (ie, their relationship to study treatment and/or the investigational 
procedure) will be assessed by the investigator(s), who in completing the relevant case report 
form must answer “yes” or “no” to the question “Do you consider that there is a reasonable 
possibility that the event may have been caused by the drug/the investigational procedure?”  
The following terms and definitions are used when assessing the causal relationship between each AE and the 
relevant trial product(s): 
1. 
Definite - There is no doubt that the incident is related  
2. 
Probable - Good reason and sufficient documentation to assume a causal relationship 
3. 
Possible- A causal relationship is conceivable and cannot be dismissed 
4. 
Unlikely - The event is most likely related to aetiology other than the trial product 
5. 
Not related – The event is not related to the trail product 
6. 
Unknown/Unclassifiable: a report suggesting an adverse event reaction, which cannot be judged because 
information is insufficient or contradictory and which cannot be supplemented or verified. 
N/A Not Applicable 
 

Clinical Study Protocol 
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Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
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Causal relationship in cases where the disease under study has deteriorated due to lack of 
effect will be classified as no reasonable possibility. 
For SAEs causal relationship will also be assessed for additional study drug 
and/or other medication and/or study procedure. Note that for SAEs that could be 
associated with any study procedure the causal relationship is implied as “yes”. 
Action taken: 
0 
None 
1 
Dose of study drug changed 
2 
Study drug temporarily stopped 
3 
Study drug stopped 
N/A Not Applicable 
 
Reporting in the Case report Form  
The following variables will be recorded in the CRF for each AE; description of the AE, the 
date and time when the AE started and stopped, maximum intensity, whether the AE is serious 
or not, causality rating (yes or no; if yes specify), action taken with regard to investigational 
product, AE caused subject to discontinue study and outcome. 
Adverse Events based on signs and symptoms 
All AEs spontaneously reported by the subject or reported in response to the open 
question from the study personnel: “Have you had any health problems since the 
previous visit?”, or revealed by observation will be collected and recorded in the 
CRF. When collecting AEs the recording of diagnoses is preferred (when 
possible) to recording a list of signs and symptoms. However, if a diagnosis is 
known and there are other signs or symptoms that are not generally part of the 
diagnosis, the diagnosis and each sign or symptom will be recorded separately. 
Follow-up – Outcome assessment 
Any AEs that are unresolved at the patient’s last AE assessment in the study are 
followed up by the investigator for as long as medically indicated, but without 
further recording in the CRF. 
The following terms and definitions are used in assessing the final outcome of an AE: 
• 
Recovered - The subject has fully recovered, or by medical or surgical treatment the condition has returned 
to the level observed at the first trial-related activity after the subject signed the informed consent. 

Clinical Study Protocol 
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Drug Substance: Levosimendan 
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• 
Recovering - This term is only applicable if the subject has completed the trial or has died from another AE. 
The condition is improving and the subject is expected to recover from the event. 
• 
Recovered with sequelae - The subject has recovered from the condition, but with lasting effect due to a 
disease, injury, treatment or procedure. If a sequela meets an SAE criterion, the AE must be reported as an 
SAE. 
• 
Not recovered - The condition of the subject has not improved and the symptoms are unchanged, or the 
outcome is not known at the time of reporting. 
• 
Fatal - This term is only applicable if the subject died from a condition related to the reported AE. 
Outcomes of other reported AEs in a subject before he/she died should be assessed as “recovered”, 
“recovering”, “recovered with sequelae” or “not recovered”. An AE with fatal outcome must be reported as 
an SAE. 
• 
Unknown - This term is only applicable if the subject is lost to follow-up 
Reporting of serious adverse events 
For studies in countries implementing the EU Clinical Trials Directive, informing Ethics 
Committees and Regulatory Authorities will be performed by the sponsor. 
All Suspected Unexpected Serious Adverse Reactions (SUSARs) have to be reported, whether 
or not considered causally related to the investigational product, or to the study procedure(s). 
The Clinical Study Serious Adverse Event Report Form will be used together with other 
relevant supporting documentation (e.g. ECG, laboratory results, autopsy report) and relevant 
CRF modules. All SUSARs have to be electronically registered in the EMEAs database. 
FURTHER GUIDELINES ON THE DEFINITION OF A SERIOUS ADVERSE EVENT 
Life threatening 
‘Life-threatening’ means that the subject was at immediate risk of death from the AE as it 
occurred or it is suspected that use or continued use of the product would result in the 
subject’s death. ‘Life-threatening’ does not mean that had an AE occurred in a more severe 
form it might have caused death (eg, hepatitis that resolved without hepatic failure). 
Hospitalisation 
Out-patient treatment in an emergency room is not in itself a serious AE, although the reasons 
for it may be (eg, bronchospasm, laryngeal oedema). Hospital admissions and/or surgical 
operations planned before or during a study are not considered AEs if the illness or disease 
existed before the subject was enrolled in the study, provided that it did not deteriorate in an 
unexpected way during the study. 

Clinical Study Protocol 
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EudraCT No: 013-003105-25  
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Important medical event or medical intervention 
Medical and scientific judgement should be exercised in deciding whether a case is serious in 
situations where important medical events may not be immediately life-threatening or result in 
death, hospitalisation, disability or incapacity but may jeopardize the subject or may require 
medical intervention to prevent one or more outcomes listed in the definition of serious. These 
should usually be considered as serious. 
Examples of such events are: 
- Angioedema not severe enough to require intubation but requiring iv hydrocortisone 
treatment 
- Hepatotoxicity caused by paracetamol (acetaminophen) overdose requiring treatment with 
N-acetylcysteine 
- Intensive treatment in an emergency room or at home for allergic bronchospasm 
- Blood dyscrasias (eg, neutropenia or anaemia requiring blood transfusion, etc) or 
convulsions that do not result in hospitalisation 
- Development of drug dependency or drug abuse 
A guide to interpreting the causality QUESTION 
The following factors should be considered when deciding if there is a “reasonable 
possibility” that an AE may have been caused by the drug. 
• Time Course. Exposure to suspect drug. Has the subject actually received the suspect 
drug? Did the AE occur in a reasonable temporal relationship to the administration of 
the suspect drug? 
• Consistency with known drug profile. Was the AE consistent with the previous 
knowledge of the suspect drug (pharmacology and toxicology) or drugs of the same 
pharmacological class? OR could the AE be anticipated from its pharmacological 
properties? 
• Dechallenge experience. Did the AE resolve or improve on stopping or reducing the 
dose of the suspect drug? 
• No alternative cause. The AE cannot be reasonably explained by aetiology such as the 
underlying disease, other drugs, other host or environmental factors. 

Clinical Study Protocol 
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• Rechallenge experience. Did the AE reoccur if the suspected drug was reintroduced 
after having been stopped? A re-challenge would not normally be recommended or 
supported. 
•  Laboratory tests. A specific laboratory investigation (if performed) has confirmed the 
relationship? 
A “reasonable possibility” could be considered to exist for an AE where one or more of these 
factors exist. 
In contrast, there would not be a “reasonable possibility” of causality if none of the above 
criteria apply or where there is evidence of exposure and a reasonable time course but any 
dechallenge (if performed) is negative or ambiguous or there is another more likely cause of 
the AE. 
In difficult cases, other factors could be considered such as: 
• Is this a recognised feature of overdose of the drug? 
• Is there a known mechanism? 
 
Ambiguous cases should be considered as being a “reasonable possibility” of a causal 
relationship unless further evidence becomes available to refute this. Causal relationship in 
cases where the disease under study has deteriorated due to lack of effect should be classified 
as no reasonable possibility. 
 
Other significant Adverse Events 
An expert will identify other significant Adverse Events (OAEs) during the evaluation of 
safety data for the Clinical Study Report. Significant adverse events of particular clinical 
importance, other than SAEs and those AEs leading to discontinuation of the subject from 
study treatment, will be classified as OAEs. Examples of these are marked haematological and 
other laboratory abnormalities, and certain events that lead to intervention (other than those 
already classified as serious), dose reduction or significant additional treatment. For each 
OAE, a narrative will be written and included in the Clinical Study Report.  
Overdose 
• An overdose with associated AEs is recorded as the AE diagnosis/symptoms on the 
relevant AE modules in the CRF. 
 

Clinical Study Protocol 
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Appendix D- Ethics and regulatory review 
 
The final study protocol, including the final version of the Written Informed Consent Form 
and other information given to subjects eg, advertisements must be approved or given a 
favourable opinion by an Ethics Committee before enrolment of any subject into the study.  
Before enrolment of any subject into the study, the final study protocol, including the final 
version of the Informed Consent Form, is approved by the national regulatory authority or a 
notification to the national regulatory authority is done, according to local regulations. 
It is the responsibility of the Principal Investigator to apply to the Ethics Committee in 
writing. The application document should:  
• 
contain the name and address of the Ethics Committee  
• 
clearly identify, by title and date, the protocol and other documents submitted for review 
• 
be dated. 
In addition, the Principal Investigator should request the Ethics Committee to provide: 
• 
their approval/opinion in a dated document identifying the Principal Investigator’s 
application 
• 
Ethics Committee composition for the meeting when the approval was given 
• 
a statement confirming that the Ethics Committee is organised and operates according to 
GCP and applicable laws and regulations. 
The Principal Investigator is responsible for informing the Ethics Committee of any 
modifications and amendments to the protocol as per local requirements. 
Progress reports and notifications of serious and unexpected adverse drug reactions will be 
provided to the Ethics Committee according to local regulations and guidelines 
All correspondence with the Ethics Committee should be filed by the Principal Investigator in 
the ISF. 
The Principle Investigator is also responsible for obtaining approvals from scientific bodies 
(eg, to use radiolabelled substances) if necessary for the study. 

Clinical Study Protocol 
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SUBJECT INFORMATION AND WRITTEN INFORMED CONSENT FORM 
The Principal Investigator(s) at each centre will ensure that the subject is given full and 
adequate oral and written information about the nature, purpose, possible risk and benefit of 
the study. Subjects must also be notified that they are free to discontinue from the study at any 
time. The subject should be given the opportunity to ask questions and allowed time to 
consider the information provided. 
The subject’s signed and dated informed consent must be obtained before conducting any 
procedure specifically for the study.  
The Principal Investigator(s) must store the original, signed Informed Consent Form in the 
Investigator’s Study File. A copy of the signed Informed Consent Form must be given to the 
subject. 
If a protocol amendment requires a change to the Informed Consent Form, the Ethics 
Committee must approve modifications that lead to a revised Informed Consent Form before 
the revised form is used. 
SUBJECT DATA PROTECTION 
The Informed Consent Form will incorporate (or, in some cases, be accompanied by a 
separate document incorporating) wording that complies with relevant data protection and 
privacy legislation. 
Extra precautions are taken to preserve confidentiality and prevent genetic or other study data 
being linked to the identity of the subject. In exceptional circumstances, however, certain 
individuals might see both the genetic, other study data and the personal identifiers of a 
subject. For example, in the case of a medical emergency or an investigator might know a 
subject’s identity and also have access to his or her data. Also Regulatory authorities may 
require access to the relevant files, though the subject’s medical information and the genetic 
files would remain physically separate 
AUDITS AND INSPECTIONS 
Authorized representatives of Sponsor, a regulatory authority, or an Ethics Committee may 
perform audits or inspections at the centre, including source data verification. The purpose of 
an audit or inspection is to systematically and independently examine all study-related 
activities and documents, to determine whether these activities were conducted, and data were 
recorded, analysed, and accurately reported according to the protocol, Good Clinical Practice 

Clinical Study Protocol 
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Drug Substance: Levosimendan 
EudraCT No: 013-003105-25  
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(GCP), guidelines of the International Conference on Harmonisation (ICH), and any 
applicable regulatory requirements. 
 
 

 
SOP-220-G T03 GEL MeMo Protocol Amendment  
1(2) 
Version 1, Date: 10 May 2005 
Study Protocol Amendment No 1 
EudraCT No. 2013-003105-25 
Study Code: MiLe-1 
Date: 2014-07-15 
The prophylactic effect of Levosimendan in reducting acute kidney 
injury postoperatively in pediatric patients undergoing corrective 
heart surgery 
 
Investigator: 
Doc. Albert Castellheim 
Sahlgrenska University Hospital 
The Queen Silvia Children’s Hospital AnOpIVA 
416 85 Gothenburg  
Study Site: The Queen Silvia Children’s Hospital AnOpIVA 
Section of protocol to be amended: 
Table 1 in Section 3 of the Study Protocol   
Previous text 
Revised text 
No previous text 
New activities in Table 1: 
Pharmacokinetics 6 hours, 24 hours, 48 
hours and 4,5 days 
 
Routine echocardiography (pre-op, after 
CPB termination, first post-op day and at 
discharge) and 30 days post-op 
 
 
 
Reason for Amendment:  
Pharmacokinetics at 4/5 days: For a more complete analyse of the time course of drug 
absorption, distribution, metabolism and excretion. 
Echocardiography at day 30: For extended safety surveillance  

MeMo Protocol Amendment Number Error! No text of specified style in document. 
Study Code Error! No text of specified style in document. 
 
 
 
There will be no negative consequences for the patients rather opposite, it will increase 
patients’ safety and give a better interpretation of the PK 
Actions to be taken: 
Table 1 in Study Protocol will be revised 
Signed agreement to the Amendment: 
I agree to the terms of this Protocol Amendment. 
Study Code: MiLe-1  
 
2014-07-15 
 
Albert Castellheim 
Date 
(day month year) 
 
Principal investigator 
 
 
 

 
SOP-220-G T03 GEL MeMo Protocol Amendment  
1(2) 
Version 1, Date: 10 May 2005 
Study Protocol Amendment No 2 
EudraCT No. 2013-003105-25 
Study Code: MiLe-1 
Date: 2014-09-05 
The prophylactic effect of Levosimendan in reducting acute kidney 
injury postoperatively in pediatric patients undergoing corrective 
heart surgery 
 
Investigator: 
Doc. Albert Castellheim 
Sahlgrenska University Hospital 
The Queen Silvia Children’s Hospital AnOpIVA 
416 85 Gothenburg  
Study Site: The Queen Silvia Children’s Hospital AnOpIVA 
Section of protocol to be amended: 
Exclusion criteria in Protocol Synopsis and in Section 4-2 of the Study Protocol   
Previous text 
Revised text 
Use of nephrotoxic drugs (like ibuprofen, 
angiotensin-converting-enzyme inhibitors, 
gentamicin, vancomycin) preoperative or 
postoperative until first post operative day 
 
7.  Use of nephrotoxic drugs (like 
ibuprofen, 
gentamicin, 
vancomycin) 
preoperative or postoperative until first 
post operative day 
 
 
 
 
Reason for Amendment:  
We have included the Ross score in to the protocol after an agreement with our pediatric 
cardiologist. Ross score grades the patients according to the severity of their heart 
failure.   
 
 

MeMo Protocol Amendment Number Error! No text of specified style in document. 
Study Code Error! No text of specified style in document. 
 
According the same agreement with pediatric cardiologist peers there will be no 
negative consequences for the patients rather the opposite; the study will include those 
patients who receive angiotensin-converting-enzyme inhibitors for their heart failure.  
 
Actions to be taken: 
Change of the exclusion criteria text  
Signed agreement to the Amendment: 
I agree to the terms of this Protocol Amendment. 
Study Code: MiLe-1  
 
2014.09.05 
 
Albert Castellheim 
Date 
(day month year) 
 
Principal investigator 
 
 
 

SOP-220-G T03 GEL MeMo Protocol Amendment  
1(2) 
Version 1, Date: 10 May 2005 
Study Protocol Amendment No 3 
EudraCT No. 2013-003105-25 
Study Code: MiLe-1 
Date: 2014-09-09 
The prophylactic effect of Levosimendan in reducting acute kidney 
injury postoperatively in pediatric patients undergoing corrective 
heart surgery 
Investigator: 
Doc. Albert Castellheim 
Sahlgrenska University Hospital 
The Queen Silvia Children’s Hospital AnOpIVA 
416 85 Gothenburg  
Study Site: The Queen Silvia Children’s Hospital AnOpIVA 
Section of protocol to be amended: 
At the end of the Study Protocol   
Previous text 
Revised text 
No previous text 
Research assistant: Maria Thorson 
 
 
 
Reason for Amendment:  
The study logistics necessitates involving of a 
research assistant.  
 
Actions to be taken: 
Adding the revised text to the section Collaborators in the Study Protocol.  
Signed agreement to the Amendment: 
I agree to the terms of this Protocol Amendment. 
Study Code: MiLe-1  
 

Study Protocol Amendment No 3 
EudraCT No. 2013-003105-25 
Study Code: MiLe-1 
Date: 2014-09-01 
 
 
2014.09.09 
 
Albert Castellheim 
Date 
(day month year) 
 
Principal investigator 
 
 
 

 
 
1(2) 
Study Protocol Amendment No 4 
EudraCT No. 2013-003105-25 
 
Study Code: MiLe-1 
 
Date: 2017.02.22 
 
 
 
The prophylactic effect of Levosimendan in reducting acute kidney 
injury postoperatively in pediatric patients undergoing corrective 
heart surgery 
 
Investigator: 
Albert Castellheim; associate professor 
Sahlgrenska University Hospital 
The Queen Silvia Children’s Hospital AnOpIVA 
416 85 Gothenburg  
Study Site: The Queen Silvia Children’s Hospital AnOpIVA 
Amendment: Definition of adverse events (AE) in MiLe-1 
 
Reason for Amendment:  
Lack of definition of AEs, in contrast to SAEs which were well-defined. 
 
Actions to be taken: 
AEs, according to following criteria, for all included patients will separately be 
registered. 
AEs Criteria:  
1. Heart rate > 200/min at these time points: 2, 6, 10-12 hours after CPB weaning, first 
postop morning at 6 o'clock and 24 hours after CPB weaning [1] 
2. Arrythmias (rhythm other than sinusrythm). Same time points as in no 1 [2,3] 
3. Inotropic score > 20 at the same time points as in no 1 [4] 
4. Thrombocytes < 50.000 on the first postoperative morning [3] 
5. Hypokalemia < 3.5 at the same time points as in no 1 [2] 

 
 
References: 
1) Advanced Paediatric Life Support (APLS) 
2) Pharmaceutical Specialities in Sweden (Farmaceutiska Specialiteter i Sverige: Fass) 
3) Hoffman TM. Cirkulation 2003;107:996-1002 
4) Gaies MG. Pediatr Crit Care Med 2010; 234-238 
Signed agreement to the Amendment: 
Study Code: MiLe-1  
 
Principal investigator: Albert Castellheim  
 
Date: 2017.02.22