Clinical trial NCT02251041
Conditions: Reperfusion Injury
Keywords: Liver Transplantation Ischemia-reperfusion Effectiveness
Sponsor: Universitaire Ziekenhuizen KU Leuven
Start date: 2014-09-01; Completion date: 2019-08-01
Enrollment: 143 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to establish the effectiveness of the combined drug approach (anti-thrombin III, infliximab, apotransferrin, human recombinant erythropoietin beta, C1-inhibitor, glutathione, alfa-tocopherol, melatonin and epoprostenol)aimed to reduce ischemia-reperfusion injury during liver transplantation in eligible recipients.

Detailed description:
This unicentric, investigator-driven, open-label randomized clinical trial with 2 parallel arms was conducted in Belgium from September 2013 through February 2018, with 1-year follow-up. Adults wait-listed for a first solitary full-size liver transplant were screened for eligibility. Exclusion criteria were acute liver failure, kidney failure, contraindication to treatment, participation in another trial, refusal, technical issues, and death while awaiting transplant. Included patients were enrolled and randomized at the time of liver offer. Data were analyzed from May 20, 2019, to May 27, 2020.

Participants were randomized to a combined drug approach with standard of care (static cold storage) or standard of care only (control group). In the combined drug approach group, following static cold preservation, donor livers were infused with epoprostenol (ex situ, portal vein); recipients were given oral α-tocopherol and melatonin prior to anesthesia and intravenous antithrombin III, infliximab, apotransferrin, recombinant erythropoietin-β, C1-inhibitor, and glutathione during the anhepatic and reperfusion phase.

The primary outcome was the posttransplant peak serum aspartate aminotransferase (AST) level within the first 72 hours. Secondary end points were the frequencies of postreperfusion syndrome, ischemia-reperfusion injury score, early allograft dysfunction, surgical complications, ischemic cholangiopathy, acute kidney injury, acute cellular rejection, and graft and patient survival.

Arms: arm 1: the group receives a combination of drugs arm 2: the group do not receive a combination of drugs, but a placebo (sodium chloride solution)
Interventions: intervention 1: Antithrombin-III intervention 2: Infliximab intervention 3: Apotransferrin intervention 4: Human recombinant erythropoietin intervention 5: C1-Inhibitor intervention 6: Glutathione intervention 7: Alfa-tocopherol intervention 8: Melatonin intervention 9: Epoprostenol intervention 10: Sodium chloride solution
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: None intervention 4: None intervention 5: None intervention 6: None intervention 7: None intervention 8: None intervention 9: None intervention 10: None

Protocol documents:
Prot_SAP_000.pdf: 
COMBINED DRUG APPROACH TO 
PREVENT ISCHEMIA-REPERFUSION 
INJURY DURING TRANSPLANTATION OF 
LIVERS (CAPITL): a first-in-men study 
Monbaliu Diethard, Meurisse Nicolas, Jochmans Ina, Aerts Raymond, Pirenne Jacques. 
Abdominal Transplant Surgery, UZ Leuven 
Herestraat 49 
3000 Leuven 
NCT number: 02251041 
Date: 08-03-2016


 


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COMBINED DRUG APPROACH TO 
PREVENT ISCHEMIA-REPERFUSION 
INJURY DURING TRANSPLANTATION OF 
LIVERS (CAPITL): a first-in-men study 
 
Monbaliu Diethard, Meurisse Nicolas, Jochmans Ina, Aerts Raymond, Pirenne Jacques. 
 
Abdominal Transplant Surgery, UZ Leuven 
Herestraat 49 
3000 Leuven 
 
Principal Investigator: Diethard Monbaliu 
 
Tel: +32 16 348727 
Fax: +32 16 348743 
 
 


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Table of Contents 
List of abbreviations ................................................................................................................................... 5 
I. TITLE ................................................................................................................................................................ 7 
II. SUMMARY OF THE TRIAL ....................................................................................................................... 8 
III. RATIONALE AND NOVELTY .............................................................................................................. 24 
3.1 RATIONALE ........................................................................................................................................ 24 
3.2 NOVELTY ............................................................................................................................................. 31 
3.3 FROM PRECLINICAL MODEL TO CLINICAL CAPITL STUDY ............................................ 32 
3.4 DESIGN ................................................................................................................................................. 36 
3.5 OBJECTIVES ........................................................................................................................................ 39 
IV. PARTICIPANTS ........................................................................................................................................ 40 
4.1 ELIGIBILITY CRITERIA ................................................................................................................... 40 
4.2 INCLUSION CRITERIA ..................................................................................................................... 40 
4.3 EXCLUSION CRITERIA .................................................................................................................... 40 
V. INTERVENTION ........................................................................................................................................ 42 
5.1 CONTROL GROUP: Standard care of treatment .................................................................... 42 
5.1.1 Surgical procedure .................................................................................................................. 42 
5.1.2 Anaesthesia ................................................................................................................................ 42 
5.1.3 Post-operative care in the intensive care unit .............................................................. 43 
5.1.4 Post-operative follow up on nursing ward .................................................................... 44 
5.1.5 Outpatient follow-up .............................................................................................................. 44 
5.1.6 Immunosuppression ............................................................................................................... 45 
5.2 TREATMENT GROUP: multifactorial modulation ................................................................ 47 
VI. TIME LINE & PARTICIPATING CENTER ........................................................................................ 49 
6.1 TIME LINE ........................................................................................................................................... 49 
6.2 PARTICIPATING CENTER .............................................................................................................. 50 
VII. PRIMARY ENDPOINT .......................................................................................................................... 51  
VIII. SECONDARY ENDPOINTS ................................................................................................................ 52 
IX. VARIABLES OF INTEREST .................................................................................................................. 54 
9.1 DONOR CHARACTERISTICS ......................................................................................................... 54 
9.2 RECIPIENT CHARACTERISTICS .................................................................................................. 54 
9.3 INTRA-OPERATIVE VARIABLES ................................................................................................. 54 
9.4 POST-OPERATIVE VARIABLES ................................................................................................... 55 
9.5 LABORATORY ASSESSMENT ....................................................................................................... 55 
9.6 HISTOLOGICAL FEATURES ........................................................................................................... 55 
X. DATA ANALYSIS ....................................................................................................................................... 56 


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10.1 REPORTING OF THE DATA ........................................................................................................ 56 
10.2 STATISTICAL METHODOLOGY ................................................................................................. 56 
10.2.1 Primary outcome ................................................................................................................... 56 
10.2.2 Sample size calculation ....................................................................................................... 56 
10.2.3 Secondary outcomes ............................................................................................................ 57 
XI. RANDOMIZATION .................................................................................................................................. 58 
XII. ALLOCATION CONCEALMENT ........................................................................................................ 58 
XIII. ETHICAL CONSIDERATION ............................................................................................................. 58 
13.1 ETHICAL COMMITTEE ................................................................................................................. 59  
13.2 PATIENT INFORMATION AND INFORMED CONSENT .................................................... 59 
13.3 PERSONAL DATA PROTECTION .............................................................................................. 59 
XIV. WITHDRAWALS AND DROPOUTS ................................................................................................ 60 
XV. INTERIM ANALYSIS ............................................................................................................................. 61 
XVI. PROCESS LEADING TO TRIAL STOPPING RULES ................................................................... 62 
16.1 DATA SAFETY MONITORING BOARD .................................................................................... 62 
16.2 DESCRIPTION OF ADVERSE EVENT ....................................................................................... 62 
16.2.1 Definition of adverse event ............................................................................................... 62 
16.2.2 Grade of severity .................................................................................................................... 62 
16.2.3 Timeframe of observation ................................................................................................. 63 
16.2.4 Predefined serious adverse events ................................................................................. 63 
16.3 Protocol-defined adverse events……………………………………………………………………64 
16.4 DRUG CAUSALITY .......................................................................................................................... 65 
16.5 INDIVIDUAL AND COHORT LEVELS ....................................................................................... 65 
XVII. REFERENCES ....................................................................................................................................... 67 
XVIII. APPENDICES ...................................................................................................................................... 69 
APPENDIX 1: BANFF CRITERIA ......................................................................................................... 69 
APPENDIX 2:  ADVERSE EVENTS ...................................................................................................... 70 
1. Definition of adverse events ...................................................................................................... 70 
2. Detection, reporting, and responsibilities ............................................................................ 71 
3. Surgical complication classification (within 30 days after transplantation) .......... 72 
4. Infection ............................................................................................................................................. 73 
5. Predefined types of adverse events ........................................................................................ 74 
APPENDIX 3: COMBINED DRUG APPROACH ................................................................................ 75 
3.1 GENERAL DESCRIPTION ........................................................................................................... 75 
3.2 CONTRA-INDICATION TO USE THE COMBINED DRUG APPROACH .....................102 
3.3 RATIONALE FOR THE CHOICE OF DOSES........................................................................105 
APPENDIX 4: SCHEDULE OF EVENTS ............................................................................................113 


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APPENDIX 5:  ETHICAL PRINCIPLES FOR MEDICAL RESEARCH INVOLVING HUMAN 
SUBJECTS ..................................................................................................................................................114 
APPENDIX 6: INFORMED CONSENT ..............................................................................................118 
 
APPENDIX 8: PATIENT INFORMATION BROCHURE for Randomized Controlled Trial
 .......................................................................................................................................................................139 
APPENDIX 9: CHRONOLOGICAL IDENTIFICATION LIST .......................................................160 
APPENDIX 10: CHRONOLOGICAL SCREENING LIST ................................................................161 
APPENDIX 11: PARTICIPATING CENTER .....................................................................................161 
APPENDIX 12: SIGNATURE PAGE ...................................................................................................162 
APPENDIX 13: ANCILLARY STUDIES 
13.1 Impact of multifactorial biomodulation on the post-reperfusion bile toxicity 
and bile duct injury/regeneration 
13.2 Impact of multifactorial biomodulation on mechanisms of ischemia 
reperfusion injury  
13.3 Impact of perioperative macrophage migration inhibitory factor (MIF) release 
on outcome of patients undergoing orthotopic liver transplantation.  
13.4 Impact of multifactorial biomodulation on kidney injury after liver 
transplantation 
13.5 Determination of changes in gene expression and altered molecular processes 
in the liver affected by the multifactorial biomodulation  
13.6 Impact of multifactorial biomodulation on the postreperfusion syndrome  
APPENDIX 14: OVERVIEW OF SAMPLE COLLECTION DURING CAPITL STUDY 
    APPENDIX 15: REPORTING POST-TRANSPLANT MEDICATION 
 
 
 


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List of abbreviations 
 
 
AE: Adverse event 
ALT: Alanine aminotransferase 
AR: Acute rejection 
AST: Aspartate aminotransferase 
cAMP: cyclic Adenosyl Mono-Phosphate 
C1-INH: C1-inhibitor 
CIT: cold ischemia time 
DCD: Donation after circulatory death 
DDI: Drug-to-drug interaction 
DSMB: Data Safety Monitoring Board 
ECD: Extended criteria donor 
E-CRF: Electronic case report form 
EPO: erytrhopoietin 
ERCP: Endoscopic retrograde cholangio-pancreatography 
GCP: Good clinical practice 
GSH: glutathione 
HCV: Hepatitis C virus 
IRI: Ischemia-reperfusion injury 
IV: Intravenous 
LTx: Liver transplantation 
MRCP: Magnetic resonance cholangio-pancreatography 
NTBI: non-transferrin-bound iron 


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PNF: primary non function 
PTC: Percutaneous transhepatic cholangiography 
RCT: Randomized control trial 
ROS: Reactive Oxygen species 
SAE: Serious adverse event 
SCD: Standard criteria donor 
SSAR: Suspected serious adverse reaction 
SUSAR: Suspected unexpected serious adverse reaction 
SSPP: Stable solution of plasma protein 
TNF-α: tumor necrosis factor- alpha 
 


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I. TITLE 
 
Combined Drug Approach to Prevent Ischemia-reperfusion injury during 
Transplantation of Livers (CAPITL study), a first-in-men study. 
 
 


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II. SUMMARY OF THE TRIAL 
 
TITLE 
 
Combined 
Drug 
Approach 
to 
Prevent 
Ischemia-reperfusion 
injury 
during 
Transplantation of Livers (CAPITL): a first-in-men study. 
 
 
TRIAL DESIGN 
 
A two-part, investigator driven, multi-center, single-blinded, adaptive study to 
assess the safety and to study the efficacy of a Combined drug Approach to Prevent 
Ischemia-reperfusion injury during Transplantation of Livers. 
 
The first part of the study (part A) was a safety study conducted in 10 patients 
undergoing a liver transplantation and was regarded to be safe by the Data and 
Safety Monitoring Board.  These results will be submitted to the local ethical 
committee.  
 
The second part of the study (part B) foresees in a randomized controlled trial 
conducted in patients undergoing a liver transplantation at the University 
Hospitals Leuven, Belgium, Centre Hospitalier Universitaire de Liège and other 
national and international centers.  
 
 
Study part A: safety study  
  
 
A phase I, safety study will be first performed (Figure 1) in 10 patients in 4 smaller 
cohorts.  The rationale is to establish the safety of the combined drug approach 
aimed to reduce ischemia-reperfusion injury during liver transplantation in 
eligible recipients. The combined drug approach, also referred as multifactorial 
modulation, foresees the sequential administration of drugs in eligible liver 
transplant recipients. Drugs will be administered as such that they will never be in 
direct contact with each other. 
  
There will be 10 patients included in Part A, conducted in 4 consecutive small 
cohorts of 2, 2, 3, and 3 patients, respectively (Figure 1).  For this safety study, all 
consecutive patients listed for LTx will be assessed for study eligibility by a senior 
staff physician at time of organ offer. The data and safety assessment of each cohort 
will then be reviewed by the Data and Safety Monitoring Board (DSMB) before 
patients are included in the next cohort (see section 16.1). 
  
Safety will be assessed by well-defined objective criteria and parameters based on 
incidence and/or severity of adverse events indicating a potential health hazard 
caused by the multifactorial modulation within 7 days post LTx (see chapter XVI: 
Process leading to trial stopping rules).  When serious adverse events are observed 
with probable causality linked to one of the components, then a withdrawal of the 


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incriminated component(s) will be investigated and implemented by the DSMB if 
possible. 
  
A positive feedback from the DSMB will allow to include the subsequent cohort of 
patients in the safety study.   On the other hand, according the harshness, the 
recurrence and the limitation of acceptable side-effect rates of severe adverse 
events, the DSMB can stop the trial (cf. chapter XVI: Process leading to trial 
stopping rules).  
  
The period of time (7 days) has been determined keeping in mind the half-life and 
pharmacokinetic data of the different components in order to have sufficient time 
to observe potential adverse events. As shown in table 1, the half-life of Anti-
thrombin III, C1-inhibitor, erythropoietin-beta, melatonin, glutathione and alpha-
tocopherol does not exceed 3 days. The half-life of infliximab is 14 days but the 
most important potentially expected side effect is an acute allergic reaction within 
the first hours following administration. No pharmacokinetic data are available for 
apotransferrin but no side effects were observed in clinical trials at the dose 
proposed in the combined drug approach. Concerning epoprostenol, an ex-vivo 
administration is foreseen through the portal vein during the surgical preparation 
of the graft, as such, no systemic absorption is anticipated. 
During the safety study, data on the pharmacokinetics of the orally administrated 
components will be obtained as requested by the local Ethical Committee.  PK 
profiles will be determined based on measurements in blood samples obtained 
after anesthesia induction, just before skin incision, immediately prior to the start 
of the anhepatic phase, immediately prior to reperfusion (defined as opening of the 
portal vein), 30 minutes, 1 h, 2 h, 6h, 12h, 24h, 48h, 72h after reperfusion, daily 
from day 4 to day 7, as foreseen by the protocol (see section 9.5). These 
measurements will be done in the Laboratorium voor Farmacotechnologie en 
Biofarmacie, KU Leuven, O&N2, Campus Gasthuisberg, Leuven. When the 
anticipated levels are not reached, a dose change can be done for the next cohort.  
  
At the end of Part A (safety study), the peak of Aspartate aminotransferase (peak 
AST) of all 10 patients will be also assessed by senior staff physicians and the 
DSMB, as requested by the local Ethical Committee. Only in case of a major increase 
compared to a historical control group, peak AST in the safety study will be then 
compared with patients from a historical matched control group. For this purpose, 
patients will be matched by the following list of variables: donor age (20-30, 31-
40, 41-50, 51-60, 61-70, 71-80, 81-90), donor type [donation after brain death 
(DBD), donation after circulatory death (DCD)], cold ischemia time (CIT) (4-6h, 6-
8h, 8-10h, 10-12h, 12-14h), Lab MELD score (6-19; 20-24; 25-29; 30-34; >35), 
cause of death (trauma vs. non-trauma). The matching will be based on a 
propensity score that is the probability to be a patient in the safety study based on 
the aforementioned list of variables. Each patient in the safety study will be 
matched with one or more patients from the control group having a similar 
propensity score, i.e. 1:N case-control match on the propensity score will be 
performed.  
  
All data and other information of each patient included in the safety study will be 
recorded in an electronic Case Report Form (e-CRF). The e-CRF will be designed to 


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allow continuous recording of variables during the study. The e-CRF and its 
maintenance are technically organized by an external company (EONIX, Mons, 
Belgium www.eonix.be). The e-CRF will be completed under the responsibility of 
the principal investigator. 
At the end of Part A (safety study), the results (including pharmacokinetic profile 
for melatonin and vitamin E as well as peak AST results) will be submitted to the 
Ethical Committee and approval for part B (randomized controlled trial) will be 
requested if appropriate.  
  
 
 


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Study part B: randomized controlled trial 
  
Following approval by Ethical Committee, the second phase of the trial will be 
continued as an investigator driven, phase III, multi-center, randomized and 
single-blinded study. 
  
For this RCT, all consecutive patients listed for LTx at the University Hospitals 
Leuven, Belgium, Centre Hospitalier Universitaire de Liège and other national and 
international centers will be assessed for study eligibility by a senior staff 
physician at time of organ offer. Patients will be randomly assigned to the 
treatment group (multifactorial modulation) or control group (standard of care 
treatment alone).  
  
Eligible patients will be randomized at time of organ offer according to computer-
based varying block size-generated randomization list by a third, independent 
party (EONIX).  
 
 
 
 
 
 
 


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Figure 1. Description of the CAPITL study. First a phase I, safety study will be initiated.  After each cohort of patients, 
the Data Safety Monitoring Board (DSMB) will analyze the data with a particular attention for safety.  Hereafter, there 
are three possibilities: (i) the study is considered to be safe and the study can continue with the following cohort (green 
arrow), (ii) When serious adverse events have been observed with causality linked to one of the components, then a 
withdrawal of the incriminated component(s) will be investigated and implemented by the DSMB if possible (orange 
arrow), or (iii) the study is considered not to be safe and will be stopped (red arrow).  After the first safety study, data 
will be submitted to the local Ethical committee and approval to continue the study requested.   Following approval of 
the local Ethical committee to continue the second phase of the trial eligible patients will be randomly assigned to the 
treatment group (multifactorial modulation) or control group (standard of care treatment alone).  
 
 
 
 


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OBJECTIVE 
 
To demonstrate  
-  the safety of  the multifactorial modulation (part A) 
- the effectiveness of the multifactorial modulation in reducing the peak AST  – a surrogate 
marker of ischemia-reperfusion injury (IRI) - after LTx (part B). 
 
 
PRIMARY ENDPOINT (part B: RCT) 
 
The primary endpoint of the RCT is the log-transformed peak AST, where peak AST is 
defined as the highest value of serum AST within 72 hours following LTx. 
 
 
PLANNED NUMBER OF PATIENTS (part B: RCT) 
 
For the RCT, 36 patients are planned to be included in each group. This number is based 
on a sample size calculation using peak AST values obtained from a historical series of 
patients that underwent LTx at the University Hospitals Leuven. 
 
 
SECONDARY ENDPOINTS (part B: RCT) 
 
- 
Graft loss at 3, 12 months after LTx.  Graft loss is defined as the need for 
retransplantation within one week post LTx due to a non-life-sustaining liver graft 
function (primary non function (PNF)) or later (other reason). 
 
- 
Recipient death at 3, 12months after LTx. 
 
- 
Early graft dysfunction as defined by Olthoff (1): the presence of one or more of the 
following postoperative laboratory analyses: bilirubin ≥ 10mg/dL on day 7, 
international normalized ratio ≥ 1.6 on day 7, and alanine aminotransferase (ALT) 
or AST > 2000 IU/L within the first 7 days. 
 
- 
Incidence of biliary strictures within 12 months post LTx: a biliary stricture is 
defined as a narrowing within the biliary tree, radiologically evident [endoscopic 
retrograde cholangio-pancreatography (ERCP) and/or magnetic resonance 
cholangiopancreatography (MRCP)] to cause clinical symptoms or biochemical 
abnormalities 
requiring 
intervention 
(ERCP, 
percutanous 
transhepatic 
cholangiographic (PTC) drainage, surgery, retransplantation). Biliary strictures 
are categorized as anastomotic or non-anastomotic based on the cholangiographic 
appearance of the biliary tree as judged by a blinded radiologist. Non-anastomotic 
strictures are defined as any strictures, dilatation, or irregularity of the intra- or 
extrahepatic bile ducts of the liver graft at a site(s) other than that of the 
anastomosis. Intra-hepatic biliary strictures are classified in 4 groups: unilateral 
focal, confluence, bilateral multifocal and diffuse necrosis (2). Beside the routine 1 
year post-transplant assessment of the biliary tree by MRCP, biliary strictures will 
be investigated in case of clinical or biochemical suspicion (based upon 
cholestasis). Other causes leading to cholestasis (e.g. hepatic artery thrombosis, 


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bile leakage, rejection or cholangitis) will be excluded based on state-of-the-art 
radiological and histological examination as part of the routine standard treatment 
of care.  These examinations include ultrasound Doppler, CT and CT angiogram, 
biopsy-proven rejection based on histology scored by 2 blinded experienced liver 
pathologists according to the Banff criteria, ERCP and MRCP. 
 
- 
IRI score: The extent of IRI will be assessed by a histological score based on the 
degree of cytoplasmic vacuolization, sinusoidal congestion, necrosis of 
parenchymal cells, apoptosis and influx of neutrophils as described by Suzuki 
score (3) and Monbaliu et al. (4). Liver biopsies will be taken before implantation 
(bench table), 1 hour after reperfusion and 1 week after transplantation.  All 
biopsies will be blindly scored by 2 blinded liver pathologists.  
 
- 
Graft rejection: graft rejection will always be biopsy-proven; a liver biopsy is taken 
after LTx and in case of clinical suspicion of acute rejection.  Clinical suspicion of 
acute rejection may be based on clinical symptoms (such as jaundice, low-grade 
fever) or sometimes nonspecific complaints (such as generalized malaise, 
decreased appetite) and/or biochemical abnormalities (usually increasing or 
plateauing levels -in an abnormal elevated range- of liver tests that were returning 
to normal values). Histological changes will be scored blindly according to the 
BANFF criteria (see appendix 1) by 2 experienced liver pathologists. 
 
- 
Surgical complications: the Clavien-Dindo classification (5) will be used to rank 
surgical complications within 30 days after LTx according to an objective, simple, 
reliable, and reproducible manner. This classification is based on the therapy 
required to treat the complication (appendix 2, adverse event). The severity of the 
biliary strictures as well will be classified using this standardized grading system. 
 
 
 
 


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ELIGIBILITY 
 
- 
Patient suffering from irreversible liver failure eligible for LTx according to 
Eurotransplant guidelines. 
- 
Patients ≥ 18 years of age at time of listing on the waiting list for LTx at the University 
Hospitals Leuven, Belgium, Centre Hospitalier Universitaire de Liège and at  other 
national and international centers. 
 
All consecutive patients listed for LTx will be assessed for study eligibility by a senior staff 
physician via the outpatient clinics (pre-transplant evaluation) or as an inpatient during 
their hospital stay. If patients are willing to participate to this multi-center randomized 
controlled trial and provide informed consent, a member of the LTx team will inform 
patients about the study, orally and in writing.  
 
 
EXCLUSION CRITERIA 
 
- 
Patients who refuse to participate in the study, 
- 
Patients suffering from  acute liver failure 
- 
History of hypersensitivity to anti-thrombin III (Atenativ®), C1-inhibitor 
(Cetor®/Cinryze®), 
melatonin 
(Circadin®), 
epoprostenol 
(Flolan®), 
recombinant human EPO (Neorecormon®), infliximab (Remicade®), glutathione 
(Tationil®), tocopferol (vitamin e suspension 100 mg/mL®) will be excluded from 
the treatment group (cfr. Appendix 3). 
- 
Conditions that prevent the use of the multifactorial modulation (cf. chapter XVIII, 
Appendix 3.2): 
- 
Administration of heparin at therapeutic dose pre-operatively: anti-
thrombin III (Atenativ®), epoprostenol (Flolan®). 
- 
Congestive heart failure arising from severe left ventricular dysfunction: 
epoprostenol (Flolan®), infliximab (Remicade®). 
- 
History of seizures (not related to the underlying liver disease or to 
metabolic disturbances secondary to liver cirrhoris and to be 
distuinghuised from e.g. hepatic encephalopathy), poorly controlled 
arterial hypertension, myocardial infarction or stroke in the month 
preceding the liver transplantation, and history of pre-existing venous 
thromboembolic disease which is not related to liver cirrhosis and 
hypercoagulability (eg. partial, complete or previous vena porta 
thrombosis/ vena mesenterica thrombosis/ vena lienalis): recombinant 
human EPO (Neorecormon®).  
- 
Unstable angina pectoris: recombinant human EPO (Neorecormon®).  
- 
Severe untreated infections such as sepsis, abscesses and opportunistic 
infections: infliximab (Remicade®).  
- 
Use of Vitamin K antagonist anticoagulation preoperatively which can not 
be reversed, women taking oral contraceptives containing oestrogens: 
tocopferol (vitamin E suspension 100 mg/mL®).  
- 
Patients with previous treatment of infliximab (Remicade®),  
 


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- 
Mental conditions rendering the subject incapable to understand the nature, scope, 
and consequences of the trial, 
- 
Combined organ transplantation, 
- 
Re-transplantation, 
- 
Patients that are dialysis-dependent prior to LTx, 
- 
LTx from a living or a split organ donation 
- 
Administration of the multifactorial modulation technically non-feasible (i.e 
impossibility to place the second central catheter required for the separate and 
sequential injection of the different components of the multifactorial modulation). 
 
 
 
 
 
 
 
 
 
 


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TREATMENT 
 
Apart from receiving the standard care as described below (cfr Chapter control group: 
Standard care of treatment), patients randomized to the treatment group receive:  
1. C1-inhibitor 
2. α-Tocopherol 
3. Glutathione 
4. Apotransferrin 
5. Human Recombinant Erythropoietin beta (EPO-β) 
6. Infliximab 
7. Antithrombin-III 
8. Epoprostenol 
9. Melatonin  
 
Detailed description on administration, dosing and treatment periods can be found in 
Table 1 and Figure 2.  
 
Briefly, C1-inhibitor, Glutathione, Apotransferrin, Human Recombinant Erythropoietin 
beta (EPO-β), Infliximab and Antithrombin-III will be infused intravenously (IV) 
sequentially and through different perfusion sets without direct contact during the 
anhepatic and the reperfusion phase. A second infusion of EPO-β is foreseen 6 hours after 
reperfusion. This IV sequential administration just before reperfusion aims to reach a 
plasmatic peak of concentration of each component directly after their infusions. As 
recommended by independent feedback of pharmacologist experts Prof Pieter Annaert 
(Laboratorium of Pharmacotechnology and Biopharmacy, KU Leuven) and Prof Paul 
Declerck , (Laboratorium of Pharmaceutical biology, KU Leuven) (Cfr Addendum 
Feedback regarding CAPITL study) the infusion of infliximab is interrupted during the 
administration of Glutathione. Melatonin and α-Tocopherol will be given orally before the 
transplantation. According to the available pharmacokinetic data, plasmatic peaks of 
concentration should be reached after reperfusion. This will be evaluated during the 
safety study where the pharmacokinetics of melatonin and α-Tocopherol will studied.  
Epoprostenol will be added to 1L of University of Wisconsin preservation solution and 
infused ex-situ directly into the liver through the vena portae during the bench table 
before the implantation. To avoid any contact between the components, a short flush of 
isotonic electrolyte solution will take place between the sequential infusions of the 
components. The infusion of Remicade® (anti-TNF-alpha antibody) starts before the 
reperfusion, is interrupted during administration of glutathione and then restarted 15 
minutes after reperfusion (period of time required to stabilize the patient after the 
reperfusion). The infusion of apotransferrine (scavenger of non-transferrin-bound iron) 
starts before the reperfusion, is interrupted for the administration of EPO-β, C1-inhibitor 
and glutathione, and then restarted 15 minutes after reperfusion. As such, the 
manufacture’s guidelines are followed (slow infusion rate for infliximab (Remicade ®) 
and larger volume for apotransferrine, respectively).  Moreover, as our preclinical studies 
have shown:  the peak of TNF-α and non-transferrin bound iron was observed 3 hours 
and 1 hour after the reperfusion, respectively.  
 
Except for Apotransferrin, all the components are registered and described in the 
MICROMEDEX Healthcare Series (6) and in the MARTINDALE (7). 
 


CAPITL 2016 
 
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Except for apotransferrin, the administration of a single dose of each drug component, at 
the dose described in Table 1, has been proven to be safe and efficient in reducing IRI in 
both animals and clinical studies. A full description of current uses, precautions and 
contraindications, proposed mechanism of action, expected adverse effects, drug 
interactions, dosage form, and packaging can be found in appendix 3.  


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
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Figure 2.  Schematic overview of the combined drug approach: sequence and way of administration of the components of the multifactorial modulation during the different phases of 
the LTx in the treatment arm. A short flush of isotonic electrolyte solution will take place between the sequential infusions of the components in order to avoid any contact between 
components.   


CAPITL 2016 
 
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Product 
(Company) 
Mode of action 
Half-life 
Dose 
Total volume 
required for 
reconstitution 
Way and duration 
of administration 
Timing of 
administration 
Reported side 
effects at 
recommended 
dose  
Main 
described side 
effects in 
literature 
(Cfr appendix 
3) 
Antithrombin III 
Atenativ® 
(Octapharma) 
 
- 
Increase the release of 
prostacyclin and NO. 
- 
Reduction 
of 
inflammatory 
cell 
migration. 
- 
Decrease 
the 
formation of ROS. 
 
72 hours 
3000 IU 
60 cc 
IV – 15 minutes 
Start of anhepatic 
phase 
No 
Allergic 
reactions, 
arterial 
hypertension 
C1-inhibitor 
Cetor®/Cinryze® 
(Sanquin/ViroPhar-
ma (taken over by 
Shire)) 
- 
Inhibition of classical 
(+++), lectine (+) and 
alternative 
(+)  
pathway 
of 
the 
complement activation 
- 
Regulation of intrinsic 
and 
fibrinolytic 
pathways 
of 
the 
coagulation cascade. 
- 
Anti-inflammatory 
protein   
42 hours 
1000 U 
10 cc 
IV – 5 minutes 
10 minutes before 
reperfusion 
No 
Allergic 
reactions, 
Anaphylactic 
shock  
EPO-β 
Neorecormon® 
(Roche) 
- 
Anti-apoptotic. 
- 
Reduction 
of 
inflammatory 
cytokines. 
- 
Antioxidant. 
12 hours 
30.000 IU 
+ 
 
30.000 IU 
0.6 cc 
+ 
 
0.6 cc 
IV – 2 minutes 
13-15 minutes 
before reperfusion  
+ 
6 hours after 
reperfusion 
No 
Thrombus, 
seizure, 
arterial 
hypertension 
Melatonin 
Circadin® 
(Nycomed) 
- 
Antioxidant. 
- 
ROS scavenger. 
4 hours 
6 mg 
3 capsules 
Orally 
On the ward 
before the 
transplantation 
No 
No 
Epoprostenol 
Flolan® 
(GlaxoSmithKline) 
- 
Vasodilatation. 
- 
Antioxidant. 
- 
Inhibition of platelet 
aggregation. 
- 
Reduction of leukocyte 
activation 
and 
adhesion. 
0.1 hour 
500 µg 
50 cc 
Flush through the 
vena porta during 
the bench table 
Ex-situ during the 
bench table before 
the implantation 
Ex-situ 
administration 
(no foreseen 
systemic 
absorption) 
In case of 
systemic 
absorption: 
headache, 
hypotension, 
arrhythmia, 
heart failure, 


CAPITL 2016 
 
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Glutathione 
Tationil 600® 
(Roche Italy) 
- 
Antioxidant. 
- 
ROS scavenger. 
0.25 hour 
3 g 
20 cc 
IV – 2 minutes 
2-4 minutes before 
reperfusion 
No 
No 
Infliximab 
Remicade® 
(Janssen Biologics) 
- 
Inhibition 
of 
inflammatory cascade 
by 
blocking 
both 
soluble 
and 
transmembrane forms 
of 
Tumor 
Necrosis 
Factor-. 
14 days 
3 mg/Kg 
7.5 cc/Kg 
IV – 3 hours 
Start of anhepatic 
phase after 
infusion of 
Antihrombin III 
 
Interruption of the 
infusion during the 
administration of 
Glutathione 
 
Restarted 15 
minutes after 
reperfusion 
Anaphylactic 
reaction first 
hours 
following 
administration 
Anaphylactic 
shock, 
hematological 
effect 
Vitamin E 
suspension 
100 mg/ml 
(Cambridge 
Laboratories) 
- 
Antioxidant. 
- 
ROS scavenger. 
- 
Increase the release of 
prostacyclin. 
53 hours 
500 mg 
5 cc 
Orally 
On the ward 
before the 
transplantation 
No 
No 
Apotransferrin 
(Sanquin) 
- 
Non-transferrine 
bound 
iron 
(redox 
active) chelator. 
/ 
170 mg/kg 
3,4 cc/Kg 
IV – 3 hours 
Start of anhepatic 
phase 
 
Interruption for 
sequential 
administration of 
erythropoietin, C1-
inhibitor and 
Glutathione 
 
Restarted 15 
minutes after 
reperfusion 
No 
No 
Table 1: List of components of the multifactorial modulation. For each component, the mode of action, the half-life, the dose, the total 
volume required for reconstitution, the way of administration, the timing of use and described side effects are described.  Reperfusion 
is defined as restoration of the hepatic inflow through the portal or the hepatic artery or by both. 


CAPITL 2016 
 
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TIME LINE 
 2012: 
 
- 
Finalising multi-factorial modulation protocol, 
 
 2013:  
 
- 
Finalising multi-factorial modulation protocol 
- 
Start recruitment of patients, 
- 
Start inclusion of patients in the first safety phase of the study,  
- 
Start inclusion of patients in RCT phase of the study,  
- 
Start data analysis of patients included in safety phase study, 
- 
Inclusion of patients in the study if safety study is considered as safe, 
- 
Start data collection, 
 
 2014:  
 
- 
Recruitment of patients in RCT phase continued, 
- 
Data collection continued. 
 
 2015:  
 
- 
Recruitment of patients in RCT phase continued, 
- 
Data collection continued. 
 
 2016:  
 
- 
Recruitment of patients in RCT phase continued, 
- 
Data collection continued. 
 
 2017:  
 
- 
Data collection continued, 
- 
Start data analysis. 
 
 2018:  
 
- 
Data analysis continued. 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
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2012 
2013 
2014 
2015 
2016 
2017 
2018  
Finalizing 
Protocol, 
submission 
to 
ethical 
committee 
and FAGG 
                                
 
 
 
 
 
 
 
 
 
 
 
 
Patient 
recruitment                                 
 
 
 
 
 
 
 
 
 
 
 
 
Data 
collection 
                                 
 
 
 
 
 
 
 
 
 
 
 
Data 
analysis 
                                 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3 Time line of the RCT 
 
 
 
 
 
 


CAPITL 2016 
 
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Pagina 24 
 
III. RATIONALE AND NOVELTY 
3.1 RATIONALE  
3.1.1 Liver transplantation and the quest to solve the donor organ shortage 
 
LTx has become the treatment of choice for liver failure offering patients both an 
improved survival and quality of life. Indeed, excellent outcome after LTx with reported 1 
and 5 years graft and recipient survival exceeding 90% and 75% are achieved in our 
institution. Furthermore, successful LTx allows recipients a return to a near-to-normal 
life style.  Currently, the annual incidence of LTx is ~20 per one million inhabitants (~100-
120 LTxs are performed every year in Flanders, ~1500 within the Eurotransplant area).   
The success of LTx has resulted in a dramatic shortage of organs, resulting in death of 
patients on the waiting list. This justifies the search for novel strategies to prevent death 
of patients who could have benefitted a life-saving transplantation.   
 
Liver grafts are usually retrieved from brain-dead donors, but due to the afore-mentioned 
worldwide shortage (8), there is an increasing interest by using less-than-optimal donors 
that offers the most immediate promise to substantially enlarge the donor pool for LTx. 
Those grafts -the so-called extended criteria donor grafts (ECD) -previously considered 
unsuitable for transplantation are increasingly used by many centers, including our 
center (9). Factors defining extended criteria liver grafts include donor age (>65 years), 
graft steatosis, prolonged cold ischemia time (>12 hours), cause of death, hemodynamic 
stability at the time of procurement, and donation after circulatory death (DCD).  
 
Grafts from these donors are known to be more sensitive for IRI (10) which is the main 
cause of early graft dysfunction. Early graft dysfunction is clinically and biochemically 
characterized by liver injury and poor function (1). Most importantly, early graft 
dysfunction is associated with an increased risk of mortality and graft loss which 
jeopardizes the short and long term outcome for liver transplant recipients.  
 
In addition, in search for objective and transparent allocation of these scarce donor 
organs, recipient allocation policies have been adopted and have brought sicker patients 
to the operation room.  Indeed, prioritizing recipients for a LTx is based on the Model of 
End-stage Liver Disease (MELD) score.  The MELD score was developed to predict survival 
and reflects severity of the end-stage liver disease based on the patient's values for serum 
bilirubin, serum creatinine, and the international normalized ratio for prothrombin time 
(INR).  Since liver grafts are more often transplanted into sicker patients, this also affects 
graft and recipient survival. 
 
This coincidence of donor and recipient factors contributing to early graft dysfunction 
justifies the strong need to investigate liver-protective strategies in LTx as well. 
 
In this context, the commonly applied strategy of organ preservation using hypothermic 
preservation solutions no longer fulfils the demand to maintain graft viability after 
transplantation. Additional new strategies are needed to overcome this increased risk of 
early graft dysfunction, and should aim to tackle IRI.  
3.1.2 Ischemia reperfusion injury in LTx and graft function 
 


CAPITL 2016 
 
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In the course of LTx, IRI represents an important non-immunologic antigen-independent 
factor that influences graft outcome, increases graft immunogenicity and host-allo-
responsiveness (11, 12, 13). Moreover, as recently assessed by our team, the severity of 
IRI as reflected by the peak AST>2000IU/l per se has a substantial impact on patient 
survival (figure 4). 
 
IRI is characterized by a complex series of interrelated events which invariably takes place 
in every graft during reperfusion in the recipient (11, 12, 13, 14) (Figure 5 and 6). 
 
Briefly, in the transplantation setting, organs become deprived from oxygen during 
procurement and preservation.  Restoration of oxygenation at the moment of graft 
reperfusion with blood enhances the ischemic injury caused at a cellular level resulting in 
an inflammatory self-amplifying loop and hepatocellular damage. The extent of injury to 
the graft on reperfusion is variable from minimal over severe to total destruction of the 
graft (12, 13) and depends on the degree of activation of key players including Kupffer 
cells, platelets and leukocytes, besides the generated pro-inflammatory response 
(oxidative stress, inflammatory cytokines, cytoplasmatic proteases, up regulation of pro-
inflammatory transcription factors…).   
 
Clinically IRI can result in immediate graft function, early graft dysfunction (considered 
to occur in 10-30% of grafts) or primary graft non-function (considered to occur in < 5% 
of grafts), respectively (15). Furthermore, the biliary tree is especially vulnerable to IRI, 
contributing to intrahepatic biliary strictures, a common reason of late graft loss (16). 
Finally, IRI also contributes to graft rejection since it activates a cascade of innate-
dominated pro-inflammatory immune responses culminating the adaptive immune 
response. 
 
Because graft dysfunction affects both short and long term graft survival, understanding 
and attenuating hepatic IRI is regarded an imperative strategy to improve short and long 
term outcome of liver grafts, especially with the increased use of less than ideal liver 
grafts. Basic research using animal models has elucidated dominant molecular pathways 
important in the pathogenesis of liver IRI (Figure 4 and 5). This knowledge has resulted 
in designing different therapeutic modalities to reduce IRI.   
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
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Figure 4: In a historical cohort of 552 LTx recipients at UZ Leuven (01/2000 – 12/2010) 
inferior recipient survival is observed in recipients with severe IRI (peak AST > 2000 
IU/L) compared to recipients with a less severe IRI (peak AST < 2000 IU/L).  
 
 


CAPITL 2016 
 
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Figure 5: Schematic overview of the pathogenic network of anoxic cell resulting in self-
amplifying loops of several injurious pathways (14).  
 


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Figure 6: Pathogenic network of the inflammatory response of ischemia reperfusion 
injury with the initiation of self-amplifying loops of several injurious pathways (14).  
 
 
3.1.3. Therapeutic strategies to attenuate ischemia reperfusion injury 
To improve outcome of ECD and DCD LTx, and to increase their safer use, attenuating the 
IRI cascade by pharmacological modulation is an interesting concept. Several drugs have 
already been tested in animal models. However, only few strategies have been tested in 
humans and very few are currently implemented as common clinical practice (17, 18, 19, 
20, 21, 22, 23).   
 
As mentioned above, the IRI cascade is notorious for its redundant pathways (Figure 4 
and 5). Hence, blocking one component of the cascade is rarely enough to decrease the 
severity of IRI. We therefore believe it is essential to combine pharmacological agents 
acting simultaneously at several steps of the IRI cascade referred to as combined drug 
approach. 
 
 


CAPITL 2016 
 
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3.1.4. Ischemia reperfusion injury leading to graft failure in a preclinical model of 
LTx at the KU Leuven  
The KU Leuven group previously studied IRI in a large animal (pig) LTx model.  More in 
particular, the IRI using livers from DCD was studied in this model inducing a well-
characterized and controllable injury of normothermic ischemia prior to procurement, 
cooling, preservation and transplantation. Using such liver grafts has been suggested a 
promising strategy to extend the donor pool, but has been found associated with a poor 
graft survival (10–15% lower compared to the traditional brain death donors) because of 
a higher rate of primary graft non-function and intrahepatic bile duct strictures or 
ischemic-biliary lesions. We therefore first investigated the tolerance of livers exposed to 
warm ischemia, provided clinical guidelines to avoid transplantation of grafts destined to 
fail and elucidated some mechanisms contributing to this graft failure (24).   
LTx in a porcine model is generally regarded to be clinically relevant because of the 
undeniable physiological and anatomical similarities between pigs and human. It is 
considered at the same time as an extremely stringent model.  Because neither 
vasopressive drugs, nor transfusions of blood derivatives are used, the natural evolution 
of the hepatic injury and inflammation in such a model remains unmasked.  We observed 
that – prior to a short fixed cold ischemic period - 15 min of warm ischemia was well-
tolerated, whereas warm ischemia ≥ 30 min and ≥ 60 min, induced a 50 or 100% PNF risk, 
respectively (24).  
In grafts destined to fail, warm ischemia on its own caused hepatocellular damage and 
activation of Kupffer cells prior to cold storage and reperfusion. In recipients with PNF, 
this resulted in an overproduction of inflammatory cytokines (in particular TNF-α and IL-
6) and a failure of some antioxidant mechanisms (especially α-tocopherol and 
glutathione).  In PNF recipients, high quantities of redox-active iron - known to catalyze 
the generation of free oxygen radicals - were released in blood (2).  Moreover in PNF 
recipients, an increased activity and decreased inhibition of serum PhospholipaseA2 were 
observed (25). Furthermore, in this preclinical transplantation model, exposure to 
incremental periods of warm ischemia was also found related to increased bile salt 
toxicity after transplantation (24).  Extending the cold ischemia - even following a short 
warm ischemia – caused graft dysfunction and inversely affected post-transplantation 
recipient survival (26).    
 
Based on these pathophysiological IRI mechanisms contributing to graft failure, we 
designed a multifactorial modulation strategy targeting these mechanisms (Table 2)(4).   
We focused on (i) optimizing graft quality during procurement and preservation and (ii) 
administering multiple biological reagents expected to attenuate the IRI in the recipient.  
Most of the agents selected had to represent natural sources (glycine, glutathione, α-
tocopherol, α1 acid glycoprotein, and apotransferrin) or known to be non-toxic under 
physiological circumstances/concentrations.  In other words, it could be anticipated that 
these drugs can be safely infused.  The use of these products had also to be supported by 
literature reports and available for clinical or preclinical use.  Any intervention in the 
donor – prior to declaration of death – was omitted in this protocol to avoid all potential 
ethical conflicts when applying this strategy clinically at a later stage.  This resulted in a 
multifactorial strategy combining several drugs and operating on different mechanisms 
and at different stages of the IRI cascade (table 2).   In addition, we speculated that the 
cumulative/synergistic effect of this multifactorial modulation would exceed the effect of 
the individual components when administered in monotherapy.    
 


CAPITL 2016 
 
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Active components 
Mechanism 
Glycine 
Kupffer cell stabilizer, hepatocyte and 
endothelial cell protector 
 
Gluthatione 
Antioxidant 
 
Apotransferrin 
Redox-active iron chelator 
 
α1-acid glycoprotein 
Antioxidant 
 
α-tocopherol 
Antioxidant 
 
FR167653 
MAPK inhibitor 
 
Table 2: components of the multifactorial modulation used in the preclinical model and 
their mechanisms of action. 
 
3.1.5. Outcome after LTx in our porcine model using our multifactorial 
pharmacological modulation protocol 
The working hypothesis was that in porcine livers exposed to long warm ischemia and 
normally destined to fail, such a multifactorial modulation strategy would reduce the IRI 
and that this would result in better liver function, and increased graft and recipient 
survival.   
 
Prior to the design of the study, no power analysis or sample calculation was performed 
since this was a seminal study for which no prior data were available.  Therefore - in 
retrospect- a one-tail power calculation with an alpha error level of 5% (DSS research, 
http://www.dssresearch.com/toolkit/spcalc/power_p2.asp) was performed using the 
described cohorts and the observed recipient and graft survivals which corresponded to 
a statistical power of 80.2% for graft and 80.5% for recipient survival, respectively.   
Porcine livers exposed to 45 min warm ischemia were cold stored, transplanted and 
either modulated (n=6) or not (controls, n=9).  In the modulation group, donor livers were 
flushed with warm Ringers (avoiding cold-induced vasoconstriction), streptokinase 
(eliminating stagnating thrombi) and epoprostenol (vasodilator, platelet aggregation 
inhibitor) prior to cold storage.  In recipients, glycine (Kupffer cell stabilizer), α1-acid-
glycoprotein (anti-inflammatory protein), MAPKinase-inhibitor (pro-inflammatory 
cytokine generation inhibitor), α-tocopherol and glutathione (anti-oxidants), and 
apotransferrin (iron chelator) were administrated intravenously.  PNF, survival, lactate, 
transaminase, TNF-α, redox-active iron, and biliary bile salt-to-phospholipid ratio were 
monitored. No PNF was observed in modulated versus 55% in control pigs (p=0.025). 
Survival was 83% in modulated versus 22% in control pigs (p=0.02).  At 180 min post-
reperfusion, lactate was lower in modulated (5.4±1.9 mmol/L) versus control pigs 
(9.4±2.2 mmol/L; p=0.011).  At 60 min post-reperfusion, there was a trend for lower AST 
in modulated versus control pigs at 60 min (939±578 versus 1683±873 IU/L; p=0.089).  
Post-reperfusion, TNF-α remained stable in modulated pigs (49±27pg/ml at 15 min and 
85±26pg/ml at 180 min; p=0.399) but increased in control pigs (107±36pg/ml at 15 min 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
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and 499±216pg/ml at 180 min; p=0.023).  At 180 min post-reperfusion, redox-active iron 
was lower in modulated pigs versus control pigs (0.21±0.18 versus 0.042±0.062 µM; 
p=0.038). Biliary bile salt-to-phospholipid ratio post-LTx was lower in modulated versus 
control pigs (1128±447 versus 4836±4619; p=0.05) (4).  
In conclusion, our preclinical study demonstrated that in a stringent model of severe IRI (as 
in DCD LTx), a multifactorial modulation containing several biological reagents targeting 
previously identified mechanisms of warm IRI remarkably improved the degree of IRI, 
eliminated PNF, reduced TNF-α, improved liver function, reduced bile salt toxicity, and 
improved survival (4).  
 
3.2 NOVELTY 
 
Due to the promising results of a multifactorial modulation strategy to attenuate IRI, as 
observed in our preclinical model, we now aim to translate this strategy clinically. Indeed, 
because of organ shortage and a persisting imbalance between LTx candidates and 
available organs, expanded criteria organs are increasingly used.  The use of these 
“extended criteria” livers has been found associated with an enhanced IRI, which is the 
main cause of delayed graft function and PNF. Therefore, a multifactorial modulation 
strategy or combined drug approach is a beneficial intervention for expanded criteria 
donor organs that could lead to a reduction of IRI, delayed graft function and consequent 
hospital stay with associated health care cost; an enlargement of the donor pool with a 
safer use of the extended criteria donor and therefore a limitation of the waiting time and 
the concomitant risk to die whilst awaiting a LTx. Any intervention that will be beneficial 
for expanded criteria donor organs is also regarded to be beneficial for all other 
normal/uncompromised organs.   
In particular, the development, after LTx using “normal livers”, of biliary complications 
such as ischemic type biliary strictures represents an additional potential target that 
might benefit of administration of the multifactorial modulation.    
 
The novelty of our study to attenuate IRI lies on the sequential administration of 
drugs acting simultaneously on several steps of the IRI cascade and its proof of 
concept in a clinically relevant stringent large animal model of LTx. Such combined 
drug approach has already been approved in other fields than LTx and is currently 
used in oncological treatment for instance.  
 
All those efforts required to set up the multifactorial modulation were presented in 
numerous national and international conferences, published in a top-ranking surgical 
magazine (Annals of Surgery) and awarded on national and international meetings (Best 
communication in basis science 2007,  Belgian Week of Gastroenterology.  Best abstract 
2007, Belgian Transplantation Society.  International young investigator award, American 
Transplant Congress, 2007). 
 
Since then, publications on the multifactorial modulation have been cited by some authors 
as a required and innovative strategy to attenuate IRI. Professor Pierre-Alain Clavien 
(Zurich, Switzerland), expert in LTx and IRI referred to our multifactorial approach as 
following: “However, from our point of  view, the future of pharmacological strategies 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
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attenuating or preventing ischemia/reperfusion injury lies more in the combination of drugs 
acting simultaneously on several steps of the ischemia/reperfusion injury cascade” (28). 
 
Finally, we learned that a research group who was previously studying a single agent 
(glycine) against hepatic IRI (Prof Dr P Schemmer, http://www.controlled-trials. 
com/ISRCTN69350312 /) is now –similar to our multi-factorial approach- also focusing 
on more than one single agent to attenuate IRI.  
 
3.3 FROM PRECLINICAL MODEL TO CLINICAL CAPITL STUDY 
 
In contrast to the preclinical study, not all components are available for a clinical study.  
FR167653, a p38MAP Kinase inhibitor that we previously obtained from Astellas is no 
longer available. The reasons for not continuing were not officially communicated.   
Moreover, the plasma protein alpha-1-glycoprotein and glycine are not readily available 
for clinical use (not in accordance with the required GMP standards), and would require 
substantial product development effort from Sanquin or CAF-DCF which is currently not 
anticipated by this company.  Finally, glycine is commercially not available.   
We looked for an appropriate alternative for all components of the multifactorial 
strategy that could not be applied in the clinical trial: 
 
- Infliximab: Neutralization of the overproduction of TNF-α, 
- Antithrombin III: vasodilatation of the microcirculation through i.a. release of   
NO and reduction of NFκB expression and pro-inflammatory cytokine production, 
- EPO: anti-apoptosis, anti-inflammation, anti-oxidant, 
- Melatonine: anti-oxidant with synergistic action with respect to glutathione and 
vitamin E. 
 
Extensive literature research learned us that some key factors of IRI as described and 
depicted in figures 4 and 5 such as the complement have been left untouched in the 
preclinical model. In addition, Vekemans et al. have demonstrated that the multifactorial 
modulation used in the preclinical model was found to suppress a range of inflammation-
regulating genes but identified other potential targets as well (27).  
 
Therefore, an intensive and up-to-date literature study has been undertaken at the start 
of this research project aiming to “update the multifactorial modulation protocol” (Table 
3). Other components (including so called “biologicals” and “small molecules”) were 
chosen on the basis of their clinical availability, short half-life, published efficiency on liver 
IRI, safety profile if administrated 1 time (except EPO) at a therapeutic and clinical 
recommended dose. Consequently, erythropoietin, C1 inhibitor, melatonin, antithrombin-
III and infliximab have been added to the protocol.  
 
Preclinical and safety studies have already been published for those additional 
components (cf appendix 3). In contrast to pharmaceutical sponsored trials that test 
entirely new compounds whose toxicity profile is not yet clear and that may have been 
stopped prematurely because of an increase in morbidity and mortality, our drugs are 
already used clinically in other indications (cf appendix 3). 
 


CAPITL 2016 
 
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It has been anticipated that administration of the individual components of the combined 
drug approach is safe: 
 
 
1. Most of the components selected represent natural sources: biological or 
endogenous compounds and are available for clinical use. 
 
2. The clinical and safe use of these products is supported by extensive literature 
reports (dose, side effect, safety regulation, contraindication). 
 
3. The components are known to be non-toxic with a single intravenously dose (or 
even repeated dose for EPO) and at the dose selected. 
 
4. Independent feedback of pharmacologist experts (Prof Pieter Annaert, 
Laboratorium of Pharmacotechnology and Biopharmacy KU Leuven;  Prof Paul 
Declerck, Laboratorium of Pharmaceutical biology KU Leuven) on the potential 
occurrence of drug-to-drug interactions (DDI) between the different components 
of the multifactorial modulation. (Cfr Addendum: Feedback regarding CAPITL 
study).  
 
a. The risk for PK type drug-drug interactions between the components is 
limited.  
b. At the given dose (which is not exceeding the normal physiological level for 
the biologicals or small molecules), it is anticipated that the 
pharmacokinetics of the components will not be affected.   
c. There is no concern for potential interactions with human serum albumin 
or with pharmaca used during the anesthesia for liver transplantation 
(chapter V, 5.1.2 Anesthesia).  
d. The different elements of the combined drug approach will be administered 
sequentially, through separate infusion sets, and without direct contact 
between the components (lines will be flushed with an isotonic electrolyte 
solution in between the administration of the components), for a detailed 
overview see figure 2.  
e. It is unlikely that the pharmacodynamics of the components will be affected 
because (except for C1 inhibitor and antithrombin-III) the components 
exert distinct effects through different mechanisms.  
 
5. As recommended by the pharmacological experts, specific attention will be given 
regarding safety issues of individual components:  
 
a. No drugs will be administered during the administration of glutathione,  
b. No heparine will be administered during the liver transplantation to avoid 
enhancement of the anticoagulant properties of Antithrombin-III; in case of 
clinically relevant problems, hemostasis can be corrected.  
c. Patients previously treated with infliximab will be excluded.   
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
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Product 
(Company) 
Mode of action 
Half-life 
Dose 
Total volume 
required for 
reconstitution 
Way and duration 
of administration 
Timing of 
administration 
Reported side 
effects at 
recommended 
dose  
Main 
described side 
effects in 
literature 
(Cfr appendix 
3) 
Antithrombin III 
Atenativ® 
(Octapharma) 
 
- 
Increase the release of 
prostacyclin and NO. 
- 
Reduction 
of 
inflammatory 
cell 
migration. 
- 
Decrease 
the 
formation of ROS. 
 
72 hours 
3000 IU 
60 cc 
IV – 15 minutes 
Start of anhepatic 
phase 
No 
Allergic 
reactions, 
arterial 
hypertension 
C1-inhibitor 
Cetor®/Cinryze® 
(Sanquin/ViroPhar-
ma (taken over by 
Shire)) 
- 
Inhibition of classical 
(+++), lectine (+) and 
alternative 
(+)  
pathway 
of 
the 
complement activation 
- 
Regulation of intrinsic 
and 
fibrinolytic 
pathways 
of 
the 
coagulation cascade. 
- 
Anti-inflammatory 
protein   
42 hours 
1000 U 
10 cc 
IV – 5 minutes 
10 minutes before 
reperfusion 
No 
Allergic 
reactions, 
Anaphylactic 
shock  
EPO-β 
Neorecormon® 
(Roche) 
- 
anti-apoptotic 
- 
Reduction 
of 
inflammatory 
cytokines. 
- 
Antioxidant. 
12 hours 
30.000 IU 
+ 
 
30.000 IU 
0.6 cc 
+ 
 
0.6 cc 
IV – 2 minutes 
13-15 minutes 
before reperfusion  
+ 
6 hours after 
reperfusion 
No 
Thrombus, 
seizure, 
arterial 
hypertension 
Melatonin 
Circadin® 
(Nycomed) 
- 
Antioxidant. 
- 
ROS scavenger. 
4 hours 
6 mg 
3 capsules 
Orally 
On the ward 
before the 
transplantation 
No 
No 
Epoprostenol 
Flolan® 
(GlaxoSmithKline) 
- 
Vasodilatation. 
- 
Antioxidant. 
- 
Inhibition of platelet 
aggregation. 
- 
Reduction of leukocyte 
activation 
and 
adhesion. 
0.1 hour 
500 µg 
50 cc 
Flush through the 
vena porta during 
the bench table 
Ex-situ during the 
bench table before 
the implantation 
Ex-situ 
administration 
(no foreseen 
systemic 
absorption) 
In case of 
systemic 
absorption: 
headache, 
hypotension, 
arrhythmia, 
heart failure, 


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Glutathione 
Tationil 600® 
(Roche Italy) 
- 
Antioxidant. 
- 
ROS scavenger. 
0.25 hour 
3 g 
20 cc 
IV – 2 minutes 
2-4 minutes before 
reperfusion 
No 
No 
Infliximab 
Remicade® 
(Janssen Biologics) 
- 
Inhibition 
of 
inflammatory cascade 
by 
blocking 
both 
soluble 
and 
transmembrane forms 
of 
Tumor 
Necrosis 
Factor-. 
14 days 
3 mg/Kg 
7.5 cc/Kg 
IV – 3 hours 
Start of anhepatic 
phase after 
infusion of 
Antithrombin III 
 
Interruption of the 
infusion during the 
administration of 
Glutathione 
 
Restarted 15 
minutes after 
reperfusion 
Anaphylactic 
reaction first 
hours 
following 
administration 
Anaphylactic 
shock, 
hematological 
effect 
Vitamin E 
suspension 
100 mg/ml 
(Cambridge 
Laboratories) 
- 
Antioxidant. 
- 
ROS scavenger. 
- 
Increase the release of 
prostacyclin. 
53 hours 
500 mg 
5 mL 
Orally 
On the ward 
before the 
transplantation 
No 
No 
Apotransferrin 
(Sanquin) 
- 
Non-transferrine 
bound 
iron 
(redox 
active) chelator. 
/ 
170 mg/kg 
3.4 cc/Kg 
IV – 3 hours 
Start of anhepatic 
phase 
 
Interruption for 
sequential 
administration of 
erythropoietin, C1-
inhibitor and 
Glutathione 
 
Restarted 15 
minutes after 
reperfusion 
No 
No 
Table 3: List of components of the multifactorial modulation. For each component, the mode of action, the half-life, the dose and the 
reported adverse event at the recommended dose are described 


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3.4 DESIGN 
 
A two-part, investigator driven, multi-center, single-blinded, adaptive study to 
assess the safety and to study the efficacy of a Combined drug Approach to Prevent 
Ischemia-reperfusion injury during Transplantation of Livers. 
 
The first part of the study (part A) was a safety study conducted in 10 patients 
undergoing a liver transplantation which was regarded to be safe by the Data and 
Safety Monitoring Board and the results will be submitted to the local ethical 
committee.  
 
The second part of the study (part B) foresees in a randomized controlled trial 
conducted in patients undergoing a liver transplantation at the University 
Hospitals Leuven, Belgium, Centre Hospitalier Universitaire de Liège and at other 
national and international centers.  
 
 
Study part A: safety study  
 
A phase I, safety study will be first performed (Figure 1) in 10 patients in 4 smaller 
cohorts.  The rationale is to establish the safety of the combined drug approach 
aimed to reduce ischemia-reperfusion injury during liver transplantation in 
eligible recipients. The combined drug approach, also referred as multifactorial 
modulation, foresees the sequential administration of drugs in eligible liver 
transplant recipients. Drugs will be administered as such that they will never be in 
direct contact with each other. 
  
There will be 10 patients included in Part A, conducted in 4 consecutive small 
cohorts of 2, 2, 3, and 3 patients, respectively (Figure 1).  For this safety study, all 
consecutive patients listed for LTx will be assessed for study eligibility by a senior 
staff physician at time of organ offer. The data and safety assessment of each cohort 
will then be reviewed by the Data and Safety Monitoring Board (DSMB) before 
patients are included in the next cohort (see section 16.1). 
  
Safety will be assessed by well-defined objective criteria and parameters based on 
incidence and/or severity of adverse events indicating a potential health hazard 
caused by the multifactorial modulation within 7 days post LTx (see chapter XVI: 
Process leading to trial stopping rules).  When serious adverse events are observed 
with probable causality linked to one of the components, then a withdrawal of the 
incriminated component(s) will be investigated and implemented by the DSMB if 
possible. 
  
A positive feedback from the DSMB will allow to include the subsequent cohort of 
patients in the safety study.   On the other hand, according the harshness, the 
recurrence and the limitation of acceptable side-effect rates of severe adverse 


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events, the DSMB can stop the trial (cf. chapter XVI: Process leading to trial 
stopping rules).  
  
The period of time (7 days) has been determined keeping in mind the half-life and 
pharmacokinetic data of the different components in order to have sufficient time 
to observe potential adverse events. As shown in table 1, the half-life of Anti-
thrombin III, C1-inhibitor, erythropoietin-beta, melatonin, glutathione and alpha-
tocopherol does not exceed 3 days. The half-life of infliximab is 14 days but the 
most important potentially expected side effect is an acute allergic reaction within 
the first hours following administration. No pharmacokinetic data are available for 
apotransferrin but no side effects were observed in clinical trials at the dose 
proposed in the combined drug approach. Concerning epoprostenol, an ex-vivo 
administration is foreseen through the portal vein during the surgical preparation 
of the graft, as such, no systemic absorption is anticipated. 
During the safety study, data on the pharmacokinetics of the orally administrated 
components will be obtained as requested by the local Ethical Committee.  PK 
profiles will be determined based on measurements in blood samples obtained 
after anesthesia induction, just before skin incision, immediately prior to the start 
of the anhepatic phase, immediately prior to reperfusion (defined as opening of the 
portal vein), 30 minutes, 1 h, 2 h, 6h, 12h, 24h, 48h, 72h after reperfusion, daily 
from day 4 to day 7, as foreseen by the protocol (see section 9.5). These 
measurements will be done in the Laboratorium voor Farmacotechnologie en 
Biofarmacie, KU Leuven, O&N2, Campus Gasthuisberg, Leuven. When the 
anticipated levels are not reached, a dose change can be done for the next cohort. 
  
At the end of Part A (safety study), the peak of Aspartate aminotransferase (peak 
AST) of all 10 patients will be also assessed by senior staff physicians and the 
DSMB, as requested by the local Ethical Committee. Only in case of a major increase 
compared to a historical control group, peak AST in the safety study will be then 
compared with patients from a historical matched control group. For this purpose, 
patients will be matched by the following list of variables: donor age (20-30, 31-
40, 41-50, 51-60, 61-70, 71-80, 81-90), donor type [donation after brain death 
(DBD), donation after circulatory death (DCD)], cold ischemia time (CIT) (4-6h, 6-
8h, 8-10h, 10-12h, 12-14h), Lab MELD score (6-19; 20-24; 25-29; 30-34; >35), 
cause of death (trauma vs. non-trauma). The matching will be based on a 
propensity score that is the probability to be a patient in the safety study based on 
the aforementioned list of variables. Each patient in the safety study will be 
matched with one or more patients from the control group having a similar 
propensity score, i.e. 1:N case-control match on the propensity score will be 
performed.  
  
All data and other information of each patient included in the safety study will be 
recorded in an electronic Case Report Form (e-CRF). The e-CRF will be designed to 
allow continuous recording of variables during the study. The e-CRF and its 
maintenance are technically organized by an external company (EONIX, Mons, 
Belgium www.eonix.be). The e-CRF will be completed on a strict daily basis under 
the responsibility of the principal investigator. 


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At the end of Part A (safety study), the results (including pharmacokinetic profile 
for melatonin and vitamin E as well as peak AST results) will be submitted to the 
Ethical Committee and approval for part B (randomized controlled trial) will be 
requested if appropriate.  
 
 
 
 
 


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Study part B: randomized controlled trial 
 
Following approval by Ethical Committee, the second phase of the trial will be 
continued as an investigator driven, phase III, multi-center, randomized and 
single-blinded study. 
 
For this RCT, all consecutive patients listed for LTx at the University Hospitals 
Leuven, Belgium, Centre Hospitalier Universitaire de Liège and at other national 
and international centers will be assessed for study eligibility by a senior staff 
physician at time of organ offer. Patients will be randomly assigned to the 
treatment group (multifactorial modulation) or control group (standard of care 
treatment alone).  
 
Eligible patients will be randomized at time of organ offer according to computer-
based permuted blocks-generated randomization list by a third, independent party 
(EONIX).  
 
 
3.5 OBJECTIVES  
 
To demonstrate  
-  the safety of  the multifactorial modulation (part A) 
- the effectiveness of the multifactorial modulation in reducing the peak AST  – a surrogate 
marker of ischemia-reperfusion injury (IRI) - after LTx (part B). 
 
 
Peak AST is widely used as a surrogate reflecting the extent of IRI. This parameter 
correlates well with the parenchymal graft injury (29) following IRI and is associated with 
subsequent initial liver function or dysfunction after transplantation (30). 
 
 
 


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IV. PARTICIPANTS 
4.1 ELIGIBILITY CRITERIA 
 
- Patient suffering from any type of irreversible liver failure eligible for LTx according to 
Eurotransplant guidelines. 
- Patients ≥ 18 years of age at time of listing on the waiting list for LTx in at the University 
Hospitals Leuven, Belgium, Centre Hospitalier Universitaire de Liège and other national 
and international centers 
 
All consecutive patients listed for LTx will be assessed for study eligibility by a senior staff 
physician via the outpatient clinics or as an inpatient during their hospital stay. 
 
4.2 INCLUSION CRITERIA 
 
- 
Older than 18 years, 
- 
Patients undergoing LTx at the University Hospitals Leuven, Belgium, Centre 
Hospitalier Universitaire de Liège and other national and international transplant 
centers 
- 
Patient must have signed the patient informed consent, 
- 
All donor types: donation after brain death (DBD), donation after cardiac death 
(DCD), standard criteria donors (SCD) and extended criteria donors (ECD) defined 
according Paris consensus (31). 
 
4.3 EXCLUSION CRITERIA 
 
- 
Patients who refuse to participate in the study, 
- 
History of hypersensitivity to anti-thrombin III (Atenativ®), C1-inhibitor 
(Cetor®/Cinryze®), 
melatonin 
(Circadin®), 
epoprostenol 
(Flolan®), 
recombinant human EPO (Neorecormon®), infliximab (Remicade®), glutathione 
(Tationil®), tocopferol (vitamin e suspension 100 mg/mL®) will be excluded from 
the treatment group (cfr. Appendix 3.2). 
- 
Conditions that prevent the use of the multifactorial modulation (cf. chapter XVIII, 
Appendix 3.2 ): 
o Administration of heparin at therapeutic dose pre-operatively: anti-
thrombin III (Atenativ®), epoprostenol (Flolan®). 
o Congestive heart failure arising from severe left ventricular dysfunction: 
epoprostenol (Flolan®), infliximab (Remicade®). 
o History of seizures (not related to the underlying liver disease or to 
metabolic disturbances secondary to liver cirrhoris and to be 
distuinghuised from e.g. hepatic encephalopathy), poorly controlled 
arterial hypertension, myocardial infarction or stroke in the month 
preceding the liver transplantation, and history of pre-existing venous 
thromboembolic disease which is not related to liver cirrhosis and 
hypercoagulability (eg. partial, complete or previous vena porta 


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thrombosis/ vena mesenterica thrombosis/ vena lienalis): recombinant 
human EPO (Neorecormon®).  
o Unstable angina pectoris: recombinant human EPO (Neorecormon®).  
o Severe untreated infections such as sepsis, abscesses and opportunistic 
infections: infliximab (Remicade®).  
o Use of Vitamin K antagonist anticoagulation preoperatively which can not 
be reversed, women taking oral contraceptives containing oestrogens: 
tocopferol (vitamin E suspension 100 mg/mL®).  
o Patients with previous treatment of infliximab (Remicade®),  
 
- 
Mental conditions rendering the subject incapable to understand the nature, scope, 
and consequences of the trial, 
- 
Patients suffering from  acute liver failure 
- 
Combined organ transplantation, 
- 
Re-transplantation, 
- 
Patients that are dialysis-dependent prior LTx, 
- 
LTx from a living or a split organ donation 
- 
Administration of the multifactorial modulation technically non-feasible (i.e 
impossibility to place the second central catheter required for the separate and 
sequential injection of the different components of the multifactorial modulation). 
 
 


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V. INTERVENTION 
5.1 CONTROL GROUP: Standard care of treatment 
5.1.1 Surgical procedure 
 
The liver is first assessed and prepared for the implantation on the bench table 
(quality of the perfusion during procurement, degree of steatosis, vascular 
anomaly,...). During this phase, 1l of a CE labelled, preservation solution [including 
all CE marked preservation solutions such as University of Wisconsin (UW), 
Histidine Trytophan Ketoglutarate (HTK), IGL-1®, Celsior®,…] preservation 
solution is directly infused through the vena portae with the aim to rinse the liver 
from remaining blood, toxic metabolites and cellular debris. The orthotopic LTx 
consists of a hepatectomy phase, an anhepatic phase and a reperfusion phase.  
Following the hepatectomy, depending on the surgeon’s and local center’s 
preference, a veno-venous bypass is installed or not.  During the anhepatic phase, 
the liver can be transplanted either by reconstructing both the supra- and 
infrahepatic cava vein (in case of veno-venous bypass) or either by a piggy-back 
technique (with or without creating a portocaval shunt). During the implantation 
of the graft, the graft may be rinsed out with 2 liters of Hartmann solution (isotonic, 
sodium 130 mmol/L, chlorine 109 mmol/L, lactate 28 mmol/L, potassium 4 
mmol/L, calcium 1,35 mmol/L, pH 6,2) at room temperature.  Alternatively, no 
rinsing of the liver is persued, according to the local center’s standard treatment of 
care. Reperfusion is defined as the moment of restoration of blood flow into the 
liver graft after completion of the supra-/infra-caval anastomosis and the portal 
vein and/or hepatic artery. Thereafter, a biliary reconstruction is performed either 
by an end-to-end duct-to-duct anastomosis or a choledocho-enterostomy.  
 
 
5.1.2 Anaesthesia 
 
Induction and maintenance of anaesthesia are performed according to the local standard 
treatment of care and typically includes curare (e.g. Cisatracurium or Rocuronium), 
analgesics (e.g. Sufentanil), a volatile anesthetic (e.g. Sevofluran) or an intravenous 
anesthetic (e.g. Propofol). A peripheral and central venous line, an arterial catheter, a 
Swan-Ganz, nasogastric tube, a urinary catheter and a temperature probe are installed. 
Administration of blood derivatives (red blood cells, fresh frozen plasma or blood 
platelets), Stable Solution of Plasma Protein (SSPP), albumin, colloids (Mannitol), and 
vasopressors are administered by the anaesthesiologist taking into account the 
hemodynamic and medical conditions. Infection prophylaxis (e.g. Amoxicillin, Cefotaxime 
and Metronidazole in case of choledocho-enterostomy) are given according the history of 
the patient prior to the intervention. In case of a known penicillin allergy, Ampicilline and 
Cefotaxime are replaced by Vancomycine and Levofloxacin.   
Regular blood samples are taken to assess arterial blood gas analysis, ionogram, glycemia 
during the transplantation according the clinical need of the recipient: 
- 
after anesthesia induction, just before skin incision, 
- 
immediately prior to the start of the anhepatic phase, 
- 
immediately prior to reperfusion (defined as opening of the portal vein), 


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- 
30 minutes, 1 hr, 2 hrs after reperfusion.  
 
Prior to, during and after LTx, the coagulation status of the patient may be severely 
disturbed and/or rapidly deteriorate, necessitating appropriate treatment by the 
anesthesiologists. Depending on the clinical need and to some extent guided by 
coagulation tests, peri-operative correction of coagulation is routinely done.  Coagulation 
can be optimized by administration of e.g. platelets, fresh frozen plasma, selective 
administration of PPSB,  or through administration of pro-coagulants  (e.g. recombinant 
factor VIIa…) or anti-fibrinolytics (e.g. transexamic acid…).  Of note, taking into account 
the well-known limitation of coagulation tests during LTx, optimizing the coagulation 
status will never be done based on the absolute values of the clotting tests alone. 
Nevertheless, the coagulation status will be routinely monitored by measuring 
prothrombin time (PT or INR), activated pro-thromboplastin time (aPTT), platelets, 
fibrinogen and through thromboelastography before LTx and 1 hour after reperfusion.  
Whenever regarded appropriate, the anesthesiologist may increase the frequency of 
monitoring the coagulation status. 
 
Dobutamine, Levophed or other inotropics are given according to the hemodynamic 
status of the recipient as assessed by an anaesthesiologist (staff member). 
5.1.3 Post-operative care in the intensive care unit 
 
Early post-operative care consists out of monitoring and stabilizing of hemodynamic and 
pulmonary function, monitoring and prevention of surgical complications and prevention 
of infection.  
 
Analgesia and sedation are adjusted according the liver function. Daily routine urine and 
blood samples are analysed to primarily monitor hematology, ionogram, liver and kidney 
function and levels of immunosuppression. 
 
Post-LTx standard treatment of care includes prophylaxis against fungal infection (e.g. 
Nystatin sirup during 3 months, 2 ml QD), CMV infection (e.g. Valganciclovir, 450 mg OD 
or adjusted to the kidney function during 3 months in case of a CMV positive donor liver 
transplanted into a CMV negative recipient), pneumocystis carinii infection (e.g. Co-
trimoxazole or Dapsone orally during 3 months). Post-operative antibiotic prophylaxis is 
continued for 48 hours (e.g. Cefotaxime, Amoxicilline, and additional Metronidazole in 
case of choledoco-enterostomy; in case of a known penicillin allergy, Amoxicilline and 
Cefotaxime are replaced by e.g. Vancomycine and Levofloxacin, respectively).  
 
Patients will receive IV anti-hepatitis-B immunoglobulin in case of transplantation for 
hepatitis B followed by a lifelong treatment with antiviral oral Lamuvidine or alternative 
anti-viral medication if needed (e.g. related to side effects of Lamuvidine).  
 
Finally, immunosuppression (cf chapter 5.6.1 Immunosuppression) is initiated and 
closely monitored.  
 
During the postoperative stay in the intensive care unit in continuity with the stay on the 
nursing ward, blood samples are routinely collected at regular time points:  
- 
6h, 12h, 24h, 48h, 72h  after reperfusion 


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- 
Daily from day post LTx 4 to day 7, and day 14 
 
Shortly after LTx, the coagulation status can still be severely disturbed, which is then 
treated accordingly by the intensivists. Postoperative optimization or correction of 
coagulation is routinely done, depending on the clinical need  by administration of 
platelets, FFP, selective administration of PPSB, clotting factors or others depending of 
the clinical need. In addition, coagulation is monitored by blood samples to anticipate on 
coagulation problem. Therefore, prothrombin time, activated pro-thromboplastin time, 
platelets and fibrinogen are as routinely assessed during ICU stay. 
 
Finally, daily production of ascites post-transplantation will be monitored via the wound 
drains as long as wound drains are present.  
 
 
5.1.4 Post-operative follow up on nursing ward 
Post-operative care on nursing ward consists of non-invasive monitoring clinical 
parameters (blood pressure, pulse, temperature, weight, intake and output), 
complications (surgical, immunological, infection and metabolic), the care for drains and 
catheters, correct administration of the medication and education of the patients.  
In continuity with the stay in ICU, daily blood samples are routinely collected every 
morning in order to assess hematology, ionogram, liver and kidney function and levels of 
immunosuppression (whole blood trough levels of Tacrolimus before administration of 
the morning dose of Tacrolimus). 
Daily urine samples are collected in order to assess diuresis and kidney function.  
Ultrasound Doppler is routinely performed weekly in order to evaluate the parenchyma, 
vascularisation, possible bile duct abnormalities or intra-abdominal fluid collections. A 
liver biopsy is taken 1 week after LTx (except in case of contra-indication, e.g. suboptimal 
coagulation) or in case of clinical suspicion of AR.   
 
 
5.1.5 Outpatient follow-up 
After discharge, patients are re-assessed weekly/every two weeks during the first 3 
months following the LTx. A clinical examination is performed (including clinical 
abdominal examination, blood pressure measurement, body weight). Blood samples are 
routinely collected in order to assess hematology, coagulation status (PT and INR) 
ionogram, liver and kidney function and levels of immunosuppression (whole blood 
trough levels of Tacrolimus). Thereafter, during the first year post LTx a monthly 
outpatient clinic assessment is scheduled.  Blood samples are routinely collected at 3and 
12 months.  
At one year follow up, a routine MRCP is planned to investigate (i) the appearance of the 
parenchyma, (ii) the hepatic vasculature and (iii) the biliary tree.    
Finally, daily production of ascites post-transplantation will be monitored via the wound 
drains as long as wound drains are present.  
 
 
 
 
 


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5.1.6 Immunosuppression  
5.1.6.1 Tacrolimus  
 
The first dose of Tacrolimus will be administered orally or via the nasogastric tube post-
LTx. The recommended daily initial dose is 0.05-0.1 mg/kg/day in 2 doses with 12 hours 
interval and will be adapted depending on the trough levels. Depending on the timing of 
the first dose, the time of the second dose is adjusted to fit the hospital routine. However, 
the second dose of Tacrolimus is not given less than 12 hours after the first dose.  
Blood for the measurement of whole blood trough levels of Tacrolimus is drawn in the 
morning, before administration of the morning dose. 
Dosing is then titrated during the first post-transplant week to give whole blood trough 
levels between 5-10 ng/mL, then, it will be adapted according to adverse events or clinical 
need.  
To avoid any bias in the potential graft survival, switch from Prograft ® and Advagraf® 
to other Tacrolimus generics is not allowed; since there is no evidence of non-inferiority 
of these alternative formulations.    
 
5.1.6.2 Mycophenolate Mofetil  
 
The first dose of Mycophenolate Mofetil is administrated orally or IV post-LTx. The 
recommended daily dose is 0.5-1 g/day in 2 doses with 12 hours interval. Depending on 
the timing of the first dose, the time of the second dose is adjusted to fit the hospital 
routine. However, the second dose of Mycophenolate Mofetil is not given less than 12 
hours after the first dose.  
Trough levels with AUC for Mycophenolate Mofetil are not measured routinely. The 
adjustment of the doses is done according to side effects such as gastro-intestinal 
complications or leucopoenia. 
 
 
 
5.1.6.3 Corticosteroids 
 
The first dose of corticosteroids is administrated IV after transplantation. The regimen of 
corticosteroid therapy is as follows: 
 
- Day 1 - 3: Methylprednisolone IV, 2 x 20 mg, 
- Day 3 - day 21: Methylprednisolone per os (po), 16 mg in 2 doses, 
- Day 21 - day 42: Methylprednisolone po, 12 mg in 2 doses, 
- Day 42 - day 63: Methylprednisolone po, 8 mg in 2 doses, 
- Day 63 - day 84: Methylprednisolone po, 4 mg in one dose, 
- Day 84 - day 105: Tapering of the Methylprednisolone dose. 
 
Steroids are tapered and withdrawn after 12 weeks under strict monitoring of liver 
function. After the end of week 12 at the latest, patient will no longer receive any steroid 
therapy unless it is clinically necessary (e.g. auto-immune hepatitis). No steroids are given 
in HCV positive recipients. 
 


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Regimen doses are adjusted according to adverse events (e.g. rejection). In case of an 
acute rejection, high-dose pulsed corticosteroid therapy (500 mg IV Methylprednisolone 
is given during 3 consecutive days) is the first treatment of choice except for HCV positive 
recipients where doses of Tacrolimus are increased to trough levels of 10-15 ng/mL.  
 
 
 
 


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5.2 TREATMENT GROUP: multifactorial modulation 
 
This approach is based on the beneficial effects of the multifactorial modulation on graft 
and recipient survival previously observed in our harsh preclinical DCD-LTx model (4). 
Moreover, this multifactorial modulation was found to suppress inflammation-regulating 
genes in IRI (24).  
 
Apart from receiving the standard care as described above, patients randomized to the 
treatment group receive:  
1. C1-inhibitor 
2. α-Tocopherol 
3. Glutathione 
4. Apotransferrin 
5. Human Recombinant Erythropoietin beta (EPO-β) 
6. Infliximab 
7. Antithrombin-III 
8. Epoprostenol 
9. Melatonin  
 
Detailed description on administration, dosing and treatment periods can be found in 
Table 1 and Figure 2.  
As recommended by independent feedback of pharmacologist experts Prof Pieter Annaert 
(Laboratorium of Pharmacotechnology and Biopharmacy, KU Leuven) and Prof Paul 
Declerck , (Laboratorium of Pharmaceutical biology, KU Leuven) (Cfr Addendum 
Feedback regarding CAPITL study) C1-inhibitor, Glutathione, Apotransferrin, Human 
Recombinant Erythropoietin beta (EPO-β), Infliximab and Antithrombin-III will be 
infused intravenously (IV) sequentially and through different perfusion sets without 
direct contact during the anhepatic and the reperfusion phase. A second infusion of EPO-
β is foreseen 6 hours after reperfusion. This IV sequential administration just before 
reperfusion aims to reach a plasmatic peak of concentration of each component directly 
after their infusions. In addition the infusion of infliximab is interrupted during the 
administration of Glutathione. Melatonin and α-Tocopherol will be given orally before the 
transplantation. According to the available pharmacokinetic data, plasmatic peaks of 
concentration should be reached after reperfusion. Epoprostenol will be added to 1L of 
preservation solution and infused ex-situ directly into the liver through the vena porta 
during the bench table before the implantation. To avoid any contact between the 
components, a short flush of isotonic electrolyte solution will take place between the 
sequential infusions of the components. The infusion of Remicade (anti-TNF-alpha 
antibody) starts before the reperfusion, is interrupted during administration of 
glutathione and then restarted 15 minutes after reperfusion (period of time required to 
stabilize the patient after the reperfusion). The infusion of apotransferrine (scavenger of 
non-transferrin-bound iron) starts before the reperfusion, is interrupted for the 
administration of EPO-β, C1-inhibitor and glutathione, and then restarted 15 minutes 
after reperfusion. As such, the manufacture’s guidelines are followed (slow infusion rate 
for infliximab and larger volume for apotransferrine, respectively).  Moreover, as our 
preclinical studies have shown:  the peak of TNF-alpha and non-transferrin bound iron 
was observed 3 hours and 1 hour after the reperfusion, respectively.  
 
 


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Except for Apotransferrin, all the components are registered and described in the 
MICROMEDEX Healthcare Series (6) and in the MARTINDALE (7). 
 
Except for Apotransferrin, the administration of a single dose of each drug component, at 
the dose described in Table 1 (cfr summary), has been proven to be safe and efficient in 
reducing ischemia-reperfusion injury in both animals and clinical studies. A full 
description of current uses, precautions and contraindications, proposed mechanism of 
action, expected adverse effects, drug interactions, dosage form, and packaging can be 
found in Appendix 3.  
 
 
 
 
 


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VI. TIME LINE & PARTICIPATING CENTER 
 6.1 TIME LINE  (Figure 3)  
 
 2012: 
 
- 
Finalising multi-factorial modulation protocol, 
 
 2013:  
 
- 
Finalising multi-factorial modulation protocol 
- 
Start recruitment of patients, 
- 
Start inclusion of patients in the first safety phase of the study,  
- 
Start inclusion of patients in RCT phase of the study,  
- 
Start data analysis of patients included in safety phase study, 
- 
Inclusion of patients in the study if safety study is considered as safe, 
- 
Start data collection, 
 
 2014:  
 
- 
Recruitment of patients in RCT phase continued, 
- 
Data collection continued. 
 
 2015:  
 
- 
Recruitment of patients in RCT phase continued, 
- 
Data collection continued. 
 
 2016:  
 
- 
Recruitment of patients in RCT phase continued, 
- 
Data collection continued. 
 
 2017:  
 
- 
Data collection continued, 
- 
Start data analysis. 
 
 2018:  
 
- 
Data analysis continued. 
 
 
 
 
 
 
 
 


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6.2 PARTICIPATING CENTER 
 
The recruitment of 10 patients for the safety study (part A) of the multifactorial 
modulation will be conducted in the University Hospitals of Leuven. 
This safety phase will be followed by a multi-center RCT (part B) at  the University 
Hospitals Leuven, Centre Hospitalier Universitaire de Liège and other national and 
international centres. 
 
 


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VII. PRIMARY ENDPOINT (part B, RCT)  
 
The primary objective of part B (RCT) this trial is to demonstrate effectiveness of the 
multifactorial modulation on the extent of IRI as reflected by the peak AST during the first 
week after LTx and later during follow-up. 
 
Peak AST is widely used as a surrogate marker reflecting the extent of IRI. This parameter 
correlates well with parenchymal graft injury (29) ensuing IRI and is associated with the 
subsequent initial liver function or dysfunction after transplantation (30). Albeit simple, 
the peak of aspartate amino transferase is internationally accepted as the best surrogate 
of IRI and is included in all definitions or scoring systems for delayed graft function after 
LTx (30). 
 
Therefore, peak AST will be assessed as the primary endpoint of this study. The peak is 
defined as the highest value of AST during the first 72 hours following the LTx. 
 
AST analysis will be performed in the central lab of the University Hospitals Leuven 
by means of a colorimetric method, detection limit 4 U/L (Hitachi/Roche Modular P, 
Roche Diagnostics, Vilvoorde, Belgium). 


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VIII. SECONDARY ENDPOINTS (part B, RCT) 
 
- 
Graft loss at 3, 12 months after LTx.  Graft loss is defined as the need for 
retransplantation within one week post LTx due to a non-life-sustaining liver graft 
function (PNF) or later (other reason). 
 
- 
Recipient death at 3, 12 months after LTx. 
 
- 
Early graft dysfunction as defined by Olthoff (1): the presence of one or more of the 
following postoperative laboratory analyses: bilirubin ≥ 10mg/dL on day 7, 
international normalized ratio ≥ 1.6 on day 7, and alanine aminotransferase (ALT) 
or AST > 2000 IU/L within the first 7 days. 
 
- 
Incidence of biliary strictures within 12 months post LTx: a biliary stricture is 
defined as a narrowing within the biliary tree, radiologically evident [endoscopic 
retrograde cholangio-pancreatography (ERCP) and/or magnetic resonance 
cholangiopancreatography (MRCP)] to cause clinical symptoms or biochemical 
abnormalities 
requiring 
intervention 
(ERCP, 
percutanous 
transhepatic 
cholangiographic (PTC) drainage, surgery, retransplantation). Biliary strictures 
are categorized as anastomotic or non-anastomotic based on the cholangiographic 
appearance of the biliary tree as judged by a blinded radiologist. Non-anastomotic 
strictures are defined as any strictures, dilatation, or irregularity of the intra- or 
extrahepatic bile ducts of the liver graft at a site(s) other than that of the 
anastomosis. Intra-hepatic biliary strictures are classified in 4 groups: unilateral 
focal, confluence, bilateral multifocal and diffuse necrosis (2). Beside the routine 1 
year post-transplant assessment of the biliary tree by MRCP and/or ERCP, biliary 
strictures will be investigated in case of clinical or biochemical suspicion (based 
upon cholestasis). Other causes leading to cholestasis (e.g. hepatic artery 
thrombosis, bile leakage, rejection or cholangitis) will be excluded based on state 
–of-the-art radiological and histological examination as part of the routine 
standard treatment of care. These examinations include ultrasound Doppler, CT 
and CT angiogram, biopsy-proven rejection based on histology scored by 2 blinded 
experienced liver pathologists according to the Banff criteria, ERCP and MRCP. 
 
- 
IRI score: The extent of IRI will be assessed by a histological score based on the 
degree of cytoplasmic vacuolization, sinusoidal congestion, necrosis of 
parenchymal cells, apoptosis and influx of neutrophils as described by Suzuki 
score (3) and Monbaliu et al. (4). Liver biopsies will be taken before implantation 
(bench table), 1 hour after reperfusion and 1 week after transplantation and 
blindly scored by 2 pathologists.  
 
- 
Graft rejection: graft rejection will always be biopsy-proven; a liver biopsy is taken 
after LTx and in case of clinical suspicion of acute rejection.  Clinical suspicion of 
acute rejection may be based on clinical symptoms (such as jaundice, low-grade 
fever) or sometimes nonspecific complaints (such as generalized malaise, 
decreased appetite) and/or biochemical abnormalities (usually increasing or 
plateauing levels -in an abnormal elevated range- of liver tests that were returning 
to normal values). Histological changes will be scored blindly according the BANFF 
criteria (see appendix 1) by 2 experienced liver pathologists. 


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- 
Surgical complications: the Clavien-Dindo classification (5) will be used to rank 
surgical complications within 30 days after LTx according to an objective, simple, 
reliable, and reproducible manner. This classification is based on the therapy 
required to treat the complication (appendix 2, adverse event). The severity of the 
biliary strictures as well will be classified using this standardized grading system. 
 
 
 
 
 


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IX. VARIABLES OF INTEREST 
9.1 DONOR CHARACTERISTICS 
 
Age, ethnicity (black versus non-black), gender, cause of death (trauma, cerebro-vascular 
accident, anoxia), weight, height, Body Mass Index (BMI), donor type (DCD,   DBD, ECD 
and SCD), diuresis last hour, duration of hypotensive period, presence of cardiac arrest, 
duration of donor warm ischemic time, time of start cold perfusion, time of hepatectomy, 
administration (yes/no) and dose (IU) of Heparin, perfusate solution type and volume, 
perfusion via aorta or portal vein or both, length of ICU stay, administration of 
vasopressors, laboratory values (peak and most recent value of AST, ALT, gamma-GT, 
creatinine, total bilirubin, sodium), CMV viral status, imported versus local liver 
procurement, calculation of the donor risk index as described by Feng et al. (32) and the 
Balance of Risk (BAR) score described by Dutkowski et al. (33), pretreatment with 
steroids (dose, nature of steroid, way of administration) (34). 
 
 
9.2 RECIPIENT CHARACTERISTICS 
 
Age, ethnicity (black versus nonblack), gender, weight, height, BMI, laboratory Model for 
End-stage Liver Disease (MELD) score the day of LTx, match MELD, time on waiting list, 
United Network  for Organ Sharing (UNOS) score, diagnosis according the Eurotransplant 
classification, additional morbidity as hypertension (defined as a documented blood 
pressure above 140/90 mmHg and/or requiring at least one anti-hypertensive drug 
within 3 months before LTx), diabetes mellitus (type I or II defined as a hyperglycaemia 
requiring oral anti-diabetic or insulin within 3 months before transplantation), 
hemodialysis at the time of the LTx, duration of hemodialysis prior to LTx and past history 
of ascites (Grade 1 defined as mild with only visible on ultrasound and CT, grade 2 defined 
as detectable with flank bulging and shifting dullness, grade 3 with directly visible and 
confirmed with fluid thrill). 
 
9.3 INTRA-OPERATIVE VARIABLES 
 
Duration of surgery, piggy-back (with or without creating a portocaval shunt) versus 
veno-venous by-pass technique, cold ischemia time (CIT) (defined as the time between 
cold flush of the liver in the donor and its leaving from the melting ice water just before 
implantation), intra-operative warm ischemia time (defined as the time between the liver 
leaving the ice and the portal vein reperfusion), arterial anastomosis time (defined as the 
time between the liver leaving the ice and arterial reperfusion), transfusion need (packed 
red blood cells, fresh frozen plasma, and platelets), cardiac output 15 minutes after 
reperfusion, invasive cardiac hemodynamics reflecting right and left ventricular function, 
hemodynamic status after 15 minutes of the reperfusion (stable > 100 mmHg of systolic 
pressure, mild 80-100 mmHg, moderate 60-80 mmHg, severe < 60 mmHg), portal and 
arterial hepatic flow, urine output from the start of surgery until start of reperfusion, 
urine output from reperfusion to 120 minutes after reperfusion, urine output from 
beginning until the end of surgery and length of veno-venous bypass. 
 
 


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9.4 POST-OPERATIVE VARIABLES 
 
Length of ICU stay, length of hospital stay,  PNF (defined as a non-life sustaining function 
of the liver graft, leading to death or re-LTx within 7 days), delayed graft-function as 
defined by Olthoff (1), 3, 12 months patient death and graft loss, date of death, biopsy 
proven rejection, adverse events (cfr. appendix 2). 
 
 
9.5 LABORATORY ASSESSMENT 
 
Blood samples are taken before, during and daily after LTx. Analysis of AST will be 
performed in the central lab of the University Hospitals Leuven.   
10 cc of plasma will be collected at the following time points: 
 
- 
After anesthesia induction, just before skin incision, 
- 
Immediately prior to the start of the anhepatic phase, 
- 
Immediately prior to reperfusion (defined as opening of the portal vein), 
- 
30 minutes, 1 hr, 2 hr after reperfusion,  
- 
6h, 12h, 24h, 48h, 72h after reperfusion, 
- 
Daily from day 4 to day 7, 
- 
Day 14  when the recipient is still an in patient, a 2 day window is allowed 
whenever the recipient has been discharged prior to day 14 and blood samples 
cannot be taken on day 14 (e.g. weekend) 
- 
3 months +- 2 weeks after transplantation, 
- 
12 months +- 4 weeks after the transplantation.  
 
 
 
9.6 HISTOLOGICAL FEATURES 
 
Liver biopsies are taken at the following time points: 
- 
Before implantation at the bench as a baseline biopsy, 
- 
Approximately 1 hour after the reperfusion, 
- 
1 week (max. 3 weeks) after the transplantation, 
- 
On indication (e.g. suspicion of acute rejection). 
Biopsies of the caudal part of the common bile duct (ring of 1 mm) will be taken and stored 
in formaline 
- 
Before implantation at the bench 
- 
After reperfusion and before closure  
 
The tissue will be fixated in formaldehyde 6% and embedded in paraffin. After 
hematoxylin and eosin staining, blind analysis will be performed by 2 experienced 
pathologists in the pathological department of the University Hospitals Leuven and 
Centre Hospitalier Universitaire de Liège in order to assess the IRI score and graft 
rejection. 
 
 
 


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X. DATA ANALYSIS 
 
10.1 REPORTING OF THE DATA (part B, RCT) 
 
An intention-to-treat analysis will be done and all participating patients will be included. 
Data will be reported according to the CONSORT criteria.  
All data and other information of each patient included in the trial will be recorded in an 
electronic Case Report Form (e-CRF). The e-CRF will be designed to allow continuous 
recording of variables during the study. The e-CRF and its maintenance are technically 
organized by an external company (EONIX). The e-CRF will be completed on a regular 
basis under the responsibility of the local principal investigator.  
  
 
10.2 STATISTICAL METHODOLOGY 
 
10.2.1 Primary outcome 
 
The log-transformed peak AST values will be compared between both groups using a 
linear model with group and centre as factors. If variances between both groups differ 
significantly (based on a likelihood-ratio test comparing the models with equal and 
unequal variances, respectively), the result from the model with unequal variances will 
be reported. 
The following analyses will be added to verify the robustness of the obtained conclusion: 
firstly, a comparison of the groups will be done after correction for MELD score and cold 
ischemic time using a linear model on the log-transformed peak AST values. Secondly, all 
analyses will be repeated as a subgroup analysis, excluding the (expected) small set of 
patients with a DCD liver. 
 
10.2.2 Sample size calculation 
 
Sample size calculation is based on peak AST values obtained from a series of patients 
(N=308) that underwent LTx at the University Hospitals Leuven between January 2007 
and October 2011. For 264 patients, peak AST value was available. These values followed 
a lognormal distribution; the log-transformed AST was normally distributed with mean 
and standard deviation equal to 6.53 and 0.93 respectively. The corresponding geometric 
mean equalled 685. Based on this distribution, 34.3%, 20.1%, and 12.6% of the subjects 
are expected to have an AST higher than 1000, 1500, and 2000, respectively. It is assumed 
that the treatment will lead to a 50% reduction of the (geometric) mean. This implies that 
12.6%, 5.7%, and 3.0% of the subjects in the treatment group are expected to have an AST 
higher than 1000, 1500, and 2000 IU/L respectively. 
 
Based on a two-sided two-sample pooled t-test of a mean ratio with lognormal data, 58 
subjects are needed in total to have 80% power (with alpha set at 5%). Anticipating a 
drop-out rate of 20% and aiming to have complete blocks in the randomisation, 72 
patients (36 per group) will be included in the study.  


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10.2.3 Secondary outcomes 
 
Kaplan-Meier estimates will be used to construct curves for graft and recipient survival, 
which will be compared between groups with a stratified log-rank test (if there are deaths 
without graft dysfunction, cumulative incidence estimates will be considered for graft 
survival).  
 
A Cox regression model, stratified on center, will be used to compare graft and recipient 
survival between both groups after correction for MELD score and ischemic time (and 
center). 
 
To compare early graft dysfunction and presence of biliary strictures between both 
groups, an exact test for the common odds ratio will be used. A correction for MELD score 
and ischemic time is considered with a logistic regression model, unless the number of 
events is too low. Deaths not known to be related to biliary strictures will not be included 
in the biliary strictures analysis (hence assuming that these are unrelated to the 
probability of biliary strictures). The severity of biliary strictures will be described in both 
groups. 
 
For all outcomes (primary and secondary), it will be verified if the difference between 
groups varies between centers. 
 
All analyses will be performed using SAS software, version 9.2 of the SAS System for 
Windows. Copyright © 2002 SAS Institute Inc. SAS and all other SAS Institute Inc. product 
or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, 
USA. 
 
 


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XI. RANDOMIZATION (part B, RCT) 
 
All consecutive patients listed for LTx will be assessed for study eligibility by a staff 
surgeon via the outpatient clinic (pre-transplant evaluation) or as an inpatient during 
their hospital stay. If patients are willing to participate to this first in man study and 
provide informed consent, a member of the LTx team will inform patients about the study, 
orally and in writing.  
 
The randomization to the control group or to the treatment group is done pre-operatively, 
immediately after the liver has been allocated to a patient. 
 
A third party – not involved in this trial – provides a distant central randomization of 
patients to the control or to the intervention group. Following inclusion in one group, the 
study coordinator will be informed by the third party randomizer with the details of the 
randomization to ensure correct verification of group allocation. 
 
In each participating centres, patients will be randomised into two groups using variable 
block size (e.g. 2, 4, 6, …). No stratification is performed on the a priori defined potential 
confounders MELD score, ischemic time and DCD. The expected set of DCD is too small to 
consider as a separate stratum and an analysis on historical data showed no evidence for 
a relation between peak AST with ischemic time and only a weak relation with MELD 
score. 
 
 
XII. ALLOCATION CONCEALMENT 
 
Distant randomization and allocation concealment of the patients to treatment is 
performed with the use of an electronic randomization table and a web-interface. The 
electronic randomization and its maintenance are technically organized by an external 
company (EONYX).  
 
 
XIII. ETHICAL CONSIDERATION 
 
The protocol is written conform the declaration of Helsinki (Appendix 5) and the 
investigators and their personnel will act according to the principles therein.  
The investigators agree to conduct this study in accordance with the International 
Conference on Harmonization (ICH) principle of Good Clinical Practice (GCP). 
The investigator will conduct all aspects of this study in accordance with all national and 
local laws of the applicable regulatory agencies. The study protocol will be approved by 
the institutional review board of the University Hospitals Leuven before the start of 
patient inclusion. 
 
 


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13.1 ETHICAL COMMITTEE 
 
The protocol, patient brochure and informed consent will be approved by the Ethical 
Committees before the start of patient inclusion. Approval will be obtained in writing, 
stating the identity of the clinical trial, the date of review, the documents reviewed and a 
list of the names and titles of the committee members. 
 
Any substantial amendment to the protocol will be submitted to the Ethical Committee. 
 
The investigators will be responsible of informing their Ethical Committee of all problems 
involving risks to patients according to national regulations. 
 
 
13.2 PATIENT INFORMATION AND INFORMED CONSENT 
 
Each patient participating in the trial will give his/her informed consent prior to entry 
into the trial. Informed consent will be given in writing after full written and verbal 
information has been provided by the investigator or a nominated representative 
(Appendix 6). In addition to the patient, the investigators or a nominated representative 
will sign the informed consent form. If necessary, it is acceptable for a patient’s witness to 
sign the informed consent form on behalf of the patient. 
 
The patient will receive the information brochure (Appendices 7 and 8 for part A, and part 
B, respectively) and can withdraw from the trial at any time without prejudice.  
 
 
13.3 PERSONAL DATA PROTECTION 
 
The investigators uphold the principle of the patient’s right to protection against invasion 
of privacy. All data recorded in the e-CRF or passed on for further evaluation will be coded 
by patient number, initials and date of birth. Identification is restricted to authorized 
persons. The data will be anonymized correspondingly in all data analyses. 
 
 
 
 


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XIV. WITHDRAWALS AND DROPOUTS 
 
To avoid withdrawal and dropout bias, an intention-to-treat analysis will be performed. 
Participants will be included in the analyses as part of the groups to which they were 
randomized, regardless of whether they completed the study or not. No imputation 
techniques will be considered for missing outcome values, but differences in 
characteristics will be explored between patients with and without a missing outcome 
value. 
 
 
 
 
The reasons for withdrawal are: 
- 
Withdrawal of consent: Every patient is free to withdraw from the study for any 
reason and at any time without giving reason for doing so and without penalty or 
prejudice. 
 
- 
The investigator is also free to terminate a patient’s involvement in the study at 
any time if warranted by the patient’s clinical condition (e.g. anaphylactic 
symptoms related to one of the substances in the multifactorial bio-modulation 
during infusion). 
 
- 
Patient lost to follow-up. 
 
 
The investigator will maintain a chronological patient identification list of all patients who 
enter into the trial containing the patient details (name, study number, initials, date of 
birth, age, sex), date and time of entry into the study (see Appendix 8). 
The number of withdrawals, dropouts and the reasons will be stated in this 
enrolment/withdrawal form. If there is no withdrawal, it will also be stated.  
 
Patients who are discontinued will be treated and followed without any disadvantage 
with regard to medical care or physician-patient relationship. 
 
The investigators will maintain a chronological patient screening list of all patients who 
will be transplanted during the study period but who will not enter the study. This list 
must contain patient details, date of transplantation, and reason for non-participation 
(see appendix 9). 
 
 


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XV. INTERIM ANALYSIS 
 
During part B (RCT), no interim analyses will be performed to stop the study early due to 
efficacy or futility reasons. 
 
 
 


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XVI. PROCESS LEADING TO TRIAL STOPPING RULES  
16.1 DATA SAFETY MONITORING BOARD 
 
An independent safety board will be composed by staff members from the departments 
of intensive care (Prof dr FERDINANDE and Prof dr em. LAUWERS), biostatistics (Kris 
Bogaerts) and chaired by Prof dr em. FEVERY (former chief of the Hepatology 
department). They will handle concerns about safety and termination of the study in its 
first or second phases in case of increased incidence and/or severity of adverse events 
indicating a potential health hazard caused by the multifactorial modulation. 
The DSMB will –independently from the investigators- decide whether to continue or to 
stop the trail based on predefined serious adverse events (see 16.2.4) and the potential 
drug causality.  
 
16.2 DESCRIPTION OF ADVERSE EVENT  
16.2.1 Definition of adverse event (AE)  and serious adverse event (SAE) 
 
According the GCP (Clinical Trial Directive 2001/20/EC), an adverse event (AE) is 
defined as any untoward medical occurrence in a patient or subject of the treated group 
during an experiment, and which does not necessarily have a causal relationship with this 
treatment. This definition includes physical signs, symptoms and laboratory test values. 
At study enrolment, laboratory values that fall outside the relevant reference range will 
not be reported as adverse events. Clinically significant modifications of values during the 
study period will be reported. 
 
A serious adverse event (SAE) is any untoward medical occurrence or effect that suggests 
a significant hazard, contraindication, side effect or a precaution for human patients. The 
term includes any event that results in: 
- 
Death, 
- 
Life threatening condition, 
- 
Persistent significant disability or incapacity, 
- 
Prolongation of initial hospitalisation or re-hospitalisation, 
- 
Requiring intervention to prevent one of above. 
 
16.2.2 Grade of severity 
 
- 
Mild (grade 1): patient is aware of symptoms but tolerates them easily. Symptoms 
does not interfere with daily activity. 
- 
Moderate (grade 2):  patient experiences discomfort that interferes with normal 
activity. No treatment is required except acetaminophen. 
- 
Severe (grade 3): patient is unable to carry out normal activity. Treatment is 
required. 
- 
Life-threatening (grade 4): emergency room visit or disabling or hospitalization. 
 
 


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16.2.3 Timeframe of observation 
 
1. During the LTx. 
 
2. Immediately after LTx until day 7. 
 
 
 
16.2.4 Predefined serious adverse events  
  
The predefined serious adverse events will be considered in the decision to continue/to 
stop the trial by the DSMB  Some of these predefined SAE’s listed are inherently related 
to LTx, regardless of the multifactorial modulation protocol (e.g. post reperfusion 
syndrome, cardiac arrhythmia…), therefore the definition of SAE for this study also 
includes “refractory to standard treatment”  whenever appropriate.  
1. During the LTx: 
 
- 
Death 
- 
Anaphylactic reaction/shock (life-threatening type of allergic reaction) 
- 
Hemodynamic effects: 
 Abnormally more frequent post reperfusion syndrome (30% decrease of 
mean arterial pressure compared to the end of the anhepatic phase during 
1 min within the 5 min after reperfusion)refractory to standard treatment 
of care 
 Severe arrythmia after reperfusion (leading to hemodynamic disturbance) 
refractory to standard treatment of care 
 
 Respiartory effects (PaO2/FiO2 ratio) (cfr SOFA score) 1 hour after 
reperfusion refractory to standard treatment of care 
 
- 
Abnormal level of platelets (cfr SOFA score) 1 hour after reperfusion  refractory to 
standard treatment of care 
- 
Metabolic effects: 
 Hypoglycemia (level of blood glucose < 45-75 mg/dL) 1 hour after 
reperfusion refractory to standard treatment of care 
 
 Hypo-/hyperkalemia (level of potassium < 3.5 and > 5.0 mEq/L 
respectively) 1 hour after reperfusion refractory to standard treatment of 
care 
 
 Abnormal hyperlactataemia (level of lactate > 5 mmol/L) 1 hour after 
reperfusion refractory to standard treatment of care 
 
 
 
 


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2. Immediately after LTx until day 7. 
 
- 
Death 
- 
PNF 
- 
Peak AST > 2000 UI/L 
- 
Kidney dysfunction (cfr SOFA score) 
- 
Sepsis (whole-body inflammatory state caused by severe infection) 
- 
Bleeding (requiring radiological and/or surgical intervention) 
- 
Myocardial infarction 
- 
Severe cardiac arrhythmia (leading to hemodynamic disturbance) 
- 
Thrombosis (clot formation in vein/artery) 
 
The SOFA score will be used to rank organ dysfunction from day 1 to day 7 after LTx in 
an objective, simple, reliable, and reproducible manner (see table 4).  
 
 
Table 4: Description of the SOFA score 
 
 
16.3 Protocol-defined adverse events 
 
The following adverse events are commonly observed during or after liver 
transplantation and are therefore not considered as adverse events for the purpose of the 
trial: 
 
- Gastrointestinal problems (nausea, constipation and/or diarrhoea) related to the 
use of immunosuppression (such as Mycophenolate acid derivatives) 
 
- Hypertension as a pre-existing disease or induced by immunosuppression 
 
- Headaches related to immunosuppression 
 
- Anaemia, leukopenia or thrombocytopenia related to immunosuppression, 
 
- Transient hyper/hypocalcemia, hyper/hyponatremia, hyper/hypokalaemia, 
hyper/hypophosphataemia, hypomagnesemia - 
Peripheral 
oedema 
and 
hypoalbuminemia in the peri-operative period related to filling status, peri-operative 
management and recovering liver function (until first 3 months after liver 
transplantation) 


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- Post- liver transplantation delirium related to pre-existing encephalopathy and 
post-operative and ICU delirium 
16.4 DRUG CAUSALITY 
 
The causality of an AE in relation to the study therapy as a whole will be categorized as 
follows: 
- 
Highly probable: Apparent relationship in time between AE and drug 
administration or drug concentration in body and fluids or tissues. Relationship 
between AE and drug is already known or expected. Reaction has occurred with 
this medication previously and there is an appropriate temporal relationship 
between therapy and AE. 
- 
Probable: Known pharmacological effect with no possible other cause and 
appropriate temporal association. 
- 
Possible: AE likely to be associated with the drug and no other medication was 
taken, or known pharmacological effect of medication that could also be associated 
with another concomitant therapy, illness or external cause. 
- 
Unlikely: Unlikely to be causally related: e.g. reaction occurred after cessation of 
drug therapy or is more likely to be due to another concomitant therapy, illness or 
external cause. 
- 
Definitely not: AE known to be caused by another concomitant therapy, illness or 
external cause. 
- 
Not assessable: Likelihood of AE not known, or relationship of AE to study therapy, 
another concomitant therapy, illness or external cause is not clear. This category 
should be used very scarcely. 
 
16.5 INDIVIDUAL AND COHORT LEVELS 
 
The safety analysis will be conducted by the independent DSMB.  
 
The description of severe adverse events for safety analysis will be prospectively 
collected according to a strict procedure (Figure 9). 
 
 
 


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Figure 9: Description of serious adverse events for safety analysis. 
 
For each patient, grade of severity, time of onset, type of adverse event, SOFA score and 
drug causality will be daily detailed. 
For each cohort of patients, number of subjects experiencing similar adverse events will 
be described.   
 
According the harshness, the recurrence and the limit of acceptable side-effect rates of 
severe adverse events, the DSMB can decide to stop the trial. 
 


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XVII. REFERENCES 
 
1. Validation of a current definition of early allograft dysfunction in liver transplant 
recipients and analysis of risk factors. Olthoff et al., Liver Transplant. 2010. 
2. Classification and prognosis of intrahepatic biliary stricture after liver transplantation. 
Won Lee et al., Liver Transplantation 2007; 13: 1736-1742. 
3. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury. 
Suzuki et al., Transplantation 1993, 55; 1265-1272. 
4. Multifactorial biological biomodulation of warm ischemia reperfusion injury in liver 
transplantation from non-heart-beating donors eliminates primary non-function and 
reduces bile salt toxicity. Monbaliu et al., Ann Surg. 2009; 250: 808-17. 
5. The Clavien-Dindo classification of surgical complications, five years experience. 
Clavien et al., Ann Surg 2009, 250: 187-196. 
6. Thomson. Micromedex. Drug Information for the Health Care Professional. 25th ed. plus 
Updates. Content Reviewed by the United States Pharmacopeial Convention, Inc. 
Greenwood Village, CO. 2005. 
7. https://www.medicinescomplete.com/mc/martindale/current/ 
8. Utilization of extended donor criteria in liver transplantation: a comprehensive review 
of the literature. Nickkholg et al., Nephrol Dial Transplant 2007, 22: 829-836 
9. Influence of marginal donors on liver preservation injury. Briceno et al., 
Transplantation 2002, 74: 522-526 
10. Preoperative factors associated with outcome and their impact on resource use in 
1148 consecutive primary liver transplants. Markmann et al., Transplantation 2001, 72:  
1113-1122 
11. Ischemia and reperfusion- from mechanism to translation. Eltzschig et al., Nature 
medicine 2011; 11: 1391-1401. 
12. Molecular mediators of liver ischemia and reperfusion injury: a brief review. 
Vardanian et al., Mol Med 2008, 14: 337-345 
13. Mechanism of hepatic ischemia/reperfusion injury and protection against reperfusion 
injury. Kang et al., Transplantation proceedings 2002, 34: 2659-2661. 
14. de Groot Rauen et al. Ischemia reperfusion injury: processes in pathogenic networks: 
a review Transpl Proc 2007; 39: 481 
15. Preservation and reperfusion injuries in liver allografts. An overview and synthesis of 
current studies. Clavien et al., Transplantation 1992, 53: 957-978. 
16. Biliary tract complications after liver transplantation: a review. Wojcicki et al., Dig 
Surg 2008; 25: 245-257. 
17. Effects of donor pretreatment with dopamine on graft function after kidney 
transplantation, a randomized control trial. Schnuelle et al., JAMA 2009, 302: 1067-1075. 
18. Clinical trial of the Pan-caspase inhibitor, IDN-6556, in human liver preservation 
injury. Baskin-Bey at al., Transplantation 2007; 7: 218-225. 
19. HEPGOL: Randomized, placebo controlled multicenter, double-blind clinical trial to 
investigate hepatoprotective effects of glycine in the postoperative phase of liver 
transplantation (ISRCTN69350312). Luntz et al., BMC Surg 2005, 5: 18. 
20. Inhaled NO accelerates restoration of liver function in adults following orthotopic liver 
transplantation. Lang et al., The Journal of clinical investigation 2007; 2583-2591. 
21. Thymoglobulin induction protects liver allografts from ischemia/reperfusion injury. 
Bogetti et al., Clin Transplant 2005, 19; 507-511. 
22. Tacrolimus as a liver flush solution to ameliorate the effects of ischemia/reperfusion 
injury following liver transplantation. St Peter et al., Liver Transpl 2003, 9; 144-149. 


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23. Impact of stable PGI(2) analog iloprost on early graft 
viability after liver transplantation: a pilot study. Bärthel E et al., Clin Transplant 2012, 
26 ; E38-E47. 
24. Monbaliu  et al. Livers from non-heart-beating donors tolerate short periods of warm 
ischemia.  Transplantation 2005. 
25. Monbaliu  et al.  Increased serum Phospholipase A2 activity after non-heart-beating 
donor LTx and association with ischemia-reperfusion injury.  J Surg Res. 2009. 
26. Monbaliu D, Liu Q, Vekemans K et al. Potentiation of adverse effects of cold by warm 
ischemia in circulatory death donors for porcine LTx. Transplant Proc 2012, 44: 2874-
2879. 
27. Improving the function of liver grafts exposed to warm ischemia by the leuven drug 
protocol: exploring the molecular basis by microarray. Vekemans et al., Liver Transplant 
2012, 18: 206-218. 
28. De Rougemeont, Dutkowski, Clavien: Biological modulation of liver ischemia-
reperfusion injury.  Current Opinion in Organ transplantation 2010, 15, 184-189 
29. Diagnosis and monitoring of hepatic injury. Performance characteristics of laboratory 
tests. Dufour et al., Clinical Chemistry 2000; 46: 2027. 
30. Analysis of initial poor graft function after orthotopic liver transplantation: experience 
of an australian single liver transplantation center. Nanashima A et al., Transplant Proc 
2002; 34:1231-1235. 
31. Report of the Paris consensus meeting on expanded criteria donors in liver 
transplantation. Durand et al., Liver Transplant 2008; 14: 1694-1707. 
32. Characteristics associated with liver graft failure: The concept of a donor risk index. 
Feng et al., Am J Transpl 2006; 6: 783-790. 
33. Are there better guidelines for allocation in liver transplantation? A novel score 
targeting justice and utility in the model for end-stage liver disease era. Dutkowski et al., 
Ann Surg 2011; 254: 745-753. 
34. Methylprednisolone therapy in deceased donors reduces inflammation in the donor 
liver and improves outcome after liver transplantation. Kotsch et al., Ann Surg 2008; 
1042-1050. 
 
 


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XVIII. APPENDICES 
 
APPENDIX 1: BANFF CRITERIA 
Blinded analysis by 2 independent pathologists will be performed and histological 
changes scored according to BANFF criteria (Banff schema for grading liver allograft 
rejection: an international consensus document. Banff working group, Hepatology 1997; 
25: 658-663). 
 
Category 
Criteria 
Score 
Portal Inflammation 
- 
Mostly lymphocytic inflammation involving, 
but not noticeably expanding, a minority of 
the triads. 
- 
Expansion of most or all of the triads, by a 
mixed infiltrate containing lymphocytes 
with occasional blasts, neutrophils and 
eosinophils. 
- 
Marked expansion of most or all of the 
triads by a mixed infiltrate containing 
numerous blasts and eosinophils with 
inflammatory spillover into the periportal 
parenchyma. 
 
 
1 
 
 
2 
 
 
3 
Bile 
duct 
inflammation 
damage 
- 
A minority of the ducts are cuffed and 
infiltrated by inflammatory cells and show 
only mild reactive changes such as 
increased nuclear: cytoplasmic ratio of the 
epithelial cells. 
- 
Most or all of the ducts infiltrated by 
inflammatory cells. More than an occasional 
duct shows degenerative changes such as 
nuclear pleomorphism, disordered polarity 
and cytoplasmic vacuolization of the 
epithelium. 
- 
As above for 2, with most or all of the ducts 
showing degenerative changes or focal 
luminal disruption. 
 
 
1 
 
 
 
 
2 
 
 
 
 
3 
Venous 
endothelial 
inflammation 
- 
Subendothelial 
lymphocytic 
infiltration 
involving some, but not a majority of the 
portal and/or hepatic venules. 
- 
Subendothelial infiltration involving most 
or all of the portal and/or hepatic venules. 
- 
As above for 2, with moderate or severe 
perivenular inflammation that extends into 
the 
perivenular 
parenchyma 
and 
is 
associated with perivenular hepatocyte 
necrosis. 
 
 
1 
 
 
2 
 
3 
 
 


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APPENDIX 2:  ADVERSE EVENTS  
1. Definition of adverse events 
 
According the GCP (Clinical Trial Directive 2001/20/EC), an adverse event (AE) is any 
untoward medical occurrence in a patient or subject of the treated group during an 
experiment, and which does not necessarily have a causal relationship with this 
treatment. This definition includes physical signs, symptoms and laboratory test values. 
At study enrolment, laboratory values that fall outside the relevant reference range will 
not be reported as adverse events. Clinically significant modifications of values during the 
study period will be reported. 
 
A serious adverse event (SAE) is any untoward medical occurrence or effect that suggests 
a significant hazard, contraindication, side effect or a precaution for human patients. The 
term includes any event that results in: 
- 
Death, 
- 
Life threatening condition, 
- 
Persistent significant disability or incapacity, 
- 
Prolongation of initial hospitalisation or re-hospitalisation, 
- 
Requiring intervention to prevent one of above. 
 
Hospitalisation is not assessed as a SAE if hospitalisation: 
- 
Ensues due to routine procedures, 
- 
Was planned before study entry or occurs without being scheduled before study 
entry for a pre-existing non worsening condition, 
- 
Is not fulfilling the criterion of untoward medical occurrence (e.g. social and/or 
convenience admissions, rehabilitation program). 
 
The severity of an AE is to be determined as follows: 
- 
Mild (grade 1): patient is aware of symptoms but tolerates them easily. Symptom 
does not interfere with daily activity. 
- 
Moderate (grade 2):  patient experiences discomfort that interferes with normal 
activity. No treatment is required except acetaminophen. 
- 
Severe (grade 3): patient is unable to carry out normal activity. Treatment is 
required. 
- 
Life-threatening (grade 4): emergency room visit or disabling or hospitalization. 
 
The causality of an AE in relation to the study therapy as a whole will be categorized as 
follows: 
- 
Highly probable: Apparent relationship in time between AE and drug 
administration or drug concentration in body and fluids or tissues. Relationship 
between AE and drug is already known or expected. Reaction has occurred with 
this medication previously and there is an appropriate temporal relationship 
between therapy and AE. 
- 
Probable: Known pharmacological effect with no possible other cause and 
appropriate temporal association. 
- 
Possible: AE likely to be associated with the drug and no other medication was 
taken, or known pharmacological effect of medication that could also be associated 
with another concomitant therapy, illness or external cause. 


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- 
Unlikely: Unlikely to be causally related. E.g. reaction occurred after cessation of 
drug therapy or is more likely to be due to another concomitant therapy, illness or 
external cause. 
- 
Definitely not: AE known to be caused by another concomitant therapy, illness or 
external cause. 
- 
Not assessable: Likelihood of AE not known, or relationship of AE to study therapy, 
another concomitant therapy, illness or external cause is not clear. This category 
should be used very scarcely. 
 
A suspected unexpected serious adverse reaction (SUSAR) is an untoward medical 
effect. The nature or severity of which is not consistent with the information on the 
experiment, and with the applicable product information, which results in death, is life-
threatening, requires hospitalisation or prolongation of existing hospitalisation, results in 
persistent or significant disability or incapacity, or when it is a congenital anomaly or 
birth defect. 
 
 
2. Detection, reporting, and responsibilities 
 
On an ongoing basis, the investigators will determine whether any clinical or laboratory 
AE has occurred. Only the moderate or severe (needing a treatment) AEs will be notified 
center and recorded in the e-CRF describing date of onset, date of cessation, maximum 
intensity, severity, causality, therapy and outcome. The assessment of the potential 
association with the drugs will be handled by the DSMB in case of safety concern. 
 
Occurrence of any SAE or any event that deserves reporting according to the investigator 
will be reported on an ongoing basis.  
SAE and SUSAR will have to be reported within 24 hours. New SAE or SUSAR occurring 
within 28 days after the patient has completed the clinical trial or 28 days after the patient 
is withdrawn from the study must be reported. 
 
The investigators will inform the competent authorities and the ethical committee (UZ 
Leuven) in the due times requested by them. They will also publish an annual safety 
report to be sent to all competent authorities and leading ethical committee, containing 
all reported suspected serious adverse reactions (SSARs). 
 
 
 


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3. Surgical complication classification (within 30 days after transplantation) 
 
The Clavien-Dindo classification will be used to rank surgical complications according to 
an objective, simple, reliable and reproducible way.  This classification is based on the 
type of therapy required to treat the complication.  
 
Grades 
 
Definition 
Grade I 
Any deviation from the normal postoperative course without the need for pharmacological treatment or surgical, 
endoscopic and radiological interventions. 
Acceptable therapeutic regimens are: drugs as antiemetics, antipyretics, analgetics, diuretics and electrolytes, and 
physiotherapy. 
This grade also includes wound infections opened at the bedside. 
Grade II 
Requiring pharmacological treatment with drugs other than such allowed for grade I complication. 
Blood transfusions and total parenteral nutrition are also included. 
Grade III 
Requiring surgical, endoscopic or radiological intervention. 
Grade III-a 
Intervention not under general anesthesia. 
Grade III-b 
Intervention under general anesthesia. 
Grade IV 
Life-threatening complication (including CNS complications) requiring IC/ICU-management. 
Grade IV-a 
Single organ dysfunction (including dialysis). 
Grade IV-b 
Multi-organ dysfunction. 
Grade V 
Death of patient. 
Suffix “d” 
If the patient suffers from a complication at the time of discharge, the suffix “d” (for disability) is added to the 
respective grade of complication. This label indicates the need for a follow-up to fully evaluate the complication. 
 CNS: brain hemorrhage, ischemic stroke, subarachnoid bleeding, but excluding transcient ischemic attack; IC: 
intermediate care; ICU: intensive care unit (Clavien-Dindo et al. 2009; 250: 187-196) 
 
 


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4. Infection 
 
Each patient will be routinely monitored for bacterial, fungal or viral infection on an 
ongoing basis (HBV, CMV, Epstein-Barr virus, and HCV recurrence). Details about any 
infection detected and therapeutic measures applied must be recorded. 
 
4.1 CMV 
 
For clarity purposes about CMV infection, the following definitions are applied. 
 
4.1.1 CMV infection 
 
CMV infection is defined as detection of: 
- 
Anti-CMV IgM antibodies in a previously seronegative patient, 
- 
or CMV early-antigen (CMV pp-65) in any body fluid (blood, urine, saliva), 
- 
or positive DEAFF-test in any body fluid (blood, urine, saliva), 
- 
or intraleukocytic CMV-DNA by PCR amplification, 
- 
or CMV DNA or antigens in tissue biopsies. 
 
4.1.2 CMV disease  
 
CMV disease is defined as: 
- 
Evidence of CMV infection as described above, and 
- 
fever > 37.9 °C for 3 days or more, 
- 
and/or leucopenia < 4000 WBC/ml, 
- 
and/or thrombocytopenia, 
- 
or invasive CMV as defined by detection of any CMV material (nucleic acids or 
antigens) in any tissue biopsy (lung including bronchiolo-alveaolar-lavage, gastro-
intestinal tract). 
 
4.1.3 CMV syndrome 
 
CMV syndrome is defined as: patient with fever, fatigue, muscular skeletal pain, headache 
and CMV infection criteria who does not fulfill the CMV disease criteria. 
Screening for CMV infection or CMV disease during follow up will be done on an ongoing 
basis. Occurrence of either CMV infection or CMV disease will be recorded with pertaining 
details. 
 
4.2 Hepatitis C 
 
As hepatitis C virus (HCV) infects the liver allograft immediately after transplantation and 
is an uncommon cause of allograft dysfunction during the first several weeks after 
transplantation, HCV recurrence will be monitored on an ongoing way in patients at risk. 
 


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5. Predefined types of adverse events 
 


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APPENDIX 3: COMBINED DRUG APPROACH 
 
3.1 GENERAL DESCRIPTION 
 
3.1.1. ANTITHROMBIN III, ATENATIV® 
 
Introduction 
Antithrombin III (ATIII) is a plasma-derived serine protease inhibitor (serpin). 
Independently and besides its potent anticoagulant activity, ATIII has a direct action on 
activated endothelial cells by increasing the release of prostacyclin and NO. Moreover, 
ATIII reduces vessel wall transmigration and subsequent tissue damage. Finally, ATIII 
decreases the formation of ROS and proteases by neutrophils (1). A potential underlying 
mechanism was recently characterized by the experimental proven reduction of nuclear 
factor kappa beta expression resulting in less systemic release of cytokines (2). 
The AT-III-mediated reduction of microcirculatory disorders and tissue injury has been 
proven as highly effective in various models of renal (3), intestinal (4) and hepatic 
ischemia-reperfusion 
(5-6). 
In 
addition, 
ATIII 
reduced 
rejection 
in 
renal 
xenotransplantation (7). In clinic, Fertmann et al. has characterized a significant 
improvement of renal allograft reperfusion by single-shot application during human 
kidney transplantation in a randomized controlled trial (8). They also observed a 
decreased peak of Tumor Necrosis Factor-alpha (TNF-alpha) after kidney transplantation 
(2). Further, the same group has described a reduction of reperfusion injury, pancreatitis 
and a prevention of graft thrombosis with a single-shot of ATIII in human pancreas-kidney 
transplantation (9). Moreover, ATIII has been shown to reduce rejection in allogenic 
cardiac transplant (10). Currently, a multi-centric protocol for clinical phase III trial 
testing pre-reperfusion ATIII application in combination with acetylcysteine is ongoing 
for simultaneous kidney and pancreas transplantation. 
Pharmacokinetic studies with ATIII have shown a mean biological half-life of about 3 days. 
Safety and adverse reaction 
Concerning the safety, Fertmann et al. (9) did not observe perioperative ATIII-mediated 
changes on standard coagulation tests (partial thromboplastin time, thromboplastin time, 
platelet counts), on the perioperative bleeding rates and on the number of packed red 
blood cell concentrates given to the patient with a single IV dose of 3000 IU. This finding 
is in agreement with the literature where long-term ATIII application also did not 
influence these standard blood coagulation tests (11). 
ATIII replacement during administration of heparin in therapeutic dosage increases the 
risk of bleeding. The effect of ATIII is enhanced by heparin. The half-life of ATIII may be 
considerably decreased with concomitant heparin treatment due to accelerated ATIII 
turnover (1.5 days).  
Hypersensitivity or allergic reactions (which may include angioedema, burning and 
stinging at the infusion site, chills, flushing, generalized urticaria, headache, hives, 
hypotension, lethargy, nausea, restlessness, tachycardia, tightness of the chest, tingling, 
vomiting, wheezing) have been observed infrequently in conjunction with the use of 
Antithrombin III concentrates and may in some cases progress to severe anaphylaxis 
(including shock). On rare occasions, fever has been observed. 


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Contra-indication linked to the use of Atenativ® 
Patient requiring therapeutic dose of heparin perioperatively will be excluded from the 
treatment group. 
  
Product characteristics 
The ATIII Atenativ® is manufactured by Octapharma. This product is prepared from 
pooled units of human plasma from normal donors and contains no preservative. It is a 
lyophilized powder in a vial and a solvent in a vial which is used for reconstitution of the 
powder. 
 
Packaging and storage 
Atenativ® is presented as powder for solution for infusion containing nominally 500, 
1000 or 1500 IU human plasma-derived antithrombin III per vial. The product contains 
approximately 50 IU/ml human plasma-derived antithrombin III when reconstituted 
with 10 (500IU), 20 (1000 IU) or 30 (1500 IU) ml of water for injection respectively. 
The vial of powder should be stored at a temperature of 2-8°C. It should stay in the outer 
carton in order to be protected from light. 
Within its shelf-life, the product may be stored at room temperature (25°C) for up to 1 
month, without being refrigerated again during this period, but must be disposed if not 
used after this.  
After reconstitution, the product should be used as soon as possible. If not used 
immediately, in-use storage times should not be longer than 24 hours at 2 to 8°C. 
Dose and administration 
According these experimental and clinical data that have proven profound effect without 
toxicological events, 1 single IV dose (3000 IU) of ATIII (Atenavit®) will be administrated 
after the start of anastomosis during the anhepatic phase (50 minutes before reperfusion) 
over 15 minutes. 
References: 
1. Antithrombin: a new look at the actions of a serine protease inhibitor. Roemisch et al., 
Blood coagulation and fibrinolysis 2002; 13: 657-670. 
2. Effects of antithrombin on cytokine secretion after human allogenic kidney 
transplantation. Fertmann et al., Transplantation 2004; 78: 222-223. 
3. Antithrombin III reduces renal ischemia-reperfusion injury in rats. Ozden et al., Res Exp 
Med 2001; 200: 195. 
4. Antithrombin III prevents and rapidly reverses leukocyte recruitment in 
ischemia/reperfusion. Ostrovsky et al., circulation 1997; 96: 2302. 
5. Antithrombin reduces IRI of rat liver by increasing the hepatic level of prostacyclin. 
Harada et al., Blood 1999; 93:157. 
6. Controlled trial of antithrombine III supplementation in fulminant hepatic failure. 
Langley et al., J hepatol 1993; 17: 326. 
7. Protective effects of recombinant human antithrombin III in pig-to-primate renal 
transplantation. Cowan et al., Am J Transpl 2002; 2: 520. 
8. High dose antithrombine therapy reduces IRI and improves graft function during 
human allogenic kidney transplantation: final results of a randomized controlled clinical 
trial. Fertmann et al.,Transplantation 2002; 74, 352. 


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9. Single-shot antithrombin in human pancreas-kidney transplantation: reduction of 
reperfusion pancreatitis and prevention of graft thrombosis. Fertmann et al., Transplant 
International 2005; 18: 40. 
10. High dose of AT induces indefinite survival of fully allogenic cardiac grafts and 
generates regulatory cells. Arakami et al., Transplantation 2003; 75: 217. 
11. Effect of long-term and high-dose antithrombin supplementation on coagulation and 
fibrinolysis in patients with severe sepsis. Hoffmann et al., Crit Care Med 2004; 32: 1851. 
 
 
 


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3.1.2. C1-ESTERASE INHIBITOR, CETOR®/Cinryze® 
 
Introduction 
C1-inhibitor (C1-INH) is a blood-derived serine protease inhibitor (serpin). C1-INH is the 
key player in the regulation of complement activity. It acts mainly by inhibiting the 
classical pathway but also with a lower effect on the lectine and alternative pathways. 
Moreover, it also balances the intrinsic pathway and the fibrinolytic system of the 
coagulation. Finally, it reduces the expression of IL-6, thereby preventing damage 
generated by neutrophil recruitment and subsequent neutrophil activation (1). 
Therefore, C1-INH has emerged as a promising agent to inhibit damage resulting from 
activation of inflammation and coagulation.  
Studies have provided evidence for the role of complement in the pathogenesis of IRI (2). 
The use of C1-INH for the inhibition of the complement in various models of myocardial 
(3), lung (4), renal, intestinal and hepatic ischemia-reperfusion (5) has been proven as 
highly effective in the reduction of inflammation and consequent tissue injury. Moreover, 
besides the routine clinical practice use of C1-INH in angioedema, clinical trials have 
focused on the decrease of vascular leakage syndrome after lung transplantation (6) and 
the reduction of myocardial IRI. Bauernschmitt et al. (7), Thielmann et al. (8) and Fattouch 
et al. (9) have all described a significant positive impact of C1-INH on the cardiac function 
during coronary surgical reperfusion. Stieh et al. has observed the same findings with a 
post-operative administration of C1-INH in open heart surgery. Finally, C1-INH may also 
be used to prevent antibody induced acute graft rejection after the transplantation. This 
however requires longer-term administration after the procedure (10, 11). 
 
Safety and adverse reaction 
Toxicity of C1-INH is very low. No side effects were observed in those clinical trials (6, 7, 
8, 9). No complications are expected in patients either with a liver dysfunction or during 
the LTx.  
Hypersensitivity or allergic reactions (which may include angioedema, burning and 
stinging at the infusion site, chills, flushing, generalized urticaria, headache, hives, 
hypotension, lethargy, nausea, restlessness, tachycardia, tightness of the chest, tingling, 
vomiting, wheezing) could be observed with C1-INH and may in some cases progress to 
severe anaphylaxis (including shock). 
Contra-indication linked to the use of Cetor®/Cinryze®  
There is no specific contraindication linked to the use Cetor®/Cinryze® in the 
multifactorial modulation (12, 13). 
Product characteristics 
The 
C1-INH 
Cetor®/Cinryze® 
is 
manufactured 
by 
Sanquin 
Blood 
Supply 
Foundation/ViroPharma (taken over by Shire). It is purified from the human plasma. It is 
a sterile, stable, lyophilized preparation. The elimination half-life has been determined at 
42 hours. The mean residence time (the time required for 62.3% of the administered dose 
of C1- inhibitor to be eliminated; comparable with the elimination half-life but calculated 
independently of the model used) is 65 hours. This applies equally to individuals with or 
without C1- inhibitor deficiency. The clearance in man is 0.053 L per hour. 
 


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Packaging and storage 
The commercial packing of Cetor®/Cinryze® consists of: 
- 
A vial of Cetor®/Cinryze®  500 U (powder for solution for IV injection), 
- 
A 5 ml vial of water for injections, 
- 
An administration set consisting of 10 ml disposable syringe, transfer needle, filter 
needle, and butterfly-wing needle. 
After dissolution in the water for injections supplied, the product contains 100 U C1-INH 
per ml; 5 ml = 500 U C1-INH. 
Cetor®/Cinryze® should be administered as soon as possible and no later than 3 hours 
after piercing the vial. The entire solution should be used in a single administration.  
The vial of product and water should be stored at a temperature of 2-8 °C. 
Dose and administration 
In this study, the IV dose of Cetor®/Cinryze® (Cinryze® will be used in September 2015 
when the expiration date of vials of Cetor® is reached)  has been determined according 
the experimental and clinical data that have proven profound effect without toxicological 
events. Thus, 1000 U will be administrated gradually, as recommended during 5 minutes 
at the end of the anhepatic phase (10 minutes before reperfusion). This time course is 
adequate according the half-life of Cetor®/Cinryze® after IV injection and we still expect 
a high plasma concentration after reperfusion. 
During administration, the solution should not be too cold. The powder also dissolves 
more readily if both vials are brought to room temperature in advance (15-25°C). 
References: 
1. Recent advances in the use of C1-inhibitor as a therapeutic agent. Bergamaschini et al., 
Mol Immunol 2003; 40: 155-158. 
2. Ischemia and reperfusion: from mechanism to translation. Eltzschig and Eckle, Nature 
medicine; 11: 1391. 
3. Intracoronary application of C1 esterase inhibitors improves cardiac function and 
reduces myocardial necrosis in an experimental model of ischemia and reperfusion. 
Horstick et al., Circulation 1997; 95: 701-708. 
4. C1-esterase inhibitor reduces reperfusion injury after lung transplantation. Scherer et 
al., Ann Thorac Surg 2002, 73: 233-238. 
5. Endothelial targeting with C1-inhibitor reduces complement activation in vitro and 
during ex vivo reperfusion of pig liver. Bergamaschimi et al., Clin Exp Immunol 2001; 126: 
412-420. 
6. C1-esterase inhibitor in graft failure after lung transplantation. Struber et al., Intensive 
care med 1999; 25: 1315-1318. 
7. Rescue therapy with C1-esterase inhibitor concentrate after emergency coronary 
surgery for failed PTCA. Bauernschmitt et al., Intensive care medicine 1998; 24: 635-638. 
8. Administration of C1-esterase inhibitor during emergency coronary artery bypass 
surgery in acute ST-elevation myocardial infarction. Thielmann et al., Eur J Cardio Thorac 
Surg 2006; 285-293. 
9. Beneficial effects of C1-esterase inhibitor in ST-elevation myocardial infarction in 
patients who underwent surgical reperfusion: a randomized double-blind study. Fattouch 
et al., Eur J Cariothorac Surg 2007; 32: 326. 
10. Recombinant human C1-inhibitor inhibits cytotoxicity induced by allo- and 
xenoantibodies. Poirier et al., Transplant Proc. 2008 Mar;40(2):581-3. 


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11. Recombinant human C1-inhibitor prevents acute antibody-mediated rejection in 
alloimmunized baboons. Tillou et al. Kidney Int. 2010 Jul;78(2):152-9. 
12. Treatment of hereditary angioedema with nanofiltered C1-esterase inhibitor 
concentrate (Cetor): Multi-center phase II and III studies to assess pharmacokinetics, 
clinical efficacy and safety. J.J. Hofstra, I. Kleine Budde, E. van Twuyver, G. Choi, M. Levi, 
F.W.G. Leebeek, J.G.R. de Monchy, P.F. Ypma, R.J. Keizer, A.D.R. Huitema, P.F.W. Strengers. 
Clin Immunol 142 (2011) 280-90. 
13. Model-Based Evaluation of Similarity in Pharmacokinetics of Two Formulations of the 
Blood-Derived Plasma Product C1 Esterase Inhibitor. R.J. Keizer, I. Kleine Budde, P.F. 
Strengers, M. Levi, J.H. Beijnen, A.D. Huitema. J Clin. Pharmacol.2011. 
 
 
 


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3.1.3. ERYTHROPOIETIN BETA, NEORECORMON® 
 
Introduction 
EPO is a cytokine known to stimulate the proliferation and the survival of erythroid 
progenitor cells. However, the expression of EPO receptors has been described in several 
organs outside the bone marrow. Recently, increasing attention has been given to the 
pleiotropic effect of EPO. It has been demonstrated that recombinant human EPO (rhEPO) 
has relevant tissue-protective effects against various injuries in animal models, including 
liver  IRI in pigs (1). The use of EPO in animal models has been reviewed by Sharples et al 
(2) and Johson et al (3). Virtually every organ that has been evaluated has been reported 
to benefit from EPO administration following IRI. The exact mechanism by which EPO 
mediates its organ-protective effects is not completely understood, but anti-apoptotic, 
anti-inflammatory and anti-oxidative properties have been demonstrated (4).  
A growing number of clinical studies are testing the tissue protection effect afforded by 
rhEPO in various acute clinical settings, including neuroprotection and cardioprotection. 
Ehrenreich et al (5) has used EPO beta at a total dose of 100.000 IU for the first 3 days 
after an acute cerebral stroke, noting that this was safe and well tolerated. Lipsic et al (6) 
has demonstrated that a single bolus injection of EPO at a dose of 60.000 IU in patient 
with an acute myocardial infarction was not associated to any significant increase in 
hemoglobin (Hb). Mocini et al (7) has described that a single injection of EPO at a dose of 
40.000 IU before cardiac surgery has no significant impact on the cardiac function and on 
the erythropoiesis. Belonje et al (8) has demonstrated in a safety pilot assessment that 
60.000 IU of EPO in patients with an acute myocardial infarction was not associated with 
a raise in blood pressure, an increase in Hb and thrombocytes or any adverse events like 
vascular thrombosis or seizure. In elective liver surgery, Mosato Kato et al (9) has 
indicated that a two time injection of 30.000 IU EPO rather than one single bolus of 60.000 
IU has a stronger inhibitory effect on IRI following the pringle maneuver during liver 
surgery. Moreover, these doses and their timing of administration did not affect blood cell 
count, hematocrit and platelets. 
Considering the results of the different species experiments and clinical studies in 
different organs, it is suggested to adapt the dose of EPO and its timing of administration 
in order to reach an optimal protective effect. This was confirmed by Ramakrishnan et al. 
(10). They have described the importance of doses, dosage regimens and routes of 
administration in physiologically mechanistic PK/PD model. Pharmacokinetic 
investigations in healthy volunteers show that the half-life of IV administrated EPO beta 
is between 3 and 12 hours. 
Notably, the receptor mediating the non-erythropoietic effects of EPO differ from the one 
responsible for hematopoiesis. The tissue-protective receptor exhibits a lower affinity for 
EPO. It is a heteromer consisting of EPO receptor monomers in association with the 
common receptor. This heteromeric receptor is expressed immediately following injury, 
whereas EPO production is delayed providing a window of opportunity for therapeutic 
intervention. Thus early administration of EPO can dramatically reduce the deleterious 
components of the local inflammatory cascade. However, a larger dose is required to 
trigger the protective effect as the affinity for the heteromer is lower than the 
hematopoietic receptor. Thus, to effectively treat injured tissue using exogenous EPO, 
high parenteral doses are required (11).  
 


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Safety and adverse reaction 
Safety data arising from clinical studies suggest that a high single IV dose of rhEPO is safe 
regarding acute as well as chronic adverse effects including vascular thrombosis, seizure 
and raise in blood pressure, Hb levels, platelets and hematocrit. Anaphylactoïd reactions 
were observed in isolated cases.  
However, as patients with a history of seizure and a level of Hb higher than 13 gr/dl were 
excluded in a few studies, this treatment (and the rest of the multifactorial modulation) 
will be avoided, for safety concerns in patients with these findings. 
The clinical results obtained so far do not indicate any interaction of NeoRecormon with 
other medicinal products. 
Contra-indication linked to the use of Neorecormon®  
Patient with a history of seizure and a pre-operative level of Hb higher than 13 g/dl will 
be excluded from the treatment group. 
Product characteristics 
The EPO-beta Neorecormon® is manufactured by Roche as pre-filled syringe. It is a 
colourless solution for injection.  
Packaging and storage 
One pre-filled syringe with 0.6 ml solution for injection contains 30.000 international 
units (IU). 
The pre-filled syringe should be stored in a refrigerator (2°C – 8°C). It should stay in the 
outer carton in order to be protected from light. 
Dose and administration 
Considering the distribution profile after IV injection, the half-life of EPO-beta (3-12h), 
the potential elimination of the administrated medication by blood loss during the 
procedure and the need to keep a high concentration for the post-reperfusion phase, a 
first dose of 30.000 IU will be administrated 13-15minutes before the reperfusion of the 
liver over 2 minutes followed by a second dose of 30.000 IU of EPO-beta 6 hours after the 
reperfusion. 
 
 
References:  
1. Shimoda et al, hepato-gastroenterology 2009; 56: 470-475. EPO strongly protects the 
liver from IRI in a pig model. 
2. Sharples et al, Nat Clin Pract Nephrol 2005; 1: 87-97. Mechanisms of disease: cell death 
in acute renal failure and emerging evidence for a protective role of EPO. 
3. Johnson et al, Nephrology 2006; 11: 306-312. Novel renoprotective action of EPO: new 
use for an old hormone. 
4. Ghezzi et al, Cell deat and differenciation 2004; 11: 37-44. EPO as an antiapoptotic, 
tissue-protective cytokine. 
5. Ehrenreich et al, Molecular medicine 2002; 8: 495-505. EPO therapy for acute stroke is 
both safe and beneficial. 
6. Lipsic et al, J Am Coll Cardiol 2006; 48: 2161-2167. Protective effect of EPO in cardiac 
ischemia: from bench to bedside. 
7. Mocini et al, Perfusion 2008; 23: 187-192. Endogenous EPO and a single bolus of 40.000 
IU do not protect the heart from IRI during extracorporeal circulation for cardiac surgery. 


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8. Belonje et al, Am Heart J 2008; 155: 817-822. Effects of EPO after an acute myocardial 
infarction: Rationale and study design of a prospective, randomized, clinical trial. 
9. Mosato Kato et al, World Journal of Gastroenterology 2010; 16: 4838-4845. EPO 
ameliorates early ischemia-reperfusion injury following the pringle maneuver. 
10. Ramakrishnan et al, J Clin Pharmacol 2004; 44: 991-1002. PK and PD modeling of 
recombinant human EPO after a single and multiple doses in healthy volunteers. 
11. Brines et al, Blood Purif 2010; 29: 86-92. The therapeutic potential of EPO for tissue 
protection: a tale of two receptors. 
 
 


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3.1.4. MELATONIN, CIRCADIN® 
 
Introduction 
Melatonin is a pineal hormone involved in the circadian rhythm of vertebrates. Besides 
this role, Melatonin and its metabolites as well are known to exhibit strong antioxidant 
properties. Among any antioxidant agents, Melatonin is described as a most powerful 
endogenous free radical scavenger being 5 - 14 times more effective at scavenging the 
highly toxic hydroxyl radicals than Glutathione and Mannitol, respectively; it is also 
reported by twice more efficient than Vitamin E in detoxifying the peroxyl radical (1). 
Moreover, Melatonin shows synergistic effects with other antioxidants. When Melatonin 
is combined with Vitamin E or Glutathione, the protective effects against iron-induced 
lipid peroxidation is dramatically enhanced (1). Furthermore, Melatonin stimulates a 
number of antioxidative enzymes such as superoxide dismutase, catalase and glutathione 
peroxidase. Finally, Melatonin promotes the efficiency of the mitochondrial electron 
transport chain leading to a reduction of electron leakage, free radical formation, and 
improvement of energy production (1).  
Via these combined beneficial actions, Melatonin has proven highly effective in reducing 
IRI in various models and clinical settings (2). In a warm ischemic model, Vairetti et al. 
has shown a significant reduction of transaminases in the liver of rats and pigs after 
preoperative administration of melatonin in a dose dependent manner (1). Chen et al. has 
described an enhancement of neutrophil apoptosis after administration of Melatonin 
during partial hepatectomy (3). More recently, Gitto et al. has shown the efficacy of IV 
Melatonin to decrease the oxidative stress status and pro-inflammatory cytokines in 
human neonates undergoing surgery for congenital malformation, observing that it was 
safe and well tolerated (4).  
The antioxidative effects of Melatonin require concentrations that are much higher than 
endogenously produced plasma concentrations. On the other hand, a very low 
concentration of exogenous Melatonin (> 0.3 mg) produces already supra-physiological 
levels in humans. After an exogenous administration of Melatonin, very high 
concentrations are found in the hepato-biliary system due to an extensive first-pass and 
high affinity binding sites in hepatocyte nuclei. The serum Melatonin peak was reached 
after approximately 15 minutes from oral administration with an elimination half-life of 
30-45 minutes. Additionally, half-life from crushed Melatonin and administrated through 
the naso-gastric tube is extended to 90 minutes (5). Indeed, the area of intestinal mucosa 
exposed to Melatonin is larger (6). This half-life is extended to approximately 100 minutes 
in cirrhotic patients (7) after oral administration. With the use of a prolonged and 
sustained release formula, the serum Melatonin peak is reached after 2-4 hours. 
Safety and adverse reaction 
Cost and toxicity of Melatonin is remarkably low. In the United States, the Food and Drug 
Administration (FDA) has designated Melatonin as an orphan product sold as a dietetic 
complement (8). No significant adverse effects were observed following administration 
of high doses (3-6.6 g/day) in humans for 15 to 35 days (9). High doses of IV Melatonin 
have been used with success in babies with sepsis syndromes without toxicological effect 
(10). IV infusion in doses between 10 and 60 mg during abdominal aortic-aneurysm 
repair has already been shown to be without immediate toxic reaction (11).  
Melatonin's metabolism is mainly mediated by CYP1A enzymes. Therefore, interactions 
between Melatonin and other active substances as a consequence of their effect on CYP1A 
enzymes are possible. By inhibiting the isoenzymes CYP1A2, Quinolone, fFuvoxamine, 


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Cimetidine and Oestrogene can increase the plasma level of Melatonin. On the other hand, 
Carbamazepin and Rifampicin may decrease the Melatonin exposure by stimulating its 
metabolism. 
Contra-indication linked to the use of Circadin® 
There is no specific contraindication linked to the use Circadin® in the multifactorial 
modulation. 
Product characteristics 
Circadin® is produced by Takeda-Nycomed. Tablets are white, round and biconvex.  
Packaging and storage 
The tablets are packed in PVC/PVDC opaque blister strips with aluminium foil backing. 
The pack consists of one blister strip containing 20 or 21 tablets, or two blister strips 
containing 15 tablets each (30 tablets). The blisters are then packed in carton boxes. 
Not all pack sizes may be marketed. 
Do not store above 25°C and keep the original package in order to protect from light. 
Dose and administration 
In this study, the oral dose of Melatonin has been determined according the experimental 
and clinical data that have proven profound effect without toxicological events (6). Thus 
6 mg of melatonin will be administrated orally at the ward just before transfer to the 
operative theatre. This time course is adequate according the pharmacokinetic data of 
melatonin after oral administration (6). 
References: 
1. Exogenous melatonin enhances bile flow and ATP levels after cold storage and 
reperfusion in rat liver: implication for liver transplantation. Vairetti et al., J Pineal Res 
2005, 38: 223-230. 
2. A unicenter, randomized, double-blind, parallel-group, placebo-controlled study of 
melatonin as an adjunct in patients with acute myocardial infarction undergoing primary 
angioplasty (MARIA). Dominguez-Rodriguez et al., Contemporary Clinical Trial 2007, 28: 
532-539. 
3. Altered neutrophil apoptosis activity is reverse by melatonin in liver ischemia 
reperfusion. Chen et al., J Pineal Research 2003, 34:260-264. 
4. Melatonin reduces oxidative stress in surgical neonates. Gitto et al., J Pediatr Surg2004, 
39: 184-189.  
5. Pharmacokinetics of orally administered melatonin in critically ill patients. Mistraletti 
et al., J Pineal Res 2010; 48: 142-147. 
6. Melatonin pharmacokinetics following two different oral surge-sustained release doses 
in older adults. Gooneratne N et al., J Pineal Res 2012; 52: 437-445. 
7. Melatonin serum levels and metabolic clearance rate in patients with liver cirrhosis. 
Igushi et al., J Clin Endocrin METAB 1982, 54: 1025-1027. 
8. Optimization of a vehicle mixture for the transdermal delivery of melatonin using 
artificial neural networks and response surface method. Kandimalla et al., J Control 
Release 1999, 61: 71-82. 
9. Melatonin and Parkinsonism. Papavasilou et al., JAMA 1972, 221: 88-89. 
10. Effects of melatonin treatment in septic newborns. Gitto et al., Ped Res 2001, 50: 756-
760. 
11. Utility of melatonin to treat surgical stress after major vascular surgery – a safety 
study. Kucukakin et al., J Pineal Res 2008, 44:426-431. 


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3.1.5. GLUTATHIONE, TATIONIL ® 
 
Introduction 
Glutathione (GSH) is one of the major endogenous antioxidants produced by the cells in 
high concentration providing a prime position to neutralize free radicals and to maintain 
the cellular redox potential. GSH participates directly in the neutralization of free radicals 
and reactive oxygen species (ROS). Additionally, GSH improves the hepatic 
microcirculation by counteracting the ROS-mediated mechanisms of vasoconstriction and 
leukocyte adherence/vascular occlusion and by preventing detachment of sinusoidal 
endothelial cells. Moreover, studies have demonstrated that GSH released through the 
GSH transporter of hepatocytes may act as an endogenous defense system against Kupffer 
cell–mediated endothelium damage (1). Finally, GSH is crucial in the modulation of the 
immune system. Indeed glutathione is required for cells in modulating antigen 
presentation to lymphocytes, thereby influencing cytokine production like leukotrienes, 
prostaglandins and cytokines. By doing so, Glutathione is involved in the regulation of cell 
proliferation, apoptosis, DNA synthesis and protein synthesis, thus maintaining control of 
the immune response. It is well known from experimental models, as well as from data 
obtained from human allograft recipients, that antioxidants are consumed during 
reperfusion injury of transplanted grafts (2, 3). 
Studies have indicated that GSH was able to react spontaneously with nearly all oxidants 
formed during inflammation (4). Shauer et al. has described an effective impact of GSH on 
rat liver models of ischemia reperfusion and transplantation with a significant reduction 
in ALT after reperfusion (5, 6). They clearly demonstrated the essential role of a 
preventive administration of GSH in protecting the parenchymal liver cells. Indeed, post-
ischemic GSH treatment can only protect cells that are not already seriously damaged 
before the onset of reperfusion. This GSH-mediated cytoprotection seems to be 
attributable to an extracellular antioxidant mechanism as no evidence of increase of the 
GSH concentration in the hepatic cells after intravenous administration has been 
described (11). In humans, recent investigations have proven the therapeutic potential of 
GSH, in particular since it has a low toxicity in humans (7) and it is cost-effective. Chawla 
et al. has described a lower concentration of GSH in the plasma of cirrhotic patients (8). 
Thus, any intervention increasing hepatic or plasma GSH levels should convey protection 
against reperfusion injury (9). As shown recently in vitro, treatment of cold preserved 
livers with 2 or 4 mM GSH upon reperfusion prevented cell damage to hepatocytes in a 
model of cell-free rat liver perfusion (10).  
 
Safety and adverse reaction 
Overall, Glutathione and precursor (acetylcysteine) appears to have a low toxicity in 
humans. Currently, there are no known side effects of GSH (7). None adverse reactions 
have been reported, neither in clinical studies nor in case reports. 
Interactions may occur between the different antioxidants. GSH maintains exogenous 
antioxidants such as Vitamins C and E in their reduced (active antioxidant) forms.  
Contra-indication linked to the use of Tationil® 
There is no specific contraindication linked to the use Tationil® in the multifactorial 
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Product characteristics 
Tationil 600® is manufactured by Teofarma Italy and will be distributed by a wholesale 
in Germany (= regular supplier of the pharmacy in UZ Leuven) . It is a white, crystalline 
powder freely soluble in water. 
Packaging and storage 
Each box contains 10x600mg of Glutathione together with 10x4ml distilled aqua for 
mixing. 
It should be stored in airtight containers and protected from light. 
In vitro studies have shown that reduced Glutathione (GSH) is oxidized to GSSG during 
storage at 4°C limiting the half-life to eight days. This can be prevented by lyophilization 
of GSH. 
Dose and administration 
Aebi et al. (7) has studied the pharmacokinetics of GSH in man after IV infusion. They have 
demonstrated that a high dose of IV GSH distributes safely in the extracellular 
compartment and is cleared from the circulation with a half-life of ±15 min.  
It must be noted that the best dosage of Glutathione has not been clearly established for 
any use; most dose schedules have been empirically chosen based on animal data. Applied 
IV concentrations vary from 2.0 to 4.8 gram in total for an adult. For example, GSH has 
been reported in randomized studies to reduce the incidence of neurotoxicity induced by 
chemotherapy at a dose of 1.5 g/m2 (10, 11, 12). IV Glutathione is also used to slow down 
the progression of Parkinson’s disease. Most patients are given 1400 mg along with saline 
for ten minutes, three times a week. It has been shown to have a critical function as 
elimination of toxic compounds and by restoring the hepatic glutathione stores in case of 
acetaminophen intoxication. In that case, an approved 20 hour IV protocol for 
acetylcysteine –precursor of glutathione- treatment has been used in the UK since 1970s. 
This treatment protocol provides a total of 300 mg/Kg over 20 hours with an initial 
loading dose of 150 mg/kg IV over 15 to 60 minutes. This treatment period is often 
extended when patients have large ingestions of acetaminophen or elevated serum 
transaminase (15). 
In this study, the IV dose of GSH has been determined according the experimental and 
clinical data that have proven profound effect without toxicological events. According the 
short half-life, 3 gr will be administrated 2-4 minutes just before the reperfusion. 
References: 
1. Neutrophil and Kupffer cell-induced oxidant stress and ischemia-reperfusion injury in 
rat liver. Jaeschke H et al., Am J Physiol. 1991;260:G355–362. 
2. Primary graft nonfunction and Kupffer cell activation after liver transplantation from 
non-heart-beating donors in pigs. Monbaliu et al., Liver Transplant 2007; 13: 239-247. 
3. Reperfusion injury, antioxidants and hemodynamics during orthotopic liver 
transplantation. Goode HF et al. Hepatology 1994; 19: 354. 
4. Glutathione metabolism in activated human neutrophils: stimulation of glutathione 
synthesis and consumption of glutathione by reactive oxygen species. Bilzer M et al., Eur 
J Clin Invest. 1991;21:316–322. 
5. Glutathione protects the rat liver against reperfusion injury after prolonged warm 
ischemia. Schauer et al., Ann Surg 2004; 239: 220-231. 
6. Intravenous administration of glutathione protects parenchymal and non-parenchymal 
liver cells against reperfusion injury following rat liver transplantation. Schauer et al., 
World J Gastroenterol 2004; 10: 864-870. 


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7. High-dose intravenous glutathione in man. Pharmacokinetics and effects on cysteine in 
plasma and urine. Aebi et al., Eur J Invest 1991; 21: 103-110. 
8. Plasma cysteine, cystine, and glutathione in cirrhosis. Chawla et al., Gastroenterology 
1984; 87: 770. 
9. Plasma membrane and mitochondrial transport of hepatic reduced glutathione. 
Fernández-Checa JCet al. Semin Liver Dis. 1996; 16:147–158. 
10. Glutathione protects the rat liver against reperfusion injury after hypothermic 
preservation. Bilzer M et al., Gastroenterology. 1999; 117:200–210.  
11. Beneficial effects of extracellular glutathione against endotoxin-induced liver injury 
during ischemia and reperfusion. Liu P et al., Circ Shock. 1994;43:64–70. 
12. Neuroprotective effect of reduced glutathione on cisplatin-based chemotherapy in 
advanced gastric cancer: a randomized double-blind placebo-controlled trial. Cascinu et 
al., J Clin Oncol 1995; 13: 26-32. 
13. Neuroprotective effect of reduced glutathione on oxaliplatin-based chemotherapy in 
advanced colorectal cancer: a randomized, double-blind, placebo-controlled trial. Cascinu 
et al., J Clin Oncol 2002; 20: 3478-83. 
14. Administration of reduced glutathione in FOLFOX4 adjuvant treatment for colorectal 
cancer: effect on oxaliplatin pharmacokinetics and neurotoxicity. Anti-Cancer Drugs 
2009; 20: 396-402. 
15. Treatment of paracetamol (acetaminophen) poisoning with N-acetylcysteine. Prescott 
LF, Park J, Ballantyne A, Adriaenssens P, Proudfoot AT. Lancet. 1977;2(8035):432. 
 
 
 


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3.1.6. INFLIXIMAB, REMICADE ® 
 
Introduction 
TNF- is a pleiotropic cytokine that induces cellular responses such as proliferation and 
production of inflammatory mediators. In the liver, TNF- is involved in the 
pathophysiology of viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease 
and IRI. 
TNF- is produced mainly by macrophages but also by a broad variety of other cell types 
including lymphoid cells and endothelial cells. TNF- is primarily produced as a 
transmembrane protein but may be released in soluble trimeric form via proteolytic 
cleavage by the metalloprotease TNF-converting enzyme (TACE). TNF- exerts its 
biological functions via interactions with cognate membrane receptors, TNF-R1 and TNF-
R2. These signalling pathways interact in a complex network at several levels and 
activation of one pathway often depends on the inactivation of another pathway, 
suggesting that cells are capable of directing the TNF--induced signal toward the 
appropriate response. In normal liver TNF-R1 expression is slow, but high TNF-R1 
expression occurs in hepatocytes, cholangiocytes, sinusoidal endothelium and 
inflammatory cells in disease states. Whereas TNF-R1 is efficiently activated by soluble 
TNF-, TNF-R2 activation requires the binding of membrane-bound TNF- (1). 
TNF- is a crucial mediator in hepatic reperfusion injury. During ischemia-reperfusion, 
Kupffer cells generate ROS subsequent to expression of MAP-kinase JNK, which in turn 
activates and enhances the secretion of TNF-. TNF- released may activate TNF- 
receptors on hepatocytes to induce JNK and Inhibitor of κB (IKK) activation as well as ROS 
production. Whereas ROS promote hepatocytes cell death; IKK activation further 
enhances leukocyte infiltration in the liver (2). 
TNF- antagonists constitute a recent class of immunomodulators. Infliximab® is a 
chimeric human-murine monoclonal antibody that binds with high affinity to both soluble 
and transmembrane forms of TNF-α. Infliximab is well established in the treatment of 
rheumatoid arthritis (I.V. 3 mg/kg at 0, 2, and 6 weeks, followed by 3 mg/kg every 8 weeks 
thereafter; doses have ranged from 3-10 mg/kg repeated at 4- to 8-week intervals), 
psoriatric arthritis (I.V.: 5 mg/kg at 0,2, and 6 weeks, followed by 5 mg/kg every 8 weeks 
thereafter), ankylosing spondylitis (I.V.: 5 mg/kg at 0, 2, and 6 weeks, followed by 5 mg/kg 
every 6 weeks thereafter), inflammatory bowel diseases (I.V.: 5 mg/kg at 0, 2, and 6 
weeks, followed by 5 mg/kg every 8 weeks thereafter; dose may be increased to 10 mg/kg 
in patients who respond but then lose their response),  and several studies have 
demonstrated a high efficacy with rapid overall patient improvement, maintenance 
treatment, and decrease in rates of hospitalizations and surgical interventions (3).  
Due to these promising results, experimental Infliximab treatment has been transferred 
to the field of organ transplantation. Experimental data have shown that inhibition of 
TNF- signalling by TNF antiserum or genetic inactivation of TNF-R1 ameliorates hepatic 
reperfusion injury and prolongs recipient survival (4, 5). Moreover, TNF- has been 
assessed in experimental studies after lung (6) and renal transplantations (7). TNF- 
inhibition resulted in a reduction of inflammatory responses and prolonged graft survival. 
Clinically, anti-TNF- monoclonal antibodies which bind directly to soluble TNF- have 
been studied extensively in the treatment of patients with sepsis (8, 9). These studies have 
revealed that anti-cytokine administration usually as a single dose is safe but not 
particularly efficacious in the management of sepsis. TNF activity, however, peaks within 
hours of insult and usually precedes the initiation of anti-TNF- therapy by hours or days 


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in these clinical studies. The most efficacious use of these agents therefore appears to be 
in clinical scenarios permitting pre-treatment. Furthermore, clinical experience with 
TNF- inhibitors after solid organ transplantation has revealed good results in terms of 
safety, reduction in toxicity and allograft survival (10). In some case reports, Infliximab 
has been shown to be a therapeutic option for refractory acute rejection after intestinal 
transplantation (11). Currently in Leuven, Infliximab at the dose of 3 mg/Kg is infused in 
patient during intestine transplantation at the same timing that foreseen in this protocol. 
Safety and adverse reaction 
Single doses up to 20 mg/kg have been administered without toxic effects. 
Infliximab is contra-indicated in patients with a history of hypersensitivity, tuberculosis 
or other severe infections such as sepsis, abcesses and opportunistic infections. Moreover, 
it is not recommended to administrate Infliximab < 5 mg/kg in patients with severe heart 
failure. 
 
Infliximab has been associated with acute infusion-related reactions, including 
anaphylactic shock and delayed hypersensitivity reactions. These reactions may occur 
during (within seconds) or within a few hours following infusion. 
 
Antibodies to Infliximab may develop and have been associated with an increased 
frequency of infusion reactions. A low proportion of the infusion reactions were serious 
allergic reactions. An association between development of antibodies to Infliximab and 
reduced duration of response has also been observed. Concomitant administration of 
immunomodulators has been associated with lower incidence of antibodies to Infliximab 
and a reduction in the frequency of infusion reactions. The effect of concomitant 
immunomodulator therapy was more profound in episodically treated patients than in 
patients given maintenance therapy. In clinical studies using single and multiple 
Infliximab doses ranging from 1 to 20 mg/kg, antibodies to Infliximab were detected in 
14% of patients with any immunosuppressant therapy, and in 24% of patients without 
immunosuppressant therapy.  
 
Reactivation of hepatitis B has occurred in patients receiving Infliximab. However, in our 
institution, each patient who is a chronic carrier or suffering of an active infection are 
treated by immunoglobulines against HBV during and after the LTx. 
 
Very rare cases of jaundice and non-infectious hepatitis, some with features of 
autoimmune hepatitis, have been observed in the post-marketing experience of 
Remicade®. Isolated cases of liver failure resulting in LTx or death have occurred.  
 
There have been reports of pancytopenia, leucopenia, neutropenia and thrombocytopenia 
in patients receiving TNF-blockers, including Remicade®. 
No interaction studies have been performed. 
Contra-indication linked to the use of Remicade® 
Patient already exposed to Remicade®, with a history of hypersensitivity, tuberculosis or 
other severe infections such as sepsis, abcesses and opportunistic infections and patients 
with severe heart failure will be excluded from the treatment group. 


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Product characteristics 
Remicade®, the antagonist of TNF- is manufactured by Janssen Biologics. It is a sterile 
freeze-dried powder for solution for infusion.  
Packaging and storage 
The shelf-life is 3 years.  
The Remicade® should be stored in a refrigerator (2°C - 8°C). 
Each vial (rubber stopper and aluminium crimp) contains 100 mg of Infliximab. After 
reconstitution each ml contains 0,4 mg of Infliximab. 
Remicade® is available in packs of 1, 2, 3, 4 or 5 vials. 
The chemical and physical in use stability of the reconstituted solution has been 
demonstrated for 24 hours at 25°C. From a microbiological point of view, the product 
should be used as soon as possible but within 3 hours of reconstitution and dilution. If not 
used immediately, in use storage times and conditions prior to use are the responsibility 
of the user and should not be longer than 24 hours at 2 to 8°C. 
Dose and administration 
Since Infliximab is a human-derived monoclonal antibody, 3 hours of infusion are 
required in order to avoid allergic reactions. 
Wen et al. (12) have determined evolution of the plasma level of TNF- during orthotopic 
LTx. An increase of TNF- plasma levels was observed in all patients after the reperfusion. 
TNF- concentrations increased rapidly at the clampage of the vena portae, peaked at 90 
minutes after the reperfusionand then decreased rapidly after 3 hours post-reperfusion 
until 24 hours after the operation. 
In this study, the IV dose of Infliximab has been determined according to the experimental 
and clinical data that have proven profound effect without toxicological events (8, 9, 10). 
Pharmacokinetic studies have described a long half-life ranging from 11 to 14 days after 
the IV administration. Thus, 3 mg/kg of Infliximab will be administrated during 3 hours 
from the beginning of the anhepatic phase after administration of Antithrombin III. 
References 
1. Tumor necrosis factor signaling. Wajant et al., Cell Death Differ 2003 ; 10 :45-65. 
2. Mechanisms of liver injury. TNF--induced liver injury: role of IKK, JNK, and ROS 
pathways. Schwabe and al., Am J Physiol Gastrointest Liver Physiol 2006; 290: 583-
589. 
3. Long-term outcome of treatment with infliximab in 614 patients with Crohn’s disease: 
results from a single-center cohort. Schnitzler et al., Gut 2009; 58: 492-500. 
4. Role of tumor necrosis factor- in the pathophyiologic alterations after hepatic 
ischemia/reperfusion injury in the rats. Colletti et al., J Clin Invest 1990; 85: 1936-
1943. 
5. Tumor necrosis factor-, but not Fas, mediates hepatocellular apoptosis in the murine 
ischemic liver. Rudiger et al., Gastroenterology 2002; 122: 202-210. 


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6. Tumor necrosis factor-alfa in a porcine bronchial model of obliterative bronchiolitis. 
Alho et al., Transplantation 2003; 76: 516-523. 
7. Inhibition of the effects of the TNF in renal allograft recipients using recombinant 
human dimeric tumor necrosis factor receptors. Eason et al., Transplantation 1995; 
59: 300-305. 
8. Hepatotoxicity of immunomodulating agents and the transplant situation. Kaplowitz 
et al., Drug-induced liver disease 2007; 662-681. 
9. Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury. 
Donnahoo et al., The Journal of Urology 1999, 162: 196-203. 
10. New monoclonal antibodies in renal transplantation. Vincenti et al., Minerva Urol 
Nefrol 2003; 55: 57-66. 
11. Intestinal transplantation: evolution in immunosuppression protocols. Pirenne et al., 
Curr Opin Organ Transplant 2009; 14: 250-255. 
12. Plasma levels of tumor necrotic factor- and IL-6, -8 during orthotopic liver 
transplantation and their relations to post-operative pulmonary complication. Wen et 
al., Hepatobiliary Pancreat Dis Int 2004; 3: 38-41. 
 
 


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3.1.7. α-TOCOPHEROL, VITAMIN E SUSPENSION 100 mg/mL®  
Introduction 
Vitamin E is the collective name for a group of fat-soluble compounds that are active 
throughout the body.  Vitamin E includes 4 Tocopherols and 4 Tocotrienols and naturally 
occurs in 8 chemical forms.  α-Tocopherol has the highest Vitamin E activity and is the 
only formulation that is recognized to meet human requirements. It has been claimed that 
α-Tocopherol is the most important lipid-soluble free radical scavenger and antioxidant 
(1). It protects cell membranes from oxidation by reacting with lipid radicals, such as ROS 
produced in the lipid peroxidation chain reaction. In addition, Vitamin E is involved in 
immune function. It has been shown that α-Tocopherol was also implied in cell signalling, 
regulation of gene expression and other metabolic processes (1). α-Tocopherol inhibits 
the activity of protein kinase C, increases the release of prostacyclin from the 
endothelium, which in turn, dilates blood vessels and inhibits platelet aggregation. Finally, 
α-Tocopherol decreases the adhesion of inflammatory cells to the endothelial cell lining 
(1). 
 
Oxidised α-tocopheroxyl radicals produced in this process may be recycled back to the 
active reduced form through reduction by other antioxidants, such as Vitamin C, Selenium, 
Vitamin B3 and GSH. Indeed, Vitamin C is required to keep Vitamin E in its metabolically 
active form; GSH is required to keep Vitamin C in its active form; and Selenium and 
Vitamin B3 are required to keep Glutathione in its active form. Moreover, α-Tocopherol 
may positively influence the total GSH content of the cells during the reperfusion period 
by blocking its decrease in the cells.  
 
It is well known from experimental models, as well as from data obtained from human 
allograft recipients, that antioxidant vitamins are consumed during reperfusion injury of 
transplanted grafts (2, 3, 4). Moreover, antioxidant status and antioxidant vitamin levels 
in patients undergoing organ transplantation have shown that the circulating levels of 
GSH, Vitamins C and E were significantly lower in these patients before transplantation 
than in normal controls (1).  
 
It has been described that lipid peroxidation observed after reperfusion can be effectively 
prevented with antioxidant vitamins, both in animal models (5) and in patients (6, 7). 
Moreover, α-Tocopherol alone has proven highly effective in reducing IRI in various 
models and clinical settings. Oda et al. described a decrease in the severity of reperfusion 
injury, an improvement of short-term allograft function, and survival in a rat model of 
pancreas transplantation (8). The same observations were demonstrated by Hower et al. 
in human kidney transplantation (7). Bartels et al. indicated that preoperative parenteral 
administration of Vitamin E in liver surgery was safe and that this treatment had 
beneficial effects by reducing the impact of IRI (9).  
 
α-Tocopherol is usually administered per os, no clinical grade preparations of pure α-
Tocopherol are known for IV administration in humans. However, α-Tocopherol is added 
to many multi-ingredient preparations, including those that are administered IV (e.g. 
Cernevit®).   
 
The oral supplementation hours before acute episodes of oxidative stress may be less 
effective (10). This might be due to the fact that oral Vitamin E is delivered to the liver via 


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chylomicrons and secreted in VLDL/LDL into the blood. Ferslew et al. (11) described that 
α-Tocopherol first appeared in the plasma in 2-4 hours after oral administration and 
peaked at 5-14 hours when administering 800 mg α-Tocopherol to healthy volunteers. 
The disappearance from the plasma was achieved with a half-life of 53 hours after 
administration. Moreover, short-term parenteral administration was shown to be 
superior to enriching endothelial cells with Vitamin E (10). 
 
Safety and adverse reaction 
This major antioxidant vitamin has excellent safety data in doses that far exceed the doses 
required to reach adequate antioxidant protection in vivo. Regarding controlled, double-
blinded studies of Vitamin E toxicity in humans, several reports confirm that Vitamin E 
has very low toxicity and no consistent adverse events have been reported (12, 13). 
However, Vitamin E at high intakes can affect the coagulation if Vitamin K deficiency is 
also present. Clearly, Vitamin E must not be given in case of anticoagulant therapy. 
Alternatively, administration of Vitamin E can be accompanied by concomitant 
administration of Vitamin K. 
Contra-indication linked to the use of Vitamin E suspension ® 
Patient requiring an anticoagulation that functions as a Vitamin K antagonist 
perioperatively will be excluded from the treatment group. 
Product characteristics 
Vitamin E suspension ®is produced by Cambridge Laboratories. The capsules must be 
orally administrated. 
Packaging and storage 
Vitamin E suspension ®should be stored below 25°C (do not freeze), protected from light. 
The product is for single use in one patient only. 
Dose and administration 
Lassnig et al. (13) have studied multiple parenteral Vitamin E infusion in patients 
undergoing elective cardiac surgery. A total dose of 1200 IU Vitamin E was administered 
at four points in time: two infusions before the start of surgery and one infusion each on 
days 1 and 2 after surgery. Based on pharmacokinetic data the dose was considered 
sufficient to maintain Vitamin E concentrations during surgery. Since no protective effect 
of Vitamin E supplementation was observed, Lassnigg et al. suggested that the supply of 
Vitamin E was inadequate and/or that not enough Vitamin E was incorporated into the 
target cells. Based on these findings and investigations, Bartels et al. (9) decided to 
administer 1800 IU (1620 mg) the day before the liver surgery and compared to a placebo 
group the extent of IRI. After three infusions of Vitamin E, there was a significant fivefold 
increase in the plasma concentration; half of this increase remained about 12 h later, prior 
to surgery. After reperfusion, the Vitamin E concentration was significantly decreased in 
both groups. It is likely that, due to severe oxidative stress, the need for and consumption 
of, α-Tocopherol was great. Six days after surgery the treated group still had plasma 
Vitamin E concentrations comparable to the baseline value, whereas the placebo group 
still had significantly reduced plasma Vitamin E concentrations. They have shown that the 
preoperative parenteral administration of 1800 IU of α-Tocopherol was safe, not only 
leads to a marked increase of plasma concentration but also prevents Vitamin E depletion 
after IRI.  


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Currently, no solutions containing Vitamin E alone are available for human use. Therefore, 
5 mL of Vitamin E suspension ®will be administrated orally at the ward before transport 
to the operative theatre. This time course is adequate according the pharmacokinetic data 
of Vitamin E after oral administration. 
References 
1. Rationale for the use of antioxidant vitamins in clinical organ transplantation. Lehr et 
al., Transplantation 1996; 62: 1197-1199.  
2. Inhibition of free radical generation and improved survival by protection of the hepatic 
microvascular endothelium by targeted erythrocytes in orthotopic liver transplantation. 
Rao PN et al.  Transplantation 1990; 49: 1055. 
3. Reperfusion injury, antioxidants and hemodynamics during orthotopic liver 
transplantation. Goode HF et al. Hepatology 1994; 19: 354. 
4. Primary graft nonfunction and Kupffer cell activation after liver transplantation from 
non-heart-beating donors in pigs. Monbaliu et al., Liver Transplant 2007; 13: 239-247. 
5. Effect of alpha tocopherol on the prevention of reperfusion injury caused by free oxygen 
radicals in the canine kidney autotransplantation model. Demirbas A et al. Transplant 
Proc 1993; 25: 2274. 
6. A multivitamin infusion prevents lipid peroxidation and improves transplantation 
performance. Rabl H et al. Kidney Int 1993; 43: 912. 
7. Assessment of oxygen radicals during human kidney transplantation: effect of radical 
scavenger. Hower R et al. Transpl Int Transpl Int 1996;9 Suppl 1:S479-82. 
8. Role of oxygen radicals and synergistic effect of superoxide dismutase and catalase on 
ischemia-reperfusion injury of the rat pancreas. Oda T et al. Transplant Proc 1992; 24: 
797. 
9. Pilot study on the effect of parenteral vitamin E on ischemia and reperfusion induced  
liver injury: a double-blind, randomized, placebo-controlled trial. Bartels et al., Clin Nutrit 
2004; 23: 1360-1370. 
10. Short-term parenteral application of tocopherol leads to increased concentration in 
plasma and tissue of the rat. Engelhart et al., Free radic res 1998; 29: 421-426 
11. Pharmacokinetics and bioavailability of the RRR and all racemic stereoisomers of 
alpha-tocopherol in humans after single oral administration. Ferslew et al., J Clin 
Pharmacol 1993; 33: 84-88. 
12. Safety of antioxidant vitamins and B-carotene. Diplock et al., Am J Clin Nutr 1995; 62: 
1510-1516. 
13. Influence of intravenous vitamin E supplementation in cardiac surgery on oxidative 
stress: a double-blinded, randomized, controlled study. Br J Anaesth 2003; 90: 148-154. 
 
 


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3.1.8. APOTRANSFERRIN 
Introduction 
Transferrin is the major iron carrier protein in human plasma and extracellular space in 
tissue of a healthy individual. Transferrin iron saturation is 20-35% and redox-active non-
transferrin-bound iron cannot be detected in the serum.  
Several studies have indicated circumstances such as malignacies associated with an 
increase in the serum total iron content, which often exceeds the iron binding capacity of 
transferrin and results in the appearance of non-transferrin-bound iron in the serum of 
patients (1, 2).  
The Rationale to the attempt binding NTBI in the patients by administering 
Apotransferrin is the prevention of iron-induced toxicity, which is thought to depend on 
iron-catalyzed formation of hydroxyl radicals (3). NTBI is effectively taken up by 
parenchymal cells, particularly in the liver and there are several lines of evidence 
suggesting that NTBI is toxic to liver cells (4). On the other hand, NTBI in the sera of 
leukaemia patients has been shown to induce lipid peroxidation and it could thus damage 
cells even without uptake into the cells (2). Ferric iron causes cytotoxicity after a few 
hours in liver cell cultures (5) and it also rapidly impairs the phagocytic activity of 
polymorphonuclear leukocytes (6). Another possible benefit of the binding of NTBI by 
Apotransferrin could be the prevention of the growth of opportunistic bacteria and fungi. 
Practically all micro-organisms are dependent on iron for growth. In normal plasma, 
transferrin keeps the level of free iron far too low to sustain the growth of micro-
organisms and only virulent bacterial species have developed mechanisms to acquire iron 
directly from transferrin (7). The increased level of free iron may thus predispose the 
neutropenic and immunosuppressed patients to septic infections by opportunistic 
bacteria and fungi that are dependent on NTBI for growth (1). Another possible benefit is 
that Apotransferrin binds NTBI into a physiological form that can be utilized by the 
recovering bone marrow.  
A potential alternative for Apotransferrin as an iron-chelating agent, desferrioxamine has 
been extensively studied in chronic iron overload diseases. Unfortunately, it has been 
shown that some of the NTBI is not effectively chelated with desferrioxamine (8) and the 
presence of NTBI in patient sera without full transferrin saturation has been reported (9). 
Moreover, this low-molecular-weight iron chelator displays dose-related toxicity (10) and 
may increase the risk of bacterial and fungal infections (11). 
Safety and adverse reaction 
Apotransferrin is purified from human plasma. Sahlstedt et al. (12) have demonstrated 
that a single dose of Apotransferrin was effective and safe on the serum iron binding 
capacity. They have shown that this effect was temporarily for variable periods from 
hours to days. Parkkinen et al. have described in a dose-finding study the highest dose 
level for the prevention of appearance of NTBI in patients with myeloablation. A total dose 
of 1040 mg/kg (115 mg/Kg/day for 9 days) was given to the patients without any related 
serious adverse event (13). 
Contra-indication linked to the use of Apotransferrin 
There is no specific contraindication linked to the use Apotransferrin in the multifactorial 
modulation. 
 


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Product characteristics 
Human Apotransferrin is manufactured by Sanquin Blood Supply Foundation. The 
product has no marketing authorisation. However, Apotransferrin is currently used in few 
patients with a congenital Apotransferrin deficiency worldwide and no toxic side effects 
have been reported so far (personal communication Sanquin). Apotransferrin consists of 
purified and iron-depleted protein prepared from human plasma. The product is supplied 
as a colourless to slightly yellowish, sterile and pyrogen-free liquid solution. 
Packaging and storage 
Apotransferrin is supplied as solution in a filling size of 40 ml containing 2 g of 
apotransferrin (50 g/l). Apotransferrin is supplied in hydrolytic glass vials with a 
bromobutyl rubber stopper, an aluminium cap and a flip-off seal.  
The primary container consists of colourless and transparent vials of type II glass (50 ml 
container). 
Apotransferrin should be stored at a temperature of 2-8 ˚C and protected from light. 
Dose and administration 
170 mg/Kg of Apotransferrin® will be infused during 3 hours from the beginning of the 
anhepatic phase. 
References: 
1. Non-transferrin bound iron and neutropenia after cytotoxic chemotherapy. Harrison et 
al., J Clin Pathol 1994; 47: 350-352. 
2. Presence of iron catalytic for free radical reactions in patients undergoing 
chemotherapy: implications for therapeutic management. Carmine et al., Cancer letters 
1995; 94: 219-226. 
3. Role of free radicals and catalytic metal ions in human disease: an overview. Halliwell 
et al. 1990, Method in enzymologie; 186: 1-85. 
4. Non-transferrin bound iron and cellular toxicity. Anderson et al. 1999; J Gastroenterol 
Hepatol; 14: 105-108. 
5. Oxidative stress and cytotoxicity induced by ferric-nitrilotriacetate in HepG2 cells that 
express cytochrome P450 2E1. Sakurai et al., Molecular Pharmacology 1998; 54: 1024-
1035. 
6. Effect of iron in the presence of various ligands on the phagocytic and metabolic activity 
of human polymorphonuclear leukocytes. Van asbeck et al., J Immunol 1984; 132: 851-
856. 
7. Iron metabolism in pathogenic bacteria. Ratledge et al., Annual review of microbiology 
2000; 54: 881-941. 
8. Desferrioxamine-chelatable iron, a component of serum non-transferrin-bound-iron, 
used for assessing chelation therapy. Breuer et al., Blood 2001; 97: 792-798. 
9. Non-transferrin bound iron in plasma from hemochromatosis patients: effect of 
phlebotomy therapy. Blood 1988; 72: 1416-1419. 
10. Kinetics of removal and reappearances of non-transferrin-bound plasma iron with 
deferoxamine therapy. Porter et al., Blood; 88: 705-713. 
11. Iron withholding: a defense against infection and neoplasia. Weiberg et al., 
Physiological reviews 1984; 64: 65-102. 
12. Effective binding of free iron by a single intravenous dose of human apotransferrin in 
haematological stem cell transplant patient. Sahlstedt et al., Br J Haemato 2002; 119: 547-
553. 


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13. Effect of repeated apotransferrin administrations on serum iron parameters in 
patients undergoing myeloablative conditioning and allogenic stem cell transplantation. 
Parkkinen et al., Br J Haemato 2006; 135: 228-234. 
 
 


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3.1.9. EPOPROSTENOL, FLOLAN® 
Introduction 
At early stages of inflammation the endothelium modifies its normal homeostasis and may 
lose the ability to synthesize NO and prostacyclin (PGI2). As a result, vasoconstriction 
takes place besides a prothrombotic phenotype favoring the expression of tissue factor 
and triggering the coagulation response. Prostacyclin is synthesized by all endothelial 
cells and in particular in the liver. PGI2, derived from arachidonic acid is a well-known 
vasodilatator and exerts anti-platelet aggregation properties (1). Additionally, PGI2 
reduces leukocyte activation and adhesion. Furthermore, PGI2 regulates TNF-α synthesis 
and tissue factor expression in macrophages (1). Finally, PGI2 has cytoprotective effects 
and an antioxidant action. The underlying mechanism acts by a cyclic Adenosyl Mono-
Phosphate (cAMP) dependant pathway (1).  
The protective effects of the prostaglandin on liver necrosis were first reported by 
Stachura et al. (2). Since then, PGI2-mediated reduction of microcirculatory disorders and 
tissue injury has been proven as highly effective in various models of hepatic ischemia-
reperfusion (3-4). In clinic, PGI2 was originally chosen for lung preservation because it 
allowed a more even distribution of cold perfusion. Its use has then been extended to 
other organs. Klein et al. has demonstrated a significant impact of the PGI2 in 
preconditioning the graft prior to the LTx (5). Indeed, an injection of 500 µg PGI2 in the 
donor has shown an improvement of the IRI due to the prevention of thrombosis and 
vasospasm after cold storage. Moreover, Muhlbacher et al. (6) has demonstrated that 
pretreatment with PGI2 (200 µg) to organ donors has also an impact on the kidney. He 
observed a reduction of the number of post-transplant dialyses and hospital care days in 
patients undergoing renal transplantation. Finally, Pirenne et al. demonstrated that donor 
treatment with PGI2 for better flushing and preserving peribiliary vascular plexus and 
biliary mucosa offers protection from biliary strictures (7).  
Safety and adverse reaction 
Flolan®, the analogue of Epoprostenol is contraindicated in patients with known 
hypersensitivity to the drug and in patients with congestive heart failure arising from 
severe left ventricular dysfunction. Due to its vasodilatator effect, hypotension may occur 
during IV Flolan infusion. 
Epoprostenol is a potent inhibitor of platelet aggregation. Therefore, an increased risk for 
haemorrhagic complications should be considered. On the other hand, Epoprostenol is 
not considered as a conventional anticoagulant. 
Facial flushing, decreased in platelet count and tachycardia have been commonly 
reported. 
The cardiovascular effects during infusion disappear within 30 minutes of the end of 
administration. Effects on platelets have been found to disappear within 2 hours of 
discontinuing the infusion.  
Its metabolites are inactive and excreted in the urine. 
Patients on Digoxin have shown elevations of Digoxin concentrations after initiation of 
therapy with Epoprostenol. This may be clinically relevant in patients prone to Digoxin 
toxicity. Monitoring of Digoxin levels is therefore advisable until Digoxin levels are 
clinically stable in patients receiving treatment with Epoprostenol and Digoxin. 
 


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Contra-indication linked to the use of Flolan® 
Patient with a congestive heart failure arising from severe left ventricular dysfunction will 
be excluded from the treatment group. 
Product characteristics 
Flolan® is manufactured by GlaxoSmithKline. It is a sterile freeze-dried powder for 
solution for infusion. It requires special handling including a constant controlled 
temperature and protection from light. Pharmacokinetic studies have described a short 
half-life ranging from 3 to 5 minutes after the IV administration. 
Glycine and Mannitol are provided as excipient.  
Packaging and storage 
The commercial packing of Flolan® consists of: 
- 
A single 0.5 mg vial of freeze-dried powder in glass vials with synthetic butyl 
rubber plugs and aluminium collars. 
- 
Single vial of diluents. 
GlaxoSmithKline Glycine Buffer Diluent contains no preservative, consequently a vial 
should be used once only and then discarded. 
The shelf-life is about 3 years. Flolan® should be stored below 25°C (do not freeze) and 
the unopened vial in an outer carton to protect from light and moisture. When 
reconstituted with GlaxoSmithKline Glycine Buffer Diluent and diluted with physiological 
saline as instructed, freshly prepared Flolan® solutions should be used within 12 hours 
at 25°C. 
Dose and administration 
Flolan® is not to be used for bolus administration and it must be reconstituted only with 
the specific sterile GlaxoSmithKline Glycine Buffer Diluent.  
To avoid any side effects in the recipient, an ex-situ administration of Flolan® has been 
chosen. Flolan® will be administrated directly through the vena porta during the bench 
table. After reconstitution, 50 cc of Flolan® will be added to a liter of preservation 
solution. 500 cc will be flushed through both the vena porta. The IV dose of Flolan® has 
been determined according our clinical data. In case of DCD procedure with liver and lung 
procurement, we routinely use a preconditioning with 65 cc of Flolan®. 38 cc of 
reconstituted Flolan® are diluted in the lung cold perfusion whereas 7 cc are directly 
administrated through the pulmonary artery before the cooling of the lungs. Moreover 20 
cc are diluted in the abdominal cold pre-flush solution. 
 
References: 
1. Ischemia and reperfusion: from mechanism to translation. Eltzschig and Eckle, Nature 
medicine 2011; 11: 1391. 
2. Prostaglandin protection of carbon tetrachloride-induced liver cell necrosis in the rat. 
Stachura et al., Gastroenterology 1981; 81: 211-217. 
3. Hepatoprotection by a PGI2 analogue in complete warm ischemia of the pig liver. 
Prostanoid release from the reperfused liver. Kim et al., Transplantation 1994; 58: 875-
879. 
4. Efficacy of prostaglandine I2 analog on liver grafts subjected to 30 minutes of warm 
ischemia. Kishida et al., Surg Today 1997; 27: 1056-1060. 


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5. Preconditioning of donor livers with prostaglandin I2 before retrieval decreases 
hepatocellular ischemia-reperfusion injury. Klein et al., Transplantation 1999; 67: 1128-
1132. 
6. Improved renal graft function after prostacyclin pretreatment. Muhlbacher et al., 
Transplant Proc 1987; 19: 4162-4163. 
7. Biliary strictures after liver transplantation: risk factors and prevention by donor 
treatment with epoprostenol. Pirenne et al., Transplant Proc 2009; 41: 3399-3402. 
 
 
3.2 CONTRA-INDICATION TO USE THE COMBINED DRUG APPROACH 
3.2.1. ANTI-THROMBIN III (Atenativ®) 
Anti-thrombin III (Atenativ®) replacement during administration of heparin in 
therapeutic dosage increases the risk of bleeding. The effect of anti-thrombin III 
(Atenativ®) is enhanced by heparin. The half-life of anti-thrombin III (Atenativ®)  may 
be considerably decreased with concomitant heparin treatment due to accelerated anti-
thrombin III (Atenativ®) turnover (1.5 days).  
Hypersensitivity or allergic reactions (which may include angioedema, burning and 
stinging at the infusion site, chills, flushing, generalized urticaria, headache, hives, 
hypotension, lethargy, nausea, restlessness, tachycardia, tightness of the chest, tingling, 
vomiting, wheezing) have been observed infrequently in conjunction with the use of anti-
thrombin III (Atenativ®) concentrates and may in some cases progress to severe 
anaphylaxis (including shock). On rare occasions, fever has been observed. 
 Patient requiring therapeutic dose of heparin pre-operatively will be excluded 
from the treatment group. 
 History of hypersensitivity to anti-thrombin III (Atenativ®) and /or drugs with a 
similar chemical structure will be excluded from the treatment group. 
 
3.2.2. C1-INHIBITOR (Cetor®/Cinryze®) 
Hypersensitivity or allergic reactions (which may include angioedema, burning and 
stinging at the infusion site, chills, flushing, generalized urticaria, headache, hives, 
hypotension, lethargy, nausea, restlessness, tachycardia, tightness of the chest, tingling, 
vomiting, wheezing) could be observed with C1-inhibitor (Cetor®/Cinryze®) and may in 
some cases progress to severe anaphylaxis (including shock). 
 History of hypersensitivity to C1-inhibitor (Cetor®/Cinryze®) and /or drugs 
with a similar chemical structure will be excluded from the treatment group. 
 
3.2.3. ERYTHROPOÏETIN BETA (Neorecormon®) 
Safety data arising from clinical studies suggest that a high single IV dose of rhEPO 
(Neorecormon®) is safe regarding acute as well as chronic adverse effects including 
vascular thrombosis, seizure and raise in blood pressure, Hb levels, platelets and 
hematocrit. However, as patients with a history of seizure and a level of Hb higher than 
13 gr/dl were excluded in a few studies. 
 Patient with a pre-operative level of Hb higher than 13 g/dl will be excluded 
from the treatment group. 


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 Patient with a history of seizure, poorly controlled arterial hypertension, 
myocardial infarction or stroke in the month preceding the liver 
transplantation, and venous thromboembolic disease will be excluded from the 
treatment group. 
 Patients with unstable angina pectoris will be excluded from the treatment 
group. 
 History of hypersensitivity to rhEPO (Neorecormon®) and /or drugs with a 
similar chemical structure will be excluded from the treatment group. 
 
 
 
3.2.4. MELATONIN (Circadin®) 
Hypersensitivity or allergic reactions could be observed with melatonin (Circadin®). 
 History of hypersensitivity to melatonin (Circadin®) and /or drugs with a similar 
chemical structure will be excluded from the treatment group. 
 
 
3.2.5. GLUTATHIONE (Tationil®) 
There is no specific contraindication linked to the use glutathione (Tationil®) in the 
multifactorial modulation. 
Overall, Glutathione and precursor (acetylcysteine) appears to have a low toxicity in 
humans. Currently, there are no known side effects of GSH. None adverse reactions have 
been reported, neither in clinical studies nor in case reports. 
 
 
3.2.6. INFLIXIMAB (Remicade®) 
 Patient already exposed to Infliximab (Remicade®) will be excluded from the 
treatment group. 
 History of hypersensitivity to infliximab (Remicade®) and /or drugs with a 
similar chemical structure and/or murine proteins will be excluded from the 
treatment group. 
 Patients with severe infections such as sepsis, abcesses and opportunistic 
infections will be excluded from the treatment group. 
 Patients with severe heart failure will be excluded from the treatment group. 
 
 
3.2.7. TOCOPHEROL (vitamin e suspension 100 mg/mL®) 
Vitamin E has been reported to increase the risk of thrombosis in patients taking 
oestrogens. This finding has not been confirmed but should be borne in mind when 
selecting patients for treatment, in particular women taking oral contraceptives 
containing oestrogens. Moreover, Vitamin E at high intakes can affect the coagulation if 


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Vitamin K deficiency is also present. Clearly, Vitamin E must not be given in case of 
anticoagulant therapy. Alternatively, administration of Vitamin E can be accompanied by 
concomitant administration of Vitamin K. 
 Patient requiring an anticoagulation that functions as a Vitamin K antagonist 
preoperatively will be excluded from the treatment group. 
 History of hypersensitivity to tocopferol (vitamin e suspension 100 mg/mL®) 
and /or drugs with a similar chemical structure will be excluded from the 
treatment group. 
 
 
3.2.8. APOTRANSFERRIN 
There is no specific contraindication linked to the use Apotransferrin in the multifactorial 
modulation. 
 
3.2.9. EPOPROSTENOL (Flolan®) 
Due to its vasodilatator effect, hypotension may occur during IV epoprostenol infusion. 
Moreover, epoprostenol (Flolan®) is a potent inhibitor of platelet aggregation. Therefore, 
an increased risk for haemorrhagic complications should be considered.  
 Patient with a congestive heart failure arising from severe left ventricular 
dysfunction will be excluded from the treatment group. 
 Patient requiring therapeutic dose of heparin pre-operatively will be excluded 
from the treatment group. 
 History of hypersensitivity to epoprostenol (Flolan®) and /or drugs with a 
similar chemical structure will be excluded from the treatment group. 
 
 
 
 
 


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3.3 RATIONALE FOR THE CHOICE OF DOSES 
 
For each component of the multifactorial modulation approach, the lowest but still 
efficient and non-toxic dose has been chosen.  A detailed listing is proved for every 
component.  
3.3.1.  ANTI-THROMBIN III (Atenativ®) 
Anti-thrombin III (Atenativ®) has already been used safely in clinic. Hoffmann et al. has 
characterized a significant improvement of renal allograft reperfusion by a single-shot of 
anti-thrombin III (Atenativ®) (4000 IU) application during human kidney 
transplantation in a randomized controlled trial (1). Further, the same group has 
described a reduction of reperfusion injury pancreatitis and a prevention of graft 
thrombosis with a single-shot of anti-thrombin III (Atenativ®) (3000 IU) in human 
pancreas-kidney transplantation (2). At this recommended dose, Fertmann et al. (2) did 
not observe perioperative anti-thrombin III (Atenativ®)-mediated changes on standard 
coagulation tests (partial thromboplastin time, thromboplastin time, platelet counts), on 
the perioperative bleeding rates and on the number of packed red blood cell concentrates 
given to the patient.  Currently, a multi-centric protocol for clinical phase III trial testing 
pre-reperfusion anti-thrombin III (Atenativ®) (3000 UI) application in combination with 
acetylcysteine is ongoing for simultaneous kidney and pancreas transplantation. 
According these clinical data that have proven profound effect without toxicological 
events, a single IV dose of 3000 IU of ATIII (Atenavit®) has been chosen. 
 
References 
1. High dose antithrombine therapy reduces IRI and improves graft function during 
human allogenic kidney transplantation: final results of a randomized controlled 
clinical trial. Hoffmann et al.,Transplantation 2002; 74, 394. 
2. Single-shot antithrombin in human pancreas-kidney transplantation: reduction of 
reperfusion pancreatitis and prevention of graft thrombosis. Fertmann et al., 
Transplant International 2005; 18: 40. 
 
3.3.2. C1-INHIBITOR (Cetor®/Cinryze®) 
Besides the routine clinical practice use of C1-inhibitor (Cetor®/Cinryze®) in 
angioedema (1000 U), clinical trials have been performed to test the protective effect of 
C1-inhibitor (Cetor®) during ischemia reperfusion injury. Different doses were used. 
Struber et al.  focused on the decrease of vascular leakage syndrome after lung 
transplantation by using 26.900 U of  C1-inhibitor (Cetor®) (1). Bauernschmitt et al. 
(2000 U) (2), Thielmann et al. (4800 U)(3) and Fattouch et al. (1000 U) (4) have all 
described a significant positive impact of C1-inhibitor (Cetor®) on the cardiac function 
during coronary surgical reperfusion. Stieh et al. has observed the same findings with a 
post-operative administration of C1-inhibitor (Cetor®) at the dose of 56000 U in open 
heart surgery. Toxicity of C1-inhibitor (Cetor®/Cinryze®) is very low. No side effects 
were observed in those clinical trials (1, 2, 3, 4). No complications are expected in patients 
either with a liver dysfunction or during the liver transplantation. 
According these clinical data that have proven profound effect without toxicological 
events, a single IV dose of 1000 U of C1-inhibitor (Cetor®/Cinryze®) has been chosen. 
 


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References 
1. C1-esterase inhibitor in graft failure after lung transplantation. Struber et al., 
Intensive care med 1999; 25: 1315-1318. 
2. Rescue therapy with C1-esterase inhibitor concentrate after emergency coronary 
surgery for failed PTCA. Bauernschmitt et al., Intensive care medicine 1998; 24: 635-
638. 
3. Administration of C1-esterase inhibitor during emergency coronary artery bypass 
surgery in acute ST-elevation myocardial infarction. Thielmann et al., Eur J Cardio 
Thorac Surg 2006; 285-293. 
4. Beneficial effects of C1-esterase inhibitor in ST-elevation myocardial infarction in 
patients who underwent surgical reperfusion: a randomized double-blind study. 
Fattouch et al., Eur J Cariothorac Surg 2007; 32: 326. 
 
3.3.3. RECOMBINANT HUMAN ERYTHROPOÏETIN BETA (Neorecormon®) 
A growing number of clinical studies are testing the tissue protection effect afforded by 
rhEPO beta (Neorecormon®) in various acute clinical settings, including neuroprotection 
and cardioprotection. Ehrenreich et al (1) has used EPO beta at a total dose of 100.000 IU 
for the first 3 days after an acute cerebral stroke, noting that this was safe and well 
tolerated. Lipsic et al (2) has demonstrated that a single bolus injection of EPO at a dose 
of 60.000 IU in patient with an acute myocardial infarction was not associated to any 
significant increase in hemoglobin (Hb). Mocini et al (3) has described that a single 
injection of EPO at a dose of 40.000 IU before cardiac surgery has no significant impact on 
the cardiac function and on the erythropoiesis. Belonje et al (4) has demonstrated in a 
safety pilot assessment that 60.000 IU of EPO in patients with an acute myocardial 
infarction was not associated with a raise in blood pressure, an increase in Hb and 
thrombocytes or any adverse events like vascular thrombosis or seizure. In elective liver 
surgery, Mosato Kato et al (5) has indicated that a two time injection of 30.000 IU EPO 
rather than one single bolus of 60.000 IU has a stronger inhibitory effect on IRI following 
the pringle maneuver during liver surgery. Moreover, these doses and their timing of 
administration did not affect blood cell count, hematocrit and platelets. 
Considering the results of clinical studies in different organs, it is suggested to adapt the 
dose of EPO and its timing of administration in order to reach an optimal protective effect. 
This was confirmed by Ramakrishnan et al. (6). They have described the importance of 
doses, dosage regimens and routes of administration in physiologically mechanistic 
PK/PD model. Pharmacokinetic investigations in healthy volunteers show that the half-
life of IV administrated EPO beta is between 3 and 12 hours. 
Notably, the receptors mediating the non-erythropoietic effects of EPO differ from the one 
responsible for hematopoiesis. The tissue-protective receptor exhibits a lower affinity for 
EPO. It is a heteromer consisting of EPO receptor monomers in association with the 
common receptor. This heteromeric receptor is expressed immediately following injury, 
whereas EPO production is delayed providing a window of opportunity for therapeutic 
intervention. Thus early administration of EPO can dramatically reduce the deleterious 
components of the local inflammatory cascade. However, a larger dose is required to 
trigger the protective effect as the affinity for the heteromer is lower than the 
hematopoietic receptor. Thus, to effectively treat injured tissue using exogenous EPO, 
high parenteral doses are required (7).  
Considering the distribution profile after IV injection, the half-life of EPO-beta (3-12h), 
the potential elimination of the administrated medication by blood loss during the 


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procedure, the need to keep a high concentration for the post-reperfusion phase and the 
absence of toxicological events, a first dose of 30.000 IU will be administrated 13-15 
minutes before the reperfusion of the liver over 2 minutes followed by a second dose of 
30.000 IU of EPO-beta 6 hours after the reperfusion. 
 
References 
1. Ehrenreich et al, Molecular medicine 2002; 8: 495-505. EPO therapy for acute 
stroke is both safe and beneficial. 
2. Lipsic et al, J Am Coll Cardiol 2006; 48: 2161-2167. Protective effect of EPO in 
cardiac ischemia: from bench to bedside. 
3. Mocini et al, Perfusion 2008; 23: 187-192. Endogenous EPO and a single bolus of 
40.000 IU do not protect the heart from IRI during extracorporeal circulation for 
cardiac surgery. 
4. Belonje et al, Am Heart J 2008; 155: 817-822. Effects of EPO after an acute 
myocardial infarction: Rationale and study design of a prospective, randomized, 
clinical trial. 
5. Mosato Kato et al, World Journal of Gastroenterology 2010; 16: 4838-4845. EPO 
ameliorates early ischemia-reperfusion injury following the pringle maneuver. 
6. Ramakrishnan et al, J Clin Pharmacol 2004; 44: 991-1002. PK and PD modeling of 
recombinant human EPO after a single and multiple doses in healthy volunteers. 
7. Brines et al, Blood Purif 2010; 29: 86-92. The therapeutic potential of EPO for tissue 
protection: a tale of two receptors. 
 
3.3.4. MELATONIN (Circadin®) 
 
The antioxidative effects of melatonin require concentrations that are much higher than 
endogenously produced plasma concentrations. On the other hand, a very low 
concentration of exogenous Melatonin (> 0.3 mg) produces already supra-physiological 
levels in humans. Cost and toxicity of melatonin melatonin is remarkably low. In the 
United States, the Food and Drug Administration (FDA) has designated melatonin as an 
orphan product sold as a dietetic complement. No significant adverse effects were 
observed following administration of high doses (3-6.6 g/day) in humans for 15 to 35 
days (1). High doses of oral melatonin have been used with success in babies with sepsis 
syndromes without toxicological effect (20 mg) (2). IV infusion in doses between 10 and 
60 mg during abdominal aortic-aneurysm repair has already been shown to be without 
immediate toxic reaction (3). In this study, the oral dose of melatonin (Circadin®) has 
been determined according the clinical data that have proven profound effect without 
toxicological events. Thus 6 mg of melatonin (Circadin®) will be administrated orally at 
the ward just before transfer to the operative theatre. This time course is adequate 
according the pharmacokinetic data of melatonin after oral administration (4). 
 
References 
1. Melatonin and Parkinsonism. Papavasilou et al., JAMA 1972, 221: 88-89. 
2. Effects of melatonin treatment in septic newborns. Gitto et al., Ped Res 2001, 50: 
756-760. 
3. Utility of melatonin to treat surgical stress after major vascular surgery – a safety 
study. Kucukakin et al., J Pineal Res 2008, 44:426-431. 


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4. Melatonin pharmacokinetics following two different oral surge-sustained release 
doses in older adults. Gooneratne N et al., J Pineal Res 2012; 52: 437-445. 
 
 
3.3.5. GLUTATHIONE (Tationil®) 
 
In humans, recent investigations have proven the therapeutic potential of glutathione, in 
particular since it has a low toxicity in humans (1) and it is cost-effective. Chawla et al. has 
described a lower concentration of GSH in the plasma of cirrhotic patients (2). Thus, any 
intervention increasing hepatic or plasma glutathione levels should convey protection 
against reperfusion injury (3). 
Overall, glutathione and precursor (acetylcysteine) appears to have a low toxicity in 
humans. Currently, there are no known side effects of glutathione (1). None adverse 
reactions have been reported, neither in clinical studies nor in case reports. 
It must be noted that the best dosage of glutathione has not been clearly established for 
any use; most reported dose schedules have been empirically chosen based on animal and 
clinical data. Applied IV concentrations vary from 2.0 to 4.8 gram in total for an adult. For 
example, glutathione has been reported in randomized studies to reduce the incidence of 
neurotoxicity induced by chemotherapy at a dose of 1.5 g/m2 (4, 5, 6). IV glutathione is 
also used to slow down the progression of Parkinson’s disease. Most patients are given 
1400 mg along with saline for ten minutes, three times a week. It has been shown to have 
a critical function as elimination of toxic compounds and by restoring the hepatic 
glutathione stores in case of acetaminophen intoxication. In that case, an approved 20 
hour IV protocol for acetylcysteine –precursor of glutathione- treatment has been used in 
the UK since 1970s. This treatment protocol provides a total of 300 mg/Kg over 20 hours 
with an initial loading dose of 150 mg/kg IV over 15 to 60 minutes. This treatment period 
is often extended when patients have large ingestions of acetaminophen or elevated 
serum transaminase (7). 
In this study, the IV dose of glutathione has been determined according the experimental 
and clinical data that have proven profound effect without toxicological events. According 
the short half-life, 3 gr will be administrated 2-4 minutes just before the reperfusion. 
 
References 
1. High-dose intravenous glutathione in man. Pharmacokinetics and effects on 
cysteine in plasma and urine. Aebi et al., Eur J Invest 1991; 21: 103-110. 
2. Plasma cysteine, cystine, and glutathione in cirrhosis. Chawla et al., 
Gastroenterology 1984; 87: 770. 
3. Plasma membrane and mitochondrial transport of hepatic reduced glutathione. 
Fernández-Checa JCet al. Semin Liver Dis. 1996; 16:147–158. 
4. Glutathione protects the rat liver against reperfusion injury after hypothermic 
preservation. Bilzer M et al., Gastroenterology. 1999; 117:200–210.  
5. Beneficial effects of extracellular glutathione against endotoxin-induced liver 
injury during ischemia and reperfusion. Liu P et al., Circ Shock. 1994;43:64–70. 
6. Neuroprotective effect of reduced glutathione on cisplatin-based chemotherapy in 
advanced gastric cancer: a randomized double-blind placebo-controlled trial. 
Cascinu et al., J Clin Oncol 1995; 13: 26-32. 


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7. Treatment of paracetamol (acetaminophen) poisoning with N-acetylcysteine. 
Prescott LF, Park J, Ballantyne A, Adriaenssens P, Proudfoot AT. Lancet. 
1977;2(8035):432 
 
3.3.6. INFLIXIMAB (Remicade®) 
 
Infliximab (Remicade®) is well established in the treatment of a few inflammatory 
diseases: rheumatoid arthritis (I.V. 3 mg/kg at 0, 2, and 6 weeks, followed by 3 mg/kg 
every 8 weeks thereafter; doses have ranged from 3-10 mg/kg repeated at 4- to 8-week 
intervals), psoriatric arthritis (I.V.: 5 mg/kg at 0,2, and 6 weeks, followed by 5 mg/kg 
every 8 weeks thereafter), ankylosing spondylitis (I.V.: 5 mg/kg at 0, 2, and 6 weeks, 
followed by 5 mg/kg every 6 weeks thereafter), inflammatory bowel diseases (I.V.: 5 
mg/kg at 0, 2, and 6 weeks, followed by 5 mg/kg every 8 weeks thereafter; dose may be 
increased to 10 mg/kg in patients who respond but then lose their response),  and several 
studies have demonstrated a high efficacy with rapid overall patient improvement, 
maintenance treatment, and decrease in rates of hospitalizations and surgical 
interventions (1). Infliximab (Remicade®) has been also studied extensively in the 
treatment of patients with sepsis (2, 3). These studies have revealed that anti-cytokine 
administration usually as a single dose is safe but not particularly efficacious in the 
management of sepsis.  
Furthermore, clinical experience with TNF- inhibitors after solid organ transplantation 
has revealed good results in terms of safety, reduction in toxicity and allograft survival 
(4). In some case reports, infliximab (Remicade®)  has been shown to be a therapeutic 
option for refractory acute rejection after intestinal transplantation (5). Currently in 
Leuven, infliximab (Remicade®)  at the dose of 3 mg/Kg is infused in patient during 
intestine transplantation at the same timing that foreseen in this protocol. 
In this study, the IV dose of Infliximab has been determined according to the experimental 
and clinical data that have proven profound effect without toxicological events (2, 3, 4). 
Thus, 3 mg/kg of Infliximab will be administrated during 3 hours from the beginning of 
the anhepatic phase after administration of Antithrombin III. 
 
 
 
References 
1.Long-term outcome of treatment with infliximab in 614 patients with Crohn’s 
disease: results from a single-center cohort. Schnitzler et al., Gut 2009; 58: 492-500. 
2.Hepatotoxicity of immunomodulating agents and the transplant situation. 
Kaplowitz et al., Drug-induced liver disease 2007; 662-681. 
3.Review article: the role of tumor necrosis factor in renal ischemia-reperfusion 
injury. Donnahoo et al., The Journal of Urology 1999, 162: 196-203. 
4.New monoclonal antibodies in renal transplantation. Vincenti et al., Minerva Urol 
Nefrol 2003; 55: 57-66. 
5.Intestinal transplantation: evolution in immunosuppression protocols. Pirenne et 
al., Curr Opin Organ Transplant 2009; 14: 250-255. 
 
3.3.7. α-TOCOPHEROL (vitamin E suspension 100 mg/ml®) 
 


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This major antioxidant vitamin has excellent safety data in doses that far exceed the 
doses required to reach adequate antioxidant protection in vivo. Regarding controlled, 
double-blinded studies of vitamin E toxicity in humans, several reports confirm that 
vitamin E has very low toxicity and no consistent adverse events have been reported (1, 
2). Ferslew et al. (3) described that α-tocopherol first appeared in the plasma in 2-4 
hours after oral administration and peaked at 5-14 hours when administering 800 mg α-
Tocopherol to healthy volunteers. This study was not associated with any adverse 
events. 
Lassnig et al. (2) have studied multiple parenteral vitamin E infusion in patients 
undergoing elective cardiac surgery. A total dose of 1080 mg of vitamin E was 
administered at four points in time: two infusions before the start of surgery and one 
infusion each on days 1 and 2 after surgery. Based on pharmacokinetic data the dose was 
considered sufficient to maintain Vitamin E concentrations during surgery. Since no 
protective effect of Vitamin E supplementation was observed, Lassnigg et al. suggested 
that the supply of Vitamin E was inadequate and/or that not enough Vitamin E was 
incorporated into the target cells. Based on these findings and investigations, Bartels et 
al. (4) decided to administer 1620 mg the day before the liver surgery and compared to a 
placebo group the extent of IRI. After three infusions of vitamin E, there was a significant 
fivefold increase in the plasma concentration; half of this increase remained about 12 h 
later, prior to surgery. After reperfusion, the vitamin E concentration was significantly 
decreased in both groups. It is likely that, due to severe oxidative stress, the need for and 
consumption of, α-Tocopherol was great. Six days after surgery the treated group still had 
plasma vitamin E concentrations comparable to the baseline value, whereas the placebo 
group still had significantly reduced plasma vitamin E concentrations. They have shown 
that the preoperative parenteral administration of 1620 mg of α-Tocopherol was safe, not 
only leads to a marked increase of plasma concentration but also prevents vitamin E 
depletion after IRI.  
Currently, no solutions containing Vitamin E alone are available for human use. Therefore, 
5 mL of Vitamin E suspension® (500 mg) will be administrated orally at the ward before 
transport to the operative theatre. This time course is adequate according the 
pharmacokinetic data of Vitamin E after oral administration. 
References 
1. Safety of antioxidant vitamins and B-carotene. Diplock et al., Am J Clin Nutr 1995; 
62: 1510-1516. 
2. Influence of intravenous vitamin E supplementation in cardiac surgery on 
oxidative stress: a double-blinded, randomized, controlled study. Br J Anaesth 2003; 
90: 148-154. 
3. Pharmacokinetics and bioavailability of the RRR and all racemic stereoisomers of 
alpha-tocopherol in humans after single oral administration. Ferslew et al., J Clin 
Pharmacol 1993; 33: 84-88. 
4. Pilot study on the effect of parenteral vitamin E on ischemia and reperfusion 
induced  liver injury: a double-blind, randomized, placebo-controlled trial. Bartels et 
al., Clin Nutrit 2004; 23: 1360-1370. 
 
3.3.8. APOTRANSFERRIN  
 
Apotransferrin is purified from human plasma. Sahlstedt et al. (1) have demonstrated that 
a single dose of Apotransferrin (100 mg/kg) was effective and safe on the serum iron 


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binding capacity. Parkkinen et al. have described in a dose-finding study the highest dose 
level for the prevention of appearance of NTBI in patients with myeloablation. A total dose 
of 1040 mg/kg (115 mg/Kg/day for 9 days) was given to the patients without any related 
serious adverse event (2). 
In this study, the IV dose of apotransferrin has been determined according to the 
experimental and clinical data that have proven profound effect without toxicological 
events (1, 2). Thus, 170 mg/Kg of Apotransferrin® will be infused during 3 hours from 
the beginning of the anhepatic phase. 
 
References 
1. Effective binding of free iron by a single intravenous dose of human apotransferrin 
in haematological stem cell transplant patient. Sahlstedt et al., Br J Haemato 2002; 
119: 547-553. 
2. Effect of repeated apotransferrin administrations on serum iron parameters in 
patients undergoing myeloablative conditioning and allogenic stem cell 
transplantation. Parkkinen et al., Br J Haemato 2006; 135: 228-234. 
 
 
3.3.9. EPOPROSTENOL (Flolan®) 
 
In clinic, recent investigations have proven the therapeutic potential of epoprostenol 
(Flolan®) in transplantation.  Epoprostenol (Flolan®) was originally chosen for lung 
preservation because it allowed a more even distribution of cold perfusion during the 
cooling of organs. Its use has then been extended to other organs. Klein et al. has 
demonstrated a significant impact of the epoprostenol (Flolan®) in preconditioning the 
graft prior to the LTx (1). Indeed, an injection of 500 µg PGI2 in the donor has shown an 
improvement of the IRI due to the prevention of thrombosis and vasospasm after cold 
storage. Moreover, Muhlbacher et al. (2) has demonstrated that pretreatment with 
epoprostenol (Flolan®) (200 µg) to organ donors has also an impact on the kidney. He 
observed a reduction of the number of post-transplant dialyses and hospital care days in 
patients undergoing renal transplantation. Finally, Pirenne et al. demonstrated that donor 
treatment with epoprostenol (Flolan®) for better flushing and preserving peribiliary 
vascular plexus and biliary mucosa offers protection from biliary strictures (3).  
To avoid any side effects in the recipient, an ex-situ administration of epoprostenol 
(Flolan®) has been chosen. Epoprostenol (Flolan®) will be administrated directly 
through the vena porta during the bench table (ex-situ). By doing so, no systemic 
absorption at the reperfusion is expected in the recipient. After reconstitution, 50 cc of 
epoprostenol (Flolan®) will be added to a liter of preservation solution. 500 cc will be 
flushed through the vena porta. The IV dose of epoprostenol (Flolan®) has been 
determined according our clinical data. In case of DCD procedure with liver and lung 
procurement, we routinely use a preconditioning with 65 cc of Flolan®. 38 cc of 
reconstituted Flolan® are diluted in the lung cold perfusion whereas 7 cc are directly 
administrated through the pulmonary artery before the cooling of the lungs. Moreover 20 
cc are diluted in the abdominal cold pre-flush solution. 
 
References 


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1. Preconditioning of donor livers with prostaglandin I2 before retrieval decreases 
hepatocellular ischemia-reperfusion injury. Klein et al., Transplantation 1999; 67: 
1128-1132. 
2. Improved renal graft function after prostacyclin pretreatment. Muhlbacher et al., 
Transplant Proc 1987; 19: 4162-4163. 
3. Biliary strictures after liver transplantation: risk factors and prevention by donor 
treatment with epoprostenol. Pirenne et al., Transplant Proc 2009; 41: 3399-3402. 
 
 
 
 
 


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APPENDIX 4: SCHEDULE OF EVENTS 
 
 
Before 
TX 
Baseline 
day 
of 
TX 
immediately 
before 
hepatectomy 
immediately 
before 
reperfusion 
30-60-120 
minutes after 
reperfusion  
6-12-24-
48-72 
hours after 
reperfusion 
Day 1 to 7 
Day 14 
Month 3 
Month 12 
Informed 
consent 
X 
 
 
 
 
 
 
 
 
 
Recipient 
details 
 
X 
 
 
 
 
 
 
 
 
Medical 
history 
 
X 
 
 
 
 
 
 
 
 
Lab 
MELD 
score 
 
X 
 
 
 
 
 
 
 
 
Donor details 
 
X 
 
 
 
 
 
 
 
 
Surgical 
procurement 
details 
 
X 
 
 
 
 
 
 
 
 
Plasma sample 
(AST) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Liver biopsy 
 
 
 
X 
(before 
implantation) 
X (1 hr after 
reperfusion) 
 
X (1 week 
after 
reperfusion) 
 
 
 
EDTA/serum 
samples 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Urine samples 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Data collection 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
Tacrolimus 
trough levels 
 
 
 
 
 
 
X 
X 
X 
X 
Adverse events 
monitoring (on 
going) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Surgical 
complications 
monitoring (on 
going) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX 5:  ETHICAL PRINCIPLES FOR MEDICAL RESEARCH INVOLVING 
HUMAN SUBJECTS 
 
 
 
Adopted by the 18th World Medical Assembly 
Helsinki, Finland, June 1964 
and amended by the 
29th World Medical Assembly, Tokyo, Japan, October 1975 
35th World Medical Assembly, Venice, Italy, October 1983 
41st World Medical Assembly, Hong Kong, September 1989 
48th General Assembly, Somerset West, Republic of South Africa, October 1996 
and the 
52nd WMA General Assembly, Edinburgh, Scotland, October 2000 
 
1. Introduction 
 
1. The World Medical Association has developed the Declaration of Helsinki as a statement 
of ethical principles to provide guidance to physicians and other participants in medical 
research involving human subjects. Medical research involving human subjects includes 
research on identifiable human material or identifiable data. 
 
2. It is the duty of the physician to promote and safeguard the health of the people. The 
physician’s knowledge and conscience are dedicated to the fulfillment of this duty. 
 
3. The Declaration of Geneva of the World Medical Association binds the physician with 
the words, "The health of my patient will be my first consideration," and the International 
Code of Medical Ethics declares that, "A physician shall act only in the patient's interest 
when providing medical care which might have the effect of weakening the physical and 
mental condition of the patient." 
 
4. Medical progress is based on research which ultimately must rest in part on 
experimentation involving human subjects. 
 
5. In medical research on human subjects, considerations related to the well-being of the 
human subject should take precedence over the interests of science and society. 
 
6. The primary purpose of medical research involving human subjects is to improve 
prophylactic, diagnostic, and therapeutic procedures and the understanding of the 
etiology and pathogenesis of disease. Even the best proven prophylactic, diagnostic and 
therapeutic methods must continuously be challenged through research for 
their effectiveness, efficiency, accessibility, and quality. 
 
7. In current medical practice and in medical research, most prophylactic, diagnostic and 
therapeutic procedures involve risks and burdens. 
8. Medical research is subject to ethical standards that promote respect for all human 
beings and protect their health and rights. Some research populations are vulnerable and 
need special protection. The particular needs of the economically and medically 


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disadvantaged must be recognized. Special attention is also required for those who cannot 
give or refuse consent for themselves, for those who may be subject to giving consent 
under duress, for those who will not benefit personally from the research and for those 
for whom the research is combined with care. 
 
9. Research Investigators should be aware of the ethical, legal, and regulatory 
requirements for research on human subjects in their own countries as well as applicable 
international requirements. No national ethical, legal or regulatory requirement should 
be allowed to reduce or eliminate any of the protections for human subjects set forth in 
this Declaration. 
 
2. Basic principles for all medical research  
 
10. It is the duty of the physician in medical research to protect the life, health, privacy 
and dignity of the human subject. 
 
11. Medical research involving human subjects must conform to generally accepted 
scientific principles, be based on a thorough knowledge of the scientific literature, other 
relevant sources of information, and on adequate laboratory and where appropriate, 
animal experimentation. 
 
12. Appropriate caution must be exercised in the conduct of research which may affect 
the environment and the welfare of animals used for research must be respected. 
 
13. The design and performance of each experimental procedure involving human 
subjects should be clearly formulated in an experimental protocol. This protocol should 
be submitted for consideration, comment, guidance, and where appropriate, approval to 
a specially appointed ethical review committee, which must be independent of the 
investigator, the sponsor or any other kind of undue influence. This independent 
committee should be in conformity with the laws and regulations of the country in which 
the research experiment is performed. The committee has the right to monitor ongoing 
trials. The researcher has the obligation to provide monitoring information to the 
committee, especially any serious adverse events. The researcher should also submit to 
the committee, for review, information regarding funding, sponsors, institutional 
affiliations, other potential conflicts of interest and incentives for subjects. 
 
14. The research protocol should always contain a statement of the ethical considerations 
involved and should indicate that there is compliance with the principles enunciated in 
this Declaration. 
 
15. Medical research involving human subjects should be conducted only by scientifically 
qualified persons and under the supervision of a clinically competent medical person. The 
responsibility for the human subject must always rest with a medically qualified person 
and never rest on the subject of the research, even though the subject has given consent. 
 
16. Every medical research project involving human subjects should be preceded by 
careful assessment of predictable risks and burdens in comparison with foreseeable 


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benefits to the subject or to others. This does not preclude the participation of healthy 
volunteers in medical research. The design of all studies should be publicly available. 
 
17. Physicians should abstain from engaging in research projects involving human 
subjects unless they are confident that the risks involved have been adequately assessed 
and can be satisfactorily managed. Physicians should cease any investigation if the risks 
are found to outweigh the potential benefits or if there is conclusive proof of positive and 
beneficial results. 
 
18. Medical research involving human subjects should only be conducted if the 
importance of the objective outweighs the inherent risks and burdens to the subject. This 
is especially important when the human subjects are healthy volunteers. 
 
19. Medical research is only justified if there is a reasonable likelihood that the 
populations in which the research is carried out stand to benefit from the results of the 
research. 
 
20. The subjects must be volunteers and informed participants in the research project. 
 
21. The right of research subjects to safeguard their integrity must always be respected. 
Every precaution should be taken to respect the privacy of the subject, the confidentiality 
of the patient’s information and to minimize the impact of the study on the subject's 
physical and mental integrity and on the personality of the subject. 
 
22. In any research on human beings, each potential subject must be adequately informed 
of the aims, methods, sources of funding, any possible conflicts of interest, institutional 
affiliations of the researcher, the anticipated benefits and potential risks of the study, and 
the discomfort it may entail. The subject should be informed of the right to abstain from 
participation in the study or to withdraw consent to participate at any time without 
reprisal. 
After ensuring that the subject has understood the information, the physician should then 
obtain the subject's freely-given informed consent, preferably in writing. If the consent 
cannot be obtained in writing, the non-written consent must be formally documented and 
witnessed. 
 
23. When obtaining informed consent for the research project the physician should be 
particularly cautious if the subject is in a dependent relationship with the physician or 
may consent under duress. In that case the informed consent should be obtained by a 
well-informed physician who is not engaged in the investigation and who is completely 
independent of this relationship. 
 
24. For a research subject who is legally incompetent, physically or mentally incapable  of 
giving consent or is a legally incompetent minor, the investigator must obtain informed 
consent from the legally authorized representative in accordance with applicable law. 
These groups should not be included in research unless the research is necessary to 
promote the health of the population represented and this research cannot instead be 
performed on legally competent persons. 
 


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25. When a subject deemed legally incompetent, such as a minor child, is able to give 
assent to decisions about participation in research, the investigator must obtain that 
assent in addition to the consent of the legally authorized representative. 
 
26. Research on individuals from whom it is not possible to obtain consent, including 
proxy or advance consent, should be done only if the physical/mental condition that 
prevents obtaining informed consent is a necessary characteristic of the research 
population. The specific reasons for involving research subjects with a condition that 
renders them unable to give informed consent should be stated in the experimental 
protocol for consideration and approval of the review committee. The protocol should 
state that consent to remain in the research should be obtained as soon as possible from 
the individual or a legally authorized surrogate. 
 
27. Both authors and publishers have ethical obligations. In publication of the results of 
research, the investigators are obliged to preserve the accuracy of the results. Negative as 
well as positive results should be published or otherwise publicly available. Sources of 
funding, institutional affiliations and any possible conflicts of interest should be declared 
in the publication. Reports of experimentation not in accordance with the principles laid 
down in this Declaration should not be accepted for publication. 
 
3. Additional principles for medical research combined with medical  
 
28. The physician may combine medical research with medical care, only to the extent 
that the research is justified by its potential prophylactic, diagnostic or therapeutic value. 
When medical research is combined with medical care, additional standards apply to 
protect the patients who are research subjects. 
 
29. The benefits, risks, burdens, and effectiveness of a new method should be tested 
against those of the best current prophylactic, diagnostic, and therapeutic methods. This 
does not exclude the use of placebo, or no treatment, in studies where no proven 
prophylactic, diagnostic or therapeutic method exists. 
 
30. At the conclusion of the study, every patient entered into the study should be assured 
of access to the best proven prophylactic, diagnostic, and therapeutic methods identified 
by the study. 
 
31. The physician should fully inform the patient which aspects of the care are related to 
the research. The refusal of a patient to participate in a study must never interfere with 
the patient-physician relationship. 
 
32. In the treatment of a patient, where proven prophylactic, diagnostic, and therapeutic 
methods do not exist or have been ineffective, the physician, with informed consent from 
the patient, must be free to use unproven or new prophylactic, diagnostic and therapeutic 
measures, if in the physician’s judgment it offers hope of saving life, reestablishing health 
or alleviating suffering. Where possible, these measures should be made the object of 
research, designed to evaluate their safety and efficacy. In all cases, new information 
should be recorded and, where appropriate, published. The other relevant guidelines of 
this Declaration should be followed. 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 118 
 
APPENDIX 6: INFORMED CONSENT 
 
Toestemmingsformulier  
 
Toediening van geneesmiddelen om  leverbeschadiging  tijdens 
transplantatie te verminderen 
 
Protocol Nr:  
 
Sponsor: UZ Leuven, Herestraat 49, 3000 Leuven 
 
Dit formulier is enkel bestemd voor de patiënt of zijn/haar wettelijke 
vertegenwoordiger. 
 
Ik, ..............................................................................................................., bevestig dat ik geïnformeerd 
ben over de klinische studie en dat ik een kopie van de ‘Patiëntenbrochure’ en het 
‘Toestemmingsformulier’ ontvangen heb. Ik heb deze informatie gelezen en begrepen. 
Mijn dokter heeft me voldoende informatie gegeven betreffende de voorwaarden, de duur 
van de studie, het effect en de bijwerkingen van de behandeling. Daarbij heb ik voldoende 
tijd gehad om deze informatie te overwegen en vragen te stellen waarop ik voldoende en 
begrijpbare antwoorden heb gekregen. 
Ik aanvaard eveneens dat weefsel-, bloed- en urinestalen, afgenomen gedurende het 
volledige verloop van de studie (vóór, tijdens en na de transplantatie), gebruikt worden 
voor wetenschappelijke doeleinden. 
Ik heb begrepen dat ik op ieder moment kan stoppen met mijn deelname aan deze studie, 
nadat ik mijn dokter hierover heb geïnformeerd en dat deze beslissing geen enkel nadeel 
zal hebben voor mij. 
Ik geef toestemming aan de verantwoordelijke onderzoekers van de studie en de 
regulerende instanties om toegang te hebben tot mijn medische gegevens. Mijn medische 
gegevens zullen strikt persoonlijk behandeld worden. Ik ben mij bewust dat mijn 
medische gegevens zullen worden verzameld, verwerkt en gebruikt binnen de context 
van deze studie. 
Ik ga akkoord bij het gebruik van mijn medische data door de onderzoeker voor andere 
onderzoeksdoelstellingen. 
Ik geef vrijwillig mijn toestemming om deel te nemen aan deze studie en mee te werken 
in alle vereiste onderzoeken. Ik ben bereid om informatie te geven omtrent mijn medische 
voorgeschiedenis, gebruik van medicijnen en deelname aan andere studies indien van 
toepassing. 
Ik ga akkoord dat alle zorgverleners, die betrokken zijn in deze studie, ingelicht zullen zijn 
over mijn deelname aan deze studie. 
 
Ik geef toestemming voor deelname aan het onderzoek. 
 
Achternaam en voornaam: 
 
Geboortedatum: 
 
Handtekening: 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 119 
 
 
Datum: 
 
 
Ondergetekende verklaart dat de hierboven genoemde persoon zowel mondeling als 
schriftelijk over het bovenvermelde onderzoek is geïnformeerd.  
 
Naam: 
 
Functie: 
 
Handtekening: 
 
Datum: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 120 
 
Consent form 
 
Combined drug approach to Prevent Liver damage during 
Transplantation  
 
Protocol Nr:  
 
Sponsor: UZ Leuven, Herestraat 49, 3000 Leuven 
 
Part which is destined only to the patient or his/her legal representative 
 
I, …………………………………………………………………………………………………………… confirm that I 
have been informed about the clinical study and that I have received a copy of the patient 
information sheet and the consent form. I have read and understood the information. My 
doctor has given me sufficient information concerning the conditions, the length of the 
study, the effect and the side effects of this treatment. In addition, I have received 
sufficient time to consider the information and to ask questions to which I have received 
satisfying answers. 
I accept that blood, urine and tissue samples that will be taken during the whole study 
(before, during and after the transplantation) will be used for scientific purposes. 
I have understood that I can stop my participation in this study at any time after having 
informed my doctor about this and that this decision will not cause any disadvantage. 
I authorize the investigators of the study and the regulating authorities to have access to 
my medical records. My medical data will be treated strictly confidential. I am aware of 
the purpose for which these data are collected, processed and used in the context of the 
study. I agree with the collection, the processing and the use of these medical data, as 
described in the patient information sheet.  
I agree with the use by the investigator of these coded medical data for other research 
purposes. 
I consent voluntarily to participate in this study and to cooperate in all the examinations 
requested. I am willing to give information concerning my medical history, use of 
medication(s) and participation in other studies if any. 
I agree that all the healthcare professionals involved in my treatment are informed about 
my participation in this study. 
 
I give permission for participation in the study. 
Last en first name : 
Date of birth : 
Signature : 
Date : 
Undersigned declares that the person above is informed oral and by letter about the study.  
Name : 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 121 
 
Function : 
Signature : 
Date : 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 122 
 
Consentement éclairé 
 
Réduction des lésions du foie en transplantation après administration 
séquentielle de médicaments (CAPITL)  
 
Protocole Nr:  
 
Promoteur: UZ Leuven, Herestraat 49, 3000 Leuven 
 
Partie réservée au patient ou à son représentant légal  
 
Je soussigné, ……………………………………………………………………………………………………………  
 
certifie avoir été informé au sujet de cette étude et avoir reçu une copie de la brochure 
informative et du présent consentement éclairé. J’ai lu et compris tous les tenant et 
aboutissant. Mon médecin m’a donné suffisamment d’informations requises pour une 
bonne compréhension des conditions de l’étude, de l’action et des potentiels effets 
secondaires des médicaments. En outre, j’ai bénéficié de suffisamment de temps 
nécessaire à la compréhension de ces informations. J’assure que les réponses concernant 
mes questions étaient adaptées et complètes pour ma compréhension.  
J’accepte par le présent consentement que des échantillons de sang, d’urine et de tissue 
soient prélevés à des fins purement scientifiques avant, durant et après la transplantation 
de  foie.  
J’ai compris que je peux à tout moment refuser de continuer de participer à l’étude après 
en avoir averti le médecin et ce sans le moindre inconvénient ou disparité pour le suivi de 
la greffe.  
J’autorise les investigateurs et les autorités compétentes à pouvoir accéder, de façon 
strictement confidentielle à mon dossier médicales. J’approuve l’analyse de ces 
informations personnelles dans le contexte de l’étude comme décrit dans la brochure 
informative.  
Je déclare participer volontairement à cette étude et à coopérer à la totalité des examens 
requis. J’accepte de donner des informations concernant mes antécédents médicaux. Je 
déclarerais ma participation à d’autre potentielle étude.  
Je permets au personnel médical et paramédical impliqué dans mon traitement d’être au 
courant de ma participation à l’étude.  
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 123 
 
Je suis d’accord de participer à l’étude. 
Nom et prénom : 
Date de naissance : 
Signature : 
Date : 
La personne sous-mentionnée declare que le patient concerné a été clairement informé 
oralement et par écrit au sujet de l’étude.  
Nom : 
Fonction : 
Signature : 
Date : 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 124 
 
Einverständniserklärung 
 
 
Medikamentöse Strategien zur Reduktion des während einer 
Lebertransplantation auftretenden Leberschadens 
 
Protokoll Nr.: 
 
Sponsor: University Hospitals Leuven, Herestraat 49, 3000 Leuven 
 
Dieses Formular ist allein für den Patienten oder seinen gesetzlichen Vertreter 
bestimmt. 
 
Ich,..............................................................................................................., bestätige, dass ich über die 
klinische Studie informiert wurde und dass ich eine Kopie der „Informationsbroschüre" 
und der "Einverständniserklärung" erhalten habe. Ich habe diese Informationen gelesen 
und verstanden. Mein Arzt hat mich umfassend informiert über die Bedingungen zur 
Teilnahme an der Studie, die Dauer der Studie, sowie die Wirkungen und 
Nebenwirkungen der Behandlung. Ich hatte ausreichend Zeit, um diese Informationen zu 
überdenken und Fragen zu stellen, auf die ich ausreichende und verständliche Antworten 
erhalten 
habe. 
Ich akzeptiere ebenfalls, dass Gewebe-, Blut- und Urinproben, die während des gesamten 
Verlaufs der Studie (vor, während und nach der Transplantation) abgenommen werden, 
für 
wissenschaftliche 
Zwecke 
verwendet 
werden. 
Ich habe verstanden, dass ich jederzeit meine Teilnahme an dieser Studie widerrufen 
kann, indem ich meinen Arzt darüber in Kenntnis setze, und dass diese Entscheidung 
keinen 
einzigen 
Nachteil 
für 
mich 
haben 
wird. 
Ich ermächtige die zuständigen Prüfarzte und die Aufsichtsbehörden zum Zugriff auf 
meine Krankenakten. Die mich betreffenden medizinischen Informationen werden streng 
vertraulich behandelt werden. Ich bin mir bewusst, dass für die Untersuchung meine 
medizinischen 
Daten 
gesammelt, 
verarbeitet 
und 
ausgewertet 
werden. 
Ich stimme zu, dass der Prüfarzt meine medizinischen Daten auch für andere 
Forschungsvorhaben 
nutzen 
kann. 
Ich gebe freiwillig meine Zustimmung, an dieser Studie und an allen erforderlichen 
Untersuchungen teilzunehmen. Ich bin bereit, Informationen zu geben über meine 
medizinische Vorgeschichte, die Einnahme von Medikamenten und die eventuelle 
Teilnahme 
an 
anderen 
Studien. 
Ich bin damit einverstanden, dass alle Gesundheitsdienstleister, die an dieser Studie 
beteiligt sind, über meine Teilnahme an dieser Studie informiert werden. 
 
Ich gebe die Einwilligung zur Teilnahme an dieser klinischen Studie. 
 
Name und Vorname: 
 
Geburtsdatum: 
 
Unterschrift: 
 
Datum: 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 125 
 
 
---------------------------------------------------------------------------------------------------------------------------------------- 
 
Ich erkläre, dass die oben genannte Person mündlich und schriftlich über die oben 
genannten Studie informiert wurde. 
 
Name: 
 
Funktion: 
 
Unterschrift: 
 
Datum: 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 126 
 
APPENDIX 7: PATIENT INFORMATION BROCHURE: Part A. Safety study 
 
Informatiebrochure  
 
 
 
 
 
Patiënten nr:  
 
 
U wordt gevraagd om deel te nemen aan een klinische studie waarbij verschillende 
medicijnen tijdens uw levertransplantatie toegediend zullen worden met als doel de 
beschadiging van de lever tijdens het transplantatie proces (zogenoemde ischemie-
reperfusie schade) te verminderen. Het is belangrijk om dit document te lezen alvorens u 
beslist om aan deze studie deel te nemen. De doelstellingen, onderzoeken, voor- en 
nadelen, risico’s en ongemakken die aan deze studie verbonden zijn, worden in de 
informatiebrochure en het toestemmingsformulier beschreven. Er kan niets worden 
beloofd noch worden gegarandeerd in verband met de resultaten van de studie. U hebt 
het recht om, om het even wanneer, vragen te stellen met betrekking tot de mogelijke 
en/of bestaande risico’s in deze studie. 
 
Doelstelling en beschrijving van de studie 
  
Tijdens het chirurgisch verwijderen, bewaren en transplanteren van een lever kan dit 
orgaan beschadigd worden. Deze beschadiging is deels het gevolg van een onvermijdbaar 
zuurstoftekort tijdens de bewaring van de lever na het chirurgisch verwijderen tot de 
transplantatie(ook ischemie genoemd).  
Deze ischemische schade kan echter na de levertransplantatie versterkt worden wanneer 
de bloeddoorstroming in de ontvanger hersteld wordt (dit noemt men ischemie-
reperfusie schade). De opgelopen orgaanschade kan minimaal maar kan ook zeer ernstig 
tot levensbedreigend zijn. Een vermindering van de ischemie-reperfusie schade is dan 
ook van groot belang omdat het de resultaten van levertransplantatie kan verbeteren. 
Het proces van ischemie-reperfusie schade bestaat uit een complexe cascade van elkaar 
versterkende processen.  
Het doel van deze studie is dan ook ischemie-reperfusie schade na levertransplantatie te 
verminderen tot minimaliseren.   
Om al deze schadelijke processen te blokkeren, voorzien we om verschillende 
geneesmiddelen toe te dienen zodat de kans op succes zo groot mogelijk is. Deze 
combinatie aan geneesmiddelen zal voor het eerst bij de mens worden gebruikt en wordt 
tijdens de levertransplantatie toegediend. Ondertussen zullen uw bloeddruk, hartritme 
en temperatuur voortdurend gemeten worden. 
Het doel van deze studie is de veiligheid van deze geneesmiddelen aan te tonen (een 
zogenaamde safety studie). Wanneer u besluit om aan deze studie deel te nemen, zal u 
naast de standaardbehandeling aan de bijkomende toegediende geneesmiddelen zoals 
voorzien in deze studie worden onderworpen. 
De geneesmiddelen die toegediend zullen worden, zijn: 
Toediening geneesmiddelen om leverbeschadiging tijdens 
transplantatie te verminderen  
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 127 
 
- 
Cetor®/Cinryze®: C1-esterase inhibitor. C1-esterase inhibitor speelt een belangrijke 
rol in de onstekingsreactie en bloedstolling. 
- 
Atenativ®: anti-stolling, anti-ontsteking 
- 
Vitamin E oplossing®: Vitamine E biedt bescherming tegen de schadelijke effecten 
van zuurstof 
- 
Tationil® 600 en Circadin®: biedt bescherming tegen de schadelijke effecten van 
zuurstof 
- 
Apotransferrine: houdt ijzer vast en biedt ook bescherming tegen de schadelijke 
effecten van zuurstof 
- 
Neorecormon®: biedt bescherming tegen celdood en de schadelijke effecten van 
zuurstof, zorgt voor een vermindering van ontstekingssignalen  
- 
Remicade®: vermindering van ontstekingssignalen 
- 
Flolan®: anti-ontsteking, blokkeren van de activatie van bloedplaatjes en 
verwijding van de bloedvaten 
 
Er zullen 10 patiënten aan deze studie deelnemen. 
 
Tijdsduur studie 
 
De safety studie zal 1 week duren. 
 
Onderzoeken 
 
- 
Zoals iedereen die een levertransplantatie ondergaat, zullen er dagelijks en 
routinematig bloed- en urinestalen worden afgenomen de eerste 14 dagen na 
levertransplantatie. Ook standaard klinische onderzoeken zullen plaatsvinden en 
dit 3, 6, 12 maanden na de transplantatie op de raadpleging. 
- 
Bovendien worden ook 2 leverbiopsies genomen: 
 1 uur voor de implantatie 
 1 uur na de reperfusie 
Vrijwillige deelname 
 
Uw deelname aan deze studie is volledig vrijwillig. U hebt het recht om deelname te 
weigeren. Wat u ook beslist, u zal behandeld en opgevolgd worden zonder enig nadelig 
gevolg voor uw  medische zorgen en voor uw relatie met uw behandelende arts (dokter-
patiënt relatie). 
Wanneer u beslist om aan deze studie deel te nemen, dan zal er aan u gevraagd worden 
het toestemmingsformulier te ondertekenen. 
Nadat u het toestemmingsformulier hebt ondertekend is het uw recht om uw deelname 
aan de studie te stoppen om het even wanneer, U moet geen reden geven waarom u uw 
toestemming voor deelname aan de studie intrekt. 
De studiedokter kan op elk moment uw deelname aan de studie stopzetten en dit zonder 
uw toestemming te vragen. 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 128 
 
 
Risico’s en ongemakken 
 
Er zal maar één dosis (twee van Neorecormon®) van ieder geneesmiddel in deze studie 
worden toegediend. De veiligheid van toediening van deze componenten wordt specifiek 
onderzocht in deze safety studie. Onverwachte interacties/potentiële risico’s eigen aan 
de combinatie kunnen niet helemaal uitgesloten worden. Het kan ook zijn dat u 
bijwerkingen tijdens de behandeling met deze medicijnen ondervindt, namelijk:    
- 
Cetor®/Cinryze®: Allergische of anafylactische reacties (bijvoorbeeld: versneld 
hartritme, hoge of lage bloeddruk, roodheid in de hals en in het gezicht, netelroos, 
kortademigheid) 
- 
Atenativ®: 
Allergische 
reacties 
of 
overgevoeligheid, 
toename 
in 
lichaamstemperatuur en verwijdering van de bloedvaten 
- 
Vitamine E oplossing®: geen gekende bijwerkingen 
- 
Tationil® 600: geen gekende bijwerkingen 
- 
Circadin®: geen gekende bijwerkingen 
- 
Apotransferrine: geen gekende bijwerkingen 
- 
Neorecormon®: vorming van een bloedklonter in een bloedvat, epilepsie en hoge 
bloeddruk 
- 
Remicade: 
huiduitslag, 
afwijkingen 
in 
de 
samenstelling 
van 
 
bloed, 
overgevoeligheid en levensbedreigende allergische reacties 
- 
Flolan®: hoofdpijn, misselijkheid, lage bloeddruk, traag hartritme, versneld 
hartritme en kortademigheid 
 
Voordelen 
 
We kunnen het therapeutisch effect van de geneesmiddelen niet verzekeren zelfs al weten 
we dat ieder geneesmiddel op zich de intensiteit van ischemie-reperfusie schade 
vermindert. 
 
Verzekering 
 
Conform de Belgische wet inzake experimenten op de menselijke persoon van 7 mei 2004 
is de sponsor van het onderzoek, zelfs foutloos, aansprakelijk voor alle schade die de 
deelnemer en/of zijn rechthebbenden oplopen en die rechtstreeks dan wel 
onrechtstreeks verband houdt met de proef. De sponsor heeft een verzekering afgesloten 
die deze aansprakelijkheid dekt. Indien u schade zou oplopen ten gevolge van uw 
deelname aan deze studie, zal die schade bijgevolg worden vergoed conform de Belgische 
wet inzake experimenten op de menselijke persoon van 7 mei 2004. 
 
Compensaties 
 
Deze studie brengt voor U geen extra kosten met zich mee. U hoeft de geneesmiddelen 
ook niet te betalen. Bovendien worden alle onderzoeken op stalen (bloed-, urine- en 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 129 
 
leverbiopsies) die niet beschouwd worden als courant en routinematig, betaald door de 
onderzoeker.  
 
Bescherming van uw privéleven 
 
Uw identiteit en deelname aan deze studie zullen strikt vertrouwelijk behandeld worden. 
U zal niet geïdentificeerd kunnen worden bij naam of op enige andere manier in 
documenten, resultaten of publicaties betreffende de studie. 
Uw medische gegevens zullen onderzocht worden door vertegenwoordigers van de 
onderzoeker en door regulerende instanties die de studie ook controleren. Dit gebeurt 
volgende de GCP (= Good Clinical Practice) richtlijnen. Hierbij wordt Uw identiteit geheim 
gehouden door een uniek patiëntennummer te gebruiken om Uw persoonlijke informatie 
aan te duiden. 
Uw persoonlijke informatie zal doorgegeven worden aan de regulerende instanties, de 
ethische commissie en andere dokters die samenwerken met de onderzoeker. 
Uw persoonlijke informatie zal elektronisch of manueel verwerkt en geanalyseerd 
worden om de resultaten van deze studie te bepalen. U hebt het recht aan de dokter van 
de studie te vragen naar uw data die verzameld zijn en de doelstelling van deze 
verzameling. U hebt ook het recht om de studiedokter te verzoeken om u toegang te geven 
tot uw persoonlijke informatie en die te verbeteren indien nodig.  
Alle reglementering betreffende de bescherming van de persoonlijke data is terug te 
vinden in de Wet van 8 december 1992 (België), betreffende de bescherming van het 
privéleven. 
 
 
Nieuwe informatie 
 
Soms is het mogelijk dat nieuwe informatie omtrent behandeling of medicinale producten 
in de context van het klinisch project verschijnt. Indien dit het geval is, zal u geïnformeerd 
worden over deze nieuwe informatie die uw bereidwilligheid voor verdere deelname in 
deze studie kan beïnvloeden. In dit geval zal u gevraagd worden de nieuwe 
informatiebrochure en het nieuwe toestemmingsformulier te ondertekenen. 
 
Ethische commissie 
 
De onafhankelijke ethische commissie van UZ Leuven heeft deze studie beoordeeld en 
goedgekeurd. 
 
 
Contactpersoon in geval van vragen omtrent de studie 
 
Wanneer u denkt dat u schade hebt opgelopen gerelateerd aan de studie, een reactie hebt 
op de studiemedicatie of vragen hebt omtrent de studie kan u nu, tijdens en/of na de 
studie contact opnemen met de hoofdonderzoeker. 
 
Hoofdonderzoeker: Prof. dr. Diethard Monbaliu 
Adres: Abdominale transplantatiechirurgie, UZ Leuven 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 130 
 
             Herestraat 49 bus 7003, 3000 Leuven 
Telefoon secretariaat: + 32 16 348727 
Fax secretariaat: + 32 16 348743 
 
Studiecoördinator: Ilse Senesael 
Adres: Abdominale transplantatiechirurgie, UZ Leuven 
 
Herestraat 49 bus 7003, 3000 Leuven 
Telefoon: + 32 16 342848 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 131 
 
Patient Information 
 
Patient Number: ____________ 
 
 
 
 
 
 
 
You are invited to take part in a research study that tries to reduce the damage to the liver 
(the so called ischemia-reperfusion injury) during the transplantation process by using a 
multi-drug conditioning. Before you decide to participate in this study, it is important that 
you read this form. In this information and consent form, the purpose, the examinations, 
the advantages, risks and inconveniences coupled with this study are described. The right 
to withdraw your consent to participate at any time is also described below. No promises 
or guarantees can be made concerning the results of the study. You have the right to ask 
questions at any time, for example concerning the possible and/or known risks contained 
in this study.  
 
Purpose and description of the study 
 
Procurement, storage and transplantation can by themselves injure the liver. This damage 
starts because of the unavoidable oxygen deficiency during the storage of the liver (= time 
between the procurement of the liver and the transplantation) and is so called the 
ischemia-reperfusion injury (IRI). The extent of IRI can cause minimal to severe injury or 
even total destruction of the graft. Decreasing IRI is therefore crucial.  
The process of IRI is composed of a whole mechanism of processes. The reduction of the 
IRI by using a multi-drug conditioning is based on the attenuation of these processes so 
that the chance is maximal to reduce the process of IRI. 
The combination of medications will be administered for the first time in men in a well-
controlled environment during your liver transplantation, whereby continuous 
monitoring of the blood pressure, rhythm and temperature is. 
The objective of this study is to demonstrate the safety of the combined drug approach. 
This is a safety study. If you accept to participate in this study, beside the standard 
treatment of care, you will receive the multi-drug conditioning. 
 
The composition of the multi-drug conditioning is defined as: 
- 
Cetor®/Cinryze®: C1-esterase inhibitor. It plays a major role in the 
inflammatory reaction and blood coagulation.  
- 
Atenativ®: anti-coagulation, anti-inflammation. 
- 
Vitamine E suspension®: protects against the damaging effects of oxygen 
- 
Tationil® 600 and Circadin®: protects against the damaging effects of oxygen  
- 
Apotransferrin:  protection against the damaging effects of oxygen and free 
iron 
- 
Neorecormon®: Protection against cell death and damaging effects of oxygen, 
diminution of inflammatory messengers  
Combined drug Approach to Prevent liver damage 
during Transplantation  
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 132 
 
- 
Remicade®: Diminution of the inflammatory messengers. 
- 
Flolan®: dilatation of the bloodvessels, anti-inflammatory and platelet 
activation inhibition.  
 
10 patients will take part in this medical-research study. 
 
Length of the study 
 
The duration of the safety study will be 1 week. 
 
Examinations 
 
- 
Blood and urine samples will routinely be taken during 14 days after the liver 
transplantation on a daily basis. Standard clinical examinations at 3, 6, 12 
months after transplantation are also foreseen at the outpatient control 
examinations during the consultation.  
- 
Likewise, 2 liver biopsies are predicted: 
 1 hour before the implantation 
 1 hour after the reperfusion 
 
 
Voluntary participation 
 
Your participation to this study is entirely voluntary. You have the right to refuse. You will 
be treated and followed without any disadvantage and any regard to medical care or 
physician-patient relationship whatever your decision.  
If you accept to participate in this study, you will be asked to sign the attached consent 
form. 
It is your right to stop your participation at the study whenever you want even after you 
have signed the informed consent. You don’t have to give a reason for withdrawing your 
consent to participate. 
The study doctor can stop your participation in this study at any moment, even without 
having to request your consent. 
 
 
Risks and inconveniences 
 
You will receive only one dose of each component (except for 2 doses for Neorecormon®). 
One single administration of these substances will be investigated in this study. 
Unexpected interactions/ potential risks due to the combination of drugs cannot be 
completely excluded. During the treatment with pharmacological products, you can 
encounter side effects:  
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 133 
 
- 
Cetor®/Cinryze®: Allergic or anaphylactic reactions (eg. Tachycardia, hyper- 
or hypotension, redness in the neck and face, hives (urticaria), dyspnea, 
headache, dizziness and nausea). 
- 
Atenativ®: Allergic reactions or hypersensitivity, increase of body 
temperature, dilatation of the blood vessels. 
- 
Vitamine E suspension®: no side effects. 
- 
Tationil® 600: no known side effects. 
- 
Circadin®: no side effects with the used dose. 
- 
Apotransferrin: no known side effects. 
- 
Neorecormon®: formation of a clot in a blood vessel, seizure and high blood 
pressure. 
- 
Remicade®: skin reaction, abnormalities in blood, hypersensibility and 
threatening allergic reactions. 
- 
Flolan®: headache, nausea, hypotension, low blood pressure, slow or rapid 
heart rate and dyspnea. 
 
 
Advantages 
 
Even if each component of the multi-drug conditioning is well known to reduce the 
severity of ischemia-reperfusion injury, we cannot certify a beneficial effect of the multi-
drug conditioning.  
 
Insurance 
 
The sponsor of the study is responsible for all the damage that you and/or your rightful 
claimants incur that is directly or indirectly related with the study. This is in accordance 
with the Belgian Law concerning experiments on the human person of May 7, 2004. The 
sponsor has closed an insurance that will cover this liability. If you would incur damage 
because of your participation to the study, this damage will be compensated by the 
sponsor of this study in accordance with the Belgian Law concerning experiments on the 
human person of May 7, 2004. 
 
Compensations 
 
There will be NO extra costs for you. You don’t have to pay components of the multidrug 
conditioning. Moreover, all the examinations on the samples (blood, urine and tissue) that 
are not considered as current and routine practices will be paid by the sponsor.  
 
 
Protection of your private life 
 
Your identity and your participation to this study will be treated as strictly confidential. 
You will not be identified by name or in any other identifying manner in files, results or 
publications concerning this study. 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 134 
 
According to the GCP (good clinical practice) guidelines, your medical records will be 
examined by representatives of the sponsor and by the regulating authorities in order to 
control the study. Your identity will remain secret since a unique patient number will only 
designate personal information. 
Your personal information might be transferred to regulating authorities, to the ethics 
committee and to other doctors that cooperate with the sponsor. 
Your personal information will be processed and analyzed electronically or manually in 
order to determine the results of this study. You have the right to ask to the study doctor 
which data are collected about you in the context of the study and what the purpose of 
that collection is. You also have the right to request the study doctor to give you access to 
your personal information and to correct it if necessary. The protection of personal data 
is legally established in the Law of December 8, 1992 (Belgium) concerning the protection 
of private life. 
 
Notification of new information 
 
Sometimes new information on the study treatment or medicinal product appears in the 
course of the research project. If this is the case, you will be informed about new 
information that might influence your willingness to further participate in this study. In 
that case you will be asked to sign new information and consent form. 
 
Ethics committee 
 
The independent ethics committee of UZ Leuven has reviewed and approved this study. 
 
Contact persons in the case of questions concerning the study 
 
If you think having incurred damage related to the study or having a reaction on the study 
medications, or if you have questions concerning the study or your rights as a participant, 
you can contact, now, during or after the study: 
Principal investigator: Prof. dr. Diethard Monbaliu 
Adress: Abdominal Transplant Surgery, UZ Leuven 
              Herestraat 49 bus 7003 
              3000 Leuven 
Phone secretariat: +32 16 348727 
Fax secretariat: +32 16 348743 
 
Coordinator of clinical trials : Ilse Senesael 
Adress: Abdominal Transplant Surgery, UZ Leuven 
               Herestraat 49 bus 7003, 3000 Leuven 
Phone: +32 16 342848 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 135 
 
BROCHURE INFORMATIVE 
 
Patient numéro: ____________ 
 
 
 
 
 
 
 
Vous êtes convié à participer à une étude clinique dans laquelle une série de médicaments 
sera administrée afin de réduire les lésions du foie liées à la préservation (appelées 
lésions d’ischémie-reperfusion). Avant de prendre une décision quant à votre 
participation, il est capital de prendre connaissance du contenu de cette brochure. Dans 
celle-ci seront successivement exposés les buts, les analyses, les avantages, les risques et 
inconvénients inhérents à l’étude mais également votre droit de vous rétracter et 
d’annuler votre accord à n’importe quel moment du projet. Aucune garantie ne peut être 
promise quant aux résultats escomptés. Vous avez le droit de poser des questions quand 
vous le désirez. Nous sommes à votre entière disposition pour vous répondre. 
 
Objectif et description de l’étude 
 
Le prélèvement d’organe, sa préservation et la transplantation engendrent des lésions au 
foie. Cette détérioration est liée à l’absence de perfusion et d’oxygénation du foie durant 
la période entre le prélèvement d’organes du donneur et la greffe chez le receveur. Ces 
dommages sont nommés «lésions d’ischémie reperfusion». L’ampleur de ces lésions varie 
et peut être responsable d’une destruction totale de la greffe, d’où l’intérêt de les 
diminuer. L’apparition des lésions d’ischémie reperfusion est secondaire à une série 
d’évènements complexes. 
L’avantage lié à l’utilisation concomitante de plusieurs médicaments résulte dans l’action 
simultanée de chaque produit sur une étape précise de cette cascade d’évènements, 
augmentant ainsi la possibilité de réduire ces lésions. 
L’administration se fera de façon bien précise et contrôlée. Le suivi de vos paramètres 
cliniques tels que la pression artérielle, le rythme cardiaque et la température sera assuré 
de façon continue. 
L’objectif de cette étude est donc de démontrer pour la première fois la diminution des 
lésions d’ischémie reperfusion grâce à l’administration séquentielle de ces médicaments.  
L’objectif ultime de cette étude est de démontrer la sécurité avec laquelle nous pouvons 
utiliser la combinaison de médicament. Si vous acceptez de participer à cette étude, outre 
le traitement standard dont bénéficient tous les patients greffés hépatiques actuellement, 
vous recevrez la combinaison de médicaments. 
 
Les médicaments sont les suivants: 
- 
Cetor®/Cinryze® : Inhibiteur du complément qui joue un rôle crucial dans la 
réaction inflammatoire et dans la coagulation. 
- 
Atenativ® : Anti-thrombine-III  qui a également un rôle prépondérant dans la 
réaction inflammatoire et dans la coagulation. 
Réduction des lésions du foie en transplantation après 
administration séquentielle de médicaments (CAPITL) 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 136 
 
- 
Vitamine E sirop®, Circadin®, Tationil® 600 : Protection contre les effets 
délétères liés à l’oxygène (antioxidant). 
- 
Apotransferrin® : l’Apotransferrine permet la chélation du fer toxique outre son 
pouvoir antioxidant. 
- 
Neorecormon® : Erythropoïetin qui permet de protéger la mort des cellules, qui 
diminue l’inflammation et possède un pouvoir antioxidant. 
- 
Remicade® : Infliximab qui diminue la réaction inflammatoire. 
- 
Flolan® : Epoprostenol qui est un vasodilatateur puissant. Il possède également 
des propriétes antioxidante et anti-aggrégante. 
 
10 patients participeront à cette étude. 
 
 
Durée de l’étude 
 
La durée de l’étude est limitée à 1 semaine. 
 
Analyses 
 
- 
Exactement comme réalisé chez tous les patients greffés du foie, des 
échantillons de sang et d’urines seront prélevés une fois par jour durant les 
14 premiers jours après la greffe de foie. Ces échantillons seront également 
réalisés comme d’habitude lors d’une consultation de contrôle à 3, 6, 12 mois 
après la transplantation.  
- 
En outre, 2 biopsies de la greffe hépatique habituellement réalisées sont 
prévues: 
  Juste avant la greffe de foie, 
 1 heure après la reperfusion de la greffe de foie, 
 
Participation volontaire 
 
Votre participation à l’étude dépend strictement de votre décision. Vous avez le droit de 
refuser d’y participer. Quelque soit votre décision, vous serez traités avec la plus grande 
attention sans aucune  faveur ou privilège de la part des corps médical et paramédical.  
Si vous acceptez de participer à l’étude, nous vous invitons à signer un consentement 
éclairé. 
Durant toute la durée de l’étude et sans devoir vous justifier, il vous appartient d’annuler 
votre participation même si vous avez signé le consentement éclairé. 
Par contre, le corps médical a également le droit de mettre fin à votre participation à 
n’importe quel moment dans devoir exiger votre accord. 
 
 
Effets secondaires  
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 137 
 
Vous allez recevoir une seule dose de chaque médicament (excepté le Néorecormon qui 
requière une seconde administration). Après une revue de la littérature importante, il 
s’est avéré que l’administration d’une dose unique de ces produits est dénuée d’effet 
secondaire sévère. Cependant, même si le risque de survenue est extrêmement faible, il 
n’est pas exclu d’en présenter. 
 
- 
Cetor®/Cinryze®: réaction allergique ou anaphylactique (accélération du 
rythme cardiaque, chute de la tension artérielle, rougeur, urticaire, nausée, 
difficulté respiratoire) 
- 
Atenativ®: réaction allergique, augmentation de la température corporelle, 
dilatation des vaisseaux sanguins. 
- 
Vitamine E sirop®: Pas d’effet secondaire connu à la dose utilisée. 
- 
Tationil 600®: Pas d’effet secondaire connu à la dose utilisée. 
- 
Melatonin®: Pas d’effet secondaire connu à la dose utilisée. 
- 
Apotransferrin®: Pas d’effet secondaire connu à la dose utilisée. 
- 
Neorecormon®: formation de caillot, épilepsie, augmentation de la pression 
artérielle. 
- 
Remicade®: Réaction cutanée, troubles sanguins, réactions allergique et 
anaphylactique. 
- 
 Flolan®: Chute de la pression artérielle, augmentation du rythme cardiaque, 
difficulté respiratoire. 
 
Finalement, des effets secondaires inattendus liés à l’administration séquentielle de ces 
médicaments ne peuvent être complètement exclus. 
 
 
Avantages 
 
Chaque médicament inclus a prouvé son efficacité dans la réduction des lésions 
d’ischémie reperfusion. Leur administration simultanée durant la greffe de foie devrait 
avoir un effet bénéfique mais que nous ne pouvons prédire avec certitude un effet 
bénéfique.  
 
 
Assurances 
 
En accord avec la loi belge concernant les études cliniques du 7 mai 2004, il va de soi que 
le promoteur de l’étude est considéré comme étant responsable de tout dommage 
encouru qu’il soit directement ou indirectement lié à l’étude.  
 
Frais 
 
Il n’y aura aucun coût supplémentaire pour vous, aussi bien pour les médicaments inclus 
que pour tout échantillon (sang, urine et biopsie) considéré comme étant en dehors de la 
pratique clinique habituelle.  
 
Anonymat-Protection de la vie privée 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 138 
 
 
En accord avec les directives GCP (Good Clinical Practice) et la loi de la protection de la 
vie privée du 8 décembre 1992, votre identité et votre participation à l’étude restera 
strictement confidentielle. Vous ne serez jamais identifié dans les données, les résultats 
ou publications liés à l’étude si ce n’est par un numéro unique qui désignera vos données 
personnelles. Toutes ces données seront traitées manuellement ou électroniquement. 
Seuls les représentants du promoteur ainsi que les autorités compétentes pourront avoir 
accès à votre dossier dans l’unique but de contrôler l’étude. Vos données personnelles 
pourront donc être transmises aux autorités compétentes tel que le comité d’éthique ou 
à d’autres médecins participant dans le cadre de l’étude. 
A tout moment, vous avez le droit de consulter vos données personnelles. 
  
Information au sujet d’une modification 
 
Vous serez informés de tout potentiel changement concernant l’étude ou les 
médicaments. Dans ce cas, vous serez invités à signer un nouveau consentement éclairé. 
 
Comité d’éthique 
 
Avant d’entreprendre cette étude, le comité d’éthique indépendant de l’UZ Leuven a 
analysé le protocole et a donné son accord pour le projet. 
  
Personnes à contacter en cas de questions liées à l’étude 
 
Si vous avez des questions concernant un potentiel dommage, vos données personnelles 
ou vos droits en tant que participant, n’hésitez pas à nous contacter que ce soit avant la 
greffe, durant ou après votre hospitalisation. 
 
Investigateur principal: Prof. Dr. Diethard Monbaliu 
Adresse: Abdominal Transplant Surgery, UZ Leuven 
              Herestraat 49 bus 7003 
              3000 Leuven 
Téléphone secrétariat: +32 16 348727 
Fax secrétariat: +32 16 348743 
 
Coordonatrice des essais cliniques : Senesael Ilse  
 
Adress : Abdominal Transplant Surgery, UZ Leuven 
               Herestraat 49 bus 7003 
 
3000 Leuven 
Téléphone : +32 16 342848 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 139 
 
APPENDIX 8: PATIENT INFORMATION BROCHURE: Part B. Randomized 
Controlled Trial 
 
 
Informatiebrochure 
 
 
 
 
 
Patiënten nr:  
 
Sponsor: UZ Leuven, Herestraat 49, 3000 Leuven 
 
 
 
U wordt gevraagd om deel te nemen aan een klinische studie waarbij verschillende 
medicijnen tijdens uw levertransplantatie toegediend zullen worden met als doel de 
beschadiging van de lever tijdens het transplantatie proces (zogenoemde ischemie-
reperfusie schade) te verminderen. Deze studie heeft UZ KU Leuven als opdrachtgever en 
zal plaatsvinden in  verschillende nationale en internationale ziekenhuizen. Het is 
belangrijk om dit document te lezen alvorens u beslist om aan deze studie deel te nemen. 
De doelstellingen, onderzoeken, voor- en nadelen, risico’s en ongemakken die aan deze 
studie verbonden zijn, worden in de informatiebrochure en het toestemmingsformulier 
beschreven. Er kan niets worden beloofd noch worden gegarandeerd in verband met de 
resultaten van de studie. U hebt het recht om, om het even wanneer, vragen te stellen met 
betrekking tot de mogelijke en/of bestaande risico’s in deze studie. 
 
Doelstelling en beschrijving van de studie 
  
Tijdens het chirurgisch verwijderen, bewaren en transplanteren van een lever kan dit 
orgaan beschadigd worden. Deze beschadiging is deels het gevolg van een onvermijdbaar 
zuurstoftekort tijdens de bewaring van de lever na het chirurgisch verwijderen tot de 
transplantatie(ook ischemie genoemd).  
Deze ischemische schade kan echter na de levertransplantatie versterkt worden wanneer 
de bloeddoorstroming in de ontvanger hersteld wordt (dit noemt men ischemie-
reperfusie schade). De opgelopen orgaanschade kan minimaal maar kan ook zeer ernstig 
tot levensbedreigend zijn. Een vermindering van de ischemie-reperfusie schade is dan 
ook van groot belang omdat het de resultaten van levertransplantatie kan verbeteren. 
Het proces van ischemie-reperfusie schade bestaat uit een complexe cascade van elkaar 
versterkende processen.  
Het doel van deze studie is dan ook ischemie-reperfusie schade na levertransplantatie te 
verminderen tot minimaliseren.   
Om al deze schadelijke processen te blokkeren, voorzien we om verschillende 
geneesmiddelen toe te dienen zodat de kans op succes zo groot mogelijk is. Deze 
combinatie aan geneesmiddelen zal voor het eerst bij de mens worden gebruikt en wordt 
Toediening geneesmiddelen om leverbeschadiging tijdens 
transplantatie te verminderen  
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 140 
 
tijdens de levertransplantatie toegediend. Ondertussen zullen uw bloeddruk, hartritme 
en temperatuur voortdurend gemeten worden. 
Het doel van deze studie is de doeltreffendheid van deze geneesmiddelen in het 
verminderen van ischemie-reperfusie schade aan te tonen. Het gaat om een 
gerandomiseerde en gecontroleerde studie. In een eerste zogenomende “safety, fase A” 
van deze studie, konden geen ernstige nevenwerkingen aangetoond worden van de 
gecombineerde toediening van al deze geneesmiddelen.  Wanneer u besluit om aan deze 
studie deel te nemen, zal u naast de standaardbehandeling willekeurig aan al dan niet de 
bijkomende toegediende geneesmiddelen zoals voorzien in deze studie worden 
onderworpen. 
De geneesmiddelen die toegediend zullen worden, zijn: 
- 
Cetor®/Cinryze®: C1-esterase inhibitor. C1-esterase inhibitor speelt een belangrijke 
rol in de onstekingsreactie en bloedstolling. 
- 
Atenativ®: anti-stolling, anti-ontsteking 
- 
Vitamin E oplossing®: Vitamine E biedt bescherming tegen de schadelijke effecten 
van zuurstof 
- 
Tationil® 600 en Circadin®: biedt bescherming tegen de schadelijke effecten van 
zuurstof 
- 
Apotransferrine: houdt ijzer vast en biedt ook bescherming tegen de schadelijke 
effecten van zuurstof 
- 
Neorecormon®: biedt bescherming tegen celdood en de schadelijke effecten van 
zuurstof, zorgt voor een vermindering van ontstekingssignalen  
- 
Remicade®: vermindering van ontstekingssignalen 
- 
Flolan®: anti-ontsteking, blokkeren van de activatie van bloedplaatjes en 
verwijding van de bloedvaten 
 
Er zullen 72 patiënten aan deze studie deelnemen. De randomisatie gebeurt door een 
derde die niet bij de studie betrokken is. Patiënten zullen in ieder deelnemend centrum 
gerandomiseerd worden in twee groepen door gebruik te maken van gepermuteerde 
blokken van variabele grootte. -  
 
Tijdsduur studie 
 
De studie zal één jaar duren. 
 
Onderzoeken 
 
- 
Zoals iedereen die een levertransplantatie ondergaat, zullen er dagelijks en 
routinematig bloed- en urinestalen worden afgenomen de eerste 14 dagen na 
levertransplantatie. Ook standaard klinische onderzoeken zullen plaatsvinden en 
dit 3, 12 maanden na de transplantatie op de raadpleging. 
- 
Bovendien worden ook 3 leverbiopsies genomen: 
 1 uur voor de implantatie 
 1 uur na de reperfusie 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 141 
 
 7 dagen na de transplantatie (deze behoort niet tot de routine) 
Naast de leverbiopsies, worden ook twee biopsies van de galwegen en 1 collectie van de 
gal afgenomen en dit tijdens de levertransplantatie.  
 
- 
Een jaar na de transplantatie zal er routinematig een MRI (Magnetic Resonance 
Imaging) gepland worden om de kwaliteit van de getransplanteerde lever na te 
gaan.  
 
Vrijwillige deelname 
 
Uw deelname aan deze studie is volledig vrijwillig. U hebt het recht om deelname te 
weigeren. Wat u ook beslist, u zal behandeld en opgevolgd worden zonder enig nadelig 
gevolg voor uw  medische zorgen en voor uw relatie met uw behandelende arts (dokter-
patiënt relatie). 
Wanneer u beslist om aan deze studie deel te nemen, dan zal er aan u gevraagd worden 
het toestemmingsformulier te ondertekenen. 
Nadat u het toestemmingsformulier hebt ondertekend is het uw recht om uw deelname 
aan de studie te stoppen om het even wanneer, U moet geen reden geven waarom u uw 
toestemming voor deelname aan de studie intrekt. 
De studiedokter kan op elk moment uw deelname aan de studie stopzetten en dit zonder 
uw toestemming te vragen. 
 
Risico’s en ongemakken 
 
Er zal maar één dosis (twee van EPO) van ieder geneesmiddel in deze studie worden 
toegediend. Na het uitvoerig raadplegen van de medische literatuur, blijkt een eenmalige 
toediening van deze componenten veilig te zijn. Dit blijkt ook uit de resultaten van de 
safety studie. Toch kunnen onverwachte interacties/potentiële risico’s eigen aan de 
combinatie niet helemaal uitgesloten worden. U kan ook bijwerkingen tijdens de 
behandeling met deze medicijnen ondervinden, namelijk: 
- 
Cetor®/Cinryze®: Allergische of anafylactische reacties (bijvoorbeeld: versneld 
hartritme, hoge of lage bloeddruk, roodheid in de hals en in het gezicht, netelroos, 
kortademigheid) 
- 
Atenativ®: 
Allergische 
reacties 
of 
overgevoeligheid, 
toename 
in 
lichaamstemperatuur en verwijdering van de bloedvaten 
- 
Vitamine E oplossing®: geen gekende bijwerkingen 
- 
Tationil® 600: geen gekende bijwerkingen 
- 
Circadin®: geen gekende bijwerkingen 
- 
Apotransferrine: geen gekende bijwerkingen 
- 
Neorecormon®: vorming van een bloedklonter in een bloedvat, epilepsie en hoge 
bloeddruk 
- 
Remicade: huiduitslag, afwijkingen in de samenstelling van  bloed overgevoeligheid 
en levensbedreigende allergische reacties 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 142 
 
- 
Flolan®: hoofdpijn, misselijkheid, lage bloeddruk, traag hartritme, versneld 
hartritme en kortademigheid 
 
Voordelen 
 
We kunnen het therapeutisch effect van de geneesmiddelen niet verzekeren zelfs al weten 
we dat ieder geneesmiddel op zich de intensiteit van ischemie-reperfusie schade 
vermindert. 
 
Verzekering 
 
Conform de Belgische wet inzake experimenten op de menselijke persoon van 7 mei 2004 
is de sponsor van het onderzoek, zelfs foutloos, aansprakelijk voor alle schade die de 
deelnemer en/of zijn rechthebbenden oplopen en die rechtstreeks dan wel 
onrechtstreeks verband houdt met de proef. De sponsor heeft een verzekering afgesloten 
die deze aansprakelijkheid dekt. Indien u schade zou oplopen ten gevolge van uw 
deelname aan deze studie, zal die schade bijgevolg worden vergoed conform de Belgische 
wet inzake experimenten op de menselijke persoon van 7 mei 2004. 
 
Compensaties 
 
Deze studie brengt voor U geen extra kosten met zich mee. U hoeft de geneesmiddelen 
ook niet te betalen. Bovendien worden alle onderzoeken op stalen (bloed-, urine- en 
weefselstalen) die niet beschouwd worden als courant en routinematig, betaald door de 
onderzoeker.  
 
Bescherming van uw privéleven 
 
Uw identiteit en deelname aan deze studie zullen strikt vertrouwelijk behandeld worden. 
U zal niet geïdentificeerd kunnen worden bij naam of op enige andere manier in 
documenten, resultaten of publicaties betreffende de studie. 
Uw medische gegevens zullen onderzocht worden door vertegenwoordigers van de 
onderzoeker en door regulerende instanties die de studie ook controleren. Dit gebeurt 
volgende de GCP (= Good Clinical Practice) richtlijnen. Hierbij wordt Uw identiteit geheim 
gehouden door een uniek patiëntennummer te gebruiken om Uw persoonlijke informatie 
aan te duiden. 
Uw persoonlijke informatie zal doorgegeven worden aan de regulerende instanties, de 
ethische commissie en andere dokters die samenwerken met de onderzoeker. 
Uw persoonlijke informatie zal elektronisch of manueel verwerkt en geanalyseerd 
worden om de resultaten van deze studie te bepalen. U hebt het recht aan de dokter van 
de studie te vragen naar uw data die verzameld zijn en de doelstelling van deze 
verzameling. U hebt ook het recht om de studiedokter te verzoeken om u toegang te geven 
tot uw persoonlijke informatie en die te verbeteren indien nodig.  
Alle reglementering betreffende de bescherming van de persoonlijke data is terug te 
vinden in de Wet van 8 december 1992 (België), betreffende de bescherming van het 
privéleven. 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 143 
 
 
 
Nieuwe informatie 
 
Soms is het mogelijk dat nieuwe informatie omtrent behandeling of medicinale producten 
in de context van het klinisch project verschijnt. Indien dit het geval is, zal u geïnformeerd 
worden over deze nieuwe informatie die uw bereidwilligheid voor verdere deelname in 
deze studie kan beïnvloeden. In dit geval zal u gevraagd worden de nieuwe 
informatiebrochure en het nieuwe toestemmingsformulier te ondertekenen. 
 
Ethische commissie 
 
De onafhankelijke Commissie Medische Ethiek van UZ KU Leuven/Onderzoek heeft deze 
studie beoordeeld en goedgekeurd, evenals de lokale Commissies voor Medische Ethiek van de 
centra waar deze studie doorgaat. 
 
Contactpersoon in geval van vragen omtrent de studie 
 
Wanneer u denkt dat u schade hebt opgelopen gerelateerd aan de studie, een reactie hebt 
op de studiemedicatie of vragen hebt omtrent de studie kan u nu, tijdens en/of na de 
studie contact opnemen met de hoofdonderzoeker. 
 
Hoofdonderzoeker: Prof. dr. Diethard Monbaliu 
Adres: Abdominale transplantatiechirurgie, UZ Leuven 
             Herestraat 49 bus 7003, 3000 Leuven 
Telefoon secretariaat: + 32 16 348727 
Fax secretariaat: + 32 16 348743 
 
Studiecoördinator: Sarah Mertens 
Adres: Abdominale transplantatiechirurgie, UZ Leuven 
 
Herestraat 49 bus 7003, 3000 Leuven 
Telefoon: + 32 16 342961 
 
 
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 144 
 
Patient Information 
 
Patient Number: ____________ 
 
Sponsor: 
University 
Hospitals 
Leuven, 
Herestraat 
49, 
3000 
Leuven 
 
 
 
 
 
 
 
 
You are invited to take part in a research study that tries to reduce the damage to the liver 
(the so called ischemia-reperfusion injury) during the transplantation process by using a 
multi-drug conditioning. This study has UZ KU Leuven as sponsor and will take place in  
different national and international hospitals. Before you decide to participate in this 
study, it is important that you read this form. In this information and consent form, the 
purpose, the examinations, the advantages, risks and inconveniences coupled with this 
study are described. The right to withdraw your consent to participate at any time is also 
described below. No promises or guarantees can be made concerning the results of the 
study. You have the right to ask questions at any time, for example concerning the possible 
and/or known risks contained in this study.  
 
Purpose and description of the study 
 
Procurement, storage and transplantation can by themselves injure the liver. This damage 
starts because of the unavoidable oxygen deficiency during the storage of the liver (= time 
between the procurement of the liver and the transplantation) and is so called the 
ischemia-reperfusion injury (IRI). The extent of IRI can cause minimal to severe injury or 
even total destruction of the graft. Decreasing IRI is therefore crucial.  
The process of IRI is composed of a whole mechanism of processes. The reduction of the 
IRI by using a multi-drug conditioning is based on the attenuation of these processes so 
that the chance is maximal to reduce the process of IRI. 
The combination of medications will be administered for the first time in men in a well-
controlled environment during your liver transplantation, whereby continuous 
monitoring of the blood pressure, rhythm and temperature is. 
The objective of this study is to demonstrate the efficacy of the combined drug approach 
in reducing the IRI. 
This is a randomized, controlled and single center study. In the safety phase, phase A of 
this study, no serious adverse events were observed.  . If you accept to participate in this 
study, beside the standard treatment of care, you will be randomly assigned to receive or 
not the multi-drug conditioning. 
 
The composition of the multi-drug conditioning is defined as: 
- 
Cetor®/Cinryze®: C1-esterase inhibitor. It plays a major role in the 
inflammatory reaction and blood coagulation.  
- 
Atenativ®: anti-coagulation, anti-inflammation. 
Combined drug Approach to Prevent liver damage 
during Transplantation  
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 145 
 
- 
Vitamine E suspension®: protects against the damaging effects of oxygen 
- 
Tationil® 600 and Circadin®: protects against the damaging effects of oxygen  
- 
Apotransferrin:  protection against the damaging effects of oxygen and free 
iron 
- 
Neorecormon®: Protection against cell death and damaging effects of oxygen, 
diminution of inflammatory messengers  
- 
Remicade®: Diminution of the inflammatory messengers. 
- 
Flolan®: dilatation of the bloodvessels, anti-inflammatory and platelet 
activation inhibition.  
 
72 patients will take part in this medical-research study. The randomization will be done 
by a third party, who is not involved in the study. In each participating center, patients 
will be randomized into two groups using permuted blocks of variable size.  
 
Length of the study 
 
The duration of the study will be 1 years. 
 
Examinations 
 
- 
Blood and urine samples will routinely be taken during 14 days after the liver 
transplantation on a daily basis. Standard clinical examinations at 3, 12 
months after transplantation are also foreseen at the outpatient control 
examinations during the consultation.  
- 
Likewise, 3 liver biopsies are predicted: 
 1 hour before the implantation 
 1 hour after the reperfusion 
 7 days after the transplantation (doesn’t belong to the routine tests) 
Besides the liver biopsies, 2 biopsies of the bile duct and 1 bile collection will be taken 
during the liver transplantation.  
 
As done routinely, an MRI (magnetic resonance imaging) scan will be planned one year 
after the transplantation to evaluate the quality of the liver, its blood supply and the bile 
ducts.  
 
Voluntary participation 
 
Your participation to this study is entirely voluntary. You have the right to refuse. You will 
be treated and followed without any disadvantage and any regard to medical care or 
physician-patient relationship whatever your decision.  
If you accept to participate in this study, you will be asked to sign the attached consent 
form. 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 146 
 
It is your right to stop your participation at the study whenever you want even after you 
have signed the informed consent. You don’t have to give a reason for withdrawing your 
consent to participate. 
The study doctor can stop your participation in this study at any moment, even without 
having to request your consent. 
 
 
Risks and inconveniences 
 
You will receive only one dose of each component (except for 2 doses for EPO). After a 
review of the literature and research to the safety in the safety study, one single 
administration of these substances is safe. However, unexpected interactions/ potential 
risks due to the combination of drugs cannot be completely excluded. During the 
treatment with pharmacological products, you can also encounter side effects: 
- 
Cetor®/Cinryze®: Allergic or anaphylactic reactions (eg. Tachycardia, hyper- 
or hypotension, redness in the neck and face, hives (urticaria), dyspnea, 
headache, dizziness and nausea). 
- 
Atenativ®: Allergic reactions or hypersensitivity, increase of body 
temperature, dilatation of the blood vessels. 
- 
Vitamine E suspension®: no side effects. 
- 
Tationil® 600: no known side effects. 
- 
Circadin®: no side effects with the used dose. 
- 
Apotransferrin: no known side effects. 
- 
Neorecormon®: formation of a clot in a blood vessel, seizure and high blood 
pressure. 
- 
Remicade®: skin reaction, abnormalities in blood, hypersensibility and 
threatening allergic reactions. 
- 
Flolan®: headache, nausea, hypotension, low blood pressure, slow or rapid 
heart rate and dyspnea. 
 
Advantages 
 
Even if each component of the multi-drug conditioning is well known to reduce the 
severity of ischemia-reperfusion injury, we cannot certify a beneficial effect of the multi-
drug conditioning.  
 
Insurance 
 
The sponsor of the study is responsible for all the damage that you and/or your rightful 
claimants incur that is directly or indirectly related with the study. This is in accordance 
with the Belgian Law concerning experiments on the human person of May 7, 2004. The 
sponsor has closed an insurance that will cover this liability. If you would incur damage 
because of your participation to the study, this damage will be compensated by the 
sponsor of this study in accordance with the Belgian Law concerning experiments on the 
human person of May 7, 2004. 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 147 
 
Compensations 
 
There will be NO extra costs for you. You don’t have to pay components of the multidrug 
conditioning. Moreover, all the examinations on the samples (blood, urine and tissue) that 
are not considered as current and routine practices will be paid by the sponsor.  
 
 
Protection of your private life 
 
Your identity and your participation to this study will be treated as strictly confidential. 
You will not be identified by name or in any other identifying manner in files, results or 
publications concerning this study. 
According to the GCP (good clinical practice) guidelines, your medical records will be 
examined by representatives of the sponsor and by the regulating authorities in order to 
control the study. Your identity will remain secret since a unique patient number will only 
designate personal information. 
Your personal information might be transferred to regulating authorities, to the ethics 
committee and to other doctors that cooperate with the sponsor. 
Your personal information will be processed and analyzed electronically or manually in 
order to determine the results of this study. You have the right to ask to the study doctor 
which data are collected about you in the context of the study and what the purpose of 
that collection is. You also have the right to request the study doctor to give you access to 
your personal information and to correct it if necessary. The protection of personal data 
is legally established in the Law of December 8, 1992 (Belgium) concerning the protection 
of private life. 
 
Notification of new information 
 
Sometimes new information on the study treatment or medicinal product appears in the 
course of the research project. If this is the case, you will be informed about new 
information that might influence your willingness to further participate in this study. In 
that case you will be asked to sign new information and consent form. 
 
Ethics committee 
 
The independent Medical Ethics Committee of the University Hospitals KU Leuven / 
Research has assessed and approved this study, as well as the local Medical Ethics 
Committees of the other participating centers where this study is taking place.  
 
Contact persons in the case of questions concerning the study 
 
If you think having incurred damage related to the study or having a reaction on the study 
medications, or if you have questions concerning the study or your rights as a participant, 
you can contact, now, during or after the study: 
Principal investigator: Prof. dr. Diethard Monbaliu 
Adress: Abdominal Transplant Surgery, UZ Leuven 
              Herestraat 49 bus 7003 
              3000 Leuven 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 148 
 
Phone secretariat: +32 16 348727 
Fax secretariat: +32 16 348743 
 
Coordinator of clinical trials : Sarah Mertens 
Adress: Abdominal Transplant Surgery, UZ Leuven 
               Herestraat 49 bus 7003, 3000 Leuven 
Phone: +32 16 342961 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 149 
 
BROCHURE INFORMATIVE 
 
Patient numéro: ____________ 
 
Promoteur: UZ Leuven, Herestraat 49, 3000 Leuven 
 
 
 
 
 
 
 
 
Vous êtes convié à participer à une étude clinique dans laquelle une série de médicaments 
sera administrée afin de réduire les lésions du foie liées à la préservation (appelées 
lésions d’ischémie-reperfusion). Cette étude a comme principal UZ KU Leuven et se 
poursuivra dans des centres hospitaliers nationaux et internationaux différents. Avant de 
prendre une décision quant à votre participation, il est capital de prendre connaissance 
du contenu de cette brochure. Dans celle-ci seront successivement exposés les buts, les 
analyses, les avantages, les risques et inconvénients inhérents à l’étude mais également 
votre droit de vous rétracter et d’annuler votre accord à n’importe quel moment du projet. 
Aucune garantie ne peut être promise quant aux résultats escomptés. Vous avez le droit 
de poser des questions quand vous le désirez. Nous sommes à votre entière disposition 
pour vous répondre. 
 
Objectif et description de l’étude 
 
Le prélèvement d’organe, sa préservation et la transplantation engendrent des lésions au 
foie. Cette détérioration est liée à l’absence de perfusion et d’oxygénation du foie durant 
la période entre le prélèvement d’organes du donneur et la greffe chez le receveur. Ces 
dommages sont nommés «lésions d’ischémie reperfusion». L’ampleur de ces lésions varie 
et peut être responsable d’une destruction totale de la greffe, d’où l’intérêt de les 
diminuer. L’apparition des lésions d’ischémie reperfusion est secondaire à une série 
d’évènements complexes. 
L’avantage lié à l’utilisation concomitante de plusieurs médicaments résulte dans l’action 
simultanée de chaque produit sur une étape précise de cette cascade d’évènements, 
augmentant ainsi la possibilité de réduire ces lésions. 
L’administration se fera de façon bien précise et contrôlée. Le suivi de vos paramètres 
cliniques tels que la pression artérielle, le rythme cardiaque et la température sera assuré 
de façon continue. 
L’objectif de cette étude est donc de démontrer pour la première fois la diminution des 
lésions d’ischémie reperfusion grâce à l’administration séquentielle de ces médicaments.  
Il s’agit d’une étude randomisée et contrôlée. Pendant la phase de cette étude de sécurité, 
phase A, aucun effet secondaire grave n’a pu être démontré. C'est-à-dire que si vous 
acceptez de participer à cette étude, outre le traitement standard dont bénéficient tous 
les patients greffés hépatiques actuellement, vous serez réparti dans un des deux groupes 
qui recevra les médicaments ou non. 
 
Réduction des lésions du foie en transplantation après 
administration séquentielle de médicaments (CAPITL) 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 150 
 
Les médicaments sont les suivants: 
- 
Cetor®/Cinryze® : Inhibiteur du complément qui joue un rôle crucial dans la 
réaction inflammatoire et dans la coagulation. 
- 
Atenativ® : Anti-thrombine-III  qui a également un rôle prépondérant dans la 
réaction inflammatoire et dans la coagulation. 
- 
Vitamine E sirop®, Circadin®, Tationil® 600 : Protection contre les effets 
délétères liés à l’oxygène (antioxidant). 
- 
Apotransferrin® : l’Apotransferrine permet la chélation du fer toxique outre son 
pouvoir antioxidant. 
- 
Neorecormon® : Erythropoïetin qui permet de protéger la mort des cellules, qui 
diminue l’inflammation et possède un pouvoir antioxidant. 
- 
Remicade® : Infliximab qui diminue la réaction inflammatoire. 
- 
Flolan® : Epoprostenol qui est un vasodilatateur puissant. Il possède également 
des propriétes antioxidante et anti-aggrégante. 
 
72 patients participeront à cette étude. La randomisation se fait par un tiers qui n'est pas 
impliqué dans l'étude. Les patients seront mis en place dans chaque centre participant, 
randomisés en deux groupes par l'utilisation de blocs permutés taille variable.  
 
 
Durée de l’étude 
 
La durée de l’étude est limitée à un année. 
 
Analyses 
 
- 
Exactement comme réalisé chez tous les patients greffés du foie, des 
échantillons de sang et d’urines seront prélevés une fois par jour durant les 
14 premiers jours après la greffe de foie. Ces échantillons seront également 
réalisés comme d’habitude lors d’une consultation de contrôle à 3, 12 mois 
après la transplantation.  
- 
En outre, 3 biopsies de la greffe hépatique habituellement réalisées sont 
prévues: 
  Juste avant la greffe de foie, 
 1 heure après la reperfusion de la greffe de foie, 
 7 jours après la transplantation (pas de façon routinière), 
- 
En plus des biopsies du foie, 2 biopsies de la voie biliaire et une collection de 
bile seront prises au cours de la transplantation du foie. 
- 
Classiquement, une résonance magnétique nucléaire (RMN) est réalisée afin 
d’étudier l’intégrité de l’arbre biliaire 1 an après la transplantation.  
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 151 
 
Participation volontaire 
 
Votre participation à l’étude dépend strictement de votre décision. Vous avez le droit de 
refuser d’y participer. Quelque soit votre décision, vous serez traités avec la plus grande 
attention sans aucune  faveur ou privilège de la part des corps médical et paramédical.  
Si vous acceptez de participer à l’étude, nous vous invitons à signer un consentement 
éclairé. 
Durant toute la durée de l’étude et sans devoir vous justifier, il vous appartient d’annuler 
votre participation même si vous avez signé le consentement éclairé. 
Par contre, le corps médical a également le droit de mettre fin à votre participation à 
n’importe quel moment dans devoir exiger votre accord. 
 
 
Effets secondaires  
 
Vous allez recevoir une seule dose de chaque médicament (excepté le Néorecormon qui 
requière une seconde administration). Après une revue de la littérature importante et 
l’analyse des résultats d’une étude évaluant la sécurité de l’administration des 
médicaments, il s’est avéré qu’une seule dose de ces produits est dénuée d’effet 
secondaire sévère. Cependant, même si le risque de survenue est extrêmement faible, il 
n’est pas exclu d’en présenter. 
 
- 
Cetor®/Cinryze®: réaction allergique ou anaphylactique (accélération du 
rythme cardiaque, chute de la tension artérielle, rougeur, urticaire, nausée, 
difficulté respiratoire) 
- 
Atenativ®: réaction allergique, augmentation de la température corporelle, 
dilatation des vaisseaux sanguins. 
- 
Vitamine E sirop®: Pas d’effet secondaire connu à la dose utilisée. 
- 
Tationil 600®: Pas d’effet secondaire connu à la dose utilisée. 
- 
Melatonin®: Pas d’effet secondaire connu à la dose utilisée. 
- 
Apotransferrin®: Pas d’effet secondaire connu à la dose utilisée. 
- 
Neorecormon®: formation de caillot, épilepsie, augmentation de la pression 
artérielle. 
- 
Remicade®: Réaction cutanée, trouble sanguin, réactions allergique et 
anaphylactique. 
- 
 Flolan®: Chute de la pression artérielle, augmentation du rythme cardiaque, 
difficulté respiratoire. 
 
Finalement, des effets secondaires inattendus liés à l’administration séquentielle de ces 
médicaments ne peuvent être complètement exclus. 
 
 
Avantages 
 
Chaque médicament inclus a prouvé son efficacité dans la réduction des lésions 
d’ischémie reperfusion. Leur administration simultanée durant la greffe de foie devrait 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 152 
 
avoir un effet bénéfique mais que nous ne pouvons prédire avec certitude un effet 
bénéfique.  
 
Assurances 
 
En accord avec la loi belge concernant les études cliniques du 7 mai 2004, il va de soi que 
le promoteur de l’étude est considéré comme étant responsable de tout dommage 
encouru qu’il soit directement ou indirectement lié à l’étude.  
 
Frais 
 
Il n’y aura aucun coût supplémentaire pour vous, aussi bien pour les médicaments inclus 
que pour tout échantillon (sang, urine et biopsie) considéré comme étant en dehors de la 
pratique clinique habituelle.  
 
Anonymat-Protection de la vie privée 
 
En accord avec les directives GCP (Good Clinical Practice) et la loi de la protection de la 
vie privée du 8 décembre 1992, votre identité et votre participation à l’étude restera 
strictement confidentielle. Vous ne serez jamais identifié dans les données, les résultats 
ou publications liés à l’étude si ce n’est par un numéro unique qui désignera vos données 
personnelles. Toutes ces données seront traitées manuellement ou électroniquement. 
Seuls les représentants du promoteur ainsi que les autorités compétentes pourront avoir 
accès à votre dossier dans l’unique but de contrôler l’étude. Vos données personnelles 
pourront donc être transmises aux autorités compétentes tel que le comité d’éthique ou 
à d’autres médecins participant dans le cadre de l’étude. 
A tout moment, vous avez le droit de consulter vos données personnelles et de demander 
que des corrections éventuelles y soient apportées.  
  
Information au sujet d’une modification 
 
Vous serez informés de tout potentiel changement concernant l’étude ou les 
médicaments. Dans ce cas, vous serez invités à signer un nouveau consentement éclairé. 
 
Comité d’éthique 
 
Le Comité d'éthique médicale indépendant de la KU/UZ Leuven, ainsi que les comités 
locaux d’éthique médicale des centres où cette étude se poursuit, ont évalué et approuvé 
l’étude. En aucun cas vous ne devez considérer cet avis favorable comme une incitation à 
participer à cette étude.  
 
Personnes à contacter en cas de questions liées à l’étude 
 
Si vous avez des questions concernant un potentiel dommage, vos données personnelles 
ou vos droits en tant que participant, n’hésitez pas à nous contacter que ce soit avant la 
greffe, durant ou après votre hospitalisation. 
 
Investigateur principal: Prof. Dr. Diethard Monbaliu 
Adresse: Abdominal Transplant Surgery, UZ Leuven 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 153 
 
              Herestraat 49 bus 7003 
              3000 Leuven 
Téléphone secrétariat: +32 16 348727 
Fax secrétariat: +32 16 348743 
 
Coordonatrice des essais cliniques : Sarah Mertens  
 
Adress : Abdominal Transplant Surgery, UZ Leuven 
                Herestraat 49 bus 7003 
 
3000 Leuven 
Téléphone : +32 16 342961 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 154 
 
Informationsbroschüre 
 
 
 
 
Patienten-Nr:  
 
Sponsor: 
University 
Hospitals 
Leuven, 
Herestraat 
49, 
3000 
Leuven 
 
 
Sie werden gebeten, sich an einer klinischen Studie zu beteiligen, bei der mehrere 
Medikamente mit dem Ziel verabreicht werden, die während einer Transplantation 
auftretenden Leberschäden (den so genannten Ischämie-Reperfusions-Schaden) zu 
reduzieren. Auftraggeber dieser Studie ist UZ KU Leuven. Die Studie wird in mehreren 
nationalen und internationalen Krankenhäusern durchgeführt. 
Lesen Sie dieses Dokument bitte sorgfältig durch, bevor Sie beschließen, ob Sie an der Studie 
teilnehmen möchten. Die Ziele, notwendigen Untersuchungen, Vor- und Nachteile, Risiken 
und Unannehmlichkeiten, die mit dieser Studie verbunden sind, werden in der 
Informationsbroschüre und in der Einwilligungserklärung ausführlich beschrieben. Die Studie 
ist ergebnisoffen. Sie haben jederzeit das Recht, Fragen zu stellen über die möglichen und 
bestehenden Risiken, die mit einer Teilnahme an dieser Studie verbunden sind. 
 
Zielsetzung und Beschreibung der Studie 
  
Während der chirurgischen Entfernung (Explantation), der Konservierung und der 
Transplantation der Leber kann dieses Organ beschädigt werden. Diese Beschädigung ist zum 
Teil das Resultat des Sauerstoffmangels (auch als Ischämie bezeichnet), der im Zeitraum der 
Konservierung der Leber (zwischen der chirurgischen Entfernung des Spenderorgans bis hin 
zur 
Transplantation) 
unvermeidbar 
ist. 
Der Ischämie-Schaden kann nach Wiederherstellung der Blutversorgung der Leber im 
Empfänger sogar noch zunehmen (dieses wird als Ischämie-Reperfusionsschaden bezeichnet). 
Der auftretende Organschaden kann minimal sein, ist aber in seltenen Fällen auch ernst oder 
sogar lebensbedrohlich. Eine Verringerung des Ischämie-Reperfusionsschadens ist daher von 
großer Bedeutung, weil es die Erfolgsaussichten der Lebertransplantation verbessern kann. 
 
Der Ischämie-Reperfusionsschaden wird durch eine komplexe Kaskade von sich gegenseitig 
verstärkenden 
Prozessen 
verursacht. 
Der Zweck dieser Studie ist es daher, den Ischämie-Reperfusionsschaden nach einer 
Lebertransplantation 
zu 
vermindern 
oder 
sogar 
minimieren. 
 
Um all diese schädlichen Prozesse zu blockieren, planen wir, verschiedene Medikamente zu 
verabreichen, so dass die Wahrscheinlichkeit für einen Erfolg so groß wie möglich ist. Diese 
Medikamenten-Kombination wird zum ersten Mal am Menschen angewendet und während 
der Lebertransplantation verabreicht werden. Während der Medikamentengabe werden Ihr 
Blutdruck, Ihre Herzfrequenz und Ihre Temperatur kontinuierlich gemessen. 
 
Medikamentöse Strategien zur Reduktion des während einer 
Lebertransplantation auftretenden Leberschadens  


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 155 
 
Das Ziel dieser Studie ist es, die Wirksamkeit dieser Medikamente zur Verringerung des 
Ischämie-Reperfusionsschadens zu demonstrieren. Es handelt sich um eine randomisierte, 
kontrollierte Studie. In einer ersten, sogenannten "Sicherheit, Phase A" dieser Studie 
konnten keine ernsthaften Nebenwirkungen der kombinierten Verabreichung dieser 
Arzneimittel beobachtet werden. 
Wenn Sie sich entscheiden sollten, an dieser Studie teilzunehmen, wird per Los entschieden 
(„Randomisation“), ob oder ob nicht bei Ihnen zusätzlich zur Standardbehandlung die 
Kombination der folgenden Medikamente verabreicht wird: 
- Cetor®/Cinryze®: C1-Esterase-Hemmstoff. Der C1-Esterase-Hemmstoff spielt eine wichtige 
Rolle in der Entzündungsreaktion und Blutgerinnung. 
- Atenativ®: Hemmstoff der Blutgerinnung, entzündungshemmend. 
- Vitamin E Lösung®: Vitamin E schützt vor den schädlichen Auswirkungen von Sauerstoff. 
- Tationil® 600 und Circadin®: Schützen vor den schädlichen Auswirkungen von Sauerstoff. 
- Apotransferrin: Bindet Eisen und bietet auch Schutz vor den schädlichen Auswirkungen von 
Sauerstoff. 
- Neorecormon®: Bietet Schutz gegen Zelltod und die schädlichen Auswirkungen von 
Sauerstoff, führt zu einer Verringerung der Entzündung-Signale. 
- Remicade®: Verringerung der Entzündung-Signale. 
- Flolan®: Entzündungshemmend, blockiert die Aktivierung von Blutplättchen und erweitert 
Blutgefäße. 
 
72 Patienten werden an dieser Studie teilnehmen. Die Randomisierung wird durch eine 
unabhängige Person vollzogen, die nicht in die Studie involviert ist. Die Patienten werden in 
jedem Teilnehmerzentrum durch die Verwendung von permutierten Blöcken variabler Größe 
in zwei Gruppen randomisiert.  
 
Zeitdauer der Studie 
 
Die Studie wird ein Jahr dauern. 
 
Untersuchungen 
 
- Wie bei jeder Lebertransplantation werden in den ersten 14 Tagen nach der 
Transplantation täglich und routinemäßig Blut- und Urinproben abgenommen werden. 3 und 
12 Monate nach der Lebertransplantation werden standardmäßig klinische Untersuchungen 
zur Nachbeobachtung durchgeführt.  
- Darüber hinaus werden auch drei Leber-Biopsien durchgeführt, die zum großen Teil 
Standard bei einer Lebertransplantation sind: 
  
• 1 Stunde vor der Implantation 
  
• von 1 Stunde nach der Reperfusion 
  
• 7 Tage nach der Transplantation (dies ist nicht Teil der Routine) 
- Zusätzlich zu den Leber-Biopsien, werden auch zwei Biopsien der Gallengänge und Probe 
des Gallensaftes abgenommen (während der Lebertransplantation). 
-Ein Jahr nach der Transplantation wird routinemäßig ein MRT (Kernspin-Tomogramm) 
durchgeführt, um die Qualität der transplantierten Leber zu überprüfen. 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 156 
 
Freiwillige Teilnahme 
 
Ihre Teilnahme an dieser Studie ist vollkommen freiwillig. Sie haben das Recht, die 
Teilnahme zu verweigern. Auch im Falle einer Ablehnung der Teilnahme erhalten Sie eine 
vollwertige medizinische Behandlung, und müssen keine nachteiligen Auswirkungen auf Ihre 
medizinische Versorgung und die Beziehung zu Ihrem behandelnden Arzt (Arzt-Patient-
Beziehung) fürchten. 
Wenn Sie sich entscheiden, an dieser Studie teilzunehmen, möchten wir sie höflich fragen, 
die Einverständniserklärung zu unterschreiben. 
Nachdem Sie die Einverständniserklärung unterschrieben haben, haben Sie das Recht, Ihre 
Teilnahme an der Studie jederzeit zu beenden. Sie müssen keinen Grund angeben, warum 
Sie Ihre Einwilligung widerrufen, an der Studie teilzunehmen. 
Der Prüfarzt kann zu jeder Zeit Ihre Teilnahme an der Studie beenden, und dies ohne Ihre 
Zustimmung. 
 
Risiken und Unannehmlichkeiten 
 
Von jedem Medikament wird nur eine Dosis (von EPO zwei Dosen) verabreicht werden. Nach 
ausführlicher Recherche der medizinischen Literatur scheint eine einmalige Verabreichung 
dieser Medikamente sicher zu sein. Dies wurde auch durch die Ergebnisse der „Safety-
Studie“ bestätigt. Dennoch können unerwartete Wechselwirkungen / potenzielle Risiken 
dieser Medikamenten- Kombination nicht vollständig ausgeschlossen werden. Folgenede 
Nebenwirkungen können während der Behandlung mit diesen Medikamenten auftreten: 
- Cetor®/Cinryze®: Allergische oder anaphylaktische Reaktionen (z.B. Herzrasen, hoher oder 
niedriger Blutdruck, Rötung am Hals und im Gesicht, Nesselsucht, Atembeschwerden) 
- Atenativ®: Allergische Reaktionen oder Überempfindlichkeit, erhöhte Körpertemperatur 
und Erweiterung von Blutgefäßen 
- Vitamin E Lösung®: keine bekannten Nebenwirkungen 
- Tationil® 600: keine bekannten Nebenwirkungen 
- Circadin®: keine bekannten Nebenwirkungen 
- Apotransferrin: keine bekannten Nebenwirkungen 
- Neorecormon®: Bildung eines Blutgerinnsels in einem Blutgefäß, Epilepsie und 
Bluthochdruck 
- Remicade®: Hautausschlag, Anomalien in der Zusammensetzung des Blutes und 
lebensbedrohliche allergische Reaktionen 
- Flolan®: Kopfschmerzen, Übelkeit, niedriger Blutdruck, langsamer Herzschlag, Herzrasen 
und Kurzatmigkeit 
 
Vorteile 
 
Wir können die therapeutische Wirkung der Medikamente nicht garantieren, auch wenn wir 
wissen, daß jedes Arzneimittel für sich den Ausmass des Ischämie-Reperfusionsschadens zu 
reduzieren vermag. 
 
Versicherung 
 
In Übereinstimmung mit der belgischen Gesetzgebung zu Untersuchungen an der 
menschlichen Person vom 7. Mai 2004 der Auftraggeber/Sponsor einer klinischen 


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Untersuchung haftbar für alle Schäden (auch im Falle einer Schuldlosigkeit), die dem 
Teilnehmer und / oder seinem Rechtsnachfolger entstanden sind und die direkt oder indirekt 
mit der Studie verbunden sind. Der Sponsor hat eine Versicherung abgeschlossen, die diese 
Haftung abdecken wird. Falls bei Ihnen als Folge Ihrer Teilnahme an dieser Studie ein 
Schaden auftreten sollte, wird Ihnen dieser Schaden daher in Übereinstimmung mit der 
belgischen Gesetzgebung zu Untersuchungen an der menschlichen Person vom 7. Mai 2004 
erstattet werden. 
 
Entschädigungen 
 
Durch diese Studie werden Ihnen keine zusätzlichen Kosten entstehen. Sie müssen die 
Studienmedikamente nicht bezahlen. Darüber hinaus werden alle Untersuchungen von Blut-, 
Urin- und Gewebeproben, die nicht routinemäßig durchgeführt werden, durch die 
Untersucher bezahlt. 
 
Der Schutz Ihrer Privatsphäre 
 
Ihre Identität und die Teilnahme an dieser Studie werden streng vertraulich behandelt 
werden. Sie werden nicht mit Ihrem Namen oder auf andere Weise in den Studien-
Dokumenten, Ergebnissen oder Veröffentlichungen identifiziert werden können. 
Ihre medizinischen Unterlagen werden von Vertretern der Prüfärzte und durch die 
Aufsichtsbehörden, die die Studie überwachen, untersucht werden. Dies erfolgt im Einklang 
mit den GCP = Good Clinical Practice - Richtlinien. Hierbei wird Ihre Identität durch eine 
einzigartige Patientennummer geheim gehalten, die lediglich Rückschluss auf wenige 
persönliche Daten erlaubt. 
Ihre personenbezogenen Daten können an die Aufsichtsbehörden, die Ethik-Kommission und 
andere Ärzte, die mit den Prüfärzten kooperieren, offen gelegt werden. 
Ihre persönlichen Daten werden elektronisch oder manuell verarbeitet und analysiert, um 
die Ergebnisse dieser Studie auszuwerten. Sie haben das Recht, vom Prüfarzt Auskunft über 
die gesammelten Daten und den Zweck dieser Daten zu erhalten. Sie haben auch das Recht 
auf Einsicht in Ihre persönlichen Daten und so nötig, zur Korrektur derselben. 
Alle Vorschriften zum Schutz personenbezogener Daten sind im Gesetz zum Schutz der 
Privatsphäre vom 8. Dezember 1992 (Belgien) beschrieben. 
 
Neue Informationen 
 
Manchmal ist es möglich, dass neue Informationen über die im Rahmen der klinischen Studie 
angewandten Therapien oder Arzneimittel bekannt werden. Wenn dies der Fall ist, werden 
Sie über diesen neuen Kenntnisstand informiert werden, wenn dies Ihre Bereitschaft zur 
weiteren Teilnahme an dieser Studie beeinflussen könnte. In diesem Fall werden Sie 
aufgefordert, eine neue Informationsbroschüre und eine neue Einverständniserklärung zu 
unterschreiben. 
 
Ethik-Kommission 
 
Die unabhängige medizinische Ethikkommission des „UZ KU Leuven / Forschung“ hat diese 
Studie geprüft und genehmigt, ebenso die lokalen Ethikkommissionen der Zentren, in denen 
diese Studie durchgeführt wird. 
 


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Kontaktperson für Fragen über diese Studie 
 
Wenn Sie glauben, Schäden durch diese Studie erlitten zu haben, eine unerwünschte 
Reaktion auf ein Studienmedikament durchgemacht zu haben oder Sie Fragen zu der Studie 
haben, können Sie sich jetzt, während und / oder nach der Studie, jederzeit an den leitenden 
Prüfarzt wenden: 
 
Principal investigator: Prof. dr. Diethard Monbaliu 
Adresse: Abdominale transplantatiechirurgie, UZ Leuven 
    Herestraat 49 bus 7003, 3000 Leuven 
Telefon Sekretariat: + 32 16 348727 
Fax Sekretariat: + 32 16 348743 
 
Studienkoordinator: Sarah Mertens 
Adresse: Abdominale transplantatiechirurgie, UZ Leuven 
                  Herestraat 49 Bus 7003, 3000 Leuven 
Telefon: + 32 16 342961 
 


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APPENDIX 9: CHRONOLOGICAL IDENTIFICATION LIST 
 
 
NAME 
UNIQUE 
NUMBER 
INITIALS 
DATE OF 
BIRTH 
AGE 
SEX 
DATE OF ENROLMENT 
WITHDRAWAL 
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  
  
 
  
  
  
  
  
  


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APPENDIX 10: CHRONOLOGICAL SCREENING LIST 
 
NAME 
INITIALS 
D.O.B 
AGE 
SEX 
DATE OF TRANSPLANTATION 
EXCLUSION CRITERIA 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  


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APPENDIX 11: PARTICIPATING CENTERS 
 
Center Country 
City 
Principal 
investigator 
1. 
Belgium 
Leuven 
D. MONBALIU 
2 
Belgium 
Liège 
N. 
Meurisse 
(local 
investigator) 
 
 


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APPENDIX 12: SIGNATURE PAGE 
 
 
Signature page 
 
 
 
“We, the signatories, confirm that this clinical trial protocol contains all the information 
and regulations necessary for the conduct of this particular trial. We sign the protocol as 
an agreement of the details of the clinical trial and the means of data recording. We 
commit ourselves to comply with all instructions, regulations and agreements as laid 
down in this clinical trial protocol and in the EC-GCP guidelines and applicable national 
laws and regulations. We certify that our local ethics committee has seen and accepted 
the protocol.” 
 
 
 
 
Name, date and signature of investigator 
 
 
 
 
 
 
 
Name, date and signature of co-investigator 
 
 


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APPENDIX 13: ANCILLARY STUDIES  
 
13.1 ANCILLARY STUDY 1: 
 
IMPACT OF MULTIFACTORIAL BIOMODULATION ON THE 
POST-REPERFUSION BILE TOXICITY AND BILE DUCT 
INJURY/REGENERATION 
 
 
 
In collaboration with Professor Robert Porte, dr. Alix Matton, Institute of Hepatobiliary 
Surgery and Liver Transplantation, University of Groningen, The Netherlands 
 
RATIONALE 
 
Non-anastomotic strictures (NAS) represent a major cause of morbidity, increased costs, 
graft loss and mortality after Liver Transplantation (LTx). NAS have been reported in 9%-
12% of LTx recipients but are less frequent after LTx using grafts from donation after 
brain death (DBD) donors: 1%-13% compared to grafts from donation after circulatory 
death (DCD) donors: 21%-33%.  The pathogenesis of NAS is complex and although not 
completely understood related to three different types of injury: (i) ischemia-related 
injury, (ii) immune-mediated injury and (iii) cytotoxic injury caused hydrophobic bile 
salts.   
 
Recent new insights on ischemia-reperfusion induced injury of the bile duct have also 
shown that considerable damages of the biliary epithelium occur already during the cold 
storage in a large proportion of liver grafts, with further injuries upon reperfusion.  
Lately, a new hypothesis of a concomitant insufficient regeneration of the bile epithelium 
after ischemia-reperfusion has been proposed as a pathophysiological mechanism. A 
niche of progenitor cells of the biliary epithelium can indeed be found in the peribiliary 
glands, from which the newly formed biliary epithelial cells can migrate and contribute to 
the restoration of the epithelial linings. The extent of the damage induced by ischemia-
reperfusion injury in this cell compartment may be related to the development of biliary 
strictures. 
 
Hepatic secretion of bile salts, phospholipids, and cholesterol is an active process 
mediated by specific hepatobiliary transporter proteins located in the canalicular cell 
membrane of hepatocytes. Although the bile salt export pump (gene ABCB11) is largely 
responsible for the secretion of bile salts, the multidrug resistance protein 3 (gene 
ABCB4) is responsible for the secretion of phospholipids. Experimental and clinical 
studies have shown that the expression and function of these transporters may be 
impaired after LTx, resulting in an altered bile composition (bile salt/phospholipid ratio) 
that has been associated with the NAS after LTx.  
A well-functioning graft immediately starts to secrete bile salts on graft reperfusion, but 
the overall biliary secretion of bile salts remains low within the first week after LTx. 
Restoration of biliary secretion of phospholipids, however, may recover even slower. 
These differences might be explained by differences in the expression and function of the 


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transporters bile salt export pump and multidrug resistance protein 3, resulting in a 
higher bile salt/phospholipid (BS/PL) ratio in the bile during the first days after LTx. The 
formation of protective mixed micelles is therefore impaired and free hydrophobic bile 
salts may cause injury of the cellular lipid membranes of cholangiocytes through their 
detergent activities. Compelling evidence that this high BS/PL ratio is correlated with the 
development of BS has been provided in experimental animal studies and prospective 
clinical trials. 
Finally, in our preclinical porcine LTx model, multifactorial biomodulation not only 
eliminated PNF but also resulted in an reduced bile toxicity or BS/PL ratio.  
We therefore assume that the multifactorial biomodulation will reduce the hepatic 
ischemia reperfusion injury and the resulting bile salt toxicity and the extent of injuries 
in the perbiliary gland niche, assuming a restoration of expression and function of the 
transporter proteins and a better epithelial regeneration response.  Finally this might 
reduce the incidence of NAS following LTx.  
 
AIM 
 
To investigate whether our multifactorial biomodulation reduces 
-  the alteration of expression and function of the transporter bile salt export pump and 
multidrug resistance protein 3 and thus resulting in a less toxic BS/PL ratio in the bile 
during the first hour after LTx,  
- reduces the injuries of the progenitor cells compartment in peribiliary glands. 
 
METHOD 
 
Collection of bile  
Bile samples will be collected within the first hour after reperfusion with a catheter 
inserted in the common bile duct. The bile duct anastomosis will be performed 
accordingly to the standard of care, therefore no changes in the surgical procedure is 
foreseen due to this ancillary study (cfr. section 5.1.1 of the main protocol). 
 
Bile analysis 
Total biliary bile salt concentration will be measured spectrophotometrically using 3α- 
hydroxysteroid dehydrogenase; biliary phospholipid concentration will be analysed using 
a commercially available enzymatic method (Wako Chemicals GmbH, Neuss, Germany). 
Bile Salt/Phospholipid ratio (BS/PL) therefore will be calculated and compared between 
the two study groups (multifactorial biomodulation vs. placebo) 
Biliary concentration of LDH, γGT (biomarkers of biliary epithelial cell injury) and bilirubin 
(biomarker of hepatocellular secretory function will measured using standard 
biochemical methods.  Biliary bicarbonate concentration  (biomarker of biliary epithelial 
cell function) will be assessed, by collecting bile samples under mineral oil and immediate 
analysis by an ABL800 FLEX analyzer (Radiometer).  
Hepatic gene expression of bile transporter 
In parallel with the measurement of bile composition, we will measure hepatic mRNA 
expression of the bile salt transporter (BSEP or ABCB11) and the phospholipid 
translocator multidrug resistance protein (MDR3 or ABCB4) on liver biopsies.  
 


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The timing of sample collections suit perfectly those required in the CAPITL protocol (S-
54348). Therefore, no extra tissue samples are needed for this ancillary study. 
Tissue will be collected exactly at the following time points:  
- 
Before the implantation at the bench. 
- 
1 hour post reperfusion  
- 
1 week after the transplantation. 
 
Tissue samples will be directly immerged in a solution of RNAlater® at room temperature 
(Ambion Corporation, Austin, TX), then stored at -80°C and they will be processed by real-
time PCR to investigate and to compare the expression of the two genes of interest (BSEP 
or ABCB11, MDR3 or ABCB4) in the two study groups (multifactorial biomodulation vs. 
placebo). 
 
Morphological Analysis of bile duct damage   
Two sample of bile duct tissue will be taken (i) at the beginning of the back-table 
preparation of the graft and (ii) after liver reperfusion, just before performing the bile 
duct anastomosis. All the samples will be anonymously labeled with the patient study 
number and date. 
 
The collected bile duct biopsies will be immediately preserved in 10% formaldehyde and 
processed for hematoxylin and eosin staining. The severity of injury of the biliary 
epithelium and of the peribiliary glands will be assessed based on the severity of 
detachment or disappearance of epithelial cells from the glands. 
 
REFERENCES 
1. Yska J M, Buis C I, Monbaliu D et al, Therole of bile salt toxicity in the pathogenesis of bile 
duct injury after non-heart-beating porcine liver transplantation. Transplantation 2008, 
85: 1625-1631. 
 
2. op den Dries S, Sutton M E, Lisman T, et al. Protection of bile ducts in liver 
transplantation, looking beyond ischemia. Transplantation 2011, 92: 373-379 
3. Sutton M E, op den Dries S, Koster M H, et al. Regeneration of human extrahepatic biliary 
epithelium: the peribiliary glands as progenitor cell compartment. Liver Int 2012, 32: 554-
559  
 
4. Karimian N, op den Dries S, Porte R J The origin of biliary strictures after liver 
transplantation: is it the amount of epithelial injury or insufficient regeneration that 
counts? Journal of Hepatology 2013, 58: 1065-1067 
 
5. Buis C I, Geuken E, Visser D S et al. Altered bile composition after liver transplantation is 
associated with the development of nonanastomotic biliary strictures. Journal of 
hepatology 2009, 50: 69-79. 
 
6. Geuken E, Visser D, Kuipers F et al. Rapid increase of bile salt secretion is associated with 
bile duct injury after human liver Transplantation. Journal of hepatology 2004, 41: 1017-
1025. 
 


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7. Hoekstra H, Porte RJ, Tian Y et al. Bile salt toxicity aggravates cold ischemic injury of bile 
ducts after liver transplantation in Mdr2 +/- mice. Hepatology 2006, 43: 1022. 
 
8. Chen G, Wang SG, Bie P et al. Endogenous bile salts are associated with bile duct injury in 
the rat liver Transplantation model. Transplantation 2009, 87: 330. 
 
9. Monbaliu D, Vekemans K, Hoekstra H et al. Multifactorial biological modulation of warm 
ischemia reperfusion injury in liver Transplantation from non-heart-beating donors 
eliminates primary nonfunction and reduces bile salt toxicity. Annals of Surgery 2009, 250: 
808. 
 
 
 


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13.2 ANCILLARY STUDY 2 
IMPACT OF MULTIFACTORIAL BIOMODULATION ON 
MECHANISMS OF ISCHEMIA REPERFUSION INJURY  
 
 
RATIONALE 
 
Ischemia reperfusion injury (IRI) is the results of the activation of a great number of 
transcription factors with variation in gene expression and signaling pathways. 
Indeed, IRI is mediated by several processes that lead to hepatocellular damage and apoptosis 
through interconnections between inflammation, coagulation, and innate/adaptive immune 
responses. This pathological condition is triggered when the liver is transiently deprived of 
oxygen during the organ procurement for transplantation and later re-oxygenated during 
reperfusion. Ischemia reperfusion activates Kupffer cells, which represent the main sources of 
vascular reactive oxygen species formation, cytokines and chemokines from reperfusion up to 
6 hours after restoration of the blood flow. Together with activated complement factors, 
these inflammatory mediators activate and recruit neutrophils into post-ischemic livers. This 
lead to enhanced reactive oxygen species production and additional proteases releases. 
Moreover, endothelin-1-mediated vasoconstriction of sinusoids promotes heterogeneous 
closure of micro-vessels, which in turn prolongs ischemia in certain areas of the liver.  
Beside the molecular mechanism of direct vascular and parenchymal injury, IRI is associated 
with an alteration in gene expression. These genes encode molecular pathways related to liver 
metabolism, inflammatory response, apoptosis, cell proliferation and liver protection. 
With the multifactorial biomodulation approach targeting several steps of the previously 
identified mechanisms of injury, we therefore assume that a reduction of inflammatory 
mediators, endothelial dysfunction markers, complement factors, redox active iron, 
apoptosis, Liver-fatty acid binding protein (L-FABP) will be observed. Moreover, a modification 
on the gene expression profile that characterizes the early response of the liver graft after the 
reperfusion is foreseen. 
 
 
AIM 
- 
To investigate whether our multifactorial biomodulation tackles specific features of 
inflammation, coagulation, complement activation, adaptive immunity, adhesion and 
apoptosis at a protein level. 
- 
To assess the impact of our multifactorial biomodulation on the gene expression 
profile that characterizes the early response of the liver graft after the reperfusion. 
 
 


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METHOD 
 
ANALYSIS OF BLOOD  
Blood samples will be taken before, during, and after transplantation. The timing of sample 
collections suit perfectly those required in the CAPITL protocol (S-54348). Therefore, no extra 
blood samples are needed for this ancillary study. 
 
Plasma will be collected at the following time points: 
- 
After anesthesia induction, just before skin incision. 
- 
Immediately prior to reperfusion (defined as opening of the portal vein). 
- 
30 minutes, 1 hr, 2 hr after reperfusion.  
- 
6h, 12h, 24h, 48, 72h and day 7 post transplantation 
 
The samples will be centrifuged and tubes with plasma will be anonymously labeled (patient 
study number, date and time) and frozen at -20°C. 
 
The following features will be assessed and compared between the two study groups 
(multifactorial biomodulation vs. placebo): 
- 
Inflammation mediators : TNF-α, IL-6, IL-8, IL-10, TGF-β, MCP-1, ICAM-1,  
HMGB-1 by ELISA. 
- 
Complement factors: C3a, C4a, C5a by ELISA. 
- 
Endothelial activation and dysfunction markers: vWF, Ang-1 and Ang-2 by 
ELISA. 
- 
Apoptosis marker: keratin 18 positive cells by ELISA.  
- 
Redox active iron will be measured by labile plasma iron assay. 
 
To avoid any discomfort to the patient regarding sample collection, blood samples will be 
taken either through a central or arterial line that is placed during anesthesia according to the 
standard of care or together with blood collections for laboratory tests performed during the 
post-operative course, according to the standard of care. 
 
ANALYSIS OF TISSUE 
No additional tissue samples are necessary besides those required for the CAPITL protocol (S-
54348). All the samples will be anonymously labeled with the patient study number and date. 
 
The timing of sample collections suit perfectly those required in the CAPITL protocol (S-54348). 
Therefore, no extra tissue samples are needed for this ancillary study. 
Tissue will be collected exactly at the following time points:  
- 
Before the implantation at the bench. 
- 
1 hour post reperfusion 
 


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The collected biopsy will be split into 3 pieces. One will be directly immerged in a solution of 
RNAlater® at room temperature (Ambion Corporation, Austin, TX) and then stored at -80°C; 
the second piece will be snap frozen in liquid nitrogen for protein preparation and the third 
will be preserved in formaldehyde 6%. 
mRNA LEVEL QUANTIFICATION 
Tissue stored in RNAlater® (Ambion Corporation, Austin, TX) at room temperature and then 
at -80°C will be processed by real-time PCR to investigate the gene expression. Genes that will 
be assessed are associated with some transcriptional alterations of liver metabolism, 
inflammatory response, apoptosis and liver protection: 
- 
SOD2: encoding factors involved in protection from oxidative stress-mediated 
injury. 
- 
STAT3: encoding for a transcription factor of hepato-cellular protection and 
regeneration. 
- 
iNOS: encoding for the protective inducible NO synthase. 
- 
COX-2: encoding for protective PTGS2. 
- 
Bcl-2: encoding for anti-caspase regulator. 
- 
Bax: encoding for pro-caspase regulator. 
- 
HO-1: encoding for a stress response protein believed to exert a protective 
function on both the development of IRI and graft rejection. 
- 
SELE: encoding for endothelial adhesion molecule 1. 
- 
IL1B: encoding for the cytokine IL-1B. 
- 
IL-6: encoding for the cytokine IL-6 
- 
CXCL2: encoding for the chemokine ligand 2.  
- 
ANGPT1: encoding for the angiopoietin 1. 
- 
PBEF1: encoding for a inflammatory cytokine that is anti-apoptotic in 
neutrophils and up-regulated in activated lymphocytes. 
- 
FosB: encoding for the redox sensitive transcription factor AP-1 
- 
JUN: involved in the TLR pathways. 
- 
IL10: encoding for the cytokine IL-10 
- 
CCL2: encoding for chemotactic agents MCP-1. 
- 
HSP70: encoding for heat shock protein. 
- 
ICAM1: encoding for adhesion molecule. 
- 
TNF- 
- 
ET-1: encoding for vasoconstrictor agent. 
 
Other genes of interest can be included and investigated accordingly to the results of the 
ancillary study number five (‘Impact of multifactorial biomodulation on gene expression 
involved in ischemia reperfusion injury by microarray’, cfr. relative protocol). 
 


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PROTEIN ASSAY 
Snap frozen biopsy will be homogenized in ice using a polytron homogenizer. Determination 
of tissue proteins will be assessed by using the plasma assay. Therefore, the tissue amount of 
TNF-, IL-6, IL-10, MCP-1 and Ang-1 will be investigated. 
 
APOPTOSIS 
The liver biopsies preserved in formaldehyde 6% will be embedded in paraffin. Apoptosis will 
be determined by staining of tissue with a monoclonal antibody against a specific cleavage 
site within cytokeratin 18 (M30 Cytodeath, Roche). 
 
REFERENCES 
1. Wang C F, Wang Z Y, Li J Y et al. Dual protective of HO-1 in transplanted liver grafts: a review 
of experimental and clinical studies. W J Gastroenterol 2011, 17: 3101. 
 
2. Borozan I, Chen L, Sun J et al. Gene expression profiling of acute liver stress during living 
donor liver Transplantation. Am J Transpl 2006, 6: 806-824. 
 
3. Conti A, Scala S, D’Agostino P et al. Wide expression profiling of ischemia reperfusion injury 
in human liver transplantation. Liver transplantation 2007, 13: 99-113. 
 
4. Defamie V, Cursio R, Le Brigand K et al. Gene expression profiling of human liver transplants 
identifies an early transcriptional signature associated with initial poor graft function. Am J 
Transpl 2008, 8: 1221-1236. 
 
 


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13.3  ANCILLARY STUDY 3 
 
INFLUENCE OF PERIOPERATIVE MACROPHAGE MIGRATION 
INHIBITORY FACTOR (MIF) RELEASE ON OUTCOME OF 
PATIENTS UNDERGOING ORTHOTOPIC LIVER 
TRANSPLANTATION 
 
In collaboration with prof. dr. Christian Stoppe, Institute of Biochemistry and Molecular Cell Biology, University 
Hospital Aachen, Germany 
 
In collaboration with prof. dr. Steffen Rex, Department of anesthesiology, University Hospitals Leuven & 
Department of Cardiovascular Sciences, KU Leuven 
 
 
RATIONALE 
 
Macrophage migration inhibitory factor (MIF) is a key proinflammatory cytokine and 
chemokine-like function cytokine that is rapidly released from various immune cells but 
also from endothelial cells, tissue macrophages, and certain parenchymal 
cells upon inflammatory and stress stimulation. MIF plays a nonredundant role in 
several inflammatory diseases, including sepsis, rheumatoid arthritis, obesity, and 
atherosclerosis. Because most of these diseases were ameliorated by genetic Mif 
deletion or MIF neutralization, anti-MIF therapies have been considered to be of 
potential clinical value. 
 
AIM 
 
- 
To investigate the influence of perioperative release of MIF, MIF-2, soluble receptor 
CD74 and MIF genotype on the severity of ischemia reperfusion injury during liver 
transplantation and the occurrence of postoperative organ function and outcome of 
patients. 
- 
To investigate the potential effect of the multifactorial modulation on the 
perioperative release of MIF. 
 
 
OUTCOME VARIABLES 
Primary outcome: 
- 
Association between circulating MIF, MIF-2 and sCD74 values and log-transformed 
peak AST (peak AST is defined as the highest value of serum AST within 72 hours days 
following LTx). 


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Secondary outcomes: 
- 
Significance of MIF genotype (low vs. high MIF expression) for organ (dys-) function 
- 
Significance of protein-kinase activation in hepatic tissue for organ (dys-)function 
- 
28 day mortality 
- 
Persistent organ dysfunction (PODS)+death a day 7 and day 28 
- 
Severe Acute Kidney Injury as defined by need for RRT 
- 
Length of stay in ICU (time to readiness of discharge, time of discharge from ICU, in 
hours and number of readmissions) 
- 
Length of stay in hospital (time postoperative until discharge from hospital, in days, 
and number of readmissions) 
- 
APACH II score during ICU stay 
- 
Severe kidney injury post-LTx as assessed by the RIFLE criteria 
 
Organ specific considerations: 
- 
Graft loss at 3 and 12months after LTx.  Graft loss is defined as the need for 
retransplantation within one week post LTx due to a non-life-sustaining liver graft 
function (primary non function) or later (other reason). 
- 
Recipient death at 3 and 12 months after LTx. 
- 
Early graft dysfunction as defined by Olthoff (1): the presence of one or more of the 
following postoperative laboratory analyses: bilirubin ≥ 10mg/dL on day 7, 
international normalized ratio ≥ 1.6 on day 7, and alanine aminotransferase (ALT) or 
AST > 2000 IU/L within the first 7 days. 
- 
Incidence of biliary strictures within 12 months post LTx: a biliary stricture is defined 
as a narrowing within the biliary tree, radiologically evident [endoscopic retrograde 
cholangio-pancreatography (ERCP) and/or magnetic resonance 
cholangiopancreatography (MRCP)] to cause clinical symptoms or biochemical 
abnormalities requiring intervention (ERCP, percutanous transhepatic 
cholangiographic (PTC) drainage, surgery, retransplantation). Biliary strictures are 
categorized as anastomotic or non-anastomotic based on the cholangiographic 
appearance of the biliary tree as judged by a blinded radiologist. Intra-hepatic biliary 
strictures are classified in 4 groups: unilateral focal, confluence, bilateral multifocal 
and diffuse necrosis (2). Beside the routine 1 year post-transplant assessment of the 
biliary tree by MRCP biliary strictures will be investigated in case of clinical or 
biochemical suspicion (based upon cholestasis). Other causes leading to cholestasis 
(e.g. hepatic artery thrombosis, bile leakage, rejection orcholangitis) will be excluded 
based on state- of-the-art radiological and histological examination as part of the 
routine standard treatment of care.  These examinations include ultrasound Doppler, 
CT and CT angiogram, biopsy-proven rejection based on histology scored by 2 blinced 
experienced liver pathologist according to the Banff criteria, ERCP and MRCP. 
- 
Ischemia/Reperfusion Injury (IRI): The extent of IRI will be assessed by a histological 
score based on the degree of cytoplasmic vacuolization, sinusoidal congestion, 
necrosis of parenchymal cells, apoptosis and influx of neutrophils as described by 
Suzuki score (3) and Monbaliu et al. (4). Liver biopsies will be taken before 
implantation (bench table), 1 hour after reperfusion and 1 week after 
transplantation.  All biopsies will be blindly scored by 2 blinded liver pathologists. 


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- 
Graft rejection: graft rejection will always be biopsy-proven; a liver biopsy is taken 
routinely 1 week after LTx and in case of clinical suspicion of acute rejection (AR).  
Clinical suspicion of AR may be based on clinical symptoms (such as jaundice, low-
grade fever) or sometimes nonspecific complaints (such as generalized malaise, 
decreased appetite) and/or biochemical abnormalities (usually increasing or 
plateauing levels -in an abnormal elevated range- of liver tests that were returning to 
normal values). Histological changes will be scored blindly according to the BANFF 
criteria by 2 experienced liver pathologists. 
- 
Surgical complications: the Clavien-Dindo classification (5) will be used to rank 
surgical complications within 30 days after LTx according to an objective, simple, 
reliable, and reproducible manner. This classification is based on the therapy 
required to treat the complication 
 
 
METHOD 
 
ANALYSIS OF BLOOD AND SERUM 
Blood and serum samples will be taken before, during, and after transplantation. The timing 
of sample collections suit perfectly those required in the CAPITL protocol (S-54348).  
 
Blood and serum samples will be collected at the following time points: 
- 
After anesthesia induction, just before skin incision. 
- 
Immediately prior to reperfusion (defined as opening of the portal vein). 
- 
30 minutes, 1 hr, 2 hr after reperfusion.  
- 
6h, 12h, 24h, 48, 72h after reperfusion. 
- 
Daily from day 4 till day 7. 
- 
3 and 12 months after transplantation.  
 
To determine MIF polymorphism, whole blood samples will be stored at -20°C until final 
transport/analysis.  
To determine MIF, MIF-2 and sCD74, serum samples will be centrifuged (3000 rpm, 10 min) 
and afterwards the supernatant will be transferred into 4 anonymously labeled cryotubes and 
frozen at -20°C until final transport/analysis.  
 
Perioperative inflammation will be assessed by the time course of: 
- 
White Blood cell count (WBC) 
- 
Procalcitonin (PCT) 
- 
C reactive protein (CRP) 
- 
interleukin (IL)-6 
- 
MIF 
- 
stromal cell-derived factor-1 (SDF-1) 
- 
D-dopachrome tautomerase (DDT, MIF-2) 
- 
soluble receptor CD74 (sCD74)  
 


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ANALYSIS OF TISSUE 
Tissue samples will be taken to assess activation of distinguished protein kinases by the 
means of western-blot (pAMPK, pAKT, pERK1/2, pJNK, p38, HIF-1alpha).  
No additional tissue samples are necessary for this ancillary study besides those required for 
the CAPITL protocol (S-54348). All the samples will be anonymously labeled with the patient 
study number and date. 
 
Tissue will be collected exactly at the following time points:  
- 
Before the implantation at the bench. 
- 
1 hour post reperfusion  
- 
1 week after the transplantation. 
 
Tissue samples will be directly immerged in a solution of RNAlater® at room temperature 
(Ambion Corporation, Austin, TX), then stored at -80°C. 
 
 
DATA COLLECTION 
Besides blood and tissue samples, a few parameters have to be collected during the 
surgery (after skin incision) and at the admission to the ICU: 
- 
Hemodynamics (SAP, DAP, MAP, HR, CVP, CO, SvO2, SPAP, DPAP, MPAP) 
- 
Total requirement of perioperative fluids/blood products (red blood cells, 
fresh frozen plasma, platelets) 
 
 
 
 
 


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13.4  ANCILLARY STUDY 4 
IMPACT OF THE MULTIFACTORIAL BIOMODULATION ON THE 
KIDNEY INJURY AFTER LIVER TRANSPLANATION 
 
 
RATIONALE 
 
Renal dysfunction is a frequent complication after liver transplantation and has a significant 
impact on recipient morbidity and mortality.  The current standard tools to detect renal 
dysfunction – creatinine and creatinine clearance – result in a delayed and inaccurate 
diagnosis of kidney injury and renal dysfunction. The observational KILT Study (S-53174) has 
assessed, in its first phase, which were the reliable plasma/urine biomarkers of interest in the 
early detection of kidney injury and renal dysfunction after the liver transplantation. 
We now want to use these biomarkers to assess whether the multifactorial biomodulation 
applied in the CAPITL study decreases acute kidney injury after liver transplantation.  
 
AIM 
To evaluate the potential influence of the multifactorial biomodulation on kidney injury and 
renal dysfunction during and after liver transplantation, measuring the release of plasma and 
urine biomarkers previously assessed in the observational KILT Study (S-53174). 
 
SAMPLE COLLECTION  
Blood samples will be taken before, during, and after transplantation. The time point samples 
suit perfectly those required in the CAPITL protocol (S-54348). Therefore, no extra blood 
samples are needed for this ancillary study. 
 
Plasma and urine will be collected at the following time points: 
- 
After anesthesia induction, just before skin incision. 
- 
Immediately prior to the start of the anhepatic phase. 
- 
Immediately prior to reperfusion (defined as opening of the portal vein). 
- 
30 minutes, 1 hr, 2 hr after reperfusion. 
- 
6h, 12h after reperfusion. 
- 
Daily until postoperative day 5. 
 


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SAMPLE ANALYSIS 
Analysis will be performed in the lab of Abdominal Transplant Surgery. For all these 
biomarkers test kits are commercially available or have been developed in the lab of 
Abdominal Transplant Surgery. 
 
The concentration of 5 biomarkers will be determined in the peri-transplant period: 
Urine:  
1. Neutrophil gelatinase associated lipocalin (NGAL): present on neutrophils and whose 
expression is markedly increased in injured kidney proximal tubular epithelium. 
2. Heart-fatty acid binding protein (H-FABP): a small cytoplasmic fatty acid binding 
protein present in the distal tubular cells and released upon cellular damage. 
3. Liver-fatty acid binding protein (L-FABP): a small cytoplasmic fatty acid binding protein 
present in the proximal tubular cells and released upon cellular damage. 
4. Kidney injury molecule-1 (KIM-1): a transmembrane protein markedly over-expressed 
in proximal tubules after an ischemic insult. 
5. N-acetyl glucosaminidase (NAG): a lysomal enzyme whose urinary concentration is 
increased in response to a proximal tubular damage. 
 
Plasma:  
1. NGAL 
2. H-FABP 
 
To avoid any discomfort to the patient regarding sample collection, blood samples will be 
taken either through a central or arterial line that is placed during anesthesia according to the 
standard of care (and thus does not require extra phlebotomies).  These blood samples are 
taken together with blood collections for laboratory tests performed during the post-
operative course, according to the standard of care. Urine samples will be collected via a 
bladder catheter already inserted during anesthesia according to the standard of care. 
 
REFERENCES 
 
1. Mishra J, Ma Q, Prada A, et al. Identification of neutrophil gelatinase-associated lipocalin 
as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol 2003; 
14:2534-2543. 
2. Mori K, Lee HT, Rapoport D, et al. Endocytic delivery of lipocalin-siderophore-iron complex 
rescues the kidney from ischemia-reperfusion injury. J Clin Invest 2005; 115:610-621. 
3. Bolignano D, Donato V, Coppolino G, et al. Neutrophil gelatinase-associated lipocalin 
(NGAL) as a marker of kidney damage. Am J Kidney Dis 2008; 52:595-605. 


CAPITL 2016 
 
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4. Pelsers MM, Hermens WT, Glatz JF. Fatty acid-binding proteins as plasma markers of tissue 
injury. Clin Chim Acta 2005; 352:15-35. 
5. Yamamoto T, Noiri E, Ono Y, et al. Renal L-type fatty acid--binding protein in acute 
ischemic injury. J Am Soc Nephrol 2007; 18:2894-2902. 
6. Han WK, Bailly V, Abichandani R, et al. Kidney Injury Molecule-1 (KIM-1): a novel 
biomarker for human renal proximal tubule injury. Kidney Int 2002; 62:237-244. 
7. Liangos O, Perianayagam MC, Vaidya VS, et al. Urinary N-acetyl-beta-(D)-
glucosaminidase activity and kidney injury molecule-1 level are associated with adverse 
outcomes in acute renal failure. J Am Soc Nephrol 2007; 18:904-912. 
8. Bonventre JV, Vaidya VS, Schmouder R, et al. Next-generation biomarkers for detecting 
kidney toxicity. Nat Biotechnol 2010; 28:436-440. 
9. Cruz DN, Ricci Z, Ronco C. Clinical review: RIFLE and AKIN--time for reappraisal. Crit Care 
2009; 13:211. 
10. O'Riordan A, Wong V, McQuillan R, et al. Acute renal disease, as defined by the RIFLE 
criteria, post-liver transplantation. Am J Transplant 2007; 7:168-176. 
11. Ferreira AC, Nolasco F, Carvalho D, et al. Impact of RIFLE classification in liver 
transplantation. Clin Transplant 2010; 24:394-400. 
12. Chen J, Singhapricha T, Hu KQ, et al. Postliver transplant acute renal injury and failure 
by the RIFLE criteria in patients with normal pretransplant serum creatinine 
concentrations: a matched study. Transplantation 2011; 91:348-353. 
13. Ricci Z, Cruz D, Ronco C. Classification and staging of acute kidney injury: beyond the 
RIFLE and AKIN criteria. Nat Rev Nephrol 2011; 14: 1-8. 
14. Olthoff KM, Kulik L, Samstein B, et al. Validation of a current definition of early allograft 
dysfunction in liver transplant recipients and analysis of risk factors. Liver Transpl 
2010;16:943-949. 
 
 
 
 


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13.4 ANCILLARY STUDY 5 
 
DETERMINATION OF CHANGES IN GENE EXPRESSION AND 
ALTERED MOLECULAR PROCESSES IN THE LIVER AFFECTED 
BY THE MULTIFACTORIAL BIOMODULATION 
 
In collaboration with prof. ir. Ing. Jos F. van Pelt, Liver Research Facility, Labo Hepatology 
KU Leuven 
 
BACKGROUND  
Livers that have been exposed to a period of warm ischemia (WI) prior to transplantation (Tx) 
have an increased risk of primary non function (PNF), graft dysfunction and ischemic type 
biliary strictures all associated with Ischemia Reperfusion Injury (IRI). The mechanisms of 
organ damage after IRI have been studied extensively and consist of complex interactions 
involving multiple inflammatory pathways. The major contributors to IRI include production 
of reactive oxygen species, release of pro-inflammatory cytokines and chemokines, and 
activation of immune cells altogether causing inflammation and resulting in tissue damage (1). 
Interventions to limit this inflammatory cascade are considered beneficial for the outcome 
after transplantation.  In small animal models, single drugs have been investigated to target 
WI damage and IRI specifically and these showed a beneficial effect for the liver (2-5). In 
humans there are only a few small controlled studies that investigate the effect of single 
compounds targeting IRI in LTx (6-8).  
Our multifactorial biological modulation approach has been shown to eliminate PNF, improve 
liver function, reduce bile salt toxicity and increase recipient survival after transplantation in 
a model of WI/IRI-damaged porcine livers (9). In this model we investigated the molecular 
mechanisms of WI (10) and those responsible for the hepatoprotective effect of the 
multifactorial drug cocktail using microarray (11). We found that the number of differentially 
expressed genes between baseline and 1 hour after reperfusion was 686 in the biomodulation 
group and 325 in controls. The extra genes in the biomodulation group belonged 
predominantly to molecular pathways related to cytokine activity, apoptosis and cell 
proliferation. We identified 7 genes (IL6, IL8, JUN, MMP1, PTGS2/COX2, SERPINE1 and STAT3) 
that were suppressed group. These genes could be linked - in part - to the drugs administered. 
The Leuven multifactorial biomodulation protocol primarily resulted in the suppression of 
inflammation regulating genes in IRI.  Furthermore, new potential drugs targets could be 
identified. 
 
AIM 
Detailed study of the changes induced in gene expression in humans treated by the CAPITL 
protocol. 
Primary question:  Can we unravel the molecular processes that are affected by this 
combination of drugs, identify pathways etc in human liver transplantation. Can we 
understand the performance of the treatment at the molecular level? 
Secondary questions are:  


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- can we link early changes in transcription (after 1 hour) to gene expression after the first 
week and to function status after the first week? 
- can we link changes in transcription (after 1 week) to functional status after the first week? 
- can we identify groups of genes and processes that are not targeted by the drugs but based 
on literature potentially important in relation to organ damage, regeneration or function? In 
other words: can we identify additional drug targets for further improvement? 
 
METHODS 
We will use a microarray approach much in the same way as was used previously by us in 
several  large animal studies (10,11,12).  The most valuable information we expect to obtain 
from the paired observation for the individual patients. Microarray has the advantage that 
from a single sample a very broad and unbiased analysis can be made of all the processes that 
are affected by the treatment. The current bioinformatics protocols and the experience that 
we have developed within our group over the last 4-6 years with this approach will enable us 
to explore optimally the vast information that will come out of the microarrays (13, 14). 
Furthermore, the microarray is expected to independently validate and complement some of 
the other ancillary studies. 
 
Sample collection 
Liver biopsies will be taken at baseline, 1 hour after reperfusion and after one week. The 
tissues will immediately be put into a container containing allprotect® solution (to prevent 
RNA breakdown), mixed and stored at -80°C.  
 
The timing of sample collections suit perfectly those required in the CAPITL protocol (S-54348). 
Therefore, no extra tissue samples are needed for this ancillary study with no extra burden for 
the patients. 
 


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Microarray and data processing  
RNA will be extracted using Qiagen protocol and microarray hybridization procedure will be 
performed on Affymetrix Primeview arrays according to the manufacturer’s instructions 
(Affymetrix Inc, Santa Clara, CA USA). Analysis of the microarray data is done in the R 
programming environment, in conjunction with the packages developed within the 
Bioconductor project. Differential expression was assessed via the moderated t-statistic, 
implemented in the Limma package (version 3.12.1). To control the false discovery rate, 
multiple testing correction is performed by Benjamini-Hochberg’s method. Analysis will be 
performed at the Nucleomics Core Facility of VIB at KU Leuven. 
Pathway analysis 
Gene enrichment and pathway analysis is performed with the internet based DAVID 
Bioinformatics Resource 6.7 program suite. To investigate known and predicted protein-
protein interactions for the differentially expressed genes we use the String 9.1 program. 
Results will be analyzed in combination with clinical and histological evaluation that is part of 
the main protocol. 
 
REFERENCES 
 
1 
Klune JR, Tsung A. Molecular biology of liver ischemia/reperfusion injury: established 
mechanisms and recent advancements. Surg Clin North Am 2010;90(4):665-77. 
1 
Li JD, Peng Y, Peng XY, Li QL, Li Q. Suppression of nuclear factor-kappaB activity in 
Kupffer cells protects rat liver graft from ischemia-reperfusion injury. Transplant Proc 
2010;42(5):1582-6. 


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3 
Freitas MC, Uchida Y, Zhao D, Ke B, Busuttil RW, Kupiec-Weglinski JW. Blockade of 
Janus kinase-2 signaling ameliorates mouse liver damage due to ischemia and reperfusion. 
Liver Transpl 2010;16(5):600-10. 
4 
Liu YX, Jin LM, Zhou L, Xie HY, Jiang GP, Wang Y, et al. Mycophenolate mofetil 
attenuates liver ischemia/reperfusion injury in rats. Transpl Int 2009;22(7):747-56 
5 
Oshima K, Yabata Y, Yoshinari D, Takeyoshi I. The effects of cyclooxygenase (COX)-2 
inhibition on ischemia-reperfusion injury in liver transplantation. J Invest Surg 
2009;22(4):239-45. 
6 
Baskin-Bey ES, Washburn K, Feng S, Oltersdorf T, Shapiro D, Huyghe M, et al. Clinical 
Trial of the Pan-Caspase Inhibitor, IDN-6556, in Human Liver Preservation Injury. Am J 
Transplant 2007;7(1):218-25. 
7 
Bogetti D, Sankary HN, Jarzembowski TM, Manzelli A, Knight PS, Thielke J, et al. 
Thymoglobulin induction protects liver allografts from ischemia/reperfusion injury. Clin 
Transplant 2005;19(4):507-11. 
8 
St Peter SD, Post DJ, Rodriguez-Davalos MI, Douglas DD, Moss AA, Mulligan DC. 
Tacrolimus as a liver flush solution to ameliorate the effects of ischemia/reperfusion injury 
following liver transplantation. Liver Transp. 2003;9(2):144-9.l 
9 
Monbaliu D, Vekemans K, Hoekstra H, Vaahtera L, Libbrecht L, Derveaux K, et al. 
Multifactorial biological modulation of warm ischemia reperfusion injury in liver 
transplantation from non-heart-beating donors eliminates primary nonfunction and  
reduces bile salt toxicity. Ann Surg 2009;250(5):808-17. 
10 
Vekemans K, Balligand E, Liu Q, Heedfeld V, Wylin T, Monbaliu D, Pirenne J, van Pelt 
J. Differential gene expression profile of porcine livers subjected to warm ischemia alone. 
Transplant Proc. 2011 Nov;43(9):3460-4. 
11 
Vekemans K, Monbaliu D, Balligand E, Heedfeld V, Jochmans I, Pirenne J, van Pelt J. 
Improving the function of liver grafts exposed to warm ischemia by the Leuven drug 
protocol: exploring the molecular basis by microarray. Liver Transpl. 2012 Feb;18(2):206-
18.  
12 
van Malenstein H, Wauters J, Mesotten D, Langouche L, De Vos R, Wilmer A, van Pelt 
J. Molecular analysis of sepsis-induced changes in the liver: microarray study in a porcine 
model of acute fecal peritonitis with fluid resuscitation. Shock. 2010 Oct;34(4):427-3 


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13 
van Malenstein H, Verslype C, Windmolders P, van Eijsden R, Nevens F, van Pelt J. 
Characterization of a cell culture model for clinically aggressive hepatocellular carcinoma 
induced by chronic hypoxia. Cancer Lett. 2012 Feb 28;315(2):178-88. 
14 
du Plessis J, van Pelt J, Korf H, Matthieu C, Lanoo M, et al. Gene expression in 
subcutaneous and visceral fat predicts liver histology in morbidly obese patients. Submitted 
 
 


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13.6 ANCILLARY STUDY 6 
 
IMPACT OF MULTI-FACTORIAL BIOMODULATION ON THE POSTREPERFUSION 
SYNDROME  
In collaboration with prof. dr. Steffen Rex, Department of anesthesiology, University 
Hospitals Leuven & Department of Cardiovascular Sciences, KU Leuven 
 
RATIONALE 
 
Despite the substantial decrease in perioperative mortality for liver transplantation (LTx) 
during the recent years, intra-operative major hemodynamic events remain a serious 
concern. These events are variable in presentation  and also depend on the phase of the 
operative procedure (e.g. pre-anhepatic, anhepatic and neo-hepatic phase).  These events 
and hemodynamic instability in particular are associated with adverse outcomes during 
the postoperative period.   
During the pre-anhepatic phase, hypovolemia might occur after incision and drainage of 
the abdominal cavity (if the patient suffers from ascites) whilst mobilization and 
dissection of the liver can lead to significant blood loss and hypovolemia.  During the 
anhepatic phase, the vascular inflow of the livers is occluded and hepatic ischemia begins.  
As a result from occluding the inferior vena cava and portal vein, venous return may 
diminish and cardiac output may decrease by 40-50%.  However, the period of greatest 
hemodynamic disturbances during LTx occurs at graft reperfusion, the so-called neo-
hepatic phase. Unclamping the portal vein and graft reperfusion leads to an abrupt 
increase in preload and at the same time a decrease of systemic blood pressure.  In 
extreme situations, hypotension (potentially aggravated by ongoing blood loss), 
bradycardia, supraventricular and ventricular arrhythmias and variable cardiac output, 
eventually with cardiac arrest, are observed.   
 
Hemodynamic instability due to the reperfusion is a commonly observed phenomenon.  
This so-called post-reperfusion syndrome (PRS) is defined as a 30% decrease of mean 
systemic blood pressure - compared to the previous value at the end of the anhepatic 
phase - for more than 1 minute during the first 5 min following graft reperfusion 
(unclamping the portal vein).    The mechanisms and pathophysiology of PRS are complex. 
The abrupt influx into the systemic and pulmonary circulation of desaturated blood, 
accumulated with high amounts of potassium, protons and metabolites as well as 


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inflammatory mediators/ vasoactive substances from the graft, minute air or thrombotic 
embolization are proposed to contribute to PRS.  This induces decreased peripheral 
vascular tone, reduced heart rat, impaired myocardial contractility.   
Moreover, ischemia reperfusion injury occurring in every liver transplant procedure 
inevitably elicits a complex inflammatory response that has been suggested to contribute 
to major hemodynamic alterations.  
 
 
AIM 
Primary outcome parameter:  
 
The incidence and severity of PRS 
Secondary objectives:  
- to determine clinical independent predictors of the occurrence of intraoperative 
PRS. 
- to investigate the link between the severity of PRS and patient and liver allograft 
short-outcome. 
PRS will be defined as a ≥30 % decrease of mean arterial blood pressure as compared to 
the pre-reperfusion values (immediately at the end of the anhepatic phase), lasting for at 
least 1 minute and upt to 5 miinutes after unclamping of the portal vein.  
 
METHOD 
No extra blood nor tissue samples are required in this ancillary study, only a detailed 
hemodynamic profile will be obtained collecting prospectively the following variables: 
 
- 
Heart rate (HR) 
- 
Mean arterial pressure (MAP) 
- 
Central venous pressure (CVP) 
- 
Mean pulmonary arterial pressure (MPAP) 
- 
Cardiac output (CO) 
- 
Stroke volume (SV) 


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- 
Systemic vascular resistance (SVR) 
- 
Pulmonary vascular resistance (PVR) 
- 
Pulmonary capillary wedge pressure (PCWP) 
- 
Mixed venous oxygen saturation 
- 
Use and doses of recue medication including vasoactive agents, inotropes, 
calcium and fluids 
 
The abovementioned variables are part of the intra-operative hemodynamic monitoring 
according to the standard of care and do not require extra or specific measuring devices.  
The timing of data collections perfectly coincides with the blood sampling in the CAPITL 
protocol (S-54348). 
Data will be therefore collected at the following time points: 
- 
After anesthesia induction, just before skin incision 
- 
Immediately prior to anhepatic phase 
- 
Immediately prior to reperfusion (defined as opening of the portal vein) 
- 
1,3,5,10, 30 minutes, 1hr, 2 hrs after reperfusion 
 
REFERENCES 
1. Reich DL, Wood RK, Emre S et al. Association of intraoperative hypotension and 
pulmonary hypertension with adverse outcomes after orthotopic liver 
transplantation. J Cardiothorac Vasc Anesth 2003, 17: 699-702. 
2. Hilmi I, Horton C, Planinsic R et al. The impact of postreperfusion syndrome on 
short-term patient and liver allograft outcome in patients undergoing orthotopic 
liver transplantation. Liver transplantation 2008, 14: 504-508. 
3. Chung IS, Kim HY, Shin YH et al., Incidence and predictors of post-reperfusion 
syndrome in living donor liver transplantation. Clin Transplant 2011. 
4. Paugam-Burtz C, Kavafyan J, Merckx P et al. Postreperfusion syndrome during liver 
transplantation for cirrhosis: outcome and predictors. 
 
 


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APPENDIX 14: OVERVIEW OF SAMPLE COLLECTION DURING CAPITL STUDY  
 
Sample collection in the donor 
studies 
type 
time 
amount 
storage 
 
 
 
 
 
 
Bile duct 
biopsy 
During 
prelevation 
procedure 
1 mm thick 
Formol: +4°C 
(will be sent to 
pathology) 
 
Sample collection during liver transplantation 
(LTx) 
studies 
type 
time 
amount 
storage 
Main protocol 
inclusive KILT 
study 
blood 
Before incision 
till 2 h post 
reperfusion 
EDTA tube of 4 
cc 
-20°C 
 
urine 
Before incision 
till 2 h post 
reperfusion  
Urine tube of 
9cc 
-20°C 
 
Liver biopsies 
Before 
implantation, 
1 h post 
reperfusion 
0,5 cm on 0,5 
cm 
Formol: +4°C 
(will be sent to 
pathology) 
Bile salt toxicity bile 
recipient 
(within 1 h 
post 
reperfusion) 
Max  10 cc 
(depending) 
First on + 4°C, 
after LTx at -
80°C 
 
Bile duct 
biopsies 
before 
implantation, 
at end bench, 
30 min post 
reperfusion 
1 mm thick 
Formol: +4°C 
(will be sent to 
pathology) 
 
Liver biopsies 
Before 
implantation, 1 
h post 
reperfusion 
0,2 cm on 0,2 
cm 
+4°C on 
AllProtect and 
afterwards on -
80°C 
Mechanisms IRI blood 
Before incision 
till 2 h post 
reperfusion 
EDTA tube of 4 
cc 
-20°C 
 
Liver biopsies  
Before 
implantation, 1 
h post 
reperfusion 
0,2 cm on 0,2 
cm 
+4°C on 
AllProtect and 
afterwards on -
80°C 
MIF 
blood 
Before incision 
till 2 h post 
reperfusion 
EDTA tube of 4 
cc 
-20°C 


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Serum  
Before incision 
till 2 h post 
reperfusion 
Serum tube of 
10 cc 
 -20°C 
 
Liver biopsies 
Before 
implantation, 1 
h post 
reperfusion 
0,2 cm on 0,2 
cm 
+4°C on 
AllProtect and 
afterwards on -
80°C 
Post 
reperfusion 
syndrome 
---- 
---- 
---- 
---- 
Micro array 
Liver biopsies 
Before 
implantation, 1 
h post 
reperfusion 
0,2 cm on 0,2 
cm 
+4°C on 
AllProtect and 
afterwards on -
80°C 
 
Sample collection post liver transplantation 
(LTx) 
studies 
type 
time 
amount 
storage 
Main protocol 
inclusive KILT 
study 
blood 
6, 12, 24, 48, 
72 h post 
reperfusion, 
day 4-7 , day 
14, 3 and 12 m 
post 
EDTA tube of 4 
cc 
-20°C 
 
urine 
6, 12, 24, 48, 
72 h post 
reperfusion, 
day 4-7 , day 
14, 3 and 12 m 
post 
Urine tube of 
9cc 
-20°C 
 
Liver biopsies 
1 week 
Piece of needle 
biopsy 
(variable 
dependent) 
+4°C on 
AllProtect and 
afterwards on -
80°C 
Bile salt toxicity Liver biopsy 
1 week post 
Piece of needle 
biopsy 
(variable 
dependent) 
+4°C on 
AllProtect and 
afterwards on -
80°C 
Mechanisms IRI 
blood 
6, 12, 24, 48, 
72 h and day 7 
post 
reperfusion 
EDTA tube of 4 
cc 
-20°C 
MIF 
blood 
6, 12, 24, 48, 
72 h post 
reperfusion, 
day 4-7 , day 
EDTA tube of 4 
cc 
-20°C 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 188 
 
14, 3 and 12 m 
post 
 
Serum  
6, 12, 24, 48, 
72 h post 
reperfusion, 
day 4-7 , day 
14, 3 and 12 m 
post 
Serum tube of 
10 cc 
-20°C 
 
Liver biopsies 
1 week post 
Piece of needle 
biopsy 
(variable 
dependent) 
+4°C on 
AllProtect and 
afterwards on -
80°C 
Post 
reperfusion 
syndrome 
---- 
---- 
---- 
---- 
Micro array 
Liver biopsies 
1 week post 
Piece of needle 
biopsy 
(variable 
dependent) 
+4°C on 
AllProtect and 
afterwards on -
80°C 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


CAPITL 2016 
 
CAPITL Protocol version 15, 20160308 
Pagina 189 
 
APPENDIX 15: Reporting post-transplant medication 
 
Based on the short half-life of the study medication (see table 1, page 21), the short and 
unlikely interaction with the post-transplant medication, the complexity of the post- liver 
transplant process, the timewindows for the primary endpoint (peak AST defined as the 
highest value of serum AST within 72 hours post-transplant) and the secondary 
endpoints, the reporting of post-transplant medication in the electronic Case Report Form 
will be as follow: 
- 
All medication will be reported from day 0 to day 7 post-transplantation  
o All post-transplant medication drips administered in the intensive care unit 
as part of the standard treatment of care (e.g. analgesia, sedation, insulin, 
etc…), will be reported using the total dose administered during 24 hours.  
o All post-transplant medication including vasopressors and inotropics, will 
be reported using the total dose administered during 24 hours. 
- 
For the reporting of the post-transplant medication, the  same medication label 
will be used as found in the source documents.