Pilot Trial: The Effects of Intravenous Heme Arginate on HO-1 Expression and Oxidative Stress in the Human Heart
Phase: PHASE2
Conditions: Myocardial Ischemia
Sponsor: Martin Andreas, M.D.

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
1 / 31 
 
 
 
CLINICAL STUDY PROTOCOL 
 
The effects of intravenous heme arginate on HO-1 expression and 
oxidative stress in the human heart 
 
Martin Andreas1, Martin Bilban2,3, Barbara Messner1, Dominik Wiedemann1, 
Guenther Laufer1, Alfred Kocher1, Michael Wolzt3 
 
1) Department of Cardiac Surgery, Medical University of Vienna 
2) Department of Laboratory Medicine, Medical University of Vienna 
3) Department of Clinical Pharmacology, Medical University of Vienna 
 
  
EudraCT: 2013-000887-27 
Version 2.0 – 8th May, 2013) 
 
Clinical investigator:  
 Prof. Dr. Alfred Kocher 
 
Sponsor: 
 Department of Cardiac Surgery, Medical University 
of Vienna 
 
Monitor: 
 TBA (Clinical Studies Support Center) 
 
Clinical Trial Centre: 
 Department of Cardiac Surgery 
 
 Medical University of Vienna 
 
 
 Währinger Gürtel 18-20, A-1090 Vienna 
 
Test Drug (IMP): 
 Heme arginate 
 
Study phase: 
 1 
 
 
 
Herr Martin Andreas
NCT number: NCT02314780

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
2 / 31 
Introduction 
 
Project background 
Ischemia and ischemia-reperfusion injury play a crucial role in today’s medicine1. 
Several approaches were developed to decrease tissue injury during or after 
ischemia in the animal model. However, despite major effort is put in new substances 
and techniques, the timely reperfusions remains the main approach to avoid 
sustained tissue damage2.  
 
Our working group focused on an already well-established concept based on data 
gathered in animal research, which was not further developed in the clinical setting 
until our first project. With the support of the Vienna Science and Technology Fund 
(Life Science Call 2007 – Therapy of ischema-reperfusion injury by heme oxygenase-
1 induction in skeletal muscle and ischemic kidney), we evaluated the induction of 
heme oxygenase – 1  (HO-1) by heme arginate. HO-1 is a well-known cellular protein 
protecting the tissue from ischemia-reperfusion injury3,4. We could provide data 
regarding HO-1 induction in peripheral blood mononuclear cells (PBMCs) as well as 
the characterisation of ischemia and ischemia-reperfusion injury in the healthy 
skeletal muscle5. Further, we established a human in-vivo model supporting the 
hypothesis of the protection induced by heme arginate in healthy human skeletal 
muscle after ischemia-reperfusion injury6.  
 
Our next step regarding the introduction of this promising therapy into daily clinical 
practice is the application of heme arginate in a distinct disease model. The human 
heart seems to be ideal as a target organ for HO-1 mediated protection. First, 
ischemic heart disease represents still one of the leading causes of death in the 
industrialist world1,7. An improved protection from ischemia may be beneficial in any 
kind of acute coronary syndromes regardless of the planned revascularisation 
strategy. In addition, several surgical procedures involving the heart and the great 
vessel necessitate some degree of myocardial ischemia, which may lead to adverse 
events. Improved protection from ischemia-reperfusion injury may therefore optimize 
the outcome of aneurysm surgery, valvular heart procedures as well as heart 
transplantation.  
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
3 / 31 
 
In conclusion, we intend to evaluate and optimize the induction of HO-1 in the human 
heart by heme arginate in this clinical project. This shall provide sufficient data to 
enable large-scale clinical trials for the evaluation of this promising therapeutic 
approach.  
 
Ischemia and Ischemia Reperfusion Injury 
Ischemic injury to muscular tissue is a common problem in cardiovascular medicine. 
Myocardial infarction and peripheral artery disease account substantially for morbidity 
and mortality in the industrialized world1,7. The most effective treatment to avoid 
ischemic damage is the rapid re-establishment of reperfusion. However, reperfusion 
itself can result in additional damage to ischemic tissue8. This phenomenon is called 
ischemia – reperfusion (IR) injury and is caused by different pathologic 
mechanisms8,9.  
One of the major pathologic mechanisms is the increased production of free oxygen 
radicals, thereby overwhelming the cellular antioxidant protection and damaging 
cellular proteins and membranes10. In a second step, neutrophil activation and 
invasion is suggested to be part of the deleterious cascade of IR injury11.  
 
Several strategies have been demonstrated to prevent IR injury in various 
experimental settings, including mechanical preconditioning and postconditioning, 
administering of antioxidant substances like vitamin C and treatment with endothelin 
receptor antagonists12-16. These experimental findings support the hypothesis that the 
reduction of reactive oxygen species is mandatory in the treatment of reperfusion 
injury. As a new approach in humans, the enzyme induction of heme oxygenase 1 
(HO – 1) may represent a potent strategy to diminish IR injury. 
 
On-pump coronary artery bypass grafting 
Coronary artery bypass grafting (CABG) is still the treatment of choice in patients 
with complex coronary artery disease or left main stenosis. The majority of cases are 
operated on-pump, leaving the heart without blood support for 1 to 2 hours. Cold 
blood cardioplegia is routinely used to protect the heart during this period. Although 
this method is very safe, regional and temporal ischemia still occurs. Therefore, we 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
4 / 31 
suggest that an antioxidant treatment like HO-1 induction prior to CABG may 
additionally protect the heart and improve early postoperative performance17. 
 
The role of heme oxygenase and bilirubin 
HO - 1 is the rate-limiting enzyme that catalyzes the degradation of heme b (Fe-
protoporphyrin-IX) into biliverdin (which is rapidly converted to bilirubin), carbon 
monoxide (CO) and iron18. It is a heat shock protein (HSP 32) and serves as a 
protective enzyme due to its anti-inflammatory, antioxidant, anti-apoptotic and 
antiproliferative actions19,20. HO-1 is expressed in several organs, in endothelial, 
epithelial, smooth muscle cells and the heart3,21-23. 
 
One hypothesis to explain the beneficial effects of induced HO-1 focuses on the 
pleiotropic cytoprotective actions of HO-1 and the products of heme degradation. 
These are potently anti-inflammatory and anti-oxidant. In addition, they modulate 
apoptotic and cell proliferative responses (but always in the direction of what appears 
to be a homeostasis-inducing response). Thus, after vascular injury, CO enhances 
the proliferation of endothelial cells (EC), which benefit return to homeostasis as the 
endothelium is more rapidly repaired, while blocking vascular smooth muscle cells 
proliferation, which prevents obliteration of the lumen of the vessel. Thus, these 
diametrically opposed actions are both homeostasis supporting. The same is true for 
apoptosis. HO-1 also impacts the immunological response by enhancing T cell 
mediated suppression (relatively more T regulatory cells) and suppressing the 
rejection response (fewer allo-aggressive T cells). 
Regarding the inflammatory cascades, IRI leads to increased leukocyte 
extravasation, which includes the production of the injurious chemokines and 
cytokines - such as tumor necrosis factor  (TNF)-α, interleukin (IL)-1β, and 
monocyte/macrophage chemoattractant protein (MCP)-1 - ultimately leading to early 
dysfunction or primary non-function of transplanted organs. In previous studies using 
animal models, induction of HO-1 resulted in diminished production of pro-
inflammatory cytokines in various IRI associated situations. We hypothesize that 
induced HO-1 expression results in diminished pro-inflammatory cytokine expression 
following IRI. Lastly, HO-1 acts as a therapeutic funnel in that a large number of 
physiologically active molecules (e.g. IL-10) appear to function by up-regulating HO-1 
with HO-1 then mediating, at least the anti-inflammatory, therapeutic actions of that 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
5 / 31 
molecule. Without HO-1, the molecule loses its anti-inflammatory effects. The 
beneficial effects of HO-1 stimulation on IRI has been evaluated in multiple organs 
studied in rodents and in pig studies in liver IRI. 
 
Pharmacologic concept 
A promising concept for the reduction of IRI is the induction of heme oxygenase 1 
(HO-1). Heme oxygenases are expressed in the cellular microsomes. HO-2 is the 
constitutively expressed form in humans. In contrast, HO-1 can be expressed in 
response to several stimuli, which are generally pro-inflammatory. As a new 
therapeutic approach in humans, the induction of the enzyme HO-1 may be 
employed to mitigate IRI20.  
Heat shock proteins’ transcriptional regulation responds to several chemical and 
biological stimuli. Although it has been shown that heme is such an inducer of HO-1 
24, the molecular steps and signal transduction pathways underlying HO-1 up-
regulation in general, and by heme, in particular, remain largely unclear 25,26. 
IRI may be attenuated by pharmacological HO-1 induction with heme arginate as 
shown in a rodent hemorrhagic shock model20,27-29. Previous data have confirmed 
dose-dependent induction of HO-1 mRNA and protein by heme arginate in venous 
blood of healthy subjects 5,30.  
There are two heme-containing drugs for intravenous human application available.  
Hemin is the ferriheme chloride. Heme arginate is the conjugation of hemin with L-
arginine in a solution of propylene glycol, ethanol and water. HA is commercially 
available from Orphan Europe Ltd. as Normosang® which is the approved form of 
heme in Europe for episodes of acute intermittent porphyria (AIP), variegate 
porphyria (VP) and hereditary coproporphyria (HCP). The usual dose of HA is 3 to 4 
mg/kg body weight given as intravenous infusion once daily over 4 to 7 days. 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
6 / 31 
Safety and tolerability 
Heme arginate has the same advantages as hemin with lesser side-effects. Most 
important, heme arginate is less harmful to the endothelium and thereby reduces 
vasculopathy and thrombotic side effects31-33. Further, no adverse effects on 
hemostasis are observed34. Acute toxicity occurs at doses of 48 to 70 mg/kg body 
weight (depending on animal species). 
 
Table 1: Known adverse reactions to heme arginate35 
frequency 
adverse reaction 
very common (> 10 %) 
difficult venous access (with repeated administration) 
common (1-10 %) 
injection site pain, injection site swelling, phlebitis at 
injection site 
uncommon (0.1-1 %) 
elevated serum ferritin concentration 
rare (0.01-0.1 %) 
fever, anaphylactoid reaction, allergic reactions 
(dermatitis medicamentosa, tongue edema) 
 
 
Pharmacokinetics 
Intravenously administered HA is bound to hemopexin and albumin and as heme-
hemopexin-complex transported to the liver. There it is degraded by HO-1 and 
biliverdin reductase to bilirubin and excreted into the bile. 
 
Table 2: Relevant pharmacokinetic parameters of heme arginate36 
Half-life (single dose) 
10.8 hours 
Volume of distribution 
3.37 litres 
Total plasma clearance 
3.7 ml/min 
 
 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
7 / 31 
 
Rationale of the study 
 
Ischemia reperfusion injury may be attenuated by HO-1 induction. Our previous data 
confirmed strong HO-1 induction in peripheral blood cells following heme arginate 
infusion in healthy humans5. Furthermore, we could demonstrate the amelioration of 
experimental ischemia reperfusion injury in the calf musculature by heme arginate in 
healthy subjects as measured by functional MRI6.  
Therefore, we propose that HO-1 induction in the human heart may be a suitable 
target to mitigate cardiac ischemia-reperfusion injury.  
The HO-1 induction will be assessed in a clinical trial by myocardial biopsy prior to 
and after aortic cross clamping in subjects with or without preceding heme arginate 
treatment in two different dosages. The HO-1 expression will also be measured in the 
clinical trials in peripheral blood mononuclear cells. As additional outcome, levels of 
myoglobin, creatine-kinase and troponin T and reactive oxygen species will be 
measured in plasma according to standard laboratory procedures. 
 
Risk/Benefit assessment 
The study drug heme arginate is approved in humans for acute episodes of hepatic 
porphyrias, used since 1985, and is well tolerated. The most commonly observed 
AEs of intravenous infusion of heme arginate are local reactions at the injection site. 
Known AEs are listed in detail in Table 1. 
Biopsies will be taken under direct vision from a superficial part of the right ventricle 
and oversewn immediately. Therefore, the risk of bleeding is very low because the 
site of biopsy can be observed after the administration of protamin until the sternum 
is closed. Right ventricular biopsies are routinely performed in heart-transplanted 
patients and are safe regarding ventricular dysfunction.  
There is a potential benefit for the participants although clinical data in humans are 
lacking until now. However, study related procedures do not lead to an increased 
length of stay for the patient. This study may provide new insights into the 
mechanisms of reperfusion injury and the protective capacity of HO-1 stimulation in 
cardiovascular pathologies. Therefore the risk / benefit assessment is acceptable. 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
8 / 31 
 
Study objectives 
1) To evaluate the effects of heme arginate administration in two different doses on 
myocardial HO – 1 induction  
2) To evaluate postoperative plasma levels of myoglobin, creatin-kinase, troponin T 
and reactive oxygen species according to treatment period.  
 
Investigational plan 
Design 
A placebo controlled, double blind study design with two treatment groups and one 
placebo group will be used for this purpose in 36 patients undergoing aortic valve 
replacement (AVR). Screening and drug administration will be performed on the day 
of hospitalisation. Subjects will be randomized to the study periods by a computer 
program (“Randomizer”). AVR will be performed 24 hours after heme arginate 
administration.  
Dosage and treatment duration 
The currently approved dose for heme arginate therapy in acute hepatic porphyrias is 
3 mg/kg once daily over 4 to 7 days. A single intravenous dose of 1 mg/kg will be 
used in this study for the first treatment cohort and a dose of 3mg/kg will be used for 
the second treatment cohort.  
Treatments 
All subjects will be randomized to receive a predefined treatment 24 hours prior to 
AVR surgery: 
 
- Placebo (12 patients): intravenous infusion of 110 ml 0.9 % NaCl solution. 
- Treatment cohort A (12 patients): intravenous infusion of 1 mg/kg heme arginate 
diluteded to 110 mL with 0.9 % NaCl. 
- Treatment cohort B (12 patients): intravenous infusion of 3 mg/kg heme arginate 
diluteded to 110 mL with 0.9 % NaCl. 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
9 / 31 
Inclusion criteria 
- 
Signed informed consent 
- 
Men and women aged between 40 and 85 years (inclusive) 
- 
Planed aortic valve replacement 
- 
Body mass index <35 kg/m2 
- 
Ability to communicate well with the investigator in the local language and to 
understand and comply with the requirements of the study 
Exclusion criteria 
Any of the following will exclude a subject from the study: 
- 
Known hypersensitivity to the study drug or any excipients of the drug 
formulation 
- 
Treatment with another investigational drug within 3 weeks prior to screening 
- 
History or clinical evidence of any disease and/or existence of any surgical or 
medical condition, which might interfere with the absorption, distribution, 
metabolism or excretion of the study drug 
- 
Severe renal failure (glomerula filtration rate < 30ml/min) 
- 
Moderately or severe impaired left ventricular function (ejection fraction < 
40%) 
- 
Moderately or severe impaired right ventricular function 
- 
Systolic pulmonary pressure > 45mmHg 
- 
Acute or recent (<7 days) myocardial infarction 
- 
Child bearing potential 
 
Description of study days 
Informed consent will be obtained after admission for AVR at the Medical University 
of Vienna. All patients are admitted with an internal approval for AVR and all 
necessary echocardiographic and laboratory data. Therefore, only a physical 
examination will be performed and subjects are directly sent for randomization and 
heme arginate or placebo infusion to the Department of Clinical Pharmacology. Blood 
pressure and heart rate will be obtained before and after drug infusion. Subjects will 
be scheduled for AVR on the day after infusion.  
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
10 / 31 
During surgery, a small biopsy (1-2mm) will be taken from the right ventricle prior to 
cross clamping and 10 minutes after protamin administration prior to chest closure. 
Biopsies will be performed under direct vision and every bleeding can be controlled 
directly. A blood sample (15 ml) will be taken prior to AVR, after chest closure as well 
as 24, 48 and 72 hours post surgery. Routine postoperative controls of creatine 
kinase, myoglobin and troponin T will be performed at admission to the ICU as well 
as 24, 48 and 72 hours post surgery. 
 
Study medication 
Substance: heme arginate (25 mg/mL NORMOSANG® vials á 10 mL) 
Manufacturer: Orphan Europe Ltd., Paris, France 
Placebo: 0.9 % NaCl solution 
 
Dosage and administration 
Dose: 1 mg/kg body weight (treatment cohort A); 3 mg/kg body weight (treatment 
cohort B) 
Preparation: the specified amount of NORMOSANG® is diluted to 100 mL with 0.9% 
NaCl solution (in glass bottles), preparation is done by the pharmacist / investigator 
or his / her designee under sterile conditions. 
Route: within 1 hour after preparation the study drug is administered intravenously in 
a cubital vein or large forearm vein via a transfusion set. 
Timing: the study drug will be administered between 9.00 and 11.00  
Administration: infusion is administered within 15 minutes, which correspond to an 
infusion rate of 400 ml/min. To ensure a correct timing an infusomat (IP 85-2, Döring, 
Munic, Germany) is used. The post-treatment infusion of a 0.9 % NaCl solution is 
administered over the same infusion line to rinse out heme arginate solution (20 mL 
filling volume of infusion line) and prevent loss of heme arginate. 
Post-treatment: infusion of 250 ml 0.9 % NaCl solution over 10-15 min. 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
11 / 31 
 
Treatment after end of study 
After end of the study further treatment will be performed at the Department of 
Cardiac Surgery until discharge from hospital. Thereafter, subjects will be sent to 
their cardiologist for routine control.  
 
Treatment assignment 
The assignment of number and code for the subject identification is based on the 
obligation for anonymity. Only their subject number, initials (first name and last name) 
and date of birth will identify subjects. 
 
Outcome Variables  
Main outcome variables 
HO – 1 mRNA and protein induction in the heart due to heme arginate pretreatment 
HO – 1 mRNA and protein induction in PMBC in patients undergoing cardiac surgery 
due to heme arginate pretreatment 
Evaluate dose-response of HO-1 induction in the heart 
 
Additional outcome 
Reactive oxygen species levels (Biopsies and blood samples) 
Postoperative plasma levels of creatine kinase, troponin T and myoglobin 
 
Safety variables 
Major cardiac adverse events 
 
Methods of evaluation 
 
HO-1 protein and mRNA levels in heart biopsies and PBMCs 
HO-1 levels will be analyzed in cardiac biopsies and peripheral blood mononuclear 
cells (PBMC). PBMC are considered effectors of oxidative stress-mediated injury and 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
12 / 31 
influenced by HO-1 expression. PBMCs will be isolated from EDTA-blood with Ficoll-
Plaque® (Amersham Biosiences, United Kingdom) prefilled tubes (Leusosep®, 
Greiner bio-one, Austria). Cell pellets and biopsy samples for HO-1 mRNA analysis 
are treated with lysis buffer (Buffer RLT, Qiagen Sciences, Maryland, USA) and 
immediately frozen on liquid nitrogen. Lysates and cell pellets are stored at -80°C 
until analysis. First-strand cDNAs will be synthesized from approximately 1 µg of total 
RNA with the use of MLV reverse transcriptase and random hexamer primers 
according to the manufacturer’s instructions (RT-PCR Core Kit; Takara Bio). For 
quantitative real-time polymerase chain reaction (RT-PCR), sense and antisense 
primers (Invitrogen, Paisley, Scotland) and fluorogenic probes (Eurogentec, Herstal, 
Belgium) for HO-1 and 18S will be used as described previously 37: for HO-1, the 
primers and probe used were 5'-GACTGCGTTCCTGCTCAACAT-3' (sense), 5'- 
GCTCTGGTCCTTGGTGTCATG-3' (antisense) and 5'-
TCAGCAGCTCCTGCAACTCCTCAAAGAG-3' (probe), generating a 75 base pair 
PCR product. For 18S, the primers and probe used were 5'-
CGGCTACCACATCCAAGG-3' (sense), 5'-CCAATTACAGGGCCTCGAAA-3' 
(antisense) and 5'-CGCGCAAATTACCCACTCCCGA-3' (probe), generating a 109 
base pair PCR fragment. The ABI PRISM 7700 (Applied Biosystems, Foster City, 
CA) will be used for PCR. Results are expressed as the target/reference ratio. The 
difference between the HO-1 mRNA levels of heme arginate and vehicle-treated cells 
are considered to reflect the capacity of cells to upregulate HO-1 mRNA, and were 
expressed as ΔHO-1 mRNA. 
For Western blot analysis the dry cell pellets will be transferred from -80°C to dry ice 
and the cells will immediately lysed with a lysis buffer (5X extraction reagent diluted 
with water) containing protease inhibitors. After centrifugation (14000 rpm for 15 min 
at 4 °C) the resulting supernatant will be used for measurements using a 
bicinchominic acid protein assay kit using a BSA standard (Thermo Scientific). 30 µL 
of a solution containing SDS loading buffer and mercaptoethanol are combined with 
50 µg of sample protein and lysis buffer up to 100 µL. Samples are heated at 93°C 
for 5 min and loaded onto a gel for protein separation. The gel will be run at 120 V for 
1.5-2 hours and then transferred to a Western Blot membrane (20 V for 1.5 hrs) 
using the semi-dry method. The membranes will be blocked in 5 % milk for 1 hour, 
washed three times for 5 min with TBST (tris-buffered saline tween-20) and 
incubated with the primary HO-1 antibody over night at 4 °C, washed and incubated 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
13 / 31 
with the second antibody for 1 hr. The membranes will be incubated for 5 min in 
substrate solution and imaged using clear X-ray films (Thermo Scientific). The 
antibody staining and development procedure are repeated again using an anit-β-
actin antibody for normalization of the results. 
 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
14 / 31 
 
Western Blot analyses 
Western blotting will performed as previously described.38 Equal amounts of protein 
will be loaded and Ponceau staining is performed to verify protein loading. Primary 
antibody used is anti-Heme-Oxygenase 1 (rabbit monoclonal antibody to heme-
oxygenase 1; Abcam, Cat. No.: 52947). Secondary antibody used is goat anti-rabbit 
IgG (Thermo Scientific, Cat. No.: 32460). Quantification of bands is performed using 
the Quantity One Software (Quantity one, Biorad).  
 
Reactive oxigen species 
The Reactive Oxygen Species (ROS) ELISA Kit (ABIN511031, Antibodies Online, 
Atlanta, USA) will be used according to the manufactorers specifications for ROS 
measurement.  
 
Statistical methodology and analysis 
Sample size considerations 
We could demonstrate a significantly improved reperfusion by 38% after 20 minutes 
ischemia in the skeletal muscle 24 hours after heme arginate administration in a 
sample size of 12 healthy subjects6. We used this data concerning the skeletal 
muscle for the sample size calculation of this trial. For a desired alpha of 0.05 and a 
power of 80%, the calculated sample size is 8. However, myocardial tissue may react 
in a different way to heme arginate administration. Therefore, the randomizer 
program will be programmed to distribute the first 24 patients evenly in all three 
groups. An intermediate evaluation of the outcome parameters will be performed by 
ANOVA (applying the Bonferroni correction). We hypothesize that the sample size of 
12 patients per group for this trial may be appropriate. If the intermediate evaluation 
provides significant results to answer the study endpoints, no additional subjects will 
be included. If not, a sample size calculation will be performed based on the results 
of the first 24 patients. If more than 36 patients are required to answer the proposed 
questions, an amendment will be formulated to the ethical board and the competent 
authority.  
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
15 / 31 
 
Safety and tolerability endpoints 
The treatment-emergent AEs will be tabulated (if possible, coded by using the 
MedDRA dictionary. Adverse events leading to premature discontinuation of study 
drug will be listed and summarized similarly to AEs. Treatment-emergent SAEs will 
be listed and summarized similarly to AEs, separately for treatment-emergent SAEs 
and SAEs occurring before study drug initiation and after study drug discontinuation. 
Reasons for premature discontinuation of study drug will be listed and summarized 
by frequency. 
Vital signs (blood pressure and pulse rate) will be summarized for the absolute 
values and for the change from baseline to each scheduled time point of 
measurement. Individual subject listings will be provided.  
 
Baseline parameters and concomitant medications 
Continuous demographic variables (age, height, weight, BMI) will be summarized by 
the usual location and scale statistics (mean, median, standard deviation, minimum, 
maximum and number of available observations). 
Qualitative demographic characteristics (race) will be summarized by counts and 
percentages. Other subject characteristics (medical history, physical examination 
findings, previous and concomitant medications) will only be listed. 
Previous and concomitant medications will be coded according to the WHO drug 
code and the ATC code. They will be summarized by type (i.e., previous and 
concomitant) by tabulating the number and percentages of subjects having received 
each treatment. 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
16 / 31 
Ethical and legal aspects 
The study will be conducted according to the principles of Good Clinical Practice and 
the Declaration of Helsinki and in agreement with Austrian laws and regulation. 
Project management and monitoring will be done by the KKS or a comparable 
institution. The study protocol shall be registered in a public database 
(clinicaltrials.gov). 
 
Informed consent of subjects 
Following comprehensive instruction regarding the nature, significance, impact and 
risks of this clinical trial, the patient must give written consent to participation in the 
study. 
During the instruction the patients are to be made aware of the fact that they can 
withdraw their consent – without giving reasons – at any time without their further 
medical care being influenced in any way. 
In addition to the comprehensive instructions given to the patients by the investigator, 
the patients also receive a written patient information sheet in comprehensible 
language, explaining the nature and purpose of the study and its progress. 
The patients must agree to the possibility of study-related data being passed on to 
relevant authorities. 
The patients must be informed in detail of their obligations in relation to the study 
insurance in order not to jeopardize insurance cover. 
 
Withdrawal and replacement of subjects 
Criteria for withdrawal 
 
Subjects may prematurely discontinue from the study at any time. Premature 
discontinuation from the study is to be understood when the subject did not undergo 
biopsy.  
Subjects must be withdrawn under the following circumstances: 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
17 / 31 
- at their own request 
- if the investigator feels it would not be in the best interest of the subject to continue 
- if the subject violates conditions laid out in the consent form / information sheet or 
disregards instructions by the study personal 
In all cases, the reason why subjects are withdrawn must be recorded in detail in the 
CRF and in the subject’s medical records. Should the study be discontinued 
prematurely, all study materials (complete, partially completed and empty CRFs) will 
be retained.   
Subjects prematurely discontinued from the study for any reason will be replaced. 
 
Follow-up of patients withdrawn from the study 
 
In case of premature discontinuation after study drug intake, the subjects will be 
closely followed until dismissal. They may request that from the time point of 
withdrawal no more data will be recorded and that all biological samples collected in 
the course of the study will be destroyed. 
 
Premature termination of the study 
 
The sponsor has the right to close this study at any time. The IEC and the competent 
regulatory authority must be informed. The trial will be terminated prematurely in the 
following cases: 
- If adverse events occur which are so serious that the risk-benefit ratio is not 
acceptable 
- If the number of dropouts is so high that proper completion of the trial cannot 
realistically be expected 
 
Safety definitions and reporting requirements 
Averse events (AEs) 
Summary of known and potential risks of the study drug 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
18 / 31 
Heme arginate has been used in humans to treat acute episodes of hepatic 
porphyrias since 1985 and is well tolerated. The most commonly observed AEs of 
intravenous infusion of heme arginate are local reactions. Known AEs are listed in 
detail in Table 2. 
 
Definition of adverse events 
 
An AE is any adverse change from the subject's baseline condition, i.e., any 
unfavourable and unintended sign including an abnormal laboratory finding, symptom 
or disease that occurs during the course of the study, whether or not considered 
related to the study drug.  
A treatment-emergent AE is any AE temporally associated with the use of a study 
drug, whether or not considered related to the study drug. 
 
 
Adverse events include: 
- Exacerbation of a pre-existing disease. 
- Increase in frequency or intensity of a pre-existing episodic disease or medical 
condition. 
- Disease or medical condition detected or diagnosed after study drug administration 
even though it may have been present prior to the start of the study. 
- Continuous persistent disease or symptoms present at baseline that worsen 
following the start of the study. 
- Lack of efficacy in the acute treatment of a life-threatening disease. 
- Events considered by the investigator to be related to study-mandated procedures. 
- Abnormal assessments, e.g., ECG and physical examination findings, must be 
reported as AEs if they represent a clinically significant finding that was not present 
at baseline or worsened during the course of the study. 
- Laboratory test abnormalities must be reported as AEs if they represent a clinically 
significant finding, symptomatic or not, which was not present at baseline or 
worsened during the course of the study or led to dose reduction, interruption or 
permanent discontinuation of study drug. 
 
Adverse events do not include:  
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
19 / 31 
- Medical or surgical procedure, e.g., surgery, endoscopy, tooth extraction, 
transfusion. However, the event leading to the procedure is an AE. If this event is 
serious, the procedure must be described in the SAE narrative. 
- Pre-existing disease or medical condition that does not worsen. 
- Situations in which an adverse change did not occur, e.g., hospitalizations for 
cosmetic elective surgery or for social and/or convenience reasons. 
- Overdose of either study drug or concomitant medication without any signs or 
symptoms. However, overdose must be mentioned in the Study Drug Log. 
 
Serious adverse events (SAEs) 
A Serious Adverse Event (SAE) is defined by the International Conference on 
Harmonization (ICH) guidelines as any AE fulfilling at least one of the following 
criteria: 
- Fatal (including fetal death). 
- Life-threatening – defined as an event in which the subject was, in the judgment of 
the investigator, at risk of death at the time of the event; it does not refer to an event 
that hypothetically might have caused death had it been more severe. 
- Requiring subject's hospitalization or prolongation of existing hospitalization – 
inpatient hospitalization refers to any inpatient admission, regardless of length of 
stay. 
- Resulting in persistent or significant disability or incapacity (i.e., a substantial 
disruption of a person’s ability to conduct normal life functions). 
- Congenital anomaly or birth defect. 
- Is medically significant or requires intervention to prevent at least one of the 
outcomes listed above.  
Life-threatening refers to an event in which the subject/subject was at risk of death at 
the time of the event. It does not refer to an event that hypothetically might have 
caused death if it were more severe. 
Important medical events that may not immediately result in death, be life-
threatening, or require hospitalization may be considered as SAEs when, based upon 
appropriate medical judgment, they may jeopardize the subject and may require 
medical or surgical intervention to prevent one of the outcomes listed in the 
definitions above. 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
20 / 31 
 
Hospitalization – Prolongation of existing hospitalization 
 
Hospitalization is defined as an overnight stay in a hospital unit and/or emergency 
room. 
An additional overnight stay defines a prolongation of existing hospitalization. 
The following is not considered an SAE and should be reported as an AE only: 
- Treatment on an emergency or outsubject basis for an event not fulfilling the 
definition of seriousness given above and not resulting in hospitalization. 
The following reasons for hospitalizations are not considered AEs, and therefore not 
SAEs: 
- Hospitalizations for cosmetic elective surgery, social and/or convenience reasons. 
- Standard monitoring of a pre-existing disease or medical condition that did not 
worsen, e.g., hospitalization for coronary angiography in a subject with stable angina 
pectoris. 
- Elective treatment of a pre-existing disease or medical condition that did not 
worsen, e.g., hospitalization for chemotherapy for cancer, elective hip replacement 
for arthritis. 
 
SAEs related to study-mandated procedures 
 
Such SAEs are defined as SAEs that appear to have a reasonable possibility of 
causal relationship (i.e., a relationship cannot be ruled out) to study-mandated 
procedures (excluding administration of study drug) such as discontinuation of 
subject's previous treatment during a washout period, or complication of a mandated 
invasive procedure (e.g., blood sampling, heart catheterization), or car accident on 
the way to the hospital for a study visit, etc. 
 
 
Suspected unexpected serious adverse reactions (SUSARs) 
 
SUSARs are all suspected adverse reactions related to the study drug that are both 
unexpected (not previously described in the SmPC) and serious.  
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
21 / 31 
Severity of adverse events 
The severity of clinical AEs is graded on a three-point scale: mild, moderate, severe, 
and reported on specific AE pages of the CRF. 
If the severity of an AE worsens during study drug administration, only the worst 
intensity should be reported on the AE page. If the AE lessens in intensity, no change 
in the severity is required.  
If an AE occurs during a washout or placebo run-in phase and afterwards worsens 
during the treatment phase, a new AE page must be filled in with the intensity 
observed during study drug administration.  
 
 
 
Mild 
Event may be noticeable to subject; does not influence daily activities; the AE 
resolves spontaneously or may require minimal therapeutic intervention;  
 
Moderate 
Event may make subject uncomfortable; performance of daily activities may be 
influenced; intervention may be needed; the AE produces no sequelae. 
 
Severe 
Event may cause noticeable discomfort; usually interferes with daily activities; subject 
may not be able to continue in the study; the AE produces sequelae, which require 
prolonged therapeutic intervention. 
 
A mild, moderate or severe AE may or may not be serious. These terms are used to 
describe the intensity of a specific event (as in mild, moderate, or severe myocardial 
infarction). However, a severe event may be of relatively minor medical significance 
(such as severe headache) and is not necessarily serious. For example, nausea 
lasting several hours may be rated as severe, but may not be clinically serious. Fever 
of 39°C that is not considered severe may become serious if it prolongs hospital 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
22 / 31 
discharge by a day. Seriousness rather than severity serves as a guide for defining 
regulatory reporting obligations. 
These definitions do not apply to clinically significant and asymptomatic laboratory 
test abnormalities or abnormal assessments (e.g., ECG findings) considered as AEs. 
The investigator should tick non-applicable on the AE page of the CRF to qualify the 
intensity of the AE. 
Relationship to study drug 
For all AEs, the investigator will assess the causal relationship between the study 
drug and the AE using his/her clinical expertise and judgment according to the 
following algorithm that best fits the circumstances of the AE: 
Unrelated: 
- May or may not follow a reasonable temporal sequence from administration of the 
study product 
- Is biologically implausible and does not follow known response pattern to the 
suspect study drug (if response pattern is previously known) 
- Can be explained by the known characteristics of the subject’s clinical state or other 
modes of therapy administered to the subject. 
 
Possibly related: 
- Follows a reasonable temporal sequence from administration of the study product. 
- May follow a known response pattern to the study drug (if response pattern is 
previously known). 
- May also be reasonably explained by the subject’s clinical state or other modes of 
therapy administered to the subject. 
 
Probably related: 
- Follows a reasonable temporal sequence form administration of the study drug. 
- May follow a known response pattern to the study drug (if response pattern is 
previously known). 
- Could not be reasonable not be reasonably explained by the known characteristics 
of the subject’s clinical state or other modes of therapy administered to the subject, if 
applicable. 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
23 / 31 
Definitely related: 
- Follows a reasonable temporal sequence form administration of the study drug. 
- Follows a known response pattern to the study drug (if response pattern is 
previously known). 
- No other reasonable cause is present. 
 
Reporting procedures 
A special section is designated to adverse events in the case report form. The 
following details must thereby be entered: 
- Type of adverse event 
- Start (date and time) 
- End (date and time) 
- Severity (mild, moderate, severe) 
- Serious (no / yes) 
- Unexpected (no / yes)  
- Outcome (resolved, ongoing, ongoing – improved, ongoing – worsening) 
- Relation to study drug (unrelated, possibly related, probably related, definitely 
related) 
Adverse events are to be documented in the case report form in accordance with the 
above mentioned criteria. 
 
Reporting procedures for SAEs  
 
In the event of serious, the investigator has to use all supportive measures for best 
patient treatment.   A written report is also to be prepared and made available to the 
clinical investigator within five days. The following details should at least be available: 
- Patient initials and number 
- Patient: date of birth, sex, ethical origin 
- The suspected investigational medical product (IMP) 
- The adverse event assessed as serious 
- Short description of the event and outcome 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
24 / 31 
Reporting procedures for SUSARs 
 
It must be remembered that the regulatory authorities, and in case of SUSARs which 
could possibly concern the safety of the study participants, also the Institutional 
Review Board / Independent Ethics Committee (IRB / IEC) are to be informed. Such 
reports shall be made by the study management and the following details should be 
at least available: 
- Patient initials and number 
- Patient: date of birth, sex, ethical origin 
- Name of investigator and investigating site 
- Period of administration 
- The suspected investigational medical product (IMP) 
- The adverse event assessed as serious and unexpected, and for which there is a 
reasonable suspected causal relationship to the IMP 
- Concomitant disease and medication 
- Short description of the event (description, onset and (if applicable) end, therapeutic 
intervention, causal relationship, hospitalization of prolongation of hospitalization, 
death, life-threatening, persistent or significant disability or incapacity 
Electronic reporting should be the expected method for reporting of SUSARs to the 
competent authority. In that case, the format and content as defined by Guidance 
{Module,  #68}  should be adhered to. The latest version of MedDRA should be 
applied. Lower level terms (LLT) should be used. 
Data handling procedures 
 
A case record form will be completed for each volunteer. Trained personnel will 
check the entries and any errors or inconsistencies will be resolved immediately. The 
results of the prestudy screening examination will be documented in the study 
masterfile. 
 
Acknowledgement / approval of the study 
The investigator will submit this protocol and any related document provided to the 
subject (such as subject information used to obtain informed consent) to the Ethics 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
25 / 31 
Committee (EC) of the Medical University of Vienna. Approval from the committee 
will be obtained before starting the study. 
Adverse events - whether serious and/or unexpected, and possibly endangering the 
safety of the study participants - are likewise to be reported to the EC. 
The clinical trial shall be performed in full compliance with the valid legal regulations 
according to the Drug Law (AMG - Arzneimittelgesetz) of the Republic of Austria. 
The study must be notified to the Austrian Agency for Health and Food Safety 
(AGES) and to the European Agency for the Evaluation of Medicinal Products 
(EMEA) and registered to the European Clinical Trial Database (EurdaCT) using the 
required forms. 
 
 
 
Protocol amendments 
 
Modifications made to the protocol after receipt of the EC/IRB approval must also be 
submitted as amendments by the investigator to the EC/IRB in accordance with local 
procedures and regulations. 
 
Finance and insurance 
During their participation in the clinical trial the patients will be insured as defined by 
legal requirements.  The principal investigator of the clinical trial will receive a copy of 
the insurance conditions of the 'proband insurance'. The sponsor is providing 
insurance in order to indemnify (legal and financial coverage) the investigator/center 
against claims arising from the study, except for claims that arise from malpractice 
and/or negligence. The compensation of the subject in the event of study-related 
injuries will comply with the applicable regulations. 
Details on the existing proband insurance are given in the patient information sheet. 
 
Confidentiality 
The information contained in this document, especially unpublished data, is the 
property of the sponsor of this study, the Department of Clinical Pharmacology, 
Medical University Vienna. It is therefore provided to you in confidence as an 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
26 / 31 
investigator, potential investigator, or consultant, for review by you, your staff, and an 
Ethics Committee or Institutional Review Board. It is understood that this information 
will not be disclosed to others without written authorization from the Department of 
Clinical Pharmacology, Medical University Vienna, except to the extent necessary to 
obtain informed consent from those persons to whom the study drug may be 
administered. 
 
Ethics and Good clinical Practice (GCP) 
The investigator will ensure that this study is conducted in full conformance with the 
principles of the "Declaration of Helsinki" (as amended at the 56th WMA General 
Assembly, Tokyo, Japan, 2004) and with the laws and regulations of the country in 
which the clinical research is conducted. 
The principal investigator of the clinical trial shall guarantee that only appropriately 
trained personnel will be involved in the study. All studies must follow the ICH GCP 
Guidelines (June 1996) and, if applicable, the Code of Federal Regulations (USA). In 
other countries in which GCP Guidelines exist, the investigators will strictly ensure 
adherence to the stated provisions. 
 
 
Documentation of study results 
A Subject Screening and Enrollment Log will be completed for all eligible or non-
eligible subjects with the reasons for exclusion. 
For each subject enrolled, regardless of study drug initiation, a CRF must be 
completed and signed by the principal investigator or co-investigator. This also 
applies to those subjects who fail to complete the study. If a subject withdraws from 
the study, the reason must be noted on the CRF. Case report forms are to be 
completed on an ongoing basis. 
All forms should be completed using a black pen and must be legible. Trained 
personnel will check the entries and any errors or inconsistencies will be checked 
immediately. Errors should be crossed out but not obliterated, the correction inserted, 
and the change initialled and dated by the investigator, co-investigator or study 
nurse. 
The monitor will collect original completed and signed CRFs at the end of the study. 
A copy of the completed and signed CRFs will remain on site. 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
27 / 31 
 
Safekeeping 
The investigator will maintain adequate and accurate records to enable the conduct 
of the study to be fully documented and the study data to be subsequently verified. 
These documents will be classified into two different categories: investigator's file, 
and subject clinical source documents. 
The investigator's file will contain the protocol/amendments, EudraCT forms, CRFs 
and data clarification and query forms, EC/IRB and Health Authority approval with 
correspondence, informed consent, drug records, staff curriculum vitae and 
authorization forms, screening and enrollment logs, and other appropriate 
documents/correspondence as per ICH/Good Clinical Practice (GCP) and local 
regulations. 
Subject clinical source documents include, but are not limited to subject 
hospital/clinic records, physician's and nurse's notes, appointment book, original 
laboratory reports, ECG, X-ray, pathology and special assessment reports, 
consultant letters, etc. 
These two categories of documents must be kept on file by the investigator for as 
long as needed to comply with national and international regulations (in Austria 15 
years after discontinuing clinical development or after the last marketing approval). 
No study document should be destroyed without prior written approval from the 
Department of Clinical Pharmacology. 
When source documents are required for the continued care of the subject, 
appropriate copies should be made for storing outside of the site. 
 
Monitoring 
The monitor will contact and visit the investigator regularly and will be allowed, on 
request, to have access to all source documents needed to verify the entries on the 
CRF and other protocol-related documents provided that subject confidentiality is 
maintained in agreement with local regulations. It will be the monitor's responsibility 
to inspect the CRF at regular intervals throughout the study, to verify the adherence 
to the protocol and the completeness, consistency and accuracy of the data being 
entered on them. The monitoring standards require full verification for the presence 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
28 / 31 
of informed consent, adherence to the inclusion/exclusion criteria, documentation of 
SAEs and the recording of the main efficacy, safety, and tolerability endpoints. 
 
Publication of study results 
The findings of this study will be published by the investigators in a scientific journal 
and presented at scientific meetings. The manuscript will be circulated to all co-
investigators before submission. 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
29 / 31 
 
Study time course 
 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
30 / 31 
 
References 
 
1. 
Cooper R, Cutler J, Desvigne-Nickens P, et al. Trends and disparities in coronary heart 
disease, stroke, and other cardiovascular diseases in the United States: findings of the national 
conference on cardiovascular disease prevention. Circulation 2000;102:3137-47. 
2. 
Morel O, Perret T, Delarche N, et al. Pharmacological approaches to reperfusion 
therapy. Cardiovasc Res 2012;94:246-52. 
3. 
Otterbein LE, Soares MP, Yamashita K, Bach FH. Heme oxygenase-1: unleashing the 
protective properties of heme. Trends Immunol 2003;24:449-55. 
4. 
Tsuchihashi S, Fondevila C, Kupiec-Weglinski JW. Heme oxygenase system in 
ischemia and reperfusion injury. Ann Transplant 2004;9:84-7. 
5. 
Doberer D, Haschemi A, Andreas M, et al. Haem arginate infusion stimulates haem 
oxygenase-1 expression in healthy subjects. Br J Pharmacol 2010;161:1751-62. 
6. 
Andreas M, Schmid AI, Doberer D, et al. Heme arginate improves reperfusion patterns 
after ischemia: a randomized, placebo-controlled trial in healthy male subjects. J Cardiovasc 
Magn Reson 2012;14:55. 
7. 
Lloyd-Jones DM, Larson MG, Beiser A, Levy D. Lifetime risk of developing coronary 
heart disease. Lancet 1999;353:89-92. 
8. 
Matsumura K, Jeremy RW, Schaper J, Becker LC. Progression of myocardial necrosis 
during reperfusion of ischemic myocardium. Circulation 1998;97:795-804. 
9. 
Eefting F, Rensing B, Wigman J, et al. Role of apoptosis in reperfusion injury. 
Cardiovasc Res 2004;61:414-26. 
10. 
Zweier JL, Talukder MA. The role of oxidants and free radicals in reperfusion injury. 
Cardiovasc Res 2006;70:181-90. 
11. 
Vinten-Johansen J. Involvement of neutrophils in the pathogenesis of lethal 
myocardial reperfusion injury. Cardiovasc Res 2004;61:481-97. 
12. 
Zhao ZQ, Vinten-Johansen J. Postconditioning: reduction of reperfusion-induced 
injury. Cardiovasc Res 2006;70:200-11. 
13. 
Pleiner J, Schaller G, Mittermayer F, Bayerle-Eder M, Roden M, Wolzt M. FFA-
induced endothelial dysfunction can be corrected by vitamin C. J Clin Endocrinol Metab 
2002;87:2913-7. 
14. 
Bayerle-Eder M, Pleiner J, Mittermayer F, et al. Effect of systemic vitamin C on free 
fatty acid-induced lipid peroxidation. Diabetes Metab 2004;30:433-9. 
15. 
Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal 
cell injury in ischemic myocardium. Circulation 1986;74:1124-36. 
16. 
Kharbanda RK, Peters M, Walton B, et al. Ischemic preconditioning prevents 
endothelial injury and systemic neutrophil activation during ischemia-reperfusion in humans 
in vivo. Circulation 2001;103:1624-30. 
17. 
De Backer D, Dubois MJ, Schmartz D, et al. Microcirculatory alterations in cardiac 
surgery: effects of cardiopulmonary bypass and anesthesia. Ann Thorac Surg 2009;88:1396-
403. 
18. 
Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to bilirubin 
by microsomal heme oxygenase. Proc Natl Acad Sci U S A 1968;61:748-55. 
19. 
Vitek L, Schwertner HA. The heme catabolic pathway and its protective effects on 
oxidative stress-mediated diseases. Adv Clin Chem 2007;43:1-57. 
20. 
Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic 
science to therapeutic applications. Physiol Rev 2006;86:583-650. 
Herr Martin Andreas

HO-1 in the Heart 
 
M. Andreas, M. Wolzt 
Department of Cardiac Surgery, MUV 
 
31 / 31 
21. 
Clark JE, Foresti R, Sarathchandra P, Kaur H, Green CJ, Motterlini R. Heme 
oxygenase-1-derived bilirubin ameliorates postischemic myocardial dysfunction. Am J 
Physiol Heart Circ Physiol 2000;278:H643-51. 
22. 
Terry CM, Clikeman JA, Hoidal JR, Callahan KS. TNF-alpha and IL-1alpha induce 
heme oxygenase-1 via protein kinase C, Ca2+, and phospholipase A2 in endothelial cells. Am 
J Physiol 1999;276:H1493-501. 
23. 
Kawamura K, Ishikawa K, Wada Y, et al. Bilirubin from heme oxygenase-1 attenuates 
vascular endothelial activation and dysfunction. Arterioscler Thromb Vasc Biol 2005;25:155-
60. 
24. 
Yoshida T, Biro P, Cohen T, Muller RM, Shibahara S. Human heme oxygenase cDNA 
and induction of its mRNA by hemin. Eur J Biochem 1988;171:457-61. 
25. 
Alam J, Killeen E, Gong P, et al. Heme activates the heme oxygenase-1 gene in renal 
epithelial cells by stabilizing Nrf2. Am J Physiol Renal Physiol 2003;284:F743-52. 
26. 
Alam J, Cook JL. Transcriptional regulation of the heme oxygenase-1 gene via the 
stress response element pathway. Curr Pharm Des 2003;9:2499-511. 
27. 
Ferenbach DA, Nkejabega NC, McKay J, et al. The induction of macrophage 
hemeoxygenase-1 is protective during acute kidney injury in aging mice. Kidney Int 
2011;79:966-76. 
28. 
Maeshima K, Takahashi T, Uehara K, et al. Prevention of hemorrhagic shock-induced 
lung injury by heme arginate treatment in rats. Biochem Pharmacol 2005;69:1667-80. 
29. 
Sasaki T, Takahashi T, Maeshima K, et al. Heme arginate pretreatment attenuates 
pulmonary NF-kappaB and AP-1 activation induced by hemorrhagic shock via heme 
oxygenase-1 induction. Med Chem 2006;2:271-4. 
30. 
Bharucha AE, Kulkarni A, Choi KM, et al. First-in-human study demonstrating 
pharmacological activation of heme oxygenase-1 in humans. Clin Pharmacol Ther 
2010;87:187-90. 
31. 
Mustajoki P, Tenhunen R, Tokola O, Gothoni G. Haem arginate in the treatment of 
acute hepatic porphyrias. Br Med J (Clin Res Ed) 1986;293:538-9. 
32. 
Herrick A, McColl KE, McLellan A, Moore MR, Brodie MJ, Goldberg A. Effect of 
haem arginate therapy on porphyrin metabolism and mixed function oxygenase activity in 
acute hepatic porphyria. Lancet 1987;2:1178-9. 
33. 
Tenhunen R, Mustajoki P. Acute porphyria: treatment with heme. Semin Liver Dis 
1998;18:53-5. 
34. 
Volin L, Rasi V, Vahtera E, Tenhunen R. Heme arginate: effects on hemostasis. Blood 
1988;71:625-8. 
35. 
German full package insert for Normosang in Austria. 
36. 
Tokola O, Tenhunen R, Volin L, Mustajoki P. Pharmacokinetics of intravenously 
administered haem arginate. Br J Clin Pharmacol 1986;22:331-5. 
37. 
Geuken E, Buis CI, Visser DS, et al. Expression of heme oxygenase-1 in human livers 
before transplantation correlates with graft injury and function after transplantation. Am J 
Transplant 2005;5:1875-85. 
38. 
Bernhard D, Skvortsov S, Tinhofer I, et al. Inhibition of histone deacetylase activity 
enhances Fas receptor-mediated apoptosis in leukemic lymphoblasts. Cell death and 
differentiation 2001;8:1014-21. 
 
 
Herr Martin Andreas