 
 
 
Protocol Version 6 / 17-May-2016  
 
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CLINICAL STUDY PROTOCOL 
 
A randomized, double-blind, placebo-controlled study to 
investigate the effects of recombinant human erythropoietin 
(NeoRecormon) on cycling performance and the occurrence of 
adverse events in well-trained cyclists. 
 
 
Short Title: 
Recombinant human erythropoietin effects on cycling performance. 
Version: 
6 
Date: 
17-May-2016 
CHDR number: 
CHDR1514 
Toetsing Online number: 
NL54516.056.15 
EudraCT number: 
2015-003269-27 
CHDR1514 
 
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CONTACT DETAILS  
Trial Site 
(visit & delivery address) 
Centre for Human Drug Research 
Zernikedreef 8 
2333 CL Leiden 
The Netherlands 
Telephone: + 31 71 5246 400 
Fax: + 31 71 5246 499 
Emergency: + 31 71 5246 444 
Principal Investigator 
 
A.F. (Adam) Cohen, MD, PhD 
Telephone: + 31 (0)71 5246404 
e-mail: ac@chdr.nl 
Co-Investigator 
 
J. (Koos) Burggraaf, MD, PhD 
Telephone: + 31 (0)71 5246418 
e-mail: kb@chdr.nl 
Co-Investigator 
Dr. J.I. (Joris) Rotmans 
Telephone: + 31 71 5262148 
e-mail: J.I.Rotmans@lumc.nl 
Co-Investigator 
Dr. J.M.A. (Hans) Daniels 
Telephone: +31 (0)20 4444782  
e-mail: j.daniels@vumc.nl 
Co-Investigator 
J. A. A. C. (Jules) Heuberger, MSc 
Telephone: +31 (0)71 5246471 
e-mail: JHeuberger@chdr.nl 
Co-Investigator 
Mr. H. (Herman) Ram 
Telephone: 010 - 2010154 
e-mail: H.Ram@dopingautoriteit.nl 
Co-Investigator 
Dr. O. (Olivier) de Hon 
Telephone: 010-2010155 
e-mail: O.Dehon@dopingautoriteit.nl 
Co-Investigator 
T. (Thijs) Zonneveld 
e-mail: info@thijszonneveld.com 
Director of Clinical Operations 
J. M. (Ria) Kroon, BSc 
Telephone: + 31 (0)71 5246498 
e-mail: rk@chdr.nl 
Statistician 
M. L. (Marieke) de Kam, MSc 
Telephone: + 31 (0)71 5246458 
e-mail: MdeKam@chdr.nl 
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INDEPENDENT PHYSICIAN 
Prof. Dr G.J. Blauw, MD, PhD 
Department of Gerontology and Geriatrics LUMC 
Postbus 9600 
2300 RC Leiden 
Telephone: + 31 71 5266 640 
LABORATORY - CHEMISTRY 
C.M. Cobbaert, PhD 
CKCL LUMC, E2-P 
Albinusdreef 2 
2333 ZA Leiden 
The Netherlands 
Contact person 
J. Verhagen 
LABORATORY - HAEMATOLOGY 
W.A.F. Marijt, MD, PhD  
CKHL LUMC, E1-Q 
Albinusdreef 2 
2333 ZA Leiden 
The Netherlands 
Contact person 
F. Reymer 
LABORATORY – MICROBIOLOGY 
A.C.M. Kroes, MD, PhD 
KML LUMC, E4-P 
Albinusdreef 2 
2333 ZA Leiden 
The Netherlands  
Contact person 
A.C.M. Kroes 
LABORATORY – DOPING 
DETECTION[ 
DoCoLab – Ugent   
Technologiepark 30 
B-9052, Zwijnaarde 
Belgium 
Contact person 
Prof. Dr. ir. Peter Van Eenoo    
Telephone: + 32-9-3313291 
e-mail: Peter.VanEenoo@Ugent.be 
LABORATORY – RNA 
EXPRESSION 
Leiden Genome Technology Center (LGTC) 
Human and Clinical Genetics 
Leiden University Medical Center, Postzone S4-P 
Postbus 9600 
2300 RC Leiden 
Nederland 
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Contact person 
Henk Buermans 
Tel: 
+31-71-526 9500/9522 
Fax: 
+31-71-526 8285 
E-mail: info@LGTC.nl 
 
PHARMACY 
(visit & delivery address) 
Apotheek LUMC, L0-P30 
Albinusdreef 2 
2333 ZA Leiden 
The Netherlands 
Telephone: 
+ 31 71 5269111 
Fax 
+ 31 71 5262611 
Pharmacist / Trial Manager 
Irene Teepe-Twiss, PhD / Linda van der Hulst 
 
 
cHDR1514
SIGNATURE PAGE . PRINCIPAL INVESTIGATOR
Study Title
A randomized, double-blind, placebo-controlled study to investigate the effects of recombinant
human erythropoietin (NeoRecormon) on cycling performance and the occurrence of adverse
events in well-trained cyclists.
I acknowledge accountability for this protocol in accordance with CHDR's current procedures.
A.F. (Adam) Cohen, MD, PhD
Principal I nvestigator
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Date (dd Mmm yyyy)
Protocol Version 6 I 17-May-2016
Page 5 of 68
cHDR1514
SIGNATURE PAGE. TRIAL SITE STAFF
Centre for Human Drug Research
Study Title
A randomized, double-blind, placebo-controlled study to investigate the effects of recombinant
human erythropoietin (NeoRecormon) on cycling performance and the occurrence of adverse
events in well-trained cyclists.
I acknowledge responsibility for this protocol in
J.A.A.C (Jules) Heuberger, MSc
Co-lnvestigator
J. M. (Ria) Kroon, BSc
Director Clinical Operations
M. L. (Marieke) de Kam, MSc
Statistieian
ce with CHDR's current procedures.
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Date (dd Mmm yyyy)
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Protocol Version 6 I 17 -May-2016
Page 6 of 68
CHDR1514 
 
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TABLE OF CONTENTS 
CONTACT DETAILS ........................................................................................................................................... 2 
SIGNATURE PAGE - PRINCIPAL INVESTIGATOR ......................................................................................... 5 
SIGNATURE PAGE - TRIAL SITE STAFF ......................................................................................................... 6 
PROTOCOL SYNOPSIS – CHDR1514 ............................................................................................................ 13 
1 
BACKGROUND AND RATIONALE ......................................................................................................... 23 
1.1 
Context............................................................................................................................................... 23 
1.1.1 
Need for research on doping ..................................................................................................... 23 
1.1.2 
Erythropoietin substances in patients and athletes ................................................................... 23 
1.1.3 
Examination of the evidence for the ergogenic properties of rHuEPO in cyclists ..................... 23 
1.1.4 
Study objectives ......................................................................................................................... 24 
1.2 
Non-clinical information ..................................................................................................................... 24 
1.2.1 
Non-clinical pharmacology ......................................................................................................... 24 
1.2.2 
Non-clinical pharmacokinetics and metabolism......................................................................... 24 
1.2.3 
Non-clinical toxicology and safety pharmacology ...................................................................... 24 
1.3 
Clinical information ............................................................................................................................ 24 
1.3.1 
Clinical pharmacology ................................................................................................................ 24 
1.3.2 
Clinical pharmacokinetics and metabolism ................................................................................ 24 
1.3.3 
Clinical toxicology and safety pharmacology ............................................................................. 24 
1.4 
Study rationale ................................................................................................................................... 24 
1.4.1 
Benefit and risk assessment ...................................................................................................... 24 
1.4.2 
Medical and regulatory background .......................................................................................... 25 
1.4.3 
Study population ........................................................................................................................ 25 
1.4.4 
Study design .............................................................................................................................. 25 
1.4.5 
Investigational drug and placebo ............................................................................................... 26 
1.4.6 
Dosing, safety margin calculations, stopping criteria ................................................................ 26 
1.4.7 
Treatment duration .................................................................................................................... 27 
1.4.8 
Endpoints ................................................................................................................................... 27 
1.4.9 
Statistical hypotheses and sample size ..................................................................................... 28 
2 
STUDY OBJECTIVES ............................................................................................................................... 30 
2.1 
Primary objective ............................................................................................................................... 30 
2.2 
Secondary objectives ......................................................................................................................... 30 
2.3 
Exploratory objectives ........................................................................................................................ 30 
3 
STUDY DESIGN ........................................................................................................................................ 31 
3.1 
Overall study design and plan ........................................................................................................... 31 
3.1.1 
Screening ................................................................................................................................... 31 
3.1.2 
Treatment and exercise tests .................................................................................................... 32 
3.1.3 
Training period ........................................................................................................................... 32 
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3.1.4 
Competition ................................................................................................................................ 32 
3.1.5 
Follow-up ................................................................................................................................... 32 
3.1.6 
Urine doping control ................................................................................................................... 32 
4 
STUDY POPULATION .............................................................................................................................. 33 
4.1 
Subject population ............................................................................................................................. 33 
4.2 
Inclusion criteria ................................................................................................................................. 33 
4.3 
Exclusion criteria ................................................................................................................................ 33 
4.4 
Concomitant medications .................................................................................................................. 34 
4.4.1 
Mandatory concomitant supplementation .................................................................................. 34 
4.4.2 
Allowed concomitant medications ............................................................................................. 34 
4.4.3 
Prohibited concomitant medications .......................................................................................... 34 
4.5 
Lifestyle restrictions ........................................................................................................................... 34 
4.6 
Study drug discontinuation and withdrawal ....................................................................................... 35 
4.6.1 
Study drug interruption or discontinuation ................................................................................. 35 
4.6.2 
Subject withdrawal ..................................................................................................................... 35 
4.6.3 
Replacement policy ................................................................................................................... 35 
5 
INVESTIGATIONAL MEDICINAL PRODUCT .......................................................................................... 36 
5.1 
Investigational drug and matching placebo ....................................................................................... 36 
5.2 
Comparative drug .............................................................................................................................. 36 
5.3 
Study drug dosing scheme ................................................................................................................ 36 
5.4 
Study drug packaging and labelling ................................................................................................... 36 
5.5 
Drug accountability ............................................................................................................................ 36 
5.6 
Treatment assignment and blinding .................................................................................................. 36 
5.6.1 
Treatment assignment ............................................................................................................... 36 
5.6.2 
Blinding ...................................................................................................................................... 37 
6 
STUDY ENDPOINTS ................................................................................................................................. 38 
6.1 
Efficacy endpoints .............................................................................................................................. 38 
6.2 
Safety endpoints ................................................................................................................................ 39 
6.3 
Exploratory endpoints ........................................................................................................................ 40 
7 
STUDY ASSESSMENTS .......................................................................................................................... 41 
7.1 
Exercise-specific screening assessments ......................................................................................... 41 
7.1.1 
Exercise test .............................................................................................................................. 41 
7.1.2 
Questionnaire ............................................................................................................................ 41 
7.2 
Safety and tolerability assessments .................................................................................................. 41 
7.2.1 
Specific safety assessments...................................................................................................... 41 
7.2.2 
Vital signs................................................................................................................................... 41 
7.2.3 
Weight and height ...................................................................................................................... 41 
7.2.4 
Physical examination ................................................................................................................. 41 
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7.2.5 
Electrocardiography ................................................................................................................... 41 
7.2.6 
Laboratory assessments ............................................................................................................ 41 
7.3 
Efficacy assessments ........................................................................................................................ 43 
7.3.1 
Exercise tests ............................................................................................................................ 43 
7.3.2 
Competition ................................................................................................................................ 43 
7.3.3 
Athlete biological passport ......................................................................................................... 43 
7.3.4 
Blood flow .................................................................................................................................. 43 
7.4 
NeoRecormon detection assessments .............................................................................................. 43 
7.4.1 
Urine doping screen ................................................................................................................... 43 
7.5 
RNA expression levels ....................................................................................................................... 43 
7.6 
Sequence of assessments and time windows ................................................................................... 44 
7.7 
Total blood volume ............................................................................................................................ 45 
8 
SAFETY REPORTING .............................................................................................................................. 46 
8.1 
Definitions of adverse events ............................................................................................................ 46 
8.1.1 
Intensity of adverse events ........................................................................................................ 46 
8.1.2 
Relationship to study drug ......................................................................................................... 46 
8.1.3 
Chronicity of adverse events ..................................................................................................... 46 
8.1.4 
Action ......................................................................................................................................... 46 
8.1.5 
Serious adverse events ............................................................................................................. 46 
8.1.6 
Suspected unexpected serious adverse reactions .................................................................... 47 
8.1.7 
Reporting of serious adverse events ......................................................................................... 47 
8.1.8 
Follow-up of adverse events ...................................................................................................... 47 
8.2 
Section 10 WMO event ...................................................................................................................... 47 
8.3 
Annual safety report or development safety update report ............................................................... 47 
9 
STATISTICAL METHODOLOGY AND ANALYSES ................................................................................ 49 
9.1 
Statistical analysis plan...................................................................................................................... 49 
9.2 
Protocol violations/deviations ............................................................................................................ 49 
9.3 
Power calculation ............................................................................................................................... 49 
9.4 
Missing, unused and spurious data ................................................................................................... 49 
9.5 
Analysis sets ...................................................................................................................................... 49 
9.5.1 
Safety set ................................................................................................................................... 49 
9.5.2 
Efficacy analysis set .................................................................................................................. 49 
9.6 
Subject disposition ............................................................................................................................. 49 
9.7 
Baseline parameters and concomitant medications .......................................................................... 50 
9.7.1 
Demographics and baseline variables ....................................................................................... 50 
9.7.2 
Medical history ........................................................................................................................... 50 
9.7.3 
Concomitant Medications .......................................................................................................... 50 
9.7.4 
Treatment compliance/exposure ............................................................................................... 50 
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9.8 
Safety and tolerability endpoints ........................................................................................................ 50 
9.8.1 
Adverse events .......................................................................................................................... 50 
9.8.2 
Vital signs................................................................................................................................... 50 
9.8.3 
ECG ........................................................................................................................................... 51 
9.8.4 
Clinical laboratory tests .............................................................................................................. 51 
9.9 
Efficacy endpoints .............................................................................................................................. 51 
9.9.1 
Efficacy ...................................................................................................................................... 51 
9.9.2 
Inferential methods .................................................................................................................... 51 
9.10 
Exploratory analyses and deviations ................................................................................................. 52 
9.11 
Interim analyses ................................................................................................................................. 52 
10 
GOOD CLINICAL PRACTICE, ETHICS AND ADMINISTRATIVE PROCEDURES ............................ 53 
10.1 
Good clinical practice ......................................................................................................................... 53 
10.1.1 
Ethics and good clinical practice ............................................................................................... 53 
10.1.2 
Ethics committee / institutional review board ............................................................................. 53 
10.1.3 
Informed consent ....................................................................................................................... 53 
10.1.4 
Insurance ................................................................................................................................... 53 
10.2 
Study funding ..................................................................................................................................... 53 
10.3 
Data handling and record keeping ..................................................................................................... 53 
10.3.1 
Data collection ........................................................................................................................... 53 
10.3.2 
Database management and quality control ............................................................................... 54 
10.4 
Access to source data and documents .............................................................................................. 54 
10.5 
Quality control and quality assurance ................................................................................................ 54 
10.5.1 
Monitoring .................................................................................................................................. 54 
10.6 
Protocol amendments ........................................................................................................................ 54 
10.6.1 
Non-substantial amendment ...................................................................................................... 54 
10.6.2 
Substantial amendment ............................................................................................................. 54 
10.7 
End of study report ............................................................................................................................ 55 
10.8 
Public disclosure and publication policy ............................................................................................ 55 
11 
STRUCTURED RISK ANALYSIS ......................................................................................................... 56 
12 
REFERENCES ...................................................................................................................................... 57 
13 
APPENDIX 1 – SUMMARY OF PRODUCT CHARACTERISTICS – ENGLISH VERSION ................. 59 
14 
APPENDIX 2 – SUMMARY OF PRODUCT CHARACTERISTICS – DUTCH VERSION .................... 60 
15 
APPENDIX 3 – EUROPEAN PUBLIC ASSESSMENT REPORT ........................................................ 61 
16 
APPENDIX 4 – SCREENING: QUESTIONNAIRE SPORT ACTIVITIES ............................................. 62 
17 
APPENDIX 5 – EXERCISE TEST ......................................................................................................... 64 
18 
APPENDIX 6 – COMPETITION ............................................................................................................ 65 
19 
APPENDIX 7 – URINE DOPING CONTROL PROCEDURE ................................................................ 67 
 
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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
ALT 
Alanine aminotransferase/Serum glutamic pyruvic transaminase (SGPT) 
ANCOVA 
Analysis of Covariance 
ANOVA 
Analysis of Variance 
ATC 
Anatomic Therapeutic Chemical 
ABP 
Athletes Biological Passport 
BMI 
Body Mass Index 
BP 
Blood Pressure 
bpm 
Beats per minute 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: Centrale 
Commissie Mensgebonden Onderzoek 
CHDR 
Centre for Human Drug Research 
CIRC 
Cycling Independent Reform Commission 
CK 
Creatine Kinase 
CRF 
Case Report Form 
EC 
Ethics Committee (also Medical Research Ethics Committee (MREC); in Dutch: 
Medisch Ethische Toetsing Commissie (METC). 
ECG 
Electrocardiogram 
EDTA 
Ethylene diamine tetra-acetic acid 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
Hb 
Haemoglobin 
Ht 
Haematocrit 
ICH 
International Conference on Harmonization 
IRB 
Institutional Review Board 
LDH 
Lactate dehydrogenase 
MedDRA 
Medical Dictionary for Regulatory Activities 
rHuEPO 
Recombinant Human Erythropoietin 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
SD 
Standard Deviation 
SEM 
Standard Error of the Mean 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
SST 
Serum Separator Tube 
SmPC 
Summary of Product Characteristics 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TTE 
Time To Exhaustion 
UCI 
Union Cycliste Internationale 
WADA 
World Anti-Doping Agency 
WHO 
World Health Organization 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen. 
 
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PROTOCOL SYNOPSIS – CHDR1514 
1. Title 
A randomized, double-blind, placebo-controlled study to investigate the effects of recombinant 
human erythropoietin (NeoRecormon) on cycling performance and the occurrence of adverse 
events in well-trained cyclists. 
 
2. Short Title 
Recombinant human erythropoietin effects on cycling performance. 
 
3. Background & Rationale 
3.1 Need for research on doping 
A recent report of the Union Cycliste Internationale gives an in-depth analysis of doping throughout 
cycling’s history, from 1890 to the present day. The report’s final conclusion is that cycling has had, 
and continues to have, a serious doping problem.[1]  
Although it could be argued that administering substances that improve performance is forbidden 
and nothing more needs to be known about it, these substances are apparently being used and 
therefore research to investigate the effects and safety of doping substances in this population is 
necessary. 
There are a number of reasons for this. First, it is often unknown if a forbidden substance really 
improves performance. If this is not the case the need for administration is strongly diminished. 
Additionally the adverse effects of such substances are often insufficiently known and athletes may 
be exposed to risks without being adequately informed about them. 
 
3.2 Erythropoietin substances in patients and athletes 
Recombinant Human Erythropoietin (rHuEPO) is used to treat patients with anemia resulting from 
chronic kidney disease or chemotherapy.[2] The correction of the anemia results in an increase in 
exercise capacity in these patients.[2] The treatment immediately attracted the attention of athletes 
because they assumed that rHuEPO would also improve their exercise performance. Due to this 
presumption, the use of rHuEPO in athletes became very common. In 1990, the use of rHuEPO was 
placed on the list of prohibited substances published by the World Anti-Doping Agency (WADA).[3] At 
the time of the first ban there was no published evidence that rHuEPO would actually improve 
sports performance. 
 
3.3 Examination of the evidence for the ergogenic properties of rHuEPO in cyclists 
The evidence for the effect of rHuEPO in well trained athletes is in fact sparse until today. A 
qualitative systematic review of the available literature was performed in 2012 to examine the 
evidence for the performance enhancing properties of rHuEPO in cyclists.[4] 
 
The review demonstrated that the characteristics of the study populations differed from the 
population suspected of rHuEPO abuse. Studies did not use well-trained cyclists, still less elite or 
world-class cyclists.[5] Most studies used a small number of untrained subjects and the quality of the 
research was often questionable. In these studies, the main studied effect was the maximal oxygen 
carrying capacity of blood (VO2, max) which only has a remote connection to performance in 
endurance sports, especially in well-trained athletes. This is in line with the knowledge that multiple 
factors affect performance, in which oxygen carrying capacity of the blood becomes less relevant 
when other factors become rate-limiting. Endurance performance may be better correlated with 
submaximal exercise factors. In addition, there is virtually no research on the potential adverse 
effects of this form of doping. 
 
To conclude, the results of this literature search showed that there is no scientific basis from which 
to conclude that rHuEPO has performance-enhancing properties in well-trained cyclists and that 
knowledge about potential side effects is lacking in full. 
 
3.4 Aim 
In the current study the effect of NeoRecormon, a rHuEPO, will be investigated in a population with 
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cycling performance abilities as close as possible to those of professional cyclists and in conditions 
closely resembling racing conditions and the required performance duration.  
 
4. Objective(s) 
4.1 Primary Objective 
- To explore the effects of NeoRecormon on well-trained cyclists and their cycling performance by 
maximal and sub-maximal exercise parameters measured during exercise tests. 
  
4.2 Secondary Objectives 
- To explore the effects of NeoRecormon on well-trained cyclists and their cycling performance by 
the performance and outcome of a race. 
- To evaluate the safety of NeoRecormon in well-trained cyclists. 
- To evaluate the performance of doping detection methods for NeoRecormon use in well-trained 
cyclists. 
 
4.3 Exploratory objectives 
 
- To explore how a standardized submaximal exercise affects gene expression patterns in well-
trained individuals. 
- To explore the difference in RNA-profiles between individuals treated with rHuEPO and placebo  
- To identify potential transcripts that can be used as biomarkers for rHuEPO use 
- To explore correlations between changes in whole blood gene expression patterns observed 
before and after a submaximal exercise test in individuals and their performance 
  
5. Design 
Randomized, double-blind, placebo-controlled study to investigate the effects and safety of 
NeoRecormon in well-trained cyclists. 
 
6. Principal Investigator & Trial Site 
 
Principal Investigator:  Prof. dr. A.F. (Adam) Cohen, MD 
Co-Investigators:     Prof. dr. J. (Koos) Burggraaf, MD 
                Dr. J.I. (Joris) Rotmans 
                Dr. J.M.A. (Hans) Daniels 
                 J.A.A.C. (Jules) Heuberger, MSc 
                Mr. H. (Herman) Ram 
                Dr. O. (Olivier) de Hon 
                T. (Thijs) Zonneveld 
 
Trial Site:          Centre for Human Drug Research, Zernikedreef 8, 2333 CL Leiden,  
                 The Netherlands 
 
7. Subjects / Groups 
A total of 48 subjects are planned to be enrolled in this study. Eligible subjects will be randomized to 
the NeoRecormon or Placebo treatment groups on a 1:1 basis. 
 
8. Sample Size Justification 
8.1 Power analysis based on VO2,max 
Based on the increase in VO2,max after administration of NeoRecormon in a previous study with 
moderately trained subjects an exploratory power analysis has been performed.[6] A sample size of 
6 in each group will have a power of 80% to detect a difference in means of 3.8 ml/min/kg, 
assuming that the common standard deviation is 1.95, using a two-tailed t-test with a 0.05 two sided 
significance level.  
 
A review of the available literature showed that, after initial years of training, well-trained athletes 
maintain a plateau in their VO2,max, but continue to improve their performance.[4] This indicates that 
the difference between effects on VO2,max between the NeoRecormon and placebo group in well-
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trained cyclists might be smaller. The smaller the size of the difference, the larger the sample size 
must be to detect a significant difference. To detect a difference of 1.5ml/min/kg with a power of 
80% a sample size of 22 is needed, assuming that the common standard deviation is 1.95, using a 
two-tailed t-test with a 0.05 two sided significance level. When taking into account a ±10% attrition 
rate, 24 subjects are needed in both the NeoRecormon and placebo group. 
 
8.2 Power analysis based on Pmax/kg 
The power calculation in section 8.1 is based on an endpoint that only has a remote connection to 
performance in well-trained cyclists. A better endpoint would be power output per kilogram (P/kg) at 
a submaximal level, such as 80% of VO2,max. Unfortunately no studies have been performed using 
this endpoint, so the effect of rHuEPO on P/kg at 80% VO2,max is still unknown. The mean P/kg at 
80% VO2,max of 11 male professional cyclists however, is 5.2 W/kg with a standard deviation of 
0.199.[7] Using a sample size of 22 (including 10% attrition rate) and a two-tailed t-test with a 0.05 
two sided significance level a difference of 0.172 W/kg can be detected with a power of 80%. This 
difference would mean that a professional cyclist weighing 75 kg would go from an average of 390 
W at 80% VO2,max to 402.9 W. On a racing bike weighing 9 kg sitting in racing position at 25 degrees 
Celsius, this would produce a speed of 43.80 km/h and 44.32 km/h respectively (calculated from 
http://bikecalculator.com). In a flat terrain of 40 km this would result in a finish time of 54 min 48 sec 
and 54 min 09 sec, a difference of 39 seconds, which is very relevant in a race like to Tour the 
France.  
 
9. Inclusion criteria 
 Well-trained (as determined by cycling history and maximal power output >4 W/kg) male 
subjects, 18 to 50 years old (inclusive); 
 Subjects must be healthy / medically stable on the basis of clinical laboratory tests, medical 
history, vital signs, and 12-lead ECG performed at screening, including exercise ECG. 
 Each subject must sign an informed consent form prior to the study. This means the subject 
understands the purpose of and procedures required for the study. 
10. Exclusion criteria 
 Any clinically significant abnormality, as determined by medical history taking and physical 
examinations, obtained during the screening visit that in the opinion of the investigator would 
interfere with the study objectives or compromise subject safety. 
 Unacceptable known concomitant diagnoses or diseases at baseline, e.g., known 
cardiovascular, pulmonary, muscle, metabolic or haematological disease, renal or liver 
dysfunction, ECG or laboratory abnormalities, etc. 
 Unacceptable concomitant medications at baseline, e.g., drugs known or likely to interact 
with the study drugs or study assessments. 
 Unacceptable potential cycling performance enhancing medications at baseline, e.g. 
Erythropoiesis-stimulating agents, Anabolic Androgenic Steroids, Growth Hormone, Insulin, 
IGF-I and Beta-Adrenergic Agents or methods, e.g. altitude tents. 
 Blood transfusion in the past three months. 
 Loss or donation of blood over 500 mL within three months. 
 Participation in a clinical trial within 90 days of screening or more than 4 times in the 
previous year. 
 Known hypersensitivity to the treatment or drugs of the same class, or any of their 
excipients.  
 Any known factor, condition, or disease that might interfere with treatment compliance, study 
conduct or interpretation of the results such as drug or alcohol dependence or psychiatric 
disease. 
 Positive urine drug test at screening. 
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 Positive alcohol breath test at screening. 
 Haemoglobin (Hb) concentration > 9.8 mmol/l at screening. 
 Hb concentration < 8 mmol/l at screening. 
 Haematocrit (Ht) ≥ 48% at screening. 
 Being subject to WADA’s anti-doping rules, meaning being a member of an official cycling 
union or other sports union for competition (such as the KNWU) or participating in official 
competition during the study.  
 Positive results from serology at screening (except for vaccinated subjects or subjects with 
past but resolved hepatitis) 
 Previous history of fainting, collapse, syncope, orthostatic hypotension, or vasovagal 
reactions.  
 Any circumstances or conditions, which, in the opinion of the investigator, may affect full 
participation in the study or compliance with the protocol.  
 
11. Concomitant medications 
 
The clinical results obtained so far do not indicate any interaction of NeoRecormon with other 
medicinal products. 
 
Mandatory supplementation: 
 
 50mg vitamin C (ascorbic acid) per day 
 200 mg iron (ferrofumerate) per day 
 
Allowed: 
 Paracetamol 
 Other medications that are discussed, approved and clearly documented by the investigator. 
 
Prohibited: 
 
 All substances (except NeoRecormon during treatment period) that are on the doping list 
and enhance cycling performance are prohibited within 6 months prior to study drug 
administration and during the course of the study (e.g. Other Erythropoiesis-stimulating 
agents, Anabolic Androgenic Steroids, Growth Hormone, Insulin, IGF-I and Beta-Adrenergic 
Agents). 
 
12. Study periods 
The total study period will be 17 weeks.  
 
Study periods 
Occasion 
Weeks 
Screening + training 
1x 
Within 6 weeks prior to treatment period 
Ramp exercise 
test*: 
1x 
Within 2 weeks before the start of the treatment 
period, but after the screening 
Time To Exhaustion 
exercise test*: 
1x 
Treatment 
Once a week 
During 8 weeks 
Ramp exercise test 
Every two weeks 
Training 
Whole period 
TTE exercise test: 
1x 
During the 7th treatment week 
Competition 
1x 
During the 9th week 
Follow-up 
1x 
30 Days after last visit 
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*Exercise tests will be performed within 2 weeks prior to the treatment period for baseline measurements. 
 
13. Investigational drug 
 
rHuEPO:        NeoRecormon (Active substance: Epoëtine beta) 
 
Dosage:        See Figure 1 for a detailed description of the dosage adjustment schedule.  
 
Administration:    Subcutaneously 
 
Dosage Rationale: NeoRecormon 2000, 5000 or ≥6000 but ≤10.000 IU/week, to be able to reach  
              the target range and adjust the dosage as soon as possible if it seems  
              necessary from Hb or Ht results 
14. Placebo 
Placebo: subcutanous injection of saline, (0.90% w/v NaCl) 
 
The investigational drug and its matching placebo are indistinguishable and will be packaged in the 
same way. Blinding will be accomplished by using the same syringes or by covering the syringes 
with aluminum foil. 
 
15. Efficacy endpoints 
Efficacy will be assessed at the time points indicated in the Visit and Assessment Schedule  
(see Table 1) in four ways: 
 
 Exercise tests will measure maximal and submaximal exercise parameters (e.g. VO2,max, 
Pmax, VE, Vt, VO2, VCO2, see  
 
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 Table 2.)  
 Subjects will participate in a competition designed in such a way that it closely resembles 
real racing conditions. Before and during the race, physiological parameters will be 
measured (e.g. power, heart rate, blood pressure).  
 Blood will be collected at predetermined stages at each clinical visit before administration of 
NeoRecormon/placebo and before and during the exercise tests and competition. This blood 
will be used for the following Clinical Laboratory Assessments: 
o Haematology (e.g. markers for the Athlete’s Biological Passport) 
o Chemistry (e.g. creatinine phosphokinase and c-reactive protein levels) 
o Coagulation (e.g. D-Dimer, F1+2, e-Selectin, p-Selectin) 
 Laser Speckle contrast imaging for blood flow measurements. 
 
16. Safety endpoints 
Safety will be assessed by:  
1. Physical examination 
2. Monitoring vital signs  
        o Pulse Rate (bpm) 
        o Systolic blood pressure (mmHg) 
        o Diastolic blood pressure (mmHg) 
        o Temperature measurements (ºC) 
3. Electrocardiogram (ECG)  
        o Heart Rate (HR) (bpm), PR, QRS, QT, QTcB        
4. Clinical Laboratory Assessments  
        o Haematology 
            Ht must be <52%. If Ht level is ≥52%, therapy should be interrupted until the Ht percentage  
           begins to fall. 
           Hb must be below a certain level (see Figure 1). If Hb exceeds that level, therapy should be  
           interrupted until the Hb concentration falls back into the range.  
        o Chemistry 
        o Urinalysis 
        o Coagulation 
5. Collection of treatment-emergent (serious) adverse events ((S)AEs)  
 
1-4 will be assessed during screening, 2-5 will be assessed at each clinical visit and before the 
competition. 2 and 3 will be assessed before and during the exercise tests. 
 
(Serious) Adverse Events ((S)AEs) will be collected throughout the study. 
 
17. Blinding 
This study will be performed in a double-blind fashion. The investigator, subjects and all study staff 
will remain blinded. A non-blinded CHDR staff member (not part of study staff) will receive report of 
Hb and Ht before dosing and will follow the instruction as described in Figure 1. This individual will 
be responsible for the dosage adjustments. 
 
Procedure for dosage adjustment: 
When the Hb concentration and/or Ht exceeds a certain value or Hb stays below a certain value 
(see Figure 1) the dose adjustment officer will issue a request for a dosage change for the subject 
that requires the change. This request will be for the subject that requires the change in treatment 
but will also be issued to a random placebo subject to preserve the blinding of the study. 
 
18. NeoRecormon detection in urine 
Urine will be collected at two predetermined periods. In the second treatment week samples will be 
taken pre-dose (day 7/8/9), two days later (day 9/10/11), at day 11/12/13/14 before and after the 
exercise test and pre-dose at day 14/15/16. Additionally, one sample will be taken before and after 
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the competition (week 9). These samples will be sent to a lab specialized in rHuEPO 
(NeoRecormon) detection in urine, according to the current protocol of the Dutch Doping Authority.  
 
19. Statistical methodology 
All efficacy endpoints will be summarized (mean and standard deviation of the mean, median, 
minimum and maximum values) by treatment and time, and will also be presented graphically as 
mean over time, with standard deviation as error bars. Change from baseline results will be utilized 
in all data summaries. All categorical efficacy endpoints will be summarized by frequencies. The PD 
endpoints will be analyzed separately by mixed model analyses of variance with treatment, time and 
treatment by time as fixed effects, with subject and subject by time as random effect, and with the 
(average) baseline value as covariate for recurring measurements and a one-way ANCOVA with 
treatment and baseline as covariates for measurements done at baseline and the end of the 
treatment period. 
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Figure 1. This dosing schedule must be applied before every  
administration of NeoRecormon/placebo during the 8 week  
treatment period. Ht = haematocrit, Hb = haemoglobin 
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Table 1. Visit and Assessment Schedule. 
 
SCR + 
training 
Baseline 
measurements 
Treatment and exercise test weeks* 
Competition week 
FU 
 
 
1, 3, 5, 7 
2, 4, 6, 8 
9 
12 
Day 
-42 to -3 
Day 
-14 to -1 
Day 0/1/2, 
14/15/16, 
28/29/30, 
42/43/44 
Ramp exercise tests: 
Day 11/12/13/14, 
25/26/27/28, 39/40/41/42, 
53/54/55/56 
 
Time To Exhaustion 
exercise test: 
46/47/48/49 
Day 7/8/9, 
21/22/23, 
35/36/37, 
49/50/51 
Weekend after last exercise test 
80 +- 7 
                            Time point                             
Assessment 
 
 
-6 to -
1h 
0h 
Before 
ET 
During 
ET 
After  
ET 
-6 to -1h 
0h 
Before 
competition 
During 
competition 
After 
competition 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion & exclusion criteria 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Height and Weight 10 
X 
 
 
 
X 
 
 
 
 
 
 
 
X 
Medical history  
X 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
Concomitant medication 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Serology 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Blood sample chemistry  
X6 
 
X 
 
X9 
 
X9 
 
 
X 
 
X 
X6 
Blood sample haematology 
X 
 
X 
 
 
 
 
 
 
X 
 
X 
X 
Blood sample coagulation 
X3 
X4 
X 
X8 
X9 
 
X9 
 
 
X 
 
X 
X3 
Blood sample biomarker 
 
 
X5 
 
X11 
 
X11 
 
 
 
 
 
 
Blood sample RNA 
 
 
 
 
X12 
 
X12 
 
 
 
 
 
 
Urinalysis 
X 
 
X 
 
 
 
 
 
 
X 
 
X 
X 
Urine Drug Screen,  
Breath Alcohol Test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
ECG 
X 
 
X 
 
 
 
 
X 
 
 
 
 
X 
General symptoms 
X 
 
X 
 
 
 
 
X 
 
X 
 
 
X 
Vital Signs (HR, BP, Temp) 
X 
 
X 
 
 
 
 
X 
 
 
 
 
X 
Blood sample haemoglobin and 
haematocrit "online" 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
Drug (-placebo) administration 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
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Ramp exercise test 
X1 
X 
 
 
 
X 
 
 
 
 
 
 
 
TTE exercise test 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
Laser Speckle (LSCI) 
 
 
X7 
 
X7 
 
X7 
 
 
 
 
 
 
Blood Sample Lactate 
 
 
 
 
 
X 
 
 
 
 
 
 
 
Urine doping control 
 
 
 
X2 
X2 
 
X2 
X2 
 
X 
 
X 
 
(S)AE/Con-meds 
 
 
<----- continuous -----> 
Training 
 
 
<----- Whole week -----> 
 
SCR = Screening, ET = Exercise Test, ECG = Electrocardiogram, BP = Blood Pressure, HR = Heart Rate, Temp = temperature, PD = Pharmacodynamics, (S)AE = (Special) Adverse Event, FU = Follow Up, * / means ‘or’  
 
 
 
                                                
 
1 Exercise test training session.  
2 Urine samples for doping control will only be collected in week 2 on day 7/8/9, at 7/8/9 +2 days, 11/12/13/14 and 14/15/16 (meaning post third dose and at day 2, 4 and 7 after that dose)  
3 Short coagulation panel at screening and follow-up, full panel for all other samples 
4 Before and after ramp exercise test 
5 Only on day 0/1/2 and 42/43/44 
6 Full chemistry panel at screening and follow-up, short panel for all other samples 
7 Only at day pre-first-dose and after exercise test on 39/40/41/42 and before exercise test on 46/47/48/49 
8 Only at day 0/1/2 post-dose, 1-3h 
9 Not at Time to Exhaustion test, except for Blood sample chemistry 
10 Height measured at screening only 
11 Before and after exercise test on day 39/40/41/42 
12 Before and after Time to exhaustion test on day 46/47/48/49 
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Table 2. Exercise parameters in endurance performance. 
Abbreviation 
Description 
Calculations / Characteristics 
VO2, max 
Maximal oxygen consumption (ml kg-1 min-1) 
 
Pmax 
Maximal power output (W) 
1W = 1J/sec 
VE 
Respiratory minute ventilation (L/min) 
 
Vt 
Tidal volume (L) 
 
Rf 
Respiratory frequency  
 
VO2 
Oxygen consumption (L/min) 
 
VCO2 
Carbon dioxide production (L/min) 
 
eqVO2 
Ventilatory equivalent for oxygen 
VE/VO2 
eqVCO2 
Ventilatory equivalent for carbon dioxide 
VE/VCO2 
EE 
Energy Expenditure (J/sec) 
([3.869 xVO2]+[1.195 x VCO2]) x (4.186/60) x 1000 
GE 
Gross Efficiency (%) 
Reflects the percentage of total chemical energy expended that contributes to external work, with the remaining 
energy lost as heat. 
(Work rate (J/sec) / Energy Expenditure (J/sec)) x 100% 
DE 
Delta Efficiency (%) 
(∆ Work Production (J/sec)/ ∆ Energy Expenditure (J/sec)) x 100% 
CE 
Cycling economy (W L-1 min-1)  
Can be defined as the submaximal VO2 per unit of body weight required to perform a given task. 
P/VO2 
Fatmax 
The highest absolute fat oxidation 
 
LT1 = AT = OBLA 
Lactate Threshold 1 = Anaerobic Threshold = 
Onset of Blood Lactate Accumulation 
The threshold where blood lactate levels raise 1mmol/L above baseline values. 
LT2 
Lactate Threshold 2 
The highest exercise intensity where lactate levels remain stable. 
VT1 
Ventilatory Threshold 1 
The first increase in VE that is proportional to the increase in VCO2 with no increase in VO2. 
Increase of end tidal O2 pressure. 
VT2 = RCP 
Ventilatory Threshold 2 = Respiratory 
Compensation Point 
Represents itself at a high work. 
VE increases far more compared to VCO2. 
End tidal CO2 pressure decreases. 
 
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1 
BACKGROUND AND RATIONALE 
1.1 Context 
1.1.1 Need for research on doping 
A recent report of the Union Cycliste Internationale (UCI) gives an in-depth analysis of doping 
throughout cycling’s history, from 1890 to the present day. The report’s final conclusion is that 
cycling has had, and continues to have, a serious doping problem.[1]  
Although it could be argued that administering substances that improve performance is forbidden 
and nothing more needs to be known about it, research to investigate the effects and safety of 
doping substances in this population is necessary. There are number of reasons for this. First, it is 
often unknown if a forbidden substance really enhances performance. If this is not the case the 
need for administration is strongly diminished. Additionally the adverse effects of such substances 
are often insufficiently known and athletes may be exposed to risks without being adequately 
informed about them. 
 
1.1.2 Erythropoietin substances in patients and athletes 
Recombinant Human Erythropoietin (rHuEPO) is used to treat patients with anemia resulting from 
chronic kidney disease.[2]  The correction of the anemia results in an increase in exercise capacity in 
these patients.[2] The treatment immediately attracted the attention of athletes because they 
assumed that rHuEPO would also improve their exercise performance. Due to this presumption, the 
use of rHuEPO in athletes became very common. In 1990, the use of rHuEPO was placed on the 
list of prohibited substances published by the World Anti-Doping Agency (WADA).[3] At the time of 
the first ban there was no published evidence that rHuEPO would actually improve sports 
performance. 
 
1.1.3 Examination of the evidence for the ergogenic properties of rHuEPO in cyclists 
The evidence for the effect of rHuEPO in well trained athletes is in fact sparse until today. A 
qualitative systematic review of the available literature was performed in 2012 to examine the 
evidence for the performance enhancing properties of rHuEPO in cyclists.[4] 
 
The review demonstrated that the characteristics of the study populations differed from the 
population suspected of rHuEPO abuse. rHuEPO studies often used untrained or moderately 
trained cyclists.[5] It cannot be assumed that effects found in these studies automatically apply to 
well-trained cyclists. Most studies used a small number of untrained subjects and the quality of the 
research was often questionable. In these studies, the main studied effect was the maximal oxygen 
carrying capacity of blood (VO2, max) which only has a remote connection to performance in 
endurance sports, especially in well-trained athletes. This is in line with the knowledge that multiple 
factors affect performance, in which oxygen carrying capacity of the blood becomes less relevant 
when other factors become rate-limiting. Endurance performance may be better correlated with 
submaximal exercise factors. There a number of findings that support this last notion. Firstly, 
research into training for endurance performance shows that moderately trained athletes are able to 
improve VO2,max by interval and/or intensive training, whereas these training regimens do not 
improve VO2,max in well-trained athletes. After initial years of training, these well-trained athletes 
maintain a plateau in their VO2,max, but continue to improve their performance. This shows that other 
factors have to play an important role in endurance performance. 
Secondly, long exercise times during consecutive days, with the finish line as a known end-point 
(contrary to the ‘ open end’  of time-to-exhaustion tests) makes it crucial for cyclists to distribute their 
power during a race. This, combined with (team) tactics, the terrain and the effects of drag force, 
means that cyclists work for only a small amount of time at their peak intensities (VO2, max), the rest of 
the time they will work at sub-maximal intensities where submaximal exercise factors are more 
important. 
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Finally, adverse events were never studied despite the fact that there is much evidence from patient 
studies that rHuEPO may cause hypertension and thrombotic events. Uncontrolled use of rHuEPO 
therefore involves risks for the users’ health, irrespective of such a substance being used legally or 
illegally.  
 
1.1.4 Study objectives 
The current study will 
- 
explore the effects of NeoRecormon on cycling performance in well-trained cyclists by  
 
performance in exercise tests  
 
performance in a competition  
 
measuring markers from the haematological module of the Athlete Biological Passport 
 
measuring blood flow  
- 
evaluate the safety of NeoRecormon in well-trained cyclists. 
- 
evaluate the performance of doping detection methods for NeoRecormon use in well-trained 
cyclists. 
 
1.2 Non-clinical information 
1.2.1 Non-clinical pharmacology 
Please refer to the respective Summary of Product Characteristics (SmPC) (see appendix 1) and 
European Public Assessment Report (EPAR) (see appendix 3). 
1.2.2 Non-clinical pharmacokinetics and metabolism 
Please refer to the respective SmPC and EPAR (see appendix 1 and 3). 
1.2.3 Non-clinical toxicology and safety pharmacology 
Please refer to the respective SmPC and EPAR (see appendix 1 and 3). 
1.3 Clinical information 
1.3.1 Clinical pharmacology 
Please refer to the respective SmPC and EPAR (see appendix 1 and 3). 
1.3.2 Clinical pharmacokinetics and metabolism 
Please refer to the respective SmPC and EPAR (see appendix 1 and 3). 
1.3.3 Clinical toxicology and safety pharmacology 
Please refer to the respective SmPC and EPAR (see appendix 1 and 3). 
 
1.4 Study rationale 
1.4.1 Benefit and risk assessment 
NeoRecormon is a registered drug. The safety profile of this compound is known. Because side 
effects might occur and anaphylactoid reactions were observed in isolated cases (≤1/10.000) (see 
SmPC, appendix 1), the study drug administrations will be done in the clinic under medical 
supervision. Subjects will be closely monitored and will only be discharged from the unit if their 
medical condition allows this.  
 
Subjects will receive a dose of 2000, 5000 or ≥6000IU with a maximum of 10.000IU/week of 
NeoRecormon once a week for 8 weeks. The dosage depends on haemoglobin (Hb) concentration 
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and haematocrit (Ht) measured prior to administration. If Ht is ≥52% dosage will be interrupted. If Ht 
is <52% the dosage depends on the Hb concentration (see Figure 1).  
The effects of NeoRecormon used in patients in an autologous blood predonation programme most 
closely resembles the effects of NeoRecormon in healthy volunteers. For the use of NeoRecormon 
in an autologous blood predonation programme, the SmPC (see appendix 1) states that the 
maximum dose should not exceed 1200IU/kg (or 90.000 IU for a 75kg subject) per week for 
subcutaneous administration. Planned doses of 2000IU, 5000IU or ≥6000IU with a maximum of 
10.000IU/week of NeoRecormon will be well below this maximum dose and are therefore 
considered safe. The risk is considered small and therefore acceptable compared to the scientific 
benefit.  
 
1.4.2 Medical and regulatory background 
The genetically engineered erythropoietin hormone, rHuEPO (in this research NeoRecormon), 
works in the same way as the natural hormone erythropoietin (EPO). EPO is a (glycoprotein) 
hormone primarily produced by the kidneys.[8] The kidneys secrete EPO in response to hypoxia in 
the renal circulation.[8] The secreted EPO binds to the EPO receptor on the erythroid progenitor cell 
surface and in this way activates intracellular signaling pathways that lead to erythropoiesis in the 
bone marrow.[8] Erythropoiesis is the proliferation and differentiation of erythroid progenitor cells to 
erythrocytes (red blood cells).[8] Erythrocytes are responsible for oxygen transport through the 
blood.[4] Due to the lack of a nucleus and other cellular machinery, erythrocytes are not able to 
repair themselves.[4] They have a lifespan of approximately 120 days in the circulation after which 
they are degraded by the spleen (2-3 million every second).[4] To keep the oxygen carrying capacity 
of the blood at a steady level, constant erythropoiesis is necessary.[4] The concentration of EPO in 
blood is relatively constant at approximately 5 pmol L-1.[4] 
 
1.4.3 Study population 
Forty-eight well-trained male subjects, 18 to 50 years of age who have a Hb level >8.0 mmol/L and 
≤ 9.8 mmol/L, a Ht <48% and a maximal power output > 4.0 W/kg will be included.  
 
Previous studies showed that professional cyclists have Hb concentrations between 8.25 and 
10.25mmol/l and Ht levels between 39.2 and 48.1%.[9], [10] The normal Hb concentration for males is 
8.75 to 11.25 mmol/l.[11;12] The rHuEPO treatment in this study will increase Hb levels of the subjects 
with 10-15%. Subjects will be excluded during the screening when they have Hb levels >9.8mmol/l, 
because the normal Hb level (11.25mmol/l) should not be exceeded (9.8 x 1.15 = 11.27 mmol/l).  
 
1.4.4 Study design 
This study will be conducted in accordance with guidelines from the International Conference on 
Harmonization (ICH) on current Good Clinical Practice (GCP) and the ethical principles that have 
their origins in the Declaration of Helsinki. 
 
In order to investigate the effects and safety of NeoRecormon in well-trained cyclists a randomized, 
double-blind, placebo-controlled study is most appropriate.  
 
The total study period will be 17 weeks. Within 6 weeks prior to the start of the treatment, subjects 
will undergo a medical screening. A ramp exercise test on a cycle ergometer will be performed 
directly after the screening to familiarize with the test and to determine the subject’s maximal power 
output. A second ramp and TTE exercise test will be performed for baseline measurements within 2 
weeks prior to the first administration, but after the screening. During the 8 week treatment period 
subjects will be administered with NeoRecormon or placebo once a week. During the treatment 
period subjects will follow their usual training program and will perform a ramp exercise test (see 
appendix 5) every two weeks. In the 7th treatment week the subjects will also perform a TTE 
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exercise test (see appendix 5). After the treatment period subjects will participate in a competition 
(see appendix 6). A follow-up visit will be scheduled, 30 days after the last dose.  
 
Please refer to the visit and assessment schedule for a more detailed description of the study 
design (see Table 1). 
 
1.4.5 Investigational drug and placebo 
 
Investigational drug:  NeoRecormon (Active substance: Epoëtine beta) 
 
Dosage:  
      2000IU, 5000IU or ≥6000IU with a maximum of 10.000IU/week, depends  
      on Ht percentage and Hb concentration (see Figure 1)          
  
Administration:      Subcutaneously 
 
Placebo:          Saline, 0.90% w/v NaCl. 
 
The investigational drug and its matching placebo are indistinguishable and will be packaged in the 
same way. Blinding will be accomplished by using the same syringes or by covering the syringes 
with aluminum foil. 
 
Eligible subjects will be randomized to one of two treatment groups on a 1:1 basis. One group will 
be treated with placebo and one group with NeoRecormon. 
 
1.4.6 Dosing, safety margin calculations, stopping criteria 
Dose selection and adjustments 
 
Please refer to Figure 1 for a detailed dosing schedule.  
 
The review of the data and the decision on the next dose level will be made by a non-blinded CHDR 
staff member unconnected to the study. 
 
Dosage Rationale 
An initial dose of 5000IU/week will be administered because previous studies showed that with this 
dose the above mentioned target range should be reached.[13-16] If the upper limit of the target range 
is exceeded the dose will be interrupted until the Hb concentration falls back into the range. At that 
point, therapy should be restarted at 2000IU/week. If treatment weeks ≥ 5 and Hb < (1.10*Initial Hb) 
the dose will be adjusted to ≥6000IU/week (maximal 10.000IU/week), so that it is still possible to 
reach the target range within 8 weeks. 
 
Safety margin calculations 
NeoRecormon is a marketed medication. The safety profile of this treatment is well-known (see 
SmPC (appendix 1) and EPAR (appendix 3)). See chapter 1.4.1 for the benefit and risk 
assessment.  
Subcutaneously administered NeoRecormon has a half-life of 12-28h. There will be one week 
between subcutaneous administrations of NeoRecormon, which is sufficient time to wash-out 
treatment from a previous occasion. 
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Stopping criteria 
Dosing will be interrupted if 
- Ht ≥ 52%  
- Hb exceeds the upper limit of the target range (see Figure 1) 
- there’s an unacceptable tolerability profile based on the nature, frequency, and intensity of 
observed AEs  
 
These criteria will be maintained by the non-blinded CHDR staff member unconnected to the study. 
 
1.4.7 Treatment duration 
In this study 24 subjects will be injected with NeoRecormon and 24 subjects will be injected with 
placebo once a week during 8 weeks.  
 
A treatment period of 8 weeks is chosen because this will provide sufficient time to reach the target 
range. If the target range is not reached after 5 weeks, the dose will be adjusted. In addition, during 
this treatment period study objectives such as assessment of safety and efficacy can be performed 
adequately. 
1.4.8 Endpoints 
 
Efficacy endpoints 
Efficacy will be assessed at the time points indicated in the Visit and Assessment Schedule (Table 
1).  
 
Efficacy will be assessed by: 
- performance in exercise tests  
- performance in a competition  
- measuring markers from the haematological module of the Athlete Biological Passport 
- measuring blood flow 
 
Exercise tests 
A review showed that most of the research investigating the effect of rHuEPO in cyclists focused on 
a parameter for maximal exercise oxygen consumption, VO2,max.[4] The review showed that besides 
this parameter, research should also focus on parameters for sub-maximal exercise. Therefore in 
this study maximal as well as sub-maximal exercise parameters (see table 2) will be measured 
during exercise tests. Subjects will perform a ramp exercise test every two weeks during their 
treatment/training period and a time to exhaustion exercise test in the 7th treatment week to see if 
the NeoRecormon/placebo treatment has an effect on their performance. Baseline measurements 
for these exercise tests will be performed one week prior to the treatment period.  
 
Before and during the exercise tests blood will be collected at predetermined stages to determine if 
certain protein concentrations are influenced by NeoRecormon. 
Please refer to appendix 5 for more detailed descriptions of the exercise tests.  
 
Competition 
Multiple factors affect cycling performance, especially in the racing conditions seen in official 
competition. Therefore, in this study the effects of NeoRecormon will also be determined by 
performance in a competition. The competition will be designed in such a way that it closely 
resembles real racing conditions. During the competition certain maximal and sub-maximal exercise 
parameters will be measured. Before and during the competition blood will be collected at 
predetermined stages to determine if certain protein concentrations are influenced by 
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NeoRecormon. After the competition urine will be collected for a doping control and for urinalysis for 
a.o. proteinuria. 
Please refer to appendix 6 for a more detailed description of the competition. 
 
Athlete Biological Passport 
According to the WADA, the athlete biological passport (ABP) is introduced to establish whether an 
athlete is manipulating his physiological variables without detecting a particular substance or 
method. The objective of this testing is to identify athletes in a haematological module and a 
steroidal module. The haematological module tests for certain markers in the body that identify the 
enhancement of oxygen transport. The steroidal module collects information on markers for steroid 
doping and aims to identify endogenous anabolic androgenic steroids. In this study only the 
haematological module will be conducted because rHuEPO only influences markers of this module.  
 
In this study an ABP will be created to determine the effect of NeoRecormon on markers of the 
haematological module and in addition to investigate if these markers together are really able to 
prove that an athlete is manipulating his physiological variables. The ABP will also be administered 
for safety, because based on Hb concentration and Ht (two of the haematological module markers) 
a dose will be selected weekly (during the treatment period). Blood to measure the haematological 
module markers will be collected before NeoRecormon/Placebo administration and before the 
competition.  
 
Blood flow  
LSCI measures: 
 
basal blood flow 
 
blood flow upon occlusion-reperfusion 
 
Blood flow after ‘exercise’  
 
1.4.9 Statistical hypotheses and sample size 
1.4.9.1 Power calculation based on VO2,max 
Null hypothesis:  
There’s no difference between the effects of NeoRecormon and placebo on cycling performance 
parameters. 
 
Based on the results of a previous study a power analysis has been performed.[6] In this study 
sixteen endurance-trained men (cyclists, runners and triathletes) were assigned randomly to either 
the rHuEPO- (n=9) or placebo- (n=7) treated groups. Both the participants and the investigators 
engaged in exercise testing and blood sampling were blind with respect to the group assignment. All 
athletes had been in regular training for several years preceding the study and continued to train 
throughout the period of the study but were forbidden to participate in sport events. The load of 
training was not different between the rHuEPO and placebo groups throughout the 4-weeks 
treatment period. Moderate doses of rHuEPO or placebo were injected s.c. 3 times a week for 4 
weeks in the morning. The two groups also received a daily oral dose of 200mg of iron sulphate 
during the 4 weeks. VO2,max was evaluated before and after rHuEPO treatment by using a ramp 
exercise test (30W/min after a 3-min baseline of warm-up at 60W) performed to the limit of tolerance 
on an electrically braked cycle ergometer that controlled external power output independent of pedal 
cadence.  
 
The mean VO2,max before treatment was similar in rHuEPO and placebo groups (63.0±1.5 vs 
64.8±2.0 ml/min/kg). VO2,max increased significantly after rHuEPO treatment in the rHuEPO group 
(68.4±1.9 ml/min/kg) and was significantly higher than in the placebo group (64.6±2.0 ml/min/kg, 
P<0.05). 
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Equation to determine sample size: [17]   
 
n = 1+ 2C(s/d)2 
 
n    =  sample size 
α    =  significance level 
1-β   =  the desired power of the experiment to detect the postulated effect 
C    =  constant dependent on the value of α and β selected (see table 4) 
s    =  standard deviation of the variable 
d    =  magnitude of the difference the investigator wishes to detect 
 
Table 4. Constant C dependent on the value of α and β selected.[17] 
 
α 
 
0.05 
0.01 
 
1-β 
0.8 
7.85 
11.68 
0.9 
10.51 
14.88 
 
Factors from the previous study to calculate sample size: [6] 
d    =   68.4 (rHuEPO-group) - 64.6 (Placebo group) = 3.8 ml/min/kg 
s    =   1.95 
1 - β  =   80% 
α    =   5% 
 
A sample size of 6 in each group will have a power of 80% to detect a difference in means of 
3.8ml/min/kg, assuming that the common standard deviations is 1.95, using a two-tailed t-test with a 
0.05 two sided significance level.  
 
A review of the available literature of the research investigating the effect of rHuEPO in cyclists 
showed that, after initial years of training, well-trained athletes maintain a plateau in their VO2,max, but 
continue to improve their performance further.[4] This indicates that the difference between effects 
on VO2,max between the NeoRecormon and placebo group in well-trained cyclists will be smaller. The 
smaller the size of the difference, the larger the sample size must be to detect a significant 
difference. To detect a difference of 1.5ml/min/kg with a power of 80% a sample size of 22 is 
needed, assuming that the common standard deviation is 1.95, using a two-tailed t-test with a 0.05 
two sided significance level. When taking into account a ±10% attrition rate, 24 subjects are needed 
in both the NeoRecormon and placebo group. 
 
1.4.9.2 Power calculation based on Pmax/kg 
A better endpoint would be power output per kilogram (P/kg) at a submaximal level, such as 80% of 
VO2,max. Unfortunately no studies have been performed using this endpoint, so the effect of rHuEPO 
on P/kg at 80% VO2,max is still unknown. The mean P/kg at 80% VO2,max of 11 male professional 
cyclists however, is 5.2 W/kg with a standard deviation of 0.199.[7] Using a sample size of 22 
(including 10% attrition rate) and a two-tailed t-test with a 0.05 two sided significance level a 
difference of 0.172 W/kg can be detected with a power of 80%. This difference would mean that a 
professional cyclist weighing 75 kg would go from an average of 390 W at 80% VO2,max to 402.9 W. 
On a racing bike weighing 9 kg sitting in racing position at 25 degrees Celsius, this would produce a 
speed of 43.80 km/h and 44.32 km/h respectively (calculated from http://bikecalculator.com). In a 
flat terrain of 40 km this would result in a finish time of 54 min 48 sec and 54 min 09 sec, a 
difference of 39 seconds, which is very relevant in a race like to Tour the France.  
 
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2 
STUDY OBJECTIVES 
2.1 Primary objective 
 
To explore the effects of NeoRecormon on cycling performance by  
 
performance in exercise tests  
 
measuring markers from the haematological module of the Athlete Biological Passport 
 
measuring blood flow 
in well-trained cyclists. 
 
2.2 Secondary objectives 
 
To explore the effects of NeoRecormon on cycling performance in a competition (road race) 
To evaluate the safety of NeoRecormon in well-trained cyclists 
To evaluate the performance of doping detection methods for NeoRecormon use in well-trained 
cyclists 
 
2.3 Exploratory objectives 
 
To explore how a standardized submaximal exercise affects gene expression patterns in well-trained 
individuals. 
To explore the difference in RNA-profiles between individuals treated with rHuEPO and placebo  
To identify potential transcripts that can be used as biomarkers for rHuEPO use 
To explore correlations between changes in whole blood gene expression patterns observed before 
and after a submaximal exercise test in individuals and their performance 
 
 
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3 
STUDY DESIGN 
3.1 Overall study design and plan 
This study will explore the effects of NeoRecormon on well-trained cyclists and their cycling 
performance during exercise tests and in a competition. It will consist of a screening, a training of 
the ramp exercise tests, 8 treatment visits, an 8 week training program, 7 exercise test visits, a 
competition and a follow-up visit which are outlined in the study Visit and Assessment Schedule 
(see Table 1). 
  
The total duration of the study for each subject will be up to 129 days divided as follows: 
 
Study periods**  
Days* 
Screening + Training ramp exercise test  
-42 to -2 days 
Ramp + TTE exercise test for baseline 
measurements 
-7 to -1 days 
Visit clinical unit: Treatment  
0/1/2, 7/8/9, 14/15/16, 21/22/23, 28/29/30, 
35/36/37, 42/43/44, 49/50/51 
Visit clinical unit: Ramp exercise tests 
11/12/13/14, 25/26/27/28, 39/40/41/42, 
53/54/55/56 
Visit clinical unit: TTE exercise test 
46/47/48/49 
Training 
0 to 55 
Competition 
57 to 60 
Follow-up 
80 +- 7  
* / means ‘or’  
** TTE = Time to exhaustion 
Dosage is spread over three days because it is logistically very difficult to perform safety 
measurements in 48 subjects and inject them afterwards with the study drug in one day. The 
exercise tests are spread over four days for the same reason.  
 
3.1.1 Screening 
The screening phase (days -42 to -2) will only be started after full written, verbal and signed 
informed consent has been obtained, according to CHDR standard operating procedures. The entire 
screening process will last approximately 2 hours. The screening will be divided into two parts: 
 
Medical screening 
- Medical interview 
- Physical examination 
- 12-lead ECG 
- Vital signs (Heart rate, blood pressure) 
- Weight 
- Height 
- Urine Drug Screen (THC, morphine, benzodiazepines, cocaine, amphetamines,  
  methamphetamines, MDMA) 
- Alcohol Breath Test 
- Blood sampling (haematology, biochemistry, serology, coagulation) 
- Urinalysis 
 
Training 
- Exercise test performed on an ergometer (see Appendix 5 – Exercise test for a more detailed 
description) 
 
During screening urine and blood samples will be collected from each patient for analysis as 
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described in section 7.2.6. 
 
3.1.2 Treatment and exercise tests 
 
The time schedule of study days is provided in general in the Visit and Assessment Schedule (see 
Table 1). On treatment days subjects will arrive at the clinical unit at a predetermined time. Before 
dosing standard safety measurements will be performed (see Table 1).  
 
Subjects will perform a ramp exercise test every two weeks during the treatment period. They also 
will perform a Time To Exhaustion exercise test in the 7th treatment week. Baseline measurements 
for these tests will be performed 2 weeks prior to the treatment period. During the exercise test 
maximal and submaximal exercise parameters will be measured. Before and during the exercise 
tests blood will be collected at predetermined stages. 
 
3.1.3 Training period 
 
During the treatment period (8 weeks) subjects will maintain their usual training programme. They 
will record their training intensity during this period in a diary. Additionally, subjects bikes will be 
mounted with a Pioneer power meter that will be used for each training activity to log the trip. This 
information will be shared with the investigators. 
 
3.1.4 Competition 
After the treatment period subjects will participate in a competition. The competition is designed in a 
way that it closely resembles real racing conditions and complies with the required performance 
duration. During the competition physiological parameters will be measured. Before and after the 
competition blood will be collected at predetermined stages.  
Please refer to appendix 6 for a detailed description of the competition. 
 
3.1.5 Follow-up 
 
A follow-up visit will be performed 30 days after the last visit, which includes a final physical 
examination, safety laboratory tests (haematology, chemistry, coagulation and urinalysis) and the 
measurement of vital signs (see Visit and Assessment Schedule, Table 1). Subjects will also be 
asked if they believe they were on active or placebo treatment. A description of all procedures and 
analyses is included in section 7.2. 
3.1.6 Urine doping control 
Urine will be collected at two predetermined periods. In the second treatment week samples will be 
taken pre-dose (day 7/8/9), two days later (day 9/10/11), at day 11/12/13/14 before and after the 
exercise test and pre-dose at day 14/15/16. Additionally, one sample will be taken before and after 
the competition (week 9). These samples will be sent to a lab (DoCoLab – Ugent, Technologiepark 
30, Zwijnaarde) specialized in rHuEPO (NeoRecormon) detection in urine, according to the current 
protocol of the Dutch Doping Authority.  
 
Please refer to appendix 7 for a detailed description of the urine doping control procedure. 
 
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4 
STUDY POPULATION 
4.1 Subject population 
A total of 48 subjects will be enrolled into the study following satisfactory completion of a screening 
visit where eligibility for the study will be checked. Subjects will be recruited via media 
advertisements and via advertisements at cycling associations. 
4.2 Inclusion criteria 
Eligible subjects must meet all of the following inclusion criteria: 
 
 Well-trained (as determined by cycling history and maximal power output >4 W/kg) male 
subjects, 18 to 50 years old (inclusive); 
 Subjects must be healthy / medically stable on the basis of clinical laboratory tests, medical 
history, vital signs, and 12-lead ECG performed at screening, including exercise ECG. 
 Each subject must sign an informed consent form prior to the study. This means the subject 
understands the purpose of and procedures required for the study. 
4.3 Exclusion criteria 
Eligible subjects must meet none of the following exclusion criteria: 
 
 Any clinically significant abnormality, as determined by medical history taking and physical 
examinations, obtained during the screening visit that in the opinion of the investigator would 
interfere with the study objectives or compromise subject safety. 
 Unacceptable known concomitant diagnoses or diseases at baseline, e.g., known 
cardiovascular, pulmonary, muscle, metabolic or haematological disease, renal or liver 
dysfunction, ECG or laboratory abnormalities, etc. 
 Unacceptable concomitant medications at baseline, e.g., drugs known or likely to interact 
with the study drugs or study assessments. 
 Unacceptable potential cycling performance enhancing medications at baseline, e.g. 
Erythropoiesis-stimulating agents, Anabolic Androgenic Steroids, Growth Hormone, Insulin, 
IGF-I and Beta-Adrenergic Agents. 
 Blood transfusion in the past three months. 
 Loss or donation of blood over 500 mL within three months. 
 Participation in a clinical trial within 90 days of screening or more than 4 times in the 
previous year. 
 Known hypersensitivity to the treatment or drugs of the same class, or any of their 
excipients.  
 Any known factor, condition, or disease that might interfere with treatment compliance, study 
conduct or interpretation of the results such as drug or alcohol dependence or psychiatric 
disease. 
 Positive urine drug test at screening. 
 Positive alcohol breath test at screening. 
 Haemoglobin (Hb) concentration > 9.8 mmol/l at screening. 
 Hb concentration < 8 mmol/l at screening. 
 Haematocrit (Ht) ≥ 48% at screening. 
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 Being subject to WADA’s anti-doping rules, meaning being a member of an official cycling 
union or other sports union for competition (such as the KNWU) or participating in official 
competition during the study.  
 Positive results from serology at screening (except for vaccinated subjects or subjects with 
past but resolved hepatitis) 
 Previous history of fainting, collapse, syncope, orthostatic hypotension, or vasovagal 
reactions.  
 Any circumstances or conditions, which, in the opinion of the investigator, may affect full 
participation in the study or compliance with the protocol.  
 
4.4 Concomitant medications 
The clinical results obtained so far do not indicate any interaction of NeoRecormon with other 
medicinal products. 
 
All medications (prescription and over-the-counter [OTC]) taken after study screening until the end 
of the study will be recorded. 
 
4.4.1 Mandatory concomitant supplementation  
 
Mandatory concomitant supplementation for all 48 subjects: 
 50mg vitamin C (ascorbic acid) per day 
 200 mg iron (ferrofumerate) per day 
 
4.4.2 Allowed concomitant medications 
 
Allowed concomitant medications: 
 
 Paracetamol 
 
 Other medications are only allowed if they are discussed, approved and clearly documented 
by the investigator. 
 
4.4.3 Prohibited concomitant medications 
 
Prohibited concomitant medications: 
 All substances (except NeoRecormon during treatment period) that enhance cycling 
performance are prohibited within 6 months prior to study drug administration and during the 
course of the study (e.g. Other Erythropoiesis-stimulating agents, Anabolic Androgenic 
Steroids, Growth Hormone, Insulin, IGF-I and Beta-Adrenergic Agents etc.). 
 
4.5 Lifestyle restrictions  
 
In the interest of the subjects’ safety and to facilitate assessment of the treatment effect, the patients 
participating in this study will be requested to agree to the following restrictions during the study: 
 
 
Alcohol will not be allowed from at least 24 hours before screening and before each 
scheduled visit, and whilst in the study unit until discharge from the study unit. 
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 
During the study subjects are not allowed to be subject to WADA’s anti-doping rules, 
meaning they are for example not allowed to be a member of an official cycling union or 
other sports union for competition (such as the KNWU). NeoRecormon treated subjects are 
not allowed to become a member of such a union for 3 months after the last dose. 
 
Subjects must maintain their usual training programme until the end of the treatment period 
(week 1-8). They will record their training activity in a diary and using the Pioneer power 
meter. Directly after the treatment period (week 9) the subjects will participate in a 
competition (see appendix 6).  
 
4.6 Study drug discontinuation and withdrawal 
4.6.1 Study drug interruption or discontinuation 
The investigator must temporally interrupt or permanently discontinue the study drug if continued 
administration of the study drug is believed to be contrary to the best interests of the subject. The 
interruption or premature discontinuation of study drug might be triggered by an Adverse Event 
(AE), a diagnostic or therapeutic procedure, an abnormal assessment (e.g., ECG or laboratory 
abnormalities), or for administrative reasons in particular withdrawal of the subject’s consent. The 
reason for study drug interruption or premature discontinuation must be documented. 
4.6.2 Subject withdrawal 
Subjects have the right to withdraw from the study at any time for any reason. Should a subject 
decide to withdraw from the study, all efforts should be made to complete and report the 
observations, particularly the follow-up examinations, as thoroughly as possible. 
4.6.3 Replacement policy 
Subjects withdrawing in the first treatment week for reasons other than adverse events or any other 
tolerability issues with the treatment will be replaced. 
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5 
INVESTIGATIONAL MEDICINAL PRODUCT 
5.1 Investigational drug and matching placebo 
Study drug or placebo will be administered to the subjects as detailed in Table 1. NeoRecormon will 
be ordered as available and its matching placebo will be manufactured. The investigational drug 
and its matching placebo are indistinguishable and will be packaged in the same way. Blinding will 
be accomplished by using the same syringes or by covering the syringes with aluminum foil. 
 
5.2 Comparative drug  
Placebo: Saline, 0.90% w/v NaCl. 
5.3 Study drug dosing scheme 
Please refer to Figure 1 for a detailed dosing schedule. 
5.4 Study drug packaging and labelling  
NeoRecormon and placebo will be acquired, packaged and labelled by the LUMC pharmacy in 
accordance with local regulations. Upon arrival at the pharmacy, the investigational products should 
be checked for damage and verify proper identity, quantity, integrity of seals and temperature 
conditions, and report any deviations or product complaints upon discovery. The dispensing of the 
study drug will be performed by the pharmacy. Study drug will be dispensed for each subject 
according to the randomization list. Study drug packaging will be overseen by the Leiden University 
Medical Centre Pharmacy and bearing a label with the identification required by local law, the 
protocol number, drug identification, and dosage.   
 
All subcutaneously administered drugs and placebos will be indistinguishable and will be packaged 
in the same way. 
 
The study drug must be stored between 2ºC and 8ºC in a secure, temperature-controlled 
(refrigerator) area with limited access. The vial must be kept in the outer carton, in order to protect it 
from light.  
 
For the purpose of ambulatory use, the unreconstituted product may be removed from the 
refrigerator and be stored at room temperature (not above 25ºC ) for one single period of up to 3 
days. Leaving the reconstituted solution outside the refrigerator should be limited to the time 
necessary for preparing the injections.  
 
5.5 Drug accountability 
Drug accountability will be maintained by the Leiden University Medical Centre Pharmacy and 
assessed by maintaining adequate study drug dispensing records.   
 
The investigator is responsible for ensuring that dosing is administered in compliance with the 
protocol. Delegation of this task must be clearly documented and approved by the investigator. All 
study drug administration will occur under medical supervision.   
 
5.6 Treatment assignment and blinding 
5.6.1 Treatment assignment 
Subjects must be randomized in a consecutive order starting with the lowest number. Replacement 
subjects will be numbered +100, e.g. subject 5 will have subject 105 as replacement, and subject 
105 will have subject 205 as replacement, etc. 
 
The randomization code will be generated SAS version 9.1.3 (or a more recent version, if available) 
by a study-independent, CHDR statistician. To reduce the variability between active and placebo, 
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block randomisation will be used, with one block of subjects aged 18-34 (inclusive) and another of 
subjects aged 34-50 (inclusive). The sample size of the blocks will be determined at day -14, when 
the included subjects have been identified. An attempt will be made to choose the highest block 
size. The randomization code will be unblinded/broken and made available for data analysis only 
after study closure, i.e., when the study has been completed, the protocol deviations determined, 
and the clinical database declared complete, accurate and locked. The randomization code will be 
kept strictly confidential. Sealed individual randomization codes, per subject and per treatment, will 
be placed in a sealed envelope containing the and labelled 'emergency decoding envelopes' will be 
kept in a safe cabinet at CHDR.  
5.6.2 Blinding 
This study will be performed in a double-blind fashion. The investigator, study staff, subjects and 
monitor will remain blinded to the treatment until study closure. The investigational drug and its 
matching placebo are indistinguishable and will be packaged in the same way. 
 
With the exceptions described in this section, the randomization list will not be available to the 
investigator, study staff, subjects and monitors.  
 
The randomization list will be made available to the pharmacist preparing the study drug, to an 
individual responsible for dosage adjustments and to statisticians or programmers involved in 
preparing blinded summaries, graphs and listings to support the dose decisions.   
The summaries, graphs and listings provided by the statisticians or programmers will be produced in 
an area to which other team members do not have access. In addition a single non blinded CHDR 
staff member unconnected to the study will receive reports on the haemoglobin and haematocrit of 
the subjects. This individual will be responsible for the dosage adjustments. 
 
The investigator will receive a set of sealed emergency codes to be broken in case of emergency 
situations. If the identity of the study drug administered needs to be known in order to manage the 
subject’s condition i.e., in case of a medical emergency or in the case a SUSAR occurs, the 
treatment emergency code for that subject may be broken and the study drug identified. All such 
occurrences should be documented in the study file. Treatment emergency codes should not be 
broken except in emergency situations and, if possible, the investigator should be contacted before 
the emergency code is opened. Just prior to database lock the unused emergency code labels will 
be checked and a statement to the effect that all are intact (or not as the case may be) will be made 
on the database lock form.  
 
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6 
STUDY ENDPOINTS 
6.1 Efficacy endpoints 
Efficacy will be assessed at the time points indicated in the Visit and Assessment Schedule (Table 
1). 
 
Efficacy will be assessed by: 
- performance in exercise tests  
- performance in a competition  
- measuring markers from the haematological module of the Athlete Biological Passport 
- measuring blood flow 
 
Exercise tests 
All subjects will breathe during the exercise test through a facemask that will be connected to an 
oxymeter to collect inspired and expired gasses for analyzing: 
   - Oxygen consumption, VO2 (L/min) 
   - Carbon dioxide production, VCO2 (L/min) 
   - Respiratory minute ventilation, VE (L/min) 
    - Tidal volume, Vt (L) 
    - Respiratory frequency, Rf 
    - Maximal oxygen consumption, VO2,max (ml kg-1 min-1) 
 
During the exercise tests blood will be collected at predetermined stages to measure: 
    - Lactate levels 
    - Tissue plasminogen activator 
    - Creatinine phosphokinase  
    - C-reactive protein levels 
 
VO2 and VCO2 will be used to calculate: 
    - Ventilatory equivalent for oxygen (VE/VO2), eqVO2 
    - Ventilatory equivalent for carbon dioxide (VE/VCO2), eqVCO2 
these values will be used to determine: 
    - Ventilatory threshold 1, VT1 
    - Ventilatory threshold 2, VT2 
 
Physiological parameters that will be determined at VT1 and VT2: 
   - Oxygen consumption, VO2 (L/min) 
   - Oxygen consumption per kg, VO2 (L/min/kg) 
    - Percentage of maximal oxygen consumption, %VO2max (L/min) 
    - Power output, P (J/s) 
    - Power output per kg, P (J/s/kg) 
 
Physiological parameters that will be determined at maximal effort: 
   - Maximal oxygen consumption, VO2max (L/min) 
   - Maximal oxygen consumption per kg, VO2max (L/min/kg) 
   - Maximal power output, Pmax (J/s) 
    - Maximal power output per kg, Pmax (J/s/kg) 
    - Lactate values 
 
Other determinations: 
   - Lactate threshold 1, LT1 
    - Lactate threshold 2, LT2 
   - Cycling economy, CE (W L-1 min-1) 
   - Gross efficiency, GE (%) 
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   - Heart rate (bpm) 
    - Systolic blood pressure (mmHg) 
    - Diastolic blood pressure (mmHg) 
 
Competition 
Exercise parameters that will be measured and calculated during the competition: 
- Result in the race 
- Total time of the race 
- Power (W) 
- Heart rate (bpm) 
- Systolic blood pressure (mmHg) 
- Diastolic blood pressure (mmHg) 
 
Before and after the competition blood will be collected to determine if certain protein concentrations 
are influenced by NeoRecormon such as: 
- Lactate 
- Tissue plasminogen activator 
- Creatinine phosphokinase  
- C-reactive protein 
- Coagulation and activation markers (D-Dimer, F1+2, p-Selectin, e-Selectin, ThromboxaneB2, 
bTG, PF4, thrombomodulin) 
 
In urine: 
- Proteinuria 
 
Athlete Biological Passport 
The following Markers are considered within the ABP Haematological Module:  
    - Haematocrit (Ht) 
    - Haemoglobin (Hb) 
    - Red blood cell (erythrocyte) count (RBC) 
       - Red blood cell mass (RCM) 
    - Reticulocytes percentage (RET%) 
    - Reticulocyte count (RET#) 
    - Mean corpuscular volume (MCV)  
    - Mean corpuscular haemoglobin (MCH) 
    - Mean corpuscular haemoglobin concentration (MCHC) 
    - Red cell distribution width (standard deviation) (RDW-SD) 
    - Immature reticulocyte fraction (IRF) 
 
Further calculated markers specific to the Haematological Module include OFF-hr Score (OFFS), 
which is a combination of Hb and RET%[18], and Abnormal Blood Profile Score (ABPS), which is a 
combination of Ht, Hb, RBC, RET%, MCV, MCH, and MCHC[19]. 
 
Blood flow  
LSCI measures: 
 
basal blood flow 
 
blood flow upon occlusion-reperfusion 
 
Blood flow after ‘exercise’  
 
6.2 Safety endpoints 
 
Safety measurements before NeoRecormon/placebo administration 
Safety will be assessed by: 
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 Monitoring vital signs  
        o Pulse Rate (bpm) 
        o Systolic blood pressure (mmHg) 
        o Diastolic blood pressure (mmHg) 
        o Temperature measurements (ºC) 
 Electrocardiogram (ECG) (at rest) 
        o Heart Rate (HR) (bpm), PR, QRS, QT, QTcB       
 Clinical Laboratory Assessments  
        o Haematology 
            Ht must be <52%. If Ht level is ≥52%, therapy should be interrupted until the    
           Ht level begins to fall. 
           Hb must be below 1.15*Initial Hb (see Figure 1). If Hb is above that level, dosage       
      should be interrupted until the Hb concentration falls back into the range. Then the  
      dosage should be restarted at 2000IU/week of the previous dose. 
        o Chemistry 
        o Urinalysis 
        o Coagulation 
In addition a single non blinded CHDR staff member unconnected to the study will receive reports 
on the Hb and Ht of the subjects. This individual will be responsible for the dosage adjustments. 
 
Procedure for dosage adjustment: 
When the Hb and/or Ht exceeds a certain value (see 5.3) the dose adjustment officer will issue a 
request for a dosage change for the subject that requires the change. This request will be for the 
subject that requires the change in treatment but will also be issued to a random placebo subject to 
preserve the blinding of the study. 
 
Additional safety measures 
A single non-blinded CHDR staff member unconnected to the study will receive reports on Hb 
concentration and Ht of the subjects. This individual will be responsible for the dosage adjustments.  
If Ht exceeds 52% in a subject, dosage will be stopped and if necessary, ±0.5L blood will be 
collected from that subject to rearrange a normal Ht percentage. This subject will then be excluded 
from the study. 
 
(Serious) Adverse Events ((S)AEs) will be collected throughout the study. 
6.3 Exploratory endpoints 
 
RNA expression levels in venous blood before and after a submaximal exercise test 
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7 
STUDY ASSESSMENTS 
See Table 1 for the time points of the assessments. 
7.1 Exercise-specific screening assessments 
7.1.1 Exercise test 
For the exercise test at screening, a ramp protocol will be followed until exhaustion. The details of 
the test will be described in the exercise manual. 
7.1.2 Questionnaire  
Please refer to appendix 4. 
7.2 Safety and tolerability assessments 
The definitions, reporting and follow-up of AEs and SAEs are described in section 8.  
7.2.1 Specific safety assessments 
A single non-blinded CHDR staff member unconnected to the study will receive reports on the 
haemoglobin (Hb) concentration and haematocrit (Ht) of the subjects. This individual will be 
responsible for the dosage adjustments.  
If Ht exceeds 52% in a subject, dosage will be stopped and if necessary, ±0.5L blood will be 
collected from that subject to correct the Ht percentage to a normal level. This subject will then be 
excluded from the study. 
7.2.2 Vital signs 
Evaluations of systolic and diastolic blood pressure, pulse rate and temperature will be performed 
throughout the study.  Pulse and blood pressure will be taken after 5 minutes in the supine position. 
Automated oscillometric blood pressures will be measured using a Dash 3000, Dash 4000, 
Dynamap 400 or Dynamap ProCare 400. Additionally, the pulse rate data provided by the pulse 
oximeter attached to the monitor.  
7.2.3 Weight and height 
Weight (kg) will be recorded at screening, before each exercise test and the follow-up visit or upon 
early termination. Height (cm) will be recorded and body mass index (BMI) calculated at screening. 
7.2.4 Physical examination 
Physical examination (i.e., inspection, percussion, palpation and auscultation) is performed during 
the course of the study. Clinically relevant findings that are present prior to study drug initiation must 
be recorded with the subject’s Medical History. Clinically relevant findings found after study drug 
initiation and meeting the definition of an AE (new AE or worsening of previously existing condition) 
must be recorded. 
7.2.5 Electrocardiography 
12-lead electrocardiographs (ECGs) will be obtained during the course of the study using Marquette 
800/5500 or Dash3000 and stored using the MUSE Cardiology Information System. The investigator 
will assess the ECG recording as 'normal', 'abnormal - not clinically significant', or 'abnormal - 
clinically significant' and include a description of the abnormality as required. The ECG parameters 
assessed will include heart rate, PR, QRS, QT, and QTcB (calculated using Bazett’s method).  
7.2.6 Laboratory assessments 
Laboratory parameters 
Blood and other biological samples will be collected for the following clinical laboratory tests: 
Lab 
Tests 
Collection & Analysis 
Haematology 
Haemoglobin [including Mean Corpuscular 
volume (MCV), Mean corpuscular 
4 mL of venous blood in a BD 
Vacutainer® K2EDTA tube. 
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haemoglobin (MCH), Mean corpuscular 
haemoglobin concentration (MCHC)], 
haematocrit, red cell count (RBC), Red cell 
distribution width (standard deviation) (RDW-
SD), reticulocyte count (RET#), reticulocytes 
percentage (RET%), immature reticulocyte 
fraction (IRF), total white cell count (WBC), 
leukocyte differential count and Platelet count. 
Differential blood count, including: basophils, 
eosinophils, neutrophils, lymphocytes, and 
monocytes. 
Samples will be analysed by 
the Central Clinical Hematology 
Laboratory (CKHL) of Leiden 
University Medical Center. 
Chemistry 
and 
electrolytes 
Sodium, potassium, calcium, inorganic 
phosphate, total protein, albumin, triglycerides, 
blood urea nitrogen (BUN), creatinine, ferritin, 
creatinine phosphokinase (CPK)*, uric acid, 
total bilirubin1, alkaline phosphatase, AST, 
ALT, gamma-GT, LDH, C-reactive protein*. 
8.5 mL or 2 mL of venous blood 
in a BD Vacutainer® SST Gel 
and Clot Activator tube. 
Samples will be analysed by 
the Central Clinical Chemistry 
Laboratory (CKCL) of Leiden 
University Medical Center. 
Coagulation 
APTT*, PT*, fibrinogen, D-dimer, F1+2, bTG, 
PF4, P-selectin, E-selectin, thrombomodulin, 
TXB2, TNF-alfa 
2.7 mL of venous blood in a  
3.2% citrate BD Vacutainer and 
4 mL in CTAD. Samples will be 
analysed by Good Biomarker 
Sciences (GBS) in Leiden and 
labs to be determined. 
RNA 
expression 
RNA profiles 
2.5 mL of venous blood in 
PAXgene vacuum tubes using 
a blood collection system 
analysed by Leiden Genome 
Technology Center (LUMC) 
Serology 
HIV1 and HIV2 antibodies, Hepatitis B antigen 
and Hepatitis C antibodies 
5 mL of venous blood in a BD 
Vacutainer® SST Gel and Clot 
Activator tube. Samples will be 
analysed by the Central Clinical 
Microbiology Laboratory 
(CKML) of the Leiden University 
Medical Center. 
Urinalysis 
Leucocytes, blood, nitrite, protein, 
urobilinogen, bilirubin, pH, specific gravity, 
ketones, glucose. If there is a clinically 
significant positive result, urine will be sent to 
the CKCL for microscopy and/or culture. 
A midstream, clean-catch urine 
specimen will be analysed by 
dipstick (Multistix® 10 SG, 
Siemens Healthcare 
Diagnostics, Frimley, UK). 
Alcohol 
Alcohol Breath Test 
The hand-held Alco-Sensor IV 
meter (Honac, Apeldoorn, the 
Netherlands) will be used to 
measure the breath ethanol 
concentrations. 
Urine drug 
screen 
Cocaine, amphetamines, opiates (morphine), 
benzodiazepines, methamphetamine, MDMA 
and cannabinoids. 
A urine specimen will be 
analysed at CHDR by test kit 
(InstAlert, Innovacon, San 
Diego, USA). 
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1Conjugated bilirubin will be reported only when total bilirubin is outside the reference range. 
*Part of the short chemistry/coagulation panel 
The results of the haematology assessment will be used for the safety report and for the athlete’s 
biological passport.  
7.3 Efficacy assessments 
7.3.1 Exercise tests 
Please refer to Appendix 5 – Exercise test for a detailed description of the exercise tests. 
7.3.2 Competition 
Please refer to Appendix 6 – Competition for a detailed description of the competition. 
7.3.3 Athlete biological passport 
This assessment has already been performed for safety measurements. The results will be used for 
the safety report and for the athlete’s biological passport.  
 
Further calculated markers specific to the Haematological Module of the ABP include OFF-hr Score 
(OFFS), which is a combination of Hb and RET%[17], and Abnormal Blood Profile Score (ABPS), 
which is a combination of Ht, Hb, RBC, RET%, MCV, MCH, and MCHC[18]. 
7.3.4 Blood flow  
LSCI measures: 
 
basal blood flow 
 
blood flow upon occlusion-reperfusion 
 
Blood flow after ‘exercise’  
 
Forearm blood flow will be measured by non-invasivce laser speckle contrast imaging (LSCI; 
PeriCam PSI System, Perimed). At baseline and after 7 weeks of dosing, the flow will be measured 
at rest, during a five minute occlusion of the brachial artery and during reperfusion after the 
occlusion. Blood flow will be measured after exercise at 6 weeks. Procedures for LSCI, brachial 
artery occlusion-reperfusion are described in SOPs. Briefly, the subject will be seated with the left 
arm placed on a (table) rest. A suitable area of the volar side of the forearm will be identified. This 
area will be 'illuminated' by the laser and the response signal will be captured.  
 
7.4 NeoRecormon detection assessments 
7.4.1 Urine doping screen 
Please refer to Appendix 7 – Urine doping control procedure for a detailed description of the urine 
doping control procedure. 
7.5 RNA expression levels 
In general, genes react to internal or external stimuli by becoming more active, less active or 
showing no response. Training stimuli induce stress resulting in changes in gene expression 
patterns. Gene expression (RNA-) profiles made from whole venous blood taken before and after 
physical exercise differ between non-trained and trained individuals. This suggests that adaptation 
to training can be assessed using gene expression analysis. Changes in gene expression patterns 
can then be used to monitor training effects. It is unclear how adaptation to training with or without 
use of rHuEPO will change gene expression profiles in well-trained individuals. We hypothesize that 
the changes in these patterns observed in samples taken before and after an exercise test are 
correlated with the performance and health status of individual athletes. The measurements of 
maximal and submaximal exercise parameters and clinical laboratory assessments of the 
participants in this study will be combined with gene expression profile data to investigate if specific 
changes in the acute response to a submaximal exercise test are correlated with better endurance 
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performance or other parameters. Comparisons of these profiles in time are expected to reveal the 
effects of specific types of training and changes in health status. 
RNA profiles from whole venous blood samples will be determined using next generation 
sequencing. Samples for RNA-profiling will be collected around a submaximal intensity test (45 
minutes, starting at 80% Power from the baseline maximal exercise test allowing changes to the 
power at any time by the subject himself if he wishes). Blood samples (2.5 ml) for RNA analysis will 
be drawn in PAXgene vacuum tubes at 2 time points, before and directly after the test. RNA-profiles 
made from both time points of each individual will be compared to identify changes in gene 
expression due to the submaximal intensity test. Profiles from the same time points will also be 
compared to determine interindividual variation and possible EPO treatment effects. 
Expression levels of the different transcripts in the RNA samples will be determined as digital counts 
using standard bioinformatics pipelines. Cluster analysis and principal component analysis will be 
performed to determine the relationships between the samples. In this way it will be possible to 
determine if: 
 
The expression patterns differ at the time points before and after submaximal exercise test 
between individuals and between groups. 
 
rHuEPO treatment does influence gene expression levels. 
Multivariate statistics will be applied to analyse and interpret complete RNA-profiles. The leukocyte 
composition of the original blood samples is correlated with cell-specific transcripts. Therefore, this 
can be derived from RNA-profiles allowing their correction for this effect. 
Comparison with other parameters measured during the study may indicate which changes in gene 
expression patterns of individuals are correlated with their performance. 
 
For all subjects RNA samples will be collected for potential analyses of gene expression. Instructions 
for collection, processing, handling and shipment of the samples will be outlined in the laboratory 
manual. Samples will be archived according to local administration regulations.  
 
Analysis will be performed outside the scope of the main study and reported separately.  
 
7.6 Sequence of assessments and time windows 
Assessments will be performed in the following order, where possible: physical examination, weight 
and height, ECG, vital signs, blood samples, dosing. 
The deviations of actual time points from the expected time points will be within ten percent, 
calculated from the zero point (time of drug administration) or the last relevant activity. Deviations of 
more than 10% will be explained in a note. Pre-dose assessments are given in indicative expected 
times. 
 
 
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7.7 Total blood volume 
 
 
Sample
Samples taken
Sample Volume*
Volume
Haematology
8
x
4 mL
=
32 mL
Chemistry
2
x
8.5 mL
=
17 mL
Chemistry short
16
x
2.5 mL
=
40 mL
Serology
1
x
5 mL
=
5 mL
Coagulation
17
x
6.2 mL
=
105.4 mL
Coagulation short
2
x
4.5 mL
=
9 mL
RNA expression
2
x
2.5 mL
=
5 mL
Biomarker
4
x
4 mL
=
16 mL
Hb/Hct (1mL tube)
8
x
1 mL
=
8 mL
Lactate (Drop)
51
x
0.8 mL
=
40.8 mL
* inclusive discarded volume
Total blood volume/subject
278.2 mL  
 
 
 
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8 
SAFETY REPORTING 
8.1 Definitions of adverse events 
An Adverse Event (AE) is any untoward medical occurrence in a subject who is participating in a 
clinical study performed. The adverse event does not necessarily have to follow the administration 
of a study drug, or to have a causal relationship with the study drug. An adverse event can therefore 
be any unfavourable and unintended sign (including an abnormal laboratory or vital sign finding), 
symptom, or disease temporally associated with the study participation, whether or not it is related 
to the study drug. 
8.1.1 Intensity of adverse events  
The intensity of clinical AEs is graded three-point scale as defined below:  
 
Mild: discomfort noticed but no disruption of normal daily activity; 
 
Moderate: discomfort sufficient to reduce or affect normal daily activity; 
 
Severe: inability to work or perform daily activity. 
8.1.2 Relationship to study drug  
For each adverse event the relationship to drug as judged by the investigator:  
 
Probable;  
 
Possible;  
 
Unlikely;  
 
Unrelated. 
8.1.3 Chronicity of adverse events 
The chronicity of the event will be classified by the investigator on a three-item scale as defined 
below: 
 
Single occasion: single event with limited duration; 
 
Intermittent: several episodes of an event, each of limited duration; 
 
Persistent: event which remained indefinitely. 
8.1.4 Action 
Eventual actions taken will be recorded. 
8.1.5 Serious adverse events 
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: 
 
Is fatal 
 
Is life-threatening  
 
Is disabling 
 
Requires or prolongs in-patient hospitalisation   
 
Causes congenital anomaly  
will be described as a SAE. Important medical events that may not be immediately life threatening 
or result in death or hospitalisation may be considered a serious adverse event when, based on 
appropriate medical judgement, they may jeopardise the subject or may require medical or surgical 
intervention to prevent one of the outcomes listed above.  
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8.1.6 Suspected unexpected serious adverse reactions 
A SUSAR (Suspected Unexpected Serious Adverse Reaction) is a serious adverse event that is 
unexpected, (nature or severity of which is not consistent with the applicable product information 
(e.g., investigator's brochure for an unauthorised investigational product or summary of product 
characteristics for an authorised product)) and suspected (a reasonable possibility of causal 
relationship with investigational drug). 
8.1.7 Reporting of serious adverse events 
SAEs and SUSAR's will be reported according to the following procedure. 
All SUSARs and SAEs must be reported to the investigator.  
 
In case of multi-centre studies of which a CHDR employee is the responsible investigator, the sub-
investigators at sites outside CHDR must notify the investigator by telephone and in writing 
preferably using a standard form (e.g. CIOMS for SUSARs). 
 
The investigator must report all SAEs and SUSAR’s to the EC that approved the study, in writing as 
soon as practical, but at least within 15 days. Fatal and life-threatening suspected SUSAR’s should 
be reported within 7 calendar days, with another 8 days for completion of the report. 
The investigator must report all SUSAR’s to the CA, in writing as soon as practical, but at least 
within 15 days. Fatal and life-threatening suspected SUSAR’s should be reported within 7 calendar 
days, with another 8 days for completion of the report. SAE's do not have to be reported to the CA. 
The investigator must furthermore report all SUSAR’s to EMA's EudraVigilance database within 15 
days. Fatal and life-threatening suspected SUSAR’s should be reported within 7 calendar days, with 
another 8 days for completion of the report. 
The investigator can prepare additional reports for other authorities (e.g. FDA). 
All SAEs and SUSAR’s must be reported on the Toetsing Online website2.  Fatal and life-
threatening SAEs and SUSARs must be reported within 7 calendar days. All other SAEs must be 
reported within 15 days. By reporting on the Toetsing Online website the EC that approved the 
study will also be informed. 
8.1.8 Follow-up of adverse events 
All adverse events will be followed until they have abated, returned to baseline status or until a 
stable situation has been reached. Depending on the event, follow up may require additional tests 
or medical procedures as indicated, and/or referral to the general physician or a medical specialist. 
8.2 Section 10 WMO event 
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and 
the EC if anything occurs, on the basis of which it appears that the disadvantages of participation 
may be significantly greater than was foreseen in the research proposal. The study will be 
suspended pending further review by the EC, except insofar as suspension would jeopardise the 
subjects’ health. The investigator will ensure that all subjects are kept informed.  
8.3 Annual safety report or development safety update report 
In addition to the expedited reporting of SUSARs, the investigator will submit, once a year 
throughout the clinical trial, a safety report to the EC, CA, MEB and CAs of the concerned Member 
States. 
 
                                                
 
2 https://www.toetsingonline.nl/ 
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This safety report consists of: 
 
a list of all suspected (unexpected or expected) serious adverse reactions, along with an 
aggregated summary table of all reported serious adverse reactions, ordered by organ system, 
per study; 
 
a report concerning the safety of the subjects, consisting of a complete safety analysis and an 
evaluation of the balance between the efficacy and the harmfulness of the medicine under 
investigation. 
 
 
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9 
STATISTICAL METHODOLOGY AND ANALYSES 
9.1 Statistical analysis plan 
All safety and statistical programming is conducted with SAS 9.4 for Windows (SAS Institute Inc., 
Cary, NC, USA). PK variable programming is conducted with R 2.12.0 for Windows (R Foundation 
for Statistical Computing/R Development Core Team, Vienna, Austria, 2010. 
A Statistical Analysis Plan (SAP) will be written and finalized before the study closure, i.e., database 
closure and unblinding of the randomization code. The SAP will provide full details of the analyses, 
the data displays and the algorithms to be used for data derivations.  
The SAP will include the definition of major and minor protocol deviations and the link of major 
protocol deviations to the analysis sets.  
9.2 Protocol violations/deviations 
Protocol deviations will be identified based on conditions related to the categories below: 
 
Protocol entry criteria 
 
Forbidden concomitant medications 
 
Missing evaluations for relevant endpoints 
 
Other protocol deviations occurring during study conduct. 
Major protocol deviations will be identified before the study closure, and listed where appropriate. 
9.3 Power calculation 
Please refer to section 1.4.9. 
 
9.4 Missing, unused and spurious data 
All missing or incomplete safety and efficacy data, including dates and times, are treated as such. 
Missing test results or assessments will not be imputed. Missing efficacy data, indicated as ‘M’ in 
the data listing, will be estimated within the statistical mixed model using SAS PROC MIXED. 
For graphical and summary purposes efficacy and safety values below the limit of quantification will 
be set to half (½) of the limit of quantification. For analysis no undetermined values will be replaced. 
The handling of missing, unused and spurious data will be documented in the study report. 
 
9.5 Analysis sets 
Data of all subjects participating in the study will be included in the analyses if the data can 
meaningfully contribute to the objectives of the study. 
9.5.1 Safety set 
The safety population will be defined as all subjects who were validated (randomised) and received 
at least 1 dose of study treatment. 
9.5.2 Efficacy analysis set 
The analysis population for efficacy is defined as all subjects who were validated (randomised), 
received at least one dose of study treatment, and have at least one post-baseline assessment of 
the parameter being analysed. 
9.6 Subject disposition 
The following subject data will be summarized by treatment and overall: 
 
Number and percentage of subjects enrolled in each analysis set for all randomized 
subjects; 
 
Number and percentage of subjects who completed the study or prematurely discontinued 
from the investigational period by reasons for discontinuation, to be tabulated for each 
analysis set. 
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Subject disposition will be listed. 
9.7 Baseline parameters and concomitant medications 
9.7.1 Demographics and baseline variables 
Continuous demographic variables (e.g., age, height, weight, BMI) will be summarized by 
descriptive statistics (n, mean, SD, median, Min, Max).Qualitative demographic characteristics (sex, 
race/ethnicity) will be summarized by counts and percentages.  
The results of the sport activities questionnaire at screening will only be listed. 
9.7.2 Medical history  
Medical history will only be listed. 
9.7.3 Concomitant Medications 
Previous and concomitant medications will be coded according to the World Health Organization 
(WHO) drug code and the anatomical therapeutic chemical (ATC) class code.  
All concomitant medications will be displayed in a listing. 
9.7.4 Treatment compliance/exposure 
Exposure to study treatment is described in terms of duration of treatment and average infusion 
rate. The average infusion rate (mL/hr) is summarized by mean, SD, median, Q1, Q3, Min, Max. 
9.8 Safety and tolerability endpoints  
The safety set is used to perform all safety analyses.  
Baseline is defined as the last value prior to dosing. Change from baseline will be calculated for all 
continuous safety parameters.  
9.8.1 Adverse events 
The AE coding dictionary for this study will be Medical Dictionary for Regulatory Activities 
(MedDRA). It will be used to summarize AEs by primary system organ class (SOC) and preferred 
term (PT). 
All adverse events will be displayed in listings. 
A treatment-emergent adverse event (TEAE) is defined as an adverse event observed after starting 
administration of the specific treatment, and prior to the start of another treatment, if any OR up to 5 
days (96 hours) after study drug administration. If a subject experiences an event both prior to and 
after starting administration of a treatment, the event will be considered a TEAE (of the treatment) 
only if it has worsened in severity (i.e., it is reported with a new start date) after starting 
administration of the specific treatment, and prior to the start of another treatment, if any. All TEAEs 
collected during the investigational period will be summarized. 
The number of and/or the number of subjects with treatment emergent AEs will be summarized by:  
1. treatment, MedDRA SOC and PT; 
2. treatment, MedDRA SOC, PT and severity; 
3. treatment, MedDRA SOC, PT and drug relatedness. 
9.8.2 Vital signs 
At each time point, absolute values and change from baseline of supine BP and HR will be 
summarized with n, mean, SD, SEM, median, Min, and Max values. The number of available 
observations and out-of-range values (absolute and in percentage) will be presented. Values 
outside the reference range will be flagged in the listing. ‘H’ and ‘L’, denoting values above or below 
the investigator reference range (when present), will flag out-of-range results.  
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9.8.3 ECG 
At each time point, absolute values and change from baseline of ECG numeric variables will be 
summarized with n, mean, SD, SEM, median, Min, and Max values. The number of available 
observations and out-of-range values (absolute and in percentage) will be presented. Values 
outside the investigator’s normal range will be flagged in the listing. ‘H’ and ‘L’, denoting values 
above or below the investigator reference range (when present), will flag out-of-range results. 
9.8.4 Clinical laboratory tests 
At each time point, absolute values and change from baseline of clinical laboratory variables will be 
summarized with n, mean, SD, SEM, median, Min, and Max values. The number of available 
observations and out-of-range values (absolute and in percentage) will be presented. All laboratory 
data (including re-check values if present) will be listed chronologically. ‘H’ and ‘L’, denoting values 
above or below the investigator reference range (when present), will flag out-of-range results.  
9.9 Efficacy endpoints 
9.9.1 Efficacy 
The final analysis will be preceded by a blind data review which consists of individual graphs per 
visit by time of all efficacy measurements by time. The graphs will be used to detect outliers and 
measurements unsuitable for analysis. The efficacy parameters will be listed by treatment, subject, 
visit and time. Individual graphs by time will be generated. 
All measured PD endpoints will be summarised (n, mean, SD, SEM, median, Min and Max values) 
by treatment and time, and will also be presented graphically as mean over time, with standard 
deviation as error bars. All efficacy endpoints will be summarised (mean, SD, SEM, median, Min 
and Max values) by treatment, and will also be presented graphically as mean in a bargraph, with 
standard deviation as error bars. 
Parameters will initially be analyzed without transformation, but if the data suggest otherwise, log-
transformation may be applied. Log-transformed parameters will be back-transformed after analysis 
where the results may be interpreted as percentage change. 
To establish whether significant treatment effects can be detected on the measured efficacy 
parameters, each parameter that is measured repeatedly will be analyzed with a mixed model 
analysis of covariance (ANCOVA) with treatment, time and treatment by time as fixed factors and 
subject, subject by treatment and subject by time as random factors and the (average) baseline 
measurement as covariate. Parameters that are only measured at baseline and at the end of the 
treatment phase will be analysed with a one-way ANCOVA with treatment and baseline as 
covariate. 
Single measured efficacy parameters will be analyzed with an unpaired t-test. 
The Kenward-Roger approximation will be used to estimate denominator degrees of freedom and 
model parameters will be estimated using the restricted maximum likelihood method. 
The general treatment effect and specific contrasts will be reported with the estimated difference 
and the 95% confidence interval, the least square mean estimates and the p-value. Graphs of the 
Least Squares Means(LSM) estimates over time by treatment will be presented with 95% 
confidence intervals as error bars, as well as change from baseline LSM estimates.  
The following contrasts will be calculated within the model: 
 
NeoRecormon - Placebo 
9.9.2 Inferential methods 
Null hypothesis:  
There’s no difference between the effects of NeoRecormon and placebo on cycling performance 
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parameters. 
 
9.10 Exploratory analyses and deviations 
Exploratory data-driven analyses can be performed with the caveat that any statistical inference will 
not have any confirmatory value. Deviations from the original statistical plan will be documented in 
the clinical study report. 
 
9.11 Interim analyses 
No interim analysis is planned. 
 
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10 GOOD CLINICAL PRACTICE, ETHICS AND ADMINISTRATIVE PROCEDURES  
10.1 Good clinical practice 
10.1.1 Ethics and good clinical practice 
The investigator will ensure that this study is conducted in full compliance with the protocol, the 
principles of the Declaration of Helsinki, ICH GCP guidelines, and with the laws and regulations of 
the country in which the clinical research is conducted.  
10.1.2 Ethics committee / institutional review board 
The investigator will submit this protocol and any related documents to an Ethics Committee (EC) 
and the Competent Authority (CA). Approval from the EC and the statement of no objection from the 
CA must be obtained before starting the study, and should be documented in a dated letter/email to 
the investigator, clearly identifying the trial, the documents reviewed and the date of approval. A list 
of EC members must be provided, including the functions of these members. If study staff were 
present, it must be clear that none of these persons voted. 
Modifications made to the protocol after receipt of the EC approval must also be submitted as 
amendments by the investigator to the EC in accordance with local procedures and regulations. 
10.1.3 Informed consent 
It is the responsibility of the investigator to obtain written informed consent from each individual 
participating in this study after adequate explanation of the aims, methods, objectives and potential 
hazards of the study. The investigator must also explain to the subjects that they are completely free 
to refuse to enter the study or to withdraw from it at any time for any reason.  
The Informed Consent and Subject Information will be provided in dutch. 
10.1.4 Insurance 
The investigator has a liability insurance which is in accordance with article 7, subsection 6 of the 
WMO. 
The investigator has an insurance which is in accordance with the legal requirements in the 
Netherlands (Article 7 WMO and the Measure regarding Compulsory Insurance for Clinical 
Research in Humans of 23rd June 2003). This insurance provides cover for damage to research 
subjects through injury or death caused by the study. 
 
€ 650,000.- (i.e., six hundred and fifty thousand Euro) for death or injury for each subject 
who participates in the Research; 
 
€ 5,000,000.- (i.e., five million Euro) for death or injury for all subjects who participate in the 
Research;  
 
€ 7,500,000.- (i.e., seven million five hundred thousand Euro) for the total damage incurred 
by the organisation for all damage disclosed by scientific research for the Sponsor as 
‘verrichter’ in the meaning of said Act in each year of insurance coverage. 
The insurance applies to the damage that becomes apparent during the study or within 4 years after 
the end of the study. 
10.2 Study funding 
CHDR is the sponsor of the study and is funding the study.   
 
10.3 Data handling and record keeping 
10.3.1 Data collection 
Data will be recorded on paper and/or electronic data collection forms and will be entered after 
quality control in a Promasys database for subsequent tabulation and statistical analysis. The data 
will be handled confidentially and if possible anonymously. 
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A Subject Screening and Enrolment Log will be completed for all eligible or non-eligible subjects 
with the reasons for exclusion. 
10.3.2 Database management and quality control 
A quality control check will be done by CHDR staff using data entry progress checks and database 
listings (blind data review). Errors with obvious corrections will be corrected before database lock.  
Results of computer tests and electronically captured questionnaires, clinical laboratory and efficacy 
analyses will be sent electronically to CHDR and loaded into the database. 
After the database has been declared complete and accurate, the database will be locked. Any 
changes to the database after that time can only be made by joint written agreement between the 
investigator and the statistician. 
10.4 Access to source data and documents 
All study data will be handled confidentially. The investigator will retain the originals of all source 
documents generated at CHDR for a period of 2 years after the report of the study has been 
finalised, after which all study-related documents will be archived (at a minimum) on micro-film 
which will be kept according to GCP regulations.  
The investigator will permit trial-related monitoring, audits, EC review and regulatory inspections, 
providing direct access to source data and documents. 
10.5 Quality control and quality assurance 
This study will be conducted according to applicable Standard Operating Procedures (SOPs). 
Quality assurance will be performed under the responsibility of CHDR’s Quality Assurance 
manager. 
10.5.1 Monitoring 
An initiation visit will be performed before the first subject is included. Monitoring visits and contacts 
will occur at regular intervals thereafter, according to a frequency defined in the study-specific 
monitoring plan. A close-out visit will be performed after study closure. 
 
10.6 Protocol amendments 
Any change to a protocol has to be considered as an amendment. 
10.6.1 Non-substantial amendment 
Administrative or logistical minor changes require a non-substantial amendment. Such changes 
include but are not limited to changes in study staff or contact details or minor changes in the 
packaging or labelling of study drug. Non-substantial amendments will be approved (signed) by the 
investigator(s) and will be recorded and filed by the investigator but will not be notified to the EC and 
the CA. 
The implementation of a non-substantial amendment can be done without notification to the 
appropriate EC or CA. It does not require their approval.  
The following amendments will be regarded as non-substantial: 
- 
change in timing of the samples; 
- 
changes in assay-type and / or institution where an assay will be performed, provided that 
validated assays will be used; 
- 
editorial changes to the volunteer information sheets; 
- 
determination of additional parameters in already collected materials, which are in 
agreement with the study objectives and do not provide prognostic or genetic information; 
- 
other statistical analyses than described in the protocol. 
10.6.2 Substantial amendment 
Significant changes require a substantial amendment. Significant changes include but are not 
limited to: new data affecting the safety of subjects, change of the objectives/endpoints of the study, 
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eligibility criteria, dose regimen, study assessments/procedures, treatment or study duration, with or 
without the need to modify the core Subject Information and Informed Consent Form. 
Substantial amendments are to be approved by the appropriate EC and the CA will need to provide 
a ‘no grounds for non-acceptance’ notification prior to the implementation of the substantial 
amendment. 
Urgent amendment 
An urgent amendment might become necessary to preserve the safety of the subjects included in 
the study. The requirements for approval should in no way prevent any immediate action being 
taken by the investigators in the best interests of the subjects. Therefore, if deemed necessary, an 
investigator can implement an immediate change to the protocol for safety reasons. This means 
that, exceptionally, the implementation of urgent amendments will occur before submission to and 
approval by the EC(s) and CA.  
10.7 End of study report 
The investigator will notify the EC and the CA of the end of the study within a period of 90 days. The 
end of the study is defined as the last subject’s last visit.  
 
In case the study is ended prematurely, the investigator will notify the EC and the CA within 15 
days, including the reasons for the premature termination. 
 
Within one year after the end of the study, the investigator will submit a final study report with the 
results of the study, including any publications/abstracts of the study, to the EC and the CA. The 
principal investigator will be the signatories for the study report.   
10.8 Public disclosure and publication policy 
In accordance with standard editorial and ethical practice, the results of the study will be published. 
The authorship guidelines of the Vancouver Protocol3  will be followed regarding co-authorship. 
 
The principal investigator will have the opportunity to review the analysis of the data and to discuss 
the interpretation of the study results prior to publication. 
                                                
 
3 http://www.icmje.org/ 
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11 STRUCTURED RISK ANALYSIS 
Please refer to the respective Summary of Product Characteristics (SmPC, see appendix 1) and the 
European Public Assessment Report (EPAR, see appendix 3). 
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12 REFERENCES 
References 
 
 
1.  Dr.Dick Marty, Mr.Peter Nicholson, Prof.Dr.Ulrich Haas. Report to the president of the Union 
Cycliste Internationale. Cycling Independent Reform Commision 2015. 
 
2.  Nimesh SA Patel, Massimo Collino, Muhammad M Yaqoob, Christoph Thiemermann. Erythropoietin 
in the intensive care unit: beyond treatment of anemia. Annals of Intensive Care 2011; 1: 1-9. 
 
3.   [online] Available at http://www.wada.-ama.org/e. World Anti-Doping Agency 2015. 
 
4.  Heuberger JA, Cohen Tervaert JM, Schepers FM, Vliegenthart AD, Rotmans JI, Daniels JM, 
Burggraaf J, Cohen AF. Erythropoietin doping in cycling: lack of evidence for efficacy and a negative 
risk-benefit. Br J Clin Pharmacol 2013; 75: 1406-1421. 
 
5.  Jeukendrup AE, Craig NP, Hawley JA. The bioenergetics of World Class Cycling. J Sci Med Sport. 
2000; 3: 414-433. 
 
6.  Connes P, Perrey S, Varray A, Prefaut C, Caillaud C. Faster oxygen uptake kinetics at the onset of 
submaximal cycling exercise following 4 weeks recombinant human erythropoietin (r-HuEPO) 
treatment. Pflugers.Arch. 2003; 447: 231-238. 
 
7.  Lucia A, Hoyos J, Perez M, Santalla A, Chicharro JL. Inverse relationship between VO2max and 
economy/efficiency in world-class cyclists. Med Sci.Sports Exerc. 2002; 34: 2079-2084. 
 
8.  Norio Suzuki. Erythropoietin Gene Expression: Developmental-Stage Specificity, Cell-Type 
Specificity, and Hypoxia Inducibility. Tohoku J.Exp.Med. 2015; 235: 233-240. 
 
9.  Lucia A, Hoyos J, Santalla A, Perez M, Chicharro JL. Curvilinear VO(2):power output relationship in 
a ramp test in professional cyclists: possible association with blood hemoglobin concentration. Jpn J 
Physiol. 2002; 52: 95-103. 
 
10.  Heinicke K, Wolfarth B, Winchenbach P, Biermann B, Schmid A, Huber G, Friedmann B, Schmidt 
W. Blood volume and hemoglobin mass in elite athletes of different disciplines. Int J Sports Med 
2001; 22: 504-512. 
 
11.  Henny H.Billett. Hemoglobin and hematocrit.In: Clinical Methods: The History, Physical, and 
Laboratory Examinations. 3rd edition. 1990: 718-719. 
 
12.   Online available at http://www.blood.ca/en/blood/hemoglobin. Canadian Blood Services 2015. 
 
13.  Wilkerson DP, Rittweger J, Berger NJ, Naish PF, Jones AM. Influence of recombinant human 
erythropoietin treatment on pulmonary O2 uptake kinetics during exercise in humans. J Physiol. 2005; 
568: 639-652. 
 
14.  Thomsen JJ, Rentsch RL, Robach P, Calbet JA, Boushel R, Rasmussen P, Juel C, Lundby C. 
Prolonged administration of recombinant human erythropoietin increases submaximal performance 
more than maximal aerobic capacity. Eur.J Appl.Physiol. 2007; 101: 481-486. 
 
15.  Lundby C, Achman-Andersen NJ, Thomsen JJ, Norgaard AM, Robach P. Testing for recombinant 
human erythropoietin in urine: problems associated with current anti-doping testing. J Appl.Physiol. 
2008; 105: 417-419. 
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16.  Birkeland KI, Stray-Gundersen J, Hemmersbach P, Hallen J, Haug E, Bahr R. Effect of rhEPO 
administration on serum levels of sTfR and cycling performance. Med Sci.Sports.Exerc. 2000; 32: 
1238-1243. 
 
17.  Ralph B.Dell, Steve Holleran, Rajasekhar Ramakrishnan. Sample Size Determination. ILAR journal 
2002; 43: 207-213. 
 
18.  Gore CJ, Parisotto R, Ashenden MJ, Stray-Gundersen J, Sharpe K, Hopkins WG, Emslie KR, Howe 
C, Trout GJ, Kazlauskas R, Hahn AG. Second-generation blood tests to detect erythropoietin abuse by 
athletes. Haematologica 2003; 88: 333-343. 
 
19.  Sottas PE, Robinson N, Giraud S, Taroni F, Kamber M, Mangin P, Saugy M. Statistical Classification 
of Abnormal Blood Profiles in Athletes. The International Journal of Biostatistics 2006; 2: 3. 
 
 
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13 APPENDIX 1 – SUMMARY OF PRODUCT CHARACTERISTICS – ENGLISH VERSION 
 
 
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14 APPENDIX 2 – SUMMARY OF PRODUCT CHARACTERISTICS – DUTCH VERSION  
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15 APPENDIX 3 – EUROPEAN PUBLIC ASSESSMENT REPORT   
 
  
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16 APPENDIX 4 – SCREENING: QUESTIONNAIRE SPORT ACTIVITIES 
Sport activities in the past 3 years: 
 
Type of 
sport 
Times a week 
Distance and duration 
per training/competition 
Mean velocity 
Since when  
1 
 
 
 
 
 
2 
 
 
 
 
 
3 
 
 
 
 
 
4 
 
 
 
 
 
5 
 
 
 
 
 
 
Sport activities in the past 6 weeks: 
 
Type of 
sport 
Times a week 
Distance and duration 
per training/competition 
Mean velocity 
Since when  
1 
 
 
 
 
 
2 
 
 
 
 
 
3 
 
 
 
 
 
4 
 
 
 
 
 
5 
 
 
 
 
 
 
 
Dutch: 
Sport activiteiten in de afgelopen 3 jaar: 
 
Type sport 
Aantal 
keer per 
week 
Afstand en duur van 
training/competitie 
Gemiddelde 
snelheid (km/u) 
Sinds 
wanneer  
1 
 
 
 
 
 
2 
 
 
 
 
 
3 
 
 
 
 
 
4 
 
 
 
 
 
5 
 
 
 
 
 
 
Sport activiteiten in de afgelopen 6 weken: 
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Type sport 
Aantal 
keer per 
week 
Afstand en duur van 
training/competitie 
Gemiddelde 
snelheid (km/u) 
Sinds 
wanneer  
1 
 
 
 
 
 
2 
 
 
 
 
 
3 
 
 
 
 
 
4 
 
 
 
 
 
5 
 
 
 
 
 
 
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17 APPENDIX 5 – EXERCISE TEST 
 
During this study the exercise tests will be performed on a cycle ergometer. 
The subjects will cycle at a self-selected pedal rate (between 70 and 90 rpm) and this pedal rate 
along with the saddle and handlebar heights will be recorded and reproduced in subsequent tests.  
 
All subjects will breathe through a facemask that is connected to an oxymeter to collect inspired and 
expired gasses for analyzing oxygen consumption, carbon dioxide production, minute ventilation, 
tidal volume and breathing frequency. Before each test, the oxymeter will be calibrated to ensure 
valid measurements. Blood pressure and heart rate will also be monitored during the tests. The 
blood pressure and heart rate measuring devices, cycle ergometer and oxymeter will be connected 
to a computer to measure and analyse all data. 
 
18.1 Ramp exercise test  
 
Protocol 
A ramp protocol will be followed until exhaustion, which will take approximately 1 hour. The details 
of the ramp exercise test will be described in the exercise test manual. 
 
 
18.2 Time to exhaustion test 
 
Protocol 
This exercise test will be performed according to a protocol looking at performance at submaximal 
intensity. It will last approximately 1 hour. The details of time to exhaustion test will be described in 
the exercise test manual. 
 
18.3 Statistics 
All data will be presented as means ± SD (or standard error). First, a two-way factor (group versus 
respectively VO2, VO2/kg, %VO2max, W and W/kg) ANOVA with repeated measures will be performed 
to determine if an interactive effect exists between both groups and mentioned parameters. These 
parameters will be analyzed at VT1, VT2 and maximal intensity. The ANOVA will be conducted to 
determine whether the factor group will significantly influence these physiological parameters. When 
a significant F-ratio is detected, comparisons for unpaired data will be performed (Student’s t-tests) 
to locate the differences (2-tailed). Maximal lactate values between both groups will also be 
compared using an unpaired Student’s t-test. VO2 at the ventilatory and lactate thresholds will be 
used to compare both thresholds by using a paired Student’s t-test. Statistical significance will be 
set at P < 0.05. 
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18 APPENDIX 6 – COMPETITION 
Subjects will climb the Mont Ventoux in an open course via Bédoin after a 150km closed course. A 
blood sample of each subject will be collected in Bédoin (at the end of the closed course) and at the 
finish. A power meter will be incorporated into each bike.  
 
Figure 2. Detailed description of the Mont Ventoux climb via Bédoin.1 
 
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Figure 3. Characteristics of the Mont Ventoux climb via Bédoin.1 
 
References 
 
1. http://www.clubcinglesventoux.org/en/roads/1-bedoin.html 
 
 
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19 APPENDIX 7 – URINE DOPING CONTROL PROCEDURE 
 
Urine collection for doping detection will be done at predetermined moments as specified in the 
Schedule of Assessments. For each sample, a minimum of 50 mL urine will be collected, which will 
be stored at -20 degrees Celcius or below before shipment to the Doping Lab.  
The doping lab will process the samples in a blind fashion as per the WADA Technical document 
published online at https://www.wada-ama.org/en/resources/science-medicine/td2014-epo.  
Samples will be analysed using two methods, being the SAR-PAGE and Isoelectrofocusing (IEF) as 
described in this technical document.   

Repeatability and predictive value of lactate threshold concepts in endurance sports.
Heuberger, Jules A A C,Gal, Pim,Stuurman, Frederik E,de Muinck Keizer, Wouter A S,Mejia Miranda, Yuri,Cohen, Adam F