A Single-arm, Observational Study to Explore and Characterize Wound Healing After Skin Punch Biopsies in Healthy Volunteers
Phase: NA
Conditions: Wound Healing
Sponsor: Centre for Human Drug Research, Netherlands

CHDR1736 
 
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CLINICAL STUDY PROTOCOL 
A single-arm, observational study to explore and characterize 
wound healing after skin punch biopsies in healthy volunteers 
 
 
 
 
 
Short Title: 
Wound healing in healthy volunteers 
Version: 
1 
Date: 
13-Sep-2017 
CHDR number: 
CHDR1736 
Toetsing Online number: 
NL63280.056.17 
EudraCT number: 
Not applicable  

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CONTACT DETAILS 
SPONSOR 
Centre for Human Drug Research 
Zernikedreef 8 
2333 CL Leiden 
The Netherlands 
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 
R. (Robert) Rissmann, PhD 
Telephone:  + 31 (0)71 5246438 
E-mail:     rrissmann@chdr.nl  
Medical Responsibility 
J. (Koos) Burggraaf, MD, PhD 
Telephone:  + 31 (0)71 5246400 
E-mail:     kb@chdr.nl 
Co-investigator 
M. (Mahdi) Saghari, MD 
Telephone:  + 31 (0)71 5246147 
E-mail:     msaghari@chdr.nl 
Co-investigator 
M.B.A. van Doorn, MD, PhD 
Telephone:  + 31 (0)71 5246400 
E-mail:     m.b.a.vandoorn@erasmusmc.nl 
Manager Operations Unit 
J. M. (Ria) Kroon, BSc 
Telephone:  + 31 71 5246 498 
E-mail:     rk@chdr.nl 
Manager Clinical Unit 
C. E. (Emilie) Jonxis, MANP 
Telephone   +31 71 5246433 
E-mail:     ejonxis@chdr.nl 
Statistician 
E. S. (Erica) Klaassen, MSc 
Telephone:  + 31 71 5246428 
E-mail:     EKlaassen@chdr.nl 
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 AND 
HAEMATOLOGY  
 
C.M. Cobbaert, PhD 
AKCL LUMC, E2-P 
Albinusdreef 2 
2333 ZA Leiden 
The Netherlands 
Contact person 
A.G. van Tilborg  

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LABORATORY 
 
Centre for Human Drug Research  
Zernikedreef 8  
2333 CL Leiden  
The Netherlands  
Contact person 
Monique van der Linden 
E-mail:     mlinden@chdr.nl 
LABORATORY – Pathology 
 
Erasmus MC 
's-Gravendijkwal 230 
3015 CE Rotterdam 
The Netherlands 
Contact person 
dr. J. Damman 
E-mail:     j.damman@erasmusmc.nl 
LABORATORY – IMMUNOLOGY 
 
Erasmus MC 
's-Gravendijkwal 230 
3015 CE Rotterdam 
The Netherlands 
Contact person 
Prof. dr. E.P. Prens 
E-mail:     e.prens@erasmusmc.nl 
LABORATORY – IMMUNOLOGY 
FibroTx LLC 
Maealuse 4 
12618 Tallinn 
Estonia 
Contact person  
P. Spee 
Telephone:  +45 42179934 
LABORATORY – Microbiome 
Baseclear BV 
Einsteinweg 
Leiden 
The Netherlands 
Contact person  
Bas Schenkenveld 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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SIGNATURE PAGE - PRINCIPAL INVESTIGATOR 
 
Study Title 
A single-arm, observational study to explore and characterize wound healing after skin punch 
biopsies in healthy volunteers. 
I acknowledge accountability for this protocol in accordance with CHDR’s current procedures. 
R. (Robert) Rissmann, RPh, PhD 
Principal investigator 
 
 
 
 
Signature 
Date (dd Mmm yyyy) 
 
J. (Koos) Burggraaf, MD, PhD 
Medical Responsibility 
 
 
 
 
 
Signature 
Date (dd Mmm yyyy) 
 

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SIGNATURE PAGE - TRIAL SITE STAFF 
Centre for Human Drug Research 
Study Title 
A single-arm, observational study to explore and characterize wound healing after skin punch 
biopsies in healthy volunteers 
I acknowledge responsibility for this protocol in accordance with CHDR’s current procedures. 
M. (Mahdi) Saghari, MD 
Co-investigator 
 
 
 
 
Signature 
Date (dd Mmm yyyy) 
M.B.A. van Doorn, MD, PhD  
Co-investigator 
 
 
 
 
Signature 
Date (dd Mmm yyyy) 
J. M. (Ria) Kroon, BSc 
Manager Operations Unit 
 
 
 
 
Signature 
Date (dd Mmm yyyy) 
C.E. (Emilie) Jonxis, MANP 
Manager Clinical Unit 
 
 
 
 
Signature 
Date (dd Mmm yyyy) 
E. S. (Erica) Klaassen, MSc 
Statistician 
 
 
 
 
Signature 
Date (dd Mmm yyyy) 

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TABLE OF CONTENTS 
CONTACT DETAILS ........................................................................................................................................... 2 
SIGNATURE PAGE - PRINCIPAL INVESTIGATOR ......................................................................................... 4 
SIGNATURE PAGE - TRIAL SITE STAFF ......................................................................................................... 5 
TABLE OF CONTENTS ...................................................................................................................................... 6 
LIST OF ABBREVIATIONS .............................................................................................................................. 10 
PROTOCOL SYNOPSIS ................................................................................................................................... 11 
1 
BACKGROUND AND RATIONALE ......................................................................................................... 16 
1.1 
Context............................................................................................................................................... 16 
1.2 
Non-clinical information ..................................................................................................................... 17 
1.3 
Clinical information ............................................................................................................................ 17 
1.4 
Study rationale ................................................................................................................................... 17 
1.4.1 
Benefit and risk assessment ...................................................................................................... 17 
1.4.2 
Study population ........................................................................................................................ 17 
1.4.3 
Study design .............................................................................................................................. 17 
1.4.4 
Comparative drug(s) and/or placebo ......................................................................................... 17 
1.4.5 
Duration of observation .............................................................................................................. 17 
1.4.6 
Primary endpoint ........................................................................................................................ 17 
1.4.7 
Statistical hypotheses and sample size ..................................................................................... 18 
2 
STUDY OBJECTIVES ............................................................................................................................... 19 
2.1 
Primary objective ............................................................................................................................... 19 
2.2 
Secondary objectives ......................................................................................................................... 19 
3 
STUDY DESIGN ........................................................................................................................................ 20 
3.1 
Overall study design and plan ........................................................................................................... 20 
3.1.1 
Screening ................................................................................................................................... 20 
3.1.2 
Observation period .................................................................................................................... 20 
4 
STUDY POPULATION .............................................................................................................................. 21 
4.1 
Subject population ............................................................................................................................. 21 
4.2 
Inclusion criteria ................................................................................................................................. 21 
4.3 
Exclusion criteria ................................................................................................................................ 21 
4.4 
Concomitant medications .................................................................................................................. 21 
4.5 
Lifestyle restrictions ........................................................................................................................... 22 
4.6 
Study  discontinuation and withdrawal .............................................................................................. 22 
4.6.1 
Study drug interruption or discontinuation ................................................................................. 22 
4.6.2 
Subject withdrawal ..................................................................................................................... 22 
4.6.3 
Replacement policy ................................................................................................................... 22 
5 
INVESTIGATIONAL MEDICINAL PRODUCT .......................................................................................... 23 
5.1 
Investigational drug ............................................................................................................................ 23 

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5.2 
Study drug up- and down-titration ..................................................................................................... 23 
5.3 
Study drug packaging and labelling ................................................................................................... 23 
5.4 
Example label in Dutch ...................................................................................................................... 23 
5.5 
Drug accountability ............................................................................................................................ 23 
5.6 
Treatment assignment and blinding .................................................................................................. 23 
5.6.1 
Randomization and treatment assignment ................................................................................ 23 
5.6.2 
Blinding ...................................................................................................................................... 23 
6 
STUDY ENDPOINTS ................................................................................................................................. 24 
6.1 
Endpoints ........................................................................................................................................... 24 
6.2 
Safety and tolerability endpoints ........................................................................................................ 24 
7 
STUDY ASSESSMENTS .......................................................................................................................... 25 
7.1 
Assessments ..................................................................................................................................... 25 
7.1.1 
Biopsy sample collection ........................................................................................................... 25 
7.1.2 
Histology .................................................................................................................................... 25 
7.1.3 
Immunohistochemistry ............................................................................................................... 25 
7.1.4 
Local biomarker sequencing ...................................................................................................... 26 
7.1.5 
Transdermal analysis patch (TAP) ............................................................................................ 26 
7.1.6 
Skin microbiome ........................................................................................................................ 26 
7.1.7 
Clinical photography .................................................................................................................. 27 
7.1.8 
Skin temperature by thermography ........................................................................................... 27 
7.1.9 
Perfusion by Laser spectrum contrast imaging (LSCI) .............................................................. 27 
7.1.10 
Trans epidermal water loss (TEWL) .......................................................................................... 27 
7.1.11 
Skin colour by colorimetry .......................................................................................................... 27 
7.1.12 
Clinical grading scales ............................................................................................................... 28 
7.1.13 
Subject reported outcomes ........................................................................................................ 28 
7.2 
Safety and tolerability assessments .................................................................................................. 29 
7.2.1 
Vital signs................................................................................................................................... 29 
7.2.2 
Weight and height ...................................................................................................................... 29 
7.2.3 
Physical examination ................................................................................................................. 29 
7.2.4 
Laboratory assessments ............................................................................................................ 29 
7.2.5 
Shipping Procedures ................................................................................................................. 30 
7.2.6 
Concomitant medications .......................................................................................................... 30 
7.2.7 
Local tolerability ......................................................................................................................... 30 
7.3 
Sequence of assessments and time windows ................................................................................... 30 
8 
SAFETY REPORTING .............................................................................................................................. 32 
8.1 
Definitions of adverse events ............................................................................................................ 32 
8.1.1 
Intensity of adverse events ........................................................................................................ 32 
8.1.2 
Relationship to the intervention ................................................................................................. 32 

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8.1.3 
Chronicity of adverse events ..................................................................................................... 32 
8.1.4 
Action ......................................................................................................................................... 32 
8.1.5 
Serious adverse events ............................................................................................................. 32 
8.1.6 
Suspected unexpected serious adverse reactions .................................................................... 33 
8.1.7 
Reporting of serious adverse events ......................................................................................... 33 
8.1.8 
Follow-up of adverse events ...................................................................................................... 33 
8.2 
Temporary halt for reasons of subject safety .................................................................................... 34 
8.3 
Annual safety report or development safety update report ............................................................... 34 
9 
STATISTICAL METHODOLOGY AND ANALYSES ................................................................................ 35 
9.1 
Statistical analysis plan...................................................................................................................... 35 
9.2 
Protocol violations/deviations ............................................................................................................ 35 
9.3 
Power calculation ............................................................................................................................... 35 
9.4 
Missing, unused and spurious data ................................................................................................... 35 
9.5 
Analysis sets ...................................................................................................................................... 35 
9.5.1 
Pharmacodynamic analysis set ................................................................................................. 35 
9.5.2 
Safety set ................................................................................................................................... 35 
9.6 
Subject disposition ............................................................................................................................. 35 
9.7 
Baseline parameters and concomitant medications .......................................................................... 36 
9.7.1 
Demographics and baseline variables ....................................................................................... 36 
9.7.2 
Medical history ........................................................................................................................... 36 
9.7.3 
Concomitant Medications .......................................................................................................... 36 
9.7.4 
Treatment compliance/exposure ............................................................................................... 36 
9.8 
Safety and tolerability endpoints ........................................................................................................ 36 
9.8.1 
Adverse events .......................................................................................................................... 36 
9.8.2 
Vital signs................................................................................................................................... 37 
9.8.3 
Clinical laboratory tests .............................................................................................................. 37 
9.9 
Endpoints ........................................................................................................................................... 37 
9.9.1 
Inferential methods .................................................................................................................... 37 
9.10 
Exploratory analyses and deviations ................................................................................................. 37 
9.11 
Interim analyses ................................................................................................................................. 37 
10 
GOOD CLINICAL PRACTICE, ETHICS AND ADMINISTRATIVE PROCEDURES ............................ 38 
10.1 
Good clinical practice ......................................................................................................................... 38 
10.1.1 
Ethics and good clinical practice ............................................................................................... 38 
10.1.2 
Ethics committee / institutional review board ............................................................................. 38 
10.1.3 
Informed consent ....................................................................................................................... 38 
10.1.4 
Insurance ................................................................................................................................... 38 
10.2 
Study funding ..................................................................................................................................... 39 
10.3 
Data handling and record keeping ..................................................................................................... 39 

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10.3.1 
Data collection ........................................................................................................................... 39 
10.3.2 
Database management and quality control ............................................................................... 39 
10.4 
Access to source data and documents .............................................................................................. 39 
10.5 
Quality control and quality assurance ................................................................................................ 39 
10.5.1 
Monitoring .................................................................................................................................. 39 
10.6 
Protocol amendments ........................................................................................................................ 39 
10.6.1 
Substantial amendment ............................................................................................................. 40 
10.6.2 
Non-substantial amendment ...................................................................................................... 40 
10.6.3 
Urgent amendment .................................................................................................................... 40 
10.7 
End of study report ............................................................................................................................ 40 
10.8 
Public disclosure and publication policy ............................................................................................ 41 
11 
STRUCTURED RISK ANALYSIS ......................................................................................................... 42 
12 
REFERENCES ...................................................................................................................................... 43 
APPENDIX A ..................................................................................................................................................... 44 
APPENDIX B ..................................................................................................................................................... 45 
 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event 
ABR 
ABR form, General Assessment and Registration form, is the application form that is 
required for submission to the accredited Ethics Committee; in Dutch, ABR = 
Algemene Beoordeling en Registratie 
ANCOVA 
Analysis of Covariance 
BMI 
Body Mass Index 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: Centrale 
Commissie Mensgebonden Onderzoek 
CHDR 
Centre for Human Drug Research 
CRF 
Case Report Form 
EC 
Ethics Committee (also Medical Research Ethics Committee (MREC); in Dutch: 
Medisch Ethische Toetsing Commissie (METC). 
EDTA 
EOS 
Ethylene diamine tetra-acetic acid 
End Of Study 
EU 
European Union 
GCP 
HE 
Good Clinical Practice 
hematoxylin and eosin 
ICH 
IL 
International Conference on Harmonization 
Interleukin 
LSCI 
Laser Speckle Contrast Image 
MedDRA 
NSAID 
NRS 
OTC 
PD 
POSAS 
qRT-PCR 
RNA-seq 
RYB 
Medical Dictionary for Regulatory Activities 
non-steroid anti-inflammatory drugs 
Numeric Rating Scale 
over-the-counter 
Pharmacodynamics 
Patient and Observer Scar Assessment Scale 
real-time reverse transcription polymerase chain reaction 
RNA sequencing 
Red-Yellow-Black wound assessment scale 
SAE 
Serious Adverse Event 
SD 
Standard Deviation 
SEM 
Standard Error of the Mean 
SOC 
System Organ Class 
SOP 
Standard Operating Procedure 
SST 
Serum Separator Tube 
SUSAR 
TAP 
TEWL 
Suspected Unexpected Serious Adverse Reaction 
Transdermal analysis patch 
Trans Epidermal Water Loss 
TNF 
Tumor Necrosis Factor 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen. 
 

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PROTOCOL SYNOPSIS 
Title 
A single-arm, observational study to explore and characterize wound healing after skin punch 
biopsies in healthy volunteers 
Short Title 
Wound healing in healthy volunteers 
 
Principal investigator & Trial Site 
R. Rissmann, PhD; Medical responsibility J. Burggraaf, MD, PhD Centre for Human Drug Research, 
Zernikedreef 8, 2333 CL Leiden, The Netherlands 
 
Background & Rationale 
The skin plays a critical role in protection where it acts as a barrier from damage and pathogens 
between the external and internal environments (Dreifke et al., 2015). Wounds compromise its 
protective role by disrupting the function and the normal structure of the skin and the underlying soft 
tissue. As a response to injury wound healing occurs in order to rapidly restore the defect. This 
process involves activation of, among others, keratinocytes, fibroblasts, endothelial cells, 
macrophages, and platelets and consists of multiple phases including hemostasis, inflammation, 
migration and cellular proliferation, and maturation and remodeling (Armstrong and Meyr, 2017). A 
simplified schematic of the course of wound healing is depicted in Figure 2. Due to the broad 
involvement of various cell types, extracellular matrix and many reactive molecules each phase in 
wound healing produces characteristic changes within the tissue. A deficiency in any part of the 
process can lead to delayed wound healing, abnormal scar formation or chronic wounds.  
To study wound healing in healthy volunteers a challenge model with skin punch biopsies has been 
described in literature previously (Greaves et al., 2014, Greaves et al., 2015, Illigens and Gibbons, 
2013, Ud-Din et al., 2012). However, the characterization of this model was not performed 
comprehensively since advanced analysis of biopsies were omitted. Furthermore, analyses 
performed in previous studies only partially described wound healing processes either by insufficient 
time points for characterization or scarce simultaneous evaluations of multiple wound healing 
modalities. In addition, novel non-invasive imaging methodologies including thermography, 3D 
photography, multispectral imaging, enable more comprehensive profiling of the wound morphology. 
The overall aim of this study is to develop a standardized model to temporarily and locally induce a 
skin trauma to investigate wound healing and monitor wound closure. This clinical model will enable 
future application as proof-of-pharmacology and proof-of concept studies as well as drug profiling in 
early drug development programs. More specifically, the objective of the trial is to explore and 
characterize the induction of well-defined skin trauma and natural wound healing process over the 
course of the different phases using a battery of dermatological assessments after skin punch 
biopsies in healthy volunteers. Furthermore, safety and tolerability will be assessed. 
Characterization and monitoring of wound healing effects following skin punch biopsies will be 
performed by means of biophysical, biochemical, imaging, clinical parameters and subject reported 
outcomes. 
 
 

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Objective(s) 
Primary Objective 
 
To characterize and monitor wound healing after a standardized, induced skin trauma  
Secondary Objectives 
 
To evaluate safety and tolerability of biopsy-induced skin trauma  
 
Design 
This is an observational, single center, single arm study. 
 
Treatments 
Biopsy sites will be treated by secondary healing intention with a non-stick gauze dressing followed 
by no treatment after 48h. 
Investigational drug 
Not applicable 
Comparative drug 
No comparative drug will be used in this study. 
Study periods 
The total duration of the study will be approximately 14 weeks: 4 weeks for screening and 10 weeks 
of observation. Subjects will visit CHDR 14 times. For a detailed outline see Table 1. 
Subjects / Groups 
A total of 18 healthy volunteers are planned to be enrolled. The study will entail 1 cohort with a 
randomized repeated biopsy collection time. Three skin punch biopsies (3 mm) of the lower back 
will be taken from each volunteer on day 0, with a distance of approximately 3-5 cm in between. All 
biopsy lesions will be treated with a gauze dressing (Jelonet®, paraffine gauze + Tegaderm®) for 
48h after which the gauze is removed. Hereafter, the biopsy lesions will remain untreated. One 
biopsy sample taken on day 0 will serve as a baseline measurement for the repeated samples 
regarding the histology, immunohistochemistry, and RNA sequencing (RNA-seq) or real-time 
reverse transcription polymerase chain reaction (qRT-PCR) assessments.  
Repeated biopsies of the same location as on day 0 will be taken on day 7, 14 or 21 (biopsy lesion 
and day randomized), and day 28, 42 or 56 (biopsy lesion and day randomized) for all subjects. The 
observation biopsy (biopsy lesion randomized) will serve as primary biopsy and followed for all 
measurements. All repeated biopsy lesions will also be treated with a gauze dressing (Jelonet®, 
paraffine gauze + Tegaderm®) for 48h after which the gauze is removed and observation 
commences. 
 
Cohort  Observation period (day 2 - day 70) 
Number of subjects 
1 
3 biopsies taken on day 0 
- 
1 repeated biopsy taken on day 7, 14 or 21 (randomized) 
- 
1 repeated biopsy taken on day 28, 42 or 56 (randomized) 
- 
1 target (primary) biopsy for observation without re-excision 
18 
 
 
 

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Inclusion criteria 
Eligible subjects must meet all of the following inclusion criteria at screening: 
1. Healthy subjects, 18 to 30 years of age (inclusive). The health status is verified by absence 
of evidence of any clinical significant active or uncontrolled chronic disease following a 
detailed medical history, a complete physical examination including vital signs, blood 
sampling of hematology, chemistry, and virology, urinalysis, urine drug and cotinine testing,  
and alcohol breath testing. In the case of uncertain or questionable results, tests performed 
during screening may be repeated before randomization to confirm eligibility or judged to be 
clinically irrelevant for healthy subjects. 
2. Body mass index (BMI) between 18 and 30 kg/m2, inclusive  
3. Fitzpatrick Skin type I-II (Caucasian type). 
4. Eligible lower back to perform biopsies (no excessive hair growth, no local skin disorder) 
5. Willing to give written informed consent and willing and able to comply with the study 
protocol. 
 
Exclusion criteria 
Eligible subjects must meet none of the following exclusion criteria at screening: 
1. History of pathological scar formation (keloid, hypertrophic scars) 
2. Any form of body modification of the lower back hindering biopsy collection of unaltered skin 
(e.g. tattoos, piercings, implants) 
3. Any disease associated with immune system impairment, including auto-immune diseases, 
HIV and transplantation patients. 
4. Requirement of immunosuppressive or immunomodulatory medication, including 
glucocorticoids, non-steroid anti-inflammatory drugs (NSAIDs), and chemotherapeutic drugs 
within 30 days prior to enrollment or planned to use during the course of the study. 
5. Have any current and/or recurrent pathologically, clinical significant relevant skin condition.  
6. Use of topical medication (prescription or over-the-counter (OTC)) within 30 days of the start 
of the study.in local treatment area. 
7. Pregnant, a positive pregnancy test, intending to become pregnant, or breastfeeding. 
8. Current smoker and/or regular user of other nicotine-containing products (e.g., patches). 
9. Average consumption of more than 14 units of alcohol per week 
10. Tanning due to sunbathing, excessive sun exposure or a tanning booth within 3 weeks of 
enrollment or planned to do so during the course of the study 
11. Participation in an investigational drug or device study within 3 months prior to screening or 
more than 4 times a year. 
12. Loss or donation of blood over 500 mL within three months prior to screening. 
13. Any (medical) condition that would, in the opinion of the investigator, potentially compromise 
the safety or compliance of the subject or may preclude the subjects’ successful completion 
of the clinical trial. 
 
Concomitant medications 
No prescription medication or OTC medications (excluding multivitamins) will be permitted within 21 
days prior to the start of the study, or less than 5 half-lives (whichever is longer), and during the 
course of the study. Exceptions are paracetamol (up to 4 g/day) in case of local pain. Use of pain 
medication will be determined by the investigator individually. Other exceptions will only be made if 
the rationale is discussed and clearly documented. 
 
 

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Endpoints 
 
Biopsy biomarkers: 
o Histology with hematoxylin and eosin (HE) staining 
o Immunohistochemistry with wound healing related biomarkers (e.g. CD31, collagen I, 
collagen III, aSMA, fibronectin) 
o RNA-seq or qRT-PCR for wound healing related biomarkers (e.g. VEGFα, TGFβ1, 
TGFβ2, TGFβ3, PDGF, CTGF, TNF, IL-1B, IL-4, GM-CSF, IL-6, IL-10, MMP1, 
MMP3, OSM, LOX) 
 
Local skin biomarkers for wound healing related biomarkers (e.g. VEGF-A, TNFα, IL-8, 
TLSP, MMP-3, IL-4) by transdermal analysis patch (TAP) 
 
Clinical imaging (e.g. 2D and 3D photography, thermography, laser speckle contrast imaging 
(LSCI), trans epidermal water loss (TEWL), colorimetry) 
 
Clinical evaluation (erythema grading scale, Red-Yellow-Black (RYB) wound assessment 
scale, the Patient and Observer Scar Assessment Scale (POSAS)) 
 
Skin microbiome (healthy and biopsy lesions) 
 
Tolerability / safety endpoints 
 
Adverse events (AEs) 
 
Local tolerance (erythema grading scale, RYB wound assessment scale, POSAS, NRS 
pruritus and pain) 
All of above mentioned pharmacodynamic and safety measurements will be performed multiple 
times during the study according to the Visit and assessment schedule (Table 1). 
 
Sample Size Justification 
A total cohort size of 18 healthy volunteers will be investigated. This is justified since the primary 
objective is to explore and monitor the wound healing model. No formal power calculation was 
performed given the exploratory character of the study. 
 
Statistical methodology 
Data listings and averages will be presented for pharmacodynamics and safety measures.  
Given the exploratory character of the study, pharmacodynamic endpoints will be primarily analyzed 
using descriptive statistics. All pharmacodynamic endpoints will be summarized (mean and standard 
deviation of the mean, median, minimum and maximum values) by time, and will also be presented 
graphically as mean over time, with standard deviation as error bars. Both Nominal results, and log-
transformed results and change from baseline results will be utilized in all data summaries. All 
categorical pharmacodynamic endpoints will be summarized by frequencies. 
.

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Table 1: Visit and schedule assessment 
                                               
A Biopsies taken after measurements are performed 
B Randomized repeated biopsy sample collection time, maximum 5 per subject over three site during the trial 
 
SCR 
Day 0 
Day 2 
Day 4 
Day 7 
Day 10 
Day 14 
Day17 
Day 21 
Day 24 
Day 28 
Day 42 
Day 56 
Day 70 
Time 
Assessment 
Up to 
-28 
days 
 
 
 
 
 
 
 
 
 
 
 
 
EOS 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demography (age, race, gender) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion and exclusion criteria 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical examination 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height and weight 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
BsHem, BsChem, BsVir, UrSamStk, 
UrPregnancy, UrDrugSc, UrCotinine, 
BreathAlc 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs (HR, BP) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Temperature 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
General symptoms 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Clinical imaging (2D and 3D 
photography, thermography, LSCI, 
TEWL, colorimetry) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Clinical assessments (erythema 
grading scale, RYB wound 
assessment scale, POSAS) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Subject assessment (pruritus, pain, 
POSAS) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
TAP and skin microbiome (healthy and 
lesions) 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Skin punch biopsyA 
 
X 
 
 
XB 
 
XB 
 
XB 
 
XB 
XB 
XB 
 
(S)AE / ConMed 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
SCR = Screening; EOS = End of study; BsHem = Hematology blood sample; BsChem = Chemistry blood sample; BsVir = Virology 
blood sample; UrSamStk = Urinalysis; UrPregnancy =  Urine sample pregnancy test for woman of childbearing potential; UrDrugScr 
= Urine drug test; UrCotinine = Urine cotinine test; BreathAlc = Alcohol breath test; HR = Heart rate; BP = Blood pressure; LSCI = 
Laser Speckle Contrast Imaging; TEWL = Transepidermal Water Loss; RYB = Red-Yellow-Black wound assessment scale; TAP = 
Transdermal Analysis Patch; (S)AE = (Serious) adverse event; ConMed = Concomitant medication 
 
 
 
 
 

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1 
BACKGROUND AND RATIONALE 
1.1 Context 
The skin plays a critical role in protection where it acts as a barrier from damage and pathogens 
between the external and internal environments (Dreifke et al., 2015). Wounds compromise its 
protective role by disrupting the function and the normal structure of the skin and the underlying soft 
tissue. As a response to injury wound healing occurs in order to rapidly restore the defect. This 
process involves activation of keratinocytes, fibroblasts, endothelial cells, macrophages, and 
platelets and consists of multiple phases including hemostasis, inflammation, migration and cellular 
proliferation, and maturation and remodeling (Armstrong and Meyr, 2017). A simplified schematic of 
the course of wound healing is depicted in Figure 2. Hemostasis occurs immediately after dermal 
injury. The inflammation phase is characterized by cellular recruitment and increased vascular 
permeability. The epithelization phase is achieved by proliferation of basal cells and migration of 
epithelial cells. The last phase is known as the maturation and remodeling phase where collagen 
cross-linking and remodeling, wound contraction, and repigmentation takes place. Due to the broad 
involvement of various cell types, extracellular matrix and many reactive molecules each phase in 
wound healing produces characteristic changes within the tissue. A deficiency in any part of the 
process can lead to delayed wound healing, abnormal scar formation or chronic wounds.  
To study wound healing in healthy volunteers a challenge model with skin punch biopsies has been 
described in literature previously (Greaves et al., 2014, Greaves et al., 2015, Illigens and Gibbons, 
2013, Ud-Din et al., 2012). However, the characterization of this model was not performed 
comprehensively since advanced analysis of biopsies were omitted. Furthermore, analyses 
performed in previous studies only partially described wound healing processes either by insufficient 
time points for characterization or scarce simultaneous evaluations of multiple wound healing 
modalities. 
 
Figure 2: Wound healing time line 
The overall aim of this study is to develop a standardized model to temporarily and locally induce a 
skin trauma to investigate wound healing and monitor wound closure. This clinical model will enable 
future application as proof-of-pharmacology and proof-of concept studies as well as drug profiling in 
early drug development programs. More specifically, the objective of the trial is to explore and 
characterize the induction of well-defined skin trauma and natural wound healing process over the 

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course of the different phases using a battery of dermatological assessments after skin punch 
biopsies in healthy volunteers. Furthermore, safety and tolerability will be assessed. 
Characterization and monitoring of wound healing effects following skin punch biopsies will be 
performed by means of biophysical, biochemical, imaging, clinical parameters and subject reported 
outcomes. 
 
1.2 Non-clinical information 
No drug or treatment with active ingredients is to be used in this study. 
 
1.3 Clinical information 
No drug or treatment with active ingredients is to be used in this study. 
 
1.4 Study rationale 
1.4.1 Benefit and risk assessment 
Small skin punch biopsies are frequently performed in both clinical care and clinical research. Skin 
punch biopsies of 3 mm and 4 mm are considered minimally invasive since the wound surface is 
limited and  wound care does usually not involve sutures. The induced wounds heal quickly and do 
not cause tremendous scarring. Subjects  with a high risk for hypertrophic scarring will be excluded. 
Therefore, the risks associated with study participation are considered minimal.  
 
1.4.2 Study population 
For this trial 18 healthy Caucasian (Fitzpatrick Skin type I-II) volunteers between the age of 18 and 
30 will be included.  
 
1.4.3 Study design 
This is an observational, single center, single arm study to monitor and characterize wound healing 
after skin punch biopsies in healthy volunteers. An extensive battery of tests of various 
dermatological assessments will advance the understanding of normal wound healing. 
 
1.4.4 Comparative drug(s) and/or placebo 
Not applicable. 
 
1.4.5 Duration of observation 
Based on previous wound healing studies an observation duration of 70 days is chosen (Greaves et 
al., 2014, Greaves et al., 2015, Illigens and Gibbons, 2013, Ud-Din et al., 2015, Ud-Din et al., 2012). 
This is appropriate since study objectives such as characterizing and monitoring wound healing can 
be assessed adequately. 
 
1.4.6 Primary endpoint 
Pharmacodynamic endpoints 
 
Biopsy biomarkers: 

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o Histology with hematoxylin and eosin (HE) staining 
o Immunohistochemistry with wound healing related biomarkers (e.g. CD31, collagen I, 
collagen III, aSMA, fibronectin) 
o RNA sequencing (RNA-seq) or qRT-PCR for wound healing related biomarkers (e.g. 
VEGFα, TGFβ1, TGFβ2, TGFβ3, PDGF, CTGF, TNF, IL-1B, IL-4, GM-CSF, IL-6, IL-
10, MMP1, MMP3, OSM, LOX) 
 
Local skin biomarkers for wound healing related biomarkers (e.g. VEGF-A, TNFα, IL-8, 
TLSP, MMP-3, IL-4) by transdermal analysis patch (TAP) 
 
Clinical imaging (e.g. 2D and 3D photography, thermography, laser speckle contrast imaging 
(LSCI), trans epidermal water loss (TEWL), colorimetry) 
 
Clinical evaluation (erythema grading scale, Red-Yellow-Black (RYB) wound assessment 
scale, the Patient and Observer Scar Assessment Scale (POSAS)) 
 
Skin microbiome (healthy and biopsy lesions) 
Tolerability / safety endpoints 
 
Adverse events (AEs) 
 
Local tolerance (erythema grading scale, RYB wound assessment scale, NRS pruritus and 
pain, POSAS) 
All of above mentioned pharmacodynamic and safety measurements will be performed multiple 
times during the study according to the Visit and Assessment Schedule (Table 1). 
 
1.4.7 Statistical hypotheses and sample size 
A total cohort size of 18 healthy volunteers will be investigated. This is justified since the primary 
objective is to explore and monitor the wound healing model. No formal power calculation was 
performed given the exploratory character of the study. 

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2 
STUDY OBJECTIVES 
 
2.1 Primary objective 
 
To characterize and monitor wound healing after a standardized, induced skin trauma  
2.2 Secondary objectives 
 
To evaluate safety and tolerability of biopsy-induced skin trauma  
 

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3 
STUDY DESIGN 
3.1 Overall study design and plan 
This is an observational, single center, single arm study to monitor wound healing, explore the 
effects and evaluate safety/tolerability following a standardized, biopsy-induced skin trauma in 
healthy volunteers. A total of three biopsy will be taken: one will be used as primary (observational) 
lesion and two will be randomized to be re-excised for histological examination and exploration of 
biomarkers. 
The total duration of the study for each subject will be up to 98 days divided as follows: 
 
Screening: Up to 28 days prior to first study day; 
 
In clinic visits: Days 0, 2, 4, 7, 10, 14, 17, 21, 24, 28, 42, 56, 70 
 
3.1.1 Screening 
Within 4 weeks prior to study baseline visit (day 0), volunteers will undergo a medical screening. 
Screening will be performed in a fasting state (≥4 hours), and consists of medical history, physical 
examination, height, weight, vital signs, temperature, blood sampling (hematology, biochemistry, 
virology), urinalysis, urine pregnancy testing (women only), urine drug and cotinine testing, and 
alcohol breath testing.  
In addition, skin types will be assessed according to the Fitzpatrick classification. 
 
Fitzpatrick skin type classification: 
I: Pale white; blond or red hair; blue eyes; freckles — always burns, never tans 
II: White; fair; blond or red hair; blue, green, or hazel eyes — usually burns, tans minimally 
III: Cream white; fair with any hair or eye color; quite common — sometimes mild burn, tans 
uniformly 
IV: Moderate brown; typical Mediterranean skin tone — sometimes mild burns, always tans well 
V: Dark brown; Middle Eastern skin types — Very rarely burns, tans very easily 
VI: Deeply pigmented dark brown to darkest brown— never burns, never tans 
 
3.1.2 Observation period 
The total observation period of the trial will be 10 weeks (70 days). Subjects will visit the clinical unit 
as specified in section 3.1. 
Assessments will be performed on day 0, pre-dose on day 2, 4, 7, 10, 14, 17, 21, 24, 28, 42, 56 and 
70. Safety and tolerability will be assessed throughout the study via questionnaires, clinical grading 
scales, and adverse event reporting. 
All study procedure performed pre-dose and during the observation period are outlined in Table 1. 
Visit variances are allowed as described in Table 2.  
 

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4 
STUDY POPULATION 
4.1 Subject population 
A total of 18 healthy Caucasian 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 advertisement or from the subjects’ database of the Centre for 
Human Drug Research, Leiden, The Netherlands. 
 
4.2 Inclusion criteria 
Eligible subjects must meet all of the following inclusion criteria at screening: 
1. Healthy subjects, 18 to 30 years of age (inclusive). The health status is verified by absence 
of evidence of any clinical significant active or uncontrolled chronic disease following a 
detailed medical history, a complete physical examination including vital signs, blood 
sampling of hematology, chemistry, and virology, urinalysis, urine drug and cotinine testing,  
and alcohol breath testing. In the case of uncertain or questionable results, tests performed 
during screening may be repeated before randomization to confirm eligibility or judged to be 
clinically irrelevant for healthy subjects. 
2. Body mass index (BMI) between 18 and 30 kg/m2, inclusive  
3. Fitzpatrick Skin type I-II (Caucasian type). 
4. Eligible lower back to perform biopsies (no excessive hair growth, no local skin disorder) 
5. Willing to give written informed consent and willing and able to comply with the study 
protocol. 
 
4.3 Exclusion criteria 
Eligible subjects must meet none of the following exclusion criteria at screening: 
1. History of pathological scar formation (keloid, hypertrophic scars) 
2. Any form of body modification of the lower back hindering biopsy collection of unaltered skin 
(e.g. tattoos, piercings, implants) 
3. Any disease associated with immune system impairment, including auto-immune diseases, 
HIV and transplantation patients. 
4. Requirement of immunosuppressive or immunomodulatory medication, including 
glucocorticoids, non-steroid anti-inflammatory drugs (NSAIDs), and chemotherapeutic drugs 
within 30 days prior to enrollment or planned to use during the course of the study. 
5. Have any current and / or recurrent pathologically, clinical significant relevant skin condition.  
6. Use of topical medication (prescription or over-the-counter (OTC)) within 30 days of the start 
of the study in local treatment area. 
7. Pregnant, a positive pregnancy test, intending to become pregnant, or breastfeeding. 
8. Current smoker and/or regular user of other nicotine-containing products (e.g., patches). 
9. Average consumption of more than 14 units of alcohol per week 
10. Tanning due to sunbathing, excessive sun exposure or a tanning booth within 3 weeks of 
enrollment or planned to do so during the course of the study 
11. Participation in an investigational drug or device study within 3 months prior to screening or 
more than 4 times a year. 
12. Loss or donation of blood over 500 mL within three months prior to screening. 
13. Any (medical) condition that would, in the opinion of the investigator, potentially compromise 
the safety or compliance of the subject or may preclude the subjects’ successful completion 
of the clinical trial. 
 
4.4 Concomitant medications 
All medications (prescription and over-the-counter (OTC)) taken within 30 days of study screening 
will be recorded. 

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No prescription medication or OTC medications (excluding multivitamins) will be permitted within 21 
days prior to the first study day, or less than 5 half-lives (whichever is longer), and during the course 
of the study. Exceptions are paracetamol (up to 4 g/day) in case of local pain. Use of pain 
medication will be determined by the investigator individually. Other exceptions will only be made if 
the rationale is discussed and clearly documented. 
 
4.5 Lifestyle restrictions  
ln the interest of their safety and to facilitate assessment, the subjects participating in this study will 
be requested to agree to the following restrictions during the study: 
 
Subjects should avoid prolonged exposure of involved skin to sunlight from three weeks prior 
to the study until EOS visit 
 
Bathing and washing of involved skin is not allowed 6 hours prior to each study visit. 
 
Alcohol will not be allowed from at least 24 hours before screening and before each 
scheduled visit. At other times throughout the study, subjects should not consume more than 
2 units of alcohol daily on average (one unit is 10 grams of alcohol). 
 
Subjects will abstain from the use of tobacco-or nicotine-containing products (including e-
cigarettes and patches) from the screening visit until the EOS visit. 
 
Female subjects must use effective contraception for the duration of the study. 
 
4.6 Study  discontinuation and withdrawal 
4.6.1 Study drug interruption or discontinuation 
The investigator must temporarily interrupt or permanently discontinue the study  if continued 
participation is believed to be contrary to the best interests of the subject. The interruption or 
premature discontinuation might be triggered by an Adverse Event (AE), a diagnostic or therapeutic 
procedure, an abnormal assessment, or for administrative reasons in particular withdrawal of the 
subject’s consent. The reason for 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 for reasons other than adverse events or any other tolerability issues might 
replaced. 

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5 
INVESTIGATIONAL MEDICINAL PRODUCT 
5.1 Investigational drug 
Not applicable. 
 
5.2 Study drug up- and down-titration 
Not applicable. 
 
5.3 Study drug packaging and labelling  
Not applicable. 
5.4 Example label in Dutch 
Not applicable. 
 
5.5 Drug accountability 
Not applicable. 
 
5.6 Treatment assignment and blinding 
5.6.1 Randomization and treatment assignment 
No treatment will be administered. Subjects in this study will be numbered sequentially from 1 to 18 
in order of inclusion. Replacements will be numbered +100 (e.g. replacement for subject 1 will 
receive number 101). 
The time of repeated biopsies will be randomized. The randomization code will be generated using 
SAS v9 by a study-independent, CHDR statistician.  
 
5.6.2 Blinding 
Not applicable. 

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6 
STUDY ENDPOINTS 
6.1 Endpoints  
Wound healing will be assessed at the time points indicated in Table 1. 
 
Biopsy biomarkers: 
o Histology with hematoxylin and eosin (HE) staining 
o Immunohistochemistry with wound healing related biomarkers (e.g. CD31, collagen I, 
collagen III, aSMA, fibronectin) 
o RNA-seq or qRT-PCR for wound healing related biomarkers (e.g. VEGFα, TGFβ1, 
TGFβ2, TGFβ3, PDGF, CTGF, TNF, IL-1B, IL-6, IL-10, MMP1, MMP3, OSM, LOX) 
 
Local skin biomarkers for wound healing related biomarkers (e.g. VEGF-A, TNFα, IL-8, 
TLSP, MMP-3, IL-4) by transdermal analysis patch (TAP) 
 
Clinical imaging (e.g. 2D and 3D photography, thermography, laser speckle contrast imaging 
(LSCI), trans epidermal water loss (TEWL), colorimetry) 
 
Clinical evaluation (erythema grading scale, Red-Yellow-Black (RYB) wound assessment 
scale, the Patient and Observer Scar Assessment Scale (POSAS)) 
 
Skin microbiome (healthy and biopsy lesion) 
 
6.2 Safety and tolerability endpoints 
Adverse events (AE) will be collected throughout the study, at every study visit. 
 
(serious) adverse events ((S)AEs). 
 
Concomitant medication 
 
Local tolerability (erythema grading scale, RYB wound assessment scale, POSAS, NRS 
pruritus and pain) 

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7 
STUDY ASSESSMENTS 
See the Visit and assessment schedule (Table 1) for the time points of the assessments. 
 
7.1 Assessments 
7.1.1 Biopsy sample collection 
Initial biopsies 
Three skin punch biopsies (3 mm) of the lower back will be taken from each volunteer on day 0, with 
a distance of approximately 3-5 cm in between. All biopsy lesions will be treated with a gauze 
dressing (Jelonet®, paraffine gauze + Tegaderm®) for 48h after which the gauze is removed. 
Hereafter, the biopsy lesions will remain untreated. One biopsy sample taken on day 0 will serve as 
a baseline measurement for the repeated samples regarding the histology, immunohistochemistry, 
and RNA sequencing (RNA-seq) or real-time reverse transcription polymerase chain reaction (qRT-
PCR) assessments.  
Repeated biopsies 
Repeated biopsies of the same location as on day 0 will be taken on day 7, 14 or 21 (biopsy lesion 
and day randomized), and day 28, 42 or 56 (biopsy lesion and day randomized) for all subjects. The 
observation biopsy (biopsy lesion randomized) will serve as primary biopsy and followed for all 
measurements. All repeated biopsy lesions will also be treated with a gauze dressing (Jelonet®, 
paraffine gauze + Tegaderm®) for 48h after which the gauze is removed and observation 
commences. 
The biopsy procedure will be performed according to the standard operating procedure for skin 
punch biopsies with local anaesthetics (CGESPBIO).  
The biopsies will be placed in sealed tubes and frozen in liquid nitrogen immediately at a 
temperature of less than -160°C. The biopsy samples will be stored at CHDR in a -80°C freezer until 
shipment. The biopsy samples will be analyzed at the Immunology/pathology Laboratory at 
Erasmus MC, Rotterdam, The Netherlands for histology and biomarkers. 
 
7.1.2 Histology 
Collected biopsy samples will be analyzed at Erasmus MC, Rotterdam, The Netherlands. A 
histopathological score of the biopsies will be obtained using HE stained tissue multiple times during 
the study (see Table 1). Skin response will be assessed using the histological parameters 
characteristics of wound healing (amount of granulation tissue, inflammatory infiltrate, collagen fiber 
orientation, pattern of collagen, amount of early collagen, amount of mature collagen, migration of 
keratinocytes, bridging of cells, keratinization, new vessel formation). 
The histopathological score for each of the patterns will be graded on a scale from 0 to 3. 
 
7.1.3 Immunohistochemistry 
Serial sections of biopsy samples will be stained with antibodies to identify wound healing related 
biomarkers (e.g. CD31, collagen I, collagen III, aSMA, fibronectin), and assessed by 
immunohistochemistry at Erasmus MC, Rotterdam, The Netherlands.   
Biomarker analyses are subject to change and additional analyses may be performed in already 
collected materials. 
 

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7.1.4 Local biomarker sequencing 
RNA-seq or qRT-PCR will be performed on collected biopsy samples for wound healing related 
biomarkers (e.g. VEGFα, TGFβ1, TGFβ2, TGFβ3, PDGF, CTGF, TNF, IL-1B, IL-6, IL-10, MMP1, 
MMP3, OSM, LOX) at Erasmus MC, Rotterdam, The Netherlands.  
Biomarker analyses are subject to change and additional analyses may be performed in already 
collected materials. 
 
7.1.5 Transdermal analysis patch (TAP) 
Skin biomarkers will be measured explorative by TAP (FibroTx, Estonia) as indicated in Table 1. 
TAP consists of a multiplex capture-antibody micro-array that is supported by a dermal adhesive 
bandage for fixture to skin. When TAP is applied to skin and left on for 20 minutes, the antibodies 
printed on the micro-array capture biomarkers from skin through immune-recognition. Biomarkers 
(e.g. VEGF-A, TNFα, IL-4, IL-8, TSLP, MMP-3) captured from skin by TAP are qualitatively and 
quantitatively analyzed by spot-ELISA by a specific TAP analyzer. Each TAP kit will be labelled and 
stored at 4 ºC overnight and after that frozen at -20ºC until shipment. 
Biomarker analyses are subject to change and additional analyses may be performed. 
 
7.1.6 Skin microbiome 
Sample collection 
Collection of skin culture samples is a non-invasive procedure where a sterile polyester flock tip 
(Puritan Sterile Polyester Tipped Applicators REF 25-3206-H 20MM) per site is passed along the 
surface of the 3 different areas. The target areas are i) regions surrounding one of the biopsy 
lesions on the lower back, ii) a control site of healthy, unaffected skin in proximity of a biopsy lesion 
and iii) a control area on the lower back with a minimum distance of 10cm from a biopsy site. The 
skin swab will be placed in a 2 ml lysis tube (REF ZY-R1103, Zymo Research) containing DNA/RNA 
shield to stabilize and preserve the DNA. The tubes will be stored in the freezer at -80°C, to be 
shipped to BaseClear at the end of the study. The microbiology samples will be analyzed at 
BaseClear Laboratories, The Netherlands. 
 
DNA extraction 
The DNA extraction will be performed using adapted DNA extraction method based on the Zymo 
Research fecal DNA extraction methodology. In short, the swabs in the 2 ml lysis tubes undergo a 
mechanical shearing procedure that lyses cells from micro-organisms captured by the swab. DNA 
will be eluted in in a volume of 50μl. 
 
Microbiome analysis 
After DNA extraction, the variable regions 3 and 4 of the 16S rRNA gene are amplified giving an 
amplicon of around 450 base pairs. This amplicon is analyzed by capillary systems using standard 
protocols, to confirm successful amplification of a PCR fragment of the expected size. PCR products 
are cleaned up by Ampure XP beads (Beckman Coulter) to remove primer-dimers and small a-
specific PCR products and the purified PCR products are quantified using the Quant-iT PicoGreen 
dsDNA kit (Life Technologies). Subsequently, the PCR products are diluted and an equal mass for 
each sample is used as template in a 2nd PCR where sample specific barcodes (Index primers 
(Nextera). XT Index kit) will be appended to the PCR products using a 2nd PCR with a limited 
number of cycles. Following an PCR purification step as above, the PCR products are equimolarly 

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pooled and sequenced on the Illumina MiSeq platform using the MiSeq v3 sequencing kit 
generating paired end 300 nt sequence reads. De-multiplexed FASTQ files are generated as output. 
Paired end reads are assembly into ‘pseudoreads’ followed by removal of chimeric sequences and 
taxonomic classification. In addition, sequence reads are clustered together based on sequence 
similarity. 
7.1.7 Clinical photography 
On every visit day a standardized set of photographs will be taken of the treatment sites under 
standardized conditions (environment, light, distance) with FotoFinder ATBM and digital camera. 
Photographs will include a label with subject initials, subject study number, and study day/occasion 
number. All the photographs will be taken with QPcard 201 for later colour correction with image J 
software.  
Pictures will also be taken and analyzed by other digital photosystems including a 3D stereo camera 
system (LifeViz QuantifiCare, Valbonne, France) and an Antera 3D camera system (Miravex, 
Dublin, Ireland).  
 
7.1.8 Skin temperature by thermography 
Skin temperature will be measured using a thermal imaging camera (FLIR X6540sc, FLIR Systems 
Inc., Breda, The Netherlands) according to the Table of Assessments (Table 1).  
 
7.1.9 Perfusion by Laser spectrum contrast imaging (LSCI) 
Cutaneous microcirculation will be assessed using the laser speckle imager (LSCI; PeriCam PSI 
System, Perimed Jäfälla, Sweden), and will be done according to the SOP of CHDR at multiple time 
points during the study (see Table 1). Measurements have to be performed in a temperature 
controlled room with a temperature around 22°C. The subject has to get accommodated to the room 
temperature for a minimum of 15 minutes prior to testing. After this, the speckle assessments can 
commence. Briefly, the subject will be resting for at least ten minutes before any measurements 
take place. A suitable area of the lower back will be identified. This area will be illuminated' by the 
laser and the response signal will be captured. Analysis of the data will be according to the 
pertaining SOP.  
 
7.1.10 Trans epidermal water loss (TEWL) 
To assess the barrier status of the skin TEWL will be measured at multiple time points during the 
study (see Table 1). The water loss will be measured non-invasively using an Aquaflux AF200 
system (Biox, London, UK) according to the standard operating procedure of CHDR. A circular area 
of 7mm diameter of skin will be enclosed by the measuring probe. The flux of water that enters the 
chamber will be measured until a steady-state flux is reached. Minimal measurement time is 90 
seconds and maximum measurement time is 200 seconds. All measurements will be performed 
under standard environmental conditions (temperature 22ºC±2ºC; relative humidity <60%), and 
subjects will be acclimatized under relaxed conditions for at least 15 minutes prior to testing. 
 
7.1.11 Skin colour by colorimetry 
Colorimetric assessments will be done and erythema grade will be calculated (a* of CIELAB, 
average of 3 measurements) by DSM II ColorMeter (Cortex Technology, Denmark). 
 

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7.1.12 Clinical grading scales 
Skin colour will be assessed using an erythema grading scale provided by the supplier of the Finn 
Chambers. Wound healing will be assessed using Red-Yellow-Black (RYB) wound assessment 
scale and the Patient and Observer Scar Assessment Scale (POSAS).  
The RYB wound assessment scale was introduced in the 1980s in order to simplify the 
assessments of wounds and to guide treatment. Classification of wounds with RYB model combined 
with the humidity of the wound is an approach used to categorize wounds and apply treatment (see 
below). 
 
Wound colour 
 
Wound humidity 
Red 
Yellow 
Black 
 
Wet 
 
Protect 
Absorb fluid 
Cleanse 
Absorb fluid 
Remove necrosis 
Absorb fluid 
 
Humid 
 
Protect 
Regulate fluid 
Cleanse 
Regulate fluid 
Remove necrosis 
Regulate fluid 
 
Dry 
 
Protect 
Apply fluid 
Cleanse 
Apply fluid 
Remove necrosis 
Apply fluid 
 
The POSAS is a questionnaire developed in 2004 to measure scar quality in the opinion of the 
patient and the observer. The POSAS observer scale is provided in English in Appendix A and in 
Dutch in Appendix B. The location of the scar can be designated in a representative drawing of the 
human body.  
The observer scale of the POSAS consists of six items (vascularity, pigmentation, thickness, relief, 
pliability and surface area). All items are scored on a scale ranging from 1 (‘like normal skin’) to 10 
(‘worst scar imaginable’). The sum of the six items results in a total score of the POSAS observer 
scale. Categories boxes are added for each item:  
 
Vascularity category: pale, pink, red, purple, mix 
 
Pigmentation category: hypo, hyper, mix 
 
Thickness category: thicker, thinner 
 
Relief category: more, less, mix 
 
Surface area category: expansion, contraction, mix 
Furthermore, an overall opinion is scored on a scale ranging from 1 to 10. 
All parameters should preferably be compared to normal skin on a comparable anatomic location. 
 
7.1.13 Subject reported outcomes 
Subjects are asked to report the pruritus and pain by a numeric rating scale of all biopsy sites. The 
pruritus and pain NRS are single-question assessment tools that will be used to assess the 
subject’s worst itch and pain in the previous time interval. Subjects will fill in the e-diary daily and be 
asked the following question; "on a scale of 0 - 100, with 0 being no itch, and 100 being the worst 
itch imaginable, how would you rate your average degree of itch of all biopsy sites combined 
experienced during the previous time interval?" and "on a scale of 0 - 100, with 0 being no pain, and 
100 being the worst pain imaginable, how would you rate your average degree of pain of all biopsy 

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sites combined experienced during the previous time interval?" Depending on visit day the time 
interval will be 2,3, 7, 14 days. Subjects will be instructed on reporting at the day 0 visit. Subjects 
will complete the pruritus and pain on every study visit through the last study visit (EOS). 
 
7.2 Safety and tolerability assessments 
The definitions, reporting and follow-up of AEs, SAEs and potential pregnancies are described in 
section 8. 
 
7.2.1 Vital signs 
Evaluation of systolic and diastolic blood pressure, pulse rate and temperature will be performed at 
screening as stated in Table 1. 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. 
 
7.2.2 Weight and height 
Weight (kg) and height (cm) will be recorded and body mass index (BMI) will be calculated at 
screening. 
 
7.2.3 Physical examination 
Physical examination (i.e., inspection, percussion, palpation and auscultation) is performed during 
screening. Clinically relevant findings that are present prior to study drug initiation must be recorded 
with the subject’s Medical History. 
 
7.2.4 Laboratory assessments 
Following clinical laboratory tests will be performed at screening: 
 
Lab 
Tests 
Collection & Analysis 
Haematology 
 
Haemoglobin [including Mean Corpuscular 
volume (MCV), Mean corpuscular 
haemoglobin (MCH), Mean corpuscular 
haemoglobin concentration (MCHC)], 
haematocrit, red cell count (RBC), total white 
cell count (WBC)and Platelet count. 
Differential blood count, including: basophils, 
eosinophils, neutrophils, lymphocytes, and 
monocytes. 
4 mL of venous blood in a BD 
Vacutainer® K2EDTA tube. 
Samples will be analysed by 
the Clinical Chemistry 
Laboratory (AKCL) of Leiden 
University Medical Center. 
Chemistry and 
electrolytes 
Sodium, potassium, calcium, inorganic 
phosphate, total protein, albumin, glucose1, 
triglycerides, blood urea nitrogen (BUN), 
creatinine, uric acid, total bilirubin2, alkaline 
phosphatase, AST, ALT gamma-GT, LDH, and 
CDT. 
8.5 mL of venous blood in a BD 
Vacutainer® SST Gel and Clot 
Activator tube. Samples will be 
analysed by the AKCL of 
Leiden University Medical 
Center. 
Serology 
HIV1 and HIV2 antigen and antibodies, 
Hepatitis B surface antigen, Hepatitis B 
antibodies 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 Microbiology 

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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 AKCL for microscopy and/or culture. 
A midstream, clean-catch urine 
specimen will be analysed by 
dipstick (Multistix® 10 SG, 
Siemens Healthcare 
Diagnostics, Frimley, UK). 
Pregnancy3 
hCG. If there is a clinically significant, positive 
result, urine will be sent to the AKCL for 
confirmation. 
A urine specimen will be 
analysed at CHDR by test kit 
(InstAlert, Innovacon, San 
Diego, USA). 
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 and cannabinoids. 
A urine specimen will be 
analysed at CHDR by test kit 
(InstAlert, Innovacon, San 
Diego, USA). 
1After 4-hours fasting. 2Conjugated bilirubin will be reported only when total bilirubin is outside the 
reference range. 3Pregnancy test for women of childbearing potential will be performed at 
screening and if pregnancy is suspected during the study. 
 
7.2.5 Shipping Procedures 
CHDR will arrange shipment of the biopsy samples. The samples must be packed securely together 
with completed shipment forms in polystyrene insulated shipping containers together with enough 
dry ice to last for 48 hours. Samples must be shipped to the Immunology Laboratory at Erasmus 
MC, Rotterdam. 
 
7.2.6 Concomitant medications 
Concomitant medications initiated, stopped, up-titrated or down-titrated for an AE will be recorded. 
 
7.2.7 Local tolerability 
The erythema grading scale, RYB wound assessment scale, POSAS, and NRS pruritus and pain will 
also be used to assess local tolerability. 
 
7.3 Sequence of assessments and time windows 
On study day 0 pharmacodynamic assessments will be performed prior to skin punch biopsies. 
The deviations of actual time points from the expected time points will be within ten percent, 
calculated from the zero point (time of skin punch biopsy) 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. 
Visit variances are given in Table 2. 

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Table 2: Visit variances 
Protocol procedure 
Approved time window 
Day 0 
Per protocol 
Day 2 
Per protocol 
Day 4 
Per protocol 
Day 7, Day 10 
May be done plus or minus one (1) calendar 
days from scheduled 
Day 14, Day 17 
May be done plus or minus two (2) calendar 
days from scheduled 
Day 21, Day 24 
May be done plus or minus two (3) calendar 
days from scheduled 
Day 28 
May be done plus or minus two (4) calendar 
days from scheduled 
Day 42 
May be done plus or minus three (5) calendar 
days from scheduled 
Day 56, Day 70 
May be done plus or minus three (7) calendar 
days from scheduled 

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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 AE does not necessarily have to follow the administration of a study 
drug, or to have a causal relationship with the study drug. An AE 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 the intervention  
For each AE the relationship to drug as judged by the investigator:  
 
Probable;  
 
Possible;  
 
Unlikely;  
 
Unrelated. 
 
8.1.3 Chronicity of adverse events 
The chronicity of the AE 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: 
 
results in death; 
 
is life threatening (at the time of the event); 
 
requires hospitalization or prolongation of existing inpatients’ hospitalization; 
 
results in persistent or significant disability or incapacity; 
 
is a congenital anomaly or birth defect; or 

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 
any other important medical event that did not result in any of the outcomes listed above due 
to medical or surgical intervention but could have been based upon appropriate judgement 
by the investigator. 
An elective hospital admission will not be considered as a SAE. 
 
8.1.6 Suspected unexpected serious adverse reactions 
A SUSAR (Suspected Unexpected Serious Adverse Reaction) is a SAE that is unexpected, (nature 
or severity of which is not consistent with the applicable product information (e.g., investigator's 
brochure for an unauthorized investigational product or summary of product characteristics for an 
authorized product)) and suspected (a reasonable possibility of causal relationship with 
investigational drug, regardless of the administered dose). 
 
8.1.7 Reporting of serious adverse events 
SAEs and SUSAR's will be reported according to the following procedure, according to CHDR 
SOPs CGEAE and CGEEMERG. 
The investigator will report the SAEs through the web portal ToetsingOnline (see 
https://toetsingonline.nl/) to the accredited EC that approved the protocol, within 7 days of first 
knowledge for SAEs that result in death or are life threatening followed by a period of maximum of 8 
days to complete the initial preliminary report. All other SAEs will be reported within a period of 
maximum 15 days after the sponsor has first knowledge of the SAE. 
SUSARS must be reported to the EC that approved the study, the CA and the Dutch Medicines 
Evaluation Board (College ter Beoordeling van Geneesmiddelen). 
The investigator will report expedited the following SUSARs through the web portal ToetsingOnline 
to the EC: 
• 
SUSARs that have arisen in the clinical trial that was assessed by the EC; 
• 
SUSARs that have arisen in other clinical trials of the same sponsor and with the same 
medicinal product, and that could have consequences for the safety of the subjects involved 
in the clinical trial that was assessed by the EC. 
The expedited reporting of SUSARs through the web portal ToetsingOnline is sufficient as 
notification to the EC, CA and the Dutch Medicines Evaluation Board, a separate notification is not 
necessary. To prevent a double notification, it must be indicated in ToetsingOnline if the SUSAR is 
reported in the EMA EudraVigilance database, this will prevent the notification of the CA and the 
Dutch Medicines Evaluation Board through the web portal ToetsingOnline. 
The investigator will report expedited all SUSARs to the competent authorities in other Member 
States, according to the requirements of the Member States.  
The expedited reporting will occur not later than 15 days after the first knowledge of the adverse 
reactions. For fatal or life threatening cases the term will be maximal 7 days for a preliminary report 
with another 8 days for completion of the report. 
 
8.1.8 Follow-up of adverse events 
All AEs 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. 
 

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8.2 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, 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 jeopardize 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 and CAs of the concerned Member States. 
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 or newer (SAS 
Institute Inc., Cary, NC, USA).  
 
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 
Due to the nature of this exploratory trial, no formal power analysis has been conducted. 
 
9.4 Missing, unused and spurious data 
All missing or incomplete safety and pharmacodynamics data, including dates and times, are 
treated as such. Missing test results or assessments will not be imputed. Missing 
pharmacodynamics 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 pharamacodynamic 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 Pharmacodynamic analysis set 
The analysis population for pharmacodynamics is defined as all subjects who were validated 
(randomized), underwent at least 1 skin punch biopsy, and have at least one post-baseline 
assessment of the parameter being analyzed. 
 
9.5.2 Safety set 
The safety population will be defined as all subjects who were validated (randomized) and 
underwent at least 1 skin punch biopsy. 
 
9.6 Subject disposition 
Subject disposition will be listed by subject. 

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The following subject data will be summarized: 
 
number and percentage of subjects screened, 
 
number and percentage of subjects enrolled, 
 
number and percentage of subjects completed, 
 
number and percentage of subjects included in safety population and 
 
number and percentage of subjects included in the pharmacodynamic analysis population. 
A subject is defined to have completed the trial when he or she has all biopsies taken. 
 
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.  
 
9.7.2 Medical history  
Medical history will only be listed. 
 
9.7.3 Concomitant Medications 
All concomitant medications will be displayed in a listing. 
 
9.7.4 Treatment compliance/exposure 
Not applicable. 
 
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 the first 
skin punch biopsy is taken. If a subject experiences an event both prior to and after the first skin 
punch biopsy, 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 the first skin punch biopsy, and prior to the 
start of another treatment, if any. All TEAEs collected during the investigational period will be 
summarized. 

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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. 
 
9.8.3 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.  
The categorical data of the urinalysis will be summarized by treatment and time in frequency tables 
by variable. 
 
9.9 Endpoints 
The final analysis will be preceded by a data review which consists of individual graphs per visit by 
time of all pharmacodynamic measurements by time. The graphs will be used to detect outliers and 
measurements unsuitable for analysis. 
The pharmacodynamic parameters will be listed by treatment, subject, visit and time. Individual 
graphs by time will be generated. 
Given the exploratory character of the study, pharmacodynamic endpoints will be primarily analyzed 
using descriptive statistics. All pharmacodynamic 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. Both Nominal 
results, and log-transformed results and change from baseline results will be utilized in all data 
summaries. All categorical pharmacodynamic endpoints will be summarized by frequencies. 
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. 
9.9.1 Inferential methods 
The study is exploratory and no formal null hypothesis is set. No adjustments for multiple 
comparisons will be applied. 
 
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 (www.wma.net), ICH GCP guidelines 
(http://www.ich.org/products/guidelines.html), 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 also 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 and five hundred thousand Euro) for the total damage 
incurred by the organization 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. 
 

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10.2 Study funding 
CHDR is the sponsor of the study and Cutanea Life Sciences is funding the study. All financial 
details are provided in the separate contract(s) between the CHDR and Cutanea Life Sciences. 
 
10.3 Data handling and record keeping 
10.3.1 Data collection 
Data will be recorded mainly on electronic data collection forms. When necessary, paper data 
collection forms can be used and data will then 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. 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 
All data from paper source will be entered into the Promasys database twice, by two different 
individuals. A quality control check will be done by CHDR staff on all data entered in the Promasys 
database, 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 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 
finalized, after which all study-related documents will be archived (at a minimum) on micro-film 
which will be kept according to GCP regulations. After 2 years the sponsor will be notified that the 
source documents can be retained with the sponsor or destroyed. 
The investigator will permit trial-related 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 
Data monitoring will be performed by CHDR procedures including source data verification, trial 
master file verification, verification of delegation of authorities and appropriate training of personnel. 
According to CHDR procedures an initiation visit (pre-study meeting) 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. 
 

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10.6.1 Substantial amendment 
Significant changes that affect subject safety and/or the scientific value of a trial require a 
substantial amendment. Examples of significant changes are given in EU guidelines on the request 
to the competent authorities for authorization of a clinical trial on a medicinal product for human use, 
the notification of substantial amendments and the declaration of the end of the trial (CT-1, 2010/C 
82/01). The need for submitting a substantial amendment is the responsibility of the sponsor. 
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. 
 
10.6.2 Non-substantial amendment 
Non substantial amendments do not affect subject safety or the scientific integrity of the trial. Non-
substantial amendments will be approved (signed) by the investigator(s) and will be recorded and 
filed by the investigator/sponsor. Non-substantial amendments will be submitted to the EC for 
information only. The CA will only be notified by changes in Eudract form and ABR form (if 
applicable) at toetsingonline. The implementation of a non-substantial amendment can be done 
immediately.  
The EU guideline CT-1 2010/C 82/01 stipulates the importance of preventing over-reporting. 
Therefore the following changes are by definition non-substantial in this study: 
 
change in amount and timing of the samples (maximum of 2 samples without a > 50 ml 
increase in the amount of blood taken and not exceed 500 ml of blood in total) 
 
changes in assay-type and / or institution where an assay will be performed, provided that 
validated assays will be used; 
 
editorial changes to documents in the submission dossier including the volunteer information 
sheets and the protocol. An editorial change is defined as a modification in the documents of 
typographical errors and other modifications that in no way alter the meaning or content of 
the document 
 
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. 
 
A change in clinical staff, including the principal investigator, when this concerns regular staff 
members of CHDR who comply with internal regulations for training and authorization. 
 
A change in dosing schedule in an ascending dose trial, provided the expected exposure of 
the subjects does not exceed the preset values indicated in this protocol. 
 
10.6.3 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 sponsor 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 

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investigator will notify the EC and the CA within 15 days, including the reasons for the premature 
termination. 
CHDR will notify the EC immediately of a temporary halt of the study, including the reason of such 
an action. 
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. 
 
                                               
3 http://www.icmje.org/ 

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11 STRUCTURED RISK ANALYSIS 
No drug or treatment with active ingredients is to be used in this study. 
 

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12 REFERENCES 
ARMSTRONG, D. & MEYR, A. 2017. Basic principles of wound healing [Online]. Available: 
https://www.uptodate.com/contents/basic-principles-of-wound-healing [Accessed 28 June 
2017]. 
DREIFKE, M. B., JAYASURIYA, A. A. & JAYASURIYA, A. C. 2015. Current wound healing 
procedures and potential care. Mater Sci Eng C Mater Biol Appl, 48, 651-62. 
GREAVES, N. S., BENATAR, B., WHITESIDE, S., ALONSO-RASGADO, T., BAGUNEID, M. & 
BAYAT, A. 2014. Optical coherence tomography: a reliable alternative to invasive histological 
assessment of acute wound healing in human skin? Br J Dermatol, 170, 840-50. 
GREAVES, N. S., IQBAL, S. A., HODGKINSON, T., MORRIS, J., BENATAR, B., ALONSO-
RASGADO, T., BAGUNEID, M. & BAYAT, A. 2015. Skin substitute-assisted repair shows 
reduced dermal fibrosis in acute human wounds validated simultaneously by histology and 
optical coherence tomography. Wound Repair Regen, 23, 483-94. 
ILLIGENS, B. M. & GIBBONS, C. H. 2013. A human model of small fiber neuropathy to study wound 
healing. PLoS One, 8, e54760. 
UD-DIN, S., GREAVES, N. S., SEBASTIAN, A., BAGUNEID, M. & BAYAT, A. 2015. Noninvasive 
device readouts validated by immunohistochemical analysis enable objective quantitative 
assessment of acute wound healing in human skin. Wound Repair Regen, 23, 901-14. 
UD-DIN, S., PERRY, D., GIDDINGS, P., COLTHURST, J., ZAMAN, K., COTTON, S., WHITESIDE, 
S., MORRIS, J. & BAYAT, A. 2012. Electrical stimulation increases blood flow and 
haemoglobin levels in acute cutaneous wounds without affecting wound closure time: 
evidenced by non-invasive assessment of temporal biopsy wounds in human volunteers. Exp 
Dermatol, 21, 758-64. 
 

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APPENDIX A 
CHDR1736 POSAS Observer Scale (English)  

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APPENDIX B 
CHDR1736 POSAS Observer Scale (Dutch)