MELADERM-trial: Melatonin Cream Against Acute Radiation Dermatitis in Patients With Early Breast Cancer: a Pivotal Phase 2, Double-blind, Randomized, Placebo-controlled Trial
Phase: PHASE2
Conditions: Radiation Dermatitis, Radiation Dermatitis Acute, Breast Cancer
Sponsor: Dennis Bregner Zetner

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MELADERM-trial: Melatonin cream against acute radiation dermatitis in 
patients with early breast cancer: a pivotal phase 2, double-blind, randomized, 
placebo-controlled trial 
 
Dennis Bregner Zetner1, Claus Kamby2, Faisal Mahmood3, Jacob Rosenberg1. 
 
1: Department of Surgery, Copenhagen University Hospital, Herlev Hospital, Herlev Ringvej 75, 
2730 Herlev, Denmark 
2: Department of Oncology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 
Copenhagen Ø, Denmark 
3: Department of Oncology, University of Southern Denmark, Odense University Hospital, Søndre 
Boulevard 29, Odense, Denmark 
 
 

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Table of Contents 
Contact information ............................................................................................................................. 3 
Background .......................................................................................................................................... 4 
Study design ......................................................................................................................................... 5 
Interventions..................................................................................................................................... 6 
Recruitment ...................................................................................................................................... 7 
Primary outcomes ............................................................................................................................ 8 
Secondary outcomes ........................................................................................................................ 9 
Participant timeline .......................................................................................................................... 9 
Randomization and blinding .......................................................................................................... 10 
Drop-outs and patient retention...................................................................................................... 11 
Conclusion of study ....................................................................................................................... 11 
Handling of drugs............................................................................................................................... 11 
Storage ........................................................................................................................................... 11 
Administration ............................................................................................................................... 12 
Statistics ............................................................................................................................................. 13 
Sample size calculations ................................................................................................................ 13 
Statistical analyses ......................................................................................................................... 15 
Data management ............................................................................................................................... 16 
Monitoring ......................................................................................................................................... 17 
Harms ................................................................................................................................................. 17 
Adverse reactions to melatonin ...................................................................................................... 17 
Adverse reactions to DMSO .......................................................................................................... 19 
Adverse events ............................................................................................................................... 19 
Ethical considerations ........................................................................................................................ 20 
Insurance ............................................................................................................................................ 21 
Approvals ........................................................................................................................................... 21 
Confidentiality ................................................................................................................................... 22 
Protocol amendments ......................................................................................................................... 22 
Funding .............................................................................................................................................. 22 
Conflicts of interests .......................................................................................................................... 23 
Dissemination policy.......................................................................................................................... 23 
References .......................................................................................................................................... 24 
 
 
 
 

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Contact information 
Primary investigator and sponsor: 
Dennis Zetner, MD 
Department of Surgery, Herlev Hospital, Herlev Ringvej 75, 2730 Herlev, Denmark 
E-mail: dennis.zetner@gmail.com 
Telephone number: +45 27 29 13 76 
 

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Background 
Radiation injury is a common serious adverse reaction of treatment of various cancers with ionizing 
radiation [1-8]. In treatment of breast cancer, the most common complication to radiation therapy is 
radiation dermatitis [9]. During the course of radiation therapy, most of the patients (74-100%) will 
experience radiation dermatitis [10]. Symptoms of acute radiation dermatitis include pruritus, 
discomfort, and local pain [11]. Radiation dermatitis can even limit the intended therapeutic dose of 
radiation delivered to the patient and lead to prolongation in treatment [9]. Furthermore, radiation 
dermatitis decreases patients’ quality of life [12].  
 
Ionizing radiation causes tissue damage by releasing free radicals leading to oxidative 
stress [13]. The ionizing irradiation of water molecules in the cells leads to aqueous radiolysis, 
creating free radicals [14]. These free radicals may react with macromolecules, such as DNA, RNA, 
proteins, and cell membranes, eventually leading to dysfunction of cells and cell death [13].  
 
The sleep-hormone melatonin reduces oxidative stress [15, 16]. Melatonin is 
amphiphilic and distributes freely between all body compartments [17-19]. Studies have 
documented that melatonin is a strong free radical scavenger. The anti-oxidative effects of 
melatonin may therefore provide protective effects against the damage of ionizing radiation in 
clinical cancer treatment and radiological imaging procedures [20]. In animal studies, melatonin 
protects the healthy tissue against radiation injury [20]. However, melatonin has also been 
documented to possess oncostatic and pro-apoptotic effects in cancers in experimental studies [21, 
22], and has been demonstrated to sensitize human breast cancer cells to ionizing radiation in cell 
culture studies [23, 24].  
A report describing the expected incidence of cancers up until 2020, predicts a yearly 
incidence of approximately 4800 cases of breast cancer in Denmark alone [25]. These numbers 
suggest that if melatonin can protect against radiation injury, a large number of patients could be 
candidates for melatonin treatment, and thereby obtain a better quality of life. A previous study 

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demonstrated the radioprotective effect of melatonin in the treatment of breast cancer [11]. This 
study included patients receiving breast-conserving surgery, and randomized them to receive either 
melatonin or placebo in a cream, which was applied twice daily. This study demonstrated a 
protective effect of melatonin against radiation dermatitis in women receiving 50 Gy of radiation 
[11]. However, this study did not list the dose of melatonin applied, and only used the RTOG scale 
as an outcome parameter. No subjective outcomes were measured in this study [11]. The authors of 
the previous study have not been able to inform us of the dose of melatonin used in the study 
(personal communication). 
Melatonin has the disadvantage of being unstable when exposed to air and light. A 
study demonstrated that melatonin in aqueous solutions at room temperature, and at neutral pH, 
degrades by 29% over 21 days [26]. The compound dimethyl sulfoxide (DMSO) has seen wide use 
both as a solvent and as an anti-inflammatory drug [27, 28]. Based on a report made available to the 
authors from the pharmaceutical company, Bioneer A/S, melatonin dissolved in DMSO undergoes 
no degradation over 45 days when stored at 25 ºC temperature, away from light. 
The aim of the present randomized double-blinded placebo-controlled clinical trial is 
to investigate if melatonin can protect against acute radiation dermatitis in patients with early breast 
cancer receiving radiation therapy, and whether this has an impact on the patients’ quality of life. 
 
Study design 
The study is registered at clinicaltrials.gov (indsæt ID, når dette er sket). This is protocol version 
1.0, 13/09/2018. The study will be a randomized, placebo-controlled, double-blinded clinical pivotal 
trial. Patients will be allocated in a ratio of 1:1 to the melatonin or placebo group. Patients will be 
stratified according to the type of surgery (lumpectomy or mastectomy). Randomization will be 
performed in blocks of randomized sizes. The study will be performed in the Department of 

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Oncology, Rigshospitalet, Denmark. Patients will be included into our study according to specific 
eligibility criteria (see Table 1). 
 
Interventions 
Eligible patients with early breast cancer receive adjuvant radiation therapy over 15 to 30 daily 
fractions (5 fractions per week) of ionizing megavoltage photon radiation to a total of 40 - 60 Gy 
within 3-5 weeks according to the guidelines of the Danish Breast Cancer Cooperative Group [29]. 
In our study, the patients will administer approximately 1 g of cream containing melatonin (25 
mg/g) and dimethyl sulfoxide (DMSO) (150 mg/g) or a placebo cream topically twice daily on the 
irradiated skin area. They are scheduled to do this every day from the first to the last fraction of 
radiation therapy, including the days where they do not receive radiation therapy. We have chosen 
placebo as our comparator due to it being safe, and in our view the most reasonable method of 
evaluating any effects of the intervention. On days where the patients receive radiation, the 
melatonin/DMSO or placebo cream will be applied no less than 2 hours prior to radiation. 
Throughout the study, the patients will meet with the primary investigator once 
weekly and; the primary investigator will monitor compliance. In patients requiring steroid cream as 
a treatment for their radiation dermatitis, the steroid usage will be monitored. The patients will be 
recommended not to apply any other topical treatments to the irradiated area during the study. The 
use of other local therapeutics will be registered. Furthermore, patients will be informed that they 
should use safe contraception during the study. Safe contraception is defined as either an 
intrauterine device or hormonal contraception. A sterile permanent partner or abstinence from 
intercourse are also considered safe contraception. Should more than five years have passed since 
menopause the patient does not need to use contraception. 
 

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Recruitment 
Patients with early breast cancer referred to radiation therapy will be screened through the 
electronic health records at Rigshospitalet (Sundhedsplatformen and Aria - Timeplanner (a program 
used locally for scheduling of radiation therapy)) by a secretary working in the Radiation  Section, 
Department of Oncology. Before patients begin radiation therapy, they are referred to a dose-
planning CT-scan in the Radiation Section, Department of Oncology, Rigshopitalet. When an 
eligible patient is identified through her referral for dose-planning, the secretary will inform them 
that we are conducting a study, and ask if an investigator can contact them. If the patient agrees, an 
investigator will be notified by the secretary. Patients will be screened by: diagnosis of early breast 
cancer, age, and previous radical tumor resection surgery.The investigator will then contact the 
patient (personally or by phone). The patients will be informed that we are conducting a study, and 
would like to invite them to participate. The patient will be offered a meeting, where she is allowed 
to bring a companion. The patient will receive both verbal and written information at this meeting. 
The patient will, before the information regarding the study is given, be informed that: 
- It is an enquiry about willingness to participate in the study. 
- The study has been approved by the Danish Data Protection Agency, the local ethics 
committee, and the Danish Medicines Agency. 
- The subject has the right to a minimum of 24 hours for consideration. 
- Choosing not to participate in the study will in no way influence the treatment they 
are offered at Rigshospitalet. 
The information will be given in a room where both the investigator and the patient can sit 
comfortably without disturbances. The subject will be given enough time to read and listen to the 
information and ask questions. Both oral and written consent will be obtained either at this meeting 
or after at least 24 hours of consideration. A screening-list of patients will be created listing their 
CPR-numbers, name, inclusion/exclusion, and reason for exclusion if relevant. 

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Primary outcomes 
The Radiation Therapy Oncology Group’s (RTOG) acute radiation morbidity scoring criteria of the 
skin will be used as the primary outcome of the study [30]. The RTOG scale is widely used as a 
measure of radiation injury in the skin [30, 31]. This scale ranges from 0 to 4, being: 
0: No change over baseline 
1: Follicular, faint or dull erythema/epilation/dry desquamation/decreased sweating 
2: Tender or bright erythema, patchy moist desquamation/moderate edema 
3: Confluent, moist desquamation other than skin folds, pitting edema 
4: Ulceration, hemorrhage, necrosis 
The score will be performed at baseline, once weekly during radiation therapy, and once weekly for 
three weeks after the last radiation fraction. 
Furthermore, an evaluation of the erythema will be performed with image analysis of 
clinical photographs after radiation exposure. Erythema has previously been evaluated by a 
validated method using software analysis (Image J, version 1.45S, National Institute of health, 
USA) of digital photos [32]. A “color space converter” function will be used to convert the clinical 
photos into grayscale in the software analysis. Erythema will be quantified by pixel color analyses 
where all white colored pixels represent erythema. An a*-value will represent degree of erythema 
(high a*-values represent a high degree of erythema). This method has previously been used in a 
study evaluating erythema developed as a result of ultraviolet radiation [33]. Photos for the analyses 
will be taken at baseline, once weekly during radiation therapy, and once weekly for three weeks 
after the last radiation fraction. Photographs will be taken with the same camera in the same lighting 
conditions for each picture.  
 
The QLQ-BR23 is a questionnaire examining the quality of life in breast cancer 
patients, developed by the European Organization for Research and Treatment of Cancer (EORTC). 

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The QLQ-BR23 is a broadly used questionnaire that has been validated in Danish [34, 35]. The 
questionnaire includes several items of interest, but specifically for the present study, we focus on 
breast symptoms as a primary outcome. The remaining items from the questionnaire will be 
considered secondary outcomes, as described below. The QLQ-BR23 questionnaire will be filled 
out by the patients once weekly for the duration of the radiation therapy, and once weekly for three 
weeks after the last radiation fraction. 
 
Secondary outcomes 
The QLQ-C30 is a questionnaire concerning the quality of life in cancer patients in general, which 
is widely used and has been validated in Danish [36]. All items in the QLQ-C30 as well as the items 
from QLQ-BR23 (above) that are not mentioned under primary outcomes will be considered 
secondary outcomes. Patients will be asked to answer both questionnaires at baseline, once weekly 
during radiation therapy, and once weekly for three weeks after the last radiation fraction. 
Patients will be asked about steroid cream usage for radiation dermatitis at baseline, 
once weekly during radiation therapy, and once weekly for three weeks after the last radiation 
fraction. 
Demographic and background variables will be collected at baseline (see table 2 for a 
full list of demographic and background variables). 
 
 
Participant timeline 
The participant timeline is outlined in table 3. At the pre-screen consent, the patients will be 
informed that they have a right to 24 hours of consideration prior to deciding to participate in the 
trial or not. Oral and written consent will be obtained by the primary investigator at the time noted 

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in table 3, or at a maximum 24 hours hereafter. There will be no study-specific post-trial care for the 
participants, other than following the standard treatment regimens offered at Rigshospitalet. 
 
Randomization and blinding 
Patients will be randomly assigned to either a control or experimental group allocated in the ratio of 
1:1 by use of a computer-generated randomization schedule. Patients will be stratified according to 
whether they have undergone lumpectomy or mastectomy. We will randomize the block size to 
between 4 and 8 patients per block. Each block will therefore consist of patients who have 
undergone either lumpectomy or mastectomy, allocated in a 1:1 ratio. Block size will be concealed 
to the investigators until the end of the trial (i.e. after data analysis and statistical evaluation). The 
allocation sequence will be generated by a staff member with no other involvement in the trial. 
Assignments will be placed in sequentially numbered, opaque, sealed envelopes with the 
randomization number printed on them. These envelopes will be packed by a staff member with no 
other involvement in the trial. Every assignment will contain a code that corresponds to tubes of the 
melatonin/DMSO or placebo preparation. The primary investigator will enroll and assign 
participants to intervention on the basis of the assignment code from the envelope. Envelopes as 
well as tubes with active and placebo cream will be stored in a locked cabinet at Rigshospitalet. 
Outcome assessment will be performed by the primary investigator, who is blinded to 
treatment allocation. Participants are also blinded to treatment allocation. The primary investigator 
will be blinded when performing data analysis and statistical evaluations. 
Should the investigator deem it necessary for the safety of the patient, the investigator 
can un-blind a patient. Treatment-allocation will be noted in a document on a secure folder on a 
drive on the server of the Region Hovedstaden. Should an investigator need to un-blind a patient, 
they can access this document from their computer. Treatment allocation will only be un-blinded if 
it is imperative to the treatment of the patient. The treatment allocation will in this case only be 

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revealed to one other investigator than the primary investigator. This investigator will oversee the 
treatment of the patient. Registration of adverse reactions is described in the “Harms” section. 
 
Drop-outs and patient retention 
We plan to promote participant retention and complete follow up by asking for contact information 
(phone number and e-mail) and preferred way of contact. We will schedule appointments with 
patients at same location, accommodating their calendar, as far as possible.  
If a patient does not wish to complete all three follow-up visits, some of the outcomes 
can be filled in electronically from home, should the patients wish to do so. These outcomes are the 
QLQ-C30 and QLQ-BR23 questionnaires, and steroid usage outcomes. All data from drop-outs will 
be used in statistical analyses. Discontinuation of treatment will only occur in the following 
eventualities: the patient withdraws consent, or if the sponsor decides to terminate the study due to 
serious adverse events. 
 
Conclusion of study 
Recruitment of patients will be concluded when 48 patients have completed the study (see sample 
size calculations below, in the “Statistics” section). The study will be concluded, when all patients 
included have completed the study. 
Handling of drugs 
Storage 
The active cream will consist of 25 mg/g melatonin as well as 150 mg/g DMSO, in a cream base. 
The placebo will consist of just the cream base. The cream base consists of polysorbate 80 (0,5%), 
cetostearyl alcohol (5%), paraffin oil (5%), glycerol monostearate 40-50 (6%), methyl 

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parahydroxybenzoate (0,1%), glycerol 85% (4%), sorbitol (7%), purified water (72,4%). The dose 
for each application will be 1 g, and the cream will be packaged in 30 g tubes. The tubes will be 
stored at room temperature, in a locked cabinet, in the office of one of the investigators. The cream 
will be produced and packaged by Glostrup Apotek in their manufacturing facility, according to EU 
GMP-guidelines. The tubes will be labelled with all required information listed in the EU GMP-
guidelines concerning investigational medical products. All drugs will be delivered directly to the 
sponsor, who will document receiving the products according to GCP-guidelines (modtagekontrol). 
Patients will be given one tube of cream at a time, and a second and third tube will be 
given to them, once the previous tube is empty or running low. Patients will be instructed to keep 
the tubes out of reach of children, away from sunlight and at room temperature. These instructions 
will also be listed on the label of the cream. 
 
Administration 
Prior to the first radiation treatment patients will receive a tube of cream with a number 
corresponding to their randomization number. They will be instructed in storage of the tube as 
described above. Patients will be seen once weekly throughout their radiation therapy. At this 
meeting, the patients will be asked how much cream they have left, and another tube of cream will 
be handed out when the patients are expected to run out of cream before the next meeting. Patients 
will receive a total of two to three tubes, depending on the number of radiation fractions they 
receive, each containing 30 g of cream. 
 
Patients will be instructed to apply the cream twice daily, once in the morning and 
once in the evening. The patients will be instructed to apply approximately 1 g of cream in the 
treatment area twice daily. On days where the patients receive radiation fractions, the patients will 
be instructed to apply the morning dose no less than 2 hours prior to radiation. As part of the 
instruction, the investigator will weigh out 1 g of cream, so that the patient can see what amount of 

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cream they should apply each time. During the weekly visits, the patients will be asked if they are 
experiencing trouble applying the cream, and the investigator will help guide them. At every visit 
the patients will be asked to bring with them the tube of cream they are currently using. When first 
handed out, and during each visit, the tube will be weighed on a high-precision scale (range 0.01 mg 
to 200 g). Usage of cream will be calculated, usage will be evaluated. Furthermore, along with the 
first tube of cream, the patients will receive a schedule with the relevant dates for the patient; the 
patient will have to fill in every time they have applied the cream.  When a patient is expected to 
run out of cream before the next visit, another tube will be handed to them. One tube of cream 
contains 30 g of cream, and the patients will apply 2 g each day they receive radiation. One tube 
should last for 15 days, assuming they administer the correct dose daily, and assuming they are able 
to completely empty the tube. Ideally patients will receive new tubs on Study visit week 2 and 4, if 
they have managed to use the instructed dose.  
Statistics 
Sample size calculations 
In order to establish the needed number of patients for the study, we have performed 3 separate 
sample size calculations. 
The sample size calculation for the RTOG acute radiation morbidity scoring criteria is based on the 
findings in a previous study examining melatonin as a radiation protector of the skin in patients 
receiving radiation therapy for breast cancer [11]. In this study, at two weeks follow-up, 41% of the 
patients receiving melatonin had toxicity grade 1-2 vs 90% of the patients receiving placebo. 
Incidence in placebo group: 90% 
Incidence in melatonin group: 41% 
Alpha: 5% 
Beta: 20% 

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Sample size needed: Two groups of 14 patients, totaling 28 patients. 
 
The sample size calculation for the image analysis is based on the study evaluating erythema after 
exposure to ultraviolet radiation [33]. Data for the sample size calculation were obtained directly 
from the authors. We make the assumption that the erythema based on ultraviolet radiation can be a 
reasonable substitute for acute radiation dermatitis based on erythema being a part of the RTOG 
acute radiation morbidity scoring criteria grade 1 and 2 [30]. Based on the study used for the RTOG 
sample size calculation, we assume that the erythema will be most pronounced 2 weeks after 
conclusion of radiation therapy. We also make the assumption that melatonin applied twice daily in 
a 2.5% cream in which it is completely dissolved, will provide an effective dose comparable to the 
12.5% used in the study evaluating ultraviolet radiation [17], due to a deposition or depot-effect in 
the skin [37]. Furthermore, we have been informed by the authors of the previous study utilizing the 
12.5% cream that the cream was not homogenous, and contained small granules of undissolved 
melatonin in both the 2.5%, 5% and 12.5% creams. This suggests that the required dose, to obtain 
an effect, is in fact smaller than 12.5%. The sample size is calculated based on the a*-values, where 
a high a*-value corresponds to a higher degree of erythema. 
Mean (SD) a*-value for placebo at all time points: 18.4 (3.47) 
Mean (SD) a*-value for 12.5% melatonin cream at all time points: 13.44 (2.36) 
Alpha: 5% 
Beta: 20% 
Sample size needed: Two groups of 8 patients, totaling 16 patients. 
 
The sample size calculation for the QLQ-BR23 breast symptom score is based on a study 
investigating quality of life following radiation therapy, comparing mastectomy to breast 
conservation therapy in patients with breast cancer [38]. The data used for the sample size-

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calculation are based on the patients receiving breast-conserving therapy, on their last day of 
radiation. The 142 patients scored a breast symptom score mean (SD) of 82.2 (15.1). 
Mean (SD) a*-value for breast symptoms: 82.2 (15.1) 
We aim to reduce the breast symptom score to 70. 
Alpha: 5% 
Beta: 20% 
Sample size needed: Two groups of 24, totaling 48 patients. 
 
Because we have three sample size calculations, resulting in required sample sizes of 28, 16, and 48 
patients, respectively, and when considering the risk of drop-outs, we estimate to include a total of 
approximately 80 patients. This is based on the inclusion schedule, where the required 48 evaluable 
patients finalizing all data for the outcome parameters have to be completed, will require an 
inclusion larger than 48 patients. This is because there will be 8 weeks from inclusion to obtaining 
the data point at two weeks after ended radiation therapy. Thus, we will include continuously in 
randomized blocks (see randomization above) until patient 48 has completed the 8 weeks data 
point. If we get less than 24 patients in one group completing the 8-weeks data point, additional 
blocks will be included, until we have at least 48 patients. The staff-member responsible for the 
randomization sequence will inform the investigators how many patients they will need to include 
to reach this point because of the randomization of the block size. During 2016, Rigshospitalet 
treated 194 patients who matched our inclusion criteria. Therefore we find it feasible to finish 
inclusion in approximately 6 months. 
 
Statistical analyses 
Normality of data will be assessed through visual inspection of histograms and Q-Q plots. Normally 
distributed data will be analyzed with parametric tests (e.g. unpaired t-tests), and not normally 

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distributed data will be analyzed with non-parametric tests (e.g. Mann-Whitney U test). Our 
primary outcomes are the RTOG scores and the image analysis two weeks after the last radiation 
fraction, as well as the QLQ–BR3 breast symptoms on the last day of radiation therapy. Our 
secondary outcomes will be analyzed as follows: We will analyze steroid usage through two 
different outcomes. Days of steroid usage will be analyzed through either parametric or non-
parametric tests. Any steroid usage (yes/no) will be analyzed through χ2 or Fisher’s Exact test. The 
RTOG scores, the quality of life questionnaires, and the image analysis values for the entire period 
will be analyzed in a mixed linear model, which takes correlation between patients into account. 
Subgroup analyses will be performed for mastectomized and lumpectomized patients respectively, 
if feasible. Other subgroup analyses may be performed based on background variables 
(fractionation schedule, histopathology and demographic data). Missing data will be taken into 
consideration when using the mixed linear model. A detailed statistical analysis plan will be 
formulated and be supervised by a bio-statistician prior to study start. 
Data management 
All outcomes will be collected by the primary investigator at time points listed in table 3. All data 
entered into the electronic case report form (eCRF) will be validated through “required fields” and 
format (e.g. date-format) where applicable. In the clinical photographs, patients will be marked with 
patient-ID written on tape, fixed to the patients’ chest. The time-stamp of the photograph will be 
noted in the eCRF. a*-values will be imported into the eCRF. 
Our data will be stored electronically in Research Electronic Data Capture (REDCap), 
an electronic database used throughout the Capital Region of Denmark. All confidential data will be 
stored in a secure server for 15 years after patient enrollment in the study. Only the investigators 
and the Good Clinical Practice (GCP) Unit will have access to these data. After conclusion of the 
study, a final anonymized dataset will be available to RepoCeuticals ApS who has sponsored the 

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study through a grant through a formalized research contract between the Capital Region of 
Denmark and the company. Ownership of data is shared between the investigators and 
RepoCeuticals ApS.  
Monitoring 
This study will be conducted in compliance with the protocol,  EU ICH-GCP guidelines and the 
applicable regulatory requirements. The standard procedures for quality control and quality 
assurance are followed in compliance with the ICH-GCP guideline, and investigator/sponsor allows 
direct access to data/documents for monitoring, auditing, and inspection from both Danish 
Medicines Agency and the Good Clinical Practice (GCP)-unit. The study will be audited by the 
GCP Unit of the University of Copenhagen. The GCP Unit will be independent from the 
investigators and sponsors, and have direct access to the electronic case report forms as well as the 
trial master file. The GCP Unit will also perform data monitoring. The GCP Unit is an independent 
unit that manages clinical trials in Denmark. Further information can be found at http://www.gcp-
enhed.dk. No interim analyses have been planned. Currently, a monitoring schedule has not been 
formulated. 
 
 
Harms 
Adverse reactions to melatonin 
Melatonin is non-toxic in both physiological and pharmacological doses [39]. In mice, rabbits, cats, 
and dogs, melatonin doses of 800 mg/kg, administered intravenously, have demonstrated no toxicity 
[40].  

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A study administered 1 g of melatonin orally to patients for 25-30 days, with 
drowsiness as the only adverse reaction [41]. These patients had substantial clinical tests 
(hematological and blood chemistry tests, urine analysis, heart rate and blood pressure monitoring 
and ECG) performed, and no reactions to melatonin were demonstrated [41]. Another study 
investigated the effect of intravenously administered melatonin in healthy subjects, as well as 
patients with Parkinson’s disease and epilepsy. They applied 1.25 mg/kg melatonin as a bolus 
injection. One of the patients, who had 1.25 mg/kg melatonin intravenously, received further two 
doses of 0.5 mg/kg. Correspondingly, melatonin was non-toxic and no adverse reactions were 
observed [42].  
Furthermore, melatonin has been used as an intravenous infusion in neonates with 
sepsis, in neonates undergoing abdominal surgery, and in preterm infants [43-45]. A total of 224 
patients were included (of who 105 received melatonin) and no adverse reactions were reported, at a 
dose of 10 mg/kg intravenously [43-45].  
Melatonin has also been given in very large doses up to 100 mg intravenously in 
adults, with no adverse reactions [46]. Other studies investigated melatonin when used as a pre-
medication for gynecological laparoscopic surgery, and demonstrated no adverse reactions at a dose 
of 5-20 mg of sublingual melatonin [47, 48]. No adverse reactions were reported in a study 
investigating pre-medication with 5 mg melatonin prior to laparoscopic cholecystectomy [49]. 
Drowsiness, confusion and depression has been described as side-effects of melatonin 
in a study examining if melatonin could augment antidepressant treatment in patients with 
depression [50]. Furthermore, a few studies report headache [51] and fatigue as side-effects of 
melatonin [52]. 
 

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Adverse reactions to DMSO 
 
Dimethyl sulfoxide (DMSO) is regularly used in the treatment of interstitial cystitis either as 
monotherapy, or serving as a vehicle for anti-inflammatory agents [27, 53-55]. DMSO has 
previously been applied as a solvent for intravesical drug administration in the treatment of bladder 
cancer [56]. An adverse reaction of DMSO is a garlic-like breath odor and taste, due to pulmonary 
elimination of a small percentage of the DMSO as dimethyl sulfide. One study demonstrated that up 
to 48% of patients with interstitial cystitis reported adverse reactions, including urethral irritation 
and nausea [53]. However, the dose in this study was a total of 25 g of DMSO (50 ml fluid of 500 
mg DMSO/ml), which is a much higher dose than we aim to apply [53]. These adverse reactions are 
self-limiting, lasting up to 24-48 hours [27, 53, 54]. DMSO has demonstrated a self-limiting mild 
burning sensation as an adverse reaction when administered topically [57], but DMSO dose locally 
was approximately 50 times larger than in current study [57], and DMSO was applied every 8 
hours, opposed to the current application two times a day [57]. 
 
 
Adverse events 
The subjects will be questioned about adverse events once weekly during treatment. An adverse 
event is defined as any medical event that leads to an unwanted effect in the subject after the 
administration of the intervention medication, without any necessary connection between the 
adverse event and the intervention. An adverse reaction is defined as harm or unwanted reaction to 
a drug, no matter the dosage. We have defined six pre-specified self-reported symptoms 
(nervousness, confusion, depressed mood, dizziness, headache, or garlic like odor), based on the 
most commonly reported adverse reactions of melatonin and DMSO available in the literature [41, 
50-53]. Patients will also be asked to report additional symptoms of adverse effects, if any.  Both 
adverse reactions and adverse events will be reported in the eCRF. 

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Serious adverse events (SAE) are defined as any medical effect that result in death, 
are life-threatening, lead to hospitalization, result in disability or permanent damage, or any other 
important medical event. All serious adverse events will be registered in EudraCT..  
 
Serious adverse drug reactions (SARs) will be reported annually. Most likely this will 
not be applicable to this study, as it will last less than a year. In this case, SARs will be reported on 
conclusion of the study.  
Sudden unexpected serious adverse reactions (SUSARs) will be reported immediately 
to the Danish Medicines Authority and the local ethics committee. Adverse event registration will 
commence on the first study day and be finalized after the last study day. In case of serious adverse 
reactions, the sponsor can decide if the study will be terminated. It is the sponsor’s responsibility to 
evaluate if an adverse event is a SUSAR. SUSARs that are lethal or life-threatening will be 
registered and reported to the Danish Medicines Agency as fast as possible and at the latest 7 days 
after the sponsor has obtained knowledge of such an adverse event. All other SUSARs will be 
reported to the Danish Medicines Agency within 15 days. Due to not expecting any serious adverse 
reactions, all SARs will be considered unexpected and therefore considered SUSARs. 
 
Ethical considerations 
The study will be performed in accordance with the Helsinki II declaration. Patients will be 
included after oral and written consent. If patients do not wish to participate in the study, or choose 
to withdraw before administration of melatonin in any of the series of radiation therapy, they will 
receive standard treatment for their cancer. Patients can withdraw from the study at any time 
without stating a reason. Withdrawal from the study will not have any consequence for further 
treatment. Should an investigator deem it necessary for the safety of the patient, they can withdraw 
the patient from the trial. It is important to clarify if melatonin has a protective effect against 

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radiation injury, due to it having a large impact on the quality of life in patients. Positive results 
from this study will guide further studies and potentially change current practice in radiation 
oncology. The protective effect of melatonin has the potential to be used in several other types of 
cancer treatment, where ionizing radiation is used, to minimize the radiation injury and therefore 
decreased quality of life in patients. All information regarding participants in this study will be kept 
protected and no personal data will be published. The anonymity of all participants will be secured 
and data kept confidential.  
 
Insurance  
For this study no additional insurance policies have been arranged that exceed the Danish Act on the 
Right to Complain and Receive Compensation within the Danish Health Service 
(Patientforsikringsloven). Patients participating in this study will be covered by the mentioned Act in 
accordance with “Bekendtgørelse af lov om klage- og erstatningsadgang inden for sundhedsvæsenet” in 
the same way as patients that are treated otherwise in the hospital. This is normal practice for 
investigator-initiated trials in Denmark. 
 
Approvals 
The study will be performed in accordance with the Helsinki II declaration. Approval from Danish 
Data Protection Agency in the Capital Region of Denmark (Datatilsynets fællesanmeldelse i Region 
H) will be sought in accordance with Data Protection Act (Databeskyttelsesloven), and the study 
will not start subject accrual before this approval has been given. The same applies for the local 
ethics committee as well as Danish Medicines Authority (Lægemiddelstyrelsen). Permission will be 

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sought to register and store data manually and electronically from participants in the study through 
their electronic health records and the Danish National Patient Register. 
 
 
Confidentiality 
All information about the patient will be noted in the eCRF, which is stored in a secure server (see 
Data management). 
The sponsor, investigators, monitors and Danish Medicines Agency will have access to required 
information (outlined in Table 2) from the patients’ electronic health records. 
 
Protocol amendments 
Currently there are no protocol amendments. Should any protocol amendments arise, these will be 
communicated to the investigators, the journal in which the protocol will be published, 
clinicaltrials.gov, the Good Clinical Practice Unit, the Danish Data Protection Agency (if relevant), 
the local ethics committee, and the Danish Medicines Authority. 
 
 
 
Funding 
A grant covering all study expenses has been donated by the pharmaceutical company 
RepoCeuticals ApS for this study. Both the active and placebo treatments used in the trial have been 

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donated by the company, which has no influence on the design, interpretation or publication of data. 
None of the investigators have any interests in RepoCeuticals, financial or otherwise. 
 
Conflicts of interests 
None of the investigators have any conflicts of interests to declare. 
 
Dissemination policy 
Three publications will be written on the basis of this study: 
- A protocol article, describing our methods 
- An article concerning the acute radiation dermatitis measured with the RTOG scale, image 
analysis and the need for steroid treatment; 
- An article concerning the impact of acute radiation dermatitis on the patients’ quality of 
life. 
The articles will be published in international peer-reviewed scientific journals. Both negative, 
positive, and inconclusive results will be published. The determination and documentation of 
authorship will be based on the authorship criteria formulated by the International Committee of 
Medical Journal Editors (ICMJE). Participants will be offered a copy of the articles when published. 

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52. Dollins AB, Lynch HJ, Wurtman RJ, Deng MH, Kischka KU, Gleason RE, et al. Effect of 
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53. Rossberger J, Fall M, Peeker R. Critical appraisal of dimethyl sulfoxide treatment for interstitial 
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54. Parkin J, Shea C, Sant GR. Intravesical dimethyl sulfoxide (DMSO) for interstitial cystitis--a 
practical approach. Urology. 1997;49(5A Suppl):105-7. 
55. Shirley SW, Stewart BH, Mirelman S. Dimethyl sulfoxide in treatment of inflammatory 
genitourinary disorders. Urology. 1978;11(3):215-20. 
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57. Bertelli G, Gozza A, Forno GB, Vidili MG, Silvestro S, Venturini M, et al. Topical 
dimethylsulfoxide for the prevention of soft tissue injury after extravasation of vesicant cytotoxic 
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Table 1: Inclusion and exclusion criteria 
Inclusion criteria 
Diagnosed with early breast cancer 
Over 49 years old 
Have had radical tumor resection surgery 
Follows treatment regimens and follow-up at Rigshospitalet 
Written informed consent after written and verbal information 
Exclusion criteria 
Inability to understand Danish, written or spoken 
Mental illness*  
Previous therapy with ionizing radiation in the thoracic or neck area 
Use of bolus for radiation therapy** 
Pregnancy*** 
* Defined as having a diagnosis and being in medical treatment, or if 
anticipated poor compliance. ** A bolus is a material which has dose 
absorption properties equivalent to tissue. It is placed on the irradiated area 
to alter dosing or target of the radiation therapy. *** Patients will be asked 
to take a pregnancy test prior to inclusion 
 
 
 

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Table 2: Demographics and background variables 
Demographics 
Source 
Age 
CPR-number1 
Weight 
Patient 
Height 
Patient 
Ethnicity 
Patient 
Bra size 
Patient 
Breast volume 
Radiation treatment plan 
Skin-type2 
Investigator evaluation 
Comorbidities 
Patient/electronic health record 
Connective tissue diseases 
Patient 
Disease and treatment 
Source 
Tumor type 
Pathology report 
Tumor size 
Surgical report 
Location of tumor 
Surgical report 
Lymph node status 
Pathology report 
Metastases 
Electronic health record 
Axillary dissection 
Surgical report 
Chemotherapy prior to radiation 
Electronic health record 
Type of chemotherapy 
Electronic health record 
Radiation dosage 
Radiation treatment plan 
Hypo-/hyperfractioning 
of radiation therapy 
Radiation treatment plan 
1Central Person Register in Denmark. 2Fitzpatrick scale.  
 
 

 
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Table 3: Participant timeline 
Activity/assessment 
Pre-study 
screening/ 
consent 
Pre-study 
baseline/ 
randomization 
First 
radiation 
fraction 
Study 
visit 
week 1 
Study 
visit 
week 2 
Study 
visit 
week 3 
Study 
visit 
week 4 
Study 
visit 
week 5 
Last 
radiation 
fraction 
Follow-
up week 
1 
Follow-
up week 
2 
Follow-
up week 
3 
Prescreening consent 
● 
 
 
 
 
 
 
 
 
 
 
 
Screening log 
● 
 
 
 
 
 
 
 
 
 
 
 
Consent form 
● 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion 
form 
● 
 
 
 
 
 
 
 
 
 
 
 
Randomization 
 
● 
 
 
 
 
 
 
 
 
 
 
Demographics  
questionnaire 
 
● 
 
 
 
 
 
 
 
 
 
 
Instruction in 
application of cream 
 
● 
 
 
 
 
 
 
 
 
 
 
Handover of 
drug/placeboa 
 
● 
 
 
● 
 
● 
 
 
 
 
 
Evaluation of 
compliance 
 
 
● 
● 
● 
● 
● 
● 
● 
 
 
 
RTOG score 
evaluationb 
 
 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
Clinical photography 
 
 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
QLQ-C30c 
 
 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
QLQ-BR23c 
 
 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
Steroid usage 
evaluation 
 
 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
Monitoring of adverse 
events 
 
 
● 
● 
● 
● 
● 
● 
● 
● 
● 
● 
Serious adverse event 
formd 
 
 
 
 
 
 
 
 
 
 
 
 
Conclusion of 
participation 
 
 
 
 
 
 
 
 
 
 
 
● 
a Patients will receive extra trial drug/placebo when required throughout study, therefore, the marked visits might vary from patient to patient depending on compliance. b RTOG score = Radiation Therapy Oncology 
Group cooperative group common toxicity criteria. c QLQ-C30 and QLQ-BR23 =European Organization for Research and Treatment of Cancer quality of life questionnaires. d Serious Adverse event form will be filled in 
as needed throughout the trial. 

 
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