Plasma Lycopene Concentration as a Dietary Compliance Biomarker - a Pilot Study
Phase: NA
Conditions: Healthy
Sponsor: Erasmus Medical Center

NL85468.078.23 / OZBS62.23545 
 
Lycopene as a dietary compliance biomarker 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Plasma lycopene concentration as a 
dietary compliance biomarker – a pilot 
study 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Version 3.1  
June 10, 2025 

NL85468.078.23 / OZBS62.23545 
 
Lycopene as a dietary compliance biomarker 
 
 
 
PROTOCOL TITLE Plasma lycopene concentration as a dietary compliance biomarker – a 
pilot study.  
 
Protocol ID 
NL85468.078.23 
OZBS62.23545 
Panama ID 11236 
Short title 
Lycopene as a dietary compliance biomarker 
EudraCT number  
Not applicable 
Version 
3.1 
Date 
June 10, 2025 
Principal investigator 
 
Dr. L.D.F. Venderbos 
Erasmus MC 
Department of Urology 
P.O. Box 2040, room NA-1520 
3000 CA Rotterdam 
Tel.: +31 (0)6 5000 1668 
Email: l.venderbos@erasmusmc.nl 
Sub-investigators 
 
 
Prof. dr. C.H. Bangma 
Department of Urology, room Na-1512 
Erasmus MC 
P.O. Box 2040 
3000 CA Rotterdam, the Netherlands 
Tel.: +31 (0)6 1090 0019 
Email: c.h.bangma@erasmusmc.nl 
 
Nicole Borggreven  
Department of Urology, room Na-1524 
Erasmus MC  
P.O. Box 2040  
3000 CA Rotterdam, the Netherlands  
Email: n.borggreven@erasmusmc.nl 
Sponsor 
Erasmus MC  
Subsidising party 
Stichting Erasmus MC Foundation 
Independent expert 
Prof. dr. K.F.M. Joosten  
Department ICU Children, room SK-3234 
Erasmus MC  
P.O. Box 2040  
3000 CA Rotterdam, the Netherlands 
Tel.: +31 (0)10-703 7029 

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Lycopene as a dietary compliance biomarker 
 
 
 
Email: k.joosten@erasmusmc.nl 
Laboratory sites 
 
 
 
 
 
 
Erasmus MC  
CKCL 
Prof. dr. R. van Schaik 
P.O. Box 2040, room Na-415 
3000 CA Rotterdam  
Tel.: +31 (0)10-703 3543 
Email: r.vanschaik@erasmusmc.nl 
 
Wageningen University & Research  
Department of Agrotechnology and Food 
Sciences, Division of Human Nutrition and Health 
Dr. Michiel Balvers 
P.O. Box 17, 124/2065 
6700 AA Wageningen 
Tel.: +31 (0)31-748 2453 
Email: Michiel.balvers@wur.nl 

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TABLE OF CONTENTS 
 
1. 
INTRODUCTION AND RATIONALE .............................................................................10 
2. 
OBJECTIVES ................................................................................................................14 
3. 
STUDY DESIGN ...........................................................................................................15 
4. 
STUDY POPULATION ..................................................................................................16 
4.1 
Population (base) ...................................................................................................16 
4.2 
Inclusion criteria .....................................................................................................16 
4.3 
Exclusion criteria ....................................................................................................16 
4.4 
Sample size calculation ..........................................................................................16 
5. 
TREATMENT OF SUBJECTS .......................................................................................18 
5.1 
Investigational product/treatment ............................................................................18 
5.2 
Use of co-intervention ............................................................................................18 
5.3 
Escape medication .................................................................................................18 
6. 
INVESTIGATIONAL PRODUCT ....................................................................................19 
6.1 
Name and description of investigational product(s) ................................................19 
6.2 
Summary of findings from non-clinical studies ........................................................19 
6.3 
Summary of findings from clinical studies ...............................................................19 
6.4 
Summary of known and potential risks and benefits ...............................................20 
6.5 
Description and justification of route of administration and dosage .........................20 
6.6 
Dosages, dosage modifications and method of administration ...............................20 
6.7 
Preparation and labelling of Investigational Medicinal Product ...............................20 
6.8 
Drug accountability .................................................................................................20 
7. 
NON-INVESTIGATIONAL PRODUCT ...........................................................................21 
7.1 
Name and description of non-investigational product(s) .........................................21 
7.2 
Summary of findings from non-clinical studies ........................................................21 
7.3 
Summary of findings from clinical studies ...............................................................21 
7.4 
Summary of known and potential risks and benefits ...............................................21 
7.5 
Description and justification of route of administration and dosage .........................21 
7.6 
Dosages, dosage modifications and method of administration ...............................21 
7.7 
Preparation and labelling of Non Investigational Medicinal Product ........................21 
7.8 
Drug accountability .................................................................................................21 
8. 
METHODS ....................................................................................................................22 
8.1 
Study parameters/endpoints ...................................................................................22 
8.1.1 
Main study parameter/endpoint .......................................................................22 
8.1.2 
Secondary study parameters/endpoints ..........................................................22 
8.1.3 
Other study parameters ...................................................................................22 

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8.2 
Randomisation, blinding and treatment allocation ..................................................22 
8.3 
Study procedures ...................................................................................................22 
8.4 
Withdrawal of individual subjects ............................................................................24 
8.4.1 
Specific criteria for withdrawal .........................................................................24 
8.5 
Replacement of individual subjects after withdrawal ...............................................24 
8.6 
Follow-up of subjects withdrawn from treatment .....................................................24 
8.7 
Premature termination of the study .........................................................................24 
9. 
SAFETY REPORTING ..................................................................................................25 
9.1 
Temporary halt for reasons of subject safety ..........................................................25 
9.2 
AEs, SAEs and SUSARs ........................................................................................25 
9.2.1 
Adverse events (AEs) ......................................................................................25 
9.2.2 
Serious adverse events (SAEs) .......................................................................25 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) .........................26 
9.3 
Annual safety report ...............................................................................................26 
9.4 
Follow-up of adverse events ...................................................................................26 
9.5 
Data Safety Monitoring Board (DSMB) / Safety Committee ....................................26 
10. 
STATISTICAL ANALYSIS ..........................................................................................27 
10.1 
Primary study parameter(s) ....................................................................................27 
10.2 
Secondary study parameter(s) ...............................................................................27 
10.3 
Other study parameters ..........................................................................................27 
10.4 
Interim analysis ......................................................................................................27 
11. 
ETHICAL CONSIDERATIONS ...................................................................................28 
11.1 
Regulation statement .............................................................................................28 
11.2 
Recruitment and consent........................................................................................28 
11.3 
Objection by minors or incapacitated subjects ........................................................28 
11.4 
Benefits and risks assessment, group relatedness .................................................28 
11.5 
Compensation for injury .........................................................................................28 
11.6 
Incentives ...............................................................................................................29 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .........................30 
12.1 
Handling and storage of data and documents ........................................................30 
12.2 
Monitoring and Quality Assurance ..........................................................................30 
12.3 
Amendments ..........................................................................................................31 
12.4 
Annual progress report ...........................................................................................31 
12.5 
Temporary halt and (prematurely) end of study report ............................................31 
12.6 
Public disclosure and publication policy ..................................................................32 
13. 
STRUCTURED RISK ANALYSIS...............................................................................33 
13.1 
Potential issues of concern .....................................................................................33 

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13.2 
Synthesis ...............................................................................................................34 
14. 
REFERENCES ..........................................................................................................35 

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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
General Assessment and Registration form (ABR form), the application 
form that is required for submission to the accredited Ethics Committee; in 
Dutch: Algemeen Beoordelings- en Registratieformulier (ABR-formulier) 
AE 
Adverse Event 
AR 
Adverse Reaction 
CA 
Competent Authority 
CCMO 
Central Committee on Research Involving Human Subjects; in Dutch: 
Centrale Commissie Mensgebonden Onderzoek 
CV 
Curriculum Vitae 
DSMB 
Data Safety Monitoring Board 
EU 
European Union 
EudraCT European drug regulatory affairs Clinical Trials  
GCP 
Good Clinical Practice 
GDPR 
General Data Protection Regulation; in Dutch: Algemene Verordening 
Gegevensbescherming (AVG) 
IB 
Investigator’s Brochure 
IC 
Informed Consent 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
METC  
Medical research ethics committee (MREC); in Dutch: medisch-ethische 
toetsingscommissie (METC) 
(S)AE 
(Serious) Adverse Event  
SPC 
Summary of Product Characteristics; in Dutch: officiële productinformatie 
IB1-tekst 
Sponsor 
The sponsor is the party that commissions the organisation or performance 
of the research, for example a pharmaceutical 
company, academic hospital, scientific organisation or investigator. A party 
that provides funding for a study but does not commission it is not 
regarded as the sponsor, but referred to as a subsidising party. 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
UAVG 
Dutch Act on Implementation of the General Data Protection Regulation; in 
Dutch: Uitvoeringswet AVG 
WMO 
Medical Research Involving Human Subjects Act; in Dutch: Wet Medisch-
wetenschappelijk Onderzoek met Mensen 
 
 

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SUMMARY 
 
Rationale: To determine the plasma lycopene concentration before and after an oral intake 
of lycopene in order to use this measurement as a life style compliance marker.  
Objective: Primary objective: to determine if a plasma lycopene concentration can serve as 
a response parameter after a single dose of dietary lycopene. 
Study design: Cross-over interventional pilot study. 
Study population: Ten male healthy volunteers 18-75 years. 
Intervention: Oral food supplement tablet 40 mg lycopene once, versus oral soup of cooked 
tomatoes equivalent to 40 mg lycopene content. In addition, the participants’ habitual diet 
and actual food intake during the intervention will be measured using a food frequency 
questionnaire and a food diary.  
Main study parameters/endpoints: Variation of plasma lycopene 1 hour before, and 
1,3,6,12,24,48,72 hours after intervention. 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness: Eight blood samples of 6 ml full venous blood obtained by vena 
puncture per intervention per individual, 2 times in a cross-over pilot study, in which 
interventions are 3 weeks apart (so 2 x 8 samples in 10 volunteers). Risk of vena puncture is 
negligible, idemque the burden.  
 

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1. INTRODUCTION AND RATIONALE 
 
Dietary study in Prostate cancer: Men with prostate cancer (PCa) often ask if the 
(composition of) their diet may contribute to the suppression of their tumor. There is indirect 
epidemiological evidence that diet, especially antioxidants like lycopene, are beneficial for an 
advantageous course of PCa [1]. Lycopene is a non-provitamin A carotenoid that exhibits 
several health benefits. Epidemiological data support a correlation between lycopene intake 
and the attenuation of several chronic diseases, including certain types of cancers and 
cardiovascular diseases. It is currently unknown whether the beneficial effects are from the 
native structure of lycopene or its metabolic derivatives [2]. 
 
Scientific proof for the impact of dietary interventions on PCa growth is extremely difficult to 
provide, as the natural course of PCa from the time of diagnosis is frequently over 20 years. 
In dietary studies many confounders are present, therefore dietary studies also need extreme 
large populations. Furthermore, the biochemical parameters needed to study lycopene 
concentrations in blood do not have defined normal values: SNPs in BCO1 and other genes 
may modulate human plasma and prostate tissue responses to dietary lycopene intake. 
These individual genetic variants related to carotenoid metabolism may partially explain 
heterogeneous human blood and tissue responses and may be critical covariates for 
population studies and clinical trials [3]. On top of that, intestinal absorption is promoted by 
co-consumption of fat [2], and 10 gram of olive oil has been identified as an optimal dose for 
lycopene uptake. The way lycopene itself is offered appears to be less of importance, as 
there was no difference of plasma concentration observed between well processed dietary 
and artificial in-tablet servings of lycopene [4].  
 
While the generally accepted opinion is that diets high in antioxidants are beneficial for 
suppressing and prevention of all cancers, and that they promote the overall health status of 
individuals, it may be difficult to change a life style that incorporates a healthy diet. Therefore 
in this study, we are searching for a parameter in body fluids that reflects the intake of 
healthy diet, and as such may motivate and sustain its continuous use (compliance marker). 
In case of prostate cancer we address lycopene as our target for reasons described above. 
Ideally, our lycopene related marker should reflect the short-term uptake of lycopene, and be 
based on a single dose consumption, in order to act as a direct feed-back to the consumers 
dietary action. Current methods of determination (see further) do not yet live up to point-of-
care applications. There is, however, evidence that single dose consumption is reflected in 
the lycopene plasma concentration. In a trial in Italian consumers on lycopene kinetics on 
single dose consumption [5], a significant increase of plasma lycopene concentration of 40 

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nmol/l was observed above an individual base level (around 400 nmol/l), with a max 
concentration at 12 hours, and a normalisation after 48 hours after consumption of 15 mg of 
lycopene. Also a biomarker reflecting the adequate lycopene consumption of a medium long 
term (e.g. one week consumption) might provide a valuable asset for consumers. In a trial in 
Swiss male consumers on an 8-day intervention of 20 mg after lycopene depletion, the 
individual lycopene plasma concentration topped to a steady state of 1000 to 1500 nmol/l, 
while two weeks after the intervention (so during lycopene depletion) a plateau phase was 
obtained on a plasma concentration of 400-500 nmol/l [4], likely due to the release of 
lycopene from liver resources. We conclude that for consumer reasons the choice for a point-
of-care testing of single dose lycopene is feasible, but restricted by current technology, and 
that feed-back on medium long term lycopene consumption is also feasible, as it is well 
reflected in plasma lycopene concentrations. 
 
Study design: We have chosen lycopene as it is related to PCa growth, it can be used as a 
dietary intervention, and it is measurable reproducibly in food and in body fluids. Here we 
study the lycopene plasma concentration dynamics after a single dose consumption in 
volunteers in a Dutch setting (which differs from the geographic and environmental setting 
and life-style in the above mentioned studies). This is to obtain the necessary information on 
technology, logistics, and food products needed to conduct the follow-up study (METC 2017-
389) in cancer patients. Once we have proven that a measurable response can be obtained 
in healthy volunteers in a Dutch population, that the assay is available and robust, and in line 
with food consumption as evaluated by validated food questionnaires, we will incorporate 
that parameter (lycopene) in the current randomised study for men with PCa and undergoing 
treatment or have undergone treatment in the past in which three dietary interventions are 
given and compliance to the intervention is currently measured by food intake questionnaires 
(METC 2017-389). 
 
Here we test plasma lycopene as a candidate compliance marker for that purpose. This is 
based on the assumption that in Dutch participants the plasma level of lycopene might be 
comparable to the Swiss population literature, that a single dose response to 40 mg lycopene 
in combination with oil will provide a measurable response of plus 10 % in individuals, and 
that a concentration between 1000-1500 nmol/l is appreciated by patients and caregivers as 
the optimal lycopene plasma concentration to be accomplished long term (1-3 months). 
 
Dietary interventions should always be analysed against the background of the normal 
individual diet. Therefore, to evaluate the lycopene intervention and related marker, it is 

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crucial to take the participant’s habitual diet, as well as actual food intake during the 
intervention into account [6]. 
 
Research questions:  
1) Can a plasma lycopene concentration serve as a response parameter after a single 
dose of dietary lycopene. 
 
We will measure plasma carotenoid concentrations (including lycopene) in response to a 
single dose dietary intervention with lycopene-rich food or a lycopene tablet in a cross-over 
study design.  
 
Plasma lycopene concentrations show variation between individuals, and within individuals. It 
is unknown if and how much a single dietary intervention with lycopene from lycopene-rich 
vegetables like tomatoes alters the plasma concentration, and therefore can be used as a 
compliance marker.  
 
As the bioavailability and plasma kinetics of lycopene is known to a limited extend (see 
studies above), a scheme for plasma determinations is followed according to previous 
marker (GABA, also found in tomatoes, serving as an exemplary study design) research from 
the same Wageningen group, including a wash out period of at least two weeks between 
interventions in the cross-over design [7].  
 
2) Is a tablet lycopene with equivalent lycopene content as effective as a single dose 
dietary intervention regarding the change of plasma lycopene concentration? 
 
Previous studies suggest equivalence, which might be of interest for consumers/patients. 
 
3) Is the prior food consumption of individuals, as measured by dietary questionnaires, 
related to the plasma lycopene levels before and after lycopene intervention?  
 
If an existing individual high intake of lycopene as analysed by a validated questionnaire 
would result in a high standard plasma lycopene concentration, or little response on single 
dose lycopene consumption, the subject might exclude for further supplementary lycopene 
consumption and life style changes. 
 
Intervention: We have chosen a single dose of 40 mg lycopene as an intervention based on 
the following. Previously, in a dose escalating study to 90 mg lycopene daily in men with 

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recurrent PCa after primary treatment, no effects were seen on the level of PSA, in 
contradiction to two studies conducted in men with metastatic PCa [8]. Also in high doses 
maintained for several weeks no toxicity was observed, while lycopene plasma levels 
corresponded with dietary dose. For chronic use, the daily dose of 30 mg is recommended 
based on a large review [9].  
 
Lycopene belongs to the chemical family of carotenoids that can be found in intensely 
coloured fruits and vegetables, like tomatoes. The uptake from the alimentary tract is 
increased by heating the natural present lycopene, initiating a chemical conversion from a 
trans to a cis-form [10]. Uptake of lycopene and its geometrical isomers is greater from heat-
processed than from unprocessed tomato juice in humans [10]. The addition of oils also are 
reported to have a beneficial influence [11]. Lycopenes are absorbed in the gut, and 
transported in chylomicrones because of their lipophylic nature [12]. Their exact biological 
action is still unknown, but regarded to be an antioxidant. 
 
Various dietary interventions have been applied previously for the study on bioavailability of 
lycopene. The lycopene content in 200g of tomato soup equals ca.33 mg of lycopene. Most 
experimental studies aim to provide 40 mg lycopene whether in a carotene-rich diet, whether 
by pill-form (supplement), in combination with a minimum of 10 g of fat [2].  
 
Assay: Standard liquid-chromatography-based technology for the analysis of carotenoids and 
similar structures including lycopene. Plasma carotenoids, including retinol, alpha- and beta-
carotene, beta-cryptoxanthin, lutein, lycopene, tocopherols and zeaxanthin, are determined 
using ultra-pressure liquid chromatography coupled to diode array detection (UPLC-DAD). In 
short, 375 µL plasma is denatured using ethanol in the presence of the internal standard 
retinyl acetate. The carotenoids are subsequently extracted using 0.01% w/v butylated 
hydroxytoluene in hexane. The extracts are dried under nitrogen in a TurboVap evaporator 
(Biotage, Uppsala, Sweden) at 35 °C, reconstituted in acetonitrile, and transferred to a LC 
vial. LC analysis is performed on a Acquity H-class UPLC coupled to an Acquity PDA 
eLambda detector (Waters, Etten-Leur, the Netherlands) using an Acquity UPLC HSS T3 
column (Waters). Gradient elution is performed using a mixture of acetonitrile-
dichloromethane-methanol (ratio 85:5:10) containing 0.1% ammonium acetate (eluens A) 
and ULC-MS grade water (eluens B) with a constant flow of 400 µL/min and a runtime of 30 
min. The detector is set at 292, 325 and 450 nm. Concentrations are calculated using a 6-
point calibration curve. A pooled plasma sample is analyzed in duplicate in each analytical 
batch to monitor the quality of the analyses. 
 

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2. OBJECTIVES 
 
Primary Objective:  
To determine whether a plasma lycopene concentration can serve as a response parameter 
after a single dose of dietary lycopene. 
 
Secondary Objectives: 
1. To determine if a food supplement tablet lycopene with equivalent lycopene content 
compared to a single dose dietary intervention provides identical plasma lycopene 
concentration responses compared to the dietary intervention. 
 
2. To determine the influence of prior food consumption, as measured by dietary 
questionnaires, on plasma lycopene levels before and after lycopene intervention. 

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3. STUDY DESIGN 
 
Cross-over interventional pilot study.  
 
Ten healthy individuals (male 18-75 years) are randomly divided in two groups (A and B) of 5 
individuals. One week before the dietary intervention a daily food questionnaire will be 
completed.  
 
One hour before the intervention a blood sample will be taken (all blood samples 6 ml EDTA 
plasma tube). All individuals will be given an infusion needle for 26 hours. Samples are 
spinned and plasma is pipetted and distributed over 1 ml Eppendorf cups for immediate 
freezing at -80 Celsius at the research lab. 
 
Group A will take 40 mg lycopene (2,5 pills of food supplement Vitabiotics Ultra Lycopene 
15mg, potent extract). Group B will take 250 ml of tomato soup (matching with 40 mg 
lycopene) with 20 ml olive oil. Blood sampling as described in all individuals from needle: 
1,3,6,12,24 hours after intervention, and after 48 and 72 hours by direct venapunction. Wash 
out period of 3 weeks, after which the identical above procedure takes place including 
questionnaires, but group A and B changed (cross-over). All samples (n=160) will be frozen 
at -80, and transported on dry ice to Wageningen University for lycopene determination. 

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4. STUDY POPULATION 
4.1 Population (base)  
Potential subjects will be recruited via distribution of flyers and publication of the flyer on 
the intranet of Erasmus MC. .  
 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a subject must meet all of the following 
criteria: 
 
Male;  
 
Healthy;  
 
18-75 yrs old. 
 
4.3 Exclusion criteria 
A potential subject who meets any of the following criteria will be excluded from 
participation in this study: 
 
 
Allergic for tomatoes;  
 
Any gastrointestinal disorder within 3 months prior to the intervention;  
 
Recent medication or supplement use;  
 
Recent substantial change in weight;  
 
Adherence to a specific diet ( for example the Moorman-diet);  
 
Using recreational drugs more than once a month;  
 
Smoking and excessive alcohol consumption (>10 standardized glasses a week). 
 
Risk of a dependency situation with the researchers.  
 
4.4 Sample size calculation 
Observational study obtaining individual lycopene concentration curves in plasma. It is 
anticipated that a measurable lycopene response will be seen in all participants. Based 
on the literature described in the introduction section it is hypothesized that in Dutch 
participants the plasma level of lycopene might be comparable to the Swiss population, 
that a single dose response to 40mg lycopene in combination with oil will provide a 
measurable response of plus 10% in all individuals. Therefore a sample size of two times 
five participants (10 participants in total) has been chosen.  
Our pilot still needs to obtain an impression that responses are observed in all individuals, 
and to confirm that our HPLC-assay is an adequate technique. We will express that in 

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percentages of the group, such as: ‘In xx percent the lycopene concentration increased 
more than 10% of the T=0 within 48 hours after intervention A’. 
 

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5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
Intervention 1: 40 mg lycopene (i.e. 2.5 food supplement pills of Vitabiotics Ultra 
Lycopene 15mg, potent extract). Tablets contain 430mg Tomato Extract which provides 
15mg pure Lycopene, no artificial colours or preservatives, made by Vitabiotics, UK. 
 
Intervention 2: 250 ml of tomato soup with 20 ml olive oil. Soup will be prepared from 5 kg 
freshly harvested tomatoes (Harvest of Health lycopene enriched tomato), blended, and 
frozen down at -20 C for max 1 week. These tomatoes have been characterised 
previously on their lycopene content which will be determined again at Wageningen 
University Human Nutrition Research Unit of Wageningen University and Research. 
 
5.2 Use of co-intervention 
Subjects are allowed to continue their normal diet without alterations.  
 
5.3 Escape medication 
Not applicable.  

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6. INVESTIGATIONAL PRODUCT 
6.1 Name and description of investigational product(s) 
Food supplement Vitabiotics Ultra Lycopene 15mg, potent extract. Tablets contain 430mg 
Tomato Extract which provides 15mg pure Lycopene, no artificial colours or 
preservatives, made by Vitabiotics, UK. 
 
Tomatoes are a source of bioactive phytochemicals and are widely consumed. The 
phytochemical pattern of tomato products can be refined by choosing breeds with 
naturally higher nutrient levels that require more intense horticultural techniques and food 
processing [13]. Producing high-nutrient tomatoes is achieved through classic and 
conventional breeding techniques, done by professional vegetable breeding companies, 
as indicated in European Union Law. This process does not include genetic modification 
processes. In this study the Harvest of Health (Proudesse) tomato will be used. Per 100g 
this tomato contains 8,4mg lycopene, which is three times higher as ‘normal’ supermarket 
tomatoes. Unique characteristics of the Proudesse tomato are its preservability, shape, 
high concentration of lycopene and its taste. 
 
Per subject 250 ml of tomato soup with 20 ml olive oil will be prepared from freshly 
harvested Harvest of Health lycopene enriched tomatoes, blended, and frozen down at -
20 °C for max one week.  
 
6.2 Summary of findings from non-clinical studies 
Food supplement Vitabiotics Ultra Lycopene 15mg, potent extract: we are not aware of 
any performed non-clinical studies. 
 
Harvest of health tomato: studies have been conducted measuring the nutrient levels 
over time, confirming that the Harvest of Health tomato contains almost 3x higher 
lycopene levels as compared to the normal supermarket tomato. This was recently 
confirmed by measurements performed by the Wageningen University.  
 
6.3 Summary of findings from clinical studies 
Food supplement Vitabiotics Ultra Lycopene, 15mg, potent extract: we are not aware of 
any performed clinical studies.  
 
Harvest of Health tomato: to the knowledge of the research group, no clinical studies 
have been done with the Harvest of Health (Proudesse) tomato.  

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6.4 Summary of known and potential risks and benefits 
Food supplement Vitabiotics Ultra Lycopene, 15mg, potent extract: according to the 
manufacturers’ frequently asked questions page ‘Ultra Lycopene has no known side-
effects when taken as directed’ (https://www.vitabiotics.com/products/ultra-lycopene-
tablets).  
 
Harvest of Health tomato: the Harvest of Health tomato contains 8,4mg lycopene per 
100gr. The acceptable daily intake of lycopene is 0,5 mg/kg bodyweight. For an average 
male participant weighing 80kg this would result in consuming a maximum of 40mg 
lycopene per day, converting into a daily uptake of ±500gr of tomatoes. It is not likely that 
participants will eat more than 500gr of tomatoes per day and therefore the risk of 
consuming too much lycopene is low. 
 
6.5 Description and justification of route of administration and dosage 
Not applicable.  
 
6.6 Dosages, dosage modifications and method of administration 
Not applicable.  
 
6.7 Preparation and labelling of Investigational Medicinal Product 
Not applicable.  
 
 
6.8 Drug accountability 
The food supplement Vitabiotics Ultra Lycopene 15mg, potent extract supplement has 
been bought in store and stored dry and at room temperature.  
 
Blended tomato fluids (soup) are frozen till consumption, and heated at normal soup 
temperature circa 60-80 degrees Celsius. 
 

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7. NON-INVESTIGATIONAL PRODUCT  
7.1 Name and description of non-investigational product(s) 
Not applicable.  
 
7.2 Summary of findings from non-clinical studies 
 
Not applicable.  
 
7.3 Summary of findings from clinical studies 
 
Not applicable.  
 
7.4 Summary of known and potential risks and benefits 
 
Not applicable.  
 
7.5 Description and justification of route of administration and dosage 
Not applicable. 
 
7.6 Dosages, dosage modifications and method of administration 
Not applicable.  
 
7.7 Preparation and labelling of Non Investigational Medicinal Product 
Not applicable.  
 
7.8 Drug accountability 
Not applicable.  
 

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8. METHODS 
8.1 Study parameters/endpoints 
8.1.1 Main study parameter/endpoint 
Raise of plasma lycopene concentration in absolute (ng/ml) and relative (percentage 
of T0-value) after single lycopene intervention. 
8.1.2 Secondary study parameters/endpoints 
1. Optimal time point of lycopene concentration response after single dietary 
intervention (hours, days). 
 
2. Influence of prior food consumption on plasma lycopene levels before and after 
lycopene interventions, as measured by dietary questionnaires. 
8.1.3 Other study parameters 
Clinical parameters at intake: age, body weight, comorbidity.  
Dietary parameters: weekly lycopene food content estimated by weekly food 
questionnaires. 
 
 
8.2 Randomisation, blinding and treatment allocation 
10 study subjects randomised for study scheme AB versus BA (cross-over) by blinded 
draw of 10 tickets by an independent statistician. The 10 study subjects will be 
randomized at once, before the start of the study.  
 
8.3 Study procedures 
Participants will be randomized into the study scheme AB versus BA (cross-over). One 
week before the intervention a food frequency questionnaire will be distributed to 
participants. Group A will take 40mg lycopene (2,5 pills of food supplement Vitabiotics 
Ultra Lycopene 15mg, potent extract). Group B will take 250 ml of tomato soup (matching 
with 40mg lycopene) with 20ml olive oil. Wash out period of 3 weeks, after which the 
identical procedure takes place including questionnaires, but group A and B changed 
(cross-over).  
  
Blood draws are taken on indicated time points (before intervention, 1, 3, 6, 12, 24, 48 
and 72 hours after intervention) in standard vacutainers (BD Vacutainer; Becton, 
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after the intervention) all participants will be given an infusion needle for 26 hours. Blood 
draws after 48 and 72 hours will be done by direct venapunction. Next, the tubes are 
centrifuged at 3000g for 10 minutes at 4 °C (Sigma 4-16K; Sigma-zentrifugen). Plasma 
samples are immediately put on dry ice for rapid freezing. At the end of each test day, the 
plasma samples are stored at −80 °C until further analysis. 
 
Standard liquid-chromatography-based technology for the analysis of carotenoids and 
similar structures including lycopene. Plasma carotenoids, including retinol, alpha- and 
beta-carotene, beta-cryptoxanthin, lutein, lycopene, tocopherols and zeaxanthin, are 
determined using ultra-pressure liquid chromatography coupled to diode array detection 
(UPLC-DAD). In short, 375 µL plasma is denatured using ethanol in the presence of the 
internal standard retinyl acetate. The carotenoids are subsequently extracted using 
0.01% w/v butylated hydroxytoluene in hexane. The extracts are dried under nitrogen in a 
TurboVap evaporator (Biotage, Uppsala, Sweden) at 35 °C, reconstituted in acetonitrile, 
and transferred to a LC vial. LC analysis is performed on a Acquity H-class UPLC 
coupled to an Acquity PDA eLambda detector (Waters, Etten-Leur, the Netherlands) 
using an Acquity UPLC HSS T3 column (Waters). Gradient elution is performed using a 
mixture of acetonitrile-dichloromethane-methanol (ratio 85:5:10) containing 0.1% 
ammonium acetate (eluens A) and ULC-MS grade water (eluens B) with a constant flow 
of 400 µL/min and a runtime of 30 min. The detector is set at 292, 325 and 450 nm. 
Concentrations are calculated using a 6-point calibration curve. A pooled plasma sample 
is analyzed in duplicate in each analytical batch to monitor the quality of the analyses. 
 
An extensive Food Frequency Questionnaire (FFQ) will be used to assess the 
participants’ habitual diet of the previous month. This extensive FFQ was validated for 
energy, macronutrients, and a number of vitamins [14-17] and administered online with 
the self-administered Dutch FFQ-tool™ [18], one week prior to the intervention. 
Participants indicate the frequency of consumed food items by selecting answers ranging 
from ‘not consumed’ to ‘7 days per week’ and portion sizes can be estimated using 
natural portions and commonly used household measures. The Dutch Food Composition 
Database (i.e., NEVO) will be used to calculate energy and nutrient content of foods. 
 
During the intervention, participants will report their food intake in a smartphone-based 
food diary on the first day of the intervention and the two following days. For this purpose, 
Traqq® will be used, an ecological momentary dietary assessment app [19]. In the 
morning, participants will receive a notification on their smartphone from Traqq®, inviting 
them to report their food intake during that day. Participants report their food intake by 

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clicking on the notification or opening the app. Next, participants can select consumed 
foods from an extensive food list based on the Dutch Food Composition Database. 
Subsequently, participants are prompted to report quantity and eating occasion, i.e., 
breakfast, lunch, dinner, snack. Quantity can be reported in household measures (e.g., 
spoon, cup), standard portion sizes (e.g., small, large) or amount in grams. Traqq® also 
contains a “My dishes” feature where participants can enter all ingredients of a recipe and 
the portion of the dish consumed, with yield and retention factors automatically being 
taken into account. The “My Dishes” feature can also be used to create frequently 
consumed product combinations (e.g., daily breakfast), which simplified reporting of these 
items and decreases (mis)calculation errors. 
 
8.4 Withdrawal of individual subjects 
Subjects can leave the study at any time for any reason if they wish to do so without any 
consequences. The investigator can decide to withdraw a subject from the study for 
urgent medical reasons. 
 
8.4.1 Specific criteria for withdrawal 
Not applicable.  
 
8.5 Replacement of individual subjects after withdrawal 
Only withdrawal of subjects before the first plasma determination (T0) will lead to 
replacement. After evaluation of results of 10 minus 3 subjects new participants will be 
reconsidered. 
 
8.6 Follow-up of subjects withdrawn from treatment 
Incomplete data will be used only with their consent, otherwise destroyed.  
 
8.7 Premature termination of the study 
There are no conditions defined for premature stopping the study. Data will be kept for 15 
years as described on the patient information leaflet. Blood samples will be kept for 2 
years to perform new analyses if needed. If, due to premature termination of the study, 
this will no longer be needed, the samples will be destroyed. 

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9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise subject 
health or safety. The sponsor will notify the accredited METC without undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending a further positive decision by the accredited METC. The investigator will take 
care that all subjects are kept informed.  
 
9.2 AEs, SAEs and SUSARs 
9.2.1 Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to the investigational product / 
trial procedure/ the experimental intervention. All adverse events reported 
spontaneously within a period of 1 week after consuming the soup or taking the food 
supplement (including blood drawing afterwards) by the subject or observed by the 
investigator or her staff will be recorded. 
9.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospitalisation or prolongation of existing inpatients’ hospitalisation; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; or 
- 
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 serious adverse 
event. 
 
The investigator will report all SAEs to the sponsor without undue delay after 
obtaining knowledge of the events, except for the following SAEs: there are no 
conditions to be considered based on swallowing a food supplement or eating 

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tomato soup or flebotomy that are likely resulting in a SAE within one week of 
consumption. 
 
The sponsor will report the SAEs through the web portal Onderzoeksportaal to the 
accredited METC 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 
serious adverse events. 
9.2.3 Suspected unexpected serious adverse reactions (SUSARs)  
Not applicable.  
 
9.3 Annual safety report 
Not applicable.  
 
9.4 Follow-up of adverse events 
All AEs will be followed until they have abated, 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. 
SAEs need to be reported till end of study within the Netherlands, as defined in the 
protocol.  
 
9.5 Data Safety Monitoring Board (DSMB) / Safety Committee 
Not applicable.  

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10. STATISTICAL ANALYSIS 
Lycopene plasma concentration curves over time will be depicted in tables and in graphs in 
absolute and relative (compared to T0) amounts. Trends in changes over time will be 
interpreted, if needed based on background information on confounding parameters. There 
are no statistical calculations planned. 
 
10.1 Primary study parameter(s) 
Lycopene plasma concentration curves over time will be depicted in tables and in graphs 
in absolute and relative (compared to T0) amounts. Trends in changes over time will be 
interpreted, if needed based on background information on confounding parameters. 
There are no statistical calculations planned. 
 
10.2 Secondary study parameter(s)  
Lycopene plasma concentration curves over time will be depicted in tables and in graphs 
in absolute and relative (compared to T0) amounts. Trends in changes over time will be 
interpreted, if needed based on background information on confounding parameters. 
There are no statistical calculations planned. 
 
FFQ and food diary data will be reported descriptively.  
 
10.3 Other study parameters 
Lycopene plasma concentration curves over time will be depicted in tables and in graphs 
in absolute and relative (compared to T0) amounts. Trends in changes over time will be 
interpreted, if needed based on background information on confounding parameters. 
There are no statistical calculations planned. 
 
10.4 Interim analysis 
No interim analysis is planned. 

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11. ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
The study will be conducted according to the principles of the Declaration of Helsinki and 
in accordance with the Medical Research Involving Human Subjects Act (WMO) and 
other relevant guidelines, regulations and Acts, such as the GDPR.  
 
11.2 Recruitment and consent 
After reading the flyer, interested volunteers can contact the principal investigator. 
Through email or mail the volunteer will receive a patient information leaflet. After careful 
consideration and time to ask questions, volunteers can decide to participate yes/no. 
Signed informed consent forms can be handed over or send back by mail to the principle 
investigator  or a member of the research team. The dedicated member of the research 
team will document and store the signed informed consent forms.  
 
11.3 Objection by minors or incapacitated subjects 
Not applicable.  
 
11.4 Benefits and risks assessment, group relatedness 
Consuming tomato soup, taking the dietary supplement and completing the food 
frequency questionnaire and the food diary do not pose any significant risks. The blood 
drawings carry a small risk of infection and may be somewhat painful.  
 
11.5 Compensation for injury 
The sponsor/investigator has a liability insurance which is in accordance with article 7 of 
the WMO. 
  
The sponsor (also) has an insurance which is in accordance with the legal requirements 
in the Netherlands (Article 7 WMO). This insurance provides cover for damage to 
research subjects through injury or death caused by the study. 
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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11.6 Incentives 
Participants will receive a remuneration of €50,- per visit cycle (cycle A, cycle B – or cycle 
B, cycle A), €100,- in total for participation in the study. Furthermore, participants will 
receive reimbursement of their travel costs. 

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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
After reading the flyer, interested volunteers can contact the principal investigator who will 
provide them with an informed consent form. Once men have returned a signed informed 
consent form they will be assigned a study number and be included in a study database 
(Castor). The designated researcher from Erasmus MC will have access to the 
identification list, all other databases shall only contain the study number assigned to an 
individual participant.  
 
In communication with the Wageningen University, who is responsible for determining the 
lycopene level, analysing the outcomes of the FFQ and the food diary, the study number 
of participants will be used. The researchers at Erasmus MC will receive a file with the 
outcomes of the analysed lycopene levels of all participants as well as the results of the 
FFQ and the food diary.  
 
Per blood draw, one tube of 6ml EDTA blood will be drawn from participants. Processed 
material will initially be stored at Erasmus MC. When all blood samples have been 
collected, half of the material per blood drawing will be send to the Wageningen 
University. The other half of the material will be stored at Erasmus MC as a back-up, and 
can be used in case something went wrong with transporting the material to the 
Wageningen University the first time.  
 
12.2 Monitoring and Quality Assurance  
Monitoring is part of a broader quality control system and an essential instrument for the 
quality assurance of research that is subject to WMO. It serves to verify that the rights 
and wellbeing of the research subjects are protected, that the study data are reported 
accurately and are fully verifiable in source documents, and that the conduct of the study 
is in accordance with the approved protocol/amendment (s), with ICH-GCP and with the 
relevant legal requirements.  
 
Monitoring will be performed in accordance with applicable law and regulation, the NFU 
Guideline ‘Quality assurance of research involving human subjects’ and the NFU risk 
classification (negligible risk/‘verwaarloosbaar risico’). The monitor will have and 
independent role in relation to the study, i.e. he/she will not have any involvement in the 
set-up of the study, the conduct of the study and the interpretation of the results. In 
accordance with the ICH-GCP guidelines, the monitor must have direct access to the 

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investigator’s study- and source documentation in order to verify compliance with the 
study protocol and check the data recorded in the CRFs for consistency.  
 
Monitoring is executed according to Erasmus MC policy standard operating procedures 
for monitoring. At the beginning of the study a study specific monitoring plan will be 
written and approved by the sponsor. The investigator agrees to cooperate with the 
monitor to ensure that any problems detected in the course of these monitoring visits are 
resolved. Prior to subject screening an initiation meeting will be held with all participating 
sites to discuss the protocol and procedures and to train the site. After data collection is 
complete, all eCRF queries have been answered and closed, a close out visit will be 
conducted. The investigator should be made aware that trial – and source documents for 
this study should be readily made available to regulatory authority or health authority 
inspectors.  
 
12.3 Amendments  
Amendments are changes made to the research after a favourable opinion by the 
accredited METC has been given. All substantial amendments will be notified to the 
METC and to the competent authority. 
 
Non-substantial amendments will not be notified to the accredited METC and the 
competent authority, but will be recorded and filed by the sponsor.  
 
12.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the 
accredited METC once a year. Information will be provided on the date of inclusion of the 
first subject, numbers of subjects included and numbers of subjects that have completed 
the trial, serious adverse events/ serious adverse reactions, other problems, and 
amendments.  
 
12.5 Temporary halt and (prematurely) end of study report 
The investigator/sponsor will notify the accredited METC of the end of the study within a 
period of 8 weeks. The end of the study is defined as the last participant’s last visit.  
 
The sponsor will notify the METC immediately of a temporary halt of the study, including 
the reason of such an action.  

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In case the study is ended prematurely, the sponsor will notify the accredited METC 
within 15 days, including the reasons for the premature termination. 
 
Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC.  
 
12.6 Public disclosure and publication policy 
Data are used to further conduct a previously started dietary study for prostate cancer 
(MEC-2017-389). 
 

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13. STRUCTURED RISK ANALYSIS  
13.1 Potential issues of concern 
There are no potential issues of concern.  
 
a. Level of knowledge about mechanism of action 
There is no adverse effect of lycopene known at a dose of 40 mg/day. 
 
b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism 
No adverse effects observed. 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo 
human cell material? 
Not in relation to life style compliance. 
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings 
- 
 
e. Analysis of potential effect 
- 
 
f. Pharmacokinetic considerations 
Previously used pharmacokinetic schedule for plasma biomarkers being used.  
 
g. Study population 
Healthy subjects. 
 
h. Interaction with other products 
Unknown. 
 
i. Predictability of effect 
The changes in plasma carotenoid concentrations are unpredictable, and are potentially 
influenced by prior food consumption. 
 
j. Can effects be managed? 
- 

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13.2 Synthesis 
Not applicable.  
 
 

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