Polyp prophylactic properties of polyacetylenes from carrots in patients with previous polypectomy—Px7 The study protocol of a multicentre binational randomised controlled trial

Abstract

Introduction

A large bowel cancer chemoprevention potential has been demonstrated by the consumption of carrots, which represent the major dietary source of polyacetylenes. Their interaction with cancer cells and enzyme systems of animals and humans has been systematically investigated over the last 15 years and has now been characterised as anti-inflammatory compounds with antineoplastic effect. Our objective is to investigate whether selected carrot species with a high content of the polyacetylenes falcarinol (FaOH) and falcarindiol (FaDOH) prevent neoplastic transformation and growth in humans, without side effects.

Methods and analysis

We will conduct a multicentre prospective binational (Denmark and Sweden) randomised controlled trial, with the aim to test the clinical effects of adjuvant treatment with carrot juice in patients who had an excision of high-risk colon adenomas. Patients from six centres will be randomised to receive either anti-inflammatory juice made of carrots high in FaOH and FaDOH or placebo. We will compare the proportion of participants with recurrent adenoma and mean size of them, found in the 1-year follow-up colonoscopy between the two randomised groups.

Ethics and dissemination

Informed written consent will be obtained from all participants before randomisation. The study was approved by the regional ethics committee in Denmark (ref. S-20230072) and Sweden (ref. 2024-04732-01). After completion of the trial, we plan to publish two articles in high-impact journals: one article on primary and secondary outcomes, respectively.

Trial registration number

NCT06335420.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• Patient adherence and compliance can be problematic.

• Data from colonoscopy reports can vary in quality.

• This trial is developed in different expert centres with a powerful combined patient capacity who attend colonoscopies.

• The study is a prospective binational randomised clinical human trial.

Introduction

Studies have shown that a diet based on vegetables and fruits reduces the risk of neoplasia.^{1 2} A development in the field of dietary prevention of cancer is the emerging focus on the observed associations between raw carrot intake and a lower risk of colorectal neoplasia. The prophylactic association is attributed to the polyacetylenes falcarinol (FaOH) and falcarindiol (FaDOH).³,¹⁴

FaOH-type polyacetylenes have shown multifaceted bioactivities, including anti-inflammatory,¹⁵,¹⁷ antiplatelet-aggregatory,¹⁶,²⁰ cytotoxic¹⁰,¹⁴¹⁶ and antineoplastic.^{11 26 27} Polyacetylenes are similar in action to aspirin (non-selective COX inhibitor) and pharmaceutical COX-2 inhibitors (celecoxib), which are known to exhibit a prophylactic effect against the formation of colon cancer in humans. These COX-2 inhibitors have been suggested as primary chemoprevention and as adjuvant treatment for patients with colorectal cancer (CRC) to prevent recurrence, but are not in general use because of adverse reactions such as upper gastrointestinal ulcers and cardiac failure.²⁸,³⁰

Research efforts have therefore been made to identify a COX-mediated antineoplastic effect by less harmful naturally bioactive compounds. Both raw carrots and purified polyacetylenes have been investigated in numerous in vitro systems on human cancer cell lines, genomics and microbiomics studies in cohort studies and healthy volunteers.^{34 12},^{18 22}

Furthermore, multiple studies of the polyacetylenes have been done over the last 15 years, and we managed to determine the polyacetylenes concentration variations between cultivars; purify and achieve a stable form for FaOH and FaDOH; measure and correlate the raw carrots juice intake with polyacetylenes serum levels; confirm through in vitro studies the polyacetylenes inhibition of the growth of human cancer cells; demonstrate in rat studies a statistically significant reduction of neoplastic developments in the forms of both aberrant crypt foci and adenomas and a reduced growth velocity of these neoplasms. Phases I–III from this study group have proven a reduction of early neoplasms in order of 35%, increasing to 80% for advanced neoplasms (large adenomas). Furthermore, we have confirmed a dose–response in the determination of the lowest therapeutic level necessary to achieve a maximal antineoplastic effect in rats.^{34 27 31},³⁵

Evidence of the adenoma preventive effects of polyacetylenes from carrots has reached a level where prospective human trials are needed. Data from preclinical trials, meta-analysis and cohort studies demonstrated their anticancer properties and did not show any significant toxic side effects, thus highlighting their potential use as adjuvant treatment in the prevention of cancer.^{3 4} The objective of this study is to investigate the possible inhibitory effects from carrot juice on the recurrence of excised adenomas and on the formation of metachronous adenomas in patients enrolled for follow-up after resection of high-risk colorectal adenomas.

Methods and analysis

Study design

Our study is a prospective double-blinded randomised controlled trial carried out in seven centres: one Danish (Department of Surgery, Odense University Hospital) and six Swedish (Södersjukhuset, Stockholm; Ersta Hospital, Stockholm; Karolinska University Hospital, Stockholm; Uppsala University Hospital, Uppsala; Sahlgrenska University Hospital, Goteborg; Capio St. Gorans Hospital, Stockholm). We will randomise patients after endoscopic resection of high-risk adenoma (≥2 cm) between two parallel arms—an intervention arm (100 mL active carrot juice daily) and a control group (100 mL placebo juice daily)—in a 1:1 allocation ratio for 1 year. We will follow-up the patients’ endoscopic results regarding recurrence or new polyps at the standard routine follow-up post-polypectomy colonoscopy. The project is structured in two work packages (WPs):

1. The clinical trial testing the effect on adenoma recurrence (WP also includes cultivation and quality control of carrots and juice production; quantification of FaOH and FaDOH in the carrot and the placebo juice and the randomised trial).

2. The assessment of patient compliance and ways to improve it (Umeå Institute of Design, Umeå University (UMU)).

Hypothesis

We hypothesise that 1-year daily intake of carrot juice high in polyacetylenes will reduce the number and growth rate (size) of recurrences after primary resection of high-risk adenomas in the large bowel.

Objectives

Our primary objective is to compare the follow-up colonoscopy results (recurrence or not) after 1 year for patients who underwent a resection of high-risk colorectal adenoma(s) and who subsequently participated in 1 year of daily consumption of 100 mL carrot juice or placebo.

Our secondary objectives are to evaluate

• The number of polyps multiplied by mean diameter of adenomas per patient.

• The number of polyps with low, medium and high degree of dysplasia and cancer.

• The compliance rate and adverse effects.

• The relation between polyp number and size, and the level of compliance.

• The user preference of carrot intake across patient profiles and carrot products.

Primary and secondary outcomes will be investigated using colonoscopy and pathology reports, questionnaires (online supplemental files 4 and 5), diaries (online supplemental file 2) and surveys.

The Umeå University team will design, redesign and deploy a variety of patient diaries so that each patient will encounter three variations in 1 year of participation. We will conduct surveys seeking feedback and information on compliance. Further, we can optionally offer recipes and food samples developed by chefs at the UMU Department of Food, Nutrition and Culinary Arts, to assist patients in diversifying their consumption of the juice and facilitate adherence and compliance.

Inclusion and randomisation

Patients assessed through colonoscopy with a high-risk adenoma who underwent a resection beginning in June 2024 in Denmark and September 2025 in Sweden will be informed about the study by their colonoscopist or project nurse (online supplemental file 1). Patients who are eligible and give written informed consent (online supplemental file 3) to participate will be enrolled. Otherwise, they will follow the standard national guidelines as usual. The randomisation is stratified by inclusion centre, each having its own block of 50/50 distribution between active and placebo arm. Each unit will receive blocks of juice marked either A or B for the active or the placebo juice. The randomisation key is generated with the software Research Electronic Data Capture (REDCap consortium, Vanderbilt, the Netherlands). The juice producer holds the key for supplementary delivery to each participant centre every 6 months.

• Inclusion criteria

  1. Any type of resection for adenomas larger than 20 mm in size (endoscopically evaluated) or endoscopically resected T1 cancers.

  2. Acceptance of the treatment and follow-up programme.

  3. Capability to understand and follow the instructions.

• Exclusion criteria

  1. Patients allocated to ‘high-risk’ group because of many small polyps (>4).

  2. Pregnancy.

  3. Known relevant allergies.

  4. Chronic consumption of COX inhibitors (diclofenac, ibuprofen, indometacin, ketoprofen, ketorolac, naproxen, piroxicam, celecoxib, aspirin) or immunosuppressive drugs (methotrexate, glucocorticoids or azathioprine).

  5. Patients with different neoplastic diseases under treatment (chemotherapy/radiotherapy/immunotherapy).

  6. Severe comorbidity New York Heart Association classes III–IV.

  7. Lynch syndrome.

Patient flow

Most high-risk adenomas will be identified at the initial colonoscopy and referred to a specialised centre for resection (Endoscopic Mucosal Resection - EMR/Endoscopic Submucosal Dissection - ESD) (figure 1). It is expected that only a few will be resected at the first colonoscopy. In both cases, the patient is informed about the study by the colonoscopist or a project nurse after the initial colonoscopy. If they give written consent for participation, at the inclusion visit they will be handed 2 months juice (8 L), a compliance diary, a calendar, participant instructions and a questionnaire.

Figure 1. Patient flowchart. EMR - Endoscopic Mucosal Resection; ESD - Endoscopic Submucosal Dissection.

The patient will collect 8.0 L of fresh juice every 2 months at a visit at the regional clinic, where they will be interviewed for compliance, intake of additional carrots/carrot containing products and possible adverse effects. Standardised questionnaires (online supplemental file 5; modified Treatment Adherence Questionnaire^{36 37}) will be filled and a compliance diary (online supplemental file 4) will be collected. Another viable option for some centres will be the delivery of the entire quantity of juice and calendars/diaries, and the follow-up will be made at monthly phone calls and through online surveys. Guidelines ensuring unanimous monthly follow-up procedures between the centres will be put in place.

All patients will be invited to undergo colonoscopy after 1 year from the resection. In addition, for the piecemeal resections we will conduct a follow-up colonoscopy after 6 months for control of the resection site. The endoscopists performing the follow-up evaluation and the anatomopathology doctors are blinded and so will not be aware of which arm the patients belong to. Information about recurrence, the number of polyps, polyp size and characterisation will be registered in the database. Polyp size will be measured with the aid of appropriate instruments, like biopsy forceps and snare for inclusion criteria and for the outcome we will use the pathology report for a standardised unified measurement. After completing the study period of 1 year, all patients will continue following the local guidelines for follow-up.

Full compliance is defined as consumption of >75% of the juice delivered (for dosage considerations, see below). Consumption data for all patients will be kept for compliance analysis.

Quality assessment

Adverse events, complications and investigation quality will be monitored by the data safety monitoring board (including the trial manager) continuously during the inclusion of participants to ensure patient safety. Patient compliance will be carefully monitored monthly, at clinic visits or phone calls. In the case of serious complications or adverse events, the trial manager will be informed immediately for consideration of stopping the trial.

Data collection and management

Data will be retrieved from patient medical records, colonoscopy forms, questionnaires (online supplemental files 4 and 5), diaries/calendars (online supplemental file 2) and surveys completed by the patients, and will be entered into a REDCap database customised for this trial by dedicated research staff. We will collect data from patient files regarding general identification characteristics, allergies, former colonoscopies with polypectomies and pathology report. We will also collect quality and technical details of both colonoscopies from colonoscopy forms entered on site by the endoscopist. Data obtained in Sweden will be entered into the Danish REDCap database under the data-sharing agreement regulations.

All data will be stored in secure databases available only to the researchers involved in the study. To promote data entry quality, questionnaire answers will be imported directly into the database. The database will specify validation formats and intervals for data that requires manual entry. All data will be handled according to the General Data Protection Regulation (GDPR) and Data Protection Act. Data will be transferred to the National Archives after publication of the results after 3 years or, if scientifically useful, prolongation until 5 years.

Statistical analysis

The primary endpoint, recurrence of adenoma after 1 year, will be estimated as proportions and compared between the intervention and placebo group using the χ² test. Categorical secondary outcomes will be estimated as proportions and the randomisation arms of the study will be compared using the χ² test. Continuous secondary outcomes will be tested using non-parametric tests.

Sample size

At the 1-year follow-up after resection of high-risk polyps, patients have a 40% risk of having new polyp/polyps at the colonoscopy.^{38 39} An absolute difference of at least 15% would be of clinical interest. The following was assumed:

1. The recurrence rate in the control arm is 40%.

2. The acceptance rate to participate is 55%.

3. The exclusion rate is 20%.

4. The total dropout rate is expected to be 20%.

Based on a two-sided χ² test, a total of 304 participants, with 152 in each arm, are needed to achieve 80% power at the 5% level of significance to detect an absolute difference of 15% or higher in recurrence rate at 1 year. To account for the assumed dropout, 380 participants have to be randomised. To further account for the assumed acceptance and exclusion rates, 863 potential participants have to be invited. However, we do not have solid information on acceptance rate, exclusion rates and dropouts. Furthermore, we expect the recurrence rate to decline due to new treatment algorithms. To also take these uncertainties into account, a total of 1000 possible candidates will be invited, with the goal of including 400 patients in the study. The combined capacity of the included centres is 1600 eligible patients per year. Therefore, we estimate that the enrolment can be completed within 12 months.

The planned start date is June 2024 for the centre in Denmark and September 2025 for the centres in Sweden. Taking into account all centres, the estimated end date of the study is December 2027.

We used the Standard Protocol Items: Recommendations for Interventional Trials checklist when writing our report.⁴⁰

Patient and public involvement

The study and content of the trial protocol were discussed with the patient representative group associated with our research unit. Corrections and clarifications were made according to the received feedback.

Product: carrot juice and placebo

We now have the knowledge, through our studies, on how to obtain a stable purified form of FaOH and FaDOH (with established conservation methods and acceptable conditions of storage).

Through the development of measuring FaOH concentration (in rats, human serum or carrot juice), the pharmacokinetics and the correlation between intake and serum levels, we have estimated a ‘dose needed’ to reach the range where the in vitro data indicate an antineoplastic effect. To reach a serum level of 0.01 µM, it requires approximately 200–400 g of carrots from selected cultivars daily corresponding to 100 mL of carrot juice (or 5 mg polyacetylenes).^{3 4 15 16 31}

These compounds found in carrots vary in concentration between different cultivars, and it has been suggested that the highest level is found in the F1 hybrid cross cultivars (purple, ‘Night Bird’; yellow, ‘Yellowstone’) rather than in the modern, sweeter commercial ones.^{3 4 15 16 18}

The cultivation of ‘Yellowstone’ carrots will be conducted by DanRoots A/S (DanRoots, Organic Vegetables, Bjerringbro, Denmark). For each kilogram of carrots, 720 mL of juice can be extracted. The juice is produced and taste-corrected by Orskov Food, Denmark, in collaboration with Naturfrisk (Naturfrisk Group/Ørbæk Brewery, Ørbæk, Denmark). Natural citrus juice is used for conservation.

The placebo juice formula is produced by matching approximately the active juice energy and micronutrients and after assuring there are no polyacetylenes traces in the juice and after testing different combinations of natural flavours for taste correction in order to prevent enabling differentiation between carrot juice and placebo.

Analysis of carrot juice for FaOH and FaDOH content will be done randomly for 10 samples leaving the factory and 10 samples collected after 30 days of storage at participants homes for each batch. All laboratory investigations are performed at the Department of Biochemistry, Svendborg Hospital.

Discussion

Despite multiple studies and improvements in screening programmes, modifiable risk factors and treatment options, CRC is still on the worldwide podium among all types of neoplasia regarding incidence and mortality rates.³⁵

Diet, as we know already, plays an important role in the prevention of the neoplastic disease.¹,⁹ A review of multiple studies published in this field, with a particular interest in carrots and especially their active compounds (FaOH and FaDOH), led to the trials we performed in vivo. The rat studies proved that this dietary supplement decreased the number of neoplastic lesions and the growth rate of polyps, suggesting a preventive effect on CRC development in azoxymethane-induced rats.³,^{727 32}

The serum level of polyacetylenes obtained in the preclinical trial can be achieved with reasonable daily intake of carrots. It is not unlikely to assume that this intake of carrot cultivars containing high concentrations of FaOH and FaDOH may expose the colon to relatively higher concentrations of polyacetylenes than in the preclinical trial.³,^{727 32}

Considering the amount of evidence piled up in this direction, we think it is time to conduct a human trial to investigate the prophylactic properties of polyacetylenes in carrot juice for patients with a resection of high-risk adenoma or R0 resected T1 cancers.

We are also aware that Yellowstone cultivars contain lutein, a carotenoid just like beta-carotene from the orange cultivars. Multiple studies have shown the potential anti-inflammatory and antineoplastic activities for these compounds.⁴¹,⁴⁴ They may exert a synergistic action, thus enhancing the potential positive action of polyacetylenes. Favourite results from our study could encourage researching this hypothesis deeper.

We aim to evaluate patient compliance and develop new ways of improving it, taking into account that procedure acceptability is critical in further implementation. We recognise the importance of food in people’s lives, and the difficulties people have in changing how they eat, even when the change is proven to be beneficial.⁴⁵ We approach the study as a complex challenge, taking into account patients changing tastes, as well as a wide diversity in their personal habits, preferences, learning styles and lived experiences.

It is our judgement that the potential gained from this may offer an important breakthrough in CRC prevention.

Strengths and limitations

We consider that one limitation of the study might be patient adherence and compliance, which we will try to address through the above-described design processes. Additionally, we will follow the patients monthly—a short standardised discussion at the outpatient clinic when they come to pick up the juice and then after 1 month via a phone call. To homogenise colonoscopy reports, we have produced a template to assist and standardise the endoscopist report. A strength of this trial is that it is developed in different expert centres with a powerful combined patient capacity who attend colonoscopies. All centres are dedicated to and hold regional referral for advanced resection of large and complex adenomas. To our knowledge, this is the first prospective randomised clinical human trial on the prophylactic properties of polyacetylenes in patients with previous polypectomies.

It is assumed that 100 mL of juice or inactive placebo will not cause major side effects. Carrot juice produced on different carrot cultivars is commercially available, as are all ingredients of the placebo. A 1-year use of the juice might add an extra calorie intake that might cause a minor weight gain, which is considered to be without health consequences. Patients with diabetes are instructed about paying extra attention at monitoring glycaemia because of the sugar content in the juice/placebo.

Ethics and dissemination

This study was approved by the regional ethics committee in Denmark (ref. S-20230072) and Sweden (ref. 2024-04732-01) and the Danish Data Protection Agency. The study will adhere to the GDPR and the Data Protection Act. The study will be conducted in accordance with the Declaration of Helsinki. The study is registered in clinicaltrials.gov (NCT06335420). The results will be published regardless of them being positive, negative or inconclusive. The main paper and all spinoff papers will be published in international open-access journals. Vancouver rules will be followed when deciding authors.

Participation is voluntary with no economic compensation, and patients are informed orally and in writing according to the information accepted by the ethics committee. They will be given at least 48 hours to consider participation. Participant consent will be given in writing using the template approved by the ethical and legal committees (online supplemental file 3). The deviation from standard follow-up periods is believed to cause no or very little additional risk to patients. Both Swedish and Danish patients are investigated earlier than the current guidelines for surveillance post-polypectomy state.