Randomized Window of Opportunity Trial of Anthocyanin Extract and Phospholipid Curcumin in Subjects With Colorectal Adenoma
Phase: NA
Conditions: Colorectal Adenoma, Risk Reduction
Sponsor: Ente Ospedaliero Ospedali Galliera

MiRACol Protocol – Version 4 of 01 October 2016 
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Blueberry Extract and Turmeric Extract 
 
EO Galliera Hospital of Genoa 
(L.833/1978, art. 41; Legislative Decree n.517/1993 art. 4 c. 12) 

Complex Structure of Medical Oncology 
Tel 010 5634508 - fax 010 57481090 
andrea.decensi@galliera.it 

PHASE II TRIAL OF BIOMARKER MODULATION IN 
SUBJECTS WITH COLON ADENOMATOSUS POLYP WITH 
BLUEBERRY EXTRACT AND TURMERIC EXTRACT 
MiRACol Study 
ClinicalTrials.gov Identifier : NCT01948661 
Sponsor 
EO Galliera Hospital 
 
Central Data Management 
SC Medical Oncology 
Clinical Research Coordination in Oncology 
EO Galliera Hospital 
 
Statistical Analysis 
Matthew Points 
Scientific Coordinator Office 
EO Galliera Hospitals 
 
Project Manager: 
 Andrea De Censi, MD 
Director of Medical Oncology 
EO Galliera Hospital, 
Cappuccine Walls 14, 16128, Genoa, Italy 
Tel 010 5634501 - fax 01057481407 
andrea.decensi@galliera.it                          
 
 
 

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INDEX 
1.0  LIST OF INVESTIGATORS OF THE COORDINATING CENTER ................................................... 4 
2.0  LIST OF PARTICIPATING CENTERS AND THEIR CO-INVESTIGATORS  ...................................... 4 
3.0  COLLABORATIONS  ................................................................................................................. 4 
4.0 STUDY SYNOPSIS  ....................................................................................................................... 5 
5.0  INTRODUCTION AND RATIONALE  ......................................................................................... 7 
6.0  AGENTS IN STUDIO  ................................................................................................................ 9 
6.1  Curcumin Extract  .............................................................................................................. 9 
6.2  Blueberry extract  ............................................................................................................ 11 
7.0  STUDY DESIGN  ..................................................................................................................... 12 
8.0  STUDY POPULATION  ........................................................................................................... 13 
8.1  Inclusion criteria  ............................................................................................................. 13 
8.2  Exclusion criteria  ............................................................................................................. 13 
9.0  GOALS  .................................................................................................................................. 14 
9.1  Primary objective  ........................................................................................................... 14 
9.2  Secondary objectives  ...................................................................................................... 14 
10.0  TREATMENT REGIME  .......................................................................................................... 15 
10.1  Name of experimental treatment and formulation  ....................................................... 15 
10.2  Dosage groups, duration of exposure and dose selection  ............................................. 15 
10.3  Supply of supplements, packaging, labelling  ................................................................. 16 
10.4  Disposal of residual supplements  ................................................................................... 17 
10.5  Treatment assignment method  ...................................................................................... 17 
11.0  STUDY PROCEDURES  ........................................................................................................... 18 
11.1  Duration of the study  ..................................................................................................... 18 
11.2  Screening  ........................................................................................................................ 18 
11.3  Baseline Visit  .................................................................................................................. 18 
11.4  Final Visit (after 4-6 weeks of treatment)  ...................................................................... 19 
11.5  Follow-up (after 1 year from the end of treatment)  ...................................................... 19 
11.6  Compliance  ..................................................................................................................... 19 
11.7  Collection and management of biological material  ....................................................... 20 
11.8  Concomitant therapies  ................................................................................................... 20 
11.9  
Food diary  .................................................................................................................. 21 
12.0  FLOW CHART  ....................................................................................................................... 22 
13.0  ETHICAL ASPECTS  ................................................................................................................ 23 
13.1  Informed consent  ........................................................................................................... 23 
13.2  Patient safety  .................................................................................................................. 23 
14.0  DATA COLLECTION AND MONITORING PLAN  ..................................................................... 25 
14.1  Data collection and monitoring procedures  ................................................................... 25 
14.2  Quality Assurance  ........................................................................................................... 25 
14.3  Original documents  ........................................................................................................ 25 
15.0  END OF STUDY CRITERIA  ..................................................................................................... 27 
15.1  Premature discontinuation of a subject  ......................................................................... 27 
16.0  TOXICITY MONITORING AND MANAGEMENT OF ADVERSE EVENTS  .................................. 27 
16.1  Definitions Adverse Events  ............................................................................................. 27 
16.2  Adverse Event Reporting (AE) Serious Adverse Events (SAE)  ......................................... 27 
 
17.0  STATISTICAL METHODS  ....................................................................................................... 29 

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17.1  Statistical design and sample calculation  ....................................................................... 29 
17.2  Interim analysis  ............................................................................................................... 29 
17.3  Statistical analysis methods  ............................................................................................ 29 
 
Acceptance of the study protocol / Investigator's responsibilities  .............................................. 31 
 
BIBLIOGRAPHY  .............................................................................................................................. 32 
 
 
 

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1.0 
LIST OF COORDINATING CENTER INVESTIGATORS 
Andrea De Censi 
Project coordinator 
Director of Medical Oncology Unit, EO Galliera Hospital, Genoa 
Gianni Coccia 
Sub-Investigator 
Director of Gastroenterology Unit, EO Galliera Hospital, Genoa 
Roberto Bandelloni 
Lab Researcher 
Director, Pathological Anatomy Unit, EO Galliera Hospital, Genoa 
Marco Mori 
Lab Researcher 
Director Lab Analysis Unit, EO Galliera Hospital, Genoa 
Mariangela Rutigliani 
Laboratory Researcher 
Pathological Anatomy Unit, EO Galliera Hospital, Genoa 
Carlotta Defferrari 
Clinical Researcher 
Medical Oncology Unit, EO Galliera Hospital, Genoa 
Matteo Puntoni 
Biostatistician 
Office of the Scientific Coordinator, EO Galliera Hospital, Genoa 
Marilena Petrera 
Study Coordinator  
Medical Oncology Unit, EO Galliera Hospital, Genoa 
Silvia Caviglia 
Administrative support and regulatory practices 
Medical Oncology Unit, EO Galliera Hospital, Genoa 
2.0 
 LIST OF PARTICIPATING CENTERS AND THEIR CO-INVESTIGATORS 
Gianni Coccia 
Principal Investigator (PI) 
Director of Gastroenterology Unit, EO Galliera Hospital, Genoa 
Bernardo Bonanni 
Principal Investigator (PI) 
Division of Prevention and Oncological Genetics, European 
Institute of Oncology, Milan 
Sebastiano Saccomanno 
Principal Investigator (PI) 
Gastroenterology Unit, Villa Scassi Hospital, ASL3 Genovese 
Marina De Mattheis 
Principal Investigator (PI) 
Emergency Gastroenterology and Digestive Endoscopy Unit, 
Hospital of Lavagna, ASL 4 Chiavari 
3.0 
COLLABORATIONS 
Paolo Morazzoni 
Scientific Director, 
Indena Spa, Milan 
Giovanna Petrangolini 
Research Scientist 
Scientific Director, Indena Spa, Milan 
Antonella Riva 
Senior Research Scientist 
Scientific Director, Indena Spa, Milan 
Massimo Ronchi 
Head of the Formulation Development Laboratory 
Indena Spa, Milan 
Elisabetta Frattini 
Research Scientist 
Formulation Development Laboratory, Indena Spa, Milan 
Harriet Johansson 
Division of Prevention and Oncological Genetics, European 
Institute of Oncology, Milan 
Francesca Mattioli 
Di.MI Section of Clinical Pharmacology and Toxicology, 
University of Genoa 
 

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4.0 
STUDY SYNOPSIS 
 
STUDY TITLE: 
Phase II Study of Biomarker Modulation in Subjects with Colonic Adenomatous Polyp with 
Bilberry Extract and Turmeric Extract - MiRACol Study 
 
PRINCIPAL INVESTIGATOR: Andrea De Censi 
 
COORDINATION CENTER: EO Galliera Hospital, Medical Oncology Unit, Mura delle Cappuccine, 
14 - 16128 Genoa, Italy                   
Agents 
Formulation 
Dose 
Regimen 
Route 
Blueberry extract 
(Mirtoselect®) 
 
TABLETS 
from 500 mg 
 
1 g/day 
(500mgx2/day) 
 
1 tablet 2 times a day 
at the same time 
(1 morning tablet + 1 
evening tablet) 
Oral 
Turmeric extract 
(Meriva®) 
TABLETS 
from 500 mg 
1 g/day 
(500mgx2/day) 
 
1 tablet 2 times a day 
at the same time 
(1 morning tablet + 1 
evening tablet) 
Oral 
PURPOSE OF THE RESEARCH : 
The study aims to verify the effect of the association of food supplements, blueberry extract 
and turmeric extract versus placebo, administered for 4 weeks, on some important surrogate 
biomarkers of colorectal cancer in subjects with colorectal adenomatous polyps candidates for 
surgical resection. 
STUDY DESIGN : 
 

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STUDY POPULATION : 
Subjects with one or more colorectal adenomatous polyps (with diameter ≥ 1 cm) aged 18 and 
85 years, ECOG performance status ≤ 1. 
Subjects with hyperplastic polyps and flat and serrated adenomas, with previous colorectal 
carcinoma, or with carcinomatous tissue in the adenoma will be excluded. 
NUMBER OF SUBJECTS : 100 divided into 2 treatment groups. 
TREATMENT :  
Group 1: Placebo A (Mirtoselect® 0 g) + Placebo B (Meriva® 0 g) / day  
Group 2: Mirtoselect® 0.5 g x2/day + Meriva® 0.5 g x2 /day 
TREATMENT DURATION : 4 weeks until surgical resection 
PRIMARY OBJECTIVE 
To evaluate the activity of the association of blueberry extract and turmeric extract on β-
catenin expression after 4 weeks of treatment between experimental and placebo arm. 
SECONDARY OBJECTIVES 
 Evaluate the tolerability of the combination of blueberry extract and turmeric extract 
and study the plasma and tissue absorption of the active ingredients (anthocyanosides 
and curcumin); 
 Evaluate the effects of treatment in the adjacent adenomatous and healthy tissue 
(perilesional) by evaluating the IHC expression of Nuclear Factor-Kβ (NFKβ), Ki-67, P53 
apoptosis, tissue expression of the EGFRs system; tissue gene expression; 
 Evaluate the modulation of some circulating growth factors in the collected serum: IGF 
system (IGF-I, IGFBP-3 and IGF-I/IGFBP-3), EGFR, inflammation markers (u-PCR). 
INCLUSION CRITERIA 
1 Age > 18 years and ≤ 85 years 
2 Written informed consent 
3 Presence of one or more adenomatous polyps in the colon-rectum (with diameter ≥ 1 cm) 
4 ECOG performance status ≤ 1. 
EXCLUSION CRITERIA 
1 Presence of hyperplastic polyps and flat and serrated adenomas; 
2 Previous colorectal cancer; 
3 Presence of carcinomatous tissue in the adenoma; 
4 Taking experimental drugs or dietary supplements containing blueberry or curcumin in the 
15 days prior to enrollment; 
5  Any other factor that, at the discretion of the investigator, may contraindicate enrollment in 
the study. 
START OF STUDY 
DURATION OF ENROLLMENT 
END OF STUDY 
Date of enrollment of the first 
subject 
48 months 
Last follow-up visit date of 
the last enrolled patient 
 
 

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5.0  INTRODUCTION AND RATIONALE 
In Western countries, the colorectal is the second most common site of tumor occurrence. 
Colorectal cancer (CRC) is the second most common malignant tumor in terms of incidence and 
mortality (1) and represents a significant burden in terms of social and economic costs. 
CRC is a multifactorial disease with approximately 75% of sporadic cases. The main risk factors 
are age, family history, the presence of colonic adenomatous polyps (PA), diffuse polyposis and 
the presence of particular hereditary conditions (HNPCC, FAP). 
A growing number of studies indicate that inflammatory activation of the tumor 
microenvironment is one of the primary causes of CRC progression. 
The efficacy of NSAIDs (nonsteroidal anti-inflammatory drugs) in preventing CRC suggests the 
concept that inflammation may play a crucial role in the development of these tumors (2-3). In 
fact, following the inflammatory process, several reactive oxygen species (ROS) are produced 
and it is now known that oxidative stress can significantly contribute to the development of 
cancer, particularly CRC (4-6). 
The colon mucosa represents the body surface most exposed to bacterial antigens, derived 
from commensal, opportunistic, or frankly pathogenic strains. Consequently, this mucosa 
presents a particular reactivity that manifests itself with evident inflammatory and immune 
reactions. In the colon, along with genetic mutations that constitute elements of initiation of 
carcinogenesis (mutation affecting APC, k-RAS, P53 and others) a continuous promotional effect 
mediated by inflammatory elements is observed. The limit to the use of aspirin or other NSAIDs 
(non-steroidal anti-inflammatory drugs) in the prevention of CRC is linked to their possible 
chronic toxicity especially at the gastrointestinal level. 
Adenomatous polyps (AP) are considered high-risk precancerous lesions: colon cancer arises in 
an adenomatous polyp in approximately 90% of cases, with a sequence that leads from AP to 
CRC in approximately 10 years (7) and is for these reasons considered a reliable intermediate 
(surrogate) marker of risk and efficacy for clinical trials of pharmacoprevention (8). 
Several case-control studies and clinical trials suggest that AP removal decreases the incidence 
of CRC; it is therefore logical to think that the regression or elimination of AP through 
pharmacoprevention strategies could reduce the incidence of CRC (9). 
The elucidation of the mechanisms inducing oxidative stress and their possible involvement in 
the development and progression of cancer represent an interesting area of study in the 
pharmacoprevention of CRC. 

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Pharmacoprevention aims to prevent the onset of cancer by using natural or synthetic drugs 
that inhibit carcinogenesis. 
Approximately 70% of the anti-cancer drugs available today are of plant origin. 
Although the main interest is for those that exert frankly antiblastic effects (e.g. vinca alkaloids, 
camptothecin and taxanes), many others are tested for their anti-inflammatory and/or 
bacteriostatic properties (e.g. salicylates, curcumin, epigallocatechin-gallate, quercetin, 
lycopene, resveratrol etc.), and it is precisely among the latter that we find the most interesting 
candidates for pharmacoprevention strategies. 
β-Catenin plays important regulatory roles, in particular it is a key element in the Wnt signaling 
pathway where it acts as a co-transcriptional activator of target genes in the cell nucleus. The 
hyperactivation of Wnt signaling essentially influenced by β-catenin functions, is a typical 
feature of colon cancer and many other tumors; furthermore β-catenin has a crucial role in 
APC-mediated colon carcinogenesis (10). 
The study aims to verify whether the association of 2 food supplements, in the form of 
standardized botanical extracts, with demonstrated modulatory properties in the process of 
colon cancer progression, is able to decrease the activation of β-catenin in AP. This is based on 
the assumption that the hyperactivation of the β-catenin signal is considered one of the earliest 
events in the sequence of genetic changes that favor the development of colon cancer (11). 
 
 

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6.0  AGENTS IN STUDIO 
6.1 Curcumin Extract 
Curcumin is the main biologically active component of Indian Saffron (Curcuma longa L.) 
belonging to the ginger family: Zingiberaceae. The root and rhizome (part of the underground 
stem) of the plant Curcuma longa L. are crushed and powdered. The powder thus obtained is 
used throughout the world as an ingredient in curry and curcumin is the polyphenol that 
characterizes its yellow color (12). 
Curcumin was introduced into European medicine by the Dutch, who imported it from their 
possessions in the East Indies. In its countries of origin, among the populations of India and 
southern China, it is widely used not only as a condiment and colorant, but also as a remedy for 
liver disease and as a diuretic. 
Curcumin has been tested for its pharmacological properties in thousands of studies, hundreds 
of which have been published in the last 4 years (13). These studies have confirmed its 
remarkable anticancer, anti-inflammatory and antioxidant properties. Unlike many other 
antioxidants, curcumin is able not only to prevent the formation of free radicals but also to 
neutralize existing free radicals. Therefore, it is considered an effective bio-protector due to this 
dual activity (13). 
As for its antitumor properties, curcumin has been shown to be potentially useful for the 
treatment of colon, prostate, lung, breast and pancreatic tumors. 
In the colon, curcumin has been shown to reduce the occurrence of aberrant foci in a rat model 
of colorectal carcinogenesis (14). 
The mechanisms of action for the antitumor activity of curcumin can be roughly summarized as 
follows: 
a) inhibition of tumor cell proliferation; 
b) induction of apoptosis; 
c) inhibition of the transformation of cells from normal to tumor; 
d) inhibition of invasiveness and metastasis; 
e) suppression of inflammation. 
Regarding the use of curcumin in clinical trials in oncology, the results, while promising, have 
shown, however, that the low bioavailability of this natural product remains a serious obstacle 
to its therapeutic use, as it involves the use of very high dosages with poor patient compliance. 

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In a phase I trial of 15 patients with advanced colon cancer and refractory to standard therapies 
(15), curcumin (0.45-3.6 g/die/4 months) reduced patients' plasma PGE2 concentration by 50% 
at one hour after treatment in the absence of side effects, the authors indicated the 3.6 g dose 
as the standard for a possible phase II trial. In a previous estimation, doses of 8 g/day (3 months) 
had been reached without significant toxicity (16). 
Curcumin was tested in association with quercetin for the chemoprevention of familial 
adenomatous polyposis in 5 patients (curcumin 480 mg and quercetin 20 mg/3 times daily/6 
months) demonstrating a significant decrease in the number (60.4%) and size (50.9%) of polyps 
(17). 
Curcumin has been tested in a phase II trial against advanced pancreatic cancer (8 g/day/8 
weeks) demonstrating once again low toxicity and demonstrating the ability to reduce some 
inflammatory markers in the PBMC of patients (NF-kB, COX-2, STAT3), 2 out of 21 evaluable 
patients also demonstrated a clinical response (18). 
A very interesting feature of curcumin is its ability to reduce EGFR expression in cells in vitro 
(19), however no one has yet evaluated whether this also occurs in vivo. 
Recently Carroll et al demonstrated a significant reduction in focal aberrant crypts (AFCs) in 44 
subjects treated with curcumin at doses of 2 and 4 g/day for 4 weeks (20). 
The dose of 4 g/day was found to be more active and plasma levels correlated significantly with 
the ACF inhibition effect, suggesting in some way that the preventive effect may be systemic 
and not direct on the rectal mucosa. 
For this study, therefore, we propose the use of Meriva®, a supplement based on a 
standardized extract of curcumin and phospholipid in a 1:2 ratio, with proven efficacy in the 
treatment of osteoarthritis and anterior uveitis (21) which, due to its pharmacokinetic 
characteristics and in particular its combination with the phospholipid part, allows us to 
overcome some of the limitations of the oral curcumin preparations tested to date, such as 
instability at intestinal pH values, low solubility in water, poor oral bioavailability and rapid 
conjugation and excretion. 
It is also interesting to note that several studies on the conjugation of curcumin with 
phosphatidylcholine increases its bioavailability by approximately 10 times (21,22). 
From the point of view of tolerability, Meriva® has proven to be safe and well tolerated. Oral 
intake in the studies cited so far has never been associated with significant side effects. 

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6.2 
Blueberry extract 
Blueberry extract (Mirtoselect®) is derived from the berries of V. myrtillus and is rich in 
anthocyanins, the polyphenols responsible for the blue and/or red pigmentation of many 
berries. The extract is characterized and standardized to contain 36% anthocyanosides. 
A large literature has demonstrated its pharmacological and clinical efficacy. In particular: 
a) increased capillary resistance 
b) reduction of abnormal vascular permeability 
c) antioxidant activity at vascular level 
d) induction of arterial vasomotility 
e) chemopreventive activity 
f) anti-inflammatory activity. 
As regards chemopreventive activity, the active component of botanical origin, anthocyanins, 
has been shown to be effective in some preclinical models of carcinogenesis, especially those of 
the gastrointestinal tract. Anthocyanins or dry extracts from fruits rich in these compounds 
have reduced the incidence of focal aberrant crypts and adenocarcinomas in rats treated with 
different chemical carcinogens (23-25). 
Furthermore, the same compounds were able to reduce the onset of intestinal adenomas in 
ApcMin mice (26), by reducing the expression of β-catenin. 
In a pilot study conducted on 25 patients with colorectal carcinoma (27), the subjects were 
treated with a dry extract containing anthocyanins for 7 days before surgery (anthocyanin 
titrated: 0.5-2g/day). Ki67 analysis on pre-operative biopsies and on the resected tumor 
demonstrated a small but systematic reduction (~7%) of tumor proliferation. 
 
 

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7.0  STUDY DESIGN 
Randomized, preoperative, double-blind, placebo-controlled study. 
 
Subjects with polyp of probable colorectal adenomatous nature will undergo biopsy of the 
polyp and healthy perilesional tissue during colonoscopy. After histological confirmation of AP 
and eligibility criteria verification, patients will be randomized to one of the two treatment 
groups. After four weeks of treatment, the second colonoscopy with polyp removal and biopsy 
of healthy perilesional tissue will be performed. The biopsy of the healthy tissue will be 
performed in the rectum if there is macroscopic inflammation in the sigma; in the absence of 
inflammation, it will be performed in the sigma. 
The determination of tissue biomarkers to verify the study hypotheses will be performed on 
adenomatous and normal tissue from adenoma biopsy taken at baseline and on the removed 
polyp and on the normal tissue biopsy after 4 weeks of treatment. Circulating biomarkers will 
be measured at baseline and at the end of treatment. 
 

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8.0  STUDY POPULATION 
A total of 100 subjects with one or more colorectal adenomatous polyps with a diameter ≥ 1 cm 
will be included in the study. 
8.1 Inclusion criteria 
1 Age > 18 years and ≤ 85 years; 
2 Written informed consent; 
3 Presence of one or more adenomatous polyps in the colon-rectum (with diameter ≥ 
1 cm); 
4 ECOG performance status ≤ 1. 
8.2  Exclusion criteria 
1 Presence of hyperplastic polyps and serrated and flat adenomas; 
2 Previous colorectal cancer; 
3 Presence of carcinomatous tissue in the adenoma; 
4 Taking experimental drugs or food supplements based on blueberry or curcumin in 
the 15 days prior to enrollment; 
5 Any other factor that, at the discretion of the investigator, may contraindicate 
enrollment in the study. 
 

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9.0 OBJECTIVE 
9.1 Primary objective 
To evaluate the effect of treatment on the reduction of β-catenin in adenomatous tissue. This 
biomarker, activator of the Wnt signaling pathway, acts as a transcription factor for some target 
genes in the nucleus that lead to neoplastic transformation of the cell, and is considered one of 
the key elements in APC-mediated colon carcinogenesis. 
9.2 Secondary objectives 
1. Evaluate the effects of treatment on: 
- 
oxidative stress and inflammation through the evaluation of IHC expression changes of 
Nuclear Factor-Kβ (NFKβ), one of the major transcription factors involved in the 
regulation of normal and neoplastic cells, closely related to the inflammatory and 
carcinogenic pathways of colon cancer; 
- 
cell proliferation (Ki-67 labeling index), P53 mutation, tissue gene expression with 
microarray; 
- 
circulating growth factors of the IGF system (IGF-I, IGFBP-3 and IGF-I/IGFBP-3) and EGFR; 
- 
markers of inflammation (u-PCR). 
 
2. Evaluate the tolerability of the treatment, as a combination of 2 food supplements and study 
the plasma and possibly tissue absorption of the active ingredients present in the botanical 
extracts (anthocyanosides and curcumin) . 
 

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10.0 
TREATMENT REGIMEN 
10.1 
Name of experimental treatment and formulation 
Bilberry extract: food supplement formulated in tablets with purple coating of 500 mg each and 
packaged in blisters.  
Composition: Bilberry extract ( Mirtoselect ®, 500 mg), microcrystalline cellulose, soy 
polysaccharides, anhydrous citric acid, croscarmellose sodium, silica dioxide, magnesium 
stearate, talc FU, purple coating for tablets. 
Turmeric extract: food supplement formulated in tablets with yellow coating of 500 mg each 
and packaged in blisters.  
Composition: turmeric extract (Meriva®, 500mg), calcium hydrogen phosphate dihydrate, 
anhydrous citric acid, soy polysaccharides, sodium croscarmellose, silica dioxide, magnesium 
stearate, talc FU, yellow coating for tablets. 
Placebo A: purple coated tablets, without active ingredients, but with excipients present in 
blueberry extract tablets. 
Placebo B: yellow-coated tablets, without active ingredients, but with excipients present in 
turmeric extract tablets. 
10.2 
Dosage groups, duration of exposure and dose selection 
Subjects will take blueberry extract (Mirtoselect®) 1 g/daily divided into 2 daily administrations 
of 500 mg or placebo A, together with turmeric extract (Meriva®) 1 g/daily divided into 2 daily 
administrations of 500 mg or placebo B for a minimum period of 4 weeks and a maximum 
period of 6 weeks. 
Mirtoselect® 500 mg, Meriva® 500 mg, placebo A or B tablets will be packaged in blisters of 9 
tablets each. The intake of the supplement or placebo will be in the order of 2 tablets per day 
of each supplement or respective placebo to be taken in the morning and evening always at the 
same time. 
The choice of a daily dose of 1 g of blueberry extract is based on a large literature regarding 
previous studies that ensure its safety even at doses much higher than 1 g. In particular, with 
blueberry extract the results on efficacy, pharmacokinetics and safety of several clinical studies 
are available starting from the dose of 160 mg with 2 daily administrations, up to the daily dose 
of 10.8 g for 7 days of treatment (27-29). 

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The choice of a daily dose of 1 g of curcumin extract is based on previous studies demonstrating 
that the complexation of curcumin with phosphatidylcholine is able to increase the 
bioavailability of the active ingredient (21,22), and this allows to lower the doses compared to 
previous studies with non-complexed curcumin (20). In fact, the supplement based on curcumin 
extract and phospholipid in a 1:2 ratio has demonstrated efficacy in the treatment of 
osteoarthritis and anterior uveitis (30,31) due to its pharmacokinetic characteristics and in 
particular for its combination with the phospholipid part which allows to overcome some limits 
of the oral preparations of curcumin, such as instability at intestinal pH values, low solubility in 
water, poor oral bioavailability and rapid conjugation and excretion. Furthermore, the curcumin 
extract has proven to be safe and well tolerated. Oral intake in the clinical studies has never 
been associated with significant side effects. 
10.3 
Supply, packaging, labelling  
The distribution of the supplements (Mirtoselect® and Meriva®) and the respective placebos, 
packaged in accordance with the regulations for food supplements by the company Indena SpA, 
is the responsibility of the coordinating center, which will provide the supply to the 
participating centers. 
The blueberry extract and the turmeric extract will be supplied in 2 different packs containing 
90 tablets (= 2 tablets per day for 6 weeks + 6 spare tablets, for each supplement). Each patient 
will receive a “patient kit” with 1 pack of blueberry extract and 1 pack of turmeric extract to 
cover the entire treatment period. The preparation and packaging will be visually identical to 
those of the respective placebos. The Indena company guarantees the correct assembly of the 
compounds (active/placebo) inside the “patient kits”. Indena will deliver the “patient kits” to 
the EO Ospedali Galliera, c/o the SC Farmacia, which will deliver them to the S.C.Oncologia 
Medica, for distribution to the enrollment centers involved. 
The “patient kit” will have an external label (study name, sponsor and patient ID) with a 
removable portion that must be applied to the appropriate Case Report Form (CRF) in order to 
ensure the correct assignment of the product. Inside the kit, the information noted on the 
boxes are: study name, sponsor, patient ID, expiration date, number of tablets, method of 
administration, warnings. 
10.4 
Disposal of residual supplements 
Residual, unused, leftover or expired supplements must be stored at the enrolling center until 
retrieval, which will be done by the monitor, assigned by the coordinating center. This 

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procedure can be implemented only after accountability verification during the monitoring 
phase. Unclassified and/or expired supplements cannot be sent independently to the 
coordinating center. 
10.5 
Treatment assignment method 
All eligible patients after signing the informed consent must be randomized before starting 
study treatment. Randomization will be centralized at the Medical Oncology Department of the 
EO Ospedali Galliera through a computerized procedure. The randomization list will be 
prepared by the biostatistician of the Scientific Coordination Office of the coordinating center 
independently: permutation blocks of different sizes will be used, stratified for the enrolling 
sites. Randomization will be performed using a paper form sent by FAX to the Medical Oncology 
Department at Galliera Hospital. Once it is ascertained that the form has been filled out 
correctly, a patient ID corresponding to the kit will be associated with the eligible patient. 
An operating manual for randomization of study subjects will be provided to centers. 
Subjects will be randomized in a strictly sequentially sequenced manner and will remain in their 
assigned arm for the duration of the study. 
The procedure for assigning treatment to randomized subjects will be kept blind according to 
the following method: the supplement packages (Mirtoselect® 500 mg, Meriva® 500 mg, 
placebo A, B) will be prepared in blisters containing 90 identical tablets, supplement and 
reference placebo, in order to allow for masking. Each pack will contain 10 blisters of 9 tablets 
for each individual product (supplement or placebo). For treatment, each subject will receive a 
“patient kit”, i.e. a box containing a pack of Mirtoselect® + a pack of Meriva®, or a pack of 
Placebo A + a pack of Placebo B. Each “patient kit” box will be associated with the subject using 
the code indicated in the randomization list. The preparation of the supplements will be carried 
out according to good practices for preparing botanical extracts and will be paid for by Indena 
SpA. Indena will identify an operator responsible for receiving the open-label (unmasked) 
randomization lists from the statistician, who will archive the lists for any future checks. The 
treatment code may be revealed to the clinician and the patient only at the end of the study or 
if it is necessary following a serious adverse event that could be related to the experimental 
treatment or in any case in cases where, for medical emergencies, it is necessary to know the 
treatment associated with the subject in the study. 
Unblinding may only be performed by the coordinating center upon specific request of the 
participating center. An operating procedure for unblinding will be provided to the centers. 

MiRACol Protocol – Version 4 of 01 October 2016 
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11.0 
STUDY PROCEDURES 
11.1 
Duration of study  
Subjects will be enrolled over 48 months, so the total study duration is expected to be 64 
months, including the determination of SEB (surrogate end-point biomarkers), statistical 
analysis and drafting of the final report. 
The study is considered concluded when the last enrolled subject has completed the follow-up 
visit one year after the end of the experimental treatment. 
11.2 
Screening  
Within the month preceding randomization, the following must be guaranteed: 
- 
signature of written informed consent; 
- 
eligibility verificaton: evaluation of inclusion/exclusion criteria. 
- 
blood tests and/or instrumental tests; 
- 
colonoscopy with evaluation of the presence of adenomatous polyp ≥ 1 cm and 
collection of biopsy material necessary for tissue determinations. 
11.3 
Baseline Visit 
- 
Clinical visit and detection of vital parameters: weight, height, blood pressure (BP), 
Performance status (PS) according to ECOG; 
- 
Blood sampling for the determination of circulating biomarkers and for the 
determination of plasma absorption of the active ingredients present in the botanical 
extracts at time zero (only for patients enrolled at the EO Ospedali Galliera). 
- 
Delivery of the food diary. 
- 
Anamnesis, concomitant pathologies and family history (I and II degree) for CRC/other 
neoplasms; 
- 
Concomitant therapies; 
- 
Randomization; 
- 
Delivery of supplement/placebo: a package containing sufficient supplement for a six-
week administration period + 6 spare tablets (90 tablets) will be delivered. The kit must 
be returned at the end of the study visit. 
11.4 
Final Visit (after 4-6 weeks of treatment) 
- Clinical visit and detection of vital parameters: weight, BP, PS according to ECOG; 

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- Blood sampling for the determination of circulating biomarkers. 
- Blood sampling to determine the plasma absorption of the active ingredients present in 
the botanical extracts, before and 1 hour after the last treatment (Only for patients 
enrolled at the EO Ospedali Galliera); 
- Concomitant therapies; 
- Adverse events and symptoms; 
- Patient kit return and compliance assessment; 
- Food diary collection and evaluation; 
- Polypectomy and biopsy for tissue determinations, to be carried out after 2-5 hours of 
taking the last dose. 
11.5 
Follow-up (1 year after the end of treatment) 
The follow-up visit, 1 year after the end of treatment, must be performed one year ± 1 month 
after the end-of-study visit. 
During the visit the following data must be collected: 
- 
Clinical visit and detection of vital parameters: weight, BP, PS according to ECOG; 
- 
Adverse events and symptoms; 
- 
CCR onset (histological evaluation/colonoscopy/polypectomies); 
- 
Relevant clinical notes. 
11.6 
Compliance   
To manage the study supplements accountability, a balance sheet will be drawn up to maintain 
traceability of the inventory of products, shipping receipts and the kit administration. This 
register will be updated regularly and, for each shipment, will identify the subject number, the 
randomization number and the quantity of supplements shipped. The accountability form will 
be stored/used in all the places where the supplements will be stored for subsequent delivery 
to patients, the main pharmacy, the pharmacies of the individual centers, the clinical unit and 
other delivery locations. The form will include, in addition to the delivery register, all other 
possible operations for the management of the supplements (receipts, transfers, returns, 
damaged packages). The register form will be provided by the coordinating center to the 
participating sites. 

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11.7 
Collection and management of biological material 
Tissue samples collected during colonoscopies for immunohistochemical determinations will be 
collected centrally at SC Anatomia Patologica, EO Ospedali Galliera, upon specific request of the 
coordinating center and at the end of the study (within the month after the last enrolled 
patient took the last dose of treatment). Transparent labeled envelopes will be provided to the 
enrolling centers. The labels will contain: name of the study, center number, randomation 
number and patient registration, date of the exam and time (1=baseline, 2=final). 
Blood samples for circulating biomarkers will be collected centrally by the Lab Analysis of the 
Prevention and Oncology Genetics Division of the IEO in Milan upon specific request and in any 
case at the end of the study (within the month after the last enrolled patient took the last dose 
of treatment). In both cases, guides for biological samples collection, preparation, storage and 
shipment to the appropriate centralized center will be provided. 
Blood samples for the determination of plasma absorption of the active ingredients will be 
performed only on the group of patients enrolled at the Gastroenterology unit of the 
coordinating center, at EO Ospedali Galliera and will be analyzed by an external laboratory. A 
guide will be provided for samples collection, preparation, storage times and shipment to the 
centralized collection center. 
11.8 
Concomitant treatment  
During the study, the intake of Indian foods is not recommended, as is the intake of H2 blockers 
(cimetidine, ranitidine, nizatidine, famotidine) and proton pump inhibitors (omeprazole, 
esomeprazole, rabeprazole, pantoprazole, lansoprazole). 
If the subject uses antacids and/or lactic ferments, he/she will be instructed to take them at 
least 2 hours before or after the experimental treatment. 
The investigator must detail in the CRF any intake of the above-described substances and any 
other concomitant drugs or food supplements. 
11.9 
Food diary 
A food diary will be provided to the patient at the beginning of the study and must be filled out 
daily by the patient. The diary is intended to monitor the intake of foods consumed by the 
subject in treatment and their possible interaction with the compounds. The diary, like the CRF, 
must be forwarded to the coordinating center 5 working days after the end-of-study visit. 
 

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12.0 FLOW CHART 
Exam/Procedure 
Screening 
Baseline Visit 
Final Visit 
Follow up 
Written Informed Consent 
X 
 
 
 
Colonoscopy/identification of polyp >1 cm (histology) 
X 
 
 
 
Blood tests1 
X 
 
 
 
Check eligibility 
X 
 
 
 
Randomization 
 
X 
 
 
Clinical Visit2 
 
X 
X 
X 
Control colonoscopy (according to clinical practice) 
 
 
 
X 
Anamnesis/clinical history/family history for CRC 
 
X 
 
 
Biomarker sampling 
 
X 
X 3 
 
Pharmacokinetic sampling 
 
X 
X 4 
 
Polypectomy and Tissue Collection for Biomarker 5 
 
X 
X 
 
Compliance 
 
 
X 
 
Adverse reactions/adverse events 
 
 
X 
X 
Concomitant treatment 
 
X 
X 
X 
1 Routine tests required for colonoscopy. 
2 Evaluation of vital parameters: weight, height, BP, PS ECOG. 
3 Post-dose sampling. 
4 To determine the absorption of the active ingredients, a sample will be taken before and 1 hour after taking the last dose of treatment. 
5 Polypectomy to be performed within 2-5 hours of taking the last dose of treatment.

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13.0 
ETHICAL ASPECTS  
This protocol complies with the principles established by the 18th World Medical Assembly in 
Helsinki, 1964 and subsequent amendments/additions and with Good Clinical Practice. 
13.1 
Informed consent 
The investigator must explain to each patient (or legal representative) the nature of the  
study, its purpose, procedures, expected duration, potential risks and benefits and any adverse 
events or reactions that may occur. Each patient must be informed that participation in the 
study is voluntary and that he/she may withdraw from it at any time, withdrawal of consent will 
not affect his/her subsequent clinical treatment or relationship with the physician. The 
informed consent will be prepared by the coordinating center by means of a standard written 
statement, using non-technical language. The patient must demonstrate that he/she has 
understood the information contained in the consent, by signing and dating it; he/she must 
also be provided with a copy of the document. If the patient is unable to read or sign the 
document, the oral and written presentation of the study may be provided to his/her legal 
representative who can sign and date the document. No patient may be enrolled in the study 
before his/her informed consent has been obtained. An original signed copy of the patient's 
written informed consent must be kept in the appropriate section of the Investigator's File of 
the enrolling center. 
13.2 
Patient safety 
Patient names will not be recorded, but each subject will be identified with a treatment 
number (randomization number) and a registration number. These numbers will identify the 
patient and must be included in all CRFs. In order to avoid misidentification, the date of birth 
must be reported in the CRFs to identify the subjects themselves. The investigators guarantee 
that all persons involved in this study will respect the confidentiality of any information 
regarding the subject participating in the trial. All parties involved in this clinical trial will 
maintain the utmost confidentiality to ensure that neither the person nor the privacy of the 
patient participant's family is violated; adequate measures will be taken to prevent 
unauthorized persons from accessing the study data. The processing of personal data of 
patients participating in the trial, and in particular the related data regarding consent, will be in 
accordance with local privacy laws:  

MiRACol Protocol – Version 4 of 01 October 2016 
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Legislative Decree 196/03 and subsequent amendments (“Privacy Code”), “Guidelines for the 
processing of personal data in the context of clinical trials of medicinal products” with 
particular reference to the profiles relating to the methods of data processing, data 
requirements, the designation of Managers and Persons in Charge and the custody and security 
of information. 
 
 

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14.0 
DATA COLLECTION AND MONITORING PLAN 
14.1 
Data collection and monitoring procedures 
Study data will be collected via a paper CRF that must be faxed to the coordinating center. CRFs 
must be sent within 5 working days of the visits. Checks on the receipt of CRFs will be carried 
out by the Coordinating center on a regular basis during the study.  
Monitoring will be carried out according to this plan: 
- 
opening of the center by teleconference (SIV) and concomitant shipment of 
supplements 
- 
visit to the center after approximately 6 months (± one month) from the inclusion of 
the first patient 
- 
closing visit of the center at the end of the study. 
Periodic telephone monitoring will be carried out to verify the correct entry of data into the 
CRF. 
It is the responsibility of the participating site investigator to prepare and maintain adequate 
and accurate CRFs for each subject enrolled in the study. All CRFs must be completed to ensure 
accurate interpretation of the data, a black ballpoint pen must be used to ensure clarity of the 
data transcription on the CRF. If a correction is necessary, the information that needs to be 
changed must not be overwritten. The corrected information must be transcribed next to the 
previous value with the reason for the correction, initialed and dated by the authorized person. 
14.2 
Quality Assurance 
The researchers of the participating centers, will have to guarantee the quality of the data, 
each information reported on the CRF will be systematically verified for coherence, 
completeness and correctness by the Coordinating Center that may ask for clarifications in case 
of doubtful data. Local quality controls will be carried out by the coordinating center as 
responsible for the monitoring plan. 
14.3 
Source documents 
The source data must be stored and recorded in a hospital medical record; if such records for 
outpatient subjects are not provided for by the practice of the facility or clinic where the trial is 
being conducted, they must be prepared for the purposes of the legislation on clinical trials of 
medicinal products. If paper CRFs are used as source documents, the principal investigator of 

MiRACol Protocol – Version 4 of 01 October 2016 
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the center must declare the methods of archiving such source documents and, after having 
signed, stamped and dated them, insert the same CRFs into the outpatient record as an integral 
part of the same. 
At the end of the trial, the documents must be kept at the center for a period of 10 years. If the 
responsible investigator changes workplace, he must forward the name of a new trial manager 
to the coordinating center. 
 

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15.0 
 END OF STUDY CRITERIA 
After 4-6 weeks of treatment, patients will stop study treatment and after all 100 subjects have 
been randomized, enrollment will be closed. The study will be considered terminated when the 
100th patient has made the follow-up visit. 
15.1 
Premature discontinuation 
Subjects who, for various reasons, do not complete the treatment period will be classified as 
having dropped out of the study early. 
Some possible reasons for early exit include: 
 
Inappropriate recruitment; 
 
Adverse event; 
 
Personal reasons: a patient may withdraw from the study at any time and for any 
reason. 
 
16.0 
 TOXICITY MONITORING AND ADVERSE EVENT MANAGEMENT 
16.1 
Adverse Event Definitions 
Adverse event (AE): is any undesirable medical event in a patient or subject included in a clinical 
trial and which does not necessarily have a causal relationship with the treatment. 
Serious adverse event (SAE) or serious adverse reaction (SAR) or suspected unexpected serious 
adverse reaction (SUSAR): any adverse event or adverse reaction that, regardless of dose, 
results in death or is life-threatening, requires hospitalisation or prolongation of hospitalisation, 
or results in severe or prolonged disability or incapacity, or involves a congenital anomaly or 
birth defect. 
16.2 
Adverse Event (AE) and Serious Adverse Events (SAE) Reporting 
All AEs must be recorded in the appropriate section of the CRF, regardless of whether or not 
they are directly related to the study treatment. 
All SAEs and SUSARs must be reported on a specific form provided by the coordinating center 
and processed in the following manner: 
SAE and SUSAR. Each investigator will ensure that all information relating to SAE is recorded on 
the appropriate phytovigilance form and notified to the Coordinating Centre within 24 hours of 
the first knowledge of the SAE by the responsible investigator (tel: 010-563.4580; fax: 
01057481407), and that subsequent relevant information is communicated within eight days of 

MiRACol Protocol – Version 4 of 01 October 2016 
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the first report. The Coordinating Centre will be responsible for sending reports of suspected 
reactions to the study treatment to the Istituto Superiore di Sanità. 
Once a year, the principal investigator will provide the Ethics Committees with a list of all SAEs 
and SUSARs observed throughout the entire period. 
The occurrence of CCR will not be considered as a SAE. 
 
 

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17.0 
STATISTICAL METHODS 
17.1 
Statistical design and sample calculation 
The primary endpoint of the study is the difference in β-catenin values [post-treatment value - 
pre-treatment value]. 
Data from our previous trial on the same study population (APAC Study) allow us to make some 
assumptions: we assume a baseline mean of β-catenin expression in adenomatous tissue equal 
to 25% (standard deviation=35) and a correlation between baseline and final β-catenin values 
equal to approximately 0.9. Given these assumptions, using an ANCOVA model (therefore 
adjusting the analysis for baseline β-catenin values), with 50 subjects per arm, for a total of 100 
subjects, we will have an 85% power to observe a mean difference between the two arms equal 
to 10%, in the levels of β-catenin expression in adenomatous tissue. The sample calculation 
takes into account 10% of subjects lost to follow-up and a 2-tailed alpha error equal to 5%. 
17.2 
Interim analysis 
At approximately halfway through the study, which will approximately coincide with 50% of 
enrolled patients completing treatment, the study monitoring committee, composed of the 
principal investigators from each center, the biostatistician, and the study coordinator, will 
perform an interim analysis to assess any adverse and/or toxic effect of the active treatment 
compared to placebo. This analysis will therefore consider 2 endpoints in the comparison arms: 
1) number of adverse events 2) size of the adenoma.  
With a total of n=50 subjects, there will be sufficient power to detect an absolute difference of 
40% in terms of frequency of adverse events and in terms of change in size of the adenoma 
between the active arm and placebo at the end of treatment (assuming an ANCOVA model 
corrected for baseline size and a correlation coefficient of 0.7 between mean baseline and final 
size). In case of a significantly greater adverse and/or toxic effect in the active arm, the 
monitoring committee will consider the possibility of prematurely stopping the study. 
17.3 
Statistical analysis methods 
All subjects enrolled in the study will be considered in the “Intention To Treat” (ITT) population, 
and the statistical analysis will be performed in accordance with this principle, therefore 
considering the randomized subjects regardless of their actual adherence to the experimental 
treatment. 

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The descriptive statistics used for continuous variables will be: mean, standard deviation, 
median, quartiles, minimum and maximum; for discrete variables they will be: absolute 
frequency and relative frequency. 
In agreement with previous results, the difference in post- and pre-treatment values of β-
catenin [post-treatment value - pre-treatment value] is a normally distributed variable. 
However, the normality of the distribution will be tested and, if necessary, the necessary 
transformations will be applied to obtain normality. 
Fisher's exact test and chi-square test will be used to compare proportions in the two 
treatment arms (toxicity: number of adverse events), while parametric (t-test) and non-
parametric (Mann-Whitney U) tests will be used to compare continuous variables. 
Parametric statistics will be used for the primary objective. Hypotheses will be tested using the 
general linear model: the null hypothesis of no treatment effect will be tested using standard 
analysis of variance (ANOVA) and covariance (ANCOVA) methods, adjusting for baseline β-
catenin and the effect of other possible confounding factors such as age, sex, body mass index 
(BMI), family history of colon cancer, smoking habits, alcohol consumption and other important 
factors (demographic and/or biological) collected. 
 
 

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Acceptance of the study protocol / Responsibilities of the investigator 
 
 
I read the study protocol with the following title: 
“ A PHASE II STUDY OF BIOMARKER MODULATION IN SUBJECTS WITH COLON ADENOMATOSUS 
POLYP WITH BLUEBERRY EXTRACT AND TURMERIC EXTRACT 
“MIRACLE STUDIO” 
 
and I agree to conduct the study as specified in the protocol and to observe the Good Clinical 
Practice (GCP) standards, I accept periodic visits by the monitor and all other data control and 
verification procedures. 
 
 
 
Name of the Center: 
 
 
____________________________________________________________________________ 
 
 
Name of Responsible Investigator: 
 
 
____________________________________________________________________________ 
 
 
 
Signature Date 
 
 
__________________________________ ___________________________________ 
 
 
Signature of Project Manager  
Date 
 
 
__________________________________  
 
01 October 2016 
Andrea De Censi 
 
 
 
 

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