WO2020161522A1

WO2020161522A1 - Fiber-based composition

Info

Publication number: WO2020161522A1
Application number: PCT/IB2019/050940
Authority: WO
Inventors: Alfredo Di Leo
Original assignee: Thd S.P.A.
Priority date: 2019-02-06
Filing date: 2019-02-06
Publication date: 2020-08-13

Abstract

A composition comprising at least one selective phytoestrogen agonist of the ER- beta receptor and at least one soluble fiber for the treatment and/or for the prevention and/or for the follow-up of inflammatory pathologies of the gastro-intestinal tract and/or adenopolyposis and/or colorectal cancer.

Description

FIBER-BASED COMPOSITION

DESCRIPTION

The present invention relates to a composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber for the treatment and/or for the prevention and/or for the follow-up of inflammatory pathologies of the gastro-intestinal tract and/or adenopolyposis and/or colorectal cancer.

BACKROUND

Adenoma-carcinoma, or carcinoma of the colon-rectum (CRC), is the third cause of death in the world. The pathogenesis of CRC includes the formation of polyps on the intestinal mucosa, considered precancerous lesions. When they are diagnosed through colonoscopy, intestinal polyps are generally removed.

Human familial adenomatous polyposis (FAP) is caused by a germ mutation of the suppressor gene of the APC cancer (Adenomatous Polyposis Coli). FAP is characterized by the development of a large number of adenomas in the colon. While the genetic substrate facilitates the start of the tumorigenic process and cannot be modified with drugs or nutrition, the promotion of the tumorigenesis can be controlled, so as to hinder the progression towards cellular de-differentiation and neoplastic transformation. The main objective of a colonoscopic screening is to monitor adenomas and their progression toward advanced forms and CRC.

It has also been noted that a state of chronic inflammation can in some cases be a predisposition to the development of cancer in the colon-rectum. In fact, it was observed that the risk of onset of CRC is increased in patients with chronic inflammatory pathologies of the intestine, such as ulcerative colitis and Crohn's disease.

The higher prevalence of CRC in males and a reduced incidence in women undergoing hormone replacement therapy (HRT) support the hypothesis that estrogens play a protective role in the tumorigenic process of the colon.

Two forms of estrogen receptors exist: alpha (ERa) and beta (ERP), which are localized differently and expressed in various anatomic locations. ERa is primarily located in the breast, in the cardiovascular system, in bones, in the urogenital system and in the central nervous system, while ERp is expressed in the prostate, salivary glands, testis, ovary, immune system and especially in the intestine.

The involvement of ERp in adenomatous polyposis and CRC is demonstrated by its reduced expression, with respect to the normal intestinal mucosa in patients who have undergone polypectomy, already in the pre-cancerous stage. Significantly, the reduction of ERp refers to an increase in proliferation and to a progressive cellular de-differentiation, demonstrating that the silencing of ERp is involved in the cancer's progression. Pre-cancerous lesions of the colon-rectum are therefore early warnings of the development of CRC in a significant part of the population. The dramatic decline of ERp is also observed in the more advanced stages of the neoplastic transformation.

In addition to genetic causes, it is known that exposure to carcinogens within the intestinal lumen or to potentially genotoxic agents for the epithelial cells of the intestinal mucosa is an epigenetic event that facilitates the inactivation of both alleles or the somatic mutation of the gene APC, both in FAP and sporadically. These dangerous substances are normally ingested with the diet or can be formed in the intestinal lumen as an effect of digestion, in particular of fats, or from the degradation of bile acids.

For this reason, a diet rich in fiber, believing that it has a shielding effect on intestinal mucosa or absorbs xenobiotics which are potentially genotoxic, is regarded as useful for the prevention of CRC. Flowever, clinical trials testing this efficacy are not always convincing.

EP2254573 and WO2013045068 describe the use of a composition based on selective phytoestrogen agonists of the ERp receptor (i.e. milk thistle) and an insoluble fiber for the treatment of adenopolyposis/adenoma-carcinoma. Said documents demonstrate the therapeutic efficacy of the composition of phytoestrogens and insoluble fiber in comparison with a composition comprising phytoestrogens and soluble fiber, which does not show significant therapeutic efficacy. Therefore, said documents teach that a composition based on phytoestrogens and selective agonists of the ERp receptor and an insoluble fiber is effective for the treatment of adenopolyposis/adenoma-carcinoma, while a composition comprising phytoestrogens and soluble fiber has not proved to be effective in the treatment of adenopolyposis/adenoma-carcinoma.

SUMMARY OF THE INVENTION

The present invention falls within this context, as it relates to a composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber for use in the treatment or in the prevention or in the follow up of inflammatory pathologies of the gastro-intestinal tract and/or adenopolyposis and/or colorectal cancer.

In fact, the Applicant has surprisingly found that the administration of the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber is capable of:

reducing inflammation in the colon-rectum, in particular in colorectal areas affected by low-grade or high-grade dysplasia and/or in the colorectal region with colorectal carcinoma;

reducing the formation of solid lesions in the colon-rectum, preferably, said composition is able to reduce the formation of solid lesions in the colorectal region affected by low-grade or high-grade dysplasia and/or colorectal carcinoma,

inducing death by apoptosis in the epithelium of the colon-rectum, preferably in colorectal carcinoma cells.

BRIEF DESCRIPTION OF THE FIGURES

The present invention is hereinafter described in detail and exemplified without any limiting purposes, also with reference to the annexed Figures as briefly described below.

Figure 1 shows the treatment schedule with a standard diet and with the THD diet of the control group;

Figure 2 shows the treatment schedule with a standard diet and with the THD diet of the group of mice in which a colorectal carcinoma was induced (AOM/DSS group); Figure 3 shows the results of the evaluation of the number of solid lesions detected in the AOM/DSS mice;

Figure 4 shows the histological score evaluated according to Yu et al. of inflammatory damage (mean ± SD) of the group of AOM/DSS mice treated with a standard diet and the THD diet;

Figure 5 shows the mean number ± SD/mouse of areas of microscopic pre-neoplastic and neoplastic lesions. CRC = colorectal cancer; HGD= high-grade dysplasia; LGD= low-grade dysplasia

Figure 6 shows the results of the expression of the ER-alpha receptors obtained following the reading of the histological preparations with an optical microscope, expressed as a labelling index (% of positive cells for the staining of the receptor - LI; LGD = low-grade dysplasia; HGD = high-grade dysplasia; CRC = colorectal cancer); in particular, Figure 6a shows the expression of the ER-alpha receptors in the AOM/DSS mice treated with a standard diet; Figure 6b shows the expression of the ER-alpha receptors in the AOM/DSS mice treated with the TFID diet; while Figure 6c shows a summary analysis of the expression of the ER-alpha receptors between the mice treated with a standard diet and those treated with the TFID diet;

Figure 7 shows the results of the expression of the ER-beta receptors obtained following the reading of the histological preparations with an optical microscope, expressed as a labelling index (% of positive cells for the staining of the receptor - LI; LGD = low-grade dysplasia; HGD = high-grade dysplasia; CRC = colorectal cancer); in particular, Figure 7a shows the expression of the ER-beta receptors in the AOM/DSS mice treated with a standard diet; Figure 7b shows the expression of the ER-beta receptors in the AOM/DSS mice treated with the THD diet; while Figure 7c shows a summary analysis of the expression of the ER-beta receptors between the mice treated with a standard diet and those treated with the THD diet;

Figure 8 shows the ratio of the expression of the ER-alpha/ER-beta receptors obtained following the reading of the histological preparations with an optical microscope, expressed as a labelling index (% of positive cells for the staining of the receptor - LI; LGD = low-grade dysplasia; HGD = high-grade dysplasia; CRC = colorectal cancer);

Figure 9 shows the results of the migration speed of the cells toward the free surface along the axis of the intestinal villi through immunofluorescence evaluated with confocal microscopy, in the AOM/DSS mice fed a standard diet and those fed the THD diet;

Figure 10 shows the results of the TUNEL staining method to evaluate epithelial apoptosis; in particular, Figure 10a shows the levels of epithelial apoptosis in the colon of the AOM/DSS mice fed a standard diet; Figure 10b shows the levels of epithelial apoptosis of the colon of the AOM/DSS mice fed the THD diet; while Figure 10c shows a summary analysis of apoptosis in the two groups of AOM/DSS mice; Figure 1 1 shows the treatment schedule with a standard diet and with the TFID diet of the group of mice with a genetically modified APC gene;

Figure 12 shows the results of the migration speed of the cells toward the free surface along the axis of the intestinal villi through immunofluorescence evaluated with confocal microscopy, in the APC mice fed a standard diet and those fed the TFID diet;

Figure 13 shows the results of the TUNEL staining method to evaluate the epithelial apoptosis in the APC mice fed a standard diet or the TFID diet.

DETAILED DESCRIPTION

DEFINITIONS

In the context of the present invention, “phytoestrogen” refers to any non-steroid molecule able to bind estrogen receptors, by imitating or by modulating its activity.

In the context of the present invention,“agonist” refers to a chemical that binds to a receptor and activates the receptor to produce a biological response.

A first aspect of the present invention relates to a composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber for use in the treatment or in the prevention or in the follow-up of inflammatory pathologies of the gastro-intestinal tract and/or adenopolyposis and/or colorectal cancer.

In fact, as shown in the example, the administration of the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber is able of inducing death by apoptosis in the epithelium of the colon- rectum, preferably in colorectal cancer cells. Therefore, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber is also useful for inducing cell death, preferably by apoptosis. Furthermore, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber is able to reduce inflammation in the colon-rectum, in particular in colorectal areas affected by low- grade or high-grade dysplasia and/or in the colorectal region with colorectal carcinoma. Therefore, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber is capable of reducing inflammation, preferably by reducing the levels of IFN-gamma and/or IL-6 and/or TNF-alpha.

The administration of the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber is moreover capable of reducing the formation of solid lesions in the colon-rectum, preferably, said composition is able to reduce the formation of solid lesions in the colorectal region affected by low-grade or by high-grade dysplasia and/or by colorectal cancer.

The at least one selective phytoestrogen agonist of the ER-beta receptor comprised in the composition of the present disclosure are phytoestrogens selective for ER-beta and with an anti-estrogenic activity, which do not induce significant proliferative activity on tissues that are classic objectives for estrogens.

In one embodiment, the composition comprises at least one phytoestrogen selective for ER-beta in a concentration comprised between 0.5 and 15.0%, preferably between 1.0 and 13.0%, even more preferably between 2.0 and 8.0% by weight.

In a preferred embodiment of the invention, the at least one phytoestrogen agonist of the ER-beta receptor is selected from a group consisting of silymarin, silybin, isosilybin, silydianin, silychristin, the isoflavone genistein, the flavonoid resveratrol, the coumestrol and mixtures thereof.

The phytoestrogen or phytoestrogens that are comprised in the mixture can be present in the composition in their purified form and/or as a vegetable extract.

In one embodiment, the phytoestrogen or phytoestrogens can be present in the composition as a vegetable extract.

In one embodiment, the at least one phytoestrogen agonist of the ER-beta receptor is silymarin, an extract of Silybum marianum comprising silybin, preferably with a content of silybin of 30-80% by weight, wherein the amount of silybin is referred to the total weight of the silymarin.

Dietary fibers are carbohydrates, preferably polysaccharides and/or oligosaccharides, resistant to digestion and absorption in the small intestine, with complete or partial fermentation in the large intestine (digestion-resistant carbohydrates).

The present invention may comprise any soluble fiber, however, especially preferred fibers are selected among digestion-resistant dextrins and/or digestion-resistant maltodextrins. Preferred digestion-resistant dextrins and/or digestion-resistant maltodextrins are those which are produced by starch hydrolysis (e.g. corn starch or potato starch, hardened inulin (fructo-oligosaccharides) produced by plants such as dahlia or chicory and partially hydrolyzed, or diversely vegetable gums fractionated as partially hydrolyzed guar gum.

In a preferred embodiment, the at least one soluble fiber is a dextrin and/or a maltodextrin which is resistant to digestion, preferably produced by the hydrolysis of corn starch.

In one embodiment, the composition comprises the at least one soluble fiber in a concentration comprised between 7 and 40%, preferably between 10 and 30%, even more preferably between 12 and 20% by weight.

In one embodiment, the composition can further comprise at least one extract of vegetable origin, such as for example a polyphenolic extract and/or a triterpenes- containing extract.

In one embodiment of the invention, the polyphenolic extract is a curcuminoid extract, preferably selected from: curcumin, demethoxycurcumin, bisdemethoxycurcumin and mixtures thereof.

In a preferred embodiment of the invention, the composition may comprise curcumin (diferuloylmethane), the polyphenolic curcuminoid principle of turmeric.

In one embodiment of the invention, the composition comprises curcumin in a concentration comprised between 0.2 and 10%, preferably between 0.5 and 8%, even more preferably between 1 and 6% by weight.

In one embodiment, the triterpenes-containing extract is an extract of Boswellia serrata comprising triterpenic acids, the triterpenic acids preferably comprising acetyl- 1 1 -keto-p-boswellic acid (AKBA), a polycyclic triterpene extracted from the gum resin of Boswellia serrata.

In one embodiment, the triterpenes-containing extract comprises 15-40% in weight of triterpenic acids, the amount of terpenic acids being referred to the total weight of the triterpenes-containing extract.

In one embodiment, the composition comprises acetyl-1 1 -keto-p-boswellic acid in a concentration comprised between 0.05 and 5%, preferably between 0.1 and 3% by weight.

In a further preferred embodiment, the composition comprises silymarin, acetyl-1 1 - keto-p-boswellic acid (AKBA), curcumin and digestion-resistant maltodextrins, more preferably in the amounts indicated above.

The composition can further comprise other phytoestrogens such as lignans derived from certain types of seeds selected from sunflower seeds, pumpkin seeds and flax seeds. In a preferred embodiment, the lignans are derived (extracted) from flax seeds.

In one embodiment, the composition comprises lignans in a concentration comprised between 0.01 and 5%, preferably between 0.05 and 3%, even more preferably between 0.1 and 2% by weight.

The composition can further comprise at least one excipient acceptable for pharmaceutical use or for cosmetic use, which is useful for the preparation of the composition and is generally biologically safe and non-toxic. According to a further embodiment, the composition further comprises a flavoring agent preferably selected from: essential oils, synthetic flavors or mixtures, including but not limited to oils derived from plants and fruits such as citrus oil, fruit essences, peppermint oil, mint oil, other mint oils, clove oil, sugar oil, anise and the like, aromatic oils such as menthol, eucalyptus, thymol, spices such as cocoa powder and ground cinnamon, and combinations thereof.

According to a further embodiment, the composition further comprises a sweetening agent preferably selected from: sucrose, dextrose, maltose, dextrin, fructose, levulose, galactose, sucralose, sorbitol, natural sweeteners such as, for example, stevia, corn syrup and the like, alone or in any combination.

In one embodiment, the composition can be formulated for oral use, preferably as tablets, capsules, bars, granular powder, oral granules, sachets, candies and drinkable formulations, for example drinkable vials or beverages.

The present disclosure refers to a food intended for special medical purposes, preferably formulated as beverage, comprising the composition for use as described in detail above.

According to an embodiment, the composition is formulated for enteral and/or parenteral administration. Preferably, the composition can be formulated in liquid form, more preferably in the form of a sterile solution, emulsion or suspension. A further aspect of the present invention relates to a method for the treatment of inflammatory pathologies of the gastrointestinal tract and/or adenopolyposis and/or colorectal cancer. Said method comprises a step of administering an effective amount of a composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber as described above in detail, to a subject in need thereof. In one embodiment, the subject in need thereof is a patient suffering from an inflammatory pathology of the gastrointestinal tract and/or by adenopolyposis and/or by a colorectal cancer.

Preferably for the medical purposes described above, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber as described above in detail, can be taken once a day.

In a one embodiment, the composition for use as described in detail above is taken at a total daily dose of equal to or less than 10000 mg/die.

In a preferred embodiment, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber is taken as a single dose or as multiple daily doses in a continuous way or according to need. In one embodiment, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber as described above in detail, can be also administered in combination with drugs used in the therapy for the treatment of inflammatory pathologies of the gastrointestinal tract. Said drugs are, for example: mesalazine, steroids, eg. cortisone, biologies anti TNF-alpha and their combinations.

In one embodiment, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber as described above in detail, can also be administered in combination with drugs used in the therapy for the treatment of adenopolyposis and/or colorectal cancer.

In one embodiment, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber as described above in detail, can also be administered in combination with drugs used in the therapy for the treatment of adenopolyposis and/or colorectal cancer and with drugs used in the therapy for the treatment of inflammatory pathologies of the gastrointestinal tract, said drugs being described above in detail.

In one embodiment, the composition comprising at least one selective phytoestrogen agonist of the ER-beta receptor and at least one soluble fiber as described above in detail, can be administered in association or in combination with a surgical treatment, preferably for the treatment of adenopolyposis and/or colorectal cancer, more preferably before and/or after a polypectomy.

EXAMPLE

Assessment of the effects of a mixture of phytoestrogens and anti-inflammatory substances in AOM/DSS and APC/MIN murine models

The effects of a mixture of phytoestrogens (hereinafter "THD mixture" or "THD diet") and anti-inflammatory substances in "AOM/DSS and APC/min” murine models, on intestinal neoplasms in murine animal models.

The study focused on the administration of the mixture (having the characteristics of a food supplement) in animals in which intestinal carcinoma was induced on both an inflammatory basis (AOM/DSS) and a genetic one (APC/min). The effect was evaluated macroscopically on the number and size of the polypoid lesions (endoscopy and then post-mortem autopsy material), histologically on sections stained with haematoxylin-eosin and through immunohistochemistry tests to evaluate the expression of the estrogen receptors, ER-alpha and ER-beta, the cell proliferation and migration along the axis of the crypts and/or villi (Brd-U assay) and apoptosis (TUNEL assay).

The objective of the study was to:

• evaluate the presence of any side effects linked to diet (fecalith, gastroenteritis, mortality);

• evaluate the effect of the THD mixture on the intestinal polypoid lesions that characterize the disease simulated by the animal model;

• monitor the intestinal inflammatory state through optical microscopy and through the dosage of inflammatory cytokines;

• monitor the mucosal expression of the alpha and beta estrogen receptors (ER), correlating them with cell proliferation and migration, as well as with the apoptosis of the intestinal epithelium.

Animal Models 150 mice were used overall: 1 10 wild type mice of the strain C57BL/6J and 40 genetically modified mice (APC/min) at 8 weeks of age and of the male sex. Of the wild type mice, 40 were used for the control group (Safety Test) and 70 were used as a model wherein intestinal carcinoma was induced on an inflammatory basis (AOM/DSS). The APC/min mice, presenting a heterozygote mutation of the APC gene (oncosuppressor), represented the model wherein a condition spontaneously develops which simulates human familial adenomatous polyposis (FAP).

Of the 1 10 animals, 40 (20 receiving standard diet and 20 receiving THD diet) were used for the preliminary assessment of the safety of dietary supplement. The remaining 70 animals (35 receiving standard diet and 35 THD diet) were used for the evaluation of anti-inflammatory and chemoprevention effect of enriched formulation. The feeding procedures, used in both phases of the study, were as follows:

i. Standard diet (Harlan Teklad Rodent diet): 18.5% proteins, 3% oils and fats, 6% fibers, 7% crude ash and 65.5% of non-nitrogenous compounds— wheat, maize, toasted soybean meal, corn gluten feed, wheat straw, fish meal, lucerne meal, mineral dicalcium phosphate, calcium carbonate, sodium chloride, whey powder, soybean oil, yeast and hazelnut skins, poly-vitamin complex (Mucedola srl, Settimo Milanese, Milan, Italy).

ii. THD diet (or THD mixture): the standard diet was enriched by a formulation containing silymarin (4 g %), AKBA (3 g %), curcumin (2 g %), maltodextrins (69.553 g %) and excipients (soluble fibers 16.667 g %, citric acid 1 g %, silicon dioxide 1 g %, lignans 0.5 g %, sucralose 0.280 g % and orange flavour 2 g %) and was administered at the cumulative dose of 22.4 mg/100 g of body weight.

For the control group, 40 male mice at 8 weeks of age were used, strain C57BL/6J (Charles River), wild type. Immediately after their arrival, the mice were kept in compliance with the established rules and left to settle for about two weeks. During this period, there was a mortality rate of 3 mice out of 40.

Once the mice were situated, the study passed to the real and proper experimental phase. The mice were divided into two groups. One group was administered the standard diet and the other was administered the mixture of phytoestrogens at the concentration of 224 mg/Kg. The schedule of the test is summarized in figure 1.

The animals were monitored daily. Upon killing, the parameters shown in table 1 were evaluated.

Table 1

In summary, after about 30 days from the start of the administration of the diet 6 mice were killed, 3 belonging to the standard diet group and 3 belonging to the special diet group. This made it possible to verify that both the standard diet and the THD diet did not provoke intestinal disorders (fecalith); the remaining mice were killed around the 100th day to exactly reproduce the control of the experimental model of the second group (azoxymethane - AOM + Dextran sulfate sodium - DSS).

AOM/DSS model

For the AOM/DSS group, 70 male mice at 8 weeks of age were used, of the strain C57BL/6J (Charles River), wild type, with the same characteristics as those of the Safety Test. These animals were subjected to treatment with Azoxymethane (AOM) + Dextran sulfate sodium (DSS) to induce the intestinal carcinoma on an inflammatory basis. Immediately after their arrival, the mice were kept in compliance with the established rules and left to settle for about 2 weeks. Once the mice were situated, the study passed to the real and proper experimental phase. The animals were divided into two groups, one group was administered the standard diet and the other group was administered the THD diet at a concentration of 224 mg/Kg.

All mice of both groups were administered an intraperitoneal injection of AOM at a concentration of 10mg/Kg. After 7 days DSS was administered in water at 2% for 7 days.

This same treatment was repeated after one week from the end of the treatment with DSS. The intraperitoneal administration of AOM was again given at a concentration of 5mg/Kg and after 7 days DSS was administered in water at 2% for 7 days. After about 2 weeks, the third and final treatment of DSS was administered at 2% for 7 days. The animals were monitored daily.

On the 100th day the endoscopic examination (colonoscopy) was performed to monitor the state of health of the colon of the two groups of mice and to view the development of solid lesions.

On the 1 10th day the animals were sacrificed and their colon, the location of the pathology, was removed. The colon was adequately cleaned from faeces, cut along the medial line and carefully observed through a lens, with 7X magnification. The number of lesions was counted, and their size was detected using a sliding calliper fitted with a scale in millimeters. Each tissue was photographed. Small portions of the tissues were removed for molecular biology investigations. The entire remaining part was appropriately treated for immunohistochemistry investigations. The experimental design is summarized in figure 2.

Endoscopy and Histology

In order to perform the endoscopic examination, the mice were suitably prepared. 24 hours before the execution of the exam, the mice were administered an electrolytic solution for gastrointestinal washing with a base of macrogol 3g, simeticon 0.29g, anhydrous sodium sulphate 0.09g, sodium bicarbonate 0.07g and sodium chloride 0.04g (SELG® 1000, Promefarm). The dose was calculated on the basis of the standard parameters used for man. Shortly before the examination, the animals were sedated by injection of Megaxilor (Xylazine) 0.5ml/kg + Zoletil (Zolazepam+Tiletamine) 0.8 ml/kg. The endoscopic examination was carried out using a STORZ paediatric endoscope.

The number of lesions and their development was detected following the animals’ killing. The entire colon was fixed in formalin and embedded in paraffin for the standard histological examination and immunohistochemistry evaluations.

RESULTS

General data

The mice of the AOM/DSS group showed a state of health which was compatible with the treatment given; toward the end of the treatment certain animals presented a prolapse of the rectum. As regards mortality, only one death was observed in the THD diet group (2.8%) and two deaths in the standard group (5.7%).

Macroscopic data

The colon of the mice that ate the standard diet appeared shorter and also more inflamed with respect to the colon of the mice that ate the THD diet (table 2).

Table 2

The group of mice that had eaten the standard diet developed on average 6.0 ± 1.9 lesions at the level of the colon, while the group of mice that had eaten the THD diet developed on average 2.08 ± 1.8 lesions (figure 3).

The average number of lesions indicated above also includes a group of animals wherein the absence of solid lesions of the colon was registered. In fact, 28% of the animals of the THD group did not present any solid lesions of the colon, compared to 0% of the standard group animals. The dimensions of the solid lesions are summarized and compared in the two groups in table 3.

Table 3: Percentage of solid lesions of the colon divided by size

P value< 0.0001 (chi-squared test for trend)

Microscopic data

The histological preparations were viewed with an optical microscope; the reading occurred in double-blind by two expert observers.

A histological score of inflammatory damage was detected which was equal to 3.6 ± 1.1 in the standard group and 2.9 ± 1.3 in the THD group (p< 0.05). (Figure 4).

The average number per animal of areas of low-grade dysplasia (LGD) was equal to 3.5 ± 0.8 in the standard group and 2.6 ± 0.5 in the THD group (p < 0.05). The average number per animal of areas of high-grade dysplasia (HGD) was equal to 1.8 ± 0.2 in the standard group and 0.30 ± 0.17 in the THD group (p < 0.001 ). The number of sites of colorectal cancer (CRC) detected was equal to 1.84 ± 0.43 in the standard group and 0.38 ± 0.21 in the THD group (p< 0.005). (Figure 5).

Finally, CRC was observed in 70.1% of the animals in the standard group and in 23.1 % of the animals in the TFID group. The results reported above are summarized in figure 5.

The mucosal expression of cytokines such as IFN-gamma, interleukin-6 and TNF- alpha is also higher in the colon of the animals in the standard group than in the animals of the TFID group, confirming that the inflammatory process, chemically induced for both groups, is attenuated by the presence of substances present in the TFID diet (table 4).

Table 4: expression of tissue cytokines evaluated through real-time PCR on tissues (fold change: mean ± SD).

Estrogen Receptors (ERs)

The data obtained as a result of the reading of the histological preparations with the optical microscope were expressed as a labelling index (% of positive cells for the staining of the receptor - LI).

The statistical analysis (ANOVA plus Bonferroni) of the expression of the alpha receptors highlighted that: N (normal tissue) = LGD< FIGD< CRC in the standard group (p O.001 ; fig. 6a) and N= LGD< FIGD= CRC in the TFID group (p O.001 , fig. 6b).

The results of the immunohistochemistry evaluation for ER-alpha are summarized in figure 6.

The statistical analysis (ANOVA plus Bonferroni) of the expression of the beta receptors showed that: N> LGD> FIGD= CRC in the standard group and N= LGD> HGD> CRC in the THD group

In particular, it was observed that the diet containing the THD mixture induces greater expression of ER-beta in the LGD areas and a lower expression of ER-beta in the CRC areas.

The results of the immunohistochemistry evaluation for ER-beta are summarized in figure 7.

As can be seen in figure 8, the ER-alpha/ER-beta ratio shows statistically significant differences only in the CRC areas.

Cell proliferation and migration

Cell proliferation was assessed by immunohistochemical staining of the nuclei after the intraperitoneal injection of an analogue of thymidine, i.e. Bromodeoxyuridine (Brd- U), which highlights the cells that are in the S phase (DNA synthesis) of the cell cycle. The animals were killed at 24, 48, 72 and 96 hours after injection of the same substance.

In both standard and THD diet groups, we observed in normal mucosa that positive cells were confined in the lower half of the crypts after 24 h, whereas a progression towards the upper half was observed after 48 h. In adddition, in the standard but not in the THD diet group, we found still few positive cells near the free surface at 72nd and 96th h.

Figure 9 illustrates the percentage of crypt axis covered by the highest labelled cell at the 48th hour after injection. This parameter was significantly higher in the THD than in the standard diet group (80.6% ± 5.1% versus 61.7% ± 5.2%, respectively; P O.001 ).

In LGD areas of the two groups, we found positive cells in both lower and upper half even at 24th hour. The same picture was seen after 48, 72 and 96 h with a decreasing LI.

In HGD and CRC, the complete derangement of the crypt architecture did not allow to evaluate the position of BrdU-positive cells.

Epithelial apoptosis

Apoptosis was evaluated through TUNEL staining in immunofluorescence with observation through confocal microscopy (figure 10 a, b, c).

The statistical analysis (ANOVA plus Bonferroni) showed: N< LGD> HGD= CRC in the standard group (p<0.0001 ; fig. 10a) and N< LGD> HGD> CRC in the THD group (p O.0001 , fig. 10b).

The THD mixture induces a significant increase in epithelial apoptosis in LGD areas (p = 0.0005), as summarized in figure 10c.

This data is in line with that of the previous paragraph concerning the migration of epithelial cells.

APC/MIN Model

40 male mice of 8 weeks of age (Charles River) were used for this study, genetically modified for the APC gene, as previously mentioned. Immediately after their arrival, the mice were kept in compliance with the established rules and left to settle for about 2 weeks. Once the mice were situated, the study passed to the real and proper experimental phase. The mice were divided into two groups, one group was administered a standard diet rich in fat (5K20, Mucedola) and the other group was administered the diet rich in fat, in addition to the THD mixture at a concentration of 224 mg/Kg.

On the 100th day the endoscopic examination (colonoscopy) was performed to monitor the state of health of the colon of the two groups of mice and to evaluate any development of solid lesions in the intestinal lumen.

On the 110th day the animals were sacrificed, and the tissues of interest were removed: the small intestine and colon. These were removed and adequately cleaned from faeces. Both the small intestine and colon were cut along the medial line and carefully observed with a 7X magnifying lens; the number of lesions was evaluated, and their size was detected using sliding callipers fitted with a scale graduated in millimetres. Each tissue was photographed. Small portions of the various tissues were removed for molecular biology investigations. The small intestine of each animal was finally entirely embedded in paraffin for the histological and immunohistochemistry investigations. The schedule of the experiment is summarized in figure 1 1.

The mice of the APC/min group showed a state of health which was compatible with the pathology under way; toward the end of the treatment certain animals presented a prolapse of the rectum. The results of the evaluations thus far carried out are reported in the tables that follow. RESULTS

General Data

The weight (m ± SD/animal) showed no significant differences among the two groups at the time of killing (table 5).

Table 5: Weight upon killing:

p not significant (p= 1)

Mortality during the course of treatment showed no significant differences between the two groups: THD diet group 4/20 (20%) - STANDARD diet group 4/20 (20%) Macroscopic data

Small intestine length: this parameter indicates fibrosis of the organ as a result of an extended inflammation over time. In this case no significant difference was observed in the length of the intestine (table 6), and the phenomenon is explained by the absence of significant inflammation in this animal model, as also detected from the data relating to the dosage of cytokines and the histological scores of inflammatory damages (reported in table 13 in the context of the microscopic findings).

Table 6: small intestine length (m ± SD/animal)

p not significant (p= 0, 168)

Expression of the inflammatory cytokines: a significant difference was not observed in the expression of cytokines, confirming that the dysplastic and neoplastic lesions do not have an inflammatory basis but a genetic one (table 7). Table 7: expression of tissue cytokines evaluated through real-time polymerase- chain-reaction (RT-PCT) on tissues

(fold-change: mean ± SD).

p not significant

Total number of solid lesions in the colon: in the animal model the solid lesions in this area are sporadic, unlike man, in whom they are so numerous that they require a colectomy (table 8). No differences were found between the two groups in relation to the number of lesions < 1 /animal.

Table 8: total number of lesions in the colon

p not significant (p=0.54) Total number of solid lesions in the small intestine: most of the lesions in the animal model develop in this area (table 9). As can be seen, the assumption of the THD diet significantly reduces the total number of solid lesions/animal (m ± SD).

Table 9: total number of solid lesions in the small intestine

Significant p (1.07^*10-⁵) Percentage of solid lesions in the small intestine divided by size: also in this case, the THD diet significantly reduces the size of the solid lesions in the small intestine.

Table 10: Percentage of solid lesions in the small intestine

divided by size

chi-squared test for trend: p<0.0001

Macroscopic data

Inflammatory damage. A histological score of inflammatory damage was detected (Changhui et al, Eur J Pharmacol 2014) which was similar in the two groups and indicative of the absence of significant inflammation (table 1 1 ).

Table 11 : histological scores of inflammatory damage

p not significant

Areas of dysplasia and/or neoplasia

The average number per animal of areas of low-grade dysplasia (LGD) was significantly higher in the THD group with respect to the control group (p < 0.05). The average number per animal of high-grade dysplasia (HGD) areas was equal to 4. 5 ± 1.04 in the standard group and 3.50 ± 1.7 in the THD group (difference not statistically significant). The number of colorectal cancer sites (CRC) detected was equal to 4.4 ± 0.54 in the standard group and 2.5 ± 0.57 in the THD group (p< 0.05). (tables 12a, 12b, 12c respectively). Table 12a: Areas of low-grade dysplasia (LGD - mean ± SD/animal)

p 0.03

Table 12b: Areas of high-grade dysplasia (HGD - mean ± SD/animal)

p = 0.28

Table 12c: Areas of neoplasia (CRC - mean ± SD/animal)

p = 0.04 Cell migration along the axis of the intestinal villi

The migration speed of cells toward the free surface along the axis of the intestinal villi was performed by immunofluorescence and the evaluation was carried out with confocal microscopy after 48 hours, at which time the marked cells are detectable not only in the crypts and in the lower part of the villi, but also all along its entire axis. In addition, the evaluation was carried out on the normal tissue areas, where the cell proliferation and migration are not subverted by the structural alterations that characterize the other stages of carcinogenesis. The evaluation was performed in accordance with the method described by Javid et al. (2005).

On 10 well-oriented villi, the marked cell located higher than the other cells was evaluated, and its position was expressed with a numeric value related to the percentage of the height of the villi corresponding to the seat of the cell itself. The results of this analysis are shown in figure 12. As can be seen from the same, this parameter is significantly higher in the THD group than in the standard group (82.22 ± 6.38 versus 65.31 ± 5.63; p < 0.001 ).

Epithelial apoptosis Apoptosis was evaluated through TUNEL staining in immunofluorescence with observation through confocal microscopy (figure 13). The statistical analysis (ANOVA plus Bonferroni) showed: N< LGD> HGD= CRC in the standard group and N< LGD> HGD> CRC in the THD group. It is clearly a significant difference of the epithelial apoptosis in the LGD areas (p = 0.0005) favoring the group of animals that ate the diet containing the THD mixture. Therefore, the THD mixture increases the phenomenon of cell apoptosis.

Claims

1. A composition comprising at least one selective phytoestrogen agonist of the ER- beta receptor and at least one soluble fiber for use in the treatment or in the prevention or in the follow-up of inflammatory pathologies of the gastro-intestinal tract and/or adenopolyposis and/or colorectal cancer.

2. The composition for use according to claims 1 , wherein the at least one phytoestrogen agonist of the ER-beta receptor is present in a concentration comprised between 0.5 and 15.0%, preferably between 1.0 and 13.0%, even more preferably between 2.0 and 8.0% by weight.

3. The composition for use according to any one of claims 1 -2, wherein the at least one phytoestrogen agonist of the ER-beta receptor is selected from a group consisting of silymarin, silybin, isosilybin, silydianin, silychristin, the isoflavone genistein, the flavonoid resveratrol, coumestrol and mixtures thereof.

4. The composition for use according to any one of claims 1 -3, wherein the at least one soluble fiber is a digestion-resistant dextrin and/or a digestion-resistant maltodextrin, preferably produced by the hydrolysis of corn starch.

5. The composition for use according to any of claims 1 -4, wherein the at least one soluble fiber is present in a concentration comprised between 7 and 40%, preferably between 10 and 30%, even more preferably between 12 and 20% by weight.

6. The composition for use according to any one of claims 1 -5, comprising a polyphenolic extract and/or a triterpenes-containing extract.

7. The composition for use according to claim 6, wherein the polyphenolic extract is a curcuminoid extract, preferably selected among: curcumin, demethoxycurcumin, bisdemethoxycurcumin and mixtures thereof, preferably is curcumin.

8. The composition for use according to any one of claims 6-7, wherein the polyphenolic extract is present in a concentration comprised between 0.2 and 10%, preferably between 0.5 and 8%, even more preferably between 1 and 6% by weight.

9. The composition for use according to any one of claims 6-8, wherein the triterpenes-containing extract is an extract of Boswellia serrata, comprising triterpenic acids, the triterpenic acids preferably comprising acetyl-1 l -keto-b- boswellic acid (AKBA), in a preferred concentration comprised between 0.05 and 5%, more preferably between 0.1 and 3% by weight.

10. The composition for use according to any one of claims 6-9, wherein the composition comprises acetyl-1 1 -keto-p-boswellic acid in a concentration comprised between 0.05 and 5%, preferably between 0.1 and 3% by weight.

1 1. The composition for use according to any one of claims 1 -10, comprising lignans derived from seeds selected among: sunflower seeds, pumpkin seeds and flax seeds, preferably derived from flax seeds.

12. The composition for use according to claim 11 , wherein the lignans are present in a concentration comprised between 0.01 and 5%, preferably between 0.05 and 3%, even more preferably between 0.1 and 2% by weight.

13. The composition for use according to any one of claims 1 -12, further comprising at least one excipient for pharmaceutical use or cosmetic use.

14. The composition for use according to any one of claims 1 -13, further comprising a flavoring agent, preferably selected among: essential oils, synthetic flavors or mixtures, citrus oil, essences of fruit, peppermint oil, mint oil, other mint oils, clove oil, sugar oil, anise and the like, aromatic oils, menthol, eucalyptus, thymol, spices such as cocoa powder and ground cinnamon, and combinations thereof.

15. The composition for use according to any one of claims 1 -14, further comprising a sweetening agent preferably selected from: sucrose, dextrose, maltose, dextrin, fructose, laevulose, galactose, sucralose, sorbitol, natural sweeteners such as, for example, stevia, corn syrup and the like, alone or in any combination.

16. The composition for use according to any one of claims 1 -15, formulated for oral use, preferably as tablets, capsules, bars, granular powder, oral granules, sachets, candies and drinkable formulations, for example drinkable vials or beverages.

17. The composition for use according to any one of claims 1 -15, formulated for enteral and/or parenteral administration, preferably in liquid form, more preferably in the form of a sterile solution, emulsion or suspension.

18. The composition for use according to any one of claims 1 -17, taken as a single dose or as multiple daily doses in a continuous way or according to need.

19. The composition for use according to any one of claims 1 -18, taken at a total daily dose of equal to or less than 10000 mg/die.

20. The composition for use according to any one of claims 1 -19, in combination with drugs used in therapy for the treatment of inflammatory pathologies of the gastrointestinal tract, preferably selected from: mesalazine, steroids, biologies anti TNF-alpha and their combinations.

21. The composition for use according to any one of claims 1 -19, in combination with drugs used in therapy for the treatment of adenopolyposis and/or colorectal cancer.

22. The composition for use according to any one of claims 1 -19, administered in association and/or in combination with a surgical treatment, preferably for the treatment of adenopolyposis and/or colorectal cancer, more preferably before and/or after a polypectomy.