DIFFERENTIATION MARKERS AND MICROBIOTA IN PATIENTS WITH

ULCERATIVE COLITIS

CROSS-REFERENCE TO RELATED APPLICATION

[001] The present application claims priority to U.S. Provisional Patent Application Serial No. 62/458,715, filed on February 14, 2017, the contents of which is incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[002] Technical Field

[003] This invention relates to the use of eicosapentaenoic acid (EPA) for the treatment of ulcerative colitis (UC), and more particularly, the use of highly purified eicosapentaenoic acid as free fatty acids (EPA-FFA) for reducing inflammation, and modifying microbiota in a subject suffering from ulcerative colitis.

[004] Related Art

[005] Patients with ulcerative colitis (UC) have an increased risk to develop colitis- associated cancer (CAC) which is proportionally related to the duration and the extent of the disease (1). Current strategies to prevent CAC development are mainly based on endoscopic surveillance in order to intercept and eradicate dysplasia which can evolve to a malignant transformation (2). However, persistent active intestinal inflammation may hamper the identification of dysplastic areas during endoscopy. Thus, despite the reduction of advanced cancer incidence rates, obtained through a regular endoscopic surveillance, critical goals for CAC prevention remain to preserve a condition of histological remission (3, 4), and to have predictive markers indicating those patients in whom endoscopic surveillance would be more effective. Fecal calprotectin (FC) is a cytosolic protein belonging to the SI 00 protein family, abundant in neutrophil granulocytes (5), which represents a good predictor of endoscopic activity also in asymptomatic UC patients (6).

[006] Several relevant molecular mechanisms contribute to the malignant epithelial transformation during chronic intestinal inflammation. Among these, aberrant activation of the signal transducer and activator of transcription 3 (STAT3), Interleukin (IL)-IO deficiency or impaired function are critically involved in the onset of CAC7, (8).

[007] Moreover, a thin and penetrable mucus layer, allowing a direct contact of bacteria with the epithelium, can lead to persistent colonic inflammation, thus promoting colon cancer development in UC patients (9). Indeed, an over-growth of mucosal and fecal bacteria in inflamed colonic mucosa has been observed in UC patients, thus supporting a critical role of the intestinal microbiota in the pathogenesis of UC and progression to CAC (10, 11).

[008] The canonical Notch signaling pathway, through the modulation of the transcriptional target hairy and enhancer of split 1 (HESl), the antagonists atonal homolog 1 (HATHl) and kruppel-like factor 4 (KLF4) target, is crucial to preserve a proper intestinal differentiation (12, 13). The present inventors recently proposed a tumor suppressor function of HESl during CAC progression (14), while the role of KLF4 in CAC is still controversial (15). The abnormal regulation of these transcriptional factors result in a compromised epithelial differentiation which can lead to an inefficient control of pathogenic microbes growth, favoring a tumor-prone microenvironment (16).

[009] The use of anti-inflammatory agents as tools for CAC prevention has been an intense focus of research (17, 18). To date, there are no uncontested chemopreventive agents for CAC. Long-standing ulcerative colitis patients are at high-risk of developing colorectal cancer (CAC). Ulcerative colitis (UC) is a chronic inflammatory condition affecting the colon, characterized by alternating periods of activity and remission. The clinical activity is always preceded by a progressive asymptomatic mucosal inflammation. Importantly, the treatment of UC may inhibit or reduce the development of cancer. [0010] Accordingly, there is a need for a treatment that has the ability to reduce and/or treat the UC with the end result of reducing inflammation and effecting both goblet cells differentiation markers and microbiota in patients suffering from ulcerative colitis.

SUMMARY OF THE INVENTION

[0011] This present invention relates to the use of the use of highly purified eicosapentaenoic acid as free fatty acids (EPA-FFA) having a purity of at least 95% purity and more preferably 99% purity for reducing inflammation in a subject suffering from ulcerative colitis, increasing levels of IL-10 of SOCS-3, induction of Hes-1 and KLF-4 associated with goblet cells differentiation and favorably modulating the microbiome of the intestinal mucosal tissue in such a subject.

[0012] In another aspect, the present invention provides a method of inducing and increasing levels of IL-10 and suppressor of cytokine signaling-3 (SOCS3), the method comprising administering to a subject suffering from ulcerative colitis, a therapeutic amount of eicosapentaenoic acid in the free fatty acid (EPA-FFA) form having purity of at least 95%, and more preferably at least 99%, wherein the therapeutic amount is in an amount from about 250 mg to 4 g per day, and more preferably in an amount from about 600 mg to about 2 g per day.

[0013] Notably with the increase of SOCS3, there is a committal partial inhibition of signal transducer and activator of transcription-3 (STAT3) activation.

[0014] Preferably the EPA-FFA is administered for about 45 days to at least 6 months and more preferably about 90 days.

[0015] In yet another aspect, the present invention provides a method to modulate the intestinal microbiota of the mucosal tissue in a subject suffering from ulcerative colitis, the method comprising administering to a subject a therapeutic amount of eicosapentaenoic acid in the free fatty acid (EPA-FFA) form having purity of at least 95%), and more preferably at least 99%, wherein the therapeutic amount is in an amount from about 250 mg to 4 g per day, and more preferably in an amount from about 600 mg to about 2 g per day, for a period of about 45 days to at least 6 months and more preferably about 90 days, wherein the levels of fecal Prevotellaceae and Porphyromonadaceae families are increased and the level of mucolytic Bacteroides spp at mucosal level is decreased.

[0016] In a still further aspect, the present invention provides for a method of inducing of KLF-4 to promote goblet cells differentiation, the method comprising the administering to a subject suffering from ulcerative colitis a therapeutic amount of eicosapentaenoic acid in the free fatty acid (EPA-FFA) form having purity of at least 95%, and more preferably at least 99%, wherein the therapeutic amount is in an amount from about 250 mg to 4 g per day, and more preferably in an amount from about 600 mg to about 2 g per day, for a period of preferably about 45 days to at least 6 months and more preferably about 90 days.

[0017] In another aspect, the present invention provides for use of eicosapentaenoic acid in the free fatty acid (EPA-FFA) form having purity of at least 95%, and more preferably at least 99% for the increase of levels of IL-10 or SOCS-3, induction of Hes- 1 and KLF-4 associated with goblet cells differentiation and favorably modulating the microbiome of the intestinal mucosal tissue in a subject suffering from UC, the EPA- FFA is in an amount from about 250 mg to 4 g per day, and more preferably in an amount from about 600 mg to about 2 g per day, for a period of preferably about 45 days to at least 6 months and more preferably about 90 days.

[0018] These and other advantages and features of the present invention will be described more fully in a detailed description of the preferred embodiments which follows.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] Figure 1 shows (a) Eicosapentaenoic acid (EPA; C20:5 n-3) percentage in RBCs and (b) FC levels ^g/g) (B) in all patients (n = 19) at TO and T3. Statistical significance was calculated using the paired two-tailed t-test. Data are shown as mean ± SEM. [0021] Figure 2 shows (a) Mayo endoscopic score and (b) Geboes histological score in compliant and responder patients (n = 15) at TO and T3. Data are presented as percentage of patients according to Mayo and Geboes cut-offs.

[0022] Figure 3 shows mRNA expression levels of (a) IL-10 and (b) SOCS3. Protein levels of (c) p-STAT3/STAT3 on homogenized sigmoid colon tissues in compliant and responder patients (n = 15) at TO and T3. Statistical significance was obtained using one-sample two-tailed t-test. Data are shown as mean of square root transformed values ± SEM. (d) Western blot representative images of p-STAT3 (Y705) and STAT3 at TO and T3 (n = 3).

[0023] Figure 4 shows protein expression levels of (a) FIES1 and (b) KLF4 on homogenized sigmoid colon tissues in compliant and responder patients (n = 15) at TO and T3. Statistical significance was measured using one-sample two-tailed t-test. Data are shown as mean of square root transformed values ± SEM. (c) Western blot representative images of FIES1 and KLF4 at TO and T3 (n = 3). (d) Alcian blue ranks and (e) representative images of goblet cells staining at TO (left panel) and T3 (right panel). Statistical significance for Alcian blue ranks was calculated using the paired two-tailed t-test. Data are shown as mean of ranks ± SEM.

[0024] Figure 5 shows median fecal microbiota composition at family level in (a) healthy adults, (b) UC patients at TO, (c) UC patient at T3 and colon biopsies of (d) UC patients at TO and (e) UC patients at T3, represented as pie chart, in available samples from compliant and responder patients. Average relative abundance of families representing at least 0.2% of the total microbiota in at least 10% of the sequenced samples are showed. Color code for the most abundant bacterial families (present at an average abundance > 1% in at least one group of samples) is reported in approximate decreasing abundance order. Mann-Whitney U test was used to test differences among median groups.

[0025] Figure 6 shows (a) ω-3 PUFAs (EPA+DPA+DHA) and b) ω-6 PUFAs (arachidonic + linoleic acids) percentage in RBCs in all patients (n=19) at TO and T3. Statistical significance was calculated using the paired two-tailed t-test. Data are shown as mean ± SEM. [0026] Figure 7 shows mRNA expression levels of IL-22 in compliant and responder patients (n =15) at TO and T3. Data are shown as mean of square root transformed values ± SEM.

[0027] Figure 8 shows protein levels of (a) IL-10 and (b) SOCS3 on homogenized sigmoid colon tissues in compliant and responder patients (n=15) at TO and T3. Data are shown as mean of square root transformed values ± SEM.

[0028] Figure 9 shows mRNA expression levels of STAT3 in compliant and responder patients (n =15) at TO and T3. Data are shown as mean of square root transformed values ± SEM.

[0029] Figure 10 shows Spearman's correlation analysis of SOCS3 mRNA with (a) IL- 10 mRNA and (b) p-STAT3/STAT3 proteins in compliant and responder patients (n=15) at T3.

[0030] Figure 11 shows mRNA expression levels of (a) FIES1 and (b) KLF4 in compliant and responder patients (n =15) at TO and T3. Data are shown as mean of square root transformed values ± SEM.

[0031] Figure 12 shows Spearman's correlation analysis of FIES1 and KLF-4 proteins in compliant and responder patients (n=15) at T3.

[0032] Figure 13 shows (a) mRNA expression levels of MUC2 in compliant and responders patients (n =15). Data are shown as mean of square root transformed values ± SEM. (b) MUC2 protein analyzed by unohistochemistry at TO and T3 in compliant and responder patients (n =15). Data are shown as percentage of MUC2 positive area/total area.

[0033] Figure 14 shows (a) Ki-67 protein analyzed by immunohistochemistry at TO and T3 in compliant and responders patients (n=15). Data are shown as percentage of Ki-67 positive/total nuclei. Expression of (b) C-MYC and (c) LGR5 mRNAs in compliant and responder patients (n=15). Data are shown as mean of square root transformed values ± SEM.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention is based on the findings that administration of EPA-FFA is effective in vivo in reducing the development of colorectal cancer in subjects suffering of ulcerative colitis. Further, the present invention provides for a modulating of the microbiome of the intestinal track. Still further the present invention provides for induction of IL-10 and SOCS3 while partially inhibiting the activation of STAT3.

[0036] Preferably, the EPA-FFA used for making the medical preparations, medicaments or compositions in accordance with the invention is of at least about 90% purity and will contain no more than minimal or pharmaceutically insignificant amounts of any other polyunsaturated fatty acids. A purity of more than 95% is recommended with the highest commercially available grade (about 99% purity), which is substantially free of any other polyunsaturated fatty acids, being the most preferred material.

[0037] Thus, according to one aspect of the present invention, highly purified eicosapentaenoic acid as a free fatty acid is used to make a medical preparation or medicament for the treatment of malignant tumors in mammals.

[0038] EPA may be found in fish oil, plants or microorganisms as free fatty acids or in conjugated forms such as acyl glycerols, phospholipids, sulfolipids or glycolipids, and may be extracted through a variety of means well-known in the art. Such means may include extraction with organic solvents, such as methanol and chloroform, sonication, supercritical fluid extraction using for example carbon dioxide, and physical means such as presses, or combinations thereof. Where desirable, the aqueous layer can be acidified to protonate negatively charged moieties and thereby increase partitioning of desired products into the organic layer. After extraction, the organic solvents can be removed by evaporation under a stream of nitrogen. When isolated in conjugated forms, the products may be enzymatically or chemically cleaved to release the free fatty acid or a less complex conjugate of interest, and can then be subject to further manipulations to produce a desired end product.

[0039] If further purification is necessary, standard methods can be employed. Such methods may include extraction, treatment with urea, fractional crystallization, HPLC, fractional distillation, silica gel chromatography, high speed centrifugation or distillation, or combinations of these techniques. Protection of reactive groups, such as the acid or alkenyl groups, may be done at any step through known techniques, for example alkylation or iodination. Protecting groups may be removed at any step. Desirably, purification of fractions containing EPA may be accomplished by initial esterfication, treatment with urea, supercritical fluid extraction and chromatography with the subsequent isolation of the free fatty acid.

[0040] In order to isolate EPA from the triglyceride it is necessary to free the fatty acids by hydrolysis or ester exchange in order that purification can be effected. Purification can be achieved by techniques such as fractional distillation, molecular distillation and chromatography. A particularly desirable chromatographic method employs super-critical fluids using, for example, carbon dioxide as the mobile phase, such as described in European Patent EP 0 712 651. It has been found that using such techniques EPA may be purified to levels approaching 100 per cent. For practical reasons the EPA may be purified as its ethyl or methyl ester and hydrolyzed back to the free fatty acid form. Purification enables a product to be prepared which is highly concentrated and free from other fatty acids that are less desirable in the finished product. In addition other chemicals entities such as mono- and di-glycerides, hydrocarbons, pesticide residues and the like can be removed. The highly purified EPA is thus suitable for human ingestion as it contains substantially reduced levels of toxins, compounds contributing to unpalatability or undesirable fatty acids such as saturated fatty acids. The free fatty acid form of EPA can be absorbed in the gut easily without need of prior enzymatic conversion. Using this method about 150 kg of unpure EPA can be converted into 50 kg of essentially pure EPA-FFA, that being at least 90% purity.

[0041] If further purification is necessary, standard methods can be employed. Such methods may include extraction, treatment with urea, fractional crystallization, HPLC, fractional distillation, silica gel chromatography, high speed centrifugation or distillation, or combinations of these techniques. Protection of reactive groups, such as the acid or alkenyl groups, may be done at any step through known techniques, for example alkylation or iodination. Protecting groups may be removed at any step. Desirably, purification of fractions containing EPA may be accomplished by initial esterfication, treatment with urea, supercritical fluid extraction and chromatography with the subsequent isolation of the free fatty acid.

[0042] A preferred EPA free fatty acid is commercially available under the tradename ALFA™ (S.L.A. Pharma, UK). This PUFA is 99% pure EPA, in a free fatty acid form and formulated into a pH-dependent, enteric-coated capsules designed to ensure release of the contents in the small intestine at pH 5.5. Other constituents include AA (<0.5%) and trace amounts of other fatty acids. Key advantages of this preparation of EPA are its high degree of purity compared with many fish oil products, its presentation as the free fatty acid maximizing systemic bioavailability, ease of dosage in 500 mg capsules and a delayed-release profile, which minimizes gastro-intestinal side-effects.

[0043] Preferably the 99% pure EPA is administered in an amount from about 250 mg to 4 g per day and more preferably from about 600 mg to about 2 g daily. The dosage may be administered daily, weekly or longer for about 1 to 12 months. Notably, the tolerability of 99% pure EPA as ALFA™ capsules is excellent and the predominant small bowel delivery of EPA minimized any unpleasant taste and smell sensations that have previously hampered therapy with other fish oil preparations.

[0044] The EPA-FFA alone or in combination with another therapeutic agent used to treat UC may be formulated in multiple delivery modes. The active agent can be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0045] A solid composition form may include a solid carrier and one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredients. In tablets, the active ingredients are mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.

[0046] Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution); alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellent.

[0047] Having described the invention in detail, it will be apparent that modifications and variations are possible without departing the scope of the invention defined in the appended claims. Furthermore, it should be appreciated that the following examples are provided as non-limiting examples.

[0048] Examples [0049] In this study, twenty patients with long-standing UC were enrolled. After baseline colonoscopy, one patient presented a clinical relapse before starting EPA-FFA supplementation, and was excluded from the trial. Nineteen patients completed the study. The clini co-pathological features of all patients (n = 19) at TO are shown in Table 1. Noteworthy, during EPA-FFA supplementation, no clinical relapse was observed.

[0050] Specifically, 20 long-standing UC patients in stable clinical remission (SCCAI = 0) and with fecal calprotectin (FC) >150 μg/g measured in stools were recruited and treated with 2 g/day of EPA-FFA for 90 days. Biopsies and biological samples were collected at the entry (TO) and at the end of the study (T3). Compliance was evaluated by EPA incorporation into red blood cell membranes. Protein levels of Jaggedl, Hesl, STAT3, phospho-STAT3 and KLF-4 were determined by western blotting. IL-22, IL- 10 and SOCS3 mRNA levels were analyzed by qRT-PCR. Goblet cells were stained by Alcian blue. Microbiota analyses were performed on fecal and biopsies DNA samples sequencing the V3-V4 region of the bacterial 16S rRNA gene; as a reference, we employed a healthy adult population previously analyzed for microbiota composition. Endoscopic and histologic disease activities were measured by Mayo and Geboes scores respectively.

[0051] Fatty acids composition was evaluated on RBC-purified membranes. Compared to TO, EPA-FFA supplementation led to a significant increase of EPA (P < 0.0001; Figure la). The mean percentage values of EPA content changed from 0.26 at TO to 2.51 at T3. Capsules counting revealed that seventeen patients were adherent to treatment with an overall compliance of 89.5%. Since EPA can be converted into the co-3 PUFA docosahexaenoic acid (DHA) in vivo through docosapentaenoic acid (DPA)(19), the overall co-3 PUFAs content was also measured including EPA, DPA and DHA, in the patients. Interestingly, the combined percentage content of EPA, DPA and DHA was significantly increased at T3 compared to TO (P < 0.0001; Figure 6a), while the percentage content of co-6 PUFAs (arachidonic + linoleic acids) was unchanged upon EPA-FFA supplementation (Figure 6b). [0052] Importantly, a significant reduction of FC at T3 was observed (P < 0.0001; Figure lb). The mean FC values changed from 230 at TO to 87.7 μg/g at T3. No side effects or serious adverse events were reported during the trial. Two patients maintained FC levels >150 μg/g at T3 after treatment and were considered non- responders. EPA-FFA treatment significantly promoted endoscopic and histological remission in compliant and responder patients (n = 15).

[0053] Indeed, compared to baseline, endoscopic improvement was observed in 8 patients while no variations were observed in 7 (P = 0.004; Figure 2a). Moreover, a resolution of histological inflammation at T3 was observed in 5 patients, while the histological score remained unchanged in 10 (P = 0.03; Figure 2b). Endoscopic and histological worsening was not observed.

[0054] EPA-FFA supplementation induces both IL-10 and SOCS3 expression reducing STAT3 activation

[0055] To elucidate the mechanisms responsible for the protective effect of EPA-FFA in patients with long-standing UC, the modulation of the IL-10/STAT3/SOCS3 axis was investigated in compliant and responder patients (n = 15). Compared to TO, a concomitant significant up-regulation of IL-10 (P = 0.03; Figure 3a) and SOCS3 (P = 0.04; Figure 3b) mRNA levels at T3 was observed associated with an increasing trend in IL-22 mRNA (Figure 7). Otherwise, no significant differences in IL-10 and SOCS3 protein expressions were observed (Figure 8a and b). Since STAT3 represents one of the major regulators of SOCS3, it was decided to characterize STAT3 activation in these patients. Treatment with EPA-FFA reduced STAT3 Tyr705 phosphorylation (P-STAT3) in 60% of patients (9/15) (Figure 3c and d), while not affecting STAT3 transcription (Figure 9). Noteworthy, 4/5 patients showing highest levels of p-STAT3 at T3 were poor compliant patients with lower percentage of EPA in RBC after supplementation. These data suggest that over-expression of SOCS3 following EPA- FFA supplementation, probably as a downstream effect of IL-10 induction, reduces STAT3 activation, in particular in patients with highest percentage of EPA in RBC membranes. Correlation analyses revealed a significant positive correlation at T3 between transcriptional levels of SOCS3 and both IL-10 mRNA levels (P = 0.02; Figure 10a) and p-STAT3 protein (P = 0.03; Figure 10b), thus supporting the proposed hypothesis.

[0056] EPA-FFA supplementation modulates HES1 and KLF4 and stimulates goblet cell differentiation

[0057] Notch signaling, through the modulation of the transcriptional targets HES1 and KLF4, is crucial to preserve a proper balance between the absorptive and the secretory cell lineages of the intestine (13, 20). Notably, a significant up-regulation of HES1 (P = 0.02; Figure 4a and c) and KLF4 proteins (P = 0.04; Figure 4b and c) was observed in patients with long-standing UC at T3 compared to TO, while no differences were observed at mRNA level (Figure 11a and b). Correlation analysis indicated that these two transcription factors positively correlate with each other (P = 0.0007; Figure 12). Importantly, although no variations in the MUC2 mRNA (Figure 13a) and protein were found (Figure 13b), compared to TO, in which goblet cells depletion was found in 20% of patients, daily supplementation of EPA-FFA for 3 months was associated with a significant increased number of goblet cells in the colon (P = 0.04; Figure 4d and e). Thus, these results unveil a role of EPA-FFA in improving secretory lineage differentiation and intestinal epithelial cells turnover through simultaneous induction of KLF4 and FIES1. No differences was found in terms of intestinal proliferation measured by Ki-67 (Figure 14a), c-MYC (Figure 14b) and LGR5 upon EPA-FFA supplementation (Figure 14c).

[0058] EPA-FFA modulates the gut microbiota composition in UC patients

[0059] Given the critical role of intestinal microbial imbalance in the pathogenesis of UC, the fecal and mucosal microbiota compositions were also assessed in the patients. To identify the main microbiota dysbioses associated with the long-term UC disease, the fecal microbiota composition of UC patients at TO was compared to that of a group of Italian healthy adults (age 22-48 years, enrolled in the same geographical area of the UC patients) (21). An enrichment of the families Clostridiaceae (4.7 vs. 1%, P = 0.003; in particular genus SMB53, P = 0.001) and Ruminococcaceae (35.7 vs. 24.1%, P = 0.008), and depletion of Verrucomicrobiaceae (0 vs. 0.4%, P = 0.002; in particular genus Akkermansia, 0 vs. 0.4%, P = 0.002), Peptostreptococcaceae (0 vs. 0.3%, P = 0.0009) and Porphyromonadaceae (0 vs. 0.5%, P = 0.006; in particular genus Parabacteroides, 0 vs. 0.5%, P = 0.006) families was found in UC patients at TO (Figure 5a (healthy) and b (UC at TO)). Noteworthy EPA-FFA supplementation increased Porphyromonadaceae (from 0 to 0.2%) and decreased Ruminococcaceae (from 35.7 to 28%) (Figure 5b (UC at TO) and c (UC at T3)) in feces of UC patients. In addition, EPA-FFA had also effects on mucosal microbiota of UC patients by decreasing the abundance of mucosal-adherent members of the Bacteroidaceae family (in particular belonging to the genus Bacteroides, 21 A vs. 14.7%) (shown in the comparison of Figure 5d (UC colon at T0)and 5e (UC colon at T3)).

[0060] Discussion

[0061] Different therapeutic approaches have been tested for CAC prevention in patients with inflammatory bowel disease (IBD) over the years. Importantly, an increasing number of data obtained from in vitro experiments, as well as, animal and clinical studies support a protective role for co-3 PUFAs (EPA and DHA) in gastrointestinal cancer prevention including CRC (as reviewed by Eltweri et al.(22)).

[0062] However, data on pharmacological and natural compounds as anticancer agents in IBD patients are elusive and still inconsistent (23). It is well known that symptoms in IBD and serum biomarkers do not always properly mirror the inflammatory degree of the mucosa (24). FC is becoming the most useful non-invasive tool for monitoring the inflammatory status of the mucosa and the response to therapy, as well as for predicting clinical relapse in IBD patients (25).

[0063] In the present invention, for the first time, the effects of EPA-FFA was tested on asymptomatic patients with long-standing UC in clinical remission who retained high FC levels (> 150 μ^) despite stable maintenance therapy. It was found that short-term EPA-FFA supplementation at a dosage of 2 g/daily (90 days) was associated with a significant increase of EPA and overall co-3 PUFAs content (EPA, DPA and DHA) into RBCs, suggesting that EPA was incorporated by most of patients (17/19) and efficiently converted into DPA and DHA. [0064] Although the primary end-point was not to test the clinical benefit of EPA-FFA but to explore its effect on mucosal inflammation and new potential chemopreventive mechanisms during long-standing UC, it was found that short-term EPA-FFA supplementation reduced mucosal inflammation (with a significant drop in FC) favoring an improvement of both endoscopic and histological inflammation in almost all patients. Since FC levels have been demonstrated to correlate with the intensity of the neutrophilic infiltrate (12), the results found herein and supported by histological evaluation, indicate that EPA-FFA improved the inflammatory state in patients with long-standing UC. It is theorized that the results found herein could be explained, at least in part, by the free-fatty acid-highly pure formulation of EPA used in the testing methods.

[0065] Previous evidence support a protective role for co-3 PUFAs intake including both EPA and DHA in the prevention of CRC in different settings (26,27,28). However, data from co-3 PUFAs supplementation in patients and murine models of UC are still controversial (29,30,31), and the impact of dietary co-3 PUFAs supplementation for CAC prevention is poorly defined.

[0066] Given the increased content of co-3 PUFAs in the patients in this study, it is reasonable to speculate that the observed protective effects may be due to both EPA and DHA. Noteworthy, no relevant differences was found in the co-6 PUFAs content upon EPA-FFA supplementation. This result could be explained by the unchanged dietary habits of enrolled patients during the study. Strikingly, the increased co-3 PUFAs content was sufficient to induce a relevant protective response in UC patients, while possibly maintaining the same co-6 PUFAs content as previously suggested (32).

[0067] In this study, in order to characterize the EPA-FFA short-term effects in longstanding UC patients, the testing first focused on the effects of EPA-FFA supplementation on IL-10/STAT3/SOCS3 signaling. The role of STAT3 during UC is actually controversial. Indeed, studies on animal models of IBD suggested both a deleterious and protective role of STAT3 hyperactivation during colitis (33, 34). Importantly, increased levels of phospho-STAT3 were detected in patients with active UC, as well as in dysplasia and cancer, while a progressive decreasing trend of SOCS3 levels was observed from low-grade dysplasia to UC-CRC (35). However, more recent evidence obtained in UC patients supported a role of SOCS3 over-expression in short- term disease relapse and mucosal inflammation impairing STAT3 activation (36, 37). In this study, a concomitant significant up-regulation of IL-10 and SOCS3 mRNA was found upon EPA-FFA supplementation with a reduction of STAT3 activation in most of the patients with highest EPA percentage levels at T3. However, no changes in IL- 10 and SOCS3 proteins were appreciated upon EPA-FFA supplementation in the patients. As previously suggested by literature data, we hypothesized that multiple post-transcriptional mechanisms may contribute to regulate SOCS3 and IL-10 proteins, thus explaining the absence of a correlation between changes in their mRNA and protein levels (38,39,40,41,42).

[0068] Importantly, considering responders, no endoscopic or histological worsening in any patient was observed. Thus, the data discussed herein support a protective role of EPA-FFA during UC remission by turning off STAT3 activation through SOCS3 transcriptional induction.

[0069] Notch signaling is also a key determinant for sustaining intestinal epithelial cells differentiation and turnover, for the integrity of the mucosal barrier, as well as for regulating malignant epithelial transformation in the colon (20). Evidence show possible oncogenic and tumor suppressor activities of HES1 and KLF4 in sporadic settings, respectively (43, 44). It has been previously shown in the AOM-DSS mouse model a loss of Notchl signaling during CAC development partially counteracted by EPA-FFA supporting a tumor-suppressor role of this pathway during inflammation- induced intestinal tumorigenesis (14). Accordingly, Garg and colleagues previously demonstrated in the same animal model, that Matrix metalloproteinase-9 (MMP-9), activating Notchl signaling and controlling p53 cascade, exerts a strong protective effect toward CAC development (45). Otherwise, in a recent in vitro work, it was observed a MMP-9-dependent activation of Notchl signaling in CRC cells exposed to a conditioned medium (CM) containing multiple pro-inflammatory cytokines secreted by activated macrophages. The activation of MMP-9/Notch signaling was associated with increased CRC cells invasiveness, suggesting a tumor-prone role of Notchl signaling in sporadic CRC. Interestingly, EPA-FFA pre-treatment of CM-exposed CRC cell lines led to reduced invasion through a Notchl signaling switch off (46). These results, as recently reviewed (47), clearly indicate that the cell response to Notch signaling activation is not univocal resulting in oncogenic or tumor-suppressive mechanisms depending on the specific pathological context.

[0070] EPA-FFA modulated intestinal differentiation inducing both HESl and KLF4 proteins and increasing the number of goblet cells.

[0071] Patients with UC in remission are generally characterized by an intact mucus layer, although a defective and penetrable intestinal barrier could be retained in some cases (48). KLF4 has a crucial role on both maturation and differentiation of goblet cells in the colon (49), and a critical role for IL-10 in the regulation of goblet cells activation during inflammation has been also previously described (50). Moreover, microbiota analysis performed in the present testing shows that the gut microbiota population constituents present in the UC group at TO were partly modulated by the EPA-FFA treatment. Indeed, the Porphyromonadaceae genus Parabacteroides, known to be decreased in UC (51), was significantly increased in T3 samples compared to TO. Also it was shown an increase in Prevotellaceae. Also EPA-FFA showed the capability to reduce the fecal amount of Clostridium spp. compared to TO. Interestingly, these proteolytic microorganisms were known to induce mucolytic metabolism in other species, i.e. Bacteroides (52). Noteworthy, mucosal-adherent members of the Bacteroides genus, known to include mucolytic species, were found to be decreased after EPA-FFA treatment, possibly contributing to the protection of the epithelium. Thus, it is theorized that the ability of EPA-FFA treatment to promote goblet cells population could be a result of multiple mechanisms including the induction of KLF-4 and IL-10, as well as the reduction of mucolytic bacteria.

[0072] In conclusion, use of EPA-FFA improved endoscopic and histological inflammation, affected the IL-10/STAT3/SOCS3 cascade, stimulated goblet cells differentiation and modulated the long-term UC-related colonic alterations of intestinal microbiota.

[0073] Methods

[0074] Study design [0075] Eligible patients were asymptomatic subjects aged 18-70 years with longstanding (> 8 years) UC, in stable clinical remission (simple clinical colitis activity index; SCCAI = 0), and FC levels higher than 150 μg/g (53). Patients were included in the study after signing the informed written consent. Concomitant stable therapies for UC (mesalamine, immunomodulators and/or biological drugs) without modifications in the previous 3 months were allowed. Exclusion criteria were: (1) recent use of steroids (< 2 months) or other experimental drugs (< 3 months); (2) concomitant use of anticoagulants; (3) probiotic use; (4) pregnancy or breast-feeding; (5) known or suspected hypersensitivity to eicosapentaenoic acid or co-3 PUFAs; and (6) severe comorbidities. Subjects were given oral supplementation of 2 g/daily (two 500 mg capsules twice a day) of EPA-FFA (ALFA™, SLA Pharma AG, Switzerland) for 90 days. During the study, subjects were asked to keep their dietary habits. Patients underwent endoscopic examination at enrollment (TO) and after 90 days of EPA-FFA supplementation (T3). Six biopsies were taken from the sigmoid colon at each time point. Blood samples were obtained for isolation of peripheral erythrocytes. Adherence to EPA-FFA supplementation was evaluated both by capsule counting and assessing EPA incorporation into red blood cell (RBC) membranes. Compliant patients were considered those who consumed at least 80% of the capsules, without interruption of the protocol for more than 14 consecutive days. The study was conducted in accordance to the Declaration of Helsinki and approved by the Ethic Committee of the S.Orsola-Malpighi Hospital (Bologna, Italy).

[0076] Fecal calprotectin dosage

[0077] Fecal samples were collected within 24 hours before endoscopy and stored at 2- 8 °C until assaying. Quantification of FC was carried out using CalFast (Eurospital, Trieste, Italy) according to the manufacturer's protocol. FC values > 150 μg/g were considered predictive of mucosal endoscopic activity as previously demonstrated (53).

[0078] Endoscopic and histological evaluation

[0079] Two investigators performed all endoscopies. According to the Mayo endoscopic sub-score, a cut-off > 1 was used to discriminate the presence of endoscopic inflammation (54). Histological activity was assessed by one expert blinded pathologist (T.B.) and scored according to the Geboes grading system (55). A Geboes cut-off score > 3.1 was assumed to define active histological inflammation (56). When biopsies showed different degrees of activity, the highest degree of inflammation was considered.

[0080] Acidic mucins quantification

[0081] Formalin-fixed and paraffin-embedded (FFPE) biopsies were de-waxed in toluene for 10 minutes, rehydrated, placed in the Alcian blue solution (Alcian blue 8GX in 3% acetic acid solution pH 2.5) for 30 minutes and counterstained with hematoxylin. For analysis, slides were placed in order of increasing Alcian blue staining intensity using a rank order scoring system (1 = lower rank; 36 = higher rank). Rank ordering method has been shown to be better than categorical scoring system to identify subtle differences between groups (57).

[0082] Immunol stochemistry

[0083] Immunohi stochemistry (IHC) was performed on FFPE colonic sections. Slides were dewaxed, subjected to endogenous peroxidase inhibition, rehydrated and treated with citrate buffer (pH 6.0) at 120 °C for 15 minutes for antigen retrieval. Then, slides were incubated overnight at +4 °C with the monoclonal antibodies against Ki-67 and MUC2 (Table 2). After incubation with secondary antibody Rabbit/Mouse (1 : 1000, DAKO EnVision™ System Peroxidase), the signal was detected with diaminobenzidine (DAB) (Sigma-Aldrich, Saint Louis, Missouri, USA). Percentages of Ki67 positive nuclei and MUC2 positive DAB areas were quantified using ImageJ software (NIH, Bethesda, MD, USA).

[0084] Membrane fatty acid analysis

[0085] Membrane fatty acids content was measured in RBCs. Lipids extraction from RBC membranes, phospholipids separation and sample preparation were performed as previously described (58). Extracted fatty acid methyl-esters were then analyzed by gas-chromatography mass-spectrometry (GC-MS). Fatty acid levels were expressed as relative percentages of total fatty acids. [0086] Western Blotting

[0087] Total protein lysates were isolated from biopsies by sonication in RTPA buffer. Forty μg of proteins for each sample were separated on a 4-12% NuPAGE Novex Bis- Tris Gels (Invitrogen™, Thermo Fisher Scientific, Waltham, Massachusetts, USA) in MOPS buffer (Novex™, Thermo Fisher Scientific) and transferred onto nitrocellulose membrane. After blocking, membranes were incubated overnight at +4 °C with primary antibodies against HESl, KLF4, phosphorylated STAT3 (Y705), STAT3, IL- 10, SOCS3 and GAPDH (Table 2). After incubation with appropriate secondary Horse-Radish-Peroxidase (HRP) conjugated antibodies (GE Healthcare Life Sciences, Little Chalfont, United Kingdom), the signal was detected with a luminol enhancer solution (WESTAR EtaC, Cyanagen, Bologna, Italy) and images were acquired using the ChemidocTM XRS + (Biorad, Hercules, CA, USA). Densitometric analysis performed using Image Lab™ software.

[0088] Gene expression analysis

[0089] Total RNA was extracted from biopsies using Trizol® (Ambion, Thermo Fisher Scientific). One μg of total RNA was converted to cDNA using the High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems™, Thermo Fisher Scientific) according to the manufacturer's instructions. qRT-PCR reactions were performed in duplicate on a MX3000p QPCR thermal cycler (Stratagene, San Diego, CA, USA) using the SYBR® Select Master Mix for CFX (Applied Biosystems™, Thermo Fisher Scientific) and the specific primers for IL-10, IL-22, LGR5, C-MYC, MUC2, HESl and KLF4. The primers sequences are listed on Table 3 (SEQ ID NOs: 1 to 16). mRNA expressions of SOCS3 and STAT3 were analyzed using a 5' nuclease probe (Assay ID: Hs.PT.58.4303529; Integrated DNA Technologies, Coralville, Iowa, USA) and the Taqman® gene expression assay (Hs00374280_ml; Thermo Fisher Scientific), respectively. Fold induction levels were obtained using the 2-ΔΔΟΐ method by normalizing against the reference gene RPS9.

[0090] Microbiota analysis [0091] Fecal samples were collected prior to the endoscopic preparation while mucosal samples were taken during endoscopy. Total bacterial DNA was extracted from feces using QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany) and from biopsies using DNeasy Blood & Tissue Mini Kit (Qiagen). Due to a poor quality or quantity of extracted DNA, data on fecal and mucosal microbiota were available from 14 and 16 of the 19 patients included in the study, respectively. For all samples the V3-V4 region of the bacterial 16S rRNA gene was amplified and sequenced using the Illumina platform (Ulumina, San Diego, CA) using a 2 x 300 bp paired-end protocol. Indexed libraries were pooled at equimolar concentrations, denatured and diluted to 6 pmol/L before loading onto the MiSeq flow cell. Raw sequences were processed using a pipeline combining PANDAseq [S6] and QIFME [S7]. High-quality reads were binned into operational taxonomic units (OTUs) at a 0.97 similarity threshold using UCLUST [S8] and a "de novo" approach. Taxonomy was assigned using the RDP classifier against the Greengenes database (May 2013 release). All singleton OTUs were removed in an attempt to discard the majority of chimera sequences. Relative abundance profiles at family or genus level were obtained and plotted. For fecal microbiota analysis, a comparison with a control population of Italian healthy adults enrolled in a previous study was also performed (21). Fecal samples from healthy subjects were collected and processed using the same procedures applied for UC patients recruited in this study.

[0092] Statistical analysis

[0093] Data were analyzed with Graphpad 5.0 Software (GraphPad Software Inc., CA, USA) and Statistix 9.0. The means of two matched groups (TO vs. T3) were compared using the paired two-tailed t-test. For statistical analysis (based on fold-changes) the mean of TO samples was assumed as 1 and two-tailed one-sample t-test was used to compare differences between TO and T3. Sign test, a test for analyzing simple +/- differences between paired comparisons (59), was used to analyze differences in the Mayo sub-score and Geboes score. Correlation analyses were carried out using Spearman's correlation coefficient (rs). For qRT-PCR and western blot analyses data were presented upon square-root transformation. For microbiota analysis, median differences among groups were tested using a non parametric approach (Mann-Whitney U test); P values were corrected for multiple comparisons using the Benjamini- Hochberg method. P values < 0.05 were considered statistically significant. References

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Table 1: Clinico-pathological characteristics of patients at baseline (TO; n= 19).

Table 2. List of primary antibodies used for IHC and western blot analyses