Treatment of intestinal inflammatory diseases

Abstract

Chitosan and / or chitin glucan for the prevention or treatment of inflammatory bowel disease affecting a mammalian patient, a corresponding kit of parts and for the protection of vitamin D, or for the prevention and / or treatment of candida biofilm or gluten induced inflammation.

Description

Technical Field

[0001] The present invention relates to the use of chitosan and / or chitoglucan for treating intestinal inflammatory diseases or conditions, or protecting vitamin D in the digestive tract. Therefore, the present invention also relates to a kit of parts comprising chitosan and / or chitoglucan, and vitamin D and / or probiotics. Background Art

[0002] Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, affect tens of millions of people worldwide, including animals such as dogs.

[0003] These diseases can cause pain, fever, local inflammation, local lesions, ulcers, and weight loss.

[0004] Additionally, the prevalence of gluten-related inflammatory conditions is increasing.

[0005] In addition to chemical molecules or antibodies that directly treat inflammation (i.e., the consequence of the underlying problem), biologics such as probiotics, vitamins, or polysaccharides aim to address the cause or inflammation in a gentler and / or more physiological way.

[0006] Finally, deficiencies in vitamin D or intestinal short-chain fatty acids become increasingly problematic.

[0007] Patent BE1024968 discloses a composition comprising chitosan and chitoglucan.

[0008] Patent application WO 2007 / 122187 and corresponding granted patents disclose chitoglucans for oral administration to produce various beneficial effects in patients.

[0009] EP2555760 discloses chitosan derivatives having at least 2% modified glucosamine residues for use in treating gastrointestinal diseases.

[0010] EP0888118 discloses the effects of chitosan oligosaccharides or β-glucan oligosaccharides and prebiotics in the intestine.

[0011] CN110179108 discloses chitosan oligosaccharides or β-glucan oligosaccharides for intestinal flora homeostasis.

[0012] EP2968651 discloses a composition for treating intestinal lesions comprising a mucoadhesive agent and a stabilizing agent; both of which may be chitosan.

[0013] EP 2 832 352 discloses a capsule comprising chitosan powder. Probiotics or vitamin D are also mentioned in this document.

[0014] Unfortunately, while the papers describe the advantages of their technology, they don't reveal how it might be used to treat inflammatory diseases that affect the intestine. Summary of the Invention

[0015] A first aspect of the present invention is a composition comprising chitosan and / or chitoglucan for use in preventing or treating an inflammatory bowel disease affecting a mammalian patient, preferably selected from irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) (preferably Crohn's disease or ulcerative colitis), and gluten intolerance or antibiotic-induced diarrhea.

[0016] Preferably, the pharmaceutical composition further comprises vitamin D.

[0017] Preferably, the pharmaceutical composition further comprises a probiotic, preferably a yeast or a bacterium, more preferably a strain selected from the group consisting of Bifidobacterium, Lactobacillus, Lacticaseibacillus, Leuconostoc or Ligilactobacillus, still more preferably Bifidobacterium breve, Lactobacillus acidophilus (such as strain NCFM (DSM33840) or strain La-14 (DSM33880)), Lacticaseibacillus paracasei (such as strain Lpc-37 (DSM32661)), or Ligilactobacillus salivarius (such as strain Ls-33 (DSM33831)).

[0018] Advantageously, the composition is administered orally and / or in the form of a food, dietary supplement or pharmaceutically acceptable formulation.

[0019] A related aspect of the invention is a kit of parts comprising chitoglucan and / or chitosan, and vitamin D and / or probiotics (preferably as described above) and / or ellagitannins (preferably in the form of an aqueous suspension comprising at least 0.1 wt% ellagitannins).

[0020] Preferably, the kit of parts is for oral administration to a mammalian patient, preferably a mammalian patient suffering from an inflammatory bowel disease, preferably a patient suffering from irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD) or diarrhea.

[0021] Another related aspect of the invention is a pharmaceutical composition comprising chitosan and / or chitoglucan for use in preventing or treating Candida sp. (Candida alba) biofilm affecting a patient, preferably for oral, topical, vaginal or rectal administration.

[0022] Advantageously, the pharmaceutical composition is in the form of a kit of parts and further comprises a probiotic (bacteria, yeast) and / or an anti-Candida active molecule as described above.

[0023] Another related aspect of the present invention is a pharmaceutical composition comprising chitosan and / or chitoglucan for use in preventing and / or treating gluten-induced inflammation, said pharmaceutical composition preferably being administered orally.

[0024] Another related aspect of the present invention is a pharmaceutical composition comprising chitosan and / or chitoglucan for increasing the production of propionate and / or butyrate in the intestine of a mammalian patient in need thereof, said pharmaceutical composition being preferably administered orally.

[0025] Another related aspect of the present invention is a pharmaceutical composition comprising chitosan and / or chitoglucan for improving intestinal absorption of vitamin D in a mammalian patient in need thereof (e.g., suffering from intestinal inflammation and / or diarrhea, and / or vitamin D deficiency), said pharmaceutical composition preferably being administered orally.

[0026] Another related aspect of the present invention is a pharmaceutical composition comprising chitosan and / or chitoglucan for use in treating bile acid malabsorption in a mammalian patient in need thereof.

[0027] Another related aspect of the invention is a pharmaceutical composition comprising ellagitannins for use in treating antibiotic-induced diarrhea.

[0028] The composition is preferably an aqueous suspension.

[0029] In the composition, ellagitannins are preferably present in a concentration of at least 0.1 wt%, preferably at least 0.2 wt%, preferably at least 0.3 or 0.4 wt%, still more preferably at least 0.5 wt%.

[0030] The composition is advantageously administered to a patient in conjunction with a chitosan and / or chitin glucan composition as described above. DETAILED DESCRIPTION

[0031] The inventors have identified the beneficial effects of chitoglucan or chitosan, particularly in combination with vitamin D and / or probiotics, in the treatment of inflammatory bowel diseases affecting mammalian patients.

[0032] Therefore, a first aspect of the present invention is a composition, preferably an edible composition, comprising chitosan and / or chitoglucan for use in preventing or treating inflammatory bowel disease.

[0033] Preferably, the composition is edible and is administered orally. When administered orally, chitosan and / or chitoglucan is advantageously formulated at 10 to 1000 mg / kg (body weight), preferably 20 to 500 mg / kg, more preferably 50 to 100 mg / kg.

[0034] Other advantageous modes of administration include topical administration, such as patches or creams, or suppositories (and / or rectal administration). In these advantageous modes of administration, suitable concentrations range from 2.5 mg / ml to 50 mg / ml, preferably from 5 to 25 mg / ml, more preferably from 10 to 20 mg / ml.

[0035] Preferred mammalian patients are humans or dogs.

[0036] In the context of the present invention, "chitosan" preferably refers to a chitin derivative in which a certain proportion of N-acetyl-D-glucosamine residues are deaminated. Preferably, the proportion of D-glucosamine (moles of D-glucosamine: sum of moles of D-amino sugars + moles of N-acetyl-D-glucosamine) is between 50% and 95%, more preferably between 75% and 90%, for example between 80% and 86%. Patent document BE 2022 / 5208, filed on March 22, 2022, describes an improved process for the production of chitosan. Preferred chitosan molecules have a glucan residue count of less than 20%, advantageously less than 10% (moles of glucan monomers: total monomers of chitosan, i.e., moles of D-glucosamine + moles of N-acetyl-D-glucosamine).

[0037] In the context of the present invention, preferred chitosan molecules have a molecular weight of 5000 to 30000 Da, more preferably 10000 to 20000 Da, such as about 15000 Da.

[0038] In the context of the present invention, "chitoglucan" preferably refers to a copolymer of chitin and beta (β) glucan. Preferably, the copolymer is extracted from a fungal source. In the copolymer, segments of N-acetyl-D-glucosamine (which may contain a small amount of D-glucosamine, less than 50%, preferably less than 30%, preferably less than 20%, preferably less than 10%: moles of D-glucosamine salt: sum of moles of D-glucosamine + moles of N-acetyl-D-glucosamine) are linked to segments consisting of beta (β) glucan (1,3); (1,3)(1,6) and / or (1,3)(1,4) linkages. Preferably, the weight ratio between chitin (moles of D-glucosamine + moles of N-acetyl-D-glucosamine) and β-glucan (moles of glucan monomers) is between 5:95 and 95:5, more preferably between 20:80 and 70:30, most preferably between 25:75 and 40:60.

[0039] Advantageously, due to its segmented structure, chitoglucan has a different 3D configuration compared to chitin or glucan or a mixture of chitin and glucan (non-segmented) polymers.

[0040] Chitosan and chitoglucan exist in many different organisms, including animal or fungal species. A convenient source of chitosan and chitoglucan is Aspergillus niger. Advantageously, the polymer is extracted as described in WO2007 / 122187 and related patents or patent EP1483299.

[0041] In the context of the present invention, preferably the inflammatory bowel disease is associated with one or more of (local) pain, local inflammation, local lesions, ulcers and / or necrosis, weight loss, presence of blood in stool samples, arthritis, chronic diarrhoea, abdominal distension, mouth ulcers, delayed growth (infants), preferably with local pain and local inflammation or local lesions (including ulcers or necrosis) or both, and / or is selected from irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), gluten intolerance and antibiotic-induced diarrhoea.

[0042] In the context of the present invention, irritable bowel syndrome (IBS) preferably refers to a "brain-gut interaction disorder" characterized by a group of symptoms that generally include abdominal pain and / or bloating and changes in bowel consistency. IBS may be characterized by diarrhea as the main symptom, or conversely, constipation as the main symptom, or mixed / altered bowel patterns, or painful as the main symptom. The main symptoms of IBS are abdominal pain or discomfort, accompanied by frequent diarrhea or constipation and changes in bowel habits.

[0043] Alternatively, or in addition, preferably, patients are considered to have IBS according to the Roma IV criteria (Mearin et al., Gastroenterology 2016) based on (i) chronic abdominal pain for more than 6 months (≥1 day / week in the previous 3 months) and (ii) at least 2 of the following 3 symptoms: painful defecation, change in stool frequency, change in stool consistency.

[0044] In practice, preferably, patients with these criteria and no known IBD (see below) are considered IBS patients for the purposes of the present invention. Similarly, IBS-like symptoms associated with intensive exercise training are also encompassed by the present invention.

[0045] In the context of the present invention, patients with such clinical symptoms and another intestinal pathology (chronic inflammatory bowel disease, celiac disease, lactose intolerance, microbial overgrowth syndrome) are preferably considered to be affected by IBS and therefore fall within the scope of and benefit from the present invention, even if, more generally, they are sometimes considered to suffer from an IBS-like condition. On the other hand, in the context of the present invention, IBD patients (see below) with IBS-like symptoms are preferably considered to be IBD patients; these cohorts include IBD patients who are treated with specific drugs (such as anti-inflammatory drugs, immune system suppressants, anti-TNF or anti-integrin α4β7 compounds, etc.) with the aim of alleviating IBD symptoms or delaying the onset of the disease, as these treated patients may still be considered to have IBS or IBS-like: the compositions of the present invention are particularly suitable to help these patients, either alone or in synergy with (i) anti-inflammatory drugs, (ii) immunosuppressants and / or (iii) blocking antibodies or small molecules (anti-TNF(α), anti-integrin α4β7 or even anti-interleukins; IL-12 and / or IL-23).

[0046] In the context of the present invention, inflammatory bowel disease (IBD) preferably refers to a group of (chronic) inflammatory diseases of the colon and small intestine. This includes chronic disease and ulcerative colitis. As mentioned above, in the context of the present invention, preferably, these patients will be considered as IBD patients if they still have IBS-like symptoms even after receiving IBD treatment.

[0047] Another group of patients suffers from gluten intolerance. As will be developed below, the condition of these patients improves thanks to the pharmaceutical composition of the invention, in particular based on chitosan.

[0048] Another group of patients suffers from diarrhea, especially after antibiotic treatment, and their condition improves thanks to the pharmaceutical composition of the present invention, especially the further inclusion of probiotics (including yeasts).

[0049] Furthermore, compositions comprising tannins (greater than 0.1 wt%, preferably at least 0.5 wt%), either alone or in combination with chitosan and / or chitoglucan, can be used to treat antibiotic-induced diarrhea. The preferred tannin is ellagitannin. The preferred composition is an aqueous suspension.

[0050] Preferably, the composition of the present invention further comprises vitamin D.

[0051] In the context of the present invention, "vitamin D" preferably refers to a group of fat-soluble steroid derivatives. These include cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). Biologically active metabolites or derivatives of vitamin D3 (e.g., calcifediol, calcitriol) are also encompassed by the present invention.

[0052] The inventors have discovered that chitoglucan and / or chitosan bind to vitamin D and protect it as it passes through the digestive tract, allowing for a gradual and smoother release into the patient's cells and tissues, and improved local benefits in the gut, particularly in the lower intestine, and / or more effective systemic release.

[0053] Preferably, in the present invention, vitamin D is administered by ingestion, advantageously together with (i) chitosan and / or chitoglucan and / or (ii) probiotics (if present; see below). Therefore, the preferred mode of administration is a combination of chitosan or chitoglucan and vitamin D. This maximizes the aforementioned "smooth" release effect. In other words, this prolongs the absorption time in the intestine and allows vitamin D to be delivered through an increased portion of the intestine.

[0054] Also contemplated are dietary supplements of vitamin D that are separate from the chitosan and / or chitosan of the present invention (i.e., not in the same tablet or capsule), particularly in the form of a kit of parts, together with (i) chitosan and / or chitoglucan and / or (ii) probiotics (if present; see below). Although such a formulation increases the complexity of the patient to some extent, it allows for a better consideration of the patient's complete profile, including potential vitamin D deficiency, the patient's age, or the level of vitamin D supplementation already administered to the patient. In this alternative mode of administration, vitamin D is advantageously administered (orally) essentially together with chitosan or chitoglucan, e.g., chitosan or chitoglucan is administered first, followed by administration of vitamin D, a full daily dose or a portion thereof, within less than 2 hours, preferably less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes.

[0055] Alternatively, vitamin D or its active metabolite can be administered by blood injection or suppository, particularly in patients with impaired metabolic (liver, kidney) function. However, preferably, at least a portion of the vitamin D (or its active metabolite) supplement is administered orally to the patient.

[0056] In the present invention, vitamin D is preferably administered in an amount effective for treatment and / or synergistically with chitosan or chitin glucan. However, this vitamin should not be administered in excess. The preferred amount ranges from 5 to 100 μg (200-4000 IU, e.g., vitamin D3) per day (for human adults), preferably 10 to 80 μg / day, and more preferably 15 to 50 μg / day.

[0057] The composition of the present invention preferably further comprises probiotics.

[0058] In the context of the present invention, "probiotic" preferably refers to a live microorganism (bacteria or yeast) that is ingested or applied topically (also rectally or vaginally) and produces a beneficial effect, such as reducing inflammatory markers and / or the colonization of pathogenic microorganisms in the mucous membranes, skin or intestine.

[0059] Preferred probiotics are selected from the genus Lactobacillus, Lactobacillus, Leuconostoc, Bifidobacterium or Lactobacillus.

[0060] Therefore, preferred probiotics are selected from Bifidobacterium breve, Lactobacillus acidophilus (such as strain NCFM (DSM33840) or strain La-14 (DSM33880)), Lactobacillus paracasei (such as strain Lpc-37 (DSM32661)), Bifidobacterium breve or Lactobacillus salivarius (such as strain Ls-33 (DSM33831)).

[0061] All four bacterial strains are commercially available from Dupont Nutrition Biosciences ApS.

[0062] All four strains have also been deposited at the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Inhoffnstr. 7B; D-38124 Braunschweig.

[0063] The strain DSM32661 (Lpc-37) was deposited on October 5, 2017 by Dupont Nutrition Biosciences ApS, Langebrogade 1, PO Box 17, 1001 K, Copenhagen, Denmark.

[0064] Strain DSM33840 (NCFM) was deposited on March 15, 2021 by DuPont Nutrition Biosciences AsP, Langebrogade 1, 1411 K, Copenhagen, Denmark.

[0065] Strain DSM33831 (Ls-33) was deposited on February 23, 2021 by DuPont Nutrition Biosciences ApS, Langebrogade 1, 1411 K, Copenhagen, Denmark.

[0066] Strain DSM33880 (La-14) was deposited on June 1, 2021 by DuPont Nutrition Biosciences ApS, Langebrogade 1, 1411 K, Copenhagen, Denmark.

[0067] Yeast (probiotics) advantageously act in synergy with chitosan and / or chitoglucan, and possibly with vitamin D, and are administered to patients with diarrhea either by ingestion or topically (e.g., for the treatment of Candida infections).

[0068] Therefore, the present invention encompasses other useful probiotics or strains from the above species and can be easily selected, for example, in in vitro tests based on the production of inflammatory cytokines and / or anti-inflammatory cytokines (such as IL-10; mRNA level) and / or expression of factors that regulate (reduce) pain (such as PPAR, such as PPARγ; mRNA level) by cell lines (preferably intestinal cell lines (such as HT-29, Caco2)), under basal conditions and / or inflammatory conditions, using potential probiotics (such as 10 7 to 10 9 CFU / ml), or the probiotic is used together with chitin glucan (e.g. 5 mg / ml). Preferred probiotics will reduce the production of one or more inflammatory cytokines (e.g. IL-1β; mRNA levels) by intestinal cell lines (preferably in the presence of chitin glucan or chitosan) under inflammatory conditions by at least two-fold, more preferably at least ten-fold, or even at least 50-fold. Conversely, or in addition, preferred probiotics will increase the production of one or more anti-inflammatory cytokines (e.g. IL-10; mRNA levels) and / or PPAR (mRNA levels, such as PPARγ) by intestinal cell lines (preferably in the presence of chitin glucan or chitosan) under inflammatory conditions by at least two-fold, more preferably at least ten-fold, or even at least 50-fold.

[0069] A related aspect of the invention is a pharmaceutical composition comprising edible chitosan and / or chitoglucan for gradual release of vitamin D in the intestinal tract of a patient in need thereof.

[0070] Advantageously, the pharmaceutical composition for gradual release of vitamins is administered orally.

[0071] Preferably, as described above, for the kit of parts, the pharmaceutical composition is administered essentially together with vitamin D, e.g., chitosan or chitoglucan is administered first, and vitamin D is taken within less than 2 hours, preferably less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes.

[0072] Preferably, there is a need for a pharmaceutical composition comprising edible chitosan and / or chitoglucan to gradually release vitamin D in the intestine of a patient suffering from or suspected of suffering from inflammatory bowel disease and / or intestinal disease or irritation.

[0073] Another related aspect of the invention is a pharmaceutical composition comprising chitosan and / or chitoglucan for use in preventing or treating Candida biofilm affecting a patient.

[0074] Preferably, the pharmaceutical composition is administered orally, topically, vaginally or rectally, advantageously at, on or near a body part that acts or may act as a reservoir for Candida (skin, rectal mucosa, genital mucosa and / or genitourinary tract mucosa). A suitable concentration of chitosan (or chitoglucan) for topical administration is at least 2.5 mg / ml, preferably at least 5 mg / ml, more preferably at least 10 mg / ml of the composition. In addition, systemic Candida infections (sometimes associated with septic shock) are advantageously treated with the chitosan or chitoglucan of the present invention, preferably by injection.

[0075] Advantageously, the pharmaceutical composition comprising chitosan and / or chitoglucan for preventing or treating Candida biofilms further comprises additional pharmaceutically acceptable carriers and / or molecules and / or probiotics (see above, bacteria or yeast) to treat Candida infections, or is administered in conjunction with these probiotics and / or molecules to treat Candida infections, such as antibiotics and / or antifungal drugs (ingestion, injection or topical application). Such carriers advantageously (additionally) comprise pharmaceutically acceptable stabilizers and / or surfactants.

[0076] In fact, the inventors have found that chitosan (and chitoglucan) interfere with Candida biofilms, a very important property because Candida embedded in biofilms can be difficult to treat and can cause long-term harmful effects, including local irritation. Such biofilms can even harbor and protect pathogenic strains of other microorganisms, which can cause additional problems for patients.

[0077] Another related aspect of the present invention is a pharmaceutical composition comprising chitosan and / or chitoglucan, preferably chitosan, for use in preventing and / or treating gluten-induced inflammation.

[0078] Preferably, the pharmaceutical composition is administered orally, advantageously substantially concurrently with a gluten-containing meal, for example a few minutes (less than an hour, less than 30 minutes, less than 15 minutes, less than 5 minutes) before the patient starts lunch with gluten-containing ingredients.

[0079] In fact, the inventors have found that chitosan (or chitoglucan) is able to bind to prolamins, neutralizing their ability to trigger an allergic reaction. This explains why chitosan or chitoglucan compositions are preferably administered essentially together with a gluten-containing meal.

[0080] Another related aspect of the present invention is an edible pharmaceutical composition comprising chitosan and / or chitoglucan for increasing the production of propionic acid and / or butyric acid by the intestinal flora of a mammalian patient in need thereof.

[0081] This is especially beneficial for elderly patients.

[0082] Another related aspect of the present invention is chitosan or chitoglucan for use in treating bile acid malabsorption, preferably an edible pharmaceutical composition and / or a composition preferably for orally administration comprising chitosan and / or chitoglucan.

[0083] Another related aspect of the present invention is a method of treating intestinal inflammation affecting a mammalian patient comprising the step of administering to the mammalian patient a pharmaceutically effective amount of chitosan and / or chitoglucan, preferably in the form of an edible composition.

[0084] Another related aspect of the present invention is a method for increasing the bioavailability of vitamin D in a mammalian patient, comprising the step of administering to the mammalian patient a pharmaceutically effective amount of chitosan and / or chitoglucan, preferably in the form of an edible composition. Advantageously, this method allows for a slower release, smoother and / or longer-lasting, and / or sustained absorption of vitamin D in the intestinal tract.

[0085] Thus, another related aspect of the present invention is a method for improving the gradual release of vitamin D in the intestine of a patient in need thereof, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising edible chitosan and / or chitin glucan, preferably orally.

[0086] Another related aspect of the present invention is a method of reducing gluten intolerance in a mammalian patient, comprising the step of administering to the mammalian patient a pharmaceutically effective amount of chitosan (and / or chitoglucan), preferably in the form of an edible composition.

[0087] Another related aspect of the invention is a method (therapeutic or cosmetic) for interfering with a Candida biofilm affecting a mammalian patient, the method comprising the step of administering to the mammalian patient a pharmaceutically effective amount of chitosan and / or chitoglucan at, on, or near the Candida biofilm.

[0088] Preferably, the method further comprises the step of administering a probiotic (see above, bacteria or yeast) topically or by ingestion.

[0089] Another related aspect of the present invention is a method for increasing the production of propionic acid and / or butyric acid in the intestine of a mammalian patient, or the blood levels of propionic acid and / or butyric acid, comprising the step of administering to the mammalian patient a pharmaceutically effective amount of chitosan and / or chitin glucan, preferably in the form of an edible composition.

[0090] Another related aspect of the present invention is a method of treating bile acid malabsorption comprising the step of (orally) administering an edible pharmaceutical composition comprising chitosan and / or chitoglucan.

[0091] Example

[0092] Example 1: Aluminum-induced colitis

[0093] Tap water was supplemented with 0.5 mg / kg of aluminum to induce visceral hypersensitivity in rats after 7 days of administration. The amount of free aluminum in the water was measured daily by inductively coupled plasma mass spectrometry. While high concentrations of chitosan and chitoglucan might chelate some aluminum, affecting the relevance of the test, the inventors found that the majority of the aluminum remained free.

[0094] Chitosan (CG) or chitosan was then administered orally by gavage twice daily at five different doses: 12.5 mg / kg, 25 mg / kg; 50 mg / kg; 75 mg / kg and 100 mg / kg for 28 days.

[0095] Addition of CG did not affect weight gain during the study period.

[0096] Visceral pain thresholds were measured by colorectal distension after 7, 14, 21, and 28 days. At D7, there was a significant reduction in pain in the 75 and 100 mg / kg dose groups (twice daily; i.e., 150 and 200 mg / kg / day). Statistically significant effects were also observed on day 28 at these doses, as well as at 25 mg / kg twice daily. On a per-animal basis, the same results showed a clear trend toward greater pain reduction compared to pain thresholds on day zero, even at the dose of 12.5 mg / kg (twice daily) and more robust at the highest doses of 75 and 100 mg / kg twice daily. CG administration relieved up to 76% of aluminum-induced pain.

[0097] The same approach was used to replace CG with chitosan, which had no effect on weight gain.

[0098] The effect on pain was less pronounced than with CG, with a trend toward a reduction at doses of 25 mg / kg (twice daily) and above, but this trend only reached statistical significance in some cases.

[0099] Example 2—Proof of Concept

[0100] Rats were pretreated for 14 days with CG, chitosan, deferoxamine, or cholestyramine; in each case, 50 mg / kg was administered twice daily. Treatment was then extended, with additional treatments, as described in Example 1, with or without the use of an aluminum sensitizer. With the exception of deferoxamine, all of these treatments reduced aluminum-induced pain by day 7, but in these experimental settings, the beneficial effects diminished by day 14.

[0101] Example 3—Synergistic Compounds; Toxicity Testing

[0102] The HT-29 and Caco-2 cell models were established.

[0103] The inventors first tested the effects of CG or chitosan (both at 10 μg to 1500 μg / ml) alone or in combination with vitamin D (0.0375 to 0.15 μg / ml) on Caco-2 cells under physiological conditions. There was no cytotoxicity (assay: CCK-8; Sigma-Aldrich).

[0104] The inventors then applied bacterial lipopolysaccharide (10 ng / ml for HT-29 and 1 mg / ml for Caco-2) to simulate inflammatory conditions and performed the same test with CG, chitosan, and vitamin D, and also found no toxicity.

[0105] Example 4—Synergistic Compounds; Inflammatory Markers

[0106] The inventors selected the CG 1500 μg / ml + vitamin D3 condition of Example 3. This condition has been supplemented with three different amounts of Bifidobacterium breve: 10 6 CFU; 10 7 CFU and 10 8 CFU. Cells were stimulated under basal conditions or after LPS prestimulation (1, 3, 6, and 24 h).

[0107] The mRNA levels of cytokines or adhesion molecules have been measured.

[0108] Without LPS pre-stimulation, the use of Bifidobacterium breve (10 8 CFU), or CG and vitamin D combined 10 6 CFU did not reduce the mRNA level of Il-1β after 1 hour. Interestingly, CG, vitamin D and 10 7 CFU or 10 8 CFU of B. breve reduced Il-1β mRNA levels by approximately 75%. All conditions containing CG, vitamin D, and B. breve reduced Il-1β mRNA at 3, 6, and 24 hours.

[0109] In contrast, CG, vitamin D, and all three B. breve concentrations consistently and very acutely increased IL-10 levels at all times measured.

[0110] TNFα; CG, vitamin D and Bifidobacterium breve can increase the level of Il-8 and decrease the level of TGFβ.

[0111] The inventors also measured the levels of molecules involved in intestinal permeability (ZO-1, Occludin, MUC2, and MUC5A).

[0112] Under CG and vitamin D conditions, ZO-1 expression levels decreased. Under all CG and vitamin D conditions, MUC5A mRNA levels increased dramatically at all three B. breve concentrations and at all measurement times. MUC2 mRNA levels remained at basal levels, except for CG, vitamin D, and 10 8 At all B. breve concentrations and at all times tested, CG and vitamin D dramatically increased MOR mRNA levels, whereas PPARγ remained unchanged, but only at 10 8 The CFU of Bifidobacterium breve increased dramatically at 24 hours. The inventors believe that these cytokines reflect the response to pain.

[0113] Similarly, after LPS pre-stimulation, the present inventors measured 7 CFU and 108 Under the presence of only 100 CFU of B. breve, IL-1β mRNA expression levels decreased at 6 and 24 h, and CG and vitamin D induced a sharp decrease in IL-10 and TNFα expression levels at all B. breve concentrations and at all times tested. TGFβ expression levels were reduced in all B. breve conditions with CG and vitamin D supplementation.

[0114] CG and vitamin D decreased ZO-1 levels under all B. breve conditions, whereas they significantly and consistently increased MUC5AC levels at all B. breve concentrations and at all times.

[0115] CG and vitamin D significantly increased MOR mRNA levels at all B. breve concentrations and at all times tested. CG and vitamin D transiently increased PPARγ levels (1 hour, 3 hours), with levels almost returning to basal levels by 6 hours.

[0116] As a control, the inventors tested the effects of CG and vitamin D on the growth of Bifidobacterium breve and Lactobacillus acidophilus (La) strain DSM 33880. There was no effect, neither stimulation (e.g., due to possible metabolism of chitosan or CG by the microorganisms) nor inhibition.

[0117] The same experiment was then repeated using Lactobacillus acidophilus (La) strain DSM33880. The same trend was observed, but the increase in MUC2 expression was much more pronounced in the absence of LPS stimulation. Following LPS pre-stimulation, both CG and vitamin D now significantly reduced IL-8 and Il-1β levels at all La concentrations tested. Similarly, TGFβ levels were also significantly reduced.

[0118] As with B. breve, PPARα and MOR levels were elevated.

[0119] The inventors then compared the effects of chitosan completely replacing CG (1500 μg / ml chitosan and 1500 μg / ml CG) or partially replacing CG (750 μg / ml CG + 750 μg / ml chitosan). The CG effect was stronger than that of chitosan.

[0120] Example 5—In vivo studies

[0121] The inventors developed a Sprague-Dauwley rat colitis model based on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis at 80 mg / kg. Colorectal distension was measured 14 days before TNBS induction and 21, 35, and 49 days after TNBS induction.

[0122] The bacteria Bifidobacterium breve and La are formulated in stick form for ingestion; their stability has been verified.

[0123] Conditioning with CG and vitamin D3 resulted in an increase in pain threshold (control group: 46.67 ± 1.44 and 53.00 ± 0.82; pressure in mm Hg), which was further enhanced by B. breve (55.00 ± 1.12), but not until day 21 with La. From day 21 onwards, strain La DSM33880 enhanced the effects of vitamin D, at least as effectively as B. breve. Other molecules used to treat IBS, such as phloroglucinol ( Also contains vitamin D) and does not increase pain threshold.

[0124] Turning to the macroscopic assessment (Wallace score), the TNBS condition resulted in a score of 2.25, which was reduced after supplementation with vitamin D (2.00) in conjunction with CG, and Bifidobacterium breve showed no improvement even with combined supplementation with vitamin D and CG. In contrast, LA supplementation synergized with CG and vitamin D, improving the Wallace score to 1.00, while It works synergistically with vitamin D, raising the score to 1.56.

[0125] Similarly, histological analysis of inflammation (Ameho) showed limited improvement with the synergistic effect of B. breve with CG and vitamin D: TNBS + vehicle: 2.00; TNBS + CG + vitamin D: 1.80, TNBS + CD + vitamin D + B. breve: 1.50. Interestingly, the result improved to 0.57 with LA supplementation. Similarly, Synergistically acting with vitamin D, the score increased to 1.56. IL-1 and IL-6 were also significantly reduced in the presence of CG, vitamin D, and LA, while Bifidobacterium breve provided only a moderate reduction.

[0126] Example 6 - Inhibition of Candida Biofilm

[0127] The inventors tested the effects of chitosan or chitin glucan on Candida biofilms and measured the disruption of these biofilms. The inventors first used Candida albicans ATCC 10231 strain in a 96-well microplate at 4.2510 3Candida biofilms were cultured in Mueller Hinton broth at an inoculum concentration of 100 CFU / ml. After 48 hours, the wells were rinsed with sterile PBS and then a composition containing chitosan was added to the cells in the wells. After 4 days, the culture medium was removed and rinsed with PBS. The microplates were then dried for crystal violet staining and the optical density was measured. Chitosan concentrations of 0.075 mg / ml, 0.15 mg / ml, 0.3 mg / ml, 0.6 mg / ml and 1.25 mg / ml had little effect on the biofilm (destruction rates were between 2% and 4%), while chitosan concentrations of 2.5, 5 and 10 mg / ml had biofilm destruction rates of 53%, 78% and 91%, respectively.

[0128] Example 7—Analysis of the production of short-chain fatty acids

[0129] The inventors used a system to stimulate the intestinal tract (ascending, transverse, and descending colon). They administered doses equivalent to 1.5 or 4.5 grams of CG per day. They noted modulation of the fermentation characteristics of intestinal microorganisms, increasing the production of propionate and butyrate. In addition to being an energy source, butyrate also protects against mucosal inflammation and oxidative stress. Propionic acid also acts as a cholesterol-lowering agent and has an impact on glycemic control.

[0130] Example 8—Effects on symbiotic intestinal flora

[0131] The inventors measured the relative abundance of bacterial strains that form the microbiota of patients with Crohn's disease, ulcerative colitis, and IBS. Stool samples from patients treated with GC, vitamin D, and La showed a trend toward recovery to unaffected levels.

[0132] Example 9 - Gluten Allergy

[0133] Prolamins (part of gluten) are responsible for the autoimmune bowel disease (celiac disease) caused by an abnormal immune response in genetically susceptible individuals. The present inventors have administered chitosan orally to patients along with a gluten-rich meal, or alone.

[0134] Chitosan supplementation reduced allergic reactions.

[0135] Example 10—IBS Treatment

[0136] Women with IBS (140 IBS with constipation, IBS-C; 60 IBS with diarrhea, IBS-D) were recruited and divided into four groups: 1.5g CG (IBS-C, 30 patients; IBS-D, 70 patients) or 3.0g CG (IBS-C, 30 patients; IBS-D, 70 patients). Both CG compositions were supplemented with 400 IU of vitamin D3. Under the supervision of a gastroenterologist, the patients were administered the above dosages for 30 days, followed by a 15-day post-treatment period.

[0137] The inventors then summarized the symptom and health improvements. 72%, 75%, 77%, and 79% of patients experienced a reduction in pain, gas, IBS discomfort, and bloating, respectively. This is quite impressive. Even sleep quality and fatigue symptoms were reduced in 65% and 60% of patients, respectively. Feelings of well-being were also greatly improved, with 73% of patients experiencing a reduction in stress, 71% in overweight, food frustration (69%), satiety (65%), energy levels (63%), mood levels (59%), well-being (58%), eating pleasure (58%), morale levels (57%), work productivity (55%), quality of life (53%), and general well-being (49%). In short, 75% of patients did feel that the treatment had a beneficial impact on their well-being. There were no statistical differences between the 1.5 and 3.0 regimens or between IBS subtypes.

[0138] Example 11 - Other probiotics

[0139] The inventors repeated these tests with varying probiotics. Among the probiotics tested, Lactobacillus acidophilus NCFM strain (DSM 33840), Lactobacillus paracasei Lpc-37 strain (DSM 32661), and Lactobacillus salivarius Ls-33 strain (DSM 33831) provided excellent results.

[0140] Example 12—BAM Treatment

[0141] The inventors have discovered that chitoglucan, when binding bile acids, can correct bile acid malabsorption and prevent bile acid accumulation in the intestine.

[0142] Example 13 - Antibiotic-Induced Diarrhea

[0143] The inventors developed a human diarrhea model based on mice infected with Citrobacter rodentium.

[0144] Mice were divided into seven groups: vehicle control, untreated mice (group 1); and infected mice in six groups: vehicle control; chitosan 1.5 g, chitoglucan 1.5 g, tannin (0.5%), chitosan 1.5 g + tannin 0.5%; chitoglucan 1.5 g + tannin (0.5%), and gelsectan. Tannin was an aqueous suspension of ellagitannins. Several parameters were monitored over nine days: daily weight gain, colon weight / size ratio, stool consistency, and histological scoring of inflammation.

[0145] Almost all treatments improved stool consistency (between the "control" and "vaccinated mice, vehicle control"). Gelsectan had the greatest effect; however, the tannin condition, including chitosan or chitoglucan supplemented with tannin, achieved almost the same effect. The "chitoglucan" treatment (without tannin) was less effective.

[0146] Chitoglucan treatment showed the greatest efficacy on inflammation, followed by Gelsectan. Chitosan and tannin groups showed intermediate effects.

[0147] The effect on weight gain (or loss) is limited.

[0148] Example 14—Pathological Candida Infection

[0149] The present inventors selected a group of Balb / C female mice and established a Candida albicans-induced colitis model under the treatment of estradiol (2.5 mg / kg; injected once every 3 days for 12 days).

[0150] Five groups were designed: control group + vehicle control, Candida infection group (5.10 7 CFU) were measured in four groups: vehicle control, chitosan plus chitosan 1.5 g (preventive), chitosan plus chitosan 1.5 g (therapeutic), and fluconazole 150 mg / kg (therapeutic). Preventive treatment began during estradiol administration, while therapeutic treatment began 2 days after Candida inoculation. Mice were sacrificed 14 days after inoculation.

[0151] Fluconazole rapidly reduced the infection, and no mice remained infected after 9 days.

[0152] Prophylactic chitosan+chitin glucan also nearly eliminated the infection (only 10% of infected mice at day 14). The other conditions were less efficient.

[0153] Similar results were obtained by measuring the presence of Candida in stool (number of CFU / 100 mg stool), for example, a decrease from 252 (control infected) to 14 (chitosan + chitin glucan 1.5 g, infected). In addition, similar results were obtained by measuring the presence of Candida in the ileum, colon, and vagina.

[0154] All treatments except fluconazole had a beneficial effect on colitis.

[0155] Example 15 IBD Model

[0156] The inventors have chosen transgenic HLA-B27 rats as a model for human ileocecal lesions such as those commonly seen in Crohn's disease.

[0157] Twelve-week-old rats (40, divided into five pools) underwent distal ileal and proximal colectomy, and inflammation levels were assessed 6 weeks later.

[0158] Treatment started at week 8 (thus 4 weeks before surgical treatment). Group 1: vehicle control; Group 2: chitosan (3 g) and vitamin D3; Group 3: chitosan (3 g), vitamin D3 and (probiotic) Saccharomyces cerevisiae (10 9 CFU); Group 4: chitin glucan (3 g), vitamin D3 and pentasa; Group 5: Pentasa.

[0159] In the first two tests and pentasa (alone) conditions, the recurrence rate after surgery was significantly reduced, and in the fourth group chitin glucan (3g), vitamin D3 + pentasa conditions, the recurrence rate was slightly reduced.

[0160] On the other hand, the anastomotic histological score improved only slightly in the first test condition and improved more significantly in the other three test conditions.

Claims

1. A composition comprising chitosan and / or chitoglucan for use in preventing or treating inflammatory bowel disease affecting a mammalian patient.

2. The composition according to claim 1, wherein the inflammatory bowel disease is selected from irritable bowel syndrome (IBS), Inflammatory bowel disease (IBD) preferably Crohn's disease or ulcerative colitis, Gluten intolerance and antibiotic-induced diarrhea.

3. The composition according to claim 1 or 2, for oral administration, wherein the chitosan and / or chitoglucan is administered at a dosage of 5 to 1000 mg / kg, preferably 10 to 500 mg / kg (body weight), preferably 20 to 200 mg / kg, more preferably 50 to 100 mg / kg.

4. The composition according to claim 1 or 2, for topical application, wherein the chitosan and / or chitoglucan concentration is between 2.5 mg / ml and 50 mg / ml, preferably between 5 and 25 mg / ml, preferably between 10 and 20 mg / ml.

5. The composition according to any one of the preceding claims, wherein the chitosan comprises 50% to 95% D-glucosamine (total of moles of D-glucosamine: moles of D-glucosamine salt + moles of N-acetyl-D-glucosamine).

6. A composition according to any one of the preceding claims, wherein the chitosan molecules have less than 20% dextran residues, advantageously less than 10% dextran residues (moles of dextran monomers: total monomers of chitosan).

7. A composition according to any preceding claim, wherein the chitosan molecule has a molecular weight of 5000 to 30000 Da, more preferably 10000 to 20000 Da, such as about 15000 Da.

8. The composition according to any one of the preceding claims, wherein the chitoglucan molecule is a copolymer of chitin and β-glucan, the weight ratio between chitin (moles of D-glucosamine + moles of N-acetyl-D-glucosamine) and β-glucan (moles of glucan monomers) being between 5:95 and 95:5, more preferably between 20:80 and 70:30, more preferably between 25:75 and 40:

60.

9. The composition of any preceding claim, further comprising vitamin D.

10. The composition according to any of the preceding claims, further comprising a probiotic, preferably a bacterium or a (probiotic) yeast.

11. The composition according to claim 10, wherein 10 7 to 10 9 The combination of probiotics at a concentration of 500 CFU / ml with chitosan and / or chitin glucan reduces the production of inflammatory cytokines (such as IL-1β; mRNA level) and / or increases the production of anti-inflammatory cytokines (such as IL-10; mRNA level) under inflammatory conditions; and / or increases the expression of factors that regulate (relieve) pain (such as PPARγ; mRNA level) in intestinal cell lines (such as HT-29, Caco2).

12. The composition according to claim 10 or 11, wherein the probiotic is selected from strains of Bifidobacterium, Lactobacillus, Lactobacillus, Leuconostoc or Lactobacillus, preferably Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus paracasei or Lactobacillus salivarius.

13. The composition of claim 12, wherein the probiotic is Lactobacillus acidophilus.

14. The composition according to any one of the preceding claims 1 to 3 and 5 to 13, which is administered orally and / or is in the form of a food, a dietary supplement or a pharmaceutically acceptable formulation.

15. A kit of parts comprising chitoglucan and / or chitosan, and Vitamin D and / or probiotics.

16. The kit of parts according to claim 15, wherein the probiotic is as described in claim 4 or 5.

17. The kit of parts according to claim 15 or 16, for oral administration to a mammalian patient, preferably a mammalian patient suffering from an inflammatory bowel disease, preferably irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD) or diarrhea.

18. A pharmaceutical composition comprising chitosan and / or chitoglucan for use in preventing or treating Candida biofilms affecting a patient and / or for treating problematic Candida colonisation, preferably Candida albicans, Candida glabrata and / or Candida krusei colonisation and / or preferably wherein the problematic colonisation involves biofilm formation or excessive mucosal colonisation.

19. The pharmaceutical composition according to claim 18, which is administered orally, topically, vaginally or rectally, preferably orally.

20. A kit of parts comprising the pharmaceutical composition according to claim 18 or 19, and further comprising an antifungal agent, preferably an anti-Candida active molecule.

21. The pharmaceutical composition according to any one of the preceding claims 18 to 20, which is in the form of a kit of parts further comprising the probiotics defined in any one of the preceding claims 10 to 12.

22. A pharmaceutical composition comprising chitosan and / or chitoglucan for use in preventing and / or treating gluten-induced inflammation, wherein the pharmaceutical composition is preferably administered orally.

23. A pharmaceutical composition comprising chitosan and / or chitoglucan for improving intestinal absorption of vitamin D in a mammalian patient in need thereof, wherein the pharmaceutical composition is preferably administered orally.

24. An edible pharmaceutical composition comprising chitosan and / or chitoglucan for use in treating bile acid malabsorption in a mammalian patient in need thereof.

25. A method for gradually releasing vitamin D in the intestine of a patient in need thereof, the method comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising edible chitosan and / or chitin glucan, wherein the pharmaceutical composition is administered orally.

26. The method of claim 25, wherein vitamin D is administered substantially together with edible chitin glucan and / or edible chitosan.

27. The method of claim 25, wherein vitamin D is administered within less than 2 hours, preferably less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes after administration of chitosan and / or chitoglucan to the patient.

28. A method of reducing gluten intolerance in a mammalian patient comprising the step of administering to the mammalian patient a pharmaceutically effective amount of chitosan in the form of an edible composition.

29. The method of claim 28, wherein the chitosan is administered substantially together with gluten ingested by the patient.

30. The method of claim 28, wherein gluten is administered to the patient less than 2 hours, preferably less than 1 hour, less than 30 minutes, less than 15 minutes, less than 5 minutes after administration of chitosan and / or chitin glucan to the patient.

31. A method of interfering with a Candida biofilm affecting a mammalian patient, said method comprising the step of administering to the mammalian patient a pharmaceutically effective amount of chitosan and / or chitoglucan.

32. A method for treating bile acid malabsorption comprising the step of orally administering an edible pharmaceutical composition comprising chitosan and / or chitoglucan.