Composition for use in the treatment of irritable bowel syndrome and / or in the treatment of intestinal hyperpermeability

Abstract

The present invention relates to a composition for use in the treatment of irritable bowel syndrome (IBS) and / or in the treatment of intestinal hyperpermeability. Said composition comprises a fish protein hydrolysate which comprises a peptide fraction comprising at least 15% peptides having a molecular weight of less than 300 Da, expressed in relation to the total weight of said peptide fraction.

Description

[0001] Description

[0002] Composition for use in the treatment of irritable bowel syndrome and / or in the treatment of intestinal hyperpermeability.

[0003] Technical field of the invention

[0004] The present invention relates to the technical field of compositions for use in the treatment of irritable bowel syndrome and / or in the treatment of intestinal hyperpermeability.

[0005] State of the art

[0006] Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder, now referred to as Disorder of the Gut-Brain Axis (GBA), with a high prevalence of approximately 11% of the global population.

[0007] HS is characterized by gastrointestinal symptoms, including abdominal pain, bloating, diarrhea, and / or constipation, which negatively impact quality of life and contribute to the high prevalence of depression.

[0008] Patients with HS often exhibit visceral hypersensitivity, as well as epithelial barrier dysfunction that contributes to pain and bowel symptoms.

[0009] In particular, intestinal hyperpermeability occurs mainly in patients with diarrhea-predominant HS (D-HS). The expression or localization of tight junction proteins in epithelial cells is altered in both the small intestinal mucosa and colonic mucosa of HS patients.

[0010] There is therefore a definite interest in identifying compositions likely to limit the low-grade colonic inflammatory response and colonic hyperpermeability in patients suffering from HS.

[0011] Presentation of the invention

[0012] In order to overcome the aforementioned drawback of the state of the art, the present invention provides a composition, advantageously intended / suitable for oral administration to a subject, for use in the treatment of irritable bowel syndrome (IBS) and / or in the treatment of intestinal hyperpermeability.

[0013] This composition comprises a fish protein hydrolysate which comprises a peptide fraction comprising at least 15% of peptides with a molecular weight of less than 300 Da, expressed as a function of the total weight of said peptide fraction. Such a composition according to the invention has properties in a subject, in particular capable of limiting irritable bowel syndrome (IBS) and / or intestinal permeability.

[0014] Other non-limiting and advantageous characteristics of the product in accordance with the invention, taken individually or in all technically possible combinations, are as follows:

[0015] - the molecular weight profile of said peptide fraction is as follows, expressed as a function of the total weight of said peptide fraction, 15 to 55% of peptides with a molecular weight of less than 300 Da, 30 to 85% (preferably 30 to 80%) of peptides with a molecular weight of between 300 and 1500 Da, less than 8% of peptides with a molecular weight of more than 1500 Da;

[0016] - said fish protein hydrolysate is derived from fish chosen from oily fish and white fish; preferably, said fish protein hydrolysate is derived from white fish chosen from cod, ling, pollock, pout, julienne, whiting and hake;

[0017] - said composition further comprises glutamine, preferably L-glutamine, and / or curcumin; preferably said composition further comprises polyunsaturated fatty acids, preferably omega-3 and vitamins, for example vitamin E and / or vitamin D3;

[0018] - the weight ratio between said fish protein hydrolyzate and glutamine is 0.01 to 0.09 and, where appropriate, the weight ratio between said fish protein hydrolyzate and curcumin is 0.1 to 6;

[0019] - said composition is administered at a daily dose, preferably in two doses, of 100 to 800 mg of said fish protein hydrolyzate and, where appropriate, of 4 to 20 g of glutamine.

[0020] The present invention also relates to the composition according to the invention, consisting of a pharmaceutical, nutraceutical, dietetic or food composition, in particular for human and / or animal use.

[0021] The present invention also relates to a non-therapeutic use of a composition comprising a fish protein hydrolysate which comprises a peptide fraction comprising at least 15% of peptides with a molecular weight of less than 300 Da, expressed as a function of the total weight of said peptide fraction, for the treatment of irritable bowel syndrome (IBS) and / or for the treatment of intestinal hyperpermeability. Of course, the various characteristics, variants and embodiments of the invention may be combined with each other in various combinations to the extent that they are not incompatible or mutually exclusive.

[0022] Detailed description of the invention

[0023] In addition, various other characteristics of the invention emerge from the appended description given with reference to the drawings which illustrate non-limiting embodiments of the invention and where:

[0024] [Fig. 1] shows plasma corticosterone levels and colonic permeability in response to acute (ARS) or chronic (CRS) stress models; (A. and D.) Plasma corticosterone levels in ng / mL (B. and E.) Lucifer yellow in ng / mL and (C. and E.) Alexa dextran concentrations in ng / mL, measured in Ussing chambers in the control groups (control) compared to the ARS or CRS groups;

[0025] [Fig. 2] illustrates the effect of each selected compound administered individually on plasma corticosterone levels (A.) and colonic permeability (B. and C.); Normalized corticosterone levels (A.) measured in plasma, plasma concentrations of Lucifer yellow (B.) and Alexa dextran (C.) measured in Ussing chambers in the CRS-vehicle (CRS-vehicle) groups vs. CRS-Glutamine (Gin), CRS-Curcumin (Cur) at high and low doses, CRS-hydrolysate (Ga);

[0026] [Fig. 3] illustrates the effect of selected combined compounds, glutamine, curcumin and hydrolysate (GCG), on plasma corticosterone levels (A.) and colonic permeability (B. and C.); corticosterone levels (A.) measured in plasma, Lucifer yellow (B.) and Alexa dextran (C.) concentrations measured in Ussing chambers in the CRS vehicle group (CRS-vehicle) vs. CRS-GCG;

[0027] [Fig. 4] represents the effect of each selected compound administered individually and GCG mixture on colon tight junction protein gene expression; (A.) Normalized occludin, (B) claudin-1 and (C) ZO-1 gene expression in CRS (CRS-vehicle) vs. CRS-Glutamine (Gin), CRS-curcumin (Cur) at high and low doses, CRS-hydrolysate (Ga) and CRS-GCG groups;

[0028] [Fig. 5] illustrates the effect of each selected compound administered individually and the GCG mixture on colon inflammation-related gene expression; (A.) Normalized CXCL1, (B), TNFa, (C.), I L1 p and (D.); IL10 gene expression in CRS (CRS-vehicle) vs. CRS-Glutamine (Gin), CRS-Curcumin (Cur) at high and low doses, CRS-hydrolysate (Ga) and CRS-GCG groups.

[0029] It should be noted that, in these figures, the structural and / or functional elements common to the different variants may have the same references. The present invention thus relates to a composition, advantageously suitable for oral administration to a subject.

[0030] The composition according to the invention comprises a fish protein hydrolysate which comprises a peptide fraction comprising at least 15%, preferably from 15 to 55%, of peptides with a molecular weight of less than 300 Da, expressed as a function of the total weight of said peptide fraction.

[0031] The present invention also advantageously encompasses a peptide fraction delimited by two values ​​chosen from 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%.

[0032] In other words, peptides with a molecular weight of less than 300 Da represent 15%, or more than 15%, of the peptide fraction, expressed as a function of the total weight of said peptide fraction.

[0033] Optionally, the composition according to the invention may also contain:

[0034] - a glutamine, preferably L-glutamine, and / or

[0035] - a curcumin.

[0036] The composition according to the invention thus comprises:

[0037] - said fish protein hydrolyzate, or

[0038] - said fish protein hydrolyzate and glutamine (possibly without curcumin), or

[0039] - said fish protein hydrolyzate and curcumin (possibly without glutamine), or

[0040] - said fish protein hydrolyzate, curcumin and glutamine.

[0041] Fish protein hydrolyzate

[0042] According to the present invention, the peptide fraction comprises peptides and amino acids.

[0043] According to a preferred embodiment of the invention, the proteins represent less than 1%, in particular less than 0.5% of the peptide fraction of the fish protein hydrolyzate. More preferably, the hydrolyzate according to the invention is free of residual proteins.

[0044] By "peptide" is meant a polymer comprising at least 2 amino acids linked together by peptide bonds. Typically, a peptide comprises less than 100 amino acids and has a molecular weight generally less than 11,000 Da, preferably less than 10,000 Da.

[0045] The molecular weight profile of said peptide fraction, expressed as a function of the total weight of said peptide fraction (also expressed as a weight ratio relative to the total weight of the peptide fraction) advantageously has a molecular profile of which:

[0046] - 15 to 55% of peptides with a molecular weight of less than 300 Da, - 30 to 85%, preferably 30 to 80%, of peptides with a molecular weight of between 300 and 1500 Da,

[0047] - less than 8% of peptides with a molecular weight greater than 1500 Da.

[0048] The present invention also advantageously encompasses the domains of peptides with a molecular weight of less than 300 Da, delimited by two values ​​chosen from 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%.

[0049] The present invention also advantageously encompasses the domains of peptides with a molecular weight of between 300 and 1500 Da, delimited by two values ​​chosen from 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%.

[0050] For example, peptides with molecular weights between 300 and 1500 Da represent 75% to 85%.

[0051] The present invention also advantageously encompasses a maximum value of peptides with a molecular weight greater than 1500 Da, chosen from 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%.

[0052] This molecular profile of the peptide fraction is advantageously determined by high-performance liquid chromatography HPLC coupled with an ultraviolet detector (HPLC-UV).

[0053] Still more generally, preferably, the hydrolyzate in accordance with the invention comprises:

[0054] (i) the peptide fraction, which represents from 60 to 80% by weight of the total weight of dry extract of said hydrolyzate,

[0055] (ii) a lipid fraction, which represents from 5 to 17% by weight of the total weight of dry extract of said hydrolyzate, and

[0056] (iii) a mineral fraction which represents from 4 to 23%, preferably from 5 to 23%, by weight of the total weight of dry extract of said hydrolyzate.

[0057] For example and not limited to, the hydrolyzate consists of a product known under the name Gabolysat (registered trademark, marketed by the company LABORATOIRE DIELEN, France).

[0058] The hydrolyzate of the invention, advantageously in the form of a paste, generally comprises a moisture content of less than 50% and preferably less than 45% relative to the total weight of the hydrolyzate. Thus, in certain embodiments, the hydrolyzate comprises at least 55% dry extract and consequently less than 45% moisture relative to the total weight of the hydrolyzate. It is then advantageously in the form of a paste, or a powder.

[0059] In certain embodiments, the hydrolyzate of the invention comprises a residual moisture content of less than or equal to 15%, in particular less than or equal to 10%, for example varying from 5 to 10%, and more preferably less than or equal to 5% by weight, relative to the total weight of the hydrolyzate. In this embodiment, the hydrolyzate is generally in the form of a powder. Typically, the lipid fraction comprises:

[0060] - at least 10% eicosapentaenoic acid (EPA) and

[0061] - at least 5% docosahexaenoic acid (DHA).

[0062] Preferably, a hydrolyzate in accordance with the invention comprises a carbohydrate content of less than 0.2% and in particular less than 0.1%, in particular less than 0.05% and particularly preferably less than 0.002% by weight relative to the total weight of dry extract of the hydrolyzate. In certain embodiments, the hydrolyzate of the invention is free of carbohydrates.

[0063] The fish protein hydrolysate according to the invention is obtained by hydrolysis, advantageously enzymatic, of at least one source of protein from at least one type (or at least one species) of fish.

[0064] Preferably, as a source of fish protein (corresponding to the raw material), tissues of at least one type of fish selected from oily fish and white fish are used.

[0065] By "white fish" we advantageously include fish from the Gadidae family, and preferably still cod, ling, pollock, pout, julienne, whiting and hake.

[0066] Whole fish or certain fish tissues are used. It is also possible to use co-products from the fish industry as raw materials.

[0067] Preferably, the fish are gutted (or gutted), meaning their abdominal wall has been opened longitudinally, the viscera removed, and the abdominal cavity cleaned. The fish may be headed.

[0068] This raw material is advantageously added with water. Acidified water can be used.

[0069] The hydrolyzate according to the invention is thus the result of a treatment during which certain peptide bonds of the proteins are broken.

[0070] Generally, the method advantageously comprises at least the following steps:

[0071] - hydrolysis of fish tissues under agitation, by adding natural fish proteases,

[0072] - separation, by filtration and / or by centrifugation,

[0073] - concentration,

[0074] - possibly drying,

[0075] - packaging of the hydrolyzate, preferably in the form of paste or powder.

[0076] The method advantageously comprises: 1) Optionally, a step of grinding said at least one source of fish protein.

[0077] 2) An enzymatic hydrolysis step of the fish protein source. This step is typically carried out under agitation.

[0078] Advantageously, the hydrolysis is carried out at a constant pH, generally for a duration ranging from 2 to 8 hours.

[0079] Hydrolysis is carried out by controlled (or gentle) enzymatic digestion under the action of endogenous and exogenous proteases.

[0080] By "endogenous protease" is meant any protease occurring naturally in the fish protein source used (typically any protease that is naturally contained in the fish flesh used).

[0081] According to the invention, the term "exogenous protease" means any exogenous enzyme, i.e. one added to the fish protein source, and capable of hydrolyzing the proteins of the selected raw materials which are subjected to the hydrolysis treatment.

[0082] In particular, it is possible to use one or more proteases of marine, plant or chemical origin.

[0083] The following enzymes or enzyme mixtures can be used: fish intestinal mucosa extract, fish pancreatic extract, alone or in mixtures, possibly in combination with chemical enzymes.

[0084] Typically the enzymes (proteases) or the mixture of exogenous enzymes are added after the heating step.

[0085] The hydrolysis is continued until the hydrolysate corresponding to the molecular profile as defined above is obtained. The enzymatic hydrolysis can be stopped by inactivating the proteases by raising the temperature of the reaction mixture to a temperature not exceeding 100°C, in particular between 85 and 95°C, preferably of the order of 90°C. This operation is generally carried out for a period of 5 to 60 minutes.

[0086] 3) A step of separation of the protein hydrolyzate obtained from the rest of the reaction mixture from step 2).

[0087] This separation can be carried out by filtration (for example on a filter of the order of 400 pm) and / or by centrifugation.

[0088] Preferably, the separation of the protein hydrolyzate is carried out by filtration of the reaction mixture followed by centrifugation.

[0089] Filtration of the reaction medium allows the elimination of solid matter.

[0090] 4) Advantageously, a step of dehydration of the hydrolyzate is implemented after step 3). This dehydration step is generally carried out by vacuum concentration. It typically makes it possible to obtain a paste. 5) Optionally, a step of low-temperature drying of the hydrolyzate, by vacuum tunnel or by atomization, can be implemented.

[0091] The hydrolyzate is thus recovered in powder form which preferably contains 15% or less, in particular 10% or less, for example between 5 and 10% and more preferably 5% or less by weight of moisture, relative to the total weight of the hydrolyzate in powder form.

[0092] Preferably, drying is carried out by atomization. The protein hydrolyzate is then typically sprayed into an enclosure in which the air has been preheated so that the water evaporates. The resulting powder is separated from the water vapor and collected at the end of the drying step.

[0093] 6) Optionally, a step of conditioning the protein hydrolysate, where appropriate in the form of paste or powder, after implementation of step 4) and / or 5).

[0094] The hydrolyzate can be packaged as a concentrated paste.

[0095] It may also be packaged in the form of a powder containing 15% or less, in particular 10% or less, for example 5 to 10% and preferably 5% or less by weight of residual moisture, relative to the total weight of the hydrolysate in powder form.

[0096] Glutamine

[0097] Glutamine is an amino acid that is regulatory considered a nutritional and / or physiological substance.

[0098] Preferably, the composition according to the invention comprises glutamine, preferably without allergens, preferably with CAS number 56-85-9.

[0099] In a particular embodiment, the composition according to the invention comprises L-glutamine, preferably in powder form.

[0100] Curcumin

[0101] Curcumin, or diferuloyl-methane, is the main pigment of turmeric (Curcuma longà), a perennial plant native to Asia that belongs to the Zingiberaceae family.

[0102] The curcumin in the combination according to the invention can be obtained by any known extraction method, for example by extraction using an organic solvent, by maceration or decoction or by extraction by supercritical fluid.

[0103] Preferably, it is an extract of turmeric rhizome obtained by a process comprising an extraction step with a solvent usable in the preparation of food supplements, for example an alcohol or an ester of ethyl acetate type.

[0104] Advantageously, curcumin is used in powder form.

[0105] For example and not limited to, curcumin consists of a product known as Curcuméga (registered trademark, marketed by the company LABORATOIRE DIELEN, France).

[0106] Curcuméga (registered trademark) contains more than 3% curcumin, for example 3 to 26%, expressed as a mass percentage. Other optional ingredients in the composition

[0107] The composition may also include:

[0108] - polyunsaturated fatty acids, preferably omega-3, and

[0109] - vitamins, for example vitamin E and / or vitamin D3.

[0110] Weight ratio

[0111] Preferably, the weight ratio of fish protein hydrolyzate to glutamine is 0.01 to 0.09.

[0112] Generally, this ratio is advantageously further delimited by two values ​​chosen from 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09.

[0113] Where appropriate, the weight ratio between said fish protein hydrolyzate and curcumin is from 0.1 to 7, preferably from 0.1 to 6, more preferably from 0.4 to 7.

[0114] Generally, this ratio is advantageously further delimited by two values ​​chosen from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0.

[0115] Application

[0116] The composition according to the invention is thus suitable for a subject (advantageously human) suffering from irritable bowel syndrome (IBS) and / or intestinal hyperpermeability.

[0117] The composition according to the invention is advantageously administered, preferably in two doses, at a daily dose:

[0118] - 100 to 800 mg of said fish protein hydrolyzate,

[0119] - if necessary, 4 to 20 g of glutamine, and / or

[0120] - if necessary, from 30 mg to 500 mg of curcumin.

[0121] Generally speaking, the daily dose of fish protein hydrolyzate is advantageously delimited by two values ​​chosen from 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg.

[0122] The daily dose of glutamine is advantageously delimited by two values ​​chosen from 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 17 g, 18 g, 19 g, 20 g.

[0123] The daily dose of curcumin is advantageously delimited by two values ​​chosen from 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg,

[0124] 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg,

[0125] 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, 500 mg.

[0126] The composition according to the invention can be used in uses chosen from a pharmaceutical composition, a nutraceutical composition or a food composition, in particular for human use.

[0127] In particular, the present invention relates to the composition which comprises a fish protein hydrolysate which comprises a peptide fraction comprising at least 15% of peptides with a molecular weight of less than 300 Da, expressed as a function of the total weight of said peptide fraction, for use in the treatment of irritable bowel syndrome (IBS) and / or in the treatment of intestinal hyperpermeability.

[0128] The invention also relates to the non-therapeutic use of a composition according to the invention, for the treatment of irritable bowel syndrome (IBS) and / or for the treatment of intestinal hyperpermeability. This non-therapeutic composition consists of a nutraceutical, dietetic or food composition, in particular for human and / or animal use.

[0129] The compositions of the present invention are advantageously presented in a form suitable for oral administration.

[0130] Generally speaking, a “pharmaceutical composition” means a preparation of the active ingredients according to the invention with other optional chemical components such as physiologically appropriate carriers and / or excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the active ingredients according to the invention to an organism.

[0131] The term "nutraceutical" refers to a composition or product made from food substances, but made available in the form of tablets, powders, potions, or other dosage forms not usually associated with food, and having a beneficial or protective physiological effect against animal or human disorders or diseases. This definition includes, in particular, dietary supplements, certain group-specific foods, or meal replacements.

[0132] By "food supplement" is meant here any composition which is formulated and administered separately from other foods and is intended to supplement the nutritional intake of a subject having a form acceptable from a galenical point of view, in particular in the form of capsules, tablets, soft capsules, sachets, stick-packs, syrup, droppers, or any other suitable form well known to those skilled in the art.

[0133] The terms "food", "food product" and "foodstuff" are used interchangeably herein and include, in addition to foods commonly consumed by humans and animals such as pets or livestock, functional foods and foods for specific groups, which in turn include foods intended for special medical purposes.

[0134] "Dietetic foods" means foods intended for special nutritional purposes that are specially processed or formulated to meet the nutritional needs of patients. They are intended to constitute the exclusive or partial diet of patients whose capacity to absorb, digest, assimilate, metabolize or excrete ordinary foods or some of their ingredients or metabolites is diminished, limited or disturbed, or whose state of health requires other special nutritional needs that cannot be met by a modification of the normal diet or by a diet consisting of foods intended for special nutritional purposes or by a combination of both.

[0135] Furthermore, "irritable bowel syndrome" (also called functional colopathy, functional intestinal disorders, or irritable bowel syndrome) means a disorder of the functioning of the intestine (of the small intestine and the colon or large intestine), which is not serious but responsible for significant discomfort.

[0136] Irritable bowel syndrome is a combination of bowel dysfunction including:

[0137] - abdominal pain;

[0138] - digestive discomfort;

[0139] - intestinal transit disorders (constipation, diarrhea or alternation of the two).

[0140] By "treatment" we mean both therapeutic and non-therapeutic treatment.

[0141] This means a reduction or even elimination of irritable bowel syndrome, or at least one of the above-mentioned bowel function disorders.

[0142] Generally speaking, "treatment" also includes alleviating the symptoms of irritable bowel syndrome.

[0143] We also include:

[0144] - a decrease in the intensity and frequency of symptoms of functional colopathy, and / or

[0145] - an improvement in daily life by reducing the severity of the disease.

[0146] Intestinal permeability refers in particular to an alteration and / or inflammation that leads to tight junctions that become distended and intestinal porosity that develops.

[0147] Such intestinal permeability encompasses the phenomenon of intestinal hyperpermeability, or Leakey Gut syndrome.

[0148] There are many factors that can trigger intestinal problems and can make the gut leaky, including:

[0149] - diet, - imbalance of intestinal flora (dysbiosis),

[0150] - long-term use of medications such as antibiotics, anti-inflammatories, corticosteroids, etc.,

[0151] - stress,

[0152] - food allergies and intolerances to FODMAPs (Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols).

[0153] By "treatment" is meant a composition according to the invention which strengthens the intestinal barrier at the cellular level, with beneficial effects on intestinal function and mucosa.

[0154] The composition according to the invention makes it possible to support intestinal function with the supply of nutrients which will relieve gastrointestinal irritations, participate in the restoration of the intestinal mucosa and therefore reduce intestinal hyperpermeability.

[0155] Of course, various other modifications may be made to the invention within the scope of the appended claims.

[0156] Examples

[0157] Effects of glutamine, curcumin, and fish bioactive peptides alone or in combination on intestinal permeability in a chronic restraint stress model Materials and methods

[0158] Chronic restraint stress (CRS)

[0159] Mice were briefly anesthetized with isoflurane and placed in restraint cages (Bioseb®, Vitrol les, France) for two hours before returning to their home cages.

[0160] For acute restraint stress (ARS), the restraint stress was performed only once.

[0161] For chronic restraint stress (CRS), restraint sessions were repeated for four consecutive days (at the same time).

[0162] Control mice were kept in their home cages during the procedure.

[0163] Treatments

[0164] All treatments were performed daily for seven days, before starting the stress procedure, and were maintained during the stress experiments.

[0165] Glutamine (Gln) was diluted in drinking water to provide 2 g. kg -1 of body weight, per day. The Gin solution was prepared and replaced daily.

[0166] Curcuméga® was administered by oral gavage so that the dose of curcumin received was 500 or 100 mg. kg -1 .day 1 according to the groups. Curcuméga® contains more than 3% curcumin, in combination with n-3 polyunsaturated fatty acids, vitamin E and vitamin D3. For 500 mg / kg of curcumin, the mice also received daily 80 mg.kg -1 of EPA, 50 mg.kg -1 of DHA, 12 mg.kg -1of vitamin E and 5 pg.kg -1 of vitamin D3.

[0167] The Curcuméga® solution was diluted in corn oil to achieve the desired concentration. Corn oil was therefore used as the vehicle treatment in the control groups.

[0168] The bioactive peptides Gabolysat® according to the invention, fish protein hydrolyzate (Ga), were administered daily at a dose of 30 mg.kg -1 by oral gavage, while control mice received water as a vehicle.

[0169] The Gabolysat® bioactive peptides according to the invention have a molecular weight profile of said peptide fraction, expressed as a function of the total weight of said peptide fraction, namely:

[0170] - 15 to 55% of peptides with a molecular weight of less than 300 Da,

[0171] - 30 to 85%, preferably 30 to 80%, of peptides with a molecular weight between 300 and 1500 Da,

[0172] - less than 8% of peptides with a molecular weight greater than 1500 Da.

[0173] Sampling

[0174] Blood samples were collected, centrifuged (4°C, 3000 rpm, 15 min), and plasma was frozen at -80°C. Fresh colon samples were collected to assess paracellular permeability in Ussing chambers.

[0175] The remaining colon samples were collected, washed with ice-cold PBS, immediately frozen in liquid nitrogen, and stored at -80°C for further analysis.

[0176] Colon permeability in Ussing chambers

[0177] Colon permeability was assessed by measuring the fluxes of lucifer yellow (LY, 457 Da) and Alexa 680-dextran (AD, 3000 Da) in Ussing chambers with an exchange surface area of ​​0.07 cm 2 (Harvard Apparatus, Holliston, USA).

[0178] The samples were kept at a temperature of 37°C.

[0179] Both fluorophores were placed in the mucosa and the medium from the serous side was collected after 3 hours and stored at -80°C.

[0180] The fluorescence level of lucifer yellow (excitation 428 nm, emission 540 nm) and Alexa dextran (excitation 665 nm, emission 710 nm) in serous medium was measured in a black 96-well plate with a Spark® multi-mode microplate reader (Tecan).

[0181] Values ​​were converted to concentration using a standard curve.

[0182] RT-qPCR

[0183] Total mucosal RNA was extracted from the samples described previously. After reverse transcription of 1 pg of total RNA into cDNA using 200 units of SuperScript™ II reverse transcriptase (LifeTechnologies, Cergy-Pontoise, France), qPCR was performed by SYBR™ Green technology on the BioRad CFX96 Real-Time PCR System (BioRad Laboratories, Marnes la Coquette, France). GAPDH was used as the endogenous reference gene.

[0184] Corticosterone dosage

[0185] Plasma corticosterone levels were measured using a commercially available ELISA kit (Abnova® KA0468)

[0186] Statistical analysis

[0187] Data were analyzed using GraphPad Prism 8.3 software (GraphPad Software Inc., San Diego, CA, USA) and expressed as mean ± standard error of the mean. Values ​​were compared by ANOVA or Kruskal-Wallis test, as appropriate, followed by appropriate post hoc tests. Results were considered significant when the p-value was less than 0.05.

[0188] Results

[0189] Acute restraint model

[0190] ARS resulted in a significant increase in plasma corticosterone levels compared to the control group (7.3-fold increase, Fig. 1A).

[0191] Furthermore, colonic permeability was decreased, as demonstrated by decreased concentrations of lucifer yellow and Alexa dextran in the serous compartment of Ussing chambers (Fig. 1 BC).

[0192] Chronic restraint stress model

[0193] An increase in plasma corticosterone was observed in the CRS group compared to the control group (2.2-fold increase, Fig. 1 D). In contrast to ARS, CRS induced an increase in lucifer yellow and Alexa dextran levels in the Ussing chamber (Fig. 1 E and 1 F), showing that this model induces an increase in colonic permeability.

[0194] Assessment of plasma corticosterone and intestinal permeability in stressed mice treated

[0195] Regarding plasma corticosterone levels, no significant changes were observed in all groups (Glutamine, Curcuméga® at low and high doses, Gabolysat®) (Fig.2A).

[0196] Overall, all treatments induced a decrease in colonic LY permeability (457 Da) compared to stressed controls (Fig. 2B) (respectively, 1.96, 1.29, 1.44 and 2.10 times more for Gin, high and low dose Curcuméga® and Gabolysat®).

[0197] AD colon permeability (3 kDa) was significantly reduced only after Gin and high-dose Curcuméga® (2.81 and 1.72 times, respectively), while low-dose Curcuméga® improved AD colonic permeability compared to the CRS vehicle group (Fig2.C). For Gabolysat® treatment, a decreasing trend was observed, but the difference did not reach a significant value (p = 0.087, Fig.20).

[0198] Taking into account the previous results, we then evaluated the effects of the combined bioactive peptides glutamine, Curcuméga® and Gabolysat® (GOG). Again, plasma corticosterone levels remained unchanged between the CRS-vehicle and CRS-GCG groups (Fig. 3A).

[0199] However, both LY and AD colonic permeability were significantly reduced compared to untreated stressed mice (2.08- and 3.19-fold, respectively, Fig. 3B and Fig. 3C), which was the most effective reduction in AD colonic permeability observed.

[0200] We assessed tight junction-related gene expression in these different groups. As shown in Figure 4, we did not observe significant changes in occludin, claudin-1, and ZO-1 mRNA levels in colonic mucosa.

[0201] Assessment of inflammation-related gene expression in the colonic mucosa of treated stressed mice

[0202] To assess the colonic inflammatory response, we evaluated gene expression of pro-inflammatory (CXCL1, TNF and IL1P) and anti-inflammatory (IL10) cytokines (Fig. 5).

[0203] For pro-inflammatory cytokines, we only observed a significant effect for CXCL1 (p (Kruskal-Wallis) = 0.0302). The changes observed for TNF and ILi p remained non-significant. Interestingly, post-tests revealed that the CXCL1 mRNA level was significantly reduced only in the CRS-GCG group compared to the CRS-vehicle group (Fig. 5A), suggesting a synergistic effect of the bioactive peptides Gin, Curcuméga® and Gabolysat® on this parameter.

[0204] Regarding the anti-inflammatory cytokine, IL10, CRS-GA showed an increase in IL10 mRNA level (Fig. 5D) which was not observed in other treated groups, including the CRS-GCG group.

[0205] Discussion

[0206] Glutamine treatment combined with CRS induced a decrease in colonic permeability, as demonstrated by the reduction in the concentration of Alexa-dextran and lucifer yellow in Ussing chambers compared to the CRS-vehicle group.

[0207] Treatment with Curcuméga® had a similar effect, except in the low-dose group where Alexan-dextran levels increased.

[0208] The CRS group treated with Gabolysat® bioactive peptides also showed a decrease in intestinal permeability, which was only significant with the Lucifer Yellow marker. Due to its smaller size, the Lucifer Yellow marker appears to be more sensitive for measuring paracellular flow in the Ussing chambers. The bioactive peptides in Gabolysat® maintain the integrity of the colonic mucosal barrier.

[0209] Finally, the mixed treatment including glutamine, Curcuméga® and Gabolysat ® (GOG) showed the most pronounced decrease in intestinal permeability, as well as a decrease in the inflammatory marker CXCL1, which was not observed with each compound administered alone.

[0210] CXCL1 and its receptor CXCR2 play a key role in neutrophil recruitment and thus regulate colonic mucosal inflammation. These results show that a synergistic effect occurs with the combination of treatments. In conclusion, we demonstrate in the present study that combined administration of glutamine, curcumin, and bioactive peptides is more effective in reducing intestinal barrier disruption and the inflammatory response than either compound alone.

Claims

Claims

1. A composition for use in the treatment of irritable bowel syndrome and / or in the treatment of intestinal hyperpermeability, which composition comprises a fish protein hydrolyzate which comprises a peptide fraction comprising at least 15% of peptides with a molecular weight of less than 300 Da, expressed as a function of the total weight of said peptide fraction.

2. Composition according to claim 1, characterized in that the molecular weight profile of said peptide fraction is as follows, expressed as a function of the total weight of said peptide fraction: - 15 to 55% of peptides with a molecular weight of less than 300 Da, - 30 to 85%, preferably 30 to 80%, of peptides with a molecular weight between 300 and 1500 Da, - less than 8% of peptides with a molecular weight greater than 1500 Da.

3. Composition according to any one of claims 1 or 2, characterized in that said fish protein hydrolyzate is derived from fish chosen from oily fish and white fish.

4. Composition according to claim 3, characterized in that said fish protein hydrolyzate is derived from white fish chosen from cod, ling, pollock, pout, julienne, whiting and hake.

5. Composition according to any one of claims 1 to 4, characterized in that said composition also comprises a glutamine, preferably L-glutamine.

6. Composition according to claim 5, characterized in that the weight ratio between said fish protein hydrolyzate and glutamine is from 0.01 to 0.

09.

7. Composition according to any one of claims 1 to 6, characterized in that said composition also comprises a curcumin.

8. Composition according to any one of claims 1 to 7, characterized in that said composition comprises said fish protein hydrolyzate, curcumin and glutamine.

9. Composition according to any one of claims 1 to 8, characterized in that said composition also comprises: - polyunsaturated fatty acids, preferably omega-3, and - vitamins, for example vitamin E and / or vitamin D3.

10. Composition according to any one of claims 1 to 9, characterized in that it consists of a pharmaceutical, nutraceutical, dietetic or food composition, in particular for human and / or animal use.

11. Non-therapeutic use of a composition which comprises a fish protein hydrolysate which comprises a peptide fraction comprising at least 15% of peptides with a molecular weight of less than 300 Da, expressed as a function of the total weight of said peptide fraction, for the treatment of irritable bowel syndrome (IBS) and / or for the treatment of intestinal hyperpermeability, which composition consists of a nutraceutical, dietetic or food composition, in particular for human and / or animal use.

12. Non-therapeutic use, according to claim 11, characterized in that the molecular weight profile of said peptide fraction is as follows, expressed as a function of the total weight of said peptide fraction: - 15 to 55% of peptides with a molecular weight of less than 300 Da, - 30 to 85%, preferably 30 to 80%, of peptides with a molecular weight between 300 and 1500 Da, - less than 8% of peptides with a molecular weight greater than 1500 Da.

13. Non-therapeutic use, according to claim 12, characterized in that said composition also comprises a glutamine and / or a curcumin.

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