Patent Description:
The human gastrointestinal (GI) tract, the largest microbial reservoir in the body, harbors about <NUM><NUM> microorganisms, predominantly bacteria. These microorganisms are collectively referred to as microbiota, while their collective genomes constitute what is known as the microbiome. The normal microbiota typically consist of <NUM>-<NUM> different species, primarily inhabiting the colon, of which Firmicutes and Bacteroidetes species represent more than <NUM>%. GI bacterial populations vary in their qualitative composition and abundance from the proximal to the distal portion, and from inner to outer lumen, and are influenced by the subject's age, dietary habits, geographical origin, type of birth, antibiotic therapies, and exposure to environmental stimuli (<NPL>).

The gut microbiota is involved in a number of physiological functions including digestion, metabolism, extraction of nutrients, synthesis of vitamins, prevention of pathogen colonization, and immune modulation. Alterations or changes in composition and biodiversity of the gut microbiota have been observed in various metabolic states and in many gastrointestinal disorders and other pathophysiological conditions. For example, microbiota disruptions or alterations have been associated or correlated with cases of obesity, inflammatory bowel disease (IBD), celiac disease, irritable bowel syndrome (IBS), colon cancer, diabetes, liver disorders, cystic fibrosis and allergies. Thus, specific microbiota profiles have been evaluated as markers for various pathophysiological states. At present, the interplay between the development of such pathologies and changes in microbiota composition and diversity is yet to be fully elucidated. Thus, studies aimed at investigating this interplay, attempting to determine whether a particular microbial alteration results from the development of a pathology of interest, and whether (and to what extent) it contributes to its etiology, are emerging as an intriguing field of study (Vajro et al. , <NUM>, ibid; <NPL>).

Various approaches of modulating the gut microbiota have been reported. Such approaches include for example probiotics (administration of live microorganisms), prebiotics (digestion resistant dietary supplements that selectively enhance the growth and/or activity of a particular resident gut microbe), antimicrobial agents (e.g. antibiotics), as well as more indirect approaches such as surgery and weight loss strategies. In view of evidence that links the disruption in the composition and diversity of the gut microbiota to the development of certain pathologies, approaches aimed at modulating the gut microbiota have been suggested as potential therapies. However, while some studies reported on favorable results, others have failed to report any therapeutic benefit, or even described aggravation or deterioration of the condition or its symptoms (Erejuwa et al. , <NUM>, ibid). Thus, research investigating the role of gut microbiota in disease development, and in particular the prospects of microbial modulation as a therapeutic approach, is still in its infancy, and necessitates more rigorous in vitro, animal, and clinical studies.

Fatty acid bile salt conjugates, referred to also as Fatty Acid Bile Acid Conjugates (FABACs), are a family of synthetic molecules that may be used to improve conditions related to bile acids or cholesterol metabolism. FABACs are believed to lower blood cholesterol concentration, reduce liver fat levels and dissolve gallstones (<NPL>; and <NPL>). FABAC include inter alia 3β-arachidylamido-7α,12α-dihydroxy-5β-cholan-<NUM>-oic acid, also known as Aramchol.

<CIT>, <CIT>, <CIT> disclose certain FABACs and their use in dissolving cholesterol gallstones in bile and treating arteriosclerosis. These and additional FABACs were disclosed in <CIT>, <CIT>and<CIT> for use in treating fatty liver, in reducing blood cholesterol levels and in treating hyperglycemia, diabetes, insulin resistance and obesity. Further therapeutic uses of FABACs are disclosed in <NPL>) and in <CIT> and <CIT>. Amine salts of certain FABACs are disclosed in <CIT>.

<CIT> and <CIT> refer to the use of purified populations of spore-forming bacteria, and to therapeutic compositions containing non-pathogenic, germination-competent bacterial spores. These publications suggest the use of various culture media supplements including Oxgall (dehydrated bovine bile, composed of fatty acids, bile acids, inorganic salts, sulfates, bile pigments, cholesterol, mucin, lecithin, glycuronic acids, porphyrins, and urea) for in vitro purposes, namely in germinating populations of bacterial spores in vitro.

<CIT> relates to preparations of glycan therapeutics, pharmaceutical compositions and medical foods thereof, optionally comprising micronutrients, polyphenols, prebiotics, probiotics or other agents, and methods of making same. Also provided are methods of using the glycan therapeutics, e.g. for the modulation of human gastrointestinal microbiota and to treat dysbioses. <CIT> further discloses that said glycan therapeutics may be used for the treatment of metabolic disorders such as NAFLD and NASH. Aramchol is mentioned merely among hitherto known treatments for NAFLD/NASH. Glycan therapeutics are disclosed as comprising branched glycans that comprise glucose, galactose, arabinose, mannose, fructose, xylose, fucose, or rhamnose glycan units, further defined by particular parameters including their average degree of branching and polymerization.

<CIT> relates to a preparation from animal, artificial, synthetic, cultured and/or fermented stool, made by suspending the stool in a liquid and by depletion of the microbiota comprised in the suspension analogue to or by filtrating the suspension with a filter having a maximum pore size of < <NUM>, for use as a medicine.

Safadi et al. discloses the reduction of liver fat content in patients with non-alcoholic fatty liver disease by the fatty acid-bile acid conjugate Aramchol (<NPL>).

There exists an unmet medical need for effective treatments specifically aimed at improving or restoring gut microbiota. In addition, there remains a need for additional and improved therapies for alleviating gastrointestinal disorders, or other conditions involving gut flora imbalance as part of their etiology.

The present invention is directed to the modulation of gut microbiota.

Specifically, the invention relates to pharmaceutical compositions for use in modifying gastrointestinal (GI) microbial populations and in alleviating GI disorders and other conditions associated with imbalance of gut flora.

The present invention thus provides a pharmaceutical composition for use in a therapeutic method of promoting the growth of beneficial microbiota in the gastrointestinal (GI) tract, for use in a therapeutic method of inhibiting the growth of detrimental microbiota in the GI tract, for use in a therapeutic method of enhancing microbiota biodiversity in the GI tract or for use in a therapeutic method of treating or preventing dysbiosis of the GI tract in a subject in need thereof, the composition comprising a therapeutically effective amount of 3β-arachidylamido-7α, 12α-dihydroxy-5β-cholan-<NUM>-oic acid (Aramchol), or a pharmaceutically acceptable salt thereof.

The term "dysbiosis" as used herein refers to a state of the microbiota of the gut or other body area in a subject, in which the normal diversity and/or function of the microbial populations is disrupted. This unhealthy state can be due to a decrease in diversity, the overgrowth of one or more pathogens or pathobionts, symbiotic organisms able to cause disease only when certain genetic and/or environmental conditions are present in a subject, or the shift to an ecological microbial network that no longer provides an essential function to the host subject, and therefore no longer promotes health. The essential functions may include enhancement of the gut mucosal barrier, direct or indirect reduction and elimination of invading pathogens, enhancement of the absorption of specific substances, and suppression of GI inflammation.

In an embodiment, the dysbiosis is associated with a gastrointestinal disease or disorder.

In an embodiment, said dysbiosis is associated with a chronic inflammatory disease, an autoimmune disease, an infection, bowel resection, and/or a condition associated with chronic diarrhea.

In an embodiment, the disease or disorder is irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), short bowel syndrome (SBS), celiac disease, small intestinal bacterial overgrowth (SIBO), gastroenteritis, leaky gut syndrome, or gastric lymphoma.

In an embodiment, the disease or disorder is associated with a bacterial, viral or parasitic infection or overgrowth, optionally the bacteria is drug resistant bacteria.

In an embodiment, the subject is human. Optionally, the human subject exhibits dysbiosis or is at risk for developing dysbiosis.

In an embodiment, said subject is under a treatment regimen with at least one of antimicrobial agents, parenteral nutrition, or immune suppressive agents.

In an embodiment, said composition comprises the Aramchol as a sole active ingredient.

In an embodiment, said composition is formulated for oral administration, as a controlled release formulation enabling jejunal, duodenal, ileal or colonic delivery, for localized jejunal, duodenal, ileal or colonic delivery by infusion, or for rectal administration.

In an embodiment, said composition is for use in combination with a diet selected from fiber-enriched, fructose-reduced, elemental, total liquid enteral, total parenteral, or peripheral parenteral diet.

In an embodiment, the pharmaceutical composition is for use in combination with at least one antibiotic, probiotic or prebiotic agent, preferably the antibiotic agent is selected from nitroimidazoles, macrolides, or beta-lactams, the probiotic agent comprises at least one of lactobacillus casei, lactobacillus plantarum, lactobacillus acidophilus, and lactobacillus rhamnosus, or a fecal microbiota transplant (FMT), and/or the prebiotic agent is selected from lactulose, lignin, cellulose, hemicelluloses, β-glucans, pectin, gums, resistant starch, dextrin, psyllium, inulin, fructooligosaccharides, or polydextrose.

The present invention also provides a kit comprising a pharmaceutical composition for use according to the present invention and instructions for use thereof, in combination with at least one antibiotic, probiotic or prebiotic agent.

In an embodiment, Aramchol is in the form of Aramchol free acid. In another embodiment Aramchol is in the form of an amine-based salt. In certain particular embodiments, the salt is a meglumine, lysine or tromethamine Aramchol salt.

In another embodiment the compositions of the invention comprise said conjugate as the active ingredient and further comprise at least one of a pharmaceutically acceptable excipient, diluent or carrier.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

The present invention is directed to the modulation of gut microbiota. Specifically, the invention relates to compositions for restoring gastrointestinal homeostasis, for modifying gastrointestinal microbiota profiles at various levels of the GI tract, and for alleviating gastrointestinal disorders and other conditions associated with imbalance of gut flora.

More specifically, the invention relates to the use of compositions comprising a therapeutically effective amount of the fatty acid bile acid conjugate (FABAC) 3β-arachidylamido-7α, 12α-dihydroxy-5β-cholan-<NUM>-oic acid (Aramchol) or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition comprising a therapeutically effective amount of Aramchol or a pharmaceutically acceptable salt thereof may be for use in preventing and/or treating a gastrointestinal pathology associated with alternation of intestinal flora balance in a subject in need thereof.

In an embodiment, the pharmaceutical composition comprising a therapeutically effective amount of Aramchol or pharmaceutically acceptable salt thereof is in combination with at least one antibiotic, probiotic or prebiotic agent.

The present invention also provides a kit comprising a pharmaceutical composition for use according to the present invention and instructions for use thereof, in combination with at least one antibiotic, probiotic or prebiotic agent. In another aspect, the invention relates to a kit comprising Aramchol or a pharmaceutically acceptable salt thereof and instructions for use thereof in combination with at least one antibiotic, probiotic or prebiotic agent, for the treatment or prevention of dysbiosis or a gastrointestinal pathology associated therewith.

As used herein, the terms "FABAC", "FABACs", "BAFACs", and "the FABACs" are used interchangeably and refer to conjugates of the Formula W - X - G (Formula I), wherein G represents a bile acid or a bile salt radical thereof, W represents one or two fatty acid radical(s) having <NUM>-<NUM> carbon atoms, and X represents a bonding member between said bile acid and the fatty acid radical(s). FABACs are known in the art, and are described, for example, in <CIT>, <CIT>, and <CIT>.

In this invention, the FABAC for use according to the present invention is Aramchol, which is presented in Formula III herein below wherein n=<NUM>.

The FABAC for use according to the invention is 3β-arachidylamido-7α, 12α, dihydroxy-5β-cholan-<NUM>-oic acid (Arachidyl Amido Cholanoic Acid; an amide conjugate of cholic acid with arachidic acid; also known as "Aramchol" or "C20 FABAC"). In another embodiment Aramchol is in the form of Aramchol free acid. In another embodiment Aramchol is in the form of an amine-based salt. In certain particular embodiments, the salt is a meglumine, lysine or tromethamine Aramchol salt.

Any suitable route may be used to administer the composition of the invention to a subject.

According to some embodiments, suitable administration routes may be systemic routes. According to some embodiments, administering is administering systemically. According to some embodiments, the composition is formulated for systemic administration.

Yet according to other advantageous embodiments, it is hereby disclosed that the pharmaceutical composition of the invention may be effective even when administered locally (e.g. topically to a GI mucous membrane or by localized administration to a particular segment or region of the GI tract), even when systemic exposure to the active ingredient is reduced or suboptimal. Thus, according to some embodiments, administering is administering locally. According to some embodiments, the composition is formulated for local administration. in other embodiments, the composition is formulated for localized administration, e.g. by infusion by enema or colonoscope or an orogastric, nasogastric, nasoduodenal or nasojejunal tube or any other GI port of entry such as percutaneous endoscopic gastrostomy (PEG) tubes and stomas.

According to other embodiments, a therapeutically effective amount refers to an amount effective to provide a beneficial effect to the subject's microbiota, e.g. when administered locally.

According to another embodiment, administration systemically is through an enteral route. According to another embodiment, administration through an enteral route is oral administration. According to some embodiments, the composition is formulated for oral administration.

According to some embodiments, oral administration is in the form of hard or soft gelatin capsules, pills, capsules, tablets, including coated tablets, dragees, elixirs, suspensions, liquids, gels, slurries or syrups and controlled release forms thereof.

Suitable carriers for oral administration are well known in the art. Compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries as desired, to obtain tablets or dragee cores. Examples of suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, and sodium carbomethylcellulose, and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).

If desired, disintegrating agents, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added. Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as it normal practice, additional substances other than inert diluents, e.g., lubricating, agents. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

The dosage forms can additionally and advantageously be prepared with a controlled release coating such as enteric coatings. The term "enteric coating", as used herein, refers to a coating which controls the location of composition absorption within the digestive system. Examples for materials used for enteric coating are fatty acids, waxes, plant fibers or plastics. In other embodiments, the dosage forms may be prepared with a controlled release matrix, controlling the location of composition absorption within the digestive system. According to various embodiments, the composition is formulated as a controlled release formulation enabling e.g. enhanced or targeted jejunal, duodenal, ileal or colonic delivery. In other words, said formulation may release the active ingredient in a controlled manner so as to provide significantly increased absorption (e.g. by at least <NUM>%, <NUM>%, <NUM>% or <NUM>%) of said active ingredient in the GI region of interest compared to its absorption from conventional immediate release formulations. In other embodiments, said composition is formulated for localized jejunal, duodenal, ileal or colonic delivery by infusion. In another embodiment said composition is formulated for oral or rectal administration. Thus, in certain embodiments, the invention is directed to the use of sustained-release or controlled release formulations of the compounds of Formula (I) as defined herein or salts thereof, including those disclosed by <CIT> For example, the composition may comprise a core coated with two films, the first inner film being a semi-permeable to water or body fluids film applied directly on said core and comprising cellulose acetate, the second outer film being a permeable to water or body fluids film comprising ethylcellulose.

For a formulation of the drug administered at a particular absorption site, the drug level achieved is the outcome of rate constants for (<NUM>) release of the drug from the formulation, (<NUM>) absorption, and (<NUM>) elimination, respectively. For immediate release dosage forms, the rate constant for drug release is far greater than the absorption rate constant. For the controlled release formulations, the opposite is true, such that the rate of release of drug from the dosage form is the rate-limiting step in the delivery of the drug to the target area. The term "controlled release" as used herein is intended to include any non-immediate release formulation, including sustained release, delayed release and pulsatile release formulations.

A controlled release formulation may comprise, for example, controlled release beads comprising the drug. A common type of controlled release beads comprises an inert core, such as a sugar sphere, coated with an inner drug-containing layer and an outer membrane layer controlling drug release from the inner layer. An example of such controlled release beads is described in <CIT> where each bead comprises (i) a core unit of a soluble or insoluble inert material, (ii) a first layer on the core unit comprising an active ingredient dispersed in a hydrophilic polymer, (iii) an optional second layer of hydrophilic polymer covering the first layer, and (iv) an outermost membrane layer effective for controlled release of the active ingredient. In the above and similar controlled release beads it is not uncommon to apply a "sealcoat" in the form of a small amount (e.g. <NUM>-<NUM>%) of a water-soluble polymer, such as hydroxypropylmethyl cellulose (HPMC) or polyvinylpyrrolidone (PVP), between the inert core and the layer containing the active ingredient. The purpose thereof is generally to isolate the drug from the core surface in the event that a drug-core chemical interaction is possible, and/or to smooth the surface of the inert core such that the surface area is more consistent from lot to lot to thereby improve the coating quality when the drug layer and the controlled release membrane layers are applied.

The cores are typically of a water-soluble or swellable material, and may be any such material that is conventionally used as cores or any other pharmaceutically acceptable water-soluble or water-swellable material made into beads or pellets. Especially, the beads are spheres of sucrose/starch (Sugar Spheres NF), sucrose crystals, or extruded and dried spheres typically comprised of excipients such as microcrystalline cellulose and lactose.

The substantially water-insoluble material in the first or sealcoat layer is generally a "GI insoluble" or "GI partially insoluble" film forming polymer (latex or dissolved in a solvent). As examples may be mentioned ethyl cellulose, cellulose acetate, cellulose acetate butyrate, polymethacrylates such as ethyl acrylate/methyl methacrylate copolymer (Eudragit NE-<NUM>-D) and ammonio methacrylate copolymer types A and B (Eudragit RL30D and RS30D), and silicone elastomers. Usually, a plasticizer is used together with the polymer. Exemplary plasticizers include: dibutylsebacate, propylene glycol, triethylcitrate, tributylcitrate, castor oil, acetylated monoglycerides, acetyl triethylcitrate, acetyl butylcitrate, diethyl phthalate, dibutyl phthalate, triacetin, fractionated coconut oil (medium-chain triglycerides).

The second layer containing the active ingredient may be comprised of the active ingredient (drug) with or without a polymer as a binder. The binder, when used, is usually hydrophilic but may be water-soluble or water-insoluble. Exemplary polymers to be used in the second layer containing the active drug are hydrophilic polymers such as polyvinylpyrrolidone (PVP), polyalkylene glycol such as polyethylene glycol, gelatine, polyvinyl alcohol, starch and derivatives thereof, cellulose derivatives, such as hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, carboxyethyl cellulose, carboxymethylhydroxyethyl cellulose, acrylic acid polymers, polymethacrylates, or any other pharmaceutically acceptable polymer.

In a particular embodiment, the composition is a controlled release formulation formulated for colonic delivery. For example, colonic delivery systems are disclosed in <CIT> and <CIT> to some of the inventors of the present invention.

Liquid dosage forms for oral administration may further contain adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides. In a particular embodiment, the composition comprises Aramchol as the sole active ingredient. For example, an effective amount for oral administration to human subjects may be a daily dose of <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM> or <NUM>-<NUM>.

The pharmaceutical compositions and kits of the invention may optionally further comprise additional active ingredients, including antimicrobial (e.g. antibiotic), probiotic and prebiotic agents. In another embodiment, the invention relates to pharmaceutical compositions and kits comprising a therapeutically effective amount of Aramchol or a pharmaceutically acceptable salt thereof in combination with at least one antibiotic, probiotic or prebiotic agent. In another embodiment said composition comprises a plurality of antibiotic, probiotic and/or prebiotic agents.

As used herein, the term "antimicrobial" refers to a compound or element that inhibits the growth or kills microorganisms. Antimicrobial agents include compounds such as antibiotics produced naturally or synthetically. As used herein, the term "antibiotic" denotes an antimicrobial agent that inhibits bacterial growth or kills bacteria. The term "antibacterial" is often used synonymously with the term antibiotic(s). The term "antimicrobial" denotes a broader range of compounds providing efficacy against the causative agents of one or more infectious diseases, including e.g. antibacterial, anti-fungal and anti-parasitic agents.

Antimicrobial agents may be produced by known synthetic methods or isolated from their natural source, and various antimicrobial drugs are available commercially. Exemplary antibiotic agents include nitroimidazoles, macrolides and beta-lactams. Various nitroimidazole drugs capable of inhibiting nucleic acid synthesis and/or disrupting microbial DNA were developed against anaerobic bacterial and parasitic infections, and include <NUM>, <NUM>- and <NUM>-nitroimidazoles (classified according to the location of the nitro functional group). Drugs of the <NUM>-nitro variety include e.g. metronidazole (Flagyl), tinidazole, nimorazole, dimetridazole, <NUM>-Amino PA824, ornidazole, megazol, and azanidazole. Drugs based on <NUM>-nitromidazoles include e.g. benznidazole. The macrolides are a class of natural products having a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. Various macrolides having protein synthesis inhibitory activity were developed as antibiotic agents (e.g. Azithromycin, Clarithromycin, Erythromycin, Fidaxomicin, and Telithromycin). Polyene antimycotics such as amphotericin B and nystatin manifests antifugal activity. β-lactam antibiotics (or beta-lactams) are a class of broad-spectrum antibiotics, consisting of all antibiotic agents that contain a β-lactam ring in their molecular structures. This includes penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. Their activity includes inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls.

As used herein, the term "probiotic" refers to a live microbial agent that is beneficial to health. Typically, this term denotes compositions comprising ingestible live microbial cultures such as bacterial cultures, which survive transit through the gastrointestinal tract and beneficially affect the host by improving its intestinal microbial balance. Exemplary probiotic agents include, lactobacillus species such as lactobacillus casei, lactobacillus plantarum, lactobacillus acidophilus, and lactobacillus rhamnosus, as well as other lactic acid-producing bacteria such as Bifidobacterium species. Probiotic agents may be formulated for oral administration e.g. in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets, pastilles, sweets, bars, syrups and corresponding administration forms, which may be in the form of a unit dose. The probiotic composition may typically comprise <NUM><NUM> to <NUM><NUM> colony forming units (cfu), preferably at least <NUM><NUM> cfu, <NUM><NUM> cfu, <NUM><NUM> cfu, or <NUM><NUM> cfu per gr dry weight of the composition. Probiotic agents may also be delivered via other enteral routes, such as by enema, colonoscopy, asogastric or nasoduodenal tube. For example, the probiotic agent may be a fecal microbiota transplant (FMT), namely a preparation of fecal matter containing bacteria (and typically further containing natural antibacterials), introduced directly into the GI tract of the recipient. FMT are typically produced by collecting a stool sample from a tested healthy donor, mixing the sample with a saline or other solution, straining the resulting solution, and placing the resulting FMT in the patient e.g. by enema, orogastric tube or by mouth in the form of a capsule containing freeze-dried material.

As used herein, the term "prebiotic" refers to a digestion-resistant food ingredient that beneficially affects a human and/or other animal that ingests the prebiotic. In preferred embodiments, prebiotics selectively stimulate the growth and/or activity of a limited number of bacterial types in the intestinal tract, such that the health of the human and/or other animal is improved. Examples for prebiotics are resistant starch, fructo-oligosaccharides, galactooligosaccharides, xylo-oligosaccharides, polydextrose, lactulose, inulin or soluble fiber (e.g. psyllium husk or acacia fibers). Other exemplary prebiotic agents are lignin, cellulose, hemicelluloses, β-glucans, pectin, gums and dextrin.

In another embodiment of the invention, said composition or kit may comprise ursodeoxycholic acid or other substances aimed at modification of the entero-hepatic cycle and cholesterol metabolism. In yet another embodiment said composition or kit does not comprise ursodeoxycholic acid. In another embodiment said composition or kit does not comprise a branched glycan comprising glucose, galactose, arabinose, mannose, fructose, xylose, fucose, or rhamnose glycan units.

According to certain embodiments, the compositions and kits of the invention are for use in treating or preventing dysbiosis in a subject in need thereof. In other embodiments, the compositions and kits of the invention are for use in for promoting the growth of beneficial microbiota in the GI tract. In other embodiments, the compositions and kits of the invention are for use in inhibiting the growth of detrimental microbiota in the GI tract. In other embodiments, the compositions and kits of the invention are for use in enhancing microbiota biodiversity in the GI tract. In other embodiments, the compositions and kits of the invention are useful in treating or preventing the appearance of symptoms of dysbiosis in a subject in need thereof. In some embodiments, symptoms of dysbiosis may include abdominal distension, regular/frequent episodes of diarrhea, frequent stools, recent onset/chronic diarrhea or diarrhea for <NUM>-<NUM> months, poor tolerance/intolerance of sugars, flatulence, rotten egg burps, meal-related bloating, and constant fatigue.

As used herein, the terms "gastrointestinal" and "GI" refer to the stomach and intestines in the digestive tract of humans and other animals. However, as also used in context herein, the term "gastrointestinal tract" ("GI tract") refers to the entire alimentary canal, from the oral cavity to the rectum. The term encompasses the tube that extends from the mouth to the anus, in which the movement of muscles and release of hormones and enzymes digest food. The gastrointestinal tract starts with the mouth and proceeds to the esophagus, stomach, small intestine, large intestine, rectum and, finally, the anus. As used herein, the terms "gastrointestinal" and "GI tract" are not intended to include accessory organs of digestion, such as the liver, gallbladder, and pancreas.

As used herein, gastrointestinal diseases and disorders, further referred to collectively as gastrointestinal pathologies, denote diseases and disorders that primarily affect the GI tract. GI pathologies to be treated, prevented or alleviated by the compositions and kits of the invention may be associated with dysbiosis or alteration of intestinal floral balance (as part of their etiology and/or pathology). Such dysbiosis or alteration may in various embodiments result from, or be associated with, chronic inflammatory reactions, autoimmune reactions, infections (e.g. bacterial, viral or parasitic), bowel resection, and/or chronic diarrhea.

In other embodiments, the compositions and kits of the invention are useful in preventing and/or treating a gastrointestinal pathology associated with alternation of intestinal flora balance in a subject in need thereof. In other embodiments, the compositions and kits of the invention are useful in treating and/or preventing a gastrointestinal pathology associated with a chronic inflammatory disease, an autoimmune disease, an infection, bowel resection, and/or a condition associated with chronic diarrhea. In other embodiments, the compositions and kits of the invention are useful in treating and/or preventing irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), short bowel syndrome (SBS), celiac disease, small intestinal bacterial overgrowth (SIBO), gastroenteritis, leaky gut syndrome, and gastrointestinal lymphoma, or a pathology associated with a bacterial, viral or parasitic infection or overgrowth. In other embodiments, the compositions and kits of the invention are useful in combination with at least one antibiotic, probiotic or prebiotic agent, for the treatment or prevention of dysbiosis or a gastrointestinal pathology associated therewith.

Chronic inflammatory GI diseases include IBS and IBD. Autoimmune GI diseases include Celiac disease. GI pathologies associated with an infection include pathologies associated with a bacterial, viral or parasitic infection or overgrowth. In an embodiment, the infection or overgrowth is of a pathogenic bacterium including Yersinia, Vibrio, Treponema, Streptococcus, Staphylococcus, Shigella, Salmonella, Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella, Klebsiella, Helicobacter, Haemophilus, Francisella, Escherichia, Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella, or Bacillus spp. In another embodiment, said infection is of drug-resistant bacteria such as antibiotic-resistant bacteria, including multi-drug resistant bacteria, Carbapenem-resistant Enterobacteriaceae (CRE), extended spectrum beta-lactam resistant Enterococci (ESBL), and vancomycin-resistant Enterococci (VRE).

Chronic diarrhea, defined as a decrease in stool consistency for more than four weeks, may be caused by various pathologies (including IBD and IBS), infection or digestive problems.

Crohn's disease is characterized by ulcerations of the small and/or large intestines, but can affect the digestive system anywhere from the mouth to the anus. Various terms are used to describe Crohn's disease, and tend to reflect the portion of the gastrointestinal tract affected. For example, involvement of the large intestine (colon) only has been termed Crohn's colitis or granulomatous colitis, while involvement of the small intestine only has been termed Crohn's enteritis. Disease in the terminal portion of the small intestine i.e. the ileum, has been termed Crohn's ileitis. When both the small intestine and the large intestine are involved, the condition has been termed Crohn's enterocolitis or ileocolitis. Ulcerative colitis is a condition related to Crohn's disease that involves only the colon, and collectively these diseases are frequently referred to as inflammatory bowel disease (IBD).

Irritable bowel syndrome (IBS) is a common disorder that has a pronounced effect on the quality of life and that accounts for a large proportion of healthcare costs. The disorder is characterized by lower abdominal pain, bloating, diarrhea, constipation, or constipation alternating with diarrhea. Altered bowel motility, visceral hyperalgesia, food allergy, bacterial overgrowth, psychosomatic factors, stress associated with the myenteric nervous system have all been proposed as playing a part in the pathogenesis of IBS. Gastrointestinal inflammation may also be associated with irritable bowel syndrome, along with stress.

Short bowel syndrome (SBS) is a malabsorption disorder caused by a lack of functional small intestine. SBS occurs in subjects having less than <NUM> of working bowel, resulting in intestinal failure (decreased intestinal function such that nutrients, water, and electrolytes are not sufficiently absorbed). The primary symptom is diarrhea, which can result in dehydration, malnutrition, and weight loss. Other symptoms may include bloating, heartburn, feeling tired, lactose intolerance, and foul smelling stool.

Celiac disease is an autoimmune disease manifested in genetically susceptible people caused by intolerance to gluten, resulting in mucosal inflammation and villous atrophy, which causes malabsorption. Symptoms usually include diarrhea and abdominal discomfort. Diagnosis is by small-bowel biopsies showing characteristic though not specific pathologic changes of villous atrophy that resolve with a strict gluten-free diet.

Small intestinal bacterial overgrowth (SIBO), also termed small bowel bacterial overgrowth syndrome, is a disorder of excessive bacterial growth in the small intestine (bacterial counts of ><NUM><NUM>/mL). SIBO can result from alterations in intestinal anatomy (e.g. due to surgery or partial obstruction) or GI motility, or from lack of gastric acid secretion. This condition can lead to vitamin deficiencies, fat malabsorption, and undernutrition. The most frequent symptoms are abdominal discomfort, diarrhea, bloating, and excess flatulence.

Gastroenteritis, is inflammation of the lining of the stomach and small and large intestines. Most cases are infectious, although gastroenteritis may occur after ingestion of drugs and chemical toxins (e.g., metals or plant substances). Symptoms include anorexia, nausea, vomiting, diarrhea, and abdominal discomfort. Treatment is symptomatic, with certain parasitic and bacterial infections requiring specific anti-microbial therapy.

Increased intestinal permeability (abnormally excessive opening of intercellular tight junctions) allows passage of microbes, microbial products, and foreign antigens into the mucosa and bloodstream, with subsequent possible development of immune and/or inflammatory reactions. Such reactions, collectively referred to as "leaky gut syndrome", may result in chronic inflammation and be associated with the development of additional GI pathologies such as celiac disease, Crohn's disease and IBS. Gastrointestinal lymphomas include primary GI tract lymphomas (typically of the non-Hodgkin type) such as mucosaassociated lymphoid tissue (MALT) lymphoma in stomach, mantle cell lymphoma in terminal ileum, jejunum and colon, as well as enteropathy-associated T-cell lymphoma in jejunum, and follicular lymphoma in duodenum.

In other embodiments, the compositions and kits of the invention comprise, or are used in combination with, at least one antibiotic, probiotic or prebiotic agent. Exemplary antibiotic agents include nitroimidazole, macrolide, and beta-lactam antibiotics. Exemplary prebiotic agents include lactulose, lignin, cellulose, hemicelluloses, β-glucans, pectin, gums, resistant starch, dextrin, psyllium, inulin, fructooligosaccharides, and polydextrose. Exemplary probiotic agents include various lactobacillus species or a fecal microbiota transplant (FMT). In other embodiments, the compositions and kits of the invention are used in combination with a specialized diet, including enteral or parentearal liquid formulations, reported to be associated with the development of dysbiosis, or a diet prescribed for the management of dysbiosis. According to exemplary embodiments, the compositions and kits of the invention are used in combination with fiber-enriched, fructose-reduced, elemental, total liquid enteral, total parenteral, and peripheral parenteral diet. Liquid enteral diet refers to the introduction of a nutritionally complete liquid formula directly into the stomach or small intestine via a designated (e.g. nasogastric) tube. Parenteral diet (parenteral nutrition) refers to the delivery of liquid nutrition into a vein. When the diet is used as the exclusive source of nutrition, it is referred to as total liquid enteral diet, or total parenteral diet, respectively. Elemental diet comprises liquid nutrients in an easily assimilated form (for example, nitrogen is provided in the form of free amino acids rather than as whole or partial protein). It is usually composed of amino acids, fats, sugars, vitamins, and minerals. Elemental diet may be administered orally or by use of a gastric feeding tube or intravenous feeding. Fiber enriched diet typically refers to specialized fiber-enriched enteral formulations (e.g. Nutrison multifibre, containing <NUM>/<NUM> of soluble and non-soluble fibers at a <NUM>:<NUM> ratio), but may also refer in some embodiments to prescribed oral diets including daily ingestion of e.g. <NUM> gr of fiber or more. Fructose-reduced diets may be e.g. low-fermentable oligo-saccharides, disaccharides, monosaccharides, and polyol (FODMAP) diet, or enteral nutrition formulations devoid of added fructose.

According to particular embodiments, the compositions of the invention are administered locally to a particular site of the GI tract, e.g. in combination with a FMT.

The subject to be treated by the compositions and kits of the invention, also referred to herein as a subject in need thereof, is a mammalian and preferably a human subject. In certain embodiments, the subject has been diagnosed as suffering from dysbiosis. In certain other embodiments, the subject is at risk for developing dysbiosis.

Various methods for diagnosing dysbiosis are known in the art, and include breathtesting methods, small-bowel culture techniques and culture-independent techniques such as high-throughput next-generation sequencing. For example, the Comprehensive Digestive Stool Analysis (CDSA, a non-invasive evaluation of gastrointestinal function that includes analyses of digestion, absorption, bacterial balance, yeast and parasites), or the GA-map Dysbiosis Test (Genetic Analysis AS, Oslo, Norway, based on DNA profiling using probes targeting variable regions of the bacterial <NUM> rRNA gene), may be used.

In various embodiments, a subject at risk of developing dysbiosis is under a treatment regimen with at least one agent selected from anti-microbial agents, parenteral nutrition, or immune suppressive agents. Immune suppressive agents are drugs that inhibit or prevent activity of the immune system, including glucocorticoids, cytostatics, antibodies or drugs acting on immunophilins.

Known indications suggested for FABAC treatment include those disclosed in <CIT>,<CIT>, <CIT>, <CIT>, <CIT> and<CIT>, as detailed herein. In contradistinction, the present application surprisingly discloses that FABAC may provide therapeutic and/or prophylactic benefits in the management of dysbiosis. It is herein disclosed for the first time that therapeutic regimens comprising Aramchol in particular are particularly advantageous in patients suffering from dysbiosis, or a condition associated therewith. In certain embodiments, the subject is not concomitantly afflicted with an additional condition (such as diabetes, non-alcoholic fatty liver disease, non-alcoholic seatohepatitis, or other known indications for FABAC treatment as detailed above). In a particular embodiment said subject is not afflicted with a liver disease.

The following example is presented in order to more fully illustrate some embodiments of the invention.

Colitis is induced in male rats weighing <NUM> by intra-colonic administration under light ether inhalation anesthesia of <NUM> of dinitrobenzensulfonic acid (DNBS) (<NUM>/ml, in ethanol <NUM> % v/v). The solution is instilled slowly over <NUM> seconds via a flexible, perforated Foley catheter, which was then immediately removed. The rats are placed in an upside down position for another <NUM> seconds. The control group is treated with saline instead of Aramchol using the same procedure.

The rats are anesthetized by ketamine (<NUM>/kg body weight) given by intraperitoneal injection and the colon is exposed through a longitudinal abdominal incision. The distal <NUM> of the colon is removed, cut open and rinsed with ice-cold saline. Euthanasia of the anesthetized rats is performed by puncturing of the chest wall.

The fresh tissues are scored for presence of (<NUM>) edema, (<NUM>) hyperemia and (<NUM>) number and areas of mucosal ulcers. If present, the total area of all ulcers is taken. They are then blotted dry, weighed and immediately frozen in liquid nitrogen. On the day of analysis the colon specimens are warmed to <NUM>, homogenized (Polytron, Kinematica GmbH, Germany) in <NUM> volumes of <NUM> phosphate-buffer, pH <NUM>, and stored at -<NUM> for biochemical analysis of inflammatory markers.

In additional experiments, Aramchol (<NUM>/kg, <NUM>/kg or <NUM>/kg) or vehicle is given by oral gavage, and colitis is induced and evaluated as described above.

Claim 1:
A pharmaceutical composition for use in a therapeutic method of promoting the growth of beneficial microbiota in the gastrointestinal (GI) tract, for use in a therapeutic method of inhibiting the growth of detrimental microbiota in the GI tract, for use in a therapeutic method of enhancing microbiota biodiversity in the GI tract or for use in a therapeutic methof of treating or preventing dysbiosis of the GI tract in a subject in need thereof, the composition comprising a therapeutically effective amount of 3β-arachidylamido-7α, 12α-dihydroxy-5β-cholan-<NUM>-oic acid (Aramchol), or a pharmaceutically acceptable salt thereof.