Treatment of inflammatory bowel diseases with 2'-fucosyllactose compounds

Abstract

To provide methods of treating inflammatory bowel diseases (IBD) (e.g., Crohn's disease (CD) or ulcerative colitis (UC)) or alleviating or reducing the risk of relapse of IBD in a subject.SOLUTION: The present invention provides a method for alleviating or reducing the risk of relapse of inflammatory bowel disease (IBD), the method comprising administering to a subject in need thereof an effective amount of a 2'-fucosyllactose compound, wherein the subject is a human IBD patient who has undergone or is on an anti-inflammatory therapy.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 482,840, filed April 7, 2017, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC) are chronic and debilitating disorders with peak incidence in the second and third decades of life. Although considerable progress has been made in optimizing anti-tumor necrosis factor (TNF) treatment to induce remission in Crohn's disease (CD) and ulcerative colitis (UC), relapses are common and often unpredictable. CD patients who experienced relapse after infliximab withdrawal to thiopurine therapy had lower baseline levels of certain taxa, including Bacteroides, Clostridium coccoides, and F. prausnitzii. Lower baseline levels of F. prausnitzii have also been associated with higher rates of UC relapse. Suppression of mucosal inflammation with infliximab, a monoclonal anti-TNF antibody, only partially corrects this dysbiosis. Thus, alternative approaches for stable maintenance of IBD remission are needed. Summary of the Invention

[0003] The present disclosure is based at least in part on the development of using 2'-fucosyllactose compounds such as 2'-fucosyllactose (2'-FL) to maintain remission in IBD patients.For example, 2'FL can be provided as a dietary supplement alone or as an adjunct to immunosuppressive therapy such as anti-inflammatory therapy.

[0004] Thus, one aspect of the present disclosure features a method for reducing or reducing the risk of recurrence of inflammatory bowel disease (IBD) by administering an effective amount of a 2'-FL compound to a subject in need thereof. For example, the subject may be a human IBD patient who has undergone or is undergoing anti-inflammatory therapy.

[0005] In another aspect, the disclosure provides a method of treating IBD by administering to a subject in need thereof a 2'-FL compound in an amount equivalent to 1 mg / day to 20 mg / day of 2'-FL.

[0006] In any of the methods described herein, the subject in need of a 2'-FL compound may be a human patient at risk of developing IBD, suspected of having IBD, or having IBD. For example, the subject in need of a 2'-FL compound may be a human patient in remission of IBD (e.g., including but not limited to, Crohn's disease (CD) or ulcerative colitis (UC)), for example, who has undergone or is undergoing anti-inflammatory therapy. The anti-inflammatory therapy may be, for example, an anti-TNF therapy, such as the use of a TNF inhibitor, including but not limited to, infliximab and / or adalimumab. The subject to be treated may be an adult or a child. The subject to be treated may be a FUT2 secretor or a FUT2 non-secretor. In some embodiments, the subject to be treated has a daily fiber intake of less than 7g / 1000kcal. In some embodiments, the subject to be treated has a daily fiber intake of 7g / 1000kcal or more. The subject being treated may not be receiving corticosteroids, antibiotics, probiotics, and / or prebiotics that are not 2'-FL compounds.

[0007] The 2'-FL compound may be administered to a subject in need thereof in an amount effective to achieve a desired clinical effect. For example, a 2'-FL compound may be administered to a human IBD patient, for example, a human IBD patient that has undergone or is undergoing anti-inflammatory therapy and / or is in remission from IBD, in an amount sufficient to increase the abundance of intestinal microorganisms that produce short chain fatty acids (e.g., Bifidobacteria, Bacteroides, and / or Parabacteroides) in the human patient. As another example, a 2'-FL compound may be administered to a human IBD patient, for example, a human IBD patient that has undergone or is undergoing anti-inflammatory therapy and / or is in remission from IBD, in an amount sufficient to decrease intestinal calprotectin in the human patient. In some embodiments, the effective amount of the 2'-FL compound administered to a subject in need thereof may correspond to 1 mg / day to 20 mg / day of 2'-FL, 1 mg / day to 15 mg / day of 2'-FL, or 1 mg / day to 10 mg / day of 2'-FL.

[0008] The 2'-FL compound can be administered to a subject via any route of administration, including, for example, oral administration. In some embodiments, the 2'-FL compound can be formulated as a pharmaceutical composition or dietary supplement suitable for, for example, oral administration. The composition can include the 2'-FL compound as the only oligosaccharide content, or can further include at least one additional oligosaccharide. An exemplary 2'-FL compound is 2'-FL. In some embodiments, the 2'-FL compound can be administered to a human patient receiving an anti-inflammatory drug as an adjuvant to anti-inflammatory therapy.

[0009] Also within the scope of the disclosure are (i) pharmaceutical compositions (e.g., as described herein) for use in treating IBD and / or reducing or decreasing the risk of relapse of IBD in a subject, comprising a 2'-FL compound as described herein and a pharma- ceutically acceptable carrier, and (ii) the use of a 2'-FL compound as described herein in the manufacture of a medicament for use in treating IBD and / or reducing or decreasing the risk of relapse of IBD in a subject. The subject can be a human IBD patient who has undergone or is undergoing anti-inflammatory therapy, e.g., a human patient in remission of IBD.

[0010] Also within the scope of the present disclosure is a dietary supplement for use in treating IBD and / or reducing or reducing the risk of recurrence of IBD in a subject, the composition comprising a 2'-FL compound as described herein.The subject may be a human IBD patient who has undergone or is undergoing anti-inflammatory therapy, for example, the human patient is in remission from IBD.

[0011] The details of one or more embodiments of the disclosure are set forth in the description below. Other features or advantages of the disclosure will become apparent from the following drawings and detailed description of certain embodiments, as well as the appended claims. [Brief description of the drawings]

[0012] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0013] [Figure 1]We show that microbial shifts and changes in rectal mitochondrial gene expression in ulcerative colitis (UC) are addressed by 2'-FL supplementation in mice. 254 rectal biopsies and 293 stool samples were collected from 371 untreated pediatric patients with UC at the time of initial diagnosis. Samples were subjected to 16S rRNA amplicon sequencing and data were analyzed to infer microbial taxonomic composition. Whole-rectum patterns of gene expression pre-therapy were determined using RNASeq in 206 UC patients and 18 healthy controls. Whole-small-intestine patterns of gene expression were determined using RNASeq in mice with and without 2'-FL supplementation after ileocecal resection. Gene set enrichment analysis identified associated biological processes. Gene signatures of mitochondrial biogenesis in untreated pediatric UC and after 2'-FL supplementation in mice are also presented in the table as shown in Figure 1. [Diagram 2] FIG. 1 is a schematic showing 2′-FL / anti-TNF combination therapy to enhance microbial butyrate production and cellular butyrate responsiveness. [Figure 3A-C] FIG. 1 is a bar graph showing taxa associated with newly diagnosed Crohn's disease (CD) and complications of B2 stenosis or B3 internal permeability disease compared with B1 inflammatory behavior. [Figure 4] FIG. 1 is a bar graph showing differentially abundant taxa associated with disease severity in newly diagnosed ulcerative colitis. [Diagram 5] FIG. 1 is a graph showing ileal gene signatures associated with disease complications in pediatric Crohn's disease. [Figure 6] FIG. 1 is a schematic showing the overall patient stratification strategy for the clinical trial involving the use of 2'-FL as a dietary supplement in pediatric and young adult IBD patients receiving stable maintenance anti-TNF therapy (left), as well as dose allocation at different stages of the clinical trial (right). [Figure 7] FIG. 1 is a schematic showing the overall study design of a clinical trial providing a pilot and feasibility study of 2'-FL as a dietary supplement in pediatric and young adult IBD patients receiving stable maintenance anti-TNF therapy. [Figure 8]Table showing the Schedule of Activities (SOA), as shown in Figure 7, outlining the study procedures and assessments to be performed at each visit during the clinical trial. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Inflammatory bowel disease (IBD) (e.g., including ulcerative colitis and Crohn's disease) is a chronic and debilitating disorder with peak incidence in the second to third decades of life. Although considerable progress has been made in optimizing pharmacotherapy, such as therapies targeting inflammatory cytokines, to achieve remission in IBD, relapses are common and unpredictable. For example, it was reported that 37% of IBD patients (from a single treatment center) receiving anti-TNF therapy with infliximab or adalimumab relapsed despite optimal therapeutic drug monitoring and administration. In addition to the negative impact on quality of life and work productivity, IBD relapse increases treatment costs. Thus, there is a need to develop alternative methods and compositions for the treatment of IBD as well as the maintenance of IBD remission to reduce the risk of IBD relapse.

[0015] The present disclosure is based at least in part on the development of using 2'-fucosyllactose (2'-FL) compounds to maintain remission in IBD patients, thus reducing the risk of IBD relapse. For example, 2'-FL can be provided as a dietary supplement or as a pharmaceutical composition, either alone or as an adjunct to immunosuppressive therapy, such as anti-inflammatory therapy. Administration of 2'-FL compounds (e.g., 2'FL) can increase microbial production of butyrate, an essential regulator of intestinal epithelial cell function. Treatment of IBD with anti-inflammatory therapy (e.g., anti-TNF inhibitors) suppresses intestinal inflammation and promotes cellular responsiveness to butyrate in intestinal endothelial cells, but does not increase the abundance of beneficial gut bacteria. Thus, 2'-FL / anti-TNF combination therapy can provide direct modulation of beneficial microbiota, increasing microbial butyrate production while promoting cellular butyrate responsiveness with anti-TNF therapy, thereby enhancing sustained clinical remission of IBD.

[0016] Thus, methods and compositions are described herein for treating IBD in subjects using 2'-FL compounds.When applied to human IBD patients (e.g., human patients in remission of IBD) who have undergone or are undergoing anti-inflammatory therapy, the treatment methods described herein are expected to reduce or reduce the risk of IBD recurrence.Furthermore, the treatment methods described herein are expected to be particularly effective in sustaining remission of IBD when 2'-FL compounds are provided as an adjuvant to anti-inflammatory therapy (e.g., anti-TNF therapy) that is administered simultaneously to patients in remission of IBD.

[0017] In some embodiments, the present disclosure relates to a method for treating IBD or alleviating or reducing the risk of recurrence of IBD using 2'-FL compounds, which can be provided as a dietary supplement or in a pharmaceutical composition. Such dietary supplements or pharmaceutical compositions can be used alone or as an adjunct to anti-inflammatory therapy for IBD in a subject in need of treatment.

[0018] I. 2'-Fucosyllactose (2'-FL) Compounds and Compositions Containing Them 2-fucosyllactose (2'-FL) compounds are oligosaccharides with a three sugar unit backbone, such as 2'-fucosyllactose (Fucαl, 2Galβ1,4Glc), in which each of the sugar units (fucose (Fuc), galactose (Gal), and glucose (Glc)) can be independently either in its native form or in a modified form. For example, a modified form of a sugar unit can be a sugar unit in which at least one or more (e.g., one, two, three or more) of the hydroxyl groups are replaced with hydrogen, methyl, ethyl, or amine groups.

[0019] In some embodiments, the 2'-FL compound is 2'-FL (Fucα1,2Galβ1,4Glc), which has a chemical structure that is identical to that of native 2'-FL found, for example, in milk (eg, breast milk).

[0020] In some embodiments, the 2'-FL compound is a modified 2'-FL that retains at least 70% or more (e.g., at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, and up to 100%) of the biological function of native 2'-FL, e.g., 2'-FL found in milk (e.g., breast milk). In some embodiments, the modified 2'-FL can provide enhanced biological function compared to native 2'-FL, e.g., 2'-FL found in milk (e.g., breast milk). Such biological functions of 2'-FL include, but are not limited to, its beneficial effects on the gut, e.g., anti-inflammatory effects, antimicrobial adhesion effects, prebiotic effects (e.g., increasing the abundance of beneficial microflora, such as short chain fatty acid producing microorganisms or butyric acid producing microorganisms). For example, in some embodiments, the modified 2'-FL is a 2'-FL in which (i) at least one of its hydroxyl groups has been replaced with a hydrogen, methyl, ethyl, or amine group, and / or (ii) the glucose at its reducing end has been replaced with N-acetylglucosamine.

[0021] The 2'-FL compounds described herein can be prepared by any method known in the art. For example, the 2'-FL compounds can be chemically synthesized, purified from milk, or produced in microorganisms. In some embodiments, the 2'-FL compounds described herein can be isolated from milk (e.g., breast milk). For example, milk is first skimmed by centrifugation to obtain nonfat milk. The nonfat milk is then mixed with organic solvents such as acetone (e.g., 50% acetone in water) and ethanol (e.g., 67% ethanol in water) to precipitate milk proteins. The supernatant is collected upon centrifugation and subjected to chromatography. The oligosaccharide-containing fractions are collected and pooled. If necessary, the oligosaccharides thus prepared can be concentrated by conventional methods, for example, dialysis or freeze-drying. Alternatively, the 2'-FL compounds can be separated from nonfat milk by passing the nonfat milk through a 30,000 MWCO ultrafiltration membrane, collecting the diffusion liquid, passing the diffusion liquid through a 500 MWCO ultrafiltration device, and collecting the retentate containing milk oligosaccharides.

[0022] In some embodiments, the 2'-FL compounds described herein can be chemically synthesized or produced in a microorganism (e.g., by fermentation of recombinant microorganisms such as Escherichia coli, yeast, and Corynebacterium glutamicum). See, for example, WO2017 / 134176, WO2016 / 153300, WO2014 / 009921, WO2010 / 115934, WO2005 / 055944, and US8652808, the relevant disclosures of which are incorporated by reference for purposes or subject matter referenced herein.

[0023] In some embodiments, the 2'-FL compounds described herein can be provided in a glycoconjugate form (e.g., glycoconjugate). As used herein, "glycoconjugate" refers to a complex that contains a sugar moiety (e.g., a 2'-FL compound) bound to another chemical species, such as a protein, peptide, lipid, nucleic acid, and sugar (e.g., an oligosaccharide or polysaccharide). The 2'-FL compound can be bound to the other chemical species through a covalent or non-covalent bond, or through other forms of association, such as entrapment (e.g., of a moiety on or within another moiety, or of one or both entities on or within a third moiety). The glycoconjugates described herein can contain one or more (e.g., 1, 2, 3 or more) 2'-FL compounds bound to a chemical species, such as a protein, peptide, lipid, nucleic acid, or sugar. In one example, the 2'-FL compound is covalently bound to a protein, peptide, lipid, nucleic acid, or sugar (e.g., an oligosaccharide or polysaccharide) through its reducing end sugar unit. For example, the reducing end sugar unit can be N-acetylglucosamine. In some embodiments where the 2'FL compound is conjugated to a saccharide (e.g., an oligosaccharide or polysaccharide), the glycoconjugate is not a naturally occurring molecule found in milk. In some embodiments, the glycoconjugate form of the 2'-FL compound is not a naturally occurring molecule.

[0024] Suitable peptide scaffolds for making the above-mentioned glycoconjugates include those with multiple glycosylation sites (e.g., asparagine, lysine, serine, or threonine residues) and low allergenicity. Examples include, but are not limited to, amylase, bile salt-stimulated lipase, casein, folate binding protein, globulin, gluten, haptocorrin, lactalbumin, lactoferrin, lactoperoxidase, lipoprotein lipase, lysozyme, mucin, ovalbumin, and serum albumin.

[0025] Usually, 2'-FL compounds can be covalently linked to serine or threonine residues via O-linkage, or linked to asparagine residues via N-linkage. To form these bonds, the sugar unit at the reducing end of 2'-FL compounds is preferably an acetylated sugar unit, such as N-acetylgalactosamine, N-acetylglucosamine, and N-acetylmannosamine. 2'-FL compounds can be linked to peptides (e.g., proteins) using standard methods. For example, see McBroom et al., Complex Carbohydrates, Part B, 28:212-219, 1972; Yariv et al., Biochem J., 85:383-388, 1962; Rosenfeld et al., Carbohydr.Res., 46:155-158, 1976, and Pazur, Adv.Carbohydr.Chem, Biochem., 39:405-447, 1981.

[0026] In one example, the 2'-FL compound is linked to the scaffold molecule via a linker.Exemplary linkers are described in WO2005 / 055944.The 2'-FL compound can be linked to the linker by enzymatic reaction, for example, glycosyltransferase reaction.Some glycosyltransferases, including fucosyltransferase, galactosyltransferase, glucosyltransferase, mannosyltransferase, galactosaminyltransferase, sialyltransferase, and N-acetylglucosaminyltransferase, can be used to generate the glycoconjugates described herein. Further details regarding these glycosyltransferases can be found in U.S. Patent Nos. 6,291,219, 6,270,987, 6,238,894, 6,204,431, 6,143,868, 6,087,143, 6,054,309, 6,027,928, 6,025,174, 6,025,173, and 6,026,113. Nos. 5,955,282, 5,945,322, 5,922,540, 5,892,070, 5,876,714, 5,874,261, 5,871,983, 5,861,293, 5,859,334, 5,858,752, 5,856,159, and 5,545,553.

[0027] Alternatively, the complex carbohydrates described herein can be purified from milk by conventional methods, such as by passage through an ultrafiltration membrane, by precipitation into a non-polar solvent, or by partitioning between immiscible solvents.

[0028] The 2'-FL compounds (either as free oligosaccharides or in glycoconjugate form as described herein) can be formulated with one or more pharma- ceutically acceptable carriers, diluents, and / or excipients to form pharmaceutical compositions. "Pharmaceutically acceptable" carriers, diluents, or excipients include those that are sterile and pyrogen-free. Suitable pharmaceutical carriers, diluents, and excipients are well known in the art. The carrier(s) must be "acceptable" in the sense of being compatible with the inhibitor and not deleterious to the recipient thereof.

[0029] Pharmaceutical compositions containing 2'-FL compounds (either as free oligosaccharides or in complex glycoconjugate form as described herein) can be formulated according to the route of administration, including, for example, parenteral, oral, buccal, sublingual, and topical administration.

[0030] In some embodiments, pharmaceutical compositions or formulations such as in the form of powders, tablets, capsules, ovules, elixirs, solutions, or suspensions, which may contain flavoring or coloring agents, are suitable for oral, buccal, or sublingual administration for immediate, delayed, or controlled release applications.

[0031] Suitable tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, and glycine, disintegrants such as starch (preferably corn, potato, or tapioca starch), sodium starch glycolate, croscarmellose sodium, and certain complex silicates, as well as granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin, and acacia. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included.

[0032] A similar type of solid composition can also be used as a filler for gelatin capsules.Preferred excipients in this regard include lactose, starch, cellulose, milk sugar, or high molecular weight polyethylene glycols.For aqueous suspensions and / or elixirs, 2'-FL compounds (either as free oligosaccharides or in complex carbohydrate form as described herein) can be mixed with various sweeteners or flavorings, colorants or dyes, emulsifiers and / or suspending agents, and diluents such as water, ethanol, propylene glycol, and glycerin, and combinations thereof.For powders, 2'-FL compounds (either as free oligosaccharides or in complex carbohydrate form as described herein) can be mixed with various sweeteners or flavorings, colorants or dyes, emulsifiers and / or suspending agents, and diluents such as water, ethanol, propylene glycol, and glycerin, and combinations thereof.

[0033] In some embodiments, the pharmaceutical composition or formulation is for parenteral administration, such as intravenous, intraarterial, intramuscular, subcutaneous, or intraperitoneal administration.

[0034] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. The aqueous solutions may be suitably buffered (preferably pH 3-9). The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0035] Alternatively, 2'-FL compounds (either as free oligosaccharides or in complex carbohydrate form as described herein) can be formulated as dietary supplements according to methods well known in the food / dietary supplement industry. In one example, dietary supplements containing 2'-FL compounds can be taken alone. In another example, dietary supplements containing 2'-FL compounds can be incorporated into food and / or beverages. Such foods and / or beverages include, but are not limited to, milk, milk powder, yogurt, cheese, ice cream, cereals, etc., among others. Such foods and / or beverages also include oral dietary supplements, nutritional drinks, and enteral nutritional preparations, for example, for tube feeding.

[0036] In some embodiments, the formulation of any aspect described herein may contain 2'-fucosyllactose compounds (either as free oligosaccharides or in complex glycoforms as described herein) as the only oligosaccharide content. In some embodiments, the formulation of any aspect described herein may contain 2'-fucosyllactose compounds (either as free oligosaccharides or in complex glycoforms as described herein) as the only oligosaccharide content that provides a prebiotic effect. In some embodiments, the formulation of any aspect described herein may contain 2'-fucosyllactose compounds (either as free oligosaccharides or in complex glycoforms as described herein) as the only oligosaccharide content that increases short-chain fatty acid-producing microorganisms in the gut and / or increases the production of short-chain fatty acids (e.g., butyric acid) by microorganisms.

[0037] In alternative embodiments, the formulations of any aspect described herein may further comprise at least one or more additional (e.g., 1, 2, 3 or more) oligosaccharides. Examples of such oligosaccharides include, but are not limited to, other non-2'-FL lactose, fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), and any combination thereof, such as those shown in Tables 1-4 below. [Table 1] [Table 2] [Table 3] [Table 4]

[0038] The formulations of any of the aspects described herein may be presented in unit-dose or multi-dose containers, for example, sealed ampoules or vials and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier immediately prior to use.

[0039] II. Subject In some embodiments, the subject treated by any of the methods described herein can be a mammal, such as a human, that has, is suspected of having, or is at risk of developing inflammatory bowel disease (IBD).IBD is a disorder that involves chronic inflammation of the digestive tract.Examples of IBD include, but are not limited to, Crohn's disease (CD) and ulcerative colitis (UC).

[0040] Symptoms of IBD may vary depending on the severity and location of inflammation. Symptoms may range from mild to severe. In some cases, IBD patients may experience periods of active disease followed by periods of remission. Signs and symptoms common to both Crohn's disease and ulcerative colitis include, but are not limited to, diarrhea, fever and fatigue, abdominal pain and cramps, blood in stool, loss of appetite, unintentional weight loss, and combinations thereof. Diagnosis of IBD is known in the art and can be determined by a skilled professional, for example, through blood tests, various endoscopic procedures, and / or imaging procedures.

[0041] In some embodiments, the subject treated by the methods described herein may be an IBD subject (e.g., a human IBD patient) who has undergone or is undergoing anti-inflammatory and / or immune system suppression therapy. In some embodiments, the subject treated by the methods described herein is undergoing anti-inflammatory and / or immune system suppression therapy. Such exemplary therapy includes, but is not limited to, anti-TNF therapy. Non-limiting examples of anti-TNF therapy include infliximab, adalimumab, golimumab, natalizumab, vedolizumab, and ustekinumab. In some embodiments, the subject treated by the methods described herein is undergoing anti-TNF therapy including infliximab and / or adalimumab.

[0042] In some embodiments, the subject treated by the methods described herein can be an IBD subject (e.g., a human IBD patient) in remission of IBD. As used herein, the term "remission" refers to the disappearance or alleviation of at least one or more symptoms associated with IBD, such as the symptoms described herein. Remission can be complete remission (e.g., disappearance of all signs or symptoms associated with IBD) or partial remission (e.g., disappearance or alleviation of at least one sign or symptom associated with IBD).

[0043] In some embodiments, the subject treated by the methods described herein can be an IBD subject (e.g., a human IBD patient) in remission of Crohn's disease (CD).For example, if the Crohn's disease activity index (CDAI) score is less than 150, the subject is determined to be in remission of CD (see, for example, Merck Manuals; Best et al. "Development of a Crohn's disease activity index" Gastroenterology (1976) 70:439-444 and Best "Predicting the Crohn's disease activity index from the Harvey-Bradshaw Index" Inflamm Bowel Dis. (2006) 12:304-310). In some embodiments, a subject is determined to be in remission of CD if they have a weighted Pediatric Crohn's Disease Activity Index (wPCDAI) score of less than 10 (see, e.g., Turner et al. "Which PCDAI Version Best Reflects Intestinal Inflammation in Pediatric Crohn's Disease?" J Pediatr Gastroenterol Nutr (2017) 64:254-260).

[0044] In some embodiments, the subject treated by the methods described herein may be an IBD subject (e.g., a human IBD patient) in remission of ulcerative colitis (UC). For example, the subject is determined to be in remission of UC according to any one of the disease activity indices provided in Travis et al. "Review article: defining remission in ulcerative colitis" Aliment. Pharmacol. Ther. (2011) 34:113-124. In some embodiments, the subject is determined to be in remission of UC if the modified ulcerative colitis disease activity index (UCDAI) score is 1 or less, the UCDAI score is 2 or less, the clinical activity index score is 4 or less, or the Mayo clinical score is 2 or less (no subscores exceed 1). In some embodiments, the subject is determined to be in remission of UC if the subject has a complete cessation of rectal bleeding, urgency, and increased bowel frequency, for example, confirmed by endoscopic appearance of mucosal healing. See, e.g., Walsh and Travis "Assessing Disease Activity in Patients with Ulcerative Colitis" Gastroenterol Hepatol (NY) (2012) 8:751-754. In some embodiments, a subject is determined to be in remission of UC if the Pediatric Ulcerative Colitis Activity Index (PUCAI) score is less than 10 (see, e.g., Turner et al. "Appraisal of the pediatric ulcerative colitis activity index (PUCAI)" Inflamm Bower Dis (2009); 15:1218-23).

[0045] In some embodiments, the subject treated by the methods described herein has a daily fiber intake of less than 7g / 1000kcal. In some embodiments, the subject treated by the methods described herein has a daily fiber intake of 7g / 1000kcal or more. To assess fiber intake, for example, Nutrition Data Systems for Research (NDSR) (Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN) software and food databases can be used to determine daily fiber intake. See, for example, Sievert et al. "Maintenance of a nutrient database for clinical trials." Control Clin Trials (1989) 10:416-25.

[0046] In some embodiments, the subject treated by the methods described herein is not receiving a corticosteroid or antibiotic indicated for the treatment of IBD.

[0047] In some embodiments, the subject treated by the methods described herein can be a FUT2 secretor. In some embodiments, the subject treated by the methods described herein can be a FUT2 non-secretor. FUT2 corresponds to the fucosyltransferase 2 gene, which is involved in the production of 2'-FL. Individuals with inactivating polymorphisms in the FUT2 gene are FUT2 non-secretors. Non-secretors of FUT2 lack the native intestinal carbohydrates that contain fucose, which increases susceptibility to microbial dysregulation and chronic inflammation.

[0048] The subject treated by the methods described herein can be of any age. In some embodiments, the subject treated by the methods described herein can be a child, such as a subject under 18 years of age, including, for example, 6 months to 18 years of age. In some embodiments, the subject can be a child 11 years of age or older, including, for example, 11 to 18 years of age. In some embodiments, the subject can be a child 5 to 10 years of age. In some embodiments, the subject can be a child under 5 years of age, including, for example, 6 months to 4 years of age.

[0049] In some embodiments, the subject treated by the methods described herein may be an adult over the age of 18, such as between 19 and 80 years old. In some embodiments, the adult subject is between 19 and 25 years old. In some embodiments, the adult subject treated by the methods described herein may be over 25 years old, such as between 25 and 80 years old. In some embodiments, the adult subject treated by the methods described herein may be elderly over 65 years old, such as between 66 and 80 years old.

[0050] In some embodiments, the subject treated by the methods described herein may be between 11 and 25 years old.

[0051] Subjects in need of the treatment described herein can be identified by routine medical examination.

[0052] III. Treatment of Inflammatory Bowel Disease (IBD) To treat IBD, e.g., Crohn's disease (CD) or ulcerative colitis (UC), any of the 2'-FL compounds (either as free oligosaccharides or in glycoconjugate form as described herein) and / or compositions comprising same, e.g., as described herein, can be administered to a subject in need thereof, e.g., as described herein. For example, in some embodiments, the subject is a human patient at risk of developing IBD, e.g., CD or UC. In some embodiments, the subject is a human patient with IBD, e.g., CD or UC. In some embodiments, the subject is a human IBD patient who has undergone or is undergoing immune system suppression and / or anti-inflammatory therapy (e.g., anti-TNF therapy). In some embodiments, the subject is a human IBD patient who is in remission of IBD and is undergoing immune system suppression and / or anti-inflammatory therapy (e.g., anti-TNF therapy).

[0053] Any of the 2'-FL compounds and / or compositions comprising same, such as those described herein, can be administered to a subject of any age in need of IBD treatment. In some embodiments, the subject to which the 2'-FL compounds and / or compositions described herein are administered can be a child, such as a subject up to 18 years of age, including 6 months to 18 years of age. In some embodiments, the subject can be a child aged 11 years or older, including 11 to 18 years of age. In some embodiments, the subject can be a child aged 5 to 10 years. In some embodiments, the subject can be a child under 5 years of age, including 6 months to 4 years of age.

[0054] In some embodiments, the subject receiving the 2'-FL compounds and / or compositions described herein can be an adult greater than 18 years of age, such as between 19 and 80 years of age. In some embodiments, the adult subject is between 19 and 25 years of age. In some embodiments, the adult subject receiving the 2'-FL compounds and / or compositions described herein can be greater than 25 years of age, such as between 25 and 80 years of age. In some embodiments, the adult subject receiving the 2'-FL compounds and / or compositions described herein can be greater than 65 years of age, such as between 66 and 80 years of age.

[0055] In some embodiments, subjects receiving the 2'-FL compounds and / or compositions described herein can be between 11 and 25 years old.

[0056] The term "treat" or "treatment" as used herein refers to the application or administration of a 2'-FL compound (e.g., as described herein, either as free oligosaccharides or in complex glycoconjugate form) as a monotherapy or combination therapy (e.g., as an adjunct to immune system suppression and / or anti-inflammatory therapy) to a subject having IBD, symptoms of IBD, or predisposed to IBD, for the purpose of curing, curing, alleviating, mitigating, altering, ameliorating, improving, or affecting the disease, symptoms of the disease, or predisposition to the disease. The term "treat" or "treatment" also includes the application or administration of a 2'-FL compound (e.g., as described herein, either as free oligosaccharides or in complex glycoconjugate form) as a monotherapy or combination therapy (e.g., as an adjunct to immune system suppression and / or anti-inflammatory therapy) to a subject in remission of IBD, for the purpose of maintaining remission and thus alleviating, reducing, preventing, or delaying the occurrence of relapse.

[0057] In some embodiments, the treatment is prophylactic. The term "prophylactic" refers to the application or administration of a 2'-FL compound (e.g., as described herein, either as a free oligosaccharide or in a glycoconjugate form as described herein) to a subject at risk of IBD, as a monotherapy or combination therapy (e.g., as an adjunct to immune system suppression and / or anti-inflammatory therapy) to prevent or delay the onset of IBD.

[0058] In some embodiments, the treatment is therapeutic. The term "therapeutic" refers to the application or administration of 2'-FL compounds (e.g., as described herein, either as free oligosaccharides or in glycoconjugate form) to a subject with IBD or symptoms of IBD, as a monotherapy or combination therapy (e.g., as an adjunct to immune system suppression and / or anti-inflammatory therapy) to improve at least one or more symptoms associated with IBD, such as reducing diarrhea, reducing blood in stool, and / or reducing the frequency of recurrence of symptoms.

[0059] For example, a treatment is therapeutic if the application or administration of a 2'-FL compound (e.g., as described herein, either as a free oligosaccharide or in a glycoconjugate form), either as a monotherapy or combination therapy (e.g., as an adjunct to immune system suppression and / or anti-inflammatory therapy), alleviates or reduces the risk of recurrence of IBD symptoms. As used herein, the term "recurrence" refers to the occurrence or worsening of at least one or more symptoms associated with IBD.

[0060] In some embodiments, a treatment is therapeutic if application or administration of a 2'-FL compound (e.g., as described herein), either as a monotherapy or combination therapy (e.g., as an adjunct to immune system suppression and / or anti-inflammatory therapy), reduces or reduces the risk of recurrence of symptoms of Crohn's disease (CD). For example, a human patient is determined to have recurrent CD when the Crohn's Disease Activity Index (CDAI) score increases to 150 or greater. In some embodiments, a human patient is determined to have recurrent CD when, for example, there is an increase of 20 or more points in the wPCDAI during the first 4 weeks of treatment.

[0061] In some embodiments, a treatment is therapeutic if application or administration of a 2'-FL compound (e.g., as described herein), either as a monotherapy or combination therapy (e.g., as an adjunct to immune system suppression and / or anti-inflammatory therapy), reduces or reduces the risk of recurrence of symptoms of ulcerative colitis (UC). For example, a human patient is determined to have recurrent UC if the modified ulcerative colitis disease activity index (UCDAI) score is greater than 1, the UCDAI score is greater than 2, the clinical activity index score is greater than 4, or the Mayo clinical score is greater than 2 (subscore greater than 1). In some embodiments, a human patient is determined to have recurrent UC if they experience rectal bleeding, urgency, and increased bowel movement frequency, for example, as confirmed by endoscopic examination of the mucosa. In some embodiments, a human patient is determined to have recurrent UC if, for example, there is an increase of 15 points or more in the PUCAI during the 4 weeks following the start of treatment.

[0062] To practice the therapeutic methods described herein, an effective amount of the 2'-FL compounds and compositions containing same can be administered to a subject in need of treatment.

[0063] "Effective amount" refers to an amount of 2'-FL compound (e.g., as described herein) that, alone or together with additional doses, produces a desired response, such as eliminating or reducing symptoms, preventing or reducing the risk of recurrence of IBD symptoms, reducing diarrhea, reducing blood in stool, reducing weight gain, abdominal pain or cramps, increasing the abundance of gut microbes that produce short chain fatty acids (e.g., butyric acid), and / or reducing gut inflammation. The desired response is to inhibit the progression or recurrence of disease symptoms. This may involve only slowing the progression of the disease temporarily, but may also involve stopping the progression of the disease permanently. In some cases, this may involve only slowing the recurrence of the disease temporarily, but may also involve preventing the recurrence of the disease permanently. This can be monitored by routine methods. The desired response to the treatment of a disease may be to delay the onset of the disease, or even to prevent the onset of the disease.

[0064] Such amount depends on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, duration of treatment, nature of concomitant treatment (if any), specific route of administration, and similar factors within the knowledge and expertise of medical practitioners. These factors are well known to those skilled in the art and can be addressed with only routine experimentation. In general, it is preferred to use the maximum dose of each component or combination thereof, that is, the highest safe dose according to sound medical judgment. However, it will be understood by those skilled in the art that a patient may insist on a lower dose or tolerated dose for medical reasons, psychological reasons, or any other practical reasons.

[0065] For example, an effective amount of a 2'-FL compound (e.g., as described herein, either as a free oligosaccharide or in a glycoconjugate form as described herein), when administered to a subject in need thereof, results in, for example, an increase in the abundance of gut microbes that produce short chain fatty acids by at least about 10% or more, including, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, compared to the abundance of short chain fatty acid producing gut microbes not administered with the 2'-FL compound (e.g., as a free oligosaccharide or in a glycoconjugate form as described herein). Examples of gut microbes that produce short chain fatty acids (e.g., butyric acid) include, but are not limited to, Bifidobacteria, Bacteroides, and / or Parabacteroides. In some embodiments, an effective amount of a 2'-FL compound (e.g., as described herein), when administered to a subject in need thereof, increases the abundance of intestinal Bifidobacteria by at least about 10% or more, including, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, compared to the abundance of intestinal Bifidobacteria without administration of a 2'-FL compound (either as a free oligosaccharide or in a complex glycoform as described herein). Such therapeutic characteristics may be determined by measuring the abundance of fecal microorganisms (e.g., Bifidobacteria, Bacteroides, and / or Parabacteroides).

[0066] In some embodiments, an effective amount of a 2'-FL compound (e.g., as described herein, either as free oligosaccharides or in a glycoconjugate form) when administered to a subject in need thereof results in an increase in microbial butyrate production by at least about 10% or more, including, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, compared to microbial butyrate production without administration of a 2'-FL compound (either as free oligosaccharides or in a glycoconjugate form as described herein). Such therapeutic characteristics can be determined, for example, by measuring the abundance of fecal short chain fatty acids, including butyrate.

[0067] In some embodiments, an effective amount of a 2'-FL compound (e.g., as described herein, either as a free oligosaccharide or in a glycoconjugate form as described herein), when administered to a subject in need thereof, results in a reduction in intestinal inflammation by at least about 10% or more, including, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, compared to intestinal inflammation without administration of a 2'-FL compound (either as a free oligosaccharide or in a glycoconjugate form as described herein). Such therapeutic characteristics may be determined, for example, by measuring the presence of fecal calprotectin, a biomarker of intestinal inflammation.

[0068] In some embodiments, an effective amount of a 2'-FL compound (e.g., as described herein, either as a free oligosaccharide or in a glycoconjugate form, as described herein), when administered to a subject in need thereof, results in a reduction in intestinal inflammation by at least about 10% or more, including, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, compared to intestinal inflammation without administration of a 2'-FL compound (either as a free oligosaccharide or in a glycoconjugate form as described herein). Such therapeutic characteristics can be determined, for example, by measuring the abundance of fecal calprotectin, a biomarker of intestinal inflammation. Alternatively, such therapeutic characteristics can be determined, for example, by measuring the abundance of pro-inflammatory microorganisms, including, but not limited to, Enterobacteriaceae.

[0069] In some embodiments, an effective amount of a 2'-FL compound (e.g., as described herein, either as a free oligosaccharide or in a complex glycoform) for use in the methods described herein is at least 0.5 mg / day of 2'-fucosyllactose, at least 1 mg / day of 2'-fucosyllactose, at least 2 mg / day of 2'-fucosyllactose, at least 3 mg / day of 2'-fucosyllactose, at least 4 mg / day of 2'-fucosyllactose, at least 5 mg / day of 2'-fucosyllactose, at least 6 mg / day of 2'-fucosyllactose, at least 7 mg / day of 2'-fucosyllactose, at least 8 mg / day of 2'-fucosyllactose. 2'-fucosyllactose, at least 9 mg / day of 2'-fucosyllactose, at least 10 mg / day of 2'-fucosyllactose, at least 11 mg / day of 2'-fucosyllactose, at least 12 mg / day of 2'-fucosyllactose, at least 13 mg / day of 2'-fucosyllactose, at least 14 mg / day of 2'-fucosyllactose, at least 15 mg / day of 2'-fucosyllactose, at least 16 mg / day of 2'-fucosyllactose, at least 17 mg / day of 2'-fucosyllactose, at least 18 mg / day of 2'-fucosyllactose, at least 19 mg / day of 2'-fucosyllactose, or at least 20 mg / day of 2'-fucosyllactose. In some embodiments, the effective amount of 2'-FL compound (e.g., as described herein) for use in the methods described herein can correspond to 20mg / day or less of 2'-fucosyllactose, 15mg / day or less of 2'-fucosyllactose, 10mg / day or less of 2'-fucosyllactose, 9mg / day or less of 2'-fucosyllactose, 8mg / day or less of 2'-fucosyllactose, 7mg / day or less of 2'-fucosyllactose, 6mg / day or less of 2'-fucosyllactose, 5mg / day or less of 2'-fucosyllactose, 4mg / day or less of 2'-fucosyllactose, 3mg / day or less of 2'-fucosyllactose, or 2mg / day or less of 2'-fucosyllactose. Combinations of the above ranges are also included.For example, in some embodiments, an effective amount of a 2'-FL compound (e.g., those described in the specification) for use in the methods described herein may be equivalent to 0.5 mg / day to 20 mg / day of 2'-fucosyllactose, equivalent to 1 mg / day to 20 mg / day of 2'-fucosyllactose, equivalent to 1 mg / day to 15 mg / day of 2'-fucosyllactose, equivalent to 1 mg / day to 10 mg / day of 2'-fucosyllactose, equivalent to 1 mg / day to 8 mg / day of 2'-fucosyllactose, or equivalent to 1 mg / day to 5 mg / day of 2'-fucosyllactose.

[0070] In some embodiments where the subject in need of treatment is between 11 and 25 years of age, an effective amount of a 2'-FL compound (e.g., as described herein, either as a free oligosaccharide or in a complex glycoform) for use in the methods described herein may be equivalent to 1 mg / day to 20 mg / day of 2'-fucosyllactose, equivalent to 1 mg / day to 15 mg / day of 2'-fucosyllactose, equivalent to 1 mg / day to 10 mg / day of 2'-fucosyllactose, equivalent to 1 mg / day to 8 mg / day of 2'-fucosyllactose, or equivalent to 1 mg / day to 5 mg / day of 2'-fucosyllactose.

[0071] In some embodiments, an effective daily amount of a 2'-FL compound (e.g., as described herein, either as a free oligosaccharide or in a glycoconjugate form) can be administered in a single daily dose or divided into multiple doses (e.g., 2-4 doses) for administration at given time intervals throughout the day. In some embodiments, an effective daily amount of a 2'-FL compound (e.g., as described herein) can be administered as a single daily dose in the morning, e.g., alone or in combination with food or beverages. Administration of a 2'-FL compound (e.g., as described herein) at any other time throughout the day is also appropriate.

[0072] 2'-FL compounds (e.g., those described herein, either as free oligosaccharides or in a glycoconjugate form) can be administered to a subject in need thereof as a single oligosaccharide for the treatment of IBD or in combination with at least one additional oligosaccharide (e.g., those described herein). In some embodiments, a 2'-FL compound (e.g., those described herein) is administered to a subject in need thereof as a single oligosaccharide, i.e., the subject is given a 2'-FL compound (e.g., those described herein) as the only oligosaccharide, which is not used simultaneously with other oligosaccharides (e.g., those described herein). In other embodiments, a 2'-FL compound (e.g., those described herein) is co-administered with at least one different oligosaccharide. "Co-administration" or "in combination with" means providing a subject with a 2'-FL compound (e.g., those described herein) containing different oligosaccharides during the course of treatment, such as simultaneously, sequentially, intermittently, or in other regimens.

[0073] 2'-FL compounds (e.g., as described herein, either as free oligosaccharides as described herein or in the glycoconjugate form as described herein) can be administered as an adjunct to immune system suppressants and / or anti-inflammatory agents, such as those taken by human IBD patients. Exemplary immune system suppressants and / or anti-inflammatory agents include, but are not limited to, anti-TNF agents. Non-limiting examples of anti-TNF agents include infliximab, adalimumab, golimumab, natalizumab, vedolizumab, and ustekinumab. In some embodiments, 2'-FL compounds (e.g., as described herein) are administered as an adjunct to anti-TNF agents, including infliximab and / or adalimumab.

[0074] As used herein, the term "adjunct" refers to a first agent being provided as a supplement to a second agent. The first agent can be administered before, simultaneously with, or after the administration of the second agent. In some embodiments, administration of 2'-FL compounds as an adjuvant to immune system suppressants and / or anti-inflammatory agents (e.g., anti-TNF agents) can provide a synergistic effect in the treatment of IBD, including, for example, reducing or decreasing the risk of recurrence of IBD. In some embodiments, administration of 2'-FL compounds as an adjuvant to immune system suppressants and / or anti-inflammatory agents (e.g., anti-TNF agents) can provide an additional effect in the treatment of IBD, including, for example, reducing or decreasing the risk of recurrence of IBD. For example, a therapeutic effect is synergistic if the mean duration of remission achieved by a combination of a 2'-FL compound (e.g., as described herein) and an immune system suppressant and / or anti-inflammatory agent (e.g., an anti-TNF agent) is significantly greater than the additive effect that occurs as a result of individual treatment with the same doses of a 2'-FL compound (e.g., as described herein) and an immune system suppressant and / or anti-inflammatory agent (e.g., an anti-TNF agent). In some embodiments, a synergistic therapeutic effect increases the mean duration of remission by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0075] When a 2'-FL compound (e.g., as described herein) is used simultaneously with a second agent (e.g., other oligosaccharides or immune system suppressants and / or anti-inflammatory agents (e.g., anti-TNF agents) described herein), it can be formulated with the second agent in a single composition, which can be in any suitable form (e.g., a powder or tablet for oral administration) as described herein. Alternatively, the 2'-FL compound (e.g., as described herein) and the second agent (e.g., other oligosaccharides or immune system suppressants and / or anti-inflammatory agents (e.g., anti-TNF agents) described herein) can be formulated separately.

[0076] Administration of the IBD treatments described herein can be accomplished by any method known in the art (see, for example, Harrison's Principle of Internal Medicine, McGraw Hill Inc., 1896). th ed., 2011). In the case of combination therapy, each agent may be administered by the same route or by different routes. Administration may be local or systemic. Administration may be, for example, parenteral (e.g., intravenous, intraperitoneal, subcutaneous, intraarterial, or intradermal) or oral. Compositions for different routes of administration are well known in the art (e.g., Remington: The Science and Practice of Pharmacy, Pharmaceutical Press, 22 nd ed., 2012). The compositions can also be formulated as modified release dosage forms, including delayed-, extended-, sustained-, extended-, pulsed-, controlled-, accelerated-, and fast-, targeted-, programmed-, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. The dosage depends on the particular condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, duration of treatment, the nature of concurrent therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. Dosages can be determined by those skilled in the art.

[0077] In some embodiments, the 2'-FL compound (e.g., those described herein) and the second agent (e.g., other oligosaccharide(s) or immune system suppressant and / or anti-inflammatory agent (e.g., anti-TNF agent) described herein) can be administered orally. Oral administration also includes buccal, lingual, and sublingual administration. In some embodiments, the composition comprising the 2'-FL compound (e.g., those described herein) can be provided in a solid, semi-solid, or liquid composition for oral administration (e.g., a pharmaceutical composition or dietary supplement). Suitable oral dosage forms include, but are not limited to, tablets, capsules, pills, troches, lozenges, pastilles, cachets, pellets, medicated chewing gums, granules, bulk powders, effervescent or non-effervescent powders or granules, solutions, emulsions, suspensions, solutions, wafers, sprinkles, elixirs, and syrups. In addition to the active ingredient(s), the compositions may contain one or more pharma- ceutically acceptable or edible carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, colorants, dye transfer inhibitors, sweeteners, and flavoring agents.

[0078] In some embodiments, 2'-FL compounds (e.g., those described herein) can be administered by injection (e.g., parenterally, such as intravenously or intraperitoneally). Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain one or more of a preservative, a wetting agent, an emulsifying agent, and a dispersing agent. The dosage forms can be sterilized, for example, by filtering the composition, irradiating the composition, or heating the composition. They can also be prepared using sterile water or other sterile injectable medium before use.

[0079] In some embodiments, the method further comprises taking measures other than or in addition to the IBD treatment described herein. In some embodiments, the method further comprises monitoring the onset of IBD symptoms in a subject at risk for IBD or monitoring the effectiveness of the treatment. Monitoring can include, for example, physical examination, endoscopy, and / or stool sample testing to evaluate gut inflammation and / or gut microbiota. If a subject does not respond to an administered dose of 2'-FL compound (e.g., as described herein, either as free oligosaccharides or as glycoconjugates), a physician can increase the dose of 2'-FL, for example, based on the subject's medical and / or physical condition, provided that the increased dose does not cause significant gastrointestinal symptoms, such as abdominal bloating, abdominal pain, nausea, loose stools, and / or gas.

[0080] IV. Kits for Use in Treating IBD Another aspect of the present disclosure relates to a kit for use in treating IBD as described herein.Thus, in some embodiments, such kit can include 2'-FL compound (e.g., as described herein, either as free oligosaccharide or in glycoconjugate form), or a pharmaceutical composition comprising it, or a dietary supplement comprising it.

[0081] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The instructions may include instructions for administration of 2'-FL compounds (e.g., as described herein, either as free oligosaccharides or in glycoconjugate form), or pharmaceutical or nutraceutical compositions comprising the same, for the treatment of IBD. Instructions for 2'-FL compounds (e.g., as described herein), or pharmaceutical or nutraceutical compositions comprising the same, generally include information regarding the dosage, administration schedule, and route of administration of the intended treatment. Such instructions may also include recommended weight-based and / or age-based dosages.

[0082] The instructions provided in the kits described herein are typically written instructions on a label or package insert (e.g., paper included in the kit), although machine-readable instructions (e.g., instructions written on a magnetic or optical storage disk) are also acceptable. The label or package insert indicates that the composition is used to treat IBD in a subject. In some embodiments, the label or package insert may indicate that the composition is suitable for use in a particular subject population, e.g., as described herein. For example, the label or package insert may indicate that the composition is suitable for use in human IBD patients (e.g., human CD or UC patients) who have undergone or are undergoing immune system suppression and / or anti-inflammatory therapy. In some embodiments, the label or package insert may indicate that the composition is suitable for use in human IBD patients (e.g., human CD or UC patients) who are undergoing stable maintenance anti-TNF therapy. Instructions for carrying out any of the methods described herein may be provided.

[0083] The 2'-FL compound (e.g., as described herein) of the kit, or the pharmaceutical or nutraceutical composition comprising it, can be appropriately packaged.Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags, or paper bags with polyethylene liner), etc.Packaging can be unit dose, bulk packaging (e.g., multi-dose packaging) or sub-unit dose.

[0084] The kit may optionally provide additional components, such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0085] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the above description. Therefore, the following specific embodiments are to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0086] Common techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Culture (RIFreshney, ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994), Current Protocols in Immunology (JEColigan et al., eds.,1991), Short Protocols in Molecular Biology (Wiley and Sons,1999), Immunobiology (CA Janeway and P. Travers,1997); Antibodies (P. Finch,1997), Antibodies: a practical approach (D. Catty.,ed.,IRL Press,1988-1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean,eds.,Oxford University Press,2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane,Cold Spring Harbor Laboratory Press,1999), The Antibodies (M. Zanetti and J. D. Capra,eds.,Harwood Academic Publishers,1995) and other publications.

[0087] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the above description. Therefore, the following specific embodiments are to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein. EXAMPLES

[0088] Example 1: Administration and efficacy of 2'-fucosyllactose in inflammatory bowel disease Inflammatory bowel disease (IBD), Crohn's disease (CD), and ulcerative colitis (UC) are chronic and debilitating disorders with peak incidence in the second and third decades of life (Kaplan et al., Gastroenterology, 152:313-321e2, 2017). Although considerable progress has been made in optimizing medications to achieve remission, relapses are common and unpredictable (Minar et al., Inflamm Bowel Dis, 22:2641-2647, 2016; Minar et al., J Pediatr Gastroenterol Nutr, 62:715-22, 2016). Alterations in the microbiota likely drive intestinal inflammation and clinical relapse (De Cruz et al., J Gastroenterol Hepatol, 30:268-78, 2015; Gevers et al., Cell Host Microbe, 15:382-92, 2014; Rajca et al., Inflamm Bowel Dis, 20:978-86, 2014; Varela et al., Aliment Pharmacol Ther, 38:151-61, 2013; Wills et al.,.PloS One, 9:e90981, 2014). Suppression of mucosal inflammation with infliximab, a monoclonal anti-TNF antibody, only partially corrects this dysbiosis (Lewis et al., Cell Host Microbe, 18:489-500, 2015).

[0089] A significant barrier to progress in the IBD field has been the lack of evidence-based approaches to directly modulate the microbiota to prevent clinical relapse (Kaplan et al., Gastroenterology, 152:313-321e2, 2017; Sartor et al., Gastroenterology, 152:327-339e4, 2017). Although a wide variety of prebiotics are commercially available, the lack of controlled dose-finding studies with appropriate clinical and microbiological endpoints precludes informed decision-making regarding their use (Ghouri et al., Clin Exp Gastroenterol, 7:473-87, 2014).

[0090] Although TNF-alpha inhibitors are effective, this therapy has high potential toxicity and does not directly address the microbial dysregulation that characterizes IBD (Lewis et al., Cell Host Microbe, 18:489-500, 2015). Presented herein is the use of the prebiotic human milk oligosaccharide, 2'-fucosyllactose (2'-FL), to maintain remission in IBD patients. A pilot dose-finding study is used to evaluate whether supplementation with 2'-FL in IBD patients is safe and well tolerated while simultaneously increasing the abundance of short-chain fatty acid (SCFA)-producing microbiota and reducing intestinal inflammation. The study utilizes state-of-the-art genomic approaches to evaluate response. The study provides important safety and efficacy data. This study supports a fundamental shift in clinical practice toward personalized microbial therapeutic interventions to maintain clinical remission in the growing global population of IBD patients.

[0091] The multicenter RISK pediatric CD and PROTECT pediatric UC initiation cohort studies were conducted to test genomic and microbial factors associated with clinical outcomes (Gevers et al., Cell Host Microbe, 15:382-92, 2014; Kugathasan et al. Lancet, 389:1710-1718, 2017; Haberman et al., J Clin Invest, 124:3617-33, 2014). They found that early anti-TNF therapy reduced progression to internal permeability complications in the RISK CD cohort, but not stenosis complications (Kugathasan et al. Lancet, 389:1710-1718, 2017). An imbalance between low expression of ileal genes regulating mitochondrial function and high expression of ileal genes driving extracellular matrix (ECM) production was seen in patients who progressed to strictures (Kugathasan et al. Lancet, 389:1710-1718, 2017). Distinct microbial taxa were similarly associated with complications (Kugathasan et al. Lancet, 389:1710-1718, 2017). Within the PROTECT UC cohort, taxa associated with disease severity were primarily from the families Ruminococcaceae and Lachnospiraceae, including two common commensals: Faecalibacterium prausnitzii, a known short-chain fatty acid (SCFA) producer, and Dorea formicigenerans, a member of Clostridium cluster XIV (Figure 1 and data not shown). Rectal-wide patterns of gene expression included induction of lymphocyte activation and associated extracellular matrix (ECM) responses, whereas gene programs regulating mitochondrial function were significantly repressed (Figure 1). Corticosteroid (CS) remission rates were inversely correlated with the degree of mitochondrial pathway dysregulation, ranging from 32% in the highest quartile of dysregulation to 71% in the lowest quartile (p=0.0004).2'-FL supplementation enhanced microbial alpha diversity, including a specific increase in SCFA-producing Parabacteroides (Mezoff et al., Am J Physiol Gastrointest Liver Physiol, 310: G427-38, 2016). The main small intestinal gene signature induced by 2'-FL was for enhanced mitochondrial function (Figure 1) (Mezoff et al., Am J Physiol Gastrointest Liver Physiol, 310: G427-38, 2016). This study seeks to evaluate whether 2'-FL supplementation in IBD patients may enhance intestinal mitochondrial function via induction of SCFA-producing microbes as a novel therapeutic target.

[0092] To define the dose-dependent safety, tolerability, and efficacy of 2'-FL as a dietary supplement in IBD. 2'-FL will be provided at 1, 5, or 10 gm as a daily dietary supplement to pediatric and young adult IBD patients in stable remission receiving infliximab or adalimumab anti-TNF therapy. Safety and tolerability will be assessed using validated clinical disease activity indices, electronic symptom trackers, fecal metabolite assays, and fecal calprotectin. Efficacy will be assessed by measuring the dose-dependent effect of 2'-FL on increasing fecal Bifidobacteria and decreasing fecal calprotectin abundance as biomarkers of mucosal inflammation.

[0093] Determine the effects of 2'-FL supplementation on the gut microbial community and associated SCFA production. Utilizing established genomic and metabolomic approaches, we will test the effects of a range of 2'-FL doses compared to glucose placebo on the gut microbial community and associated metabolic functions with a focus on SCFA production.

[0094] research design We will conduct a single-center randomized dose-ranging study of 2'-FL as a dietary supplement in pediatric and young adult IBD patients receiving stable maintenance infliximab or adalimumab (anti-TNF) therapy. The primary objective of this study is to obtain 2'-FL dose-dependent safety and efficacy data to guide the design of a larger multicenter placebo-controlled RCT. Inclusion criteria include male and female CD and UC patients aged 11 years or older who are currently in corticosteroid-free remission receiving stable anti-TNF maintenance therapy.

[0095] Patients will be excluded if they have experienced a clinical relapse in the past 6 months or have received antibiotics, probiotics, or prebiotics in the previous month. A 3-day food diary will be used to determine whether differences in usual diet are associated with differences in response, and participants will be encouraged to maintain a stable diet. The study timeline and procedures are summarized in Table 1. [Table 5]

[0096] Coordinator calls will occur prior to each of the four study visits and at weeks 5, 6, or 7. Data from the Orchestra symptom tracker will be obtained weekly, except for weeks 4-8 when they will be obtained daily. Safety labs will include CBC, CMP, PT / INR, and U / A.

[0097] Patients complete a 4-week run-in period to collect baseline data on gastrointestinal (GI) symptoms, plasma cytokines, fecal calprotectin, and fecal microbial community. A smartphone-based symptom tracker is utilized to track patient-reported indicators of 2'-FL tolerability, including abdominal pain, nausea, loose stools, and gas. These parameters were modestly increased in healthy adults receiving a 20 g dose of 2'-FL in a recent randomized controlled trial (RCT), but did not change with lower doses of 2'-FL (Elison et al., Br J Nutr, 116:1356-1368, 2016). The severity of each of these four symptoms is reported on a 10-point Likert scale ranging from (1) no symptoms to (10) severe symptoms, and the mean scores are calculated for each participant for the time periods baseline to week 4, week 4 to week 8, and week 8 to week 20. Participants will be randomized to receive one of three daily doses of 2'-FL for 4 weeks. A recent dose-ranging RCT of 2'-FL in healthy adults found doses of 5, 10, and 20 g / day to be safe and well tolerated over 2 weeks with no change in fecal calprotectin as a biomarker of intestinal inflammation (Elison et al., Br J Nutr, 116:1356-1368, 2016). A three-fold increase in Bifidobacterium was observed at the 10 g dose (Elison et al., Br J Nutr, 116:1356-1368, 2016). Thus, 1, 5, and 10 g / day of 2'-FL will be tested in five CD and five UC subjects per treatment group for 4 weeks. 2'-FL powder will be provided in a single-dose packet. Participants will be asked to dissolve the powder in water each morning just before consuming breakfast. Patient self-report data will be obtained on daily symptoms and 2'-FL intake during weeks 4 through 8 when patients are asked to record their 2'-FL intake. Patients and their parents will also be provided with free, automatically generated mobile phone text and / or email reminder prompts to improve adherence to 2'-FL to tolerated levels, defined as intake of a randomized dose on at least 24 of 30 treatment days.A 12-week follow-up period will then be completed to determine the stability of any changes detected in clinical disease activity, self-reported GI symptoms, plasma cytokines, and fecal calprotectin or microbiota over the 4-week supplementation period.

[0098] Study endpoints The primary safety endpoint was clinical relapse using validated measures of disease activity, the weighted Pediatric Crohn's Disease Activity Index (wPCDAI) for CD patients and the Pediatric Ulcerative Colitis Activity Index (PUCAI) for UC patients (Turner et al., Inflamm Bower Dis, 15:1218-23, 2009; Turner et al., J Pediatr Gastroenterol Nutr, 64:254-260, 2017). Clinical relapse was defined as an increase of more than 20 points in the wPCDAI or more than 15 points in the PUCAI at weeks 4 to 8 (Turner et al., Inflamm Bower Dis, 15:1218-23, 2009; Turner et al., J Pediatr Gastroenterol Nutr, 64:254-260, 2017). If 3 or more subjects in a dose group experience clinical relapse or an overall increase in GI symptom tolerability scores is observed, the dose is concluded to be unsafe and poorly tolerated. Secondary safety endpoints are tolerable GI symptom scores and fecal calprotectin collected using a symptom tracker. The primary efficacy endpoint is the increase in fecal Bifidobacterium abundance with 2'-FL supplementation within each dose group at weeks 4-8. 16S-DNA profiles of an average depth of 20,000 paired-end filtered reads per sample are generated using the Illumina MiSeq platform, using a primer set targeting the V4(515F / 806R) region (Gevers et al., Cell Host Microbe, 15:382-92, 2014). Read processing and error correction will be performed on a high-performance computing cluster using the DADA2 package and algorithms in R, which have been shown to be more sensitive and specific than percent similarity (i.e., OTU) clustering methods (Callahan et al., Nat Methods, 13:581-3, 2016). A secondary efficacy endpoint is reduction of fecal calprotectin as a biomarker of intestinal inflammation at weeks 4-8.

[0099] statistical analysis Primary analyses are per protocol and include only patients who received at least 24 of the 30 randomized 2'-FL doses (Elison et al., Br J Nutr, 116:1356-1368, 2016). Differences in Bifidobacterium abundance, fecal calprotectin, GI symptom tolerability scores, and plasma cytokines before and after supplementation are tested using mixed ANOVA (or nonparametric equivalents) with Bonferroni's multiple comparison correction. Models are fitted by mixed effects regression with within-subject contrasts comparing changes in the primary response of interest from weeks 4 to 8. Fisher's exact test is used to compare differences in clinical relapse rates at week 8 between each of the 2'-FL intervention groups. Within-dose comparisons of the number of relapses at weeks 4 to 8 are performed using exact tests for paired data. Based on a recent RCT in healthy adults (Elison et al., Br J Nutr, 116:1356-1368, 2016), we expect to detect a two-fold increase in fecal Bifidobacterium abundance and a two-fold decrease in fecal calprotectin at week 8 following 2'-FL supplementation at a dose of 10 g. We will use high-dimensional data classification and statistical learning approaches to identify differences in microbial community structure in response to 2'-FL supplementation.

[0100] Examination of relevant biological variables Biological variables that may affect the safety and efficacy of 2'-FL include age, sex, race / ethnicity, FUT2 secretor status, IBD diagnosis of CD or UC, mucosal inflammation as measured by fecal calprotectin, and baseline microbial community (Lewis et al., Cell Host Microbe, 18:489-500, 2015; Currier et al., Clin Infect Dis, 60:1631-8, 2015; Payne et al., JAMA Pediatr, 169:1040-5, 2015; Tong et al., ISME J, 8:2193-206, 2014; Wacklin et al., PLoS One, 6:e20113, 2011). Equal numbers of men and women aged 11 years or older, and Caucasian (90%) and African American (10%) subjects proportional to the overall CCHMC IBD population will be enrolled. Younger children will be excluded until any unexpected safety signals are identified.The effects of age, sex, race, CD vs. UC diagnosis, fecal calprotectin and microbiota at week 4, and FUT2 secretor status will be tested in an exploratory fashion to guide the design of a multicenter RCT.

[0101] Sample size The sample size of 10 participants per 2'-FL dose group is based on a recent dose-finding RCT in healthy adults in which mean (SD) fecal Bifidobacterium relative abundance increased from 7(2) at baseline to 20(4) after 2 weeks at a dose of 10 g (Elison et al., Br J Nutr, 116:1356-1368, 2016). Based on recent reports, we expect to observe greater variation in fecal Bifidobacterium abundance in CD patients (Gevers et al., Cell Host Microbe, 15:382-92, 2014; Kugathasan et al. Lancet, 389:1710-1718, 2017). Thus, with 10 participants per 2'-FL dose group, there is 80% power at α=0.0167 to detect a mean increase of 5(4) in fecal Bifidobacterium abundance within each dose group considering multiple trials. If larger variations in Bifidobacterium abundance were observed, with the SD of the mean difference being twice as large as expected, 80% power would be retained to detect a mean increase of 10 in relative abundance. A 10% dropout rate is assumed, so 33 participants will be enrolled.

[0102] Recruitment, follow-up, and retention Subjects will be enrolled from the IBD population aged 11 years or older who are currently in sustained remission receiving infliximab or adalimumab maintenance therapy. Patient visits will be required at baseline and at weeks 4, 8, and 20. Study coordinators will contact patients by telephone prior to each study visit and at weeks 5, 6, or 7 to assist with maintenance and compliance with study procedures. If new signals of lack of tolerability are detected for a 2'-FL dose, early stopping of randomization to that dose will be implemented.

[0103] Example 2: Pilot and feasibility study of 2'-FL as a dietary supplement in pediatric and young adult IBD patients receiving stable maintenance anti-TNF therapy Prebiotics studied in previous IBD RCTs included oligofructose-enriched inulin (OF-IN), fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and psyllium (Benjamin et al., Gut, 60:923-9, 2011). A 4-week RCT of 15 g of FOS in 103 active CD patients showed no benefit compared to placebo on clinical response (Benjamin et al., Gut, 60:923-9, 2011).

[0104] Microbial SCFA production and epithelial mitochondrial function Butyrate, a microbial SCFA metabolite, regulates intestinal epithelial cell (IEC) function through two mechanisms: as an energy source for oxidative phosphorylation and ATP production, and as a regulator of gene transcription via histone deacetylase (HDAC) activity (Donohoe et al., Cell Metab,13:517-26,2011; Kaiko et al., Cell,167:1137, 2016). Colonocytes isolated from germ-free mice show reduced oxidative phosphorylation and ATP production (Donohoe et al., Cell Metab,13:517-26,2011). Consequences include diarrhea and poor weight gain. Defects in colonocyte mitochondrial function can be rescued by single conjugation with Butyrivibrio fibrisolve, a butyrate-producing bacterial strain (Donohoe et al., Cell Metab,13:517-26,2011). The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1A) is a central regulator of mitochondrial biogenesis in the intestinal epithelium (Cunningham et al., J Biol Chem, 291:10184-200, 2016). Targeted IEC PGC1A deletion causes barrier dysfunction and increased severity of colitis after dextran sulfate sodium administration (Cunningham et al., J Biol Chem, 291:10184-200, 2016). In IBD patients who undergo mucosal healing, inflammatory suppression of EC genes regulating butyrate transport, signaling, and mitochondrial oxidation is significantly corrected by infliximab anti-TNF therapy (De Preter et al., Inflamm Bowel Dis, 4:e30, 2012). However, suppression of mucosal inflammation by infliximab was not associated with increased Bifidobacterium abundance in a recent pediatric CD study (Lewis et al., Cell Host Microbe, 18:489-500, 2015).The studies discussed herein will determine whether 2′-FL administration in CD and UC patients in stable remission on anti-TNF therapy is safe, well tolerated, and exerts a dose-dependent effect on the butyrate-producing microbiota, as illustrated in Figure 2 .

[0105] Although several IBD therapies targeting inflammatory cytokines have reached the clinic, no mechanism-based approach that directly modulates the microbiota has been used (Sartor et al., Gastroenterology, 152:327-339e4, 2017). This study challenges the current clinical practice paradigm by testing adjunctive microbial therapy for the first time in the context of anti-TNF immunosuppression. Basic and translational research has solidified the role of the microbiota and associated metabolites in the pathogenesis of IBD (Sartor et al., Gastroenterology, 152:327-339e4, 2017). Currently, environmental factors contributing to the rising incidence of disease worldwide are primarily responsible for the shift in pro-inflammatory microbes that interact with host genetic variation to drive chronic mucosal inflammation. 1 It is understood that it does so by driving the immune system. In this study, 2'-FL, a prebiotic trisaccharide synthesized by the FUT2 gene enzyme, is used as a therapeutic agent for IBD. Polymorphisms in the FUT2 gene (inactivating mutations, or non-secretor status) are associated with increased risk of CD, and therefore also lend themselves to personalized clinical trials where optimal doses of 2'-FL associated with FUT2 secretor status can be determined to account for patient sources of variation in response (McGovern et al., Hum Mol Genet, 19:3468-76, 2010). State-of-the-art electronic symptom trackers, high-throughput microbial sequencing methods, and fecal metabolite assays are utilized to accurately assess response (Integrative HMPRNC., Cell Host Microbe, 16:276-89, 2014). The results of the study discussed herein support a fundamental shift in clinical practice toward personalized microbial therapeutic interventions.

[0106] Preliminary Study At 95 centers participating in the ImproveCareNow (ICN) Pediatric IBD Quality Improvement (QI) Network, 47% of patients relapsed in the past year (improvecarenow.org). At a Cincinnati center, 37% of patients receiving infliximab or adalimumab anti-TNF therapy relapsed despite optimal therapeutic drug monitoring and administration (Minar et al., Inflamm Bowel Dis; 2016; Minar et al., J Pediatr Gastroenterol Nutr, 62:715-22, 2016). In addition to negative impacts on quality of life, and school and work productivity, relapses increase the cost of treatment. Based on a recent cost-effectiveness analysis, the annual pharmacy costs of adalimumab or infliximab therapy in the United States range from $27,664 to $92,300 per year (Yokomizo et al., BMJ Open Gastroenterol, 3:e000093, 2016). At the Cincinnati center, median annual treatment costs increased by $16,862 among patients receiving anti-TNF therapy who relapsed. In comparison, the annual cost of 2'-FL at a daily dose of 10 g is estimated to be approximately $500, providing a highly cost-effective adjuvant treatment option.

[0107] 2'-FL promotes the growth of Bifidobacterium, which enhances butyrate production by acetate symbiosis with F. prausnitzii and other beneficial microorganisms (Rios-Covian et al., FEMS Microbial Lett, 362, 2015; Yu ZT et al., Glycobiology, 23: 1281-92, 2013). Conversely, 2'-FL does not support the growth of Enterobacter spp. or Escherichia, which are increased in IBD patients with more severe symptoms (Gevers et al., Cell Host Microbe, 15: 382-92, 2014; Morgan et al., Genome Biol, 13: R79, 2012; Yu ZT et al., Glycobiology, 23: 1281-92, 2013). In addition to these prebiotic effects, 2'-FL also exerts direct anti-inflammatory effects in the gut by inhibiting pathogen adhesion and suppressing epithelial inflammatory responses to bacterial production (He et al., Gut, 65:33-46, 2016; Yu et al., J Nutr, 146:1980-1990, 2016). 2'-FL was reported to promote weight gain in mice after ileocecal resection (Mezoff et al., Am J Physiol Gastrointest Liver Physiol, 310:G427-38, 2016). This was associated with the expansion of Parabacteroides and induction of an intestinal gene signature of mitochondrial function (Mezoff et al., Am J Physiol Gastrointest Liver Physiol, 310:G427-38, 2016). Preclinical safety studies of 2'-FL in rats established a no observed adverse effect level (NOAEL) of 5 gm / kg bw / day in both males and females (Goulet et al., Regul Toxicol Pharmacol, 68:59-69, 2014).Infants fed 2'-FL supplemented formula had improved growth velocity and reduced plasma cytokine profiles compared to infants fed control formula (Goehring et al., J Nutr,146:2559-2566,2016; Marriage et al., J Pediatr Gastroenterol Nutr;61:649-58,2015). A recent RCT in healthy adults reported that 2'-FL was safe and well tolerated at doses ranging from 5 gm to 20 gm daily and promoted the expansion of Bifidobacterium and the reduction of Proteobacteria (Elison et al., Br J Nutr,116:1356-1368,2016). However, it is unclear whether 2'-FL exerts similar benefits in the IBD population. Previous studies have shown a significant shift in 2'-FL target microbiota, as well as reduced colonic butyrate absorption and oxidative metabolism in the setting of active mucosal inflammation (Gevers et al., Cell Host Microbe, 15:382-92, 2014; Morgan et al., Genome Biol, 13:R79, 2012; De Preter et al., Inflamm Bowel Dis, 18:1127-36, 2012). These changes in the microbial niche and host butyrate metabolism in the setting of active mucosal inflammation may reduce the benefit of 2'-FL treatment. This may explain the mixed results of local butyrate enema therapy in the setting of active colitis (Scheppach et al., Gastroenterology, 103:51-6, 1992). Therefore, a more effective approach would be to first suppress mucosal inflammation with anti-TNF therapy and then utilize 2'-FL supplementation to promote the expansion of SCFA-producing microbiota and enhance the maintenance of remission (Figure 2).

[0108] Microbial shifts associated with disease complications and treatment response in CD The multicenter CCFA-supported RISK Inception Cohort study was undertaken to examine clinical, demographic, genomic, microbial, and immune factors associated with early treatment response and subsequent occurrence of disease complications during 36 months of follow-up in 913 pediatric CD patients enrolled at diagnosis prior to therapy (Gevers et al., Cell Host Microbe,15:382-92,2014, (Kugathasan et al. Lancet,389:1710-1718,2017, (Haberman et al., J Clin Invest,124:3617-33,2014). Ileal and rectal global patterns of gene expression, as well as ileal, rectal, and fecal microbial communities, were determined using high-throughput sequencing in a representative subset of 243 and 22 CD patients, respectively (Gevers et al., Cell Host Microbe,15:382-92,2014, (Kugathasan et al. Lancet,389:1710-1718,2017, (Haberman et al., J Clin Invest,124:3617-33,2014). In untreated ileum and treated rectum, an expansion of proinflammatory genera including Veillonellaceae was identified (Figure 3A) (Gevers et al., Cell Host Microbe, 15:382-92, 2014, (Kugathasan et al. Lancet, 389:1710-1718, 2017, (Haberman et al., J Clin Invest, 124:3617-33, 2014) along with a contraction of butyrate-producing Blautia, Parabacteroides, and Roseburia. (Gevers et al., Cell Host Microbe, 15:382-92, 2014, (Kugathasan et al. Lancet, 389:1710-1718, 2017, (Haberman et al., J Clin Invest,124:3617-33,2014). Distinct taxa were similarly associated with progression to B2 stenosis or B3 internal permeability complications, suggesting a role in regulating host biology (Figures 3B and 3C). Multivariate logistic regression models including baseline microbial abundance outperformed those including only clinical, demographic, and genomic factors in predicting steroid- and surgery-free remission (SFR) 6 months after diagnosis (Haberman et al.,J Clin Invest,124:3617-33,2014).In this model, the relative abundance of Blautia and Veillonellaceae was associated with the likelihood of achieving SFR after accounting for anti-TNF exposure (Haberman et al., J Clin Invest, 124:3617-33, 2014). Importantly, the data supported a model in which these pro- and anti-inflammatory microbes co-exclude each other and concurrent antibiotic use exacerbates dysbiosis (Gevers et al., Cell Host Microbe, 15:382-92, 2014).

[0109] Microbial shifts associated with disease severity in UC Following the success of the RISK study, we conducted the NIH / NIDDK-supported PROTECT initiation cohort study to examine clinical, demographic, genomic, microbial, and immune factors associated with achievement of SFR with mesalamine alone in 431 pediatric UC patients enrolled at diagnosis prior to therapy. Rectal global patterns of gene expression, as well as rectal and fecal microbial communities, were determined using high-throughput sequencing in a representative subset of 206 and 371 UC patients, respectively. In total, 48 operational taxonomic units (OTUs) were associated with disease severity and showed continuous increases or decreases with increasing disease severity (FDR threshold: 0.5) (Figure 4). The majority of OTUs were negatively correlated with disease severity, indicating that loss of these bacterial taxa was associated with worsening UC. Primarily, these OTUs were from the genera Ruminococcaceae and Lachnospiraceae and included two common symbionts: F. prausnitzii (a known SCFA producer) and Dorea formicigenerans (a member of Clostridium cluster XIV). Increases in six OTUs representing many Veillonellaceae organisms, such as Veillonella dispar and Megasphaera, were associated with increased severity. These data from a large prospective initiation cohort study support the examination of a dose-dependent effect of 2'-FL in modulating the SCFA-producing microbiota in IBD.

[0110] An ileal gene signature of mitochondrial dysfunction is associated with disease complications in pediatric CD Although there was considerable heterogeneity in the overall ileal patterns of gene expression within the RISK cohort, comparisons between groups revealed striking differences in gene expression (Kugathasan et al. Lancet, 389:1710-1718, 2017; Haberman et al., J Clin Invest, 124:3617-33, 2014). Analysis identified enrichment of mitochondrial function gene signatures in patients who were otherwise at high risk for B2 stricture complications and remained complication-free (B1 protected, Figure 5). Conversely, enrichment of extracellular matrix (ECM) gene signatures was detected in predicted low-risk patients who nevertheless progressed to stricture (B2 low probability). Genes involved in mitochondrial respiratory chain (GO pathway:0022900 and GO pathway:0045333; dark shading) were upregulated in "B1 protected" and genes involved in ECM production (GO pathway:0005201; light shading) were upregulated in "B2 low probability". Multivariate logistic regression models of disease complications including these gene signatures outperformed those including only clinical, demographic and serological factors (Kugathasan et al. Lancet,389:1710-1718,2017). Higher expression of ileal mitochondrial function gene signatures was associated with a reduced likelihood of developing stenotic complications in the model (HR(95 th Cl: 0.69 (0.51, 0.94), p = 0.019), but higher expression of the ECM gene signature was associated with an increased likelihood of developing stenotic complications (HR (95 th Cl): 1.7 (1.12, 2.57), p=0.012). These data indicate that approaches to increase intestinal epithelial cell (IEC) mitochondrial function, such as expansion of the butyrate-producing microbiota with 2'-FL, may improve CD treatment response and outcome.

[0111] A rectal gene signature of mitochondrial dysfunction associated with clinical severity and treatment response in pediatric UC is modulated by 2'-FL supplementation in mice. A similar analysis of rectal gene expression was performed on patients enrolled in the PROTECT UC cohort study. Genes that were differentially expressed between UC patients and controls with a fold change of ≥1.5 and a false discovery rate (FDR) of 0.001 were used to define pathogenic processes. These included lymphocyte activation and associated extracellular matrix (ECM) responses (Figure 1-Table). The master regulator of mitochondrial biogenesis PGC1A was suppressed 4-fold in UC, in conjunction with genes regulating mitochondrial biogenesis and ATP production. Strikingly, these same epithelial energy pathways were induced in mice by supplementation with 2'-FL (Figure 1-Table) (Mezoff et al., Am J Physiol Gastrointest Liver Physiol,310:G427-38,2016). Among moderate-to-severe patients treated with corticosteroids, remission rates at week 4 were inversely correlated with the degree of mitochondrial pathway dysregulation, ranging from 32% in the highest quartile of dysregulation to 71% in the lowest quartile (p=0.0004). These data indicate that approaches to increase rectal mitochondrial function, such as expansion of butyrate-producing microbiota with 2'-FL, can improve UC treatment response. Together, these studies show that a gene signature of epithelial mitochondrial function induced by 2'-FL in mice is suppressed in association with a shift in butyrate-producing microbiota and poor response to therapy in pediatric CD and UC patients (Kugathasan et al. Lancet,389:1710-1718,2017; Mezoff et al.,Am J Physiol Gastrointest Liver Physiol,310:G427-38,2016). These data support 2'-FL supplementation in IBD by enhancing epithelial mitochondrial function via induction of butyrate-producing microbes as a novel therapeutic target.

[0112] Risk / benefit assessment Known and Potential Risks Risks associated with this study are associated with 2'-FL or glucose supplementation and venipuncture. Risks associated with 2'-FL supplementation include a potential dose-dependent increase in GI symptoms including abdominal pain, nausea, loose stools, and / or gas. Risks associated with peripheral blood sampling (venipuncture) are pain, bruising, fainting (rare), and / or infection (rare).

[0113] Known Potential Benefits Based on the results of preclinical and clinical studies, nutritional supplementation with prebiotic 2'-FL may be effective in maintaining IBD remission, for example, by increasing beneficial microbes, inhibiting harmful microbes, and suppressing pro-inflammatory cytokines. It has been shown to be safe and well-tolerated in healthy infants and adults.

[0114] The results of this study provide valuable information on whether 2'-FL administration is safe and well tolerated in CD and UC patients in stable remission and exerts dose-dependent effects on the SCFA-producing microbiota that promote gut health and stable remission. This knowledge will aid in the design of phase III randomized clinical trials. Ultimately, this knowledge can be utilized to improve clinical practice.

[0115] Assessment of potential risks and benefits The risk to which participants will be exposed is likely mild to moderate. The study will mitigate the risks associated with 2'-FL by having the study monitored by an experienced gastroenterologist familiar with the adverse reaction profile of this patient population. A dose range of 2'-FL that has been well tolerated in a healthy adult population without systemic (plasma cytokine) changes or mucosal inflammation (fecal calprotectin) will be utilized. In addition, an adaptive dosing study design will be utilized to ensure that participants randomized to the higher dose (5 g / day and 10 g / day doses) do not begin taking the drug until at least 10 participants have completed the lower dose.

[0116] Reduce the risks associated with venipuncture by having the procedure performed by a professional experienced in caring for pediatric patients.

[0117] The potential benefits of participating in the study outweigh the potential risks to participants. Objectives and Endpoints [Table 6] TIFF2025041753000008.tif154159

[0118] research design Overall Design This study evaluates whether 2'-FL supplementation in IBD is safe and well tolerated while increasing fecal Bifidobacterium abundance and butyrate in a dose-dependent manner. To that end, we will conduct a two-center, phase I / IIa, double-blind, randomized, placebo-controlled, dose-ranging clinical trial. There will be two study arms, one randomized to receive 2'-FL and the other randomized to a placebo arm. Patients will be randomized to placebo or treatment arms within strata using a stepwise approach that allows for the evaluation of safety in the lowest dose arm before randomization begins at the next highest dose. Given that the duration of the study is short and the inclusion of patients randomized to placebo and low dose arms at each stage allows for the testing and consideration of any observed cohort / time effects, the likelihood of bias in randomizing patients to later higher dose arms is low.

[0119] Supplementation with 2'-FL or glucose placebo will occur over a 4-week period. Daily doses will be tracked and patient-reported symptoms will be captured weekly. The study will require 4 visits: 1) a screening / baseline visit with a 4-week run-in period, 2) a randomization visit, 3) a completion visit, and 4) a 12-week follow-up visit.

[0120] This study is the first dose-finding randomized clinical trial (RCT) of 2'-FL prebiotics in IBD, utilizing state-of-the-art metagenomic and metabolomic approaches to evaluate response. Because polymorphisms in the FUT2 gene are associated with CD risk (McGovern et al., Hum Mol Genet, 19:3468-76, 2010), optimal doses of 2'-FL with respect to FUT2 secretor status will be determined to also inform future personalized clinical trials. These studies will have a significant impact on the field by providing important phase I / IIa safety and efficacy data that will support future phase III RCTs to test the efficacy of 2'-FL in directly modulating beneficial microbiota, thereby enhancing sustained clinical remission. Ultimately, these studies will facilitate a fundamental shift in clinical practice toward personalized microbial therapeutic interventions.

[0121] dose A recent dose-ranging RCT of 2'-FL in healthy adults found it to be safe and well tolerated at doses of 5, 10, and 20 g / day over 2 weeks (Elison et al., Br J Nutr,116:1356-1368,2016). A three-fold increase in Bifidobacteria and a reduction in Proteobacteria were observed at the 10 g dose (Elison et al., Br J Nutr,116:1356-1368,2016). A slight increase in GI symptoms, including abdominal bloating and loose stools, was reported at the 20 g dose, but no participants discontinued 2'-FL (Elison et al., Br J Nutr,116:1356-1368,2016). A previous RCT in adults with CD showed efficacy in reducing clinical disease activity and increasing butyrate production by utilizing 20 g of a different prebiotic, oligofructose-enriched inulin, daily, but this was associated with high rates of GI intolerance (mainly cramps and flatulence) and discontinuation of the study drug (De Preter et al., Clin Transl Gastroenterol, 18:1127-36, 2013). Thus, in some cases, a 20 g / day 2'-FL dose may not be well tolerated. In some cases, a 10 g / day 2'-FL dose may be effective in increasing Bifidobacterium abundance and butyrate production. Thus, 1, 5, and 10 g / day 2'-FL will be evaluated over a 4-week period in 20 UC and 20 CD subjects. 2 g / day glucose will be utilized as placebo in 20 UC and 20 CD subjects.

[0122] Differences in baseline microbial communities and levels of mucosal inflammation, as measured by fecal calprotectin, between CD and UC patients may affect the response to 2'-FL supplementation. Usual dietary fiber intake may also affect the response to 2'-FL (Holscher et al., J Nutr, 145:2025-32, 2015). A previous study of agave inulin prebiotic supplementation in healthy adults showed a positive correlation between total dietary fiber + agave inulin intake and fecal butyrate (Holscher et al., J Nutr, 145:2025-32, 2015). Furthermore, a study involving 142 pediatric IBD patients at three centers found that the mean (SD) dietary fiber intake was only 14(7) grams per day, at the lower end of the range of intakes associated with appreciable fecal butyrate in healthy adults (Holscher et al., J Nutr, 145:2025-32, 2015). Furthermore, GI intolerance of 2'-FL in IBD patients may occur at lower doses than detected in recent RCTs in healthy adults (Elison et al., Br J Nutr, 116:1356-1368, 2016; De Preter et al., Clin Transl Gastroenterol, 18:1127-36, 2013). Therefore, enrollment will be balanced for disease phenotype (CD or UC), and usual dietary fiber intake and treatment arms will be randomized within strata of these factors.

[0123] Definition of Exam Completion Participants were considered to have completed the study if they completed all study visits, including the final visit, as shown in the Schedule of Activities (SoA) listed in Figure 8 .

[0124] Study population Inclusion criteria To be eligible to participate in this study, individuals had to meet all of the following criteria: (1) provide a signed and dated informed consent form, (2) express willingness to complete all study procedures and be available for the duration of the study, (3) be male or female, aged 11–25 years, (4) be diagnosed with Crohn's disease or ulcerative colitis, (5) have disease in remission, (6) are not receiving corticosteroids, (7) are receiving a stable anti-TNF maintenance dose of adalimumab or infliximab, and (8) agree not to significantly change their diet throughout the study period, which could include changing their usual diet to a vegan diet, a specific carbohydrate diet (SCD), or an exclusive enteral nutrition (EEN) diet.

[0125] Exclusion criteria Individuals who met any of the following criteria would be excluded from participating in the study: (1) having experienced a clinical relapse during the past 6 months, defined as wPCDAI or PUCAI>10, (2) use of either antibiotics, probiotics, or prebiotics during the previous month, (3) diagnosis of celiac disease, diabetes, or other comorbidities determined to be exclusive, (4) treatment with another investigational drug or other intervention within 4 weeks, (5) problems with lactose digestion, or (6) pregnancy.

[0126] Participants of childbearing potential will be required to use effective contraception throughout the study and for at least 30 days after stopping the last dose of the study supplement.

[0127] Participants who meet the initial criteria will undergo additional screening and a 4-week run-in period during which baseline data will be collected. Participants with any of the following additional exclusion criteria will be excluded from further participation in the study: Abnormal baseline screening laboratory results defined as: WBC>1.5ULN ○CMP w LFT>2.0ULN GI symptoms with a mean baseline total score of >3 on the GSRS symptom questionnaire wPDCAI and PUCAI values ​​≥ 10 at baseline and week 4

[0128] Lifestyle Considerations During this study, participants will be asked to maintain a stable diet over the course of the study.

[0129] Administration of study interventions Description of study intervention 2'-Fucosyllactose powder (2'-FL) is a human milk oligosaccharide prebiotic. According to the 2006 FDA Guidance for Industry on Complementary and Alternative Medicine Products and Their Regulation by the Food and Drug Administration, prebiotics are non-digestible food ingredients that beneficially affect the host by stimulating the growth and / or activity of colonic bacteria in a selective manner. According to the National Center for Complementary and Integrative Health (NCCIH), prebiotics are classified as biologically-based practices. In this study, it is used as a complementary dietary supplement to anti-TNF therapy.

[0130] Glucose powder is utilized as a placebo control. Glucose is the primary source of energy and occurs naturally in its free state in fruits and other parts of plants.

[0131] Dosage and Administration A total of 160 participants will be randomized to receive one of three daily doses of 2'-FL or a glucose placebo for four weeks. Eighty participants will have Crohn's disease and eighty participants will have ulcerative colitis. Participants will be instructed that any missed doses may be taken later on the day of the missed dose.

[0132] In the first group, 120 participants will receive 2'-FL in one of three groups: ●Group 1: 1g / day; n=40 (20 CD / 20 UC) ●Group 2: 5g / day; n=40 (20 CD / 20 UC) After 10 people completed group 1 ●Group 3: 10g / day; n=40 (20 CD / 20 UC) After 10 people completed group 2

[0133] In the second group, 40 participants, 20 CD / 20 UC, will receive 2 g glucose placebo.

[0134] A stratified stepwise randomization will be used. Strata will be defined by study center, disease phenotype (e.g., CD / UC), and usual fiber intake (< or >= 7g / 1000kcal / day), resulting in 8 equally balanced strata of 20 participants. This will allow for the evaluation of safety in the lowest dose group before randomization begins at the next highest dose. If a dose / disease phenotype group experiences sufficient safety issues, allocation to that group and all higher dose groups will be discontinued.

[0135] After completing dosing at week 8, participants will complete a 12-week follow-up period to determine the stability of any changes detected in clinical disease activity, self-reported GI symptoms, plasma cytokines, and fecal calprotectin, microbiota, or metabolites during the supplementation period.

[0136] Formulation and Packaging 2'-Fucosyllactose (2'-FL) is a white homogeneous powder, neutral to slightly sweet, without any off-odor. It accounts for 96% dry matter, with a moisture content of 4%. The results of the analysis show that the total content is 93% 2'-fucosyllactose, 3% other sugars, and 4% moisture. The packaging consists of a multi-layer paper bag with a polyethylene liner and a capacity of 25 kg net. The name of the drug is indicated on the label. Glucose is a white powder with a sweet taste.

[0137] Participants will be instructed to consume 2'-FL or glucose each morning at breakfast by adding the required amount to a beverage or food. A food diary will be kept by participants to record the foods or beverages with which the products are consumed.

[0138] Measures to minimize bias: randomization and blinding The overall enrollment and randomization schema is depicted diagrammatically in Figure 6. A double-blind, placebo-controlled, stratified stepwise randomization will be used.

[0139] Strata were defined by study center, disease phenotype (e.g., CD / UC), and usual fiber intake (< or >= 7 g / 1000 kcal / day), resulting in eight equally balanced strata of 20 participants.

[0140] Patients will be randomized to placebo or treatment arms within strata using a stepwise approach, with a total of 20 CD and 20 UC participants randomized to each dose arm, with randomization in stage 1 in a 1:1 ratio to placebo 1g, stage 2 in a 1:1:2 ratio to placebo 1g, 5g, and stage 3 in a 1:1:2:4 ratio to placebo 1g, 5g, 10g. Sequential stepping with shifting allocation ratios allows for the evaluation of safety and tolerability of the lowest dose arm before randomization begins at the next highest dose. If a dose / disease phenotype arm experiences sufficient safety or intolerance issues, allocation to that arm and all higher dose arms will be discontinued. Natural block sizes of 2, 4, and 8 will be used to randomize patients to dose arms within strata in stages 1, 2, and 3, respectively, to select the optimal randomization in each stage to ensure counterbalance.

[0141] Compliance with study interventions Patient self-reported data on 2'-FL intake using a gastrointestinal symptom rating scale will be obtained at weeks 4, 5, 6, 7, and 8, when participants are asked to record symptoms and daily 2'-FL intake. Coordinators will call participants weekly during the refill period to improve 2'-FL adherence to tolerable levels, defined as intake of the randomized dose on at least 24 of 28 treatment days.

[0142] Combination therapy Concomitant medications, including prescription, over-the-counter, and dietary supplements, will be recorded at all study visits. Corticosteroids, antibiotics, probiotics, prebiotics (other than 2'-FL), and other investigational drugs will not be allowed during the study. If clinical remission is lost, participants will be treated according to standard of care.

[0143] Study assessment and procedures Efficacy assessment To obtain efficacy endpoint data, the following assessments and procedures will be performed: Stool sample collection and sequencing: Stool samples are collected from participants for DNA and RNA extraction. DNA and RNA are isolated with the AllPrep DNA / RNA Mini Kit (QIAGEN) plus mechanical lysis. RNA is subsequently reverse transcribed to DNA and samples are quantified by Quant-iT PicoGreen dsDNA Assay (Life Technologies) and normalized to a concentration of 50 pg / ml. Whole-genome shotgun sequencing libraries are prepared using the Nextera XT DNA Library Preparation Kit (Illumina) with 100–250 pg of input DNA according to the manufacturer's instructions. Libraries are pooled by transferring equal amounts of each library using a Labcyte Echo550 liquid handler. The concentration and insert size range of each pooled library are confirmed using an Agilent Bioanalyzer DNA1000 kit (Agilent Technologies). Libraries are then sequenced on an Illumina HiSeq2000 platform in paired-end mode (2x101bp) targeting 2.5Gb of sequence per sample (Kugathasan et al. Lancet,389:1710-1718,2017; Schirmer et al., Cell,167:1125-1136 e8,2016).

[0144] Metagenomic and metatranscriptomic analysis: If a sample had sufficient sequence reads, the sample was included in the analysis. Reads were first processed using KneadData (huttenhower.sph.harvard.edu / kneaddata), which included quality trimming (trimmomatic parameters: MAXINFO:90:0.5), read filtering based on a minimum read length of 60 bp, and removal of potential human contamination by filtering reads aligned to the human genome (reference genome hg19). Quality-controlled paired-end reads were aligned against a database of unique clade-specific marker genes using Bowtie2, and taxonomic profiles were inferred with MetaPhlAn2.2 (Segata et al., 2012). Subsequent analyses took into account the species and genus composition of the sample. Functional profiling was performed using HUMANnN2 (huttenhower.sph.harvard.edu / humann2). Briefly, reads are mapped against a customized database of functionally annotated pan-genomes, and only organisms identified during the taxonomic profiling step are considered. Functional annotation of protein sequences within the pan-genome to their respective UniRef50 families is provided by the software. Reads that cannot be mapped are then aligned against the full UniRef50 database. Community sums are calculated for each protein family (RPK) and converted to relative abundances. For subsequent downstream analysis, these tens of thousands of gene families were further grouped into broader functional categories: MetaCyc metabolic pathways and informative GO categories, focusing on molecular functions and biological processes.

[0145] Specifically, the selected GO terms each annotated >2,000 proteins in UniRef50, and all their descendant (more specific) terms annotated <2,000 proteins.

[0146] Metabolomic Analysis: Data are acquired using an LC-MS system consisting of a Nexera X2 U-HPLC system (Shimadzu Scientific Instruments; Marlborough, MA) and a Q Exactive / Exactive Plus orbitrap mass spectrometer (Thermo Fisher Scientific; Waltham, MA) using negative ion mode MS analysis of polar metabolites. LC-MS samples are prepared from stool homogenates (30 μL) by protein precipitation with the addition of four volumes of 80% methanol containing inosine-N4 (Rios-Covian et al., FEMS Microbial Lett; 2015), thymine-d4, and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). Samples are centrifuged (10 min, 9,000xg, 4°C) and the supernatant is injected directly onto a 150 x 2.0 mm Luna NH2 column (Phenomenex; Torrance, CA). The column is eluted at a flow rate of 400 µL / min with initial conditions of 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide in water) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v / v acetonitrile / methanol), followed by a 10 min linear gradient to 100% mobile phase A. MS analysis is performed using electrospray ionization mass spectrometry in negative ion mode, using full scan analysis from m / z 60 to 750, at a resolution of 70,000 and a 3 Hz data acquisition rate. Additional MS settings are as follows: ion spray voltage, -3.0 kV; capillary temperature, 350 °C; probe heater temperature, 325 °C; sheath gas, 55; auxiliary gas, 10; and S-lens RF level, 40.

[0147] Data Processing: Raw LC-MS data are collected on a data acquisition computer connected to each LC-MS system and then stored in a robust redundant file storage system (Isilon Systems) accessed via the Broad Institute's internal network. Data processing is performed using one of five Dell Precision T7600 workstations, each equipped with an eight-core XEON E5-2687W processor, 32 GB of DDR3 RAM, and 2 TB of storage in a RAID0 array of four 600 GB SAS hard drives. Untargeted data are processed using Progenesis CoMet software (v2.0, Nonlinear Dynamics) for peak detection and de-isotope, alignment of chromatographic retention times, and integration of peak areas. Peaks of unknown identity are tracked by method, m / z, and retention time. Identification of LC-MS peaks of non-targeted metabolites is performed initially by i) matching measured retention times and masses to a mixture of reference metabolites analyzed in each batch, ii) matching an internal database of >600 compounds characterized using Broad Institute methods, and iii) matching exact masses only to an external database of >40000 metabolites (Human Metabolome Database v3) (Wishart et al., Nucleic Acids Res, 41:D801-7, 2013). Compounds matching the external database are confirmed by analyzing reference standards, if available.

[0148] Demographics: Information about participants' age and sex will be collected as part of the medical record review conducted for screening (see below). Race will be collected at Visit 1.

[0149] Fecal calprotectin: Fecal calprotectin is measured using a monoclonal antibody-based ELISA (Bohlmann Laboratories, Switzerland) that has shown excellent linearity over a wide dynamic range (Burri., et al. Clin Chim Acta, 416:41-7, 2013).

[0150] Plasma cytokines: Thirteen plasma cytokines representing innate and adaptive immune responses are measured using a highly sensitive bead-based multiplex assay (Milliplex Multiplex Assay using Luminex) as previously reported (Dorn et al., Psychosom Med, 78:646-56, 2016).

[0151] Fiber Intake: Three unannounced 24-hour dietary recall interviews were conducted at baseline, and at weeks 4 and 8 to allow for randomization of patients within strata of usual fiber intake (high / low) and to determine whether differences in usual diet are associated with differences in response to 2'FL. Dietary recalls were conducted by expert interviewers using the USDA Automated Multipass Method (AMPM) to ensure accurate and consistent recording of foods and quantities reported by participants (Moshfegh et al., Am J Clin Nutr, 88:324-32, 2008). Nutrition Data Systems for Research (NDSR) (Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN) software and food databases were used to assess daily total energy, macronutrient, and fiber intake, as well as food group items consumed (Sievert et al., Control Clin Trials, 10:416-25, 1989).

[0152] FUT2 secretor status: FUT2 secretor status was associated with both infectious and inflammatory conditions, with opposing trends: FUT2+ (secretor) individuals were at increased risk for rotavirus and norovirus gastroenteritis (Currier et al., Clin Infect Dis, 60:1631-8, 2015; Payne et al., JAMA Pediatr, 169:1040-5, 2015), whereas FUT2- (nonsecretor) individuals were at increased risk for CD (McGovern et al., Hum Mol Genet, 19:3468-76, 2010). Furthermore, FUT2 nonsecretors may show a reduction in 2'-FL target microbiota, including Bifidobacterium, even in the absence of mucosal inflammation (Rausch et al., Proc Natl Acad Sci USA, 108:19030-5, 2011; Tong et al., ISME J, 8:2193-206, 2014; Wacklin et al., PLoS One; 2011, 6:e20113; Wacklin et al., PLoS One, 9:e94863, 2014). Secretor status can be measured genotypically or phenotypically. Genotyping in the United States involves the analysis of a single nucleotide 428G>A polymorphism in the FUT2 gene (rs601338). 23% of the US population is homozygous for this inactivating mutation, which results in a deficiency of fucosylated intestinal carbohydrates. Phenotypically, non-secretor status can be measured by testing whole saliva samples for "secretor carbohydrates" using the Ulex europaeus-1 (UEA-1) lectin enzyme immunoassay. The UEA-1 immunoassay detects the alpha 1,2-fucose-linked product of the FUT2 gene enzyme (Kazi et al., J Infect Dis, 215:786-789, 2017; Morrow et al., J Pediatr, 158:745-51, 2011). Studies have shown that FUT2+ secretor individuals, who are genetically capable of synthesizing secretor carbohydrates, produce less secretor carbohydrates and appear phenotypically similar to non-secretor individuals. Thus, in this study, both the FUT2 genotype and phenotype are measured.

[0153] Safety and other evaluations Prior to enrollment, the following will be conducted for screening purposes:

[0154] Medical Record Review: Existing information on potential participants will be reviewed to determine gender, age, diagnosis, current medications, medication history, comorbidities, and allergies.

[0155] To obtain safety data, eligibility for study participation (patient population) will be determined and the following assessments and procedures will be performed throughout the study:

[0156] Physical Examination and Vital Signs: A physical examination will be performed at each study visit. Vital signs will be collected, including temperature, heart rate, respiratory rate, and blood pressure. Weight will also be collected at these visits.

[0157] Blood collection: A blood sample is taken and analyzed for CBC, CMP, and ESR.

[0158] Urine collection: Female participants of childbearing potential will undergo a urine pregnancy test at the baseline visit.

[0159] Saliva sample: Saliva samples will be collected at Visit 1 to determine FUT2 phenotype secretor status.

[0160] Weighted Pediatric Crohn's Disease Activity Index (wPCDAI) and Pediatric Ulcerative Colitis Activity Index (PUCAI): The wPCDAI and PUCAI are utilized to measure clinical disease activity in the CD and UC groups, respectively. They have been validated in pediatric IBD populations with well-established cut points for clinical remission and relapse. wPCDAI and PUCAI scores are obtained at baseline and at weeks 4, 12, and 20 (Turner et al., Inflamm Bower Dis, 15:1218-23, 2009; Turner et al., Gastroenterology, 133:423-32, 2007). For both the wPCDAI and PUCAI, values ​​of <10 are required at baseline and week 4 to meet the entry criteria for stable clinical remission.

[0161] IMPACT III: The IMPACT-III questionnaire is used to measure quality of life (QOL) at baseline and at weeks 4, 8, and 20. The IMPACT-III has been validated in IBD populations with good reliability of the total score (Otley et al., J Pediatr Gastroenterol Nutr, 35:557-63, 2002; Otley et al., Inflamm Bowel Dis, 12:684-91, 2006). A score of 144 or higher is used to indicate good quality of life.

[0162] GSRS Survey: The GSRS questionnaire is utilized to track patient-reported indicators of 2'-FL tolerability, including abdominal pain, nausea, loose stools, and gas. These parameters were modestly increased in healthy adults receiving a 20 g dose of 2'-FL in a recent RCT, but did not vary with lower doses of 2'-FL. Each participant is provided with a GSRS questionnaire at each study visit. Each participant then receives a GSRS questionnaire weekly during the treatment (weeks 4–8) phase. The severity of 15 gastrointestinal symptoms is reported on a 7-point Likert scale ranging from (1) no symptoms to (7) severe symptoms, and the mean scores for each participant are calculated for the time periods baseline–week 4, week 4–week 8, and week 8–week 20.

[0163] A typical diet, including daily fiber intake: Three unannounced 24-hour dietary recall interviews were conducted at baseline, and at weeks 4 and 8 to allow for randomization of patients within strata of usual fiber intake (high / low) and to determine whether differences in usual diet are associated with differences in response to 2'FL. Dietary recalls were conducted by expert interviewers using the USDA Automated Multipass Method (AMPM) to ensure accurate and consistent recording of foods and quantities reported by participants (Moshfegh et al., Am J Clin Nutr, 88:324-32, 2008). Nutrition Data Systems for Research (NDSR) (Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN) software and food databases were used to assess daily total energy, macronutrient, and fiber intake, as well as food group items consumed (Sievert et al., Control Clin Trials, 10:416-25, 1989). Patients are encouraged to maintain a stable diet over the course of the study.

[0164] Plasma cytokines and fecal calprotectin: Plasma cytokines and fecal calprotectin are measured to assess systemic and mucosal inflammation, respectively. Thirteen plasma cytokines representing innate and adaptive immune responses are measured using a highly sensitive bead-based multiplex assay. Fecal calprotectin is measured using a monoclonal antibody-based ELISA that showed excellent linearity over a wider dynamic range than other available assay kits.

[0165] Statistical Considerations The objective 1 study will determine whether administration of 2'-FL is safe and well tolerated in CD and UC patients in stable remission receiving maintenance anti-TNF therapy. The objective 2 study will focus on indicators of 2'-FL's efficacy in shifting the microbial community toward greater Bifidobacterium abundance and butyrate production, and reducing systemic and mucosal inflammation as measured by plasma cytokines and fecal calprotectin, respectively. This will include metagenomics, metatranscriptomics, and metabolomics of fecal microbes. These will be examined at weeks 4, 8, and 20. The same methodology will be used as in the current PROTECT, RISK, and HMP2 studies (Integrative HMP RNC., Cell Host Microbe, 16:276-89, 2014).

[0166] Objective 1: To define the dose-dependent safety and tolerability of 2'-FL as a dietary supplement in IBD. 2'-FL is expected to be safe and well-tolerated as a dietary supplement in patients with IBD in remission.

[0167] Primary Objective 1 Endpoint: Change in GSRS symptom scores and tolerability of 2'-FL or glucose supplementation within each treatment group at weeks 4-8.

[0168] Secondary objective 1 endpoint(s): Clinical relapse using wPCDAI for CD patients and PUCAI, plasma cytokines, and fecal calprotectin for UC patients.

[0169] Aim 2: Define the dose-dependent efficacy of 2'-FL as a dietary supplement for IBD. 2'-FL is expected to increase fecal Bifidobacterium abundance and butyrate in a dose-dependent manner.

[0170] Primary Objective 2 Endpoint: Change in fecal Bifidobacterium abundance with 2'-FL or glucose supplementation within each treatment group from weeks 4 to 8.

[0171] Secondary Objective 2 Endpoints: Changes in fecal SCFAs including butyrate, pro-inflammatory taxa including Enterobacteriaceae, plasma cytokines, and fecal calprotectin with 2'-FL or glucose supplementation within each treatment group at weeks 4–8.

[0172] Sample size determination As described in Objective 2, the sample size of 20 participants per 2'-FL dose group in CD or UC is based on the primary efficacy endpoint, an increase in fecal Bifidobacterium. The primary endpoint for Objective 1 is the mean change in GSRS tolerability score in each of the 2'-FL dose groups and the glucose placebo group. In a recent 2'-FL RCT in healthy adults, 10 participants per dose group were sufficient to demonstrate an increase in mild GI symptoms in the 20 g 2'-FL group compared with the 2 g glucose placebo group. This included an increase in mean (SD) daily frequency of bowel movements from 1.3 (0.3) to 1.6 (0.4) in the 20 g 2'-FL group. Assuming there are 20 CD or UC participants per 2'-FL treatment group, a baseline total GSRS score of 2.5, and no change with placebo, with a=0.05 (2-sided) and power=0.80, a difference between treatment groups for linear change in total GSRS score as small as 0.2 units with 1 g 2'-FL, 0.7 units with 5 g 2'-FL, and 1.25 units with 10 g 2'-FL should be detected. In addition, if the mean difference / standard deviation (i.e., standardized effect size) does not exceed 1.25, statistical power is sufficient to allow detection of a 2-fold increase in total GSRS score after the first 10 subjects are randomized to placebo and 1 g 2'-FL. For example, statistical power should be sufficient to detect a difference of 2.5 in total GSRS score between placebo and 1 g 2'-FL if the pooled standard deviation does not exceed SD=2. Given the larger numbers randomized to placebo, greater power will be realized in stage 2. As a 20% discontinuation or non-evaluable rate is assumed, 100 CD and 100 UC participants will be enrolled.

[0173] Examination of shotgun metagenomic sequencing data from pediatric and young adult CD patients receiving infliximab and with fecal calprotectin levels <250 μg / g indicates that baseline variation in Bifidobacterium abundance in this study could reach SD=10.6% (Lewis et al., Cell Host Microbe, 18:489-500, 2015). Sample size determination was performed based on the following assumptions: baseline mean Bifidobacterium abundance of 6.8%, expected linear dose response in mean Bifidobacterium abundance of 6.8% for placebo, 7.6% for 1 g 2-FL, 10.7% for 5 g 2-FL, and 14.6% for 10 g 2-FL, and pooled standard deviation of Bifidobacterium abundance of 10.6%, as well as equal allocation to treatment arms. Thus, with a = 0.05 (2-sided) and power = 0.80, a total of 20 patients in each group would allow detection of a mean difference in response to the intervention at levels corresponding to those reported for adults. Greater power would be achieved if the variation in Bifidobacterium abundance was closer to that observed in healthy adults.

[0174] Examination of relevant biological variables Biological variables that may affect the safety and efficacy of 2'-FL include age, sex, race / ethnicity, FUT2 secretor status, IBD diagnosis of CD or UC, dietary fiber intake, mucosal inflammation as measured by fecal calprotectin, and baseline microbial community (Lewis et al., Cell Host Microbe, 18:489-500, 2015; Currier et al., Clin Infect Dis, 60:1631-8, 2015; Tong et al., ISME J, 8:2193-206, 2014; Wacklin et al., PLoS One, 6:e20113, 2011). Of these, IBD diagnosis of CD or UC and dietary fiber intake may have the greatest effect. Therefore, we will enroll enough participants with CD or UC in each dosing group to assess these independently, and enrollment will be balanced across the four groups for usual dietary fiber intake. Equal numbers of men and women aged 11 years and older, and Caucasian (90%) and African American (10%) subjects proportional to the overall IBD population will be enrolled.

[0175] Analysis population The primary analysis was per protocol and included only patients who received at least 24 of the 30 randomized doses of 2'-FL (Elison et al., Br J Nutr, 116:1356-1368, 2016). The secondary analysis was based on an intention-to-treat (ITT) scheme, with each patient included in the group to which they were randomized.

[0176] statistical analysis General approach Descriptive statistics and graphical analyses will be used to describe GSRS tolerability scores, clinical relapse rates, Disease Activity Index scores, plasma cytokines, fecal calprotectin, and QoL across the four groups at each time point.

[0177] Analysis of primary endpoints The primary safety outcome utilizes descriptive statistics, and graphical analysis is used to describe clinical relapse rates across the four groups at each time point, disease activity index scores, plasma cytokines, fecal calprotectin, and tolerability scores for abdominal pain, nausea, loose stools, and gas. The primary determination of tolerability is based on the Gastrointestinal Symptom Rating Scale (GSRS), the same measure of tolerability utilized in a recent 2'-FL RCT in healthy adults (Elison et al., Br J Nutr, 116:1356-1368, 2016). Clinical relapse rates and changes in GSRS within the glucose placebo group at weeks 4-8 are utilized to evaluate the safety and tolerability of each dose of 2'-FL. If a 2-fold increase in GSRS is observed in the 2'-FL-treated group in CD or UC compared to the glucose placebo group, it is concluded that the dose was not well tolerated. If two additional subjects in the 2'-FL-treated group with CD or UC experience clinical relapse beyond the clinical relapse rate observed in the glucose placebo group, it would be concluded that the dose is not safe.

[0178] The primary efficacy outcome will be the difference between dose groups in the mean change in fecal Bifidobacterium abundance before and after supplementation. Differences in Bifidobacterium abundance will be examined using linear mixed-effects regression with a time-by-treatment interaction term providing tests for mean change as described in Objective 1. Post-hoc tests for differences between specific dose groups will be compared using linear contrasts focused on identifying linear trends with increasing 2-FL dose. Tests will be conducted separately for CD and UC patients. Classification of high-dimensional data and statistical learning approaches will be used to identify differences in microbiome structure in response to 2'-FL supplementation.

[0179] Analysis of secondary endpoints Secondary safety and tolerability outcomes examine differences between dose groups in mean changes in disease activity index scores, plasma cytokines, fecal calprotectin, tolerability scores, and QoL before and after supplementation. Differences are examined using linear mixed effects regression (LMER) with a time-by-treatment interaction term providing tests for mean changes. Post-hoc tests for differences between specific dose groups are compared using linear contrasts focused on identifying whether there is an impact on safety and tolerability at higher dose levels. To assess differences in response to treatment by disease phenotype, tests are conducted separately for CD and UC patients. If substantial differences are observed, formal tests of interactions are conducted. Safety and tolerability measures collected at week 12 are incorporated into an LMER framework to examine stability of symptoms at follow-up. The LMER framework is used to test for differences in weekly rates of change in tolerability from the GSRS by nesting observations within subjects and testing for differences in slopes according to dose group. Potential nonlinear associations with time are identified using graphical approaches and model fit statistics and modeled using polynomial terms or restricted cubic splines as appropriate. Fisher's exact test will be used to compare the difference in clinical relapse rates at week 8 between each of the 2'-FL intervention groups. Within-dose comparisons of the number of relapses at weeks 4 to 8 will be performed using exact tests for paired data. Based on a recent 2'-FL RCT in healthy adults, each of the three doses of 2'-FL are expected to be safe and well tolerated compared to glucose placebo in both CD and UC groups. FUT2 secretors and non-secretors are expected to show similar profiles of safety and tolerability for each dose of 2'-FL.

[0180] LMER will also be used to test mean differences in secondary efficacy outcomes including fecal calprotectin, GI symptom tolerability scores, plasma cytokines, and Enterobacteriaceae before and after supplementation. Based on a recent RCT in healthy adults, we expect to detect a two-fold increase in fecal Bifidobacterium abundance at week 8 after supplementation with a 10 g dose of 2'-FL, as well as a significant reduction in taxa within the Proteobacteria phylum, including Enterobactericae. A recent pediatric CD study showed no change in overall mean (SD) fecal Bifidobacterium abundance over 8 weeks of anti-TNF-inducing therapy (baseline: 6.5 (14.8) vs. week 8: 6.8 (10.6)), despite a reduction in mucosal inflammation measured by fecal calprotectin. Thus, we expect no change in fecal Bifidobacterium abundance with glucose supplementation indicating specificity of the 2'-FL response, and less change with doses of 1 g and 5 g 2'-FL indicating dose-dependent response. Consistent with an increase in fecal SCFA-producing microorganisms, SFCA analysis is expected to detect a significant increase in fecal butyrate concentrations with 2'-FL supplementation. Because 2'-FL supplementation of infant formula has been shown to reduce circulating plasma cytokines in healthy newborns, 2'-FL supplementation is expected to also reduce both plasma cytokines and fecal calprotectin in a dose-dependent manner, with no change with glucose supplementation. FUT2 non-secretors are expected to show a trend toward greater benefit from 2'-FL supplementation.

[0181] Safety analysis Based on data from IBD patient population management reports collected for the ImproveCareNow (ICN) Quality Improvement Collaborative, it is expected that 10% or less of patients on stable maintenance anti-TNF therapy will experience a clinical relapse over a 4-week period (Minar et al., Inflamm Bowel Dis, 22:2641-2647, 2016; Minar et al., J Pediatr Gastroenterol Nutr, 62:715-22, 2016). The number of subjects experiencing a clinical relapse is determined for each of the three doses of 2-FL and the glucose placebo. Within the glucose placebo group for CD or UC, the safety and tolerability of each dose of 2-FL is assessed utilizing clinical relapse rates and changes in GI symptom tolerability scores collected using the GSRS questionnaire. If two additional subjects experience a clinical relapse in the CD or UC dose group beyond the clinical relapse rate observed in the placebo group, it is concluded that the dose is not safe. Similarly, if a significant 2-fold increase in GI symptom tolerability scores collected using the GSRS questionnaire is observed compared with the placebo group, it can be concluded that the dose was not well tolerated in the experience of the CD or UC treatment group.

[0182] Baseline descriptive statistics Descriptive statistics and graphical analyses will be used to describe clinical and demographic characteristics across the four groups at study entry, FUT2 secretor status and dietary fiber intake, baseline GSRS tolerability scores, plasma cytokines, fecal calprotectin, fecal microbial community and function, and Qol.

[0183] Mid-term planning analysis Interim analyses will assess the safety and tolerability of each 2'-FL dose before randomizing participants to the next highest dose. Patients will be randomized to placebo or treatment arms within strata using a stepwise approach, with randomization in phase 1 in a 1:1 ratio to placebo or 1g, in phase 2 in a 1:1:2 ratio to placebo 1g, 5g, and in phase 3 in a 1:1:2:4 ratio to placebo 1g, 5g, 10g. A total of 20 CD and 20 UC participants will be randomized to each dose arm. Sequential stepping with shifting allocation ratios will allow for the evaluation of safety and tolerability of the lowest dose arm before randomization begins at the next highest dose. If a dose / disease phenotype arm experiences sufficient safety or intolerance issues, allocation to that arm and all higher dose arms will be discontinued. Natural block sizes of 2, 4, and 8 will be used to randomize patients to dose arms within strata in phases 1, 2, and 3, respectively, to select the optimal randomization at each stage to ensure counterbalance. An independent statistician generates the randomization and provides a computer-generated list to the pharmacy for dispensing. The advantage of this approach is that it allows for an assessment of safety before moving to higher doses and for balancing across factors that may affect response to treatment. Given the short duration of the study, there is no potential for bias in randomizing patients to higher dose groups at later time points. The inclusion of patients randomized to placebo and lower dose groups at each stage allows for testing and consideration of any observed cohort / time effects.

[0184] subgroup analysis The analysis for primary objective 1 will be per protocol and will include only patients who received at least 24 of the 28 randomized 2'-FL doses (Elison et al., Br J Nutr, 116:1356-1368, 2016). Secondary analyses will be based on an intention-to-treat (ITT) schema, with each patient included in the group to which they were randomized. Descriptive statistics and graphical analyses will be used to describe GSRS tolerability scores, clinical relapse rates, disease activity index scores, plasma cytokines, fecal calprotectin, and Qol across the four groups at each time point. Safety and tolerability outcomes will examine differences in mean changes between treatment groups in GSRS, disease activity index scores, plasma cytokines, fecal calprotectin, and Qol before and after supplementation. Clinical relapse is defined as an increase of 20 points or more in the wPCDAI and 15 points or more in the PUCAI at weeks 4-8 (Haberman et al., J Clin Invest, 124:3617-33, 2014; Holscher et al., J Nutr;2015, 145:2025-32; Schirmer et al., Cell, 167:1125-1136 e8, 2016; Wishart et al., Nucleic Acids Res, 41:D801-7, 2013). Primary patient-reported outcome (PRO) components of pain and stool for CD, and stool and blood for UC are also examined separately. The primary measure of tolerability is the mean change between treatment arms on the GSRS. Differences are examined using linear mixed-effects regression (LMER) with a time-by-treatment interaction term providing a test for mean change. For primary analyses testing changes at weeks 4-8 (i.e., pre-post-test interventions), Kenward-Roger corrections are used to obtain the correct degrees of freedom for F-tests and the specified unstructured correlation structure. Post-hoc tests for differences between specific dose groups are compared using linear contrasts focused on identifying whether there is an impact on safety and tolerability at higher dose levels. To assess differences in response to treatment by disease phenotype, tests are performed separately for CD and UC patients. If substantial differences are observed, formal tests of interaction are performed.Safety and tolerability measures collected at week 12 are incorporated into an LMER framework to examine stability of symptoms at follow-up. Using the LMER framework, differences in weekly rate of change in GSRS tolerability scores are tested by nesting observations within subjects and testing for differences in slopes according to dose group. Potential nonlinear associations with time are identified using graphical approaches and model fit statistics and modeled using polynomial terms or restricted cubic splines as appropriate. Fisher's exact test is used to compare differences in clinical relapse rates at week 8 between each of the 2'-FL intervention groups. Within-dose comparisons of the number of relapses at weeks 4 to 8 are performed using exact tests for paired data. Based on a recent 2'-FL RCT in healthy adults, each of the three doses of 2'-FL are expected to be safe and well tolerated compared to glucose placebo in both CD and UC groups. This is determined by showing no differences in mean change in GSRS tolerability scores, or clinical relapse rates, between each of the 2'-FL treatment and glucose placebo groups.

[0185] Analyses for primary objective 2 will be per protocol and will include only patients who completed all study procedures, including supplementation with at least 24 of the 28 randomized doses. Secondary analyses will be based on an intention-to-treat (ITT) schema, with each patient included in the group to which they were randomized. Descriptive statistics will be used to show differences in taxonomic and functional profiles of the microbiota, fecal SCFAs, plasma cytokines, as well as fecal calprotectin, between the four groups. The primary efficacy outcome will be the difference in mean change between dose groups in fecal Bifidobacterium abundance before and after supplementation. Differences in Bifidobacterium abundance will be examined using linear mixed-effects regression (LMER) with a time-by-treatment interaction term providing a test for mean change as described in objective 1. Post-hoc tests for differences between specific dose groups will be compared using linear contrasts focused on identifying linear trends of increasing 2-FL dose. In addition to comparing the relative abundance of Bifidobacterium between the different dose groups, differences in intrapatient variance of Bifidobacterium will also be assessed using the Fligner-Killeen test of homogeneity of variance. These relative changes are then compared to changes in absolute abundance measurements by qPCR. Testing is performed separately for CD and UC patients. Furthermore, principal coordinate analysis with Bray-Curtis distance is used to investigate differences in the overall community composition. Following this, specific taxonomic and functional microbial features associated with the response to 2'-FL supplementation are identified. Towards this end, a linear modeling system fitted to the microbial community data is used, controlling for major factors influencing the microbiome such as age, ethnicity, and gender, and considering multiple samples from the same patient (MaAslin: huttenhower.sph.harvard.edu / maaslin). Furthermore, strain-level differences between patient groups are examined by comparing SNP profiles of species with sufficient coverage and their functional implications. LMER is also used to test mean differences in secondary efficacy outcomes including fecal calprotectin, GSRS tolerability scores, plasma cytokines, SCFAs, and Enterobacteriaceae before and after supplementation.Based on a recent RCT in healthy adults, we expect to detect a two-fold increase in fecal Bifidobacterium abundance at 8 weeks after supplementation with a 10 g dose of 2'-FL, as well as a significant reduction in taxa within the Proteobacteria phylum, including Enterobactericae. A recent pediatric CD study showed no change in fecal Bifidobacterium abundance over 8 weeks of anti-TNF-inducing therapy, despite a reduction in mucosal inflammation measured by fecal calprotectin (Lewis et al., Cell Host Microbe, 18:489-500, 2015). Thus, in contrast to patients with 2'-FL supplementation showing specificity of the 2'-FL response, patients with glucose supplementation will experience a smaller degree of variation in fecal Bifidobacterium abundance, with less change at 1 g and 5 g doses of 2'-FL. Metabolite analysis is expected to detect a significant increase in fecal butyrate concentration with 10 g of 2'-FL supplementation. 2'-FL supplementation of infant formula was recently shown to reduce circulating plasma cytokines in healthy newborns, so it is expected that 2'-FL supplementation will also reduce both plasma cytokines and fecal calprotectin in a dose-dependent manner, with no change with glucose supplementation (Goehring et al., J Nutr, 146:2559-2566, 2016). This will be followed by hierarchical all-against-all significance testing (HAllA: huttenhower.sph.harvard.edu / halla) to identify groups of bacterial species and functions associated with changes in SCFAs, fecal calprotectin, and plasma cytokines. FUT2 non-secretors are expected to show a trend toward greater benefit from 2'-FL supplementation.

[0186] Exploratory analysis The effects of age, sex, race, fecal calprotectin and microbiota at week 4, usual fiber intake, and FUT2 secretor status will be tested in an exploratory fashion to guide the design of a multicenter RCT. Descriptive statistics will be calculated and models will be fitted separately for each of these subgroups, allowing for formal tests of interaction if important differences are observed.

[0187] Other embodiments All of the features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced with an alternative feature serving the same, equivalent, or similar purpose. Thus, unless otherwise stated, each feature disclosed is merely an example of a generic series of equivalent or similar features.

[0188] From the above description, those skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt it to various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the claims.

[0189] Equivalent Although several inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application(s) for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Thus, the foregoing embodiments have been presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0190] All definitions and those used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0191] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, in some cases including the entire document.

[0192] As used herein in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless clearly indicated otherwise.

[0193] The term "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising," a reference to "A and / or B" may refer in one embodiment to only A (optionally including elements other than B), in another embodiment to only B (optionally including elements other than A), and in yet another embodiment to both A and B (optionally including other elements).

[0194] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, i.e., including at least one, but not more than one, of a number of elements or a list of elements, and optionally including additional unlisted items. Only terms clearly indicated otherwise, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," refer to the inclusion of exactly one element of a number of elements or a list of elements. In general, the term "or" as used herein should only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both) when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0195] As used herein in the specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. Also, this definition allows for the optional presence of elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") may refer in one embodiment to at least one, optionally including more than one A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally including more than one B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements);

[0196] It should also be understood that, unless expressly stated otherwise, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

Claims

[Claim 1] A method for reducing or alleviating the risk of recurrence of inflammatory bowel disease (IBD), comprising administering to a subject in need thereof an effective amount of a 2'-fucosyllactose compound, wherein the subject is a human IBD patient who has received or is receiving anti-inflammatory therapy.

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