Point-of-care test diagnostic markers and adjuvant treatments for gastrointestinal diseases

Abstract

Described herein is a method of diagnosing and / or treating irritable bowel syndrome (IBS) in a subject. The method comprises determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a first reference profile indicating an absence of IBS or a second reference profile indicating IBS. Also described herein is a composition for treating IBS, which comprises two or more of a Monoglobaceae bacterium, a Lachnospiraceae bacterium; and a Ruminococcaceae bacterium, as well as a method of treating IBS using the same.

Description

[0001] POINT-OF-CARE TEST DIAGNOSTIC MARKERS AND ADJUVANT TREATMENTS FOR GASTROINTESTINAL DISEASES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority under 35 U. S. C. § 119(e) to U.S. Provisional Application No. 63 / 542,468, filed October 4, 2023, and U. S. Provisional Application No. 63 / 542,557, filed October 5, 2023, both of which are herein incorporated by reference in their entireties.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The present application contains a Sequence Listing in XML. The XML text file named “37043 l-1052WOl_Seq Listing. xml” created on July 10, 2024 comprising 2,910 bytes, is hereby incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Diseases and disorders of the gastrointestinal (GI) tract affect over 60 million people in the United States and over 2 billion people worldwide, many of which do not have reliable diagnostic methods. Irritable bowel syndrome (IBS) affects 10-15% of the world’s population, and it can significantly impact the quality of life of patient. In addition, this disease has a significant impact on the economics of health care with over $30 billion in health care costs per year due to physician visits for IBS.

[0008] Currently, there are no reliable diagnostic methods for IBS. This is in part due to the complex etiology, multifactorial pathophysiology, and heterogeneity of clinical manifestations of IBS, which results in significant diagnostic testing that must be done to rule out other conditions. In addition, effective treatment of IBS is limited, as well.

[0009] Accordingly, there is a need for methods of diagnosing IBS, as well as treatment thereof. The present invention addresses this need.

[0010] SUMMARY

[0011] In some aspects, the present invention is directed to the following non-limiting embodiments: Method of Diagnosing IBS

[0012] In some aspects, the present invention is directed to a method of diagnosing irritable bowel syndrome (IBS) in a subject.

[0013] In some embodiments, the method comprises: determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a first reference profile indicating an absence of IBS or a second reference profile indicating IBS.

[0014] In some embodiments, the subject is diagnosed to have IBS when a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile.

[0015] In some embodiments, the subject is diagnosed to have IBS when a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile.

[0016] In some embodiments, the method further comprises obtaining the stool sample from the subject.

[0017] In some embodiments, the subject is diagnosed with IBS when a level of one or more saturated fatty acids in the fatty acid profile is higher than that in the first reference profile.

[0018] In some embodiments, the subject is diagnosed with IBS when a level of one of more polyunsaturated fatty acids in the fatty acid profile is lower than that in the first reference profile.

[0019] In some embodiments, a level of one or more saturated fatty acids in the second profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when a level of one or more saturated fatty acids in the fatty acid profile is higher than or equal to that in the second reference profile.

[0020] In some embodiments, a level of one of more polyunsaturated fatty acids in the second profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of one or more polyunsaturated fatty acids in the fatty acid profile is equal to or lower than that in the second reference profile.

[0021] In some embodiments, the at least one fatty acid comprises palmitic acid (16:0), margaric acid (17:0), linoleic acid (18:2n6), gamma-linoleic acid (18:3n6), and / or alphalinolenic acid (18:3n3). In some embodiments, the subject is diagnosed with IBS when a level of palmitic acid (16:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0022] In some embodiments, the subject is diagnosed with IBS when a level of margaric acid (17:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0023] In some embodiments, the subject is diagnosed with IBS when a level of linoleic acid (18:2n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

[0024] In some embodiments, the subject is diagnosed with IBS when a level of gammalinoleic acid (18:3n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

[0025] In some embodiments, the subject is diagnosed with IBS when a level of alphalinolenic acid (18:3n3) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

[0026] In some embodiments, a level of palmitic acid (16:0) in the second reference profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of palmitic acid (16:0) in the fatty acid profile is equal to or higher than that of the second reference profile.

[0027] In some embodiments, a level of margaric acid (17:0) in the second reference profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of margaric acid (17:0) in the fatty acid profile is equal to or higher than that of the second reference profile.

[0028] In some embodiments, a level of linoleic acid (18:2n6) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of linoleic acid (18:2n6) in the fatty acid profile is equal to or lower than that of the second reference profile.

[0029] In some embodiments, a level of gamma-linoleic acid (18:3n6) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of gamma-linoleic acid (18:3n6) in the fatty acid profile is equal to or lower than that of the second reference profile.

[0030] In some embodiments, a level of alpha-linolenic acid (18:3n3) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of alpha-linolenic acid (18:3n3) in the fatty acid profile is equal to or lower than that of the second reference profile.

[0031] In some embodiments, the second reference profile comprises an IBS-D profile indicating irritable bowel syndrome-diarrhea (IBS-D), an IBS-M profile indicating irritable bowel syndrome-mixed (IBS-M), and / or an IBS-C profile indicating irritable bowel syndrome-constipation (IBS-C).

[0032] In some embodiments, determining a fatty acid profile in the stool sample comprises analyzing the stool sample with a chromatography method, a mass spectrometry method, a fluorometric method, or a colorimetric method.

[0033] Method of Treating IBS

[0034] In some embodiments, the present invention is directed to a method of treating IBS in a subject in need thereof.

[0035] In some embodiments, the method comprises: confirming that the subject suffers from IBS, and administering to the subject a treatment for IBS.

[0036] In some embodiments, the subject is diagnosed with IBS with the diagnostic method herein.

[0037] In some embodiments, the treatment for IBS comprises an anti diarrhoeal, a gut antispasmodic, a laxative, a nerve pain medication, an antibiotic, a dietary supplement, a prebiotic, and / or a probiotic.

[0038] In some embodiments, the treatment for IBS comprises alosetron, amitriptyline, atropine, bisacodyl, bismuth subsalicylate, dicyclomine, diphenoxylate, doxepin, eluxadoline, hyoscyamine, a Lactobacillus (optionally Lactobacillus acidophilus), lactulose, linaclotide, loperamide, lotronex, lubiprostone, polyethylene glycol 3350, psyllium seed husk, and / or rifaximin.

[0039] Composition

[0040] In some aspects, the present invention is directed to a composition.

[0041] In some embodiments, the composition comprises at least one selected from the group consisting of a Monoglobaceae bacterium, a Lachnospiraceae bacterium, and a Ruminococcaceae bacterium; and a pharmaceutically acceptable carrier.

[0042] In some embodiments, the composition comprises two or more of the group consisting of the Monoglobaceae bacterium, the Lachnospiraceae bacterium, and the Ruminococcaceae bacterium. Method of Treating IBS with Probiotic Composition

[0043] In some aspects, the present invention is directed to a method of treating IBS in a subject in need thereof.

[0044] In some embodiments, the method comprises administering to the subject an effective amount of the composition herein.

[0045] In some embodiments, the method further comprises administering to the subject an effective amount of an antidiarrhoeal, a gut antispasmodic, a laxative, a nerve pain medication, an antibiotic, a dietary supplement, a prebiotic, and / or a probiotic.

[0046] In some embodiments, the method further comprises administering to the subject an effective amount of alosetron, amitriptyline, atropine, bisacodyl, bismuth subsalicylate, dicyclomine, diphenoxylate, doxepin, eluxadoline, hyoscyamine, a Lactobacillus (optionally Lactobacillus acidophilus), lactulose, linaclotide, loperamide, lotronex, lubiprostone, polyethylene glycol 3350, psyllium seed husk, and / or rifaximin.

[0047] BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The following detailed description of exemplary embodiments will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating, non-limiting embodiments are shown in the drawings. It should be understood, however, that the present invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0049] Figs. 1A-1B: Microbiome beta diversity of healthy controls versus IBS and IBS subtypes, in accordance with some embodiments. Fig. 1 A: Un-weighted UniFrac PCoA plots are shown comparing healthy control participants (green, n = 12) and IBS patient samples (purple, n = 33) and between (Fig. IB) healthy control subjects (green, n = 12) and IBS subtypes: IBS-D (red, n = 10), IBS-M (purple, n = 13), and IBS-C (blue, n = 10). Percentages along each axis show the portion of phylogenetic variance across samples captured by that axis. Each point represents the phylogenetic composition of one sample. Ellipses represent 95% confidence intervals of the group’s PCoA coordinates. Abbreviations: IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome-diarrhea; IBS-M, irritable bowel syndrome, mixed; IBS-C, irritable bowel syn-drome-constipation, principal coordinates analysis axis (PC), principal coordinates analysis (PCoA). Figs. 2A-2B: Taxa bar plot of healthy controls versus IBS and IBS subtypes, in accordance with some embodiments. Taxa bar plot shows relative abundances of phyla in each sample. Each vertical bar represents one sample, and the value on the y-axis represents the relative abundance of each taxon (separated by color) in that sample. Names of taxa corresponding to taxa are shown to the right of the plot, with bold headers indicating the phylum-level and non-bold text indicating the genus-level taxonomic assignment. Abbreviations: IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome-diarrhea; IBS-M, irritable bowel syndrome, mixed; IBS-C, irritable bowel syndrome-constipation.

[0050] Figs. 3 A-3D: Differentially abundant taxa in IBS and IBS subtypes versus healthy controls, in accordance with some embodiments. Taxonomic differences in the microbiomes of healthy control participants and IBS patients were analyzed via ANCOM analysis (Figs. 3A-3B) and module network analysis (Figs. 3C-3D). ANCOM analyses of healthy control subjects versus IBS patients (Fig. 3A) and of healthy control subjects versus IBS subtypes (IBS-D, IBS-M, and IBS-C) (Fig. 3B) are shown. The summed relative abundances of modules (module 9; Fig. 3C and module 11; Fig. 3D) identified from co-occurrence networks within the dataset using SCNIC are shown in the lower panel. Abbreviations: ANCOM, analysis of compositions of microbiomes; IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome-diarrhea; IBS-M, irritable bowel syndrome, mixed; IBS-C, irritable bowel syndrome-constipation; SCNIC, sparse correlation network investigation in compositional data (SCNIC).

[0051] Figs. 4A-4B: Linear discriminant analysis effect size (LEfSe) scores for taxa enriched in healthy control participants or IBS patients (Fig. 4A) and IBS-C or IBS-D subtypes (Fig. 4B), in accordance with some embodiments. Negative values in Fig. 4A represent taxa that were enriched in healthy control participants, whereas positive values represent taxa that were enriched in IBS patients. Negative values in Fig. 4B represent taxa that were enriched in IBS-D patients, whereas positive values represent taxa that were enriched in IBS-C patients. Color rep-resents the phylum. Abbreviations: IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome-diarrhea; IBS-M, irritable bowel syndrome, mixed; IBS-C, irritable bowel syndrome-constipation; LEfSe, linear discriminant analysis effect size.

[0052] Figs. 5A-5D: HFCS consumption (g / per month) of healthy control participants and IBS subtypes (Fig. 5A) and phylogenetic isometric log-ratio transformation (PhILR) analysis of taxa associated with HFCS consumption (Figs. 5B-5D), in accordance with some embodiments. Fig. 5A: Boxplots show estimated HFCS consumption, separated into IBS subtypes. Horizontal lines represent the median. Boxes indicate the 1st through 3rd quartiles of monthly HFCS consumption in each IBS subtype, and whiskers represent the range up to 1.5x the interquartile range. Diamonds represent monthly consumption for an individual outside of 1.5 x the interquartile range. Fig. 5B: A phylogenetic tree constructed using 16S amplicon sequence variants is highlighted to show PhILR balances associated with HFCS consumption. Balances were constructed at each branching point in the phylogenetic tree, using the isometric log-ratio of one subtree to the other. As such, the numerator and denominator of each balance are signified using “num” and “denom”. Balances shown were associated with HFCS consumption, per LASSO regression. Figs. 5C-5D:: The relationship between phylogenetic balances associated with HFCS consumption are shown using a scatter plot, where each point depicts that balance’s value on the y-axis as a function of HFCS consumption on the x-axis, and each point represents one participant’s sample, and samples from all participants were included. Abbreviations: IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome-diarrhea; IBS-M, irritable bowel syndrome, mixed; IBS-C, irritable bowel syndrome-constipation; LASSO, least absolute squares shrinkage and selection operator; PhILR, phylogenetic isometric log-ratio transformation.

[0053] Figs. 6A-6B: Stacked lipids bar plot of healthy controls versus IBS and IBS subtypes, in accordance with some embodiments. Each bar represents one sample, and the total height of the bar represents the total concentration of all detected lipids. Colors representing each group (unsaturation position) of lipids are indicated as saturated (purple), omega 9 (green), omega 6 (blue), omega 3 (orange), and other (gray). Abbreviations: IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome-diarrhea; IBS-M, irritable bowel syndrome, mixed; IBS-C, irritable bowel syndrome-constipation.

[0054] Figs. 7A-7D: Lipidome analyses of healthy controls versus IBS and IBS subtypes, in accordance with some embodiments. Compositional changes in lipids that were significantly different (per Kruskal-Wallis test) across healthy control participants and various IBS subtypes are presented via a spider plot (Fig. 7A). Each lipid’s concentrations were Z-score transformed, and the mean Z-score for each IBS subtype is plotted on each axis. Lipids that were significantly different (per Mann-Whitney U test) between healthy control participants and all IBS patients are presented in a volcano plot (Fig. 7B). Each point represents one lipid, the x-axis represents log-fold change from healthy controls to participants with IBS, and the y-axis represents the negative log of the p value. Points above the horizontal dashed line had a p < 0.05, and points outside of the vertical dashed lines had greater than a 2-fold change. Concentrations of significantly differing lipids, separated by IBS status and IBS subtype, respectively, are presented as boxplots with overlaid scatterplots in (Figs. 7C-7D), respectively. Individual points represent the concentration of the lipid in one sample. Horizontal lines represent the median. Boxes indicate the 1st through 3rd quartiles of the lipid’s concentration in its IBS / subtype group, and whiskers represent the range up to 1.5x the interquartile range. Abbreviations: IBS, irritable bowel syndrome; IBS-D, irritable bowel syndrome-diarrhea; IBS-M, irritable bowel syndrome, mixed; IBS-C, irritable bowel syndrome-constipation.

[0055] Figs. 8A-8B: Procrustes-transformed Bray-Curtis multi-dimensional scaling of microbiome and lipidome, in accordance with some embodiments. Fig. 8A: Blue triangles indicate untransformed Bray-Curtis PCoA coordinates for the micro-biome data, and orange circles indicate Procrustes-transformed Bray-Curtis PCoA coordinates of the lipidome. Each point indicates one participant’s sample, and lines are drawn between each participant’s microbiome and lipidome. Fig. 8B: Histogram shows distribution of Procrustes disparity scores across 10,000 permutations of the dataset, and the vertical red line indicates the Procrustes disparity of the dataset. Abbreviations: PC, principal coordinates analysis axis; PCoA, principal coordinates analysis.

[0056] Figs. 9A-9D: Faith’s phylogenetic alpha diversity of the microbiome as a function of socioeconomic factors, in accordance with some embodiments. Figs. 9A and 9C show the relationship between phylogenetic diversity and (Fig. 9A) alcohol consumption or (Fig. 9C) the number of household occupants. Each point represents one participant’s microbiome sample, the line represents the line of best fit, and the shaded region indicates the 95% confidence interval of a linear regression on Faith’s phylogenetic diversity as a function of alcohol consumption or the number of household occupants, respectively. Boxplots with overlaid scatter-plots in Figs. 9B and 9D depict the relationship between phylogenetic diversity and (Fig. 9B) household in-come group or (Fig. 9D) marital status. Individual points represent the phylogenetic diversity of one participant’s sample. Horizontal lines represent the median. Boxes indicate the 1st through 3rd quartiles of the phylogenetic diversity of each group, and whiskers represent the range up to 1.5x the interquartile range.

[0057] Figs. 10A-10B: Microbiome beta diversity as a function of socioeconomic factors, in accordance with some embodiments. Fig. 10A: Unweighted UniFrac PCoA plots are shown comparing the phylogenetic composition of samples across household income groups and (Fig. 10B) marital status. Percentages along each axis show the portion of phylogenetic variance across samples captured by that axis. Each point represents the phylogenetic composition of one sample. Ellipses represent 95% confidence intervals of each group’s PCoA co-ordinates. Abbreviations: PC, principal coordinates analysis axis; PCoA, principal coordinates analysis.

[0058] DETAILED DESCRIPTION

[0059] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0060] The pathophysiology of these diseases often involves dysregulation of the bacteria in the GI tract, called the gut microbiome, as well as dysfunction of the neurological and immunological components of the GI tract. As a result, the onset of disease causes a shift in the gut microbiome and its metabolites that is specific to the disease.

[0061] IBS is one of several gastrointestinal diseases which has been shown to be impacted by the composition or dysregulation of the gut microbiome (the collection of microorganisms which colonize the gastrointestinal tract). Some bacterial species have been identified to play a role in this dysregulation, while other species have been shown to exert a protective effect. Current treatment modalities for IBS include lifestyle and dietary modifications, pharmacotherapy for symptomatic management, and probiotics (treatments of beneficial bacteria).

[0062] In the examples described herein, the relationships between the gut microbiome / metabolites and the disease status of irritable bowel syndrome (IBS) (e.g., presence and absence, as well as types of IBS) were investigated. It was discovered that levels of various fatty acids in stool samples can be used to determine the presence and absence of IBS, as well as the types of IBS (e.g., IBS with constipation / IBS-C, IBS with diarrhea / IBS-D, or mixed type IBS / IBS-M). Accordingly, in some aspects, the present invention is directed to a method of diagnosing and / or treating IBS.

[0063] In other aspects, it was further discovered that the relative abundances of several beneficial bacteria in patients are lower. It was expected that the administration of these bacteria in the form of compositions would be beneficial to IBS patients.

[0064] Accordingly, in some aspects, the present invention is directed to compositions for treating IBS, as well as methods of using the same.

[0065] Definitions

[0066] As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Generally, the nomenclature used herein are those well-known and commonly employed in the art. It should be understood that the order of steps or order for performing certain actions is immaterial, so long as the present teachings remain operable. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0067] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0068] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0069] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0070] A disease or disorder is “alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

[0071] As used herein, the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0072] The term “independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3are independently selected from noble gases” would include the scenario where, for example, X1, X2, and X3are all the same, where X1, X2, and X3are all different, where X1and X2are the same but X3is different, and other analogous permutations.

[0073] The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0074] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, ie., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0075] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.

[0076] Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2- hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, P-hydroxybutyric, salicylic, galactaric and galacturonic acid.

[0077] Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N,N’-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

[0078] As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound(s) described herein. Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0079] The term “room temperature” as used herein refers to a temperature of about 15 °C to 28 °C, such as 20 °C or 25 °C.

[0080] The term “solvent” as used herein refers to a liquid that can dissolve a solid, liquid, or gas. Non-limiting examples of solvents are silicones, organic compounds, water, alcohols, ionic liquids, and supercritical fluids.

[0081] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term “substantially free of’ can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0082] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0083] The terms “treat,” “treating” and “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.

[0084] Diagnostics and Treatments of Irritable Bowel Syndrome (IBS)

[0085] In some aspects, the present invention provides a method of diagnosing IBS.

[0086] In some embodiments, the method comprises determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a first reference profile indicating an absence of IBS. In some embodiments, the fatty acid profile is compared with a second reference profile indicating IBS. In some embodiments, the fatty acid profile is compared with both the first reference profile and the second reference profile. In some embodiments, the subject is determined to have IBS if a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile.

[0087] In some embodiments, the first reference profile includes reference level ranges of one or more fatty acids. In some embodiments, if the level of a fatty acid in the stool sample is outside the reference level range of that fatty acid in the first reference profile, the level of the fatty acid is considered different than the level of the fatty acid in the first reference profile.

[0088] In some embodiments, the subject is determined to have IBS if a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile.

[0089] In some embodiments, the second reference profile includes reference level ranges of one or more fatty acids. In some embodiments, if the level of a fatty acid in the stool sample is within the reference level range of that fatty acid in the second reference profile, the level of the fatty acid is considered matching or equal to the level of the fatty acid in the second reference profile.

[0090] In some embodiments, obtaining the stool sample from the subject is not considered part of the method herein. In some embodiments, obtaining the stool sample is part of the method.

[0091] In some embodiments, the subject is diagnosed with IBS when a level of one or more saturated fatty acids in the fatty acid profile is higher than that in the first reference profile.

[0092] In some embodiments, the subject is diagnosed with IBS when a level of one of more polyunsaturated fatty acids in the fatty acid profile is lower than that in the first reference profile.

[0093] In some embodiments, a level of one or more saturated fatty acids in the second profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when a level of one or more saturated fatty acids in the fatty acid profile is higher than or equal to that in the second reference profile.

[0094] In some embodiments, a level of one of more polyunsaturated fatty acids in the second profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of one or more polyunsaturated fatty acids in the fatty acid profile is equal to or lower than that in the second reference profile. In some embodiments, the first reference profile and / or the second reference profile include reference levels or reference level ranges of palmitic acid (16:0), margaric acid (17:0), linoleic acid (18:2n6), gamma-linoleic acid (18:3n6), and / or alpha-linolenic acid (18:3n3).

[0095] In some embodiments, the subject is diagnosed with IBS when a level of palmitic acid (16:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0096] In some embodiments, the subject is diagnosed with IBS when a level of margaric acid (17:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level.

[0097] In some embodiments, the subject is diagnosed with IBS when a level of linoleic acid (18:2n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level,

[0098] In some embodiments, the subject is diagnosed with IBS when a level of gammalinoleic acid (18:3n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

[0099] In some embodiments, the subject is diagnosed with IBS when a level of alphalinolenic acid (18:3n3) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

[0100] In some embodiments, a level of palmitic acid (16:0) in the second reference profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of palmitic acid (16:0) in the fatty acid profile is equal to or higher than that of the second reference profile.

[0101] In some embodiments, a level of margaric acid (17:0) in the second reference profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of margaric acid (17:0) in the fatty acid profile is equal to or higher than that of the second reference profile.

[0102] In some embodiments, a level of linoleic acid (18:2n6) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of linoleic acid (18:2n6) in the fatty acid profile is equal to or lower than that of the second reference profile.

[0103] In some embodiments, a level of gamma-linoleic acid (18:3n6) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of gamma-linoleic acid (18:3n6) in the fatty acid profile is equal to or lower than that of the second reference profile.

[0104] In some embodiments, a level of alpha-linolenic acid (18:3n3) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of alpha-linolenic acid (18:3n3) in the fatty acid profile is equal to or lower than that of the second reference profile.

[0105] In some embodiments, the second reference profile comprises an IBS-D profile indicating irritable bowel syndrome-diarrhea (IBS-D), an IBS-M profile indicating irritable bowel syndrome-mixed (IBS-M), and or an IBS-C profile indicating irritable bowel syndrome-constipation (IBS-C). In some embodiments, each profile of the plurality of second reference profiles includes one or more reference levels or reference level ranges of fatty acids that indicates a specific type of IBS.

[0106] The methods of determining the fatty acid profile, such as determining the levels of fatty acids in the stool sample are not limited. In some embodiments, the fatty acid profile in the stool sample is analyzed with a chromatography method, a mass spectrometry method, a fluorometric method or a colorimetric method.

[0107] In some aspects, the present invention is directed to a method of treating IBS in a subject in need thereof.

[0108] In some embodiments, the method comprises confirming that the subject suffers from IBS, and administering to the subject a treatment for IBS. In some embodiments, that the subject suffers from IBS is confirmed with a diagnostic method herein.

[0109] In some embodiments, the treatment for IBS comprises an anti diarrhoeal, a gut antispasmodic, a laxative, a nerve pain medication, an antibiotic, a dietary supplement, a prebiotic, and / or a probiotic.

[0110] In some embodiments, the treatment for IBS comprises alosetron, amitriptyline, atropine, bisacodyl, bismuth subsalicylate, dicyclomine, diphenoxylate, doxepin, eluxadoline, hyoscyamine, a Lactobacillus (optionally Lactobacillus acidophilus), lactulose, linaclotide, loperamide, lotronex, lubiprostone, polyethylene glycol 3350, psyllium seed husk, and / or rifaximin.

[0111] In some embodiments, the treatment for IBS comprises administering to the subject a composition herein.

[0112] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Composition Useful for Treating Irritable Bowel Syndrome (IBS) and Methods of Using the Same

[0113] In some aspects, the present invention is directed to a composition.

[0114] In some embodiments, the composition is a composition for treating IBS which, when administered to a subject suffering from IBS alone or in combination with conventional IBS treatments, cures or reduces the severity of IBS in the subject.

[0115] In some embodiments, the composition comprises at least one selected from the group consisting of: a Monoglobaceae bacterium; a Lachnospiraceae bacterium; and a Ruminococcaceae bacterium. In some embodiments, the composition comprises at least two of the bacteria from the group.

[0116] In some embodiments, the present invention provides a probiotic formulation composed of Lachnospiraceae group ND3007 and / or Ruminococcaceae which can be used as an adjuvant treatment for IBS or for general digestive health. Bacteria of the Lachnospiraceae family produce short chain fatty acids (SCFAs), which have been found to be the main nutritional source for colonic epithelial cells. Additionally, SCFAs can decrease local expression of inflammatory markers and promote proliferation of regulatory T cells, which are important for decreasing inflammation and the potential for autoimmunity. Butyrate, an SCFA produced by Lachnospiraceae, has also been shown to promote gut serotonin release, which can alter the sensory activity typically disrupted in IBS. In addition to its effects on inflammation, Lachnospiraceae are protective against neoplastic processes by promoting tumor immune surveillance. An abundance of Ruminococcaceae leads to increased efficacy of immunotherapy as well as a decreased risk of antibiotic-associated diarrhea. The inventors conducted a pilot study which found that patients with IBS had less relative abundance of Lachnospiraceae group ND3007 and Ruminococcaceae. Multiple analytical methods confirmed these results. Given that these bacteria have shown to have anti-inflammatory properties and were less prevalent in patients with IBS, these bacteria represent an adjuvant treatment for IBS, as well as for maintenance of general digestive health in healthy individuals.

[0117] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0118] In some aspects, the present invention is directed to a method of treating IBS in a subject in need thereof. In some embodiments, the method comprising administering to the subject an effective amount of the composition herein.

[0119] In some embodiments, the subject is further administered with an effective amount of an anti diarrhoeal, a gut antispasmodic, a laxative, a nerve pain medication, an antibiotic, a dietary supplement, a prebiotic, and / or a probiotic.

[0120] In some embodiments, the subject is further administered with an effective amount of alosetron, amitriptyline, atropine, bisacodyl, bismuth subsalicylate, dicyclomine, diphenoxylate, doxepin, eluxadoline, hyoscyamine, a Lactobacillus (optionally Lactobacillus acidophilus), lactulose, linaclotide, loperamide, lotronex, lubiprostone, polyethylene glycol 3350, psyllium seed husk, and / or rifaximin.

[0121] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0122] Pharmaceutical Compositions

[0123] In another aspect, the present disclosure provides a pharmaceutical composition comprising a composition of the present disclosure and at least one pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises at least one adjuvant.

[0124] Such a pharmaceutical composition may consist of at least one composition of the invention, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one composition, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one composition of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0125] In certain embodiments, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In other embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng / kg / day and 1,000 mg / kg / day.

[0126] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0127] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically- based formulations.

[0128] In certain embodiments, the compositions of the invention are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.

[0129] The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

[0130] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.

[0131] As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0132] In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMB UMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0133] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0134] Formulations may be employed in admixtures with conventional excipients, z.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and / or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, “additional ingredients” include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.

[0135] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid.

[0136] The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0137] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or / / -propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

[0138] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an “oily” liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0139] A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally- occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

[0140] Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (z.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.

[0141] Administration / Dosing

[0142] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0143] Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of therapeutic (z.e., composition) necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular therapeutic employed; the time of administration; the rate of excretion of the composition; the duration of the treatment; other drugs, compounds or materials used in combination with the composition; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic composition of the disclosure is from about 0.01 mg / kg to 100 mg / kg of body weight / per day of active agent (z.e., nucleic acid). One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic composition without undue experimentation.

[0144] The composition may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of composition dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.

[0145] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0146] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0147] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic composition to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic composition and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic composition for the treatment of a disease or disorder in a patient.

[0148] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.

[0149] The amount of active agent of the composition(s) of the disclosure for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 pg to about 6,000 mg, about 100 pg to about 5,500 mg, about 200 pg to about 5,000 mg, about 400 pg to about 4,000 mg, about 800 pg to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in-between.

[0150] In some embodiments, the dose of active agent (z.e., nucleic acid) present in the composition of the disclosure is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of active agent present in the composition of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0151] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of the composition of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

[0152] The term “container” includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, z.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.

[0153] Administration

[0154] Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0155] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein. Parenteral Administration

[0156] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

[0157] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In certain embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (z.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0158] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a nontoxic parenterally acceptable diluent or solvent, such as water or 1,3 -butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0159] Examples

[0160] The instant specification further describes in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless so specified. Thus, the present invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0161] Example 1: Gut Microbiome and Lipidome Signatures in Irritable Bowel Syndrome Patients from a Low-Income, Food-Desert Area: A Pilot Study

[0162] In Example 1 section, a study (“the first study” or “the pilot study”) on the relationship between gut microbiome / lipidome signatures and irritable bowel syndrome (IBS) disease states is described.

[0163] Irritable bowel syndrome (IBS) is a common gastroenterological disorder with triggers such as fructose. The first study showed that the IBS patients suffering from socioeconomic challenges have a significantly high consumption of high-fructose corn syrup (HFCS). Here, the first study characterizes gut microbial dysbiosis and fatty acid changes, with respect to IBS, HFCS consumption, and socioeconomic factors. Fecal samples from IBS patients and healthy controls were subjected to microbiome and lipidome analyses. The first study assessed phylogenetic diversity and community composition of the microbiomes, and used linear discriminant analysis effect size (LEfSe), analysis of compositions of microbiomes (ANCOM) on highly co-occurring subcommunities (modules), least absolute shrinkage and selection operator (LASSO) on phylogenetic isometric log-ratio transformed (PhILR) taxon abundances to identify differentially abundant taxa. Based on a Procrustes randomization test, the microbiome and lipidome datasets correlated significantly (p = 0.002). Alpha diversity correlated with economic factors (p < 0.001). Multiple subsets of the phylogenetic tree were associated with HFCS consumption (p < 0.001). In IBS patients, relative abundances of potentially beneficial bacteria such as Monoglobaceae, Lachnospiraceae, and Ruminococcaceae were lower (p = 0.007), and Eisenbergiella, associated with inflammatory disorders, was higher. In IBS patients, certain saturated fatty acids were higher and unsaturated fatty acids were lower (p < 0.05). The study aims first to underscore the influence of HFCS consumption and socioeconomic factors on IBS pathophysiology, and provides new insights that inform patient care.

[0164] Example 1-1:

[0165] The gastrointestinal tract along with the microbiome that inhabits it is a complex organ system that has functions beyond digestion and absorption such as immunological and neurological functions. The dysregulation of these functions leads to the development of several digestive disorders. One of the most common of these disorders is irritable bowel syndrome (IBS). Common symptoms of IBS include abdominal pain, bloating, and altered bowel habits. It is one of the most common gastroenterological disorders, affecting 10-15% of the global population with varied regional prevalence. Direct and indirect costs of IBS in the United States result in healthcare costs of more than USD 30 billion / year. IBS is considered a debilitating disorder as it significantly affects the quality of life of patients. The precise etiology and pathophysiology of IBS are yet to be resolved; however, altered gut motility, visceral hypersensitivity, inflammation, altered gut microbiome, psychiatric disorders, and neurological disturbances are frequently associated with IBS. The complex etiology, multifactorial pathophysiology, involvement of psychological comorbidities, heterogeneity of clinical manifestations, and lack of effective diagnostic methods for IBS pose difficulty in the development of more efficacious treatment strategies. There are now increasing numbers of reports that the CO VID-19 pandemic has further worsened the gastrointestinal and psychological symptoms of IBS patients.

[0166] Food triggers have been reported for IBS. Among the possible associated causes of IBS are fructose intolerance and an elevated consumption of fructose products. Up to one third of patients with IBS may have dietary fructose intolerance or fructose malabsorption. High-fructose com syrup (HFCS) is a major source of fructose in American diets, representing up to 40% of caloric sweeteners added to foods and beverages. Recently, a significantly high consumption of HFCS in the IBS patients was reported. These patients suffer from socioeconomic challenges. The hospital is located in Camden, New Jersey. It is estimated that 37.4% of Camden residents live below the poverty line, with a median household income of USD 26,105. This is less than half of the median household income in the United States. Other highlights of this community include high unemployment rate, low education levels, and “food desert” status. The lack of access to nutritious and affordable food items leads to limited dietary choices for the residents. Readily available HFCS- containing inexpensive food products thus become staple foods.

[0167] Studies have been carried out to elucidate the gut microbiome profile of IBS compared to that of healthy control participants. Kim et al. carried out a case-control study and a cross-cohort analysis of gut dysbiosis in IBS. A meta-analysis by Pittayanon et al. reported a higher prevalence of Firmicutes and a lower prevalence of Bacteroidetes in IBS patients. Wang et al. carried out a systematic review and meta-analysis of 6333 case-control studies of gut microbiome dysbiosis in IBS. Some studies have focused on the brain-gut axis aspect of the disease (i.e., signaling from the brain to the gut). As dietary modification is one of the major methods of management for IBS patients, a few groups have also studied the effect of certain diets on IBS. A study showed that interventions such as a low-FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) diet for IBS patients altered their gut microbiome’s taxonomy and functional potential to more closely resemble healthy controls, and that IBS patients with “pathogenic” baseline microbiomes had a greater reduction in symptoms from the low-FODMAP diet than IBS patients with healthy- like microbiomes. Another study analyzed the gut microbiomes of IBS patients on exclusion (including gluten-free, dairy-free, or low-FODMAP) diets. Vandeputte and Joossens discussed the effects of low- and high-FODMAP diets on gut microbiome composition with respect to GI diseases. Van Lanen et al. carried out a systematic review and meta-analysis of the efficacy of a low-FODMAP diet in adult IBS.

[0168] This study was undertaken to characterize the gut microbiome and changes in fatty acid concentrations with respect to IBS phenotypes. These changes were then evaluated with respect to a high consumption of HFCS-containing foods in the IBS patient population as well as other dietary, lifestyle, and socioeconomic factors such as alcohol consumption, household income, persons per household, etc., that may influence the manifestation and progression of IBS. This is the first study to underscore the influence of HFCS consumption and socioeconomic factors in IBS pathophysiology based on distinct microbiome and lipidome differences. These correlations are relevant from a clinical standpoint, especially when helping underserved communities. This study aims to help guide further research and improve IBS patient care regarding dietary interventions to avoid the exacerbation of gastrointestinal and coexisting psychological comorbidities.

[0169] Example 1-2: Materials and Methods

[0170] Patients and Sample Collection Our study included patients that presented to the Cooper University Hospital (CUH) in Camden, New Jersey. IBS patients at least 18 years of age with ongoing care at CUH were eligible for this study. Exclusion criteria included age <18 years, non-English-speaking patients, pregnancy, and antibiotics in last three months. IBS patients are identified in the electronic medical record system (EPIC) with ICD-10 codes of K58.0, K58.1, K58.2, K58.8, and K58.9. ROME IV criteria were used to determine IBS subtypes as: (i) IBS with constipation (IBS-C); (ii) IBS with diarrhea (IBS-D); (iii) mixed IBS (IBS-M), or (iv) unclassified IBS (Schmulson et al., J. Neurogastroenterol. Motil. 2017;23: 151-163; and Mearin et al., Gastroenterology. 2016; 150: 1393-1407.E5.). Control participants were recruited from a healthy population from the same area, who reported to CUH for wellness visits. Screening was carried out to confirm that the control subjects did not have a previous history or current documentation of IBS. They also did not have any Gl-related symptoms before recruitment. Informed consent was obtained as per the IRB guidelines. The medical records of the participants were reviewed to confirm demographic and clinical details. Surveys were also collected from the participants regarding their demographic information and dietary history. In brief, the survey included questions about demographic variables (sex, gender, ethnicity / race), lifestyle factors (residence, education level, marital status, household income, persons per household, alcohol, and tobacco use), dietary factors (consumption of HFCS-rich foods), and clinical data (height, weight, type of IBS, colonoscopy / endoscopy, medications, hypertension, diabetes, cholesterol, and psychological comorbidities). A total of 398 food items listed as high in HFCS were included in the surveys; these were identified using the United States Department of Agriculture (USDA) database. Patients were asked to provide a stool sample. Samples were collected in Norgen Stool Nucleic Acid Collection and Preservation Tubes (Norgen Biotek Corp., Thorold, ON, Canada) and were frozen at -80 °C as described in White et al., Wo ld J. Gastroenterol. 2021;27:5575-5594), O’Neill et al. (Obes. Surg. 2022;32:480-488. doi: 10.1007 / sl 1695-021-05805-z) and Awan et al. Pediatr. Gastroenterol. Hepatol. Nutr. 2023;26:99-115).

[0171] 16S rRNA Gene Sequencing

[0172] Genomic bacterial DNA collected from fecal samples was extracted using a Qiagen DNeasy PowerSoil HTP extraction kit (Qiagen, Redwood City, CA, USA). Manufacturer’s instructions were followed for the procedure. The marker genes in the extracted DNA were amplified via PCR using GoTaq Master Mix (Promega, Fitchburg, WI, USA). High- throughput sequencing was conducted using the Golay barcode primers 515 F (5'- GTGCCAGCMGCCGCGGTAA-3', SEQ ID NO:1) and 806 R (5'- GGACTACHVGGGTWTCTAAT-3', SEQ ID NO:2), respectively. The Golay barcode primers target the V4 hypervariable region of the 16S rRNA gene, which has been found to be highly conserved and useful for the taxonomic profiling of the gut microbiome. PCR amplification methods include heating at 94 °C for 3 min followed by 35 * 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 1.5 min, with the final extension being carried out at 72 °C for 10 min. To purify and normalize the PCR products, the SequelPrep Normalization Kit (Cat. No. A1051001, ThermoFisher, Waltham, MA, USA) was used. Library preparation and gene sequencing for the 16S rRNA gene were carried out using the V2 300-cycle Illumina MiSeq System.

[0173] Analysis of Lipids

[0174] Stool samples were homogenized, and aliquots (100 mg) were analyzed via gas chromatography-mass spectrometry (GC-MS). Fatty acids were extracted through liquid / liquid extraction, which also removes the nucleic acid preservative. An aliquot (250 pL) of each extract was transferred to a fresh analysis tube. Removal of the solvent was carried out through evaporation under a stream of nitrogen. Internal standard solution was added to the tubes that contained dried sample extracts, quality controls (QCs), and calibration standards. Evaporation under nitrogen was used to remove the solvent. Processing of the dried samples and QCs by methylation / transmethylation with methanol / sulfuric acid resulted in the formation of the corresponding fatty acid methyl esters (FAME) of free fatty acids and conjugated fatty acids. The reaction mixture was neutralized and extracted with hexane, which was then analyzed using 7890A / 5975C GC / MS in the single-ion monitoring (SIM) positive mode with electron ionization. Linear and quadratic regression analyses were generated from fortified calibration standards prepared immediately prior to each run. Quantitation was performed using both these analyses. Agilent MassHunter GC / MS Acquisition B.07.04.2260 and Agilent MassHunter Workstation Software Quantitative Analysis for GC / MS B.09.00 / Build 9.0.647.0 were used for collection and processing of raw data. The total fecal content of 30 fatty acids was measured after conversion into their corresponding FAMEs. Concentrations are provided in weight-corrected pg / g of fecal dry mass. Values below the lower limit of quantification were treated as a concentration of 0.001 pg / mL.

[0175] Microbiome Data Processing and Statistical Analysis Data generated through 16S rRNA gene sequencing were analyzed using Quantitative Insights Into Microbial Ecology (QIIME) 2 2020.11 as well as Python-based packages (Python 3.6.12) with packages in accordance with the QIIME 2 2020.11 environment. Sequences were de-multiplexed, filtered, and clustered into amplicon sequence variants (ASVs) using QIIME 2 DADA2. The phylogenetic tree was created using SATe-enabled phylogenetic placement (SEPP) via QIIME 2. A naive Bayes classifier trained on the latest SILVA version 138 16S rRNA gene database (March 2021) was used to assign the taxonomy via the QIIME 2 interface. Additional Python packages (SciPy, Statsmodels, Scikit-bio) were used for statistical tests on QIIME 2-generated data. Diversity analyses were performed in QIIME 2, rarefied to an even sampling depth of 54,000 reads per sample. Faith’s phylogenetic diversity (Faith’s PD) was considered the primary metric for microbiome richness, and unweighted UniFrac was considered the primary metric for beta diversity. PERMANOVA was used to assess group-based differences in microbiome community composition (beta diversity). Stacked bar plots were created using the R package microshades vl.10.

[0176] Procrustes Randomization Test

[0177] Principal coordinate analysis (PCoA, using the Python package Scikit-learn) was performed on Bray-Curtis distance matrices from both the fecal lipidome and fecal microbiome datasets (using the Python package SciPy. di stance), and a Procrustes test was performed on the lipidome and microbiome PCoA coordinates. Following the protocol from Peres-Neto and Jackson, a Procrustes randomization test (PROTEST) was performed by randomly permuting the PCoA coordinates and performing a Procrustes test on the permuted samples 10,000 times. The p value was calculated based on the portion of randomized Procrustes tests with resulting m2(Gower’s statistic) scores lower than the Procrustes m2score for the observed datasets.

[0178] Differential Abundance Testing

[0179] To identify differentially abundant taxa based on IBS status and subtype, the first study used Analysis of Compositions of Microbiomes (ANCOM, via Scikit-bio). Further, the first study also created a co-occurrence network of taxa and lipids, from which features were grouped into highly correlated modules, which were summed for analysis to identify differential groups of features. Specifically, rare taxa were filtered with total read counts <500 and average read counts <3. Taxa were center log-ratio transformed to account for compositionality, while lipids were untransformed. Pearson’s correlation coefficients were calculated pairwise between all features. Edges were drawn only between features with a Pearson’s rho > 0.5 and p < 0.05. The Louvain modularity maximization algorithm was applied to identify modules with many strong connections within modules and few connections between modules. Relative abundances of all features in each module were summed, and IBS-based differences in module abundances were assessed via a Kruskal- Wallis test.

[0180] Next, the first study utilized a phylogenetic isometric log-ratio transformation (PhILR) to identify taxa associated with HFCS consumption to test the assumption that HFCS consumption causes a phylogenetic shift in the gut microbiome. The PhILR transformation considers ratios between subtrees of the phylogenetic tree to use phylogeny to transform compositional microbiome data outside the simplex, such that each feature can change independently of the others. At each branching point in the tree, the isometric logratio of the summed read counts in one subtree of the branch to the other subtree was calculated to create the “balances”. Least absolute shrinkage and selection operator (LASSO) regression, with lambda (penalty parameter) = 3000, was used to identify the top 5 balances associated with HFCS consumption. In R, PhILR was performed using the philr package vl.24.0, LASSO was performed using the glmnet package v4.7, and balances were visualized using ggtree v3.6.2.

[0181] Ethics Approval

[0182] This study received approval from the Cooper Health System Institutional Review Board (IRB) (17-079EX), and all the steps were carried out as per the standards set by the IRB. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and / or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

[0183] Example 1-3: Patient Demographics

[0184] The demographic data for the study participants, collected via surveys and confirmed by chart review, are presented in Table 1. As expected, a majority of the IBS patients that participated in this study were females (78.8%), >40 years of age (63.6%), and Caucasian (84.8%). Most IBS patients (96.7%) did not smoke. A majority of the IBS patients did not have diabetes, high cholesterol, or hypertension. Psychological comorbidities such as depression (12 patients; 36%) and anxiety (13 patients; 39%) were noted in IBS patients. Control subjects did not have depression or anxiety. All participants in this study were included for all analyses; no outliers were removed from any statistical testing or figures.

[0185] Table 1 : Patient demographics and clinical characteristics.

[0186] Example 1-4: Gut Microbiome Analysis

[0187] There were no differences in the richness of the gut microbiome between IBS patients and controls or across IBS subtypes, as assessed via a Kruskal-Wallis test on the Faith’s phylogenetic diversity of each sample (7 / (3) = 4.1, / ? = 0.25). IBS patients had a mean Faith’s phylogenetic diversity of 48.7 (95% confidence interval 44.9-52.5), whereas healthy control participants had a mean of 47.8 (95% confidence interval 42.8-52.8).

[0188] Association of the Community Composition of the Gut Microbiome with IBS Group Unweighted UniFrac principal coordinate analysis (PCoA) (Figs. 1 A-1B) shows the ordination of the healthy control participants and IBS patients. PCoA plots are shown comparing healthy control participants and IBS patient samples (Fig. 1A) and healthy control subjects and IBS subtypes (Fig. IB). Each point represents the phylogenetic composition of one sample, and shaded regions represent 95% confidence intervals of the first two PCoA axes for each group. Points that are close together have similar phylogenetic composition, and points that are far apart have dissimilar phylogenetic composition. The proportion of variance explained by each principal coordinate axis is denoted in the corresponding axis label; PCI explains 14.8% of the variation and PC2 explains 7.6% of the variation across samples. IBS patients had significantly different overall community composition, compared to control participants, based on unweighted UniFrac PERMANOVA (pseudo-F = 1.63, p = 0.021; Fig. 1A). Additionally, there were significant differences in microbiome community composition across healthy controls and IBS subtype (pseudo-F= 1.21, p = 0.020; Fig. IB). A stacked bar plot in Figs. 2A-2B shows the taxonomic composition of the healthy control and IBS subtype samples. Bacteroidota and Firmicutes were predominant in all groups of samples.

[0189] Example 1-5: Association of Differentially Abundant Gut Microbiome Taxa with IBS Groups

[0190] Next, the differences in the specific taxa (ANCOM) and groups of taxa (Kruskal- Wallis on summed network modules) were assessed in the microbiomes of the healthy control participants versus the IBS patients. The relative abundance of Lachnospiraceae group ND3007, which ANCOM identified as differentially abundant (W= 42 / 254), was lower in participants with IBS than healthy controls (Fig. 3 A). Additionally, the relative abundance of Eisenbergiella, which ANCOM identified as differentially abundant (W= 150 / 254), was higher in participants with IBS-C and IBS-M, relative to healthy controls and participants with IBS-D (Fig. 3B). ANCOM is a conservative but robust method using pairwise log-ratios to account for the compositionality of the data; thus, the outcomes usually include only few differentially abundant taxa.

[0191] The network module analysis (Louvain modularity maximization) identified eleven modules, or highly co-occurring subcommunities, of bacteria. The summed relative abundances of two modules within the dataset were different based on IBS status (Figs. 3C- 3D). The relative abundance of module 9, which contained members of the class Clostridia (including the families Monoglobacea, Lachnospiraceae, and Ruminococcaceae), was lower in participants with IBS, relative to healthy controls (7 / (1) = 7.1, / ? = 0.007) (Fig. 3C). The relative abundance of module 11, which contained different members of the class Clostridia, was highest in participants with IBS-C ( / (3) = 13.7, p = 0.003) (Fig. 3D).

[0192] Next, linear discriminant analysis effect size (LEfSe) analysis was carried out. LEfSe scores for taxa enriched in healthy control participants or IBS patients are shown in Fig. 4A, whereas LEfSe scores for taxa enriched in IBS-C versus IBS-D subtypes are shown in Fig. 4B. As seen from Fig. 4 A, purportedly beneficial bacteria belonging to Ruminococcaceae were highest in the healthy control participants, while the genera Bulyricimonas and Parabacteroides and the family Tannerellaceae within the Bacteroidota phylum were enriched in IBS patients as compared to healthy control participants (Fig. 4A). The relative abundances of the genus Alislipes and family Rikenellaceae within the Bacteroidota phylum were higher in IBS-C patients than in IBS-D patients (Fig. 4B).

[0193] Example 1-6: Monthly HFCS Consumption with Respect to IBS Groups

[0194] Previously, a higher consumption of HFCS-containing food items in IBS patient population was observed. Consistent with that observation, a higher estimated consumption of HFCS in all IBS subtypes relative to control participants was also observed in the present study using ANOVA followed by Dunn’s post hoc tests (F(3,41) = 3.4, / ? = 0.026, Fig. 5A). The participants in the current study are different to those mentioned in the previous study. All IBS subtypes had significantly higher estimated HFCS consumption than the controls. No significant difference was observed among the IBS subtypes.

[0195] Example 1-7: Phylogenetic Groups of Taxa in the Gut Microbiome with Respect to Monthly HFCS Consumption

[0196] As diet can influence the growth of microorganisms, it can be hypothesized that a higher consumption of HFCS will cause a phylogenetic shift in the gut microbiome. Thus, the first study analyzed phylogenetic isometric log-ratio transformed (PhILR) abundances to identify taxa associated with HFCS consumption (Figs. 5B-5D). LASSO regression on PhILR-transformed microbiome data identified five phylogenetic balances of taxa that were associated with estimated HFCS consumption (adjusted R2= 0.388, F(5,39) = 6.6, / ? < 0.001). One of the most strongly positively associated phylogenetic balances was the ratio of the family Erysipelatoclostridiaceae to a broad section of the phylogenetic tree predominantly including the classes Clostridia and Bacteroidia (balance nl4). Additionally, HFCS was negatively associated with a ratio between members of the family Lachnospiraceae ( Eu bacte r i um ventriosum and an ASV within the genus Anaer sporobacter) and a broad section of the phylogenetic tree predominantly including the classes Clostridia and Bacteroidia (n55). Within the Erysipelatoclostridiaceae family, the first study identified a negative association between estimated HFCS consumption and the ratio of the genera Coprobacillus to Catenibacterium (balance nl5). The ratio of taxa in the phylum Firmicutes and class Clostridia was positively associated with estimated HFCS consumption (nl 19). Finally, the first study identified a negative association between estimated HFCS consumption and a balance within the order Burkholderiales, which was the ratio of two taxa (one Sutterella ASV and one within the family Oxalob acteraceae) to another Sutterella ASV, supporting that within-genus (and potentially strain-level) differences are associated with dietary patterns.

[0197] Example 1-8: Association of Fecal Lipids with IBS Groups

[0198] To evaluate the impact of IBS on fatty acid composition, the first study performed a targeted lipidomics analysis of a panel of 30 fatty acids. These fatty acids included long-chain fatty acids (LCFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs). The stacked bar plot in Figs. 6A-6B shows the concentrations of detected lipids in fecal samples. Saturated fatty acids were predominant in all groups of samples. Please note that saturated fatty acids typically exist in higher concentrations, but unsaturated fatty acids can be highly bioactive in low concentrations. Concentrations of multiple lipids were significantly different (per Kruskal-Wallis test) across healthy control participants and various IBS subtypes (Fig. 7A). A spider chart (also known as a radar plot) shows these lipids in Fig. 7A, where each lipid has its own axis; all axes are joined in the center of the figure. Each lipid’s concentrations were Z-score transformed, and the mean Z-score for each IBS subtype is plotted on each axis. Mean Z-scores of significantly different lipids are shown for each group. Gamma linolenic acid (18:3n6) was significantly higher in the control participants than in IBS patients. Palmitic acid (16:0) was highest in the IBS-C patients, while margaric acid (17:0) was higher in the participants with IBS-M and IBS-C. Lipids that were significantly different (per Mann-Whitney U test) between healthy control participants and all IBS patients are presented in a volcano plot in Fig. 7B. A positive log-fold change indicates higher concentration in the IBS patient group. Gamma linolenic acid (18:3n6) showed a level more than 2-fold higher in control participants than in IBS patients. Based on uncorrected Mann-Whitney U tests (p value < 0.05), certain saturated fatty acids (palmitic acid, 16:0; margaric acid, 17:0) were higher in IBS patients, while certain unsaturated fatty acids (linoleic acid,18:2n6; gamma-linolenic acid, 18:3n6; alpha-linolenic acid, 18:3n3) were lower in IBS patients. Gamma-linolenic acid (18:3n6) was the lipid with the largest fold change between groups. Concentrations of significantly differing lipids, separated by IBS status and IBS subtypes, respectively, are presented in boxplots with overlaid scatterplots in Figs. 7C and 7D, respectively.

[0199] Example 1-9: Link between the Gut Microbiome and the Fecal Lipidome Compositions via Procrustes Randomization Test

[0200] A Procrustes randomization test was carried out to explore the multivariate association between each participant’s microbiome and lipidome. As seen from Fig. 8 A, the microbiome and lipidome datasets associated much better than expected by random chance (empirical p = 0.002). A histogram showing the distribution of Procrustes disparity scores across 10,000 permutations of the dataset is presented in Fig. 8B. The Procrustes disparity score for the data (shown by the red line in Fig. 8B) was lower than 99.8% of disparity scores when the first study randomly sampled the data 10,000 times. This suggests that the microbiome and lipidome datasets fit together better than randomly matching the microbiome-lipidome samples 99.8% of the time.

[0201] Example 1-10: Association of Socioeconomic Factors with Diversity and Community Composition of the Gut Microbiome

[0202] As the manifestation of IBS symptom ology is influenced by various socioeconomic factors, the first study also explored the influence of (i) alcohol consumption, (ii) household income, (iii) number of household occupants, and (iv) marital status using Faith’s phylogenetic alpha diversity analysis. The first study observed that a higher alcohol consumption was associated with a higher alpha diversity (Fig. 9A). A higher income was associated with a higher alpha diversity, while a lower income corresponded to a lower alpha diversity (Fig. 9B). An increased number of occupants (Fig. 9C) and married status (Fig. 9D) were associated with a higher alpha diversity. Additionally, household income (Fig. 10A; pseudo-F = 1.64, / ? < 0.001) and marital status (Fig. 10B; pseudo-F = 1.32, / ? = 0.028) were associated with differences in beta diversity (as measured using unweighted UniFrac).

[0203] Example 1-11:

[0204] ANCOM, the network module method, and LEfSe all showed that the Lachnospiraceae group ND3007 was lower in participants with IBS than in healthy controls. In many studies, identified differentially abundant taxa are not robust across various differential abundance methods, so the finding of the present study should be particularly considered robust due to the consensus across differential abundance methods. Members of the Lachnospiraceae ND3007 group are short-chain fatty acid (SCFA) producers and have been linked to decreases in the blood glucose levels in rats with diabetes. In general, bacteria belonging to the family Lachnospiraceae are considered important butyrate producers. Butyrate and other short-chain fatty acids are shown to modify gut motility, maintain the integrity of the intestinal barrier, and also play a role in the inhibition of intestinal inflammation. SCFAs are essential in maintaining a homeostatic immune environment, and butyrate particularly has anti-inflammatory and immunoregulatory effects. Additionally, butyrate stimulates serotonin release via enterochromaffin cells, altering sensory neuron activity, which is disrupted in individuals with IBS. It has also been discussed that environmental and psychological stressors reduce the proportion of Lachnospiraceae. This may, in turn, lead to a reduction in mucus and SCFA production, as well as reductions in tight junction protein expression resulting in intestinal inflammation. The observation that the Lachnospiraceae group ND3007 was abundant in healthy control participants and was less abundant in IBS, which has inflammatory pathophysiology in many cases, is consistent with these findings.

[0205] It is likely that dietary differences in individuals with and without IBS may impact the relative abundance of Lachnospiraceae. There is an association between diet quality and the relative abundance of Lachnospiraceae; specifically, persons with a higher healthy eating index showed higher levels of Lachnospiraceae ND3007 and Ruminococcaceae. The dietary inclusion of fiber, antioxidants, and phytochemicals, for example, through the consumption of flaxseed and grapes, led to higher relative abundances of Lachnospiraceae ND3007. Moreover, it is evident that HFCS in the diet may alter the relative abundance of particular taxa. A modest decrease in Ruminococcaceae was found with a high HFCS intake in an adolescent mouse model. The decrease in Ruminococcaceae observed in the IBS patients in the present study may, thus, at least partly be due to the high consumption of HFCS in these patients.

[0206] ANCOM also showed that Eisenbergiella was higher in IBS patients, especially those with IBS-C and, to a certain extent, IBS-M, relative to healthy controls. The first study is the first report of an alteration of genus Eisenbergiella in IBS patients. The exact mechanism underlying these observations is not known, but it is suspected that certain bacteria including Eisenbergiella is associated with inflammatory factors. Higher levels of Eisenbergiella spp. were also observed in persons following a high-saturated-fatty-acid diet, which often co-occurs in foods with HFCS. Consistent with the effects of dietary saturated fatty acids on Eisenbergiella, the participants with IBS (particularly IBS-C) had high fecal concentrations of saturated fatty acids, including palmitic and margaric acids. It is suggested that though HFCS and saturated fatty acids often co-occur in foods, HFCS consumption may alter lipid metabolism and could be another mechanism explaining the higher fecal concentration of palmitic acid in IBS patients. Lipidome analysis of the fecal samples showed lower concentrations of fecal gamma linolenic acid in all subtypes of IBS patients than in healthy controls. Gamma linolenic acid has anti-inflammatory properties in multiple human tissues, and there could be multiple reasons for its lower concentration in IBS patients, including dietary differences, microbial metabolism of gamma linolenic acid / its precursors, or changes in enterocyte absorption / metabolism.

[0207] The present study found a number of important and meaningful observations, which led to new insights into IBS pathophysiology. The robust statistical analyses using a number of different approaches, the significant statistical differences observed in the IBS patient and healthy control cohorts, and the consistency of the key observations with the reported literature from a number of different groups supported the validity of the outcomes. As mentioned above, the study underscored the influence of HFCS consumption and socioeconomic factors in IBS pathophysiology based on distinct microbiome and lipidome differences. Observations from this study may thus inform IBS patient care.

[0208] Example 2:

[0209] The role of gastrointestinal tract along with the microbiome extends beyond digestion and absorption to affect immunological and neurological functions. Dysregulation of these functions leads to the development of several digestive disorders, one of the most common being irritable bowel syndrome (IBS). IBS affects 10-15% of the global population with varied regional prevalence. In addition to affecting a significant portion of the population, IBS also affects health-related economics - physician office visits for IBS result in healthcare costs of more than $30 billion / year. IBS is considered a debilitating disorder as it significantly affects patient’s quality of life. Symptoms of IBS can mainly be manifested as constipation (IBS-constipation; IBS-C), diarrhea (IBS-diarrhea; IBS-D), or mixed constipation and diarrhea (IBS-mixed; IBS-M). The precise etiology and pathophysiology of IBS are unresolved.

[0210] The lack of effective diagnostic methods, complex etiology, multifactorial pathophysiology, and heterogeneity of clinical manifestations of IBS are all barriers to development of more accurate diagnostic strategies. Therefore, any improvements in diagnostic development will require a more thorough understanding of the biological and environmental drivers of the disease. IBS is a functional gastrointestinal disorder in that it is characterized by persistent and recurring GI symptoms and is not caused by structural (e.g. tumors or masses) or biochemical abnormalities. Thus, routine diagnostic medical tests such as X-rays, CT scans, blood tests and endoscopic exams show essentially normal results for IBS, making proper diagnosis challenging. A major knowledge gap exists in diagnostic indicators for this disease. To address this gap, a first study (N=45) (described in Example 1 section) was carried out to decipher differences in gut lipidomic profiles of healthy controls and IBS subtype patients by gas chromatography-mass spectrometry (GC-MS). It was observed that saturated fatty acids (e.g. palmitic acid) were significantly higher and polyunsaturated fatty acids (e.g. gamma-linolenic acid) were significantly lower in IBS patients compared to healthy controls. For example, palmitic acid and gamma-linolenic acid can be used as biomarkers of IBS. In the study described in Example 2 section (“the second study”), a study with strong statistical power (N=300) is designed to elucidate the potential of these two biomarkers and yield additional fatty acids that can be used as diagnostic biomarkers of IBS.

[0211] The local community suffers from a number of socioeconomic challenges and is declared a ‘food desert’ due to the lack of access to fresh, healthy, and affordable food items. Residents of such communities gravitate to readily-available and inexpensive food products that contain the artificial sweetener, high fructose com syrup (HFCS). Fructose is present in high amount in HFCS. Fructose malabsorption has been implicated in the development of IBS. HFCS can thus exacerbate IBS. Using dietary surveys, it was found that the IBS patients have a significantly high consumption of HFCS. Importantly, HFCS consumption is not limited to food desert communities as it is present in several common foods. According to the USDA Sugar and Sweeteners outlook January 2023 report, HFCS remains the second largest component of sweeteners in the U.S. market, averaging 31 percent of the total sweetener deliveries in the past 2 years. Driven by these findings, the differences in the lipidomic profiles in the healthy controls and IBS subtype patients are driven by HFCS consumption.

[0212] Example 2-1: Specific Aims

[0213] Specific Aim 1: Determine the lipidomic profiles of IBS patients and healthy controls The second study collects demographic and medical information, dietary surveys based on USDA database and stool samples (N=300; n=75 each IBS-C, IBS-D, IBS-M, controls). The second study carries blinded lipidome analyses of all samples stratified for changes in the fatty acids and correlate that to the presence / absence of IBS to address (i) if palmitic acid and gamma-linolenic acid are differentially detected in this large cohort using GC-MS and (ii) if additional fatty acids (FAs) are differentially detected in this large cohort using GC-MS.

[0214] Specific Aim 2: Development of fatty acids as IBS diagnostic markers

[0215] The lipidome analyses in Aim 1 is carried out using sensitive method, GC-MS. To test usefulness of the emerging FAs as diagnostic markers, the second study optimizes colorimetric assays to test if these are present in stool at levels sufficient for detection to indicate presence of IBS and if these are influenced by patient diet, e.g. HFCS. Assays results are checked against (i) medical data to confirm accuracy (e.g. false negatives / positives) and (II) dietary surveys.

[0216] Example 2-2: Clinical challenges

[0217] Irritable bowel syndrome (IBS) is the most common gastrointestinal (GI) disorder characterized by chronic abdominal pain and altered bowel habits in the absence of any organic cause. It affects 10-15% of the global population with varied regional prevalence. IBS also presents an economic challenge - physician office visits for IBS cost more than $30 billion per year. IBS is considered a debilitating disorder as it significantly affects patient’s quality of life. It is the second leading cause, after the common cold, for missing work or school, having a wide-ranging impact on both the patients and their communities. These health, economic, and social costs of IBS are driven primarily by the main challenge that there is lack of effective diagnostic method. IBS is a functional GI disorder that occurs as a result of abnormal functioning of the GI tract, it is characterized by persistent and recurring GI symptoms and is not caused by structural (e.g. tumors or masses) or biochemical abnormalities. Thus, routine diagnostic medical tests such as X-rays, CT scans, blood tests and endoscopic exams show essentially normal results for IBS, making proper diagnosis challenging. A major knowledge gap exists in diagnostic indicators for this disease. The proposal addresses this challenge by identifying and evaluating if specific fatty acids can serve as diagnostic markers.

[0218] Example 2-3: Preliminary data

[0219] HFCS consumption by IBS patients and lipidome data The present study carried out a retrospective chart review of 969 IBS patients for demographics and medical comorbidities. The present study then collected information about dietary HFCS intake from a subset of these IBS patients and healthy controls using USDA- database-driven-surveys that included 398 HFCS-rich food items. Controls lacked history of GI symptoms or disorders. 84% of IBS patients were female and 82% were >40 years old. Critically, overall HFCS intake was significantly higher in IBS patients compared to control subjects (p>0.0001). This was also seen for ail the three specific subtypes, IBS-C, IBS-D. and IBS-M (p-0.0033, p<0.0001, p-0.0005, respectively). The present study was the first to show a significantly high consumption of HFCS in IBS patients and underscored the urgent need for provider-driven patient education regarding dietary interventions. Next, a pilot study (see Example 1) in which stool samples and dietary surveys were collected, was carried out (N=45, IBS-D and IBS-C: n=10 each, IBS-M: n=13; controls: n=12). IBS subtypes were determined based on the ROME IV criteria. Consistent with other IBS studies, significantly higher estimated consumption of HFCS in all IBS subtypes relative to controls was also observed in the pilot study using ANOVA followed by Dunn’s post hoc tests F(3,4i)=3.4, p = 0.026 (Fig. 5A). Stool samples were subjected to fatty acids (FA) analysis by gas chromatography-mass spectrometry (GC-MS).

[0220] Concentrations of multiple FAs were significantly different across healthy controls and IBS subtypes. A spider / radar chart (Fig. 7A) shows these lipids, where each lipid has its own axis, all axes are joined in the center of the figure. Each lipid’s concentrations were Z- score transformed, and the mean Z-score for each IBS subtype is plotted on each axis. Mean Z scores of significantly different lipids are shown. Gamma linolenic acid (18:3n6) is significantly lower in IBS patients compared to the control participants, while palmitic acid (16:0) is highest in the IBS-C patients. FAs that were significantly different (per Mann- Whitney U test) between healthy control subjects and overall IBS patients are presented in a Volcano plot in Figure 7B. Positive log fold change indicates higher concentration in the IBS patient group. Palmitic acid was significantly higher in IBS patients (p value<0.05); margaric acid (17:0) was also higher in IBS patients. Gamma linolenic acid (18:3n6) showed a greater than 2-fold lower level (p value < 0.05) in IBS patients as compared to control subjects, while other unsaturated FAs such as linoleic acid, and alpha-linolenic acid, were also lower. Concentrations of significantly differing FAs, separated by IBS status and IBS subtypes, respectively, are presented in boxplots with overlaid scatterplots in Figs. 7C and 7D, respectively. Results of boxplots were similar to those with the Volcano plot in that palmitic acid and margaric acid were higher and linoleic acid, gamma-linolenic acid, and alpha- linolenic acid were lower in IBS patients. In summary, independent analytical methods showed consistent differences in the lipidomic profiles of IBS patients and healthy controls. These results indicate that IBS patients have difference in their fecal FAs, palmitic acid and gamma linolenic acid being the most significant ones, which can be explored as diagnostic markers for IBS. The levels of these two FAs in turn may be related to the consumption of HFCS.

[0221] Example 2-4:

[0222] Hypersensitivity to certain foods is a contributing factor to IBS, e.g. exacerbation of IBS symptoms has been observed due to fructose malabsorption. Fructose is mainly absorbed through carrier-mediated facilitative diffusion and GLUT-5. Even small magnitude increases in the dietary fructose can overwhelm the transporter capacity. Malabsorption of fructose leads to water influx into the lumen due to osmotic pressure. This results in rapid propulsion of bowel contents into the colon. Unabsorbed fructose is fermented by colonic bacteria which results in the production of certain metabolites, hydrogen, carbon dioxide, and trace gases. This can result in symptoms including abdominal pain, excessive gas, diarrhea, and bloating. Several mechanisms have been proposed to explain how unabsorbed fructose may lead to symptoms seen in IBS such as, (i) osmotic effect leading to increase in the gastrointestinal motility, (ii) bacterial fermentation of unabsorbed fructose leading to the production of short chain FAs, hydrogen, carbon dioxide, and trace gases and (ill) local irritation caused due to physical contact of fructose with the intestinal tract. As dietary modifications are used to manage IBS, a few groups have also studied the effect of certain diets on gut microbiome of IBS patients. Changes in gut microbiome are often reflected in changes in metabolome. However, thus far, compared to gut microbiome studies, only few gut metabolome studies are reported in IBS. The present study observed significant HFCS consumption, and significantly higher levels of saturated FAs (e.g. palmitic acid) and lower levels of polyunsaturated FAs (e.g. gamma-linolenic acid) in IBS patients. Interestingly, significantly elevated levels of palmitic acid were reported in mice exposed to HFCS for 6 weeks. It is interesting to note that IBS has inflammatory pathophysiology and saturated FAs have been linked to inflammation, while gamma linolenic acid has been reported to have anti-inflammatory properties. These findings are thus supportive.

[0223] Our pilot study described in Example 1 identified that saturated fatty acids (e.g. palmitic acid) were significantly higher and polyunsaturated fatty acids (e.g. gamma-linolenic acid) were significantly lower in IBS patients compared to healthy controls (Figs. 7A-7D). Therefore, these lipids can be used for the diagnosis of IBS. The second study addresses if higher level of palmitic acid and lower level of gamma-linolenic acid can serve as diagnostic biomarkers of IBS. The present study also analyzes the influence of HFCS consumption on the manifestation of the disease using these biomarkers.

[0224] Example 2-5: Approach

[0225] A human subject study including IBS-C, IBS-D and IBS-M patients and healthy control participants is proposed. IBS patients with ongoing care at the Cooper University Hospital (CUH) in Camden, New Jersey, are recruited. IBS patients are identified in the electronic medical record system (EPIC) by ICD-10 codes of K58.0, K58.1, K58.2, K58.8 and K58.9. IBS subtypes are determined based on the ROME IV criteria as (I) with constipation (IBS-C); (ii) with diarrhea (IBS-D); (iii) mixed IBS (IBS-M), or (iv) unclassified IBS. IBS patients are mostly indicated as belonging to the first three categories in the medical system so the study includes these three categories. Control participants are recruited from a healthy population, also seen at CUH for well visits. The control subjects are screened for lack of previous history and current documentation for IBS as well as lack of any Gl-related symptoms before recruitment. Exclusion criteria: (i) vulnerable population such as people who are unable to consent including those who are <18 years age, pregnant women (also pregnancy is a compounding factor) and prisoners, and (ii) patients with antibiotics history for three months (antibiotics can change gut flora and consequently gut chemicals profiles).

[0226] Specific Aim 1: Determine the lipidomic profiles of IBS patients and healthy controls.

[0227] The present study carries out blinded lipidome analyses of 300 samples using GC-MS stratified for changes in the FAs and correlate that to the presence / absence of IBS to address

[0228] (i) if palmitic acid and gamma-linolenic acid are differentially detected in IBS samples and

[0229] (ii) if additional FAs are differentially detected.

[0230] Collection of samples and surveys

[0231] The present study collects stool samples, demographic and medical information and implement a USDA-based dietary survey. The survey includes 398 food items listed as high in HFCS and is streamlined to be completed in less than ten minutes. It includes questions pertinent to sex, gender, ethnicity / race, height, weight, type of IBS, colonoscopy / endoscopy, medications, hypertension, diabetes, cholesterol, psychological comorbidities, smoking, alcohol intake, and known food triggers for IBS. Medical records are reviewed to confirm clinical details. The first study has Cooper IRB (#17-079EX) and Rowan IBC (#2017-03) approvals for the proposed experiments. The present study has optimized a stool collection system that preserves the highly sensitive need for sterility and sample integrity, while maintaining a patient-centered, user-friendly approach. Patients are given kits that include a handout with simple and concise picture-based instructions, stool collection kit, gloves, and zip lock bags for discarding materials. Stool samples are collected using Norgen Biotek preservation tubes and also on cotton swabs and stored at -80°C.

[0232] Sample size computations

[0233] Study design has been and data analyses are reviewed for statistical validity. A two- sample independent means power analysis study using the pilot study data indicated that a sample size of 75 in each of the four groups (IBC-M, IBC-D, IBC-M and controls) would be sufficient to equip the analysis with at least 80% power to detect a 35% change in the fatty acids of interest between each of the IBS groups and the control group at a significance level alpha=0.05.

[0234] Lipidomic profiling of patient samples

[0235] There is a significant knowledge gap in the diagnostic markers for IBS. As IBS causes gut dysbiosis, it poses that metabolic profile of IBS patients will differ from that of healthy controls. Studies have tried to analyze various metabolites that are differentially produced in IBS patients for diagnosis, e.g. measurement of volatile organic compounds (VOC) in feces. However, detection of VOC requires gas chromatography and thus cannot be readily used as a routine diagnostic method. The present study focuses on FA changes in IBS patients as these metabolites often exhibit changes in response to gut microbiome changes. To analyze fatty acids of the collected patient samples, the second study uses a 30-fatty acid panel that includes short-chain fatty acids (SCFA), long-chain fatty acids (LCFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) ranging from 2 to 22 carbons in length. These fatty acids can be readily and accurately measured in stool samples. The first study identified a specific pattern of FA levels (Figs. 7A-7D) with respect to IBS subtypes.

[0236] Statistical analysis

[0237] The amount of HFCS per food item is quantified using Glinsmann’s coefficient and HFCS consumption for various groups are assessed using analysis of variance (ANOVA) on ranks. Quantitation is performed using both linear and quadratic regression analysis generated from fortified calibration standards prepared immediately prior to each run. Raw data are collected and processed using Agilent MassHunter GC / MS Acquisition B.07.04.2260 and Agilent MassHunter Workstation Software Quantitative Analysis for GC / MS B.09.00 / Build 9.0.647.0. Data reduction is performed using Microsoft Office 365 ProPlus Excel. Differences between groups for numerical variables are assessed using ANOVA / independent t-test for normally distributed data, or Kruskal-Wallis ANOVA / Wilcoxon for non-normal data. Where appropriate, analyses are followed up with multiple pairwise comparisons adjusting for multiplicity using Tukey’s post hoc Honest Significant Difference (HSD) test. Mann-Whitney U test is also used to differentiate between FAs. A p-value of < 0.05 is considered statistically significant. Radar, volcano and boxplot analyses will be presented as in Figs. 7A-7D.

[0238] Expected outcomes, potential challenges and alternative approaches

[0239] Quantification of FAs performed in this Aim provides a chemical profile associated with IBS. The proposed blinded analysis of lipidomic profiles with the strong statistical power of 300 samples illustrates that palmitic acid and gamma-linolenic acid can serve as IBS diagnostic biomarkers in that their levels in the blinded samples positively identify IBS patients. The measured number of IBS office visits per year at CUH ensures that the second study has sufficient cohort for recruitment. The second study established efficient procedures for sample and data transfer for metabolite analysis with Metabolon, LLC. The methods have been standardized for reproducible results with samples stored for prolonged time at minus 80°C. Apart from palmitic acid and gamma linolenic acid, few other possible targets were observed in the first study (Figs. 7A-7D), e.g. linoleic acid. The second study then evaluates those new targets in a manner similar to that used for palmitic acid and gamma linolenic acid. Another challenge is sampling and regional biases. Previous chart review of 969 IBS patients showed aspects common in general IBS population, such as predominance of females (84%) and age (>40). The second study makes an effort to keep inclusion of participants from major ethnic / racial groups comparable.

[0240] Specific Aim 2: Development of fatty acids as IBS diagnostic markers

[0241] The lipidome analyses in Aim 1 are via a highly sensitive detection method, GS-MS. To test usefulness of these targets as diagnostic markers that can be evaluated rapidly, colorimetric assays is optimized to test if these are present in stool at levels sufficient for detection to indicate presence of IBS. Assay results are checked against (i) medical data to confirm accuracy (e.g. false negatives / positives) and (ii) dietary surveys. The large number (N=300) of stool samples and surveys collected help in testing accuracy of assay outcomes.

[0242] (A) Colorimetric assays to detect fatty acids

[0243] The second study uses an assay designed by Mackenzie et al for detection of palmitic acid and linolenic acid at different wavelengths (Mackenzie et al., Journal of lipid research, 8, 589-597). In this assay, rhodamine B and uranyl ion are used to form a colored complex with free FAs. This complex is extractable into benzene or toluene and can be measured spectrophotometrically. The method was described for plasma samples. The present study successfully modified this assay to work with stool samples. Fresh stool samples (100 mg) of healthy controls (n=3) and IBS patients (n=3) were tested. A palmitic acid (PA) assay was performed and found that IBS patients had higher levels of PA when measured by this assay. This result is further validated using a larger pool of patient samples

[0244] (B) Assay optimization

[0245] In the case where the assay results are not reproducible or the sensitivity is low, assay parameters, e.g. reagent concentrations, are changed. Palmitic acid could be a better diagnostic biomarker for IBS-C and IBC-M patients than IBS-D based on the difference as compared to controls. However, gamma-linolenic acid could be a good biomarker to discern IBS-D from controls (Fig. 7D). For practical purposes, the second study collects the samples in Norgen stabilizer and store them at -80°C until analyzed. These samples are suitable for GC-MS, but stabilizer may interfere with the assays. Thus, the second study also collects samples on cotton swabs (without stabilizer), which have been found to give reproducible results. The second study uses both sets of samples for the assays. In addition, the second study uses a paper-based colorimetric assay for the detection of linoleic and linolenic acids (Zhang et al., Sensors (Basel), 18, 2018). This test is based on the combination of the highly selective binding of Ag+to olefinic bonds and Ag+mediated color variation of 3, 3’, 5,5’,- tetramethylbenzidine. The second study standardizes it for fecal samples.

[0246] (C) Assessment of assay outcomes for accuracy and influence of HFCS consumption

[0247] Colorimetric assay results are assessed against the respective patient data for presence / absence of IBS. The large number (N=300) of samples helps the evaluation of the accuracy of the assay. The FA levels are also checked against the collected surveys for the relationship to HFCS consumption. In the first study, it was observed that HFCS intake was significantly higher in IBS patients compared to control subjects (p>0.0001). Which foods contributed to HFCS in individual IBS subtypes was also identified. For example, certain beverages contributed to the highest HFCS consumption in the IBS-diarrhea patients.

[0248] The assay is rapid with simple steps; a large number of samples can thus be readily assayed. Based on the first study, it is expected that palmitic acid and gamma-linolenic acid are consistently and significantly changed across IBS samples. It is also expected that stratifying the assay outcomes for the consumption of HFCS will have practical value for the use of these FAs as diagnostic biomarkers as their levels are likely influenced by dietary habits.

[0249] ENUMERATED EMBODIMENTS

[0250] In some aspects, the present invention is directed to the following non-limiting embodiments:

[0251] Embodiment 1: A method of diagnosing irritable bowel syndrome (IBS) in a subject, the method comprising: determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a first reference profile indicating an absence of IBS or a second reference profile indicating IBS, wherein the subject is diagnosed to have IBS when: a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile; or a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile.

[0252] Embodiment 2: The method of Embodiment 1, further comprising: obtaining the stool sample from the subject.

[0253] Embodiment 3: The method of any one of Embodiments 1-2, wherein the subject is diagnosed with IBS when a level of one or more saturated fatty acids in the fatty acid profile is higher than that in the first reference profile, and / or a level of one of more polyunsaturated fatty acids in the fatty acid profile is lower than that in the first reference profile.

[0254] Embodiment 4: The method of any one of Embodiments 1-3, wherein a level of one or more saturated fatty acids in the second profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when a level of one or more saturated fatty acids in the fatty acid profile is higher than or equal to that in the second reference profile, and / or a level of one of more polyunsaturated fatty acids in the second profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of one or more polyunsaturated fatty acids in the fatty acid profile is equal to or lower than that in the second reference profile.

[0255] Embodiment 5: The method of any one of Embodiments 1-4, wherein the at least one fatty acid comprises palmitic acid (16:0), margaric acid (17:0), linoleic acid (18:2n6), gamma-linoleic acid (18:3n6), and / or alpha-linolenic acid (18:3n3).

[0256] Embodiment 6: The method of any one of Embodiments 1-5, wherein the subject is diagnosed with IBS when a level of palmitic acid (16:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level, a level of margaric acid (17:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level, a level of linoleic acid (18:2n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level, a level of gamma-linoleic acid (18:3n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level, and / or a level of alpha-linolenic acid (18:3n3) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

[0257] Embodiment 7: The method of any one of Embodiments 1-6, wherein a level of palmitic acid (16:0) in the second reference profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of palmitic acid (16:0) in the fatty acid profile is equal to or higher than that of the second reference profile, a level of margaric acid (17:0) in the second reference profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of margaric acid (17:0) in the fatty acid profile is equal to or higher than that of the second reference profile, a level of linoleic acid (18:2n6) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of linoleic acid (18:2n6) in the fatty acid profile is equal to or lower than that of the second reference profile, a level of gamma-linoleic acid (18:3n6) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of gamma-linoleic acid (18:3n6) in the fatty acid profile is equal to or lower than that of the second reference profile, and / or a level of alpha-linolenic acid (18:3n3) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of alpha-linolenic acid (18:3n3) in the fatty acid profile is equal to or lower than that of the second reference profile.

[0258] Embodiment 8: The method of any one of Embodiments 1-7, wherein the second reference profile comprises an IBS-D profile indicating irritable bowel syndrome-diarrhea (IBS-D), an IBS-M profile indicating irritable bowel syndrome-mixed (IBS-M), and or an IBS-C profile indicating irritable bowel syndrome-constipation (IBS-C).

[0259] Embodiment 9: The method of any one of Embodiments 1-8, wherein determining a fatty acid profile in the stool sample comprises analyzing the stool sample with a chromatography method, a mass spectrometry method, a fluorometric method, or a colorimetric method.

[0260] Embodiment 10: A method of treating IBS in a subject in need thereof, comprising: confirming that the subject suffers from IBS using the method of any one of Embodiments 1-9, and administering to the subject a treatment for IBS.

[0261] Embodiment 11 : The method of Embodiment 10, wherein the treatment for IBS comprises an anti diarrhoeal, a gut antispasmodic, a laxative, a nerve pain medication, an antibiotic, a dietary supplement, a prebiotic, and / or a probiotic.

[0262] Embodiment 12: The method of any one of Embodiments 10-11, wherein the treatment for IBS comprises alosetron, amitriptyline, atropine, bisacodyl, bismuth subsalicylate, dicyclomine, diphenoxylate, doxepin, eluxadoline, hyoscyamine, a Lactobacillus (optionally Lactobacillus acidophilus), lactulose, linaclotide, loperamide, lotronex, lubiprostone, polyethylene glycol 3350, psyllium seed husk, and / or rifaximin.

[0263] Embodiment 13: A composition comprising: at least one selected from the group consisting of a Monoglobaceae bacterium, a Lachnospiraceae bacterium, and a Ruminococcaceae bacterium; and a pharmaceutically acceptable carrier. Embodiment 14: The composition of Embodiment 13, comprising two or more of the group consisting of the Monoglobaceae bacterium, the Lachnospiraceae bacterium, and the Ruminococcaceae bacterium.

[0264] Embodiment 15: A method of treating IBS in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of Embodiments 13-14.

[0265] Embodiment 16: The method of Embodiment 15, further comprising administering to the subject an effective amount of an anti diarrhoeal, a gut antispasmodic, a laxative, a nerve pain medication, an antibiotic, a dietary supplement, a prebiotic, and / or a probiotic.

[0266] Embodiment 17: The method of any one of Embodiments 15-16, further comprising administering to the subject an effective amount of alosetron, amitriptyline, atropine, bisacodyl, bismuth subsalicylate, dicyclomine, diphenoxylate, doxepin, eluxadoline, hyoscyamine, a Lactobacillus (optionally Lactobacillus acidophilus), lactulose, linaclotide, loperamide, lotronex, lubiprostone, polyethylene glycol 3350, psyllium seed husk, and / or rifaximin.

[0267] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A method of diagnosing irritable bowel syndrome (IBS) in a subject, the method comprising: determining a fatty acid profile in a stool sample of the subject; and comparing the fatty acid profile with a first reference profile indicating an absence of IBS or a second reference profile indicating IBS, wherein the subject is diagnosed to have IBS when: a level of at least one fatty acid in the fatty acid profile is different than a level of the at least one fatty acid in the first reference profile; or a level of at least one fatty acid in the fatty acid profile matches a level of the at least one fatty acid in the second reference profile.

2. The method of claim 1, further comprising: obtaining the stool sample from the subject.

3. The method of claim 1 or 2, wherein the subject is diagnosed with IBS when a level of one or more saturated fatty acids in the fatty acid profile is higher than that in the first reference profile, and / or a level of one of more polyunsaturated fatty acids in the fatty acid profile is lower than that in the first reference profile.

4. The method of any one of claims 1-3, wherein a level of one or more saturated fatty acids in the second profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when a level of one or more saturated fatty acids in the fatty acid profile is higher than or equal to that in the second reference profile, and / or a level of one of more polyunsaturated fatty acids in the second profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of one or more polyunsaturated fatty acids in the fatty acid profile is equal to or lower than that in the second reference profile.

5. The method of any one of claims 1-4, wherein the at least one fatty acid comprises palmitic acid (16:0), margaric acid (17:0), linoleic acid (18:2n6), gamma-linoleic acid (18:3n6), and / or alpha-linolenic acid (18:3n3).

6. The method of any one of claims 1-5, wherein the subject is diagnosed with IBS when a level of palmitic acid (16:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level, a level of margaric acid (17:0) in the fatty acid profile is higher than that in the first reference profile and / or matches that in the second reference level, a level of linoleic acid (18:2n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level, a level of gamma-linoleic acid (18:3n6) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level, and / or a level of alpha-linolenic acid (18:3n3) in the fatty acid profile is lower than that in the first reference profile and / or matches that in the second reference level.

7. The method of any one of claims 1-6, wherein a level of palmitic acid (16:0) in the second reference profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of palmitic acid (16:0) in the fatty acid profile is equal to or higher than that of the second reference profile, a level of margaric acid (17:0) in the second reference profile is higher than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of margaric acid (17:0) in the fatty acid profile is equal to or higher than that of the second reference profile, a level of linoleic acid (18:2n6) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of linoleic acid (18:2n6) in the fatty acid profile is equal to or lower than that of the second reference profile, a level of gamma-linoleic acid (18:3n6) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of gamma-linoleic acid (18:3n6) in the fatty acid profile is equal to or lower than that of the second reference profile, and / ora level of alpha-linolenic acid (18:3n3) in the second reference profile is lower than that in the first reference profile, and the subject is diagnosed with IBS when the level of the level of alpha-linolenic acid (18:3n3) in the fatty acid profile is equal to or lower than that of the second reference profile.

8. The method of any one of claims 1-7, wherein the second reference profile comprises an IBS-D profile indicating irritable bowel syndrome-diarrhea (IBS-D), an IBS-M profile indicating irritable bowel syndrome-mixed (IBS-M), and or an IBS-C profile indicating irritable bowel syndrome-constipation (IBS-C).

9. The method of any one of claims 1-8, wherein determining a fatty acid profile in the stool sample comprises analyzing the stool sample with a chromatography method, a mass spectrometry method, a fluorometric method, or a colorimetric method.

10. A method of treating IBS in a subject in need thereof, comprising: performing the method of any one of claims 1-9 to determine that the subject has IBS, and administering to the subject a treatment for IBS.

11. The method of claim 10, wherein the treatment for IBS comprises an anti diarrhoeal, a gut antispasmodic, a laxative, a nerve pain medication, an antibiotic, a dietary supplement, a prebiotic, and / or a probiotic.

12. The method of any one of claims 10-11, wherein the treatment for IBS comprises alosetron, amitriptyline, atropine, bisacodyl, bismuth subsalicylate, dicyclomine, diphenoxylate, doxepin, eluxadoline, hyoscyamine, a Lactobacillus (optionally Lactobacillus acidophilus), lactulose, linaclotide, loperamide, lotronex, lubiprostone, polyethylene glycol 3350, psyllium seed husk, and / or rifaximin.

13. A composition comprising: a bacterium selected from the group consisting of a Monoglobaceae bacterium, a Lachnospiraceae bacterium, and a Ruminococcaceae bacterium; and a pharmaceutically acceptable carrier.

14. The composition of claim 13, comprising two or more of the bacteria.

15. A method of treating IBS in a subject in need thereof, comprising administering to the subject an effective amount of the composition of any one of claims 13-14.

16. The method of claim 15, further comprising administering to the subject an effective amount of an anti diarrhoeal, a gut antispasmodic, a laxative, a nerve pain medication, an antibiotic, a dietary supplement, a prebiotic, and / or a probiotic.

17. The method of any one of claims 15-16, further comprising administering to the subject an effective amount of alosetron, amitriptyline, atropine, bisacodyl, bismuth subsalicylate, dicyclomine, diphenoxylate, doxepin, eluxadoline, hyoscyamine, a Lactobacillus (optionally Lactobacillus acidophilus), lactulose, linaclotide, loperamide, lotronex, lubiprostone, polyethylene glycol 3350, psyllium seed husk, and / or rifaximin.

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