Immune-modulatory effects of branched chain fatty acids

Abstract

The present disclosure provides products and methods for treating disorders associated with aberrant inflammation in a subject, including in the brain and periphery of a subject. The subject may be administered a branched chain fatty acid (BCFA) in an effective amount for promoting an anti-inflammatory response in a tissue of the subject. The tissue may be one that is not part of the gastrointestinal tract. In some aspects the products are useful for slowing the progression of or preventing the development of or for treating neurodegenerative disorders such as Alzheimer's Disease, cardiovascular disease, rheumatoid arthritis or infectious disease.

Description

[0001] IMMUNE-MODULATORY EFFECTS OF BRANCHED CHAIN FATTY ACIDS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of U.S. Provisional Application Serial No. 63 / 678,146, entitled “IMMUNE-MODULATORY EFFECTS OF BRANCHED CHAIN FATTY ACIDS” and filed on August 1, 2024, the entire contents of which are incorporated herein by reference

[0004] BACKGROUND

[0005] Short chain fatty acids (SCFAs) are bacterial fermentation byproducts which play a role in human health. Bacterial fermentation of dietary fibers are the major source of SCFAs, however, other dietary nutrients such as proteins can also be metabolized by bacteria to form SCFAs (Kim C. (2021 ) Cell Mol Immunol 18:1161-1171). SCFAs have been associated with various health benefits, including anti-inflammatory effects (Vinolo et al., (2011) Regulation of Inflammation by Short Chain Fatty Acids. Nutrients 3:858-876), as well as supporting brain health and cognition (Han et al., (2014) Neuropharmacology 81:292-302; Liu et al., (2017). J Neurol Sci 381:176-181; Sun et al., (2019) Transl Psychiatry 9:1-13).

[0006] SUMMARY

[0007] Branched chain fatty acids (BCFAs), e.g., isobutyric and isovaleric acid, and 2- methylbutyric acid are generated by bacterial fermentation of branched amino acids in the body from undigested protein. The effect of BCFAs in health and disease, however, unlike the effect of SCFAs, is not fully understood.

[0008] In aspects of the current disclosure, a method for promoting an anti-inflammatory response in a subject in need thereof is provided. The method involves administering to the subject a branched chain fatty acid (BCFA) in an effective amount for promoting an antiinflammatory response in a tissue of the subject, wherein the tissue is not a tissue of the gastrointestinal tract, wherein the BCFA is not conjugated to another active agent and, optionally wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2- methylbutyric acid, derivatives, analogs and isomers and salts thereof. In some embodiments the BCFA comprises isobutyric acid, isovaleric acid, and / or 2-methylbutyric acid.

[0009] In some embodiments the tissue is brain tissue, and wherein the subject has a neurodegenerative disorder such as Alzheimer’s disease or Parkinson’s disease. In some embodiments the subject has cardiovascular disease, optionally wherein the cardiovascular disease is atherosclerosis. In some embodiments the subject has rheumatoid arthritis, Type I diabetes mellitus, or an infectious disease.

[0010] In some embodiments the subject is identified as APOE4 positive prior to administration of the BCFA.

[0011] In some embodiments the BCFA is administered in a composition and wherein the composition comprises a pharmaceutically acceptable carrier. In some embodiments the composition is a medical grade food composition, a food supplement, a sustained release formulation, or a capsule or tablet.

[0012] In some embodiments the BCFA is administered in a composition comprising a BCFA producing microorganism. In some embodiments the BCFA producing microorganism is an engineered microorganism having at least one heterologous gene. In some embodiments the BCFA producing microorganism is of the genus Escherichia or Lactobacillus, and optionally wherein the BCFA producing microorganism is Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus or Escherichia coli Nissle. In some embodiments the BCFA producing microorganism is administered at a dose of about IxlO11to about 2xl012live microorganism once per day, twice per day or three times per day.

[0013] In some aspects of the disclosure a method for treating a neurodegenerative disorder, by administering to a subject having a neurodegenerative disorder a branched chain fatty acid (BCFA) in an effective amount for treating the disorder is provided. In some embodiments the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

[0014] A method for treating atherosclerosis, comprising, consisting of or consisting essentially of administering to a subject having atherosclerosis a branched chain fatty acid (BCFA) in an effective amount for treating the disorder, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid is provided in other aspects of the disclosure.

[0015] In some aspects a method for treating an inflammatory disorder comprising, consisting of or consisting essentially of administering to a subject having a non- gastrointestinal (GI) inflammatory disorder a branched chain fatty acid (BCFA) in an effective amount for treating the non-GI inflammatory disorder, and optionally, wherein the non-GI inflammatory disorder is selected from rheumatoid arthritis, Type I diabetes mellitus, infectious disease, or a metabolic disorder characterized by chronic inflammation and / or lipid dysregulation, such as obesity is provided. In some embodiments the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid, derivatives, analogs and isomers and salts thereof.

[0016] In some aspects a pharmaceutical composition comprising, consisting of or consisting essentially of isobutyric acid, isovaleric acid, and 2-methylbutyric acid in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition comprises a medical food or dietary supplement, and optionally the isobutyric acid, isovaleric acid, and 2-methylbutyric acid are formulated as a BCFA producing microorganism, wherein the BCFA producing microorganism is an engineered microorganism having at least one heterologous gene encoding one or more proteins involved in BCFA synthesis or maintenance, or prevention of degradation of BCFA is provided.

[0017] In some aspects a method for treating an inflammatory disorder, comprising administering to a subject having an inflammatory disorder selected from a neurodegenerative disorder, atherosclerosis, rheumatoid arthritis, Type I diabetes mellitus, or an infectious disease a branched chain fatty acid (BCFA) in an effective amount for treating the disorder, wherein the BCFA is administered in a composition comprising a BCFA producing microorganism is provided.

[0018] In some embodiments the BCFA producing microorganism is an engineered microorganism having at least one heterologous gene. In some embodiments the BCFA producing microorganism is of the genus Escherichia or Lactobacillus, optionally wherein the BCFA producing microorganism is Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus or Escherichia coli Nissle. In some embodiments the BCEA producing microorganism is administered at a dose of about IxlO11to about 2xl012live microorganism once per day, twice per day or three times per day.

[0019] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims. BRIEF DESCRIPTION OF DRAWINGS

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIGs. 1A-1D: Data demonstrating that branched chain fatty acids (BCFAs) have immune-modulatory effects in peripheral mouse bone marrow derived macrophages. 1A is a diagram depicting in vitro experiment using wildtype primary mouse bone-marrow derived macrophages (BMDMs) stimulated withlOO ng / mL of bacterial LPS, to stimulate immune responses, then with or without 20 pM SCFAs for 24 hours (Illustration created in BioRender.com). IB is a set of graphs showing results of an ELISA assay demonstrating that 24 hours after the treatment with BCFAs secretion of both IL-6 and CCL2 were decreased. FIG. 1C shows a set of graphs showing results of q-RT PCR demonstrating that BCFAs decreased proinflammatory genes (TNF-alpha, iNOS), and increased antiinflammatory / homeostatic gene markers (IL-10, CX3R1). FIG. ID shows a set of bar graphs demonstrating that the Alzheimer’s disease relevant genes APOE and TREM2 were decreased by 2-methylbutyric acid. PBS = control, B = isobutyric acid, V = isovaleric acid, 2-M = 2-methylbutyric acid.

[0021] FIGs. 2A-2D: Bulk RNA sequencing of treated macrophages revealed potent immune-modulatory effects in multiple gene sets. 2A shows a diagram depicting an in vitro experiment using bone marrow derived macrophages (Illustration created in BioRender.com). Macrophage cultures were challenged using LPS with or without 20 pM of branched chain fatty acid. RNA was isolated after 24 hours and subjected to bulk RNA sequencing. 2B depicts the results of a gene set enrichment analysis (GSEA), revealing that in comparing the BCFA treated condition to the control condition, there was an across-the-board significant decrease in gene sets related to multiple immune, infection, and disease / disorder pathways. 2C shows a Volcano plot of the bulk RNA sequencing gene dataset showing the False Discovery Rate (FDR) corrected p value (all genes above the horizontal bar have a corrected p value less than or equal to 0.05). Comparisons are showing those genes that were significantly decreased (dark gray dots surrounding left vertical dashed line) or significantly increased (dark gray dots surrounding right vertical dashed line) in the BCFA treated condition versus the control condition. Overall, 11696 genes were detected and 134 genes significantly altered by the BCFA treatment (dark gray dots). All non-significant genes are shown in light gray. 2D is a Table showing a list of the top 10 significant differentially expressed genes (DEGs) that were either increased or decreased in the BCFA condition versus control condition, and include decrease of immune-regulatory genes (e.g. 116), and increase of genes related to cholesterol homeostasis (e.g. Ncehl). FIGs. 3A-3D: BCFAs exert an immune-modulatory effect in microglia, the resident macrophages of the brain. 3A shows a diagram depicting an in vitro experiment using human inducible microglia-like cells (iMGLs) (Illustration created in BioRender.com). 3B depicts a study using qRT-PCR to demonstrate that isovaleric acid increased gene expression of the homeostatic markers ARG1, CX3CR1, P2RY12, and increased the anti-inflammatory cytokine IL-10. 3C depicts data demonstrating that gene expression of Alzheimer’s and Parkinson’s disease relevant genes APOE, TREM2, LRRK2, and inflammatory / immune related genes MMP9 and TYROBP were all decreased by isovaleric acid treatment. N.D. = not detectable, suggesting potent inhibition of gene transcription. 3D shows a graph demonstrating that both isovaleric acid and 2-methylbutyric acid significantly increased levels of secreted total cholesterol, suggesting restored cholesterol homeostasis during LPS stimulation.

[0022] FIGs. 4A-4B: BCFAs reduce lipid droplet accumulation in activated peripheral macrophages. An in vitro experiment using primary mouse bone marrow-derived macrophages (BMDMs) stimulated with 100 ng / mL of bacterial LPS was run, followed by treatment with individual or combined BCFAs (isobutyric acid, isovaleric acid, or 2- methylbutyric acid) for 24 hours. Immunocytochemistry of Plin2+ lipid droplets across treatment groups was performed. FIGs. 4A-4B show graphs demonstrating the number of Plin2+ lipid droplets per nucleus (4A) and the mean volume of Plin2+ droplets (4B), shown as a percent of control. Iso-B = isobutyric acid, Iso-V = isovaleric acid, 2-M = 2- methylbutyric acid, Iso-B+Iso-V+2-M = combination BCFAs. One-way ANOVA with Tukey’s multiple comparisons.

[0023] DETAILED DESCRIPTION

[0024] In contrast to SCFAs, little is known about the function of branched chain fatty acids (BCFAs) outside of the gut. Surprisingly, it has been discovered that BCFAs are able to modulate cells from the central nervous system and the periphery. The data disclosed herein demonstrates that BCFAs promoted expression of homeostatic markers and antiinflammatory molecules, whilst concomitantly decreasing disease-associated genes and pro- inflammatory molecules. These fatty acids have immune modulatory effects that could be used to target aberrant immune activation and inflammation, as well as cholesterol homeostasis and lipid disruption in the periphery and the central nervous system.

[0025] A “Branched Chain Fatty Acid” (“BCFA”), as used herein, refers to a fatty acid containing a carbon constituent branched off the carbon chain. Typically, the branch is an alkyl branch, especially a methyl group, but ethyl and propyl branches are also known. BCFAs are an important component in the human gut for establishing bacteria during colonization, as BCFAs are prominent membrane component of many bacterial species. However, BCFAs are only found in trace amounts in foods.

[0026] BCFAs include but are not limited to isobutyric acid, isovaleric acid, and 2- methylbutyric acid, derivatives, analogs and isomers and salts thereof.

[0027] Isobutyric acid is also known as 2-methylpropanoic acid or isobutanoic acid. It is a short-chain fatty acid comprised of a carboxylic acid with structural formula (CHahCHCOOH. Isobutyric acid is an isomer of butyric acid. Isobutyric acids, as used herein include Isobutyric acid, derivatives, analogs and isomers and salts thereof. It is produced by the oxidation of isobutyraldehyde, which is a byproduct of the hydroformylation of propylene and has the following structure:

[0028] Isovaleric acid is also known as 3 -methylbutanoic acid or P -methylbutyric acid. It is a short-chain fatty acid comprised of an alkyl carboxylic acid with structural formula (CHa CHCFhCC H. Isovaleric acids, as used herein include isovaleric acid, derivatives, analogs and isomers and salts thereof. Isovaleric acid is produced by hydroformylation of isobutylene with syngas, forming isovaleraldehyde, which is oxidized to the final product and has the following structure:

[0029] 2-methylbutyric acid is also known as 2-Methylbutanoic acid or Methylethylacetic acid. It is a methylbutyric acid comprising a butyric acid core carrying a 2-methyl substituent with structural formula C5H10O2. 2-methylbutyric acid is produced by the oxidation of isobutyraldehyde, which is a byproduct of the hydroformylation of propylene. 2- methylbutyric acids, as used herein include 2-methylbutyric acid, derivatives, analogs and isomers and salts thereof. 2-methylbutyric acid has the following structure:

[0030] A BCFA analog, such as an analog of Isobutyric acid, Isovaleric acid, and 2- methylbutyric acid include structural modifications and thus mimic the structure of naturally occurring fatty acids but have been chemically modified. In some embodiments the BCFA analogs do not significantly alter the function of the BFCA. Analogs include, for instance, fluorescent fatty acid analogs, which have a fluorophore linked within the fatty acid chain or, more commonly, at the terminal (omega) carbon atom that is furthest from the carboxylate moiety. Other analogs include structures having different saturation or functional groups.

[0031] In some embodiments the BCFA in the composition is purified. The term “purified” when used in relation to BCFA such as a “purified BCFA” refers to BCFAs that are separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified BCFA is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method. In some embodiments, the BCFA composition comprises a formulation wherein the BCFA(s) are the only fatty acids in the composition. In some embodiments, the BCFA composition is not an enriched lipid composition or fraction. In some embodiments, the BCFA composition comprises a formulation wherein the BCFA(s) are the only lipids in the composition. In some embodiments, the BCFA composition comprises a formulation wherein the BCFA(s) are the only active agent in the composition. In some embodiments, the BCFA is not conjugated to an active agent. An active agent in this context refers to an agent that has the ability to produce a specific effect or action on the same target as the BCFA. In some embodiments, the BCFA is not conjugated to any agent. The term “conjugated” as used herein refers to a covalent linkage. In some embodiments, the BCFA is not linked to an active agent or to any agent. The term “linked” as used herein refers to a covalent or non-covalent linkage.

[0032] Methods for formulating the BCFAs into a pharmaceutical composition, medical food or dietary supplement are encompassed within the disclosure. Multiple methods for processing are available and may include any one or more of the following steps: formulating the preparation into a drug product or a food supplement, packaging the composition, and / or labeling the packaged composition. Methods for formulating lipids into a drug product or food supplement are available and may include one or more of the following steps: (i) removing unwanted constituents from the preparation, (ii) reducing the volume of the preparation, (iii) sterilizing the preparation, (iv) admixing the preparation with a pharmaceutically acceptable excipient or carrier, (v) admixing the preparation with a second drug or pharmaceutical agent, (vi) formulating the preparation into a suitable consistency, such as, e.g., aqueous diluted solution, a syrup or a solid, (vii) formulating the preparation into a suitable dosage form, e.g. into a tablet, pill or capsule.

[0033] In some embodiments, the BCFAs may be further processed to a food product, therapeutic syrup or powder, or encapsulated in a gel or similar ingestible. Provided herein are pharmaceutical compositions, medical foods and dietary supplements comprising therapeutic preparations of BCFAs. Optionally, the pharmaceutical compositions, medical foods and dietary supplements comprising BCFA therapeutic preparations further comprise two or more BCFAs and / or additional therapeutic agents for treating a disease as disclosed herein. In some embodiments it is particularly desirable to formulate the BCFAs to neutralize any undesirable odors and / or tastes. In one embodiment this is achieved by encapsulating the BCFA, using known techniques.

[0034] In some embodiments, the pharmaceutical compositions and medical foods and dietary supplements comprising BCFA therapeutic preparations further comprise a micronutrient. In some embodiments, the micronutrient is selected from the group consisting of a trace mineral, choline, a vitamin, and a polyphenol. In some embodiments, the micronutrient is a trace metal. Trace minerals suitable as a micronutrient include, but are not limited to, boron, cobalt, chromium, calcium, copper, fluoride, iodine, iron, magnesium, manganese, molybdenum, selenium, and zinc. In some embodiments, the micronutrient is a vitamin.

[0035] In some embodiments, the composition of BCFAs comprises a mixture of two or more BCFAs selected isobutyric acid, isovaleric acid, and 2-methylbutyric acid. In some embodiments the two or more BCFAs may be included in any desired amount (e.g. anywhere from 1-99% of one BCFA and 99-1% of another BCFA, not exceeding 100%). In some embodiments the isobutyric acid, isovaleric acid, and 2-methylbutyric acid are included in the composition in one of the following ratios (isobutyric acid:isovaleric acid:2-methylbutyric acid): 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, :8:1, 1:9:1, 0:0:1, 0:1:0, 0:1:1, 0:1:4, 0:1:9, 0:2:3, 0:3:2, 0:3:7, 0:4:1, 0:7:3, 0:9:1, 1:0:0, 1:0:1, 1:0:4, 1:0:9, 1:1:0, 1:1:3, 1:1:8,

[0036] 1:2:2, 1:2:7, 1:3:1, 1:3:6, 1:4:0, 1:4:5, 1:5:4, 1:6:3, 1:7:2, 1:8:1, 1:9:0, 2:0:3, 2:1:2, 2:1:7,

[0037] 2:2:1, 2:3:0, 2:3:5, 2:5:3, 2:7:1, 3:0:2, 3:0:7, 3:1:1, 3:1:6, 3:2:0, 3:2:5, 3:3:4, 3:4:3, 3:5:2,

[0038] 3:6:1, 3:7:0, 4:0:1, 4:1:0, 4:1:5, 4:3:3, 4:5:1, 5:1:4, 5:2:3, 5:3:2, 5:4:1, 6:1:3, 6:3:1, 7:0:3,

[0039] 7:1:2, 7:2:1, 7:3:0, 8:1:1, 9:0:1, 9:1:0.

[0040] In some embodiments, the BCFA composition may undergo further processing to produce either BCFA syrup or powder. For example, in one variation, the BCFA composition is concentrated to form a syrup or other gel-like substance (especially in the case of encapsulation). Any suitable methods known in the art to concentrate a solution may be used, such as the use of a vacuum evaporator. In another embodiment, the BCFA composition may be spray dried to form a powder. Any suitable methods known in the art to spray dry a solution to form a powder may be used.

[0041] Provided herein are pharmaceutical compositions, medical foods and dietary supplements comprising BCFA compositions. The pharmaceutical compositions and medical foods and dietary supplements comprising the BCFA composition may include one or more excipients or carriers, including diluents, binders, disintegrants, dispersants, lubricants, glidants, stabilizers, surfactants, flavoring agents, and colorants.

[0042] Further, if desired, the pharmaceutical compositions and medical foods and dietary supplements comprising BCFA compositions may comprise therapeutically active agents. Alternatively, or in addition, therapeutically active agents may be administered separately (e.g. prior to, concurrent with or after administration of the BCFA composition) and not as a part of the pharmaceutical composition or medical food or dietary supplement (e.g. as a coformulation) of BCFA.

[0043] Pharmaceutically acceptable excipients (carriers) including buffers, are well known in the art. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™ (polysorbate), PLURONICS™ (poloxamers) or polyethylene glycol (PEG).

[0044] In one embodiment, pharmaceutical formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, and intraarticular, intraparenchymal), inhalation (including fine particle dusts or mists which may be generated by means of various types of metered doses, pressurized aerosols, nebulizers or insufflators), rectal and topical (including dermal, transdermal, transmucosal, buccal, sublingual, and intraocular) administration, although the most suitable route may depend upon, for example, the condition and disorder of the recipient.

[0045] The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

[0046] Exemplary compositions for oral administration include suspensions which can contain, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavoring agents such as those known in the art; and immediate release tablets which can contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and / or lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the art. The BCFA can also be delivered through the oral cavity by sublingual and / or buccal administration. Molded tablets, compressed tablets, gel encapsulations, or freeze-dried tablets are exemplary forms which may be used. Exemplary compositions include those formulating the present BCFAs with fast dissolving diluents such as mannitol, lactose, sucrose and / or cyclodextrins. Also included in such formulations may be high molecular weight excipients such as celluloses (avicel) or polyethylene glycols (PEG). Such formulations can also include an excipient to aid mucosal adhesion such as hydroxy propyl cellulose (HPC), hydroxy propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleic anhydride copolymer (e.g., Gantrez), and agents to control release such as polyacrylic copolymer (e.g. Carbopol 934). Lubricants, glidants, flavors, coloring agents and stabilizers may also be added for ease of fabrication and use. Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents, thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example saline or water-for-injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Exemplary compositions for parenteral administration include injectable solutions or suspensions which can contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3- butanediol, water, Ringer's solution, an isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids, including oleic acid, or Cremophor.

[0047] An aqueous carrier may be, for example, an isotonic buffer solution at a pH of from about 3.0 to about 8.0, preferably at a pH of from about 3.5 to about 7.4, for example from 3.5 to 6.0, for example from 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate / acetic acid buffers. Excipients that can be included are, for instance, non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0048] Exemplary compositions for nasal aerosol or inhalation administration include solutions in saline, which can contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and / or other solubilizing or dispersing agents such as those known in the art. Conveniently in compositions for nasal aerosol or inhalation administration the BCFA of the disclosure is delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoro-methane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated to contain a powder mix of the BCFA and a suitable powder base, for example lactose or starch. In one specific, nonlimiting example, a BCFA of the disclosure is administered as an aerosol from a metered dose valve, through an aerosol adapter also known as an actuator. Optionally, a stabilizer is also included, and / or porous particles for deep lung delivery are included.

[0049] Formulations for rectal administration may include a retention enema or a suppository with carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycol. Such carriers are typically solid at ordinary temperatures but liquefy and / or dissolve in the rectal cavity to release the drug.

[0050] Formulations for topical administration in the mouth, for example buccally or sublingually, include lozenges comprising the active ingredient in a flavored composition including ingredients such as sucrose and acacia or tragacanth, and pastilles having an active ingredient such as gelatin and glycerin or sucrose and acacia. Exemplary compositions for topical administration include a topical carrier such as mineral oil gelled with polyethylene.

[0051] Unit dosage formulations contain an effective dose. In addition to the disclosed ingredients, the formulations may include other agents conventional in the art, for example those suitable for oral administration may include flavoring agents.

[0052] The BCFAs may also be administered as sustained-release systems. Sustained-release systems may be administered orally; rectally; parenterally; intracistemally; intravaginally; intraperitoneally; topically, for example as a powder, ointment, gel, drop or transdermal patch; bucally; or as an oral or nasal spray.

[0053] The BCFAs may be delivered by way of a pump or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The key factor in selecting an appropriate dose is the result obtained, as measured by improvements in one or more symptoms of neurodegenerative or other disorders of interest, or by other criteria for measuring control or prevention of one or more symptoms of disorders of interest, as are deemed appropriate by the practitioner. Implantable drug infusion devices are used to provide patients with a constant and long term dosage or infusion of a drug or any other therapeutic agent. Such devices may be categorized as either active or passive. The BCFAs may be formulated as a depot preparation. Such a long acting depot formulation can be administered by implantation, for example subcutaneously or intramuscularly; or by intramuscular injection. Methods of Use

[0054] BCFA compositions and formulations are useful for a variety of therapeutic purposes, including methods for promoting an anti-inflammatory response and treating neurodegenerative diseases or disorders, including but not limited to Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, multiple sclerosis, amyotrophic lateral sclerosis (ALS), other conditions involving neuroinflammation, cardiovascular disease such as atherosclerosis, rheumatoid arthritis, Type I diabetes mellitus, metabolic disorders characterized by inflammation and lipid dysregulation, such as obesity, and infectious disease. The BCFAs may be administered using any means known in the art, including inhalation, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, or dermally.

[0055] In some aspects of the disclosure, BCFA compositions are administered to a tissue of a subject in an effective amount for promoting an anti-inflammatory response in a tissue of the subject. The tissue may, in some embodiments, be any tissue other than a tissue of the gastrointestinal tract. A tissue of the gastrointestinal tract, as used herein, is the epithelium lining the gastrointestinal tract, including the pharynx, esophagus, stomach, small intestine, colon, rectum. Other tissues include epithelial tissue of the skin, connective tissue, muscle tissue, and nervous tissue.

[0056] In some aspects of the disclosure, BCFA compositions are administered to a tissue of a subject in an effective amount for promoting an anti-inflammatory response in order to treat an inflammatory disorder. In some embodiments an inflammatory disorder includes but is not limited to a fatty liver disease, endometriosis, Type 1 or 2 diabetes mellitus, inflammatory bowel disease (IBD), asthma, rheumatoid arthritis, obesity, neurogenerative disorders such as Alzheimer’s and Parkinson’s diseases. In some embodiments, inflammatory disorder excludes inflammatory gastrointestinal disorders such as IBD, and is thus a non-GI inflammatory disorder.

[0057] As used herein, the term treat, treated, or treating when used with respect to a disorder refers to a prophylactic treatment which increases the resistance of a subject to development of the disease or, in other words, decreases the likelihood that the subject will develop the disease as well as a treatment after the subject has developed the disease in order to fight the disease, prevent the disease from becoming worse, or slow the progression of the disease compared to in the absence of the therapy. In some embodiments, the methods are directed to treating or managing neurodegenerative diseases or disorders. In a non-limiting example, the BCFA compositions are administered to a subject diagnosed as having or at risk for developing an amyloid-related neurodegenerative disease or disorder such as Alzheimer's disease, cerebral amyloid angiopathy (CAA), mild cognitive impairment, moderate cognitive impairment, and combinations thereof. The term “amyloidosis,” as used herein, refers to a group of diseases and disorders caused by or associated with amyloid or amyloid- like proteins and includes, but is not limited to, diseases and disorders caused by the presence or activity of amyloid-like proteins in monomeric, fibril, or polymeric state, or any combination of the three, including by amyloid plaques.

[0058] The BCFA of the invention are useful in treating Alzheimer’s disease. Alzheimer's disease is a degenerative brain disorder characterized by cognitive and noncognitive neuropsychiatric symptoms, which accounts for approximately 60% of all cases of dementia for patients over 65 years old. Psychiatric symptoms are common in Alzheimer's disease, with psychosis (hallucinations and delusions) present in many patients.

[0059] In some embodiments the subjects are identified as having or at risk of developing myelination degeneration based on genotype, whether they are APOE4 positive and successfully treated with the compounds described herein. If the subject is APOE4 positive, those subjects are at risk of developing disorders such as CAA or Alzheimer’s disease.

[0060] In some embodiments, the methods are directed to treating or managing diseases or disorders in which inflammation occurs in the brain, such as those diseases associated with learning and / or memory or Alzheimer’s disease. In a non-limiting example, the BCFAs disclosed herein are administered to a patient diagnosed as having or at risk for developing Alzheimer's disease, cerebral amyloid angiopathy (CAA), mild cognitive impairment, moderate cognitive impairment, and combinations thereof.

[0061] The subject may have been diagnosed with or at risk for a disease, such as Alzheimer’s disease. In some embodiments the subject can be treated following diagnosis, at varying stage of the disease, or as a prophylactic measure in instances where genetic traits, family history, or other factors put the patient at risk for the neurodegenerative disease or disorder. Successful dosage amounts and schedules may be established and monitored by metrics indicative of effective treatment, for example the extent of inhibition, delay, prevention or reduction of symptoms such as cognitive decline, loss of myelination in the brain, and neurodegeneration which are detected following the initiation of treatment. In some embodiments the subject is determined to be APOE4 positive. A number of genetic factors in early- and late-onset Alzheimer's disease have been documented. The ApoE4 allele is strongly associated with late-onset familial and sporadic Alzheimer's disease, with a reported allele frequency of 50%-65% in patients with Alzheimer's disease, which is approximately three times that in the general population and for other neurologic disorders. In addition to Alzheimer's disease, the ApoE4 allele has been implicated in other amyloid- forming disorders, including CAA.

[0062] Thus, in some embodiments the methods disclosed herein are useful for treating Alzheimer’s disease. The methods of treatment may alleviate the pathological symptoms of Alzheimer's disease, including and not limited to amyloidp accumulation or aggregation, brain cell aging, and (3) synapse loss. As used herein, the inhibiting accumulation and / or aggregation encompasses inhibiting aggregation by suppressing the production or synthesis of amyloid P and / or inhibiting accumulation by degrading already produced amyloidp.

[0063] The deposition of extracellular amyloid plaques in the brain is a hallmark pathologic finding in Alzheimer’s disease. These amyloid plaques are primarily composed of Abeta peptides generated by the sequential cleavage of amyloid precursor protein ("APP") via P and y-secretase activity. Techniques and tools have been developed to visualize the presence of plaques in patients. For example, position emission tomography ("PET") scans using imaging agents, such18F-florbetapir, that detect amyloid-beta can be used to detect the presence of amyloid in the brain.

[0064] The methods of treatment provided herein can be applied to subjects suffering from Alzheimer's disease. The subject may, in some embodiments have mild to moderate Alzheimer's disease. In other embodiments the subject may have moderate to severe Alzheimer's disease. The severity of the disease can be assessed using a number of diagnostic criteria known in the art, such as biomarkers. For instance, mild Alzheimer's disease or Stage 1 disease may be an asymptomatic patient characterized by PET or CSF positive for amyloid P, a Stage 2 disease may show downstream neurodegeneration biomarkers such as tau, FDG- PET, or structural MRI, and Stage 3 disease may present as amyloidosis plus neuronal injury and cognitive / behavioral decline.

[0065] In some aspects, the methods provided herein are methods of reducing or slowing decline due to Alzheimer's disease in patients suffering from early, mild, or mild to moderate Alzheimer's disease. In some embodiments, the decline is one or more of: clinical decline, cognitive decline, and functional decline. In some embodiments, the decline is a decline in cognitive capacity or cognitive decline. In some embodiments, the decline comprises a decline in functional capacity or functional decline. Various tests and scales have been developed to measure cognitive capacity (including memory) and / or function. In various embodiments, one or more test is used to measure clinical, functional, or cognitive decline. A standard measurement of cognitive capacity is the Alzheimer's Disease Assessment Scale Cognitive (ADAS-Cog) test, for example, the 12-item ADAS-Cog or ADAS-Cogl2, or the 13-item ADAS-Cog or ADAS-Cog- 13. Thus, in some embodiments, the reduction or slowing in decline in cognitive capacity (or cognitive decline) in patients being treated with the BCFAs of the disclosure is determined using the ADAS-Cogl2 test. An increase in ADAS- Cog 12 score is indicative of worsening in a patient's condition. In some embodiments, the reduction or slowing in cognitive decline in patients being treated with the BCFAs of the disclosure is determined by a Clinical Dementia Rating Scale / Sum of Boxes (CDR-SB) score. In some embodiments, reduction or slowing in functional decline (or decline in functional ability) in patients being treated with the BCFAs of the disclosure is determined using the Instrumental Activities of Daily Living (iADL) scale. In some embodiments, decline of one or more types is assessed and one or more of the foregoing tests or scales is used to measure reduction or slowing in decline.

[0066] Amyloid-positive subjects or patients may have brain amyloid load consistent with that seen in patients diagnosed with Alzheimer's disease. A subject suffering from mild cognitive impairment or Alzheimer's disease or having preclinical Alzheimer's disease, prodromal Alzheimer's disease, early or mild Alzheimer's disease, are typically subjects with an MMSE score of 20 or above (e.g., 20-30, 20-26, 24-30, 21-26, 22-26, 22-28, 23-26, 24-26, or 25-26) or with a Clinical Dementia Rating-Global Score (CDR-GS) of 0.5 or 1.0, and subjects with a Free and Cued Selective Reminding Test-Immediate Recall (FCSRT-IR) Cueing Index of 0.67 or above and a total free recall score of 27 or greater.

[0067] Several Alzheimer's disease -risk genes are expressed in cells that constitute the brain and may directly influence the accumulation and clearance of Ap. In particular, Apolipoprotein E (APOE) protein is highly expressed in astrocytes and microglia of the brain. In humans, there are three genetic polymorphisms of APOE, e2, e3, and e4. The e4 isoform of APOE (APOE4) is the most significant known risk factor for CAA and sporadic Alzheimer's disease. In some embodiments, subjects are carriers of at least one ApoE4 allele (“ApoE4 carriers”). As shown in the Examples herein, BCFAs have been demonstrated to reduce expression of significant Alzheimer’s associated genes such as APOE. Alleviating a neurodegenerative disease such as Alzheimer’s Disease, includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method.

[0068] The terms reduce, interfere, inhibit, and suppress refer to a partial or complete decrease in activity levels relative to an activity level typical of the absence of the BCFAs. For instance, the decrease, which may be observed in both RNA and / or protein levels, may be by at least 10%, 20%, 50%, 70%, 85%, 90%, 100%, 150%, 200%, 300%, or 500%, or by 2- fold, 10-fold, 20-fold, 50-fold, 100-fold, or 1000-fold.

[0069] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a neurodegenerative disease includes initial onset and / or recurrence.

[0070] The BCFA may be administered to the brain or other tissue of the patient, either directly or indirectly by administration to the tissue directly or by systemic administration such as oral administration. For instance, the BCFA may be administered directly by intracerebroventricular, intrathecal, intracardiac, or intraparenchymal injection. The BCFA may be administered indirectly to the desired tissue by administration through any route that delivers a BCFA to a body of a subject, including oral administration. In some embodiments the BCFA is administered as an immediate release formulation. In some embodiments the BCFA is administered as a sustained release formulation. In some embodiments the BCFA is administered as a composition comprising for instance a capsule or tablet.

[0071] In some embodiments the BCFA may be administered in a composition comprising a BCFA producing microorganism. The BCFA producing microorganism may be a naturally occurring microorganism that naturally produces BCFA or may be an engineered microorganism that has been manipulated or modified to produce BCFA or to produce higher levels of BCFA. A microorganism, as used herein, includes for instance, bacteria, yeast, viruses etc. The microorganism may be delivered alone or in a composition such as a pharmaceutical composition thereof or a food based composition. In some embodiments, the microorganism, has been genetically engineered to comprise heterologous gene sequences encoding one or more proteins involved in BCFA synthesis or maintenance, or prevention of degradation of BCFA. Proteins involved in BCFA synthesis, maintenance or prevention of degradation play an important role in biological processes that can enhance the therapeutic benefit achieved by the BCFA when delivered in conjunction with the BCFA. Proteins involved in BCFA synthesis include but are not limited to branched-chain a-keto acid dehydrogenase complex (BCKD, a multi-enzyme complex including multiple subunits including decarboxylase (El), dihydrolipoamide acyltransferase (E2), and dihydrolipoamide dehydrogenase (E3)), methylbutyryl-CoA synthetase (MbcS), fatty acid synthase (FAS), and 3-ketoacyl-ACP synthase III (FabH). Proteins involved in BCFA maintenance / regulation include but are not limited to BCAA transporters, BCAA aminotransferases (BCAT), BCKD Kinase (BCKDK), BCKD Phosphatase (PP2Cm), and mTOR signaling pathway enzymes. Proteins involved in prevention of BCFA degradation (inhibition of BCAA catabolism) include but are not limited to BCKD Kinase (BCKDK), ECHDC1, Stringent factor (RelA / SpoT homolog), Sestrin2, and Leucyl-tRNA synthetase (LRS).

[0072] In some embodiments the microorganism is a bacteria such as a bacteria of the genus Escherichia or Lactobacillus. In some embodiments the BCFA producing microorganism is Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus or Escherichia coli Nissle. The microorganism may be administered using any known route for administration or any known dosage. In some embodiments the BCFA producing microorganism is administered at a dose of about IxlO11to about 2xl012live microorganism once per day, twice per day or three times per day. In some embodiments, the microorganism is administered at a dose of about IxlO11live bacteria, about 2xlOnlive bacteria, about 3xlOnlive bacteria, about 4xlOnlive bacteria, about 4.5x 1011live bacteria, about 5xlOnlive bacteria, about 6xlOnlive bacteria, about IxlO12live bacteria, or about 2xl012live bacteria, or any range therebetween, such as about IxlO11- IxlO12, IxlO11-2xl012, IxlO11-2xlOn, IxlO11-3xlOn, 2xlOn-2xl012, 2xlOn-3xlOn, 2xlOn-4xlOn, IxlO11-4xlOn, IxlO11-5xlOn, IxlO11-6xlOn, 6xlOn-2xl012, 4.5x 1011-2xl012, or4.5x 1011-6xlOnlive bacteria. In one embodiment, the administering is once per day. In another embodiment, the administering is twice per day. In another embodiment, the administering is three times per day. The administration may be oral, with or without meals. In some embodiments the bacteria is included in a food or dietary supplement.

[0073] The BCFA compositions disclosed herein are useful in some embodiments for treating infectious disease. Infectious diseases include but are not limited to disease characterized by chronic inflammatory processes that are caused by infectious agents such as viruses, bacteria, fungi, protozoa and parasites. Viral diseases treatable using the BCFA include, but are not limited to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, herpes virus such as herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, cytomegalovirus, echovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, and polio virus, or Epstein Barr virus. In some embodiments the virus, after infecting the subject, remains dormant in cells of the subject, leading to chronic infection and inflammatory conditions. Bacterial diseases include but are not limited to borrelia.

[0074] In some embodiments the BCFA is useful in a method of treating cardiovascular disease, such as atherosclerosis. It was discovered that BCFA are able to restore deficient cholesterol transport, leading to increases in extracellular cholesterol. BCFA treatment was also shown to significantly elevate Ncehl, a gene involved in cholesterol and lipid droplet homeostasis. The BCFA may be used to prevent, reduce or inhibit the formation of cells carrying excess cholesterol, one of the contributors to the development and worsening of atherosclerosis.

[0075] In some embodiments the BCFA is useful in a method of treating rheumatoid arthritis. Rheumatoid arthritis is an autoimmune disease that occurs when the body's immune system mistakenly identifies as a foreign body the synovium that secretes joint synovial fluid. Inflammation results and damage or destruction of cartilage and tissue in and around the joint. In severe cases, this inflammation can spread to other joint tissues and surrounding cartilage, eroding or destroying bone and cartilage, resulting in joint deformity. The body replaces damaged tissue with scar tissue, which results in a narrow normal space within the joint and the bones fuse together. Rheumatoid arthritis causes stiffness, swelling, fatigue, anemia, weight loss, fever, and often disability pain. Some common symptoms of rheumatoid arthritis include joint stiffness that lasts for more than an hour upon awakening; swelling of certain fingers or wrists; swelling of soft tissue around the joint; and swelling on both sides of the joint. Rheumatoid arthritis, including juvenile rheumatoid arthritis, is a type of inflammatory arthritis related to inflammation in a joint.

[0076] In some embodiments the BCFA is useful in a method of treating Type I diabetes mellitus. Diabetes is a chronic disease characterized by either the inability of the body to produce insulin (Type I) or the failure to respond to it (Type II). Type I, also known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition. In this condition, the pancreas makes little or no insulin. Type 1 diabetes may be caused by an autoimmune reaction. Thus, the anti-inflammatory properties of BCFA may help reduce the body’s autoimmune reaction.

[0077] A “subject” herein is typically a human. In certain embodiments, a subject is a nonhuman mammal. Exemplary non-human mammals include laboratory, domestic, pet, sport, and stock animals, e.g., mice, cats, dogs, horses, and cows. In one embodiment, such eligible subject or patient is one that is experiencing or has experienced one or more signs, symptoms, or other indicators of a neurodegenerative disease such as an amyloid disease or has been diagnosed with a neurodegenerative disease, whether, for example, newly diagnosed, previously diagnosed or at risk for developing a disease such as Alzheimer's disease. Diagnosis of disease may be made based on clinical history, clinical examination, and established imaging modalities. A "patient" or "subject" herein includes any single human subject eligible for treatment who is experiencing or has experienced one or more signs, symptoms, or other indicators of disease. Intended to be included as a subject are any subjects involved in clinical research trials, or subjects involved in epidemiological studies, or subjects once used as controls.

[0078] The BCFAs may be administered directly to a subject in the absence of any additional carriers or formulation agents. In other embodiments the BCFAs may be combined with pharmaceutically acceptable carriers or excipients for administration to a subject.

[0079] A therapeutically effective amount of the BCFAs may be administered as a single dose or administration or multiple doses or administrations over the course of a single day, multiple days, weeks, months or years. A dose may be administered, for instance, as a pulse dose, as a bolus dose, or as pulse doses administered over time. Thus, in pulse doses, a bolus administration of a BCFA of the disclosure is provided, followed by a time period wherein no BCFA of the disclosure is administered to the subject, followed by a second bolus administration. In specific, non-limiting examples, pulse doses of a BCFA of the disclosure are administered during the course of a day, during the course of a week, or during the course of a month.

[0080] Therapeutically effective amount of the BCFAs will be dependent on the lipid utilized, the subject being treated, the severity and type of the affliction, and the manner and route of administration. In some embodiments the daily dose administered to the patient is between 0.1 and 500 mg, 0.1 and 1,000 mg, 0.1 and 100 mg, 0.1 and 50 mg, 1.0 and 500 mg, 1.0 and 1,000 mg, 1.0 and 100 mg, 1.0 and 50 mg, 10 and 500 mg, 10 and 1,000 mg, 10 and 500 mg, 10 and 100 mg, 10 and 50 mg, 100 and 500 mg, 100 and 1,000 mg, 100 and 150mg, or 1 and 50 mg and the dose is administered once or more daily, weekly, biweekly, or monthly. For example, a therapeutically effective amount of the BCFAs may vary from about 0.001 mg / Kg to about 2000 mg / Kg body weight. In one embodiment, a therapeutically effective amount of the BCFAs may vary from about 0.01 mg / Kg to about 1 mg / Kg body weight. In one embodiment, a therapeutically effective amount of the BCFAs may be present in the composition or formulation in an amount from about 0.003 g / 100 kcal to about 6.1 g / 100 kcal. In other embodiments, the BCFAs may be present in the composition or formulation in an amount from about 2.2 g / 100 kcal to about 4.3 g / 100 kcal. In yet another embodiment the BCFAs may be present in the composition or formulation in an amount from about 3.5 g / 100 kcal to about 5.7 g / 100 kcal.

[0081] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0082] EXAMPLES

[0083] Example 1: Demonstration of the effects of branched chain fatty acids in peripheral macrophages.

[0084] The effects of branched chain fatty acids on immune cells that were activated by the bacterial endotoxin lipopolysaccharide (LPS) was examined. In order to determine whether the SCFAs alter immune cell phenotypes, in vitro experiments using peripheral macrophages and brain microglia were conducted and levels of inflammatory proteins and gene expression were assessed. Following established protocols (Toda et al., 2021, Preparation and culture of bone marrow -derived macrophages from mice for functional analysis. STAR Protoc 2:100246) primary mouse bone marrow derived macrophages (BMDMs) from wildtype mice (the Jackson Labs, strain #000664) were cultured. LPS was used to potently induce inflammation (100 ng / mL; Sigma-Aldrich, #L-2654) and was provided to the cells along with either vehicle control solution (phosphate buffered saline, “PBS”), 20 pM isobutyric acid (“B”), 20 pM isovaleric acid (“V”), or 20 pM 2-methylbutyric acid (“2-M”) for 24-hours (schematic of experimental protocol is shown in Fig 1A). Sources of SCFAs include Isovaleric acid: Fisher Scientific AC156690100; Isobutyric acid: Fisher Scientific AC122520250; and 2-methylbutyric acid: Sigma-Aldrich 193070-25G. After the 24-hour treatment, conditioned media was collected to assess secreted cytokine protein levels, and cellular RNA was harvested to determine gene expression. To assess the cellular secretion of inflammatory molecules into the media, the pro-inflammatory cytokines IL-6 and CCL2 were probed using enzyme-linked immunosorbent assay (ELISA). For IL-6 levels in the media, both isobutyric acid and isovaleric acid significantly decreased the amount of the inflammatory cytokine IL-6 when compared to control. In terms of CCL2, a trending decrease in levels of this inflammatory molecule in the isovaleric acid treated condition was observed (Fig IB, only isovaleric acid and PBS were tested in this particular assay). Using quantitative real-time polymerase chain reaction (qRT-PCR), the expression of genes related to inflammation, homeostasis, as well as certain disease-relevant genes were observed. The proinflammatory genes tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS), as well as the anti-inflammatory cytokine IL- 10, and the macrophage homeostatic marker CX3CR1 were assessed. Interestingly, it was found that expression of TNF-alpha and iNOS were both decreased by isovaleric acid and 2-methylbutyric acid, whilst isovaleric acid increased the anti-inflammatory gene IL- 10, and that 2-methylbutyric acid potently increased CX3CR1 (FIG. 1C), a homeostatic marker. To assess relevance to neurodegenerative diseases, two genes that are implicated in Alzheimer’s disease: APOE and TREM2 were assessed. It was found that 2-methylbutyric acid decreased expression of APOE and TREM2 (FIG. ID).

[0085] In view of these results, a more comprehensive analysis of the transcriptome using bulk RNA sequencing in macrophages that were stimulated with LPS + / - the branched-chain fatty acid isovaleric acid was conducted (FIG 2A). The RNA sequencing data was subjected to a gene set enrichment analysis (GSEA), which revealed an across-the-board decrease in several gene sets (FIG 2B). The GSEA revealed a significant down-regulation of gene sets related to the following terms: “inflammatory bowel disease (IBD)”, “IL- 17 signaling pathway”, “Type I diabetes mellitus”, “Rheumatoid arthritis”, and “Cellular senescence”, as well as many infection-related terms, including “Salmonella infection”, Epstein-Barr virus infection”, “Leishmaniasis”, “Toll-like receptor signaling pathway”, and more. In looking at the specific genes altered by the BCFA treatment compared to control, some of the top significant decreased genes were ILla, Slamfl, 1L6, ILlb, Ccl3, Cxcl3, and Csf3 (FIGs. 2C- D). Notably, the interleukin-related genes, ILla, 1L6, ILlb represent a class of cytokines involved in inflammatory processes. Relatedly, Slamfl has been shown to enhance inflammatory responses in macrophages; thus, a reduction of the interleukin-related genes and Slamfl, as shown here with BCFA treatment, represents an approach for regulating cytokine production in the context of aberrant immune responses. Conversely, genes that were increased by the BCFA treatment compared to control included Pltp, Ncehl, Bcl6, and Steap3 (FIGs. 2C-D). Phospholipid transfer protein (Pltp') has been shown to bind, transfer, and neutralize LPS, and importantly, it has also been shown that Pltp inhibits nuclear factor kappa-B levels. Another gene increased by BCFA treatment was Bcl6, which is a transcription repressor, and of particular interest. Prior work has shown that mice that are devoid of Bcl6 display a lethal inflammatory response. Steap3 has recently been shown to regulate both iron homeostasis and toll-like receptor 4 mediated inflammatory responses in macrophages. Loss of Steap3 causes dysregulation of iron sequestration and distribution, suggesting that promoting levels of Steap3 would restore normal iron homeostasis and also innate immune cell responses. In summary, the BCFA treatment resulted in an overall decrease in pro-inflammatory genes and an increase in anti-inflammatory genes / pathways. Together, these significant genes changes support BCFAs as a therapeutic target for inflammatory diseases including, but not limited to, rheumatoid arthritis and infectious disease.

[0086] Example 2: Demonstration of the effects of short chain fatty acids on microglia, the resident macrophages of the brain.

[0087] The effects of the branched chain fatty acid isovaleric acid on human inducible microglia-like cells (iMGLs) were assessed. The cells were cultured similar to an established protocol (Abud et al., 2017, iPSC-derived human microglia-like cells to study neurological diseases. Neuron 94:278-293. e9). The goal of these experiments was to examine the effect of BCFA treatment on the resident macrophages of the brain in order to assess their relevance to brain health and disease. To this end, 100 ng / mL of LPS was added to the cultures with either control vehicle solution (PBS) or 20 pM isovaleric acid for 24 hours (FIG. 3A). Bar graphs represent mean values with individual data points and SEM error bars. All analyses consisted of unpaired student t test, or one-way ANOVA with post-hoc Tukey’s tests (cholesterol assay).

[0088] Using qRT-PCR, the samples were probed for the homeostatic markers ARG1, CX3CR1, P2RY12, and the anti-inflammatory molecule IL-10 (FIG. 3B). Consistently, isovaleric acid increased expression of the three homeostatic markers and increased IL-10 gene expression. Disease relevance was also assessed by probing for genes associated with Alzheimer’s disease and Parkinson’s disease. It was found that isovaleric acid decreased expression of the disease associated genes (APOE, TREM2, LRRK2) as well as those associated with aberrant inflammatory / immune signaling (MMP9, TYROBP) (FIG. 3C). Cholesterol buildup has been observed within glial cells in Alzheimer’s disease (Blanchard et al., 2022, APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes. Nature 611:769-779). The levels of cholesterol in the conditioned media, a proxy of cholesterol release, was examined. Of relevance, in atherosclerosis, macrophages accumulate excessive amounts of cholesterol, leading to the formation of dysfunctional macrophages known as foam cells. As such, an in vitro cholesterol assay measuring the amount of total cholesterol in the media obtained after 24 hours of treatment of LPS + / - 20 pM of BCFAs was conducted. It was found that both isovaleric acid and 2-methylbutyric acid increased the amount of total cholesterol in the media compared to PBS control (FIG. 3D). This suggests that the BCFAs restored cholesterol efflux from the cells, which may help protect microglia from transforming into foam-like cells over-burdened by lipid droplets. Recently, it has been shown that microglia that accumulate lipid droplets promote aberrant inflammation in the aging brain, suggesting that therapies targeted to reduce excessive accumulation of cholesterol within glial cells would be protective. Of particular interest, the bulk RNA sequencing of the bone-marrow derived macrophages in the prior experiment (FIGs. 2C-D) revealed that one of the top increased genes in the BCFA treated cells versus control treated cells was Ncehl, or neutral cholesterol ester hydrolase 1. Ncehl catalyzes the hydrolysis of cholesterol ester in macrophages. Previous work has shown that genetic knockout of this gene promotes lipid droplet formation and leads to the development of atherosclerosis in mice, suggesting that increasing its expression, as seen here in the case of BCFA treatment, would promote healthy cholesterol / lipid homeostasis. To further assess this, lipid droplet accumulation in LPS -stimulated macrophages was examined using Plin2 immunocytochemistry. It was found that BCFA treatment significantly reduced both the number and average volume of lipid droplets per nucleus (FIGs. 4A-4B). Given that lipid droplet accumulation contributes to inflammatory and metabolic pathology across diseases, this observation supports a beneficial role for BCFAs in promoting lipid clearance. Together with the increased Ncehl in the macrophage experiment (FIG. 2D), and the enhanced cholesterol efflux in the BCFA-treated microglia (FIG. 3D), these findings provide evidence for the utility of BCFAs in treating lipid, immune, and metabolic disorders, including atherosclerosis and Alzheimer’s disease.

[0089] In summary, isovaleric acid increased homeostatic markers while decreasing inflammatory / diseases markers in microglia, and both isovaleric acid and 2-methylbutyric acid promoted cholesterol efflux. These data provide evidence for the role of BCFAs in ameliorating inflammatory immune responses as well as restoring cholesterol homeostasis in diseases associated with inflammation and aging such as Alzheimer’s disease and atherosclerosis.

[0090] Embodiments:

[0091] Embodiment 1. A method for promoting an anti-inflammatory response in a subject in need thereof, comprising, consisting of or consisting essentially of: administering to the subject a branched chain fatty acid (BCFA) in an effective amount for promoting an anti-inflammatory response in a tissue of the subject, wherein the tissue is not a tissue of the gastrointestinal tract.

[0092] Embodiment 2. The method of Embodiment 1, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

[0093] Embodiment 3. The method of any one of Embodiments 1-2, wherein the BCFA comprises isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

[0094] Embodiment 4. The method of any one of Embodiments 1-3, wherein the tissue is brain tissue.

[0095] Embodiment 5. The method of Embodiment 4, wherein the subject has Alzheimer’s disease or Parkinson’s disease. Embodiment 6. The method of Embodiment 4, wherein the subject is identified as APOE4 positive prior to administration of the BCFA.

[0096] Embodiment 7. The method of any one of Embodiments 1-4, wherein the subject has cardiovascular disease.

[0097] Embodiment 8. The method of Embodiment 7, wherein the cardiovascular disease is atherosclerosis.

[0098] Embodiment 9. The method of any one of Embodiments 1-4, wherein the subject has rheumatoid arthritis.

[0099] Embodiment 10. The method of any one of Embodiments 1-4, wherein the subject has Type I diabetes mellitus.

[0100] Embodiment 11. The method of any one of Embodiments 1-4, wherein the subject has an infectious disease.

[0101] Embodiment 12. The method of any one of Embodiments 1-11, wherein the BCFA is not linked to another compound.

[0102] Embodiment 13. The method of any one of Embodiments 1-11, wherein the BCFA is administered in a composition and wherein the composition comprises a pharmaceutically acceptable carrier.

[0103] Embodiment 14. The method of Embodiment 13, wherein the composition is a medical grade food composition.

[0104] Embodiment 15. The method of Embodiment 13, wherein the composition is a food supplement. Embodiment 16. The method of Embodiment 13, wherein the composition comprises a sustained release formulation.

[0105] Embodiment 17. The method of Embodiment 13, wherein the composition comprises a capsule or tablet.

[0106] Embodiment 18. The method of any one of Embodiments 1-11, wherein the BCFA is administered in a composition comprising a BCFA producing microorganism.

[0107] Embodiment 19. The method of Embodiment 18, wherein the BCFA producing microorganism is an engineered microorganism having at least one heterologous gene.

[0108] Embodiment 20. The method of Embodiment 18, wherein the BCFA producing microorganism is of the genus Escherichia or Lactobacillus.

[0109] Embodiment 21. The method of Embodiment 20, wherein the BCFA producing microorganism is Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus or Escherichia coli Nissle.

[0110] Embodiment 22. The method of Embodiment 18, wherein the BCFA producing microorganism is administered at a dose of about IxlO11to about 2x1012 live microorganism once per day, twice per day or three times per day.

[0111] Embodiment 23. A method for treating a neurodegenerative disorder, comprising, consisting of or consisting essentially of: administering to a subject having a neurodegenerative disorder a branched chain fatty acid (BCFA) in an effective amount for treating the disorder.

[0112] Embodiment 24. The method of Embodiment 23, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

[0113] Embodiment 25. A method for treating atherosclerosis, comprising, consisting of or consisting essentially of: administering to a subject having atherosclerosis a branched chain fatty acid (BCFA) in an effective amount for treating the disorder, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

[0114] Embodiment 26. A method for treating rheumatoid arthritis, comprising, consisting of or consisting essentially of: administering to a subject having rheumatoid arthritis a branched chain fatty acid (BCFA) in an effective amount for treating the rheumatoid arthritis.

[0115] Embodiment 27. The method of Embodiment 26, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

[0116] Embodiment 28. A method for treating Type I diabetes mellitus, comprising, consisting of or consisting essentially of: administering to a subject having Type I diabetes mellitus a branched chain fatty acid (BCFA) in an effective amount for treating the diabetes.

[0117] Embodiment 29. The method of Embodiment 28, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

[0118] Embodiment 30. A method for treating an infectious disease, comprising, consisting of or consisting essentially of: administering to a subject having infectious disease a branched chain fatty acid (BCFA) in an effective amount for treating the infectious disease.

[0119] Embodiment 31. The method of Embodiment 30, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

[0120] Embodiment 32. A pharmaceutical composition comprising, consisting of or consisting essentially of isobutyric acid, isovaleric acid, and 2-methylbutyric acid in a pharmaceutically acceptable carrier. Embodiment 33. The composition of Embodiment 32, wherein the pharmaceutical composition comprises a medical food or dietary supplement.

[0121] Embodiment 34. A method for treating a disorder, comprising: administering to a subject having a disorder selected from a neurodegenerative disorder, atherosclerosis, rheumatoid arthritis, Type I diabetes mellitus, or an infectious disease a branched chain fatty acid (BCFA) in an effective amount for treating the disorder, wherein the BCFA is administered in a composition comprising a BCFA producing microorganism.

[0122] Embodiment 35. The method of Embodiment 34, wherein the BCFA producing microorganism is an engineered microorganism having at least one heterologous gene.

[0123] Embodiment 36. The method of Embodiment 34, wherein the BCFA producing microorganism is of the genus Escherichia or Lactobacillus.

[0124] Embodiment 37. The method of Embodiment 36, wherein the BCFA producing microorganism is Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus or Escherichia coli Nissle.

[0125] Embodiment 38. The method of Embodiment 35, wherein the BCFA producing microorganism is administered at a dose of about IxlO11to about 2xl012live microorganism once per day, twice per day or three times per day.

[0126] EQUIVALENTS AND SCOPE

[0127] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the present disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0128] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims. In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0129] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for promoting an anti-inflammatory response in a subject in need thereof, comprising, consisting of or consisting essentially of: administering to the subject a branched chain fatty acid (BCFA) in an effective amount for promoting an anti-inflammatory response in a tissue of the subject, wherein the tissue is not a tissue of the gastrointestinal tract, wherein the BCFA is not conjugated to another active agent and, optionally wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid, derivatives, analogs, isomers and / or salts thereof.

2. The method of claim 1, wherein the BCFA comprises isobutyric acid, isovaleric acid, and 2-methylbutyric acid, optionally in one of the following ratios (isobutyric acid:isovaleric acid:2-methylbutyric acid): 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, :8:1, 1:9:1, 0:0:1, 0:1:0, 0:1:1, 0:1:4, 0:1:9, 0:2:3, 0:3:2, 0:3:7, 0:4:1, 0:7:3, 0:9:1, 1:0:0, 1:0:1, 1:0:4, 1:0:9, 1:1:0, 1:1:3, 1:1:8, 1:2:2, 1:2:7, 1:3:1, 1:3:6,1:4:0, 1:4:5, 1:5:4, 1:6:3, 1:7:2, 1:8:1, 1:9:0, 2:0:3, 2:1:2, 2:1:7, 2:2:1, 2:3:0, 2:3:5,2:5:3, 2:7:1, 3:0:2, 3:0:7, 3:1:1, 3:1:6, 3:2:0, 3:2:5, 3:3:4, 3:4:3, 3:5:2, 3:6:1, 3:7:0,4:0:1, 4:1:0, 4:1:5, 4:3:3, 4:5:1, 5:1:4, 5:2:3, 5:3:2, 5:4:1, 6:1:3, 6:3:1, 7:0:3, 7:1:2,7:2:1, 7:3:0, 8:1:1, 9:0:1, 9:1:0.

3. The method of any one of claims 1-2, wherein the tissue is brain tissue, and wherein the subject has a neurodegenerative disorder such as Alzheimer’s disease or Parkinson’s disease.

4. The method of claim 3, wherein the subject is identified as APOE4 positive prior to administration of the BCFA.

5. The method of any one of claims 1-2, wherein the subject has cardiovascular disease, optionally wherein the cardiovascular disease is atherosclerosis.

6. The method of any one of claims 1-2, wherein the subject has rheumatoid arthritis, Type I diabetes mellitus, or an infectious disease.

7. The method of any one of claims 1-6, wherein the BCFA is administered in a composition and wherein the composition comprises a pharmaceutically acceptable carrier.

8. The method of claim 7, wherein the composition is a medical grade food composition, a food supplement, a sustained release formulation, or a capsule or tablet.

9. The method of any one of claims 1-6, wherein the BCFA is administered in a composition comprising a BCFA producing microorganism.

10. The method of claim 9, wherein the BCFA producing microorganism is an engineered microorganism having at least one heterologous gene.

11. The method of claim 10, wherein the BCFA producing microorganism is of the genus Escherichia or Lactobacillus, and optionally wherein the BCFA producing microorganism is Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus or Escherichia coli Nissle.

12. The method of claim 9, wherein the BCEA producing microorganism is administered at a dose of about IxlO11to about 2xl012live microorganism once per day, twice per day or three times per day.

13. A method for treating a neurodegenerative disorder, comprising, consisting of or consisting essentially of:administering to a subject having a neurodegenerative disorder a branched chain fatty acid (BCFA) in an effective amount for treating the disorder.

14. The method of claim 13, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

15. A method for treating atherosclerosis, comprising, consisting of or consisting essentially of: administering to a subject having atherosclerosis a branched chain fatty acid (BCFA) in an effective amount for treating the disorder, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid.

16. A method for treating an inflammatory disorder comprising, consisting of or consisting essentially of: administering to a subject having a non-gastrointestinal (GI) inflammatory disorder a branched chain fatty acid (BCFA) in an effective amount for treating the non-GI inflammatory disorder, and optionally, wherein the non-GI inflammatory disorder is selected from rheumatoid arthritis, Type I diabetes mellitus, infectious disease, or a metabolic disorder characterized by chronic inflammation and / or lipid dysregulation, such as obesity.

17. The method of claim 16, wherein the BCFA is one or more of isobutyric acid, isovaleric acid, and 2-methylbutyric acid, derivatives, analogs and isomers and salts thereof.

18. A pharmaceutical composition comprising, consisting of or consisting essentially of isobutyric acid, isovaleric acid, and 2-methylbutyric acid in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition comprises a medical food or dietary supplement, and optionally the isobutyric acid, isovaleric acid, and 2-methylbutyric acid are formulated as a BCFA producing microorganism, wherein the BCFA producing microorganism is an engineered microorganism having at least one heterologous gene encoding one or more proteins involved in BCFA synthesis or maintenance, or prevention of degradation of BCFA.

19. A method for treating an inflammatory disorder, comprising: administering to a subject having an inflammatory disorder selected from a neurodegenerative disorder, atherosclerosis, rheumatoid arthritis, Type I diabetes mellitus, or an infectious disease a branched chain fatty acid (BCFA) in an effective amount for treating the disorder, wherein the BCFA is administered in a composition comprising a BCFA producing microorganism.

20. The method of claim 19, wherein the BCFA producing microorganism is an engineered microorganism having at least one heterologous gene.

21. The method of claim 19, wherein the BCFA producing microorganism is of the genus Escherichia or Lactobacillus, optionally wherein the BCFA producing microorganism is Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus or Escherichia coli Nissle.

22. The method of claim 20, wherein the BCEA producing microorganism is administered at a dose of about IxlO11to about 2xl012live microorganism once per day, twice per day or three times per day.

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