Methods for treating or improving autism and related disorders

Abstract

To provide methods for treating or improving autism and related disorders. [Solution] The present disclosure relates in part to a method for treating or preventing a disease using a bacterial formulation comprising probiotic bacteria, biocompatible microspheres and / or prebiotics. The present invention provides, for example, a method for treating or improving autism spectrum disorder, wherein the method comprises administering to a subject in need of such treatment an effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and, optionally, prebiotics.

Description

Technical Field

[0001] Related Applications This application claims priority based on U.S. Patent Application No. 62 / 960,328, filed on January 13, 2020, and U.S. Patent Application No. 62 / 960,331, filed on January 13, 2020, the contents of which are incorporated herein by reference.

Background Art

[0002] Background Probiotics are live microorganisms that, when ingested in sufficient amounts, confer health benefits on the host (Food and Agriculture Organization of the United Nations and World Health Organization, "Health and Nutritional Properties of Probiotics in Food Including Powdered Milk with Live Bacteria" (2001)). As a result, probiotics have gained traction as a viable option for treating diseases (Hemarajata, P. et al. (2013); Therap Adv Gastroenterol.; 6(1):39-51). Bacteria are more likely to colonize and persist when they are already in an activated form of biofilm (surface and / or self-adhesive community), in contrast to being plankton-like (free-living). The positive effects of probiotic bacteria can be enhanced by providing them in an activated state. This can be easily achieved by exposing the bacteria to the surface of biocompatible and non-toxic microspheres.

[0003] Oxytocin is a hypothalamic-derived, posterior pituitary-conserved nonapeptide that has recently attracted attention as an important psychoneurotropic and metabolic hormone beyond its classical role in lactation and childbirth. Oxytocin is central to the recognition of complex social cognition and behaviors such as attachment, social seeking, and cognition. In healthy humans, oxytocin binds to receptors in social brain regions such as the amygdala and anterior cingulate cortex (Boccia, M. et al. (2013); Neuroscience.; 253, 155-164). It is a key component of the network that modulates social brain functions such as the regulation of social stress, emotion recognition, and memory formation (Meyer-Lindenberg, A. et al. (2011); Nat. Rev. Neruosci.; 12, 524). In autism spectrum disorder, several studies have reported that intranasal administration of oxytocin improves emotion recognition. Furthermore, oxytocin does not cross the blood-brain barrier, which facilitates delivery by intranasal administration. However, intranasal oxytocin cannot achieve sustained physiological levels of pulsatile oxytocin signaling. Individuals with autism may also have an increased incidence of gastrointestinal disorders.

[0004] Furthermore, in many patients, antibiotic use is accompanied by gastrointestinal side effects ranging from mild (e.g., stomach upset, nausea, diarrhea, and vomiting) to severe (e.g., abdominal pain, pseudomonas colitis). These side effects may be even more pronounced in pediatric and elderly (patients over 65 years of age) populations receiving antibiotic treatment. These gastrointestinal side effects associated with antibiotic regimens have been reported to result from the elimination of beneficial bacteria necessary to promote the normal digestive process of the intestines. Scientific studies have shown that antibiotic use leads to the disruption of the normal intestinal ecosystem, causing a decrease in the population of beneficial bacteria and an abnormal and harmful increase in harmful bacteria. Therefore, there is a need for safe and effective treatments to treat or improve autism, as well as gastrointestinal disorders associated with autism and / or antibiotic use. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hemarajata, P. et al. (2013);Therap Adv Gastroenterol.;6(1):39-51 [Non-Patent Document 2] Boccia, M. et al. (2013); Neuroscience.; 253, 155-164. [Non-Patent Document 3] Meyer-Lindenberg, A. et al. (2011);Nat.Rev.Neruosci.;12,524 [Overview of the project] [Means for solving the problem]

[0006] Abstract This disclosure relates in part to a method for treating or improving autism spectrum disorder, comprising administering to a subject in need of such treatment a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and, optionally, prebiotics (e.g., a composition comprising dextranomer microparticles; L. reuteri having deposit #ATCC23272; and prebiotics), wherein the autism spectrum disorders intended for treatment by the provided method may include autism spectrum disorder, Asperger syndrome, Heller syndrome, Rett syndrome, and pervasive developmental disorder not otherwise specified (PDD-NOS).

[0007] Provided herein is a method for treating developmental disorders and / or autism that may be associated with premature birth in premature infants, comprising administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier to a patient in need, for example, a premature infant. In certain embodiments, the composition comprises dextranomer microparticles, L. reuteri, ATCC23272, and a prebiotic. In certain embodiments, administration of at least one daily dose of the composition to a subject results in upregulation of oxytocin levels.

[0008] In one embodiment, a method for treating gastroenteritis in a patient due to the administration of an antibiotic regime is provided herein, comprising administering to the patient a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and / or prebiotics. For example, a method for treating a patient who has or is expected to have gastroenteritis associated with the administration of an oral antibiotic regime is provided herein, comprising administering to the patient a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and / or prebiotics. Such a composition may be administered before, during, and / or after the administration of the antibiotic regime. A method for substantially preventing or reducing adverse effects associated with oral antibiotics is also provided, in one embodiment, which involves administering a therapeutically effective amount of a composition containing Lactobacillus reuteri and biocompatible microspheres to a subject in need thereof, wherein the subject is being treated with oral antibiotics.

[0009] Methods for treating depression or anxiety disorders, wherein the method comprises administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics to a subject in need thereof. For example, methods are provided herein for treating one or more of the following using the disclosed methods: clinical depression, postpartum depression, obsessive-compulsive disorder, post-traumatic stress disorder, bipolar disorder, atypical depression, Melanchol's depression, psychotic major depression (PMD), catatonic depression, seasonal affective disorder (SAD), dysthymia, double depression, depressive personality disorder (DPD), relapsing short-term depression (RBD), mild depressive disorder, bipolar disorder or manic-depressive disorder, treatment-resistant depression, treatment-resistant depression, suicidal ideation, suicidal thoughts and suicidal behavior. In certain embodiments, the patient has postpartum depression.

[0010] In some embodiments, the treatment methods contemplated herein may be intended for male or female subjects who require them. In some embodiments, the treatment methods contemplated herein may be intended for infants, children, or adults. In certain embodiments, the treatment methods contemplated herein involve administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier, wherein the composition is administered as a unit dose. In certain other embodiments, the composition is administered in multiple doses. In certain embodiments, the composition is administered at least daily. In certain embodiments, the composition is administered as a single dose. [Brief explanation of the drawing]

[0011] [Figure 1] Figures 1A and 1B show the dry mass of stomach contents in female and male rat pups treated with physiological saline, respectively. Figure 1C shows the total stomach calories of male pups on day 17 (D17) and day 21 (D21). Figures 1D and 1E show the calorie density in female and male pups treated with physiological saline, respectively.

[0012] [Figure 2-1] Figures 2A and 2B compare the dry mass of stomach contents of offspring treated with formulation A and offspring treated with Rx saline (data from female and male offspring of D21 are shown in Figures 2A and 2B, respectively). Figures 2C and 2D compare the stomach calorie content of offspring treated with formulation A and offspring treated with Rx saline (data from female and male offspring of D21 are shown in Figures 2C and 2D, respectively). [Figure 2-2] Figures 2E to 2F compare the gastric lactose content of pups treated with formulation A and pups treated with Rx saline (data from female and male pups of D21 are shown in Figures 2E to 2F, respectively).

[0013] [Figure 3] Figure 3A shows the gastric content pH of female rat pups treated with physiological saline. Figures 3B and 3C compare the gastric content pH of pups treated with formulation A and pups treated with Rx physiological saline (data for female pups from D20 and male pups from D21 are shown in Figures 3B and 3C, respectively).

[0014] [Figure 4] Figure 4A shows blood glucose levels and jejunal glucose levels. Figure 4B shows jejunal protein concentrations in female rat pups.

[0015] [Figure 5] Figures 5A to 5C compare serum peptidoglycan levels in 16-day-old female rat pups at (24 hours) (Figure 5A), (48 hours) (Figure 5B), and (7 days) (Figure 5C) after administration of Rx saline or formulation A. Figure 5D shows serum peptidoglycan levels in adult female rats.

[0016] [Figure 6]Figures 6A and 6B compare the concentration of vitamin B12 in the ileum of female rat pups on day 2 (Figure 6A) and day 3 (Figure 6B) after treatment with either Rx saline or preparation A. Figures 6C and 6D compare the concentration of vitamin B12 in the ileum of male rat pups on day 2 (Figure 6C) and day 3 (Figure 6D) after treatment with either Rx saline or preparation A.

[0017] [Figure 7] Figures 7A to 7C compare the concentrations of various SCFAs (cecal acetate shown in Figure 7A, cecal propionate shown in Figure 7B, and cecal butyrate shown in Figure 7C) in female offspring treated with Rx saline or formulation A two days after administration of Rx saline or formulation A. Figures 7D to 7F compare the concentrations of various SCFAs (cecal acetate shown in Figure 7D, cecal propionate shown in Figure 7E, and cecal butyrate shown in Figure 7F) in male offspring treated with Rx saline or formulation A two days after administration of Rx saline or formulation A.

[0018] [Figure 8] Figures 8A and 8B compare the distal colon fecal mass of female rats (Figure 8A) and male rats (Figure 8B) treated with Rx saline or formulation A on day 2 and day 3 after administration of Rx saline or formulation A. Figures 8C and 8D compare the fecal water mass of female rats (Figure 8C) and male rats (Figure 8D) treated with Rx saline or formulation A on day 2 and day 3 after administration of Rx saline or formulation A.

[0019] [Figure 9] Figure 9A shows serum oxytocin concentrations in female parents after pup weaning. Figure 9B compares serum oxytocin levels in 16-day-old male pups 24 hours after administration of Rx saline or formulation A. Figure 9C compares serum oxytocin levels in 20-day-old male rat pups 48 hours after administration of Rx saline or formulation A.

[0020] [Figure 10]Figure 10 is a graph showing plasma oxytocin levels in female rats after treatment with formulation B, plankton-like L. reuteri (Lr), or saline. Oxytocin levels are shown as mean ± standard error mean for the first three days after treatment (indicated by the bars above). Groups were compared using ANOVA, followed by Tukey multiple comparisons.

[0021] [Figure 11] Figure 11A is a graph showing plasma oxytocin levels in female offspring after treatment with formulation B, plankton-like L. reuteri (Lr), or saline. Figure 11B is a graph showing plasma oxytocin levels in male rat offspring after treatment with formulation B, plankton-like L. reuteri (Lr), or saline. Values ​​are shown as the mean ± standard error mean over the first three days after treatment (days indicated by the bars above). Groups were compared using ANOVA, followed by Tukey's multiple comparison test.

[0022] [Figure 12] Figure 12 shows the experimental study design for the Rx Lactobacillus reuteri ("L. reuteri") formulation after administration of prescription-grade azithromycin (Rx azithromycin).

[0023] [Figure 13] Figures 13A to 13D show the dry mass of gastric contents, gastric water content, gastric calorie content, and pH of gastric contents in female rats treated with physiological saline and female rats treated with formulation A, respectively.

[0024] [Figure 14-1] Figures 14A and 14B show the fecal calprotectin and fecal lactotransferrin levels in female rats treated with physiological saline and female rats treated with formulation A, respectively. [Figure 14-2]Figures 14C to 14D show the fecal IL-22 and fecal IL-6 levels in female rats treated with physiological saline and female rats treated with formulation A, respectively. Data were collected on days 3–4, 4–5, and 5–6 after azithromycin administration.

[0025] [Figure 15] Figures 15A to 15C show the IL-6, IL-10, and IL-22 levels in the ileum of female rats treated with Rx saline and female rats treated with formulation A, respectively, three days after administration of either Rx saline or Rx saline.

[0026] [Figure 16] Figure 16A shows the glucagon-like peptide-2 (GLP-2) levels in the proximal colon of female rats treated with Rx saline and female rats treated with formulation A, three days after administration of either Rx saline or formulation A. Figure 16B compares serum peptidoglycan levels in female rats three days after administration of Rx saline or formulation A.

[0027] [Figure 17] Figure 17 shows fecal sIgA levels measured on days 1, 2, and 3 (or days 4, 5, and 6 after azithromycin administration) in female rats to evaluate intestinal protection.

[0028] [Figure 18-1] In Figures 18A-18C, the concentrations of various SCFAs (fecal acetate shown in Figure 18A, fecal propionate shown in Figure 18B, and fecal butyrate shown in Figure 18C) in fecal pellets collected on days 3-4, 4-5, and 5-6 from female rats treated with Rx saline or Rx preparation A are compared. [Figure 18-2]In Figures 18A-18C, the concentrations of various SCFAs (fecal acetate shown in Figure 18A, fecal propionate shown in Figure 18B, and fecal butyrate shown in Figure 18C) in fecal pellets collected on days 3-4, 4-5, and 5-6 from female rats treated with Rx saline or Rx preparation A are compared.

[0029] [Figure 19] Figures 19A to 19D compare the concentrations of various SCFAs (fecal acetate shown in Figure 19A, fecal propionate shown in Figure 19B, fecal butyrate shown in Figure 19C, and fecal isovalerate shown in Figure 19D) in female rats treated with either Rx saline or preparation A, three days after administration of either Rx saline or preparation A. [Modes for carrying out the invention]

[0030] Detailed explanation This disclosure relates, in part, to methods for treating or preventing diseases or disorders using bacterial formulations, including probiotic bacteria, biocompatible microspheres, and / or prebiotics.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure pertains. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0032] The implementation of this technology shall, unless otherwise indicated, utilize conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the scope of the skills of those skilled in the art. For example, Sambrook and Russell (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. (eds.) (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane (eds.) (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins (eds.) (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed.See (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker (eds.) (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. (eds.) (1996) Weir's Handbook of Experimental Immunology.

[0033] All numerical values, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary (+) or (-) by increments of 1.0 or 0.1 as needed, or alternatively by + / - 15%, or alternatively by 10%, or alternatively by 5%, or alternatively by 2%. It should be understood that, although not always explicitly stated, the term "approximately" precedes all numerical specifications. Also, although not always explicitly stated, it should be understood that the reagents described herein are merely illustrative, and equivalents of such reagents are known in the art.

[0034] As used herein and in the claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. For example, the term "bacteria" includes multiple bacteria, including mixtures thereof.

[0035] As used herein, the term “formulation” may be used interchangeably with “composition.”

[0036] The following definitions are included for the purpose of understanding this subject matter and for the purposes of forming the appended claims. Abbreviations used herein have their conventional meanings in the fields of chemistry and biology. definition

[0037] As used herein, terms such as “to treat” and “treatment” are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents the disorder or its signs or symptoms, and / or therapeutic in that it partially or completely cures the disorder and / or adverse effects resulting from the disorder.

[0038] "Preventing" means intending to prevent damage or effect in a system or subject that is susceptible to damage or effect, either in vitro or in vivo. An example of such prevention is preventing obesity by achieving satiety in a subject.

[0039] As used herein, “Subject” or “Patient” refers to any animal, including but not limited to mammals, primates, and humans, that is at risk of, suffering from, or diagnosed with a disease or disorder (e.g., autism spectrum disorder, depression, anxiety disorder, or developmental disorder). In certain embodiments, the subject may be a non-human mammal, such as a cat, rat, dog, or horse. In certain embodiments, the subject is a human subject. In certain embodiments, the subject is male or female. In some embodiments, the subject is an infant, child, or adult. In certain embodiments, the subject may be a postpartum woman, a pregnant woman, or a lactating woman.

[0040] "Prebiotics" refers to nutritional supplements containing probiotic bacteria. Prebiotics are food components, such as oligosaccharides, that are not digested by the subject (e.g., by mammals such as humans) and stimulate the growth or activity of one or more beneficial bacteria and / or inhibit the growth or activity of one or more pathogenic bacteria. Prebiotics may selectively stimulate the growth and / or activity of one or a limited number of bacteria in the subject.

[0041] "Microspheres" are intended to be particulate or granular materials that are porous and / or semipermeable biofilm-supported and / or compound-supported (e.g., drug-supported) within the enumerated specific size ranges. As used herein, microspheres consist of particles with a diameter of 50 millimeters or less, and a diameter of about 1 micron or more (e.g., about 1 to about 100, or about 1 to about 75 microns, or about 1 to about 50, or about 1 to about 25, or about 1 to about 10 microns, or about 0.5 to about 200 microns, or about 0.5 to about 700 microns, or about 1 to about 600 microns, or less than about 700 microns, or less than about 600 microns, or less than 500 microns, or less than about 400 microns, or less than about 300 microns, or less than about 200 microns, or less than about 100 microns). Non-limiting examples of such are hollow microspheres that are porous and / or semi-permeable and, in some embodiments, may contain pharmaceuticals or drugs, microcapsules (where excipients form a skin or shell that surrounds and contains cargo such as drugs, chemical reducing agents, or absorbent or adsorbent molecules), and fine particles. These terms are used as a general term for any particles within the enumerated size range, whether spherical or not, and these terms are typically used in the art.

[0042] "Biodegradable polymers" are intended to be polymers that are biocompatible, can be broken down in vivo by bodily processes, and can be easily disposed of by the body, making them products that should not accumulate in the body.

[0043] "Biocompatible" means that the components of a delivery system will not cause tissue damage or injury to the human biological system. To confer biocompatibility, polymers and excipients with a history of safe use in humans or under GRAS (Generally Recognized as Safe) conditions are preferred. Biocompatibility means that the components and excipients used in a composition are ultimately "bioabsorbed" or removed by the body without causing any adverse effects on the body. For a composition to be considered biocompatible and non-toxic, it must not cause toxicity to cells. Similarly, the term "bioabsorbable" refers to microspheres made from materials that are bioabsorbed in vivo over a period such that long-term accumulation of the material in a patient is avoided. Biocompatible nanoparticles may be bioabsorbed over periods of less than two years, less than one year, or, for example, less than six months. The bioabsorption rate is related to particle size, the material used, and other factors well recognized by those skilled in the art. Mixtures of bioabsorbable, biocompatible materials can be used to form microspheres in formulations used in this disclosure.

[0044] "Pharmacologically acceptable carriers" refers to any diluent, excipient, or carrier that may be used in the compositions of the present disclosure. Examples of pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference in the art. They are preferably selected with respect to the intended dosage form, i.e., oral tablets, capsules, elixirs, syrups, etc., and are consistent with conventional pharmaceutical practices.

[0045] "Administration" refers to the delivery of a substance to an animal or human subject. Administration can be carried out in a single dose, continuously or intermittently throughout the course of treatment. Methods for determining the most effective means of administration and dosage are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of treatment, and the age, health condition, or sex of the subject being treated. Single or multiple doses may be administered, with the dose level and pattern selected by the treating physician, or, in the case of pets and animals, by the treating veterinarian. Appropriate dosage formulations and methods for administering drugs are known in the art. The route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary depending on the composition used for treatment, the purpose of treatment, the health condition or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include oral administration, vaginal administration, enteral administration, nasal administration (inhalation), injection, topical application, and suppository administration.

[0046] The term "effective dose" refers to the amount sufficient to achieve a beneficial or desired outcome or effect. In the context of therapeutic or prophylactic application, the effective dose depends on the type and severity of the symptom in question, as well as the characteristics of the individual subject, such as general health status, age, sex, weight, and tolerance to the pharmaceutical composition. In relation to therapeutic compositions, in some embodiments, the effective dose is sufficient to produce a protective response against a pathogen or to support a state of health. In some embodiments, the amount is sufficient to achieve one or more of the following: 1) eliminate a pathogen, 2) restore a healthy microbiome, 3) modulate the immune system, 4) maintain metabolism and metabolic pathways, 5) reduce calorie intake, or 6) improve social behavior.

[0047] For in vitro or ex vivo applications, in some embodiments, the effective dose depends on the size and nature of the application in question. It also depends on the nature and sensitivity of the in vitro target and the method in use. Those skilled in the art can determine the effective dose based on these and other considerations. The effective dose may comprise one or more doses of the composition, depending on the embodiment.

[0048] The drugs and compositions can be used in the manufacture of pharmaceuticals, as well as for the treatment of humans and other animals, by administration in accordance with conventional procedures, such as the administration of the active ingredient in the pharmaceutical composition. composition

[0049] The compositions for use in the disclosed manner may comprise pharmaceutically acceptable excipients, e.g., microspheres, probiotic bacteria (e.g., L. reuteri), and prebiotics, the prebiotics comprising probiotic bacterial nutritional supplements. In one embodiment, the composition further comprises one or more of the following: biofilms, prebiofilms, coatings of therapeutic drugs or agents on the surface of the microspheres, chemical reducing agents, molecules that promote adsorption, and molecules that assist absorption. The disclosed microspheres may have a solid core, a hollow core, or a porous core, and in one embodiment, the microspheres encapsulate prebiotics within the hollow or porous core. The microspheres may be biocompatible and / or semipermeable. In one embodiment, the microspheres include a biofilm layer or coating on the outer surface of the microspheres. In one embodiment, the microspheres activate bacteria that enhance persistence and function.

[0050] The intended biocompatible microspheres may comprise materials selected from the group of biodegradable polymers and / or non-biodegradable polymers, and such disclosed microspheres may have dimensions from about 0.5 microns to about 1000 microns. Further preferred ranges are described herein and incorporated herein by reference. The microspheres may be porous and / or semipermeable.

[0051] Non-limiting examples of biodegradable polymers include dextran, dextranomers, e.g., Sephadex (dextran crosslinked with epichlorohydrin), Sephadex G-25, poly(lactic acid-co-glycolic acid) or PLGA, polycaprolactone or PLC, chitosan, gelatin, acetalized dextran, poly(lactide), poly(glycolide), poly(lactide-co-glycolide), poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), poly(lactic acid) / poly(ethylene glycol) copolymer, poly(glycolide) / poly(ethylene glycol) copolymer, poly(lactic acid) / poly(ethylene glycol) copolymer, poly(glycolic acid) / poly(ethylene glycol) copolymer, poly(lactic acid) / poly(ethylene glycol) copolymer, poly (Caprolactone), poly(caprolactone) / poly(ethylene glycol) copolymer, poly(orthoester), poly(phosphazene), poly(hydroxybutyrate), poly(hydroxybutyrate), poly(lactide-co-caprolactone), polycarbonate, polyesteramide, polyandride, poly(dioxanone), poly(alkylene alkylate), polyethylene glycol / polyorthoester copolymer, polyurethane, poly(amino acid), polyether ester, polyacetal, polycyanoacrylate, poly(oxyethylene) / poly(oxypropylene) copolymer, Sephadex® copolymer and / or combinations thereof, selected from one or more of the following:

[0052] Non-biodegradable polymers are selected from one or more of the following: poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulose polymers and cellulose derivatives, acyl-substituted cellulose acetates and their derivatives, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxides, and copolymers and blends thereof.

[0053] The compositions intended for use in the disclosed manner may comprise one or more of the following prebiotics: water-soluble carbohydrates, inulin, oligosaccharides, oligofructose, fructooligosaccharides, galactooligosaccharides, glucose, starch, maltose, maltodextrin, polydextrose, amylose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, carbonates, thiamine, choline, histidine, trehalose, nitrogen, sodium nitrate, ammonium nitrate, beta-glucan, phosphorus, phosphates, hydroxyapatite, potassium, potassium carbonate, sulfur, homopolysaccharides, heteropolysaccharides, cellulose, chitin, vitamins, and combinations thereof.

[0054] In another embodiment, the prebiotic is selected from one or more of the following: trehalose, nitrogen such as in sodium nitrate, phosphorus such as in phosphates such as ammonium nitrate and hydroxyapatite, potassium such as in potassium carbonate, sulfur, oligosaccharides, homopolysaccharides, heteropolysaccharides, cellulose, chitin, glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonate, and combinations thereof. For example, a composition comprising maltose and / or L. reuteri is disclosed herein.

[0055] The compositions for use in the disclosed manner may comprise probiotic bacteria selected to provide one or more of the following: support for antimicrobial immunity, enhancement or support for the health status of the subject, enhancement or support for the gastrointestinal barrier, or antagonism of disease-associated bacterial infections. In another embodiment, the probiotic bacteria are selected to limit an excessive inflammatory response by preventing pathogen colonization and / or limiting and / or eliminating pathogens and / or downregulating cytokine and chemokine production. In certain embodiments, the probiotic bacteria are selected to induce hormones that have a role in social bonding, energy metabolism, and wound healing, which contribute to good physical, mental, and social health. Non-exclusive examples of probiotic bacteria include L. acidophilus, L. crispatus, L. gasseri, L. delbrueckii, L. salivarius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, and B. dragescenti. B. adolescentis, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophiles, Pseudomonas fluorescens, P. protegens, P. brassicacearum, P. aeruginosa; Azospirillum brabrasilense, A. lipferum, A. illakense (A.irakense); Acetobacter diazotrophicus, Herbaspirillum seropedicae; Bacillus subtilis, Pseudomonas stutzeri, fluorescens, P. putida, P. cepacian, P. vesicularis, P. paucimobilis; Bacillus cereus, B. thuringiensis, B. sphaericus; Shewanella oneidensis; Geobacter bemidiensis G. bemidjiensis), G. metalliclreducens, G. sulfurreducens, G. uraniireducens, G. lovleyi; Serratia marcescens, Desulfovibrio vulgaris, Dechloromonas, Dechloromonas aromatic, Deinococcus radiodurans, Methylibium petroleiphilum, Alcanivorax borkumensis, Archaeglobus fulgidus This includes one or more of the following: *L. fulgidus*, *Haloferax sp.*, *Halobacterium* species, and combinations thereof. In certain embodiments, the composition for use in the disclosed treatment method includes *L. reuteri* (ATCC® 23272).

[0056] The disclosed compositions may include further agents, such as agents selective against pathogens that may compete with probiotic organisms. Such complementary agents may be present in the core or on the surface of microspheres in compositions containing microspheres. Such non-limiting examples include molecules and / or surfaces (inside or on the surface of the microspheres) that promote the adsorption of chemical reducing agents; and molecules and / or surfaces (inside the core or on the surface of the microspheres) that promote absorption. In one embodiment, the chemical reducing agents and molecules and / or surfaces that promote absorption are coated onto the surface of the microspheres.

[0057] For example, the disclosed composition may include, for example, colonies of activated bacteria that can form a biofilm layer on the outer surface of fine particles containing L. reuteri. Such layers may have a depth of about 0.5 microns to about 1 millimeter, and may range, for example, from about 1 micron to about 500 microns, from about 1 micron to about 250 microns, from about 1 micron to about 200 microns, from about 1 micron to about 100 microns, from about 1 micron to about 50 microns, from about 1 micron to about 40 microns, from about 1 micron to about 30 microns, from about 2 microns to about 100 microns, from about 2 microns to about 50 microns, from about 2 microns to about 40 microns, from about 2 microns to about 30 microns, from about 3 microns to about 100 microns, from about 3 microns to about 50 microns, from about 3 microns to about 40 microns, from about 3 microns to about 30 microns, from about 5 microns to about 100 microns, from about 5 microns to about 50 microns, from about 5 microns to about 40 microns, and from about 5 microns to about 30 microns. In other embodiments, the composition to be considered includes a scaffold or matrix to have the ability to assist in the activation of bacteria (e.g., L. reuteri) upon administration to a target or treated environment.

[0058] As contemplated herein, the compositions for use in the disclosed methods can include one or more microsphere compositions in combination with a carrier, such as a pharmaceutically acceptable carrier or a biocompatible scaffold. Non-limiting examples of pharmaceutically acceptable carriers include diluents, excipients, or carriers that can be used in the compositions of the present disclosure. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0059] The disclosed compositions can be formulated into dosage forms of biofilm-forming activating probiotic bacteria or an effective amount of microsphere composition for final use, such as about 1×10 9 , , 8 , 10 , 7 , 7 ~1×10 11 CFU / ml, or alternatively about 1×10 5 ~ about 1×10 11 CFU / ml, or about 1×10 5 ~ about 1×10 9 CFU / ml, or about 1×10 6 ~ about 1×10 11 CFU / ml, or about 1×10 6 ~ about 1×10 9 CFU / ml, or about 1×10 7 ~ about 1×10 11 CFU / ml, or about 1×10 7 ~ about 1×10 10 CFU / ml, or about 1×10 7 ~ about 1×10 9 CFU / ml, or about 1×10 8 CFU / ml can be provided.

[0060] In certain embodiments, the bacterial preparation (e.g., L. reuteri preparation) may be administered over approximately 6, 12, 18, 24, 36, 48, and 72 hours, or as a single dose for treatment. In certain embodiments, the bacterial preparation (e.g., L. reuteri preparation) may be administered orally, vaginally, topically, by inhalation, intravenously, intramuscularly, or as a suppository. They may be administered in any suitable formulation.

[0061] The composition may be formulated or processed, for example, by freezing, lyophilizing, suspending (as a suspension formulation), or powdering, to facilitate administration, storage, and application, and may be made into suppositories, tablets, liquids, suspensions, pills, capsules, or sustained-release formulations.

[0062] The effective dose of a therapeutic composition is determined based on the intended goal. The term “unit dose” or “dosage” refers to a physically distinct unit suitable for use in the subject, each unit containing a predetermined amount of the composition calculated to produce the desired response described above in relation to its administration, i.e., the appropriate route and regimen. The amount administered depends on the desired outcome and / or protection, depending on both the number of treatments and the unit dose. The exact amount of composition also depends on the practitioner's judgment and is unique to each individual. Factors influencing the dose include the subject’s physical and clinical condition, the route of administration, the intended treatment goal (alleviation of symptoms versus cure), and the potency, stability, and toxicity of the particular composition. Once formulated, the solution will be administered in a manner compatible with the administered formulation, in an amount that is therapeutically or prophylactically effective. The formulation is readily administered in various dosage forms, such as the types of injection solutions described above.

[0063] In certain embodiments, the present disclosure provides a method of treatment by administering a therapeutically effective dose of a bacterial preparation (e.g., a L. reuteri preparation). In certain embodiments, the bacterial preparation includes Lactobacillus reuteri, biocompatible microspheres, and / or prebiotics. method

[0064] This specification provides a method for treating or preventing a disease or disorder that is appropriately treated by a bacterial formulation in a subject where it is needed. This method involves administering an effective amount of a bacterial formulation disclosed herein, having components selected for a particular treatment, to a subject. In one embodiment, this disclosure provides a method for inducing oxytocin by administering a therapeutically effective amount of a bacterial formulation (e.g., a L. reuteri formulation). In a particular embodiment, oxytocin is induced in a subject by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and / or prebiotics. In a particular embodiment, the administration of a therapeutically effective amount of a bacterial formulation (e.g., a L. reuteri formulation) activates oxytocin receptors.

[0065] In certain embodiments, the Disclosure provides a method for treating, improving, and / or preventing autism spectrum disorders, including but not limited to autism disorder, Asperger syndrome, Heller syndrome, Rett syndrome, and pervasive developmental disorder not otherwise specified (PDD-NOS), by administering a pharmaceutically acceptable composition of a bacterial preparation (e.g., a L. reuteri preparation) to a subject.

[0066] Methods for treating or preventing diseases or disorders (e.g., gastroenteritis) by administering antibiotic regimens appropriately treated with bacterial formulations in the target area where needed are also provided herein. These methods involve administering an effective amount of a bacterial formulation disclosed herein, having components selected for a specific treatment, to the target area. Non-limiting examples of diseases include gastrointestinal disorders, e.g., gastroenteritis, or diseases or disorders involving bowel motility dysfunction caused by antibiotic use, where pathogenic bacteria replace healthy bacteria, or chronic and / or recurrent diseases antagonizing disease-associated bacterial infections.

[0067] This disclosure provides, in one embodiment, a method for treating gastroenteritis in a patient caused by the administration of an antibiotic regimen (e.g., azithromycin) by administering a therapeutically effective amount of a bacterial preparation (e.g., a L. reuteri preparation). In a particular embodiment, the gastroenteritis is reduced or alleviated in the subject by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics.

[0068] In certain embodiments, the Disclosure provides a method for treating patients who have or are expected to have gastroenteritis associated with the administration of an oral antibiotic regimen by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier to the patient. In certain embodiments, gastroenteritis is significantly reduced by the administration of a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics. Such oral antibiotic regimens may include oral antibiotic administrations of the following common classes, e.g., sulfonamides; folic acid analogs; beta-lactams including penicillin and cephalosporins; tetracyclines; macrolides; lincosamides; streptogramin; quinolones including fluoroquinolones; polypeptides such as polymyxins; aminocyclitols; glycopeptides; oxazolidinones; etc. "Antibiotics" means antibiotics, antimicrobial agents, antibacterial agents, anti-infective agents, etc.

[0069] Examples of antibiotics include amoxicillin, amoxicillin and potassium clavulanate, ampicillin, ampicillin and sulbactam, atovaquone, azithromycin, carbenicillin, cefaclor, cefdinir, cefonisid, ceftibuten, cefotetan, cefpodoxime, ceftriaxone, cefuroxime, cephalexin, cephalothin, cephamycin, chlortetracycline, ciprofloxacin, clindamycin, clarithromycin, cycloserine, dalfopristin, dicloxacillin, and doxyl This includes cyclines, erythomycin, levofloxacin, linezolid, moxifloxacin, mupirocin, oxytetracycline, penicillin, rifampin, quinupristin, a combination of dalfopristin and quinupristin, spectinomycin, sulfadiazine, sulfamethoxazole, sulfamol, sulfamoxol, sulfarene, sulfanilamide, tetracycline, trimethoprim, a combination of trimethoprim and sulfamethoxazole, vancomycin, and combinations including at least one of the aforementioned. In certain embodiments, the antibiotic regimen includes azithromycin.

[0070] In certain embodiments, the Disclosure provides a method for treating a patient for a gastrointestinal disorder, comprising administering a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics. In certain embodiments, the gastrointestinal disorder is one or more of constipation, abdominal pain, flatulence, and diarrhea.

[0071] In certain embodiments, the Disclosure provides a method for treating a patient for gastrointestinal disorders, wherein the patient is on the autism spectrum or is autistic.

[0072] A method for substantially preventing or reducing adverse effects associated with oral antibiotics is provided herein, the method for using a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics in a subject requiring such prevention or reduction.

[0073] In certain embodiments, the bacterial formulations described herein (e.g., L. reuteri formulations) may be used to prevent and / or improve and / or treat adverse gastrointestinal (GI) side effects (e.g., from the administration of another drug having GI side effects, e.g., an antibiotic) including but not limited to pseudomembranous colitis (inflammation of the colon due to the overgrowth of Clostridium difficile), nausea, convulsions, diarrhea, and vomiting. These are some of the side effects associated with antibiotics. However, the effect of the formulations used in the context of this disclosure on these conditions may be mediated whole or partially through an effect on body weight, or may be unrelated thereto. In certain embodiments, the bacterial formulations of the disclosure (e.g., L. reuteri formulations) may be used as pharmaceuticals for the prevention of the aforementioned disorders and / or diseases. In some embodiments, administration may cause one or more of the following: reduction or elimination of one or more symptoms, prevention of an increase in the severity of one or more symptoms, and / or reduction, prevention, or elimination of further diseases or symptoms.

[0074] In certain embodiments, administration of a pharmaceutically acceptable composition of a bacterial preparation described herein (e.g., a L. reuteri preparation) causes a reduction in gastrointestinal inflammation in the subject. In certain embodiments, administration causes a reduction of at least 5% of gastrointestinal inflammation in the patient (e.g., at least 7%, 10%, 20%, 30%, 50%, 75%, or more).

[0075] In certain embodiments, the bacterial composition (e.g., L. reuteri preparation) is administered before, during, and / or after the administration of an antibiotic regimen.

[0076] This disclosure provides, in one embodiment, a method for treating depression or anxiety disorders in a subject by administering a pharmaceutically acceptable composition of a bacterial preparation (e.g., a L. reuteri preparation) to the subject. In certain embodiments, depression or anxiety disorders include clinical depression, postpartum or post-delivery depression, postpartum obsessive-compulsive disorder, post-traumatic stress disorder, e.g., postpartum post-traumatic stress disorder, postpartum bipolar disorder, atypical depression, depressive depression, psychotic major depressive disorder (PMD), catatonic depression, seasonal affective disorder (SAD), dysthymia, double depression, depressive personality disorder (DPD), relapsing short-term depression (RBD), mild depressive disorder, bipolar or manic-depressive disorder, treatment-resistant depression, treatment-refractory depression, suicidal ideation, suicidal thoughts, and suicidal behavior. In some embodiments, the subject suffers from an anxiety disorder. In some embodiments, the subject suffers from depression. In some embodiments, the subject suffers from postpartum or post-delivery depression.

[0077] This disclosure provides, in one embodiment, a method for treating developmental disorders associated with premature birth by administering a pharmaceutically acceptable composition of a bacterial preparation (e.g., a L. reuteri preparation) to a premature infant. In a particular embodiment, the bacterial preparation used for treatment comprises a therapeutically effective amount of a composition containing Lactobacillus reuteri and a pharmaceutically acceptable carrier.

[0078] In certain embodiments, the present disclosure provides methods for treating developmental disorders associated with premature birth, including but not limited to bronchopulmonary dysplasia (BPD); intellectual disability, cerebral palsy, white matter disease; and retinopathy of prematurity.

[0079] In certain embodiments, the disclosure provides a method for inducing oxytocin in premature infants who require it by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier to the infant. In certain embodiments, oxytocin is induced in premature infants by administration of a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics.

[0080] In certain embodiments, administration of at least one daily dose of a bacterial formulation (e.g., a L. reuteri formulation) to a subject results in an upcontrol of oxytocin levels. In certain embodiments, oxytocin is upcontrolled by at least 5% in the subject after administration (e.g., at least 5%, 10%, 20%, 30%, 50%, 75%, or more).

[0081] In certain embodiments, the Disclosure provides a method for enhancing lactation in a subject in need, comprising administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and / or prebiotics to the subject. In certain embodiments, the enhancement of lactation after administration of the bacterial formulation of the Disclosure (e.g., the L. reuteri formulation) is attributed to an increase in oxytocin levels, which are known to be involved in lactation and secretion (see Pang, WW et al.; (2007); J Mammary Gland Biol Neoplasia 12, 211-221).

[0082] In certain embodiments, administration of a bacterial formulation of the Disclosure (e.g., L. reuteri formulation) increases oxytocin levels (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 220%, about 240%, about 260%, about 280%, or about 300%). In certain embodiments, a single dose of a bacterial formulation (e.g., L. reuteri formulation) increases oxytocin levels over several hours (e.g., about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours). In certain embodiments, multiple doses of a bacterial formulation (e.g., L. reuteri formulation) increase oxytocin levels over several hours (e.g., about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours). In certain embodiments, administration of a bacterial formulation of the Disclosure (e.g., L. reuteri formulation) increases prolactin levels (e.g., levels (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 140%, about 160%, about 180%, about 200%, about 220%, about 240%, about 260%, about 280%, or about 300%)).

[0083] In some embodiments, a single dose of the bacterial preparation (e.g., L. reuteri preparation) is administered to the subject. In other embodiments, multiple doses are administered over two or more time points, divided into several hours, days, or weeks (e.g., approximately 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours). In some embodiments, the bacterial preparation (e.g., L. reuteri preparation) is administered over a long period (e.g., chronically), for example, over several months or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more). In certain embodiments, the bacterial preparation (e.g., L. reuteri preparation) may be taken regularly over a long period (e.g., daily, weekly, etc.).

[0084] In some embodiments, the composition of the method is approximately 1 × 10 7 ~Approx. 1×10 10 CFU / ml (for example, approximately 1.5 × 10⁻⁶) 7 , about 2×10 7 , about 2.5×10 7 , about 3×10 7 , about 3.5×10 7 , about 4×10 7 , about 4.5×10 7 , about 5×10 7 , about 5.5×10 7 , about 6×10 7 , about 6.5×10 7 , about 7×10 7 , about 7.5×10 7 , about 8×10 7 , about 8.5×10 7 , about 9×10 7 , about 9.5×10 7 , about 1×10 8 , about 1.5×10 8 , about 2×10 8 , about 2.5×10 8 , about 3×10 8 , about 3.5×10 8 , about 4×10 8 , about 4.5×10 8 , about 5×10 8 , about 5.5×10 8 , about 6×10 8 , about 6.5×10 8 , about 7×10 8 , about 7.5×10 8 , about 8×10 8 , about 8.5×10 8 , about 9×10 8 , about 9.5×10 8 , 1 x 10 9 , about 1.5×10 9 , about 2×10 9 , about 2.5×10 9 , about 3×10 9 , about 3.5×10 9 , about 4×10 9 , about 4.5×10 9 , about 5×10 9 , about 5.5×10 9 , about 6×10 9, about 6.5×10 9 , about 7×10 9 , about 7.5×10 9 , about 8×10 9 , about 8.5×10 9 , 9×10 9 , or approximately 9.5 x 10 9 ) is administered to provide activated biofilm-forming probiotic bacteria.

[0085] In certain embodiments, the bacterial formulations of the disclosure used in the context of this disclosure (e.g., L. reuteri formulations) may be administered as part of a combination therapy with at least one other agent for treating a disease or disorder discussed in this disclosure.

[0086] The generally accepted description of this disclosure is included solely for the purpose of describing specific aspects and embodiments of this disclosure and is not intended to limit this disclosure in any way, and will be more readily understood by referring to the following examples. [Examples]

[0087] The following examples are illustrative and are not intended to limit the scope or content of this disclosure. Example 1 - Formulation of Lactobacillus reuteri

[0088] The formulation is provided as a powder for reconstitution into a liquid and administered orally. Each mL of formulation A contains 1.9 × 10¹⁶ units in 74 mM maltose. 9 It contains the colony-forming unit L. reuteri and 18.5 mg of Sephadex. The dose is 1.9 × 10⁴ in 74 mM maltose. 10 This formulation contains 10 mL of the colony-forming unit L. reuteri and 185 mg of Sephadex, administered once daily. Example 2 - Study of Lactobacillus reuteri formulations

[0089] We conducted research on L. reuteri formulations. The preclinical safety study design for L. reuteri formulations involved 24 μmol of prebiotic maltose and 6 × 10⁶ 9 Formulation A contains 60 mg of dextranomer microparticles (cross-linked dextran containing epichlorohidin) loaded with CFU L. reuteri (ATCC23272). Briefly, naturally delivered neonatal rats were divided into the following treatment groups: (1) formulation-grade saline (Rx saline) and (2) Formulation A. Each treatment group consisted of 24 males and 24 females. On day 15, each pup received a single enteral dose of either Rx saline or Formulation A. Throughout the study, the pups remained with their mothers (female parents). The pups were breastfed for nutrition. As the pups approached day 21, only those treated with Formulation A began consuming solid feed provided for their mothers. The pups (N=8 from each treatment group (8 males and 8 females)) were euthanized 24 hours, 48 ​​hours, and 7 days after administration. Histological studies were performed on the esophagus, stomach, duodenum, jejunum, ileum, cecum, proximal colon, distal colon, rectum, lungs, kidneys, and liver. Histopathology of the entire GI duct appeared normal in both pups treated with Rx saline and formulation A. Light microscopy imaging showed that microspheres were removed intact in the feces by 48 hours after oral enteral feeding. Preclinical toxicology studies demonstrated that formulation A formulation is safe. Macroscopic evaluation of pups showed that pups treated with formulation A consumed solid food faster than those treated with saline, but their calories were comparable on days 17 and 21. On day 17, pups consumed less at a higher calorie density, yet their calories were equal to those on day 21, when they had a higher calorie density but a higher food intake (total mass). This suggests an established hypothalamic setpoint-regulated energy balance. These photographs show pups that were supplemented with solid food in addition to breast milk. Example 3 - Spontaneous weaning: Effects of administering L. reuteri preparations to newborn rats

[0090] The design for the weaning study of neonatal rat pups was as follows: Briefly, naturally delivered pups were divided into the following treatment groups: (1) prescription-grade saline (Rx saline) and (2) formulation A. Each treatment group consisted of 20 males and 20 females. On day 18, each pup received a single enteral dose of either Rx saline or formulation A. Pups (N=10 from each treatment group (10 males and 10 females)) were euthanized 48 and 72 hours after administration. Trunk blood, stomach, jejunum, ileum, cecum, proximal colon, and distal colon were collected on days 20 and 21. In addition, body composition, thymus mass, and spleen mass were analyzed for each pup. Effects on calorie intake

[0091] Figures 1A and 1B show the dry mass of stomach contents in female and male offspring treated with saline, respectively. Figure 1C shows the total stomach calories of male offspring on day 17 (D17) and day 21 (D21). Total stomach calories were the same in female offspring. Total calories were maintained as the offspring transitioned from milk to solid feed. Figures 1A and 1C show that calories are equivalent on D17 and D21. On day 17, even though the offspring consumed less at a higher calorie density, their calories were equal to those on day 21, when there was a lower calorie density and more food intake (total mass). This suggests an established hypothalamic setpoint-regulated energy balance. Figures 1D and 1E show the calorie density in female and male offspring treated with saline, respectively. Figures 1A and 1E show that as weaning approaches, the mass of stomach contents increases while the calorie density decreases in saline-treated offspring. Figures 2A and 2B compare the dry mass of stomach contents of offspring treated with formulation A and offspring treated with Rx saline (data from female and male offspring of D21 are shown in Figures 2A and 2B, respectively). Figures 2C and 2D compare the stomach calorie content of offspring treated with formulation A and offspring treated with Rx saline (data from female and male offspring of D21 are shown in Figures 6C and 6D, respectively). Figures 2E and 2F compare the stomach lactose content of offspring treated with formulation A and offspring treated with Rx saline (data from female and male offspring of D21 are shown in Figures 2E and 2F, respectively). Figures 2A and 2F show that formulation A reduces food intake despite established calorie intake setpoints. Figures 2A and 2F also suggest that formulation A treatment may accelerate the transition from milk to solid feed in males. Effects on stomach acid

[0092] The effect of L. reuteri formulations on gastric acid was tested by measuring intragastric pH. Briefly, animals were euthanized and gastric contents were removed for the assay. Data from days 16–19 and 22 pups were obtained from the safety study described in Example 2. The data shown in Figure 3A are from saline-treated or untreated pup rats. Figure 3A shows the gastric content pH of female pup rats treated with saline. Figures 3B–3C compare the gastric content pH of pups treated with formulation A and pups treated with Rx saline (data for female pups from D20 and male pups from D21 are shown in Figures 3B–3C, respectively). Figures 3A–3C show that gastric acid production increases after 18 days of age. Figures 3A–3C also show that administration of formulation A does not promote weaning by increasing gastric acid production. Effects on jejunal protein concentration

[0093] The jejunum is specialized for nutrient absorption. Briefly, the jejunum was isolated from euthanized pups. The contents of the jejunum were collected in vials and stored at -20°C until assay. Figure 4A shows blood glucose levels and jejunal glucose levels. Figure 4B shows jejunal protein concentrations in female rat pups. Figure 4A also shows that blood glucose is dependent on jejunal glucose concentration. Figure 4A shows that the function of intestinal glucose transporters (SGLT-1, GLUT-2) and fructose transporter (GLUT5) is active in rat pups from day 16. Figure 4B shows that jejunal protein concentrations are dependent on gastric acid production and consequently decrease after 18 days of age. Effect on serum peptidoglycan concentration

[0094] Serum peptidoglycan levels were measured in female pups treated with formulation A. Briefly, the pups were euthanized by decapitation, trunk blood was collected in a tube, and coagulated in ice. The serum was separated by centrifugation and stored at -20°C until assayed for peptidoglycan by enzyme-linked immunosorbent assay (ELISA). Figures 5A to 5C compare serum peptidoglycan levels in 16-day-old female pups (24 hours) (Figure 5A), 17-day-old pups (48 hours) (Figure 5B), and 21-day-old pups (7 days) (Figure 5C) after administration of Rx saline or formulation A. Figure 5D shows serum peptidoglycan levels in adult female rats. Effects on vitamin B12 production

[0095] Ileal vitamin B12 content was measured in female and male offspring on days 2 and 3 after treatment with either formulation A or Rx saline. Briefly, the ileum was isolated from euthanized offspring. The contents of the ileum were collected in vials and stored at -20°C until assay. Vitamin B12 was measured by ELISA. Figures 6A and 6B compare the concentrations of vitamin B12 in the ileum of female rat offspring on days 2 (Figure 6A) and 3 (Figure 6B) after treatment with either Rx saline or formulation A. Figures 6C and 6D compare the concentrations of vitamin B12 in the ileum of male rat offspring on days 2 (Figure 6C) and 3 (Figure 6D) after treatment with either Rx saline or formulation A. Figures 6A and 6C show that administration of formulation A increases the concentration of vitamin B12 in the ileum of rat offspring (male and female). Vitamin B12 is synthesized in the ileum by L. reuteri. Figure 6B shows a decrease in vitamin B12 concentration in the ileum of female rat pups treated with formulation A. Figure 6D shows no statistically significant difference in vitamin B12 concentration in male pups treated with Rx saline or formulation A. These graphs indicate that vitamin B12 is removed from the ileum by day 3. Vitamin B12 is also used as a marker for reuterin, a potent antimicrobial compound produced by L. reuteri. Effects on short-chain fatty acids (SCFAs)

[0096] SCFA (acetate, propionate, and butyrate) levels were measured by gas chromatography-mass spectrometry (GC / MS). Briefly, the cecum was isolated from euthanized pups. Cecal contents were collected in vials and stored at -20°C until assay. Samples were sonicated in 0.5% phosphoric acid (200 mg / ml), centrifuged to remove solid unextracted material, and filtered through a 0.6 μm filter to remove large particles. These samples were injected (1 μl) into a GC / MS equipped with a Stabilwax-DA column. Samples were calibrated using standard solutions of six short-chain fatty acids. Figures 7A–7C compare the concentrations of various SCFAs (cecal acetate shown in Figure 7A, cecal propionate shown in Figure 7B, and cecal butyrate shown in Figure 7C) in female pups treated with Rx saline or formulation A two days after administration of Rx saline or formulation A. Figures 7D to 7F compare the concentrations of various SCFAs (cecal acetate shown in Figure 7D, cecal propionate shown in Figure 7E, and cecal butyrate shown in Figure 7F) in male offspring treated with Rx saline or formulation A two days after administration of Rx saline or formulation A. Figures 7A to 7F show that administration of formulation A did not alter SCFA production in the cecum of offspring rats (male and female), and therefore the weaning effect is not mediated through SCFA production. Impact on fecal volume

[0097] The distal colon was isolated from euthanized offspring. Fecal pellets from the distal colon were collected in vials and stored at -20°C until assay. To measure the dry and watery masses, the fecal pellets were freeze-dried to remove water. The difference between the wet and dry masses was defined as the watery mass. Figures 8A and 8B compare the distal colon fecal masses of female (Figure 8A) and male (Figure 8B) offspring treated with Rx saline or formulation A on days 2 and 3 after administration of Rx saline or formulation A. Figure 8B shows an increase in fecal volume in male offspring treated with formulation A. The data obtained from Figure 8B also indicates that irregularity can be treated using formulation A. Figures 8C and 8D compare the fecal watery masses of female (Figure 8C) and male (Figure 8D) offspring treated with Rx saline or formulation A on days 2 and 3 after administration of Rx saline or formulation A. Figures 8C to 8D show that administration of formulation A does not cause diarrhea or constipation in male or female rat pups.

[0098] The data presented in Example 3 indicates that Formulation A promotes weaning and satiety at the hypothalamic level rather than altering intestinal function. Example 4 - Effects of administering L. reuteri preparations to adult rats

[0099] Figure 9A shows serum oxytocin concentrations in female mothers after weaning. The graph shows that serum oxytocin levels remained elevated for approximately 11 days after weaning of nursing offspring in adult female mothers treated with formulation A. The data show that the elevated oxytocin levels observed during breastfeeding decreased approximately 11 days after weaning of the offspring from the female mothers. These results suggest that formulation A may modulate postpartum depression in female rats. Figure 9B compares serum oxytocin levels in 16-day-old male offspring 24 hours after administration of Rx saline or formulation A. Figure 9C compares serum oxytocin levels in 20-day-old male offspring 48 hours after administration of Rx saline or formulation A. Figures 9B–9C show that serum oxytocin levels increased in male offspring treated with formulation A 24 hours after administration, suggesting that increased oxytocin levels may help modulate food intake and social and exploratory behaviors in childhood. The data shown in Figures 9B and 9C were obtained after a single dose of formulation A. Multiple doses of formulation A may help maintain elevated oxytocin levels. Example 5 - Activation of L. reuteri by dextranomer microparticles (DM)

[0100] L. reuteri formulations containing maltose and DM exhibit enhanced biological activity. In vitro activation mimics the activation in the gastrointestinal tract when a microorganism binds to mucin glycans. DM is a biocompatible, porous, semipermeable solid particle containing dextran, which is similar to mucin O-linked glycans. The L. reuteri strains of this disclosure express the extracellular glucosyltransferase (GTF) enzyme. The in vitro activation process is a simulation of the biological changes induced when L. reuteri comes into contact with and binds to mucin glycans in the ileum. Thus, activation offers advantages over inactivated L. reuteri, leading to better initiation of colony formation. Example 6 - Effect of L. reuteri administration on plasma oxytocin levels

[0101] Freeze-dried L. reuteri (ATCC23272 strain) in 1.7 × 10⁻⁶ 11 L. reuteri was supplied to vials containing CFU / g. 1 mL of sterile saline was added to the lyophilized bacteria, rehydrated at room temperature for 5 minutes, and then added to a DM-maltose slurry. The DM-maltose slurry was prepared by weighing autoclaved dried Sephadex G25 Superfine microspheres (DM) and adding them to a filter-sterilized 1 M maltose solution (25% w / v suspension). To initiate the activation process, the mixture of L. reuteri and DM-maltose slurry was incubated together at room temperature for 60 minutes before use. The final dose per administration of the L. reuteri preparation was 500 μL of 2 × 10⁶ units. 9 The preparation consisted of CFU L. reuteri, 20 mg of DM, and 28.8 mg of maltose (in 1 mL of physiological saline) ["Preparation B"]. Plankton-like L. reuteri was prepared similarly, but without the addition of the DM-maltose slurry.

[0102] To investigate the effect of L. reuteri formulations on plasma oxytocin levels, adult Sprague-Dawley female rats were given a single oral dose of either plankton-like L. reuteri (Lr) or saline two days after a single dose of prescription-grade azithromycin. A niche for L. reuteri was created using (45 mg / kg, administered via oral enteral nutrition) (a two-day washout period was used between antibiotic administration and treatment). On days 1, 2, and 3 post-administration, blood was collected in tubes containing aprotinin (500 kallikrein inactivator units: KIU) / mL of blood; used due to the short half-life of oxytocin in the blood), and plasma oxytocin levels were determined by enzyme immunoassay (EIA; assay design). Figure 10 is a graph showing plasma oxytocin levels in female rats after treatment with formulation B, plankton-like L. reuteri (Lr), or saline. Oxytocin levels are shown as mean ± standard error mean for the first three days after treatment (indicated by the bars above). Groups were compared using ANOVA followed by Tukey multiple comparisons. As shown in Figure 10, plasma oxytocin levels on day 1 were significantly higher in animals treated with formulation B than in animals treated with planktonic L. reuteri or saline. Oxytocin levels between groups plateaued by day 3. This study demonstrated that formulation B is effective in stimulating circulating oxytocin on day 1 and maintaining it on day 2. Furthermore, planktonic L. reuteri showed a tendency in its ability to stimulate oxytocin levels on day 2, but the levels were not significantly different from those of the saline-treated group.

[0103] To investigate the effect of L. reuteri preparations on the plasma oxytocin levels of offspring, the offspring were administered saline, L. reuteri, or 300 μL of preparation B (prepared as described above). Immature rat offspring had low baseline circulating oxytocin levels, and there was no sex difference in response to baseline levels or preparation B. All animals were treated once by oral enteral nutrition at 15 days of age. Blood was collected on days 1, 2, and 3 post-treatment, and plasma oxytocin was measured using the method described above. Figure 11A is a graph showing plasma oxytocin levels in female offspring after treatment with preparation B, plankton-like L. reuteri (Lr), or saline. Figure 11B is a graph showing plasma oxytocin levels in male offspring after treatment with preparation B, plankton-like L. reuteri (Lr), or saline. Values ​​are shown as mean ± standard error mean over the first three days post-treatment (days indicated by the bars above). Groups were compared using ANOVA, followed by Tukey's multiple comparison test. This study confirmed that formulation B can increase circulating oxytocin levels. Example 7 - Evaluation of L. reuteri formulation

[0104] This example describes the measurement of lactic acid, histamine, vitamin B-12, and glycerol production by L. reuteri in formulations, as well as the effects of various preparation methods on L. reuteri formulations, including their sugar utilization. Two L. reuteri formulations are used in the example: 1) Formulation B (2 × 10⁻⁶) 9 CFU L. reuteri, 20 mg DM and 28.8 mg maltose (in 1 mL of physiological saline); and 2) Preparation C (same amount of L. reuteri as Preparation B (2 × 10) 9 The formulations included CFUs, but with 10x dilutions of DM and maltose (200 mg of DM and 288 mg of maltose). The formulation preparation process was carried out as described in Example 6, except that the bacteria and DM-maltose slurry were incubated at various time points (1 hour, 6 hours, 24 hours, and 96 hours) before evaluating the various analytes in the culture medium. The results of these studies are summarized in Table 1. [Table 1] Example 8 - Identification of L. reuteri formulations for increasing plasma oxytocin levels

[0105] Using the pup rat model described in Example 6, identify the optimal L. reuteri formulation and administration strategy for increasing plasma oxytocin levels. Treatment with L. reuteri formulations of various concentrations and regimens will be performed to identify the minimum effective dose that defines a significant stimulation of plasma oxytocin during the first 48 hours compared to animals treated with physiological saline. The optimal L. reuteri formulation and dose will then be evaluated with an activated L. reuteri formulation, and it will be determined whether the same efficacy as seen with formulations containing intact L. reuteri is achieved using a protocol for dissolving L. reuteri. The formulation providing the minimum effective dose will be tested in adult non-pregnant rats to ensure efficacy in mature females.

[0106] Fifteen-day-old male and female immature rat pups were randomized to receive one of the following treatments: 5 male and 5 female pups were randomly assigned to each group. 1) saline, 2) planktonic L. reuteri, or 3) L. reuteri preparation. The L. reuteri preparation and planktonic L. reuteri were prepared as described in Example 6, and 2 × 10⁻⁶ doses were used. 9 Contains CFU L. reuteri / 1 mL of physiological saline. 2 × 10 9 Along with CFU L. reuteri / 1 mL saline, the L. reuteri preparation also contains 10 mg DM with 14.4 mg maltose, 20 mg DM with 28.8 mg maltose, 100 mg DM with 144 mg maltose, or 200 mg DM with 288 mg maltose. As described in Example 6, the L. reuteri preparation is activated for 1 hour, and the dose is administered once by oral enteral nutrition at 300 μL. This study involved 2 × 10⁶ 9A tapering approach is used, including an initial evaluation of formulations containing CFU / mL of L. reuteri, 200 mg of DM, and 288 mg of maltose. After administration, formulations containing lower levels of DM and maltose are used, and the formulation is reduced until no statistically significant efficacy in stimulating plasma oxytocin levels is observed. Blood is collected by tail snip at 24 and 48 hours and oxytocin is assayed (see Grewen, KM et al. (2010); Psychophysiology 47, 625-632). The groups are compared using one-way ANOVA and Tukey's test for multiple comparisons. The least effective L. reuteri formulation is then evaluated using a protocol to lyse activated L. reuteri formulations, followed by L. reuteri. The lysis protocol utilizes STET buffer containing lysozyme to cleave cell membranes and walls; sucrose to maintain osmotic pressure; Triton X to aid in cell wall cleavage; ethylenediaminetetraacetic acid (EDTA) as a chelating agent; and Tris HCl for buffering. Using immature male and female pup rats (5 males and 5 females per group) as described in previous studies, and animals receiving saline or saline with STET buffer as controls, intact L. reuteri formulations will be compared with dissolved L. reuteri formulations. The least effective L. reuteri formulation (dissolved or intact) providing a statistically significant increase in oxytocin levels will then be evaluated in adult female Sprague Dawley rats to confirm that the activity observed in pup rats is converted to adults. Eight adult female rats per group will be treated with L. reuteri formulations, plankton-like L. reuteri, or saline via oral enteral nutrition (500 μl), and oxytocin levels will be evaluated at 24, 48, and 72 hours post-administration, as described in Example 6. The least effective dose is a significant oxytocin stimulation at one of the two time points (24 or 48 hours). The oxytocin effect can last for 72 hours with formulations containing the highest levels of DM and maltose (maximum effective dose).Oxytocin levels will be compared to the saline-treated group using one-way ANOVA and Tukey's test for multiple comparisons. Example 9 - Effect of L. reuteri administration after azithromycin administration

[0107] Figure 12 shows the results of 400 μmol of prebiotic maltose and 1 × 10⁶ doses on the morning of day 3 after administration of prescription-grade azithromycin (Rx azithromycin). 9 This study design evaluates the efficacy of administering an L. reuteri preparation containing 100 mg of dextranomer microparticles (cross-linked dextran with epichlorolysine) loaded with CFU L. reuteri (ATCC 23272) [Preparation A]. Briefly, female Sprague-Dawley rats (170 g body weight) fed Teklad 2920X solid feed were administered azithromycin (45 mg / kg, orally) on day 0 of the study. On the morning of day 3 after azithromycin administration, six female rats were administered a single enteral dose of prescription-grade saline (reconstructed saline), and seven female rats were administered a single enteral dose of prescription-grade Preparation A (Rx Preparation A). Fecal pellets were collected from the female rats 4, 5, and 6 days after azithromycin administration, but 1, 2, and 3 days after either saline or Preparation A administration. Blood glucose was measured on days 4, 5, and 6. Female rats were euthanized on days 5 and 6. Tissue samples (stomach, jejunum, ileum, cecum, proximal colon, and distal colon) were collected on day 6. Evaluation of gastric contents

[0108] Figures 13A to 13D show the dry mass of gastric contents, gastric water content, gastric calorie content, and gastric content pH on the morning of day 3 after administration of either azithromycin or Rx saline to female rats treated with Rx saline and female rats treated with formulation A, respectively. Figure 13B shows that administration of formulation A to female rats after azithromycin administration increased gastric water content. Evaluation of enteritis by analysis of fecal samples

[0109] Intestinal inflammation can be assessed by analyzing fecal calprotectin and lactotransferrin levels. Figures 14A and 14B show fecal calprotectin and lactotransferrin levels in female rats treated with saline and female rats treated with formulation A, respectively. Figure 14A shows the decrease in fecal calprotectin levels in female rats treated with formulation A compared to female rats treated with saline two days after administration. Figure 14B shows the decrease in fecal lactotransferrin levels in female rats treated with formulation A compared to female rats treated with saline three days after administration. Figures 14C and 14D show fecal IL-22 and IL-6 levels in female rats treated with saline and female rats treated with formulation A, respectively. Figure 14C shows the decrease in fecal IL-22 levels in female rats treated with formulation A compared to female rats treated with saline two days after administration. Figure 14D shows that fecal IL-6 levels did not change after administration of formulation A. The data shown in Figures 14A to 14D were collected between days 3–4, 4–5, and 5–6 after administration of Rx azithromycin. Evaluation of pro-inflammatory and anti-inflammatory cytokines in the ileum

[0110] Ileal inflammation can be assessed using fecal markers of inflammation. IL-10 and IL-22 are anti-inflammatory cytokines. Briefly, the ileum was isolated from euthanized female rats. Ileal contents were collected in vials and stored at -20°C until assay. Figures 15A–15C show the levels of IL-6, IL-10, and IL-22 in the ileum of female rats treated with Rx saline and female rats treated with formulation A, respectively, three days after administration of either saline or saline. Figure 15A shows that administration of formulation A causes a decrease in the level of the pro-inflammatory cytokine IL-6 in the ileum of female rats treated with formulation A. Figure 15B shows that administration of formulation A causes a decrease in the level of the anti-inflammatory cytokine IL-10 in the ileum of female rats treated with formulation A. Figure 15C shows that administration of formulation A causes an increase in the level of the anti-inflammatory cytokine IL-22 in the ileum of female rats treated with formulation A. Evaluation of intestinal repair

[0111] Glucagon-like peptide 2 (GLP-2) promotes tight junctions and stimulates transporters for small and large intestinal growth, as well as glucose and fatty acid uptake. To evaluate intestinal repair, GLP-2 levels were measured in the proximal colon. The proximal colon was obtained from female rats euthanized three days after administration of either formulation A or saline. Figure 16A shows GLP-2 levels in the proximal colon of female rats treated with saline and female rats treated with formulation A. Figure 16A shows that administration of formulation A causes an increase in GLP-2 levels in the proximal colon. Evaluation of bowel leakage

[0112] Serum peptidoglycan levels were measured in female rats treated with formulation A and female rats treated with physiological saline. Briefly, adult female rats were euthanized by decapitation three days after administration of either Rx physiological saline or formulation A, and trunk blood was collected in a tube and allowed to coagulate on ice. The serum was separated by centrifugation and stored at -20°C until assayed for peptidoglycan by ELISA. Figure 16B compares serum peptidoglycan levels in female rats three days after administration of Rx physiological saline or formulation A. Figure 16B shows that administration of formulation A did not alter the elevated serum peptidoglycan levels caused by azithromycin administration. Evaluation of intestinal protection

[0113] Fecal secretory immunoglobulin A (sIgA) provides protection against potentially pathogenic microorganisms due to its resistance to enzymatic degradation and its ability to survive in harsh environments such as the gastrointestinal tract. Patients with low IgA levels are at higher risk of mucosal surface infections, food allergies, celiac-like enteropathy, and autoimmune disorders. Fecal sIgA levels were measured in female rats treated with formulation A and female rats treated with saline to assess intestinal protection. Figure 17 shows fecal sIgA levels measured on days 1, 2, and 3 (or days 4, 5, and 6 after azithromycin administration) in female rats to assess intestinal protection. Figure 17 shows an increase in fecal sIgA levels in female rats on day 3 after administration of formulation A. This increase in fecal sIgA levels in the intestinal lumen of adult female rats after administration of formulation A provides evidence of intestinal protection. Evaluation of SCFA production

[0114] SCFA (acetate, propionate, and butyrate) were measured by gas chromatography-mass spectrometry (GC / MS). Briefly, fecal pellets were collected from both female rats treated with formulation A and female rats treated with physiological saline between days 3–4, 4–5, and 5–6. The fecal pellets were stored at -20°C until assay. The samples were sonicated in 0.5% phosphoric acid (200 mg / ml), centrifuged to remove solid unextracted material, and filtered through a 0.6 μm filter to remove large particles. These samples were injected (1 μl) into a GC / MS equipped with a Stabilwax-DA column. The samples were calibrated using standard solutions of six short-chain fatty acids. Figures 18A to 18C compare the concentrations of various SCFAs (fecal acetate shown in Figure 18A, fecal propionate shown in Figure 18B, and fecal butyrate shown in Figure 18C) in female rats treated with Rx saline or formulation A. Figures 18A and 18C show increases in acetate and butyrate levels in excreted fecal pellets between day 2 and day 3 after administration of formulation A, respectively. As shown in Figure 18B, there is no statistically significant difference in propionate levels between adult female rats treated with formulation A and adult female rats treated with saline.

[0115] In addition to measuring SCFAs from collected fecal pellets, various SCFA levels were also measured in the distal colon feces of euthanized female rats three days after administration of formulation A or saline. Briefly, fecal pellets from the distal colon were collected in vials and stored at -20°C until assay. Figures 19A to 19D compare the concentrations of various SCFAs (fecal acetate shown in Figure 19A, fecal propionate shown in Figure 19B, fecal butyrate shown in Figure 19C, and fecal isovalerate shown in Figure 19D) in female rats treated with Rx saline or formulation A three days after administration of either saline or formulation A. As shown in Figures 19A and 19D, acetate and isovalerate levels increased in female rats three days after administration of formulation A. Figure 19B shows that propionate levels decreased in female rats three days after administration of formulation A. As shown in Figure 19C, there was no statistically significant difference in butyrate levels between adult female rats treated with formulation A and adult female rats treated with physiological saline.

[0116] The data presented in Example 3 demonstrate that a single dose of Formulation A significantly reduced intestinal inflammation 2-3 days after administration and was effective in mitigating the side effects of azithromycin. Example 10 - Study to evaluate L. reuteri formulations

[0117] This example describes a randomized, double-blind, placebo-controlled single-dose study to evaluate the safety and tolerability of a single-dose L. reuteri formulation (Sephadex® and L. reuteri containing maltose) in healthy adult volunteers. Such a study would monitor the elimination of L. reuteri and Sephadex® from the gastrointestinal (GI) tract in healthy adults and include evaluation of biomarkers of microbiome function, immunomodulation, and digestion after a single oral administration of an L. reuteri formulation in healthy adults.

[0118] L. reuteri Kandler (ATCC23272) containing Sephadex® and maltose, 2 x 10 units containing 200 mg Sephadex® and 1 M maltose 10 Administer orally in a final volume of 10 mL at the dose of colony-forming units (CFUs) ["Formulation B"]. L. reuteri Kandler is provided as a lyophilized powder for reconstitution in sterile saline. After reconstitution, mix L. reuteri Kandler with a slurry of Sephadex® and maltose.

[0119] Thirty eligible subjects will be randomly assigned in a 1:1 ratio to receive either formulation B or a placebo (saline solution). Subjects will be screened within 14 days prior to administration of either formulation B or placebo. All subjects will be required to provide written informed consent before any study-specific procedures are performed. Subject eligibility for the study will be determined at screening by evaluating inclusion and exclusion criteria. Subjects will be asked to provide consent to allow biobanking of their biological samples for future analysis. Eligible subjects will be provided with a stool sampling kit to collect a pre-treated stool sample within 48 hours prior to administration of formulation B or placebo (the timing of collection will be determined by the subject's bowel movement frequency).

[0120] Participants will return to the study site on day 1 after an 8-hour overnight fast. After confirming eligibility criteria, participants will receive a single oral dose of either Formulation B or placebo according to their assigned treatment group, followed by 100 mL of water. Fasting will be maintained for 1 hour after administration of either Formulation B or placebo. Participants will undergo study-specific procedures on day 1 according to the event schedule. Vital signs (pulse rate, systolic and diastolic blood pressure, respiratory rate, and oral temperature) will be measured before and 1 hour after administration of either Formulation B or placebo. Baseline stool samples will be collected 48 hours prior to the first dose to determine the presence of L. reuteri and Sephadex® microspheres and to perform exploratory biomarker analysis. Participants will be discharged after all day 1 evaluations are completed. Participants will be instructed to collect four stool samples at home according to the following schedule. Participants will be provided with a sampling kit for collecting stool samples at home. The kit includes labels for the sample containers to record the date and time the samples were obtained. Participants are instructed to return all stool samples using pre-labeled transport containers. Alternative strategies for returning stool samples may also be discussed by the researcher and participants.

[0121] Follow-up visits will be conducted on days 8 and 15. Participants will return to the hospital on day 30 for the end of the study.

[0122] Eligible subjects will receive a single dose of either Formulation B or placebo according to their assigned treatment group. Either Formulation B or placebo will be administered on day 1 under fasting conditions at the study site. Following administration, subjects will be given 100 mL of water to drink. Both Formulation B and placebo will be provided to subjects in opaque plastic syringes so that the contents are not visible in order to maintain blinding of the study. Subjects must fast for at least 8 hours prior to administration of either Formulation B or placebo. Subjects must refrain from using other probiotics and any antibiotics throughout their study involvement. Safety endpoints will include the incidence and severity of AEs occurring under treatment (TEAEs), serious AEs (SAEs), AESIs, and AEs leading to discontinuation of the study. This includes laboratory results (hematology [complete blood count with fractionation and erythrocyte sedimentation rate], biochemistry and urinalysis), vital signs, and physical examination findings; incidence of L. reuteri Kandler, Sephadex® microspheres and leukocytes in stool samples; Bristol Stool chart scores; and incidence of symptomatic bacteremia with positive L. reuteri identification. Other endpoints for the study include biomarkers for microbiome function: short-chain fatty acids (acetate, butyrate, propionate), lactate, and pH; biomarkers for immunomodulation: pro-inflammatory and anti-inflammatory cytokines (interferon-γ, interleukin [IL]-2, IL-4, IL-5, IL-6, IL-10, IL-22, chemokine ligand 1, tumor necrosis factor-α), as well as calprotectin, lactoferrin, and secretory immunoglobulin A; and biomarkers for digestion: fecal content of glucose, triglycerides, total bile acids, proteins, and lactose.

[0123] Demographic and baseline characteristics (including age, sex, race, ethnicity, weight, height, and body mass index) will be summarized using descriptive statistics, and the levels of L. reuteri, Sephadex® microspheres, and leukocytes in stool samples will be qualitatively assessed, with the results summarized using descriptive statistics.

[0124] The entire study period includes a one-month active enrollment period and a one-month follow-up period. The order and maximum duration of the study periods are as follows: Screening: 14 days; Treatment: 1 day; Follow-up: 30 days. The maximum study period for each subject is approximately six weeks. The maximum treatment period for each subject is one day.

[0125] Example 11 - Effects of administering L. reuteri preparations on autism spectrum disorder

[0126] This example details a study design to evaluate the efficacy of L. reuteri administration in the treatment of autism disorder. The target population's gastrointestinal (GI) system is far more developed than that of premature infants at risk of developing NEC, who are born with an immature and underdeveloped GI system (see Yatsunenko, T. et al. (2012); Nature.; 486, 222-227). However, GI symptoms, including constipation, abdominal pain, flatulence, and diarrhea, are often associated with autism disorder with a prevalence ranging from 23% to 70% (see Chaidez, V. et al. (2014); Journal of autism and developmental disorders.; 44(5), 1117-1127; Holingue, Calliope et al. (2018); Autism research: official journal of the International Society for Autism Research.; 11(1), 24-36). Neither GI pathology after histological examination nor changes in stool consistency or fecal volume were observed in preclinical studies using L. reuteri preparations. Furthermore, a decrease in fecal calprotectin levels, a clinical biomarker of GI inflammation, was observed, suggesting that L. reuteri preparations may reduce clinical or subclinical enteritis and improve GI symptoms commonly associated with autism spectrum disorder.

[0127] After initial screening, participants are randomized to one of two groups of eight participants each. Group A receives L. reuteri for 28 days, while Group B receives a placebo (saline). Following the initial dose, there is a 14-day washout period during which participants receive neither L. reuteri nor placebo. After the washout period, Group A receives placebo (saline) for 28 days, and Group B receives L. reuteri. The primary outcome is to evaluate the safety and tolerability of L. reuteri administered daily for 28 days, as assessed by the frequency and severity of adverse events and laboratory abnormalities. Secondary outcomes include evaluation of the effects of L. reuteri on circulating oxytocin levels, systemic and fecal levels of host and microbial metabolites, blood and fecal biomarkers of enteritis, and validated instruments for behavior in association with autism spectrum disorder. The total study duration is approximately 70 days.

[0128] Participants are eligible to participate in the study if they have a diagnostic confirmation of autism disorder, as confirmed by a gold-standard clinical interview using DSM-5 criteria and administration of the Autism Diagnostic Observation Schedule-2. L. reuteri Kandler will be administered orally in a 10 mL final volume dose containing 2 × 10¹⁰ colony-forming units with 185 mg Sephadex® and 74 mM maltose. Participants will receive a single dose daily for 28 days. L. reuteri Kandler will be provided as a lyophilized powder for reconstitution in (provided) saline. After reconstitution, L. reuteri Kandler will be mixed with a slurry of Sephadex® and maltose. The reference product / placebo is saline. Participants will receive a single oral dose of 10 mL of the corresponding saline for 28 days.

[0129] Eligible participants will receive a single dose of either L. reuteri Kandler or placebo daily for 28 days, according to their assigned treatment group. The 28 doses will be provided to participants to prepare at home and combine with their preferred beverage or soft food (e.g., applesauce, yogurt, pudding). The first dose of IP will be administered in a clinic. Participants are required to refrain from using probiotics throughout their study involvement.

[0130] Clinicians and caregivers report that outcome scale analysis consists of two paired t-tests: L. Reuteri Kandler versus placebo. Measures for each subject are changes in continuous scales, including CGI-S, CGI-I, ABC, CFQL-2, WJ3 Spatial Relations and Auditory Attention subtests, KiTap, RBANS, Eye Tracking, and psychophysical scales. For each subject, the difference between the change in continuous outcome scale score from the change relative to placebo is derived. A one-sample(pairs) t-test is performed on these within-subject differences. Results are considered statistically significant if the two-sided p-value is less than 0.05 after multiplicity correction using the false detection rate (FDR) approach. Furthermore, a linear mixed model with a two-treatment crossover design is analyzed. The response to this model is the continuous outcome scale of interest at the end of the period. Fixed-effect covariates are treatment, duration, sex, and carryover.

[0131] The order and maximum duration of the study periods are as follows: Screening: 14 days; Treatment Period 1: 28 days; Washout: 14 days; Treatment Period 2: 28 days; Follow-up: 30 days; The study period for each subject is approximately 17 weeks. Eye-tracking measurements are performed either during screening or baseline visits for participants with ASD. ADOS-2 = Autism Diagnostic Observation Schedule Module 3 or 4; WASI-II = Wechsler Abbreviated Scale of Intelligence Scale-Second Edition, SCQ = Social Communication Questionnaire; CGI-S = Clinical Global Impression Severity Scale, CGI-I = Clinical Global Impression Improvement Scale; Vineland-3 = Vinland Adaptive Behavior Scale, Third Edition; PK = Pharmacokinetics; EEG = Electroencephalogram Protocol; WJ3 = Woodcock Johnson's Spatial Relations and Auditory Attention Subtest; RBANS = Reproducible Battery of Neuropsychological States; KiTap = Computerized Test of Attentional Behavior in Children; ABC = Abnormal Behavior Checklist; CFQL-2 = Quality of Life for Children and Families, Second Edition Equal parts

[0132] While specific embodiments of this disclosure have been described, the above specification is illustrative and not limiting. Many variations of this disclosure will become apparent to those skilled in the art by examining this specification. The full scope of this disclosure should be determined by referring to the claims, the full scope of its equivalents, and the specification, as well as such variations.

[0133] Unless otherwise indicated, all figures used herein and in the claims, representing quantities of components, reaction conditions, etc., should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties to be obtained by this disclosure. Embedding by reference

[0134] The entire contents of all patents, published patent applications, websites, and other references cited herein are expressly incorporated herein by reference. The present invention provides, for example, the following items: (Item 1) A method for treating or improving autism spectrum disorder, wherein the method comprises administering to a subject in need of such treatment a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and optionally prebiotics. (Item 2) The method according to item 1, wherein the autism spectrum disorder is selected from the group consisting of autism spectrum disorder, Asperger syndrome, Heller syndrome, Rett syndrome, and pervasive developmental disorder, and pervasive developmental disorder not otherwise specified (PDD-NOS). (Item 3) The method according to any one of items 1 to 2, wherein the composition comprises dextranomer microparticles, L. reuteri, ATCC23272, and a prebiotic. (Item 4) The method according to any one of items 1 to 3, wherein, upon administration of at least one daily dose of the composition to the subject, the subject has up-control of oxytocin levels. (Item 5) The method according to any one of items 1 to 4, wherein the subject is a pediatric patient. (Item 6) The method described in any one of items 1 to 5, wherein the subject also has gastrointestinal disorders. (Item 7) A method for treating a patient with gastrointestinal disorders, comprising administering a composition comprising dextranomer microparticles, L. reuteri, ATCC23272, and prebiotics. (Item 8) The method according to item 7, wherein the patient is on the autism spectrum or has autism. (Item 9) The method according to item 7 or 8, wherein the gastrointestinal disorder is related to antibiotic administration. (Item 10) A method for treating an autistic patient suffering from a gastrointestinal disorder, comprising administering a composition comprising dextranomer microparticles, L. reuteri, ATCC23272, and a prebiotic. (Item 11) The method according to any one of items 7 to 10, wherein the gastrointestinal disorder is one or more of constipation, abdominal pain, flatulence, and diarrhea. (Item 12) A method for treating developmental disorders associated with premature birth in a premature infant, the method comprising administering to the premature infant a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier. (Item 13) A method for inducing oxytocin, wherein in a premature infant requiring oxytocin, the method comprises administering to the premature infant a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier. (Item 14) The method according to item 12 or 13, wherein the composition further comprises biocompatible microspheres and / or prebiotics. (Item 15) The method according to any one of items 12 to 14, wherein the composition comprises dextranomer microparticles, L. reuteri, ATCC23272, and a prebiotic. (Item 16) The method according to any one of items 12 to 14, wherein, upon administration of at least one daily dose of the composition to the premature infant, the premature infant has upregulated oxytocin levels. (Item 17) A method for treating gastrointestinal inflammation in a patient caused by the administration of an antibiotic regimen, comprising administering to the patient a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and / or prebiotics. (Item 18) A method for treating a patient who has or is expected to have gastroenteritis associated with the administration of an oral antibiotic regimen, comprising administering to the patient a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and / or prebiotics. (Item 19) The method according to item 17 or 18, wherein the composition is administered before, during, and / or after the administration of the antibiotic regimen. (Item 20) A method for substantially preventing or reducing adverse effects associated with oral antibiotics, comprising administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri and biocompatible microspheres to a subject in need thereof, wherein the subject has been treated with the oral antibiotic. (Item 21) A method for treating depression or anxiety disorder, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics. (Item 22) The method according to item 21, wherein depression is selected from the group consisting of clinical depression, postpartum or post-delivery depression, obsessive-compulsive disorder, post-traumatic stress disorder, bipolar disorder, atypical depression, Melanchol's depression, psychotic major depressive disorder (PMD), catatonic depression, seasonal affective disorder (SAD), dysthymia, double depression, depressive personality disorder (DPD), relapsing short-term depression (RBD), mild depressive disorder, bipolar disorder or manic-depressive disorder, treatment-resistant depression, treatment-resistant depression, suicidal ideation, suicidal thoughts and suicidal behavior. (Item 23) The method according to item 21, wherein the patient has postpartum or post-delivery depression. (Item 24) The method according to any one of items 1 to 23, wherein the composition is administered in a single dose. (Item 25) The method according to any one of items 1 to 23, wherein the composition is administered daily. (Item 26) The method according to items 1 to 25, wherein the prebiotic comprises a water-soluble carbohydrate, the water-soluble carbohydrate comprising one or more of the following: inulin, oligofructose, fructooligosaccharide, galactooligosaccharide, glucose, starch, maltose, maltodextrin, polydextrose, amylose, sucrose, fructose, lactose, isomaltulose, polyol, glycerol, carbonate, thiamine, choline, histidine, trehalose, nitrogen, sodium nitrate, ammonium nitrate, phosphorus, phosphate, hydroxyapatite, potassium, potassium carbonate, sulfur, homopolysaccharide, heteropolysaccharide, cellulose, chitin, vitamin, or a combination thereof. (Item 27) The method according to any one of items 1 to 26, wherein the prebiotic is maltose.

Claims

[Claim 1] The invention described in the specification.

Images