Methods of treating or ameliorating autism and related conditions

Abstract

The present application relates to methods of treating or ameliorating autism and related conditions. The present disclosure relates, in part, to methods of treating or preventing disease using bacterial formulations comprising probiotics, biocompatible microspheres, and / or prebiotics.

Description

[0001] This application is a divisional application of the Chinese invention patent application with an application date of January 13, 2021, a priority date of January 13, 2020, an application number of 202180020245.9, and an invention name of “Methods for treating or improving autism and related disorders”.

[0002] Related Applications

[0003] This application claims priority to USSN 62 / 960,328 filed on January 13, 2020 and USSN 62 / 960,331 filed on January 13, 2020, the contents of which are incorporated herein by reference. Background of the Invention

[0005] 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)). Therefore, probiotics are gaining attention as a viable option for treating diseases (Hemarajata, P. et al. (2013); Therap Adv Gastroenterol.; 6(1): 39-51). Bacteria are more likely to establish and persist when they are already in an activated form of biofilm (surface and / or self-adherent communities) rather than in a planktonic state (free-living). Providing probiotics in an activated state can enhance the positive effects of probiotics; this can be easily achieved by exposing the bacteria to the surface of biocompatible and non-toxic microspheres.

[0006] Oxytocin is a nonapeptide derived from the hypothalamus and stored in the posterior pituitary gland. In addition to its classic role in lactation and childbirth, it has recently attracted people's interest as an important neuropsychiatric hormone and metabolic hormone. Oxytocin is the core of recognizing complex social cognition and behavior (such as attachment, social exploration and recognition). In healthy people, oxytocin binds to receptors in social brain areas 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 regulates social brain functions (such as regulating social stress, emotion recognition and memory formation) (Meyer-Lindenberg, A. et al. (2011); Nat. Rev. Neurosci.; 12, 524). In autism, some studies report that intranasal administration of oxytocin can improve emotion recognition. In addition, oxytocin does not cross the blood barrier to cause childbirth by intranasal administration. However, intranasal oxytocin cannot reach the sustained physiological level of pulsatile oxytocin signaling. People with autism may also have an increased incidence of gastrointestinal disorders.

[0007] In addition, in many patients, the use of antibiotics is accompanied by side effects of gastrointestinal disorders ranging from mild (such as stomach upset, nausea, diarrhea and vomiting) to severe (such as abdominal pain, pseudomonal colitis). These side effects are more significant in children and the elderly (patients over 65 years old) who receive antibiotic treatment. It is reported that the gastrointestinal side effects associated with these antibiotic regimens are due to the elimination of beneficial flora necessary to promote normal digestive processes in the intestines. Scientific studies have shown that the use of antibiotics disrupts the normal ecology of the intestines, resulting in a decrease in the number of beneficial bacteria and an abnormal and harmful increase in harmful bacteria.

[0008] Therefore, there is a need for safe, effective therapies to treat or ameliorate autism and gastrointestinal disorders associated with autism and / or antibiotic use. SUMMARY OF THE INVENTION

[0010] The present disclosure relates in part to a method for treating or ameliorating autism spectrum disorders in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, and optionally a prebiotic (e.g., comprising polysaccharanhydride microparticles; Lactobacillus reuteri having a deposit number of ATCC23272; and a composition of prebiotics). The expected autism spectrum disorders treated using the provided methods may include autism, Asperger's syndrome, Heller's syndrome, Rett syndrome, and pervasive developmental disorder not otherwise specified (PDD-NOS).

[0011] Provided herein is a method for treating developmental disorders and / or autism, for example, the developmental disorders and / or autism may be, for example, related to premature birth of a premature infant, the method comprising administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier to a patient (e.g., a premature infant) in need thereof. In certain embodiments, the composition comprises polysaccharanhydride microparticles, Lactobacillus reuteri, ATCC 23272 and a prebiotic. In certain embodiments, after administering at least one daily dose of the composition to a subject, the subject has an increase in oxytocin levels.

[0012] In embodiments, provided herein is a method for treating gastrointestinal inflammation caused by the administration of an antibiotic regimen in a patient, comprising administering to the patient a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics. For example, provided herein is a method for treating a patient suffering from or expected to suffer from gastrointestinal inflammation 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. Such compositions may be administered before, during and / or after the administration of an antibiotic regimen. Also provided in embodiments is a method for substantially preventing or reducing adverse reactions associated with oral antibiotics in a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres, wherein the subject is undergoing oral antibiotic therapy.

[0013] It is also contemplated and provided herein that there is a method for treating depression or anxiety in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics. For example, provided herein is a method for treating one or more of the following using the disclosed method: clinical depression, postnatal or postpartum depression, obsessive-compulsive disorder, post-traumatic stress disorder, bipolar disorder, atypical depression, melancholic depression, psychotic major depression (PMD), tension depression, seasonal affective disorder (SAD), dysthymia, double depression, depressive personality disorder (DPD), recurrent brief depression (RBD), mild depression, bipolar disorder or manic depression, treatment-resistant depression, refractory depression, suicidal tendencies, suicidal ideation and suicidal behavior. In certain embodiments, the patient suffers from postnatal or postpartum depression.

[0014] In some embodiments, the therapeutic methods contemplated herein may be directed to male or female subjects in need thereof. In some embodiments, the therapeutic methods contemplated herein may be directed to infants, children, or adults. In certain embodiments, the therapeutic methods contemplated herein include 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 once. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A -B show the dry mass of stomach contents of saline-treated female and male rat pups, respectively. Figure 1C Total gastric calories of male pups at day 17 (D17) and day 21 (D21) are shown. Figure 1D -E show the caloric density of saline-treated female and male pups, respectively.

[0016] Figure 2A -B compares pups treated with formulation A and The dry weight of stomach contents of saline-treated pups (data from D21 female pups and D21 male pups are shown in Figure 2A and 2B middle). Figure 2C -D compares pups treated with formulation A and Gastric caloric content of saline-treated pups (data from D21 female pups and D21 male pups are shown in Figure 2C and 2D middle). Figure 2E -F compares pups treated with formulation A and Gastric lactose content of saline-treated pups (data from D21 female pups and D21 male pups are shown in Figure 2E and 2F middle).

[0017] Figure 3A Shown is the pH of gastric contents of saline-treated female rat pups. Figure 3B -C compares pups treated with formulation A and Gastric pH of saline-treated pups (data from D20 female pups and D21 male pups are shown in Figure 3B and 3C middle).

[0018] Figure 4A Blood glucose and jejunal glucose levels are shown. Figure 4B Jejunal protein concentrations in female rat pups are shown.

[0019] Figure 5A -C compares the application After saline or formulation A (24 hours) ( Figure 5A )、(48 hours)( Figure 5B ) and (7 days)( Figure 5C ) in serum peptidoglycan levels in 16-day-old female rat pups. Figure 5D Serum peptidoglycan levels in adult female rats are shown.

[0020] Fig. 6A -B compares the The next day after saline or formulation A treatment ( Fig. 6A ) and the third day ( Figure 6B ) Vitamin B12 concentration in the ileum of female rat pups. Figure 6C -D compares the The next day after saline or formulation A treatment ( Figure 6C ) and the third day ( Fig.6D ) Vitamin B12 concentration in the ileum of male rat pups.

[0021] Fig. 7A -C compares the 2 days after saline or formulation A, Various SCFAs ( Fig. 7A Cecal acetate as shown in ; Figure 7B Cecal propionate shown in ; Figure 7C Cecal butyrate concentrations as shown in . Fig.7D -F compares the 2 days after saline or formulation A, Various SCFAs ( Fig.7D Cecal acetate as shown in ; Fig. 7E Cecal propionate shown in ; Figure 7F Cecal butyrate concentrations as shown in .

[0022] Fig. 8A -B compared the 2 and 3 days after saline or formulation A, Female rat pups treated with saline or Formulation A ( Fig. 8A ) and male rat pups ( Figure 8B ) of the distal colon stool mass. Figure 8C -D compares the 2 and 3 days after saline or formulation A, Female pups treated with saline or Formulation A ( Figure 8C ) and male cubs ( Fig.8D ) of fecal water quality.

[0023] Fig.9A Serum oxytocin concentrations of dams after pup withdrawal are shown. Fig. 9B Compared with the application Serum oxytocin levels in 16-day-old male pups 24 hours after saline or Formulation A. Fig. 9C Compared with the application Serum oxytocin levels in 20-day-old male rat pups 48 hours after saline or Formulation A.

[0024] Fig.10 is a graph showing plasma oxytocin levels in female rats treated with Formulation B, planktonic Lactobacillus reuteri (Lr), or saline. Oxytocin levels are shown as mean ± standard error mean for the first three days after treatment (days are marked above the bars). Groups were compared using ANOVA followed by Tukey's multiple comparisons.

[0025] Fig.11A is a graph showing plasma oxytocin levels in female pups treated with Formulation B, planktonic Lactobacillus reuteri (Lr), or saline. Fig. 11B is a graph showing plasma oxytocin levels in male rat pups after treatment with Formulation B, planktonic Lactobacillus reuteri (Lr), or saline. The values ​​are shown as mean ± standard error mean for the first three days after treatment (days are indicated above the bars). Groups were compared using ANOVA followed by Tukey's multiple comparisons.

[0026] Fig.12 The use of prescription-grade azithromycin ( After azithromycin, Experimental study design of Lactobacillus reuteri ("L. reuteri") formulations.

[0027] Fig.13A -D show the gastric content dry mass, gastric water content, gastric caloric content and gastric content pH of saline-treated and Formulation A-treated female rats, respectively.

[0028] Fig.14A -B shows fecal calprotectin and fecal lactotransferrin levels of saline-treated and Formulation A-treated female rats, respectively. Fig. 14C -D show fecal IL-22 and fecal IL-6 levels in female rats treated with saline and formulation A, respectively. Data are Collected between days 3 and 4, between days 4 and 5, and between days 5 and 6 after azithromycin administration.

[0029] Fig.15A -C displays in Formulation A or IL-6, IL-10, and IL-22 levels in the ileum of saline-treated and Formulation A-treated female rats on day three after saline administration.

[0030] Fig.16A Show in Formulation A or Glucagon-like peptide 2 (GLP-2) levels in the proximal colon of saline-treated and Formulation A-treated female rats on day 3 after saline administration. Fig. 16B Comparison Salt water or Serum peptidoglycan levels in female rats on day 3 after administration of Formulation A.

[0031] Fig.17 showed that in female rats, Formulation A or 1, 2 and 3 days after saline administration (or Fecal sIgA levels were measured on days 4, 5, and 6 after azithromycin administration to assess intestinal protection.

[0032] Fig.18A -C compared the changes in the number of days between days 3 and 4, between days 4 and 5, and between days 5 and 6. Salt water or Various SCFAs in fecal pellets collected from female rats treated with Formulation A ( Fig.18A Fecal acetate as shown in ; Fig.18B Fecal propionate as shown in ; Fig.18C Fecal butyrate concentrations as shown in .

[0033] Fig.19A -D compares Salt water or On the third day after administration of Formulation A Salt water or Various SCFAs ( Fig.19A Fecal acetate as shown in ; Fig.19B Fecal propionate as shown in ; Fig.19C Fecal butyrate as shown in ; Fig.19D Fecal isovalerate concentrations as shown in . DETAILED DESCRIPTION OF THE INVENTION

[0035]

[0013] The present disclosure relates in part to methods of treating or preventing a disease or condition using bacterial formulations comprising probiotics, biocompatible microspheres, and / or prebiotics.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0037] Unless otherwise indicated, the practice of the present technology will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art. See, for example, Sambrook and Russell, eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; Ausubel et al., eds. (2007) Current Protocols in Molecular Biology series; Methods in Enzymology (Academic Press, Inc., NY) series; 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; Edited by Gait (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Edited by Hames and Higgins (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; edited by Hames and Higgins (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; edited by Miller and Calos (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); edited by Makrides (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.

[0038] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate and may vary (+) or (-) in increments of 1.0 or 0.1, as appropriate, or alternatively by + / -15%, or alternatively 10%, or alternatively 5%, or alternatively 2%. It should be understood that all numerical designations are preceded by the term "about", although not always expressly stated. It should also be understood that the agents described herein are exemplary only and equivalents thereof are known in the art, although not always expressly stated.

[0039] As used in the specification and claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a bacterium" includes a plurality of bacteria, including mixtures thereof.

[0040] As used in the specification, the term "formulation" may be used interchangeably with "composition".

[0041] For purposes of understanding the present subject matter and for purposes of constructing the appended patent claims, the following definitions are included. Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.

[0042] definition

[0043] As used herein, the terms "treat", "treatment" and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of complete or partial prevention of a condition or its signs or symptoms thereof, and / or therapeutic, in terms of partial or complete cure of a condition and / or adverse effects attributable to the condition.

[0044] "Prevention" means preventing a condition or reaction in an in vitro or in vivo system or subject susceptible to the condition or reaction. An example of this is preventing obesity by achieving satiety in a subject.

[0045] As described herein, "subject" or "patient" refers to any animal at risk of, suffering from, or diagnosed with a disease or condition (e.g., autism spectrum disorder, depression, anxiety, or developmental disorder), including but not limited to mammals, primates, and humans. In certain embodiments, the subject can 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 can be a postpartum woman, a pregnant woman, or a lactating woman.

[0046] "Prebiotics" means nutritional supplements for probiotics. Prebiotics are food ingredients, such as oligosaccharides, that are not digestible by a subject (e.g., a mammal such as a human) and that 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 can selectively stimulate the growth and / or activity of one or a limited number of bacteria in a subject.

[0047] "Microsphere" means a porous and / or semipermeable microparticle or granular material carrying a biofilm and / or carrying a compound (e.g., carrying a drug) within the specified size range. As used herein, microspheres are composed of particles having a diameter of 50 mm or less and a diameter of about 1 micron or more (e.g., about 1 to about 100 microns or alternatively, or alternatively about 1 to about 75 microns, or alternatively about 1 to about 50 microns, or alternatively about 1 to about 25 microns, or alternatively about 1 to about 10 microns, or alternatively about 0.5 to about 200 microns, or alternatively about 0.5 to about 700 microns, or alternatively about 1 to about 600 microns, or alternatively less than 700 microns, or alternatively less than about 600 microns, or alternatively less than 500 microns, or alternatively less than about 400 microns, or alternatively less than about 300 microns, or alternatively less than about 200 microns, or alternatively less than about 100 microns). Non-limiting examples of this class include: hollow microspheres that are porous and / or semipermeable and in some aspects may contain a drug or medication, microcapsules (in which an excipient forms a sheath or shell surrounding and containing a cargo, such as a drug, a chemical reducing agent, or an absorbent or adsorbent molecule), and microparticles, which are used as a general term for any particle within the recited size range, whether spherical or not, as these terms are commonly used in the art.

[0048] "Biodegradable polymer" refers to a polymer that is biocompatible and can be degraded in vivo by bodily processes into products that are easily discarded by the body and should not accumulate in the body.

[0049] "Biocompatible" means that the components of the delivery system will not cause tissue damage or cause damage to the human biological system. In order to impart biocompatibility, polymers and excipients with a history of safe use in the human body or with GRAS (generally recognized as safe) status are preferably used. Biocompatibility means that the ingredients and excipients used in the composition will eventually be "bioabsorbed" or removed by the body without adverse reactions to the body. For a composition to be biocompatible and considered non-toxic, it must not cause toxicity to cells. Similarly, the term "bioabsorbable" refers to microspheres made of materials that undergo bioabsorption in the body over a period of time, thereby avoiding long-term accumulation of materials in the patient's body. Biocompatible nanoparticles can be bioabsorbed in less than 2 years, less than 1 year, or, for example, less than 6 months. The bioabsorption rate is related to the particle size, the materials used, and other factors well known to those skilled in the art. A mixture of bioabsorbable, biocompatible materials can be used to form the microspheres in the formulation used in the present disclosure.

[0050] "Pharmaceutically acceptable carrier" refers to any diluent, excipient or carrier that can be used in the compositions of the present disclosure. Pharmaceutically acceptable carriers include ion exchangers, aluminum oxide, 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in the standard reference text Remington's Pharmaceutical Sciences, Mack Publishing Company in the art. They are preferably selected according to the expected form of administration, i.e. oral tablets, capsules, elixirs, syrups, etc. and consistent with conventional pharmaceutical practice.

[0051] "Administering" means delivering a substance to a subject, such as an animal or a human. Administration can be achieved in a single dose, continuous or intermittent manner throughout the treatment process. The method of determining the most effective mode of administration and dosage is known to those skilled in the art, and will vary with the composition used for therapy, the purpose of therapy, and the age, health or sex of the subject being treated. Single or multiple administrations can be performed, and the dosage level and mode are selected by the treating physician, or in the case of pets and animals, by the treating veterinarian. Suitable dosage formulations and methods of administering medicaments are known in the art. The route of administration can also be determined, and the method of determining the most effective route of administration is known to those skilled in the art, and will vary with the composition used for treatment, the purpose of treatment, the subject being treated, and the health status or disease stage of the target cell or tissue being treated. Non-limiting examples of route of administration include oral administration, vaginal administration, enteral administration, nasal administration (inhalation), injection, topical application, and by suppository.

[0052] The term "effective amount" refers to an amount sufficient to achieve a beneficial or desired result or effect. In the case of therapeutic or preventive applications, the effective amount will depend on the type and severity of the condition in question and the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the case of therapeutic compositions, in some embodiments, an effective amount is an amount sufficient to cause a protective response against a pathogen or, alternatively, to support a healthy state of survival. In some embodiments, the amount is sufficient to achieve one or more of the following: 1) clear pathogens; 2) restore a healthy microbiome; 3) modulate the immune system; 4) maintain metabolism and metabolic pathways; 5) reduce caloric intake; 6) improve social behavior.

[0053] In the case of in vitro or ex vivo applications, in some embodiments, the effective amount will depend 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 used. The skilled person will be able to determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may include one or more administrations of the composition.

[0054] The agents and compositions may be used in the manufacture of medicaments and in the treatment of humans and other animals by administration according to conventional procedures (as active ingredients in pharmaceutical compositions).

[0055] Composition

[0056] The composition used for the disclosed method can include pharmaceutically acceptable excipients such as microspheres, probiotics (e.g., Lactobacillus reuteri) and prebiotics, wherein the prebiotics include nutritional supplements for probiotics. In one aspect, the composition further includes one or more of the following: biofilm, prebiofilm, coating on the surface of the microspheres, therapeutic drugs or agents, chemical reducing agents, molecules that promote adsorption, molecules that support absorption. The disclosed microspheres can have a solid core, a hollow core or a porous core, wherein in one aspect, the microspheres encapsulate the prebiotics in the hollow core or the porous core. The microspheres can be biocompatible and / or semi-permeable. In one aspect, the microspheres include a coating on a biofilm layer or the outer surface of the microspheres. In one aspect, the microspheres activate bacteria, enhancing persistence and function.

[0057] The contemplated biocompatible microspheres may include a material selected from the group consisting of a biodegradable polymer and / or a non-degradable polymer, and such disclosed microspheres may have a diameter of about 0.5 microns to about 1000 microns. Additional preferred ranges are described herein and incorporated herein by reference. The microspheres may be porous and / or semi-permeable.

[0058] Non-limiting examples of biodegradable polymers are selected from one or more of the following: dextran; polyanhydrides; such as Sephadex (dextran cross-linked with epichlorohydrin), Sephadex G-25; poly(lactic-co-glycolic acid) or PLGA; polycaprolactone or PLC; chitosan; gelatin; acetylated dextran; poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); poly(lactic acid-co-glycolic acid); poly(lactide) / poly(ethylene glycol) copolymer; poly(glycolide) / poly(ethylene glycol) copolymer; poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer; poly(lactic acid) / poly(ethylene glycol) copolymer; poly(glycolic acid) / poly(ethylene glycol) copolymer. ) copolymers; poly(lactic acid-co-glycolic acid) / poly(ethylene glycol) copolymers; poly(caprolactone); poly(caprolactone) / poly(ethylene glycol) copolymers; poly(orthoesters); poly(phosphazenes); poly(hydroxybutyrate); poly(hydroxybutyrate); poly(lactide-co-caprolactone); polycarbonates; polyesteramides; polyanhydrides; poly(dioxanone); poly(alkylene alkylate); polyethylene glycol / polyorthoester copolymers; polyurethanes; poly(amino acids); polyetheresters; polyacetals; polycyanoacrylates; poly(oxyethylene) / poly(oxypropylene) copolymers; Copolymers and / or combinations thereof.

[0059] Non-limiting examples of 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 cellulosic 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 oxide, and copolymers and blends thereof.

[0060] Contemplated compositions for use in the disclosed methods may include prebiotics, such as one or more of the following: water-soluble carbohydrates, inulin, oligosaccharides, fructooligosaccharides, fructooligosaccharides, galacto-oligosaccharides, glucose, starch, maltose, maltodextrin, polydextrose, amylose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, carbonates, thiamine, choline, histidine, trehalose, nitrogen, sodium nitrate, ammonium nitrate, β-glucan, phosphorus, phosphate, hydroxyapatite, potassium, potash, sulfur, homopolysaccharides, heteropolysaccharides, cellulose, chitin, vitamins, and combinations thereof.

[0061] In another aspect, the prebiotic is selected from one or more of the following: trehalose; nitrogen such as sodium nitrate, ammonium nitrate, phosphorus such as phosphates such as hydroxyapatite, potassium such as potash, sulfur, oligosaccharides, homopolysaccharides, heteropolysaccharides, cellulose, chitin, glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonates and combinations thereof. For example, disclosed herein is a composition comprising maltose and / or Lactobacillus reuteri.

[0062] The composition for the disclosed method may include probiotics selected to provide one or more of: supporting antibacterial immunity, enhancing or supporting the health status of the subject, enhancing or supporting the gastrointestinal barrier, or antagonizing disease-related bacterial infections. On the other hand, the probiotics are selected to prevent pathogen colonization and / or limit and / or remove pathogens and / or limit excessive inflammatory responses by downregulating cytokine and chemokine production. In certain embodiments, the probiotics are selected to induce hormones that play a role in social bonding, energy metabolism, wound healing, thereby contributing to good physical, mental and social health. Non-limiting examples of probiotics are one or more of the following: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus rhamnosus, Bifidobacterium adolescentis, B. angulation, B. bifidum. bifidum), Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Streptococcus thermophilus, Pseudomonas fluorescens, Pseudomonas protegens, Pseudomonas brassicacearum, Pseudomonas aeruginosa; Azospirillum brabrasilense, Azospirillum lipferum, Azospirillum halopraeferens, Azospirillum iraqiensis.irakense; Acetobacter diazotrophicus; Herbaspirillum seropedicae; Bacillus subtilis, Pseudomonas stutzeri, fluorescein, P. putida, P. cepacian, P. vesicularis, P. paucimobilis; Bacillus cereus, B. thuringiensis, B. sphaericus; Shewanella oneidensis; Geobacter bemidjiensis, G.metallireducens, G.sulfurreducens, G.uraniireducens, G.lovleyi; Serratiamarcescens, Desulfovibrio vulgaris, D.desulfuricans, Dechloromonas aromatic, Deinococcus radiodurans, Methylibiumpetroleiphilum, Alcanivorax borkumensis, Archaeglobusfulgidus, Haloferax sp., Halobacterium sp., and combinations thereof. In certain embodiments, the compositions provided for use in the disclosed methods of treatment include Lactobacillus reuteri. 23272 TM ).

[0063] The disclosed compositions may include additional agents, such as agents selective for pathogens that may compete with probiotics. Such complementary agents may be in the core or on the surface of the microspheres in the composition comprising microspheres. Non-limiting examples of this type include chemical reducing agents; molecules and / or surfaces that promote adsorption (in the microsphere core or on the surface); molecules and / or surfaces that promote absorption (in the microsphere core or on the surface). In one aspect, the chemical reducing agent and the molecules and / or surfaces that promote absorption are coated on the surface of the microspheres.

[0064] For example, the disclosed compositions may include activated bacterial colonies that may form a biofilm layer on the outer surface of the microparticles, such as a composition comprising Lactobacillus reuteri. The depth of such a layer may be from about 0.5 micrometer to about 1 millimeter, and in the following ranges: for example, from about 1 micrometer to about 500 micrometers, from about 1 micrometer to about 250 micrometers, from about 1 micrometer to about 200 micrometers, from about 1 micrometer to about 100 micrometers, from about 1 micrometer to about 50 micrometers, from about 1 micrometer to about 40 micrometers, from about 1 micrometer to about 30 micrometers, from about 2 micrometers to about 100 micrometers, from about 2 micrometers to about 50 micrometers, from about 2 micrometers to about 40 micrometers, from about 2 micrometers to about 30 micrometers, from about 3 micrometers to about 100 micrometers, from about 3 micrometers to about 50 micrometers, from about 3 micrometers to about 40 micrometers, from about 3 micrometers to about 30 micrometers, from about 5 micrometers to about 100 micrometers, from about 5 ...5 micrometers to about 40 micrometers, and from about 5 micrometers to about 30 micrometers. In other embodiments, contemplated compositions include a scaffold or matrix for supporting the activation of bacteria (eg, Lactobacillus reuteri) when administered to a subject or environment to be treated.

[0065] As contemplated herein, the composition for the disclosed method may include one or more microsphere compositions in combination with a carrier, such as a pharmaceutically acceptable carrier or a biocompatible stent. 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, aluminum oxide, 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0066] The disclosed compositions can be formulated into a dosage form of biofilm-forming, activated probiotics, for example, or provide an effective amount of microsphere composition for end use, for example, about 1×10 5 Up to 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 7About 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.

[0067] In certain embodiments, bacterial formulations (e.g., Lactobacillus reuteri formulations) can be administered at about 6, 12, 18, 24, 36, 48, and 72 hours, or can be administered in a single dose for treatment. In certain embodiments, bacterial formulations (e.g., Lactobacillus reuteri formulations) can be administered orally, vaginally, topically, by inhalation, intravenously, intramuscularly, or by suppositories. They can be administered in any suitable formulation.

[0068] The composition can be formulated or processed for ease of administration, storage and use, for example frozen, lyophilized, suspended (suspension formulations) or powdered; and processed into suppositories, tablets, solutions, suspensions, pills, capsules, sustained-release formulations.

[0069] The therapeutic composition of effective amount is determined based on the expected target. The term "unit dose" or "dose" refers to a physically discrete unit suitable for a subject, each unit containing a predetermined amount of a composition that is calculated to produce the desired response discussed above related to its administration (i.e., appropriate route and regimen). Depending on the number of treatments and the unit dose, the amount of administration depends on the desired result and / or protection. The precise amount of the composition also depends on the practitioner's judgment and is specific to each individual. Factors affecting the dosage include the physical and clinical state of the subject, the route of administration, the expected therapeutic target (symptom relief and cure), and the efficacy, stability, and toxicity of the specific composition. When formulated, the solution will be administered in a manner compatible with the dosage formulation and in an amount effective for treatment or prevention. The formulation is easily administered in a variety of dosage forms, such as administered in the above-mentioned injection solution type.

[0070] In certain embodiments, the present disclosure provides a method of treatment by administering a therapeutically effective amount of a bacterial formulation (eg, a Lactobacillus reuteri formulation). In certain embodiments, the bacterial formulation comprises Lactobacillus reuteri, biocompatible microspheres, and / or prebiotics.

[0071] method

[0072] Provided herein is a method for treating or preventing a disease or condition appropriately treated by a bacterial formulation in a subject in need thereof. The method includes administering an effective amount of a bacterial formulation disclosed herein to a subject, which has a component selected for a specific therapy. In one embodiment, the disclosure provides a method for inducing oxytocin by administering a therapeutically effective amount of a bacterial formulation (e.g., a Lactobacillus reuteri formulation). In certain embodiments, oxytocin is induced in a subject by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics. In certain embodiments, administering a therapeutically effective amount of a bacterial formulation (e.g., a Lactobacillus reuteri formulation) activates the oxytocin receptor.

[0073] In certain embodiments, the present disclosure provides in an embodiment a method of treating, ameliorating and / or preventing autism spectrum disorders in a subject by administering a pharmaceutically acceptable composition of a bacterial formulation (e.g., a Lactobacillus reuteri formulation) to the subject, including but not limited to autism, Asperger's syndrome, Heller's syndrome, Rett syndrome, and pervasive developmental disorder not otherwise specified (PDD-NOS).

[0074] Also provided herein is a method for treating or preventing a disease or condition (e.g., gastrointestinal inflammation) caused by the administration of an antibiotic regimen appropriately treated by a bacterial formulation in a subject in need thereof. The method includes administering to the subject an effective amount of a bacterial formulation disclosed herein having a component selected for a specific therapy. Non-limiting examples of diseases include gastrointestinal disorders such as gastrointestinal inflammation, or any chronic and / or recurrent disease caused by the use of antibiotics and resulting in pathogens replacing healthy bacteria, diseases or conditions involving intestinal dysmotility, or bacterial infections associated with antagonistic diseases.

[0075] In an embodiment, the present disclosure provides a method for treating gastrointestinal inflammation caused by the administration of an antibiotic regimen (e.g., azithromycin) in a patient by administering a therapeutically effective amount of a bacterial formulation (e.g., a Lactobacillus reuteri formulation). In certain embodiments, the gastrointestinal inflammation of a subject is reduced or alleviated by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or a prebiotic.

[0076] In certain embodiments, the present disclosure provides a method for treating a patient suffering from or expected to suffer from gastrointestinal inflammation associated with the administration of an oral antibiotic regimen, the method being performed by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier to a patient in need thereof. In certain embodiments, gastrointestinal inflammation is significantly reduced by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or prebiotics. Such oral antibiotic regimens can include the administration of oral antibiotics of the following general categories, such as sulfonamides; folic acid analogs; beta-lactams, including penicillins and cephalosporins; tetracyclines; macrolides; lincosamides; streptogramins; quinolones, including fluoroquinolones; polypeptides such as polymyxins; aminocyclols; glycopeptides; oxazolidinones; etc. "Antibiotics" refers to antibiotics, antibacterial agents, antimicrobial agents, anti-infective agents, etc.

[0077] Exemplary antibiotics include amoxicillin, a combination of amoxicillin and clavulanate potassium, ampicillin, a combination of ampicillin and sulbactam, atovaquone, azithromycin, carbenicillin, cefaclor, cefdinir, cefonicid, ceftibuten, cefotetan, cefpodoxime, ceftriaxone, cefuroxime, cephalexin, cephalothin, cephalosporin, chlortetracycline, ciprofloxacin, clindamycin, clarithromycin, cycloserine, dalfopristin, , dicloxacillin, doxycycline, erythromycin, levofloxacin, linezolid, moxifloxacin, mupirocin, oxytetracycline, penicillin, rifampicin, quinupristin, a combination of dalfopristin and quinupristin, spectinomycin, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfadimethoxazole, sulfamethoxazole, sulfamethoxazole, tetracycline, trimethoprim, a combination of trimethoprim and sulfamethoxazole, vancomycin, a combination comprising at least one of the foregoing, etc. In certain embodiments, the antibiotic regimen includes azithromycin.

[0078] In certain embodiments, the present disclosure provides a method of treating a gastrointestinal disorder in a patient comprising administering a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or a prebiotic. In certain embodiments, the gastrointestinal disorder is one or more of constipation, abdominal pain, bloating, and diarrhea.

[0079] In certain embodiments, the present disclosure provides methods of treating a gastrointestinal disorder in a patient, wherein the patient is on the autism spectrum or has autism.

[0080] Provided herein are methods of substantially preventing or reducing adverse effects associated with oral antibiotics in a subject in need thereof using a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or a prebiotic.

[0081] In certain embodiments, the bacterial formulations described in the present disclosure (e.g., Lactobacillus reuteri formulations) can be used to prevent and / or improve and / or treat adverse gastrointestinal (GI) side effects (e.g., from another treatment with GI side effects, e.g., antibiotic administration), including but not limited to pseudomembranous colitis (inflammation of the large intestine due to overgrowth of Clostridium difficile), nausea, cramps, diarrhea, and vomiting. These are some of the side effects associated with antibiotics. However, the effects of the formulations used in the context of the present disclosure on these conditions can be mediated in whole or in part by the effects on body weight, or can be mediated independently of body weight. In certain embodiments, the disclosed bacterial formulations (e.g., Lactobacillus reuteri formulations) can be used as agents for preventing the above-mentioned conditions and / or diseases. In some embodiments, administration causes one or more of the following: reducing or eliminating one or more symptoms of a condition, preventing the severity of an increase in one or more symptoms of a condition, and / or reducing, preventing, or eliminating further diseases or conditions.

[0082] In certain embodiments, administration of a pharmaceutically acceptable composition of a bacterial formulation (e.g., a Lactobacillus reuteri formulation) described in the present disclosure results in a reduction in gastrointestinal inflammation in a subject. In certain embodiments, administration results in at least a 5% reduction in gastrointestinal inflammation in a patient (e.g., at least a 7%, 10%, 20%, 30%, 50%, 75% or more reduction).

[0083] In certain embodiments, the bacterial composition (eg, a Lactobacillus reuteri formulation) is administered prior to administration of an antibiotic regimen, during administration of an antibiotic regimen, and / or after administration of an antibiotic regimen.

[0084] In embodiments, the present disclosure provides a method for treating depression or anxiety of a subject by applying a pharmaceutically acceptable composition of a bacterial formulation (e.g., a Lactobacillus reuteri formulation) to the subject. In certain embodiments, depression or anxiety include clinical depression, postnatal or postpartum depression, postpartum obsessive-compulsive disorder, posttraumatic stress disorder such as postpartum posttraumatic stress disorder, postpartum bipolar disorder, atypical depression, melancholic depression, psychotic major depression (PMD), tension depression, seasonal affective disorder (SAD), dysthymia, double depression, depressive personality disorder (DPD), recurrent brief depression (RBD), mild depression, bipolar disorder or manic depression, treatment-resistant depression, refractory depression, suicidal tendency, suicidal ideation and suicidal behavior. In some embodiments, the subject suffers from anxiety. In some embodiments, the subject suffers from depression. In some embodiments, the subject suffers from postnatal or postpartum depression.

[0085] In an embodiment, the present disclosure provides a method for treating developmental disorders associated with preterm birth in a premature infant by administering a pharmaceutically acceptable composition of a bacterial formulation (e.g., a Lactobacillus reuteri formulation) to the premature infant. In certain embodiments, the bacterial formulation for treatment comprises a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier.

[0086] In certain embodiments, the present disclosure provides methods of treating developmental disorders associated with prematurity, including but not limited to bronchopulmonary dysplasia (BPD); intellectual disability, cerebral palsy, white matter disease; and retinopathy of prematurity.

[0087] In certain embodiments, the present disclosure provides a method of inducing oxytocin in a premature infant in need thereof by administering to the premature infant a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier. In certain embodiments, oxytocin is induced in a premature infant by administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or a prebiotic.

[0088] In certain embodiments, administration of at least one daily dose of a bacterial formulation (e.g., a Lactobacillus reuteri formulation) to a subject results in upregulation of oxytocin levels. In certain embodiments, oxytocin is upregulated by at least 5% (e.g., at least 5%, 10%, 20%, 30%, 50%, 75% upregulated, or more) in a subject following administration.

[0089] In certain embodiments, the present disclosure provides a method for enhancing lactation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or a prebiotic. In certain embodiments, the enhancement of lactation after administration of a bacterial formulation of the present disclosure (e.g., a Lactobacillus reuteri formulation) is due to an increase in oxytocin levels known to be associated with lactation and secretion (see Pang, WW et al.; (2007); J Mammary Gland Biol Neoplasia 12, 211-221).

[0090] In certain embodiments, administration of a bacterial formulation of the present disclosure (e.g., a Lactobacillus 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., a Lactobacillus reuteri formulation) increases oxytocin levels for 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., a Lactobacillus reuteri formulation) increase oxytocin levels for 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 (e.g., a Lactobacillus reuteri formulation) of the present disclosure increases prolactin 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%).

[0091] In some embodiments, a single dose of bacterial formulation (e.g., Lactobacillus reuteri formulation) is administered to a subject. In other embodiments, multiple doses are administered at two or more time points, at intervals of hours, days, weeks (e.g., after about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours). In some embodiments, a bacterial formulation (e.g., Lactobacillus reuteri formulation) is administered over a long period of time (e.g., chronically), for example, for a period of several months or several years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months or years). In certain embodiments, a bacterial formulation (e.g., Lactobacillus reuteri formulation) can be taken regularly (e.g., daily, weekly, etc.) over the duration of an extended period of time.

[0092] In some embodiments, the composition of the method is administered to provide about 1×10 7 About 1×10 10 CFU / ml (e.g., about 1.5×10 7 , about 2×10 7 , about 2.5×10 7 , about 3×10 7 , about 3.5×107 , 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 ,1×10 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 about 9.5×10 9 ) activated biofilm-forming probiotics.

[0093] In certain embodiments, the disclosed bacterial formulations (eg, Lactobacillus reuteri formulations) employed in the context of the present disclosure may be administered as part of a combination therapy with at least one other agent for treating a disease or condition discussed in the present disclosure.

[0094] Having now generally described the disclosure, the disclosure will be more readily understood by reference to the following examples, which are included merely to illustrate certain aspects and embodiments of the disclosure and are not intended to limit the disclosure in any way. Example

[0095] The following examples are illustrative only and are not intended to limit the scope or content of the present disclosure in any way.

[0096] Example 1 - Lactobacillus reuteri formulation

[0097] The formulation is provided in powder form for reconstitution into a liquid for oral administration. Each mL of formulation A contains 1.9x10 9 reuteri colony forming units and 18.5 mg Sephadex in 74 mM maltose. The dose is 10 mL of formulation once daily, with a daily dose of 1.9 x 10 10 reuteri colony forming units and 185 mg Sephadex in 74 mM maltose.

[0098] Example 2 - Study of Lactobacillus reuteri Formulations

[0099] The preclinical safety study design of Lactobacillus reuteri formulation included loading of 24 μmole prebiotic maltose and 6x10 9 60 mg of polyanhydride microparticles (dextran cross-linked with epichlorohydrin) containing CFU of Lactobacillus reuteri (ATCC 23272). [Formulation A] Briefly, naturally delivered newborn rats were divided into the following treatment groups: (1) prescription saline ( Saline) and (2) Formulation A. Each treatment group consisted of 24 male pups and 24 female pups. On day 15, each pup received a single enteral dose of: Saline or Formulation A. Throughout the study, pups stayed with their mothers (dams). Pups were breastfed for nutrition. As the pups approached day 21, only pups treated with Formulation A began to eat food prepared for the dams. Pups (N=8 (8 males and 8 females) per treatment group) 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 tract appeared normal in saline and Formulation A treated pups. Light microscopy imaging demonstrated intact clearance of microspheres in feces 48 hours after oral gavage. Preclinical toxicology studies indicate that the Formulation A formulation is safe. Macroscopic evaluation of rat pups demonstrated that pups treated with Formulation A began eating earlier than saline treated pups, while days 17 and 21 were calorically equivalent. On day 17, even though the pups consumed less mass and a higher caloric density, they were calorically equivalent to day 21, when food intake (total mass) was higher and caloric density was lower. This suggests that a hypothalamic set point regulating energy balance is established. These photos show pups fed breast milk supplemented with solid food.

[0100] Example 3 - Spontaneous weaning: Effect of administering a Lactobacillus reuteri formulation to neonatal rats

[0101] The weaning study design for neonatal rats was as follows. Briefly, naturally delivered pups were divided into the following treatment groups: (1) prescription saline ( Saline) and (2) Formulation A. Each treatment group consisted of 20 male pups and 20 female pups. On day 18, each pup received a single enteral dose of: Saline or Formulation A. Pups (N=10 (10 males and 10 females) per treatment group) were euthanized 48 and 72 hours after administration. Trunk blood, stomach, jejunum, ileum, cecum, proximal colon, and distal colon were harvested on days 20 and 21. In addition, body composition, thymus mass, and spleen mass of each pup were analyzed.

[0102] Effect on caloric intake

[0103] Figure 1A -B shows the dry mass of stomach contents of saline-treated female and male pups, respectively. Figure 1C Total stomach calories are shown for male pups at day 17 (D17) and day 21 (D21). Total stomach calories were the same for female pups. Total calories remained constant during the transition from breast milk to food. Figure 1A -C shows caloric equality between D17 and D21. On D17, even though pups consume less mass and a higher caloric density, they are calorically equivalent to D21, when food intake (total mass) is higher and caloric density is lower. This suggests that a hypothalamic set point regulating energy balance is established. Figure 1D -E show the caloric density of saline-treated female and male pups, respectively. Figure 1A -E shows that as saline-treated pups approach weaning, their stomach contents increase in mass and decrease in caloric density. Figure 2A -B compares pups treated with formulation A and The dry weight of stomach contents of saline-treated pups (data from D21 female pups and D21 male pups are shown in Figure 2A and 2B middle). Figure 2C -D compares pups treated with formulation A and Gastric caloric content of saline-treated pups (data from D21 female pups and D21 male pups are shown in Figure 6C and 6D middle). Figure 2E -F compares pups treated with formulation A and Gastric lactose content of saline-treated pups (data from D21 female pups and D21 male pups are shown in Figure 2E and 2F middle). Figure 2A -F shows that formulation A reduced food intake despite establishing a caloric intake set point. Figure 2A -F also showed that Formulation A treatment may accelerate the males' earlier transition from breast milk to food.

[0104] Effects on gastric acid

[0105] Intragastric pH measurements were performed to test the effect of the Lactobacillus reuteri formulation on gastric acid. Briefly, animals were euthanized and gastric contents were removed for assay. Data for 22 pups on days 16-19 were obtained from the safety study described in Example 2. Figure 3A The data described in are from saline-treated rat pups or untreated rat pups. Figure 3A The stomach content pH of saline-treated female pups is shown. Figure 3B -C compares pups treated with formulation A and Gastric pH of saline-treated pups (data from D20 female pups and D21 male pups are shown in Figure 3B and 3C middle). Figure 3A -C shows the increase in gastric acid production after 18 days of age. Figure 3A -C also shows that administration of Formulation A does not promote weaning by increasing gastric acid production.

[0106] Effects on jejunal protein concentration

[0107] The jejunum is specialized for nutrient absorption. Briefly, the jejunum was isolated from euthanized pups. The contents of the jejunum were collected into vials and stored at -20 °C until assayed. Figure 4A Blood glucose and jejunal glucose levels are shown. Figure 4B Jejunal protein concentrations in female rat pups are shown. Figure 4A Blood glucose was also shown to be dependent on jejunal glucose concentration. Figure 4AIt was shown that starting from day 16, the intestinal glucose transporters (SGLT1, GLUT2) and fructose transporter (GLUT5) of rat pups were functionally active. Figure 4B showed that jejunal protein concentration is dependent on gastric acid production and therefore decreases after 18 days of age.

[0108] Effects on serum peptidoglycan concentrations

[0109] Serum peptidoglycan levels were measured in female pups treated with Formulation A. Briefly, pups were euthanized by decapitation and trunk blood was collected into tubes and allowed to clot while placed on ice. Serum was separated by centrifugation and stored at -20°C until assayed for peptidoglycan by enzyme-linked immunosorbent assay (ELISA). Figure 5A -C compares the After saline or formulation A, 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 ) serum peptidoglycan levels. Figure 5D Serum peptidoglycan levels in adult female rats are shown.

[0110] Effects on Vitamin B12 Production

[0111] In female and male pups, the Ileal vitamin B12 content was measured on the second and third day after saline treatment. Briefly, the ileum was isolated from euthanized pups. The contents of the ileum were collected into vials and stored at -20 °C until assayed. Vitamin B12 was assayed by ELISA. Fig. 6A -B compares the The next day after saline or formulation A treatment ( Fig. 6A ) and the third day ( Figure 6B ) Vitamin B12 concentration in the ileum of female rat pups. Figure 6C -D compares the The next day after saline or formulation A treatment ( Figure 6C ) and the third day ( Fig.6D ) Vitamin B12 concentration in the ileum of male rat pups. Fig. 6A and 6C Administration of Formulation A was shown to increase the concentration of vitamin B12 in the ileum of rat pups (male and female). Vitamin B12 is synthesized in the ileum by Lactobacillus reuteri. Figure 6B Formulation A treated female rat pups showed decreased vitamin B12 concentrations in the ileum. Fig.6D Show in There were no statistical differences in vitamin B12 concentrations in male pups treated with saline or Formulation A. These figures show that vitamin B12 is cleared from the ileum on day 3. Vitamin B12 is also used as a marker for reuterin, a potent antimicrobial compound produced by Lactobacillus reuteri.

[0112] Effects on short-chain fatty acids (SCFA)

[0113] Measurement of SCFA (acetate, propionate and butyrate) was performed by gas chromatography-mass spectrometry (GC / MS). Briefly, the cecum was isolated from euthanized pups. The contents of the cecum were collected into vials and stored at -20°C until assayed. The samples were sonicated in 0.5% phosphoric acid (200 mg / ml), centrifuged to remove unextracted solid matter, and filtered through a 0.6 μm filter to remove any large particles. These samples (1 μl) were injected into a GC / MS with a Stabilwax-DA column. Standard solutions of 6 short-chain fatty acids were used to calibrate the samples. Fig. 7A -C compares 2 days after administration of saline or formulation A, Various SCFAs ( Fig. 7A Cecal acetate as shown in ; Figure 7B Cecal propionate shown in ; Figure 7C Cecal butyrate concentrations as shown in . Fig.7D -F compares the 2 days after saline or formulation A, Various SCFAs ( Fig.7D Cecal acetate as shown in ; Fig. 7E Cecal propionate shown in ; Figure 7F Cecal butyrate concentrations as shown in . Fig. 7A -F shows that administration of formulation A did not alter SCFA production in the cecum of rat pups (male and female), thus the weaning effect was not through SCFA production.

[0114] Effects on fecal excretion

[0115] The distal colon was isolated from euthanized pups. Fecal pellets in the distal colon were collected into vials and stored at -20 °C until assayed. To measure dry mass and water mass, fecal pellets were lyophilized to remove water. The difference between wet mass and dry mass is the water mass. Fig. 8A -B compared the 2 and 3 days after saline or formulation A, Female pups treated with saline or Formulation A ( Fig. 8A ) and male cubs ( Figure 8B) of the distal colon stool mass. Figure 8B Male pups treated with Formulation A showed increased fecal output. Figure 8B The data obtained also showed that Formulation A can be used to treat irregularity. Figure 8C -D compares the 2 and 3 days after saline or formulation A, Female pups treated with saline or Formulation A ( Figure 8C ) and male cubs ( Fig.8D ) of fecal water quality. Figure 8C -D shows that administration of Formulation A did not cause diarrhea or constipation in male rat pups or female rat pups.

[0116] The data presented in Example 3 demonstrate that Formulation A promotes weaning and satiety at the hypothalamic level without altering intestinal function.

[0117] Example 4 - Effects of administering a Lactobacillus reuteri formulation to adult rats

[0118] Fig.9A Serum oxytocin concentrations of mothers after pup withdrawal are shown. The figure shows that in adult female mothers treated with Formulation A, serum oxytocin levels remain elevated for about 11 days after their nursing pups are withdrawn. The data show that the elevated oxytocin levels observed during breastfeeding decrease after about 11 days after the mothers' pups are withdrawn. These results suggest that Formulation A can modulate postpartum depression in female rats. Fig. 9B Compared with the application Serum oxytocin levels in 16-day-old male pups 24 hours after saline or Formulation A. Fig. 9C Compared with the application Serum oxytocin levels in 20-day-old male pups 48 hours after saline or Formulation A. Fig. 9B -C shows an increase in serum oxytocin levels in male pups treated with Formulation A 24 hours after administration and suggests that elevated oxytocin levels may help regulate food intake and social and exploratory behaviors during early childhood. Data after one dose of Formulation A are shown in Fig. 9B -C. Multiple doses of Formulation A may help maintain elevated oxytocin levels.

[0119] Example 5 - Activation of Lactobacillus reuteri using polyanhydride microparticles (DM)

[0120] Lactobacillus reuteri formulations comprising maltose and DM show enhanced bioactivity. In vitro activation simulates activation in the gastrointestinal tract when microorganisms bind to mucin glycans. DM is a biocompatible porous semipermeable solid particle containing dextran, which is similar to the O-linked glycans of mucin. The Lactobacillus reuteri strains disclosed herein express an extracellular glucosyltransferase (GTF) enzyme. The in vitro activation process is a simulation of the biological changes induced when Lactobacillus reuteri contacts and binds to mucin glycans in the ileum. Therefore, activation provides an advantage over non-activated Lactobacillus reuteri to better initiate colonization.

[0121] Example 6 - Effect of administration of Lactobacillus reuteri formulations on plasma oxytocin levels

[0122] Lactobacillus reuteri (ATCC 23272 strain) was cultured in vials containing freeze-dried Lactobacillus reuteri at 1.7 x 10 11 CFU / g is provided. 1mL sterile saline was added to the lyophilized bacteria and rehydrated at room temperature for 5 minutes and then added to DM-maltose syrup. DM-maltose syrup was prepared by weighing autoclaved dry Sephadex G25 Superfine microspheres (DM) and adding them to a filter-sterilized 1M maltose solution (25% w / v suspension). To start the activation step, the mixture of Lactobacillus reuteri and DM-maltose syrup was incubated together at room temperature for 60 minutes before use. The final dose of each application of Lactobacillus reuteri formulation was 500μL 2x10 9 CFU L. reuteri, 20 mg DM and 28.8 mg maltose in 1 mL saline ["Formulation B"]. Planktonic L. reuteri was prepared similarly but without the addition of DM-maltose syrup.

[0123] To investigate the effects of Lactobacillus reuteri formulations on plasma oxytocin levels, adult female Sprague-Dawley rats were given a single oral dose of planktonic Lactobacillus reuteri (Lr) or saline 2 days after a single dose of prescription-grade azithromycin (45 mg / kg, administered by oral gavage) to create a niche for Lactobacillus reuteri (a 2-day washout period was used between antibiotics and treatments). On days 1, 2, and 3 post-dose, blood was collected in tubes containing aprotinin (500 kallikrein inactivator units (KIU) / mL blood; used due to the short half-life of oxytocin in the blood), and plasma oxytocin levels were determined by enzyme immunoassay (EIA; Assay Designs). Fig.10 is a graph showing plasma oxytocin levels in female rats treated with Formulation B, planktonic Lactobacillus reuteri (Lr), or saline. Oxytocin levels are shown as mean ± standard error mean for the first three days after treatment (days are indicated above the bars). Groups were compared using ANOVA followed by Tukey's multiple comparisons. Fig.10 As shown, day 1 plasma oxytocin levels in animals treated with Formulation B were significantly higher than those treated with either planktonic L. reuteri or saline. Oxytocin levels between groups leveled off on day 3. This study demonstrates that Formulation B effectively stimulated circulating oxytocin on day 1 and maintained this on day 2. In addition, planktonic L. reuteri showed a trend toward an ability to stimulate oxytocin levels on day 2, but levels were not significantly different from the saline treated group.

[0124] To investigate the effects of Lactobacillus reuteri formulations on plasma oxytocin levels in pups, pups were dosed with saline, Lactobacillus reuteri, or 300 μL of Formulation B (prepared as described above). Immature rat pups have low baseline circulating oxytocin levels, and there were no sex differences in baseline levels or responses to Formulation B. All animals were treated once by oral gavage at 15 days of age. Blood was collected on days 1, 2, and 3 post-treatment, and plasma oxytocin was measured using the methods described above. Fig.11A is a graph showing plasma oxytocin levels in female pups treated with Formulation B, planktonic Lactobacillus reuteri (Lr), or saline. Fig. 11B is a graph showing plasma oxytocin levels in male pups after treatment with Formulation B, planktonic Lactobacillus reuteri (Lr), or saline. The values ​​are shown as mean ± standard error mean for the first three days after treatment (days are indicated above the bars). Groups were compared using ANOVA followed by Tukey's multiple comparisons. This study demonstrates that Formulation B is able to increase circulating oxytocin levels.

[0125] Example 7 - Evaluation of Lactobacillus reuteri Formulations

[0126] This example describes the effects of various preparation methods on Lactobacillus reuteri formulations, including measuring the production of lactic acid, histamine, vitamin B-12, and glycerol by Lactobacillus reuteri in the formulations, as well as its utilization of sugars. These examples describe the evaluation of the following two Lactobacillus reuteri formulations: 1) Formulation B (2x10 9 CFU Lactobacillus reuteri, 20 mg DM and 28.8 mg maltose (in 1 mL saline); and 2) Formulation C (containing the same amount of Lactobacillus reuteri as Formulation B (2 x 10 9 CFU), but 10 times the DM and maltose (200 mg DM and 288 mg maltose). The formulation preparation process was carried out as described in Example 6, except that the bacteria were incubated with DM-maltose syrup at different time points (1 hour, 6 hours, 24 hours and 96 hours) before evaluating various analytes in the culture medium. The results of these studies are summarized in Table 1.

[0127] Table 1. Summary of evaluation of two Lactobacillus reuteri formulations (Formulation B and Formulation C)

[0128]

[0129] Example 8 - Identification of Lactobacillus reuteri formulations to increase plasma oxytocin levels

[0130] The rat pup model described in Example 6 was used to determine the optimal Lactobacillus reuteri formulation and dosing strategy to increase plasma oxytocin levels. Treatment with various concentrations and regimens of Lactobacillus reuteri formulations was performed to determine the minimum effective dose, which determined significant stimulation of plasma oxytocin within the first 48 hours compared to animals treated with saline. The optimal Lactobacillus reuteri formulation and dose were then evaluated with an activated Lactobacillus reuteri formulation, and then a regimen of lysing Lactobacillus reuteri was used to determine whether similar efficacy was achieved to that seen with formulations containing intact Lactobacillus reuteri. The formulation that provided the minimum effective dose was tested in adult non-pregnant rats to ensure efficacy in mature female rats.

[0131] Male and female immature rat pups at 15 days of age were randomly assigned to receive the following treatments. Five male and five female rat pups per group were randomly assigned to: 1) saline; 2) planktonic Lactobacillus reuteri; or 3) a Lactobacillus reuteri formulation. The Lactobacillus reuteri formulation and planktonic Lactobacillus reuteri were prepared as described in Example 6 and included 2x10 9 CFU Lactobacillus reuteri / 1mL saline. Together with 2x10 9 CFU Lactobacillus reuteri / 1 mL saline, the Lactobacillus reuteri formulations also included 10 mg DM and 14.4 mg maltose, 20 mg DM and 28.8 mg maltose, 100 mg DM and 144 mg maltose, or 200 mg DM and 288 mg maltose. As described in Example 6, activation of the Lactobacillus reuteri formulations was performed for 1 hour and a single dose of 300 μL was given by oral gavage. This study used a step-down approach, first evaluating a 2x10 9CFU / mL Lactobacillus reuteri, 200mg DM and 288mg maltose formulation. After administration, a formulation with lower levels of DM and maltose was used, and the formulation was reduced until the efficacy of stimulating plasma oxytocin levels was not observed statistically. Blood was collected and oxytocin was determined by tail cutting at 24 hours and 48 hours (see Grewen, KM et al. (2010); Psychophysiology 47, 625-632). Multiple comparisons were performed using one-way ANOVA and Tukey's test to compare each group. The minimal effective Lactobacillus reuteri formulation was then evaluated using a scheme for lysing Lactobacillus reuteri with an activated Lactobacillus reuteri formulation. The lysis scheme uses STET buffer, which contains lysozyme to lyse cell membranes and cell walls; sucrose to maintain osmotic pressure; Triton X helps to lyse cell walls, ethylenediaminetetraacetic acid (EDTA) as a chelating agent, and TrisHCl for buffering. The complete Lactobacillus reuteri formulation was compared to the lysed Lactobacillus reuteri formulation, using male and female immature rat pups (5 male rat pups and 5 female rat pups per group) as described in previous studies, and animals receiving saline or saline containing STET buffer as controls. The minimally effective Lactobacillus reuteri formulation (lysed or intact) that provided a statistically significant increase in oxytocin levels was then evaluated in mature female Sprague Dawley rats to confirm that the activity observed in rat pups was translated to adulthood. Eight female adult rats per group were treated with Lactobacillus reuteri formulations, planktonic Lactobacillus reuteri, or saline by oral gavage (500 μl), and oxytocin levels were assessed at 24 hours, 48 ​​hours, and 72 hours after dosing as described in Example 6. The minimal effective dose was a significant stimulation of oxytocin at 1 of the 2 time points (24 hours or 48 hours). Oxytocin stimulation could be sustained for 72 hours using the formulation containing the highest levels of DM and maltose (maximal effective dose).Oxytocin levels were compared to the saline treated group using one-way ANOVA and Tukey's test for multiple comparisons.

[0132] Example 9 - Effect of administration of a Lactobacillus reuteri formulation following administration of azithromycin

[0133] Fig.12 The design of an experimental study evaluating the effects of administering a Lactobacillus reuteri formulation comprising prescription-grade azithromycin ( On the morning of the third day after administration of azithromycin, 100 mg of polyanhydride microparticles (dextran cross-linked with epichlorohydrin) loaded with 400 μmole of the prebiotic maltose and 1×10 9CFU Lactobacillus reuteri (ATCC 23272) [Formulation A]. Briefly, on day 0 of the experimental study, female Sprague-Dawley rats (weight 170 g) fed Teklad 2920X were administered Azithromycin (45 mg / kg, orally administered). On the morning of the third day after azithromycin administration, six female rats were given a single enteral dose of prescription-grade saline ( saline), and 7 female rats were administered a single enteral dose of formulation A ( Formulation A). Fecal pellets were collected from female rats on days 4, 5, and 6 after azithromycin administration, but on days 1, 2, and 3 after saline or Formulation A administration. Blood glucose was measured on days 4, 5, and 6. Female rats were euthanized on days 5 and 6. Tissues (stomach, jejunum, ileum, cecum, proximal colon, and distal colon) were collected on day 6.

[0134] Evaluation of gastric contents

[0135] Fig.13A -D show administration of 20 mg / ml of saline-treated and 20 mg / ml of Formulation A-treated female rats, respectively. Azithromycin or The dry mass of gastric contents, gastric water content, gastric caloric content and pH of gastric contents on the morning of the 3rd day after normal saline administration. Fig. 13B Administration of Formulation A to female rats following administration of azithromycin was shown to increase gastric water content.

[0136] Assessing intestinal inflammation by analyzing stool samples

[0137] Intestinal inflammation can be assessed by analyzing fecal calprotectin and lactotransferrin levels. Fig.14A -B shows fecal calprotectin and fecal lactotransferrin levels in saline-treated and Formulation A-treated female rats, respectively. Fig.14A It was shown that on the second day after administration, the fecal calprotectin levels of female rats treated with Formulation A were reduced compared to female rats treated with saline. Fig. 14B It was shown that on the third day after administration, the fecal lactotransferrin levels of female rats treated with Formulation A were reduced compared to female rats treated with saline. Fig. 14C -D show fecal IL-22 and fecal IL-6 levels of saline-treated and Formulation A-treated female rats, respectively. Fig. 14C It was shown that on the second day after administration, the fecal IL-22 levels of female rats treated with Formulation A were reduced compared to saline-treated female rats. Fig.14D Fecal IL-6 levels were shown to be unchanged by administration of Formulation A. Fig.14A -D displays the data in Collections were made between days 3 and 4, between days 4 and 5, and between days 5 and 6 after azithromycin administration.

[0138] Assessment of pro-inflammatory and anti-inflammatory cytokines in the ileum

[0139] Inflammation in the ileum 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. The contents of the ileum were collected into vials and stored at -20 °C until assayed. Fig.15A -C shows IL-6, IL-10 and IL-22 levels in the ileum of saline-treated female rats and Formulation A-treated female rats on the third day after administration of Formulation A or saline, respectively. Fig.15A Administration of Formulation A was shown to result in decreased IL-6 (a pro-inflammatory cytokine) levels in the ileum of Formulation A treated female rats. Fig. 15B Administration of Formulation A was shown to result in a decrease in IL-10 (anti-inflammatory cytokine) levels in the ileum of Formulation A treated female rats. Fig. 15C Administration of Formulation A was shown to result in increased levels of IL-22 (anti-inflammatory cytokine) in the ileum of Formulation A treated female rats.

[0140] Gut Repair Assessment

[0141] Glucagon-like peptide 2 (GLP-2) promotes tight junctions and stimulates growth of the small and large intestine, as well as transporters for glucose and fatty acid uptake. To assess intestinal repair, GLP-2 levels were measured in the proximal colon. The proximal colon was obtained from euthanized female rats on the third day after administration of Formulation A or saline. Fig.16A Shown are GLP-2 levels in the proximal colon of saline-treated and Formulation A-treated female rats. Fig.16A It was shown that administration of Formulation A resulted in an increase in GLP-2 levels in the proximal colon.

[0142] Leaky Gut Assessment

[0143] Serum peptidoglycan levels were measured in Formulation A-treated and saline-treated female rats. Briefly, adult female rats were administered Saline or Formulation A Euthanasia was performed by decapitation 3 days later and trunk blood was collected into tubes and allowed to clot while placed on ice. Serum was separated by centrifugation and stored at -20°C until assayed for peptidoglycan by ELISA. Fig. 16B Compared with female rats administered Salt water or Serum peptidoglycan levels on day 3 after Formulation A. Fig. 16BAdministration of Formulation A was shown to not alter serum peptidoglycan levels that were elevated due to administration of azithromycin.

[0144] Gut protection assessment

[0145] Fecal secretory immunoglobulin A (sIgA) provides protection against potentially pathogenic microorganisms because it is able to resist enzymatic degradation and survive harsh environments such as the gastrointestinal tract. Patients with low IgA levels are at increased risk for developing mucosal surface infections, food allergies, celiac-like enteropathy, and autoimmune disorders. Fecal sIgA levels were measured in Formulation A-treated and saline-treated female rats to assess intestinal protection. Fig.17 In female rats, Formulation A or 1, 2 and 3 days after saline administration (or Fecal sIgA levels were measured on days 4, 5, and 6 after azithromycin administration to assess intestinal protection. Fig.17 The fecal sIgA levels of female rats were shown to increase on the third day after administration of Formulation A. The increase in fecal sIgA levels in the intestinal lumen of adult female rats after administration of Formulation A provides evidence of intestinal protection.

[0146] Assessment of SCFA production

[0147] The measurement of SCFA (acetate, propionate and butyrate) was performed by gas chromatography-mass spectrometry (GC / MS). In brief, fecal pellets were collected from female rats treated with formulation A and saline between the 3rd and 4th days, the 4th and 5th days, and the 5th and 6th days. The fecal pellets were stored at -20°C until assayed. The samples were ultrasonically treated in 0.5% phosphoric acid (200 mg / ml), centrifuged to remove unextracted solid matter, and filtered through a 0.6 μm filter to remove any large particles. These samples (1 μl) were injected into a GC / MS with a Stabilwax-DA chromatographic column. The samples were calibrated using standard solutions of 6 short-chain fatty acids. Fig.18A -C compares the Various SCFAs ( Fig.18A Fecal acetate as shown in ; Fig.18B Fecal propionate as shown in ; Fig.18C Fecal butyrate concentrations as shown in . Fig.18A and 18C Increased acetate and butyrate levels were shown in fecal pellets excreted between days 2 and 3 after administration of Formulation A, respectively. Fig.18B As shown, there was no statistical difference in propionate levels between Formulation A-treated and saline-treated adult female rats.

[0148] In addition to measuring SCFAs from collected fecal pellets, various SCFA levels were also measured in feces from the distal colon of euthanized female rats on the third day after administration of Formulation A or saline. Briefly, fecal pellets in the distal colon were collected into vials and stored at -20 °C until assayed. Fig.19A -D compares the Normal saline or On the third day after formulation A, Various SCFAs ( Fig.19A Fecal acetate as shown in ; Fig.19B Fecal propionate as shown in ; Fig.19C Fecal butyrate as shown in ; Fig.19D Fecal isovalerate concentrations as shown in Fig.19A and 19D As shown, on the third day after administration of Formulation A, acetate and isovalerate levels increased in female rats. Fig.19B The results showed that propionate levels in female rats decreased on the third day after administration of Formulation A. Fig.19C As shown, there was no statistical difference in butyrate levels between Formulation A-treated and saline-treated adult female rats.

[0149] The data presented in Example 3 demonstrate that a single dose of Formulation A significantly reduced intestinal inflammation after 2-3 days of administration and effectively mitigated the side effects of azithromycin.

[0150] Example 10 - Study evaluating Lactobacillus reuteri formulations

[0151] This example describes a randomized, double-blind, placebo-controlled, single-dose study to evaluate a single dose of a Lactobacillus reuteri formulation (Lactobacillus reuteri and and maltose) in healthy adult volunteers. Such studies monitor the presence of Lactobacillus reuteri and The aim of this study was to evaluate biomarkers of microbiota function, immune regulation, and digestion after a single oral administration of a Lactobacillus reuteri formulation in healthy adults.

[0152] Lactobacillus reuteri Kandler (ATCC 23272) and and maltose at 2x10 10 Colony forming units (CFU), with 200mg and 1 M maltose in a final volume of 10 mL ["Formulation B"] was administered orally. Lactobacillus reuteri Kandler was provided as a lyophilized powder for reconstitution in sterile saline. After reconstitution, Lactobacillus reuteri Kandler was mixed with Mix with slurry of maltose.

[0153] 30 eligible subjects were randomly assigned in a 1:1 ratio to receive treatment with Formulation B or placebo (saline). Subjects were screened within 14 days prior to the administration of Formulation B or placebo. All subjects were required to provide written informed consent before any study-specific procedures were performed. The study eligibility of the subject was determined by evaluating the inclusion and exclusion criteria at the time of screening. Subjects were asked to provide consent to allow biobanking of biological samples for future analysis. Eligible subjects were provided with a stool sampling kit to collect pre-treatment stool samples within 48 hours prior to receiving Formulation B or placebo (collection time was determined by the frequency of the subject's bowel movements).

[0154] Subjects returned to the study site on Day 1 after an 8-hour overnight fast. After confirmation of eligibility criteria, subjects received a single oral dose of Formulation B or placebo, followed by 100 mL of water, depending on the treatment group to which they were assigned. Fasting was maintained until 1 hour after administration of Formulation B or placebo. Subjects underwent 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) were measured before and 1 hour after administration of Formulation B or placebo. Baseline stool samples were collected within a 48-hour window before the first dose to determine the presence of Lactobacillus reuteri and Presence of microspheres and exploratory biomarker analysis. Subjects left after completing all Day 1 assessments. Subjects were instructed to collect 4 stool samples at home according to the following schedule: Subjects were provided with a sampling kit to collect stool samples at home. The kit included labeled sample containers with the time and date the samples were obtained. Subjects were instructed to return all stool samples using pre-labeled shipping containers; the investigator and subject could also negotiate an alternative strategy for returning stool samples.

[0155] Follow-up visits were conducted on Days 8 and 15. Subjects attended an end-of-study visit on Day 30.

[0156] Eligible subjects received a single dose of Formulation B or placebo according to the treatment group to which they were assigned. Formulation B or placebo was administered on Day 1 at the study site under fasting conditions. After administration, subjects were provided with 100 mL of water to drink. Both Formulation B and placebo were provided to subjects in opaque plastic syringes so that the contents were not visible to keep the study blinded. Subjects were required to fast for at least 8 hours prior to administration of Formulation B or placebo. Subjects had to avoid the use of other probiotics and any antibiotics during their participation in the study. Safety endpoints included treatment-emergent AEs (TEAEs), serious AEs (SAEs), the incidence and severity of AESIs, and AEs leading to study discontinuation; laboratory results (hematology [complete blood count, differential, and erythrocyte sedimentation rate], biochemistry, and urinalysis), vital sign results, and physical examination results; the presence of Lactobacillus reuteri Kandler in stool, The incidence of microspheres and leukocytes; Bristol stool chart score; and the incidence of symptomatic bacteremia with positive identification of Lactobacillus reuteri. Other endpoints of the study included: biomarkers of microbiota function: short-chain fatty acids (acetate, butyrate, propionate), lactate, and pH; biomarkers of immunomodulation: pro- and anti-inflammatory cytokines (interferon-γ, interleukin [IL]-2, IL-4, IL-5, IL-6, IL-10, IL-22, chemokine ligand 1, and tumor necrosis factor-α), as well as calprotectin, lactoferrin, and secretory immunoglobulin A; and biomarkers of digestion: fecal content of glucose, triglycerides, total bile acid, protein, and lactose.

[0157] Demographic and baseline characteristics (including age, sex, race, ethnicity, weight, height, and body mass index) were summarized using descriptive statistics, and the presence of Lactobacillus reuteri, Microsphere and leukocyte levels were assessed qualitatively, and the results were summarized using descriptive statistics.

[0158] The total study duration includes 1 month of active enrollment and 1 month of follow-up. 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 duration for each subject is approximately 6 weeks. The maximum treatment duration for each subject is 1 day.

[0159] Example 11 - Effect of administration of Lactobacillus reuteri formulation on autism

[0160] This example details the study design for evaluating the effect of administering a Lactobacillus reuteri formulation in the treatment of autism. Compared with premature infants who are born at risk for developing NEC with immature and underdeveloped gastrointestinal (GI) systems, the target population has a better developed 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, 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). In preclinical studies using Lactobacillus reuteri formulations, neither GI pathology after histological examination nor changes in stool consistency and stool output were observed. In addition, reduced levels of fecal calprotectin, a clinical biomarker of GI inflammation, have been observed, suggesting that Lactobacillus reuteri formulations may reduce clinical or subclinical intestinal inflammation and may improve GI symptoms commonly associated with autism.

[0161] After preliminary screening, subjects were randomly assigned to one of 2 groups, 8 subjects in each group. Group A received Lactobacillus reuteri formulation for 28 days, while Group B received placebo (saline). After initial administration, there was a 14-day washout period, during which subjects neither received Lactobacillus reuteri formulation nor placebo. After the washout period, Group A received placebo (saline) for 28 days, while Group B will receive Lactobacillus reuteri formulation. The primary outcome was to assess the safety and tolerability of the Lactobacillus reuteri formulation administered daily over a 28-day period, assessed by the frequency and severity of adverse events and laboratory abnormalities. Secondary outcomes included assessing the effects of Lactobacillus reuteri formulation on circulating oxytocin levels, systemic and fecal levels of host and microbial derived metabolites, blood and fecal biomarkers of intestinal inflammation, and behavioral tools validated in the context of autism. The total study duration was approximately 70 days.

[0162] Subjects were eligible for study participation if they had a confirmed diagnosis of autism as confirmed using a gold standard clinical interview for DSM-5 criteria and administration of the Autism Diagnostic Observation Schedule 2. Lactobacillus reuteri Kandler was administered at 2 × 10 10 colony forming units, and 185mg The subjects received a single dose daily for 28 days. Lactobacillus reuteri Kandler was provided as a lyophilized powder for reconstitution in saline (provided). After reconstitution, Lactobacillus reuteri Kandler was mixed with and maltose slurry. The reference product / placebo was saline. Subjects received a single oral dose of 10 mL of matching saline for 28 days.

[0163] Eligible subjects received a daily dose of Lactobacillus reuteri or placebo for 28 days, depending on the treatment group assigned. Subjects were provided with 28 doses, prepared at home and mixed with a preferred beverage or soft food (e.g., applesauce, yogurt, pudding). The first dose was administered IP in the clinic. Subjects had to refrain from using probiotics during their participation in the study.

[0164] Analysis of clinician- and caregiver-reported outcome measures consisted of two paired t-tests between L. reuteri Kandler and placebo. The measures for each subject were changes in continuous measures, including CGI-S, CGI-I, ABC, CFQL-2, WJ3 spatial relations and auditory attention subtests, KiTap, RBANS, eye tracking, and psychophysical measures. For each subject, the difference between the change in continuous outcome measure scores and the change in placebo was derived. A single-sample (paired) t-test was performed on these within-subject differences. Results were considered statistically significant if the two-sided p-value was less than 0.05 and multiplicity was corrected using the false discovery rate (FDR) method. In addition, a linear mixed model of a two-treatment crossover design was analyzed. The response of the model was the continuous outcome measure of interest at the end of the period. The fixed effect covariates were treatment, period, sex, and carry-over.

[0165] The order and maximum duration of the study periods were as follows: screening: 14 days; treatment period 1: 28 days; washout: 14 days; treatment period 2: 28 days; follow-up: 30 days; the maximum study duration for each subject was approximately 17 weeks. Eye tracking measurements were performed at the screening or baseline visit for participants with ASD. ADOS-2 = Autism Diagnostic Observation Schedule Module 3 or 4; WASI-II = Wechsler Brief Intelligence Scale-Second Edition, SCQ = Social Communication Questionnaire; CGI-S = Clinical Global Impression Severity Scale, CGI-I = Clinical Global Impression Improvement Scale; Vineland-3 = Vineland Adaptive Behavior Inventory, 3rd Edition; PK = pharmacokinetics; EEG = electroencephalogram protocol; WJ3 = Woodcock Johnson Spatial Relations and Auditory Attention Subtest; RBANS = Repeated Battery of Neuropsychological Status; KiTap = Computerized Test of Attentional Performance in Children; ABC = Aberrant Behavior Checklist; CFQL-2 = Child and Family Quality of Life, 2nd Edition.

[0166] Equivalent

[0167] Although specific embodiments of the present disclosure have been discussed, the above description is illustrative rather than restrictive. After reading this description, many changes of the present disclosure will become apparent to those skilled in the art. The full scope of the present disclosure should be determined by reference to the full scope of the claims and their equivalents and the description and such changes.

[0168] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure.

[0169] Incorporated by Reference

[0170] All patents, published patent applications, websites, and other references cited herein are expressly incorporated by reference in their entirety.

Claims

1. A method for treating or ameliorating autism spectrum disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and optionally a prebiotic.

2. The method of claim 1, wherein the autism spectrum disorder is selected from autism, Asperger's syndrome, Heller syndrome, Rett syndrome, and pervasive developmental disorder not otherwise specified (PDD-NOS).

3. The method of any one of claims 1-2, wherein the composition comprises dextran microparticles; Lactobacillus reuteri, ATCC 23272; and a prebiotic.

4. The method of any one of claims 1-3, wherein the subject has upregulation of oxytocin levels after administering at least one daily dose of the composition to the subject.

5. The method of any one of claims 1-4, wherein the subject is a pediatric patient.

6. The method of any one of claims 1-5, wherein the subject also suffers from a gastrointestinal disorder.

7. A method of treating a gastrointestinal disorder in a patient, comprising administering a composition comprising dextranomer microparticles; Lactobacillus reuteri, ATCC 23272; and a prebiotic.

8. The method of claim 7, wherein the patient is on the autism spectrum or suffers from autism.

9. The method of claim 7 or 8, wherein the gastrointestinal disorder is associated with antibiotic administration.

10. A method of treating an autistic patient suffering from a gastrointestinal disorder, comprising administering a composition comprising: polyanaplastic anhydride microparticles; Lactobacillus reuteri, ATCC 23272; and a prebiotic.

11. The method of any one of claims 7-10, wherein the gastrointestinal disorder is one or more of constipation, abdominal pain, flatulence, and diarrhea.

12. A method of treating a developmental disorder associated with preterm 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.

13. A method of inducing oxytocin in a premature infant in need thereof, the method comprising administering to the premature infant a therapeutically effective amount of a composition comprising Lactobacillus reuteri and a pharmaceutically acceptable carrier.

14. The method of claim 12 or 13, wherein the composition further comprises biocompatible microspheres and / or prebiotics.

15. The method of any one of claims 12-14, wherein the composition comprises dextran microparticles, Lactobacillus reuteri, ATCC 23272, and a prebiotic.

16. The method of any one of claims 12-14, wherein upon administration of at least one daily dose of the composition to the premature infant, the premature infant has upregulation of oxytocin levels.

17. A method of treating gastrointestinal inflammation caused by administration of an antibiotic regimen in a patient, comprising administering to the patient a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or a prebiotic.

18. A method of treating a patient suffering from or expected to suffer from gastrointestinal inflammation associated with 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 a prebiotic.

19. The method of claim 17 or 18, wherein the composition is administered before administration of the antibiotic regimen, during administration of the antibiotic regimen, and / or after administration of the antibiotic regimen.

20. A method of substantially preventing or reducing adverse effects associated with oral antibiotics in a subject in need thereof, comprising administering a therapeutically effective amount of a composition comprising Lactobacillus reuteri and biocompatible microspheres, wherein the subject is undergoing treatment with the oral antibiotic.

21. A method of treating depression or anxiety in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising Lactobacillus reuteri, biocompatible microspheres and / or a prebiotic.

22. The method of claim 21, wherein the depression is selected from the group consisting of clinical depression, postnatal or postpartum depression, obsessive-compulsive disorder, post-traumatic stress disorder, bipolar disorder, atypical depression, melancholic depression, psychotic major depression (PMD), catatonic depression, seasonal affective disorder (SAD), dysthymia, double depression, depressive personality disorder (DPD), recurrent brief depressive disorder (RBD), minor depression, bipolar disorder or manic depression, treatment-resistant depression, refractory depression, suicidal tendencies, suicidal ideation, and suicidal behavior.

23. The method of claim 21, wherein the patient suffers from postnatal or postpartum depression.

24. The method of any one of claims 1-23, wherein the composition is administered once.

25. The method of any one of claims 1-23, wherein the composition is administered daily.

26. The method of claims 1-25, wherein the prebiotic comprises a water-soluble carbohydrate, wherein the water-soluble carbohydrate comprises one or more of: inulin, fructooligosaccharides, fructooligosaccharides, galacto-oligosaccharides, glucose, starch, maltose, maltodextrin, polydextrose, amylose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, carbonate, thiamine, choline, histidine, trehalose, nitrogen, sodium nitrate, ammonium nitrate, phosphorus, phosphate, hydroxyapatite, potassium, potash, sulfur, homopolysaccharides, heteropolysaccharides, cellulose, chitin, vitamins, or a combination thereof.

27. The method of any one of claims 1-26, wherein the prebiotic is maltose.

Images