WO2026030281A1 - Compositions et méthodes pour le traitement des crises d'épilepsie et des troubles associés - Google Patents

Compositions et méthodes pour le traitement des crises d'épilepsie et des troubles associés

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Publication number
WO2026030281A1
WO2026030281A1 PCT/US2025/039607 US2025039607W WO2026030281A1 WO 2026030281 A1 WO2026030281 A1 WO 2026030281A1 US 2025039607 W US2025039607 W US 2025039607W WO 2026030281 A1 WO2026030281 A1 WO 2026030281A1
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WIPO (PCT)
Prior art keywords
bacteria
fiber
subject
bifidobacterium
genera
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PCT/US2025/039607
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English (en)
Inventor
Elaine Y. HSIAO
Ezgi OZCAN
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Publication of WO2026030281A1 publication Critical patent/WO2026030281A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani

Definitions

  • Epilepsy is characterized by recurrent seizures that can lead to loss of awareness, loss of consciousness, and/or disturbances of movement, autonomic function, sensation (including vision, hearing and taste), mood, and/or mental function. Epilepsy afflicts 1-2% of the population in the developed world.
  • the low-carbohydrate, high-fat ketogenic diet is a treatment for refractory epilepsy, wherein more than one-third of epileptic individuals do not respond to existing anticonvulsant medications.
  • the efficacy of the KD is supported by multiple retrospective and prospective studies, which estimate that -30% of patients become seizure-free, and -60% experience significant benefit.
  • use of the KD remains low due to difficulties with implementation, dietary compliance and adverse side effects.
  • epileptic patient retention on the KD is only an estimated 12% by the third year of dietary therapy.
  • the gut microbiome is emerging as an important modulator of the anti-seizure effects of the classic ketogenic diet.
  • many variations of the ketogenic diet are used clinically to treat refractory epilepsy, and how different dietary formulations differentially modify the gut microbiome in ways that impact seizure outcome is poorly understood.
  • Clinically prescribed ketogenic infant formulas vary in macronutrient ratio, fat source, and fiber content and also in their ability to promote resistance to 6-Hz psychomotor seizures in mice. By screening specific dietary variables for their effects on a model human infant microbial community, the instant disclosure demonstrates that particular bacteria in the gut microbiome drives substantial metagenomic shifts.
  • Distinct subsets of metagenomic responses in the model human infant microbial community have been identified that correspond with increased seizure resistance in mice.
  • Supplementation with seizure- protective fibers and bacteria enriches microbial representation of genes related to L-alanine biosynthesis, queuosine biosynthesis, and preQo biosynthesis, which is also seen in seizure- protected mice that are fed fiber-containing ketogenic infant formulas.
  • the present disclosure shows that different formulations of clinical ketogenic diets, and dietary fiber content in particular, differentially impact seizure outcome in mice through modification of the gut microbiome. Leveraging interactions between dietary components of the ketogenic diet, the gut microbiome, and host susceptibility to seizures informs novel microbiome-guided approaches to treat refractory epilepsy.
  • compositions for preventing or treating a condition responsive to a ketogenic diet in a subject comprising administering to the subject a composition comprising bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and/or Clostridium genera.
  • the composition comprises Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088).
  • the composition comprises Bifidobacterium longum (e.g., Bifidobacterium longum sups, longum ATCC BAA-999). In some embodiments, the composition comprises Bifidobacterium breve (e.g., Bifidobacterium breve DSM 20213). In some embodiments, the composition comprises Bacteroides fragilis (e.g., Bacteroides fragilis ATCC 25285). In some embodiments, the composition comprises Clostridium perfringens (e.g., Clostridium perfringens ATCC 13124). Also provided herein are combinations of the agents/bacteria disclosed herein for use in the methods described herein.
  • the method comprises a combination method comprising administering a bacteria of the Deferribacteres phylum and a bacteria of the Coriobacteriia class. In some embodiments, the method further comprising administering one or more types of dietary fiber, such as FOS, inulin, gum arabic and/or cellulose. In some embodiments, the method further comprising administering a mixture of dietary fiber, such as a mixture comprising FOS, inulin, gum arabic and cellulose.
  • compositions for preventing or treating a condition responsive to a ketogenic diet in a subject comprising administering to the subject an agent that decreases levels of one or more bacteria in the subject’s gut, wherein the one or more bacteria comprise bacteria of the Actinobacteria phylum, Erysipelotrichia class, Escherichia genera, Enterococcus genera, Mammaliicoccus genera, Bifidobacterium species, Bacteroides species, and/ or Klebsiella species.
  • the agent decreases levels of Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the agent decreases levels of Bacteroides vulgatus (e.g., Bacteroides vulgatus ATCC8482). In some embodiments, the agent decreases levels of Klebsiella pneumoniae (e.g., Klebsiella pneumoniae subsp. pneumoniae ATCC 13883).
  • the method comprises a combination method comprising administering lactose or a suppressing agent. In some embodiments, the method further comprising administering one or more types of lactose. In some embodiments, the method further comprising administering a mixture of lactose.
  • compositions for preventing or treating a condition responsive to a ketogenic diet in a subject comprising administering to the subject a composition comprising L-alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and sucrose and/or lactose.
  • the condition is seizures, optionally wherein subject has a neurodevelopmental condition, e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome.
  • a neurodevelopmental condition e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome.
  • the subject has a condition selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines, and traumatic brain injury (TBI).
  • a neurodevelopmental condition e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome.
  • the subject has a condition selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines,
  • the method further comprises administering to the subject a fiber supplement (e.g., inulin, fructooligosaccharides, acacia fiber, gum arabic, cellulose, and/or orange fiber).
  • a fiber supplement e.g., inulin, fructooligosaccharides, acacia fiber, gum arabic, cellulose, and/or orange fiber.
  • the method further comprises administering to a subject a composition comprising L-alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and sucrose and/or lactose.
  • compositions may be self-administered.
  • the composition may be a food product, such as yogurt or fiber supplement.
  • the composition is a synbiotic product, such as one that combines bacteria (e.g., probiotic, such as bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and/or Clostridium genera) and a fiber mixture that supports the growth of bacteria or bacterial mixture.
  • the composition is infant formula.
  • the food product is a fiber supplement or mixture containing one or more of the dietary fiber types disclosed herein.
  • the subject is on a diet (e.g., a ketogenic diet) that includes at least one dietary fiber (e.g., a dietary fiber disclosed herein) BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1A-1C shows the different formulations of medical ketogenic diets (KD) and the resulting differential responses to 6-Hz seizures in mice who receive the diets.
  • FIG. 1A shows macronutrient composition without fiber (for determining KD fat ratio), macronutrient composition with fiber, absence/presence of fat sources, and percent carbohydrate composition for the commercial KD infant formulas KD4: 1, KD3: 1, and MCT2.5: 1, relative to standard infant formula as control diet (CD).
  • FIG. 1A shows macronutrient composition without fiber (for determining KD fat ratio), macronutrient composition with fiber, absence/presence of fat sources, and percent carbohydrate composition for the commercial KD infant formulas KD4: 1, KD3: 1, and MCT2.5: 1, relative to standard infant formula as control diet (CD).
  • the line at y 10 s represents threshold for scoring seizures.
  • FIG. 2A-2E shows that medical KDs induce differential alterations in the gut microbiome that associate with resistance vs. susceptibility to 6-Hz seizures.
  • FIG. 2B shows principal coordinates analysis (PCoA) of Bray-Curtis dissimilarity (left) and weighted UniFrac distance (right) based on fecal metagenomic sequencing data after dietary treatment.
  • PCoA principal coordinates analysis
  • FIG. 3A-3E shows the addition of dietary fiber to KDs enriches metagenomic features associated with seizure protection in a model human infant gut community and restores resistance to 6-Hz seizures in mice.
  • FIG. 3A shows experimental design: Fiber mix containing inulin, gum arabic, cellulose, and fructooligosaccharide (FOS), or lactose as a non-fiber carbohydrate control, was added to KD-based synthetic culture media for anaerobic culture of a model human infant gut microbial community.
  • the line at y 10 s represents threshold for scoring seizures.
  • FIG. 4-4E shows the addition of excess dietary fiber to fiber-containing KD4: 1 further potentiates seizure resistance.
  • the line at y 10 s represents threshold for scoring seizures.
  • FIG. 4C shows experimental design: 13 dietary fiber sources and types were supplemented to KD4: 1 infant formula for anaerobic culture of a model human infant gut microbial community.
  • FIG. 4C shows experimental design: 13 dietary fiber sources and types were supplemented to KD4: 1 infant formula for anaerobic culture of a model human infant gut microbial community.
  • FIG. 4D shows a heatmap of 15 fiber-induced differential metagenomic pathways (q ⁇ 0.05) that were similarly seen in seizure-protected mice fed KD4: 1 or MCT2.5: 1 (
  • the line at y 10 s represents threshold for scoring seizures.
  • FIG. 5A-5D shows medical KDs administered as solid diets phenocopy differential seizure responses seen with liquid diets.
  • FIG. 6A-6E shows the effects of KDs on taxonomic and metagenomic signatures of the fecal microbiome in mice.
  • FIG. 7A-7I shows the effects of fat ratio, fat source/type, and carbohydrate source for KD-based synthetic culture media on metagenomic profiles of a model human infant microbial community.
  • FIG. 7A shows bacterial species comprising the model human infant microbial community, as compared to published data from human infants.
  • FIG. 7C shows the experimental design: KD-based synthetic culture media was formulated with differing fat ratios for anaerobic culture of a model human infant gut microbial community.
  • FIG. 7A-7I shows the effects of fat ratio, fat source/type, and carbohydrate source for KD-based synthetic culture media on metagenomic profiles of a model human infant microbial community.
  • FIG. 7A shows bacterial species comprising the model human infant microbial community, as compared to published data from human infants.
  • FIG. 7B shows change in bacterial species abundance
  • FIG. 7E shows the experimental design: KD-based synthetic culture media was formulated with differing fat sources that vary in level of saturation for anaerobic culture of a model human infant gut microbial community.
  • FIG. 7G shows the experimental design: KD-based synthetic culture media was formulated with differing fat types for anaerobic culture of a model human infant gut microbial community.
  • FIG. 9A-9B shows the addition of fiber to KD3 : 1 as a solid diet phenocopies increases in seizure resistance seen with liquid diet.
  • FIG. 10A-10C shows the addition of excess fiber to KD4: 1 as a solid diet phenocopies increases in seizure resistance seen with liquid diet.
  • the line at y 10 s represents threshold for scoring seizures.
  • FIG. 11A-11B shows that SCFA supplementation does not phenocopy effects of fiber supplementation on KD-induced response to 6-Hz seizures.
  • the line at y 10 s represents threshold for scoring seizures.
  • the line at y 10 s represents threshold for scoring seizures.
  • compositions for preventing or treating a condition responsive to a ketogenic diet in a subject comprising administering to the subject a composition comprising bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and/or Clostridium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Proteobacteria phylum. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Deferribacteres phylum. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Streptococcaceae family.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Coriobacteriia class. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Bacteroidetes phylum and a bacteria of the Clostridium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Deferribacteres phylum.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Streptococcaceae family.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Coriobacteriia class.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Proteobacteria phylum and a bacteria of the Clostridium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Streptococcaceae family.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Coriobacteriia class.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Bifidobacterium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Deferribacteres phylum and a bacteria of the Clostridium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Streptococcaceae family and a bacteria of the Coriobacteriia class.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Streptococcaceae phylum and a bacteria of the Bifidobacterium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Streptococcaceae phylum and a bacteria of the Bifidobacterium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Streptococcaceae phylum and a bacteria of the Clostridium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Coriobacteriia class and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Coriobacteriia class and a bacteria of the Bifidobacterium genera. In some embodiments, the methods described herein may include administering a combination of bacteria, such a bacteria of the Coriobacteriia class and a bacteria of the Clostridium genera.
  • the methods described herein may include administering a combination of bacteria, such a bacteria of the Bifidobacterium genera and a bacteria of the Clostridium genera.
  • the composition comprises Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the composition comprises Bifidobacterium longum (e.g., Bifidobacterium longum sups, longum ATCC BAA- 999). In some embodiments, the composition comprises Bifidobacterium breve e.g., Bifidobacterium breve DSM 20213). In some embodiments, the composition comprises Bacteroides fragilis (e.g., Bacteroides fragilis ATCC 25285). In some embodiments, the composition comprises Clostridium perfringens (e.g., Clostridium perfringens ATCC 13124).
  • Bifidobacterium infantis e.g., Bifidobacterium longum sups, infantis DSM 20088
  • the composition comprises Bifidobacterium longum (e.g., Bif
  • compositions for preventing or treating a condition responsive to a ketogenic diet in a subject comprising administering to the subject an agent that decreases levels of one or more bacteria in the subject’s gut, wherein the one or more bacteria comprise bacteria of the Actinobacteria phylum, Erysipelotrichia class, Escherichia genera, Enterococcus genera, Mammaliicoccus genera, Bifidobacterium species, Bacteroides species, and/ or Klebsiella species.
  • the agent is a small molecule that specifically decreases the level of bacteria of the Actinobacteria phylum. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Erysipelotrichia class. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Escherichia genera. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Enterococcus genera. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Mammaliicoccus genera.
  • the agent is a small molecule that specifically decreases the level of bacteria of the Bifidobacterium breve species. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Bifidobacterium infantis. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the Bacteroides vulgatus species. In some embodiments, the agent is a small molecule that specifically decreases the level of bacteria of the and/ or Klebsiella pneumoniae.
  • the agent is an antibiotic specific for a particular bacterial phylum. In some embodiments, the agent is an antibiotic that specifically reduces levels of bacteria of the Actinobacteria phylum, Erysipelotrichia class, Escherichia genera, Enterococcus genera, Mammaliicoccus genera, Bifidobacterium species, Bacteroides vulgatus species, or Klebsiella species.
  • the agent decreases levels of Escherichia coli (e.g., Escherichia coli K-12 ATCC 10798). In some embodiments, the agent decreases levels of Enterococcus faecalis (e.g., Enterococcus faecalis ATCC 19433). In some embodiments, the agent decreases levels of Mammaliicoccus sciuri. In some embodiments, the agent decreases levels of Bifidobacterium breve e.g., Bifidobacterium breve DSM 20213).
  • the agent decreases levels of Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the agent decreases levels of Bacteroides vulgatus (e.g., Bacteroides vulgatus ATCC8482). In some embodiments, the agent decreases levels of Klebsiella pneumoniae (e.g., Klebsiella pneumoniae subsp. pneumoniae ATCC 13883).
  • the methods further comprise administering a bacteria or a molecule that causes alterations in the pathways related to L-alanine biosysnthesis, preQO biosynthesis, sucrose degradation and partial TCA cycle in the gut microbiome, such as L- alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2- oxoglutarate), citrate, succinate and sucrose and/or lactose.
  • a bacteria or a molecule that causes alterations in the pathways related to L-alanine biosysnthesis, preQO biosynthesis, sucrose degradation and partial TCA cycle in the gut microbiome such as L- alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2- oxoglutarate), citrate, succinate and sucrose and/or lactose.
  • compositions for preventing or treating a condition responsive to a ketogenic diet in a subject comprising administering to the subject a composition comprising L-alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and sucrose and/or lactose.
  • the condition is seizures, optionally wherein subject has a neurodevelopmental condition, e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome.
  • a neurodevelopmental condition e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome.
  • the subject has a condition selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines, and traumatic brain injury (TBI).
  • a neurodevelopmental condition e.g., selected from epilepsy, autism spectrum disorder, Rett syndrome, attention deficit disorder, and fragile X syndrome.
  • the subject has a condition selected from Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines,
  • the method further comprises administering to the subject a fiber supplement (e.g., inulin, fructooligosaccharides, acacia fiber, gum arabic, cellulose, and/or orange fiber).
  • a fiber supplement e.g., inulin, fructooligosaccharides, acacia fiber, gum arabic, cellulose, and/or orange fiber.
  • the method further comprises administering to a subject a composition comprising L-alanine, queuosine, preQo, guanosine, guanosine triphosphate, alpha-ketoglutarate (2-oxoglutarate), citrate, succinate and/or sucrose and/or lactose.
  • compositions may be self-administered.
  • the composition may be a food product.
  • the composition is infant formula.
  • the subject may be a pediatric subject (e.g., the subject is under 18 years of age).
  • the subject may be an adult (e.g., 18 years old or older).
  • the subject is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 years of age.
  • the condition disclosed herein may be a condition in children.
  • the condition may be a pediatric condition.
  • the child may be less than about 1 week old. The child may be less than about 1 month old. The child may be less than about 6 months old. The child may be less than about 12 months old. The child may be less than about 2 years old. The child may be less than about 3 years old. The child may be less than about 4 years old. The child may be less than about 5 years old. The child may be less than about 6 years old. The child may be less than about 7 years old. The child may be less than about 8 years old. The child may be less than about 9 years old. The child may be less than about 10 years old. The child may be less than about 12 years old.
  • a or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more.
  • agent is used herein to include, but is not limited to, a chemical compound, a small molecule, and/or a mixture of chemical compounds. “Agent” includes, but is not limited to, microbially produced metabolites that are product of microbial activity from carbohydrates, proteins and fats. The activity of such agents may render them suitable as a “therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
  • ketogenic diet is used herein to include, but is not limited to, a high-fat diet, and/or a low-carbohydrate diet.
  • the ketogenic diet may comprise additional fiber supplements (e.g., inulin, fructooligosaccharides, acacia fiber, gum arabic, cellulose, and/or orange fiber).
  • the ketogenic diet may comprise the components of Table 1.
  • the diet may consist of more than 60-75% fats.
  • the diet may consist of less than 5-10% carbohydrates.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydro
  • prevention of seizures includes, for example, reducing the number of seizures in a population of patients receiving a prophylactic treatment relative to an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • prophylactic or therapeutic treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
  • the unwanted condition e.g., disease or other unwanted state of the host animal
  • small molecule is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.
  • subject refers to a mammal, including, but not limited to, a human or nonhuman mammal, such as a bovine, equine, canine, ovine, or feline.
  • a “therapeutically effective amount” of a compound with respect to the subject method of treatment refers to an amount of the compound(s) in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment.
  • treating includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject's condition.
  • a ketogenic diet (KD) with fiber induces substantial changes in the gut microbiome, and enriching KD-associated bacteria via probiotic administration, fecal transplant, or selective microbial reconstitution of the native microbiome mimics the beneficial effects of such a diet.
  • KD ketogenic diet
  • the methods and compositions described herein can be used separately or in conjunction with the KD diet or another diet disclosed herein in the treatment or prevention of a condition described herein. Therefore, in some embodiments, the subject is on a ketogenic diet that includes at least one dietary fiber (e.g., a dietary fiber disclosed herein).
  • the subject is given antibiotics or antimicrobial agents to deplete the subject’s gut microbiota.
  • the methods treat or prevent seizures in a subject.
  • methods for preventing or treating a condition responsive to a ketogenic diet in a subject comprising administering to the subject a composition comprising particular bacterial species.
  • methods for increasing the expression of genes or products related to L-alanine biosynthesis, queuosine biosynthesis, and/or preQo biosynthesis comprising administering to the subject a composition comprising particular bacterial species.
  • the subject has epilepsy (e.g., pediatric epilepsy; refractory or non-refractory epilepsy).
  • the disclosed epilepsy disorder may be benign Rolandic epilepsy, frontal lobe epilepsy, infantile spasms, juvenile myoclonic epilepsy (JME), juvenile absence epilepsy, childhood absence epilepsy (e.g.
  • pyknolepsy febrile seizures, progressive myoclonus epilepsy of Lafora, Lennox-Gastaut syndrome, Landau -KI effner syndrome, Dravet syndrome, Generalized Epilepsy with Febrile Seizures (GEFS+), Severe Myoclonic Epilepsy of Infancy (SMEI), Benign Neonatal Familial Convulsions (BFNC), West Syndrome, Ohtahara Syndrome, early myoclonic encephalopathies, migrating partial epilepsy, infantile epileptic encephalopathies, Tuberous Sclerosis Complex (TSC), focal cortical dysplasia, Type I Lissencephaly, Miller-Dieker Syndrome, Angelman's syndrome, Fragile X syndrome, epilepsy in autism spectrum disorders, subcortical band heterotopia, Walker-Warburg syndrome, Alzheimer's disease, posttraumatic epilepsy, progressive myoclonus epilepsies, reflex epilepsy, Rasmussen's syndrome, temporal lobe epilepsy, limbi
  • Epilepsy disorder may be Dravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, or Ohtahara Syndrome.
  • the epilepsy disorder may be Dravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, or Ohtahara Syndrome, or a pediatric epilepsy disorder.
  • the pediatric epilepsy disorder may be benign childhood epilepsy, Benign Neonatal Familial Convulsions (BFNC), febrile seizures, Dravet Syndrome, Lennox-Gastaut Syndrome, infantile spasm, Ohtahara Syndrome, juvenile myoclonic epilepsy, juvenile absence epilepsy, childhood absence epilepsy (e.g. pyknolepsy), infantile spasms.
  • the epilepsy disorder is Dravet Syndrome.
  • the epilepsy disorder is Dravet syndrome or Lennox-Gastaut syndrome.
  • the disclosed pediatric epilepsy disorder may be benign childhood epilepsy.
  • the disclosed pediatric epilepsy disorder may be Benign Neonatal Familial Convulsions (BFNC).
  • the disclosed pediatric epilepsy disorder may be febrile seizures.
  • the pediatric epilepsy disorder is Dravet Syndrome.
  • the pediatric epilepsy disorder is Lennox-Gastaut Syndrome.
  • the disclosed pediatric epilepsy disorder may be infantile spasm.
  • the disclosed pediatric epilepsy disorder may be Ohtahara Syndrome.
  • the disclosed pediatric epilepsy disorder may be juvenile myoclonic epilepsy.
  • the disclosed pediatric epilepsy disorder may be juvenile absence epilepsy.
  • the disclosed pediatric epilepsy disorder may be childhood absence epilepsy (e.g. pyknolepsy).
  • the disclosed pediatric epilepsy disorder may be infantile spasms.
  • the epilepsy disorder may be a result of a neurological disease or injury such as, for example, encephalitis, cerebritis, abscess, stroke, tumor, trauma, genetic, tuberous sclerosis, cerebral dysgenesis, or hypoxic-ischemic encephalophathy.
  • the epilepsy disorder may be associated with a neurodegenerative disease such as, for example, Alzheimer's disease or Parkinson's Disease.
  • the epilepsy disorder may be associated with autism.
  • the epilepsy disorder may be associated with a single gene mutation.
  • the epilepsy disease may be associated with compulsive behaviors or electrographic seizures.
  • the epilepsy disorder may be an epilepsy disorder which is non-responsive to treatment with an antiepileptic drug (AED).
  • AED antiepileptic drug
  • the AED may be acetazolamide.
  • the AED may be benzodiazepine.
  • the AED may be cannabadiols.
  • the AED may be carbamazepine.
  • the AED may be clobazam.
  • the AED may be clonazepam.
  • the AED may be eslicarbazepine acetate.
  • the AED may be ethosuximide.
  • the AED may be ethotoin.
  • the AED may be felbamate.
  • the AED may be fenfluramine.
  • the AED may be fosphenytoin.
  • the AED may be gabapentin.
  • the AED may be ganaxolone.
  • the AED may be huperzine A.
  • the AED may be lacosamide.
  • the AED may be lamotrigine.
  • the AED may be levetiracetam.
  • the AED may be nitrazepam.
  • the AED may be oxcarbazepine.
  • the AED may be perampanel.
  • the AED may be piracetam.
  • the AED may be phenobarbital.
  • the AED may be phenytoin.
  • the AED may be potassium bromide.
  • the AED may be pregabalin.
  • the AED may be primidone.
  • the AED may be retigabine.
  • the AED may be rufinamide.
  • the AED may be valproic acid.
  • the AED may be sodium valproate.
  • the AED may be stiripentol.
  • the AED may be tiagabine.
  • the AED may be topiramate.
  • the AED may be vigabatrin.
  • the subject has a neurodevelopmental disorder.
  • Representative neurodevelopmental disorders include autism spectrum disorder, Rett syndrome, fragile X, attention deficit disorder, and attention-deficit/hyperactivity disorder.
  • the neurodevelopmental disorder is a disorder known to be comorbid with seizures.
  • a condition “responsive to a ketogenic diet” includes, but is not limited to, epilepsy, autism spectrum disorder, Rett syndrome, fragile X, attention deficit disorder, attention- deficit/hyperactivity disorder, seizures, autism spectrum disorder, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease (e.g., diabetes or obesity), a mitochondrial disorder, depression, migraines (e.g., chronic migraines), or traumatic brain injury (TBI).
  • the subject is refractory to anti-conversant drug or anti-seizure drug.
  • anti-seizure drug used interchangeably herein and according to their common and ordinary meaning and include compositions for reducing or eliminating seizures.
  • Anticonvulsants include, but are not limited to acetazolamide, benzodiazepine, cannabidiols, carbamazepine, clobazam, clonazepam, eslicarbazepine acetate, ethosuximide, ethotoin, felbamate, fenfluramine, fosphenytoin, gabapentin, ganaxolone, huperzine A, lacosamide, lamotrigine, levetiracetam, nitrazepam, oxcarbazepine, perampanel, piracetam, phenobarbital, phenytoin, potassium bromide, pregabalin, primidone, retigabine, rufinamide, valproic acid, sodium valproate, stiripentol, tiagabine, topiramate, vigabatrin, or zonisamide.
  • the agents and/or compositions described herein may be administered conjointly.
  • a composition described herein may be conjointly administered with an anticonvulsant.
  • the subject has a condition responsive to a ketogenic diet.
  • the condition may be Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease, a mitochondrial disorder, depression, migraines (e.g., chronic migraines), or traumatic brain injury (TBI).
  • the methods and compositions comprise administering to the subject a composition provided herein.
  • the condition is epilepsy, seizures, autism spectrum disorder, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, a metabolic disease (e.g., obesity or diabetes), a mitochondrial disorder, depression, migraines (e.g., chronic migraines), Rett syndrome, attention deficit disorder, fragile X syndrome, or traumatic brain injury (TBI).
  • a metabolic disease e.g., obesity or diabetes
  • a mitochondrial disorder e.g., depression, migraines (e.g., chronic migraines), Rett syndrome, attention deficit disorder, fragile X syndrome, or traumatic brain injury (TBI).
  • TBI traumatic brain injury
  • the compositions and methods provided herein are useful in treating or preventing aging or aging-associating conditions.
  • compositions and methods provided herein can replace the ketogenic diet in the treatment or prevention of a condition described herein; in other embodiments, the compositions and methods provided herein can be combined with the ketogenic diet. More information on conditions may be found in Stafstrom et al. (2012) Front. Pharmacol. 3:59, hereby incorporated in its entirety.
  • the composition may be formulated for oral delivery.
  • the composition may comprise probiotics.
  • the compositions disclosed herein are food products.
  • the composition may be in the form of a pill, tablet, or capsule.
  • the subject may be a mammal (e.g., a human).
  • the composition is self-administered.
  • the above methods include reducing the amount of pathogenic bacteria in a subject (i.e., in the gastrointestinal tract of the subject) prior to administration of an agent disclosed herein. In some embodiments, this includes any such therapy that achieves the same goal of reducing the number of pathogenic organisms, when used in combination with the compositions described herein, would lead to replacement of the pathogenic microflora involved in the diseased state with microflora associated with a non- diseased state, or less pathogenic species occupying the same ecological niche as the type causing a disease state.
  • a subject may undergo treatment with antibiotics (e.g., antimicrobial compounds) or a composition comprising antibiotics to target and decrease the prevalence of pathogenic organisms, and subsequently be treated with a composition described herein.
  • antibiotics e.g., antimicrobial compounds
  • the treatment may also comprise an antifungal or anti-viral compound.
  • Suitable antimicrobial compounds include capreomycins, including capreomycin IA, capreomycin IB, capreomycin IIA and capreomycin IIB; carbomycins, including carbomycin A; carumonam; cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefime, ceftamet, cefmenoxime, cefmetzole, cefminox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefpimizole, cefpiramide, cefpirome, cefprozil, cefroxadine, cefsulodin, ceftazidime, ce
  • Suitable anti-fungal compounds include ketoconazole, miconazole, fluconazole, clotrimazole, undecylenic acid, sertaconazole, terbinafine, butenafine, clioquinol, haloprogin, nystatin, naftifine, tolnaftate, ciclopirox, amphotericin B, or tea tree oil and analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms thereof.
  • compositions e.g., compositions comprising an agent disclosed herein and a pharmaceutically acceptable carrier.
  • the composition may comprise bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and/or Clostridium genera.
  • the composition may comprise a pharmaceutically acceptable carrier.
  • the composition may comprise probiotics.
  • the compostion may comprise one or more types of bacteria disclosed herein, and optionally further comprising and agent disclosed herein and/or a fiber disclosed herein.
  • the pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including orally, buccally, sublingually, parenterally, and rectally, as by powders, ointments, drops, liquids, gels, tablets, capsules, pills, or creams.
  • the pharmaceutical compositions are delivered generally (e.g., via oral administration).
  • the compositions disclosed herein are delivered rectally.
  • the pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier may be one or more substance that may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide
  • a pharmaceutical composition can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin).
  • the compound may also be formulated for inhalation.
  • a compound may be simply dissolved or suspended in sterile water.
  • Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • Compositions or compounds may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents,
  • pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro- encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could prescribe and/or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of the compositions and methods of the invention will be that amount of the composition that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the effective daily dose of the composition may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the composition may be administered two or three times daily. In preferred embodiments, the composition will be administered once daily.
  • the patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines, cattle, swine, sheep, cats, and dogs; poultry; and pets in general.
  • compositions of the invention may be used alone or conjointly administered with another type of therapeutic agent.
  • bacterial compositions comprising bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Deferribacteres phylum, Streptococcaceae family, Coriobacteriia class, Bifidobacterium genera, Bacteroides genera, and/or Clostridium genera, or a combination thereof.
  • substantially all of the bacteria in the bacterial composition are selected from the species of Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Bacteroides fragilis, and/or Clostridium perfringens.
  • the bacterial formulation comprises a bacterium and/or a combination of bacteria described herein and a pharmaceutically acceptable carrier.
  • At least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the bacteria in the bacterial composition are selected from bacteria of the Bacteroidetes phylum, Proteobacteria phylum, Def
  • substantially all of the bacteria in the bacterial composition are selected from the species of Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium breve, Bacteroides fragilis, and/or Clostridium perfringens.
  • the composition comprises Bifidobacterium infantis (e.g., Bifidobacterium longum sups, infantis DSM 20088). In some embodiments, the composition comprises Bifidobacterium longum (e.g., Bifidobacterium longum sups, longum ATCC BAA- 999). In some embodiments, the composition comprises Bifidobacterium breve e.g., Bifidobacterium breve DSM 20213). In some embodiments, the composition comprises Bacteroides fragilis (e.g., Bacteroides fragilis ATCC 25285). In some embodiments, the composition comprises Clostridium perfringens (e.g., Clostridium perfringens ATCC 13124).
  • Bifidobacterium infantis e.g., Bifidobacterium longum sups, infantis DSM 20088
  • the composition comprises Bifidobacterium longum (e.g., Bif
  • the bacterial composition comprises at least 10 colony forming units (CFUs), at least 100 colony forming units (CFUs), at least 1 x 10 3 colony forming units (CFUs), at least 1 x 10 4 colony forming units (CFUs), at least 1 x 10 5 colony forming units (CFUs), at least 5 x 10 5 colony forming units (CFUs), at least 1 x 10 6 colony forming units (CFUs), at least 2 x 10 6 colony forming units (CFUs), at least 3 x 10 6 colony forming units (CFUs), at least 4 x 10 6 colony forming units (CFUs), at least 5 x 10 6 colony forming units (CFUs), at least 6 x 10 6 colony forming units (CFUs), at least 7 x 10 6 colony forming units (CFUs), at least 8 x 10 6 colony forming units (CFUs), at least 9 x 10 6 colony forming units (CFUs), at least
  • the bacterial composition comprises 1 x 10 9 to 1 x 10 11 colony forming units of bacteria.
  • the selected dosage level will depend upon a variety of factors including the subject’s diet, the route of administration, the time of administration, the residence time of the particular microorganism being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the bacterial composition required.
  • the physician or veterinarian could prescribe and/or administer doses of the bacteria employed in the composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • the bacterial formulation comprises an enteric coating (e.g., for duodenal release at pH 5.5) or micro encapsulation.
  • the enteric coating or micro encapsulation improves targeting to a desired region of the gastrointestinal tract.
  • the bacterial composition comprises an enteric coating and/or microcapsules that dissolve at a pH associated with a particular region of the gastrointestinal tract.
  • the enteric coating and/or microcapsules dissolve at a pH of about 5.5 - 6.2 to release in the duodenum, at a pH value of about 7.2 - 7.5 to release in the ileum, and/or at a pH value of about 5.6 - 6.2 to release in the colon.
  • Exemplary enteric coatings and microcapsules are described, for example, in U.S. Pat. Pub. No. 2016/0022592, which is hereby incorporated by reference in its entirety.
  • the composition is a food product (e.g., a food or beverage) such as an infant formula, a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food product e.g., a food or beverage
  • an infant formula e.g., a health food or beverage
  • a food or beverage for infants e.g., a food or beverage for infants
  • a food or beverage for pregnant women e.g., athletes, senior citizens or other specified group
  • a functional food e.g., a beverage
  • a beverage e.g., a food or beverage for specified health use
  • a dietary supplement e.g., a food or beverage for patients, or an animal feed.
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like.
  • beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages
  • the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.
  • the composition may be a fermented food product, such as, but not limited to, a fermented milk product.
  • fermented food products include kombucha, sauerkraut, pickles, miso, tempeh, natto, kimchi, raw cheese, and yogurt.
  • the composition may also be a food additive, such as, but not limited to, an acidulent (e.g., vinegar). Food additives can be divided into several groups based on their effects.
  • Non-limiting examples of food additives include acidulents (e.g., vinegar, citric acid, tartaric acid, malic acid, fumaric acid, and lactic acid), acidity regulators, anticaking agents, antifoaming agents, foaming agents, antioxidants (e.g., vitamin C), bulking agents (e.g., starch), food coloring, fortifying agents, color retention agents, emulsifiers, flavors and flavor enhancers (e.g., monosodium glutamate), flour treatment agents, glazing agents, humectants, tracer gas, preservatives, stabilizers, sweeteners, and thickeners.
  • acidulents e.g., vinegar, citric acid, tartaric acid, malic acid, fumaric acid, and lactic acid
  • acidity regulators e.g., anticaking agents, antifoaming agents, foaming agents, antioxidants (e.g., vitamin C), bulking agents (e.g., starch)
  • food coloring fort
  • Example 1 Dietary fiber content in clinical ketogenic diets modifies the gut microbiome and seizure resistance in mice
  • KD low-carbohydrate, high-fat ketogenic diet
  • KD therapies have variable effectiveness in reducing seizures, ranging from 45 to 85% in infants and children that exhibit high compliance and with substantially lower rates in adults.
  • Recent reports highlight a key role for the gut microbiome in mediating effects of the KD on various host physiologies, including glucose and lipid metabolism, immune function, brain activity, and behavior.
  • the KD alters the gut microbiome across several human and animal epilepsy studies, and relationships are seen between the gut microbiome and seizure resistance in various rodent epilepsy models.
  • Findings from the field are converging upon the notion that variation in the gut microbiome may contribute to variability in patient responsiveness to the KD, and that microbiome- targeted interventions could be used to promote the efficacy of the KD in treating refractory epilepsy.
  • KD is commonly administered as a 4: 1 or 3 : 1 fat to carbohydrate and protein ratio, depending on patient tolerance.
  • MCT medium chain triglyceride
  • the Modified Atkins Diet (MAD), which does not require strict weighing of food or fluids, and Low Glycemic Index Treatment (LGIT), which focuses on carbohydrates with low glycemic index rather than removal, are additional less restrictive variations of the KD that are frequently used in older children and adults.
  • KD regimens vary widely in nutritional content and are often tailored to the particular individual’s needs and tolerability, making it difficult to identify standard regimens.
  • KD infant formulas - KD4: 1, KD3: 1, and MCT2.5: 1 - were used due to their reproducible composition, direct clinical relevance, frequent prescription, and importance for infants as an especially vulnerable subset of refractory epilepsy patients for which improved interventions are needed.
  • the three KD infant formulas Compared to a standard infant formula as a control diet (CD), the three KD infant formulas all exhibit high fat content relative to carbohydrate and protein, but they display nuanced differences in formulation (FIG. 1A, Table 1).
  • fat source varies between the formulations, where KD4: 1 contains soy lecithin but lacks coconut oil and linoleic acid, KD3 : 1 contains linoleic acid but lacks soy lecithin and coconut oil, and MCT2.5: 1 contains coconut oil but lacks soy lecithin and linoleic acid.
  • KD4: 1 and MCT2.5: 1 contain corn syrup solids, high amylose com starch, chicory root inulin, gum arabic (acacia fiber), cellulose, fructooligosaccharides (FOS), soy fiber, and maltodextrin
  • KD3: 1 contains only lactose and corn syrup solids, with none of the dietary fibers.
  • the CD contains lactose and less than 2% dietary fiber comprised of galactooligosaccharides, which differs from the types of fibers included in KD4 : 1 and MCT2.5: 1.
  • Vitamins (ug/kcal)
  • Vitamin A 3000.0 1750.0 1640.0
  • Vitamin D (lU/kcal) 600.0 569.0 863.0
  • Vitamin E (lU/kcal) 15.0 23.0 17.0
  • Vitamin B6 600.0 1000.0 900.0 Folic Acid 150.0 165.0 135.0
  • mice were fed the KD4: 1, KD3: 1, MCT2.5: 1, or CD formula as liquid diet for 1 week, and then tested for susceptibility to 6-Hz psychomotor seizures (FIG. IB).
  • Juvenile mice were selected to mimic the typical use of the KD to treat pediatric epilepsy, to align the timing of mouse brain development to early brain development in humans, and to preclude effects of pre-weaning treatment, where effects of the diets on maternal behavior and physiology would confound their direct effects on offspring.
  • One week of feeding was selected based on prior longitudinal characterization, which indicated that KD chow shifts the gut microbiome and confers seizure protection by day 4 of treatment in mice.
  • 6-Hz seizure assay was selected as a benchmark model of refractory epilepsy that is used to screen for new anti-seizure medications and involves low-frequency corneal stimulation to induce complex partial seizures related to human temporal lobe epilepsy.
  • KD chow protects against 6-Hz seizures, as indicated by increases in current intensity required to elicit a seizure in 50% of the subjects tested (CC50, seizure threshold).
  • KD4 1 infant formula increased seizure thresholds compared to controls fed a CD infant formula (FIG. 1C).
  • MCT2.5: 1 also increased seizure thresholds albeit to a lesser degree than KD4: 1, which may be due to its comparatively lower fat ratio or different fat source.
  • KD3 1 infant formula yielded decreased seizure thresholds compared to all other groups, including CD-fed controls, suggesting that the KD3: 1 formulation increases susceptibility to 6-Hz seizures in mice.
  • seizure threshold There was no correlation of seizure threshold with average calories consumed for the different KDs or with degree of ketosis as assessed by serum levels of beta-hydroxybutyrate (FIG. 5A, B).
  • KD3 1 on Shannon diversity levels, despite comparable increases across all KD formula groups in species richness of the fecal microbiota. This suggests that the main driver of a-diversity differences between the KD groups is differential alteration in species evenness — indeed, KD3: 1 yielded fecal microbiota with significantly reduced Pielou’s evenness compared to KD4: 1 and MCT2.5: 1 groups.
  • KD4 1 and MCT2.5: l shared statistically significant decreases in Erysipelotrichia and increases in Streptococcaceae, Coriobacleriia. and Deferribacteres
  • KD3 : 1 exhibited no significant changes in these taxa compared to CD (FIG. 6A-C).
  • KD3 : 1 showed significantly increased relative abundance of Proteobacteria, Escherichia coli, Enterococcus faecalis, and Mammaliicoccus sciuri compared to CD, KD4: 1, and/or MCT2.5:1 controls (FIG. 6A-C).
  • the seizure susceptible KD3: 1 group also exhibited decreased representation of the top 10 most abundant metagenomic superclass pathways (FIG. 2C), suggesting that the KD3: 1 limits the presence of microbial taxa associated with prevalent functions and/or enriches the representation of previously rare metagenomic pathways.
  • the relative abundance of superclass pathways related to amino acid, carbohydrate, and nucleoside and nucleotide biosyntheses were significantly lower in KD3: 1 relative to MCT2.5: 1, CD, and/or KD4:1 groups.
  • superclass pathways related to carboxylic acid, fatty acid and lipid, and secondary metabolite degradation were significantly elevated in KD3 : 1 compared to other groups.
  • KD4: 1 and MCT2.5:l similarly induced significant metagenomic increases in select pathways related to carbohydrate biosynthesis (UDP-N-acetyl-D- galactosamine II and UDP-N-acetyl-D-glucosamine biosynthesis II), carboxylic acid degradation (biotin-dependent malonate degradation), and cofactor, carrier, and vitamin biosynthesis (biotin biosynthesis), and decreases in select pathways related to carbohydrate degradation (hexitol and galactitol degradation, sucrose, lactose, galactose degradation, and Entner-Doudoroff pathway), amino acid biosynthesis (L-lysine and L-alanine biosynthesis), carbohydrate biosynthesis (UDP-N
  • KD3: 1 displayed the most differentially abundant metagenomic pathways compared to CD, which were distinct from those seen in the other KD groups (FIG. 6D). The majority of differentially abundant pathways were elevated by KD3: 1 and related to amide, amidine, amine, and polyamine degradation, fatty acid and lipid biosynthesis, carboxylic acid degradation, and fermentation (FIG. 6D). In particular, pathways for phospholipid remodeling, lactate fermentation, and biosynthesis of octanoyl and myristate, and degradation of erythronate, threonate, galactitol, and allantoin were all significantly increased by KD3: 1, decreased by KD4: 1 and MCT2.5: 1 (FIG.
  • Fiber content in the KD drives microbial alterations and promotes seizure resistance
  • the gut microbiome is shaped by changes in host diet and can be responsive to the presence, abundance, and sources of dietary macronutrients.
  • various dietary parameters were screened for their effects on a model human infant microbial community.
  • Nine bacterial strains were selected based on their prevalence and relative abundances across multiple large studies of the infant gut microbiome (FIG. 7A, Table 2). All community members were confirmed to grow stably together in a rich complex medium as a positive control (FIG. 7B).
  • the model infant gut microbial community was cultured in synthetic media representing KD4: 1, KD3 : 1, or MCT2.5: 1, each using sunflower oil (6% saturated fat), soy lecithin (23% saturated fat and dominant in KD4: 1 infant formula), or palm oil (50% saturated fat), as fat sources with different levels of saturation (FIG. 7E).
  • the media prepared with soy lecithin increased the absolute abundance of B. infantis, B.fragilis, and C. perfringens, resulting in distinct separation along PCoAl from the sunflower and palm oil groups (PERMANOVA, p ⁇ 0.05; FIG. 7F). This may be due to the presence of free sugars (8%) in the commercial soy lecithin and/or the emulsifying properties of soy lecithin, compared to the other fat sources. There were no statistically significant differences between the sunflower and palm oil groups across all media conditions (FIG. 7F), suggesting that the differential effects of soy lecithin are driven by its fat source rather than saturation level.
  • KD-based media were also prepared with addition of MCT, dominant in MCT2:5: 1 infant formula, or linoleic acid, dominant in KD3: 1 infant formula (FIG. 7G).
  • addition of linoleic acid decreased the absolute abundance of B.
  • the model infant gut microbial community was cultured in synthetic media representing KD4: 1, KD3: 1, and MCT2.5: 1 and containing either lactose or a fiber mix, comprised of equal amounts of FOS, inulin, cellulose, and gum arabic, as the fiber sources that distinguish KD4: 1 and MCT2.5: 1 infant formula from KD3: 1 and CD formulas (FIG. 3A).
  • the presence of dietary fiber led to substantial shifts in the model infant gut microbial community across all media conditions, with particular enrichment of B. fragilis and decreases in B. breve and B. infantis (FIG. 71).
  • fiber- induced decreases in pentose phosphate pathways pathways related carbohydrate degradation (sucrose, glucose, xylose, and glycogen degradation), carbohydrate biosynthesis (UDP-N- acetyl-D-glucosamine biosynthesis and UDP -glucose derived O-antigen building blocks biosynthesis), amino acid biosynthesis (L-alanine, L-lysine and L-aspartate and L-asparagine biosynthesis), partial TCA cycle, and methylerythritol phosphate pathway were also shared with mouse metagenomes of KD4: 1 and MCT2.5: 1 groups (FIG. 3C and 2E).
  • the fiber mix was supplemented into the KD3 : 1 infant formula to match reported fiber levels in KD4: 1 infant formula, and mice were tested for seizure susceptibility at 7 days after dietary treatment (FIG. 3D).
  • mice fed liquid KD3.1 exhibited decreased seizure threshold compared to CD controls (FIG. 3E).
  • addition of fiber to the KD3 : 1 elevated seizure thresholds to levels that exceeded those seen in CD controls.
  • the fiber supplementation was further repeated using the solid diet paradigm, where the same infant formulas were dehydrated and administered as chow instead of liquid diet.
  • the fiber-containing KD4: 1 infant formula which yielded the highest seizure thresholds of all KD variants (FIG. 1), was supplemented with the dietary fiber mix that is already existing in the formula and tested mice for resistance to 6-Hz seizures after 7 days of feeding with the liquid diet (FIG. 4A).
  • the additional fiber added to KD4: 1 formula increased fiber content from 5.3% to -10.3%.
  • Dietary fiber supplementation significantly increased seizure thresholds to levels that exceeded those seen with KD4: 1 alone (FIG. 4B). There were no significant differences between groups in dietary consumption (FIG. 10A).
  • Group la consisted of fiber mix, FOS, and orange fiber and was characterized by increases in genes related to preQO biosynthesis and L-alanine biosynthesis, with reductions in sucrose degradation and partial TCA cycle (FIG. 4D).
  • Group lb consisting of pea, acacia, and psyllium husk fibers, clustered together with Group la and exhibited a similar general pattern of metagenomic features but with reductions in L-alanine biosynthesis and less substantial shifts in preQO biosynthesis and sucrose degradation (FIG. 4D).
  • Group 2 consisted of inulin, cellulose, and gum arabic, which was characterized by significant decreases in genes related to 5-7 pathways (glycogen and sucrose degradation, L-alanine, L- lysine, L-aspartate, L-asparagine, and UDF-N-acetyl-D-glucosamine biosynthesis, partial TCA cycle, and methylerythritol phosphate pathway) and significant increases in preQO biosynthesis genes (FIG. 4D).
  • Group 3 consisting of oat, potato, wheat, and apple fibers, was characterized by notable increases in representation of L-alanine biosynthesis and UDP- glucose-derived O-antigen building blocks biosynthesis, with decreases in queuosine biosynthesis (FIG. 4D).
  • one representative fiber condition per primary grouping (Group 1 : fiber mix, Group 2: gum arabic, Group 3: oat fiber) was selected to test for causal effects on seizure resistance.
  • Representative fibers from each group were supplemented into KD4: 1 infant formula to raise fiber content from 5.3% to -10.3%, and tested mice for resistance to 6- Hz seizures after 7 days of feeding in paste form.
  • KD4 1 paste with fiber mix significantly increased resistance to 6-Hz seizures
  • KD4: 1 and MCT2.5: 1, but not KD3: l reduce representation of select genes related to carbohydrate degradation, which were significantly associated with the presence of dietary fiber and similarly induced by fiber supplementation to a cultured infant gut microbial community.
  • Adding a fiber mixture to the KD3 : 1 to match levels present in KD4: 1 and MCT2.5: 1 restores seizure protection in mice.
  • supplementing the fiber mixture to the already protective KD4: 1 infant formula further enhances seizure resistance in mice.
  • a human study of KD therapy in children with refractory epilepsy reported changes in 29 metagenomic pathways, including the reduction of seven pathways involved in carbohydrate metabolism and fermentation such as fructooligosaccharides (FOS) and raffinose utilization, sucrose utilization, glycogen metabolism, lacto-N-biose I and galacto-N-biose metabolic pathway; lactate, pentose phosphate pathway; and formaldehyde assimilation: ribulose monophosphate pathway.
  • FOS fructooligosaccharides
  • raffinose utilization sucrose utilization, glycogen metabolism, lacto-N-biose I and galacto-N-biose metabolic pathway
  • lactate pentose phosphate pathway
  • formaldehyde assimilation ribulose monophosphate pathway.
  • Dietary fibers are resistant to digestion by the host and specifically fermented by gut bacteria that together encode hundreds of glycoside hydrolases with varying specificity for different fiber types. As such, not only does the gut microbiome degrade fiber, it also responds to and is shaped in composition and function by dietary fiber.
  • the disclosure demonstrates that supplementing mice with SCFAs, as the microbial byproducts of fiber fermentation, fails to phenocopy the beneficial effects of fiber supplementation on potentiating seizure protection in mice fed the KD4: 1. This may align with prior human studies reporting that epilepsy is associated with deficient levels of SCFA-producing bacteria, which are further reduced by KD therapy to promote seizure control.
  • Different fiber types and sources can vary greatly in their chemical structure, fermentability, and effects on the gut microbiome.
  • the in vitro screening approach was expanded to include 13 different soluble or insoluble fiber types and sources, as supplemented directly into the commercial KD4: 1 infant formula (rather than a diet-based synthetic culture medium).
  • KD4 1 infant formula
  • key fiber-associated metagenomic features to stratify microbial responses to the 13 fiber conditions, a specific subset of fibers that potentiates the seizure protective effects of the KD4: 1 in mice was identified.
  • This subgroup including fiber mix (inulin, FOS, gum arabic, cellulose), FOS alone, and orange fiber, is characterized by metagenomic enrichment of pathways related to preQo biosynthesis and L-alanine biosynthesis, and decreases in metagenomic representation of sucrose degradation and partial TCA cycle.
  • PreQo is a deazapurine nucleoside.
  • microbial preQo biosynthesis was associated with alterations in hippocampal expression of genes related to neuron generation and migration protection.
  • L-alanine is an essential amino acid that is modulated by ketosis and regulates the function of glutamatergic neurons and astrocytes.
  • L- alanine levels were diminished significantly in the cerebrospinal fluid of children after four months of KD therapy, and genes related to L-alanine metabolism were elevated in imputed microbial metagenomic pathways from epileptic individuals relative to healthy controls.
  • Microbial sucrose utilization is a carbohydrate pathway reduced after KD therapy in humans, likely due to the low availability of carbohydrates in the diet.
  • the TCA pathway fuels aerobic respiration, wherein acetyl-CoA is converted to intermediate organic acids such as citrate, 2-oxoglutarate, and succinate.
  • mice All mouse experiment protocols were approved by the UCLA Institutional Animal Care and Use Committee. Juvenile (4-week old) specific pathogen free (SPF), male Swiss Webster (Taconic Farms) mice were used for all animal experiments, fed standard chow (Labdiet 5010, 28.7%: 13.1%: 58.2% protein: fat: carbohydrate by calories), and housed in sterile caging under a 12 h: 12 h light:dark cycle with standard temperature and humidity control.
  • mice were fed commercially available KD infant formulas (KetoCai, Nutricia North America, FIG. la,) or a popular commercially available, standard infant formula as control diet (Abbott Nutrition, FIG. la) for 7 days.
  • liquid diet paradigm 90 g of powder formula or 90 mL of liquid formula was mixed with 600 mL water at 60°C. Before adding to cages, the diet solution was brought to IL and each cage containing 3-4 mice was supplemented with liquid diets in water bottles. The water bottles were filled with liquid diets and the cages were changed every 1-2 days.
  • 90 g of powder formula was mixed with 600 mL water and dehydrated using a food dehydrator (CASORI). The diets were administered in sterile petri dishes and cages were provided with standard sterile water.
  • pasted diet paradigm 30 g of powder was mixed with water and administered as a paste in sterile petri dishes.
  • the 6-Hz psychomotor seizure test was conducted as previously described in Olson, C. A. et al. The Gut Microbiota Mediates the Anti-Seizure Effects of the Ketogenic Diet. Cell 173, 1728-1741. el3 (2016).
  • One drop ( ⁇ 50 ul) of 0.5% tetracaine hydrochloride ophthalmic solution was applied to the corneas of each mouse 10-15 min before stimulation.
  • Corneal electrodes were coated with a thin layer of electrode gel (Parker Signagel).
  • a current device (ECT Unit 57800, Ugo Basile) was used to deliver current at 3 s duration, 0.2 ms pulse-width and 6 pulses/s frequency.
  • CC50 the intensity of current required to elicit seizures in 50% of the experimental group
  • CC50 the intensity of current required to elicit seizures in 50% of the experimental group
  • Locomotor behavior was recorded using a camera and quantitative measures for stunned fixture, falling, tail dorsiflexion (Straub tail), forelimb clonus, eye/vibrissae twitching, and behavioral remission were scored manually.
  • Latency to exploration time elapsed from when an experimental mouse is released into the observation cage (after corneal stimulation) to its normal exploratory behavior) was scored manually with an electronic timer. Mice were blindly scored as protected from seizures if they did not show seizure behavior and resumed normal exploratory behavior within 10 s.
  • Seizure threshold (CC50) was determined, using the average log interval of current steps per experimental group, where sample n is defined as the subset of animals displaying the less frequent seizure behavior. Data used to calculate CC50 are also displayed as latency to explore for each current intensity, where n represents the total number of biological replicates per group regardless of seizure outcome.
  • Frozen stool samples from mice pre- and post-dietary treatment were subjected to DNA extraction using the ZymoBIOMICS DNA Miniprep kit (Zymo), with bead beating used to lyse cells. Briefly, the samples were transferred into PowerBead tubes containing lysis solution and bead beaded at maximum speed for 1 min five times with 1 min of ice incubation in between cycles. The rest of the protocol followed the manufacturer’s instructions. The DNA was eluted in 60 pL elution buffer provided by the kit. Purified DNAs were sent to Novogene Corporation Inc for paired end (PE) metagenomic sequencing. Sequencing was performed on the Illumina NovaSeq platform with PE reads of 150 bp for each sample averaging around 3GB data.
  • PE paired end
  • Metagenomic data was analyzed using HUMAnN3 and MetaCyc database to profile gene families and pathway abundance.
  • MetaPhlAn4 was used for metagenomic taxonomic profiling, a-diversity indexes for taxonomic profiling were determined by Shannon’s index, richness, and Pielou's Evenness using vegan v2.6-4 in R. For P-diversities, calculate diversity.R script were run within the MetaPhlAn4. For Unifrac distances, mpa_vOct22_CHOCOPhlAnSGB_202212.nwk was used for SGB-level phylogenetic tree as reference.
  • Blood was collected via a capillary tube from the medial canthus of the eye, allowed to clot 30 min at room temperature, and spun through SST vacutainers (Becton Dickinson) at 1500g for 90 sec for serum separation. Samples were immediately snap frozen in liquid nitrogen and stored at -80°C until further processing. BHB levels were quantified by colorimetric assay according to the manufacturer’s instructions (Cayman Chemical).
  • Vitamin Ki (HiMedia) 0.002
  • Vitamin B 12 0.1 p-Aminobenzoic acid 5
  • Synthetic KDs with different ratios, fat and carbohydrate source were prepared using sunflower oil (Baja Precious), vegetable shortening (Crisco), palm oil (Okonatur), soy lecithin (Modernist Pantry), linoleic acid (Sigma-Aldrich), and Medium Chain Triglycerides (MCT, Nutriticia) as fat sources, whey protein isolate (Bulk Supplements) as protein source, and lactose (modernist pantry) and dietary fiber mixture of fructooligosaccharides (Sigma- Aldrich), inulin from chicory (Sigma-Aldrich), crystalline cellulose (Sigma-Aldrich), and gum arabic from Acacia Tree (Sigma-Aldrich) as carbohydrate sources.
  • wheat, pea, potato, and apple fiber from J. Rettenmaier USA LP, orange (citrus) fiber from Citri-Fi Naturals, oat (NuNaturals), acacia (Nutricost organic), and psyllium husk (It’s just) were used.
  • the powders were ultraviolet (UV)-sterilized and confirmed to be sterile by aerobic and anaerobic culture. They were then mixed with simulated saliva solution, gastric solution, and intestinal fluid as described in INFOGEST model without enzymatic solution to simulate the gastric and intestinal bolus entering to the colon and was subjected to an in vitro batch culture fermentation.
  • the representative bacterial strains were mixed in a minimal media at the dilution factor (1 : 100) needed to achieve a ratio of 21% Actinobacteria, 14% of Bacteroidetes, 28% of Firmicutes, and 37% of Proteobacteria, reflective of relative abundances seen in a typical infant gut (Table 2).
  • Species that comprise Actinobacteria, Bacteroidetes and Firmicutes were mixed at 1 : 1 ratio, whereas Proteobacteria consists of 57 % of Escherichia coli and 42% of Klebsiella pneumoniae.
  • the bacterial mixture was then mixed with each diet bolus (1 : 1 v/v) and subjected to 24-hour anaerobic culture. After 24 hours, the bacterial pellets were separated from the media and stored at -80°C until further analysis. The pellets from pre- fermentation were also collected as a control.
  • Total DNA was extracted from the pellets collected after fermentation, following standard procedures for the ZymoBIOMICS DNA Miniprep kit.
  • the microbial composition was determined using quantitative RT-PCR with species specific primers and respective qPCR conditions. DNA extracted from individual overnight cultures were used to generate a standard curve. The copy numbers for each sample were calculated based on the standard curve and normalized to DNA concentration of the original sample. Absolute quantification of growth after anaerobic culture of each sample was determined by subtracting the prefermentation quantities and presented as log values. Any species that exhibited negative values after subtraction were regarded as zero or no growth. Data are presented in bar plots as a mean of each bacteria. PCoA plots were created using cmdscale from the distance matrix created using Euclidean distances in vegan package in R.
  • the genome fastq files for each species were obtained from ATCC.org. Open source BBMap v38.94 randomreads. sh plugin was used to randomly produce paired reads at 150 bp length from each genome based on the qPCR absolute quantification multiplied by a million.
  • Metagenomes were analyzed using Humann3 and significant pathway associations were determined with MaAsLin2 package in R as described above.

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Abstract

La présente invention concerne des méthodes et des compositions pour traiter ou prévenir les crises d'épilepsie.
PCT/US2025/039607 2024-07-30 2025-07-29 Compositions et méthodes pour le traitement des crises d'épilepsie et des troubles associés Pending WO2026030281A1 (fr)

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