Probiotics and methods for suppressing appetite

Abstract

Methods and compositions for suppressing appetite are provided herein. In some embodiments, a method for suppressing appetite in a subject is provided, comprising the step of administering a composition comprising Bacteroides parabacteroides and Akkermansia to the subject.

Description

Technical Field

[0001] Related Applications and Incorporation by Reference This application claims priority to and relates to U.S. Provisional Application No. 63 / 520,053, filed Aug. 16, 2023, the entire contents of which are incorporated herein by reference.

[0002] Methods and compositions for suppressing appetite in a subject are disclosed herein. In some embodiments, a method for suppressing appetite in a subject is provided herein by administering to the subject a composition comprising Parabacteroides and Akkermansia bacteria.

Background Art

[0003] Epilepsy is characterized by recurrent seizures that can result in loss of awareness, loss of consciousness, and / or impairment of movement, autonomic function, sensation (including vision, hearing, and taste), mood, and / or mental function. 1-2% of the population in developed countries suffers from epilepsy.

[0004] The low-carbohydrate, high-fat ketogenic diet (KD) is a treatment for refractory epilepsy, although more than one-third of epilepsy patients do not respond to existing antiepileptic medications. The effectiveness of KD has been supported by multiple retrospective and prospective studies, which estimate that approximately 30% of patients become seizure-free and approximately 60% experience significant benefits. However, despite its value as a treatment for epilepsy, and its increasing application to other disorders including autism, Alzheimer's disease, Parkinson's disease, metabolic syndrome, and cancer, the use of KD remains low due to difficulties in implementation, dietary compliance, and adverse side effects. In fact, even with effective seizure reduction, only an estimated 12% of epilepsy patients maintain KD by the third year of dietary therapy. Furthermore, the mechanisms underlying the beneficial effects of KD are not well understood, and there is a lack of molecular and / or cellular targets for intervention. The success of diet in managing various types of symptoms when medication fails suggests that diet enhances intrinsic neuroprotective pathways that are not targeted by existing medications. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent No. 6,461,607 [Non-patent literature]

[0006] [Non-Patent Document 1] Stafstrom et al. (2012) Front. Pharmacol. 3:59 [Non-Patent Document 2] Reikvam et al., PloS one (6), 2011 [Non-Patent Document 3] van der Stel, Frontiers in Microbiology (6), 567 (2015) [Overview of the project] [Means for solving the problem]

[0007] Methods and compositions for suppressing appetite in a subject are provided herein. In some embodiments, the method involves applying to the subject at least 1 × 10 11 Colony-forming units (CFUs) of Akkermansia (Akk) fungi, and at least 1 × 10⁻⁶ 10 The process includes administering CFU of Parabacteroides (Pb) bacteria. In some embodiments, the bacteria are administered orally or rectally to the subject. In some embodiments, the bacteria are administered in pill form or as part of food. In some embodiments, the subject is a human subject. In some embodiments, Akk bacteria include Akkermansia muciniphila. In some embodiments, Pb bacteria include Parabacteroides merdae, Parabacteroides distasonis, and / or Parabacteroides johnsonii. In some embodiments, Akk bacteria and Pb bacteria are administered to the subject at least daily. In some embodiments, the bacteria are administered to the subject in a ratio of Akkermansia bacteria to Parabacteroides bacteria of at least 1:1, 3:1, 6:1, or 10:1.

[0008] at least 1 × 10 11 Colony-forming units (CFUs) of Akk bacteria, and at least 1 × 10⁻⁶ 10Bacterial compositions containing CFU of Pb bacteria are also provided herein. In some embodiments, the compositions are formulated for oral or rectal delivery. In some embodiments, the compositions are part of a food, which may be yogurt. In some embodiments, the compositions further include prebiotics. In some embodiments, the bacteria are in a ratio of at least 1:1, 3:1, 6:1, or 10:1 of Akkermansia bacteria to Parabacteroides bacteria. In some embodiments, the bacteria include Akkermansia muciniphila, Parabacteroides meldae, Parabacteroides johnsonii, Parabacteroides distasonis, or any combination thereof.

[0009] The use of any one of the embodiments of the present disclosure in suppressing appetite in a subject is also provided herein.

[0010] Methods and compositions for mimicking the effects of a ketogenic diet by administering probiotic compositions to subjects are also provided herein. In certain embodiments, the methods and compositions are intended for the treatment or prevention of seizures in subjects (e.g., subjects having neurodevelopmental disorders, e.g., autism spectrum disorder, Rett syndrome, fragile X, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), intractable epilepsy, and / or intractable epilepsy). In other embodiments, the methods and compositions are intended for the prevention or treatment of conditions in subjects (e.g., epilepsy, seizures, autism spectrum disorder, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, metabolic disorders (e.g., diabetes or obesity), mitochondrial diseases, depression, migraines (e.g., chronic migraines), Rett syndrome, attention deficit disorder, fragile X syndrome, or traumatic brain injury (TBI)). Preferably, the method includes administering to a subject a composition comprising bacteria of the genera Akkermansia (Akk) and Parabacteroides (Pb), or a plurality of compositions comprising bacteria of the genera Akkermansia (Akk) and Parabacteroides (Pb) together. In some embodiments, the composition comprises bacteria of the genera Akkermansia (Akk) and Parabacteroides (Pb). In some embodiments, the bacteria of the genus Akkermansia (Akk) comprises Akkermansia muciniphila. In some embodiments, the bacteria of the genus Parabacteroides (Pb) comprises Parabacteroides meldae, Parabacteroides johnsonii, and / or Parabacteroides distasonis. In some embodiments, the method includes the steps of removing the target intestinal microbiota and administering to the target a composition containing bacteria of the genera Akkermansia (e.g., Akkermansia muciniphila) and Parabacteroides (e.g., Parabacteroides meldae, Parabacteroides johnsonii, or Parabacteroides distasonis). In some embodiments, the composition includes a ratio of Akkermansia to Parabacteroides of 6:1, 6:2, 6:3, 6:4, 6:5, 6:6, 1:6, 2:6, 3:6, 4:6, or 6:6. In some embodiments, the composition includes a ratio of Akkermansia to Parabacteroides of 6:2.In some embodiments, Akkermansia bacteria are present in the composition at about 1×10. 5 , 5×10 5 , 1×10 6 , 5×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , 1×10 9 , 5×10 9 , 1×10 10 , 5×10 10 , 1×10 11 , 5×10 11 , 1×10 12 , 5×10 12 , 1×10 13 , 5×10 13 , 1×10 14 , 5×10 14 , 1×10 15 , 5×10 15 , 1×10 20 , 5×10 20 colony-forming units (CFU), or in any abundance between 1×10 5 and 5×10 20 colony-forming units (CFU).

[0011] In some embodiments, the subject is on a dietary restriction, which can be a control diet, a ketogenic diet, a high-fat diet, or a low-carbohydrate diet. The composition can be formulated for oral or rectal delivery. The composition can be a food product. In some embodiments, the food product is a dairy product (e.g., yogurt). In some embodiments, the composition contains probiotics. In some embodiments, the composition is self-administered. In some embodiments, the composition contains a fecal sample (e.g., a fecal sample from a fecal bank) containing bacteria of the genus Akkermansia (e.g., Akkermansia muciniphila) and bacteria of the genus Parabacteroides (Pb) (e.g., Parabacteroides merdae, Parabacteroides johnsonii, or Parabacteroides distasonis). In some embodiments, the subject is given an antibiotic to deplete the subject's gut microbiota. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] [Figure 1A] This study demonstrates that seizure protection and ketone production in response to a ketogenic diet correlate with changes in the gut microbiota. The left figure shows seizure thresholds in response to 6Hz stimulation in independent cohorts of mice fed a control diet (CD) or a ketogenic diet (KD) for 2, 4, 8, 10, or 14 days. n=8, 6, 9, 20, 6 (CD); 8, 7, 12, 21, 5 (KD). The right figure shows the behavior of a representative cohort of seizure test mice 14 days after dietary intervention. The dashed line at y=10 seconds represents the seizure scoring threshold, and the triangle at 24mA represents the starting current in the experimental cohort. n=16. [Figure 1B] This study demonstrates that seizure protection and ketone production in response to a ketogenic diet correlate with changes in the gut microbiota. Serum glucose levels are shown in independent cohorts of mice fed CD or KD for 2, 4, 8, 10, or 14 days. Data are normalized to serum glucose levels observed in SPF CD mice at each time point. n=8, 5, 8, 8, 19 (CD); 8, 8, 8, 7, 19 (KD). [Figure 1C] This study demonstrates that seizure protection and ketone production in response to a ketogenic diet correlate with changes in the gut microbiota. Serum beta-hydroxybutyrate (BHB) levels are shown in independent cohorts of mice fed CD or KD for 2, 4, 8, 10, or 14 days. n=8, 13, 8, 8, 37 (CD); 8, 16, 8, 7, 38 (KD). [Figure 1D] This study demonstrates that seizure protection and ketone production in response to a ketogenic diet correlate with changes in the gut microbiota. It shows principal coordinate analysis (PCoA) of weighted (left) and unweighted (right) UniFrac distance matrices based on 16S rDNA profiling of fecal samples from independent cohorts of mice fed CD or KD for 0, 4, 8, or 14 days. n=3 cages (9 mice) / group. [Figure 1E]This study demonstrates that seizure protection and ketone production in response to a ketogenic diet correlate with changes in the gut microbiota. Alpha diversity is shown from fecal 16S rDNA sequencing data of mice fed CD or KD for 14 days. n=3 cages / group. [Figure 1F] This study demonstrates that seizure protection and ketone production in response to a ketogenic diet correlate with changes in the gut microbiota. Relative abundances of Ackermansia muciniphila and Parabacteroides are shown from fecal 16S rDNA sequencing data. n=3 cages (9 mice) / group. Data are presented as mean ± sem. Two-way ANOVA using Bonferroni (Figures 1A-1C, 1E), Kruskal-Wallis using Bonferroni (Figure 1F): P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns = not statistically significant. SPF = Specific pathogen elimination (conventionally-colonized), CD = control diet, KD = ketogenic diet, CC50 = current intensity that induces seizures in 50% of tested mice, BHB = beta-hydroxybutyrate, OTU = operational taxonomic unit. [Figure 2A] This shows that a ketogenic diet enriches specific bacterial species that distinguish the gut microbiota of KD (ketogenic diet) from that of CD (clear vein thrombocytopenia). Alpha diversity based on 16S rDNA sequencing of fecal gut microbiota at days 0, 4, 8, and 14 after treatment with KD (lower figure) versus CD (upper figure). n=3 per time point. [Figure 2B] This study demonstrates that a ketogenic diet enriches specific bacterial species that distinguish the gut microbiota of KD (ketogenic diet) from that of CD (coagulant diet). The relative abundances of specific bacterial taxa enriched in SPF mice fed KD (top) or CD (bottom) are shown. n=3. Data are presented as mean ± sem. Kruskal-Wallis using Bonferroni: *P<0.05, **P<0.01, ***P<0.0001. ns = not statistically significant. CD = control diet, KD = ketogenic diet. [Figure 3A]This study shows the relationship between gut microbiota and the anti-seizure effect of a ketogenic diet. It presents seizure thresholds in response to 6Hz stimulation in SPF, GF, or conventionalized GF mice fed CD or KD. n=13, 18, 12, 6. [Figure 3B] This figure shows the relationship between the gut microbiota and the anti-seizure effect of a ketogenic diet. Serum BHB levels (left figure) and glucose levels (right figure) are shown in SPF, GF, or normalized GF mice fed CD or KD. n=37, 38, 19, 8. [Figure 3C] This paper shows the relationship between gut microbiota and the anti-seizure effect of a ketogenic diet. It also shows the seizure threshold in response to 6Hz stimulation in SPF mice treated with pre-meal preparation of vehicle or Abx. n=13, 18, 13. [Figure 3D] This study shows the relationship between the gut microbiota and the anti-seizure effect of a ketogenic diet. Serum BHB levels (left) and glucose levels (right) are shown in SPF mice treated with pre-meal preparation of Vehicle or Abx. n=18, 18, 19 (BHB); n=12, 11, 11 (glucose). Data are shown as mean ± sem. One-way ANOVA using Bonferroni: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns = not statistically significant. SPF = specific pathogen removal (normally established state), GF = sterile, GF-conv = sterile normalized with SPF microbiota, CD = control diet, KD = ketogenic diet, CC50 = current intensity that causes seizures in 50% of tested mice. BHB = beta-hydroxybutyrate, veh = vehicle, Abx = antibiotic (ampicillin, vancomycin, neomycin, metronidazole [AVNM]). [Figure 4A]This shows that KD-related bacteria adequately mediated the anti-seizure effect of the ketogenic diet. The left figure shows seizure thresholds in SPF mice pre-treated with vehicle or Abx and colonized with Parabacteroides species (P. meldae and P. distasonis), Ackermansia muciniphila, or Bifidobacterium longum, in response to 6Hz stimulation. n=13, 18, 15, 6, 8, 5, 5. The right figure shows behavior in a representative cohort of seizure-test mice. The dashed line at y=10 seconds represents the seizure scoring threshold, and the triangle at 24mA represents the onset current in the experimental cohort. n=12, 16, 8, 25. [Figure 4B] This shows that KD-related bacteria adequately mediated the anti-seizure effect of the ketogenic diet. The upper figure shows the seizure threshold in response to 6Hz stimulation in GF mice colonized with Parabacteroides species (P. meldae and P. distasonis) and / or Ackermansia muciniphila. n=15, 4, 9, 9. The lower figure shows the behavior in seizure test mice. The dashed line at y=10 seconds represents the seizure scoring threshold, and the triangle at 24mA represents the onset current in the experimental cohort. n=17, 19. Data are shown as mean ± sem. One-way ANOVA using Bonferroni: **P<0.01, ***P<0.001, ****P<0.0001. SPF = Suppressed Specified Pathogens (normal colonization state), GF = Sterile, CD = Control diet, KD = Ketogenic diet, CC50 = Current intensity that induces seizures in 50% of tested mice, veh = Vehicle, Abx = Pre-treatment with antibiotics (ampicillin, vancomycin, neomycin, metronidazole [AVNM]), Pb = Parabacteroides species (P. meldae and P. distasonis), Akk = Akkermansia muciniphila, AkkPb = A. muciniphila, P. meldae and P. distasonis, Bf = Bifidobacterium longum. [Figure 5A]This study demonstrates that KD-associated microbiomes provide seizure protection in mice fed a control diet. The left figure shows the seizure threshold in Abx-treated SPF mice, transplanted with CD or KD microbiomes (CD-FMT) or KD microbiomes (KD-FMT), in response to a 6Hz stimulus. n=6, 5, 5. The right figure shows behavior in a representative cohort of seizure-test mice. The dashed line at y=10 seconds represents the seizure scoring threshold, and the triangle at 24mA represents the onset current in the experimental cohort. n=12. [Figure 5B] This study demonstrates that the KD-associated microbiome provides seizure protection in mice fed a control diet. It shows seizure thresholds in response to 6Hz stimulation in SPF mice pre-treated with vehicle or Abx and colonized with Parabacteroides species (P. meldae and P. distasonis), Ackermansia muciniphila, or Bifidobacterium longum (left figure). n=13, 18, 9, 8, 6, 6. [Figure 5C]This study demonstrates that the KD-associated microbiome provides seizure protection in mice fed a control diet. The left figure shows seizure thresholds in response to 6Hz stimulation in SPF mice orally administered Akkermansia muciniphila, P. meldae, and P. distasonis (AkkPb), A. muciniphila alone (Akk), or heat-sterilized Akkermansia muciniphila and Parabacteroides (hk-AkkPb). n=6, 6, 4, 3. Data are presented as mean ± sem. One-way ANOVA using Bonferroni: *P<0.05, ***P<0.001, ****P<0.0001. SPF = SPF (Specific Pathogen Removal) (normal colonization state), CD = Control diet, KD = Ketogenic diet, CC50 = Current intensity that induces seizures in 50% of tested mice, CD-FMT = Transplantation of CD microbiome, KDFMT = Transplantation of KD microbiome, veh = Vehicle, Abx = Pretreatment with antibiotics (ampicillin, vancomycin, neomycin, metronidazole [AVNM]), Pb = Parabacteroides species (P. meldae and P. distasonis), Akk = Akkermansia muciniphila, AkkPb = A. muciniphila, P. meldae and P. distasonis, Bf = Bifidobacterium longum, hk-AkkPb = Heat-sterilized A. muciniphila, P. meldae and P. distasonis. [Figure 6A] This study demonstrates the reversal of the KD microbiome and KD-related seizure protection depending on the control diet. It shows principal coordinate analysis (PCoA) of weighted UniFrac distance matrices based on longitudinal 16S rDNA profiling of fecal samples from SPF mice fed CD for 28 days (CD), mice fed KD for 28 days (KD), or mice fed KD for 14 days followed by CD for 14 days (KD-CD). n=3 cages / group. [Figure 6B] This shows the regression of the KD microbiome and protection against KD-related seizures depending on the control diet. The seizure thresholds in response to 6Hz stimulation are shown in SPF mice fed CD for 28 days (CD), mice fed KD for 28 days (KD), or mice fed KD for 14 days followed by CD for 14 days (KD-CD) (left figure). n=4. [Figure 6C]This shows the regression of the KD microbiome and protection against KD-related seizures in response to a control diet. It shows the seizure threshold in response to 6Hz stimulation in SPF mice 21 days after probiotic treatment with Akkermansia muciniphila, P. meldae and P. distasonis (AkkPb), A. muciniphila alone (Akk), or heat-sterilized Akkermansia muciniphila and Parabacteroides species (hk-AkkPb) (left figure). n=8. [Figure 6D] This study shows the regression of the KD microbiome and KD-related seizure protection in response to a control diet. It shows the seizure threshold in response to 6Hz stimulation in SPF mice that were force-fed either vehicle or Akkermansia muciniphila, P. meldae, and P. distasonis (AkkPb) for 4 days. n=6, 7, 7, 7. Data are presented as mean ± sem. One-way ANOVA using Bonferroni: *P<0.05, **P<0.01, ****P<0.0001. ns = not statistically significant. SPF = specific pathogen elimination (normal colonization state), CD = control diet, KD = ketogenic diet, KD-CD = KD for 14 days followed by CD for 14 days. CC50 = Current intensity that causes seizures in 50% of tested mice, veh = Vehicle, Akk = Ackermansia muciniphila, AkkPb = A. muciniphila, P. meldae and P. distasonis, hk-AkkPb = Heat-sterilized A. muciniphila, P. meldae and P. distasonis. [Figure 7A] This study demonstrates that KD-associated bacteria mediate protection against tonic-clonic seizures in response to a ketogenic diet. We present principal coordinate analysis (PCoA) of the weighted UniFrac distance matrix based on 16S rDNA profiling of feces from Kcna1- / - mice fed CD or KD for 14 days. n=5 cages / group. [Figure 7B] This study demonstrates that KD-related bacteria mediate protection against tonic-clonic seizures in response to a ketogenic diet. The relative abundances of Ackermansia muciniphila and Parabacteroides species from fecal 16S rDNA sequencing data are shown (right figure). n=5 cages / group. [Figure 7C]This demonstrates that KD-associated bacteria mediate protection against tonic-clonic seizures in response to a ketogenic diet. A representative EEG trace showing the stages used to define the seizures quantified in Figure 7D is shown. [Figure 7D] This study demonstrates that KD-associated bacteria mediate protection against tonic-clonic seizures in response to a ketogenic diet. The average daily number of seizures (left figure) and total daily seizure duration (right figure) are shown in SPF Kcna1- / - mice treated with vehicle or Abx, either colonized with A. muciniphila and Parabacteroides or left uncolonized, and given CD or KD. n=2, 8, 6, 12, 9, 3. [Figure 7E] This study demonstrates that KD-associated bacteria mediate protection against tonic-clonic seizures in response to a ketogenic diet. The graph shows the mean number of seizures per day (left), mean duration per seizure (center), and total seizure duration per day (right) in SPF CD Kcna1- / - mice treated with GGsTop. Data for SPF CD mice are the same as in (D). n=6, 4. Data are presented as mean ± sem. Analysis was performed using the Kruskal-Wallis method with Bonferroni (A, B), a nonparametric one-way nested ANOVA with Dunn (D), and a nonparametric Kolmogorov-Smirnov t-test (E). SPF = Specific pathogen elimination (normal colonization state), CD = control diet, KD = ketogenic diet, veh = vehicle, Abx = pre-treatment with antibiotics (ampicillin, vancomycin, neomycin, metronidazole [AVNM]), AkkPb = A. muciniphila, P. meldae and P. distasonis. [Figure 8A] This study demonstrates the association between reduced peripheral gamma-glutamyl amino acid levels and elevated hippocampal GABA / glutamate ratios with diet- and microbiome-dependent seizure protection. Principal component analyses of colonic lumen metabolites (upper figure) and serum metabolites (lower figure) from SPF mice given CD, SPF mice given KD, Abx-treated mice given KD, and AkkPb-immobilized mice given KD are shown. n=8 cages / group. [Figure 8B]This study demonstrates the association between reduced peripheral gamma-glutamyl amino acids and increased hippocampal GABA / glutamate ratios with diet- and microbiome-dependent seizure protection. It also shows levels of gamma-glutamyl amino acids and cysteine ​​in the colonic lumen contents of SPF mice given CD, SPF mice given KD, Abx-treated mice given KD, and AkkPb-immobilized mice given KD. n=8 cages / group. [Figure 8C] This study demonstrates the association between reduced peripheral gamma-glutamyl amino acids and increased hippocampal GABA / glutamate ratios with diet- and microbiome-dependent seizure protection. Serum levels of gamma-glutamyl amino acids and glutamine are shown from SPF mice given CD, SPF mice given KD, Abx-treated mice given KD, and AkkPb-immobilized mice given KD. n=8 cages / group. [Figure 8D] This study demonstrates the association between reduced peripheral gamma-glutamyl amino acid levels and increased hippocampal GABA / glutamate ratios with diet- and microbiome-dependent seizure protection. It shows GABA / glutamate (left figure) and glutamine (right figure) levels in the hippocampus of SPF mice given CD, SPF mice given KD, Abx-treated mice given KD, and AkkPb-immobilized mice given KD. n=5. Data are presented as mean ± sem. Two-way ANOVA comparison (Figures 8A-8C), one-way ANOVA using Bonferroni (Figure 8D): *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns = not statistically significant. CD = control diet, KD = ketogenic diet, SPF = specific pathogen elimination (normal colonization state), veh = vehicle, Abx = pre-treatment with antibiotics (ampicillin, vancomycin, neomycin, metronidazole [AVNM]), AkkPb = A. muciniphila, P. meldae and P. distasonis, au = arbitrary unit. [Figure 9A] This study illustrates the regulation of the colonic lumen and serum metabolome by a ketogenic diet and microbiome status. It shows the number of statistically significant metabolite changes among the 622 metabolites detected in the colonic lumen and the 670 metabolites detected in serum. Values ​​in black text represent the total number of changed metabolites; green values ​​indicate upregulation, and red values ​​indicate downregulation. n=8 cages / group. [Figure 9B] This study illustrates the regulation of the colon lumen and serum metabolome by a ketogenic diet and microbiome status. The glucose levels (left panel) and BHB levels (right panel) detected by metabolomics screening of the colon lumen (top panel) and serum (bottom panel) are shown. n=8 cages / group. [Figure 9C] This study demonstrates the regulation of the colon lumen and serum metabolome by ketogenic diet and microbiome status. It shows the levels of non-gamma glutamylated amino acids in the colon lumen contents of SPF mice given CD, SPF mice given KD, Abx-treated mice given KD, and AkkPb-immobilized mice given KD. n=8 cages / group. [Figure 9D] This study demonstrates the regulation of the colon lumen and serum metabolome by ketogenic diet and microbiome status. It shows levels of non-gamma-glutamylated amino acids in serum from SPF mice given CD, SPF mice given KD, Abx-treated mice given KD, and AkkPb-immobilized mice given KD. n=8 cages / group. Data are presented as mean ± sem. Two-way ANOVA comparison: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns = not statistically significant. CD = control diet, KD = ketogenic diet, SPF = specific pathogen elimination (normal colonization state), veh = vehicle, Abx = pre-treatment with antibiotics (ampicillin, vancomycin, neomycin, metronidazole [AVNM]), AkkPb = A. muciniphila, P. meldae and P. distasonis, au = arbitrary unit, BHB = beta-hydroxybutyrate. [Figure 10A] This study demonstrates that a ketogenic diet and bacterial nutritional symbiosis reduce gamma-glutamyl transpeptidase (GGT) activity, providing sufficient seizure protection. The left figure shows the 6Hz seizure threshold in SPF mice given CD, in response to forced oral administration of the GGT inhibitor, GGsTop. n=6, 9. The right figure shows the behavior of seizure test mice. The dashed line at y=10 seconds represents the seizure scoring threshold, and the triangle at 24mA represents the onset current in the experimental cohort. n=16. [Figure 10B]This study demonstrates that a ketogenic diet and bacterial nutritional symbiosis reduce gamma-glutamyl transpeptidase (GGT) activity and provide sufficient seizure protection. The left figure shows the 6Hz seizure threshold in Abx-treated SPF mice enriched with A. muciniphila and Parabacteroides, in response to ketogenic amino acid supplementation. n=5, 6. The right figure shows the behavior of seizure test mice. The dashed line at y=10 seconds represents the seizure scoring threshold, and the triangle at 24mA represents the onset current in the experimental cohort. n=12. [Figure 10C] This study demonstrates that a ketogenic diet and bacterial nutrient symbiosis reduce gamma-glutamyl transpeptidase (GGT) activity, providing significant seizure protection. The left panel shows total GGT activity per 100 mg of feces from SPF CD, SPF KD, AkkPb KD, or AkkPb CD mice, and the right panel shows inhibition by GGsTop. n=5. [Figure 10D] This study demonstrates that a ketogenic diet and bacterial symbiosis reduce gamma-glutamyl transpeptidase (GGT) activity, providing significant seizure protection. The images show total GGT activity per 100 mg of feces (left) and inhibition by GGsTop (right) from SPF CD mice treated bi-daily for 28 days with vehicle, A. muciniphila and Parabacteroides probiotics, or heat-sterilized bacteria. n=5. [Figure 10E] This study demonstrates that a ketogenic diet and bacterial nutrient symbiosis reduce gamma-glutamyl transpeptidase (GGT) activity, providing sufficient protection against seizures. The levels of live A. muciniphila (Akk) after incubation in KD culture medium for C. chlorophyll (CD) or in CD or KD agar layered with (0)M9 minimal medium containing or without live Parabacteroides meldae (PbM). n=3. [Figure 10F] This study demonstrates that a ketogenic diet and bacterial nutrient symbiosis reduce gamma-glutamyl transpeptidase (GGT) activity, providing sufficient protection against seizures. The levels of live PbM after incubation in M9 minimal medium overlaid on (0)CD or KD agar, with or without Akk, are shown. n=5. [Figure 10G] This study demonstrates that a ketogenic diet and bacterial nutrient symbiosis reduce gamma-glutamyl transpeptidase (GGT) activity, providing sufficient protection against seizures. It shows GGT activity at t=24 hours and inhibition of GGT activity by GGsTop in P. meldae grown in M9 medium overlaid on CD agar with or without A. muciniphila. n=5. [Figure 10H] This study demonstrates that a ketogenic diet and bacterial nutrient symbiosis reduce gamma-glutamyl transpeptidase (GGT) activity, providing sufficient protection against seizures. It shows GGT activity at t=24 hours and inhibition of GGT activity by GGsTop in P. meldae grown in M9 medium overlaid on KD agar with or without A. muciniphila. n=5. Data are shown as mean ± sem. Student's t-test (Figures 10A-10B), two-way ANOVA using Bonferroni (Figures 10C-10D), one-way ANOVA using Bonferroni (Figures 10E-10H): **P<0.01, ***P<0.001, ****P<0.0001. SPF = SPF (Specific Pathogen Removal) (normal colonization state), CD = Control diet, KD = Ketogenic diet, CC50 = Current intensity that induces seizures in 50% of tested mice, AA = Amino acids, veh = Vehicle, Abx = Pre-treatment with antibiotics (ampicillin, vancomycin, neomycin, metronidazole [AVNM]), AkkPb = A. muciniphila, P. meldae and P. distasonis, GGsTop = GGT inhibitor, PbM = Parabacteroides meldae, Akk = Akkermansia muciniphila, M9 = Minimal medium, GGT = Gamma glutamyl transpeptidase, AU = Absorbance unit. [Figure 11A] This study demonstrates the effects of amino acids on seizures, bacterial GGT activity, and dietary regulation of bacterial genes related to amino acid metabolism. GGT activity is shown in conventionally cultured Parabacteroides meldae (left panel) and A. muciniphila (right panel) treated with GGsTop or vehicle. n=5. [Figure 11B]This study shows the effects of amino acids on seizures, bacterial GGT activity, and dietary regulation of bacterial genes related to amino acid metabolism. It also shows the levels of A. muciniphila (Akk) after 0, 7, or 24-hour incubation in CD agar overlaid with P. meldae (PbM) pretreated with vehicle in M9 minimal medium or GGsTop. n=3. Data are shown as mean ± sem. One-way ANOVA using Bonferroni (Figure 11A): *P<0.05, Two-way ANOVA using Bonferroni (Figure 11B): **P<0.01, PbM=Parabacteroides meldae, Akk=Akkermansia muciniphila, GGsTop=GGT inhibitor, GGT=Gamma-glutamyl transpeptidase, AU=Absorbance unit. [Figure 12] As detailed in Example 8 below, a non-limiting schematic diagram of a dosage regimen for administering Ackermansia muciniphila and Parabacteroides meldae to human subjects is shown. [Figure 13] As detailed in Example 8 below, a non-limiting schematic diagram of the quantitative determination of bacteria present in human subjects over a 6-week period according to the administration plan is shown. [Figure 14A] As detailed in Example 8 below, this shows a non-limiting, constant amount of bacteria present in human subjects over a 6-week period according to the administration plan. The linear relative abundance of bacteria is shown over time. [Figure 14B] As detailed in Example 8 below, this shows a non-limiting, constant amount of bacteria present in human subjects over a 6-week period according to the administration plan. The concentric logarithmic ratio of bacterial abundance is shown over time. [Figure 15] As detailed in Example 16 below, a non-limiting schematic diagram of a high-dose human control cohort used for fecal sample collection followed by fecal transplantation into germ-free mice is shown. [Figure 16] As detailed in Example 17 below, a non-limiting schematic diagram of an experimental protocol for collecting fecal samples from healthy humans, transferring them to germ-free mice on day 0, and testing seizure protection on day 4 is shown. [Figure 17A]As detailed in Example 9 below, this shows a non-limiting amount of seizure protection in mice following fecal bacterial transplantation. It shows the percentage of mice that exhibited seizures at a fixed current of 32 mA. [Figure 17B] As detailed in Example 9 below, this shows a non-limiting amount of seizure protection in mice following fecal bacterial transplantation. CC50 in mA is shown for mice either before or after fecal transplantation. [Figure 18] Following fecal transplantation, we present a non-limited quantitative analysis of bacteria present in a mouse. [Modes for carrying out the invention]

[0013] Methods and probiotic compositions for suppressing appetite in a subject are provided herein. In some embodiments, the method involves applying at least 1 × 10 to the subject. 11 Colony-forming units (CFUs) of Akkermansia (Akk) fungi, and at least 1 × 10⁻⁶ 10 The process includes administering CFU-derived Parabacteroides (Pb) bacteria.

[0014] Methods and compositions for mimicking the effects of a ketogenic diet by administration of probiotic compositions are also provided herein. In certain embodiments, the methods and compositions are intended for the treatment or prevention of seizures in subjects (e.g., subjects having neurodevelopmental disorders, e.g., autism spectrum disorder, Rett syndrome, fragile X, attention deficit disorder (ADD), attention deficit hyperactivity disorder (ADHD), intractable epilepsy, and / or intractable epilepsy). In other embodiments, the methods and compositions are intended for the prevention or treatment of conditions in subjects (e.g., autism spectrum disorder, epilepsy, seizures, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, metabolic disorders (e.g., obesity or diabetes), mitochondrial diseases, depression, migraines (e.g., chronic migraines), Rett syndrome, attention deficit disorder, fragile X syndrome, or traumatic brain injury (TBI)). In preferred embodiments, the method includes administering to a subject a composition comprising bacteria of the genus Akkermansia (e.g., Akkermansia muciniphila) and the genus Parabacteroides (e.g., Parabacteroides meldae, Parabacteroides johnsonii, or Parabacteroides distasonis), or a plurality of compositions comprising bacteria of the genus Akkermansia (e.g., Akkermansia muciniphila) and the genus Parabacteroides (e.g., Parabacteroides meldae, Parabacteroides johnsonii, or Parabacteroides distasonis) together. In some embodiments, the method and composition are intended for the treatment of neurological disorders or disorders in the subject. In some embodiments, the method and composition are intended for the treatment of Dravet syndrome and / or amyotrophic lateral sclerosis (ALS) in the subject requiring treatment. In some embodiments, the method and composition suppress appetite in the subject. In some embodiments, the method and composition are part of a weight loss program. In some embodiments, the methods and compositions are intended to induce ketosis in a subject. In other embodiments, the methods and compositions alter neurotransmitter biosynthesis in a subject. In specific embodiments, the methods and compositions alter serum ketogenic amino acids in a subject. In other embodiments, the methods and compositions reduce gamma-glutamyl transpeptidase activity in a subject.In certain embodiments, the method and composition reduce glutamine synthase activity in a subject. In other embodiments, the method and composition reduce gamma-glutamyl amino acid levels in a subject. In certain embodiments, the method and composition increase the GABA / glutamate ratio in a subject. In other embodiments, the method and composition increase glutamine levels in a subject. In other embodiments, the method includes the steps of removing the intestinal microbiota of a subject and administering to the subject a composition containing bacteria of the genera Akkermansia and Parabacteroides, or a plurality of compositions containing Akkermansia and Parabacteroides together.

[0015] One embodiment of the composition disclosed herein, "BL-001," was initially designed to replicate the antiepileptic effects of a ketogenic diet that modulates gamma-aminobutyric acid (GABA) and other major bioenergy pathways. BL-001 contains two reasonably selected human intestinal microorganisms that have been shown in both cell-based assays and animal studies to eliminate hyperexcitatory activity, increase hippocampal GABA, and significantly reduce or eliminate both seizure frequency and duration.

[0016] BL-001 aims to potentially slow or halt disease progression by focusing on underlying oxidative stress, a decisive factor in ALS progression. In preclinical ALS trials, BL-001 has been shown to attenuate motor neuron loss and enhance lifespan and motor coordination. BL-001 is also believed to influence the gut-brain axis in the subjects.

[0017] definition As used herein, “a” or “an” may mean one or more. As used herein in the claims, the words “a” or “an,” when used in conjunction with the word “comprising,” may mean one or more. As used herein, “another” may mean at least one more.

[0018] As used herein, the expression “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and does not harm the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and acetylcellulose; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, e.g., cocoa butter and suppository wax; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and (10) Soybean oil; (11) Glycols, e.g., propylene glycol; (12) Polyols, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (13) Esters, e.g., ethyl oleate and ethyl laurylate; (14) Agar; (15) Buffers, e.g., magnesium hydroxide and aluminum hydroxide; (16) Alginic acid; (17) Pyrogen-free water; (18) Isotonic saline solution; (19) Ringer's solution; (20) Ethyl alcohol; (21) Phosphate buffer; and (21) Other non-toxic and suitable substances used in pharmaceutical formulations.

[0019] The term “prevent” is approved in the field and, when used in relation to a condition, for example, local recurrence, is well understood in the field, and includes the administration of a composition that reduces the frequency of symptoms of a medical condition in a subject compared to a subject not administered with the composition. Accordingly, prevention of seizures includes, for example, a reduction in the number of seizures in a group of patients treated with prophylactic treatment compared to an untreated control group, and / or a delay in the appearance of detectable lesions in the treated group compared to an untreated control group, by a statistically and / or clinically significant amount.

[0020] The terms “preventive” or “therapeutic” treatments are approved in the art and involve the administration of one or more subject compositions to a host. If a subject composition is administered prior to the clinical manifestations of an undesirable condition (e.g., disease or other undesirable condition in a host animal), the treatment is preventive (i.e., protects the host from the undesirable condition); if administered after the onset of an undesirable condition, the treatment is therapeutic (i.e., intended to reduce, improve or stabilize an existing undesirable condition or its side effects).

[0021] The term "subject" refers to, but is not limited to, mammals, including humans and non-human mammals, such as cattle, horses, dogs, sheep, or cats.

[0022] The “therapeutically effective amount” of a compound in the subject treatment method refers to the amount of the compound in a preparation that, when administered (to mammals, preferably humans) as part of a desired administration plan, alleviates symptoms, improves a condition, or slows the onset of a disease state in accordance with clinically acceptable criteria for the disorder or condition to be treated, or for cosmetic purposes, for example, in a reasonable benefit / risk ratio applicable to any medical treatment.

[0023] As used herein, the terms “treating” or “treatment” include reducing, mitigating, or preventing the underlying pathology of symptoms, clinical signs, and conditions in a manner that improves or stabilizes the condition in question.

[0024] Treatment method This disclosure relates, in part, to the discovery that a ketogenic diet (KD) induces substantial changes in the gut microbiome, and to the discovery that the enrichment of KD-related bacteria through probiotic administration, fecal transplantation, or selective microbial reconstruction of the natural microbiome mimics the beneficial effects of KD. Methods and compositions that can substitute for a KD diet in the treatment and prevention of the conditions described herein are provided herein. The methods and compositions described herein may be used separately or in combination with a KD diet in the treatment or prevention of the conditions described herein.

[0025] In certain embodiments, the method treats or prevents seizures in a subject. In some embodiments, the method includes the step of administering a composition containing bacteria of the genera Ackermansia and Parabacteroides. In other embodiments, the method alters neurotransmitter biosynthesis in a subject. In certain embodiments, the method alters serum ketogenic amino acids in a subject. In other embodiments, the method reduces gamma-glutamyl transpeptidase activity in a subject. In certain embodiments, the method reduces glutamine synthase activity in a subject. In other embodiments, the method reduces gamma-glutamyl amino acids in a subject. In certain embodiments, the method increases the GABA / glutamate ratio in a subject. In other embodiments, the method increases glutamine levels in a subject. In other embodiments, the method provided herein includes the steps of removing the target intestinal microbiota and administering to the target a composition comprising bacteria of the genera Ackermansia (e.g., Ackermansia muciniphylla) and Parabacteroides (e.g., Parabacteroides meldae, Parabacteroides johnsonii, or Parabacteroides distasonis). In some embodiments, the target has epilepsy (e.g., refractory or intractable epilepsy). In some embodiments, the target has a neurodevelopmental disorder. Typical neurodevelopmental disorders include autism spectrum disorder, Rett syndrome, fragile X, attention deficit disorder, and attention deficit hyperactivity disorder. In some embodiments, the neurodevelopmental disorder is a disorder known to be associated with seizures.

[0026] In other embodiments, the subject has a condition that responds to a ketogenic diet. This condition may be Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, metabolic disorders, mitochondrial diseases, depression, migraines (e.g., chronic migraines), or traumatic brain injury (TBI). In some embodiments, the methods and compositions include the step of administering the compositions provided herein to the subject. In some embodiments, the condition may be epilepsy, seizures, autism spectrum disorder, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), cancer, stroke, metabolic disorders (e.g., obesity or diabetes), mitochondrial diseases, depression, migraines (e.g., chronic migraines), Rett syndrome, attention deficit disorder, fragile X syndrome, or traumatic brain injury (TBI). In some embodiments, the compositions and methods provided herein are useful in treating or preventing aging or age-related conditions. In some embodiments, the compositions and methods provided herein can substitute for a ketogenic diet in the treatment or prevention of the conditions described herein, and in other embodiments, the compositions and methods provided herein can be used in combination with a ketogenic diet. Further information regarding the conditions can be found in Stafstrom et al. (2012) Front. Pharmacol. 3:59 (Non-Patent Literature 1), which is incorporated herein in its entirety.

[0027] The compositions may be formulated for oral delivery. In some embodiments, the compositions may contain probiotics. In some embodiments, the compositions disclosed herein are food products. The compositions may be in the form of pills, tablets, or capsules. In some embodiments, the target may be a mammal (e.g., human). In some embodiments, the compositions are self-administered. A single composition containing all the bacteria to be administered is preferred, but it is recognized that, with respect to any of the various embodiments described herein, combinations of bacteria may be administered in multiple compositions containing the combinations of bacteria together. For example, the present invention further provides a kit comprising multiple compositions containing bacteria of the genera Akkermansia (e.g., Akkermansia muciniphila) and Parabacteroides (e.g., Parabacteroides meldae, Parabacteroides johnsonii, or Parabacteroides distasonis) together.

[0028] In some embodiments, the composition is formulated for rectal delivery (e.g., fecal sample). In some embodiments, the subject receives a fecal microbiota transplant containing the composition disclosed herein. Fecal microbiota transplantation (FMT), also commonly known as "fecal bacteriotherapy," is a therapeutic protocol that enables the reconstruction of the colonic microbiome. The method involves the transplantation of fecal bacteria from a healthy individual to a recipient. FMT restores the intestinal flora by introducing a healthy bacterial flora obtained from a healthy donor, for example, through the injection of a fecal sample via enema, an oral gastric tube, or orally in the form of a capsule containing lyophilized material. In some embodiments, the fecal sample is from a fecal bank.

[0029] In some embodiments, bacterial DNA in the target gut microbiota is sequenced. The target gut bacterial DNA may be sequenced prior to administration of the composition. For example, a sample containing bacterial DNA may be obtained from the target, and the bacterial DNA is then sequenced with respect to Akkermansia (Akk) and / or Parabacteroides DNA, thereby determining the presence or level of Akkermansia and / or Parabacteroides in the target gut microbiota. The compositions disclosed herein may then be administered to the target if the levels of Akkermansia and / or Parabacteroides are low. In some embodiments, a subject is considered to have low levels of Akkermansia and / or Parabacteroides if less than 0.0001%, less than 0.001%, less than 0.01%, less than 0.02%, less than 0.03%, less than 0.04%, less than 0.05%, less than 0.06%, less than 0.07%, less than 0.08%, less than 0.09%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 5%, less than 7%, less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the bacteria in the sample are Akkermansia and / or Parabacteroides DNA. The bacterial DNA to be sequenced may be obtained by any means known in the art, including, but not limited to, obtaining a fecal sample from the subject and isolating the bacterial DNA. Bacterial DNA sequencing by any known technique in the art includes, but is not limited to, the Maxam-Gilbert method, Sanger method, shotgun method, bridge PCR, or next-generation sequencing methods, such as large-scale parallel signature sequencing (MPSS), Polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single-molecule sequencing, single-molecule real-time (SMRT) sequencing, or nanopore DNA sequencing.

[0030] In some embodiments, the method described above acts directly to reduce the amount of pathogenic bacteria in the subject (i.e., in the gastrointestinal tract of the subject). In some embodiments, it includes any such therapy that achieves the same objective of reducing the number of pathogens, which, when used in combination with the compositions described herein, would result in the substitution of pathogenic microflora involved in a disease state with natural microflora associated with a non-disease state, or less pathogenic species that occupy the same ecological niche as the types that cause the disease state. For example, the subject may be treated with an antibiotic (e.g., an antimicrobial compound) or a composition containing an antibiotic to target and reduce the spread of pathogens, and then treated with the compositions described herein. The treatment may also include antifungal or antiviral compounds.

[0031] Suitable antimicrobial compounds include capreomycin, including capreomycin IA, capreomycin IB, capreomycin IIA, and capreomycin IIB; carbomycin, including carbomycin A; carmonam; cefaclor, cefadroxil, cefamandol, cefatoridine, cefazedone, cefazolin, cefbuperazone, cefcapene pivoxil, cefclizine, cefdinir, cefditoren, cefim, ceftamet, cefmenoxime, cefmetazole, cefminox, cefozidime, cefonisid, cef Pellazone, cefolanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefpimisole, cefpyramide, cefpirom, cefprodil, ceffloxazine, cefsulodine, ceftazidime, cefteram, ceftezol, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, ceffuroxime, cefzonam, cephalexin, cephaloglysin, cephaloridine, cephalosporin C, cephalothin, cefapillin, cefamicin C, cefamicin, cefradiin, chlortetracycline;Clarithromycin, clindamycin, clometocillin, chromocycline, cloxacillin, cyclacillin, danofloxacin, demeclocycline, destomycin A, dicloxacillin, zithromycin, doxycycline, epicillin, erythromycin A, ethambutol, fenbenicillin, flomoxef, florfenicol, floxacillin, flumequin, forthymicin A, forthymicin B, fosfomycin, flaltadone, fusidic acid, gentamicin, glyconiazid, guamecycline, hetacillin, ida Rubicin, imipenem, isepamycin, josamycin, kanamycin, rhumycin (like rhumycin 1), lincomycin, lomefloxacin, loracalbef, rimescycline, meropenam, methampicillin, metacycline, methicillin, mezlocillin, micronomycin, midekamicin (like midekamicin A1), mikamicin, minocycline, mitomycin (like mitomycin C), moxalactam, mupirocin, nafcillin, netiricin, norcardian (like norcardian A), oleandmycin , oxytetracycline, panipenum, pazufloxacin, penamecillin, penicillin G, penicillin N and penicillin-like penicillins, penylic acid, pentylpenicillin, peplomycin, pheneticillin, pipaciclin, piperacillin, pirurimycin, pivampicillin, pibcephalexin, porphyromycin, propialin, quinacillin, ribostamycin, rifabutin, rifamide, rifampin, rifamycin SV, rifapentin, rifaximin, litipenem, rekitamycin, lolitetracycline, ro This includes salamicin, roxithromycin, sancycline, shisomycin, sparfloxacin, spectinomycin, streptozocin, sulbenicillin, sultamicillin, tarampicillin, teicoplanin, temocillin, tetracycline, tostrepton, thiamrin, ticarcillin, tigemonam, tilmicosin, tobramycin, tropospectromycin, trovafloxacin, tyrosine, and vancomycin, as well as their analogues, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms.

[0032] Suitable antifungal compounds include ketoconazole, miconazole, fluconazole, clotrimazole, undecylenic acid, sertaconazole, terbinafine, butenafine, cryoquinol, haloprozin, nystatin, naphthifine, tolnaphthate, cyclopirox, amphotericin B, or tea tree oil, as well as their analogues, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms.

[0033] Appropriate antiviral agents include acyclovir, azidouridine, anisomycin, amantadine, bromovinyldeoxusidine, chlorovinyldeoxusidine, cytarabine, delaviridine, didanosine, deoxynojirimycin, dideoxycytidine, dideoxyinosine, dideoxynucleoside, desciclovir, deoxyacyclovir, efavirenz, enviroxime, phiacitabine, foscarnet, phialuridine, and fluorothymidine. This includes phloxuridine, ganciclovir, hypericin, idoxuridine, interferon, interleukin, isethionate, nevirapine, pentamidine, ribavirin, rimantadine, stabudine, salglamostim, suramin, tricosanthine, tribromothymidine, trichlorothymidine, trifluorothymidine, trisodium phosphonoformate, vidarabine, zidovudine, zalcitabine, and 3-azido-3-deoxythymidine, as well as their analogues, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms.

[0034] Other suitable antiviral agents include 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG), 2',3'-dideoxycytidine (ddC), 2',3'-dideoxythymidine (ddT), 2'3'-dideoxy-dideoxythymidine (d4T), 2'-deoxy-3'-thiacytosine (3TC or lamivudine), 2',3'-dideoxy-2'-fluoroadenosine, and 2',3'-dideoxy-2'-fluoroidenosine. These include 2',3'-dideoxy-2'-fluorothymidine, 2',3'-dideoxy-2'-fluorocytosine, 2'3'-dideoxy-2',3'-didehydro-2'-fluorothymidine (Fd4T), 2'3'-dideoxy-2'-beta-fluoroadenosine (F-ddA), 2'3'-dideoxy-2'-beta-fluoroinosine (F-ddI), and 2',3'-dideoxy-2'-beta-fluorocytosine (F-ddC). In some embodiments, the antiviral agent is selected from trisodium phosphonoformate, ganciclovir, trifluorothymidine, acyclovir, 3'-azido-3'-thymidine (AZT), dideoxyinosine (ddI), and doxuridine, as well as their analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms.

[0035] composition In some embodiments, the present invention relates to compositions (e.g., food or pharmaceutical compositions) comprising bacteria of the genera Akkermansia (Akk) and Parabacteroides (Pb). The compositions may include a pharmaceutically acceptable carrier. The compositions may include probiotics. The pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including oral, buccal, sublingual, parenteral, and rectal, in the form of powders, ointments, intravenous infusions, liquids, gels, tablets, capsules, pills, or creams. In certain embodiments, the pharmaceutical compositions are delivered systemically (e.g., by oral administration). In certain other embodiments, the compositions disclosed herein are delivered rectally.

[0036] In certain embodiments, the present invention provides a kit comprising multiple compositions that together contain bacteria of the genera Akkermansia (e.g., Akkermansia muciniphylla) and Parabacteroides (e.g., Parabacteroides meldae, Parabacteroides johnsonii, or Parabacteroides distasonis) (for example, that when combined, would result in compositions described somewhere in this section).

[0037] The composition may, but is not limited to, contain any of the species of Parabacteroides, including P. chartae, P. chinchillae, P. distasonis, P. faecis, P. goldsteinii, P. gordonii, P. johnsonii, or P. meldae. In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, 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%, or at least 95% of the bacteria in the composition are Parabacteroides (Pb) bacteria. The Akkermansia bacteria in the composition may include Akkermansia muciniphila. In some embodiments, the compositions disclosed herein may contain at least 1%, at least 5%, at least 10%, at least 15%, 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%, or at least 95% of Akkermansia bacteria.

[0038] In some embodiments, the composition comprises a ratio of Akkermansia to Parabacteroides of 6:1, 6:2, 6:3, 6:4, 6:5, 6:6, 1:6, 2:6, 3:6, 4:6, or 6:6. In some embodiments, the composition comprises a ratio of Akkermansia to Parabacteroides of 6:2. In some embodiments, Akkermansia is present in the composition in a quantity of about 1 × 10⁻⁶. 5 , 5×10 5 , 1 x 10 6 , 5×10 6 , 1 x 10 7 , 5×10 7 , 1 x 10 8 , 5×10 8 , 1 x 10 9 , 5×10 9 , 1 x 10 10 , 5×10 10 , 1 x 10 11 , 5×10 11 , 1 x 10 12 , 5×10 12 , 1 x 10 13 , 5×10 13 , 1 x 10 14 , 5×10 14 , 1 x 10 15 , 5×10 15 , 1 x 10 20 , 5×10 20 In terms of colony-forming units (CFU), or 1 × 10⁻⁶ 5 From 5x10 20 It exists in any abundance between the colony-forming units (CFUs).

[0039] The compositions described herein may be used for oral administration to the gastrointestinal tract, with the aim of achieving the introduction of bacteria (e.g., the bacteria disclosed herein) into the tissues of the gastrointestinal tract. Formulations for the compositions of the present invention (e.g., probiotic compositions) may also include other probiotic agents or nutrients that promote spore germination and / or bacterial growth. One exemplary material is a bifidobacteria-promoting oligosaccharide that promotes the growth of beneficial probiotic bacteria. In some embodiments, the probiotic bacteria are administered together with a therapeutically effective dose of an antibiotic or antifungal agent (preferably a broad-spectrum agent). In some embodiments, the compositions described herein are encapsulated in enteric-coated sustained-release capsules or tablets. The enteric coating allows the capsule / tablet to remain intact (i.e., undissolved) for a certain period of time and / or until it reaches a specific portion of the GI tubule (e.g., the small intestine) as it passes through the gastrointestinal tract. The sustained-release component prevents the “release” of the probiotic bacteria in the compositions described herein over a predetermined period of time.

[0040] The composition may be a food product, for example, a dairy product, but is not limited to that. The dairy product may be fermented or unfermented (e.g., milk) dairy product. Non-limited examples of fermented dairy products include yogurt, cottage cheese, sour cream, kefir, buttermilk, etc. Dairy products also frequently contain various specialized dairy ingredients, such as whey, dried skim milk, whey protein concentrate, etc. Dairy products may be processed in any way known in the art to achieve desired qualities, such as flavor, thickening power, nutrition, specific microorganisms, and other properties, such as mold growth control. The composition of the present invention may also contain known antioxidants, buffers, other agents, such as colorants, flavorings, vitamins, or minerals.

[0041] In some embodiments, the compositions of the present invention are used in combination with a carrier (e.g., a pharmaceutically acceptable carrier) that is physiologically compatible with the gastrointestinal tissue to be administered. The carrier may consist of a solid-based dry material for formulation into tablets, capsules, or powders, or it may consist of a liquid or gel-based material for formulation into liquids or gels. The specific type of carrier and final formulation depends in part on the chosen route of administration. The therapeutic compositions of the present invention may also include various carriers and / or binders. In some embodiments, the carrier is microcrystalline cellulose (MCC) added in an amount sufficient to produce a total dose weight of 1 gram. The carrier may be a solid-based dry material for formulations in tablet, capsule, or powder form, or a liquid or gel-based material for formulations in liquids or gels, and their forms depend in part on the route of administration. Typical carriers for dry formulations include, but are not limited to, trehalose, maltodextrin, rice flour, microcrystalline cellulose (MCC), magnesium stearate, inositol, FOS, GOS, dextrose, sucrose, and similar carriers. Suitable liquid or gel-based carriers include, but are not limited to, water and physiological saline; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); and glycols (e.g., ethylene glycol, propylene glycol, etc.). Preferably, water-based carriers have a neutral pH value (i.e., pH 7.0). Other carriers or agents for administration of the compositions described herein are known in the art, for example, in U.S. Patent No. 6,461,607 (Patent Document 1).

[0042] In some embodiments, the composition further comprises other bacteria or microorganisms known to colonize the digestive tract. For example, the composition may include species belonging to the phyla Firmicutes, Proteobacteria, Tenericutes, Actinobacteria, or combinations thereof. Further examples of bacteria and microorganisms that may be included in the subject composition include, but are not limited to, Saccharomyces, Bacteroides, Eubacterium, Clostridium, Lactobacillus, Fusobacterium, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, and Staphylococcus and Peptostreptococcus. In certain embodiments, the composition is substantially free of bacteria that increase the risk of seizures or otherwise impair the effects of the ketogenic diet. Such bacteria include Bifidobacteria. Accordingly, in some embodiments, the composition is substantially free of Bacteroides. The composition is substantially free of a particular bacterial type if that bacterial type constitutes less than 10%, preferably less than 5%, more preferably less than 1%, most preferably less than 0.5%, or even 0% of the bacteria in the composition.

[0043] In some embodiments, the composition comprises a fecal sample containing at least one Akkermansia (Akk) and at least one Parabacteroides (Pb). In some embodiments, the fecal sample is from a fecal bank. In some embodiments, the composition may be added to the fecal sample prior to administration to the subject.

[0044] In some embodiments, methods for treating or preventing a condition, such as a seizure, are provided herein, which involve administering a composition (e.g., a fecal sample) containing at least one Akkermansia (Akk) and at least one Parabacteroides (Pb) to a subject. A fecal sample is concentrated if at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, or 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 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the bacteria in the fecal sample are Akkermansia (Akk). In some embodiments, the fecal sample is concentrated if at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, or 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 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the bacteria in the fecal sample are Parabacteroides (Pb). In some embodiments, the fecal sample is from a fecal bank. In some embodiments, the fecal sample is from a donor.

[0045] The composition may further contain nutrients. In some embodiments, the nutrients assist in the growth of bacteria (e.g., bacteria disclosed herein). In some embodiments, the nutrients are components listed in Figure 12. In some embodiments, the nutrients are lipids (e.g., linoleic acid, stearic acid, or palmitic acid). In some embodiments, the nutrients may be administered in conjunction with the compositions disclosed herein. As used herein, “co-administration” refers to any form of administration of two or more different drugs (e.g., the compositions disclosed herein and the nutrients disclosed herein) such that the second drug is administered while the previously administered drug is still effective in the body. For example, the compositions disclosed herein and the nutrients disclosed herein may be administered incidentally or sequentially, either in the same formulation or in separate formulations.

[0046] The actual dose level of the active ingredient in a pharmaceutical composition can be modified to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0047] The selected dose level depends on a variety of factors, including the activity of the specific drug being used; the route of administration, time of administration, elimination rate or metabolic rate of the specific compound being used; the duration of treatment; other drugs, compounds and / or substances used in combination with the specific composition being used; the age, sex, weight, condition, overall health and medical history of the patient being treated; and similar factors well known in the medical field.

[0048] A physician or veterinarian with ordinary skills in the field can easily determine and prescribe the required effective amount of a pharmaceutical composition. For example, a physician or veterinarian may prescribe and / or administer the dose of a compound used in a pharmaceutical composition at a lower level than required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0049] The present invention is described in general terms here and will be more readily understood by referring to the following examples, which are included solely for the purpose of illustrating specific aspects and embodiments of the invention and are not intended to limit the invention. [Examples]

[0050] (Example 1) Materials and methods Animals and food Three-to-four-week-old SPF wild-type Swiss Webster mice (Taconic Farms), GF wild-type Swiss Webster mice (Taconic Farms), and SPF C3HeB / FeJ KCNA1 KO mice (Jackson Laboratories) were reared at the UCLA Health Science Center Barrier Facility. The animals were fed "breeder" diet (Lab Diets 5K52). Experimental animals were fed standard diet (Lab Diet 5010), 6:1 ketogenic diet (Harlan Teklad TD.07797.PWD), or the same vitamin and mineral control diet (Harlan Teklad TD.150300). Young mice were used for three reasons: i) to mimic the typical use of KD for treating pediatric and adolescent epilepsy patients; ii) to align the timing of mouse brain development with early human brain development (neural developmental milestones in 3-week-old mice are comparable to those in 2-3 year-old human brains); and iii) to eliminate pre-weaning feeding treatments, which would confound maternal behavior and maternal physiology. Mice were randomly assigned to experimental groups. All animal experiments were approved by the UCLA Animal Experiments Committee.

[0051] 6Hz psychomotor seizure assay A 6Hz test was performed. Preliminary tests revealed no sexual dimorphism in seizure threshold. All subsequent experimental cohorts included male mice. One drop (approximately 50 μl) of 0.5% tetracaine hydrochloride eye drops was applied to the cornea of ​​each mouse 10–15 minutes before stimulation. Corneal electrodes were coated with a thin layer of electrode gel (Parker Signagel). A constant current device (ECT Unit 57800, Ugo Basile) was used to deliver current with a duration of 3 seconds, a pulse width of 0.2 ms, and a frequency of 6 pulses / s. CC50 (the current intensity required to induce a seizure in 50% of the experimental group) was measured as a criterion for seizure susceptibility. Preliminary experiments identified 24 mA as the CC50 for SPF wild-type Swiss Webster mice. Since each mouse was tested for seizures only once, at least n>6 mice were used to ensure sufficient power for each experimental group. To determine the CC50 for each experimental group, the first mouse in each group per cohort was given a 24 mA current, which was then steadily increased or decreased in 2 mA intervals. During stimulation, the mouse was held down by hand and then released into a new cage for behavioral observation. Motor behavior was recorded using Ethovision XT software (Noldus), and quantitative measurements of falls, caudal dorsiflexion (Straub's tail elevation), forelimb clonus, oculo / triciliac spasms, and behavioral remission were manually scored. For each behavioral parameter, no correlation was found between the percentage incidence during 24 mA seizures and the microbiome state or group seizure susceptibility, suggesting that the microbiome has a major effect on the incidence of seizures, rather than the presentation or form of the seizures. The delay time to exploration (the time elapsed from when the experimental mouse was released into the observation cage (after corneal stimulation) until its first horizontal movement) was manually scored using an electronic timer with Ethovision. Blinding was performed within the diet group. True blinding between different dietary groups was impossible due to the changes in stool color caused by diet. However, preliminary results did not show significant differences between the results obtained from the blinded experimental group and the results obtained from the same experimental group in the open-label experimental group.Mice were scored as protected from seizures if they did not exhibit seizure behavior and resumed normal searching behavior within 10 seconds. The seizure threshold (CC50) was determined for each experimental group using the mean log interval of the current step, with sample n defined as a subset of mice exhibiting low-frequency seizure behavior. The data used to calculate CC50 also shows the delay time to searching for each current intensity, with n representing the total number of biological replicas per group, regardless of seizure outcome.

[0052] Glucose measurement Blood samples were collected via cardiac puncture and spun using an SST vacuum tanker (Becton Dickinson) for serum separation. Glucose levels were detected in serum by a colorimetric assay as per the manufacturer's instructions (Cayman Chemical). Data collected across multiple experiments are expressed as glucose concentrations normalized to the SPF control within each group.

[0053] Beta-hydroxybutyrate (BHB) measurement Blood was collected by cardiac puncture and spun using an SST vacuum tanker (Becton Dickinson) for serum separation. The colon was lavaged and rinsed with PBS to remove luminal contents. The prefrontal cortex, hippocampus, hypothalamus, and cerebellum were microdissected, and the liver was recovered and washed with PBS. Tissue samples were sonicated at 10s intervals on ice in RIPA lysis buffer (Thermo Scientific) at 20mV. BHB levels were detected in serum by a colorimetric assay as per the manufacturer's instructions (Cayman Chemical). Data were normalized to total protein content detected by BCA assay (Thermo Pierce). Data collected across multiple experiments are expressed as BHB concentrations normalized to the SPF control within each group.

[0054] 16S rDNA microbiome profiling Bacterial genomic DNA was extracted from mouse fecal samples or colonic lumen contents using the MoBio PowerSoil kit, where sample n represents an independent cage containing 3 mice per cage. A library was generated. The V4 region of the 16S rDNA gene was PCR amplified using individually barcoded universal primers and 30 ng of the extracted genomic DNA. The PCR reaction was set up in triplicate configuration, and the PCR product was purified using the Qiaquick PCR purification kit (Qiagen). The purified PCR product was pooled at equimolar concentrations quantified using the Kapa Library Quantification Kit (Kapa Biosystems, KK4824), and sequenced by Laragen, Inc. using the Illumina MiSeq platform and 2 × 250 bp reagent kit for paired-end sequencing. Operational taxa units (OTUs) were selected by open reference OTU picking based on 97% sequence similarity to the Greengenes 13_5 database. Taxonomic assignment and rarefaction were performed using QIIME 1.8.0 with 85,134 reads per sample. The metagenomics were estimated from a closed reference OTU table using PICRUSt. The results were filtered to display the top 72 genes related to amino acid metabolism.

[0055] Normalization of microflora Fresh fecal samples were collected from adult SPF Swiss Webster mice and homogenized in 1 ml of pre-reduced PBS per pellet. 100 μl of the precipitated suspension was administered to recipient GF mice by forced oral administration. In mock treatment, mice were force-fed in pre-reduced PBS.

[0056] Antibiotic treatment SPF mice were force-fed with a solution of vancomycin (50 mg / kg), neomycin (100 mg / kg), and metronidazole (100 mg / kg) every 12 hours for 7 days, according to the method previously described by Reikvam et al., PloS one (6), 2011 (Non-Patent Literature 2). Ampicillin (1 mg / ml) was administered via free drinking water. In the mock treatment, mice were force-fed with ordinary drinking water every 12 hours for 7 days. Kcna1 - / - In mice, vancomycin (500 mg / ml), neomycin (1 mg / ml), and ampicillin (1 mg / ml) were supplemented in their drinking water for one week to eliminate the stress of forced oral administration in seizure-prone mice.

[0057] Gnathobiotic colonization and bacterial concentration in antibiotic-treated mice A. muciniphila (ATCC BAA845) was cultured under anaerobic conditions in Brain Heart Infusion (BHI) medium supplemented with 0.05% porcine stomach mucin type III (Sigma Aldrich). P. merdae (ATCC 43184) and P. distasonis (ATCC 8503) were grown under anaerobic conditions in reinforced Clostridia medium (RCM). 10 9 CFU of bacteria were suspended in 200 µl of pre-reduced PBS and force-administered orally to antibiotic-treated or germ-free mice. When co-administered as "A. muciniphila and Parabacteroides," a ratio of 2:1:1 for A. muciniphila:P. meldae:P. distasonis was used. In mock treatment, mice were force-fed in pre-reduced PBS. Preliminary tests, measured for bacterial load, did not reveal any significant differences in fecal DNA concentration or 16S rDNA amplification between colonization groups. Mice were kept in micro-isolate cages and handled aseptically. Mice were seizure-tested 14 days after colonization.

[0058] Fecal microbiota transplantation Fresh fecal samples were collected from donor mice that had been given KD or CD for 14 days and suspended in pre-reduced PBS at 50 mg / ml. Antibiotic-treated mice were immobilized by forced oral administration of 100 μl of the suspension. For mock treatment, mice were force-fed in pre-reduced PBS. Mice were housed in micro-isolate cages and handled aseptically. Seizure tests were performed 4 days after transplantation.

[0059] Bacteriological treatment A. muciniphylla, P. meldae, and P. distasonis were cultured fresh under anaerobic conditions as described above, then washed, pelletized, and 5 × 10⁻⁶ in pre-reduced PBS. 9 The bacteria were resuspended at cfu / ml. A. muciniphila was prepared in a 2:1:1 ratio with Bacteroides species. For heat sterilization, the bacteria were placed at 95°C for 10 minutes. Mice were force-fed in 200 μl bacterial suspension or sterile pre-reduced PBS as a vehicle control, every 12 hours for 28 days.

[0060] Kcna1 seizure record EEG implantation and recovery. EEG is implanted in male and female Kcna1 6-7 week old. - / - Recorded from mice. Kcna1 + / +Lactate offspring were used as controls. No significant differences were observed between males and females in seizure frequency or duration. The data presented include both sexes. Mice were anesthetized with isoflurane (5% induction, 2% maintenance) and eye ointment was applied to each eye. Fur was removed along the head, and the area was cleaned by scrubbing three times with chlorhexidine and 70% isopropanol. In a safety cabinet, the mice were placed in a stereotactic device (Harvard Biosciences), and 1 mg / kg of lidocaine + 1 mg / kg of bupivacaine was applied topically along the incision site. Using sterile surgical instruments, a 2 cm incision was made along the dorsal midline, from the posterior edge of the eye to the midpoint between the scapulae. A subcutaneous pocket was created along the dorsal lateral side, and the pocket was irrigated with sterile saline. A wireless telemetry transmitter was inserted with a bi-potential lead facing cephalically. The skull was cleansed with 3% hydrogen peroxide followed by 70% isopropanol. A 1.0 mm micro-drill bit was used to create two small holes 1–2 mm from the sagittal suture, midway between the bregma and lambda plexus, in the skull. Bilateral EEG recording electrodes (PhysioTel, ETA-F10, Data Sciences International (DSI)) were implanted epidurally in the frontoparietal cortex. Sterile acrylic was applied to the dry area. The incision site was closed with absorbable 5-0 sutures and cleansed with 3% hydrogen peroxide followed by 70% ethanol. Mice were individually housed in autoclaved micro-isolator cages and allowed to recover for 3–5 days before recording began.

[0061] Data collection and analysis During EEG recording, mice were allowed to move freely and maintained on a laboratory diet. EEG traces were collected over three days using the DSI Ponemah V5.1 data acquisition system. Coinciding video recordings of behavioral seizures correlated with EEG recordings and scored based on the Adaptive Racine Scale, defined on a 5-point scale: 1) myoclonus reflex, 2) cephalic stereotypic and facial clonus, 3) bilateral alternating forelimb / hindlimb clonus, 4) rearing and falling, and 5) generalized rigid-clonic episodes. Data were analyzed by blinded researchers using Neuroscore CNS software (DSI). EEG signals were filtered using a 10 Hz high-pass filter, and seizure events were detected using blinded manual scoring. Seizures were defined as high-frequency, high-amplitude synchronized heterogeneous spike-and-slow waveform patterns lasting longer than 6 seconds, with an amplitude at least twice that of the background. Spike frequency was determined as the number of spikes above baseline in a given seizure, and spike interval was analyzed as a function of time between spikes for five representative seizures at each stage for each mouse. The duration of the maximum spike amplitude was determined as the percentage of time spent on spikes that were three times larger than baseline for five representative seizures at each stage for each mouse.

[0062] Metabolomics of the colon lumen and serum Samples were collected from mice housed in independent cages containing at least two mice per cage. Colonic lumen contents were collected from the final mouse dissection, immediately snap-frozen in liquid nitrogen, and stored at -80°C. Blood samples were collected by cardiac puncture, separated using an SST vacuum tanker, and frozen at -80°C. Samples were prepared using an automated MicroLab STAR system (Hamilton Company) and analyzed on GC / MS, LC / MS, and LC / MS / MS platforms provided by Metabolon, Inc. Protein fractions were removed by stepwise extraction with organic aqueous solvents, concentrated using a TurboVap system (Zymark), and vacuum-dried. For LC / MS and LC-MS / MS, samples were reconstituted in acidic or basic LC-compatible solvents containing >11 implantation standards and run through Waters ACQUITY UPLC and Thermo-Finnigan LTQ mass spectrometers equipped with linear ion trap front-ends and Fourier transform ion cyclotron resonance mass spectrometer back-ends. For GC / MS, samples were derivatized under dry nitrogen using bistrimethylsilyltrifluoroacetamide and analyzed using Thermo-Finnigan Trace DSQ fast scan single quadrupole mass spectrometers employing electron impulse ionization. Chemical entities were identified by comparison with metabolomics library entries of purified standards. For each compound, data were analyzed using one-way ANOVA, following log transformation and imputation with minimum observed values, and group effects were tested. P and q values ​​were calculated based on two-way ANOVA comparisons. Principal component analysis was used to visualize the variance distribution. Supervised random forest analysis was performed to identify metabolomics predictive accuracy.

[0063] Hippocampal metabolomics Hippocampal tissue was homogenized in 1 ml of cold 80% MeOH, vigorously mixed on ice, and then centrifuged (1.3 × 10⁻⁶). 4(rpm, 4°C). 5 μg of supernatant was transferred to a glass vial, 5 nmol of D / L-norvaline was added, and the mixture was dried down under vacuum and finally resuspended in 70% acetonitrile. For mass spectrometry-based analysis, the sample was injected onto a Luna NH2 (150 mm × 2 mm, Phenomenex) column. The sample was analyzed using an UltiMate 3000RSLC (Thermo Scientific) connected to a Q Exactive mass spectrometer (Thermo Scientific). The Q Exactive was operated in full scan mode in the 70–1050 m / z range with polarity switching (+4.00 kV / -4.00 kV). Separation was achieved using A) 5 mM NH4AcO (pH 9.9) and B) ACN. The gradient started at 15% A) and progressed to 90% A) over 18 minutes, followed by a 9-minute isocratic step, and then returned to the initial 15% A) over 7 minutes. Metabolites were quantified using TraceFinder 3.3 with precise mass measurement (≤3 ppm), retention time of pure standards, and MS2 fragmentation patterns. Data analysis, including principal component analysis and hierarchical clustering, was performed using R.

[0064] Amino acid supplementation As described in the method above, 4-week-old Swiss Webster SPF mice were treated with antibiotics, colonized with A. muciniphila and Bacteroides species, and given KD for 14 days. Starting on the evening of day 11, mice were intraperitoneally injected every 12 hours for 3 days with a ketogenic amino acid cocktail (Sigma Aldrich) in sterile PBS: L-leucine (2.0 mg / kg), L-lysine (2.0 mg / kg), L-tyrosine (2.4 mg / kg), L-tryptophan (1.6 mg / kg), and L-threonine (3.1 mg / kg). The concentrations were based on the physiological levels reported for each amino acid in mouse blood and the fold change observed between control SPF KD mice and AkkPb KD mice in our metabolomics dataset for each amino acid. Vehicle-treated mice were injected with PBS (200 μl / 30 g mouse). On the 14th day, two hours after the amino acid injection in the morning of the final day, and after a one-hour adaptation period in the behavioral testing laboratory, the mice were subjected to a 6Hz seizure test.

[0065] GGsTop treatment For wild-type mice: 4-week-old SPF Swiss Webster mice were given free access to CD for 14 days. Starting on the evening of day 11, mice were force-fed 13.3 mg / kg of 3-[[(3-amino-3-carboxypropyl)methoxyphosphinyl]oxy]benzeneacetic acid (GGsTop, Tocris Bioscience) in sterile water every 12 hours. Vehicle-treated mice were force-fed in sterile water (200 μl / 30 g mice). On day 14, two hours after the GGsTop force-fed on the morning of the final day, and after a one-hour adaptation period in the behavioral testing laboratory, the mice were subjected to a 6 Hz seizure test. For Kcna1 mice: 3-4 week-old Kcna1 mice - / - Mice were given free access to CD for 23 days. On day 15, an EEG transmitter was implanted as described in the Kcna1 seizure record section above. On the evening of day 18, the mice were force-fed GGsTop at a dose of 13.3 mg / kg every 12 hours until the morning of day 21. Seizures were recorded by EEG for 3 days, starting 2 hours after the final force-feeding.

[0066] Nutritional symbiosis in vitro assay Nutritional symbiosis was measured as previously described. A. muciniphila were placed in 5 ml of pre-reduced CD or KD-based liquid medium, supplemented with 1% agar, at the bottom of an anaerobic tube, in a mixture of 2 × 10⁶ molecules. 6 The cells were embedded in cfu / ml, and then P. meldae were placed on top of them in 5 ml of pre-reduced M9 minimal medium, 6 × 10⁴ 6 The culture medium was stratified with cfu / ml. The diet-based medium was prepared by aseptically suspending the CD diet for the mouse KD diet in M9 medium at 2 kcal / ml. Preliminary experiments confirmed that embedded A. muciniphila did not ectopically migrate from the agar compartment to the M9 liquid compartment. At each time point, a fixed amount was taken from the upper and lower compartments and plated in a diluted series on nutrient-rich media (RCM for P. meldae and BHI + 0.05% mucin for A. muciniphila), and the colonies were counted. For the GGsTop pretreatment experiment, P. meldae was incubated with a vehicle for GGsTop at 500 μM in RVM at 37°C for 2 hours, and then washed with sterile medium. Preliminary experiments revealed that GGsTop pretreatment had no significant effect on the viability of P. meldae.

[0067] GGT Activity Assay GGT activity was measured as previously described in van der Stel, Frontiers in Microbiology (6), 567 (2015) (Non-Patent Literature 3). For anaerobic culture, bacteria were placed in CD-based medium and KD-based medium in 3 × 10⁶ units. 5The bacteria were seeded at cfu / ml. 1 ml of the bacterial suspension was pelletized and frozen at -80°C for 1 hour. For subsequent data normalization by bacterial cfu, a separate constant volume of the same suspension was plated on BHI mucin agar or RCM and incubated at 37°C in a Coy anaerobic chamber. The pellets were then resuspended in 250 μl of lysis buffer (50 mM Tris-HCl containing 1 μg / ml lysozyme) and incubated on ice for 30 minutes. For fecal samples, the pellets were weighed and homogenized in 1 ml of lysis buffer. The bacterial and fecal suspensions were then sonicated (QSonica 125) and centrifuged at 12000 × g for 10 minutes at 4°C. 20 μl of supernatant was mixed with 180 μl of substrate buffer (2.9 mM L-gamma-glutamyl-3-carboxy-4-nitroanilide (Gold Bio), 100 mM glycylglycine (Sigma Aldrich), 100 mM Tris-HCl), and 500 μM GGsTop (if specified). Using an automated multimode microplate reader (Biotek Synergy H1), absorbance at 405 nm, indicating 3-carboxy-4-nitroaniline production, was measured every minute for 1 hour at 37°C.

[0068] Intestinal permeability assay The experiment began at 7 a.m., after a 4-hour fasting period, mice were force-fed with 0.6 g / kg of 4 kDa FITC dextran (Sigma Aldrich). Four hours after force-fed, serum samples were collected by cardiac puncture, diluted 3-fold in water, and the fluorescence intensity at 521 nm was read in pairs using a Synergy H9 multimode plate reader (Biotek). This was then compared to the standard dilution series of stock FITC dextran in normal mouse serum diluted 3-fold in water.

[0069] statistical analysis Statistical analysis was performed using Prism software (GraphPad). Data were evaluated for normal distribution and plotted as mean ± s.e.m in the figures. For each figure, n = the number of independent biological replicates. No sample or mouse was excluded from the analysis. Differences between two treatment groups were evaluated using Student's independent two-sided t-test with Welch's correction. Differences between >2 groups with a single variable were evaluated using one-way ANOVA with Bonferroni's post-test. Data from Kcna1 mice were analyzed by non-parametric one-way ANOVA with Dunn's post-test. For ≥2 groups with two variables (e.g., seizure time course, BHB time course, metabolomics data, bacterial growth curve), two-way ANOVA with Bonferroni's post-test was used. For the GGT assay, repeated measures one-way ANOVA with Bonferroni's post-test was used. Significant differences revealed from the above tests were indicated as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 in the figures. Near-significant differences (0.5<P<0.1) were shown in the figures. Significant non-significant (and non-significant trend) differences were indicated as "n.s." in the figures.

[0070] (Example 2) The ketogenic diet alters the gut microbiota and confers protection against seizures The 6 Hz psychomotor seizure model of intractable epilepsy involves low-frequency corneal stimulation that induces focal awareness-impairing seizures reminiscent of human temporal lobe epilepsy. KD protects against 6 Hz seizures, as shown by an increase in the current intensity (CC50, seizure threshold) required to induce seizures in 50% of the subjects tested. Specific pathogen-free (SPF) Swiss Webster mice were fed a 6:1 fat:protein KD or an isovitamin and isomineral control diet (CD). Mice fed KD showed an increase in seizure threshold in response to 6 Hz stimulation (Figure 1A), a decrease in serum glucose (Figure 1B), and an increase in serum β-hydroxybutyrate (BHB; Figure 1C) compared to CD controls. There were no significant differences in terms of food intake or weight gain between the KD and CD groups.

[0071] In addition to an elevated seizure threshold, the ketogenic diet altered the composition of the gut microbiota (Figure 1D), decreased α-diversity (Figure 1E), and increased the relative abundance of Ackermansia muciniphila (Figure 1F). Similarly, Parabacteroides, Sutterella, and Erysipelotrichaceae increased in KD-fed mice, while Allobaculum, Bifidobacterium, and Desulfovibrio increased in control-fed mice (Figures 2A-2B). These results demonstrate that the composition of the gut microbiota changed rapidly and significantly in response to KD.

[0072] (Example 3) The gut microbiota provides the anti-seizure effect of the ketogenic diet. To determine whether gut microbiota was necessary for the anti-seizure effect of the ketogenic diet, the 6Hz psychomotor seizure threshold was measured in germ-free (GF) and antibiotic-treated (Abx) SPF mice.

[0073] Compared to CD controls (SPF CD), SPF mice given 14 days of KD (SPF KD) showed elevated seizure thresholds and altered microbiomes (Figures 3A and 1D). This was not observed in GF mice (Figure 3A) and Abx-treated SPF mice (Figure 3C), indicating that the gut microbiome was necessary for KD-mediated seizure protection. Normalization of GF mice by the SPF gut microbiome restored KD-related seizure protection to the level seen in natural SPF KD mice (Figure 3A), suggesting that microbial mediation of KD seizure resistance was independent of pre-weaning microbiome colonization and that the microbiome actively mediated KD-induced seizure protection. In particular, microbial regulation of KD-related seizure resistance did not correlate with changes in serum BHB or glucose levels (Figures 3B and 3D). Similarly, there were no significant differences in gut, liver, or brain BHB levels between groups. Overall, these data demonstrate that the gut microbiota was necessary for the anti-seizure effect of KD in the 6Hz seizure model, and further suggest that the gut microbiota regulated seizure susceptibility through a mechanism that did not involve changes in BHB levels.

[0074] To determine whether specific bacterial taxa mediated seizure protection in response to KD, Abx-treated SPF mice were colonized with specific KD-associated bacteria, administered KD, and then tested for 6Hz seizures (Figures 4A-4B). 9CFU bacteria were force-fed with: i) A. muciniphila; ii) Parabacteroides meldae and P. distasonis in a 1:1 ratio (Figure 1D), which are representative enteric Parabacteroides species from the human microbiome that have the highest homology to the parabacteroides operational taxonomic unit reads enriched by KD; or iii) A. muciniphila, P. meldae, and P. distasonis in a 2:1:1 ratio. Fourteen days after force-fed, 16S rDNA sequencing of the colonic lumen contents revealed that mice treated with A. muciniphila had a relative abundance of 43.7 ± 0.4% of A. muciniphila. Mice force-fed with Parabacteroides had a relative abundance of 70.9±4.0%, and mice force-fed with both taxa contained 49.0±4.1% A. muciniphila and 22.5±5.4% Parabacteroides. Consistent with this, FISH treatment of colon sections from mice treated with A. muciniphila and Parabacteroides showed increased hybridization of the A. muciniphila probe MUC1437 and the Bacteroides and Parabacteroides probe BAC303, although the mean distance from BAC303-positive cells to the nearest MUC1437-positive cells was 0.64±0.09 microns. Both A. muciniphila and Parabacteroides were localized in the lumen of the mouse colon, rather than in the mucosal interstitial spaces. There were no significant differences in body weight, serum glucose levels, or enrichment of A. muciniphila and Parabacteroides between mice given CD and mice given KD. Bacterial hemoglobin (BHB) was similarly induced in the KD-fed group, regardless of colonization and seizure status. These data demonstrate that forced oral administration of exogenous bacteria, following microbiome depletion by Abx treatment, leads to sustained intestinal enrichment up to 14 days post-inoculation.

[0075] Treatment with KD alone increased the seizure threshold by 24.5%, from 19.4±0.8 mA in SPF CD mice to 24.2±0.3 mA in SPF KD mice. On the other hand, co-administration of A. muciniphila and Parabacteroides restored protection against 6 Hz seizures in Abx-treated mice given KD, increasing the threshold by 36.0%, from 19.9±0.3 mA in Abx KD mice to 27.0±0.5 mA in AkkPb KD mice (Figure 4A). The seizure-protective effect due to bacterial concentration was specific to A. muciniphila with P. meldae added, because mice force-fed with A. muciniphila and P. distasonis did not show restoration of seizure protection. The fact that there was no significant difference in seizure threshold after enrichment of either A. muciniphila or Bacteroides species alone (Figure 4A) indicates that both were necessary to mediate the anti-seizure effect of the ketogenic diet. Treatment with Bifidobacterium longum, which increased in CD-treated mice (Figure 1F), also had no effect (Figure 4A). Furthermore, co-colonization of A. muciniphila and Bacteroides species in GF mice promoted seizure protection in response to KD compared to colonization of Bacteroides or A. muciniphila alone in GF mice (Figure 4B). Overall, these findings reveal that A. muciniphila and Bacteroides species increased in response to the ketogenic diet and mediated the protective effect of the ketogenic diet in a 6 Hz seizure model.

[0076] (Example 4) The gut microbiota provides sufficient seizure protection to mice fed a control diet. To determine whether KD-associated gut microbiota also exert an anti-seizure effect on mice fed a control diet, Abx-treated mice were transplanted with the KD microbiome from SPF mice, replacing the CD microbiome. They were then fed either CD or KD, and their susceptibility to 6Hz seizures was tested 4 days after dietary intervention. Abx-treated mice were used to mimic a clinical fecal approach, including Abx pretreatment to remove the natural microbiome. Day 4 was selected based on i) KD's ability to induce significant changes in the microbiome by that time (Figures 1D, 1F, and 5A), and ii) evidence that the KD microbiome shows an incomplete reversion to the CD profile 4 days after switching from KD to CD (Figure 6A). Mice transplanted with the CD microbiome and fed KD for 4 days showed an elevated seizure threshold compared to CD-fed controls (Figure 5A). Abx-treated mice transplanted with the KD microbiome but fed CD for 4 days also showed seizure protection. This suggested that colonization of the KD microbiome increased seizure thresholds in mice treated with CD. However, seizure protection was lost, particularly after a complete reversal from the KD microbiome to the CD profile on day 28 (Figure 6B), suggesting that sustained interaction between the KD microbiome, diet, and neuronal activity was necessary. Similar anti-seizure effects were observed in Abx-treated SPF mice treated with CD after enrichment of A. muciniphila and B. parabacteroides compared to controls of B. parabacteroides, A. muciniphila, or B. longum (Figure 5B). However, the increased seizure threshold in SPF CD mice treated with Abx alone, compared to SPF CD controls, confounded the interpretation of these results (Figure 5B). To clarify this ambiguity, a bacterial treatment approach was used to investigate whether exogenous treatment with A. muciniphila and B. parabacteroides induced anti-seizure effects in CD-treated mice. SPF CD mice were fed twice a day for 28 days. 9CFUs of A. muciniphila and Bacteroides were force-fed with either a vehicle or other means. This bacterial treatment increased the seizure threshold compared to the vehicle-fed control group (Figure 6C). Consistent with experiments using mice fed a ketogenic diet (Figure 4), this seizure protection was not observed in mice treated with A. muciniphila alone, indicating that co-administration of A. muciniphila and Bacteroides was necessary for seizure protection (Figure 5C). Furthermore, treatment with heat-sterilized bacteria lowered the seizure threshold compared to the vehicle-treated control group, suggesting that live bacteria were necessary to provide an anti-seizure effect, and that the release of bacterial cell surface factors and / or intracellular factors increased sensitivity to 6Hz seizures. The elevated seizure threshold disappeared after 21 days of treatment cessation, indicating that sustained exposure to A. muciniphila and Bacteroides was necessary (Figure 6C). Furthermore, no seizure protection was observed in mice treated for only four days (Figure 6D), suggesting that long-term exposure was necessary. In summary, these findings demonstrate that fecal transplantation of the KD microbiome, and bacterial treatment with KD-related taxa, specifically A. muciniphila and Parabacteroides species, provided protection against 6Hz psychomotor seizures in mice fed a control diet.

[0077] (Example 5) KD-related bacteria include Kcna1 - / - Reduced rigid-clonic seizures in mice. Epilepsy is a heterogeneous disorder with diverse clinical presentations. To determine whether the microbiome influenced different seizure types, the role of the gut microbiome in regulating generalized rigid-clonic seizures was investigated in relation to temporal lobe epilepsy and sudden epileptic death (SUDEP) in Kcna1. - / - Tested in a mouse model. Kcna1 - / - The mouse contains a null mutation in the voltage-gated potassium channel Kv1.1 alpha subunit, mimicking the association of the human KCNA1 gene variant with epilepsy, recurrent paroxysmal ataxia, and SUDEP. - / - Mice develop severe, spontaneous, recurrent seizures, which are reduced by 54% with KD. Kcna1 - / -SPF C3HeB / FeJ mice were treated with Abx or vehicle for one week, force-fed with vehicle or A. muciniphila and Parabacteroides bacteria, and given KD or CD for three weeks. Seizure frequency and duration were recorded by EEG over three days, and seizures were identified on the EEG based on a five-stage characteristic epileptic spike pattern (Figure 7C): A) low-frequency background with low-amplitude spikes, B) synchronous high-frequency high-amplitude spikes, C) high-frequency high-amplitude spikes, D) asynchronous high-frequency high-amplitude spikes, and E) high-frequency burst spikes. Furthermore, the EEG seizure patterns were supported by typical seizure behavior identified by the five stages. There were no significant differences in weight gain or food intake between mice given KD and mice given CD. No differences in survival were observed between the groups. CD-fed Kcna1 - / - Compared to the control, KD-fed Kcna1 - / - Mice showed alterations in their gut microbiota profile (Figure 7A), accompanied by increases in A. muciniphila and Parabacteroides species. In particular, these changes were mild and not statistically significant compared to the KD-induced enrichment observed in Swiss-Webster mice (Figure 1F), highlighting the effect of host genotype on baseline microbiota composition and the response to KD. Vehicle-treated Kcna1 - / - The mice exhibited seizures lasting 15–180 seconds, with an average maximum spike amplitude of 490 ± 26 uV (Figure 7C). CD-fed Kcna1 - / - Compared to the control, KD-fed Kcna1 - / - In mice, a reduction in seizure incidence and duration was observed (Figure 7D), which was consistent with the KD-mediated seizure protection described above. Kcna1 was pretreated with Abx to remove the gut microbiota. - / - The mice were treated with a vehicle and given KD (Kcna1). - / - Compared to the control group, there was a significant increase in the number of seizures per day and the total duration of seizures (Figure 7D). There were no significant differences in spike frequency, spike interval, and average duration per seizure, suggesting that Abx treatment and gut microbiota removal had the primary effect on seizure occurrence. Furthermore, Abx treatment Kcna1 - / - Colonization of mice with A. muciniphila and Parabacteroides species is achieved through vehicle treatment, KD feeding, and Kcna1- / - Compared to the levels observed in the control group, seizure frequency and total seizure duration were reduced (Figure 7D). This suggests that treatment with A. muciniphila and Parabacteroides bacteria similarly provided seizure protection in mouse strains with different baseline and diet-altered microbiomes (in this case, C3HeB / FeJ compared to C57Bl / 6). In summary, these findings support the view that specific bacterial species from the innate gut microbiome mediated the anti-seizure effects of KD across various seizure types and models.

[0078] (Example 6) The microbiome regulated the metabolome of the gut, serum, and brain. Metabolomics profiling was used to identify candidate microbiome-dependent molecules in the colonic lumen contents and serum of SPF mice given CD, SPF mice given KD, Abx-treated SPF mice, and mice enriched with A. muciniphila and parabacteroides (Figures 8A and 9A). Metabolomics profiles in the colonic lumen contents and serum distinguished the seizure-protected group (vehicle-treated SPF mice given KD, and A. muciniphila and parabacteroides-enriched mice given KD) from the seizure-susceptible group (vehicle-treated SPF mice given CD, and Abx-treated SPF mice given KD), with predictive accuracy of 94% for colonic lumen metabolites and 87.5% for serum. The majority of metabolites that contributed significantly to group differentiation were related to amino acid metabolism and included derivatives of lysine, tyrosine, and threonine. Furthermore, colonic lumen contents (Figure 8C) and serum (Figure 8D) from the seizure-protected group showed widespread decreases in a subset of ketogenic gamma-glutamyl amino acids—gamma-glutamyl (GG)-leucine, GG-lysine, GG-threonine, GG-tryptophan, and GG-tyrosine—compared to the seizure-susceptible group. This suggests that the gut microbiota regulated gamma-glutamylation itself, or the selective metabolism of ketogenic GG amino acids, and that an increase in ketogenic GG amino acids was associated with seizure susceptibility. Supporting this view, complementary metagenomics predicted KD-related changes in bacterial genes associated with amino acid metabolism. These data highlight the significant effect of the gut microbiota on the gut and systemic metabolome responses to KD and further elucidate the association between KD-induced seizure protection and microbiota-dependent changes in ketogenic GG amino acid levels.

[0079] The brain relies on the active import of essential amino acids for neurotransmitter biosynthesis and is therefore sensitive to fluctuations in peripheral amino acid bioavailability. In particular, GG-amino acids are hypothesized to exhibit enhanced transport characteristics compared to non-gamma-glutamylated forms. Based on data revealing that diet- and microbiome-dependent changes in serum ketogenic amino acids link amino acid import and brain GABA levels, and a strong hypothesis that GABA contributes to the anti-seizure effect of KD, we investigated bulk levels of GABA and glutamate in the hippocampus, a major region of seizure propagation. The hippocampal metabolite profile distinguishes seizure-susceptible mouse samples from seizure-protected mice. In KD-fed SPF mice, the hippocampal GABA / glutamate ratio was significantly elevated compared to CD-fed controls (Figure 8D, left panel). This elevation was abolished in Abx-treated mice given KD and restored upon addition of A. muciniphila and Parabacteroides to Abx-treated mice (Figure 8D). Similar changes were observed with respect to hippocampal levels of glutamine, a precursor of glutamate and GABA (Figure 8D, right panel). Overall, these results reveal diet- and microbiome-dependent regulation of glutamine bioavailability and the preferential elevation of hippocampal GABA levels relative to glutamate in seizure-protected mice.

[0080] (Example 7) Bacterial gamma-glutamylation affected seizure susceptibility. Based on the finding that ketogenic essential GG-amino acids were reduced in the colon lumen and serum of the seizure protection group compared to the seizure susceptibility group, we hypothesized that the microbiome-dependent restriction of ketogenic GG-amino acids is important for mediating the anti-seizure effect of KD. The gamma-glutamylated form of the amino acid was generated by peptide transfer of the GG portion from glutathione to the amino acid. To determine whether gamma-glutamylation of the amino acid affects seizure susceptibility, SPF CD mice were force-fed with GGsTop, a selective irreversible inhibitor of GGT, for 3 days. SPF CD mice treated with GGsTop showed an elevated seizure threshold towards the levels observed in SPF KD mice (Figure 10A). Similarly, CD-fed SPF Kcna1 mice treated with GGsTop - / - EEG recordings of mice showed a significant reduction in seizures per day (Figure 7E). This demonstrated that peripheral inhibition of gamma-glutamylation and restriction of GG amino acids promoted seizure protection, consistent with the observation of a reduction in the metabolome of ketogenic GG amino acids in the colonic lumen and serum from the seizure-protected group compared to the seizure-susceptible control group. To determine whether amino acid restriction, rather than glutathione catabolism, was necessary for the anti-seizure effect of the KD microbiome, KD-fed A. muciniphila and parabacteroides-enriched mice were supplemented with a combination of leucine, lysine, threonine, tryptophan, and tyrosine by intraperitoneal injection twice daily for 3 days, and then tested for 6 Hz seizures. Physiologically relevant amino acid concentrations were calculated based on serum metabolomics data, with each dose restoring blood levels to those seen in vehicle-treated SPF CD controls. Increased systemic levels of ketogenic amino acids lowered the seizure threshold to levels seen in vehicle-treated SPF CD controls (Figure 10B). This suggests that restriction of peripheral ketogenic amino acids was necessary to mediate a microbiome- and KD-dependent increase in seizure tolerance.

[0081] Both host cells and specific bacterial species exhibit GGT activity. To gain insight into whether KD and the interaction between A. muciniphila and Parabacteroides inhibited bacterial in vivo gamma-glutamylation, GGT activity was measured in fecal samples collected from SPF, or mice enriched with A. muciniphila and Parabacteroides fed with CD or KD. KD feeding in SPF mice reduced fecal GGT activity compared to CD controls (Figure 10C). A similar reduction in fecal GGT activity was observed in CD-fed mice after enrichment with A. muciniphila and Parabacteroides. Furthermore, enrichment with A. muciniphila and Parabacteroides, and KD feeding, further reduced fecal GGT activity compared to the activity observed in SPF KD and SPF CD mice. Exposure of all fecal samples to the GGT inhibitor GGsTop eliminated the detected signal, confirming that the measurement reflected GGT activity. Consistent with this, treatment of CD-fed SPF mice with A. muciniphila and parabacteroides reduced fecal GGT activity compared to vehicle-treated controls and mice treated with heat-sterilized bacteria (Figure 10D). Overall, these data reveal that enrichment of A. muciniphila and parabacteroides, or exogenous treatment with A. muciniphila and parabacteroides, reduces fecal GGT activity, which may explain the low levels of colonic and serum GG-amino acids observed in seizure-protected mice.

[0082] To investigate whether bacterial gamma-glutamylation is affected by interactions between A. muciniphila and Parabacteroides species, GGT activity was measured in bacteria grown in an in vitro vegetative symbiosis system. When A. muciniphila was embedded in CD or KD-based agar and P. meldae in M9 minimal medium was overlaid on the agar, both bacteria showed improved growth (Figures 10E and 10F), suggesting that A. muciniphila released soluble factors to enable P. meldae growth, and conversely, P. meldae enhanced the growth of A. muciniphila. Preliminary experiments revealed that A. muciniphila in M9 medium did not grow when overlaid on P. meldae embedded in KD or CD agar, suggesting that A. muciniphila cannot rely solely on vegetative symbiosis from P. meldae for survival.

[0083] P. meldae exhibited high GGT activity, which was abolished upon the addition of A. muciniphila embedded in CD or KD agar (Figures 10G and 10H). To determine whether the decrease in GGT activity in P. meldae promoted the growth of A. muciniphila, P. meldae were pre-treated with a vehicle or GGsTop, and GGT activity was pharmacologically inhibited before testing in a nutrient symbiosis assay. A. muciniphila exposed to GGsTop-pre-treated P. meldae showed increased growth 24 hours after incubation compared to A. muciniphila exposed to vehicle-treated P. meldae (Figure 11B). In summary, these findings suggest that A. muciniphila can metabolize components from KD and CD diets, supporting the growth of P. meldae, and that their synergistic interaction reduced GGT activity. Subsequently, a decrease in GGT activity in P. meldae promoted the proliferation of A. muciniphila. This was consistent with the finding that enrichment of A. muciniphila and Parabacteroides reduced fecal GGT activity, colonic lumen GG-amino acids, and serum GG-amino acids. This demonstrated that amino acid restriction is necessary for seizure protection, and that inhibition of GGT promotes seizure protection. This was consistent with previous studies that associated GGT activity with changes in seizure severity. In a study of 75 epilepsy patients, 84.5% of patients showed higher serum GGT activity compared to controls. In a rat seizure model, GGT activity increased after daily electroshocks for 5 consecutive days. Decreases in various peripheral amino acids are associated with KD-mediated seizure suppression in humans and animals.

[0084] Based on the data and existing literature concerning the role of peripheral amino acids as peripheral substrates for neurotransmitter biosynthesis, we hypothesized that bacterial regulation of GG-amino acids altered the brain importation of amino acids that supply raw materials for GABA / glutamate metabolism (Figure 8). In particular, while some gut bacteria have been reported to de novo synthesize GABA, circulating GABA exhibits limited transport across the blood-brain barrier. Furthermore, changes in the gut microbiota were associated with changes in brain GABA levels, but the molecular mechanisms involved remain unclear. Further research is needed to determine whether GG-amino acids affect amino acid brain transport and local synthesis of GABA for glutamate.

[0085] Overall, this study demonstrated a novel role for specific KD-associated enterobacteria (A. muciniphila and Bacteroides species) in mediating and conferring seizure protection in a mouse model of refractory epilepsy. Increases in A. muciniphila were similarly observed during fasting in humans, hamsters, squirrels, and pythons, as well as in response to calorie restriction and high polyunsaturated fat diets in mice. A. muciniphila and Bacteroides species are also positively associated with increased ketosis and ketogenic diets in humans. The data herein reveal a possible pathway in which KD facilitated interactions between specific microorganisms that reduced host levels of ketogenic GG-amino acids and increased the total bioavailability of GABA to glutamate in the hippocampus. Pharmacological inhibition of gamma-glutamylation increased the seizure threshold, suggesting that reduced GGT activity was important in mediating the anti-seizure effects of the KD-associated gut microbiota in mice. In particular, considering that the absence of bacterial GGT activity under GF conditions was associated with seizure susceptibility, it appears that A. muciniphila and Bacteroides species may have contributed to seizure protection in addition to suppressing GGT activity.

[0086] (Example 8) Bacterial efficacy in Phase 1 human trials In a Phase 1 clinical trial involving healthy subjects, compound BL-001, an orally delivered live biotherapeutic product (LBP), demonstrated favorable safety, tolerability, and strain kinetics profiles.

[0087] The Phase 1 trial of BL-001 was a randomized, double-blind, placebo-controlled, single-center repeated-dose escalation study in healthy subjects (NCT05818306). The primary objective of the trial was to investigate the safety and tolerability of BL-001 over 28 consecutive days in healthy subjects. In the trial, 32 healthy adult participants were administered either BL-001 or placebo across four dose cohorts (BL-001 n=24; placebo n=8). The exact dose range administered to each subject is shown in Figure 12. The colony-forming units of each bacterium were 10 7 ~10 11 It reached that point.

[0088] Thirty-two subjects were given daily treatment with various doses of BL-001 in pill form for four weeks, followed by a two-week follow-up period. Subjects were screened for cross-dose safety and tolerability of the BL-001 composition, as well as for the gut microbiota dynamics of the BL-001 strain, over a one-month period. Clinicians further monitored changes in the gut microbiota, the subjects' health status, and any metabolic changes, and provided subjective assessments of product intake and subject satisfaction.

[0089] All subjects completed the trial. BL-001 demonstrated a favorable safety and tolerability profile in healthy subjects, supporting a 10-fold increase in dose compared to the effective dose in animal models. Furthermore, dose-dependent pharmacokinetics of BL-001 were observed.

[0090] BL-001 demonstrated a favorable safety profile and was well-tolerated in all four dose cohorts without any serious adverse events (SAEs). A list of all reported adverse events is provided in Table 1 below.

[0091]

Table 1

[0092] From the safety data listed in Table 1 (Table 1), four doses of BL-001 (1×10 8 , 1×10 9 , 1×10 10 , and 1×10 11 CFU) administered to four cohorts of eight healthy subjects (six for the active substance and two for the placebo) under fasting conditions for 28 days were safe and well tolerated. Overall, there were 28 cases in the active substance treatment group and 5 cases in the placebo treatment group of treatment-emergent adverse events (TEAEs), of which only 14 cases were considered treatment-related. Nine out of 24 (37%) subjects in the BL-001 treatment group experienced TEAEs compared to two out of eight (25%) in the placebo group. All TEAEs were transient. Five out of 24 treated subjects had TEAEs considered related, and only 1 / 24 was related and moderate (fatigue / tiredness). All adverse events (AEs) were mild (29) or moderate (4) in terms of severity and did not require any clinical intervention. No serious adverse events or AEs leading to treatment discontinuation were recorded. Gastrointestinal disorders (7 AEs in 4 subjects) and headache (3 episodes in 2 subjects) were the most frequent AEs. The mean change from baseline in ECG parameters was not considered clinically significant in any treatment cohort.

[0093] Strain dynamics were also monitored in the subjects, and the results are shown in Figure 13. The BL-001 strain composition was found to engraft rapidly and sustainably in human subjects. The BL-001 strain was observed to engraft rapidly and consistently in the treated subjects, and its abundance increased in a dose-dependent manner. At least one BL-001 strain or closely related strain was absent at baseline in all subjects, while administration introduced the BL-001 strain and, in some cases, replaced the natural congener. None of the other strains showed a significant increase in kinetics from baseline in the treated subjects compared to the placebo subjects. The overall microbiome composition was highly subject-specific and did not change significantly with BL-001 administration. The precise time-course quantification of strains in the subjects is plotted in Figures 14A-14B. Overall, 10 11 The highest dose tested using each bacterium of the CFU was well tolerated (Figure 15).

[0094] The BL-001 composition strain exhibited favorable strain dynamics that increased in a dose-dependent manner. All treatment-related adverse events (AEs) were mild, with the exception of one subject at the highest dose who experienced moderate fatigue that resolved without intervention, and treatment was continued. In the highest-dose cohort of BL-001, the most common treatment-related adverse event (AE) was decreased appetite (3 / 6 subjects), and no other treatment-related AEs occurred in two or more individuals. All treatment-related AEs were transient, with the exception of decreased appetite that persisted to the follow-up period in two subjects, and all resolved without intervention. No AEs led to discontinuation of the study drug, and all participants completed the study. No clinically significant ECG abnormalities were reported in any individual across all treatment cohorts.

[0095] Similarly, elevated ketone concentrations were detected in human urine, consistent with the onset of ketosis in the subjects. Increased urinary ketones were present in subjects at three high-dose levels (1 / 6 subjects in Cohort 1; 1 / 6 subjects in Cohort 3; 2 / 6 subjects in Cohort 4; none in the placebo group). Urinary ketones were measured at multiple time points for subjects in Cohorts 3 and 4, consistently showing elevated levels compared to the placebo group.

[0096] (Example 9) Human gut microbiota provides protection in mice. As shown in Figure 16, the gut microbiota was collected from human subjects after the experiment in Example 8 and transferred to 14 germ-free mice via fecal transplantation. Fecal transplantation was performed on day 0. On day 4, a standard 6Hz psychomotor seizure model was used for each mouse, and on day 5, the mice were dissected.

[0097] Each mouse showed elevated levels of both AkM and PbM bacteria (Figure 18). The mice were then exposed to a fixed current of 32 mA, and the percentage of mice exhibiting seizures was quantified (Figure 17A). The current was increased to CC50 (the critical current intensity required to induce seizures in 50% of mice); CC50 was highest in fecal bacteria-transplanted mice (Figure 17B). In other words, fecal bacteria transplantation demonstrated enhanced seizure protection compared to controls. This data suggests that administration of compositions containing AkM and PbM bacteria is useful and effective in protecting against seizures.

[0098] Embedding by reference All publications and patents referenced herein are incorporated herein in whole by reference, to the same extent that each individual publication or patent is explicitly and individually incorporated by reference. In any conflict, this application shall be compared, including any definition herein.

[0099] Equivalents While specific embodiments of the subject invention have been considered, the above specification is illustrative and not limiting. Many variations of the invention will become apparent to those skilled in the art upon examination of this specification and the following claims. The full scope of the invention is determined by reference to the claims, together with the full scope of their equivalents, and to the specification, together with such variations.

Claims

1. The target has at least 1 × 10 11 Colony-forming units (CFUs) of Akkermansia (Akk) fungi, and at least 1 × 10⁻⁶ 10 A method for suppressing appetite in a subject, comprising the step of administering CFU-derived Bacteria parabacteroides (Pb).

2. The method according to claim 1, wherein the bacteria are administered orally or rectally to the subject.

3. The method according to claim 1 or 2, wherein the bacteria are administered as a liquid, as pills, or as part of food.

4. The method according to any one of claims 1 to 3, wherein the subject is a human subject.

5. The method according to any one of claims 1 to 4, wherein the Akk fungus includes Akkermansia muciniphila.

6. The method according to any one of claims 1 to 5, wherein the Pb bacteria include Parabacteroides meldae and / or Parabacteroides johnsonii.

7. The method according to any one of claims 1 to 6, wherein the Akk bacteria and Pb bacteria are administered to the subject at least daily.

8. The method according to any one of claims 1 to 7, wherein the bacteria are administered to the subject in a ratio of at least 1:1, 3:1, 6:1, or 10:1 of Akkermansia to Parabacteroides.

9. at least 1 × 10 11 Colony-forming units (CFUs) of Akk bacteria, and at least 1 × 10⁻⁶ 10 A bacterial composition containing CFU and Pb bacteria.

10. The bacterial composition according to claim 9, formulated for oral or rectal delivery.

11. The bacterial composition according to claim 9 or 10, which may be part of a food product that is yogurt.

12. A bacterial composition according to any one of claims 9 to 11, further comprising a prebiotic.

13. The bacterial composition according to any one of claims 9 to 12, wherein the bacteria are in a ratio of at least 1:1, 3:1, 6:1, or 10:1 of Akkermansia bacteria to Parabacteroides bacteria.

14. The bacterial composition according to any one of claims 9 to 13, wherein the bacteria comprises Ackermansia muciniphila, Parabacteroides meldae, Parabacteroides johnsonii, or any combination thereof.

15. Use of the bacterial composition according to any one of claims 9 to 14 in suppressing appetite in a subject.

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