Microbiota compositions and methods for treating disorders

Abstract

The present invention relates to a composition for storing microbiota. The invention also relates to dosage forms and methods for treating conditions and diseases by administering said compositions to a patient in need thereof.

Description

Technical Field

[0001] This invention relates to compositions for storing microbiota. The invention also relates to dosage forms and methods for treating conditions and diseases by administering said compositions to patients in need. Background Technology

[0002] The following discussion of the background art is intended only to facilitate understanding of the invention. This discussion is not an admission or confirmation that any of the materials mentioned were, or were, part of common general knowledge at the priority date of this application.

[0003] Gut microbiota

[0004] The human gut microbiota comprises trillions of microorganisms, including at least 100 prevalent bacterial species and at least 1,000 less common bacterial species, carrying more than 100 times the number of genes present in the human genome. While primarily composed of bacteria, the gut microbiota also contains archaea, fungi, yeasts, protozoa, and viruses. The microbiota performs vital functions essential for maintaining health, including processing food, digesting complex, indigestible polysaccharides, and synthesizing vitamins. It also secretes bioactive metabolites with a wide range of functions, from inhibiting pathogens and metabolizing toxic compounds to regulating host metabolism.

[0005] Cultured microbiome therapy

[0006] Cultured microbiome therapies consist of one or more microorganisms that can be administered to patients to treat or prevent disease or enhance the effectiveness of another therapy. Cultured microbiome therapies are currently being developed for the treatment of many diseases. However, these efforts face the challenge of loss of viable microorganisms, which limits stability, storage, transportation, and delivery conditions, and thus restricts the clinical use of these therapies. Sampling, storing, transporting, and delivering viable and effective microbiome samples to patients presents challenges. There is also a need in the art for effective treatments for diseases associated with gut microbiome loss or dysbiosis.

[0007] Storage microbiota

[0008] Efforts have been made to store microbial communities in the form of freeze-drying or lyophilization. Freeze-drying, also known as lyophilization, is a low-temperature dehydration process that involves freezing the product and reducing pressure, thereby removing ice through sublimation. This contrasts sharply with dehydration in most conventional methods that use heat to evaporate water. Freeze-drying microbial communities is a useful method for long-term preservation. However, one of the main challenges is the loss of cell viability due to the effects of freeze-thaw damage and osmotic pressure changes on the function and structural integrity of microorganisms. In the prior art, attempts have been reported to develop suitable cryoprotectants to protect the function and structural integrity of microorganisms during freeze-drying and thawing processes; however, many of these attempts have failed. There is a need in the art for effective cryoprotectants that maintain cell viability and preserve the function and structural integrity of microorganisms during freeze-drying and thawing processes.

[0009] The purpose of this invention is to overcome one or more problems indicated by the prior art. Summary of the Invention

[0010] On one hand, the present invention broadly relates to a composition for the prevention or treatment of a disease or ailment in a subject in need, said composition comprising at least one microbial strain.

[0011] The microorganisms mentioned therein are selected from the group consisting of bacteria, yeast, or archaea; and

[0012] excipient.

[0013] In a preferred embodiment, the excipient is a cryoprotectant. In a preferred embodiment, the excipient is inulin or an analogue or variant thereof. In a preferred embodiment, the inulin is selected from the group consisting of: α-D-glucopyranosyl-[β-D-fructofuranosyl](n-1)-D-fructofuranosyl; β-D-fructofuranosyl-[D-fructofuranosyl](n-1)-D-fructofuranosyl; fructooligosaccharides; fructooligosaccharides containing 2 to 70 fructose units; fructooligosaccharides containing 1 to 500 fructose units; fructooligosaccharides containing 1 to 300 fructose units; fructooligosaccharides containing 1 to 200 fructose units; fructooligosaccharides containing 1 to 100 fructose units; or analogues, variants, or combinations thereof.

[0014] In a preferred embodiment, the excipient is maltodextrin or its analogues or variants. In a preferred embodiment, the maltodextrin is selected from the group consisting of: maltodextrin with a length selected from 3 to 17 glucose units; corn syrup with a length of 20 glucose units or more; corn syrup solids; modified corn starch; modified rice starch; modified cassava starch; modified wheat starch; or its analogues or variants or combinations thereof.

[0015] In a preferred embodiment, the composition comprises an analogue of inulin or a variant thereof, the concentration of which is selected from the group consisting of: 0.01% w / v to 20% w / v; 0.1% w / v to 20% w / v; 0.1% w / v to 10% w / v; 1% w / v to 10% w / v; 2% w / v to 9% w / v; 3% w / v to 8% w / v; 4% w / v to 7% w / v; 4% w / v to 6% w / v; 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v.

[0016] In a preferred embodiment, the composition comprises an analogue of maltodextrin or a variant thereof, the concentration of which is selected from the group consisting of: 0.01% w / v to 20% w / v; 0.01% w / v to 20% w / v; 0.1% w / v to 10% w / v; 1% w / v to 10% w / v; 2% w / v to 9% w / v; 3% w / v to 8% w / v; 4% w / v to 7% w / v; 4% w / v to 6% w / v; 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v.

[0017] In another preferred embodiment, the composition comprises inulin and maltodextrin.

[0018] In a preferred embodiment, the composition comprises inulin and maltodextrin, the concentrations of which are selected from the group consisting of: inulin (1% w / v) and maltodextrin (1% w / v); inulin (2% w / v) and maltodextrin (2% w / v); inulin (3% w / v) and maltodextrin (3% w / v); inulin (4% w / v) and maltodextrin (4% w / v); inulin... Inulin (5% w / v) and maltodextrin (5% w / v); inulin (6% w / v) and maltodextrin (6% w / v); inulin (7% w / v) and maltodextrin (7% w / v); inulin (8% w / v) and maltodextrin (8% w / v); inulin (9% w / v) and maltodextrin (9% w / v); and inulin (10% w / v) and maltodextrin (10% w / v).

[0019] In a preferred embodiment, the composition comprises inulin and maltodextrin, the concentrations of which are selected from the group consisting of: (1) the concentration of inulin being selected from the group consisting of: 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v; and (2) the concentration of maltodextrin being selected from the group consisting of: 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v.

[0020] In a preferred embodiment, the composition comprises inulin and maltodextrin, the concentrations of which are selected from the group consisting of: inulin (1% w / v) and maltodextrin (1% w / v); inulin (2% w / v) and maltodextrin (2% w / v); inulin (3% w / v) and maltodextrin (3% w / v); inulin (4% w / v) and maltodextrin (4% w / v); inulin... Inulin (5% w / v) and maltodextrin (5% w / v); inulin (6% w / v) and maltodextrin (6% w / v); inulin (7% w / v) and maltodextrin (7% w / v); inulin (8% w / v) and maltodextrin (8% w / v); inulin (9% w / v) and maltodextrin (9% w / v); and inulin (10% w / v) and maltodextrin (10% w / v).

[0021] In a preferred embodiment, the composition is in lyophilized form.

[0022] In a preferred embodiment, the composition is in liquid form.

[0023] In a preferred embodiment, the excipient is selected from the group consisting of: inulin; inulin and maltodextrin; inulin and dextran 70k; inulin and pectin; inulin and sucrose; inulin and trehalose; inulin and maltodextrin and sucrose; inulin and maltodextrin and dextran 70k; inulin and maltodextrin and pectin; inulin and maltodextrin and sucrose; and inulin and maltodextrin and pectin.

[0024] In a preferred embodiment, the excipient is selected from the group consisting of: inulin (10% w / v); inulin (5% w / v); inulin (5% w / v) and maltodextrin (5% w / v); inulin (5% w / v) and dextran 70k (5% w / v); inulin (5% w / v) and pectin (5% w / v); inulin (5% w / v) and sucrose (5% w / v); inulin (5% w / v) and trehalose (5% w / v); inulin (5% w / v) And maltodextrin (5% w / v) and sucrose (5% w / v); inulin (5% w / v) and maltodextrin (5% w / v) and dextran 70k (5% w / v); inulin (5% w / v) and maltodextrin (5% w / v) and pectin (5% w / v); inulin (5% w / v) and maltodextrin (5% w / v) and sucrose (5% w / v); and inulin (5% w / v) and maltodextrin (5% w / v) and pectin (5% w / v).

[0025] In a preferred embodiment, the moisture concentration of the composition is less than that selected from the group consisting of: 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v.

[0026] In a preferred embodiment, the moisture concentration of the composition is selected from the group consisting of: between 1% w / v and 5% w / v; between 0.5% w / v and 2% w / v; between 0.9% w / v and 1.2% w / v; between 0.99% w / v and 1.09% w / v; and 1.093% w / v.

[0027] In a preferred embodiment, the Young's modulus of the composition is selected from the group consisting of: between 1 and 5; between 2 and 4; between 2.1 and 3.9; between 2.5 and 3.8; 2; 3; 4; 5; 2.98; 2.19; 3.51; 3.23; less than 2; less than 3; less than 4; less than 5; greater than 2; greater than 3; greater than 4; greater than 5.

[0028] In a preferred embodiment, the maximum stress (kPa) measure of the composition at the fracture point is selected from the group consisting of: between 20 and 40; between 21 and 39; between 20 and 30; between 20 and 29; between 25 and 30; 20; 21; 22; 23; 24; 25; 26; 27; 28; 29; 30; 29.36; 29.01; 22.68; less than 20; less than 21; less than 22; less than 23; less than 24; less than 25; less than 26; less than 27; less than 28; less than 29; less than 30; greater than 20; greater than 21; greater than 22; greater than 23; greater than 24; greater than 25; greater than 26; greater than 27; greater than 28; greater than 29; and greater than 30.

[0029] In a preferred embodiment, the composition comprises additional excipients and a carrier.

[0030] In a preferred embodiment, the microorganism is fecal microorganism or colonic microorganism.

[0031] In a preferred embodiment, the microorganism is non-inflammatory.

[0032] In a preferred embodiment, the microorganisms are cultured from fecal or colonic biopsy samples.

[0033] In a preferred embodiment, the consortium comprises a microbial cell community derived from feces or biopsies from one or more human donors.

[0034] In a preferred embodiment, the microbial cell community comprises cultured microbial cells.

[0035] In a preferred embodiment, the cultured microbial cells are derived from multiple human donors.

[0036] In a preferred embodiment, the microbial cell community comprises uncultured microbial cells.

[0037] In a preferred embodiment, the uncultured microbial cells are derived from a single human donor.

[0038] In a preferred embodiment, the composition is a fecal transplantation microbiome composition.

[0039] In a preferred embodiment, the composition comprises a purified or reconstituted mixture of fecal bacteria.

[0040] In a preferred embodiment, the composition is lyophilized.

[0041] In a preferred embodiment, the composition is a liquid.

[0042] In a preferred embodiment, after being stored at the storage temperature for at least 4 weeks, the composition is able to maintain at least 50% cell viability relative to the initial cell viability immediately prior to storage.

[0043] In a preferred embodiment, after being stored at the storage temperature for at least 4 weeks, the composition is able to maintain about 60% to about 80% cell viability relative to the initial cell viability immediately prior to the start of the storage.

[0044] In a preferred embodiment, after storage at the storage temperature for at least 2, 4, 8, 12, 16, or 20 weeks, the composition is able to maintain at least about 5%, 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% cell viability relative to the initial cell viability immediately prior to storage.

[0045] In a preferred embodiment, after being stored at the storage temperature for at least 8, 12, 16, 20, 50, 75, 100, 150 or 200 weeks, the composition maintains at least 50% cell viability relative to the initial cell viability immediately prior to storage.

[0046] In a preferred embodiment, after at least 12 weeks at storage temperature, the composition maintains 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 40% to 80%, 50% to 70%, 55% to 65%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, or 70% to 80% cell viability relative to the initial cell viability immediately prior to storage.

[0047] In a preferred embodiment, the storage temperature is selected from the group consisting of: ambient temperature or below; -70°C; -50°C; -47°C; -30°C; -20°C; -8°C; -4°C; below zero; 2°C; 4°C; 8°C; between 2°C and 8°C; 18°C; 25°C; room temperature; and ambient temperature.

[0048] In a preferred embodiment, cell viability is measured by methods selected from the group consisting of: imaging assays using membrane permeability; staining with a combination of membrane-permeable and impermeable DNA dyes; staining with SYTO and propidium iodide to distinguish between live and dead bacteria; viability assays combined with fluorescent Gram staining; colorimetric methods; assessing bacterial cell viability using BactoBox or other impedance flow cytometry tools; assessing bacterial cell viability by counting the number of colonies on an agar plate; and assessing cell viability by molecular viability analysis.

[0049] In a preferred embodiment, the composition comprises a prebiotic.

[0050] In a preferred embodiment, the composition comprises a carrier.

[0051] In a preferred embodiment, the composition comprises insoluble fibers, buffer solution, penetrant, defoamer and / or preservative.

[0052] In a preferred embodiment, the composition comprises a chemostat culture medium.

[0053] In a preferred embodiment, the composition comprises a brine composition.

[0054] In a preferred embodiment, the composition comprises resistant starch.

[0055] In a preferred embodiment, the composition is lyophilized together with a pharmaceutically acceptable excipient.

[0056] In a preferred embodiment, the composition contains additional stabilizers and / or additional cryoprotectants.

[0057] In a preferred embodiment, the additional cryoprotectant is selected from the group consisting of: trehalose; mannitol; sucrose; glycerol; sorbitol; DMSO; propylene glycol; ethylene glycol; saccharose; galactose-lactose; and any combination thereof.

[0058] In a preferred embodiment, the additional cryoprotectant further comprises a compound selected from the group consisting of: glycerol; polyethylene glycol (PEG); glycerol; erythritol; arabinitol; xylitol; sorbitol; glucose; lactose; ribose; and any combination thereof.

[0059] In a preferred embodiment, the additional cryoprotectant is trehalose at a concentration of 2% to 15% in the freeze-dried formulation.

[0060] In a preferred embodiment, the additional cryoprotectant is trehalose at a concentration of at least 5% in the freeze-dried formulation.

[0061] In a preferred embodiment, the additional cryoprotectant is trehalose at a concentration of at least 10% in the freeze-dried formulation.

[0062] In a preferred embodiment, the composition is a pharmaceutical composition.

[0063] In a preferred embodiment, the at least one microbial strain is diluted with an inert powdered diluent.

[0064] In a preferred embodiment, the composition comprises one or more pharmaceutically acceptable carriers or excipients.

[0065] In a preferred embodiment, the composition is formulated as a gel sheet, pill, enema, microcapsule, capsule, or tablet.

[0066] In a preferred embodiment, the capsule or tablet is enteric-coated, pH-dependent, sustained-release, and / or gastric-tolerant.

[0067] In a preferred embodiment, the composition is suitable for oral or rectal administration.

[0068] In a preferred embodiment, the composition comprises one or more, two or more, three or more, four or more, or five or more isolated, purified or cultured microorganisms.

[0069] In a preferred embodiment, the microorganism is selected from members of the phylum, family, genus, and species taxonomic units listed in the group consisting of: Table 1, Table 2, Table 3, Table 4, Table 5, Table 13, Table 14, Table 15, and any combination thereof.

[0070] For example, the composition contains one or more microorganisms selected from Table 3.

[0071] In another example, the composition comprises one or more microorganisms selected from the BB265 complex as listed in Table 4.

[0072] For example, the composition comprises one or more microorganisms selected from the BB265 complex of 143 isolates listed in Table 5.

[0073] In a preferred embodiment, the composition is further supplemented with at least one microorganism selected from the group consisting of: Table 1, Table 2, Table 3, Table 4, Table 5, Table 13, Table 14, Table 15 and any combination thereof.

[0074] In a preferred embodiment, the composition lacks one or more of the microorganisms listed in the group consisting of: Table 1, Table 2, Table 3, Table 4, Table 5, Table 13, Table 14, Table 15 and any combination thereof.

[0075] Please note that in Tables 1 to 5, * indicates taxa identified in the analysis that are considered for inclusion in BB265 but are not present in the BB265 complex.

[0076] Table 1 - List of classification units by door.

[0077] Phylum Actinomycetota (formerly known as Actinobacteria) Firmicutes (formerly known as Bacillota) Bacteroidetes (formerly known as Bacteroidetes) *Campylobacterota *Pseudomonadota (formerly known as Proteobacteria) Phylum Thermodesulfobacteriota Verrucomicrobiota

[0078] Table 2 - List of taxonomic units by genus.

[0079]

[0080]

[0081]

[0082] Table 3 - A broad list of families (bacteria, archaea, and viruses).

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] Table 4 - List of taxa identified by species / 16S, including the 16S sequence of each species in the BB265 complex or identified as a sulfidogen.

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Table 5 - List of isolates containing the BB265 complex of 143 isolates.

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] In a preferred embodiment, the enterococcus is selected from the group consisting of: Enterococcus faecalis; Enterococcus faecium; Enterococcus CC00149, which was deposited at the National Measurement Institute of Australia on September 9, 2019, under the accession number V19 / 018754. Enterococcus CC00259, deposited at the Australian National Metrology Institute (ANI) on September 9, 2019, under the accession number V19 / 018755; Enterococcus CC00620, deposited at the ANI on June 29, 2021, under the accession number V21 / 013048; Enterococcus CC00064, deposited at the ANI on June 29, 2021, under the accession number V21 / 013046; Enterococcus CC00619, deposited at the ANI on June 29, 2021, under the accession number V21 / 013047; Enterococcus CC00262, deposited at the ANI on March 18, 2020, under the accession number V20 / 006238; and Enterococcus CC0002, deposited at the ANI on July 20, 2021, under the accession number V21 / 014119.

[0106] In a preferred embodiment, the lactobacillus is selected from the group consisting of: *Lactobacillus rhamnosus* (e.g., strain GG (ATCC 53103), CGMCC 1.3724, or SP1 (DSM 21690)); *Lactococcus lactis*; *Lactococcus cremoris*; *Lactococcus diacetylactis*; *Lactobacillus paracasei*; *Lactobacillus reuteri* (e.g., strain ATCC 55730 or DSM 17938); *Lactobacillus acidophilus*; *Lactobacillus murinus*; *Lactobacillus helveticus*; *Lactobacillus bulgaricus*. Lactobacillus bulgaricus; Lactobacillus casei; Lactobacillus salivarius; Lactobacillus plantarum; Lactobacillus fermentum; Lactobacillus taiwanensis; Lactobacillus animalis; Lactobacillus johnsonii (e.g., strains NCC533; CNCM 1-1225); and Lactobacillus gasseri.

[0107] In a preferred embodiment, the Bifidobacterium is selected from the group consisting of: Bifidobacterium lactis (e.g., strains BB-12, BI-04, or CNCM 1-3446 (Bb12)); Bifidobacterium longum (e.g., strains NCC3001, ATCC BAA-999 (BB536)); Bifidobacterium shortum (e.g., strains Bb-03, M-16V, or R0070); Bifidobacterium infantis; Bifidobacterium animalis; Bifidobacterium bifidum; and Bifidobacterium adolescentis.

[0108] In a preferred embodiment, the streptococcus is selected from the group consisting of: Streptococcus thermophilus, Streptococcus thermophilus ST-21, and Streptococcus salivarius.

[0109] In a preferred embodiment, the Clostridium is selected from the group consisting of: Clostridium difficile; Clostridium halys; Clostridium scintillans; and Flavobacterium prevalence.

[0110] In a preferred embodiment, the microorganism is yeast.

[0111] In a preferred embodiment, the yeast is *Saccharomyces boulardii*.

[0112] In a preferred embodiment, the microorganism is an archaea.

[0113] In a preferred embodiment, the archaea are selected from the group consisting of: Methanobrevibacter spp; Methanobrevibacter smithii; and Methanosphaera sp; Methanosphaera stadtmaniae.

[0114] In a preferred embodiment, the composition comprises a fecal microbiota with a Shannon Diversity Index greater than or equal to 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, or higher. 2.2, greater than or equal to 2.3, greater than or equal to 2.4, greater than or equal to 2.5, greater than or equal to 3.0, greater than or equal to 3.1, greater than or equal to 3.2, greater than or equal to 3.3, greater than or equal to 3.4, greater than or equal to 3.5, greater than or equal to 3.6, greater than or equal to 3.7, greater than or equal to 3.8, greater than or equal to 3.9, greater than or equal to 4.0, greater than or equal to 4.1, greater than or equal to 4.2, greater than or equal to 4.3, greater than or equal to 4.4, greater than or equal to 4.5, or greater than or equal to 5.0.

[0115] In a preferred embodiment, the composition comprises a fecal microbiota with a Shannon diversity index ranging from 0.1 to 3.0, 0.1 to 2.5, 0.1 to 2.4, 0.1 to 2.3, 0.1 to 2.2, 0.1 to 2.1, 0.1 to 2.0, 0.4 to 2.5, 0.4 to 3.0, 0.5 to 5.0, 0.7 to 5.0, 0.9 to 5.0, and 1. Between 1 and 5.0, between 1.3 and 5.0, between 1.5 and 5.0, between 1.7 and 5.0, between 1.9 and 5.0, between 2.1 and 5.0, between 2.3 and 5.0, between 2.5 and 5.0, between 2.7 and 5.0, between 2.9 and 5.0, between 3.1 and 5.0, between 3.3 and 5.0, between 3.5 and 5.0, between 3.7 and 5.0, between 3.9 and 5.0, or between 4.1 and 5.0.

[0116] In a preferred embodiment, the Shannon diversity index is calculated at the level of groups selected from the following: phylum level, family level, genus level, and species level.

[0117] In a preferred embodiment, the composition comprises a formulation containing a microbial community in proportions similar to those found in the fecal microbiota of a normal healthy person.

[0118] In a preferred embodiment, the composition comprises fecal bacteria from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different families.

[0119] In a preferred embodiment, the composition comprises a fecal microbiota comprising no more than 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of the weight of inanimate material / biological material.

[0120] In a preferred embodiment, the composition comprises a fecal microbiota comprising no more than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the weight of inanimate material / biological material.

[0121] In a preferred embodiment, the composition comprises particles of inanimate material and / or biological material of a fecal sample passing through a screen, filter media, or similar filtration device with a size of 2.0 mm, 1.0 mm, 0.5 mm, 10 mm, 0.25 mm, 0.212 mm, 0.101 mm, 0.180 mm, 0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053 mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, 0.01 mm, or 0.2 mm.

[0122] In a preferred embodiment, the composition comprises substantially isolated or purified fecal microbiota or a complete (or substantially complete) microbiota, which is (or comprises) at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% isolates of isolated or purified fecal microbiota or non-fecal microbiota material having no more than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0% or more; or substantially isolated, purified, or substantially complete microbiota, as described in WO2012 / 122478 or as described in WO 2012 / 016287.

[0123] In a preferred embodiment, the weight ratio of the inanimate material derived from feces to the biological material derived from feces in the composition is no greater than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 5%, 8%, 10%, 15%, 20%, 30%, 40%, or 50%.

[0124] In a preferred embodiment, each 200 mg of the composition contains a pharmacologically active dose of microbial cells or spores selected from the group consisting of: 10 3 Up to 10 14 ;10 4 Up to 10 14 ;10 5 Up to 10 14 ;10 6 Up to 10 14 ;10 7 Up to 10 14 ;10 8 Up to 10 14 ;10 4 Up to 10 13 ;10 5 Up to 1012 ;10 6 Up to 10 11 ;10 7 Up to 10 10 ;10 8 Up to 10 9 ;10 3 Up to 10 13 ;10 3 Up to 10 12 ;10 3 Up to 10 11 ;10 3 Up to 10 10 ;10 3 Up to 10 9 ;10 3 Up to 10 8 ;10 3 Up to 10 7 ;10 3 Up to 10 6 ;10 3 Up to 10 5 and 10 3 Up to 10 4 Colony forming units (CFU) or total cell count.

[0125] In a preferred embodiment, the composition comprises microbial cells or spores at a pharmacologically active dose selected from the group consisting of: 10 million to 100 billion cfu / mL, 10 million to 50 million cfu / mL, more preferably 50 million to 100 million cfu / mL, 100 million to 500 million cfu / mL, 500 million to 1 billion cfu / mL, 1 billion to 5 billion cfu / mL, 5 billion to 10 billion cfu / mL, 10 billion to 15 billion cfu / mL, 15 billion to 20 billion cfu / mL, 20 billion to 25 billion cfu / mL, 25 billion to 30 billion cfu / mL, 30 0 billion to 35 billion cfu / mL, 35 billion to 40 billion cfu / mL, 40 billion to 45 billion cfu / mL, 45 billion to 50 billion cfu / mL, 50 billion to 55 billion cfu / mL, 55 billion to 60 billion cfu / mL, 60 billion to 65 billion cfu / mL, 65 billion to 70 billion cfu / mL, 70 billion to 75 billion cfu / mL, 75 billion to 80 billion cfu / mL, 80 billion to 85 billion cfu / mL, 85 billion to 90 billion cfu / mL, 90 billion to 95 billion cfu / mL, 95 billion to 100 billion cfu / mL.

[0126] In a preferred embodiment, the composition comprises a pharmacologically active dose of microbial cells or spores, wherein the concentration of the microbial cells or spores as dry microorganisms is selected from the group consisting of: 5 w / w% to 50 w / w%, 1 w / w% to 75 w / w%, 0.1 w / w% to 100 w / w%, and 1 w / w% to 100 w / w.

[0127] In a preferred embodiment, the disease or symptom is inflammation of the gastrointestinal mucosa.

[0128] In a preferred embodiment, the disease or symptom is characterized by a reduction in gut microbial diversity.

[0129] In a preferred embodiment, the disease or symptom is characterized by a decrease in gut microbial function.

[0130] In a preferred embodiment, the disease or symptom is characterized by a loss of gut microbial ecology.

[0131] In a preferred embodiment, the disease or symptom is ecological imbalance.

[0132] In a preferred embodiment, the ecological imbalance is associated with one or more conditions selected from the group consisting of: inflammatory bowel disease (IBD), pouchitis, irritable bowel syndrome (IBS), intestinal bacterial infection, metabolic disease, neuropsychiatric disorder, autoimmune disease, allergic disease, hepatic encephalopathy, or cancer.

[0133] In a preferred embodiment, the disease or ailment is a malignant disease or cancer.

[0134] In a preferred embodiment, the disease or condition is hepatic disease or liver disease.

[0135] In a preferred embodiment, the disease or symptom is a gastrointestinal symptom.

[0136] In a preferred embodiment, the gastrointestinal condition is inflammatory bowel disease.

[0137] In a preferred embodiment, the inflammatory bowel disease is selected from the group consisting of: ulcerative colitis; Crohn's disease; gastroenteritis; colitis; and pouchitis.

[0138] In a preferred embodiment, the gastrointestinal condition is selected from the group consisting of: irritable bowel syndrome; gastrointestinal ulcers; and gastrointestinal cancers.

[0139] In a preferred embodiment, the composition reduces the level of endogenous sulfides in the colon of patients in need.

[0140] In a preferred embodiment, the composition reduces the load of sulfides and nitric oxide on epithelial cells that cause metabolic damage by inhibiting cellular respiration.

[0141] In a preferred embodiment, the composition: reduces the relative abundance and / or metabolic activity of sulfide-producing microbiota; directly reduces sulfide levels in the colon by consumption / absorption; reduces sulfide levels in the colon by competition with metabolic substrates; and / or reduces sulfide levels in the colon by consuming hydrogen.

[0142] In a preferred embodiment, the composition reduces the relative abundance and / or metabolic activity of sulfide-producing microbiota by reducing the amount of metabolizable sulfur substrates; and reduces the release of sulfur amino acids (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

[0143] In a preferred embodiment, the composition induces apoptosis in colon cells in the lesion to disrupt the induced stable inflammatory state.

[0144] In a preferred embodiment, the composition reduces unwanted inflammation.

[0145] In a preferred embodiment, the composition prevents or reduces activation of the mucosal immune system in a natural killer T cell-driven IL-13 and IL-5 dependent, TH2-mediated immune response.

[0146] In a preferred embodiment, the composition reduces inflammation in a subject when measured by parameters selected from the group consisting of: TNFα signaling via NF-κB; IFNα signaling; IFNγ signaling; IL6 JAK STAT3 signaling; activation of pro-apoptotic pathways; and initiation of unfolded protein responses.

[0147] In a preferred embodiment, the composition downregulates genes associated with pro-apoptotic pathways and unfolded protein responses, including genes selected from the group consisting of CHAC1, CEBPB, TRIB3, PPP1R15A, DDIT3, ATF4, and XBP1.

[0148] In a preferred embodiment, the composition is free of excipients selected from the group consisting of: inulin HP-gel (Orafti) (10% w / v); inulin (5% w / v), maltodextrin-free; inulin (10% w / v), maltodextrin-free; inulin (15% w / v), maltodextrin-free; trehalose (2.5% w / v) and inulin (2.5% w / v); trehalose (5% w / v) and inulin (5% w / v); trehalose (7.5% w / v) and inulin (7.5% w / v); inulin (15% w / v) and tocopherol (10 μL / L); inulin (15% w / v) and tocopherol (100 μL / L); inulin (15% w / v) and ascorbic acid (4 mg / L); and inulin (15% w / v) and ascorbic acid (40 mg / L).

[0149] In a preferred embodiment, the microorganism is not Lactobacillus acidophilus MJLA1.

[0150] In a preferred embodiment, the composition is free of excipients selected from the group consisting of: inulin HP-gel (Orafti) (10% w / v); inulin (5% w / v), maltodextrin-free; inulin (10% w / v), maltodextrin-free; inulin (15% w / v), maltodextrin-free; trehalose (2.5% w / v) and inulin (2.5% w / v); trehalose (5% w / v) and inulin (5% w / v). The following are the microorganisms: (w / v); trehalose (7.5% w / v) and inulin (7.5% w / v); inulin (15% w / v) and tocopherol (10 μL / L); inulin (15% w / v) and tocopherol (100 μL / L); inulin (15% w / v) and ascorbic acid (4 mg / L); and inulin (15% w / v) and ascorbic acid (40 mg / L); and the microorganisms are not Lactobacillus acidophilus MJLA1.

[0151] In a preferred embodiment, the composition contains excipients selected from the group consisting of: inulin HP-gel (Orafti) (10% w / v); inulin (5% w / v), maltodextrin-free; inulin (10% w / v), maltodextrin-free; inulin (15% w / v), maltodextrin-free; trehalose (2.5% w / v) and inulin (2.5% w / v); trehalose (5%) and inulin (5% w / v); trehalose (7.5% w / v) and inulin (7.5%); inulin (15% w / v) and tocopherol (10 μL / L); inulin (15% w / v) and tocopherol (100 μL / L); inulin (15% w / v) and ascorbic acid (4 mg / L); and inulin (15% w / v) and ascorbic acid (40 mg / L).

[0152] In a preferred embodiment, the microorganism is Lactobacillus acidophilus MJLA1.

[0153] In a preferred embodiment, the composition contains excipients selected from the group consisting of: inulin HP-gel (Orafti) (10% w / v); inulin (5% w / v), maltodextrin-free; inulin (10% w / v), maltodextrin-free; inulin (15% w / v), maltodextrin-free; trehalose (2.5% w / v) and inulin (2.5% w / v); trehalose (5% w / v) and inulin (5% w / v) % w / v); trehalose (7.5% w / v) and inulin (7.5% w / v); inulin (15% w / v) and tocopherol (10 μL / L); inulin (15% w / v) and tocopherol (100 μL / L); inulin (15% w / v) and ascorbic acid (4 mg / L); and inulin (15% w / v) and ascorbic acid (40 mg / L); and wherein the microorganism is Lactobacillus acidophilus MJLA1.

[0154] In a preferred embodiment, the composition does not contain excipients selected from the group consisting of: inulin (2% w / v); maltodextrin (2% w / v); sucrose (2% w / v).

[0155] In a preferred embodiment, the composition does not contain excipients selected from the group consisting of: inulin (10% w / v); maltodextrin (10% w / v); and sucrose (10% w / v).

[0156] In a preferred embodiment, the microorganism is not Lactobacillus plantarum.

[0157] In a preferred embodiment, the composition contains excipients selected from the group consisting of: inulin (2% w / v); maltodextrin (2% w / v); and sucrose (2% w / v).

[0158] In a preferred embodiment, the composition contains excipients selected from the group consisting of: inulin (10% w / v); maltodextrin (10% w / v); and sucrose (10% w / v).

[0159] In a preferred embodiment, the microorganism is Lactobacillus plantarum.

[0160] In another aspect, the present invention is a biotherapeutic composition comprising the composition of the first aspect of the invention, and an acceptable diluent or carrier.

[0161] In a preferred embodiment, the carrier is 0.9% sterile saline.

[0162] In another aspect, the present invention is a pharmaceutical composition comprising the composition of the first aspect of the invention, and a pharmaceutically acceptable diluent or carrier.

[0163] In another aspect, the present invention is a method for treating and / or preventing a disease or ailment in a patient in need, the method comprising administering to a subject an effective amount of the composition of the first aspect of the present invention.

[0164] In a preferred embodiment, the disease or symptom is inflammation of the gastrointestinal mucosa.

[0165] In a preferred embodiment, the disease or symptom is characterized by a reduction in gut microbial diversity.

[0166] In a preferred embodiment, the disease or symptom is characterized by a decrease in gut microbial function.

[0167] In a preferred embodiment, the disease or symptom is characterized by a loss of gut microbial ecology.

[0168] In a preferred embodiment, the disease or symptom is ecological imbalance.

[0169] In a preferred embodiment, the ecological imbalance is associated with one or more conditions selected from the group consisting of: inflammatory bowel disease (IBD), pouchitis, irritable bowel syndrome (IBS), intestinal bacterial infection, metabolic disease, neuropsychiatric disorder, autoimmune disease, allergic disease, hepatic encephalopathy, or cancer.

[0170] In a preferred embodiment, the disease or ailment is a malignant disease or cancer.

[0171] In a preferred embodiment, the disease or condition is a hepatic disease or liver disease. For example, the disease or condition is primary sclerosing cholangitis.

[0172] In a preferred embodiment, the disease or symptom is a gastrointestinal symptom.

[0173] In a preferred embodiment, the gastrointestinal condition is inflammatory bowel disease.

[0174] In a preferred embodiment, the inflammatory bowel disease is selected from the group consisting of: ulcerative colitis; Crohn's disease; gastroenteritis; colitis; and pouchitis.

[0175] In a preferred embodiment, the gastrointestinal condition is selected from the group consisting of: irritable bowel syndrome; gastrointestinal ulcers; and gastrointestinal cancers.

[0176] In a preferred embodiment, the composition is administered orally or rectally.

[0177] In a preferred embodiment, the composition is administered in combination with or as a support for immunotherapy.

[0178] In a preferred embodiment, the composition is administered to the patient using a dosing regimen selected from the group consisting of: every hour; every 2 hours; every 3 hours; every 4 hours; every 5 hours; every 6 hours; every 12 hours; once daily; twice daily; every 2 days; every 3 days; every 4 days; every 5 days; every 6 days; weekly; twice weekly; every 2 weeks; every 3 weeks; every 4 weeks; every 5 weeks; every 6 weeks; once monthly; twice monthly; every 2 months; every 3 months; every 4 months; every 5 months; every 6 months; annually; twice annually; every 2 years; every 3 years; every 4 years; and every 5 years.

[0179] In a preferred embodiment, the composition reduces the level of endogenous sulfides in the colon of patients in need.

[0180] In a preferred embodiment, the composition reduces the load of sulfides and nitric oxide on epithelial cells that cause metabolic damage by inhibiting cellular respiration.

[0181] In a preferred embodiment, the composition reduces nitric oxide production in the colon and / or lowers nitric oxide levels in the colon.

[0182] In a preferred embodiment, the composition: reduces the relative abundance and / or metabolic activity of sulfide-producing microbiota; directly reduces sulfide levels in the colon by consumption / absorption; reduces sulfide levels, relative abundance, and / or metabolic activity of sulfide-producing microbiota by competition with metabolic substrates; and / or reduces sulfide levels, relative abundance, and / or metabolic activity of sulfide-producing microbiota by consuming hydrogen.

[0183] In a preferred embodiment, the composition reduces the relative abundance and / or metabolic activity of sulfide-producing microbiota by decreasing the amount of metabolizable sulfur substrates in the colon; and / or by reducing protein fermentation to reduce the release of sulfur amino acids (methionine, cysteine, homocysteine, taurine) into the colon, thereby reducing the relative abundance and / or metabolic activity of sulfide-producing microbiota.

[0184] In a preferred embodiment, the composition induces apoptosis in colon cells in the lesion to disrupt the induced stable inflammatory state.

[0185] In a preferred embodiment, the method reduces unwanted inflammation.

[0186] In a preferred embodiment, the composition prevents or reduces activation of the mucosal immune system in a natural killer T cell-driven IL-13 and IL-5 dependent, TH2-mediated immune response.

[0187] In a preferred embodiment, the method reduces inflammation in a subject when measured by method parameters selected from the group consisting of: TNFα signaling via NF-κB; IFNα signaling; IFNγ signaling; IL6 JAKSTAT3 signaling; activation of pro-apoptotic pathways; and initiation of unfolded protein responses.

[0188] In a preferred embodiment, the method downregulates genes associated with pro-apoptotic pathways and unfolded protein responses, including genes selected from the group consisting of CHAC1, CEBPB, TRIB3, PPP1R15A, DDIT3, ATF4, and XBP1.

[0189] In another aspect, the present invention is a method for preparing the biotherapeutic composition of the present invention, the method comprising mixing the composition of the first aspect of the present invention with an acceptable diluent or carrier.

[0190] In another aspect, the present invention is a method for preparing a pharmaceutical composition of the present invention, the method comprising mixing a composition of the first aspect of the present invention with a pharmaceutically acceptable excipient, diluent or carrier.

[0191] In a preferred embodiment, the excipient, diluent, or carrier is sterilized.

[0192] In a preferred embodiment, the cryoprotectant is sterilized.

[0193] In another aspect, the invention relates to the use of the composition of the first aspect of the invention for preparing a medicament for alleviating or preventing a disease or ailment of a subject.

[0194] In another aspect, the present invention is a dosage form comprising the composition of the first aspect of the present invention.

[0195] A kit comprising the dosage form of the present invention and its instructions for use.

[0196] Further features of the invention are described more fully in the following description of several non-limiting embodiments thereof. This description is included for illustrative purposes only and should not be construed as a broad generalization, disclosure, or limitation of the invention described above. Attached Figure Description

[0197] The following is a brief description of each of the figures and diagrams.

[0198] Figure 1The results of MicroPress analysis of three samples (Analysis 1, 2, and 3) of freeze-dried FMT cakes using a formulation containing 5% inulin and 5% maltodextrin cryoprotectant are shown. MicroPress analysis was used to quantitatively determine the strength and physical properties of in-situ freeze-dried cakes.

[0199] Figure 2 The mean folding loss, expressed in CFU / mL, is shown for FMT prepared with eight different cryoprotectant formulations when anaerobically plated on non-selective media after lyophilization. Error bars represent 1 S.D.

[0200] Figure 3 The mean CFU / mL of the intermediate product is shown for FMT prepared using eight different cryoprotectant formulations, after lyophilization and anaerobic plated on non-selective media. Error bars indicate 1 S.D.

[0201] Figure 4 Bar graphs showing the intact cell count (ICC) per gram of feces from eight batches of donor fecal material, when combined and when comparing net feces with the combined intermediate (feces homogenized in a cryoprotectant formulation), are presented. ICC was determined using a BactoBox (SBT Instruments).

[0202] Figure 5 Bar graphs showing the complete cell count (ICC) per gram of feces as determined by BactoBox (SBT Instruments, Inc.), including net feces (eight separate batches combined), intermediate products (combined feces homogenized in a cryoprotectant formulation), lyophilized products (after grinding), and packaged lyophilized products 'T0'.

[0203] Figure 6 Bar graphs are shown comparing changes in the intact cell count (ICC) per gram of feces in packaged lyophilized products as a factor of storage time (1 week, 2 weeks, 4 weeks, 2 months, 6 months) and temperature (-80°C, -20°C, 4-8°C, and 20-25°C). ICC was determined using a BactoBox (SBT Instruments). Detailed Implementation

[0204] For convenience, the following sections provide a general overview of the various meanings of the terms used herein. Following this discussion, general aspects concerning the uses and methods of the compositions, pharmaceuticals, and methods of the invention are discussed, followed by specific examples demonstrating the nature of the various embodiments of the invention and how they can be used.

[0205] Those skilled in the art will understand that variations and modifications may be made to the invention described herein, other than those specifically described. The invention includes all such variations and modifications. The invention also includes all steps, features, formulations, and compounds individually or collectively mentioned or indicated in this specification, and any and all combinations of any two or more of said steps or features.

[0206] Every document, reference, patent application, or patent cited in this article is expressly incorporated herein in its entirety by reference, meaning that the reader should read and consider it as part of this article. The documents, references, patent applications, or patents cited herein are not repeated here for reasons of brevity only. However, the cited material or the information contained therein should not be construed as common knowledge.

[0207] Manufacturer's instructions, descriptions, product specifications, and product lists of any products mentioned herein or in any document incorporated herein by reference are hereby incorporated by reference and may be used in the practice of this invention.

[0208] This invention is not limited in scope to any of the specific embodiments described herein. These embodiments are intended for illustrative purposes only. Functionally equivalent products, formulations, and methods as described herein are clearly within the scope of this invention.

[0209] 1. Definition

[0210] The following provides the meanings of certain terms and phrases used in this specification, examples, and appended claims. If there is a significant discrepancy between the terminology used in the art and the definitions provided herein, the definitions provided herein shall prevail.

[0211] Except where specified in operational examples or otherwise indicated, all figures used herein to represent amounts of components or reaction conditions should be understood to be modified by the term “about.” When used with percentages, the term “about” may mean ±1%.

[0212] The invention described herein may include one or more value ranges (e.g., size, concentration, etc.). A value range will be understood to include all values ​​within the range, including the value defining the range and values ​​adjacent to the range that produce the same or substantially the same result as the value immediately adjacent to the boundary defining the range. For example, those skilled in the art will understand that a 10% variation in the upper or lower limit of the range may be perfectly suitable and covered by this invention. More specifically, the variation in the upper or lower limit of the range will be 5% or, as is generally accepted in the art, whichever is greater.

[0213] In this application, unless otherwise expressly stated, the singular includes the plural. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not restrictive. Furthermore, unless otherwise specifically stated, terms such as "element" or "component" cover both elements and components comprising one unit and elements and components comprising more than one subunit. Furthermore, the use of the term "part" can include a portion or the entire part.

[0214] Throughout this specification, unless the context otherwise requires, the word “comprise” or variations such as “comprises” or “comprising” should be understood to imply inclusion of the stated whole or group of wholes, but not to exclude any other whole or group of wholes.

[0215] As used herein, the term "therapeuticly effective amount" in relation to treatment methods and, in particular, drug dosage, should mean the dose that provides a specific pharmacological response when administered to a large number of subjects requiring such treatment. It should be emphasized that a "therapeuticly effective amount" administered to a specific subject in a specific situation is not always effective in treating the disease described herein, even if such a dose is considered "therapeuticly effective" by those skilled in the art. It should be further understood that, in a specific situation, drug dosage is measured as an oral dose or with reference to drug levels measured in the blood. The effective amount for this purpose will depend on: the desired therapeutic effect; the potency of the bioactive material; the desired duration of treatment; the stage and severity of the disease being treated; the patient's weight and general health condition; and the judgment of the prescribing physician. Treatment dosages need to be adjusted to optimize safety and efficacy. Those skilled in the art will understand that the appropriate dose level for treatment will therefore vary in part depending on the indication for use of the active agent, the route of administration, and the patient's body size (weight, body surface or organ size) and condition (age and general health condition). Therefore, clinicians may titrate the dosage and modify the route of administration to obtain the best therapeutic effect. Based on the factors described above, the typical dosage range can be from about 0.1 μg / kg to at most about 100 mg / kg or more. In other embodiments, the dosage range can be from 0.1 μg / kg to at most about 100 mg / kg; or from 1 μg / kg to at most about 100 mg / kg; or from 5 μg / kg to at most about 100 mg / kg.

[0216] The frequency of administration will depend on the pharmacokinetic parameters of the active agent and formulation used. Typically, clinicians will administer the composition until a dose is reached to achieve the desired effect. Therefore, the composition may be administered as a single dose or in two or more doses over time (which may or may not contain the same amount of the desired molecule) or as a continuous infusion via an implantable device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of their usual duties. An appropriate dosage can be determined by using suitable dose-response data.

[0217] As used herein, “carrier” can be any solvent, diluent, excipient or other medium, dispersant or suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant, etc., as long as it is suitable for the desired specific dosage form.

[0218] As used herein, the term "pharmaceutically acceptable carrier" component can refer to a component that is not biologically or otherwise undesirable, i.e., a component that can be incorporated into the compositions of the present invention and administered to subjects as described herein without causing any significant undesirable biological effects or interacting in a harmful manner with any other components of formulations containing it. The component generally meets the requirements of toxicological and manufacturing standards.

[0219] As used herein, the term "subject" generally includes mammals such as: humans; farm animals such as sheep, goats, pigs, cows, horses, and llamas; companion animals such as dogs and cats; primates; birds such as chickens, geese, and ducks; fish; and reptiles. Humans are preferred subjects.

[0220] As used herein, the "gastrointestinal tract" refers to the passage from the mouth to the anus, including all organs of the digestive system such as the esophagus, stomach, pancreas, liver, gallbladder, small intestine (including the ileum), cecum, large intestine, colon, and rectum. The strains of this invention are applicable to at least the terminal ileum, cecum, or rectum for conditions.

[0221] As used herein, "non-inflammatory strain" refers to the strain of the present invention, which, when present in the gastrointestinal tract of a subject (preferably a human), is associated with a non-inflammatory state. The non-inflammatory strain of the present invention exhibits minimal or no cytotoxicity to cultured mammalian epithelial cells. In one embodiment, said strain causes cell death in cultured mammalian epithelial cells of less than 15%, less than 10%, or less than 5%.

[0222] As used herein, "inflammatory strain" refers to the strain of the present invention, which, when present in the gastrointestinal tract of a subject (preferably a human), is associated with an inflammatory state. The inflammatory strain of the present invention is cytotoxic to cultured mammalian epithelial cells (such as Caco 2 cells). In one embodiment, said strain causes at least 40%, at least 45%, or at least 50% cell death in cultured mammalian epithelial cells.

[0223] As used herein, the term “bacterial therapy” refers to the use of bacterial isolates to treat or prevent a disease or condition in a subject or to provide a health benefit.

[0224] As used herein, the term “biotherapy” refers to microorganisms, such as bacterial isolates, that can be used to treat or prevent a disease or condition in a subject or to provide a health benefit.

[0225] As used herein, the term "biothermal composition" refers to a formulation comprising a biological therapeutic agent, which is formulated together with one or more other prescription ingredients to obtain a finished formulation suitable for delivery to a subject.

[0226] As used herein, the term "treat" and its grammatical variations refer to subjecting an individual subject to a protocol, therapy, procedure, or treatment in which a physiological response or outcome is expected in the subject. Because each treated subject may not respond to a particular protocol, therapy, procedure, or treatment, treatment is not required to achieve the desired physiological response or outcome in every subject or group of subjects. Therefore, a given subject or group of subjects may not respond to treatment or may respond inadequately.

[0227] As used herein, the term “prevent, prevented, or preventing” in relation to the treatment of gastrointestinal mucosal inflammation refers to preventive treatment that increases a subject’s resistance to gastrointestinal mucosal inflammation, in other words, reducing the likelihood of a subject developing gastrointestinal mucosal inflammation, and treatment after the onset of gastrointestinal mucosal inflammation to combat the inflammation, such as reducing or eliminating the inflammation or preventing it from worsening.

[0228] As used herein, the term “relief” or variations thereof refers to the reduction, but not necessarily the complete elimination, of gastrointestinal mucosal inflammation in the subject.

[0229] As used herein, the term "sample" refers to a collection of biological material obtained from a subject or the subject's surrounding environment (such as soil or water in the area where the subject resides). In some embodiments, the sample is obtained directly from the subject. For example, the sample may be a fecal sample or a sample obtained during a colonoscopy. The sample may be in a form obtained directly from the subject or the surrounding environment, or it may be at least partially purified to remove at least some non-nucleic acid material. Purification may be minimal, such as a total concentration not exceeding that of a solid, or reducing the sample cells to a smaller volume or separating some or all of the cells from the remainder of the sample. In some embodiments, nucleic acids are isolated from the sample. Such isolated preparations include reverse transcription products and / or PCR amplification products of the nucleic acids in the sample. In some embodiments, the primary nucleic acid is DNA. The nucleic acid preparation may be a pure or partially purified nucleic acid preparation. Many techniques are available for isolating nucleic acids from samples, including complex samples, and are well known in the art.

[0230] The unit "cfu" stands for "colony forming unit," which is the number of live microbial cells revealed by microbial counting on an agar plate.

[0231] The unit "%w / v" refers to the weight / volume percentage concentration. It is also known as the mass / volume percentage concentration. Weight / volume percentage concentration is also abbreviated as w / v(%), w / v%, (w / v)%, %(w / v), or %w / v. Mass / volume percentage concentration is also abbreviated as m / v(%), m / v%, (m / v)%, %(m / v), or %m / v.

[0232] Other definitions of the selected terms used herein may be found in the specific embodiments of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0233] The features of the invention will now be discussed with reference to the following non-limiting description and examples.

[0234] 2. Example

[0235] Composition

[0236] The present invention provides a composition for the prevention or treatment of a condition in a subject in need, the composition comprising at least one bacterial, archaea, or fungal strain.

[0237] In another preferred embodiment, the composition is selected from the group consisting of: therapeutic compositions; pharmaceutical compositions; cosmetic compositions; and veterinary compositions.

[0238] Preferably, the composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be used for human or animal purposes). Suitable carriers and diluents include isotonic saline solutions, such as phosphate-buffered saline. As used herein, "pharmaceuticalally acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is contemplated, except in cases where any conventional culture medium or agent is incompatible with the active ingredient. Complementary active ingredients may also be incorporated into the composition. See, for example, Remington's Pharmaceutical Sciences, 19th edition (1995, Mack Publishing Co., Easton, PA.) and Remington's The Science and Practice of Pharmacy, 23rd edition (2020, Mack Publishing Co., Easton, PA.), which are incorporated herein by reference.

[0239] The composition may contain formulation materials for modifying, maintaining, or preserving, for example, the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or permeability. Suitable formulation materials include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); swelling agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); compounding agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring agents; flavoring agents and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); and low molecular weight polymers. Peptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, dehydrated sorbitol esters, such as polysorbate 20, polysorbate 80, etc., triton, tromethamine, lecithin, cholesterol, or tyloxapol); stability enhancers (sucrose or sorbitol); tension enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride); delivery mediators; diluents; excipients and / or pharmaceutical adjuvants.

[0240] The optimal composition will be determined by those skilled in the art based on, for example, the intended route of administration, delivery format, and desired dosage. Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the biotherapeutic active substances of the present invention. The preferred form of the pharmaceutical composition depends on the intended administration mode and therapeutic application.

[0241] The primary medium or carrier in the composition is inherently aqueous. Suitable mediums or carriers may be water for injection, physiological saline solutions, and possibly supplemented with other materials. Neutral buffered saline or saline mixed with serum albumin are other exemplary mediums. Other exemplary pharmaceutical compositions comprise a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, which may further comprise sorbitol or a suitable alternative thereof. In one embodiment of the invention, a pharmaceutical composition for storage can be prepared by mixing a selected composition having the desired purity with an optional formulation in aqueous solution form.

[0242] The formulation components are present at concentrations acceptable at the application site. For example, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8.

[0243] Other compositions will be apparent to those skilled in the art, including formulations of the present invention in sustained-release or controlled-release formulations. Techniques for formulating a variety of other sustained-release or controlled-release methods, such as liposome carriers, biodegradable microparticles or porous beads and depot injections, are also known to those skilled in the art. Further examples of sustained-release formulations include semi-permeable polymer matrices in the form of molded articles, such as films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0244] Compositions intended for in vivo administration can be filtered to remove unwanted components. This can be achieved through filtration using a membrane filter. Additionally, the composition is typically placed in a sealed container to minimize oxygen exposure. Once the pharmaceutical composition is prepared, it can be stored in a sealed container.

[0245] As used herein, the term "sequence homology %" can be calculated, for example, as follows: The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al., Nucleic Acids Research, 22:4673-4680 (1994)). The comparison is made within a window corresponding to one of the aligned sequences (e.g., the shortest sequence). In some cases, the window may be defined by the target sequence. In other cases, the window may be defined by the query sequence. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have the same correspondence with the target sequence is reported as the sequence homology %.

[0246] In one embodiment, the polynucleotide identity percentage is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG procedure), where a vacancy production penalty = 5 and a vacancy extension penalty = 0.3. Preferably, the GAP analysis compares the two sequences over their entire length.

[0247] The bacterial strains used in this invention can be cultured using microbiological techniques detailed in, for example, the following literature: Browne et al., “Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation,” *Nature* (2016), Vol. 533, pp. 543-546; *Handbook of Microbiological Media*, 4th Edition (2010), Ronald Atlas, CRC Press; *Maintaining Cultures for Biotechnology and Industry* (1996), Jennie C. Hunter-Cevera, Academic Press; and the use of yeast extract, casein, and fatty acid (YCFA) media detailed in the examples.

[0248] In yet another preferred embodiment, the composition further comprises water.

[0249] In yet another preferred embodiment, the composition is a liquid, such as an aqueous solution.

[0250] In yet another preferred embodiment, a pharmaceutically acceptable carrier is further included.

[0251] In yet another preferred embodiment, the composition retains its effective biological activity for a period of time selected from the group consisting of: greater than 24 hours; greater than 36 hours; and greater than 48 hours. Preferably, the composition is stable for a period of time selected from the group consisting of: 6 months, 1 year, and 2 years. In one example, the composition is stable at temperatures selected from the group consisting of: -4°C, 4°C, 18°C, and 25°C.

[0252] Pharmaceutical compositions and therapeutic compositions are within the scope of this invention.

[0253] The therapeutic compositions of the present invention may comprise pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the isolated bacteria present in the therapeutic composition. The exact nature of the pharmaceutically acceptable excipients or other materials will depend on the route of administration, which may be, for example, oral or rectal. Many methods for preparing therapeutic compositions are known to those skilled in the art (see, for example, Robinson, ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978).

[0254] The therapeutic compositions of the present invention may contain prebiotics, carriers, insoluble fibers, buffers, penetrants, defoamers and / or preservatives.

[0255] The therapeutic composition may be prepared or provided in a chemostat medium. Alternatively, the therapeutic composition may be prepared or provided in saline (e.g., 0.9% saline). It should be understood that any carrier or solution that does not impair the viability of the bacteria present in the therapeutic composition and is compatible with individual administration may be used.

[0256] The therapeutic composition can be prepared or provided under a reducing atmosphere, i.e., under anaerobic conditions. Synthetic fecal preparations can be prepared or provided under N2, CO2, H2, or mixtures thereof, optionally with control of the partial pressure levels of N2:CO2:H2.

[0257] The therapeutic composition can be prepared or provided under an oxygen-containing atmosphere.

[0258] The therapeutic composition can be used for oral or rectal administration to an individual. When used for oral administration, the therapeutic composition can be in the form of capsules or tablets. When used for rectal administration, the therapeutic composition can be in the form of enemas or suppositories. The preparation of suitable capsules, tablets, suppositories, and enemas is well known in the art. Capsules or tablets may contain a coating to protect them from gastric acid. For example, capsules or tablets may be enteric-coated, pH-dependent, sustained-release, and / or gastric-tolerant. Such capsules and tablets are intended, for example, to minimize dissolution in the stomach but allow dissolution in the small intestine.

[0259] For example, in addition to live microorganisms, formulations for oral administration may contain inert compression aids (such as microcrystalline cellulose or oligosaccharides), flow aids (such as silica gel), or lubricants (such as magnesium stearate (plant-derived) or stearic acid (plant-derived)).

[0260] The compositions disclosed herein can be used, for example, as food supplements, edible products, or pharmaceutical products. When the composition is a food supplement, it may further comprise conventional food supplement fillers and / or extenders. The compositions disclosed herein may also be included in any edible product, such as dairy products, including, for example, dairy products, milk, yogurt, curd, ice cream, condiments, and cheese, beverage products, meat products, and baked goods.

[0261] For example, suppository formulations for rectal use may contain, in addition to the composition, ingredients such as cocoa butter, polyethylene glycol, glycerin, or gelatin.

[0262] The composition may contain a disintegrant, a flow aid, and / or a lubricant. When placed in a fluid environment, the disintegrant helps to disintegrate the compacted material. The disintegrant can be any suitable disintegrant, for example, a disintegrant selected from the group consisting of: croscarmellose sodium, croscarmellose, gellan gum, hydroxypropyl cellulose, starch, and sodium glycolate starch. The flow aid can be any suitable flow aid, for example, a flow aid selected from the group consisting of: silica, colloidal silica, and talc. Lubricants are generally always used to prevent the compressed powder material from adhering to the equipment during the tableting or encapsulation process when preparing dosage forms by direct compression. The lubricant can be any suitable lubricant, for example, a lubricant selected from the group consisting of: calcium stearate, magnesium stearate, stearic acid, sodium stearate fumarate, and plant-based fatty acids. In the compositions and methods of the present invention, the carrier may be present in the composition in the range of about 30% w / w to about 98% w / w; this weight percentage is a cumulative weight percentage taking into account all components present in the carrier.

[0263] Coating can be used to control the solubility of a composition. Examples of coatings include carrageenan, cellulose acetate phthalate, ethyl cellulose, gellan gum, maltodextrin, methacrylates, methyl cellulose, microcrystalline cellulose, and shellac.

[0264] The composition may contain one or more preservatives. Exemplary preservatives include antioxidants, chelating agents, antifungal preservatives, alcohol preservatives, acid preservatives, and other preservatives.

[0265] Exemplary antioxidants include alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0266] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzyl chloride, benzyl alcohol, bromonitrol, cetrimonium bromide, hexadecylpyridine chloride, chlorhexidine, chlorobutanol, chlorocresol, xyloxyphenol, cresol, ethanol, glycerol, hexetidine, imidureurium, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0267] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, and phenylethyl alcohol.

[0268] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0269] Other preservatives include tocopherol, tocopheryl acetate, deteroximemesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolon, Kathon, and Euxyl.

[0270] The therapeutic composition may be lyophilized. A lyophilized therapeutic composition may contain one or more stabilizers and / or cryoprotectants. The lyophilized therapeutic composition may be reconstituted with a suitable diluent prior to individual administration.

[0271] The therapeutic compositions according to the invention can be administered alone or simultaneously or sequentially in combination with other treatments, or as a combination formulation with one or more other therapeutic agents, for the treatment of dysbiosis or dysbiosis-related diseases as described herein. For example, the strains of the invention can be used in combination with existing therapeutic agents for inflammatory bowel disease, irritable bowel syndrome, metabolic diseases, neuropsychiatric disorders, autoimmune diseases, allergic diseases, cancer, or hepatic encephalopathy.

[0272] For example, in cases where the therapeutic composition is used to treat cancer-related dysbiosis, the composition may optionally be administered to the individual in combination with cancer immunotherapy, such as an immune checkpoint inhibitor. Examples of checkpoint inhibitors that may be used in this context include programmed cell death protein 1 (PD-1) inhibitors, programmed death ligand 1 (PD-L1) inhibitors, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors. It has been shown that the manipulation of the gut microbiota in combination with immune checkpoint inhibitor therapy can enhance the efficacy of immune checkpoint inhibitors in the treatment of cancer. In a preferred embodiment, the cancer in this context is lung cancer or melanoma.

[0273] In another embodiment, the compositions of the present invention further comprise an immunomodulatory compound. In other embodiments, the immunomodulatory compound is a cytokine, chemokine, or complement component that enhances the expression of immune system helper or adhesion molecules, their receptors, or combinations thereof. In some embodiments, the immunomodulatory compound includes interleukins (e.g., interleukin 1 to 15), interferon α, β, or γ, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), chemokines (such as neutrophil activating protein (NAP)), macrophage chemical inducers and activators (MCAF), RANTES, macrophage inflammatory peptides MIP-1a and MIP-1b, complement components, or combinations thereof. In other embodiments, immunomodulatory compounds stimulate or enhance the expression of OX40, OX40L (gp34), lymphocyte chemokines, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2 or 3, cytokine receptors or combinations thereof.

[0274] In another embodiment, the immunomodulatory compound induces or enhances the expression of co-stimulatory molecules involved in the immune response. In some embodiments, the co-stimulatory molecules include CD40 or its ligand, CD28, CTLA-4, or B7 molecules. In another embodiment, the immunomodulatory compound induces or enhances the expression of heat-stable antigen (HSA), chondroitin sulfate-modified MHC constant chain (Ii-CS), or intracellular adhesion molecule 1 (ICAM-1).

[0275] The therapeutic compositions of the present invention can be administered to individuals, preferably human individuals. They can be administered at a “therapeutic effective dose” sufficient to demonstrate benefit to the individual. Such benefit may be improvement of at least one symptom. Therefore, “treatment” for a particular disease refers to improvement of at least one symptom. The actual dosage, as well as the rate and timing of administration, will depend on the nature and severity of the disease being treated, the specific patient being treated, the individual patient’s clinical condition, the cause of the dysbiosis, the site of delivery of the composition, the type of therapeutic composition, the method of administration, the timing of administration, and other factors known to the practicing physician. Treatment prescriptions (e.g., decisions regarding dosage, etc.) are the responsibility of general practitioners and other physicians and may depend on the severity of symptoms and / or the progression of the disease being treated. The therapeutic effective dose or appropriate dosage of the therapeutic compositions of the present invention can be determined by comparing their in vitro and in vivo activities in animal models or Phase 0 human studies. Methods for extrapolating effective doses from mice and other experimental animals to humans are known. The precise dosage will depend on many factors, including whether the therapeutic composition is for prevention or treatment.

[0276] The prescription ingredients may be brought into contact with and mixed or prepared until a formulation is obtained. As will be apparent to those skilled in the art, the formulation conditions are generally designed to preserve viable microorganisms. High temperatures, such as those exceeding 40°C, should be avoided in particular.

[0277] As those skilled in the art will appreciate, the amount of live microorganisms included in the composition can be varied and can be adjusted and optimized. For example, such optimization can be achieved by preparing a series of different doses of live microorganisms. The bacterial concentration in the composition can be, for example, 10 million CFU / mL to 100 billion CFU / mL, 10 million to 50 million CFU / mL, more preferably 50 million to 100 million CFU / mL, 100 million to 500 million CFU / mL, 500 million to 1 billion CFU / mL, 1 billion to 5 billion CFU / mL, 5 billion to 10 billion CFU / mL, 10 billion to 15 billion CFU / mL, 15 billion to 20 billion CFU / mL, 20 billion to 25 billion CFU / mL, 25 billion to 30 billion CFU / mL, or 30 billion to 35 billion CFU / mL. 35 billion to 40 billion CFU / mL, 40 billion to 45 billion CFU / mL, 45 billion to 50 billion CFU / mL, 50 billion to 55 billion CFU / mL, 55 billion to 60 billion CFU / mL, 60 billion to 65 billion CFU / mL, 65 billion to 70 billion CFU / mL, 70 billion to 75 billion CFU / mL, 75 billion to 80 billion CFU / mL, 80 billion to 85 billion CFU / mL, 85 billion to 90 billion CFU / mL, 90 billion to 95 billion CFU / mL, 95 billion to 100 billion CFU / mL.

[0278] In one embodiment, the strain of the present invention may be administered at a dose of, for example, 0.01 to 100 x 10¹¹ cells / body, 0.1 to 10 x 10¹¹ cells / body, or 0.3 to 5 x 10¹¹ cells / body. Furthermore, for example, the daily intake by the microorganism may be 0.01 to 100 x 10¹¹ cells / 60 kg body weight, 0.1 to 10 x 10¹¹ cells / 60 kg body weight, or 0.3 to 5 x 10¹¹ cells / 60 kg body weight.

[0279] The content of at least one bacterial, archaea, or fungal strain contained in the oral intake composition of the present invention can be appropriately determined according to the application. As dried microorganisms, it can be, for example, 5 to 50 w / w%, 1 to 75 w / w%, 0.1 to 100 w / w%, or 1 to 100 w / w%.

[0280] In one embodiment, the composition is a controlled-release composition. As used herein, the term "controlled release" refers to the release or administration of the strain of the invention from a given dosage form in a controlled manner to achieve desired in vivo pharmacokinetic characteristics. One aspect of "controlled" delivery is the ability to manipulate the formulation and / or dosage form in order to establish desired release kinetics.

[0281] Methods for preparing the tablets, capsules, and other forms of compositions of the present invention are known to those skilled in the art and include, but are not limited to, wet granulation, dry granulation, and direct compression (for tablets and capsules).

[0282] Wet granulation and dry granulation are used to prepare tablets, capsules, or pouches. For granulation technology, chilsonation is used to prepare powders for dosage forms. A chilsonator consists of grooved rotating rollers that are hydraulically pressed together. Raw materials are placed into the hopper of the chilsonator and fed into the rollers by a system of horizontal and vertical screws. As the material passes through the grooves on the rollers, it is compacted under very high pressure and discharged from the chilsonator as a dense sheet. The sheet is ground into a fine powder using a Fitz mill and then passed through a sieve to produce uniform, free-flowing particles. The density of the finished powder produced by the chilsonation process is two to four times that of the starting material, a characteristic that allows these ingredients to be formulated into the desired dosage form.

[0283] For dry granulation, the powder can be incorporated into gelatin capsules, or it can be mixed with gelatin to form tablets or capsules. For wet granulation, the powder is wetted, thus creating large "lumps" of material, which are then dried and ground to transform these lumps into particles of the desired size for the manufacturing process. Once the desired particle size is obtained, the particles are incorporated into gelatin capsules or mixed with gelatin to form tablets or capsules.

[0284] General considerations in formulation and / or preparation can be found, for example, in the following literature: Remington Pharmaceutical Science and Practice, Vol. 23 rd ed. (2020, Mark Publishing, Easton, PA). The cited reference is incorporated by way of citation.

[0285] prebiotics

[0286] The compositions of the present invention may contain prebiotics. Because the chemical structure of prebiotics is resistant to digestion in the digestive tract, they reach the colon in their intact molecular form, where they can trigger systemic physiological functions and act as fermentable substrates for the colonic microbiota. When prebiotics are combined with biotherapeutic agents, the resulting compositions are sometimes referred to as "synbiotics".

[0287] Examples of suitable prebiotics include, but are not limited to, oligosaccharides, such as fructooligosaccharides and P95. Examples include galacto-oligosaccharides, xyloo-oligosaccharides, isomaltooligosaccharides, human milk oligosaccharides, inulin oligosaccharides, mannan oligosaccharides, caramelized dextrin, fructans, maltotriose, pectin oligosaccharides, bimuno-galacto-oligosaccharides, arabinoylxylan, fucoidan, and resistant starch. Fructooligosaccharides can be extracted from, for example, chicory, artichokes, asparagus, dandelion, dahlia, endive, garlic, leeks, lettuce, and onions.

[0288] In one embodiment, the prebiotic comprises amino acids such as one or more of the following: alanine, aspartic acid, glutamic acid, glycine, leucine, isoleucine, proline, serine, threonine, and valine.

[0289] In one embodiment, the prebiotic contains a simple sugar, which may be a monosaccharide (such as glucose, galactose, or fructose) and / or a disaccharide (such as sucrose, maltose, or lactose).

[0290] In one embodiment, the prebiotic comprises about 5% (w / w) to about 50% (w / w), about 7.5% (w / w) to about 30% (w / w), or about 10% (w / w) to about 15% (w / w) of the composition.

[0291] Other microorganisms

[0292] To achieve the desired health benefits for the subjects, it may be advantageous to include one or more additional biotherapeutic microorganisms in the composition. Therefore, in addition to the strains of the present invention, the composition may contain more than one microbial species / strain, such as two, three, four, five, or more microbial species / strains. Non-limiting examples of biotherapeutic agents are suitable strains selected from Table 3. It should be understood that the foregoing list is for illustrative purposes only and is not a limiting representation of biotherapeutic agents that may be included in the compositions of the present invention. In this respect, any additional biotherapeutic species may also be used in the compositions of the present invention.

[0293] In one embodiment, enterococci include Enterococcus faecalis and / or Enterococcus faecium.

[0294] In one embodiment, the lactobacilli are selected from the group consisting of: Lactobacillus rhamnosus (e.g., strain GG (ATCC53103), CGMCC 1.3724, or SP1 (DSM 21690)); Lactococcus lactis; Streptococcus lactis; Lactococcus diacetyl lactis; Lactobacillus paracasei; Lactobacillus reuteri (e.g., strain ATCC 55730 or DSM 17938); Lactobacillus acidophilus; Lactobacillus murineis; Lactobacillus helveticus; Lactobacillus bulgaricus; Lactobacillus casei; Lactobacillus salivarius; Lactobacillus plantarum; Lactobacillus fermentum; Lactobacillus taiwanensis; Lactobacillus animalis; Lactobacillus johnsonii (e.g., strain NCC533; CNCM 1-1225); and Lactobacillus gasseri.

[0295] In one embodiment, the Bifidobacterium is selected from the group consisting of: Bifidobacterium lactis (e.g., strains BB-12, BI-04, or CNCM 1-3446 (Bb12)), Bifidobacterium longum (e.g., strains NCC3001, ATCC BAA-999 (BB536)), Bifidobacterium breve (e.g., strains Bb-03, M-16V, or R0070), Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium chainii, Bifidobacterium dendritum, Bifidobacterium pseudofidum, and Bifidobacterium adolescentis.

[0296] In one embodiment, streptococci include thermophilic streptococci, such as Streptococcus thermophilus ST-21, Streptococcus pasteurellii, and Streptococcus salivarius.

[0297] In one embodiment, the clostridium includes Clostridium difficile, Clostridium butyricum, Clostridium halys, Clostridium scintillans, Clostridium C1, Clostridium spiralis, and Flavobacterium prevalence.

[0298] Some yeasts can also be used as biotherapeutic agents and are sometimes included in compositions. A non-limiting example of a yeast used for biotherapy is *Bula spp.*

[0299] Some archaea can also be used as biotherapeutic agents and are sometimes included in compositions. Non-limiting examples of archaea used for biotherapy are methanogenic shortbacteria, including *Methanobrevibacter stadtmanae* and methanogenic cocci, including *Methanobrevibacter stadtmanae*.

[0300] Dosage form

[0301] Dosage forms are within the scope of this invention. In a preferred embodiment, the invention provides dosage forms comprising the compositions described in the first aspect of the invention. Preferably, the dosage forms are stored in sealed and sterile containers.

[0302] Methods of treatment

[0303] Methods for treating diseases or conditions are within the scope of this invention.

[0304] Methods for treating gastrointestinal disorders are also within the scope of this invention.

[0305] In a preferred embodiment, the present invention provides a method for treating a condition associated with gut microbiota loss or dysbiosis, wherein the method comprises administering a therapeutically effective amount of a composition as described in the first aspect of the invention to a patient in need.

[0306] Fecal microbiota transplantation

[0307] Fecal microbiota transplantation (FMT) involves administering the human colonic microbiota into a patient's gut. While initially designed to treat Clostridium difficile infection, FMT is currently being explored for the treatment of many other conditions, including ulcerative colitis. However, these efforts are challenged by the loss of viable microbiota samples due to poor sampling, storage, transportation, and delivery conditions that lead to the loss of live cells. Sampling, storing, transporting, and delivering viable and effective microbiota samples to patients presents challenges. There is also a need in the field for effective treatments for diseases associated with gut microbiota loss or dysbiosis.

[0308] Therapies based on cultured or second-generation microbiome

[0309] Clinical trials have demonstrated the efficacy and safety of FMT for a variety of diseases, including but not limited to Clostridium difficile infection, ulcerative colitis, and irritable bowel syndrome. Therefore, there is a need to identify the microorganisms for therapies based on defined culture-based microbiomes. Compared to FMT, culture-based therapies offer the advantage of being produced in bioreactors (non-human donor source), and are therefore more compositionally consistent, more scalable in production, and have more predictable safety profiles. Ideally, second-generation microbiome-based therapies would initially be cultured and isolated from human fecal material. Many of these organisms are highly sensitive to oxygen environments and will not survive in storage unless frozen or lyophilized (freeze-dried) at very low temperatures. Sampling, storing, transporting, and delivering viable and effective microbiome samples to patients presents challenges. There is also a need in the art for effective treatments for diseases associated with gut microbiome loss or dysbiosis.

[0310] In another preferred embodiment, the dosage form is administered in an amount sufficient to at least partially treat a gastrointestinal symptom.

[0311] Subjects who can be treated with this invention will include humans as well as other mammals and animals.

[0312] The effects of the applied therapeutic composition can be monitored through standard diagnostic procedures.

[0313] The method of the present invention can be used to treat or prevent gastrointestinal dysbiosis in subjects. In the context of the present invention, "dysbiosis" refers to a state in which the normal diversity and / or function of the microbiome, particularly the human gastrointestinal microbiome, is disrupted. Any disruption of the normal state of the microbiome in a healthy individual can be considered dysbiosis, even if this dysbiosis does not cause a detectable decline in the individual's health. In a preferred embodiment, dysbiosis may be associated with one or more pathological symptoms. For example, "dysbiosis" may refer to a reduction in the microbial diversity of the microbiome. Alternatively or additionally, "dysbiosis" may refer to an increase in the abundance of one or more bacteria (e.g., one or more pathogenic bacteria) in an individual's microbiome relative to the abundance of said one or more bacteria in the microbiome of a healthy individual (i.e., an individual without dysbiosis). The pathogenic bacteria present during dysbiosis are typically Proteobacteria and resistant to one or more antibiotics. Examples of Proteobacteria include Escherichia coli, Salmonella, Campylobacter, Vibrio, Helicobacter pylori, and Yersinia.

[0314] Ecological imbalance can be associated with intestinal bacterial infections, such as gastrointestinal infections caused by pathogenic bacteria. Many bacteria are known to cause gastrointestinal infections in humans, including both Gram-positive and Gram-negative bacteria. Pathogenic bacteria are preferably pathogenic species of the genera *Clostridium*, *Escherichia coli*, *Enterococcus*, *Klebsiella*, *Enterobacter*, *Proteus*, *Salmonella*, *Shigella*, *Staphylococcus*, *Vibrio*, *Aeromonas*, *Campylobacter*, *Pseudomonas*, *Bacillus*, *Helicobacter pylori*, *Listeria*, or *Yersinia*. Preferred examples of such pathogens include Clostridium difficile, Clostridium perfringens, Clostridium botulinum, Escherichia coli, Salmonella typhi, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Campylobacter fetus, Campylobacter jejuni, Aeromonas hydrophila, Plesiomonas shigelloides, Bacillus cereus, Helicobacter pylori, Listeria monocytogenes, and Yersinia enterocolitica. More preferably, the pathogen is a pathogenic species of the genus Clostridium or Escherichia. Most preferably, the pathogen is Clostridium difficile or Escherichia coli.

[0315] The method of the present invention can be used to reduce or prevent gastrointestinal mucosal inflammation in subjects using the compositions of the present invention.

[0316] In one embodiment, the subject had or was susceptible to inflammatory bowel disease (IBD). IBD is an increasingly prevalent and currently incurable condition believed to result from an abnormal immune response to the resident gut microbiota in genetically susceptible individuals. The term IBD encompasses ulcerative colitis (UC), Crohn's disease (CD), and enteritis. UC is characterized by chronic, non-granulomatous inflammation confined to the colonic mucosa, typically involving the rectum and a continuously variable proximal colon. CD is characterized by transmural inflammation, usually granulomatous, that can involve any part of the gastrointestinal tract from the mouth to the anus.

[0317] As used herein, the term “inflammatory bowel disease (IBD)” has its general meaning in the art and refers to a group of inflammatory diseases of the colon and small intestine, such as those revised in World Health Organization classification (ICD-10) K20-K93, including Crohn's disease (e.g., granulomatous colitis; small bowel Crohn's disease; large bowel Crohn's disease; granulomatous and regional colitis; Crohn's disease of the colon, large intestine and rectum; small bowel and large intestine Crohn's disease), ulcerative colitis (e.g., ulcerative (chronic) pancolitis; reflux ileitis; ulcerative (chronic) proctitis; ulcerative (chronic) rectosigmoiditis; inflammatory polyps; left colitis; left hemicolitis) and non-inflammatory bowel disease. Infectious gastroenteritis and colitis (radiation-induced gastroenteritis and colitis; toxic gastroenteritis and colitis; allergic and dietary gastroenteritis and colitis; food allergy gastroenteritis or colitis; indeterminate colitis; specific non-infectious gastroenteritis and colitis, such as collagenous colitis; eosinophilic gastritis or gastroenteritis; lymphocytic colitis, microscopic colitis (collagenous colitis or lymphocytic colitis); non-infectious gastroenteritis and colitis, such as diarrhea; enteritis; ileitis; jejunitis; sigmoid colitis) and postoperative conditions of the digestive system, such as haustra colitis. In one embodiment, IBD is pediatric IBD.

[0318] In one embodiment, the subjects had or were susceptible to all forms of irritable bowel syndrome (IBS) as detailed in the Rome IV criteria. These include, but are not limited to, diarrhea-predominant IBS-D, constipation-predominant IBS-C, or mixed IBS-M. This includes subjects whose symptoms responded to a low-FODMAP diet, a gluten-free diet, or other dietary restrictions.

[0319] In one embodiment, the subject has or is susceptible to malignant diseases or cancers. These include, but are not limited to, melanoma, lung cancer, colorectal cancer, stomach cancer, esophageal cancer, oral cancer, hepatocellular carcinoma, hematologic malignancies, breast cancer, lymphoma, sarcoma, germ cell tumors, carcinoma, kidney cancer, prostate cancer, pancreatic cancer, ovarian cancer, thyroid cancer, brain malignancies, skin cancer, melanoma, bladder cancer, or testicular cancer.

[0320] In one embodiment, the subject has or is susceptible to liver disease or liver disorders. These include, but are not limited to, cirrhosis, hepatic encephalopathy, alcoholic hepatitis, infectious hepatitis, autoimmune hepatitis, or ascites.

[0321] In one embodiment, the subject had or was susceptible to diseases associated with dysbiosis or reduced gut microbial diversity or reduced gut microbial ecology.

[0322] In another aspect, the present invention also relates to fecal microbiota transplantation compositions comprising one or more strains of the present invention. The term "fecal microbiota transplantation composition" has its general meaning in the art and refers to any composition capable of restoring the fecal microbiota.

[0323] Human administration includes administration by a healthcare professional and self-administration. Typically, to obtain health benefits, multiple doses of the composition are administered, such as daily administration for a period of at least one week, at least two weeks, at least three weeks, at least six weeks, at least nine weeks, or at least twelve weeks. In one embodiment, the composition may be administered for the remainder of the subject's life.

[0324] Device

[0325] The apparatus is within the scope of this invention. In a preferred embodiment, the invention provides an apparatus comprising: (1) a composition as described in the first aspect of the invention; and (2) an applicator, container, or material.

[0326] Use of the composition in pharmaceutical preparation

[0327] Uses are within the scope of this invention. In a preferred embodiment, the invention provides the use of the composition in the preparation of a medicament for treating gastrointestinal disorders.

[0328] Methods for stabilization

[0329] Methods for stabilizing the compositions of the present invention are within the scope of the present invention.

[0330] In another preferred embodiment, the method protects the compositions of the present invention from degradation.

[0331] In yet another preferred embodiment, the composition of the present invention retains its effective biological activity for a period of time selected from the group consisting of: greater than 24 hours; greater than 36 hours; greater than 48 hours.

[0332] From a safety perspective, adding approved pharmaceutical excipients to stabilize the composition of the solutions of the present invention is preferred because simpler methods may produce less variation, and the choice of excipients can be limited to those generally regarded as safe (GRAS). Based on the chemical properties and mechanisms of action of excipients, excipients used for protein solution stabilization can be classified into four main categories: salts, sugars, polymers, or proteins / amino acids. Salts (e.g., chlorides, nitrates) stabilize the tertiary structure of proteins by shielding charges through ionic interactions. Sugars (e.g., glycerol, sorbitol, fructose, trehalose) increase the surface tension and viscosity of the solution to prevent protein aggregation. Similarly, polymers (e.g., polyethylene glycol, cellulose derivatives) stabilize the tertiary structure of proteins by increasing the viscosity of the solution to prevent protein aggregation and intramolecular and intermolecular electrostatic interactions between amino acids in the protein. Proteins (e.g., human serum albumin) can stabilize the structure of other proteins through ionic, electrostatic, and hydrophobic interactions. Similarly, small amino acids with no net charge, such as alanine and glycine, stabilize proteins by forming weak electrostatic interactions.

[0333] As discussed above, the pharmaceuticals of the present invention may include one or more pharmaceutically acceptable carriers. The use of such media and agents in pharmaceutical preparation is well known in the art. Their use in the preparation of pharmaceutical compositions according to the present invention is contemplated, except in cases where any conventional media or agent is incompatible with pharmaceutically acceptable materials. Pharmaceutically acceptable carriers according to the present invention may include one or more of the following examples:

[0334] a. Surfactants and polymers, including but not limited to polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, crosslinked polyvinylpyrrolidone, polyvinylpyrrolidone-polyvinyl acrylate copolymer, cellulose derivatives, HPMC, hydroxypropyl cellulose, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose phthalate, polyacrylates and polymethacrylates, urea, sugars, polyols and their polymers, emulsifiers, gums, starches, organic acids and their salts, vinylpyrrolidone and vinyl acetate; and / or

[0335] b. Adhesives, such as various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose; and / or (3) fillers, such as lactose monohydrate, anhydrous lactose, microcrystalline cellulose and various starches; and / or

[0336] c. Fillers, such as lactose monohydrate, anhydrous lactose, mannitol, microcrystalline cellulose, and various starches; and / or

[0337] d. Lubricants, such as agents that enhance the ability of a dosage form to be ejected from the packaging cavity, and / or

[0338] e. Sweeteners, such as any natural or artificial sweeteners, including sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame K; and / or

[0339] f. Flavorings; and / or

[0340] g. Preservatives, such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parabens, such as butylparaben, alcohols, such as ethanol or benzyl alcohol, phenolic chemicals, such as phenol, or quaternary ammonium compounds, such as benzalkonium chloride; and / or

[0341] h. Buffers; and / or

[0342] i. Diluents, such as pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dicalcium phosphate, sugars and / or any mixtures thereof; and / or

[0343] j. Absorption enhancers, such as trinitroglycerin; and / or

[0344] k. Other pharmaceutically acceptable excipients.

[0345] The medicines of the present invention, applicable to animals and especially humans, must generally be sterile and stable under the conditions of preparation and storage.

[0346] Methods for detection

[0347] The strains of this invention can be detected using a variety of known techniques. Conveniently, nucleic acid-based detection systems can be used to detect the strains.

[0348] In one embodiment, nucleic acid sequencing is used. Illustrative and non-limiting examples of nucleic acid sequencing technologies include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. In some embodiments, the technologies provided herein can be used for second-generation (also known as next-generation or next-gen), third-generation (also known as next-next-generation), or fourth-generation (also known as N3) sequencing technologies, including but not limited to pyrosequencing, ligation sequencing, single-molecule sequencing, sequencing by synthesis (SBS), massively parallel cloning, massively parallel single-molecule SBS, massively parallel single-molecule real-time sequencing, and massively parallel single-molecule real-time nanopore technology.

[0349] In some embodiments, hybridization is used in the detection method of the present invention. Illustrative and non-limiting examples of nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarrays, and Southern or Northern blotting. In one embodiment, FISH assay is used. In other embodiments, nucleic acid amplification is used. Nucleic acids may be amplified before or simultaneously with detection. Performing one or more amplification reactions may comprise one or more PCR-based amplifications, non-PCR-based amplifications, or combinations thereof. Illustrative and non-limiting examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), nested PCR, linear amplification, multiple substitution amplification (MDA), real-time SDA, rolling circle amplification, loop-to-loop amplification, transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand substitution amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Those skilled in the art will recognize that some amplification techniques (e.g., PCR) require reverse transcription of RNA to DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

[0350] Nucleic acids, whether amplified or unamplified, can be detected by any conventional method. For example, nucleic acids can be detected by hybridizing with a detectably labeled probe and measuring the resulting hybrid. In another example, nucleic acids are detected by sequencing. This document describes illustrative and non-limiting examples of detection methods.

[0351] "Real-time" assessment of the amplification process involves continuously or periodically measuring the amount of amplicon in the reaction mixture during the amplification reaction and using the measured values ​​to calculate the amount of the target sequence initially present in the sample. Various methods for determining the amount of the initial target sequence present in a sample based on real-time amplification are well known in the art. These include the methods disclosed in U.S. Patents 6,303,305 and 6,541,205. Another method for determining the amount of the target sequence initially present in a sample (but not based on real-time amplification) is disclosed in U.S. Patent 5,710,029.

[0352] Amplification products can be detected in real time using a variety of self-hybridization probes, most of which have stem-loop structures. These self-hybridization probes are labeled so that they emit different detectable signals depending on whether the probe is in a self-hybridization state or a state altered by hybridization with a target sequence. As a non-limiting example, a "molecular torch" is a type of self-hybridization probe that includes different self-complementary regions (referred to as "target-binding domains" and "target-closing domains") connected by linker regions (e.g., non-nucleotide linkers) and hybridizing with each other under predetermined hybridization assay conditions. In a preferred embodiment, the molecular torch contains a single-stranded base region in the target-binding domain, which is 1 to about 20 bases in length and readily hybridizes with the target sequence present in the amplification reaction under strand substitution conditions. Under strand substitution conditions, hybridization of the two complementary regions of the molecular torch is advantageous; these two complementary regions can be fully or partially complementary unless a target sequence is present, which will bind to the single-stranded region present in the target-binding domain and replace all or part of the target-closing domain. The target-binding domain and target-sealing domain of the molecular torch include detectable markers or a pair of interacting markers (e.g., luminescent / quenching agents) positioned such that the signal generated when the molecular torch self-hybridizes differs from the signal generated when the molecular torch hybridizes with the target sequence, thereby allowing the detection of probe-target duplexes in the test sample in the presence of an unhybridized molecular torch. Molecular torches and various types of interacting marker pairs are disclosed in U.S. Patent No. 6,534,274, which is incorporated herein by reference in its entirety.

[0353] Another example of a self-complementary detection probe is a "molecular beacon." A molecular beacon comprises a nucleic acid molecule having a target complementary sequence, an affinity pair (or nucleic acid arm) that maintains the probe in a closed conformation in the absence of the target sequence during the amplification reaction, and a tag pair that interacts when the probe is in the closed conformation. Hybridization of the target sequence and the target complementary sequence causes the members of the affinity pair to separate, thereby transforming the probe into an open conformation. The transition to the open conformation can be detected due to reduced interaction between the tag pairs, which can be, for example, fluorophores and quenchers (e.g., DABCYL and 25EDANS). Molecular beacons are disclosed in U.S. Patents 5,925,517 and 6,150,097.

[0354] In one embodiment, the method includes quantifying the amount of bacterial strains present in the sample.

[0355] The invention will now be described with reference to the following non-limiting examples. These examples are not intended to limit the scope of the preceding paragraphs of this specification; however, they are provided to illustrate the methods and compositions of the invention.

[0356] Example

[0357] For those skilled in the art of grinding and pharmaceuticals, numerous enhancements and modifications can be made to the methods described above without departing from the basic concept of the invention. For example, in some applications, bioactive materials may be pretreated and supplied to the process in a pretreated form. All such modifications and enhancements are considered to be within the scope of the invention, the nature of which will be determined based on the foregoing description and the appended claims. Furthermore, the following examples are provided for illustrative purposes only and are not intended to limit the scope of the methods or compositions of the invention.

[0358] Example 1 - Study of Blending Materials

[0359] A.1 Research Objectives

[0360] The purpose of this experiment was to develop a cyclic lyophilization process for fecal suspension samples. Eight excipient formulations were added to samples with negative and positive controls. The specific objectives were as follows: (1) to collect data on the selected excipients and their concentrations to achieve an acceptable lyophilized product that does not kill most cells; and (2) to achieve a moisture content of less than 10% w / w.

[0361] A.2 Materials, Methods and Results

[0362] A.2.1 equipment

[0363] The following equipment was used in the preparation and freeze-drying steps of the formulation.

[0364] Table 6 - Equipment List

[0365]

[0366]

[0367] A.2.2 Excipients and methods

[0368] Prepare excipient solutions at twice the working concentration so that the correct final concentration can be achieved when the twice-strength excipient mixture is added to the FMT sample. Add the excipient stock solution w / v to the FMT solution to make a final volume of 3 ml per vial. Prepare 17 vials of each formulation (formulations 1 through 8) with a filling volume of 3 ml. Prepare 4 vials of each mixture for positive and negative controls.

[0369] Table 7: List of Original Excipients

[0370]

[0371] Table 8. Final excipient concentration

[0372]

[0373]

[0374] The material was filtered using a 0.101mm filter. The feces were homogenized and filtered during sample processing and after the addition of cryoprotectant and brine. This effectively filtered out large pieces of plant matter.

[0375] 0.9% sterile saline solution was used as a carrier for cryoprotectant samples.

[0376] A research batch record (BB374) was created to document the production of the excipients and the resulting 10 different formulations. Each cryoprotectant formulation was produced in 16 vials, with 4 vials each for positive and negative controls. The inventors retained one vial of each formulation (including controls) for testing purposes. 126 vials were to be sent to BioPharma Process Systems Ltd, Biopharma House, Winnall Valley Road, Winchester SO23 0LD, United Kingdom.

[0377] The research team reported the following results for the different cryoprotectants being tested. Inulin itself dissolves well when mixed by hand and is a very fine powder. Sucrose crystals are larger than inulin crystals and require a smaller volume to achieve the same weight. It dissolves under vortexing, but not by hand. Maltodextrin and dextran 70K are similar in texture to inulin, but they clump together when attempted to dissolve by hand and require vortexing to dissolve. Pectin is a very fine powder and difficult to weigh indoors. When used in formulations, it coagulates into a gel-like state and cannot be filtered for sterilization.

[0378] A.3 Analysis

[0379] The analysis includes:

[0380] Lyostat analysis: Two 2µl samples, a positive control skim milk powder and 10% inulin, were analyzed using Lyostat analysis. Lyostat analysis involves microscopic observation of the freeze lines of the product. This indicates the optimal operating temperature for the lyophilizer.

[0381] Appearance: The appearance of the freeze-dried products was visually evaluated by taking photographs on a scale of 1-5 (1 = worst, 5 = best).

[0382] Moisture content: The moisture content of the two formulations in three vials was analyzed by Karl Fischer titration.

[0383] Mechanical Properties: Two formulations were selected for mechanical property analysis using MicroPress. MicroPress contains a load cell with an actuator indenter. This indenter presses down to break the surface of the freeze-dried cake. The applied pressure as it presses down is displayed on the corresponding graph. The lower the maximum stress (kPa), the crisper the freeze-dried cake.

[0384] mDSC: Two formulations were selected for solid-state mDSC analysis.

[0385] Plate CFU: Plate CFU analysis was performed by comparing non-lyophilized and lyophilized samples of all formulations. The frozen non-lyophilized samples were thawed at room temperature outside an anaerobic environment for 1.5 to 2 hours. The lyophilized samples were rehydrated with the same amount of liquid removed during the lyophilization process and allowed to stand for 1 hour. The lyophilized samples were vortexed for 2 minutes to dissolve the lyophilized product into the solution. Then, 1 ml was aliquoted into 1.5 mL epi tubes and vortexed for 5 minutes. Serial dilutions were made in PBS (100 μL FMT diluted in 900 mL PBS), ranging from 10⁻³ to 10⁻⁸. 50 μL of the dilution was then plated in triplicate on WCA and immersed in agar. The plates were then inverted and incubated anaerobically at 37 °C for 48 hours. After 48 hours of incubation, the plates were checked to see if they were the “best” group among the triplicate plates with 30–300 non-confluent colonies. The plates were photographed and colony counts were performed.

[0386] A.4 Results

[0387] A.4.1 LYOSTAT analysis

[0388] Both skim milk powder (a) and 10% inulin (C) showed good cryogenic structure at -50.0°C. Skim milk powder (B) began to collapse at -47.0°C, while 10% inulin (D) also began to collapse at -30.5°C. This indicates that temperatures below -30.5°C are suitable for the sublimation of water when inulin is used as one of the cryoprotectants.

[0389] A.4.2 Appearance

[0390] Except for 10 negative control waters, all formulations that used a visually permeable cryoprotectant were treated as 5 / 5 for drying. Partial drying was observed. Due to difficulties in use and lack of consistency, pectin was not included in the analysis.

[0391] A.4.3 Moisture content

[0392] Moisture content determination of 5% inulin / 5% maltodextrin yielded an average moisture content of 1.093%, indicating that a moisture content (<5%) suitable for microbial storage can be obtained using the combination of 5% inulin and 5% maltodextrin. The results are presented in Table 9.

[0393] Table 9: Moisture content.

[0394]

[0395] A.4.4 Mechanical properties

[0396] MicroPress revealed that the sample was brittle. The results were compared to the negative control sample (highly brittle). See Table 10. See also... Figure 1 .

[0397] Table 10 - Maximum stress at the fracture point.

[0398]

[0399]

[0400] A.4.5 MDSC

[0401] mDSC results showed an initial Tg of 51.13 °C. Some studies have shown that the sample should be stable at approximately 50 °C below the initial Tg, suggesting that storage conditions of 2–8 °C may be suitable for the sample.

[0402] Table 11-mDSC

[0403]

[0404] A.4.6 CFU (Cardboard Unit)

[0405] Pectin addition showed the smallest decrease in CFU. The CFU of the pre-lyophilized pectin sample was also the lowest, and after lyophilization, it was similar to most other samples. 10% sucrose showed the second lowest CFU reduction before and after lyophilization, and the highest CFU. This is likely due to sucrose increasing CFU as a nutrient before plating. 5% inulin / 5% maltodextrin showed the smallest CFU reduction after lyophilization, and the second highest CFU. Based on both CFU and the lyophilization process, this was considered the preferred formulation. See also Figure 2 and 3 .

[0406] A.5 Discussion

[0407] The combination of inulin and maltodextrin was selected as the optimal cryoprotectant for freeze-drying. Inulin is a fiber that can ferment rapidly to moderately. The inventors obtained materials with a residual moisture content of less than 5%, resulting in freeze-dried cakes that were crisp and could be ground for further processing, with minimal cell loss observed by CFU plating. As can be seen from the above experiments, inulin and maltodextrin are superior to all other materials because both inulin and maltodextrin are beneficial during the freeze-drying process. The logarithmic change in CFU of inulin and maltodextrin was also minimal when comparing freeze-dried and non-freeze-dried materials. Inulin exhibited the ability to maintain the frozen structure without collapse at temperatures up to -30.5°C, allowing for temperature increases when water sublimates from the frozen freeze-dried cake. This is relatively close to the cake collapse temperature of around -30°C compared to trehalose. The inventors have demonstrated that, in a preferred embodiment, the compositions of the present invention can be freeze-dried faster than sucrose, resulting in shorter run times while still maintaining the viability within the sample. Maltodextrin also contributes to the stability of the freeze-dried sample during storage. It can stabilize the microorganisms within the freeze-drying matrix and help control the entry of moisture into the freeze-dried product.

[0408] Example B - Stability Study

[0409] B.1 Research Objectives

[0410] The objectives of this study were: (1) to determine whether the inulin / maltodextrin cryoprotectant formulation maintained intact cell counts (ICC) during short-term storage of intermediate (liquid) products; (2) to determine the effect of lyophilization on ICC; (3) to evaluate the effect of the inulin / maltodextrin cryoprotectant formulation on the ICC of packaged lyophilized FMT products over a 6-month period; and (4) to determine the optimal storage temperature for lyophilized packaged FMT. The primary focus was on determining how the cryoprotectant formulation's effectiveness in preserving ICC over time varied under different storage conditions, particularly -80°C, -20°C, 4-8°C, and 20-25°C.

[0411] B.2 Materials, Methods and Results

[0412] B.2.1 Excipient addition and lyophilization

[0413] The intermediate products (liquid) were processed from eight fecal donations from a single healthy donor over a one-month period. These intermediate products consisted of donor feces homogenized in an excipient solution containing 5% inulin and 5% maltodextrin, dissolved in 0.9% saline (NaCl) at a ratio of 1:2.6 w / v. The intermediate products were combined, and the resulting material was homogenized and filtered. The homogenized material was then stored and frozen at -80°C until lyophilized. The samples were then lyophilized according to the protocol and procedure described in Example 1. After lyophilization, the samples were ground, packaged, and stored in inductively sealed vials at the appropriate test temperature.

[0414] Table 12 - List of Original Excipients

[0415]

[0416] B.2.2 Sampling and complete cell counting

[0417] Integrity cell counts (ICCs) were determined using BactoBox (SBT Instruments) from the following samples: (1) net feces from each batch, combined according to the ratio of intermediate products (merged); (2) merged intermediate products (immediately pre-lyophilized); and (3) after lyophilization at the appropriate temperatures at multiple time points (ground powder, packaged product ('TO')), week 1, week 2, week 4, month 2, and month 6. All results were normalized to ICC per gram of feces.

[0418] B.3 Results

[0419] Eight batches of intermediate product were compared with net feces to determine whether the inulin / maltodextrin cryoprotectant formulation was sufficient as a short-term cryoprotectant for the intermediate product. Figure 4 Overall, at the time of testing, the ICC in the net fecal samples was not statistically significantly different from that of the intermediate products, except for batch 23, whose ICC was significantly lower than that of net fecal samples from the same donation. When the combined net fecal samples were compared with the combined intermediate products, the results were comparable (net fecal 1.97 x 10⁻⁶). 9 ICC / g (SD 1.17 x 10⁻⁶) 8 ) relative to the intermediate product 1.91 x 10 9 ICC / g (SD 2.14 x 10) 8 (p = 0.729)

[0420] To determine the viability loss caused by the freeze-drying process, the fecal ICC / g in pre-frozen clean feces and intermediate products was compared with that in freeze-dried samples after grinding and encapsulation of freeze-dried powder. Figure 5As mentioned above, there was no difference between the pre-freeze-dried feces and the intermediate product; however, there was a significant difference between the pre-freeze-dried feces (combined) and the intermediate product (containing both ground powder and packaged product). The average value of the intermediate product was determined to be 1.91 x 10⁻⁶. 9 ICC / g (SD 2.14 x 10) 8 The powder was ground to a density of 9.7 x 10⁻⁶. 8 ICC / g (SD 6.99 x 10^7) (p = <0.0001); the average value of the packaged product was 2.81 x 10^7 compared to the intermediate product. 8 ICC / g feces (SD 1.33 x 10^8) (p = 0.0007).

[0421] The encapsulated product serves as 'T0' for subsequent viability studies to compare the effects of storage time and temperature. Figure 6 No significant reduction in ICC was observed between T0 and any follow-up time point. Furthermore, storage temperature had no effect on ICC over time.

[0422] B.4 Discussion

[0423] The ability of the 5% w / v inulin and 5% w / v maltodextrin cryoprotectant formulation as a cryoprotectant for intermediate products, and its ability as a lyophilization protectant for fecal microbiota (FMT) via lyophilization of the intermediate products, was tested. Inulin and maltodextrin were suitable as cryoprotectants for the test timeframe, with ICC comparable to that of net feces. A significant decrease in cell viability throughout the lyophilization process was expected. The ICC decreased by approximately 0.78 log, within the acceptable and expected range of approximately 1 log reduction. Furthermore, the ICC remained consistent throughout the 28-week test period regardless of storage temperature, indicating that the inulin / maltodextrin cryoprotectant formulation maintained microbial stability at the storage time and temperatures tested in this study.

[0424] In summary, the efficacy of inulin and maltodextrin as cryoprotectants / lyophilization protectants for FMT has been demonstrated.

[0425] Example C 3 - Study on the formulation of BB265, a live biotherapy product for complex communities

[0426] C.1 Research Objectives

[0427] The aim of this study is to develop a lyophilization cycle for the complex community biotherapy product BB265. The specific objectives are as follows: (1) to collect data on the optimal excipients and excipient concentrations as determined in Example 1 to achieve an acceptable lyophilized product that does not kill most cells; and (2) to achieve a moisture content of less than 10% w / w.

[0428] C.2 Materials, Methods and Results

[0429] C.2.1 Composition of BB265

[0430] Table 13 - List of Classification Units in BB265 by Door

[0431]

[0432]

[0433] Table 14 - List of taxonomic units in BB265 by genus

[0434] genus *Bacillus* spp. of fecal cocci Marseille microbiota Akkermania Dorrylia Megamonas genus *Alternaria* The patient's weight exceeded the normal range for bacterial species. fecal bacteria AMED Microbes Enterobacteriaceae Osmotherium Anaerobic butyric acid-producing bacteria Clostridium erythropoiesis Vibrio family Anaerobic Corynebacterium Eubacterium Parabacterium Anaerobic Corynebacterium Bacillus faecalis Clostridium parasiticum Anaerobic small woody fungi spp. Maritime City Cocci Anaerobic clumps Fingoldella Porphyromonas spp. Bacillus genus *Flintibacterium* Prevotella Bacteroides genus Bacillus Micromonas spp. Barnes Holdmannii Rombutzella Bifidobacterium Hungarian bacteria spp. of Roseola Broutella Enterococcus spp. Rumenococcus Butyric Vibrio genus *Anaerobic bacillus* Russian bacillus Christensenella genus *Trichophyton* fecal monocytogenes Clostridium Teardrop-shaped Sporogens Alone spp. Collins Lactococcus Streptococcus Bacillus spp. Lactobacillus Thomas spp. Fecal bacteria Long chain bacteria Micromonas

[0435] Table 15 - List of taxonomic units in BB265 by species

[0436]

[0437]

[0438] C.2.2 equipment

[0439] The following equipment was used in the preparation and freeze-drying steps of the formulation.

[0440] Table 16 - Equipment List

[0441]

[0442]

[0443] C.2.3 Excipients and methods

[0444] An excipient solution containing 5.0% w / v inulin and 5.0% w / v maltodextrin dissolved in 0.9% saline (NaCl) was prepared and added to the granulated microbial particles collected from the BB265 complex at a ratio of 2.6:1 v / w. The mixture was then homogenized. The sample was immediately frozen at -80°C and transported frozen to BioPharma Process Systems Ltd., Winchester SO23 0LD, Winnal Valley Road, UK, for analysis.

[0445] Table 17 - List of Original Excipients

[0446]

[0447] C.2.4 analyze

[0448] The analysis includes:

[0449] Lyostat analysis: A 2 μL sample was analyzed using Lyostat analysis. Lyostat analysis involves observing the freeze lines of the product under a microscope. This indicates the appropriate operating temperature for the lyophilizer.

[0450] Appearance: The appearance of the freeze-dried products was visually evaluated by taking photographs on a scale of 1-5 (1 = worst, 5 = best).

[0451] Moisture content: The moisture content of the three vials was analyzed by Karl Fischer titration.

[0452] Mechanical Properties: Mechanical property analysis was performed using MicroPress. MicroPress contains a load cell with an actuator indenter. This indenter presses down to break the surface of the freeze-dried cake. The applied pressure as it presses down is displayed on the corresponding graph. The lower the maximum stress (kPa), the crisper the freeze-dried cake.

[0453] mDSC: Modulated differential scanning calorimetry (mDSC) is used to determine the thermal properties of formulations during freeze-drying.

[0454] C.3 Results

[0455] Lyostat analysis showed a good frozen structure at -50.0°C. Collapse began at -38.5°C, indicating that temperatures below -38.5°C would be suitable for water sublimation when using inulin and maltodextrin as cryoprotectants for BB265.

[0456] Appearance. All vials scored 5 / 5 in terms of appearance and structure after freeze-drying.

[0457] Moisture content. The measured average residual moisture content was 2.13% w / w, indicating that an appropriate moisture content (<5%) can be achieved for microbial storage using a combination of 5% inulin and 5% maltodextrin.

[0458] Table 18 - Residual Moisture Content

[0459]

[0460] Mechanical properties. MicroPress analysis indicates the robustness of the sample, with a maximum stress of 296.544 kPa at the fracture point and a Young's modulus of 4.80E.

[0461] Modulated differential scanning calorimetry (mDSC) was used. The mDSC results showed an initial Tg of 53.89 °C. Some studies have shown that the sample should be stable at approximately 50 °C below the initial Tg, suggesting that storage conditions of 2–8 °C may be suitable for the sample.

[0462] Table 19-mDSC

[0463]

[0464] C.4 Discussion

[0465] A combination of inulin and maltodextrin was selected as the optimal cryoprotectant for the freeze-drying of FMT materials and was used to evaluate the cryoprotection of the complex community biotherapeutic product BB265. The inventors obtained materials with a residual moisture content of less than 5%, resulting in a robust freeze-dried cake that could be milled for further processing. Based on the experiments presented above, the results for the inulin and maltodextrin combination are similar to those shown for FMT, indicating optimal cryoprotection parameters for the freeze-drying of BB265. The mixture demonstrated the ability to maintain its frozen structure without collapse at temperatures up to -38.5°C, allowing for temperature increases as water sublimates from the frozen freeze-dried cake. This is relatively close to the cake collapse temperature of around -30°C compared to trehalose.

Claims

1. A composition for use in preventing or treating a disease or condition in a subject in need thereof, the composition comprising at least one microbial strain, wherein the microorganism is selected from the group consisting of a bacterium, a yeast, or an archaeon; and an excipient.

2. The composition of claim 1, wherein the excipient is a cryoprotectant.

3. The composition of any one of claims 1-2, wherein the excipient is inulin or an analog or variant thereof.

4. The composition of claim 3, wherein the inulin is selected from the group consisting of a-D-glucopyranosyl-[b-D-fructopyranosyl](n-1)-D-fructofuranoside; b-D-fructopyranosyl-[D-fructopyranosyl](n-1)-D-fructofuranoside; fructooligosaccharide; fructooligosaccharide containing 2 to 70 fructose units; fructooligosaccharide containing 1 to 500 fructose units; fructooligosaccharide containing 1 to 300 fructose units; fructooligosaccharide containing 1 to 200 fructose units; fructooligosaccharide containing 1 to 100 fructose units; or an analog or variant or combination thereof.

5. The composition of any one of claims 1-4, wherein the excipient is maltodextrin or an analog or variant thereof.

6. The composition of any one of claim 5, wherein the maltodextrin is selected from the group consisting of a maltodextrin having a length selected from the group consisting of 3 to 17 glucose units; corn syrup having a length of 20 glucose units or more; corn syrup solids; modified corn starch; modified rice starch; modified tapioca starch; modified wheat starch; or an analog or variant or combination thereof.

7. The composition of any one of claims 1-6, wherein the composition comprises inulin or a variant analog thereof at a concentration selected from the group consisting of 0.01% w / v to 20% w / v; 0.1% w / v to 20% w / v; 0.1% w / v to 10% w / v; 1% w / v to 10% w / v; 2% w / v to 9% w / v; 3% w / v to 8% w / v; 4% w / v to 7% w / v; 4% w / v to 6% w / v; 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v.

8. The composition according to any one of claims 1 to 7, wherein the composition comprises an analogue of maltodextrin or a variant thereof, the concentration of which is selected from the group consisting of: 0.01% w / v to 20% w / v; 0.01% w / v to 20% w / v; 0.1% w / v to 10% w / v; 1% w / v to 10% w / v; 2% w / v to 9% w / v; 3% w / v to 8% w / v; 4% w / v to 7% w / v; 4% w / v to 6% w / v; 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v.

9. The composition according to any one of claims 1 to 8, wherein the composition comprises inulin and maltodextrin.

10. The composition according to any one of claims 1 to 9, wherein the composition comprises inulin and maltodextrin, the concentrations of said inulin and maltodextrin selected from the group consisting of: inulin (1% w / v) and maltodextrin (1% w / v); inulin (2% w / v) and maltodextrin (2% w / v); inulin (3% w / v) and maltodextrin (3% w / v); inulin (4% w / v) and maltodextrin ... 4% w / v); inulin (5% w / v) and maltodextrin (5% w / v); inulin (6% w / v) and maltodextrin (6% w / v); inulin (7% w / v) and maltodextrin (7% w / v); inulin (8% w / v) and maltodextrin (8% w / v); inulin (9% w / v) and maltodextrin (9% w / v); and inulin (10% w / v) and maltodextrin (10% w / v).

11. The composition according to any one of claims 1 to 10, wherein the composition comprises inulin and maltodextrin, the concentrations of said inulin and maltodextrin being selected from the group consisting of: (1) the concentration of inulin being selected from the group consisting of: 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v; and (2) the concentration of maltodextrin being selected from the group consisting of: 1% w / v; 2% w / v; 3% w / v; 4% w / v; 5% w / v; 6% w / v; 7% w / v; 8% w / v; 9% w / v; and 10% w / v.

12. The composition of any one of claims 1 to 11, wherein the composition comprises inulin and maltodextrin at a concentration selected from the group consisting of: inulin (1% w / v) and maltodextrin (1% w / v); inulin (2% w / v) and maltodextrin (2% w / v); inulin (3% w / v) and maltodextrin (3% w / v); inulin (4% w / v) and maltodextrin (4% w / v); inulin (5% w / v) and maltodextrin (5% w / v); inulin (6% w / v) and maltodextrin (6% w / v); inulin (7% w / v) and maltodextrin (7% w / v); inulin (8% w / v) and maltodextrin (8% w / v); inulin (9% w / v) and maltodextrin (9% w / v); and inulin (10% w / v) and maltodextrin (10% w / v).

13. A biotherapeutic composition comprising the composition of any one of claims 1 to 12, and an acceptable diluent or carrier.

14. A pharmaceutical composition comprising the composition of any one of claims 1 to 12, and a pharmaceutically acceptable diluent or carrier.

15. A method of treating and / or preventing a disease or disorder in a patient in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 1 to 12.

16. A method of preparing the biotherapeutic composition of claim 13, the method comprising mixing the composition of any one of claims 1 to 12 with an acceptable diluent or carrier.

17. A method of preparing the pharmaceutical composition of claim 14, the method comprising mixing the composition of any one of claims 1 to 12 with a pharmaceutically acceptable excipient, diluent or carrier.

18. Use of the composition of any one of claims 1 to 12 in the manufacture of a medicament for reducing or preventing a disease or disorder in a subject.

19. A dosage form comprising the composition of any one of claims 1 to 12.

20. A kit comprising the dosage form of claim 19 and instructions for its use.

Images