Microbiota compositions and methods for treating diseases

Abstract

The present invention relates to compositions for preserving the microbiome. The present invention also relates to dosage forms and methods for treating disorders and diseases by administering compositions to patients who require treatment for disorders and diseases.

Description

[Technical Field]

[0001] The present invention relates to compositions for preserving the microbiome. The present invention also relates to dosage forms and methods for treating disorders and diseases by administering compositions to patients who require treatment for disorders and diseases. [Background technology]

[0002] The following discussion of background technology is intended solely to facilitate understanding of the present invention. This discussion does not acknowledge or acknowledge that any of the referenced materials are, or were, part of the general knowledge as of the priority date of this application.

[0003] intestinal microbiota The human gut microbiota comprises trillions of microorganisms, including at least 100 epidemic bacterial species and at least 1,000 less common bacterial species, possessing more than 100 times the number of genes found in the human genome. While primarily composed of bacteria, the gut microbiota also includes archaea, fungi, yeasts, protozoa, and viruses. The microbiota plays vital roles essential to maintaining health, including food processing, digestion of complex, indigestible polysaccharides, and vitamin synthesis. It secretes bioactive metabolites with diverse functions ranging from pathogen inhibition and the metabolism of toxic compounds to the regulation of host metabolism.

[0004] Cultured microbiome therapy Cultured microbiome therapies consist of one or more microorganisms that can be administered to patients for the purpose of treating or preventing disease, or enhancing the effects of other therapies. Cultured microbiome therapies are currently being developed as treatments for many diseases. However, these efforts are hindered by limitations in stability, storage, transport, and delivery conditions, as well as the loss of viable microorganisms, thereby limiting the clinical use of these therapies. There are challenges in sampling, storing, and shipping and delivering viable and effective microbiome samples to patients. Furthermore, there is a need in this technology for effective treatments of diseases associated with the loss or dysbiosis of gut microbiota.

[0005] Storage of the microbiome Efforts have been made to preserve microbiomes in freeze-dried or lyophilized form. Freeze-drying, also known as lyophilization, is a low-temperature dehydration process that involves freezing the product, reducing the pressure, and removing the ice by sublimation. This is in contrast to dehydration by most conventional methods that use heat to evaporate water. Freeze-dried microbiomes are a useful method for long-term storage. However, one of the main challenges is the loss of cell viability due to freeze-thaw damage, as well as the effect of osmotic changes on the functional and structural integrity of microorganisms. Attempts to develop suitable cryoprotective agents to protect the functional and structural integrity of microorganisms during freeze-drying and thawing processes have been reported in the prior art, however, many of these attempts have failed. There is a need in the art for effective cryoprotective agents that maintain cell viability and preserve the functional and structural integrity of microorganisms during freeze-drying and thawing processes.

[0006] The objective of this invention is to overcome one or more problems foreseeable by the prior art. [Overview of the project]

[0007] In a first aspect, the present invention is broadly a composition for performing it in a subject that needs to prevent or treat a disease or disorder, the composition being at least one strain of a microorganism, the microorganism being at least one strain selected from the group consisting of bacteria, yeast, or archaea, and an excipient, and is present in the composition.

[0008] In a preferred embodiment, the excipient is a cryoprotectant. In a preferred embodiment, the excipient is inulin or an analog or variant thereof. In a preferred embodiment, inulin is alpha-D-glucopyranosyl-[beta-D-fructofuranosyl](n-1)-D-fructofuranoside, beta-D-fructopyranosyl-[D-fructofuranosyl](n-1)-D-fructofuranoside, fructo-oligosaccharide, fructo-oligosaccharide containing 2 to 70 fructose units, fructo-oligosaccharide containing 1 to 500 fructose units, fructo-oligosaccharide containing 1 to 300 fructose units, fructo-oligosaccharide containing 1 to 200 fructose units, fructo-oligosaccharide containing 1 to 100 fructose units, or an analog or variant or combination thereof selected from the group consisting of.

[0009] In a preferred embodiment, the excipient is maltodextrin or an analog or variant thereof. In a preferred embodiment, maltodextrin is maltodextrin having a length selected from the group consisting of 3 to 17 glucose units, corn syrup having a length of 20 or more glucose units, corn syrup solids, modified corn starch, modified rice starch, modified tapioca starch, modified wheat starch, or an analog or variant or combination thereof selected from the group consisting of.

[0010] In a further preferred embodiment, the composition comprises an inulin or an analogue of a variant 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.

[0011] In a further preferred embodiment, the composition comprises a maltodextrin or an analogue of a variant thereof at a concentration 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.

[0012] In a further preferred embodiment, the composition comprises inulin and maltodextrin.

[0013] In preferred embodiments, the composition includes 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% The product contains inulin and maltodextrin at concentrations selected from the group consisting of w / v inulin and maltodextrin (10% w / v).

[0014] In a preferred embodiment, the composition comprises (1) inulin at a concentration 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) maltodextrin at a concentration 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

[0015] In preferred embodiments, the composition includes 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% The product contains inulin and maltodextrin at concentrations selected from the group consisting of w / v inulin and maltodextrin (10% w / v).

[0016] In a preferred embodiment, the composition is in a freeze-dried form.

[0017] In a preferred embodiment, the composition is in liquid form.

[0018] 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.

[0019] In preferred embodiments, the excipients include 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% The group is selected from those consisting of (w / v) pectin (5% w / v), inulin (5% w / v), maltodextrin (5% w / v), and sucrose (5% w / v), and inulin (5% w / v), maltodextrin (5% w / v), and pectin (5% w / v).

[0020] In a preferred embodiment, the composition has a water concentration less than a concentration 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.

[0021] In a preferred embodiment, the composition has a water content selected from the group consisting of 1-5% w / v, 0.5-2% w / v, 0.9-1.2% w / v, 0.99-1.09% w / v, and 1.093% w / v.

[0022] In a preferred embodiment, the composition has a Young's modulus scale selected from the group consisting of 1-5, 2-4, 2.1-3.9, 2.5-3.8, 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.

[0023] In a preferred embodiment, the composition has a maximum stress (kPa) on a fracture point scale selected from the group consisting of 20-40, 21-39, 20-30, 20-29, 25-30, 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.

[0024] In a preferred embodiment, the composition comprises a further excipient and a carrier.

[0025] In preferred embodiments, the microorganisms are fecal or colonic microorganisms.

[0026] In preferred embodiments, the microorganisms are non-inflammatory.

[0027] In a preferred embodiment, the microorganisms are cultured from fecal or colon biopsy samples.

[0028] In a preferred embodiment, the consortium includes a community of microbial cells derived from the stool or biopsy of one or more human donors.

[0029] In a preferred embodiment, the community of microbial cells includes cultured microbial cells.

[0030] In a preferred embodiment, the cultured microbial cells are derived from multiple human donors.

[0031] In a preferred embodiment, the community of microbial cells includes uncultured microbial cells.

[0032] In a preferred embodiment, the uncultured microbial cells are derived from a single human donor.

[0033] In a preferred embodiment, the composition is a fecal microbiota composition.

[0034] In a preferred embodiment, the composition comprises a purified or reconstituted mixture of fecal bacteria.

[0035] In a preferred embodiment, the composition is freeze-dried.

[0036] In a preferred embodiment, the composition is a liquid.

[0037] In a preferred embodiment, after storage at the storage temperature for at least four weeks, the composition is capable of maintaining at least 50% of the cell viability compared to the initial cell viability immediately before storage.

[0038] In a preferred embodiment, after storage at a storage temperature for at least four weeks, the composition can maintain a cell viability of approximately 60% to approximately 80% compared to the initial cell viability immediately before the start of said storage.

[0039] In a preferred embodiment, after storage at storage temperature for at least 2, 4, 8, 12, 16, or 20 weeks, the composition is capable of maintaining a cell viability of at least about 5%, 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the initial cell viability immediately before storage.

[0040] In a preferred embodiment, after storage at a storage temperature for at least 8, 12, 16, 20, 50, 75, 100, 150, or 200 weeks, the composition maintains at least 50% of the cell viability compared to the initial cell viability immediately prior to storage.

[0041] In a preferred embodiment, after at least 12 weeks at storage temperature, the composition maintains a cell viability of 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%, 80%-90%, 40%-80%, 50%-70%, 55%-65%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, or 70%-80% compared to the initial cell viability immediately before storage.

[0042] In a preferred embodiment, the storage temperature is selected from the group consisting of below ambient temperature, -70°C, -50°C, -47°C, -30°C, -20°C, -8°C, -4°C, less than zero, 2°C, 4°C, 8°C, 2-8°C, 18°C, 25°C, room temperature, and ambient temperature.

[0043] In preferred embodiments, cell viability is measured by a method selected from the group consisting of using an imaging assay to measure membrane permeability, using a combination of membrane permeable and impermeable DNA dye staining, SYTO and propidium idodide are used to stain and differentiate live and dead bacteria, viability determination is combined with fluorescence Gram staining, colorimetric method, bacterial cell viability is assessed by using BactoBox or other impedance flow cytometry tools, bacterial cell viability is assessed by counting the number of colonies on an agar plate, and cell viability is assessed via molecular viability analysis.

[0044] In a preferred embodiment, the composition includes a prebiotic.

[0045] In a preferred embodiment, the composition includes a carrier.

[0046] In preferred embodiments, the composition comprises insoluble fibers, a buffer, an osmotic agent, an antifoaming agent, and / or a preservative.

[0047] In a preferred embodiment, the composition includes a chemostat medium.

[0048] In a preferred embodiment, the composition comprises a physiological saline composition.

[0049] In preferred embodiments, the composition includes resistant starch.

[0050] In a preferred embodiment, the composition is freeze-dried together with a pharmaceutically acceptable excipient.

[0051] In preferred embodiments, the composition comprises further stabilizers and / or further cryoprotectants.

[0052] In preferred embodiments, further cryoprotectants are selected from the group consisting of trehalose, mannitol, sucrose, glycerol, sorbitol, DMSO, propylene glycol, ethylene glycol, saccharose, galactose-lactose, and any combination thereof.

[0053] In preferred embodiments, further cryoprotectants include compounds selected from the group consisting of glycerol, polyethylene glycol (PEG), glycerin, erythritol, arabitol, xylitol, sorbitol, glucose, lactose, ribose, and any combination thereof.

[0054] In a preferred embodiment, the further cryoprotectant is trehalose at a concentration of 2% to 15% in the lyophilized formulation.

[0055] In a preferred embodiment, the further cryoprotectant is trehalose at a concentration of at least 5% in the lyophilized formulation.

[0056] In a preferred embodiment, the further cryoprotectant is trehalose at a concentration of at least 10% in the lyophilized formulation.

[0057] In a preferred embodiment, the composition is a pharmaceutical composition.

[0058] In a preferred embodiment, at least one of the microbial strains is diluted with an inert powder diluent.

[0059] In preferred embodiments, the composition comprises one or more pharmaceutically acceptable carriers or excipients.

[0060] In preferred embodiments, the composition is formulated as a gel tab, tablet, enema, microcapsule, capsule, or tablet.

[0061] In preferred embodiments, the capsule or tablet is enterically coated, pH-dependent, sustained-release, and / or gastric-tolerant.

[0062] In preferred embodiments, the composition is suitable for oral or rectal administration.

[0063] In preferred embodiments, the composition comprises one or more, two or more, three or more, four or more, or five or more isolated, purified, or cultured microorganisms.

[0064] In a preferred embodiment, the microorganism is a member of a phylum, family, genus, or species taxonomy selected from those listed in the groups consisting of Table 1, Table 2, Table 3, Table 4, Table 5, Table 13, Table 14, Table 15, and any combination thereof.

[0065] For example, the composition contains one or more microorganisms selected from Table 3.

[0066] In another example, the composition comprises one or more microorganisms selected from the BB265 composite consortium listed in Table 4.

[0067] For example, the composition contains one or more microorganisms selected from the 143 isolates of the BB265 composite consortium listed in Table 5.

[0068] In a preferred embodiment, the composition is further supplemented with at least one microorganism from the group listed in Tables 1, 2, 3, 4, 5, 13, 14, 15, and any combination thereof.

[0069] In a preferred embodiment, the composition lacks one or more microorganisms selected from those 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] In Tables 1-5, please note that * indicates a taxonomic group identified in the analysis that is included in BB265 but is not considered to exist within the BB265 composite consortium. [Table 1] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 4-1] [Table 4-2] [Table 4-3] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0071] In preferred embodiments, the Enterococcus species is Enterococcus faecalis; Enterococcus faecium; Enterococcus species CC00149 deposited with the National Measurement Institute, Australia under V19 / 018754 on September 9, 2019; Enterococcus species CC00259 deposited with the National Measurement Institute, Australia under V19 / 018755 on September 9, 2019; Enterococcus species CC00620 deposited with the National Measurement Institute, Australia under V21 / 013048 on June 29, 2021; Enterococcus species CC00064 deposited with the National Measurement Institute, Australia under V21 / 013046 on June 29, 2021; National Measurement The group is selected from the following species: Enterococcus species CC00619 deposited with the National Measurement Institute, Australia; Enterococcus species CC00262 deposited with the National Measurement Institute, Australia on March 18, 2020, under V20 / 006238; and Enterococcus species CC0002 deposited with the National Measurement Institute, Australia on July 20, 2021, under V21 / 014119.

[0072] In a preferred embodiment, the Lactobacillus species is Lactobacillus rhamnosus (such as strain GG (ATCC 53103), CGMCC 1.3724, or SP1 (DSM 21690)), Lactococcus lactis, Lactococcus cremoris, Lactococcus diacetylactis, Lactobacillus paracasei, Lactobacillus reuteri (such as strain ATCC55730 or DSM17938), Lactobacillus acidophilus, Lactobacillus murinus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus taiwanensis, Lactobacillus animalis, Lactobacillus The selection is made from the group consisting of johnsonii (strain NCC533, CNCM1-1225, etc.) and Lactobacillus gasseri.

[0073] In a preferred embodiment, the Bifidobacterium species is selected from the group consisting of Bifidobacterium lactis (e.g., strain BB-12, BI-04, or CNCM1-3446 (Bb12)), Bifidobacterium longum (e.g., strain NCC3001, ATCC BAA-999 (BB536)), Bifidobacterium breve (e.g., strain Bb-03, M-16V, or R0070), Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, and Bifidobacterium adolescentis.

[0074] In a preferred embodiment, the Streptococcus species is selected from the group consisting of Streptococcus thermophilus, Streptococcus thermophilus ST-21, and Streptococcus salivarius.

[0075] In a preferred embodiment, the Clostridium species is selected from the group consisting of Clostridium difficile, Clostridium hylemonae, Clostridium scindens, and Flavinofractor plautii.

[0076] In a preferred embodiment, the microorganism is yeast.

[0077] In a preferred embodiment, the yeast is Saccharomyces boulardii.

[0078] In a preferred embodiment, the microorganism is an archaea.

[0079] In a preferred embodiment, the archaea are selected from the group consisting of the species Methanobrevibacter, Methanobrevibacter smithii, and the species Methanosphaera, Methanosphaera stadtmaniae.

[0080] In preferred embodiments, the composition comprises a fecal microbiota containing a Shannon diversity index of 0.3 or higher, 0.4 or higher, 0.5 or higher, 0.6 or higher, 0.7 or higher, 0.8 or higher, 0.9 or higher, 5 or higher, 1.0 or higher, 1.1 or higher, 1.2 or higher, 1.3 or higher, 1.4 or higher, 1.5 or higher, 1.6 or higher, 1.7 or higher, 1.8 or higher, 1.9 or higher, 2.0 or higher, 2.1 or higher, 2.2 or higher, 2.3 or higher, 2.4 or higher, 2.5 or higher, 3.0 or higher, 3.1 or higher, 3.2 or higher, 3.3 or higher, 3.4 or higher, 3.5 or higher, 3.6 or higher, 3.7 or higher, 3.8 or higher, 3.9 or higher, 4.0 or higher, 4.1 or higher, 4.2 or higher, 4.3 or higher, 4.4 or higher, 4.5 or higher, or 5.0 or higher.

[0081] In preferred embodiments, the composition comprises a fecal microbiota with Shannon diversity indices of 0.1-3.0, 0.1-2.5, 0.1-2.4, 0.1-2.3, 0.1-2.2, 0.1-2.1, 0.1-2.0, 0.4-2.5, 0.4-3.0, 0.5-5.0, 20, 0.7-5.0, 0.9-5.0, 1.1-5.0, 1.3-5.0, 1.5-5.0, 1.7-5.0, 1.9-5.0, 2.1-5.0, 2.3-5.0, 2.5-5.0, 2.7-5.0, 2.9-5.0, 3.1-5.0, 3.3-5.0, 3.5-5.0, 3.7-5.0, 3.9-5.0, or 4.1-5.0.

[0082] In a preferred embodiment, the Shannon diversity index is calculated at a level selected from a group consisting of the phylum level, family level, genus level, and species level.

[0083] In a preferred embodiment, the composition comprises a flora preparation in a proportionate amount similar to that of a normal, healthy human fecal flora.

[0084] In a preferred embodiment, the composition contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different families of fecal bacteria.

[0085] In preferred embodiments, the composition comprises a fecal microbiome containing 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% by weight of non-biological material / by weight of biological material.

[0086] In preferred embodiments, the composition comprises a fecal microbiome containing 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% by weight of non-biological material / by weight of biological material.

[0087] In a preferred embodiment, the composition comprises particles of non-biological and / or biological material of a fecal sample passing through a sieve, column, or similar filtration device having sieve sizes, exclusion sizes, or particle filter sizes of 2.0 mm, 1.0 mm, 0.5 mm, 10, 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.

[0088] In preferred embodiments, the composition contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% isolated or pure non-fecal flora material, or less 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 of non-fecal flora material, or substantially isolated or purified fecal flora, or substantially isolated or purified fecal flora, or substantially entirely (or substantially entirely) microbiome, as described in WO2012 / 122478 or WO2012 / 016287.

[0089] In preferred embodiments, the composition comprises a weight ratio of 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% or less of fecal-derived non-biological material to fecal-derived biological material.

[0090] In a preferred embodiment, 10 per 200 mg of composition 3 ~10 14 , 10 4 ~10 14 , 10 5 ~10 14 , 10 6 ~10 14, 10 7 ~10 14 , 10 8 ~10 14 , 10 4 ~10 13 , 10 5 ~10 12 , 10 6 ~10 11 , 10 7 ~10 10 , 10 8 ~10 9 , 10 3 ~10 13 , 10 3 ~10 12 , 10 3 ~10 11 , 10 3 ~10 10 , 10 3 ~10 9 , 10 3 ~10 8 , 10 3 ~10 7 , 10 3 ~10 6 , 10 3 ~10 5 , and 10 3 ~10 4 comprising a pharmacologically active dose of microbial cells or spores selected from the group consisting of colony forming units (cfu) or total cell numbers of 10

[0091] In preferred embodiments, the composition is 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, 30 billion to 35 billion cfu / mL, and 35 billion to 40 billion cfu / mL. The mixture contains microbial cells or spores in a pharmacologically active dose selected from the group consisting of L, 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, and 95 billion to 100 billion cfu / mL.

[0092] In preferred embodiments, the composition comprises a pharmacologically active dose of microbial cells or spores, wherein the concentration of microbial cells or spores as dried microorganisms is selected from the group consisting of 5-50 w / w%, 1-75 w / w%, 0.1-100 w / w%, and 1-100 w / w%.

[0093] In a preferred embodiment, the disease or disorder is inflammation of the gastrointestinal mucosa.

[0094] In a preferred embodiment, the disease or disorder is characterized by a reduction in intestinal microbial diversity.

[0095] In a preferred embodiment, the disease or disorder is characterized by a reduction in intestinal microbial function.

[0096] In a preferred embodiment, the disease or disorder is characterized by the loss of the intestinal microbial ecosystem.

[0097] In a preferred embodiment, the disease or disorder is dysbiosis.

[0098] In preferred embodiments, dysbiosis is associated with one or more disorders selected from the group consisting of inflammatory bowel disease (IBD), pouchitis, irritable bowel syndrome (IBS), enterobacterial infection, metabolic disorders, neuropsychiatric disorders, autoimmune diseases, allergic diseases, hepatic encephalopathy, or cancer.

[0099] In a preferred embodiment, the disease or disorder is a malignant tumor or cancer.

[0100] In a preferred embodiment, the disease or disorder is a liver disease or hepatic disorder.

[0101] In a preferred embodiment, the disease or disorder is a gastrointestinal disorder.

[0102] In a preferred embodiment, the gastrointestinal disorder is inflammatory bowel disease.

[0103] In a preferred embodiment, the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, gastroenteritis, colitis, and pouchitis.

[0104] In a preferred embodiment, the gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, gastrointestinal ulcers, and gastrointestinal cancer.

[0105] In a preferred embodiment, the composition reduces endogenous sulfide levels in the colon of patients requiring treatment.

[0106] In preferred embodiments, the composition reduces sulfide and nitric oxide loads on epithelial cells, which result in metabolic lesions through inhibition of cellular respiration.

[0107] In preferred embodiments, the composition reduces the relative abundance and / or metabolic activity of sulfide microbiota, directly reduces sulfide levels in the colon through ingestion / assimilation, reduces sulfide levels in the colon through metabolic substrate competition, and / or reduces sulfide levels in the colon by ingestion of hydrogen.

[0108] In preferred embodiments, the composition reduces the relative abundance and / or metabolic activity of sulfide-producing microbiota by reducing metabolizable sulfur substrates, thereby reducing the release of sulfur amino acids (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

[0109] In a preferred embodiment, the composition induces colonic cell apoptosis in the lesion, thereby disrupting the progression of the induced stable inflammatory state.

[0110] In a preferred embodiment, the composition suppresses undesirable inflammation.

[0111] In preferred embodiments, the composition prevents or reduces the activation of the mucosal immune system in a natural killer T cell-propelled IL-13 and IL-5-dependent TH2-mediated immune response.

[0112] In preferred embodiments, the composition reduces inflammation in the subject when measured by parameters selected from the group consisting of NF-κB-mediated TNFα signaling, IFNα signaling, IFNγ signaling, IL6 JAK STAT3 signaling, activation of the pro-apoptotic pathway, and initiation of an unfolded protein response.

[0113] In preferred embodiments, the composition downregulates genes associated with pro-apoptotic pathways and unfolded protein responses, comprising genes selected from the group consisting of CHAC1, CEBPB, TRIB3, PPP1R15A, DDIT3, ATF4, and XBP1.

[0114] In preferred embodiments, the composition includes: inulin HP-Gel (Orafti) (10% w / v); maltodextrin-free inulin (5% w / v); maltodextrin-free inulin (10% w / v); maltodextrin-free inulin (15% w / v); 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 tocopheral (10 μL / L); inulin (15% w / v) and tocopheral (100 μL / L); inulin (15% w / v) and ascorbic acid (4 mg / L); and inulin (15% It does not contain excipients selected from the group consisting of w / v and ascorbic acid (40 mg / L).

[0115] In preferred embodiments, the microorganism is not Lactobacillus acidophilus MJLA1.

[0116] In preferred embodiments, the composition includes: inulin HP-Gel (Orafti) (10% w / v); maltodextrin-free inulin (5% w / v); maltodextrin-free inulin (10% w / v); maltodextrin-free inulin (15% w / v); 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 tocopheral (10 μL / L); inulin (15% w / v) and tocopheral (100 μL / L); inulin (15% w / v) and ascorbic acid (4 mg / L); and inulin (15% It does not contain an excipient selected from the group consisting of w / v and ascorbic acid (40 mg / L), and the microorganism is not Lactobacillus acidophilus MJLA1.

[0117] In preferred embodiments, the composition includes: inulin HP-Gel (Orafti) (10% w / v); maltodextrin-free inulin (5% w / v); maltodextrin-free inulin (10% w / v); maltodextrin-free inulin (15% w / v); 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 tocopheral (10 μL / L); inulin (15% w / v) and tocopheral (100 μL / L); inulin (15% w / v) and ascorbic acid (4 mg / L); and inulin (15% It contains an excipient selected from the group consisting of w / v and ascorbic acid (40 mg / L).

[0118] In a preferred embodiment, the microorganism is Lactobacillus acidophilus MJLA1.

[0119] In preferred embodiments, the composition includes: inulin HP-Gel (Orafti) (10% w / v); maltodextrin-free inulin (5% w / v); maltodextrin-free inulin (10% w / v); maltodextrin-free inulin (15% w / v); 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 tocopheral (10 μL / L); inulin (15% w / v) and tocopheral (100 μL / L); inulin (15% w / v) and ascorbic acid (4 mg / L); and inulin (15% The product contains an excipient selected from the group consisting of w / v and ascorbic acid (40 mg / L), and the microorganism is Lactobacillus acidophilus MJLA1.

[0120] In preferred embodiments, the composition does not contain an excipient selected from the group consisting of inulin (2% w / v), maltodextrin (2% w / v), and sucrose (2% w / v).

[0121] In preferred embodiments, the composition does not contain an excipient selected from the group consisting of inulin (10% w / v), maltodextrin (10% w / v), and sucrose (10% w / v).

[0122] In a preferred embodiment, the microorganism is not Lactobacillus plantarum.

[0123] In preferred embodiments, the composition contains an excipient selected from the group consisting of inulin (2% w / v), maltodextrin (2% w / v), and sucrose (2% w / v).

[0124] In a preferred embodiment, the composition contains an excipient selected from the group consisting of inulin (10% w / v), maltodextrin (10% w / v), and sucrose (10% w / v).

[0125] In a preferred embodiment, the microorganism is Lactobacillus plantarum.

[0126] In a further embodiment, the present invention is a biological therapeutic composition comprising a composition of the first embodiment of the present invention together with an acceptable diluent or carrier.

[0127] In a preferred embodiment, the carrier is 0.9% sterile physiological saline.

[0128] In a further embodiment, the present invention is a pharmaceutical therapeutic composition comprising a composition of the first embodiment of the present invention together with a pharmaceutically acceptable diluent or carrier.

[0129] In a further embodiment, the present invention is a method for treating and / or preventing a disease or disorder in a patient who requires such treatment, the method comprising administering an effective amount of the composition described in the first embodiment of the present invention to the subject.

[0130] In a preferred embodiment, the disease or disorder is inflammation of the gastrointestinal mucosa.

[0131] In a preferred embodiment, the disease or disorder is characterized by a reduction in intestinal microbial diversity.

[0132] In a preferred embodiment, the disease or disorder is characterized by a reduction in intestinal microbial function.

[0133] In a preferred embodiment, the disease or disorder is characterized by the loss of the intestinal microbial ecosystem.

[0134] In a preferred embodiment, the disease or disorder is dysbiosis.

[0135] In preferred embodiments, dysbiosis is associated with one or more disorders selected from the group consisting of inflammatory bowel disease (IBD), pouchitis, irritable bowel syndrome (IBS), enterobacterial infection, metabolic disorders, neuropsychiatric disorders, autoimmune diseases, allergic diseases, hepatic encephalopathy, or cancer.

[0136] In a preferred embodiment, the disease or disorder is a malignant tumor or cancer.

[0137] In a preferred embodiment, the disease or disorder is a liver disease or hepatic disorder. For example, the disease or disorder is primary sclerosing cholangitis.

[0138] In a preferred embodiment, the disease or disorder is a gastrointestinal disorder.

[0139] In a preferred embodiment, the gastrointestinal disorder is inflammatory bowel disease.

[0140] In a preferred embodiment, the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, gastroenteritis, colitis, and pouchitis.

[0141] In a preferred embodiment, the gastrointestinal disorder is selected from the group consisting of irritable bowel syndrome, gastrointestinal ulcers, and gastrointestinal cancer.

[0142] In preferred embodiments, the composition is administered orally or rectally.

[0143] In preferred embodiments, the composition is administered in conjunction with or to support immunotherapy.

[0144] 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 12 hours, once a day, twice a day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, weekly, twice a week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, once a month, twice a month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, annually, twice a year, every 2 years, every 3 years, every 4 years, and every 5 years.

[0145] In a preferred embodiment, the composition reduces endogenous sulfide levels in the colon of patients requiring treatment.

[0146] In preferred embodiments, the composition reduces sulfide and nitric oxide loads on epithelial cells, which result in metabolic lesions through inhibition of cellular respiration.

[0147] In a preferred embodiment, the composition reduces nitric oxide production in the colon and / or reduces nitric oxide levels.

[0148] In preferred embodiments, the composition reduces the relative abundance and / or metabolic activity of sulfide-producing microbiota, directly reduces sulfide levels in the colon through ingestion / assimilation, reduces sulfide levels, relative abundance and / or metabolic activity of sulfide-producing microbiota through metabolic substrate competition, and / or reduces sulfide levels, relative abundance and / or metabolic activity of sulfide-producing microbiota by ingestion of hydrogen.

[0149] In preferred embodiments, the composition reduces the relative abundance and / or metabolic activity of sulfide-producing microbiota by reducing metabolizable sulfur substrates in the colon, and / or by reducing the release of sulfur amino acids (methionine, cysteine, homocysteine, taurine) into the colon by reducing protein fermentation.

[0150] In a preferred embodiment, the composition induces colonic cell apoptosis in the lesion, thereby disrupting the induced stable inflammatory state.

[0151] In a preferred embodiment, the method suppresses undesirable inflammation.

[0152] In preferred embodiments, the composition prevents or reduces the activation of the mucosal immune system in a natural killer T cell-propelled IL-13 and IL-5-dependent TH2-mediated immune response.

[0153] In a preferred embodiment, the method reduces inflammation in the subject when measured by a parameter selected from the group consisting of NF-κB-mediated TNFα signaling, IFNα signaling, IFNγ signaling, IL6 JAK STAT3 signaling, activation of the pro-apoptotic pathway, and initiation of an unfolded protein response.

[0154] 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.

[0155] In a further embodiment, the present invention relates to a method for preparing a biological therapeutic composition of the present invention, the method comprising mixing a composition of the first embodiment of the present invention with an acceptable diluent or carrier.

[0156] In a further embodiment, the present invention relates to a method for preparing a pharmaceutical composition of the present invention, the method comprising mixing a composition of the first embodiment of the present invention with a pharmaceutically acceptable excipient, diluent, or carrier.

[0157] In a preferred embodiment, the excipient, diluent, or carrier is sterilized.

[0158] In a preferred embodiment, the cryoprotectant is sterilized.

[0159] In a further embodiment, the present invention relates to the use of a composition of the first embodiment of the present invention in the manufacture of a pharmaceutical product for reducing or preventing a disease or disorder in a subject.

[0160] In a further embodiment, the present invention is a dosage form comprising a composition of the first embodiment of the present invention.

[0161] A kit comprising the dosage form of the present invention, along with instructions for use.

[0162] Further features of the present invention are described more fully in the following description of some non-limiting embodiments thereof. This description is included solely for illustrative purposes of the invention and should not be understood as a limitation to the broad summary, disclosure, or description of the invention set forth above.

[0163] The following is a brief explanation of each of the figures and drawings. [Brief explanation of the drawing]

[0164] [Figure 1] The results of MicroPress analysis (Analysis 1, 2, and 3) of three samples of lyophilized FMT cake treated with a cryoprotective formulation of 5% inulin and 5% maltodextrin are shown. MicroPress analysis was used to quantitatively determine the strength and physical characteristics of the lyophilized cake in situ. [Figure 2] This chart shows the average loss factor of intermediate products in CFU / mL when FMT prepared with eight different cryoprotective agent formulations was anaerobically plated onto non-selective media after lyophilization. Error bars represent 1 standard deviation (S.D). [Figure 3] This graph shows the mean CFU / mL of intermediate products obtained when FMT prepared with eight different cryoprotective agent formulations was anaerobically plated onto non-selective media after lyophilization. Error bars represent 1 standard deviation (S.D.). [Figure 4] Individual bar plots of raw cell count (ICC) per gram of stool material for eight batches of donor stool material are shown, compared to pooled stool and to an intermediate product (stool homogenized in a cryoprotective agent formulation) of undiluted stool. ICC was determined using BactoBox (SBT Instruments). [Figure 5] The bar plots show the raw cell count (ICC) per gram of stool for undiluted stool (8 individual pooled batches), intermediate product (pooled stool homogenized in cryoprotectant formulation), lyophilized product (after grinding), and encapsulated lyophilized product "T0," as determined by BactoBox (SBT Instruments). [Figure 6] This bar plot compares the change in raw cell count (ICC) per gram of encapsulated lyophilized product 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 BactoBox (SBT Instruments). [Modes for carrying out the invention]

[0165] For convenience, the following sections outline the various meanings of terms used herein. Following this discussion, general aspects of the compositions, pharmaceuticals, and methods of the present invention will be considered, followed by specific examples demonstrating the characteristics of various embodiments of the present invention and how they can be used.

[0166] Those skilled in the art will understand that the present invention as described herein is susceptible to modifications and alterations other than those specifically described. The present invention includes all such modifications and alterations. The present invention also includes all of the steps, features, formulations, and compounds referred to or shown herein, individually or collectively, and any and all combinations thereof, or any two or more of the steps or features.

[0167] Each document, reference, patent application, or patent cited herein is expressly incorporated herein by reference in its entirety, meaning that it should be read and considered by the reader as part of this text. The absence of repetition of any document, reference, patent application, or patent cited herein is solely for the sake of brevity. However, neither the cited material nor any information contained herein should be understood as general knowledge.

[0168] Manufacturer's instructions, manuals, product specifications, and product sheets for any product described in this specification or any document incorporated herein by reference may be incorporated herein by reference and used in the practice of the present invention.

[0169] The present invention is not limited in scope by any of the specific embodiments described herein. These embodiments are intended for illustrative purposes only. Functionally equivalent products, formulations, and methods are clearly within the scope of the present invention as described herein.

[0170] 1.Definition The meanings of certain terms and phrases used in this specification, the examples, and the appended claims are provided below. In the event of any apparent conflict between the use of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0171] Unless otherwise indicated in the operating examples or otherwise specified, all numbers representing the quantities of components or reaction conditions used herein should be understood to be modified in all cases by the term "approximately." When used in relation to percentages, the term "approximately" may mean ±1%.

[0172] The invention as described herein may include one or more ranges of values ​​(e.g., size, concentration, etc.). A range of values ​​will be understood to include all values ​​within the range, including the value that defines the range, and adjacent values ​​to the range that produce the same or substantially the same result as the values ​​directly adjacent to that value that define the boundary for the range. For example, a person skilled in the art will understand that a 10% variation at the upper or lower limit of a range may be perfectly appropriate and is included in the invention. More specifically, the variation at the upper or lower limit of a range may be 5%, or whichever is greater, as is generally recognized in the art.

[0173] In this application, unless otherwise specifically stated, the use of the singular form also includes the plural form. In this application, the use of “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “including,” and other forms such as “includes” and “included,” is not limited. Also, terms such as “element” or “component” include both elements and components containing one unit, and elements and components containing two or more subunits, unless otherwise specifically stated. Also, the use of the term “part” may include a part of a part or the whole of a part.

[0174] Throughout this specification, unless otherwise required by context, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of the integer or group of integers described, but not the exclusion of any other integer or group of integers.

[0175] As used herein with respect to methods of treatment, particularly drug administration, “therapeutic dose” means a dosage of the drug that provides a specific pharmacological response to a significant number of subjects requiring such treatment. In particular cases, it is emphasized that the “therapeutic dose” administered to a particular subject may be considered a “therapeutic dose” by those skilled in the art, but is not necessarily effective in treating the diseases described herein. It is further understood that drug dosages are measured in particular cases as oral doses or by reference to drug levels measured in the blood. The amount effective for such use will depend on the desired therapeutic effect, the potency of the biologically active material, the desired duration of treatment, the stage and severity of the disease being treated, the patient’s weight and general health, and the judgment of the prescribing physician. To optimize safety and efficacy, therapeutic dosages should be titrated. Accordingly, those skilled in the art will understand that the appropriate dosage level for treatment will vary in part depending on the indications for use of the activator, the route of administration, and the patient’s size (weight, body surface area, or organ size) and condition (age and general health). Therefore, clinicians may titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages may range from approximately 0.1 μg / kg to a maximum of approximately 100 mg / kg or more, depending on the factors described above. In other embodiments, the dosage may range from 0.1 μg / kg to a maximum of approximately 100 mg / kg, or from 1 μg / kg to a maximum of approximately 100 mg / kg, or from 5 μg / kg to a maximum of approximately 100 mg / kg.

[0176] The frequency of administration will depend on the pharmacokinetic parameters of the activator and formulation used. Typically, clinicians will administer the composition until they reach a dosage that achieves the desired effect. Therefore, the composition may be administered as a single dose, or as two or more doses over time (with or without the same amount of the desired molecule), or as a continuous infusion via an implantable device or catheter. Further adjustment of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of tasks routinely performed by those skilled in the art. Appropriate dosage can be confirmed through the use of appropriate dose-response data.

[0177] As used herein, “carrier” may be any solvent, diluent, excipient or other vehicle, dispersing or suspending agent, surfactant, isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant, etc., suitable for a particular desired dosage form.

[0178] As used herein, the term “pharmaceutically acceptable carrier” component may refer to a biologically undesirable or undesirable component, i.e., a component that can be incorporated into the composition of the present invention and administered to a subject described herein without causing any significant undesirable biological effect or without interacting in a harmful manner with any of the other components of the formulation containing it. The component generally meets the required toxicological and manufacturing standards.

[0179] As used herein, the term “subject” generally includes humans; domestic animals such as sheep, goats, pigs, cattle, horses, and llamas; companion animals such as dogs and cats; mammals such as primates; birds such as chickens, geese, and ducks; fish; and reptiles. The subject is preferably humans.

[0180] As used herein, “gastrointestinal tract” refers to the tube 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 the present invention are at least useful for conditions of the terminal ileum, cecum, or rectum.

[0181] As used herein, “non-inflammatory strain” refers to the strain of the present invention that, when present in a subject, preferably the human gastrointestinal tract, is associated with a non-inflammatory state. The non-inflammatory strains of the present invention are little to no cytotoxic to mammalian epithelial cells in a culture. In one embodiment, the strain causes cell death of less than 15%, less than 10%, or less than 5% of mammalian epithelial cells in a culture.

[0182] As used herein, “inflammatory strain” refers to the strain of the present invention that, when present in a subject, preferably the human gastrointestinal tract, is associated with an inflammatory condition. The inflammatory strains of the present invention are cytotoxic to mammalian epithelial cells in a culture, such as Caco2 cells. In one embodiment, the strain causes cell death of at least 40%, at least 45%, or at least 50% of the mammalian epithelial cells in the culture.

[0183] As used herein, the term “bacteriological 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.

[0184] As used herein, the term “biological therapeutic agent” refers to a microorganism, such as a bacterial isolate, that is useful for treating or preventing a disease or condition in a subject, or for providing a health benefit.

[0185] As used herein, the term “biological therapeutic composition” refers to a formulation comprising a biological therapeutic preparation formulated with one or more additional formulation components to obtain a finished formulation suitable for delivery to a subject.

[0186] As used herein, the terms “to treat,” “to treat,” and “treatment,” and their grammatical variations, mean to administer a protocol, regimen, process, or treatment to an individual subject, in which it is desirable to obtain a physiological response or outcome in that subject. Since not all subjects being treated will respond to a particular treatment protocol, regimen, process, or treatment, treating does not require that a desirable physiological response or outcome be achieved in all subjects or subject groups. Therefore, a given subject or subject group may not respond to treatment, or may not respond adequately.

[0187] As used herein, and as used in relation to the treatment of mucosal inflammation of the gastrointestinal tract, the terms “prevent,” “prevent,” or “prevent” mean a preventive treatment that increases the subject’s resistance to mucosal inflammation of the gastrointestinal tract, in other words, a treatment that reduces the subject’s likelihood of developing mucosal inflammation of the gastrointestinal tract, as well as a treatment that combats inflammation after it has started, for example, by reducing or eliminating the inflammation together or preventing the inflammation from worsening.

[0188] As used herein, the term “reduce,” or variations thereof, refers to a reduction in gastrointestinal mucosal inflammation in the subject, but does not necessarily mean complete elimination.

[0189] As used herein, the term “sample” refers to a collection of biological material obtained from a subject or from the surrounding environment of the subject, such as soil or water, in the area in which the subject resides. In some embodiments, the sample is obtained directly from the subject. For example, the sample may be a fecal sample or one obtained during a colonoscopy. The sample may be in a form taken 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, for example, a small amount less than the concentration of a solid or cellular isolate of the sample into a smaller volume, or a cellular isolate from some or all of the rest of the sample. In some embodiments, nucleic acids are isolated from the sample. Such isolated preparations include reverse transcripts and / or PCR amplification products of 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. Numerous techniques exist for isolating nucleic acids from samples, including composite samples, and are well known in the art.

[0190] The unit "cfu" refers to "colony-forming unit," which is the number of viable microbial cells revealed by microbiological counting on an agar plate.

[0191] The unit "%w / v" refers to weight / volume percentage concentration. This is also known as mass / volume percentage concentration. Weight / volume percentage concentration is also abbreviated as w / v(%), w / v%, or (w / v)%, or %(w / v), or %w / v. Mass / volume percentage concentration is also abbreviated as m / v(%), or m / v%, or (m / v)%, or %(m / v), or %m / v.

[0192] Other definitions of the selected terms used herein are found in the detailed description of the invention and may apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention belongs.

[0193] The features of the present invention are discussed here with reference to the following non-limiting description and examples.

[0194] 2. Embodiments composition The present invention provides a composition for preventing or treating a disease in a subject where such treatment is required, wherein the composition comprises at least one strain of bacteria, archaea, or fungi.

[0195] In a further preferred embodiment, the composition is selected from the group consisting of therapeutic compositions, pharmaceutical compositions, cosmetic compositions, and veterinary compositions.

[0196] Preferably, the composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, such as phosphate-buffered saline. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retardants. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is intended for use in the therapeutic composition unless it is incompatible with the active ingredient. Auxiliary active ingredients may also be incorporated into the composition. For example, see Remington's Pharmaceutical Sciences, 19th Ed. (1995, Mack Publishing Co., Easton, Pa.) and Remington's The Science and Practice of Pharmacy, 23, which are incorporated herein by reference. rd Please refer to Edition (2020, Mack Publishing Co., Easton, Pa.).

[0197] The composition may contain, for example, formulation materials for modifying, maintaining, or preserving the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterilization, stability, dissolution or release rate, adsorption or osmosis. 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); and bulking agents. Agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers; monosaccharides, disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulin); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methyl phosphate). Examples include parabens, propylparabens, chlorhexidine, sorbic acid, or hydrogen peroxide; solvents (glycerin, propylene glycol, or polyethylene glycol, etc.); sugar alcohols (mannitol or sorbitol, etc.); suspending agents; surfactants or wetting agents (polysorbates such as Pluronic acid, PEG, sorbitan esters, polysorbate 20, polysorbate 80, etc., Triton, tromethamine, lecithin, cholesterol, tyroxapole, etc.); stability enhancers (sucrose or sorbitol); tension enhancers (alkali metal halides, preferably sodium chloride or potassium chloride, etc.); delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants.

[0198] The optimal composition will be determined by those skilled in the art, for example, depending on the intended route of administration, delivery format, and desired dosage. Such a composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the biological therapeutic agent of the present invention. The preferred form of the pharmaceutical composition depends on the intended mode of administration and therapeutic application.

[0199] The primary vehicle or carrier in the composition is essentially aqueous. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, supplemented with other materials. Neutral buffered saline, or saline mixed with serum albumin, are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at approximately pH 7.0–8.5, or acetate buffer at approximately pH 4.0–5.5, which may further include sorbitol or a suitable substitute. In one embodiment of the present invention, a pharmaceutical composition for storage may be prepared by mixing a selected composition having a desired degree of purity with an optional formulation in the form of an aqueous solution.

[0200] The formulation components are present at the administration site at an acceptable concentration. For example, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0201] Additional compositions, including the formulation of the present invention in sustained-release or controlled-release formulations, will be apparent to those skilled in the art. Techniques for formulating various other sustained-release or controlled-release means, such as liposome carriers, biodegradable microparticles, or porous beads, and depot injections, are also known to those skilled in the art. Additional examples of sustained-release preparations include semipermeable polymer matrices in the form of molded articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. The sustained-release composition may also comprise liposomes, which can be prepared by any of several methods known in the art.

[0202] Compositions used for in vivo administration can be filtered to remove undesirable components. This can be achieved by filtration through a filtration membrane. In addition, compositions are generally placed in sealed containers to reduce exposure to oxygen. Once the pharmaceutical composition is formulated, it can be stored in a sealed container.

[0203] When used here, 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 performed across a window corresponding to one of the shortest aligned sequences. 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 identical counterparts in the target sequence is reported as sequence homology %.

[0204] In one embodiment, the identity percentage of polynucleotides is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. Preferably, the GAP analysis aligns the two sequences over their entire length.

[0205] The bacterial strains used in this invention can be cultured using microbiological techniques detailed in, for example, Browne et al. Culturing of 'unculturable' human microbiota reveals novel taxa and extensive sporulation. Nature (2016) Volume 533, pages 543-546 (2016), Handbook of Microbiological Media, Fourth Edition (2010) Ronald Atlas, CRC Press, and Maintaining Cultures for Biotechnology and Industry (1996) Jennie C. Hunter-Cevera, Academic Press. Similarly, methods using yeast extract, cassitone, and fatty acid (YCFA) media are detailed in the examples.

[0206] In a further preferred embodiment, the composition further comprises water.

[0207] In yet another preferred embodiment, the composition is a liquid, such as an aqueous solution.

[0208] In even more preferred embodiments, the carrier further includes a pharmaceutically acceptable carrier.

[0209] In a further preferred embodiment, the composition retains its effective biological activity for a period selected from the group consisting of more than 24 hours, more than 36 hours, and more than 48 hours. Preferably, the composition is stable for a period 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.

[0210] Pharmaceutical compositions and therapeutic compositions fall within the scope of the present invention.

[0211] The therapeutic compositions of the present invention may contain 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 potency of isolated bacteria present in the therapeutic composition. The exact properties of the pharmaceutically acceptable excipients or other materials will depend, for example, on the route of administration, which may be 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).

[0212] The therapeutic composition of the present invention may comprise a prebiotic, a carrier, insoluble fibers, a buffer, an osmotic agent, an antifoaming agent, and / or a preservative.

[0213] The therapeutic composition may be prepared or provided in chemostat medium. Alternatively, the therapeutic composition may be prepared or provided in physiological saline, for example, 0.9% physiological saline. It will be understood that any carrier or solution may be used that does not impair the viability of bacteria present in the therapeutic composition and is suitable for administration to an individual.

[0214] Therapeutic compositions may be prepared or provided under a reducing atmosphere, i.e., in the absence of oxygen. Synthetic stool preparations may optionally be prepared or provided under N2, CO2, H2, or mixtures thereof, while controlling the levels of the partial pressures of N2:CO2:H2.

[0215] The therapeutic composition may be prepared or provided under an oxygen-containing atmosphere.

[0216] The therapeutic composition may be for oral or rectal administration to an individual. If the therapeutic composition is for oral administration, it may be in the form of a capsule or tablet. If the therapeutic composition is for rectal administration, it may be in the form of an enema or suppository. The preparation of suitable capsules, tablets, suppositories, and enemas is well known in the art. Capsules or tablets may include a coating to protect them from gastric acid. For example, capsules or tablets may have an enteric coating, be pH-dependent, sustained-release, and / or gastric-resistant. Such capsules and tablets are used, for example, to minimize dissolution of the capsule or tablet in the stomach but to allow dissolution in the small intestine.

[0217] Oral formulations may, for example, include, in addition to viable microorganisms, inert compression aids such as microcrystalline cellulose or oligosaccharides, flow aids such as silica gel, or lubricants such as magnesium stearate (plant source) or stearic acid (plant source).

[0218] The compositions disclosed herein can be used, for example, as food supplements, edible products, or pharmaceutical products. If used as a food supplement, the composition may further include conventional food supplement fillers and / or bulking agents. The compositions disclosed herein may also be incorporated into any edible product, such as dairy products including dairy products, milk, yogurt, curds, ice cream, dressings, and cheese, beverage products, meat products, and baked goods.

[0219] For example, suppository formulations for rectal use may contain, in addition to the composition, any of the following: cocoa butter, polyethylene glycol, glycerin, or gelatin.

[0220] The composition may include a disintegrant, a flow enhancer, and / or a lubricant. The disintegrant assists in the disintegration of the compressed mass when placed in a fluid environment. The disintegrant may be any suitable disintegrant selected from the group consisting of, for example, croscarmellose sodium, crospovidone, gelan gum, hydroxypropyl cellulose, starch, and sodium starch glycolate. The flow enhancer may be any suitable flow enhancer selected from the group consisting of, for example, silicon dioxide, colloidal silicon dioxide, and talc. Lubricants are generally always used in the production of dosage forms by direct compression to prevent the compressed powder mass from sticking to the equipment during the tableting or encapsulation process. The lubricant may be any suitable lubricant selected from the group consisting of, for example, calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumerate, and vegetable fatty acids. In the composition and method of the present invention, the carrier may be present in the composition in a range of approximately 30% w / w to approximately 98% w / w, and this weight percentage is the cumulative weight percentage considering all components present in the carrier.

[0221] Coatings can be used to control the solubility of the composition. Examples of coatings include carrageenan, cellulose phthalate acetate, ethylcellulose, gellan gum, matodextrin, methacrylate, methylcellulose, microcrystalline cellulose, and shellac.

[0222] The composition may contain one or more preservatives. Examples of preservatives include antioxidants, chelating agents, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0223] Exemplary antioxidants include alpha-tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium pyrosulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium pyrosulfite, and sodium sulfite.

[0224] Examples of 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. Examples of antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidourea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, and thimerosal.

[0225] Examples of antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Examples of alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and phenylethyl alcohol.

[0226] Examples of acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0227] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium pyrosulfite, potassium sulfite, potassium pyrosulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.

[0228] The therapeutic composition may be lyophilized. The lyophilized therapeutic composition may contain one or more stabilizers and / or cryoprotectants. The lyophilized therapeutic composition may be reconstituted with a suitable diluent before administration to an individual.

[0229] The therapeutic compositions according to the present invention may be administered alone, simultaneously or sequentially in combination with other therapies, or as combination preparations with other therapeutic agents or multiple therapeutic agents for the treatment of dysbiosis or diseases associated with dysbiosis as described herein. For example, the strains of the present invention may be used in combination with existing therapeutic agents for inflammatory bowel disease, irritable bowel syndrome, metabolic disorders, neuropsychiatric disorders, autoimmune diseases, allergic diseases, cancer, or hepatic encephalopathy.

[0230] For example, if a therapeutic composition is for the treatment of dysbiosis associated with cancer, the therapeutic composition may, optionally, be administered to an 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 cell death ligand 1 (PD-L1) inhibitors, and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitors. Manipulation of the gut microbiota in combination with immune checkpoint inhibitor therapy has been shown to improve the efficacy of immune checkpoint inhibitors in treating cancer. In preferred embodiments, cancer in this context may be lung cancer or melanoma.

[0231] In another embodiment, the composition of the present invention further comprises an immunomodulatory compound. In other embodiments, the immunomodulatory compound is a cytokine, chemokine, or complement component that enhances the expression of immune system accessories or adhesion molecules, their receptors, or combinations thereof. In some embodiments, the immunomodulatory compound may be an interleukin, e.g., interleukin 1-15, interferon alpha, beta, or gamma; tumor necrosis factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF); neutrophil-activating protein (NAP); macrophage chemoattractant and activator (MCAF); RANTES; macrophage inflammatory peptides MIP-1a and MIP-1b; complement components, or combinations thereof. In other embodiments, the immunomodulatory compound stimulates the expression or enhancement of expression of OX40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1, B7.2, TRAP, ICAM-1, 2, or 3, cytokine receptors, or combinations thereof.

[0232] In another embodiment, the immunomodulatory compound induces or enhances the expression of costimulatory molecules participating in the immune response, including CD40 or its ligand, CD28, CTLA-4, or B7 molecules, in some embodiments. In yet another embodiment, the immunomodulatory compound induces or enhances the expression of heat-stable antigens (HSAs), chondroitin sulfate-modified MHC invariant chains (Ii-CS), or intracellular adhesion molecule 1 (ICAM-1).

[0233] The therapeutic compositions of the present invention may be administered to an individual, preferably a human individual. The administration may be a “therapeutic effective dose,” which is sufficient to demonstrate a beneficial effect on the individual. Such a benefit may be the improvement of at least one symptom. Thus, “treatment” of a particular disease refers to the improvement of at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated, the specific patient being treated, the clinical condition of the individual patient, the cause of dysbiosis, the site of delivery of the composition, the type of therapeutic composition, the method of administration, the administration schedule, and other factors known to the physician. The determination of the prescription of treatment, e.g., dosage, is the responsibility of the general practitioner and other physicians and may depend on the severity and / or progression of the symptoms of the disease being treated. The therapeutic effective dose or preferred dose of the therapeutic composition of the present invention can be determined by comparing its in vitro and in vivo activities in animal models or Phase 0 human studies. Methods for extrapolating effective doses in mice and other test animals to humans are known. The exact dose will depend on several factors, including whether the therapeutic composition is for prevention or treatment.

[0234] The formulation components may be brought into contact with the preparation and mixed or prepared until a formulation is obtained. As will be apparent to those skilled in the art, the formulation conditions will generally be such that viable microorganisms are maintained. In particular, high temperatures, such as those above 40°C, should be avoided.

[0235] The amount of viable microorganisms contained in the composition can vary, be adjusted and optimized, as will be understood by those skilled in the art. Such optimization can be achieved, for example, by preparing a series of different doses of viable 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, 30 billion to 35 billion cfu / mL, 35 The possible cfu / mL concentrations are 0 to 40 billion cfu / mL, 40 to 45 billion cfu / mL, 45 to 50 billion cfu / mL, 50 to 55 billion cfu / mL, 55 to 60 billion cfu / mL, 60 to 65 billion cfu / mL, 65 to 70 billion cfu / mL, 70 to 75 billion cfu / mL, 75 to 80 billion cfu / mL, 80 to 85 billion cfu / mL, 85 to 90 billion cfu / mL, 90 to 95 billion cfu / mL, and 95 to 100 billion cfu / mL.

[0236] In one embodiment, the strain of the present invention can be administered in doses of, for example, 0.01 to 100 × 10¹¹ cells / body, 0.1 to 10 × 10¹¹ cells / body, or 0.3 to 5 × 10¹¹ cells / body. Furthermore, for example, the daily intake as a microorganism may be 0.01 to 100 × 10¹¹ cells / 60 kg body weight, 0.1 to 10 × 10¹¹ cells / 60 kg body weight, or 0.3 to 5 × 10¹¹ cells / 60 kg body weight.

[0237] The content of at least one strain of bacteria, archaea, or fungi contained in the orally ingested composition of the present invention can be appropriately determined depending on its application. As dried microbial material, it may be, for example, 5-50 w / w%, 1-75 w / w%, 0.1-100 w / w%, or 1-100 w / w%.

[0238] In one embodiment, the composition is a controlled-release composition. As used herein, the term “controlled-release” refers to the controlled release or administration of the strain of the present invention from a given dosage form in order to achieve a desired pharmacokinetic profile in vivo. One aspect of “controlled” delivery is the ability to manipulate the formulation and / or dosage form to establish a desired release rate.

[0239] Procedures for preparing the compositions of the present invention in the form of tablets, caplets, capsules, and other forms are known to those skilled in the art and are not limited to, but include wet granulation, dry granulation, and (in the case of tablets and caplets) direct compression.

[0240] Wet and dry granulation are used to manufacture tablets, caplets, or capsules. Granulation techniques using Chilsonation produce powders for dosage forms. A Chilsonator houses grooved rotating rollers that are pressed tightly together by hydraulic pressure. The raw materials are placed in the Chilsonator's hopper and fed to the rollers by a system of horizontal and vertical screws. As the material passes through the grooves of the rollers, it is compressed under very high pressure and discharged from the Chilsonator as a high-density sheet. The sheet is ground into fine granular powder using a Fitz mill, and then passes through a screen to produce uniform, free-flowing granules. The Chilsonation process produces a finished powder that is 2 to 4 times denser than the starting material, which is a characteristic feature that allows the ingredients to be formed into the desired dosage form.

[0241] In dry granulation, the powder can be incorporated into gelatin capsules or mixed with gelatin to form tablets or caplets. In wet granulation, the powder is moistened, thus creating large "lumps" of material, which are then dried and pulverized to convert the lumps into particles of the desired size for the manufacturing process. Once particles of the desired size are obtained, the particles can be incorporated into gelatin capsules or mixed with gelatin to form tablets or caplets.

[0242] General considerations in formulation and / or manufacturing are incorporated, for example, by reference, Remington's The Science and Practice of Pharmacy, 23 rd It can be found in Edition. (2020, Mack Publishing Co., Easton, Pa.).

[0243] Prebiotics The compositions of the present invention may include prebiotics. Because prebiotics have a chemical structure that resists digestion through the digestive tract, they can reach the colon as intact molecules, where they can elicit systemic physiological functions and function as fermentable substrates for the colonic microbiota. When prebiotics are combined with biological therapeutic agents, the resulting compositions are sometimes referred to as "synbiotics."

[0244] Suitable prebiotics include, but are not limited to, fructo-oligosaccharides, P95 Nutraflora®, and oligosaccharides such as galactooligosaccharides, xylooligosaccharides, isomaltoligosaccharides, human milk oligosaccharides, inulin oligosaccharides, mannan oligosaccharides, pyrodextrins, levan, maltotriose, pectic oligosaccharides, vimno-galactooligosaccharides, arabinoxylan, fucoidan, and resistant starch. Fructoligosaccharides can be extracted from chicory, artichoke, asparagus, dandelion, dahlia, endive, garlic, leek, lettuce, and onion.

[0245] In one embodiment, the prebiotics include amino acids such as one or more or all of alanine, aspartic acid, glutamic acid, glycine, leucine, isoleucine, proline, serine, threonine, and valine.

[0246] In one embodiment, the prebiotics may include simple sugars that are monosaccharides (such as glucose, galactose, or fructose) and / or disaccharides (such as sucrose maltose or lactose).

[0247] In one embodiment, the prebiotics constitute 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.

[0248] Other microorganisms To obtain desirable health benefits for the target, it may be advantageous to include one or more additional biological therapeutic microorganisms in the composition. Therefore, the composition may contain two or more species / strains of microorganisms, such as two, three, four, five, or more species / strains of microorganisms, in addition to the strain of the present invention. Non-limiting examples of biological therapeutics are preferred strains selected from Table 3. It should be understood that the aforementioned list is intended to be merely illustrative and not an exhaustive expression of biological therapeutics that may be included in the composition of the present invention. In this regard, any additional biological therapeutic species may also be used in the composition of the present invention.

[0249] In one embodiment, the Enterococcus species include Enterococcus faecalis and / or Enterococcus faecium.

[0250] In one embodiment, the Lactobacillus species is Lactobacillus rhamnosus (such as strain GG (ATCC 53103), CGMCC 1.3724, or SP1 (DSM 21690)), Lactococcus lactis, Lactococcus cremoris, Lactococcus diacetylactis, Lactobacillus paracasei, Lactobacillus reuteri (such as strain ATCC55730 or DSM17938), Lactobacillus acidophilus, Lactobacillus murinus, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus taiwanensis, Lactobacillus animalis, Lactobacillus The selection is made from the group consisting of johnsonii (strain NCC533, CNCM1-1225, etc.) and Lactobacillus gasseri.

[0251] In one embodiment, the Bifidobacterium species is selected from the group consisting of Bifidobacterium lactis (e.g., strain BB-12, BI-04, or CNCM1-3446 (Bb12)), Bifidobacterium longum (e.g., strain NCC3001, ATCC BAA-999 (BB536)), Bifidobacterium breve (e.g., strain Bb-03, M-16V, or R0070), Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium pseudocatenulatum, and Bifidobacterium adolescentis.

[0252] In one embodiment, examples of Streptococcus species include Streptococcus thermophilus such as Streptococcus thermophilus ST-21, Streptococcus pasteurianus, and Streptococcus salivarius.

[0253] In one embodiment, examples of Clostridia include Clostridium difficile, Clostridium butyricum, Clostridium hylemonae, Clostridium scindens, Clostridium species C1, Clostridium spiroforme, and Flavinofractor plautii.

[0254] Some yeasts are also useful as biological therapeutics and are sometimes included in the composition. A non-limiting example of yeast used in biological therapeutics is Saccharomyces boulardii.

[0255] Some archaea are also useful as biological therapeutics and are sometimes included in the composition. Non-limiting examples of archaea used in biological therapeutics are Methanobrevibacter species including Methanobrevibacter smithii, and Methanosphaera species including Methanobrevibacter stadtmanae.

[0256] [[ID=J]] Dosage form Dosage forms are within the scope of the present invention. In a preferred embodiment, the present invention provides a dosage form comprising the composition according to the first aspect of the present invention. Preferably, the dosage form is stored in a sealed sterile container.

[0257] [[ID=J]] Method for treatment A method for treating a disease or disorder is within the scope of the present invention.

[0258] Methods for treating gastrointestinal disorders are also within the scope of the present invention.

[0259] In a preferred embodiment, the present invention provides a method for treating a disorder associated with loss of or dysbiosis of the intestinal microbiota, the method comprising administering a therapeutically effective amount of the composition described in a first aspect of the present invention to a patient in need of treatment.

[0260] Fecal microbiota transplantation Fecal microbiota transplantation (FMT) is the administration of human colon microbiota to a patient's intestines and was originally designed to treat Clostridium difficile infection. Currently, FMT is being explored as a treatment for many other diseases, including ulcerative colitis. However, these efforts are hampered by the loss of viable microbiota samples due to inadequate sampling, storage, shipping, and delivery conditions that result in the loss of viable cells. There are challenges in sampling, storing, shipping, and delivering viable and effective microbiota samples to patients. Furthermore, there is a need in this field for effective treatments of diseases associated with intestinal microbial loss or dysbiosis.

[0261] Culture or second-generation microbiome-based therapies Clinical trials have demonstrated the efficacy and safety of FMT for many diseases, including, but not limited to, C. difficile infection, ulcerative colitis, and irritable bowel syndrome. Therefore, there is a need to identify microorganisms for use in defined cultured microbiome-based therapies. Culture therapy is advantageous over FMT produced in bioreactors (not derived from human donors) because it can have greater consistency in composition, greater scalability in production, and a more predictable safety profile. An ideal second-generation microbiome-based therapy would be cultured and isolated from human fecal matter. Many of these organisms are highly sensitive to oxygen environments and would not survive storage at very low temperatures without freezing or lyophilization (lyophilization). There are challenges in sampling, storing, and shipping and delivering feasible and effective microbiome samples to patients. Furthermore, there is a need in this art for effective treatments of diseases associated with gut microbiota loss or dysbiosis.

[0262] In a further preferred embodiment, the dosage form is administered in an amount that at least partially treats the gastrointestinal disorder.

[0263] The subjects that can be treated by this invention include humans, as well as other mammals and animals.

[0264] The effects of the administered therapeutic composition can be monitored by standard diagnostic procedures.

[0265] The methods of the present invention can be used to treat or prevent gastrointestinal dysbiosis in a subject. In the context of the present invention, “dysbiosis” refers to a condition in which the normal diversity and / or function of the microbiome, particularly the human gastrointestinal microbiome, is disrupted. Any disruption from the normal state of the microbiome in a healthy individual can be considered dysbiosis, even if the dysbiosis does not result in a detectable decrease in the individual's health. In preferred embodiments, dysbiosis may be associated with one or more pathological symptoms. For example, “dysbiosis” may refer to a decrease in the microbial diversity of the microbiome. In addition or alternatively, “dysbiosis” may refer to an increase in the abundance of one or more bacteria, e.g., one or more pathogenic bacteria, in the microbiome of an individual compared to the abundance of such or more bacteria in the microbiome of a healthy individual, i.e., an individual without dysbiosis. Pathogenic bacteria present in dysbiosis are often Proteobacteria, which are resistant to one or more antibiotics. Examples of Proteobacteria include Escherichia, Salmonella, Campylobacter, Vibrio, Helicobacter, and Yersinia species.

[0266] Dysbiosis can be associated with intestinal bacterial infections, such as infections of the gastrointestinal tract caused by pathogenic bacteria. Many bacteria are known to cause gastrointestinal infections in humans, including Gram-positive and Gram-negative bacteria. Pathogenic bacteria are preferably pathogenic species of the genera Clostridium, Escherichia, Enterococcus, Klebsiella, Enterobacter, Proteus, Salmonella, Shigella, Staphylococcus, Vibrio, Aeromonas, Campylobacter, Plesiomonas, Bacillus, Helicobacter, Listeria, or Yersinia. Preferred examples of such pathogenic bacteria 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 pathogenic bacteria are pathogenic species of the genera Clostridium or Escherichia. Most preferably, the pathogenic bacteria are Clostridioides difficile or Escherichia coli.

[0267] The method of the present invention can be used to reduce or prevent inflammation of the gastrointestinal mucosa in a subject using the composition of the present invention.

[0268] In one embodiment, the subjects have or are prone to having inflammatory bowel disease (IBD). IBD is an increasingly prevalent and incurable current condition thought to be caused by an abnormal immune response to the commensal gut microbiome in genetically predisposed patients. The term IBD encompasses both ulcerative colitis (UC), Crohn's disease (CD), and pouchitis. UC is characterized by chronic non-granulomatous inflammation confined to the colonic mucosa, typically involving a variable proximal range of the rectum and continuous colon. CD is characterized by transmural, often granulomatous inflammation and can involve any part of the digestive tract from mouth to anus.

[0269] As used herein, the term “inflammatory bowel disease (IBD)” has its general meaning in the art, and includes Crohn’s disease (granulomatous colitis, Crohn’s disease of the small intestine, Crohn’s disease of the large intestine, granulomatous and focal colitis, Crohn’s disease of the colon, large intestine, and rectum, Crohn’s disease of both the small and large intestines, etc.), ulcerative colitis (ulcerative (chronic) pancreatitis, ulcerative colitis with inflammation beyond the ileocecal valve (backwash ileitis), ulcerative (chronic) proctitis, ulcerative (chronic) rectosigmoid colitis, inflammatory polyps, left-sided colitis, left hemicolitis (left IBD refers to a group of inflammatory diseases of the colon and small intestine, such as backwash ileitis, as revised in the World Health Organization classification K20-K93 (ICD-10), including postoperative complications of the digestive system, such as hemicolitis, non-infectious gastroenteritis and colitis (radiation-induced gastroenteritis and colitis, toxic gastroenteritis and colitis, allergic and nutritional gastroenteritis and colitis, food irritation gastroenteritis or colitis, colitis that is difficult to differentiate, certain non-infectious gastroenteritis and collagenous colitis, eosinophilic gastritis or gastroenteritis, lymphocytic colitis, microcolitis (collagenous colitis or lymphocytic colitis), colitis such as non-infectious gastroenteritis and diarrhea, enteritis, ileitis, jejunal colitis, sigmoid colitis), and postoperative complications of the digestive system such as pouchitis. In one embodiment, IBD is pediatric IBD.

[0270] In one embodiment, subjects have or are prone to having any form of irritable bowel syndrome (IBS) as detailed in the Rome IV criteria. These include, but are not limited to, diarrhea-dominant IBS-D, constipation-dominant IBS-C, or mixed IBS-M. These subjects may respond to a low-FODMAP diet, a gluten-free diet, or other dietary restrictions.

[0271] In one embodiment, the subjects are individuals who have or are prone to having malignant tumors or cancer. These include, but are not limited to, melanoma, lung cancer, intestinal cancer, gastric cancer, esophageal cancer, oral cancer, hepatocellular carcinoma, hematological malignancies, breast cancer, lymphoma, sarcoma, germ cell tumor, carcinoma, kidney cancer, prostate cancer, pancreatic cancer, ovarian cancer, thyroid cancer, brain malignancies, skin cancer, melanoma, bladder cancer, or testicular cancer.

[0272] In one embodiment, the subjects are individuals who have or are prone to liver disease. These include, but are not limited to, cirrhosis, hepatic encephalopathy, alcoholic hepatitis, infectious hepatitis, autoimmune hepatitis, or ascites.

[0273] In one embodiment, the subjects have or are prone to having a disease associated with dysbiosis, or a reduction in gut microbial diversity, or a reduction in the gut microbial ecosystem.

[0274] In a further embodiment, the present invention also relates to a fecal microbiota transplantation composition 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.

[0275] Administration to humans includes administration by medical professionals and self-administration. Generally, to achieve health benefits, multiple doses of the composition are administered daily for, for example, 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.

[0276] Device The device is within the scope of the present invention. In a preferred embodiment, the present invention provides a device comprising: (1) a composition according to the first aspect of the present invention; and (2) an applicator, a container, or a material.

[0277] Use of the composition in the manufacture of a medicament The use is within the scope of the present invention. In a preferred embodiment, the present invention provides the use of a composition in the manufacture of a medicament for treating gastrointestinal disorders.

[0278] Method for stabilization A method for stabilizing the composition of the present invention is within the scope of the present invention.

[0279] In a further preferred embodiment, the method protects the composition of the present invention from degradation.

[0280] In an even further preferred embodiment, the composition of the present invention retains its effective biological activity for a period selected from the group consisting of greater than 24 hours, greater than 36 hours, and greater than 48 hours.

[0281] Since simpler methodologies are more likely to result in less variability in outcomes, and the selection of excipients can be limited to those with generally recognized as safe (GRAS) status, the addition of approved pharmaceutical excipients to stabilize the composition of the solution of the present invention is preferable from a safety standpoint. Excipients for stabilizing protein solutions can be classified into four broad categories based on their chemical properties and mechanisms of action: salts, sugars, polymers, or protein / amino acid. Salts (e.g., chlorides, nitrates) stabilize the tertiary structure of proteins by shielding charges through ionic interactions. Sugars (e.g., glycerol, sorbitol, fructose, trehalose) prevent protein aggregation by increasing the surface tension and viscosity of the solution. Similarly, polymers (e.g., polyethylene glycol, cellulose derivatives) stabilize the tertiary structure of proteins by increasing the viscosity of the solution, thereby preventing 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 through the formation of weak electrostatic interactions.

[0282] As discussed above, the pharmaceuticals of the present invention may comprise one or more pharmaceutically acceptable carriers. The use of such media and agents for the manufacture of pharmaceuticals is well known in the art. Unless any conventional media or agent is incompatible with the pharmaceutically acceptable materials, their use in the manufacture of the pharmaceutical compositions according to the present invention is intended. The pharmaceutically acceptable carriers according to the present invention include one or more of the following examples: a. Surfactants and polymers, including but not limited to polyethylene glycol (PEG), polyvinylpyrrolidone, polyvinyl alcohol, crospovidone, polyvinylpyrrolidone-polyvinyl acrylate copolymer, cellulose derivatives, HPMC, hydroxypropylcellulose, carboxymethylethylcellulose, hydroxypropylmethylcellulose phthalate, polyacrylate, and polymethacrylate, urea, sugars, polyols and their polymers, emulsifiers, sugar gum, starch, organic acids and their salts, vinylpyrrolidone, and vinyl acetate, and / or b. Various celluloses and binders such as cross-linked polyvinylpyrrolidone, microcrystalline cellulose, and / or (3) fillers such as lactose monohydrate, anhydrous lactose, microcrystalline cellulose, and various starches, and / or c. Fillers such as lactose monohydrate, anhydrous lactose, mannitol, microcrystalline cellulose, and various starches, and / or d. Lubricants such as agents that act to increase the capacity of the dosage form to be discharged from the packaging cavity, and / or e. Sweeteners such as any natural or artificial sweetener, including sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame K, and / or f. Fragrances, and / or g. Preservatives such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic chemicals such as phenol, or quaternary compounds such as benzalkonium chloride, and / or h. Buffer, and / or i. Diluents such as pharmaceutically acceptable inert fillers, including microcrystalline cellulose, lactose, calcium dibasic phosphate, sugars, and / or mixtures of any of the above, and / or j. Absorption enhancers such as nitroglycerin, and / or k. May contain other pharmaceutically acceptable excipients.

[0283] The pharmaceuticals of the present invention, which are suitable for use in animals, particularly humans, must typically be sterile and stable under manufacturing and storage conditions.

[0284] Method for detection The strains of the present invention can be detected using a wide variety of known techniques. Conveniently, the strains are detected using nucleic acid-based detection systems.

[0285] In one embodiment, nucleic acid sequencing is used. Exemplary, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, linkage stop sequence (Sanger) sequencing and dye stop sequence sequencing. In some embodiments, the techniques provided herein have been found to be used in second-generation (also known as next-generation or Next-Gen), third-generation (also known as Next-Next-Gen), or fourth-generation (also known as N3-Gen) sequencing techniques, and include, but are not limited to, pyrosequencing, ligation sequencing, single-molecule sequencing, sequence-by-synthesis (SBS), large-scale parallel cloning, large-scale parallel single-molecule SBS, large-scale parallel single-molecule real-time, and large-scale parallel single-molecule real-time nanopore techniques.

[0286] In some embodiments, hybridization is used in the detection method of the present invention. Exemplary, 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, a FISH assay is used. In other embodiments, nucleic acid amplification is used. Nucleic acids may be amplified before detection or simultaneously with detection. Performing one or more amplification reactions may include one or more PCR-based amplifications, non-PCR-based amplifications, or a combination thereof. Exemplary, 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, circle-to-circle 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 certain amplification techniques (e.g., PCR) require the reverse transcription of RNA into DNA before amplification (e.g., RT-PCR), while other amplification techniques (e.g., TMA and NASBA) amplify RNA directly.

[0287] Nucleic acids, whether unamplified or amplified, can be detected by any conventional means. For example, nucleic acids can be detected by hybridization with a detectably labeled probe and measurement of the resulting hybrid. In another example, nucleic acids are detected by sequencing. Exemplary, non-limiting examples of detection methods are described herein.

[0288] The evaluation of the amplification process in "real time" involves determining the amount of amplified material in the reaction mixture either continuously or periodically during the amplification reaction, and using the determined value to calculate the amount of the target sequence initially present in the sample. A variety of 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 initial target sequence present in a sample but not based on real-time amplification is disclosed in U.S. Patent 5,710,029.

[0289] The amplified product can be detected in real time through the use of various self-hybridization probes, most of which have a stem-loop structure. Such self-hybridization probes are labeled to emit different detectable signals depending on whether the probe is in a self-hybridization state or in a state altered through hybridization to a target sequence. As a non-limiting example, a “molecular torch” is a type of self-hybridization probe that contains separate self-complementary regions (referred to as “target-binding domains” and “target-closing domains”) linked by a junction region (e.g., a non-nucleotide linker) and hybridizes to one another under given hybridization assay conditions. In a preferred embodiment, the molecular torch contains a single-stranded base region in the target-binding domain that is 1 to about 20 nucleotides long and accessible for hybridization to a target sequence present during the amplification reaction under strand-displacement conditions. Under chain substitution conditions, except in the presence of the target sequence, hybridization of two complementary regions of the molecular torch, which may be completely or partially complementary, is preferred, binding to a single-stranded region present in the target-binding domain and substituting all or part of the target-closing domain. The target-binding domain and target-closing domain of the molecular torch include a detectable label or a pair of interacting labels (e.g., luminescent / quencher) positioned such that a different signal is generated when the molecular torch is self-hybridized than when the molecular torch is hybridized to the target sequence, thereby enabling detection of the probe:target double strand in the test sample in the presence of an unhybridized molecular torch. Molecular torches and various types of interacting label pairs are disclosed in U.S. Patent No. 6,534,274, which is incorporated herein by reference in its entirety.

[0290] 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 holds the probe in a closed conformation in the absence of the target sequence present during the amplification reaction, and a label pair that interacts when the probe is in the closed conformation. Hybridization of the target sequence and the target complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. This shift to an open conformation is detectable due to a reduction in the interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and 25EDANS). Molecular beacons are disclosed in U.S. Patents 5,925,517 and 6,150,097.

[0291] In one embodiment, the method includes quantifying the amount of strain present in the sample.

[0292] The present invention is described here with reference to the following non-limiting examples. The description of the examples is not intended to limit the preceding paragraphs of this specification, but is provided for illustrative purposes of the methods and compositions of the present invention. [Examples]

[0293] Those skilled in the art of grinding and pharmaceuticals will see that numerous improvements and modifications can be made to the above process without departing from the basic concept of the present invention. For example, in some applications, the biologically active material may be pre-treated and supplied to the process in a pre-treated form. All such modifications and improvements are considered to be within the scope of the present invention, and their properties are determined from 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 processes or compositions of the present invention.

[0294] Example 1 - Formulation Research A.1 Research Objectives The objective of this experiment was to perform a development lyophilization cycle on fecal suspension samples. Eight formulations of excipients were added to samples including negative and positive controls. The specific objectives were (1) to collect data on excipients and excipient concentrations selected to achieve an acceptable lyophilized product that does not kill most cells, and (2) to achieve a water content of less than 10% w / w.

[0295] A.2 Materials, Methods, and Results A.2.1 Equipment The following equipment was used in the preparation and freeze-drying steps of the formulation. [Table 6]

[0296] A.2.2 Excipients and Methods Excipient solutions were prepared at twice the working concentration so that the correct final concentration would be achieved when twice the amount of excipient mixture was added to the FMT sample. Excipient stock solution w / v was added to the FMT solution to constitute a final volume of 3 ml per vial. Seventeen vials (formulation numbers 1-8) were prepared per formulation, filled to a volume of 3 ml. Four vials were prepared per mixture for positive and negative controls. [Table 7] [Table 8]

[0297] The material was filtered using a 0.101 mm filter. During sample processing, and after adding cryoprotectant and physiological saline, the stool was homogenized and filtered. This effectively filters out large plant matter.

[0298] 0.9% sterile physiological saline was used as the carrier for the cryoprotection agent sample.

[0299] A research batch record (BB374) was created to document the production of the excipients and the 10 different formulations produced. Sixteen vials of each cryoprotective formulation were produced, with four each required for positive and negative controls. For testing purposes, the inventor retained one vial of each formulation, including the controls. (126 vials were sent to BioPharma Process Systems Ltd, Biopharma House, Winnall Valley Road, Winchester SO23 0LD, United Kingdom.)

[0300] The team reported the following results for the different cryoprotectants tested: Inulin itself dissolved well when mixed by hand and was a very fine powder. Sucrose had larger crystals than inulin and required a smaller volume to reach the same weight. It dissolved under vortexing but not by hand. Maltodextrin and dextran 70K were similar in texture to inulin, but they clumped together when attempted to dissolve by hand and required vortexing to dissolve. Pectin was a very fine powder and difficult to weigh in the chamber. When used in formulations, it solidified into a gel-like state that made it impossible to sterilize the filter.

[0301] A.3 Analysis The analysis included the following: Lyostat Analysis: Two 2µl samples were analyzed using a positive control skim milk powder and 10% inulin for Lyostat analysis. Lyostat analysis is a microscopic examination that looks at the freezing line of the product. This is an indicator of the temperature that a freeze-dryer can operate at. Appearance: The appearance of the freeze-dried product was visually evaluated on a scale of 1 to 5 (1=worst, 5=best) by taking photographs. Water content: The water content of three vials of two formulations was analyzed by Karl Fischer titration. Mechanical Properties: Two formulations were selected for mechanical property analysis using MicroPress. MicroPress consists of a load cell with an actuator indenter. This indenter descends while breaking the surface of the cake. As it is pressed down, the applied pressure is displayed on the corresponding graph. The lower the maximum stress (kPa), the more brittle the freeze-dried cake is. mDSC: Two formulations were selected for analysis using solid-state mDSC. CFU Plating: Plate CFU analysis was performed by comparing non-freeze-dried samples with lyophilized samples of all formulations. Frozen non-freeze-dried samples were thawed at room temperature outside the anaerobic chamber for 1.5–2 hours. Lyophilized samples were rehydrated with the same amount of liquid removed during the lyophilization process and allowed to rest for 1 hour. Lyophilized samples were vortexed for 2 minutes to dissolve the lyophilized product in solution. Then, 1 ml was divided equally into 1.5 mL epitubes and vortexed for 5 minutes. Serial dilutions were made in PBS in the range of 10⁻³ to 10⁻⁸ (100 μL of FMT to 900 mL of PBS). Then, 50 μL of the dilution was spread three times on a 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 examined for the "best" three sets of colonies, counting 30 to 300 non-confluent colonies. Photographs of the plates were taken, and colony counts were performed.

[0302] A.4 Results A.4.1 LYOSTAT analysis Both skim milk powder (a) and 10% inulin (C) showed good frozen structures at -50.0°C. Disintegration occurred at -47.0°C for skim milk powder (B), and disintegration began at -30.5°C for 10% inulin (D). This indicates that temperatures below -30.5°C are suitable for water sublimation when using inulin as one of the cryoprotective agents.

[0303] A.4.2 Exterior All formulations using the expected cryoprotective agent were visually assessed as 5 / 5 for dryness, excluding 10 negative control water samples. Partial drying was observed. Pectin was not included in the analysis due to the difficulty of the procedure and lack of consistency.

[0304] A.4.3 Moisture content When moisture content was measured using 5% inulin / 5% maltodextrin, an average moisture content of 1.093% was obtained, indicating that using a combination of 5% inulin and 5% maltodextrin can achieve a moisture content (less than 5%) suitable for microbial storage. The results are shown in Table 9. [Table 9]

[0305] A.4.4 Mechanical properties MicroPress indicated that the sample was brittle. The results were compared with a negative control sample (highly brittle). See Table 10 and Figure 1. [Table 10]

[0306] A.4.5 MDSC mDSC results showed Tg initiation at 51.13°C. Several studies have shown that the sample should be stable at approximately 50°C below Tg initiation, suggesting that storage conditions of 2–8°C may be suitable for the sample. [Table 11]

[0307] A.4.6 CFU Plating Pectin addition showed the smallest decrease in CFU. The pectin sample also had the lowest CFU before lyophilization and was similar to most of the other samples after lyophilization. 10% sucrose showed the second lowest reduction in CFU and the highest CFU both before and after lyophilization. This may be due to sucrose acting as a nutrient that increases CFU before plating. 5% inulin / 5% maltodextrin showed the next smallest reduction and the second highest CFU after lyophilization. This suggests that it is a preferred formulation based on both CFU and the lyophilization process. See also Figures 2 and 3.

[0308] A.5 Consideration A combination of inulin and maltodextrin was selected as the optimal freeze-protective agent for freeze-drying. Inulin is a rapidly to moderately fermentable fiber. The inventors achieved a material with less than 5% residual moisture, the resulting freeze-dried cake was brittle and could be pulverized for further processing, and minimal cell loss was observed via CFU plating. From the experiments presented above, inulin and maltodextrin were preferred over all others, as both were beneficial to the freeze-drying process. Inulin and maltodextrin also showed the smallest logarithmic change in CFU when freeze-dried material was compared to non-freeze-dried material. Inulin demonstrated the ability to maintain its frozen structure without collapse down to -30.5°C, allowing for a rise in temperature when sublimating water from the frozen freeze-dried cake. Compared to trehalose, it is relatively close to the same cake collapse temperature of around -30°C. The inventors have demonstrated that, in preferred embodiments, the compositions of the present invention can freeze-dry faster than sucrose, producing a shorter runtime while still maintaining viability within the sample. Maltodextrin also aids in the time-dependent stability of freeze-dried samples during storage. It contributes to the stabilization of microorganisms within the freeze-dried matrix and helps control moisture intrusion into the freeze-dried product.

[0309] Example B, 2 - Stability Study B.1 Research Objectives The objectives of this study were to (1) determine whether the inulin / maltodextrin cryoprotective formulation maintained the original cell count (ICC) during short-term storage of the intermediate (liquid) product, (2) determine the effect of lyophilization on ICC, (3) evaluate the effect of the inulin / maltodextrin cryoprotective formulation on the ICC of encapsulated lyophilized FMT products over a period of 6 months, and (4) determine the optimal storage temperature for lyophilized encapsulated FMT. The main focus was on confirming the efficacy of the cryoprotective formulation in storing ICC under different time-dependent storage conditions, specifically at -80°C, -20°C, 4-8°C, and 20-25°C, as a time factor.

[0310] B.2 Materials, Methods, and Results B.2.1 Addition of excipients and freeze-drying Intermediate products (liquids) were processed from eight stool samples from a single healthy donor over a one-month period. These intermediate products consisted of donor stool homogenized in an excipient solution containing 5% inulin and 5% maltodextrin dissolved in 0.9% saline (NaCl) at a 1:2.6 w / v ratio. The intermediate products were pooled, the resulting material was homogenized, and filtered. The homogenized material was then stored and frozen at -80°C until freeze-dried. The samples were then freeze-dried according to the protocol and procedures presented in Example 1. After freeze-drying, the samples were pulverized, encapsulated, and stored in induction-sealed bottles at their respective test temperatures. [Table 12]

[0311] B.2.2 Sample collection and raw cell count The undiluted cell count (ICC) was determined via BactoBox (SBT Instruments) from the following samples: (1) undiluted stool from each batch combined in ratios based on the intermediate product (pooled), (2) pooled intermediate product (immediately before lyophilization), and (3) after lyophilization at multiple time points at different temperatures (ground powder, encapsulated product ("T0")), week 1, week 2, week 4, month 2, and month 6. All results were normalized to ICC per gram of stool.

[0312] B.3 Results Eight batches of intermediate products were compared to undiluted stool samples to determine whether the inulin / maltodextrin cryoprotective formulation was sufficient as a short-term cryoprotective agent for the intermediate products (Figure 4). Overall, with the exception of batch 23, which had significantly lower ICCs than undiluted stool samples from the same donor, there were no statistically significant differences in ICCs between the intermediate products at the time of testing and the undiluted stool samples. When pooled undiluted stool samples were compared to pooled intermediate products, the results were comparable (undiluted stool 1.97 x 10⁻¹⁰). 9 ICC / g(SD1.17x10 8 ) vs. intermediate product 1.91x10 9 ICC / g(SD2.14x10 8 p=0.729).

[0313] To determine the loss of viability due to the freeze-drying process, the ICC / g of undiluted stool before freeze-drying and stool in the intermediate product was compared with the freeze-dried samples after grinding and freeze-dried powder encapsulation (Figure 5). As shown above, there was no difference between the undiluted stool before freeze-drying and the intermediate product; however, there were significant differences between both the undiluted stool (pooled) and the intermediate product, as well as between the ground powder and the encapsulated product. The intermediate product had an average ICC / g of 1.91 × 10⁻⁶. 9 ICC / g(SD.2.14×10 8 ), and pulverized powder 9.7 × 10 8 It was determined that the encapsulated product had an ICC / g (SD 6.99 × 10^7) (p = less than 0.0001), and compared to the intermediate product, the encapsulated product had an average ICC of 2.81 × 10^7. 8It has ICC / fecal g (SD 1.33 × 10^8) (p = 0.0007).

[0314] In a follow-up survival study to compare the effects of storage time and temperature, the encapsulation product was used as "T0" (Figure 6). No significant reduction in ICC was observed between T0 and any given follow-up time point. Furthermore, storage temperature did not affect ICC over time.

[0315] B.4 Consideration Cryoprotective formulations of 5% w / v inulin and 5% w / v maltodextrin were tested for their ability to act as cryoprotective agents for intermediate products and as lysis-protective agents for FMT intermediate products through lyophilization. Inulin and maltodextrin were suitable cryoprotective agents within the time frame tested, and the ICC was equivalent to that of undiluted stool. A significant reduction in cell viability occurred throughout the lyophilization process, which was expected. This logarithmic reduction of approximately 0.78 in the ICC was within the acceptable and expected range of a logarithmic reduction of approximately 1 in the ICC. Furthermore, the ICC was determined to be consistent throughout the entire 28-week period tested, regardless of storage temperature, indicating that the inulin / maltodextrin cryoprotective formulations maintained microbial stability regardless of the storage time and temperature tested herein.

[0316] In conclusion, inulin and maltodextrin demonstrate efficacy as cryoprotective / freeze-drying protective agents for FMT.

[0317] Example C3 - Formulation study of the complex community biobiological therapeutic product BB265 C.1 Research Objectives The objective of this study was to perform a lyophilization cycle for the development of the complex community biological therapy product BB265. Specifically, the objectives were (1) to collect data on the optimal excipients and excipient concentrations to achieve an acceptable lyophilized product that does not kill most cells, as determined in Example 1, and (2) to achieve a water content of less than 10% w / w.

[0318] C.2 Materials, Methods, and Results C.2.1 Composition of BB265 [Table 13] [Table 14] [Table 15]

[0319] C.2.2 Equipment The following equipment was used in the preparation and freeze-drying steps of the formulation. [Table 16]

[0320] C.2.3 Excipients and Method An excipient solution containing 5.0% w / v inulin and 5.0% w / v maltodextrin dissolved in 0.9% physiological saline (NaCl) was prepared and added to microbial pellets of pelletized harvest from the BB265 composite consortium in a ratio of 2.6:1 v / w. The mixture was then homogenized. The sample was immediately frozen at -80°C and sent frozen to BioPharma Process Systems Ltd, Biopharma House, Winnall Valley Road, Winchester SO23 0LD, United Kingdom for analysis. [Table 17]

[0321] C.2.4 Analysis The analysis included the following: Lyostat Analysis: A 2 μL sample was analyzed using Lyostat analysis. Lyostat analysis is a microscopic examination that examines the freezing line of the product. This is an indicator of the temperature that a freeze-dryer can operate at. Appearance: The appearance of the freeze-dried product was visually evaluated on a scale of 1 to 5 (1=worst, 5=best) by taking photographs. Moisture content: The moisture content from the three vials was analyzed by Karl Fischer titration. Mechanical Properties: Mechanical properties analysis was performed using MicroPress. MicroPress consists of a load cell with an actuator indenter. This indenter descends while breaking the surface of the cake. As pressure is applied, the applied pressure is displayed on the corresponding graph. The lower the maximum stress (kPa), the more brittle the freeze-dried cake is. mDSC: Modulated differential scanning calorimetry (mDSC) was used to determine the thermal properties of the formulation during lyophilization.

[0322] C.3 Results Lyostat analysis. A good frozen structure was observed at -50.0°C. Disintegration occurred at -38.5°C, indicating that temperatures below -38.5°C are suitable for water sublimation when using inulin and maltodextrin as cryoprotectants for BB265.

[0323] Appearance. All vials scored 5 / 5 for the visual appearance of the structure after drying and freeze-drying.

[0324] Moisture content. The average residual moisture content was determined to be 2.13% w / w, indicating that a moisture content suitable for microbial storage (less than 5%) can be achieved by using a combination of 5% inulin and 5% maltodextrin. [Table 18]

[0325] Mechanical properties. MicroPress analysis demonstrated the robustness of the sample, showing that the instantaneous maximum stress at the fracture point was 296.544 kPa and the Young's modulus was 4.80E.

[0326] Modulated differential scanning calorimetry (mDSC). The mDSC results showed Tg onset at 53.89°C. Several studies have indicated that the sample should be stable approximately 50°C below Tg onset, suggesting that storage conditions of 2–8°C may be suitable for the sample. [Table 19]

[0327] C.4 Consideration As the optimal cryoprotectant for freeze-drying FMT materials, we selected a combination of inulin and maltodextrin, which was used here to evaluate the cryoprotection of the complex community biological treatment product BB265. We achieved a material with less than 5% residual moisture, and the resulting freeze-dried cake is robust and can be pulverized for further processing. From the experiments presented above, the combination of inulin and maltodextrin is in line with those shown for FMT and exhibits optimal cryoprotection parameters when used for freeze-drying BB265. The mixture demonstrated the ability to maintain its frozen structure without collapse down to a maximum of -38.5°C, allowing for a rise in temperature when sublimating water from the frozen freeze-dried cake. Compared to trehalose, it is relatively close to the same cake collapse temperature of around -30°C.

Claims

1. A composition for preventing or treating a disease or disorder in a subject where such treatment is necessary, wherein the composition comprises at least one strain of a microorganism. The aforementioned microorganism is at least one strain selected from the group consisting of bacteria, yeast, or archaea. A composition comprising an excipient.

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

3. The composition according to claim 1 or 2, wherein the excipient is inulin, or an analog or variant thereof.

4. The composition according to claim 3, wherein the inulin is selected from the group consisting of alpha-D-glucopyranosyl-[beta-D-fructofuranosyl](n-1)-D-fructofuranosyl, beta-D-fructopyranosyl-[D-fructofuranosyl](n-1)-D-fructofuranosyl, fructo-oligosaccharide, fructo-oligosaccharide containing 2 to 70 fructose units, fructo-oligosaccharide containing 1 to 500 fructose units, fructo-oligosaccharide containing 1 to 300 fructose units, fructo-oligosaccharide containing 1 to 200 fructose units, fructo-oligosaccharide containing 1 to 100 fructose units, or analogs, variants, or combinations thereof.

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

6. The composition according to any one of claims 5, wherein the maltodextrin is selected from the group consisting of maltodextrin having a length selected from the group consisting of 3 to 17 glucose units, corn syrup having a length of 20 or more glucose units, corn syrup solid, modified corn starch, modified rice starch, modified tapioca starch, modified wheat starch, or analogs, variants, or combinations thereof.

7. The composition according to any one of claims 1 to 6, wherein the composition contains inulin or an analogue of a variant 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 contains maltodextrin or an analogue of its variant at a concentration 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 aforementioned composition includes 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% A composition according to any one of claims 1 to 9, comprising inulin and maltodextrin at a concentration selected from the group consisting of (w / v) and maltodextrin (10% w / v).

11. The composition according to any one of claims 1 to 10, wherein the composition comprises (1) inulin at a concentration 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) maltodextrin at a concentration 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 inulin and maltodextrin at a concentration 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 aforementioned composition includes 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% A composition according to any one of claims 1 to 11, comprising inulin and maltodextrin in a concentration selected from the group consisting of w / v and maltodextrin (10% w / v).

13. A biological therapeutic composition comprising the composition according to any one of claims 1 to 12, together with an acceptable diluent or carrier.

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

15. A method for treating and / or preventing a disease or disorder in a patient who requires treatment and / or prevention, the method comprising administering an effective amount of the composition according to any one of claims 1 to 12 to the subject.

16. A method for preparing the biological therapeutic composition according to claim 13, wherein the method comprises mixing the composition according to any one of claims 1 to 12 with an acceptable diluent or carrier.

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

18. Use of the composition according to any one of claims 1 to 12 in the manufacture of a pharmaceutical product for reducing or preventing a disease or disorder in a target.

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

20. A kit comprising the dosage form described in claim 19, together with instructions for use thereof.

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