Improved compositions and methods for fecal microbiota transplantation and microbiota transplant therapies

Abstract

The present disclosure provides freeze-dried compositions that include an extract of human feces and a flavoring agent, where the compositions, upon reconstitution with a liquid, have no objectionable odor and has no objectionable taste. Also provided are methods for making and using such compositions, including methods for replacing or supplementing or modifying a subject's colon microbiota, and methods for treating a disease. In some embodiments, the subject can be an individual with reduced ability to swallow, such as pill dysphagia, or an individual refractory to administration of a liquid having an objectionable odor or objectionable taste.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 616,001, filed December 29, 2023, which is incorporated by reference herein in its entirety.

[0003] BACKGROUND

[0004] Over the last decade, fecal microbiota transplantation (FMT) has emerged as a highly effective treatment for recurrent Clostridioides difficile infections (rCDI) that fail traditional treatment with antibiotics alone. As initially envisioned and practiced, FMT involved administration of a fecal slurry that was prepared from stool of a healthy donor, typically identified by the patient, via nasogastric tube or enema (Brandt and Aroniadis, Gastrointestinal endoscopy. 2013;78:240-249; Aas et al., Clinical infectious diseases. 2003;36:580-585). However, these treatments lacked standardization and presented formidable logistical difficulties for routine implementation into clinical practice. These problems were solved in large part by the development of formal criteria for use of standardized donors, protocols for separating microbiota from stool, cryopreservation using glycerol as a cryoprotectant, and dose quantification in terms of viable bacterial counts (Hamilton et al, The American journal of gastroenterology. 2012;107:761-767). While colonoscopy was initially one of the most common methods of administering of the purified liquid suspension of fecal microbiota, an encapsulated preparation of purified and freeze-dried fecal microbiota was more recently introduced and shown to have comparable efficacy in treatment of rCDI, relative to freshly prepared materials (Staley et al., The American journal of gastroenterology. 2017;112:940-947; Vaughn et al., Clin Gastroenterol Hepatol. 2023 May;21(5): 1330-1337.e2). The use of oral capsules eliminated the burden, costs, and intrinsic risks associated with colonoscopy and enabled more mainstream practice of FMT for C. difficile indications. Although administration of donor microbiota via FMT was originally developed to repair gut microbes decimated by antibiotics, as is the case in C. difficile infections, many other indications have since been, and continue to be, investigated with the goal of restructuring the gut microbiome. These indications, e.g., typically require repeated dosing, which is best accomplished using oral administration, which is much less burdensome relative to colonoscopy or even enema (Kang et al., Microbiome. 2017;5:10; Haifer et al., Lancet Gastroenterol Hepatol. 2022;7:141-151). The term acknowledging the complexity and intensity of these dosing regimens is microbiota transplant therapy or MTT (Kang et al., Microbiome. 2017).

[0005] SUMMARY OF THE APPLICATION

[0006] A primary exclusion criterion for MTT with oral capsules is pill dysphagia, the inability to swallow capsules or tablets. Pill dysphagia is not rare and is especially common among the elderly patients with esophageal dysmotility and strictures. The inability to swallow capsules is also a challenge in very young patients and in children and adults with developmental disabilities. While other means of placement of microbiota into recipients might be plausible, the freeze-dried microbiota currently contained within the capsules is not organoleptically acceptable in a liquid suspension, in large part due to the residual odor and foul taste associated with the partially purified fecal material that results from using standard preparation methods (Hamilton et al., The American journal of gastroenterology. 2012;107:761-767; Staley et al., The American journal of gastroenterology. 2017; 112:940947; Sadowsky et al., WO 2012 / 122478; Sadowsky et al., WO 2014 / 152484).

[0007] Described herein is the development and clinical evaluation of a drinkable liquid (oral liquid preparation) preparation for microbiota replacement therapies, where the preparation overcomes obstacles in patients with pill dysphagia, difficulties in swallowing capsules, and those who are not candidates for colonoscopy. The microbial preparation includes a flowable, dissolvable powder that can be easily administered to the patient using a carrier liquid. Optionally, the powder is also flavored. Since the final preparation was organoleptically acceptable to the tested patients, displaying no objectionable odors or taste, the formulation will be useful for certain pediatric populations, and pediatric and adult populations with conditions that limit the ability to swallow, such as pill dysphagia.

[0008] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein, and their meanings, are set forth below.

[0009] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0010] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not imply that the use of "or" in other instances may not mean "and / or."

[0011] The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.

[0012] As used herein, "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" or the like are used in their open ended inclusive sense, and generally mean "include, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0013] It is understood that wherever embodiments are described herein with the language "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" and the like, otherwise analogous embodiments described in terms of "consisting of' and / or "consisting essentially of' are also provided. The term "consisting of means including, and limited to, whatever follows the phrase "consisting of." That is, "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of' indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0014] Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0015] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0016] In the description herein particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

[0017] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0018] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0019] BRIEF DESCRIPTION OF THE FIGURES

[0020] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings. FIG. 1 shows changes in the microbial community structure following oral administration of liquid suspension of powdered fecal microbiota. Patient 1 stool was sampled at the following time points: 1=4 days prior to FMT; 2=1 week post-FMT; 3 = 3 weeks post-FMT; 4 = 4 weeks post-FMT. FIG. 1A. Bacterial genera are shown in different colors and donor similarity, as determined by using the SourceTracker program, is represented by empty circles. FIG. IB. The PCoA plot shows the movement of the bacterial composition in the patient’s stool with respect to the donor sample. FIG. IC. Shannon diversity index of the microbiota within donor and patient samples at different time points.

[0021] Schematic drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.

[0022] DETAILED DESCRIPTION

[0023] Provided herein are compositions that include microbes (microbiota) recovered from feces of human donors. As used herein, the term “fecal microbes” refers to microorganisms that are present in the gut, intestine, colon, or feces of a normal healthy adult human. The term gut microbiome refers to the totality of genomic material present in all microbiota present in the gut. Such a composition may be prepared by processing fecal material. As used herein, the term “fecal material” refers to human stool. Unprocessed fecal material contains non-living material and biological material. The “non-living material” refers to the nonliving material in fecal material, and may include, but is not limited to, dead bacteria, shed host cells, proteins, carbohydrates, fats, minerals, mucus, bile, undigested fiber and other foods, and other compounds resulting from food and metabolic ingestion and waste products and partial or complete digestion of food materials. “Non-living material” does not include an excipient, e.g., a pharmaceutically inactive substance, such as a cryoprotectant or lyoprotectant, added to a processed fecal material. “Biological material” refers to the living material, also referred to as microorganisms, in fecal material (e.g., all possible microorganisms present in a human gut), and includes microbes including prokaryotic cells, such as bacteria and archaea (e.g., living prokaryotic cells and spores that can sporulate to become living prokaryotic cells), eukaryotic cells such as protozoa and fungi, and viruses. In one embodiment, "biological material” refers to the living material, e.g., the microbes, eukaryotic cells, and viruses, which are present in the colon of a normal healthy human.

[0024] A composition described herein can be liquid or freeze-dried. As used herein, “liquid" refers to a composition having the characteristics described herein and further having an aqueous component, typically water, milk, or another liquid carrier. As used herein, “freeze-dried” refers to a composition having the characteristics described herein and further having a small amount of moisture dictated by the atmosphere of the preparation room, substantially no water present, and in one embodiment, no detectable water. Methods for freeze-drying a composition are known and routinely used. The word freeze drying is used synonymously with lyophilization. A method for freeze-drying a composition may include one or more pretreatments (e.g., concentrating, addition of a cryoprotectant or lyoprotectant, increasing the surface area of a composition), freezing the composition, and drying (e.g., exposing the composition to a reduced atmospheric pressure to result in sublimation of the water present in the composition).

[0025] Prokaryotic cells that may be present in a composition described herein include the normal members of the intestinal microbiota present in any healthy human. Examples of prokaryotic cells that may be present cells include those that are members of the class Actinobacteria, such as the subclass Actinobacteridae and subclass Coriobacteridae. Examples of the subclass Actinobacteridae include members of the order Actinomycetales, and members of the order Bifidobacteriales. Members of the order Bifi dob act eri ales include members of the family Bifidobacteriaceae. Examples of the subclass Coriobacteridae include members of the order Coriobacteriales. Members of the order Coriobacteriales include members of the family Coriobacteriaceae.

[0026] Other examples of prokaryotic cells include members of the phylum Bacteroidetes, such as class Bacteroidia. Members of class Bacteroidia include order Bacteroidales. Members of order Bacteroidales include members of the family Bacteroidaceae, members of the family Porphyromonadaceae, members of the family Prevotellaceae (such as members of the genus Prevotelid), and members of the family Rikenellaceae.

[0027] Other examples of prokaryotic cells include members of the phylum Firmicutes, such as class Bacilli, Clostridia, Erysipelotrichi, and Negativicutes. Examples of the class Bacilli include members of the order Bacillales (including members of the family Paenibacillaceae and members of the family Planococcaceae) and the order Lactobacillales (including members of the family Aerococcaceae, Enterococcaceae, Lactobacillaceae, and Streptococcaceae). Examples of the class Clostridia include members of the order Clostridiales, and examples of the order Colstridiales include the family Catabacteriaceae, Peptococcaceae, Peptostreptococcaceae, Ruminococcaceae, Clostridiaceae, Eubacteriaceae, and Lachnospiraceae. Examples of the class Erysipelotrichi include members of the family Erysipelotrichaceae. Examples of the class Negativicutes include members of the family Veillonellaceae. Other examples of the order Bacillales include Bacillales Family XI. Incertae Sedis, and Bacillaceae 1. Other examples of the order Clostridiales include Clostridiales Family XI. Incertae Sedis, Clostridiales Family XIII. Incertae Sedis, and Clostridiaceae.

[0028] Other examples of prokaryotic cells include members of the phylum Proteobacteria, such as class Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, and Gammaproteobacteria. Examples of the class Alphaproteobacteria include members of the order Rhizobiales, and examples of members of the order Rhizobiales includes members of the family Rhodobiaceae, members of the family Brucellaceae, and members of the family Hyphomicrobiaceae. Examples of the class Betaproteobacteria include members of the order Burkholderiales, and examples of members of the order Burkholderiales include members of the family Alcaligenaceae, members of the family Burkholderiaceae, and members of the family Sutterellaceae. Examples of the class Deltaproteobacteria include members of the order Desulfovibrionales, and examples of members of this order include members of the family Desulfovibrionaceae and Desulfomicrobiaceae. Examples of the class Epsilonproteobacteria include members of the order Desulfobacterales, and examples of members of this order include members of the family Desulfobacteraceae. Examples of the class Gammaproteobacteria includes members of the order Alteromonadales and Enterobacteriales. Examples of members of the order Alteromonadales include members of the family Shewanellaceae, and examples of members of the order Enterobacteriales include members of the family Enterobacteriaceae.

[0029] Other examples of prokaryotic cells include members of the phylum Mycoplasmatota, formerly Tenericutes. Examples of members of the phylum Mycoplasmatota include members of the class Mollicutes. Examples of the class Mollicutes include members of the order Entomoplasmatales, and members of the order Entomoplasmatales include members of the family Spiroplasmataceae.

[0030] Other examples of prokaryotic cells include members of the class Verrucomicrobiae. Examples of members of the class Verrucomicrobiae include members of the order Verrucomicrobiales, and examples of members of the order Verrucomicrobiales includes members of the family Verrucomicrobiaceae. Other examples of prokaryotic cells include members of the family Fusobacteriaceae.

[0031] In one embodiment a composition described herein may include prokaryotic bacteria that are members of at least 1 phylum, at least 2 phyla, at least 3 phyla, at least 4 phyla, at least 5 phyla, at least 6 phyla, at least 7 phyla, at least 8 phyla, at least 9 phyla, or at least 10 phyla. In one embodiment a composition of the present disclosure may include prokaryotic bacteria that are members of at least 1 class, at least 2 classes, at least 3 classes, at least 4 classes, at least 5 classes, at least 6 classes, or at least 7 classes. In one embodiment a composition of the present disclosure may include prokaryotic bacteria that are members of at least 1 order, at least 2 orders, at least 3 orders, at least 4 orders, a least 5 orders, at least 6 orders, or at least 7 orders. In one embodiment a composition of the present disclosure may include prokaryotic bacteria that are members of at least 1 family, at least 2 families, at least 3 families, at least 4 families, at least 5 families, at least 6 families, at least 7 families. In one embodiment a composition of the present disclosure may include at least 5, at least 10, at least 20, or at least 30 different genera of prokaryotic bacteria. In one embodiment a composition of the present disclosure may include at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 different species of prokaryotic bacteria.

[0032] In one embodiment a freeze-dried composition described herein includes, when reconstituted with water such as a buffer, no greater than 10 % weight of non-living material / weight biological material (wt / wt), no greater than 5 % (wt / wt), no greater than 2.5 % (wt / wt), no greater than 1 % (wt / wt), no greater than 0.1 % (wt / wt), no greater than 0.01 % (wt / wt), or no greater than 0.001 % (wt / wt) non-living material. In one embodiment, the amount of non-living material in a composition of the present disclosure is undetectable using currently available techniques. For instance, living material can be stained for biological activity, electron transport, DNA, and RNA, and DNA and RNA for specific genes.

[0033] In one embodiment, the fecal material present in a liquid composition described herein, and a freeze-dried composition described herein when reconstituted with water, do not include particles (e.g., particles of non-living material and / or particles of biological material) having a size of greater than 2.0 millimeters (mm), greater than 1.0 mm, greater than 0.5 mm, greater than 0.4 mm, greater than 0.3 mm, greater than 0.25 mm, greater than 0.212 mm, greater than 0.180 mm, greater than 0.150 mm, greater than 0.125 mm, greater than 0.106 mm, greater than 0.090 mm, greater than 0.075 mm, greater than 0.063 mm, greater than 0.053 mm, greater than 0.045 mm, greater than 0.038 mm, greater than 0.032 mm, greater than 0.025 mm, greater than 0.020 mm, or greater than 0.01 mm. Non-fecal material present in a composition may include particles having a size of greater than 2.0 mm, greater than 1.0 mm, greater than 0.5 mm, greater than 0.4 mm, greater than 0.3 mm, greater than 0.25 mm, greater than 0.212 mm, greater than 0.180 mm, greater than 0.150 mm, greater than 0.125 mm, greater than 0.106 mm, greater than 0.090 mm, greater than 0.075 mm, greater than 0.063 mm, greater than 0.053 mm, greater than 0.045 mm, greater than 0.038 mm, greater than 0.032 mm, greater than 0.025 mm, greater than 0.020 mm, or greater than 0.01 mm. In one embodiment, the fecal material present in a composition of the present disclosure consists of, or consists essentially of, particles of non-living material and / or biological material having a size that will pass through a sieve having a sieve size of 2.0 mm, 1.0 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.25 mm, 0.212 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, or 0.01 mm. Thus, in such an embodiment, the fecal material present in a composition has a size that is less than or equal to 2.0 mm, less than or equal to 1.0 mm, less than or equal to 0.5 mm, less than or equal to 0.4 mm, less than or equal to 0.3 mm, less than or equal to 0.25 mm, less than or equal to 0.212 mm, less than or equal to 0.180 mm, less than or equal to 0.150 mm, less than or equal to 0.125 mm, less than or equal to 0.106 mm, less than or equal to 0.090 mm, less than or equal to 0.075 mm, less than or equal to 0.063 mm, less than or equal to 0.053 mm, less than or equal to 0.045 mm, less than or equal to 0.038 mm, less than or equal to 0.032 mm, less than or equal to 0.025 mm, less than or equal to 0.020 mm, or less than or equal to 0.01 mm. The sieve size may be based on the US Standard sieve sizes of, for instance, 10, 18, 35, 60, 70, 80, 100, 120, 140, 170, 200, 230, 270, 325, or 400.

[0034] A composition of the present disclosure can include a flavor agent. As used herein, a "flavor agent" or "flavoring agent" is a compound or composition that can be added to a composition described herein, e.g., a liquid composition or a freeze-dried composition, to alter the flavor of the liquid composition, or to alter the flavor of a freeze-dried composition after it is reconstituted with an aqueous solution. A flavor agent can be liquid or powder. Examples of flavor agents include, but are not limited to, chocolate flavor, peppermint, methyl salicylate (wintergreen), or orange flavoring. Liquid and powder forms of chocolate flavor are commercially available.

[0035] A composition of the present disclosure may optionally include a cryoprotectant, a lyoprotectant, or both a cryoprotectant and a lyoprotectant. A cryoprotectant is a compound that maintains the viability of fecal microbes during freezing. A lyoprotectant is a compound that maintains the viability of fecal microbes during drying. Cryoprotectants and lyoprotectants are known in the art and used routinely to protect microbes when exposed to freezing and / or drying conditions. Examples include, but are not limited to, amino acids such as alanine, glycine, proline; simple sugars such as sucrose, glucose, lactose, ribose, mannitol, and trehalose; and other compounds such as dimethyl sulfoxide (DMSO), and glycerol. The amount of cryoprotectant and / or lyoprotectant present in a composition described herein may vary depending on the cryoprotectant and / or lyoprotectant used, the temperature to be used for freezing (e.g., -20 °C,-80 °C, or a different temperature), and the conditions used for drying. The amount of cryoprotectant and / or lyoprotectant that can be used is known to the skilled person or may be easily determined using routine experimentation. In one embodiment, a composition of the present disclosure may include trehalose or mannitol at a concentration of about 8% when prepared for use as a colonoscopic product.

[0036] A freeze-dried composition described herein includes a cryoprotectant and / or lyoprotectant. Cryoprotectants and lyoprotectants useful in freeze drying microbes are known and include, for instance, D-Mannitol, D-Sorbitol, D-Glucose, sucrose, trehalose, casein hydrolysate, sucrose, gelatin, non-fat skim milk, starch hydrolysate, fetal calf serum, bovine serum albumin, or combinations of 1, 2, 3, or 4 of the above cryoprotectants and lyoprotectants. Other cryoprotectants and lyoprotectants are also known. A cryoprotectant useful herein maintains the viability of fecal microbes when subjected to freeze-drying conditions, milling or grinding, and / or when stored as a freeze-dried composition. Milling, also referred to as grinding, is a process that physically changes a material into smaller particles. Methods for milling freeze-dried compositions are known to the skilled person, and can occur at various temperatures, e.g., at or below 0 °C., or above 0 °C. A cryoprotectant and / or lyoprotectant useful herein results in a freeze-dried composition that is friable. As used herein, a "friable” composition refers to a composition that can be easily milled to result in a fine powder. In one embodiment, a freeze-dried composition described herein that is friable is one that results in a powder that can be subsequently used to produce a oral liquid composition. In one embodiment, a freeze-dried composition described herein that is friable is one that results in a powder that can be subsequently used to produce a tablet. In one embodiment, a useful powder may have size, density, flow, and compression characteristics suitable for production of tablets or encapsulation.

[0037] Useful cryoprotectants and / or lyoprotectants include those that result in a composition that is friable, does not crystallize during freeze-drying, and maximizes survival of microbes. In one embodiment, examples of useful cryoprotectants include, but are not limited to, skim milk, gelatin, and mannitol, and sucrose. In one embodiment, more than one cryoprotectant may be used, such as the combination of sucrose (5 %) and skim milk (5 %), trehalose and mannitol, or the combination of sucrose (10 %) and gelatin (0. 1 %).

[0038] In one embodiment, a useful cryoprotectant and / or lyoprotectant is not sucrose alone, which may crystallize and hardens during freeze-drying, or glycerol, which unexpectedly results in an oily and viscous composition upon freeze drying with a fecal material as described herein.

[0039] The total cryoprotectant and / or lyoprotectant used to produce a freeze-dried composition may be 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, or 30 % (vol / vol) of the final concentration of a mixture of fecal microbes and the cryoprotectant and / or lyoprotectant before freeze drying the composition. For instance, to produce a composition having a cryoprotectant and / or lyoprotectant at a final concentration of 10 %, equal volumes of a 20 % solution of the cryoprotectant and / or lyoprotectant and a mixture of microbes derived from fecal material can be combined and mixed, and then freeze-dried.

[0040] In one embodiment a composition of the present disclosure, e.g., a liquid composition or a freeze-dried composition, does not include pathogenic biological material. In one embodiment, fecal material is from a person that has undergone a clinical evaluation, including medical history, a physical examination, and laboratory testing. The clinical evaluation may include, but is not limited to, risk of infectious agents, carriage of multidrug resistant organisms, presence of gastrointestinal co-morbidities, factors that can or do affect the composition of the intestinal microbiota, and systemic medical conditions. Exclusion criteria regarding risk of infectious agents may include, but are not limited to, known viral infection with Hepatitis B, C or HIV; known exposure to HIV or viral hepatitis at any time; high risk behaviors including sex for drugs or money, any past use of intravenous drugs or intranasal cocaine, history of incarceration; tattoo or body piercing within 12 months; travel to areas of the world where risk of traveler's diarrhea is higher than the US; and current communicable disease, e.g., upper respiratory viral infection.

[0041] Exclusion criteria regarding gastrointestinal comorbidities include, but are not limited to, history of irritable bowel syndrome, wherein specific symptoms may include frequent abdominal cramps, excessive gas, bloating, abdominal distension, fecal urgency, diarrhea, constipation; history of inflammatory bowel disease such as Crohn's disease, ulcerative colitis, microscopic colitis, chronic diarrhea; chronic constipation or use of laxatives; history of gastrointestinal malignancy or known colon polyposis; history of any abdominal surgery, e.g., gastric bypass, intestinal resection, appendectomy, cholecystectomy, and the like; use of probiotics or any other over the counter aids used by the potential donor for purpose of regulating digestion, but yogurt and kefir products may be allowed if taken merely as food rather than nutritional supplements.

[0042] Exclusion criteria regarding factors that can, or do affect, the composition of the intestinal microbiota include, but are not limited to, antibiotics for any indication within the preceding 6 months, and any prescribed immunosuppressive or anti-neoplastic medications.

[0043] Exclusion criteria regarding systemic medical conditions include, but are not limited to, established or emerging metabolic syndrome, where criteria used for definition here are stricter than established criteria, including history of increased blood pressure, history of diabetes or glucose intolerance; known systemic autoimmunity, e.g., connective tissue disease, multiple sclerosis; known atopic diseases including asthma or eczema, chronic pain syndromes including fibromyalgia, chronic fatigue syndrome; ongoing (even if intermittent) use of any prescribed medications, including inhalers or topical creams and ointments; neurologic, neurodevelopmental, and neurodegenerative disorders including autism, Parkinson's disease.

[0044] Exclusion criteria on physical examination may include, but are not limited to, general, such as body mass index < 30 kg / m2, central obesity defined by waist: hip ratio > 0. 90 (male) and > 0. 85 (female); blood pressure >135 mmHg systolic and > 85 mmHg diastolic; skin — presence of a rash, tattoos or body piercing placed within a year, jaundice; enlarged lymph nodes; wheezing on auscultation; hepatomegaly or stigmata of liver disease; swollen or tender joints; muscle weakness; abnormal neurologic examination.

[0045] Exclusion criteria on laboratory testing may include, but is not limited to, detection of Clostridium difficile toxin B, Escherichia coli O157:H7, Shiga toxins, Enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia, Campylobacter, Pleisomonas, and Vibrio, Giardia, Cryptosporidium, Cyclospora, and Cystoisospora (previously Isospora)', Rotavirus, Norovirus I and II, and adenovirus by PCR; detection of multi-drug resistant organisms, including, but not limited to, MRSA, ESBL, CRE, VRE using PCR or culture in sele tive media; positive screening for any circulating viral pathogens, including HIV 1 and 2, Hepatitis A, Hepatitis B, Hepatitis C, Cytomegalovirus, Covid, Epstein-Barr Virus; any abnormal liver function tests including alkaline phosphatase, aspartate aminotransaminase, alanine aminotransferase; raised serum triglycerides > 150 mg / dL; HDL cholesterol < 40 mg / dL (males) and < 50 mg / dL (females); high sensitivity CRP > 2. 4 mg / L; raised fasting plasma glucose (> 100 mg / dL).

[0046] The compositions of the present disclosure may be included in a diversity of pharmaceutically acceptable formulations. In one embodiment, a formulation may be a liquid composition. Liquid compositions include, but are not limited to, solutions, suspensions, dispersions, and the like. In one embodiment, a formulation may be a solid composition. Solid compositions include, but are not limited to, powder (e.g., a friable freeze-dried powder), granule, compressed tablet, pill, capsule, chewing gum, microsphere, wafer, and the like. Those formulations may include a pharmaceutically acceptable carrier to render the composition appropriate for administration to a subject. As used herein “pharmaceutically acceptable carrier” includes pharmacologically inactive compounds compatible with pharmaceutical administration. A pharmaceutically acceptable carrier can be, and typically is, United States Pharmacopeia (USP) grade. Examples of pharmaceutically acceptable carriers include flavor agents and gut-transit protectants. The compositions of the present disclosure may be formulated to be compatible with its intended route of administration. In one embodiment, a composition is formulated for oral administration as a liquid.

[0047] A composition of the present disclosure may be administered by any method suitable for depositing in the gastrointestinal tract, preferably the colon, of a subject. Examples of routes of administration include rectal administration (e.g., by suppository, enema, upper endoscopy, upper push enteroscopy, flexible sigmoidoscopy, or colonoscopy), intubation through the nose or the mouth (e.g., by nasogastric tube, nasoenteric tube, or nasal jejunal tube), or oral administration (e.g., by a solid such as a pill, tablet, or capsule, or by liquid). In embodiments where a liquid form of the composition is delivered to a subject, the freeze-dried composition can be reconstituted with an aqueous solution, such as by adding water, saline, milk, or plant-based milk, a flavor agent, and / or a gut-transit protectant, or by exposing the freeze-dried composition to a body fluid. In one embodiment, a composition is formulated for oral administration as a liquid that is swallowed by the subject. Such a composition is also referred to herein as an oral liquid preparation.

[0048] For therapeutic use in the method of the present disclosure, a composition described herein may be conveniently administered in a form containing one or more pharmaceutically acceptable carriers. Suitable carriers are well known in the art and vary with the desired form and mode of administration of the composition. For example, they may include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, glidants, lubricants, and the like. Typically, the carrier may be a solid (including powder), liquid, or combinations thereof. Each carrier is preferably "acceptable” in the sense of being compatible with the other ingredients in the composition and not injurious to the subject. The carrier is preferably biologically acceptable and inert, i.e., it permits the composition to maintain viability of the biological material until delivered to the appropriate site.

[0049] Compositions formulated for oral administration may include an inert diluent or an edible carrier. For purposes of oral therapeutic administration, in one embodiment a liquid composition, e.g., an oral liquid preparation, can include a flavor agent, a gut-transit protectant, or both a flavor agent and a gut-transit protectant. A flavor agent and / or a guttransit protectant can be present in a freeze-dried composition that is reconstituted to result in a liquid composition, or a flavor agent and / or a gut-transit protectant can be added to the composition at the time of reconstitution. Flavor agents are described herein. A gut-transit protectant is a compound or composition that protects fecal microbes during gut transit, e.g., transit through the high acidity and digestive enzymes present in the stomach and / or intestine. Examples of gut-transit protectants include, but are not limited to, a dairy product such yogurt, a milk or powdered milk. Examples of milk include regular or chocolate milk, and whole milk or milk having reduced fat, such as 1% or 2% milk fat. Other examples of milk include a plant-based milk such as, but not limited to. almond, oat, and soy. Examples of powdered milk, also referred to as milk powder, dried milk, and dry milk, include but are not limited to nonfat skimmed milk, whole milk, buttermilk, and whey. Gut-transit protectants can be added to a liquid composition to a final concentration of at least 1 % w / w, at least 5 % w / w, at least 10 % w / w, or at least 15 % w / w, and no greater than 20 % w / w, no greater than 15 % w / w, no greater than 10 % w / w, or no greater than 5 % w / w. Exemplary ranges include but are not limited to at least 1 % w / w to no greater than 20 % w / w, or at least 5 % w / w to no greater than 15 % w / w. In one embodiment, for the purpose of oral therapeutic administration a freeze-dried composition can be incorporated with excipients and used in the form of tablets, or capsules, e.g., gelatin or hypromellose capsules. Oral compositions can also be prepared by combining a composition of the present disclosure with a food. In one embodiment a food used for administration is chilled, for instance, ice cream or milk. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; or a sweetening agent such as sucrose or saccharin. Other ingredients may be added to a formulation to provide desired characteristics such as flow, compression, hardness, and taste.

[0050] In one embodiment, the freeze-dried composition may be present in a formulation that permits passage to the small intestine or colon. For instance, when the composition is to be administered orally using capsules, the dosage form may be formulated so the composition is not exposed to conditions prevalent in the gastrointestinal tract before the small intestine or colon, e.g., high acidity and digestive enzymes present in the stomach and / or intestine. In one embodiment, the dosage form may be formulated so the composition passes through the stomach and is released in conditions that include a pH of greater than 5. 5, greater than 6, greater than 6. 5, or greater than 7. In one embodiment an enteric coating is acid-resistant to protect the composition from the low pH of the stomach and break down when exposed to a pH greater than present in the stomach. The encapsulation of compositions in an enteric coating for therapeutic use is routine in the art. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. Examples include, but are not limited to, methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, enteric coatings (hydroxypropyl methylcellulose (HPMC), hydroxy propyl methyl cellulose acetate succinatelate (PVAP), methyl methacrylate-methacrylic acid copolymers, cellulose acetate trimellitate, and sodium alginate. Encapsulation may include hard-shelled capsules, which may be used for dry, powdered ingredients, or soft-shelled capsules. Capsules may be made from aqueous solutions of gelling agents such as animal protein (e.g., gelatin), plant polysaccharides or derivatives like carrageenans and modified forms of starch and cellulose. Other ingredients may be added to a gelling agent solution such as plasticizers (e.g., glycerin and or sorbitol), coloring agents, preservatives, disintegrants, lubricants, and surface treatment. Enteric coated capsules can be co-combined to provide for release of the freeze-dried composition within the large bowel or colon.

[0051] Useful diluents include aqueous solutions that are routinely used for manipulating microbes, eukaryotic cells, and / or viruses. Useful diluents may include constituents to maintain physiological buffer, osmolarity, and the like. The diluent is preferably sterile and / or non-allergenic. An example of a diluent includes, but is not limited to, saline or phosphate buffered saline at pH 7. In one embodiment, 1 part donor feces may be combined with 5 parts diluent (e.g., 50 grams of donor feces may be combined with 250 ml diluent) and blended. In one embodiment, the oxygen in the blending chamber may be decreased or removed by purging with an inert gas such as nitrogen or argon prior to blending. Such anaerobic conditions may be useful to maintain viability of most anaerobic bacteria present in a colon. The sample may be blended multiple times and / or more diluent may be added until a consistency is achieved that will permit the following steps to occur. In one embodiment, anaerobic conditions are not used in steps following the blending. It was found that anaerobic conditions were not necessary in the steps following the blending, and this was unexpected and surprising since a substantial percentage of prokaryotic cells in fecal material are strict anaerobes, and exposure to oxygen kills them. After the blending, the solutions used for washing and resuspension did not need to be purged of oxygen, and manipulation of the microbiota in an oxygen free cabinet or glove box was not needed.

[0052] In one embodiment, a blended sample may be prepared by obtaining a fecal sample from an appropriate donor and blending with a diluent as described in Sadowsky et al. (WO 2012 / 122478, WO 2014 / 152484). In another embodiment, a composition may be prepared by obtaining a fecal sample from an appropriate donor and using ballistic disruption with a horizontal or vertical shaker, a suitable diluent and stainless steel beads of 3. 2 mm in diameter. The mixture is shaken to break up the sample and the beads are removed from the suspension by filtration a stainless steel strainer. The suspension is centrifuged at a suitable speed to pellet the microbes, for instance, 500-1, 000 rpm, the supernatant poured off and the resulting microbial fraction obtained by selective filtration.

[0053] Not all microbes and eukaryotic cells present in an individual's colon can be cultured, thus, in one embodiment conditions for preparing a composition of the present disclosure include the use of temperatures that decrease the replication of the microbes and eukaryotic cells. In one embodiment, the conditions used for preparation are maintained below 37 °C. For instance, the conditions used for preparation are maintained at a temperature of no greater than 30 °C., no greater than 20 °C., no greater than 10 °C., or no greater than 5 °C. In one embodiment, conditions are used such that replication of the microbes and eukaryotic cells is undetectable, and preferably does not occur. When the conditions used to prepare a composition of the present disclosure include lower temperatures to minimize replication and cell death, the biological material present in a composition includes a population of microbes, eukaryotic cells, and viruses that is essentially identical to a population of microbes, eukaryotic cells, and viruses present in the colon or feces of a normal healthy human, e.g., the donor from whom the fecal sample was obtained. In one embodiment, the conditions used for preparation decrease exposure of the microbes and eukaryotic cells to oxygen, both before and after purification of microbiota.

[0054] As described in Sadowsky et al. (WO 2012 / 122478, WO 2014 / 152484) removal of nonliving material may be achieved by selective filtration, e.g., passing the blended sample through a series of sieves with a sieve size of no greater than 2. 0 mm, no greater than 1. 0 mm, no greater than 0. 5 mm, no greater than 0. 25 mm, no greater than 0. 212 mm, no greater than 0. 180 mm, no greater than 0. 150 mm, no greater than 0. 125 mm, no greater than 0. 106 mm, no greater than 0. 090 mm, no greater than 0. 075 mm, no greater than 0. 063 mm, no greater than 0. 053 mm, no greater than 0. 045 mm, no greater than 0. 038 mm, no greater than 0. 032 mm, no greater than 0. 025 mm, no greater than 0. 020 mm, no greater than 0. 01 mm, or no greater than 0. 2 mm. In one embodiment, the blended sample is prepared by passing it through a sieve with a sieve size of 0. 25 mm and collecting the filtrate. In one embodiment, the blended sample is passed through sieves with progressively smaller sieve sizes until final passage through a sieve size of 0. 25 mm. For instance, if a total of four sieves are used the sieve size of the first sieve may be 2 mm, followed by 1 mm, followed by 0. 5 mm, and then followed by 0. 25 mm. The final filtrate may be collected in a centrifuge tube, and centrifuged at a speed sufficient to pellet the biological material, for instance, 5, OOOxg for 10 minutes at 4 °C. The supernatant is removed, the cells are resuspended in diluent, optionally centrifuged again, for instance at 5,OOOxg for 10 minutes at 4 °C. The final supernatant is discarded, and the cells are resuspended in an aqueous solution (e.g., diluent). In one embodiment, the volume of the blended mixture is decreased through the steps of sieving and washing. For instance, in one embodiment, the volume is decreased to 14 % of the volume used in the blending (e.g., from 250 mis to 35 mis). In one embodiment, the volume of the blended mixture is decreased through the steps of sieving and washing to result in between IxlO10 and 5xl010 cells in a volume that is subsequently administered to a subject. The final filtrate may also be collected in a centrifuge tube, washed, and the cells resuspended in an aqueous solution (e.g., diluent, cryoprotectant, and the like, or a combination thereof). In one embodiment, the volume of the blended mixture is decreased through the steps of sieving and washing to result in at least IxlO10 cells in a volume that is subsequently freeze-dried. Since most biological material is difficult or impossible to culture, a hemocytometer may be used to determine the number of cells. This process results in an extract of feces that is highly enriched for all colon microbiota that are able to pass through a sieve as described above, and can be centrifuged at 10,000xg for 10 minutes. As used herein, “enriched” refers to increasing the abundance of biological material relative to non-living material, such that biological material constitutes a significantly higher proportion compared to the fecal material before the enrichment. The term “enriched” refers to those situations in which a person has intervened to elevate the proportion of biological material.

[0055] The amount of aqueous solution added may be in an amount to result in a single dosage having an appropriate number of cells. In one embodiment, a single dosage may include from IxlO12 cells to 8xl012 cells, for instance, 5xl012 cells. Since most biological material is difficult or impossible to culture, a hemocytometer may be used to determine the number of cells.

[0056] In one embodiment the resulting pellet may be suspended in half the original volume of diluent containing 10 % glycerol. The sample may be used immediately, or may be frozen, for instance, at -80 °C, for later use. When freezing, the sample may be left in a centrifuge tube, or may be in a different container. In one embodiment, the container is one that increases the surface area of the sample. For instance, the sample may be placed in a cryo bag. When the frozen sample is to be used, it may be thawed on ice and then transplanted into the recipient. It was found that freezing the compositions described herein did not result in destruction of its curative potential. In one embodiment the sample resulting from centrifugation may be processed for long term storage of 1 year or longer. The ability to store such a sample provides a level of flexibility that was not possible with other methods. For instance, it was necessary to quickly identify a donor, rapidly process a fecal sample from the donor, and use it immediately. Examples of useful processing methods include, but are not limited to, freezing, and freeze drying or lyophilization. Processing of a composition of the present disclosure may include the production of a powder following any drying procedure.

[0057] The use of sieves to extract biological material from fecal material as described in Sadowsky et al. (WO 2012 / 122478, WO 2014 / 152484) resulted in compositions that were nearly free of fiberous material; however, the liquid forms of the compositions (e.g., a freeze-dried composition reconstituted in an aqueous solution) had the organoleptically unacceptable property of foul taste and odor. Moreover, while some of the odor of the liquid forms of the compositions (e.g., a freeze-dried composition reconstituted in an aqueous solution) was substantially reduced relative to feces the residual odor was at a level that also made liquid forms of the compositions unusable for oral administration using an oral liquid preparation.

[0058] In considering how to achieve a liquid composition derived from fecal material that had no objectionable odor or and no objectionable taste that could be used for patients with, e.g., pill dysphagia, the inventors expected the removal of compounds responsible for the undesirable organoleptic properties to be difficult. For instance, skatole, indoles, and shortchain fatty acids such as butyrate would need to be reduced significantly in view of the great sensitivity of the human nose for these fecal odorants. Moreover, the viability of many fecal microbes is reduced by manipulation of fecal material. Initial proposed approaches included, for instance, the use of column chromatography to adsorb the biological material in fecal material. Surprisingly, adding more washing steps resulted in a liquid composition that had no discernable four odor and a taste that was also undiscernible or could be masked by use of a flavor agent. In addition, the added steps did not result in a significant decrease in viability. Accordingly, making a composition disclosed herein can include at least four separation / washing steps. Typically, the final filtrate obtained after selective filtration, e.g., passing the blended sample through a series of sieves, is subjected to a force to cause separation of material from liquid and, after removal of the liquid, the resulting material is resuspended in diluent. This process of separation, removal of supernatant, and resuspension is repeated at least 3 additional times for a total of four separations and washes. The force to cause separation of material from liquid can be centrifugal force from centrifugation. The speed of a centrifugation step can be at any speed that causes the separation of material from liquid but does not cause loss of therapeutic efficacy of the composition in FMT or MTT. Care should be taken as damage of the fecal microbes can be increased at greater speeds, and the fecal microbes can be more difficult to resuspend. Examples of suitable conditions include speeds resulting in at least 3,000 xg, at least 4,000 xg, or at least 5,000 xg. Other examples of suitable conditions include speeds resulting in no greater than 9,000 xg, no greater than 8,000 xg, or no greater than 7,000 xg. Suitable ranges include at least 3,000 xg to no greater than 9,000 xg, at least 4,000 xg to no greater than 8,000 xg, and at least 5,000 xg to no greater than 7,000 xg. Typically, the volume of diluent added back to the material after removal of the supernatant is reduced to cause concentration of the material. For instance, the diluent added back to the material can be one half the volume present before the separation step. In one embodiment, the material is concentrated to result in at least IxlO12 cells, for instance, at least 2.5xl012 cells, at least 3xl012 cells, at least 3.5xl012 cells, at least 4xl012 cells, at least 4.5xl012 cells, at least 5xl012 cells, or at least 5.5xl012 cells, at least 6xl012 cells, at least 6.5xl012 cells, at least 7xl012 cells, at least 7.5xl012 cells, or at least 8xl012 cells in a volume that is subsequently freeze-dried. In one embodiment, the material is concentrated to result in no greater than 9xl012 cells in a volume that is subsequently freeze-dried.

[0059] The ability to produce a composition from feces that has no objectionable odor and no objectionable taste is a significant advantage as it permits the production of liquid compositions for oral administration. This in turn results in the advantage of now being able to easily administer fecal microbiota transplantation (FMT) or microbiota transplant therapies (MTT) to target populations of subjects that were incapable of ingesting a composition for FMT or MTT in tablet form or via colonoscopy. As used herein, an "objectional odor" means a liquid composition has an odor that is so unacceptable and offensive to a subject that the subject will refuse to drink the composition. As used herein, an "objectional taste" means a liquid composition has a tase that is so unacceptable and offensive to a subject that the subject will refuse to drink the composition. Whether a liquid composition described herein has an objectionable odor or objectionable taste can be determined by comparing the odor and taste of a liquid composition described herein with a placebo composition, e.g., a composition having the same components as a liquid composition described herein except the fecal microbes. In some embodiments, a liquid composition described herein is indistinguishable from a composition having the same components as a liquid composition described herein except the fecal microbes.

[0060] In one embodiment, a composition described herein of fecal microbes is freeze-dried to form solid dried powder. A composition of fecal microbes is mixed with a cryoprotectant and / or lyoprotectant and subjected to conditions that result in freeze-drying. In those embodiments where the freeze-dried composition will be used to make a liquid solution for oral consumption, a composition of fecal microbes can be mixed with a flavor agent, and in other embodiments a flavor agent is added when the freeze-dried composition is reconstituted. Conditions that result in freeze-drying typically include freezing the sample, and reducing the pressure surrounding the frozen sample to remove water from the sample. Once freeze-dried, the composition may be further processed by subjecting the dried material to force sufficient to break up the material into a powder that can be easily stored until used. In one embodiment, the powder may be used to form granules, compressed tablets, pills, capsules, wafers, and the like. In one embodiment, the freeze-dried material can be formulated such that it is released from a capsule into the small or large intestine or the colon, and not the stomach.

[0061] In one embodiment, a freeze-dried composition described herein is reconstituted to result in a liquid suspension. A freeze-dried composition is mixed with an aqueous diluent. The freeze-dried composition can include a flavor agent and / or a gut-transit protectant, or a flavor agent and / or a gut-transit protectant can be added at the time of reconstitution. In one embodiment, the aqueous diluent includes a dairy product such yogurt or a milk product. Examples of milk include whole milk or milk having reduced fat, such as 1% or 2% milk fat, and the milk can be regular or chocolate milk, or vegetable-based milks. Typically, the reconstitution occurs using conditions that maintain the efficacy of the composition, e.g., conditions that reduce inactivating the microbes present. Appropriate conditions can include gentle mixing to dissolve or suspend the freeze-dried composition in the aqueous diluent.

[0062] The present disclosure is further directed to methods of using the freeze-dried compositions described herein. One method includes administering to a subject in need thereof an effective amount of a composition described herein. The subject can be anyone in need thereof. In some embodiments, the subject can be an individual with reduced ability to swallow, such as pill dysphagia, or an individual refractory to administration of a liquid having an objectionable odor or objectionable taste. Examples of refractory individuals include those developmentally disabled. An individual can be a pediatric or an adult. The administering is done under conditions suitable for deposition of the composition in a region of the large or small intestine such that the biological material in the composition colonizes the small intestine and colon. For instance, administration may be into upper gastrointestinal tract, as well as lower gastrointestinal tract, e.g., the terminal ileum, cecum, colonic areas containing diverticulosis, and rectum. In one embodiment the administering may be oral, such as by tablet. In one embodiment the administering may be by intubation, such as by nasogastric tube, of a freeze-dried composition that has been reconstituted. In one embodiment the administering may be rectal, for instance by a colonoscope, enema, or suppository. Conditions that are "suitable” for an event to occur, or " suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and / or are conducive to the event. As used herein, an “effective amount” relates to a sufficient amount of a composition described herein, to provide the desired effect. For instance, in one embodiment an “effective amount” is an amount effective to alleviate one or more symptoms and / or signs of a disease or a condition as described herein. In some embodiments, an effective amount is an amount that is sufficient to effect a reduction in a symptom and / or sign associated with a disease or condition, such as diarrhea or C. difficile. A reduction in a symptom and / or a sign is, for instance, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, or at least 100 % in a measured sign as compared to a control, a non-treated subject, or the subject prior to administration of the composition. In one embodiment, an effective amount is an amount sufficient to result in at least IxlO12, at least 1.5xl012, at least 2xl012, or at least 2.5xl012 cells administered to the subject. In one embodiment, an effective amount is an amount sufficient to result in at least IxlO12, at least 1.5xl012, at least 2xl012, at least 3xl012, at least 4xl012, or at least 5xl012 cells delivered to the colon. In one embodiment, an effective amount is an amount sufficient to result in IxlO12 to 5.0xl012 cells delivered to the colon, or 1.5xl012 to 3xl012 cells delivered to the colon. In some embodiments, an initial high dosage followed by one or more lower maintenance doses. An initial high dosage can include an amount sufficient to result in at least IxlO12, at least 1.5xl012, at least 2xl012, or at least 2.5xl012 cells administered to the subject, or an amount sufficient to result in at least 1x1012, at least 1.5x1012, at least 2x1012, at least 3xl012, at least 4x1012, or at least 5x1012 cells delivered to the colon. It will be understood, however, that the total dosage of the compositions as disclosed herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type and extent of disease or condition being treated.

[0063] In one embodiment, a method of the present disclosure includes treating certain diseases in a subject in need of treatment. The subject may be a mammal, such as a human. In some embodiments animal models may be used, such as a mammal, including a rat, a mouse, a hamster, a gerbil, or a primate. As used herein, the term "disease” refers to any deviation from or interruption of the normal structure or function of a part, organ, or system, or combination thereof, of a subject that is manifested by a characteristic symptom or clinical sign. Diseases include those characterized by dysfunctional composition of intestinal microbiota. Such diseases include, but are not limited to, colitis, including autoimmune colitis (e.g., inflammatory bowel disease, ulcerative colitis, Crohn's disease, microscopic colitis), irritable bowel syndrome, and infectious colitis. Examples of infectious colitis include, but are not limited to Clostridioides difficile colitis (e.g., acute C. difficile colitis, relapsing C. difficile colitis, or severe / fulminant C. difficile colitis) and enterohemorrhagic colitis (e.g., a colitis caused by Shigella spp. or E. coll). Other examples of diseases include, but are not limited to, chronic diarrhea; chronic constipation; metabolic syndrome and obesity; atopic diseases including asthma, eczema, food allergies, eosinophilic disorders of the GI tract; systemic autoimmunity including rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis, etc.; chronic pain disorders such fibromyalgia, chronic fatigue syndrome, neurodegenerative disorders, eating disorders, and malnutrition. As used herein, the term "condition” refers to an illness or other medical problem. Examples of conditions include but are not limited to autism spectrum disorder, Pitt-Hopkins Syndrome, Parkinson's disease, alcohol use disorder, obesity, Type II diabetes, and presence of multi-drug resistant organisms. The term "condition” also refers cancers that can be treated using immune-oncology, such as optimization of anti-cancer checkpoint immunotherapy, treatment of checkpoint inhibitor colitis, and treatment or prevention of complications of hematopoietic stem cell transplantation, such as graft-versus-host disease, or prevention of complications due to abdominal surgeries.

[0064] As used herein, the term “symptom” refers to subjective evidence of disease or condition experienced by the patient and caused by disease. As used herein, the term “clinical sign, " or simply “sign,” refers to objective evidence of a disease present in a subject. Symptoms and / or signs associated with diseases or conditions referred to herein and the evaluation of such signs are routine and known in the art. Typically, whether a subject has a disease or condition, and whether a subject is responding to treatment, may be determined by evaluation of signs associated with the disease or condition.

[0065] Treatment of a disease or condition can be prophylactic or, alternatively, can be initiated after the development of a disease or condition. Treatment that is prophylactic, for instance, initiated before a subject manifests signs of a disease or condition, is referred to herein as treatment of a subject that is at risk” of developing a disease or condition. An example of a subject that is at risk of developing a disease or condition is a person having a risk factor. An example of a risk factor for C. difficile colitis is antibiotic therapy of the gastrointestinal tract. Treatment can be performed before, during, or after the occurrence of the diseases described herein. Treatment initiated after the development of a disease may result in decreasing the severity of the signs of the disease, or completely removing the signs.

[0066] In one embodiment, a method of the present disclosure includes transplanting a microbiota from a donor to a recipient.

[0067] In one embodiment, a method of the present disclosure includes increasing the relative abundance of members of the phylum Firmicutes, such as a non-pathogenic member of the class Clostridia, and / or members of the phylum Bacteroidetes, in a recipient's colon. In one embodiment, a method of the present disclosure includes increasing the relative abundance of members of the family Prevotellaceae, such as members of the genus Prevotella. The phrase "relative abundance" refers to the number of members of a phylum or class compared to the number of members of all other taxa in a recipient's colon. Such a comparison can be expressed as a percent. In one embodiment, the relative abundance of non-pathogenic members of the class Clostridia in a recipient's colon after the administration may be increased by at least 5 %, at least 10 %, at least 20 %, or at least 50 %, compared to the recipient's colon before the administration. In one embodiment, the relative abundance of members of the phylum Firmicutes in a recipient's colon after the administration may be increased by at least 5 %, at least 10 %, at least 20 %, or at least 50 % compared to the recipient's colon before the administration. The change in the abundance may be determined at, for instance, 3 days, 10 days, 15 days, or 25 days after the administration of fecal microbiota.

[0068] In one embodiment, a method of the present disclosure includes decreasing the relative abundance of members of the phylum Proteobacteria in a recipient's colon. In one embodiment, the relative abundance of members of the phylum Proteobacteria in a recipient's colon after the administration may be decreased by at least 10 %, at least 20 %, at least 30 %, or at least 40 % compared to the recipient's colon before the administration. The change in the abundance of members of the phylum Proteobacteria may be determined at, for instance, 3 days, 10 days, 15 days, or 25 days after the administration.

[0069] In one embodiment, the existing microbiota does not need to be cleared prior to administration of a freeze-dried composition of the present disclosure. In other embodiments clearance of the microbiota may be necessary. Methods for clearance of existing microbiota are known and routine. In one example, clearance can be accomplished by administering a cocktail of antibiotics for one week until a day prior to transplant. An example of a useful cocktail is Metronidazole (1000 mg twice daily), Rifaximin (550 mg twice daily), Vancomycin (500 mg twice daily), and Neomycin (1000 mg twice daily).

[0070] EXAMPLES

[0071] The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.

[0072] Example 1

[0073] Successful treatment of recurrent Clostridioides difficile infection using a novel, drinkable oral formulation of fecal microbiota

[0074] Materials and Methods

[0075] Production of purified fecal microbiota. The product was manufactured by the University of Minnesota Microbiota Therapeutics Program in accordance with the initial procedures and protocols, including donor testing, as previously described [3,4] and detailed in the Investigational New Drug Application (IND) 15071 approved by the US Food and Drug Administration (FDA). The volunteer stool donor program was overseen by the University of Minnesota Institutional Review Board. Stool donations were obtained in a supervised bathroom in the research clinic from volunteer “standardized” donors and transported on ice into the manufacturing facility (Molecular and Cellular Therapeutics Facility, St. Paul campus of the University of Minnesota), where it was processed within two hours of collection.

[0076] The fecal material was weighed and homogenized (typically 1 part donor feces is combined with 5 parts sterile 0.85% NaCl diluent) in a commercial blender (under N2 gas) in a dedicated biological cabinet. The slurry was passed through 2.0 mm, 1.0 mm, 0.5 mm, and 0.25 mm stainless steel laboratory sieves (W. S.Tyler, Inc., Mentor, OH) to remove undigested food, fiber, and small particulate material. The resulting material passing through the 0.25 mm sieve was centrifuged at 6,000 x g for 15 min in a Sorvall SS-34 rotor and resuspended to one half the original volume in sterile 0.85% NaCl saline. This procedure was repeated 3 times for a total of 4 centrifugations and washes.

[0077] Preparation of freeze-dried, flowable, potable, powdered microbiota. The final washed cell suspension was freeze-dried using 10% trehalose as the lyoprotectant [4], The lyophilizer (LyoStar II, Stone Ridge, NY or Genesis 35EL, SP, Warminster, PA) used a shelf temperature of -20 °C for 36 hr followed by 6 h at +30 °C. Following lyophilization, the freeze-dried material was mixed to break up clumps and the resulting power was hand mixed with USP grade chocolate flavoring powder (Sensient Technologies, St. Louis) to a final concentration of 1.0% (w / w). To facilitate microbial cell protection during gut transit, Nestle KLIM Fortificada Dry Whole Milk Powder (Nestle USA, Arlington, VA) was added as an admix to the flavored microbiota preparation to a final concentration of 10% (w / w). Cell numbers in the final product were determined by using the Bacteria Counting Kit (ThermoFisher, Waltham, MA), a Zeiss epi fluorescence microscope, equipped with a 470490 nm excitation filter and 500 nm cut-on long pass emission filter, and a Petroff Hauser counting chamber. A membrane integrity assay was used to directly enumerate microbes present in a diluted sample and to calculate the percentage of microbes with intact membranes. This was done using a BacLight live / dead membrane integrity assay (ThermoFisher, Waltham, MA) and the microscope and counting chamber.

[0078] Storage and preparation for patient use of the flavored microbiota. The mixed final product was manually added to a mylar pouch (1.3" Wx3.3"L, Impak Corp, Los Angeles, CA), to obtain a final cell number of l.Ox to 5.0xl0n cells / sachet, and heat sealed using an impulse sealer. Sealed pouches were placed in a sterile 100 ml FreeStyleTm Wide-Mouth clear plastic bottle (Drug Plastics, Boyertown, PA), containing a 2 % x 1” silica gel pack and a 3.17 cm, grade SAE 304 stainless steel, shaker mixing whisk ball (Yofan, Amazon, Seattle, WA). The bottles were closed with a lined cap, and induction sealed. The final product was stored at -80 °C.

[0079] The suspension of microbiota for administration to a patient was prepared by the clinical provider. The bottles containing the microbiota were transferred from the -80 °C freezer to 4 °C refrigerator overnight and transported to the clinic the next day at room temperature. The silica gel pack and the sachet were removed from the bottle, while the mixing ball stayed within the bottle (due to size limitations it could not easily be removed). The bottle was filled with ~ 50 mL of chocolate milk (commercial grade, ~ 2% milk fat), the sachet was tom-open, and the powdered contents were poured into the bottle. The bottle cap was replaced, and the bottle was gently mixed, 10-times by inversion, to dissolve the freeze-dried microbiota powder mixture. This preparation was taken orally by the patient after transfer to a drinking glass.

[0080] Engraftment of human fecal bacteria into antibiotic-treated mice. The viability of lyophilized human fecal bacteria used in an encapsulated preparation by engraftment into germ-free mice was previously confirmed [4], Here similar testing was performed with the highly purified and flavored microbiota to ensure that viability was not lost due to the additional washing steps and addition of a flavoring agent. This was done using the model of antibiotic-treated mice previously shown to be equivalent to germ-free mice in the ability to achieve short-term engraftment of human microbiota [8], Briefly, female 8-week-old C57BL / 6 mice (The Jackson Laboratory, Bar Harbor, ME) were administered alternating mixtures of antibiotics in their drinking water at concentrations of 1 mg / mL for a period of 3 weeks. During weeks one and three they received ertapenem sodium (Merck and Co., Inc., Whitehouse Station, NJ), neomycin sulfate (Fagron, Inc., St. Paul, MN), and vancomycin hydrochloride (Mylan Institutional LLC, Rockford, IL), and during week two they received ampicillin (WG Critical Care, LLC, Paramus, NJ, USA), cefoperazone sodium salt (Sigma-Aldrich Co., St. Louis, MO), and clindamycin hydrochloride (Fagron, Inc.). The fecal microbiota separated from stool, done using the originally described procedure [4] or the enhanced purification procedure described here, was administered to the mice via oral gavage, 1010 bacteria suspended in 250 pL of normal saline per dose, 24 hr after completing antibiotics. Fecal pellets were collected from individual mice 7 days after the oral gavage.

[0081] Treatment of recurrent Clostridioides difficile infections (rCDI) patients using powdered fecal microbiota. Three adult rCDI patients who were unable to swallow capsules were offered the powdered fecal microbiota as an alternative to colonoscopy. The patients consented to this option after being informed about the novelty of the product as well as all the infectious and non-infectious disease potential risks associated with FMT that are mitigated, but not eliminated, by donor screening and testing. The treatment was done under the FDA’s enforcement discretion policy for use of FMT in the treatment of CDI that is not responsive to standard therapies and did not constitute a formal clinical trial. The clinical outcome data was captured into a prospective registry approved by the University of Minnesota Institutional Review Board (IRB) [5], Patient 1 provided stool samples for microbiome analysis under a separately approved University of Minnesota IRB protocol.

[0082] The patients were administered powdered fecal microbiota suspended in ~ 2% chocolate milk in the clinic and under direct observation of the treating physician. Antibiotics used to treat rCDI were discontinued ~ 2 days prior to the clinic visit. No purgative preparation was used to wash-out residual antibiotics. Production of stomach acid was suppressed using omeprazole or famotidine prior to the clinic visit. The patients were instructed to have no solid food on the morning of the treatment and to remain on clear liquids for two hours afterwards.

[0083] DNA sequence analyses and bioinformatics. DNA extraction from the mouse and human fecal samples was done as previously described [8], Amplification of the V4 hypervariable region of the 16S rRNA gene was done using the 515F / 806R primer set [9] by the University of Minnesota Genomics Center, with dual indexing

[10] , Paired-end sequencing was performed on the Illumina MiSeq platform (Illumina, Inc., San Diego, CA) at a read length of 301 nucleotides (nt). Raw data are deposited in the sequence read archive under accession number PRJNA1000588.

[0084] Sequence processing was done using mothur ver. 1.41.1

[11] and our previously published sequence analysis pipeline

[12] , Briefly, samples were trimmed to the first 170 nt, paired-end joined, and aligned against the SILVA database (ver. 1381)

[13] , Chimeras were identified and removed using UCHIME

[14] , Operational taxonomic units (OTUs) were classified at 99% similarity and annotated using the Ribosomal Database Project (release 18)

[15] , For statistical comparisons, samples from the first mouse experiment were rarefied to 10,000 reads per sample while the second mouse and human datasets were rarefied to 5,000 reads. Alpha and beta diversity were also evaluated in mothur. Bray-Curtis dissimilarities were used to describe beta diversity and were visualized by ordination via principal coordinate analysis (PCoA).

[0085] SourceTracker2

[16] was used to measure differences in community composition from baseline. This program takes OTU tables as inputs and determines the proportion of a community in user-defined sink samples that are attributable to user-defined sources (i.e. baseline sample) using an iterative Bayesian process. Default parameters were used and the script was implemented in a Python 3.6 working environment.

[0086] Results

[0087] Development of baseline protocol and organoleptic analysis of the purified, powdered fecal microbiota. The fecal microbiota contained in our original, encapsulated formulation [4] retains some fecal odor that makes it non-palatable to administer as a liquid suspension. To overcome this problem, we tested whether an organoleptically acceptable product could be made using additional centrifugation washes and the addition of a flavoring agent. Initial studies were done to determine the minimal number of centrifugation steps needed to produce an organoleptically acceptable drinkable product without loss of viable cells. The potential viability was determined by live dead staining. We also tested whether it was influenced by the addition of flavoring agent, prior to or after lyophilization (Table 1). Our experiments showed that the additional processing steps needed to make an odor-free and organoleptically acceptable fecal microbiota preparation did not decrease the percentage of viable bacterial cells, as measured by the membrane integrity assay, when compared with the more limited processing in the original preparation. In addition, investigators in the study conducted two blinded tastings of the new preparation from two different donors suspended in chocolate milk and did not detect any organoleptically objectional taste, smell, texture, flavor, or odors, relative to chocolate milk alone. The final protocol developed included a total of four centrifugation steps, the addition of the flavoring agent, and the whole milk powder to aid in stomach passage. Number of washes during processing Addition of 1% flavoring Total bacterial cells / mL % Live bacterial cells Experiment 1 (Donor 099) 2 None 4.950 x 1011 74.1 4 None 4.725x 10” 68.8 4 Before lyophilization 4.050 x 10” 50.3 4 After lyophilization 4.525 x 10” 67.2 Experiment 2 (Donor 070) 2 None 3.925 x 10” 69.7 4 None 3.550 x 10” 73.4 4 After lyophilization 3.375 x 10” 75.6 Table 1. Two independent experiments were done with preparations from two different donors. Two washes following fdtration are part of the older protocol used to manufacture encapsulated fecal microbiota. The new protocol requires four washes. Chocolate flavoring was added either before or after the lyophilization step. The cells were counted using microscopy and live / dead membrane integrity assay after lyophilization of microbiota.

[0088] Engraftment of human fecal microbiota preparations into antibiotic-treated mice. To ensure engraftability, we also conducted two experiments with preparations from two different human donors, in antibiotic-treated mice using the human microbiota association protocol reported previously (Table 2) [8], Analysis of engraftment (donor microbiota similarity) was done using SourceTracker, where the source mouse community was taken at the preantibiotic time point. Taken together, these results indicated that the procedure used to produce orally-consumed microbiota to treat dysphagic patients had no loss of viability and engraftability relative to the original encapsulated version. w -U Day Preparation 0 None 7 Saline 7 Control 7 New 7 New 7 New Flavoring added None None None None Prior to lyophilization After lyophilization Experiment 1. % control engraftment ± SEM 1.053 ±0.53 0.15 ±0.08 100.0 ±6.73 164.3 ± 8.7 98.5 ±24.4 148.8 ± 13.3 Experiment 2. % control engraftment ± SEM 6.25 ±0.16 ND 100.0 ±4.92 44.5 ±9.2 ND 146.8 ± 38.9 Table 2. Engraftment of human donor microbiota into antibiotic treated mice. Two independent experiments were done with different human donor preparations. The engraftment human bacteria in the new formulation (4 washes) with or without flavoring was measured using SourceTracker and normalized for the engraftment achieved with the old formulation (2 washes). WO 2025 / 144772                                   PCT / US2024 / 061651

[0089] Patient Histories and Treatment Outcomes. Four patients who were unable to swallow capsules were treated with the new, drinkable fecal microbiota formulation. They did not have a diagnostic indication for a colonoscopy and multiple medical comorbidities presented an increased risk that could be associated with the endoscopic procedure. The basic demographics, essential elements of clinical history, and treatment details are summarized in Table 3. Patient 4 had previously failed two treatments with Rebyota, a commercial FMT-based product. All patients swallowed the liquid preparation without any difficulty or noted organoleptically unacceptable properties. Following treatment, none of the patients had relapse of diarrhea at 1 week and 1, 2, 6, and 12 months of follow-up. Patient 1 developed transient constipation after the first week, which subsequently resolved spontaneously. No serious adverse events associated with the treatment were reported. One patient, an 8-year-old boy with was treated with MTT using the drinkable fecal microbiota formulation under a compassionate case single patient Investigational New Drug application approved by the FDA. In this protocol the patient swallows a high (loading) dose of donor microbiota on days 1-4 and continued with a maintenance daily dose for 15 weeks. The preparation was well tolerated without any organoleptic barriers or concerns. The loading dose of fecal microbiota is the same as the one used for prevention of C. difficile infection recurrence, which is > 5 x 1011 bacterial cells. The same drinkable fecal microbiota formulation is currently being tested in a placebo-controlled clinical trial for Pitt-Hopkins Syndrome, which is a genetic condition associated with a severe form of autism. The MTT treatment used for Pitt-Hopkins Syndrome is nearly identical as for the autism patient above - 4 days of the loading daily dose of fecal microbiota followed by 12 weeks of the daily maintenance dose. This trial is a follow-up on a previous clinical trial, which showed significant benefit for gastrointestinal symptoms, such as abdominal pain, in these patients. This earlier trial used an encapsulated preparation of fecal microbiota. Unfortunately, the majority of otherwise eligible patients could not swallow the capsules. So far, 9 patients have received the treatment. Since the trial is 1:1 placebo-controlled, we know that at least 4 patients received the active (donor fecal microbiota) treatment. All patients have been able to swallow the drinkable formulation, and none have been able to detect any organoleptically objectional taste, smell, texture, flavor, or odors. Patient 1 Patient 2 Patient 3 Patient 4 Age Sex Swallowing Problem Duration of rCDI prior to FMT Number of CDI episodes Prior rCDI antibiotic treatments 79 Male Dementia; chewing all medications 10 months >4 Three pulse / taper regimens of vancomycin 91 Female Pill dysphagia 15 months >4 At least two pulse / taper regimens of vancomycin 85 Female Vagal nerve injury with dysphagia to solids and gastroparesis 8 months >4 Three pulse / taper regimens of vancomycin 89 Female Pill dysphagia 2 years >4 Multiple courses of vancomycin, and tw o administrations of Rebvota Table 3. Demographics and clinical metadata of rCDI patients. Charlson Comorbidity Index Prior abdominal surgical history History of hospitalization for CDI Gastric acid suppression 7 None No Prilosec (liquid) x 5 days 7 • Hysterectomy • Appendectom y • Bladder repair No Famotidine 10 • Cholecystecto my • Gastric fundoplicatio n • Hiatal hernia repair • Hysterectomy No Famotidine 6 None Yes Mylanta WO 2025 / 144772                                   PCT / US2024 / 061651

[0090] Changes in the microbial community structure. Stool samples at 1-,3-, and 4-weeks following administration of the drinkable microbiota suspension from patient 1, and the corresponding donor sample, were analyzed by 16S rRNA gene profiling. Results of sequence analysis (FIG. 1) demonstrated that the gut of this patient was rapidly colonized by donor microbiota. The recipient’s gut bacteria shared greatest similarity to the donor bacteria at 1 week, and then slightly declined (FIG. 1A, B). Microbial diversity, as measured by the Shannon index (FIG. IC), rapidly increased via treatment. However, while improved microbial diversity was maintained throughout the study period, the similarity to donor decreased. Similarly, the PCoA plot (FIG. IC) showed that while the microbiota in the patient following treatment was initially more similar to that of the donor, it diverged over time.

[0091] Discussion

[0092] Although several standardized commercial and investigational formulations of FMT-based products are now available, there remains a need for patients who can benefit from a drinkable liquid formulation of microbiota. These include adult and pediatric patients, who have difficulties in swallowing capsules, especially in protocols for non-C. difficile indications that require multiple, repeated dosing. Here we presented initial data with a new, drinkable, and flavored preparation of fecal microbiota that has shown promise in curing rCDI.

[0093] It has been a working hypothesis among microbiologists and clinicians that stomach acid negatively impacts the viability of donor microbiota during its transit through the gastrointestinal tract following oral administration. This problem was somewhat mitigated in our formulation by the addition of dry whole milk powder, containing 10% fat, to the lyophilized microbiota preparation and using a chocolate milk carrier that contained ~ 2% milk fat. The patients were treated with acid suppressing medications prior to the treatment visit in clinic. Patients were also restricted from solid food prior to the administration of the microbiota suspension to facilitate its rapid exit from the stomach. Since ingestion of food is needed to activate gastric proton pumps, proton pump inhibitors are probably not ideal suppressors of stomach acid to be used in this context. Two of the patients described here 38 used histamine 2 receptor antagonists. However, another simple option may be use of calcium carbonate or magnesium / aluminum hydroxide antacids that can be administered just prior to the microbiota suspension. These have the advantage of rapid onset of action and likely have sufficient duration of gastric acid neutralization during the transit of microbiota through the stomach

[17] ,

[0094] During the intestinal transit, the donor microbiota are also subjected to other perils, such as bile acids and digestive enzymes. Therefore, the pharmacokinetics of this liquid preparation may differ from that of the encapsulated preparation and further study would be appropriate. It is also likely that the microbiota in the liquid, drinkable formulation interacts with the host immune receptors in the small bowel differently from encapsulated or colonoscopically administered preparations. Microbiota-host interactions in the small bowel may have important systemic effects on the host energy metabolism and the immune responsiveness of therapeutic interest [18,19], Small bowel mucosa is also more vulnerable to bacterial translocation and caution should be exercised in immunosuppressed patients, especially those with damaged or stressed bowel mucosa. To date, pharmacologic investigations of all microbiota-based preparations remain very limited. In fact, the methodology for studying microbiota-based therapeutics is still in the very early stages of development

[20] , However, there is a great need for short- and long-term comparisons of different preparations with respect to their pharmacokinetics, pharmacodynamics, toxicity, and drug-drug interactions in this young but rapidly developing field of therapeutics.

[0095] Citations for Example 1

[0096] 1 Brandt LJ, Aroniadis OC. An overview of fecal microbiota transplantation: techniques, indications, and outcomes Gastrointestinal endoscopy. 2013;78:240-249.

[0097] 2 Aas J, Gessert CE, Bakken JS. Recurrent Clostridium difficile colitis: case series involving 18 patients treated with donor stool administered via a nasogastric tube Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 2003;36:580-585.

[0098] 3 Hamilton MJ, Weingarden AR, Sadowsky MJ, Khoruts A. Standardized frozen preparation for transplantation of fecal microbiota for recurrent Clostridium difficile infection The American journal of gastroenterology. 2012;107:761-767.

[0099] 4 Staley C, Hamilton MJ, Vaughn BP et al.. Successful Resolution of Recurrent Clostridium difficile Infection using Freeze-Dried, Encapsulated Fecal Microbiota; Pragmatic Cohort Study The American journal of gastroenterology. 2017;l 12:940-947.

[00100] 5 Vaughn BP, Fischer M, Kelly CRet al.. Effectiveness and safety of colonic and capsule fecal microbiota transplantation for recurrent Clostridioides difficile infection Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association. 2022.

[00101] 6 Kang DW, Adams JB, Gregory ACet al. Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study Microbiome. 2017;5:10.

[00102] 7 Haifer C, Paramsothy S, Kaakoush NOet al. Lyophilised oral faecal microbiota transplantation for ulcerative colitis (LOTUS): a randomised, double-blind, placebo-controlled trial Lancet Gastroenterol Hepatol. 2022;7:141-151.

[00103] 8 Staley C, Kaiser T, Beura LKet al. Stable engraftment of human microbiota into mice with a single oral gavage following antibiotic conditioning Microbiome. 2017;5:87.

[00104] 9 Caporaso JG, Lauber CL, Walters WAet al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms The ISME journal. 2012;6:1621-1624.

[00105] 10 Gohl DM, Vangay P, Garbe Jet al. Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies Nature biotechnology. 2016;34:942-949.

[00106] 11 Schloss PD, Westcott SL, Ryabin Tet al. Introducing mothur: open-source, platformindependent, community-supported software for describing and comparing microbial communities Applied and environmental microbiology. 2009;75:7537-7541.

[00107] 12 Staley C, Kaiser T, Vaughn BPet al. Predicting recurrence of Clostridium difficile infection following encapsulated fecal microbiota transplantation Microbiome. 2018;6:166.

[00108] 13 Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Glockner FO. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB Nucleic acids research. 2007;35:7188-7196.

[00109] 14 Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R UCHIME improves sensitivity and speed of chimera detection Bioinformatics. 2011;27:2194-2200.

[00110] 15 Cole JR, Wang Q, Cardenas Eet al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis Nucleic acids research. 2009;37:D141-145.

[00111] 16 Knights D, Kuczynski J, Charlson ESet al. Bayesian community-wide cultureindependent microbial source tracking Nature methods. 2011;8:761 -763.

[00112] 17 Dettmar PW, Strugala V, Fisher J, Woodcock AD. Antacid Efficacy of Maalox in Comparison to a Range of Antacids J Pharm Sci Therap. 2019;5:301-325.

[00113] 18 Martinez-Guryn K, Hubert N, Frazier Ket al. Small Intestine Microbiota Regulate Host Digestive and Absorptive Adaptive Responses to Dietary Lipids Cell host & microbe. 2018;23:458-469.e455.

[00114] 19 de Vos P, Mujagic Z, de Haan BJet al. Strains Can Enhance Human Mucosal and Systemic Immunity and Prevent Non-steroidal Anti-inflammatory Drug Induced Reduction in T Regulatory Cells Frontiers in immunology. 2017;8:1000.

[00115] 20 Khoruts A, Staley C, Sadowsky MJ. Faecal microbiota transplantation for Clostridioides difficile: mechanisms and pharmacology Nature reviews. Gastroenterology & hepatology. 2021;18:67-80.

[00116] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and / or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

[00117] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[00118] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[00119] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

1. A freeze-dried composition comprising a fecal extract, wherein the composition comprises:biological material, a cryoprotectant or lyoprotectant or both cryoprotectant and lyoprotectant, and a flavoring agent,wherein the freeze-dried composition is friable,wherein the freeze-dried composition, upon reconstitution with a liquid, has no objectionable odor and has no objectionable taste, andwherein the biological material comprises human gut, colon, or intestinal fecal microbes, and optionally the biological material comprises human gut, colon or intestinal bacteria.

2. The freeze-dried composition of claim 1, wherein the lyoprotectant comprises trehalose.

3. The freeze-dried composition of claim 1, wherein the flavoring agent comprises a chocolate flavoring powder.

4. The freeze-dried composition of claim 1, wherein the composition further comprises a gut-transit protectant.

5. The freeze-dried composition of claim 4, wherein the gut-transit protectant comprises a powdered milk.

6. The freeze-dried composition of claim 1, wherein the composition comprises no greater than 0.05%, 0.1 % , 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% , 0.9%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% weight non-living material / weight biological material.

7. The freeze-dried composition of claim 1, wherein the composition comprises 1x1010 to 5xl012 cells.

8. A composition obtained by reconstituting a freeze-dried mixture with a liquid, wherein the freeze-dried mixture comprises biological material, a cryoprotectant or lyoprotectant or both cryoprotectant and lyoprotectant, and a flavoring agent, and wherein the composition has no objectionable odor and has no objectionable taste, and wherein the biological material comprises human gut, colon, or intestinal fecal microbes, and optionally the biological material comprises human gut, colon or intestinal bacteria.

9. The composition of claim 8, wherein the lyoprotectant comprises trehalose.

10. The composition of claim 8, wherein the flavoring agent comprises a chocolateflavoring powder.

11. The composition of claim 8, wherein the composition further comprises a guttransit protectant.

12. The composition of claim 11, wherein the gut-transit protectant comprises a powdered milk.

13. The composition of claim 8, wherein the liquid comprises a dairy product.

14. The composition of claim 13, wherein the dairy product comprises chocolate milk.

15. The composition of claim 8, wherein the freeze-dried mixture comprises no greater than 0.05%, 0.1 % , 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% , 0.9%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% weight non-living material / weight biological material.

16. The freeze-dried composition of claim 8, wherein the composition comprises l.OxlO11 to 5.0xl0n cells.

17. A method for making a freeze-dried composition comprising a fecal extract, the method comprising:filtering a fecal sample with a filter medium, wherein the filter medium comprises at least one sieve size of no greater than 2.0 mm, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 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, or 0.01 mm to result in or to generate a filtrate;applying a centrifugal force to the filtrate to form a pellet and resuspending the pellet in a reduced volume compared to the filtrate before application of the centrifugal force;repeating the applying and the resuspending at least three times to result in a processed filtrate;adding a lyoprotectant or cryoprotectant or both lyoprotectant and cyroprotectant to the processed filtrate to result in a mixture;freeze-drying the mixture to result in a freeze-dried composition; andconverting the freeze-dried composition into a powder;wherein the composition comprises a biological material, and optionally the biological material comprises bacteria, wherein optionally the composition comprises a pharmaceutically acceptable carrier, and optionally the composition is a formulation for oral administration.

18. The method of claim 17, wherein the method further comprises adding a flavoring agent to the powder, adding a gut-transit protectant to the powder, or adding both a flavoring agent and a gut-transit protectant to the powder.

19. The method of claim 18, wherein the flavoring agent comprises a chocolate flavoring powder.

20. The method of claim 18, wherein the gut-transit protectant comprises a powdered milk.

21. The method of claim 17, the method further comprising reconstituting the freeze-dried composition with a liquid.

22. The method of claim 21, wherein the liquid comprises a milk.

23. The method of claim 21, wherein the milk comprises (i) a dairy product such aschocolate milk, or (ii) a plant-based milk.

24. The method of claim 21, wherein the reconstituted composition has no objectionable odor and has no objectionable taste.

25. The method of claim 21, wherein the reconstituted composition comprises l.OxlO11 to S.OxlO11 cells.

26. A method of treating a disease or condition in a subject, the method comprising administering to the subject the reconstituted composition of claim 8 or 22.

27. The method of claim 26, wherein the subject has reduced ability to swallow.

28. The method of claim 27, wherein the reduced ability to swallow comprises adysphagia.

29. The method of claim 28, wherein the dysphagia comprises pill dysphagia, dysphagia to solids, or a combination thereof.

30. The method of claim 27, wherein the reduced ability to swallow comprises an esophageal dysmotility and strictures.

31. The method of claim 26, wherein the subject is refractory to administration of a liquid having an objectionable odor or objectionable taste.

32. The method of claim 26, wherein the subject is a pediatric subject, a subject with a developmental disability, or a combination thereof.

33. The method of claim 26, wherein the disease or condition comprises autism spectrum disorder, Pitt-Hopkins Syndrome, Parkinson's disease, alcohol use disorder, obesity, Type II diabetes, presence of multi-drug resistant organisms, and cancers that can be treated using immune-oncology, such as optimization of anti-cancer check-point immunotherapy, treatment of checkpoint inhibitor colitis, treatment or prevention of complications of hematopoietic stem cell transplantation, such as graft-versus-host disease, and prevention of complications of surgical intestinal resections.

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