Methods for treating hepatic encephalopathy

Abstract

The present disclosure provides freeze-dried and liquid compositions that include an extract of human feces. Also provided are methods for making and using such compositions, including methods for replacing or supplementing or modifying a subject's intestinal microbiota, and methods for treating a disease or condition such as hepatic encephalopathy.

Description

METHODS FOR TREATING HEPATIC ENCEPHALOPATHY

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 650,036, filed May 21, 2024, which is incorporated by reference herein in its entirety.

[0003] GOVERNMENT FUNDING

[0004] This invention was made with government support under 2101CX001076 awarded by the Department of Veterans Affairs. The government has certain rights in the present invention.

[0005] BACKGROUND

[0006] Cirrhosis, the end-stage of liver injury, is widely prevalent globally due to high rates of steatotic liver disease, viral hepatitis, and alcohol misuse (Devarbhavi et al., J Hepatol 2023;79(2):516-537. DOI: 10.1016 / j .jhep.2023.03.017). Cirrhosis often results in hepatic encephalopathy (HE), which is pathophysiologically associated with an altered gut-liverbrain axis and forms a major clinical, psychosocial, and financial burden (Vilstrup et al., Hepatology 2014;60(2):715-35. DOI: 10.1002 / hep.27210; Bloom et al., J Hepatol 2021;75(6): 1452-1464. DOI: 10.1016 / j .jhep.2021.08.004). In the United States HE is primarily treated with lactulose and rifaximin, but despite these therapies, patients remain at risk for recurrent episodes (Bajaj et al., J Hepatol 2020. DOI: 10.1016 / j.jhep.2020.01.017; Kibble et al., United European Gastroenterol J 2024. DOI: 10.1002 / ueg2.12530). HE recurrences are linked with hospital readmissions, cumulative and irreversible cognitive impairment, and mortality, and are not prioritized for liver transplant (Kim et al., Gastroenterology 2021 ; 161 (6): 1887- 1895 e4. DOI: 10.1053Zj.gastro.2021.08.050).

[0007] SUMMARY OF THE APPLICATION

[0008] Provided herein are methods for using a fecal extract. In one embodiment, a method can be for treating a patient. A method for treating can include administering to a patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier. The patient can have cirrhosis, hepatic encephalopathy (HE), or can be at risk for a recurrent episode of HE.

[0009] In one embodiment, a method can be for increasing the relative abundance of at least one microbe in a patient. This type of method can include administering to the patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier. The patient can have cirrhosis, HE, or can be at risk for a recurrent episode of HE. The at least one microbe can be one or more members of the order Bifidobacteriales, one or more members of the family Lachnospiraceae one or more members of the family Riiminococcaceae, one or more members of the family Clostridiaceae, one or more members of the family Rikenellaceae, one or more members of the family Bacteroidaceae, one or more members of the family Desulfovibrionaceae, or a combination thereof. The relative abundance is increased in the patient after the administering.

[0010] In one embodiment, a method can be for decreasing the relative abundance of at least one microbe in a patient. This type of method can include administering to the patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier. The patient can have cirrhosis, HE, or can be at risk for a recurrent episode of HE. The at least one microbe can be one or more members of the family Lactobacillaceae, one or more members of the family Prevotellaceae, one or more member of the family Aerococcaceae, one or more member of the family Fusobacteriaceae, one or more member of the family Coriobacteriaeae, or a combination thereof. The relative abundance is decreased in the patient after the administering.

[0011] 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 examplescan 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.

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings.

[0014] FIG. 1A-D shows study flow and major procedures (FIG. 1A), CONSORT Diagram (FIG. IB), clinical outcomes between the four groups (FIG. 1C) (data are presented at percentage and Fisher and Chi-square test results are shown), and linear mixed methods analysis between baseline (0) and day 60 (post 2 FMTs) (FIG. ID). Psychosocial and Physical SIP showed time group interactions that were significant but not PHES and Stroop. Gp 1 : Group 1 assigned to active capsule & enema FMT at baseline and active capsules at day 30, Gp 2: Group 2 assigned to capsule enema at baseline and day 30 and placebo enema at baseline, Gp 3: Group 3 assigned to active enema at baseline and placebo capsules at baseline and day 30, Gp 4: Group 4 assigned to placebo capsules and enema at baseline and placebo capsules at day 30. PHES: psychometric hepatic encephalopathy score (high indicates good performance), SIP: Sickness impact profile, QOL: quality of life, AE: adverse events, Rx: treatment, FMT: fecal microbiota transplant, hosp: hospitalizations, HE: hepatic encephalopathy

[0015] FIG. 2A-D shows baseline and overall changes in microbiome in those whose HE recurred versus did not. N=10 overall and N=4 within FMT and N=6 within all placebo groups. FIG. 2A: Baseline comparisons between families among all subjects. “Yes”: Those who recurred, “No”: those who did not recur. *=p<0.05 Kruskal-Wallis test.FIG. 2B: LEfSe of patients who recurred (HE) versus did not (no HE) using samples at all timepoints. FIG. 2C: LEfSe at baseline of the 6 placebo patients who recurred (red) versus the 9 who did not recur. FIG. 2D: LEfSe at baseline of the 4 FMT patients who recurred (red) versus the 41 who did not recur.

[0016] FIG. 3A-D shows relative abundance of Lachnospiraceae and higher Lactobacillaceae (FIG. 3 A), relative abundance of Lachnospiraceae, Clostridiaceae, Rikenellaceae,Bacteroidaceae, and Desulfovibrionaceae, and Prevotellaceae, Aerococcaceae, Fusobacteriaceae, Coriobactericaeae, and Lactobacillaceae (FIG. 3B), engraftment in FMT groups who recurred compared to those who did not, (FIG. 3C), and baseline Lachnospiraceae and engraftment (FIG. 3D).

[0017] FIG. 4A-E show microbiota changes within groups, FIG. 4A: Shannon diversity changes within groups, FIG. 4B: LEfSe of group 1 showing higher Subdoligranulum, Fusobacterium, and Bifidobacterium post-FMT day 60, FIG. 4C : LEfSe of group 2 showing higher Enterobacteriaceae members at baseline that reduced at post-FMT day 60, FIG. 4D: LEfSe of group 3 showing higher Fusobacterium post-FMT day 60, and FIG. 4E: LEfSe of Group 4 showed higher Bifidobacteria at baseline that reduced at day 60.

[0018] FIG. 5 shows improvement in SIP values using linear mixed models over baseline and 2 months but not in PHES or Stroop. Gp=group, SIP: Sickness Impact Profile, PHES: psychometric hepatic encephalopathy score.

[0019] FIG. 6 shows engraftment rates at days 15, 30 and month 2 based on vegan or omnivorous donor were statistically similar. Data are presented as individual points with median and 95% CI. Comparison performed using Mann-Whitney U test. Omni: omnivorous donor. DI 5: day 15, D30: day 30, 2M: 2 months after enrollment.

[0020] FIG. 7 shows engraftment dynamics across the 3 FMT groups. D15: day 15, D30: day 30, M=months after enrollment.

[0021] FIG. 8 shows acid production from fermentation of lactulose by fecal microbiota from individual healthy donors as measured in vitro.

[0022] The 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.

[0023] DETAILED DESCRIPTION

[0024] Hepatic encephalopathy (HE) is driven by neuroactive toxins that a cirrhotic liver is not able to neutralize. Some of the toxins are products of microbial metabolism in the intestine, which cannot be processed by diseased liver or simply bypass the liver entirely due to altered blood circulation caused by liver scarring and portal hypertension. All current standard therapies for HE target the gut microbiota, which can facilitate toxin neutralization and elimination (Bloom et al., Journal of hepatology 2021; 75:1452-64; Bajaj and Khoruts, Journal of hepatology 2020; Kibble and Shawcross, United European Gastroenterol J 2024; 12:187-93). In the United States these therapies include lactulose, which is a nonabsorbable sugar that is fermented exclusively by gut microbiota, and the antibiotic rifaximin, which modulates the composition of microbiota.

[0025] Ammonia is a major neurotoxin, long recognized to play an important role in HE pathogenesis. Both lactulose and rifaximin lower ammonia levels in the body. Fermentation of lactulose by gut microbes results in production of short-chain fatty acids, including acetate and lactate, which lower the intraluminal pH in the colon and trap ammonia as ammonium. The latter is positively charged, cannot be reabsorbed into circulation, and is excreted in stool. Rifaximin lowers ammonia production in the intestine by certain groups of bacteria, such as members of the Enter ohacteriaceae family, which is increased in patients with liver cirrhosis (Acharya C, Bajaj JS. Clinical Gastroenterology and Hepatology, 2019; 17:307-321, doi: 10.1016 / j.cgh.2018.08.008).

[0026] The problem in relying on gut microbes to eliminate ammonia is the high prevalence of gut dysbiosis in patients with liver disease, characterized by decreased microbial diversity, increased producers of ammonia such as Enter obacteriaceae, and decreased beneficial microbial taxa such as Lachnospiraceae and Ruminococcacea . (Acharya and Bajaj, Clinical Gastroenterology and Hepatology, 2019; 17:307-321, doi:10.1016 / j.cgh.2018.08.008; Xu et al., World J Hepatol 2025; 17: 100377). Fecal microbiota transplantation (FMT), also known as microbiota transplant therapy (MTT), has the potential to repair the microbial community structure and optimize the ability of the gut microbes to eliminate neurotoxins such as ammonia. However, the inventors haveidentified methods and compositions that are useful in treating and managing HE. Regimens that include multiple doses of donor microbiota help to achieve effective engraftment - also referred to as colonization - of donor microbiota, especially when used without prior conditioning that may include purgatives and / or antibiotics. Donor microbiota is highly heterogeneous among healthy individuals with respect to its ability to ferment lactulose (or other non-absorbable and fermentable polysaccharides, such as lactitol) and produce acids; donor microbiota containing the greatest potential to ferment lactulose and dietary fiber into acid products can be the most effective at treating HE. Patients most likely to benefit from microbiota transplant therapy are those whose gut microbiota is least able to produce acids from therapeutic sugar substrates, such as lactulose, or dietary fiber.

[0027] Provided herein are methods and compositions for aiding individuals having HE or at risk of recurrent HE. The compositions for use in the methods 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. 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. As used herein, a “fecal extract” is a composition derived from a fecalsample, but includes predominantly biological material, including bacteria, and little nonliving material, e.g., undigested fiber and other foods.

[0028] 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).

[0029] 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 include 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 Bifidobacteriales include members of the family Bifidobacteriaceae. Members of the family Bifidobacteriaceae include members of the genus Bifidobacterium (also referred to as Bifidobacteria). Examples of the subclass Coriobacteridae include members of the order Coriobacteriales. Members of the order Coriobacteriales include members of the family Coriobacteriaceae. 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 Prevotella), and members of the family Rikenellaceae.

[0030] 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 , Enter ococcaceae, Lactobacillaceae, and Streptococcaceae). Examples of the class Clostridia include members of the order Clostridiales, and examples of the order Colstri diales include the family Catabacteriaceae Peptococcaceae, Peptostreptococcaceae, Ruminococcaceae, (also known as the family Oscillospiraceae), Clostridiaceae, Eubacteriaceae, and Lachnospiracea . Examples of the family Ruminococcaceae include members of the genera Ruminococcus and Faecalibacterium. Examples of the family Lachnospiraceae include members of the genera Blautia, Coprococcus, Dorea, Lachnospira, Oribacterium, and Roseburia. 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.

[0031] 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 members of the family Desulfomicrobiaceae . Examples of the class Epsilonproteobacteria include members of the order Desulfobacterales, and examples of members of this order includemembers 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 Shewaiiellaceae, and examples of members of the order Enterobacteriales include members of the family Enterobacteriaceae .

[0032] 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 .

[0033] 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 .

[0034] 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 least5 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, at least 5 orders, at least6 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, atleast 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.

[0035] In one embodiment a freeze-dried composition described herein includes, when reconstituted with water such as a buffer, no greater than 0.05%, no greater than 0.1 %, no greater than 0.2%, no greater than 0.3%, no greater than 0.4%, no greater than 0.5%, no greater than 0.6%, no greater than 0.7%, no greater than 0.8%, no greater than 0.9%, no greater than 1 %, no greater than 2%, no greater than 3%, no greater than 4%, no greater than 5%, no greater than 6%, no greater than 7%, no greater than 8%, no greater than 9% or no greater than 10% weight of non-living material / weight biological material (wt / wt). 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, RNA, and DNA and RNA for specific genes.

[0036] In one embodiment, the fecal material present in a liquid composition described herein, and the fecal material present in a freeze-dried composition described herein when reconstituted with water does 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 0.7 mm, greater than 0.65 mm, greater than 0.6 mm, greater than 0.55 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 includes, consists essentially of, or consists 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. 7 mm, greater than 0.65 mm, greater than 0.6 mm, greater than 0.55 mm, greater than 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 materialpresent 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. 7 mm, greater than 0.65 mm, greater than 0.6 mm, greater than 0.55 mm, greater than 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.

[0037] 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.

[0038] A composition of the present disclosure may optionally include a cryoprotectant, a lyoprotectant, or both a cryoprotectant and a lyoprotectant. Cryoprotectants and lyoprotectants useful in freeze-drying microbes are known and include, for instance, amino acids such as alanine, glycine, proline; simple sugars such as sucrose, glucose, lactose, ribose, mannitol, and trehalose; other compounds such as dimethyl sulfoxide (DMSO), glycerol, casein hydrolysate, sucrose, gelatin, non-fat skim milk, starch hydrolysate, fetal calf serum, bovine serum albumin, or combinations of 1, 2, 3, 4, or more 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. A lyoprotectant is a compound that maintains the viability of fecalmicrobes during drying. 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 tablet. In one embodiment, a useful powder may have size, density, flow, and compression characteristics suitable for production of tablets or encapsulation, or double encapsulation.

[0039] Useful cryoprotectants and / or lyoprotectants include those that result in a composition that is friable, does not crystallize during freeze-drying, and maximize survival of microbes. 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. In one embodiment, more than one cryoprotectant may be used, such as the combination of sucrose (e.g., 5 %) and skim milk (e.g., 5 %), trehalose and skim milk (e.g., 5%), trehalose and dextran (e.g., 3- 10%), trehalose and mannitol, or the combination of sucrose (e.g., 10 %) and gelatin (e.g., 0. 1 %).

[0040] In one embodiment, a useful cryoprotectant and / or lyoprotectant is not sucrose alone, which may crystallize and harden during freeze-drying, or glycerol, which unexpectedly results in an oily and viscous composition upon freeze-drying with a fecal material as described herein.

[0041] The total cryoprotectant and / or lyoprotectant used to produce a freeze-dried composition may be at least 1 %, at least 2 %, at least 3 %, at least 4 %, at least 5 %, at least 6 %, at least 7 %, at least 8 %, at least 9 %, at least 10 %, at least 11 %, at least 12 %, at least 13 %, at least 14 %, at least 15 %, at least 16 %, at least 17 %, at least 18 %, at least 19 %, at least 20 %, at least 21 %, at least 22 %, at least 23 %, at least 24 %, at least 25 %, at least26 %, at least 27 %, at least 28 %, at least 29 %, at least 30 % (vol / vol), or more 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.

[0042] In one embodiment a composition of the present disclosure, e.g., a freeze-dried composition, does not include pathogenic biological material. In one embodiment, fecal material is from a person, referred to herein as a donor, 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 multi-drug resistant organisms, presence of gastrointestinal co-morbidities, factors that can or do affect the composition of the intestinal microbiota, and systemic medical conditions.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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 Isosporaf, 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 seletive 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 < 40mg / dL (males) and < 50 mg / dL (females); high sensitivity CRP > 2. 4 mg / L; raised fasting plasma glucose (> 100 mg / dL).

[0049] In one embodiment, a donor is an omnivore or an herbivore. An example of a herbivore is a person practicing a vegan diet, e.g., does not consume meat, eggs, dairy products, or any other animal-derived substances. In one embodiment, a donor has a higher microbiota content of microbes producing acidic compounds. Examples of acidic compounds include, but are not limited to, short-chain fatty acids (SCFAs), and lactic acid. Examples of SCFA- producing microbes include, but are not limited to, members of the family Lachnospiraceae , and members of the family Ruminococcaceae . Examples of lactic acidproducing microbes include, but are not limited to, members of the order Bifidobacteriales, members of the family Bifidobacteriaceae, and members of the genus Bifidobacterium. In one embodiment, the stool of a donor has a pH of no greater than 6.2, no greater than 6.1, no greater than 6, no greater than 5.9, or no greater than 5.8.

[0050] A donor having a higher microbiota content of microbes producing acidic compounds can be identified by measuring the ability of the donor’s biological material to produce one or more SCFAs, lactic acid, or a combination thereof. Methods for measuring the amount of SCFAs and lactic acid in a donor’s biological material are routine and known in the art.

[0051] A donor having a higher microbiota content of microbes producing acidic compounds can be identified by measuring the ability of the donor’s biological material to produce acids in response to a substrate. Examples of substrates include, but are not limited to, polysaccharides that can be fermented by, but not absorbed by, the human gastrointestinal tract. Examples of useful polysaccharides are two carbohydrates (a di saccharide), three carbohydrates (a trisaccharide), or more than three carbohydrates. Useful non-absorbable fermentable polysaccharides include, but are not limited to, lactulose and lactitol.

[0052] Methods for identifying a donor having a higher microbiota content of microbes producing acidic compounds can include separating fecal microbes from non-biological material, suspending the biological material at a predetermined concentration, adding a nonabsorbable fermentable polysaccharide, and measuring acid production at time intervals.

[0053] Any method can be used to separate fecal microbes from non-biological material, including any method described herein. In one embodiment, a rapid method for preparation of small samples can be used (see Example 2). This rapid method includes mixing a sample of, for instance, one gram of a stool sample and four milliliters of phosphate buffered saline with a Vortex and then filtering through a filter that has been fitted into a syringe. In one embodiment, the filter has pores of 0.6 mm. The material passing through the filter can be centrifuged to result in a pellet of microbiota and resuspended in one ml of an aqueous solution. Optionally, the pellet of microbiota can be washed once, and then resuspended in 1 ml of an aqueous solution. Optionally, the resuspended microbiota can be cryopreserved using a cryoprotectant.

[0054] Multiple samples of the microbiota can be prepared for the assay. The microbiota can be resuspended at any concentration, and in one embodiment is suspended at a concentration of 7 ± 0.5 x 1010bacteria in 2 mL. The fermentable non-absorbable polysaccharide is added to each tube to be tested, and then the tubes are incubated. The pH is measured at time intervals. Samples from multiple donors are compared for acid production resulting from fermentation of the non-absorbable polysaccharide. Microbiota that produce acid is most desirable for use in the methods of the present disclosure, thus, microbiota from the donors producing a greater amount of acid are selected for use in the methods described herein. In one embodiment, a microbiota from the top 75% of donors, the top 50% of donors, or the top 25% of donors producing the most acid is selected for use in the methods described herein. Accordingly, in some embodiments a microbiota used in a method of the present disclosure produces acid at a level greater than 25% of the population, a level greater than 50% of the population, or a level greater than 75% of the population. A microbiota producing acid at a level greater than 25% of the population, a level greater than 50% of the population, or a level greater than 75% of the population is referred to herein as a high acid-producing microbiota. A fecal extract derived from a high acid-producing microbiota is a high acid-producing fecal extract. When determining if a microbiota produces acid at a level greater than 25% of the population, a level greater than 50% of the population, or a level greater than 75% of the population, microbiota from at least eight donors can be compared.

[0055] 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 and liquid formulations include, but are not limited to, solutions, suspensions, dispersions, and the like. In one embodiment, a formulation may be a solid composition. Solid compositions and solid formulations 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.

[0056] A composition of the present disclosure may be administered by any method suitable for depositing in the gastrointestinal tract of a subject. Examples of routes of administration include oral administration (e.g., by a solid such as a pill, tablet, or capsule), or by liquid, via intubation through the nose or the mouth (e.g., by nasogastric tube or nasal jejunal tube, upper endoscopy, upper push enteroscopy, balloon enteroscopy), or rectal administration (e.g., by suppository, enema, flexible sigmoidoscopy, or colonoscopy). 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. A gut-transit protectant may include an acid suppressant medication, such as antacid, an Eh-blocker, a proton pump inhibitor, or potassium-competitive acid blocker. 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.

[0057] For therapeutic use in the methods 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.

[0058] 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. A gut-transitprotectant may also include an acid suppressant medication, such as antacid, an H2-blocker, a proton pump inhibitor, or potassium-competitive acid blocker.

[0059] 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.

[0060] 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.

[0061] 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, 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.

[0062] 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 ahorizontal 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.

[0063] 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.

[0064] 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 thefiltrate. 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 at least and 5x1010cells 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 IxlO10cells 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, OOOxg 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.

[0065] 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 at least 5xl010cells, at least 5.5xl010cells, at least 6xlO10cells, at least 6.5xl010cells, at least 7xlO10cells, at least 7.5xlO10cells, at least 8xl010cells, at least 8.5xl010cells, at least 9xlO10cells, at least 9.5xlO10cells, at least IxlO11cells, at least 2.5xlOncells, at least3xl Oncells, at least 3.5xlOncells, at least 4xlOncells, at least 4.5xlOncells, at least5xlOncells, at least 5.5xlOncells, at least 6xlOncells, at least 6.5xlOncells, at least7xlOncells, at least 7.5xlOncells, at least 8xlOncells, at least 8.5xlOncells, at least9xlOncells, at least 9.5xlOncells, at least IxlO12cells, at least 1.5xl012cells, at least2xl012cells, at least 2.5xl012cells, at least 3xlO12cells, at least 3.5xlO12cells, at least4xl012cells, at least 4.5xl012cells, at least 5xl012cells, at least 5.5xl012cells, at least6xl012cells, at least 6.5xl012cells, at least 7xl012cells, at least 7.5xl012cells, or at least8xl012cells. Since most biological material is difficult or impossible to culture, a hemocytometer may be used to determine the number of cells.

[0066] 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.

[0067] In some embodiments, 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 andwashes. 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 5xl010cells, , at least 5.5xl010cells, at least 6xlO10cells, at least 6.5xlO10cells, at least 7xlO10cells, at least 7.5xlO10cells, at least 8xl010cells, at least 8.5xl010cells, at least 9xlO10cells, at least 9.5xlO10cells, at least IxlO11cells, at least 2.5xlOncells, at least 3xl0ncells, at least 3.5xl0ncells, at least 4xlOncells, at least 4.5xlOncells, at least 5xl0ncells, or at least 5.5xl0ncells, at least 6xlOncells, at least 6.5xlOncells, at least 7xlOucells, at least 7.5xl0ncells, at least 8xl0ncells, at least 8.5X1011cells, at least 9xl0ncells, at least 9.5xlOncells, at least IxlO12cells, at least 1.5xl012cells, at least 2xl012cells, at least 2.5xl012cells, at least 3xl012cells, at least 3.5xl012cells, at least 4xl012cells, at least 4.5xl012cells, at least 5xl012cells, or at least 5.5xl012cells, at least 6xl012cells, at least 6.5xl012cells, at least 7xl012cells, at least 7.5xl012cells, or at least 8xl012cells in a volume that is subsequently freeze-dried. In one embodiment, the material is concentrated to result in no greater than 9xl012cells in a volume that is subsequently freeze-dried.

[0068] In some embodiments, making a composition disclosed herein can include homogenizing a sample. As used herein, “homogenizing” refers to conditions that promote the break-up of non-living and living material present in a fecal sample and increase the release of living biological material from the non-living material. A fecal sample can be present in a container in which homogenization can occur. An example of such a container is aflexible sterile plastic bag. In one embodiment, the oxygen in a container may be decreased or removed by purging with an inert gas such as nitrogen or argon prior to homogenization. Such anaerobic conditions may be useful to maintain viability of strict anaerobic bacteria present in a colon. The sample may be homogenized multiple times and / or additional diluent may be added until a consistency is achieved that will permit the following steps to occur. After the homogenizing, the solutions used for washing and resuspension can be treated to remove oxygen. Manipulation of the microbiota may occur in an oxygen free cabinet or anaerobic glove box chamber.

[0069] In one embodiment, a method disclosed herein (optionally referred to as a pressurized filter press method) can use a container such as a flexible sterile plastic bag that is impervious to liquid. Examples of such containers are described in, but not limited to, US 5,180,229, US 2024 / 0009908, and US 2024 / 0017888, and those available from WHIRL-PAK® Filtration Group (Pleasant Prairie, WI). Care should be taken during the methods described herein to reduce exposure to oxygen, and the transfer can include using nitrogen gas to replace any air that may be present.

[0070] A cell extraction buffer is added to the fecal sample. The use of gelatin in combination with ammonium phosphate aids in the release of biological material from the non-living material present in a fecal sample, accordingly, in some embodiments, a cell extraction buffer includes gelatin and ammonium phosphate.

[0071] In one embodiment a cell extraction buffer includes, but is not limited to a buffer, or both a buffer and a gelatin. In one embodiment, 1 part donor feces may be combined with 5 parts cell extraction buffer (e.g., 50 grams of donor feces may be combined with 250 ml cell extraction buffer) and homogenized. An example of a buffer includes, but is not limited to, (NH4)2HPO4. The buffer, for instance (NH^HPCh, can be present at a concentration of at least 0.01 M, at least 0.05 M, or at least 0.1 M, and at a concentration of no greater than 1 M, no greater than 0.5 M, or no greater than 0.1 M. Examples of ranges of a buffer include, but are not limited to, 0.01 M to 1 M, and 0.05 M to 0.5 M. Gelatin is typically a mixture of peptides and proteins commonly derived from collagen. In one embodiment, the gelatin can be partially hydrolyzed. In one embodiment, the gelatin can be USP grade.Gelatin can be present at a concentration of at least 0.01% volume / volume (v / v), at least 0.05% v / v, or at least 0.1% v / v, and at a concentration of no greater than 1% v / v, no greater than 0.5% v / v, or no greater than 0.1% v / v. In one embodiment, a 10% v / v gelatin solution in water is adjusted to pH 10.0 and is partially hydrolyzed by autoclaving for 10 min at 15 PSI (Kingsley and Bohlool , Appl Environ Microbiol. 1981 Aug;42(2):241-8. doi: 10.1128 / aem.42.2.241-248.1981). Examples of ranges of gelatin in a cell extraction buffer include, but are not limited to, 0.01% v / v to 1% v / v, and 0.05% v / v to 0.5% v / v.

[0072] A cell extraction buffer can also include other components, including but not limited to a nonionic surfactant and emulsifier. Examples of a nonionic surfactant and emulsifier include, but are not limited to, polysorbate 80 (available under the trade name TWEEN- 80®). A polysorbate 80 can be present at a concentration of at least 0.001%, at least 0.005%, or at least 0.01%, and at a concentration of no greater than 0.1%, no greater than 0.05%, or no greater than 0.01%.

[0073] The fecal sample in the container is subjected to conditions that homogenize the sample. In one embodiment, such conditions can be obtained through use of a paddle homogenizer, also known in the art as a stomacher. Typically, the sample is homogenized until it is a slurry, e.g., a mixture of solids suspended in a liquid. The slurry is then fdtered with a pressurized fdter press. An example of a pressurized fdter press useful in the method includes a cylinder, a lid, and a plunger. The side of the plunger in contact with the surface of the cylinder fits tightly to prevent seepage of the slurry, and the surface of the plunger in contact with the slurry includes at least one filter medium. An example of a pressurized filter press is a French Press, modified to include a filter as described herein.

[0074] The filter medium present at the surface of the plunger in contact with the slurry includes at least one, at least two, at least three, or at least four filters. In one embodiment, the surface of the plunger in contact with the slurry includes no greater than six filters. The one or more filters can be described by sieve size, also referred to as pore size, in millimeters (mm) or sieve size based on the US Standard sieve sizes. The sieve size of one or more of the filters can be no greater than 2 mm, no greater than 1 mm, no greater than 0.7 mm, no greater than 0.65 mm, no greater than 0.6 mm, no greater than 0.55 mm, no greater than 0.5mm, no greater than 0.25 mm, no greater than 0.212 mm, no greater than 0. 18 mm, no greater than 0. 15 mm, no greater than 0. 125 mm, no greater than 0. 106 mm, no greater than 0. 09 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. 033 mm, no greater than 0. 025 mm, no greater than 0. 02 mm, or no greater than 0. 01 mm. The sieve size of one or more filters can be no less than 0.212 mm, no less than 0.25 mm, no less than 0.3 mm, no less than 0.400 mm, no less than 0.5 mm, no less than 0.55 mm, no less than 0.6 mm, no less than 0.65 mm, no less than 0.7 mm, or no less than 0. mm. Sieve size of one or more of the filters can be, when described as US Standard sieve sizes, no greater than 400, no greater than 325, no greater than 270, no greater than 230, no greater than 200, no greater than 170, no greater than 140, no greater than 120, no greater than 100, no greater than 80, no greater than 70, no greater than 60, no greater than 35, no greater than 18, or no greater than 10. In one embodiment, the filter medium present at the surface of the plunger in contact with the slurry includes at least one, at least two, at least three, or at least four filters, each of which has a sieve size of no greater than 0.15 mm or no greater than US Standard sieve size 100. In one embodiment, the filter medium present at surface of the plunger in contact with the slurry includes at least four filters, each of which has a sieve size of 0.15 mm or US Standard sieve size 100.

[0075] Pressure can be applied manually to push the liquid component and biological material through the filter while preventing passage of the non-living material and biological material that exceeds the pore size of the smallest filter. The liquid component containing biological material is removed (filtrate) and the solids (unfiltered material) resuspended in a diluent, such as a saline solution. Saline solutions useful in maintaining the viability of biological material present in a fecal sample are known, and in one embodiment includes phosphate buffered saline at pH 7.0. This suspension is then filtered with the pressurized filter press to yield a second liquid component (second filtrate) containing biological material. The first and second liquid components are combined in a container suitable for centrifugation. The container can be purged with an inert gas or gas mixture, such as nitrogen gas, to maintain conditions for anaerobes. The force of a centrifugation step can be at any speed that causes the separation of biological material from liquid but does not reduce ease of resuspension in a suitable buffer, and does not cause loss of therapeuticefficacy of the composition in 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 biological 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. The washing procedure of centrifugation followed by removal of the supernatant and resuspending the pellet in a reduced volume of diluent is repeated at least once for a total of four washes.

[0076] In another embodiment, a method disclosed herein (optionally referred to as a linear filtration method) can use a container such as a flexible sterile plastic bag that is impervious to liquid, and has been further modified to include one or more linear filters. In one embodiment, a linear filter in a container is sealed to both sides and bottom of the container, resulting in two compartments that share one wall that is the filter. For instance, a container such as those described in, but not limited to, US 5,180,229, US 2024 / 0009908, and US 2024 / 0017888, can be modified to include one or more filters. Useful containers containing a filter are available from WHIRL-PAK® Filtration Group (e g., WHIRL- PAK® Filter Sterilized Bags). A fecal sample can be placed on either side, and the liquid component with biological material will transfer through the filter, while the non-living materials remain separate and contained.

[0077] In another embodiment, a fecal sample can be placed in a bag that includes one or more walls that are made of a linear filter. The bag can be sealed and then placed in a container such as a flexible sterile plastic bag that is impervious to liquid. For instance, a container such as those described in, but not limited to, US 5,180,229, US 2024 / 0009908, and US 2024 / 0017888. Useful containers containing a filter are available from WHIRL-PAK® Filtration Group (e.g., WHIRL-PAK® Filter Sterilized Bags). The liquid component of the fecal sample with biological material will transfer through the filter, while the non-livingmaterials remain separate and contained. The inventors have found that the method of using this double filtration system resulted in surprisingly efficient production of a homogenized sample that can be further processed to result in a fecal extract. Without intending to be limited by theory, it is possible the homogenization step causes the fecal sample to pass through the filter multiple times.

[0078] The linear filter can be described by sieve size, also referred to as pore size, in millimeters (mm) or sieve size based on the US Standard sieve sizes. The sieve size of the linear filter can be no greater than 2 mm, no greater than 1 mm, no greater than 0.7 mm, no greater than 0.65 mm, no greater than 0.6 mm, no greater than 0.55 mm, no greater than 0.5 mm, no greater than 0.33 mm, no greater than 0.25 mm, no greater than 0.212 mm, no greater than 0. 18 mm, no greater than 0. 15 mm, no greater than 0. 125 mm, no greater than 0. 106 mm, no greater than 0. 09 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. 02 mm, or no greater than 0. 01 mm. The sieve size of one or more filters can be no less than 0.212 mm, no less than 0.25 mm, no less than 0.3 mm, no less than 0.400 mm, no less than 0.5 mm, no less than 0.55 mm, no less than 0.6 mm, no less than 0.65 mm, no less than 0.7 mm, or no less than 0. mm. Sieve size of one or more of the filters can be, when described as US Standard sieve sizes, no greater than 400, no greater than 325, no greater than 270, no greater than 230, no greater than 200, no greater than 170, no greater than 140, no greater than 120, no greater than 100, no greater than 80, no greater than 70, no greater than 60, no greater than 35, no greater than 18, or no greater than 10. In one embodiment, the pores can be distributed at 200 to 300 pores per cm2.

[0079] A cell extraction buffer can be added to the fecal sample. In one embodiment a cell extraction buffer includes, but is not limited to a buffer, or both a buffer and a gelatin. In one embodiment, 1 part donor feces may be combined with 5 parts cell extraction buffer (e.g., 50 grams of donor feces may be combined with 250 ml cell extraction buffer) and homogenized. An example of a buffer includes, but is not limited to, (NHfhHPCM. The buffer, for instance, (NHTriHPC can be present at a concentration of at least 0.01 M, at least 0.05 M, or at least 0.1 M, and at a concentration of no greater than 1 M, no greaterthan 0.5 M, or no greater than 0.1 M. Examples of ranges of a buffer include, but are not limited to, 0.01 M to 1 M, and 0.05 M to 0.5 M. The gelatin is typically a mixture of peptides and proteins commonly derived from collagen. In one embodiment, the gelatin can be partially hydrolyzed. In one embodiment, the gelatin can be USP grade. The gelatin can be present at a concentration of at least 0.01% volume / volume (v / v), at least 0.05% v / v, or at least 0.1% v / v, and at a concentration of no greater than 1% v / v, no greater than 0.5% v / v, or no greater than 0.1% v / v. In one embodiment, a 10% v / v gelatin solution in water is adjusted to pH 10.0 and is partially hydrolyzed by autoclaving for 10 min at 15 PSI (Kingsley and Bohlool , Appl Environ Microbiol. 1981 Aug;42(2):241-8. doi: 10.1128 / aem.42.2.241-248.1981). Examples of ranges of gelatin in a cell extraction buffer include, but are not limited to, 0.01% v / v to 1% v / v, and 0.05% v / v to 0.5% v / v. The inventors have observed that the use of gelatin in combination with ammonium phosphate aided in the release of biological material from the non-living material present in a fecal sample, accordingly, in some embodiments, a cell extraction buffer includes gelatin, ammonium phosphate, or both gelatin and ammonium phosphate.

[0080] A cell extraction buffer can also include other components, including but not limited to a nonionic surfactant and emulsifier. Examples of a nonionic surfactant and emulsifier include, but are not limited to, polysorbate 80 (available under the trade name TWEEN- 80®), Tween 60, and Nonident P40. A polysorbate 80 can be present at a concentration of at least 0.001%, at least 0.005%, or at least 0.01%, and at a concentration of no greater than 0.1%, no greater than 0.05%, or no greater than 0.01%.

[0081] The fecal sample in the container can be subjected to conditions that homogenize the sample. In one embodiment, such conditions can be obtained through use of a paddle blender, also known in the art as a stomacher. Typically, the sample is homogenized until it is a slurry. Force can then be applied to the slurry to push the liquid component and biological material through the linear filter while preventing passage of the non-living material. The liquid component containing biological material is removed and the nonliving material (the filtered material) resuspended in a diluent, such as a saline solution. Saline containing solutions useful in maintaining the viability of biological material present in a fecal sample are known, and in one embodiment includes phosphate buffered saline atpH 7.0. This can be subjected to conditions that homogenize the sample, and force applied to the resulting slurry to push the liquid component and biological material through the linear fdter while preventing passage of the non-living material. The second liquid component containing biological material is removed and combined in a container suitable for centrifugation. The container can be purged with nitrogen gas to maintain conditions for anaerobes. The force of a centrifugation step can be at any speed that causes the separation of biological material from liquid but does not cause loss of therapeutic efficacy of the composition in 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 biological 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. The washing procedure of centrifugation followed by removal of the supernatant and resuspending the pellet in a reduced volume of diluent is repeated at least twice for a total of four washes.

[0082] The final pellet resulting from either the pressurized filter press method or the linear filtration method is resuspended in a diluent. The diluent used can be a volume that is no greater than 50% of the starting volume (e.g., the volume present when the fecal sample and cell extraction buffer are combined), no greater than 40% of the starting volume, no greater than 30% of the starting volume, no greater than 20% of the starting volume, or no greater than 10% of the starting volume. The final volume is typically dictated by the final numbers of bacteria desired after processing as described herein (e.g., adding a cryoprotectant or freeze drying). Typically, resuspending the final pellet in an aqueous composition results in a final slurry. The final slurry can be referred to herein as a fecal extract.

[0083] Cell numbers in a fecal extract can be determined. Methods for determining cell numbers include any combination of the Bacteria Counting Kit (ThermoFisher, Waltham, MA), an epifluorescence microscope, equipped with a 470-490 nm excitation filter and 500 nm cut- on long pass emission filter, and a Petroff Hauser counting chamber. A membrane integrity assay can be used to directly enumerate microbes present in a diluted sample and to calculate the percentage of microbes with intact membranes, thereby indicating potential viability. An example of a membrane assay is the BacLight live / dead membrane integrity assay (ThermoFisher, Waltham, MA) and a microscope and counting chamber.

[0084] 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.

[0085] 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 thecomposition, 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.

[0086] 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. In one embodiment, the freeze-dried composition is reconstituted. The subject can be anyone in need thereof. In some embodiments, the subject is an individual that has a disease or condition such as cirrhosis, HE, has a risk of HE, or a combination thereof. A subject can be pediatric or adult. A 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. In one embodiment, a composition includes a high acid-producing fecal extract microbiota that produces acid from a polysaccharide substrate, such as lactulose and lactitol. In one embodiment, a composition includes a high acid-producing fecal extract. In some embodiments, a microbiota used in a method of the present disclosure produces acid at a level greater than 25% of the population, a level greater than 50% of the population, or a level greater than 75% of the population.

[0087] In one embodiment, a subject is an individual having a lower microbiota content of microbes producing acidic compounds. It is expected that a subject having a lower microbiota content of microbes producing acidic compounds is more likely to benefit from the methods described herein. A subject having a lower microbiota content of microbes producing acidic compounds can be identified by measuring the ability of the subject’s biological material to produce acids in response to a substrate. Methods for this type of measuring are the same as described herein for measuring a donor’s microbiota for acid production.

[0088] In one embodiment, a subject is expected to be more likely to benefit from the methods described herein if their microbiota is producing acid at a level less than 25% of the population, a level less than 50% of the population, or a level less than 75% of the population. Accordingly, in some embodiments a subject that is the recipient of acomposition described herein has a microbiota that produces acid at a level less than 25% of the population, a level less than 50% of the population, or a level less than 75% of the population. A microbiota producing acid at a level less than 25% of the population, a level less than 50% of the population, or a level less than 75% of the population is referred to herein as a low acid-producing microbiota. When determining if a microbiota of a subject produces acid at a level less than 25% of the population, a level less than 50% of the population, or a level less than 75% of the population, microbiota from at least eight individuals can be compared. In one embodiment, the individuals have cirrhosis, HE, or are at risk of HE.

[0089] 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 / or colon. For instance, administration may be into an upper gastrointestinal tract, as well as a lower gastrointestinal tract, e.g., the terminal ileum, cecum, colonic areas containing diverticulosis, or rectum. In one embodiment the administering may be oral, such as by a capsule or a tablet. In one embodiment the administering may be by intubation, such as by nasogastric tube or post-pyloric nasojejunal feeding 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. In some embodiments, a desired effect is the reduction of a symptom and / or a sign of HE, or a reduction of the incidence of HE.

[0090] In one embodiment an “effective amount” is an amount effective to alleviate one or more symptoms and / or signs of HE or a reduction of the incidence of HE. In some embodiments, an effective amount is an amount that is sufficient to result in a reduction in a symptom and / or sign associated with HE and / or incidence of HE. 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 measuredsign 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 5xlO10cells, at least 5.5xlO10cells, at least 6xlO10cells, at least 6.5xlO10cells, at least 7xlO10cells, at least 7.5xlO10cells, at least 8xlO10cells, at least 8.5xlO10cells, at least 9xlO10cells, at least 9.5xlO10cells, at least IxlO11cells, at least 2.5xlOncells, at least 3xlOncells, at least 3.5x1011cells, at least 4xlOncells, at least 4.5xlOncells, at least 5xlOncells, at least 5.5xlOncells, at least 6xlOncells, at least 6.5xlOncells, at least 7xlOucells, at least 7.5xlOncells, at least 8xlOncells, at least 8.5xlOncells, at least 9xlOncells, at least 9.5x1011cells, at least IxlO12cells, at least 1.5xl012cells, at least 2xl012cells, at least 2.5xl012cells, at least 3xl012cells, at least 3.5xl012cells, at least 4xl012cells, at least 4.5xl012cells, at least 5xl012cells, at least 5.5xl012cells, at least 6xl012cells, at least 6.5xl012cells, at least 7xl012cells, at least 7.5xl012cells, or at least 8xl012cells administered to the subject. In one embodiment, an effective amount is an amount sufficient to result in at least 5xlO10cells, at least 5.5xlO10cells, at least 6xlO10cells, at least 6.5xlO10cells, at least 7xlO10cells, at least 7.5xlO10cells, at least 8xlO10cells, at least 8.5xlO10cells, at least 9xlO10cells, at least 9.5xlO10cells, at leastIxlO11cells, at least 2.5xlOncells, at least 3xlOncells, at least 3.5xlOncells, at least4xlOncells, at least 4.5xlOncells, at least 5xlOncells, at least 5.5xlOncells, at least6xlOncells, at least 6.5xlOucells, at least 7xlOncells, at least 7.5xlOncells, at least8xlOncells, at least 8.5xlOncells, at least 9xlOncells, at least 9.5xlOncells, at leastIxlO12cells, at least 1.5xl012cells, at least 2xl012cells, at least 2.5xl012cells, at least3xl012cells, at least 3.5xl012cells, at least 4xl012cells, at least 4.5xl012cells, at least5xl012cells, at least 5.5xl012cells, at least 6xl012cells, at least 6.5xl012cells, at least7xl012cells, at least 7.5xl012cells, or at least 8xl012cells delivered to the desired location in the gastrointestinal tract.

[0091] In some embodiments, an initial high dosage is followed by one or more lower maintenance doses. An initial high dosage can include an amount sufficient to result in at least 5xlO10cells, at least 5.5xlO10cells, at least 6xlO10cells, at least 6.5xlO10cells, at least7xlO10cells, at least 7.5xlO10cells, at least 8xlO10cells, at least 8.5xlO10cells, at least9xlO10cells, at least 9.5xlO10cells, at least IxlO11cells, at least 2.5xlOncells, at least3xlOncells, at least 3.5xlOucells, at least 4xlOncells, at least 4.5xlOncells, at least5xl Oncells, at least 5.5xlOncells, at least 6xlOncells, at least 6.5xlOncells, at least7xlOncells, at least 7.5xlOncells, at least 8xlOncells, at least 8.5xlOncells, at least9xlOncells, at least 9.5xlOncells, at least IxlO12cells, at least 1.5xl012cells, at least2xl012cells, at least 2.5xl012cells, at least 3xlO12cells, at least 3.5xlO12cells, at least4xl012cells, at least 4.5xl012cells, at least 5xlO12cells, at least 5.5xlO12cells, at least6xl012cells, at least 6.5xl012cells, at least 7xl012cells, at least 7.5xl012cells, or at least8xl012cells administered to the subject or delivered to the desired location in the gastrointestinal tract. 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.

[0092] The inventors were surprised to find that repeated administration of a composition described herein is desirable to achieve engraftment of the introduced microbiota in the gastrointestinal tract. Administration can occur for at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, at least 10 doses, at least 11 doses, at least 12 doses, at least 13 doses, or at least 14 doses. Administration can occur one or more times per day for one or more days. Administration can occur one or more times per week, such as weekly, two times a week, three times a week, four times a week, five times a week, six times a week, or every day of a week. Administration can occur over one or more weeks. In one embodiment, a composition described herein is administered for at least one week, at least two weeks, at least three weeks, or at least four weeks, where the subject receives the composition every other day. In one embodiment, a composition described herein is administered at least once each day for at least one week, at least two weeks, at least three weeks, or at least four weeks.

[0093] In one embodiment, a method of the present disclosure includes treating a disease or condition in a subject in need of treatment. The disease or condition can be cirrhosis or HE, risk of HE, or a combination thereof. 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 manifestssigns 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. Examples of risk factors for HE include cirrhosis, alcohol intoxication, infections, sedatives, surgery, complications from transjugular intrahepatic portosystemic shunt placement, dehydration, and electrolyte imbalances. Treatment can be performed before, during, or after the occurrence of a disease or condition 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.

[0094] The method can further include administration of one or more fermentable non-absorbable polysaccharides to the subject. Examples of suitable polysaccharides include but are not limited to di saccharides, such as lactulose and lactitol. Without intending to be limiting, fermentable non-absorbable polysaccharides are believed to aid decreasing levels of ammonia in a gastrointestinal tract by reducing ammonia to ammonium. Optionally, the method can further include administration of one or more antibiotics. Examples of suitable antibiotics include rifaximin. Without intending to be limiting, the use of antibiotics is helpful in lowering ammonia production in the intestine by certain groups of bacteria, such as members of the Enterobacteraceae family.

[0095] 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.

[0096] In one embodiment, a method of the present disclosure includes transplanting a microbiota from a donor to a recipient.31

[0097] In one embodiment, a method of the present disclosure includes increasing the relative abundance of certain prokaryotic cells in a recipient, such as a recipient’s intestine, after administration of a composition described herein. In one embodiment, a method of the present disclosure includes increasing the relative abundance of members of the order Bifidobacteriales, such as members of the family Bifidobacteriaceae, including members of the genus Bifidobacterium, in a recipient's intestine. In one embodiment, a method of the present disclosure includes increasing the relative abundance of members of the family Lachnospiraceae, such as members of the genera Blautia, Coprococcus, Dorea, Lachnospira, Oribacterium, Roseburia, or a combination thereof, in a recipient's intestine. In one embodiment, a method of the present disclosure includes increasing the relative abundance of members of the family Ruminococcaceae, such as members of the genera Ruminococcus and / or Faecalibacterium, in a recipient's intestine. In one embodiment, a method of the present disclosure includes increasing the relative abundance of members of the family Clostridiaceae, the family Rikenellaceae , the family Bacteroidaceae, the family Desulfovibrionaceae , or a combination thereof, in a recipient's intestine. In one embodiment, the relative abundance in a recipient's colon after the administration may be increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 a composition described herein.

[0098] In one embodiment, a method of the present disclosure includes decreasing the relative abundance of certain prokaryotic cells in a recipient, such as a in a recipient’s colon, after administration of a composition described herein. In one embodiment, a method of the present disclosure includes decreasing the relative abundance of members of the family Lactobacillaceae in a recipient's colon. In one embodiment, a method of the present disclosure includes decreasing the relative abundance of members of the family Prevotellaceae (such as members of the genus Prevotelid), the family Aerococcaceae, the family Fusobacteriaceae, the family Coriobacteriaeae, or a combination thereof, in a receipient’s colon. In one embodiment, the relative abundance in a recipient's colon after the administration may be decreased by at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, 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 a composition described herein.

[0099] In one embodiment, the existing microbiota present in a subject 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 in a subject are known and routine.

[0100] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. The meaning of some terms used herein are set forth herein.

[0101] As used herein, "relative abundance" refers to the number of members of a phylum, class, order, family, genus, or species compared to the number of members of all other taxa in a recipient's colon or stool. Such a comparison can be expressed as a percent. For instance, the number of members of a phylum, class, order, family, genus, or species in a recipient’s colon after an administration can be increased or decreased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, compared to the recipient’s colon or stool before administration of a composition described herein. The change in the abundance may be determined at, for instance, 3 days, 10 days, 15 days, or 25 days after the administration of a composition described herein.

[0102] As used herein, a short-chain fatty acid (SCFA) refers to fatty acids of two to six carbon atoms. Examples of SCFAs include, but are not limited to, acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, 2-ethylbutyric acid, and 4- methylvaleric acid.

[0103] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0104] 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."

[0105] The words "preferred" and "preferably" refer to embodiments of the disclousre 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.

[0106] 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."

[0107] 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.

[0108] 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.

[0109] 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 inat 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] The invention is defined in the claims. However, below there is provided a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0114] Exemplary Aspects

[0115] Aspect 1. A method for treating a patient, comprising: administering to the patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier, wherein the patient has cirrhosis, has hepatic encephalopathy (HE), or is at risk for a recurrent episode of HE.

[0116] Aspect 2. The method of any of Aspects 1 or 3-26, wherein the treatment further comprises administration of a non-absorbable polysaccharide.

[0117] Aspect 3. The method of any of any of Aspects 1-2 or 4-26, wherein the polysaccharide is a disaccharide.

[0118] Aspect 4. The method of any of Aspects 1-3 or 5-26, wherein the disaccharide comprises lactulose or lactitol.

[0119] Aspect 5. The method of any of Aspects 1-4 or 6-26, wherein the fecal extract is a high acid-producing fecal extract.

[0120] Aspect 6. The method of any of Aspects 1-5 or 7-26, wherein the composition is administered with at least 7 doses.

[0121] Aspect 7. The method of any of Aspects 1-6 or 8-26, wherein the composition is administered with at least 14 doses.

[0122] Aspect 8. The method of any of Aspects 1-7 or 9-26, wherein the composition is administered over a week, over two weeks or over three weeks,

[0123] Aspect 9. The method of any of Aspects 1-8 or 10-26, wherein the composition comprises from IxlO10to 5xl012microbes.

[0124] Aspect 10. The method of any of Aspects 1-9 or 11-26, wherein the relative abundance of one or more members of the order Bifidobacteriales, one or more members of the family Lachnospiraceae, one or more members of the family Ruminococcaceae, one or more members of the family Clostridiaceae, one or more members of the family Rikenellaceae, one or more members of the family Bacteroidaceae, one or more members of the familyDesulfovibrionaceae, or a combination thereof, is increased in the patient after the administering.

[0125] Aspect 11. The method of any of Aspects 1-10 or 12-26, wherein the more members of the order Bifidobacteriales comprises one or more members of the family Bifidobacteriaceae.

[0126] Aspect 12. The method of any of Aspects 1-11 or 13-26, wherein the one or more members of the family Bifidobacteriaceae comprises one or more members of the genus Bifidobacterium.

[0127] Aspect 13. The method of any of Aspects 1-12 or 14-26, wherein the more members of the family Lachnospiraceae comprises one or more members of the genus Blautia, the genus Coprococcus, the genus Dorea, the genus Lachnospira, the genus Oribacterium, the genus Roseburia, or a combination thereof.

[0128] Aspect 14. The method of any of Aspects 1-13 or 15-26, wherein the more members of the family Ruminococcaceae comprises one or more members of the genus Ruminococcus, one or more members of the genus Faecalibacterium, or a combination thereof.

[0129] Aspect 15. The method of any of Aspects 1-14 or 16-26, wherein the increase comprises an increase in the recipient’s intestine.

[0130] Aspect 16. The method of any of Aspects 1-15 or 17-26, wherein the relative abundance is increased by at least 1%, at least 5%, or at least 10%.

[0131] Aspect 17. The method of any of Aspects 1-16 or 18-26, wherein the increase is an increase compared to the patient before the administering.

[0132] Aspect 18. The method of any of Aspects 1-17 or 19-26, wherein the relative abundance of one or more members of the family Lactobacillaceae, one or more members of the family Prevotellaceae, one or more member of the family Aerococcaceae, one or more member of the family Fusobacteriaceae, one or more member of the family Coriobacteriaeae, or a combination thereof, is decreased in the patient after the administering.

[0133] Aspect 19. The method of any of Aspects 1-18 or 20-26, wherein the one or more members of the family one or more members of the family Prevotellaceae comprises one or more members of the genus Prevotella.

[0134] Aspect 20. The method of any of Aspects 1-19 or 21-26, wherein the decrease comprises an increase in the recipient’s intestine.

[0135] Aspect 21. The method of any of Aspects 1-20 or 22-26, wherein the relative abundance is decreased by at least 1%, at least 5%, or at least 10%.

[0136] Aspect 22. The method of any of Aspects 1-21 or 23-26, wherein the decrease is a decrease compared to the patient before the administering.

[0137] Aspect 23. The method of any of Aspects 1-22 or 24-26, wherein the administering comprises at least one rectal administration or at least one oral administration.

[0138] Aspect 24. The method of any of Aspects 1-23 or 25-26, wherein the at least one rectal administration comprises an enema.

[0139] Aspect 25. The method of any of Aspects 1-24 or 26, wherein the oral administration comprises a solid formulation of the fecal extract.

[0140] Aspect 26. The method of any of Aspects 1-25, wherein the solid formulation comprises a capsule or pill.

[0141] Aspect 27. A method for increasing relative abundance of at least one microbe in a patient, comprising: administering to the patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier, wherein the patient has cirrhosis, has hepatic encephalopathy (HE), or is at risk for a recurrent episode of HE, and wherein the at least one microbe is one or more members of the order Bifidobacteriales, one or more members of the family Lachnospiraceae, one or more members of the family Ruminococcaceae, one or more members of the family Clostridiaceae, one or more members of the family Rikenellaceae, one or more members of the family Bacteroidaceae, one or more members of the family Desulfovibrionaceae, or a combination thereof, and the relative abundance is increased in the patient after the administering.

[0142] Aspect 28. The method of any of Aspects 27 or 29-34, wherein the more members of the order Bifidobacteriales comprises one or more members of the family Bifidobacteriaceae.

[0143] Aspect 29. The method of any of Aspects 27-28 or 30-34, wherein the one or more members of the family Bifidobacteriaceae comprises one or more members of the genus Bifidobacterium.

[0144] Aspect 30. The method of any of Aspects 27-29 or 31-34, wherein the more members of the family Lachnospiraceae comprises one or more members of the genus Blautia, the genus Coprococcus, the genus Dorea, the genus Lachnospira, the genus Oribacterium, the genus Roseburia, or a combination thereof.

[0145] Aspect 31. The method of any of Aspects 27-30 or 32-34, wherein the more members of the family Ruminococcaceae comprises one or more members of the genus Ruminococcus, one or more members of the genus Faecalibacterium, or a combination thereof.

[0146] Aspect 32. The method of any of Aspects 27-31 or 33-34, wherein the increase comprises an increase in the recipient’s colon.

[0147] Aspect 33. The method of any of Aspects 27-32 or 34, wherein the relative abundance is increased by at least 1%, at least 5%, or at least 10%.

[0148] Aspect 34. The method of any of Aspects 27-33, wherein the increase is an increase compared to the patient before the administering.

[0149] Aspect 35. A method for decreasing relative abundance of at least one microbe in a patient, comprising: administering to the patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier, wherein the patient has cirrhosis, has hepatic encephalopathy (HE), or is at risk for a recurrent episode of HE, and wherein the at least one microbe is one or more members of the family Lactobacillaceae, one or more members of the family Prevotellaceae, one or more member of the family Aerococcaceae, one or more member of the family Fusobacteriaceae, one or more member of the family Coriobacteriaeae, or a combination thereof, and the relative abundance is decreased in the patient after the administering.

[0150] Aspect 36. The method of any of Aspects 35 or 37-39, wherein the one or more members of the family one or more members of the family Prevotellaceae comprises one or more members of the genus Prevotella.

[0151] Aspect 37. The method of any of Aspects 35-36 or 38-39, wherein the decrease comprises an increase in the recipient’s intestine.

[0152] Aspect 38. The method of any of Aspects 35-37 or 38, wherein the relative abundance is decreased by at least 1%, at least 5%, or at least 10%.

[0153] Aspect 39. The method of any of Aspects 35-38, wherein the decrease is a decrease compared to the patient before the administering.

[0154] Aspect 40. The method of any of Aspects 27-39 or 41-51, wherein the method further comprises administration of a non-absorbable polysaccharide.

[0155] Aspect 41. The method of any of Aspects 27-40 or 42-51, wherein the polysaccharide is a disaccharide.

[0156] Aspect 42. The method of any of Aspects 27-41 or 43-51, wherein the disaccharide comprises lactulose or lactitol.

[0157] Aspect 43. The method of any of Aspects 27-42 or 44-51, wherein the fecal extract is a high acid-producing fecal extract.

[0158] Aspect 44. The method of any of Aspects 27-43 or 45-51, wherein the composition is administered with at least 7 doses.

[0159] Aspect 45. The method of any of Aspects 27-44 or 46-51, wherein the composition is administered with at least 14 doses.

[0160] Aspect 46. The method of any of Aspects 27-45 or 47-51, wherein the composition is administered over a week, over two weeks or over three weeks,

[0161] Aspect 47. The method of any of Aspects 27-46 or 48-51, wherein the composition comprises from IxlO10to 5xl012microbes.

[0162] Aspect 48. The method of any of Aspects 27-47 or 49-51 , wherein the administering comprises at least one rectal administration or at least one oral administration.

[0163] Aspect 49. The method of any of Aspects 27-48 or 50-51, wherein the at least one rectal administration comprises an enema.

[0164] Aspect 50. The method of any of Aspects 27-51 or 51, wherein the oral administration comprises a solid formulation of the fecal extract.

[0165] Aspect 51. The method of any of Aspects 27-50, wherein the solid formulation comprises a capsule or pill.

[0166] EXAMPLES

[0167] 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.

[0168] Example 1

[0169] The impact of different FMT doses as oral, enema, or both, in patients with cirrhosis and HE already on lactulose and rifaximin.

[0170] Background and Aims: Patients with cirrhosis and hepatic encephalopathy (HE) already on lactulose and rifaximin have few options to prevent further HE. Fecal microbiota transplant (FMT) was helpful in small studies, but route / dosage remains unclear. Aim: to compare 2 FMT routes (oral capsule & enema) with dose-ranging in HE prevention.

[0171] Method: A double-blind, placebo controlled RCT with 2 modes of FMT administration in cirrhosis patients on lactulose & rifaximin was completed under FDA IND. 4 groups (Gpl: both oral+enema active, Gp2:oral active + placebo enema, Gp3:oral placebo+ active enema, Gp4: both oral+enema placebo) received oral+enema FMT at baseline & a 3rd oral dose at day 30. 2 donors were used for FMT products. Patients with recent infections, other antibiotics, MELD>22, transplant & immunosuppression were excluded. We needed 60 total (15 / gp) for >90% power across groups at 6 months on ITT analysis. The primaryoutcome was safety, especially HE recurrence defined as >Grade2 on West-Haven criteria. Secondary outcomes were other adverse events, changes in infections / cirrhosis severity / cognition (PHES / Stroop) & patient-reported outcomes (Sickness Impact Profile (SIP), total / physical / psych; high=worse). Regression for HE-recurrence was performed.

[0172] Results: 60 pts (15 per group) with similar MELD (13, 12, 12, 12, p=0.5) & age(65, 63, 61, 63, p=0.6) on lactulose+rifaximin were included. Last prior HE episode duration was similar (8-13 mths prior, p=0.51). Baseline cognition, SIP, & cirrhosis severity were similar between gps.

[0173] Course: All were followed till death or 6 months in-person / remotely. 6 pts dropped out (2 Gpl patients died after falls in rehab), 1 Gp2 died after a seizure), 1 Gp2 & 2 Gp 4 did not return for visits), but ITT analysis was performed. 2 pts in Gp 2 & 1 in Gp 1 did not receive the day30 active dose. 5 patients missed some visits due to COVID-19, but were seen remotely. 4 pts developed infections (SBP, cholecystitis & 2 cellulitis), all unrelated to FMT.

[0174] Primary outcome: HE recurrence was highest in Gp 4 (both placebo: 40%) vs other (Gp 1 : 13%, Gp 2: 13%, Gp3 :0%, p=0.03). Both Gp Ipatients who died had HE prior to death.

[0175] Secondary outcomes: liver-related hospitalizations tended higher in Gp 4 vs rest (47% vs Gpsl-3: 7- 20%, p=0.12). MELD / PHES / Stroop did not change, but SIP total / physical & psych improved with FMT (p=0.003) on RMANOVA. Regression in all pts: HE recurrence was related to dose number (OR 0.27, 95% CI 0.10-0.79, p=0.02), male sex (OR 0.16, 0.03-0.89, p=0.04) & Physical SIP (OR 1.05, 1.01- 1.10, p=0.05). Dose / route / donor: Within FMT recipients neither dose, route, nor FMT donor affected HE recurrence.

[0176] Conclusion: HE recurrence was significantly lower in FMT (enema or oral capsule) recipients versus placebo in a phase 2 placebo-controlled, double-blind, dose-ranging RCT in pts with cirrhosis and HE on lactulose and rifaximin. The FMT route, donor, and dose range did not affect HE recurrence.

[0177] Methods:

[0178] A double-blind, placebo-controlled randomized clinical trial with 2 modes of administration and up to 3 FMT doses in patients with cirrhosis and HE on lactulose and rifaximin was completed under FDA-approved IND (FIG. 1A and 1C. All subjects underwent informed consent and evaluation for eligibility criteria, including baseline minimental status examination (MMSE). Our main inclusion criteria were age>21 years, confirmed diagnosis of cirrhosis, on treatment for with lactulose and rifaximin, and able to consent. Moreover, we required the subject to notify us of individual(s) whom we may contact to determine if they had an HE episode during the study.

[0179] Cirrhosis was diagnosed in a patient with chronic liver disease by either liver biopsy; radiologic evidence of varices, cirrhosis or portal hypertension; laboratory evidence of platelet count <100,000 or AST / ALT ratio>l; endoscopic evidence of varices or portal gastropathy; or firbroscan values suggestive of cirrhosis. Ability to give written, informed consent was demonstrated by MMSE>25 at the time of consenting. Women of childbearing potential had to agree to use effective contraception for the duration of the study and for 10 days prior and 30 days after the study. Women of childbearing age had to test negative in a pregnancy test.

[0180] Exclusion criteria were applied: MELD score >22; WBC count <1000 cells / mm3; platelet count < 25,000 / mm3; TIPS in place for less than a month; currently on antibiotics apart from rifaximin; infection at the time of the FMT (diagnosed by blood culture positivity, urinalysis, paracentesis as applicable); hospitalization for any non-elective cause within the last 1 month; patients who are aged >75 years; patients who were pregnant or nursing (checked using a urine pregnancy test); patients who were incarcerated; patients who were incapable of giving their own informed consent; patients on renal replacement therapy; patients with untreated, in-situ colorectal cancer; patients with a history of chronic intrinsic GI diseases such as inflammatory bowel disease (ulcerative colitis, Crohn’s disease or microscopic colitis), eosinophilic gastroenteritis, or celiac disease; major gastro-intestinal or intra-abdominal surgery in the last three months; and patients who had received FMT within last 6 months.

[0181] Also excluded were patients who were immuno-compromised due to: HIV infection (any CD4 count); inherited / primary immune disorders; current or recent (<3 months) treatment with anti-neoplastic agent; or current or recent (<3 months) treatment with any immunosuppressant medications including but not limited to monoclonal antibodies to B and T cells, anti-TNF agents, glucocorticoids, antimetabolites (azathioprine, 6- mercaptopurine), calcineurin inhibitors (tacrolimus, cyclosporine), and mycophenolate mofetil. Subjects who were otherwise immunocompetent and had discontinued any immunosuppressant medications 3 or more months prior to enrollment were eligible to enroll.

[0182] Enema-related exclusion criteria were a platelet count<25,000, and grade IV hemorrhoids. Safety-related exclusion criteria were dysphagia; history of aspiration, gastroparesis, or intestinal obstruction; ongoing antibiotic use (except for Rifaximin); severe anaphylactic food allergy; allergy to ingredients Generally Recognized As Safe in the FMT capsules (glycerol, sodium chloride, hypromellose, gellan gum, titanium dioxide, theobroma oil); adverse event attributable to prior FMT; ASA Class IV or V; pregnant or nursing patients; acute illness or fever within 48 hours of the day of planned FMT; immunocompromised due to medical conditions; probiotics use within the last 48 hours of the day of planned FMT; and any condition that the physician investigators deemed unsafe, including other conditions or medications that the investigator determined would put the participant at greater risk from FMT.

[0183] We did not require patients to have MHE for enrollment. All subjects swallowed a placebo capsule before proceeding further to screen for pill dysphagia.

[0184] Once eligibility was confirmed, patients were randomized into 4 groups using a random number generator by the investigational pharmacy. Subjects received 3 administrations (60ml enema and 5 capsules at baseline and 5 more capsules at day 30, FIG. 1C. Group 1 received active enema and capsules at baseline and active capsules at day 30, Group 2 received placebo enema and active capsules at baseline and active capsules at day 30, Group 3 received active enema at baseline and placebo capsules at both baseline and day 30, while Group 4 received placebo enema and capsules.

[0185] Stool donations from two healthy donors were used to manufacture the FMT products. The two formulations of FMT used in the study were liquid / frozen fecal microbiota (compound MTP-101LR) and encapsulated, freeze-dried microbiota (compound MTP-101C). Both compounds contain fecal microbiota extracted from the stool of healthy human donors using Good Manufacturing Practice protocols detailed in IND 15071 (Microbiota Therapeutics Program; Principal Investigator: Alexander Khoruts, MD). The stool was donated in a supervised bathroom from two qualified donors, placed on ice, and transported with documented chain of custody to the manufacturing facility, where it was processed within two hours of arrival. The microbiota extraction processing steps included blending of stool in phosphate buffered saline and purification via repeated filtration and centrifugation steps, as described previously (see 13). Liquid / frozen suspension of fecal microbiota was cryopreserved with 10% glycerol in phosphate buffered saline and packaged in cryobags (^ 5.0 x 1011and2.0 x 1012). Alternatively, the fecal microbiota was lyopreserved with trehalose and double encapsulated into DRcaps (Lonza, Morristown, N ), as described previously (see 14). Each capsule contained1.0 x 1011and2.0 x 10A11bacteria.[00186J The donors were qualified using detailed questionnaires, in-person clinical history and physical exams, and laboratory testing. The screening process, which was maintained on a continuous basis, ensured that the donors qualified as blood donors and were also in excellent health, including: had no history of antibiotic exposure for at least 6 months; took no prescription medications; had no gastrointestinal disorders, e.g., irritable bowel syndrome, inflammatory bowel disease, food intolerances, etc.; had no allergies or atopic conditions; had no metabolic disorders or any individual diagnostic criteria for metabolic syndrome (e.g., increased blood pressure, increased body mass index); had no neurologic or psychiatric disorders; were not currently using illicit drugs as defined by Federal law; were not pregnant or lactating; and did not have increased risk factors for MDRO colonization (including healthcare worker at a hospital, ambulatory clinic, nursing home, hospice, or a similar facility; person who has been hospitalized at a long-term care facility; person who regularly attends outpatient medical or surgical clinics; person who has everengaged in medical tourism; and person who has been taking care of sick people at home or having contact with sick patients in the hospital or care facilities).

[0187] Metabolic fitness was documented by body mass < 25 kg / m2, normal hip / waist ratio, normal blood pressure, normal lipid panel, liver function tests, and fasting glucose. Negative serologic infectious disease testing was documented bracketing a 3-month period of stool donations and negative serologic testing for infectious disease was documented at least 15 days following the last donation. Required negative donor testing included NAAT testing for HIV, HAV, HBV, HCV, CMV, and EBV; and serologic testing for syphilis. Every batch of stool was tested for enteric pathogens and multidrug resistant organisms (MDRO). Enteric pathogen testing included 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. MDRO testing included Methicillin-Resistant Staphylococcus aureus (MRSA), Extended Spectrum Beta-Lactamase-resistant bacteria (ESBL), vancomycin-resistant Enterococci (VRE), and Carbapenem-Resistant Enterobacterales (CRE).

[0188] The fecal microbiota compounds were kept at quarantined -80°C until reviewed and released by the facility Quality Assurance Department. The compounds were sent on dry ice to the Investigational Drug Pharmacy in Virginia. The stool donor program has been approved by the University of Minnesota Institutional Review Board and monitored by auditors employed by the Clinical and Translational Science Institute at the University of Minnesota. Two donors participated in this trial: donors #67 and #70, respectively. Both donors were male, bom vaginally, and were breast fed. Donor #67 has maintained a longterm vegan diet, whereas donor #70 was an omnivore.

[0189] All FMT material (enema or capsules) came from the same one donor for individual recipients. Both administration routes delivered 2.5 x 1012bacteria and both donors had higher content of short-chain fatty acid (SCFA)-producing Lachnospiraceae and Ruminococcaceae relative to the recipients. Enema was provided via a rectal tube and was retained >30 minutes under observation. We performed dietary recall, sample collection,adverse event (AE) analysis, quality of life (QOL) using Sickness Impact profile (SIP; psychosocial, and physical components), and cognitive testing using psychometric hepatic encephalopathy score (PHES) and EncephalApp Stroop. Adverse events were evaluated, and samples collected at each visit, while QOL and cognitive testing were performed at baseline, 2 months (1 month after all FMT / placebo administrations) and at study end at 6 months.

[0190] Cognitive testing: EncephalApp Stroop is a validated smartphone App version of the Stroop test (see 35). This involves presentation of an easier “Off’ Stage in which subjects must identify the color of the pound-signs presented on the phone and a more difficult “On” stage in which subjects must correctly identify the color of a discordant word presented. For example, the word “GREEN” will be presented in blue colored letters and the correct response would be blue and not green. The App has two practice runs and requires 5 correct runs in the Off and On Stage. The total time required for 5 correct On and 5 correct Off stage runs is the “OffTime+OnTime” which is of relevance in HE (see 2). A low OffTime+OnTime indicates better cognition (see 35). Psychometric hepatic encephalopathy score (PHES) is a validated five test paper-pencil battery which tests visuo- motor coordination, psychomotor speed and reaction time (see 36). It consists of the number connection test- A, number connection test-B, digit symbol test, serial dotting test and line tracing test (has two components; time and errors). Based on population control values, the standard deviations are calculated for each sub-test and the total is added to give one value (see 2). A low score indicates better cognition.

[0191] QOL: Sickness impact profile (SIP) is a 136-question survey that inquires about health- related concerns over the last 24 hours. It has a psychosocial and physical domain, and a high score indicates poor health-related quality of life (see 37).

[0192] HE-recurrence and infections were specific AEs of interest. Specifically, infections possibly or potentially related to FMT needed reporting even if not hospitalized. HE recurrence was defined as grade ^2 on the West-Haven criteria that needed therapy change or treatment (see 2). All SAEs were reported to the central data monitoring committee (DMC) of the VHA per protocol. All subjects were followed till 6 months oruntil death or transplant. The trial was registered at clinicaltrials.gov (NCT03796598) before the first enrollment.

[0193] Statistical analysis: Groups were compared at baseline for clinical, demographic, and cognitive / QOL variables using parametric and non-parametric tests as appropriate. Within and between group analyses were performed using Chi-square and Fisher exact for nonparametric (HE episodes, hospitalizations, death / transplant), for continuous variables [MELD, ammonia, SIP and cognitive tests], and specialized bio-informatics analyses for the microbiota. We also performed repeated measures analyses for cognitive tests, and QOL one-way and linear mixed models for group and time interactions. For the effect size, we used the Cramer’s V (V) and the odds ratio (OR) for the 2X4 (all groups) and 2X2 (only placebo vs any FMTs) comparisons respectively.

[0194] Microbiome analysis: Donor microbiota was studied vis-a-vis recipients to determine engraftment using SourceTracker and EMU strain analysis (see 38). Linear discriminant analysis effect size (LefSe) was used to determine changes in microbiota between and within groups (see 39). Dose-response of FMT and specific donors were studied with respect to the primary outcome. Finally, logistic regression for HE-recurrences were performed. The last measurements were carried forward in those who missed appointments or had missing data. Statistical analyses were performed using the R software package, v4.3.2 while power calculations were performed using nQuery v9.3.1 assuming an a = 0.05 significance level.

[0195] Microbiome analysis from samples from patients and donors was performed using 16S rRNA gene sequence data using published techniques (see 15). The primary outcome was safety and tolerability related to FMT, especially HE-related recurrences. Secondary outcomes were all-cause and liver-related hospitalizations, changes in cognition and QOL, microbial changes and engraftment of donors into recipients.

[0196] Sample size: Based on prior studies with rectal FMT9, we found that standard of care patients had an 80% chance of hospitalizations (mostly HE) at 6 months, while it was 20% hospitalization in FMT-randomized patients. Given that we had used antibiotics for this study, we will assume that rectal FMT alone will have a 30% chance of hospitalization ratewithout antibiotics. We also assumed that oral FMT alone will have a similar HE-related event rate, and their combination will have a 10% HE-related event rate. Using a significance level of a=0.05, a sample size of 15 per group would give a power of 94%.

[0197] Results:

[0198] We considered 112 patients of whom 60 were randomized (FIG. 1C). Of the remaining 72 patients, 14 refused study participation, while the rest did not meet eligibility criteria (19 patients with MELD>22, 15 with unclear prior HE episodes, 15 on SBP prophylaxis or other chronic antibiotics, 7 with autoimmune diseases or immuno-suppressants, 6 with food allergies, 6 with low WBC or platelet counts, and 5 with inflammatory bowel diseases). As shown in Table 1, at baseline all groups were evenly matched with respect to baseline features.

[0199] Table 1 : Baseline Comparison between groups

[0200] Clinical course: The first patient was enrolled in December 2019 and the last patient was enrolled in June 2023. Enrollment was halted and in-person visits were curtailed during the pandemic from March 2020 through January 2021. Enrollment resumed February 2021. As shown in the FIG. 1C, all subjects were followed for 6 months unless they died (n=3). The visits were a combination of in-person or remote in part due to the pandemic-era restrictions and patients’ unwillingness to travel during those time periods. All initial interventions (enemas and capsules) for FMT vs placebo were administered per protocol and patients retained the enema for 30 minutes. All subjects apart from 2 patients in group 2 and 1 person in group 2 received their second active capsule dose at day 30. This was due to hospitalizations in the case of one patient each in groups 1 and 2 and due to COVID-19 restrictions in the second group 1 patient.

[0201] Primary outcome:

[0202] HE recurrences were seen in 10 (17%) of the 60 patients (FIG. 1C). Group 4 (all placebo) had the highest (6 of 15, 40%) compared to FMT groups (4 of 45, 8.8%, groups 1 and 2, n=2 each and 0 in group 3 FIG. 1C). All HE episodes were associated with hospitalizations as described herein. The median time from randomization to the first HE episodes were similar (Group 1, 1 month, Group 2, 1.5 months and group 4, 1.5 months). There were no other FMT -related SAEs noted, which the DMC agreed with. HE recurrences in placebo compared to any FMT was significantly higher (40% vs 8.8%,p 0.01 ). Using a Fisher’s Exact test, we found a significant difference among the four groups (p = 0.027). This difference persisted even when the doses of actual FMT received (since 3 patients did notreceive their 2ndFMT capsule dose) were compared, 40% (6 of 15) recurrence in placebo- only or zero FMTs, vs 0% (0 of 18) recurrence in those who received only one dose, and 17% (4 of 23) who received =?:2 FMT doses (p=0.004). Within the FMT-receiving groups, however, the route or dose and subsequent HE recurrence was not significant. V for the 2 x 4 table is 0.39 indicating a large effect size for all groups with FMT while for any FMT vs placebo the OR for HE recurrence with FMT is 0.146 (95% CI: 0.034, 0.628).[00203J Of the group 4 patients, 4 had one episode each (2 unclear precipitants, 1 cellulitis, 1 respiratory infection), while 2 had >1 episodes (one patient with 2 and one with 3 due to falls and cellulitis). Of the two group 1 patients with recurrence, one had pneumonia that precipitated it while another had a fall when they were not confused that resulted in hospitalization and HE during that hospitalization. Of the two Group 2 patients who developed HE, one patient had one episode after a fall without confusion that resulted in HE post-hospitalization, and one had 2 episodes due to cellulitis.

[0204] Secondary outcomes:

[0205] Ammonia levels and safety laboratories: No changes in MELD score or ammonia levels were seen throughout the study (Tables 2 and 3).

[0206] Table 2: Baseline comparison of those who experienced HE recurrence versus not in the entire cohort

[0207] Data are presented as mean ± SD or as N (%) unless mentioned otherwise. Comparisons performed using t-tests, Mann-Whitney tests, or Chi-square and Fisher exact tests as appropriate.

[0208] Table 3: Changes in safety laboratory values over time

[0209] Other hospitalizations and deaths: 22 patients were hospitalized at least once during the study. 4 patients died during the 6-month period; 1 each in groups 1 & 2 died after falls and were transferred to hospice. One patient in group 3 developed seizure recurrence (already had one before) and was found deceased at home and one patient in group 4 died to a fall as well. None were considered related to FMT by the DMC.

[0210] Six (40%) each from groups 1 (median 2.5 IQR 1-4 months) and 4 (median 1.5 IQR 1-2 months), and 5 (33%) each in groups 3 (median 3 IQR 2.5-3.5 months) and 2 (median 2 IQR 1-2 months post-randomization) (p=0.96) developed hospitalizations other than due to HE. Liver-related hospitalizations were seen in 2 patients (13%) in groups 1, 2 and 3, and 6 (40%) in group 4 (p=0.21). Both liver-related hospitalizations in groups 1 and 2 were from the two patients with HE. Both group 2 patients had HE-related hospitalizations. The two Group 3 patients with liver-related hospitalizations had SBP and portal-hypertensive gastropathy-related bleeding respectively that was considered unrelated to FMT. The SBP did not grow any organism. Liver-unrelated hospitalizations in four remaining group 1 patients were due to a fractured femur, fractured shoulder, fractured arm, and foot blisters respectively that were considered unrelated to FMT; none of these resulted in HE or further decompensation during the hospitalization. The three patients with liver-unrelated hospitalizations in group 2 were admitted due to a seizure, chest pain and cholecystitis respectively. Blood culture of the patient with cholecystitis grew resistant Acinetobacter and therefore not related to FMT (which excluded MDROs). The patients recovered and were followed successfully later. Of the three patients with liver-unrelated hospitalizations in group 3, two were due to chest pain and one due to generalized weakness.

[0211] Cognitive testing and QOL analysis: There were some improvements in SIP and PHES in groups 1 and 2. 2 patients each in groups 1 and 2, and 1 each in groups 3 and 4 did not perform month 2 cognitive testing. Within group RMANOVA showed improvement in PHES and Stroop in group 1 but not others (Tables 4 and 5). However, in the mixed effect model, there was no time group interaction for either PHES (p=0.36) or Stroop (p=0.78, FIG. 5) for baseline and month 2 timepoints. SIP psychosocial and physical domains significantly improved (lowered) in groups 1 and 2 within groups but not in other groups (Tables 4 and 5). On mixed-effects analysis, there was a significant time group interactionfor both psychosocial (p=0.003) and physical SIP (p=0.04, FIG. ID) for baseline and month 2 timepoints.

[0212] Table 4: Summary of Adverse Events throughout the study

[0213] Data presented as number of unique events and average CTCAE score in parentheses. None of these was considered related to FMT by the Central VA Data Monitoring Committee.

[0214] Table 5: Changes in cognitive performance and QOL between and within groups at 3 timepoints

[0215] Only SIP variables showed a significant time group interaction across the 3 timepoints between groups on linear mixed models; RMANOVA p values shown are within groups and ANOVA comparison p values shown are between groups.

[0216] Other AEs were relatively minor and did not require changing therapy. Proportion of liver- related hospitalizations were lowest in all FMT patients FMT versus placebo (13% vs 40%, p=0.02), while all-cause hospitalizations and death were similar.

[0217] Microbiome analysis:

[0218] Within groups: There was no difference at baseline on Shannon diversity (Table 1), which changed over time without a specific pattern (FIG. 2A). Microbial change patterns varied on LEfSe by group. In placebo, there was reduced Bifidobacteriaceae at day 60 versus baseline. All FMT groups individually either showed higher beneficial taxa post-FMT (Bifidobacteria and Subdoligranuhim post-FMT in group 1) or lower potential pathobionts (Enterobacteriaceae genera in group 2, Fusobacterium post-FMT in group 3).

[0219] Impact of the individual donors and engraftment (FIG. 6 and FIG. 7): Two separate donors (one vegan and one omnivore) were used. 31 subjects received material from the omnivore while 14 patients received from the vegan donor. Engraftment rates in recipient stool of donor microbiota were highest in group 1, intermediate in group 2 and lowest in group 3 (FIG. 4A). This pattern continued over the 6 months (FIG. 4B). Low baseline Lachnospiraceae relative abundance was associated with higher engraftment post-FMT (FIG. 4C). Every person had variable engraftment patternsd (FIG. 4D-E).

[0220] Comparisons between donors: Engraftment rates between those who received material from vegan or omnivore donor were also similar at day 15 (median vegan 6 vs 10%, p=0.24), day 30 (vegan 9 vs 14%, p=0.27), and month 2 (vegan 10% vs 12%, p=0.61). A non-significant trend towards lower HE recurrence from the vegan donor 0% (0 of 14) compared to the omnivore donor 13% (4 of 31) was seen (p=0.29). This trend continuedwith liver-related hospitalization as well with higher rates in the omnivore donor 19% (6 of 31) compared to the vegan one, 0% (0 of 14, p=0.15) and for all hospitalizations with omnivore 41% (13 of 31) versus vegan 21% (3 of 14, p=0.31).

[0221] HE recurrence:

[0222] The only clinical difference at baseline between those who recurred (apart from being randomized into placebo vs FMT) was serum sodium (Table 2). Those who recurred had a trend towards worse physical SIP impairment. There was no significant difference in baseline microbial diversity of those who recurred versus not. However, patients who developed HE recurrence were more likely to have lower relative abundance of Lachnospiraceae and higher Lactobacillaceae (FIG. 3A, Table 3). At the end of all FMTs, those who recurred had lower Bifidobacteriaceae than those who remained HE-free (Table 3). Across all visits, in those whose HE never recurred, there was a higher relative abundance of Lachnospiraceae, Clostridiaceae, Rikenellaceae, Bacteroidaceae, and Desulfovibrionaceae while the reverse pattern was seen with Prevotellaceae, Aerococcaceae, Fusobacteriaceae, Coriobacteriaceae, and Lactobacillaceae (FIG. 3B). Within the placebo group, those who recurred had higher Lactobacillaceae at baseline, while within the FMT groups, those who recurred had a higher relative abundance of Prevotellaceae, Leuconostocaceae, and Bacteroidales compared to those who did not (FIG. 3C-D). The four HE recipients who recurred had a lower engraftment (2.7% vs 11.3%, p=0.020) at day 15 compared to those whose HE did not recur. At this timepoint, none of these patients had developed HE recurrence; after this additional treatment for recurrent HE was administered.

[0223] Logistic regression:

[0224] In the entire group we studied the potential factors associated with HE recurrence. We included demographics, cognitive variables, SIP results, and FMT and doses received. A subsequent model added baseline microbiome families that were significant on univariable analysis (Lachnospiraceae and Lactobacillaceae, Table 6). When all patients were included, HE recurrence was predicted by any FMT receipt (OR 0.07, 95% CI 0.01-0.50, p=0.008), baseline serum sodium (OR 0.72, 95% CI 0.051-0.99, p=0.04) and Physical SIP (OR 1.06,95% CI 1 .01 -1.10, p=0.04). This meant that those with worse physical quality of life at baseline were more likely to develop HE recurrence while those who received FMT doses and had higher sodium were less likely to develop recurrence. When baseline microbial families were added, Lachnospiraceae were associated with lower HE recurrence (OR 0.87, 95% CI 0.76-0.98, p=0025) as was serum sodium (OR 0.67, 95% CI 0.49-0.90, p=0.009).

[0225] Table 6: Median relative abundances of specific microbial families of interest

[0226] Discussion:

[0227] This phase 2 study of standardized FMT using different doses and routes was safe and showed a significant reduction in HE recurrence and liver-related hospitalizations compared to placebo in patients with cirrhosis and HE on lactulose and rifaximin regardless of route of administration, donor, or doses received. HE recurrence and engraftment were associated with the pre-existing low relative abundance of Lachnospiraceae in the recipients. Patients who recurred despite an FMT had a lower donor engraftment compared to those who did not recur. Our analysis showed that FMT was safe and HE recurrencerates were lower in FMT-randomized groups compared to placebo in patients on lactulose and rifaximin than previously published regardless of route, donor, or number of doses.

[0228] We used a definition of HE as grade^2 on West-Haven criteria which required change in medications, and the investigators and treating clinicians remained blinded to the assignment (see 2). The specific protective impact of FMT against HE, which formed most of the liver-related but not all-cause hospitalizations underline the unique impact of gut microbial change on HE. Other hospitalizations seen were largely related to falls, chest pain, and cholecystitis.

[0229] We included patients on rifaximin and lactulose to ensure equipoise (see 4 and 20). The high HE recurrence rate (40% of the placebo-assigned and 17% of the total cohort) despite maximal therapy reiterates the unmet need and could be due to our advanced cohort (MELDL- 22, on lactulose and rifaximin) that are currently underserved (see 21). In contrast to FMT studies for C. difficile recurrence prevention where antibiotics are discontinued, or in ulcerative colitis where antibiotic were used for pre-FMT conditioning (see 22 and 23), we continued rifaximin throughout (see 24). Despite rifaximin use, donor engraftment was detectable across FMT groups. Stool donor engraftment was higher with those who received an FMT enema versus capsules only likely due to a greater change in the upper intestine from capsules, consistent with our prior Phase 1 study (see 8). More importantly, we found lower donor engraftment in the four subjects who received FMT but had recurrence versus the rest.

[0230] Overall, we found that a low Lachnospiraceae and high Lactobacillaceae relative abundance was predictive of HE and any FMT resulted in higher Lachnospiraceae and lower Lactobacillaceae. Furthermore, the few FMT recipients who did develop HE did not experience these microbiome changes. While the donors were selected for high Lachnospiraceae due to the SCFA-generating potential, we also found increased levels of beneficial Bifidobacteriaceae post-FMT (see 9, 15, and 25). Lactobacillaceae are linked with poor outcomes in previous studies (see 26 and 27). We also found higher relative abundance of potential pathobionts such as Enterob acteriaceae decreased after FMT, butpersistent in the placebo group and was higher in patients who experience HE recurrence (see 28).

[0231] A non-significant impact on ammonia levels was seen after FMT in our patients already on rifaximin and lactulose (see 8, 9, and 29). Therefore the impact of FMT could be due to ammonia-unrelated mechanisms such as higher SCFA production due to high Lachnospiraceae (see 30) and points towards the relative abundance of Lachnospiraceae as a biomarker to screen those who respond best to FMT.

[0232] We found improvement in both physical and psychosocial aspects of QOL. While no change in PHES or Stroop within groups on linear mixed models was seen, cognitive performance improved within some FMT recipient groups compared to baseline performance. There is often a disconnect between cognitive performance and QOL perception in cirrhosis due to cognitive reserve differences, which is unaffected by microbiome (see 31). Pre-existing cognitive impairment not being an inclusion and cognitive re-testing at 2 months, which was after HE recurrence could also explain these findings. Sodium levels, which were associated with HE recurrence affect brain function, increases susceptibility to further HE, and affect QOL (see 32-34). Interestingly, the contribution of sodium was independent of the microbiota change in predicting HE recurrence, which points towards the multi-faceted nature of HE pathogenesis. Ultimately, the improved QOL that is consistent with protection from HE recurrence in FMT groups extend the clinical outcomes into the patient-reported outcome domain.

[0233] Several patients due to COVID-19 or other reasons did not follow-up in person, but regardless, we used an ITT analysis. We did not restrict ourselves to patients with preexisting cognitive impairment but focused on patient-reported outcomes. We also only performed 16SrRNA sequencing without functional analyses or metagenomics.

[0234] The results of this Phase 2, randomized, double-blind, placebo-controlled dose-ranging clinical trial show that FMT is safe and is associated with lower rates of HE recurrence in patients with cirrhosis already on lactulose and rifaximin. This is regardless of dose, donor, and route of administration and is affected by baseline microbiota composition in the recipient.

[0235] Citations for Example 1 :1. Devarbhavi H, Asrani SK, Arab JP, Nartey YA, Pose E, Kamath PS. Global burden of liver disease: 2023 update. J Hepatol 2023;79(2):516-537. DOI: 10.1016 / j.jhep.2023.03.017.2. Vilstrup H, Amodio P, Bajaj J, et al. Hepatic encephalopathy in chronic liver disease: 2014 Practice Guideline by the American Association for the Study of Liver Diseases and the European Association for the Study of the Liver. Hepatology 2014;60(2):715-35. DOI: 10.1002 / hep.27210.3. Bloom PP, Tapper EB, Young VB, Lok AS. Microbiome therapeutics for hepatic encephalopathy. J Hepatol 2021 ;75(6): 1452-1464. DOI: 10.1016 / j.jhep.2021.08.004.4. Bajaj JS, Khoruts A. Microbiota changes and Intestinal Microbiota Transplantation in Liver Diseases and Cirrhosis. J Hepatol 2020. DOI: 10.1016 / j .jhep.2020.01.017.5. Kibble H, Shawcross DL. The assessment and management of cirrhotic patients with encephalopathy. United European Gastroenterol J 2024. DOI:10.1002 / ueg2.12530.6. Kim WR, Mannalithara A, Heimbach JK, et al. MELD 3.0: The Model for End- Stage Liver Disease Updated for the Modem Era. Gastroenterology 2021;161(6):1887-1895 e4. DOI: 10.1053 / j.gastro.2021.08.050.7. Lai JC, Dodge JL, Sen S, Covinsky K, Feng S. Functional decline in patients with cirrhosis awaiting liver transplantation: Results from the functional assessment in liver transplantation (FrAILT) study. Hepatology 2016;63(2):574-80. DOI: 10.1002 / hep.28316.8. Bajaj JS, Salzman NH, Acharya C, et al. Fecal Microbial Transplant Capsules are Safe in Hepatic Encephalopathy: A Phase 1, Randomized, Placebo-Controlled Trial. Hepatology 2019. DOI: 10.1002 / hep.30690.9. Bajaj JS, Kassam Z, Fagan A, et al. Fecal Microbiota Transplant from a Rational Stool Donor Improves Hepatic Encephalopathy: A Randomized Clinical Trial. Hepatology 2017. DOI: 10.1002 / hep.29306.10. Bloom PP, Donlan J, Torres Soto M, Daidone M, Hohmann E, Chung RT. Fecal microbiota transplant improves cognition in hepatic encephalopathy and its effect varies by donor and recipient. Hepatol Commun 2022;6(8):2079-2089. DOI: 10.1002 / hep4.1950.11. Kao D, Roach B, Park H, et al. Fecal microbiota transplantation in the management of hepatic encephalopathy. Hepatology 2016;63(l):339-40. DOI:10.1002 / hep.28121.12. Schnabl B, Brenner DA. Interactions between the intestinal microbiome and liver diseases. Gastroenterology 2014; 146(6): 1513-24. DOI: 10.1053 / j.gastro.2014.01.020.13. Hamilton MJ, Weingarden AR, Sadowsky MJ, Khoruts A. Standardized frozen preparation for transplantation of fecal microbiota for recurrent Clostridium difficile infection. Am J Gastroenterol 2012; 107(5): 761 -7. (In eng). DOI: 10.1038 / ajg.2011.482.Staley C, Hamilton MJ, Vaughn BP, et al. Successful Resolution of Recurrent Clostridium difficile Infection using Freeze-Dried, Encapsulated Fecal Microbiota; Pragmatic Cohort Study. Am J Gastroenterol 2017; 112(6):940-947. (In eng). DOI: 10.1038 / ajg.2017.6. Bajaj JS, Fagan A, White MB, et al. Specific Gut and Salivary Microbiota Patterns Are Linked With Different Cognitive Testing Strategies in Minimal Hepatic Encephalopathy. Am J Gastroenterol 2019; 114(7): 1080-1090. DOI:10.14309 / ajg.0000000000000102. Smits LP, Kootte RS, Levin E, et al. Effect of Vegan Fecal Microbiota Transplantation on Carnitine- and Choline-Derived Trimethylamine-N-Oxide Production and Vascular Inflammation in Patients With Metabolic Syndrome. J Am Heart Assoc 2018;7(7). DOI: 10.1161 / J AHA.117.008342. Witjes JJ, Smits LP, Pekmez CT, et al. Donor Fecal Microbiota Transplantation Alters Gut Microbiota and Metabolites in Obese Individuals With Steatohepatitis. Hepatol Commun 2020;4(l l): 1578-1590. DOI: 10.1002 / hep4.1601. Bajaj JS, Shamsaddini A, Acharya C, et al. Multiple bacterial virulence factors focused on adherence and biofilm formation associate with outcomes in cirrhosis. Gut Microbes 2021 ; 13( I): 1993584. DOI: 10.1080 / 19490976.2021.1993584. Bajaj JS, Kamath PS, Reddy KR. The Evolving Challenge of Infections in Cirrhosis. N Engl J Med 2021 ;384(24):2317-2330. DOI: 10.1056 / NEJMra2021808. DeFilipp Z, Bloom PP, Torres Soto M, et al. Drug-Resistant E. coli Bacteremia Transmitted by Fecal Microbiota Transplant. N Engl J Med 2019;381 (21 ):2043- 2050. DOI: 10.1056 / NEJMoal910437. Bass NM, Mullen KD, Sanyal A, et al. Rifaximin treatment in hepatic encephalopathy. N Engl J Med 2010;362( 12): 1071-81. DOI: 10.1056 / NEJMoa0907893. Narula N, Kassam Z, Yuan Y, et al. Systematic Review and Meta-analysis: Fecal Microbiota Transplantation for Treatment of Active Ulcerative Colitis. Inflamm Bowel Dis 2017;23(10): 1702-1709. DOI: 10.1097 / MIB.0000000000001228. Kelly CR, Yen EF, Grinspan AM, et al. Fecal Microbiota Transplantation Is Highly Effective in Real-World Practice: Initial Results From the FMT National Registry. Gastroenterology 2021 ; 160(1): 183-192 e3. DOI: 10.1053 / j.gastro.2020.09.038. Patel VC, Lee S, McPhail MJW, et al. Rifaximin-alpha reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial. J Hepatol 2022;76(2):332-342. DOI:10.1016 / j.jhep.2021.09.010. Odenwald MA, Lin H, Lehmann C, et al. Bifidobacteria metabolize lactulose to optimize gut metabolites and prevent systemic infection in patients with liver disease. Nat Microbiol 2023;8(l l):2033-2049. DOI: 10.1038 / s41564-023-01493-w. Sung CM, Lin YF, Chen KF, et al. Predicting Clinical Outcomes of Cirrhosis Patients With Hepatic Encephalopathy From the Fecal Microbiome. Cell Mol Gastroenterol Hepatol 2019;8(2):301-318 e2. DOI: 10.1016 / j.jcmgh.2019.04.008. Bajaj JS, Heuman DM, Hylemon PB, et al. Altered profile of human gut microbiome is associated with cirrhosis and its complications. J Hepatol 2014;60(5):940-7. DOI: 10.1016 / j jhep.2013.12.019.28. Hsu CL, Schnabl B. The gut-liver axis and gut microbiota in health and liver disease. Nat Rev Microbiol 2023 ;21 ( 11 ): 719-733. DOI: 10.1038 / s41579-023- 00904-3.29. Eriksen PL, Djernes L, Vilstrup H, Ott P. Clearance and production of ammonia quantified in humans by constant ammonia infusion - the effects of cirrhosis and ammonia-targeting treatments. J Hepatol 2023;79(2):340-348. DOI: 10.1016 / j.jhep.2023.03.042.30. Mann ER, Lam YK, Uhlig HH. Short-chain fatty acids: linking diet, the microbiome and immunity. Nat Rev Immunol 2024. DOI: 10.1038 / s41577-024- 01014-8.31. Patel AV, Wade JB, Thacker LR, et al. Cognitive reserve is a determinant of health- related quality of life in patients with cirrhosis, independent of covert hepatic encephalopathy and model for end-stage liver disease score. Clin Gastroenterol Hepatol 2015; 13(5):987-91. DOI: 10.1016 / j.cgh.2014.09.049.32. Soriano G, Roman E, Cordoba J, et al. Cognitive dysfunction in cirrhosis is associated with falls: a prospective study. Hepatology 2012;55(6): 1922-30. DOI: 10.1002 / hep.25554.33. Alukal JJ, John S, Thuluvath PJ. Hyponatremia in Cirrhosis: An Update. Am J Gastroenterol 2020; 115(11): 1775-1785. DOI: 10.14309 / ajg.0000000000000786.34. Renneboog B, Musch W, Vandemergel X, Manto MU, Decaux G. Mild chronic hyponatremia is associated with falls, unsteadiness, and attention deficits. Am J Med 2006; 119(1):71 el-8. DOI: 10.1016 / j.amjmed.2005.09.026.35. Allampati S, Duarte-Rojo A, Thacker LR, et al. Diagnosis of Minimal Hepatic Encephalopathy Using Stroop EncephalApp: A Multicenter US-Based, Norm- Based Study. Am J Gastroenterol 2016;111 :78-86.36. Weissenbom K, Ennen JC, Schomerus H, et al. Neuropsychological characterization of hepatic encephalopathy. J Hepatol 2001;34:768-73.37. Bergner M, Bobbitt RA, Carter WB, Gilson BS. The Sickness Impact Profile: development and final revision of a health status measure. Med Care 1981 ; 19:787- 805.38. Knights D, Kuczynski J, Charlson ES, et al. Bayesian communi ty-wi de cultureindependent microbial source tracking. Nat Methods 2011 ; 8 :761 -3.

[0236] Example 2

[0237] Identification of acid production by fecal microbiota

[0238] A method was developed to screen donor and recipient microbiota for their potential to produce acids in response to a substrate, such as lactulose. The method involves purifying the fecal microbes away from stool through filtration, suspending the purified microbes at aspecific concentration in unbuffered saline, and measuring acid production at different intervals of incubation (30, 60, 120, 240 minutes) with the substrate, such as lactulose, relative to no substrate. Lactulose was used as the substrate in these experiments, but other non-absorbable and fermentable compounds can be used.

[0239] The fecal microbiota from different healthy donors was purified using the method described by Hamilton et al. (Hamilton et al., American journal of gastroenterology 2012; 107:761-7). The purification ensured that the assay was standardized in terms of the number of bacteria per test and that any residual non-microbial components of stool did not contribute to production of microbial metabolites as substrates. The experiments were conducted in an anaerobic chamber.

[0240] Procedure:

[0241] Purified microbiota was suspended in unbuffered saline at a concentration of 7 ± 0.5 x 1010bacteria in 2 mL in multiple test tubes. Each tube was used for one individual pH measurement. Baseline pH was obtained. Lactulose (0.8 g ) was added per tube (or no addition to the control tubes). Each tube was mixed using a Vortex for 10 seconds. The test tubes were incubated at 37°C for 30, 60, 120, and 240 minutes. pH was measured in triplicate at each time point. Aliquots from each were also used for targeted (specific acids) and untargeted metabolite measurements.

[0242] Results:

[0243] The results of testing individual donors of fecal microbiota are shown in FIG. 8. Measurements shown were obtained at 240 minutes after addition of lactulose. No acid production was seen in absence of lactulose. Each point represents an individual experiment. Donors 67 and 122 were both vegans. Donor 67, highlighted by the black column, participated in the THEMATIC clinical trial for HE (Bajaj et al., Journal of hepatology 2025. an 10:S0168-8278(25)00005-4. doi: 10.1016 / j.jhep.2024.12.047). Recipients of microbiota 67 did not experience any HE events.

[0244] In selecting donors for use in treating the HE indication, microbiota in the top 50% of acid production potential in response to lactulose were selected. Thus, in the example shown inFIG. 8, donors 67, 108, 119 were considered eligible, and donors 71, 109, 121, 122 to be ineligible. It should be recognized that microbiota from all donors could be used to treat HE, however, microbiota from donors 67, 108, 119 was more desirable due to increased acid production.

[0245] 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.

[0246] 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.

[0247] 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 examplesare 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.

[0248] 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

CLAIMS1. A method for treating a patient, comprising: administering to the patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier, wherein the patient has cirrhosis, has hepatic encephalopathy (HE), or is at risk for a recurrent episode of HE.

2. The method of claim 1, wherein the treatment further comprises administration of a non-absorbable polysaccharide.

3. The method of claim 2, wherein the polysaccharide is a disaccharide.

4. The method of claim 3, wherein the disaccharide comprises lactulose or lactitol.

5. The method of any one of claims 1-4, wherein the fecal extract is a high acidproducing fecal extract.

6. The method of claim 1, wherein the composition is administered with at least 7 doses.

7. The method of claim 1, wherein the composition is administered with at least 14 doses.

8. The method of any one of claims 6-7, wherein the composition is administered over a week, over two weeks or over three weeks,9. The method of claim 1, wherein the composition comprises from IxlO10to 5xl012microbes.

10. The method of claim 1, wherein the relative abundance of one or more members of the order Bifidobacteriales, one or more members of the family Lachnospiraceae one ormore members of the family Ruminococcaceae, one or more members of the family Clostridiaceae, one or more members of the family Rikenellaceae, one or more members of the family Bacteroidaceae, one or more members of the family Desulfovibrionaceae , or a combination thereof, is increased in the patient after the administering.

11. The method of claim 10, wherein the more members of the order Bifidobacteriales comprises one or more members of the family Bifidobacteriaceae .

12. The method of claim 11, wherein the one or more members of the family Bifidobacteriaceae comprises one or more members of the genus Bifidobacterium.

13. The method of claim 10, wherein the more members of the family Lachnospiraceae comprises one or more members of the genus Blautia, the genus Coprococcus, the genus Dorea, the genus Lachnospira, the genus Oribacterium, the genus Roseburia. or a combination thereof.

14. The method of claim 10, wherein the more members of the family Ruminococcaceae comprises one or more members of the genus Ruminococcus, one or more members of the genus Faecal ibac ter ium, or a combination thereof.

15. The method of claim 10, wherein the increase comprises an increase in the recipient’s intestine.

16. The method of any one of claims 10-15, wherein the relative abundance is increased by at least 1%, at least 5%, or at least 10%.

17. The method of claim 16, wherein the increase is an increase compared to the patient before the administering.

18. The method of claim 1, wherein the relative abundance of one or more members of the family Lactobacillaceae, one or more members of the family Prevotellaceae, one ormore member of the family Aerococcaceae , one or more member of the family Fusobacteriaceae one or more member of the family Coriobacteriaeae , or a combination thereof, is decreased in the patient after the administering.

19. The method of claim 18, wherein the one or more members of the family one or more members of the family Prevotellaceae comprises one or more members of the genus Prevotella.

20. The method of claim 19, wherein the decrease comprises an increase in the recipient’s intestine.

21. The method of any one of claims 18-20, wherein the relative abundance is decreased by at least 1%, at least 5%, or at least 10%.

22. The method of claim 21, wherein the decrease is a decrease compared to the patient before the administering.

23. The method of any one of claims 1-4, wherein the administering comprises at least one rectal administration or at least one oral administration.

24. The method of claim 23, wherein the at least one rectal administration comprises an enema.

25. The method of claim 23, wherein the oral administration comprises a solid formulation of the fecal extract.

26. The method of claim 23, wherein the solid formulation comprises a capsule or pill.

27. A method for increasing relative abundance of at least one microbe in a patient, comprising:administering to the patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier, wherein the patient has cirrhosis, has hepatic encephalopathy (HE), or is at risk for a recurrent episode of HE, and wherein the at least one microbe is one or more members of the order Bifidobacteriales, one or more members of the family Lachnospiraceae, one or more members of the family Ruminococcaceae, one or more members of the family Clostridiaceae, one or more members of the family Rikenellaceae, one or more members of the family Bacteroidaceae, one or more members of the family Desulfovibrionaceae , or a combination thereof, and the relative abundance is increased in the patient after the administering.

28. The method of claim 27, wherein the more members of the order Bifidobacteriales comprises one or more members of the family Bifidobacteriaceae .

29. The method of claim 28, wherein the one or more members of the family Bifidobacteriaceae comprises one or more members of the genus Bifidobacterium.

30. The method of claim 27, wherein the more members of the family Lachnospiraceae comprises one or more members of the genus Blautia, the genus Coprococcus, the genus Dorea, the genus Lachnospira, the genus Oribacterium, the genus Roseburia or a combination thereof.

31. The method of claim 27, wherein the more members of the family Ruminococcaceae comprises one or more members of the genus Ruminococcus, one or more members of the genus Faecalibacterhim, or a combination thereof.

32. The method of claim 27, wherein the increase comprises an increase in the recipient’s colon.

33. The method of any one of claims 27-32, wherein the relative abundance is increased by at least 1%, at least 5%, or at least 10%.

34. The method of claim 33, wherein the increase is an increase compared to the patient before the administering.

35. A method for decreasing relative abundance of at least one microbe in a patient, comprising: administering to the patient at least two doses a composition comprising a fecal extract and a pharmaceutically acceptable carrier, wherein the patient has cirrhosis, has hepatic encephalopathy (HE), or is at risk for a recurrent episode of HE, and wherein the at least one microbe is one or more members of the family Lactobacillaceae, one or more members of the family Prevotellaceae, one or more member of the family Aerococcaceae, one or more member of the family Fusobacteriaceae , one or more member of the family Coriobacteriaeae, or a combination thereof, and the relative abundance is decreased in the patient after the administering.

36. The method of claim 35, wherein the one or more members of the family one or more members of the family Prevotellaceae comprises one or more members of the genus Prevotella.

37. The method of claim 36, wherein the decrease comprises an increase in the recipient’s intestine.

38. The method of any one of claims 35-37, wherein the relative abundance is decreased by at least 1%, at least 5%, or at least 10%.

39. The method of claim 38, wherein the decrease is a decrease compared to the patient before the administering.

40. The method of claim 27 or 35, wherein the method further comprises administration of a non-absorbable polysaccharide.

41. The method of claim 40, wherein the polysaccharide is a disaccharide.

42. The method of claim 41, wherein the disaccharide comprises lactulose or lactitol.

43. The method of claim 27 or 35, wherein the fecal extract is a high acid-producing fecal extract.

44. The method of claim 27 or 35, wherein the composition is administered with at least 7 doses.

45. The method of claim 27 or 35, wherein the composition is administered with at least 14 doses.

46. The method of claim 44, wherein the composition is administered over a week, over two weeks or over three weeks,47. The method of claim 27 or 35, wherein the composition comprises from IxlO10to 5xl012microbes.

48. The method of claim 27 or 35, wherein the administering comprises at least one rectal administration or at least one oral administration.

49. The method of claim 48, wherein the at least one rectal administration comprises an enema.

50. The method of claim 48, wherein the oral administration comprises a solid formulation of the fecal extract.

51. The method of claim 48, wherein the solid formulation comprises a capsule or pill.

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