WO2019213595A1

WO2019213595A1 - Microbiota restoration therapy (mrt) compositions and methods of manufacture

Info

Publication number: WO2019213595A1
Application number: PCT/US2019/030700
Authority: WO
Inventors: Gregory J. FLUET; Kenneth F. Blount; Lee A. Jones
Original assignee: Ferring B.V.
Priority date: 2018-05-04
Filing date: 2019-05-03
Publication date: 2019-11-07

Abstract

Microbiota restoration therapy compositions and methods for making and using microbiota restoration therapy compositions are disclosed. An example microbiota restoration therapy composition may include a processed bacterial suspension including a first population of bacteria derived from a first human fecal sample and a second population of bacteria derived from a source other than the first human fecal sample.

Description

MICROBIOTA RESTORATION THERAPY (MRT) COMPOSITIONS AND METHODS OF MANUFACTURE

Field

[0001] The present disclosure pertains to compositions and methods for treating patients.

Background

[0002] A wide variety of compositions and methods have been developed for treating diseases and/or conditions, for example diseases and/or conditions of the digestive track, liver and immune system. Of the known compositions and methods, each has certain advantages and disadvantages. There is an ongoing need to provide alternative compositions and methods for treating diseases and/or conditions.

Brief Summary

[0003] This disclosure provides design, material, manufacturing method, and use alternatives for compositions and methods for treating patients. An example microbiota restoration therapy composition is disclosed. The microbiota restoration therapy composition comprises: a processed bacterial suspension including a first population of bacteria derived from a first human fecal sample and a second population of bacteria derived from a source other than the first human fecal sample.

[0004] Alternatively or additionally to any of the embodiments above, the first population of bacteria, the second population of bacteria, or both are suspended in a cryoprotectant solution comprising 30-90 g/L polyethylene glycol in saline.

[0005] Alternatively or additionally to any of the embodiments above, the second population of bacteria is derived from a second human fecal sample.

[0006] Alternatively or additionally to any of the embodiments above, the second population of bacteria is derived from a bacterial culture.

[0007] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the family Ruminococcaceae

[0008] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the genus Bacteroides [0009] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes Bacteroides fragilis.

[0010] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes Bacteroides thetaiotaomicron.

[0011] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the family Bifldobacteriaceae.

[0012] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the genus Prevotella.

[0013] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the genus Fusobacterium.

[0014] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes Lactobacillus plantarum.

[0015] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the classes Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli.

[0016] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes a bacteriophage.

[0017] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes a spore forming bacteria.

[0018] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes a non-spore-forming bacteria.

[0019] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of bacteria from the family Ruminococcaceae and a carrier.

[0020] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of bacteria from the genus Bacteroides.

[0021] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Bacteroides fragilis and a carrier.

[0022] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Bacteroides thetaiotaomicron and a carrier.

[0023] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of bacteria from the genus Fusobacterium and a carrier. [0024] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of bacteria from the genus Prevotella and a carrier.

[0025] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of bacteria from the family Bifldobacteriaceae and a carrier.

[0026] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Lactobacillus plantarum and a carrier.

[0027] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of bacteria from the classes Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli.

[0028] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the genus Akkermansia.

[0029] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes Akkermansia muciniphila.

[0030] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Akkermansia muciniphila.

[0031] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Akkermansia muciniphila and a carrier.

[0032] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists of Akkermansia muciniphila.

[0033] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists of Akkermansia muciniphila and a carrier.

[0034] Alternatively or additionally to any of the embodiments above, further comprising a fungus.

[0035] Alternatively or additionally to any of the embodiments above, further comprising mesalamine.

[0036] Alternatively or additionally to any of the embodiments above, further comprising infliximab.

[0037] Alternatively or additionally to any of the embodiments above, wherein the second population of bacteria includes genetically modified bacteria.

[0038] Alternatively or additionally to any of the embodiments above, wherein the genetically modified bacterial include a gene coding for antibacterial resistance. [0039] A microbiota restoration therapy composition is disclosed. The microbiota restoration therapy composition comprises: a bacterial suspension derived from a first human fecal sample, the bacterial suspension comprising 30-90 g/L polyethylene glycol in saline; a bacterial additive mixed with the bacterial suspension; and wherein the bacterial additive includes one to four classes of bacteria.

[0040] Alternatively or additionally to any of the embodiments above, the bacterial additive is derived from a second human fecal sample different from the first human fecal sample.

[0041] Alternatively or additionally to any of the embodiments above, the bacterial additive includes cultured bacteria.

[0042] Alternatively or additionally to any of the embodiments above, the bacterial additive includes bacteria from the family Ruminococcaceae .

[0043] Alternatively or additionally to any of the embodiments above, the bacterial additive includes bacteria from the genus Bacteroides.

[0044] Alternatively or additionally to any of the embodiments above, the bacterial additive includes Bacteroides fragilis.

[0045] Alternatively or additionally to any of the embodiments above, the bacterial additive includes Bacteroides thetaiotaomicron.

[0046] Alternatively or additionally to any of the embodiments above, the bacterial additive includes bacteria from the family Bifldobacteriaceae .

[0047] Alternatively or additionally to any of the embodiments above, the bacterial additive includes a bacteria from the genus Prevotella.

[0048] Alternatively or additionally to any of the embodiments above, the bacterial additive includes bacteria from the genus Fusobacterium.

[0049] Alternatively or additionally to any of the embodiments above, the bacterial additive includes Lactobacillus plantarum.

[0050] Alternatively or additionally to any of the embodiments above, further comprising mesalamine.

[0051] Alternatively or additionally to any of the embodiments above, further comprising infliximab.

[0052] Alternatively or additionally to any of the embodiments above, the bacterial additive includes bacteria from the classes Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli. [0053] Alternatively or additionally to any of the embodiments above, the bacterial additive consists essentially of bacteria from four classes and a carrier.

[0054] Alternatively or additionally to any of the embodiments above, the bacterial additive consists essentially of bacteria from three classes and a carrier.

[0055] Alternatively or additionally to any of the embodiments above, the bacterial additive consists essentially of bacteria from two classes and a carrier.

[0056] Alternatively or additionally to any of the embodiments above, the bacterial additive includes a bacteriophage.

[0057] Alternatively or additionally to any of the embodiments above, further comprising a fungus.

[0058] Alternatively or additionally to any of the embodiments above, the bacterial additive consists essentially of bacteria from one class and a carrier.

[0059] A method for manufacturing a microbiota restoration composition is disclosed. The method comprises: manufacturing a full-spectrum base composition, the method for manufacturing the full-spectrum base composition comprising: collecting a fresh human fecal sample, adding a diluent to the fresh human fecal sample to form a diluted sample, wherein the diluent includes 30-90 g/L polyethylene glycol in saline, and filtering the diluted sample to form a filtrate comprising the full-spectrum base composition; and adding a bacterial additive to the full-spectrum base composition, the bacterial additive including one to four classes of bacteria.

[0060] A microbiota restoration therapy composition is disclosed. The microbiota restoration therapy composition comprises: a bacterial suspension derived from a fresh human fecal sample, the bacterial suspension comprising 30-90 g/L polyethylene glycol in saline; a cultured additive mixed with the bacterial suspension; and wherein the cultured additive comprises one or more of a cultured bacteria, a bacteriophage, and a fungus.

[0061] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes genetically modified bacteria.

[0062] Alternatively or additionally to any of the embodiments above, the genetically modified bacterial include a gene coding for antibacterial resistance.

[0063] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the genus Akkermansia.

[0064] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes Akkermansia muciniphila. [0065] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Akkermansia muciniphila.

[0066] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Akkermansia muciniphila and a carrier.

[0067] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists of Akkermansia muciniphila.

[0068] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists of Akkermansia muciniphila and a carrier.

[0069] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes bacteria from the family Enterococcaceae.

[0070] Alternatively or additionally to any of the embodiments above, the second population of bacteria includes Enterococcus hirae.

[0071] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Enterococcus hirae.

[0072] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists essentially of Enterococcus hirae and a carrier.

[0073] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists of Enterococcus hirae.

[0074] Alternatively or additionally to any of the embodiments above, the second population of bacteria consists of Enterococcus hirae and a carrier.

[0075] A microbiota restoration therapy composition is disclosed. The microbiota restoration therapy composition comprises: a bacterial suspension derived from a human fecal sample, the bacterial suspension comprising 30-90 g/L polyethylene glycol in saline; and a drug additive mixed with the bacterial suspension, the drug additive including mesal amine.

[0076] The above summary of some embodiments is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The Figures and Detailed Description which follow more particularly exemplify these embodiments.

Brief Description Of The Drawings

[0077] The disclosure may be more completely understood in consideration of the following detailed description of various embodiments of the disclosure in connection with the accompanying drawings, in which: [0078] FIG. 1 is a flowchart depicting an example process for manufacturing a microbiota restoration therapy (MRT) composition.

[0079] FIG. 2 is a flowchart depicting an example process for manufacturing an MRT composition.

[0080] While the disclosure is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the disclosure to the particular embodiments described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure.

Detailed Description

[0081] For the following defined terms, these definitions shall be applied, unless a different definition is given in the claims or elsewhere in this specification.

[0082] All numeric values are herein assumed to be modified by the term“about,” whether or not explicitly indicated. The term“about” generally refers to a range of numbers that one of skill in the art would consider equivalent to the recited value (i.e., having the same function or result). In many instances, the terms“about” may include numbers that are rounded to the nearest significant figure.

[0083] The recitation of numerical ranges by endpoints includes all numbers within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0084] As used in this specification and the appended claims, the singular forms“a”, “an”, and“the” include plural referents unless the content clearly dictates otherwise. 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.

[0085] It is noted that references in the specification to“an embodiment”,“some embodiments”,“other embodiments”, etc., indicate that the embodiment described may include one or more particular features, structures, and/or characteristics. However, such recitations do not necessarily mean that all embodiments include the particular features, structures, and/or characteristics. Additionally, when particular features, structures, and/or characteristics are described in connection with one embodiment, it should be understood that such features, structures, and/or characteristics may also be used in connection with other embodiments whether or not explicitly described unless clearly stated to the contrary. [0086] The following detailed description should be read with reference to the drawings in which similar elements in different drawings are numbered the same. The drawings, which are not necessarily to scale, depict illustrative embodiments and are not intended to limit the scope of the disclosure.

[0087] “Mammal” as used herein refers to any member of the class Mammalia, including, without limitation, humans and nonhuman primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.

[0088] The term“cry opreservation,” as used herein, refers to the process of cooling and storing biological cells, tissues, or organs at very low temperatures to maintain their viability. As a non-limiting example, eiyopreservation can be the technolog} of co ing and storing cells at a temperature below the freezing point (e.g., 196 1C) that permits high rates of survivability of the cells upon thawing.

[0089] The term“cryoprotectant,” as used herein, refers to a substance that is used to protect biological cells or tissues from the effects of freezing.

[0090] As used herein, the term“microbiota” can refer to the human microbiome, the human microbiota or the human gut microbiota. The human microbiome (or human microbiota) is the aggregate of microorganisms that reside on the surface and in deep layers of skin, in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal, genito urinary, and/or vaginal tracts of humans inclusive of microorganisms related to reproductive health such as the placental microbiome. The human microbiome is comprised of bacteria but may also include fungi, phages, viruses, archaea, and the like. Some of these organisms perform tasks that are useful for the human host, but the function of the majority of the organisms that make up the human microbiome is unknown. Under normal circumstances, these microorganisms do not cause disease to the human host, but instead participate in maintaining health. Hence, this population of organisms is frequently referred to as“normal flora.”

[0091] The population of microorganisms living in the human gastrointestinal tract is commonly referred to as“gut flora” or“gut microbiota.” The microbial flora of the human gut encompasses a wide variety of microorganisms that may aid in digestion, the synthesis of vitamins, and creating enzymes (and/or other metabolites) including those not produced by the human body.

[0092] The phrase“microbiota restoration therapy” (MRT), as used herein, refers to a composition which may include, but is not limited to, human fecal material containing viable gut flora from a patient or donor, a diluent, and a cryoprotectant. Additional compositions include equivalent freeze-dried and reconstituted feces, a“synthetic” fecal composition, or a“cultured” fecal composition. The human fecal material is screened for the presence of pathogenic microorganisms prior to its use in the microbiota restoration therapy. The human fecal material is screened for the presence of Clostridium species including C. difficile, Norovirus, Adenovirus, enteric pathogens, antigens to Giardia species, Cryptosporidia species and other pathogens, including acid-fast bacteria, enterococci, including but not limited to vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), as well as any ova or parasitic bodies, or spore-forming parasites, including but not limited to Isospora, Clyslospora, and Cryptospora.

[0093] Within the field of microbiome targeted therapies, there are multiple divergent approaches including live microbial compositions, bacteriophages, microbial metabolites as well as genetically modified microorganisms. Generally, these approaches have been advanced in isolation and within each of these categories there may be different divergent approaches. For example, under the category live microbial compositions there are two very different approaches. One approach is to source the microbial community from harvesting the microbes from human fecal donations to create a formulation consisting of either all or a subset of the microbiota of the donor. The other is to isolate specific bacterial species or strains and then culture these microbes in an artificial environment creating a formulation made exclusively of cultured pure bacteria, either from a single species or a mix of cultured species.

[0094] A benefit of the human sourced approach is that there is a significant amount of clinical evidence demonstrating not only the impact of microbial therapies on disease, such as C. difficile infection, but also research that demonstrates the ability of the microbial community to impact the pre-treatment microbiome of the patient and shift its relative composition to a consortium more closely resembling the therapeutic consortium. However, the relative proportions of taxonomic classes in human sourced compositions is generally limited to the natural range of diversity found in a healthy human population. [0095] With cultured microbial consortia, the field is in its infancy and there is little clinical evidence of the therapeutic impact of a cultured consortia on disease and likewise relatively limited data on the ability to engraft the micro-organisms in the patient’s dysbiotic microbiome and sustain a therapeutically beneficial population of the cultured organism and long term resolution of dysbiosis. In addition, while there is research connecting certain strains to specific disease conditions, typically these strains correlate with relatively abundant commensal populations found in patients with the correlative disease. What is not well characterized is the contribution of less abundant populations to the metabolism and population health of the more abundant species. The high-abundance species that correlate with various health states may require populations of less abundant microbes, phages or fungi to have the same clinical effect on a patient. Therefore, combining a human sourced diverse consortium of commensal bacteria with a second population of bacteria may have greater systemic effect than each separately.

[0096] The process of fecal bacteriotherapy may include introducing an MRT composition (e.g., that may be a processed fecal drug-product derived from a fecal sample from a healthy donor or a donor having one or more desired characteristics) into a gastrointestinal tract of a patient to repopulate a healthy or desirable gut microbiota. The MRT composition may be introduced directly into the lower gastrointestinal tract (e.g., via enema or colonoscope), directly into the upper gastrointestinal tract (e.g., via endoscope, duodenoscope, gastro-nasal tube, etc.), orally (e.g., which may include a MRT composition encapsulated in a suitable capsule), or in another suitable manner. In at least some instances, the aim of the intervention is to restore the microbiota of the patient, treat a disease or condition, or otherwise improve the health of the patient.

[0097] In some instances, the presence of particular organisms in the MRT composition, and/or variation in the relative quantities of the particular organisms in the MRT compositions, may improve the treatment of particular diseases, infections, or conditions. For example, it may be desirable to combine the features and benefits of a broad or full-spectrum MRT composition (e.g., a MRT composition derived from a human fecal sample without selection of or enhancement of any particular organisms) with that of a collection of a relatively small number of organisms that may be correlated with a particular disease condition. In other instances, it may be desirable to combine a first population of organisms (e.g., bacteria), including a population that is not considered to be full-spectrum MRT composition with a second population of organisms (e.g., bacteria). In other instances, the processes used to prepare a Microbiota Restoration Therapy may reduce or eliminate the population of certain desirable organisms and it would be desirable to restore or increase the population of affected bacteria by adding back a cultured population of that bacteria post processing. Disclosed herein are MRT compositions that include a first population of organisms (e.g., a full-spectrum MRT composition, a population of organisms that is not considered to be a full-spectrum MRT, etc.) combined with or doped with one or more additional organisms that are correlated with a particular disease target. In addition, disclosed herein are MRT compositions that include a population of organisms (e.g., a full-spectrum MRT composition, a population of organisms that is not considered to be a full-spectrum MRT, etc.) combined with or doped with one or more drugs.

[0098] The MRT compositions disclosed herein may be understood as including a first population of bacteria and/or a bacterial suspension and/or a first population of organisms. In general, the first population of bacteria/organisms may include a full-spectrum drug product that may be manufactured by processing a fresh human fecal sample. The process for manufacturing the full-spectrum drug product may be similar to those processes disclosed in U.S. Patent No. 9,675,648 and U.S. Patent Application Pub. No. US 2016/0361263, the entire disclosures of which are herein incorporated by reference. Alternatively, the first population of bacteria/organisms may be a population of organisms that is not considered to be full-spectrum (e.g., the first population of bacteria/organisms are a subset of a full-spectrum product, a conditioned or processed full-spectrum product that includes a particular population of bacteria/organisms, etc.). The MRT compositions disclosed herein also include an additive so that the MRT compositions include both a full- spectrum material (e.g., a first population of bacteria and/or a bacterial suspension formed/derived from a fresh human fecal sample) and an additive that includes a second population of bacteria/organism(s). In some instances, the resultant MRT composition may be termed a“hybrid” MRT composition, a“doped” MRT composition, a two-part or multi part MRT composition, or simply an MRT composition (e.g., with the features disclosed herein). In some of these and in other instances, the additive may include a drug.

[0099] FIG. 1 is a flow chart summarizing one example process. Broadly, the process may include collecting a fresh human fecal sample 10, adding a diluent to the fresh human fecal sample 12, and purifying and/or concentrating the sample 14 (e.g., which may include mixing the sample, filtering the sample, etc.) to define the first population of bacteria and/or a bacterial suspension. In at least some instances, the diluent may include a cryoprotectant such as polyethylene glycol. For example, the diluent/cryoprotectant may include about 10-150 g/L or about 20-100 g/L, or about 30-90 g/L polyethylene glycol in saline. When used with the first population of bacteria, the first population of bacteria may be suspended in the diluent (e.g., the first population of bacteria may be suspend in a saline solution that includes 30-90 g/L polyethylene glycol). These are just examples. Other diluents, cryoprotectants, quantities/concentrations, etc. are contemplated.

[0100] The process may also include adding an additive 16. The additive may take a number of different forms. In some instances, the additive may include a population of bacteria (e.g., a second population of bacteria and/or a second population of organisms). In some of these and in other instances, the additive may include one or more bacteriophages, one or more fungi, and/or other suitable organisms. The additive may take the form of a suspension (e.g., where the organism(s) that are part of the additive are suspended in a material such as the diluent, for example as described above). In some instances, the organism(s) that make up part of the additive may be derived from a fresh human fecal sample. In other instances, the organism(s) that make up part of the additive may be derived from a culture (e.g., a bacterial culture). In some of these and in other instances, the additive may include a material suitable to stimulate the growth of one or more populations/sub- populations of bacteria. In some of these and in other instances, the additive may include a drug.

[0101] The process may also include processing the MRT composition and/or one or more components of the MRT composition. This processing step may take place before, during, or after the addition of the additive. For example, the MRT composition and/or one or more components thereof may be treated with an alcohol, an anti-viral formulation, combinations thereof, or the like. This may destroy one or more components of the MRT composition (e.g., including unwanted organisms, non-beneficial organism, beneficial organisms, etc.). In some instances, following this process the additive (e.g., which may contain one or more the beneficial organisms derived either from culturing the organisms or from alternative processing methods) could be reintroduced to the MRT composition to increase the population of the beneficial organism. In some instances this could be beneficial to overexpress a single strain or species within a higher order taxa. By depopulating the taxa in the human sourced composition it would give a re-introduced or “doped” strain or species more ability to survive in a competitive ecosystem. [0102] In at least some instances, the full-spectrum MRT composition may provide a “base” that helps establish the desirable effects of the additive. For example, administration of the additive alone (e.g., where the additive is not combined with the full-spectrum MRT composition) may not provide the desired effect or may not be able to establish a sustainable population in the dysbiotic microbiome of a patient. The use of the full- spectrum MRT composition may help the additive more efficiently fit in within the microbiota environment so that the additive can have the desired effect or restore the dysbiotic microbiome to a state that is more conducive for the additive and enable it to have a beneficial impact. In some instances, the presence of both the full-spectrum MRT composition and the additive in a hybrid or doped MRT composition may have a cooperative or synergistic influence on the desired outcome. In addition or in the alternative, the MRT composition may help to increase the functionality of human systems including the immune system, diseases connected to the functioning of the immune system (auto-immune disease, UC, etc.), and other therapies that rely on the functioning of the immune system (immune-oncology treatments, t-cell inhibitors, etc.). For example, an increase in the immune system functionality could increase the effectiveness of other therapies targeting other diseases that use the human immune system as a weapon to attach these diseases. Accordingly, a physician planning to treat a patient with cancer with an immuno-oncology drug could first treat the patient with MRT to improve the capabilities of the patient’s immune system to respond to the immune-oncology and increase the effectiveness of this therapy in treating cancer.

[0103] The second population of organisms (e.g., the additive) may be added at a suitable time during manufacturing. In some instances, the additive may be added prior to processing the (e.g., full-spectrum) MRT composition. In some instances, the additive may be added after initial process then followed by processing steps to create a largely homogenous mixture, such as mixing, before storing the combined composition. In other instances, the additive may be added prior to storage/freezing of the MRT composition, after storage/freezing the MRT composition, after thawing the MRT composition, prior to use/administration, etc.

[0104] A number of example populations of organisms (e.g.,“second” populations of bacteria/organisms and/or additives) are contemplated for the additive. For example, in some instances, the second population of bacteria includes bacteria from the family Ruminococcaceae. In some instances, the second population of bacteria consists essentially of bacteria from the family Ruminococcaceae and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from the family Ruminococcaceae. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with bacteria from the family Ruminococcaceae may increase the ability of the MRT composition to treat, attenuate, or otherwise improve cancer. For example, including or otherwise doping a full-spectrum MRT drug product with bacteria from the family Ruminococcaceae may improve the capabilities of the patient’s immune system to respond to the immune-oncology and increase the effectiveness of this therapy in treating cancer.

[0105] In some instances, the second population of bacteria includes bacteria from the genus Bacteroides. In some instances, the second population of bacteria consists essentially of bacteria from the genus Bacteroides and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from the genus Bacteroide . In at least some instances, including or otherwise doping a full-spectrum MRT drug product with bacteria from the genus Bacteroides may increase the ability of the MRT composition to treat, attenuate, or otherwise improve the health of the patient’s immune system. This improvement in the immune system could treat conditions directly, such as auto-immune disease, allergies, inflammatory bowel disease, GI inflammatory side effects caused by checkpoint therapies for cancer, irritable bowel syndrome, colitis caused by checkpoint therapies, etc.

[0106] In some instances, the second population of bacteria includes Bacteroides fragilis. In some instances, the second population of bacteria consists essentially of Bacteroides fragilis and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full- spectrum MRT drug-product) combined or doped with Bacteroides fragilis. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with Bacteroides fragilis may increase the ability of the MRT composition to treat, ahenuate, or otherwise improve cancer. For example, including or otherwise doping a full-spectrum MRT drug product with Bacteroides fragilis may improve the capabilities of the patient’s immune system to respond to the immune-oncology and increase the effectiveness of this therapy in treating cancer. For example, including or otherwise doping a full-spectrum MRT drug product with Bacteroides fragilis may improve the capabilities of the patient’s immune system to respond to the immune-oncology and increase the effectiveness of this therapy in treating cancer.

[0107] In some instances, the second population of bacteria includes Bacteroides thetaiotaomicron. In some instances, the second population of bacteria consists essentially of Bacteroides thetaiotaomicron and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with Bacteroides thetaiotaomicron. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with Bacteroides thetaiotaomicron may increase the ability of the MRT composition to treat, attenuate, or otherwise improve cancer. For example, including or otherwise doping a full-spectrum MRT drug product with Bacteroides thetaiotaomicron may improve the capabilities of the patient’s immune system to respond to the immune- oncology and increase the effectiveness of this therapy in treating cancer. Furthermore, such a MRT composition may help the patient’s immune system by colonizing the patient’s gut with organisms that can outcompete other organisms and/or reduce/prevent colonization of the gut by pathogens.

[0108] In some instances, the second population of bacteria includes bacteria from the family Bifldobacteriaceae . In some instances, the second population of bacteria consists essentially of bacteria from the family Bifldobacteriaceae and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from the family Bifldobacteriaceae. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with bacteria from the family Bifldobacteriaceae may increase the ability of the MRT composition to treat, attenuate, or otherwise improve cancer. For example, including or otherwise doping a full-spectrum MRT drug product with bacteria from the family Bifldobacteriaceae may improve the capabilities of the patient’s immune system to respond to the immune-oncology and increase the effectiveness of this therapy in treating cancer.

[0109] In some instances, the second population of bacteria includes bacteria from the genus Prevotella. In some instances, the second population of bacteria consists essentially of bacteria from the genus Prevotella and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from the genus Prevotella. In at least some instances, including or otherwise doping a full- spectrum MRT drug product with bacteria from the genus Prevotella may increase the ability of the MRT composition to treat, attenuate, or otherwise improve cancer. For example, including or otherwise doping a full-spectrum MRT drug product with bacteria from the genus Prevotella may improve the capabilities of the patient’s immune system to respond to the immune-oncology and increase the effectiveness of this therapy in treating cancer.

[0110] In some instances, the second population of bacteria includes bacteria from the genus Fusobacterium. In some instances, the second population of bacteria consists essentially of bacteria from the genus Fusobacterium and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from the genus Fusobacterium. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with bacteria from the genus Fusobacterium may increase the ability of the MRT composition to treat, attenuate, or otherwise improve ulcerative colitis.

[0111] In some instances, the second population of bacteria includes Lactobacillus plantarum. In some instances, the second population of bacteria consists essentially of Lactobacillus plantarum and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with Lactobacillus plantarum. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with Lactobacillus plantarum may increase the ability of the MRT composition to treat, attenuate, or otherwise improve sepsis, pneumonia, combinations thereof, or the like.

[0112] In some instances, the second population of bacteria includes bacteria from the class Clostridia. In some instances, the second population of bacteria consists essentially of bacteria from the class Clostridia and a carrier. In at least some instances, the bacteria from the class Clostridia are bacteria other Clostridia difficile. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from the class Clostridia (other Clostridia difficile). In at least some instances, including or otherwise doping a full-spectrum MRT drug product with bacteria from the class Clostridia (other Clostridia difficile) may increase the ability of the MRT composition to treat, attenuate, or otherwise improve Clostridia difficile infections.

[0113] In some instances, the second population of bacteria includes bacteria from the family Enter ococcaceae. In some instances, the second population of bacteria consists essentially of bacteria from the family Enterococcaceae and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from the family Enterococcaceae. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with bacteria from the family Enterococcaceae may increase the ability of the MRT composition to treat, attenuate, or otherwise improve cancer. For example, including or otherwise doping a full-spectrum MRT drug product with bacteria from the family Enterococcaceae may improve the capabilities of the patient’s immune system to respond to the immune-oncology and increase the effectiveness of this therapy in treating cancer.

[0114] In some instances, the second population of bacteria includes Enterococcus hirae. In some instances, the second population of bacteria consists essentially of Enterococcus hirae and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full- spectrum MRT drug-product) combined or doped with Enterococcus hirae. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with Enterococcus hirae may increase the ability of the MRT composition to treat, attenuate, or otherwise improve cancer. For example, including or otherwise doping a full-spectrum MRT drug product with Enterococcus hirae may improve the capabilities of the patient’s immune system to respond to the immune-oncology and increase the effectiveness of this therapy in treating cancer.

[0115] In some instances, the second population of bacteria includes bacteria from the classes Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli. In some instances, the second population of bacteria consists essentially of bacteria from the classes Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from four classes (e.g., only four classes), for example, Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli. Other MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from three classes (e.g., only three classes). Other MRT compositions are contemplated that include a first population of bacteria (e.g., a full- spectrum MRT drug-product) combined or doped with bacteria from two classes (e.g., only two classes). Other MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with bacteria from a single class (e.g., only a single class).

[0116] In some instances, the second population of bacteria includes Akkermansia muciniphila. In some instances, the second population of bacteria consists essentially of Akkermansia muciniphila and a carrier. The carrier may be a diluent (e.g., as described above) or another suitable material such as saline, water, bacterial culture media, or the like. Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with Akkermansia muciniphila. In at least some instances, including or otherwise doping a full-spectrum MRT drug product with Akkermansia muciniphila may increase the ability of the MRT composition to treat, attenuate, or otherwise improve a number of conditions including diabetes, obesity, alcoholic liver disease (ALD), fatty liver, other hepatic disorders, inflammation, cancer, GI inflammatory side effects caused by checkpoint therapies for cancer, and the like. [0117] In some instances, the second population of bacteria includes spore forming bacteria. In some of these and in other instances, the second population of bacteria includes non-spore forming bacteria. For example, the second population could include cultured organisms that are non-spore forming bacteria. In such instances, if the MRT is processed with the use of chemicals or in an environment that is not conducive to survival of non spore forming bacteria, it may be desirable to supplement the resultant MRT with populations of non-spore forming bacteria.

[0118] In some instances, the second population of organisms may include one or more bacteriophages. In some of these and/or in other instances, the second population of organisms may include one or more fungi. In some instances, the MRT composition may include a drug. For example, the MRT composition may include mesalamine. In some of these and in other instances, the MRT composition may include infliximab. If a drug is added to the MRT composition, the drug may be added prior to storage/freezing of the MRT composition, after storage/freezing the MRT composition, after thawing the MRT composition, prior to use/administration, etc.

[0119] In some instances, the second population of bacteria may include bacteria that are genetically modified or otherwise transformed by a nucleic acid (e.g., DNA). For example, a population of bacteria may be transfected with a plasmid carrying a gene of interest. The gene of interest may be a gene that codes for antibiotic resistance (e.g., resistance to one or more antibiotics). Thus, MRT compositions are contemplated that include a first population of bacteria (e.g., a full-spectrum MRT drug-product) combined or doped with genetically modified bacteria. In some instances, patients with an infection may be treated with such a MRT composition prior to treatment with an antibiotic. Following treatment with the MRT composition, the patient may be treated with an antibiotic. The antibiotic may help to reduce the population of undesirable or unwanted organisms in the patient while allowing more desirable organisms (e.g., genetically modified bacteria in the MRT composition) to desirable impact the microbiome of the patient.

[0120] The relative proportions or quantities of the first population of bacteria (e.g., a full-spectrum MRT drug-product) to the second population of bacteria/organisms and/or additives may vary. In some instances, the number of organisms in the first population of bacteria (e.g., a full-spectrum MRT drug-product) may be quantified using a suitable technique (e.g., qPCR such as the process disclosed in U.S. Patent Application Pub No. US 2017/0327862, the entire contents of which is herein incorporated by reference), the number of organisms in the second population of bacteria/organisms and/or additives may be quantified using a suitable technique (e.g., qPCR), and a ratio may be utilized that corresponds to of the number of organisms in the first population to the number of organisms in the second population. In some instances, the ratio of the first population of organisms to the second population of organisms may be on the order of about 1: 1 to 10,000: 1, or about 5: 1 to 1000:1, or about 10: 1 to 750: 1, or about 20:1 to 500: 1.

[0121] FIG. 2 is a flow chart summarizing another example process. This process may form a full-spectrum MRT composition (e.g., a first population of bacteria and/or a bacterial suspension) doped with an additive and that is suitable for oral delivery. The process may include manufacturing a full-spectrum MRT composition 18. An additive may be added 20. The additive may be similar to those described herein. The new MRT composition may be stabilized 22. Once the purified sample has been purified and stabilized in an aqueous suspension which may be suitable for delivery via a gastro-nasal tube or an enema, the sample may be further processed to be suitable for an oral delivery, such as in the form of tablets, troches, or capsules. For example, the liquid MRT composition may be converted to a solid 24. This may include lyophilization or another suitable process. Once the sample has been dried/lyophilized, the sample may be further processed and the sample may be encapsulated 26.

[0122] Other processes are contemplated where two separate freeze drying processes may be utilized. For example, a full spectrum MRT composition may be converted to a solid (e.g., lyophilized) and may be ground into particulates. In this example, the full spectrum MRT composition may include a first population of bacteria and/or a bacterial suspension (e.g., without and additive). The additive (e.g., a second position of bacteria/organisms) may also be freeze dried during a parallel lyophilization process. The lyophilized additive may also be ground into particulates. The lyophilized full spectrum product and the lyophilized additive may be mixed together and encapsulated (e.g., in a manner similar to what is disclosed herein). This may include adding a desired excipient and/or other materials.

[0123] In addition to what is disclosed herein, the present disclosure is directed to compositions, methods of manufacture and methods of treatment utilizing MRT composition for the treatment of a number of different conditions. For example, the present disclosure pertains to compositions, methods of manufacture and methods of treatment utilizing MRT composition for the treatment of Clostridium difficile infections (CDI). CDI is a common nosocomial infection and is frequently associated with severe morbidity and mortality, especially in elderly patients. While CDI treatment is one example use for the MRT compositions disclosed herein, this is not intended to be limiting. Other diseases and/or conditions are contemplated. Some of the medical conditions that may be desirably impacted by treatment with MRT compositions may include cardiovascular and/or peripheral vascular disease, allergies, obesity, hypoglycemia, constipation, celiac sprue (e.g., celiac disease), cancer including gastrointestinal cancer (e.g. gastrointestinal cancer is at least one of stomach cancer, esophageal cancer, colon cancer gallbladder cancer, liver cancer, pancreatic cancer, colorectal cancer, anal cancer, melanoma, lunch cancer, bladder cancer, prostate cancer, and gastrointestinal stromal tumors), hepatic encephalopathy, myoclonus dystonia, sacrolileitis, spondyloarthropathy, spondylarthritis, proximal myotonic myopathy; an autoimmune disease nephritis syndrome, autism, travelers’ diarrhea, small intestinal bacterial overgrowth, chronic pancreatitis, a pancreatic insufficiency, chronic fatigue syndrome, benign myalgic encephalomyelitis, chronic fatigue immune dysfunction syndrome, Parkinson’s Disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), degenerative neurological diseases, Grand mal seizures or petitmal seizures, Steinert’s disease, chronic infectious mononucleosis, epidemic myalgic encephalomyelitis, idiopathic thrombocytopenic purpura (ITP), an acute or chronic allergic reaction obesity, anorexia, irritable bowel syndrome (IBS or spastic colon) Crohn’s disease, inflammatory bowel disease (IBD), colitis, ulcerative colitis or Crohn’s colitis, chronic infectious mononucleosis, epidemic myalgic encephalomyelitis, acute or chronic urticarial, lupus, rheumatoid arthritis (RA) or juvenile idiopathic arthritis (JIA), pre-diabetic syndrome, fibromyalgia (FM), Type I or Type II diabetes, asthma, acute or chronic insomnia, migraines, urinary tract infections (UTIs), and attention deficit/hyperactivity disorder (ADHD).

[0124] In the case of humans, the present disclosure encompasses methods of treatment of chronic disorders associated with the presence of abnormal enteric microflora. Such disorders include but are not limited to those conditions in the following categories: gastro intestinal disorders including irritable bowel syndrome or spastic colon, functional bowel disease (FBD), including constipation predominant FBD, pain predominant FBD, upper abdominal FBD, nonulcer dyspepsia (NUD), gastro-oesophageal reflux, inflammatory bowel disease including Crohn’s disease, colitis (including colitis and/or GI inflammatory side effects caused by checkpoint therapies for cancer), ulcerative colitis, indeterminate colitis, collagenous colitis, microscopic colitis, chronic Clostridium difficile infection, pseudemembranous colitis, mucous colitis, antibiotic associated colitis, idiopathic or simple constipation, diverticular disease, AIDS enteropathy, small bowel bacterial overgrowth, celiac/coeliac disease, polyposis coil, colonic polyps, chronic idiopathic pseudo obstructive syndrome; chronic gut infections with specific pathogens including bacteria, viruses, fungi and protozoa; viral gastrointestinal disorders, including viral gastroenteritis, Norwalk viral gastroenteritis, rotavirus gastroenteritis, AIDS related gastroenteritis; liver disorders such as primary biliary cirrhosis, primary sclerosing cholangitis, fatty liver or cryptogenic cirrhosis; rheumatic disorders such as rheumatoid arthritis, non-rheumatoid arthritidies, non-rheumatoid factor positive arthritis, ankylosing spondylitis, Lyme disease, and Reiter’s syndrome; immune mediated disorders such as glomerulonephritis, haemolytic uraemic syndrome, juvenile diabetes mellitus, mixed cryoglobulinaemia, polyarteritis, familial Mediterranean fever, amyloidosis, scleroderma, systemic lupus erythematosus, and Behcets syndrome; autoimmune disorders including systemic lupus, idiopathic thrombocytopenic purpura, Sjogren’s syndrome, haemolytic uremic syndrome or scleroderma: neurological syndromes such as chronic fatigue syndrome, migraine, multiple sclerosis, amyotrophic lateral sclerosis, myasthenia gravis, Guillain-Barre syndrome, Parkinson’s disease, Alzheimer’s disease, Chronic Inflammatory Demyelinating Polyneuropathy, and other degenerative disorders; psychiatric disorders including chronic depression, schizophrenia, psychotic disorders, manic depressive illness; regressive disorders including, Asbergers syndrome, Rett syndrome, attention deficit hyperactivity disorder (ADHD), and attention deficit disorder (ADD); the regressive disorder, autism; sudden infant death syndrome (SIDS), anorexia nervosa; dermatological conditions such as chronic urticaria, acne, dermatitis herpetiformis and vasculitis disorders; and cardiovascular and/or vascular disorders and diseases.

[0125] Globally, the increase in the prevalence of drug resistant organisms has created many challenges for clinicians that may pose public health risks. Infections by drug resistant organisms (e.g., vancomycin-resistant Enterococcus (VRE)) and Clostridium difficile infection share similar risk factors. VRE is a nosocomial pathogen that can be a complication among transplant and immune compromised patients. VRE carriers may also be at increased risk for infection due to VRE and also be a potential source of VRE transmissions to others. VRE shedding in stool increases with antimicrobial exposures and decreases with normalization of the intestinal microbiota after antimicrobials are discontinued. Accordingly, normalization of intestinal microbiota may not only be useful for treating Clostridium difficile infections (including chronic infections), these treatments may also be useful for treating infections by drug resistant organisms (e.g., VRE and/or other drug resistant organisms including those disclosed herein).

[0126] In some instances, the microbiota restoration therapy compositions (and/or fecal bacteriotherapy compositions) disclosed herein may be used to treat patients with infections by drug resistant organisms and/or multi-drug resistant organisms (MDRO). The drug resistant organisms may be resistant to antimicrobial agents (e.g., antibiotics, antivirals, antifungals, antiparasitics, other drugs, combinations thereof, and the like) and may include drug resistant micro-organisms such as bacteria, viruses, fungi, parasites, etc. The infections that can be treated by the microbiota restoration therapy compositions disclosed herein may be along the digestive tract or along other systems of the patient.

[0127] The microbiota restoration therapy compositions may be used to treat infections by a variety of drug resistant organisms such as vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum b-lactamase producing gram-negative bacteria, Klebsiella pneumoniae carbapenemase producing gram negative bacteria, multi-drug resistant gram negative rods bacteria (e.g., such as Enterobacter species, E.coli , Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa ), drug resistant Enterobacter species, multi-drug resistant tuberculosis (e.g., Mycobacterium tuberculosis), drug resistant staphylococci, drug resistant enterococci, drug resistant gonococci, drug resistant streptococci (e.g., including Streptococcus pneumoniae), drug resistant salmonella, drug resistant gram negative bacteria, drug resistant Candida, drug resistant HIV, drug resistant influenza virus, drug resistant cytomegalovirus, drug resistant herpes simplex virus, drug resistant malaria, drug resistant Plasmodium vivax, drug resistant Plasmodium falciparum, drug resistant Toxoplasma gondii, and the like, and/or other drug resistant organisms. These are just examples.

[0128] Treatment of infections by drug resistant organisms with the microbiota restoration therapy compositions disclosed herein may include treating patients with no prior history of infection by or caused by a drug resistant organism, treating patients with a single prior infection by a drug resistant organism, treating patients with two or more (e.g., two, three, four, five, six, or more) prior infections by a drug resistant organism, etc. In some instances, the microbiota restoration therapy compositions may be used to treat a patient with three prior infections by a drug resistant organism. In other instances, the microbiota restoration therapy compositions may be used to treat a patient with two prior infections by a drug resistant organism if the prior infections resulted in hospitalization, if the prior or current infections require treatment with toxic drugs, or if the prior infections were all from the same organism.

[0129] In some instances, MRT compositions can be administered to a patient using an enema or other suitable technique. However, it may be desirable to orally administer an MRT composition. In order to prepare an MRT composition in a form suitable for oral administration, a number of steps may be carried out. Generally, these steps may include collecting a fecal sample, processing the fecal sample, lyophilizing or“freeze-drying” the processed fecal sample (or otherwise converting the processed fecal sample from a liquid to a solid), adding one or more additives and/or excipients, and forming an oral form of the MRT composition from the lyophilized material and additives (e.g., a tablet, capsule, liquid preparation, or the like). Some additional details regarding at least some of these steps are disclosed herein.

[0130] Also disclosed herein are MRT compositions that include a first population of bacteria/organisms (e.g., as described and/or disclosed herein) and a drug additive. The drug additive may include a drug such as mesalamine. Other drugs are contemplated including rectally administered drugs, antibodies, infliximab, and/or the like. In some instances, the MRT composition including the first population of bacteria/organisms and mesalamine may be administered rectally via an enema. Other routes of administration are contemplated including oral routes of administration. In some instances, treatment regimens are contemplated that include administering an MRT composition that includes a first population of bacteria/organisms (e.g., as described and/or disclosed herein) and a drug additive to a patient. For example, the treatment regimen may include administering an MRT composition that includes a first population of bacteria/organisms (e.g., as described and/or disclosed herein) and mesalamine to a patient with ulcerative colitis. The mesalamine may help to ameliorate/attenuate the inflammatory response so that the first population of bacteria/organisms can efficiently engraft or otherwise populate/repopulate the gut of the patient. As needed/desired, the patient may subsequently be administered an MRT composition (e.g., an MRT composition, alone, without a drug and/or additive). Additional treatment regimens may include administering an MRT composition to a patient (e.g., an MRT composition, alone, without a drug and/or additive) and, if the patient demonstrates an inflammatory response, the patient may be subsequently treated with an MRT composition that includes a first population of bacteria/organisms (e.g., as described and/or disclosed herein) and mesalamine. Additional treatment regimens may include administering an MRT composition that includes a first population of bacteria/organisms (e.g., as described and/or disclosed herein) and mesalamine to a patient previously treated for IBD that later develops a C. difficile infection (e.g., treating a C. difficile infection that develops after a patient with IBD is treated for IBD). In such cases, the MRT composition may help reduce the possibility that the patient has an IBD flare after being treated for the C. difficile infection.

[0131] U.S. Patent No. 9,629,881 is herein incorporated by reference.

[0132] U.S. Patent Application Pub. No. US 2018/0289750 is herein incorporated by reference.

[0133] U.S. Patent Application No. 62/666,904 is herein incorporated by reference.

[0134] Examples

[0135] The disclosure may be further clarified by reference to the following Examples, which are prophetic in nature and serve to exemplify some embodiments, and not to limit the disclosure in any way.

[0136] Example 1 - Example process for manufacturing a first population of bacteria (e.g. a full-spectrum MRT drug product)

[0137] NOTE: Steps 1-3 may be performed within a Type II Class B2 biosafety cabinet. All instruments used to manipulate drug substance, excipient solution or drug product formulation (e.g., tongue depressors, serological pipettes) are single use instruments provided pre-sterilized by the manufacturer. Filter bags, closure bags and EVA bags are also provided pre-sterilized by the manufacturer.

[0138] Step 1 - Collect Donor Pooled Drug Substance Test Sample and Drug Substance Reserve Samples

[0139] A fresh human fecal sample can be collected from a pre-screened donor. In some instances, multiple samples from the same donor are collected and pooled. The pooled sample may be stored at 5°C ± 3°C in a sterile microbiology container. One or more additional samples, and/or portions of the pooled sample, may be stored as a reserve samples in a sterile microbiology container in a -80°C freezer.

[0140] Step 2 - Dispense Drug Substance [0141] A 50 ± lOg sample (e.g., the drug substance) may be taken from the pooled sample and disposed in a filter bag assembly. The filter bag assembly may include a filter bag within an outer closure bag.

[0142] Step 3 - Dispense Excipient Solution

[0143] An excipient solution (e.g., which also may be understood to be a diluent, cryoprotectant, or other solution) may be added to the drug substance. The excipient solution may include 30g/L polyethylene glycol (e.g., polyethylene glycol 3350 powder) in 0.9% sodium chloride. The excipient solution may be added at a ratio of 3mL of the excipient solution per lg of drug substance. If the weight of the remainder of the pooled sample is 50 ± lOg or more, steps 2 and 3 may be repeated. If the weight of the remainder of the pooled sample is less than 50 ± lOg, the remainder of the pooled sample may be discarded and the filter bag assemblies may be closed.

[0144] Step 4 - Mixing and Filtration

[0145] One at a time, closed filter bag assemblies containing drug substance and excipient solution are placed into the paddle mixer and processed for 2 minutes at a speed of 230 RPM. The paddle mixer run time and speed are electronically controlled, settings are verified prior to manufacturing every batch.

[0146] NOTE: Steps 5-6 are performed within a Type II Class B2 biosafety cabinet.

[0147] Step 5 - Collect Drug Product Quality Control and Reserve Samples

[0148] The first filter bag processed in the paddle mixer is opened and the filtrate may be withdrawn and filled into cryovials. The cryovials can be submitted to quality control (QC) and stored in a -80°C freezer. QC samples are only collected from the first dose manufactured for each batch.

[0149] Step 5A - Drug Product Quality Control Release Testing

[0150] Quality Control drug product release samples are tested in a QC laboratory.

Reserve samples may be stored in a -80°C freezer.

[0151] Step 6 - Filling

[0152] The fill tube cap of an ethylene vinyl acetate (EVA) enema bag may be removed and 150 ± 30g of the microbiota suspension (e.g., the filtered drug substance and excipient) is withdrawn from the filter bag assembly and filled into the EVA bag through the fill port. When filling is complete, the fill tube cap is replaced, sealing the EVA bag prior to removal from the biosafety cabinet.

[0153] Step 7 - Seal EVA Bag [0154] The fill tube on the EVA bag may be sealed between the bag and fill cap using a tube sealer to prevent inadvertent opening of the container-closure.

[0155] Step 8 - Atach Batch Identification Tags and Quarantine Labels

[0156] A tag labeled with drug product batch number, and a“quarantine” batch status sticker is atached to every EVA enema bag.

[0157] Step 9 - Refrigerate Drug Product at 5°C ± 3°C

[0158] The in-process drug product is refrigerated at 5°C ± 3°C prior to freezing in a - 80°C freezer. The drug product can be held at 5°C ± 3°C for up to 24 hours prior to freezing in a -80°C freezer.

[0159] Step 10 - Freeze Drug Product in a -80°C Freezer

[0160] The drug product (e.g., contained within the sealed enema bag) may be transferred from refrigerated storage to a designated -80°C drug product quarantine freezer. Quarantined drug product remains in this location until it is dispositioned by QC.

[0161] Step 11 - Drug Product Disposition

[0162] If all donor and QC test results are acceptable the batch will be dispositioned as released. If results are not acceptable the batch will be dispositioned as rejected and discarded.

[0163] Step 12 - Release Drug Product

[0164] Drug product dispositioned as accepted will be removed from the -80°C quarantine freezer, labelled“Accepted” and transferred to a designated released drug product -80°C freezer. The accepted, released drug product may be thawed and administered to a patient (e.g., via enema).

[0165] Example 2: Example process for manufacturing a second population of bacteria/organisms and/or additives

[0166] Bacteria from the class Bacterioidia can be cultured using standard bacterial culture techniques. The bacteria can be collected by centrifugation and suspended in 30- 90 g/L polyethylene glycol in saline at a concentration of 1 x 10⁷ to 1 x 10¹⁰ CFU/mL.

[0167] Example 3: Another example process for manufacturing a second population of bacteria/organism and/or additives

[0168] Bacteroides thetaiotaomicron, Bacteroides ovatus, and Bacteroides caccae can be cultured using standard bacterial culture techniques in separate cultures. The bacteria from each strain can be cultured and mixed together in equal proportions. The mixture of bacteria can be collected by centrifugation and suspended in 30-90 g/L polyethylene glycol in saline at a concentration of 1 x 10⁷ to 1 x 10¹⁰ CFU/mL.

[0169] Example 4: Example process for manufacturing a Doped MRT composition by combining a first population of bacteria with a second population of bacteria/organisms and/or additives.

[0170] A first population of bacteria (e.g., the full-spectrum MRT drug product) may be manufactured according to Example 1. Fifty milliliters of the first population of bacteria (e.g., the full-spectrum MRT drug product) may have a bacterial count in the range of 1 x 10⁷ to 1 x 10¹⁰ CFU/mL

[0171] A second population of bacteria/organisms and/or additives may be manufactured according to Example 2. Fifty milliliters of the second population of bacteria/organisms and/or additives may have a bacterial count in the range of 1 x 10⁷ to 1 x 10¹⁰ CFU/mL.

[0172] Ninety milliliters of the first population of bacteria (e.g., the full-spectrum MRT drug product) may be combined with ten milliliters of the second population of bacteria/organisms and/or additives to form a Doped MRT composition.

[0173] Example 5: Another example process for manufacturing a Doped MRT composition bv combining a first population of bacteria with a second population of bacteria/organisms and/or additives.

[0174] A first population of bacteria (e.g., the full-spectrum MRT drug product) may be manufactured according to Example 1. Fifty milliliters of the first population of bacteria (e.g., the full-spectrum MRT drug product) may have a bacterial count in the range of 1 x 10⁷ to 1 x 10¹⁰ CFU/mL.

[0175] A second population of bacteria/organisms and/or additives may be manufactured according to Example 3. Fifty milliliters of the second population of bacteria/organisms and/or additives may have a bacterial count in the range of 1 x 10⁷ to 1 x 10¹⁰ CFU/mL.

[0176] Ninety milliliters of the first population of bacteria (e.g., the full-spectrum MRT drug product) may be combined with ten milliliters of the second population of bacteria/organisms and/or additives to form a Doped MRT composition.

[0177] Example 6: Another example process for manufacturing a first population of bacteria (e.g. a full-spectrum MRT drug product)

[0178] Step 1 - Thaw Suspended Intermediate [0179] Batch manufacturing of an oral MRT composition begins with selection of multiple bags of released drug product (e.g., where each bag of released drug product contains a frozen suspended intermediate manufactured as described in Example 1). Each of the bags of released drug product may be from the same donor. Alternatively, the bags of released drug product may be from different donors. The selected bags are thawed at room temperature for at least 2 hours and 500 g of suspended intermediate is transferred into 1 -liter centrifuge bottles.

[0180] Step 2 - Add Diluent

[0181] Phosphate Buffered Saline (PBS) may be added to the Suspended Intermediate in a 1 : 1 ratio by weight, mixed by intermittent gentle shaking, and held for 30 minutes at 4°C.

[0182] Step 3 - Differential Centrifugation

[0183] The diluted intermediate may be centrifuged for 2 min at l,400xg and 4°C ± 3°C. The supernatant from this slow speed spin may be transferred into new 1L centrifuge bottles containing bottle liners for further processing, and the pelleted material is discarded. The bottles containing the collected supernatant may then centrifuged at l0,000xg for 45 mins at 4°C ± 3°C. After the high-speed spin, the supernatant may discarded and the recovered microbiota are retained for further processing.

[0184] Step 4 - Resuspend the recovered microbiota with lyophilization excipient solution

[0185] A lyophilization excipient/cryoprotectant may be added to the recovered microbiota in a 1 :1 ratio (w/w). The lyophilization excipient may include 2.3 % polyethylene glycol (e.g., PEG 3350), 10% trehalose, 10% sucrose, and 1% glycerin in purified water. The mixture may be mixed using a paddle mixer to form a uniform suspension.

[0186] Step 5 - Lyophilization

[0187] The resuspended microbiota solution is aliquoted into 96-well plates (200 pl per well) and placed into a qualified lyophilizer. Lyophilization steps are described in the following table. [0188] Table 1: Lyophilization Cycle Details

[0189] Step 6 - Milling Operation

[0190] Within a NF -grade-nitrogen purged glove box workstation, lyophilized drug pellets are milled through a low energy mill.

[0191] Example 7: Another example process for manufacturing a second population of bacteria/organisms and/or additives

[0192] The bacteria from Example 2 may be lyophilized according to the process of Example 7.

[0193] Example 8: Another example process for manufacturing a second population of bacteria/organism and/or additives

[0194] The bacteria from Example 3 may be lyophilized according to the process of Example 7.

[0195] Example 9: Example process for manufacturing a Doped MRT composition by combining a first population of bacteria with a second population of bacteria/organisms and/or additives.

[0196] A first population of bacteria (e.g., the full-spectrum MRT drug product) may be manufactured according to Example 6. 400 milliliters of the first population of bacteria (e.g., the full-spectrum MRT drug product) may have a bacterial concentration in the range of 1 x 10⁷ to 1 x 10¹⁰ CFU/mL. [0197] A second population of bacteria/organisms and/or additives may be manufactured according to Example 7. 100 milliliters of the second population of bacteria/organisms and/or additives may have a bacterial count in the range of 1 x 10⁹ to 1 x 10¹¹ CFU.

[0198] 400 mL of the first population of bacteria (e.g., the full-spectrum MRT drug product) may be combined with 100 mL of the second population of bacteria/organisms and/or additives to form a Doped MRT composition.

[0199] Example 10: Another example process for manufacturing a Doped MRT composition by combining a first population of bacteria with a second population of bacteria/organisms and/or additives.

[0200] A first population of bacteria (e.g., the full-spectrum MRT drug product) may be manufactured according to Example 6. 200-250 grams of the first population of bacteria (e.g., the full-spectrum MRT drug product) may have a bacterial count in the range of 2.5 x 10⁹ to 2.5 x 10¹² CFU.

[0201] A second population of bacteria/organisms and/or additives may be manufactured according to Example 8. 200-250 grams of the second population of bacteria/organisms and/or additives may have a bacterial count in the range of 1 x 10¹² CFU.

[0202] 250 milligrams of the first population of bacteria (e.g., the full-spectrum MRT drug product) may be combines with 50 milligrams of the second population of bacteria/organisms and/or additives to form a Doped MRT composition.

[0203] Example 11 : Example process for treating graft versus host disease

[0204] The composition of the fecal stool in hematopoietic stem cell transplant (HCT) patients may correlate with the likelihood of subsequent development of severe graft versus host disease (GvHD). For example, the presence of Bacteroides thetaiotaomicron, Bacteroides ovatus, and Bacteroides caccae negatively correlated with severe GvHD, whereas Bacteroides dorei may be positively correlated with severe GvHD. Fecal transplant may be a possible treatment to improve outcomes in HCT, including to prevent GvHD development.

[0205] A doped composition may be manufactured according to Example 5. The doped composition may be administered to a patient to improve outcomes in HCT, including to prevent GvHD development.

[0206] Example 12: Example process for treating graft versus host disease [0207] The composition of the fecal stool in hematopoietic stem cell transplant (HCT) patients may correlate with the likelihood of subsequent development of severe graft versus host disease (GvHD). For example, the presence of Bacteroides thetaiotaomicron, Bacteroides ovatus, and Bacteroides caccae negatively correlated with severe GvHD, whereas Bacteroides dorei may be positively correlated with severe GvHD. Fecal transplant may be a possible treatment to improve outcomes in HCT, including to prevent GvHD development.

[0208] A doped composition may be manufactured according to Example 10. The doped composition may be administered to a patient to improve outcomes in HCT, including to prevent GvHD development.

[0209] It should be understood that this disclosure is, in many respects, only illustrative. Changes may be made in details, particularly in matters of shape, size, and arrangement of steps without exceeding the scope of the disclosure. The invention’s scope is, of course, defined in the language in which the appended claims are expressed.

Claims

What is claimed is:

1. A microbiota restoration therapy composition, comprising:

a processed bacterial suspension including a first population of bacteria derived from a first human fecal sample and a second population of bacteria derived from a source other than the first human fecal sample.

2. The microbiota restoration therapy composition of claim 1, wherein the first population of bacteria, the second population of bacteria, or both are suspended in a cryoprotectant solution comprising 30-90 g/L polyethylene glycol in saline.

3. The microbiota restoration therapy composition of any one of claims 1-2, wherein the second population of bacteria is derived from a second human fecal sample.

4. The microbiota restoration therapy composition of any one of claims 1-3, wherein the second population of bacteria is derived from a bacterial culture.

5. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria includes bacteria from the family Ruminococcaceae.

6. The microbiota restoration therapy composition of any one of claims 1-5, wherein the second population of bacteria includes bacteria from the genus Bacteroides.

7. The microbiota restoration therapy composition of any one of claims 1-6, wherein the second population of bacteria includes Bacteroides fragilis.

8. The microbiota restoration therapy composition of any one of claims 1-7, wherein the second population of bacteria includes Bacteroides thetaiotaomicron.

9. The microbiota restoration therapy composition of any one of claims 1-8, wherein the second population of bacteria includes bacteria from the family Bifldobacteriaceae.

10. The microbiota restoration therapy composition of any one of claims 1-9, wherein the second population of bacteria includes bacteria from the genus Prevotella.

11. The microbiota restoration therapy composition of any one of claims 1-10, wherein the second population of bacteria includes bacteria from the genus Fusobacterium.

12. The microbiota restoration therapy composition of any one of claims 1-11, wherein the second population of bacteria includes Lactobacillus plantarum.

13. The microbiota restoration therapy composition of any one of claims 1-12, wherein the second population of bacteria includes bacteria from the classes Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli.

14. The microbiota restoration therapy composition of any one of claims 1-13, wherein the second population of bacteria includes a bacteriophage.

15. The microbiota restoration therapy composition of any one of claims 1-14, wherein the second population of bacteria includes a spore forming bacteria.

16. The microbiota restoration therapy composition of any one of claims 1-15, wherein the second population of bacteria includes a non-spore-forming bacteria.

17. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of bacteria from the family Ruminococcaceae and a carrier.

18. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of bacteria from the genus Bacteroides.

19. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of Bacteroides fragilis and a carrier.

20. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of Bacteroides thetaiotaomicron and a carrier.

21. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of bacteria from the genus Fusobacterium and a carrier.

22. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of bacteria from the genus Prevotella and a carrier.

23. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of bacteria from the family Bifldobacteriaceae and a carrier.

24. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of Lactobacillus plantarum and a carrier.

25. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of bacteria from the classes Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli.

26. The microbiota restoration therapy composition of any one of claims 1-25, further comprising a fungus.

27. The microbiota restoration therapy composition of any one of claims 1-26, further comprising mesalamine.

28. The microbiota restoration therapy composition of any one of claims 1-27, further comprising infliximab.

29. A microbiota restoration therapy composition, comprising:

a bacterial suspension derived from a first human fecal sample, the bacterial suspension comprising 30-90 g/L polyethylene glycol in saline;

a bacterial additive mixed with the bacterial suspension; and

wherein the bacterial additive includes one to four classes of bacteria.

30. The microbiota restoration therapy composition of claim 29, wherein the bacterial additive is derived from a second human fecal sample different from the first human fecal sample.

31. The microbiota restoration therapy composition of any one of claims 29-30, wherein the bacterial additive includes cultured bacteria.

32. The microbiota restoration therapy composition of any one of claims 29-31, wherein the bacterial additive includes bacteria from the family Ruminococcaceae.

33. The microbiota restoration therapy composition of any one of claims 29-32, wherein the bacterial additive includes bacteria from the genus Bacteroides.

34. The microbiota restoration therapy composition of any one of claims 29-33, wherein the bacterial additive includes Bacteroides fragilis.

35. The microbiota restoration therapy composition of any one of claims 29-34, wherein the bacterial additive includes Bacteroides thetaiotaomicron.

36. The microbiota restoration therapy composition of any one of claims 29-35, wherein the bacterial additive includes bacteria from the family Bifidobacteriaceae.

37. The microbiota restoration therapy composition of any one of claims 29-36, wherein the bacterial additive includes a bacteria from the genus Prevotella.

38. The microbiota restoration therapy composition of any one of claims 29-37, wherein the bacterial additive includes bacteria from the genus Fusobacterium.

39. The microbiota restoration therapy composition of any one of claims 29-38, wherein the bacterial additive includes Lactobacillus plantarum.

40. The microbiota restoration therapy composition of any one of claims 29-39, further comprising mesalamine.

41. The microbiota restoration therapy composition of any one of claims 29-40, further comprising infliximab.

42. The microbiota restoration therapy composition of any one of claims 29-41, wherein the bacterial additive includes bacteria from the classes Clostridia, Bacterioidia, Gammaproteobacteria, and Bacilli.

43. The microbiota restoration therapy composition of any one of claims 29-42, wherein the bacterial additive consists essentially of bacteria from four classes and a carrier.

44. The microbiota restoration therapy composition of any one of claims 29-42, wherein the bacterial additive consists essentially of bacteria from three classes and a carrier.

45. The microbiota restoration therapy composition of any one of claims 29-42, wherein the bacterial additive consists essentially of bacteria from two classes and a carrier.

46. The microbiota restoration therapy composition of any one of claims 29-45, wherein the bacterial additive includes a bacteriophage.

47. The microbiota restoration therapy composition of any one of claims 29-46, further comprising a fungus.

48. The microbiota restoration therapy composition of any one of claims 29-47, wherein the bacterial additive consists essentially of bacteria from one class and a carrier.

49. A method for manufacturing a microbiota restoration composition, the method comprising:

manufacturing a full-spectrum base composition, the method for manufacturing the full-spectrum base composition comprising:

collecting a fresh human fecal sample,

adding a diluent to the fresh human fecal sample to form a diluted sample, wherein the diluent includes 30-90 g/L polyethylene glycol in saline, and filtering the diluted sample to form a filtrate comprising the full-spectrum base composition; and

adding a bacterial additive to the full-spectrum base composition, the bacterial additive including one to four classes of bacteria.

50. A microbiota restoration therapy composition, comprising:

a bacterial suspension derived from a fresh human fecal sample, the bacterial suspension comprising 30-90 g/L polyethylene glycol in saline;

a cultured additive mixed with the bacterial suspension; and

wherein the cultured additive comprises one or more of a cultured bacteria, a bacteriophage, and a fungus.

51. The microbiota restoration therapy composition of any one of claims 1-28, wherein the second population of bacteria includes genetically modified bacteria.

52. The microbiota restoration therapy composition of claim 51, wherein the genetically modified bacterial include a gene coding for antibacterial resistance.

53. The microbiota restoration therapy composition of any one of claims 1-16, wherein the second population of bacteria includes bacteria from the genus Akkermansia.

54. The microbiota restoration therapy composition of any one of claims 1-16, wherein the second population of bacteria includes Akkermansia muciniphila.

55. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of Akkermansia muciniphila.

56. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of Akkermansia muciniphila and a carrier.

57. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists of Akkermansia muciniphila.

58. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists of Akkermansia muciniphila and a carrier.

59. The microbiota restoration therapy composition of any one of claims 1-16, wherein the second population of bacteria includes bacteria from the family Enter ococcaceae.

60. The microbiota restoration therapy composition of any one of claims 1-16, wherein the second population of bacteria includes Enterococcus hirae.

61. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of Enterococcus hirae.

62. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists essentially of Enterococcus hirae and a carrier.

63. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists of Enterococcus hirae.

64. The microbiota restoration therapy composition of any one of claims 1-4, wherein the second population of bacteria consists of Enterococcus hirae and a carrier.

65. A microbiota restoration therapy composition, comprising:

a bacterial suspension derived from a human fecal sample, the bacterial suspension comprising 30-90 g/L polyethylene glycol in saline; and

a drug additive mixed with the bacterial suspension, the drug additive including mesal amine.