Software Technology for “Microbiota Restoration Therapy (MRT), compositions, and methods of manufacturing”

Infectious Diseases – Lee A. Jones, Courtney R. Jones, Edwin J. Hlavka, Ryan D. Gordon, Rebiotix Inc

Abstract for “Microbiota Restoration Therapy (MRT), compositions, and methods of manufacturing”

The invention discloses microbiota therapy compositions and methods of manufacturing, processing and/or delivering them. A microbiota restoration treatment composition could be made by taking a sample of human feces and then adding a diluent. This will create a diluted sample. The diluent could also include a cryoprotectant. Mixing the diluted samples with a mixer and filtering the filtrate may be part of the method. Filtering can form a filtrate. This may include placing the filtrate in a bag to collect it and sealing the bag.

Background for “Microbiota Restoration Therapy (MRT), compositions, and methods of manufacturing”

“A variety of methods and compositions have been created to treat diseases or conditions of the digestive track. Each of the existing compositions and methods has its advantages and disadvantages. It is important to continue developing alternative compositions and methods of treating conditions and diseases of the digestive tract.

The invention discloses microbiota therapy compositions and methods of manufacturing, processing and/or delivering them. A microbiota restoration treatment composition could be made by taking a sample of human feces and then adding a diluent. This will create a diluted sample. The diluent could also include a cryoprotectant. Mixing the diluted samples with a mixer and filtering the filtrate may be part of the method. Filtering can form a filtrate. This may include placing the filtrate in a bag to collect it and sealing the bag.

A method of manufacturing, processing and packaging a microbiota-restoration therapy composition might include taking a sample from a prescreened donor, then transferring it to a filter bag and adding a diluting agent to the bag. A cryoprotectant may be included in the diluent. This may include sealing the filter bag and then transferring it to a mixer. Finally, transfer the filtrate from filter bag into a sample bag. The microbiota restoration therapy content within the sample bag can be determined by transferring the filtrate from the filter bags to a sample bag. This may include sealing the bag and cooling the bag before transferring the cool bag to a temperature controlled storage device. The microbiota restoration treatment composition may be frozen before being transferred to the controlled temperature storage device. This may include freezing the microbiota restoration treatment composition and packaging it in an insulated bag. Finally, the package is shipped to a treatment facility.

A pre-screened donor may give a sample of feces to be used as a guideline for medical treatment. The sample is then transferred to a bag and diluent added to it. A cryoprotectant may be included in the diluent. This may include sealing the filter bag and then transferring it to a mixer. Finally, transfer the filtrate from filter bag into a sample bag. The microbiota restoration treatment composition may be defined by the transfer of the filtrate from the filterbag to a sample bag. This may include sealing the bag and cooling the bag before transferring the cool bag to a temperature controlled storage device. The microbiota restoration treatment composition may be frozen after being transferred to the controlled temperature storage device. This may include freezing the frozen microbiota restore therapy composition, wrapping the sample bag in an insulation system, and shipping the bag to a treatment facility to administer the microbiota therapy composition to the patient.

“The summary of some embodiments does not include every embodiment disclosed or every implementation of this disclosure. These embodiments are more clearly illustrated by the Figures and Detailed Description which follow.

“For the following terms, these definitions shall apply, except where a different definition is given elsewhere in this specification or in the claims.”

“All numeric values are assumed to be modified by?about?. This is true regardless of whether it is explicitly stated. The term “about” refers to a range of numbers that can be considered equivalent to the recited value. The term?about? refers to a set of numbers that one skilled in the art would consider equivalent or equal to the recited value (e.g. having the same function, result). In many cases, the term ‘about’ may include numbers that are rounded to the nearest significant figure. Many times, the terms?about? may contain numbers that have been rounded to nearest significant number.

“The recitation by endpoints of numerical ranges includes all numbers within the range (e.g. 1-5 includes 1, 1.5, 2.75, 3.80, 4, and 5).

“The singular forms?a,?an, and??the as used in this specification and the attached claims are the ones that are used. If the content clearly indicates otherwise, plural referents should be included. The term “or” is used in the specification and the attached claims. The term?or?, as used in this specification and the appended claims, is commonly used in its full meaning including?and/or’ Unless the content clearly indicates otherwise,

“It should be noted that references to?an embodiment? or?some embodiments?”, etc., may indicate that an embodiment described may contain one or more specific features, structures, or characteristics. These citations don’t necessarily mean all embodiments have the same features, structures, or characteristics. If certain features, structures and/or characters are mentioned in connection to one embodiment, it is important to understand that these features, structures and/or attributes may be used in connection with other embodiments, even if they are not specifically described.

“The following detailed description should only be read in conjunction with the drawings, in which identical elements are numbered the exact same in all drawings. These drawings are not to scale and illustrate illustrative embodiments. They do not limit the invention’s scope.

“?Mammal? “?Mammal” as used herein refers any member of the Mammalia class, including humans and nonhuman primates like chimpanzees and other ape species, farm animals such cattle, sheeps, pigs goats and horses, domestic mammals such dogs and cats, rodents such mice, rats, guinea-pigs, and others. This term does not indicate a specific age or sex. This term includes both adult and newborn subjects as well as female and male fetuses.

“Cryoprotectant” is a term that refers to a substance used to protect biological tissues or cells from the effects of freezing.

“The term “microbiota” as used herein refers to the human microbiome, the human microbiota or the human gut microbiota. The term?microbiota’ can be used to refer to the human human microbiome or human microbiota. The human microbiome, also known as the human microbiota, is a collection of microorganisms found in the skin’s surface and deep layers. It can also be seen in saliva, oral mucosa and the conjunctiva. The human microbiome includes bacteria, fungi and viruses as well as archaea. At least one of these organisms can perform useful tasks for the human host. These microorganisms are not harmful to the host but rather help maintain health. This is why this group of organisms is often called “normal flora.”

“The human gastrointestinal tract’s microorganism population is often referred to as “microbial flora”, “gut flora” and/or “gut microbiota”. The human microbial flora includes a variety of microorganisms that help in digestion, synthesis of vitamins and the creation of enzymes not made by the body.

“Microbiota restoration therapy” is a term that refers to a composition that may contain human feces containing viable gut bacteria from a donor or patient, as well as a diluent and a cryoprotectant. Other compositions include freeze-dried, reconstituted feces and a?synthetic’. Fecal composition. Before it is used in microbiota restoration therapy, the human fecal matter is tested for pathogenic microorganisms. 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.”

“More that 1000 species of bacteria live in healthy GI tracts. Clostridia is anaerobic and spore-forming bacteria. Clostridium difficile (?C diff?) is a pathogen that produces toxins that can cause harm to humans. Clostridium difficile (?C diff?) Clostridium difficile (?C diff?) is one type of Clostridium. If it becomes too abundant in the GI tract, it can release toxic substances that can cause many symptoms including constipation, diarrhea and inflammation. In some cases, this can even lead to death.

Clostridium difficile spores can survive extreme conditions that other active bacteria cannot. Clostridium difficile does not thrive in a healthy GI system. Clostridium difficile can be overgrown if antibiotics are used. Clostridium difficile can be resistant to some antibiotics in certain cases. Clostridium difficile spores can be resistant to antibiotics once the normal intestinal flora has been cleared. This results in a large population.

According to the Centers for Disease Control and Prevention (CDC), approximately 337,000 cases (CDI) of Clostridium difficile are reported annually in the United States. This results in roughly 14,000 deaths. Current standard care is antibiotic treatment, usually with vancomycin and metronidazole. About 25% of patients who have received initial antimicrobial treatment experience a recurrence of symptoms. About 45 to 65 percent of these patients developed persistent recurrent CDI. High mortality and morbidity are associated with persistent recurrent CDI. Clostridium difficile incidence in the United States is estimated to be between 1.5 and 2.7 million per year. These numbers are increasing. According to one estimate, Clostridium difficile hospital discharges doubled between 2001 and 2005. This is a compound annual growth rate of 5% to 25%. Clostridium difficile patients with overpopulation are more likely to experience prolonged hospital stays of 3 to 36 days. Nearly 20% of patients infected will be readmitted to the hospital within 180 days. Each patient is more likely to be discharged into long-term care facilities than those not affected. Clostridium difficile has a financial impact estimated at $1-3 billion per year. Clostridium difficile overpopulation is responsible for 300 deaths per day, with a mortality rate between 1 and 7.7%. This number is increasing.

Clostridium difficile antibiotics can be used to treat diarrhea in the first instance. This is a treatment that works well within 2 to 4 days and at rates of approximately 90%. Clostridium difficile can recur after the first occurrence, which is typically within a few days to 12 weeks of ceasing antibiotics. This happens at an estimated 20% rate (15-30%). The rate of recurrence increases dramatically with each subsequent occurrence. It is an average 40% rate (e.g., 15%-30%) after the second recurrence and an additional 60% or more thereafter. It is estimated that about 5% of patients will have six or more recurrences.

Clostridium difficile treatment is usually different for each case. Metronidazole can also be taken orally for the first mild to moderate recurrence (e.g. 500 mg three times daily,?TID?) For a period of 10-14 days. Vancomycin can also be given orally in tapered doses or pulsed doses to prevent a second recurrence (e.g., at doses of 125 mg four times per day (?QID?)). For 14 days, at a dose 125 mg twice daily (?BID?) For 7 days, at a dose 125 mg once daily (?BID?) For 7 days, at a dose 125mg, once daily (?QD?) You can apply vancomycin at higher doses for a third recurrence (e.g., at doses of 125 mg four times per day (?QID?). For 14 days, combined with any other treatment options such as intravenous immunoglobulin (e.g. at a dose 125 mg four times daily (?QID?)). for 14 days), etc.”

Recurrent CDI patients may have to resort to fecal transplantation (FT) as a last resort. FT is used to reestablish a healthy microbial mixture in the patient’s gastrointestinal microbiota after antimicrobial treatment. There have been over 480 cases with a?90% success rate in curing recurrent CDI. The FT material has not caused any adverse reactions. Current institutional practice is to collect fecal samples from relatives or volunteers in the hospital for transplantation. This method of treatment has a problem in that the FT material isn’t standardized. While donors are usually selected if they are healthy at the time of donation it is not sufficient to ensure the viability and quality of the microbes that will be transplanted. The donor may not know of a disease that affects the fecal material. The quality of the raw fecal matter can also affect the viability and quality of the FT material that is given to patients. Each institution must also handle raw fecal material. This is not desirable.

A standardized, pre-processed MRT product is needed to ensure quality and viability for patients at time of delivery. A MRT product that can be easily handled and delivered to the appropriate facility is desirable. This will eliminate the need to handle raw feces material at each institution. These improvements will make MRT a viable and desirable option for CDI, rather than being a last resort treatment at a few institutions.

This document discusses, among others, the receipt of multiple donor fecal specimens from a plurality, and the storage and indexing of each donor fecal sample using at least one characteristic. The donor fecal samples can be processed and screened for use in fecal therapy to eliminate pathogenic or unwanted organisms from the digestive tract of patients with healthy or desirable microbiota.

“The disclosure describes a composition of microbiota restoration therapy that includes a combination of effective amounts of fecal microbiota as well as an effective amount a cryoprotectant. Polyethylene glycol is an example of a cryoprotectant. The disclosure also states that the concentration of polyethylene glycol in the microbiota recovery therapy composition is between 5-60 g/ml and 5-30 g/ml. It should not exceed 30 g/ml. As a diluent, the composition may also contain saline. Polyethylene glycol with an average molecular mass of 600 to 20000 may be included in the disclosed compositions. PEG-3150, which has an average molecular mass of 3150, can be used. Certain embodiments of the microbiota therapy compositions for microbiota restoration contain fecal microbiota from one or more human stool samples.

“Other cryoprotectants can be used, such as betaine, dextrose and glycine.

“In other embodiments, viability of the microbiota compositions disclosed may be confirmed by culturing (and/or filtrate and/or an bacteriotherapy composition) on Bacteroides Bile Esculinagar (BBE), plate (available at Becton, Dickinson and Company, catalogue number 221836, BBL). Bacteroides Bile Esculin Plate BBE, or a Center for Disease Control plate (available at Becton, Dickinson and Company, catalogue number 221733, BL) CDC Anaerobe 5% sheep blood agar plates), or both. At least in some instances, viability of the microbiota contained in the compositions disclosed may be confirmed by a BBE or CDC plate. This is done by a presence of a colony forming units (CFU) count of approximately 30 to 300 CFU at a serial dilution (10?5) or a CFU count between 30 and 300 CFU at a 10?6 serial dilution (10?6). Microbiota restoration therapy compositions are also provided where the microbiota concentration is at least 107 microbes/ml. The methods of producing a microbiota therapy composition could also include the steps of pre- and/or post-donor screening using the human stool sample taken in the time period between.

Plating tests are able to confirm viability and diversity of living microbes. The quality of the stool sample from humans and the MRT product that was made from it is also determined by the mix or diversity of microbes. The CDC plates and BBE plates can be used together or separately to measure quality through diversity, as described in this document.

The disclosure also includes methods to ascertain the quality and quantity of a human stool specimen to be used in microbiota therapy. These methods include identifying a donor of human stool; conducting pre-donation screening, which involves a health history questionnaire, daily diet questionnaire, and at most one blood test. Collecting a stool sample from the donor and processing it to make one or more microbiota therapy compositions. Keeping in quarantine any or all of the microbiota therapy compositions that were made from the donated stool samples; The likelihood of obtaining a healthy sample may be increased by pre- and post screening. This interval was chosen to allow enough time for any disease or other factor present at the time human stool is collected to manifest symptoms or be positive in a serum or human stool test at the post-screening. The composition can also be stored in a quarantine until the pre- or post-screening confirms that donor health has been confirmed.

“The disclosure may also include at least one step to test the stool sample for infectious disease. Additionally, the methods of the disclosure may further include the step of testing the human stool sample for a constituent selected from the group consisting of: C. difficile; Norovirus; Adenovirus; Enteric Pathogens; Giardia antigen; Cryptosporidium antigen; Acid-fast staining (Clyslospora, Isospora); ova and parasites; Vancomycin-resistant enterococci (VRE); Methicillin-resistant Staphylococcus aureus (MRSA) and combinations thereof. The disclosure could include the testing of blood from the donor. This includes at least one test for HIV, Hepatitis A, Hepatitis B, Hepatitis C and combinations thereof.

A representative way to make a microbiota therapy composition using a human stool sample is to collect a sufficient amount of stool from the patient, add saline and a cryoprotectant (e.g. polyethylene glycol), then mix the mixture. The filtrate containing microbes can be then filtered. You can take a portion of the filtrate for testing, and you can freeze the rest to be tested. Testing will be done using the results of pre-screening and post-screening.

The present disclosure relates to compositions, manufacturing methods, and treatment methods that utilize microbiota restoration therapy for the gastrointestinal tract. This involves replacing pathogenic or ineffective organisms by healthy, effective bacteria. Clostridium difficile infection (IBS), Crohn?s disease (UC), ulcerative colitis resulting in a staphylococcus/C. diff infection, inflammatory bowel disorder (IBD), ulcers and diabetes as well as constipation and obesity are some examples of conditions that can be treated.

“In some cases, compositions, manufacturing methods, and treatment methods utilizing microbiota therapy (MRT), for Clostridium difficile infection (CDI) treatment are shown. CDI is a common nosocomial disease and is often associated with high mortality and severe morbidity, particularly in older patients.

Each person has a unique gut microbiota, with 500-5000 species of bacteria, viruses, microorganisms and archaea. These microorganisms provide a host of beneficial symbiotic functions. They include aiding in digestion, nutrition for the colon, production of vitamins, and stimulating the immune system. However, an improperly balanced or functioning gut microbiota may play a role in certain diseases or afflictions, such as pseudomembranous colitis, Clostridium difficile colitis, antibiotic-associated diarrhea (AAD), ulcerative colitis (UC), pouchitis, irritable bowel syndrome (IBS), obesity, among others.”

According to the inventor, there are systems and methods that provide bacteriotherapy for treating afflictions related to the gut microbiota. This includes Clostridium difficile colitis. The system or method can be used to displace pathogenic organisms from the digestive track of patients with healthy bacterial bacteria, or to benefit an individual suffering from an affliction related to the gut microbiota. The systems and methods described in this invention can be used to provide bacteriotherapy to patients by integrating with the existing facilities and procedures of hospitals and clinics. Similar treatment may be successful for patients suffering from IBS, Crohn?s disease, ulcerative colitis, IBD, IBD, IBS, and other gastrointestinal, metabolic or digestive tract-related diseases. Bacteriotherapy can also be used to help with weight loss by replacing inefficient microbiota with more productive. Bacteriotherapy can also be used to treat peripheral and cardiovascular disease.

“For example, bacteriotherapy can be used to treat Clostridium difficile and other digestive tract diseases. It may include a combination antibiotics and the repopulation of healthy or desired mammalian bacteria flora. Fecal bacteriotherapy or fecal transplant can be used in certain cases to repopulate bacterial flora.

“Fecal bacteriotherapy is the process of inserting a fecal specimen from a healthy donor or one of its desired characteristics into a patient’s gastrointestinal tract to restore a healthy or desirable microbiota. Some cases may require that antibiotics be administered to the patient before the fecal sample is introduced. This will ensure that the desired or healthy gut microbiota can quickly populate the gastrointestinal tract. Some of the methods described herein include the administration of a pre-treatment antibiotic and the administration to the patient of a microbiota restore therapy composition (e.g. the microbiota recovery therapy compositions disclosed herein). The pre-treatment antibiotic may include vancomycin, fidaxomicin, rifaximimin, linezolid, daptomycin, quinupristin-dalfopristin, tigecycline, ceftaroline, ceftobiprole, televancin, teicoplanin, dalbavancin, metronidazole, ortivancin, or a combination thereof. Pre-treatment antibiotics can be given at any dose, from 0.01 mg per daily to approximately 5000 mg per days. Sometimes, the pretreatment antibiotic can be administered in one dose. Sometimes, the pre-treatment antibiotic may be administered in a single dose. The antibiotics that are used to treat multiple doses of antibiotics can be different or the same. Pre-treatment with antibiotics may not be required or desirable in some cases. Methods are considered that don’t include pre-treatment with antibiotics (e.g., pretreatment is deliberately omitted).

After administering the microbiota restore therapy composition, the methods of treating a gastrointestinal problem may include the administration of a post-treatment antibiotic to the patient. The post-treatment antibiotic can be different or the same as the pre-treatment antibiotic. Sometimes, it may not be necessary or desirable to continue treatment with antibiotics after the initial treatment. Methods are considered that don’t include antibiotic post-treatment (e.g., treatment is deliberately omitted).

A second dose of microbiota restoration therapy may be administered to a patient with a gastrointestinal disorder. You may also receive additional doses. Multiple doses may be given of the microbiota restore therapy composition in combination with one or several doses a pretreatment antibiotic (e.g. as disclosed herein), or in conjunction one or more dosages of a second-line antibiotic (e.g. as disclosed herein), or in absence of any other antibiotic.

“In one case, the patient receives a single dose (pre-treatment) of antibiotics. Then, the primary dose of microbiota restoration treatment composition is administered to him. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another example, the pre-treatment antibiotics are administered to the patient. A secondary dose is then administered to them. Finally, the primary dose of the microbiota-restoration therapy composition is given to the patient. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another example, the pre-treatment antibiotic is given to the patient. The primary dose of microbiota restore therapy composition is then administered to him. A secondary dose is also administered to him. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another example, the pre-treatment antibiotic is given to the patient. A secondary dose is then administered to him. The patient is then administered a primary dose and secondary doses of the microbiota therapy composition. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another instance, the primary dose of the microbiota restore therapy composition is given to the patient without administering any pre-treatment antibiotics to it. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another case, the primary and secondary doses of the microbiota restore therapy composition are administered to patients. In the absence of administering pre-treatment antibiotics to the patient, the primary and secondary doses are administered. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“The appropriate routes for administering antibiotics and/or microbiota restoration therapy compositions may include oral, enema, or suppository administrations.

“The microbiota restoration therapy uses human fecal material that is optionally filtered.”

“In some embodiments, the composition is a standard fecal microbiological preparation from pre-screened donors. Common infectious diseases are tested in serum and stool from donors. This includes both laboratory testing and a review of the donor?s medical history. After the donor is qualified, he/she must undergo rescreening at specific intervals. The re-screening can be performed every one to three months, with an example of one interval being approximately every three months. Some systems for collecting normal or quality fecal material will require that donors contact the collector immediately after any changes in their health. This information can also be obtained at every donation, between the complete screenings via questionnaires or other means. As long as the donor passes their screening and meets all health requirements, he/she can remain a qualified donor.

“Collected human stool can then be processed according to the instructions and frozen before being quarantined. After both the pre-collection as well as post-collection medical screenings (for example, blood tests and human stool) are completed, the product can be released. This ensures that the quality of the samples collected during screenings is maintained. The product can be thawed and shipped in a temperature-controlled container to the institution or alternatively shipped frozen and thawed at the institution.”

The present composition is especially suitable for patients with recurrent CDI. Recurrent CDI refers to a patient with at least one episode of CDI after their primary episode. They must have received at least two oral antibiotics to treat their CDI. This composition can also be used to treat a primary episode.

A patient can receive an oral antibiotic regimen of 10-14 days, with at least 7 days worth of vancomycin, and then a washout period of 24-48 hours. Enema can then be used to introduce the MRT composition. If CDI symptoms are resolved within 60 days of administration of the first enema, then treatment is considered successful. If symptoms return within 60 days, patients may be able to have a second MRT enema. Patients who are successful can see results within 60 days.

“Human fecal materials are by their nature varied between donors. Even within the same donor, they can vary from day to day. A single sample of human stool may contain more than 1000 microbes, making it difficult to identify and test the whole fecal microbiome and to determine the effects of each microbe on a particular disease like CDI. The present disclosure has shown that a standard or normal sample can be identified from fecal material and processed into a MRT composition. The present composition, which is made from a variable raw material, uses donor screening methods, fecal test methods, fecal material handling methods, processed material preservation methods and testing methods to create a consistent, reproducible and quality-controlled composition of human feces that can be used to treat various diseases and conditions caused by microbial imbalance in the GI tract.

The present composition can be started with what is known as a normal human stool sample. To determine if a human stool sample is normal, a patient can be screened using a questionnaire or other means. Blood and/or stool testing can also be used to verify or check other characteristics. The human stool sample can be processed in controlled conditions and the final product can be tested to verify that it was normal. Select testing can also be done to confirm viability. This is another way to determine if the stool is healthy. This allows for the identification of donor-to-donor variation as well as within-distributor variation to be used in the definition of normal human stool. It is also possible to confirm the validity of the handling and processing techniques used. Combining a normal human stool with validated processing and hand-holding techniques creates a standard composition. To ensure successful treatment, a standardized composition must be stored and managed from production through administration. Successful treatment requires a validated process that maintains the viability of the standard product from production to administration. You will see that it is possible to track samples during processing to make medicaments. It may be useful to use suitable tags to identify the sample, extract, and medicine and to allow them to be linked with each other and the patient being treated. RFID tags and DNA analysis of donor samples can be used to achieve this. A skilled person in the art will be familiar with the other tags that can be used to track medicaments disclosed, such as bar codes.

“Health history data, blood analysis, and human stool analysis are all useful in characterizing the human stool sample. A constant set of processing parameters can also be used to create a product that can easily be cultured on selected media. This will confirm the viability and diversity of the stool sample. You can use the processing parameters to create a product that can easily be cultured on certain media to confirm the absence of specific microbes or groups of microbes. This section of the processing protocol can be used for safety testing of the stool sample to ensure that it is safe to use in the MRT product.

The handling and processing of the human stool sample can include consistent and reliable collection and handling techniques, filtering procedures, controlled homogenization, addition of selected liquids, and amounts. The standardized composition, or product, can be cultured using selected media to verify viability and diversity of the microbes.

The standardized product must remain viable from the moment it is produced to the time it is administered to the patient. To ensure viability, this can be done using cryoprotectant and frozen storage techniques. The applicants discovered that polyethylene glycol can be used as a cryoprotectant for MRT-products. Viability is also affected by the time of storage, thawing, shipping, and handling of the thawed products. These factors are described here. These techniques can be used starting at the time that stool donation is received until the production of the standard product and the administration to patients. These techniques allow for the viability and confirmation of the MRT product’s microbiota. Protocols are provided for the confirmation of the viability, diversity and presence of selected microbes or groups of microbes using selected media. These techniques can also be used to confirm that selected microbes or groups of microbes are present, viable, and diverse at all stages of the process. This includes collection, processing, storage, post-thaw, administration to patients, and every step in between. One embodiment allows the cryoprotectant polyethylenegl (PEG), to be added to the human stool sample along with isotonic solution at the time of processing. PEG can be added to the solution at a concentration of about 0.1g/ml. To about 70 g/ml or between about 2 g/ml and about 68.8 g/ml or between about 4 g/ml and 65.5 g/ml or from approximately 5 g/ml up to 60 g/ml. PEG can range in molecular weight from 600 to 20000. Some embodiments of the PEG have a molecular mass of 2000 to 4000. For example, PEG 3350 has a 3350 molecular weight.

The following methods can be used to grow cultures that define normal human stool samples. However, it is important to recognize that raw stool cannot always be cultured. To create a bacterial suspension, a processed sample product was created using a 50-gram (g) sample of human stool. The mixture consisted of approximately 2 to 4 mL PEG/saline and 1 g of human stool in an sterile mixing/filter bag. The bacterial suspension may be filtered according to industry standards before being culturable. The bacterial suspension was incubated on standard growth media according to industry standards. These protocols produced consistent results for the count of colony-forming units (CFU).

To culture the bacterial suspension, there are two culturing media. The Center for Disease Control (CDC), plate is commonly referred as?CDC Anaerobe 5% sheep blood agar plate. This is a general anaerobic microbe plat that allows the cultivation and isolation of both fastidious and slow-growing obligately aerobic bacteria. The Bacteroides Bile Esculin Agar plate (BBE), is a specific indicator media for Bacteroides. Both media types are available through lab suppliers such as Fisher Scientific and Becton, Dickinson and Co.

“Applicants can measure the diversity of microbes in the human stool, product produced, and product being administered by culturing on select media. Any stage of a stool sample can be measured using the techniques described above. The CDC plates can grow up to seven different types of microbes, which is more than what is possible in MRT materials. One example is that a CDC plate containing a bacterial suspension must contain at least three distinct genera of microbes to allow the bacterial suspension be processed and/or used in MRT therapy. The BBE plate provides an additional measure in microbial diversity for the MRT product. Multiple species of Bacteroides microbes can be grown on the BBE plate. The BBE plates with sufficient CFUs indicate the diversity of the genus in the MRT product. The CDC plate and the BBE plates can both be used as a measure for diversity in certain embodiments. Other embodiments allow for the use of both the CDC and BBE plates together to increase diversity at the microbe genus and species levels within a microbe genus.

Step three could involve the transport of the sample to a processing center. You can see that the sample does not need to be transported if it is collected at the processing center. It may be possible to collect the sample at the facility to establish the chain of custody. A donor profile will be created once the first stool donation has been received. A human stool test can be performed on any subsequent stool samples to confirm and match the identity of the donors. A human stool profile is created for each donor based on previous collected samples. This profile can be kept or improved over multiple donations. To confirm that the donor is the same, any new sample will be compared to this profile. Based on the presence of Bacteroides species within the stool, it is possible to differentiate donor identity. The processing facility collects the stool samples that were used to create the profile. This is to confirm donor identity. Another non-limiting example is that the base set stool samples used in creating the profile can also be collected at other locations than the processing facility. Donor identity assurance protocols are appropriate for the specific situation.

“Step 4 of the process may involve labeling the donation with?Quarantine? The donation should be kept in quarantine for at least 24 hours before it is processed. If the temperature indicator is activated and the time between receipt and donation exceeds 24 hours, donations may be rejected. The human stool test results must be consistent with the donor profile, if applicable. The donor will be disqualified if the results of the human stool test do not match the donor’s profile.

“The human stool sample is processed in one method of disclosure within 24 hours of collection. Another method of applying the disclosure records the time at which the stool sample arrived at the processing facility. Inspecting the stool donation may be step six. The stool sample can be visually inspected upon arrival at the processing center. In the event the human stool sample is loose, unformed, is not of sufficient weight (e.g., less than about 50 g), or for any other reason, including but not limited to evidence indicating poor sample quality or concerns about donor health, the sample may be rejected, labeled ?Inspection?Rejected? The donation is discarded. Trained personnel can also review the answers to any questions regarding the collection of human stool. Some answers on the collection form might require extensive rejection. If the sample is accepted, it may be labeled ?Inspection?Accepted? It may then be sent to a manufacturing facility.

FIG. 5 shows another example of screening donors, collecting stool samples from human beings, and then processing the stool samples into a MRT product. 2. Step one could include recruiting potential donors. Step two could include requesting potential donors to complete an Initial Donor History Questionnaire (IDHHQ). This questionnaire could be used to screen potential donors for blood donation. It may also include additional screening questions if necessary. Failing to meet the criteria will result in the potential donor being removed from the donor pool. The potential donor will be removed from the donor pool if they fail to meet the criteria. A donor may be given a “fail” result. The conditions under which a potential donor would fail a Red Cross screening are similar to those that result in a?fail? A score of?pass? The donor is accepted for further testing if they are deemed to pass. The fourth step may include further testing and review. If the donor fails to pass, the donor is removed from the pool. Donors are routinely screened for common infections and other conditions. Regular screening will include a review of the donor?s medical history by trained personnel and repeat screening tests. The constituents in Table 1 can be included in screening.

“TABLE 1nDonor screening testsnTest name Material Tested Acceptance CriterianC. difficile B via PCR and GDH Stool Negative for C. difficile\nGDH result is not a pass/fail\ncriterion – for information only\nNorovirus via PCR Stool Negative\nRotovirus via PCR Stool Negative\nAdenovirus via PCR Stool Negative\nEnteric Pathogens (Shigella, Stool Negative for Shigella,\nSalmonella, Campylobactoer, Salmonella, Camphylobacter,\nsorbitol-negative E. coli., sorbitol-negative E. coli.,\nAeromonas, Yersinia, Aeromonas, Yersinia, and\nPlesiomonas, Shiga toxins) Plesiomonas.\nNo Shiga toxins detected\nGiardia Antigen Stool Negative\nCryptosporidium Antigen Stool Negative\nAcid-fast Staining (Clyslospora, Stool Negative\nIsospora)\nOva and Parasites Stool No detection/identification of:\nGiardia, Entamoeba histolytica\n(amoeba), Helminth eggs, protozoa,\nlarval worms and segments\nVancomycin-resistant Stool No VRE isolated\nenterococci (VRE)\nMethicillin-resistant Stool No MRSA isolated\nStaphylococcus aureus (MRSA)\nVibrio Stool Non-reactive\nListeria Blood (Serum) Negative\nHuman Immunodeficiiency Virus Blood (Serum) Non-reactive\n(HIV)\nHepatitis A (IgG); must be + or Blood (Serum) Non-reactive\nelse be vaccinated\nHepatitis B: Anti-Hepatitis B Blood (Serum) Non-reactive\nsurface antigen must be + or else\nbe vaccinated\nHepatitis C Antibody Blood (Serum) Non-reactive\nTreponema Antibody Blood (Serum) Negative\nThese are just examples. You may also use other tests.

Step five could involve the acceptance of a donor to the donor pool. Step six could involve the delivery of a stool collection kit to the donor. Step seven could involve regular stool sample collection from the donor. Step eight may include manufacturing a drug product using the stool samples. Step nine could involve the quarantining of the drug product. Step ten may include rescreening the donor stool sample every 45 days. All drug products made from the donor’s stool sample in the past 45 days are thrown out if the sample fails screening. The drug product is released from quarantine if it passes screening (step 11).

“Another exemplary way is to weigh a sample of human stool and then transfer 45 to 75 grams of it into a sterilized filter bag. As a diluent, a mixture of saline (e.g., PEG 3350) and a cryoprotectant can be added to the human stool sample. The terms “saline/PEG combination” are used herein. ?diluent? They can be interchanged. The PEG concentration for the diluent is approximately 30-90 g/liter. The PEG concentration can be anywhere from 25 to 75 g/liter. As an example, the ratio between stool sample and saline/PEG mix is 2:1. This means that 2 mL of saline/PEG mix will be required to make 1 gram of stool. For 50g of stool, you can use approximately 100mL of the saline/PEG combination. Although saline/PEG can be used as a diluent and/or cryoprotectant, it is not recommended to be considered limiting. You can also use other cryoprotectants. Other cryoprotectants include, for example, dextrose and betaine, sucrose, polyvinylalcool, Pluronic F127, manitol, Mannitol 80, ethylene, 1,3-propanediols, hydroxypropylcellulose, glycerols, PEG/glycerol mixtures, propylene glycols, or combinations thereof. These materials can be used in combination with solvents such as saline or alone.

The bag containing the sample of human stool is sealed once the diluent has been added. The stool sample is mixed using a paddle mixer with the diluent. A bacterial preparation is the product of the diluent and stool sample.

“The bacterial preparation may be filtered using standard methods. The filtrate can then be taken from the bag and transferred to sterile pouches and bags. Some embodiments of the bacterial preparation are not filtered.

“In other embodiments each bag containing the bacteria preparation (filtered, not filtered), is equivalent to one dose MRT product and treated as such. Other embodiments allow the bags to be stored in accordance with proper protocol and then aliquoted into smaller quantities at a later time. Other embodiments allow the bags to be combined according to appropriate protocol to make one or multiple doses. One or more bags of the bacterial preparation may be kept for Quality Control (QC).

“In an exemplary test of quality of the manufactured MRT products (including viability of microbes as processed), a stool sample or fecal donation from one qualified donor can be processed. Some embodiments prohibit the pooling of samples from different donors or individuals. Other embodiments allow for sample preparations from a single donor to be pooled or combined. Other embodiments combine sample preparations from multiple donors.

A sample of the MRT product or processed human stool can be diluted, plated and incubated to count according to industry standards. A CDC plate can count Anaerobic CFU, while a BBE plate can count Bacteroides CFU. The Quality Control standards will be used to verify that plate counts are within acceptable limits. This includes testing results from previous donations and other appropriate standards. The MRT product will only be accepted if the counts fall within the acceptable range. All bags of MRT product made from the same human stool samples will be removed from the freezer and destroyed if they are outside the acceptable range. The final acceptable counts range from 30 to 300 CFU at a serial dilution of 10?6 on CDC plates to 30 to 300 CFU on the BBE plates. This is before freezing the sample preparation or the MRT product.

“FIG. “FIG.5” 5 is another example of a delivery process in a flowchart. The product is ordered and received. It is then inspected. If the product fails to pass inspection, the product is discarded. A report will be sent to the processing centre and a request for a reorder. A report is sent to processing center if the product passes inspection. The product is then either taken to the patient or kept in a secure location before being given to them for treatment. The primary packaging of the product is discarded when it is given to the patient.

“FIG. 7 illustrates sample bag 12. Bag 12 can be made from ethyl vinylcetate. There are other materials that can be used. Bag 12 could include a polyethylene-terephthalate polyester movie, which is a material that is substantially impervious against gases and other polymers. Bag 12 could look like an intravenous bag. Optionally, bag 12 might include an attachment that allows bag 12 to hang on a stand (e.g. to be placed/hung above an endoscope).

Bag 12 may hold between 25 and 250 ml (e.g. 50 ml). Bag 12 may include a fill port 24 to allow for the transport of the MRT composition into bag 12. To facilitate the transportation of the MRT composition, fill port 24 can include a luer (or another type of adapter). Once you have filled bag 12 with the MRT mixture, port 24 can be sealed. This seals the bag’s MRT composition. Bag 12 may also contain a spike port 26. The spike port 26 can be used to extract the MRT from bag 12.

“FIG. 8 illustrates tube assembly 16. Tube assembly 16 might include a spike member 28, which is used to attach the spike port 26 to bag 12. Tube assembly 16 could also include a tube body 30, with a step adapter 32. A step adapter 32 could allow tube 16 and a delivery tube 34 to be combined.

“Tube assembly 16 could include additional features. A clamp 36 could be attached to delivery tube 34. A visual marker 38 can also be placed along delivery tube 34. An optional spike cover 40 can be placed along spike member 28.

The MRT compositions described in the present disclosure could include bacteria belonging to at minimum one phylum, at most 2 phylas, at best 3 phylas, at worst 4 phylas, at worst 5 phylas, at lowest 6 phylas, at maximum 7 phylas, at most 8 phylas, at-most 9 phylas, or at-most 10 phyla. At least some embodiments may contain bacteria belonging to at least one class, at most 2 classes or more, at minimum 3 classes, at the least 4 classes and at the least 5 classes, 6 classes, or 7 classes in the MRT compositions. At least some embodiments may contain bacteria members of at minimum one order, at most 2 orders or 3 orders, at the least 4 orders and at the least 5 orders, 6 orders, or 7 orders in the MRT compositions. At least some embodiments may contain bacteria from at least one family, at most 2 families, at minimum 3 families or at the very least 4 families, as well as at the very least 5 families, 6 families, and at the very least 7 families. At least some embodiments may contain bacteria from at least 5 or 10 families, at most 20 or 30 different genera. The MRT compositions described in at most some embodiments may contain at least 10, 50, 100, 200, 300, or 400 different species of bacteria.

“For example, MRT compositions could contain viable bacteria from one or more orders, or two or more orders, including Clostridiales and Bacteroidales. Some embodiments could contain about 20-95% or 30-85% or 40-60% of viable bacteria in MRT compositions. Some of these, and other embodiments, may contain about 10-150%, or approximately 20-60%, or 30-40%, of viable bacteria in the MRT compositions.

“The MRT compositions can also include bacteria from five or more families or approximately 6-12 families or approximately 7-10 families. It could include bacteria from Bacteroidaceae and Burkholderiales, Clostridiaceae and Clostridiales. Some embodiments could have as much as 20-84% or 30-50% or 36-48% of viable bacteria in the MRT compositions. Some of these, as well as other embodiments, may contain about 0.5-22% or 1% viable bacteria in the MRT formulations. Some of these, and other embodiments, may contain about 1-10% or about 1-8% or approximately 2-7% viable bacteria in the MRT formulations. Some of these, and other embodiments, may contain about 1-22% or 3-22% or about 1-8% or about 4-7% viable bacterial in MRT compositions. Some of these, as well as other embodiments, may contain about 1-10% or about 1-9% or approximately 4-8% viable bacteria in the MRT formulations. Some of these, as well as other embodiments, may contain 0.5-2% to 1% viable bacteria in the MRT formulations. Some of these, and other embodiments, may contain 0.5-23% or about 1- 10% or approximately 4-9% viable bacteria in the MRT compositions. Some of these, as well as other embodiments, may contain 0.5-8% or approximately 1-5% or about 1-3% viable bacteria in the MRT formulations. Some of these, as well as other embodiments, may contain 0.5-2% to 1% viable bacteria in the MRT formulations. Some of these, and other embodiments, may contain about 1-30% or about 1-52% or approximately 4-23% viable bacteria in the MRT formulations. Some of these, as well as other embodiments, may contain about 5-30% or 8-25% or 10-18% viable bacteria in the MRT compositions. Some of these, as well as other embodiments, may contain 0.5-22% or 1% viable bacteria from the family Streptococcaceae.

“The MRT compositions in the present disclosure could have a Shannon Diversity Index between 0.4-2.5 or about 1.0-2, or approximately 1.08-1.89 or 1.25-1.75. These numbers are calculated at a?family? level. level. Calculations at higher levels (e.g. species, phyla etc.) will produce different numbers. Different numbers would be generated (e.g. 1-8). The Shannon Diversity Index could therefore be around 1-8 when calculated at different levels (phyla, species or other).

“The MRT composition described in this disclosure can be administered using a method that is suitable for depositing in a subject’s gastrointestinal tract, preferably their colon. There are many routes for administration, including colonoscopy, colonoscopy and suppository. Intubation through the nose, mouth, or throat by a nasogastric tube or nasoenteric tub, or nasal jejunal tubes, may also be used. Oral administration may be done by tablet, pill, suspension, geltab, semisolid, or tablet. It can also be used as an enteral formula or reformulated to final delivery as liquid, gel, geltab or semisolid. You can use the compositions to treat or untreat fecal bacteria, whole (or substantially) entire microbiota, or isolate, purify, or process fecal fauna into a powder.

“A composition can be administered conveniently in a form that contains one or more pharmaceutically approved carriers for therapeutic purposes in accordance with the method disclosed in this disclosure. The art is well-versed in the selection of suitable carriers. They can be modified to suit the form and method of administration of the composition. They may contain diluents, such as binders or wetting agents, disintegrators and surface-active agents, fillers, binders and glidants, or lubricants. The carrier can be either a solid (including a powder), liquid, or a combination thereof. Acceptable is the preferred way to describe each carrier. In the sense that it is compatible with other ingredients and does not cause harm to the subject. “Biologically acceptable and inert means that the carrier allows the composition to continue to be viable until it is delivered to the proper site.

Oral compositions can include an inert carrier or a diluent. The active compound may be combined with excipients to form tablets, troches, capsules, or other forms for oral therapeutic administration. Combining the composition of this disclosure with food can make oral compositions. One embodiment of the invention includes ice cream as a food for administration. The composition can include pharmaceutically compatible binding agents and/or adjuvant material. Tablets, pills, capsules and troches can contain any one of the following ingredients or compounds. These examples are intended to provide an example and not limit your options.

The active compounds may also be prepared as suppositories, e.g. with conventional suppository base bases like cocoa butter or other glycerides, or retention enemas to provide rectal delivery. You can prepare active compounds with carriers that protect it from rapid elimination from your body. This includes implants and controlled release formulations. You can use biodegradable and biocompatible polymers such as polyanhydrides polyethylene vinyl acetate, ethylene vinyl acid, polyglycolic acids, collagen, polyorthoesters and polylactic acid. These formulations can be made using standard methods. These materials can also easily be purchased commercially at Nova Pharmaceuticals, Inc. and Alza Corporation. Liposomal suspensions are also available for use as pharmaceutically acceptable carriers. These materials can be prepared using techniques that are well-known to the skilled.

A composition can be encapsulated. When the composition is to go orally, for example, the dosage form must be formulated so that it is not exposed to the conditions of the gastrointestinal tract, such as high acidity or digestive enzymes. It is a common practice to encapsulate compositions for therapeutic purposes. There are many options for encapsulation, including hard-shelled capsules that can be used to encapsulate dry, powdered ingredients and soft-shelled caps. Capsules can be made from aqueous gelling agents, such as animal protein (e.g. gelatin), plant polysaccharides and derivatives like carrageenans or modified forms of starch. A gelling agent solution may also contain other ingredients such as coloring agents, disintegrants or lubricants, and even surface treatment.

Gels, creams and lotions are suitable for rectal administration. Preferably, the formulations are provided as unit-dose capsules that contain the active ingredient in one of the solid carriers that form the suppository base. These formulations can be carried in petroleum jelly, lanolin or polyethyleneglycols as well as alcohols and combinations thereof. Colonic washes containing the rapid colonization deployment agent described in the present disclosure could also be prepared for rectal administration.

For oral administration, formulas can be prepared in discrete units such as capsules, tablets, cachets or syrups. They also come with elixirs and chewing gum. Formulations, microemulsions solutions, suspensions and gel-coated ampules containing predetermined amounts of active compound.

“Formulations that are suitable for transmucosal administration, such a by sublingual or buccal administration, include lozenges patches and tablets, which contain the active compound and typically a flavor base such as sugar and/or tragacanth, and pastilles that contain the active compound in an inert basis such as gelatin, glycerine, or sucrose acacia.”

Summary for “Microbiota Restoration Therapy (MRT), compositions, and methods of manufacturing”

“A variety of methods and compositions have been created to treat diseases or conditions of the digestive track. Each of the existing compositions and methods has its advantages and disadvantages. It is important to continue developing alternative compositions and methods of treating conditions and diseases of the digestive tract.

The invention discloses microbiota therapy compositions and methods of manufacturing, processing and/or delivering them. A microbiota restoration treatment composition could be made by taking a sample of human feces and then adding a diluent. This will create a diluted sample. The diluent could also include a cryoprotectant. Mixing the diluted samples with a mixer and filtering the filtrate may be part of the method. Filtering can form a filtrate. This may include placing the filtrate in a bag to collect it and sealing the bag.

A method of manufacturing, processing and packaging a microbiota-restoration therapy composition might include taking a sample from a prescreened donor, then transferring it to a filter bag and adding a diluting agent to the bag. A cryoprotectant may be included in the diluent. This may include sealing the filter bag and then transferring it to a mixer. Finally, transfer the filtrate from filter bag into a sample bag. The microbiota restoration therapy content within the sample bag can be determined by transferring the filtrate from the filter bags to a sample bag. This may include sealing the bag and cooling the bag before transferring the cool bag to a temperature controlled storage device. The microbiota restoration treatment composition may be frozen before being transferred to the controlled temperature storage device. This may include freezing the microbiota restoration treatment composition and packaging it in an insulated bag. Finally, the package is shipped to a treatment facility.

A pre-screened donor may give a sample of feces to be used as a guideline for medical treatment. The sample is then transferred to a bag and diluent added to it. A cryoprotectant may be included in the diluent. This may include sealing the filter bag and then transferring it to a mixer. Finally, transfer the filtrate from filter bag into a sample bag. The microbiota restoration treatment composition may be defined by the transfer of the filtrate from the filterbag to a sample bag. This may include sealing the bag and cooling the bag before transferring the cool bag to a temperature controlled storage device. The microbiota restoration treatment composition may be frozen after being transferred to the controlled temperature storage device. This may include freezing the frozen microbiota restore therapy composition, wrapping the sample bag in an insulation system, and shipping the bag to a treatment facility to administer the microbiota therapy composition to the patient.

“The summary of some embodiments does not include every embodiment disclosed or every implementation of this disclosure. These embodiments are more clearly illustrated by the Figures and Detailed Description which follow.

“For the following terms, these definitions shall apply, except where a different definition is given elsewhere in this specification or in the claims.”

“All numeric values are assumed to be modified by?about?. This is true regardless of whether it is explicitly stated. The term “about” refers to a range of numbers that can be considered equivalent to the recited value. The term?about? refers to a set of numbers that one skilled in the art would consider equivalent or equal to the recited value (e.g. having the same function, result). In many cases, the term ‘about’ may include numbers that are rounded to the nearest significant figure. Many times, the terms?about? may contain numbers that have been rounded to nearest significant number.

“The recitation by endpoints of numerical ranges includes all numbers within the range (e.g. 1-5 includes 1, 1.5, 2.75, 3.80, 4, and 5).

“The singular forms?a,?an, and??the as used in this specification and the attached claims are the ones that are used. If the content clearly indicates otherwise, plural referents should be included. The term “or” is used in the specification and the attached claims. The term?or?, as used in this specification and the appended claims, is commonly used in its full meaning including?and/or’ Unless the content clearly indicates otherwise,

“It should be noted that references to?an embodiment? or?some embodiments?”, etc., may indicate that an embodiment described may contain one or more specific features, structures, or characteristics. These citations don’t necessarily mean all embodiments have the same features, structures, or characteristics. If certain features, structures and/or characters are mentioned in connection to one embodiment, it is important to understand that these features, structures and/or attributes may be used in connection with other embodiments, even if they are not specifically described.

“The following detailed description should only be read in conjunction with the drawings, in which identical elements are numbered the exact same in all drawings. These drawings are not to scale and illustrate illustrative embodiments. They do not limit the invention’s scope.

“?Mammal? “?Mammal” as used herein refers any member of the Mammalia class, including humans and nonhuman primates like chimpanzees and other ape species, farm animals such cattle, sheeps, pigs goats and horses, domestic mammals such dogs and cats, rodents such mice, rats, guinea-pigs, and others. This term does not indicate a specific age or sex. This term includes both adult and newborn subjects as well as female and male fetuses.

“Cryoprotectant” is a term that refers to a substance used to protect biological tissues or cells from the effects of freezing.

“The term “microbiota” as used herein refers to the human microbiome, the human microbiota or the human gut microbiota. The term?microbiota’ can be used to refer to the human human microbiome or human microbiota. The human microbiome, also known as the human microbiota, is a collection of microorganisms found in the skin’s surface and deep layers. It can also be seen in saliva, oral mucosa and the conjunctiva. The human microbiome includes bacteria, fungi and viruses as well as archaea. At least one of these organisms can perform useful tasks for the human host. These microorganisms are not harmful to the host but rather help maintain health. This is why this group of organisms is often called “normal flora.”

“The human gastrointestinal tract’s microorganism population is often referred to as “microbial flora”, “gut flora” and/or “gut microbiota”. The human microbial flora includes a variety of microorganisms that help in digestion, synthesis of vitamins and the creation of enzymes not made by the body.

“Microbiota restoration therapy” is a term that refers to a composition that may contain human feces containing viable gut bacteria from a donor or patient, as well as a diluent and a cryoprotectant. Other compositions include freeze-dried, reconstituted feces and a?synthetic’. Fecal composition. Before it is used in microbiota restoration therapy, the human fecal matter is tested for pathogenic microorganisms. 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.”

“More that 1000 species of bacteria live in healthy GI tracts. Clostridia is anaerobic and spore-forming bacteria. Clostridium difficile (?C diff?) is a pathogen that produces toxins that can cause harm to humans. Clostridium difficile (?C diff?) Clostridium difficile (?C diff?) is one type of Clostridium. If it becomes too abundant in the GI tract, it can release toxic substances that can cause many symptoms including constipation, diarrhea and inflammation. In some cases, this can even lead to death.

Clostridium difficile spores can survive extreme conditions that other active bacteria cannot. Clostridium difficile does not thrive in a healthy GI system. Clostridium difficile can be overgrown if antibiotics are used. Clostridium difficile can be resistant to some antibiotics in certain cases. Clostridium difficile spores can be resistant to antibiotics once the normal intestinal flora has been cleared. This results in a large population.

According to the Centers for Disease Control and Prevention (CDC), approximately 337,000 cases (CDI) of Clostridium difficile are reported annually in the United States. This results in roughly 14,000 deaths. Current standard care is antibiotic treatment, usually with vancomycin and metronidazole. About 25% of patients who have received initial antimicrobial treatment experience a recurrence of symptoms. About 45 to 65 percent of these patients developed persistent recurrent CDI. High mortality and morbidity are associated with persistent recurrent CDI. Clostridium difficile incidence in the United States is estimated to be between 1.5 and 2.7 million per year. These numbers are increasing. According to one estimate, Clostridium difficile hospital discharges doubled between 2001 and 2005. This is a compound annual growth rate of 5% to 25%. Clostridium difficile patients with overpopulation are more likely to experience prolonged hospital stays of 3 to 36 days. Nearly 20% of patients infected will be readmitted to the hospital within 180 days. Each patient is more likely to be discharged into long-term care facilities than those not affected. Clostridium difficile has a financial impact estimated at $1-3 billion per year. Clostridium difficile overpopulation is responsible for 300 deaths per day, with a mortality rate between 1 and 7.7%. This number is increasing.

Clostridium difficile antibiotics can be used to treat diarrhea in the first instance. This is a treatment that works well within 2 to 4 days and at rates of approximately 90%. Clostridium difficile can recur after the first occurrence, which is typically within a few days to 12 weeks of ceasing antibiotics. This happens at an estimated 20% rate (15-30%). The rate of recurrence increases dramatically with each subsequent occurrence. It is an average 40% rate (e.g., 15%-30%) after the second recurrence and an additional 60% or more thereafter. It is estimated that about 5% of patients will have six or more recurrences.

Clostridium difficile treatment is usually different for each case. Metronidazole can also be taken orally for the first mild to moderate recurrence (e.g. 500 mg three times daily,?TID?) For a period of 10-14 days. Vancomycin can also be given orally in tapered doses or pulsed doses to prevent a second recurrence (e.g., at doses of 125 mg four times per day (?QID?)). For 14 days, at a dose 125 mg twice daily (?BID?) For 7 days, at a dose 125 mg once daily (?BID?) For 7 days, at a dose 125mg, once daily (?QD?) You can apply vancomycin at higher doses for a third recurrence (e.g., at doses of 125 mg four times per day (?QID?). For 14 days, combined with any other treatment options such as intravenous immunoglobulin (e.g. at a dose 125 mg four times daily (?QID?)). for 14 days), etc.”

Recurrent CDI patients may have to resort to fecal transplantation (FT) as a last resort. FT is used to reestablish a healthy microbial mixture in the patient’s gastrointestinal microbiota after antimicrobial treatment. There have been over 480 cases with a?90% success rate in curing recurrent CDI. The FT material has not caused any adverse reactions. Current institutional practice is to collect fecal samples from relatives or volunteers in the hospital for transplantation. This method of treatment has a problem in that the FT material isn’t standardized. While donors are usually selected if they are healthy at the time of donation it is not sufficient to ensure the viability and quality of the microbes that will be transplanted. The donor may not know of a disease that affects the fecal material. The quality of the raw fecal matter can also affect the viability and quality of the FT material that is given to patients. Each institution must also handle raw fecal material. This is not desirable.

A standardized, pre-processed MRT product is needed to ensure quality and viability for patients at time of delivery. A MRT product that can be easily handled and delivered to the appropriate facility is desirable. This will eliminate the need to handle raw feces material at each institution. These improvements will make MRT a viable and desirable option for CDI, rather than being a last resort treatment at a few institutions.

This document discusses, among others, the receipt of multiple donor fecal specimens from a plurality, and the storage and indexing of each donor fecal sample using at least one characteristic. The donor fecal samples can be processed and screened for use in fecal therapy to eliminate pathogenic or unwanted organisms from the digestive tract of patients with healthy or desirable microbiota.

“The disclosure describes a composition of microbiota restoration therapy that includes a combination of effective amounts of fecal microbiota as well as an effective amount a cryoprotectant. Polyethylene glycol is an example of a cryoprotectant. The disclosure also states that the concentration of polyethylene glycol in the microbiota recovery therapy composition is between 5-60 g/ml and 5-30 g/ml. It should not exceed 30 g/ml. As a diluent, the composition may also contain saline. Polyethylene glycol with an average molecular mass of 600 to 20000 may be included in the disclosed compositions. PEG-3150, which has an average molecular mass of 3150, can be used. Certain embodiments of the microbiota therapy compositions for microbiota restoration contain fecal microbiota from one or more human stool samples.

“Other cryoprotectants can be used, such as betaine, dextrose and glycine.

“In other embodiments, viability of the microbiota compositions disclosed may be confirmed by culturing (and/or filtrate and/or an bacteriotherapy composition) on Bacteroides Bile Esculinagar (BBE), plate (available at Becton, Dickinson and Company, catalogue number 221836, BBL). Bacteroides Bile Esculin Plate BBE, or a Center for Disease Control plate (available at Becton, Dickinson and Company, catalogue number 221733, BL) CDC Anaerobe 5% sheep blood agar plates), or both. At least in some instances, viability of the microbiota contained in the compositions disclosed may be confirmed by a BBE or CDC plate. This is done by a presence of a colony forming units (CFU) count of approximately 30 to 300 CFU at a serial dilution (10?5) or a CFU count between 30 and 300 CFU at a 10?6 serial dilution (10?6). Microbiota restoration therapy compositions are also provided where the microbiota concentration is at least 107 microbes/ml. The methods of producing a microbiota therapy composition could also include the steps of pre- and/or post-donor screening using the human stool sample taken in the time period between.

Plating tests are able to confirm viability and diversity of living microbes. The quality of the stool sample from humans and the MRT product that was made from it is also determined by the mix or diversity of microbes. The CDC plates and BBE plates can be used together or separately to measure quality through diversity, as described in this document.

The disclosure also includes methods to ascertain the quality and quantity of a human stool specimen to be used in microbiota therapy. These methods include identifying a donor of human stool; conducting pre-donation screening, which involves a health history questionnaire, daily diet questionnaire, and at most one blood test. Collecting a stool sample from the donor and processing it to make one or more microbiota therapy compositions. Keeping in quarantine any or all of the microbiota therapy compositions that were made from the donated stool samples; The likelihood of obtaining a healthy sample may be increased by pre- and post screening. This interval was chosen to allow enough time for any disease or other factor present at the time human stool is collected to manifest symptoms or be positive in a serum or human stool test at the post-screening. The composition can also be stored in a quarantine until the pre- or post-screening confirms that donor health has been confirmed.

“The disclosure may also include at least one step to test the stool sample for infectious disease. Additionally, the methods of the disclosure may further include the step of testing the human stool sample for a constituent selected from the group consisting of: C. difficile; Norovirus; Adenovirus; Enteric Pathogens; Giardia antigen; Cryptosporidium antigen; Acid-fast staining (Clyslospora, Isospora); ova and parasites; Vancomycin-resistant enterococci (VRE); Methicillin-resistant Staphylococcus aureus (MRSA) and combinations thereof. The disclosure could include the testing of blood from the donor. This includes at least one test for HIV, Hepatitis A, Hepatitis B, Hepatitis C and combinations thereof.

A representative way to make a microbiota therapy composition using a human stool sample is to collect a sufficient amount of stool from the patient, add saline and a cryoprotectant (e.g. polyethylene glycol), then mix the mixture. The filtrate containing microbes can be then filtered. You can take a portion of the filtrate for testing, and you can freeze the rest to be tested. Testing will be done using the results of pre-screening and post-screening.

The present disclosure relates to compositions, manufacturing methods, and treatment methods that utilize microbiota restoration therapy for the gastrointestinal tract. This involves replacing pathogenic or ineffective organisms by healthy, effective bacteria. Clostridium difficile infection (IBS), Crohn?s disease (UC), ulcerative colitis resulting in a staphylococcus/C. diff infection, inflammatory bowel disorder (IBD), ulcers and diabetes as well as constipation and obesity are some examples of conditions that can be treated.

“In some cases, compositions, manufacturing methods, and treatment methods utilizing microbiota therapy (MRT), for Clostridium difficile infection (CDI) treatment are shown. CDI is a common nosocomial disease and is often associated with high mortality and severe morbidity, particularly in older patients.

Each person has a unique gut microbiota, with 500-5000 species of bacteria, viruses, microorganisms and archaea. These microorganisms provide a host of beneficial symbiotic functions. They include aiding in digestion, nutrition for the colon, production of vitamins, and stimulating the immune system. However, an improperly balanced or functioning gut microbiota may play a role in certain diseases or afflictions, such as pseudomembranous colitis, Clostridium difficile colitis, antibiotic-associated diarrhea (AAD), ulcerative colitis (UC), pouchitis, irritable bowel syndrome (IBS), obesity, among others.”

According to the inventor, there are systems and methods that provide bacteriotherapy for treating afflictions related to the gut microbiota. This includes Clostridium difficile colitis. The system or method can be used to displace pathogenic organisms from the digestive track of patients with healthy bacterial bacteria, or to benefit an individual suffering from an affliction related to the gut microbiota. The systems and methods described in this invention can be used to provide bacteriotherapy to patients by integrating with the existing facilities and procedures of hospitals and clinics. Similar treatment may be successful for patients suffering from IBS, Crohn?s disease, ulcerative colitis, IBD, IBD, IBS, and other gastrointestinal, metabolic or digestive tract-related diseases. Bacteriotherapy can also be used to help with weight loss by replacing inefficient microbiota with more productive. Bacteriotherapy can also be used to treat peripheral and cardiovascular disease.

“For example, bacteriotherapy can be used to treat Clostridium difficile and other digestive tract diseases. It may include a combination antibiotics and the repopulation of healthy or desired mammalian bacteria flora. Fecal bacteriotherapy or fecal transplant can be used in certain cases to repopulate bacterial flora.

“Fecal bacteriotherapy is the process of inserting a fecal specimen from a healthy donor or one of its desired characteristics into a patient’s gastrointestinal tract to restore a healthy or desirable microbiota. Some cases may require that antibiotics be administered to the patient before the fecal sample is introduced. This will ensure that the desired or healthy gut microbiota can quickly populate the gastrointestinal tract. Some of the methods described herein include the administration of a pre-treatment antibiotic and the administration to the patient of a microbiota restore therapy composition (e.g. the microbiota recovery therapy compositions disclosed herein). The pre-treatment antibiotic may include vancomycin, fidaxomicin, rifaximimin, linezolid, daptomycin, quinupristin-dalfopristin, tigecycline, ceftaroline, ceftobiprole, televancin, teicoplanin, dalbavancin, metronidazole, ortivancin, or a combination thereof. Pre-treatment antibiotics can be given at any dose, from 0.01 mg per daily to approximately 5000 mg per days. Sometimes, the pretreatment antibiotic can be administered in one dose. Sometimes, the pre-treatment antibiotic may be administered in a single dose. The antibiotics that are used to treat multiple doses of antibiotics can be different or the same. Pre-treatment with antibiotics may not be required or desirable in some cases. Methods are considered that don’t include pre-treatment with antibiotics (e.g., pretreatment is deliberately omitted).

After administering the microbiota restore therapy composition, the methods of treating a gastrointestinal problem may include the administration of a post-treatment antibiotic to the patient. The post-treatment antibiotic can be different or the same as the pre-treatment antibiotic. Sometimes, it may not be necessary or desirable to continue treatment with antibiotics after the initial treatment. Methods are considered that don’t include antibiotic post-treatment (e.g., treatment is deliberately omitted).

A second dose of microbiota restoration therapy may be administered to a patient with a gastrointestinal disorder. You may also receive additional doses. Multiple doses may be given of the microbiota restore therapy composition in combination with one or several doses a pretreatment antibiotic (e.g. as disclosed herein), or in conjunction one or more dosages of a second-line antibiotic (e.g. as disclosed herein), or in absence of any other antibiotic.

“In one case, the patient receives a single dose (pre-treatment) of antibiotics. Then, the primary dose of microbiota restoration treatment composition is administered to him. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another example, the pre-treatment antibiotics are administered to the patient. A secondary dose is then administered to them. Finally, the primary dose of the microbiota-restoration therapy composition is given to the patient. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another example, the pre-treatment antibiotic is given to the patient. The primary dose of microbiota restore therapy composition is then administered to him. A secondary dose is also administered to him. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another example, the pre-treatment antibiotic is given to the patient. A secondary dose is then administered to him. The patient is then administered a primary dose and secondary doses of the microbiota therapy composition. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another instance, the primary dose of the microbiota restore therapy composition is given to the patient without administering any pre-treatment antibiotics to it. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“In another case, the primary and secondary doses of the microbiota restore therapy composition are administered to patients. In the absence of administering pre-treatment antibiotics to the patient, the primary and secondary doses are administered. This may include administering one or more doses to the patient of a post-treatment antibiotic.

“The appropriate routes for administering antibiotics and/or microbiota restoration therapy compositions may include oral, enema, or suppository administrations.

“The microbiota restoration therapy uses human fecal material that is optionally filtered.”

“In some embodiments, the composition is a standard fecal microbiological preparation from pre-screened donors. Common infectious diseases are tested in serum and stool from donors. This includes both laboratory testing and a review of the donor?s medical history. After the donor is qualified, he/she must undergo rescreening at specific intervals. The re-screening can be performed every one to three months, with an example of one interval being approximately every three months. Some systems for collecting normal or quality fecal material will require that donors contact the collector immediately after any changes in their health. This information can also be obtained at every donation, between the complete screenings via questionnaires or other means. As long as the donor passes their screening and meets all health requirements, he/she can remain a qualified donor.

“Collected human stool can then be processed according to the instructions and frozen before being quarantined. After both the pre-collection as well as post-collection medical screenings (for example, blood tests and human stool) are completed, the product can be released. This ensures that the quality of the samples collected during screenings is maintained. The product can be thawed and shipped in a temperature-controlled container to the institution or alternatively shipped frozen and thawed at the institution.”

The present composition is especially suitable for patients with recurrent CDI. Recurrent CDI refers to a patient with at least one episode of CDI after their primary episode. They must have received at least two oral antibiotics to treat their CDI. This composition can also be used to treat a primary episode.

A patient can receive an oral antibiotic regimen of 10-14 days, with at least 7 days worth of vancomycin, and then a washout period of 24-48 hours. Enema can then be used to introduce the MRT composition. If CDI symptoms are resolved within 60 days of administration of the first enema, then treatment is considered successful. If symptoms return within 60 days, patients may be able to have a second MRT enema. Patients who are successful can see results within 60 days.

“Human fecal materials are by their nature varied between donors. Even within the same donor, they can vary from day to day. A single sample of human stool may contain more than 1000 microbes, making it difficult to identify and test the whole fecal microbiome and to determine the effects of each microbe on a particular disease like CDI. The present disclosure has shown that a standard or normal sample can be identified from fecal material and processed into a MRT composition. The present composition, which is made from a variable raw material, uses donor screening methods, fecal test methods, fecal material handling methods, processed material preservation methods and testing methods to create a consistent, reproducible and quality-controlled composition of human feces that can be used to treat various diseases and conditions caused by microbial imbalance in the GI tract.

The present composition can be started with what is known as a normal human stool sample. To determine if a human stool sample is normal, a patient can be screened using a questionnaire or other means. Blood and/or stool testing can also be used to verify or check other characteristics. The human stool sample can be processed in controlled conditions and the final product can be tested to verify that it was normal. Select testing can also be done to confirm viability. This is another way to determine if the stool is healthy. This allows for the identification of donor-to-donor variation as well as within-distributor variation to be used in the definition of normal human stool. It is also possible to confirm the validity of the handling and processing techniques used. Combining a normal human stool with validated processing and hand-holding techniques creates a standard composition. To ensure successful treatment, a standardized composition must be stored and managed from production through administration. Successful treatment requires a validated process that maintains the viability of the standard product from production to administration. You will see that it is possible to track samples during processing to make medicaments. It may be useful to use suitable tags to identify the sample, extract, and medicine and to allow them to be linked with each other and the patient being treated. RFID tags and DNA analysis of donor samples can be used to achieve this. A skilled person in the art will be familiar with the other tags that can be used to track medicaments disclosed, such as bar codes.

“Health history data, blood analysis, and human stool analysis are all useful in characterizing the human stool sample. A constant set of processing parameters can also be used to create a product that can easily be cultured on selected media. This will confirm the viability and diversity of the stool sample. You can use the processing parameters to create a product that can easily be cultured on certain media to confirm the absence of specific microbes or groups of microbes. This section of the processing protocol can be used for safety testing of the stool sample to ensure that it is safe to use in the MRT product.

The handling and processing of the human stool sample can include consistent and reliable collection and handling techniques, filtering procedures, controlled homogenization, addition of selected liquids, and amounts. The standardized composition, or product, can be cultured using selected media to verify viability and diversity of the microbes.

The standardized product must remain viable from the moment it is produced to the time it is administered to the patient. To ensure viability, this can be done using cryoprotectant and frozen storage techniques. The applicants discovered that polyethylene glycol can be used as a cryoprotectant for MRT-products. Viability is also affected by the time of storage, thawing, shipping, and handling of the thawed products. These factors are described here. These techniques can be used starting at the time that stool donation is received until the production of the standard product and the administration to patients. These techniques allow for the viability and confirmation of the MRT product’s microbiota. Protocols are provided for the confirmation of the viability, diversity and presence of selected microbes or groups of microbes using selected media. These techniques can also be used to confirm that selected microbes or groups of microbes are present, viable, and diverse at all stages of the process. This includes collection, processing, storage, post-thaw, administration to patients, and every step in between. One embodiment allows the cryoprotectant polyethylenegl (PEG), to be added to the human stool sample along with isotonic solution at the time of processing. PEG can be added to the solution at a concentration of about 0.1g/ml. To about 70 g/ml or between about 2 g/ml and about 68.8 g/ml or between about 4 g/ml and 65.5 g/ml or from approximately 5 g/ml up to 60 g/ml. PEG can range in molecular weight from 600 to 20000. Some embodiments of the PEG have a molecular mass of 2000 to 4000. For example, PEG 3350 has a 3350 molecular weight.

The following methods can be used to grow cultures that define normal human stool samples. However, it is important to recognize that raw stool cannot always be cultured. To create a bacterial suspension, a processed sample product was created using a 50-gram (g) sample of human stool. The mixture consisted of approximately 2 to 4 mL PEG/saline and 1 g of human stool in an sterile mixing/filter bag. The bacterial suspension may be filtered according to industry standards before being culturable. The bacterial suspension was incubated on standard growth media according to industry standards. These protocols produced consistent results for the count of colony-forming units (CFU).

To culture the bacterial suspension, there are two culturing media. The Center for Disease Control (CDC), plate is commonly referred as?CDC Anaerobe 5% sheep blood agar plate. This is a general anaerobic microbe plat that allows the cultivation and isolation of both fastidious and slow-growing obligately aerobic bacteria. The Bacteroides Bile Esculin Agar plate (BBE), is a specific indicator media for Bacteroides. Both media types are available through lab suppliers such as Fisher Scientific and Becton, Dickinson and Co.

“Applicants can measure the diversity of microbes in the human stool, product produced, and product being administered by culturing on select media. Any stage of a stool sample can be measured using the techniques described above. The CDC plates can grow up to seven different types of microbes, which is more than what is possible in MRT materials. One example is that a CDC plate containing a bacterial suspension must contain at least three distinct genera of microbes to allow the bacterial suspension be processed and/or used in MRT therapy. The BBE plate provides an additional measure in microbial diversity for the MRT product. Multiple species of Bacteroides microbes can be grown on the BBE plate. The BBE plates with sufficient CFUs indicate the diversity of the genus in the MRT product. The CDC plate and the BBE plates can both be used as a measure for diversity in certain embodiments. Other embodiments allow for the use of both the CDC and BBE plates together to increase diversity at the microbe genus and species levels within a microbe genus.

Step three could involve the transport of the sample to a processing center. You can see that the sample does not need to be transported if it is collected at the processing center. It may be possible to collect the sample at the facility to establish the chain of custody. A donor profile will be created once the first stool donation has been received. A human stool test can be performed on any subsequent stool samples to confirm and match the identity of the donors. A human stool profile is created for each donor based on previous collected samples. This profile can be kept or improved over multiple donations. To confirm that the donor is the same, any new sample will be compared to this profile. Based on the presence of Bacteroides species within the stool, it is possible to differentiate donor identity. The processing facility collects the stool samples that were used to create the profile. This is to confirm donor identity. Another non-limiting example is that the base set stool samples used in creating the profile can also be collected at other locations than the processing facility. Donor identity assurance protocols are appropriate for the specific situation.

“Step 4 of the process may involve labeling the donation with?Quarantine? The donation should be kept in quarantine for at least 24 hours before it is processed. If the temperature indicator is activated and the time between receipt and donation exceeds 24 hours, donations may be rejected. The human stool test results must be consistent with the donor profile, if applicable. The donor will be disqualified if the results of the human stool test do not match the donor’s profile.

“The human stool sample is processed in one method of disclosure within 24 hours of collection. Another method of applying the disclosure records the time at which the stool sample arrived at the processing facility. Inspecting the stool donation may be step six. The stool sample can be visually inspected upon arrival at the processing center. In the event the human stool sample is loose, unformed, is not of sufficient weight (e.g., less than about 50 g), or for any other reason, including but not limited to evidence indicating poor sample quality or concerns about donor health, the sample may be rejected, labeled ?Inspection?Rejected? The donation is discarded. Trained personnel can also review the answers to any questions regarding the collection of human stool. Some answers on the collection form might require extensive rejection. If the sample is accepted, it may be labeled ?Inspection?Accepted? It may then be sent to a manufacturing facility.

FIG. 5 shows another example of screening donors, collecting stool samples from human beings, and then processing the stool samples into a MRT product. 2. Step one could include recruiting potential donors. Step two could include requesting potential donors to complete an Initial Donor History Questionnaire (IDHHQ). This questionnaire could be used to screen potential donors for blood donation. It may also include additional screening questions if necessary. Failing to meet the criteria will result in the potential donor being removed from the donor pool. The potential donor will be removed from the donor pool if they fail to meet the criteria. A donor may be given a “fail” result. The conditions under which a potential donor would fail a Red Cross screening are similar to those that result in a?fail? A score of?pass? The donor is accepted for further testing if they are deemed to pass. The fourth step may include further testing and review. If the donor fails to pass, the donor is removed from the pool. Donors are routinely screened for common infections and other conditions. Regular screening will include a review of the donor?s medical history by trained personnel and repeat screening tests. The constituents in Table 1 can be included in screening.

“TABLE 1nDonor screening testsnTest name Material Tested Acceptance CriterianC. difficile B via PCR and GDH Stool Negative for C. difficile\nGDH result is not a pass/fail\ncriterion – for information only\nNorovirus via PCR Stool Negative\nRotovirus via PCR Stool Negative\nAdenovirus via PCR Stool Negative\nEnteric Pathogens (Shigella, Stool Negative for Shigella,\nSalmonella, Campylobactoer, Salmonella, Camphylobacter,\nsorbitol-negative E. coli., sorbitol-negative E. coli.,\nAeromonas, Yersinia, Aeromonas, Yersinia, and\nPlesiomonas, Shiga toxins) Plesiomonas.\nNo Shiga toxins detected\nGiardia Antigen Stool Negative\nCryptosporidium Antigen Stool Negative\nAcid-fast Staining (Clyslospora, Stool Negative\nIsospora)\nOva and Parasites Stool No detection/identification of:\nGiardia, Entamoeba histolytica\n(amoeba), Helminth eggs, protozoa,\nlarval worms and segments\nVancomycin-resistant Stool No VRE isolated\nenterococci (VRE)\nMethicillin-resistant Stool No MRSA isolated\nStaphylococcus aureus (MRSA)\nVibrio Stool Non-reactive\nListeria Blood (Serum) Negative\nHuman Immunodeficiiency Virus Blood (Serum) Non-reactive\n(HIV)\nHepatitis A (IgG); must be + or Blood (Serum) Non-reactive\nelse be vaccinated\nHepatitis B: Anti-Hepatitis B Blood (Serum) Non-reactive\nsurface antigen must be + or else\nbe vaccinated\nHepatitis C Antibody Blood (Serum) Non-reactive\nTreponema Antibody Blood (Serum) Negative\nThese are just examples. You may also use other tests.

Step five could involve the acceptance of a donor to the donor pool. Step six could involve the delivery of a stool collection kit to the donor. Step seven could involve regular stool sample collection from the donor. Step eight may include manufacturing a drug product using the stool samples. Step nine could involve the quarantining of the drug product. Step ten may include rescreening the donor stool sample every 45 days. All drug products made from the donor’s stool sample in the past 45 days are thrown out if the sample fails screening. The drug product is released from quarantine if it passes screening (step 11).

“Another exemplary way is to weigh a sample of human stool and then transfer 45 to 75 grams of it into a sterilized filter bag. As a diluent, a mixture of saline (e.g., PEG 3350) and a cryoprotectant can be added to the human stool sample. The terms “saline/PEG combination” are used herein. ?diluent? They can be interchanged. The PEG concentration for the diluent is approximately 30-90 g/liter. The PEG concentration can be anywhere from 25 to 75 g/liter. As an example, the ratio between stool sample and saline/PEG mix is 2:1. This means that 2 mL of saline/PEG mix will be required to make 1 gram of stool. For 50g of stool, you can use approximately 100mL of the saline/PEG combination. Although saline/PEG can be used as a diluent and/or cryoprotectant, it is not recommended to be considered limiting. You can also use other cryoprotectants. Other cryoprotectants include, for example, dextrose and betaine, sucrose, polyvinylalcool, Pluronic F127, manitol, Mannitol 80, ethylene, 1,3-propanediols, hydroxypropylcellulose, glycerols, PEG/glycerol mixtures, propylene glycols, or combinations thereof. These materials can be used in combination with solvents such as saline or alone.

The bag containing the sample of human stool is sealed once the diluent has been added. The stool sample is mixed using a paddle mixer with the diluent. A bacterial preparation is the product of the diluent and stool sample.

“The bacterial preparation may be filtered using standard methods. The filtrate can then be taken from the bag and transferred to sterile pouches and bags. Some embodiments of the bacterial preparation are not filtered.

“In other embodiments each bag containing the bacteria preparation (filtered, not filtered), is equivalent to one dose MRT product and treated as such. Other embodiments allow the bags to be stored in accordance with proper protocol and then aliquoted into smaller quantities at a later time. Other embodiments allow the bags to be combined according to appropriate protocol to make one or multiple doses. One or more bags of the bacterial preparation may be kept for Quality Control (QC).

“In an exemplary test of quality of the manufactured MRT products (including viability of microbes as processed), a stool sample or fecal donation from one qualified donor can be processed. Some embodiments prohibit the pooling of samples from different donors or individuals. Other embodiments allow for sample preparations from a single donor to be pooled or combined. Other embodiments combine sample preparations from multiple donors.

A sample of the MRT product or processed human stool can be diluted, plated and incubated to count according to industry standards. A CDC plate can count Anaerobic CFU, while a BBE plate can count Bacteroides CFU. The Quality Control standards will be used to verify that plate counts are within acceptable limits. This includes testing results from previous donations and other appropriate standards. The MRT product will only be accepted if the counts fall within the acceptable range. All bags of MRT product made from the same human stool samples will be removed from the freezer and destroyed if they are outside the acceptable range. The final acceptable counts range from 30 to 300 CFU at a serial dilution of 10?6 on CDC plates to 30 to 300 CFU on the BBE plates. This is before freezing the sample preparation or the MRT product.

“FIG. “FIG.5” 5 is another example of a delivery process in a flowchart. The product is ordered and received. It is then inspected. If the product fails to pass inspection, the product is discarded. A report will be sent to the processing centre and a request for a reorder. A report is sent to processing center if the product passes inspection. The product is then either taken to the patient or kept in a secure location before being given to them for treatment. The primary packaging of the product is discarded when it is given to the patient.

“FIG. 7 illustrates sample bag 12. Bag 12 can be made from ethyl vinylcetate. There are other materials that can be used. Bag 12 could include a polyethylene-terephthalate polyester movie, which is a material that is substantially impervious against gases and other polymers. Bag 12 could look like an intravenous bag. Optionally, bag 12 might include an attachment that allows bag 12 to hang on a stand (e.g. to be placed/hung above an endoscope).

Bag 12 may hold between 25 and 250 ml (e.g. 50 ml). Bag 12 may include a fill port 24 to allow for the transport of the MRT composition into bag 12. To facilitate the transportation of the MRT composition, fill port 24 can include a luer (or another type of adapter). Once you have filled bag 12 with the MRT mixture, port 24 can be sealed. This seals the bag’s MRT composition. Bag 12 may also contain a spike port 26. The spike port 26 can be used to extract the MRT from bag 12.

“FIG. 8 illustrates tube assembly 16. Tube assembly 16 might include a spike member 28, which is used to attach the spike port 26 to bag 12. Tube assembly 16 could also include a tube body 30, with a step adapter 32. A step adapter 32 could allow tube 16 and a delivery tube 34 to be combined.

“Tube assembly 16 could include additional features. A clamp 36 could be attached to delivery tube 34. A visual marker 38 can also be placed along delivery tube 34. An optional spike cover 40 can be placed along spike member 28.

The MRT compositions described in the present disclosure could include bacteria belonging to at minimum one phylum, at most 2 phylas, at best 3 phylas, at worst 4 phylas, at worst 5 phylas, at lowest 6 phylas, at maximum 7 phylas, at most 8 phylas, at-most 9 phylas, or at-most 10 phyla. At least some embodiments may contain bacteria belonging to at least one class, at most 2 classes or more, at minimum 3 classes, at the least 4 classes and at the least 5 classes, 6 classes, or 7 classes in the MRT compositions. At least some embodiments may contain bacteria members of at minimum one order, at most 2 orders or 3 orders, at the least 4 orders and at the least 5 orders, 6 orders, or 7 orders in the MRT compositions. At least some embodiments may contain bacteria from at least one family, at most 2 families, at minimum 3 families or at the very least 4 families, as well as at the very least 5 families, 6 families, and at the very least 7 families. At least some embodiments may contain bacteria from at least 5 or 10 families, at most 20 or 30 different genera. The MRT compositions described in at most some embodiments may contain at least 10, 50, 100, 200, 300, or 400 different species of bacteria.

“For example, MRT compositions could contain viable bacteria from one or more orders, or two or more orders, including Clostridiales and Bacteroidales. Some embodiments could contain about 20-95% or 30-85% or 40-60% of viable bacteria in MRT compositions. Some of these, and other embodiments, may contain about 10-150%, or approximately 20-60%, or 30-40%, of viable bacteria in the MRT compositions.

“The MRT compositions can also include bacteria from five or more families or approximately 6-12 families or approximately 7-10 families. It could include bacteria from Bacteroidaceae and Burkholderiales, Clostridiaceae and Clostridiales. Some embodiments could have as much as 20-84% or 30-50% or 36-48% of viable bacteria in the MRT compositions. Some of these, as well as other embodiments, may contain about 0.5-22% or 1% viable bacteria in the MRT formulations. Some of these, and other embodiments, may contain about 1-10% or about 1-8% or approximately 2-7% viable bacteria in the MRT formulations. Some of these, and other embodiments, may contain about 1-22% or 3-22% or about 1-8% or about 4-7% viable bacterial in MRT compositions. Some of these, as well as other embodiments, may contain about 1-10% or about 1-9% or approximately 4-8% viable bacteria in the MRT formulations. Some of these, as well as other embodiments, may contain 0.5-2% to 1% viable bacteria in the MRT formulations. Some of these, and other embodiments, may contain 0.5-23% or about 1- 10% or approximately 4-9% viable bacteria in the MRT compositions. Some of these, as well as other embodiments, may contain 0.5-8% or approximately 1-5% or about 1-3% viable bacteria in the MRT formulations. Some of these, as well as other embodiments, may contain 0.5-2% to 1% viable bacteria in the MRT formulations. Some of these, and other embodiments, may contain about 1-30% or about 1-52% or approximately 4-23% viable bacteria in the MRT formulations. Some of these, as well as other embodiments, may contain about 5-30% or 8-25% or 10-18% viable bacteria in the MRT compositions. Some of these, as well as other embodiments, may contain 0.5-22% or 1% viable bacteria from the family Streptococcaceae.

“The MRT compositions in the present disclosure could have a Shannon Diversity Index between 0.4-2.5 or about 1.0-2, or approximately 1.08-1.89 or 1.25-1.75. These numbers are calculated at a?family? level. level. Calculations at higher levels (e.g. species, phyla etc.) will produce different numbers. Different numbers would be generated (e.g. 1-8). The Shannon Diversity Index could therefore be around 1-8 when calculated at different levels (phyla, species or other).

“The MRT composition described in this disclosure can be administered using a method that is suitable for depositing in a subject’s gastrointestinal tract, preferably their colon. There are many routes for administration, including colonoscopy, colonoscopy and suppository. Intubation through the nose, mouth, or throat by a nasogastric tube or nasoenteric tub, or nasal jejunal tubes, may also be used. Oral administration may be done by tablet, pill, suspension, geltab, semisolid, or tablet. It can also be used as an enteral formula or reformulated to final delivery as liquid, gel, geltab or semisolid. You can use the compositions to treat or untreat fecal bacteria, whole (or substantially) entire microbiota, or isolate, purify, or process fecal fauna into a powder.

“A composition can be administered conveniently in a form that contains one or more pharmaceutically approved carriers for therapeutic purposes in accordance with the method disclosed in this disclosure. The art is well-versed in the selection of suitable carriers. They can be modified to suit the form and method of administration of the composition. They may contain diluents, such as binders or wetting agents, disintegrators and surface-active agents, fillers, binders and glidants, or lubricants. The carrier can be either a solid (including a powder), liquid, or a combination thereof. Acceptable is the preferred way to describe each carrier. In the sense that it is compatible with other ingredients and does not cause harm to the subject. “Biologically acceptable and inert means that the carrier allows the composition to continue to be viable until it is delivered to the proper site.

Oral compositions can include an inert carrier or a diluent. The active compound may be combined with excipients to form tablets, troches, capsules, or other forms for oral therapeutic administration. Combining the composition of this disclosure with food can make oral compositions. One embodiment of the invention includes ice cream as a food for administration. The composition can include pharmaceutically compatible binding agents and/or adjuvant material. Tablets, pills, capsules and troches can contain any one of the following ingredients or compounds. These examples are intended to provide an example and not limit your options.

The active compounds may also be prepared as suppositories, e.g. with conventional suppository base bases like cocoa butter or other glycerides, or retention enemas to provide rectal delivery. You can prepare active compounds with carriers that protect it from rapid elimination from your body. This includes implants and controlled release formulations. You can use biodegradable and biocompatible polymers such as polyanhydrides polyethylene vinyl acetate, ethylene vinyl acid, polyglycolic acids, collagen, polyorthoesters and polylactic acid. These formulations can be made using standard methods. These materials can also easily be purchased commercially at Nova Pharmaceuticals, Inc. and Alza Corporation. Liposomal suspensions are also available for use as pharmaceutically acceptable carriers. These materials can be prepared using techniques that are well-known to the skilled.

A composition can be encapsulated. When the composition is to go orally, for example, the dosage form must be formulated so that it is not exposed to the conditions of the gastrointestinal tract, such as high acidity or digestive enzymes. It is a common practice to encapsulate compositions for therapeutic purposes. There are many options for encapsulation, including hard-shelled capsules that can be used to encapsulate dry, powdered ingredients and soft-shelled caps. Capsules can be made from aqueous gelling agents, such as animal protein (e.g. gelatin), plant polysaccharides and derivatives like carrageenans or modified forms of starch. A gelling agent solution may also contain other ingredients such as coloring agents, disintegrants or lubricants, and even surface treatment.

Gels, creams and lotions are suitable for rectal administration. Preferably, the formulations are provided as unit-dose capsules that contain the active ingredient in one of the solid carriers that form the suppository base. These formulations can be carried in petroleum jelly, lanolin or polyethyleneglycols as well as alcohols and combinations thereof. Colonic washes containing the rapid colonization deployment agent described in the present disclosure could also be prepared for rectal administration.

For oral administration, formulas can be prepared in discrete units such as capsules, tablets, cachets or syrups. They also come with elixirs and chewing gum. Formulations, microemulsions solutions, suspensions and gel-coated ampules containing predetermined amounts of active compound.

“Formulations that are suitable for transmucosal administration, such a by sublingual or buccal administration, include lozenges patches and tablets, which contain the active compound and typically a flavor base such as sugar and/or tragacanth, and pastilles that contain the active compound in an inert basis such as gelatin, glycerine, or sucrose acacia.”

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