Patent Application
20200147151
The present invention relates generally to the fields of microbiology and medicine. More particularly, it concerns compositions of fecal microbiota and uses thereof.
CDI recurrence is associated with reduced diversity of fecal microbiota and impaired microbial community resilience and colonization resistance (1). In a study of elderly hospitalized patients with CDI, gut microbiota showed lower proportions for those taxa previously shown to be associated with diverse healthy intestinal microbiota, Bacteroides, Alistipes, Lachnospira or Barnesiella, when compared with elderly patients receiving antibiotics in the hospital but without CDI (2).
Fecal microbiota transplantation (FMT) has become standard for therapy of recurrent CDI with clinical cure rates ≥80% (3-8) with most centers using frozen fecal products (5, 8-10). Preliminary data suggest that lyophilized FMT product given orally to patients with recurrent CDI could reduce subsequent infections (11, 12). However, there is an unmet need for lyophilized FMT product with fresh and frozen fecal FMT products, with follow-up of recipients for five months for clinical response and for one month after FMT for microbiome composition in a subset of subjects.
Embodiments of the present disclosure provide compositions comprising human fecal microbiota and methods of use thereof. In a first embodiment, there is provided a composition comprising an extract of human feces comprising fecal microbiota and at least one sugar. In some aspects, the extract of human feces comprises viable fecal microbiota. In particular aspects, the human feces is initially derived from an individual screened to have a normal, healthy or wild type population of fecal flora. In some aspects, the extract of human feces is from a donor who has not been exposed to antibiotics for at least three months prior to sample collection.
In some aspects, the sugar is mannitol or sucrose. In certain aspects, the at least one sugar is present at a concentration of 0.5% to 5% (vol/vol), such as 1% to 3% (vol/vol). In some aspects, the at least one sugar is present at a concentration of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (vol/vol). In particular aspects, the at least one sugar, such as mannitol, is present at a concentration of about 2% (vol/vol).
In certain aspects, the composition comprises at least 4 different phyla of bacteria selected from the group consisting of Bacteroidetes, Firmicutes, Proteobacteria, Verrucomicrobiae, and Actinobacteria. In some aspects, the composition comprises at least 5, 6, 7, 8, 9, or 10 different classes of bacteria selected from the group consisting of Actinobacteria, Bacteroidia, Bacilli, Clostridia, Erysipelotrichi, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Mollicutes, and Verrucomicrobiae. In certain aspects, the composition comprises at least 4, 5, 6, 7, 8, 9, or 10 different families of bacteria selected from the group consisting of Lachnospiraceae, Enterobacteriaceae, Bacteroidaceae, Ruminococcaceae, Verrucomicrobiaceae, Bifidobacteriaceae, and Veillonellaceae. In additional aspects, the composition is enhanced with one or more additional bacterial species, such as those selected from the group consisting of a Bacteroides species, a Firmicutes species, and Bacillus thuringiensis.
In some aspects, the fecal microbiota essentially consists of particles that will pass through a sieve having a sieve size of 2.0 mm, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180 mm, 0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053 mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, or 0.01 mm. In some aspects, the composition comprises at least 1×1010, 1×1012, 1.5×1012, 2×1012, 2.5×1012, or more bacteria.
In certain aspects the composition is a formulation for oral administration, administration by nasogastric tube, or administration by colonoscopy. In particular aspects, the composition is formulated for oral administration, such as in capsules. In some aspects, the composition is lyophilized. In other aspects, the composition is frozen. In some aspects, the composition is a solid. In some aspects, the composition has been frozen or lyophilized for at least 1 week, 2 weeks, 3 weeks, one month, 2 months, 3 months, 6 months, or longer. In specific aspects, the composition is in the form of a tablet, a troche, or a capsule, particularly a capsule with an acid-resistant enteric coating. In some aspects, the coating comprises hypromellose (HPMC) or hypromellose phthalate (HPMCP). In certain aspects, the coating comprises a mixture of HPMC and HPMCP. In some aspects, the capsule is formulated to release its contents in the small intestines. In some aspects, the capsule has a volume of between 0.2 and 2 mls, between 0.2 and 1.5 mls or between 0.5 and 1.5 mls. In certain aspects, the composition is capable of being re-formulated for final delivery as comprising a liquid, a suspension, a gel, a geltab, a semisolid, a tablet, a sachet, a lozenge, a capsule, or as an enteral formulation. In some aspects, the composition is formulated for multiple administrations.
In some aspects, the composition has a water content of less than 5%. In certain aspects, the composition has a water content of less than 4%, 3%, 2%, 1%, or 0.5%. In some aspects, the composition is frozen. In some aspects, the composition is solid.
In additional aspects, the composition further comprises a saline, a defoaming agent, a surfactant agent, a lubricant, an acid neutralizer, a marker, a cell marker, a drug, an antibiotic, a contrast agent, a dispersal agent, a buffer or a buffering agent, a sweetening agent, a debittering agent, a flavoring agent, a pH stabilizer, an acidifying agent, a preservative, a desweetening agent, coloring agent, at least one vitamin, mineral supplement, a dietary supplement, a prebiotic nutrient or any combination thereof.
In some embodiments, the present disclosure provides an enteric-coated capsule comprising a lyophilized extract of human feces comprising at least 1×1010 viable fecal microorganisms, a sugar, and a salt, wherein the enteric-coated capsule is formulated to release its contents in the small intestines upon oral administration to a human subject. In some aspects, the sugar comprises mannitol. In further aspects, the sugar (e.g., mannitol) is present at about 0.5% to 5%, 1% to 5%, or 1% to 3%. In some aspects, the salt comprises sodium chloride. In certain aspects, the salt (e.g., sodium chloride) is present at about 0.1% to 2%, 0.25% to 1%, or 0.5% to 1%. In specific aspects, prior to lyophilization human feces are blended with a salt solution and a sugar, filtered to remove all solids larger than 0.5 mm, or frozen for at least one hour. In other aspects, prior to lyophilization human feces are blended with a salt solution and a sugar, filtered to remove all solids larger than 0.5 mm, and frozen for at least one hour. In some aspects, prior to lyophilization the human feces are blended with a saline solution at a volume ratio of from 1:2 to 1:10 (feces to saline) and mannitol to a final concentration of 0.5 to 5%. In specific aspects, prior to lyophilization human feces are blended with a saline solution at a volume ratio of 1:5 (feces to saline) and mannitol to a final concentration of 2%. In some aspects, the enteric coating comprises a mixture of HPMC and HPMCP.
In another embodiment, the composition is for therapeutic use to treat a patient having a disease characterized by dysfunctional microbiota. In some aspects, the disease is selected from the group consisting of a Clostridium difficile colitis, a metabolic syndrome, obesity, asthma, eczema, an eosinophilic disorder of the gastrointestinal tract, inflammatory bowel disease, irritable bowel syndrome, ulcerative colitis, Crohn's disease, enterohemorrhagic colitis, chronic diarrhea, chronic constipation, an eating disorder, malnutrition, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, fibromyalgia, chronic fatigue syndrome, nonalcoholic fatty liver disease, and a neurodegenerative disorder. In one particular aspect, the neurodegenerative disorder is Parkinson's disease. In specific aspects, the disease is a Clostridium difficile infection, such as acute Clostridium difficile colitis, relapsing Clostridium difficile colitis, or severe Clostridium difficile colitis.
In addition to the exemplified indication of recurrent CD, the disclosed compositions and methods are being used to treat or alleviate symptoms associated with Irritable Bowel Syndrome (IBS); Fatty Liver disease; and Parkinson's Disease. Additional indications for which these compositions and methods can be used include, but are not limited to, metabolic syndrome, diabetes, obesity, neurodegenerative disorders, neurodevelopmental disorders, autism, minimal hepatic encephalopathy, atherosclerosis, pancreatitis, fibromyalgia, autoimmune diseases or disorders, allergic diseases or disorders, asthma, eczema, an eosinophilic disorder of the gastrointestinal tract, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, fibromyalgia, chronic fatigue syndrome, nonalcoholic fatty liver disease.
A further embodiment provides a composition of the embodiments (e.g., an extract of human feces comprising fecal microbiota and at least one sugar) for therapeutic use to replace a patient's microbiota. In some aspects, some, most, or substantially all of the patient's colon, gut or intestinal microbiota are removed prior to the administering of the composition. In certain aspects, a single dosage of the composition comprises between 1×1010 and 5×1010 cells.
In another embodiment, there is provided a method of preparing a composition comprising an extract of human feces comprising fecal microbiota, the method comprising: blending a fecal sample from a fecal donor with a diluent, filtering the blended fecal sample, and adding at least one sugar to the blended fecal sample. In another embodiment, there is provided a method of preparing a composition comprising an extract of human feces comprising fecal microbiota, the method comprising: blending a fecal sample from a fecal donor with a diluent and at least one sugar and filtering the blended fecal sample. In particular aspects, the diluent is NaCl, such as 0.8% to 0.9% NaCl, particularly 0.85% NaCl. In some aspects, the blending is performed at a 1:2 to 1:10 dilution (fecal sample to diluent). In certain aspects, the blending is performed at a 1:4 to 1:6 dilution (fecal sample to diluent), such as a 1:5 dilution (fecal sample to diluent). In particular aspects, the diluent does not comprise an antibacterial preservative. In some aspects, the blending is performed using a paddle blender, e.g., Stomacher® 80 Master.
In certain aspects, filtering comprises passing the fecal sample through a sieve, such as sterile gauze. The sterile gauze may be composed of multiple layers, such as 1, 2, 3, 4, 5, 6, or more layers, particularly 5 layers. The sterile gauze may be moistened, such as with saline. In some aspects, the sieve comprises a sieve size of no greater than 2.0 mm, 1.0 mm, 0.5 mm, 0.25 mm, 0.212 mm, 0.180 mm, 0.150 mm, 0.125 mm, 0.106 mm, 0.090 mm, 0.075 mm, 0.063 mm, 0.053 mm, 0.045 mm, 0.038 mm, 0.032 mm, 0.025 mm, 0.020 mm, or 0.01 mm. In particular aspects, filtering is performed more than once, such as 2, 3, 4, or more times.
In further aspects, solids can be removed from the sample by sedimenting solids via centrifugation (e.g., at 500-3000×g). In some aspects, solids are removed from a sample by both filtering and centrifugation of the sample.
In some aspects, the at least one sugar is mannitol. In certain aspects, the at least one sugar is present at a concentration of 0.5% to 5% (vol/vol), such as 1% to 3% (vol/vol), particularly at a concentration of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (vol/vol).
In additional aspects, the method further comprises centrifuging a filtrate from the filtering step. In some aspects, the method further comprises freezing or lyophilizing the composition. In certain aspects, the method further comprises reconstituting the composition with an aqueous solution.
In some aspects, the method further comprises freezing the composition for at least 30 minutes, 1 hour, 2 hours, 3 hours, or 4 hours. In some aspects, the sample is frozen for no more than 1 month, 1 week, 3 days, or 1 day. In some aspects, the method further comprises lyophilizing the frozen composition. In particular aspects, the method further comprises formulating the lyophilized composition in enteric capsules. In some aspects, the enteric capsules are acid resistant. In some aspects, the capsules comprise HPMC and HPMCP. In particular aspects, the capsules are Acid Resistant AR Caps® (see the world wide web at farmacapsulas.com/en/productos/ar-caps-2/). In specific aspects, the method further comprises storing the enteric capsules at between 10° C. and 1° C., such as at about 4° C.
A further embodiment provides a method for replacing, supplementing, or modifying a subject's colon microbiota, the method comprising administering to the subject a composition of the embodiments (e.g., an extract of human feces comprising fecal microbiota and at least one sugar). In some aspects, the method further comprises reconstituting the composition with an aqueous solution. In certain aspects, the method further comprises removal of some, most, or substantially all of the subject's colon, gut or intestinal microbiota prior to administering the composition. In some aspects, the composition is administered more than once. In certain aspects, the composition is administered daily, weekly, or monthly. In some aspects, the composition is administered for two, three, or four months. In certain aspects, the composition is administered orally or by colonoscopy.
In yet another embodiment, there is provided a method for treating a subject having a disease characterized by dysfunctional microbiota, the method comprising administering to the subject in need thereof an effective amount of the composition of the embodiments (e.g., an extract of human feces comprising fecal microbiota and at least one sugar). In some aspects, the method further comprises removal of some, most, or substantially all of the subject's colon, gut or intestinal microbiota prior to administering the composition. In certain aspects, the disease is selected from the group consisting of a Clostridium difficile colitis, a metabolic syndrome, obesity, asthma, eczema, an eosinophilic disorder of the gastrointestinal tract, inflammatory bowel disease, irritable bowel syndrome, ulcerative colitis, Crohn's disease, enterohemorrhagic colitis, chronic diarrhea, chronic constipation, an eating disorder, malnutrition, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, fibromyalgia, chronic fatigue syndrome, nonalcoholic fatty liver disease, and a neurodegenerative disorder. In some aspects, the neurodegenerative disorder is Parkinson's disease. In particular aspects, the Clostridium difficile colitis is recurrent. In some aspects, the composition has been frozen or lyophilized for at least 1 week, 2 weeks, 3 weeks, one month, 2 months, 3 months or 6 months prior to the administration.
In certain aspects, the composition is administered more than once. In some aspects, the composition is administered daily, weekly, or monthly. In certain aspects, the composition is administered for two, three, or four months. In specific aspects, the composition is administered orally or by colonoscopy.
In additional aspects, the further comprises evaluating the subject by microbiome sequencing. In some aspects, administering the composition results in increased microbial diversity in the subject. In certain aspects, administering the composition results in a decrease in the proportion of Proteobacteria and/or in increase in the proportion of Firmicutes, Actinobacteria, Bacteroidetes and/or Verrucomicrobia in the subject's microbiota.
As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
As used herein in the specification and claims, “a” or “an” may mean one or more. As used herein in the specification and claims, when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein, in the specification and claim, “another” or “a further” may mean at least a second or more.
As used herein in the specification and claims, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating certain embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
The present studies confirmed the activity of both frozen and lyophilized fecal product for fecal matter transplantation (FMT) in patients with recurrent Clostridium difficile infection (CDI) without obvious adverse experiences. Thus, the frozen and lyophilized product with a sugar, such as 2% mannitol, as a cryoprotectant is provided in the present disclosure. In particular, the fecal composition can be encapsulated in enteric-coated capsules for oral use. In addition to recurrent CDI infections, the fecal composition provided herein may be used for other disorders in which the subject has a dysfunctional microbiota.
PRIM-DJ2727 (microbiota suspension) is an intestinal microbial suspension prepared from stool obtained from carefully and thoroughly screened healthy human donors. PRIM-DJ2727 capsules are administered orally. PRIM-DJ2727 is prepared from a standard amount of healthy human stool mixed with 0.85% NaCl (normal saline) and 2% mannitol. Product in capsule formulation is stored at the University of Texas School of Public Health at 4° C. The product is given to the subjects orally at the clinic with drinking water. PRIM-DJ2727 can be delivered, inter alia, by colonoscopy, retention enema, or oral administration of lyophilized capsules, comprising for example lyophilized product with 2% mannitol.
In a preferred embodiment, subjects/patients with multiple (more than 3) recurrent C. difficile infections (RCDI) can be treated with multiple or weekly oral administration of lyophilized and encapsulated PRIM-DJ2727 (5-6 capsules of microbiota suspension). RCDI patients treated with PRIM-DJ2727 are expected to demonstrate improved flora diversity and a reduction in the symptoms or RCDI, such as diarrhea, nausea, vomiting, abdomen pain, fever or other potential flora mediated disorders, as well as reductions in the presence of C. difficile toxins.
In another preferred embodiment, subjects/patients with Nonalcoholic fatty liver disease (NAFLD) can be treated with weekly oral administration of lyophilized and encapsulated PRIM-DJ2727 (5-6 capsules of microbiota suspension). It is believed that intestinal flora may play an active role in the development of insulin resistance and probiotics can have a favorable effect in the management of NAFLD in animals and humans, in some cases reducing proinflammatory cytokines. NAFLD patients receiving therapy are expected to improve with regards to metabolic function, as determined by blood studies; increasing BMI; changes in diabetic control; and liver function studies In addition, NAFLD patients receiving therapy are expected to experience decreased symptoms of fatty liver disease: feeling tired, loss of weight or appetite, weakness, nausea, confusion, poor judgement or trouble concentrating.
In yet another preferred embodiment, subjects/patients with Parkinson's Disease can be treated with weekly oral administration of lyophilized and encapsulated PRIM-DJ2727 (5-6 capsules of microbiota suspension). Abnormal microbiota, dysbiosis and small bowel bacterial overgrowth may contribute to the pathogenesis of Parkinson's disease through effects on cytokine levels of the gut, local mucosal inflammation, motility regulation and alteration of mucosal permeability and constipation. Parkinson's Disease patients receiving therapy are expected to improve with regards to bowel habits, as well as neurologic symptoms as measured on the Unified Parkinson's Disease Rating Scale which measures mentation, behavior, mood, activities of daily living and motor manifestations as well as changes to anti-PD medications and patient assessments of global improvement in PD and quality of life.
In yet another preferred embodiment, subjects/patients with Irritable Bowel Syndrome (IBS) can be treated with weekly oral administration of lyophilized and encapsulated PRIM-DJ2727 (5-6 capsules of microbiota suspension) for 8 weeks. Irritable Bowel Syndrome (IBS) patients receiving therapy are expected to improve with regards to bowel habits, symptoms such as, but not limited to, diarrhea, constipation, abdomen pain as well a reduction in overall IBS symptom severity scale (IBS-SSS questionnaire),
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
72 subjects with ≥3 bouts of CDI were randomized in a double-blind study to receive fresh, frozen or lyophilized FMT product via colonoscopy from 50 g of stool per treatment from eight healthy donors. Recipients provided stools pre- and 7, 14 and 30 days post-FMT for C. difficile toxin and, in a subset, microbiome composition by 16S rRNA gene profiling.
Enrollment criteria included a history of ≥3 separate bouts of CDI in the past 12 months in non-pregnant adults ≥18 years of age. A study exclusion was previous receipt of an FMT. Subjects were instructed to stop all anti-CDI medications 48 hours before the FMT procedure. Subjects were enrolled and randomized by computer table to receive fresh, frozen or lyophilized donor fecal microbiota on a 1:1:1 allocation. The subjects were contacted daily by telephone after FMT for safety and clinical features and after one week contacted by telephone weekly for 5 months and instructed to report health issues. Subjects provided written subject diary of symptoms at regular intervals for five months after FMT for adverse events, stool pattern and CDI episodes. Recipients provided four stools each: 1-2 days before and 7, 14 and 30 days after FMT. When diarrhea occurred, the subjects underwent C. difficile toxin testing and if positive, antimicrobial treatment was given at the discretion of their physicians with the recommendation that they take two weeks of vancomycin followed by tapering doses of the drug.
Donors were thoroughly evaluated by history, serum and stool studies using previously published methods (13) and were required to have all negative results and to have normal body BMI.
Preparation of FMT Product—Individual stool samples from donors of ≥50 g were processed within 4 hours of passage, diluted in 0.85% NaCl (1:10) with a total volume of 1,500 mL and mixed in a Stomacher® 80 Master (Seward Laboratory System Inc., Davie, Fla.) in a sterilized bag, then filtered through moistened 5-layer sterile gauze in a funnel (both sterilized) under biological safety cabinet. Stools remained at room temperature less than 8 hours after which bacterial content would be expected to be reduced (14).
Fresh aliquots were used within 2 hours of preparation. Frozen aliquots were stored at −80° C. and used within 6 months after preparation. For the product to be lyophilized, the 50 g filtered solution was frozen at −80° C. for at least 6 hours, then placed under a Freeze Dry System (Labconco, Kansas City, Mo.) enabling the water in the product to vaporize without passing through the liquid phase (sublimation). After lyophilization, the product was maintained as powder at 4° C. in sealed 50 mL conical tubes and used within 6 months after preparation. Prior to the study the inclusion of the 10% glycerol with the fecal extract was evaluated but a dry product by lyophilization could not be obtained so no cryoprotectant was used in the study.
On the day of FMT, 250 mL of sterilized 0.85% NaCl (Thermo Fisher Scientific, Waltham, Mass.) was added to the vials of lyophilized donation products. After reconstitution, the product was kept at 4° C. and used within 4 hours.
Transplantation was performed via colonoscopy after polyethylene glycol colonoscopy bowel preparation the night before. Subjects took a single dose of 4 mg loperamide 3 hours before the procedure. Two gastroenterologists performed all FMT administrations infusing 50% of the product in the proximal colon with the remaining 50% infused in progressively decreasing amounts until reaching the rectum where the last portion of product was delivered.
Study Blinding—Only the laboratory director, was aware of the type of product assigned to subjects. Syringes with product prepared in the laboratory and identified by number were taken to the endoscopy suite at a university hospital in Houston for blind product administration. The treating gastroenterologist, the nurses following up the subjects and the recipients were unaware of the product assignment.
Laboratory Studies—All stools (donor and recipient) were examined for form and tested for presence of C. difficile toxin by commercial enzyme immunoassay (Remel, Lenexa, Kans.). Microbiota composition and relative abundance were evaluated by 16S rRNA gene compositional analysis in a subset of subjects. Using methods developed for the Human Microbiome Project, DNA isolation and microbiome sequencing were conducted at the Baylor College of Medicine's Alkek Center for Metagenomics and Microbiome Research (15, 16). Briefly, genomic bacterial DNA was extracted from fecal samples using the MO BIO PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, Calif.). One aliquot of each frozen stool sample was thawed, and 500 μL of stool was transferred to a MO BIO PowerSoil DNA Extraction PowerBead Tube. Samples were incubated at 95° C. for 10 minutes, then at 65° C. for 10 minutes, followed by genomic DNA extraction using the MO BIO PowerSoil DNA Extraction Kit protocol, and DNA samples were stored at −20° C.
Microbiome sequence analysis was performed on the first 52 subjects providing all four stool samples. DNA extraction, polymerase chain reaction and sequencing were performed using a published protocol (17). Briefly, the 16S rRNA gene V4 region was amplified by PCR and sequenced in the MiSeq platform (Illumina, San Diego, Calif.) using the 2×250 bp paired-end protocol yielding pair-end reads. The primers used for amplification contain adapters for MiSeq sequencing and single-end barcodes to permit pooling and direct sequencing of polymerase chain reaction products (17). Read pairs were demultiplexed based on the unique molecular barcodes, and reads were merged using USEARCH v7.0.1090 (18) allowing zero mismatches and a minimum overlap of 50 bases. Merged reads were trimmed at first base with Q5. In addition, a quality filter was applied to the resulting merged reads and reads containing more than 0.05 expected errors were discarded. 16S rRNA gene sequences were clustered into Operational Taxonomic Units at a similarity cutoff value of 97% using the UPARSE algorithm (19, 20). Operational Taxonomic Units were mapped to an optimized version of the SILVA Database (21) containing only the 16S v4 region to assign taxonomies. Demultiplexed reads were mapped to UPARSE Operational Taxonomic Units to recover abundances.
Statistical Methods—For quantitative description of changes in microbiota, diversity and richness of microbiota were first measured in each sample. Shannon diversity (Shannon entropy) (22) was used to measure diversity and total number of Operational Taxonomic Units with mapped reads to measure richness. Box plots were generated by grouping samples into five groups: donors, recipients before FMT, and recipients 7, 14, and 30 days post-FMT. Each sample group was then divided into the sub-groups of fresh, frozen, and lyophilized depending on the FMT delivery method. To determine whether significant differences in diversity or richness measures between different groups of samples existed, the Kruskal-Wallis test was used. The Kruskal-Wallis test is a non-parametric rank-based method to test whether samples in two or multiple groups are drawn from the same distribution without assuming a specific underlying distribution (23).
Relative abundances of each phylum and family were then aggregated relative to the donor product using stacked bar plots. In addition to diversity and richness measures, 2-dimensional plots of individual samples were generated to visualize relative similarity and discrepancy between individual samples. Beta microbiota diversity was used to compare the microbial composition between different samples by using the Bray-Curtis (BC) index in the Phyloseq (24) package with R version 3.3.1 for the PCoA analysis. All other analyses were performed in Stata version 14.
Sample Size Calculation—In developing a sample size, it was assumed each FMT treatment would be of comparable efficacy in preventing post-treatment recurrences. Because of ethical considerations wanting subjects to receive effective treatment, the Institutional Review Board approved a 100 subject study and in view of the large cost of the studies it was decided to perform an interim analysis after enrolling at least 20 subjects/group. When a statistical difference was found between two of the treatment groups enrollment was stopped.
Study Approval and Registry—The study used an approved Institutional Review Board protocol from the University of Texas Health Science Center and Baylor St. Luke's Medical Center (IRB # HSC-SPH-13-0119) and was registered in ClinicalTrials.gov (Number: NCT02318992).
Results
Of 11 potential donors screened, 8 (5 males and 3 females) were included in the study. Their median age was 58 years (range 36-77). For the 3 excluded donors, 2 were positive for fecal C. difficile toxin while for the third donor serum tested positive for serum hepatitis B surface antibody. Eight of the donors provided all stools needed for FMT products.
Seventy-three subjects were enrolled with one being lost to follow-up after FMT. Demographics and clinical features were similar in the 3 treatment groups (Table 1 below). Overall 63 of 72 (87%) subjects undergoing FMT achieved clinical wellness with no subsequent bouts of CDI during the two months following FMT. The cure rate was highest for the group randomized to receive fresh product, 25/25 (100%) and lowest for the group of subjects randomized to receive lyophilized product, 18/23 (78%), with intermediate response seen in the group receiving frozen product, 20/24 (83%) (p=0.041). The group receiving fresh product showed a statistically significant increase in efficacy compared with the group receiving lyophilized material (p=0.022); differences between groups receiving fresh vs. frozen did not reach significance (see Table 2 below). All failures of FMT occurred in the absence of receiving antibiotics during the observation period. One patient had recurrence of CDI at 150 days after FMT with lyophilized product and was considered cured in the analysis. The failures were all treated with a tapering dose of vancomycin and not followed further.
Twelve of 14 (86%) subjects with pre-existent inflammatory bowel disease were cured from their CDI. Six of the 10 recurrences of CDI occurred 7 days after FMT; one subject each was diagnosed with post-FMT CDI at 14 days, 41 days, 41 days and 150 days.
Cure rates in recipients were not significantly associated with specific donors (p=0.633). Cure rates for each donor were: 3/3 (100%), 2/3 (67%), 19/21 (90%), 5/6 (83%), 17/18 (83%), 7/8 (88%), 6/8 (75%), and 3/5 (60%).
Laboratory Studies—There was no difference in frequency of 2 month post-FMT CDI development between subjects with fecal C. difficile toxin at enrollment 3/11 (15%) compared with the toxin-negative group 6/61 (10%; p=0.108).
A subset of 52 subjects providing all four stool samples were included in this part of the study. These samples generated 1,558,302 high-quality, filtered, Illumina pair-end reads and were analyzed at a depth (rarefied) of 1,556 reads per sample. The mean/median diversity of fecal samples from recipients before FMT was notably lower than the average diversity of donors as measured by the Shannon diversity index (
Among the three FMT products, the Shannon diversity index was slightly lower for the group randomized to receive frozen product (P=0.0636). The median Shannon diversity indices 7 days post-FMT were lower in the lyophilized group compared with the fresh and frozen groups although three-way Kruskal-Wallis test statistic was not significant (p=0.0917). The median Shannon diversity index of the lyophilized group was 3.6, notably lower than that in the fresh and frozen groups (4.0 and 3.9, respectively). Diversity measures in the lyophilized group remained significantly lower at 14 days after FMT in a three-group test (p=0.0305). Thirty days after FMT, the median diversity in the lyophilized group increased to 3.9, similar to the other two groups (p=0.259). Total observed Operational Taxonomic Units in the lyophilized group remained significantly lower than other groups at 7, 14, and 30 days after FMT, but the median values gradually increased from 45 to 51 (
Taxonomic compositions of microbial population in subjects with recurrent CDI patients were also remarkably different from those of donors before FMT but resembled the composition of donors after FMT (
Post-FMT in the recipients, it was found that 10 microbial families represented more than 90% of the overall microbial population in all groups (
The microbiome composition of FMT recipients shifted over the study time points as demonstrated by PCoA of Bray-Curtis distances (
Adverse Events (AEs)—There were no serious adverse events. There were no observed differences in proportions of subject adverse events in the three groups. During the 48 hours after FMT a majority of subjects complained of nausea, mild diarrhea and transient abdominal discomfort (62/72, 86%). Two subjects experienced subjective fever, one at 2 days and the other 7 days after FMT; both of these failed FMT. Other complaints were fatigue (6/72, 8%), headache (4/72, 6%) and weight gain 2/72, 3%). One subject reported a gain of 7 pounds body weight 7 days after FMT. The other reported a gain of 8 pounds body weight 30 days after FMT, which brought their weight up to pre-CDI levels.
Overall resolution of CDI was 86% during 5 months of follow-up after FMT. Stool samples collected from subjects with CDI before FMT had significantly decreased bacterial diversity with a high proportion of Proteobacteria compared to donors. Cure rates were highest for the group receiving fresh product seen in 25/25 (100%), lowest for the lyophilized product 16/23 (78%; p=0.022 vs. fresh and 0.255 vs. frozen) and intermediate for frozen product 20/24 (p=0.233 vs fresh). Microbial diversity was reconstituted by day 7 in the subjects receiving fresh or frozen product. Improvement in diversity was seen by day 7 in those randomised to lyophilized material with reconstitution by 30 days.
Comparative efficacy in FMT was observed in subjects receiving fresh or frozen fecal product from the same donors. Lyophilized product with slightly lowered efficacy compared with fresh product resembled other treatments in microbial restoration one month after FMT.
PRIM-DJ2727 is prepared from 150 g of human stool from a healthy, screened donor mixed with 750 mL 0.85% NaCl (normal saline) with 2% mannitol, which is added to reduce fluffiness of the product. It will contain normal microbiota. The product was designed and developed by the principal investigators from the University of Texas Health Science Center in Houston School of Public Health, University of Houston School of Pharmacy and Compounding Shop (License No. 29601) in Houston, Tex.
Donor Screening and Testing—Each donor must be disease- and enteric pathogen-free before donations begin. Donors are stable employees of the Texas Medical Center.
Since the product being used in the studies is derived from fecal matter obtained from a small number of volunteers, donor screening is critical to assure subject health to minimize safety concerns. A standard donor qualification process has been implemented. Donors sign an informed consent form and are screened at the Center for Infectious Disease, the University of Texas School of Public Health. Each donor must meet inclusion/exclusion criteria (see Table 3 below.
Potential donors complete a comprehensive initial health, family history and lifestyle questionnaire and then provide blood and stool sample for a comprehensive analysis for potential pathogens, including HIV, Hepatitis B, C and syphilis in the blood and a wide variety of pathogens in the stool. (see, Table 4 below)
Clostridium difficile toxin A/B
Shigella spp.
Campylobacter spp.
Escherichia coli
Staphylococcus aureus
Enterococcus spp.
Enterobacteriaceas
Aeromonas spp
Plesiomonas spp
Yersinia spp.
Vibrio spp
Cryptosporidum
Entamoeba histolytica
Cyclospora
Isospora
On the day of donation, 200 g of stool per transplant are obtained (all stools greater than 200 grams will be accepted and used in totality) with stool passage not more than 2 hours with ice package before submitting for processing. Stool will be kept in an Igloo cooler with ice pack during transportation, see the protocol # UT-SPH-CID-04-040A. Stools less than 200 g will be discarded. No more than 10 donation products will be made within each lot number. An additional stool from the same donor will be collected within one month screened for the second form of FMT.
Processing of the sample will be as follows:
Storage—UTSPH-FMT-D-XXX-FRESH Intestinal Bacteria will be kept in an igloo color with ice pack and delivered to the procedure room and used within 4 hours after completion the process. UTSPH-FMT-D-XXX-FROZEN Intestinal Bacteria will be kept at −80° C. for up to 6 months at the University of Texas Health Science Center at Houston School of Public Health room 804 RAS (CID-0066-CAP). UTSPH-FMT-D-XXX-Lyophilized products containing bacteria from ≥200 g stool will be kept in a sterilized vial (50 mL conical tube) at 4° C. at the University of Texas Health Science Center at Houston School of Public Health room 320 RAS (CID-0068-CAP) for up to 6 months in a test tube rack clearly labelled with the lot # and expiration date. This FMT product will be delivered via colonoscopy. On the day of FMT, 500 mL of sterilized 0.85% NaCl (Thermo Fisher Scientific, Waltham, Mass.) will be added to the sterilized vials with lyophilized donation products (concentrated from 100 g stool). After reconstitution, the product will be used with 4 hours and kept at 4° C. During the transportation (15 minutes driving distance), the product will be kept in an igloo color with ice pack. The process for reconstitution will be completed at the University of Texas Science Center at Houston School of Public Health in room 320 RAS. UTSPH-FMT-D-XXX-Lyophilized Capsules containing bacteria from 100 g stool will be kept in a sterilized vial at 4° C. at the University of Texas Health Science Center at Houston School of Public Health room 320 RAS (CID-0068-CAP) for up to 6 months in a test tube rack clearly labelled with the lot # and expiration date. Upon completion of the process, stool samples must be recorded into the Donor Specimens Log.
Aside from the addition of 0.85% NaCl and 2% mannitol (Fisher Scientific), which is added to increase the density of the sample during processing as described above, the sample is not manipulated (i.e., no strains are purified from the sample.)
PRIM-DJ2727 (intestinal flora suspension) is obtained from healthy, screened donors. It contains normal microbiota. PRIM-DJ2727 will be labeled with lot number and date and time processed in addition with D-001-LIB-R-001.
Placebo will be identical to the investigational product but will not contain intestinal bacteria. Placebo will consist of Lactose (spray-dried USP 64.385 gm), and food color: powdered Black (0.847 gm), Brown (3.384 gm), and Yellow (3.384 gm); in the enteric capsules (size 00). Placebo will be made at Compounding shop, 11851-A Wilcrest, Houston, Tex. 77031 (License No. 29601).
Placebo products will be tested for bioburden testing according FDA standards regarding the microbial examination of non-sterile products, see Protocol # UT-SPH-CID-04-039A.
Environmental Assessment—PRIM-DJ2727 (intestinal flora suspension) is obtained from healthy, screened donors. It will contain normal fecal microbiota which produces no harmful effects to the environment, including air, soil or water. Since donors will be carefully screened, there should be no known enteric pathogens in the product (PRIM-DJ2727) with current knowledge and laboratory technology.
A double-blind, placebo-controlled, randomized 2:1 pilot study evaluating the effects of lyophilized PRIM-DJ272 microbiota on bowel function will be performed with randomly selected individuals suffering from irritable bowel syndrome (IBS). IBS is defined as following Rome III criteria IBS, having abdominal pain intensity with a weekly average of worst daily (in past 24 hours) abdominal pain score of ≥3.0 on a 0 to 10 point pain scale. Moderate to severe IBS is defined by a score of ≥175 overall on the IBS symptom severity scale (IBS-SSS).
Studies have shown that fecal flora disturbances occur in IBS. This pilot study is intended to characterize the microbiome in this group of subjects, and evaluate microbiota replacement treatment as a means of flora restoration. This study will evaluate the effects of lyophilized PRIM-DJ2727 microbiota on bowel function, as well as on abdominal pain in IBS subjects. Further, it will characterize the intestinal flora in subjects with IBS and determine the safety and trend in individuals with 50% improvements in diversity of fecal microbiome following administration of lyophilized PRIM-DJ2727 microbiota.
This study will compare the effect of treatment with lyophilized PRIM-DJ2727 microbiota to placebo, and evaluate its effect in individuals on the overall IBS symptom severity scale (IBS-SSS questionnaire) and a health survey questionnaire (SF-36), as well as evaluate the product safety of PRIM-DJ2727 given orally each week for 8 weeks, including whether the patient suffers from diarrhea, constipation, or abdomen pain. 45 individuals will be included, 30 treated with FMT and 15 with placebo.
This study will also characterize the microbiome (diversity and genera) and microbiome stability in subjects with IBS treated with fecal microbiota transplantation (FMT) for 8 weeks during an 8 month study. This study will compare the proportions of subjects who have adequate relief of global IBS symptoms for at least 2 weeks during the 8 weeks of therapy in the lyophilized PRIM-DJ2727 microbiota group and the placebo group, and compare the effect of lyophilized PRIM-DJ2727 microbiota on the stool form (Bristol Stool Scale) and stool frequency. Additionally, the study will compare the proportions of subjects who experience a decrease in the weekly average of worst abdominal pain in the past 24 hours score of at least 30% compared with baseline in the lyophilized PRIM-DJ2727 microbiota group and the placebo group.
The study will consist of 3 parts: the baseline, treatment and safety assessment periods. Subjects will have a screening evaluation visit with a study physician to include history and physical exam, a review of medical records, and any necessary screening tests required for inclusion if ordered. If the subject qualifies, their physician will refer the subject to a research coordinator at Kelsey-Seybold Clinic who will consent the subject, draw blood serum, provide a stool sample collection kit for the subject, and, if applicable, a urine pregnancy test.
After consent, subjects begin the 2-week run-in period. During the screening period (Days 1-14), participants will complete daily diary questionnaires as well as SF36 and IBS-SSS at the end of each week. An aliquot (2 mL) of each stool sample collected will be stored at −80° C. for future analysis (e.g. microbiome studies). The serum sample will be stored for future study, possibly related to cytokine and chemokine levels.
After completion of the screening phase, the eligible participants will be randomized to 8 weekly treatments of either lyophilized PRIM-DJ2727 microbiota or placebo. Treatment will be administered as twice filtered fecal microbiota product diluted in saline to 250 mL from a screened healthy donor, lyophilized and contained within enteric-coated capsules (5-6 capsules) in a weekly dose for 8 consecutive weeks. In the treatment phase, participants will continue to keep daily diaries for all bowel movements. The IBS-SSS and SF-36 and Hospital Anxiety and Depression Scale (HADS) questionnaires will be completed on the day of enrollment and at all evaluation appointments, including the end of study evaluation. Compliance and adverse effects also will be monitored through the daily diaries that are collected weekly. Follow up for each subject will be 6 months.
Based on 90% clinical efficacy in curing recurrent Clostridium difficile infection (CDI) with the lyophilized product, it is hypothesized there will be a 50% improvement in subjects with IBS treated with weekly FMT, compared to the placebo group. The 50% improvement will be determined by analyzing patients' scores on the IBS Symptom Severity Scale (IBS-SSS), the Health Related Quality of Life SF36 Questionnaire (SF36), and the Hospital Anxiety and Depression Scale (HADS). Using the 2:1 allocation ratio, it is expected that there is more than 80% power to detect the statistical difference when recruiting a total of 45 subjects (30 with FMT and 15 with placebo) in this preliminary study. Failure of the treatment is defined as a subject that has less than 50% improvement at the end of study.
To ensure safe study monitoring during the study, an independent, non-blinded, external Data and Safety Monitoring Board (DSMB) will be established to perform safety evaluations on an ongoing basis. A DSMB charter will be developed and will detail the review of the safety data. The schedule of assessments and procedures is provided in Section 9.0. The study will be discontinued if the DSMB identifies a trend of more severe illness in the active study participants.
The inclusion and exclusion criteria for this study are presented below, in Table 5.
A double-blind, placebo-controlled, randomized 2:1 pilot study evaluating the effects of lyophilized PRIM-DJ272 microbiota on bowel function will be performed with randomly selected individuals suffering from Parkinson's Disease (PD).
Limited studies have shown that fecal flora disturbances occur in PD. This pilot study will completely characterize the microbiome in this group of subjects, and evaluate microbiota replacement treatment as a means of flora restoration in PD subjects. This study will characterize the intestinal flora in subjects with PD and determine safety and trends in improvements in diversity of colonic microbiome following administration of PRIM-DJ2727.
This study will examine improvements in flora diversity by oral administration of a fecal suspension from healthy donors, determining said improvements by comparing data from treated individuals with untreated controls. The study will also seek to determine whether there is improvement in bowel patterns in PD subjects, focusing on bowel habits following treatment.
Additionally, the study seeks to understand the neurological state of PD subjects as a result of fecal microbiome changes. The study will look at improvement in neurologic picture using the Unified Parkinson's Disease Rating Scale measuring mentation, behavior, mood, activities of daily living and motor manifestations as a result of PRIM-DJ2727 treatment. The subjects will also be monitored for change in required anti-PD medications following PRIM-DJ2727 treatment. The study will also ask subjects for assessments of global improvement in PD, as well as quality of life.
Individuals in the study will be carefully monitored and assessed for complications of PD including worsening of symptoms or other potential flora-mediated disorders. These data will be collected through the 16 weeks of treatment and an additional 8 weeks after treatment via in-clinic assessment. The study would seek to provide data that will be used to determine appropriateness of designing a properly powered clinical trial of microbial restoration treatment in this population.
Forty-five eligible subjects with Parkinson's disorder will be randomly assigned to receive either PRIM-DJ2727 in orally administered enteric coated capsules (30 subjects) or placebo capsules (15 subjects). Subjects meeting inclusion criteria will be randomized in a 2:1 ratio and given either active or placebo fecal microbial transplant and treated weekly for 16 weeks. A physical examination will be performed at the time of enrollment and both stool and blood samples will be collected. Stool and serum will also be collected during the run-in period, as well as during weekly treatments. Subjects will not be required to follow a special diet during this study. 2 mL of each stool sample collected will be stored at −80° C. for future analysis such as microbiome studies. Inclusion and exclusion criteria are listed below, in Table 6.
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
This application claims the benefit of U.S. Provisional Patent Application No. 62/452,325, filed Jan. 30, 2017, the entirety of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/015915 | 1/30/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62452325 | Jan 2017 | US |
