EP4577226A1 - Compositions et méthodes de réduction du sulfure endogène chez des patients atteints de maladies inflammatoires de l'intestin - Google Patents

Compositions et méthodes de réduction du sulfure endogène chez des patients atteints de maladies inflammatoires de l'intestin

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Publication number
EP4577226A1
EP4577226A1 EP23783935.2A EP23783935A EP4577226A1 EP 4577226 A1 EP4577226 A1 EP 4577226A1 EP 23783935 A EP23783935 A EP 23783935A EP 4577226 A1 EP4577226 A1 EP 4577226A1
Authority
EP
European Patent Office
Prior art keywords
composition
sulphide
colon
billion
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23783935.2A
Other languages
German (de)
English (en)
Inventor
Reuben Zachary WHEELER
Wanniarachchige Isiri Adhiwarie PERERA
Mian Chee GOR
Maria Rosita Icban CHUA
Remy Blair YOUNG
Lisa Michelle KIRKLAND
Samuel Paul COSTELLO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cost-Bry Pty Ltd
Original Assignee
Cost-Bry Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2022902716A external-priority patent/AU2022902716A0/en
Application filed by Cost-Bry Pty Ltd filed Critical Cost-Bry Pty Ltd
Publication of EP4577226A1 publication Critical patent/EP4577226A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Definitions

  • UC results from an energy deficiency state in colonocytes (colonic epithelial cells) induced by high levels of sulphide and nitric oxide (NO) depleting Coenzyme A (CoA) and inhibiting 0-oxidation of butyrate. Without energy in the form of adenosine triphosphate (ATP), colonocytes are unable to maintain the normal mucosal barrier that separates the luminal contents of the colon from the mucosal immune system. The loss of the mucus and tight junction barrier then allows activation of the mucosa immune system in a natural killer T-cell driven IL-13- and IL-5-dependent, TH2-mediated, immune response.
  • NO sulphide and nitric oxide
  • CoA Coenzyme A
  • ATP adenosine triphosphate
  • the process is as follows: (1 ) high levels of NO are damaging to human tissue; (2) the source of colonic NO is from anaerobic bacterial respiration or activated immune cells; (3) nitrosation of CoA then produces S-nitroso CoA (inactive) and (a) prevents the acetylation required for lipogenesis and (b) diminishes 0-oxidation of butyrate in colonocytes; (4) colonocyte energy deficiency follows with the inability to synthesis mucus, lipids and proteins;
  • Butyrate is the primary energy source of colonocytes
  • SRB sulphate-reducing bacteria
  • SO X sulphur oxides
  • the management of UC involves both induction therapy (to induce remission) and maintenance therapy (to prevent further flares).
  • the goal of treatment is maintenance of remission without steroids.
  • Induction therapy is usually high dose oral 5-aminosalicylic acid compounds (5-ASAs) with or without topical 5-ASAs via enema or suppository. More severe flares require systemic corticosteroids (tapered over time and discontinued). Maintenance therapy choice in ulcerative colitis is determined by disease extent, severity, frequency of flares and past treatment history.
  • the mainstays of maintenance therapy are the 5-ASAs used orally or topically.
  • thiopurines should be used.
  • new biological agents have demonstrated efficacy for the maintenance of remission in UC. These are the anti-TNFa agents, infliximab, adalimumab and golimumab and the anti- integrin agent, vedolizumab. However, these agents are expensive and have incomplete efficacy.
  • the invention broadly resides in a composition for preventing or treating a gastrointestinal disorder in a subject in need thereof, said composition comprising at least one strain of a microorganism, wherein the microorganism is selected from the group consisting of: bacteria, archaea and yeast.
  • the microorganism comprises a phenotype and/or genes that are responsible for reducing the taxonomic diversity, number of species, relative abundance and or metabolic activity of sulphidogenic microbiota.
  • the microorganism comprises a phenotype and/or genes that are responsible for reducing sulphide levels, taxonomic diversity, number of species, relative abundance and or metabolic activity of sulphidogenic microbiota by reducing release of metabolizable sulphur substrates.
  • the microorganism comprises a phenotype and/or genes that are responsible for reducing colonic protein fermentation; reducing nitric oxide production in the colon; and reducing nitric oxide levels in the colon.
  • the microorganism comprises a phenotype or genes that are responsible for the phenotype of sulphide consumption.
  • the sulphide is present in a form of sulphide selected from the group consisting of: a sulphide ion (S 2 ); a hydrosulphide (HSj ion; a bisulphide ion (SH ), hydrogen sulphide (H2S); an organic compound containing the group R- SH (a thiol); sulphide metals (including but not limited to iron, copper, and molybdenum sulphides.
  • the microorganism comprises a gene or several of the genes selected from the group consisting of: genes responsible for the use of sulphide in the biosynthesis of methionine and cysteine. [0051] In a further embodiment, the microorganism comprises a gene or several of the genes selected from the group consisting of: genes responsible for the anaerobic oxidation of sulphide.
  • the microorganism comprises a gene or several of the genes selected from the group consisting of: genes responsible for sulphide utilisation in the pathway of anaerobic ethanolamine utilisation.
  • the microorganism is selected from a phyla, selected from a genus, a species or an isolate selected from the groups consisting of: a species selected from the group listed in Figure 5 and any combination thereof of these species; an isolate selected from the group listed in Table 1 and any combination thereof of these isolates; a phyla selected from the group listed in Table 2 and any combination thereof of these phylum; a genus selected from the group listed in Table 3 and any combination thereof of these genera; a species selected from the group listed in Table 4 and any combination thereof of these species; an isolate selected from the group listed in Table 5 and any combination thereof of these isolates; an isolate selected from the group listed in Table 8 and any combination thereof of these isolates; and an isolate selected from the group listed in Table 9 and any combination thereof of these isolates.
  • the microorganism comprises a 16S ribosomal RNA (rRNA) gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs 1 to 206.
  • rRNA ribosomal RNA
  • the microorganism comprises whole genome sequences having a nucleotide sequence selected from the group consisting of: SEQ ID NOs 207 to 412. [0059] In a further preferred embodiment, the microorganism comprises whole genome sequences having a nucleotide sequence selected from the group consisting of: SEQ ID NOs 413-9193.
  • the microorganism comprises a 16S ribosomal RNA (rRNA) gene or contigous whole genome sequence having a sequence identity to one or more SEQ ID NOs 1 to 9193, wherein the sequence identity is selected from the group consisting of: at least 99.9%; at least 99.5%; at least 99%; at least 98.5%; at least 98%; at least 97.5%; at least 97%; at least 96.5%; at least 96%; at least 95.5%; at least 95%; at least 94.5%; at least 94%; at least 93.5%; at least 93%; at least 92.5%; at least 92%; at least 9 .5%; at least 91 %; at least 90.5% and at least 90%.
  • rRNA 16S ribosomal RNA
  • the microorganism is a faecal or colonic microorganism.
  • the microorganism is non-inflammatory.
  • the microorganism is cultured from a faecal or colonic biopsy sample.
  • the microorganism comprises a community of microorganism cells derived from a stool or biopsy of one or more human donors.
  • the community of microorganism cells comprises cultured microorganism cells.
  • the cultured microorganism cells are derived from a multiple of human donors.
  • the community of microorganism cells comprises uncultured microorganism cells.
  • the composition is a faecal transplant microbiota composition.
  • the composition is lyophilized.
  • the composition is a liquid. [0072] In a further preferred embodiment, after at least 4 weeks of storage at room temperature, said composition is capable of maintaining at least 50% cell viability relative to the initial cell viability immediately prior to storage.
  • said composition is capable of maintaining about 60% to about 80% cell viability relative to the initial cell viability immediately prior to the start of said storage.
  • the composition reduces the rate or concentration of sulphide produced in the colon by increasing the taxonomic diversity, the number of species, relative abundance and/or metabolic activity of sulphide consuming/assimilating/degrading isolates.
  • the composition reduces the rate or concentration of sulphide produced in the colon by increasing the taxonomic diversity, number of species, relative abundance and/or metabolic activity of sulphide consuming/assimilating/degrading isolates relative to the number of species, the taxonomic diversity or relative abundance of sulphidogenic microbiota.
  • the method drives down undesired inflammation.
  • the composition comprises a strain selected from the group consisting of: Flavinofractor spp.; Flavinofractor plautii; Christensenella spp.; Christensenella minuta; Anaerobutyricum spp.; Anaerobutyricum hallii; Escherichia-Shigella spp.; and Escherichia coli.
  • the composition comprises at least one strain of a microorganism selected from the group consisting of: Flavinofractor spp. [00152] In a further preferred embodiment, the composition comprises at least one strain of a microorganism selected from the group consisting of: Flavinofractor plautii.
  • the composition comprises at least one strain of a microorganism selected from the group consisting of: Christensenella spp.
  • the composition comprises at least one strain of a microorganism selected from the group consisting of: Christensenella minuta.
  • the composition comprises at least one strain of a microorganism selected from the group consisting of: Escherichia-Shigella spp.
  • the composition comprises at least one strain of a microorganism selected from the group consisting of: Escherichia coli.
  • the composition is administered with dietary fibre.
  • dietary fibre is selected from the group consisting of: FOS, inulin, maltodextrin, and starch.
  • the dietary fibre is administered concurrently or sequentially with the composition.
  • the invention broadly resides in a method of reducing endogenous sulphide levels in the colon of a patient in need thereof.
  • the method reduces sulphide and nitric oxide load on epithelial cells leading to a metabolic lesion via inhibition of cellular respiration.
  • the method reduces relative abundance and or metabolic activity of sulphidogenic microbiota.
  • the method reduces sulphide levels in the colon directly through consumption/assimilation/degradation.
  • the method reduces sulphide levels, taxonomic diversity, number of species, relative abundance and/or metabolic activity of sulphidogenic microbiota in the colon by metabolic substrate competition.
  • the method reduces sulphide levels, taxonomic diversity, number of species, and relative abundance and/or metabolic activity of sulphidogenic microbiota ir the colon by consuming hydrogen.
  • the method reduces sulphide levels, taxonomic diversity, number of species, and relative abundance and/or metabolic activity of sulphidogenic microbiota by reducing metabolisable sulphur substrates in the colon.
  • the method reduces sulphide levels, the taxonomic diversity, numoer of species, relative abundance and/or metabolic activity of sulphidogenic microbiota by reducing sulphur amino acid release (methionine, cysteine, homocysteine, taurine) into the colon.
  • the method reduces sulphide levels, the taxonomic diversity, number of species, relative abundance and/or metabolic activity of sulphidogenic microbiota in the colon by reducing protein fermentation.
  • the invention broadly resides in a method of preparing the biotherapeutic composition according to the second aspect of the invention, the method comprising mixing the composition according to the first aspect of the invention with an acceptable diluent or carrier.
  • the invention broadly resides in a method of preparing the pharmaceutical composition according to the third aspect of the invention, the method comprising mixing the composition according to the first aspect of the invention with a pharmaceutically acceptable diluent or carrier.
  • the invention broadly resides in the use of the composition according to the first aspect of the invention in the manufacture of a medicament for reducing or preventing gastrointestinal disorder in a subject.
  • the invention broadly resides in a dosage form comprising the composition of the first aspect of the invention.
  • the invention broadly resides in a kit comprising the dosage form according to the tenth aspect of the invention together with instructions for its use.
  • the invention broadly resides in a nucleotide sequence selected from the group consisting of: SEQ ID NOs 1 to 9193.
  • the invention is a nucleotide sequence having a sequence identity to one or more SEQ ID NOs 1 to 9193, wherein the sequence identity is selected from the group consisting of: at least 99.9%; at least 99%; at least 98%; at least 97%; at least 96%; at least 95%; at least 94%; at least 93%; at least 92%; at least 91%; and at least 90%.
  • the nucleotide sequence is substantially purified or isolated.
  • the invention is a micro-organism comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOs 1 to 9193.
  • the invention is a micro-organism comprising a nucleotide sequence having a sequence identity to one or more SEQ ID NOs 1 to 9193, wherein the sequence identity is selected from the group consisting of: at least 99.9%; at least 99%; at least 98%; at least 97%; at least 96%; at least 95%; at least 94%; at least 93%; at least 92%; at least 91 %; and at least 90%.
  • the micro-organism is substantially purified or isolated.
  • Figure 1 shows tie diagrammatical representation of the processes that lead to ulcerative colitis induced by high levels of sulphide (either as a sulphide ion (S 2- ), a bifsulphide ion (SHj, or hydrogen sulphide (H 2 S)) and nitric oxide (NO).
  • S 2- sulphide ion
  • SHj bifsulphide ion
  • NO nitric oxide
  • Figure 2 shows a graphical representation of hydrogen competition between methanogens, acetogens and sulphate-reducing bacteria. Competition for hydrogen by methanogens and acetogens leads to a decrease in the activity and abundance of sulphate- reducing bacteria, resulting in decreased production of sulphide.
  • Figure 3 shows the BB265 drug discovery process detailing lead selection and BB265 mechanistic validation prior to clinical validation in a phase 1 trial.
  • Figure 7 shows a table of the full-length 16S rRNA gene sequences taken from the isolates listed in Figure 5. These sequences are SEQ ID No 207-412 (a total of 206 sequences). These sequences are used for taxonomic classification of microbial species because the 16S rRNA gene, which is a small ribosomal subunit, is conserved amongst microbial species whilst containing hypervariable regions, such as the V3-V4 region, which provides sufficient variation to determine species-level discrimination between 16 rRNA gene sequences. The sequences presented are DNA sequences encocing the ribosomal RNA.
  • Figure 12a shows box and whisker plots representing the changes in the number of BB265 species in patients who achieved remission from FMT in a clinical trial.
  • Week 0 indicates the number of species prior to remission when patient disease state was considered active;
  • Week 12 is post FMT treatment during remission.
  • the invention described herein may include one or more range of values (e.g. size, concentration etc.).
  • a range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.
  • a person skilled in the field will understand that a 10% variation in upper or lower limits of a range can be totally appropriate and is encompassed by the invention. More particularly, the variation in upper or lower limits of a range will be 5% or as is commonly recognised in the art, whichever is greater.
  • “Therapeutically effective amount” as used herein with respect to methods of treatment and in particular drug dosage shall mean that dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment. It is emphasized that “therapeutically effective amount,” administered to a particular subject in a particular instance will not always be effective in treating the diseases described herein, even though such dosage is deemed a “therapeutically effective amount” by those skilled in the art. It is to be further understood that drug dosages are, in particular instances, measured as oral dosages, or with reference to drug levels as measured in blood.
  • Amounts effective for such a use will depend on: the desired therapeutic effect; the potency of the biologically active material; the desired duration of treatment; the stage and severity of the disease being treated; the weight and general state of health of the patient; and the judgment of the prescribing physician. Treatment dosages need to be titrated to optimize safety and efficacy.
  • the appropriate dosage levels for treatment will thus vary depending, in part, upon the indication for which the active agent is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titre the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the active agent and the formulation used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.
  • a “carrier” can be any solvents, diluents, excipients or other vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • the term "pharmaceutically acceptable carrier” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a composition of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained.
  • the component has generally met the required standards of toxicological and manufacturing.
  • the term “subject” generally includes mammals such as: humans; farm animals such as sheep, goats, pigs, cows, horses, llamas; companion animals such as dogs and cats; primates; birds, such as chickens, geese and ducks; fish; and reptiles.
  • the subject is preferably human.
  • the “gastrointestinal tract” refers to the tract from the mouth to the anus which includes all the organs of the digestive system such as the esophagus, stomach, pancreas, liver, gallbladder, small intestine (including the ileum), caecum, large intestine, colon and rectum. Strains of the invention are at least useful for conditions of the terminal ileum, caecum or rectum.
  • a “non-inflammatory strain” refers to a strain of the invention which, when present in the gastrointestinal tract of a subject, preferably a human, is associated with a non-inflamed state.
  • an “inflammatory strain” refers to a strain of the invention which, when present in the gastrointestinal tract of a subject, preferably a human, is associated with an inflamed state. Inflammatory strains of the invention have cytotoxicity against mammalian cells in culture. In an embodiment, the strain results at least 40%, at least 45% or at least 50% of cell death of the mammalian cells in culture.
  • bacteriotherapy refers to the use of a bacterial isolate to treat or prevent a disease or a condition, or provide a health benefit, in a subject.
  • biotherapeutic refers to a microorganism or combination thereof, such as bacterial isolate, that is useful for treating or preventing a disease or a condition, or provide a health benefit, in a subject.
  • biotherapeutic composition refers to a formulation comprising a biotherapeutic preparation formulated together with one or more additional formulary ingredients to obtain a finished formulation suitable for delivery to a subject.
  • the terms "treat,” “treating,” “treatment” and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject. Since every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population. Accordingly, a given subject or subject population may fail to respond or respond inadequately to treatment.
  • the term “prevent”, “prevented”, or “preventing” when used with respect to the treatment of mucosal inflammation in the gastrointestinal refers to a prophylactic treatment which increases the resistance of a subject to mucosal inflammation in the gastrointestinal, in other words, decreases the likelihood that the subject will develop mucosal inflammation in the gastrointestinal as well as a treatment after mucosal inflammation in the gastrointestinal has begun in order to fight the inflammation, e.g., reduce or eliminate it altogether or prevent it from becoming worse.
  • reducing refers to a reduction but not necessarily a complete abolition of gastrointestinal tract mucosal inflammation in a subject.
  • sample refers to a collection of biological material obtained from a subject or a subject's surrounding environment, such as soil or water in the area that the subject inhabits.
  • the sample is obtained directly from the subject.
  • the sample can be a faecal sample or obtained during a colonoscopy.
  • the sample may be in a form taken directly from the subject or surrounding environment, or it may be at least partially purified to remove at least some non-nucleic acid material. The purification may be slight, for instance amounting to no more than the concentration of the solids, or cells, of the samole into a smaller volume or the separation of cells from some or all of the remainder of the sample.
  • nucleic acids are isolated from the sample. Such isolated preparations include reverse transcription products and/or PCR amplification products of the nucleic acids in the sample. In some embodiments, the predominant nucleic acid is DNA.
  • the nucleic acid preparations can be pure or partially purified nucleic acid preparations. Techniques for the isolation of nucleic acid from samples, including complex samples, are numerous and well known in the art.
  • the unit “cfu” refers to "colony forming unit", which is the number of bacterial cells as revealed by microbiological counts on agar plates.
  • the present invention provides a composition for preventing or treating a gastrointestinal disorder in a subject in need thereof, said composition comprising at least one strain of bacteria.
  • the composition is selected from the group consisting of: a therapeutic composition; a pharmaceutical composition; a cosmetic composition; and a veterinary composition.
  • compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use).
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate- buffered saline.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • formulation materials for modifying, maintaining or preserving for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulphite or sodium hydrogen-sulphite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetra acetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin), fillers; monosaccharides, disaccharides; and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); colouring, flavouring and diluting agents; emulsifying agents;
  • the optimal composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the biotherapeutic actives of the invention.
  • the preferred form of the pharmaceutical composition depends on the intended mode of administration and therapeutic application.
  • the primary vehicle or carrier in a composition is aqueous in nature.
  • a suitable vehicle or carrier may be water for injection, physiological saline solution, possibly supplemented with other materials.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof.
  • pharmaceutical compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of an aqueous solution.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • compositions will be evident to those skilled in the art, including formulations of the invention in sustained- or controlled-delivery formulations.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.
  • Additional examples of sustained-sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, for example, films, or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, ethylene vinyl acetate or poly- D(-)-3-hydroxybutyric acid.
  • Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.
  • composition to be used for in vivo administration can be filtered to remove undesirable components. This may be accomplished by filtration through filtration membranes.
  • the compositions generally are placed into a sealed container to reduce exposure to oxygen. Once the pharmaceutical composition has been formulated, it may be stored in sealed containers.
  • % sequence homology may for example be calculated as follows.
  • the query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)).
  • a comparison is made over the window corresponding to one of the aligned sequences, for example the shortest.
  • the window may in some instances be defined by the target sequence. In other instances, the window may be defined by the query sequence.
  • the nucleic acid residues (nucleotides) at each position are compared, and the percentage of positions in the query sequence that have identical correspondences in the target sequence is reported as % sequence homology, or percent identification.
  • GAP analysis aligns two sequences over their entire length.
  • the bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for instance, Handbook of Microbiological Media, Fourth Edition (2010) Ronald Atlas, CRC Press, Maintaining Cultures for Biotechnology and Industry (1996) Jennie C. Hunter-Cevera, Academic Press, as well as how detailed in the Examples using YCFA medium.
  • the composition further comprises water.
  • the composition is a liquid, such as an aqueous solution.
  • composition further comprises a pharmaceutically acceptable carrier.
  • the composition retains its effective biological activity for a period selected from the group consisting of; greater than 24 hours; greater than 36 hours; and greater than 48 hours.
  • the composition is stable for periods selected from the group consisting of: 6 months, 1 year and 2 years.
  • the composition is stable at temperatures selected from the group consisting of: -80°C, -20 °C ,-4°C, 4°C, 18°C and 25°C.
  • compositions are within the scope of the invention.
  • the therapeutic composition of the invention may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the isolated bacteria present in the therapeutic composition.
  • the precise nature of the pharmaceutically acceptable excipient or other material will depend on the route of administration, which may be, for example, oral or rectal. Many methods for the preparation of therapeutic compositions are known to those skilled in the art (see e.g. Robinson ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978).
  • the therapeutic composition of the invention may comprise a prebiotic, an antioxidant, a carrier, insoluble fibre, a buffer, an osmotic agent, an anti-foaming agent and/or a preservative.
  • the therapeutic composition may be made or provided in chemostat medium.
  • the therapeutic composition may be made or provided in saline, e.g., 0.9% saline. It will be understood that any carrier or solution which does not impair viability of the bacteria present in the therapeutic composition and is compatible with administration to an individual may be used.
  • the therapeutic composition may be made or provided under reduced atmosphere, i.e., in the absence of oxygen.
  • a synthetic stool preparation may be made or provided under N2, CO2, H2, or a mixture thereof, optionally with controlled levels of partial pressure of N2:CO2:H2.
  • the therapeutic composition may be for oral or rectal administration to the individual. Where the therapeutic composition is for oral administration, the therapeutic composition may be in the form of a capsule, or a tablet. Where the therapeutic composition is for rectal administration, the therapeutic composition may be in the form of an enema or through colonoscopic delivery.
  • the preparation of suitable capsules, tablets and enema is well-known in the art.
  • the capsule or tablet may comprise a coating to protect the capsule or tablet from stomach acid.
  • the capsule or tablet may be enteric-coated, pH dependent, slow-release, and/or gastro-resistant. Such capsules and tablets are used, for example, to minimize dissolution of the capsule or tablet in the stomach but allow dissolution in the small intestine.
  • Orally dosed formulations can, in addition to the viable microorganisms comprise, inert compression aids, such as microcrystalline cellulose or oligosaccharide, flow aids, such as a silica gel, or a lubricant of, for example magnesium stearate (vegetable source) or stearic acid (vegetable source).
  • inert compression aids such as microcrystalline cellulose or oligosaccharide
  • flow aids such as a silica gel
  • a composition disclosed herein can be used as, for example, a food supplement, an edible product or pharmaceutical product.
  • the composition can further comprise a conventional food supplement filler and/or an extender.
  • the composition disclosed herein can also be included in any edible products, such as dairy products, including for example, a milk product, milk, yogurt, curd, ice-cream, dressing, and cheese, beverage products, meat products, and baked goods
  • Suppository formulations for example, either for rectal use, can in addition to the compositions, comprise, for example, cocoa butter, polyethylene glycol, glycerin or gelatine.
  • Lubricants are generally always used in the manufacture of dosage forms by direct compression in order to prevent the compacted powder mass from sticking to the equipment during the tableting or encapsulation process.
  • the lubricant may be any suitable lubricant such as for example, a lubricant selected from the group consisting of calcium stearate, magnesium stearate, stearic acid, sodium stearyl fumarate, and vegetable based fatty acids.
  • the carrier may be present in the composition in a range of approximately 30% w/w to approximately 98% w/w; this weight percentage is a cumulative weight percentage taking into consideration all ingredients present in the carrier.
  • Coatings can be used to control the solubility of the composition. Examples of coatings include carrageenan, cellulose acetate phthalate, ethylcellulose, gellan gum, maltodextrin, methacrylates, methylcellulose, microcrystalline cellulose, and shellac.
  • the composition may comprise one or more preservatives.
  • preservatives include antioxidants, chelating agents, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.
  • antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabi sulfite, propionic acid, glutathione, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabi sulfite, and sodium sulfite.
  • Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof.
  • EDTA ethylenediaminetetraacetic acid
  • salts and hydrates thereof e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like
  • citric acid and salts and hydrates thereof e.g., citric acid mono
  • antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.
  • Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxy benzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.
  • Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
  • the therapeutic composition may be lyophilized.
  • the lyophilized therapeutic composition may comprise one or more stabilisers and/or cryoprotectants.
  • the lyophilized therapeutic composition may be reconstituted using a suitable diluent prior to administration to the individual.
  • the therapeutic composition may optionally be administered in combination with a cancer immunotherapy, such as an immune check-point inhibitor, to the individual.
  • a cancer immunotherapy such as an immune check-point inhibitor
  • check-point inhibitors which may be employed in this context include Programmed cell death protein 1 (PD-1 ) inhibitors, Programmed death-ligand 1 (PD-L1 ) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors.
  • PD-1 Programmed cell death protein 1
  • PD-L1 Programmed death-ligand 1
  • CTL-4 cytotoxic T-lymphocyte-associated protein 4
  • Manipulation of the gut microbiota in combination with immune check-point inhibitor treatment has been shown to improve efficacy of immune check-point inhibitors in treating cancer.
  • the cancer in this context is colorectal cancer.
  • the cancer is renal cancer, lung cancer or melanoma.
  • the immunomodulating compound include interleukins, for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoattractant and activating factor (MCAF), RANTES, macrophage inflammatory peptides MIP-1a and MIP-1 b, complement components, or combinations thereof.
  • interleukins for example interleukins 1 to 15, interferons alpha, beta or gamma, tumour necrosis factor, granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), chemokines such as neutrophil activating protein (NAP), macrophage chemoat
  • the immunomodulating compound stimulate expression, or enhanced expression of 0X40, OX40L (gp34), lymphotactin, CD40, CD40L, B7.1 , B7.2, TRAP, ICAM-1 , 2 or 3, cytokine receptors, or combination thereof.
  • the immunomodulatory compound induces or enhances expression of co-stimulatory molecules that participate in the immune response, which include, in some embodiments, CD40 or its ligand, CD28, CTLA-4 or a B7 molecule.
  • the immunomodulatory compound induces or enhances expression of a heat stable antigen (HSA), chondroitin sulfate-modified MHC invariant chain (li-CS), or an intracellular adhesion molecule 1 (ICAM-1).
  • HSA heat stable antigen
  • li-CS chondroitin sulfate-modified MHC invariant chain
  • ICM-1 intracellular adhesion molecule 1
  • the therapeutic compositions of the invention may be administered to an individual, preferably a human individual. Administration may be in a "therapeutically effective amount", this being sufficient to show benefit to the individual. Such benefit may be at least improvement or amelioration of at least one symptom. Thus “treatment” of a specified disease refers to amelioration of at least one symptom.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular patient being treated, the clinical condition of the individual patient, the cause of the dysbiosis, the site of delivery of the composition, the type of therapeutic composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g.
  • a therapeutically effective amount or suitable dose of a therapeutic composition of the invention can be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the therapeutic composition is for prevention or for treatment.
  • Formulary ingredients can be contacted with the preparation and mixed or prepared until a formulation is obtained.
  • formulation conditions will generally be such that viable microorganisms are retained. In particular high temperatures, for example temperatures in excess of 40°C are avoided.
  • the amount of viable microorganisms included in a composition can vary and can be adjusted and optimized as will be appreciated by those of skill in the art. Such optimization may, for example, be achieved by preparing a series of different doses of a viable microorganism.
  • the bacterial concentration in the composition can be, for example, from 10 1 cfu/mL to 10 16 cfu/mL, 1 cfu/mL to 10 cfu/mL, 100 cfu/mL to 1 thousand cfu/mL, 10 thousand cfu/mL to 100 thousand cfu/mL, 1 million cfu/mL to 10 million cfu/mL, from 10 million cfu/mL to 100 billion cfu/mL, from 10 million to 50 million cfu/mL, more preferably from 50 million to 100 million cfu/mL, from 100 million to 500 million cfu/mL, from 500 million to 1 billion cfu/mL, from 1 billion to 5 billion cfu/mL, from 5 billion to 10 billion cfu/mL, from 10 billion to 15 billion cfu/mL, from 15 billion to 20 billion cfu/mL, from 20 billion to 25 billion
  • the strain of the invention can be administered at, for example, a dosage of 0.01 to 100 x 10 11 cells/body, 0.1 to 10 x 10 11 cells/body or 0.3 to 5 x 10 11 cells/body.
  • the amount ingested per day as the bacteria can be 0.01 to 100 x 10 11 cells/60 kg body weight, 0.1 to 10 x 10 11 cells/60 kg body weight or 0.3 to 5 x 10 11 cells/60 kg body weight.
  • the content of at least one strain of bacteria contained in the orally ingested composition of the present invention may be determined as appropriate depending on its application form.
  • a dry microbial body it can be, for example, 5 to 50 w/w %, 1 to 75 w/w %, 0.1 to 100 w/w % or 1 to 100 w/w %.
  • the composition is a controlled-release composition.
  • controlled-release refers to release or administration of a strain of the invention from a given dosage form in a controlled fashion in order to achieve the desired pharmacokinetic profile in vivo.
  • An aspect of "controlled” delivery is the ability to manipulate the formulation and/or dosage form in order to establish the desired kinetics of release.
  • Procedures for preparing tablets, caplets, capsules and other forms of compositions of the invention are known to those of ordinary skill in the art and include without limitation wet granulation, dry granulation, and direct compression (for tablets and caplets).
  • a chilsonation is used to manufacture the powder for the dosage forms.
  • a chilsonator houses grooved, rotating rollers that are pressed tightly against one another by hydraulic pressure.
  • Raw materials are placed into the hopper of the chilsonator and are fed by a system of horizontal and vertical screws into the rollers.
  • the sheets are milled into a fine granular powder using a Fitz mill and then passed through a screen to produce a uniform free flowing granule.
  • the chilsonation process results in a finished powder that is two to four times denser than the starting material, a feature that permits the ingredients to be fashioned into the desired dosage form.
  • the powder With dry granulation, the powder may be incorporated into a gelatin capsule or it may be mixed with gelatin to form a tablet or caplet. With wet granulation, the powder is moistened thus creating large "chunks" of material that are subsequently dried and milled to convert the chunks to particles of a desired size for the manufacturing process. Once the particles of a desired size are obtained, the particles are incorporated into a gelatin capsule or mixed with gelatin to form a tablet or caplet.
  • a composition of the invention can comprise a prebiotic. Because prebiotics have a chemical structure that resists digestion through the alimentary tract, they reach the colon as intact molecules where they are able to elicit systemic physiological functions and act as fermentable substrates for colonic microflora. Where a prebiotic is combined with a biotherapeutic, the resulting composition is sometimes referred to as a "synbiotic.”
  • oligosaccharide such as fructooligosaccharides, P95 Nutraflora®, for example, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, quercetin, human milk oligosaccharides, inulin oligosaccharides, mannan oligosaccharides, pyrodextrin, levan, maltotriose, pectic oligosaccharides, bimuno-galactooligosaccharides, arabinoxylan, fucoidan and resistant starches.
  • Fructooligosaccharides can be extracted from, for example, chicory, artichokes, asparagus, dandelions, dahlias, endive, garlic, leeks, lettuce, and onions.
  • the prebiotic comprises amino acids such as one or more or all of alanine, aspartic acid, glutamic acid, glycine, leucine, isoleucine, proline, serine, threonine and valine.
  • the prebiotic comprises simple sugars which can be a monosaccharide (such as glucose, galactose or fructose) and/or a disaccharide (such as sucrose maltose or lactose).
  • a monosaccharide such as glucose, galactose or fructose
  • a disaccharide such as sucrose maltose or lactose
  • the prebiotic comprises from about 0.01 % (w/w) to about 75% (w/w), 5% (w/w) to about 50% (w/w), about 7.5% (w/w) to about 30% (w/w) or about 10% (w/w) to about 15% (w/w) of the composition.
  • the composition may comprise more than one species/strain of microorganisms in addition to the strain of the invention, such as two, three, four, five or a higher plurality of species/strains of microorganisms.
  • biotherapeutics are suitable strains of the genera Absiella, Acetobacterium, Adlercreutzia, Aerococcus, Agathobaculum, Akkermansia, Alistipes, Allobaculum, Amedibacterium, Anaerobutyricum, Anaerofustis, Anaerostipes, Anaerotignum, Anaerotruncus, Bacillus, Bacteroides, Barnesiella, Bifidobacterium, Blautia, Butyricimonas, Carnobacterium, Christensenella, Ciostridium, Collinsella, Coprobacillus, Coprobacter, Coprococcus, Dorea, Enorma, Enterocloster, Enterococcus, Erysipelatoclostridium, Escherichia, Eubacterium, Faecalibacillus, Faecalibacterium, Finegoldia, Flavonifractor, Flintibacter, Gemmiger, Holdemanella, Hungatella,
  • biotherapeutics that may be included in the composition of the present invention.
  • any additional biotherapeutic species may also be used in the compositions of the present invention.
  • yeasts are also useful as biotherapeutics and are sometimes included in the compositions.
  • One non-limiting example of a yeast used in biotherapeutics is Saccharomyces boulardii.
  • Method for treating a gastrointestinal disorder are within the scope of the invention.
  • the invention provides a method for treating a gastrointestinal disorder, wherein said method comprises the administration to a patient in need thereof a therapeutically effective amount of the composition as described in the first aspect of this invention.
  • the dosage form is administered at an amount to at least partially treat a gastrointestinal disorder.
  • Methods of the invention can be used to treat or prevent a dysbiosis of the gastrointestinal tract in a subject.
  • Dysbiosis in the context of the present invention refers to a state in which the normal diversity, relative proportions of species and/or function of the microbiota or microbiome, in particular the human gastrointestinal microbiota, is disrupted. Any disruption from the normal state of the microbiota in a healthy individual can be considered a dysbiosis, even if the dysbiosis does not result in a detectable decrease in health in the individual.
  • the dysbiosis may be associated with one or more pathological symptoms.
  • Administration to humans includes administration by a medical professional and self-administration.
  • single or multiple doses of the composition are administered, for example once-off, daily for a period of at least one week, at least two weeks, at least three weeks, at least six weeks, at least nine weeks, or at least twelve weeks.
  • the compositions can be administered for the remaining duration of a subject's life.
  • the said method protects the compositions of the invention against degradation.
  • composition of the invention retains its effective biological activity for a period selected from the group consisting of; greater than 24 hours; greater than 36 hours; greater than 48 hours.
  • compositions of the invention solutions are preferred from a safety standpoint, as the simpler methodology is likely to produce a less variable outcome and the choice of excipient can be limited to those with Generally Regarded as Safe (GRAS) status.
  • Excipients for the stabilisation of protein solutions can be classified into four broad categories: salts, sugars, polymers or protein/amino acids, based on their chemical properties and mechanism of action. Salts (e.g. chlorides, nitrates) stabilise the tertiary structure of proteins by shielding charges through ionic interactions.
  • Sugars e.g.
  • glycerol, sorbitol, fructose, trehalose increase the surface tension and viscosity of the solution to prevent protein aggregation.
  • polymers e.g. polyethylene glycol, cellulose derivatives
  • stabilise the protein tertiary structure by increasing the viscosity of the solution to prevent protein aggregation and intra- and inter-molecular electrostatic interactions between amino acids in the protein.
  • Proteins e.g. human serum albumin
  • small amino acids with no net charge, such as alanine and glycine stabilise proteins through the formation of weak electrostatic interactions.
  • the medicaments of the present invention may include one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers may include one or more of the following examples: a.
  • surfactants and polymers including, however not limited to, polyethylene glycol (PEG), polyvinylpyrrolidone , polyvinylalcohol, crospovidone, polyvinylpyrrolidone-polyvinylacrylate copolymer, cellulose derivatives, HPMC, hydroxypropyl cellulose, carboxymethylethyl cellulose, hydroxypropylmethyl cellulose phthalate, polyacrylates and polymethacrylates, urea, sugars, polyols, and their polymers, emulsifiers, sugar gum, starch, organic acids and their salts, vinyl pyrrolidone and vinyl acetate; and/or b.
  • PEG polyethylene glycol
  • polyvinylpyrrolidone polyvinylalcohol
  • crospovidone polyvinylpyrrolidone-polyvinylacrylate copolymer
  • cellulose derivatives HPMC, hydroxypropyl cellulose, carboxymethylethyl cellulose,
  • binding agents such as various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose; and/or (3) filling agents such as lactose monohydrate, lactose anhydrous, microcrystalline cellulose and various starches; and/or c. filling agents such as lactose monohydrate, lactose anhydrous, mannitol, microcrystalline cellulose and various starches; and/or d. lubricating agents such as agents that act on the increased ability of the dosage form to be ejected from the packaging cavity, and/or e.
  • sweeteners such as any natural or artificial sweetener including sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame K; and/or f. flavouring agents; and/or g. preservatives such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxy benzoic acid such as butyl paraben, alcohols such as ethyl or benzyl alcohol, phenolic chemicals such as phenol, or quarternary compounds such as benzalkonium chloride; and/or h. buffers; and/or i.
  • preservatives such as potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxy benzoic acid such as butyl paraben, alcohols such as ethyl or benzyl alcohol, phenolic chemicals such as phenol, or quarternary
  • diluents such as pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing; and/or j. absorption enhancer such as glyceryl trinitrate; and/or k. other pharmaceutically acceptable excipients.
  • pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing
  • absorption enhancer such as glyceryl trinitrate
  • k other pharmaceutically acceptable excipients.
  • Medicaments of the invention suitable for use in animals and in particular in human beings typically must be sterile and stable under the conditions of manufacture and storage.
  • a strain of the invention can be detected using a wide variety of known techniques. Conveniently the strain is detected using a nucleic acid-based detection system.
  • nucleic acid sequencing is used.
  • Illustrative non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing.
  • the technology provided herein finds use in a Second Generation (a.k.a. Next Generation or Next- Gen), Third Generation (a.k.a. Next-Next-Gen), or Fourth Generation (a.k.a. N3-Gen) sequencing technology including, but not limited to, pyrosequencing, sequencing-by-ligation, single molecule sequencing, sequence-by-synthesis (SBS), massive parallel clonal, massive parallel single molecule SBS, massive parallel single molecule real-time, massive parallel single molecule real-time nanopore technology.
  • SBS sequence-by-synthesis
  • hybridization is employed in a detection method of the invention.
  • nucleic acid hybridization techniques include, but are not limited to, in situ hybridization (ISH), microarray, and Southern or Northern blot.
  • ISH in situ hybridization
  • FISH FISH assay
  • nucleic acid amplification is used. Nucleic acids may be amplified prior to or simultaneous with detection. Conducting one or more amplification reactions may comprise one or more PCR-based amplifications, non- PCR based amplifications, or a combination thereof.
  • nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), nested PCR, linear amplification, multiple displacement amplification (MDA), real-time SDA, rolling circle amplification, circle-to-circle amplification transcription-mediated amplification (TMA), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA).
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • MDA multiple displacement amplification
  • TMA circle-to-circle amplification transcription-mediated amplification
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • RNA be reversed transcribed to DNA prior to amplification e.g., RT-PCR
  • other amplification techniques directly amplify RNA (e.g., TMA and NASBA).
  • Non-amplified or amplified nucleic acids can be detected by any conventional means.
  • the nucleic acids can be detected by hybridization with a detectably labeled probe and measurement of the resulting hybrids.
  • the nucleic acids are detected by sequencing. Illustrative non-limiting examples of detection methods are described herein.
  • Evaluation of an amplification process in "real-time” involves determining the amount of amplicon in the reaction mixture either continuously or periodically during the amplification reaction and using the determined values to calculate the amount of target sequence initially present in the sample.
  • a variety of methods for determining the amount of initial target sequence present in a sample based on real-time amplification are well known in the art. These include methods disclosed in U.S. Pat. Nos. 6,303,305 and 6,541 ,205.
  • Another method for determining the quantity of target sequence initially present in a sample, but which is not based on a real-time amplification, is disclosed in U.S. Pat. No. 5,710,029.
  • Amplification products may be detected n real time through a combination of primers specific to the region of interest and a non-specific fluorescent DNA binding dye.
  • primers are designed so that they specifically target a region unique to the microbe of interest, leading to amplification of only the designed target.
  • the primer design incorporates two individual primers that complementarily bind up and downstream of the targeted DNA site. During amplification the primers will bind up and downstream of the target of interest, generating an additional copy through polymerase chain reaction, with the DNA-binding dye being incorporated into the newly generated amplicon. Post amplification, the DNA-binding dye is excited via a specific wavelength of light and the emission at a different wavelength is measured.
  • Binding of the fluorescent DNA-bindinc dye to the exponentially increasing amplicon of interest can be correlated with standards of known concentration through linear regression to determine the original copy number of the target region amplified.
  • the methodology of using DNA-binding dyes is disclosed n U.S. Pat. No. 6,174,670.
  • the specific use of the fluorescent DNA-binding dye SYBR green I is disclosed in U.S. Pat. No. 5,338,671 and 5,587,287.
  • Amplification products may be detected in real-time through the use of various self-hybridizing probes, most of which have a stem-loop structure.
  • Such self-hybridizing probes are labeled so that they emit differently detectable signals, depending on whether the probes are in a self-hybridized state or an altered state through hybridization to a target sequence.
  • “molecular torches” are a type of self-hybridizing probe that includes distinct regions of self-complementarity (referred to as “the target binding domain” and “the target closing domain") which are connected by a joining region (e.g., nonnucleotide linker) and which hybridize to each other under predetermined hybridization assay conditions.
  • molecular torches contain single-stranded base regions in the target binding domain that are from 1 to about 20 bases in length and are accessible for hybridization to a target sequence present in an amplification reaction under strand displacement conditions.
  • hybridization of the two complementary regions, which may be fully or partially complementary, of the molecular torch is favored, except in the presence of the target sequence, which will bind to the singlestranded region present in the target binding domain and displace all or a portion of the target closing domain.
  • the target binding domain and the target closing domain of a molecular torch include a detectable label or a pair of interacting labels (e g., luminescent/quencher) positioned so that a different signal is produced when the molecular torch is self-hybridized than when the molecular torch is hybridized to the target sequence, thereby permitting detection of probe- -target duplexes in a test sample in the presence of unhybridized molecular torches.
  • a detectable label or a pair of interacting labels e g., luminescent/quencher
  • Molecular beacons include nucleic acid molecules having a target complementary sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of a target sequence present in an amplification reaction, and a label pair that interacts when the probe is in a closed conformation. Hybridization of the target sequence and the target complementary sequence separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to reduced interaction of the label pair, which may be, for example, a fluorophore and a quencher (e.g., DABCYL and 25 EDANS). Molecular beacons are disclosed in U.S. Pat. Nos. 5,925,517 and 6,150,097.
  • the method includes quantifying the amount of strain present in the sample
  • the aim of the therapy is to induce and maintain remission by reducing sulphide levels in the colon of patients with ulcerative colitis Materials, Methods and Results
  • compositions of this invention targets the proximal cause of colonocyte barrier disruption that results in immune activation.
  • Reducing luminal sulphide can be accomplished by “turning off the tap” of sulphide production or by removing sulphide from the colon. Each of these mechanisms can be targeted using a live biotherapeutic product.
  • Sulphidogenic microbiota use specific substrates for metabolism.
  • SRB sulphate-reducing bacteria
  • Non-sulphidogenic microbiota which utilise these substrates, compete with sulphidogenic microbiota; such competition would limit the growth of sulphidogenic microbiota and lessen the production of luminal sulphide.
  • Microbiota also compete with sulphidogenic organisms for electron acceptors.
  • H2 molecular hydrogen
  • Methanogenic archaea and/or acetogenic bacteria divert hydrogen (H 2 ) away from sulphidogenic microbiota via competitive H 2 uptake.
  • Reducing luminal sulphide can be accomplished by competitive H 2 uptake by methanogenic archaea, acetogenic bacteria or other microorganisms (Smith, N. W., Shorten, P. R., Altermann, E., Roy, N. C. & McNabb, W. C. Competition for Hydrogen Prevents Coexistence of Human Gastrointestinal Hydrogenotrophs in Continuous Culture. Front. Microbiol. 11 , 1073 (2020)).
  • Methanogenic archaea such as Methanobrevibacter smithii and acetogenic bacteria such as Alistipes spp., Blauti spp.and Roseburia spp. are diminished in ulcerative colitis and associated with clinical remission in FMT.
  • Figure 2 shows a diagrammatic representation of hydrogen uptake by acetogens and methanogens that reduce sulphide production by SRB. Competition for metabolic substrates necessary for the growth and/or metabolism of sulphidogenic microbiota offers a therapeutic target to reduce endogenous sulphide in the colon.
  • Sulphidogenic microbiota require sulphur substrates (such as inorganic sulphate, sulphite, thiosulphate, and tetrathionate, and organic cysteine, methionine, and taurine) to produce sulphide.
  • substrates such as inorganic sulphate, sulphite, thiosulphate, and tetrathionate, and organic cysteine, methionine, and taurine
  • these substrates come from, but are not limited to, dietary sulphur sources, such as meat and cruciferous vegetables, host mucin, bile acids, and endogenous proteins (Wolf, P. G. et al. Diversity and distribution of sulphur metabolism in the human gut microbiome and its association with colorectal cancer.
  • sulphur is ubiquitously present in the colon, much of it is bound in complex molecules inaccessible to sulphidogenic microbiota.
  • other microbiota are often required to biotransform these compounds into accessible forms.
  • SRB depend on saccharolytic bacteria, such as Bacteroides spp., to remove sulphate from host mucin (Tsai, H. H., Sunderland, D., Gibson, G. R., Hart, C. A. & Rhodes, J. M.
  • colonic bacteria ferment carbohydrates in preference to protein in the human colon and thus adequate amounts of dietary fibre reduce the amount of sulphur-containing amino acids released into the colon.
  • a high protein diet results in faecal microbiota changes that increase sulphide production and decrease SCFA production (Teigen, L. M. et al. Dietary Factors in Sulfur Metabolism and Pathogenesis of Ulcerative Colitis. Nutrients 11, (2019)).
  • dietary fibres have been demonstrated to attenuate sulphide gas production in bioreactors containing stool from ulcerative colitis patients (Yao, C. K. et al.
  • Bacterial species can consume sulphide thereby reducing colonic sulphide levels.
  • An example of sulphide consumption is through assimilation of sulphide to cysteine.
  • Cysteine is synthesized from serine in a two-step process, commencing with transfer of the acetyl group from acetyl-CoA to serine, forming O-acetylserine (OAS), catalyzed by serine acetyltransferase (SAT).
  • OAS O-acetylserine
  • SAT serine acetyltransferase
  • the second step a condensation of OAS with sulphide to yield cysteine and acetate, catalyzed by O-acetylserine(thiol)lyase (OASTL) (Figure 4).
  • Direct consumption i.e., the biotransformation of sulphide, either metabolically or otherwise, can therefore reduce colonic levels of sulph
  • BB265 is an encapsulated bioreactor-derived microbial consortium comprising specific microbial strains that reduce colonic sulphide which drives the onset of the chemical lesion seen in ulcerative colitis and can optionally include a collection of the globally most prevalent taxa or isolates (global core microbiome), taxa or isolates which best discriminate between health and ulcerative colitis, keystone taxa or isolates, and phylogenetically diverse taxa or isolates.
  • the BB265 drug discovery process detailing lead selection and validation prior to consortium validation in a phase 1 study is presented in Figure 3.
  • Colonic microorganisms live in an ecosystem with many syntrophic relationships. Individual strains require many other species to sustain them via synergistic interactions. Keystone species are those that have a particularly critical role for the survival of many other species in the ecosystem, and in ecosystem stability and have outsized importance on the ecosystem often through uniquely held functions or functions that have limited redundancy within the system. (Banerjee, S., Schlaeppi, K. & van der Heijden, M. G. A. Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2016)). In addition, keystone species are also those linked with clinical efficacy, identified by their presence as a predictive factor for whole community recovery post-antibiotic treatment.
  • the inventors performed a global metagenomic meta-analysis to identify the most prevalent species shared across different geographical regions. This global core microbiome was then supplemented with the addition of previously identified keystone species and those identified informatically as keystones through multi-correlative network analysis. These bacterial isolates were drawn from the inventor’s culture collection and progressed to pilot process development to co-culture the defined community of species in bioreactors. This community supports the defined therapeutic candidate organisms, disrupts the existing community, and provides therapeutic potential themselves given the community contains many health associated organisms and has the emergent property of bolstering the effect of sulphide reduction by candidate microorganisms in ulcerative colitis.
  • Bacteria or archaea with the following characteristics were identified as Leads considered for inclusion in the BB265 complex consortium: (1 ) globally prevalent taxa or isolates commonly found in healthy human faecal microbiomes, (2) taxa or isolates that best discriminate between the faecal microbiomes of health and ulcerative colitis (health associated), (3) keystone taxa or isolates, (4) phylogenetically diverse taxa or isolates, (5) sulphide consuming (or assimilating) taxa or isolates, (6) putative H2 consuming taxa or isolates such as methanogenic archaea and homoacetogenic bacteria, and (7) taxa or isolates that divert metabolic substrates away from sulphide production.
  • Bacteria or archaea with the following characteristics were identified as Targets considered for exclusion from the BB265 complex consortium: (1) taxa or isolates that best discriminate between the faecal microbiomes of health and ulcerative colitis (disease- associated), (2) and taxa or isolates identified as sulphidogens.
  • the inventors have developed unique phenotypic assays that identify sulphide producing microbial isolates; the inventor’s bacterial culture collection has been screened for sulphide production.
  • the inventors have additionally developed unique phenotypic assays that identify sulphide consuming (or assimilating) microbial isolates; isolates identified as Leads with the ability to consume sulphide were considered candidate therapeutic organisms.
  • Unique phenotypic assays were developed to quantify sulphide production by the community, this was paired with measurements of real-time rates of production of H2S using H2S-specific commercial electrochemical sensors.
  • the inventors further supported the mechanism of action of BB265 by identifying an increase in taxa comprising the BB265 consortium in patients with active ulcerative colitis who successfully went into remission due to receiving faecal microbiota transplantation in a randomized, controlled trial.
  • This increase in BB265-associated taxa with remission was paired with a decrease in taxa identified as putative sulphidogens, supporting the hypothesis of sulphide reduction as a therapeutic target.
  • the inventors assayed stool from patients prior to receiving faecal microbiota transplantation and then from stool post remission and identified a significant reduction in the sulphide-producing capacity of the community post-remission.
  • the final candidate consortium will be further tested in a phase 1 human trial.
  • This final drug product will contain the enriched bacterial strains which target the underlying cause of the metabolic lesions in ulcerative colitis, further supported by a community comprising the most prevalent bacteria in healthy humans globally in addition to known keystone species that sustain important synergistic links in the colonic ecosystem.
  • the phase 1 human trial will show that the inventor’s live biotherapeutic product acts early in the disease pathway to prevent barrier disruption by limiting the NO- and H2S-induced metabolic lesion.
  • the phase 1 human trial will have the ability to assess important mechanistic endpoints e.g. therapeutic candidate’s ability to reduce sulphide in a patient with ulcerative colitis.
  • MPC 'Most Prevalent Community
  • Microbial species found to be globally prevalent in healthy human faecal microbiomes were determined via publicly available Illumina paired-end shotgun metagenomes from faeces of individuals with a reported healthy phenotype downloaded from the European Nucleotide Archive (ENA). This analysis resulted in the generation of priority lists from 1 to 2, and 4 to 6 depending on global span and read coverage. Metagenomes with over five million reads were retained and the 882 remaining metagenomes were assigned to geographic regions based on their origin, including Africa, South America, North America, Europe and Asia-Pacific.
  • Sequenced reads were trimmed using Trimmomatic version 0.39 and taxonomic classification was performed using Kraken version 2.1.2 through comparison to a custom database built from all available complete genomes in the National Center for Biotechnology Information (NCBI) Reference Sequence Database (RefSeq release 209). Reads mapping to Homo sapiens were removed and low abundance taxa with less than 0.001 % of total reads were removed to filter out spurious read classifications from each sample.
  • NCBI National Center for Biotechnology Information
  • CO-OCCURRENCE INFORMED MOST PREVALENT COMMUNITY COIMPC
  • Microbial communities contain a plethora of complex interactions both between and within species and these synergistic relationships are integral to a functioning ecosystem (Sharon, I. et al. The Core Human Microbiome: Does It Exist and How Can We Find It? A Critical Review of the Concept. Nutrients 14, (2022)).
  • One way of considering these interactions is via co-occurrence analyses.
  • a co-occurrence informed; most prevalent community structure approach overlays co-occurrence data onto the prevalence data to maximise the likelihood of forming functional communities.
  • Co-occurrence suggests a mutually beneficial relationship among bacteria of the MPC and more globally transient bacteria.
  • Bioinformatic analyses were performed to identify species co-occurring with globally prevalent species in the complex consortium. Specifically, network analysis was performed on the global metagenomic classification output with SpiecEasi v1.1.2 using Meinshausen-Buhlmann neighbourhood selection with 50 repetitions of the Stability Approach to Regularisation Selection (StARS), an nlambda value of 20 and a lambda. min.ratio of 0.01 . Species directly connected in the network to existing MPC taxa were selected for inclusion in the consortium based on this co-occurrence. These species have statistically inferred interactions with the complex consortium taxa, which implies a supportive role in the community.
  • StARS Stability Approach to Regularisation Selection
  • Microbial taxa have varying levels of connectivity within their ecological community, with some taxa contributing more to community functioning and stability than others, referred to as hubs. Hubs have a high level of connectivity to other community members and are often essential for community structure and metabolic cycling (Banerjee, S., Schlaeppi, K. & van der Heijden, M. G. A. Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev. Microbiol. 16, 567-576 (2018)). A method of identifying hub taxa in the gastrointestinal microbiome is through network analyses of sequencing data. Hubs are defined by their level of connectedness to other nodes in the network, inferring their importance in community functioning.
  • Bioinformatic network analyses were performed to identify statistically inferred hubs in the global metagenomic classification output. These analyses were performed using SpiecEasi with Meinshausen-Buhlmann neighbourhood selection and NetCOMI with SPRING association estimation, to identify taxa with the highest degree of centrality. These methods identify nodes with the highest connectivity, determining potential hubs within the network. Taxa identified as hubs, with high closeness, betweenness or eigenvector centrality were selected for inclusion. The species identified through these methods may have key roles in community structure determination and metabolic precesses.
  • 16S rRNA gene sequence-based similarity comparisons were used to identify culture collection isolates from BiomeBank that were phylogenetically distinct from the existing complex consortium. Isolates with a 16S sequence less than 98% identity, determined by NCBI-BLAST (blastn) (v2.13.0) comparison to any species or strain in the consortium, were iteratively included as they may fill ecological niches not covered by the initial community. Phylogenetic analyses were performed to evaluate phylogenetic coverage of the complex consortium with the addition of these diversity inclusion isolates.
  • Faecal microbiota transplant has emerged as a potential treatment for ulcerative colitis, showing induction of remission in UC patients ).
  • the clinical success of FMT is varied, possibly due to variation in preparation, delivery and donor . Since some donors have a greater observed success in recipient remission , it has been suggested that diversity or the presence of a specific combination of microbes may be responsible.
  • this analysis aims to define a consortium of taxa that discern between active UC, and healthy controls from a diverse array of publicly available faecal metagenomes, for the purpose of determining microbial targets associated with disease, or therapeutic bacteria and communities for disease treatment.
  • the inventors selected isolates for inclusion in the BB265 consortium that showed a negative phenotype for sulphide production. However, where this could not occur, rather than dismissing these sulphide producing isolates outright, the inventors considered their potential role in ulcerative colitis management due to their prevalence in healthy individuals, keystone characteristics and anti-correlation with ulcerative colitis datasets. This decision was driven due to the relationship between sulphide and ulcerative colitis being nuanced and multifaceted. While the inventors posit sulphide as the deleterious agent in the context of ulcerative colitis, the human gut microbiome is a diverse and complex ecosystem, and microbial diversity is crucial for health (Mosca, A., Leclerc, M.
  • sulphide production was identified as being a phylogenetically diverse phenotype, both present in taxa associated with health and ulcerative colitis. Isolates chosen for inclusion in the BB265 complex consortium were preferentially selected if they showed a negative phenotype for sulphide production; however, where this could not occur, isolates were still considered due to evidence for their association with health, importance as keystones, anticorrelation with ulcerative colitis, and hypothesised mechanisms of action, such as H2 consumption, which target sulphide production at the community level.
  • the amount of methylene blue produced is proportional to the original concentration of aqueous sulphide.
  • the presence of methylene blue can be determined spectrophotometrically via absorption of light at 667 nm
  • Frozen isolates were thawed at 37 °C in the anaerobic cabinet, diluted 1 :10 in prereduced YCFA broth and left to incubate overnight. In triplicate, 100 pL overnight culture was used to inoculate 3.85 mL of modified YCFA broth (lacking L-cysteine) in a sterile cuvette spiked with 0.1 % L-serine.
  • Sulphide is a cytotoxic metabolite produced in excess by resident colonic microbiota in patients with inflammatory bowel disease.
  • Sulphide in this experiment is measured in the form of a sulphide ion (S 2 ‘); a hydrosulphide (HS j ion; a bisulphide ion (SH' ), and hydrogen sulphide (H2S).
  • Microbiota-derived sulphide in combination with nitric oxide plays a direct role in ulcerative colitis (UC) pathogenesis.
  • UC ulcerative colitis
  • These gases lead to sequestration of CoA, and depletion of glutathione and an inability of colonocytes (colonic epithelial cells) to produce energy via p-oxidation of butyrate.
  • This biochemical lesion in ulcerative colitis results in an energy-deprived state for colonocytes and contributes to a loss of epithelial barrier function and ensuing mucosal inflammation.
  • sulphide in faecal samples has been found to be 3-4 fold higher in ulcerative colitis than in control cases. This is likely due to the relatively high levels of sulfidogenic microbiota, including SRB and cysteine degrading bacteria in patients with inflammatory bowel disease in both active and quiescent disease. Nitric oxide is produced by both inflamed colonocytes and the colonic microbiota.
  • Faecal microbiota transplantation has demonstrated efficacy in the induction of remission of ulcerative colitis, indicating that the colonic microbiota are involved in disease pathogenesis and that modulation of the colonic microbiota can ameliorate disease.
  • modulation of the colonic microbiome in a way that reduces colonic levels of sulphide offers a promising therapeutic target.
  • the final list of isolates comprising the BB265 consortium consisted of 143 microbial isolates (Table 2).
  • the BB265 complex consortium list is presented using the following taxonomic classifications.
  • NB Due to the ephemeral nature of taxonomic names, taxon IDs are also provided for each isolate in the BB265 consortium, including also those isolates not in the BB265 consortium but identified with a sulphide consumption phenotype ( Figure 5). All past, present and future taxon names describing each species are to be covered here, including all homotypic, heterotypic, subjective, objective, nomenclatural and invalid synonyms, along with misapplied names.
  • Table 4 List of taxa by Species / 16S identification including 16S sequences for each species in the BB265 complex consortium or identified as sulphidogens
  • GC gas chromatography
  • GC vials were removed from the anaerobic chamber and 200 pL of the mixture was transferred to cuvettes containing 3.8 mL of RO water.
  • 345 pL of a solution containing FeCI3 (37.0 mM) and N, N-dimethyl-p-phenylenediamine sulphate salt (17.1 mM) dissolved in HCI (6 M) was added, the cuvette was immediately capped and inverted once to mix. Cuvettes were left for 10 min to allow for colour development and reaction to run to completion. 1 mL of the samples were transferred to Eppendorf tubes and centrifuged at 15,000 x g for 5 min.
  • Results [00451] Analysis of the metagenomes from phase 1 of the clinical trial dataset, identified that patients who went into remission (Clinical remission) had a significant increase in the number of BB265 species from week 0 to week 12. For patients who did not go into remission, the number of BB265 species did not change significantly between baseline and remission samples ( Figure 12a). Patients who did not enter clinical remission also had a significant decrease in sulphide consumer abundance from baseline to week 12 ( Figure 12b).
  • BB265 species are important gut microbiota in the induction of remission for ulcerative colitis.
  • the number of BB265 species and sulphide consumer species significantly increased from baseline to week 12 for patients that went into remission. Additionally, the statistically significant reduction in the relative abundance of consumer species in patients who did not enter remission also supports those isolates identified as sulphide consumers as a potential therapeutic for the treat ulcerative colitis.
  • the results of the modified methylene blue assay correlated directly with the clinical data, where a reduction of aqueous sulphide in vitro corresponded to a decrease in total Mayo score in clinical observation.
  • the total Mayo score reduced from 3 in week 0 to 0 in week 52 and the modified methylene blue assay testing showed a significant decrease in the levels of aqueous sulphide in the clinical sample from week 0 to week 52.
  • this correlation was also observed for Patient E, where the total Mayo score reduced from 8 in week 0 to 0 in week 12. This corresponded to elevated sulphide levels in the week 0 clinical sample and a significantly reduced level of hydrogen sulphide in the week 12 sample where the patient had achieved clinical remission.
  • microbiota comprising the BB265 complex consortium have therapeutic potential in ulcerative colitis by modulating the dynamics of both sulphide production and consumption, resulting in a net reduction in sulphide levels when these taxa are present in the faecal samples of ulcerative colitis patients.
  • Two ulcerative colitis stool samples (Patient A and C) were used in this study.
  • Different concentrations of faecal slurry were prepared per donor to measure adequate sulphide; concentration was determined prior by assaying faecal slurries for sulphide production at various concentrations using the modified methylene blue assay.
  • 7 g of stool sample from Patient A and 5 g of stool sample from Patient C were dissolved in 70 mL and 100 mL of pre-reduced Solution I, respectively.
  • the modified methylene blue assay is a highly specific assay to detect total sulphides (H2S, HS and S 2 ) in the aqueous phase of our bioreactor.
  • This method allows endpoint data to be collected, when fermentation has run to completion and concentrations of sulphide between the aqueous phase and headspace have equilibrated.
  • It does not allow real-time monitoring of the production of H2S in the bioreactor.
  • This study aims to utilise commercial H 2 S microsensors to monitor the production of H 2 S in a closed bioreactor system in real-time whilst serving as a validation method for the results seen using the modified methylene blue assay.
  • the sensor was inserted into a sterile vial of pure water to remove any residual hydrogen sulphide.
  • the millivolt (mV) signals corresponding to the respective concentrations of calibration points were then integrated into the curve, and the software was configured to create a standard curve, thereby preparing it for subsequent measurements.
  • the temperature sensor was connected to the fx-6 pA UniAmp amplifier and the SensorTrace Suite software was opened.
  • the temperature sensor was placed in the known temperatures of 0°C, 22°C and 81 °C until the signal was stable. These temperatures were determined against a calibrated alcohol thermometer.
  • the millivolt (mV) signals to the respective temperatures of calibration points were then integrated into the curve, and the software was configured to create a standard curve, thereby preparing it for subsequent measurements
  • the H2S needle-type sensors and temperature sensor were connected to the amperometry and temperature channels of the Fx-6 UniAmp, respectively. Subsequently, the sensors were initiated by scanning through the SensorTrace Logger software. Then, all sensors were securely placed inside a 37°C incubator and left for a few min until a stable signal was observed. Bottles containing samples were pierced with the H2S needle-type sensors until sensor tips were immersed in the stool suspension and incubated at 37°C. The recording time interval was adjusted to 300 seconds, and recording commenced while these samples incubated at 37°C for 24 hours. Following the incubation period, the recording was stopped, and the data was saved as an .xls file.
  • the use of individual H 2 S consumers may not consistently facilitate H 2 S reduction in some ulcerative colitis patients, as their effectiveness appears to depend on the inter-variability between patient samples.
  • the BB265 complex consortium consistently demonstrates the same phenotypic response in both samples, underscoring its beneficial role in supporting H 2 S reduction within the context of ulcerative colitis stool.
  • These results highlight the pivotal role played by the BB265 complex consortium in the scope of ulcerative colitis treatment.
  • the BB265 consortium exhibits the capacity to substantially mitigate the rate of H 2 S production, a factor of paramount importance within the colon environment of ulcerative colitis patients. This reduction total and rate of H 2 S holds significant therapeutic promise by effectively limiting the concentration of H 2 S generated at any given moment, potentially offering substantial benefits to individuals affected with ulcerative colitis and also IBD.
  • NO nitric oxide
  • mammalian cells such as colonocytes
  • intraluminal NO has been observed to be increased, working alongside sulphide to inhibit P-oxidation to starve the colonocyte of cellular energy ( Figure 1 ).
  • NO 2 ‘ Direct measurements of NO in stool are challenging due to its high reactivity and short half-life. As a result, measurements of NO 2 ‘ are frequently used as a surrogate marker for NO.
  • Nitrite is an intermediate product in the NO 3 "-NO 2 '-NO pathway, which is a common route for NO production in the gut.
  • NO 3 ⁇ is a stable and abundant molecule in the human diet, and it can be reduced to nitrite by certain commensal bacteria present in the gut. Subsequently, NO 2 " can be further converted to NO under specific conditions, such as low oxygen levels, acidic pH, or in the presence of microbial enzymes like nitrite reductases.
  • NO 2 ⁇ and NO exist in a dynamic equilibrium, with the conversion between the two influenced by environmental factors and enzymatic activities.
  • higher levels of NO 2 " in stool may indicate an increased potential for NO generation, further supporting the utility of nitrite measurement as a proxy for NO levels.
  • Patient samples were selected based on a positive sulphide phenotype observed in previous bioreactor experiments.
  • Patient C was chosen due to the observation that sulphide concentration in the stool sample was reduced when incubated with BB265 complex consortium and an individual sulphide consumer.
  • Patient D and E were selected as they showed clinical response and remission to FMT, and a significant reduction of sulphide concentrations in remission samples compared to baseline (active) samples.
  • GC vials containing 5% faecal slurry of Patient C were spiked with 100 pL washed BB265 complex consortium and bb0214. Control samples were spiked with 100pL of Solution I only. Samples of Patient D and E were left unspiked to compare the difference in NO Z ⁇ produced by the autochthonous mic’obial communities in baseline and remission samples.. Once inoculated, the GC vials were capped immediately and incubated anaerobically at 37 °C overnight. The experiment for Patient C was performed in triplicate whilst the experiment for Patient D and E was performed in duplicate due to limited sample weight.

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Abstract

La présente invention concerne des compositions pour le traitement d'une maladie inflammatoire de l'intestin. La présente invention concerne également des formes pharmaceutiques et des méthodes de traitement d'une maladie inflammatoire de l'intestin par l'administration de la composition à un patient en ayant besoin.
EP23783935.2A 2022-09-20 2023-09-20 Compositions et méthodes de réduction du sulfure endogène chez des patients atteints de maladies inflammatoires de l'intestin Pending EP4577226A1 (fr)

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