WO2022187678A1 - Compositions and methods for the diagnosis and treatment of alzheimer's disease or other neurodegenerative disorders - Google Patents

Abstract

Described herein are compositions and method for the diagnosis and treatment of Alzheimer's disease or other neurodegenerative disorders, in one embodiment, the composition augments a subject's gut microbial flora and reduces one or more biomarkers associated with Alzheimer's Disease. In one aspect, the composition comprises one or more bacteria species selected from Lactobacillus, Erysipelatociostridium, Faecalitalea, Erysypeiotrichaceae, Anaerostipes, Blautia, Ruminiclostridium, or combinations thereof and one or more pharmaceutically acceptable excipients.

Description

COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF ALZHEIMER’S DISEASE OR OTHER NEURODEGENERATIVE DISORDERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 63/156,425, filed on March 4, 2021 , and U.S. Provisional Patent Application No. 63/276,718, filed on November s, 2021 , each of which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

Described herein are compositions and method for the diagnosis and treatment of Alzheimer’s disease or other neurodegenerative disorders. In one embodiment, the composition augments a subject’s gut microbial flora and reduces one or more biomarkers associated with Alzheimer’s Disease, in one aspect, the composition comprises one or more bacteria species selected from Lactobacillus, Erysipeiatociostridium, Faecalitalea, Erysypeiotrichaceae, Anaerostipes, Biautia, Ruminiclostndium, or combinations thereof and one or more pharmaceutically acceptable excipients.

BACKGROUND

Growing evidence links alterations in the gut-microbiota-brain axis to Alzheimer’s disease (AD) and potential mechanisms such as direct bacterial brain invasion, production of toxins, inflammation and immune modulation are being investigated. Blood levels of neurofilament light (NfL) and phosphorylated tau (pTau), are promising biomarkers of neurodegeneration in AD as they correlate with atrophy, pathology and may predict future decline.

What is needed are compositions and methods for the diagnosis and treatment of Alzheimer’s disease or other neurodegenerative disorders.

SUMMARY

One embodiment described herein is a pharmaceutical composition for augmenting gut microbial flora in a subject and reducing one or more biomarkers associated with a neurodegenerative disorder comprising: one or more bacterial species selected from Lactobacillus, Eryslpelaiociostridlum, Faecalitalea, Erysypeiotrichaceae, Anaerostipes, Biautia, or Ruminiclostndium; and one or more pharmaceutically acceptable excipients. In one aspect, the one or more bacterial species is in an amount of from about 1G⁶ CFU to about 1G¹² CFU. In another aspect, the pharmaceutical composition further comprises rifaximin. In another aspect, the rifaximin is in an amount of from about 100 mg to about 1 ,100 mg. In another aspect, the pharmaceutically acceptable excipient is a binding agent, a filler, a lubricant, a disintegrant, a wetting agent, or combinations thereof, in another aspect, the pharmaceutical composition comprises a dosage form comprising a tablet, a capsule, or a lozenge.

Another embodiment described herein is a method of treating or preventing a neurodegenerative disorder in a subject, the method comprising: (a) obtaining or having obtained a biological sample from the subject; (b) performing or having performed a diagnostic assay on the biological sample to determine the level of one or more biomarkers associated with the neurodegenerative disorder, wherein the subject has or is at risk of developing the neurodegenerative disorder when the levei of the one or more biomarkers is greater than a control; and, (c) administering a composition to the subject having levels of the one or more biomarkers greater than the control, wherein the composition increases bacterial species from the Genera of the Phylum Firmicutes in the gut of the subject, and wherein neurodegeneration is decreased or prevented in the subject following administration of the composition. In one aspect, the one or more biomarkers associated with the neurodegenerative disorder is pTau, GFAP, IL- 6, IL-13, Neurofilament Light (NfL), or combinations thereof. In another aspect, the one or more biomarkers is selected from pTau181 , GFAP, NfL or combinations thereof. In another aspect, the composition comprises one or more bacterial species selected from Lactobacillus, Erysipelatodostridium, Faecalitalea, Erysypelotnchaceae, Anaerostipes, B!autia, or Ruminiclostridium. In another aspect, the composition comprises rifaximin. in another aspect, the bacterial species from the Genera of the Phylum Firmicutes is one or more of bacterial species selected from Lactobacillus, Erysipelatodostridium, Faecalitalea, Erysypelotnchaceae, Anaerostipes, Blautia, and Ruminiclostridium. In another aspect, the neurodegenerative disorder is Alzheimer’s Disease. In another aspect, the level of one or more biomarkers associated with the neurodegenerative disorder is about 10% to about 200% greater than the control. In another aspect, the biological sample is blood, serum, plasma, or cerebral spinal fluid (CSF). In another aspect, the diagnostic assay is an immunoassay. In another aspect, the increase in bacterial species from the Genera of the Phylum Firmicutes in the gut of the subject is detected from a fecal sample from the subject. In another aspect, the composition is administered 1 week to 3 years. In another aspect, neurodegeneration is decreased or prevented when the level of one or more biomarkers associated with the neurodegenerative disorder is decreased significantly as compared to the level of the one or more biomarkers before administration of the composition, in another aspect, the significant decrease is from about 5% to about 200% as compared to the level of the one or more biomarkers before administration of the composition. Another embodiment described herein is a method for treating or preventing a neurodegenerative disorder in a subject, the method comprising administering to the subject a composition comprising one or more bacterial species from the Genera of the Phylum Firmicutes to the subject thereby preventing or treating the neurodegenerative disorder in the subject. In one aspect, the composition is administered orally to the subject. In another aspect, the method further comprises administering to the subject one or more additional therapeutic agents. In another aspect, the one or more additional therapeutic agents comprise rifaximin. In another aspect, the one or more additional therapeutic agents comprise an agent that Increases the numbers of one or more of the bacterial species selected from Lactobacillus, Erysipelatodosiridium, Faecalitalea, Erysypeloirichaceae, Anaerostlpes , Blaulia , Ruminlclostridium , or combinations thereof, in another aspect, the composition is administered 1 week to 3 years. In another aspect, the neurodegenerative disorder is Alzheimer’s Disease, In another aspect, the bacterial species from the Genera of the Phylum Firmicutes is one or more of bacterial species selected from Lactobacillus, Eiysipeiatodosiridium, Faecalitalea, Erysypeloirichaceae, Anaerostlpes, Biautia, or Ruminlclostridium.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a box plot illustrating the mean (SD) reductions from baseline in serum NfL, pTau181 , il-6, and lL-3.

FIG. 2 shows a chart illustrating significant increases in the abundance of 7 genera of Phylum Firmicutes following rifaximin treatment in accordance with one embodiment of the present disclosure. Lactobacillus (p = 0.019), Erysipelatodosiridium (p = 0.00035), Faecalitalea (p = 0.02), Erysypeloirichaceae (p = 0.0008), Anaerostlpes { p = 0.038), Biautia (p = 0.004), Ruminlclostridium 9 (p = 0.027).

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, ceil and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. in case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein. As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” "vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, orY) are used herein.

As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising,” The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

As used herein, the term “or” can be conjunctive or disjunctive.

As used herein, the term “substantially” means to a great or significant extent, but not completely.

As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term "about” can mean within an order of magnitude, in some embodiments within 5-foid, and in some embodiments within 2-fold, of a value. As used herein, the symbol

means “about” or “approximately.”

All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.

As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect. As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference" or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including ceils, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation" and “composition” are used interchangeably herein.

As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or ce!i(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

As used herein, the term “subject” refers to an animal. Typically, the subject Is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non- human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate, in one embodiment, the subject is a human.

As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term "treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a ceil, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a ceil, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

As used herein, the term “biomarker” refers to a naturally occurring biological molecule present In a subject at varying concentrations useful in predicting the risk or Incidence of a disease or a condition, such as Alzheimer^'s Disease or other neurodegenerative disorder. For example, the biomarker can be a protein present in higher or lower amounts in a subject at risk for Alzheimer’s Disease or other neurodegenerative disorders. The biomarker can include metabolites, bacteria flora, nucleic acids, or polypeptides used as an indicator or marker for Alzheimer’s Disease or other neurodegenerative disorders. In some embodiments, the biomarker is a protein, in some embodiments, the biomarker is selected from pTau (including pTau181), GFAP, iL-6, IL-13, Neurofiiament Light (NfL), or combinations thereof.

The term “biological sample” as used herein includes, but is not limited to, a sample containing tissues, ceils, or biological fluids obtained from or isolated from a subject. Examples of biological samples include, but are not limited to, tissues, cells, biopsies, blood, lymph, serum, plasma, urine, saliva, mucus, and tears. A biological sample may be obtained directly from a subject (e.g., by blood or tissue sampling) or from a third party (e.g., received from an intermediary, such as a healthcare provider or lab technician).

The term “disease” as used herein includes, but is not limited to, any abnormal condition or disorder of a structure or a function that affects a part of an organism, it may be caused by an external factor, such as an infectious disease, or by internal dysfunctions, such as Alzheimer’s Disease or other neurodegenerative disorder, and the like.

As used herein, the terms “other neurological diseases” or “other neurological disorders” are used interchangeably and refer to a host of undesirable conditions affecting neurons in the brain of a subject. Representative examples of such conditions include, without limitation and including all stages of the following diseases, Parkinson’s disease, Huntington’s disease, Pick’s disease, Kuf’s disease, Lewy body disease, neurofibrillary tangles, Rosenthal fibers, Mallory’s hyaline, senile dementia, myasthenia gravis, Giiles de la Touretie’s syndrome, multiple sclerosis (MS), amyoirophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), epilepsy, Creutzfeldt-Jakob disease, deafness-dystonia syndrome, Leigh syndrome, Leber hereditary optic neuropathy (LHON), parkinsonism, dystonia, motor neuron disease, neuropathy-ataxia and retinitis pigmentosa (NARP). maternal inherited Leigh syndrome (MILS), Friedreich ataxia, hereditary spastic paraplegia, Mohr-Tranebjaerg syndrome, Wilson disease, sporadic Alzheimer’s disease, familial Alzheimer’s disease, preclinicai Alzheimer’s disease (e.g., mild cognitive impairment (MCI); subjects at risk carrying susceptibility genes or pathological changes), age- associated cognitive impairment, !ong-CGV!D, neuro-COVID, sporadic amyotrophic lateral sclerosis, sporadic Parkinson’s disease, autonomic function disorders, hypertension, sleep disorders, neuropsychiatric disorders, dystonia, depression, schizophrenia, schizoaffective disorder, Korsakoff^'s psychosis, mania, anxiety disorders, phobic disorder, learning or memory disorders, amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, obsessive-compulsive disorder, psychoactive substance use disorders, panic disorder, bipolar affective disorder, severe bipolar affective (mood) disorder (BP- 1), migraines, hyperactivity and movement disorders.

Compositions

One embodiment described herein is a composition comprising, consisting of, or consisting essentially of, one or more of a bacteria species selected from Lactobacillus, Erysipelatoc!ostridium, Faecalitalea, Erysypelotnchaceae, Anaerostipes , Blaulia ,

Ruminiclostridium, or combinations thereof.

Another embodiment described herein is a composition comprising one or more of a bacteria species selected from consisting of Lactobacillus, Erysipelatoclostridium, Faecalitalea, Erysypelotnchaceae, Anaerostipes, Blaulia, Ruminiclostridium, or combinations thereof and an appropriate pharmaceutical carrier, excipient, or diluent. The exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use.

When used to treat or prevent a disease, such as Alzheimer’s Disease or other neurodegenerative disorders, the compositions described herein may be administered singiy, as mixtures of one or more compounds or in mixture or combination with other agents (e.g., therapeutic agents) useful for treating such diseases and/or the symptoms associated with such diseases. Such agents may include, but are not limited to, rifaximin, cholinesterase inhibitors (e.g., Aricept®, Exelon®, Razadyne®), memantine (e.g., Namenda®), drugs such as Amantadine (SYMMETREL), Apomorphine (APOKYN), anti-tau and anti-amyloid agents such as Aducanumab (ADUHELM®), Baclofen (LIGRESAL), Carbidopa (LODGSYN), Carbidopa/levodopa (SiNEMET, ATAMET, others; orally disintegrating tablet, PARCOPA), Dantrolene (DANTRIUM), Donepezil (ARICEPT), Entacapone (COMTAiM; fixed combination with carbidopa/levodopa, STAVELO), Gaiantamine (NIVALIN, others), Levodopa (L-DQPA, LARODOPA), Pramipexole (MiRAPEX), Rasagiline (AZILECT), Riluzole (RILUTEK), Rlvastigmine (EXELON), Ropinlroie (REOUIP), Selegiline (ELDEPRYL; oral disintegrating tablet, EMSAM; transderma! patch, ZELAPAR), Tacrine (COGNEX), Tetrabenazine (XENAZINENITOIViAN), Tizanidine (ZANAFLEX), Tolcapone (TASMAR), to name a few. The compositions may be administered in the form of compositions per se, or as pharmaceutical compositions comprising a compound.

The amount of an agent will typically be in the range of from about 50 mg to about 1 ,500 mg, about 100 mg to about 1 ,500 mg, about 200 mg to about 1 ,500 mg, about 300 mg to about 1 ,500 mg, about 400 mg to about 1 ,500 mg, about 500 mg to about 1 ,500 mg, about 600 mg to about 1 ,500 mg, about 700 mg to about 1 ,500 mg, about 800 mg to about 1 ,500 mg, about 900 mg to about 1 ,500 mg, about 1 ,000 mg to about 1 ,500 mg, about 50 mg to about 1 ,400 mg, about 50 mg to about 1 ,300 nig, about 50 mg to about 1 ,200 mg, about 50 mg to about 1 , 100 mg, about 50 mg to about 1 ,000 mg, about 50 mg to about 900 mg, about 50 mg to about 800 mg, about 50 mg to about 700 mg, about 50 mg to about 600 mg, about 50 mg to about 500 mg, about 50 mg to about 400 mg, or about 50 mg to about 300 mg. in some embodiments, the agent may be in the range of from about 100 mg to about 1 ,100 mg.

Pharmaceutical compositions comprising one or more bacteria as provided herein may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping or lyophilization processes. The compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.

The compositions may be formulated in the pharmaceutical composition per se, or in the form of a hydrate, solvate, A/-oxide or pharmaceutically acceptable salt, as previously described. Typically, such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.

Pharmaceutical compositions may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, fecal transplant, etc., or a form suitable for administration by inhalation or insufflation.

For topical administration, the compositions may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations Include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneai injection, as well as those designed for transdermal, transmucosai oral or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions, or emulsions of the active composition(s) in aqueous or oily vehicles. The compositions may also contain formulating agents, such as suspending, stabilizing, or dispersing agent. The formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives. Alternatively, the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free wafer, buffer, dextrose solution, etc., before use. To this end, the active compositions may be dried by any art- known technique, such as lyophilization, and reconstituted prior to use.

For transmucosai administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

For oral administration, the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents {e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methy!cellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch orsodium starch glycolate); or wetting agents (e.g., sodium !aury! sulfate). The tablets may be coated by methods well known in the art with, for example, sugars, films, or enteric coatings.

Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophore™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoaies or sorbic acid). The preparations may also contain buffer salts, preservatives, flavoring, coloring, or sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the composition, as is well known. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For rectal and vaginal routes of administration, the compGsition(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.

For nasal administration or administration by inhalation or insufflation, the composition(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas. in the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges for use in an inhaler or insufflator (for example capsules and cartridges comprised of gelatin) may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

For ocular administration, the composition(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye. A variety of vehicles suitable for administering compounds to the eye are known in the art.

For prolonged delivery, the composition(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection. The composition(s) may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt. Alternatively, transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the compositon(s) for percutaneous absorption may be used. To this end, permeation enhancers may be used to facilitate transdermal penetration of the compositlon(s).

Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver compositlon(s). Certain organic solvents such as dimethyl sulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more-unit dosage forms containing the compound(s). The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The composition(s) described herein, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder. Therapeutic benefit also generally includes halting or stowing the progression of the disease, regardless of whether improvement is realized.

The amount of composition(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailabiliiy of the particular composition(s) the conversation rate and efficiency into active drug compound under the selected route of administration, etc.

Determination of an effective dosage of composition(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of composition for use in animals may be formulated to achieve a circulating blood or serum concentration of the metabolite active compound that is at or above an IC₅o of the particular composition as measured in as in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailabiliiy of the particular compound via the desired route of administration is well within the capabilities of skilled artisans, initial dosages of composition can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavaiiability and/or metabolism of composition into active metabolites are also well-known. Ordinarily skilled artisans can routinely adapt such Information to determine dosages of particular compounds suitable for human administration.

Dosage amounts will typically be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active compound, the bioavaiiability of the composition, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the composition(s) and/or active metabolite composition(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of eomposition(s) and/or active metabolite composifion(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation.

Methods

The compositions described herein may also be used in many different methods for the diagnosis, treatment, or prevention of Alzheimer^'s Disease or other neurodegenerative disorder. Accordingly, one embodiment described herein is a method of treating or preventing a neurodegenerative disorder in a subject, the method comprising (a) obtaining or having obtained a biological sample from the subject; (b) performing or having performed a diagnostic assay on the biological sample to determine the level of one or more biomarkers associated with the neurodegenerative disorder, wherein the subject has or is at risk of developing the neurodegenerative disorder when the level of the one or more biomarkers is greater than a control; and, (c) administering a composition to the subject having levels of the one or more biomarkers greater than the control, wherein the composition increases bacterial species from the Genera of the Phylum Firmicutes In the gut of the subject, and wherein neurodegeneration is decreased or prevented in the subject following administration of the composition. in another embodiment, the therapy comprises a composition comprising one or more of a bacteria species selected from Lactobacillus._, Erysipelatoclostridium, Faecalitalea, Erysypelotrichaceae, Anaerostipes, Blautia, Ruminiclostridium, or combinations thereof, or a pharmaceutical composition thereof, as described herein.

In one embodiment, the one or more biomarkers associated with Alzheimer’s Disease or other neurodegenerative disorder is selected pTau (including pTau181), GFAP, !L~6, IL-13, Neurofilamenf Light (NfL), or combinations thereof.

In another embodiment, the level of one or more biomarkers associated with the neurodegenerative disorder is from about 10% to about 200% greater than the control.

In some embodiments, the neurodegeneration is decreased or prevented when the level of one or more biomarkers associated with the neurodegenerative disorder is decreased by about 5% to about 200% as compared to the level of the one or more biomarkers before administration of the composition. In the art, a 20% decrease in neurodegeneration as compared to pre- treatment levels is considered to be clinically significant. Another embodiment described herein is a method for treating or preventing a neurodegenerative disorder in a subject, the method comprising administering to the subject a composition comprising one or more bacterial species from the Genera of the Phylum Firmicutes to the subject thereby preventing or treating the neurodegenerative disorder in the subject.

Another embodiment described herein is a method of preventing and/or treating Alzheimer’s Disease or other neurodegenerative disorders in a subject, the method comprising, consisting of, or consisting essentially of administering to the subject a composition comprising one or more of a bacteria species selected from Lactobacillus._, Erysipelatociostridium, Faecalltalea._, Erysype!otrichaceae, Anaerostipes, Blautia, Ruminiclostridium, or combinations thereof, ora pharmaceutical composition thereof, to the subject thereby preventing and or treating the Aizheimer’s Disease or other neurodegenerative disorder in the subject.

In another embodiment described herein the compositions, or pharmaceutical compositions thereof, are administered orally or by other means suitable for increasing the numbers of the bacteria species in the subject.

In yet another embodiment, the methods provided herein may further comprise administering to the subject one or more additional therapeutic agents, in some embodiments, the therapeutic agents are suitable for treating Alzheimer’s Disease or other neurodegenerative disorder, in certain embodiments, the therapeutic agent comprises Rifaximin. in other embodiments, the one or more additional therapeutic agents comprise a medication that increases the numbers of one or more of the bacteria species selected from Lactobacillus, Erysipelatociostridium, Faecaiitalea, Erysypeiotrichaceae, Anaerostipes , Blautia, Ruminiclostridium, or combinations thereof.

In some embodiments, the at least one additional therapeutic agent is administered prior to, concurrently, or after the administration of the composition or pharmaceutical composition as provided herein.

In another embodiment, the is administered for about 1 week to about 3 years. Some neurodegenerative disorders require a short treatment time frame (e.g., weeks) and others require lifelong therapy. The compositions as described herein may also be administered intermittently such as one week every month to avoid adverse effects of long term antibiotic therapy.

The present disclosure is based, in part, on the identification by the inventors of seven Genera of the Phylum Firmicutes whose abundance in the gut increased significantly in ten patients with Aizheimer’s Disease following three-months of oral administration of rifaximin. The seven Genera identified were Lactobacillus, Erysipelatociostridium, Faecaiitalea, Erysypeiotrichaceae, Anaerostipes, Blautia, and Ruminiclostridium. The same group of patients were also found to have had a significant reduction in serum Neurofilament Light, a biomarker for neurodegeneration. It is believed that the increase in numbers of one or a number of, or all of these organisms in the gut caused a reduction of neurodegeneration in the brain either by translocation of the organisms or their metabolites via the gut-brain axis (venous system or vagal nerve pathway) or by reducing the virulence of other microbes in the gut. Reduction of virulence of other microbes implies that the other organisms produce less neurotoxic metabolites or that they and their metabolites are less likely to translocate to the brain by the gut brain axis.

One embodiment described herein is a composition comprising, consisting of, or consisting essentially of one or more of a bacteria species selected from Lactobacillus, Erysipeiatodosiridium, Faecaiitalea, Erysypeiotrichaceae, Anaerostipes , Blautia, Ruminic!ostridium , or combinations thereof. The composition can be formulated for oral or rectal administration and can be used to increase the gut population of these species of bacteria.

Another aspect of the present disclosure provides a pharmaceutical composition comprising, consisting of, or consisting essentially of a composition comprising one or more of a bacteria species selected from Lactobacillus, Erysipeiatodosiridium, Faecaiitalea , Erysypeiotrichaceae, Anaerostipes, Blautia, Ruminiclostridium, or combinations thereof and one or more pharmaceutically acceptable carriers, excipients, diluents, or combinations thereof.

One embodiment described herein is a pharmaceutical composition for augmenting gut microbial flora in a subject and reducing one or more biomarkers associated with a neurodegenerative disorder comprising: one or more bacterial species selected from Lactobadiius, Erysipeiatoclostridium, Faecaiitalea, Erysypeiotrichaceae, Anaerostipes , Blautia, or Ruminiclostridium: and one or more pharmaceutically acceptable excipients, in one aspect, the one or more bacterial species is in an amount of from about 10⁶ CFU to about 10¹² CFU. In another aspect, the pharmaceutical composition further comprises rifaximin. In another aspect, the rifaximin is in an amount of from about 100 mg to about 1 ,100 mg. In another aspect, the pharmaceutically acceptable excipient is a binding agent, a filler, a lubricant, a disintegrant, a wetting agent, or combinations thereof. In another aspect, the pharmaceutical composition comprises a dosage form comprising a tablet, a capsule, or a lozenge.

Another embodiment described herein is a method of treating or preventing a neurodegenerative disorder in a subject, the method comprising: (a) obtaining or having obtained a biological sample from the subject; (b) performing or having performed a diagnostic assay on the biological sample to determine the level of one or more biomarkers associated with the neurodegenerative disorder, wherein the subject has or is at risk of developing the neurodegenerative disorder when the level of the one or more biomarkers is greater than a control; and, (c) administering a composition to the subject having levels of the one or more biomarkers greater than the control, wherein the composition increases bacterial species from the Genera of the Phylum Firmicutes in the gut of the subject, and wherein neurodegeneration is decreased or prevented in the subject following administration of the composition, in one aspect, the one or more biomarkers associated with the neurodegenerafive disorder is pTau, GFAP, IL- 6, IL-13, Neurofiiament Light (NfL), or combinations thereof, in another aspect, the one or more biomarkers is selected from pTau181 , GFAP, NfL or combinations thereof, in another aspect, the composition comprises one or more bacterial species selected from Lactobacillus, Erysipelatociostridium, Faecaiitalea, Erysypelotnchaceae, Anaerostipes, Biautia, or Ruminiclostridium. in another aspect, the composition comprises rifaximin. in another aspect, the bacterial species from the Genera of the Phylum Firmicutes is one or more of bacterial species seiected from Lactobacillus , Erysipelatociostridium, Faecaiitalea, Erysypelotnchaceae, Anaerostipes, Biautia, and Ruminiclostridium. In another aspect, the neurodegenerafive disorder is Alzheimer's Disease. In another aspect, the level of one or more biomarkers associated with the neurodegenerative disorder is about 10% to about 200% greater than the control. In another aspect, the biological sample is blood, serum, plasma, or cerebral spinal fluid (CSF). In another aspect, the diagnostic assay is an immunoassay. In another aspect, the increase in bacterial species from the Genera of the Phylum Firmicutes in the gut of the subject is detected from a fecal sample from the subject. In another aspect, the composition is administered 1 week to 3 years. In another aspect, neurodegeneration is decreased or prevented when the level of one or more biomarkers associated with the neurodegenerative disorder is decreased significantly as compared to the level of the one or more biomarkers before administration of the composition. In another aspect, the significant decrease is from about 5% to about 200% as compared to the level of the one or more biomarkers before administration of the composition.

Another embodiment described herein is a method for treating or preventing a neurodegenerative disorder in a subject, the method comprising administering to the subject a composition comprising one or more bacterial species from the Genera of the Phylum Firmicutes to the subject thereby preventing or treating the neurodegenerative disorder in the subject. In one aspect, the composition is administered oraily to the subject. In another aspect, the method further comprises administering to the subject one or more additional therapeutic agents. In another aspect, the one or more additional therapeutic agents comprise rifaximin. In another aspect, the one or more additional therapeutic agents comprise an agent that increases the numbers of one or more of the bacterial species selected from Lactobacillus, Erysipelatociostridium, Faecaiitalea, Erysypelotnchaceae, Anaerostipes, Biautia, Ruminiclostridium, or combinations thereof. In another aspect, the composition is administered 1 week to 3 years, in another aspect, the neurodegenerative disorder is Alzheimer’s Disease, In another aspect, the bacterial species from the Genera of the Phylum Firmicuies is one or more of bacterial species selected from Lactobacillus, Erysipeiatociostridium, Faecalitalea, Erysypelotrichaceae, Anaerostlpes, Blautla, or Ruminiclostridium.

It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components In the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases In this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

Clause 1 . A pharmaceutical composition for augmenting gut microbial flora in a subject and reducing one or more biomarkers associated with a neurodegenerative disorder comprising: one or more bacterial species selected from Lactobacillus, Erysipeiatocbstridium, Faecalitalea, Erysypelotrichaceae , Anaerostlpes, Blautla, or Ruminiclostridium·, and one or more pharmaceutically acceptable excipients.

Clause 2. The pharmaceutical composition of clause 1 , wherein the one or more bacterial species is in an amount of from about 10⁶ CFU to about 10¹² CFU. Clause 3. The pharmaceutical composition of clause 1 or 2, wherein the pharmaceutical composition further comprises rifaximin.

Clause 4. The pharmaceutical composition of clause 3, wherein the rifaximin is in an amount of from about 100 mg to about 1 , 100 mg.

Clause s. The pharmaceutical composition of any one of clauses 1-4, wherein the pharmaceutically acceptable excipient is a binding agent, a filler, a lubricant, a disintegrant, a wetting agent, or combinations thereof.

Clause 6. The pharmaceutical composition of any one of clauses 1-5, wherein the pharmaceutical composition comprises a dosage form comprising a tablet, a capsule, or a lozenge.

Clause 7. A method of treating or preventing a neurodegenerative disorder in a subject, the method comprising:

(a) obtaining or having obtained a biological sample from the subject;

(b) performing or having performed a diagnostic assay on the biological sample to determine the level of one or more biomarkers associated with the neurodegenerative disorder, wherein the subject has or is at risk of developing the neurodegenerative disorder when the level of the one or more biomarkers is greater than a control; and,

(c) administering a composition to the subject having levels of the one or more biomarkers greater than the control, wherein the composition increases bacterial species from the Genera of the Phyium Firmicutes in the gut of the subject, and wherein neurodegeneration is decreased or prevented in the subject following administration of the composition.

Clause 8. The method of clause 7, wherein the one or more biomarkers associated with the neurodegenerative disorder is pTau, GFAP, IL-6, !L-13, Neurofilament Light (NfL), or combinations thereof.

Clause 9. The method of clause 8, wherein the one or more biomarkers is selected from pTau181 , GFAP, NfL or combinations thereof.

Clause 10. The method of any one of clauses 7-9, wherein the composition comprises one or more bacterial species selected from Lactobacillus , Erysipelatociostridium, Faecalltalea , Erysypeiotrichaceae, Anaerostipes , Blautia, or Ruminiclostridium.

Clause 11. The method of any one of clauses 7-10, wherein the composition comprises rifaximin. Clause 12. The method of any one of clauses 7-11 , wherein the bacterial species from the Genera of the Phylum Firmicutes is one or more of bacterial species selected from Lactobacillus , Erysipelatoclostridium, Faacalitalea, Erysypetotrichacaae , Anaerostipes, Biautia, and Ruminiclostridium.

Clause 13. The method of any one of clauses 7-12, wherein the neurodegenerative disorder is Alzheimer’s Disease.

Clause 14. The method of any one of clauses 7-13, wherein the level of one or more biomarkers associated with the neurodegenerative disorder is about 10% to about 200% greater than the control.

Clause 15. The method of any one of clauses 7-14, wherein the biological sample is blood, serum, plasma, or cerebral spinal fluid (CSF).

Clause 18. The method of any one of clauses 7-15, wherein the diagnostic assay is an immunoassay.

Clause 17. The method of any one of clauses 7-16, wherein the Increase in bacterial species from the Genera of the Phylum Firmicutes in the gut of the subject is defected from a fecal sample from the subject.

Clause 18. The method of any one of clauses 7-17, wherein the composition is administered 1 week to 3 years.

Clause 19. The method of any one of clauses 7-18, wherein neurodegeneration is decreased or prevented when the level of one or more biomarkers associated with the neurodegenerative disorder is decreased significantly as compared to the level of the one or more biomarkers before administration of the composition.

Clause 20. The method of clause 19, wherein the significant decrease is from about 5% to about 200% as compared to the level of the one or more biomarkers before administration of the composition.

Clause 21 . A method for treating or preventing a neurodegenerative disorder in a subject, the method comprising administering to the subject a composition comprising one or more bacterial species from the Genera of the Phylum Firmicutes to the subject thereby preventing or treating the neurodegenerative disorder in the subject.

Clause 22. The method of clause 20, wherein the composition is administered orally to the subject.

Clause 23. The method of clause 20 or 21 , wherein the method further comprises administering to the subject one or more additional therapeutic agents. Clause 24. The method of clause 22, wherein the one or more additional therapeutic agents comprise rifaximin.

Clause 25. The method of clause 22, wherein the one or more additional therapeutic agents comprise an agent that increases the numbers of one or more of the bacterial species selected from Lactobacillus, Erysipelatoclostndium, Faecaiitalea, Erysypeiotrichaceae, Anaerosiipes, Blautia, Ruminiclostridium, or combinations thereof.

Clause 28. The method of any one of clauses 20-24, wherein the composition is administered 1 week to 3 years.

Clause 27. The method of any one of clauses 20-25, wherein the neurodegenerative disorder is Alzheimer’s Disease.

Clause 28. The method of any one of clauses 20-28, wherein the bacterial species from the Genera of the Phylum Firmicutes is one or more of bacterial species selected from Lactobacillus, Erysipeiatociosiridium , Faecaiitalea, Erysypeiotrichaceae, Anaerosiipes, Biautia, or Ruminiclostridium.

EXAMPLES Example 1

Probing Gut-Brain Links in Alzheimer’s Disease with Rifaximin

Gut-microbiome-inflammation interactions have been linked to neurodegeneration in Alzheimer’s disease (AD) and other disorders. It was hypothesized that treatment with rifaximin, a minimally absorbed gut-specific antibiotic, may modify the neurodegenerative process by changing gut flora and reducing neurotoxic microbial drivers of inflammation. In a pilot, open- label trial, 10 subjects with mild to moderate probable AD dementia (MMSE = 17 ± 3) were treated with rifaximin for 3 months. Treatment was associated with a significant reduction in serum neurofilament-iight levels (p <0.004) and a significant increase in fecal phylum Firmicutes microbiota. Serum pTau181 and GFAP levels were reduced (effect sizes of -0.41 and -0.48 respectively) but did not reach significance. There was also a non-significant downward trend in serum cytokine IL-6 and IL-13 levels (FIG. 1). Increases in stool Erysipelatoclostndium were correlated significantly with reductions in serum pTau and serum GFAP. Insights from this pilot trial are being used to design a larger placebo-controlled clinical trial to determine if specific microbial flora/products underlie neurodegeneration, and whether rifaximin is clinically efficacious as a therapeutic.

The study described herein was based on the hypothesis by the inventors that reducing exposure to neurotoxic bacterial products (such as LPS or certain bile acids) would reduce neuroinflammation and neurodegeneration in AD. One potential approach to probe such links is through the use of antibiotics or probiotics. For example, colonization of germ-free APP-PS1 mice with microbiota from aged wild type mice leads to increased cerebral AD pathology and treatment with an antibiotic that reduces such pathology. Rifaximin was identified as a drug that may be well suited to test this hypothesis in AD. Rifaximin is a gut-selective antibiotic that binds to the beta-subunit of the bacterial DNA-dependent RNA polymerase. Animal models, culture studies and metagenomic analyses have demonstrated that rifaximin improves the integrity of the gut epithelial barrier and decreases virulence, motility, and adhesion of pathogens. Rifaximin has also been shown to down-regulate epithelium damaging metalloproteinase and to increase the abundance of beneficial gut bacteria such as Lactobacillus. It has a relatively good safety profile, lack of drug interactions, and oral dosing. Human studies have demonstrated neurological benefits for treating and preventing the recurrence of hepatic encephalopathy. Hence, for these studies it was repurposed for use in AD and other neurodegenerative disorders.

In a pilot biomarker trial, 10 subjects were treated with mild to moderate AD (MMSE 17.3 ± 3.3) for 12 weeks with rifaximin 500 mg bid. Rifaximin treatment was associated with a significant reduction in serum neurofilament-light (Nfl) levels as well as non-significant reductions in serum tau and inflammatory cytokines. Although the trial was too short to detect cognitive benefits, Nfl is a biomarker for axonal neurodegeneration and hence reductions in NfL may potentially be indicative of a future cognitive benefit. Rifaximin treatment in this trial also significantly and selectively increased the abundance of microbiota in seven genera of the phylum Flrmicutes - with increases in one of these genera also significantly correlating with reductions in pTau181 and GFAP. Flrmicutes may play a vital role in multiple gut and brain functions such as preserving the gut integrity (through synthesis of short chain fatty acids), neurotransmitter metabolism, polyphenol absorption, and reducing risk for microbial toxicity. Prior studies have noted that the abundance of Flrmicutes is reduced in AD and hence these findings also suggest a potential neuroprotective effect. Rifaximin was relatively well tolerated, with transient diarrhea being the most common side effect.

Overall, the study provides preliminary support to the hypothesis that microbiome modulation can alter markers of neurodegeneration. Because of the limitations of a small pilot study, there are still many unknowns including the mechanisms involved and the clinical significance of the observed changes. A larger (N = 100, 12-month), placebo-controlled study is being designed to more definitively examine the effects of rifaximin on microbiome composition and toxicity (by measuring neurotoxic products such as IPS and bile acids) in relation to neuropathoiogical and cognitive changes. Consolidated Results and Study Design

This study was approved by the Duke IRB and all subjects/legai representatives gave written informed consent (Ciinicaltrials.gov identifier NCT03856359). An IND exemption was obtained for the study. Ten subjects with probable mild to moderate AD were recruited. Key inclusion criteria were MMSE score of 10-23 and stable health; key exclusion criteria were recent history of antibiotic use, cirrhosis, and diarrhea. All subjects were treated with rifaximin 550 mg twice daily for 3 months. Following the end of treatment, subjects were monitored for an additional month for any adverse effects.

Serum samples at baseline and at 3 months were analyzed for neural (NfL), pathological (total tau, plau181) and glial (glial fibrillary acidic protein (GFAR) markers as well as cytokines. Baseline and post-treatment fecal samples underwent microbiome analyses. Serum biomarker analysis was done using ultrasensitive SIMOA assay (Quanterix, Billerica, MA) whereas cytokines were assayed using high sensitivity T-cell panel (Millipore Sigma, Saint Louis, MO). Baseline and post-treatment fecal samples underwent microbiome 16s sequencing using the Qiagen PowerSoilPro DNA Kit (Qiagen, Germantown, MD). Bacterial community composition was characterized by amplification of the V4 variable region of the 16S rRNA gene by polymerase chain reaction using the forward primer 515 and reverse primer 806 following the Earth Microbiome Project protocol. Concentrations of the PCR products were accessed using a Qubit dsDNA MS assay kit (Thermo Fisher, Waltham MA) and a Promega G!oMax plate reader. Equimolar 16S rRNA Polymerase Chain Reaction products from all samples were pooled prior to sequencing. Sequencing was performed by the Duke Sequencing and Genomic Technologies shared resource on a MiSeq (lliumina, San Diego, CA) instrument configured for 250 base-pair paired-end sequencing runs. Raw reads were processed into Amplicon Sequence Variant count tables via the Qiime2 pipeline (2020.2). Raw sequence data was demultiplexed and quality filtered using the q2-demux plugin followed by denoising with Divisive Amplicon Denoising Algorithm 2. Reads were trimmed at the beginning or truncated at the end if the median base quality fell below a score of 30 as determined by the fastx quality stats tool from Fastx-too!kit (vQ.0,14). All amplicon sequence variants were aligned with Mafft (via q2-a!ignment) and used to construct a phylogeny with RAxML version 8 (via q2-phyiogeny). Taxonomy was assigned to Amplicon Sequence Variants using the q2- feature-classifier classify-skiearn naive Bayes taxonomy classifier against the SILVA 132 database. Changes from baseline to endpoint (intent to treat) in biomarker and cognitive test scores were analyzed using two-tailed paired t-tests and the Spearman test examined correlations (Rv3,5.2), Ten subjects (9 F, mean [SD] age 72.5 ± 5.8 years) with mild to moderate AD dementia (MMSE 17.3 ± 3.3) were enrolled. Serum NfL levels were significantly reduced following rifaximin treatment (p < 0.004, effect size = -1 ,21) (FIG. 1). pTau and GFAP levels were reduced (effect sizes of -0.41 and -0.48 respectively) but did not reach significance (p = 0.23 and p = 0.17 respectively). Cytokine levels were lowered non-significantly for I L-6 (p = 0.07) and IL-13 (p = 0.09). The mean changes for other cytokines (serum IL-1 beta, !L-2, IL-4, IL-5, I L-6, IL-8, IL-10, IL-13, TNF-aipha) were not significant. The mean 12-week change in ADAS~Cog~11 (p = 0.60) and MMSE was not significant. There was a significant increase in abundance of seven genera of the phylum Firmicutes following treatment (see FIG. 2): Lactobacillus ( p = 0.019), Erysipelatoc!ostridium (p = 0.00035), Faecalilalea (p = 0.02), Erysipe!atotrichaceae (p -0.0008), Anaerostipes (p = 0.038), Blautia (p = 0.004), and Ruminoclostridium 9 (p = 0.027). There was a significant inverse correlation between increases in Erysipelatoc!ostridium and reduction in pTau (Rho = -0.883, p = 0.003) and GFAP (Rho - -0.35, p = 0.036). Rifaximin was relatively well tolerated, with the most common side effect being transient diarrhea (N = 4). One subject discontinued early due to worsening dementia. There were no serious adverse events.

There are relatively few gut microbiome modulating drugs in the late stage pipeline for treatment of AD dementia. Of the 17 disease modifying therapies in Phase 3 clinicai trials, only two are known to directly target the gut-brain axis. Atuzaginstat is a bacterial protease inhibitor that targets glngipain, a Porphyromonas gingivalis byproduct, that has been associated with neuroinfiammation and hippocampal degeneration. Sodium oiigomannate is an alga derived oligosaccharide that alters the gut microbiome to reduce peripheral and central inflammation, A number of other candidates are in eariy-stage development including probiotics, bile acids, neuroprotective microbes, and antibodies against toxic microbial polysaccharide antigens.

Rifaximin was repurposed for use in AD because of its high gut-specificity and favorable safety profile over decades of use. Rifaximin is U.S. FDA approved for the treatment of hepatic encephalopathy, travelers’ diarrhea, and inflammatory bowel disease. Less than 0.4% of this antibiotic is absorbed, making it weil-toierated for treating gastrointestinal infections, it reduces the virulence of gut pathogens and significantly lowers blood IPS levels. Rifaximin may also fortify the host gut epithelial barrier by blocking the ability of pathogens to attach to the gut epithelial cell surface or to be internalized by the epithelial cells. This significantly reduces translocation of pathogens from the gut iumen Into the lamina propria and mesenteric lymph nodes. Rifaximin, via gut epithelial cell pregnane X receptor (PXR), may also promote the transcription of genes for detoxification enzymes and cytokines. These mechanisms have the potential to benefit not just AD but other neurodegenerative disorders. The preliminary findings support the hypothesis that bacterial products may contribute to neurodegeneration and that rifaximin could prove useful to probe and potentially treat these underlying mechanisms. The significant lowering of Nfl levels along with decreases in serum IL~ 8 support the hypothesis that neuroinf!ammatory changes may mediate microbiome effects on neurodegeneration. Prior studies have noted increased expression of IL-6 in AD brains prior to the deveiopment of neuritic plaques suggesting inflammation may be a cause rather than a response.

There are several microbial products that could potentially lead to neurotoxic or neuroinfiammatory effects in AD. This includes ammonia, IPS, and secondary bile acids. Bacterial products can cross the gut epithelial barrier into the lamina propria and gain access into the brain via either the vagus nerve or a compromised blood-brain barrier. No changes in blood ammonia was detected in the study suggesting that in subjects with healthy livers this may not be a major contributor. LPS, a cell wall component of gram-negative gut pathogens, is elevated in the blood and brains of AD patients and may induce Ab production and myelin destruction through cytokines. In mouse models, Kahn et al showed that peripheral LPS-induced inflammation can cause cognitive deficits and significantly increased hippocampal beta amyloid. LPS was not measured but plans to do so are being considered for the study.

Microbiota in the Firmicutes genera ( Lactobacillus , Erysipelatoclostridium, Anaerostipes and B!autia) were significantly increased in abundance in the patients following treatment. Prior studies have noted a lower abundance of fecal Firmicutes in AD and this correlated with elevated CSF biomarkers of neurodegeneration. Firmicutes genera have previously been reported to have neuroprotective effects involving multiple mechanisms. Lactobacillus is involved in production of neurotransmitters and metabolites, including GABA, serotonin, acetylcholine, histamine, dopamine, and short chain fatty acids. Erysipelatoclostridium promotes gut po!yphenolic compound absorption which can cross into the brain and protect against nitric oxide production and pro-inflammatory cytokines. Polyphenollc compounds are also anticholinesterase inhibitors and may have direct cognitive benefits. A prior study found a correlation between abundance of Erysipelatoclostridium in the gut and cognitive function in APP/PS1 mice. Anaerostipes production of butyrate helps to maintain a healthy gut epithelial barrier by fueling enterocytes. Based on these data, the current hypothesis is that rifaximin may also have potential probiotic effects through increasing the abundance of beneficial neuroprotective Firmicutes strains. These findings are also supported by two additional studies. In a trial of 15 patients with irritable bowel syndrome, rifaximin increased bacterial diversity, the Firmicutes! Bacteroidetes ratio and the abundance of Faeca!ibaclerium prausnitzii, a butyrate producer with strong anti-inflammatory properties, in another report on 20 patients with a variety of gastrointestinal and liver disorders, rifaximin increased the abundance of Lactobacilli. Additional studies in larger samples are needed to test this hypothesis and determine if the microbial changes correlate with biomarker/clinical benefits. Animal models may also help elucidate mechanisms involved.

Limitations in the study include open design, short duration, and multiplicity of tests (Type 1 error) which may reduce the likelihood that these findings reflect a true effect. Also, strict control for diet or time of stool collection was not provided hence fecal microbiome analyses should be viewed as preliminary, in addition, blood for the laboratory assay was not collected in the morning or after fasting. There also remain many unknowns in the microbial hypothesis of AD, such as the exact microbial organisms and/or products implicated in neurodegeneration, how they enter the brain to cause neurodegeneration, whether biomarker changes observed {e.g., NfL) translate into meaningful clinical benefits as well as the optimal dosing of rifaximin in AD and other neurodegenerative disorders.

Despite such limitations, this is the first study to demonstrate that microbiome modulation alters a marker of neurodegeneration in human AD subjects. The intent was to generate pilot biomarker data to refine the hypothesis and power a larger placebo-controlled study to more definitively examine the effects of rifaximin on microbial composition, microbial products (e.g., IPS) and neurodegeneration markers along with cognitive and safety measures. Since rifaximin is already approved for other indications, a next study is planned under an IND exemption. Additional dose finding studies are needed to assess whether lower doses or intermittent dosing is safer for long-term use or whether higher doses prove more beneficial. Because the drug is relatively free of drug-drug interactions it can also be used with existing AD therapies in combination. Efforts to determine whether the specific microbes identified in this study could be developed into a therapeutic for AD. Lastly, the findings may also have relevance for other neurodegenerative disorders such as MCI, ALS, or PD and studies may be warranted in those disorders.

Example 2

Exemplary Plan of Treatment

An exemplary plan of treatment comprises: (1) pre-treat the gut microbiome with a course of rifaximin (e.g., 12-24 weeks) to eliminate the toxic species and promote beneficial species; (2) augment the gut microbiome with a cocktail of beneficial microbes as described herein (e.g., Lactobacillus, Erysipelatoclostridium, Faecaiitalea, Erysypelotrichaceae, Anaerostipes , Blautia, or Ruminiciostridium\) - the cocktail would be tailored for each disease and would be given for aboui 6 months to about 36 months; (3) re-dose with rifaximin periodically, e.g., once every six months, to dear out toxic overgrowth; (4) monitor therapy every 3 months using biomarkers and cognitive changes; (5) monitor beneficial and harmful microblome changes every 3 months using stool analyses; (6) monitor for Clostridium difficile overgrowth as an adverse effect.

Claims

CLAIMS What is claimed:

1. A pharmaceutical composition for augmenting gut microbial flora in a subject and reducing one or more biomarkers associated with a neurodegenerative disorder comprising: one or more bacterial species selected from Lactobacillus , Erysipelatoclostridium, Faecalitalea , Erysypelotrichaceae , Anaerostipes_: Blautia, or Ruminidostridium ; and one or more pharmaceutically acceptable excipients.

2. The pharmaceuticai composition of claim 1 , wherein the one or more bacterial species is in an amount of from about 1Q⁶ CFU to about 10¹² CFU.

3. The pharmaceutical composition of claim 1 , wherein the pharmaceuticai composition further comprises rifaximin.

4. The pharmaceutical composition of claim 3_: wherein the rifaximin is in an amount of from about 100 mg to about 1 , 100 mg.

5. The pharmaceutical composition of claim 1 , wherein the pharmaceutically acceptable excipient is a binding agent, a filler, a lubricant, a disintegrant, a wetting agent, or combinations thereof.

6. The pharmaceutical composition of claim 1 , wherein the pharmaceuticai composition comprises a dosage form comprising a tablet, a capsule, or a lozenge.

7. A method of treating or preventing a neurodegenerative disorder in a subject, the method comprising:

(a) obtaining or having obtained a biological sample from the subject;

(b) performing or having performed a diagnostic assay on the biological sample to determine the level of one or more biomarkers associated with the neurodegenerative disorder, wherein the subject has or is at risk of developing the neurodegenerative disorder when the level of the one or more biomarkers is greater than a control; and, (c) administering a composition to the subject having levels of the one or more biomarkers greater than the control, wherein the composition increases bacterial species from the Genera of the Phylum Firmicutes in the gut of the subject, and wherein neurodegeneration is decreased or prevented in the subject following administration of the composition.

8. The method of claim 7, wherein the one or more biomarkers associated with the neurodegenerative disorder is pTau, GFAP, 11-6, 11.-13, Neurofilament Light (NfL), or combinations thereof.

9. The method of claim 8, wherein the one or more biomarkers is selected from pTau181 , GFAP, NfL or combinations thereof.

10. The method of claim 7, wherein the composition comprises one or more bacteriai species selected from Lactobacillus , Erysipe!atoclostridium , Faecalitalea, Erysypeio trich a ce a e , Anaerostipes, Blautia , or Ruminiclostridium.

11 . The method of claim 7, wherein the composition comprises rifaximin.

12. The method of claim 7, wherein the bacterial species from the Genera of the Phylum

Firmicutes is one or more of bacterial species selected from Lactobacillus ,

Erysipelatoc!ostridium, Faecalitalea, Erysypeloirichaceae, Anaerostipes, Blautia, or Ruminiclostridium .

13. The method of claim 7, wherein the neurodegenerative disorder is Alzheimer’s Disease.

14. The method of claim 7, wherein the level of one or more biomarkers associated with the neurodegenerative disorder is about 10% to about 200% greater than the control.

15. The method of claim 7, wherein the biological sample is blood, serum, plasma, or cerebral spinal fluid (CSF).

16. The method of claim 7, wherein the diagnostic assay is an immunoassay.

17. The method of claim 7, wherein the increase in bacterial species from the Genera of the Phylum Firmicutes in the gut of the subject is detected from a fecal sample from the subject.

18. The method of claim 7, wherein the composition is administered 1 week to 3 years.

19. The method of claim 7, wherein neurodegeneration is decreased or prevented when the level of one or more biomarkers associated with the neurodegenerative disorder is decreased significantly as compared to the level of the one or more biomarkers before administration of the composition.

20. The method of claim 19, wherein the significant decrease is from about 5% to about 200% as compared to the level of the one or more biomarkers before administration of the composition.

21 . A method for treating or preventing a neurodegenerative disorder in a subject, the method comprising administering to the subject a composition comprising one or more bacterial species from the Genera of the Phylum Firmicutes to the subject thereby preventing or treating the neurodegenerative disorder in the subject.

22. The method of claim 20, wherein the composition is administered orally to the subject,

23. The method of claim 20, wherein the method further comprises administering to the subject one or more additional therapeutic agents.

24. The method of claim 22, wherein the one or more additional therapeutic agents comprise rifaximin.

25. The method of claim 22, wherein the one or more additional therapeutic agents comprise an agent that increases the numbers of one or more of the bacterial species selected from Lactobacillus, Erysipeiatociosiridium , Faecaiitaiea, Erysypeiotrichaceae, Anaerostipes, Biautia, Ruminiclostridium, or combinations thereof.

26. The method of claim 20, wherein the composition is administered 1 week to 3 years.

27. The method of claim 20, wherein the neurodegenerative disorder is Alzheimer^'s Disease.

28. The method of claim 20, wherein the bacterial species from the Genera of the Phylum Firmicutes is one or more of bacterial species selected from Lactobacillus, En/sipelatoc!ostridium, Faecaiitaiea, Erysype!oirichaceae, Anaerostipes, Blautia, or Ruminiclostridium.