Probiotics and methods to reduce weight

Abstract

Provided herein are methods and compositions related to reducing / suppressing appetite. In some aspects, provided herein are methods of reducing / suppressing appetite in a subject by administering to the subject a composition comprising Parabacteroides and Akkermansia bacteria.

Description

BSCT.006WO PCT ApplicationPROBIOTICS AND METHODS TO REDUCE WEIGHTRELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63 / 713,872, filed October 30, 2024, which is hereby incorporated by reference in its entirety.RELATED FIELD

[0002] Aspects of the present disclosure relate to methods and compositions for reducing weight and / or preventing weight gain in an overweight or obese subject. In some aspects, provided herein are methods of reducing weight in an overweight or obese subject by administering to the subject a composition comprising Parabacteroides and Akkermansia bacteria.BACKGROUND

[0003] Excess weight is typically associated with the presence of excess fat deposits in an individual. In contrast, obesity is attributed to excess fat that can impair health. Obesity can lead to an increased risk of many diseases, including type 2 diabetes, heart disease, cancer, high blood pressure, depression, and gallbladder disease. It can affect bone health and reproduction, and can reduce an individual’s capability for sleep or movement.

[0004] The diagnosis of overweight and obesity is made by measuring people’s weight and height and by calculating the body mass index (BMI): weight (kg)Zheight2(m2). The BMI categories for defining obesity vary by age and gender in infants, children and adolescents. For adults over 20 years old, BMI falls into one of these categories: below 18.5 is considered underweight, 18.5-24.9 is considered normal, 25.0-29.9 is considered overweight, and 30.0 and above is considered obese.SUMMARY

[0005] Provided herein are methods and compositions for reducing weight and / or preventing weight gain in a subject. In some embodiments, the subject is overweight and / or obese. In some embodiments, the subject has a BMI that is at least about 20, 21, 22, 23, 24, or 25. In some embodiments, the method comprises administering to the subject at least IxlO8colony forming units (CFUs) of Akk bacteria and at least 1x107CFUs of Pb bacteria is administered to the subject. In some embodiments, the method comprises administering to the subject at least IxlO11CFUs of Akkermansia (Akk) bacteria and at least IxlO10CFUs of Parabacteroides (Pb) bacteria. In some embodiments, the bacteria is administered to the subject orally or rectally. In some embodiments, the bacteria is administered in a pill, a capsule, a tablet, a minitablet, a powder, or as part of a food product. In some embodiments, the subject is a human subject. In some embodiments, the Akk bacteria comprises Akkermansia muciniphila. In some embodiments, the Pb bacteria comprises Parabacteroides merdae, Parabacteroides distasonis, and / or Parabacteroides johnsonii. In some embodiments, the Akk and Pb bacteria are administered to the subject at least daily. In some embodiments, the bacteria is administered to the subject at a ratio of at least 1 : 1, 2: 1, 3: 1, 6: 1, or 10: 1 Akkermansia to Parabacteroides bacteria.

[0006] Also provided herein is a bacterial composition, comprising an at least IxlO8CFUs of Akk bacteria and at least IxlO7CFUs of Pb bacteria. In some embodiments, the composition comprises an at least IxlO11CFUs of Akk bacteria and at least IxlO10CFUs of Pb bacteria. In some embodiments, the composition is formulated for oral or rectal delivery. In some embodiments, the composition is part of a food product, optionally wherein the food product is a yogurt. In some embodiments, the composition further comprises a prebiotic. In some embodiments, the bacteria is at a ratio of at least 1 : 1, 2: 1, 3 : 1, 6: 1, or 10: 1 Akkermansia to Parabacteroides bacteria. In some embodiments, the bacteria comprises Akkermansia muciniphila, Parabacteroides merdae, Parabacteroides johnsonii, Parabacteroides distasonis, or any combination thereof.

[0007] Also provided herein is a use of the bacterial composition of any one of the embodiments of the present disclosure in reducing weight in a subject. Also provided herein is a use of the bacterial composition of any one of the embodiments of the present disclosure in preventing weight gain in a subject. In some embodiments, the subject is overweight and / or obese. In some embodiments, the subject has a BMI that is at least about 20, 21, 22, 23, 24, or 25. In some embodiments, the subject has a BMI that is at least about 30.

[0008] In some embodiments, the method or use disclosed herein comprises administering to the subject a composition comprising bacteria of the Akkermansia (Akk) and Parabacteroides (Pb) genera, or multiple compositions that together comprise bacteria of theAkkermansia (Akk) and Parabacteroides (Pb) genera. Tn some embodiments, the compositions comprise bacteria of the Akkermansia (Akk) and Parabacteroides (Pb) genera. In some embodiments, the bacteria of the Akkermansia (Akk) genus comprise Akkermansia muciniphila. In some embodiments, the bacteria of the Parabacteroides (Pb) genus comprise Parabacteroides merdae, Parabacteroides johnsonii, and / or Parabacteroides distasonis. In some aspects, the methods comprise depleting the gut microbiota of the subject and administering a composition comprising bacteria of Akkermansia genus (e.g., Akkermansia muciniphila) and Parabacteroides genus (e g., Parabacteroides merdae, Parabacteroides johnsonii, or Parabacteroides distasonis to the subject. In some embodiments, the composition comprises a 6:1, 6:2, 6:3, 6:4, 6:5, 6:6, 1 :6, 2:6, 3:6, 4:6, or 6:6 ratio of Akkermansia to Parabacteroides bacteria. In some embodiments, the composition comprises a 6:2 ratio of Akkermansia to Parabacteroides bacteria. In some embodiments, the Akkermansia bacteria is present in the composition at about lxlOe, 5xl05, IxlO6, 5xl06, IxlO7, 5xl07, IxlO8, 5xl08, IxlO9, 5xl09, IxlO10, 5xlO10, IxlO11, 5xlOn, IxlO12, 5xl012, IxlO13, 5xl013, IxlO14, 5xl014, IxlO15, 5xl013, IxlO20, 5xlO20, or any abundancy that is between IxlO5and 5xlO20, colony forming units (CFUs).

[0009] In some embodiments, the subject is on a diet, and the diet may be a control diet, a ketogenic diet, a high fat diet, or a low carbohydrate diet. The composition may be formulated for oral or rectal delivery. The composition may be a food product. In some embodiments, the food product is a dairy product (e.g., yogurt). In some embodiments, the composition comprises probiotics. In some embodiments, the composition is selfadministered. In some embodiments, the composition comprises a fecal sample (e.g., a fecal sample from a fecal bank) comprising bacteria of the Akkermansia genus (e.g., Akkermansia muciniphila) and bacteria of the Parabacteroides (Pb) genus (e.g., Parabacteroides merdae, Parabacteroides johnsonii, o Parabacteroides distasonis'). In some embodiments, the subject is given antibiotics to deplete the subject’s gut microbiota.

[0010] Also provided herein is a use of a bacterial composition comprising Akkermansia bacteria and Parabacteroides bacterial in the preparation of a drug product for reducing weight in an individual. In some embodiments, the bacterial composition is the composition of any one of the embodiments of the present disclosure. In some embodiments, the individual has a BMI above 25. In some embodiments, the individual is obese.

[0011] Also provided herein is a use of a bacterial composition comprising Akkermansia bacteria and Parabacteroides bacterial for inducing ketosis in an individual. In some embodiments, ketosis is induced for at least about 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years. In some embodiments, the bacterial composition is the composition of any one of the embodiments of the present disclosure. In some embodiments, the individual has a BMI that is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 . In some embodiments, the individual is obese.

[0012] Also provided herein is a use of a bacterial composition of any one of the embodiments of the present disclosure for reducing appetite in an individual. In some embodiments, the bacterial composition is the composition of any one of the embodiments of the present disclosure. In some embodiments, the individual has a BMI that is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 . In some embodiments, the individual is obese.

[0013] Also provided herein is a use of a bacterial composition of any one of the embodiments of the present disclosure for increasing expression of at least one metabolic hormone in an individual. In some embodiments, the at least one hormone comprises at least 1, 2, or 3 of: GLP1, Leptin, PYY, or any combination thereof. In some embodiments, the bacterial composition is the composition of any one of the embodiments of the present disclosure. In some embodiments, the individual has a BMI that is at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 . In some embodiments, the individual is obese.

[0014] Also provided herein is a method for changing the concentration of a metabolic compound in a subject, the method comprising administering to the subject the bacterial composition of any one of the embodiments of the present disclosure. In some embodiments, the metabolic compound comprises a metabolic hormone, a metabolite, or any combination thereof. In some embodiments, the metabolic hormone comprises at least one of: leptin, PYY, GLP1, or any combination thereof. In some embodiments, the metabolic hormone comprises at least one of: leptin L, PYY, GLP1, or any combination thereof. In some embodiments, the metabolite comprises at least one of: BHB, acetoacetate, a-ketobutyrate, ascorbic acid 3-sulfate, cysteine s-sulfate, a-ketoglutaramate, butyrylcarnitine, isobutyrylcarnitine, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), or any combination thereof. In some embodiments, the metabolic hormone increases in concentration following administration of the bacterial composition to the subject. In some embodiments, themetabolite increases in concentration following administration of the bacterial composition to the subject. In some embodiments, the metabolite decreases in concentration following administration of the bacterial composition to the subject. In some embodiments, BHB, acetoacetate, a-ketobutyrate, ascorbic acid 3-sulfate, butyryl carnitine, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), or any combination thereof, increases in concentration following administration of the bacterial composition to the subject. In some embodiments, cysteine s-sulfate, a-ketoglutaramate, isobutyrylcamitine, or any combination thereof, decreases in concentration following administration of the bacterial composition to the subject. In some embodiments, the changed concentration of the at least one metabolic compound persists for at least about 7, about 14, about 21, about 28, about 35, or about 42 days. In some embodiments, the changed concentration of the at least one metabolic compound is detectable through collecting and measuring its presence in the plasma of the subject.

[0015] The above description discloses several methods and materials. This disclosure is susceptible to modifications in the methods and materials, as well as alterations in the fabrication methods and equipment. Such modifications will become apparent to those skilled in the art from a consideration of the disclosure disclosed herein. Consequently, it is not intended that embodiments described herein be limited to the specific embodiments disclosed herein, but that it covers all modifications and alternatives coming within the true scope and spirit of the disclosure.

[0016] In another embodiment of the methods described herein, any of the methods described herein can be used alone, or any of the methods described herein can be used in combination with any other method or methods described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to describe the manner in which the above-recited and other advantages and features of embodiments described herein can be obtained, a more particular description will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments and are not therefore to be considered to be limiting of its scope, the disclosure will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

[0018] Figure 1 shows a non-limiting example cartoon schematic of a dosing regimen for administering Akkermansia muciniphila and Parabacteroides merdae to human subjects, as detailed in the below Example 2.

[0019] Figure 2 shows a non-limiting cartoon schematic of the quantification of bacteria present in human subjects over six weeks following a dosing regimen, as detailed in the below Example 2.

[0020] Figures 3A and 3B show a non-limiting quantification of bacteria present in human subj ects over six weeks following a dosing regimen, as detailed in the below Example 2. Figure 3A shows the linear relative abundance of bacteria over time. Figure 3B shows the center-log ratio of bacteria abundance over time.

[0021] Figure 4 shows a non-limiting example cartoon schematic of the high dose human subject cohorts used to collect fecal samples, followed by a fecal transplant to germ- free mice, as detailed in the below Example 2.

[0022] Figure 5 shows a non-limiting quantification of the percentage of subjects (both overall and overweight) that had a non-zero metabolic change (MC) score following treatment with BL-001, as detailed in the below Example 4.

[0023] Figure 6 shows a non-limiting quantification of the average MC score in subjects (both overall and overweight) following treatment with BL-001, as detailed in the below Example 3.

[0024] Figure 7 shows a non-limiting quantification of the average weight change in subjects (both overall and overweight) following treatment with BL-001, as detailed in the below Example 3.

[0025] Figure 8 shows a non-limiting quantification of the percentage weight change as pooled by treatment and BMI status, as detailed in the below Example 5.

[0026] Figure 9 shows a non-limiting quantification of the mean weight change from baseline for treated subjects and treated subjects with a metabolic change, as detailed in the below Example 4.

[0027] Figures 10A-10B show a non-limiting quantification of the least squares means change from baseline for GLP1 concentration in subjects after 28 days of treatment. Figure 10A compared GLP1 changes in those given a placebo, subjects that did not lose weight (the “treatment no weight loss” group, or “T No WL”), and subjects that did lose weight (the“treatment and weight loss” group, or “T WL”). Figure 10B compared GLP1 changes in those given a placebo, those that had a healthy weight BMI (“HW”), and those that had a high BMI (the “over weight” group, or “OW”).

[0028] Figures 11A-11B show a non-limiting quantification of the least squares means change from baseline for Leptin L concentration in subjects after 28 days of treatment. Figure 11A compared Leptin L changes in those given a placebo, subjects that did not lose weight (the “treatment no weight loss” group, or “T No WL”), and subj ects that did lose weight (the “treatment and weight loss” group, or “T WL”). Figure 11B compared Leptin L changes in those given a placebo, those that had a healthy weight BMI (“HW”), and those that had a high BMI (the “over weight” group, or “OW”).

[0029] Figures 12A-12B show a non-limiting quantification of the least squares means change from baseline for PYY concentration in subjects after 28 days of treatment. Figure 12A compared PYY changes in those given a placebo, subjects that did not lose weight (the “treatment no weight loss” group, or “T No WL”), and subjects that did lose weight (the “treatment and weight loss” group, or “T WL”). Figure 12B compared PYY changes in those given a placebo, those that had a healthy weight BMI (“HW”), and those that had a high BMI (the “over weight” group, or “OW”).

[0030] Figures 13A-13C show a non-limiting quantification of the log-change in concentration compared to baseline of BHB (Figure 13A) and acetoacetate (Figure 13B) in the plasma of subjects over 42 days of treatment. Figure 13C is a non-limiting cartoon schematic demonstrating where BMB and acetoacetate (ACAC) production occurs during metabolism.

[0031] Figures 14A-14D show a non-limiting quantification of the log-change in concentration compared to baseline of a-ketobutyrate (Figure 14A), ascorbic acid 3-sulfate (Figure 14B), and cysteine s-sulfate (Figure 14C) in the plasma of subjects over 42 days of treatment. Figure 14D is a non-limiting cartoon schematic demonstrating where production of each metabolite occurs during the trans-sulfuration pathway.

[0032] Figure 15A shows a non-limiting quantification of the log-change in concentration compared to baseline of a-ketoglutaramate in the plasma of subjects over 42 days of treatment.

[0033] Figure 15B is a non-limiting cartoon schematic demonstrating where a- ketoglutaramate participates as part of the glutamine synthesis and degradation pathways.

[0034] Figures 16A-16C show a non-limiting quantification of the log-change in concentration compared to baseline of butyrylcamitine (Figure 16A), and isobutyrylcarnitine (Figure 16B) in the plasma of subjects over 42 days of treatment. Figure 16B is a non-limiting cartoon schematic demonstrating where production of each metabolite occurs during cholesterol and fatty acid synthesis.

[0035] Figure 17 shows a non-limiting quantification of the log-change in concentration compared to baseline of N-acetylglucosamine (GlcNAc) and N- acetylgalactosamine (GalNAc) in the plasma of subjects over 42 days of treatment.DETAILED DESCRIPTION

[0036] Although the disclosure is described in various exemplary alternatives and implementations as provided herein, it should be understood that the various features, aspects, and functionality described in one or more of the individual alternatives are not limited in their applicability to the particular alternative with which they are described. Instead, they can be applied alone or in various combinations to one or more of the other alternatives of the present disclosure, whether the alternatives are described or whether the features are presented as being a part of the described alternative. The breadth and scope of the present disclosure should not be limited by any exemplary alternatives described or shown herein.

[0037] Provided herein are methods and compositions for reducing weight and / or preventing weight gain in a subject by administering to the subject a formulation comprising Akkermansia (Akk) and Parabacteroides (Pb) bacteria. In some embodiments, the subject is overweight and / or obese. In some embodiments, the subject has a BMI that is at least about 20, 21, 22, 23, 24, or 25. In some embodiments, the method comprises administering to the subject at least IxlO8colony forming units (CFUs) of Akk bacteria and at least IxlO7CFUs of Pb bacteria. In some embodiments, the method comprises administering to the subject at least IxlO11CFUs of Akk bacteria and at least IxlO10CFUs of Pb bacteria. In some embodiments, the weight reduction is durable, and is significantly maintained in the subject for at least 1, 2, 3, 4, 5, or 6 weeks following treatment. In some embodiments, the weight reduction is significantly maintained in the subject for at least 1, 2, 3, 4, 5, or 6 months following treatment.

[0038] Also provided herein are methods and compositions for mimicking the effects of a ketogenic diet by administering probiotic compositions including Akk and Pb bacteria to the subject. One embodiment of the composition disclosed herein, “BL-001,” was first designed to replicate the antiepileptic effect of the Ketogenic Diet to modulate gamma aminobutyric acid (GABA) and other key bioenergetic pathways. BL-001 contains two rationally selected human gut microbes, Akkermansia muciniphila and Parabacteroides merdae that have been shown in both cell-based assays and animal studies to eliminate hyper- excitatory activity, increase GABA in the hippocampus and significantly reduce or eliminate both seizure frequency and duration. In some embodiments, the subject enters ketosis after being administered the composition of any one of the embodiments of the present disclosure.

[0039] Also provided herein are methods and compositions for long-term and / or durable weight loss in a subject by administering to the subject a formulation such as BL-001 comprising Akkermansia (Akk) and Parabacteroides (Pb) bacteria. In some embodiments, the subject is overweight and / or obese. In some embodiments, the subject has a BMI that is at least about 20, 21, 22, 23, 24, or 25. In some embodiments, the method comprises administering to the subject at least IxlO8colony forming units (CFUs) of Akk bacteria and at least IxlO7CFUs of Pb bacteria is administered to the subject. In some embodiments, the method comprises administering to the subject at least IxlO11CFUs of Akk bacteria and at least IxlO10CFUs of Pb bacteria is administered to the subject. In some embodiments, the weight reduction is significantly maintained in the subject for at least 1, 2, 3, 4, 5, or 6 weeks following treatment. In some embodiments, the weight reduction is significantly maintained in the subject for at least 1, 2, 3, 4, 5, or 6 months following treatment.Terms

[0040] The following definitions are provided to facilitate understanding of the alternatives or alternatives described herein.

[0041] As used herein in the specification, "a" or "an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words"a" or "an" may mean one or more than one. As used herein “another” may mean at least a second or more.

[0042] The term “comprising” as used herein is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0043] As used herein, “about" and “approximately" in reference to a numeric value, including, for example, whole numbers, fractions, and percentages, generally refers to a range of numerical values (e.g., + / - 5 % to 10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result).

[0044] The phrase “pharmaceutically acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. It is preferred that a carrier be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically- acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0045] The term “preventing' is art-recognized, and when used in relation to a condition, such as a local recurrence, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of seizures includes, for example, reducing the number of seizures in a populationof patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the appearance of detectable lesions in a treated population versus an untreated control population, e.g., by a statistically and / or clinically significant amount.

[0046] The term "''prophylactic ” or “therapeutic'" treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0047] The term “subject" refers to a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

[0048] A “therapeutically effective amount" of a compound with respect to the subject method of treatment refers to an amount of the compound(s) in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0049] As used herein, the term “treating" or “treatment" includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject's condition.

[0050] The term “body mass index,” or “BMI” refers to the value as calculated of the weight (kg) / height2(m2) for a given individual or subject. The term “overweight” refers to a subject with a BMI of at least about 25. The term “obese” refers to a subject with a BMI of at least about 30. The term “not overweight” or “healthy weight” refers to a subject with a BMI that is less than about 25.Therapeutic Methods

[0051] The disclosure herein, relates, in part, to the discovery that the ketogenic diet (KD) induces substantial changes in the gut microbiome, and that enriching KD-associated bacteria via probiotic administration, fecal transplant, or selective microbial reconstitution ofthe native microbiome mimics the beneficial effects of the KD. Provided herein are methods and compositions that can replace the KD diet in the treatment overweight individuals. The methods and compositions described herein can be used separately or in conjunction with the KD diet in the treatment or prevention of a condition described herein.

[0052] In certain embodiments, the methods treat or prevent diseases or disorders associated with excess weight in a subject. In some embodiments, the methods comprise administering a composition comprising bacteria of Akkermansia and Parcibacteroides genera.

[0053] The composition may be formulated for oral delivery. In some embodiments, the composition may comprise probiotics. In some embodiments, the compositions disclosed herein are food products. The composition may be in the form of a pill, tablet, minitablet, powder, or capsule. In some embodiments, the subject may be a mammal (e.g., a human). In some embodiments, the composition is self-administered. While it is preferred for a single composition to comprise all the bacteria to be administered, it will be recognized that for any of the various embodiments described herein, the combination of bacteria can similarly be administered in multiple compositions that together comprise the combination of bacteria. For example, aspects of the disclosure further provides kits comprising multiple compositions that together comprises bacteria of Akkermansia genus.

[0054] In some embodiments, the composition is formulated for rectal delivery (e.g., a fecal sample). In some embodiments, the subject undergoes fecal microbiota transplant, wherein the transplant comprises a composition disclosed herein. Fecal microbiota transplantation (FMT), also commonly known as 'fecal bacteriotherapy' represents a therapeutic protocol that allows the reconstitution of colon microbial communities. The process involves the transplantation of fecal bacteria from a healthy individual into a recipient. FMT restores colonic microflora by introducing healthy bacterial flora through infusion of a fecal sample, e.g., by enema, orogastric tube or by mouth in the form of a capsule containing freeze-dried material, obtained from a healthy donor. In some embodiments, the fecal sample is from a fecal bank.

[0055] In some embodiments, the bacterial DNA in subject’s gut microbiota is sequenced. The subject’s gut bacterial DNA may be sequenced prior to administration of the composition. For example, a sample comprising bacterial DNA may be obtained from the subject, and the bacterial DNA is then sequenced for Akkermansia and / or ParabacteroidesDNA, therefore measuring the presence or level of Akkermansia and / or P ar abacter aides in the subject’s gut microbiota. The composition disclosed herein may then be administered to the subject if the level of Akkermansia and / or Parabacteroides is low. In some embodiments, the subject is deemed to have low levels of Akkermansia and / or Parabacteroides if less than about .0001%, less than .001%, less than .01%, less than .02%, less than .03%, less than .04%, less than .05%, less than .06% less than .07%, less than .08%, less than .09%, less than .1%, less than .2%, less than .3% less than .4%, less than .5%, less than .6%, less than .7%, less than .8%, less than .9%, less than 1%, less than 2%, less than 3%, less than 5%, less than 7%, less than 10%, less than 20%, less than 30%, less than 40%, or less than about 50% of the bacteria in the sample is Akkermansia and / or Parabacteroides DNA. Bacterial DNA to be sequenced may be obtained through any means known in the art, including, but not limited to, obtaining a fecal sample from the subject and isolating the bacterial DNA. Bacterial DNA sequencing by any known technique in the art, including, but not limited to, Maxam Gilbert sequencing, Sanger sequencing, shotgun sequencing, bridge PCR, or next generation sequencing methods, such as massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, or nanopore DNA sequencing.

[0056] In some embodiments, the above methods directly act to reduce the amount of pathogenic bacteria in a subject (i.e., in the gastrointestinal tract of the subject). In some embodiments, this includes any such therapy that achieves the same goal of reducing the number of pathogenic organisms, when used in combination with the compositions described herein, would lead to replacement of the pathogenic microflora involved in the diseased state with natural microflora associated with a non-diseased state, or less pathogenic species occupying the same ecological niche as the type causing a disease state. For example, a subject may undergo treatment with antibiotics (e.g., antimicrobial compounds) or a composition comprising antibiotics to target and decrease the prevalence of pathogenic organisms, and subsequently be treated with a composition described herein. The treatment may also comprise an antifungal or anti-viral compound.

[0057] Suitable antimicrobial compounds include capreomycins, including capreomycin IA, capreomycin IB, capreomycin IIA and capreomycin ITB; carbomycins,including carbomycin A; carumonam; cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefbuperazone, cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefime, ceftamet, cefmenoxime, cefmetzole, cefminox, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam, cefoxitin, cefpimizole, cefpiramide, cefpirome, cefprozil, cefroxadine, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephalexin, cephalogycin, cephaloridine, cephalosporin C, cephalothin, cephapirin, cephamycins, such as cephamycin C, cephradine, chlortetracycline; chlarithromycin, clindamycin, clometocillin, clomocycline, cloxacillin, cyclacillin, danofloxacin, demeclocyclin, destomycin A, dicloxacillin, dirithromycin, doxycyclin, epicillin, erythromycin A, ethanbutol, fenbenicillin, flomoxef, florfenicol, floxacillin, flumequine, fortimicin A, fortimicin B, forfomycin, foraltadone, fusidic acid, gentamycin, glyconiazide, guamecycline, hetacillin, idarubicin, imipenem, isepamicin, josamycin, kanamycin, leumycins such as leumycin Al, lincomycin, lomefloxacin, loracarbef, lymecycline, meropenam, metampicillin, methacycline, methicillin, mezlocillin, micronomicin, midecamycins such as midecamycin Al, mikamycin, minocycline, mitomycins such as mitomycin C, moxalactam, mupirocin, nafcillin, netilicin, norcardians such as norcardian A, oleandomycin, oxytetracycline, panipenam, pazufloxacin, penamecillin, penicillins such as penicillin G, penicillin N and penicillin O, penillic acid, pentylpenicillin, peplomycin, phenethicillin, pipacyclin, piperacilin, pirlimycin, pivampicillin, pivcefalexin, porfiromycin, propiallin, quinacillin, ribostamycin, rifabutin, rifamide, rifampin, rifamycin SV, rifapentine, rifaximin, ritipenem, rekitamycin, rolitetracycline, rosaramicin, roxithromycin, sancycline, sisomicin, sparfloxacin, spectinomycin, streptozocin, sulbenicillin, sultamicillin, talampicillin, teicoplanin, temocillin, tetracyclin, thostrepton, tiamulin, ticarcillin, tigemonam, tilmicosin, tobramycin, tropospectromycin, trovafloxacin, tylosin, and vancomycin, and analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms thereof.

[0058] Suitable anti-fungal compounds include ketoconazole, miconazole, fluconazole, clotrimazole, undecylenic acid, sertaconazole, terbinafme, butenafine, clioquinol, haloprogin, nystatin, naftifine, tolnaftate, ciclopirox, amphotericin B, or tea tree oil and analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms thereof.

[0059] Suitable antiviral agents include acyclovir, azidouridine, anismoycin, amantadine, bromovinyldeoxusidine, chlorovinyldeoxusidine, cytarabine, delavirdine, didanosine, deoxynojirimycin, di deoxy cytidine, dideoxyinosine, dideoxynucleoside, desciclovir, deoxyacyclovir, efavirenz, enviroxime, fiacitabine, foscamet, fialuridine, fluorothymidine, floxuridine, ganciclovir, hypericin, idoxuridine, interferon, interleukin, isethionate, nevirapine, pentamidine, ribavirin, rimantadine, stavudine, sargramostin, suramin, trichosanthin, tribromothymidine, trichlorothymidine, trifluorothymidine, trisodium phosphomonoformate, vidarabine, zidoviridine, zalcitabine and 3-azido-3-deoxythymidine and analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms thereof.

[0060] Other suitable antiviral agents include 2',3'-dideoxyadenosine (ddA), 2', 3'- di deoxy guanosine (ddG), 2', 3 '-dideoxy cytidine (ddC), 2',3'-dideoxythymidine (ddT), 2'3'- dideoxy-dideoxythymidine (d4T), 2'-deoxy-3 '-thia-cytosine (3TC or lamivudime), 2', 3'- dideoxy-2'-fluoroadenosine, 2',3'-dideoxy-2'-fluoroinosine, 2',3'-dideoxy-2'-fluorothymidine, 2',3'-dideoxy-2'-fluorocytosine, 2'3'-dideoxy-2',3'-didehydro-2'-fluorothymidine (Fd4T), 2'3'- dideoxy-2'-beta-fluoroadenosine (F-ddA), 2'3'-dideoxy-2'-beta-fluoro-inosine (F-ddI), and 2',3'-dideoxy-2'-beta-flurocytosine (F-ddC). In some embodiments, the antiviral agent is selected from trisodium phosphomonoformate, ganciclovir, trifluorothymidine, acyclovir, 3'- azido-3 '-thymidine (AZT), di deoxyinosine (ddl), and idoxuridine and analogs, derivatives, pharmaceutically acceptable salts, esters, prodrugs, and protected forms thereof.Compositions

[0061] In some aspects, the disclosure relates to a composition (e.g., a food product or a pharmaceutical composition) comprising bacteria of Akkermansia and Parabacteroides genera. The composition may comprise a pharmaceutically acceptable carrier. The composition may comprise probiotics. The pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including orally, bucally, sublingually, parenterally, and rectally, as by powders, ointments, drops, liquids, gels, tablets, minitablets, capsules, pills, or creams. In certain embodiments, the pharmaceutical compositions are delivered generally (e.g., via oral administration). In certain other embodiments, the compositions disclosed herein are delivered rectally.

[0062] In certain embodiments, the disclosure provides kits comprising multiple compositions that together comprises bacteria of Akkermansia genus (e.g., Akkermansia muciniphila) and Parabacteroides genus. The composition may comprise any species of Parabacteroides, including, but not limited to, P. chartae, P. chinchillas, P. distasonis, P. faecis, P. goldsteinii, P. gordonii, P. johnsonii, or P. merdae. In some embodiments, at least about 1%, at least about 5% at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, , at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, of the bacteria in the composition are Parabacteroides bacteria. The bacteria of Akkermansia in the composition may comprise Akkermansia muciniphila. In some embodiments, the compositions disclosed herein may comprise at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, , at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% Akkermansia bacteria.

[0063] In some embodiments, the composition comprises a 6:1, 6:2, 6:3, 6:4, 6:5, 6:6, 1 :6, 2:6, 3:6, 4:6, or 6:6 ratio of Akkermansia to Parabacteroides bacteria. In some embodiments, the composition comprises a 6:2 ratio of Akkermansia to Parabacteroides bacteria. In some embodiments, the composition comprises a 1 : 1, 2:1, 3: 1, 6:1, or 10: 1 ratio of Akkermansia to Parabacteroides bacteria. In some embodiments, the Akkermansia bacteria is present in the composition at about IxlO5, 5xl05, IxlO6, 5xl06, IxlO7, 5xl07, IxlO8, 5xl08, IxlO9, 5xl09, IxlO10, 5xl010, IxlO11, 5xl0n, IxlO12, 5xl012, IxlO13, 5xl013, IxlO14, 5xl014, IxlO15, 5xl015, IxlO20, about 5xl020, or any abundancy that is between about IxlO5and about 5xlO20, colony forming units (CFUs).

[0064] Compositions described herein may be used for oral administration to the gastrointestinal tract, directed at the objective of introducing the bacteria (e.g., the bacteria disclosed herein) to tissues of the gastrointestinal tract. The formulation for a composition (e.g., a probiotic composition) of the present disclosure may also include other probiotic agents or nutrients which promote spore germination and / or bacterial growth. An exemplary material isa bifidogenic oligosaccharide, which promotes the growth of beneficial probiotic bacteria. In some embodiments, the probiotic bacterial composition is administered with a therapeutically- effective dose of an (preferably, broad spectrum) antibiotic, or an anti-fungal agent. In some embodiments, the compositions described herein are encapsulated into an enterically-coated, time-released capsule or tablet. The enteric coating allows the capsule / tablet to remain intact (i.e., undissolved) as it passes through the gastrointestinal tract, until after a certain time and / or until it reaches a certain part of the GI tract (e.g., the small intestine). The time-released component prevents the “release” of the probiotic bacterial strain in the compositions described herein for a pre-determined time period.

[0065] The composition may be a food product, such as, but not limited to, a dairy product. The dairy product may be cultured or a non-cultured (e.g., milk) dairy product. Nonlimiting examples of cultured dairy products include yogurt, cottage cheese, sour cream, kefir, buttermilk, etc. Dairy products also often contain various specialty dairy ingredients, e g. whey, non-fat dry milk, whey protein concentrate solids, etc. The dairy product may be processed in any way known in the art to achieve desirable qualities such as flavor, thickening power, nutrition, specific microorganisms and other properties such as mold growth control. The compositions of the present disclosure may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins, or minerals.

[0066] In some embodiments, the compositions of the present disclosure are combined with a carrier (e g., a pharmaceutically acceptable carrier) which is physiologically compatible with the gastrointestinal tissue of the subject(s) to which it is administered. Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule or powdered form; or the carrier can be comprised of liquid or gel-based materials for formulations into liquid or gel forms. The specific type of carrier, as well as the final formulation depends, in part, upon the selected route(s) of administration. The therapeutic composition of the present disclosure may also include a variety of carriers and / or binders. In some embodiments, the carrier is micro-crystalline cellulose (MCC) added in an amount sufficient to complete the one gram dosage total weight. Carriers can be solid-based dry materials for formulations in tablet, capsule or powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes of administration. Typical carriers for dry formulations include, but are not limited to: trehalose, malto-dextrin, rice flour,microcrystalline cellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and like carriers. Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like). Preferably, water-based carriers possess a neutral pH value (i.e., pH 7.0). Other carriers or agents for administering the compositions described herein are known in the art, e.g., in U.S. Patent No. 6,461,607.

[0067] In some embodiments, the compositions of the present disclosure comprise one or more fillers. In some embodiments, the filler comprises any 1, 2, or 3 of: mannitol, magnesium stearate, and silica. In some embodiments, the concentration of filler is adjusted based on the dosage and starting CFUs of the composition. In some embodiments, the filler is present in the composition at about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 0.75%, about 1%, about 2%, about 3%, about 4%, about 5%, about 7.5%, about 10%, or any value that is between about 0.01% and about 10% of the total composition.

[0068] In some embodiments, the composition further comprises other bacteria or microorganisms known to colonize the gastrointestinal tract. For example, the composition may comprise species belonging to the Firmicutes phylum, the Proteobacteria phylum, the Teneri cutes phylum, the Actinobacteria phylum, or a combination thereof. Examples of additional bacteria and microorganisms that may be included in the subject compositions include, but are not limited to, Saccharomyces, Bacteroides, Eubacterium, Clostridium, Lactobacillus, Fusobacterium, Propionibacterium, Streptococcus, Enteroccus, Lactococcus and Staphylococcus, Peptostreptococcus. In certain embodiments, the composition is substantially free of bacteria that increase the risk of seizures or otherwise detract from the effect of a ketogenic diet. Such bacteria include Bifidobacterium bacteria. Thus, in some embodiments, the composition is substantially free of Bacteroides bacteria. A composition is substantially free of a bacterial type if that type makes up less than 10% of the bacteria in a composition, preferably less than 5%, even more preferably less than 1%, most preferably less than 0.5%, or even 0% of the bacteria in the composition.

[0069] In some embodiments, the composition comprises a fecal sample comprising at least one species of Akkermansia and at least one species of P ar abacter aides . In some embodiments, the fecal sample is from a fecal bank. In some embodiments, the compositions may be added to a fecal sample prior to administration to the subject.

[0070] In some embodiments, provided herein are methods of treating or preventing a condition, such as seizures, by administering a composition (e.g., a fecal sample) that is enriched for at least one species of Akkermcmsia and at least one species of P ar abacter aides to the subject. The fecal sample is enriched if at least about .01%, at least .02%, at least .03%, at least .04%, at least .05%, at least .06%, at least .07%, at least .08%, at least .09%, at least 0. 1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, or at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least about 50% of the bacteria in the fecal sample is Akkermansia. In some embodiments, fecal sample is enriched if at least about .01%, at least .02%, at least .03%, at least .04%, at least .05%, at least .06%, at least .07%, at least .08%, at least .09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, or at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least about 50% of the bacteria in the fecal sample is and Parabacteroides. In some embodiments, the fecal sample is from a fecal bank. In some embodiments, the fecal sample is from a donor.

[0071] The composition may further comprise a nutrient. In some embodiments, the nutrient aids in the growth of bacteria (e.g., bacteria disclosed herein). In some embodiments, the nutrient is a lipid (e.g., lineoleic acid, stearic acid, or palmitic acid). In some embodiments, the nutrient may be conjointly administered with a composition disclosed herein. As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different agents (e g., a composition disclosed herein and a nutrient disclosed herein) such that the second agent is administered while the previously administered agent is still effective in the body. For example, the compositions disclosed herein and the nutrients disclosed herein can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially.

[0072] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0073] The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0074] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and / or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0075] It will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.EXEMPLIFICATION

[0076] While the present disclosure has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the present disclosure.Example 1 : Materials and MethodsBacterial Treatment

[0077] A. muciniphila, P. merdae and P. distasonis were freshly cultured in anaerobic conditions, and then washed, pelleted and re-suspended at 5 x 109cfu / ml in prereduced PBS. A. muciniphila with Parabacteroides spp. were prepared at a 2: 1 :1 ratio. For heat-killing, bacteria were placed at 95°C for 10 min.Statistical Analysis

[0078] Statistical analysis was performed using SAS software. Analyses of continuous outcomes, e.g., weight change, were assessed using Mixed effect Models with Repeated Measures (MMRM) with SAS PROC MIXED. MMRM allows for fixed effects for treatment, visits, as well as any potential covariates such as BMI Status, as well as exploring potential interactions between these terms. Binary outcomes were assessed using Barnard’s 2x2 unconditional exact test for the risk (proportion) difference with SAS PROC FREQ with the EXACT BARNARD options.Example 2: Bacterial Effect in Phase 1 Fluman Studies

[0079] In a Phase 1 clinical study of healthy volunteers, the compound BL-001, an orally-delivered Live Biotherapeutic Product (LBP), demonstrated a favorable safety, tolerability, and strain kinetics profile.

[0080] The Phase 1 study of BL-001 was a randomized, double-blind, placebo- controlled, single-center, multiple ascending dose study in healthy volunteers (NCT05818306). The primary objective of the study was to investigate the safety and tolerability of BL-001 in healthy volunteers for 28 consecutive days. In the study, BL-001 or placebo was administered to 32 healthy adult participants (BL-001 n=24; placebo n =8) across four dose cohorts. The exact dosage range given to each subjects is as shown in Figure 1. The colony forming units for each bacteria ranged from 107, to 1011.

[0081] The 32 human subjects were given a daily treatment of the BL-001 composition at various dosages in pill form for four weeks, and then allowed a two week follow-up period. The subjects were screened over a month for safety and tolerability of the BL-001 compositions across dosage, as well as the intestinal strain kinetics of the microbiota with BL-001 component strains. Clinicians further monitored the changes in the intestinal microbiota, the subject’s well-being, and any metabolic changes, as well as performed a subjective evaluation of the product intake and the subject’s satisfaction.

[0082] All subjects completed the trial. BL-001 was demonstrated to have a favorable safety and tolerability profile in healthy volunteers that supported dosing up to 10- fold higher than the efficacious dose level in animal models. Furthermore, dose-dependent strain kinetics of BL-001 were observed.

[0083] BL-001 demonstrated a favorable safety profde and was well tolerated with no Serious Adverse Events (SAEs) across all four dose cohorts. The list of total reported adverse effects are as listed in the below Table 1.Table 1: List of Adverse Effects in Human Subjects

[0084] From the safety data listed in Table 1, it was concluded that the four doses of BL-001 (1 x 108, 1 x 109, 1 x IO10, and 1 x 1011CFUs of Akk bacteria, and 5 x 107, 5 x 108, 5 x 109, and 5 x IO10CFUs of Pb bacteria), administered under fasting conditions for 28 days to four cohorts of 8 healthy volunteers (6 on active, 2 on placebo), were safe and well tolerated. Overall, there were 28 Treatment Emergent Adverse Events (TEAEs) in the active and 5 in the placebo treated subjects, of which only 14 were deemed treatment related. Nine of 24 (37%) of BL-001 treated subjects experience a TEAE compared to two of 8 (25%) for the placebo. All TEAEs were transient. Five of the 24 treated subjects had TEAE’ s deemed related and only 1 / 24 was related and moderate (fatigue / tiredness). All adverse effects (AEs) were mild (29) or moderate (4) in severity and did not require any clinical intervention. No Serious AdverseEvents or AEs leading to discontinuation of treatment were recorded. Gastrointestinal disorders (7 AEs in 4 subjects) and headache (3 episodes in 2 subjects) were the most frequent AEs. The mean changes in ECG parameters from baseline were not considered clinically significant in any treatment cohort.

[0085] Strain kinetics were also monitored in subjects, the results of which are depicted in Figure 2. It was found that the BL-001 composition of strains rapidly and durably engrafted in the human subjects. The BL-001 strains were observed to rapidly and consistently engraft in treated subjects, with abundance increasing in a dose-dependent manner. At least one BL-001 strain or closely related strain was not present in all subjects at baseline, while dosing enabled BL-001 strains to appear and in some cases displace native conspecifics. No other strains showed significant kinetic increases from baseline in treated vs placebo subjects. The overall microbiome community composition was highly subject-specific and recalcitrant to large changes from BL-001 dosing. The exact quantification of strain density in subjects over time is as plotted in Figures 3A-3B. In all, the highest dose tested, with 1011CFU of each bacteria, was well tolerated (Figure 4).

[0086] Favorable strain kinetics were observed with BL-001 component strains increasing in a dose dependent manner. All treatment-related Adverse Events (AEs) were mild with the exception of one subject in the highest dose who experienced moderate fatigue that resolved without intervention and they continued on treatment . In the highest dose cohort of BL-001, the most common treatment-related adverse event (AE) was decreased appetite (3 / 6 subjects) with no other treatment-related AEs occurring in more than a single individual. All treatment-related AEs were transient, with the exception of decreased appetite which persisted until the follow-up period for two subjects, and all treatment-related AEs resolved without intervention. No AEs led to study drug withdrawal and all participants completed the study. There were no clinically significant ECG abnormalities reported in any individual across all treatment cohorts.

[0087] There were also high ketone concentrations detected in the urine of humans, consistent with subjects entering ketosis. The elevated urine ketones were present in subjects at the three highest dose levels (1 / 6 subjects in Cohort 1; 1 / 6 subjects in Cohort 3; 2 / 6 subjects in Cohort 4; none in placebo groups). The urinary ketones were measured over multiple timepoints for subjects in Cohorts 3 and 4, and consistently showed elevated levels compared to the placebo group.Example 3: Weight Loss and Ketogenic / Metabolic Changes in Humans

[0088] In this placebo-controlled, healthy volunteer Phase 1 trial, BL-001 was tested across four dose groups (6 subjects each) and one placebo group (8 subjects). The dosage cohort groups are as shown in Figure 1. The subjects and their initial respective BMI levels were as shown in the below Table 2, wherein “not overweight” is classified as any BMI below 25, and “overweight” is classified as a BMI of at least 25.Table 2: List of Human Subjects and their Weight Groups

[0089] Weight loss over time was indicated as a reduction of at least 1 kg from baseline. A total ketogenic change was indicated by either the presence of urinary ketones atany time during treatment, or as a reported TEAE of decreased appetite. A metabolic change (MC) score was reported as an average of two scores: the K score and the A score. The A score was either 0.5 (if a subject experienced decreased appetite for 24 hours or less) or 1.0 (if a subject experienced decreased appetite for more than 24 hours). The K score was given 1 point for each “+” on a urinary ketone test observed during the treatment period. If they have two time points with athen that is 2, and similarly, if they have one time point with athat is also 2. The K score then is the number of +’s that an individual has divided by 4 (as that is the maximum observed score in this dataset which is one individual who had two “++” scores. The metabolic change score is then calculated as:(A score + K score) / 2 , resulting in a number between 0 and 1.

[0090] The highest dose group (Cohort 4) had 66% (N=4) of subjects showing indicators of metabolic change (Figure 5). Three of the six subjects reported decreased appetite, and two had urinary ketones detected during treatment. One subject exhibited both effects. Notably, all three (100%) of the overweight subjects in this group (BMI >= 25; N=3 / 6) exhibited at least one indicator of metabolic change (Figure 5). In contrast, no placebo subjects showed any such effects during the treatment period (Figure 5). Further, across all treated subjects, 5 out of the 6 subjects with a positive metabolic change experienced weight loss, and no one else in the study, whether on placebo or BL-001, experienced any weight loss.

[0091] Subjects that were reported to have weight loss and / or a ketogenic change were also reported to have adverse effects (AEs), including headache, tiredness, drowsiness, or weakness. Similarly, most GI related AEs also occurred in this group, such as flatulence, loose stools, nausea, and vomiting. One individual reported acid reflux.

[0092] A non-zero MC score was observed primarily in subjects with a BMI of 25 or greater (Figure 5). Of those, the average value of the MC score increased with the higher dosage (Figure 6), suggesting that higher levels of BL-001 correlated with decreased appetite and / or increased urinary ketones, mainly in overweight individuals. There was also a clear trend of weight loss across these overweight individuals, which was more significant at higher dosages of BL-001 (Figure 7).Example 4: Weight Change Analysis using Mixed Model Repeated Measures (MMRM)

[0093] Statistical analysis was conducted on the results from Example 4 using Mixed Model Repeated Measures (MMRM). The results are as shown in the below Tables 3- 6. Analysis was also conducted by plotting percentage weight change as pooled by treatment and BMI status (Figure 8), or treatment and metabolic change (Figure 9).Table 3: Type 3 Tests of Fixed Effects for Pooled BL-001 Treatment vs Placebo (dosegroupc) and by BMI StatusTable 4: Differences of Least Squares Means for Pooled BL-001 Treatment vs Placebo and byBMI Status

[0094] Percent weight change was significantly different in overweight vs healthy weight treated individuals (p=0.0002) (Table 4). Percent weight change as a continuous outcome comparing BL-001 to placebo is statistically significant when BMI Status is in the analysis model (the treatment effect, i.e., treated vs placebo, is p=0.0424 and the effect of BMI Status, i.e., Healthy vs Overweight, is p=0.010) (Table 3).

[0095] The BMI effect is driven by a change in how Overweight vs Healthy Weight individuals responded to BL-001 treatment (p=0.0002, LS mean change >2% body weight change in 28 days) and not within the Placebo group (Table 4).

[0096] Given the observation that the BMI Status significantly impacted the BL- 001 treated group but not placebo, the placebo group could be pooled to understand the mean differences better and to account for small numbers, as shown in the below Tables 5-6.Table 5: Type 3 Tests of Fixed Effects for Pooled BL-001 Treated Overweight vs Treated Healthy Weight vs PlaceboTable 6: Differences of Least Squares Means for Pooled BL-001 Treated Overweight vs Treated Healthy Weight vs Placebo

[0097] In this analysis, there are three groups: BL-001 Treated Overweight, BL- 001 Treated Healthy Weight, and Placebo. Percent weight change between BL-001 Treated Overweight vs Healthy Weight individuals was still highly statistically significant (p=0.0003, LS mean change >2% body weight change). The percent weight change of the BL-001 Treated vs All Placebo Subjects was also highly statistically significant (p=0.0007, LS mean change >2% body weight change). It was found that the BL-001 Treated individuals who started with a Healthy Weight did not have a significant percent weight change as compared to Placebo (p=0.8422).Example 5: Weight Change Analysis using Barnard’s Binary Outcome

[0098] Next, metabolic change was evaluated as a binary outcome. Overall weight loss (not accounting for the subject’s initial BMI) was insignificant, as shown in the below Tables 7-8.Table 7: Statistic Analysis for Overall Weight LossTable 8: Barnard’s 2x2 Analysis for Overall Weight Loss

[0099] Weight loss analysis by BMI status was highly statistically significant, with a p-value of 0.0076 using Barnard's Test within the BL-001 treated group. Analysis performed was as shown in the below Tables 9-10.Table 9: Statistic Analysis for Weight Loss by BMI GroupTable 10: Barnard’s 2x2 Analysis for Weight Loss by BMI Group

[0100] Similarly, analyzing weight loss (as a binary) in Dose 4 vs placebo was found to be significant at p = 0.0246. Analysis performed was as shown in the below Tables 11-12.Table 11 : Statistic Analysis for Weight Loss in High Dose vs PlaceboTable 12: Barnard’s 2x2 Analysis for Weight Loss in High Dose vs Placebo

[0101] Overall, it was found that treatment with BL-001 resulted in weight loss in individuals who had an BMI of at least 25. This effect was observed in greater levels with an increased dosage of BL-001.Example 6: Maintenance of Weight Change after completion of Dosing

[0102] The weight of test subjects was monitored after treatment concluded. The percent weight loss between the end of treatment (week 4) and the follow-up visit 2 weeks later (week 6) for subjects who demonstrated weight loss during the study is as shown in the below Table 13. Week 6 measurements represent a two- week washout period following the last-dose.Table 13: Percentage Weight Loss from Week 4 to Week 6 in Subjects Who Demonstrated Weight Loss During Treatment.

[0103] Of the 5 subjects that lost weight during the 4 week treatment period, 2 maintained their weight loss and even continued to lose more weight (these were the two overweight Dose 4 subjects), 1 regained a small part, but not all weight (the overweight Dose 3 subject), 1 regained a little more but not all of their weight (Dose 4 Healthy weight), and 1 regained all of their weight (Overweight Dose 1 subject).

[0104] In conclusion, 100% of the overweight subjects in Dose 4 that lost weight (N=2) maintained this weight loss and even continued to lose more weight after stopping treatment. The durability of weight loss was dose-dependent and strongest within the overweight group. Further, the three higher dose subjects who were overweight and lost weight all lost at least 3% of their body weight by the end of the follow-up period (2 weeks after having treatment).Example 7: Gastrointestinal Hormone Levels after completion of Dosing

[0105] The concentration of gastrointestinal hormones present in the plasma of test subjects was assessed through a Mesoscale after 28 days of treatment with either BL-001 or a placebo. The change in concentration compared to baseline was quantified and compared across groups of placebo, those that responded to treatment (i.e. had weight loss), and thosethat did not respond to treatment (i.e. had no significant weight loss). Subjects that lost weight following BL-001 treatment showed a significant increase in GLP1, leptin L, and PYY compared to subjects that did not lose weight (Figures 10A, 11A, and 12A).

[0106] The change in concentration compared to baseline was also quantified and compared across groups of placebo, those that were of healthy weight, and those that were overweight. Subjects that were overweight also showed a significant increase in GLP1, leptin L, and PYY compared to subjects of healthy weight, following treatment (Figures 10B, 11B, and 12B).Example 8: Metabolic Hallmarks after completion of Dosing

[0107] The concentration of metabolites present in the plasma of test subjects was assessed over 42 days of treatment with either BL-001 or a placebo. The log-transformed change in concentration compared to baseline was quantified and compared across groups of placebo, those that responded to treatment (i.e. had weight loss), and those that did not respond to treatment (i.e. had no significant weight loss). Subjects that lost weight following BL-001 treatment showed a significant elevation in the products of the fatty acid oxidation pathway, acetoacetate and beta- hydroxybutyrate (Figures 13A-13C). These signals in the absence of a ketogenic diet suggest that BL-001 treatment induced a similar metabolic shift towards increased fatty acid catabolism.

[0108] The log-transformed change in concentration of metabolites in the transsulfuration pathway were quantified and compared across groups of placebo, those that responded to treatment (i.e. had weight loss), and those that did not respond to treatment (i.e. had no significant weight loss). Subjects that lost weight following BL-001 treatment showed a significant increase in concentration of a-ketobutyrate and ascorbic acid 3 -sulfate, and a decrease in concentration of cysteine s-sulfate (Figures 14A-14C). Increased alphaketobutyrate is a biomarker of KD-induced changes, as it is a byproduct in the production of cysteine in the transsulfuration pathway (Figure 14D). The increased production of cysteine may lead to downstream increases in glutathione (GSH); the KD has been shown to upregulate GSH2. An increased GSH may explain an increased conversion of ascorbic acid to ascorbic acid 3-sulfate. Increased GSH may also reduce the availability of sulfites, leading to the observed decrease in circulating cysteine s-sulphate, a potent NMDA receptor agonist.

[0109] The log-transformed change in concentration of metabolites in glutamine synthesis and degradation were quantified and compared across groups of placebo, those that responded to treatment (i.e. had weight loss), and those that did not respond to treatment (i.e. had no significant weight loss). Subjects that lost weight following BL-001 treatment showed a significant decrease in a-ketoglutaramate (Figure 15 A). Alpha-ketoglutaramate (KGM) is a key mediator in glutamine synthesis and degradation (Figure 15B). Formed by transamidation of glutamine, the subsequent hydrolysis of KGM to alpha-ketoglutarate provides an anaploretic input to the tricarboxylic acid (TCA) cycle. A decrease in KGM is consistent with reduced glutamine transamination and increased use of anaploresis to feed the TCA cycle during ketosis. This also aligns with the KD’s tendency to dampen excitatory tone and favor GABAergic stability.

[0110] The log-transformed change in concentration of metabolites involved in cholesterol and fatty acid synthesis were quantified and compared across groups of placebo, those that responded to treatment (i.e. had weight loss), and those that did not respond to treatment (i.e. had no significant weight loss). Subjects that lost weight following BL-001 treatment showed a significant increase in levels of butyrylcarnitine (Figure 16A), and a decrease in isobutyrylcarnitine (Figure 16B). A drop in isobutyryl carnitine is related to reduced branched-chain amino acid breakdown and a rise in butyrylcarnitine is related to increased fatty acid oxidation (Figure 16C). Together, they signal the metabolic reprogramming that BL-001 is designed to create — mimicking the ketogenic diet’s seizure- protective energy state without carbohydrate restriction.[OHl] The log-transformed change in concentration of metabolites involved in glycosylation were quantified and compared across groups of placebo, those that responded to treatment (i.e. had weight loss), and those that did not respond to treatment (i.e. had no significant weight loss). Subjects that lost weight following BL-001 treatment showed a significant increase in levels of N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) (Figure 17). GlcNAc and GalNAc are involved in microbial and host glycosylation processes, especially in gut epithelial cell surface glycans. While the de novo production of amino sugars from glucose is reduced, the ketogenic diet may enhance the salvage pathways for GlcNAc and GalNAc. Studies show that the levels of these compounds can increase, possibly due to altered cellular recycling and utilization. The KD is known to increase BL-001strains A. muciniphila and P. merdae, both of which are mucin degraders capably of cleaving GLcNAc and GalNAc, explaining the increase in levels.Incorporation by Reference

[0112] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0113] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.Equivalents

[0114] While specific example embodiments have been discussed, the above specification is illustrative and not restrictive. Variations of any embodiment of the present disclosure will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the present disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

[0115] With respect to the use of plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0116] It will be understood by those of skill within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” theterm “includes” should be interpreted as “includes but is not limited to,” etc.). Tt will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0117] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is alsothereby described in terms of any individual member or subgroup of members of the Markush group.

[0118] Any of the features of an embodiment of the first through second aspects is applicable to all aspects and embodiments identified herein. Moreover, any of the features of an embodiment of the first through third aspects is independently combinable, partly or wholly with other embodiments described herein in any way, e.g., one, two, or three or more embodiments may be combinable in whole or in part. Further, any of the features of an embodiment of the first through third aspects may be made optional to other aspects or embodiments.

Claims

WHAT TS CLAIMED:

1. A method for reducing weight and / or preventing weight gain in a subject, the method comprising administering to the subject at least IxlO8colony forming units (CFUs) of Akkermansia (Akk) bacteria and at least 1x107CFUs of Parabacteroides (Pb) bacteria, wherein the subject has a BMI that is at least about 20.

2. The method of claim 1, wherein the bacteria is administered to the subject orally.

3. The method of any one of the preceding claims, wherein the bacteria is administered as a liquid, in a pill, in a tablet or minitablet, in a capsule, or as part of a food product.

4. The method of any one of the preceding claims, wherein the subject is a human subject.

5. The method of any one of the preceding claims, wherein the Akk bacteria comprises Akkermansia muciniphda.

6. The method of any one of the preceding claims, wherein the Pb bacteria comprises Parabacteroides merdae and / or Parabacteroides johnsonii.

7. The method of any one of the preceding claims, wherein the Akk and Pb bacteria are administered to the subject at least daily.

8. The method of any one of the preceding claims, wherein the bacteria is administered to the subject at a ratio of at least 1 : 1, 2:1, 3: 1, 6:1, or 10: 1 Akkermansia to Parabacteroides bacteria.

9. The method of any one of the preceding claims, wherein the subject is overweight and / or obese.

10. The method of any one of the preceding claims, wherein the subject has a BMI that is at least about 30.

11. The method of any one of the preceding claims, the method comprising administering to the subject at least IxlO11CFUs of Akk bacteria and at least IxlO10CFUs of Pb bacteria.

12. A bacterial composition for use in reducing weight and / or preventing weight gain in a subject, comprising an at least IxlO8colony forming units (CFUs) of Akkermansia bacteria and at least IxlO7CFUs of Parabacteroides bacteria.

13. The bacterial composition of claim 12, wherein the composition is formulated for oral delivery.

14. The bacterial composition of claim 12 or 13, wherein the composition is part of a liquid, a pill, a tablet or minitablet, a powder, a capsule, or as part of a food product.

15. The bacterial composition of claim 14, wherein the food product is a yogurt.

16. The bacterial composition of any one of claims 12-15, wherein the composition further comprises a prebiotic.

17. The bacterial composition of any one of claims 12-16, wherein the bacteria is at a ratio of at least 1 : 1, 2: 1, 3: 1, 6: 1, or 10: 1 Akkermansia to Parabacteroides bacteria.

18. The bacterial composition of any one of claims 12-17, wherein the bacteria comprises Akkermansia muciniphda and Parabacteroides merdae.

19. The bacterial composition of any one of claims 12-18, wherein the subject is overweight and / or obese.

20. The bacterial composition of any one of claims 12-19, wherein the subject has a BMI that is at least about 20.

21. The bacterial composition of claim 20, wherein the subject has a BMI that is at least about 30.

22. Use of a bacterial composition comprising Akkermansia bacteria and Parabacteroides bacterial in the preparation of a drug product for reducing weight in an individual.

23. The use of claim 22, wherein the individual has a BMI above 25.

24. The use of claim 22, wherein the individual is obese.

25. Use of a bacterial composition comprising Akkermansia bacteria andParabacteroides bacterial in the preparation of a drug product for inducing ketosis in an individual.

26. The use of claim 25, wherein the individual has a BMI above 25.

27. The use of claim 25, wherein the individual is obese.

28. A method for changing the concentration of a metabolic compound in a subject, the method comprising administering to the subject the bacterial composition of any one of claims 12-21.

29. The method of claim 28, wherein the metabolic compound comprises a metabolic hormone, a metabolite, or any combination thereof.

30. The method of claim 29, wherein the metabolic hormone comprises at least one of: leptin, PYY, GLP1, or any combination thereof.

31. The method of claim 29, wherein the metabolite comprises at least one of: BHB, acetoacetate, a-ketobutyrate, ascorbic acid 3-sulfate, cysteine s-sulfate, a-ketoglutaramate, butyrylcarnitine, isobutyrylcamitine, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), or any combination thereof.

32. The method of claim 29, wherein the metabolic hormone increases in concentration following administration of the bacterial composition to the subject.

33. The method of claim 29, wherein the metabolite increases in concentration following administration of the bacterial composition to the subject.

34. The method of claim 29, wherein the metabolite decreases in concentration following administration of the bacterial composition to the subject.

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