Microbial therapy

Abstract

The present invention relates to compositions comprising microorganisms for use in reducing blood urate concentration. These compositions can be used in the therapeutic and prophylactic treatment of subjects at risk of, or suffering from, elevated blood urate levels. The compositions can be pharmaceutical or nutraceutical compositions for the treatment and prevention of urate and hyperuricemia related diseases such as cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease, chronic kidney disease, gout, insulin resistance, hypertension, dyslipidemia, renal insufficiency, obesity, prediabetes and diabetes.

Description

Technical Field

[0001] This invention relates to compositions comprising a variety of microorganisms for lowering blood urate concentrations and thus for therapeutic and preventative treatment of subjects at risk of or suffering from elevated blood urate levels, and their use in pharmaceutical compositions for the treatment and prevention of urate and hyperuricemia-related diseases. Background Technology

[0002] Urate is the end product of purine nucleoside metabolism. It is secreted by both the kidneys and the intestines. The final step in purine metabolism to urate is a target for many drugs, and this reaction is catalyzed by the enzyme xanthine oxidase. Xanthine oxidase is primarily expressed in the liver, small intestine, and via the gut microbiome. In most mammals, urate is further metabolized to allantoin by the enzyme uricase. Humans do not express the gene for uricase, and the human gut microbiome is responsible for metabolizing urate to allantoin.

[0003] Urate is the monovalent sodium salt of uric acid. Urate has low solubility and forms crystals in serum at a concentration of 6.8 mg / dL (405 μmol / L) (Burns et al., 2012). Chapter 359. Disorders of purine and pyrimidine metabolism. Harrison's Principles of Internal Medicine, 18th New York, NY: McGraw-Hill. Crystals can deposit throughout the body and cause inflammatory reactions, as seen in patients with gout, or kidney damage, as seen in patients with chronic kidney disease.

[0004] An abnormally high concentration of urate or uric acid in the blood, also known as hyperuricemia, is known to be associated with many diseases and conditions, including cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease, chronic kidney disease, gout, insulin resistance, hypertension, dyslipidemia, renal insufficiency, obesity, prediabetes, and diabetes, especially type II diabetes.

[0005] Elevated urate levels may result from increased uric acid production via xanthine oxidase or decreased urate excretion via the intestines, kidneys, or a combination of both. In healthy individuals, the kidneys excrete two-thirds of total urate. The intestines supplement the kidneys and excrete one-third of total urate. Endogenous urate excretion via the intestines provides an opportunity for local treatment of hyperuricemia, thus avoiding systemic adverse effects.

[0006] Several drugs are known to lower serum urate levels. Xanthine oxidase inhibitors (such as allopurinol and febuxostat) limit the formation of urate in the body by inhibiting xanthine oxidase, while uricosuric agents (such as probenecid) increase the elimination of urate through the kidneys. Unfortunately, treatment with xanthine oxidase inhibitors and uricosuric agents is often accompanied by side effects, including rash, nausea, stomach pain, kidney stones, and decreased liver function. Furthermore, these drugs show low efficacy, and only 30-40% of patients treated with allopurinol achieve their treatment goals.

[0007] Other treatment strategies that have been considered include multivitamins, such as vitamins B2, B9, and C, which have been associated with lowering or preventing elevated serum uric acid levels.

[0008] The use of bacteria for uric acid degradation has been considered in some early studies. Nine aerobic bacterial cultures capable of growing on selective media containing uric acid as the sole source of carbon, nitrogen, and energy were identified (Rouf MA and Lomprey RF Jr. 1968. "Degradation of uric acid by certain aerobic bacteria"). J Bacteriol. , September;96(3): 617-22). However, no studies have been conducted on its effect in lowering urate levels.

[0009] Therefore, new treatment strategies with fewer side effects and improved efficacy are needed.

[0010] This invention overcomes the shortcomings of the prior art by addressing the problem of excessive urate production and insufficient excretion.

[0011] The microbial therapy according to the invention can reduce the concentration of urate in the blood by locally treating hyperuricemia through optimizing the metabolism of the human gut microbiome and by optimizing the intestinal host-microbiome interaction. More specifically, the therapy modulates two pathophysiological pathways; (1) by reducing excessive uric acid production by inhibiting xanthine oxidase, and (2) by increasing urate excretion by increasing the degradation of urate. Summary of the Invention

[0012] This invention relates to a composition containing bacterial strains for use in lowering blood urate concentrations.

[0013] In one aspect, the present invention relates to a composition comprising multiple microorganisms for use in a method for treating or preventing elevated serum urate levels, wherein the composition increases the metabolic capacity of uricase oxidase in the gut microbiota and decreases the metabolic capacity of xanthine oxidase.

[0014] In one embodiment, the multiple microorganisms are multiple bacterial strains. In a specific embodiment, the multiple bacterial strains include at least one bacterial strain with xanthine oxidase inhibitory activity and at least one bacterial strain with urate oxidase activity. In some embodiments, the multiple bacterial strains have Generally Recognized As Safe (GRAS) status and / or Qualified Presumption of Safety (QPS) status. In a further embodiment, the multiple bacterial strains are isolated from blood. In some embodiments, the at least one bacterial strain with xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase. In some embodiments, the metabolite is a flavonoid. In a further embodiment, the multiple bacterial strains have a synergistic effect in reducing serum urate levels. In a further embodiment, the multiple bacterial strains have a synergistic effect in reducing serum urate levels.

[0015] In some implementation schemes, multiple bacterial strains are selected from the genus Bifidobacterium (Bifidobacterium). Bifidobacterium ), Bacillus spp. Bacillus ) and Lactobacillus genus ( Lactobacillus In the specific implementation plan, various bacterial strains belonging to the genus Bifidobacterium are selected from the species Bifidobacterium breve (Bifidobacterium breve). Bifidobacterium breve Bifidobacterium longum ( Bifidobacterium longum Bifidobacterium adolescentis ( ) Bifidobacterium adolescentis ) and Bifidobacterium bifidum ( Bifidobacterium bifidum In a further specific implementation, multiple bacterial strains from the genus *Lactobacillus* are selected from the species *Lactobacillus plantarum*. Lactobacillus plantarum Lactobacillus rhamnosus ( Lactobacillus rhamnosus Lactobacillus bulgaricus ( Lactobacillus bulgaricus ), Lactobacillus fermentum ( Lactobacillus fermentum ) and Lactobacillus casei ( Lactobacillus casei In a further specific implementation, multiple bacterial strains from the genus Bacillus are selected from the species Bacillus subtilis (…). Bacillus subtilis ).

[0016] In another embodiment, the multiple microorganisms are multiple fungal strains. In some embodiments, the multiple fungal strains include at least one fungal strain with xanthine oxidase inhibitory activity and at least one fungal strain with uric acid oxidase activity. Preferably, the at least one fungal strain with xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase. In some embodiments, the metabolite is a phenolic compound and its oxidized derivative. In a more specific embodiment, the multiple fungal strains are selected from the species *Aspergillus niger* (…). Aspergillus niger ) and Darjeeling thymol ( M. darjeelingensis ).

[0017] In another embodiment, the multiple microorganisms are multiple yeast strains. In some embodiments, the multiple yeast strains include at least one yeast strain with xanthine oxidase inhibitory activity and at least one yeast strain with uric acid oxidase activity. Preferably, the at least one yeast strain with xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase. In some embodiments, the metabolite is a phenolic compound and its oxidized derivative.

[0018] In another embodiment, the plurality of microorganisms includes any combination of at least one bacterial strain and / or at least one fungal strain and / or at least one yeast strain. In a specific embodiment, at least one of the bacterial strain and / or yeast strain and / or fungal strain exhibits xanthine oxidase inhibitory activity, and at least one of the bacterial strain and / or yeast strain and / or fungal strain exhibits uric acid oxidase activity. Preferably, at least one of the bacterial strain and / or yeast strain and / or fungal strain expresses a metabolite that inhibits xanthine oxidase. In cases where the plurality of microorganisms includes at least one bacterial strain that expresses a metabolite that inhibits xanthine oxidase, said metabolite is, in some embodiments, a flavonoid compound.

[0019] In some embodiments, the composition is co-formulated and / or co-administered with one or more additives, including micronutrients, amino acids, prebiotics, foods, food additives, dietary supplements, or medical foods.

[0020] In some embodiments, the composition is formulated and / or co-administered with one or more micronutrients. In some embodiments, the composition is formulated and / or co-administered with one or more trace elements. In some embodiments, the composition is formulated and / or co-administered with one or more amino acids. In some embodiments, the composition is formulated and / or co-administered with one or more prebiotics.

[0021] In some embodiments, the composition is co-formulated and / or co-administered in combination with further agents such as therapeutic agents and / or therapies.

[0022] In another aspect, the present invention relates to compositions according to the invention in the form of pharmaceutical compositions.

[0023] In a further aspect, the present invention relates to novel strains of Lactobacillus species deposited in DSMZ as detailed herein, for use in methods of treating or preventing conditions of elevated serum urate. In some embodiments, the strains increase the uricase metabolic capacity of the gut microbiota and decrease the xanthine oxidase metabolic capacity of the gut microbiota.

[0024] In a further implementation, elevated serum urate is selected from conditions such as cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), chronic kidney disease, gout, insulin resistance, hypertension, hyperuricemia, dyslipidemia, renal insufficiency, obesity, prediabetes, and diabetes. Attached Figure Description

[0025] Figure 1 : Figure 1 The fluorescence (measured in fluorescence units using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm) after subjecting the corresponding bacterial strains to the Amplex® Red Xanthine / Xanthine Oxidase Assay Kit (catalog No. A22182, Molecular Probes) assay is shown, as described in detail in Example 2; for detailed descriptions of bacterial strain abbreviations, see Table 1; Bifidobacterium breve strain ATCC 15701 was used as a positive control, and Bifidobacterium animalis (… Bifidobacterium animalis (BB12®) was used as a negative control.

[0026] Figure 2 : Figure 2 The fluorescence (measured in fluorescence units using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm) after subjecting the corresponding bacterial strains to the Amplex® Red Uric Acid / Uric Acid Oxidase Assay Kit (catalog No. A22181, Molecular Probes) is shown as described in detail in Example 3; for detailed descriptions of the abbreviations for the bacterial strains, see Table 1; 20 mM H2O2 solution was used as a positive control and 0 mM uricate oxidase solution was used as a negative control.

[0027] Figure 3 : Figure 3The fluorescence of the bacterial strain *Lactobacillus casei* (BEO #109) grown on media containing 0% and 2% uric acid after undergoing the Amplex® Red Uric Acid / Uric Acid Oxidase Assay Kit (catalog No. A22181, Molecular Probes) is shown (measured in fluorescence units using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm), as described in detail in Example 5.

[0028] Figure 4 : Figure 4 The fluorescence at different time points after subjecting the corresponding bacterial strains to the Amplex® Red Xanthine / Xanthine Oxidase Assay Kit (catalog No. A22182, Molecular Probes) is shown (measured in fluorescence units using excitation at 530 ± 12.5 nm and detection at 590 ± 17.5 nm), as described in detail in Example 6; for detailed descriptions of bacterial strain abbreviations, see Table 1; Bifidobacterium breve strain ATCC 15701 was used as a positive control and Bifidobacterium animalis (BB12®) was used as a negative control.

[0029] Figure 5 : Figure 5 The fluorescence at different time points after subjecting the corresponding bacterial strains to the Amplex® Red Uric Acid / Uric Acid Oxidase Assay Kit (catalog No. A22181, Molecular Probes) is shown (measured in fluorescence units using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm), as described in detail in Example 7; for detailed descriptions of bacterial strain abbreviations, see Table 1; 20 mM H2O2 solution was used as a positive control and 0 mM uricase solution was used as a negative control.

[0030] Figure 6 : Figure 6 The fluorescence at different time points after subjecting the corresponding bacterial strains to the Amplex® Red Uric Acid / Uric Acid Oxidase Assay Kit (catalog No. A22181, Molecular Probes) is shown (measured in fluorescence units using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm), as described in detail in Example 7; for detailed descriptions of bacterial strain abbreviations, see Table 1; 20 mM H2O2 solution was used as a positive control and 0 mM uricase solution was used as a negative control.

[0031] Figure 7 : Figure 7The study showed a statistically significant reduction in serum uric acid in mice treated with single or combined test products. Allopurinol was used as a reference product (x-axis: control (A), allopurinol (B), and test products, namely single strains #104 (C), #110 (D), #227 (E), and strain combinations #110 and #227 (F); y-axis: uric acid (UA) units / ml, where one unit corresponds to 10 µmol / L; the normal physiological range is 40–65 µmol / L).

[0032] Figure 8 : Figure 8 The study showed a statistically significant additive effect of the combination of strains #110 and #227 in reducing serum uric acid in mice compared with the administration of a single strain (x-axis: single strains #110, #227, and the combination of strains #110 and #227; y-axis: uric acid (UA) units / ml, where one unit corresponds to 10 µmol / L; normal physiological range is 40–65 µmol / L).

[0033] Figure 9 : Figure 9 The efficacy of bacterial treatment with the combination of strains #110 and #227 (black solid circles) compared to standard drug treatment with allopurinol (black solid squares) is shown. The group treated with the strain combination #110 / #227 maintained serum urate concentrations within the normal range of 40–65 mmol / L over a 23-hour treatment period, while the allopurinol-treated control group showed large fluctuations in serum urate concentrations outside the normal range (x-axis: time (hours), y-axis: serum urate concentration (µmol / L)). Detailed Implementation

[0034] Unless otherwise specified, the following definitions apply.

[0035] As used in this invention, the term "microorganism" refers to a single-celled organism and includes prokaryotes, such as bacteria, and eukaryotes (such as fungi and yeast). The term "bacteria" includes both Gram-positive and Gram-negative bacteria, and includes, but is not limited to, strains from the genera *Bifidobacterium*, *Bacillus*, and *Lactobacillus*, such as strains from the species *Bifidobacterium breve*, *Bifidobacterium longum*, *Bifidobacterium adolescentis*, *Bifidobacterium bifidum*, *Bacillus subtilis*, *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus bulgaricus*, *Lactobacillus fermentum*, and *Lactobacillus casei*. The selection of one or more suitable microorganisms is within the knowledge of a person skilled in the art.

[0036] The term "strain" refers to the offspring of a pure, isolated culture, as well as subsequent offspring that can be cultured from it without contamination. A strain is a subvariety of a microorganism that is distinguishable from other microorganisms in terms of phenotype and / or genotype.

[0037] As used in this article, “hyperuricemia” is characterized by abnormally high concentrations of urate or uric acid in the blood. Hyperuricemia is known to be associated with many diseases and conditions, including cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), chronic kidney disease, gout, insulin resistance, hypertension, dyslipidemia, renal insufficiency, obesity, prediabetes, and diabetes, particularly type II diabetes.

[0038] The term "micronutrients" includes, for example, vitamins and trace elements. Vitamins are organic substances that the body does not synthesize and are essential for normal metabolism. They are classified as water-soluble or fat-soluble and as having or not having coenzyme functions. Typical vitamins used in this invention include, but are not limited to, vitamin B2, vitamin B9, and vitamin C. Trace elements are metals present in the body in extremely small amounts. They are essential for normal metabolic functions and are generally cofactors of enzymes or essential components in the formation of specific enzyme structures. Typical trace elements used in this invention include, but are not limited to, zinc, manganese, iron, copper, molybdenum, chlorine, nickel, and boron.

[0039] The term "amino acid" refers to any one of the twenty standard amino acids: glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid, and glutamic acid, their single stereoisomers, and their racemic mixtures. The term "amino acid" may also refer to known non-standard amino acids, such as 4-hydroxyproline, ε-N,N,N-trimethyllysine, 3-methylhistidine, 5-hydroxylysine, O-phosphoserine, γ-carboxyglutamic acid, γ-N-acetyllysine, ω-N-methylarginine, N-acetylserine, N,N,N-trimethylalanine, N-formylmethionine, γ-aminobutyric acid, histamine, dopamine, thyroxine, citrulline, ornithine, β-cyanalanine, homocysteine, diazoserine, and S-adenosylmethionine. In some embodiments, the amino acid is glutamic acid, glutamine, lysine, tyrosine, or valine. In some embodiments, the amino acid is arginine and citrulline.

[0040] The term "prebiotic" is used for components that undergo selective fermentation, resulting in specific changes in the composition and / or activity of the gastrointestinal microbiota, thereby conferring one or more health benefits to the host. The term "prebiotic" includes, but is not limited to, fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), resistant starch, pectin, beta-glucan, and xylooligosaccharides.

[0041] The term pharmaceutical composition refers to a dosage form comprising the composition of the present invention and pharmaceutically acceptable excipients.

[0042] The term "food" means any processed, semi-processed, or raw material intended for consumption by mammals, such as animals or humans. It does not include substances intended solely for medicinal purposes. As used herein, "food additive" means additives added to, mixed with, or incorporated into food during food production or for the purpose of processing or storing food, such as water binders, gelling agents, thickeners, antioxidants, dyes, flavor enhancers, acidifiers, and sweeteners (including additives assigned an E (European) number).

[0043] The term "dietary supplement" refers to a dosage form containing the composition of the present invention as well as nutrients or substances having nutritional or physiological effects, intended to supplement a normal diet, such as herbs or other plants; metabolites, extracts.

[0044] The term "medicinal food" refers to a dosage form of food comprising the compositions of the present invention and intended for use under the supervision of a medically trained person for the dietary management of a condition or disease. Medical foods generally meet regulatory requirements, particularly those under the Federal Food, Drug, and Cosmetic Act and those defined by the European Food Safety Authority as "foods for specific medical purposes." Medical foods are generally specifically processed or formulated and intended for use under medical supervision. Medical foods include, but are not limited to, oral rehydrated products, nutritionally incomplete formulas, nutritionally complete formulas, and formulas for metabolic disorders.

[0045] As used herein, the term "co-formulation" refers to combining the compositions of the present invention with one or more additives to form a single pharmaceutical composition. Co-formulation is therefore intended for simultaneous administration.

[0046] As used herein, the term "co-application" refers to the combined application of the composition of the present invention with one or more additives. The term "co-application" refers to both simultaneous application and sequential application (regardless of order).

[0047] The abbreviation "CFU" stands for "colony forming unit," which is commonly used in microbiology to estimate the number of viable cells of a microorganism, such as a bacterial strain, in a given sample. Determining a CFU count generally involves counting at least a portion of the colonies of a microorganism, such as a bacterial strain, on a culture dish after an optional initial dilution, and estimating the total number of viable cells in the given sample based on said count.

[0048] Enzyme metabolic capacity corresponds to the ability to convert at least one substrate of an enzyme into at least one corresponding product, and can be defined, for example, as the number of molecules of at least one substrate converted into at least one corresponding product within a given time period. Enzyme metabolic capacity is particularly affected by the amount of enzyme present, the activity of the present enzyme or isoenzyme, and the presence and / or absence of enzyme activators and / or inhibitors.

[0049] In a first aspect, the present invention relates to a composition comprising multiple microorganisms for use in a method for treating or preventing elevated serum urate levels, wherein the composition increases the metabolic capacity of uricase in the gut microbiota and decreases the metabolic capacity of xanthinecase.

[0050] In a specific embodiment, the composition increases the metabolic capacity of uricase oxidase in the gut microbiota and decreases the metabolic capacity of xanthine oxidase in the gut microbiota.

[0051] In one embodiment, the multiple microorganisms are multiple bacterial strains. In a specific embodiment, the multiple bacterial strains include at least one bacterial strain exhibiting xanthine oxidase inhibitory activity and at least one bacterial strain exhibiting uric acid oxidase activity. Preferably, the at least one bacterial strain exhibiting xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase. In some embodiments, the metabolite is a flavonoid.

[0052] In a further embodiment, the uricase metabolic capacity of the gut microbiota is enhanced in the presence of uric acid. In some embodiments, the enhancement is due to the induction of uricase expression in the presence of uric acid. In a preferred embodiment, the enhancement is 2-10 times, particularly 3-8 times.

[0053] In the presence of uric acid, the enhanced metabolic capacity of the gut microbiota, specifically uricase, is beneficial. This is because it implies that the metabolic capacity of uricase in the absence of uric acid is minimal, thus minimizing undesirable side effects associated with uric acid deficiency, which is related to enhanced safety. Generally, the bacteria used have been granted QPS (Quality Permitted) status by the European Food Safety Authority (EFSA) and / or GRAS (Generally Recognized As Safe) status by the FDA.

[0054] In a more specific implementation, multiple bacterial strains are selected from the genera Bifidobacterium, Bacillus, and Lactobacillus.

[0055] In some preferred embodiments, the compositions of the present invention comprise one or more bacterial strains selected from species Bifidobacterium breve, Bifidobacterium longum, Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum, and one or more intestinal bacteria or other strains of bacteria derived from food sources, including strains of other bacterial species.

[0056] In other preferred embodiments, the bacterial strains from the genus Bifidobacterium are selected from species Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum, particularly species Bifidobacterium breve ATCC 15701.

[0057] In other preferred embodiments, strains of bacteria from the genus *Lactobacillus* are selected from species *Lactobacillus plantarum*, *Lactobacillus rhamnosus*, *Lactobacillus bulgaricus*, *Lactobacillus fermentum*, and *Lactobacillus casei*, preferably strains DSM 33579, DSM 33580, and DSM 33581 deposited on July 14, 2020 at DSMZ (International Depositary Authority: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7 B, 38124 Braunschweig, Germany).

[0058] In other preferred embodiments, various bacterial strains from the genus Bacillus are selected from the species Bacillus subtilis.

[0059] Generally, bacterial strains are isolated from food. They can be in liquid, frozen, or dried form.

[0060] In another embodiment, the multiple microorganisms are multiple fungal strains. In some embodiments, the multiple fungal strains include at least one fungal strain with xanthine oxidase inhibitory activity and at least one fungal strain with uric acid oxidase activity. Preferably, the at least one fungal strain with xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase.

[0061] In another embodiment, the multiple microorganisms are multiple yeast strains. In some embodiments, the multiple yeast strains include at least one yeast strain with xanthine oxidase inhibitory activity and at least one yeast strain with uric acid oxidase activity. Preferably, the at least one yeast strain with xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase.

[0062] In some implementations, the metabolites are phenolic compounds and / or their oxidized derivatives.

[0063] In some embodiments, the multiple microorganisms include a combination of at least one bacterial strain and at least one fungal strain. In some embodiments, the multiple microorganisms include a combination of at least one bacterial strain and at least one yeast strain.

[0064] In some implementations, the metabolites are phenolic compounds and / or their oxidized derivatives.

[0065] In some embodiments, the compositions of the present invention may be in the form of pharmaceutical compositions.

[0066] Suitable dosage forms include, but are not limited to, tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of an active ingredient, soft capsules, hard capsules, gel-capsule tablets, tablets, suppositories, solutions, or packaged powders. Pharmaceutically acceptable excipients suitable for formulating the dosage forms of the present invention include, but are not limited to, disintegrants, diluents, plasticizers, binders, flow aids, lubricants, sweeteners, flavoring agents, anti-caking agents, antimicrobial agents, defoamers, emulsifiers, surfactants, buffers, and colorants, or mixtures thereof. Suitable excipients include, for example, PBS, glycerin, cocoa butter, or polyethylene glycol.

[0067] In some embodiments, the composition is formulated and / or administered in combination with one or more additives, said additives including micronutrients, amino acids, prebiotics, foods, food additives, dietary supplements, or medicinal foods. In some embodiments, the compositions of the present invention, specifically the pharmaceutical compositions of the present invention, may be formulated and / or administered in combination with one or more micronutrients, specifically with one or more vitamins and / or one or more trace elements. In some embodiments, the compositions of the present invention, specifically the pharmaceutical compositions of the present invention, may be formulated and / or administered in combination with one or more vitamins, preferably selected from vitamin B2, vitamin B9, and vitamin C. In some embodiments, the compositions of the present invention, specifically the pharmaceutical compositions of the present invention, may be formulated and / or administered in combination with one or more trace elements, such as zinc, manganese, iron, copper, molybdenum, chlorine, nickel, and boron.

[0068] In some embodiments, the compositions of the present invention, specifically the pharmaceutical compositions of the present invention, may be formulated and / or administered in combination with one or more amino acids.

[0069] In some embodiments, the compositions of the present invention, specifically the pharmaceutical compositions of the present invention, may be formulated and / or administered in combination with one or more prebiotics, preferably selected from fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), resistant starch, pectin, β-glucan, and xylooligosaccharides.

[0070] In some embodiments, the compositions of the present invention may be formulated and / or administered (in combination or alone, sequentially or simultaneously) with further agents such as therapeutic agents. Further agents, such as therapeutic agents, refer to agents used in addition to a variety of microorganisms. Suitable agents that can be used as further therapeutic agents include, but are not limited to, those for use with other microorganisms.

[0071] - Known compounds for treating gout, such as nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, oral corticosteroids, and / or...

[0072] - Urate-lowering medications such as allopurinol, febuxostat, probenecid, pegloticase, benzbromarone, sulfinpyrazone, and / or

[0073] - Compounds used to treat hypertension and chronic kidney disease, such as compounds selected from thiazide diuretics, beta-blockers, angiotensin II receptor blockers, calcium channel blockers, renin inhibitors, or angiotensin-converting enzyme (ACE) inhibitors, and / or

[0074] - Compounds used to treat dyslipidemia, such as statins, fibrates, niacin, bile acid polychelators, and cholesterol absorption inhibitors.

[0075] In other embodiments, the compositions of the present invention may be administered in combination with further therapies.

[0076] The compositions of the present invention can be used in subjects having blood urate concentrations within the normal range and in subjects having blood urate concentrations above the normal range to treat or prevent an increase in the concentration of urate in the blood of the subjects in question.

[0077] In specific embodiments, the compositions of the present invention can be used to treat, prevent, or reduce the risk of elevated urate concentrations in the blood of patients and / or subjects diagnosed with or suffering from cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), chronic kidney disease, gout, insulin resistance, hypertension, hyperuricemia, dyslipidemia, renal insufficiency, obesity, prediabetes, and diabetes.

[0078] In further embodiments, the compositions of the present invention, when formulated and / or administered (in combination or alone, sequentially or simultaneously) with further agents such as therapeutic agents, can be used to treat, prevent or reduce the risk of elevated urate concentrations in the blood of patients and / or subjects diagnosed with or suffering from cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), chronic kidney disease, gout, insulin resistance, hypertension, hyperuricemia, dyslipidemia, renal insufficiency, obesity, prediabetes, and diabetes.

[0079] The composition of the present invention for lowering blood urate concentration can be prepared in 1 x 10⁻⁶ units. 6 Administer at a concentration of CFU / day or higher. A preferred concentration is at least 1 x 10⁻⁶ CFU / day. 9 CFU / day, at least 1 x 10 10 CFU / day, at least 1 x 10 11 CFU / day, at least 1 x 10 12 CFU / day, at least 1 x 10 13 CFU / day, at least 1 x 10 14 CFU / day, at least 1 x 10 15 CFU / day.

[0080] Example

[0081] Materials and methods

[0082] The urate-lowering effect of this invention can be evaluated by measuring the urate concentration in blood, urine, or saliva samples. The analysis can be performed as a home analysis using a test stick (Berkeley Fit saliva test).

[0083] MRS refers to De Man, Rogosa, and Sharpe agar medium. It is used as is, according to the manufacturer's instructions, as is available commercially from Sigma-Aldrich. BHI refers to Brain Heart Infusion medium. It is used as is, according to the manufacturer's instructions, as is available commercially from Sigma-Aldrich. Anaerobic environment generating bag refers to a reagent anaerobic environment generating bag, which is used as is, according to the manufacturer's instructions, as is available commercially from Millipore. Saline refers to an aqueous solution of sodium chloride, and is used as is, as is, available from Sigma-Aldrich.

[0084] Table 1 shows the abbreviations used for each bacterial strain.

[0085] abbreviation Bacterial species Beo #101 Lactobacillus casei Beo #102 Bacillus subtilis Beo #103 Lactobacillus casei Beo #104 Bacillus subtilis Beo #105 Lactobacillus plantarum Beo #106 Lactobacillus plantarum Beo #107 Lactobacillus casei Beo #108 Lactobacillus casei Beo #109 Lactobacillus casei Beo #110 Lactobacillus casei DSM 33579 Beo #111 Lactobacillus casei Beo #112 Lactobacillus casei Beo #113 Lactobacillus casei Beo #115 Lactobacillus plantarum Beo #116 Lactobacillus plantarum Beo #117 Lactobacillus casei Beo #118 Lactobacillus plantarum Beo #119 Lactobacillus plantarum Beo #120 Lactobacillus plantarum Beo #121 Lactobacillus plantarum Beo #122 Lactobacillus plantarum Beo #123 Lactobacillus plantarum Beo #124 Lactobacillus plantarum Beo #125 Lactobacillus plantarum Beo #126 Bacillus subtilis Beo #127 Bacillus subtilis Beo #128 Bacillus subtilis Beo #129 Lactobacillus plantarum Beo #130 Lactobacillus plantarum Beo #207 Bifidobacterium longum Beo #209 Bifidobacterium longum Beo #210 Bifidobacterium longum Beo #215 Bifidobacterium longum Beo #216 Bifidobacterium longum Beo #224 Lactobacillus plantarum DSM 33580 Beo #227 Lactobacillus plantarum DSM 33581 Beo #228 Lactobacillus plantarum Beo #229 Lactobacillus plantarum Beo #230 Lactobacillus plantarum

[0086] Example 1. Culture of bacteria used for screening

[0087] Pre-culture the strain in 1 ml of deep-well plates under the following conditions (see Table 2).

[0088] Table 2

[0089] Lactobacillus species MRS 37 No shaking 20 Bifidobacterium MRS + 0.05% cysteine 37 Anaerobic bottle with anaerobic environment generating bag 72 Bacillus species BHI 37 No shaking 20

[0090] The 5% cysteine ​​solution was freshly prepared, filtered and sterilized, and then added to the culture medium prior to inoculation.

[0091] Glycerin stock solution was prepared by mixing 120 µl of growth culture with 40 µl of 60% sterile glycerol solution in a microtiter plate. The resulting glycerol stock solution was stored at -80°C.

[0092] Example 2. Inhibition of xanthine oxidase

[0093] The ability of bacterial strains to inhibit the catalytic activity of xanthine oxidase was evaluated. Prior to the assay, 1 / 100 volume of the stock glycerol solution (Example 1) was added to a microtiter plate containing an appropriate culture medium (see Overview). Culture conditions (time, temperature, medium, conditions) were selected for different species as described in Table 2. The growth of the bacterial strains was assessed visually before harvesting for fluorescence measurement. After incubation, 100 µl of the appropriate culture medium containing the corresponding species was transferred to a new microtiter plate for the enzyme activity assay.

[0094] Enzyme assay: Amplex® Red Xanthine / Xanthine Oxidase Assay Kit; Catalogue No. A22182; Molecular Probes, used according to manufacturer's instructions. Fluorescence readout was determined after 60 minutes using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. *Bifidobacterium breve* strain ATCC 15701 was used as a positive control, and *Bifidobacterium animalis* (BB12®) was used as a negative control.

[0095] Figure 1 The observed fluorescence indicates that the corresponding bacterial strain inhibited xanthine oxidase activity.

[0096] Example 3. Bacterial uricase activity

[0097] The catalytic activity of uric acid oxidase in bacterial strains was evaluated. Prior to assay, 1 / 100 volume of the stock glycerol solution (Example 1) was added to a microtiter plate containing appropriate culture medium (see Overview). Culture conditions (time, temperature, culture medium, aerobic or anaerobic) depend on the species and can be found in Table 1. Uric acid was dissolved in the culture medium to 2%, and the medium was filtered and sterilized (so that uric acid was immediately present in the medium upon incubation). The growth of the bacterial strain was assessed visually (viscosity level of the culture medium) before harvesting for fluorescence measurement. After incubation, 100 µl of the appropriate culture medium containing the corresponding species was transferred to a new microtiter plate for enzyme activity assay.

[0098] Enzyme assay, Amplex® Red Uric Acid / Uric Acid Oxidase Assay Kit; Catalogue No. A22181; Molecular Probes, used according to the manufacturer's instructions. Fluorescence readout was determined after 30 minutes using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. 20 mM H₂O₂ solution was used as a positive control, and 0 mM uricate oxidase solution was used as a negative control.

[0099] Figure 2 The observed fluorescence indicates enhanced uricase activity in the corresponding bacterial strain.

[0100] Example 4. Bacterial species activity

[0101] As described in Examples 2 and 3, the activity of the strains listed in Table 1 was determined as the percentage of maximum fluorescence from the positive controls (Bifidobacterium breve strain ATCC 15701 was used as a positive control for screening according to Example 2, and 20 mM H2O2 solution was used as a positive control for screening according to Example 3). The strains were then classified according to their species, and the average activity of all strains belonging to each species was calculated. Tables 3 and 4 show the average xanthine oxidase inhibitory activity and average urate oxidase inhibitory activity for each species, respectively, where the average inhibitory activity was classified as low (5-30% of the positive controls), medium (25-75% of the positive controls), and high (50-100% of the positive controls).

[0102] Table 3. Inhibition of xanthine oxidase:

[0103] Species Inhibitory activity Bifidobacterium breve ATCC 15701 Low Bifidobacterium longum middle Lactobacillus plantarum high

[0104] Table 4. Uric acid oxidase activity

[0105] Species Inhibitory activity Lactobacillus casei high Bacillus subtilis middle Lactobacillus plantarum Low

[0106] Example 5. Uric acid-induced uricase activity

[0107] The bacterial strain *Lactobacillus casei* (BEO #109) was cultured under the conditions described in Example 1. Uric acid was dissolved in the culture medium at 0% to 2% and then filtered and sterilized (so that it was immediately present in the medium upon incubation). The growth of the bacterial strain was assessed by visual inspection before harvesting for fluorescence measurement. After incubation, 100 µl was transferred to a new microtiter plate for enzyme activity measurement.

[0108] Enzyme assay (Amplex® Red Uric Acid / Uric Acid Oxidase Assay Kit; Catalogue No. A22181; Molecular Probes) was performed according to the manufacturer's instructions. Fluorescence readout was determined after 60 minutes using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm.

[0109] Figure 3 The observed fluorescence indicates that the addition of uric acid to the growth medium induces uricase activity.

[0110] Example 6. Xanthine oxidase inhibition is associated with microbial growth.

[0111] The experiment was conducted as described in Example 2. Fluorescence readouts were measured every 6 minutes from time 0 to 72 minutes.

[0112] Enzyme assay, Amplex® Red Xanthine / Xanthine Oxidase Assay Kit; Catalogue No. A22182; Molecular Probes, use according to manufacturer's instructions. Fluorescence readout was determined using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. *Bifidobacterium breve* strain ATCC 15701 was used as a positive control, and *Bifidobacterium animalis* (BB12®) was used as a negative control.

[0113] Figure 4 The fluorescence observed at various time points is shown.

[0114] Example 7. Uric acid oxidase activity is related to microbial growth.

[0115] The experiment was conducted as described in Example 3. Fluorescence readouts were measured every 6 minutes from time 0 to 30 minutes.

[0116] Enzyme assay, Amplex® Red Uric Acid / Uric Acid Oxidase Assay Kit; Catalogue No. A22181; Molecular Probes, use according to manufacturer's instructions. Fluorescence readout is determined using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. 20 mM H₂O₂ solution is used as a positive control, and 0 mM uricate oxidase solution is used as a negative control.

[0117] Figure 5 The fluorescence observed at various time points is shown.

[0118] Example 8. Inhibition of xanthine oxidase by supernatant

[0119] The bacterial strain *Lactobacillus plantarum* (BEO #227) was used in the experiment. The experiment was conducted as described in Example 2, except that after culturing the corresponding bacterial strain, the supernatant was separated by centrifugation (3000 rpm, 10 minutes, 4°C). The same volume of whole cells (WC) and supernatant (SUP) was then used in the same assays as in Example 2. Fluorescence was measured after 0, 25, 45, 60, 90, and 110 minutes.

[0120] Enzyme assay, Amplex® Red Xanthine / Xanthine Oxidase Assay Kit; Catalogue No. A22182; Molecular Probes, use according to manufacturer's instructions. Fluorescence readout is determined using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm.

[0121] Figure 6 The fluorescence observed at various time points for whole cells (WC) and supernatant (SUP) is shown.

[0122] Example 9. Mouse model of hyperuricemia

[0123] Mice aged 6-10 weeks were randomly divided into the following groups, with 10 mice in each group. For the test product, 1.0 x 10⁻⁶ μL was used. 8 -10 10 CFU:

[0124] 1) Control group

[0125] 2) Allopurinol treatment group

[0126] 3) Microbial therapy group 1: The test product was bacterial strain BEO #104

[0127] 4) Microbial therapy group 2: The test product was bacterial strain BEO #110

[0128] 5) Microbial therapy group 4: The test product was bacterial strain BEO #227

[0129] 6) Microbial therapy group 6: The test product is a mixture of bacterial strains BEO #110 and BEO #227.

[0130] The control group was fed a normal diet (unrestricted laboratory food and water) and treated with intragastric administration of saline (0.5 ml, sodium chloride solution) once daily. Other groups were fed a high-purine diet (unrestricted laboratory high-purine food and water) and received intraperitoneal injection of potassium oxonate 300 mg / kg once daily. The microbial therapy group received approximately 1.0 × 10⁻⁶ mg / kg intragastric administration daily. 9CFU / day treatment. All tested strains were QPS qualified and were produced and formulated under laboratory conditions.

[0131] Mice were weighed on day -1 to determine the average body weight of the animals in the experiment and the composition of the groups. Groups were mixed in cages to avoid cage effects (except for group 1, which was kept in a separate cage). Disease was induced on day 0 by intraperitoneal injection of oxazine (300 mg / kg).

[0132] Serum was collected weekly and at the end of the experiment. Serum was collected on day 0, 6 hours after administration of the test product; on day 7, 4 hours after administration of the test product; and on day 14, 4 hours after administration of the test product and 23 hours after administration of the test product when the mice were sacrificed. The collected blood was centrifuged, and the plasma was separated and frozen until further analysis. Serum uric acid concentration was analyzed. The strain combination BEO #110+BEO #227 showed a beneficial reducing effect, i.e., complete normalization of serum urate concentration.

[0133] Example 10. Preparation of the bacterial composition

[0134] The cultured bacteria were separated from the used culture medium by centrifugation at 3000 rpm for 10 minutes at 4°C. The harvested bacteria were redissolved in a formulation / low-temperature buffer. The formulation / low-temperature buffer consisted of 25-50 mM buffer and 10-30% sugar. Alternative formulations were tested by including additives such as 1-6% ascorbate, arginine, and 0.3-1.0 mg folate in the formulation buffer.

[0135] Example 11. Inhibition of xanthine oxidase by yeast and fungi

[0136] The ability of fungi and yeast to inhibit the catalytic activity of xanthine oxidase was evaluated. Fungi and yeast were cultured according to conditions recommended by DSMZ prior to the assay. Bacterial strains were assessed by visual inspection for growth. After culturing, 100 µl was transferred to a new microtiter plate for the enzyme activity assay.

[0137] Enzyme assay (Amplex® Red Xanthine / Xanthine Oxidase Assay Kit; Catalogue No. A22182; Molecular Probes) was performed according to the manufacturer's instructions. Fluorescence readout was determined after 60 minutes using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm.

[0138] Example 12. Uric acid oxidase activity by yeast and fungi

[0139] The catalytic activity of uricase in fungi and yeast was evaluated. Fungi and yeast were cultured according to conditions recommended by DSMZ prior to the assay. Bacterial strain growth was assessed by visual inspection. Uric acid was dissolved in the culture medium and sterilized by filtration (so that it was immediately present in the medium upon incubation). Bacterial strain growth was assessed by visual inspection before harvesting for fluorescence measurement. After incubation, 100 µl was transferred to a new microtiter plate for the enzyme activity assay.

[0140] Enzyme assay (Amplex® Red Uric Acid / Uric Acid Oxidase Assay Kit; Catalogue No. A22181; Molecular Probes) was performed according to the manufacturer's instructions. Fluorescence readout was determined after 60 minutes using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm.

Claims

1. A pharmaceutical composition comprising multiple bacterial strains for use in a method of treating or preventing elevated serum urate levels, wherein the multiple bacterial strains include *Lactobacillus plantarum*, deposited on July 14, 2020, at DSMZ with accession number DSM 33580, as at least one bacterial strain possessing xanthine oxidase inhibitory activity. Lactobacillus plantarum The strain and *Lactobacillus plantarum* deposited at DSMZ with accession number DSM 33581 on July 14, 2020. Lactobacillus plantarum At least one strain of *Lactobacillus casei*, and at least one bacterial strain possessing uricase activity, deposited on July 14, 2020, at DSMZ with accession number DSM 33579. Lactobacillus casei ( ) strains, wherein the composition increases the uricase metabolic capacity of the intestinal microbiota and decreases the xanthine oxidase metabolic capacity.

2. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for the treatment or prevention of hyperuricemia.

3. The use according to claim 2, wherein the uricase metabolic capacity of the gut microbiota is enhanced in the presence of uric acid.

4. Use of the pharmaceutical composition of claim 1 and one or more micronutrients in the preparation of a medicament for the treatment or prevention of hyperuricemia.

5. The use according to claim 4, wherein the one or more micronutrients are selected from one or more vitamins of vitamin B2, vitamin B9 and vitamin C, and / or one or more amino acids, and / or one or more prebiotics.

6. The use according to claim 5, wherein one or more amino acids are arginine and / or citrulline.

7. The use according to claim 5, wherein the one or more prebiotics are selected from fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), resistant starch, pectin, β-glucan, and xylooligosaccharides.

8. Use of the pharmaceutical composition of claim 1 and further reagents for the preparation of a medicament for the treatment or prevention of hyperuricemia.

9. The use according to claim 8, wherein the further reagent is a second reagent for treating cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease, chronic kidney disease, gout, insulin resistance, hypertension, hyperuricemia, dyslipidemia, renal insufficiency, obesity, prediabetes, and diabetes.

10. The use of the pharmaceutical composition of claim 1 and allopurinol, febuxostat, probenecid, preclinique, benzbromarone or sulfinpyrazone in the preparation of a medicament for the treatment or prevention of hyperuricemia.

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