Patent Application
20230285477
The present invention relates to compositions comprising a plurality of microorganisms for use in the reduction of blood urate concentration and thus for the therapeutic and the preventive treatment of subjects at risk or suffering from elevated blood urate levels and their use in form of pharmaceutical compositions or nutraceutical compositions for treatment and prevention of urate and hyperuricemia associated diseases.
Urate is the final product of purine nucleoside metabolism. It is secreted both via the renal system and via the intestines. The final steps in the metabolism of purines into urate is the target of multiple drugs and the reactions are catalysed by the enzyme xanthine oxidase. Xanthine oxidase is primarily expressed in the liver, small intestines and by the gut microbiome. Urate is in most mammals further metabolised to allantoin by the enzyme uricase. Humans do not express the gene for uricase, and the human intestinal microbiome is responsible for metabolising urate to allantoin.
Urate is the monovalent sodium salt of uric acid. Urate has low solubility and forms crystals in serum at concentrations 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, 18e New York, N.Y.: McGraw-Hill). The crystals can be deposited throughout the body and give rise to inflammatory responses as seen in gout patients or kidney damage as seen in patients with chronic kidney disease.
Abnormally high concentrations of urate or uric acid in blood, also referred to as hyperuricemia, is known to be associated with a number of diseases and conditions, including cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease, chronic kidney disease, gout, insulin resistance, hypertension, dyslipidaemia, renal insufficiency, obesity, prediabetes and diabetes, particularly type II diabetes.
Elevated levels of urate can be the result of an increased production of uric acid by xanthine oxidase or a decreased urate excretion via the intestines, the renal system, or a combination of both. The renal system excretes two thirds of the total urate in healthy humans. The intestines complement the renal system and excrete one-third of the total urate. The excretion of endogenous urate through the intestinal tract is an opportunity for treating hyperuricemia locally thereby avoiding the systemic adverse reactions.
A variety of drugs are known to lower urate serum levels. Xanthine oxidase inhibitors (e.g., allopurinol and febuxostat) limit the formation of urate in the body by inhibiting xanthine oxidase and uricosuric agents (e.g., probenecid) increase urate elimination by the kidneys. Unfortunately, the treatment of patients with xanthine oxidase inhibitors and uricosuric agents are associated with side effects, including rashes, nausea, stomach pain, kidney stones and reduced liver function. Further, the drugs show low efficacy and only 30-40% of patients treated with allopurinol reach the treatment target.
Other treatment strategies that have been considered include a number of vitamins, such as vitamin B2, B9, C that have been associated with reduction or prevention of elevated serum uric acid.
The use of bacteria for degradation of urate has been considered in some early studies. Nine cultures of aerobic bacteria capable of growing on an elective medium containing uric acid as the only source of carbon, nitrogen and energy were identified (Rouf M A and Lomprey R F Jr. 1968. “Degradation of uric acid by certain aerobic bacteria.” J Bacteriol., September; 96(3): 617-22). However, the urate lowering effects of the were not studied.
Consequently, there is a need for new treatment strategies with fewer side-effects and improved efficacy.
The present invention is able to overcome the drawbacks of the prior art by addressing both the over-production and the under-excretion of urate.
The microbial treatment according to the present invention can reduce urate concentration in blood by treating hyperuricemia locally by optimising the metabolism of the human gut microbiome and by optimising the intestinal host—microbiome interactions. More specifically, the treatment modulates two pathophysiological pathways; (1) it reduces the overproduction of uric acid by inhibiting xanthine oxidase and (2) it increases the excretion of urate by increasing the degradation of urate.
The present invention relates to compositions comprising bacterial strains for use in the reduction of blood urate concentration.
In one aspect, the present invention is directed towards a composition comprising a plurality of microorganisms for use in a method of treating or preventing the condition of elevated serum urate, wherein the composition increases the gut microbiota uricase metabolic capacity and reduces xanthine oxidase metabolic capacity.
In one embodiment, the plurality of microorganisms is a plurality of bacterial strains. In a specific embodiment the plurality of bacterial strains comprises at least one bacterial strain with xanthine oxidase inhibitory activity and at least one bacterial strain with uricase activity. In some embodiments, the plurality of bacterial strains has a Generally Recognized As Safe (GRAS) status and/or Qualified Presumption of Safety (QPS) status. In further embodiments, the plurality of bacterial strains is 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 further embodiments, the plurality of bacterial strains has a synergistic effect in lowering serum urate levels. In further embodiments, the plurality of bacterial strains has a synergistic effect in lowering serum urate levels.
In some embodiments, the plurality of bacterial strains is selected from the genus Bifidobacterium, Bacillus, and Lactobacillus. In specific embodiments, the plurality of bacterial strains from the genus Bifidobacterium is selected from the species Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum. In further specific embodiments, the plurality of bacterial strains from the genus Lactobacillus is selected from the species Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus fermentum, and Lactobacillus casei. In further specific embodiments, the plurality of bacterial strains from the genus Bacillus is selected from the species Bacillus subtilis.
In another embodiment, the plurality of microorganisms is a plurality of fungal strains. In some embodiment the plurality of fungal strains comprises at least one fungal strain with xanthine oxidase inhibitory activity and at least one fungal strain with uricase activity. Preferably, at least one fungal strain with xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase. In some embodiment the metabolites are phenolics and oxidative derivatives thereof. In a more specific embodiment, the plurality of fungal strains is selected from the species Aspergillus niger and M. darjeelingensis.
In another embodiment, the plurality of microorganisms is a plurality of yeast strains. In some embodiment the plurality of yeast strains comprises at least one yeast strain with xanthine oxidase inhibitory activity and at least one yeast strain with uricase activity. Preferably, the at least one yeast strain with xanthine oxidase inhibitory activity ex-presses a metabolite that inhibits xanthine oxidase. In some embodiment the metabolites are phenolics and oxidative derivatives thereof.
In another embodiment, the plurality of microorganisms comprises 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 the yeast strain and/or the fungal strain displays xanthine oxidase inhibitory activity and at least one of the bacterial strain and/or the yeast strain and/or the fungal strain displays uricase activity. Preferably, at least one of the bacterial strain and/or the yeast strain and/or the fungal strain expresses a metabolite that inhibits xanthine oxidase. In case the plurality of microorganisms comprises at least one bacterial strain which expresses a metabolite that inhibits xanthine oxidase, the metabolite is in some embodiments a flavonoid.
In some embodiments, the composition is co-formulated and/or co-administered with one or more additives including micronutrients, amino acids, prebiotics, food, food additive, dietary supplement, or medical food.
In some embodiments, the composition is co-formulated and/or co-administered with one or more micronutrients. In some embodiments, the composition is co-formulated and/or co-administered with one or more trace elements. In some embodiments, the composition is coformulated and/or co-administered with one or more amino acids. In some embodiments, the composition is co-formulated and/or co-administered with one or more prebiotics.
In some embodiments, the composition is co-formulated and/or co-administered in combination with a further agent, e.g., a therapeutic agent and/or therapy.
In another aspect, the present invention is directed towards the composition according to the invention in form of a pharmaceutical composition or nutraceutical composition.
In a further aspect, the invention is directed towards new strains of the species Lactobacillus, deposited with the DSMZ as specified, which are for use in a method of treating or preventing the condition of elevated serum urate. In some embodiments, the strains increase the gut microbiota uricase metabolic capacity and reduces the gut microbiota xanthine oxidase metabolic capacity.
In a further embodiment, the condition of elevated serum urate is selected from cardio-vascular disease, metabolic syndrome, non-alcoholic fatty liver disease (such as nonalcoholic steatohepatitis (NASH)), chronic kidney disease, gout, insulin resistance, hypertension, hyperuricemia, dyslipidemia, renal insufficiency, obesity, pre-diabetes and diabetes.
The following definitions apply unless indicated otherwise.
The term “microorganism” as used for the present invention refers to a single cell organism and includes a prokaryotic organism, e.g., bacteria, and a eukaryotic organism (e.g., fungi, yeast). The term bacteria includes both gram positive bacteria and gram negative bacteria, and includes but is not limited to strains from the genus Bifidobacterium, Bacillus, and Lactobacillus, e.g. 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 microorganism lies within the knowledge of the skilled per-son.
The term “strain” refers to progenies of a pure, isolated culture and the succeeding descendants that can be cultured from it without contamination. A strain refers to a sub-variety of a microbe that is phenotypically and/or genotypically distinguishable from other microbes.
The term “hyperuricemia” as used herein characterizes abnormally high concentrations of urate or uric acid in blood. Hyperuricemia is known to be associated with a number of diseases and conditions, including cardiovascular disease, metabolic syndrome, nonalcoholic fatty liver disease (such as non-alcoholic steatohepatitis (NASH)), chronic kidney disease, gout, insulin resistance, hypertension, dyslipidaemia, renal insufficiency, obesity, pre-diabetes and diabetes, particularly type II diabetes.
The term “micronutrient” includes e.g., vitamins and trace elements. Vitamins are organic substances not synthesized by the body and necessary for normal metabolism. They are divided into water soluble or fat soluble and those with or without coenzyme function. Typical vitamins for use in the present invention include, but are not limited to, vitamin B2, vitamin B9, vitamin C. Trace elements are metals present in very minute quantities in the body. They are essential for normal metabolic functions and are typically cofactors of enzymes or form an integral part of the structure of specific enzymes. Typical trace elements for use in the present invention include, but are not limited to zinc, manganese, iron, copper, molybdenum, chlorine, nickel, boron.
The term “amino acid” refers any of the twenty standard amino acids, i.e., glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid, single stereoisomers thereof, and racemic mixtures thereof. The term “amino acid” can also refer to the known non-standard amino acids, e.g., 4-hydroxyproline, ε-N,N,N-trimethyllysine, 3-methylhistidine, 5-hydroxylysine, O-phosphoserine, γ-carboxyglutamate, γ-N-acetyllysine, ω-N-methylarginine, N-acetylserine, N,N,N-trimethylalanine, N-formylmethionine, γ-aminobutyric acid, histamine, dopamine, thyroxine, citrulline, ornithine, β-cyanoalanine, homocysteine, azaserine, and S-adenosylmethionine. In some embodiments, the amino acid is glutamate, glutamine, lysine, tyrosine or valine. In some embodiments, the amino acids are arginine and citrulline.
The term “prebiotics” is used for selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health. The term “prebiotics” includes but is not limited to fructooligosaccharides (FOS), inulins, galactooligosaccharides (GOS), resistant starch, pectin, beta-glucans, and xylooligosaccharides.
The term pharmaceutical composition refers to a dosage form comprising a composition of the invention together with a pharmaceutically acceptable excipient.
The term “nutraceutical composition” refers to a dosage form comprising a composition of the invention together with a food, food additive, dietary supplement, or medical food.
The term “food” refers to any processed, semi-processed, or raw substance, which is intended for consumption by mammals, e.g., animals or humans. It does not include substances intended only as pharmaceuticals. The term “food additive” as used in the present invention refers to an additive that is, added to, mixed with, or infiltrated into a food in the process of manufacturing the food or for the purpose of processing or storing the food, such water-binding agents, gelling agents, thickeners, antioxidants, dyes, flavor enhancers, acidulants and sweeteners (including additives to which an E (Europe) number is assigned).
The term “dietary supplement” refers to a dosage form comprising a composition of the invention together with a nutritional substance or a substance with a nutritional or physiological effect whose purpose is to supplement the normal diet, such as an herb or other botanical; a metabolite, an extract.
The term “medical food” refers to a dosage form comprising a composition of the invention together with a food intended for the dietary management of a disorder or disease to be administered under the supervision of medically trained people. Medical foods typically fulfil regulatory requirements, including, among others, those of the Federal Food, Drug, and Cosmetic Act and those established for “foods for special medical purposes” by the European Food Safety Authority. Medical foods are typically specially processed or formulated and intended to be used under medical supervision. Medical foods include, but are not limited to, oral rehydration products, nutritionally incomplete formulas, nutritionally complete formulas and formulas for metabolic disorders.
The term “co-formulation” as used herein refers to combining a composition of the invention with one or more additives to form a single pharmaceutical composition or single nutraceutical composition. A co-formulation is thus intended for a simultaneous administration.
The term “co-administration” as used herein refers to a combined administration of a composition of the invention with one or more additives. The term “combined” refers to both a simultaneous and a sequential administration (independent of the order).
The abbreviation “CFU” denotes the unit “colony-forming unit”, which is commonly used in microbiology to estimate the number of viable cells of a microorganism, e.g., a bacterial strain, in a given sample. Determination of the CFU count typically involves, after an optional initial dilution, counting at least part of the number of colonies of a microorganism, e.g., a bacterial strain, on a petri dish and, based on this count, approximating the total number of viable cells in the given sample.
An enzyme metabolic capacity corresponds to the ability to provide for the conversion of at least one of the enzyme's substrates into at least one of the corresponding products and may, for example, be defined as the number of molecules of at least one substrate converted into at least one of the corresponding products in a given period of time. The enzyme metabolic capacity is influenced, among many other factors, by the amount of the enzyme present, the activity of the enzyme or isozyme present and the presence and/or absence of activators and/or inhibitors of the enzyme.
In a first aspect, the present invention is directed towards a composition comprising a plurality of microorganisms for use in a method of treating or preventing the condition of elevated serum urate, wherein the composition increases the gut microbiota uricase metabolic capacity and reduces xanthine oxidase metabolic capacity.
In a specific embodiment, the composition increases the gut microbiota uricase metabolic capacity and reduces the gut microbiota xanthine oxidase metabolic capacity.
In one embodiment, the plurality of microorganisms is a plurality of bacterial strains. In a specific embodiment the plurality of bacterial strains comprises at least one bacterial strain with xanthine oxidase inhibitory activity and at least one bacterial strain that displays uricase activity. Preferably, the at least one bacterial strain with xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase. In some embodiment the metabolite is a flavonoid.
In further embodiments, the gut microbiota uricase metabolic capacity is enhanced in the presence of uric acid. In some embodiments, the enhancement is due to induction of expression of uricase in the presence of uric acid. In preferred embodiments, this enhancement amounts to a factor of 2-10, particularly 3-8.
The enhancement of the gut microbiota uricase metabolic capacity in the presence of uric acid is advantageous because it means that uricase metabolic capacity is only minimal in the absence of uric acid, thus minimizing undesired side effects in the absence of uric acid, which is associated with enhanced safety. Typically, the bacteria employed have been granted QPS (qualified presumption of safety) status by the European Food Safety Authority (EFSA) and/or GRAS (generally recognized as safe) status by the FDA.
In a more specific embodiment, the plurality of bacterial strains is selected from the genus Bifidobacterium, Bacillus, and Lactobacillus.
In some preferred embodiments, the compositions of the present invention comprise one or more bacterial strains selected from the species Bifidobacterium breve, Bifidobacterium longum, Bacillus subtilis, Lactobacillus casei, Lactobacillus plantarum and one or several other strains of intestinal bacteria or bacteria derived from food-sources, including strains of other bacterial species.
In other preferred embodiments, the plurality of bacterial strains from the genus Bifidobacterium is selected from the species Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum, particularly the species Bifidobacterium breve ATCC 15701.
In other preferred embodiments, the plurality of bacterial strains from the genus Lactobacillus is selected from the species Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus fermentum, and Lactobacillus casei, preferably the strains DSM 33579, DSM 33580, DSM 33581 deposited with the DSMZ (International Depositary Authority: Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstrasse 7 B, 38124 Braunschweig, Germany) on Jul. 14, 2020.
In other preferred embodiments, the plurality of bacterial strains from the genus Bacillus is selected from the species Bacillus subtilis.
Typically, the bacterial strains are isolated from food. They may be in liquid, frozen or dried form.
In another embodiment, the plurality of microorganisms is a plurality of fungal strains. In some embodiment the plurality of fungal strains comprises at least one fungal strain with xanthine oxidase inhibitory activity and at least one fungal strain with uricase activity. Preferably, the at least one fungal strain with xanthine oxidase inhibitory activity expresses a metabolite that inhibits xanthine oxidase.
In another embodiment, the plurality of microorganisms is a plurality of yeast strains. In some embodiment the plurality of yeast strains comprises at least one yeast strain with xanthine oxidase inhibitory activity and at least one yeast strain with uricase 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/or an oxidative derivative thereof.
In some embodiment, the plurality of microorganisms comprises a combination of at least one bacterial strain and at least one fungal strain. In some embodiment, the plurality of microorganisms comprises a combination of at least one bacterial strain and at least one yeast strain.
In some embodiment the metabolite is a phenolic compound and/or an oxidative derivative thereof.
In some embodiments the compositions of the invention may be in form of a pharmaceutical composition or a nutraceutical composition.
Suitable dosage forms include but are not limited to a tablet, pill, powder, lozenge, sachet, cachet, elixir, suspension, emulsion, solution, syrup, aerosol (as a solid or in a liquid medium), ointment containing, for example, up to 10% by weight of the active component, soft capsule, hard capsule, gel-cap, tablet, suppository, solution, or packaged powder. Pharmaceutically acceptable excipients suitable for formulating the dosage form of the present invention include, but are not limited to, disintegrants, diluents, plasticizers, binders, glidants, lubricants, sweeteners, flavoring agents, anti-caking agents, anti-microbial agents, antifoaming agents, emulsifiers, surfactants, buffering agents and coloring agents and the like or mixtures thereof. Suitable excipients include, for example, PBS, glycerol, cocoa butter, or polyethylene glycol.
In some embodiments, the composition is co-formulated and/or co-administered with one or more additives including micronutrients, amino acids, prebiotics, food, food additive, dietary supplement, or medical food, In some embodiments, the composition of the invention, specifically the pharmaceutical composition or nutraceutical composition of the invention may be co-formulated and/or co-administered with one or more micro-nutrients, specifically with one or more vitamin and/or one or more trace element. In some embodiments, the composition of the invention, specifically the pharmaceutical composition or nutraceutical composition of the invention may be co-formulated and/or co-administered with one or more vitamins, preferably selected from vitamin B2, vitamin B9 and vitamin C. In some embodiments, the composition of the invention, specifically the pharmaceutical composition or nutraceutical composition of the invention may be co-formulated and/or co-administered with one or more trace elements, such as zinc, manganese, iron, copper, molybdenum, chlorine, nickel and boron.
In some embodiments, the composition of the invention, specifically the pharmaceutical composition or nutraceutical composition of the invention may be co-formulated and/or co-administered with one or more amino acids. In some embodiments, the composition of the invention, specifically the pharmaceutical composition or nutraceutical composition of the invention may be co-formulated and/or co-administered with one or more amino acids, such as arginine and/or citrulline.
In some embodiments, the composition of the invention, specifically the pharmaceutical composition or nutraceutical composition of the invention may be co-formulated and/or co-administered with one or more prebiotics, preferably selected from fructooligosaccharides (FOS), inulins, galactooligosaccharides (GOS), resistant starch, pectin, beta-glucans, and xylooligosaccharides.
In some embodiments, the compositions of the present invention may be co-formulated and/or co-administered (in combination or separately, sequentially or at the same time) with a further agent, e.g., a therapeutic agent. The further agent, e.g., therapeutic agent, denotes an agent used in addition to the plurality of microorganisms. Suitable agents that may be used as further therapeutic agents include, but are not limited to:
In other embodiments, the compositions of the present invention may be administered in combination with a further therapy.
The compositions of the present invention may be used for subjects having a urate blood concentration within the normal range as well as for subjects having a urate blood concentration above the normal range to treat or to prevent an elevation of the urate concentration in blood for the subject in question.
In specific embodiments, the compositions of the present invention may be used for treating, preventing or reducing the risk of an elevation of the urate concentration in blood of a patient and/or subject diagnosed with or suffering from cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease (such as non-alcoholic steatohepatitis (NASH)), chronic kidney disease, gout, insulin resistance, hypertension, hyperuricemia, dyslipidaemia, renal insufficiency, obesity, pre-diabetes and diabetes.
In further embodiments, the compositions of the present invention, co-formulated and/or co-administered (in combination or separately, sequentially or at the same time) with a further agent, e.g., a therapeutic agent, may be used for treating, preventing or reducing the risk of an elevation of the urate concentration in blood of a patient and/or subject diagnosed with or suffering from cardiovascular disease, metabolic syndrome, non-alcoholic fatty liver disease (such as non-alcoholic steatohepatitis (NASH)), chronic kidney disease, gout, insulin resistance, hypertension, hyperuricemia, dyslipidaemia, renal insufficiency, obesity, pre-diabetes and diabetes.
The composition for use in the reduction of urate concentration in blood of the present invention may be administered in a concentration of 1×106 CFU/day or more. Preferred concentrations are at least 1×109 CFU/day, at least 1×1010 CFU/day, at least 1×1011 CFU/day, at least 1×1012 CFU/day, at least 1×1013 CFU/day, at least 1×1014 CFU/day, at least 1×1015 CFU/day.
The urate lowering effect of the present invention may be evaluated by measuring urate concentration in a blood sample, a urine sample or a saliva sample. The analysis may be in the form of a home analysis with a test stick (BerkeleyFit saliva test).
MRS refers to the De Man, Rogosa and Sharpe agar medium. It was used as commercially available from Sigma-Aldrich and was used according to the instructions of the manufacturer. BHI refers to the Brain Heart Infusion Broth. It was used as commercially available from Sigma-Aldrich and was used according to the instructions of the manufacturer. Anaerocult refers to the reagent anaerocult, which was used as commercially available from Millipore and according to the instructions of the manufacturer. Saline refers to a sodium chloride solution in water and was used as obtained from Sigma-Aldrich.
Table 1 shows the abbreviations used for the respective bacterial strains.
Lactobacillus casei
Bacillus subtilis
Lactobacillus casei
Bacillus subtilis
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus casei
Lactobacillus casei
Lactobacillus casei
Lactobacillus casei DSM 33579
Lactobacillus casei
Lactobacillus casei
Lactobacillus casei
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus casei
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
Lactobacillus plantarum
Lactobacillus plantarum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Bifidobacterium longum
Lactobacillus plantarum DSM 33580
Lactobacillus plantarum DSM 33581
Lactobacillus plantarum
Lactobacillus plantarum
Lactobacillus plantarum
Strains were precultured in 1 ml in a deep-well plate under the following conditions (see Table 2).
Lactobacillus sp.
Bifidobacterium
Bacillus sp.
5% Cystein solution was freshly prepared, filter sterilized and afterwards added to the medium prior to inoculation.
Glycerol stocks were prepared in a microtiter plate by mixing 120 μl grown culture and 40 μl 60% sterile glycerol solution. The resulting glycerol stocks were stored at −80° C.
Bacterial strains were evaluated for their ability to inhibit xanthine oxidase catalytic activity. Prior to the assay, 1/100th volume from the glycerol stocks (example 1) was added to the microtiter plate containing the appropriate medium (see overview). Culturing conditions for the different species (time, temperature, medium, condition) were chosen as set out in Table 2. Growth of the bacterial strains was assessed by visual examination before harvesting for fluorescence measurements. After cultivation, 100 μl of the respective medium containing the respective species was transferred into a new microtiter plate to use in the enzymatic activity assays.
The enzyme assay, Amplex® Red Xanthine/Xanthine Oxidase Assay Kit; Catalog No. A22182; Molecular Probes, was used according to the instructions of the manufacturer. Fluorescence readouts were determined after 60 min using excitation at 530±12.5 nm and fluorescence detection at 590±17.5 nm. Bifidobacterium breve strain ATCC 15701 was used as positive control and Bifidobacterium animalis (BB12®) was used as negative control.
Bacterial strains were evaluated for uricase catalytic activity. Prior to the assay, 1/100th volume from the glycerol stocks (example 1) was added to the microtiter plate containing appropriate medium (see overview). Culturing conditions (time, temperature, medium, aerobic or anaerobic) depend on species and can be found in Table 1. Uric acid was dissolved in medium to 2% and the medium was filter sterilized (therefore uric acid was immediately present in the medium upon culturing). Growth of the bacterial strains was assessed by visual examination (the level of viscosity of the medium) before harvesting for fluorescence measurement. After cultivation, 100 μl of the respective medium containing the respective species was transferred into a new microtiterplate to use in the enzymatic activity assays.
The enzyme assay, Amplex® Red Uric Acid/Uricase Assay Kit; Catalog No. A22181; Molecular Probes, was used according to the instructions of the manufacturer. Fluorescence readouts were determined after 30 min using excitation at 530±12.5 nm and fluorescence detection at 590±17.5 nm. 20 mM H2O2 solution was used as positive control and 0 mM uricase solution was used as negative control.
The activity of the strains listed in Table 1 was determined, as described in Examples 2 and 3, as percentage of maximal fluorescence from positive controls (Bifidobacterium breve strain ATCC 15701 was used as positive control for the screems according to Example 2 and 20 mM H2O2 solution was used as positive control for the screems according to Example 3). The strains were subsequently categorized according to their species and the mean average activity of all strains belonging to each species was calculated. Tables 3 and 4 show the average xanthine oxidase inhibitory activity and the average uricase inhibitory activity of the respective species, respectively, wherein the average inhibitory activities were categorized as low (5-30% of positive control), medium (25-75% of positive control) and high (50-100% of positive control).
Bifidobacterium breve ATCC 15701
Bifidobacterium longum
Lactobacillus plantarum
Lactobacillus casei
Bacillus subtilis
Lactobacillus plantarum
The bacterial strain Lactobacillus casei (BEO #109) was cultured according to the conditions described in Example 1. 0% to 2% uric acid was dissolved in medium and filter sterilized (therefore immediately present in the medium upon culturing). Growth of the bacterial strains were assessed by visual examination before harvesting for fluorescence measurement. After cultivation, 100 μl is transferred into a new microtiter plate for enzymatic activity measurements.
The enzyme assay Amplex® Red Uric Acid/Uricase Assay Kit; Catalog No. A22181; Molecular Probes, was used according to the instructions of the manufacturer. Fluorescence readouts were determined after 60 min using excitation at 530±12.5 nm and fluorescence detection at 590±17.5 nm.
This experiment was conducted as described in Example 2. The fluorescence readouts were measured every 6 minutes from time 0 to 72 minutes.
The enzyme assay, Amplex® Red Xanthine/Xanthine Oxidase Assay Kit; Catalog No. A22182; Molecular Probes, was used according to the instructions of the manufacturer. Fluorescence readouts were determined using excitation at 530±12.5 nm and fluorescence detection at 590±17.5 nm. Bifidobacterium breve strain ATCC 15701 was used as positive control and Bifidobacterium animalis (BB12®) was used as negative control.
This experiment was conducted as described in Example 3. The fluorescence readouts were measured every 6 minutes from time 0 to 30 minutes.
The enzyme assay, Amplex® Red Uric Acid/Uricase Assay Kit; Catalog No. A22181; Molecular Probes, was used according to the instructions of the manufacturer. Fluorescence readouts were determined using excitation at 530±12.5 nm and fluorescence detection at 590±17.5 nm. 20 mM H2O2 solution was used as positive control and 0 mM uricase solution was used as negative control.
Bacterial strain Lactobacillus plantarum (BEO #227) was used in this experiment. The experiment was conducted as described in example 2 except that, after cultivation of the respective bacterial strain, the supernatant is isolated by centrifugation (3000 rpm, 10 min, temperature 4° C.). The same volume of whole cells (WC) and supernatant (SUP) was then used in the same assay as the one used in example 2. The fluorescence was measured after 0, 25, 45, 60, 90 and 110 minutes.
The enzyme assay, Amplex® Red Xanthine/Xanthine Oxidase Assay Kit; Catalog No. A22182; Molecular Probes, was used according to the instructions of the manufacturer. Fluorescence readouts were determined using excitation at 530±12.5 nm and fluorescence detection at 590±17.5 nm.
6-10 weeks old mice are randomly divided into groups of 10 mice as follows, using 1.0×108-1010 CFU for the test product:
The control group is fed a normal diet (laboratory chow and water ad libitum) and treated with saline (0.5 ml, sodium chloride solution in water) by intragastric administration once a day. The other groups are fed a high-purine diet (laboratory high-purine chow and water ad libitum) and administered an intraperitoneal injection of potassium oxonate 300 mg/kg once a day. The microbial treatment groups are treated daily with approximately 1.0×109 CFU/day by intragastric administration. The strains tested all have QPS status and they are produced and formulated under laboratory conditions.
Mice are weighed day −1 for determination of mean weight of animals in the experiment and for composition of groups. Groups are mixed within cages to avoid cage effects (except for group 1 that is in a separate cage). Disease is induced day 0 by an intraperitoneal injection of oxonate (300 mg/kg).
Serum is collected once weekly and at the end of experiment. On day 0, serum was collected 6 hours after dosing of the test product; on day 7, serum was collected 4 hours after dosing of the test product; on day 14, serum was collected 4 hours after dosing of the test product and 23 hours after dosing of the test product when the mice were sacrificed. The collected blood is centrifuged, and plasma separated and frozen until further analysis. Serum is analysed for uric acid concentration. The strain combination BEO #110+BEO #227 shows a beneficial lowering effect, i.e., full normalisation of the serum urate concentration.
Cultured bacteria are separated from spent media by centrifugation at 3000 rpm for 10 min at 4° C. Harvested bacteria is re-dissolved in formulation/cryo buffer. The formulation/cryo buffer is composed of 25-50 mM buffer, 10-30% sugar. Alterative formulations are tested by including additives in the formulation buffer such as 1-6% ascorbate, arginine and 0.3-1.0 mg folate.
Fungi and yeast are evaluated for their ability to inhibit xanthine oxidase catalytic activity. Prior to the assay fungi and yeast are cultured according to conditions recommended by DSMZ. Growth of the bacterial strains are assessed by visual examination. After cultivation, 100 μl is transferred into a new microtiter plate for enzymatic activity assaying.
The enzyme assay Amplex® Red Xanthine/Xanthine Oxidase Assay Kit; Catalog No. A22182; Molecular Probes, was used according to the instructions of the manufacturer. Fluorescence readouts were determined after 60 min using excitation at 530+12.5 nm and fluorescence detection at 590+17.5 nm.
Fungi and yeast are evaluated for uricase catalytic activity. Prior to the assay fungi and yeast are cultured according to conditions recommended by DSMZ. Growth of the bacterial strains is assessed by visual examination. Uric acid is dissolved in medium and filter sterilized (therefore immediately present in the medium upon culturing). Growth of the bacterial strains is assessed by visual examination before harvesting for fluorescence measurement. After cultivation, 100 μl is transferred into a new microtiter plate for enzymatic activity assaying.
The enzyme assay Amplex® Red Uric Acid/Uricase Assay Kit; Catalog No. A22181; Molecular Probes, was used according to the instructions of the manufacturer. Fluorescence readouts were determined after 60 min using excitation at 530+12.5 nm and fluorescence detection at 590+17.5 nm.
| Number | Date | Country | Kind |
|---|---|---|---|
| 00923/20 | Jul 2020 | CH | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/070685 | 7/23/2021 | WO |
