# Patent

## Title
Composition and method for preventing or treating gout or hyperuricemia

## Abstract

A composition composed of a Lactobacillus bacterium, Bifidobacterium longum, and xylooligosaccharide is provided for use in reducing uric acid levels in the blood and preventing or treating hyperuricemia or gout.

## Background
This application is a continuation-in-part application of U.S. Ser. No. 13/602,386, filed Sep. 4, 2012, now U.S. Pat. No. 8,481,025, which is a continuation of U.S. Ser. No. 12/407,201 filed Mar. 19, 2009, now U.S. Pat. No. 8,257,693, which is a continuation of U.S. Ser. No. 10/936,262 filed Sep. 8, 2004, now abandoned, which is a continuation-in-part of U.S. Serial No. PCT/US2002/007554 filed Mar. 13, 2002.


INTRODUCTION
Background of the Invention
One of the main functions of the normal, healthy kidney, besides its regulatory, endocrine, and metabolic functions, is the disposal of waste products. Any impairment of excretory function can lead to the accumulation of a variety of nitrogenous waste products including, urea, creatinine and uric acid. High concentrations of waste products in the blood stream can exacerbate renal failure and promote kidney stones. Moreover, nitrogenous solutes in the circulating blood promote osmotic diffusion into the lumen because of the concentration gradient across the intestinal wall. This diffusion mechanism led to the concept of oral sorbents to augment gut-based clearance of nitrogenous waste products. Sorbents or microbes have demonstrated their ability to remove various compounds and nitrogenous wastes within the large bowel.
Urea-specific sorbents such as synthetic polymers and modified polysaccharides have been evaluated for the removal of urea and other nitrogenous wastes via the gut. Other sorbents such as oxidized starch, activated charcoal, and carob flour have also been investigated for the in vivo elimination of uremic toxins with some success. Prakash & Chang ((1996) Nature Medicine 2:883-88) demonstrated that microencapsulated, genetically-engineered E. coli DH5 are effective in removing urea and ammonia in an in vitro system. The same researchers obtained similar results in oral administration of E. coli DH5 cells in a uremic rat animal model. Bliss et al. ((1996) Am. J. Clin. Nutr. 63:392-398) have demonstrated that supplemental gum arabic fiber increases fecal nitrogen excretion and lowers urea nitrogen concentration in chronic renal failure patients consuming a low protein diet. Reinhart et al. ((1998) Rec. Adv. In Canine and Feline Nutr. Iams Nutrition Symposium Proceedings. Vol. 11:395-404) found that canine renal patients fed a diet containing a fermentable fiber blend improved clinical end-stage renal disease status, suggesting that specific nutritional alteration allows repartitioning of nitrogen excretion away from the kidney and into the feces by colonic fermentation or additional bacterial growth.
U.S. Pat. No. 5,756,088 teaches a prescription diet for the prevention and treatment of dog and cat dermatosis comprising a composition containing a poly-unsaturated fatty acid such as γ-linolenic acid, γ-linolenic acid and docosahexaenoic acid, and/or biotin, and an antiflatulent such as a lactic acid bacterium, a Bifidobacterium, a Lactobacillus, a butyric acid bacterium or a Bacillus, and optionally an oligosaccharide.
U.S. Pat. No. 7,993,903 teaches a composition for inhibiting cholesterol absorption in the intestinal tract, wherein the composition includes Bifidobacterium, and optionally a Lactobacillus bacterium and carbohydrate.
US 2011/0171283 teaches a composition containing at least one nutrient, at least one disinfecting or decontaminating and/or at least one proteases inhibiting substance and/or complex of substances incorporated in an absorbent dressing for external care and/or treatment of wounds to a human or animal. In one embodiment, the protease inhibiting substance includes non-pathogenic acid producing micro-organisms (e.g., bifidobacteria, lactococci, or lactobacilli) and/or synbiotics (e.g., xylooligosaccharide).
US 2009/0252709 teaches a preventive or therapeutic agent for gastritis or ulcer, which includes as an active ingredient Bifidobacterium bifidum. This reference teaches that other microorganisms (e.g., Bifidobacterium or Lactobacillus bacteria), as well as sugars such as xylooligosaccharide.
WO 2007/140622 teaches a probiotic composition containing a mixture of a Propionibacterium, a Lactobacillus, a Bifidobacterium and a Streptococcus, wherein said composition can further include a prebiotic.
SUMMARY OF THE INVENTION
The present invention provides methods for reducing uric acid levels in the blood and preventing or treating hyperuricemia or gout by administering an effective amount of a composition containing a Lactobacillus bacterium, a Bifidobacterium bacterium, and xylooligosaccharide to a subject.
The present invention is also a composition consisting essentially of a Lactobacillus bacterium (e.g., Lactobacillus acidophilus), Bifidobacterium bacterium (e.g., Bifidobacterium longum), and xylooligosaccharide.


DETAILED DESCRIPTION OF THE INVENTION
Nitrogenous waste products accumulating in the blood stream have detrimental affects on health. Removal of nitrogenous wastes by diverting them into the colon is a viable approach to decrease the negative impact that waste product accumulation has on an individual's physiology. The present invention combines the properties of probiotic and prebiotic components into a synbiotic product to effectively reduce the blood concentration of nitrogenous waste products and has the added beneficial effect of promoting the growth of desirable intestinal microflora.
A probiotic component of the present invention refers to a mono or mixed culture of live or freeze-dried microorganisms which, when applied to man or animal, beneficially affects the host by improving the properties of the indigenous microflora, such as bifidobacterium organisms that metabolize undigested carbohydrates and are beneficial to an individual. Probiotic components of the present invention are selected for their ability to exert a beneficial effect on the host, survive transit through the intestinal tract, to adhere to intestinal epithelial cell lining, to produce of anti-microbial substances towards pathogens and/or to stabilize the intestinal microflora. Furthermore, a probiotic component should have a good shelf-life. Synbiotic products of the present invention generally contain a large number of viable cells at the time of consumption, and are non-pathogenic and nontoxic. In particular embodiments of this invention, the probiotic component of the invention includes a Bifidobacterium spp. (e.g., bifidum, longum, infantis) and a Lactobacillus spp. (e.g., bulgaricus, acidophilus, lactis, helveticus, casei, plantarum, reuteri, delbrueckii, chamnosus, johnsonii, paracasei). In addition to a Bifidobacterium spp. and a Lactobacillus spp. the probiotic component can also include a Streptococcus spp. (e.g., thermophilus, diacetilactis, cremoris, durans, faecalis), Saccharomyces spp. (e.g., pombe, boulardii), Leuconostoc spp. (e.g., citrovorum, dextranicum) and Bacillus sp. (e.g., pasteurii).
Microorganisms also useful in the invention are those that have the ability, either through natural selection or by genetic manipulation, to catabolize various nitrogenous compounds (e.g., urea, creatinine, uric acid and ammonia) by expressing or overexpressing one or more cognate catabolic enzymes. Exemplary microorganisms are those having an elevated level of urease or creatininase secretion.
A microorganism exhibiting elevated levels of catabolic enzyme secretion can be selected or trained by exposing a selected microorganism on increasing amounts of the metabolite of interest (e.g., urea, creatinine, uric acid and ammonia). For example, it has been found that a standard strain of Streptococcus thermophilus can be trained to express elevated levels of urease by sequential passage of the strain on increasing amounts of urea, e.g., a single colony growing on 0.5% urea is selected and applied to medium containing 1.0% urea, a single colony growing on 1.0% urea is selected and applied to medium containing 2.0% urea, etc. Using such a method, a S. thermophilus strain having the ability to grow on 5% urea was isolated. This strain proliferated in artificial intestinal fluid (AIF, US Pharmacopeia) in the pH range of 5.5 to 7.5, characteristic of the colon environment; used urea as a sole nitrogen source; and catabolized urea in the presence of other nitrogen sources. It was found that urea hydrolysis was growth- and pH-dependent and that urea concentrations could be reduced by this strain from 300 mg/dL to 20 mg/dL within 24 hours at pH 6.3 when inoculated in AIF at an initial density of 109 cfu/mL. Moreover, this strain survived 3 hours in acidic pH 3.0 with only a one-log loss in cfu and was able to pass through bile. In addition, this strain did not appear to exhibit any resistance to eight commonly used antibiotics. Therefore, these data indicate that a specifically selected or trained bacterial isolate can be used as a urea-targeted component in a synbiotic product of the present invention. Accordingly, certain embodiments of this invention include a Bifidobacterium bacterium and/or Lactobacillus bacterium that, by selection or training, is capable of substantially reducing urea concentrations within 24 hours to about 50%, 40%, 30%, 20% or 10% of the starting amount of urea.
Elevated levels of secretion can also be obtained by overexpressing the gene of interest (e.g., via multiple copies or a promoter driving high levels of expression) in a prokaryotic microorganism of interest such as Bifidobacterium, Lactobacillus, Streptococcus, Leuconostoc or Bacillus, or a eukaryotic microorganism such as Saccharomyces. The gene of interest can be under the regulatory control of an inducible or constitutive promoter. Promoters for use in recombinant prokaryotic expression vectors are well-established in the art and can include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al. (1978) Nature 275:615; Goeddel et al. (1979), Nature 281:544), a tryptophan (trp) promoter system (Goeddel et al. (1980) Nucleic Acids Res. 8:4057; EPO App. Publ. No. 36,776) and the tac promoter (De Boer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21). While these are commonly used promoters which are commercially available, one of skill in the art can appreciate that any other suitable microbial promoter can be used as well. Nucleic acid sequences encoding suitable prokaryotic promoters have been published thereby enabling one of skill in the art to readily isolate these promoters (e.g., by standard cloning or PCR methodologies) for cloning into plasmid or viral vectors (Siebenlist et al. (1980) Cell 20:269). The promoter and Shine-Dalgarno sequence (for prokaryotic host expression) are operably-linked to the DNA encoding the gene of interest, i.e., they are positioned so as to promote transcription of the messenger RNA from the DNA, and subsequently introduced into a suitable host cell.
Eukaryotic microbes such as yeast cultures can also be transformed with suitable protein-encoding vectors. See e.g., U.S. Pat. No. 4,745,057. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available. Yeast vectors can contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the desired protein, sequences for polyadenylation and transcription termination, and a gene encoding for a selectable marker. An exemplary plasmid is YRp7, (Stinchcomb et al. (1979) Nature 282:39; Kingsman et al. (1979) Gene 7:141; Tschemper et al. (1980) Gene 10:157). This plasmid contains the trp1 gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones (1977) Genetics 85:12). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al. (1980) J. Biol. Chem. 255:2073) or other glycolytic enzymes (Hess et al. (1968) J. Adv. Enzyme Reg. 7:149; Holland et al. (1978) Biochemistry 17:4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast expression are commercially available and further described in Hitzeman et al., EP 73,657.
As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode a degradative enzyme of interest, e.g., a urease or creatininase, can be designed to contain signal sequences which direct secretion of enzyme of interest through a prokaryotic or eukaryotic cell membrane. Such signal sequences are well-established in the art and can be taken from other enzymes/proteins known to be secreted into the extracellular environment.
Transforming the microorganisms as defined herein, describes a process by which exogenous DNA is introduced into and changes a recipient cell. It can occur under natural or artificial conditions using various methods well-known in the art. Transformation can rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the type of host cell being transformed and can include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. Such “transformed” cells include stably-transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. This also includes cells which transiently express the inserted DNA or RNA for limited periods of time.
As will be appreciated by the skill artisan, a microorganism can also be exposed to a mutagen to cause changes in the genetic structure of the organism so that it expresses elevated levels of a catabolic enzyme of interest.
Transformed or mutagenized strains are subsequently selected for the ability to grow in the presence of the metabolite which is degraded by the catabolic enzyme of interest. By way of example, a strain transformed with nucleic acid sequences encoding a urease is selected for high levels of urease secretion by growing said strain on high levels of urea. Levels of urease secretion can also be detected using standard enzymatic assays. As disclosed herein, the strain can be sequentially subcultured on increasing levels of urea to further enhance urease secretion. In one embodiment, a urease-secreting strain is a Bifidobacterium bacterium and/or Lactobacillus bacterium that is capable of substantially reducing urea concentrations within 24 hours to about 50%, 40%, 30%, 20% or 10% of the starting amount of urea.
The probiotics according to the invention can be obtained by fermentation and can be stored after fermentation and before addition to the synbiotic composition of the present invention for a time and at a temperature that prevents substantial loss of probiotic cfu. For example, the probiotic component can be fermented until a final concentration of 106 to 5×1010, or 107 to 1010, or 108 to 109 cfu per mL of fermented medium is achieved.
When the probiotic component is a mono culture, said mono culture is 100% of the probiotic component. When the probiotic component is composed of at least two microorganisms, each microorganism can be 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90% of the probiotic component, wherein the total of all microorganisms is 100%. An exemplary probiotic component is composed of about 30-70% Lactobacillus bacterium (e.g., L. acidophilus) and about 30-70% Bifidobacterium bacterium (e.g., B. longum). In particular embodiments, the probiotic component is composed of about 30-70% Lactobacillus bacterium and about 50% Bifidobacterium bacterium.
As used herein, the probiotic component is one component or additive to a food product or to an enterically coated tablet, capsule, powder, soft gel, gelcap, or liquid. Accordingly, the probiotic component is included at a concentration of 108 cfu/mL, 109 cfu/mL, 1010 cfu/mL, 1011 cfu/mL, or 1012 cfu/mL when added as a liquid or 108 cfu/g, 109 cfu/g, 1010 cfu/g, 1011 cfu/g, or 1012 cfu/g when added as a freeze-dried powder. In one embodiment, the probiotic component is about 20% to about 70% of the total synbiotic product weight. In particular embodiments, the probiotic component is about 50% of the total synbiotic product weight.
A prebiotic component of the present invention refers to a non-digestive food that beneficially affects the host by selectively stimulating the growth and/or activity of one or more non-pathogenic bacteria in the colon. Prebiotic components of the present invention are considered to have anti-carcinogenic, anti-microbial, hypolipidemic and glucose modulatory activities. They can also improve mineral absorption and balance. Furthermore, bacteria belonging to the Bifidobacterium and Lactobacillus families are stimulated by the presence of the prebiotic component and proliferate. Pharmacokinetically, the prebiotic components reach the colon largely intact. An exemplary prebiotic component includes, but is not limited to, an oligosaccharide such as xylooligosaccharide. In addition to a xylooligosaccharide, the prebiotic component of the invention can also include fructo-oligosaccharide, inulin, isomaltose oligosaccharide, trans-galacto-oligosaccharide, or soy-oligosaccharide; a pyrodextrin such as arabinogalactan, lactilol, lactosucrose, or lactulose; or a fiber source such as oat gum, pea fiber, apple fiber, pectin, guar gum, psyllium husks, glucomannan or guar gum hydrolysate (BeneFiber, Novartis Pharmaceuticals). In one embodiment, the prebiotic component xylooligosaccharide.
When the prebiotic component is a single non-digestive food, said non-digestive food is 100% of the prebiotic component. When the prebiotic component is composed of two or more non-digestive foods, each non-digestive food can be 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90% of the prebiotic component, wherein the non-digestive food total is 100%.
The amount of prebiotic component added to the probiotic component is 100 milligrams per serving, 500 milligrams per serving, 1 gram per serving, 5 grams per serving or 10 grams per serving. In one embodiment, the prebiotic component is not less than 100 milligrams and not more than 10 grams per serving. In one embodiment, the prebiotic component is about 20% to about 70% of the total synbiotic product weight. In particular embodiments, the prebiotic component is about 50% of the total synbiotic product weight. When the synbiotic product further includes addition additives, the percent of the prebiotic and/or probiotic component can be decreased to accommodate the additional additive. In particular embodiments, the percent of the prebiotic component is decreased to accommodate the additional additive.
A synbiotic product combining the beneficial properties of probiotic and prebiotic components can include a food product, dietary supplement, comestible medical food or pharmaceutical product. In the context of the present invention, “synbiotic” refers to a mixture of at least one probiotic and at least one prebiotic components to promote health enhancing effects (Gibson and Roberfroid (1995) J. Nutr. 125:1401-1412). The ingestion of said synbiotic product reduces the blood concentration of nitrogenous waste products that accumulate in the circulating blood stream. These waste products of the present invention can be of an endogenous origin such as normal or abnormal metabolic routes or bacterial putrefaction. Furthermore, the waste products can be of an exogenous origin as in dietary intake of proteins and amino acids. Furthermore, repeated ingestion of the synbiotic product will have a highly beneficial effect upon the intestinal microflora by localization and colonization in the large intestine of microbes known to promote a healthy intestinal microenvironment. In some embodiments of this invention, the synbiotic product comprises a Lactobacillus bacterium, a Bifidobacterium bacterium and xylooligosaccharide. In particular embodiments of this invention, the synbiotic product consists essentially of a Lactobacillus bacterium, a Bifidobacterium bacterium and xylooligosaccharide, wherein the term “consisting essentially of” or “consists essentially of” means that the activity of the synbiotic is attributed to the Lactobacillus bacterium, a Bifidobacterium bacterium and xylooligosaccharide and not other, non-essential ingredients such as fillers, additives, excipients, flavors, sweetening agents, binders or bulking agents. In specific embodiments, the synbiotic product consists essentially of Lactobacillus acidophilus, Bifidobacterium longum and xylooligosaccharide.
As indicated herein, a synbiotic product of the present invention can take the form of a food product including, but is not limited to, a health bar, health drink, yogurt, dahi, or sachet or an enterically coated tablet, capsule, powder, soft gel, gelcap, or liquid. In addition to containing the prebiotic and probiotic components, the synbiotic product of the present invention can further containing various fillers or additives.
Optional additives of the present composition include, without limitation, pharmaceutical excipients such as magnesium stearate, talc, starch, sugars, fats, antioxidants, amino acids, proteins, nucleic acids, electrolytes, vitamins, derivatives thereof or combinations thereof. In one embodiment, an additive of the synbiotic product is carob flour, for example, locust bean gum. In another embodiment, an additive is a mushroom extract from Agaricus bisporus. In particular embodiments, a gel cap contains fillers such as magnesium stearate, talc and starch.
Further, to increase the palatability of a food product containing a prebiotic and probiotic, it may be desirable to add flavors, sweetening agents, binders or bulking agents.
Flavors which can optionally be added to the present compositions are those well-known in the pharmaceutical art. Examples include, but are not limited to, synthetic flavor oils, and/or oils from plants leaves, flowers, fruits and so forth, and combinations thereof are useful. Examples of flavor oils include, but are not limited to, spearmint oil, peppermint oil, cinnamon oil, and oil of wintergreen (methylsalicylate). Also useful are artificial, natural or synthetic fruit flavors such as citrus oils including lemon, orange, grape, lime, and grapefruit, and fruit essences including apple, strawberry, cherry, pineapple and so forth.
Sweetening agents can be selected from a wide range of materials such as water-soluble sweetening agents, water-soluble artificial sweeteners, and dipeptide-based sweeteners, including salts thereof and mixtures thereof, without limitation.
Binders can be selected from a wide range of materials such as hydroxypropylmethylcellulose, ethylcellulose, or other suitable cellulose derivatives, povidone, acrylic and methacrylic acid co-polymers, pharmaceutical glaze, gums (e.g., gum tragacanth), milk derivatives (e.g., whey), starches (e.g., corn starch) or gelatin, and derivatives, as well as other conventional binders well-known to persons skilled in the art. Examples of bulking substances include, but are not limited to, sugar, lactose, gelatin, starch, and silicon dioxide.
When the above-mentioned additives are included in the synbiotic product of the present invention, they are generally less than 15% of the total synbiotic product weight. In particular embodiments, they are less than 5 to 10% of the total synbiotic product weight.
To facilitate targeting of the synbiotic product of the present invention to the gastrointestinal tract, controlled release formulations are useful, preferably for oral administration. These include, but are not limited to, osmotic pressure-controlled gastrointestinal delivery systems; hydrodynamic pressure-controlled gastrointestinal delivery systems; membrane permeation-controlled gastrointestinal delivery systems, which include microporous membrane permeation-controlled gastrointestinal delivery devices; gastric fluid-resistant intestine targeted controlled-release gastrointestinal delivery devices; gel diffusion-controlled gastrointestinal delivery systems; and ion-exchange-controlled gastrointestinal delivery systems, which include cationic and anionic drugs. Additional information regarding controlled release drug delivery systems can be found in Yie W. Chien, Novel Drug Delivery Systems, 1992 (Marcel Dekker, Inc.).
For example, enteric coatings are applied to tablets to prevent the release of drugs in the stomach either to reduce the risk of unpleasant side effects or to maintain the stability of the drug which might otherwise be subject to degradation of expose to the gastric environment. Most polymers that are used for this purpose are polyacids that function by virtue or the fact that their solubility in aqueous medium is pH-dependent, and they require conditions with a pH higher than normally encountered in the stomach. One desirable type of oral controlled release structure is enteric coating of a solid or liquid dosage form. Enteric coatings promote the compounds remaining physically incorporated in the dosage form for a specified period when exposed to gastric juice. Yet the enteric coatings are designed to disintegrate in intestinal fluid for ready absorption. Delay of absorption is dependent on the rate of transfer through the gastrointestinal tract, and so the rate of gastric emptying is an important factor. Some investigators have reported that a multiple-unit type dosage form, such as granules, may be superior to a single-unit type.
Typical enteric coating agents include, but are not limited to, hydroxypropylmethylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate (Hasegawa, (1985) Chem. Pharm. Bull. 33:1615-1619). Various enteric coating materials can be selected on the basis of testing to achieve an enteric-coated dosage form designed ab initio to have a preferable combination of dissolution time, coating thicknesses and diametral crushing strength. (Porter et al. (1970) J. Pharm. Pharmacol. 22:42p). It is contemplated that the enteric coating can be either food grade or pharmaceutical material which is generally used in the production of various drug or dietary supplements.
Depending on whether the synbiotic product is to be consumed by an adult human, child or animal (e.g., companion animal or livestock), it can be produced in various sizes and with various ingredients suitable for the intended recipient. For example, while a gel cap size of 0 or 1 may be suitable for humans, a gel cap size of 2, 3, 4, or 5 may be more suitable for a companion animal.
Further, because the probiotic and prebiotic components of the present invention are generally recognized as safe, they can be consumed one, two or three times daily or more.
The present invention also relates to a method for reducing uric acid levels in the blood of a subject. The method of this invention involves administering an effective amount of a synbiotic product of the present invention so that the levels of uric acid in the blood are decreased or reduced, desirably to a normal range. For example, normal uric acid levels in males and females is in the range of 2.1 to 8.5 mg/dL and 2.0 to 7.0 mg/dL, respectively. As one of skill in the art can appreciate, means for determining the levels of uric acid are well-known to the skilled laboratory clinician.
As a synbiotic product of the present invention reduces the levels of uric acid in the blood, this composition is useful in a method for preventing or treating hyperuricemia or gout. Such a method involves administering a synbiotic product of the present invention to a subject having or at risk of having hyperuricemia or gout. Subjects having or at risk of having hyperuricemia or gout are those in the upper range of normal uric acid levels (e.g., 6 to 8.5 mg/dL for men and 5 to 7 mg/dL for women) or above the normal uric acid levels (e.g., more than 8.5 mg/dL for men and 7 mg/dL for women). Desirably, an effective amount of a synbiotic product for preventing or treating hyperuricemia or gout is an amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an effective amount of a synbiotic product is one which results in the alleviation or amelioration of one or more symptoms associated with hyperuricemia or gout (e.g., crystal deposits in joints or tendons), delay or slowing of disease progression (i.e., a delay of crystal deposits in joints or tendons), or preventing crystal deposits in joints or tendons of subject with a history of hyperuricemia or gout.
The invention will be further described in the following example, which does not limit the scope of the invention described in the claims.
Example 1
Yogurt Food Product
A yogurt food product can be prepared from one gallon of commercially available whole, homogenized, pasteurized milk which is heated to boiling and quickly allowed to cool to approximately 45° C. To this is added approximately one ounce of yogurt starter culture containing lactic acid bacteria of the genus Lactobacillus and Bifidobacteria. The mixture is mixed well and allowed to ferment at 37° C. for 10 to 12 hours. Xylooligosaccharide, whole fruit additives, flavoring, sweetening agents, binders, or other additives can be combined and added to the yogurt to obtain a product of desired consistency or to suit the palette of the prospective consumer. In one embodiment of the present invention, a food product comprises components to meet the special dietary needs of individuals with renal insufficiency.
Example 2
Health Bar
Health bars can be prepared by combining various excipients, such as binders, additives, flavorings, colorants and the like, along with the probiotic (i.e., Lactobacillus and Bifidobacteria) and prebiotic component (i.e., xylooligosaccharide), and mixing to a plastic mass consistency. The mass is then either extruded or molded to form “candy bar” shapes that are then dried or allowed to solidify to form the final product.
Example 3
Medical Food
A medical food can be prepared by combining rolled oats, dehydrated apples, honey, inulin, carob flour, cinnamon, sugar, vanilla extract, and lyophilized cultures of L. acidophilus and a Bifidobacteria (108-1010 cfu each). These ingredients are mixed in appropriate proportions with xylooligosacchraide and formed into a rectangular bar approximately 12.5 to 15 centimeters in length, 3 to 4 centimeters in width and 1 centimeter in height and placed into a sterile vacuum oven for 12 to 24 hours to obtain an edible food product of the desired consistency.
Example 4
Dietary Supplement
A dietary supplement of the present invention can be prepared by combining the lactic acid bacteria Lactobacillus acidophilus (40 to 60%) and Bifidobacteria longum (40 to 60%), aseptically freeze-drying the bacteria and combining the processed bulk bacteria (e.g., about 50% of the total synbiotic product weight) with the prebiotic xylooligosaccharide component, wherein the final prebiotic component is about 45% of the total synbiotic product weight. Fillers such as magnesium stearate, talc and starch (e.g., about 5% of the total synbiotic product weight) are added to the prebiotic and probiotic components and enterically coated gel caps are produced according to the method of Kim et al ((1988) J. Indust. Microbiol. 3:253-257). Approximately 10 to 25 billion CFU of the freeze-dried microorganism is contained in each capsule (i.e., approximately 30 to 75 billion CFU microorganisms per gram) that is enterically coated with hydroxy-propylmethyl cellulose phthalate by spraying over a fluidized bed of capsules. The resulting dietary supplement has a low surface area, is relatively non-porous and can protect the contents therein from low pH as is found in the gastric environment for several hours, and will release the contents into the bowel wherein the pH is relatively neutral or slightly alkaline. Advantageously, approximately 90-95% of the microorganisms can survive to be released into the gastric environment.
Example 5
Pharmaceutical Product
A pharmaceutical product for persons suffering from gout or hyperuricemia can be prepared by aseptically freeze-drying a Lactobacillus and a Bifidobacterium, combining the processed bulk microorganisms with the prebiotic component, and preparing the synbiotic product as enterically coated capsules according to the method of Kim et al ((1988) supra) or tablets, powders, soft gels, gelcaps, or liquids according to standard methods. For example, the prebiotic and probiotic components in each capsule are enterically coated with hydroxy-propylmethyl cellulose phthalate by spraying over a fluidized bed of capsules. The resulting pharmaceutical product has a low surface area, is relatively non-porous and can protect the contents therein from low pH as is found in the gastric environment for several hours, and will release the contents into the bowel wherein the pH is relatively neutral or slightly alkaline.
Example 6
Urease-Secreting Strains of Streptococcus thermophilus 
This example discloses the isolation and selection of a high level urease-secreting strain of Streptococcus thermophiluss. Three isolates of gram-positive, lactic acid-producing non-pathogenic cocci of Streptococcus thermophilus were isolated from various sources and designated KB4, KB19, and KB25. KB4 was isolated from a probiotic product, KB19 was isolated from a commercial yogurt product and KB25 from Dahi yogurt (from India).
Growth rates and urea hydrolysis of these bacteria in the intestinal pH range (pH 5.5, 6.3 and 7.5) were determined by transferring exponentially growing cultures of KB19, KB4 and KB25 into modified Artificial Intestinal Fluid M2 (AIF, US Pharmacopeia) supplemented with 100 mg/dL filter-sterilized urea, 100 μM NiCl2, 10% MRS broth, dextrose to final concentration of 1%, and 0.3% yeast extract, wherein the initial cell density was 109 cfu/mL. Pancreatin was omitted from the recipe to allow the evaluation of bacterial growth by direct OD600 nm measurement. Urea concentration in the supernatants (% of control) and growth (OD600 nm) were measured every 4 hours.
Concentration of urea in the supernatants of bacterial cultures was measured using the protocol and standards supplied with the Blood Urea Nitrogen Reagent Kit (535, Sigma, St. Louis, Mo.). Urea hydrolysis was monitored by comparing urea-nitrogen concentrations in bacterial supernatants to appropriate control medium incubated in the same conditions and expressed as percent of control. Four to nine independent experiments were conducted and Student t-test was used for statistical analysis.
Under similar assay conditions, exponentially growing cultures of KB19, KB4 and KB25 were inoculated into AIF M2, pH 6.3, supplemented with 100 mg/dL urea and with or without 100 μM NiCl2 at initial cell density of 109 cfu/mL to determine whether the growth and rates of urea hydrolysis by these strains was dependent on the additional Ni++. Urea concentration in the supernatants as a % of control and growth (OD600 nm) were measured every 4 hours. Four to nine independent experiments were conducted and Student t-test was used for statistical analysis.
Similarly, it was determined whether the growth and rate of urea hydrolysis of these strains was dependent on urea concentration. Under similar growth conditions exponentially growing cultures of KB19, KB4 and KB25 were inoculated into AIF M2, pH 6.3, supplemented with 100 μM NiCl2 and 100, 200, or 300 mg/dL urea. Urea concentration in the supernatants as a % of the control and growth (OD600 nm) were measured every 4 hours. Four to nine independent experiments were conducted and Student t-test was used for statistical analysis.
The survivability of these KB19, KB4 and KB25 was determined in artificial gastric juice in the presence and absence of urea and dextrose. The average loss in viable cell count after exposure to artificial gastric juice (logs cfu/mL) is shown in Table 1.











TABLE 1






PH/Additive
KB19
KB4
KB25








1.4
7
7
7



2.0
7
7
7



2.5
3
4
4



2.5/Urea
3
4
4



2.5/Dextrose
3
3
3



2.5/Urea + Dextrose
3
3
3



3.0
2
2
3



3.0/Urea
2
3
3



3.0/Dextrose
1
2
3



3.0/Urea + Dextrose
1
2
2









Initial cell density was 107 cfu/mL. Urea and dextrose concentrations were 10 mg/mL and 1%, respectively.
Further it was determined whether the nutrient composition and availability had an affect on growth and urea hydrolysis by KB19, KB4, and KB25. Each strain was grown for 24 hours at 37° C. and pH 6.0 in the presence or absence of urea and combinations of nitrogen and carbon sources. This analysis indicated that urea hydrolysis was growth- and pH-dependent and that urea concentrations could be reduced by strain KB19 from 300 mg/dL to 20 mg/dL within 24 hours at pH 6.3 when inoculated in AIF at an initial density of 109 cfu/mL.
Further analysis of S. thermophilus KB19 indicated that this strain could survive a 3 hour exposure to gastric juice, pH 3.0, followed by a 3 hour exposure to 0.3% oxgal, pH 6.0, with only 1 log loss in viability. Remaining viable cells were able to proliferate in AIF M2, pH 6.0, supplemented with 230 mg/dL urea and completely hydrolyzed the urea within less than 18 hours (n=4). All test solutions were supplemented with 230 mg/dL urea and 1% dextrose.
Collectively, these analyses indicated that all three strains studied proliferated in the fed state AIF medium in the pH range from 5.5 to 7.5, characteristic of colon environment; they could all use urea as a sole nitrogen source; and they each catabolized urea in the presence of other nitrogen sources. Urea hydrolysis was growth and pH dependent. Under the conditions tested, the rate of urea hydrolysis was strain-dependent in tests of pH stability: KB19=KB25>KB4; Ni requirement: KB25>KB19>KB4; urea hydrolysis for over 300 mg/dL: KB19=KB25>KB4; and specific nutrients: KB19>KB25>KB4. Further, there was strain-dependent results relating to survivability, wherein in tests of gastric juice stability: KB19>KB4>KB25; and bile stability: KB19>KB4>KB25.
In view of the desirable traits exhibited by the selected S. thermophilus strains, the same methodology can be used to select or train strains of Lactobacillus and Bifidobacterium for increased urease activity. Therefore, in certain embodiments, the Lactobacillus and/or Bifidobacterium of this invention are selected for the ability to reduce urea concentrations from 300 mg/dL to 20 mg/dL within 24 hours at pH 6.3.
Example 7
Growth of Lactobacillus and Bifidobacterium on Oligosaccharides
Pure strains of L. acidophilus and B. longum were grown on growth medium containing the prebiotics xylooligosaccharide and arabinogalactan as the carbon source. The control medium had dextrose as carbon source. After 3 days of incubation at 37° C., bacterial colonies were counted. The results of this analysis indicated that xylooligosaccharide supported the growth of both L. acidophilus and B. longum. 
Example 8
Reduction in Uric Acid Levels in Patients with Chronic Kidney Disease (CKD)
A synbiotic product composed of Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and psyllium husks was orally administered to CKD patients in a cross-over experiment, i.e., Group A was provided with the synbiotic product for three months (period 1) and then switched to a placebo for three months (period 2), whereas Group B was provided with a placebo for three months (period 1) and then switched to the synbiotic product for three months (period 2). Biochemical uremic markers (creatinine, uric acid, BUN and CRP) were measured at the end of period 1 and period 2 for each group. The percent of patients showing improvement in uric acid levels upon receiving the synbiotic product is presented in Table 2.











TABLE 2







No of
No. of





Patients with
Patients with




No. of
Decreased Levels
Improved Quality of



Site
Patients
of Uric Acid (%)
Life Ratings (%)




















Argentina
8
4
(50)
7
(88)


Canada
13
4
(31)
11
(85)


Nigeria
15
5
(33)
13
(87)


USA
10
2
(20
8
(80)


Totals
46
15
(33)
39
(85)









In a similar crossover study, stage 3 and 4 chronic kidney disease patients were provided dietary supplementation with a synbiotic product composed of Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium longum and psyllium husks. A total of 13 patients completed the study. Blood was drawn from each patient at every monthly visit. Subsequent to the study completion, relative changes in uric acid levels were calculated for both treatment periods and for each patient. Based on this cumulative data from all patients, relative changes based on the administered treatment—symbiotic product or placebo—were pooled and average relative changes for were calculated.
This analysis indicated that there was a significant mean change in uric acid concentration during the synbiotic treatment period (−24.70 μmol/L) versus during the placebo period (50.62 μmol/L, p=0.05). Therefore, a synbiotic product can effectively reduce uric acid levels in the blood.

## Claims

1. A method for reducing uric acid levels in the blood comprising administering an effective amount of a composition comprising a Lactobacillus bacterium, Bifidobacterium bacterium, and xylooligosaccharide to a subject in need of treatment thereby reducing the subject's uric acid levels.

2. A method for treating or reducing the risk of hyperuricemia or gout comprising administering an effective amount of a composition comprising a Lactobacillus bacterium, Bifidobacterium bacterium, and xylooligosaccharide to a subject in need of treatment thereby treating or reducing the risk of the subject's hyperuricemia or gout.