Pharmaceutical compositions comprising oxalate-reducing bacteria

[0001] The present invention relates to compositions and methods for the treatment and prevention of oxalate related conditions. More specifically, the invention relates to compositions and uses comprising bacteria that degrade oxalate or reduce oxalate. BACKGROUND [0002] Disease with stones in the kidneys and urinary tract (urolithiasis) is a major health problem in the world. Most stones associated with urolithiasis consist of calcium oxalate alone or calcium oxalate together with calcium phosphate. Other disease states are also associated with excess oxalate. These include vulvodynia, oxalose associated with end-stage renal disease, cardiac conduction disorders, Crohn's disease, and other bowel conditions.

[0003] Oxalic acid and / or its salt, oxalate, is found in a wide variety of food products and is therefore found in many components of animal and human diets. Increased absorption of oxalate may occur with the consumption of foods containing increased amounts of oxalic acid. It is well known that foods such as spinach and rhubarb contain large amounts of oxalate, but many other foods and drinks also contain oxalate. Because oxalate is found in so many different foods, it's difficult to formulate diets that are low in oxalate and are still tasty. In addition, adherence to a low oxalate diet is often problematic.

[0004] Endogenous oxalate is also produced metabolically by normal tissue enzymes. Oxalate, which includes food oxalate, which is absorbed, as well as oxalate, which is metabolized, is not further metabolized by tissue enzymes and must therefore be excreted. This excretion is mainly through the kidneys. Oxalate concentration in kidney fluids is key, with increased oxalate concentrations causing an increased risk of calcium oxalate crystal formation and thus further kidney stone formation.

[0005] The risk of kidney stone formation is associated with many factors that are not yet fully understood. Disease with stones in the kidneys or urinary tract occurs in up to 12% of the population in Western countries, and about 70% of these stones are composed of calcium oxalate or calcium oxalate together with calcium phosphate. Some individuals (e.g. patients with bowel disease such as Crohn's disease, inflammatory bowel disease or fatty stools, as well as patients who have undergone fasting-ileal anastomosis surgery absorb more oxalate from their diets than others. For these individuals, the incidence of oxalate urolithiasis increases significantly. The increased incidence of this disease is due to increased levels of oxalate in the kidneys and urine, and this, the most common syndrome associated with hyperoxaluria in men, is called intestinal hyperoxaluria. Oxalate is also a problem in patients with end-stage renal disease and recent evidence exists (Solomons, CC, MH Melmed, MS Heitler [1991] "Calcium citrate for vulvar vestibulitis" Journal of Reproductive Medicine 36: 879-882), that elevated urinary oxalate levels are also involved in vaginitis (vulvodynia).

[0006] Bacteria that degrade oxalate have been isolated from human feces (Allison, MJ, HM Cook, DB Milne, S. Gallagher, RV Clayman [1986] "Oxalate degradation by gastrointestinal bacteria from humans" J. Nutr. 116: 455- 460). These bacteria were found to be similar to oxalate-reducing bacteria that were isolated from the intestinal content of many animal species (Dawson, KA, MJ Allison, PA Hartman [1980] "Isolation and some characteristics of anaerobic oxalate-degrading bacteria the rumen" Appl. Environ. Microbiol. 40: 833-839; Allison, MJ, HM Cook [1981] "Oxalate degradation by microbes of the large bowel of herbivores: the effect of dietary oxalate" Science 212: 675-676; Daniel, SL, PA Hartman, MJ Allison [1987] "Microbial degradation of oxalate in the gastrointestinal tracts of rats" Appl. Environ. Microbiol. 53: 1793-1797). These bacteria differ from any previously described organisms and have been given both a new species name and a new genus name (Allison, MJ, KA Dawson, WR Mayberry, JG Foss [1985] "Oxalabacter formigenes gen. Nov., Sp. Nov. : oxalatedegrading anaerobes that inhabit the gastrointestinal tract "Arch. Microbiol. 141: 1-7).

[0007] Not all people are carriers of the O. formigenes population in their deer tract (Allison, MJ, SL Daniel, NA Comick [1995] "Oxalate-degrading bacteria" In: Khan, SR (ed.), Calcium Oxalate in Biological Systems CRC Press; Doane, LT,

M. Liebman, DR Caldwell [1989] "Microbial oxalate degradation: effects on oxalate and calcium balance in humans" (Nutrition Research 9: 957-964). Oxalate-decomposing bacteria are present at low concentrations or are completely absent from stool samples from individuals after fasting-ileal anastomosis surgery (Allison et al. [1986] "Oxalate degradation by gastrointestinal bacteria from humans" J. Nutr. 116: 455-460 ). Some people and animals may also maintain O colonies. formigenes, but nevertheless have elevated levels of oxalate for reasons that are not well understood.

[0008] There is a need for methods of treating humans and animals to reduce oxalate levels in their bodies such that oxalate related conditions are treated or prevented. Desirable methods would include administering oxalate reducing compositions.

SUMMARY OF THE INVENTION [0009] The present invention includes compositions for the treatment and prevention of oxalate related conditions. The compositions of the present invention include pharmaceutical compositions containing microorganisms that reduce the amount of oxalate. The methods of the present invention include methods of making pharmaceutical compositions. One embodiment includes methods that reduce the risk of developing oxalate related disorders by reducing the amount of oxalate in the gastrointestinal tract. This reduction in the gastrointestinal tract thus leads to a decrease in systemic oxalate levels, thereby promoting good health.

[0010] In one embodiment of the present invention, a reduction in oxalate absorption is achieved by providing oxalate degrading bacteria to the gastrointestinal tract. In an embodiment, these bacteria are Oxalobacter formigenes. These bacteria use oxalate as a substrate. This use reduces the concentration of soluble oxalate in the intestine, and thus the amount of oxalate available for absorption. Reducing the amount of oxalate in the gastrointestinal tract can also lead to the removal of oxalate from the circulatory system. The uses of the present invention take into account the overall reduction of oxalate load in a subject.

[0011] In a particular embodiment, the present invention provides methods and compositions for delivering live O. formigenes to the gastrointestinal tract of individuals who are at increased risk of oxalate-related disease. Bacteria remove oxalate from the intestinal tract, thereby reducing the amount of oxalate available for absorption and leading to increased excretion of oxalate from the blood to the intestines.

[0012] According to the information of the present invention, oxalate-decomposing microorganisms other than O. formigenes that use oxalate as a substrate can also be used to achieve therapeutic oxalate degradation, thereby reducing the risk of urolithiasis and other oxalate-related disorders. Such other microorganisms can be, for example, bacteria such as clostridia or pseudo-3 monads. In addition, the present invention includes methods and compositions for providing exogenous polynucleotide sequences capable of conferring oxalate-reducing function to microorganisms that naturally do not produce oxalate-reducing enzymes. Such polynucleotide sequences can be used to convert such native microorganisms, initially unable to reduce oxalate, into microorganisms capable of reducing oxalate. These transformed microorganisms can be used in the methods and compositions of the present invention, and are contemplated herein.

[0013] In one embodiment of the present invention, the compositions contain microorganisms that degrade oxalate and produce enzymes that give these microorganisms the ability to degrade oxalate. In an alternative embodiment, the compositions may contain microorganisms that have been transformed with polynucleotide sequences that confer on the transformed microorganisms the ability to degrade oxalate. Polynucleotide sequences that encode oxalate-reducing genes and proteins are contemplated by the present invention. Polynucleotide sequences encoding enzymes found in oxalate reducing microorganisms such as bacteria or fungi, or other oxalate reducing enzymes can be used in the methods of the present invention. Polynucleotide sequences can be used to transform microorganisms or cells, such that the microorganisms or cells have more oxalate-reducing activity, the same oxalate-reducing activity, or less oxalate-reducing activity than naturally occurring oxalate-reducing microorganisms. Polynucleotide sequences can also be used in synthetic or ex vivo systems to provide proteins having oxalate reducing activity. Such microorganisms or enzymes may be provided in compositions that are provided as pharmaceutical compositions and preparations described herein, wherein the microorganisms or enzymes may be provided in pharmaceutical preparations containing excipients and other pharmaceutical carriers known in the art. In addition, such pharmaceutical compositions contain delivery vehicles, in the form of powders, for delivery to the gastrointestinal tract of humans or animals.

[0014] Enzymes involved in oxalate degradation can be used in the methods and compositions of the present invention and include, but are not limited to, formyl-CoA transferase, oxalyl-CoA decarboxylase, oxalate oxidase, oxalate decarboxylase and other enzymes, cofactors and coenzymes, which they are surrogate components of oxalate degradation pathways or are involved in oxalate metabolic pathways, particularly oxalate reduction.

[0015] Uses and compositions containing enzymes for reducing oxalate levels for the treatment or prevention of oxalate related conditions are also described. For example, a reduction in oxalate levels is achieved by administering enzymes that lead to oxalate degradation. These enzymes can be isolated and purified or can be administered as cell lysate. Cell lysate can be made from any microorganism that has oxalate-reducing functions, for example O. formigenes. In a particular embodiment, the enzymes that are administered are one or more enzymes of the invention, such as, but not limited to, oxalate decarboxylase, oxalate oxidase, formyl-CoA transferase and oxalyl-CoA decarboxylase. Optionally, additional agents that improve enzymatic activity may be administered. These additional factors may be, for example, oxalyl-CoA, MgCl2 and TPP (thiamine diphosphate, the active form of vitamin B1). Pharmaceutical compositions containing enzymes contain one or more enzymes and optionally cofactors, coenzymes and other agents that increase enzymatic activity, alone or in combination, and are provided with pharmaceutically acceptable carriers and excipients.

[0016] Reduction of oxalate levels is achieved by administering oxalate-degrading enzymes produced by a recombinant microorganism, such as Escherichia coli, which has been transformed to express oxalate-degrading enzymes. The recombinant host can be administered in live or non-live form. Another aspect of the present invention relates to pharmaceutical compositions and / or dietary supplements for oral administration. These compositions release oxalate degrading microorganisms or oxalate degrading enzymes in the intestines of humans or animals. The compositions of the present invention include pharmaceutically acceptable preparations. For example, the methods and compositions of the present invention include a dose delivery system that delivers the compositions to desired sites, such as to deliver the composition to the recipient's gastrointestinal tract. The compositions of the present invention may be administered as an ingredient in food products such as milk, meat and yogurt.

[0017] In a further embodiment of the present invention, a reduction in oxalate absorption is achieved in domestic, farmed or exotic animals deficient in oxalate degrading bacteria by administering oxalate degrading microorganisms.

[0018] The uses of the present invention include the treatment or prophylaxis of oxalate related conditions in humans and animals by administering an effective amount of oxalate reducing compositions containing one or more oxalate reducing microorganisms, one or more oxalate reducing enzymes or combinations thereof, and mixture. Conditions associated with oxalate include, but are not limited to, hyperoxaluria, primary hyperoxaluria, idiopathic diseases involving calcium oxalate kidney stones (urolithiasis), intestinal hyperoxaluria, vulvodynia, oxalosis associated with end-stage renal disease, conduction heart disease, inflammatory bowel disease , Crohn's disease and ulcerative colitis.

BRIEF DESCRIPTION OF THE FIGURES [0019]

FIG. 1A is a graph of data for high calcium diet.

FIG. 1B is a graph of data for the low-calcium diet.

FIG. 2A is a graph of excreted oxalate.

FIG. 2B is a graph of excreted oxalate.

FIG. 2C is a graph of excreted oxalate.

FIG. 3A-C are graphs of excreted oxalate.

FIG. 4 is a graph of excreted oxalate.

DETAILED DISCLOSURE OF THE INVENTION [0020] The present invention includes compositions for reducing oxalate. The compositions of the present invention contain microorganisms that are capable of reducing oxalate. The compositions contain microorganisms that are capable of reducing oxalate. Such microorganisms include, but are not limited to, Oxalobacter formigenes, Pseudomonas, Clostridia, Lactobacilli, Bifidobacteria, some or all of which are capable of reducing oxalate, but also include microorganisms such as bacteria or fungi that have been transformed with exogenous polynucleotide sequences, such that they were given the ability to reduce oxalate. Additionally, the microorganisms of the present invention include microorganisms that have been transformed with one or more oxalate-reducing vectors containing endogenous or exogenous polynucleotide sequences that encode oxalate-reducing enzymes or related activities such that the microorganisms are "superreductors". Superreductors have enhanced natural oxalate-reducing abilities, for example, in the Oxalobactor for5 migenes transformation, additional oxalate-reducing sequences, or are microorganisms that initially have no oxalate-reducing activity that has been transformed with one or more oxalate-coding peptide-reducing sequences oxalate reducing activity. Sequences coding for oxalate reducing activity may or may not be inserted into the genome or other vectors found in the microorganism. Such transformation may involve providing gene sequences that encode oxalate-reducing proteins or peptides, or may provide blocking nucleotides such as antisense or iRNA. Techniques for introducing polynucleotide sequences and transforming microorganisms are known in the art.

[0021] As used herein, the terms oxalate-degrading enzymes and oxalate-reducing enzymes are interchangeable and both refer to enzymes involved in reducing oxalate amount or degradation in any organism, or active fragments or recombinant proteins containing active fragments capable of reducing oxalate amount or degradation.

[0022] Also described herein are polynucleotide sequences that encode peptides or proteins that are involved in oxalate reduction pathways. Such polynucleotide sequences can be derived from any source and can be used in methods known to those skilled in the art, such as the transformation of cells of microbial, plant or animal origin, including entire organisms.

[0023] The compositions of the present invention also include pharmaceutical compositions containing live bacteria that reduce oxalate and optionally pharmaceutical excipients or carriers in the delivery vehicle. The compositions also include pharmaceutical compositions containing one or more purified oxalate-reducing enzymes, including, but not limited to, enzymes purified from natural sources of such enzymes, recombinantly or synthetically produced, and optionally pharmaceutical excipients or carriers in a delivery vehicle.

[0024] The pharmaceutical compositions of the present invention comprise oral delivery vehicles in the form of powders. Such oral delivery vehicles are used to deliver live bacteria and oxalate reducing enzymes at the dosages and methods given herein. Such pharmaceutical compositions are stable. The compositions may provide live bacteria and enzymes showing activity for at least 12 months, with minimal loss of cfu and enzymatic activity.

[0025] Also described herein are plants and animals that have an altered oxalate reducing function. For example, such plants include plants that have been transformed using polynucleotide compositions such that the amount of oxalate in the plant is reduced or the amount of oxalic acid produced is increased compared to a non-transformed plant. The compositions of the present invention also include animals that have an enhanced ability to reduce oxalate. For example, animals having enhanced oxalate reducing ability can be used as in vivo models to study oxalate related conditions.

[0026] The methods of the present invention include preparing the compositions of the present invention. The methods of the present invention include transforming cells, plants and animals by methods known to those skilled in the art to introduce exogenous polynucleotide sequences. Such polynucleotide sequences can be derived from any source and can be used in methods known to those skilled in the art, such as the transformation of cells of microbial, plant or animal origin, including entire organisms. The methods also include preparing compositions comprising cell lysates having oxalate-reducing activity, compositions containing one or more enzymes having oxalate-reducing activity, and compositions containing dietary components made from plants or microorganisms having altered oxalate levels. The methods also include preparing stable oral pharmaceutical compositions containing live bacteria that reduce oxalate.

[0027] The uses of the present invention include the use of the compositions of the present invention. Such applications include providing polynucleotide sequences to cells to enhance or inhibit the ability of cells to reduce oxalate, as well as methods of dietary supplementation, such that the compositions of the present invention are administered to plants or animals at food or fertilizer sources or simultaneously with food or fertilizer sources to change oxalate levels in food, when digesting food, or when being absorbed by plants.

[0028] The present invention relates to the introduction of compositions containing one or more bacteria and / or enzymes that degrade oxalate into the gastrointestinal tract of a human or animal, wherein the activity of the composition reduces the amount and / or concentration of oxalate present, thereby reducing the risk of disease caused by oxalate.

[0029] The present invention includes compositions for the treatment and prophylaxis of oxalate related conditions in humans and animals by administering a composition comprising one or more oxalate reducing enzymes. Such compositions may be administered once or more times a day for one or more days, depending on the severity of the oxalate-related condition or the amount of oxalate in the intestine or body fluids of a human or animal. Treatment may be continued for as long as undesirable levels or oxalate are present in the human or animal. For example, the enzyme composition may be administered once or more times daily for a period of time ranging from one day to years. For humans or animals with chronic oxalate related conditions, the composition may be administered throughout the remaining life of the human or animal.

[0030] Applications for the treatment and prevention of oxalate-related conditions may include the administration of a composition comprising an effective amount of oxalate-reducing enzymes or enzymatic activity to reduce oxalate. Effective amount means the amount of oxalate-reducing enzyme activity units that will reduce the proportion of oxalate present or the level of oxalate-reducing enzyme activity unit that starts reducing oxalate or maintains a reduced amount of oxalate in the subject compared to the amount of oxalate present prior to administration of the composition. The number of oxalate-reducing enzymatic activity units that can be used in a single dose of the composition may range from 0.0001 units to 5000 units, from 5 units to 100 units, from 0.05 to 50 units, to 0.5 to 500, from 0.01 units to 50 units, from 0.01 units to 5 units, from 1 unit to 100 units, from 25 units to 50 units, from 30 units to 100 units, from 40 units to 120 units, from 60 units to 15 from 50 units to 100 units, from 100 units to 500 units, from 100 units to 300 units, from 100 units to 400 units, from 100 units to 5000 units, from 1000 units to 5000 units, from 2500 units up to 5000 units, from 0.001 units to 2000 units and in all ranges covered in these. The composition may further include other enzymes, cofactors, substrates, coenzymes, minerals and other agents that are useful in reducing oxalate. The enzyme unit is the amount of enzyme that will degrade one micromole of oxalate per minute at 37 ° C.

[0031] In a particular embodiment, the present invention relates to methods for producing compositions comprising cells of oxalate-degrading oxalobacter formigenes bacteria into the gastrointestinal tract of a human or animal, wherein microbial activity reduces the amount of oxalate present in the intestine, thereby reducing oxalate concentration in kidneys and other cell fluids. In another embodiment, the present invention includes methods of making and administering compositions comprising one or more oxalate-degrading enzymes from any source into the gastrointestinal tract of a human or animal, wherein the activity of one or more enzymes reduces the amount of oxalate present in the intestine and leads to a reduction of oxalate in the kidneys and other cell fluids. The introduced cells or enzymes break down oxalate, and bacteria may or may not reproduce in the intestinal environment, so that the progeny of the initial cells colonize the intestine and continue to remove oxalate. The presence of oxalate-reducing bacteria reduces the risk of kidney stone formation as well as other complications of diseases caused by excess oxalic acid. In some form for human use, the specific O strains used. formigenes are strains isolated from human intestinal samples. Strains are therefore part of the normal intestinal bacterial flora in humans. However, because they are not present in all people or are not present in sufficient numbers, the introduction of these organisms corrects the deficiency that occurs in some people.

[0032] Although not wishing to be bound by any particular theory, it is believed that enriching the intestinal contents with one or more species of oxalate-degrading bacteria or oxalate-reducing enzymes causes a decrease in the amount of oxalate in the intestinal content. Some of the bacteria or enzymes administered break down oxalate at or near the absorption site. The activity of bacteria or administered enzymes reduces the level of absorption of food oxalate. A decrease in oxalate concentration in the intestines can also lead to the removal of oxalate from the cells or general circulation. More specifically, a decrease in oxalate concentration in the intestine can also lead to enhanced oxalate secretion into the intestine from the blood, and thus reduce the amount of oxalate that must be excreted in the urine. Thus, the methods of the present invention for administering oxalate-reducing bacteria or oxalate-reducing enzymes, in addition to treating food hyperoxaluria, can be used to treat or prevent oxalate related conditions such as primary hyperoxaluria. The compositions and methods of the present invention are particularly beneficial in promoting healthy oxalate levels in humans and animals.

[0033] Pharmaceutical and nutraceutical compositions for introducing oxalate-degrading bacteria or one or more oxalate-degrading enzymes, alone or in combination, into the gastrointestinal tract include bacteria or enzymes that have been lyophilized or frozen in liquid or paste form, and can be delivered through an oral delivery vehicle. The released composition then converts the oxalate present in the intestine to harmless products. Pharmaceutical or nutraceutical carriers can be combined with bacteria or enzymes. These may include, for example, physiological saline and phosphate buffer or a bicarbonate buffer. The methods of the present invention include administering oxalate reducing compositions to the gastrointestinal tract of humans or animals.

[0034] Other uses of administering these compositions containing one or more microorganisms, one or more oxalate reducing enzymes or combinations and mixtures to the intestines include adding the composition directly to the food source. One or more bacteria can be added as freshly harvested cells, freeze dried cells or other protected cells. One or more enzymes may be added as lyophilized proteins, enzymes complexed with other materials to maintain enzyme activity, and other methods known to those skilled in the art for adding active enzymes to the composition. Food products can be supplemented with oxalate decomposing compositions without affecting their taste and appearance. These food products may be, for example, yogurt, milk, peanut butter or chocolate. After ingestion, when food products are digested and absorbed through the intestines, oxalate degrading compositions containing one or more microorganisms, one or more enzymes or combinations, degrade oxalate present in the intestines, thereby reducing the absorption of oxalate into the bloodstream.

[0035] As noted above, many food products can be supplemented with oxalate degrading compositions. Methods for making such food products containing oxalate reducing compositions include mixing the food material with an oxalate reducing composition. For example, oxalate-reducing microorganisms can be cultured in media and isolated from media for example by centrifugation. Traditional yogurt cultures obtained from a commercial dairy can be mixed with a culture of oxalate-degrading microorganisms. This mixture of cultures can then be added to a regular dairy yogurt masterbatch without adversely affecting the taste and texture. Yogurt can then be made and packaged using traditional commercial procedures. In another example, oxalate-degrading bacteria can be added to existing yoghurts. In a similar method, the oxalate reducing composition comprising one or more oxalate reducing enzymes can be added to a yogurt bacterial culture or to a food product in the form of yogurt.

[0036] Another example of the uses of the present invention is the addition of a composition reducing oxalate to milk after homogenizing and sterilizing it. Currently, such a method is used in the dairy industry for adding Lactobacillus acidophilis to milk. Any source of food containing bacteria can be used by supplementing with oxalate-degrading bacteria. These food products include cheese or meat products to which the desired microorganisms are added during processing. Food products containing oxalate reducing compositions containing oxalate reducing enzymes are not limited to those foods that contain microorganisms but include any food source to which active enzymes can be added. Materials commonly considered as food materials can be used as a carrier material for enzymes, such that the enzymes are active with oxalate present in the food material at any stage of production or growth of the food material, or at any stage or digestion by human or animal, or with oxalate present in intestine.

[0037] In one embodiment, the bacterial strains, for example O. formigenes, used in accordance with the present invention are pure cultures that are isolated from anaerobic cultures that have been inoculated with dilutions of intestinal contents from healthy people or, when used in animals, from healthy animals . Special media containing calcium oxalate can be used to detect oxalate-degrading colonies. In one embodiment, the purity of each strain can be ensured by using at least two consecutive repeated cloning steps.

[0038] O. formigenes strains useful in accordance with the present invention have been characterized based on several tests, which include: fatty acid cellular patterns, cellular protein, DNA and RNA patterns (Jensen, NS, MJ Allison (1995) "Studies on the diversity among anaerobic oxalate degrading bacteria now in the species Oxalobacter formigenes "Abstr. to the General Meeting of the Amer. Soc. Microbiol., 1-29) and responses to oligonucleotide probes (Sidhu et al. 1996). Two groups of these bacteria have been described (Groups I and II, both existing within this species description). The strains used were selected based on the oxalate degradation capacity and evidence of human colonic tract colonization ability. Selected strains include representatives of both Groups I and II species.

[0039] One embodiment of the present disclosure includes procedures for selecting, preparing and administering appropriate oxalate-degrading bacteria to different individuals. Mostly, but not exclusively, they are people or animals that do not have these bacteria in their intestines. These uncolonized or weakly colonized individuals or animals are identified using tests that allow O to be detected quickly and definitively. formigenes, even when the organisms are at relatively low concentrations in mixed populations of bacteria such as those found in intestinal contents. The methods of the present invention can also be used to treat individuals or animals in which the number of bacteria that degrade oxalate has decreased due to, for example, antibiotic treatment or in postoperative situations. The methods of the present invention may also be used to treat individuals or animals that have colonies of oxalate degrading bacteria, but who still have unhealthy oxalate levels due to, for example, oxalate sensitivity and / or excessive production of endogenous oxalate.

[0040] Bacteria that can be used in accordance with the present invention can be identified by at least two methods:

1) oligonucleotide probes specific for these bacteria can be used; and / or

2) in a culture-based test in which an anaerobic environment with 10 mM oxalate is inoculated and after incubation at 37 ° C for 1 to 7 days, oxalate loss is determined.

[0041] Methods for making pharmaceutical compositions are provided herein, and methods for growing bacteria are generally known to those skilled in the art. For example, pure cultures of O. formigenes strains can be grown in batch fermenters in large fermenters and cells can be harvested using techniques known to those skilled in the art. Cells from a selected single strain or from mixtures of known strains can be processed as needed (e.g. freeze-dry with trehalose or glycerol) to maintain viability. Bacterial cells, either fresh from fermentation or from frozen stock preparations, can be mixed with carriers or excipients and then lyophilized. The delivery carrier with the powder composition is then introduced.

[0042] The pharmaceutical compositions provided herein are taken in dosages and amounts and at intervals determined for the needs of the individuals. In some cases, a single or periodic dose may be all you need, and in other cases regular intake (e.g. with meals) may be required. Dosages are given to obtain an effective amount. Pharmaceutical compositions contain live bacteria that reduce oxalate and / or enzymes that reduce oxalate alone or in combination with physiologically acceptable excipients or carriers or pharmaceutical carriers or excipients, such terms are used interchangeably herein. The dose of the pharmaceutical composition of the present invention may be less than, equal to or greater than the amount of oxalate constitutively produced and / or taken by the subject in an effective amount of oxalate reducing dose administered over a period of time. For some oxalate-related conditions, the amount of oxalate-reducing activity administered in the methods and compositions of the present invention may be less than the amount of oxalate ingested or constitutively produced, and may only require complementing or supplementing the low level of oxalate reduction in the patient. For other conditions, more oxalate reducing activity may be required.

[0043] For example, in primary hyperoxaluria (PH), which is a genetic disease and the most severe form of hyperoxaluria, patients produce about 100-300 mg of oxalate per day. Methods of treating PH and preventing the sequelae of PH include administering an amount of oxalate reducing composition that is effective to reduce the amount of oxalate by at least 100-300 mg per day or at least 200 mg per day, or 300 mg per day, or over 300 mg per day. daily, or 400 mg daily. Such a regimen of administration can be carried out by the oral route of administration. For example, if provided by any free oral delivery medium disclosed herein containing a composition of lyophilized oxalate-reducing bacteria, the oral delivery medium may be delivered at least once a day, at least twice a day, at least three times a day, which at least four times a day or when needed to provide an effective amount of oxalate reducing activity.

[0044] In the case of powder formulations, the carriers may be dry materials based on solids.

[0045] Typical carriers for dry preparations include trehalose, maltodextrin, rice flour, microcrystalline cellulose (MCC), magnesium stearate, inositol, FOS, (fructose oligosaccharides), gluco-oligosaccharides (GOS), dextrose, sucrose and similar carriers.

[0046] Suitable carriers include aqueous and oily carriers such as, for example, white petrolatum, isopropyl myristate, lanolin or lanolin alcohols, mineral oil, sorbitan monooleate, propylene glycol, cetyl stearyl alcohol (together or in various combinations), hydroxypropyl cellulose (MW = 100,000 to 1,000,000), detergents (e.g., polyoxyl stearate or sodium lauryl sulfate) and are mixed with water to form a lotion, gel, cream or semi-solid composition. Other suitable carriers include water-in-oil or oil-in-water emulsions and mixtures of emulsifiers and emollients with solvents such as sucrose stearate, sucrose salt and coconut oil acids, sucrose distearate, mineral oil, propylene glycol, 2-ethyl-1,3 - hexanediol, polyoxypropylene ether-stearyl and water. For example, emulsions containing water, glycerol stearate, glycerin, mineral oil, synthetic spermaceti, cetyl alcohol, butylparaben, propylparaben and methylparaben are commercially available. Preservatives may also be included in the carrier, including methylparaben, propylparaben, benzyl alcohol and ethylenediaminetetraacetic acid salts. Well known flavors and / or colors may also be included in the carrier. The composition may also contain a plasticizer such as glycerol or polyethylene glycol (MW = 800 to 20,000). The carrier composition can vary as long as it does not significantly harm the viability of oxalate reducing bacteria or oxalate reducing enzymes in the composition.

[0047] A typical composition of this invention may further comprise any of the following inactive ingredients: acacia, aspartame, citric acid, D&C Yellow No. 10, FD&C Yellow No. 6, flavoring agent (natural and / or artificial), polysorbate 80, propylene glycol alginate, colloidal silicon dioxide as well as sucrose and xanthan gum.

[0048] The composition may also contain the following inactive ingredients: aspartame, beta carotene, citric acid, flavoring (natural and / or artificial), glycerin, maltol, mannitol and methylcellulose.

[0049] In the methods disclosed herein for making pharmaceutical compositions O. formigenes for oral delivery to the gastrointestinal tract, comprising growing bacteria using fermentation methods known to those skilled in the art, optionally freezing bacterial cells, thawing frozen cells and lyophilizing bacterial cells, optionally mixing in excipient solution, followed by screening the lyophilized cells to obtain a powder and providing a powder in a delivery medium for pharmaceutical preparation.

[0050] The invention further relates to the administration to the human or animal's gastrointestinal tract of oxalate degrading products or oxalate degrading products made from oxalate-reducing organisms such as O. formigenes cells or from other sources, or by such methods as using recombinant agents. In one embodiment, the oxalate degrading enzymes can be purified and prepared as a pharmaceutical or nutraceutical composition for oral ingestion. In a preferred embodiment, these enzymes are produced recombinantly. DNA sequences encoding these enzymes are known to those skilled in the art and are described, for example, in WO 98/16632. These sequences, or other sequences encoding oxalate degrading proteins, can be expressed in a suitable host. The host can be, for example, E. coli or Lactobacillus. The transformed host would contain appropriate regulatory and transport signals. The expressed protein can be isolated, purified and administered as described herein. Alternatively, a recombinant host expressing the desired oxalate degrading proteins can be administered. The recombinant host can be administered either live or non-live. In another preferred embodiment, the enzymes are coated or otherwise formulated or modified to protect the enzymes such that they are not inactivated in the stomach and are available to exert oxalate-degrading activity in the small intestine. Examples of such formulations are known to those of skill in the art and are described, for example, in US Patent No. 5,286495.

[0051] The term oxalate degrading enzymes as used herein includes all enzymes involved in oxalate pathways and includes, but is not limited to, oxalate oxidase, oxalate decarboxylase, formyl-CoA transferase and oxalyl-CoA decarboxylase. Oxalate oxidase is expressed in higher plants and catalyzes the oxygen-dependent oxidation of oxalate to CO2 while producing H2O2. Oxalate oxidases have been purified from many sources, for example, the roots and leaves of barley seedlings; beet stalks and leaves; wheat germ, sorghum leaves and banana peel. A quick three-step purification procedure was developed for obtaining oxalate oxidase from barley roots. The gene encoding barley root oxalate oxidase has been cloned, sequenced and expressed.

[0052] Oxalate decarboxylase is mainly present in mushrooms. Bacterial oxalate decarboxylase has recently been found in B. subtilis and it is encoded by the yvrk gene. Oxalate decarboxylases catalyze the decomposition of free oxalate into CO2 and formate. This enzyme has been found in many fungi, including Myrothecium, verrucaria, certain Aspergillus niger strains and the wood rot fungus, Coriolus versicolor. The gene encoding Flammulin velutipes oxalate decarboxylase was cloned and sequenced; See WO 98/42827.

[0053] Oxalyl-CoA decarboxylase is active on the activated CoA substrate and converts it to formyl-CoA. Formyl-CoA transferase then works and exchanges formate and oxalate at CoA. These enzymes have been studied in oxalate-degrading bacteria, Pseudomonas oxalaticus, present in soil and in Oxalobacter formigenes, inhabiting the gastrointestinal tract of vertebrates, including humans. It has been shown that O. formigenes is in a symbiotic relationship with the host by regulating the absorption of oxalic acid in the intestine as well as plasma oxalic acid levels. As a result, it was found that the absence of this bacterium is a risk factor in oxalate-related disorders such as recurrent idiopathic urolithiasis due to calcium oxalate and intestinal hyperoxaluria secondary to fasting-ileal anastomosis, cystic fibrosis and inflammatory bowel disease.

[0054] Patent descriptions describing various oxalate-degrading enzymes and genes encoding these enzymes include US Patent Nos. 5,912,125; 6090628 and 6214980. The term oxalate degrading enzyme includes, but is not limited to, oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase and formyl-CoA transferase, and includes enzymes that are capable of interacting with oxalate or oxalic acid. These enzymes may be derived from natural sources or be synthesized using recombinant means known in the art and include all fragments such as binding sites, active sites or fragments capable of interacting with oxalate or oxalic acid. The term also includes, but is not limited to, all necessary cofactors, coenzymes, metals or binding materials or substrates that the enzyme needs to interact with oxalate or oxalic acid. Any binding partners for these enzymes are also contemplated in accordance with the present invention, and includes antibodies and antibody fragments that bind or interact with enzymes.

[0055] The use of O. formigenes is particularly advantageous because it is an anaerobic that does not grow in aerobic environments in tissues and does not produce any compounds that are toxic to humans or animals. As an alternative to O. formigenes or a recombinant host, other oxalate-degrading bacteria such as Clostridium, Bacillus subtilis, Pseudomonas, Lactobacilli, Bifidobacteria can be used. Oxalate-degrading enzymes made from such an alternative bacterium may be administered, or the entire microorganism may be administered.

[0056] In addition, all of the above-mentioned forms are suitable for use in domestic animals, farmed or kept in a zoo, suffering from a deficiency in the number of bacteria decomposing oxalate, as well as in humans. For example, oxalate and / or microbial degrading enzymes can be administered to domestic animals such as dogs, cats, rabbits, ferrets, guinea pigs, hamsters and gebrils, as well as breeding animals such as horses, sheep, cows and pigs, or to wild animals kept for farming purposes, such as otters. Many animals that are able to reduce the amount of oxalate lose this ability upon capture. The present invention includes methods and compositions for restoring lost or reduced oxalate reducing activity. One aspect of the present invention includes the treatment of animals obtained from the wild which have lost or have reduced oxalate-reducing activity with the compositions provided herein.

[0057] The present invention includes compositions and uses for administering compositions containing one or more bacteria, one or more enzymes or a combination of bacteria and oxalate-degrading enzymes to the human or animal gastrointestinal tract. Such compositions and uses are effective in reducing the amount and / or concentration of oxalate present. Such uses and compositions are effective in the treatment and prevention of oxalate related conditions. An aspect of the present invention includes compositions and methods for introducing oxalate degrading enzymes into the gastrointestinal tract of a human or animal. The present invention includes uses for delivering one or more oxalate-degrading enzymes to the human or animal's gastrointestinal tract as pharmaceutical and / or nutraceutical carrier compositions. Such enzymes include, but are not limited to, oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl-CoA transferase. These enzymes may be derived from sources known to those skilled in the art. For example, the plant enzyme, oxalate oxidase (OXO), can be purified from barley seedlings, and oxalate decarboxylase can be purified from bacterial or fungal sources.

[0058] Alternatively, oxalate-degrading enzymes can be obtained using recombinant agents. For example, recombinant agents such as cloning, expression and purification can be used to obtain oxalate-reducing enzymes, for example, the B. subtilis oxalate decarboxylase enzyme. Such recombination methods are known to those skilled in the art. For example, cloning and expression of the oxalate decarboxylase (YvrK) B gene is generally disclosed. subtilis: The gene for oxalate decarboxylase (YvrK) protein has been cloned into the plasmid pET-9a and pET-14b (Novagen, WI), under the control of the strong bacteriophage T7 promoter, to overexpress as a soluble cytosolic protein. The expression host was strain E. coli BL 21 (DE3) pLysS, λDE3 lysogen with protease deficiency and which contains a chromosomal copy of the T7-RNA polymerase gene under lacUV5 control. In addition, this strain carries a pET-compatible plasmid that encodes T7 lysozyme, a bifunctional enzyme that cleaves binding in the cell wall peptidoglycan layer and inhibits T7 RNA polymerase. This allows greater control of uninduced basal expression and allows the use of methods that destroy the intima, such as freeze-thaw, or mild detergents, etc. for effective cell lysis. Gene expression is induced by the addition of isopropyl eD-thiogalactopyranoside (IPTG). Accordingly, an aspect of the present invention includes methods comprising administering oxalate-degrading enzymes that have been produced by a recombinant microorganism. Various expression and host vectors can be used to produce oxalate-degrading enzymes, and such methods are known to those skilled in the art.

[0059] Another aspect of the present invention includes uses for reducing oxalate absorption by providing oxalate-degrading bacteria to the human or animal's gastrointestinal tract. Such bacteria may include, but are not limited to, Oxalobacter formigenes, Clostridium, Lactobacilli, Bifidobacteria and Pseudomonas. O. formigenes was isolated from human stool samples and cloned by selection of individual colonies. This includes the HC-1 isolate that was originally obtained from Ixion Biotechnology in 1996 from Dr. Milton Allison. For example, frozen stock preparations of the human HC-1 strain may be used. The methods of the present invention include enriching the intestines with one or more species of oxalate-degrading bacteria, reducing the amount of oxalate in the intestinal contents generally, reducing the absorption of oxalate in the intestines, reducing the concentration of oxalate in the blood and kidney fluids, and reducing the harmful effects on the body caused by the presence of oxalate.

[0060] Accordingly, an aspect of the present invention includes compositions and uses for providing oxalate-reducing bacteria and oxalate-degrading enzymes that can reduce the amount of oxalate in the gastrointestinal tract of individuals at increased risk of oxalate related diseases and / or conditions. Such diseases and conditions include, but are not limited to, hyperoxaluria, primary hyperoxaluria, idiopathic diseases involving calcium oxalate kidney stones (urolithiasis), intestinal hyperoxaluria, vulvodynia, oxalosis associated with end-stage renal disease, conduction disorders, inflammatory bowel disease , Crohn's disease, ulcerative colitis, people who have undergone an operation to develop fasting-ileal anastomosis, people with insufficient concentrations of bacteria that degrade oxalate, and other intestinal disease states. Humans and animals after antibiotic, chemotherapeutic or other treatments that alter the intestinal flora are treated with the compositions and methods of the present invention. The present invention is used to restore the ability to reduce oxalate in humans and animals with altered intestinal flora. Increased urinary oxalate excretion promotes the formation of kidney stones, contributes to scarring of the kidneys, and can even cause kidney failure. Accordingly, an aspect of the present invention includes compositions and uses for reducing kidney stone formation.

[0061] Reduction of overall oxalate concentrations in the intestine can also lead to the removal of oxalate from the cells and general circulation. More specifically, a decrease in oxalate concentration in the intestine can also lead to increased secretion of oxalate into the intestine from the blood. Although without wishing to be bound by any particular theory, it is now believed that there is a transepithelial gradient for the elimination of oxalate in the intestines. Accordingly, an aspect of the present invention includes compositions and methods for lowering oxalate levels in the blood and increasing oxalate excretion by promoting the excretion of oxalate from the blood through a transepithelial oxalate gradient for excreting oxalate in the colon. The method of the present invention includes providing to the intestines of a human or animal a composition for lowering the concentration or level of oxalate in a human or animal. Such lowering may include lowering the amount of oxalate found in the intestines, blood, serum, tissue fluids and other body fluids.

[0062] One composition of the present invention comprises an O. formigenes paste prepared for oral administration. For each lot of O. formigenes paste, a single vial of HC-1 stock preparation is used to produce a seed culture to initiate growth in large-scale production fermentation. Bacteria from each fermentation are harvested by centrifugation and mixed with cryoprotective excipients that provide protection against freeze-drying. The cell paste can also be subjected to freeze drying or spray drying or vacuum drying to obtain a fine powder with a concentration in the range of 10⁷ up to 10⁹ CFU / gram.

[0063] The compositions of the present invention include compositions made from extracts of one or more oxalate reducing bacteria in the range of³ up to 10¹² CFU / gram, from 10³ up to 10¹⁰ CFU / gram, from 10⁵ up to 10¹² CFU / gram, from 10⁵up to 10¹⁰ CFU / gram, from 10⁷ up to 10⁹ CFU / gram, from 10⁷ up to 10⁸ CFU / gram and all ranges in between.

[0064] Also described herein are compositions comprising one or more enzymes that have oxalate reducing activity. An aspect of the invention includes administering an effective amount of the enzyme composition to the gastrointestinal tract of a human or animal. An effective amount of the enzyme composition is capable of reducing the proportion of oxalate in the intestines or reducing the concentration of oxalate in a human or animal relative to the level measured prior to administration of the composition. Such a measurement can be a measurement of oxalate present in the intestine from food sources or it can be a level measured in a body fluid such as blood or urine.

[0065] The present invention includes the use for the treatment or prevention of oxalate related conditions by administering compositions containing O. formigenes to the gastrointestinal tract of a human or animal. Individuals can be dosed₃ gastro-resistant capsules containing> 10 CFU / g live O. formigenes cells. This dosage can be done at least twice a day with meals. The present invention also includes uses for administering oxalate reducing compositions containing one or more oxalate reducing microorganisms, one or more oxalate reducing enzymes, or combinations thereof. The use of the present invention comprises administering at least once a day an effective amount of an oxalate reducing composition, wherein the oxalate reducing composition comprises one or more oxalate reducing enzymes. The uses also include administering such compositions more than once daily, more than twice daily, more than three times daily, and in a range of 1 to 15 times daily. Such administrations can be continuous, such as daily for days, weeks, months or years, or can be made at specific times for the treatment or prevention of oxalate related conditions. For example, a person or animal may be administered oxalate reducing compositions at least once a day for years for the treatment or prophylaxis of oxalate related conditions, or a person or animal may be administered oxalate reducing compositions at least once a day only when taken. food products containing oxalate or for a limited period of time, such as days or weeks, after treatments or therapies, which disrupt the normal bacterial flora. Such administration can be accomplished by routes known for administration of pharmaceutical agents. Administration by oral or enteral route or in combination with food materials are contemplated by the present invention.

[0066] Also described herein is a therapeutic system for reducing oxalate comprising a container containing a label and a therapeutic composition according to the present invention, said label providing instructions for using the composition for reducing oxalate.

[0067] Typically, the system is in the form of a package containing the therapeutic composition of this invention or in combination with a packaging material. The packaging material includes a label or instructions for use of the packaging components. The instructions indicate the contemplated use of the packaging component as described herein for the methods or compositions of the invention. For example, the system may contain one or more unit doses of the therapeutic composition of the invention. Alternatively, the system may contain large amounts of the therapeutic composition. The label contains instructions for the use of the therapeutic composition in unit dosage form or in a large amount, if appropriate, and may contain information regarding storage of the composition, disease indications, doses, routes of administration and the like.

[0068] The present invention includes pharmaceutical compositions and uses for reducing oxalate in humans and animals. The composition for reducing oxalate concentration in a human or animal comprises an oral delivery vehicle containing an oxalate reducing composition comprising, a) from 0.5% to 95% of an oxalate reducing bacterium; b) from 0.1% to 50% disaccharide; c) from 3% to 85% of maltodextrin; d) from 0.5% to 25% of alginate; and e) from 1.0% to 60% oligofructose. The composition may contain an oral delivery vehicle.

[0069] The compositions may contain an oxalate-reducing bacterium, which is Oxalobacter formigenes, Pseudomonas, Clostridia, Lactobacilli, Bifidobacteria or a bacterium transformed with one or more vectors containing exogenous or endogenous polynucleotide sequences encoding oxalate-reducing enzymes, or in which the compositions are the oxalate reducing bacterium is Oxalobacter formigenes or compositions, wherein the oxalate-reducing bacterium is Oxalobacter formigenes of the HC1 strain. Oxalate reducing compositions may be a lyophilized powder. The powder may have a particle size of 10 microns to 2,000 microns or from 100 microns to 1,000 microns, or from 500 microns to 1,500 microns, or from 500 microns to 1,000 microns, 500 to 1,500 microns, or in any ranges within these or near ones.

[0070] The composition may contain disaccharide, trehalose, or the alginate present therein is sodium alginate. The oxalate reducing composition may contain at least 1E + 03 to 1E + 13 cfu / g oxalate reducing bacteria. The oxalate-reducing composition may have an oxalate-reducing enzyme activity of at least from 2 mg of oxalate degraded / h to 2500 mg of oxalate degraded / h.

[0071] The composition for reducing oxalate concentration in a human or animal may comprise an oral delivery vehicle containing an oxalate reducing composition comprising, a composition comprising a delivery vehicle containing a composition comprising, a) from 3% to 25% oxalate reducing bacteria; b) from 1.5% to 6% disaccharide; c) from 45% to 60% maltodextrin; d) from 4% to 6% of alginate; and e) from 20% to 35% oligofructose.

[0072] The composition for reducing oxalate comprises an effective amount of oxalate-reducing activity that will reduce the proportion of oxalate present, comprising a) from 0.5% to 95% of a live, lyophilized oxalate-reducing bacterium; and b) from 95% to 0.5% of a pharmaceutically acceptable excipient, and further comprises a pharmaceutical delivery vehicle. The pharmaceutical delivery vehicle may be a powder. An effective amount of oxalate-reducing activity can be provided by oxalate-reducing bacteria, which can be Oxalobacter formigenes, Pseudomonas, Clostridia, Lactobacilli, Bifidobacteria, or a bacterium transformed with one or more vectors containing exogenous or endogenous polynucleotide sequences encoding enzymes that reduce the amount of oxalate it may be Oxalobacter formigenes or which may be Oxalobacter formigenes of the HC1 strain. The composition may be provided in the form of a lyophilized powder. The powder may have a particle size of 10 microns to 2,000 microns or from 100 microns to 1,000 microns, or from 500 microns to 1,500 microns, or from 500 microns to 1,000 microns, 500 to 1,500 microns, or in any ranges within these or near ones. The oxalate reducing composition may contain at least from about 1E + 03 to about 1E + 13 cfu / g oxalate reducing bacteria. The oxalate reducing composition may have an enzymatic activity that reduces the amount of oxalate / g from at least 2 mg of decomposed oxalate / h to 2500 mg of decomposed oxalate / h.

[0073] The uses of the present invention include reducing oxalate concentrations in humans and animals, treating oxalate related conditions in humans and animals, prophylaxis of oxalate related conditions in humans and animals, and methods for preparing oxalate reducing compositions. The present invention also includes systems for reducing oxalate. The use for reducing oxalate concentration in a human or animal comprises administering to the human or animal an effective amount of a composition comprising a delivery vehicle comprising an oxalate reducing composition comprising a) from 0.5% to 95% oxalate reducing bacteria; b) from 0.1% to 50% disaccharide; c) from 3% to 85% of maltodextrin; d) from 0.5% to 25% of alginate; and e) from 1.0% to 60% oligofructose. The delivery substrate is a powder. The oxalate-reducing bacterium can be Oxalobacter formigenes, Pseudomonas, Clostridia, Lactobacilli, Bifidobacteria or a bacterium transformed with one or more vectors containing exogenous or endogenous polynucleotide sequences encoding oxalate-reducing enzymes, it can be Oxalobacter formigenes, or it can be an Oxobacter formigenal strain . The composition may be provided in the form of a lyophilized powder. The powder may have a particle size of 10 microns to 2,000 microns or from 100 microns to 1,000 microns, or from 500 microns to 1,500 microns, or from 500 microns to 1,000 microns, 500 to 1,500 microns, or in any ranges within these or near ones. The composition may contain disaccharide, trehalose, or the alginate present therein is sodium alginate. The oxalate reducing composition may contain at least from about 1E + 03 to about 1E + 13 cfu / g oxalate reducing bacteria. The oxalate reducing composition may exhibit an enzymatic activity that reduces the amount of oxalate / g from at least 2 mg of decomposed oxalate / h to 2500 mg of decomposed oxalate / h when administered by the oral route of administration.

[0074] Applications may also include prophylaxis of an oxalate-related condition involving the administration of the compositions provided herein. Such uses may also include treating an oxalate related condition comprising administering the compositions provided herein. Conditions associated with oxalate include, but are not limited to, hyperoxaluria, primary hyperoxaluria, idiopathic diseases involving calcium oxalate kidney stones (urolithiasis), intestinal hyperoxaluria, vulvodynia, oxalosis associated with end-stage renal disease, conduction heart disease, inflammatory bowel disease , ulcerative colitis, Crohn's disease, fatty stools, patients after gastrointestinal tract surgery, such as surgery to create an anastomotic bypass or after antibiotic treatment. Uses include administering the compositions given herein more than once a day for a period of time until the oxalate level is sufficiently reduced or for an indefinite period of time to monitor oxalate levels continuously. The use includes prophylaxis of a oxalate-related condition comprising administering to a human or animal an effective amount of oxalate-reducing activity that will reduce the proportion of oxalate present, including: a) from 0.5% to 95% of a live, lyophilized oxalate-reducing bacterium; and b) from 95% to 0.5% of a pharmaceutically acceptable excipient, and further comprising a pharmaceutical delivery vehicle. The pharmaceutical delivery vehicle may be a powder. The oxalate-reducing bacterium can be Oxalobacter formigenes, Pseudomonas, Clostridia, Lactobacilli, Bifidobacteria or a bacterium transformed with one or more vectors containing exogenous or endogenous polynucleotide sequences encoding oxalate-reducing enzymes, it can be Oxalobacter formigenes, or it can be an Oxygen strain HC1. The composition may be provided in the form of a lyophilized powder. The powder may have a particle size of 10 microns to 2,000 microns or from 100 microns to 1,000 microns, or from 500 microns to 1,500 microns, or from 500 microns to 1,000 microns, 500 to 1,500 microns, or in any ranges within or around these. The composition may contain disaccharide, trehalose, or the alginate present therein is sodium alginate. The oxalate reducing composition may contain at least 1E + 03 to 1E + 13 cfu / g oxalate reducing bacteria. The oxalate reducing composition may have an enzymatic activity that reduces the amount of oxalate / g from at least 2 mg of decomposed oxalate / h to 2500 mg of decomposed oxalate / h. The composition may be administered by oral routes of administration. Oxalate-related conditions that can be treated or prevented include hyperoxaluria, primary hyperoxaluria, idiopathic diseases involving calcium oxalate kidney stones (urolithiasis), intestinal hyperoxaluria, vulvodynia, oxalosis associated with end-stage renal disease, conduction disorders in the heart , inflammatory bowel disease, ulcerative colitis, Crohn's disease, fatty stools, patients who have undergone gastrointestinal tract surgery, such as surgery to bypass the iliac artery or after antibiotic treatment.

[0075] Methods for producing a oxalate reducing pharmaceutical composition include providing oxalate reducing bacteria at a concentration of at least from about 1E + 03 to about 1E + 13; optionally mixing the oxalate reducing bacterium with one or more pharmaceutically acceptable excipients; lyophilization of bacteria; and loading or delivering the bacteria to a pharmaceutical delivery vehicle. Such excipients may contain one or more substances from disaccharide, maltodextrin, alginate or oligofructose. The oxalate-reducing bacterium can be Oxalobacter formigenes, Pseudomonas, Clostridia, Lactobacilli, Bifidobacteria, or a bacterium transformed with one or more vectors containing exogenous or endogenous polynucleotide sequences encoding oxalate-reducing enzymes, it can be an Oxalobacter formigenes strain, or it can be an Oxalobacter formigenes strain HCl. The pharmaceutical delivery vehicle may be a powder.

[0076] It should be noted that the singular forms used in this description and the appended claims include references to the plural, unless the context clearly indicates otherwise. [0077] The following are examples that illustrate procedures for practicing the invention.

EXAMPLE 1

Treatment of high-risk patients [0078] Patients with primary hyperoxaluria were administered enteric-coated capsules containing freeze-dried O. formigenes powder twice daily, preferably with their two large meals per day. Each size 2 capsule contained an eye₈ 137 mg of freeze-dried loose powder, containing at least 10⁸ colony forming units (CFU) / gram.

[0079] For high-risk individuals, this may constitute lifelong treatment. It has been shown that in clinical trials, colonization decreased after stopping treatment. In the clinical trial, the treatment was carried out for 4 weeks and there was a biweekly catamnesis period. The 4-week treatment resulted in a significant decrease in blood and urine oxalate levels compared to baseline. However, during the catamnesis period, Oxalobacter levels in the stool dropped, and plasma and urine oxalate levels began to increase. Thus, it is suggested that continuous administration of oxalate reducing compositions will be needed to provide a reduced oxalate state. Compositions containing bacteria that colonize and persist in the intestine could lead to a reduction in the need for oxalate reducing compositions.

[0080] Enteric-coated capsules with O. formigenes cells can be taken by patient populations at high risk of oxalate-related diseases. They include:

1. People who produce too much endogenous oxalate, for example due to a genetic defect such as primary hyperoxaluria

2. People at risk of urolithiasis with high levels of oxalate in the urine due to an intestinal disease (intestinal hyperoxaluria).

3. People with a history of urolithiasis with many episodes of idiopathic disease with stones.

4. People with high levels of serum oxalate due to end-stage renal disease.

5. People with vaginitis.

6. People whose diet contains high levels of oxalate, as is the case in certain areas and seasons in India and Saudi Arabia. This will also include potential individuals who prefer high oxalate foods, such as spinach.

[0081] Any of the above described persons or animals are provided a composition of the present invention. For example, a person with higher than normal endogenous oxalate levels is treated twice a day with a capsule designed to deliver its contents to the large intestine, the capsule containing about 10⁶ cfu O. formigenes. The capsule is preferably administered with food.

EXAMPLE 2

Treatment of low-risk patients:

[0082] Enterally protected O. formigenes cells, such as those provided in enteric-coated capsules, can be taken by individuals in populations with a lower risk of oxalate-related disease. It would be desirable to colonize these patients with one or two administrations containing oxalate reducing material compositions, such as oxalate reducing bacteria. These patients could also routinely receive treatment with oxalate-reducing materials in the form of supplements or as food additives such as milk or yogurt. They include:

1. People who lost populations of normal oxalate-degrading bacteria as a result of: treatment with oral antibiotics or attacks of diarrhea disease.

2. Infants can be vaccinated so that a normal protective Oxalabacter population will be much easier to produce than later in life when competitive exclusion rules are in force.

[0083] Low-risk persons or animals are administered a capsule twice daily to deliver its contents to the large intestine, the capsule containing ₇ at least 10 cfu of one or more oxalate reducing organisms such as O. formigenes. The capsule is preferably administered with food. EXAMPLE 3

Use of Oxalobacter formigenes degrading oxalate enzymes to combat hyperoxaluria [0084] The study was conducted to evaluate the effectiveness of Oxalobacter formigenes degrading oxalate enzymes in controlling hyperoxaluria.

[0085] Animals used: Male Sprague Dawley rats: body weight 250-300 g [0086] Diet used: Normal Diet (ND): Harlan Teklad TD 89222; 0.5% Ca, 0.4% P [0087] Drug used: A lyophilized mixture of Oxalobacter formigenes lysate (enzyme source) with Oxalyl-CoA, MgCl2 and TPP.

[0088] Drug delivery system (Capsules): Size 9 capsules for preclinical studies in rats (Capsu-Gel). Eudragit L-100-55 enteric coating (Hulls America, Inc.). Baseline urine collection from 24 h. Analysis of feces for Oxalobacter formigenes - rats were not colonized with Oxalobacter formigenes.

Experimental protocol:

A. Long-term studies:

Animal protocol:

[0089] Group I (n = 4): Oxalate diet with lysate was administered. Rats received two capsules each day at 16:00 and an oxalate diet overnight. Diet discontinued during the day (8:00 am to 4:00 pm) Group II (n = 4): Oxalate diet was administered as described for Group I (control rats with hyperoxaluria).

[0091] 24 h urine samples were collected on Day 7 and Day 9 of the above treatment.

[0092] Data on the average urinary oxalate concentration for the two groups of rats shown above indicated that administration of Oxalobacter lysate reduced the urinary oxalate concentration in Group I rats compared to hyperoxaluria control rats (Group II). Enzymes cannot be active for a long time in the gastrointestinal tract; therefore short-term studies were performed as described below.

B. Short-term studies:

Animal protocol:

[0093] Group I (n = 4): 1 capsule was administered at 8:00; oxalate diet for two hours (rats were fasted overnight to eat well during this period) and 1 capsule at 10:00.

[0094] Group II (n = 4): Oxalate diet for two hours as for Group I. [0095] Urine was collected from all animals for the next five hours and analyzed for oxalate concentration.

[0096] This was done on days 11, 12 and 15 of this study.

[0097] The results of this study show that administration of Oxalobacter lysate causes a significant decrease in urinary oxalate levels within 5 hours after oxalate and drug administration in Group I rats compared to the hyperoxaluria control group (Group II). At this point, a cross-over study was performed between two groups of rats.

C. Cross-examination:

Animal protocol:

[0098] Group I: The oxalate diet was administered twice a day between 8:00 and 10:00 and 15:00 and 17:00.

[0099] Group II: 1 capsule was administered twice daily prior to administration of the oxalate diet as for Group I.

[0100] Short-term studies of the effect of administering Oxalobacter lysate on urinary oxalate levels were performed as described in the above part B on day 2 and day 5 after crossing the groups.

[0101] Alternate studies showed that Group II rats that previously had hyperoxaluria and were administered Oxalobacter lysate showed a decrease in urinary oxalate levels. In contrast, rats in Group I developed hyperoxaluria after withdrawal of the drug.

EXAMPLE 4

Treatment of the rat with Oxalobacter formigenes cells [0102] The study was performed to assess the fate of food oxalate when Oxalobacter formigenes cells were included in the diet.

methods:

[0103] Male Wistar rats were administered a high oxalate diet (0.5%) with normal calcium content (1%) or a low calcium (0.02%), high oxalate (0.5%) diet in two separate experiments. On day 1 and again on day 7, testing was given¹⁴C-oxalate (2.0 LiCi). Oxalobacter formigenes cells (380 mg / day) were administered in drinking water for rats on days 5-11. Moose¹⁴C from oxalate was measured based on analysis ¹⁴C in faeces, urine and exhaled air. Rats served as self controls and measurements during the control period (prior to administration of Oxalobacter cells) were made on days 1-4; measurements during the experimental period (when bacterial cells were given) were taken on days 7-11.

Results:

[0104]

1. When rats were given a diet with a normal calcium content (1%) less than 1% of the administered dose ¹⁴C from oxalate was recovered in exhaled air (as carbon dioxide produced from ¹⁴C oxalate in the intestine, absorbed into the blood and then exhaled) but more in all cases ¹⁴C was recovered during the period when rats were administered Oxalobacter cells (FIG. 1a). This contradicts the results obtained when the diet was low in calcium (0.02%), when over 50%¹⁴C from oxalate was recovered as carbon dioxide in exhaled air during the experimental period when rats were administered Oxalobacter cells (FIG. 1b). These results are strikingly different from very small amounts¹⁴C (less than 5%) recovered during the control period (before administration of Oxalobacter cells). Thus, administration of Oxalobacter formigenes cells to rats noticeably increased the amount of food oxalate that was broken down in the intestinal tract.

2. Administration of Oxalobacter cells also reduced the amount ¹⁴C-oxalate that was excreted in the urine. The values ​​from the collection for 4 days during both the control and experimental period and for one day in each of these periods are shown in FIG. 2a and 2b. The amounts of oxalate recovered in the feces of rats were also lower during the experimental period (when Oxalobacter cells were given) than the amounts found during the control period (FIG. 2c).

[0105] Most laboratory rats are not carriers of Oxalobacter in their intestinal tract (they are not colonized). These results showed that intentional administration of these oxalate-degrading bacteria in rats caused the degradation of a large portion of food oxalate and that as a result less oxalate from the diet was excreted in urine.

[0106] The effect of dietary calcium on oxalate degradation was clear. Calcium forms complexes with oxalate, so that its solubility and availability for attack by Oxalobacter is limited, and the amount that is broken down when rats are given a high calcium diet is much smaller than the amounts broken down when the calcium content in the diet is low.

EXAMPLE 5

Effect of administration of O. formigenes on urinary excretion of oxalate in pigs [0107] Pigs are naturally colonized with Oxalobacter. Decolonization was achieved in experimental pigs by supplementing the diet with antibiotics. Pigs were given Oxalobacter in a culture broth, which they willingly consumed. Pigs were fed soy / maize based feed supplemented with 1300 mg oxalate / kg. The initial diet contained 680 mg oxalate / kg. The results are shown in FIG. 3a-c for three individual pigs.

[0108] Urine oxalate levels in all three pigs clearly decreased during ingestion of Oxalobacter. Oxalate excretion levels in these pigs fell to a minimum of around 6 mg / g creatinine in all three pigs. This should be compared with a level of 8-10 mg / g creatinine, which was observed in people taking diets from oxalate mixtures. This level is equal to endogenous synthesis in humans because dietary delivery has been eliminated. It appears that this level reflects endogenous synthesis in pigs and that absorption in the intestines has been eliminated by treatment with Oxalobacter. In addition, these results indicate that ingested Oxalobacter were able to remove both added crystalline oxalate and oxalate derived from food that was bioavailable.

[0109] In this experiment, 1.0 g of cell paste was administered to each pig with po₈ an early meal. At O.D600 0.6, the number of viable cells is 2.1.x10⁸ cells / ml, 13 which can be extrapolated to 2.1.x10 cells per 100 liters. A series of 100 liters of fermenter provides on average 50-60 g of wet cell mass. Thus, in 1 g of moist cell mass there are about 3.5 x 10¹¹ live cells.

[0110] 3.5 x 10 dose¹¹ viable cells as indicated above could eliminate intestinal absorption of about 2.0 g oxalate present per kg diet (1300 mg added oxalate + 680 mg present in the diet). Animals consumed 1 kg of diet per meal.

[0111] The pig's body weight is about 200 pounds and the digestive system of pigs is thought to be very similar to human. In humans, the average daily oxalate intake is approximately 100-400 mg depending on the composition of the diet, which is further divided into three meals / 10, thus an average daily dose of 10⁸ up to 10¹⁰ live cells would be sufficient to prevent absorption of food oxalate.

EXAMPLE 6

Effect of O. formigenes supplementation on urinary excretion of oxalate in rats given high oxalate diet.

[0112] The study was conducted to determine the effect of IxOC-3 on the colonization status and urinary oxalate levels after administration of the high oxalate diet. IxOC-3 contains freeze dried live cells of oxalate-reducing bacteria such as O. formigenes. The preparation contains about 10⁶-10⁷ CFU / gram per dose. The preparation also contains cryoprotective agents such as trehelose and maltodextrin.

methods:

[0113] Male Harlan Sprague Dawley rats were randomly assigned to 3 groups (6 animals / group). Group 1 animals served as a control group and were given a placebo enteric-coated formulation of size 9 twice daily by oral gavage at a dose of 10⁰ Colony forming units (CFUs). Animals of Groups 2 and 3 were administered Oxalobacter formigenes IxOC in the form of an enteric-coated capsule size 9 twice daily by oral gavage at a dose of 10⁶ and 10⁷ CFU. In all three groups, the capsule tube was rinsed with autoclaved tap water. After the initial acclimatization period, all groups were given a standard diet supplemented with 1% oxalate per gram.

[0114] Test materials and placebo control materials were prepared according to a standardized protocol. Before use, representative samples from each test material were analyzed to confirm the identity, purity and potency of the test capsules, as well as to confirm the absence of Oxalobacter formigenes in the placebo control material during the dosing period.

The diet was limited to two one-hour periods per day starting 15 minutes after gavage capsules in the morning and evening to ensure that they were given to an empty stomach. Water was provided ad libitum. Food intake was recorded twice daily. Fecal and urine samples from 24 hours were taken on Day 1 (before supplementing with oxalate diet), followed by weekly. Urine data were analyzed by repeated measurement analysis for differences in mean urine parameters across the dose groups and over time. The interaction group of the dose group versus time was also included to evaluate any possible interactions between dose groups and time.

Results:

[0115] The results of the analysis indicated that there was a statistically significant interaction between dose groups and time (p <0.0001) for all parameters, indicating that the profile of urine parameters during this period was different among the dose groups. To facilitate interpretation of this interaction, a data analysis was performed by time point for each parameter to determine if there was a difference between dose groups with respect to mean urine parameters. This analysis showed that for the low dose and high dose groups, the urinary oxalate level increased from baseline to 7 days (p <0.0001 in both groups), but there was no increase from 7 days to 28 days (p = 0.1094 for low dose and p = 0.6910 for high dose). However, the placebo group increased from baseline to 28 days (p = 0.0010). Also on day 21 and day 28, the average urinary oxalate levels in Group I placebo were significantly higher than in the low (Group II) and high (Group III) dose groups, but without a significant difference between the low dose and high dose. Thus, there was an overall significant decrease in urinary oxalate excretion in treated rats compared to placebo-treated rats. EXAMPLE 7 [0116] The effect of oral administration of O. formigenes on urinary oxalate levels in patients suffering from primary hyperoxaluria (PH).

methods:

[0117] Nine patients with primary hyperoxaluria (PH) confirmed by biopsy participated in the study. After obtaining initial baseline assessments, all subjects were administered 1 g of Oxalobacter forimigenes cell paste (> 10¹⁰ CFU / gram) twice a day (bid) with their main meals for 4 weeks. During this period of time, all patients continued to take their normal medication, they were asked to eat their normal diet and keep their fluid intake as high as normal. With the exception of spinach and rhubarb, high oxalate foods were not banned. Oxolobacter colonization and its effect on urinary and plasma oxalate levels were measured at weeks 5 and 6. Treatment efficacy was observed in terms of urinary excretion of oxalate in subjects with normal renal function and plasma oxalate levels in subjects with end-stage renal disease (ESRD).

Results:

[0118]

1. Treatment showed a significant decrease in urinary oxalate levels in subjects with normal urine function. Plasma oxalate levels dropped significantly in seven out of nine individuals. There was a sharp decrease in plasma oxalate levels in two individuals with ESRD, which confirms the elimination of endogenous oxalate into the intestine in the intestines against a transepithelial gradient.

2. Consumption of O. formigenes strain HC-1 at doses ranging from 0.25 g to 2.0 g per meal was well tolerated by normal, healthy volunteers receiving a diet containing average or high oxalate levels. Dosage of 1.0 g cell paste twice a day for 28 days was well tolerated by PH patients.

EXAMPLE 8

Treatment of high-risk patients with oxalate-reducing enzyme compositions [0119] Patients with primary hyperoxaluria are administered one or more enteric-coated capsules containing a lyophilized oxalate-reducing enzyme composition containing oxalate decarboxylase and / or oxalate oxidase, twice daily preferably with two main meals of the day. An effective amount of the enzyme composition is administered. For example, each size 2 capsule contains about 5-100 units of each enzyme.

[0120] For high risk individuals, this is continuous administration over an extended period of time, probably lifelong treatment. Colonization will decrease when treatment is stopped.

[0121] Enteric-coated capsules of oxalate-reducing compositions containing oxalate-reducing enzymes can be administered to patient populations at high risk of oxalate-related disease. They include:

1. People who produce too much exogenous oxalate, for example due to a genetic defect such as primary hyperoxaluria

2. People at risk of urolithiasis with high levels of oxalate in the urine due to an intestinal disease (intestinal hyperoxaluria)

3. People with a history of urolithiasis with many episodes of idiopathic disease with stones.

4. People with high levels of serum oxalate due to end-stage renal disease.

5. People with vaginitis.

6. People whose diet contains high levels of oxalate, as is the case in certain areas and seasons in India and Saudi Arabia. This will also include potential individuals who prefer high oxalate foods, such as spinach.

[0122] Any of the above described persons or animals are provided a composition of the present invention. For example, a person with higher than normal endogenous oxalate levels is treated twice a day with a capsule designed to deliver its contents to the large intestine, the capsule containing about an equivalent effective amount of the enzyme composition with enzymatic activity similar to that provided by⁷ cfu oxalate reducing bacteria such as O. formigenes. The capsule is preferably administered with food.

EXAMPLE 9

Treatment of low risk patients with oxalate reducing enzyme compositions [0123] Enterally protected oxalate reducing compositions containing a mixture of oxalate reducing enzymes, oxalate decarboxylase and / or oxalate oxidase, such as those provided in enteric coated capsules, may be administered to subjects in lower populations the risk of oxalate related disease or the risk of oxalate related conditions. An effective amount of the enzyme composition is administered in the desired treatment regimen.

[0124] It would be desirable to administer the composition to these patients either at shorter times when they are at risk of oxalate related conditions, or simultaneously with materials that contribute to the oxalate related condition. These patients could also routinely receive treatment with oxalate reducing compositions, in the form of supplements or as food additives such as milk and yogurt. They include individuals who have lost normal oxalate degrading populations as a result of oral antibiotic treatment or attacks of diarrheal disease, or infants.

[0125] Low-risk individuals or animals are administered a capsule twice daily to deliver its contents to the large intestine, the capsule containing an effective amount of the enzyme composition. For example, each size 2 capsule contains about 5-100 units of each enzyme. The capsule is preferably administered with food. Example 10 [0126] Method for producing enteric-coated capsules containing lyophilized Oxalobacter formigenes.

[0127] 200 grams of Oxalobacter formigenes cell paste was used. Cell paste may be fresh, from fermentation, or it may be from previously thawed frozen pre24 basic parats. A 100 mM trehalose solution as a cryopreservative or cryoprotective agent was mixed with the cell paste. The solution was constantly stirred. This mix was then mixed with the excipient mixture. The excipient mixture was Maltodextrin M500 and sodium alginate mixed together and added to a 79% solution of Raftilose P95. The cell paste mixture was then poured into the lyophilization tray (s). The filled trays were then freeze-dried in an Edwards Lyofast S24 dryer (can be used with any suitable type dryer) for 40-65 hours, which can be changed for different production runs. After freeze-drying, the dried cake was ground by hand and forced through a 20 mesh sieve as per US notation. In this way, a powder with a particle size <850 μm was obtained.

[0128] After the dried powder was sieved, it was ready to be filled into capsules. The capsules were filled using a manual capsule filling machine, but any capsule filling method such as automatic filling machines can be used. Generally, size 2 capsules were used. The capsules were coated with enteric coating polymers such as Eudragit (obtained by purchase from Rhom Pharma (Degussa) using an aqueous coating process, alternatively a solvent coating process can be used. This company produces many different types of Eudragit polymers that specifically dissolve at different pH.

[0129] Coating was performed using standard techniques. Eudragit polymers are polymers and copolymers of methacrylic acid. For example, Eudragit L100-55 is a type C methacrylic acid copolymer, Eudragit L30 is a dispersion of methacrylic acid copolymer, and Eudragit S100 is a type B methacrylic acid copolymer. These and other enteric coatings are known in the art.

Coating in an aqueous environment [0130] Eudragit FS30D film-forming agent and Eudragit L30D55 ​​film-forming agent were used, and other materials together with plasticizers, release agents and carriers such as water and those known in the pharmaceutical art.

[0131] 800 gram capsules were coated with a coating suspension to obtain homogeneously coated capsules with a USP disintegration profile. Disintegration profile: no decomposition in simulated gastric juice (pH 1.2) in one hour and complete disintegration in simulated intestinal fluid (pH 6.8) in one hour.

Solvent coating process:

Film forming agent Eudragit L100-55, [0132] The film forming agent Eudragit S100 was used and other materials such as plasticizers, release agents and carriers such as water and those known in the pharmaceutical field can be used with it.

[0133] 800 gram capsules were coated with a coating suspension containing to obtain uniformly coated capsules with a USP disintegration profile. Disintegration profile: no decomposition in simulated gastric juice (pH 1.2) in one hour and complete disintegration in simulated intestinal fluid (pH 6.8) in one hour. Example 11.

[0134] Using the oral delivery capsules prepared by the method of Example 1 to seven different experiments for more than 1 year of testing, the following data was obtained. The stability of the pharmaceutical compositions thus prepared was evaluated in terms of bacterial viability and oxalate-degrading activity. The following table, Table 1, shows 7 production series and stability of enteric-coated pharmaceutical compositions containing live oxalate-reducing bacteria, especially O. formigenes.

Table 1

<td>No experience</td><td>0 months</td><td> 1</td><td> 2</td><td> 3</td><td> 3,4</td><td> 6</td><td> 9</td><td> 12</td>

<td> 1</td><td>3,6E + 08</td><td>2,2E + 06</td><td></td><td>1,6E + 06</td><td></td><td>1,1E + 06</td><td>1.2E + 06</td><td>5,1E + 05</td>

<td> 2</td><td>3,6E + 08</td><td>1,8E + 07</td><td></td><td></td><td>3,9E + 06</td><td>1,5E + 06</td><td>2,2E + 06</td><td>6,0E + 05</td>

<td> 3</td><td>1,5E + 08</td><td>1,8E + 07</td><td></td><td>4,2E + 06</td><td></td><td>2,0E + 06</td><td>1,8E + 06</td><td>1,1E + 06</td>

<td> 4</td><td>1,5E + 08</td><td></td><td>1.2E + 07</td><td>3,5E + 06</td><td></td><td>4,1E + 06</td><td>1,7E + 06</td><td>1,1E + 06</td>

<td> 5</td><td>1,5E + 08</td><td></td><td>5,9E + 06</td><td></td><td></td><td></td><td></td><td></td>

<td> 6</td><td>2,4E + 08</td><td>2,9E + 07</td><td>1,7E + 07</td><td></td><td></td><td>3,4E + 06</td><td>3,7E + 06</td><td>2,3E + 05</td>

<td> 7</td><td>2,4E + 08</td><td>7,1E + 07</td><td>1.2E + 07</td><td></td><td></td><td>2,0E + 06</td><td>1.2E + 06</td><td>1,5E + 05</td>

[0135] Another example is shown in the data in Table 2 below.

Table 2

<td>Time in months</td><td>Oxalate / capsule degrading activity (mg / h / capsule)</td>

<td> 0</td><td> 2,9</td>

<td> 1</td><td> 3,9</td>

<td> 3</td><td> 3,48</td>

<td> 6</td><td> 3,6</td>

<td> 9</td><td> 1,6</td>

<td> 12</td><td> 2,5</td>

[0136] Data regarding the stability of the lyophilized powder containing the oxalate reducing bacterium prepared by the method of Example 10 from Oxalobacter formigenes is shown in Table 2A.

Table 2A

<td>Time in months</td><td>CFU / g powder</td>

<td> 0</td><td>1.8 E + 8</td>

<td> 3</td><td>8.45 E + 9</td>

<td> 6</td><td>1.06 E + 10</td>

<td> 126</td><td>7,1E + 9</td>

[0137] The following data was obtained for the next production run:

Table 3

<td>Month</td><td>CFU / g powder</td><td>CFU / capsule</td><td>Activity / g powder</td><td>Activity / capsule</td>

<td> 0</td><td>2,4E + 08</td><td>3,3E + 07</td><td> 18,6</td><td> 2,55</td>

<td> 1</td><td>2,9E + 07</td><td>4,0E + 06</td><td> 42,9</td><td> 5,88</td>

<td> 2</td><td>1,7E + 07</td><td>2,3E + 06</td><td> 39,1</td><td> 5,36</td>

<td> 6</td><td>3,4E + 06</td><td>4,7E + 05</td><td> 23,2</td><td> 3,18</td>

<td> 9</td><td>3,7E + 06</td><td>5,1E + 05</td><td> 24,8</td><td> 3,40</td>

<td> 12</td><td>2,3E + 05</td><td>3,2-e + 04</td><td> 22,2</td><td> 3,04</td>

[0138] Activity is given in mg oxalate decomposed per hour.

Example 13 [0139] Formulations for oral delivery of pharmaceutical compositions with live Oxalobacter formigenes

Preparation 1 [0140]

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Ox.formigenes cell paste (dry)</td><td> 24,00</td><td> 6%</td>

<td>D (+) Trehalose (Cryoprotective agent)</td><td> 11,34</td><td> 3%</td>

<td>Maltodextrin QD M-500</td><td> 240,00</td><td> 57%</td>

<td>Sodium Alginate</td><td> 16,00</td><td> 4%</td>

<td>Raftilose P95 or oligofructose</td><td> 126,24</td><td> 30%</td>

[0141] For example, trehalose can be obtained from Sigma Co. Trehalose is a disaccharide, and therefore the formulation according to the invention consists of a disaccharide such as maltose, lactose, cellobiose, sucrose, diglucose or trehalose. Maltodextrin QD M-500 has a DE value

10 and is a white powder or granular white powder. It is a unsweetened, nutritious saccharide polymer composed of D-glucose units connected mainly by alpha-1-4 bonds. DE is the dextrose equivalents, a quantitative measure of the degree of hydrolysis of the starch polymer. The higher the DE, the greater the degree of starch hydrolysis. The components of the formulation are also stabilizing agents such as sodium alginate, which is also used as a stabilizing agent, thickening agent, gelling agent or emulsifier. Sodium alginate is a natural algae refined amylose carbohydrate. It is widely used in food products, medicines, textiles, printing and dyeing, papermaking and household chemistry as a thickener, emulsifier, stabilizer and binding agent, etc. The molecular formula is C6H7O6Na) and is not white or pale yellow, with vagiform strength, no smell, no taste, it dissolves in water, insoluble in ethanol and ether. Raftilose P95 is a powder with 95% oligofructose DP2 to DP7, and sugars: glucose, fructose and sucrose (5%).

Preparation 2 [0142]

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Ox.formigenes cell paste (dry)</td><td> 41,7,00</td><td> 10%</td>

<td>D (+) Disaccharide</td><td> 11,34</td><td> 3%</td>

<td>Maltodextrin QD M-500</td><td> 221,00</td><td> 53%</td>

<td>Sodium Alginate</td><td> 16,00</td><td> 4%</td>

<td>oligofructose</td><td> 126,24</td><td> 30%</td>

Preparation 3 [0143]

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Ox.formigenes cell paste (dry)</td><td> 24,00</td><td> 6%</td>

<td>D (+) Disaccharide (Maltose)</td><td> 11,34</td><td> 3%</td>

<td>Maltodextrin QD M-500</td><td> 240,00</td><td> 57%</td>

<td>Sodium Alginate</td><td> 16,00</td><td> 4%</td>

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Oliogofruktoza</td><td> 126,24</td><td> 30%</td>

Preparation 4 [0144]

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Ox.formigenes cell paste</td><td> 83,4</td><td> 20%</td>

<td>D (+) Disaccharide</td><td> 22,6</td><td> 6%</td>

<td>Maltodextrin QD M-500</td><td> 196,26</td><td> 47%</td>

<td>Sodium Alginate</td><td> 16,00</td><td> 4%</td>

<td>Raftilose P95</td><td> 95,91</td><td> 23%</td>

Preparation 5 [0145]

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Ox.formigenes cell paste (dry)</td><td> 62,64</td><td> 15%</td>

<td>D (+) Disaccharide</td><td> 25,05</td><td> 6%</td>

<td>Maltodextrin QD M-500</td><td> 212,97</td><td> 51%</td>

<td>Sodium Alginate</td><td> 16,00</td><td> 4%</td>

<td>oligofructose</td><td> 100,22</td><td> 24%</td>

Preparation 6 [0146]

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Ox.formigenes cell paste (dry)</td><td> 12,52</td><td> 3%</td>

<td>D (+) Disaccharide</td><td> 6,26</td><td> 1,5%</td>

<td>Maltodextrin QD M-500</td><td> 240,00</td><td> 57%</td>

<td>Sodium Alginate</td><td> 25,05</td><td> 6%</td>

<td>oligofructose</td><td> 137,80</td><td> 33%</td>

Preparation 7 [0147]

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Ox.formigenes cell paste (dry)</td><td> 104,40</td><td> 25%</td>

<td>D (+) Trehalose</td><td> 25,05</td><td> 6%</td>

<td>Maltodextrin QD M-500</td><td> 187,91</td><td> 45%</td>

<td>Sodium Alginate</td><td> 16,00</td><td> 4%</td>

<td>oligofructose</td><td> 83,52</td><td> 20%</td>

Preparation 8 [0148]

<td>Ingredient</td><td>Quantity (g)</td><td> %</td>

<td>Ox.formigenes cell paste (dry)</td><td> 396,04</td><td> 95%</td>

<td>Excipient from disaccharide</td><td> 8,34</td><td> 2%</td>

<td>Excipient mixture-Maltodextrin, Na alginate, Oligofructose</td><td> 12,5</td><td> 3%</td>