Pharmaceutical compositions and methods for treating or preventing oxalate-related disease

[0001] The present invention relates to compositions and methods for the treatment and prevention of conditions associated with oxalates. In particular, the invention relates to compositions and methods including the use of oxalate-decreasing bacteria or reducing oxalates.

BACKGROUND [0002] Kidney stones-urinary tract (urolithiasis) is a serious health problem around the world. Most stones associated with urolithiasis consist of calcium oxalate alone or calcium oxalate and calcium phosphate. Other disease states are also associated with an excess of oxalates. These include vulvodynia, oxalosis associated with end-stage renal disease, impaired cardiac conduction, Crohn's disease and other intestinal morbidity.

[0003] Oxalic acid and / or its salts, oxalates, are found in many types of food and are therefore a component of many elements of human and animal diets. Increased oxalate absorption may occur after ingestion of foods containing increased amounts of oxalic acid. It is well known that foods such as spinach and rhubarb contain large amounts of oxalates, but many other foods and drinks also contain oxalates. Because oxalates occur in so many different foods, it is difficult to form diets which are low-sodium diets and which are also tasty. In addition, adherence to the low-sodium diet is often problematic.

[0004] Endogenous oxalates are also produced in metabolic processes by normal tissue enzymes. Oxalates, including oxalates absorbed from the diet, as well as oxalates produced in metabolic processes, are not further metabolised by tissue enzymes and must therefore be excreted. This excretion takes place mainly through the kidneys. The concentration of oxalates in renal fluids is of key importance, because increased oxalate concentrations increase the risk of calcium oxalate crystals forming and thus the formation of kidney stones.

[0005] The risk of kidney stones is associated with many factors that are not yet fully understood. Kidney stone or urinary tract occurs even in 12% of the population in Western countries, and about 70% of these stones consist of calcium oxalate or calcium oxalate and calcium phosphate. Some individuals (e.g., patients with intestinal disease such as Crohn's disease, inflammatory bowel disease or fat diarrhea, as well as patients who have undergone colonic bypass surgery) absorb more oxalate in the diet than others. In these individuals, the incidence of oxalate urolithiasis is significantly increased. The increased incidence of the disease is due to increased levels of oxalate in the kidneys and urine, and the most common hyperoxaluria syndrome in humans is known as intestinal hyperoxaluria. Oxalate is also a problem in patients with end-stage renal disease and recent evidence (Solomons, CC, MH Melmed,

SM Heitler [1991] 'Calcium cittate for vulvarvestibulltis' Jouirnal of Reproductive Medicine 36: 879-882) showed that elevated oxalate in the urinary tract also occurs in vulval vulvalisation (wulwodynia).

[0006] Bacteria that break down oxalates 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, which 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, S.

L., 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 organism and have been given both new species and genus (Allison, MJ, KA Dawson, WR Mayberry, JG Foss [1985] "Oxalabacter formigenes gen. Nov., Sp. Nov .: oxalate- degrading anaerobes that inhabit the gastrointestinal tract "Arch. Microbiol. 141: 1-7).

[0007] Not all people in the gastrointestinal tract are populations of O. formigenes (Allison, MJ, SL Daniel, NA Comick [1995] "Oxalate-degradating bacteria" in Khan, SR (eds.), 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). In patients after coronary bypass surgery, low concentrations or complete absence of oxalate-degrading bacteria are found in stool samples (Allison et al. [1986] "Oxalate degradation by gastrointestinal bacteria from humans" J. Nutr. 116: 455 -460). In addition, some people and animals may have colonies of O. formigenes, but they may still have excessive levels of oxalates for reasons that are not yet fully understood. There is a need for methods for treating humans and animals to reduce oxalate levels in their organisms so as to enable treatment or prevention of oxalate related conditions. Desired methods may include the administration of oxalate reducing compositions. A coated enteric formulation containing bacteria that break down oxalates is disclosed. US20040234514A1 describes compositions comprising bacteria or enzymes that degrade oxalates that have been lyophilized or frozen as a liquid or paste and encapsulated in a gel capsule or otherwise protected for enteral administration. However, the present inventors have found that there is a need for further compositions for oral administration designed to deliver intestinal oxalytic bacteria to the intestine, i.e. such a composition should allow the bacteria to break down the oxalate-degrading bacteria into the gut without loss of activity during passage through the stomach. In addition, there is a need to develop such compositions that also have an acceptable shelf life under storage conditions.

SUMMARY OF THE INVENTION [0008] The present invention encompasses compositions and methods for the treatment and prevention of conditions associated with oxalates. Compositions of the present invention include pharmaceutical compositions containing Oxalobacter formigenes that reduce oxalates. In particular, the present invention provides a composition for oral administration to a human or animal, which composition comprises an orally administrable vehicle containing a oxalate degrading composition comprising a) from 3% to 25% of oxalate-degrading bacteria, b) from 1.5% to 6% of disaccharide , c) from 45% to 60% maltodextrin, d) from 4% to 6% alginate and e) from 20% to 35% oligofructose; for the delivery of oxalate-degrading bacteria to the gut of a human or animal after oral administration, wherein the oxalobacterformigenes are oxalytic bacteria that break down oxalates. The composition is designed to have an adequate shelf life and additionally allows the enteralysis of bacteria that break down oxalates. In the present invention, it has been contemplated that in addition to bacteria, one or more suitable oxalate degrading enzymes may be added to the composition, provided that the enzyme is active in the intestinal environment, e.g., such as about pH 6.8 and above. At present, according to the best knowledge of the inventors, only the native oxalyl-CoA decarboxylase is active at such pH, ​​but this enzyme also requires formyl-CoA transferase to activate oxalyl-CoA oxalate, a oxalyl-CoA decarboxylase substrate. In the future, modified enzymes may be developed. The use of purified enzyme will provide additional benefits in terms of activity, purity, etc.

[0009] The methods of the present invention include the administration of pharmaceutical compositions to treat or prevent oxalate related conditions and methods of preparing such pharmaceutical compositions. One embodiment includes methods that reduce the risk of developing oxalate related disorders by reducing the amount of oxalates in the gastrointestinal tract. This reduction in the gastrointestinal tract leads to a reduction in systemic oxalate levels, thereby promoting good health.

[0010] In one embodiment of the invention of interest, a decrease in the absorption of oxalates is achieved by providing bacteria that break down oxalates into the gastrointestinal tract.

[0011] These bacteria are Oxalobacterformigenes. These bacteria use oxalates as a substrate. This use reduces the concentration of soluble oxalate in the intestine and therefore the amount of oxalates available for absorption. Oxalate reduction in the gastrointestinal tract can also lead to oxalate removal from the circulatory system. In the methods of the present invention, overall reduction of oxalate loading in a subject is considered.

[0012] In a particular embodiment of the invention of interest, there are provided methods and compositions for delivering viable O. formigenes to the gastrointestinal tract of individuals who are at an increased risk of disease associated with oxalates. Bacteria remove oxalates from the gastrointestinal tract, thereby reducing the amount of oxalates available for absorption and leading to increased secretion of oxalates from the blood into the intestines.

[0013] The compositions contain microorganisms that break down oxalates and produce enzymes that give these microorganisms the ability to break down oxalates.

[0014] Such microorganisms or enzymes may be provided in compositions that are provided as formulations of the formulations and pharmaceuticals described herein, wherein microorganisms or enzymes may be provided in pharmaceutical formulations containing excipients and other pharmaceutical carriers known in the art. In addition, such pharmaceutical compositions contain excipients that facilitate administration, such as powders, capsules, pills, granules or tablets, for delivery to the gastrointestinal tract of humans or animals.

[0015] 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 co-enzymes that they are the substituents of oxalate degradation pathways or participate in metabolic pathways of oxalates, especially in oxalate reduction.

[0016] The present invention also encompasses methods and compositions containing enzymes for reducing oxalate levels to treat or prevent conditions related to oxalates. For example, reducing oxalate levels can be achieved by administering enzymes that break down oxalates. These enzymes may be administered in the form of a cell lysate. The cell lysate is made from a microorganism that has the oxalate reduction ability, O. formigenes. In a particular embodiment, the enzymes to be administered are one or more of the enzymes of the present invention, such as, but not limited to, oxalate oxidase, oxalate decarboxylase, formyl-CoA transferase, oxalyl-CoA decarboxylase. Optionally, additional agents that increase the enzyme activity may be administered. These additional factors can be for example, oxalyl-CoA, MgCl2 and TPP (thiamine diphosphate, active form of vitamin B1). Pharmaceutical compositions containing enzymes contain one or more enzymes and optionally cofactors, coenzymes and other agents that increase the activity of the enzyme, alone or in combination, and are provided with pharmaceutically acceptable carriers and excipients.

[0017] Another aspect of the invention of interest relates to pharmaceutical compositions and / or nutritional supplements for oral administration. These compositions release microbes that break down oxalates or enzymes that break down oxalates in the intestines of humans or animals. The compositions of the present invention contain pharmaceutically acceptable formulations. For example, the methods and compositions of the present invention comprise a dose-providing system that provides compositions to a desired site, for example providing compositions to the recipient's gastrointestinal tract. The compositions of the present invention may be administered as a food ingredient, e.g. milk, meats and yogurt.

[0018] In a further embodiment of the invention of interest, the reduction of oxalate absorption is achieved in domesticated, agricultural or exotic animals deficient in bacteria that break down oxalates by administering microorganisms, plants and enzymes that break down oxalates, either alone or in combination. [0019] The methods of the present invention comprise the treatment or prevention of oxalate related conditions in humans and animals by administering an effective amount of a oxalate reducing composition comprising one or more microorganisms reducing oxalates, one or more oxalate reducing enzymes or combinations and mixtures thereof. Conditions associated with oxalates include, but are not limited to, hyperoxaluria, primary hyperoxaluria,

BRIEF DESCRIPTION OF THE FIGURES [0020]

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

FIG. 1B is a graph of data about a low calcium diet.

FIG. 2A is a graph of secreted oxalates.

FIG. 2B is a graph of secreted oxalates.

FIG. 2C is a graph of secreted oxalates.

FIGS. 3A-C is a graph of secreted oxalates.

FIG. 4 is a graph of secreted oxalates.

FIG. 5 is a CFU chart / capsule coated with respect to storage for weeks at 4 ° C and -20 ° C.

FIG. 6 is a graph showing the average loss in coated capsules versus weekly storage at 4 ° C and -20 ° C.

FIG. 7 is a graph showing the average loss in gelatin capsules versus HPMC capsules stored within weeks.

FIG. 8 is a graph showing the average loss in coated water-based capsules compared to organic ones.

FIG. 9 is a graph showing the average loss depending on the coating and the type of capsule.

FIG. 10 is a graph showing the average loss with or without Avicel®.

FIG. 11 is a graph showing the average loss in packaging in the form of polypropylene tubes relative to blisters.

FIG. 12 is a graph showing the average loss in packages in the form of polypropylene tubes relative to blisters without error bars.

DETAILED DISCLOSURE OF THE INVENTION [0021] The present invention includes methods and compositions for reducing oxalates. The compositions of the present invention contain bacteria, but in some embodiments may contain microorganisms, enzymes, polynucleotide sequences, vectors, cells, plants or animals that are capable of reducing oxalates. The compositions contain microorganisms that are capable of reducing oxalates. Such microorganisms include Oxalobacter formigenes.

[0022] The compositions also contain enzymes that are part of the oxalate reduction pathway. Such compositions contain one or more enzymes and optionally contain cofactors, coenzymes and other agents needed or desirable for enzyme activity. The compositions contain one or more enzymes, including but not limited to oxalate reducing enzymes and other enzymes involved in the metabolism of oxalates found in plants, animals or humans. The compositions contain one or more of the oxalate reducing enzymes described herein. As used herein, the term "one or more enzymes" means that one type of enzyme may be present, for example a formyl-CoA transferase or more than one type of enzyme, for example a composition comprising, for example, oxalyl-CoA decarboxylase and formyl-CoA transferase; oxalate decarboxylase and oxalate oxidase or a combination of a wild-type enzyme and a mutant enzyme are present in the composition. As is known in the art, this term does not mean one enzyme molecule, but many molecules of one or more types of enzymes.

[0023] As used herein, the terms oxalate degradation enzymes and oxalate reducing enzymes are used interchangeably and both refer to enzymes involved in the reduction or degradation of oxalates in any organism or active fragments or recombinant proteins containing active fragments capable of oxalate reduction or degradation.

[0024] The compositions of the present invention also contain pharmaceutical compositions containing live oxalate reducing bacteria and, optionally, pharmaceutical excipients or carriers in an excipient for ease of administration. The compositions also contain pharmaceutical compositions containing one or more purified oxalate reducing enzymes, including, but not limited to, purified from natural sources of such enzymes, recombinantly produced or synthetically produced enzymes and, optionally, pharmaceutical excipients or carriers, in an excipient for ease of administration .

[0025] The pharmaceutical compositions of the present invention contain oral excipients for administration, including but not limited to powders, capsules, pills, granules and tablets that can be coated to resist harsh environmental conditions such as the stomach. Such oral excipients for administration are used to provide live bacteria that reduce oxalates and enzymes at dosages and methods described herein. Such pharmaceutical compositions are stable. The compositions may provide live bacteria and, if applicable, enzymes having activity for at least 12 months with minimal loss of cfu (colony forming units) and enzyme activity. [0026] More specifically, the composition according to the invention can be one in which the oral vehicle-facilitating administration comprises a gel capsule. In a particular embodiment, the gel capsule is further strengthened to exclude penetration of the gastric juice into the capsule during passage through the stomach. It has been found that a suitable reinforcement consists in the assembly of a gel capsule by joining the edges of two parts of the capsule with a suitable material. The present inventors have found that when a gelatin capsule is made of gelatin, a suitable material for connection is gelatin, and when the gel capsule is made of hydroxypropyl methylcellulose (HPMC), the appropriate material for attachment is HPMC. Connection or use of other materials with similar properties may also be appropriate. The oxalate degrading bacteria present in the composition of the invention may be in the form of a cell paste, lyophilized powder, micro- or nanoparticles, micro- or nanoparticle emulsions, etc. [0027] A feature of the composition of the present invention is its ability to suppress the adverse effects of the acidic stomach environment (as well as the adverse effects of enzymes present in the stomach). One method is to provide an enteric coating composition. In those cases where the gel capsule is provided with the anastomosis, the enteric coating is delivered after the fusion process. Suitable materials for enteric coatings are usually polymeric materials, such as e.g. materials conventionally used in the pharmaceutical industry for the production of enteric coatings. These include materials mentioned in Remington's Pharmaceutical Science, namely cellulose derivatives including cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate, methacrylic acid polymers, including methacrylic acid copolymers such as Eudragit® L and S, available from Rohm GmbH, Germany; and polyvinyl acetate phthalate and the like.

In a composition according to the present invention in which the oxalate degradation activity is provided by a cell paste, the oxalate degrading composition has cfu / g of at least about 1x10³ to about 1x10¹³from about 1x10⁵ to about 1x10¹² Such a cell paste, not in the delivery composition of the present invention, typically has a higher level of cfu / g and each process step may contribute to a reduction in the cfu / g of the final composition, which has to be taken into account during manufacture. Typically, the final oxalate degrading composition of the invention comprises a cfu / unit dosage form from about 5x10⁵ to about 1x101 ° or from about 5x10⁵ up to around 5x10⁷ The composition of the invention is conveniently in a unit dosage form, such as e.g. capsules, sachets, tablets or the like. In an interesting form, the composition is in the form of a capsule. The tablets are also of interest, but may be associated with the problem of lower durability and activity, namely the risk of loss of activity during the tableting process. In addition, with each coating provided on the surface of the tablet, there may be a risk of direct contact with bacteria and therefore an increased risk of loss of activity and stability.

[0030] As shown herein in the examples, the composition provided in the invention has acceptable shelf life. Therefore, the loss of oxalate-degrading bacteria in the composition of the invention after storage for 6 months at 4 ° C is at most 3 logs, such as, e.g., at most 2 logs, at most 1 log or at most 0.5 logs / or the loss of oxalate decay forming bacteria in the composition of the invention after storage for 12 months at 4 ° C is at most 3 logs, such as e.g. at most 2 logs, at most 1 log or at most 0.5 logs.

[0031] Additionally or alternatively, the loss of colony forming units of oxalate-degrading bacteria in the composition of the invention after storage for 6 months at -20 ° C is at most 2 logs, such as e.g. at most 1.5 logs, at most 1 log or at most 0.5 log and / or loss of colony-forming bacteria-containing colonies in the composition of the invention after storage for 12 months at -20 ° C is at most 2 logs, such as e.g. at most 1.5 logs, at most 1 log or at most 0.5 log.

[0032] Permissible stability can also be expressed in enzymatic activity. Thus, the oxalate degrading composition according to the present invention has a oxalate degrading enzyme activity / g of at least about 2 mg of decomposed oxalates / h to about 2500 mg of decomposed oxalates / h, such as e.g. about 60 to about 250 mg / h or about 20 to about 100 mg / h.

[0033] In some situations, it is contemplated that delivery of said composition to the intestine may lead to the colonization of bacteria that break down oxalates in the intestines. Bacteria can become part of the normal intestinal flora, as shows the analysis of fecal material after treatment with oxalate reducing bacteria. In some cases colonization in the gut is transient. Previous experience in human studies shows that bacteria could be detected in a stool sample one week after stopping treatment with oxalate reducing bacteria, but were not present in a sample taken two weeks after treatment.

[0034] The composition of the present invention is usually in solid dosage form. Accordingly, the oxalate reducing composition contains a lyophilized powder, respectively. For this purpose, the presence of a cryopreservation agent is suitable, particularly during the preparation of the composition. Suitable cryopreservatives are carbohydrates, amino acids, polymers, polyols and salts of organic acids. In a particular embodiment, the cryopreservative agent is a disaccharide, such as e.g. trehalose. [0035] The preservative may be a carbohydrate selected from the group consisting of trehalose, glucose, fructose, sucrose, lactose, maltose, sucrose, di-glucose, raffinose, starch, including corn starch, potato starch, rice starch, tapioca starch and wheat starch. or it may be a sugar alcohol,

[0036] As is well known in the pharmaceutical industry, pharmaceutically acceptable excipients may be used in the formulation of pharmaceutical compositions to adjust the technical properties of the composition (e.g., flowability of the powder to fill the capsule or tabletting apparatus, addition of bulking agents to increase the weight of the individual dosage form) addition of binders, fillers, diluents, etc.). In the present invention, it is usually necessary to add excipients to increase the weight of each dosage form. In an interesting form, the excipient may also have other suitable properties, such as, e.g., increasing the flowability of the filling powder, e.g. capsules, increasing stability, or may act as a cryopreservation agent or a moisture scavenger. Accordingly, in one embodiment, the composition of the invention comprises one or more excipients that are pharmaceutically acceptable excipients. Namely, such an auxiliary substance may be a bulking agent. In some cases, the excipient also has cryopreservation properties.

[0037] Examples of such excipients for use in the present invention include, but are not limited to, maltodextrin, raftilose / oligofructose and alginate or gelatin, cellulose derivatives, lactose or starches. In a particular embodiment, the excipient is an alginate, such as e.g. an alkali metal or alkaline earth metal salt and alginic acid, including sodium alginate, potassium alginate or calcium alginate.

[0038] The composition of the invention may also contain one or more moisture scavengers, such as, for example, celluloses, cellulose derivatives, silica and silica derivatives. Particular examples are cellulose, microcrystalline cellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose or silica, including colloidal silicon dioxide. Namely, microcrystalline cellulose may be Avicel ™, and colloidal silicon dioxide may be Cabosil ™.

[0039] It is contemplated that the specific surface area of ​​one or more moisture scavengers is important because of their effect. Accordingly, in one embodiment, the one or more moisture scavengers have a specific surface area of ​​at least 0.6 m²/ g, such as e.g. at least 0.7 m²/ g or at least 1 m²/ G.

[0040] The excipient for administering administration comprises from about 3% to about 25% of the oxalate-degrading bacteria, b) from about 1.5% to about 6% of disaccharide, c) from about 45% to about 60% of maltodextrin, d) from about 4 % to about 6% alginate and e) from about 20% to about 35% oligofructose.

[0041] In cases where a lyophilized powder is used, the powder typically has a particle size of about 10 microns to about 2000 microns, such as, e.g., from about 500 microns to about 1500 microns, from about 600 microns to about 1000 microns, such like about 800 microns.

The composition administered is usually in solid form, e.g. in the form of particles or in a solid dosage form, e.g. in the form of sachets, capsules or tablets (e.g., the particles are further processed in a suitable dosage form by methods well known to a person skilled in the art). For this purpose, suitable pharmaceutically acceptable excipients may be added, e.g., fillers, binders, disintegrants, colorants, flavors, pH adjusting agents, stabilizing agents, buffering agents, solubilizing agents, preservatives, enzyme cofactors, etc. Furthermore, one or more further substances that act therapeutically and / or prophylactically and / or other enzymes, cofactors, substrates, co-enzymes, minerals and other agents may be added,

[0043] Examples of suitable pharmaceutically acceptable excipients include: dextrins, maltodextrins, dextrose, fructose, glucose, lactose, cellulose derivatives including carboxymethylcellulose calcium, carboxymethylcellulose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose (HPMC), microcrystalline cellulose (e.g., various types of Avicel® ), starches or modified starches (e.g. potato starch, maize starch, rice starch, pre-gelatinized starch), polyvinyl acetate, polyvinylpyrrolidone, agar, sodium alginate, croscarmellose sodium, calcium hydrogen phosphate, calcium phosphate (e.g. basic calcium phosphate, calcium hydrogen phosphate) , calcium sulfate, carboxyalkylcellulose, dextra, dibasic calcium phosphate, gelatin, gummi arabicum, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose,polyethylene glycol, polyethylene oxide and as lubricating agents: talc, magnesium stearate, calcium stearate, stearic acid, hydrogenated vegetable oils and the like.

[0044] The methods of the present invention comprise the preparation and use of the compositions of the present invention. The methods also include the preparation of compositions comprising cell lysates having oxalate reducing activity, compositions comprising one or more enzymes having oxalate reducing activity and compositions containing nutrients derived from plants or microorganisms having altered oxalate levels. The methods also include the preparation of stable oral pharmaceutical compositions containing live reducing oxalate bacteria.

[0045] Sposoby według niniejszego wynalazku obejmują stosowanie kompozycji według niniejszego wynalazku. Niniejszy wynalazek obejmuje sposoby podawania kompozycji według niniejszego wynalazku roślinom lub zwierzętom dla zmiany poziomów szczawianów w roślinie lub u zwierzęcia. Sposoby obejmują także sposoby suplementacji pokarmowej, takie że kompozycje według niniejszego wynalazku są podawane roślinom lub zwierzętom w pokarmie lub w źródłach nawozu lub równocześnie z pokarmem lub źródłem nawozu, aby zmienić poziomy szczawianów w pokarmie, w trakcie trawienia pokarmu lub w trakcie absorbcji przez rośliny.

[0046] The methods of the present invention include methods of treating or preventing oxalate related conditions. The methods comprise administering the compositions of the present invention in amounts effective to alter the level of oxalates in the body. Such methods are effective for the treatment of conditions associated with oxalates in humans and animals, including, but not limited to, hyperoxaluria, primary hyperoxaluria, idiopathic nephrolithiasis with calcium oxalate (urolithiasis), intestinal hyperoxaluria, vulvodynia, oxalosis associated with end-stage renal disease. , cardiac conduction disorders, inflammatory bowel disease, Crohn's disease, fatty diarrhea, patients undergoing gastrointestinal surgery, for example, intestinal bypass surgery,

The invention of interest relates to the introduction of compositions comprising one or more bacteria and / or enzymes degrading oxalates to the human or animal gastrointestinal tract, whereby the activity of the composition reduces the amount and / or concentration of oxalates present, thus reducing the risk of disease related to oxalates.

[0048] The present invention includes methods and compositions for the treatment and prevention of conditions associated with oxalates in humans and animals. A method of treating conditions associated with oxalates comprises administering a composition comprising one or more oxalate reducing enzymes. Such compositions may be administered one or more times a day for one or more days depending on the severity of the condition associated with oxalates or the amount of oxalates in the intestine or body fluids of a human or animal. Treatment can be carried out as long as undesirable levels or oxalates are present in a human or animal. For example, the enzyme composition may be administered one or more times a day for a period ranging from one day to one year.

[0049] Methods of treating and preventing conditions associated with oxalates may include administering a composition containing an effective amount of oxalate reducing enzymes or enzymatic activity to reduce oxalate. An effective amount includes the amount of units of the oxalate reducing enzyme activity that will reduce some of the oxalate present or the level of oxalate reducing enzyme activity that will initiate oxalate reduction or cause the oxalate to be reduced in the subject compared to the amount of oxalate present prior to administration of the composition. The number of units of the oxalate reducing enzyme activity that can be used in a single dose composition can range from about 0.0001 units to about 5,000 units, from about 5 units to 100 units, from 0.05 to 50 units, to 0, 5 to 500, from about 0.01 units to about 50 units, from about 0.01 units to about 5 units, from about 1 units to about 100 units, from about 25 units to about 50 units, from about 30 units to about 100 units, from about 40 units to about 120 units, from about 60 units to about 15 from about 50 units to about 100 units, from about 100 units to about 500 units, from about 100 units to about 300 units, from about 100 units up to about 400 units, from about 100 units to about 5,000 units, from about 1,000 units to about 5,000 units, from about 2,500 units to about 5,000 units, from about 0.001 units to about 2,000 units and all ranges covered by them. The compositions may further contain other enzymes, co-products, substrates, co-enzymes, minerals and other agents, which are helpful in reducing oxalates. The enzyme unit is the amount of enzyme that breaks down one micromole of oxalate per minute at 37 ° C.

[0050] In a particular embodiment, the invention of interest relates to methods of producing and administering compositions comprising oxalobacter formigenes oxalate-degrading bacterial cells to the human or animal gastrointestinal tract, where the microbial activity reduces the amount of oxalates present in the intestine, thus resulting in a decrease in oxalate concentrations. in kidneys and other cellular fluids. In another embodiment, the present invention encompasses methods of making and administering compositions comprising one or more oxalate degrading enzymes, from any source, into the human or animal gastrointestinal tract, where the activity of one or more enzymes reduces the amount of oxalates present in the intestine, which leads to a decrease in oxalate concentrations in the kidneys and other cellular fluids. Introduced cells or enzymes break down oxalates, and bacteria may or may not multiply in the intestinal environment, so that the offspring of the initial cells colonize the intestine and continue to remove the oxalates. The presence of oxalate reducing bacteria reduces the risk of kidney stones and other complications caused by excess oxalic acid. In the human application form, the particular strains of O. formigenes used are strains isolated from human intestinal samples. Thus, the strains are part of the normal intestinal bacterial flora of a human being. However, because they are not present in all individuals or are present in insufficient amounts, the introduction of these organisms corrects the deficiency,

[0051] Although, without intending to be bound by any particular theory, it is believed that enriching the intestinal contents with one or more species of bacteria that break down oxalates or oxalate reducing enzymes results in oxalate reduction in the intestinal content. Some of the bacteria or enzymes administered distribute oxalates at or near the absorption site. The activity of bacteria or administered enzymes reduces the absorption of oxalates from the diet. Decreasing the oxalate concentration in the intestines can also lead to the removal of oxalate from the cells and general circulation. In particular, a decrease in oxalate concentration in the gut may also lead to increased oxalate secretion to the intestine from the blood stream and thus reduce the amount of oxalates that must be excreted in the urine. Therefore, The methods of the invention of interest for the administration of oxalate reducing bacteria or oxalate reducing enzymes may be used to treat or prevent conditions associated with oxalates, such as primary hyperoxaluria in addition to the treatment of dietary hyperoxaluria. The compositions and methods of the invention of interest are particularly advantageous in promoting healthy levels of oxalates in humans and animals.

[0052] Pharmaceutical and nutriceutical compositions for incorporating 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 freeze-dried or frozen as a liquid or paste and can be delivered using an oral excipient for administration, such as a gel capsule or other antiseptic for gastric juice. The material of the gel cap is preferably a polymeric material from which a pill or delivery capsule is made that is resistant to degradation by gastric acid and enzymes in the stomach but decomposes, with the simultaneous release of oxalate degrading compositions, through higher pH and bile acid content. intestine. The released composition then transforms the oxalates present in the intestine into harmless products. Pharmaceutical or nutriceutical carriers can also be combined with bacteria or enzymes. They may contain, for example, a phosphate salt solution or a bicarbonate buffer. The methods of the present invention comprise administering oxalate reducing compositions to the human or animal gut.

[0053] Oxalate reducing compositions comprising one or more oxalate reducing bacteria or one or more oxalate reducing enzymes or combinations of bacteria and enzymes to be administered can be provided in the form of capsules or microcapsules designed to protect the composition against adverse effects of gastric acid . One or more different coating methods for preventing gastric juice can be used. The description of such enteric coatings includes the use of cellulose acetate phthalate (CAP) (Yacobi, A., EH Walega, 1988 or even sustained release formulations: Dosing and evaluation, Pergammon Press). Other descriptions of encapsulation technology are included in US Patent No. 5,284,959.

[0054] Other methods for administering these compositions into the intestine include adding one or more microorganisms, one or more oxalate reducing enzymes, or combinations and mixtures directly to the food sources. One or more bacteria may be added in the form of freshly harvested cells, lyophilized cells or cells otherwise protected. One or more enzymes may be added in the form of lyophilized proteins, encapsulated or microencapsulated enzyme compositions, enzymes complexed with other materials to maintain enzyme activity, and other methods for adding active enzymes to the compositions known to those skilled in the art. Food can be enriched with compositions that break down oxalates without affecting their taste or appearance. These foods may be, for example, yogurt, milk, peanut butter or chocolate. After ingestion, when food products are digested and absorbed by the intestines, compositions that break down oxalates, containing one or more microorganisms, one or more enzymes or combinations, break down oxalates present in the intestine, thereby reducing the absorption of oxalates into the bloodstream.

[0055] As mentioned above, various foods may be enriched with oxalate degradation compositions. Methods for preparing such foods containing oxalate reducing compositions include mixing the food material with the oxalate reducing composition. For example, reducing oxalate microorganisms can be cultured on a substrate and separated from the substrate by, for example, centrifugation. Traditional yoghurt cultures obtained from commercial dairies can be mixed with the growth of microorganisms that break down oxalates. This culture mixture can then be added to the basic premix of dairy yogurt without adversely affecting the taste or consistency. Yoghurt can then be produced and packaged using traditional commercial procedures. In another example, bacteria that break down oxalates can be added to the yoghurts already made. In a similar method, a oxalate reducing composition containing one or more oxalate reducing enzymes may be added to a yogurt bacterial culture or to a yogurt food product.

[0056] Another example of the methods of the present invention is to add a reducing composition of the oxalate to milk after it has been homogenized and sterilized. Such a method is currently used in the dairy industry to add Lactobacillus acidophilis to milk. Any food source containing bacteria can be used by enriching it with bacteria that break down oxalates. These food products include cheese or meat products that contain the desired microorganisms added during processing. Foods 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 may be added.

[0057] In one embodiment, the O. formigenes bacterial strains used according to the invention of interest are pure cultures isolated from anaerobic cultures that have been inoculated with dilutions of the intestinal contents of healthy people or, when used in animals, from healthy animals. A special substrate 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.

[0058] O. formigenes strains useful according to the invention of interest have been characterized based on a series of tests, including: cellular fatty acid standards, cellular protein, DNA and RNA standards (Jensen, NS,

MJ Allison (1995) "Studies on the diversity of 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 are described (groups I and II, both included in the present description of the species). The strains used were selected on the basis of oxalate degradation capacity and experimental data indicating colonization capacity in the human gastrointestinal tract. Selected strains include representatives of both groups I and II.

[0059] One embodiment of the present invention includes procedures for the selection, production and administration of suitable oxalate-degrading bacteria to various individuals. What is important, but not exclusively, are people or animals that are not the habitat of these bacteria in their intestines. These non-colonized or poorly colonized individuals or animals can be identified using tests that allow rapid and unambiguous detection of O. formigenes even when organisms are present at relatively low concentrations in mixed bacterial populations such as those found in intestinal contents. The methods of the invention of interest can also be used to treat individuals or animals that have reduced the number of bacteria that break down oxalates as a result of, for example, antibiotic treatment or post-operative situations.

[0060] Bacteria that can be used according to the invention of interest can be detected by at least two methods:

1) Oligonucleotide probes specific for these bacteria and / or may be used

2) Culture test in which an anaerobic medium containing 10 mM oxalate is inoculated and after incubation at 37 ° C for 1 to 7 days oxalate loss is determined.

[0061] Methods for preparing pharmaceutical compositions and methods for culturing bacteria are generally known to those skilled in the art. For example, pure cultures of O. formigenes strains can be grown in large batch cultures in a fermenter, and the cells can be harvested using techniques known to those skilled in the art. Cells from a selected single strain or mixture of known strains can be treated as needed (e.g., lyophilised with trehalose or glycerol) to preserve viability and then placed in capsules designed to protect cells during their passage through the acidic stomach environment ( capsules with enteric coating). Bacterial cells, fresh from fermentation or from frozen stocks, may be mixed with carriers or auxiliaries and then lyophilized. Subsequently, the powdered composition is placed in the excipient to facilitate administration. For example, for delivering compositions of live bacteria reducing oxalates to the intestines of a human or animal, a stable pharmaceutical composition may contain an excipient to facilitate enteric coating, and a powdered lyophilized composition of live oxalate reducing bacteria is encapsulated in the capsule.

[0062] The pharmaceutical compositions described herein are consumed in doses and amounts, and at intervals determined according to the needs of the subjects. In some cases, a single dose or a periodic dose may be sufficient, and in other cases, systematic consumption (eg with meals) may be necessary. Dosages of effective amounts are described herein. The pharmaceutical compositions comprise live oxalate reducing bacteria and / or oxalate reducing enzymes, alone or in combination with physiologically acceptable excipients or pharmaceutical carriers or carriers or excipients: such terms are used interchangeably herein. The dose of the pharmaceutical composition according to the present invention may be smaller, equal to or greater than the amount of constitutively produced oxalates and / or consumed by the subject, in an effective amount of the dose of reducing oxalate administered over a particular period of time. In some oxalate-related conditions, the dose amount of oxalate-reducing activity administered in the methods and compositions of the present invention may be less than the amount of oxalate consumed or constitutively produced, and it may be necessary to supplement or enrich the low level of oxalate reduction in the patient. In other states, it may be necessary to provide more reducing activity of the oxalate. the dose amount of oxalate-reducing activity administered by the methods and in the compositions of the present invention may be less than the amount of oxalate consumed or constitutively produced, and it may be necessary to supplement or enrich the low level of oxalate reduction ability in the patient. In other states, it may be necessary to provide more reducing activity of the oxalate. the dose amount of oxalate-reducing activity administered by the methods and in the compositions of the present invention may be less than the amount of oxalate consumed or constitutively produced, and it may be necessary to supplement or enrich the low level of oxalate reduction ability in the patient. In other states, it may be necessary to provide more reducing activity of the oxalate.

[0063] For example, in primary hyperoxaluria (PH), which is a genetic disease and the most severe form of hyperoxaluria, patients produce about 100 - 300 mg oxalates per day. Methods of treating PH and preventing sequelae of PH include administering an amount of a reducing oxalate composition effective to reduce at least 100-300 mg of oxalate per day or at least 200 mg per day or 300 mg per day or over 300 mg per day or 400 mg per day. Such a delivery regimen may include an oral route of administration. For example, if provided by an orally administrable oral vehicle disclosed herein, such as an enteric coated capsule containing a composition of lyophilized oxalate reducing bacteria, the capsule may be provided at least once a day, at least two times a day, at least three times a day . at least four times a day or, if desired, to provide a dosage amount with an effective reducing activity of the oxalate. For patient convenience, a dosage regimen may include oral administration of an enteric coated capsule containing a composition containing lyophilized oxalate reducing bacteria containing 5x10⁵ up to 5x10⁷cfu / capsule and this dose is given two to three times a day during a meal. Each capsule has a reducing oxalate activity of at least 6.5 - 10 mg oxalate / h or 120 - 240 mg / day, and two or three such capsules can provide the maximum amount of oxalates produced by the patient with PH over a 24-hour period.

[0064] Carriers may be dry solid-based materials for preparations in the form of a tablet, capsule or powder and may be liquid or gel materials for liquid or gel preparations, which forms are partially dependent on the routes of administration.

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 the like.

[0066] Suitable liquid or gel carriers are well known in the art, such as water and physiological saline solutions, urea, alcohols and glycols such as methanol, ethanol, propanol, butanol, ethylene glycol and propylene glycol, and the like. Preferably, the water-based carriers have a pH close to neutral.

[0067] Suitable carriers include aqueous and oily vehicles, such as, for example, white petrolatum, isopropyl myristate, lanolin or lanolin alcohols, mineral oil, sorbitan monooleate, propylene glycol, cetyl alcoholostearyl (together or in different combinations), hydroxypropylcellulose (MW = 100,000 to 1,000,000), detergents (e.g., polyoxyl stearate or sodium lauryl sulfate) and 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 cocoate, sucrose distearate, mineral oil, propylene glycol, 2-ethyl-1,3-hexanediol, polyoxypropylene ether. 15-stearyl and water. For example, water-containing emulsions, glycerol stearate, glycerol, mineral oil, synthetic spermacet, cetyl alcohol, butyl paraben, propyl paraben and methyl paraben are commercially available. The carrier may also contain preservatives, including methyl paraben, propyl paraben, benzyl alcohol and ethylenediaminetetraacetate salts. The carrier may also contain well-known flavors and / or colorants. The composition may also contain a plasticizer, such as glycerol or polyethylene glycol (MW = 800 to 20,000). The composition of the carrier may vary, as long as it does not interfere with the viability of bacteria reducing oxalates or oxalate reducing enzymes in the composition.

[0068] A typical composition of the present invention may additionally contain any of the following inactive ingredients: gum arabic, aspartame, citric acid, D & C yellow No 10, D & C yellow No. 6, flavor (natural and / or artificial), polysorbate 80, propylene glycol alginate , colloidal silicon dioxide and sucrose, and xanthan gum.

[0069] The composition may also contain the following inactive ingredients: aspartame, beta carotene, citric acid, flavor (natural and / or artificial), glycerin, maltol, mannitol and methylcellulose.

In the methods disclosed herein for the preparation of O. formigenes pharmaceutical formulations for oral delivery to the gastrointestinal tract, including culturing the bacteria using fermentation methods known to those skilled in the art, freezing bacterial cells, thawing frozen cells and lyophilizing bacterial cells, optionally mixed in a solution of the substance auxiliary, and subsequent screening of lyophilized cells into the powder and delivery of the powder in an excipient to facilitate administration of the pharmaceutical preparation.

[0071] The invention further relates to administering to the gastrointestinal tract of a human or animal products or enzymes that degrade oxalates made from oxalate reducing organisms, such as O. formigenes cells.

[0072] Enzymes are coated or otherwise formulated or modified to protect enzymes so that they are not inactivated in the stomach and are able to exert the oxalate-degrading effect in the small intestine. Examples of such preparations are known to those skilled in the art and are described in, for example, US Patent No. 5,284,959.

[0073] Oxalate degrading enzymes as used herein include all enzymes involved in the oxalate pathway and include, but are not limited to, oxalate oxidase, oxalate decarboxylase, formyl-CoA transferase and oxalyl-CoA decarboxylase. Oxalate oxidase is expressed in higher plants and catalyzes oxygen-dependent oxidation of oxalate to CO 2 with the simultaneous formation of H2O2. Oxalate oxydases have been purified from many sources, for example from the roots of seedlings and leaves of barley, stems and leaves of beetroot, wheat germ, sorghum leaves and banana peel. A quick three-step purification procedure was developed to obtain oxalate oxidase from barley roots. The gene coding oxalate oxidase of barley roots has been cloned, sequenced and expressed.

[0074] Oxalate decarbosyllase is mainly present in fungi. Recently, bacterial oxalate decarboxylase in B. subtilis has been described, which is encoded by the yvrk gene. Oxalate decarboxylases catalyze the decomposition of free oxalate to CO 2 and formate. This enzyme has been described in various fungi, including Myrothecium, verrucaria, specific strains of Aspergillus niger and white rot fungus, Coriolus versicolor. The gene coding for the Flammulina velutipes oxalate decarboxylase has been cloned and sequenced; see WO 98/42827.

[0075] Oxalyl-CoA decarboxylase acts on the activated CoA substrate and converts it to formyl-CoA. Then, Formyl-CoA transferase works by exchanging formate and oxalate on CoA. These enzymes were tested in oxalate-decomposing bacteria, Pseudomonas oxalaticus, found in soil and in Oxalobacterformigenes, found in the digestive tract of vertebrates, including humans. O. formigenes has been shown to enter into a symbiotic relationship with the host organism, regulating the absorption of oxalic acid in the intestine as well as levels of oxalic acid in the plasma. As a result, it was found that the lack of these bacteria is a risk factor in oxalate related disorders, such as relapsing idiopathic nephrolithiasis with calcium oxalate deposition and intestinal hyperoxaluria secondary to intestinal bypass surgery.

Patents describing various oxalate degrading enzymes and genes encoding these enzymes include U.S. Patent Nos. 5912125, 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 oxalates or oxalic acid. These enzymes may be derived from natural sources or be synthesized by recombinant means known in the art and include all fragments, such as binding sites, active sites or fragments capable of interacting with oxalates or oxalic acid. This term also includes, but is not limited to, all necessary cofactors, coenzymes, metals or binding materials or substrates,

[0077] Zastosowanie O. formigenes jest szczególnie korzystne, ponieważ jest to bakteria beztlenowa, która nie rośnie w tlenowym środowisku tkanek i nie wytwarza żadnych związków, które są toksyczne dla ludzi lub zwierząt. Enzymy rozkładające szczawiany otrzymane z takich bakterii mogą być podawane lub cały drobnoustrój może być podawany.

[0078] In addition, all of the above-mentioned forms can be administered to domesticated animals, farm animals or kept in the zoo, suffering from a deficiency of bacteria that break down oxalates and humans. For example, enzymes and / or oxalate-degrading microbes may be administered to domestic animals, such as dogs, cats, rabbits, ferrets, guinea pigs, hamsters and gerbils, and to farm animals such as horses, cows and pigs or wild animals kept for breeding purposes, such as river otters. Many animals taken prisoner may lose their oxalate reduction ability. The present invention includes methods and compositions for restoring lost or reduced oxalate reduction activity. One aspect of the present invention includes treatment with the compositions described herein,

[0079] The present invention includes compositions and methods for administering compositions comprising one or more oxalate degrading bacteria or combinations of bacteria and enzymes to the human or animal gut. Such compositions and methods are effective in reducing the amount and / or concentration of oxalates present. Such methods and compositions are effective in the treatment and prevention of conditions associated with oxalates. The present invention encompasses methods of delivering one or more oxalate-degrading enzymes to the human or animal gastrointestinal tract in the form of a pharmaceutical and / or nutriceutical carrier composition. Such enzymes include, but are not limited to, oxalate oxidase, oxalate decarboxylase, oxalyl-CoA decarboxylase, and formyl-CoA transferase. These enzymes can be obtained from sources known to those skilled in the art. For example, a plant enzyme oxalate oxidase (OXO) can be purified from barley seedlings and oxalate decarboxylase can be purified from bacterial or fungal sources.

[0080] Alternatively, oxalate degrading enzymes can be obtained by recombinant means. For example, recombinant methods 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, the cloning and expression of the oxalate decarboxylase gene (YvrK) of B. subtilis: the oxalate decarboxylase gene (YvrK) gene has been cloned in the plasmid pET-9a and pET-14b (Novagen, WI), under the control of the strong T7 bacteriophage promoter to obtain overexpression as a soluble cytosolic protein. The expression host was the E. coli strain BL 21 (DE3) pLysS, XDE3 lysogen with protease deficiency, which contains the chromosomal copy of the T7-RNA polymerase gene under the control of lacUV5. In addition, this strain carries a pET-conforming plasmid that encodes a T7 lysozyme, a difunctional enzyme that cleaves the binding in the peptidoglycan cell wall and inhibits T7 RNA polymerase. This allows greater control of the non-induced basal expression and allows the use of methods that damage the inner membrane, such as freeze-thaw or mild detergents, etc. for efficient cell lysis. Expression of the gene product is triggered by the addition of isopropyl-PD-thiogalactopyranoside (TPTG). Accordingly, an aspect of the present invention includes methods comprising administering oxalate-degrading enzymes that have been produced by the recombinant microorganism.

[0081] Another aspect of the present invention includes methods for reducing the absorption of oxalates by providing bacteria that break down oxalates into the human or animal gut. Such bacteria include Oxalobacterformigenes. 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 by Ixion Biotechnology in 1996 from Dr. Milton Allison. For example, frozen stocks of human HC-1 strain can be used. The methods of the present invention involve enriching the intestines with one or more species of bacteria that break down oxalates, a general reduction of oxalates in the intestinal content, a reduction in the absorption of oxalates in the gut,

[0082] Accordingly, an aspect of the present invention includes compositions and methods for providing oxalate reducing bacteria and oxalate-degrading enzymes that can reduce oxalates to the gut of individuals at elevated risk for diseases and / or conditions associated with oxalates. Such diseases and conditions include, but are not limited to, hyperoxaluria, primary hyperoxaluria, idiopathic nephrolithiasis with calcium oxalate deposition (urolithiasis), intestinal hyperoxaluria, vulvodynia, oxalosis associated with end-stage renal disease, cardiac conduction disorders, inflammatory bowel disease, Crohn's disease, ulcerative colitis, people after undergoing a jejunal bypass surgery, people with insufficient concentrations of bacteria that break down oxalates and other intestinal diseases. People and animals that have undergone antibiotic therapy, chemotherapy or other therapies that alter the intestinal flora are treated with the compositions and methods of the present invention. The present invention is used to restore the oxalate reduction ability in humans or animals with altered intestinal flora. Increased levels of oxalate excretion in the urine favor the formation of kidney stones, contribute to scarring of the kidneys, and can even lead to kidney failure. Accordingly, an aspect of the present invention includes compositions and methods for reducing the formation of kidney stones. they are treated with the compositions and methods of the present invention. The present invention is used to restore the oxalate reduction ability in humans or animals with altered intestinal flora. Increased levels of oxalate excretion in the urine favor the formation of kidney stones, contribute to scarring of the kidneys, and can even lead to kidney failure. Accordingly, an aspect of the present invention includes compositions and methods for reducing the formation of kidney stones. they are treated with the compositions and methods of the present invention. The present invention is used to restore the oxalate reduction ability in humans or animals with altered intestinal flora. Increased levels of oxalate excretion in the urine favor the formation of kidney stones, contribute to scarring of the kidneys, and can even lead to kidney failure. Accordingly, an aspect of the present invention includes compositions and methods for reducing the formation of kidney stones.

[0083] The reduction of total oxalate concentrations in the intestine can also lead to oxalate removal from the cells and general circulation. In particular, a reduction in oxalate concentration in the gut may also lead to increased secretion of oxalate from the blood into the intestine. Without intending to be bound by any particular theory, it is now believed that there is a transepithelial gradient in the intestinal elimination of oxalates. Accordingly, an aspect of the present invention includes compositions and methods for lowering blood oxalate levels and increasing oxalate excretion by promoting the excretion of oxalates from the blood through the transepithelial gradient of oxalate for excretion of oxalate from the colon. The method of the present invention comprises providing a human or animal gut composition with a composition for lowering the concentration or level of oxalates in a human or animal. Such a reduction may include a reduction in the amount of oxalate present in the gut, in the blood, in the serum, in tissue fluids, and in other body fluids.

[0084] One composition of the present invention contains a O. formigenes paste prepared for oral administration. For each batch of O. formigenes, a single vial is used with the HC-1 starting material to produce seeding cultures to start the culture during large-scale fermentation. Bacteria from each fermentation are harvested by centrifugation and mixed with cryoprotectant excipients that provide protection during lyophilization. Cell paste can also be lyophilized or spray dried or vacuum dried, resulting in a fine powder that has a strength in the range of 10⁷ up to 10⁹ CFU / gram. The powder obtained is placed in gelatin capsules or in other capsules, such as HPMC capsules, which are enteric coated to safely deliver bacteria to the small intestine.

[0085] The compositions of the present invention contain compositions made from extracts of one or more oxalate reducing bacteria in the range of about 10³ to around 10¹² cfu / gram, from about 10³ to around 10¹⁰ cfu / gram, from about 10⁵ to around 10¹² cfu / gram, from about 10⁵ up to about 101 ° cfu / gram, from about 107 to about 109 cfu / gram, from about 107 to about 10⁸ cfu / gram and all compartments contained therein.

[0086] The compositions of the present invention also contain one or more enzymes that have oxalate reduction activity. The effective amount of the enzyme composition is able to reduce some oxalate in the intestine or reduce the oxalate concentration in a human or animal compared to the level measured prior to administration of the composition. Such measurement may be a measurement of oxalates present in the intestines from food sources or it may be a level measured in body fluid such as blood or urine.

[0087] The present invention encompasses methods of treating or preventing conditions associated with oxalates by administering compositions comprising O. formigenes to the human or animal gastrointestinal tract. Subjects can be administered enteric capsules containing> 103 cfu / g viable O. formigenes cells. Such dosages may take place at least twice a day during meals. The present invention also includes methods of administering oxalate reducing compositions comprising one or more microorganisms reducing oxalates, one or more oxalate reducing enzymes, or combinations thereof. The method of the present invention comprises the administration, at least once daily, of an effective amount of the oxalate reducing composition, wherein the oxalate reducing composition contains one or more oxalate reducing enzymes. The methods also include administering such compositions more frequently than once a day, more frequently than twice daily, daily, more than three times daily and in the range of 1 to 15 times per day. Such administration may be continuous, such as daily for days, weeks, months or years, or may take place at specific time points in the treatment or prevention of conditions associated with oxalates. For example, a person or animal can be administered oxalate reducing compositions at least once a day for the treatment or prevention of oxalate related conditions, or the person or animal can be administered oxalate reducing compositions at least once a day only at time points when foods containing oxalates, or for a limited period of time, such as days or weeks, after treatments or therapies that interfere with normal bacterial flora. Such administration may take place via known routes for administering pharmaceuticals. The present invention contemplates administration by oral or parenteral routes or in combination with food.

[0088] The invention further contemplates a therapeutic system for reducing oxalate, comprising a container containing a label and a therapeutic composition of the present invention, wherein said label includes instructions for using the oxalate reduction composition.

[0089] Typically, the system is in the form of a pack comprising a therapeutic composition of the present invention or in combination with packaging material. The packaging material includes a label or instructions for using the package components. The instruction indicates the contemplated use of a packet element, as described herein, in 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 mass amounts of the therapeutic composition. The label includes instructions for using the therapeutic composition in a unit dose or in bulk forms as desired, and may include information on composition storage, indications for use, dosages, routes of administration, and similar information.

[0090] The present invention includes pharmaceutical compositions and oxalate reduction methods in humans and animals. The composition may contain an oral vehicle to facilitate administration comprising a capsule, pill, granule or tablet. The composition may further comprise an enteric coating on an oral vehicle to facilitate administration. The enteric coating may be a polymeric material. Such polymeric materials may be one of a wide variety of Eudragit or other polymeric materials known to those skilled in the art for use as enteric coatings.

[0091] The compositions may contain oxalate reducing bacteria, i.e. Oxalobacter formigenes, or compositions in which the oxalate reducing bacteria are Oxalobacter formigenes strain HCl. Oxalate reducing compositions may contain a lyophilized powder. The powder may have a particle size of about 10 microns to about 2000 microns or from about 100 microns to about 1000 microns or from about 500 microns to about 1500 microns or from about 500 microns to about 1000 microns, 500 to about 1500 microns or in any compartment included in in them or around.

[0092] The composition may contain disaccharide, trehalose or be a composition wherein the alginate is sodium alginate. The oxalate reducing composition may have a cfu / g of at least from about 1E + 03 to about 1E + 13 of oxalate reducing bacteria. The oxalate reducing composition may have oxalate reducing enzyme activity / g of at least about 2 mg of oxalate cleavages / h to about 2500 mg of oxalate / hastergenic composition. [0093] The oxalate reducing composition in human or animal composition contains an oral vehicle to facilitate administration containing a oxalate reducing composition containing a composition comprising an excipient to facilitate administration comprising a composition comprising a) from about 3% to about 25% of oxalate reducing bacteria;

b) from about 1.5% to about 6% disaccharide; c) from about 45% to about 60% of an inertrose; d) from about 4% to about 6% alginate and e) from about 20% to about 35% oligofructose. [0094] The oxalate reduction composition contains an effective dosage amount with a reducing activity of oxalate that reduces a portion of the oxalates present and further comprises a pharmaceutical excipient to facilitate administration. The pharmaceutical excipient for ease of administration may include powder, pill, granule, suppository or tablet. Optionally, an enteric coating is provided on the delivery aid, and the enteric coating is essentially a polymeric material. An effective dosage amount having a reducing oxalate activity may be provided by oxalate reducing bacteria, which may be Oxalobacter formigenes or which may be Oxalobacter formigenes strain HCl. The composition may be provided in the form of a lyophilized powder. The powder may have a particle size of about 10 microns to about 2000 microns or from about 100 microns to about 1000 microns or from about 500 microns to about 1500 microns or from about 500 microns to about 1000 microns, 500 to about 1500 microns or in any compartment included in in them or around. The oxalate reducing composition may have a cfu / g of at least from about 1E + 03 to about 1E + 13 of oxalate reducing bacteria. The oxalate reducing composition may have oxalate reducing enzyme activity / g of at least about 2 mg of oxalate / h to about 2500 mg of oxalate / h. The powder may have a particle size of about 10 microns to about 2000 microns or from about 100 microns to about 1000 microns or from about 500 microns to about 1500 microns or from about 500 microns to about 1000 microns, 500 to about 1500 microns or in any compartment included in in them or around. The oxalate reducing composition may have a cfu / g of at least from about 1E + 03 to about 1E + 13 of oxalate reducing bacteria. The oxalate reducing composition may have oxalate reducing enzyme activity / g of at least about 2 mg of oxalate / h to about 2500 mg of oxalate / h. The powder may have a particle size of about 10 microns to about 2000 microns or from about 100 microns to about 1000 microns or from about 500 microns to about 1500 microns or from about 500 microns to about 1000 microns, 500 to about 1500 microns or in any compartment included in in them or around. The oxalate reducing composition may have a cfu / g of at least from about 1E + 03 to about 1E + 13 of oxalate reducing bacteria. The oxalate reducing composition may have oxalate reducing enzyme activity / g of at least about 2 mg of oxalate / h to about 2500 mg of oxalate / h. The oxalate reducing composition may have a cfu / g of at least from about 1E + 03 to about 1E + 13 of oxalate reducing bacteria. The oxalate reducing composition may have oxalate reducing enzyme activity / g of at least about 2 mg of oxalate / h to about 2500 mg of oxalate / h. The oxalate reducing composition may have a cfu / g of at least from about 1E + 03 to about 1E + 13 of oxalate reducing bacteria. The oxalate reducing composition may have oxalate reducing enzyme activity / g of at least about 2 mg of oxalate / h to about 2500 mg of oxalate / h.

[0095] The methods of the present invention include methods for reducing oxalate concentrations in humans and animals, methods for treating oxalate related conditions in humans and animals, methods for preventing oxalate related conditions in humans and animals, and methods for preparing oxalate reducing compositions. The present invention also includes oxalate reduction systems. A method for reducing oxalate concentration in a human or animal comprises administering to a human or animal an effective amount of a composition containing a vehicle-facilitating excipient comprising a oxalate reducing composition comprising a) from about 3% to about 25% of oxalate reducing bacteria; b) from about 1.5% to about 6% disaccharide; c) from about 45% to about 60% maltodextrin; d) from 4% to about 6% alginate and e) from about 20% to about 35% oligofructose. An excipient for administering may include a suppository, powder, pill, granule or tablet, which may further comprise an enteric coating on an excipient for ease of administration. The enteric coating may be a polymeric material. Oxalate reducing bacteria may be Oxalobacter formigenes or may be Oxalobacter formigenes strain HCl. The composition may be provided in the form of a lyophilized powder. The powder may have a particle size of about 10 microns to about 2000 microns or from about 100 microns to about 1000 microns or from about 500 microns to about 1500 microns or from about 500 microns to about 1000 microns, 500 to about 1500 microns or in any compartment included in in them or similar. The composition may contain disaccharide, trehalose or be a composition in which the alginate is sodium alginate. The oxalate reducing composition may have a cfu / g of at least from about 1E + 03 to about 1E + 13 of oxalate reducing bacteria. The oxalate reducing composition may have oxalate reducing enzyme activity / g of at least about 2 mg of oxalate / h to about 2,500 mg of dispersed oxalate / h. The method may comprise oral administration. The methods may also include preventing the oxalate related condition, including administering the compositions described herein. Such methods may also include treatment of the oxalate-related condition, including administration of the compositions described herein. Conditions associated with oxalates include, but are not limited to, hyperoxaluria, primary hyperoxaluria, idiopathic nephrolithiasis with calcium oxalate deposition (urolithiasis), intestinal hyperoxaluria, and vulwodynia. oxalosis associated with end-stage renal disease, cardiac conduction disorders, inflammatory bowel disease, ulcerative colitis, Crohn's disease, fat diarrhea, patients after gastrointestinal surgery, such as the operation of jejunal bypass surgery or antibiotic therapy. The methods include administering the compositions described herein more frequently than once a day for a period until sufficient oxalate reduction is achieved or for an indefinite time for continuous regulation of oxalate levels. The method includes a method of preventing an oxalate related condition comprising administering to a human or animal an effective amount of a dose with a reducing activity of oxalate which will reduce part of the oxalate present, comprising a) from about 3% to 25% of 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% of oligofructose and further comprising a pharmaceutical excipient to facilitate administration. The pharmaceutical excipient for ease of administration may include powder, pill, granule, suppository or tablet. A pharmaceutical excipient for ease of administration may include a capsule. Any of these excipients to facilitate administration may include an enteric coating. The enteric coating may be a polymeric material. Oxalate reducing bacteria may be Oxalobacterformigenes or may be Oxalobacterformigenes strain HCl. The composition may be provided in the form of a lyophilized powder. The powder may have a particle size of about 10 microns to about 2000 microns or from about 100 microns to about 1000 microns or from about 500 microns to about 1500 microns or from about 500 microns to about 1000 microns, 500 to about 1500 microns or in any compartment included in in them or around. The composition may contain disaccharide, trehalose or be a composition in which the alginate is sodium alginate. The oxalate reducing composition may have a cfu / g of at least from about 1E + 03 to about 1E + 13 of oxalate reducing bacteria. The oxalate reducing composition may have oxalate reducing enzyme activity / g of at least about 2 mg of oxalate / h to about 2,500 mg of dispersed oxalate / h. The method may comprise oral administration. Conditions associated with oxalates that can be treated or prevented include hyperoxaluria,

[0096] Methods for preparing a pharmaceutical reducing oxalate composition include providing a reduction of oxalate bacteria at a concentration of at least about 1E + 03 to about 1E + 13, optionally mixing the reducing bacteria with one or more pharmaceutically acceptable excipients; freeze-drying of bacteria; and placing or delivering bacteria in a pharmaceutical excipient to facilitate administration. Such excipients may include one or more compounds from disaccharide, maltodextrin, algicides or oligofructose. Oxalate reducing bacteria may be Oxalobacter formigenes or may be Oxalobacter formigenes strain HCl. The pharmaceutical excipient for ease of administration may be a powder, pill, granule, suppository or tablet. The enteric excipient may be included in the excipient to facilitate administration. The enteric coating may be a polymer coating, such as a coating containing Eudragit.

[0097] It should be noted that as used throughout this specification and the appended claims, singular forms include plural references unless the context clearly dictates otherwise.

[0098] The following are examples that illustrate the procedures for carrying out the invention. These examples should not be viewed as limiting the scope of the present invention. [0099] Examples 1 to 9 do not form part of the invention.

EXAMPLE 1

Treatment of high-risk patients [0100] Patients with primary hyperoxaluria were given enteric coated capsules containing lyophilized O. formigenes powder twice daily, preferably during two large meals per day. Each size 2 capsule contained approximately 137 mg of freeze dried bulk powder containing at least 10⁸ colony forming units (CFU) / gram.

[0101] For high-risk individuals this may be life-long treatment. Subjects in clinical trials have shown a reduction in colonization after discontinuation of therapy. In a clinical trial, treatment was continued for 4 weeks and two-week observation was used. The four-week treatment led to a significant reduction in oxalate levels in blood and urine compared to baseline levels. However, during the observation period, the number of Oxalobacter bacteria in the faeces decreased and the oxalate values ​​in plasma and urine began to increase. Thus, it has been proposed that continuous administration of oxalate reducing compositions will be necessary to provide oxalate reduction conditions. Compositions containing bacteria that can colonize and colonize continuously may lead to less frequent administration of oxalate reducing compositions.

[0102] Capsules containing enteric-coated O. formigenes cells can be consumed by populations of high-risk patients associated with oxalate-related diseases. These groups include:

1. Persons who produce too many endogenous oxalates due to, for example, a genetic disorder such as primary hyperoxaluria.

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

3. People who have urolithiasis in an interview with numerous episodes of idiopathic myeloma.

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

5. People with vulval vulvalisation.

6. People who use diets containing high levels of oxalates, such as are used in specific areas and seasons in India and Saudi Arabia. This group may also include individuals who prefer foods such as spinach, which contain a lot of oxalates.

[0103] Any of the above-described persons or animals may be provided with the composition of the present invention. For example, a person having higher than normal levels of endogenous oxalates 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 taken with food.

EXAMPLE 2

Treatment of low-risk patients [0104] O. formigenes, gastric secretion cells, such as those provided in enteric-coated capsules, may also be consumed by individuals in populations at lower risk for diseases associated with oxalates. It may be desirable to colonize these patients with one or two therapies containing compositions of oxalate reducing materials, such as oxalate reducing bacteria. These patients may also routinely receive therapies with oxalate reducing materials, in the form of supplements or food additives, such as milk or yogurt. These groups include:

1. People who have lost populations of normal bacteria that break down oxalates due to: oral antibiotics or relapses of diarrheal disease.

2. Infants may be vaccinated so that the normal protective population of Oxalobacter may be more easily formed than in the later life period when the principle of competitive exclusion operates.

[0105] Low risk individuals or animals can be treated twice daily by a capsule designed to deliver their contents to the large intestine, wherein the capsule contains at least 10⁷ cfu of one or more oxalate reducing organisms, such as O. formigenes. The capsule is preferably administered during a meal. EXAMPLE 3 [0106] Use of oxalobacterformigenes breaking enzymes to control hyperoxaluria.

[0107] A study was carried out to evaluate the efficacy of oxalobacter formigenes oxalate de-enzymes to control hyperoxaluria. Animals used: male Sprague Dawley rats: body weight: 250-300 g. Diets used: normal diet (ND): Harlan Teklad TD 89222; 0.5% Ca, 0.4% P. Drug used: lyophilized mixture of Oxalobacter formigenes lysate (source of enzymes) with oxalyl-CoA, MgCl2 and TPP. Drug delivery system (capsules): Size 9 capsules for preclinical studies on rats (gel capsules). Enteric coating Eudragit L-100-55 (Hulls America, Inc.). Basic 24h urine collection. Analysis of faeces for Oxalobacter formigenes - oxalobacter formigenes did not colonize in rats.

Experimental protocol:

A. Long-term research:

Experiment report on animals:

[0108]

Group I (n = 4): fed with oxalate diet with lysate. The rats were given two capsules daily at 16:00 and the oxalate diet overnight. The diet was removed during the day (from 8:00 to 16:00).

Group II (n = 4): fed with the oxalate diet as described for Group I (hyperoxaluric controls). 24 h urine collection was performed on day 7 and on day 9 of the above treatment.

[0109] Data for the average urinary oxalate concentration for the two groups of rats shown above indicate that feeding with Oxalobacter lysate resulted in a decrease in urinary oxalate concentrations in rats in Group I compared to hyperoxaluric controls (Group II). Enzymes are not active for a long time in the gastrointestinal tract, and short-term studies have been carried out as described below.

B. Short-term research:

Experiment report on animals:

[0110]

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

Group II (n = 4): Oxalate diet for two hours as in Group I.

Urine was collected from all animals for a further five-hour period and analyzed for oxalate concentrations. This was done on days 11, 12 and 15 of the study.

[0111] The results of this study indicate that feeding with Oxalobacter lysate induces a significant reduction in urinary oxalate levels in the 5-hour period following oxalate and drug administration in rats in Group I compared to the hyperoxaluric control group (Group II). At this point, a cross-over study was conducted between two groups of rats.

C. Badamaw uUradaienodro-namnnnm:

Experiment report on animals:

[0112]

Group I: fed the oxalate diet twice a day between 8:00 am and 10:00 am

15:00 - 17:00.

Group II: fed 1 capsule twice a day before feeding the oxalate diet, as in Group I.

[0113] Short-term studies on the effects of feeding Oxalobacter lysate on urinary oxalate levels were performed as described in Part B above at day -2 and day -5 after interchanging.

[0114] Studies in an alternation system showed that the previously hyperoxaluric Group II rats that were fed with Oxalobacter lysate had reduced levels of oxalate in the urine. In contrast, in rats in Group I, hyperoxaluria recurred after discontinuation of the drug.

EXAMPLE 4

Treatment of rats with Oxalobacter formigenes cells [0115] A study was conducted to assess the fate of oxalates consumed in the diet when Oxalobacter formigenes cells were added to the diet.

[0116] Methods: Male Wistar rats were fed a diet containing a normal amount of calcium (1%), a large amount of oxalate (0.5%) or a diet containing a small amount of calcium (0.02%), a large amount of oxalates (0.5%) during two separate experiments. ¹⁴C-oxalate (2.0 pCi) was administered on day 1 and again on day 7 of the study. Oxalobacter formigenes cells (380 mg / d) were administered to rats in drinking water on days 5-11. Moose^HC oxalate was determined based on analysis ^MC in feces, urine and expired air. The rats acted as internal controls and measurements during the control period (before feeding the cells

Oxalobacter) were made on days 1-4; during the experimental period (when cells were fed with bacteria) measurements were made on days 7-11.

Results:

[0117]

1. When the rats were fed a diet containing a normal amount of calcium (1%), less than 1% of the administered dose ¹⁴C oxalate was recovered in exhaled air (in the form of carbon dioxide produced from ^HC oxalate in the intestine, absorbed into the blood and then exhaled), but in all cases more ^HC was recovered during the period when the rats were fed Oxalobacter cells (FIG 1 a). The opposite results were obtained when the diet contained a small amount of calcium (0.02%) when more than 50% from^MC oxalate was recovered in the form of carbon dioxide in exhaled air during the experimental period when the cells were fed with Oxalobacter cells (FIG 1b). These results are strikingly different from very small amounts^HC (less than 5%) recovered during the control period (prior to feeding the rats with Oxalobacter cells). Thus, administration of Oxalobacter formigenes cells to rats significantly increased the amount of oxalate consumed, which was broken down in the gastrointestinal tract.

2. Feeding rats with Oxalobacter cells also led to a reduction in the amount of 1⁴C-oxalate, which was excreted in the urine. Values ​​for the 4-day collection in the control and experimental period, and for one day in each of these periods are depicted in FIG. 2a and 2b. The amounts of oxalate recovered in rat feces were also lower during the experimental period (when Oxalobacter cells were administered) from those found during the control period (FIG. 2c).

[0118] Most laboratory rats do not have Oxalobacter in their digestive tract (they are not colonized). These results indicate that the targeted administration of these oxalate-degrading bacteria to rats led to the breakdown of a large portion of the oxalate consumed, and as a result, less oxalate dietary intake was excreted in the urine.

[0119] The effect of the amount of calcium consumed on oxalate degradation is significant. Calcium forms complexes with oxalates, so that their solubility and availability for attack by Oxalobacter is limited, and the amount to be broken down when rats are fed high calcium diet is much less than the decomposed amounts when the calcium content in the diet is low.

EXAMPLE 5

Effect of O. formigenes feeding on urinary oxalate in pigs [0120] Pigs are naturally colonized Oxalobacter. Removal of the colonies was obtained in experimental pigs by enriching the diet with antibiotics. Pigs were given Oxalobacter in a culture broth, which the pigs eagerly ate. The pigs were fed with soy / maize-based feed enriched with 1300 mg oxalate / kg. The basic diet contained 680 mg oxalate / kg. The results are shown in FIG. 3a-c for three individual pigs. [0121] In all three pigs, the amount of oxalate in the urine dramatically decreased while consuming Oxalabacter. The level of oxalate excretion in these three pigs decreased to a minimum value of approximately 6 mg / g creatinine in all three pigs. This level should be compared with the level of 8-10 mg / g creatinine, which has been found in people using anxiety diets. This level is equal to endogenous synthesis in humans, as the dietary charge has been eliminated. It seems that this level reflects endogenous synthesis in pigs, and that intestinal absorption has been eliminated by Oxalobacter treatment. In addition, these results indicate that consumption of Oxalobacter enabled removal of both added crystalline oxalates and oxalates consumed that were bioavailable.

[0122] In this experiment, each swine was fed 1.0 g of cell paste together with a morning meal. At an OD600 of 0.6, the number of viable cells is 2.1x10⁸ cells / ml, corresponding to 2.1x10¹³ cells per 100 l. One 100 l fermenter cycle provides an average of 50-60 g wet cell mass. Thus 1 g of wet cell mass corresponds to approximately 3.5 x 1011 to live cells.

[0123] Dose 3.5 x 10ⁿ viable cells, as stated above, may lead to the elimination of intestinal absorption of about 2.0 g of oxalate present in 1 kg of feed (1300 mg of added oxalate + 680 mg present in the diet). The animals consumed 1 kg of food for one meal.

[0124] The pigs' body weight is about 200 pounds, and it is believed that the digestive system of pigs is very close to the digestive system of humans. In humans, the average daily intake of oxalates is around 100-400 mg depending on the composition of the diet, which also decomposes into three meals a day, so the average daily dose is 10⁸ up to 101 ° viable cells should be sufficient to prevent the absorption of oxalate consumed. EXAMPLE 6

Effect of O. formigenes supplementation on urinary oxalate excretion in rats fed a high-volume diet [0125] A study was conducted to determine the effect of IxOC-3 on the colonization status and urinary oxalate levels after using a high-sodium diet. IxOC-3 contains lyophilized live cells of oxalate reducing bacteria such as O. formigenes. This preparation contains approximately 10⁶-10⁷ cfu / gram per dose. The formulation also contains cryopreservatives such as trehalose and meltodextrin.

methods:

[0126] Male Harlan Sprague Dawley rats were randomly assigned to 3 groups (6 animals per group). Group 1 animals served as a control group and were administered a placebo size 9 intestinal patch twice daily by oral gavage at a dose level of 100 colony forming units (CFU). Animals from Groups 2 and 3 were dosed with Oxalobacter formigenes IxOC-3 in an enteric coated capsule, twice daily, by oral gavage, at a dose level of 106 and 107 CFU, respectively. After the administration of the capsule by gavage, all three groups were given autoclaved tap water for drinking. After the initial acclimatization period, all groups were fed with standard feed enriched with 1% oxalate per gram. [0127] The test materials and the control placebo material were prepared according to a standardized protocol. Before use, representative samples of each material tested 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 administration period.

[0128] Feeding was limited to two 1-hour periods per day, beginning 15 minutes after morning and evening capsule administration, to ensure that the capsules were administered on an empty stomach. Water was provided ad libitum. Food intake was recorded twice a day. Fecal samples were collected and a 24 h urine collection was performed on day 1 (before administration of oxalate enriched diet) and then once a week. Urine data were analyzed using repeated analysis for differences in mean urine parameters for individual dose groups and time. The analysis also included the term for the relationship between the dose group and the interaction time to assess potential relationships between the dose group and time. Results:

[0129] The results of the analysis indicate that there is a statistically significant relationship between the dose groups and the time (p <0.0001) for all parameters, indicating that the urine parameter profile over time was different in the individual dose groups. To help in the interpretation of this relationship, data was analyzed at individual time points for each parameter to determine if there was a difference between dose groups in terms of mean urine parameters. This analysis revealed that there was an increase in the amount of oxalate in the urine between baseline and day 7 (p <0.0001 for both groups) in the low dose group and high dose group, while there was no increase between day 7 and day 28 (p. = 0.1094 low dose and p = 0.6910 high dose). In the placebo group, there was an increase between the starting point and day 28 (p = 0.0010). Also on day 21 and day 28, the average levels of urinary oxalate in Group I placebo were significantly higher than in the small group (Group II) and high (Group III) dose, but with no significant difference between low dose and high dose. Thus, there was a general significant reduction in urinary oxalate excretion in treated rats compared to rats fed with placebo.

EXAMPLE 7 [0130] Effect of O. formigenes oral administration on urinary oxalate levels in patients suffering from primary hyperoxaluria (PH)

methods:

[0131] Nine patients with primary hyperoxaluria (PH) confirmed by biopsy participated in the study. After initial baseline evaluations, 1 g of Oxalobacterformigenes cell culture was administered to all subjects (> 10¹⁰ cfu / gram) during main meals for 4 weeks. During this time all patients continued to take their normal drugs, were asked to use their normal diet and maintain normal fluid intake. Consumption of foods containing large amounts of oxalates, except for spinach and rhubarb, was not prohibited. Oxalobacter colonization and its effect on urinary and plasma oxalate levels were measured at weeks 5 and 6. The efficacy of treatment was checked by urinary oxalate excretion in subjects with normal renal function and plasma oxalate levels in subjects with end-stage renal disease (ESRD).

Results:

[0132] 1. Treatment showed a significant reduction in urinary oxalate in subjects with normal renal function. The oxalate concentration dropped significantly in seven out of nine individuals. There has been dramatic depletion of plasma oxalate levels in two ESRD subjects, which provides evidence for intestinal elimination of endogenous oxalates into the intestine against the transepithelial gradient.

[0133] Consumption of O. formigenes strain HC-1 at doses ranging from 0.25g to 2.0g per meal was well tolerated by normal, healthy volunteers using diets containing medium or high levels of oxalates. The 1.0 g dose of the cell paste twice daily for 28 days was well tolerated by patients with PH. EXAMPLE 8

Treatment of high-risk patients with oxalate reducing enzymes [0134] Patients with primary hyperoxaluria were administered with one or more enterically coated capsules containing a lyophilized oxalate reducing enzyme composition containing oxalate decarboxylase and / or oxalate oxidase, twice daily, preferably in two main phases. meals of the day. An effective amount of the enzyme composition was administered. For example, each size 2 capsule contains about 5-100 units of each enzyme.

[0135] For high risk individuals, this is continuous, for a long time, possibly life-long treatment. Colonization decreases after discontinuation of treatment. [0136] Capsules of enteric coating reducing compositions containing oxalate reducing enzymes may be administered to populations of high-risk patients associated with oxalate related diseases. These populations include:

1. Persons who produce too many endogenous oxalates due to, for example, a genetic disorder such as primary hyperoxaluria.

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

3. People who have urolithiasis in an interview with numerous episodes of idiopathic myeloma.

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

5. People with vulval vulvalisation.

6. People who use diets containing high levels of oxalates, such as are used in specific areas and seasons in India and Saudi Arabia. This group may also include people who prefer foods such as spinach, which contain a lot of oxalates.

[0137] Any of the above-described persons or animals may be provided with a composition of the present invention. For example, a person having higher than normal levels of endogenous oxalates is treated twice daily with a capsule designed to deliver its contents to the large intestine, wherein the capsule contains an approximately equivalent effective amount of the enzyme composition having enzyme activity similar to that provided by⁷ cfu of a reducing oxalate, such as O. formigenes. The capsule is preferably administered with food.

EXAMPLE 9

Treatment of low-risk patients with oxalate-reducing enzymes [0138] The oxalate-reduced oxalate-containing compositions containing a mixture of oxalate reducing oxalate decarboxylase and / or oxalate oxidase, such as delivered in enteric-coated capsules, may also be administered to individuals in populations lower risk of diseases related to oxalates or the risk of conditions associated with oxalates. The effective amount of the enzyme composition is administered in the desired treatment regimen.

[0139] It may be desirable to administer the composition to these patients for a shorter time when they are at risk for conditions associated with oxalates or concurrent with materials that contribute to the oxalate related condition. These patients may also routinely receive treatment with oxalate-reducing compositions in the form of supplements or food additives such as milk or yogurt. These include people who have lost populations of normal bacteria that break down oxalates due to: oral antibiotic treatment or relapses of diarrheal disease or infants.

[0140] Low risk individuals or animals are treated twice daily with a capsule designed to deliver their contents to the large intestine, the capsule containing an effective amount of the enzyme composition. For example, each size 2 capsule contains approximately

5-100 units of each enzyme. The capsule is preferably administered during a meal. EXAMPLE 10

Process for the manufacture of enteric coated capsules containing lyophilized Oxalobacter formigenes [0141] Oxalobacterformigenes cell paste in an amount of 200 grams (activity of the oxalate decomposing enzyme / g in the range of about 60 to about 1600 mg / g) was used. The cellular paste may be fresh, fermented or may originate from frozen materials that have been thawed. The cell paste was mixed with 100 mM trehalose as a cryopreservative or cryoprotectant solution. The solution was mixed continuously. This mixture was then mixed with a mixture of excipients. The mixture of excipients contained maltodextrin M500 and sodium alginate mixed together and added to a 79% raftilose P95 solution. The cell paste mixture was then poured onto a lyophilization tray (s). The filled trays were then lyophilized in an Edwards Lyofast S24 dryer (any suitable type of dryer can be used) for 40-65 hours, which can be changed for different production cycles. After lyophilization, the dried cake was ground by hand and passed through a US 20 mesh screen. A powder with a particle size of <850 μ m was thus obtained.

[0142] After sieving the dried powder, it was ready to be filled into capsules. The capsules were filled using a manual capsule filling apparatus, but any method of filling capsules, such as automatic filling apparatus, could be used.

Capsules of size 2 were generally used. The capsules were coated with enteric polymers, such as Eudragit (commercially available from Rhom Pharma (Degussa) using a water-based coating method, alternatively a solvent coating method could be used.) This company produces various types of Eudragit polymers that specifically they dissolve at different pH.

[0143] Coating was carried out using standard techniques. Eudragit polymers are polymers and copolymers of methacrylic acid. Eudragit L100-55, for example, is a copolymer of methacrylic acid type C, Eudragit L30 is a methacrylic acid copolymer dispersion, and Eudragit S100 is a type B metacrylic acid copolymer. These and other enteric coatings are known in the art.

Water-based coating [0144] Eudragit FS30D, a thin film forming agent and Eudragit L30D55, a thin film forming agent together with plasticizers, anti-sticking agents and carriers such as water and those known in the pharmaceutical field were used.

[0145] 800 gram capsules were coated with a coating suspension to form homogeneously coated capsules with a USP disintegration profile. Disintegration profile: no breakdown in the simulated gastric fluid (pH 1.2) in one hour and complete disintegration in the simulated intestinal fluid (pH 6.8) in one hour.

The process of solvent coating

A thin film Eudragit L100-55 agent. [0146] Eudragit S100, a thin film forming agent, was used along with other materials such as plasticizers, anti-sticking agents and carriers, such as water and those known in the pharmaceutical art.

[0147] 800 gram capsules were coated with a coating suspension to form homogeneously coated capsules with a USP disintegration profile. Disintegration profile: no breakdown in the simulated gastric fluid (pH 1.2) in one hour and complete disintegration in the simulated intestinal fluid (pH 6.8) in one hour.

EXAMPLE 11 [0148] For the oral delivery of capsules obtained by the methods of Example 10 for seven different experiments, the following data was obtained during 1 year of testing. Can we add the contents of the capsules ?? The pharmaceutical compositions prepared in this way were evaluated for stability in terms of bacterial viability and oxalate degradation activity. In the table below, Table 1, there are 7 cycles of preparation and stability of pharmaceutical compositions containing enteric coated capsules containing live reducing oxalate bacteria, in particular, O. formigenes.

TABLE 1

<td>Experience number</td><td>0 months</td><td>One month</td><td>2 months</td><td>3 months</td><td>3.4 month</td><td>6 months</td><td>9 months</td><td>12 months</td>

<td>1</td><td>3,6E + 0</td><td>2,2E + 0</td><td></td><td>1,6E + 0</td><td></td><td>1,1E + 0</td><td>1.2E + 0</td><td>5,1E + 0</td>

<td></td><td>8</td><td>6</td><td></td><td>6</td><td></td><td>6</td><td>6</td><td>5</td>

<td>2</td><td>3,6E + 0</td><td>1,8E + 0</td><td></td><td></td><td>3,9E + 0</td><td>1,5E + 0</td><td>2,2E + 0</td><td>6,0E + 0</td>

<td></td><td>8</td><td>7</td><td></td><td></td><td>6</td><td>6</td><td>6</td><td>5</td>

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

<td></td><td>8</td><td>7</td><td></td><td>6</td><td></td><td>6</td><td>6</td><td>6</td>

<td>4</td><td>1,5E + 0</td><td></td><td>1.2E + 0</td><td>3,5E + 0</td><td></td><td>4,1E + 0</td><td>1,7E + 0</td><td>1,1E + 0</td>

<td></td><td>8</td><td></td><td>7</td><td>6</td><td></td><td>6</td><td>6</td><td>6</td>

<td>5</td><td>1,5E + 0</td><td></td><td>5,9E + 0</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>8</td><td></td><td>6</td><td></td><td></td><td></td><td></td><td></td>

<td>6</td><td>2,4E + 0 8</td><td>2,9E + 0 7</td><td>1,7E + 0 7</td><td></td><td></td><td>3,4E + 0 6</td><td>3,7E + 0 6</td><td>2,3E + 0 5</td>

<td>7</td><td>2,4E + 0 8</td><td>7,1E + 0 7</td><td>1.2E + 0 7</td><td></td><td></td><td>2,0E + 0 6</td><td>1.2E + 0 6</td><td>1,5E + 0 5</td>

[0149] Another Example is presented in the form of data in below Table 2.

TABLE 2

<td>Time in months</td><td>Act. OX distribution / capsule (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>

[0150] In another manufacturing cycle, the following data was obtained:

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>

[0151] The activity is given in mg of oxalates spread over an hour.

EXAMPLE 13 [0152] Formulations for the oral delivery of a pharmaceutical composition of viable Oxalobacter formigenes

Preparation 1

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

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

<td>D (+) Trehalose (cryoprotectant)</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>Raftiloza P95 or oligofructose</td><td>126.24</td><td>thirty%</td>

[0153] For example, trehalose can be obtained from Sigma Co. Trehalose is a disaccharide, whereby the preparation of the present invention contains a disaccharide, such as maltose, lactose, cellobiose, sucrose, diglucose or trehalose. The QD M-500 maltodextrin has a DE value of 10 and is a white powder or a granular white powder. Is a non-sweet, nutrient saccharide polymer composed of D-glucose units connected mainly by alpha-1-4 bonds. DE is dextrose equivalents and is a quantitative measure of the degree of hydrolysis of a starch polymer. The higher the DE value, the greater the degree of hydrolysis of starch. Stabilizers are also components of the formulation, such as sodium alginate, which is also used as a stabilizer, thickener, gelling agent or emulsifier. Sodium alginate is a natural amylose carbohydrate distilled from algae. It is widely used in food, medicines, textiles, printing and dyeing, paper production and daily chemicals such as, for example, a thickener, emulsifier, stabilizer and binder, etc. Molecular formula is COOHNO3 and is a white or slightly yellow powder, odorless, tasteless, soluble in water, not soluble in ethanol and ether. Raftiloza P95 is a powder with a content of 95% oligofructose DP2 to DP7 and sugars of glucose, fructose and sucrose (5%). not soluble in ethanol and ether. Raftiloza P95 is a powder with a content of 95% oligofructose DP2 to DP7 and sugars of glucose, fructose and sucrose (5%). not soluble in ethanol and ether. Raftiloza P95 is a powder with a content of 95% oligofructose DP2 to DP7 and sugars of glucose, fructose and sucrose (5%).

Preparation 2

<td>Component</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>thirty%</td>

Preparation 3

<td>Component</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>oligofructose</td><td>126.24</td><td>thirty%</td>

Preparation 4

<td>Component</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>Raftiloza P95</td><td>95.91</td><td>23%</td>

Preparation 5

<td>Component</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

<td>Component</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

<td>Component</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

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

<td>Ox.formigenes cell paste (dry)</td><td>396.04</td><td>95%</td>

<td>Disaccharide - excipient</td><td>8.34</td><td>2%</td>

<td>A mixture of excipients - Maltodextrin, Na alginate, oligofructose</td><td>12.5</td><td>3%</td>

EXAMPLE 14

Stability study of the compositions of the invention [0154] Material from 13 different coating cycles (7 aqueous + 6 organic, using 8 different lots of the formulation of Example 10) was stored in a refrigerator (5 ± 3 ° C) and storage conditions -20 ° C. Six of the 13 cycles had data up to a 36-week time point; in 3 with 13 cycles up to 24 weeks; in 2 of 13 cycles with data up to 12 weeks, in the other 2 data were available up to a 4-week time point. The only variables studied in these studies were:

Storage temperature (in fridge and -20 ° C)

Capsule type (gelatin to HPMC)

Coating type (aqueous to organic) [0155] With or without additional stabilizing agent (± Avicel at a concentration of 1-5% w / w)

Packaging (polypropylene tubes (PP) against blisters) [0156] The results are shown in Figures 5-12.

[0157] Figure 5 shows the results of an enteric stability test of capsules stored at 4 ° C and -20 ° C, respectively. The results showed that storage at 20 ° C leads to a smaller decrease in cfu / capsule than storage at 4 ° C. The data is presented in the table below. Figure 5 is a graph showing the average number of cfu / capsule in coated capsules in which diamonds are capsules stored at 4 ° C and squares are capsules stored at -20 ° C.

[0158] The data in the table below is shown in Figure 6, which depicts the logarithm of the defect versus storage time. Figure 6 is a graph depicting the average logarithm of loss in coated capsules wherein the diamonds are capsules stored at 4 ° C and squares are capsules stored at -20 ° C.

TABLE 4

<td colspan="7">The average logarithm of loss in coated capsules</td>

<td>Time in weeks</td><td>1</td><td>4</td><td>8</td><td>12</td><td>24</td><td>36</td>

<td>Logarithm of the defect @ 4 ° C</td><td>0.43</td><td>0.62</td><td>0.96</td><td>1.25</td><td>1.81</td><td>2.25</td>

<td>Logarithm of the defect @ -20 ° C</td><td>0.33</td><td>0.25</td><td>0.30</td><td>0.37</td><td>0.75</td><td>0.77</td>

<td>Log loss values ​​@ 4 ° C (+ SD)</td><td>0.26</td><td>0.28</td><td>0.37</td><td>0.40</td><td>0.48</td><td>0.64</td>

<td>Log loss values ​​@ -20 ° C (+ SD)</td><td>0.31</td><td>0.31</td><td>0.56</td><td>0.33</td><td>0.40</td><td>0.32</td>

[0159] Figure 7 shows the difference in mean losses in gelatin capsules and with HPMC (hydroxypropyl methylcellulose), with an enteric coating. Up to about 36 weeks of storage, it seemed that the composition contained in the enteric-coated gelatin capsules was more stable than the composition contained in the enteric-coated HPMC capsule. Figure 7 is a graph showing the logarithm of a defect over time wherein diamonds are coated gelatin capsules and squares represent coated HPMC capsules.

[0160] Gelatin or HPMC capsules may be coated using a water-based coating composition or an organic solvent. Figure 8 shows the effect of the average loss dependent on the coating composition, and Figure 9 shows the results for different types of capsules. Figure 8 is a graph showing the mean logarithm of a loss in water-based coated capsules and organic compounds. Diamonds means water-based capsules stored at 5 ± 3 ° C; squares means capsules coated with a solvent based on organic compounds, stored at 5 ± 3 ° C; triangles means water-based capsules stored at -20 ° C and cross-strikers (x) are capsules coated with an organic solvent, stored at -20 ° C. Figure 9 is a graph showing the logarithm of the loss for different coatings in different types of capsules. Diamonds are water-based gelatin capsules; squares means water-based HPMC capsules; triangles means solvent-coated gelatin capsules based on organic compounds, and cross-strikethroughs (x) are HPMC solvent-coated capsules based on organic compounds, all stored at 5 ± 3 ° C.

[0161] Avicel® can be added as a scavenger or stabilizer. Figure 10 shows the effect of Avicel® on the average loss. Figure 10 is a graph showing the mean logarithm of the loss in Avicel® coated capsules and without. Diamonds means Avicel® coated capsules, stored at 5 ± 3 ° C; squares means coated capsules without Avicel®, stored at 5 ± 3 ° C; triangles are Avicel® coated capsules, stored at -20 ° C, and cross-strikers (x) are Avicel® coated capsules, stored at -20 ° C.

[0162] Figures 11 and 12 relate to the packaging of capsules. The capsules were packaged in propylene tubes or blister packs. However, the results do not accurately reflect the user's situation due to the fact that the tubes were opened only when the sample was removed. The situation in normal life is different from this situation, because the patient normally opens the tube every time he takes a new dose, i.e. the other capsules are much more exposed to the environment than during the experience described here. Figure 11 is a graph depicting the average logarithm of activity loss for coated capsules packed into polypropylene tubes and blister packs. Diamonds means coated capsules packed in polypropylene tubes, stored at 5 ± 3 ° C; squares means coated capsules packed in blister packs, stored at 5 ± 3 ° C; triangles means coated capsules packed in polypropylene, stored at -20 ° C, and cross-strikers (x) are coated capsules packed in blister packs, stored at -20 ° C. Figure 12 is a graph showing the average logarithm of activity loss for coated capsules, packaged in polypropylene tubes and blister packs, and is identical to Figure 11, but does not include error bars. Diamonds means coated capsules packed in polypropylene, stored at 5 ± 3 ° C; squares means coated capsules packed in blister packs, stored at 5 ± 3 ° C; triangles means coated capsules packed in polypropylene, stored at -20 ° C, and cross-strikers (x) are coated capsules packed in blister packs, stored at -20 ° C.

EXAMPLE 15

Changes in the coating process [0163] Capsules (size 2) containing a lyophilized powder containing the bacteria and excipients of the Example are provided with a coating composition as described in the table below. Coating was carried out using an aqueous coating composition as described in Table 4 below. The coating formulation used the following: Eudragit® 30 D-55 (Rohm America, Piscataway NJ), triethyl citrate (Morflex Inc., Greensboro, NC), glycerol monostearate (Imwitor 900K, Sasol, Germany) and polysorbate 80 (Merck KGaA, Germany).

[0164] The final theoretical polymer weight gain of Eudragit L30 D-55 for each trial was 14 mg / cm² This corresponds to the total weight gain of the capsule of 31.1% w / w.

TABLE 5

<td>Excipient</td><td>Function</td><td>Quantity (G)</td><td>Dry substance (g)</td><td>% of dry polymeric substance</td>

<td>Eudragit® L30 D-55</td><td>Polymer</td><td>804.9</td><td>241.5</td><td></td>

<td>Triethyl citrate</td><td>plasticizer</td><td>48.3</td><td>48.3</td><td>20%</td>

<td>Glycerol monostearate</td><td>Slip agent</td><td>16.9</td><td>16.9</td><td>7%</td>

<td>Polysorbate 80</td><td>emulsifier</td><td>6.8</td><td>6.8</td><td>40% GMS</td>

<td>Deionized water</td><td>Medium</td><td>376.8</td><td>55</td><td></td>

<td>SUM</td><td></td><td>1253.7</td><td>313.4</td><td></td>

Total solids 25.0% w / w

Procedure for preparing a coating dispersion [0165]

1. Add 45% water to a clean container and heat to 70 ° C.

2. Add polysorbate 80, triethyl citrate and glyceryl monostearate to the heated water from step 1.

3. Stop heating the dispersion from step 2 and homogenize with a high speed mixer for 10 minutes.

4. Add the remaining amount of water to the emulsion from step 3 and allow to cool to room temperature.

5. Add another Eudragit® L 30 D-55 to the second clean dish and start gently mixing with a low-speed mixer.

6. Slowly add the emulsion from step 4 to Eudragit® L 30 D-55 from step 5 and continue mixing for 30 minutes.

7. Sieve the dispersion from step 6 through a 60 mesh (250 μm) screen.

8. Continue mixing the dispersion from step 7 with a low-speed mixer for the duration of the coating process.

[0166] Thin coat dispersions (thin film coating) were applied to:

a. 600 g total batch size of capsules in a 15-inch coating machine with perforated Compu-Lab drum (Thomas engineering, IL)

b. 800 g total batch size of capsules in a GPCG 1 fluidisation system (Glatt air Techniques, NJ) equipped with a 6-inch Wurster and an air distribution plate type

D. The height of the partition was 25 mm.

[0167] The process parameters are shown in the following table:

TABLE 6

<td>Coating system</td><td>Drum</td><td>Fluidized bed</td>

<td>Spray nozzle (mm)</td><td>1.0</td><td>1.2</td>

<td>Air flow</td><td>180 CFM</td><td>150 m³/ h</td>

<td>Drum speed (rpm)</td><td>15-16</td><td></td>

<td>Atomization pressure</td><td>26 psi</td><td>2 Bar</td>

<td>Air temperature at inlet (° C)</td><td>28-40</td><td>30-34</td>

<td>Outlet air temperature (° C)</td><td>25-28</td><td>26-28</td>

<td>Product temperature (° C)</td><td>21-28</td><td>25-28</td>

<td>Spraying speed (g / min / kg)</td><td>6</td><td>3-10</td>

<td>Process duration (min)</td><td>180</td><td>146</td>

[0168] Samples were placed in appropriate containers for packaging and labeling.

Conclusion [0169] The enteric formulation was applied to the capsules both in a drum and fluidized coater without any process problems.

EXAMPLE 16

Fusion of Enteric-Coated Capsules [0170] To avoid leakage of the capsules and the passage of the acidic medium (gastric fluid) after oral administration, the following encapsulation experiments were performed. Fusion means applying a seal at the edges where the two covers of the capsule overlap and leave space (connecting the capsule). As stated here early, in some circumstances, the place of contact between one capsule shell and the other can lead to the diffusion of gastric fluid into the capsule, i.e. a process that is definitely undesirable due to bacterial instability in the acidic environment.

[0171] In addition, disintegration studies have been carried out.

materials:

[0172]

Pharmacoat 606 (HPMC (Shin Etsu)

Ethanol (Spectrum)

Red dye 40 (Sensient)

Polysorbate 80 (PS-80) (Qualicaps)

Gelatin, NF (Qualicaps)

Eudragit L100 (Degussa)

Triethyl citrate (TEC) (Morflex)

Talk (Whittaker)

Isopropyl alcohol (IPA) (Univar)

Eudragit L100-55 (Degussa)

Eudragit S100 (Degussa)

Test [0173] The apparatus for decomposition evaluation was in agreement with USP. A suitable number of vessels was filled with approximately 900 ml of pH 1.2 buffer and maintained at 37 ° C. One capsule was placed in each tube of the basket for each series and covered with a wire mesh. The camera was started for one hour and the number of remaining capsules was recorded. The baskets were then transferred to new vessels filled with approximately 900 ml of pH 6.8 buffer maintained at 37 ° C. After an hour, the number of capsules that were broken up was registered.

Preparation of the preparation

Assembly [0174] Both HPMC and gelatin capsules were fused in accordance with the methods in the following table using a Schaefer Technologies STI Laboratory Capsule Bander.

AND)

TABLE 7

<td>Component</td><td>Series number</td><td>% wt./wind.</td><td>g / batch</td>

<td>PS-80</td><td>N0601316</td><td>1.20%</td><td>4.6</td>

<td>Gelatin, NF</td><td>N0602132</td><td>28.31%</td><td>108.5</td>

<td>Deionized water</td><td>ON</td><td>70.23%</td><td>269.2</td>

<td>B-1 dye</td><td>AM9123</td><td>0.26%</td><td>1</td>

<td>Sum</td><td></td><td>100.00%</td><td>383.3</td>

1) Gently mix water and surfactant

2) Add B-1 dye. Gently mix by hand.

3) Gently mix in gelatin, NF. Allow to swell for 1-2 hours.

4) Tightly cover. Place in an oven or a bath at 55 ° C to melt the gelatin.

B)

<td></td><td colspan="3">fABEL 8</td>

<td>Component</td><td>Series number</td><td>% wt./wind.</td><td>g / batch</td>

<td>Pharmacoat 606 (HPMC)</td><td>5106041</td><td>16.0%</td><td>80</td>

<td>Ethanol</td><td>VN0458</td><td>50.4%</td><td>252</td>

<td>Deionized water</td><td>ON</td><td>33.1%</td><td>165.5</td>

<td>Red dye 40</td><td>AM8564</td><td>0.5%</td><td>2.5</td>

<td>Sum</td><td>-</td><td>100.00%</td><td>500.0</td>

1) Add HPMC to ethanol and mix.

2) Add the dye to deionized water and mix

3) Add (2) to (1) and mix.

[0175] Table. A) A method of combining a capsule for the anastomosis of gelatin capsules. B) The method of combining the capsule for the anastomosis of HPMC capsules.

[0176] Typically, the gelatin capsules were fused with the gelatin-containing composition, and the HPMC capsules were fused with a composition containing HPMC. The assembly was carried out before the capsules were coated with an enteric coating. In addition to strengthening the attachment of the capsule, it seems that the fusion allowed better and more homogeneous enteric coating.

Coating tests [0177] Various coating trials of gelatin capsules and composites have been carried out

HPMC. Coating was tested both in a fluidized bed dryer and in a perforated drum coat machine. Two different organic coating formulations were tested in drum coating trials.

Drum coating - Test 1 [0178] Test 1 was performed to evaluate the viability of a new solvent formulation and to determine treatment parameters. The following coating formulation and coating parameters were used:

TABLE 9

<td colspan="6">Preparation for coating</td>

<td>Component</td><td>Series number</td><td>% w.</td><td>g / batch</td><td>g solid ingredients / batch</td><td>% solids / batch</td>

<td>Eudragit L 100</td><td>B040503013</td><td>6.00%</td><td>600</td><td>600</td><td>6.00%</td>

<td>Triethyl citrate (TEC)</td><td>000006306</td><td>0.60%</td><td>60</td><td>60</td><td>0.60%</td>

<td>Talc</td><td>H05015</td><td>1.80%</td><td>180</td><td>180</td><td>1.80%</td>

<td>Isopropyl alcohol (ipa)</td><td>MV016821523</td><td>86.54%</td><td>8654</td><td>0</td><td>0,00%</td>

<td>Water</td><td></td><td>5.06%</td><td>506</td><td>0</td><td>0,00%</td>

<td>Sum</td><td>-</td><td>100.00%</td><td>10000</td><td>840</td><td>8.40%</td>

Procedure:

[0179]

1) Dispense Eudragit in 6000 g IPA.

2) Add water to (1).

3) Mix TEC, talc and remaining IPA (2654g).

4) Connect (2) and (3) and mix for 1 hour.

Coating parameters:

[0180]

Product temperature = 23-27 ° C. Atomizing air = 25-27 psi

Air flow = 250 cfm Spray rate = 52-56 g / min

Drum rotation speed = 15 rpm [0181] About 800 g capsules were loaded into a 15-inch coating pan. Then the drum was started and the process conditions were set. Almost immediately after starting, the product temperature reached the desired range and spraying started at approximately

52 g / min. It seems that io.ccęake.w ^ xt after &lt;^^ \ caas of spattering, probably related to the increase in pressure in the ducts, led to a slight sticking, however, sticking or nailing was not observed later in the cycle. Samples were taken at 8, 12, 15 and 20% of theoretical weight gain.

Test 2 [0182] Test 2 was performed as described below.

TABLE 10

<td colspan="6">Capsules with enteric coating</td>

<td>Component</td><td>Series number</td><td>% w.</td><td>g / batch</td><td>g solid ingredients / batch</td><td>% solids / batch</td>

<td>Eudragit L100-55</td><td>B041004023</td><td>7.18%</td><td>107.7</td><td>107.7</td><td>7.18%</td>

<td>Eudragit S100</td><td>B03005052</td><td>5.85%</td><td>87,75</td><td>87,75</td><td>5.85%</td>

<td>Talc</td><td>H05015</td><td>5.00%</td><td>75</td><td>75</td><td>5.00%</td>

<td>TEC</td><td>000006306</td><td>1.30%</td><td>19.5</td><td>19.5</td><td>1.30%</td>

<td>Water</td><td></td><td>5.00%</td><td>75</td><td>0</td><td>0,00%</td>

<td>IPA</td><td>MV016821523</td><td>75.70%</td><td>1135.5</td><td>0</td><td>0,00%</td>

<td>Sum</td><td></td><td>100,03%</td><td>1500</td><td>289.95</td><td>19.33%</td>

1) Procedure:

2) Mix talc and 200 g IPA.

3) Mix in the bag Eudragit L100-55 and Eudragit S100,

4) Mix the remaining IPA (935.5 g) and water.

5) Add (2) to (3).

6) Add TEC and (1) to (4).

Process parameters:

Product temperature = 25-28 ° C Atomizing air = 38-42 psi Air flow = 250 cfm Spray rate = 17-20 g / min Drum speed = 15-16 rpm [0183] Test 2 was performed similarly to Test 1, however, sticking has been observed from the beginning. The spray rate was dramatically reduced to prevent further sticking and the coating test proceeded without further problems. The capsules were coated to give a 20% weight gain.

Results [0184] The disintegration of capsules from Trial 1 and 2 was carried out. The following results were obtained:

TABLE 11 Results of the study of the disintegration of capsules from Test 1 and Test 2.

<td colspan="2"></td><td colspan="2">The number of capsules that disintegrated within 1 hour</td>

<td>Capsule type</td><td>No. attempts</td><td>Buffer 1.2</td><td>Buffer 6.8</td>

<td>Placebo gelatine bonded, 20% weight gain</td><td>Attempt 2</td><td>0</td><td>6</td>

<td>Placebo combined HPMC, 20% weight gain</td><td>Attempt 2</td><td>0</td><td>6</td>

<td>Placebo gelatine bonded, 20% weight gain</td><td>Attempt 1</td><td>0</td><td>4</td>

<td>Placebo combined HPMC, 20% weight gain</td><td>Attempt 1</td><td>0</td><td>6</td>

<td>Placebo fused with gelatin, 15% weight gain</td><td>Attempt 1</td><td>0</td><td>6</td>

<td>Placebo combined HPMC, 15% weight gain</td><td>Attempt 1</td><td>0</td><td>6</td>

<td>Placebo fused with gelatin, 8% weight gain</td><td>Attempt 1</td><td>0</td><td>6</td>

<td>Placebo combined HPMC, 8% weight gain</td><td>Attempt 1</td><td>0</td><td>6</td>

Conclusion [0185] Both Trials 1 and 2 successfully lead to enteric-coated capsule production at low temperature and low moisture conditions. However, as recommended, Sample 1 seemed better for several reasons. Since the formulation had a lower solids content, the coating uniformity was theoretically better due to the longer coating period. In addition, a lower content of solid ingredients reduces the possibility of sticking capsules. Furthermore, only one type was used in the Trial 1 method

Eudragit, which simplifies the production of the solution. Finally, the formulation and method used in Sample 1 lead to capsules that undergo intestinal disintegration with only 8% weight gain.

[0186] Test 1 was seen as the most effective trial for a number of reasons. Since a lower solids content was used in this method, the coating uniformity was theoretically dictated because of the longer coating period. In addition, a lower content of solid ingredients reduces the possibility of sticking capsules. In addition, only one type of Eudragit was used in the Trial 1 process, which simplifies the preparation of the solution. Finally, the formulation and the process used in Sample 1 lead to capsules that undergo intestinal disintegration with only 8% weight gain.