MXPA98009158A - Salt of bismuto de sialiloligosacarido and a metodopara treat and inhibit gastric and duodenal ulceras with the mi - Google Patents

Abstract

A method for treating and / or inhibiting gastric and duodenal ulcers is described, comprising administering a pharmaceutical composition comprising a bismuth salt of an oligosaccharide of formula (I) :( NeuAc-a (2-3) -pGal-B (1) - (-X-) m - (- Y-) n-) pZ, where X = a chemical ligation or a group capable of binding the p-galactose to either the linking group Y or the multivalent support Z, wherein the Glycosylic oxygen C1 of galactose can be replaced by N, S or C, Y = a linking group, Z = a multivalent support, m = 0 or 1, n = 0 or 1, and p = an integer of 2-1,000. Also disclosed is a method for treating and / or inhibiting gastric and duodenal ulcers, comprising administering a pharmaceutical composition comprising a bismuth salt of an oligosaccharide of the formula (III): NeuAc-a (2-3) -pGal-B (1 ) -A, where A = a group capable of binding to p-galactose, where the glycosidic oxygen C1 of galactose can be replaced by N, S or

Description

SALT OF BISMUTO OF SIALILOLIGOSACARIDO AND A METHOD TO TREAT AND INHIBIT GASTRIC ULCERAS AND DUODENALES WITH THE SAME BACKGROUND OF THE INVENTION Field of the Invention: The present invention relates to a method for treating and inhibiting gastric and duodenal ulcers in a patient, by administering a bismuth salt of a sialyl oligosaccharide, and a composition for practicing the same.

Discussion of background: Infection by the microaerophilic, spiral, gram-negative bacterium Helicobacter pylori (H. pylori), formerly known as Campylobacter pylori (C. pylori), is a primary cause of non-autoimmune gastritis, it is a factor in peptic ulcer disease and is more common in patients with gastric carcinoma. First isolated by Warren (Lancet (1983) 1: 1273) and Marshall (Lancet (1983) 1: 1273-5), H. pylori has been isolated in gastric tissue biopsies from patients around the world. Although the precise mechanism of inflammation is not well understood, H. pylori was found in association with the apical surfaces of the cells that secrete gastric mucosa. Due to the specificity of the binding site, it has been suggested that there are specific binding sites for H. pylori which exists in cells that secrete gastric and duodenal mucosa. Numerous studies have been undertaken to try to identify the specific binding site of H. pylori. Zopf et al U.S. 5,515,660, describe a method for treating and preventing ulcers in mammals by administering a sialyl oligosaccharide of the formula I (NeuAc-a (2-3) -pGal-β (1) - (- X-) m - (- Y-) "-) pZ wherein X = a chemical ligation or a group capable of binding the p-galactose to either the linking group Y or the multivalent support Z; wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C; Y = a link group; Z = a multivalent support; m = 0 or 1; n = 0 or 1; and p = an integer of 2-1, 000. The specific use of a bismuth salt of a sialyl oligosaccharide is not reported. Evans et al (Infection and Immunitv (1988) 56: 2896-2906) reported that ligation of H. pylori to an erythrocyte receptor, as measured by haemagglutination inhibition, is preferably inhibited by N-acetyl neuraminol (2). 3) -Gal ß1? 4 Glc (hereinafter NeuAc (2? 3) -lactose) as compared to N-acetylneuraminyl-a (2? 6)? 1 - > 4 Glc (hereinafter NeuAc (2-6) -lactose). The sialoproteins which contain the NeuAc (2- »3) Gal isomer of NeuAc-lactose, that is, human erythrocyte glycoforin A, fetuin, and human a2-macroglobulin, also inhibited H. pylori ligation, but at concentrations higher (mg / ml) than those observed for NeuAc (2- 3) -lactose, although it was not observed for the corresponding asialoglycoproteins. Evans et al ibid, measured the ability to inhibit haemagglutination (HIA) of several compounds containing a structure of NeuAc-lactose. Based on the haemagglutination inhibition activity, the investigations determined that, in order to produce 100% HAI, 1,000 mg / ml a2-macroglobulin was needed, 0.500 mg / ml fetuin was needed, 0.250 mg / ml was needed. Glycoforin A and 0.078 mg / ml of bovine NeuAc-lactose were needed. Based on their haemagglutination inhibition studies, the findings show that fetuin is about 2 times as effective as a2-Macroglobulin, but only 0.156 times as effective as bovine NeuAc-lactose, which comprises approximately 80% NeuAc ( 2? 3) -lactose and 20% NeuAc (2-6) -lactose. Evans et al fl nfection and Immunitv (1989) 57: 2272-2278) have also observed that H. pylori binds to monolayers of Y-1 mouse adrenal cells. But, this adhesion can be prevented by pre-treating Y-1 cells with neuraminidase and is blocked by fetuin. However, it should be noted that there is no relationship between the adrenal cells of mouse Y-1 and gastric tissue. Lingwood et al [(Lancet (1989) 2: 238-241) have reported the isolation of a gastric glycerolipid material, which has been observed to behave as a receptor for H. pylori. The material was isolated from erythrocytes, and scraped from pig stomach mucus and human stomach. The researchers postulated that the material was a sulfated alkylacylglycerolipid, but the current structure of this material has not been reported. Subsequent investigations (Linowood et al., Infection and Immunitv (1992) 60: 2470-2474) showed that this receptor is phosphatidylethanolamine. Lmawood et al .. Infection and Immunitv (1992) 61: 2472-2478 report that Helicobacter pylori specifically recognizes phosphatidylethanolamine, gangliotriaosylceramide and gangliotetraosylceramide and the isolation of an S-adhesin, which is believed to be responsible for the binding specificity of lipid of this organism. However, none of the compounds that are reported as specifically recognized by H. pylori are the sialylated oligosaccharides. Tzovelekis et al (Infection and Immunitv (1991) 59: 4252-4253) reported the inhibition of H. pylori binding to Hep-2 cells by gastric mucin. The researchers observed that purified mucin showed the greatest inhibition of H. pylori ligation, while asialomucin exhibited some decreased inhibition and periodate-oxidized mucin exhibited the lowest level of ligation. In these observations, the researchers concluded that sialic acids are at least partially responsible for the binding interaction between H. pylori and human gastric mucin. However, it should be noted that mucin contains a variety of different saccharide and linker groups.

Boren et al (Science (1993) 262: 1892-1895) have reported that the Lewisb blood group and H type I antigens mediate the binding of H. pylori to the human gastric mucosa. Fauchere et al Microbial Pathogenesis, 1990 9 427-439 report that the adhesion of H. pylori can be assessed by microtitre assays and involves a bacterial surface material, which is co-purified with urease and is different from the hemoglutinin that binds N-acetyl-neuraminil-lactose. Robinson et al report in J. Med. Microbio !. (1990) 33 277-284 that the pretreatment of human erythrocytes with neuraminidase from Arthrobacter ureafaciens and Clostridium perfringens suppresses haemagglutination by soluble haemagglutinin, but not associated with the cell, which suggests that sialic acid is not involved in the inhibition of H. pylori ligation. Dunn et al. Reviews of Infectious Diseases 1991; 3 (Suppl 8) :( S657-64) report studies of ligation inhibition by means of Mean Fluorescence Intensity by treatment of materials with a neuraminidase. The researchers report a 16.8% decrease in MFI over the treatment of N-acetylneuraminylactose neuraminidase of 16.8%, a 29.8% reduction with fetuin and an 8.6% reduction in asialofetuin. However, the researchers report a 30% increase over the treatment of KATO cells with neuraminidase. Such results call into question the role of sialylation in the specific site ligation of H. pylori.

Saitoh et al report a glycerolipid containing sulfate as a ligand, which is specifically recognized by H. pylori. Although there are numerous studies on compounds with the inhibition of H. pylori ligation, the literature is replete with conflicting evidence. Furthermore, there is even a lack of a consensus regarding the significance of methods for testing the inhibition of H. pylori binding. Haemagglutination assays have been used by many different investigators, for example, Evans et al (Infection and Immunitv (1988) 56: 2896-2906). however, Fiaueroa et al report in Journal of Infection (1992) 24 263-267, a mechanism of adherence, which does not depend on the expression of specific hemagglutinin antigen. This report openly questions the relationship between the inhibition of haemagglutination and the inhibition of H. pylori ligation. Additionally, many of the cell surface adhesion systems, used to test the inhibition of H. pylori ligation, have no relationship to gastric tissue at all. In addition to the numerous studies of inhibition of ligation, methods have been pursued to treat patients with gastric and duodenal ulcers. Colloidal bismuth subcitrate (CBS) has been used successfully to treat both gastric and duodenal ulcer diseases (for review, see Lambert in Reviews of Infectious diseases (1 991) 1_3 (Suppl 8): S691-5. has been shown to be effective as an H2 histamine antagonist and has been associated with lower relapse rates after cessation of therapy attributed to the ability of CBS to eradicate H. pylori, and bismuth subsalicylate (BSS) has also been observed. H. pylori inhibits, Coleman et al (U.S. Patent No. 4,935,406) reported a method for improving gastrointestinal disorder, resulting from the H. pylori colony, through the administration of phosphated or sulfated saccharide compositions of bismuth. of saccharides according to this method are simple phosphates and sulfates of aldose and ketose monosaccharides, clinical trials have been reported (Evans et al, Ann. Internal Med. (1991) Agos. to 15, 115 (4): 266-9) to treat H. pylori using ranitidine together with a "triple therapy" of amoxicillin or tetracycline, metronidazole (an antiprotozoal), and BSS. Clinical studies suggested that ulcer healing was faster in patients who received ranitidine plus "triple therapy" than in patients who only received ranitidine. The strong role of H. pylori in peptic ulcers has led to an announcement in February 1994 by an independent expert advisory panel convened by the National Institutes of Health, to recommend that patients diagnosed with peptic ulcers and H. pylori be treated for two weeks with a combination of antibiotics. A copy of the Consensus Development Conference Statement Helicobacter oylori in Peptic Ulcer Disease is available from the National Institutes of Health. There was no recommendation for any other therapy. However, the long-term eradication of this organism has been difficult with some of these therapies. The approach of antibiotics runs the risk of the development of new species resistant to antibiotics. In addition, there are side effects associated with long-term antibiotic therapy, which are unpleasant and in combination with such make a treatment regimen more difficult. Thus, a method to treat H. pylori with good long-term eradication has not been developed yet. Based on the inventors 'studies, it has now been discovered that a bismuth salt of a sialyl oligosaccharide, such as 3' sialyl lactose, is a surprisingly effective inhibitor for H. pylori in mammals.

BRIEF DESCRIPTION OF THE INVENTION According to this, an object of the present invention is a method for treating and / or preventing gastric and / or duodenal ulcers with a bismuth salt of a sialyl oligosaccharide. Another objective of the present invention is a method for inhibiting infection and / or reinfection of Helicobacter pylori to mammalian tissue, including removing Helicobacter pylori from the stomach and / or duodenum of a patient in need thereof. Another objective of the present invention is to provide a pharmaceutical composition for inhibiting infection or reinfection of Helicobacter pylori from mammalian tissue, including removing Helicobacter pylori from the stomach and / or duodenum of a patient in need thereof and for treating and / or preventing ulcers. gastric and / or duodenal. The inventors have found that all of the above objectives of the present invention and other objects, which are apparent from the description of the invention given below, are met by administering a composition of a bismuth salt of an oligosaccharide of Formula I (NeuAc-a (2-3) -pGal-ß (1) - (- X-) m - (- Y -) "-) pZ where X = a chemical ligation or a group capable of binding p galactose to either the link group Y or the multivalent support Z; wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C; Y = a link group; Z = a multivalent support; m = 0 or 1; n = 0 or 1; and p = an integer of 2-1,000, wherein the bismuth salt is formed of an acid group of said oligosaccharide. Preferably, the bismuth salt is formed with the carboxylic acid group of NeuAc. The present invention is also provided by a bismuth salt of an oligosaccharide composition of Formula III NeuAc-a (2-3) -pGal-β (1) -A wherein A = a group capable of binding to p-galactose; wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C. In addition, the inventors of the present invention have discovered that a multivalent presentation of an oligosaccharide (i.e., the oligosaccharide of Formula I) is unexpectedly superior, in a molar base based on the oligosaccharide groups, that the monovalent presentation of the same oligosaccharide. In addition, the inventors have found that a method in which a pharmaceutical composition comprising the oligosaccharide of Formula I and / or Formula III alone is administered, or in combination with a H2 blocker, an antibiotic, oligosaccharide compounds and / or a antiulcerative compound to a mammal, is effective to inhibit the binding of Helicobacter pylori to the gastric and duodenal mucosa and improve the effects of gastric and duodenal ulcers.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The following abbreviations are used throughout the text: "Gal" for galactose, "Glc" for glucose, "NeuAc" for N-acetylneuraminic acid The designation "p" for specific saccharide groups is indicative of the pyranose ring structure The oligosaccharide compound of Formula I (NßuAc-a (2-3) -pGal-β (1) - (- X-) m - (- Y-) n-) pZ in where X = a chemical ligation or a group capable of binding the p-galactose to either the Y-linking group or the multivalent Z-support, wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C; link group, Z = a multivalent support, m = 0 or 1; n = 0 or 1; and p = an integer of 2-1, 000 is administered according to the present method. For example, X can be a substituted C1.20 alkyl group, a substituted C.sub.0-20 alkyl carboxylic ester group, a substituted C1.20 alkyl carboxy amide group, a hydroxy terminated polyether, an amine terminated polyether, inositol, an oligosaccharide, a disaccharide or a monosaccharide with the terminal reducing end of the oligosaccharide, disaccharide or monosaccharide in the open chain or pyranose form, an azaoligosaccharide, an azadisaccharide or a azamonosaccharide with the terminal reducing end of the azaoligosaccharide, azadisaccharide or azamonosaccharide in the form open chain or pyranose, wherein said substitution is capable of reacting with the linking group of the multivalent support, such as a hydroxyl group or an amine group. Preferably, the group X is a hexose group of monosaccharide such as glucose, N-acetylglucosamine, galactose, N-acetylgalactosamine, mannose, fucose, allose, altrose, gulose, idose, talose and rhamnose. In addition, a suitable X group is a reduced form of the hexose groups identified above, such as glucitol. When the group X is able to bind directly to the multivalent support, then n is 0. When the glycosidic oxygen C1 of galactose is able to bind directly to the multivalent support group, then both m and n are 0.

A suitable linking group has a terminal portion of the group Y capable of binding to the group X, while the other terminal end is capable of binding to the multivalent support. The chemistry necessary to link the group X and the linking group Y, and to link the linking group Y to the multivalent support is well known in the field of link chemistry. For example, a bond between X and Y can be formed when X is a saccharide such as an oligosaccharide, a disaccharide or a monosaccharide, by reacting an aldehyde or carboxylic acid to Ci of group X or any aldehyde or carboxylic acid group introduced on the group X by oxidation, with the group Y, to form a suitable ligation such as -NH-, -N (R) - wherein R is C1-20 alkyl, a hydroxyalkylamine, an amide, an ester, a thioester, a thioamide . When X is a saccharide such as an oligosaccharide, a disaccharide or a monosaccharide, a ligation can be formed between X and Y by reacting the hydroxyl group of Ci, in the pyranose form with an acylating agent and a molecular halide, followed by the reaction with a nucleophile to form a suitable ligation, such as -NH-, -N (R) - where R is C? 20 alkyl, -S- and -O-. This type of binding chemistry is described by Stowell et al Advances in Carbohydrate Chemistry and Biochemistry, 37 (1980) p 225+. A bismuth salt of the compound of Formula I can be prepared by analogous methods such as those described in L. Vanino, cited in Mellor's vol. IX 598 (1929), C.J. McLoughlin et al DE 2,501, 787, P.J. H Bos et al EP 75,992 and U.S. 4,801, 608, the complete contents of which are incorporated herein by reference. For example, the bismuth salt can be prepared by mixing bismuth nitrate and an oligosaccharide of formula I, in a solvent such as glycol as described by Schmitz, Pharm 5, 517 (1950). The present invention also allows the administration of a composition, which is a mixture of a bismuth salt of a sialyl oligosaccharide of Formula I, in admixture with the free carboxylic acid of the sialyl oligosaccharide of Formula I or a pharmaceutically acceptable non-bismuth salt of the sialyl oligosaccharide of Formula I. The pharmaceutically suitable salts are described below. The amount of Bi * 3, which is present in the composition to be administered, is not particularly limited, but is preferably from 0.001 - 40% by weight of the total weight of the sialyl oligosaccharide salt, more preferably from 0.01 - 20% by weight of the total weight of the sialyl oligosaccharide salt, most preferably from 0.1-15% by weight of the total weight of the sialyl oligosaccharide salt. In another preferred embodiment, a stoichiometric salt of 2 moles of Bi3 + and 3 moles of sialyl oligosaccharide of Formula I is formed. Within the scope of the present invention, the NeuAc group of the Formula I or Formula I I can be defined as a sialic acid of Formula I I; where Rβ, R l Rβ and Rio are each independently H, acyl of C? -6, lactyl, alkyl C, sulfate, phosphate, anhydrous, a sialic acid of Formula II, (a-1) Fuc, (b-) 1) Glc or (ß-1) Gal; R "is NH-C1.6 acyl, glycoliamido, amino or hydroxyl; and A is H or a cation: The group of Formula II is a sialic acid, which is a family of carboxylated sugars of 9 carbons related to neuraminic acid. The carboxylic acid may be in the form of a free acid, when A is H or a salt, including bismuth, when A is a cation. Suitable cations, in addition to bismuth, include alkali metals, alkaline earth metals or ammonium. Any suitable pharmaceutically acceptable cation can be used, including the cations of conventional non-toxic salts including a metal salt, such as an alkali metal salt (eg, sodium salt, potassium salt, etc.) or a metal salt alkaline earth (for example, calcium salt, magnesium salt, etc.), an ammonium salt, an organic base salt (for example trimethylamine salt), salt of triethylamine, pyridine salt, picoline salt, dicyclohexylamine salt, salt of N.N'-dibenzylethylenediamine, etc.), an organic acid salt (for example, formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate , benzenesulfonate, toluenesulfonate, etc.), an inorganic acid salt (eg, hydrochloride, hydrobromide, sulfate, phosphate, etc.), a salt with an amino acid (eg, arginine salt, aspartic acid salt, glutamic acid, etc.), and the like.

Preferably, the group of Formula II is selected from the group consisting of N-acetyl-neuraminic acid, N-glycolyl-neuraminic acid, keto-deoxy-nonulosonic acid, 9-O-acetyl N-acetyl-neuraminic acid, -O-acetyl N-glycolyl-neuraminic acid, 9-O-acetyl-keto-deoxy-nonulosonic acid, 7-O-acetyl-N-acetyl-neuraminic acid, 7-O-acetyl-N-glycolyl-neuraminic acid, 4 -O-acetyl-N-acetyl-neuraminic acid, 4-O-acetyl-N-glycolyl-neuraminic acid, 7,9-di-O-acetyl-N-acetyl-neuraminic acid, 8,9-di-O- acid acetyl-N-acetyl-neuraminic acid, 7,9-di-O-acetyl-N-glycolyl-neuraminic acid, 8,9-di-O-acetyl-N-glycolyl-neuraminic acid, 4,9-di-O- acetyl-N-acetyl-neuraminic acid, 7,8,9-tri-O-acetyl-N-acetyl-neuraminic acid, 7,8,9-tri-O-acetyl-N-glycolyl-neuraminic acid, 9-O-lactyl -N-acetyl-neuraminic acid, 9-O-lactyl-N-glycolyl-neuraminic acid, 4-O-acetyl-9-O-lactyl-N-acetyl-neuraminic acid, 4-O-acetyl-9-O- acid N-glycolyl-neuraminic lactyl, 8-O-methyl-N-acetyl-neu acid raminic, 8-O-methyl-N-glycolyl-neuraminic acid, 8-O-methyl-9-O-acetyl-N-glycolyl-neuraminic acid, 8-O-methyl-7,9-di-O-acetyl acid -N-glycolyl-neuraminic, 8-O-sulfo-N-glycolyl-neuraminic acid, 8-O-phosphorus-N-acetyl-neuraminic acid, 2,3-didehydro-2,6-anhydro-N-acetyl-neuraminic acid, acid 9-O-acetyl-2,3-didehydro-2,6-anhydro-N-acetyl-neuraminic acid, 9-O-lactyl-2,3-didehydro-2,6-anhydro-N-acetyl-neuraminic acid, 2,3-didehydro-acid 2, 6 anhydro-N-glycolyl-neuraminic acid, 9-O-acetyl-2,3-didehydro-2,6-anhydro-N-glycolyl-neuraminic acid, 9-O-lactyl-2,3-didehydro-2,6-anhydro-N-glycolyl acid -neuraminic acid, 8-O-methyl-2,3-didehydro-2,6-anhydro-N-glycolyl-neuraminic acid, 2,7-anhydro-N-acetyl-neuraminic acid, 2,7-anhydro-N-glycolyl-neuraminic acid, acid 8-O-methyl-2,7 anhydro-N-glycolyl-neuraminic, 4,8-anhydro-N-acetyl-neuraminic acid and salts thereof. More preferably, the sialic acid of Formula II is N-acetyl-neuraminic acid or N-glycolyl-neuraminic acid. These sialic acids are described in A. Varki Glicobioloav v2, (1992) p25-40. Accordingly, the reference describes sources of the sialic acids as well as the appropriate sialyltransferase necessary for enzymatic synthesis of oligosaccharides of Formula I. When the group of Formula II is substituted with a sialic acid, the substitution is preferably in the R 7 position. . A suitable multivalent support is a compound with multiple binding sites for a terminal end of the linking group, which is not linked to the group X of the linking group, with multiple binding sites for the group X, or with multiple binding sites. for the glycosidic oxygen of Ci of galactose. Examples include, but are not limited to, a polio, a polysaccharide, polylysine, avidin, a polyacrylamide, dextran, lipids, lipid emulsions, liposomes, a dendritimer, human serum albumin, bovine serum albumin or a cyclodextrin. The oligosaccharide is provided as a multivalent molecule according to Formula I. In this embodiment, the oligosaccharide portion is linked to a multivalent support using known techniques, in order to produce a conjugate in which more than one single molecule of the oligosaccharide is covalently linked through a linker to the multivalent support. The multivalent support is sufficiently long to provide a multivalent molecule leaving between 2-1, 000 (ie, p = an integer of 2-1, 000), preferably 2-100, more preferably, 2-30 molecules of the portion of oligosaccharide linked to the multivalent support. The oligosaccharide portion can be linked to the multivalent support via the free anomeric carbon of group X. Alternatively, the oligosaccharide portion can be linked via a phenethylamine-isothiocyanate derivative as described by Smith et al. Complex Carbohydrates part C, Methods in Enzymology, volume L, Ed by V. Ginsburg (1978), p 169-171. It is preferable that the oligosaccharide of formula I remain soluble in water, however it is also possible to administer the oligosaccharide of formula I in the form of polymer particles. For example, the oligosaccharide portion of Formula I can be ligated to a support to form an account, wherein the surface of the bead is linked to the oligosaccharide portion of Formula I. The oligosaccharide composition of Formula III NeuAc-a ( 2-3) -pGal-ß (1) -A where A = a group capable of binding to p-galactose; wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C. is administered according to the present method. For example, A can be an alkyl group of C? -20, an alkyl carboxyl ester group of C? 20, an alkyl carboxy amide group of C? .2o, a polyether, inositol, an oligosaccharide, a disaccharide or a monosaccharide with the terminal reducing end of the oligosaccharide, disaccharide or monosaccharide in the open chain or pyranose form, an azaoligosaccharide, an azadisaccharide or a azamonosaccharide with the terminal reducing end of the azaoligosaccharide, azadisaccharide or azamonosaccharide in the form of an open chain or pyranose. Preferably, group A is a hexose monosaccharide group such as glucose, N-acetylglucosamine, galactose, N-acetylgalactosamine, mannose, fucose, allose, altrose, gulose, idose, talose and rhamnose. In addition, a suitable group A is a reduced form of the hexose groups identified above, such as glucitol. The corresponding N and S glycosides of galactose can be prepared by conventional methods known to those of ordinary skill in the art from galactose followed by the attachment of a sialyl acid group in the 3-position by conventional methods. The corresponding galactose glycoside c can be made by conventional synthetic organic techniques, followed by the attachment of a group of sialic acid in the 3-position by conventional methods. Any known pharmaceutically acceptable cation, in addition to bismuth, can be used with the oligosaccharides of formula I and Formula ll, to form a salt of the carboxylic acid group. Suitable cations include conventional non-toxic salts including a metal salt, such as an alkali metal salt (e.g., sodium salt, potassium salt, etc.) or an alkaline earth metal salt (e.g., calcium salt) magnesium salt, etc.), an ammonium salt, an organic base salt (for example, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N'- salt). dibenzylethylenediamine, etc.), an organic acid salt (e.g., formate, acetate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, toluenesulfonate, etc.) an inorganic acid salt (e.g., hydrochloride, hydrobromide, sulfate, phosphate , etc.), a salt with an amino acid (eg, arginine salt, salt of aspartic acid, salt of glutamic acid, etc.), and the like.

The oligosaccharides of the present invention can be obtained using any known method, including (1) enzymatically, using one of the methods of the inventor described in published international application WO 91/16449, (2) synthetically, using classical organic chemistry, (3) ) by degradation of a naturally occurring oligosaccharide, glycolipid, or glycopeptide or (4) isolation from a natural source, such as bovine colostrum. The isolation of 3 'sialyl lactose from bovine colostrum is described in Veh et al, Journal of Chromatography, 212, (1981) 313-322. 3 'sialyl lactose can also be isolated from a stream of cheese processing waste as described by Brian et al in US Serial No. 08/337, 181, filed at the US Patent Office on November 7. of 1994, the complete contents of which are incorporated herein by reference. The bismuth salt of sialyl oligosaccharides of formula I and / or Formula 11 can be administered together with a known proton pump inhibitor or a known H2 receptor antagonist. A representative proton pump inhibitor is omeprazole, and representative H2 antagonists include cimetidine, ranitidine, nizatidine and famotidine. The amount of proton pump inhibitor and H antagonist administered together with the present bismuth salt of a sialyl oligosaccharide is approximately the same amount administered by its known therapy. Accordingly, the effective dosages of the proton pump inhibitor and H2 can be determined by routine experimentation. Alternatively, a known antiulcerative compound may be used in conjunction with or as a replacement for the H2 receptor antagonist. Suitable antiulceratives include aluminum aceglutamide complex, zinc salt of e-acetamidocaproic acid, acetoxolone, arbaprostil, debennexate hydrochloride, busmuto subcitrate sol, bismuth subsalicylate, carbenoxolone, cetraxate, cimetidine, enprostil, esaprazole, famotidine, phthaxidide, gefarnate, guaiazulene, irsoglidine, misoprostol, nazatidine, ornoprostil,? -oryzanol, pifarnine, pirenzepine, plaunotol, ranitidine, rioprostil, rosaprostol, rotraxate, roxatidine acetate, sofalcone, spizofurone, sucralfate, teprenone, trimoprostil, tritiozine, troxipid, and zolimidine . The amount of antiulcerative administered together with the present oligosaccharide is approximately the same amount administered for its known therapy. According to this, the effective dosage of the antiulcerative can be determined by routine experimentation. Alternatively, the bismuth salt of a sialyl oligosaccharide of Formula I and / or formula II can be administered together with an antibiotic with activity against H. pylori. Suitable antibiotics include metronidazole, tetracycline, bismuth, erythromycin, a macrolide, a quinolone, a cephalosporin and amoxicillin. The amount of antibiotic administered together with the bismuth salt present of a sialyl oligosaccharide is approximately the same amount administered for its known therapy. According to this, the effective dosage of the antibiotic can be determined by routine experimentation. Alternatively, the bismuth salt of a sialyl oligosaccharide of formula I and / or formula III can be administered together with a Lewis blood type antigen or H-type 1 or an oligosaccharide, such as NeuAc-a (2-6) - Gal ß1 - 4 Glc. Suitable blood group Lewisb and H-type 1 antigens are reported in Boren et al (Science (1993) 262: 1892-1895). The anti-H compositions. pylori of the present invention contains the bismuth salt of an oligosaccharide of formula I and / or Formula III in association with any suitable liquid or solid, carrier or pharmaceutically acceptable excipient, preferably in a form suitable for oral or enteral administration. In addition, the pharmaceutical compositions of the present invention are preferably, free of pyrogen. The pharmaceutical compositions are usually administered as a mixture with a suitably selected carrier depending on the route for administration using standard formulations. For example, the compound of the present invention can be administered in the form of tablets, which can be prepared using known techniques by adding an excipient such as starch, lactose, sucrose, glucose to a powder of the active ingredient of the present invention. , crystalline cellulose, calcium carbonate or kaolin, a hydroxypropyl cellulose, a glucose solution, a solution of sucrose, water or ethanol, a disintegrator such as starch, agar, gelatin powder, calcium carboxymethylcellulose (CMC-Ca), carboxymethylcellulose sodium (CMC-Na), crystalline cellulose, calcium carbonate or sodium acid carbonate, or a lubricant such as magnesium stearate, calcium stearate, talcum, macrogoal 4,000, macrogoal 6,000 or stearic acid. The mixture is then subjected to compression molding by a conventional tabletting method, and if necessary, applying a sugar coating by means of a concentrated sugar solution containing for example, gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or oxide. of titanium, apply a film coating by means of a film-forming agent composed of, for example, polyvinyl acetal diethylaminoacetate, hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose or polyvinylpyrrolidone or enteric coating by means of a film-forming agent composed of, by example, ethylcellulose phthalate, cellulose acetate phthalate or hydroxypropylmethylcellulose phthalate. These pharmaceutical compositions can be in the form of granules or fine granules, which can be prepared by adding to the active ingredient of the present invention, a binder such as starch, gelatin, gum arabic, methylcellulose, sodium carboxymethylcellulose, heavy salicylic anhydride or light silicic anhydride, followed by kneading and granulation by usual methods; or as a powder of the active ingredient of the present invention by itself; or as capsules which can be prepared by adding to the active ingredient of the present invention, an excipient such as lactose, starch or crystalline cellulose and / or a lubricant such as magnesium stearate, calcium stearate or talc, and filling the mixture in capsules A solution or suspension can be prepared by adding any customary diluent used in the technique. For example, suitable diluents include water, ethyl alcohol, propylene glycol, polyoxyethylene sorbitol, and sorbitan esters. The sodium chloride, glucose or glycerol can be incorporated into such liquid preparation in an amount sufficient to prepare an isotonic solution. The therapeutic composition may also contain ordinary solvent auxiliaries, buffers, pain relieving agents, preservatives of the art, and optionally coloring agents, fragrances, flavors, sweeteners and other pharmacologically active agents, such are well known in the art. Suitable compositions may take the form of a solution, suspension, tablet, coated tablet or any suitable pharmaceutically acceptable form for delivery to the stomach or duodenum. According to a preferred embodiment of the present invention, the bismuth salt of a sialyl oligosaccharide or pharmaceutical compositions are administered orally or enterally to a patient in need thereof in order to inhibit H. pylori ligation or eliminate H colonies. pylori of the patient's stomach and / or duodenum. Typically, patients are human. However, the present method is also applicable to the treatment of animals, including but not limited to, mammals such as pigs, cows, horses, sheep, goats, dogs, cats, rodents and non-human primates. The method of the present invention is suitable for preventing and treating patients with duodenal ulcers, gastric ulcers and the prevention of gastric cancers in patients. Suitable amounts of the pharmaceutical composition containing the bismuth salt of a sialyl oligosaccharide of Formula I and / or formula II to be administered include those which produce an effective stomach concentration of bismuth salt of a sialyl oligosaccharide from 1 μg to 10,000 mg / ml per dose, preferably 10 μg to 1,000 mg / ml, more preferably 0.5 mg to 50 mg / ml, most preferably 1 to 10 mg / ml. For example, based on an average human stomach volume of 500 ml, a dose of 3 g would produce an effective stomach concentration of approximately 6 mg / ml. The administration of the pharmaceutical composition comprising the bismuth salt of a sialyl oligosaccharide of Formula II is preferably carried out to achieve a continuous effective stomach concentration from 1 μg to 10,000 mg / ml per dose, preferably 10 μg to 1,000. mg / ml, more preferably 0.5 mg to 50 mg / ml, most preferably 1 to 10 mg / ml. This can be achieved by administration, at least daily, preferably twice a day, more preferably three times a day and most preferably four times a day. When a pharmaceutical composition is administered as a multivalent molecule, a pharmaceutical composition comprising the bismuth salt of a sialyl oligosaccharide of Formula I is administered, in order to achieve a continuous effective stomach concentration from 1 μg to 1,000 mg / ml per doses, preferably 10 μg to 100 mg / ml, more preferably 50 μg to 5 mg / ml, most preferably 10 μg to 2 mg / ml. This can be achieved by administration, at least daily, preferably twice a day, most preferably three times a day and most preferably four times a day. When co-administering a proton pump inhibitor, H2 antagonist, or antiulcer, the composition is formulated to provide between 10-500 mg, preferably 100-300 mg of the proton pump inhibitor., H2 l antagonist, or antiulcerative daily. For example, appropriate therapies include the administration of tetracycline (500 mg four times a day), bismuth subsalicylate (two tablets four times a day, with meals and at bedtime), and metronidazole (250 mg three times a day). , with meals) taken during a period of 14 days each. Dosage forms include such unit dosage forms, such as, tablets, capsules, solutions or suspensions. After eradication of H. pylori infection or ulcer treatment, maintenance procedures are administered in order to achieve a continuous effective stomach concentration from 1 μg to 1,000 mg / ml per dose, preferably 10 μg. at 100 mg / ml, more preferably 50 μg at 5 mg / ml, most preferably 10 μg at 2 mg / ml. This can be achieved by administration, at least daily, preferably twice a day, more preferably three times a day and most preferably four times a day.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments, which are given by way of illustration of the invention and are not intended to be limiting thereof. * * * EXAMPLE 1 Cell cultures, to test the effectiveness of H. pylori ligation inhibition were prepared from stomach cancer epithelial cells of human HuTu-80 carcinomas obtained from American Type Culture Collection Rockville, MD, according to a modified procedure of that reported in Fauchere et al Microbial Pathogenesis 1990; 9 427-439. The cultures were maintained in Basal medium Eagle containing 10% fetal calf serum in flasks T = 75, at 37 ° C and at an atmosphere of 5% CO2. Cells were harvested by trypsin / EDTA release and plated on 96-well flat bottom microtiter plates. The microtiter plates were incubated for 2-3 days until the monolayers grew to confluence. Before the inhibition tests, the monolayer was washed with solution of Hanks Balanced Salts (HBSS) containing Ca2 * and Mg2 +, 0.1% BSA, 50 mM HEPES, 0.01 phenol red or HBHPR. Isolates of H. pylori bacteria were obtained from B. Marshall (from the University of Virginia) and grown on sheep blood agar, harvested at 48 h, washed and suspended in a ligation buffer of HBSS + 0.1. % bovine serum albumin + 50mM HEPES buffer + 0.01% phenol red or HBHPR. In order to test the inhibition of H. pylori binding, the concentration of H. pylori, which bound to the monolayer was assigned an intermediate OD5ss (optical density at 595 nm) (approximately 0.4 OD units). The same concentration of bacteria and the test compound were combined for 10 minutes, then transferred onto the monolayer. The ligature was allowed to occur for 20 min at room temperature under gentle agitation. The unbound bacteria were washed with 1 wash of HBHPR, then 2 washes of the same buffer without HEPES buffer (HBPR). The amount of bacterial adhesion to the monolayer was measured by incubating with 50 μl of phenol-urea red solution (UPR) (0.2% urea, 0.03% phenol red in 0.85% NaCl). The presence of bound bacteria is indicated by the presence of bacterial urease which generates NH3, which raises the pH and changes the color to purple, close to ODs9S. ICS0 in mg / ml was determined for each compound tested. The test data is reported below in Table 1: Table 1 approximately 20 molecules of 3-sialyl lactose linked to HSA. 2 relative to 3 'sialyl lactose The data reveal that 3-siallyl lactose, when tested in a multivalent manner, was 290 times more effective on a molar basis than 3 siallyl lactose.

Example 2: The fetuin ligation inhibitory activity was determined as follows: The commercially available fetuin from Sigma Chemical was purified on a SEPHACRYL S-100 column (from Pharmacia) in 0.15M aqueous NaCl plus 0.05M Tris-HCl, pH 7.0 plus 0.02% NaN3 and the ICso was determined for each of the isolated peaks. The IC50s were determined using the monolayers of HuTu-80 cell lines. The results are shown later in Table 2, where fraction # 3 corresponds to pure fetuin and fractions # 1 and # 2 correspond to unidentified high molecular weight impurities.

Table 2 * Means of inhibition were not observed even at the highest tested concentration of 2 mg / ml.

In vivo test with animals: Gnotobiotic derived piglets (delivered by cesarean section and housed in a germ-free environment) were orally treated with 100 mg of 3-sialil lactose in 5.0 ml of water.

Experiment A: Six-day gnotobiotic piglets were orally treated with seven doses of 100 mg each of 3 'sialyl lactose, at approximately 8 hour intervals. As a control, water was administered to the piglets.

The third administration of 3 'sialyl lactose and control was accompanied with 2 x 109 of H. pylori alive. Two piglets were administered with 3'sailyl lactose and 2 piglets were administered with control. The results are shown later in Table 3.

Experiment B: Twenty-one day old gnotobiotic piglets were treated orally with seven doses of 100 mg each of 3 'sialyl lactose, at approximately 8 hours intervals. As a control, water was administered to the piglets. The third administration of 3 'sialyl lactose and control was accompanied with 4 x 109 of live H. pylori. Four piglets were administered with 3 'sialyl lactose and 2 piglets were administered with the control. The results are shown in Table 3 below. The piglets were evaluated by determining bacterial colonies in blood-agar as colony forming units / gram of gastric epithelium (CFU / g). Gastric epithelial homogenates were platinized on agar in serial dilutions of 1: 10 and bacterial colonies were counted on the plates, with 20-200 colonies / plate after 5 days.

Table 3 Example 3. An anthelicobacter composition is prepared by suspending 1 g of a stoichiometric Bi * 3 salt of 3'-sialyl lactose in a mixture of water and propylene glycol.

Example 4. An anti-Helicobacter composition is prepared by mixing 1 g of a stoichiometric Bi * 3 salt of 3 'sialyl lactose with 250 mg of the ranitidine H2 receptor antagonist. The mixture is then suspended in a mixture of water and propylene glycol.

Example 5. An anti-Helicobacter composition is prepared to mix 1 g of a stoichiometric Bi * 3 salt of 3 'sialyl lactose with 250 mg of the proton pump inhibitor, omeprazole. The mixture is then suspended in a mixture of water and propylene glycol Example 6. An anti-Helicobacter composition is prepared by mixing 1 g of a stoichiometric Bi * 3 salt of 3 'sialyl lactose with 500 mg of a tetracycline. The mixture was then suspended in a mixture of water and propylene glycol.

Example 7 As a therapeutic treatment, a patient infected with H. pylori is treated with the composition of Example 3. The patient is orally treated four times daily giving each dosage an effective stomach concentration of 2 mg / ml. The therapy is continued for two weeks, after which the examination showed eradication of H. pylori bacteria. After eradication, maintenance therapy with the composition of the present invention is continued to prevent recurrence.

Obviously, the numerous modifications and variations of the present invention are possible in light of the above teachings.

Therefore, it will be understood that within the scope of the appended claims, the invention may be practiced in a manner other than that specifically described herein.

Claims (14)

1. A pharmaceutical composition comprising, together with a carrier or excipient suitable for oral or enteral administration, a bismuth salt of an oligosaccharide of Formula I (NeuAc-a (2-3) -pGal-β (1) - (-X-) m - (- Y-) n-) pZ wherein X = a chemical ligation or a group capable of binding the p-galactose to either the linking group Y or the multivalent support Z; wherein the glycosidic oxygen of galactose d can be replaced by N, S or C; Y = a link group; Z = a multivalent support; m = 0 or 1; n = 0 or 1; and p - an integer of 2-1, 000.

2. A pharmaceutical composition comprising, together with a carrier or excipient suitable for oral or enteral administration, a bismuth salt of an oligosaccharide of formula III NeuAc-a (2-3) -pGal-β (1) -A wherein A = a group capable of binding to p-galactose; where the glycosidic oxygen Ct of galactose can be replaced by N, S or C.

3. The pharmaceutical composition of claim 1, further comprising an element selected from the group consisting of an H2 blocker, an antiulcerative compound, a proton pump inhibitor, an antibiotic, an active oligosaccharide of Lewis5 blood group, an oligosaccharide and an mix of them.

4. The pharmaceutical composition of claim 2, further comprising an element selected from the group consisting of an H2 blocker, an antiulcerative compound, a proton pump inhibitor, an antibiotic, an active oligosaccharide of Lewisb blood group, an oligosaccharide and a mixture of them. A method for treating or preventing an ulcer in the stomach or duodenum of a mammalian patient in need thereof, comprising administering to the stomach or duodenum of said mammalian patient an effective amount to produce an effective stomach concentration of a bismuth salt of an oligosaccharide from 1 μg to 10,000 mg / ml per dose, of a composition comprising a bismuth salt of an oligosaccharide of formula I (NeuAc-a (2-3) -pGal-β (1) - (- X-) m - (- Y-) n-) pZ wherein X = a chemical ligation or a group capable of binding the p-galactose to either the linking group Y or the multivalent support Z; wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C; Y = a link group; Z = a multivalent support; m = O or 1; n = 0 or 1; and p = an integer of 2-1, 000. 6. A method for treating or preventing an ulcer in the stomach or duodenum of a mammalian patient in need thereof, comprising administering to the stomach or duodenum of said mammalian patient, an effective amount to produce an effective stomach concentration of a bismuth salt of an oligosaccharide from 1 μg to 10,000 mg / ml per dose, of a composition comprising a bismuth salt of an oligosaccharide of Formula III NeuAc-a (2-3) -pGal-β (1) -A where A = a group capable of binding to p-galactose; wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C. 7. A method for inhibiting an infection or reinfection of H. pylori in the stomach or duodenum of a mammalian patient in need thereof, comprising administering to the stomach or duodenum of said mammalian patient, an amount effective to produce an effective stomach concentration of a bismuth salt of an oligosaccharide of Formula I (NeuAc-a (2-3) -pGal-β (1) - (-X-) m - (- Y -) "-) pZ wherein X = a chemical ligation or a group capable of binding the p-galactose to either the linking group Y or the multivalent support Z; wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C; Y = a link group; Z = a multivalent support; m = 0 or 1; n = 0 or 1; and p = an integer of 2-1, 000. A method for inhibiting an infection or reinfection of H. pylori in the stomach or duodenum of a mammalian patient in need thereof, comprising administering to the stomach or duodenum of said mammalian patient an effective amount to produce an effective stomach concentration of a bismuth salt of an oligosaccharide from 1 μg to 10,000 mg / ml per dose, of a composition comprising a bismuth salt of an oligosaccharide of Formula III NeuAc-a (2-3) -pGal-β (1) -A where A = a group capable of binding to p-galactose; wherein the glycosidic oxygen Ci of galactose can be replaced by N, S or C. The pharmaceutical composition of claim 1, wherein X is selected from the group consisting of glucose, N-acetylgiucosamine, galactose, N-acetylgalactosamine, mañosa, fucosa, alosa, altrosa, gulosa, idosa, talosa, rhamnose and glucitol. The pharmaceutical composition of claim 2, wherein A is selected from the group consisting of glucose, N-acetylglucosamine, galactose, N-acetylgalactosamine, mannose, fucose, allose, altrose, gulose, iodine, talose, rhamnose and glucitol. The pharmaceutical composition of claim 1, wherein Z is selected from the group consisting of a polyol, a polysaccharide, polylysine, avidin, a polyacrylamide, dextran, lipids, lipid emulsions, liposomes, a dendritimer, human serum albumin , bovine serum albumin or a cyclodextrin. The pharmaceutical composition of claim 2, wherein said oligosaccharide of formula III is NeuAc-a (2-3) -pGal-β (1-4) Glc. The pharmaceutical composition of claim 1, wherein X is 4-glucitol, m is 1, Y is phenethylamine-isothiocyanate, n is 1, p is 12-20, and Z is human serum albumin. The method of claim 5, wherein said bismuth salt is a stoichiometric salt.