Patent 6,828,298

An inhibitor of Helicobacter pylori colonization in the stomach comprises as an active ingredient a glycoprotein which specifically binds to H. pylori urease. This glycoprotein is isolated and purified from a glycoprotein-containing substance, especially that derived from bovine milk whey or albumen of chicken eggs by affinity chromatography using a column on which H. pylori urease is immobilized. The glycoprotein is able to effectively inhibit H. pylori colonization, so is useful for the prevention or treatment of diseases caused by infection of H. pylori such as peptic ulcers. A food and medicament comprising the inhibitor are also provided.

Description of the Invention

TECHNICAL FIELD

The present invention relates to a glycoprotein which is capable of eradicating from the stomach Helicobacter pylori, which is associated with the occurrence of peptic ulcers. It also relates to an inhibitor of the colonization of Helicobacter pylori comprising the glycoprotein, and a medicament and food comprising the inhibitor.

BACKGROUND OF THE INVENTION

At present it is believed that eradication of H. pylori from the stomach is essential for fully treating peptic ulcers. The combination of an antibiotic and an inhibitor of gastric acid secretion has been generally proposed as a therapy for eradication of H. pylori as described below.

H. pylori is a gram-negative spiral rod-shaped bacterium having flagella at one end and colonizing the human gastric mucosa. B. J. Marshall and J. R. Warren in Australia reported in 1983 that this bacterium was frequently detected in stomach biopsy specimens from patients with gastritis or gastric ulcers. At that time, this bacterium was named Campylobacter pylori since it resembles Campylobacter in morphology and growth characteristics. Later, it was found that the bacterium is different from Campylobacter in the fatty acid composition of its outer membrane and sequence of ribosome 16S-RNA. Therefore, the bacterium is now referred to as Helicobacter pylori and belongs to the newly established genus of Helicobacter.

Since then, many reports have been published based on epidemiological studies, indicating that this bacterium causes gastritis, gastric ulcers, and duodenal ulcers and is associated with diseases such as gastric cancer. Once H. pylori colonizes gastric mucosa, it survives and persists in the stomach and cannot be eradicated, although the immune response to infection thereof is strong, i.e., the antibody titer is high. Therefore, unless H. pylori is completely eliminated from the stomach by antibiotic therapy, the infection will return to the same level as before treatment within about a month after the administration of antibiotics is stopped. Additionally, the pH of the stomach is maintained very low by HCl, which is a strong acid, and therefore most antibiotics tend to be inactivated. For this reason, the combination of an antibiotic and a proton pump inhibitor which strongly suppresses the secretion of gastric acid is utilized for eradication of H. pylori.

However, the administration of antibiotics for a long time has the serious problems of increasing antibiotic-resistant strains as well as causing side effects.

Japanese Patent Application Kokai No. 11-263731 discloses that milk fat globule membrane fraction is effective for prevention of H. pylori infection. However, that publication merely teaches the ability to inhibit haemagglutination of H. pylori as evidence of prevention of H. pylori infection. Additionally, that publication states that milk fat globule membrane contains various components and does not state that which component is effective. Also, Sun Hirmo et al. states that gastric mucin and milk glycoprotein, specifically far globule membranes prepared from bovine buttermilk inhibit sialic acid-specific haemagglutination of H. pylori (FEMS Immunol. Medical Microbiology 20 (1998), pp. 275-281. However, it has been reported that there was no correlation between expression of haemagglutininins by H. pylori bacteria and the ability to bind gastric mucosa cells (M. Clyne & B. Drumm, Infection and Immunity, October 1993, pp. 4051-4057. Accordingly, the above-mentioned patent publication and article do not teach or suggest a substance which is capable of inhibiting the adherence of H. pylori to gastric mucosa.

Furthermore, the above patent publication and article have not elucidated an adhesin for adherence of H. pylori to gastric mucosa and a receptor therefor on gastric mucosa, which are important targets for inhibition of H. pylori infection.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an effective and safe inhibitor of H. pylori colonization which is associated with the occurrence of peptic ulcers, which inhibitor is capable of inhibiting the colonization of H. pylori effectively without the disadvantages of side effects and increase of drug-resistant strains which are associated with the use of antibiotics, and to provide a medicament and food useful for treating or preventing peptic ulcers.

Other objects and advantages as well as the nature of the present invention will be apparent from the following description.

Generally, the first step for establishment of an infection by a bacterium is adherence of the bacterium to a host cell and colonization of the bacterium by growing there. For the bacterium to adhere to the host cell, an adhesin has to bind to a receptor on the surface of the host cell. The specificity of the infective site of the bacterium is determined by this adhesin and the receptor. If the receptor molecule exists when the bacterium adheres to the host cell, competitive inhibition occurs and an infection is not established.

An adhesin of H. pylori and a receptor on human gastric mucosa are thought to be target molecules for inhibition of H. pylori infection. The present inventors clarified by studies on the mechanism of adherence of H. pylori that the adhesin of H. pylori, which had not been elucidated, is urease produced by H. pylori (Japanese Patent Application Kokai No. 10-287585).

The present inventors have studied substances capable of inhibiting the adherence of urease to gastric mucosa and have found that glycoproteins such as glycoprotein derived from the milk of a cow or glycoprotein derived from the albumen of a chicken egg are able to eliminate colonized H. pylori in the stomach by specifically binding to urease which is an adhesin localized on the surface layer of an H. pylori cell, and furthermore have found that the use of glycoprotein capable of specifically binding to urease even in a small amount, which glycoprotein is isolated and purified from these glycoprotein-containing substances by utilization of specific binding to urease, enables remarkably effective elimination of H. pylori.

According to the present invention, glycoprotein which is capable of specifically binding to urease is isolated and purified from a glycoprotein-containing substance by the affinity column technique which utilizes the specific binding to H. pylori urease, and the isolated and purified glycoprotein is used as an inhibitor of H. pylori colonization.

In one aspect, the present invention provides a glycoprotein which specifically binds to urease of Helicobacter pylori the glycoprotein being obtained by isolation and purification using a method utilizing specific adsorption to Helicobacter pylori urease.

In another aspect, the present invention provides an inhibitor of Helicobacter pylori colonization, comprising the above-mentioned glycoprotein as an active ingredient. The present invention also provides a pharmaceutical composition suitable for preventing or treating diseases caused by or associated with Helicobacter pylori in mammals including humans such as peptic ulcers, comprising the above-mentioned glycoprotein and a pharmaceutically acceptable carrier or diluent. Furthermore, the present invention provides a food which prevents or treats diseases caused by or associated with Helicobacter pylori in mammals including humans such as peptic ulcers when consumed in an effective amount, comprising the above-mentioned glycoprotein.

The method utilizing specific adsorption to Helicobacter pylori urease used in the present invention is preferably affinity chromatography using a column on which Helicobacter pylori urease is immobilized. The urease which is immobilized on the column may be recombinant urease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present invention, a glycoprotein which specifically binds to urease is isolated and purified from a glycoprotein-containing substance by a method utilizing specific adsorption to H. pylori urease.

Glycoprotein-containing substances used in the present invention may be any glycoprotein-containing substance such as the milk of a mammal or the albumen, chalaza, vitelline membrane or yolk of eggs of a fowl. Preferably, the whey of bovine milk and the albumen of chicken eggs, particularly high-molecular-weight whey protein concentrate and high-molecular-weight albumen protein concentrate are used.

Glycoprotein is a conjugated protein in which sugar chains consisting of about 2-6 types of monosaccharides are bound covalently to proteins. It is distributed widely in organisms. The monosaccharides contained in glycoprotein are N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-mannose, D-galactose, L-fucose, sialic acid, etc. There are various types of glycoproteins having different molecular weights and configurations. From the standpoint of forms of linkage between sugar chains and proteins, there are generally two types of glycoproteins, i.e., N-linked glycoproteins and O-linked glycoproteins (mucin type). Types of sugar chains, molecular weights and configurations of glycoproteins as well as functions or physiological activities vary depending on the location of glycoproteins existed.

Glycoproteins contained in bovine milk include lactoferin, secretory IgA, IgG, IgM, free secretory component (FSC), milk mucin and the like. Glycoproteins contained in the albumen of a chicken egg include ovomucoid, ovalbumin, ovotransferrin, phosvitin, ovomucin and the like.

In preparing a glycoprotein-containing substance, any known method can be used. A glycoprotein-containing substance may be prepared from bovine milk, for example, by removing milk fat and casein from milk in a conventional manner to obtain whey followed by fractionational concentration of the whey by appropriate means such as ultrafiltration membrane treatment to obtain high-molecular-weight whey protein concentrate (glycoprotein-containing substance). A glycoprotein-containing substance may be also prepared by removing lipoprotein from the whey, optionally followed by concentration and dialysis, and subsequently purifying the resulting material by suitable means such as gel filtration using a Sepharose column, etc., and treatment with a membrane. Optionally, further treatment such as protease treatment, alkali hydrolysis, etc. may be performed in order to obtain low-molecular-weight glycoprotein. Bovine milk used in the present invention may be either colostrum or milk produced following colostrum.

A glycoprotein-containing substance may be prepared from the albumen of chicken eggs by the following procedures, for example. Thick albumen is separated from collected albumen. A gelatinous portion is recovered by ultracentrifugation and is solubilized by techniques such as ultrasonic wave treatment or homogenization. The resulting solubilized substance is treated by gel filtration, membrane treatment or any other techniques to obtain a glycoprotein-containing substance. The thus obtained glycoprotein-containing substance may be further purified, if necessary, by a procedure such as gel filtration.

A glycoprotein-containing substance from the mucous membrane or gel layer thereof in the alimentary canal may be usually recovered by solubilizing glycoprotein by homogenization or ultrasonic wave treatment and then isolating the high molecular weight fraction by gel filtration or ethanol precipitation. Solubilization of a glycoprotein-containing substance may be performed by extraction with guanidine hydrochloride, urea, a salt solution, or a surfactant or treatment with a reducing reagent or protease. Some kinds of glycoprotein-containing substances may be recovered by forming an insoluble complex with a quaternary ammonium salt or by precipitation under acidic conditions.

Advantageouly, bovine milk or the albumen of chicken eggs is used as a starting material of a glycoprotein-containing substance, since these materials can be obtained inexpensively and in large quantities, and the preparation of a glycoprotein-containing substance therefrom can be carried out easily and by a simple procedure. Also, in preparing a glycoprotein-containing substance from milk, milk whey can be used. In the past, milk whey has been discarded since there was no effective way of using it, although it is produced in large amounts as by-product during a process for preparing cheese and the like. Therefore, a glycoprotein-containing substance from whey can be prepared in large amounts industrially, and the use of a glycoprotein-containing substance from milk is very advantageous with respect to cost and practicality.

Additionally, glycoprotein in bovine milk or the albumen of chicken eggs is of high stability and does not lose its physiological activity due to heat or at a low pH, and therefore it can be readily recovered and purified from a starting material, and it is advantageous with respect to formulation into a food or medicament, processing, and storing.

Any method which utilizes specific adsorption to urease can be used for isolation and purification of glycoprotein which specifically binds to H. pylori urease from a glycoprotein-containing substance. Preferably, affinity chromatography using a column on which H. pylori urease is immobilized is used. As urease which is immobilized on a column, recombinant urease may be preferably used because of the availability of homogeneous urease in large amounts.

Recombinant urease may be prepared in a conventional way. For example, genomic DNA of H. pylori can be extracted, and a gene coding urease molecule can be amplified by PCR method to obtain amplified DNA, which can be subsequently integrated into expression vector for E. coli (e.g. pKK233-2) by a known method. The obtained vector can be incorporated into a suitable host, E. coli (e.g. E. coli XL1-Blue) to produce recombinants. The recombinants can be cultured in a suitable culture medium, thereby expressing urease. Recombinant urease can be obtained by recovering the expressed urease. In preparing recombinant urease, expression systems using yeasts, mammal cells and insect cells may be used. Procedures for preparing recombinant urease are described, for example, in Molecular Cloning, Laboratory Mannual (2nd ed.) (Cold Spring Harbor Press), and in DNA Cloning 2 (2nd ed.) (IRL Press).

Immobilization of urease on a column may be performed using a ligand-immobilizing carrier which is capable of binding an amino group (--NH₃), carboxyl group (--COOH), thiol group (--SH), or hydroxyl group (--OH) contained in urease (e.g. NHS-activated Sepharose 4 Fast Flow). Isolation and purification of glycoproteins by a urease-immobilized column may be carried out by passing a sample containing a glycoprotein-containing substance through this column, followed by washing away non-specifically adsorbed proteins, and then eluting glycoproteins, which have specifically adsorbed to urease, from the column with an appropriate eluting solution.

According to the above-mentioned method, only glycoprotein which specifically binds to urease can be isolated and purified efficiently from various types of glycoprotein-containg substances. The thus obtained glycoprotein can inhibit the adherence of urease produced by H. pylori to mucin of gastric mucosa as demonstrated in the following examples. Since urease is localized on the surface of H. pylori cells, the glycoprotein produced by the above-mentioned method which specifically binds to urease (hereinafter referred to as the glycoprotein of the present invention) masks the adhesin i.e., urease, by predominantly binding to urease in the stomach and thereby inhibits the adherence of H. pylori to the receptor on gastric mucosa. This was confirmed in animal experiments, and the effect of the glycoprotein of the present invention on elimination of H. pylori from the stomach was observed. Also, the glycoprotein of the present invention is naturally-occurring and is very safe. Therefore, the glycoprotein of the present invention can be used as an inhibitor of H. pylori colonization in the stomach and is useful for preventing or treating diseases caused by or associated with H. pylori such as peptic ulcers.

Accordingly, the glycoprotein of the present invention can be used as an inhibitor of H. pylori colonization to be formulated into a medicament or food. Especially, the glycoprotein from milk or albumen of chicken eggs has been eaten in the past, so it can be formulated into foods such as foods for specified health use having anti H. pylori activity, foods for special dietary uses including foods for the aged or foods for the ill, or dietary supplement foods or health foods having anti H. pylori activity.

When the glycoprotein of the present invention is added to foods to be used as foods for specified health use or as foods for special dietary uses, the glycoprotein may be added to foods, usually in an amount of about 0.005-0.5% by weight, and preferably 0.01-0.1% by weight of the food. Foods for specified health uses to which the glycoprotein of the present invention is added include milk, dairy products, meat products, mayonnaise, dressings, beverages, ice cream, tofu (soybean curd), daily dishes, tsukudani (preserved foods boiled down in soy sauce), bean jams, flour paste, instant noodles, powdered food to be sprinkled over rice, pickled vegetables, powdered soups, dehydrated soups, confections, canned foods, retort pouched foods, frozen foods, and the like. Among these, foods which can be consumed continuously are preferred, but not required. When added to foods for the ill such as low sodium food, low energy food, or low protein food, the glycoprotein of the present invention may be added to soups, beverages, liquid diets, etc. to prepare foods in various forms.

Dietary supplement foods may be prepared, for example by adding to the glycoprotein of the present invention excipients such as dextrin, adhesives such as sodium caseinate, and, if necessary, nutrients (e.g. vitamins, minerals), emulsifiers, stabilizers, flavors, and the like to prepare a liquid diet.

When the glycoprotein of the present invention is utilized as a heath food, the glycoprotein may be contained as an active ingredient in an amount of about 0.1-3% by weight of the food. The glycoprotein may be formulated together with excipients such as lactose, corn starch, crystalline cellulose, or PVP, or with binders, and optionally with nutrients such as vitamins and minerals to form various forms of foods such as fine particles, tablets, and granules.

The glycoprotein of the present invention can be used alone or along with conventional additives as a pharmaceutical composition for prevention or treatment of peptic ulcers, etc. The glycoprotein alone or along with additives may be formed by a conventional method into a preparation for oral administration such as tablets, granules, powders, capsules or liquid preparations. The additives which may be used include excipients, binder, disintegrators, lubricants, antioxidants, coloring materials, corrigents, and the like.

Excipients which can be used in a pharmaceutical composition include sodium carboxymethylcellulose, agar, light anhydrous silicic acid, gelatin, crystalline cellulose, sorbitol, talc, dextrin, starch, lactose, sucrose, glucose, mannitol, magnesium metasilicate aluminate, calcium hydrogenphosphate, and the like.

Binders which can be used include gum arabic, sodium alginate, ethanol, ethyl cellulose, sodium caseinate, sodium carboxymethylcellulose, agar, purified water, gelatin, starch, tragacanth, lactose, hydroxycellulose, hydroxymethycellulose, hydroxypropylcellulose, polyvinylpyrrolidon, and the like.

Disintegrators which can be used include carboxymethylcellulose, sodium carboxymethylcellulose, calcium carboxymethylcellulose, crystalline cellulose, starch, hydroxypropylstarch, and the like.

Lubricants which can be used include stearic acid, calcium stearate, magnesium stearate, talc, hydrogenated oil, sucrose fatty acid ester, wax, and the like.

Antioxidants which can be used include tocopherol, gallic acid ester, dibutyl hydroxy toluene (BHT), butyl hydroxy anisol (BHA), ascorbic acid, and the like.

Other additional additives or agents may be added if desired, such as antacids (e.g., sodium hydrogencarbonate, magnesium carbonate, precipitated calcium carbonate, synthetic hydrotalsite), agents for protection of gastric mucosa (e.g., synthetic aluminum silicate, sucralfate, and sodium copper chlorophyllin) and digestive enzymes (e.g., biodiastase or lipase). The administration of a pharmaceutical composition for prevention or treatment of peptic ulcers, etc. may be by an oral route. The dosage of the glycoprotein of the present invention will be usually 2-30 mg and preferably 5-20 mg (as a dry weight) per day for an adult.

Additionally, the above-mentioned pharmaceutical composition for prevention or treatment of peptic ulcers, etc. may further comprise an inhibitor of gastric acid secretion. The combination of the glycoprotein and the inhibitor of gastric acid secretion is more effective in eliminating H. pylori from the stomach. Examples of an inhibitor of gastric acid secretion which can be used include H₂ blockers such as famotidine, nizatidine, roxatidine, ranitidine or cimetidine and proton pump inhibitors such as omeprazol, lansoprazol or sodium rabeprazole. The dosage of the inhibitor of gastric acid secretion is preferably 20-30 mg per day for an adult.

Claim 1 of 16 Claims

What is claimed is:

1. An inhibitor composition of Helicobacter pylori colonization, consisting essentially of a glycoprotein which is prepared by contacting a glycoprotein-containing substance from whey of bovine milk or albumen of chicken eggs with Helicobacter pylori urease and isolating and purifying the glycoprotein specifically bound to the urease.

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