JP2008156293A - Helicobacter pylori infection-preventing and treating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a medicine, and food and beverage effectively eradicating Helicobacter pylori living in stomach, effective for the prevention and treatment of Helicobacter pylori infection, gastritis and peptic ulcer, having high safety and eatable over a long period of time, and a method for preventing and treating the Helicobacter pylori infection, gastritis and peptic ulcer. <P>SOLUTION: This Helicobacter pylori infection, gastritis or peptic ulcer-preventing and treating agent is provided by having a component reducing urea in stomach as an active ingredient, and the method for preventing and treating the Helicobacter pylori and the method for preventing and treating the gastritis and peptic ulcer by reducing the urea in the stomach are also provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a preventive / therapeutic agent for Helicobacter pylori infection or a preventive / therapeutic agent for gastritis or peptic ulcer comprising an ingredient that reduces urea in the stomach as an active ingredient.

Helicobacter pylori (Helicobacter pylori) is a gastric mucosa from isolated Gram-negative bacilli of gastric ulcer patients (Non-Patent Document 1), it is an important virulence factor of chronic gastritis and peptic ulcers have been reported so far ( Non-patent document 2), and it is also suggested to be involved in the development of gastric cancer and lymphoma. Helicobacter pylori is reported to exert its pathogenicity by firstly degrading urea in the stomach with urease produced by it, and neutralizing gastric acid with the resulting ammonia as a first step. Patent Document 3).

As a method for treating gastric mucosal lesions caused by Helicobacter pylori, eradication therapy using a combination of an antibiotic and a proton pump inhibitor or an H ₂ -receptor antagonist has been clinically performed. Recently, amoxicillin has been used in Europe and the United States. , Metronidazole, and proton pump inhibitor triple therapy, and in Japan, triple therapy using clarithromycin instead of metronidazole is frequently practiced in the clinic. However, these therapies sometimes have strong side effects on the human body, and the emergence of antibiotic-resistant bacteria is regarded as a problem.

Therefore, in recent years, a search for a substance having a sterilizing action of Helicobacter pylori and a method for removing it has been vigorously conducted. Acidophilus, L. Casei, L. Various lactic acid bacteria such as gasseri (Patent Documents 1 and 2), chemical substances such as acetohydroxamic acid or flurofamide or their salts (Patent Document 3), and methods using Helicobacter pylori-derived urease as a vaccine (Patent Document 4) Has been. However, sufficient effects have not yet been obtained for any of these.
JP 2001-258549 A Japanese Patent Laid-Open No. 2001-143 JP 2002-29966 A Japanese National Patent Publication No. 7-503255 Lancet, Vol.1, p1273-1275 (1983) Lancet, Vol.1, p1311-1314 (1984) Lancet, Vol.2, p15-17 (1986)

  Accordingly, an object of the present invention is to prevent Helicobacter pylori infection and treatment used for pharmaceutical preparations, foods and drinks, etc. that are effective for sterilization of Helicobacter pylori inhabiting the stomach and can be taken for a long time. It is to provide an agent or a preventive / therapeutic agent for gastritis or peptic ulcer and a preventive / therapeutic method.

  As a result of diligent studies to solve the above problems, the present inventors have found that reducing the gastric urea with a component that reduces the gastric urea can suppress the infection of Helicobacter pylori, thereby completing the present invention.

  That is, the present invention provides an agent for preventing and treating Helicobacter pylori infection, which comprises an ingredient that reduces urea in the stomach as an active ingredient.

  The present invention also provides a prophylactic / therapeutic agent for gastritis or peptic ulcer comprising an ingredient that reduces gastric urea as an active ingredient.

  The present invention also provides a method for preventing and treating Helicobacter pylori infection, characterized by reducing gastric urea.

  Furthermore, the present invention provides a method for preventing or treating gastritis or peptic ulcer characterized by reducing urea in the stomach.

The component for reducing urea in the stomach of the present invention has an excellent eradication action of Helicobacter pylori, and is effective as a therapeutic agent for Helicobacter pylori infection prevention or treatment for gastritis or peptic ulcer. It is.
In particular, urease, which exhibits an optimum pH in the acidic region, has excellent Helicobacter pylori sterilization action, and these components are highly safe without side effects and the emergence of antibiotic-resistant bacteria. -It can be effectively and safely used for the purpose of preventing or treating H. pylori infection, gastritis or peptic ulcer.
Further, by using the method of the present invention, it is possible to effectively and safely prevent and treat Helicobacter pylori infection, gastritis or peptic ulcer.

The component for reducing the urea in the stomach used in the present invention (hereinafter also referred to as a stomach urea reducing component) may be any substance that has an action of reducing the urea present in the stomach. Specific examples include substances that specifically bind to urea, substances that decompose urea, and substances that convert urea into other substances.
The gastric urea-reducing component can reduce urea in the stomach, producing ammonia from the gastric urea, neutralizing the gastric acid and infecting it, and suppressing infection with Helicobacter pylori that exerts its pathogenicity In addition, Helicobacter pylori in the stomach can be sterilized.

  Examples of the substance that specifically binds to urea include urea adsorption resins (Japanese Patent Laid-Open Nos. 62-33539 and 63-59353), and examples of substances that decompose urea include decomposition of a metal complex urea. Examples thereof include resins (Japanese Patent Laid-Open No. 63-61007), urease, and the like, preferably urease.

The urease used in the present invention may be any urease exhibiting an optimum pH in the alkaline region, neutral region or acidic region, and may exhibit pH stability, substrate specificity, Km indicating other general properties of the enzyme. There are no particular restrictions on the value, molecular weight and the like.
Among these, urease showing the optimum reaction pH in the acidic region (hereinafter referred to as acidic urease) is most preferable, and the acidic urease is highly reactive in the acidic region of pH 1 to 4 in the same state as in the human stomach. It can exhibit urease activity better than urease which shows a pH optimum for reaction from neutral to slightly alkaline, which is usually often observed, and can improve sterilization of Helicobacter pylori. In addition, the optimum pH of Helicobacter pylori-derived urease is around 7.5, and it is a urease that exhibits neutral to slightly alkaline reaction optimum pH that does not exhibit activity at pH 4.5 or less.
The acidic urease used in the present invention refers to those having an optimum pH of activity in the acidic region of 1 to 6, and those having an optimum pH of 4 or less are particularly preferred.
In the present invention, 1 unit (1 U) of acidic urease is the amount of enzyme that liberates 1 μmol of ammonia from urea per minute under the conditions of pH 4 and 37 ° C.

  As the acidic urease used in the present invention, for example, commercially available products such as enzyme preparations and natural extracts may be used as they are, or those subjected to various treatments such as heating and ultraviolet rays may be used, and the production of microorganisms containing acidic urease may be used. You may use a microbial cell, a dead cell, a microbial cell processed material, a culture, etc. Examples of commercially available products include Nagapsin (Nagase ChemteX Corporation), Takemate-AU (Takeda Pharmaceutical Company Limited), and the like.

The acidic urease is not particularly limited by the origin of natural products or microorganisms, but those derived from microorganisms are preferred.
Examples of urease derived from microorganisms include, for example, urease derived from Lactobacillus fermentum (Japanese Patent Publication No. 7-83705), urease derived from bacteria belonging to the genus Escherichia or Staphylococcus (Japanese Patent Laid-Open No. 64-51083), Arthrobacter Urease derived from genus bacteria (Japanese Patent Publication No. 8-8867), urease derived from Pseudomonas bacteria (Japanese Patent Laid-Open No. 1-191684), urease derived from the genus Zooglea (Japanese Patent Laid-Open No. 1-222281), Lactobacillus genus or Streptococcus genus Bacteria-derived urease (JP-A-1-240187), Rhizopus bacteria-derived urease (JP-A-3-27287), filamentous fungi-derived urease (JP-A-3-43081), Bifidobacterium genus urease (Japanese Patent Fair 7-837 5 No.), and the like, safety, and because of high activity, Lactobacillus fermentum urease is preferred.

  As the acidic urease used in the present invention, a natural product containing acidic urease, a produced microorganism or the like can be used as it is, and may be separated and purified by a known separation / purification means.

The microorganism producing acid urease is not particularly limited to the genus name, species name and strain name of the fungus as long as it can produce acid urease having the desired properties of the present invention, and has been mutated by conventional means. A thing may be used.
For example, Escherichia spp. (Escherichia) bacteria, Staphylococcus (Staphylococcus) bacteria, Arthrobacter (Arthrobacter) bacterium, Pseudomonas (Pseudomonas) bacteria, Zooglea genera (zoogloea) bacteria, Lactobacillus (Lactobacillus) bacteria belonging to the genus Streptococcus genus (Streptococcus) bacteria, genus Rhizopus (Rhizopus) bacteria, bifidobacteria (Bifidobacterium) bacteria, include filamentous fungi etc., Lactobacillus fermentum of the genus Lactobacillus bacteria (Lactobacillus fermentum) is more preferable. The Lactobacillus fermentum range also includes Lactobacillus reuteri classified as Lactobacillus reuteri by analysis of the 16S rDNA base sequence (Int. J. Syst. Evol. Microbiol). 32, 266-268, 1982).
Examples of Lactobacillus fermentum producing acid urease include TK1214 (JP-B-7-83705), B-1112 (JP-A-2-39886), JCM5867, JCM5868, JCM5869, etc., particularly JCM5867 strain Can be suitably used.

  In addition, fermented foods and beverages such as fermented milk, lactic acid bacteria beverages, fermented soy milk, fermented fruit juice, fermented plant fluids, etc. produced using microorganisms such as Lactobacillus fermentum that produce acidic urease have good sensory properties and long-term Since it can withstand various intakes, it can be suitably used.

  Among the components for reducing urea in the stomach of the present invention, when using a substance that decomposes urea, such as acidic urease, it is possible to use a substance or microorganism that removes or reduces ammonia generated as a decomposition product of urea, It is preferable for the reason of flavor improvement. Bifidobacterium bifidum (JP-A-4-166079) can be suitably used as a substance or microorganism for removing or reducing ammonia.

  The component for reducing urea in the stomach of the present invention has a sterilizing action against Helicobacter pylori, so it can be used as an agent for preventing or treating infection with Helicobacter pylori. It can be used as a prophylactic / therapeutic agent for peptic ulcer, especially gastric / duodenal ulcer.

  The agent for preventing and treating Helicobacter pylori infection and the agent for preventing and treating gastritis or peptic ulcer of the present invention can be used either orally or parenterally, but oral administration is preferred. In administration, the component for reducing gastric urea, which is an active ingredient, can be mixed with a solid or liquid nontoxic pharmaceutical carrier suitable for the administration method and administered in the form of a conventional pharmaceutical preparation.

Examples of the preparation include solid agents such as tablets, granules, scattered powders, and capsules, solutions such as solutions, suspensions, and emulsions, and freeze-dried agents. The preparation can be prepared by conventional means on the preparation.
Examples of the non-toxic pharmaceutical carrier include starch, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water, and physiological saline. It is done. In addition, conventional additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents, excipients and the like can be appropriately added as necessary.
In addition, the gastric urea-reducing component, which is an active ingredient, is administered as a single agent, and the gastric urea-reducing component and an H ₂ -receptor antagonist such as antibiotics, cimetidine, ranitidine, famotidine, and proton pumps such as omeprazole You may administer 1 or more types, such as an inhibitor and a gastric mucosa protective agent, simultaneously or separately.

In addition, the Helicobacter pylori infection preventive / therapeutic agent, gastritis or peptic ulcer preventive / therapeutic agent of the present invention can be used not only as a pharmaceutical preparation as described above but also as a food or drink. When used as a food or drink, it may be contained in the food or drink as it is or after adding various nutritional components.
This food and drink can be used as a health food or food material useful for the prevention, improvement and treatment of Helicobacter pylori infection, or the prevention, improvement and treatment of gastritis and peptic ulcer. You may attach the display to the effect of said. Specifically, when the Helicobacter pylori infection preventive / therapeutic agent of the present invention, the preventive / therapeutic agent for gastritis or peptic ulcer is blended in foods and drinks, additives that can be used as food and drinks are appropriately used, and conventional means are used. It may be used in a form suitable for edible use, such as granules, granules, tablets, capsules, pastes, etc., and various foods such as ham, sausage and other processed meat products, fishery products such as kamaboko and chikuwa It may be used by being added to processed foods, bread, confectionery, butter, powdered milk, fermented foods and drinks, or added to beverages such as water, fruit juice, milk, soft drinks and tea drinks. The food and drink includes animal feed.

  Among the foods and drinks mentioned above, fermented foods and beverages such as fermented milk, lactic acid bacteria beverages, fermented soymilk, fermented fruit juice, and fermented plant juice produced using microorganisms such as Lactobacillus fermentum that produce acidic urease are functional It is suitable because it can withstand long-term intake. Production of these fermented foods and drinks may be carried out in accordance with conventional methods. For example, fermented milk is inoculated and cultured with microorganisms producing acid urease alone or simultaneously with other microorganisms in a sterilized milk medium, and this is homogenized and fermented. Get a milk base. Subsequently, a separately prepared syrup solution is added and mixed, homogenized with a homogenizer or the like, and flavor is further added to obtain a final product. The fermented milk thus obtained can be a product of any form such as a plain type, a soft type, a fruit flavor type, a solid form, a liquid form, or a frozen form.

Although there is no limitation on the dose when using the gastric urea reducing component which is an active ingredient of the Helicobacter pylori infection preventive or therapeutic agent for gastritis or peptic ulcer of the present invention, the preferred dose is It is 10 to 3000 U per day, and 20 to 1200 U is particularly preferable.
In addition, when Lactobacillus fermentum is used as a microorganism derived from acid urease, it can be used in any form such as live cells, dead cells, treated cells, cultures, etc. Such a dose is 10 ⁶ to 10 ¹⁴ cfu per day as the number of bacteria, and 10 ⁸ to 10 ¹² cfu is particularly preferable.

  Hereinafter, the content of the present invention will be described in more detail with reference to test examples and examples, but the present invention is not limited by these.

Test Example 1: Effects of acidic urease administration on Helicobacter pylori infection and gastritis onset The infection inhibitory effect of Lactobacillus fermentum-derived urease on Helicobacter pylori infection was confirmed by MGS / Sea gerbils.
As the lactase derived from Lactobacillus fermentum, Nagapsin (manufactured by Nagase Chemtech), which is a urease preparation for food addition, was used. Twenty gerbils (MGS / Sea, 6-week-old male, about 50 g) were purchased and preliminarily raised for 1 week with CE-2 solid feed (CLEA Japan). The animals were divided into 3 groups as shown in Table 1. The control group was given ion-exchanged water, and the administration group was given nagapsin 50 ppm solution (acid urease concentration 2.5 ppm, acid urease activity 0.1 U / mL) by drinking water from 2 days before infection.

Helicobacter pylori ATCC 43504 (American Type Culture Collection) was used for infection. This strain was cultured with shaking at 37 ° C. for 24 hours in Brucella medium (Becton Dickinson) supplemented with 10% inactivated horse serum (Japan Bio Supply). The cultured bacterial solution was diluted 100-fold with an uninoculated medium and used for Helicobacter pylori bacterial infection for animal infection.
After the above-prepared control group and nagapsin administration group were fasted for 24 hours on the day before infection, 0.5 mL / animal of the bacterial solution (3.5 × 10 ⁶ CFU / mL) was forcibly orally administered to each of these groups. Fasted and watered.
Non-inoculated medium was administered to the Helicobacter pylori non-infected group (no oral administration).
During the experimental period, food and water were freely consumed, and groups of 4 cages were kept.

  The animals were sacrificed 6 weeks after oral administration of Helicobacter pylori under ether anesthesia by whole blood collection of the abdominal aorta, and the stomach was collected. The stomach was incised from the large buttocks, washed twice with physiological saline, weighed, and then visually evaluated for gastritis (existence of edema and bleeding). Thereafter, the right half of the stomach was scraped and immersed in 3 mL of PBS and stored in ice to obtain a sample for measuring the number of bacteria. Gastric samples in PBS were homogenized and serially diluted with PBS. 100 μL of the diluted solution was applied to Helicobacter pylori detection medium (day water), cultured at 37 ° C. for 5 days under microaerobic condition, colony count was performed, and the number of bacteria in the stomach was measured.

Tests for infection rate and gastritis incidence were performed using Fisher's exact test.
The stomach weight and the number of Helicobacter pylori bacteria in the stomach were analyzed by variance, and the stomach weight was subjected to Dunnett's multiple test using the Helicobacter pylori infection control group as a control.

Six weeks after oral administration of Helicobacter pylori, the infection rate in the control group of Helicobacter pylori infection (oral administration) was 87.5% (7/8), and 25% (2/8) in the nagapsin administration group (Table 2). . In addition, the number of Helicobacter pylori bacteria in the stomach showed a significantly lower value (p <0.05) in the administration group compared to the Helicobacter pylori infection control group, confirming the infection inhibitory effect of acid urease administration (Fig. 1).
The incidence of gastritis was 87.5% in the Helicobacter pylori infection control group and 25% in the administration group, and the stomach weight as an index of gastritis was significant in the administration group compared to the Helicobacter pylori infection control group (p <0.05). ) Showed a low value (FIG. 2).

Test Example 2: Verification of the mechanism of action of Helicobacter pylori eradication by acid urease (1)
The effect of reducing gastric urea by administration of Lactobacillus fermentum-derived urease was confirmed in mice. That is, C57BL / 6J mice (6 weeks old, female, Japan Claire) were purchased and preliminarily raised on AIN-93G diet for 1 week. A mouse (n = 3 in each group) was administered with 100 μL of gaspsone with a Nagapsin 500 ppm solution (acid urease concentration 25 ppm, acid urease activity 1 U / mL) or distilled water, 30 minutes after administration, and killed by cervical dislocation with ether anesthesia. The stomach was removed. After incision of the excised stomach, the inside of the stomach was washed with 500 μL of distilled water, and the urea nitrogen and ammonia nitrogen concentration in the washing solution were measured with ammonia test Wako (Wako Pure Chemical) and urea nitrogen B-Test Wako (Wako Pure). And the gastric content was calculated. As a result, the amount of urea nitrogen in the stomach and the ratio of urea nitrogen (ratio of the amount of urea nitrogen to the total amount of nitrogen) decreased (Table 3). As a result, administration of Lactobacillus fermentum derived urease It was confirmed that it was decomposed into ammonia and the amount of urea was reduced.

Test Example 3: Verification of action mechanism of Helicobacter pylori eradication by acid urease (2)
In an in vitro experiment, the Helicobacter pylori bactericidal action of Lactobacillus fermentum-derived urease was examined. In the experiment, nagapsin was used as a urease derived from Lactobacillus fermentum. Since this product is a mixture of urease and lactose in a ratio of 5:95, lactose was used as a control. That is, Helicobacter pylori ATCC 43504 was cultured with shaking in Brucella medium (Becton Dickinson) supplemented with 10% inactivated horse serum (Japan Bio Supply) at 37 ° C. for 24 hours. Lactose or urease solution was added to 10 mL of the bacterial solution (1.6x10 ⁸ CFU / mL) to a final concentration of 50 or 500 ppm, shaken at 37 ° C under slight aerobic condition for 6 hours (140 rpm), The number of Helicobacter pylori was measured using a Helicobacter pylori detection medium (Nissui). As a result, the number of Helicobacter pylori in the urease-added bacterial solution did not decrease as compared to the lactose-added bacterial solution (FIG. 3). Therefore, acidic urease and lactose were directly bactericidal against Helicobacter pylori. Was not shown.

  From the above, the effect of administration of Nagapsin, which is the acidic urease confirmed in Test Example 1, is the result of the successful degradation of stomach urea into ammonia by Nagapsin, resulting in a decrease in the concentration of urea in the stomach, resulting in Helicobacter pylori in the stomach. This was thought to be due to the inability to neutralize gastric acid using urea in the stomach.

  In other words, if a component that reduces gastric urea, such as acid urease, is used, Helicobacter pylori can decrease the gastric urea used to neutralize gastric acid. Producing ammonia from urea and neutralizing gastric acid as a first step, the bacteria cannot exert pathogenicity, can suppress Helicobacter pylori infection, and can eliminate bacteria in the stomach I found it. It was also found that the incidence of gastritis with Helicobacter pylori as a causative bacterium can be reduced, and gastritis and peptic ulcers can be prevented and treated.

Example 1 Manufacture of tablets Various ingredients were mixed according to the following formulation, granulated, dried and sized, and then tableted to produce tablets.
(Prescription) (mg)
Nagapsin 100
Microcrystalline cellulose 100
Lactose 80
Magnesium stearate 0.5
Methylcellulose 12

Example 2 Manufacture of soft drinks After being pasteurized with heat and pasteurized, the brown bottle was hot-packed to obtain soft drinks.
(Prescription) (g)
Lactobacillus fermentum 0.8
Bacteria cell dried product of JCM5867 ¹⁾
Fragrance 0.8
Citric acid 0.2
Fructose 4
Sucralose 0.001
Water 94.199
(The total amount should be 100)
¹⁾ : Obtained by freeze-drying Lactobacillus fermentum JCM5867 live cells. The bacterial count of the dried bacterial cell was 1 × 10 ¹² cfu / g.

Example 3 Production of Fermented Dairy Products After adding 3% glucose to 15% skimmed milk and sterilizing at 120 ° C. for 3 seconds, inoculated with 1% inoculum of Lactobacillus fermentum JCM5867, until pH 3.6 at 37 ° C. After culturing, 210 g of yogurt base was obtained. On the other hand, 97 g of sugar and 0.2 g of iron citrate were dissolved in water, water was added to make a total amount of 790 g, and this solution was sterilized at 110 ° C. for 3 seconds to obtain a syrup. The yogurt base and syrup obtained as described above were mixed, 1 g of flavor was added, and then homogenized at 15 MPa and filled into a container to obtain a fermented milk product. The initial bacterial count of Lactobacillus fermentum JCM5867 in this fermented dairy product was 2.1 × 10 ⁸ cfu / mL.

It is a graph which shows the influence which acidic urease administration has on the number of Helicobacter pylori in the gerbil stomach. It is a graph which shows the influence which acidic urease administration has on the stomach weight of a gerbil. It is a graph which shows the influence which lactose and nagapsin addition have on the viable count of Helicobacter pylori.

Claims (6)

  An agent for preventing and treating Helicobacter pylori infection, which contains an ingredient that reduces urea in the stomach as an active ingredient.   The preventive / therapeutic agent for Helicobacter pylori infection according to claim 1, wherein the urea-reducing component in the stomach is urease having an optimum pH in the acidic region.   A prophylactic / therapeutic agent for gastritis or peptic ulcer comprising an ingredient that reduces urea in the stomach as an active ingredient.   The preventive / therapeutic agent for gastritis or peptic ulcer according to claim 3, wherein the component for reducing urea in the stomach is urease having an optimum pH in the acidic region.   A method for preventing and treating Helicobacter pylori infection, characterized by reducing urea in the stomach.   A method for preventing and treating gastritis or peptic ulcer, comprising reducing urea in the stomach.