JP2003012550A - Prophylactic and therapeutic agent for infectious disease with helicobacter pylori - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain both a medicine and a food effective for treating and preventing an infectious disease with Helicobacter pylori, being selective about Helicobacter pylori and having high safety. SOLUTION: This prophylactic, this therapeutic agent, this food and this beverage are characterized by comprising a catalase inhibitor against Helicobacter pylori as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a Helicobacter
Preventive and therapeutic agent for Helicobacter pylori infection characterized by containing a Helicobacter pylori infection as an active ingredient, especially effective for the prevention and treatment of diseases targeted for Helicobacter pylori eradication such as gastric / duodenal ulcer and gastric cancer Pharmaceutical products and foods.

[0002]

BACKGROUND OF THE INVENTION In 1983, Australian pathologist J. R. Warren and Gastroenterologist B. J. Mars
Hall is currently used in the biopsy tissue of gastric mucosa in patients with gastritis or peptic ulcer, and is currently used in Helicobacter pylori.
A bacterium named "ter pylori" was found at a high rate, and succeeded in isolation culture of this bacterium (Lancet, 1).
273-5, 1983). Helicobacter pylori is a Gram-negative, microaerobic, spiral-shaped bacillus that infects the gastric mucosa, and its strong urease activity decomposes host-derived urea into ammonia to neutralize gastric acid and to promote the growth of other bacteria. It lives predominantly in the difficult stomach. It is estimated that about half of the world's population is infected with this bacterium. Since the discovery of this bacterium, it became clear that the infection of this bacterium is involved in the development of upper gastrointestinal tract diseases such as gastritis, gastric / duodenal ulcer, and gastric cancer, and eradication therapy of this bacterium has been used to treat these diseases. It is done worldwide.

For the eradication therapy of this bacterium, β-lactam agents, macrolide agents, aminoglycoside antibiotics, tetracycline antibiotics, metronidazole used as an antiprotozoal drug, and antibacterial agents such as bismuth preparations are used. To be done. Currently, a combination therapy of three antibacterial agents (two β-lactam agents and macrolide agents) and one antiulcer agent (H2 blocker or proton pump inhibitor) (three-drug combination therapy) is the mainstream, and in Japan, , Triple combination therapy of amoxicillin + clarithromycin + proton pump inhibitor is the first-line drug (guideline for diagnosis and treatment of H. pylori infection, Japan Helicobacter Society Guideline Development Committee, Helicobacter Res)
search, 4,444-454,2000).

As described above, eradication treatment of Helicobacter pylori infection has been carried out for the purpose of treatment of upper gastrointestinal tract diseases caused by Helicobacter pylori infection and prevention of recurrence. However, as the main side effects of these eradication treatments, the amount of antibacterial agent used is larger than that used for the usual treatment of infectious diseases, and the effect on indigestive tract indigenous bacteria other than Helicobacter pylori is large. Have been done (guidelines above). Furthermore, as a problem related to eradication, it has been shown that the number of antimicrobial-resistant bacteria tends to increase (the above guideline). Therefore, there is a need to develop a new substance that is effective and highly safe for the prevention and treatment of Helicobacter pylori infection.

[0005]

SUMMARY OF THE INVENTION In view of the above situation, an object of the present invention is to provide a drug and a food which are effective against Helicobacter pylori and are highly safe and effective in preventing and treating Helicobacter pylori infection. To do.

[0006]

The present invention is a preventive and / or therapeutic agent for Helicobacter pylori infection, which comprises a Helicobacter pylori catalase inhibitor as an active ingredient. Further, the present invention is a food and drink for preventing and treating Helicobacter pylori infection, which comprises a Helicobacter pylori catalase inhibitor as an active ingredient. The present invention is described in detail below.

As a result of extensive studies to solve the above problems, the present inventor has found that the infection of Helicobacter pylori can be suppressed by suppressing the catalase of Helicobacter pylori, and has completed the present invention.

Helicobacter pylori catalase is 2
It is known to have a tetrameric structure having a molecular weight of 00 kDa and four subunits having a molecular weight of 50 kDa (J. Gen. Microbiol.
(1991), 137, 57-61). It is believed that Helicobacter pylori catalase plays an important role in escaping the attack of neutrophil phagocytosis, which is one of the human immune mechanisms, when Helicobacter pylori is infected and colonized in the gastric mucosa. Therefore, in the research and development of a vaccine for protection against Helicobacter pylori infection, an attempt has been made to protect against infection by administering catalase as an antigen and promoting antibody production against catalase (Japanese Patent Publication No. 8-5111282). 10-500958). However, there were problems such as not being able to obtain a sufficient infection protection effect. In view of this point, it could not be predicted at all that the administration of the catalase inhibitor of Helicobacter pylori reduced the number of Helicobacter pylori-infected bacteria.

The catalase-inhibiting substance of the present invention is not only a substance that inhibits the enzyme activity of catalase, but even if it does not inhibit the enzyme activity, it interacts with catalase and acts necessary for infection establishment of Helicobacter pylori. There is no particular limitation as long as it is a substance that suppresses Examples thereof include amitrole which is a known catalase inhibitor, an amino acid hydroxamic acid derivative having a catalase inhibitory activity, (−)-epigallocatechin gallate, and an antibody which immunoreacts with catalase.

As a disease in which the drug or food containing the catalase inhibitor of the present invention is used for prevention, health or treatment, a disease expected to be effective by eradicating Helicobacter pylori and reducing the number of infectious bacteria. Any disease may be used, but gastritis, gastric / duodenal ulcer, etc. may be mentioned.

The drug containing the catalase inhibitor of the present invention may be used alone. Further, it may be mixed with a general pharmaceutical carrier and administered in various forms. The dosage form is preferably, for example, powder, granules, tablets, capsules, syrups, etc., which can be safely administered orally. According to the conventional method, these various preparations are prepared according to a conventional method such as an excipient, a binder, a disintegrating agent, a coating agent, a lubricant, a stabilizer, a flavoring agent, a solubilizing agent, a suspending agent, a diluent and the like. It is possible to formulate with known auxiliary agents which can be usually used in the field. The dose varies depending on the target disease and the degree of illness, but for example, for adults, 1 mg to 2000 mg per day depending on the symptom
It can be administered once or in several divided doses.

The food containing the catalase inhibitor of the present invention may be used as it is. In addition, the catalase inhibitor can be added to any food or drink and can be ingested on a daily basis. The amount to be added to the food or drink may be arbitrary, but may be appropriately determined depending on the purpose of use such as prevention and health, and is usually 0.000.
A range of 1% to 10% is suitable.

Next, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these. Example 1 Helicobacter pylori catalase inhibitory action of amitrole The specific catalase inhibitor amitol (3-amino-1H-1,2,4-triazole) was tested for the helicobacter pylori catalase inhibitory action. Helicobacter pylori catalase was purified by the following method.

(1) Purification of Helicobacter pylori catalase Helicobacter pylori (ATCC 4350410 ⁸ CF was added to 330 sheets of Brain Heart Infusion (BHI) agar medium (manufactured by Difco) supplemented with 5% horse defibrinated blood.
(U / mL) was applied at 25 μL / sheet, and cultured at 37 ° C. under microaerobic conditions for 4 days. The obtained bacterial cells were scraped off with a platinum loop and suspended in phosphate buffered saline (PBS). Helicobacter pylori cells centrifuged at 4 ° C, 2,000 (g for 15 minutes, were suspended in 0.5% formalin and left overnight at 4 ° C to inactivate. The inactivated cells were centrifuged. (4 ° C., 3000 rpm, 15 minutes), washed 3 times with PBS, and then resuspended in PBS Using an ultrasonic disrupter (Model 7250, manufactured by Seiko Denshi Kogyo Co., Ltd.) , Output
The cells were sonicated for 30 minutes under the condition of 4, 50% duty cycle to crush the cells. The disrupted cell product was subjected to ultracentrifugation (4 ° C., 30,000 rpm, 30 minutes) to obtain 40 mL of supernatant (cell-soluble fraction) (protein concentration 4.9 mg / mL).

The anti-Helicobacter pyloricatalase monoclonal antibody 21G2 (hybridoma 21G2 FE was prepared by the method described in Amersham Pharmacia Biotech Affinity Chromatography Handbook).
A column in which a monoclonal antibody produced from RM BP-7336) was immobilized on CNBr-activated Sepharose 4B (manufactured by Amersham Pharmacia Biotech) was prepared (10 mL). 20 mL of the Helicobacter pylori cell soluble fraction was loaded onto this column (flow rate of about 4.5 mL / h). PBS 60mL (Flow rate about 30mL /
The column was washed with h) and eluted with 60 mL of 0.2 M glycine HCl buffer (pH 3.0) (flow rate about 30 mL / h). Both washing and elution were fractionated by 10 mL each. Catalase activity was examined by adding 100 μL of PBS containing 3% H ₂ O ₂ and 0.1% Tween 20 to 5 μL of each fraction and observing generation of bubbles. As a result, a strong catalase activity was observed in the eluted fractions (fractions 9 and 10).
These fractions were combined and concentrated to 5 mL to obtain Helicobacter pyloricatalase. (Protein concentration 0.8
mg / mL).

Helicobacter pyloricatalase was mixed with an equal volume of sample buffer (2% SDS, 5% mercaptoethanol) and boiled for 5 minutes. Using 5 μL of this solution, SDS-polyacrylamide gel electrophoresis (4-2
0% acrylamide) was performed. The molecular weight markers used were phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and α-lactalbumin. After the migration, the gel was stained with Silver Stain KANTO III (manufactured by Kanto Chemical Co., Inc.). Helicobacter pylori catalase is 59k
It was a single protein of Da.

(2) Helicobacter Pyloricatalase Inhibitory Activity of Amitrole To a well of a 96-well microplate, 25 μL of a Helicobacter pyloricatalase solution (0.6 μg / mL) and 25 μL of an amitrole solution of each concentration were added and left for 2 hours. After that, 3% hydrogen peroxide-0.1% Tween 8
50 μL of 0 solution was added, and the degree of foaming was visually confirmed. Amitrol 12.5 at 25 mg / mL was the control (PB
Foaming was suppressed as compared with S), and no foaming was observed at 50 mg / mL, and catalase inhibition was confirmed.

Example 2 Infection protection test of Helicobacter pylori in mice by amitrol Helicobacter pylori TN2GF4 (clinical isolate) was incubated at 37 ° C. in BHI agar medium supplemented with 5% horse defibrinated blood.
Microaerobically cultivated for 5 days, and collect the resulting colonies.
The suspension was suspended in to obtain a bacterial solution for infection. ICR mice (male, 6 weeks old) were bred for 5 days in drinking water containing amoxicillin (0.2 mg / mL) and vancomycin (10 μg / mL), and sterilized in the stomach. Then, instead of the drinking water in sterile water fasted overnight, for infection bacterial suspension 0.5mL (3 × 10 ⁸ /
Mouse) was administered by a sonde. After 30 minutes, amitrol was orally administered. Two days after infection, the animals were sacrificed and their stomachs were collected. The stomach was incised to remove the gastric contents, and then BHI liquid medium was added to homogenize. This is appropriately diluted with BHI liquid medium, and agar medium for detecting Helicobacter pylori (5% horse defibrinated blood, trimethoprim 10 μg / mL, bacitracin 10 μg / mL, vancomycin 20 μg / m
L, amphotericin 10 μg / mL, nalidixic acid 20
37 μg / mL on BHI agar medium)
The cells were microaerobically cultivated at 4 ° C for 4 days, and the number of infectious bacteria was examined. Control (P
In the group (BS administration), infection was confirmed in 4 out of 5 animals (average number of infected bacteria: 10 ^3.7 / g stomach tissue). On the other hand, amitrol 2
No infection was observed in any of the 4 groups that received mg / mL and the 5 groups that received 20 mg / mL.

Example 3 Search for Helicobacter Pyloricatalase Inhibitory Substances Using the method shown in Example 1 (2), substances having Helicobacter pyloricatalase inhibitory activity were searched.

Example 4 Infection protection test of Helicobacter pylori in mice with L-glutamic acid γ-monohydroxamic acid Helicobacter pylori SS1 (Gastroenterolo) was applied to BHI agar medium supplemented with 5% horse defibrinated blood.
gy (1997) 112,1386-1397) to 37
Micro aerobically cultivated for 5 days at ℃, collect the resulting colonies,
The suspension was suspended in BS to give a bacterial solution for infection. 0.5 mL of bacterial solution for infection in ICR mice (male, 6 weeks old) fasted for 24 hours
(3 × 10 ⁸ / mouse) was administered with a sonde. L-glutamic acid γ-monohydroxamic acid was orally administered twice, 30 minutes later and 6 hours later. Two days after infection, the animals were sacrificed and their stomachs were collected. The stomach was incised to remove the gastric contents, and then BHI liquid medium was added to homogenize. This is appropriately diluted with BHI liquid medium, applied to Helicobacter pylori agar medium (Nissui Pharmaceutical Co., Ltd.), and microaerobically cultured at 37 ° C. for 4 days,
The number of infectious bacteria was examined. Average number of infectious bacteria of 4 in control group 10
^{It was 6.3} / g stomach tissue. On the other hand, L-glutamic acid γ
-The average number of infectious bacteria in the group of 4 animals administered with monohydroxamic acid 40 mg / mL was remarkably suppressed to 10 ^4.6 / g gastric tissue.

Example 5 Preparation of egg yolk antibody against Helicobacter pyloricatalase Helicobacter pyloricatalase was mixed in an equal amount with Freund's complete adjuvant, and 1 mL was intramuscularly injected into laying hens (white leghorn strain). Immunization was performed once every two weeks for a total of three times. Yolk antibodies were prepared from 6 immunized eggs that were laid 9 to 11 weeks after the initial immunization. After the immune eggs were broken and the yolks were taken out, an equal amount of purified water was added (total amount 156 mL). This was mixed with a double amount (312 mL) of 0.15% λ-carrageenan aqueous solution, and the mixture was mixed at room temperature for 3
After leaving it for 0 minutes, it was centrifuged at 10,000 rpm for 15 minutes. Ammonium sulfate was added to the obtained supernatant so as to be 40% saturated and stirred, and then a precipitate was obtained by centrifugation. The precipitate was dissolved in PBS, dialyzed against the same buffer, and the dialysate was recovered (anti-Helicobacter pyloricatalase egg yolk antibody 288 mg protein).

The reactivity between the anti-Helicobacter pyloricatalase egg yolk antibody and Helicobacter pyloricatalase was confirmed by enzyme-linked immunosorbent assay (ELISA). 0.1 mg of Helicobacter pylori catalase in PBS
After diluting to / mL, 0.1 mL was added to each well of the 96-well ELISA plate, and the mixture was allowed to stand overnight at 4 ° C for immobilization. After washing with PBS, 0.25 mL of 0.1% skim milk-PBS was added to each well, and the mixture was left at 4 ° C. for 1 hour for masking. 0.1 in each well of the ELISA plate on which Helicobacter pyloricatalase is immobilized
0.1 mL of anti-Helicobacter pyloricatalase egg yolk antibody appropriately diluted with% skim milk-PBS was added, and the mixture was allowed to stand at room temperature for 1 hour. 0.05% Tween 20-PB
After washing 5 times with S (washing solution), peroxidase-labeled anti-IgY (manufactured by Boston Express Co., 1:10, 0)
00) 0.1 mL was added and left at room temperature for 1 hour. After washing 5 times with a washing solution, 0.1 mL of a substrate solution (tetramethylbenzidine + hydrogen peroxide, manufactured by BioFX) was added to each well and reacted at room temperature for 10 minutes. After reaction, 1N in each hole
Sulfuric acid was added by 50 μL each to stop the enzymatic reaction, and the absorbance (450 nm-630 nm) was measured. As a control, an egg yolk antibody prepared by the same method from eggs laid before immunization was used. As a result, the anti-Helicobacter pyloricatalase egg yolk antibody strongly reacted with Helicobacter pyloricatalase (Table 1).

[0023]

[Table 1]

Example 6 Effect of anti-Helicobacter pyloricatalase egg yolk antibody on Helicobacter pylori Anti-Helicobacter pyloricatalase egg yolk antibody 25 μ described in Example 5 in wells of U-bottom 96-well microplate
L and Helicobacter pylori SS1 bacterial solution (10 ⁷ / m
L) 25 μL was added, and after standing at 37 ° C. for 18 hours, visual observation was performed. As a result, it was found that the anti-Helicobacter pylori catalase egg yolk antibody exhibits the aggregating action of Helicobacter pylori cells. The pre-immune yolk antibody used as a control showed no agglutination. Therefore, it was suggested that the anti-Helicobacter pylori catalase egg yolk antibody inhibits colonization of Helicobacter pylori infection.

[0025]

EFFECTS OF THE INVENTION Since the present invention has the above-mentioned constitution, it is intended to provide a drug and a food which are effective for the prevention and treatment of upper gastrointestinal tract diseases such as gastritis, gastric / duodenal ulcer and gastric cancer caused by Helicobacter pylori infection. You can

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/00 A61K 31/198 4C206 // A61K 31/198 31/4196 31/4196 39/395 D 39 / 395 A23L 2/00 F (72) Inventor Haruhisa Hirata 1-5-3 Nihombashi Muromachi, Chuo-ku, Tokyo F-term in Wakamoto Pharmaceutical Co., Ltd. (reference) 4B017 LC03 LK21 LL09 4B018 LB08 MD85 ME14 MF01 4C084 AA02 AA03 AA17 BA44 MA23 MA35 MA37 MA41 MA43 MA52 NA14 ZA682 ZB262 ZB352 ZC202 4C085 AA13 BA20 CC07 CC26 DD38 DD41 DD73 DD86 DD88 EE01 GG08 4C086 AA01 AA02 BC60 MA01 MA04 MA23 MA35 MA37 MA41 MA14 MA14 MA14 MA14 MA14 MA14 A14 A06 A02 A16 A02 A16 A02 A06 A02 MA61 MA63 MA72 NA14 ZA68 ZB26 ZB35 ZC20

Claims (2)

[Claims] 1. A preventive and therapeutic agent for Helicobacter pylori infection, comprising a catalase inhibitor of Helicobacter pylori as an active ingredient. 2. A food or drink for the prevention and treatment of Helicobacter pylori infection, which contains a Helicobacter pylori catalase inhibitor as an active ingredient.