JPH1045602A - Adhesion inhibitor of helicobacter pylori or production inhibitor of interleukin-8 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inhibitor useful for inhibition of damage to gastric mucosa by suppressing adhesion of Helicobacter pylori on the gastric mucosa, which is a first stage of the damage of the gastric mucosa, by including a ganglioside as an active ingredient. SOLUTION: This inhibitor includes a ganglioside, preferably one kind selected from the group of GD3 , GD1a , GD1b , GT1b , GM1 , GM1 inner ester, asialo GM1 , GM3 , O-acetyl-GD3 and GT1a . The ganglioside is a glucide containing sialic acid, has a sugar chain comprising sialic acid and monosaccharide as a hydrophilic head part and a ceramide comprising a sphingosine base and a fatty acid as a hydrophobic tail part and has structures of the formulas. The ganglioside is administrated in a daily dose of 0.1-1mg for an adult in 1-3 divided portions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a helicobacter
The present invention relates to a therapeutic agent and an interleukin-8 production inhibitor effective for ameliorating gastric disease caused by H. pylori gastric mucosal infection.

[0002]

2. Description of the Related Art Helicobacter pylori
pylori ) is a gram-negative spiral bacillus found in the stomach in 1982 and causes chronic gastritis or atrophic gastritis in humans. The fungus also causes the development of duodenal ulcers or the recurrence of gastric ulcers. More recently, its involvement in gastric cancer has also been demonstrated, and this bacterium has important implications, especially for gastric diseases. Helicobacter
As a mechanism of gastric mucosal damage caused by H. pylori, since this bacterium has a high urease activity, it is considered that the gastric mucosa is damaged by ammonia produced by urease.
Helicobacter pylori also stimulates gastric mucosal epithelial cells to increase interleukin-8 production, promotes neutrophil migration, and causes long-lasting inflammation to cause atrophy of the gastric mucosa. In addition, between the hypochlorous acid released from neutrophils and the ammonia produced by Helicobacter pylori, extremely toxic monochloramines form, causing damage. In addition, the harmful effects of cytotoxins such as cagA and vagA that the bacterium itself has can be considered. The first stage of these disorders caused by Helicobacter pylori is initiated by the fungus adhering to gastric mucosal epithelial cells.

At present, the treatment of gastric diseases caused by Helicobacter pylori is, for example, a combination of a conventional antibiotic such as amoxicillin or clarithromycin with an acid secretion inhibitor such as a proton pump inhibitor (PPI). Has been conducted as a clinical trial. However, this treatment has the drawback that antibiotics are administered in twice the usual dose, resulting in side effects such as diarrhea. Further, as an interleukin-8 production inhibitor, mucosta (Otsuka Pharmaceutical Co., Ltd.), an anti-ulcer agent, has an interleukin-8 production inhibitory action (described in JP-A-8-12578), and excludes Helicobacter pylori. Although clinical trials have been conducted as fungicides, there are currently no inhibitors of interleukin-8 production due to Helicobacter pylori. Thus, there has been a demand for a therapeutic agent for gastric diseases which overcomes the above-mentioned drawbacks and has a better therapeutic effect than conventional therapeutic agents.

On the other hand, as for ganglioside, there is no drug containing ganglioside as an active ingredient at present.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a first step of gastric mucosal damage caused by Helicobacter pylori, which suppresses gastric mucosal damage by suppressing adhesion of bacteria to mucosa. It is to provide a H. pylori adhesion inhibitor.

[0006] Another object of the present invention is to provide an interleukin-8 production inhibitor that reduces the degree of inflammation by inhibiting interleukin-8 production.

Another object of the present invention is to provide a therapeutic agent for treating gastric ulcer or gastritis, which is a gastric disease caused by Helicobacter pylori.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that a therapeutic agent containing ganglioside as an active ingredient has an excellent effect. It was just.

The present invention relates to a medicine containing ganglioside as an active ingredient. The present invention further provides a Helicobacter pylori adhesion inhibitor containing ganglioside as an active ingredient, an interleukin-8 production inhibitor containing ganglioside as an active ingredient, an antiulcer agent containing ganglioside as an active ingredient, and a treatment for gastritis containing ganglioside as an active ingredient. Agent.

[0010]

DETAILED DESCRIPTION OF THE INVENTION Ganglioside is a glycolipid containing sialic acid, which has a sugar chain consisting of sialic acid and monosaccharide as a hydrophilic head, and a ceramide consisting of sphingo base and fatty acid as a hydrophobic tail. Have.

The ganglioside used in the present invention is not particularly limited, but G _D3 , G _D1a , G _D1b , G
_T1b, G _M1, G _M1 inner ester, asialo G _M1, G
_M3, O - acetyl _{-G D3, G T1a, G D2} , G M2, G M1b or G _Q1b are preferred. In particular, G _D3 , G _D1a , G _D1b ,
G _T1b , G _M1 , G _M1 inner ester, Asialo G _M1 ,
G _M3, O - acetyl -G _D3 or G _T1a is preferred.

The chemical structures of the gangliosides are as shown in Table 1.

[0013]

[Table 1]

[0014]

[Table 2]

As for the abbreviations used in the chemical structures shown in Table 1, Gal is galactose, GalNAc is N-acetylgalactosamine, Glc is glucose, and NeuAc is N-acetyl.
Acetylneuraminic acid and Cer indicate ceramide.

The gangliosides are commercially available, for example, Research Biochemicals International (Snow Brand Milk Products Co., Ltd.)
s International) (USA), EY Laboratories (USA), Sigma
a) (USA) Advanced Immuno Chemical (Advan)
Commercial products such as ce Immuno Chemical (USA) may be used.

With respect to the mechanism of adhesion of Helicobacter pylori to gastric mucosal epithelial cells, the theory that Helicobacter pylori may specifically adhere to sulfatide, an acidic glycolipid of gastric mucosal epithelial cells (The University of Tokyo,
(By Sugano et al.) Is promising, but has not yet been elucidated. Helicobacter pylori adhesion in the present invention,
In a broad sense, it means that Helicobacter pylori attaches to gastric mucosal epithelial cells.

The adhesion inhibitor of the present invention, interleukin-
8 Production inhibitor, anti-ulcer agent and gastritis therapeutic agent are each prepared in the form of general pharmaceutical preparations. The formulations are prepared in a conventional manner using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and lubricants which are conventionally used. These pharmaceutical preparations can be selected in various forms depending on the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, injections (Solutions, suspensions, etc.), aerosols and syrups. Further, it can be used by increasing the sustained release by blending it with a resin or the like.

The method of administration of the medicament of the present invention is not particularly limited, unless it is specifically selected for a particular therapeutic purpose, and includes various preparation forms, patient age, gender and other conditions, degree of disease and the like. Is administered according to the method. For example, tablets, pills, powders, solutions, suspensions, emulsions, granules, syrups and capsules are orally administered. In the case of an injection, it is administered intravenously, alone or as a mixture with a normal replenisher such as glucose or amino acid.If necessary, it is administered alone intramuscularly, intradermally, subcutaneously or intraperitoneally. You. In the case of suppositories, they are administered rectally.

In order to achieve the desired therapeutic effect, the dose of the active ingredient is appropriately selected depending on the usage, the age, sex and other conditions of the patient, the degree of the disease, and the like.
Usually, it is in the range of 0.01 to 10 mg per adult per day,
Preferably, it is selected from 0.1 to 1 mg. The unit dosage form can be administered one to three times a day.

Further, the G _D3 mice were administered 30 mg / kg there was no deaths.

The Helicobacter pylori adhesion inhibitor of the present invention makes it possible to suppress gastric mucosal damage caused by adhesion of Helicobacter pylori to gastric mucosal epithelial cells.
Such gastric mucosal disorders include atrophy and inflammation of the gastric mucosa caused by interleukin-8 produced from gastric mucosal epithelial cells by stimulation of Helicobacter pylori adhesion.

The interleukin-8 production inhibitor of the present invention is useful for the prevention or treatment of acute or chronic inflammatory diseases irrespective of Helicobacter pylori infection. Specific examples of such acute or chronic inflammatory diseases are ,
(1) Inflammatory keratoses such as inflammatory keratosis such as psoriasis, atopic dermatitis and contact dermatitis, (2) Chronic such as rheumatoid arthritis, systemic lupus erythematosus (SLE) and Behcet's disease Autoimmune diseases that are inflammatory diseases, (3)
Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, (4)
Inflammatory liver diseases such as hepatitis B, hepatitis C, alcoholic hepatitis, drug allergic hepatitis, (5) inflammatory kidney diseases such as glomerulonephritis, (6) inflammatory respiratory diseases such as bronchitis, ( 7) stomatitis, (8) vocal corditis, and (9) inflammation that occurs when using an artificial organ or an artificial blood vessel.

Next, the effects of the medicament of the present invention will be described with reference to test examples.

[Test Examples] Test Example 1 Helicobacter pylori adhesion inhibition test Bacteria used: Helicobacter pylori standard strain NCTC 11637 37 ° C, microaerobic condition, Brain Heart Infusion agar medium containing 10% horse deciliated blood (JP (Obtained from Mizu Pharmaceutical Co., Ltd.) for 3 to 4 days.

Cells used: Human gastric cancer cell line MKN45 (obtained from Institute for Immunity and Biology) 37 ° C., 5% CO ₂ , 10% FCS-containing RPMI
The cells were cultured in 1640 medium (obtained from Nissui Pharmaceutical Co., Ltd.).

Gangliosides used: G _D1a , G _D1b , G
_T1b, G _M1 inner esters and G _{M 1} (manufactured by Research Biochemicals International, Inc.), (manufactured by Snow Brand Milk Products (Ltd.)) G _D3 and G _M3, asialo G _M1 (Sigma), O - acetyl -G _D3 the (Advanced immuno Chemical Co.) and G _T1a (manufactured by E. Y. Laboratories) human gastric cancer cell line MKN45, 10 ⁴ ~10 ⁵ cells / well and made as Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co., And inoculated into a 96-well microtiter plate. This is carried out at 37 ° C. and 5% CO ₂ under
After culturing for one day, the medium was replaced with RPMI1640 medium.
Helicobacter pylori 10 ⁷ -10 ⁸ CFU and 12
Each solution mixed with a ganglioside solution at 5, 250 or 500 μg / ml was preincubated at 37 ° C. for 30 minutes. 100 μl of the ganglioside solution containing Helicobacter pylori was added to each well, and the cells were cultured at 37 ° C. and 5% CO ₂ for 4 hours. The culture supernatant was used for measurement of the amount of interleukin-8 produced in Test Example 2, and the remaining plate was used for an adhesion inhibition test. First, each well was washed with PBS and fixed with a formalin solution for 1 hour. After washing with PBS, 100 μl of a Helicobacter pylori antibody prepared by immunizing a mouse was added, and the mixture was incubated at 37 ° C. for 1 hour. This is P
After washing with BS, 50 μl of peroxidase-labeled mouse immunoglobulin (manufactured by CAPPEL, USA) was added, and the mixture was allowed to stand at room temperature for 1 hour. Next, each well was thoroughly washed with PBS, and then TMB peroxidase
Substrate kit (TMBPeroxidase Substrate Ki
t, Bio-Rad (manufactured by Bio-Rad), and the absorbance at 450 nm was measured.

The inhibition rate (%) was calculated by the following equation (I).

[0029]

(Equation 1)

In the formula, the absorbance of ganglioside refers to the absorbance when a ganglioside solution containing Helicobacter pylori is added to a well. The absorbance when using a pylori solution (containing 10 ⁷ to 10 ⁸ CFU, excluding ganglioside) is shown. The absorbance of PBS is the absorbance when using PBS instead of the ganglioside solution containing Helicobacter pylori. Is shown.
Therefore, the absorbance of ganglioside is the MKN45 of Helicobacter pylori when both ganglioside and Helicobacter pylori are added to MKN45 cells.
The degree of adhesion to cells, that is, the degree of inhibition of ganglioside adhesion, the absorbance of Helicobacter pylori refers to the degree of adhesion of Helicobacter pylori to MKN45 cells, and the absorbance of PBS refers to the non-specificity of a solvent (PBS). Means the degree of typical adhesion.

The results are shown in Table 2.

[0032]

[Table 3]

As shown in Table 2, ganglioside was dose-dependently determined from the Helicobacter pylori human gastric cancer cell line MK.
Inhibition of adhesion to N45 cells.

Test Example 2 Measurement of Interleukin-8 Production Amount The culture supernatant of the microtiter plate obtained in Test Example 1 was centrifuged (10,000 g, 10 minutes), and the supernatant was recovered. Interleukin-8 production in Quanticaine Human Elisa Kit (Quan
Using tikine Human ELISA KIT (trade name, manufactured by R & D systems)
It was measured by the SA method. At the same time, the absorbance of the interleukin-8 standard was measured, and the supernatant was used to determine the amount of interleukin-8 produced in the culture supernatant (μg
/ Ml).

The inhibition rate (%) was calculated by the following equation (II).

[0036]

(Equation 2)

In the formula, the production amount of ganglioside refers to the production amount of interleukin-8 from MKN45 cells when a ganglioside solution containing Helicobacter pylori is added to a well, and the production amount of Helicobacter pylori is Instead of a ganglioside solution containing Helicobacter pylori, a Helicobacter pylori solution (10
⁷ to 10 ⁸ CFU-containing, excluding ganglioside), the production of interleukin-8 when PBS was used.
Means the amount of interleukin-8 produced when PBS is used instead of the ganglioside solution containing Helicobacter pylori. Therefore, the amount of ganglioside produced was determined by adding MKN45 cells together with ganglioside and Helicobacter pylori.
The amount of interleukin-8 produced from N45 cells, that is, the degree of inhibition of ganglioside interleukin-8 production, and the amount of production of Helicobacter pylori is the amount of interleukin-8 produced from MKN45 cells by stimulation of Helicobacter pylori. And the amount of PBS produced means the amount of interleukin-8 produced from MKN45 cells by PBS stimulation.

The results are shown in Table 3.

[0039]

[Table 4]

As shown in Table 3, ganglioside inhibited the production of interleukin-8 from human gastric cancer cell line MKN45 in a dose-dependent manner.

Test Example 3 Interleukin-8 Production Inhibition Test Cell used: Human gastric cancer cell line MKN45 Cultured in RPMI1640 medium containing 10% FCS.

Ganglioside to be used: _GD3 human gastric cancer cell line MKN45 was prepared in Dulbecco's modified Eagle's medium at a concentration of 10 ⁴ to 10 ⁵ cells / well.
Test Example 1 by inoculating a 6-well microtiter plate
The culture was performed in the same manner as described above. Two days later the medium was changed and 125, 250 and 500 μl preincubated for 30 minutes.
Add 100 μl of each _gD3 solution at g / ml to each well,
The cells were cultured at 37 ° C. and 5% CO ₂ for 4 hours. The culture supernatant of the microtiter plate is centrifuged (10,000
g, 10 minutes), and the supernatant was collected. The amount of interleukin-8 produced in the supernatant was determined by ELISA using the Quanticain Human Elisa Kit as in Test Example 2.
It was measured by Method A. At the same time, the absorbance of the interleukin-8 standard was measured, and the supernatant was used to determine the amount of interleukin-8 produced in the culture supernatant (μg /
ml).

An inhibition rate (%) was produced by the following equation (III).

[0044]

(Equation 3)

[0045] In the formula, the production amount of G _D3 shows the interleukin-8 production amount when adding the G _D3 solution to the well, the production amount of PBS, use of PBS instead of the G _D3 solution 3 shows the amount of interleukin-8 produced when assembling. Therefore, production amount by G _D3 denotes interleukin-8 production level from MKN45 cells by stimulation of G _D3, a production amount by PBS means interleukin-8 production level from MKN45 cells by stimulation of PBS .

Table 4 shows the results.

[0047]

[Table 5]

[0048] Table 4 in G _D3 as shown, even if not applied Helicobacter pylori, in a dose-dependent manner inhibited interleukin-8 production from human gastric cancer cell line MKN45.

[0049]

EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Capsules were prepared by filling capsules with a mixed ingredient containing 0.2 mg of the active ingredient according to the following formulation.

(Components) (mg) G _D3 0.2 Lactose 100 Corn Starch 60 Crystalline Cellulose 18 Magnesium Stearate 2

[0052]

According to the present invention, it is possible to inhibit the adhesion of bacteria to the mucous membrane, which is the first stage of gastric mucosal damage caused by Helicobacter pylori, and further inhibit the production of interleukin-8.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location A61K 31/70 ACK A61K 31/70 ACK ACL ACL ACS ACS ACV ACV ADA ADA AED AED

Claims (13)

[Claims] 1. A pharmaceutical comprising ganglioside as an active ingredient. 2. The inhibitor of Helicobacter pylori adhesion according to claim 1, which comprises ganglioside as an active ingredient. 3. The method of claim 1, wherein the ganglioside is G _D3 , G _D1a ,
_{G D1b, G T1b, G M1} , G M1 inner esters, asialo G _M1, G _M3, O - acetyl _{-G D3, G T1a, G D2} ,
Helicobacter pylori adhesion inhibitor according to claim 2, wherein selected from the group consisting of G _M2, G _M1b and G _Q1b. 4. The method of claim 1, wherein the ganglioside is G _D3 , G _D1a ,
_{G D1b, G T1b, G M1} , G M1 inner esters, asialo G _M1, G _M3, O - Helicobacter pylori adhesion inhibitor according to claim 2, wherein is selected from the group consisting of acetyl -G _D3 and G _T1a. 5. The interleukin-8 production inhibitor according to claim 1, comprising ganglioside as an active ingredient. 6. The method of claim 1, wherein the ganglioside is G _D3 , G _D1a ,
_{G D1b, G T1b, G M1} , G M1 inner esters, asialo G _M1, G _M3, O - acetyl _{-G D3, G T1a, G D2} ,
G _M2, interleukin-8 production inhibitor of claim 5 selected from the group consisting of G _M1b and G _Q1b. 7. The method according to claim 7, wherein the ganglioside is G _D3 , G _D1a ,
_{G D1b, G T1b, G M1} , G M1 inner esters, asialo G _M1, G _M3, O - interleukin-8 production inhibitor of claim 5 selected from the group consisting of acetyl -G _D3 and G _T1a. 8. The anti-ulcer agent according to claim 1, comprising ganglioside as an active ingredient. 9. The method of claim 8, wherein the ganglioside is G _D3 , G _D1a ,
_{G D1b, G T1b, G M1} , G M1 inner esters, asialo G _M1, G _M3, O - acetyl _{-G D3, G T1a, G D2} ,
Antiulcer agent according to claim 8, wherein is selected from the group consisting of G _M2, G _M1b and G _Q1b. 10. The method of claim 1, wherein the ganglioside is G _D3 , G _D1a , G
_D1b , G _T1b , G _M1 , G _M1 inner ester, Asialo G
_M1, G _M3, O - antiulcer agent according to claim 8, wherein is selected from the group consisting of acetyl -G _D3 and _{G T1a.} 11. The therapeutic agent for gastritis according to claim 1, comprising ganglioside as an active ingredient. 12. The method of claim 11, wherein the ganglioside is G _D3 , G _D1a ,
_{G D1b, G T1b, G M1} , G M1 inner esters, asialo G _M1, G _M3, O - acetyl _{-G D3, G T1a, G D2} ,
Gastritis therapeutic agent according to claim 11, wherein selected from the group consisting of G _M2, G _M1b and G _Q1b. 13. The method of claim 11, wherein the ganglioside is G _D3 , G _D1a , G
_D1b , G _T1b , G _M1 , G _M1 inner ester, Asialo G
_M1, G _M3, O - gastritis therapeutic agent according to claim 11, wherein selected from the group consisting of acetyl -G _D3 and G _T1a.