JP2019026629A - Anti-helicobacter pylori agent - Google Patents

Abstract

To provide a new anti-Helicobacter pylori agent.SOLUTION: An anti-Helicobacter pylori agent contains platinum colloid.SELECTED DRAWING: None

Description

  The present invention relates to an anti-pylori agent and a preventive agent, a therapeutic agent, or a recurrence preventive agent for diseases involving H. pylori.

  Helicobacter pylori (hereinafter referred to as “H. pylori”) is a gram-negative microaerobic spiral gonococci that infects the human gastric mucosa and causes causes such as gastritis, gastric ulcer, duodenal ulcer, gastric cancer, and duodenal cancer. It is attracting attention as one.

  It has been reported that the pathogenic protein CagA derived from Helicobacter pylori plays an important role in the mechanism by which Helicobacter pylori causes an inflammatory reaction and cell proliferation in the human gastric mucosa (Non-patent Document 1). .

  Specifically, the pathogenic protein CagA possessed by Helicobacter pylori is injected into gastric epithelial cells via a needle-like IV secretion device on the surface of Helicobacter pylori. CagA is then tyrosine phosphorylated by intracellular Src family tyrosine kinase, which forms a complex with SHP2 (Src homology 2-containing protein tyrosine phosphatase) known as a human cancer protein, and activates SHP2. It has been reported that activated SHP2 generates an abnormal proliferation signal via the Erk-MAP kinase pathway, disturbs intracellular signals, and promotes canceration (Non-patent Documents 2 and 3). ).

Cell Host Microbe 12 (1): 20-33 (2012) Cell Host Microbe 15 (3): 306-316 (2014) Scientific Reports 5: 15749 (2015)

  Generally, antibiotics such as penicillin, cephalosporin, tetracycline, and macrolide are known as antibacterial agents against H. pylori. However, although these antibiotics have high antibacterial activity, they may cause side effects such as hypersensitivity, diarrhea, stomatitis, and taste disorders. It may cause serious side effects such as In addition, problems such as the emergence of Helicobacter pylori that has acquired resistance to these antibiotics have arisen.

  In view of such circumstances, development of a new anti-pylori agent is awaited. Therefore, an object of the present invention is to provide a new anti-pylori agent.

  As a result of diligent investigations, the present inventors have found that platinum colloid has an anti-pylori action, and further, by continuously administering an anti-pylori agent containing platinum colloid in vivo, in the gastric mucosal epithelium. It was found that the expression ratio of tyrosine phosphorylated CagA can be suppressed, and the present invention has been completed.

That is, the present invention provides the following [1] to [7].
[1] An anti-pylori agent containing platinum colloid as an active ingredient.
[2] The anti-pylori agent according to [1], which is a liquid agent having a platinum colloid concentration of 0.08 to 16 mM.
[3] The anti-pylori agent according to [1], further comprising a palladium colloid.
[4] The anti-pylori agent according to [3], wherein the molar ratio of platinum colloid to palladium colloid is 1 to 10: 1 to 30.
[5] The anti-pylori agent according to [3], which is a liquid preparation having a platinum colloid concentration of 0.08 to 16 mM and a palladium colloid concentration of 0.1 to 30 mM.
[6] The anti-H. Pylori agent according to [1] to [5], which is a prophylactic agent, a therapeutic agent, or a recurrence preventive agent for a disease involving H. pylori.
[7] The anti-pylori agent according to [6], wherein the disease associated with H. pylori is gastric ulcer, duodenal ulcer, gastritis, gastric cancer or duodenal cancer.

  The anti-pylori agent of the present invention exhibits an anti-pylori action and further reduces the expression rate of tyrosine-phosphorylated CagA that is a pathogenic protein in the gastric mucosa by continuous administration in vivo.

It is a photograph of the viable count plate at the start of storage of the control solution. It is a photograph of the viable count plate after 1 minute of storing the test solution. It is a photograph of the viable count plate after 1 minute of storing the control solution. It is a photograph of the viable count plate 3 minutes after storing the test solution. It is a photograph of the viable count plate 3 minutes after storing the control solution. It is a photograph of the viable count plate after 5 minutes of storing the test solution. It is a photograph of the viable count plate after 5 minutes of storing the control solution. It is the graph which showed the expression rate reduction effect of the tyrosine phosphorylated CagA in the gastric mucosa after continuous administration of platinum palladium colloid to a rat for 28 days.

  Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

  The anti-pylori agent which concerns on one Embodiment of this invention contains a platinum colloid.

  Various methods for producing platinum colloids are known (for example, JP-B-57-43125, JP-B-59-120249, JP-B-2-43801, JP-A-9-225317, JP-A-10 No. 176207, etc.), those skilled in the art can appropriately prepare platinum colloid by referring to the above method. The platinum colloid production method is not particularly limited as long as it is pharmacologically or physiologically acceptable. For example, it is known as a chemical method known as a precipitation method or a metal salt reduction reaction method, or as a combustion method. The physical method etc. which can be used can be utilized. As a method for producing platinum colloid, it is preferable to use a combustion method from the viewpoint of enhancing the antibacterial action of Helicobacter pylori.

  As the combustion method, for example, a mixed solution of a chloroplatinic acid solution and a lower alcohol, and hydrogen gas are sent from separate supply systems, and the burned hydrogen gas flame is mixed with the above mixed solution 830 to 830. A method of producing a platinum colloid by burning at 870 ° C. and blowing the combustion flame into a liquid dispersion medium in which a vortex that reaches near the bottom of the colloid production tank is generated.

In the case where the anti-pylori agent according to the present embodiment is a liquid agent, the concentration of the platinum colloid is not particularly limited, and is appropriately determined depending on the type of platinum colloid used, the use of the anti-pylori agent, the formulation form, the method of use, etc. Is set.
The concentration of the colloidal platinum in the solution is preferably 0.08 to 16 mM from the viewpoint of exhibiting anti-H. Pylori action in vivo and less likely to cause side effects even when continuously administered. 10 mM is preferable, and 0.4 to 8 mM is more preferable.

  The anti-pylori agent which concerns on another embodiment of this invention contains a platinum colloid and a palladium colloid.

  Various methods for producing palladium colloids are known (for example, JP-B-57-43125, JP-B-59-120249, JP-B-2-43801, JP-A-9-225317, JP-A-10 No. 176207, etc.), those skilled in the art can appropriately prepare palladium colloid by referring to the above method. The method for producing palladium colloid is not particularly limited as long as it is pharmacologically or physiologically acceptable. For example, it is known as a chemical method known as a precipitation method or a metal salt reduction reaction method, or a combustion method. The physical method etc. which can be used can be utilized. As a method for producing palladium colloid, it is preferable to use a combustion method from the viewpoint of preventing deterioration due to oxidation of platinum.

  As a combustion method, for example, a mixed solution of a palladium chloride solution and a lower alcohol, and hydrogen gas are sent from separate supply systems, and the burned hydrogen gas flame is mixed with the above mixed solution, 630-670. Examples include a method of producing palladium colloid by burning at a temperature of 0 ° C. and blowing the combustion flame into a liquid dispersion medium that generates a vortex that reaches the bottom of the colloid production tank.

  The molar ratio of the platinum colloid and the palladium colloid contained in the anti-pylori agent according to the present embodiment is not particularly limited, and the type and concentration of the platinum colloid and the palladium colloid used, the use of the anti-pylori agent, and the preparation form It is set as appropriate according to the method of use. The molar ratio of the platinum colloid and the palladium colloid contained in the anti-pylori agent is preferably platinum colloid: palladium colloid = 1-10: 1-30, from the viewpoint of effectively exhibiting anti-pylori action. Platinum colloid: palladium colloid = 1-3: 1-9 is more preferable.

In the case where the anti-pylori agent according to this embodiment is a liquid agent, the concentration of palladium colloid is not particularly limited, the type of palladium colloid used, the type and concentration of platinum colloid used together, It is appropriately set depending on the use, formulation form, method of use and the like.
The concentration of the colloidal palladium in the liquid is preferably 0.1 to 30 mM, preferably 0.4 to 16 mM, from the viewpoint of preventing deterioration due to oxidation of platinum and preventing side effects even when continuously administered. It is more preferable that it is 0.8-12 mM.

  The anti-pylori agent according to this embodiment may further contain a surfactant.

  The surfactant is not particularly limited as long as it is pharmacologically or physiologically acceptable, and is a nonionic surfactant, an amphoteric surfactant, an anionic surfactant, or a cationic surfactant. Any of these may be used. As the surfactant to be contained in the anti-pylori agent according to this embodiment, it is preferable to contain a nonionic surfactant from the viewpoint of effectively exhibiting the anti-pylori effect.

Nonionic surfactants include, for example, monolauric acid POE (20) sorbitan (polysorbate 20), monopalmitic acid POE (20) sorbitan (polysorbate 40), monostearic acid POE (20) sorbitan (polysorbate 60), tri PEO sorbitan fatty acid esters such as stearic acid POE (20) sorbitan (polysorbate 65), monooleic acid POE (20) sorbitan (polysorbate 80); POE (40) hydrogenated castor oil (polyoxyethylene hydrogenated castor oil 40), POE ( 60) POE hydrogenated castor oil such as hydrogenated castor oil (polyoxyethylene hydrogenated castor oil 60); POE alkyl ether such as POE (9) lauryl ether; POE-POP alkyl ether such as POE (20) POP (4) cetyl ether POE (196) POP (67) glycol (poloxamer 407, Pluronic F127), POE (200) POP (70) glycol and other polyoxyethylene / polyoxypropylene block copolymers; polyoxyethylene castor oil 35, polyoxyethylene Examples include castor oil 40, polyoxyethylene castor oil 50, polyoxyethylene castor oil 60, and the like.
In the anti-pylori agent according to this embodiment, the nonionic surfactant may be used singly or in combination of two or more.

  The anti-pylori agent according to this embodiment may have any shape such as an individual, a liquid, a paste, and the like, and includes tablets (plain tablets, dragees, effervescent tablets, film-coated tablets, chewable tablets, lozenges, and the like. ), Capsules, pills, powders (powder), fine granules, granules, liquids (including suspensions, emulsions, syrups, etc.), pastes and the like. If these various preparations are pharmacologically or physiologically acceptable, additives such as excipients, lubricants, binders, disintegrants, stabilizers and suspending agents are mixed, It can be manufactured by a known method.

  The anti-pylori agent according to this embodiment may be administered or ingested to a human or a non-human mammal. The dose (intake amount) of the anti-pylori agent according to the present embodiment is preferably 0.06 to 6 mg per day per adult in terms of the mass of platinum colloid, for example 0.1 to 4 mg. It is more preferable that it is 0.3-3 mg. The dose can be appropriately determined according to the state, age, etc. of the individual.

  The anti-pylori agent according to this embodiment may be administered orally (ingested) or parenterally, but is preferably administered orally. As long as the value converted into the mass of the platinum colloid per day is in the above range, the anti-pylori agent may be administered once a day or may be divided into a plurality of times a day.

  When the anti-pylori agent according to the present embodiment is continuously administered (taken), the effect of reducing the expression ratio of CagA in which tyrosine is phosphorylated is further enhanced. The anti-pylori agent according to this embodiment is preferably administered (ingested) continuously for 2 weeks or more, more preferably administered (ingested) continuously for 3 weeks or more, and continuously for 4 weeks or more. More preferably, it is administered (taken).

  The anti-pylori agent according to this embodiment can reduce the expression rate of tyrosine phosphorylated CagA, which is a pathogenic protein that causes inflammatory reaction, cell proliferation, etc. in human gastric mucosal epithelium, resulting in gastritis, gastric ulcer, duodenal ulcer It is effective as a preventive agent, therapeutic agent, or recurrence preventive agent for gastric cancer, duodenal cancer and the like.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Test Example 1: Anti-H. Pylori action]
<Test strain>
As a test strain, Helicobacter pylori JCM 12093 was used.

<Culture conditions of test bacteria>
The test bacterium was treated with 5% equine defibrinated blood added Blood Agar Base No. 2 (manufactured by Oxoid) was cultured at 37 ° C. ± 1 ° C. over 3-4 days. Then, equine defibrinated blood added Blood Agar Base No. 2 was further added, followed by further microaerobic culture over 3 to 4 days at 37 ° C. ± 1 ° C. The test bacteria were suspended in physiological saline and prepared so that the number of bacteria was 10 ⁸ to 10 ⁹ / mL, and this was used as a test bacterial solution.

<Test operation>
Sample: Platinum palladium colloid solution (PAPLAL (registered trademark) (1.2 mg of platinum colloid and 1.8 mg of palladium colloid are contained in 6 ml of aqueous solution): manufactured by Toyo Kosei Pharmaceutical Co., Ltd.)
Control: physiological saline 10 mL of a sample was inoculated with 0.1 mL of a test bacterial solution, and this was used as a test solution. As a control, 10 mL of the control was inoculated with 0.1 mL of the test bacterial solution, and this was used as a control solution.
The test solution and the control solution are stored at 20 ° C. ± 1 ° C., respectively, and after 1 minute, 3 minutes, and 5 minutes, the stored test solution and control solution are diluted 10 times with SCDLP medium (manufactured by Nippon Pharmaceutical Co., Ltd.). The viable cell count was measured.

<Test results>
Table 1 is a table showing the results of measuring the viable cell counts after 1 minute, 3 minutes and 5 minutes after storing the test solution and the control solution, respectively.
Further, the reduction rate (%) in Table 1 is a value indicating the ratio of the number of viable bacteria in the test solution, assuming that the number of viable bacteria in the control solution is 100%.

  From Table 1, when a specimen containing platinum colloid and palladium colloid is inoculated into H. pylori, which is a test bacteria, H. pylori is observed in any of the test solutions after 1 minute, 3 minutes and 5 minutes after storage. It can be seen that the number of viable bacteria decreases.

[Test Example 2: Tyrosine phosphorylated CagA expression level reduction effect]
<Animal preparation>
Ten 6-week-old male PMSS1 (H. pylori PMSS1 strain) positive SD rats (Charles River) were acclimated for 7 days in a test environment. As a breeding environment, 5 animals are housed in metal cages at room temperature 25 ° C ± 1 ° C, humidity 20% ± 2% or less, and 12 hours a day. It was.

<Composition of test group>
Seven-week-old rats were used for the test. Ten rats were divided into a control group and a treatment group, each with 5 rats. Saline was administered to the control group, and PAPLAL 600 μl / KgBW was administered to the administration group as a test substance once a day, and this was continued for 28 days.
After 28 days, the treated group compared with the control group in general condition, mortality, body weight change, hematological examination, blood biochemical examination, organ weight, gross pathological examination, histological pathological examination There were no adverse events and no toxicological or non-physiological effects of normal administration.

<Expression ratio of tyrosine phosphorylated CagA>
The gastric mucosa tissues excised from the rats of the control group and the administration group were individually homogenized according to a conventional method and then centrifuged, and the supernatant (soluble fraction) was collected.
Anti-CagA polyclonal antibody (Austral Biologicals, San Ramon, Ca, USA) and anti-phosphotyrosine antibody (Upstate Biotechnology Inc., Lake Placid, NY, USA) were used as primary antibodies for immunoprecipitation and immunoblotting. .
The supernatant was adsorbed on Sepharose 4B capable of adsorbing protein, and after washing, anti-CagA antibody or anti-phosphotyrosine antibody was added and reacted at 4 ° C. for 60 minutes. After thorough washing, the mixture was reacted with an HRP-labeled second antibody for 1 hour, developed with hydrogen peroxide-added DAB (3,3′-diaminobenzidine), and the color intensity was measured. Moreover, the evaluation of the expression rate of tyrosine phosphorylated CagA was evaluated from the color development rate when using an anti-CagA antibody and an anti-phosphorylated tyrosine antibody (FIG. 3).
The results are shown in Table 3.

From Table 3, it can be seen that in the PAPLAL administration group, the expression ratio of tyrosine phosphorylated CagA in the whole CagA is significantly reduced as compared with the control group.
If the platinum colloid only exhibits anti-H. Pylori action in vivo, the total amount of CagA injected from H. pylori is only reduced. However, in Test Example 2, in the PAPLAL administration group, the expression ratio of tyrosine-phosphorylated CagA in the entire CagA is decreased, and it can be seen that the platinum colloid also has an action to suppress tyrosine phosphorylation of CagA.

  Tyrosine phosphorylated CagA acts as a pathogenic protein such as gastritis, gastric ulcer, duodenal ulcer, gastric cancer, and duodenal cancer. For this reason, the platinum colloid which has the effect | action which reduces the expression rate of the tyrosine phosphorylated CagA which occupies for the whole CagA in the living body is effective as a preventive agent of the said disease in which tyrosine phosphorylated CagA is concerned, a therapeutic agent, or a recurrence preventive agent. .

Claims (7)

  An anti-pylori agent containing platinum colloid as an active ingredient.   The anti-pylori agent of Claim 1 which is a liquid agent whose density | concentration of a platinum colloid is 0.08-16 mM.   Furthermore, the anti-pylori agent of Claim 1 containing a palladium colloid.   The anti-pylori agent of Claim 3 whose molar ratio of a platinum colloid and a palladium colloid is 1-10: 1-30.   The anti-pylori agent of Claim 3 which is a liquid agent whose density | concentration of a platinum colloid is 0.08-16 mM, and whose density | concentration of a palladium colloid is 0.1-30 mM.   The anti-H. Pylori agent according to any one of claims 1 to 5, which is a prophylactic agent, a therapeutic agent, or a recurrence preventive agent for a disease involving H. pylori.   The anti-H. Pylori agent according to claim 6, wherein the disease associated with Helicobacter pylori is gastritis, gastric ulcer, duodenal ulcer, gastric cancer or duodenal cancer.