WO2000054800A1

WO2000054800A1 - Preventives/remedies for digestive diseases

Info

Publication number: WO2000054800A1
Application number: PCT/JP2000/001442
Authority: WO
Inventors: Shigeki Kimura
Original assignee: Amano Enzyme Inc.
Priority date: 1999-03-12
Filing date: 2000-03-09
Publication date: 2000-09-21
Also published as: JP2003040799A

Abstract

Preventives/remedies for digestive diseases containing, as the active ingredient, at least one of an enzyme hydrolyzing sugar chains having fucose and another enzyme decomposing ammonia. A bacteriostatic method comprising inhibiting or preventing the growth of Helicobacter pylori in a strongly acidic environment by using the above preventives/remedies. As the above enzymes, fucosidase and histidase are particularly preferable. Thus, the conditions for fixation or growth of H. pylori in the strongly acidic environment are inhibited and thus the growth of H. pylori can be inhibited or prevented.

Description

Description Title of the Invention Gastrointestinal Disease Prevention / Treatment Technical Field

The present invention relates to a preparation for preventing or treating gastrointestinal diseases caused by Helicobacter pylori (hereinafter, referred to as "H. pylori"). The prophylactic / therapeutic agent for gastrointestinal diseases of the present invention targets inflammation or ulcer of the digestive tract in a human or non-human mammal. Background art

As a therapeutic drug for digestive ulcer, H ₂ - blow Tsu car in a caulking histidinol down, Ranichiji down, off § Mochijin like are utilized. In addition, omeprazole II, which is a protein pump inhibitor (PPI), and a gastric mucosa protective agent are also used. Due to the superior effects of these treatments, gastrointestinal ulcer surgery has been dramatically reduced.

However, gastrointestinal ulcers often recur once they have been healed, and many studies have been made to determine the cause. As a result, J. R. Warren, B. J. Marshal and colleagues found a high rate of H. pylori in the gastric mucosa of patients with gastrointestinal ulcers and gastritis, and succeeded in isolating and culturing them (Lanset). , 1273-5, 1983).

Helicobacter pylori is a gram-negative, slightly aerobic bacillus that produces ammonia from urea due to its strong protease activity. The reason that H. pylori can live in the strongly acidic environment in the stomach is thought to be that the produced ammonia neutralizes the stomach acid. This ammonia can cause many disorders of the gastric mucosa and may be associated with gastritis, gastric ulcer, gastric cancer, and malignant lymphoma of the stomach. From the above, it has been recognized that removing H. pylori is an effective treatment for gastrointestinal ulcers. In the past, antibiotics were administered, for example, to remove H. pylori from the digestive tract. Some antibiotics were used alone, and some were used in combination with PPIs. Bismuth preparations, tetracycline and metronidazole were sometimes used in combination. Japanese Patent Application Laid-Open No. 7-13816 / 66 discloses that fucoli as an active ingredient for removing H. pylori A method of utilizing idan is disclosed. Japanese Patent Application Laid-Open No. 9-241173 discloses a method using lactic acid as an active ingredient for the same purpose.

However, in the method of administering antibiotics, the dosage tends to be large and the administration period tends to be long. Therefore, there are concerns about the emergence of antibiotic-resistant bacteria and side effects such as taste disturbance, vomiting, and diarrhea. The effects of using fucoidan or lactic acid bacteria as active ingredients have not yet been confirmed. In addition, each of the above methods aims to remove H. pylori that has already settled in the stomach, and it is not known whether it is effective in preventing H. pylori from colonizing the stomach.

On the other hand, as a substance that inhibits colonization of H. pylori in the stomach, Futuin II-secreted IgA is known. Futuin inhibits H. pylori from colonizing the gastric mucosa via sialic acid. Secreted IgA inhibits H. pylori from colonizing the gastric mucosa via fucosylated sugar chains. However, it is still difficult to put these fixation inhibitors to practical use due to the shortage of supply. Disclosure of the invention

The present invention provides a prophylactic / therapeutic agent for gastrointestinal diseases that is effective for preventing colonization of H. pylori on the digestive tract or removing already established H. pylori. It also provides a bacteriostatic method for H. pylori.

According to the study of the present inventors, sugar chains containing fucose appearing on the gastric mucosa are strongly related to colonization of H. pylori in the stomach. As described above, the H. pylori in the stomach requires ammonia produced by the H. pylori itself. The present inventors have found that removal of fucose monmona using an enzyme significantly suppresses colonization and inhabitation of H. pylori in the stomach. When the colonization and inhabitation of H. pylori in the stomach is suppressed, the colonization and inhabitation of H. pylori in other digestive organs such as the duodenum are suppressed as a result. Enzymes that are proteins have few harmful side effects such as antibiotics, and there is no possibility that resistant bacteria will appear.

The prophylactic / therapeutic agent for gastrointestinal diseases of the present invention comprises, as an active ingredient, an enzyme capable of removing H. pylori from the digestive organ or a composition thereof. Further, the agent for preventing or treating gastrointestinal diseases of the present invention comprises an enzyme capable of removing fucose or ammonia or a composition thereof as an active ingredient. this As these enzymes, bacteria, molds, yeasts, actinomycetes, basidiomycetes, plants or animals can be used. One condition of enzymes that can be used is to maintain sufficient enzyme activity in the low pH range in the stomach. Another condition of the enzymes that can be used is that they do not cause adverse side effects on the digestive tract of human or non-human mammals. One group of enzymes used in the present invention are hydrolases, particularly enzymes that hydrolyze sugar chains, especially enzymes that hydrolyze sugars containing fucose. Fucosidase can be exemplified as the most suitable enzyme. Fucosidase is an enzyme that has the action of hydrolyzing and releasing fucose located at the non-reducing end of a sugar chain, regardless of its binding position. As the fucosidase, for example, those derived from the internal organs of animals such as the liver or the liver of a marine snail or the liver of a mouse can be used. Fucosigsease derived from microorganisms such as Penicillium multicolor (IFO-6042) can be more preferably used because it is easily available.

Another group of enzymes for use in the present invention is the ammonia-degrading enzymes, especially ammonia lyases, especially histidases. Histidase is an enzyme that catalyzes the reaction to produce histidine from peroxidate and ammonia. Histidase is derived, for example, from the liver of animals or from microorganisms such as Pseudomonas fluorescence [The Enzyme (3rd ed.), Vol. 7, 75-166 (1972)]. Can be used.

Each of the above enzymes derived from microorganisms can be obtained by culturing the microorganism by a known method and purifying the enzyme from the culture. The degree of purification may be such that it has an enzyme activity suitable for the purpose of use, ie, exerts its action in the digestive tract.

The preventive / therapeutic agent for gastrointestinal diseases of the present invention comprises one or more of the above-mentioned hydrolase groups, and / or one or more of the above-mentioned ammoniolytic enzymes, or As an active ingredient.

The above-mentioned hydrolase and Z or ammonia degrading enzyme can be used alone or in combination with other pharmaceutical preparations or by preparing a mixture with other medicines. Can also be taken. And the above other pharmaceutical, H ₂ - blow Tsu car in a sheet main Ji di emissions, Ranichiji down or off § waxy di emissions, PFI der Ruome bra tetrazole, Ru can be exemplified a Ineba film-protecting agent . The enzyme composition or a combination thereof with another drug can be made into various dosage forms according to a usual method. For example, they can be used in the form of powders, granules, liquids, solids, capsules, etc., and powders, tablets, capsules, and syrups are particularly preferred. In addition, enzymes and other drugs mentioned above are used as main agents, and excipients, binders, disintegrants, coating agents, lubricants, stabilizers, flavoring agents, flavoring agents, solubilizing agents, Commonly used auxiliaries such as suspending agents and diluents may be added. The enzyme composition can also be taken in advance by mixing it with various foods and the like.

The bacteriostatic method of the present invention comprises the steps of: administering a prophylactic / therapeutic agent for a gastrointestinal disease, which is a combination with the above-mentioned enzyme, enzyme composition or other medicine, to the digestive organ of a human or non-human mammal. It acts on bacteria. Preferably, the prophylactic / therapeutic agent for gastrointestinal disorders is taken before, between or after meals. This bacteriostatic method can prevent the colonization of H. pylori in the digestive tract of a human or non-human mammal, or can remove the colonized H. pylori. This bacteriostatic method is particularly effective in preventing or treating gastric or duodenal inflammation, ulcer or tumor caused by H. pylori.

The dosage of the above-mentioned hydrolase or ammonia-degrading enzyme varies depending on the type of human or non-human mammal and the type or degree of the target disease. For example, if humans take fucosidase and histidase, the dose can be 100 to 100,000 units per day, respectively.

Regarding the activity of fucosidase, the amount of enzyme that hydrolyzes 1 mol of PNPF (p-nitrophenol-L-fucopyrenoside) per minute at 37 ° C is 1 The unit was used. Regarding the activity of histidase, the amount of the enzyme that decomposes 1 g of L-histidine per minute at 37 ° C. was defined as 1 unit. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the best mode for carrying out the present invention will be described together with comparative examples. The present invention is not limited to these embodiments.

(Example 1)

In this example, fucosidase derived from Penicillium multicolor I F0-6042 or histidase derived from Shudomonas fluorescein were used. The dosage of the enzyme, both fucosidase and histidase, is 200 units / kg / mouse body weight / day. In the group receiving the histidase, 100 g of lococanet was administered per mouse per day.

The non-human mammal used was a male Danryu's mouse weighing 250-270 g. Five mice were prepared for the first group, and five mice for the fucosidase-administered group and the histidase-administered group were prepared for the second and third groups, respectively. Group 1: A group that receives an enzyme-free diet from before H. pylori inoculation until just before slaughter.

Group 2: A group that receives an enzyme-free diet before inoculation with H. pylori and an enzyme-containing diet until immediately before slaughter.

Group 3: A group that receives the enzyme-containing diet from 2 weeks before inoculation of H. pylori to immediately before slaughter.

After the mice in each of the above groups were anesthetized with ether, 20% acetic acid was injected into the mucosal epithelium of the stomach to form ulcers. 5 days after ^{lxl 0 8 CFU (colony forming unit} ) pyro Li bacteria of the (ATCC-43504) was inoculated into the stomach Ri by the oral intubation. After a further 7 days, the mice were sacrificed and their stomachs removed.

The excised stomach was immersed in 20 mL of 0.1 M phosphate buffer (pH 7) and washed. After the stomach was spread on the plate to confirm the occurrence of ulcer, the stomach was returned to the same buffer and homogenized.

The homogenized solution of each example in each of the above groups was diluted 10-fold with the same buffer, and a part of the diluted solution (0.1 ml in each case) was inoculated on Brucella agar medium. This Brucella agar medium contains 10% horse serum, 2.5 g ZmL of amphotericin B, 9 g ZmL of noncomicin, and 0.32 μg Polymixin B and 2,3,5—Including triphenyltetrachloride chloride. After inoculation into the medium, the cells were cultured at 37 ° C for 7 days, and the number of colonies of H. pylori was counted.

As a result, the number of colonies in the first group was 58,000-27,000. This number of colonies is defined as “fixing rate 100%”, and is used as a standard for evaluation. The number of colonies in the second group taking fucosidase was 27,000 = 6,900, and the retention rate was 47.212. / ₀ . The number of colonies in the histidase-administered group of the second group was 32,000, The fixing rate was 55 ± 8%. The number of colonies in the third group, fucosidase administration group, was 15, 00, 24, 600, and the retention rate was 26 = 8%. The number of colonies in the histidase-dose group of the third group was 29,000 to 26,900, and the retention rate was 50 = 12%.

(Example 2)

In the present example, male sand rats weighing 40 to 50 g were used. As the H. pylori, XCTCC11673: ATCC was used. The types of fucosidase and histidase used, and the dosages of those enzymes, are the same as in Example 1.

In the same manner as in Example 1, these sand rats were prepared by a predetermined number of solids in groups 1 to 3 to form ulcers, and 1 × 10 ⁸ CFU of the H. pylori described above was removed. I was inoculated. In this example, all the sand rats were fasted for one day before the inoculation of H. pylori, fasted for 4 hours after the inoculation, and were not ingested with water. Two weeks later, the rats were killed, and the same process as in Example 1 was performed thereafter. However, culture on Brucella agar medium was performed at 37 ° C. for 4 weeks.

In the result of counting the number of colonies, the number of colonies in the first group is 250, 0 00 Sat 80, 0 0 0 (Note: The numbers in Table 2 are crushed and cannot be read. Please check. This number of colonies is defined as “fixation rate 100%”. The number of colonies in the fucosidase treatment group in the second group was 145,000 ± 43,000, and the colonization rate was 58%, 11%. The number of colonies in the second group, histidase administration group, was 132,000 ± 36,000, and the retention rate was 532-14%. In the third group, the number of colonies in the fucosidase treatment group was 112,000 ± 41,000, and the retention rate was 45 = 10%. The number of colonies in the histidase-administered group of the third group was 133,000,29,000, and the retention rate was 55 ± 4%.

(Example 3)

In this example, KAT03 cells were used as cultured cells. Cultured cells were pre-incubated with the same fucosidase 1 unit / mL as in Example 1 and incubated for 30 minutes as Group 1, and incubated for 30 minutes without fucosidase. The result was designated as the second group. To 1 × 10 ⁶ cultured cells, 1 × 10 ⁸ H. pylori used in Example 2 was added, and the mixture was incubated at 37 ° C. for 30 minutes. Then 15% Sucrose solution was added to remove free H. pylori.

Next, a primary antibody (anti-H. Pylori-ephedra antibody) and a secondary antibody (FITC-anti-peacock-pig antibody) were added. After washing, the sample was subjected to a flow cytometry to measure the fluorescence intensity. As a result, the fluorescence intensity of the first group (n = 3) is 18, 0 00 = 3, 300, and the fluorescence intensity of the second group is 30, 0, 00-2, 600,000. Met. These fluorescence intensities are considered to be proportional to the degree of implantation of H. pylori on cultured cells, that is, the colonization rate. Assuming that the fixing rate of the second group is 100%, the fixing rate of the first group is 60%.上の Business availability

As described above, the prophylactic / therapeutic agent for gastrointestinal diseases and the method of taking the same according to the present invention can effectively treat or prevent inflammation or ulcer of the digestive tract in a human or non-human mammal. It can be.

Claims

The scope of the claims

1. A prophylactic or therapeutic agent for gastrointestinal diseases, characterized in that at least one of an enzyme that hydrolyzes a sugar chain containing fucose and an enzyme that degrades ammonia is contained as an active ingredient. Prevention and treatment of digestive diseases.

2. The method according to claim 1, wherein the enzyme that hydrolyzes a sugar chain containing fucose is a sugar chain hydrolase, and Z or the enzyme that degrades ammonia is ammonia lyase. The agent for preventing or treating digestive diseases according to claim 1.

3. The preventive / therapeutic agent for a gastrointestinal disease according to claim 2, wherein the sugar chain hydrolase is fucosidase and / or the ammonia lyase is a histidase.

4-The agent for preventing or treating gastrointestinal diseases according to any one of claims 1 to 3 inhibits reproduction of Helicicobacter pylori in a strongly acidic environment. A bacteriostatic method characterized by suppressing or preventing.