JP5035787B2 - α-1,4-linked N-acetylglucosamine-containing O-glycan-type sugar chain secretagogue and therapeutic / prophylactic agent for pathological conditions caused by Helicobacter pylori containing the same - Google Patents

Description

The present invention relates to the secretion of O-glycan-type sugar chains from cells, which are O-glycan-type sugar chains, and N-acetylglucosamine is bound to the non-reducing end portion by α-1, type 4 glycoside bonds. As a component that promotes the secretion of an α-1,4-linked N-acetylglucosamine-containing O-glycan-type sugar chain containing at least one selected from glucosamine and / or a glucosamine derivative as an active ingredient, and It relates to infection therapeutic prophylactic agent for Helicobacter pylori, containing.

  Helicobacter pylori (hereinafter referred to as Helicobacter pylori) is a gram-negative spiral short bacterium inhabiting the stomach reported by Marshall, Warren in 1983. The infection rate of Helicobacter pylori in Japan is estimated at about 50%, and more than 70% of people are carriers, especially in middle-aged and elderly people. H. pylori is known to be a major factor in the occurrence and recurrence of gastritis, gastric ulcer, duodenal ulcer and the like.

  For eradication of Helicobacter pylori, sterilization therapy with antibiotics is approved as a health practice. This drug is a combination of antibiotics, proton pump inhibitors, bismuth preparations and the like.

  Moreover, the component which shows the disinfection effect of Helicobacter pylori has also been found in food components. For example, Patent Document 1 below describes that glucuronic acid-containing fucoidan contained in seaweed and the like is useful as an adhesion inhibitor for Helicobacter pylori, and Patent Document 2 described below contains mucin derived from milk milk or chicken egg white as an active ingredient. And Helicobacter pylori fixation inhibitors are described.

  Furthermore, the following Patent Document 3 discloses a composition containing a polysaccharide having receptor activity against Helicobacter pylori, and optionally, an oligosaccharide receptor for Helicobacter pylori or an analog or derivative thereof and / or a gastric epithelial protective compound. A composition for use in the treatment or prevention of a disease state caused by the presence of Helicobacter pylori is described, and the use of chitosan as a polysaccharide is described.

H. pylori is found in the gastric mucosa of infected people. The gastric mucosa is composed of a mucus cell layer and a surface mucus gel layer composed of mucus secreting mucin secreted from the mucus cells, and the surface mucus gel layer is composed of mucus secreted from the surface mucus cells and the middle to deep layers It has a layered structure in which the mucus secreted from the gland mucous cells present in is stacked alternately without intermingling with each other. In the mucus layer composed of mucus secreted from the surface mucus cells, Le ^b (Lewisb sugar chain antigen) and sialyl Le ^x (sialyl Lewis x sugar chain antigen), which are known to have an affinity for H. pylori, are added. It is thought that Helicobacter pylori is used as a foothold for colonization in the gastric mucosa. On the other hand, Helicobacter pylori is rarely colonized in the mucus layer composed of the mucus secreted from the gland mucus cells existing in the middle to deep layers of the gastric mucosa, but the core bifurcated O-glycan type is in this mucus layer There is a unique sugar chain having a GlcNAcα1 → 4Galβ residue at the non-reducing end of the sugar chain. The O-glycan structure having this α-linked N-acetylglucosamine (αGlcNAc) at the non-reducing end is a very rare structure as a sugar chain, and the sugar chain having this structure is present in the gastric mucosa and the duodenal mucosa. It is hardly expressed in normal tissues other than Brunner's gland.

Recently, the present inventors have found that a unique sugar chain is present in mucus secreted from glandular mucus cells existing in the middle to deep layers of the gastric mucosa, and has α-1, type 4 glycoside linkage at the non-reducing end portion thereof. Newly discovered that the O-glycan-type sugar chain bound with N-acetylglucosamine has a strong antibacterial activity against H. pylori (see Non-Patent Document 1 below), and this unique sugar in the gastric mucosa. We reported that the chain acts as a protective factor against Helicobacter pylori infection.
JP 10-287571 A JP 2000-229865 A Special table 2004-537538 gazette M. Kawakubo et al., Science, Vol. 305, (2004) p1003-1006

  In the conventional technology for eradicating H. pylori with antibiotics, there are problems such as a decrease in the sterilization effect due to the appearance of resistant bacteria and the appearance of side effects due to drug administration.

Moreover, although the technique of the said patent documents 1-3 using the component in a food material is excellent in the point which can be taken in comfort, since it utilizes the effect | action which adsorb | sucks and discharge | releases Helicobacter pylori, However, it did not exhibit sufficient action and effect for the purpose of sterilizing H. pylori colonized in the gastric mucosa through the receptor molecular structures such as Le ^b and sialyl Le ^x having an affinity for.

  On the other hand, the technique using an O-glycan type sugar chain in which N-acetylglucosamine is bonded to the non-reducing end part by α-1, type 4 glycosidic bond in the non-reducing terminal part is expressed in the gastric mucosa. It is excellent in that it can be taken with peace of mind, but the method of administering it exogenously is not efficient for the purpose of demonstrating the inherent mucosal defense mechanism of the living body. There wasn't. In addition, mass production technology for O-glycan-type sugar chains in which N-acetylglucosamine is bonded to the non-reducing end portion by α-1, type 4 glycoside linkage, and α-1, type 4 glycoside bonding to the non-reducing end portion. Foods rich in O-glycan type sugar chains bound with N-acetylglucosamine are not well known.

  Therefore, the object of the present invention is to effectively suppress the effect of the inhibitory action on H. pylori of O-glycan type sugar chain in which N-acetylglucosamine is bonded to the non-reducing end part by α-1, type 4 glycoside bond in mucosal tissue. Therefore, it is intended to provide a technique for promoting secretion from the production cells, and to provide an agent for treating and preventing infection of H. pylori that is safer and more effective for the human body.

  As a result of diligent research to achieve the above object, the present inventors have found that glucosamine or a glucosamine derivative is an O-glycan type sugar chain, and a non-reducing end portion thereof has α-1, type 4 glycoside bonds. It has been found that the O-glycan-type sugar chain bound with N-acetylglucosamine has an action of promoting the secretion from cells, and the present invention has been completed.

That is, the α-1,4 linked N-acetylglucosamine-containing O-glycan-type sugar chain secretion promoter of the present invention is an O-glycan-type sugar chain, and α-1, As a component that promotes the secretion of O-glycan-type sugar chains bound to N-acetylglucosamine by a type 4 glycoside bond from cells, glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, N -Acetylglucosamine, N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, N-acetylchitohexaose, lactosamine, N-acetyllactosamine, galactosyllactosamine , At least one selected from galactosyl-N-acetyllactosamine Of glucosamine and / or a glucosamine derivative as an active ingredient.

  According to the α-1,4 linked N-acetylglucosamine-containing O-glycan type sugar chain secretion promoter of the present invention, the glucosamine and / or glucosamine derivative as an active ingredient is α-1,4 linked N -Acts on a cell that secretes an acetylglucosamine-containing O-glycan-type sugar chain to promote secretion from the cell.

  On the other hand, the therapeutic / prophylactic agent for a disease state caused by Helicobacter pylori according to the present invention contains the above-mentioned α-1, 4-linked N-acetylglucosamine-containing O-glycan-type sugar chain secretion promoter.

  According to the therapeutic and prophylactic agent for a pathological condition caused by Helicobacter pylori of the present invention, the α-1,4 linked N-acetylglucosamine whose secretion is promoted by the action of the glucosamine and / or glucosamine derivative as an active ingredient. O-glycan-type sugar chain contained in antibacterial action against Helicobacter pylori, causing pathologies caused by Helicobacter pylori, such as gastritis, gastric ulcer, duodenal ulcer, or related digestive system and other organs Can be treated or prevented.

  According to the present invention, from an O-glycan-type sugar chain, which is an O-glycan-type sugar chain and N-acetylglucosamine is bound to the non-reducing end portion thereof by α-1, type 4 glycosidic linkage. The defense mechanism against H. pylori inherent in humans can be effectively activated.

  The glucosamine and / or glucosamine derivative used in the present invention can be supplied in large quantities as a pharmaceutical / food material, and is chemically stable and highly safe. Therefore, it is possible to supply anti-H. Pylori preparations in various forms.

The glucosamine and / or glucosamine derivative used in the present invention, glucosamine, chitobiose, chitotriose, chitotetraose, Quito maltopentaose, Quito maltohexaose, N- acetylglucosamine, N- acetylchitobiose, N- acetyl Quito tri Orth, N- acetyl chitotetraose, N- acetyl Quito maltopentaose, N- acetyl Quito maltohexaose, lactosamine, N- acetyllactosamine, galactosyl lactosamine include galactosyl -N- acetyllactosamine down. In addition, according to the result of the Example mentioned later, it is clear that N-acetylglucosamine, glucosamine, N-acetylchitobiose, and N-acetyllactosamine can be used particularly preferably.

  The glucosamine and / or glucosamine derivative used in the present invention may be prepared by chemical, enzymatic, or genetic engineering techniques in accordance with known conventional techniques.

  For example, the chitosan oligosaccharide (chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose) and chitin oligosaccharide (N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N -Acetylchitopentaose, N-acetylchitohexaose) can be obtained by chemically or biochemically treating chitin prepared from crustacean shells such as crabs and shrimps by conventional methods. . In that case, according to the method disclosed in Japanese Patent Application Laid-Open No. 2003-212889 or Japanese Patent Application Laid-Open No. 10-2875726, it can be more suitably manufactured.

  In the present invention, the chitosan oligosaccharide and chitin oligosaccharide obtained by the above method can be used as they are as a mixture of oligosaccharides having various different chain lengths. By fractionating and purifying by a method such as fractionation, a composition having a high degree of polymerization can be obtained and used in the present invention.

  As a commercially available chitosan oligosaccharide, for example, “COS-YS” (trade name, manufactured by Yaizu Suisan Kagaku Kogyo Co., Ltd.) is obtained by the above-described method, and chitobiose, chitotriose, chitotetraose, chitopentaose, chito It is a composition containing hexaose or the like and can be preferably used in the present invention.

  Moreover, as a commercially available chitin oligosaccharide, for example, “NA-COS-YS” (trade name, manufactured by Yaizu Suisan Chemical Co., Ltd.) is obtained by the above method, and N-acetylglucosamine and N-acetyl are obtained. It is a composition containing chitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, N-acetylchitohexaose and the like and can be preferably used in the present invention.

  Furthermore, N-acetyllactosamine and galactosyl N-acetyllactosamine are, for example, as described in Japanese Patent No. 2819312, lactose and N-acetylglucosamine as substrates, and β-galactosidase to act on sugar transfer. It can manufacture by the method of performing reaction. Lactosamine and galactosyl-lactosamine can be produced by a method in which lactose and glucosamine are used as substrates and β-galactosidase is allowed to act to carry out a transglycosylation reaction. At this time, as described in Japanese Patent No. 2819313, the reaction may be performed in the presence of a salting-out agent. The enzyme reaction solution is heated to deactivate the enzyme, and can be used in the present invention as it is or after separation, purification and concentration by activated carbon column chromatography or reverse osmosis membrane as necessary.

  The glucosamine and / or glucosamine derivative used in the present invention may be a free form, or may form a food or a pharmaceutically acceptable salt thereof. Examples of the salt include hydrochloride, acetate, lactate, citrate, sulfate, nitrate and the like.

  In the present invention, “an O-glycan-type sugar chain, which is an O-glycan-type sugar chain in which N-acetylglucosamine is bonded to the non-reducing end portion by α-1, type 4 glycosidic bond (hereinafter referred to as α -1,4-linked N-acetylglucosamine-containing O-glycan-type sugar chain) ”, for example, as described above, the core bifurcated type contained in mucus secreted from gland mucous cells of the gastric mucosa It means a sugar chain having an α-linked N-acetylglucosamine at the non-reducing end of an α-1,4-linked N-acetylglucosamine-containing O-glycan type sugar chain. The “O-glycan type sugar chain” means a sugar chain bonded to a serine / threonine residue of a polypeptide chain of a glycoprotein.

  The α-1,4 linked N-acetylglucosamine-containing O-glycan type sugar chain secretion promoter of the present invention is an active ingredient, wherein the glucosamine and / or glucosamine derivative is α-1,4 linked N-acetyl. It can act on cells that secrete glucosamine-containing O-glycan-type sugar chains to promote their secretion.

  Further, as described above, it has been clarified that α-1,4 linked N-acetylglucosamine-containing O-glycan type sugar chain acts antibacterial on H. pylori (see Non-Patent Document 1 above). .

  Therefore, the therapeutic / prophylactic agent for a pathological condition caused by Helicobacter pylori of the present invention, when taken orally, the glucosamine and / or glucosamine derivative as an active ingredient is converted into mucus cells of the gastric mucosa or duodenal mucosa. Pathology caused by Helicobacter pylori, which acts on and promotes the secretion of α-1,4-linked N-acetylglucosamine-containing O-glycan-type sugar chains. For example, it can be suitably used for the purpose of treating or preventing gastritis, gastric ulcer, duodenal ulcer and the like.

  In the present invention, in order to effectively obtain the anti-H. Pylori effect, it is preferable to take 0.01 to 15 g orally as the above glucosamine and / or glucosamine derivative per day for an adult. It is more preferable to take 5 g.

  In the α-1,4-linked N-acetylglucosamine-containing O-glycan-type sugar chain secretion promoter and the therapeutic and prophylactic agent for the pathological condition caused by Helicobacter pylori of the present invention, the above-mentioned glucosamine is used as an active ingredient of the above-mentioned effects. And / or glucosamine derivatives and additives such as excipients, stabilizers, preservatives, preservatives, brighteners, thickeners, colorants, minerals, vitamins, sugars, fragrances, fats and oils, amino acids, etc. Can be appropriately blended. Fucoidan, lactic acid bacteria, catechin, cocoa polyphenol, hop polyphenol, other polyphenols, cocoa free fatty acid, seaweed extract, plum extract, cinnamon, cranberry, garlic, turmeric In combination with components already known to have anti-pylori effects such as anti-pylori effects, a synergistic effect as an anti-pylori agent can be expected.

  The product form of the α-1,4-linked N-acetylglucosamine-containing O-glycan secretion promoter and the therapeutic / preventive agent for the pathological condition caused by Helicobacter pylori of the present invention is not particularly limited, but is suitable for oral administration, for example. Examples thereof include tablets, powders, granules, solutions, capsules and the like.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

<Example 1>
Human α-1, 4-linked N-acetylglucosaminyltransferase (α4GnT) cDNA (α4GnT cDNA; NCBI database reference number NM — 016161) was introduced into human gastric cancer-derived cultured cells AGS according to a conventional method, AGS-α4GnT, a transfection cell line that is constitutively expressed, was prepared.

  This transgenic cell line (AGS-α4GnT) is Dulbecco's Modified Eagle Medium (hereinafter referred to as DMEM), 10% Fetal Bovine Serum (Equitec Bio), 1% Penicillin-Streptomycin, liquid (Invitrogen), 0.2 mg / ml G418 Cultured and maintained in a cell culture medium consisting of (Sigma).

Moreover, as carbohydrate, ND-acetylglucosamine, D-glucosamine hydrochloride,
N-acetylchitobiose (GlcNAc (β1-4) GlcNAc), N-acetyllactosamine (Gal (β1-4) GlcNAc) (all the above carbohydrates are manufactured by Yaizu Suisan Chemical Co., Ltd.) to a concentration of 400 mM. As above, it was dissolved in DMEM, sterilized by filtration with MILLEX-GV (0.22 μm filter Unit), and prepared as a saccharide solution of 10, 100, 200 mM with ultrapure water.

The AGS-α4GnT cells were seeded in one well of a 48-well plate (manufactured by Falcon) at a cell concentration so that 2 × 10 ⁴ cells were contained in 0.45 ml of the culture solution. With the seeding date as day 0, 0.05 ml of each of the above saccharide solutions at the above concentration was added to the wells on the first day after sowing, and the final 0.5 ml of saccharide in the culture solution had a final concentration of 1 each. It added so that it might become 10, 20 mM. In addition, only DEME was added as a control containing no carbohydrate.

  The culture broth 1 to 5 days after cell seeding was collected in separate eppendorf tubes in 3 wells, and the cells were centrifuged at 1000 rpm × 3 min at 4 ° C., and the supernatant was collected in a new eppendorf tube.

  The secretory mucin concentration in the culture supernatant was measured using “ELISA kit for measuring gastric gland mucus cell mucin” (trade name, Kanto Chemical Co., Inc.) and “ELAST ELISA Amplification Kit” (Perkin Elmer Japan Co., Ltd.). The antibody used in the above kit was measured using an antibody that specifically recognizes a sugar chain in which N-acetylglucosamine is bound to the non-reducing end portion by α-1, type 4 glycoside bond. Moreover, since it hardly recognizes mucin secreted from AGS cells into which α4GnT is not introduced, the concentration of α-1,4-GlcNAc-containing Α-1, 4-linked N-acetylglucosamine-containing O-glycan-type sugar chain It was considered. Moreover, the measured value was calculated as an average value of the concentration contained in the culture supernatant of 2 wells out of 3 wells.

  FIG. 1 shows the results of the effects of ND-acetylglucosamine on the production of α-1,4-linked N-acetylglucosamine-containing O-glycans.

  As can be seen from FIG. 1, when 1 mM ND-acetylglucosamine was added, approximately 3 times as much Α-1, 4-linked N-acetylglucosamine-containing O-glycan secretion as the control was observed on the 5th day of seeding. Promoted. On the other hand, in the 10 mM and 20 mM addition groups, α-1,4 linked N-acetylglucosamine-containing O-glycan secretion was lower than the control, and a concentration of about 1 mM was α-1,4 linked N-acetylglucosamine-containing O-glycan. It was suggested to be effective for secretion.

  FIG. 2 shows the results of the effect of D-glucosamine on the production of α-1,4-linked N-acetylglucosamine-containing O-glycan.

  As can be seen from FIG. 2, when 1 mM and 20 mM of D-glucosamine was added, secretion of α-1,4-linked N-acetylglucosamine-containing O-glycan was promoted. When 1 mM was added, the α-1,4 linked N-acetylglucosamine-containing O-glycan concentration was maximum.

  FIG. 3 shows the results of the influence of N-acetylchitobiose on the production of α-1,4-linked N-acetylglucosamine-containing O-glycan.

  As can be seen from FIG. 3, when 1 mM or 10 mM of N-acetylchitobiose was added, about 4 times as much α-1 and 4-linked N-acetylglucosamine-containing O-glycan secretion as that of the control on the 5th day of seeding. Promoted. In the 20 mM addition group, α-1,4-linked N-acetylglucosamine-containing O-glycan secretion was lower than that of the control, and a concentration of about 1 to 10 mM resulted in α-1,4-linked N-acetylglucosamine-containing O-glycan secretion. It was suggested to be effective.

  FIG. 4 shows the results of the influence of N-acetyllactosamine on the production of α-1,4-linked N-acetylglucosamine-containing O-glycan.

  As can be seen from FIG. 4, when 1, 10, 20 mM of N-acetyllactosamine was added, about 4 to 5 times as much α-1 and 4-linked N-acetylglucosamine-containing O as the control on the 5th day of seeding. -Promoted glycan secretion. In all the addition groups, the α-1,4 linked N-acetylglucosamine-containing O-glycan concentration increased, suggesting that it was a good accelerator.

  As described above, D-glucosamine hydrochloride, ND-acetylglucosamine, N-acetylchitobiose (GlcNAc (β1-4) GlcNAc), N-acetyllactosamine (Gal (β1-4) GlcNAc) It was revealed that secretion of -1,4-linked N-acetylglucosamine-containing O-glycan-type sugar chains from cells was promoted.

  The present invention is useful for the treatment and prevention of pathologies caused by Helicobacter pylori.

It is a figure which shows the result of having measured about the influence which ND-acetylglucosamine exerts on the secretion amount from the cell of (alpha) -1, 4-linked N-acetylglucosamine containing O-glycan type sugar chain. It is a figure which shows the result of having measured about the influence which the D-glucosamine hydrochloride has on the secretion amount from the cell of (alpha) -1, 4-linked N-acetylglucosamine containing O-glycan type sugar chain. It is a figure which shows the result of having measured about the influence which N-acetylchitobiose has on the secretion amount from the cell of (alpha) -1, 4-linked N-acetylglucosamine containing O-glycan type sugar chain. It is a figure which shows the result of having measured about the influence which N-acetyllactosamine has on the secretion amount from the alpha-1 and 4-bond type N-acetylglucosamine containing O-glycan type sugar chain.

Claims (2)

O-glycan-type sugar chain, a component that promotes secretion from cells of O-glycan-type sugar chains in which N-acetylglucosamine is bonded to the non-reducing end portion by α-1, type 4 glycoside bonds As glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, N-acetylglucosamine, N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchito Containing at least one glucosamine and / or glucosamine derivative selected from pentaose, N-acetylchitohexaose, lactosamine, N-acetyllactosamine, galactosyllactosamine, galactosyl-N-acetyllactosamine as an active ingredient Α-1,4 bonds characterized by Secretory promoter of type N-acetylglucosamine-containing O-glycan type sugar chain . Containing secretion promoters alpha-l, 4-linked N- acetylglucosamine-containing O- glycan-type sugar chain of claim 1 Symbol placement, therapeutic prophylactic agent for disease states caused by Helicobacter pylori.