WO2007077812A1

WO2007077812A1 - Pharmaceutical having anti-histamine activity

Info

Publication number: WO2007077812A1
Application number: PCT/JP2006/325862
Authority: WO
Inventors: Hiroyuki Fukui; Jun Kojima; Kiyotaka Nishita
Original assignee: The University Of Tokushima; Kowa Pharmaceutical Co. Ltd.
Priority date: 2005-12-27
Filing date: 2006-12-26
Publication date: 2007-07-12
Also published as: JP2009067679A

Abstract

Disclosed is a pharmaceutical having an excellent anti-histamine activity relying on a novel mechanism. The pharmaceutical comprises a cell of a bifidobacterium (e.g., a cell of Bifidobacterium infantis and/or a cell of Bifidobacterium longum) as an active ingredient.

Description

Specification

Medicine with antihistamine action

Technical field

[0001] The present invention relates to a medicament having an antihistamine effect. More specifically, the present invention relates to a novel drug that has an anti-histamine action based on a new mechanism that includes an anaerobic bacterium, Bifidobacterium, as an active ingredient.

Background art

[0002] Histamine, a biological amine, is a kind of otacoid, exists in various animal and plant tissues, and is biosynthesized from L-histidine by the action of histidine decarboxylase (HDC). Histamine accumulated in mast cells and basophils, which are the largest storage sites, is released by stimulation with antigen-1 gE antibody complex and induces immediate immune response via histamine HI receptor (H1R). . Antigens that induce the release of histamine include pollen, dust, and mites from plants such as cedar. As a result of the immediate immune reaction, allergic symptoms such as atopic dermatitis, hay fever, bronchial asthma and urticaria appear.

[0003] Many antihistamines have been developed to alleviate and cure allergic symptoms from histamine. However, previously developed antihistamines, to varying degrees, cross the brain barrier and act on central nervous system H1R or cholinergic receptors, resulting in sedation, drowsiness, fatigue, or throat. There are problems with side effects such as whispering. Accordingly, there is an urgent need to provide an antihistamine that does not have these side effects.

[0004] As a means for providing an antihistamine drug with no side effects, a method of screening a drug that causes inflammation and / or acts only on a site can be employed. For example, antihistamines for the purpose of preventing or treating hay fever can be screened using the degree of attenuation of signal transduction via H1R in the nasal mucosa tissue as an indicator. Unlike conventional H1R antagonists, drugs that directly reduce the number of H1R in the inflammatory site (e.g., histamine-H1R-mediated signal attenuation as an index) Drugs that can suppress the amount of H1R induced in nasal mucosal tissues by antigen stimulation with pollen, etc.) can be provided as completely different types of new antihistamines. Alternatively, drugs that reduce the amount of histamine synthesis, which is the signal itself (such as drugs that suppress the production of HDC, a histamine synthesis enzyme), can also be a new type of antihistamine that has no side effects.

[0005] The present inventors should be able to screen for a new type of antihistamine as described above, and have succeeded in producing a rat model of toluene 2,4-diisocyanate (TDI) -induced nasal hypersensitivity (Acta Otolaryngol). , 124, pp.1053-1058, 2004). This model mouse is known as the causative agent of occupational allergy, and TDI is highly responsive to histamine! It can be produced by repeatedly applying it to the nasal mucosa of Brown Norwegian rats. In this model mouse, the expression level of both messenger RNA (H1R m-RNA) that generates H1R and HDC m-RNA, which is the m-RNA that generates HDC, increases, resulting in an increase in the amount of H1R and HDC. Severe sneezing or runny nose leakage occurs. In this model mouse, a substance that can suppress the increased expression level of H1R m-RNA and HDC m-RNA induced by TDI is a new substance that directly blocks or attenuates H1R-mediated signals that are amplified at the site of inflammation. Based on the U ヽ mechanism, it may be a new antihistamine without any side effects.

[0006] On the other hand, Bifidobacterium, an anaerobic bacterium, has become a dominant bacterium in the intestine of healthy breastfeeding infants. However, it is known that the incidence of enteric infectious diseases increases when Escherichia coli, enterococcus, etc. increase. Breastfeeding infants are said to be highly resistant to infections such as bacteria and viruses due to the mother's immunity. In adults, it is also confirmed that bifidobacteria exist as one of the dominant bacteria in the intestine, and that bifidobacteria are reduced or eliminated in an unhealthy state. . From this point of view, various pharmaceuticals using bifidobacteria or health foods such as fermented milk drinks and soft drinks are proposed and marketed.

[0007] By ingesting bifidobacteria, a large amount of acetic acid, lactic acid, and other volatile acids are produced in the intestine, resulting in a decrease in intestinal pH. As a result, the growth of pathogenic bacteria such as Escherichia coli is suppressed. The promotion of peristaltic movement improves the symptoms of constipation and diarrhea associated with abnormal gut microbiota. Thus, bifidobacteria are extremely effective in maintaining human health, and various preparations containing bifidobacteria are provided. For example, various additives are added to the culture solution of Bifidobacterium. In addition to being prepared as a healthy drink as a fermented product, after collecting and drying the cells of bifidobacteria, it is mixed with an appropriate excipient such as starch, lactose, or sucrose to form a powder or tablet. It is also used as

In recent years, research on probiotics using bifidobacteria and various lactic acid bacteria has been actively conducted. By using not only medicinal products but also bifidobacteria fermented yogurt, hay fever improvement effects and atopic dermatitis of lactic acid bacteria fermentation broth Various pharmacological effects such as improvement effects, immunostimulatory effects, or cancer recurrence suppression effects have been reported (Probiotics and Biogenitas, Chapter 3: Allergy reduction, May 9, 2005, N'T'S issue). In addition, an intestinal microflora improving composition (Japanese Patent Laid-Open No. 7-265064), an antiallergic agent containing bifidobacteria as an active ingredient (Japanese Patent Laid-Open No. 10-309178), and an antiallergic agent containing living intestinal bacteria An agent (JP 2000-86524 A) has been reported. However, the anti-allergic effect of these biobiotics is that the probiotic Bifidobacteria and various lactic acid bacteria act on the host intestinal immunity to balance the balance of allergic Th2 cells and anti-allergic Thl cells. By dominating the cell, increasing the production of various anti-allergenic site-specific ins produced by Thl cells, and simultaneously reducing the amount of allergic site-in produced by Th2 cells. (Int. Arch. Allergy Immunol, 135, pp. 205-215, 2004), and it is implied in these publications that bifidobacteria itself has antihistaminic activity. I don't teach it.

Non-Patent Document 1: Journal of Pharmacology of Japan (Folia Pharmacol. Jpn.), 125, pp.245-250, 2005

Non-Patent Document 2: Acta Otolaryngol, 124, pp.1053--1058, 2004

Non-Patent Document 3: Probiotics and Biojetus, Chapter 3: Allergy Reduction, May 9, 2005, published by ENTS Co., Ltd.

Non-Patent Document 4: Int. Arch. Allergy Immunol, 135, pp.205-215, 2004

Patent Document 1: Japanese Patent Laid-Open No. 7-265064

Patent Document 2: JP-A-10-309178

Patent Document 3: Japanese Patent Laid-Open No. 2000-86524

Disclosure of the invention

Problems to be solved by the invention [0009] An object of the present invention is to provide a medicament having an antihistaminic action. More specifically, a new drug that is different from conventional antihistamines and can exhibit excellent antihistamine action based on the mechanism, while reducing or eliminating side effects such as sleepiness and fatigue. It is an object of the present invention to provide

Means for solving the problem

[0010] In order to solve the above problems, the present inventors screened antihistamine substances based on a new mechanism using TDI-induced nasal hypersensitivity model rats. As a result, oral administration of Bifidobacteria cells can suppress the increased expression of H1R m-RNA and HDC m-RNA, and helper T2 (Th2) cells, which are inflammatory immune cells It was also found that the expression amplification of interleukin (IL) -4, IL-5, and IL-13, which is an inflammatory site force-in produced by M. coli, can be reduced at the site of inflammation. Furthermore, we have found that the ability to use bifidobacteria of a specific bacterial species as bifidobacteria, or a combination of two or more specific species of bifidobacteria, can achieve an extremely excellent antihistamine effect. . The present invention has been completed based on these findings.

[0011] That is, according to the present invention, there is provided a medicament having an antihistamine action, which comprises a bifidobacteria cell as an active ingredient.

According to a preferred embodiment of the present invention, the above-mentioned medicament containing at least Bifidobacterium infantis as bifidobacteria; the above-mentioned medicament containing at least bifidobacterium longum as bifidobacteria Medicinal medicine; The above-mentioned medicine comprising two kinds of bifidobacterium 'Infantes' and Bifidobatterium' longum as bifidobacteria; The above-mentioned medicine in which bifidobacteria can be any mixture of the above two bacterial species; The above-mentioned medicine containing the above two kinds of bacterial species at a mass ratio of 7: 3 is provided.

[0012] Further, according to the present invention, the above medicament based on the inhibitory histamine HI receptor messenger RNA expression according to the present invention; the above medicament based on the antihistamine decarboxylase messenger RNA expression inhibitory action; Antihistamine action is based on histamine HI receptor messenger RNA expression inhibitory action and histidine decarboxylase messenger RNA expression inhibitory drug as described above; and antihistamine action is helper T2 cell site force-in Or interleukin 13) The above-mentioned medicine with an action to suppress messenger RNA expression is provided.

[0013] Another aspect of the present invention is that, according to the present invention, a histamine HI receptor messenger RNA expression inhibitor containing bifidobacteria cells as an active ingredient; histidine decarboxylase containing bifidobacteria cells as an active ingredient Messenger RNA expression inhibitor; inhibitor of histamine HI receptor messenger RNA expression and histidine decarboxylase messenger RNA expression containing bifidobacteria cells as active ingredients; and helper T2 containing bifidobacteria cells as active ingredients Cell site force messenger RNA expression inhibitor is provided. In addition to these, a drug that reduces the amount of histamine synthesized at the onset of allergic symptoms, a drug that contains Bifidobacteria cells as an active ingredient; an action that reduces the amount of histamine HI receptor at the time of allergic symptoms A medicine containing Bifidobacteria cells as an active ingredient; a sneezing inhibitor containing Bifidobacteria cells as an active ingredient; and a rhinorrhea inhibitor containing Bifidobacteria cells as an active ingredient Provided by the invention.

[0014] From still another aspect, according to the present invention, the use of a cell of Bifidobacteria for the production of the pharmaceutical or expression inhibitor described above; a method for suppressing the action of histamine in the living body of mammals including humans A method comprising the step of orally administering a bifidobacterial cell to a mammal; a method for inhibiting the expression of histamine HI receptor messenger RNA in a living body of a mammal, including a human, comprising: A method comprising the step of orally administering a fungal cell body to a mammal; a method for suppressing histidine decarboxylase messenger RNA expression in a living body of a mammal, including a human, comprising: A method comprising oral administration to a mammal; histamine HI receptor messenger RNA expression and histology in a mammal, including a human, in vivo A method for suppressing messenger RNA expression of decarboxylase, comprising a step of orally administering a bifidobacterial cell to a mammal; and a helper T2 cell site in a mammal including a human. A method for suppressing expression of force messenger RNA, comprising the step of orally administering a bifidobacteria cell to a mammal.

BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The medicament of the present invention is a medicament having an antihistamine action, and has bifidobacteria cells. It is characterized by containing as an active ingredient. Bifidobacteria are anaerobic bacteria and generally have a biophysical bifidobatterium 'Bifidobacterium infantis', bifidobatterium' B.longum ', and Bifidobatterium' Strains such as B. breve and Bifidobacterium bifidum are used. Any kind of strain can be used for the production of the medicament of the present invention. For the production of the medicament of the present invention, it is preferred to use at least Bifido Batterium 'infantes, or at least Bifido Batterium longum. More preferably, it is preferable to use a combination of at least Bifido Batterium 'Infantes' and Bifido Batterium' Longam. In a more preferred embodiment, it is preferable that the cell mass (which may be either a wet mass or a dry mass) contains bifido butterium 'infantes and bifidobacterium longum in a ratio of 7: 3.

[0016] The bifidobacteria are cultured in a liquid medium containing, for example, a carbon source, a nitrogen source, an inorganic substance, and other additives such as peptone, yeast extract, glucose, and phosphate. It is preferable to collect the bacteria and freeze-dry with an appropriate cryoprotectant. The type of the liquid medium is not particularly limited, and any medium can be used as long as it is normally used for culturing bifidobacteria. The obtained bacterial cells may be mixed with an appropriate excipient such as starch, lactose, sucrose, or a mixture thereof. A single bacterial strain may be cultured alone, or two or more bacterial strains may be mixed and cultured. Alternatively, a culture of a single strain can be prepared, and then mixed at an appropriate ratio to produce a cell mixture. Further, bifidobacteria may be mixed with various lactic acid bacteria, butyric acid bacteria, or gonococci. Methods for producing pharmaceuticals and foods using bifidobacteria are well known to those skilled in the art. For example, refer to JP-A-7-265064, JP-A-10-309178, and JP-A-2000-86524. Therefore, it can be easily manufactured by those skilled in the art.

[0017] The powder of bifidobacteria obtained as a powder may be diluted with a suitable pharmaceutically acceptable diluent to produce a powder, and further suitable excipients and lubricants. Can be kneaded into condyles and tablets. Any of these preparations can be prepared according to the method described in “General Rules for Preparations” of the Japanese Pharmacopoeia. Addition of pharmaceuticals such as carriers, excipients, lubricants, fluidizers, disintegrants, binders, isotonic agents, or stabilizers used in formulation The kind of product is not particularly limited, and various additives that are widely used in pharmacology can be appropriately selected and used. Two or more pharmaceutical additives may be used in combination. The medicament of the present invention can exert an antihistamine action by oral administration. The dose and administration period of the bifidobacteria cells are not particularly limited, but it is desirable to select them appropriately according to the sex, age, weight, and symptoms of the patient to be administered. In general, it is sufficient to select a dosage and a dosage period at which a sufficient antihistamine action is exerted while confirming improvement of allergic symptoms due to histamine release.

The medicament of the present invention has an antihistaminic action. The antihistamine action of the medicament of the present invention can be determined, for example, by using an experimental animal such as a toluene 2,4-diisocyanate (TDI) -induced nasal hypersensitivity model rat (Acta Otolar yngol, 124, pp.1053-1058, 2004). Those skilled in the art can easily confirm this. It has been confirmed that an increase in H1R mRNA levels observed in TDI-sensitized rats increases HIR expression levels, and an increase in HDC mRNA levels increases HDC expression levels (Japanese pharmacology). (Folia Pharmacol. Jpn.), 125, pp.245-250, 2005). Bifidobacteria, which is an active ingredient of the medicament of the present invention, can suppress the increase in H1R m-RNA level and HDC m-RNA level in nasal mucosal cells induced by TDI sensitization, and as a result Demonstrate histamine action. Regardless of the balance (Thl / Th2) of helper Tl (Thl) cells and helper T2 (Th2), which are inflammatory immune cells, a drug that acts to suppress histamine signaling via H1R, In particular, a bifidobacteria preparation has not been known. In addition, IL-4, IL-5, and IL-13 m-RNA, which are inflammatory Th2-derived site-force ins, develop inflammation and the expression level is amplified in the nasal mucosa! However, the medicament of the present invention has an action of suppressing the expression level of these cyto force-ins! Based on these antihistamine actions, the medicament of the present invention is effective in preventing and / or treating various symptoms of allergy associated with histamine release. For example, it is known that sneezing and Z or rhinorrhea are caused by histamine, and the medicament of the present invention is a medicament for prevention and Z or treatment of sneezing and Z or rhinorrhea accompanying histamine release. Useful as. In particular, it is extremely useful as a medicine for prevention and Z or treatment of sneezing and Z or rhinorrhea in patients with allergic rhinitis. On the other hand, leucotriene is mainly associated with nasal congestion, and anti-leukotrienes are often effective for nasal congestion. Since the drug is based on an antihistamine action, its effectiveness is often not expected in severe cases that have progressed to nasal congestion.

Example

Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.

Example 1: Antiallergic effect in TDI-induced allergy model rats

A 7: 3 mixed cell powder of B. infantis and B. longum prepared by the method described in the Japanese Pharmacopoeia Standards for Pharmaceuticals 2002 edition was administered to TDI-induced allergic model rats for 3 weeks. Biff chair, viable cell count of the scan bacterial cell powder was 1.0 X 10 ¹¹ cells / g, and suspended in 1/15 M phosphate buffer pH 6.8 in such the fungus powder becomes 20 mg / mL 2 mL of this was orally administered to rats daily. The cell suspension was prepared at the time of use. Rats are brown norway rats that are highly responsive to histamine. One hour after administration, cells are sensitized by applying 10 L of 10% TDI solution with 10% TDI dissolved in ethyl acetate to the nasal mucosa of mice. Went. After an interval of 2 days for 5 consecutive days as the initial sensitization, TDI was applied for another 5 days as the late sensitization. One week later, the final provocation was performed, and the amount of nasal discharge was observed and the number of sneezing was measured for 10 minutes. Four hours after TD stimulation, the nasal mucosa was recovered by slaughter.

[0020] Total m-RNA was extracted from rat nasal mucosa, and cDNA was synthesized using reverse transcriptase (Invitrogen). PCR reaction was performed using two types of primers and TaqMan probe (Hokkaido System Science Co., Ltd.), and PCR product amplification curves were measured in real time for analysis and quantification (real-time PCR quantification system Sequence Detection System 5700, Applied Used by Biosystems). Dalyceraldehyde-3-phosphate dehydrogenase (Applied by Systems) was used as an internal standard. The test used four rats per group. The results are shown in Table 1.

[0021] [Table 1] Untreated TDI treatment Aspergillus administration TDT treatment ÷ B bacteria administration Nose discharge leak score 0 2.75 ± 0.25 * 0 1.7 ± 0.25 ** Sneezing count 0.3 ± 0.23 29.0 ± 1.4 * 0.5 丄 0.29 17.7 ± 2.7

HIR m-RNA level 1.18 ± 0.33 2.99 ± 0.22 0.78 ± 0.05 1.76 ± 0.24

HDC m-RNA level 1.09 ± 0.31 3.05 ± 0.38 * 0.61 ± 0.05 2.16 ± 0.06

IL-4 m-RNA level 0.42 ± 0.03 1.41 ± 0.20 * 0.39 ± 0.03 0.78 ± 0.11

IL-5 m-RNA level 1.11 ± 0.54 24.0 ± 3 · 89 * 0.91 ± 0.14 10.7 ± 1.73 **

TL-3 m-RNA level 0.05 ± 0.00 3.65 ± 0.46 * 0.05 ± 0.01 3.94 ± 0.73

IL 13 m-RNA repel 0.51 ± 0.10 12.3 ± 1.11 * 1.40 ± 0.07 * 6.25 ± 1.30

IL 18 m-RNA repel 0.43 ± 0.05 0.59 0.09 0.34 ± 0.03 0.41 ± 0.02 Notation: Mean value standard deviation. *, **: Significant difference.

Each m-RNA level was controlled by the glyceraldehyde-3-phosphate dehydrogenase m-RNA level.

The runny nose leakage score 1 was defined as 1 when the runny nose was observed in the nose, 3 when the runny spilled from the nose, and 2 between the two.

[0022] As shown in Table 1, oral administration of bifidobacteria significantly reduced the amount of nasal discharge in nasal hypersensitivity model rats, and also significantly decreased the number of sneezing. In addition, both the increase in the levels of HIR mRNA and HDC mRNA induced by continuous application of TDI nasal mucosa were significantly suppressed. Increased H 1 R m-RNA levels observed in TDI-induced nasal hypersensitivity model rats can increase H 1 R expression levels, and increased HDC m-RNA levels can increase HDC expression levels. Are known. Therefore, the above results indicate that oral ingestion of bifidobacteria reduces histamine synthesis at the onset of allergic symptoms and reduces histamine sensitivity due to decreased receptor levels, thereby causing nasal leakage observed in model rats. This is thought to be causing a decrease in volume and a decrease in the number of sneezes.

[0023] In addition, the medicament of the present invention also significantly suppressed IL-4, IL-5, and IL-13 mRNA levels produced by Th2 cells, which are allergic helper T cells. Enhanced production of IL-4 and IL-13 is known to induce the release of IgE, which is a B cell force, in cooperation with IL-2. Ingestion of fungi has an effective antihistamine effect in the body, and as a result, it is effective in preventing and / or alleviating symptoms when allergic symptoms occur.

[0024] Example 2 Bifidobacteria 3 times a day (2 g / day) for patients with or suspected perennial allergic rhinitis who have typical nasal allergy symptoms (sneezing attacks, rhinorrhea, and nasal congestion) The daily dose was 6 g) for 14 days, and the effects on sneezing attacks, rhinorrhea, and nasal congestion were examined. As a test drug, a preparation containing 10 mg of bifidobacteria in 1 g of the preparation (“Lucky Fine N”) was used. Subjective symptoms were investigated and recorded as scores before the start of the study and at the end of the study for the three items of sneezing, rhinorrhea, and nasal congestion. 25th of "Rhino-Allergic Practice Guidelines", Annual Rhinitis and Pollen Allergy, Nose Allergy Practice Guidelines Preparation Committee, 2005 (5th revised edition), Life'Science Co., Ltd., November 2005) Severity was scored for sneezing attacks, rhinorrhea, and nasal congestion in each case according to the disease type and severity assessment described on page 27.

[0025] Statistical significance of mean scores between the treatment group (2 men, 15 women, age 19-85 years) and the non-treatment group (1 man, 9 women, age 22-62 years) Difference test was performed by T-test. The results are shown in the table below. From these results, it can be seen that the medicament of the present invention has a remarkable ameliorating effect on sneezing attacks and rhinorrhea among nasal allergic symptoms, while it is less effective for nasal congestion. Since sneezing attacks and rhinorrhea are caused by histamine release, while nasal congestion is caused by leukotrienes (supra, “Nonallergic Allergy Guidelines”, pp. 54-56), the medicament of the present invention is antihistamine. It was shown to have improved sneezing attacks and rhinorrhea based on its pharmacological action.

[0026] [Table 2]

(Administration group)

T-test: a: 0.17; b: 1.00; c: 0.73 3] (Non-administration group)

T-test: d: 0. 17; e: 1. 00;

Industrial applicability

The medicament provided by the present invention can exhibit an excellent antihistaminic action in the living body of mammals including humans, and the side effects such as sleepiness and fatigue that frequently occur in conventional antihistamines are reduced. If you talk about it!

Claims

The scope of the claims

[I] A medicine having an antihistaminic action and containing Bifidobacteria cells as an active ingredient.

[2] The medicament according to claim 1, comprising at least Bifidobacterium 'infantes and / or Bifidobacterium longum as bifidobacteria.

[3] The medicament according to claim 1, comprising bifidobatterum 'infantes and bifidobatterium' longam in combination as bifidobacteria.

[4] The medicament according to claim 3, comprising biflud bateratum 'infantes and biflud bateratum' longum in a mass ratio of 7: 3.

[5] Antihistaminic activity suppresses histamine HI receptor messenger RNA expression and

The medicament according to claim 1, which is based on a messenger RNA expression inhibitory action of Z or histidine decarboxylase.

[6] The medicament according to claim 5, wherein the antihistaminic action is accompanied by a messenger RNA expression inhibitory action of helper T2 cell site force-in.

[7] Histamine HI receptor messenger R containing bifidobacteria as an active ingredient

Inhibitor of NA expression and Z or histidine decarboxylase messenger RNA expression.

[8] A drug that reduces the amount of histamine synthesis at the onset of allergic symptoms, and that contains bifidobacteria as an active ingredient.

[9] A drug having an action of reducing the amount of histamine HI receptor at the onset of allergic symptoms, comprising a Bifidobacteria cell body as an active ingredient.

[10] A medicine for preventing and / or treating sneezing containing Bifidobacterium as an active ingredient.

[II] A medicine for prevention and Z or treatment of sneezing in allergic rhinitis containing Bifidobacterium as an active ingredient.

[12] A medicine for preventing and / or treating rhinorrhea comprising bifidobacteria as an active ingredient

[13] Prevention and prevention of rhinorrhea in allergic rhinitis containing bifidobacteria as an active ingredient And z or medicine for treatment.