JP2020132558A - Pharmaceutical compositions for preventing or treating diseases accompanying increase in peripheral serotonin or intestinal aromatic amines and methods for screening therapeutic agents for treating or preventing diseases accompanying increase in peripheral serotonin or intestinal aromatic amines - Google Patents

Abstract

To provide pharmaceutical compositions for preventing or treating diseases accompanying increase in peripheral serotonin or intestinal aromatic amines and methods for screening therapeutic agents for treating or preventing diseases accompanying increase in peripheral serotonin or intestinal aromatic amines.SOLUTION: Disclosed is a pharmaceutical composition for preventing or treating a disease accompanying increase in peripheral serotonin or intestinal aromatic amines which comprises an agent that inhibits aromatic amino acid decarboxylase of intestinal bacteria as an active ingredient. In an embodiment, the agent that inhibits aromatic amino acid decarboxylase of intestinal bacteria is carbidopa or benserazide.SELECTED DRAWING: None

Description

The present invention relates to a pharmaceutical composition for preventing or treating a disease associated with an increase in peripheral serotonin or intestinal aromatic amine, and a method for screening a drug for preventing or treating a disease associated with an increase in peripheral serotonin or intestinal aromatic amine. Is.

Serotonin, one of the neurotransmitters, has a distribution in the body of 5% in the brain and 95% in the periphery, and it is known that peripheral serotonin is biosynthesized by enterochromaffin cells (EC cells) in the intestinal tract. There is. Peripheral serotonin has been suggested to be associated with various diseases, specifically, osteoporosis patients (Non-Patent Documents 1 and 2), celiac disease patients (Non-Patent Document 3), hypersensitive bowel disease patients and ulcerative colitis. It has been shown that patients with inflammation (Non-Patent Document 4) have higher peripheral serotonin concentrations than healthy subjects. Furthermore, in a mouse model, obesity and metabolic deficiency were improved by inhibiting the biosynthesis of peripheral serotonin, indicating that it also contributes to lipid burning in obesity and brown adipocytes (Non-Patent Document 5).

It has been reported that serotonin biosynthesis in EC cells is promoted by metabolites of intestinal bacteria (Non-Patent Document 6). Non-Patent Document 6 evaluates the ability to promote serotonin biosynthesis by administering metabolites of various intestinal bacteria to the intestinal tract of sterile mice, finds activity in some metabolites, and is one of the aromatic amines. It is shown that a certain tyramine has the ability to promote serotonin biosynthesis. On the other hand, phenylethylamine and tyramine, which are one of the aromatic amines, have been reported to be higher in the feces of Crohn's disease patients and ulcerative colitis patients, respectively, suggesting their involvement in these diseases (non-healthy). Patent Document 7).

Lavoie B, et al., Gut-derived serotonin contributes to bone deficits in colitis. Pharmacol Res., S1043-6618 (18) 30234-2 (2018) Vijay K Yadav, et al., Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis. Nat Med., 16, 308-312 (2010) Challacombe DN, et al., Increased tissue concentrations of 5-hydroxytryptmaine in the duodenal mucosa of patients with coeliac disease. Gut., 18, 882-886 (1977) Yu FY, et al., Comparison of 5-hydroxytryptophan signaling pathway characteristics in diarrhea-predominant irritable bowel syndrome and ulcerative colitis. World J Gastroenterol., 22, 3451-3459 (2016) Justin D Crane, et al., Inhibition peripheral serotonin synthesis reduces obesity and metabolic dysfunction by promoting brown adipose tissue thermogenesis. Nat Med., 21, 166-172 (2015) Jessica M. Yano, et al., Indigenous Bacteria from the Gut Microbiota Regulate Host Serotonin Biosynthesis. Cell., 161, 264-276 (2015) Maria Laura Santoru, et al., Cross sectional evaluation of the gut-microbiome metabolome axis in an Italian cohort of IBD patients. Sci Rep., 7, 9523 (2017)

An object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease associated with an increase in peripheral serotonin or intestinal aromatic amine. Another object of the present invention is to provide a method for screening a drug for preventing or treating a disease associated with an increase in peripheral serotonin or intestinal aromatic amine.

The present invention includes the following inventions in order to solve the above problems.
[1] A pharmaceutical composition for preventing or treating a disease associated with an increase in peripheral serotonin or intestinal aromatic amine, which contains a drug that inhibits the aromatic amino acid decarbonase of intestinal bacteria as an active ingredient.
[2] The pharmaceutical composition according to the above [1], wherein the drug that inhibits the aromatic amino acid decarboxylase of enterobacteria is carbidopa or benserazide.
[3] The pharmaceutical composition according to the above [1] or [2], wherein the disease associated with an increase in peripheral serotonin or intestinal aromatic amine is osteoporosis, irritable intestinal disease, ulcerative colitis, celiac disease or Crohn's disease. object.
[4] A method for screening a drug for preventing or treating a disease associated with an increase in peripheral serotonin or an intestinal aromatic amine, which comprises a step of selecting a test substance that inhibits an aromatic amino acid decarbonase of an intestinal bacterium.
[5] Step 1 of culturing an intestinal bacterium having an aromatic amino acid decarbonizing enzyme gene in the presence or absence of a test substance in a medium to which an aromatic amino acid has been added, and the aromatic amine in the medium. 2 and a step 3 of selecting a test substance that reduces the amount of aromatic amine in the medium as compared with the amount of aromatic amine in the medium when cultured in the absence of the test substance. The screening method according to the above [4].
[6] The human intestinal bacterium having an aromatic amino acid decarboxylase gene is a human intestinal bacterium selected from the group consisting of Enterococcus faecalis, Ruminococcus gnavus, Blartia hansenii, Clostridium nexile and Clostridium asparagiforme. Screening method.
[7] The screening method according to the above [5] or [6], wherein the aromatic amino acid is phenylalanine and the aromatic amine is phenethylamine.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a pharmaceutical composition for preventing or treating a disease associated with an increase in peripheral serotonin or intestinal aromatic amine. Since an aromatic amino acid decarboxylase inhibitor that has already been clinically used can be used as an active ingredient, a highly safe drug can be provided. In addition, the screening method of the present invention can obtain a substance useful for the prevention or treatment of diseases associated with an increase in peripheral serotonin or intestinal aromatic amine, thereby increasing the amount of novel peripheral serotonin or intestinal aromatic amine. Pharmaceuticals for the prevention or treatment of associated diseases can be provided.

It is a figure which shows the result of culturing 32 kinds of human intestinal bacteria dominant species, and measuring the phenethylamine in the culture supernatant. It is a figure which shows the result of having confirmed the aromatic amine production profile of the human intestinal bacterium which produces phenethylamine. It is a figure which shows the result of having confirmed the phenethylamine production ability of the wild type | It is a figure which shows the result of having measured the serotonin level in the large intestine tissue in the mouse which made the aromatic amino acid decarboxylase gene deficient strain and the aromatic amino acid decarbonizing enzyme gene complementary strain of Enterococcus faecalis the dominant flora in the intestine, respectively. It is a figure which shows the result of having confirmed the correlation between the number of copies of an aromatic amino acid decarboxylase gene in human feces and the ability to produce phenethylamine. It is a figure which shows the result of having investigated the suppression of the phenethylamine production of Enterococcus faecalis by the aromatic amino acid decarboxylase inhibitor.

[Pharmaceutical composition]
The present invention provides a pharmaceutical composition for preventing or treating a disease associated with an increase in peripheral serotonin or an intestinal aromatic amine, which comprises an agent that inhibits an aromatic amino acid decarbonase of an intestinal bacterium as an active ingredient. The active ingredient of the pharmaceutical composition of the present invention is not particularly limited as long as it is a drug that inhibits the aromatic amino acid decarboxylase of enterobacteria. For example, the drug that inhibits the aromatic amino acid decarbonase of the intestinal bacterium may be a drug that inhibits the expression of the aromatic amino acid decarbonase of the intestinal bacterium, and the aromatic amino acid decarbonase of the intestinal bacterium It may be a drug that inhibits the function of. The active ingredient of the pharmaceutical composition of the present invention may be a substance selected by the screening method of the present invention described later, or may be a known peripheral aromatic amino acid decarboxylase inhibitor. Known peripheral aromatic amino acid decarbonase inhibitors that inhibit the aromatic amino acid decarbonase of enterobacteria include, for example, carbidopa, benserazide and the like.

Carbidopa is a well-known ingredient in the treatment of Parkinson's disease in combination with Levodopa. The combination drug of levodopa and carbidopa has been approved as a prescription drug in Japan and is listed in the NHI price list. It is listed as carbidopa hydrate in the Japanese Pharmacopoeia, and the carbidopa used in the present invention may be carbidopa hydrate. The information provided in the Japanese Pharmacopoeia is as follows.

Generic name: Carbidopa Hydrate
Chemical name: (2S) -2 (-3,4-Dihydroxybenzyl) -2-hydrazinopropanoic acid monohydrate
Molecular formula: C ₁₀ H ₁₄ N ₂ O ₄ · H ₂ O
Molecular weight: 244.24
Structural formula:

Benserazide is a well-known ingredient in the treatment of Parkinson's disease in combination with Levodopa. The combination drug of levodopa and benserazide has been approved as a prescription drug in Japan and is listed in the NHI price list. It is listed as benserazide hydrochloride in the Japanese Pharmacopoeia, and the benserazide used in the present invention may be benserazide hydrochloride. The information provided in the Japanese Pharmacopoeia is as follows.

Generic name: Benserazide Hydrochloride
Chemical name: (2RS) -2-Amino-3-hydroxy-N'-(2,3,4-trihydroxybenzyl) propanoylhydrazide monohydrochloride
Molecular formula: C ₁₀ H ₁₅ N ₃ O ₅ · HCl
Molecular weight: 293.70
Structural formula:

Diseases associated with an increase in peripheral cellotonin or intestinal aromatic amines include, for example, osteoporosis, irritable bowel disease, ulcerative colitis, celiac disease, Crohn's disease and the like. Peripheral serotonin levels in osteoporosis patients (see Non-Patent Documents 1 and 2), celiac disease patients (see Non-Patent Document 3), irritable bowel disease patients and ulcerative colitis patients (see Non-Patent Document 4) compared to healthy subjects It has been reported that the amount of aromatic amines (phenylethylamine, tyramine) in the feces of Crohn's disease patients and ulcerative colitis patients is higher than that of healthy subjects (see Non-Patent Document 6). .. Therefore, these diseases can be prevented, ameliorated or treated by administering the pharmaceutical composition of the present invention containing a drug that inhibits the aromatic amino acid decarboxylase of enterobacteria as an active ingredient.

The pharmaceutical composition of the present invention can be formulated by appropriately blending the above-mentioned active ingredient with a pharmaceutically acceptable carrier and an additive. Specifically, oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions and emulsions; parenteral preparations such as injections, infusions, suppositories, ointments and patches. can do. The blending ratio of the carrier or the additive may be appropriately set based on the range usually adopted in the pharmaceutical field. The carriers or additives that can be blended are not particularly limited, but for example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; excipients, binders, pH adjusters, disintegrants, absorptions, etc. Examples include various additives such as accelerators, lubricants, colorants, flavoring agents and fragrances.

Additives that can be mixed with tablets, capsules, etc. include, for example, binders such as gelatin, cornstarch, tragant, gum arabic, excipients such as crystalline cellulose, cornstarch, gelatin, alginic acid and the like. Binders, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavors such as peppermint, reddish oil or cherry are used. When the dispensing unit form is a capsule, the above-mentioned type of material can further contain a liquid carrier such as fat or oil. Sterile compositions for injection can be prepared according to routine formulation practices (eg, dissolving or suspending the active ingredient in a solvent such as water for injection, natural vegetable oil, etc.). As the aqueous solution for injection, for example, a physiological saline solution, an isotonic solution containing glucose or other adjuvant (for example, D-sorbitol, D-mannitol, sodium chloride, etc.) is used, and an appropriate solubilizing agent is used. , For example, alcohol (eg, ethanol), polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant (eg, polysorbitol 80, HCO-50) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and may be used in combination with benzyl benzoate, benzyl alcohol and the like as solubilizing agents. Also, buffers (eg, phosphate buffer, sodium acetate buffer), soothing agents (eg, benzalkonium chloride, prokine hydrochloride, etc.), stabilizers (eg, human serum albumin, polyethylene glycol, etc.), storage. It may be blended with an agent (for example, benzyl alcohol, phenol, etc.), an antioxidant, or the like. The formulations thus obtained are safe and have low toxicity and therefore, for example, against humans and non-human mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). It can be administered orally or parenterally.

The pharmaceutical composition of the present invention can contain 0.001 to 50% by mass, preferably 0.01 to 20% by mass, and more preferably 0.1 to 10% by mass of the active ingredient. The dose of the pharmaceutical composition of the present invention is appropriately set in consideration of the purpose, the type of disease, the severity of the disease, the age, weight, sex, medical history, type of active ingredient, and the like of the patient. When an average human having a body weight of about 65 to 70 kg is targeted, about 0.02 mg to 4000 mg per day is preferable, and about 0.1 mg to 500 mg is more preferable. The total daily dose may be a single dose or a divided dose.

The present invention includes the following inventions.
(A) A method for preventing or treating a disease associated with an increase in peripheral serotonin or intestinal aromatic amine, which comprises the step of administering an effective amount of an agent that inhibits the aromatic amino acid decarbonase of intestinal bacteria.
(B) An agent that inhibits the aromatic amino acid decarbonase of intestinal bacteria for use in the prevention or treatment of diseases associated with an increase in peripheral serotonin or intestinal aromatic amine.
(C) Use of a drug that inhibits the aromatic amino acid decarbonase of gut flora to produce a pharmaceutical composition for the prevention or treatment of diseases associated with an increase in peripheral serotonin or intestinal aromatic amine.

[Screening method]
The present invention provides a method for screening a prophylactic or therapeutic agent for a disease associated with an increase in peripheral serotonin or intestinal aromatic amine or a candidate substance thereof. The screening method of the present invention may include a step of selecting a test substance that inhibits the aromatic amino acid decarboxylase of an endobacterium. Diseases associated with an increase in peripheral cellotonin or intestinal aromatic amines include the aforementioned osteoporosis, irritable bowel disease, ulcerative colitis, celiac disease, Crohn's disease and the like.

The test substance used in the screening method of the present invention is not particularly limited, and nucleic acids, peptides, proteins, non-peptidic compounds, synthetic compounds, fermented products, cell extracts, cell culture supernatants, plant extracts, mammals The tissue extract, plasma, etc. may be used. The test substance may be a novel substance or a known substance. These test substances may form salts. As the salt of the test substance, a salt with a physiologically acceptable acid or base is preferable.

The screening method of the present invention may include, for example, the following steps 1 to 3.
Step 1: A step of culturing an intestinal bacterium having an aromatic amino acid decarbonase gene in the presence or absence of a test substance in a medium supplemented with an aromatic amino acid.
Step 2: Detecting aromatic amines in the medium, and Step 3: Reduce the amount of aromatic amines in the medium compared to the amount of aromatic amines in the medium when cultured in the absence of the test substance. The step of selecting the test substance to be subjected to.

The intestinal bacterium having the aromatic amino acid decarbonase gene used in step 1 is not particularly limited, and may be an intestinal bacterium having a known aromatic amino acid decarbonase gene, and the aromatic amino acid decarbonase to be discovered in the future It may be an intestinal bacterium having a carbonate enzyme gene. The gut flora having the aromatic amino acid decarbonase gene may be a human gut flora or a non-human mammalian gut flora. Examples of human intestinal bacteria having an aromatic amino acid decarboxylase gene include Enterococcus faecalis, Ruminococcus gnavus, Blartia hansenii, Clostridium nexile, Clostridium asparagiforme and the like. The human intestinal bacterium used in the screening method of the present invention may be Enterococcus faecalis or Ruminococcus gnavus, or may be Enterococcus faecalis.

Enterobacteriaceae carrying the aromatic amino acid decarboxylase gene are usually cultured under anaerobic conditions (anaerobic culture). When an intestinal bacterium having an aromatic amino acid decarboxylase gene is anaerobically cultured, a known anaerobic culture medium can be used as the medium. Examples of the medium for anaerobic culture include Gifu anaerobic medium (GAM). The medium used in the screening method of the present invention may be a liquid medium. The method of anaerobic culture is not particularly limited, and can be appropriately selected from known anaerobic culture methods according to the intestinal bacteria to be used.

Aromatic amino acids, which are substrates for aromatic amino acid decarboxylase, are added to the medium used in the screening method of the present invention. The aromatic amino acid may be any of phenylalanine, tyrosine and tryptophan, and may be phenylalanine. The amount of the aromatic amino acid added is not particularly limited, but may be, for example, about 0.1 mM to about 10 mM, about 0.5 mM to about 5 mM, or about 0.8 mM to about 2 mM. It may be about 1 mM.

The amount of the test substance added may be an amount that does not significantly suppress the growth of the intestinal bacteria as compared with the growth level of the intestinal bacteria in the absence of the test substance, and is compared with the growth level of the intestinal bacteria in the absence of the test substance. The amount may not suppress the growth. The culturing time is not particularly limited, and may be 6 hours or more, 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more. May be good. The upper limit of the culturing time is not particularly limited, but may be 48 hours or less, 36 hours or less, or 24 hours or less.

In step 2, the aromatic amine in the medium is detected. That is, the aromatic amine decarboxylated by the decarboxylation of the substrate aromatic amino acid is detected by the aromatic amino acid decarboxylase expressed by the intestinal bacteria used. When phenylalanine is used as the substrate, phenethylamine is produced, when tyrosine is used as the substrate, tyramine is produced, and when tryptophan is used as the substrate, tryptamine is produced. The method for detecting aromatic amines is not particularly limited, and can be appropriately selected from known methods for detecting aromatic amines. The method for detecting aromatic amines used in the screening method of the present invention may be the HPLC method used in the examples described later.

In step 3, a test substance that reduces the amount of aromatic amines in the medium may be selected as compared with the amount of aromatic amines in the medium when cultured in the absence of the test substance. If the amount of aromatic amine in the medium is reduced when cultured in the presence of the test substance, it can be reasonably understood that the test substance inhibited the aromatic amino acid decarbonase of the enterobacteria. .. The degree to which the amount of aromatic amine is reduced is not particularly limited, but for example, it is 90% or less, 80% or less, 70% or less as compared with the amount of aromatic amine in the medium when cultured in the absence of the test substance. , 60% or less, 50% or less, 40% or less, 30% or less may be selected.

The test substance selected by the screening method of the present invention is useful as an active ingredient of the above-mentioned pharmaceutical composition of the present invention, and is suitably used for the prevention or treatment of diseases associated with an increase in peripheral serotonin or intestinal aromatic amine. Can be done. In addition, the test substance selected by the screening method of the present invention is useful as a candidate substance for the active ingredient of the pharmaceutical composition of the present invention, and after further screening, it becomes the active ingredient of the pharmaceutical composition of the present invention. Has the potential to become.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto.

[Example 1: Identification of human intestinal bacteria producing phenethylamine]
1. 1. Experimental method (1) Medium
Gifu anaerobic medium (GAM bouillon "Nissui", Nissui Pharmaceutical, hereinafter abbreviated as "GAM") is sterilized in an autoclave (115 ° C, 15 min), and when the temperature reaches 95 ° C, the medium is taken out and the anaerobic chamber (INVIVO2) is taken out. 400, Ruskinn Technologies) was allowed to stand overnight to remove dissolved oxygen. In the anaerobic chamber, 500 μL was dispensed into 1 mL 96 deep-well plate.

(2) Strain and culture conditions
We selected 32 predominant human gut flora species that can be cultivated in GAM (see Fig. 1). 5 μL of glycerol stock of each strain was inoculated into 500 μL of GAM in a 96 deep-well plate, a Gas permeable moisture barrier sheel (4titude) was attached to the 96 deep-well plate, and the cells were precultured at 37 ° C for 24-48 hours. The preculture was suspended in an anaerobic chamber using a multi-channel pipette, and then inoculated into 500 μL of GAM in a 96 deep-well plate using a copy plate 96 (Tokken). A gas permeable moisture barrier sheel (4titude) was attached, and main culture was performed at 37 ° C in an anaerobic chamber. After the start of culturing, OD600 was measured over time using 20 μL of the main culture solution to create a growth curve, and the OD600 became constant after the growth phase and the time when the value showed about half of the maximum OD600. The time was defined as the stationary period.

(3) Detection of phenethylamine in the culture supernatant (3-1) Sample preparation The 96 deep-well plate containing the main culture solution was collected over time and centrifuged (4,400 rpm, 4 ° C, 40 min) to culture supernatant. Got 1/10 amount of 100% (w / v) Trichloroacetic acid (TCA) was added to the obtained culture supernatant. After the addition, the mixture was thoroughly mixed and subjected to centrifugation (18,900 × g, 4 ° C, 10 min). After centrifugation, the obtained supernatant was filtered through a Cosmonice filter (0.45 μm, PVDF), and the filtrate was subjected to high performance liquid chromatography (HPLC).

(3-2) High Performance Liquid Chromatography (HPLC)
The post-column derivatization method with orthophthalaldehyde (o-phthalaldehyde) was used for the analysis of phenethylamine. A # 2619 PH (4.6 x 50 mm, Hitachi) was used for the separation column, and the temperature was maintained at 67 ° C. The flow velocity of the mobile phase was 0.4 mL / min, and mobile phase A (45.2 mM sodium citrate, 63.3 mM sodium chloride, and 60.9 mM citrate) and mobile phase B (200 mM sodium citrate, 2 M sodium chloride,) Using 5% ethanol, and 5% 1-propanol), increase mobile phase B concentration to 50-85% over 0-6 minutes, hold at 85% for up to 12 minutes, and increase to 100% over 12-18 minutes. , Held at 100% for up to 45 minutes, then reduced to 50% and held at 50% for up to 60 minutes. Derivatization with phthalaldehyde is performed by using reaction solution 1 (400 mM NaOH) and reaction solution 2 (234 mM boric acid, 0.05% Brij-35, 5.96 mM orthophthalaldehyde, 0.2% 2-mercaptoethanol) at 0.35 mL / min, respectively. This was done by pouring and mixing with column eluate in a column oven. Detection was performed with a fluorescence detector (λ ex 340 nm, λ em 435 nm).

2. 2. result
Among 32 species of human gut flora, 5 species of Ruminococcus gnavus, Blartia hansenii, Clostridium nexile, Clostridium asparagiforme and Enterococcus faecalis were found to produce phenethylamine.

[Example 2: Aromatic amine production profile of phenethylamine-producing bacteria]
1. 1. Experimental method (1) Medium GAM was prepared in the same manner as in Example 1. A medium (main culture medium) with an aromatic amino acid (phenylalanine, tyrosine, tryptophan) concentration of 1 mM was added with GAM at 10% (v / v) based on the composition of the minimum medium of Bacteroides bacteria. It was prepared by adding each aromatic amino acid solution so that the total amount of each aromatic amino acid contained in GAM was 1 mM. The prepared medium was filter sterilized (0.22 μm, PVDF), 30 mL each was dispensed into a 50 mL centrifuge tube, and allowed to stand overnight in an anaerobic chamber (INVIVO2 400, Ruskinn Technologies) to remove dissolved oxygen. The composition of the main culture medium is shown below.
10% (v / v) GAM, 0.5% (w / v) glucose, 100 mM potassium dihydrogen phosphate, 15 mM sodium chloride, 8.5 mM ammonium sulfate, 4 mM L-cysteine, 1.9 μM hematine, 200 μM L-histidine , 1 μg / mL Vitamin K3, 5 ng / mL Vitamin B12, 100 μM Magnesium Chloride, 1.4 μM Iron Sulfate, 50 μM Calcium Chloride

(2) Strains and culture conditions Five species of human gut flora confirmed to produce phenethylamine in Example 1 were inoculated into 3 mL of GAM and placed in an anaerobic chamber (INVIVO2 400, Ruskinn Technologies) at 37 ° C. Inoculated 18 hours before. 1 mL of the preculture solution was centrifuged (3,400 g, 25 ° C., 3 min), the supernatant was removed, and the cells were collected. The obtained bacterial cells were suspended in 1 mL of the main culture medium and centrifuged again (3,400 g, 25 ° C., 3 min) to wash the bacterial cells. The washed cells were resuspended in 1 mL of the main culture medium, and a part of the cells was used to measure OD600. Based on the measured OD600, the cells were inoculated into 30 mL of the main culture medium so that the OD600 was 0.03. After inoculation, the cells were cultured in an anaerobic chamber, and the culture solution was sampled 6, 12, 18, 24, and 48 hours after the start of the culture to detect aromatic amines.

(3) Detection of aromatic amines in culture supernatant (3-1) Sample preparation
1 mL of the medium was collected, and the culture supernatant was obtained by centrifugation (18,900 × g, 4 ° C., 10 min). 1/10 amount of 100% (w / v) Trichloroacetic acid (TCA) was added to the obtained culture supernatant. After the addition, the mixture was thoroughly mixed and subjected to centrifugation (18,900 × g, 4 ° C, 10 min). After centrifugation, the resulting supernatant was filtered through a Cosmonice filter (0.45 μm, PVDF) and the filtrate was subjected to high performance liquid chromatography (HPLC).

(3-2) High Performance Liquid Chromatography (HPLC)
The analysis of phenethylamine was performed in the same manner as in Example 1.
For HPLC analysis of tyramine and tryptamine, Discovery HS-F5 (4.6 x 150 mm, Supelco) was used as a separation column and kept at 30 ° C. 10 mM ammonium formate (pH 3.0) and acetonitrile were used as the mobile phase, and the flow rate was 0.4 mL / min. The gradient program increased the acetonitrile concentration from 3% to 27% over 0-22 minutes, increased to 66% over 22-80 minutes, increased to 100% over 80-81 minutes, held at 100% for up to 86 minutes, 87 The program was reduced to 3% over minutes and held at 3% for up to 102 minutes. Detection was performed with a fluorescence detector (λ ex 280 nm, λ em 325 nm).

2. 2. Results The results are shown in FIG. (A) is the result of B. hansenii, (B) is the result of R. gnavus, (C) is the result of C. nexil, (D) is the result of C. asparagiforme, and (E) is the result of E. faecalis. is there. B. hansenii, R. gnavus and C. nexil produced the highest amount of tryptamine. C. asparagiforme and E. faecalis produced the highest amount of tyramine.

[Example 3: Examination of phenethylamine-producing ability by wild strain of E. faecalis, aromatic amino acid decarboxylase gene-deficient strain, and aromatic amino acid decarboxylase gene complementary strain]
1. 1. Experimental method (1) Strain used
The wild strain SK947 of Enterococcus faecalis, the aromatic amino acid decarboxylase gene (aadc) -deficient strain SK981, and the aromatic amino acid decarboxylase gene (aadc) complementary strain SK982 were used.

The method for producing each strain used is as follows. The strains used are shown in Table 1, the plasmids used are shown in Table 2, and the primers used are shown in Table 3.

(1-1) Preparation of wild strain SK947 of E. faecalis
The wild strain of E. faecalis (WT strain, SK947) was prepared based on E. faecalis V583. Specifically, E. faecalis V583 with empty vector pLZ12 (J. Perez-Casal, MG Caparon, and JR Scott, “Mry, a trans-acting positive regulator of the M protein gene of Streptococcus pyogenes with similarity to the receptor proteins) SK947 was obtained by introducing "of two-component regulatory systems", J. Bacteriol., Vol. 173, no.8, pp. 2617-2624, 1991.) by electroporation.

(1-2) Preparation of temperature-sensitive plasmid pLT06-Δaadc with aadc-deficient cassette Next, temperature-sensitive plasmid pLT06 (LR Thurlow, VC Thomas, and LE Hancock, “Capsular polysaccharide production in Enterococcus faecalis and contribution of CpsF to capsule serospecificity” , J. Bacteriol., Vol. 191, no.20, pp.6203-6210, 2009.), and pLT06-Δaadc having an aadc-deficient cassette was prepared. A region of about 1 kbp upstream and downstream of the aadc gene ORF on the genome of the E. faecalis V583 strain was amplified by PCR. During amplification, primers for_Del_ddc_1_F and for_Del_ddc_2_5P_R were used for amplification in the upstream region, and primers for_Del_ddc_3_5P_F and for_Del_ddc_4_R were used for amplification in the downstream region. Both amplification products were ligated and PCR was performed again for full-length amplification of the addc-deficient cassette on a DNA fragment of about 2 kbp. As primers, pLT06-EcoRI_D_ddc_F and pLT06-EcoRI_D_ddc_R were used. The amplified fragment was cloned into pLT06 cleaved by EcoRI by the infusion method to prepare pLT06-Δaadc.

(1-3) Preparation of pLZ12-aadc + having an aadc gene region Next, pLZ12-aadc + derived from pLZ12 and having an aadc gene region was prepared. A 2,550 bp region containing the aadc gene and its upstream 500 bp on the genome of the E. faecalis V583 strain was amplified by PCR. DNA of E. faecalis V583 was used as a template, and C_ddc + 0.5k_F_pLZ_Bam and C_ddc + 0.5k_R_pLZ_Bam were used as primers. The obtained amplification product was cloned into pLZ12 cleaved with BamHI to prepare pLZ12-aadc +.

(1-4) Preparation of aadc-deficient strain SK977 An aadc-deficient strain (SK977) was prepared by introducing the pLT06-Δaadc prepared in (1-2) above into the E. faecalis V583 strain. Specifically, pLT06-Δaadc was introduced into the E. faecalis V583 strain by electroporation, cultured, and then incubated at 42 ° C. to remove the temperature-sensitive plasmid pLT06-Δaadc. 10 μg / mL chloramphenicol (CF) and 120 μg / mL 5-bromo-4-chloro-3-indrill-β-D- were introduced into the plasmid by single crossover near the aadc region on the genome. Screening was performed on THB agar medium supplemented with galactopyranoside (X-gal). For colonies grown on THB agar medium, it was confirmed by PCR that gene recombination occurred correctly in the target region. Then, by performing two cultures in a medium containing no CF, the plasmid introduced into the genome was removed by loop-out, and 120 μg / mL X-gal and 10 μg / mL p-chlorophenylalanine were added. Strains with successful plasmid removal were screened on MM9YEG agar containing. It was confirmed by PCR that the plasmid region was correctly removed from the genome of the white colonies that showed resistance to p-chlorophenylalanine. Furthermore, the recombinant region was subjected to PCR amplification and base sequence analysis by the direct sequence method, and it was confirmed that no unintended mutation was introduced.

(1-5) Preparation of aadc-deficient strain SK981 pLZ12 was introduced into the SK977 prepared in (1-4) above by electroporation to prepare aadc-deficient strain SK981.

(1-6) Preparation of aadc complementary strain SK982.
PLZ12-aadc + was introduced into the obtained SK977 by electroporation to prepare an aadc complementary strain SK982.

(1-7) Confirmation of each strain
The presence or absence of the aadc gene and pLZ12-aadc + on the genomes of SK947, SK981 and SK982 was confirmed by the following method. Using E. faecalis genomic DNA as a template and Conf_Del_ddc_F and Conf_Del_ddc_R as primers, the region (4,084 bp) containing the aadc gene on the genome was amplified. In addition, pLZ12_MCS_FWD and pLZ12_MCS_COMPL were used as primers to amplify the region containing pLZ12-aadc + (2,850 bp). The PCR reaction solution was prepared using DNA polymerase (PrimeSTAR Max DNA Polymerase, Takara Bio). PCR uses a thermal cycler (TaKaRa PCR Thermal Cycler Dice Gradient), 98 ° C 10 seconds, 50 ° C 30 seconds, 72 ° C 4 minutes, 35 cycles or 98 ° C 10 seconds, 56 ° C 30 seconds, 72 ° C. The reaction was carried out in 30 cycles for 3 minutes. After the PCR reaction, electrophoresis was performed using an agarose gel (Agarose S, Fuji Film Wako Pure Chemical Industries, Ltd.), and the region containing the aadc gene on the genome was at the position of 4,084 bp, and pLZ12-aadc + was at the position of 2,850 bp with or without a band. It was confirmed.

(2) Detection of phenethylamine in medium, culture conditions, and culture supernatant The wild strain SK947, aadc-deficient strain SK981 and aadc complementary strain SK982 of E. faecalis were cultured in the same medium and culture conditions as in Example 2. Detection of phenethylamine in the culture supernatant by HPLC was performed in the same manner as in Example 1.

2. 2. Results The results are shown in FIG. Phenethylamine was detected in the culture supernatants of the wild-type strain and the aadc complementary strain, but no phenethylamine was detected in the culture supernatant of the aadc-deficient strain. From this result, it was clarified that E. faecalis produced phenethylamine using an aromatic amino acid decarboxylase.

[Example 4: Examination of serotonin levels in colon tissue in mice in which the aromatic amino acid decarboxylase gene disrupted strain and the aromatic amino acid decarbonizing enzyme gene complementary strain of E. faecalis were used as the predominant intestinal flora]
1. 1. Experimental method (1) Mouse used
A 6-week-old female BALB / cCrSlc (Japan SLC) was used.

(2) Feed
CLEA Rodent Diet CE-2 (Clear Japan) and AIN-93G as a basic combination, and a special combination feed that does not contain tyrosine and whose amino acid composition is changed so that the phenylalanine content is 10 times the basic composition. used. The composition of the feed based on AIN-93G is shown below.
L-alanine 3.70 g / kg, L-arginine 5.13 g / kg, L-aspartic acid 9.22 g / kg, L-cystine 3.00 g / kg, L-glutamic acid 10.29 g / kg, glycine 2.52 g / kg, L-histidine 3.66 g / kg, L-isoleucine 6.84 g / kg, L-leucine 12.36 g / kg, L-lysine-HCl 13.05 g / kg, L-methionine 3.62 g / kg, L-phenylalanine 87.00 g / kg, L-proline 16.52 g / kg, L-serine 7.58 g / kg, L-threonine 5.36 g / kg, L-tryptophan 1.75 g / kg, L-tyrosine 0.00 g / kg, L-valine 8.03 g / kg, sucrose 100 g / kg , Corn starch 380.456 g / kg, Jietroz 145 g / kg, soybean oil 70 g / kg, tBHQ 0.014 g / kg, cellulose 50 g / kg, salt mix # 210030 35 g / kg, sodium bicarbonate 7.4 g / kg, vitamin mix # 310025 10 g / kg, choline heavy tartrate 2.5 g / kg (total weight 1 kg)

(3) Antibiotic treatment In order to remove the intestinal bacteria of mice as much as possible, drinking water was prepared by adding two kinds of antibiotics, doripenem and vancomycin, to sterile tap water. Antibiotic concentrations were 0.25 g / L doripenem (Finivax intravenous infusion 0.5 g, Shionogi Pharmaceutical Co., Ltd.) and 0.5 g / L vancomycin (vancomycin hydrochloride intravenous infusion 0.5 g, Shionogi Pharmaceutical Co., Ltd.). is there.

(4) Strains used E. faecalis aadc-deficient strain SK981 and aadc complementary strain SK982 used in Example 3 were used. Bacteria were floated in Lactobacillus MRS (LMRS) Broth supplemented with 10 μg / mL chloramphenicol (CF) with 50% (v / v) glycerol and stored at -20 ° C until use.

(5) Medium
GAM with 10 μg / mL chloramphenicol (CF) added was used. Just before use, CF was added to a concentration of 10 μg / mL. After autoclaving, 1.6% GAM Agar was cooled to 50 ° C in a constant temperature water bath set at 50 ° C, CF was added to a concentration of 10 μg / mL, and the mixture was dispensed into a petri dish and solidified. This was put into an anaerobic jar together with Aneropack Kenki (trade name) to remove dissolved oxygen and used.

(6) Preparation of bacterial suspension
Stock strains stored at -20 ° C were seeded in 5 mL of 10 μg / mL CF-added GAM and pre-cultured at 37 ° C for 12 hours under anaerobic conditions. From the bacterial solution after preculture, 5 μL (1.4 × 10 ⁹ CFU / mL) was seeded in 5 mL of 10 μg / mL CF-added GAM Broth, and main-cultured at 37 ° C. for 12 hours under anaerobic conditions. After the main culture, 1 mL of the culture solution was dispensed into an Eppen tube, centrifuged (6,000 × g, 25 ° C, 5 min), and the supernatant was removed to collect the bacterial cells. 1 mL of 10 μg / mL CF-added PBS was added to the collected cells, suspended, centrifuged (6,000 × g, 25 ° C, 5 min), and the operation of removing the supernatant was repeated twice to wash the cells. After the second wash, the cells were suspended in 5 mL of PBS, diluted with this suspension and smeared with 10 μg / mL CF-added GAM Agar, and cultured in an anaerobic jar at 37 ° C. After culturing, the number of bacteria was measured by colony counting, and the bacterial concentration was adjusted to 1 × 10 ⁸ CFU / 200 μL.

(7) Experimental protocol The experiments were conducted by dividing 10 mice into 2 groups (aadc-deficient strain administration group and aadc complementary strain administration group). Enterococcus faecalis Mice were treated with antibiotics for 2 weeks to reduce the number of bacteria in the intestinal tract by a factor of 1,000,000 in order to have a predominant flora. The start date of drinking water administration of antibiotics to mice was set to Day 0, and CE-2 was given as feed to start the experiment. On Day 13, the diet was changed from CE-2 to a special formulation of AIN-93G to increase the amount of phenylalanine in the intestinal tract and at the same time decrease tyrosine. Antibiotic administration was completed on Day 14, and drinking water was changed to sterile water. Bacterial solution was administered on Day 15. Under anesthesia, 1 × 10 ⁸ CFU / 200 μL of E. faecalis was orally administered to each mouse using an oral sonde (5202K, Fuchigami instrument). Three days after the administration of E. faecalis (Day 18), the feces, colon tissue, and cecal contents of the mice were collected. Specifically, 10 feces (about 10 mg) were collected per mouse, then euthanized and laparotomized, and the colon tissue and the contents of the cecum were removed. The collected fecal sample was anaerobicized with Aneropack Kenki (trade name) and stored at -80 ° C. The colon tissue was placed in a 50 mL centrifuge tube containing 15 mL of PBS, kept dry until sample processing, and ice-cooled together with the centrifuge tube. More than 100 μL of the cecal contents were collected, transferred to an Eppen tube, and stored at -80 ° C. During the experimental period (Day 0-18), diurnal changes in body weight and food intake were measured.

(8) Treatment of large intestine tissue
The colon tissue that had been ice-cooled in a PBS-containing centrifuge tube was taken out on a petri dish, and the intestinal tract was opened by making a notch in the longitudinal direction with dissecting scissors, and feces in the intestinal tract were removed with tweezers. Then, it was washed twice with 10 mL of PBS. Transfer the washed large intestine to a centrifuge tube containing 10 mL of PBS, and repeat the cycle of ultrasonic transmission for 30 seconds and standing for 60 seconds with an ultrasonic homogenizer (Model 250, BRANSON) 5 times per sample to remove the large intestine tissue. Homogenized. The homogenized colon sample was stored at -25 ° C as a centrifuge tube.

(9) Serotonin measurement A cryopreserved colon sample was thawed, centrifuged (15,000 rpm, 4 ° C, 5 min), and 1 mL of the supernatant was collected. 100 μL was subjected to ELISA (Serotonin ELISA Kit, Enzo Life science) for quantification of serotonin. ELISA was performed according to the manufacturer's protocol. From the calculated serotonin concentration and the weight of the large intestine tissue, the amount of serotonin per 1 g of the large intestine tissue was calculated. The serotonin level was determined as the amount of serotonin per 1 g of colon tissue of each individual in the aadc complementary strain-administered group, with the average amount of serotonin per 1 g of colon tissue in the aadc-deficient strain-administered group as 1. The Mann-Whitney U test was used for statistical processing.

2. 2. Results The results are shown in FIG. The serotonin level in the colon tissue of the aadc complementary strain-administered group was significantly higher than that of the aadc-deficient strain-administered group.

[Example 5: Examination of the number of copies of the aromatic amino acid decarboxylase gene and the ability to produce phenethylamine in human feces]
1. 1. Experimental method (1) Measurement of phenethylamine in feces
Feces were collected from 9 healthy adults. Approximately 100 mg of stool was weighed, and 9 times the amount of PBS or PBS containing 1 mM phenylalanine was added to prepare a stool suspension. The prepared fecal suspension was incubated in an anaerobic chamber at 37 ° C. for 6 hours. After incubation, the mixture was centrifuged (18,900 × g, 4 ° C, 10 min) and the supernatant was collected. 1/10 amount of 100% (w / v) Trichloroacetic acid (TCA) was added to the obtained supernatant. After the addition, the mixture was thoroughly mixed and subjected to centrifugation (18,900 × g, 4 ° C, 10 min). After centrifugation, the obtained supernatant was filtered through a Cosmos pin filter H (0.45 μm, PVDF), and the filtrate was subjected to HPLC. Detection of phenethylamine in the culture supernatant by HPLC was performed in the same manner as in Example 1.

(2) Number of aadc gene copies in stool Approximately 20 mg of stool collected from the above 9 healthy adults was weighed and suspended in 95 μL of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Add 5 μL of 300 mg / mL lysozyme solution (lysozyme dissolved in TE buffer) and 11 μL of 20,000 U / mL achromopeptidase solution (acromopeptidase dissolved in TE buffer) at 37 ° C. 15 Incubated for minutes. After incubation, 12 μL of 20% (w / v) sodium dodecyl sulfate solution was added, mixed for 5 minutes, and then incubated at 60 ° C. for 5 minutes. After incubation, DNA extraction was performed using the QIAamp Fast DNA Stool Mini Kit (QIAGEN) according to the manufacturer's protocol. The extracted DNA concentration was calculated from the absorbance at 260 nm using a μDrop plate (Thermo Fisher Scientific).

Based on the measured DNA concentration, a 1 ng / μL DNA solution was prepared using sterile water and subjected to quantitative PCR. Quantitative PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio) in a reaction system with a total volume of 20 μL. The reaction composition is 10 μL of 2 x TB qPCR mix, 9.2 μL of 1 ng / μL DNA solution, and 0.8 μL of 17.5 μM primer mixture. The primer mixture is Rgna_aadc_qPCR2_Fw (5'-AACCGGGCTTGCTGACAGTA-3', SEQ ID NO: 13) and Rgna_aadc_qPCR2_Rv (5'-CGTACGTCTGGAAGAGCCATTT-3', SEQ ID NO: 14) (100 μM each) mixed with sterile water at a ratio of 1.75: 1.75: 6.5. It was done. PCR was performed for 40 cycles of [95 ° C for 5 seconds → 60 ° C for 60 seconds] after 30 seconds at 95 ° C. pCDF23 (Nakagawa A, Matsumura E, Koyanagi T, Katayama T, Kawano N, Yoshimatsu K, Yamamoto K, Kumagai H, Sato F, Minami H, “Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli.” Nat Commun., A calibration line for calculating the number of copies was prepared using a plasmid in which the aadc gene of R. ganvus was introduced into vol. 7, Article number: 10390, 2016). Based on the copy number calculated from the calibration curve, the aadc gene copy number of R. ganvus per 1 g of stool was calculated.

2. 2. Results The results are shown in FIG. When the amount of phenethylamine produced when phenylalanine was mixed with feces was plotted against the number of copies of the aromatic amino acid decarboxylase gene (aadc) per 1 g of human feces, a significant positive correlation (p = 0.0066) was found. there were. From this result, it was shown that AADC in human feces is likely to convert phenylalanine to phenethylamine. Therefore, it was shown that the amount of phenethylamine produced can be suppressed by inhibiting the aromatic amino acid decarboxylase of enterobacteria.

[Example 6: Suppression of E. faecalis phenethylamine production by an aromatic amino acid decarboxylase inhibitor]
1. 1. Experimental method (1) Medium The same medium as the main culture medium used in Example 2 was used in Example 2. It was prepared by the same method as described in (1). Carbidopa Monohydrate (Tokyo Kasei), known as an aromatic amino acid decarboxylase (aadc) inhibitor, and methyldopa (3- (3,4-Dihydroxyphenyl) -2-methyl-L-alanine Sesquihydrate) are added to this main culture medium. Tokyo Kasei) and benserazide Hydrochloride (Tokyo Kasei) were added to a final concentration of 1.5 mM, respectively, to prepare three types of aadc inhibitor-containing media.

(2) Strain and culture conditions
Enterococcus faecalis was inoculated into 3 mL of GAM and cultured under the same culture conditions as in Example 2. The culture solution was sampled 24 hours after the start of anaerobic culture in the main culture medium to detect phenethylamine.

(3) Detection of phenethylamine in culture supernatant A sample was prepared by the same method as in Example 2 and subjected to HPLC. Analysis of phenethylamine by HPLC was performed in the same manner as in Example 1.

2. 2. Results The results are shown in FIG. Carbidopa and benserazide markedly suppressed the production of phenethylamine in E. faecalis. On the other hand, methyldopa did not suppress the production of phenethylamine in E. faecalis.

The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments may be appropriately combined. The obtained embodiments are also included in the technical scope of the present invention. In addition, all the academic and patent documents described in the present specification are incorporated herein by reference.

Claims (7)

A pharmaceutical composition for preventing or treating a disease associated with an increase in peripheral serotonin or intestinal aromatic amine, which comprises an active ingredient that inhibits an aromatic amino acid decarbonase of an intestinal bacterium. The pharmaceutical composition according to claim 1, wherein the agent that inhibits the aromatic amino acid decarboxylase of enterobacteria is carbidopa or benserazide. The pharmaceutical composition according to claim 1 or 2, wherein the disease associated with an increase in peripheral serotonin or intestinal aromatic amine is osteoporosis, irritable intestinal disease, ulcerative colitis, celiac disease or Crohn's disease. A method for screening a prophylactic or therapeutic agent for a disease associated with an increase in peripheral serotonin or intestinal aromatic amine, which comprises the step of selecting a test substance that inhibits the aromatic amino acid decarbonase of the intestinal bacteria. Step 1 of culturing an intestinal bacterium having an aromatic amino acid decarboxylase gene in the presence or absence of a test substance in a medium supplemented with an aromatic amino acid.
Step 2 to detect aromatic amines in the medium,
The screening according to claim 4, further comprising step 3 of selecting a test substance that reduces the amount of aromatic amines in the medium as compared to the amount of aromatic amines in the medium when cultured in the absence of the test substance. Method. The screening method according to claim 5, wherein the intestinal bacterium having an aromatic amino acid decarboxylase gene is a human intestinal bacterium selected from the group consisting of Enterococcus faecalis, Ruminococcus gnavus, Blartia hansenii, Clostridium nexile and Clostridium asparagiforme. The screening method according to claim 5 or 6, wherein the aromatic amino acid is phenylalanine and the aromatic amine is phenethylamine.