JP7321495B2 - Pharmaceutical composition for prevention or treatment of diseases associated with increased peripheral serotonin or intestinal aromatic amines, and screening method for agents for prevention or treatment of diseases associated with increased peripheral serotonin or intestinal aromatic amines - Google Patents

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating diseases associated with increased peripheral serotonin or intestinal aromatic amines, and a screening method for drugs for preventing or treating diseases associated with increased peripheral serotonin or intestinal aromatic amines. is.

The biodistribution of serotonin, one of the neurotransmitters, is 5% in the brain and 95% in the periphery, and it is known that peripheral serotonin is biosynthesized by enterochromaffine cells (EC cells) in the intestinal tract. there is Peripheral serotonin has been suggested to be associated with various diseases. It has been shown that inflammation patients (Non-Patent Document 4) have higher peripheral serotonin levels than healthy subjects. Furthermore, in mouse models, inhibition of peripheral serotonin biosynthesis ameliorated obesity and metabolic dysfunction, indicating a contribution 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 intestinal bacterial metabolites (Non-Patent Document 6). Non-Patent Document 6 evaluates the ability to promote serotonin biosynthesis by administering various intestinal bacterial metabolites to the intestinal tract of germ-free mice, finds activity in some metabolites, and This indicates that a certain tyramine has the ability to promote serotonin biosynthesis. On the other hand, phenylethylamine and tyramine, which are aromatic amines, have been reported to be higher in the feces of patients with Crohn's disease and ulcerative colitis than in healthy subjects, suggesting their involvement in these diseases (non 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 diseases associated with increased peripheral serotonin or intestinal aromatic amines. 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 amines.

The present invention includes the following inventions in order to solve the above problems.
[1] A pharmaceutical composition for preventing or treating diseases associated with increased peripheral serotonin or intestinal aromatic amines, containing as an active ingredient a drug that inhibits aromatic amino acid decarboxylase of intestinal bacteria.
[2] The pharmaceutical composition according to [1] above, wherein the drug that inhibits aromatic amino acid decarboxylase of intestinal bacteria is carbidopa or benserazide.
[3] The pharmaceutical composition of [1] or [2] above, wherein the disease associated with increased peripheral serotonin or intestinal aromatic amines is osteoporosis, irritable bowel disease, ulcerative colitis, celiac disease or Crohn's disease. thing.
[4] A method for screening a drug for prevention or treatment of a disease associated with an increase in peripheral serotonin or intestinal aromatic amines, comprising the step of selecting a test substance that inhibits aromatic amino acid decarboxylase of intestinal bacteria.
[5] Step 1 of culturing intestinal bacteria having an aromatic amino acid decarboxylase gene in a medium supplemented with an aromatic amino acid in the presence or absence of a test substance; and step 3 of selecting a test substance that reduces the amount of aromatic amine in the medium compared to the amount of aromatic amine in the medium when cultured in the absence of the test substance. The screening method according to [4] above.
[6] The above [5], wherein the enteric bacterium having an aromatic amino acid decarboxylase gene is a human enteric bacterium selected from the group consisting of Enterococcus faecalis, Ruminococcus gnavus, Blaartia hansenii, Clostridium nexile and Clostridium asparagiforme. screening method.
[7] The screening method according to [5] or [6], wherein the aromatic amino acid is phenylalanine and the aromatic amine is phenethylamine.

INDUSTRIAL APPLICABILITY The present invention can provide a pharmaceutical composition for preventing or treating diseases associated with increased peripheral serotonin or intestinal aromatic amines. 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, by the screening method of the present invention, it is possible to obtain a substance useful for the prevention or treatment of a disease accompanied by an increase in peripheral serotonin or intestinal aromatic amines. A medicament for the prevention or treatment of associated diseases can be provided.

FIG. 2 is a diagram showing the results of culturing 32 types of human intestinal bacteria dominant species and measuring phenethylamine in the culture supernatant. FIG. 2 is a diagram showing the results of confirming the aromatic amine production profile of human intestinal bacteria that produce phenethylamine. FIG. 3 shows the results of confirming the phenethylamine-producing ability of a wild strain, an aromatic amino acid decarboxylase gene-deficient strain, and an aromatic amino acid decarboxylase gene-complemented strain of Enterococcus faecalis, which is a phenethylamine-producing human intestinal bacterium. FIG. 2 shows the results of measuring serotonin levels in large intestine tissue in mice with an aromatic amino acid decarboxylase gene-deficient strain and an aromatic amino acid decarboxylase gene-complemented strain of Enterococcus faecalis as dominant intestinal flora. FIG. 2 shows the results of confirming the correlation between the number of copies of the aromatic amino acid decarboxylase gene in human feces and the ability to produce phenethylamine. [ Fig. 2] Fig. 2 is a diagram showing the results of examining the suppression of phenethylamine production in Enterococcus faecalis by an aromatic amino acid decarboxylase inhibitor.

[Pharmaceutical composition]
The present invention provides a pharmaceutical composition for preventing or treating diseases associated with increased peripheral serotonin or intestinal aromatic amines, containing as an active ingredient a drug that inhibits aromatic amino acid decarboxylase of intestinal bacteria. The active ingredient of the pharmaceutical composition of the present invention is not particularly limited as long as it is a drug that inhibits aromatic amino acid decarboxylase of intestinal bacteria. For example, the agent that inhibits the aromatic amino acid decarboxylase of intestinal bacteria may be an agent that inhibits the expression of aromatic amino acid decarboxylase of intestinal bacteria. 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, which will be described later, or a known peripheral aromatic amino acid decarboxylase inhibitor. Examples of known peripheral aromatic amino acid decarboxylase inhibitors that inhibit aromatic amino acid decarboxylase of intestinal bacteria include carbidopa and benserazide.

Carbidopa is well known as an ingredient in Parkinson's disease drugs in combination with Levodopa. A combination drug of levodopa and carbidopa has been approved as an ethical drug in Japan, and is listed in drug prices. It is listed as carbidopa hydrate in the Japanese Pharmacopoeia, and the carbidopa used in the present invention may be carbidopa hydrate. The information described in the Japanese Pharmacopoeia is as follows.

Generic Name: Carbidopa Hydrate
Chemical name: (2S)-2(-3,4-Dihydroxybenzyl)-2-hydrazinopropanoic acid monohydrate
Molecular _{formula : C10H14N2O4 - H2O}
Molecular weight: 244.24
Structural formula:

Benserazide is a well-known component of Parkinson's disease drugs in combination with Levodopa. A combination drug of levodopa and benserazide has been approved as an ethical drug in Japan, and is listed in the NHI drug 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 described 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} : _C10H15N3O5・_HCl
Molecular weight: 293.70
Structural formula:

Diseases associated with increased peripheral serotonin or intestinal aromatic amines include, for example, osteoporosis, irritable bowel disease, ulcerative colitis, celiac disease, and Crohn's disease. Patients with osteoporosis (see Non-Patent Documents 1 and 2), patients with celiac disease (see Non-Patent Document 3), patients with irritable bowel disease and patients with ulcerative colitis (see Non-Patent Document 4) have peripheral serotonin concentrations It has been reported that the aromatic amines (phenylethylamine, tyramine) in the feces of Crohn's disease patients and ulcerative colitis patients are higher than those 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, as an active ingredient, a drug that inhibits the aromatic amino acid decarboxylase of intestinal bacteria.

The pharmaceutical composition of the present invention can be formulated by appropriately blending the above active ingredient with a pharmaceutically acceptable carrier and further additives. Specifically, oral agents such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; parenteral agents such as injections, infusions, suppositories, ointments, and patches; can do. The blending ratio of the carrier or additive may be appropriately set based on the range normally employed in the pharmaceutical field. 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, absorption Various additives such as accelerators, lubricants, coloring agents, corrigents, and perfumes are included.

Examples of excipients that can be mixed in tablets, capsules and the like include binders such as gelatin, cornstarch, tragacanth and gum arabic, excipients such as crystalline cellulose, cornstarch, gelatin, alginic acid and the like. Bulking agents, lubricants such as magnesium stearate, sweetening agents such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, redwood oil or cherry may be used. When the dosage unit form is a capsule, the above type of material may further contain a liquid carrier such as oil. Sterile compositions for injection can be prepared according to conventional pharmaceutical practice (eg, dissolving or suspending the active ingredient in a solvent such as water for injection, natural vegetable oil, etc.). Aqueous solutions for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). , for example, alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50), and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and they may be used in combination with dissolution aids such as benzyl benzoate and benzyl alcohol. In addition, buffers (e.g., phosphate buffers, sodium acetate buffers), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives agents (eg, benzyl alcohol, phenol, etc.), antioxidants, and the like. Since the preparations thus obtained are safe and have low toxicity, they can be used for humans and mammals other than humans (e.g., 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, more preferably 0.1 to 10% by mass of the active ingredient. The dosage of the pharmaceutical composition of the present invention is appropriately set in consideration of the purpose, type of disease, severity of disease, patient's age, body weight, sex, medical history, type of active ingredient, and the like. For an average human with a body weight of about 65 to 70 kg, the dose is preferably about 0.02 mg to 4000 mg, more preferably about 0.1 mg to 500 mg per day. The total daily dose may be given in single or divided doses.

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 amines, comprising the step of administering an effective amount of a drug that inhibits aromatic amino acid decarboxylase of intestinal bacteria.
(B) An agent that inhibits intestinal bacterial aromatic amino acid decarboxylase for use in the prevention or treatment of diseases associated with increased peripheral serotonin or intestinal aromatic amines.
(C) Use of an agent that inhibits aromatic amino acid decarboxylase of intestinal bacteria for manufacturing a pharmaceutical composition for preventing or treating diseases associated with increased peripheral serotonin or intestinal aromatic amines.

[Screening method]
INDUSTRIAL APPLICABILITY The present invention provides prophylactic or therapeutic drugs for diseases associated with increased peripheral serotonin or intestinal aromatic amines, or screening methods for candidate substances thereof. The screening method of the present invention may include the step of selecting a test substance that inhibits the aromatic amino acid decarboxylase of internal bacteria. Diseases associated with increased peripheral serotonin or intestinal aromatic amines include the aforementioned osteoporosis, irritable bowel disease, ulcerative colitis, celiac disease, Crohn's disease, and the like.

The test substance to be subjected to the screening method of the present invention is not particularly limited, and includes nucleic acids, peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, cell culture supernatants, plant extracts, mammals. tissue extract, blood plasma, or the like. A test substance may be a novel substance or a known substance. These test substances may form salts. The salt of the test substance is preferably a salt with a physiologically acceptable acid or base.

The screening method of the present invention may include, for example, steps 1 to 3 below.
Step 1: step of culturing enteric bacteria having an aromatic amino acid decarboxylase gene in the presence or absence of a test substance using a medium supplemented with an aromatic amino acid;
Step 2: A step of 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. selecting a test substance to induce

The intestinal bacterium having an aromatic amino acid decarboxylase gene used in step 1 is not particularly limited, and may be any known intestinal bacterium having an aromatic amino acid decarboxylase gene. It may be an intestinal bacterium having a carbonic enzyme gene. The enteric bacterium having the aromatic amino acid decarboxylase gene may be a human enteric bacterium or a non-human mammalian enteric bacterium. Human intestinal bacteria having aromatic amino acid decarboxylase genes include, for example, Enterococcus faecalis, Ruminococcus gnavus, Blaartia hansenii, Clostridium nexile, Clostridium asparagiforme, and the like. The human enteric bacterium used in the screening method of the present invention may be Enterococcus faecalis, Ruminococcus gnavus, or Enterococcus faecalis.

Intestinal bacteria having an 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 anaerobicly cultured, a known anaerobic culture medium can be used as the medium. Anaerobic culture media include, for example, Gifu anaerobic medium (GAM). The medium used in the screening method of the present invention may be a liquid medium. The anaerobic culture method is not particularly limited, and can be appropriately selected from known anaerobic culture methods according to the intestinal bacteria to be used.

An aromatic amino acid, which is a substrate for aromatic amino acid decarboxylase, is 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 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. may be about 1 mM.

The amount of test substance to be added should be an amount that does not significantly inhibit the growth of intestinal bacteria compared to the growth level of intestinal bacteria in the absence of the test substance. It may be an amount that does not inhibit proliferation. The culture time is not particularly limited, and may be 6 hours or longer, 12 hours or longer, 24 hours or longer, 36 hours or longer, or 48 hours or longer. good too. The upper limit of the culture time is not particularly limited, but may be 48 hours or less, 36 hours or less, or 24 hours or less.

Step 2 detects aromatic amines in the medium. That is, an aromatic amine produced by decarboxylation of an aromatic amino acid as a substrate is detected by an aromatic amino acid decarboxylase expressed by the intestinal bacteria used. Phenethylamine is produced when phenylalanine is used as the substrate, tyramine is produced when tyrosine is used as the substrate, and tryptamine is produced when tryptophan is used as the substrate. A method for detecting aromatic amines is not particularly limited, and can be appropriately selected from known aromatic amine detection methods and used. The method for detecting aromatic amines used in the screening method of the present invention may be the HPLC method used in Examples described later.

In step 3, a test substance that reduces the amount of aromatic amine in the medium compared to the amount of aromatic amine in the medium when cultured in the absence of the test substance may be selected. If the amount of aromatic amine in the medium decreases when cultured in the presence of the test substance, it can be rationally understood that the test substance inhibits the aromatic amino acid decarboxylase of intestinal bacteria. . The extent to which the amount of aromatic amine is reduced is not particularly limited, but for example, 90% or less, 80% or less, or 70% or less compared to 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.

A test substance selected by the screening method of the present invention is useful as an active ingredient of the pharmaceutical composition of the present invention, and is preferably used for the prevention or treatment of diseases associated with increased peripheral serotonin or intestinal aromatic amines. 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. have the potential to become

EXAMPLES The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these.

[Example 1: Identification of human intestinal bacteria that produce phenethylamine]
1. Experimental method (1) Medium
Gifu anaerobic medium (GAM bouillon "Nissui", Nissui Pharmaceutical, hereinafter abbreviated as "GAM") was sterilized in an autoclave (115°C, 15 min). 400, Ruskinn Technologies) overnight to remove dissolved oxygen. In an anaerobic chamber, each 500 μL was dispensed into a 1 mL capacity 96 deep-well plate.

(2) Strains and culture conditions
We selected 32 human intestinal bacteria dominant species that can be cultured 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, and the 96 deep-well plate was affixed with a gas permeable moisture barrier sheet (4titude) and pre-incubated at 37°C for 24-48 hours. After suspending the preculture solution in an anaerobic chamber using a multichannel pipette, it was inoculated into 500 μL of GAM in a 96 deep-well plate using Copy Plate 96 (TOKKEN). A gas permeable moisture barrier sheet (4titude) was attached, and main culture was performed at 37°C in an anaerobic chamber. After starting the culture, OD600 was measured over time using 20 μL of the main culture medium to create a growth curve. The stationary period was defined as the time

(3) Detection of phenethylamine in culture supernatant (3-1) Sample preparation Collect the 96 deep-well plate containing the main culture medium over time, centrifuge (4,400 rpm, 4°C, 40 min) and remove the culture supernatant. got 1/10 amount of 100% (w/v) Trichloroacetic acid (TCA) was added to the obtained culture supernatant. After addition, the mixture was thoroughly mixed and subjected to centrifugation (18,900 xg, 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)
A post-column derivatization method with o-phthalaldehyde was used for the analysis of phenethylamine. #2619PH (4.6 x 50 mm, Hitachi) was used for the separation column and kept at 67°C. The mobile phase flow rate was 0.4 mL/min. Mobile phase A (45.2 mM trisodium citrate, 63.3 mM sodium chloride, and 60.9 mM citric acid) and mobile phase B (200 mM trisodium citrate, 2 M sodium chloride, 5% ethanol, and 5% 1-propanol), increasing mobile phase B concentration from 50-85% from 0-6 minutes, holding at 85% from 12 minutes, increasing to 100% from 12-18 minutes. , held at 100% for up to 45 minutes, then lowered to 50% and held at 50% for up to 60 minutes. Derivatization with ortho-phthalaldehyde was performed at 0.35 mL/min for Reaction 1 (400 mM NaOH) and Reaction 2 (234 mM boric acid, 0.05 % Brij-35, 5.96 mM ortho-phthalaldehyde, 0.2 % 2-mercaptoethanol). It was carried out by flowing and mixing with the column eluate in a column oven. Detection was performed with a fluorescence detector (λ ex 340 nm, λ em 435 nm).

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

[Example 2: Aromatic amine production profile of phenethylamine-producing bacteria]
1. Experimental method (1) Medium GAM was prepared in the same manner as in Example 1. The medium (primary culture medium) with aromatic amino acids (phenylalanine, tyrosine, tryptophan) at a concentration of 1 mM was added with GAM at 10% (v/v) based on the composition of the minimal medium for bacteria of the genus Bacteroides. It was prepared by adding each aromatic amino acid solution so that the total of each aromatic amino acid contained in GAM was 1 mM. The prepared medium was filter sterilized (0.22 μm, PVDF), 30 mL was dispensed into 50 mL centrifuge tubes, and left 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 hematin, 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) Bacterial strains and culture conditions Five human intestinal bacteria 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. It was pre-incubated for 18 hours. 1 mL of the preculture was centrifuged (3,400 g, 25°C, 3 min), the supernatant was removed, and the cells were collected. The obtained cells were suspended in 1 mL of the main culture medium and centrifuged again (3,400 g, 25°C, 3 min) to wash the cells. The washed cells were resuspended in 1 mL of main culture medium and an aliquot 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, culture was performed in an anaerobic chamber, and the culture solution was sampled 6, 12, 18, 24, and 48 hours after the start of culture, and aromatic amines were detected.

(3) Detection of aromatic amines in culture supernatant (3-1) Sample preparation
1 mL of medium was recovered and centrifuged (18,900 xg, 4°C, 10 min) to obtain a culture supernatant. 1/10 amount of 100% (w/v) Trichloroacetic acid (TCA) was added to the obtained culture supernatant. After addition, the mixture was thoroughly mixed and subjected to centrifugation (18,900 xg, 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)
Analysis of phenethylamine was performed in the same manner as in Example 1.
For the HPLC analysis of tyramine and tryptamine, Discovery HS-F5 (4.6 x 150 mm, Supelco) was used as the separation column and kept at 30°C. A mobile phase of 10 mM ammonium formate (pH 3.0) and acetonitrile was used at a flow rate of 0.4 mL/min. The gradient program was to increase the acetonitrile concentration from 3% to 27% from 0 to 22 minutes, to 66% from 22 to 80 minutes, to 100% from 80 to 81 minutes, hold at 100% until 86 minutes, and increase to 87%. It was programmed to decrease to 3% over minutes and hold at 3% until 102 minutes. Detection was performed with a fluorescence detector (λ ex 280 nm, λ em 325 nm).

2. Results The results are shown in FIG. (A) results for B. hansenii, (B) results for R. gnavus, (C) results for C. nexil, (D) results for C. asparagiforme, (E) results for E. faecalis. be. B. hansenii, R. gnavus and C. nexil were the highest tryptamine producers. C. asparagiforme and E. faecalis produced the most tyramine.

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

The method for preparing 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, respectively.

(1-1) Preparation of E. faecalis wild strain SK947
A wild strain of E. faecalis (WT strain, SK947) was generated based on E. faecalis V583. Specifically, E. faecalis V583 was transfected with the 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 of two-component regulatory systems”, J. Bacteriol., vol. 173, no.8, pp. 2617-2624, 1991.) was introduced by electroporation to obtain SK947.

(1-2) Preparation of temperature-sensitive plasmid pLT06-Δaadc containing aadc-deficient cassette ”, J. Bacteriol., vol. 191, no.20, pp.6203-6210, 2009.), and pLT06-Δaadc having an aadc deletion cassette was constructed. A region of approximately 1 kbp each upstream and downstream of the aadc gene ORF on the genome of E. faecalis strain V583 was amplified by PCR. During amplification, primers for_Del_ddc_1_F and for_Del_ddc_2_5P_R were used for upstream amplification, and primers for_Del_ddc_3_5P_F and for_Del_ddc_4_R were used for downstream amplification. Both amplified products were linked by ligation, and a DNA fragment of about 2 kbp was again subjected to PCR for amplifying the full-length addc-deficient cassette. The primers used were pLT06-EcoRI_D_ddc_F and pLT06-EcoRI_D_ddc_R. The amplified fragment was cloned into EcoRI-cut pLT06 by the infusion method to generate pLT06-Δaadc.

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

(1-4) Preparation of aadc-deficient strain SK977 An aadc-deficient strain (SK977) was prepared by introducing pLT06-Δaadc prepared in (1-2) above into the E. faecalis V583 strain. Specifically, pLT06-Δaadc was introduced into E. faecalis V583 strain by electroporation, cultured, and then incubated at 42° C. to remove the temperature-sensitive plasmid pLT06-Δaadc. A strain in which the plasmid was introduced by a single crossover near the aadc region on the genome was treated with 10 μg/mL chloramphenicol (CF) and 120 μg/mL 5-bromo-4-chloro-3-indolyl-β-D- Screening was performed on THB agar medium supplemented with galactopyranoside (X-gal). For colonies grown on the THB agar medium, it was confirmed by PCR that gene recombination had occurred correctly in the target region. Next, the plasmid introduced onto the genome was removed by loop-out by culturing twice using medium without CF, and 120 μg/mL of X-gal and 10 μg/mL of p-chlorophenylalanine were added. Strains that successfully cleared the plasmid were screened on MM9YEG agar medium containing . For white colonies showing resistance to p-chlorophenylalanine, it was confirmed by PCR that the plasmid region was correctly removed from the genome. Furthermore, the recombination region was analyzed by PCR amplification and direct sequencing to confirm that no unintended mutation was introduced.

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

(1-6) Generation of aadc-complementing strain SK982.
pLZ12-aadc+ was introduced into the obtained SK977 by electroporation to prepare an aadc-complementing 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, a region (4,084 bp) containing the aadc gene on the genome was amplified. A region containing pLZ12-aadc+ (2,850 bp) was also amplified using pLZ12_MCS_FWD and pLZ12_MCS_COMPL as primers. A PCR reaction solution was prepared using a DNA polymerase (PrimeSTAR Max DNA Polymerase, Takara Bio). PCR was performed using a thermal cycler (TaKaRa PCR Thermal Cycler Dice Gradient, Takara Bio), 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. Reactions were run for 3 minutes for 30 cycles. After the PCR reaction, electrophoresis was performed using agarose gel (Agarose S, Fujifilm Wako Pure Chemical Industries, Ltd.). The presence or absence of a band at 4,084 bp for the region containing the aadc gene on the genome and at 2,850 bp for pLZ12-aadc+. It was confirmed.

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

2. Results The results are shown in FIG. Phenethylamine was detected in the culture supernatant of the wild strain and the aadc-complemented strain, but not in the culture supernatant of the aadc-deficient strain. These results revealed that E. faecalis produced phenethylamine using aromatic amino acid decarboxylase.

[Example 4: Examination of the serotonin level in the large intestine tissue of mice in which an aromatic amino acid decarboxylase gene-disrupted strain and an aromatic amino acid decarboxylase gene-complemented strain of E. faecalis are dominant in the gut flora]
1. Experimental method (1) Mouse used
Six-week-old female BALB/cCrSlc (Japan SLC) were used.

(2) Feed
There are two types of feed: CLEA Rodent Diet CE-2 (Clea Japan) and a special formula feed that uses AIN-93G as a basic formula and does not contain tyrosine and has a phenylalanine content that is 10 times that of the basic formula. 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 , cornstarch 380.456 g/kg, dietroz 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, vitamins mix #310025 10 g/kg, choline bitartrate 2.5 g/kg (total weight 1 kg)

(3) Antibiotic Treatment In order to remove as many intestinal bacteria as possible from mice, two kinds of antibiotics, doripenem and vancomycin, were added to sterilized tap water and used as drinking water. Antibiotic concentrations were 0.25 g/L doripenem (0.5 g for Finibax for intravenous infusion, Shionogi & Co., Ltd.) and 0.5 g/L vancomycin (0.5 g for vancomycin hydrochloride for intravenous infusion, Shionogi & Co., Ltd.). be.

(4) Strains used E. faecalis aadc-deficient strain SK981 and aadc-complementing strain SK982 used in Example 3 were used. The bacteria were suspended in 50% (v/v) glycerol of Lactobacillus MRS (LMRS) Broth containing 10 µg/mL chloramphenicol (CF) and stored at -20°C until use.

(5) Medium
GAM supplemented with 10 μg/mL chloramphenicol (CF) was used. CF was added to 10 μg/mL immediately before use. After autoclaving, 1.6% GAM Agar was cooled to 50°C in a constant temperature water bath set at 50°C, added with CF so as to have a concentration of 10 µg/mL, and then poured into petri dishes and solidified. This was placed in an anaerobic jar together with Anaeropack Kenki (trade name) to remove dissolved oxygen and used.

(6) Preparation of bacterial suspension
Stock strains stored at -20°C were inoculated into 5 mL of GAM supplemented with 10 µg/mL CF, and precultured at 37°C for 12 hours under anaerobic conditions. 5 μL (1.4×10 ⁹ CFU/mL) of the pre-cultured bacterial solution was seeded in 5 mL of 10 μg/mL CF-added GAM Broth, and main culture was carried out at 37° C. for 12 hours under anaerobic conditions. After the main culture, 1 mL of the culture medium was dispensed into an Eppendorf tube, centrifuged (6,000×g, 25° C., 5 min), and the supernatant was removed to collect the cells. The recovered cells were suspended by adding 1 mL of PBS containing 10 μg/mL CF, centrifuged (6,000×g, 25° C., 5 min), and the supernatant was removed twice to wash the cells. After the second washing, the cells were suspended in 5 mL of PBS, diluted with this bacterial suspension, smeared on 10 μg/mL CF 4 -added GAM Agar, and cultured at 37° C. in an anaerobic jar. After culturing, the number of bacteria was measured by colony counting, and the concentration of bacteria was adjusted to 1×10 ⁸ CFU/200 μL.

(7) Experimental Protocol Experiments were performed by dividing mice into two groups (aadc-deficient strain administration group, aadc-complementing strain administration group) of 10 mice each. To make Enterococcus faecalis a dominant flora, mice were treated with antibiotics for 2 weeks to reduce the number of bacteria in the gut by a factor of 1,000,000. The starting date of administration of antibiotics to mice in drinking water was 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 combination of AIN-93G to increase the amount of phenylalanine in the intestine and decrease tyrosine. Antibiotic administration ended 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 per mouse using an oral probe (5202K, Fuchigami Kikai). Three days after administration of E. faecalis (Day 18), mouse feces, large intestine tissue, and cecal contents were collected. Specifically, 10 feces (approximately 10 mg) were collected from each mouse, then euthanized, the abdomen was opened, and large intestine tissue and contents of the cecum were removed. The collected stool samples were anaerobicized with Anaeropack Kenki (trade name) and stored at -80°C. The large intestine tissue was placed in a 50 mL centrifuge tube containing 15 mL of PBS, kept from drying until sample processing, and ice-cooled together with the centrifuge tube. More than 100 µL of the contents of the cecum were collected, transferred to an Eppendorf tube, and stored at -80°C. Daily changes in body weight and food intake were measured during the experimental period (Day 0 to Day 18).

(8) Treatment of large intestine tissue
The large intestine tissue that had been placed in a centrifuge tube containing PBS and cooled on ice was taken out on a petri dish, the intestine was cut open in the longitudinal direction with scissors for dissecting, and feces in the intestine were removed with forceps. After that, it was washed twice with 10 mL of PBS. After washing, the large intestine was transferred to a centrifuge tube containing 10 mL of PBS, and the large intestine tissue was isolated by repeating the cycle of transmitting ultrasonic waves for 30 seconds and standing for 60 seconds with an ultrasonic homogenizer (Model 250, BRANSON) 5 times per sample. homogenized. The colon sample after homogenization was stored at -25°C as it was in the centrifuge tube.

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

2. Results The results are shown in FIG. The serotonin level in the large intestine tissue of the aadc-complementing strain administration group was significantly higher than that of the aadc-deficient strain administration group.

[Example 5: Examination of aromatic amino acid decarboxylase gene copy number and phenethylamine production ability in human feces]
1. Experimental method (1) Measurement of phenethylamine in feces
Feces were collected from 9 healthy adults. About 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, centrifugation (18,900×g, 4° C., 10 min) was performed to collect the supernatant. 1/10 volume of 100% (w/v) Trichloroacetic acid (TCA) was added to the resulting supernatant. After addition, the mixture was thoroughly mixed and subjected to centrifugation (18,900 xg, 4°C, 10 min). After centrifugation, the resulting supernatant was filtered through Cosmospin filter H (0.45 μm, PVDF), and the filtrate was subjected to HPLC. The detection of phenethylamine in the culture supernatant by HPLC was performed in the same manner as in Example 1.

(2) aadc gene copy number in feces About 20 mg of feces 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) and incubate at 37°C for 15 minutes. Incubate for 1 minute. After incubation, 12 μL of 20% (w/v) sodium dodecyl sulfate solution was added, mixed for 5 minutes, and 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 μDrop plate (Thermo Fisher Scientific).

Based on the measured DNA concentration, a 1 ng/μL DNA solution was prepared using sterilized 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 composition of the reaction solution is 10 µL of 2x 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 was 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) and sterilized water at a ratio of 1.75:1.75:6.5. It is what I did. PCR was performed at 95°C for 30 seconds, followed by 40 cycles of [95°C for 5 seconds → 60°C for 60 seconds]. 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., vol. 7, Article number: 10390, 2016), a standard curve for calculating the copy number was created using a plasmid into which the aadc gene of R.ganvus was introduced. Based on the copy number calculated from the calibration curve, the R.ganvus aadc gene copy number per 1 g of feces was calculated.

2. Results The results are shown in FIG. Plotting the number of copies of the aromatic amino acid decarboxylase gene (aadc) per gram of human feces versus the amount of phenethylamine produced when phenylalanine was mixed with feces showed a significant positive correlation (p = 0.0066). there were. This result indicated that AADC in human feces most likely converts phenylalanine to phenethylamine. Therefore, it was shown that the amount of phenethylamine produced can be suppressed by inhibiting the aromatic amino acid decarboxylase of intestinal bacteria.

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

(2) Strains 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, and phenethylamine was detected.

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

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

The present invention is not limited to the above-described embodiments and examples, and can be modified in various ways within the scope of the claims, by appropriately combining technical means disclosed in different embodiments. The resulting embodiment is also included in the technical scope of the present invention. In addition, all scientific and patent documents mentioned in this specification are incorporated herein by reference.

Claims (1)

A pharmaceutical composition for preventing or treating irritable bowel disease accompanied by an increase in peripheral serotonin or intestinal aromatic amines, comprising as an active ingredient a drug that inhibits aromatic amino acid decarboxylase of intestinal bacteria , A pharmaceutical composition, wherein the agent that inhibits aromatic amino acid decarboxylase of intestinal bacteria is carbidopa or benserazide .