WO2022080249A1

WO2022080249A1 - Agent for preventing or ameliorating inflammatory bowel disease

Info

Publication number: WO2022080249A1
Application number: PCT/JP2021/037311
Authority: WO
Inventors: 道夫鬼澤; 守渡辺; 剛人浅川; 弘正大平
Original assignee: 国立大学法人東京医科歯科大学; 公立大学法人福島県立医科大学
Priority date: 2020-10-12
Filing date: 2021-10-08
Publication date: 2022-04-21
Also published as: JPWO2022080249A1

Abstract

The present invention addresses the problem of providing an agent for preventing or ameliorating inflammatory bowel disease, said agent having high safety upon administration, being able to be orally administered, and having a relatively low production cost.　In the present invention, an agent comprising D-serine, a derivative thereof or a physiologically acceptable salt of the same is used as an agent for preventing or ameliorating inflammatory bowel disease.

Description

Preventive or ameliorating agent for inflammatory bowel disease

The present invention contains inflammatory bowel disease containing D-serine or a derivative thereof or a physiologically acceptable salt thereof (hereinafter, these may be collectively referred to as "D-serines") as an active ingredient. And / or related to the preventive agent and / or the improver.

Inflammatory bowel disease is an intractable disease accompanied by inflammation of the intestinal tract, and is mainly classified into ulcerative colitis and Crohn's disease. Ulcerative colitis is a disease showing non-specific chronic inflammation that affects the large intestine mucosa more diffusely and continuously than the rectum, whereas Crohn's disease is inflammation and deep ulcers in various parts of the gastrointestinal tract. It is a disease that shows full-thickness symptoms such as perforation. All diseases are considered to be multifactorial diseases in which various factors such as pathogenic bacteria and viral infections, drugs such as antibiotics, environmental factors, psychosomatic medical problems, congenital abnormalities, and immune abnormalities are involved in a complicated manner. There is.

Drug therapy, nutrition therapy, surgery, etc. are known as methods for treating inflammatory bowel disease. In the case of drug therapy for inflammatory bowel disease, immunosuppressants, corticosteroids, molecular biologics (antibody drugs), aminosalicylic acid preparations and the like are mainly used. However, the problem with these existing drugs is that there are many cases in which the effect is ineffective from the time of introduction of treatment, the effect is diminished and becomes ineffective as the administration is continued, and there are many cases in which it is difficult to continue the administration due to side effects. .. Furthermore, it is also a problem that these biologics cannot be orally administered and cause physical distress. Further, in surgical treatment, there is a problem of the onset of ileal pouchitis after total colectomy for ulcerative colitis, and a problem that the relapse rate after lesion resection is high in Crohn's disease.

On the other hand, among the 20 kinds of amino acids constituting the protein, 19 kinds of amino acids other than glycine are known to have optical isomers (D-form and L-form). Although D-amino acids are energetically equivalent to L-amino acids, L-amino acids are predominantly used in the vital activities of many organisms.

In 1986, the presence of free D-aspartic acid was first revealed in the living body of mammals. In 1992, it was reported that D-serine accounted for 25% of the total serine in the rat cerebral cortex (Non-Patent Document 1), after which serine racemase was converted from L-serine to D-serine. It has been reported that D-serine is biosynthesized by this (Non-Patent Document 2). In addition, it has been reported that high concentrations of D-serine are present in the cerebral cortex also in humans, and in the cerebral cortex, shades of D-serine are observed according to the Brodmann classification (Non-Patent Document 3). , The correlation between the function of the cerebral cortex and the concentration of D-serine has been suggested. In fact, a decrease in D-serine in the cerebral cortex causes a decline in NMDA (N-Methyl-D-aspartate) receptor function, which is one of the ionotropic glutamate receptors, and is part of schizophrenia. The association with symptoms has been reported (Non-Patent Document 4).

It has been reported that a composition containing 11 kinds of essential L-amino acids and L-arginine has an anti-inflammatory effect (Patent Document 1), but D-serine has a preventive and therapeutic effect on inflammatory bowel disease. It was not previously known to have.

Japanese Unexamined Patent Publication No. 2012-214451

An object of the present invention is to provide a preventive agent or an ameliorating agent for inflammatory bowel disease, which is highly safe when ingested, can be ingested orally, and has a relatively low manufacturing cost.

The present inventors are continuing diligent research to solve the above problems. In the process, it was found that the D-form (ie, D-serin), not the L-form of serin, has the effect of effectively preventing the onset of inflammatory bowel disease. Furthermore, it was confirmed that D-serine has an excellent therapeutic effect on inflammation after the onset of inflammatory bowel disease. The present invention has been completed based on these findings.

That is, the present invention is as follows.
[1] A prophylactic or ameliorating agent for inflammatory bowel disease, which comprises D-serine or a derivative thereof or a physiologically acceptable salt thereof.
[2] The agent for preventing or ameliorating inflammatory bowel disease according to the above [1], wherein the derivative of D-serine is a compound that changes to D-serine after administration.
[3] The preventive or ameliorating agent according to the above [1] or [2], which is orally ingested.
[4] Weight loss; diarrhea; bloody stool; damage to the intestinal mucosa; increased level of leukocyte infiltration in the intestinal mucosa; and crypt tumor; The preventive or ameliorating agent according to any one of [3].

Further, as another embodiment of the present invention,
A method for preventing or treating inflammatory bowel disease, which comprises the step of administering D-serines to a subject (patient) in need of prevention or treatment of inflammatory bowel disease;
D-serines for use as a prophylactic or therapeutic agent for inflammatory bowel disease;
D-serines for use in the prevention or treatment of inflammatory bowel disease;
Use of D-serines to produce prophylactic or therapeutic agents for inflammatory bowel disease;
Can be mentioned.

D-serines have an effect of effectively preventing the onset of inflammatory bowel disease and also have an excellent therapeutic effect on inflammation after the onset of inflammatory bowel disease. Further, since D-serines are amino acids that are also present in the living body, they are highly safe when ingested, can be ingested orally, and can be produced at a relatively low cost.

Rag2 ^-/- Mice of two types of drinking water (distilled water [“ _H2O ” in the figure] or distilled water containing 1.5% D-serine [“D-Ser” in the figure]) Free drinking water was started. Naive CD4-positive T cells were transferred into Rag2 ^-/- mice 1 week (7th day) after the start of free drinking, and Rag2 ^-/- mouse weight (1-10 weeks after the start of free drinking). It is a figure which shows the result of having analyzed the clinical score value (FIG. 1B) of FIG. 1A) and enteritis. The body weight is shown as a relative value when the value of the body weight immediately before the start of free drinking water is set to 100. The horizontal axis of FIGS. 1A and 1B represents the number of weeks calculated from the start date of free drinking water. Free drinking with the above two types of drinking water was continuously performed up to each time point. In all the drawings of the present application, "*", "**" and "***" in the drawings are statistically significantly different by Dunnett's test (p <0.05, p <0, respectively). It is shown that 0.01 and p <0.001) (the same applies to drawings other than FIG. 1). “N.s.” in the figure indicates that there is no statistically significant difference (p ≧ 0.05) by Dunnett's test (the same applies to drawings other than FIG. 1). Rag2 ^-/- Mice of two types of drinking water (distilled water [“ _H2O ” in the figure] or distilled water containing 1.5% D-serine [“D-Ser” in the figure]) Free drinking water was started. Naive CD4-positive T cells were transferred into Rag2 ^-/- mice 1 week (7th day) after the start of free drinking, and colon weight (Fig. 2A), CD3 ⁺ CD4 ⁺ 10 weeks after free drinking. It is a figure which shows the result of having analyzed the LPL number (FIG. 2B), and the histological score value (FIG. 2D) of enterocolitis. The histological score of enteritis was measured based on a hematoxylin-eosin (HE) stained colon tissue sample (FIG. 2C). Further, the "colon weight" in FIG. 2A indicates the weight of the colon (mg) per 1 cm (hereinafter, the same applies). Free drinking with the above two types of drinking water was continued until the 10th week after the start of free drinking. Rag2 ^-/- mouse contains three types of drinking water (distilled water [“ _H2O ” in the figure], distilled water containing 1.5% D-serine [“D-Ser” in the figure], or Free drinking of distilled water containing 1.5% L-serine [“L-Ser” in the figure]) was started. Naive CD4-positive T cells were transferred into Rag2 ^-/- mice 1 week (7th day) after the start of free drinking, and body weight (Fig. 3A) and enteritis 1-10 weeks after the start of free drinking. It is a figure which shows the result of having analyzed the clinical score value (FIG. 3B). The body weight is shown as a relative value when the value immediately before the start of free drinking is set to 100. The horizontal axis of FIGS. 3A and 3B represents the number of weeks calculated from the start date of free drinking water. Free drinking with the above three types of drinking water was continuously performed up to each time point. Rag2 ^-/- mouse contains three types of drinking water (distilled water [“ _H2O ” in the figure], distilled water containing 1.5% D-serine [“D-Ser” in the figure], or Free drinking of distilled water containing 1.5% L-serine [“L-Ser” in the figure]) was started. Naive CD4-positive T cells were transferred into Rag2 ^-/- mice 1 week (7th day) after the start of free drinking, and colon weight (Fig. 4A), CD3 10 weeks after the start of free drinking. It is a figure which shows the result of having analyzed the ⁺ CD4 ⁺ LPL number (FIG. 4B), and the histological score value of enterocolitis (FIG. 4D). The histological score of enteritis was measured based on a HE-stained colorectal tissue sample (FIG. 4C). Free drinking with the above three types of drinking water was continuously performed from 1 to 10 weeks after the start of free drinking. Wild type (WT) mice, 3 types of drinking water (distilled water [“ _H2O ” in the figure], distilled water containing 1.5% D-serine [“D-Ser” in the figure], Alternatively, free drinking of distilled water containing 1.5% L-serine [“L-Ser” in the figure]) was continuously performed until the 7th week. It is a figure which shows the result of having analyzed the body weight (FIG. 5A) and the clinical score value of enteritis (FIG. 5B) 1 to 7 weeks after the start of free drinking water. The body weight is shown as a relative value when the value immediately before the start of free drinking (week 0) is set to 100. Free drinking with the above three types of drinking water was continued until each time point. Three types of drinking water (distilled water [“ _H2O ” in the figure], distilled water containing 1.5% D-serine [“D-Ser” in the figure], or 1.5 in WT mice. 7 weeks after the start of free drinking of distilled water containing% L-serine [“L-Ser” in the figure]), the weight of the colon (Fig. 6A) and the number of CD3 ⁺ CD4 ⁺ LPL (Fig. 6B) It is a figure which shows the result of analysis and the result of HE staining (FIG. 6C) of a colon tissue sample. Free drinking with the above three types of drinking water was continued until the time of analysis. Rag2 ^-/- mouse contains 4 types of drinking water (distilled water [“H ₂ O” in the figure], distilled water containing 0.5% D-serine [“D-Ser 0.5%” in the figure”. ], Distilled water containing 1.0% D-serine [“D-Ser 1.0%” in the figure], or distilled water containing 1.5% D-serine [“D-Ser1.” In the figure. 5% "],) free drinking water was started. Naive CD4-positive T cells were transferred into Rag2 ^-/- mice 1 week (7th day) after the start of free drinking, and body weight (Fig. 7A) and enteritis 1-9 weeks after the start of free drinking. It is a figure which shows the result of having analyzed the clinical score value (FIG. 7B). The body weight is shown as a relative value when the value immediately before the start of free drinking (day 0) is set to 100. The horizontal axis of FIGS. 7A and 7B represents the number of weeks calculated from the start date of free drinking water. Free drinking with the above four types of drinking water was continuously performed up to each time point. Rag2 ^-/- mouse contains 4 types of drinking water (distilled water [“H ₂ O” in the figure], distilled water containing 0.5% D-serine [“D-Ser 0.5%” in the figure”. ], Distilled water containing 1.0% D-serine [“D-Ser 1.0%” in the figure], or distilled water containing 1.5% D-serine [“D-Ser” in the figure. 1.5% "],) free drinking was started. Naive CD4-positive T cells were transferred into Rag2 ^-/- mice 1 week (7th day) after the start of free drinking, and colon weight (Fig. 8A), CD3 9 weeks after the start of free drinking. It is a figure which shows the result of having analyzed the ⁺ CD4 ⁺ LPL number (FIG. 8B), and the histological score value of enterocolitis (FIG. 8D). The histological score of enteritis was measured based on a HE-stained colorectal tissue sample (FIG. 8C). Free drinking with the above four types of drinking water was continued until the 9th week after the start of free drinking. A predetermined concentration of D-serine or L-serine was added to a culture medium of naive CD4-positive T cells isolated from WT mice, and the cells were cultured for 2 days under the condition of stimulating CD3 and CD28, and the cell viability was achieved. Is shown at the ATP level (FIG. 9A). 20 mM of D-serine or other amino acids was added to the culture medium of naive CD4-positive T cells isolated from WT mice, and the cells were cultured for 2 days under the condition of stimulating CD3 and CD28 to determine the cell viability. The result of evaluation at the ATP level is shown (FIG. 9B). Naive CD4-positive T cells isolated from WT mice are stained with CFSE (carboxyfluorescein succinimidyl ester), and a predetermined concentration of D-serine or L-serine is added to the culture medium to stimulate CD3 and CD28. The cells were cultured for 3 days under the added conditions. The results of flow cytometric analysis of CFSE-stained live cells are shown (FIG. 9C). Naive CD4-positive T cells isolated from WT mice were co-cultured with 20 mM D-serine for 72 hours under Th1-polarization, Th17-polarization or Treg-polarization conditions. 72 hours after stimulation, the results of flow cytometric measurement of IFNγ-positive cells, IL-17A-positive cells, and Foxp3-positive cells are shown (FIG. 9D). As representative data of FACS, the number of positive cells and the percentage of positive cells (%) are shown (FIG. 9E). Distilled water containing 1.5% D-serine is free only from 7 days before transfer of naive CD4 positive T cells to 4 weeks after transfer (ie, 0-5 weeks on the horizontal axis of FIGS. 10A and B). Drinking Rag2 ^-/- mice (“D-Ser → _H2O ” in the figure) and distilled water containing 1.5% D-serine were added from 4 weeks after the transfer of naive CD4-positive T cells. (That is, from the 5th week on the horizontal axis of FIGS. 10A and 10) For each of the free-drinking Rag2 ^-/- mice (“ _H2O → D—Ser” in the figure), 1 to 9 weeks after the start of the experiment. It is a figure which shows the result of having analyzed the body weight (FIG. 10A) and the clinical score value of enteritis (FIG. 10B) in the eyes (that is, the 1st to 9th weeks on the horizontal axis of FIGS. 10A and B). The body weight is shown as a relative value when the value immediately before the start of free drinking (day 0) is set to 100. As a comparative control, distilled water (“ _H2O ” in the figure) or distilled water containing 1.5% D-serine was continuously used from 7 days before the transfer of naive CD4 positive T cells to each time point (in the figure). The results of the same analysis are shown for Rag2 ^-/- mice that were allowed to freely drink "D-Ser"). Only from 7 days before transfer of Naive CD4 positive T cells to 4 weeks after transfer (that is, 0 to 5 weeks after the start of the experiment), Rag2- free ^- drinking distilled water containing 1.5% D-serine. ^/ -From 4 weeks after transfer of naive CD4 positive T cells (that is, from 5 weeks after the start of the experiment) with mice ("D-Ser → H ₂ O" in the figure), 1.5% D- For Rag2 ^-/- mice (“ _H2O → D—Ser” in the figure) that started free drinking of distilled water containing serine, 9 weeks after the start of the experiment (that is, after the transfer of naive CD4 positive T cells). 8 weeks), the weight of the colon (FIG. 11A), the number of CD3 ⁺ CD4 ⁺ LPL (FIG. 11B), and the histological score of enteritis (FIG. 11D) are analyzed. The histological score of enteritis was measured based on a HE-stained colorectal tissue sample (FIG. 11C). As a comparative control, distilled water (“H ₂ O” in the figure) or 1. The results of the same analysis are shown for Rag2 ^-/- mice free to drink distilled water containing 5% D-serine (“D-Ser” in the figure).

The prophylactic or ameliorating agent for inflammatory bowel disease of the present invention is a D-serine (that is, D-serine or a derivative thereof or a physiological thereof) specified for the purpose of "preventing or ameliorating inflammatory bowel disease". It is an agent containing an agent (hereinafter, may be referred to as "preventive / ameliorating agent"). As the preventive / improving agent, D-serines may be used alone as a livestock feed, a food or drink or a pharmaceutical product (formulation), or an additive may be further mixed to form a composition (livestock feed). It may be used as a composition, a food or drink composition or a pharmaceutical composition). Examples of the food and drink include health foods (functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally adjusted foods, supplements, etc.), health functional foods (specified health foods, nutritional functional foods, functional foods, etc.). Labeled foods, etc.) can be mentioned. Suitable embodiments of the agent for preventing or ameliorating inflammatory bowel disease of the present invention include pharmaceuticals and pharmaceutical compositions for preventing or ameliorating inflammatory bowel disease.

As used herein, "inflammatory bowel disease" means a disease that causes inflammation (preferably both inflammation and ulcer) in the mucous membranes of the large intestine and small intestine of mammals, mainly ulcerative colitis and Crohn's disease. It is roughly divided into.

As used herein, "treating inflammatory bowel disease" means the disappearance of an inflammatory symptom or condition in the intestine of a mammal; the suppression or reduction of the aggravation of the symptom or condition; the active phase of inflammatory bowel disease ( For example, it means one or two or more selected from shortening the period of relapse (relapse period, acute period); and prolonging the remission period. Such "symptoms or conditions of inflammation in the intestine" include, for example, weight loss; diarrhea; bloody stools; damage to the intestinal mucosa (eg, localized lymphoepithelial lesions, diffuse crypt extension, widespread crypt elongation, mucosal erosion). / Erosion); Increased levels of leukocyte infiltration in the intestinal mucosa (including increased numbers of CD3-positive and CD4-positive lamina propria lymphocytes); crypt tumors; etc.

As used herein, "preventing inflammatory bowel disease" means suppressing the onset (onset) of inflammatory bowel disease; delaying the onset of inflammatory bowel disease; reducing the risk of developing inflammatory bowel disease; and inflammatory bowel. Delay or suppress disease recurrence; means one or more selected from.

The factors of the inflammatory bowel disease are not particularly limited, and are, for example, infections by viruses (eg, norovirus, influenza virus, rotavirus, coronavirus) and pathogenic bacteria (eg, mycoplasma, diarrheagenic Escherichia coli); immune cells. (For example, lymphoid cells such as T cells, natural killer cells, and B cells; antigen-presenting cells such as monocytes, macrophages, and dendritic cells; granules such as neutrophils, neutrophils, basal spheres, and mast cells. Abnormalities of spheres;); drugs such as anticancer agents and antibiotics;

In the present specification, the mammals include humans and non-human mammals (eg, monkeys, mice, rats, dogs, cats, domestic animals [eg, rabbits, pigs, horses, cows, sheep, goats, deer]) and the like. Can be mentioned, and humans can be mentioned favorably.

In the present specification, "D-serine" means an optical isomer of L-serine, which is one of the amino acids constituting a protein.

The above-mentioned derivative of D-serine has a physiology substantially equivalent to that of D-serine, such as one that changes to D-serine after administration (more specifically, one that is metabolized in vivo to produce D-serine). Anything that has activity may be used. As an example of the derivative of D-serine, a compound in which the carboxy group, amino group or hydroxyl group of D-serine is protected / substituted can be mentioned. The carboxy group can be esterified, amidated and the like, for example. Amino groups can be amidated. Hydroxyl groups can be etherified and esterified. Derivatives of D-serine include, for example, peptides containing O-acetyl-D-serine, DO-phosphoserine, D-serine methyl ester, D-serine ethyl ester, O-benzyl-D-serine, D-serine and the like. Can be mentioned. The peptide containing D-serine may be composed only of D-serine, or in addition to D-serine, other amino acids such as alanine, glycine, valine, leucine, isoleucine, threonine, cysteine, methionine, etc. It may be composed of aspartic acid, glutamic acid, asparagine, glutamine, lysine, arginine, phenylalanine, tyrosine, tryptophan, histidine, L-serine and the like. These amino acids other than D-serine may be L-form or D-form. D-serine residues and D-serine produced by decomposition may bring about a preventive or ameliorating effect on inflammatory bowel disease. Examples of peptides containing D-serine include D-serine dipeptides, D-serine tripeptides, and glycyl-D-serine (ie, dipeptides consisting of glycine and D-serine).

As used herein, "physiologically acceptable salt" is, within reasonable medical, pharmaceutical, or biological judgment, excessively toxic to use in contact with mammalian tissue. Means a salt that is suitable for a reasonable benefit / risk ratio, without irritation, allergic response, and other problems or complications.

As used herein, the term "physiologically acceptable salt" includes, for example, hydrochlorides such as ethyl hydrochloride, benzyl hydrochloride, benzyl ester hydrochloride, and acetyl hydrochloride; sulfate; nitrate; sodium salt, potassium salt. , Metal salts such as calcium salts; ammonium salts; and the like.

D-serines can be produced by any known method such as chemical synthesis, production by microorganisms, production by enzymes, etc., but commercially available products can also be used. Examples of such commercially available products include D-serine (manufactured by Peptide Institute), D-serine methyl ester hydrochloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and O-benzyl-D-serine benzyl ester hydrochloride (Fujifilm sum). (Manufactured by Kojun Yakuhin Co., Ltd.) and the like.

The preventive / ameliorating agent includes L-serine and 19 amino acids other than serine among the 20 types constituting the protein (specifically, arginine [L-arginine, D-arginine], glycine, alanine [ L-alanine, D-alanine], tyrosine [L-tyrosine, D-tyrosine], cysteine [L-cysteine, D-cysteine], asparagine [L-asparagin, D-asparagin], glutamine [L-glutamine, D- Glutamine], proline [L-proline, D-proline], aspartic acid [L-aspartic acid, D-aspartic acid], glutamic acid [L-glutamic acid, D-glutamic acid], valine [L-valine, D-valine], Isoleucine [L-isoleucine, D-isoleucine], methionine [L-methionine, D-methionine], lysine [L-lysine, D-lysine], phenylalanine [L-phenylalanine, D-phenylalanine], tryptophan [L-tryptophan, D-tryptophan], threonine [L-threonine, D-threonine], histidine [L-histidine, D-histidine]) may contain one or more (preferably all). Those that do not are preferable. The preventive / improving agent may contain L-serine, but preferably does not contain L-serine.

The target of administration of the preventive / ameliorating agent is not particularly limited, and is usually a mammal (preferably human) in need of prevention or improvement of inflammatory bowel disease, and for example, has a high risk of developing inflammatory bowel disease. Examples include mammals (preferably humans), mammals suffering from inflammatory bowel disease (preferably humans) and the like.

As a method of ingesting (administering) the preventive / improving agent, for example, both an oral ingestion method (oral ingestion) and a parenteral ingestion method (parenteral ingestion) are possible, and parenteral ingestion (administration). Examples of the method for this include intravenous administration and local administration. As a method of ingesting the preventive / ameliorating agent, oral ingestion can be preferably exemplified in consideration of its convenience and its effect being demonstrated in the present examples described later.

The dose of D-serine in the preventive / ameliorating agent is appropriately determined according to age, body weight, gender, symptom, sensitivity to the drug, etc. For example, the concentration when converted to D-serine is 0.1 μg. The dose ranges from ~ 200 mg / kg (body weight) / day. The preventive / improving agent may be administered once or in a plurality of times (for example, 2 to 4 times) per day.

As used herein, the additives include conventional physiologically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonic agents, additives and coatings. , Solubilizers, lubricants, lubricants, solubilizers, lubricants, flavoring agents, sweeteners, solvents, gelling agents, nutrients and other compounding ingredients can be exemplified. Specific examples of such compounding ingredients include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, and polyalkylene glycol. Polyvinyl alcohol and glycerin can be exemplified.

The preventive / ameliorating agent may contain an anti-inflammatory component other than D-serine, but since D-serine alone exerts an excellent anti-inflammatory effect, it is an anti-inflammatory agent other than D-serine. Those containing no inflammatory components (eg, proteins, amino acids, DNA, RNA, plant-derived extracts, polymers) are preferred.

Hereinafter, the present invention will be described in more detail by way of examples, but the technical scope of the present invention is not limited to these examples.

1. 1. Materials and methods [Experimental animals]
Wild-type C57BL / 6 mice (obtained from Claire Japan, Inc.) (sometimes referred to as "WT mice" in the present specification), and C57BL / 6 background Rag2-deficient (T-cell and B-cell-deficient) mice ( ^{Rag2- /} -Mice) (obtained from Taconic) were bred at the animal breeding facility of Tokyo Medical & Dental University using general feed (CE-2 [manufactured by Claire Japan]) in a state of not being infected with a specific pathogen. 8-12 week old mice were used as donor and recipient mice. All animal experiments were approved by the Animal Care and Use Committee of Tokyo Medical & Dental University and were conducted according to the university guidelines.

[Amino acid administration method]
Distilled water containing 3 types of drinking water (distilled water; 1.5 [w / v]% L-serine [manufactured by Peptide Institute]; and 0.5 [w / v]%, 1.0 [w] Distilled water) containing / v]% or 1.5 [w / v]% of D-serine [manufactured by Peptide Institute]) was allowed to freely drink in WT mice or Rag2 ^-/- mice.

[Induction of naive CD4-positive T cell-introduced colitis model mice]
First, in order to prepare naive CD4 positive T cells, spleen mononuclear cells were obtained from WT mice, and then CD4 positive T cells were isolated using anti-CD4 (L3T4) MACS magnetic beads (manufactured by Miltenyi Biotec). bottom. From the isolated CD4 positive T cells, four types of antibodies (anti-CD4-APC antibody [100516], anti-TCRb-Pacific Blue antibody [109226], anti-CD44-FITC antibody [103006], and anti-CD62L-PE antibody. [104408] [all manufactured by BioLegend]) and a cell sorter (BD FACSAria, manufactured by Becton Dickinson), TCRb-positive, CD4-positive, CD44-negative, and CD62L-positive cells (that is, naive CD4-positive T). Cells) were isolated. Then, 5 × 10 ⁵ naive CD4 positive T cells were intraperitoneally transferred into 8- to 12-week-old Rag2 ^-/- mice to induce (create) naive CD4 + T cell-introduced colitis model mice. ..

[Clinical score of enteritis]
The clinical score (CS) value of enteritis was measured by using the weight loss level, stool characteristics, and the presence or absence of bloody stool as indicators (see Table 1). Specifically, regarding the body weight loss, which is an indicator of the debilitating level, a weight loss of less than 1% was observed in each mouse based on the body weight immediately before the start of free drinking of drinking water. 0 points in case; 1 point if 1-5% decrease in body weight from the standard; 2 points if 5-10% decrease in body weight from the standard; the standard If a decrease of 10 to 20% was observed from the body weight of the above-mentioned body weight, 3 points; and if a decrease of 20% or more from the standard body weight was observed, 4 points; Regarding stool properties, 0 points for normal stool (good-shaped stool pellets); among diarrhea symptoms, loose stools (paste-like stools that do not adhere to the anus and half-shaped pellets) 2 points for watery stools (liquid stools adhering to the anus) and 4 points for watery stools. Furthermore, when a small amount of bloody stool or a large amount of bloody stool was observed, 2 points and 4 points were given, respectively. The total of these score values (minimum value; 0 [health] to maximum value; 12 [severe colitis] range) was defined as the CS value of enteritis.

[Histopathological examination of enteritis]
After a predetermined number of weeks have passed since the transfer of Naive CD4-positive T cells, colon tissue samples from the proximal and distal colons were collected from Rag2 ^-/- mice (colitis model mice) and 10% neutral buffered formarin. After fixing with the solution, a 5 μm paraffin-embedded tissue section was prepared and stained with HE. Inflammation levels were measured based on stained tissue sections using mucosal damage levels, cell (leukocyte) infiltration levels, and crypt tumor levels as indicators (see Table 2). Specifically, regarding the level of mucosal damage, 0 points for normal appearance (no damage); 1 point for discrete lymphoepithelial lesions; diffuse crypts. 2 points were given when diffuse crypt elongation was observed; and 3 points were given when extensive crypt elongation or mucosal erosion / ulcer was observed. Regarding the cell infiltration level, 0 points when normal (no cell infiltration) or when leukocytes are occasionally observed; 1 point when leukocytes are scattered over a wide area or local aggregation of leukocytes is observed. 2 points were given when leukocyte infiltration in the submucosal tissue with local disappearance of the muscle layer was observed; and 3 points were given when leukocyte infiltration beyond the muscle layer was observed. Regarding the crypt tumor level, 0 points were given when there was no crypt tumor; and 1 point was given when there was crypt tumor. The total of these score values (minimum value; 0 [no colitis] to maximum value; 14 [severe colitis] range) was taken as the histological score (HS; Histological Score) value of enteritis.

[Isolation of LP lymphocytes]
Lamina Propria (LPL) lymphocytes (LPL) were isolated from healthy or colitis mice. The entire length of the colon was taken from WT or Rag2 ^-/- mice, made a longitudinal incision, washed with PBS, and then cut into small pieces. The incised tissue was incubated in HBSS (Hanks' Balanced Salt Solution) containing 1 mM DTT (manufactured by Sigma-Aldrich) and free of calcium and magnesium ions for 20 minutes to remove mucus, and then above. The skin layer was treated with collagenase (Sigma-Aldrich) and 0.01% DNase (Sigma-Aldrich) three times for 30 minutes. The cells were precipitated, washed twice with PBS, and then subjected to density gradient centrifugation using HBSS containing 40-75% isotonic Percoll (manufactured by GE Healthcare Bio-Sciences) to isolate LPL. bottom. After that, two kinds of antibodies (anti-CD3-PerCP / Cy5.5 antibody [100218] and anti-CD4-APC antibody [100516] [all manufactured by BioLegend]) and a cell sorter (BD FACSAria, manufactured by Becton Dickinson) were used. The number of CD3 positive and CD4 positive LPL (CD3 ⁺ CD4 ⁺ LPL) was measured.

[In vitro T cell assay]
Naive CD4-positive T cells were prepared from the spleen of mice using the Naive CD4 + T Cell Isolation Kit (manufactured by Miltenyi Biotec). A 96-well plate with 2.5 μg / mL anti-CD3e antibody (17A2, manufactured by TONBO biosciences) and 5 μg / mL anti-CD28 antibody (37.51, manufactured by TONBO biosciences) bound to 10% deactivated fetal bovine serum. RPMI 1640 medium supplemented with serum, 500 U / mL penicillin, 100 mg / mL streptomycin (Sigma-Aldrich), 10 mM HEPES, 1% non-essential amino acids and 50 mM 2-mercaptoethanol (Invitrogen). -Naive CD4-positive T cells were cultured in 96-well plates containing (Aldrich) and stimulated the naive CD4-positive T cells in vitro. For Th1 polarization conditions, mouse IL-12 (20 ng / ml, manufactured by Peprotech) and anti-IL-4 antibody (10 μg / ml, 11B11: manufactured by TONBO biosciences) were added to the medium, and for Th17 polarization conditions. , Human TGF-β (5 ng / ml, manufactured by Peprotech), Mouse IL-6 (20 ng / ml, manufactured by Peprotech), anti-IL-4 antibody (10 μg / ml) and anti-IFNγ antibody (10 μg / ml). Was added to. In addition, IL-2 (5 ng / mL, manufactured by Peprotech) and human TGF-β (5 ng / ml, manufactured by Peprotech or R & D) were added to the medium in order to induce inhibitory T cells (Treg). .. In some experiments, the cell viability assay was performed using the CellTiter-Glo® luminescent cellviability assay (Promega) according to the attached usage.

[Flow cytometry]
The cells were analyzed by flow cytometry using the following antibodies for surface staining. Anti-CD4-PEcy7 antibody [100528], anti-CD4-APC antibody [100516], anti-CD8-PEcy7 antibody [100722], anti-TCRb-Pacific Blue antibody [109226], anti-CD3-PerCP / Cy5.5 antibody [100218], Anti-CD44-FITC antibody [103006], anti-CD62L-PE antibody [104408]. All of these antibodies were purchased from BioLegend. Living cells were distinguished by DAPI (4', 6-diamidino-2-phenylindole) or Fixable Viability Dye efluor 780® (manufactured by eBioscience). For intracellular cytokine staining, the cells were stained with PMA (12-O-tetradecylholball 13-acetate; 50 ng / ml, manufactured by Sigma-Aldrich) and ionomycin (250 ng / ml, manufactured by Sigma-Aldrich). After incubating for 2 hours with, BD GolgiStop® (1: 100, manufactured by BD Biosciences) was added and incubated for another 2 to 3 hours. The cells were then collected and used with CytoFix / CytoPerm® (BD Biosciences) and PermWash® (BD Biosciences) to anti-IFNγ-PE (BD Bioscience), anti-IL17A-Alexa647 ( The cells were stained with an antibody (manufactured by BD Bioscience). For Foxp3 staining, cells were immobilized and permeabilized using the Foxp3 Transcription Factor stainingbuffer kit (manufactured by eBioscience). Data were collected using FACSCanto® II flow cytometer (BD Biosciences) and analyzed using FlowJo® software (Tree Star).

2. 2. Results [Preventive effect of D-serine on the onset of enteritis]
Rag2 ^-/- Mice contain two types of drinking water (distilled water [“H ₂ O” in FIGS. 1 and 2]] or distilled water containing 1.5% D-serine [“D-Ser” in the figure. "]) Free drinking water has started. Naive CD4-positive T cells were transferred into Rag2 ^-/- mice 1 week (7th day) after the start of free drinking, and body weight (see FIG. 1A) and enteritis 1-10 weeks after the start of free drinking. Clinical score values (see FIG. 1B) were analyzed. In addition, after 10 weeks of free drinking, colon weight (mg / cm) (see Figure 2A), CD3 ⁺ CD4 ⁺ laminapropria lymphocyte (LPL) count (see Figure 2B), and histological score for enteritis (Figure 2B). 2D) was analyzed. Free drinking with the above two types of drinking water was continued up to each time point even after the transfer of naive CD4 positive T cells.
As a result, Rag2 ^{-/-mice transplanted with naive CD4-positive T cells (that is, naive CD4-positive T cell-transferred colitis model mice) were transferred to Rag2-/-} mice 6 weeks after the start of free drinking (that is, Rag2 ^-/- mice). Weight decreased from (5 weeks after transfer of Naive CD4 positive T cells) (see “H ₂ O” in FIG. 1A), and an increase in inflammation score for colitis was observed (“H ₂ O” in FIG. 1B). reference). On the other hand, in the naive CD4-positive T cell-transferred colitis model mice in which D-serine was continuously ingested before the transfer of naive CD4-positive T cells, such weight loss was not observed (“D-Ser” in FIG. 1A). (See), and no increase in the inflammation score of enteritis was observed (see "D-Ser" in FIG. 1B).

In addition, the colonitis model mice ingested with D-serine for 10 weeks had a significantly reduced colon weight (mg / cm) compared with the mice without D-serine (see FIG. 2A), and CD3 ⁺ CD4. ⁺ The number of LPLs decreased significantly (see FIGS. 2B), and the histological score of colitis also decreased significantly (see FIGS. 2C and D).

These results show that D-serine exerted a preventive effect on inflammatory bowel disease, resulting in weight loss associated with enterocolitis, as well as increased weight per unit length of the colon (such an increase indicates intestinal edema). , Shows that the increase in the number of inflammatory lymphocytes (CD3 ⁺ CD4 ⁺ LPL) was suppressed.

[Preventive effect of serine D body-specific onset of enteritis]
Next, it was verified by the same analysis whether or not the preventive effect of D-serine on the onset of enteritis was also observed in L-serine. Ingestion of L-serine into enterocolitis model mice did not suppress weight loss or increase in inflammation score of enterocolitis, unlike colitis model mice ingested with D-serine (see FIGS. 3A and 3B). There was also no reduction in colon weight, CD3 ⁺ CD4 ⁺ LPL count, or histological score for enterocolitis (see FIGS. 4A-4D).

These results indicate that the preventive effect of serine on the onset of enteritis is a D-form specific effect (not the L-form).

In addition, when D-serine was ingested by WT mice (not colitis model mice), the body weight was almost the same as that of WT mice without D-serine, and no increase in the clinical score value of enterocolitis was observed. (See FIGS. 5A and 5), and colon weight, CD3 ⁺ CD4 ⁺ LPL numbers, and intestinal condition remained almost unchanged (see FIGS. 6A-C).
These results indicate that the preventive effect of D-serine on the onset of enteritis does not occur in WT mice.

[Concentration-dependent preventive effect of D-serine on the onset of enteritis]
Next, the concentration dependence of the preventive effect of D-serine on the onset of enteritis was examined.
Rag2 ^-/- mouse contains 4 types of drinking water (distilled water [“H ₂ O” in FIGS. 7 and 8]] and distilled water containing 0.5% D-serine [“D-Ser0” in the figure. .5% "], distilled water containing 1.0% D-serine [" D-Ser 1.0% "in the figure], or distilled water containing 1.5% D-serine [in the figure. "D-Ser 1.5%"],) free drinking was started. Naive CD4-positive T cells were transferred into Rag2 ^-/- mice 1 week (7th day) after the start of free drinking, and body weight (see FIG. 7A) and enteritis 1-9 weeks after the start of free drinking. Clinical score values (see FIG. 7B) were analyzed. Free drinking with the above four types of drinking water was continued up to each time point even after the transfer of naive CD4 positive T cells. In addition, 9 weeks after the start of free drinking, the weight of the colon of Rag2 ^-/- mice (see FIG. 8A), the number of CD3 ⁺ CD4 ⁺ LPL (see FIG. 8B), and the histological score value of enterocolitis (see FIG. 8B). 8D) was analyzed.
As a result, when 0.5 (w / v)% to 1.5 (w / v)% of D-serine was ingested into naive CD4-positive T cell-introduced colitis model mice, D-serine was not ingested. Compared with Naive CD4-positive T cell-introduced colitis model mice, weight loss and increase in inflammation score of enterocolitis were suppressed (see FIGS. 7A and 7B), and the weight of the colon was significantly reduced in a D-serine concentration-dependent manner. However, the number of CD3 ⁺ CD4 ⁺ LPL decreased significantly (see FIG. 8B), and the histological score of enterocolitis also decreased significantly (see FIGS. 8C and D). It was also shown that distilled water containing 1.5 (w / v)% D-serine can completely suppress the pathogenicity of the large intestine.

These results show that the preventive effect on the onset of enteritis is exerted in a concentration-dependent manner of D-serine, and the D-serine concentration is preferably 1% rather than 0.5%, more preferably 1.5%. It is shown that.

[D-serine suppresses the proliferation of CD4-positive T cells and the differentiation of Th1 and Th17 cells]
Previous experiments have shown that D-serine reduces the number of CD4 + T cells in the lamina propria and prevents the development of enteritis in a naive CD4 + T cell transfer colitis model mouse in a concentration-dependent manner. Was done. Therefore, it was decided to verify whether D-serine exerts a direct effect on the transferred CD4-positive T cells, and as a result, exerts a preventive effect on the onset of enteritis.

D-serine or L-serine was added to the culture medium of naive CD4-positive T cells isolated from WT mice, and the cells were cultured under conditions of stimulation of CD3 and CD28 to evaluate the cell viability. As a result, D-serine showed a concentration-dependent inhibitory effect on the proliferation of naive CD4-positive T cells (see FIG. 9A), but L-serine did not show such an inhibitory effect (Fig. 9A). See 9A). Moreover, even when compared with other amino acids, only D-serine specifically showed such an inhibitory effect (see FIG. 9B). A similar inhibitory effect on the proliferation of naive CD4-positive T cells was also confirmed by CFSE (carboxyfluorescein succinimidyl ester) staining (see FIG. 9C).

Since it has been reported that Th1 and Th17 immune responses are associated with Naive CD4-positive T cell-introduced colitis model mice, how D-serine affects the differentiation of CD4-positive T cells? We decided to verify.
Specifically, naive CD4 positive T cells were cultured in the presence of vehicle or D-serine under Th1 polarification conditions, Th17 polarization conditions or Treg polarization conditions. As a result, under all of the modified conditions, D-serine reduced the number and proportion of Th1 and Th17 cells (see FIGS. 9D and E). D-serine also reduced the number of Treg cells, but not the proportion of Treg cells (see FIGS. 9D, E). These data indicate that D-serine inhibits the differentiation into Th1 cells and Th17 cells, but does not inhibit the differentiation into Treg cells, and also inhibits the proliferation of Th1 cells, Th17 cells and Treg cells. Shows. It is considered that the protective action of D-serine against enteritis is due to the suppression of effector T cells while maintaining the differentiation into Treg cells. Furthermore, since the addition of D-serine after polarization also inhibited the differentiation into Th1 cells and Th17 cells (data not shown), D-serine caused enteritis after the transfer of naive CD4-positive T cells. It was thought to prevent.

[Preventive effect of D-serine on the onset of enteritis and therapeutic effect of enteritis]
Next, we analyzed the preventive effect of D-serine on the onset of enteritis when the continuous intake of D-serine was stopped halfway. In the case of Naive CD4-positive T cell-introduced enterocolitis model mice, when continuous ingestion with D-serine was stopped 4 weeks after the transfer (that is, when D-serine was ingested 0 to 5 weeks after the start of the experiment: FIG. 10 (Refer to "D-Ser → _H2O "), and when D-serine is continued thereafter (see "D-Ser" in FIG. 10), weight loss and increase in inflammation score of enterocolitis are suppressed (see "D-Ser"). (See “D-Ser → _H2O ” in FIGS. 10A and 10), the weight of the colon is significantly reduced (see “D-Ser → _H2O ” in FIG. 11A), and the number of CD3 ⁺ CD4 ⁺ LPL is reduced. (See “D-Ser → H ₂ O” in FIGS. 11B), and the histological score of enterocolitis also decreased significantly (see “D-Ser → H ₂ O” in FIGS. 11C and D).

These results indicate that the preventive effect of D-serine on the onset of enteritis is maintained even after the intake of D-serine is stopped after the onset of enteritis is induced.

Furthermore, the therapeutic effect of ingesting D-serine after the onset of enteritis (see “ _H2O → D-Ser” in FIG. 10) was analyzed. In naive CD4-positive T cell-introduced colitis model mice without D-serine (see " _H2O " in FIG. 10), weight loss and an increase in enterocolitis inflammation score were observed. , D-serine was continuously ingested into naive CD4 + T cell-transferred colitis model mice from 4 weeks after the transfer of naive CD4-positive T cells (that is, from the 5th week after the start of the experiment), and such weight loss was observed. (See “H ₂ O → D-Ser” in FIG. 10A), and the increase in the inflammation score value of enterocolitis was also suppressed (see “H ₂ O → D-Ser” in FIG. 10B).

In addition, a naive CD4 + T cell transfer colitis model mouse (“H ₂ ” in FIG. 11) in which D-serine was continuously ingested from the 4th week after the transfer of the naive CD4 positive T cell (that is, from the 5th week after the start of the experiment). O → D-Ser ”) significantly reduced the weight of the colon (see FIG. 11A) compared to the naive CD4 + T cell-introduced colitis model mice without D-serine (see“-” in FIG. 11). (See “H ₂ O → D-Ser” in FIG. 11B), the number of CD3 ⁺ CD4 ⁺ LPL is significantly reduced (see “H ₂ O → D-Ser” in FIG. 11B), and the histological score value of enteritis is also Significantly decreased (see "H ₂ O → D-Ser" in FIGS. 11C and 11C).

These results showed that D-serine exerted a therapeutic (improving) effect on inflammatory bowel disease, and as a result, the increase in the number of inflammatory lymphocytes (CD3 ⁺ CD4 ⁺ LPL) and the weight loss associated with enteritis were suppressed. It is shown that.

The present invention contributes to the prevention and / or treatment of inflammatory bowel disease.

Claims

A prophylactic or ameliorating agent for inflammatory bowel disease, which comprises D-serine or a derivative thereof or a physiologically acceptable salt thereof.
The preventive or ameliorating agent for inflammatory bowel disease according to claim 1, wherein the derivative of D-serine is a compound that changes to D-serine after administration.
The preventive or ameliorating agent according to claim 1 or 2, which is orally ingested.
Any one of claims 1 to 3, wherein one or more selected from weight loss; diarrhea; bloody stool; damage to the intestinal mucosa; increased level of leukocyte infiltration in the intestinal mucosa; and crypt tumor; The preventive or ameliorating agent described in.