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WO2022080249A1 - Agent pour prévenir ou améliorer les affections inflammatoires de l'intestin - Google Patents

Agent pour prévenir ou améliorer les affections inflammatoires de l'intestin Download PDF

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WO2022080249A1
WO2022080249A1 PCT/JP2021/037311 JP2021037311W WO2022080249A1 WO 2022080249 A1 WO2022080249 A1 WO 2022080249A1 JP 2021037311 W JP2021037311 W JP 2021037311W WO 2022080249 A1 WO2022080249 A1 WO 2022080249A1
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serine
cells
positive
mice
inflammatory bowel
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PCT/JP2021/037311
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English (en)
Japanese (ja)
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道夫 鬼澤
守 渡辺
剛人 浅川
弘正 大平
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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
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Priority to JP2022556908A priority Critical patent/JPWO2022080249A1/ja
Publication of WO2022080249A1 publication Critical patent/WO2022080249A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants

Definitions

  • 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.
  • D-serines a physiologically acceptable salt thereof
  • 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.
  • immunosuppressants, corticosteroids, molecular biologics (antibody drugs), aminosalicylic acid preparations and the like are mainly used.
  • 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. ..
  • these biologics cannot be orally administered and cause physical distress.
  • 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.
  • Non-Patent Document 1 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).
  • Non-Patent Document 4 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).
  • NMDA N-Methyl-D-aspartate
  • Patent Document 1 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.
  • 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.
  • the D-form ie, D-serin
  • the L-form of serin has the effect of effectively preventing the onset of inflammatory bowel disease.
  • 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.
  • a prophylactic or ameliorating agent for inflammatory bowel disease which comprises D-serine or a derivative thereof or a physiologically acceptable salt thereof.
  • 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.
  • 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.
  • "*", “**” 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).
  • 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).
  • HE hematoxylin-eosin
  • 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.
  • 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.
  • FIG. 8B The histological score value of enterocolitis 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).
  • 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).
  • CFSE carboxyfluorescein succinimidyl ester
  • 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 IFN ⁇ -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).
  • 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.
  • 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.
  • the histological score of enteritis was measured based on a HE-stained colorectal tissue sample (FIG. 11C).
  • 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").
  • 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.
  • 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.
  • 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 limba lymphocytes); crypt tumors; etc.
  • 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.
  • viruses eg, norovirus, influenza virus, rotavirus, coronavirus
  • 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;
  • 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.
  • non-human mammals eg, monkeys, mice, rats, dogs, cats, domestic animals [eg, rabbits, pigs, horses, cows, sheep, goats, deer]
  • domestic animals eg, rabbits, pigs, horses, cows, sheep, goats, deer]
  • 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.
  • 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.
  • D-serine 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).
  • 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.
  • 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], ly
  • 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.
  • oral ingestion an oral ingestion method
  • parenteral ingestion parenteral ingestion
  • parenteral ingestion parenteral ingestion
  • parenteral ingestion administration
  • intravenous administration intravenous administration
  • local administration parenteral ingestion
  • 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.
  • the concentration when converted to D-serine is 0.1 ⁇ g.
  • 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.
  • 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.
  • 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.
  • 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.
  • naive CD4-positive T cell-introduced colitis model 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.
  • CS 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.
  • 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.
  • Lamina Propria (LPL) lymphocytes 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.
  • HBSS Horts' Balanced Salt Solution
  • 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.
  • 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 ⁇ g / mL anti-CD3e antibody (17A2, manufactured by TONBO biosciences) and 5 ⁇ g / 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.
  • mouse IL-12 (20 ng / ml, manufactured by Peprotech
  • anti-IL-4 antibody 10 ⁇ g / ml, 11B11: manufactured by TONBO biosciences
  • Human TGF- ⁇ (5 ng / ml, manufactured by Peprotech), Mouse IL-6 (20 ng / ml, manufactured by Peprotech), anti-IL-4 antibody (10 ⁇ g / ml) and anti-IFN ⁇ antibody (10 ⁇ g / ml).
  • IL-2 5 ng / mL, manufactured by Peprotech
  • human TGF- ⁇ 5 ng / ml, manufactured by Peprotech or R & D
  • the cell viability assay was performed using the CellTiter-Glo® luminescent cellviability assay (Promega) according to the attached usage.
  • 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.
  • PMA 12-O-tetradecylholball 13-acetate
  • ionomycin 250 ng / ml, manufactured by Sigma-Aldrich
  • the cells were then collected and used with CytoFix / CytoPerm® (BD Biosciences) and PermWash® (BD Biosciences) to anti-IFN ⁇ -PE (BD Bioscience), anti-IL17A-Alexa647 ( The cells were stained with an antibody (manufactured by BD Bioscience).
  • CytoFix / CytoPerm® BD Biosciences
  • PermWash® BD Biosciences
  • the cells were stained with an antibody (manufactured by BD Bioscience).
  • 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).
  • 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.
  • 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 intestinal 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.
  • 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).
  • CFSE carboxyfluorescein succinimidyl ester
  • 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.
  • 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).
  • 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.
  • 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).
  • 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).
  • the present invention contributes to the prevention and / or treatment of inflammatory bowel disease.

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Abstract

La présente invention aborde le problème de la fourniture d'un agent destiné à prévenir ou à améliorer les maladies inflammatoires de l'intestin, ledit agent présentant une sécurité élevée lors de son administration, pouvant être administré par voie orale et ayant un coût de production relativement faible.　Dans la présente invention, un agent comprenant de la D-sérine, un dérivé de celle-ci ou un sel physiologiquement acceptable de celle-ci, est utilisé en tant qu'agent pour prévenir ou améliorer une maladie inflammatoire de l'intestin.
PCT/JP2021/037311 2020-10-12 2021-10-08 Agent pour prévenir ou améliorer les affections inflammatoires de l'intestin WO2022080249A1 (fr)

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