JP2020029412A - Antiinflammatory agent - Google Patents

Abstract

To provide an antiinflammatory agent useful for prevention, improvement or treatment of inflammation-derived diseases including type 2 diabetes mellitus, high blood pressure, atherosclerosis.SOLUTION: An antiinflammatory agent has a glutaminase inhibitor such as 6-diazo-5-oxo-L-norleucine as an active ingredient.SELECTED DRAWING: None

Description

  The present invention relates to an anti-inflammatory agent having an excellent effect of suppressing inflammation.

  Inflammation is a local transient reaction that a living body shows when a substance (an inflammatory substance) causing some harmful stimulus acts on a living tissue, and is a kind of a biological defense reaction. However, inflammation also damages living tissues, and when it becomes chronic and becomes chronic inflammation, an excessive inflammatory reaction is rather harmful.

  Chronic inflammation is a basic disease state such as an age-related disease including a lifestyle-related disease and cancer (Non-Patent Documents 1 and 2). Chronic inflammation is accompanied by damage and structural change (deformation) of the affected tissue over time, and impairs the original function of the affected tissue, and increases blood sugar, blood pressure, arthritis, and neurodegeneration. It may cause associated dementia.

  As conventional anti-inflammatory agents, steroidal anti-inflammatory agents, non-steroidal anti-inflammatory agents, anti-inflammatory agents using naturally derived components, and the like have been put to practical use.

Ichiro Manabe, Chronic inflammation and aging-related diseases, Journal of Geriatrics Society, Vol. 54, No. 2, April 2017, pp. 105-113 Maya E. Kotas and Ruslan Medzhitov, Homeostasis, Inflammation, and Disease Susceptibility, Cell, 160, February 26, 2015, 816-827.

  However, conventional anti-inflammatory drugs have drawbacks such as a high risk of side effects due to long-term use, a weak anti-inflammatory effect alone, requiring a large amount of administration, and a need for combined use with other anti-inflammatory drugs. is there. Therefore, development of an anti-inflammatory agent exhibiting an excellent anti-inflammatory action is desired.

  Therefore, an object of the present invention is to provide an anti-inflammatory agent effective for preventing, improving or treating an inflammatory effect.

  The present inventors have found that a glutaminase inhibitor has an anti-inflammatory effect, and have completed the present invention.

The present invention is as follows.
1. An anti-inflammatory agent comprising a glutaminase inhibitor as an active ingredient.
2. 2. The anti-inflammatory agent according to the above 1, wherein the glutaminase inhibitor is DON or CB-839.
3. 3. The anti-inflammatory agent according to the above 1 or 2, which is used for prevention, improvement or treatment of a disease derived from inflammation.
4. The disease is at least one selected from type 2 diabetes, hyperglycemia, insulin resistance, hypertension, atherosclerosis, NAFLD, fatty liver, dyslipidemia, arthritis, Alzheimer's, inflammatory bowel disease, asthma and heart disease. The anti-inflammatory agent according to any one of the above 1 to 3.
5. At least one of blood sugar level, blood pressure, blood triglyceride, arteriosclerosis, arthritis, chronic knee joint pain or dementia suppression, prevention or improvement, lipid metabolism improvement, memory learning function and cognitive function 3. The anti-inflammatory agent according to the above 1 or 2, which has at least one action selected from potentiating or improving actions.
6. A food or drink comprising the anti-inflammatory agent according to any one of the above 1 to 5.
7. A medicine comprising the anti-inflammatory agent according to any one of the above 1 to 5.

  Since the anti-inflammatory agent of the present invention can exert an excellent anti-inflammatory effect, it is effective for preventing, ameliorating or treating diseases or symptoms caused by inflammation.

FIG. 1A shows a representative image of mice and a graph of body weight for a control group (ND + CV) and a CB-839-administered group (ND + CB) when a normal diet (hereinafter abbreviated as ND) is ingested. FIG. 1B is a representative image and a graph of body weight of mice in a control group (HFD + CV) and a CB-839-administered group (HFD + CB) when a high-fat diet (hereinafter abbreviated as HFD) is ingested. Is shown. * P <0.05; ** p <0.01; *** p <0.001 in comparison with the control group. Data are means ± SEM. D is shown. FIG. 2A shows the ratio of food intake to body weight and water intake to body weight in a control group (ND + CV) and a CB-839 administration group (ND + CB) when ND was ingested. FIG. 2B shows the ratio of food intake to body weight and water intake to body weight in a control group (HFD + CV) and a CB-839-administered group (HFD + CB) when HFD was taken. Data are means ± SEM. D is shown. 3A to 3D show images of each tissue and the mass ratio of each tissue to body weight in a control group (HFD + CV) and a CB-839 administration group (HFD + CB) when HFD was taken. 3A shows the results of visceral adipose tissue (eWAT), FIG. 3B shows the results of subcutaneous adipose tissue (iWAT), FIG. 3C shows the results of brown adipose tissue (BAT), and FIG. 3D shows the results of liver. * P <0.05 compared to HFD + CV. Data are means ± SEM. D is shown. FIG. 4A shows the weight of mice in the control group (ND + CV) and the DON-administered group (ND + DON) when ND was taken. FIG. 4B shows the weight of mice in the control group (HFD + CV) and the DON-administered group (HFD + DON) when HFD was taken. * P <0.01; ** p <0.001 in comparison with HFD + CV. Data are means ± SEM. D is shown. 5A to 5E show the ratio of each mRNA expression level and 18S gene expression used as an internal standard in the control group (CV) and the CB-839 administration group (CB) when HFD was ingested was analyzed by RT-PCR. The results are shown. 5A shows the results of TNF-α / 18S, FIG. 5B shows the results of MCP-1 / 18S, FIG. 5C shows the results of F4 / 80 / 18S, FIG. 5D shows the results of CD11c / 18S, and FIG. 5E shows the results of CD206 / 18S. * P <0.05 compared to CV. Data are means ± SEM. D is shown.

  As used herein, “anti-inflammatory” is a concept that includes both preventing inflammation and improving inflammation.

  The present invention relates in one aspect to an anti-inflammatory agent comprising a glutaminase inhibitor. The glutaminase inhibitor may be any compound that inhibits glutaminase, which is an enzyme that produces glutamic acid from glutamine. The mode of inhibition is not particularly limited.

  Glutaminase inhibitors are not particularly limited, and include known glutaminase inhibitors. For example, 6-diazo-5-oxo-L-norleucine [(S) -2-amino-6-diazo-5-oxocaproic acid or a salt thereof (DON)], CB-839bis-2- (5-phenyl) Acetamide-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), ebselen (Ebselen), Compound 968, GlutaDON (registered trademark) (PEG-PGA + DON) (New Medical Enzymes AG), GlutaChemo (GlutaChemo) −PGA + ideal candidate) (manufactured by New Medical Enzymes AG). The glutaminase inhibitor may be used alone or in combination of two or more.

  Glutaminase inhibitors have TNFα expression inhibitory action, fat inflammation inhibitory action, macrophage (eg, macrophage-1, macrophage-2) inhibitory action, etc., and therefore have anti-inflammatory agents [eg, food compositions, pharmaceuticals, Health-promoting agents, nutritional supplements (eg, supplements), food additives, etc.]. Glutaminase inhibitors can be applied to non-human animals and humans (eg, administration, ingestion, inoculation, etc.) as such or as anti-inflammatory agents with conventional ingredients.

  The anti-inflammatory agent according to the present invention can be used for preventing, ameliorating or treating diseases caused by inflammation. Examples of diseases caused by inflammation include type 2 diabetes, hyperglycemia, insulin resistance, hypertension, atherosclerosis, NAFLD, fatty liver, dyslipidemia, arthritis, Alzheimer, inflammatory bowel disease, asthma and heart disease And the like.

  In addition, the action of the anti-inflammatory agent according to the present invention includes, for example, an increase in blood glucose level, an increase in blood pressure, an increase in blood triglyceride, arteriosclerosis, arthritis, chronic knee joint pain and dementia (for example, foreign matter in the brain or brain). At least one of dementia accompanied by accumulation of internal waste), an effect of improving lipid metabolism, and an effect of enhancing or improving at least one of a memory learning function and a cognitive function.

  Examples of the action of improving lipid metabolism include an action of lowering blood neutral fat level, an action of lowering small dense LDL cholesterol, and an action of lowering the proportion of small dense LDL cholesterol in total LDL.

  The application form of the anti-inflammatory agent of the present invention is not particularly limited. For example, it can be used in any application form such as oral, transdermal, enteral, transmucosal, intravenous, transarterial, subcutaneous, and intramuscular. Since the anti-inflammatory agent of the present invention can exert an anti-inflammatory effect when used in any application form, it can be applied to various products such as food and drink, medicine, feed, pet food and the like. The anti-inflammatory agent of the present invention may be ingested (administered) as a supplement or the like, or may be used as an additive for imparting an anti-inflammatory effect to compositions such as food and drink.

  The dosage form of the anti-inflammatory agent of the present invention may be any of a solid form, a semi-solid form, a liquid form and the like, and is appropriately set according to the type and use of the product. The anti-inflammatory agent of the present invention includes water, fats and oils, waxes, hydrocarbons, fatty acids, higher alcohols, esters, and plants, depending on the form and the like, as long as the effects of the present invention are not impaired. Extracts, water-soluble polymers, surfactants, metal soaps, alcohols, polyhydric alcohols, pH adjusters, antioxidants, UV inhibitors, preservatives, fragrances, powders, thickeners, pigments, chelating agents And the like.

  Further, the anti-inflammatory agent of the present invention may contain other anti-inflammatory components within a range not impairing the effects of the present invention, depending on the form, use, and the like. Examples of such anti-inflammatory components include vitamin C, squalane, niacin, niacinamide, long-chain hyaluronic acid, placenta extract, sorbitol, chitin, chitosan, various plant extracts, and the like. The amounts of these components are not limited as long as the effects of the present invention are not impaired.

  When the anti-inflammatory agent of the present invention is used as a food or drink, the glutaminase inhibitor is adjusted to a desired form as it is or in combination with other food materials or additives, and provided as a food or drink exhibiting the desired effect. .

  Such foods and drinks include, in addition to general foods and drinks, health foods such as health foods, functional foods, dietary supplements, and foods that include supplements and are labeled with a disease risk reduction label (eg, specific insurance products). Foods, nutritionally functional foods and functionally labeled foods), and foods for the sick.

  The form of these foods and drinks is not particularly limited, and specifically, liquid foods such as drinks, soups, non-alcoholic drinks, alcoholic drinks, jelly-like drinks and functional drinks; semi-solid forms such as jelly and yogurt Foods; Fermented foods such as miso and fermented beverages; Western confectionery such as cookies and cakes; Japanese confectionery such as buns and yokan; And products containing oils such as margarine; foods containing carbohydrates such as rice, rice cakes, noodles, breads and pasta; processed livestock foods such as ham and sausage; processed fishery products such as kamaboko, dried and salted vegetables; pickles, etc. Vegetable processed foods; retort products such as curry, red bean paste and Chinese soup; instants such as instant soup and instant miso soup Goods; microwavable food; eggs processed article using, and fish or meat processed products; seasonings; and the like. Furthermore, health foods and drinks prepared in the form of powder, granules, tablets, capsules, liquids, pastes or jellies are also included. These foods and drinks can be used for the above-mentioned uses. Further, the food for the sick is required for patients in need of prevention, improvement or treatment of inflammatory symptoms and inflammatory diseases, or in the case of prevention, improvement or treatment of diseases in which chronic inflammation is the underlying condition. It is provided for patients who do.

  When the anti-inflammatory agent of the present invention is used in foods and drinks, the amount of the glutaminase inhibitor to be added to the food and drink varies depending on the form of the food and drink, but depends on the glutaminase inhibitor used by those skilled in the art. It can be set appropriately while predicting the effect based on known information or animal tests. The amount of the glutaminase inhibitor that can be set in such a manner is preferably 0.00001 to 100% by mass, more preferably 0.0001 to 100% by mass, and still more preferably 0.001 to 100% by mass. Especially preferably, the range which becomes 0.01 to 100 mass% is mentioned.

  Furthermore, when the anti-inflammatory agent of the present invention is used in foods and drinks, the anti-inflammatory agent of the present invention can be provided alone or in combination with other components as a food additive for anti-inflammatory. When the anti-inflammatory agent of the present invention is used as a food additive, the content of the glutaminase inhibitor in the food additive, the amount of the food additive added to the food or beverage, etc. It may be appropriately set so that the content of the glutaminase inhibitor therein can be satisfied.

  When the anti-inflammatory agent of the present invention is used in a medicine, the anti-inflammatory agent of the present invention may be used alone or in combination with other pharmacologically active ingredients, pharmaceutically acceptable bases and additives, and the like. To provide the desired effect.

  The form of such a medicament is not particularly limited, and specific examples thereof include oral preparations such as tablets, granules, powders, capsules, soft capsules, and syrups; liquids, ointments, creams, and gels. And transmucosal or transmucosal preparations such as sprays, patches, inhalants, suppositories and the like; injections and the like.

  When the anti-inflammatory agent of the present invention is used for a medicine, the mixing ratio of the glutaminase inhibitor to the medicine varies depending on the form of the medicine. For example, in the case of an oral administration preparation or an injection, preferably 0%. 0.000001 to 100% by mass, more preferably 0.0001 to 100% by mass, still more preferably 0.001 to 100% by mass, and particularly preferably 0.01 to 100% by mass.

  The application (for example, administration, ingestion, inoculation, etc.) of the anti-inflammatory agent of the present invention is not particularly limited as long as it is an effective amount. Generally, the amount of the glutaminase inhibitor of the active ingredient is preferably 10 mg to 10 mg per day. The amount is 30 g, more preferably 50 mg to 10 g, and still more preferably 200 mg to 5 g. It is preferable to administer the above-mentioned application amount once a day or more (for example, 1 to 3 times a day), and it can be appropriately increased or decreased according to age, disease state and symptoms.

  Examples are shown below, but the present invention is not limited to the following examples.

[Example 1] Hierarchical clustering analysis and KEGG pathway analysis For wild-type C57BL / 6 male mice (manufactured by Sankyo Laboratories), a normal diet group (hereinafter abbreviated as ND) was ingested (20 animals). A high-fat diet group (20 animals) fed a high-fat diet (hereinafter abbreviated as HFD) having a fat content of 60 kcal% (D12492, manufactured by Research Diets) was prepared. On days 0, 3, 6, and 10, exhaustive expression fluctuation genes were extracted from adipose tissue of each of 5 mice (no obesity or impaired glucose tolerance), and hierarchical clustering analysis was performed using a microarray. As a result, Group 12 capable of sorting the normal diet group and the high fat diet group was detected in the initial stage (day 3).

  83 KEGG pathways hit Group 12. A search for “obesty” (obesity) and “diabetes (diabetes)” in PubMed for the 83 hit KEGG pathways revealed that the pathway for “glutaminase” was included. There has been no report on the pathway of “glutaminase” in the area of obesity and diabetes, and the relationship between gluminase and obesity was found for the first time.

[Example 2] Analysis of effect by administration of CB-839 using animal experiment The effect of administration of CB-839 was analyzed by animal experiment. Five-week-old male wild-type C57BL / 6 mice (manufactured by Sankyo Lab Service) were maintained at a controlled temperature with a constant 12-hour light-dark cycle. Mice had free access to food and water.

  ND or HFD was used as food. Mice fed HFD as food (hereinafter abbreviated as obese C57BL / 6 mice) were allowed to access HFD freely, starting at 5 weeks of age.

  Body weight and food and water intake after oral administration of CB-839 (200 mg / kg) (manufactured by Arctom Chemicals) or a control vehicle twice a week were measured. The control vehicle consisted of 25% (w / v) hydroxypropyl-β-cyclodextrin (HPBCD; Fujifilm Wako Pure Chemical Industries) in 10 mmol / L citrate (pH 2). CB-839 was formulated as a 20 mg / mL (w / v) solution as a control [Rossmeisl M, et al. , Diabetes, 2003, 52 (8): 1958-1966. ]. The experiments were conducted with animal ethics in mind after approval by the Animal Experiment Committee of the University of Toyama.

  FIG. 1A shows a representative image of mice and a graph of body weight for a control group (ND + CV) and a CB-839-administered group (ND + CB) when ND was ingested as food. FIG. 1B shows a representative image of mice and a graph of body weight for a control group (HFD + CV) and a CB-839-administered group (HFD + CB) when HFD was ingested.

  As shown in FIG. 1A, when ND was taken, no difference in body weight was observed between the control group and the CB-839-administered group. In addition, as shown in FIG. 1B, the increase in body weight induced by ingestion of HFD was significantly reduced by CB-839. From these results, it was found that the administration of CB-839 attenuated the weight gain of the mice that had consumed the high fat diet.

  FIG. 2A shows the ratio of food intake to body weight and water intake to body weight in a control group (ND + CV) and a CB-839-administered group (ND + CB) when ND was taken as food. FIG. 2B shows the ratio of food intake to body weight and water intake to body weight in a control group (HFD + CV) and a CB-839-administered group (HFD + CB) when HFD was taken.

  As shown in FIG. 2A and FIG. 2B, the CB-839 administration group did not show a significant difference in food intake with respect to body weight and water intake with respect to body weight as compared with the control group.

  FIGS. 3A to 3D show images of each tissue and the mass ratio of each tissue to body weight in the control group (HFD + CV) and the CB-839 administration group (HFD + CB) when HFD was ingested as food.

  As shown in FIGS. 3A, 3B, and 3D, the ratio of eWAT and iWAT to body weight was significantly lower in the CB-839-administered group than in the control group in the mice fed HFD.

  In addition, as shown in FIGS. 3A to 3C, suppression of hypertrophy in eWAT, iWAT, and BAT was observed in the CB-839 administration group as compared with the control group. Furthermore, as shown in FIG. 3D, the liver was in a more transparent red state in the CB-839 administration group than in the control group.

[Example 3] Analysis of effect by administration of DON using animal experiment The effect of administration of 6-diazo-5-oxo-L-norleucine (DON) was analyzed by an animal experiment. Four-week-old wild-type C57BL / 6 male mice (manufactured by Sankyo Lab Service) were maintained at a controlled temperature with a constant 12-hour light-dark cycle. Mice had free access to food and water.

  Obese C57BL / 6 mice had free access to HFD starting at 4 weeks of age.

  After intraperitoneal injection of a control vehicle consisting of DON (Sigma) or PBS, body weight and food and water intake were measured. DON was formulated as a 2.5 mg / mL (w / v) solution. The dose for all groups was 0.5 mg / mouse. These experiments were carried out with animal ethics in mind after approval of the Animal Experiment Committee of the University of Toyama.

  FIG. 4A shows the weight of mice in the control group (ND + CV) and the DON-administered group (ND + DON) when ND was taken. FIG. 4B shows the body weights of the mice in the control group (HFD + CV) and the DON-administered group (HFD + DON) when HFD was ingested.

  As shown in FIG. 4A, when ND was taken as food, the body weight of the DON-administered group was significantly lower than that of the control group. Further, as shown in FIG. 4B, when HFD was taken as food, the body weight of the DON-administered group was significantly lower than that of the control group.

[Example 4] Real-time RT-PCR
The immunological effects of CB-839 were examined by RT-PCR. Total RNA was isolated from frozen eWAT using TRISure (manufactured by Nippon Genetics). Tissue (100 mg) was homogenized with 300 μL of TRISure. TRISure (600 μL) and chloroform (200 μL) were added and mixed well.

  After incubation for 3 minutes at room temperature, the mixture was centrifuged at 12000 rpm for 15 minutes at 4 ° C. After centrifugation, RNA was in the aqueous phase, DNA and protein were in the interphase and phenol phase. The aqueous phase was transferred to a fresh microtube, mixed with 500 μL of isopropanol and centrifuged at 12000 rpm at 4 ° C. for 20 minutes.

  The supernatant was removed and the RNA pellet was washed by adding 75% ethanol. The pellet was centrifuged again at 7500 rpm at 4 ° C. for 5 minutes. The supernatant was removed and the RNA pellet was dried at room temperature (about 15 minutes). The RNA pellet was dissolved in 20 μL of RNAse-free water.

  The extracted total RNA was confirmed with NanoDrop 2000 (manufactured by Thermo Fisher Scientific), and diluted to 250 ng / μL with RNase-free water. cDNA synthesis was performed using the PrimerScript RT reagent kit. Real-time PCR (RT-PCR) was performed using MX3000P and MX3005P (manufactured by Agilent Technologies).

  Table 1 shows the sequences of the primers used for RT-PCR. After the denaturation step, 40-50 cycles of PCR amplification at 95 ° C for 10 seconds and annealing at 62 ° C for 20 seconds, followed by an extension reaction at 72 ° C for 15 seconds. The results were analyzed using Mx pro software version 4.10.

  5A to 5E show the ratio of each mRNA expression level and 18S gene expression used as an internal standard in the control group (CV) and the CB-839 administration group (CB) when HFD was ingested was analyzed by RT-PCR. The results are shown. FIG. 5A shows the result of TNF-α (Tumor Necrosis Factor-α), FIG. 5B shows the result of MCP-1 (Monocyte Chemotactic Protein-1), FIG. 5C shows the result of F4 / 80, FIG. 5D shows the result of CD11c, and FIG. It is. The results were compared between the control group and the CB-839-administered group using a two-tailed Student's t-test. Data are means ± SEM. Shown as E. Differences were considered statistically significant when p <0.05.

  As shown in FIGS. 5A to 5E, the expression ratios of mRNAs for TNF-α, MCP1 / 18s, F4 / 80 / 18s, CD11c / 18s, and CD206 / 18s in the eWAT were respectively different from those of the CB-839 administration group and the control group. Were significantly different.

  As shown in FIG. 5A, the expression ratio of TNF-α was significantly lower in the CB-389 administration group than in the control group, suggesting that ingestion of CB-389 inhibited inflammation.

  Further, as shown in FIG. 5B, the expression ratio of MCP1 / 18s was significantly lower in the CB-389 administration group than in the control group, suggesting that ingestion of CB-839 suppressed fat inflammation.

  Furthermore, as shown in FIGS. 5C to 5E, the expression ratios of F4 / 80 / 18s, CD11c / 18s and CD206 / 18s were significantly lower in the CB-389 administration group than in the control group, and macrophages and macrophages were increased by ingestion of CB-839. It was suggested that the expression of -1 and macrophage-2 decreased respectively.

  Therefore, these results indicated that CB-839 could inhibit inflammation in adipose tissue.

Claims (7)

  An anti-inflammatory agent comprising a glutaminase inhibitor as an active ingredient.   The anti-inflammatory agent according to claim 1, wherein the glutaminase inhibitor is DON or CB-839.   The anti-inflammatory agent according to claim 1, which is used for prevention, improvement, or treatment of a disease derived from inflammation.   The disease is at least one selected from type 2 diabetes, hyperglycemia, insulin resistance, hypertension, atherosclerosis, NAFLD, fatty liver, dyslipidemia, arthritis, Alzheimer's, inflammatory bowel disease, asthma and heart disease. The anti-inflammatory agent according to any one of claims 1 to 3.   At least one of suppression of blood sugar, increase in blood pressure, increase in blood triglyceride, arteriosclerosis, arthritis, chronic knee joint pain or dementia, prevention or improvement, lipid metabolism, memory learning function and cognitive function The anti-inflammatory agent according to claim 1 or 2, which has at least one action selected from the action of enhancing or improving the activity.   A food or drink comprising the anti-inflammatory agent according to any one of claims 1 to 5.   A medicament containing the anti-inflammatory agent according to any one of claims 1 to 5.