WO2015098928A1 - Inhibitor of il-1 and tnf activities - Google Patents

Abstract

[Problem] The present invention addresses the problem of providing an inflammatory cytokine activity inhibitor which is non-invasive and satisfies requirements such as safety, convenience and economic performance, for the purpose of treating diabetes and preventing the development of diabetic complications and as a therapy method for altering the natural history of diabetes, or as an effective means for preventing the progression of diabetes. The present invention also addresses the problem of providing a therapeutic or prophylactic agent for autoinflammatory diseases for which an activity of inhibiting the activity of inflammatory cytokines is effective. [Solution] The inflammatory cytokine activity inhibitor is characterized by containing, as an active ingredient, at least one compound selected from an alkaloid originated from a plant belonging to the family Menispermaceae, the genus Stephania, a derivative of the alkaloid and a pharmaceutically acceptable salt of the alkaloid or the derivative.

Description

IL-1 and TNF activity inhibitors

The present invention relates to diabetes, disease states and conditions characterized by insulin resistance, decreased functional pancreatic β-cell mass, hyperglycemia, hyperlipidemia, obesity, metabolic syndrome, arteriosclerosis, autoinflammatory disease (gout Etc.) and for the treatment or prevention of rheumatoid arthritis, inflammatory bowel disease, sepsis.

Diabetes is a group of diseases with chronic hyperglycemia due to lack of insulin action and various characteristic metabolic abnormalities. As the disease progresses, it diminishes in various organs including eyes, kidneys, nerves, and blood vessels. Causes severe debilitating complications.
It is thought to develop due to genetic predisposing factors such as high fat diet, lack of exercise, obesity, and other living environment factors that increase insulin resistance.
Interleukin 1 (IL-1) is an inflammatory cytokine produced and secreted from many cells such as macrophages, monocytes, fibroblasts, vascular endothelial cells, and synovial cells (Non-patent Document 1).
It is known that IL-1 released from production cells induces local and systemic inflammatory effects via IL-1 receptors expressed in various cells.
IL-1 also causes a series of biological actions mediated through the induction of other inflammatory mediators such as prostaglandin E2 (PGE2), tumor necrosis factor (TNF, IL-6, IL-8, corticotropin).
The IL-1 family of cytokines is composed of 11 members, but the effects of IL-1α, IL-1β, IL-1Ra (IL-1 receptor antagonist), and IL-18 are inflamed in humans and experimental animals. Has been studied in sexually transmitted disease states.
IL-1α, IL-1β and IL-1Ra bind to the IL-1 receptor with similar affinity, but are expressed by different genes, have different primary amino acid sequences, and have different physiological activities.
In recent years, many studies showing that IL-1β is involved in the onset, worsening of diabetes, impaired insulin secretion, and insulin resistance have been reported (for example, Non-Patent Document 2).
Metabolic syndrome is a collection of hyperinsulinemia, impaired glucose tolerance, obesity, visceral fat accumulation, hypertension, and lipid metabolism abnormalities characterized by hyperlipidemia.
Insulin resistance is closely related to these disease states and is a powerful risk factor for developing type 2 diabetes, heart attacks, strokes, and arteriosclerosis in the future.
Inflammatory cytokines including IL-1 have been shown to mediate inflammation in adipose tissue that is thought to be involved in insulin resistance (Non-Patent Document 6).
Although TNFα and IL-6 were known to make adipocytes insulin resistant, IL-1 also inhibited insulin signaling by phosphorylating the serine residue of IRS-1 (Insulin Receptor Substrate 1). It has been reported to induce insulin resistance (Non-patent Document 7).
Autoinflammatory disease is a general term for syndromes in which the involvement of autoantibodies, such as that seen in autoimmune diseases, is denied, among the main features of symptoms derived from inflammation such as fever and skin rash.
In recent years, it has been understood that many of them are caused by abnormal function of inflammasome.
Activation of caspase 1 by inflammasome and subsequent enhancement of active IL-1β production is thought to cause systemic inflammation.
At present, anti-IL-1 drugs are used for medical care, and all have remarkable effects.
Many autoinflammatory diseases are inherited diseases, but the disruption of the balance system of IL-1 and IL-1Ra is considered to be the cause of the development of autoinflammatory diseases. Some osteoarthritis, gout, and type 2 diabetes It is proposed to classify as an autoinflammatory disease (Non-patent Document 8).
Regarding the involvement of inflammatory cytokines in autoimmune diseases, analysis is proceeding mainly in rheumatoid arthritis model mice and rheumatoid arthritis patients, and IL-1 receptor antagonists as IL-1 inhibitor therapy, anti-TNF antibodies as TNF inhibitor therapy, IL-6 receptor-inhibiting antibodies have been approved as therapeutic agents for rheumatoid arthritis and are used in clinical practice as IL-6 inhibitory therapies, all of which are more effective than conventional standard drug therapies.

Patent Document 1 discloses an NF-κβ activity inhibitor containing as an active ingredient an alkaloid derived from a plant belonging to the genus Stefania belonging to the family Rubiaceae.
However, as described later in the present invention, improvement of pancreatic β-cell function, preservation of functional pancreatic β-cell amount, improvement of insulin resistance, treatment of diabetes, prevention of diabetes onset, improvement and prevention of obesity, and inflammation The object and action differ from the invention disclosed in Patent Document 1 in that it has been found useful for the treatment and prevention of autoinflammatory diseases involving cytokines.

Japanese Laid-Open Patent Publication No. 11-180873

The present invention treats diabetes and prevents the onset of complications, as well as a therapeutic method that changes the natural history of diabetes, as an effective means for preventing progression, is non-invasive, safe, simple, economical, etc. An object is to provide a inflammatory cytokine activity inhibitor that is satisfied.
It is another object of the present invention to provide a therapeutic / preventive agent for autoinflammatory diseases that is effective in inhibiting inflammatory cytokine activity.

Inhibitors of interleukin-1 (IL-1) and tumor necrosis factor (TNF), which are inflammatory cytokines according to the present invention, are alkaloids derived from plants of the genus Stefania and their derivatives and their pharmaceuticals It is characterized by containing at least one or more kinds of active ingredients among the chemically acceptable salts.
Here, the alkaloid is a bisbenzylisoquinoline compound, and specifically, may be any one or more of cephalanthin, berbamine, isotetrandrine, tetrandrine, cyclanine, and E6-berbamine.
These compounds include those chemically synthesized.

Among the alkaloids, structural formulas of cephalanthin, berbamine, isotetrandrine, and cyclanine are shown in (1) to (4), respectively.

Examples of the plant belonging to the genus Stefania belonging to the genus Stefoniaceae used in the present invention include Stephania cepharantha, Stephania merrilli, Stephania crispa, Stephania venosa and the like. .
As the active ingredient of the present invention, it is preferable to use alkaloids derived from Tamatsuki Rafuji.
As an active ingredient of the present invention, there can be used an alkaloid-containing fraction from Satsuma mushroom, a crystal obtained by separation and purification by a conventional method, the alkaloid derivative produced by a known method, and the like.
For example, using the rhizomes, stems, leaves, seeds, etc., of the Japanese red snapper, extract with a solvent such as methanol, ethanol, acetone, ethyl acetate, concentrate the extract, The alkaloid fraction can be separated by dissolving in an acidic aqueous solution such as an oxalic acid aqueous solution and collecting the precipitate formed by making the solution alkaline.
The obtained fraction may be used after purifying alkaloids by known methods such as various types of chromatography and recrystallization.
Examples of the alkaloid derivatives used in the present invention include acyl derivatives, alkyl derivatives, and carbamoyl derivatives.

When administering the drug of the present invention, the active ingredient may be used as it is, or it may be formulated into tablets, powders, granules, capsules and administered orally.
Furthermore, it may be formulated into a suppository, an injection, an instillation, an eye drop, an external preparation and administered parenterally, but it is desirable to administer as an oral preparation.
For oral preparations, tablets, powders, granules, and capsules are produced by conventional methods with the addition of binders, lubricants, disintegrants, coloring agents, flavoring agents, and the like as necessary.
Moreover, antiseptic | preservative, antioxidant, a stabilizer, etc. can be added as needed.
The dose of the drug of the present invention varies depending on the disease, symptom, age, therapeutic treatment used in combination, etc., but when administered to humans as an oral preparation, 0.1 to 5 mg of the alkaloid, a derivative thereof, or a salt thereof. / Kg is administered once to several times a day.
The agents of the present invention can be used alone or in combination with at least one or more active agents that work in different modes of action.
Active drugs include 1) sulfonylurea, 2) biguanide, 3) α-glucosidase inhibitor, 4) insulin sensitizer, 5) glucagon-like peptide (GLP-1), 6) DPP-4 inhibitor 7) Insulin 8) SGLT inhibitor.
In yet another embodiment, the active agent used in combination may be a hyperlipidemia therapeutic agent, a non-steroidal anti-inflammatory agent, or a steroid agent.
The method of using the drug of the present invention includes treatment of type 1 diabetes, type 2 diabetes, obesity, hyperglycemia, insulin secretion disorder, insulin resistance and disease states and conditions characterized by insulin resistance or prevention of progression of the disease state, Can be used in mammals to prevent the occurrence of complications.
Furthermore, it can be used as a therapeutic / prophylactic agent for inflammatory diseases effective in inhibiting IL-1 and TNFα activity.
Since the drug according to the present invention can be used for the improvement or prevention of obesity and the like as described above, it can also be an agent for improving / preventing metabolic syndrome.
It can also be a food or drink containing the drug according to the present invention, in which case it means all foods and drinks, but in addition to general foods including so-called health foods, for specified health use specified in the health functional food system of the Ministry of Health, Labor and Welfare It also includes health foods such as foods and functional foods, and supplements, feeds, food additives, and the like are also included in the food and drink of the present invention.
In order to use the active ingredient of the present invention in foods and drinks, the foods and drinks can be produced as they are or mixed with raw materials of foods and drinks such as processed meat and soft drinks together with various nutritional components.
Further, when the active ingredient of the present invention is used as a health food, a dietary supplement, etc., it can be prepared in the form of tablets, capsules, powders, granules, suspensions, syrups, etc., using, for example, conventional means. it can.
The compounding amount of the active ingredient of the present invention in the above-mentioned food and drink varies depending on the addition form and administration form and can be selected from a wide range, but it is usually desirable to blend 0.01 to 1% by weight.

An IL-1 receptor antagonist (anakinra) is an expensive protein preparation that needs to be injected subcutaneously every day, and is not suitable as a long-term treatment for diabetes.
Patients cause problems with adherence to the treatment system by self-injection and have issues such as reduced efficacy, safety and simplicity.
In contrast, the medicament according to the present invention is a non-invasive, anti-diabetic agent / preventive agent for preserving pancreatic β-cell function that satisfies safety, convenience, economy, and the like, and has an inhibitory action on IL-1 and TNFα activity. Is an effective therapeutic / preventive agent for inflammatory diseases.
The active ingredient of the present invention has effects such as pancreatic β-cell protection or β-cell function maintenance, carbohydrate metabolism improvement, lipid metabolism improvement, and hyperlipidemia improvement. Furthermore, since it is derived from a natural product, it is excellent in safety, and can be routinely used in the form of a food or drink as a preventive / improving agent for metabolic syndrome.

An evaluation system for an IL-1 inhibitor compound is shown. The cell death by human IL-1β and mouse IL-1β of human cell lines, the inhibitory action of human IL-1 receptor antagonist, and the effect of human IL-1β neutralizing antibody are shown. 2 shows the inhibitory effect of plant-derived alkaloid compounds on cell death by human IL-1β. 2 shows cell death by human TNFα and human IL-1α and MB5C (cephalanthin) inhibitory action. 2 shows the inhibitory action of MBMX (Takatsuki tsumugi extract alkaloid preparation) on cell death by human IL-1β. 2 shows the inhibitory action of MBMX on pancreatic β cell (MIN6) death by IL-1β. 2 shows the inhibitory action of a bisbenzylisoquinoline compound on pancreatic β cell (MIN6) death caused by IL-1β. The effect (prophylactic administration test) of MBMX in a high fat diet-fed obesity model is shown. The effect (prophylactic administration test) of MBMX in a high fat diet-fed obesity model is shown. The effect (prophylactic administration test) of MBMX in a high fat diet-fed obesity model is shown. The effect (therapeutic administration test) of MBMX in a high fat diet-fed obesity model is shown. The effect (therapeutic administration test) of MBMX in a high fat diet-fed obesity model is shown. The effect (therapeutic administration test) of MBMX in a high fat diet-fed obesity model is shown. The effect (therapeutic administration test) of MBMX in a high fat diet-fed obesity model is shown. 2 shows the inhibitory action of MBMX on Prostaglandin E2 production by IL-1β stimulation. 2 shows the inhibitory action of MBMX on Prostaglandin E2 production by IL-1α stimulation. The effect (weight change) of MBMX in a type 2 obese diabetic mouse (db / db) is shown. 2 shows the effect of MBMX on improving glucose metabolism in type 2 obese diabetic mice (db / db). The effect (glucose tolerance test) of MBMX in type 2 obese diabetic mice (db / db) is shown. The effect of MBMX on the feeding behavior of type 2 obese diabetic mice (db / db) is shown. The effect (weight change) of the MBMX-containing diet feeding in type 2 obese diabetic mice (db / db) is shown. The effect (blood glucose level change) of the MBMX combination diet feeding in a type 2 obese diabetic mouse (db / db) is shown. The effect (feeding amount change) of the MBMX-containing diet feeding in type 2 obese diabetic mice (db / db) is shown. The effect (a glucose tolerance test) of the MBMX-containing diet feeding in type 2 obese diabetic mice (db / db) is shown. The effect (insulin response ability test) of the MBMX-containing diet feeding in type 2 obese diabetic mice (db / db) is shown. The insulin content in the pancreas in MBMX-containing diet fed db / db mice is shown.

Next, it will be described that the active ingredient according to the present invention was tested to have an inhibitory effect on the activity of IL-1 and TNFα against cell death and to have an obesity preventing effect and an obesity improving effect.
In addition, an inhibitory effect on prostaglandin E2 production by IL-1α and IL-1β stimulation was also confirmed.

First, the reagents and test conditions used in the confirmation test will be described.
<Reagents and pharmaceuticals>
Human and mouse interleukin 1α (IL-1α), interleukin 1β (IL-1β), human tumor necrosis factor (TNF-α), interleukin receptor antagonist (IL-1Ra) are available from R & D Systems Inc. Recombinant products manufactured by (Minneapolis, MN) and Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan) were used.
Anti-human IL-1β neutralizing antibody was obtained from Otsuka Pharmaceutical Co., Ltd. (Tokushima, Japan).
Alkaloid bulk powder (hereinafter referred to as MBMX), cephalanthin, isotetrandrine, berbamine, cicleanine, and its derivatives E6-berbamine are WAKO Chemicals (Osaka, Japan) and Funakoshi Co, Ltd (Tokyo, Japan) )
Measurement kits for glucose, triglyceride, cholesterol, free fatty acids, GOT and GPT in blood are WAKO Chemicals (Osaka, Japan) and Funakoshi Co, Ltd (Tokyo, Japan), PGE2 measurement ELISA kit (R & D Systems Inc.) Obtained from Hirano Junyaku Kogyo.
<Cell line>
The evaluation of IL-1 inhibitory compounds was performed by a cell culture method using IL-1 sensitive cell line A375S2 (Nakai S, Hirai Y, et al. Biochem. Biophy. Res. Commun. 154, 1189, 1988). .
MIN6 mouse pancreatic β cells were obtained from Dr. Miyazaki, Graduate School of Medicine, Osaka University.
<High fat diet feeding test with MBMX-20 weeks administration test->
(A) Animal Mice (C57BL / 6J strain, 5-6 weeks old, male) were purchased from Sankyo Lab Service and subjected to the test after one week of preliminary breeding.
(B) Feeding high-fat diet to mice The high-fat diet combined with the test substance is a high-fat diet (D12492, 60% Kcal% Fat, Research Diets) or 0.05% MBMX (Kakensei) or 0.5 % Metformin (Wako Pure Chemical Industries, Ltd.) was uniformly mixed and then formed into pellets and stored in a refrigerator.
Feeding was performed every two days, and the amount of food intake was measured.
In the control group, a normal diet (D12450B, 10% Kcal% Fat, Research Diets) was fed.
Mice were fed the test compound diet for 20 weeks and then dissected. Mice were laparotomized under ether anesthesia, blood was collected from the abdominal vena cava, and blood glucose, triglyceride, cholesterol, free fatty acid, GOT, GPT and the like were measured using a commercially available kit (Wako Pure Chemical Industries).
(C) Insulin responsiveness test Feeding high fat diet with test compound 19-week-old mice were fasted for 4 hours, blood was collected from the tail vein, and insulin (0.8 IU / kg, bovine pancreas-derived, sigma) solution was intraperitoneally injected Blood was collected at 30, 60 and 120 minutes, and blood glucose level was measured.
<MBMX high fat diet feeding test-8-week administration test->
(A) Animal Mice (C57BL / 6J strain, 5-6 weeks old, male) were purchased from Sankyo Lab Service and subjected to the test after one week of preliminary breeding.
(B) Feeding high-fat diet to mice The test compound-containing high-fat diet is a high-fat diet (D12492, 60% Kcal% Fat, Research Diets) and 0.05% MBMX (Tamazakitsu Rafuji plant extract alkaloid) Cephalantin powder, Kaken Seiyaku) or 0.02% pioglitazone (Funakoshi) were uniformly mixed and then formed into pellets, stored in a refrigerator, and fed every two days.
In addition, the normal diet (D12450B, 10% Kcal% Fat, Research Diets) was fed in the same manner.
After the mice were fed a high fat diet for 12 weeks, the mice were replaced with a high fat diet containing a test substance, and further fed for 8 weeks, and then subjected to dissection.
Mice were laparotomized under ether anesthesia, blood was collected from the abdominal vena cava, and blood glucose, triglyceride, cholesterol, free fatty acid, GOT and GPT were measured using a commercially available kit (Wako Pure Chemical Industries).
(C) Insulin responsiveness test Fast-fed 7-week-fed mice with test compound-containing high-fat diet, blood was collected from the tail vein, and insulin (0.8 IU / kg, bovine pancreas-derived, sigma) solution was intraperitoneally Blood was collected at 30, 60 and 120 minutes, and blood glucose level was measured.
<MBMX combination feeding test-Diabetes onset prevention test->
(A) Animals Spontaneously diabetic mice (db / db strain, 5 weeks old, male) were purchased from Sankyo Lab Service and subjected to the test after one week of preliminary breeding.
(B) Feeding the test compound diet to db / db mice The test compound diet is 0.05% MBMX or 0.02% pioglitazone (Funakoshi) on a normal diet (Lab MR Stock Powder, Nippon Agricultural Products). After uniform mixing, the animals were fed and the food intake was weighed every two days.
Mice were fed a diet for 10 weeks and then dissected.
Mice were laparotomized under ether anesthesia, blood was collected from the abdominal vena cava, and blood glucose, triglyceride, cholesterol, free fatty acid, GOT and GPT were measured using a commercially available kit (Wako Pure Chemical Industries).
(C) Glucose Tolerance Test The 9-week-old mice fed with the test compound were fasted overnight, blood was collected from the tail vein, and then a glucose solution (2 g / kg) was orally administered, and blood was collected after 30, 60 and 120 minutes, respectively. The blood glucose level was measured.
<Prostaglandin E2 production test>
A375S2 cells were cultured for 18 hours in the presence of IL-1β (1 ng / ml) or IL-1α (2 ng / ml), and the amount of PGE2 in the produced culture medium was measured using an ELISA kit (R & D Systems Inc.). did.

First, the evaluation system related to this test was confirmed.
The graph shown in FIG. 1 shows the effect of IL-1β on the number of days of culture of human cell lines, the vertical axis, and the increase in the amount of living cells.
Specifically, the test was conducted as follows.
Cell culture medium or IL-1β (2.5 ng / ml) is added to each well of the tissue culture plate.
After culturing for 2 to 5 days, 0.05% neutral red solution is added to each well, followed by culturing in a cell culture vessel for 2 hours to allow the living cells to incorporate the dye.
Wash the dye that was not taken up into the cells.
Next, the dye eluate is added to each well and stirred on a plate shaker at room temperature to elute the dye incorporated into the living cells. OD540nm is measured with an automatic absorbance meter for microplate. The amount of living cells is calculated from the measured absorbance.
As a result, it was confirmed that IL-1β caused cell death in human cell lines and the amount of living cells did not increase.
Next, human IL-1 receptor antagonist (hIL-1Ra) and human IL-1β neutralization against cell death of human cell lines of human IL-1β (hIL-1β), mouse IL-1β (mIL-1β) The results of confirming the action of the antibody (hIL-1β Ab) are shown in the graph of FIG.
Human IL-1β (hIL-1β) is inhibited when human IL-1Ra (hIL-1Ra) is added to the cell death evaluation system for human cell lines using mouse IL-1β (mIL-1β).
Human IL-1β neutralizing antibody (hIL-1β Ab) inhibits cell death by human IL-1β but not mouse IL-1β.
Using this evaluation system, it was confirmed that compounds having IL-1Ra-like activity could be screened.

FIG. 3 is a graph showing the results of test evaluation of the inhibitory effect of various plant-derived alkaloid compounds on cell death by human IL-1β.
In the graph, MB5C: cephalanthin, MB1D: E6-berbamine, MB2D: berbamine, MB7R: isotetrandrine, MB1R: cyclanine, IL-1Ra: IL-1 receptor antagonist are shown.
It was confirmed that the alkaloid compound having a bisbenzylisoquinoline chemical structure inhibits cell death by IL-1β in a dose-dependent manner.

The results of testing the inhibitory effect of cephalanthin (MB5C) on human TNFα and human IL-1α are shown in the graph of FIG.
As a result, it can be seen that cephalanthin inhibits cell death by human TNFα and human IL-1α.
Although the graph is omitted, the other alkaloids shown in FIG. 3 have the same inhibitory action.

FIG. 5 is a graph showing the results of an inhibitory action test of an alkaloid preparation (MBMX) extracted from Satsuma tsumugi on cell death caused by human IL-1β.
For MBMX, cephalanthin bulk powder sold by Kaken Seiyaku Co., Ltd. was used.
The main alkaloid components of MBMX are cephalanthin, isotetrandrine, berbamine and cicleanine.
MBMX showed an inhibitory effect on cell death by human IL-1β in a dose-dependent manner.

The test result of the inhibitory action of MBMX on pancreatic β cell (MIN6) death by IL-1 is shown in the graph of FIG.
(Method) 1 × 10 ⁵ pancreatic β cells (MIN6), mouse IL-1β (5 ng / ml) and MBMX are added to each well of a tissue culture plate and cultured. After 48 hours of culture, the amount of viable cells was calculated by taking the neutral red dye into the cells.
As a result, as shown in the graph of FIG. 6, although IL-1β caused pancreatic β-cell death, MBMX was confirmed to suppress pancreatic β-cell death due to IL-1 and to have a pancreatic β-cell protective effect. .

The results of the inhibitory action of MB-1 on the pancreatic β cell (MIN6) death of the main alkaloid components of MBMX, cephalanthin, isotetrandrine, berbamine and cyclanine are shown in the graph of FIG.
(Method) 1 × 10 5 MIN6 cells, mouse IL-1β (5 ng / ml) and each alkaloid compound are added to each well of a tissue culture plate and cultured. After culturing for 72 hours, the amount of viable cells was calculated by taking the neutral red dye into the cells.
As a result, as shown in the graph of FIG. 7, IL-1β causes MIN6 cell death, but each of the main components of MBMX suppresses MIN6 cell death in a concentration-dependent manner and has a pancreatic β-cell protective effect. It could be confirmed.

The effect of alkaloid preparation (MBMX) on high fat diet fed obesity model was tested.
The weight change is shown in the graph of FIG. 8, and the insulin responsiveness test result is shown in the graph of FIG.
The blood biochemical values are shown in the table of FIG.
Normal diet (ND, Research diet D12450B 10% Kcal fat) or high fat diet (HFD, Research diet D12492 60% Kcal fat) was fed to C57BL / 6J mice (♂ 6 weeks old, group N = 6) for 20 weeks .
The test drug was 0.05% MBMX (500 μg / g) and the diabetic drug metformin (Met) 0.5% (5 mg / g) as a positive control was mixed in a high fat diet and fed for 20 weeks.
Body weight and blood glucose level were measured every 4 weeks.
An insulin responsiveness test was performed at 19 weeks after the start of the test, and a blood biochemical test and tissue weight were measured at 20 weeks. MBMX was effective in suppressing obesity caused by feeding a high fat diet.
Mice fed a high fat diet for 19 weeks had a lower ability to respond to insulin than mice fed a normal diet.
In mice fed with a high-fat diet containing 0.05% MBMX, the insulin response ability was equivalent to that of a normal diet-fed mouse, and an effect of preventing a decrease in insulin response ability was obtained.
The results showed that mice fed with high fat diet increased triglycerides, free fatty acids, cholesterol, and abnormal liver function due to fatty liver compared to mice fed normal diet.
In mice fed with a high fat diet containing 0.05% MBMX, the effects of suppressing increases in triglycerides, free fatty acids and cholesterol and preventing deterioration of liver function were obtained.

Next, a therapeutic administration test was conducted in an obese model fed with a high fat diet.
FIG. 11 shows a graph of changes in body weight of mice, and FIG. 12 shows the results of an insulin responsiveness test.
Further, FIG. 13 and FIG. 14 show blood biochemical test results.
C57BL / 6J mice (♂ 6 weeks old, group N = 6) were fed a normal diet or a high fat diet for 12 weeks.
Thereafter, mice fed with a high fat diet were fed a high fat diet or a high fat diet containing a test drug for 8 weeks.
0.05% MBMX (500 μg / g) was fed as a test drug, and 0.02% pioglitazone (Pio, diabetes drug) was fed as a positive control drug.
Body weight and blood glucose level were measured every 4 weeks.
An insulin responsiveness test was performed at 19 weeks after the start of the test, and a blood biochemical test and tissue weight were measured at 20 weeks.
The effect of improving obesity was obtained in MBMX-containing high-fat diet-fed mice.
In mice fed with a high fat diet, decreased insulin response (insulin resistance) was observed.
In mice fed with a high fat diet containing 0.05% MBMX for 7 weeks, an effect of improving insulin responsiveness was obtained.
In mice fed with a high fat diet, an increase in insulin level and an increase in leptin level were observed.
In 8 weeks, mice with high fat diet containing 0.05% MBMX were able to improve insulin and leptin levels.
Serum lipid levels were elevated in mice fed a high fat diet.
In mice fed with a high fat diet containing 0.05% MBMX, an effect of improving the serum lipid profile was obtained.

The results of evaluating the inhibitory action of the alkaloid preparation (MBMX) on prostaglandin E2 (PGE2) production by IL-1β and IL-1α stimulation are shown in the graphs of FIGS.
The graph in FIG. 15 is for IL-1β stimulation, and the graph in FIG. 16 is for IL-1α stimulation.
When the A375S2 cell line is stimulated with IL-1β (1 ng / ml) and IL-1α (2 ng / ml) for 24 hours, about 50 times as much PGE2 is produced.
In the presence of IL-1Ra (100 ng / ml), PGE2 production was suppressed to 10% or less. MBMX also suppressed PGE2 production in a concentration-dependent manner.

FIG. 17 to FIG. 20 show the results (part 1) of the test of the effect of the alkaloid preparation (MBMX) on type 2 obese diabetic mice.
FIG. 17 shows changes in body weight, FIG. 18 shows changes in blood glucose level, FIG. 19 shows glucose tolerance test results, and FIG. 20 shows changes in food intake.
db / db mice (6 weeks old, 1 group, N = 6) were fed with 0.05% (500 ug / g) of MBMX and 0.02% (200 ug / g) of pioglitazone.
In the MBMX feeding group, weight gain was suppressed from around the 2nd week.
In the pioglitazone group, weight gain (fat accumulation, water retention, etc.) was observed due to the versatility of the PPARγ agonist action.
In the MBMX group, the blood glucose level began to decrease from the third week of feeding, and decreased to 200 mg / dl or less by the eighth week.
In the pioglitazone group, no increase in blood glucose level was observed after the start of feeding, and the value was maintained at around 150 mg / dl.
Fasting blood glucose levels at 8 weeks after the start of the test were significantly lower in the MBMX group and the pioglitazone group than in the control group.

FIG. 21 to FIG. 25 show the results (part 2) of the test of the effect of the alkaloid preparation (MBMX) on type 2 obese diabetic mice.
21 shows changes in body weight, FIG. 22 shows changes in blood glucose level, FIG. 23 shows the amount of food intake, FIG. 24 shows the glucose tolerance test results, and FIG. 25 shows the insulin response ability test.
(Method) A db / db mouse having an abnormality in the leptin receptor, which is an appetite suppressive hormone, is a model animal for diabetic condition that causes pancreatic β-cell atrophy and necrosis due to overeating, obesity, and insulin resistance.
Eight-week-old male db / db mice were fed a diet containing 0.01% or 0.05% MBMX for 10 weeks.
A glucose tolerance test was conducted at 10 weeks after the start of the test, an insulin responsiveness test was conducted at the 11th week of the test, and an insulin amount in the pancreas was measured by dissecting at the 12th week of the test.
Glucose tolerance test and insulin response test were performed in the usual way after fasting

In the 0.05% MBMX feeding group, body weight gain was suppressed as compared to the control group, and the blood glucose level was significantly lower as needed.
In the 0.05% MBMX mixed diet group, the blood glucose level was significantly lower than that in the control group from the second week of feeding, and decreased to 200 mg / ml or less in the ninth week.
Further, in the 0.05% MBMX feeding group, although the mechanism is unknown, an effect of suppressing appetite of obese animals was also observed. In the glucose tolerance test conducted on week 10 of 0.05% MBMX feeding, the fasting blood glucose level was significantly lower than that in the control group, and a significant improvement effect on pancreatic β-cell function was observed.
In the insulin response ability test conducted on the 11th week after feeding with 0.05% MBMX, a significant improvement in insulin sensitivity was observed compared to the control group.

The result of pancreatic β-cell protective action of MBMX using db / db obese type 2 diabetes model mouse is shown in FIG.
(Method) A db / db mouse having an abnormality in the leptin receptor, which is an appetite suppressive hormone, is a disease state model animal that causes pancreatic β-cell atrophy and necrosis due to overeating, obesity, and insulin resistance.
In order to clarify the protective effect of MBMX on pancreatic β cells, the insulin content in the pancreas of db / db mice was measured at the 12th week of the start of the test.
That is, male db / db mice were fed with a diet containing 0.01% or 0.05% MBMX. After 12 weeks, it was dissected and the amount of insulin in the pancreas was measured.
1 ml of an acid alcohol mixed solution (75% alcohol, 23.5% distilled water, 1.5% concentrated hydrochloric acid) was added to the pancreas placed in an Eppendorf tube and crushed with a pellet mixer. After leaving still at 4 degreeC overnight and extracting insulin, the amount of insulin was measured using the insulin ELISA measuring kit.
As a result, as shown in FIG. 26, in the db / db mice having an abnormality in the leptin receptor, which is an appetite suppressing hormone, a marked decrease in the insulin content in the pancreas was observed.
It was shown that db / db mice fed the 0.05% MBMX diet maintained the pancreatic insulin content.
This is thought to be because MBMX protected the exhaustion and cell death of pancreatic β cells.

The present invention relates to an inflammatory disease (diabetes, obesity-related disease, dementia, autoinflammatory disease, rheumatoid arthritis, inflammatory bowel disease, sepsis, etc.) effective in inhibiting inflammatory cytokines IL-1 and TNFα activity It can be widely used as a therapeutic agent and a preventive agent.

Claims (10)

1. Inflammatory activity containing at least one or more of alkaloids derived from plants of the genus Stefania family and their derivatives (including those chemically synthesized) and pharmaceutically acceptable salts thereof as active ingredients Activity inhibitors of cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF).
2. The activity of interleukin 1 (IL-1) and tumor necrosis factor (TNF) according to claim 1, wherein the alkaloid is at least one of cephalanthin, berbamine, isotetrandrine, tetrandrine, cyclanine, and E6-berbamine. Inhibitor.
3. Interleukin 1 (IL-) which is an inflammatory cytokine containing as an active ingredient at least one of alkaloids derived from plants belonging to the genus Stefania and their derivatives and pharmaceutically acceptable salts thereof 1) A therapeutic or prophylactic agent for inflammatory diseases in which the activity inhibitory action of tumor necrosis factor (TNF) is effective.
4. The therapeutic or prophylactic agent for inflammatory diseases according to claim 3, wherein the alkaloid is at least one of cephalanthin, berbamine, isotetrandrine, tetrandrine, cyclanine, and E6-berbamine.
5. An agent for the treatment or prevention of diabetes containing as an active ingredient at least one of alkaloids derived from plants belonging to the genus Stefania belonging to the family Rhizomaceae and their derivatives and pharmaceutically acceptable salts thereof.
6. The agent for treating or preventing diabetes according to claim 5, wherein the alkaloid is at least one of cephalanthin, berbamine, isotetrandrine, tetrandrine, cyclanine, and E6-berbamine.
7. The agent for treating or preventing diabetes according to claim 5 or 6, wherein the agent is based on pancreatic β-cell protective action or β-cell function maintenance action.
8. An agent for improving / preventing metabolic syndrome containing at least one or more of alkaloids derived from plants belonging to the genus Stefania belonging to the genus Rubiaceae and derivatives thereof and pharmaceutically acceptable salts thereof as active ingredients.
9. The agent for improving / preventing metabolic syndrome according to claim 8, wherein the alkaloid is at least one of cephalanthin, berbamine, isotetrandrine, tetrandrine, cyclanine, and E6-berbamine.
10. A food or drink containing any one of the activity inhibitor, therapeutic or preventive agent and ameliorating / preventing agent according to any one of claims 1 to 9.

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