WO2015098928A1 - Inhibiteur d'activités il-1 et tnf - Google Patents

Inhibiteur d'activités il-1 et tnf Download PDF

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WO2015098928A1
WO2015098928A1 PCT/JP2014/084076 JP2014084076W WO2015098928A1 WO 2015098928 A1 WO2015098928 A1 WO 2015098928A1 JP 2014084076 W JP2014084076 W JP 2014084076W WO 2015098928 A1 WO2015098928 A1 WO 2015098928A1
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agent
berbamine
alkaloid
mbmx
test
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Japanese (ja)
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聖志 高津
嘉勝 平井
良憲 長井
孝之 松永
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国立大学法人 富山大学
富山県
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Publication of WO2015098928A1 publication Critical patent/WO2015098928A1/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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4741—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/59—Menispermaceae (Moonseed family), e.g. hyperbaena or coralbead
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to diabetes, disease states and conditions characterized by insulin resistance, decreased functional pancreatic ⁇ -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.
  • IL-1 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).
  • 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).
  • PGE2 prostaglandin E2
  • TNF tumor necrosis factor
  • IL-6 tumor necrosis factor
  • IL-8 corticotropin
  • the IL-1 family of cytokines is composed of 11 members, but the effects of IL-1 ⁇ , IL-1 ⁇ , 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-1 ⁇ , IL-1 ⁇ 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.
  • 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 TNF ⁇ 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.
  • Non-patent Document 8 Non-patent Document 8
  • 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- ⁇ activity inhibitor containing as an active ingredient an alkaloid derived from a plant belonging to the genus Stefania belonging to the family Rubiaceae.
  • improvement of pancreatic ⁇ -cell function, preservation of functional pancreatic ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the alkaloid derivatives used in the present invention include acyl derivatives, alkyl derivatives, and carbamoyl derivatives.
  • the active ingredient 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.
  • 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.
  • 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) ⁇ -glucosidase inhibitor, 4) insulin sensitizer, 5) glucagon-like peptide (GLP-1), 6) DPP-4 inhibitor 7) Insulin 8) SGLT inhibitor.
  • 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 TNF ⁇ 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.
  • 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.
  • the active ingredient of the present invention when 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.
  • the medicament according to the present invention is a non-invasive, anti-diabetic agent / preventive agent for preserving pancreatic ⁇ -cell function that satisfies safety, convenience, economy, and the like, and has an inhibitory action on IL-1 and TNF ⁇ activity. Is an effective therapeutic / preventive agent for inflammatory diseases.
  • the active ingredient of the present invention has effects such as pancreatic ⁇ -cell protection or ⁇ -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-1 ⁇ and mouse IL-1 ⁇ of human cell lines, the inhibitory action of human IL-1 receptor antagonist, and the effect of human IL-1 ⁇ neutralizing antibody are shown.
  • 2 shows the inhibitory effect of plant-derived alkaloid compounds on cell death by human IL-1 ⁇ .
  • 2 shows cell death by human TNF ⁇ and human IL-1 ⁇ and MB5C (cephalanthin) inhibitory action.
  • 2 shows the inhibitory action of MBMX (Takatsuki tsumugi extract alkaloid preparation) on cell death by human IL-1 ⁇ .
  • 2 shows the inhibitory action of MBMX on pancreatic ⁇ cell (MIN6) death by IL-1 ⁇ .
  • 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.
  • 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-1 ⁇ stimulation.
  • 2 shows the inhibitory action of MBMX on Prostaglandin E2 production by IL-1 ⁇ 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 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.
  • the active ingredient according to the present invention was tested to have an inhibitory effect on the activity of IL-1 and TNF ⁇ against cell death and to have an obesity preventing effect and an obesity improving effect.
  • an inhibitory effect on prostaglandin E2 production by IL-1 ⁇ and IL-1 ⁇ stimulation was also confirmed.
  • IL-1 ⁇ Human and mouse interleukin 1 ⁇
  • IL-1 ⁇ interleukin 1 ⁇
  • TNF- ⁇ human tumor necrosis factor
  • IL-1Ra interleukin receptor antagonist
  • MBMX Alkaloid bulk powder
  • cephalanthin isotetrandrine
  • berbamine berbamine
  • cicleanine and its derivatives E6-berbamine
  • WAKO Chemicals Osaka, Japan
  • 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.
  • IL-1 inhibitory compounds were evaluated 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).
  • IL-1 sensitive cell line A375S2 Non-IL-1 sensitive cell line
  • MIN6 mouse pancreatic ⁇ 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.
  • 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.
  • 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.
  • 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.
  • test compound diet 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).
  • the graph shown in FIG. 1 shows the effect of IL-1 ⁇ on the number of days of culture of human cell lines, the vertical axis, and the increase in the amount of living cells.
  • the test was conducted as follows. Cell culture medium or IL-1 ⁇ (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-1 ⁇ caused cell death in human cell lines and the amount of living cells did not increase.
  • human IL-1 receptor antagonist (hIL-1Ra) and human IL-1 ⁇ neutralization against cell death of human cell lines of human IL-1 ⁇ (hIL-1 ⁇ ), mouse IL-1 ⁇ (mIL-1 ⁇ ) The results of confirming the action of the antibody (hIL-1 ⁇ Ab) are shown in the graph of FIG.
  • Human IL-1 ⁇ (hIL-1 ⁇ ) is inhibited when human IL-1Ra (hIL-1Ra) is added to the cell death evaluation system for human cell lines using mouse IL-1 ⁇ (mIL-1 ⁇ ).
  • Human IL-1 ⁇ neutralizing antibody (hIL-1 ⁇ Ab) inhibits cell death by human IL-1 ⁇ but not mouse IL-1 ⁇ . 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-1 ⁇ .
  • MB5C cephalanthin
  • MB1D E6-berbamine
  • MB2D berbamine
  • MB7R isotetrandrine
  • MB1R cyclanine
  • IL-1Ra IL-1 receptor antagonist
  • 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-1 ⁇ .
  • MBMX alkaloid preparation
  • 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-1 ⁇ in a dose-dependent manner.
  • the test result of the inhibitory action of MBMX on pancreatic ⁇ cell (MIN6) death by IL-1 is shown in the graph of FIG. (Method) 1 ⁇ 10 5 pancreatic ⁇ cells (MIN6), mouse IL-1 ⁇ (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-1 ⁇ caused pancreatic ⁇ -cell death, MBMX was confirmed to suppress pancreatic ⁇ -cell death due to IL-1 and to have a pancreatic ⁇ -cell protective effect. .
  • the results of the inhibitory action of MB-1 on the pancreatic ⁇ 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-1 ⁇ (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-1 ⁇ 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 ⁇ -cell protective effect. It could be confirmed.
  • MBMX alkaloid preparation
  • 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 test drug was 0.05% MBMX (500 ⁇ g / 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.
  • mice fed a high fat diet for 19 weeks had a lower ability to respond to insulin than mice fed a normal diet.
  • 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.
  • 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.
  • FIG. 11 shows a graph of changes in body weight of mice
  • FIG. 12 shows the results of an insulin responsiveness test.
  • FIG. 13 and FIG. 14 show blood biochemical test results.
  • 0.05% MBMX 500 ⁇ g / g
  • 0.02% pioglitazone was fed as a positive control drug.
  • mice fed with a high fat diet decreased insulin response (insulin resistance) was observed.
  • mice fed with a high fat diet containing 0.05% MBMX for 7 weeks an effect of improving insulin responsiveness was obtained.
  • mice fed with a high fat diet an increase in insulin level and an increase in leptin level were observed.
  • 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-1 ⁇ and IL-1 ⁇ stimulation are shown in the graphs of FIGS.
  • the graph in FIG. 15 is for IL-1 ⁇ stimulation
  • the graph in FIG. 16 is for IL-1 ⁇ stimulation.
  • 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
  • FIG. 20 shows changes in food intake.
  • weight gain (fat accumulation, water retention, etc.) was observed due to the versatility of the PPAR ⁇ agonist action.
  • 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.
  • 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
  • FIG. 25 shows the insulin response ability test.
  • 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 ⁇ -cell atrophy and necrosis due to overeating, obesity, and insulin resistance.
  • mice 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
  • 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 ⁇ -cell atrophy and necrosis due to overeating, obesity, and insulin resistance.
  • 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.
  • 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 TNF ⁇ activity It can be widely used as a therapeutic agent and a preventive agent.
  • an inflammatory disease diabetes, obesity-related disease, dementia, autoinflammatory disease, rheumatoid arthritis, inflammatory bowel disease, sepsis, etc.

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Abstract

La présente invention vise à fournir un inhibiteur d'activité de cytokines inflammatoires, qui est non-invasif et satisfait des exigences telles que la sécurité, la commodité et la performance économique, de façon à traiter un diabète et à prévenir le développement de complications diabétiques, et un procédé de thérapie pour modifier l'historique naturel de diabète, ou un moyen efficace pour prévenir la progression de diabète. La présente invention vise également à fournir un agent thérapeutique ou prophylactique pour des maladies auto-inflammatoires pour lesquelles une activité pour inhiber l'activité de cytokines inflammatoires est efficace. L'invention concerne un inhibiteur d'activité de cytokines inflammatoires, qui est caractérisé par le fait qu'il contient, comme principe actif, au moins un composé choisi parmi un alcaloïde provenant d'une plante appartenant à la famille des Menispermaceae, le genre Stephania, un dérivé de l'alcaloïde et un sel pharmaceutiquement acceptable de l'alcaloïde ou du dérivé.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109771426A (zh) * 2019-02-26 2019-05-21 沈锋 千金藤素作为asph的酶活抑制剂的应用

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