WO2015105094A1 - 糖尿病治療薬 - Google Patents
糖尿病治療薬 Download PDFInfo
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- WO2015105094A1 WO2015105094A1 PCT/JP2015/050122 JP2015050122W WO2015105094A1 WO 2015105094 A1 WO2015105094 A1 WO 2015105094A1 JP 2015050122 W JP2015050122 W JP 2015050122W WO 2015105094 A1 WO2015105094 A1 WO 2015105094A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
Definitions
- the present invention relates to a novel use of 8- [2- (2-pentyl-cyclopropylmethyl) -cyclopropyl] -octanoic acid (DCP-LA), and more specifically as a therapeutic agent for diabetes based on the promotion of intracellular glucose uptake.
- DCP-LA 8- [2- (2-pentyl-cyclopropylmethyl) -cyclopropyl] -octanoic acid
- Diabetes is a disease in which insulin functions poorly, blood sugar increases, and sugar is also produced in urine. Insulin is produced and secreted by ⁇ cells in the pancreas, promotes synthesis of glucose to glycogen (stored glucose) in the liver and muscles, suppresses degradation of glycogen to glucose in the liver, and increases blood sugar levels Suppress. Patients are increasing mainly due to overeating, lack of exercise, obesity, stress, and genetic predisposition. Diabetes is broadly classified into type 1 diabetes that is absolutely deficient in insulin and type 2 diabetes that is relatively deficient. Type 1 treatment is based on insulin injection, and type 2 treatment is based on diet and exercise therapy, but drug control is necessary when blood glucose control is poor. As drug therapy, oral antidiabetic drugs or insulin are used, but all of them are required to be improved in terms of side effects, QOL, and the like.
- metabolic syndrome a state in which lifestyle-related diseases such as hypertension, hyperlipidemia, and diabetes have developed due to visceral fat accumulation is called metabolic syndrome, and increases the risk of developing arteriosclerotic diseases (myocardial infarction, cerebral infarction, etc.) Widely recognized as a thing. Therefore, if diabetes can be prevented and treated, it is inevitably effective in preventing and treating metabolic syndrome.
- DCP-LA 8- [2- (2-pentyl-cyclopropylmethyl) -cyclopropyl] -octanoic acid
- LTP long-term potentiation
- Patent Document 1 8- [2- (2-pentyl-cyclopropylmethyl) -cyclopropyl] -octanoic acid
- Non-patent Document 1 DCP-LA selectively and directly activates PKC- ⁇
- Non-patent Document 2 DCP-LA improves cognitive impairment in senescence-accelerated mice
- Non-patent Document 3 DCP -LA increases the release of ⁇ -aminobutyric acid from hippocampal neurons
- Non-Patent Document 4 DCP-LA improves cognitive dysfunction in amyloid ⁇ -peptide or scopolamine-treated rats
- DCP-LA promotes hippocampal synaptic transmission targeting the ⁇ 7 nicotinic acetylcholine receptor expressed in glutamatergic presynaptic cells
- Non-patent Document 5 DCP-LA has an action of suppressing neuronal cell death induced by oxidative stress
- Kanno T et al. J Lipid Res., 2006, 47 (6): 1146-56. Yaguchi T et al., Neuroreport, 2006, 23; 17 (1): 105-8. Kanno T et al., J Neurochem., 2005, 95 (3): 695-702. Nagata et al. T, Psychogeriatrics, 2005, 5: 122-126. Yamamoto et al., Neuroscience 2005, 130 (1): 207-213.
- the object of the present invention is to elucidate the pharmacological action of DCP-LA and the influence on the living body, and to provide a new use.
- the present invention is as follows. [1] A prophylactic and / or therapeutic agent for diabetes containing DCP-LA as an active ingredient. [2] The preventive and / or therapeutic agent according to the above [1], wherein diabetes is type 1 diabetes.
- DCP-LA for use in the prevention and / or treatment of diabetes.
- a method for promoting sugar uptake into cells which comprises treating cells with DCP-LA.
- DCP-LA has an action of promoting glucose uptake into cells and an action of lowering blood glucose level, and is useful as various reagents (agents) based on these actions and as a preventive and / or therapeutic agent for diabetes. Since the preventive and / or therapeutic agent of the present invention has a mechanism of action different from that of existing drugs, side effects that have been problematic in existing drugs can be avoided.
- the agent of the present invention can also be used as a research reagent that can be a useful tool for the development of such preventive / therapeutic agents.
- FIG. 1 is a graph showing the effect of DCP-LA on glucose uptake into 3T3L1-GLUT4myc adipocytes.
- A shows the results of measuring the extracellular glucose concentration by HPLC after incubating fat cells in PBS containing glucose in the presence or absence of DCP-LA. P value compared to control, Dunnett ’s test.
- B shows the results of measuring the extracellular glucose concentration by HPLC after incubating adipocytes in glucose-containing PBS in the presence or absence of DCP-LA and / or GF109203X (GF).
- FIG. 2 is a diagram showing that the action of DCP-LA to promote sugar uptake into cells is not due to stimulation of GLUT4 translocation to the cell membrane.
- 3T3L1-GLUT4myc adipocytes treated with DCP-LA or untreated 3T3L1-GLUT4myc adipocytes (Control) were lysed and separated into a cytoplasmic fraction (C) and a cell membrane fraction (M). Each fraction was subjected to Western blotting using an anti-c-myc antibody.
- FIG. 3 is a graph showing advantageous effects of DCP-LA in type 1 diabetes (DM) model mice (A) and type 2 DM model mice (B).
- DM type 1 diabetes
- B type 2 DM model mice
- C57BL / 6J mice treated with streptozotocin (STZ) were used as type 1 DM mice
- C57BL / KsJ-lepr db / lepr db mice were used as type 2 DM mice.
- P value Fisher's PLSD (protected least significant difference) method
- DCP-LA can be produced, for example, by the method shown in WO02 / 50013.
- DCP-LA has four optical isomers ( ⁇ , ⁇ -DCP-LA, ⁇ , ⁇ -DCP-LA, ⁇ , ⁇ -DCP-LA, ⁇ , ⁇ -DCP-LA), All such isomers and mixtures thereof are included within the scope of the present invention. These isomers can be produced, for example, by the method shown in WO2012 / 067111.
- DCP-LA may also be used as a salt thereof.
- a salt is not particularly limited, but is preferably a pharmaceutically or food acceptable salt, for example, an inorganic base (eg, alkali metals such as sodium and potassium; alkaline earth metals such as calcium and magnesium; aluminum and ammonium), organic Base (eg, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N, N-dibenzylethylenediamine), inorganic acid (eg, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphorus) Acid), organic acid (eg, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p
- a subject can be a mammal.
- mammals include primates (eg, humans, monkeys, chimpanzees), rodents (eg, mice, rats, guinea pigs), pets (eg, dogs, cats, rabbits), working animals or livestock.
- rodents eg, mice, rats, guinea pigs
- pets eg, dogs, cats, rabbits
- working animals or livestock eg, cows, horses, pigs, sheep, goats
- cows, horses, pigs, sheep, goats are preferred.
- DCP-LA has (1) an action of promoting glucose uptake into cells, and (2) an action of lowering blood glucose level in diabetes model mice.
- (1) Glucose uptake promoting action into cells Glucose uptake into cells is carried out via GLUT4 (glucose transporter 4) expressed on the cell membrane surface of adipocytes and skeletal muscle cells.
- the blood glucose level is lowered by taking glucose into cells.
- DCP-LA can lower the blood glucose level by promoting the intracellular uptake of glucose.
- the “cell” is not particularly limited as long as it is a cell that can take up glucose, and examples thereof include fat cells, skeletal muscle cells, hepatocytes, and the like.
- DCP-LA Blood glucose level lowering action
- the present invention is useful as a preventive and / or therapeutic agent for diabetes and as a preventive and / or therapeutic agent for metabolic syndrome (hereinafter also referred to as the pharmaceutical agent of the present invention).
- prevention means preventing the manifestation of symptoms in a subject who does not exhibit symptoms
- treatment refers to in subjects who exhibit symptoms. It means reducing the symptom or preventing or delaying worsening of the symptom.
- the present invention provides a method for promoting glucose uptake into cells, a method for preventing and / or treating diabetes (type 1 diabetes, type 2 diabetes), and prevention and / or metabolic syndrome.
- a treatment method (hereinafter also simply referred to as the method of the present invention) can be provided.
- the cells to be treated with DCP-LA are not particularly limited as long as they express GLUT4 and can take up glucose, but they are not limited to fat cells, skeletal muscle cells, livers. Examples thereof include cells. These cells may be derived from progenitor cells by a method known per se. For example, adipocytes can be induced to differentiate from 3T3L1 fibroblasts.
- treatment refers to bringing the cells into contact with DCP-LA for a necessary and sufficient time, and the time varies depending on the desired effect and the type of cells used, but is generally 0.1-6.
- the time is preferably about 0.5 to 2 hours. Conveniently, it is carried out by culturing the cells in a culture solution containing DCP-LA.
- the medicament of the present invention varies depending on the age and condition of each individual patient to be treated, but in the case of intravenous administration, the daily dose of DCP-LA is 0.001 per kg of human or animal body weight. ⁇ 100 mg, for intramuscular administration, the daily dose of DCP-LA is 0.001-10 mg / kg body weight of human or animal, for oral administration, the daily dose of DCP-LA is human or animal 0.01-100 mg / kg body weight is generally given for the prevention and / or treatment of diabetes or for the prevention and / or treatment of metabolic syndrome.
- the medicament of the present invention can contain any additive such as a pharmaceutically acceptable carrier in addition to DCP-LA which is an active ingredient.
- pharmaceutically acceptable carriers include sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate and other excipients, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone.
- the medicament of the present invention can be formulated as a preparation suitable for oral administration.
- Preparations suitable for oral administration include solutions in which an effective amount of a substance is dissolved in a diluent such as water or physiological saline, capsules, granules, powders or tablets containing the effective amount of the substance as solids or granules.
- a diluent such as water or physiological saline
- capsules such as water or physiological saline
- granules, powders or tablets containing the effective amount of the substance as solids or granules.
- a suspension in which an effective amount of a substance is suspended in an appropriate dispersion medium an emulsion in which a solution in which an effective amount of a substance is dissolved is dispersed in an appropriate dispersion medium, and the like.
- the medicament of the present invention can be formulated as a preparation suitable for parenteral administration.
- Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which include antioxidants, Buffers, antibacterial agents, isotonic agents and the like may be included.
- Aqueous and non-aqueous sterile suspensions are also included, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
- the preparation can be enclosed in a container in unit doses or multiple doses like ampoules and vials.
- the active ingredient and a pharmaceutically acceptable carrier can be lyophilized and stored in a state that may be dissolved or suspended in a suitable sterile vehicle immediately before use.
- DCP-LA can be provided as food.
- DCP-LA which is an active ingredient is used for mammals (eg, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans, etc.) (1) sugar uptake into cells It has a promoting action and (2) a blood glucose level lowering action, and can be provided as a functional food effective for the prevention / treatment of diabetes or the prevention / treatment of metabolic syndrome.
- “food” means all foods and drinks other than drugs and quasi drugs. Examples include, but are not limited to, foods for specified health use, nutritional functional foods, and so-called supplements.
- the medicine or food of the present invention is a product in which the unit intake or divided amount of the medicine or food is individually packaged or filled, or a large number of unit intake or divided amounts are packaged or filled comprehensively. Can be a thing.
- the unit intake of the medicine or food or its divided amount is the unit intake of DCP-LA or its divided amount.
- the unit intake or its divided amount is converted into an ordinary package (for example, a PTP (press through packaging) sheet, a paper container, What was packed or filled separately in a film (for example, plastic film) container, a glass container, a plastic container) is mentioned.
- an ordinary package for example, a PTP (press through packaging) sheet, a paper container, What was packed or filled separately in a film (for example, plastic film) container, a glass container, a plastic container
- Such individually packaged or filled pharmaceuticals or foods can be further combined and packaged together in one container (eg, paper container, film (eg, plastic film) container, glass container, plastic container). It may be filled.
- a medicine or food in which a large number of unit intakes or divided amounts thereof are comprehensively packaged or filled for example, a single container (eg, paper container, film (eg, , Plastic film) containers, glass containers, plastic containers) or the like packed or filled.
- the medicament or food of the present invention can also contain a unit intake or a divided amount thereof in a sufficient number for long-term intake.
- a unit intake or a divided amount thereof in a sufficient number for long-term intake.
- it is 3 days or more, preferably 7 days, 10 days, It may be included in numbers sufficient for ingestion for 14 days or 21 days or more, 1 month, 2 months, 3 months or more.
- DCP-LA has (1) an action of promoting sugar uptake into cells and (2) an action of lowering blood glucose level, and therefore can be provided as various reagents.
- Specific examples of the reagent include an agent for promoting sugar uptake into cells.
- the reagent is a useful tool for developing a preventive and / or therapeutic agent for diabetes and metabolic syndrome having an unprecedented new mechanism of action, reduced side effects, and / or enhanced efficacy.
- Example 1 Verification of promoting action of sugar uptake into cells (materials and methods) 1.
- a 3T3L1-GLUT4myc fibroblast cell line expressing cell culture GLUT4myc was used.
- the cell is constructed by inserting a human c-MYC epitope (14 amino acids) into the first ectodomain.
- the migration of GLUT4 to the cell membrane surface can be followed with an anti-c-myc antibody.
- the cells were treated with 5% CO in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% (v / v) bovine serum, penicillin (final concentration, 100 U / ml) and streptomycin (final concentration, 0.1 mg / ml).
- DMEM Dulbecco's Modified Eagles Medium
- Glucose Uptake Assay 3T3L1-GLUT4myc Adipocytes containing 0.2% (w / v) bovine serum albumin (BSA) and supplemented with 10 mM glucose Krebs-Ringer-HEPES buffer [136 mM NaCl, 4.7 mM KCl, 1.25 mM Incubated in CaCl 2 , 1.25 mM MgSO 4 and 20 mM HEPES, pH 7.5] at 37 ° C. for 1 hour. Cells were treated with DCP-LA and / or GF109203X in phosphate buffered saline (PBS) containing 10 mM glucose for 2 hours at 37 ° C.
- PBS phosphate buffered saline
- ABEE paraaminobenzoic acid ethyl ester
- HPLC high performance liquid chromatography
- 3T3L1-GLUT4myc adipocytes were incubated for 1 hour at 37 ° C. in Krebs-Ringer-HEPES buffer containing 0.2% (w / v) BSA and supplemented with 10 mM glucose. Cells were treated with DCP-LA for 20 minutes. The cells were then lysed in ice-cold mitochondrial buffer [210 mM mannitol, 70 mM sucrose, 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM HEPES, pH 7.5] containing 1% (v / v) protease inhibitor cocktail. Homogenized by nication.
- EDTA ethylenediaminetetraacetic acid
- the homogenate was centrifuged at 3,000 rpm for 5 minutes at 4 ° C.
- the supernatant was further centrifuged at 11,000 rpm for 15 minutes at 4 ° C.
- the collected supernatant was ultracentrifuged at 100,000 g for 60 minutes at 4 ° C., and separated into a cytoplasmic fraction and a cell membrane fraction.
- the supernatant was used as the cytoplasm fraction and the pellet was used as the cell membrane fraction.
- cytoplasmic component and the cell membrane component were successfully separated was confirmed by Western blot analysis using an antibody against lactate dehydrogenase (LDH) which is a marker of cytoplasmic component and an antibody against cadherin which is a cell membrane marker.
- LDH lactate dehydrogenase
- cadherin which is a cell membrane marker.
- the protein concentration of each fraction was measured using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).
- the protein of the cell membrane fraction was resuspended in mitochondrial buffer containing 1% (w / v) sodium dodecyl sulfate (SDS).
- SDS-PAGE SDS-polyacrylamide gel electrophoresis
- the blotting membrane was blocked with TBS-T [150 mM NaCl, 0.1% (v / v) Tween20 and 20 mM Tris, pH 7.5] containing 5% (w / v) BSA, and anti-c-myc antibody (Merck Millipore , Darmstadt, Germany) followed by horseradish peroxidase (HRP) conjugated goat anti-mouse IgG antibody.
- Immunoreactivity was detected using an ECL kit (Invitrogen) and visualized using a chemiluminescence detection system (GE Healthcare, Piscataway, NJ, USA). The signal density was measured using image analysis software (Image Healthcare).
- glucose-containing (10 mM) PBS in the presence or absence of 0.1 ⁇ M DCP-LA and / or in the presence or absence of 0.1 ⁇ M GF109203X (GF)
- the amount of glucose uptake into cells was calculated from the difference in extracellular glucose concentration before and after incubation.
- the results are shown in FIG. 1B. From the results of FIG. 1A, it was shown that DCP-LA has an action of promoting sugar uptake into adipocytes in a bell-shaped concentration-dependent manner.
- FIG. 1A From the results of FIG. 1A, it was shown that DCP-LA has an action of promoting sugar uptake into adipocytes in a bell-shaped concentration-dependent manner.
- Example 2 Verification of blood glucose level lowering effect of DCP-LA (materials and methods)
- a glucose tolerance test type 1 diabetes model was prepared by intraperitoneally administering streptozotocin (STZ) (250 mg / kg) to C57BL / 6J mice (8 weeks old) (Japan SLC Inc .; Shizuoka, Japan).
- An oral glucose tolerance test (OGTT) was performed 4 days after STZ administration.
- Type 2 diabetes model C57BL / KsJ-lepr db / lepr db mice (female, 8 weeks old) (CLEA Japan; Tokyo, Japan ) was used. OGTT was performed on mice starved for 12 hours.
- Glucose (2 g / ml / kg body weight) was forcibly administered orally at that time.
- DCP-LA (0, 0.001, 0.01, 0.1 mg / kg) was orally administered 30 minutes before administration of glucose.
- Blood (10 ⁇ l) was collected from the tail vein at 0, 30, 60, and 90 minutes, and each plasma sample prepared from the resulting blood and labeled with ABEE was analyzed by a high performance liquid chromatography (HPLC) system (LC- 10ATvp; Shimadzu Co., Kyoto, Japan). The glucose concentration was calculated from a calibration curve of peak area / concentration prepared using a standard glucose solution. (result)
- FIG. 3A shows the results when using a type 1 diabetes model mouse, and FIG.
- 3B shows the results when using a type 2 diabetes model mouse. From these results, it is shown that DCP-LA has an effect of lowering blood glucose level in a dose-dependent manner in type 1 and type 2 diabetes model mice. Therefore, the present invention suggests the possibility of becoming a therapeutic agent for type 1 and type 2 diabetes.
- DCP-LA has an action of promoting glucose uptake into cells and an action of lowering blood glucose level, and is useful as various reagents (agents) based on these actions and as a preventive and / or therapeutic agent for diabetes. Since the preventive and / or therapeutic agent of the present invention has a mechanism of action different from that of existing drugs, side effects that have been problematic in existing drugs can be avoided.
- the agent of the present invention can also be used as a research reagent that can be a useful tool for the development of such preventive / therapeutic agents.
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Abstract
Description
過食、運動不足、肥満、ストレス、遺伝的素因が主たる理由で患者が増えている。
糖尿病は絶対的にインスリンが不足する1型糖尿病と相対的に不足する2型糖尿病に大別される。1型の治療にはインスリン注射が基本であり、2型の治療には食事、運動療法が基本であるが、血糖のコントロールが悪い場合には薬物療法が必要になる。薬物療法としては経口糖尿病治療薬あるいはインスリンが用いられるが、いずれも副作用の点、QOLの点等から改善が求められている。
DCP-LAについてはまた、幾つかの報告がなされている。例えば、DCP-LAが選択的かつ直接的にPKC-εを活性化すること(非特許文献1)、DCP-LAが老化促進マウスの認知機能障害を改善すること(非特許文献2)、DCP-LAが海馬神経細胞からのγアミノ酪酸の放出を増加させること(非特許文献3)、DCP-LAがアミロイドβペプチドあるいはスコポラミン処理ラットの認知機能障害を改善すること(非特許文献4)、DCP-LAがグルタミン酸作動性シナプス前細胞に発現するα7ニコチン性アセチルコリン受容体を標的として海馬シナプス伝達を促進させること(非特許文献5)が報告されている。さらに、近年DCP-LAに酸化ストレスによって誘導される神経細胞死を抑制する作用があることが報告されている(特許文献2)。
[1]DCP-LAを有効成分として含有する、糖尿病の予防及び/又は治療薬。
[2]糖尿病が、1型糖尿病である、上記[1]記載の予防及び/又は治療薬。
[3]糖尿病が、2型糖尿病である、上記[1]記載の予防及び/又は治療薬。
[4]細胞への糖取込みを促進させることを特徴とする、上記[1]~[3]のいずれかに記載の予防及び/又は治療薬。
[5]DCP-LAを有効成分として含有する、細胞への糖取込み促進剤。
[6]研究用試薬である、上記[5]記載の剤。
[7]有効量のDCP-LAをそれを必要とする対象に投与することを特徴とする、糖尿病の予防及び/又は治療方法。
[8]糖尿病が、1型糖尿病である、上記[7]記載の方法。
[9]糖尿病が、2型糖尿病である、上記[7]記載の方法。
[10]細胞への糖取込みを促進させることを特徴とする、上記[7]~[9]のいずれかに記載の方法。
[11]糖尿病の予防及び/又は治療に使用するためのDCP-LA。
[12]糖尿病が、1型糖尿病である、上記[11]記載のDCP-LA。
[13]糖尿病が、2型糖尿病である、上記[11]記載のDCP-LA。
[14]細胞への糖取込みを促進させることを特徴とする、上記[11]~[13]のいずれかに記載のDCP-LA。
[15]DCP-LAで細胞を処理することを特徴とする、細胞への糖取込みを促進させる方法。
本発明において有効成分として用いられる8-[2-(2-ペンチル-シクロプロピルメチル)-シクロプロピル]-オクタン酸(本明細書中、必要に応じてDCP-LAと省略)は、以下の構造式を有する。
(1)細胞への糖取込み促進作用
グルコースの細胞内への取込みは、脂肪細胞や骨格筋細胞の細胞膜表面上に発現するGLUT4(グルコーストランスポーター4)を介して行われる。グルコースを細胞内へ取り込むことにより血糖値をさげる。DCP-LAはこのグルコースの細胞内取込みを促進することによって血糖値を低下させることができる。
ここで、「細胞」とはグルコースを取込むことができる細胞であれば特に限定されないが、脂肪細胞、骨格筋細胞、肝細胞等が挙げられる。
(2)血糖値低下作用
DCP-LAはインスリンを必要とせずに血糖値を低下させることができるので、2型糖尿病の治療に有用である。
これらの優れた薬理作用により、本発明は糖尿病の予防及び/又は治療薬として、またメタボリックシンドロームの予防及び/又は治療薬として有用である(以下、本発明の医薬とも称する)。本明細書中で用いられる場合、「予防」とは、症状を示さない被験体において、該症状が顕在化するのを防ぐことを意味し、「治療」とは、症状を示す被験体において、該症状を軽減すること、あるいは該症状の悪化を防ぐこと又は遅延させることを意味する。
本発明の医薬あるいは食品が、単一の製剤あるいは食品として提供される場合には、該医薬あるいは食品の単位摂取量又はその分割量は、DCP-LAの単位摂取量又はその分割量である。
(材料と方法)
1.細胞培養
GLUT4mycを発現する3T3L1-GLUT4myc線維芽細胞株を用いた。当該細胞は、ヒトc-MYCエピトープ(14アミノ酸)を第1エクトドメインに挿入して構築される。GLUT4の細胞膜表面への移行を抗c-myc抗体で追跡することが可能になる。
当該細胞を10%(v/v)ウシ血清、ペニシリンン(最終濃度,100 U/ml)及びストレプトマイシン(最終濃度,0.1 mg/ml)を添加したDulbecco’s Modified Eagles Medium(DMEM)中、5%CO2及び95%空気の湿気のある環境で37℃で培養した。細胞がコンフルエントな状態に到達したら(0日目)、線維芽細胞から脂肪細胞へと分化させるために、培地を、10%(v/v)ウシ胎仔血清(FBS),1μMデキサメサゾン,0.5mM 3-イソブチル-メチル-キサンチン及び0.1mg/mlインスリンを添加したDMEMに交換した。3日目、7日目及び11日目で、10%(v/v)FBSを添加したDMEMに培地交換した。14日目で、脂肪細胞へと分化した細胞を実験に使用した。
2.グルコース取込みアッセイ
3T3L1-GLUT4myc脂肪細胞を0.2%(w/v)ウシ血清アルブミン(BSA)を含有し、10mMグルコースを添加したKrebs-Ringer-HEPES buffer[136 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 1.25 mM MgSO4及び20 mM HEPES, pH 7.5]中、37℃で1時間インキュベートした。細胞を、10mMグルコースを含有するリン酸緩衝生理食塩水(PBS)中DCP-LA及び/又はGF109203Xで37℃で2時間処理した。処理後、細胞外溶液を回収しグルコースをパラアミノ安息香酸エチルエステル(ABEE)で標識した。次いで、ABEE標識した溶液(5μl)を高速液体クロマトグラフィー(HPLC)システムのカラム(150×4.6 mm)に注入した。ABEE標識したグルコースは、蛍光検出器を用いて、励起波長305nm及び蛍光波長360nmで検出した。細胞に取込まれたグルコース量は、2時間インキュベーションした後の細胞外グルコース濃度を最初の細胞外グルコース濃度(10mM)から差し引いて算出する。
3.GLUT4動員のモニタリング
3T3L1-GLUT4myc脂肪細胞を0.2%(w/v)BSAを含有し、10mMグルコースを添加したKrebs-Ringer-HEPES buffer中、37℃で1時間インキュベートした。細胞を、DCP-LAで20分間処理した。次いで、細胞を1%(v/v)プロテアーゼインヒビターカクテルを含む氷冷したミトコンドリアバッファー[210 mM マンニトール, 70 mM シュクロース, 1 mMエチレンジアミン四酢酸(EDTA), 10 mM HEPES, pH 7.5]中、ソニケーションによりホモジナイズした。続いて、ホモジネートを4℃で5分間、3,000rpmで遠心分離した。上清をさらに4℃で15分間、11,000rpmで遠心分離した。回収した上清を4℃で60分間、100,000gで超遠心分離し、細胞質画分と細胞膜画分に分離した。上清を細胞質画分として、ペレットを細胞膜画分としてそれぞれ用いた。細胞質成分及び細胞膜成分が首尾よく分離できたかどうかは細胞質成分のマーカーである乳酸脱水素酵素(LDH)に対する抗体と、細胞膜マーカーであるカドヘリンに対する抗体とを用いたウェスタンブロット解析によって確認した。
各画分のタンパク質濃度はBCA蛋白質アッセイキット(Thermo Fisher Scientific, Waltham, MA, USA)を用いて測定した。細胞膜画分のタンパク質を1%(w/v)ドデシル硫酸ナトリウム(SDS)を含有するミトコンドリアバッファーに再懸濁した。各画分のタンパク質をSDS-ポリアクリルアミドゲル電気泳動(SDS-PAGE)によって分離し、ポリビニリデンジフルオライド膜に転写した。ブロッティング膜は5%(w/v)BSAを含むTBS-T[150 mM NaCl, 0.1% (v/v) Tween20及び20 mM Tris, pH7.5]でブロッキングし、抗c-myc抗体(Merck Millipore, Darmstadt, Germany)と反応させ、その後ホースラディッシュペルオキシダーゼ(HRP)コンジュゲートヤギ抗マウスIgG抗体と反応させた。免疫反応性は、ECLキット(Invitrogen)を用いて検出し、化学発光検出システム(chemiluminescence detection system; GE Healthcare, Piscataway, NJ, USA)を用いて可視化した。シグナル密度は画像解析ソフト(Image Gauge software; GE Healthcare)を用いて測定した。
所定の濃度のDCP-LA存在下、あるいは非存在下で3T3L1-GLUT4myc脂肪細胞をグルコース含有(10mM)PBS中で2時間インキュベートし、ついで細胞外グルコース濃度をHPLCを用いて測定した。インキュベーション前後の細胞外グルコース濃度の差から細胞へのグルコース取込み量(nmol/μgタンパク質/分)を算出した。結果を図1Aに示す。
さらに別途に、0.1μMのDCP-LA存在下あるいは非存在下、及び/又は0.1μMのGF109203X(GF)の存在下あるいは非存在下で3T3L1-GLUT4myc脂肪細胞をグルコース含有(10mM)PBS中で2時間インキュベートし、ついで細胞外グルコース濃度をHPLCを用いて測定した。インキュベーション前後の細胞外グルコース濃度の差から細胞へのグルコース取込み量(nmol/μgタンパク質/分)を算出した。結果を図1Bに示す。
図1Aの結果より、DCP-LAが脂肪細胞への糖取込みを釣鐘型の濃度依存的な様式で促進させる作用を有することが示された。また、図1Bの結果よりPKCε阻害剤であるGF109203X存在下でもDCP-LAの糖取込み促進作用が阻害されなかったことから、DCP-LAの糖取込み促進作用はDCP-LAが有するPKCε活性化作用とは無関係であることがわかった。
次に、100nMのDCP-LAで20分間処理した3T3L1-GLUT4myc脂肪細胞及び未処理の3T3L1-GLUT4myc脂肪細胞(control)を溶解し細胞質画分(C)及び細胞膜画分(M)を調製した。抗c-myc抗体を用いてGLUT4の細胞膜表面への移行の程度を調べた。結果を図2に示す。
図2の結果より、DCP-LAで処理してもGLUT4の細胞膜移行の有意な促進は認められなかった。従って、DCP-LAによる細胞内への糖取込み促進作用はGLUT4の細胞膜への移行刺激によるものではないことがわかった。
(材料と方法)
グルコース耐性テスト
1型糖尿病モデルは、C57BL/6Jマウス(8週齢)(Japan SLC Inc.; Shizuoka, Japan)にストレプトゾトシン(STZ)(250mg/kg)を腹腔内投与することによって作製した。STZ投与して4日後に経口グルコース耐性テスト(OGTT)を行った。
2型糖尿病モデルは、C57BL/KsJ-leprdb/leprdbマウス(雌性、8週齢)(CLEA Japan; Tokyo, Japan)を用いた。
OGTTは12時間飢餓状態のマウスで行なった。グルコース(2 g/ml/kg body weight)を強制経口投与し、その時点を0hとした。グルコースを投与する30分前に、DCP-LA(0, 0.001, 0.01, 0.1mg/kg)を経口投与した。0、30、60、及び90分の時点で尾静脈から血液(10μl)を回収し、得られた血液から調製しABEEで標識した、各血漿サンプルを高速液体クロマトグラフィー(HPLC)システム(LC-10ATvp; Shimadzu Co., Kyoto, Japan)に負荷した。グルコース濃度は、標準グルコース溶液を用いて作成されたピーク面積/濃度の検量線から算出した。
(結果)
1型糖尿病モデルマウスを用いた場合の結果を図3Aに、2型糖尿病モデルマウスを用いた場合の結果を図3Bに示す。これらの結果から、DCP-LAが1型、2型糖尿病モデルマウスに対して用量依存性に血糖値を下げる作用があることを示している。従って、本発明は1型、2型糖尿病の治療薬となる可能性を示唆している。
Claims (11)
- 8-[2-(2-ペンチル-シクロプロピルメチル)-シクロプロピル]-オクタン酸を有効成分として含有する、糖尿病の予防及び/又は治療薬。
- 糖尿病が、1型糖尿病である、請求項1記載の予防及び/又は治療薬。
- 糖尿病が、2型糖尿病である、請求項1記載の予防及び/又は治療薬。
- 細胞への糖取込みを促進させることを特徴とする、請求項1~3のいずれか1項に記載の予防及び/又は治療薬。
- 8-[2-(2-ペンチル-シクロプロピルメチル)-シクロプロピル]-オクタン酸を有効成分として含有する、細胞への糖取込み促進剤。
- 研究用試薬である、請求項5記載の剤。
- 有効量のDCP-LAをそれを必要とする対象に投与することを特徴とする、糖尿病の予防及び/又は治療方法。
- 細胞への糖取込みを促進させることを特徴とする請求項7記載の方法。
- 糖尿病の予防及び/又は治療に使用するためのDCP-LA。
- 細胞への糖取込みを促進させることを特徴とする請求項9記載のDCP-LA。
- DCP-LAで細胞を処理することを特徴とする、細胞への糖取込みを促進させる方法。
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Citations (5)
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WO2002050013A1 (en) | 2000-12-19 | 2002-06-27 | Fujisawa Pharmaceutical Co., Ltd. | Carboxylic acid compound having cyclopropane ring |
JP2008143819A (ja) | 2006-12-08 | 2008-06-26 | Tomoyuki Nishizaki | 酸化ストレス誘導細胞死の抑制剤 |
WO2012067111A1 (ja) | 2010-11-16 | 2012-05-24 | 株式会社西崎創薬研究所 | PKC-ε活性化剤 |
WO2013071282A1 (en) * | 2011-11-13 | 2013-05-16 | Blanchette Rockefeller Neurosciences Institute | Pkc activators and combinations thereof |
WO2014126191A1 (ja) * | 2013-02-15 | 2014-08-21 | 株式会社西崎創薬研究所 | シクロプロパン環を有する不飽和脂肪酸誘導体を含むリン脂質化合物 |
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- 2015-01-06 WO PCT/JP2015/050122 patent/WO2015105094A1/ja active Application Filing
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- 2015-01-06 EP EP15735079.4A patent/EP3093013A4/en not_active Withdrawn
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WO2002050013A1 (en) | 2000-12-19 | 2002-06-27 | Fujisawa Pharmaceutical Co., Ltd. | Carboxylic acid compound having cyclopropane ring |
JP2008143819A (ja) | 2006-12-08 | 2008-06-26 | Tomoyuki Nishizaki | 酸化ストレス誘導細胞死の抑制剤 |
WO2012067111A1 (ja) | 2010-11-16 | 2012-05-24 | 株式会社西崎創薬研究所 | PKC-ε活性化剤 |
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WO2014126191A1 (ja) * | 2013-02-15 | 2014-08-21 | 株式会社西崎創薬研究所 | シクロプロパン環を有する不飽和脂肪酸誘導体を含むリン脂質化合物 |
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JP6372931B2 (ja) | 2018-08-15 |
US20160324815A1 (en) | 2016-11-10 |
JPWO2015105094A1 (ja) | 2017-03-23 |
EP3093013A1 (en) | 2016-11-16 |
US9889104B2 (en) | 2018-02-13 |
EP3093013A4 (en) | 2017-09-13 |
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