WO2017023131A2 - Composition pharmaceutique contenant, comme principe actif, un inhibiteur d'activité ou d'expression de chrebp, permettant de prévenir ou de traiter le diabète - Google Patents

Composition pharmaceutique contenant, comme principe actif, un inhibiteur d'activité ou d'expression de chrebp, permettant de prévenir ou de traiter le diabète Download PDF

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WO2017023131A2
WO2017023131A2 PCT/KR2016/008618 KR2016008618W WO2017023131A2 WO 2017023131 A2 WO2017023131 A2 WO 2017023131A2 KR 2016008618 W KR2016008618 W KR 2016008618W WO 2017023131 A2 WO2017023131 A2 WO 2017023131A2
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mtor
expression
chrebp
txnip
cells
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WO2017023131A3 (fr
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엄성희
칵 쨔우쨔
임동욱
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성균관대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

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  • the present invention relates to a pharmaceutical composition for preventing or treating diabetes, comprising ChREBP expression or activity inhibitor as an active ingredient.
  • Diabetes mellitus is a type of metabolic disease, such as a lack of insulin secretion or normal function, characterized by high blood glucose high blood sugar, causing a number of symptoms and signs due to high blood glucose and excreted glucose from urine.
  • Type 1 diabetes is a disease caused by the destruction of pancreatic ⁇ -cells of the pancreas that secrete insulin. It is also called insulin-dependent diabetes because it needs to be administered, and it usually occurs in children and adolescents.
  • the cause is not known precisely, but is known to be caused by genetics, the environment, viruses, chemicals and the like. Symptoms usually appear suddenly, and severe symptoms can include increased thirst, frequent urination, weight loss, extreme hunger, vomiting, abdominal pain, or fatigue.
  • Type 1 diabetes accounts for 5-10% of all diabetes incidences and is reported in 80,000 children each year worldwide, but the exact number of patients is unknown. It is estimated that there are one million to three million patients in the United States alone, and in Korea, one patient occurs every 100,000 people, and the incidence rate is gradually increasing.
  • pancreatic beta cells in type 1 diabetes results in the deletion of insulin and eventually destroys glucose homeostasis in the body.
  • Insulin acts to lower blood sugar by moving glucose in the blood into the cells and converting glucose into glycogen in the liver, so the lack of insulin due to the destruction of beta cells leads to the destruction of glucose homeostasis in the blood, resulting in hyperglycemia and the resulting metabolic disorders.
  • oxidative stress in pancreatic beta cells has been reported to affect beta cell death and loss of function in the progression of type 1 diabetes.
  • Type 2 diabetes is a condition in which insulin is relatively low, and is characterized by insulin resistance. Type 2 diabetes accounts for the majority of diabetic patients in the world, and unlike type 1, which occurs mainly in children, it occurs in adults. The cause of the disease seems to be caused by environmental factors such as high calorie, high fat, high protein diet, lack of exercise, and stress due to westernization of diet, diabetes can also be caused by a specific gene defect.
  • mTOR mimmalian Target Of Rapamycin
  • mTOR complex 1 mTOR complex 1
  • mTORC2 mTOR complex 2
  • mTORC1 mTOR complex 1
  • mTORC2 mTOR complex 2
  • mTORC1 mTOR complex 1
  • mTORC2 mTOR complex 2
  • mTORC1 regulates mRNA transcription
  • protein synthesis protein synthesis
  • lipid synthesis lipid synthesis
  • autophagy mTORC2
  • mTORC2 is responsible for cell survival, cell cycle progression, and insulin induced assimilation. It is known to be involved in the regulation of insulin-induced anabolism.
  • the present inventors attempted to identify the role of mTOR that is directly related to the survival of pancreatic beta cells, and thus the regulation of insulin secretion, and the novel target (ChREBP) that is the core of the role of mTOR and its mechanism of action.
  • ChREBP novel target
  • the present invention is made by the support of the 2014R1A2A1A11050734 project of the Ministry of Science, ICT and Future Planning.
  • the present inventors have conducted extensive studies to investigate the role and role of mTOR in the survival and control of insulin secretion of pancreatic beta cells, and through the deletion or overexpression of mTOR, the mTOR is directly involved in beta cell survival and insulin secretion.
  • mTOR is deleted or expression is inhibited, ChREBP expression is increased, Mlx and ChREBP binding affinity is markedly increased, and nuclear migration of ChREBP / Mlx complex is involved in beta cell death.
  • the expression of TXNIP was observed to increase.
  • mTOR binds to the ChREBP / Mlx complex to inhibit the transcriptional activity of ChREBP and thereby completed the present invention by elucidating that the expression of TXNIP involved in beta cell death is inhibited.
  • an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diabetes, comprising ChREBP expression or activity inhibitor as an active ingredient.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of diabetes comprising ChREBP (Carbohydrate responsive element-binding protein) expression or activity inhibitor as an active ingredient.
  • ChREBP Carbohydrate responsive element-binding protein
  • the expression or activity inhibitor of ChREBP is characterized in that the mTOR (mammalian Target Of Rapamycin) gene or mTOR protein.
  • the mTOR gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
  • the mTOR protein is characterized in that consisting of the amino acid sequence of SEQ ID NO: 2.
  • composition is characterized by inhibiting the expression of Thiedoedoxin-interacting protein (TXNIP) by inhibiting the expression or activity of ChREBP.
  • TXNIP Thiedoedoxin-interacting protein
  • the present invention also provides a method for preventing or treating diabetes, comprising administering the pharmaceutical composition to a subject.
  • the present invention provides a composition for the prevention or treatment of diabetes.
  • the pharmaceutical composition according to the present invention has a beta cell protective effect by inhibiting the expression or activity of TXNIP involved in pancreatic beta cell death by inhibiting the expression or activity of ChREBP, a transcription factor of TXNIP, and thus is useful for the prevention or treatment of diabetes. It is available.
  • Figure 1a is the result of confirming the mTOR deletion by Western blot in the mTOR knock-out mice ( ⁇ mTORKO) specific to pancreatic beta cells only
  • Figures 1b to 1e is fasting blood glucose levels according to mTOR deletion in diabetic conditions by STZ treatment Increase (FIG. 1B), glucose intolerance (FIG. 1C), increase insulin resistance (FIG. 1D), and decrease insulin secretion (FIG. 1E) were confirmed.
  • Figure 2a and Figure 2b is a decrease in the size of the pancreatic islets according to mTOR deletion (Fig. 2a) and the number of beta cells using mTOR knock-out mice ( ⁇ mTORKO) specific to pancreatic beta cells only in diabetic conditions by STZ treatment (Fig. 2a) 2b) is confirmed.
  • Figures 3a and 3g is to examine the effect of mTOR on the survival and proliferation of beta cells in diabetic conditions by STZ treatment
  • Figures 3a and 3b is increased beta cell death (m 3a) and proliferation due to mTOR deletion
  • the decrease (FIG. 3B) was confirmed through immunohistochemical staining
  • FIG. 3C to FIG. 3E show increased expression of apoptosis-related proteins (cleaved PARP, BAX) following mTOR deletion and mTOR expression inhibition (FIG. 3C and FIG. 3D)
  • FIG. 3e shows increased expression of apoptosis-related proteins (cleaved PARP, BAX) following mTOR deletion and mTOR expression inhibition
  • Figure 4a to 4f is to investigate the decrease in insulin secretion by mTOR deletion or inhibition of expression in insulin secretion conditions by low or high glucose stimulation, mouse-derived pancreatic islets according to mTOR deletion (Fig. 4a), mTOR expression is inhibited Reduced insulin secretion and increased ADP / ATP ratios in INS-1 cells (FIG. 4B) or INS-1 cells treated with rapamycin or PP242, an mTOR inhibitor (FIG. 4C), respectively. It is the result of confirming the decrease of intracellular calcium ion (Ca 2+ ) concentration (FIG. 4D), the reduction of mitochondrial membrane potential (FIG. 4E) and the increase of ROS production (FIG. 4F) due to mTOR deletion or expression inhibition in the deleted islets.
  • Ca 2+ intracellular calcium ion
  • FIG. 4E the reduction of mitochondrial membrane potential
  • ROS production FIG. 4F
  • Figures 5a and 5b is a result confirming the increased expression of TXNIP mRNA (Fig. 5a) and protein (Fig. 5b) in mouse pancreatic islet mTOR-deleted mINS and mTOR expression-inhibited INS-1 cells under STZ treatment.
  • FIG. 6A and 6B show TXNIP mRNA (FIG. 6A) and protein (FIG. 6B) inducing pancreatic beta cell death in mouse pancreatic islets lacking mTOR and mTOR expression-inhibited INS-1 cells at low or high glucose stimulation conditions (FIG. 6B).
  • FIG. 6C to 6E show that TXNIP and ChREBP protein expression decreased by mTOR overexpression (FIG. 6C), apoptosis inhibitory protein (Bcl-2) expression and inducible protein in diabetic condition by STZ treatment. Decreased expression of cleaved-PARP and BAX) (FIG. 6D), and a decrease in cell cycle SubG1 stage ratio (FIG. 6E).
  • Figures 7a to 7f is an increase in the expression of ChREBP mRNA (Figs. 7a and 7d) and protein (Figs. 7b and 7e) according to the inhibition of the expression of mTOR in diabetic conditions and glucose stimulation conditions by STZ treatment, ChREBP and TXNIP promoter ChoRE Results show an increase in binding affinity of the sites (FIGS. 7C and 7F).
  • FIG. 8A to 8C show increased binding of TXNIP and ChREBP in diabetic conditions by STZ treatment (FIG. 8A), direct binding between mTOR and ChREBP or mTOR and Mlx under the same conditions (FIG. 8B), and ChREBP and Mlx upon mTOR expression inhibition
  • FIG. 8c The increase in binding
  • Figures 8d to 8f is a result confirming the inhibition of migration in the nucleus of the complex by the ChREBP / Mlx complex binding of mTOR under STZ treatment conditions.
  • Figure 9 is a diagram showing the synthesis of the inhibition of the expression of TXNIP inducing pancreatic beta cell death through ChREBP inhibition of mTOR.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, including an inhibitor of ChREBP expression or activity that regulates the expression of TXNIP that induces pancreatic beta cell death.
  • the diabetes of the present invention is a kind of metabolic disease such as insufficient insulin secretion or normal function, and means a disease characterized by high blood glucose with high blood glucose concentration.
  • the diabetes includes both type 1 and type 2 diabetes, and more preferably, type 1 diabetes.
  • prevention means any action that inhibits or delays the onset of diabetes by administration of the pharmaceutical composition according to the invention.
  • treatment used in the present invention means any action that improves or advantageously changes the symptoms caused by diabetes by administration of the pharmaceutical composition according to the present invention.
  • the term "activity inhibitor” means to cause a decrease in the function of the target protein, preferably by means that the function of the target protein becomes undetectable or present at an insignificant level.
  • the inhibitor of expression or activity of ChREBP of the present invention may be an mTOR gene or mTOR protein.
  • the mTOR gene may be composed of the nucleotide sequence of SEQ ID NO: 1 or 3 from human or mouse, the mTOR protein may be composed of the amino acid sequence of SEQ ID NO: 2 or 4 from human or mouse (mouse) And, more preferably, it may be composed of the human-derived nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.
  • ChREBP also called MLX-interacting protein-like (MLXIPL)
  • MLXIPL MLX-interacting protein-like
  • the ChREBP protein functions to activate transcription by binding to a carbohydrate response element (CHORE) present in the promoter of triglyceride synthesis genes depending on glucose.
  • CHORE carbohydrate response element
  • the Mlx (Max like protein) is one of the basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor family is a protein involved in cell proliferation, crystallization and differentiation by forming a heterodimer with Mad protein.
  • the Mlx protein is known to interact with MNT, MXD1, and ChREBP (MLXIPL).
  • mTOR is important for maintaining glucose homeostasis through increased glucose, glucose intolerance and insulin resistance due to mTOR deletion in diabetic condition by STZ treatment in mice with mTOR deletion only in pancreatic beta cells. It was confirmed to play a role (see Example 2).
  • the result of verifying whether insulin secretion abnormality is caused by beta cell death due to mTOR deletion or expression inhibition results in a decrease in insulin secretion, an increase in ADP / ATP ratio, intracellular intracellular glucose secretion conditions
  • mTOR plays an important role in insulin secretion (see Example 5).
  • pancreatic beta due to mTOR deletion in diabetic conditions and glucose stimulation conditions by STZ treatment TXNIP expression was increased to induce cell death.
  • the expression of TXNIP and its transcription factor, ChREBP, and the inhibition of apoptosis were confirmed when overexpression of mTOR resulted in pancreatic beta cell death in the regulation of beta cell survival. Inducing TXNIP was found to be regulated by mTOR expression (see Example 6).
  • TXNIP Thioredoxin-interacting protein
  • ChREBP known as a transcription factor of TXNIP
  • the expression of ChREBP and thus the expression of TXNIP is increased. It was confirmed that the decreased expression of mTOR further increased the binding affinity between the ChREBP and TXNIP promoter ChoRE sites (see Example 7).
  • mTOR inhibits the expression and transcriptional activity of ChREBP in diabetic conditions and consequently inhibits the expression of TXNIP, which induces pancreatic beta cell death, thereby directing beta cell protection through beta cell survival and insulin secretion control. It was found to be involved.
  • the pharmaceutical composition according to the present invention may include an inhibitor of the expression or activity of ChREBP as an active ingredient and may include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included.
  • diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature.
  • the pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.
  • composition according to the invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level refers to the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts.
  • the composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight is administered daily or every other day or 1 It can be administered in 1 to 3 times a day.
  • the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
  • composition of the present invention can be used in a variety of drugs, foods and beverages for the prevention and improvement of diabetes diseases, and can be used in the form of powders, granules, tablets, capsules or beverages.
  • the present invention provides a method for preventing or treating diabetes, comprising administering the pharmaceutical composition to a subject.
  • “individual” means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, and the like. Means mammals.
  • the present invention provides a use of the pharmaceutical composition for the prevention or treatment of diabetes.
  • mTOR flox / flox mice were crossed with transgenic mice carrying a rip (Rat insulin promoter) cre recombinase.
  • Beta-cell specific mTOR knockout mice were constructed, which were purchased from the Jackson Laboratory (Bar Harbor, Maine, USA).
  • mTOR flox / flox mice with normal mTOR genes were used.
  • INS-1 a rat-derived insulinoma beta cell, RPMI1640 medium with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, and 0.05 mM 2-mercaptoethanol from Christopher Newgard (Duke University, Durham, NC, USA) (Welgene, Daegu, South Korea) was incubated under 5% CO 2 , 37 °C conditions.
  • FBS fetal bovine serum
  • 1 mM sodium pyruvate 1 mM sodium pyruvate
  • 2 mM L-glutamine 10 mM HEPES
  • 0.05 mM 2-mercaptoethanol from Christopher Newgard (Duke University, Durham, NC, USA) (Welgene, Daegu, South Korea) was incubated under 5% CO 2 , 37 °C conditions.
  • INS-1 cells were transfected with pRK5-based expression vectors myc-mTOR (Addgene) and myc-mTOR kinase dead (myc-mTOR KD, Addgene), and the plasmid at each transfection. Total amount of DNA was corrected with pRK5 vector.
  • the mTOR and ChREBP siRNA sequences used in this example are shown in Table 1 below.
  • Pancreas was isolated from 8-week-old mTOR flox / flox mice and beta cell-specific mTOR knockout mice, and HBSS (Welgene) mixed with 0.8 mg / ml collagenase P (Roche Diagnostics, Indianapolis, IN, USA) ) Solution was injected and shaken regularly and incubated at 37 ° C. for 15 minutes.
  • the solution from which the pancreatic tissue was degraded was filtered through a mesh, and density gradient centrifugation was performed using Ficoll gradients (Biochrom AG, Berlin, Germany) to separate the intermediate layer, and the pancreatic islets were separated under a microscope.
  • the isolated islets were then incubated overnight at 5% CO 2 , 37 ° C. in RPMI1640 medium containing 10% FBS and penicillin / streptomycin.
  • GTT Glucose Tolerance Test
  • mice After 12-week-old male mice were maintained on an empty stomach for 16 hours, blood was collected from the tail vein and basal blood glucose (mg / dl) was measured by AccuChek II glucometer (Roche Diagnostics). Thereafter, D-glucose (2 g / kg body weight) was mixed with sterilized phosphate buffer (PBS) and intraperitoneally administered to mice, and blood glucose was measured at 15, 30, 60, 90, and 120 minutes after administration.
  • PBS sterilized phosphate buffer
  • the cells were then incubated with KRBH buffer containing 3 mM or 25 mM glucose for 1 hour at 37 ° C., and then the supernatant was recovered and measured for insulin secretion using an ELISA kit (ALPCO Immunoassays, Salem, NH, USA). .
  • Insulin secretion of pancreatic islets was measured in the same manner as the INS-1 cells after pre-culture of 10 pancreatic islets similar in size separated from mice according to the method of Example 1-3, containing 2.8 mM or 16.7 mM of glucose. Incubated with Krebs-Ringer buffer and insulin secretion was measured.
  • RT-PCR Reverse Transcription Polymerase Chain Reaction
  • qRT-PCR Real-time quantitative PCR (qRT-PCR) using primers specific for genes to be confirmed for expression of DNA precipitated by cDNA or ChIP analysis and SYBR Green PCR Master Mix (Applied Biosystems) was performed.
  • RIPA buffer 150 mM NaCl, 50 mM Tris-Cl, pH7.4 in beta cells or mTOR siRNA and non-silencing siRNA transfected mouse pancreatic islets isolated by the method of Example 1-3
  • Cell lysate was added by adding 1 mM EDTA (ethylenediaminetetraacetic acid), 1% NP-40 (NonidetP-40), 0.25% Na-deoxycholate, 0.1 M NaF, 2 mM Na 3 VO 4 ) and protease inhibitors (Roche Diagnostics). Got it.
  • SDS-polyacrylamide gel electrophoresis, transfer, and blocking were performed according to methods known in the art, and the primary antibodies used to confirm the expression level of each protein are as follows.
  • Anti-mTOR, Bax, Bcl-2, PARP, p-S6 (S235 / 236), p-S6 (S240 / 244), P-Akt (S473), and pS6K (T389) antibodies are described in Cell Signaling Technology (Beverly, MA, USA), TXNIP, ChREBP, Mlx, Trx-1, ⁇ -tubulin and ⁇ -actin antibodies were purchased from Santa-Cruz Biotechnology (USA).
  • IP Immunoprecipitation
  • Example 1-2 cells treated with mTOR siRNA or non-silencing siRNA and glucose-treated cells were treated with 1% formaldehyde (fixaldehyde) for 10 minutes to obtain a final concentration of 0.125 M. Glycine was added to terminate the fixation reaction.
  • the immobilized cells were washed with phosphate buffer (PBS), lysed with SDS lysis buffer and sonicated with Fisher Scientific Sonic Dismembrator 500 for 30 seconds to allow the intracellular DNA to be cut to 200-300 base pairs. Thereafter, the reaction was carried out at 4 ° C. for 16 hours using 4 ⁇ g of ChREBP antibody to allow DNA / protein complexes to be immunoprecipitated.
  • PBS phosphate buffer
  • SDS lysis buffer lysed with SDS lysis buffer
  • Fisher Scientific Sonic Dismembrator 500 for 30 seconds to allow the intracellular DNA to be cut to 200-300 base pairs. Thereafter, the reaction was carried out at 4 ° C. for 16 hours using 4 ⁇ g of ChREBP antibody
  • the protein / DNA complex was 60 ⁇ l of 50% (vol./vol.) Protein A or G agarose ( Amersham) / salmon sperm DNA slurry was captured and washed for 1 hour at 4 ° C.
  • Cross-linked DNA and protein were released by heat treatment at 65 ° C. for 4 hours in Tris-HCl pH 6.5, 5 M NaCl, and 0.5 M EDTA, followed by extracting the DNA with phenol / CHCl 3 and precipitating with ethanol.
  • the primers shown in Table 3 below were used to quantify the real-time polymerase chain reaction.
  • Intracellular reactive oxygen species (ROS) concentration was measured using a fluorescent probe 2 ', 7'-dichlorofluorosane diacetate (H2DCF-DA, C6827, Invitrogen). Mitochondrial membrane potential was measured using tetramethylrhodamine, ethyl ester (TMRE, Invitrogen).
  • TOR-1 cells were transfected with mTOR siRNA for 72 hours and cultured in glucose-stimulated insulin secretion (GSIS) conditions. Cells were then washed twice with phosphate buffer and incubated for 30 minutes at 37 ° C. with phosphate buffer containing 10 ⁇ M H2DCF-DA or 10 nM TMRE while blocking light. Intracellular ROS concentration and mitochondrial membrane potential were quantified by flow cytometry with BD Canto II cell sorter (BD Biosciences).
  • mice were prepared, and experiments were conducted using the control group (WT) and the mTOR knockout mouse ( ⁇ mTORKO).
  • WT control group
  • ⁇ mTORKO mTOR knockout mouse
  • the islets and the hypothalamus were separated, and then the expression level of the mTOR protein was measured by Western blot. As shown, it was confirmed that mTOR was expressed at the same level as the control group in the hypothalamus but not in the pancreatic islets.
  • diabetes mellitus was induced by WZ and ⁇ mTORKO mice twice a week for 14 days in an amount of 100 mg / kg, and the blood glucose concentration of the mice was measured in an empty state for 8 weeks after the administration. .
  • the blood glucose level was increased in STZ-induced diabetic mice, especially in mice knocked out of mTOR (KO STZ) compared to normal mice (WT STZ). It was confirmed that the increase significantly.
  • the glucose tolerance test was performed by the method of Example 1-4 to determine the effect of mTOR on the maintenance of blood glucose homeostasis.
  • glucose tolerance was remarkable in mTOR knock-out mice (KO STZ) administered STZ compared to normal mice (ST STZ) administered STZ. It was found to remain high.
  • mTOR knocked-out mice are accompanied with insulin resistance, and the insulin in the mTOR knock-out mice administered STZ compared to normal mice administered STZ as a result of measuring blood insulin secretion through the results of FIG. 1E. It was confirmed that secretion decreased significantly.
  • mTOR plays an important role in maintaining glucose homeostasis in diabetic conditions by STZ administration.
  • pancreatic islets As a result of H & E staining, as shown in FIG. 2A, the size of pancreatic islets was significantly decreased when mTOR was deleted (KO), especially in mTOR knockout mice administered STZ compared to normal mice treated with STZ. It was confirmed that the 51-56% decrease.
  • Example 2 The results correspond to those of Example 2, which means that mTOR plays a very important role in maintaining beta cell and pancreatic islet size in diabetic condition by STZ administration.
  • Example 3 confirmed the decrease of the beta cell number due to mTOR deletion, in order to verify whether mTOR is directly involved in the survival and proliferation of beta cells, the pancreas was first isolated from each mouse to immunohistochemical staining method. Was performed.
  • Example 4-1 Since the results of animal experiments may be affected by the difference in hormone levels in each mouse, it was intended to verify the results of Example 4-1 under conditions of deletion or expression inhibition of mTOR at the cellular level.
  • STZ treatment was performed on pancreatic islets of mTOR knocked out mice ( ⁇ mTORKO) or STZ treatment at 0.5 or 1 mM for 6 hours in INS-1 cells transfected with mTOR siRNA (si-mTOR).
  • Western blotting was performed to confirm the expression of apoptosis-related proteins, a mechanism of apoptosis.
  • FIGS. 3C and 3D expression of cleaved PARP (Poly (ADP-ribose) polymerase) and BAX (Bcl-2 associated X protein), which are apoptosis-related proteins, in STZ-treated mTOR knockout islet cells Increased significantly.
  • the expression of the proteins increased in proportion to the concentration of STZ in INS-1 cells that inhibited the expression of mTOR.
  • STZ-treated cells treated with mTOR expression were suppressed compared to the case where STZ was treated in normal cells. In the case of subG1 ratio was confirmed to increase significantly.
  • the mitochondrial membrane potential and reactive oxygen species (ROS) production were measured after STZ treatment.
  • ROS reactive oxygen species
  • beta cell death causes insulin production and secretion abnormalities, and thus the purpose of this study was to investigate the effects on beta cell insulin secretion when mTOR was deleted or expression was inhibited.
  • mTOR deletion was used to isolate islets from mTOR knockout mice infected with adenovirus expressing Cre.
  • mTOR-deleted islet beta cells and Mitochondrial membrane potential is measured after treatment with low (3 mM) or high (25 mM) glucose in INS-1 cells which inhibited mTOR expression.
  • the mitochondrial membrane potential was significantly decreased when mTOR was deleted (KO) or expression was inhibited (si-mTOR).
  • the amount of ROS produced in the cells inhibited mTOR expression as shown in Figure 4f, it was confirmed that the amount of ROS produced significantly increased compared to the control cells (si-NS).
  • mTOR plays an important role in insulin secretion through regulation of ADP / ATP ratio, maintenance of mitochondrial membrane potential, increased calcium ion influx, and inhibition of ROS production in pancreatic beta cells.
  • STZ treatment was performed on mouse islet beta cells lacking mTOR and INS-1 cells that inhibited mTOR expression, followed by free radicals in beta cells.
  • the expression levels of mRNA and protein of TXNIP, the major regulators of the redox system, inducing species production and apoptosis were observed.
  • Example 6-1 Based on the results of Example 6-1, mRNA and protein expression levels of TXNIP were verified upon mTOR deletion or inhibition of expression of beta cells under glucose stimulation conditions.
  • TXNIP is a novel target of mTOR in the regulation of beta cell survival.
  • Trx-1 Thioredoxin-1
  • Glucose acts as a major inducer of TXNIP expression in INS-1 cells and pancreatic islets of human pancreas
  • Example 6 confirmed the increased expression of TXNIP under STZ treatment and glucose stimulation conditions
  • ChREBP showed the increased expression of TXNIP in various cells. It is known as a transcription factor that regulates expression. Based on this, it was verified whether mTOR regulates the expression of TXNIP by controlling the transcription of ChREBP-mediated TXNIP in beta cells. To this end, the mRNA and protein expression levels of ChREBP in INS-1 cells inhibited mTOR expression under STZ treatment or glucose stimulation conditions were observed.
  • ChIP Chromatin immuno-precipitation assay was performed according to the method of Examples 1-8 to perform the ChREBP and TXNIP promoters.
  • the binding affinity of the ChoRE site was observed.
  • Figure 7c when the expression of mTOR was inhibited, the binding affinity between the promoter ChoRE site of ChREBP and TXNIP was increased, especially when the STZ treatment was further increased.
  • Max like protein is known to act as a transcriptional regulator of TXNIP by forming a complex with ChREBP during glucose stimulation in INS-1 cells.
  • TXNIP and ChREBP increased under STZ treatment or glucose stimulation conditions.
  • immunoprecipitation was performed by the method of Example 1-7. (Immunoprecipitation; IP) was performed. As a result, as shown in Figure 8a, it was confirmed that the binding of ChREBP and Mlx increased under the STZ treatment conditions.
  • ChREBP acts as a transcription factor that regulates the transcription of TXNIP. Since Example 7 confirmed that mTOR inhibited the expression of TXNIP, IP was performed to determine whether mTOR directly binds to ChREBP. As a result, as shown in Fig. 8b, it was confirmed that mTOR and ChREBP are bonded under STZ treatment conditions. Furthermore, as a result of checking whether mTOR is coupled to Mlx, it can be seen that mTOR is directly coupled to Mlx as shown in FIG. 8B. The results indicate that mTOR binds to and interacts with the ChREBP / Mlx complex under apoptotic stress conditions such as STZ treatment.
  • mTOR is the nucleus of the ChREBP / Mlx complex to induce the transcription of TXNIP under STZ treatment conditions.
  • the purpose of this study was to examine how it affects the movement into the inner circle. As a result, as shown in Figure 8d to 8f, in the control cell (Veh) mTOR, ChREBP, and Mlx is not bound in the cytoplasm, while mTOR binds to ChREBP and Mlx in the cytoplasm under STZ treatment conditions It was confirmed. However, these bonds did not occur in the nucleus.
  • the pharmaceutical composition according to the present invention shows a prophylactic or therapeutic effect of diabetes mellitus through pancreatic beta cell protective effect, it may be usefully used in the medical industry for the development of a therapeutic agent for diabetes.

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Abstract

La présente invention concerne une composition pharmaceutique contenant, comme principe actif, un inhibiteur d'activité ou d'expression de ChREBP, permettant de prévenir ou de traiter le diabète. La composition pharmaceutique selon la présente invention inhibe l'expression ou l'activité de ChREBP, qui est un facteur transcriptionnel de TXNIP, afin d'inhiber l'expression de TXNIP impliquée dans l'apoptose de cellules bêta du pancréas, en ayant ainsi un effet de protection de cellules bêta et, de ce fait, la composition pharmaceutique peut être favorablement utilisée dans la prévention ou le traitement du diabète.
PCT/KR2016/008618 2015-08-06 2016-08-04 Composition pharmaceutique contenant, comme principe actif, un inhibiteur d'activité ou d'expression de chrebp, permettant de prévenir ou de traiter le diabète WO2017023131A2 (fr)

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KR1020150110960A KR101625333B1 (ko) 2015-08-06 2015-08-06 ChREBP의 발현 또는 활성 억제제를 유효성분으로 포함하는 제1형 당뇨병의 예방 또는 치료용 약학적 조성물
KR10-2015-0110960 2015-08-06

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