KR101602203B1 - A pharmaceutical composition for prevention or treatment of diabetes comprising n-(2-hydroxyethyl)-3-(6-(4-(trifluoromethoxy)phenylamino)pyrimidin-4-yl)benzamide or pharmaceutically acceptable salts thereof as an effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof, And to a health functional food and a pharmaceutical composition for preventing or treating diabetes. (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof, Type 1 diabetes and type 2 diabetes, and the compound may also be used as a health functional food for preventing or ameliorating diabetes.

Description

N (2-hydroxyethyl) 3 (6 (4 (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof as an active ingredient for the prevention or treatment of diabetes mellitus [0001] The present invention relates to a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound is a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof, or a pharmaceutically acceptable salt, solvate or prodrug thereof. COMPONENT}

The present invention relates to a pharmaceutical composition comprising N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof, And to a health functional food and a pharmaceutical composition for preventing or treating diabetes.

The in vivo tyrosine kinase signaling pathway is important for cell proliferation, differentiation, metabolism, and cell migration regulation, and this pathway abnormality is known to be closely related to the occurrence of diseases such as cancer.

In particular, imatinib (Gleevec or Glivec), which is a Bcr-Abl tyrosine kinase inhibitor (TKIs), has been recognized as a novel therapeutic agent for the treatment of chronic myelogeneous leukemia (CML).

(2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide, known as GNF-5, is a non-targeting target for the myristate- binding pocket near the Abl kinase domain c- -ATP-competitive inhibitors, known as Bcr-Abl inhibitors (Mian et al., 2012). The TKI-mediated mechanism of action of TKI, known as Imatinib, Nilotinib and Dasatinib, is known to inhibit autophosphorylation and substrate phosphorylation of Bcr-Abl oncoprotein by binding to Bcr-Abl kinase (Mokhtari and Welsh, 2010). TKI targets known to date include Arg (Abl-related gene), SCF (stem cell factor) recptor c-Kit, PDGFR (platelet-derived growth factor receptor), DDR1 / 2 (discoidin domain receptor 1/2) and oxidoreductase NQO2 (NADPH dehydrogenase quinone 2) have been reported (Hantschel et al., 2008).

Most TKI drugs, including Imatinib, were found to be excellent candidates for treatment of CML and to inhibit the growth of various cancer cells, including gastrointestinal stromal tumors (GISTs). In particular, the use of Imatinib in patients with chronic myeloid leukemia (CML) associated with type 2 diabetes improved not only CML but also diabetes mellitus (Breccia et al., 2004; Veneri et al., 2005) Of diabetic patients. Studies on the anti-diabetic activity of Imatinib have shown that streptozotocin (STZ) -induced protection against diabetes and diabetic improvement in db / db mice as well as non-obese diabetic mice (Han et al., 2009).

Since TKI, such as Nilotinib, Sunitinib, Dasatinib, Erlotinib, and Fasudil, has been shown to be associated with the inhibition of beta-cell apoptosis and decreased insulin resistance in type 1 or type 2 diabetes mellitus, . The mechanism of action of TKI on the antidiabetic effect is known to inhibit pancreatic beta-cell apoptosis through inhibition of protein kinase C (PKC) delta activation and stress-activated protein kinase pathway through an Imatinib study (Hagerkvist et al., 2006).

However, it is known that Imatinib, Nilotinib, and Dasatinib, which target Bcr-Abl used for CML treatment, do not function when there is a point mutation at the ATP binding site of the Abl kinase domain.

 Targeting Bcr-Abl by combining allosteric with ATP-binding-site inhibitors. Zhang J. et al., Nature. 2010, 463 (7280): 501-6  A comparative target selectivity survey of the BCR-ABL kinase inhibitor INNO-406 by kinase profiling and chemical proteomics in chronic myeloid leukemia cells. Rix U., Leukemia. 2010, 24 (1): 44-50  Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL T315I. Afsar Ali Mian et al., BMC Cancer 2012, 12: 411  Potential utility of small tyrosine inhibitors in the treatment of diabetes. Mokhtari D. et al., Clin Sci (Lond). 2009, 118 (4): 241-7  The Ins and Outs of Bcr-Abl Inhibition. E. Premkumar Reddy et al., Genes & Cancer 3 (5-6) 447-454  Response of prostate cancer during imatinib therapy in a patient with chronic myeloid leukemia. Breccia et al., Haematologica. 2004, 89 (7): ECR20  Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice. Nicole M Agostino et al., J Oncol Pharm Pract. 2011, 197-202  Imatinib improves insulin sensitivity and glucose disposal rates in rats fed a high-fat diet. Hagerkvist et al., Clin Sci (Lond). 2008, 114, 65-71

DISCLOSURE OF THE INVENTION The present invention has been made based on the above-described conventional techniques, and it is an object of the present invention to provide N- (2-hydroxyethyl) -3- (6- (4- The present invention also provides a pharmaceutical composition for preventing or treating diabetes, which comprises as an active ingredient, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof.

Another object to be solved in the present invention is to provide a process for the preparation of N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- And a pharmaceutically acceptable salt thereof, for the prevention or amelioration of diabetes mellitus.

In order to solve the above problems, the present invention provides a process for the preparation of N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- There is provided a pharmaceutical composition for the prevention or treatment of diabetes containing a pharmaceutically acceptable salt as an active ingredient.

The diabetes is preferably type 1 diabetes or type 2 diabetes.

The above N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide, or a pharmaceutically acceptable salt thereof, To inhibit cell apoptosis.

The above N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide, or a pharmaceutically acceptable salt thereof, it is preferable to inhibit the production of the species.

The N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof inhibits JNK phosphorylation. .

The N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof, .

The above N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide, or a pharmaceutically acceptable salt thereof, It is desirable to inhibit activation.

The present invention also provides a method for the prevention of diabetes comprising N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- Or health functional food for improvement.

The diabetes is preferably type 1 diabetes or type 2 diabetes.

(2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof, Type 1 diabetes and type 2 diabetes, and the compound may also be used as a health functional food for preventing or ameliorating diabetes.

FIG. 1 shows the cytotoxicity (A) of GNF-5 and the protective effect (B) of GNF-5 of the pancreatic beta cell line INS-1 treated with streptozotocin.
Figure 2 shows the inhibitory effect of GNF-5 on the accumulation of intracellular ROS induced by streptozotocin.
Figure 3 shows the inhibitory effect of GNF-5 on JNK activity induced by streptozotocin.
Figure 4 shows the inhibitory effect of GNF-5 on PKC? And caspase-3 activity induced by streptozotocin.
5 is in 5 shows the protective effect of GNF-5 on streptozotocin-induced hyperglycemia through vivo experiments.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention have found that N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) -naphthalenic acid, which is known as GNP-5, an ATP-noncompetitive allosteric Bcr-Abl kinase inhibitor, Pyrimidin-4-yl) < / RTI > benzamide in vitro and in vivo , and that GNF-5 can be used for the prophylactic or therapeutic use of diabetes.

Thus, the present invention relates to the use of N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof, A pharmaceutical composition for preventing or treating diabetes containing as an active ingredient is provided.

The diabetes is preferably type 1 diabetes or type 2 diabetes.

The above N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide, or a pharmaceutically acceptable salt thereof, To inhibit cell apoptosis.

The above N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide, or a pharmaceutically acceptable salt thereof, it is preferable to inhibit the production of the species.

The N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof inhibits JNK phosphorylation. .

The N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof, .

The above N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide, or a pharmaceutically acceptable salt thereof, It is desirable to inhibit activation.

Pharmaceutically acceptable salts of the compounds of the present invention are useful as acid addition salts formed by free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol in water (e.g., glycol monomethyl ether) may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the free acid, an organic acid and an inorganic acid can be used. As the inorganic acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acid include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, But are not limited to, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, , Carbonic acid, vanillyric acid, and hydroiodic acid can be used.

In addition, the base may be used to make a pharmaceutically acceptable metal salt. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention include acidic or basic salts which may be present in the compounds of the present invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate (Salicylate) salts, which are known in the art and which are known in the art, such as, for example, ≪ / RTI >

The pharmaceutical composition of the present invention may be formulated into various forms such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, injections of sterilized injection solutions, Can be used.

Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

In a preferred embodiment, the solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, for example starch, calcium carbonate, Sucrose, lactose, gelatin and the like are mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used.

Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, Perfumes, preservatives, and the like.

As a preferable specific example, the preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

In a preferred embodiment, the effective amount of the compound in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and is generally 1 to 5,000 mg, preferably 100 to 3,000 mg per kg of body weight per day, It can be administered every other day or one to three times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The pharmaceutical composition of the present invention can be administered to a subject through various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell.

In the present invention, the term "object" includes, but is not limited to, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig , Preferably a mammal, more preferably a human.

The present invention also provides a method for the prevention of diabetes comprising N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- Or health functional food for improvement.

The diabetes is preferably type 1 diabetes or type 2 diabetes.

The health functional food containing the compound of the present invention includes, for example, various foods, beverages, gums, tea, and vitamin complex. It may also be added to foods or beverages for the purpose of preventing diabetes. At this time, the amount of the compound in the food or beverage may be 0.01 to 15% by weight of the whole food, and the beverage may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml.

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids.

The health functional food of the present invention is not particularly limited to ingredients other than those containing the above-mentioned desirable ratio of the above-mentioned compounds, and may contain various flavors or natural carbohydrates such as ordinary foods or beverages as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors and synthetic flavors can also be advantageously used as flavors other than those described above. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the compound of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compounds of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples.

[ Example ]

1. Materials and Methods

1.1. Cell culture

INS-1 cells, a mouse insulinoma cell line, were cultured in RPMI 1640 medium (Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum, 1 mM pyruvate, 10 mM HEPES, 50 mM 2-mercaptoethanol, 100 units / mL penicillin, and 100 μg / mL streptomycin. The incubation conditions were 5% CO ₂ and 37 ° C, and INS-1 cells used in this experiment had a passage number between 20 and 30.

1.2. INS -1 for cell proliferation GNF -5 cytotoxicity assay

The effect of GNF-5 on INS-1 cell growth was examined using the WST-1 cell viability assay kit (Dojindo Laboratories, Kumamoto, Japan).

1.3. Western Blot  Method ( Western blot 분석 )

Was washed three times with 4 x 10 ⁶ cultured cells with ice-cold PBS and then, the cell lysis buffer (lysis buffer: 137 mM NaCl, 15 mM EGTA, 1 mM sodium orthovanadate, 15 mM MgCl 2, 0.1% Triton X- 100, 25 mM PMSF, 2.5 μg / ml proteinase inhibitor E-64, pH 7.2), disrupted with an ultrasonic grinder, reacted at 4 ° C for 30 minutes and centrifuged at 14,000 rpm for 20 minutes to recover the supernatant. The same amount of protein was separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein was electrotransferred to Immobilon-P membrane. The membrane was incubated with a blocking solution (TBS solution containing 3% nonfat milk and 0.1% Tween 20) for 1 hour to block non-specific binding of the antibody. The antibody to the protein to be identified was added thereto for 1 to 2 hours Lt; / RTI > Followed by washing with TBST solution containing 0.1% Tween 20 three times for 10 minutes each, and then reacted with a secondary antibody. After the reaction with the ECL system, proteins were identified on an X-ray film (Kodak, USA).

1.4. Mitochondrial membrane potential  Measurement method

FACS analysis was performed as follows. The cells to be analyzed were allowed to stand in 67% ethanol at 4 ° C for 1 hour to fix the cells. Fixed cells (1 × 10 ⁶ ) were washed twice with PBS solution and suspended in 250 ml of a solution of 12.5 mg of RNase (contained in 1.12% sodium citrate buffer (pH 8.45)) and reacted at 37 ° C for 30 minutes. RNase-treated cells were treated with 250 ml of propidium iodide (PI, 50 mg / ml) for 20 min. The stained cells were analyzed by FACScan flow cytometer for relative amount of DNA.

1.5. Mitochondrial cytochrome  c release  black

The cytotoxic effect of GNF-5 on streptozotocin treatment in INS-1 cells was assayed by mitochondrial cytochrome c release inhibition. Cells treated with the control, streptozotocin treatment, streptozotocin + GNF-5, and streptozotocin + Imatinib were washed twice with cold PBS, suspended in CE1 buffer (Qproteome cell compartment kit (Qiagen) After the reaction was completed, the supernatant was centrifuged at 1,000 g (4 ° C) for 10 minutes, and the supernatant was collected into the cytoplasmic fraction to confirm the presence of mitochondrial cytochrome c release.

1.6. In vivo  Experiment of pancreatic beta cell protection activity assay

In the animal study, diabetic rats were challenged with a single dose of streptozotocin (150 mg / kg) in a group fed with GNF-5 (60 mg / kg) and a group not fed with C57BL / 6 The protective activity of beta cell islets was measured by immunohistochemistry.

1.7. Immunostaining of pancreatic tissue

PBS, GNF-5 and Imatinib were administered at a dose of 150 mg / kg at 2 hours after the first day of gavage with 60 mg / kg / day. And then gavage at 60 mg / kg / day for 13 days. For immunohistochemical staining, pancreas was isolated from C57BL / 6 mouse, fixed (10% formalin solution in PBS), and blocked with Paraffin. Pancreatic section Paraffin block paraffin was removed and reacted with anti-insulin (H86) (Santa Cruz Biotechnology, USA) antibody at 4 ° C overnight. Antibody complexes were detected by streptavidin-peroxidase reaction kit and 3,3'-diaminobenzidine from Chromogen (ABC kit, Vector Labs, Burlingame, California, USA). H & E staining was performed with tissue quality control.

1.8. Statistical processing

The data of this experiment were repeated at least 3 times and the data were expressed as mean ± standard deviation. Statistical analysis was performed with Student's t- test. p <0.05 was considered statistically significant.

2. Results and discussion

2.1. GNF -5 rat pancreatic  β- cell  Protective action

GNF-5 is an allosteric Bcr-Abl kinase inhibitor, unlike Imatinib, Nilotinib and Dasatinib, which are ATP-noncompetitive. The anti-diabetic activity of GNF-5 was assayed using rat pancreatic β-cell line (Ins-1) cells. First, the cytotoxicity of GNF-5 to INS-1 cells was measured. As shown in Fig. 1, even when GNF-5 was treated at a concentration of 20 μM for 48 hours, it did not affect cell proliferation at all A). In contrast, cell death induced by treatment with 1 mM of streptozotocin was 50% or more protected at a concentration of 10 μM of GNF-5 (FIG. 1B).

2.2. GNF -5 ROS ( reactive oxygen species Influence on generation

The active oxygen is generated through the metabolism of the living body. The active oxygen is unstable and acts on the DNA and proteins of the surrounding cells to induce degeneration or denaturation, causing cell damage and inducing apoptosis. Streptozotocin is a powerful DNA alkylating agent and is known to act as a free radical donor in the pancreas (Larsen and Grude, 1978; Newsholme et al., 2007). In other words, the diabetes model induced by streptozotocin treatment causes ROS generation by streptozotocin, and ROS produced by insulin attacks pancreatic β-cell to induce β-cell apoptosis, resulting in insulin deficiency, leading to diabetes . Therefore, whether the cytoprotective activity of GNF-5, which protects INS-1 cells from streptozotocin, is related to inhibition of ROS production induced by streptozotocin is shown by the inhibitory effect on DCF production, a fluorescent form of DHCF-DA Respectively. As shown in FIG. 2A, when ROS produced by streptozotocin treatment was 100%, it was confirmed that ROS generation was inhibited by 78.4% when pretreating GNF-5 for 1 hour and treating streptozotocin. The inhibition of ROS formation by GNF-5 was confirmed by PI and DAPI staining (Fig. 2B), which correlated with inhibition of DNA fragmentation, and it was confirmed by DiO6 staining that the mitochondrial membrane potential of the cells was also inhibited 2 of C).

2.3. GNF -5 stress - activated protein kinase ( SAPK )

Apoptosis is a mechanism by which cell death is caused by cell shrinkage, mitochondrial depolarization, proteolytic degradation, chromosome condensation, and DNA fragmentation following exposure to stress from the outside of the cell. The mitogen-activated protein kinase family (MAPK) and protein kinase C (PKC) are known to be apoptotic signal transduction systems. Bcr-Abl phosphorylation is known to induce apoptosis by activating stress-activated protein kinase (JNK and p38MAP-kinases), activating caspase-9, and inhibiting the phosphorylation of nonreceptor protein kinase c-Abl Imatinib has also been reported to inhibit JNK phosphorylation in stress-activated protein kinases, thereby protecting against apoptosis of pancreatic β-cells and thus possessing anti-diabetic activity (Hagerkvist et al., 2006). The effect of ATP-independent Bcr-Abl kinase inhibitor GNF-5 on the JNK pathway was investigated. As shown in FIG. 3, treatment of INS-1 cells with 1 mM of streptozotocin (STZ) for 8 hours resulted in almost no change in total JNK-1 and JNK-2, but phosphorylation of JNK-1 and -2 was rapidly induced . However, activation of JNK was significantly suppressed by pretreatment with GNF-5 and JNK inhibitor SP600125 at 10 μM for 1 hour. These results suggest that GNF-5 also inhibits JNK phosphorylation and inhibits apoptosis in pancreatic beta cells, like Imatinib.

2.4. GNF -5 protein kinase  C delta  Impact on activation

When a cell receives genotoxic stress, cell survival and death are determined according to the degree of damage, and it is recovered or removed through cell death. Although the complete mechanism of apoptotic cell death is unknown, the protein kinase C δ isoform is activated by phosphorylation of c-Abl, followed by PKCδ cleavage by activated caspase-3, which induces apoptosis (Ghayur et al., 1996). Therefore, as shown in FIG. 4, cleaved PKCδ and caspase-3 were activated when treated with streptozotocin 1 mM for 8 hours as shown in FIG. 4 as a result of the changes of PKCδ cleavage and caspase-3 upon treatment with streptozotocin in INS-1 cells, Their activation was inhibited by GNF-5 treatment at 10 μM.

In summary, GNF-5 inhibits JNK phosphorylation and PKCδ activation in INS-1 cells by oxidative stress induced by streptozotocin treatment, suggesting that GNF-5 inhibits pancreatic β- It was confirmed that it suppressed the death.

2.5. In vivo  Through experiments GNF By -5 Anti-diabetic  Active black

In In vitro experiments showed that GNF-5 inhibited JNK activation, PKCδ and caspase-3 activation on genotoxic stress induced by streptozotocin, thereby inhibiting INS-1 cell apoptosis. Thus, the anti-diabetic activity in connection with this oxidative stress protective activity of the same pancreatic β- cells using C57BL / 6J mice in vivo experiments were performed.

First, GNF-5 (60 mg / kg) dissolved in PBS was gavaged in C57BL / 6J mice and streptozotocin was administered at 150 mg / kg 2 hours after gavage to induce diabetes. Anti-diabetic activities were compared by using the same method of replacing GNF-5 with the same amount of PBS and Imatinib gavage group as negative and positive control, respectively.

The same amount of PBS, GNF-5 and Iamtinib was gavaged once daily for 14 days after the first gavage. On day 14, the pancreatic islet section of each mouse was separated and stained with hematoxylin-eosin (H & E) (Fig. 5A) and the destruction of pancreatic islets by streptozotocin and the protective activity of GNF- Immunochemical staining.

As shown in FIG. 5B, it was confirmed that the GNF-5-treated group was protected from beta cell destruction by streptozotocin treatment, whereas Vehicle + streptozotocin was severely damaged by pancreatic islets.

In summary, GNF-5, an ATP noncompetitive Bcr-Abl kinase, inhibits the phosphorylation and PKCδ activation induced by oxidative stress and inhibits apoptosis of pancreatic β-cells. The protective effect of pancreatic beta -cells against apoptosis has been shown to exert an excellent effect on diabetes mellitus induced by streptozotocin treatment, i.e. type 1 diabetes mellitus.

Claims (9)

A pharmaceutical composition comprising as an active ingredient N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin-4-yl) benzamide or a pharmaceutically acceptable salt thereof A pharmaceutical composition for the prevention or treatment of type 1 diabetes. delete 2. The method of claim 1 wherein said N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- Wherein the salt inhibits apoptosis of pancreatic? -Cells. 2. The method of claim 1 wherein said N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- Wherein the salt inhibits the production of reactive oxygen species (ROS). 2. The method of claim 1 wherein said N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- Wherein the salt inhibits JNK phosphorylation. 2. The method of claim 1 wherein said N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- Wherein the salt inhibits the activation of PKC [delta]. 2. The method of claim 1 wherein said N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- Wherein the salt inhibits the activation of &lt; RTI ID = 0.0 &gt; caaspase-3. &Lt; / RTI &gt; Prevention of Type 1 diabetes including N- (2-hydroxyethyl) -3- (6- (4- (trifluoromethoxy) phenylamino) pyrimidin- Or health functional food for improvement. delete

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