KR20190141007A - Novel use of 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivative - Google Patents

Abstract

The present invention provides for the use of pancreatic beta cell protection of 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivative. The 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivatives according to the present invention cause apoptosis of pancreatic beta cells due to excitatory reactions of the conventionally developed diabetes treatments, thereby preventing insulin secretion from being regulated. Resolve side effects that can cause secondary failures. 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivative according to the present invention promotes insulin secretion only when glucose concentration is increased, thereby protecting pancreatic beta cells from apoptosis and pancreatic beta cells. Restores insulin secretion function. Thus, the 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivatives according to the invention not only when administered as the first choice of diabetes, but also cause secondary failure as the first choice of diabetes. When other drugs are used to administer pancreatic beta cells to patients with impaired pancreatic beta cell protection. In addition, it restores the insulin secretion function of pancreatic beta cells, and consequently, is useful for treating type II diabetes in patients whose blood glucose is not normally controlled by insulin secretagogues.

Description

Novel use of 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivative

The present invention relates to novel uses of 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivatives. Specifically, the present invention is directed to the pancreatic beta cell protection of 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivative and to the treatment of type II diabetes in patients whose blood glucose is not normally controlled by insulin secretagogues. It is about.

Sulfonylurea used in type II diabetes binds to ATP-sensitive K ⁺ channels or SUR-1 receptors on pancreatic beta cell membranes, depolarizes the cell membranes to increase Ca ²⁺ uptake, and due to increased intracellular Ca ²⁺ Promotes insulin secretion

However, insulin secretagogues, such as sulfonylureas, have side effects upon long term administration. Since sulfonylureas secrete insulin regardless of blood glucose levels, they are damaged by beta cell hyperexcitation, and excittotoxic reactions induce beta cell apoptosis. . Eventually, a decrease in the amount of beta cells results in a decrease in insulin secretion, leading to impaired blood glucose control.

The secondary failure of these sulfonylurea drugs is a major clinical difficulty in the treatment of diabetes mellitus, and thus, there is a need for the development of new drugs capable of secreting glucose-dependent insulin with low risk of these side effects.

Recently, free fatty acid receptor 1 (FFAR1) / G-protein coupled receptor (GPR40) has been shown to improve glucose control by glucose-stimulated insulin secretion (GSIS) in pancreatic beta cells. Turned out to be. Applicants have studied activators for GPR40 in this regard, wherein 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivatives, pharmaceutically acceptable salts thereof or optical isomers thereof are active against GPR40. It has been revealed that the increase in intracellular calcium concentration, and shows an excellent hypoglycemic effect (Patent Document 1: 10-2014-0126248 A).

The present invention provides the use of 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivatives for the protection of pancreatic beta cells and the treatment of type II diabetes in patients whose blood glucose is not normally controlled by insulin secretagogues. .

In the course of clinical studies of 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivatives, the inventors found that the substance exhibits an excellent effect on the protection of pancreatic beta cells as well as hypoglycemic effects. Discovered and completed the present invention.

3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivative according to the present invention means a compound of formula 1, an optical isomer thereof or a pharmaceutically acceptable salt thereof.

[Formula 1]

The compound of formula 1 is (3S) -3- (4- (3- (1,4-dioxaspiro [4,5] dec-7-en-8-yl) benzyloxy) phenyl) hex-4- Inoic acid.

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt.

For example, bases can be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg silver nitrate).

Furthermore, pharmaceutically acceptable salts can be prepared using amino acids having amino groups attached to organic acids, and examples of the amino acid salts include natural amino acids such as glycine, alanine, phenylalanine, valine, lysine and glutamic acid. It is pharmaceutically suitable to prepare, most preferably it is pharmaceutically suitable to prepare L-lysine.

In addition, the compound according to the present invention includes not only the compound of Formula 1 and pharmaceutically acceptable salts thereof, but also solvates, optical isomers, hydrates, and the like that can be prepared therefrom.

The present invention provides for the use of pancreatic beta cell protection of a compound of formula (1), its optical isomers or pharmaceutically acceptable salts thereof, and the insulin secretagogue of the compound of formula (1), its optical isomers or pharmaceutically acceptable salts thereof. It provides a use for treating type II diabetes in a patient who does not.

Through the following examples, the present inventors first confirmed that the compound of Formula 1 has a good action on human GPR40 through the in vitro experiment, and is a drug having excellent insulin secretion ability by glucose stimulation (Example 1 ). Oral Glucose Tolerance test (OGTT) and glucose-stimulated insulin secretion (GSIS) tests were also performed on GK (Goto-Kakizaki) rats with type II diabetes with secondary failure and apoptosis of pancreatic beta cells. Was performed (Examples 2 and 3). As a result, the compound of Formula 1 maintains excellent glycemic control and insulin secretion ability even after prolonged repeated administration through the protection of pancreatic beta cells in GK rats with already damaged pancreas, while frequently causing secondary failure in diabetes treatment. Glymepyride, a sulfonylurea-based drug, confirmed that the beta cell function was reduced (insulin secretion decreased), thereby confirming that the compound of Formula 1 has a protective effect on pancreatic beta cells, and from immunohistochemical staining of rat pancreatic tissue. This effect was confirmed again (Example 4).

GK rats are an animal model in which the pancreas is already injured. However, repeated administration of the compound of formula 1 according to the present invention cannot increase the amount of pancreatic beta cells that have already been damaged, but the addition of pancreatic islets It prevents damage, preserves it, and enhances pancreatic beta-cell functionality.

The inventors of the present invention tried to confirm whether the pancreatic beta-cell protective effect of the compound of Formula 1 was shown to be the same in Takeda's TAK-875 (Patent Document 2: WO2008-001931), which is known as a GPR40 activator. Secretion control capacity was evaluated (Example 6). As a result, although the compound of Formula 1 was administered at a low dose, the insulin secretion ability was superior to that of the same GPR40 activator TAK-875. Since abnormality of insulin secretion capacity is caused by damage of pancreatic beta cells, this result shows that GPR40 activator does not prevent pancreatic beta cell damage, that is, secondary failure, by repeated administration. The pancreatic beta cell protective ability exhibited by the compound of formula 1 means that it is an unexpected and beneficial effect that only the compound of the invention exhibits.

Based on the results of previous experiments on GK rats with already damaged pancreas, the present inventors have found that additional pancreatic beta cells of patients already on the diabetic agent whose pancreatic beta cell protective effect of the compound of formula 1 causes secondary failures such as glymepiride The aim of this study was to determine whether it can prevent the damage and restore the functionality of pancreatic beta cells.

Therefore, we evaluated the protective effect of pancreatic beta cells after repeated administration of the drug in Wistar die-induced obesity (DIO) rats (Examples 7 and 8). As a result, after 4 weeks of repeated administration of glymepiride, It was also confirmed that even in the test group in which the test drug was changed to a compound and additionally repeatedly administered, the pancreatic beta cells were protected from further damage and the pancreatic beta cells were able to restore the insulin secretion ability.

This pancreatic beta cell protection of the compound of Formula 1 prevents damage to the pancreatic beta cells, a secondary failure, which is a side effect in patients with type II diabetes who take long-term diabetes treatments to stimulate insulin secretion, and prevents pancreatic beta cells from functioning. It helps to exercise. Therefore, when the compound of Formula 1 is used as a primary therapeutic agent for type II diabetes, it can provide a sustained and excellent hypoglycemic effect and insulin secretion without damaging pancreatic beta cells.

In addition, when administered to patients with type II diabetes who have already suffered a second type of diabetes treatment, the pancreatic beta cells not only prevent further damage but also restore the insulin secretion function of the pancreatic beta cells. Particularly useful for the treatment of type II diabetes in patients whose blood glucose is not normally controlled.

Accordingly, the present invention provides a pharmaceutical composition for pancreatic beta cell protection, comprising a compound of formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient, a compound of formula 1 for preparation of a pharmaceutical composition for protecting pancreatic beta cells, A method of protecting pancreatic beta cells comprising the use of an optical isomer thereof or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, is administered to a subject. .

In one embodiment, the present invention provides a pharmaceutical composition for protecting pancreatic beta cells in a type II diabetic patient comprising a compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. In this embodiment, type II diabetic patients include all patients who are at risk of pancreatic beta cell damage or are likely to damage pancreatic beta cells.

In another embodiment, the pharmaceutical composition for protecting pancreatic beta cells according to the present invention may be administered alone or in combination with other drugs for the treatment of type II diabetes. That is, the pharmaceutical composition for protecting pancreatic beta cells according to the present invention may be used as a single therapeutic agent, but may also be used in combination with another type II diabetes therapeutic agent. There is no particular limitation on the type of drugs that can be administered in combination, and any drug used as a known type II diabetes treatment can be exemplified. Known type II diabetes therapeutics include, for example, oral hypoglycemic agents such as biguanides such as metformin, buformin, phenformin or derivatives thereof. Or alpha-glucosidase inhibitors such as acarbose, voglibose, sulfonylureas that act as insulin secretagogues, and meglitinide drugs. Include. The combination administration includes both sequential or simultaneous administration. In the embodiment of the present invention, since pancreatic cell protection was also confirmed in the group II diabetes-treated group, in which secondary failure was already induced, it is expected that the pancreatic beta cells may be prevented from being damaged due to the type II diabetes-administered combination.

In another embodiment, the present invention provides a pharmaceutical composition for pancreatic beta cell protection in patients with type II diabetes who has injured pancreatic beta cells containing the compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. to provide.

The cause and extent of the damage to the pancreatic beta cells of "type II diabetic patients with pancreatic beta cell damage" are not particularly limited. If the blood sugar is high, but the insulin is specifically lower than normal, blood glucose control is not controlled, which means that the pancreatic beta cells responsible for insulin secretion do not play a role, which is determined to be beta cell damage. Therefore, beta cells by confirming the function of pancreatic beta cells using a method of confirming insulin secretion (eg, GSIS, ASIS, etc.), or a method of confirming insulin synthesis (eg, method of confirming insulin content in the pancreas). You can verify whether the

The damage of pancreatic beta cells is accelerated according to the progression of type II diabetes, and the use of anti-diabetic agents that promote the secretion of insulin indiscriminately regardless of glucose concentration may be a major cause of pancreatic beta cell damage. Examples of insulin secretagogues having a mechanism of action that promotes insulin secretion in pancreatic beta cells include sulfonylurea-based drugs and meglitinide-based drugs. Representative examples of sulfonylurea-based drugs include glymepiride, glyclazide, glybenclamide, glipizide, and the like. nateglinide) and repaglinide.

In one embodiment of the present invention, a type II diabetic patient with pancreatic beta cell damage may be a patient receiving an insulin secretagogue, such as a sulfonylurea drug, and / or a meglitinide drug.

In addition, the present invention is an insulin secretagogue comprising the compound of formula 1, its optical isomer or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutical composition for treating type II diabetes in a patient in whom blood glucose is not normally controlled, insulin Use of a compound of formula 1, an optical isomer thereof or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of a compound of formula 1 for the preparation of a pharmaceutical composition for treating type II diabetes in a patient in which blood glucose is not normally controlled by a secretagogue The present invention provides a method for treating type II diabetes, comprising administering an optical isomer thereof or a pharmaceutically acceptable salt thereof to a patient whose blood glucose is not normally controlled with an insulin secretagogue.

A patient whose blood glucose is not normally controlled by the insulin secretagogue may be a patient receiving the insulin secretagogue. As described above, patients receiving insulin secretagogues may be damaged in the pancreatic beta cells upon prolonged administration.

In addition, a patient whose blood glucose is not normally regulated by an insulin secretagogue may be a patient who is co-administered with an insulin secretagogue and another hypoglycemic agent.

Clinically, patients with type II diabetes are given a biguanide drug as the first choice, and if the blood glucose control is not adequately controlled by the biguanide drug, they are given an insulin secretagogue or an insulin secretion. Often, accelerators and biguanide-based drugs are used in combination. Patients who received insulin-promoting agents, either alone or in combination with insulin-promoting agents and biguanide-based drugs, were unable to control their blood sugar normally due to pancreatic beta-cell damage. Is likely to be.

In one embodiment, the insulin secretagogue may be a sulfonylurea drug, and / or a meglitinide drug.

In addition, the hypoglycemic agent administered in combination with the insulin secretagogue may be a biguanide drug.

A pharmaceutical composition according to the invention comprising a compound of formula 1, an optical isomer thereof or a pharmaceutically acceptable salt thereof, may be administered alone or in combination with other drugs for the treatment of type II diabetes. That is, it may be used as a single therapeutic agent according to the present invention, but may also be used in combination administration with other types of diabetes treatment agents. The kind of drug that can be administered in combination is not particularly limited, and any drug used as a known type II diabetes treatment agent may be exemplified. Known type II diabetes therapies include, for example, oral hypoglycemic agents, for example, biguanide-based drugs such as metformin, buformin, phenformin or derivatives thereof, or alpha glucosidase such as acarbose, bogglibose and the like. Sulfonylurea-based drugs that act as inhibitors, insulin secretagogues, meglitinide-based drugs, and the like. The combination administration includes both sequential or simultaneous administration.

In particular, the pharmaceutical composition according to the present invention may be administered in combination with a biguanide drug.

For example, in patients with type II diabetes who take sulfonylurea-based drugs as the first agent biguanide drug and the second agent, when the glycemic control is poor and a change in the second agent drug is necessary, sulfonylurea-based drugs are required. By co-administering the pharmaceutical composition according to the present invention instead of a drug with a biguanide drug, type II diabetes can be treated in a patient whose blood glucose is not normally controlled by an insulin secretagogue.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, it may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

Formulations of the pharmaceutical compositions and pharmaceutically acceptable carriers can be appropriately selected according to techniques known in the art, for example, see Urquhart et al., Lancet, 16: 367, 1980. ]; Lieberman et al., PHARMACEUTICAL DOSAGE FORMS-DISPERSE SYSTEMS, 2nd ed., Vol. 3, 1998; Ansel et al., PHARMACEUTICAL DOSAGE FORMS & DRUG DELIVERY SYSTEMS, 7th ed., 2000; Martindale, THE EXTRA PHARMACOPEIA, 31 st ed .; Remington's PHARMACEUTICAL SCIENCES, 16th-20th editions; THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Goodman and Gilman, eds., 9th ed., 1996; Wilson and Gisvolds' TEXTBOOK OF ORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers, eds., 10th ed., 1998. In addition, the principles for formulating pharmaceutical compositions are described, for example, in Platt, Clin Lab Med, 7: 289-99, 1987; Aulton, PHARMACEUTICS: THE SCIENCE OF DOSAGE FORM DESIGN, Churchill Livingstone, NY, 1988; [EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP, 1998], "Drug Dosage," J Kans Med Soc, 70 (1): 30-32, 1969, and the like.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage may be based on the condition and weight of the patient, and the degree of disease. Depending on the drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

An effective amount of the active ingredient of the pharmaceutical composition of the present invention generally means an amount required to achieve treatment of a disease. In the present invention, the use of the compound of formula 1 is for the protection of pancreatic beta cells or the treatment of type II diabetes in patients whose blood glucose is not normally regulated by insulin secretagogues, so that the pharmaceutical composition of the present invention An effective amount of an active ingredient means an amount required to protect pancreatic beta cells, or an amount required to control blood glucose to a normal range in type II diabetes patients whose blood glucose is not normally controlled by an insulin secretagogue. Effective dose levels are based on factors such as the type of disease, severity, activity of the drug, sensitivity to the drug, time of administration, route and route of administration, duration of treatment, factors including concomitant medication, and other well-known factors in the medical field. Can be determined accordingly. The pharmaceutical 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 administered as a 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.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary according to the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general, Although not limited, the compound of Formula 1 may be administered from 0.001 mg to 200 mg, preferably from 0.01 mg to 100 mg, more preferably from 0.2 mg to 50 mg per kg of body weight, once or several times daily. However, since the dose may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention by any method.

As used herein, "subject" refers to a subject in need of treatment for a disease, and more specifically, a human or non-human primate, mouse, dog, cat, Mean mammals such as horses and cattle.

The 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivatives according to the present invention cause apoptosis of pancreatic beta cells due to excitatory reactions of the conventionally developed diabetes treatments, thereby preventing insulin secretion from being regulated. Resolve side effects that can cause secondary failures. 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivative according to the present invention promotes insulin secretion only when glucose concentration is increased, thereby protecting pancreatic beta cells from apoptosis and pancreatic beta cells. Restores insulin secretion function. Thus, the 3- (4- (benzyloxy) phenyl) hex-4-inoic acid derivatives according to the invention not only when administered as the first choice of diabetes, but also cause secondary failure as the first choice of diabetes. When other drugs are used to administer pancreatic beta cells to patients with impaired pancreatic beta cell protection. In addition, it restores the insulin secretion function of pancreatic beta cells, and consequently, is useful for treating type II diabetes in patients whose blood glucose is not normally controlled by insulin secretagogues.

1A shows the results of measuring the action activity of Compound A on human GPR40 in vitro.
Figure 1B shows the result of confirming insulin secretion capacity (GSIS) by the glucose stimulation of Compound A.
2A and 2B show oral glucose tolerance test (OGTT) results on day 1 of administration, and FIGS. 2C and 2D show oral glucose tolerance test (OGTT) results on week 12 of administration. 2A and 2C show glucose levels and FIGS. 2B and 2D show glucose AUC reduction levels.
3A, 3B, and 3C show the results of oral glucose tolerance test (OGTT) and blood insulin concentration measurement on day 1 of administration, and FIGS. 3D, 3E and 3F show the results of oral glucose tolerance test (OGTT) and blood insulin concentration measurement on week 4 of administration. 3A and 3D show glucose levels, FIGS. 3B and 3E show glucose AUC reduction levels, and FIGS. 3C and 3F show Insulin secretion index (SI).
FIG. 4A shows the islet form immunostained with anti-insulin antibody in 12-week-old GK rats after 4 weeks of repeated administration, and FIG. 4B is islet form immunostained with anti-insulin antibody in 40-week-old GK rats after 14 weeks of repeated administration. Shows.
FIG. 5 is a graph representing the insulin secretion index (SI) of the test compound Compound A and the control TAK-875 administered group at a ratio compared to the vehicle administered group. FIG.
Figure 6 shows the results of measuring insulin secretion capacity in the fasting state after repeated drug administration in GK rats.
7 is a graph showing the glucose AUC reduction level as a result of oral glucose tolerance test (OGTT) of the four-week administration group.
8 is a graph showing glucose AUC reduction levels as a result of oral glucose tolerance test (OGTT) of the 8-week administration group.
9 and 10 are graphs showing the insulin secretion index (SI) as a result of insulin secretion ability (GSIS) experiments of the 4-week and 8-week administration group, respectively.
FIG. 11 shows islet morphology immunostained with anti-insulin antibody in Wistar rats after 4 weeks of repeated administration of test substance.
Figure 12 shows pancreatic morphology immunostained with anti-insulin antibody in Wistar rats after 10 weeks of repeated administration of the test substance.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

EXAMPLE

Test substance used as an example of the compound of formula 1 according to the present invention is as follows.

Compound A : (3S) -3- (4- (3- (1,4-dioxaspiro [4,5] des-7-en-8-yl) benzyloxy) phenyl) hex-4-ino Iksan L-Lysine Salt

Example 1 Pharmacological Activity Test of Compound A

Example 1-1 1. Activity Activity Test of Compound A on Human GPR40

CHO cells stably expressing human GPR40 were incubated overnight at 37 ° C., 5% CO ₂ . Cells were then incubated in loading buffer (50 ml Ham'F-12 (containing 1% FBS) + 50 ml Fluo-4 solution (Invitrogen, F10471)) at 37 ° C. for 2 hours. After adding various concentrations of Compound A to the cells, the increase in intracellular calcium ion concentration was monitored for 100 seconds with Flexstation3 (Molecular Devices).

The functional activity of Compound A on human GPR40 is expressed by the following formula.

(A / B) × 100 (increased intracellular Ca ²⁺ concentration)

A: cell added Compound A, B: cell added vehicle

As a result, as can be seen in FIG. 1A, the action of Compound A on human GPR40 in vitro was very good (EC ₅₀ = 15 nM).

Example 1-2. Confirmation of Insulin Secretion Capacity (GSIS) by Glucose Stimulation of Compound A

For insulin secretion testing, INS-1 pancreatic cells were placed in 24-well plates of ⁵ × 10 ⁵ cells / well. After 48 hours, cells were harvested in 1 mM glucose-KRB buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH ₂ PO ₄ , 1.16 mM MgCl ₂ , 10 mM HEPES, 2.5 mM CaCl ₂ , 25.5 mM NaHCO ₃ , 0.2% BSA, pH 7. 4) and incubated for 2 hours with 1 mM glucose-KRB buffer for starvation. After starvation, cells were washed twice with 1 mM glucose-KRB buffer and incubated for 1 hour with desired concentrations of Compound A in 3 mM or 16 mM glucose-KRB buffer. Supernatants were collected and clarified by centrifugation, and insulin levels were measured using the insulin ELISA kit (Millipore, EZRMI-13K) (* p <0.05: Student t-test).

As a result, as shown in FIG. 1B, glucose-stimulated insulin secretion was promoted concentration-dependently in INS-1 pancreatic beta cells.

Example 2. Confirmation of pancreatic beta cell protective effect of Compound A_26-week-old GK (Goto-Kakizaki) rat

In order to confirm the pancreatic beta cell protective effect of Compound A, its efficacy was evaluated after repeated administration of Compound A in 26-week-old GK rats.

The experimental animal was a non-obese model, and GK rats having characteristics of type II diabetes such as hyperglycemia, hypoinsulinemia, and normal blood lipid concentration from 2 weeks of age were used. GK rats are known as type II diabetes models with secondary failure and pancreatic beta cell death. Five-week-old males of GK rats were purchased and reared to 26-week-old (old) with ad libitum to induce weight loss of the pancreas and used for experiments. According to the body weight and blood sugar, 8 animals were used only by the individuals whose diabetes incidence was confirmed by randomized complete block design, GK rat, vehicle, Glimepiride group (Glimepiride, 10mg / kg) and Compound A group (1, 3, 10mg / kg) and Wistar rats (26-week old male) to the vehicle group was administered as a normal group (WT), a total of six groups at 5ml / kg for 12 weeks oral administration It was. The solvent used for vehicle and compound A was a 0.5% CMC solution, and the glymepyride administration group used a solvent containing 1% PEG and 1% Tween 80 in 0.25% CMC. Oral glucose tolerance test (OGTT) was performed after 16-18 hours fasting on the 1st day and 12th week of administration. Sixty minutes after the administration of the sample, glucose (4 g / kg) was orally administered at a dose of 5 ml / kg. Blood glucose was measured by puncture of the microvenous at 0, 20, 40, 60 and 120 minutes after fasting blood glucose and glucose administration using a blood glucose meter (G-doctor AGM-4000). The blood glucose level during oral glucose tolerance test (OGTT) was analyzed by one-way ANOVA using the prism statistical program, and then proceeded to Dunnett's test without treatment group. The area under the curve (AUC) was converted into a percentage of blood sugar drop per group on the basis of no treatment. Statistical significance compared to vehicle treatment is indicated as * p <0.05, ** p <0.01 and *** p <0.001.

2A and 2B show oral glucose tolerance test (OGTT) results on day 1 of administration, and FIGS. 2C and 2D show oral glucose tolerance test (OGTT) results on week 12 of administration. 2A and 2C show glucose levels and FIGS. 2B and 2D show glucose AUC reduction levels.

As a result of the experiment, when 26 weeks of repeated administration of Compound A and glymepiride to GK rats for 12 weeks, the group of glymepiride showed a tendency to decrease the hypoglycemic effect, but Compound A did not. Since GK rats are a type II diabetes model with secondary failure and pancreatic beta-cell death, these results suggest that Compound A is capable of regulating blood glucose levels even after prolonged repeated administrations through protection of pancreatic beta cells in GK rats with already damaged pancreas. It suggests that it is maintained.

Example 3. Confirmation of the Pancreatic Beta-cell Protective Effect of Compound A_8-week-old GK Rat

In order to evaluate the pancreatic beta cell protective effect of Compound A, its efficacy was evaluated after repeated administration of Compound A in 8-week-old GK rats.

Six-week-old males of GK rats were purchased and raised up to eight weeks of age (free). According to the body weight and blood glucose, only 8 individuals were found to be inducing diabetes by the ingot method, untreated group (GK rat, Vehicle, Veh.), Glimepiride group (Glimepiride, 10mg / kg) and Compound A group (3, 10mg / kg), Wistar rats (8-week-old males) to the vehicle administered to the normal group (WT), and a total of five groups were administered orally for 5 weeks at 5ml / kg. The solvent used in the vehicle and the compound A was a 0.5% CarboxyMethyl Celluluse (CMC) solution, and the glymepyride administration group used a solvent containing 1% polyethylene glycol (PEG) and 1% Tween 80 in 0.25% CMC. Oral glucose tolerance test (OGTT) and blood insulin levels were measured after 16-18 hours of fasting on the 1st and 4th week of administration. Sixty minutes after the administration of the sample, glucose (4 g / kg) was orally administered at a dose of 5 ml / kg. Blood glucose was measured by taking blood from the tail vein after 0, 30, 60, and 120 minutes of fasting glucose and glucose administration using a blood glucose meter (G-doctor AGM-4000). The blood glucose level during oral glucose tolerance test (OGTT) was analyzed by one-way ANOVA using the prism statistical program, and then proceeded to Dunnett's test without treatment group. The lower area (AUC) value was converted into a percentage of blood sugar drop per group based on the untreated group. Statistical significance compared to vehicle treatment is indicated as * p <0.05, ** p <0.01 and *** p <0.001. On the other hand, about 60 μl of blood was collected through orbital vein collection from GK rats after fasting and 30 minutes after glucose administration to measure blood insulin concentration. Serum was separated by centrifuging the collected blood for 10 minutes at 3,000 rpm (4 ℃) and rat / mouse insulin ELISA kit (Millipore merck) was used. Insulin secretion index (SI) was obtained by dividing insulin concentration 30 minutes after glucose administration by the fasting insulin concentration. (Secretion index (SI) = insulin at 30 min / fasting insulin)

3A, 3B and 3C show oral glucose tolerance test (OGTT) results on day 1 of administration, and FIGS. 3D, 3E and 3F show oral glucose tolerance test (OGTT) results on week 4 of administration. 3A and 3D show glucose levels, FIGS. 3B and 3E show glucose AUC reduction levels, and FIGS. 3C and 3F show the insulin secretion index (SI).

The experimental results showed that after four weeks of repeated administration, the glycepiride group showed a hypoglycemic effect and a decrease in beta cell function (insulin secretion), while Compound A increased blood insulin concentration and maintained blood glucose control ability. This suggests that Compound A has a pancreatic beta cell protective effect.

Example 4. Confirmation of the pancreatic beta-cell protective effect of Compound A (immunohistochemical staining in GK rat pancreatic tissue)

In order to evaluate the pancreatic beta-cell protective effect of Compound A, GK rats used in Examples 2 and 3 were removed from the pancreatic tissue for each test group, placed in a tissue cassette, and fixed in a 10% neutral formalin solution. Make the tissue transparent by inserting it into the automatic paraffin penetrator and infiltrating the paraffin from the tissue embedding device to make a block, cutting it to a thickness of 5μm with a tissue microtomycer, attaching it to the slide, and then performing insulin immunostaining. Observed by.

FIG. 4A is a pancreatic morphology immunostained with anti-insulin antibody in the rats used in Example 3, ie, 12-week-old GK rats after 4 weeks of repeated administration, and FIG. 4B further shows in GK rats used in Example 2 After two more weeks of dosing, 14-weeks of repeated administration, 40-week-old GK rats show pancreatic islet immunostained with anti-insulin antibody.

As can be seen from FIG. 4, the islet of wild-type rats maintains morphology, but GK rats show overall morphological damage. The relative amounts of beta cells in GK rats were not significantly different from 4 and 14 weeks of repeated administration of each drug. The glycemic control effect by the compound A suggests that the effect of the beta cells due to beta cell protection, rather than the effect on the mass (beta) of beta cells.

In summary, the results of Examples 1 to 4 can be summarized as follows. Compound A had excellent activity in vitro and increased glucose-stimulated insulin secretion (GSIS) in INS-1 pancreatic beta cells. Compound A and glimepiride administration in young GK rats improved glycemic control and insulin secretion, while chronic administration of glimepiride caused loss of glycemic control efficacy while Compound A did not. After chronic administration, the glycepiride group had lower glycemic control and insulin secretion during oral glucose tolerance test (OGTT) than the compound A group. In the case of administration of Compound A and glymepiride to old GK rats, Compound A-administered group continued to maintain blood glucose control ability, but not to glymepiride-treated group.

Therefore, repeated administration of Compound A has the effect of preserving pancreatic islets and enhancing beta cell function.

Example 5 Insulin Secretion Evaluation of Compound A

Seven week-old male Sprague Dawley (SD) rats were obtained, followed by a 1-week purifying period, followed by glucose-stimulated insulin secretion (GSIS) testing using only healthy subjects. After 12-16 hours of fasting, randomly separated the group, vehicle (0.5% CMC), test compound A, and control TAK-875 were orally administered at a dose of 10 mg / kg, respectively.

Glucose (2 g / kg) was intraperitoneally administered 60 minutes after vehicle or test substance administration. Twenty minutes later, whole blood was collected through a capillary tube after puncture of the vein, and plasma was separated and measured using a Morinaga Ultra Sensitive Mouse / Rat Insulin ELISA Kit (MIoBS). Insulin secretion index (SI) was calculated by the following equation.

Secretion Index (SI) = insulin at 20 min / insulin at 0 min

The test scores were expressed as mean and standard error (Mean ± SE), and the significance test of the difference between the control group and the experimental group was one-way ANOVA (Dunnett's) of 'GraphPad Prism 4' software (Graphpad co., La Jolla, CA, USA). test) and *** p <0.001 vs. The vehicle was determined to be statistically significant.

FIG. 5 is a graph representing the insulin secretion index of the test compound Compound A and the control TAK-875 administered group in a ratio compared to the vehicle administered group. FIG.

As can be seen in Figure 5, the insulin secretion ability of Compound A was superior to the control TAK-875 in the glucose-stimulated insulin secretion (GSIS) test using SD rats.

Example 6 Evaluation of Insulin Secretion Capacity of Compound A

20-week-old male GK rats received vehicle (0.5% CMC), test compound Compound A (1, 3 mg / kg) and control TAK-875 (10 mg / kg) for 4 weeks. 12 ~ 16 hours before insulin measurement, whole blood was collected through capillary tube after puncture of the vein. Plasma was isolated and insulin measured using a Morinaga Ultra Sensitive Mouse / Rat Insulin ELISA Kit (MIoBS).

The test scores were expressed as mean and standard error (Mean ± SE), and the significance test of the difference between the control group and the experimental group was the t-tests and one-test of 'GraphPad Prism 4' software (Graphpad co., La Jolla, CA, USA). way ANOVA (Dunnett's test), # p <0.05 vs. WT or * p <0.05 vs. The vehicle was determined to be statistically significant.

Figure 6 shows the results of measuring insulin secretion capacity in the fasting state after repeated drug administration in GK rats. Many of the antidiabetic agents exhibit an insulin secretion dysregulation that results in excessive insulin secretion regardless of glucose concentration upon repeated administration. As a result, even though Compound A was administered at a low dose, insulin secretion was superior to TAK-875, the same GPR40 activator. Since abnormalities in insulin secretion capacity are caused by damage to pancreatic beta cells, it can be judged that Compound A has excellent pancreatic beta cell protective ability.

Example 7. Confirmation of the Pancreatic Beta-cell Protective Effect of Compound A_ Wistar DIO Rat

The experimental animals were purchased from Wistar die-induced obesity (DIO) rats and four-week-old females, and the high fat diet (Research diets, D12492) was used for the test after 37 weeks of free feeding. According to the weight and blood sugar, the group was divided into the following table 1 by the ingot method.

4 week dose group Normal group (Wistar rat, Lean) Untreated Group (Wistar DIO Rat, Vehicle) Glimepiride treated group (Glimepiride, 10mg / kg) Compound A administration group (1 mg / kg) 8-week administration group Normal group (Wistar rat, Lean) Untreated Group (Wistar DIO Rat, Vehicle) Glimepiride treated group (Glimepiride, 10mg / kg) Compound A alone group (1 mg / kg) Glimepiride (Glymepiride, 10mg / kg) after 4 weeks of administration of Compound A 4 weeks (1mg / kg)

The 4 week group was divided into normal group (Wistar rat, Lean), untreated group (Wistar DIO rat, Vehicle), glymepiride group (Glimepiride, 10mg / kg) and Compound A group (1mg / kg) at 5ml / kg. Oral administration was given for a week.

The 8-week administration group included the normal group (Wistar rat, Lean), the untreated group (DIO rat, Vehicle), the glimepiride group (Glimepiride, 10mg / kg), the compound A group alone (1mg / kg) and the glimepiride (10mg / kg). After 4 weeks, Compound A was divided into 4 weeks (1mg / kg) and administered orally for 5 weeks at 5ml / kg.

The solvent used for vehicle and Compound A was a 0.5% CMC solution, and the glymepyride administration group used a solvent containing 1% PEG and 1% Tween 80 in 0.25% CMC.

Oral glucose tolerance test (OGTT) and glucose-stimulated insulin secretion (GSIS) tests were performed after 4 and 8 weeks of administration.

In the 4-week oral glucose tolerance test (OGTT), glucose (4 g / kg) was orally administered at a dose of 5 ml / kg 60 minutes after the sample was administered. Blood glucose was measured by blood collected from the orbital vein after 0, 30, 60, and 120 minutes of fasting glucose and glucose administration using a blood glucose meter (ACCU-CHEK active strip, Roche). Blood glucose levels during oral glucose tolerance test (OGTT) were analyzed by one-way ANOVA using the prism statistics program, followed by Dunnett's test or t-test based on the untreated group. The area under the blood glucose curve (AUC) value was converted into a percentage of blood sugar drop per group on the basis of the untreated group.

In the eight-week oral glucose tolerance test (OGTT), glucose (4 g / kg) was orally administered at a dose of 5 ml / kg 60 minutes after the sample was administered. Blood glucose was measured by puncture of the microvein at 0, 20, 40, 60, and 120 minutes after fasting blood glucose and glucose administration standards using an ACCU-CHEK active strip (Roche). Blood glucose levels during oral glucose tolerance test (OGTT) were analyzed by one-way ANOVA using the prism statistics program, followed by Dunnett's test or t-test based on the untreated group. The area under the blood glucose curve (AUC) value was converted into a percentage of blood sugar drop per group on the basis of the no treatment group.

For the glucose-stimulated insulin secretion (GSIS) test, approximately 60 μl of blood was drawn from orbital vein blood collection in DIO rats after 0, 30 and 120 minutes of fasting and glucose administration criteria. Serum was isolated by centrifuging the collected blood for 10 minutes at 3,000 rpm (4 ℃) to measure the blood insulin concentration. Rat / mouse Insulin ELISA kit (Millipore merck) was used. The insulin secretion index (SI) was calculated by dividing the insulin concentration in the fasted state by the insulin concentration 30 minutes after the glucose administration. ('Secretion Index (SI) = insulin at 30 min / fasting insulin')

7 is a graph showing the glucose AUC reduction level as a result of oral glucose tolerance test (OGTT) of the four-week administration group.

After four weeks of repeated administration, the glycepiide-administered group induces a second failure, thereby lowering glycemic control ability, whereas the compound A-administered group has excellent blood glucose control ability. The induction of secondary failure is indicative of damage to pancreatic beta cells, which in turn suggests that Compound A exhibits good glycemic control without damaging pancreatic beta cells.

On the other hand, Figure 8 is an oral glucose tolerance test (OGTT) results of the 8-week administration group, a graph showing the glucose AUC reduction level.

As with the 4-week repeated administration, the glycemicidation group was found to have a secondary failure due to its poor glycemic control. On the other hand, Compound A alone group showed the best glycemic control ability. After 4 weeks of glymepiride (10 mg / kg), Compound A 4 week group (1 mg / kg) showed lower glycemic control ability than compound A alone group, which showed long-term administration of sulfonylureas such as glymepiride When Compound A is administered to a patient with pancreatic beta cell damage, the pancreatic beta cells may be protected from further damage and further restore the pancreatic beta cell insulin secretion potential. This was further confirmed by the following glucose-stimulated insulin secretion (GSIS) data and immunohistochemical staining results.

9 and 10 are graphs showing the insulin secretion index (SI) as a result of glucose-stimulated insulin secretion (GSIS) test of the 4-week and 8-week administration groups, respectively. The compound A administration group showed excellent insulin control ability, whereas the glymepiride administration group showed poor insulin control ability. However, after four weeks of glymepiride (Glimepiride, 10mg / kg) administration of Compound A 4 weeks (1mg / kg) it can be seen that the insulin control ability is restored to a level similar to that of Compound A administration. This may protect against additional damage to pancreatic beta cells by restoring pancreatic beta cells due to long-term administration of sulfonylurea-based drugs and further restore the pancreatic beta cells' insulin secretion potential. The result confirms that

Example 8. Confirmation of pancreatic beta cell protective effect of Compound A (Immunohistochemical staining results in Wistar DIO rat pancreas tissue)

Rats belonging to the four-week administration group tested in Example 4 were sacrificed after the end of the test, and pancreatic tissue was removed for each test group. Placed in a tissue cassette and fixed in 10% neutral formalin solution. The tissue was infiltrated into an automatic paraffin infiltrator and the tissue was infiltrated and paraffin was infiltrated in the tissue trapping device to make a block. The slides were scanned with .Z1 [Carl Zeiss, Germany]. The results were shown at 2.5x using ZEN Imaging Software.

On the other hand, the rats belonging to the 8-week administration group tested in Example 6 was further administered for two weeks under the same conditions, and immunostaining was performed by the same method after a total of 10 weeks of administration.

FIG. 11 shows islet morphology immunostained with anti-insulin antibody in Wistar rats after 4 weeks of repeated administration of test substance. Pancreatic islet cells of wild-type Wistar rats (Lean) retain their morphology, but DIO rats show some morphological damage. The glimepiride group showed overall morphological damage, suggesting a second failure. The compound A administration group showed no significant difference even after 4 weeks of repeated administration.

Figure 12 shows pancreatic morphology immunostained with anti-insulin antibody in Wistar rats after 10 weeks of repeated administration of the test substance. Compound A administration group showed no morphological changes even after repeated administration for 10 weeks. In case of additional administration of Compound A and glymepiride for 6 weeks in DIO rats, which were induced secondary failure after 4 weeks of glymepiride administration, the glymepiride group (glymepiride 10-week group) intensified morphological damage while the compound A group (Glimepiride 10mg) / kg pre-treatment (4w) + Compound A, 1mg / kg (6w)) The degree of damage was suppressed. This may protect against additional damage to pancreatic beta cells by restoring pancreatic beta cells due to long-term administration of sulfonylurea-based drugs and further restore the pancreatic beta cells' insulin secretion potential. The result confirms that

Claims (16)

A pharmaceutical composition for protecting pancreatic beta cells comprising a compound of Formula 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

The method of claim 1,
The pharmaceutical composition for pancreatic beta cell protection pharmaceutical composition for pancreatic beta cell protection in patients with type II diabetes. The method of claim 1,
Wherein the pharmaceutical composition is administered alone or in combination with other drugs for the treatment of type II diabetes pancreatic beta cell protective pharmaceutical composition. The method of claim 1,
The pharmaceutical composition is for pancreatic beta cell protection pharmaceutical composition for pancreatic beta cell protection in patients with type II diabetes suffered from pancreatic beta cells. The method of claim 4, wherein
Type II diabetic patients with pancreatic beta-cell injury are pancreatic beta-cell protective pharmaceutical compositions which are administered insulin secretagogues. The method of claim 4, wherein
Type II diabetic patients with pancreatic beta-cell damage are pancreatic beta cell protective pharmaceutical compositions which are administered in combination with insulin secretagogues and other hypoglycemic agents. The method of claim 5,
Insulin secretagogue is a sulfonylurea-based drug, and / or meglitinide-based drug for pancreatic beta cell protection pharmaceutical composition. The method of claim 6,
The hypoglycemic agent is a biguanide drug
Pharmaceutical composition for protecting pancreatic beta cells. The method according to any one of claims 1 to 8,
Pharmaceutically acceptable salts of compounds of formula 1 are (3S) -3- (4- (3- (1,4-dioxaspiro [4,5] des-7-en-8-yl) benzyloxy ) Phenyl) hex-4-inoic acid L-lysine salt for pancreatic beta cell protection pharmaceutical composition. Comprising a compound of formula (1), its optical isomers or pharmaceutically acceptable salts thereof as an active ingredient,
A pharmaceutical composition for treating type II diabetes in a patient whose blood glucose is not normally controlled by an insulin secretagogue.
[Formula 1]

The method of claim 10,
Patients whose insulin levels are not normally controlled by insulin secretagogues are patients who have received insulin secretagogues.
A pharmaceutical composition for treating type II diabetes in a patient whose blood glucose is not normally controlled by an insulin secretagogue. The method of claim 10,
Patients whose insulin levels are not normally controlled by insulin secretagogues are patients who have been co-administered with insulin secretagogues and other hypoglycemic agents.
A pharmaceutical composition for treating type II diabetes in a patient whose blood glucose is not normally controlled by an insulin secretagogue. The method of claim 10,
Insulin secretagogues are sulfonylurea drugs, and / or meglitinide drugs
A pharmaceutical composition for treating type II diabetes in a patient whose blood glucose is not normally controlled by an insulin secretagogue. The method of claim 12,
The hypoglycemic agent is a biguanide drug
A pharmaceutical composition for treating type II diabetes in a patient whose blood glucose is not normally controlled by an insulin secretagogue. The method of claim 10,
The pharmaceutical composition is administered alone or in combination with other drugs for the treatment of type II diabetes
A pharmaceutical composition for treating type II diabetes in a patient whose blood glucose is not normally controlled by an insulin secretagogue. The method of claim 10,
The pharmaceutical composition is administered in combination with a biguanide drug
A pharmaceutical composition for treating type II diabetes in a patient whose blood glucose is not normally controlled by an insulin secretagogue.

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