WO2020189853A1 - Pharmaceutical composition for prevention or treatment of vesicle stress-related disease comprising myricetin as active ingredient - Google Patents

Abstract

The present invention relates to a composition for treating vesicle stress-related diseases and protecting pancreatic cells by inhibiting a vesicle stress response by myricetin, wherein the myricetin inhibits an activation of cyclin-dependent phosphatase 5 in a cell induced to have vesicle stress, significantly increases the expression of anti-apoptotic proteins, and maintains an insulin secretion function of pancreatic beta cells, and therefore can be used effectively for the treatment of vesicle stress-related diseases and the protection of pancreatic cells.

Description

Pharmaceutical composition for preventing or treating diseases related to endoplasmic reticulum stress containing paroxetine as an active ingredient

The present invention relates to a composition for treating endoplasmic reticulum stress-related diseases and protecting pancreatic cells by inhibiting the endoplasmic reticulum stress response with paroxetine.

The endoplasmic reticulum (ER) is an organelle that is responsible for the transformation and folding of proteins after protein is translated from genes. The endoplasmic reticulum is a structure of a membrane component that looks like branching out of the nuclear membrane. There are two types of vesicles: rough vesicles with ribosomes attached and smooth vesicles without ribosomes. About one-third of intracellular proteins are translated from mRNA to protein in rough vesicles, such as post-translational modifications, such as folding and assembly, glycosylation, and disulfide bonds. Through the process, it becomes a protein structure that takes on an active form. In addition, smooth vesicles are the synthesis site of lipids and steroid hormones and play an important role in regulating the intracellular calcium concentration as a calcium reservoir.

The occurrence of a disorder in the endoplasmic reticulum function is called ER stress, and diabetes is the most frequent disease caused by endoplasmic reticulum stress.

Currently, there are about 2.85 million diabetic patients in Korea (as of 2017, data from the open health and medical system), and the incidence of diabetes complications rapidly increases as the world enters an aging society and the age of diabetes continues to decrease. have. In recent years, the prevalence of diabetes in Korea has exceeded 10%, and the number of diabetic patients is explosively increasing as the onset of diabetes is pulled to young adults and lifespan is extended.

Diabetes is most often caused by a decrease in the secretion of insulin due to a decrease in the function of beta cells in the pancreas and death, and the beta cells in the pancreas are important cells that regulate blood sugar in the body by secreting insulin into the blood. In type 1 and type 2 diabetes, one of the major causes of pancreatic beta cell function decline and death is oxidative stress. In particular, endoplasmic reticulum stress (ER stress) is believed to occupy a large proportion of oxidative stress. Accordingly, it is possible to treat diabetes by reducing oxidative stress accumulated in pancreatic beta cells and protecting beta cells. Accordingly, researches on protecting beta cells in the pancreas from oxidative stress and preventing death are being actively conducted, but among the treatments for diabetes to date, preventing dysfunction and death of beta cells in the pancreas has yet to be confirmed.

The present inventors completed the present invention by confirming that the beta cells of the pancreas induced by endoplasmic reticulum stress were treated with paroxetine, a kind of natural flavonol, to protect the beta cells and to promote the expression and secretion of insulin.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating diseases related to endoplasmic reticulum stress and a composition for protecting or inhibiting death of pancreatic cells by using paroxetine having excellent endoplasmic reticulum stress inhibitory effect.

In order to achieve the above object, an aspect of the present invention provides a pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases, including paroxetine as an active ingredient.

As used herein, the term "myricetin" is a flavonol substance having the structure of the following formula (1), and is known to have a strong antioxidant effect and an anticancer effect.

[Formula 1]

As used herein, the term "ER stress" refers to impaired endoplasmic reticulum function due to the introduction of immature proteins beyond the ability of the endoplasmic reticulum to process due to the physiological or pathological environment or the depletion of calcium within the endoplasmic reticulum. Say what to do. When endoplasmic reticulum stress occurs, cells have a defense mechanism to survive, which is called ER stress response. The induced endoplasmic reticulum stress response restores the function of the endoplasmic reticulum and reduces the accumulation of unfoled proteins. However, if the accumulation of unfolded proteins continues and the endoplasmic reticulum stress is not relieved, the apoptosis pathway is activated, resulting in apoptosis.

In the present invention, the diseases caused by endoplasmic reticulum stress are type 1 diabetes, type 2 diabetes, glutamine multimer-induced aggregation disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis. lateral sclerosis, ALS), Huntington's disease, Kreutzfeldt-Jakob disease, Walcott-Rallison syndrome, Wolfram syndrome, ischemic disease, cardiovascular disease, Neurodegenerative disease, bipolar disorder, arteriosclerosis, inflammation, ischemia, heart disease, liver disease, pancreatic disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and cancer, but may be selected from the group consisting of, but is not limited thereto. Preferably, the endoplasmic reticulum stress-related disease may be type 1 diabetes or type 2 diabetes, and more preferably, diabetes caused by loss of pancreatic beta cells.

Therefore, in the case of controlling endoplasmic reticulum stress, it is expected to be able to prevent and treat the occurrence of diseases related to endoplasmic reticulum stress, and there is a need to develop a target capable of effectively controlling endoplasmic reticulum stress.

As a result of researching this, the present inventors found that paroxetine inhibits the activation of cyclin dependent kinase 5, and anti-cell death proteins Bcl-2 (B-cell lymphoma 2) and Mcl-1 ( The present invention was completed by increasing the expression of myeloid cell leukemia-1) and BCL-XL (B-cell lymphoma-extra large) proteins to suppress the endoplasmic reticulum stress response.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used at the time of formulation, and are lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, silicic acid. Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It is not. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably may be a composition for parenteral administration, particularly intraperitoneal administration.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity. Can be. Meanwhile, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1,000 mg/kg (body weight) per day.

The pharmaceutical composition of the present invention is prepared in a unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the art Alternatively, it may be manufactured by placing it in a multi-volume container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

Another aspect of the present invention uses a composition for protecting pancreatic cells or suppressing apoptosis comprising paroxetine as an active ingredient.

In the present invention, since the composition for protecting or inhibiting death of pancreatic cells uses paroxetine as an active ingredient, contents overlapping with the pharmaceutical composition will be omitted to avoid excessive description of the specification.

In one embodiment of the present invention, the "pancreatic cell protection or apoptosis inhibition" is to protect the pancreatic cells from oxidative stress, more specifically endoplasmic reticulum stress, to maintain and activate the insulin secretion function, and inhibit the death of pancreatic cells. Means that.

In one embodiment of the present invention, the pancreatic cells may be beta cells.

As a result of inducing endoplasmic reticulum stress after pretreatment of INS-1 cells secreting insulin, the present inventors confirmed that expression of anti-apoptotic proteins increased and apoptosis decreased, so that paroxetine protects pancreatic cells. Or it can be usefully used for the purpose of suppressing death.

The paroxetine of the present invention inhibits the activation of cyclin-dependent kinase 5 in cells in which endoplasmic reticulum stress is induced, remarkably increases the expression of anti-apoptotic proteins, and maintains the insulin secretion function of pancreatic beta cells. It can be usefully used for treating diseases and protecting pancreatic cells.

1 is a result of confirming changes in levels of p35, P-CDK5, and CDK5 after treatment with tapsigargin (TG) for a certain period of time in INS-1 cells, which are rat insulinoma cell lines.

FIG. 2 is a result of confirming changes in the levels of P-PERK and P-eIF2α after treating INS-1 cells with tapsigargin (TG) for a certain period of time.

FIG. 3 is a result of confirming changes in the levels of Bcl-2, Mcl-1, and Bcl-XL after treating INS-1 cells with tapsigargin (TG) for a certain period of time.

4 is a result of confirming the intracellular reactive oxygen production level (A), mitochondrial membrane potential change (B), and apoptosis level (C) in INS-1 cells treated with tapsigargin (TG).

5 is a result of confirming changes in levels of p35, P-CDK5, and CDK5 after treatment with tapsigargin (TG) in INS-1 cells pretreated with pre-treatin (Myr).

6 is a result of confirming changes in the levels of Bcl-2, Mcl-1, and Bcl-XL after treatment with tapsigargin (TG) in INS-1 cells pretreated with pretrein (Myr).

7 is a result of confirming the intracellular active oxygen production level (A) and mitochondrial membrane potential change (B) after treatment with tapsigargin (TG) in INS-1 cells pretreated with pretrein (Myr).

FIG. 8 is a result of confirming the change in the level of cytochrome c release and the level of caspase-3 after treatment with tapsigargin (TG) in INS-1 cells pretreated with pre-treatin (Myr).

FIG. 9 is a result of confirming changes in the levels of P-PERK and P-eIF2α after treatment with tapsigargin (TG) on INS-1 cells pretreated with pre-treatin (Myr).

FIG. 10 is a result of confirming the mRNA level of PDX-1 after treatment with tapsigargin (TG) in INS-1 cells pretreated with pre-treatin (Myr).

11 is a result of confirming the degree of apoptosis after treatment with tapsigargin (TG) on INS-1 cells pretreated with pretrein (Myr).

12A is a result of confirming the mRNA level of insulin after treatment with tapsigargin (TG) in INS-1 cells pretreated with pre-treatin (Myr), and FIG. 12B is a medium containing various concentrations of glucose This is the result of measuring the insulin secretion level after culturing INS-1 cells in

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Experimental method

1. Reagents and cell culture

Myricetin, roscovitine, thapsigargin and DiOC ₆ were purchased from Sigma-Aldrich (USA). INS-1 cells, a rat insulinoma cell line, were cultured at 37°C in 5% CO ₂ /95% air using RPMI-1640 medium (Gibco BRL, USA). Real-time reverse transcriptase PCR (RT-PCR) was performed with Light Cycler (Roche, Germany) using the following primers. PDX-1 stands for pancreatic duodenal homeobox-1.

Insulin (forward) (SEQ ID NO: 1): 5'-ACC CAA GTC CCG TCG TGA AGT-3'; And

Insulin (reverse) (SEQ ID NO: 2): 5'-CCA GTT GGT AGA GGG AGC AGA TG-3'

PDX-1 (forward) (SEQ ID NO: 3): 5'-GGC TTA ACC TAA ACG CCA CA-3'

PDX-1 (reverse) (SEQ ID NO: 4): 5'-GGG ACC GTC CAA GTT TGT AA-3'

2. Measurement of intracellular ROS, mitochondrial membrane potential and cell death

Intracellular reactive oxygen species (ROS) levels were measured using 2,7-dichlorodihydrofluorescein diacetate (DCF-DA; Molecular Probes, USA).

The mitochondrial membrane potential was measured as follows. The cells were treated with or without precetin (20 μM) at a concentration of 1 μM with thapsigargin. After the treatment time elapsed, the cells were labeled with DiOC ₆ dye for 5 minutes at 37°C. Thereafter, the cells were washed twice and suspended in PBS, and then confirmed by flow cytometry.

The level of apoptosis was confirmed according to the manufacturer's protocol using the TUNEL In-situ cell death detection kit (Roche, Switzerland), and the cell image was obtained with a fluorescence microscope.

3. Immunoblot analysis

Cell lysates were prepared by treating cells with a lysis buffer containing a protease inhibitor and a phosphatase inhibitor. PDX-1, CASPASE-3, Bcl-2 (B-cell lymphoma 2), BCL-XL (B-cell lymphoma-extra large), phosphorylated JNK (phospho c-Jun N-terminal kinase), p38 MAPK (mitogen -activated protein kinase), ERK (extracellular-signal-regulated kinase), PERK (phospho ERK), and eIF2α (eukaryotic Initiation Factor 2) antibodies were purchased from Cell Signaling Technology (USA). CDK5 (cyclin dependent kinase 5) and p35 antibodies were purchased from Santa Cruz (USA), Mcl-1 antibodies were purchased from LSBio (Seattle, WA, USA), p-p66Shc, p66Shc and actin antibodies were purchased from Abcam (UK). . Antibodies were used at the concentrations suggested by each manufacturer, and proteins were visualized with ECL solution (ECL Plus; Amersham, GE Healthcare Life Sciences, UK).

4. Insulin secretion experiment

The effect of paroxetine on glucose-stimulated insulin secretion (GSIS) in INS-1 cells was confirmed by modifying the method described in Youl et al. (J Pharmacol. 2010; 161:799-814). .

INS-1 cells were pretreated with pre-treatment and incubated for 1 hour (5% CO2, 37°C), and the cells were HEPES-balanced Krebs-Ringer bicarbonate buffer (KRBB) (114 mmol/L NaCl, 4.4 mmol/L KCl, Washed _{twice with} 1.28 mmol/L CaCl ₂ , 1 mmol/L MgSO ₄ , 29.5 mmol/L NaHCO ₃ , 10 mmol/L HEPES, 2.8 mmol/L glucose, and 0.1% BSA, pH 7.4). Thereafter, the cells were cultured in KRBB for 1 hour in the state of preserine treatment or non-treatment with glucose at a base concentration (2.8 mmol/L) or a normal concentration (8.4 mM). At the end of the culture, the supernatant was carefully collected and analyzed with a rat insulin radioimmunoassay (RIA; Linco Research, USA) kit.

The cultured cells were cultured in KRBB for 1 hour in the state of preserine treatment or non-treatment with glucose at a base concentration (2.8 mmol/L) or stimulation concentration (5.6 mmol/L and 11.2 mmol/L). Then, the cAMP Assay Kit (BioVision, USA) was used to confirm the level of cyclic AMP (cAMP).

5. Analysis of results

All experiments were independently repeated at least three times, and the experimental results were described as mean±standard deviation values. As a result of statistical analysis, it was judged that there was statistical significance when p <0.05.

Experiment result

One. Thapsigargin Treatment effect

As a result of treatment of INS-1 cells with tapsigargin (TG), as shown in FIG. 1, expression of p35 protein known as a positive regulator of CDK5 activity was induced, thereby increasing the kinase activity of CDK5 protein, and It was found that the level of tyrosine phosphorylation was increased.

In addition, as shown in FIG. 2, it was confirmed that the expression levels of P-PERK and P-eIF2α, which are endoplasmic reticulum (ER) stress markers, were significantly increased in a time-dependent manner by the treatment of tapsigargin.

According to previous studies, it is known that activation of CDK5 in neurons and pancreatic β-cells contributes to mitochondrial dysfunction and apoptosis. In addition, neurotoxic substances overactivate CDK5 to induce phosphorylation of myeloid cell leukemia-1 (Mcl-1) in nerve cells, resulting in the degradation of Mcl-1. Therefore, the level of Mcl-1 in INS-1 cells treated with tapsigargin was investigated.

As a result, it was confirmed that the expression of anti-apoptotic proteins Mcl-1, Bcl-2, and Bcl-LX was decreased by the treatment of thapsigargin as shown in FIG. 3. In addition, as a result of confirming whether mitochondrial dysfunction caused by inhibition of the expression of anti-apoptotic proteins was confirmed, as shown in FIG. 4, the production of active oxygen in the cells increased (A) by tapsigargin treatment, and as a result, mitochondrial membrane potential ( membrane potential) decreased (B), and apoptosis increased (C).

2. Thapsigargin And Paroxetine Treatment effect

2-1. CDK5 Inhibition of activation

Pretreatment of INS-1 cells with pretrein (Myr) at a concentration of 20 μM was incubated for 3 hours, treated with TAXIGARGIN (TG) and cultured for 1 hour, and then protein expression changes were confirmed. As a result, as shown in FIG. 5, it was found that the effect of increasing the expression of p35 protein induced by tapsigargin (TG) treatment and increasing the phosphorylation level of CDK5 was significantly reduced by the precetin treatment. The effect of paroxetine was similar or slightly lower than that of roscovitin (a CDK5 inhibitor) used as a positive control. The above experimental results indicate that paroxetine inhibits the activation of CDK5, thereby suppressing the damage to β-cells caused by the treatment of thapsigargin.

2-2. Mcl -1 decomposition inhibition

When pre-treatment of INS-1 cells with pre-treatment (Myr), it was confirmed whether there was a change in the effect of reducing the expression of Mcl-1, Bcl-2, and Bcl-LX by tapsigargin treatment. As a result of confirmation, when only tapsigargin was treated in INS-1 cells, the expression levels of Mcl-1, Bcl-2, and Bcl-LX were significantly reduced, but when tapsigargin and paroxetine (Myr) were treated together, the above It was confirmed that the expression reducing effect was reversed (see FIG. 6). In addition, when INS-1 cells were treated with thapsigargin and myricetin (Myr) together, the production of free radicals decreased (Fig. 7A), and the effect of reducing mitochondrial membrane potential, which is evidence of mitochondrial-dependent cell death, is offset. It was found that (Fig. 7B).

2-3. Inhibition of mitochondrial dysfunction

As shown in FIG. 7, when INS-1 cells are treated with only thapsigargin, mitochondrial membrane potential decreases, which activates permeability transition pores, thereby releasing cytochrome c (cytochrome c). And activation of caspase-3. Therefore, the effect of the preserine treatment was confirmed.

As a result, as shown in FIG. 8, when INS-1 cells were treated with thapsigargin and precetin (Myr), the level of cytochrome c released into the cytoplasm from mitochondria significantly decreased, and caspase-3 It was found that the activation level was also significantly decreased.

In addition, as a result of checking the expression levels of P-PERK and P-eIF2α, which are endoplasmic reticulum stress markers, it was found that the expression of P-PERK and P-eIF2α was suppressed to the control level by co-treatment of thapsigargin and myricetin (Myr) Could be (Fig. 9).

The results of this experiment indicate that paroxetine can effectively inhibit the endoplasmic reticulum-mitochondrial stress response.

2-4. PDX Of -1 mRNA Level increase , Inhibit apoptosis

Since PDX-1 functions as an important transcription factor in β-cells, the effect of tapsigargin and precetin treatment was confirmed. As a result, as shown in FIG. 10, when only tapsigargin was treated, the mRNA level of PDX-1 was significantly reduced compared to the control, but when tapsigargin and paroxetine (Myr) were treated together, PDX-1 It was confirmed that the mRNA level of was recovered to a degree similar to that of the control group. In addition, when thapsigargin and myricetin (Myr) were treated together, apoptosis of INS-1 cells was significantly reduced (FIG. 11).

2-5. insulin mRNA Increased levels and secretion

Insulin mRNA level also showed a similar tendency to PDX-1, so when only tapsigargin was treated, insulin expression was significantly reduced, but it was found that when treated with paroxetine (Myr), it recovered to a normal level (Fig. 12). Of A).

In addition, as a result of culturing INS-1 cells in a medium containing various concentrations of glucose and then treatment with paroxetine, it was found that insulin secretion was increased depending on the treatment concentration of paroxetine (FIG. 12B). This result means that paroxetine not only inhibits the endoplasmic reticulum stress response induced by tapsigargin, but also promotes insulin secretion even in a non-stress situation.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (7)

1. A pharmaceutical composition for the prevention or treatment of diseases related to endoplasmic reticulum stress, comprising paroxetine as an active ingredient.
2. The method of claim 1, wherein the endoplasmic reticulum stress-related disease is type 1 diabetes, type 2 diabetes, polyglutamine-induced aggregation disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis. (amyotrophic lateral sclerosis, ALS), Huntington's disease, Kreutzfeldt-Jakob disease, Walcott-Rallison syndrome, Wolfram syndrome, ischemic disease, cardiovascular Diseases, neurodegenerative diseases, bipolar disorder, arteriosclerosis, inflammation, ischemia, heart disease, liver disease, pancreatic disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and cancer. Pharmaceutical composition for prophylaxis or treatment.
3. The pharmaceutical composition for preventing or treating endoplasmic reticulum stress-related diseases according to claim 2, wherein the endoplasmic reticulum stress-related disease is type 1 diabetes or type 2 diabetes.
4. The method of claim 1, wherein the paroxetine inhibits the activation of cyclin dependent kinase 5, and anti-apoptotic proteins Bcl-2 (B-cell lymphoma 2), Mcl-1 (myeloid cell). leukemia-1) and BCL-XL (B-cell lymphoma-extra large) to increase the expression of the protein, a pharmaceutical composition for the prevention or treatment of endoplasmic reticulum stress-related diseases.
5. A composition for protecting or inhibiting death of pancreatic cells comprising paroxetine as an active ingredient.
6. The composition of claim 5, wherein the paroxetine protects pancreatic cells from endoplasmic reticulum stress.
7. A method of treating diabetes, comprising administering paroxetine to an individual in need of treatment.

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