KR101481036B1 - Pharmaceutical composition containing arachidonyl dopamine for inhibiting the differentiation of adipocyte and lowering levels of lipid droplet in adipocyte - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting adipocyte differentiation containing arachidonyl dopamine and a pharmaceutical composition for reducing lipid droplet level in adipocyte containing arachidonyl dopamine. Since arachidonyl dopamine used in the present invention not only differentiates adipocytes but also reduces lipid levels in differentiated adipocytes, the pharmaceutical composition containing arachidonyl dopamine is useful for preventing or treating obesity or lipoma Not only is it useful, but it is also useful for reducing fat tissue for cosmetic or cosmetic purposes.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for inhibiting adipocyte differentiation and reducing lipid levels in adipocytes containing arachidonyldopamine, and a pharmaceutical composition for reducing lipid levels in adipocytes. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to a pharmaceutical composition for inhibiting adipocyte differentiation containing arachidonyl dopamine and a pharmaceutical composition for reducing lipid level in adipocytes.

Endocannabinoids play a variety of important roles in human organisms, functioning as lipid messengers in the cells that secrete in one cell and activate receptors in adjacent cells. In particular, the mechanism of retrograde signaling, which is secreted in postsynaptic cells and acts on receptors in presynaptic cells, temporarily reducing the release of general neurotransmitters, . To date, three cannabinoids have been identified: the phytocannabinoids, which are unique to cannabis plants; the endogenous cannabinoids and synthetic cannabinoids that occur in the human and animal body. In addition, there are two major receptors involved, one is cannabinoid receptor and the other is transient receptor potential vanilloid 1 (TRPV1) receptor.

Mammalian tissues have two cannabinoid receptor types associated with G proteins, the CB1 and CB2 receptors. CB1 receptors are mainly distributed at the nerve endings and are known to be involved in inhibiting the release of neurotransmitters. CB2 receptors are mainly expressed in immune cells and are involved in anti-inflammatory actions such as regulating the release of cytokines (Lambert et al. , J. Med. Chem., 48: 5059-5087, 2005). On the other hand, the TRPV1 receptor is a channel protein that is opened by non-selective ligands and is activated by various internal and external stimuli. TRPV1 receptors are mainly expressed in the central and parasympathetic nervous systems and have been shown to be involved in the integration of pain transmission and regulation and various pain stimuli (Caterina et al. , Annu. Rev. Neurosci, 24: 487-517, 2001).

Among the three cannabinoids, all of the endogenous cannabinoids are of the eicosanoid series. Among them, arachidonoyl ethanolamide (AEA), 2-arachidonoyl glycerol (2-AG) And arachidonyldopamine are well known. Endogenous cannabinoids also bind to G-protein coupled cannabinoid receptors, CB1 and CB2, as well as capsaicin and heat-activated TRPV1 receptors. For example, arachidonyl ethanolamide binds to the CB1 receptor, resulting in inhibition of the release of neurotransmitters in the sympathetic nervous system. In addition, it binds to the TRPV1 receptor to activate sympathetic nerves, resulting in the release of neurotransmitters, (Ralevic et al. , Eur. J. Pharmacol ., 472: 1-21, 2003). 2-arachidonoyl glycerol has also been shown to bind better to the CB1 receptor with similar affinity and to be present in the brain at higher concentrations than arachidonyl ethanolamide (Stella et al. , Nature, 388: 773-778, 1997). Arachidonyldopamine also reacts better to the CB1 receptor and is known as an agent for the vanilloid receptor subtype 1 (VR1) (Bisogno et al. , Biochem. J. 351: 817-824 , 2000).

Recently, it has been shown that CB1 and CB2 are also expressed in cell membranes in human adipocytes, and when CB1 is activated, intracellular cAMP is elevated, suggesting that CB receptors regulate the inflammatory response of adipocytes and maintain energy balance (Roche et al. , Histochem. Cell. Biol., 126: 177-187, 2006).

It is known that various abnormalities such as obesity or lipomas occur when these adipocytes are abnormally differentiated or have an abnormal lipid droplet content in adipocytes. Therefore, in order to cure these diseases, studies on lipid differentiation and lipid body content in adipocytes are actively conducted, and in this study, not only preadipocyte but also mesenchymal stem cells . In particular, mesenchymal stem cells have been widely used for the study of adipocyte differentiation because they can be differentiated into adipocytes by using a method almost similar to the method of differentiating the adipocyte precursor cells collected from subcutaneous tissues into complete adipocytes (Xu et al. , Exp Cell Res., 312: 1856-1864, 2006; and Pittenger et al. , Science., 284: 143-146, 1999).

As described above, mesenchymal stem cells that can be differentiated into adipocytes are distributed in most tissues of the body, and they are known to be able to differentiate not only into adipocytes but also into other types of cells such as cartilage cells and bone cells (Meirelles et al ., J. Cell Sci, 119: . 2204-2213, 2006). Mesenchymal stem cells are generally known to have similar properties or characteristics, although there are subtle differences in their properties or characteristics depending on which tissue it is from (Sakaguchi et al. , Arthritis Rheumat. , 52: 2521- 2529, 2005).

Therefore, the inventors of the present invention have conducted studies on endogenous endocannabinoids recognized as the same property. As a result, it has been found that human mesenchymal stem cells are treated with arachidonylethanolamide, 2-arachidonoylglycerol and arachidonyldopamine , Confirming that, unlike the other two endocannabinoids, only arachidonyldopamine effectively inhibited the differentiation of mesenchymal stem cells into adipocytes, and also decreased lipid levels in already differentiated adipocytes. It was completed.

[Literature 1] Lambert et al. , J. Med. Chem., 48: 5059-5087, 2005

[Document 2] Caterina et al. , Annual. Rev. of Neuroscience, 24: 487-517, 2001

[Literature 3] Ralevic et al. , Eur. J. Pharmacol., 472: 1-21, 2003

[Document 4] Stella et al. , ≪ / RTI > Nature, 388: 773-778,1997

[Literature 5] Bisogno et al. , Biochem. J., < / RTI > 351: 817-824,2000

[Literature 6] Roche et al. , Histochem. Cell. Biol., 126: 177-187. 2006

[Literature 7] Xu et al. , Exp. Cell Res., 312: 1856-1864, 2006

[Literature 8] Pittenger et al. , ≪ / RTI > Science, 284: 143-146, 1999

[Literature 9] Meirelles et al. , J. Cell Sci., 119: 2204-2213, 2006

[Document 10] Sakaguchi et al. , Arthritis Rheumat. , 52: 2521-2529. 2005

It is therefore an object of the present invention to provide the use of arachidonyl dopamine in inhibiting adipocyte differentiation.

It is another object of the present invention to provide the use of arachidonyl dopamine in reducing lipid bodies in adipocytes.

In order to achieve the above object, the present invention provides a pharmaceutical composition for inhibiting adipocyte differentiation, which comprises arachidonyl dopamine.

To achieve these and other objects, the present invention provides a pharmaceutical composition for reducing lipid levels in adipocytes, comprising arachidonyl dopamine.

As described above, since arachidonyldopamine has the effect of inhibiting adipocyte differentiation and reducing lipid levels in adipocytes, the composition containing arachidonyldopamine can prevent or prevent obesity or lipoma It is useful not only for treatment but also for reducing fat tissue for cosmetic or cosmetic purposes.

The present inventors have found that when mesenchymal stem cells are treated with arachidonyldopamine, not only adipocyte differentiation is inhibited but also lipid levels in already differentiated adipocytes are reduced. In addition, the present inventors have found that when adipocyte differentiation is inhibited by arachidonyldopamine and lipid levels in differentiated adipocytes are reduced, genes associated with adipocyte differentiation such as fatty acid binding protein 4 (FABP4), leptin The level of transcription of the leptin receptor, adiponectin, and g-type peroxisome proliferators-activated receptor gamma (PPAR g) is reduced (see FIG. 4). Furthermore, as compared with arachidonyl ethanolamide (AEA) known as CB1 receptor, it was confirmed that the genes involved in adipocyte differentiation were relatively decreased in arachidonyldopamine (see FIG. 5).

Accordingly, the present invention provides a pharmaceutical composition for inhibiting adipocyte differentiation, which comprises arachidonyl dopamine.

The pharmaceutical composition exhibits an effect of preventing disease associated with hypertrophy of adipose tissue. Diseases associated with hypertrophy of the adipose tissue include, but are not limited to, obesity and adipose tissue. The adipocyte differentiation is the differentiation of adipose precursor cells or mesenchymal stem cells into adipocytes of an animal, preferably a mammal, more preferably the differentiation of human mesenchymal stem cells into adipocytes, Is the differentiation of mesenchymal stem cells derived from bone marrow into adipocytes.

The present invention also provides a pharmaceutical composition for reducing lipid levels in adipocytes, comprising arachidonyl dopamine.

The pharmaceutical composition reduces fat tissue. Due to the reduction of the adipose tissue, therapeutic effects of obesity and lipomas can be obtained, although this is not so. In addition, cosmetic or cosmetic purposes can be achieved due to the reduction of the adipose tissue.

The pharmaceutical composition of the present invention may further contain a preservative, a stabilizer, a wetting agent or an emulsifying accelerator, a pharmaceutical adjuvant such as a salt and / or a buffer for controlling osmotic pressure, and other therapeutically useful substances, May be formulated in a variety of oral or parenteral dosage forms.

Examples of formulations for oral administration include tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, granules and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, Sucrose, mannitol, sorbitol, cellulose and glycine), excipients such as silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents. Tablets can be prepared by conventional mixing, granulating or coating methods.

Also typical of parenteral administration formulations are isotonic aqueous solutions or suspensions which are suitable for injectable use.

As an active ingredient, arachidonyldopamine can be administered to mammals, including humans, once or twice a day, locally to a site of parenteral route or adipose tissue at a dose of 0.1 mg / kg to 1 mg / kg per day have.

It should be understood, however, that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the severity of the symptoms, the selected route of administration, the age, sex, weight and health status of the subject, Are not intended to limit the scope of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example 1: Effect of arachidonyldopamine on the differentiation of human bone marrow mesenchymal stem cells into adipocytes

(1) Confirming the ability of human bone marrow mesenchymal stem cell cultures and arachidonyl dopamine to inhibit adipocyte differentiation

Human bone marrow mesenchymal stem cells (hBM-MSCs) were purchased from Lonza, Inc. (Walkersville, MD, USA) and cultured according to Lonza's guidelines. Adipocyte differentiation was carried out according to the method recommended by RON Corporation except that troglitazone (TRO) was used instead of indomethacin to induce differentiation of mesenchymal stem cells into adipocytes.

Specifically, mesenchymal stem cells were cultured in the presence of 1 μg / ml insulin, 1 μM dexamethasone (DEXA), 0.5 mM isobutylmethylxanthine (IBMX) and 1 μM troglitazone (TRO) And in the experimental group, 10 μM arachidonyldopamine was added to the medium at the induction of adipocyte differentiation. After 7 days or 15 days after the mesenchymal stem cells started to differentiate into adipocytes, the cell culture medium was removed and the cells were washed with 10 ml of phosphate buffered saline (PBS), and the cells were washed with 10% formalin / PBS solution Cells were fixed for 10 minutes using 0.2 ml / cm < ^{2 &} gt ;. The fixed cells were washed once with a 60% isopropanol solution (0.5 ml / cm ² ), and the lipid in the adipocyte was stained with 0.2 ml / cm ² of a dye solution obtained by dissolving Oil Red O in 60% isopropanol Respectively. Subsequently, the cells were re-stained with hematoxylin, and the nuclei were easily distinguished. The cells were observed with a microscope and the results are shown in FIG. As can be seen from Fig. 1, arachidonyl dopamine inhibited the differentiation into adipocytes without inducing apoptosis of mesenchymal stem cells to a significant level.

(2) Quantitative measurement of inhibitory effect of arachidonyldopamine on adipocyte differentiation of human mesenchymal stem cells

(Group 1), and treated with only 1 μg / ml of insulin (control group) in group A, and 1 μg / ml of insulin and DEXA 1 μM, IBMX 0.5 mM (IDX) and TRO 1 μM, and C, D and E groups treated with 10 μM, 3 μM and 1 μM arachidonyldopamine, respectively, in addition to IDX and TRO 1 μM

To measure the degree of adipocyte differentiation, the adipocyte was stained with Oil Red Oto as in (1), the stained oil red oe was eluted with 100% isopropanol solution, and the absorbance was measured at a wavelength of 500 nm As shown in Fig.

As shown in FIG. 2, in the case of induction of differentiation into adipocytes by TRO, an experimental group treated with 10 μM and 3 μM of arachidonyldopamine compared to the group not treated with arachidonyldopamine (group B) C and D), the differentiation of mesenchymal stem cells into adipocytes was reduced by 73% and 23%, respectively. There was little difference when 1 μM arachidonyl dopamine was treated. From these results, it was confirmed that arachidonyldopamine inhibits the differentiation of human bone marrow mesenchymal stem cells into adipocytes into a statistically significant level in a dose-dependent manner.

Example 2: Effect of arachidonyl dopamine on lipid levels in differentiated adipocytes

Human bone marrow mesenchymal stem cells were cultured in the same manner as in (1) of Example 1, and adipocyte dopamine 10 μM and 1 μM were treated respectively after inducing adipocyte differentiation. Specifically, human myeloid mesenchymal stem cells were cultured in the same manner as in Example 1 (1), and adipocyte differentiation was induced for 15 days, and at the time when 50% or more cells were differentiated into adipocytes, arachidonyldopamine Lt; / RTI > for 7 days. In order to quantitatively measure the lipid levels in differentiated adipocytes and observe with a microscope, oil red staining / hematoxylin re-staining and absorbance measurement were performed in the same manner as in (1) and (2) The results are shown in Fig.

As shown in Figure 3, arachidonyl dopamine did not induce apoptosis of mesenchymal stem cells at significant levels, but decreased lipid levels in adipocytes by 54% at a concentration of 10 μM arachidonyldopamine (See Fig. 3). These results suggest that arachidonyldopamine reduces lipid levels in already differentiated adipocytes to significant levels.

Example 3: Effect of arachidonyl dopamine on expression levels of genes associated with adipocytes

As shown in Examples 1 and 2, the differentiation of human bone marrow mesenchymal stem cells into adipocytes is inhibited by arachidonyldopamine, and lipid levels in the differentiated adipocytes are decreased. Here, The following experiment was conducted to investigate the change of the expression pattern of

Specifically, in accordance with the same method as in (1) of Example 1, arachidonyldopamine was added to human bone marrow mesenchymal stem cells, which induced the differentiation of human bone marrow mesenchymal stem cells into adipocytes, (Trizol reagent, Invitrogen, Carlsbad, Calif., USA) to separate the RNA in the cells. The separated RNA was further purified with an RNA kit (Qiagen RNeasy kit, Qiagen, Valencia, Calif.) From Agilent Technologies, Inc. (Agilent 2100 BioAnalyzer, Agilent Technologies, Santa Clara, Calif. Were used to confirm the quality of the RNA. CDNA was synthesized from the RNA using a reverse transcription kit (Superscript Reverse Transcriptase (RT) II kit, Invitrogen, Carlsbad, Calif.), And the cDNA was synthesized using real time reverse transcription polymerase chain reaction Q-RT-PCR). Mesenchymal stem cells, adipocytes differentiation suppression during and differentiation of fat lipid material level decreases when you, in cells fat cells related to the expression pattern changes in implied Biosystems Inc. taekmaen gene expression system of the gene (TaqMan of ^® gene expression assay kit, Applied Biosystems, Foster City, CA): leptin receptor (Hs00174497_m1), adiponectin (Hs00605917_m1), FABP4 (Hs0060 9791_m1) and PPARg (Hs00233423_m1).

When treated with 10 mM arachidonyldopamine, the adiponectin (ADIPOQ), leptin receptor (LEPR), FABP4 and PPARγ transcription levels of the adipocyte related genes decreased (see FIG. 4). From the results shown in Fig. 4, it was found that arachidonyldopamine regulates the intracellular specific mechanism involved in adipocyte-related genes, thereby suppressing differentiation into adipocytes and significantly reducing the level of lipid in the differentiated adipocytes .

On the other hand, 10 mM arachidonyl ethanolamide and 2-arachidonoyl glycerol, which are known as arachidonyldopamine and other CB1 agonists, were respectively treated to express adiponectin, leptin receptor, FABP4 and PPAR gamma Respectively. In the case of arachidonyldopamine, it was confirmed that the level of transcription of adiponectin and FABP4 was markedly reduced, in particular, compared to arachidonyl ethanolamide and 2-arachidonoyl glycerol (see FIG. 5).

FIG. 1 shows that when arachidonyldopamine was treated with human mesenchymal stem cells, adipocyte differentiation was inhibited. When lipid bodies of adipocytes were stained with an oil-red staining solution, The results show that arachidonyldopamine inhibits adipocyte differentiation,

FIG. 2 is a graph showing the results of staining of mesenchymal stem cells, which induces differentiation into adipocytes, by staining with an oil red staining solution, dissolving them in isopropanol, measuring the absorbance at a wavelength of 500 nm, N-Dopamine has been shown to inhibit adipocyte differentiation in a dose-dependent manner,

FIG. 3 shows the result of quantitatively showing a reduced lipid body when human mesenchymal stem cells were induced to differentiate into adipocytes for 15 days and treated with arachidonyldopamine at a concentration of 10 μM for 7 days,

FIG. 4 shows that human mesenchymal stem cells were induced to differentiate into adipocytes for 15 days, treated with arachidonyldopamine at a concentration of 10 μM for 7 days, and adiponectin, leptin Receptor, FABP4 and PPARγ mRNA expression was quantitatively analyzed by Q-RT-PCT,

FIG. 5 shows the results of culturing human mesenchymal stem cells for 15 days, inducing differentiation into adipocytes, treating arachidonyl ethanolamide and 2-arachidonoyl glycerol at a concentration of 10 μM for 7 days, The results of quantitative analysis of mRNA expression of adiponectin, leptin receptor, FABP4 and PPARγ, which are adipocyte related genes, in Q-RT-PCT.

Claims (8)

A pharmaceutical composition for preventing or treating obesity or lipoma containing arachidonyl dopamine. delete delete delete delete delete delete delete

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