KR101704914B1 - A pharmaceutical composition comprising dammarenediol-ii for preventing or treating diseases by diabetic vascular leakage - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating diseases by diabetic vascular leakage. According to the present invention, it is possible to prevent or treat diseases by diabetic vascular leakage comprising diabetic retinopathy, and to be used for preventing or treating various diabetic vascular complications.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by diabetic keratocyte leukemia comprising daraendenol-II. The present invention relates to a pharmaceutical composition for prevention or treatment of diabetic keratocyte-

The present invention relates to a pharmaceutical composition for preventing or treating diseases caused by diabetic vessel leakage comprising dimarenendiol-II represented by the following formula 1:

[Chemical Formula 1]

.

.

Diabetes is a multiple metabolic disorder characterized by hyperglycemia characterized by insulin secretion defects or insulin deficiencies. When glucose is abnormally elevated in the blood for a long period of time, chronic metabolic disturbances Various complications occur due to impairment and subsequent chronic vascular injury. After 10 years of diabetes, almost all organs in the body are damaged and complications are caused. In particular, diabetic patients exhibit abnormal blood vessel leakage, and diabetic vessel leakage causes complications such as diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic angiopathy, and diabetic inflammation.

Such diabetic complications are associated with overexpression of VEGF (vascular endothelial growth factor). VEGF acts as a vascular leak factor, and it is known that increased expression of VEGF in the retina during diabetes can induce angiogenesis and macular edema.

Meanwhile, the conventional treatment of complications due to diabetic and diabetic vessel leakage has been performed through the treatment of chronic diabetic hyperglycemia by administering a drug such as insulin to control blood sugar. However, such a passive form of treatment has limitations in the treatment of vascular injury, vascular leakage and complications due to chronic diabetic hyperglycemia.

In addition, some anti-inflammatory or anti-obesity effects of some components extracted from natural products such as ginseng have been reported and many studies have been made, but studies for direct prevention or treatment of diabetic vascular complications are lacking.

Korean Patent Laid-Open Publication No. 2014-0108621

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-

A pharmaceutical composition capable of directly treating a disease caused by diabetic vessel leakage by injecting a substance capable of inhibiting vascular injury and blood vessel leakage by overcoming the limit of treatment of conventional passive diabetic complication, .

Another object of the present invention is to provide a pharmaceutical composition comprising a natural origin-derived component, i.e., a tamarind diol-II, into a body to minimize side effects that may occur upon administration of the drug and to prevent diabetic vessel leakage And to provide a pharmaceutical composition capable of preventing or treating a disease caused by the disease.

In order to solve the above problems,

The present invention provides a pharmaceutical composition for preventing or treating diseases caused by diabetic vascular leakage comprising Dammarenediol-II represented by the following formula 1:

[Chemical Formula 1]

.

.

According to the pharmaceutical composition of the present invention, it is possible to prevent or treat diseases caused by diabetic vascular leakage including diabetic retinopathy, and to effectively prevent or treat other diabetic vascular complications.

FIG. 1 is a diagram showing a series of procedures for obtaining the diamandiol-II contained in the pharmaceutical composition of the present invention.
Fig. 2 is a photograph showing the result of Example 1. Fig.
FIG. 3 is a graph showing that the tamarenendiol-II inhibits the production of VEGF-inducible ROS in a concentration-dependent manner.
Figure 4 is a photograph showing that tamarenendiol-II inhibits VEGF-induced stress fiber formation.
FIG. 5A is a photograph showing that tamarenendiol-II suppresses the collapse of the VEGF-inducible adhesive junction, and FIG. 5B shows a change in the level of collapse of the adhesive junction in a histogram showing the relative fluorescence intensity.
FIG. 6 is a graph showing the results of Example 1 of the present invention. FIG. 6 is a graph showing the results of measuring the fluorescence intensity of whole retinal tissues to quantify retinal leakage (n = 8) to be. * p < 0.001.

The present inventors have conducted various studies to develop a method for the prevention or treatment of diseases caused by diabetic vascular leakage in diabetic patients. As a result, VEGF has been shown to produce intracellular ROS through intracellular calcium ion levels, It was found that the dimalendiol-II inhibitory effect of VEGF-induced intracellular ROS increases.

Accordingly, the present invention relates to a pharmaceutical composition for preventing or treating diseases caused by diabetic vascular leakage comprising Damaranediol-II, Lt; / RTI >

[Chemical Formula 1]

.

.

Hereinafter, the present invention will be described in detail.

The above-mentioned diamandiol-II is a precursor of saponin extracted from ginseng (Panax ginseng).

In one embodiment of the present invention,

The above-mentioned diamandion-II can be extracted from natural products such as ginseng (Panax ginseng) using a known method. However, because of its trace amounts, it is preferred to obtain by using the transformation method using the tobacco mosaic virus as described in Plant Cell Physiol 53: 173-182., Plant Cell Rep. 33: 225-233.

 The above-mentioned diamandiol-II can be extracted from at least one selected from the group consisting of ginseng (Panax ginseng) and Cacalia atriplicifolia,

Although it is preferable to extract it from ginseng, the range of the natural product is not limited as long as it is a natural product containing darmandiol-II.

For the purpose of the present invention, the above-mentioned daimarendiol-II is used as an active ingredient of the pharmaceutical composition of the present invention, which can cause not only human but also diabetic vascular leakage diseases It can be applied to all animals.

According to an embodiment of the present invention,

It has been found that darenendiol-II can inhibit extracellular efflux of diabetic mice, and thus compositions comprising darenendiol-II can be used for the prevention or treatment of diabetic vessel leakage.

Specifically, the preventive or therapeutic effect of such a method or composition of the present invention on the disease caused by diabetic vascular leakage is due to the action of damarendiol-II to inhibit intracellular ROS formation, A stress-fiber formation inhibitory action, and an adherens junction protein degradation inhibition action of damarindiol-II.

The prevention or treatment of diabetic vascular leakage of dimalandiol-II of the present invention can be equally applied to not only human but also all animals in which diabetes can occur. In addition, since the above effect is exhibited by inhibiting the decomposition of VEGF-induced VE cadherin induced by diabetes, the above effect can be applied not only to the retinal region confirmed in the embodiment of the present invention, but also to the peripheral blood vessel region of other tissues .

That is, in the present invention, the diabetic blood vessel leakage may be retinal blood vessel leakage or other peripheral blood vessel leakage.

Accordingly, the methods or compositions of the present invention are useful for the treatment of diabetes mellitus such as diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic vascular dysfunction, diabetic inflammation, Can be used to prevent or treat the accompanying blood vessel leakage in the onset of complications.

Meanwhile, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent as a pharmaceutical composition. "Pharmaceutically acceptable carrier, excipient or diluent" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. As the liquid preparation for oral administration, suspensions, solutions, emulsions, syrups and the like may be used. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, . Formulations for non-oral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, and freeze-drying agents. As the non-aqueous preparation and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used.

Meanwhile, the administration route of the composition containing the dimalendiol-II in the method or composition of the present invention can be administered through any ordinary route as long as it can reach the target tissue, but osmotic pump It is preferable to be administered by subcutaneous injection, intradermal injection, intravein injection, intraperitoneal injection or intravitreal injection which is administered by eye injection More preferable.

In addition, the dimarenendiol-II of the present invention may be administered in an "effective amount" or a "pharmaceutically effective amount ". Effective amount "or" pharmaceutically effective amount "means an amount sufficient to exhibit a prophylactic or therapeutic effect and an amount not causing side effects or a serious or excessive immune response, The severity of the disorder, the activity of the particular compound, the route of administration, the rate of elimination, the duration of treatment, the drugs used in combination or concurrently, the age, weight, sex, diet, general health status of the subject, It depends on various factors including arguments. Various general aspects considered in determining such "effective amount" or "pharmaceutically effective amount" are known to those skilled in the art and are described, for example, in Gilman et al., Eds., Goodman and Gilman's: The Pharmacological Bases of Therapeutics, Ed., Pergamon Press, 1990 and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.

In addition, the method or composition of the present invention can be administered as an individual therapeutic agent or in combination with another therapeutic agent, and can be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by a person skilled in the art. The composition may also be administered by any device capable of transferring the active agent to the target cell.

Hereinafter, the present invention will be described in more detail by way of non-limiting examples. The embodiments of the present invention described below are by way of example only and the scope of the present invention is not limited to these embodiments. The scope of the present invention is indicated in the claims, and moreover, includes all changes within the meaning and range of equivalency of the claims.

Example

Preparation Example  One. Damarain Dior -II Preparation

Dimarinediol-II of formula 1 was obtained using the method described in Plant Cell Physiol 53: 173-182, Plant Cell Rep 33: 225-233:

 [Chemical Formula 1]

.

.

The series of steps is shown in Fig.

Preparation Example  2. Preparation of experimental animals

Six-week old male C57BL / 6 mice were obtained from Nara Koech (Pyungtaek, Korea). The mice were housed in a thermostatic box at a 12-hour light / dark cycle. All experiments were carried out in accordance with the guidelines of the Experimental Ethics Committee of Kangwon National University.

Preparation Example  3. Cell culture

HUVECs were isolated from human umbilical cord blood according to known methods and 3-6 cells were used in the experiment. The cells were cultured in 2% gelatin-coated coverslips, dishes or plates containing M199 culture medium (20% FBS, 3 ng / ml bFGF, 5 U / ml heparin, 100 U / ml penicillin and 100 ug / ml streptomycin) Inoculated and cultured in a 5% CO ₂ incubator maintained at 37 ° C.

When used in the experiment, the cells were cultured for 6 hours in a low serum medium (M199 culture medium or M199 culture medium containing only 1% FBS) and pretreated with 1 μg / ml of demineraldiol-II for 30 minutes And then treated with 10 ng / ml VEGF for an appropriate period of time.

Example  One. Damarain Dior -II to inhibit microvascular leakage in the retina of diabetic mice

First, mice of Preparation Example 2 were injected with 100 mM citrate buffer (pH 4.5) containing streptozotocin by single intraperitoneal injection (150 mg / kg body weight) to prepare diabetic model mice, and 10% sucrose . Two days after injection of streptozotocin, hyperglycemia was induced in the mice using an Accu-Chek active blood glucose monitor (Roche Diagnostics GmbH, Mannheim, Germany). Thereafter, at the passage of one week, a mouse having a non-fasting blood glucose level of 16 mM or more and being polyuria and glucosuria was obtained and used in the experiment. Hereinafter, the diabetic mouse Quot;

Two microliters (58 nmole) of the solution containing the dimarenendiol-II of Preparation Example 1 was injected into one eye of the diabetic mouse and 2 μl of PBS was injected into the eye of the other eye. At 24 hours after ocular injection, fluorescein angiography was used to quantitate the level of retinal leakage. To quantitate the level of retinal leakage, anesthetized with 2.5% avertin solution, 1.25 mg of 500-kDa FITC-dextran (Sigma-Aldrich) was injected into the left ventricle of the mouse and stained with blood circulation for 5 minutes. The eyeballs were removed and immediately fixed with 4% paraformaldehyde for 45 minutes. The retina was cut from the fixed eyeball and cut in a Maltese cross shape. The cut retina was placed in a slide glass and observed using a confocal microscope. The intensity of FITC-dextran seeping from the entire retinal tissue (8 per group) was measured and analyzed by quantitative analysis using FV-300 software to measure the level of retinal leakage. The experimental results are shown in Fig.

FIG. 2 is a photograph showing that the tamarenendiol-II inhibits diabetic-induced retinal leakage. In one eye of a diabetic model mouse, 2 쨉 l of darenendiol-II was injected into the eyes (diabetic + demarrenediol-II; (Diabetic + Dam-II, n = 8), and the other eye was injected with the same volume of PBS (Diabetic, n = 8) and the retina was observed with a confocal microscope. FIG. 2 is a representative fluorescence image of the retina, in which a rectangular region is shown enlarged at the bottom of each image. 2, the extracellular flux level of FITC-dextran showed a remarkably high level in the retina (n = 8) of the diabetic model mouse, and this leakage was observed in the retina of the opposite eye in which the damarendiol- n = 8).

6 is a graph showing the results of the experiment obtained by measuring the fluorescence intensity of the whole retinal tissue and quantifying retinal leakage (n = 8) in terms of mean + standard deviation. * p < 0.001.

The inhibitory effect of dimalendiol-II on blood vessel leakage in the retina of the diabetic model mouse was quantitatively analyzed by confirming the fluorescence intensity of FITC-dextran in the whole retinal tissue (n = 8) (Fig. 6).

Example  2. VEGF - inducible intracellular ROS (reactive oxygen species) production Damarain Dior -II

In diabetes, increased expression of VEGF in retinal endothelial cells is associated with retinal vascular leakage through the production of intracellular ROS. Thus, the inhibitory effect of dimalendiol-II on VEGF-induced ROS formation and the effect of inhibiting vascular leakage were evaluated using HUVECs prepared in Preparation Example 3. [

In order to confirm dose-dependent inhibition of dimalendiol-II on VEGF-induced ROS formation, HUVECs were preincubated with a suitable concentration of dimalendiol-II for 30 min and treated with 10 ng / ml VEGF Is shown in Fig.

Damarenendiol-II inhibited VEGF-induced intracellular ROS production in a dose-dependent manner and completely inhibited the production of ROS at 1 μg / ml.

Example  3. VEGF - Inductive stress on fiber formation Damarain Dior -II

We investigated the effect of D-Marendiol-II on the formation of VEGF-induced stress fibers and degradation of junctional junctions in HUVECs, since VEGF activates the breakdown of VE-cadherin, which is responsible for stress fiber formation and vascular leakage.

Increased levels of VEGF in the retinal endothelial cells can induce stress fiber formation through increased ROS production. Actin filaments in HUVECs prepared by Preparation Example 3 were visualized by staining with rhodamine-phalloidin to confirm the effect of damarenendiol-II on VEGF-induced stress fiber formation.

Specifically, HUVECs were cultured on a gelatin coated circular cover slide in a 12-well culture plate, pre-treated with damarene II (Dam-II) for 30 minutes, and then incubated with 10 ng / ml VEGF And cultured. Cells were rapidly washed with PBS and fixed with PBS containing 3.7% formaldehyde for 30 minutes. Cells were then treated with PBS containing 0.2% Triton X-100 for 30 minutes and stained with PBS containing rhodamine-palloidin (Molecular Probes) for 1 hour. The stained cells were placed in a slide glass using a mounting solution, and actin filaments were observed using a confocal microscope.

The experimental results are shown in Fig. Figure 4 is a photograph showing that tamarenendiol-II inhibits VEGF-induced stress fiber formation. Figure 4 shows that tamarenendiol-II inhibits VEGF-induced stress fiber formation, either by treating HUVECs with 10 ng / ml VEGF or 1 ug / ml dimalendiol-II alone, or with 1 ug / ml After treatment with 10 ng / ml VEGF in the presence of dimalendiol-II for 1 hour, the microfilaments were stained with rhodamine-phalloidin and observed with a confocal microscope.

Referring to FIG. 4, it can be seen that VEGF is able to form stress fibers, but this is inhibited by treatment with dimalendiol-II.

In other words, considering the fact that the tamarenendiol-II confirmed from the results of Example 3 suppresses the production of VEGF-inducible ROS, damarendiol-II inhibits VEGF-induced stress fiber And inhibit the formation.

Example  4. Affects the decomposition of adhesive joint Damarain Dior -II

 In order to elucidate the mechanism of D-Marendiol-II related to diabetic-induced vascular leakage, we investigated the effect of D-Marendiol-II on the VEGF-induced changes of the adhesive conformational protein VE-cadherin as a leukocyte in HUVECs Respectively.

Specifically, saturating cultured cell layers (HUVECs) in 6 well plates were treated with dimalendiol-II for 30 minutes and treated with VEGF at 37 캜 for 90 minutes. The treated cells were washed with PBS and fixed with PBS containing 3.7% formaldehyde for 30 minutes. Cells were treated with PBS containing 0.2% Triton X-100 for 30 minutes and stained overnight at 4 ° C with PBS containing monoclonal VE-cadherin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). Then, the cells were reacted with chlorine anti-mouse FITC (fluorescein isothiocyanate; Sigma, St. Louis, MO), and VE-cadherin was observed using a confocal microscope.

The experimental results are shown in Fig. Figures 5a and 5b show that tamarenendiol-II inhibits VEGF-induced efflux, either by treating HUVECs with 10 ng / ml VEGF or 1 ug / ml dimalendiol-II alone, or with 1 ug / After treatment with 10 ng / ml VEGF and incubation for 90 minutes (n = 3) in the presence of dlMalendiol-II, VE-cadherin was stained and visualized by confocal microscopy.

5a, VEGF stimulated the degradation of VE-cadherin, and this degradation was inhibited by damareneniol-II. Furthermore, the change in the level of degradation of VE-cadherin was determined by relative fluorescence intensity RFI), it can be seen that the decrease in VEGF-inducibility in the adhesive junction fluorescence intensity is restored by the treatment of the dimarenendiol-II. Thus, it was found that dimalendiol-II inhibits the production of VEGF-stimulated VE-cadherin by inhibiting intracellular ROS production in endothelial cells.

As a result, it was found that blood vessel leakage was inhibited in the retina of the diabetic mouse when the pharmaceutical composition containing the dimalendiol-II was intraocularly injected into the retina of the diabetic mouse.

Claims (6)

A pharmaceutical composition for prevention or treatment of diseases caused by diabetic vascular leakage comprising Damarrenediol-II represented by the following formula (1)
The disease caused by vascular leakage is at least one selected from the group consisting of diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, diabetic vascular dysfunction, and diabetic inflammation , Pharmaceutical composition:

[Chemical Formula 1]

. delete delete The method according to claim 1,
Wherein said pharmaceutical composition is administered by intravitreal injection. ≪ RTI ID = 0.0 > 11. < / RTI > The method according to claim 1,
Wherein said diamandiol-II is extracted from at least one selected from the group consisting of ginseng (Panax ginseng) and Cacalia atriplicifolia. The method according to claim 1,
The prevention or treatment of diseases caused by diabetic vascular leakage can be effectively prevented by inhibiting the intracellular ROS formation inhibitory action of damarindiol-II, the stress-fiber formation inhibitory action of damarindiol-II, Diol-II, < / RTI > and adherens junction protein proteolysis.

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