KR20030075017A - Angiogenesis inhibitor comprising arsenic trioxide - Google Patents

Abstract

PURPOSE: An angiogenesis inhibitor containing arsenic trioxide having an inhibiting effect on proliferation, movement, immersion and vasculogenesis of endothelial cells in vitro is provided. The angiogenesis inhibitor is widely used for treatment of diseases that give rise to angiogenesis. CONSTITUTION: An angiogenesis inhibitor contains arsenic trioxide having an anti-angiogenic activity as an effective ingredient. Therefore, it can be used in treatment of diseases that give rise to angiogenesis, such as diabetics, a solid tumor, arthritis, psoriasis, pyogenic granuloma, renovascular glaucoma, etc. The arsenic trioxide is administered at a dosage of about 5 to 50mg/kg, prior to or during radiation therapy.

Description

Angiogenesis inhibitor comprising arsenic trioxide

The present invention relates to a new use of arsenic trioxide, and more particularly, to a novel angiogenesis inhibitor comprising arsenic trioxide as an active ingredient.

Angiogenesis refers to a multi-step process in which existing blood vessels germinate by stimulation to form new blood vessels. This process rarely occurs outside normal physiological conditions such as embryonic development, corpus luteum formation, and wound healing. However, pathological growth of neovascularization typically occurs in many diseases, including tumor development and metastasis, hemangiomas, psoriasis, diabetes, and arthritis. These diseases are classified as angiogenic diseases.

Excessive angiogenesis of angiogenic diseases implies an increased need for nutrition in proportion to abnormal cell expansion. Such abnormal angiogenesis may be inhibited by antiangiogenic factors. Angiostatin, endostatin, thrombospondin, etc. are all representative fragments of proteins and are known representative angiogenesis inhibitors to date. However, these protein-derived angiogenesis inhibitors present a potential host immune response, pharmaceutical mass production, and the cost of treatment for long-term administration. In this context, the development of low molecular weight materials with excellent angiogenesis inhibitory effects is urgent.

Traditional oriental medicine has used arsenic compounds to treat cancer, psoriasis, and arthritis. Arsenic trioxide has been developed as a traditional cancer treatment in China and has been reported to have an excellent effect on the treatment of acute leukemia. In vitro experiments showed that arsenic trioxide reduced the expression of Bcl-2 protein and induced apoptosis induced by the activation of caspase in acute leukemia-inducing cells as well as other types of leukemia-inducing cells. It was found to induce. In addition to inducing apoptosis, arsenic trioxide has been reported to have antitumor vascular effects in solid tumors planted in rats. In vitro experiments have been reported to inhibit angiogenic differentiation of vascular endothelial cells, but the fact that arsenic trioxide has antiangiogenic activity has not been reported yet.

In other words, it has been reported that arsenic trioxide blocks the mechanism for making vasculature, but it has not been found that it can inhibit the entire angiogenesis, that is, the proliferation and migration of endothelial cells, invasion, angiogenesis and in vivo.

In order to solve the above problems, the present inventors aim to provide a new anti-angiogenic use of arsenic trioxide.

1 is a graph showing the inhibition of proliferation of BCE cells by arsenic trioxide,

2 is a graph showing the results of FACS analysis of BCE cells,

Figure 3a is a graph showing the effect of arsenic trioxide on the movement of BCE cells,

3b is a 100-fold magnification of migrated BCE cells,

4 is a graph showing the effect of arsenic trioxide on the infiltration of BCE cells,

5 is a diagram showing the effect of arsenic trioxide on MMP-2 secretion of BCE cells,

6 is a photograph showing the effect of arsenic trioxide on angiogenesis formed in culture on three-dimensional fibrin gel of ECV304 cells,

7 shows inhibition of angiogenesis on rat cornea by arsenic trioxide.

The present invention, in order to achieve the above object, provides an angiogenesis inhibitor comprising arsenic trioxide as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present inventors first found that arsenic trioxide has antiangiogenic activity in this experiment using in vitro and in vivo angiogenesis measurement techniques to complete the present invention.

Arsenic compounds are recently attracting new attention due to the successful use of arsenic trioxide in the clinical treatment of acute leukemia. In traditional oriental medicine, arsenic compounds have been used to treat diseases such as psoriasis and arthritis in addition to cancer.

Recent studies suggest anti-angiogenic effects of arsenic trioxide but have not been directly identified. In this study, the inventors directly revealed that arsenic trioxide strongly inhibited angiogenesis in vitro and in vivo .

In other words, using in vitro angiogenic assays, the inventors found that arsenic trioxide effectively inhibits the multi-step processes required for angiogenesis such as bFGF-induced growth, migration, invasion, and angiogenesis. Arsenic trioxide also showed anti-angiogenic effects in in vivo experiments using rat corneal micropocket assay.

In this study, we investigated the antiangiogenic effects of arsenic trioxide in vitr o and in vivo . In vitro studies show that arsenic trioxide is the growth of bovine capillary endothelial (BCE) cells induced by basic fibroblast growth factor (bFGF) (IC ₅₀ = 336.4 nM). , Migration (IC ₅₀ = 199.1 nM), and invasion (IC ₅₀ = 199.1 nM) were inhibited and markedly inhibited at a concentration of 2 μM that was clinically attainable for angiogenesis of immortalized endothelial cells (ECV304). It was. Arsenic trioxide also induced the growth factor-induced cell cycle to stay in G2 / M phase at 0.5 μM concentration and inhibited the release of matrix metalloproteinase-2 (MMP-2) by capillary endothelial cells at 2 μM concentration. It was. In vivo oral administration of arsenic trioxide to rats at a concentration of 50 mg / kg significantly inhibited neovascularization by growth factors in corneal micropocket analysis.

From these results, it was confirmed that arsenic trioxide can be effectively used as an angiogenesis inhibitor without side effects.

When the arsenic trioxide of the present invention is used as a medicament, various known methods can be applied to the pharmaceutical preparation and its administration method.

That is, the arsenic trioxide of the present invention can be administered parenterally, orally, and can be in any dosage form suitable for pharmaceutical preparations, including injections, powders, granules, tablets, and the like. When arsenic trioxide is formulated into a pharmaceutical composition of a radiotherapy enhancer, various auxiliaries used in medicine, such as carriers or other additives such as stabilizers, emollients, emulsifiers, etc., may be used if necessary, so long as they do not adversely affect the active ingredient. have.

In pharmaceutical formulations, the content of arsenic trioxide can vary widely depending on the formulation and is in accordance with conventional methods.

In addition, the dosage and method of administration of arsenic trioxide may vary according to the condition of the patient and the amount of cancer cells, but 5 to 50 mg / kg per day before or during radiation treatment is appropriate.

Based on these in vitro and in vivo studies, arsenic trioxide showed potent antiangiogenic activity in clinically viable conditions and is expected to be effectively used for the treatment of angiogenic diseases.

Hereinafter, the angiogenesis inhibitory effect of arsenic trioxide will be revealed in more detail through the following experimental example.

Experimental Example

Experimental animals used, experimental materials and experimental methods are as follows.

1) Experimental Animal

Male Sprague-Dawley rats weighing about 200 g were used for corneal micropocket assay.

2) reagents

Arsenic trioxide (Sigma, St. Louis, Mo.) was dissolved in 1 N NaOH at a concentration of 50 mM and used by refrigeration. The solution was free of endotoxin and it was confirmed that the highest concentration of NaOH added to the Limulus Amebocyte Lysate (LAL) test kit (Bio-Whittaker, Walkersyille, MD) medium did not affect endothelial cell growth.

3) Cell culture

Bovine capillary endothelial (BCE) cells include 10% calf serum (CS) in DMEM medium containing antibiotics and 3 ng / ml growth factor (basic fibroblast growth factor (bFGF); R & D Systems, Menneapolis, MN) was added to incubate.

The BCE cells used in this experiment showed pebble-shaped morphology, typical endothelial cells, and showed positive response in immunofluorescence staining for factor VIII antigen, a marker specific for endothelial cells, and deacetylated low-lipoprotein ( Diaceylated low density lipoproteins were ingested and vasculature was formed in vitro . Capillary endothelial cells were incubated at 37 ° C. under 10% CO ₂ . Cells were used at 10-15 passages.

Immortalized human endothelial cells, ECV304, were added with 10% fetal bovine serum (FBS) to M199 medium and used for angiogenesis analysis in three-dimensional fibrin gel culture.

4) Statistical Analysis

All results are expressed as mean ± standard deviation. For statistical evaluation of the data, Student's t- test, Non-parametric and two-tailed paired t- test were applied to in vitro data and Mann-Whitney test to in vivo data.

The following effects were measured with the experimental materials as described above.

Experimental Example 1

Inhibition of Endothelial Cell Proliferation by Arsenic Trioxide

To investigate whether arsenic trioxide inhibits the growth of BCE cells, arsenic trioxide was pretreated at a concentration of 0.01 to 10 μM, incubated for 72 hours in the same manner as described above with and without addition of 3 ng / ml of bFGF. The proliferation of BCE cells was measured.

Proliferation of endothelial cells was measured by Cao et al. BCE cells were cultured in DMEM medium with 10% CS and 3 ng / ml recombinant human bFGF. Cells cultured on gelatin coated plates were detached by treatment with 0.05% trypsin solution and suspended in DMEM medium with 10% CS. About 12,500 cells were also transferred to gelatin-coated 24-well plates and incubated for 24 hours at 37 ° C., 10% CO ₂ . After incubation, the medium was removed and replaced with 0.25 ml of fresh DMEM medium with 5% CS added, and then various concentrations of arsenic trioxide solution were added to each well. After incubation for about 1 hour, 0.25 ml of medium to which 3 ng / ml bFGF was added was added to the same medium, and finally adjusted to 0.5 ml, followed by incubation for 72 hours. The cultured cells were treated with trypsin, separated and counted with a hemocytometer. The results are expressed as mean ± standard deviation value.

The data is expressed in terms of percentage of 100 control cells cultured without adding bFGF. This experiment was repeated three times in triplicate. *, 0.01 <P <0.05, **, 0.001 <P <0.01, ***, P <0.001

As shown in Figure 1, arsenic trioxide significantly inhibited the growth of bCE cells induced by bFGF concentration-dependently (IC ₅₀ = 336.4 nM). As can be seen in Figure 1, arsenic trioxide also inhibited the growth of BCE cells in the absence of bFGF, but formed an IC ₅₀ value at a very high concentration (IC ₅₀ = 578.2 nM).

From these results, the inventors determined that arsenic trioxide had a specific inhibitory effect on cell growth induced by bFGF. When measuring the effect of suppression of proliferation, the concentration of arsenic trioxide up to 2 μM showed no typical cell morphology related to apoptosis such as cell detachment, circularization, and chromosome cleavage, indicating that cell growth inhibition was not caused by apoptosis. there was. Significant apoptosis of BCE cells was observed above 5 μM.

Experimental Example 2

Effect of Arsenic Trioxide on Cell Cycle Progression of Endothelial Cells

To investigate the mechanism related to the growth inhibition of endothelial cells by arsenic trioxide, FACS analysis was performed to investigate the effect of arsenic trioxide on cell cycle progression induced by bFGF.

To determine the amount of DNA, BCE cells were incubated for 24 hours in a 60 mm culture dish with or without bFGF (3 ng / ml) or with 0.5 μM arsenic trioxide in a complete medium. Trypsinized cells were detached and suspended in PBS. Cells were stained with propidium iodide using the CycleTestTM Plus (Becton Dickinson, San Jose, Calif.) Kit, and the DNA amount was measured using a FACStar flow cytometer (Becton Dickinson).

Treatment of bFGF (3 ng / ml) as shown in Figure 2 (a) facilitated the entry of BCE cells from the G0 / G1 stage to the S stage. On the other hand, as shown in (b) of Figure 2, the treatment of arsenic trioxide (0.5 μM) bound the bFGF-stimulated cells in the G2 / M phase. These results can be thought that arsenic trioxide ultimately inhibits cell growth by inhibiting cell cycle progression of endothelial cells stimulated by growth factors at the G2 / M stage.

Experimental Example 3

Effect of Arsenic Trioxide on Endothelial Cell Migration, Invasion, and MMP-2 Secretion

Endothelial cell migration and infiltration through the basement membrane is a pivotal process for the formation of new blood vessels.

(1) Measurement of endothelial cell migration

First, wound-migration analysis was performed to determine the effect of arsenic trioxide on the migration of capillary endothelial cells in vitro . BCE cells were incubated in a gelatin-coated 12-well plate and scraped off with a 200-1000 μl micropipette tip to remove wounds and washed with medium without serum to remove fallen cells. Each well was replaced with medium without serum with or without bFGF (10 ng / ml) followed by the addition of arsenic trioxide by concentration. The plate was incubated overnight, washed twice with PBS, stained with Diff-Quick (International Reagents co., Japan) solution, dried, and counted inwardly at 1 cm long wound end on a microscope. Measured.

Cell migration of wells not treated with arsenic trioxide was used as a positive (10 ng / ml bFGF treated) or negative (bFGF not treated) control. The data were expressed as mean ± standard deviation, with the values calculated from three independent experiments with control values of 100. IC ₅₀ here refers to the concentration required for 50% inhibition.

As shown in Figure 3a arsenic trioxide significantly inhibited the migration of BCE cells by bFGF (IC ₅₀ = 236.6 nM). In addition, a 100-fold magnification of BCE cells migrated to the wounded region is shown in FIG.

(2) Invasion of endothelial cells

Another angiogenic process in parallel with the migration of endothelial cells is infiltration. Using the Matrigel-coated Boyden chamber, the effect of arsenic trioxide on the infiltration of endothelial cells induced by bFGF was investigated as follows.

That is, the infiltration measurement was performed using a modified Boyden chamer, where the precoated filter (6.5 mm diameter, 8 μm pore-size, Matrigel 100 μg / cm ² ) was rehydrated with 250 μl of medium. 1 × 10 ⁵ cells were suspended in 200 μl of medium and cultured with or without arsenic trioxide. The lower chamber was incubated with or without addition of 10 ng / ml of bFGF as a chemoattractant to DMEM without serum with 1 ml of 0.1% bovine serum albumin. After 6 hours of incubation at 37 ° C. and 10% CO ₂ , the lower membrane portion of the upper chamber was stained with Diff-Quick solution, dried, and randomly selected 5 fields on the microscope to measure the number of infiltrated cells.

Cells not treated with arsenic trioxide were used as positive (bFGF 10 ng / ml treatment) or negative (medium) controls. The data were expressed as mean ± standard deviation, with the values calculated from three independent experiments with control values of 100.

As shown in FIG. 4, bFGF treatment in BCE cells resulted in a significant increase in infiltration. Treatment with arsenic trioxide inhibited this phenomenon in a concentration-dependent manner (IC ₅₀ = 199.1 nM).

(3) MMP-2 secretion measurement

An essential element of infiltration is the infiltration of cells by the degradation of extracellular matrix (ECM) components. Cultures of BCE cells grown without serum with and without various concentrations of arsenic trioxide were analyzed by gelatin zymogram as follows.

That is, SDS-PAGE was performed as follows to measure the gelatinase activity of BCE cells cultured with or without arsenic trioxide. Cultures were obtained by treating cells for 18 hours, and these 20 μl of the cultures were mixed in a non-denaturing loading buffer and subjected to 10% SDS-PAGE containing 0.1% gelatin as a substrate. The gel was washed with 2.5% Triton X-100 solution for 1 hour at room temperature to remove SDS and 30 with room temperature buffer (50 mM Tris, 150 mM NaCl, 100 mM CaCl ₂ , and 0.01% NaN ₃ , pH 7.5). After washing with water, the reaction was carried out overnight at 37 ℃ shaking water bath. After the reaction, the gel was dyed with a decolorizing solution (10% 2-propanol, 10% acetic acid) added with 0.5% coomassie brilliant blue, and then decolorized with a decolorizing solution to confirm the clear zone. It was.

The results are shown in FIG. As can be seen from FIG. 5, BCE cells always secreted MMP-2 and treatment with arsenic trioxide inhibited the secretion of MMP-2 at a concentration of 2 μm. When 2 μM of arsenic trioxide was added to the Zymogram reaction buffer, it was determined that arsenic trioxide had no direct inhibitory effect on MMP-2 itself.

Experimental Example 4

Effect of Arsenic Trioxide on the Differentiation of Endothelial Cells

Angiogenesis is the completion of vascular endothelial cells connected to each other to form a tubular structure. In order to investigate the effect of arsenic trioxide, ECV304 cells were cultured in a three-dimensional fibrin gel to investigate angiogenesis as follows.

First, fibrin gels were prepared as described above. That is, 2.5 mg / ml of fibrinogen (Calbiochem, San Diego, CA) was dissolved in M-199 medium, and the cultured ECV304 cells were suspended to a final concentration of 1 × 10 ⁵ cells / ml, followed by thrombin (0.5 unit / ml, Sigma). ) Was added to the 48-well plate in 500 [mu] l aliquot and the gel was hardened. After the gel was formed, various concentrations of arsenic trioxide were added to 500 μl of medium with or without bFGF (10 ng / ml), followed by incubation for 48 to 96 hours, followed by vascular formation.

Figure 6 (A) in the control group without bFGF stimulation, (B) shows angiogenesis in the group to which 10 ng / ml bFGF was added. The appearance of angiogenesis was observed with a phase contrast microscope and photographed. (C) and (D) show the inhibition of angiogenesis when treated with 0.1 and 1 μM arsenic trioxide, respectively. This experiment was repeated three times and the photographing fold was 40 times.

As shown in (B), when bFGF (10 ng / ml) was added, ECV cells were connected to each other to form a tube to form a complex network structure compared to the control. On the other hand, as shown in (D), the addition of arsenic trioxide significantly inhibited angiogenesis at 1 μM concentration. These results suggest that arsenic trioxide inhibits the differentiation that occurs in three-dimensional culture of endothelial cells.

Experimental Example 5

Inhibition of angiogenesis in vivo by arsenic trioxide

The inventors investigated whether arsenic trioxide actually has antiangiogenic activity in vivo . The measurement was conducted by examining whether or not angiogenesis induced by cornea was inhibited by 50 ng of bFGF when arsenic trioxide was orally administered to rats at 50 mg / kg / day for 7 days as described below.

In other words, rat corneal micropocket analysis was performed with a slight modification. Briefly, male Sprague-Dawley was anesthetized with ketamine and a cataract knife was used to make a pocket in the cornea 1-1.5 mm away from the limbus. The pellet was mixed with 30 μl saline added with 2 μg bFGF and 300 μg sucralate (Sigma) and suspended and 30 μl of 12% poly (2-hydroxyethyl methacrylate, Sigma) ethanol solution. Mixed with. This mixture was dipped in 3 μl of Teflon tape and dried to make 17 pellets to which finally about 50 ng of bFGF was added. The resulting pellets were placed in the corneal sac of anesthetized rats and sutured with ointment added with erythromycin.

Animals were orally administered arsenic trioxide powder at a concentration of 50 mg / kg daily, and the control group was given water only. Angiogenesis of the cornea was evaluated after 7 days. The area of blood vessels born in the retina was calculated by the length of blood vessels (L) and the number of hours (C) from the limbus, and was calculated by the formula C / 12 × 3.1416 [r2- (r-L) 2]. At this time, r means the measured diameter of the rat cornea, r = 5 mm.

Figure 7 (A) is a rat cornea of the control group not administered arsenic trioxide, it can be observed that a broad network of capillaries extending from the cornea to the bFGF-containing pellets. (B) is a cornea administered orally with 50 mg / kg of arsenic trioxide daily. Only the formation of weak blood vessels was observed, indicating that the angiogenesis of the cornea induced by bFGF was significantly reduced.

The lengths of the vessels, the perimeter of the vessels, and the area of the vessels are shown in (C), (D), and (E), respectively. The length of blood vessels (C) and the extent of neovascularization (D) were also reduced by 60% or more in 16 corneas compared to 16 control corneas (P = 0.0049, Mann-Whitney test). Treated rats were found to inhibit the overall angiogenesis area by about 90% (E) (P = 0.0071).

The experiment was conducted on 16 corneas of 8 rats each of the control and the sample. *, 0.01 <P <0.05, **, 0.001 <P <0.01.

In addition, corneal blood vessel concentration was also significantly lower in the arsenic trioxide treated group. Rats treated with arsenic trioxide did not show any weight loss or abnormal behavior within the experimental period, so the concentration of arsenic trioxide used in the experiment was higher than that of Spraugue. -Darwley) Rats are not considered to be very toxic.

result

As is clear from the above experiments, the present invention clearly clarifies the use of arsenic trioxide to strongly inhibit angiogenesis both in vtro and in vivo , and by using in vitro angiogenesis measurement methods, arsenic trioxide is induced by bFGF. It has been clarified that it effectively inhibits the multistage processes required for angiogenesis such as cell growth, migration, infiltration, and angiogenesis. Arsenic trioxide also showed anti-angiogenic effects in in vivo experiments using rat cornial micropocket assay.

One of the most important stages of angiogenesis is the growth of endothelial cells. The present invention found that low concentrations of arsenic trioxide treatment strongly inhibited the growth of BCE cells induced by bFGF, one of the potent angiogenesis factors (IC ₅₀ = 336.4). Since arsenic trioxide is generally known to be non-cytotoxic up to a concentration of 2 μM, the antiangiogenic effect of arsenic trioxide was not due to apoptosis or necrosis of endothelial cells.

In the present invention, it can be seen that arsenic trioxide stagnates cell cycle progression of BCE cells induced by bFGF at the G2 / M stage. However, in the absence of bFGF-induced arsenic trioxide, the G2 / M phase was very low, indicating that arsenic trioxide only stagnated the growth factor, i.e., cell cycle progression induced by bFGF, in the G2 / M phase. do.

Endothelial cell migration and local infiltration are important steps in the creation of new blood vessels. Endothelial migration and differentiation between cells on the ECM are important factors in completing angiogenesis. In this study, arsenic trioxide inhibited the migration and invasion of bFGF-induced BCE cells and inhibited angiogenesis of ECV304 cells on three-dimensional fibrin gels. This inhibitory effect of arsenic trioxide was also confirmed on in vivo mouse corneal micropocket analysis. All these data suggest that arsenic trioxide has a strong antiangiogenic effect.

Matrix metalloproteinases (MMPs) collectively refer to endopeptidases secreted extracellularly. These MMPs certainly act on the angiogenesis process. Among many MMPs, MMP-2 plays an important role in decomposing the basement membrane to promote endothelial invasion to achieve angiogenesis. Furthermore, endothelial angiogenesis has been reported to be dependent on the activity of MMP-2. Using the gelatin zymogram analysis, BCE cells secreted MMP-2 constitutively and treatment with arsenic trioxide inhibited MMP-2 secretion. These results suggest that arsenic trioxide inhibits bFGF-induced endothelial cell invasion and angiogenesis to some extent from inhibition of MMP-2 secretion.

Toxicity Test

Rats administered orally with arsenic trioxide at 50 mg / kg / day for 7 days showed no toxicity at all. Although the pharmacological toxicity of arsenic trioxide needs to be further studied, it can be said that arsenic trioxide has high therapeutic index.

According to the present invention, arsenic trioxide has been found to inhibit the proliferation, migration, invasion and angiogenesis of endothelial cells in vitro and to act as an antiangiogenic factor because of its inhibitory effect in an in vivo rat angiogenesis model. Thus, arsenic trioxide may be widely used for the treatment of diseases caused by angiogenesis.

Claims (1)

Angiogenesis inhibitor comprising arsenic trioxide as an active ingredient.