KR20070113532A - Parmaceutical composition for preventing restenosis comprising berbamine as active ingredient - Google Patents

Abstract

A composition comprising berbamine is provided to prevent restenosis after stent implantation without side effects by inhibiting propagation of vascular smooth muscle cell due to berbamine, thereby being widely utilized as a safer treatment of coronary artery diseases. A pharmaceutical composition for preventing restenosis comprises berbamine represented by the formula(1) as an effective ingredient and a pharmaceutically acceptable carrier. The composition is in the form of a capsule agent, a liquid agent, an injection agent, a soft capsule agent, a granule agent and a tablet.

Description

{Parmaceutical Composition for Preventing Restenosis Comprising Berbamine as Active Ingredient}

Figure 1a is a graph showing the cell proliferation inhibitory effect according to the treatment concentration of verbamin to aberrant proliferation of aortic smooth muscle cells by platelet-derived growth factor.

FIG. 1B is a graph showing the proliferation inhibitory effect of the verbamin-treated cells over time on the abnormal growth of aortic smooth muscle cells by platelet-derived growth factors.

Figure 2 is a graph showing the effect of inhibiting the DNA synthesis according to the concentration of the verbamin on the DNA synthesis of platelet-derived growth factor treated aortic smooth muscle cells.

The present invention relates to a vascular restenosis preventive agent comprising verbamin as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition for preventing vascular restenosis, which includes verbamin as an active ingredient and a pharmaceutically acceptable carrier, which can prevent vascular restenosis occurring after stent surgery. will be.

Heart diseases such as heart failure, coronary artery disease, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, and angina pectoris and cardiovascular diseases such as stroke and peripheral vascular disease occur in various age groups. It can also lead to death. In particular, due to the westernized diet, the incidence of coronary artery disease is rapidly increasing, and efforts to effectively treat coronary artery disease continue.

Treatments for coronary artery disease developed to date include drug therapy, gene therapy, percutaneous transluminal coronary angioplasty and stenting (PTCA), and coronary artery bypass graft (CABG). Revascularization therapy).

Recently, percutaneous coronary angioplasty and stent implantation (PTCA) have been widely used, but there is a problem that restenosis occurs in about 40% of patients after 3 to 6 months (see: Ryan et al., J. Am. Coll. Cardiol., 22: 2033-2054, 1993). The vascular restenosis promotes the migration and proliferation of vascular smooth muscle cells by producing and secreting various cytokines from vascular endothelial cells, activated platelets or macrophages damaged by balloon cavities, thereby promoting extracellular epilepsy. It is known that the production and secretion of the extracellular matrix (ECM) is promoted, which causes thickening of the vascular endothelium and is caused (Godfried et al, Am. Heart J., 129: 203-210, 1995).

On the other hand, vascular smooth muscle cells in a normal state does not proliferate, but the endothelium of the endothelial cells is damaged through stent surgery and the like, division, migration and proliferation of vascular smooth muscle cells are induced through various stages of signal transmission. The mechanisms of vascular smooth muscle cell proliferation include the elimination of proliferation inhibitory factor and the activation of proliferation-inducing factors due to normal endothelial cell damage, transmission of proliferation-induced signals through receptors on the surface of vascular smooth muscle cells, and proliferation delivered into the nucleus of vascular smooth muscle cells. And a change in the cell cycle caused by the trigger signal. Normal endothelial cells secrete substances that inhibit the proliferation of vascular smooth muscle cells.When endothelial cells are damaged, their secretion is suppressed, and platelet derived growth factor, which is released from activated platelets, is contained in plasma. It is known that proliferation of vascular smooth muscle cells is caused by various cytokines.

Various methods have been studied to prevent vascular restenosis after such a stent procedure. For example, Herdeg et al. Reported that Taxol may inhibit the development of vascular restenosis that occurs after stent implantation (Herdeg et al., Zeischrift fur Kardiologie, 89, 390-397, 1999). ), Korean Patent Registration No. 478671 discloses a pharmaceutical composition for preventing and treating vascular restenosis comprising clotrimazole as an active ingredient, and Korean Patent Registration No. 516026 discloses 3'-deoxyadenosine (3'-deoxyadenosine). The present invention discloses a composition for preventing and treating vascular stenosis as an active ingredient, and Korean Patent Publication No. 2001-110793 discloses an agent for preventing and treating vascular stenosis as an active ingredient of a compound having Rho kinase inhibitory activity. , Republic of Korea Patent Publication No. 2003-46314 discloses such as graft angiopathy, restenosis, in-stent restenosis and pulmonary hypertension with antithrombin as an active ingredient. A composition for preventing and treating vascular proliferative diseases is disclosed, and Korean Patent Laid-Open No. 2005-23249 discloses vascular restenosis after percutaneous coronary angioplasty using an endovascular stent containing retinoid and a retinoid action modifier. Or disclosed is a pharmaceutical composition for the prevention and / or treatment of vascular diseases, such as re-closure, the Republic of Korea Patent Publication No. 2005-43183 discloses a pharmaceutical composition for preventing and treating vascular restenosis comprising curcumin (curcumin) have.

However, in addition to preventing vascular restenosis, these vascular restenosis inhibitors have various side effects, such as inhibiting wound regeneration, vascular damage, hepatotoxicity, kidney damage, and increased bleeding by platelet aggregation. Although research is being actively conducted to develop a substance capable of inhibiting vascular restenosis derived from the various proven natural products, no research results have been reported.

Therefore, there is a constant need to develop materials that can safely and effectively inhibit vascular restenosis.

Accordingly, the present inventors have made a thorough research to develop a material that can safely and effectively inhibit vascular restenosis, and as a result, berbamin contained in some natural products inhibits the proliferation of vascular smooth muscle cells, and as a result, a stent procedure It was confirmed that the occurrence of vascular restenosis occurring later can be effectively suppressed, and the present invention has been completed.

After all, the main object of the present invention is to provide a pharmaceutical composition for preventing vascular restenosis with the active ingredient verbamin.

The present inventors have treated a wide variety of natural extracts in aortic smooth muscle cells of rats, search for substances which are capable of inhibiting the growth of said cells, goldthread (Coptis chinesis French.), Sobaek (Berberis amurensis Ruper . ), Stephania tetrandria S. Moore , Stephania japonica It was confirmed that hot water extracts of natural products such as Miers ) can inhibit the growth of the cells. Thus, as a result of analyzing the components included in each of the hydrothermal extract, it was found that each hydrothermal extract commonly includes verbamin having the following structural formula.

Therefore, the inventors of the present invention treated verbamin to aortic smooth muscle cells of rats to determine whether verbamin can effectively inhibit vascular restenosis occurring after stent implantation. Proliferation can be inhibited, DNA synthesis in aortic smooth muscle cells of rats can be suppressed in a concentration-dependent manner, and the ratio of cells in the resting phase (G ₀ / G ₁ ) in the cell division cycle can be increased. As a result, it was found that verbamin can effectively prevent vascular restenosis occurring after the stent procedure.

Therefore, the pharmaceutical composition for preventing vascular restenosis after the stent treatment of the present invention is a verbamin as an active ingredient and includes a pharmaceutically acceptable carrier.

At this time, the verbamin contained as an active ingredient of the prevention of vascular restenosis of the present invention is a pharmaceutically acceptable binder (eg, polyvinylpyrrolidone, hydroxypropyl cellulose), disintegrant (eg, carboxymethyl cellulose calcium, starch Sodium glycolate), diluents (e.g. corn starch, lactose, soybean oil, crystalline cellulose, mannitol), glidants (e.g. magnesium stearate, talc), sweeteners (e.g. white sugar, fructose, sorbitol, aspartame), stabilizers (carboxy) Sodium methylcellulose, alpha or beta cyclodextrin, vitamin C, citric acid, white lead), preservatives (e.g. methyl paraoxybenzoate, propyl paraoxybenzoate, sodium benzoate) and flavorings (e.g. ethyl vanillin, masking flavor, menthol flavono) , Herbal flavors) can be prepared into pharmaceutical preparations such as tablets, capsules, soft capsules, solutions, ointments or injections.

Verbamin Acute Toxicity Test

The mortality rate within 24 hours was examined by oral administration of sorbamine to 6-7 week-old non-rodent beagle dogs (beagle), with females 6-8 kg and males 7-9 kg. 8 animals each. As a result, dead individuals do not occur up to 5 g / kg, the verbamin of the present invention is safe enough to observe acute toxicity even up to 5 g per kg, it can be safely administered in vivo as a preventive agent for vascular restenosis.

Effective amount

In the present invention, the dosage of verbamin may be divided into 10 to 100 mg / kg body weight once or three times per day, depending on the age, sex, symptoms, administration method or prevention of the patient. Dosage levels for patients with specific symptoms may vary by those skilled in the art depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of disease, and the like.

The pharmaceutical composition for preventing vascular restenosis of the present invention can prevent vascular restenosis occurring after stent surgery without side effects, and thus may be widely used for the treatment of coronary artery disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Search for Natural Products Showing Vascular Restenosis Prevention Effect

Hot water extracts were obtained from various natural products that proved safety, and the extracts showing the effect of preventing vascular restenosis were searched.

First, immerse 1 kg of powder, ogapi, rhubarb, dermis, nabokjapan, Schisandra chinensis, bokbunja, longan meat, donkey, sobaek, tofu, sokdan, bunbanggi, baekpjap, brown root, embroider, gilyeong and health in 10ℓ of water, It heated at 90 degreeC for 5 hours, and it filtered and obtained the hot water extract. Subsequently, the electric hot water extract was concentrated and lyophilized to prepare a powder, and then dissolved in water at a concentration of 10 mg / ml to confirm the cell proliferation inhibitory effect as follows.

3.0 x 10 ⁴ aortic smooth muscle cells were incubated for 24 hours using DMEM medium containing 0.5% (v / v) fetal calf serum in 12 well plates. Subsequently, 10% (v / v) of the solution of each extract was added to the medium volume, incubated for another 24 hours, and then platelet-derived growth factor (PDGF-) at a concentration of 50 ng / ml. BB, Sigma Chem. Co., USA), and incubated again for 24 hours. After the incubation, the cultured cells were dispersed by trypsin treatment in each cultured cell, and the number of cells was measured by using a cell counter (see Table 1). At this time. As a positive control, cells cultured by treating only platelet-derived growth factors without treatment with natural hydrothermal extracts were used. As negative controls, cells cultured without treatment with both natural hydrothermal extracts and platelet-derived growth factors were used. .

Smooth muscle cell proliferation inhibitory effect of extract solution from each natural product (unit: x 10 ⁴ cells / ml) Natural products Cell count Natural products Cell count Positive Control Negative Control Acid Potion Ogapi Humiliation Dermis Locust Schisandra Bokbunja Longan Meat 36 16 33 30 20 31 34 33 35 35 Donkey sobaek baekchung quick split machine 33 21 32 30 19 34 35 18 33 31

As shown in Table 1, the extract solution from most natural products did not affect the proliferation of smooth muscle cells, but the extract solution from rye, sobaek, spinneret and disintegration significantly reduced the proliferation of smooth muscle cells. Could.

Therefore, using a variety of literature, as a result of searching for a component commonly contained in the barberry, cattle white, radiator and emollient group, it was confirmed that the natural product commonly includes berbamin (berbamine), It was predicted that abnormal growth of smooth muscle cells could be suppressed.

Example 2 Effects of Verbamin on Aortic Smooth Muscle Cell Proliferation

In Example 1, it was predicted that verbamin could inhibit aberrant proliferation of aortic smooth muscle cells. To confirm this, the treatment concentration of verbamin and the effect of inhibiting cell growth over time were compared.

Example 2-1 : Cell Proliferation Inhibitory Effect According to the Treatment Concentration of Verbamin

Incubated in the same manner as in Example 1, except that various concentrations (0.1, 1.0, 5.0 and 10.0 μM) of Verbamin (Sigma Chem. Co., USA) were added to the culture instead of the extract solution of each natural product. The cell number of smooth muscle cells was measured (see FIG. 1A). Figure 1a is a graph showing the cell proliferation inhibitory effect according to the treatment concentration of verbamin to aberrant proliferation of aortic smooth muscle cells by platelet-derived growth factor. As shown in Figure 1a, it was found that as the treatment concentration of verbamin increases, the degree of proliferation by platelet-derived growth factor of aortic smooth muscle cells is suppressed.

Example 2-2 : Cell Proliferation Inhibitory Effect of Verbamin Over Time

In order to confirm that the cell proliferation inhibitory effect of verbamin shown in Example 2-1 is continuously maintained, 10.0 μM of verbamin was added to the culture medium instead of the extract solution from each natural product, and the platelet-derived growth factor was treated. After that, except for culturing the cells for up to 72 hours, the cell numbers of the smooth muscle cells cultured in the same manner as in Example 1 were measured (see FIG. 1B). In this case, as a control, smooth muscle cells cultured without treatment with verbamin were used. Figure 1b is a graph showing the proliferation inhibitory effect over the cultivation time of the verbamin-treated cells for aberrant growth of aortic smooth muscle cells by platelet-derived growth factor, (○) represents a control, (◆) Represents an experimental group treated with verbamin. As shown in Figure 1b, it can be seen that the growth inhibitory effect of verbamin is continuously maintained over time.

Example 3 Inhibitory Effect of Verbamin on DNA Synthesis of Aortic Smooth Muscle Cells

It was intended to confirm whether the cell proliferation inhibitory effect of verbamin shown in Example 2 is related to the synthesis of DNA.

That is, after treating the platelet-derived growth factor and incubating for 20 hours, the same method as in Example 2-1 was applied except that 1 μl of [ ³ H] thymidine was added to the culture solution and cultured again for 4 hours. Aortic smooth muscle cells were cultured. Subsequently, the cultured cells were washed with PBS, and 500 μl of TCA (trichloroacetic acid) was added thereto and reacted for 30 minutes. Then, TCA was removed, washed with a washing solution (ethanol / ether, 1: 1 (v / v)), and 500 μl of NaOH was added to lyse the cells. Then, 5 ml of a scintillation cocktail (scintillation cocktail) was added to the cell lysate, and the radiation dose was measured to compare the relative amounts of the synthesized DNA (see FIG. 2). Figure 2 is a graph showing the effect of inhibiting the DNA synthesis according to the concentration of the verbamin on the DNA synthesis of platelet-derived growth factor treated aortic smooth muscle cells. As shown in Figure 2, it can be seen that the amount of DNA synthesis of aortic smooth muscle cells decreases as the amount of treatment of verbamin increases.

Therefore, it was found that the inhibition of proliferation of aortic smooth muscle cells by the verbamin shown in Examples 2-1 and 2-2 occurs because the verbamin inhibits DNA synthesis of the cells.

Example 4 Effect of Verbamin on the Development of Vascular Restenosis After Stent Surgery

It was confirmed whether the effect of the verbamin shown in Examples 2 and 3 can be applied to the actual stent procedure.

A stent (Humed, Korea) was inserted into four coronary vessels of 10 pigs 3 months old after birth, and a stent was inserted into 4 coronary vessels of 10 pigs 3 months after birth. Stents were inserted into four coronary blood vessels of the experimental group 1 and 10-month-old pigs at the dose of 100 mg / kg body weight / day of Verbamin of the present invention. Experiment group 2, in which the used taxol was administered at a dose of 1 mg / kg body weight / day, was bred for 4 weeks. After 4 weeks, the average weight change of each pig was measured, the stent-embedded coronary artery was extracted from the control and experimental pigs, and then the incidence of vascular restenosis was measured and compared with each other (see: Table 2). At this time, the average weight change was calculated as the average value of the weight of the pig after 4 weeks minus the weight of the pig before the stent procedure.

Comparison of inhibitory effects of vascular restenosis on the prevention of vascular restenosis Experimental group Incidence of Vascular Restenosis Average weight change (kg) Control Experimental Group 1 Experimental Group 2 68% 23% 55% 61 57 24

As shown in Table 2, the control group that was not administered the vascular restenosis preventive agent showed a vascular restenosis rate of about 68% after 4 weeks, but the experimental group 1 administered the verbamin of the present invention was about 23% after 4 weeks. The vascular restenosis rate was shown, and Experimental Group 2, which had been used as a conventional vascular restenosis prevention agent, showed a vascular restenosis rate of about 55% after 4 weeks. Therefore, it was found that the verbamin of the present invention exhibits superior vascular restenosis prevention effects than conventional vascular restenosis inhibitors.

In addition, since the pigs in the growing phase (3 months old) were used for 4 weeks of breeding, the weight of about 60 kg should be increased as in the control group, and the experimental group 1 administered the verbamin of the present invention was similar to the control group. Although the weight was increased, the experimental group 2 administered Taxol, which has been used as a conventional vascular restenosis preventive agent, was found to significantly reduce the increase in body weight, which was predicted to be due to side effects of Taxol.

Therefore, it was found that the verbamin of the present invention can safely and effectively prevent vascular restenosis.

Example 5 Formulation of a Pharmaceutical Composition for Preventing Vascular Restenosis

Verbamin of the present invention was mixed with auxiliaries such as excipients, binders, lubricants, disintegrants, diluents and the like to prepare a pharmaceutical formulation as follows.

Example 5-1 Preparation of Tablets

Verbamin 10 mg, lactose 20 mg, starch 20 mg and magnesium stearate appropriate amount was compressed into 50 mg tablets according to a conventional tablet production method to prepare a pharmaceutical composition for preventing tablet vascular restenosis.

Example 5-2 Preparation of Capsule

Verbamin 10 mg, lactose 20 mg, starch 19 mg, talc 1 mg and magnesium stearate are appropriately packed into a 50 mg capsule according to the method for preparing a capsule, thereby preparing a pharmaceutical composition for preventing vascular restenosis of the capsule formulation.

Example 5-3 Preparation of Liquid

Verbamin 100mg, isomerized sugar 10g, honey 500mg, nicotinic acid amide (pharmaceutical) 20mg, caffeine anhydrous (pharmaceutical) 30mg and sodium benzoate 70mg were prepared according to a conventional liquid preparation method, and filled into a 100ml brown bottle, By pasteurizing to prepare a pharmaceutical composition for preventing vascular restenosis in liquid form.

Example 5-4 Preparation of Injections

Verbamin 6 mg and a suitable amount of sterile purified water were prepared according to a conventional injection method, filled in 2 ml ampoules, and then sealed and sterilized to prepare a pharmaceutical composition for preventing vascular restenosis in the injection formulation.

Example 5-5 Preparation of Soft Capsules

In a shell composition prepared by using 52% by weight of gelatin, 32% by weight of glycerin, 35/7012% by weight of ANIDRISORB, and 5% by weight of water, 10 mg of verbamin, 230 mg of polyethylene glycol, and 13 mg of glycerin were added to a conventional soft capsule manufacturing method. Therefore, a pharmaceutical composition for preventing vascular restenosis of the soft capsule formulation was prepared.

Example 5-6 Preparation of Granules

10 mg of verbamin and 25 mg of lactose were extruded with a conventional extrusion granulator to prepare a pharmaceutical composition for preventing vascular restenosis in the form of granules.

As described and demonstrated in detail above, the present invention provides a pharmaceutical composition for preventing vascular restenosis, which includes verbamin as an active ingredient and a pharmaceutically acceptable carrier, which can prevent vascular restenosis occurring after stent surgery. To provide a composition. The pharmaceutical composition for preventing vascular restenosis of the present invention can prevent vascular restenosis occurring after stent surgery without side effects, and thus may be widely used for the treatment of coronary artery disease.

Claims (2)

A pharmaceutical composition for preventing vascular restenosis after a stent procedure, comprising verbamin as an active ingredient and a pharmaceutically acceptable carrier. A pharmaceutical for preventing vascular restenosis after stent surgery, which has verbamin as an active ingredient and includes a pharmaceutically acceptable carrier, and has one dosage form selected from capsules, solutions, injections, soft capsules, granules, and tablets. Composition.

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