KR100478671B1 - Pharmaceutical composition and stent for preventing and treating coronary restenosis comprising clotrimazole - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating vascular restenosis comprising clotrimazole as an active ingredient, and a stent to which the composition is applied. The pharmaceutical composition according to the present invention has aortic vascular smooth muscle cell proliferation inhibition and vasodilatation activity, which is useful for preventing and treating artery restenosis and also providing a drug-coated stent according to the present invention to facilitate coronary artery restenosis. Can be prevented and treated.

Description

Pharmaceutical composition and stent for preventing and treating coronary restenosis comprising clotrimazole}

The present invention relates to a pharmaceutical composition for preventing and treating vascular restenosis comprising clotrimazole as an active ingredient, and a stent to which the composition is applied.

Coronary artery disease is a condition in the blood vessels surrounding the heart that causes blood supply to the heart muscle. Atherosclerosis is the most common cause of coronary artery disease. Plasma, which is a combination of cholesterol, other fats, and other components in the blood, increases in the coronary artery, causing coronary artery stenosis. The blood supply is reduced, resulting in a lack of nutrients and oxygen. This can result in chest pain (angina) or myocardial infarction and, in severe cases, even death.

Percutaneous coronary angioplasty is a method of dilatating a narrowed coronary artery without surgery. The methods include percutaneous coronary balloon dilatation and percutaneous coronary stent insertion. Percutaneous coronary balloon dilatation involves the insertion of a guiding conduit through the femoral or arm arteries, and then through the aorta, the catheter is placed at the inlet of the coronary artery with the lesion. The catheter with the balloon attached to the end is placed in the stenosis of the coronary artery, and then the balloon is expanded. An expanded balloon is a method of squeezing plaque and expanding the narrowed coronary artery to improve blood flow of the coronary artery. In addition, the stent implantation is to place a wire mesh coated balloon in the stenosis, and then expand the balloon to apply a wire mesh to the inner wall of the coronary arteries. Since it has a characteristic of supporting the inner wall of blood vessels, it is used for the treatment of complications during balloon expansion.

The interventional procedure using coronary alloplasty is simple and can reduce the risk of general anesthesia and has a high success rate. In the case of blood vessels, patients with terminal cancer of the gastrointestinal tract or biliary tract may be preferentially applied when surgery or anesthesia itself is at high risk due to age, heart disease, respiratory disease, etc. It can play a role in improving the quality of life by improving the condition.

However, in the case of coronary angioplasty, the vascular endothelial hyperplasia is a process of vascular endothelial hyperplasia that is induced by injury. Restenosis occurs due to proliferation of cells in the inner membrane (neointima), accumulation of extracellular matrix (reendothelialization), apoptoisis, lysis, and the like. Restenosis may occur due to thrombosis or vasospasm in 6.8% of patients, more severe stenosis may occur in 3 to 6 months after vasodilation, and in 40% of patients, restenosis at balloon dilatation site (Herrman J-PR et al, Drugs, vol. 46, pp. 18-52, 1993). Vascular blockage may occur due to secondary changes in the vessels in the arterial and venous bypass areas and in 20% of endarterectomy sites of the coronary and femoral vessels (Volteas N et al., Int. Angiol , 2:; 13 (2), pp.143-7, 1994] In some cases, restenosis may occur due to fibrotic hyperplasia after the procedure, requiring vasodilation or bypass. Old age, recently started angina or unstable angina (Leimgruber PP et al., Circulation, vol. 73, p. 710, 1986).

In animals, unlike in animals, restenosis of blood vessels is associated with glucocorticoids, antiplatelet agents, antiserotonin agents, and anticoagulants. Anti-inflammatory agents, antihypertensive agents, antiproliferative agents, antithrombin agents similar to hirudin (Serruys PW et al., N Engl J Med, vol. 12 (333), pp.757-763, 1995), platelet IIb / IIIa complexes ( ReoPro) is known to have little response to antibodies, etc. [The ERASER Investigators. Acute platelet inhibition with abciximab does not reduce in-stent restenosis (ERASER study). Circulation 100, pp. 799-806, 1999).

New PTCA equipment has been introduced to prevent coronary restenosis, including atherostomy, laser angioplasty, rotablator, cutting balloon angioplast, and irradiation. Molecular biological therapies have been developed and attempted, such as various systemic and topical drug therapies such as antiplatelet agents, antithrombotic agents, vasodilators, cytostatic agents, lipid metabolism enhancers, antioxidants, and gene therapy. Of these, systemic drug therapies such as oral or intravenous administration have been reported to be the most easily applicable treatments, or have been reported to prevent restenosis in animal experiments, and do not reach the desired drug concentration at the site of PTCA. The side effects of the drug did not prevent restenosis in most clinical trials. Theoretically, restenosis occurs only in the coronary arteries of the localized site where PTCA has been applied, so local drug therapies can be used to prevent high levels of restenosis. therapy is more useful.

Recently, in order to directly administer the drug to the PTCA site, a double balloon catheter, a dispatch or a microporous balloon has been developed and used in clinical practice, and to deliver the drug to the PTCA procedure for a long time. Attempts are being made to treat slow-release microspheres or stents with drugs. However, to date no pharmaceutical composition has been reported that significantly reduces restenosis occurring after PTCA.

Conventionally, as a composition for preventing and treating coronary artery stenosis, the composition relates to catechin, which is a green tea extract described in Korean Laid-Open Publication No. 2001-84811. In addition, currently used coatings are rapamycin (rapamycin), paclitaxel (paclitaxel), silorimus (silorimus), verapamil (verapamil), these drugs are expensive and did not have vasodilating activity.

Therefore, the cost is low, solubility in organic solvents is easy to apply to the actual stent, etc., the situation is required for the prevention and treatment of vascular restenosis prevention and treatment excellent vascular stenosis as well as cell growth inhibitory effect.

In order to solve the problems of the prior art as described above, an object of the present invention is to provide a composition for preventing and treating vascular restenosis comprising clotrimazole having excellent vascular growth inhibition and vascular dilatation as an active ingredient.

It is also an object of the present invention to provide a stent to which the composition is applied.

It is also an object of the present invention to provide a quantitative method for preventing restenosis applied to the stent.

In order to achieve the above technical problem, the present invention relates to a pharmaceutical composition for preventing and treating coronary artery restenosis comprising clotrimazole as an active ingredient.

The present invention also relates to an angioplasty stent to which the pharmaceutical composition is applied.

The present inventors have used various methods to prevent restenosis occurring after angioplasty procedure, but to solve the problem that the restenosis rate is still high, clotrimazole, which is widely known as an antifungal agent, inhibits the growth of vascular smooth muscle cells and decreases vasodilation activity. The present invention was completed to prepare a composition for preventing and treating coronary artery restenosis containing the clotrimazole as an active ingredient and to develop a stent coated with the composition.

Hereinafter, the present invention will be described in detail.

The active ingredient of the composition for preventing and treating vascular restenosis of the present invention is clotrimazole, which is a representative antifungal agent. In the present invention, clotrimazole may be purchased and used in a commercially available product, or may be prepared directly by a conventional manufacturing method. The composition for preventing and treating vascular restenosis according to the present invention may be determined in consideration of the purpose of preventing and treating vascular restenosis, and treatment, a condition of a patient, a period of time required, and the like, and may include 5 uM or more of clotrimazole. In one embodiment of the present invention, the content of clotrimazole in the composition can inhibit cell growth at 10-30 μM in vitro, and is about 50-100 μg / stent in vivo. Cell death may be a problem in vitro if the content is out of the range, may cause inflammation in vivo to increase restenosis.

Clotrimazole is an imidazole derivative and is a blocker of Ca ⁺⁺ influx pathway promoted by voltage and ligand in nucleated cells (Villalobos C. et al., FASEB, J6, pp. 2742-2747, 1992). the serves to prevent Ca ^++, K ⁺ go into it makes a K ⁺ channel that is stimulated by Ca ⁺⁺ cells. Clotrimazole is known to prevent cell growth by depleting intracellular Ca ⁺⁺ storage in several cancer cells as well as normal cells and by inhibiting somatic cell stimulating agents induced by cytosolic Ca ⁺⁺ . It has been reported to reduce the metastatic area of melanoma cells (Alvarez J et al., J Biol Chem, vol. 267, pp.11789-11793, 1992), and the effect on clotrimazole is not only in vitro but also in vivo. It has been proven effective. Another view on the effect of growth inhibition by clotrimazole is that clotrimazole is known to inhibit cell growth by blocking the expression of EGF (Epidermal Growth Factor), which promotes DNA synthesis as an inhibitor of cytochrome p-450. (Benzaquen LR et al., Nature Med, vol. 1, pp. 534-541, 1995).

Clotrimazole is selected as a stent coating material to prevent restenosis, and clotrimazole is used to inhibit the growth of vascular smooth muscle cells (VSMC) in vitro. In addition, there is an effect of inhibiting cell growth even when treated in cells in the resting state before the vascular wall is damaged or in the state of stopping cell division after restenosis occurs.

Clotrimazole may be useful for preventing and treating vascular restenosis. First, clotrimazole has the advantage of strongly inhibiting the growth of normal cells as well as cancer cells. It is considered to be able to suppress. Second, clotrimazole may have an effect of vasodilation because of its vasodilation effect. This is a function that has not been in the coating material so far, the clotrimazole will have a better effect due to two effects, growth inhibition and vasodilation. Third, clotrimazole is less than 50 to 1000 times cheaper than currently used coatings (e.g. rapamycin, paclitaxel, silorimus, verapamil). High solubility in organic solvents such as ethanol, dimethyl sulfoxide and the like can easily coat a large amount of drug has advantages in the manufacturing method.

The pharmaceutical composition of the present invention can be used for the prevention and treatment of coronary artery stenosis caused by balloon angioplasty such as percutaneous coronary angioplasty, stent insertion, coronary artery bypass surgery, arterial-vein anastomosis, and the like. For example, the pharmaceutical composition of the present invention may be used in the same way as a general medicament or applied to a stent.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, and may be prepared as a medicament by adding 1 to 100 parts by weight to one or more pharmaceutically acceptable carriers based on the total weight of the composition. The carrier may include, but is not limited to, saline, buffered saline, water, glycerol and ethanol, and any suitable agent known in the art (Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA) may be used. .

The pharmaceutical composition may be prepared in the form of a liquid, a liquid extract, an emulsion, a suspension, a cream, and the like, and may be used parenterally. Dosage of the composition is a conventional dosage of the anti-coronary anti-stenosis agent, for example, it may be used clotrimazole of 10 ug / stent to 50 ug / stent per day. The dosage is not limited thereto, and it is preferable to be applied differently according to the age, sex, condition of the patient, absorbance of the active ingredient in the body, inactivation rate, combined drugs, and the like.

The present invention also relates to a stent to which the pharmaceutical composition for preventing and treating vascular restenosis, which includes the clotrimazole as an active ingredient, is applied.

In the present invention, the term stent is intended to mean a general device for endoluminal application, for example, intravascular application. Preferably the vascular stent is described in Eric J Topol, "Textbook of Interventional Cardiology", Saunders Company, 1994.

The method for coating the pharmaceutical composition according to the present invention on the stent is intended to include all conventional coating methods known to those skilled in the art. For example, dip-coated and polymer coated together with polymer (Polymer cocoated) is the method, dip coating is the simplest coating method, because only the pharmaceutical composition is coated to observe the biological effect of the drug only It is easy to

In order to quantify the active ingredient coated on the stent, a wavelength representing the maximum absorbance of clotrimazole is obtained, and at this maximum wavelength, the absorbance of each concentration of clotrimazole is calculated to draw a standard curve, and the clotrimazole coated on the stent. It is a method of measuring the absorbance by releasing and quantifying using the standard curve. All compounds show a change in absorbance according to the wavelength. In the case of a substance having an aromatic functional group, the wavelength showing the maximum absorbance shifts toward the longer wavelength. Clotrimazole has a large number of aromatic functional groups, so maximum absorption occurs at 230 nm.

The present invention will be described in more detail with reference to the following examples, but the following examples are provided only to illustrate the present invention and are not intended to limit the scope of the present invention to the following examples.

Example 1

Effect of Clotrimazole on VSMC Cell Growth

Proliferation was inhibited.

1-1: VSMC Culture in Rett Aorta

After 4 weeks of SD male euthanasia, the midline of the chest was opened and the vessels from the base of the aorta pointing down to the left side of the spine to the bottom of the kidney were separated and placed in cold DMEM / 12 medium. The outer membrane was removed and washed with forceps in a 100 mm dish. Add 5 ml of the enzyme mixture (1mg / ml Collagenase type II, 0.25mg / ml Elastase, 1mg / ml Trypsin inhibitor, 2mg / ml BSA) warmed to 37 ℃ and incubate at 37 ℃ for 5 minutes. The reaction envelope was taken out again and the envelope was stripped. After washing the blood vessels remaining only in the interlayer, and finely chopped to 2 mm interval, 10-15 ml of the enzyme mixture was added and incubated for 30 minutes at 37 ℃. After the decomposition was completed, the pellet was centrifuged at 1000 rpm for 5 minutes, suspended in DMEM / F12 + 10% FBS + 0.1mg / ml streptomycin / penicillin medium, and then dispensed into a 25T flask. After 3 days, exchanged with DMEM / F12 + 10% FBS medium and on day 5-6, VSMC (vascular smooth muscle cell) was removed with trypsin-EDTA solution and dispensed 1: 3. Frozen up to five passages were used and stored for up to ten passages.

1-2: Changes in the growth curve of VSMC by clotrimazole by date

The number of cells was measured daily to see how 10 uM clotrimazole, known to block cell cycles, affects VSMC cell growth for three days.

2 * 10 ⁴ cells (24-well standard) per well were dispensed and incubated in a 37 ° C. incubator in DMEM / F12 + FBS 10% medium for one day to allow cells to adhere to the flask. After washing the medium with PBS, DMEM / F12 + FBS 10% medium with 10 μM of clotrimazole was added, and DMSO corresponding to the volume of DMEM / F12 + FBS 10% medium and clotrimazole was added to the control group. . The number of cells was observed using a hematocytometer (hemacytometer) every day for 3 days, and the results are shown in FIG. 1.

As shown in FIG. 1, in the case of the control vascular smooth muscle cells, the growth rate was slowed by filling the 24-well with 200% cell growth after 2 days, but the growth rate even after 3 days in the group treated with clotrimazole 70% of the growth slowed down.

Example 2

Variation of VSMC Growth Curve with Clotrimazole Concentration

In order to determine the growth of clotrimazole concentration, the growth of clotrimazole concentration in DMEM / F12 + FBS 10% medium was measured.

2 * 10 ⁴ cells (24 wells) per well were dispensed and incubated in DMEM / F12 + FBS 10% medium 37 ° C. 0.5% CO ₂ incubator for one day to allow the cells to adhere to the flask. The medium was washed with PBS, and then treated with clotrimazole 1uM, 10uM, 30uM, 50uMm and 100uM, and DMSO corresponding to the volume of clotrimazole dissolved in the medium was added to the control group. Two days later, the number of cells was observed using a hemocytometer, shown in the graph of FIG. 2A. In addition, micrographs of the control group 2b, the control group 2c after 2 days, and the experimental group 2d after 2 days of treatment with 30 uM of clotrimazole are shown in FIGS. 2b to 2d.

Clotrimazole is less than 1uM cell growth inhibitory effect is small, but the cell growth inhibitory effect increases with increasing concentration, the cell number tends to decrease based on about 30uM. At 50 uM and 100 uM all cells were killed.

Example 3

3-1: Effects on resting cells

We observed the effect of clotrimazole on cells in the resting phase before the vessel wall was damaged or in the state of stopping cell division after restenosis. After dispensing the cells, the cells were cultured to allow the cells to settle for one day, followed by rinsing the medium and replacing with DMEM / F12 + BSA 0.2% or DMEM / F12 + FBS 0.5% medium to incubate the cells for two days. The medium was washed, the same medium was treated, and clotrimazole was administered at the same concentration as in Example 2. After 2 days, the number of cells was measured and a growth curve was drawn according to the concentration. As shown in Figure 3, all cells were killed at clotrimazole concentration of 10 uM or more.

3-2: in vivo  Effect of Clotrimazole in a Mimic Model

When cells in the resting phase are damaged, growth is stimulated by stimulation of growth factors such as platelet-derived growth factor (PDGF), which is similar to 2% fetal bovine serum rather than normal cell culture condition of 10% fetal bovine serum. Resting cells were made for one day prior to injecting the drug and then activated with 2% fetal bovine serum and treated with clotrimazole at the same concentration as in Example 2. After dispensing the cells, the cells were allowed to settle for one day, followed by rinsing the medium and replacing with DMEM / F12 + BSA 0.2% or DMEM / F12 + FBS 0.5% medium to incubate for 2 days. Damage was created to create conditions similar to when growth was accelerated (DMEM / F12 + FBS 2%). Clotrimazole was treated at a concentration of 1-100 uM. After two days, the cell number was measured, and a growth curve according to the concentration change was drawn and shown in FIG. 3.

Cell growth was inhibited at a concentration of clotrimazole of about 10 uM and all cells were killed at a concentration of 30 uM or higher.

Example 4: Clotrimazole Coated Stents

According to the dip coating method, 50 mg / μl of clotrimazole was dissolved in alcohol, soaked the stent for 30 seconds, and then dried. The drug coating was confirmed by observing the stent surface using a scanning electron microscope (SEM). SEM photographs of the stent surface 20 and 1500 times before coating the drug are shown in FIGS. 4A and 4B, and SEM photographs of 500 times the clotrimazole coated stent are shown in FIG. 4C. As a result of tip-coating clotrimazole, it was confirmed that the whole stent was not evenly coated in the form of a layer, but was coated in the form of granules.

Example 5: Quantitative Clotrimazole Coating on Stents

All compounds show a change in absorbance according to the wavelength. In the case of a substance having an aromatic functional group, the wavelength showing the maximum absorbance shifts toward the longer wavelength. The absorbance curve for each wavelength was obtained by experimenting with a UV-spectrophotometer and is shown in FIG. 5A. Clotrimazole had a large number of aromatic functional groups, resulting in maximum absorption at 230 nm. Clotrimazole having a concentration of 500 µg / ml was obtained at 230 nm, and a standard standard curve was obtained by FIG. 5B.

After coating the stent at various concentrations by the dip coating according to Example 4, chlorotrimazole was released by dipping the stent in 2 ml of ethanol. 200 μl of the ethanol solution was taken to measure absorbance at 230 nm, and the absorbance values observed using the standard curve were used to quantify clotrimazole coated on the stent.

 The composition of the present invention has aortic vascular smooth muscle cell proliferation inhibition and vasodilatory activity to prevent artery restenosis and to be useful in the treatment, and also provides a drug-coated stent according to the present invention to easily prevent coronary artery restenosis and It can be cured.

1 is a graph showing VSMC cell growth according to elapsed time after treating clotrimazole according to Example 1. FIG.

2A is a graph showing VSMC cell growth according to the concentration of clotrimazole treated according to Example 2, and FIGS. 2B to 2D are micrographs of the control group, the control group after 2 days, and the clotrimazole treatment group after 2 days of curing. Represents a picture.

Figure 3 is a graph showing the growth of resting cells according to the treatment concentration of clotrimazole according to Example 3.

4a to 4c are SEM images of the surface of the stent coated according to Example 4, and FIGS. 4a and 4b are 20 times and 1500 times SEM images of the stent before coating, and FIG. 4c is 500 of the stent coated with clotrimazole. Magnification SEM picture.

Figure 5a is a graph showing the absorbance size of clotrimazole according to the wavelength of light according to Example 5, Figure 5b is a graph showing the absorbance size according to the concentration of clotrimazole.

Claims (4)

delete delete   A stent coated with a pharmaceutical composition for preventing and treating coronary artery stenosis containing clotrimazole as an active ingredient. The stent of claim 3, wherein the coating of the composition is dip-coating or polymer co-coating.