US 2008O139668A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0139668A1 KWOn et al. (43) Pub. Date: Jun. 12, 2008

(54) PHARMACEUTICAL COMPOSITION FOR (73) Assignees: Korea Research Institute of Bioscience and Biotechnology, OBOVATOL FOR THE PREVENTION AND Daejeon (KR); Chungbuk National TREATMENT OF RESTENOSIS University Industry-Academic Cooperation Foundation, (75) Inventors: Byoung Mog Kwon, Daejeon (KR): Chungcheongbuk-do (KR) Yeo-Pyo Yun, Chungcheongbuk-Do (21) Appl. No.: 11/951,710 (KR); Yong Lim, Daejeon (KR): (22) Filed: Dec. 6, 2007 Dong-Woon Kim, (30) Foreign Application Priority Data Chungcheongbuk-Do (KR): Dec. 12, 2006 (KR) ...... 10-2006-O126449 Jin-Sook Kwon, Chungcheongbuk-Do (KR): Publication Classification Seung-ho Lee, Chungbuk (KR): (51) Int. Cl. A63L/05 (2006.01) Jin-Tae Hong, A6IP 9/00 (2006.01) Chungcheongbuk-Do (KR) (52) U.S. Cl...... S14f736 (57) ABSTRACT Correspondence Address: Disclosed herein is a pharmaceutical composition for the LUCAS & MERCANTI, LLP prevention and treatment of restenosis following a blood ves 475 PARKAVENUE SOUTH, 15TH FLOOR sel injury procedure, comprising obovatol as an active ingre NEW YORK, NY 10016 dient.

Patent Application Publication Jun. 12, 2008 Sheet 1 of 10 US 2008/O139668A1

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FGURE 9

-- control d -- obovatol ll -- obovatol 31 M --- obovatol 51

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PHARMACEUTICAL COMPOSITION FOR helial cells are injured, the secretion is restrained while the OBOVATOL FOR THE PREVENTION AND proliferation of vascular smooth muscle cells is induced by TREATMENT OF RESTENOSIS platelet-derived growth factors, secreted from activated plate lets and by various cytokines present in plasma. BACKGROUND OF THE INVENTION 0009 Various methods for preventing restenosis follow 0001 1. Field of the Invention ing stenting (“post-PTCA restenosis) have been studied. For 0002. The present invention relates to PHARMACEUTI example, Herdeg et al. reported that taxol is effective for the CAL COMPOSITION FOR OBOVATOL FOR THE PRE prevention of restenosis following angioplasty (Herdegetal. VENTION AND TREATMENT OF RESTENOSIS. More Zeischrift fur Kardiologie, 89,390-397, 1999). Korean Patent particularly, the present invention relates to a pharmaceutical No. 478671 discloses a pharmaceutical composition for the composition useful in the prevention and treatment of rest prevention and treatment of restenosis, comprising clotrima enosis following a stenting procedure, comprising obovatol Zole as an active ingredient. Also, disclosed area composition as an active ingredient and a pharmaceutically acceptable for the prevention and treatment of restenosis comprising carrier. 3'-deoxyadenosine in Korean Patent No. 516026, an anti 0003 2. Description of the Related Art restenosis composition comprising an Rhokinase inhibitor in 0004 Cardiovascular diseases, such as cardiac failure, Korean Patent Laid-Open Publication No. 2001-1 10793, and coronary artery disease, hypertensive heart disease, arrhyth antithrombin for the prevention and therapy of vasculoprolif mia, congenital heart defects, myocardial infarction, angina erative disorders, such as restenosis, in-stent restenosis and pectoris, apoplexy, and peripheral vascular (arterial) disease, pulmonary hypertension, in Korean Patent Laid-Open Publi afflict persons of various ages and, unless treated appropri cation No. 2003-46314. Korean Patent Laid-Open Publica ately, leave serious sequelae or lead to death. Particularly, the tion No. 2005-23249 provides medicament for prophylactic morbidity of coronary artery diseases has recently sharply and/or therapeutic treatment of a vascular disease such as increased with the westernization of the Korean diet. Thus, vascular restenosis and/or reocclusion after percutaneous many attempts have been made to develop effective therapy transluminal coronary angioplasty using an intravascular for coronary artery diseases. stent, which comprises as an active ingredient a retinoid oran 0005 Examples of the therapies for coronary artery dis agent for controlling the action of retinoids. Another pharma eases developed thus far include chemical therapy, gene ceutical composition for the prevention and treatment of res therapy, and revascularization therapy. Such as non-Surgical tenosis comprising curcumin is described in Korean Patent percutaneous transluminal coronary angioplasty and stenting Laid-Open Publication No. 2005-43183. (PTCA) and surgical coronary artery bypass graft (CABG) 0010. However, the above-mentioned anti-restenosis 0006 Thanks to advantages in that it is less invasive and agents Suffer from the disadvantages of wound healing Sup more cost effective, percutaneous transluminal coronary pression, vascular injury, hepatotoxicity, nephrotoxicity, and angioplasty and stenting (PTCA) has become a widespread hemorrhage increase by platelet aggregation inhibition. technique for the treatment of coronary artery disease. How Accordingly, active study has been conducted into the devel ever, the utility of percutaneous transluminal coronary angio opment of anti-restenosis agents from various natural mate plasty (PTCA) is limited by a high incidence of restenosis rials confirmed to be safe to humans. No outstanding results following the procedure (post-PTCA restenosis), which have been reported thus far. occurs in as many as 40% of cases within 3 to 6 months of the 0011. Therefore, there is a need for natural materials that procedure (Ryan et al., J. Am. Coll. Cardiol., 22. 2033-2054. can effectively prevent restenosis and are safe for the human 1993). body. 0007. It is well documented that chronic or acute injury 0012. Of the natural materials, obovatol was found to be (such as from a balloon used in PTCA) to the arterial wall able to inhibit the proliferation of vascular smooth muscle induces the expression of a variety of growth factors and cells, as a result of the study of the present inventors. A inflammatory cytokines that stimulate Smooth muscle cell pharmaceutical composition comprising obovatol was (SMC) proliferation and migration from the media into the already disclosed in Korean Patent Publication No. 2006 intima, with the synthesis and secretion of extracellular 115454, issued to the present inventors, but is directed to the matrix (ECM), resulting in neointimal formation and even prevention and treatment of anxiety. tual restenosis (Godfried et al. Am. Heart J., 129, 203-210, 0013 Leading to the present invention, intensive and thor 1995). ough research into a safe anti-restenosis material, conducted 0008 While not proliferating under normal conditions, by the present inventors, resulted in the finding that naturally vascular Smooth muscle cells are induced to differentiation, migration and proliferation by signals transduced through occurring obovatol can inhibit the proliferation of vascular multiple stages when the medial endothelial cells are injured Smooth muscle cells, thus being useful in the prevention of by, for example, stenting. The removal of cell proliferation restenosis following stenting. inhibitors and the activation of cell proliferation-stimulating factors, which occur upon the injury of normal endothelial SUMMARY OF THE INVENTION cells, may explain the mechanism of vascular Smooth muscle cell proliferation. For the mechanism, the transduction of 0014. Accordingly, the present invention has been made proliferation-stimulating signals through receptors on Vascu keeping in mind the above problems occurring in the prior art, lar Smooth muscle cell and the change in cell cycle induced by and an object of the present invention is to provide an agent the proliferation-stimulating signals transferred to the nuclei effective in the prevention and treatment of restenosis and of vascular smooth muscle cells are also responsible. Normal safe for the human body. endothelial cells secrete factors inhibiting the proliferation of 0015. In order to accomplish the above object, the present vascular Smooth muscle cells. It is known that when endot invention provides a pharmaceutical composition for the pre US 2008/0139668A1 Jun. 12, 2008

vention and treatment of restenosis following a blood vessel fractionation (Harborne J. B. Phytochemical methods: A injury procedure, comprising obovatol as an active ingredi guide to modern techniques of plantanalysis., 3rd Ed., pp 6-7, ent. 1998). 0016. The blood vessel injury procedure includes percu 0032 For instance, the purified, non-polar solvent extract taneous transluminal coronary angioplasty, balloon angio plasty, stent insertion, coronary artery bypass graft Surgery, is concentrated in a vacuum, and the concentrate was frac and/or arteriovenous anastomosis. tioned in a mixture of 1:1 ethylacetate:water. The organic 0017. The pharmaceutical composition is in the dosage layer thus formed is concentrated and the residue is purified form of a capsule, a liquid, an injection, a soft capsule, a by silica gel column chromatography using a mixture of granule or a tablet. and and eluted with an elution solvent 0018. The obovatol useful in the present invention is of various ratios (9:1–6:4) of chloroform and methanol. The derived from an extract from leaves of Magnolia obovata. resulting eluate was purified by C18 column chromatography, The compound may be isolated from the leaves of Magnolia preferably using a solvent mixture of 4:1 methanol: water, and obovata as will be described below, or may be synthesized then by HPLC on a Phenomenex Ultracarb 10 ODS column according to a method well known in the art. (250x21.2 mm) using an elution solvent mixture of 4:1 methanol: water to afford obovatol, which is represented by BREIF DESCRIPTION OF THE DRAWINGS the following 1. 0019. The above and other objects, features and other advantages of the present invention will be more clearly Chemical Formula 1 understood from the following detailed description taken in CH2 n. OH conjunction with the accompanying drawings, in which: 0020 FIG. 1 shows the injured carotid arteries of control rat in cineangiography (x100), OH 0021 FIG. 2 shows the injured carotid arteries of obova tol-treated rat in cineangiography (x100) O 0022 FIG. 3 shows the injured carotid arteries of control rat in cineangiography (X.400), 0023 FIG. 4 shows the injured carotid arteries of obova tol-treated rat in cineangiography (x400), 0024 FIG. 5 is photograph showing cross sections of the injured carotid arteries of a control rat (x40), 0025 FIG. 6 is photograph showing cross sections of the injured carotid arteries of an obovatol-treated group, respec CH2 tively (x40) 0026 FIG. 7 is photograph showing cross sections of the injured carotid arteries of a control rat (x400), 0033. In order to examine whether obovatol can effec 0027 FIG. 8 is photograph showing cross sections of the tively Suppress restenosis following a stenting procedure, a injured carotid arteries of an obovatol-treated group (x400). histopathological analysis was conducted with a rat carotid 0028 FIG. 9 is a graph showing the inhibitory effect of artery injury model in which obovatol was applied topically obovatol on the platelet-derived growth factor-induced to an injured locus of the exima, showing that neointimal hyperplasia of the arterial Smooth muscle cells in a dose hyperplasia was prevented in obovatol-treated groups in con dependent over time, trast to a control group. When it was applied to arterial Smooth 0029 FIG. 10 is a graph showing the inhibitory effect of muscle cells, which play an important role in restenosis, obovatol on DNA synthesis in arterial smooth muscle cells obovatol was observed to inhibit the proliferation of vascular treated with platelet-derived growth factor. Smooth muscle cells in a dose-dependent manner. It was also observed that obovatol inhibited DNA synthesis in arterial DESCRIPTION OF THE PREFERRED Smooth muscle cells in a dose-dependent manner and EMBODIMENTS increased the proportion of cells in a resting state (Go/G) in the cell cycle. Therefore, obovatol is proven to inhibit vascu 0030 Below, a detailed description will be given of the lar Smooth muscle cell proliferation following stenting pro present invention. cedure, thereby being able to prevent and treat restenosis 0031 Obovatol can be prepared from Magnolia obovata. effectively. For this, leaves of Magnolia obovata are dried in a shady 0034 Leaves of Magnolia obovata have been used as a place, sliced, and added to 2 to 20 volumes of a non-polar diet or for their therapeutic or medicinal value. Accordingly, Solvent, such as hexane, chloroform, ethyl acetate, , etc., or a mixture of 1:1.0 to 1:10 of water and a non-polar extracts or compounds from the leaves are not toxic and cause solvent, and preferably to 2 to 20 volumes of chloroform, no side effects. followed by extraction at 25°C. for 24 hours. The extraction 0035. In accordance with the present invention, there is can be conducted by cold precipitation, reflux condensation, provided a pharmaceutical composition for the prevention or ultrasonication, and is preferably conducted by cold pre and treatment of restenosis, comprising the obovatol com cipitation. The resulting extract, which is soluble in the non pound isolated from the leaves of Magnolia Obovata. polar solvent, is fractionated and washed many times with 0036. For use in the prevention and treatment of restenosis distilled water and purified, optionally followed by typical following a stenting procedure, the pharmaceutical composi US 2008/0139668A1 Jun. 12, 2008 tion according to the present invention comprises obovatol as organic fractions were pooled, along with the organic layer an active ingredient in combination with a pharmaceutically The resulting organic pool was concentrated in a vacuum to acceptable carrier. form 180g of a concentrate. This concentrate was dissolved 0037 Obovatol, serving as an active ingredient, may usu in 500 ml of methanol and adsorbed into 500 g of C18, ally be formulated in combination with various pharmaceu followed by elution with 1 liter of a solvent mixture of 4:1 tically acceptable carriers or excipients into tablets, capsules, methanol: water to give an active fraction. After this active Soft capsules, liquids, ointments, or injections. Examples of fraction was concentrated in a vacuum, 100 of the concentrate the pharmaceutically acceptable carriers or excipients useful was dissolved in methylene chloride. This solution was in the present invention include binders (e.g., polyvinylpyr loaded onto a column (4.5x40 cm) filled with 1 kg of silica rolidone, hydroxypropylcellulose), disintegrants (e.g., cal gel (Merck 93.85 Silica Gel) along with a solvent mixture of cium carboxymethyl cellulose, Sodium starch glycolate), 9:1 hexane:ethyl acetate, followed by two rounds of silica gel diluents (e.g., corn starch, lactose, bean oil, crystalline cellu column chromatography using a solvent mixture of hexane lose, mannitol), lubricants (e.g., magnesium Stearate, talc), and ethyl acetate (ratio varying from 9:1 to 6:4) as an eluent. Sweeteners (e.g. white Sugar, Sucrose, Sorbitol, aspartame), Further purification of the active eluent through HPLC stabilizers (e.g., Sodium carboxymethyl cellulose, alpha or yielded 1 g of an obovatol compound showing the following beta cyclodextrin, Vitamin C, citric acid, beeswax), preserva physical properties. tives (e.g. paraoxybenzoic acid methyl, paraoxybenzoic acid 0045. Obovatol propyl, Sodium benzoate) and/or flavorings (e.g. ethyl Vanil 0046 Empirical Formula: C.H.O. lin, masking flavor, flavono, herb flavor, etc.) 0047. Mass: M*=282 0038. Ovobatol may be administered at a dosage of 0.0001 0048 H-NMR (400 MHz, CDC1) d ppm: 6.28(H-4, d. to 100 mg per kg of body weight in one dose or in two or three J=1.8 Hz), 6.56 (H-6, d. J=1.8 Hz), 3.18 (H-7, d. J=6.61 Hz), doses a day, depending on various factors including patients 5.97(H-8 and H-8', m), 5.09 (H-9 and H-9', m), 6.93 (H-2 and age, sex and symptom, administration route, and administra H-6', d, J=4.3 Hz), 7.14 (H-3' and 5', d, J=4.3 Hz), 3.36 (H-7', tion purpose. It will be apparent to those skilled in the art that d, J=6.6 Hz); 'C-NMR(100 MHz, CDC1) dippm: 143 (C-1) the suitable total daily dose may be determined by an attend 132.93 (C-2), 144.77 (C-3), 110.68 (C-4), 132.47 (C-5), ing physician within the scope of Sound medical judgment. 11.17(C-6), 39.60 (C-7), 137.33(C-8), 115.85(C-9), 15498 The specific therapeutically effective dosage level for any (C-1'), 117.84 (C-2 and 6'), 129.82 (C-3' and 5'), 135.18(C- particular patient may vary depending on a variety of factors, 4"), 39.38 (C-7), 137.18 (C-8), 115.75 (C-9'). including the kind and degree of a desired reaction, the spe 0049. In the following Examples, values are expressed as cific composition, including the use of any other agents meantstandard errors (meantS.E.). For a significance test according to the intended use, the patient’s age, weight, gen of data, an unpaired Students T-test was used. Each test was eral state of health, gender, and diet, the time of administra independently conducted at least three times. Values of p-0. tion, route of administration, and rate of excretion of the 05 were considered statistically significant. composition; the duration of the treatment; other drugs used in combination or coincidentally with the specific composi EXAMPLE 2 tion; and other factors well known in the medical arts. 0039. The pharmaceutical composition in accordance Test of Obovatol for Prevention of Restenosis in with the present invention can prevent or treat the restenosis Injured Carotid Artery following vascular injury-accompanied procedures including 0050 (Step 1). Preparation of Obovatol-Containing Plu stenting, without side effects, and thus can find various appli ronic Gel cations in the treatment of coronary arterial diseases. 0051. For the topical application of obovatol in vivo, plu 0040 Abetter understanding of the present invention may ronic gel was employed. F-127 pluronic gel (Sigma Chemical be obtained through the following examples, which are set Company, Germany) was dissolved in cold, deionized water forth to illustrate, but are not to be construed as the limit of the to give 40% gel one day before the experiment, and was present invention. allowed to stand at 4°C. for 12 hours to dissolve powdered F-127 pluronic gel completely. On the day of the experiment, EXAMPLE1 obovatol was dissolved in a concentration of 10 g/L in Isolation and Purification of Obovatol 100% ethanol and 10 uL of the obovatol solution (10 ug/uL) was mixed with 90 uL of the 40% F-127 pluronic gel to afford 0041 1-1. Preparation of Extract from Leaf of Magnolia 100 u, of obovatol-pluronic gel, comprising 100 ug of obo Obovata Vatol. A control was prepared in the same manner except that 0042 Leaves of Magnolia obovata were collected, dried no obovatol was contained therein. in a dark place, and finely sectioned. To 3 kg of the sectioned 0.052 (Step 2) Surgery for Carotid Artery Injury leaves was added 20 liters of a mixture solvent of 1:1 chloro 0053. After rats were anaesthetized by abdominal injec form:acetone, followed by extraction at 25°C. for about 24 tion with ketamine (50 mg/kg) and Xylazine (6.7 mg/kg) and hours in a water bath using a reflux condenser. A pool of the incised to expose the common carotid artery, the external resulting extracts was concentrated in a vacuum to afford 200 carotid artery and the internal carotidartery were exteriorized g of the non-polar solvent extract. through a ventral right line neck incision. While blood flow 0043. 1-2. Isolation and Purification of the Final Com was temporarily halted by occluding the artery with pound microvascular clamps (Acland, S&T, Switzerland) at the 0044) 200g of the non-polar solvent extract obtained from proximal region of the common carotid artery and the distal leaves of Magnolia obovata in Example 1-1 was fractioned region of the internal carotid artery, arteriotomy was per into an aqueous layer and an organic layer. The aqueous layer formed on the external carotid artery. A 2F Fogarty arterial was washed three times with 1 liter of ethyl acetate and the embolectomy catheter (Baxter Healthcare Corporation, US 2008/0139668A1 Jun. 12, 2008

USA) was inserted into the lumen of the right common the obovatol-treated group and the control (p<0.05) while a carotid artery through the incised region and the balloon was significant decrease in intimal area was found in the obovatol inflated to a size larger than the diameter of the common treated group, compared to the control group (p<0.05). As for carotid artery so as to generate slight arterial wall resistance. the intimal-medial area ratio and the Stenosis degree, statis The catheter was advanced a predetermined distance and then tically significant differences were found between the obova withdrawn. This procedure was repeated a total of three times tol-treated group and the control group (p<0.05, p<0.01, to induce endothelial denudation, after which the catheter (the respectively). balloon?) was removed from the arterial lumen. Then, the microvascular clamps were removed to allow blood to flow EXAMPLE 4 through the artery. Secretions and blood were completely removed from the exterior of the carotid artery before the Inhibitory Effect of Obovatol on Arterial Smooth obovatol-pluronic gel or the obovatol-lacking pluronic gel Muscle Cell Proliferation were topically applied in an amount of 100LL to upper loci of the carotid artery, followed by ligation of the artery with a 0060 Obovatol was examined for inhibitory activity suture. Two weeks after the arteriotomy, a carotid artery against the proliferation of arterial Smooth muscle cells in angiography was conducted and a carotid artery sample was rats. Arterial smooth muscle cells were plated at a density of taken and analyzed histopathologically. 3.0x10" cells/well onto 12-well plates containing a DMEM 0054 (Step 3) Carotid Artery Angiography medium supplemented with 0.5% (V/V) fetal bovine serum, 0055 14 days after the arteriotomy, the rats were put under and incubated for 24 hours. After the addition of obovatol (1, general anesthesia by abdominal injection with ketamine (50 3, and 5 uM) to the plates, the cells were incubated for 24 mg/kg) and Xylazine (6.7 mg/kg), followed by Ventral midline hours. Treatment with 50 ng/ml of platelet-derived growth incision. A 4F vascular cannula (Cook, USA) was inserted factor (PDGF-BB, Sigma Chem. Co., USA) was followed by into the ventral aorta and the catheter was advanced toward incubation for 24, 48, and 72 hours. Thereafter, the cells were the head to a locus as close as possible to a branch between the trypsinized and counted using a cell counter. common carotid artery and the transverse aorta. After the 0061. The results are graphed in FIG. 9, in which cell injection of a contrast media (VisipaqueTM, Amersham counts are plotted against culture time periods according to Health, Cork, Ireland), mean luminal diameters (MLD) were the concentrations of obovatol, proving that obovatol is measured using computerized coronary angiography (DCI inhibitory of the growth of arterial smooth muscle cells. Videodensitometry, Phillips, Netherlands)(See FIG. 1-FIG. 8). EXAMPLE 5 0056 Carotid artery angiography was also conducted in the rats which did not undergo the arteriotomy. They were Inhibitory Effect of Obovatol on DNA Synthesis in measured for the inner diameter of the carotid artery using a Arterial Smooth Muscle Cells 5F coronary catheter, and the mean value thereof was used as 0062. This experiment was undertaken to examine a control. whether the inhibitory effect of obovatol on cell proliferation 0057 The inner diameters were calculated to be 0.63+0. shown in Example 4 was attributed to the inhibition of DNA 12 mm for the control and 0.78+0.06 mm (p<0.01) for an synthesis. obovatol-treated group, which indicated that treatment with 0063 Arterial smooth muscle cells were cultured in the obovatol prevented restenosis. same manner as in Example 4, with the exception that the cells were cultured for 20 hours after treatment with platelet EXAMPLE 3 derived growth factor, and then cultured for 4 hours in the Histopathological Test presence of 1 Jul of 3H thymidine. The cultured cells were washed with PBS and incubated with 500 ul of TCA (trichlo 0058. The control and the obovatol-treated group were roacetic acid) for 30 min. After the removal of TCA, the cells analyzed for intimal thickness, medial thickness, intimal were washed with a mixture of 1:1 ethanol:ether (v/v) and area, intimal-medial area ratio and restenosis degree (%) and disrupted with 500 ul of NaOH. The cell lysate was mixed the results are summarized in Table 1. with 5 ml of a scintillation cocktail and measured for radio activity to analyze relative amounts of newly synthesized TABLE 1. DNA. FIG. 10 is a graph in which amounts of newly synthe sized DNA in PDGF-treated arterial smooth muscle cells are Obovatol Control Treated PValues plotted against concentrations of obovatol. As seen in the graph, the DNA synthesis of the arterial smooth muscle cells Intimal O.18 OO6 0.23 + 0.02 P < 0.05 (0.011) decreases with the increasing concentration of obovatol. Thickness (mm) Medial Area (mm) O.12 O.O2 0.11 + 0.10 P & 0.05 (0.669) 0064. Therefore, the inhibitory effect of obovatol on arte Intimal Area O.18 OO6 0.13 + 0.03 P & 0.05 (0.022) rial smooth muscle cell proliferation is attributed to the fact (mm) that obovatol suppresses DNA synthesis therein. Intimal-Medial 1.54 - 0.48 1.12 + 0.23 P & 0.05 (0.011) Area Ratio Stenosis (%) 50.10 + 13.25 34.94 + 7.23 P & 0.01 (0.0017) EXAMPLE 6 Acute Toxicity Assay 0059. As seen in Table 1, similarity was found in medial thicknesses between the obovatol-treated group and the con 0065 ICR mice (weighing 25+5 g) and SPF Sprague trol, indicating that there was no cytotoxicity in obovatol. Dawley rats (weighing 235+10 g) were separately divided There is a significant difference in medial thickness between into four groups of three and abdominally injected with the US 2008/0139668A1 Jun. 12, 2008

obovatol prepared in Example 2 at doses of 1000 mg/kg, 100 water to afford a soft capsule medicine useful in the preven mg/kg, and 10 mg/kg and monitored for toxicity over 24 tion and treatment of restenosis. hours. 0066 Death was observed in none of the four groups, with PREPARATION EXAMPLE 6 no abnormality observed in any of the animals, such as in body weight, food intake, etc., compared to the control. Granules Therefore, the compound of the present invention was proven 0073. Using a typical granulation extruder, 10 mg of obo to be safe to the body. Vatol and 25 mg of lactose were formulated into granules useful in the prevention and treatment of restenosis. EXAMPLE 7 0074 As explained and proven hitherto, a pharmaceutical Formulation of Pharmaceutical Compositions for composition comprising obovatol as an active ingredient is Prevention and Treatment of Restenosis provided for the prevention and treatment of restenosis fol lowing a stenting procedure, in accordance with the present 0067 Obovatol in accordance with the present invention invention. Being useful in the prevention of restenosis fol was formulated in combination with auxiliary agents, such as lowing a blood injury procedure including a stent, the phar excipients, binders, lubricants, disintegrants, diluents, etc., maceutical composition of the present invention is applicable into pharmaceutical preparations as follows. to the treatment of various vascular coronary artery diseases. 0075 Although the preferred embodiments of the present PREPARATION EXAMPLE 1. invention have been disclosed for illustrative purposes, those Tablet skilled in the art will appreciate that various modifications, additions and Substitutions are possible, without departing 0068. Using a conventional tabletting process, 10 mg of from the scope and spirit of the invention as disclosed in the obovatol. 20 mg of lactose, 20 mg of starch and a suitable accompanying claims. amount of magnesium Stearate were formulated into a 50 mg What is claimed is: tablet useful in the prevention and treatment of restenosis. 1. A pharmaceutical composition for the prevention and PREPARATION EXAMPLE 2 treatment of restenosis following a blood vessel injury pro cedure, comprising obovatol, represented by the following Capsule Chemical Formula 1, as an active ingredient: 0069. Using a conventional process, 10 mg of obovatol. 20 mg of lactose, 19 mg of starch, 1 mg of talc and a Suitable amount of magnesium Stearate were loaded into a capsule to Chemical Formula 1 afford a capsule medicine useful in the prevention and treat CH N OH ment of restenosis.

PREPARATION EXAMPLE 3 OH Liquid 0070 According to a typical process, 100 mg of obovatol, 10g of isomerized Sugar, 500 mg of honey, 20 mg of nicotinic acid amide (pharmacopoeia), 30 mg of anhydrous caffeine (pharmacopoeia) 30 mg, and 70 mg of sodium benzoate were formulated and loaded in a 100 ml brown container which was then tightly sealed and pasteurized to afford a liquid preparation useful in the prevention and treatment of resteno sis. CH PREPARATION EXAMPLE 4 2. The pharmaceutical composition according to claim 1, Injection wherein the pharmaceutical composition is in a dosage 0071. According to a typical injection preparation pro form selected from a group consisting of a capsule, a cess, 6 mg of obovatol was formulated in combination with a liquid, an injection, a Soft capsule, a granule and a tablet. Suitable amount of sterile water and loaded in a 2 ml ampule 3. The pharmaceutical composition according to claim 1, which was then tightly sealed and sterilized to afford an wherein the blood vessel injury procedure is percutaneous injection useful in the prevention and treatment of restenosis. transluminal coronary angioplasty, balloon angioplasty, stent insertion, coronary artery bypass graft Surgery, or PREPARATION EXAMPLE 5 arteriovenous anastomosis. Soft Capsule 4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered at a 0072 According to a typical process, 10 mg of obovatol, dosage of 0.0001~100 mg per kg of weight in one dose or in 230 mg of polyethylene glycol and 13 mg of glycerin were two or three doses a day. loaded into an envelope made of 52 wt % of gelatin, 32 wt % of glycerin, 12 wt % of ANIDRISORB35/70 and 5 wt % of c c c c c