WO2006019204A1

WO2006019204A1 - Pharmaceutical composition for treating and preventing artery restenosis containing thalidomide

Info

Publication number: WO2006019204A1
Application number: PCT/KR2004/002321
Authority: WO
Inventors: Hyo-Soo Kim; Bon-Kwon Koo; Hyun-Jae Kang; Seung-Jung Park; Byung-Doo Kwon
Original assignee: Stentechinc; Seoul National University Industry Foundation
Current assignee: Stentechinc; Seoul National University Industry Foundation
Priority date: 2004-08-16
Filing date: 2004-09-14
Publication date: 2006-02-23
Anticipated expiration: 2007-02-16

Abstract

Pharmaceutical compositions containing thalidomide for the treatment and prevention of arterial stenosis are described. Pharmaceutical compositions of the present invention for the treatment and prevention of vascular stenosis, inhibiting significantly neointimal hyperplasia through its anti-inflammatory effect and its anti-proliferative action, can be applied as an useful anti-restenotic agent for the treatment and prevention of vascular stenosis.

Description

PHARMACEUTICAL COMPOSITION FOR TREATING AND PREVENTING ARTERY RESTENOSIS CONTAINING THALIDOMIDE

Technical Field i

This present invention relates to pharmaceutical compositions for the treatment and prevention of all sorts of vascular stenosis, restenosis and atherosclerosis in all circulatory systems, specifically by systemic or local administration of thalidomide alone or in combination with other agents which have a inhibitory effect on neointimal hyperplasia.

Background Art

In spite of the recent advances in strategies to prevent neointimal growth after angioplasty, it still remains the major limitation of percutaneous coronary interventions. Though the pathogenic mechanisms have not been completely resolved, there is an accumulating body of evidence which suggests that inflammation plays a key role in the development of restenosis (Welt FG, Rogers C, Arterioscler Thromb Vase Biol 2002; 22:1769-76) , and that its severity is also influenced by the intensity of inflammation (Danenberg HD, Welt FG, Walker M 3rd, Seifert P, Toegel GS, Edelman ER., Circulation 2002; 105:2917-22) .

Evidences that the inflammatory response activated after vessel injury induces restenosis have been presented at the cellular, molecular and genetic levels. Mechanical injury during coronary interventions is known to activate vascular smooth muscle cells (VSMCs) and leukocytes (Tanaka H, Sukhova G, Schwartz D, Libby P., Arterios^'cler Thromb Vase Biol 1996; 16:12-8, Serrano CV Jr, Ramires JA, Venturinelli M, Arie S, D'Amico E, Zweier JL, Pileggi F, da Luz PL., J Am Coll Cardiol 1997; 29: 1276-83) .

Activated VSMCs and leukocytes are known to release various types of cytokines and growth factors (Clausell N, de Lima VC, Molossi S, Liu P, Turley E, Gotlieb AI, Adelman AG, Rabinovitch M. _r Br Heart J 1995;73:534-9, Lowe HC, Chesterman CN, Hopkins AP, Juergens CP, Khachigian LM. , Thromb Haemost 2001; 85: 574-576.), which stimulate migration and the proliferation of VSMCs leading to neointimal hyperplasia (LindnerV, ReidyMA., CircRes 1993; 73:589-95) .Moreover, genetic predispositions to restenosis have been reported in polymorphism studies upon many kinds of molecules such as CD 18, interleukin (IL) -1 receptor antagonist andmatrix metalloproteinases (Koch W, Bottiger C, Mehilli J, von Beckerath N, Neumann FJ, Schomig A, Kastrati A. , Am J Cardiol 2001;88:1120-4, Humphries S, Bauters C, Meirhaeghe A, Luong L, Bertrand M, Amouyel P., Eur Heart J 2002;23: 721-5) , which are important components of the inflammatory process .

On the other hand, initially developed as a sedative, thalidomide has since been shown to have potent anti-inflammatory and immunomodulatory properties (Raje N, Anderson K., N Engl J Med 1999; 341:1606-9) . Presently, a wide spectrum of diseases including cutaneous lupus, Crohn's disease, rheumatoid arthritis, multiple myeloma and Graft versus host disease, are being treated with thalidomide (Raje N, Anderson K.^*, N Engl J Med 1999; 341:1606-9) . Although the mechanism of biological action of thalidomide has not been fully elucidated, the majority of the anti-inflammatory properties of thalidomide havebeen attributedto change inproduction and release of a wide range of cytokines and growth factors. Currently, perhaps the best described effect of thalidomide is of its inhibitory action upon tumor necrosis factor (TNF) -α (Raje N, Anderson K., N Engl J Med 1999; 341 :1606-9) . In some vitro studies suggest that 'thalidomide makes short the half-life of TNF-α mRNA (Moreira AL, Sampaio EP, Zmuidzinas A, Frindt P, Smith KA, Kaplan G. , J Exp Med 1993; 177:1675-80) . Based on its anti-inflammatory effect, thalidomide was recently administered to patients with chronic symptomatic congestive heart failure, who showed reduced TNF-α level and increased left ventricular ejection fraction after 6 months of thalidomide therapy(Gullestad L, Semb AG, Holt E, Skardal R, Ueland T, ϋndestad A, Froland SS, Aukrust P., Am Heart J 2002/144:847-50) . Another cytokine, that may be attenuatedby thalidomide is bFGF, a pleotrophic molecule, (Raje N, Anderson K., N Engl J Med 1999; 341:1606-9) whose mRNA is overexpressed in cytoplasms and nuclei of proliferating VSMCs and endothelial cells following balloon injury (Lindner V, ReidyMA., Circ Res 1993; 73:589-95) . After being released, it is internalized into the cytoplasm by low affinity receptors and then translocated to and accumulated in the nucleus (Bouche G, Gas N, Prats H, Baldin- V, Tauber JP, Teissie J, Amalric F., Proc Natl Acad ScI USA 1987/ ^'84: 6770-4, Hawker JR Jr, Granger HJ., Am J Physiol 1992; 262:H1525-37) . This translocation of bFGF has been correlated to cell-cycle progression and proliferation via stimulation of protein kinase CKII and exposure of the VSMCs to bFGF has been shown to induce neointimal proliferation (Lindner V, Reidy MA., Circ Res 1993; 73:589-95) , whereas blockade of bFGF activity with anti-bFGF antibody reduced medial muscle cell proliferation after arterial injury (LindnerV, ReidyMA., ProcNatl Acad ScI USA 1991; 88:3739-43) . One of the proposed mechanisms of the anti-bFGF property of thalidomide is that it intercalates into DNA (Finnell RH, Waes JG, Eudy JD, Rosenquist TH., Annu Rev Pharmacol Toxicol 2002;42:181-208) , which is supported by considerable evidence (Huang PH, McBride WG., Teratog Carclnog Mutagen 1997;17:l-5) . The promoter regions of the bFGF gene lack typical TATA boxes, and instead contain multiple GGGCGG sequences called GC boxes (Jonsson NA. , Acta Pharm Suec 1972; 9:543-62) . Thalidomide specifically binds to these GC promoter sites because it has a greater affinity for guanine, which is due to the fact that the structure of guanine more resemble thalidomide than that of adenine (Jonsson NA., Acta Pharm Suec 1972; 9:543-62) . Thus the blocking of these GC boxes by thalidomide results in the down-regulation of bFGF transcription which leads to low bFGF level. The molecules that are regulated by GC boxes instead of TATA or CCAAT boxes include manyproteins such as Insulin-like growth factor (IGF) -1, IGF-I receptor, bFGF rececptor and αv β3 integrin. All of theses molecules are closely related to the pathogenesis of neointimal formation (Panda D, Kundu GC, Lee BI, Peri A, Fohl D, Chackalaparampil I, Mukherjee BB, Li XD, Mukherjee DC, Seides S, Rosenberg J, Stark K, Mukherjee AB., Proc Natl Acad Sci USA 1997; 94:9308-13) .

In addition, thalidomide has been reported to reduce the synthesis of other pro-inflammatory cytokines such as IL-I, IL-6 and IL-8 (Raje N, Anderson K., N Engl J Med 1999;341 :1606-9, Finnell RH, Waes JG, Eudy JD, Rosenquist TH., Annu Rev Pharmacol Toxicol 2002/42:181-208) , but it enhances levels of the anti-inflammatory cytokine IL-10 (Gullestad L, Semb AG, Holt E, Skardal R, Ueland T, ϋndestad A, Froland SS, Aukrust P., Am Heart J 2002;144:847-50) . Thalidomide was also found to reduce the expression of endothelial adhesion molecules including integrin, ICAM and VCAM, (Raje N, Anderson K., N Engl J Med 1999; 341:1606-9, Settles B, Stevenson A, Wilson K, Mack C, Ezell T, Davis MF, Taylor LD., Cell MoI Biol 2001; 47:1105-14) as a consequence, the interactions between endothelial cells and circulating leukocytes are inhibited. Moreover, The activation of NF-κB, an important mediator of inflammation, cell proliferation and restenosis, was also found to be blocked with thalidomide due to its inhibitory effect upon I-κB degradation (Bergmeister H, Kadi A, Baumgartl G, Steurer S, Xu Z, Koshelnick Y, Lipp J, de Martin R, Losert U, Lammer J, Binder BR. , Circulation 2002/105: 633-8, Majumdar S, Lamothe B, Aggarwal BB., J Immunol 2002/168:2644-51, Breuss JM, Cejna M) .

However, thalidomide has never been reported to exert a significant inhibitory effect on neointimal growth through either animal experiments or clinical trials.

Therefore, based upon these properties of thalidomide described above; anti-inflammatory, ant-angiogenic and immunomodulatory, investigation was performed to see whether thalidomide inhibits neointimal hyperplasia after balloon angioplasty. After the observation of the unexpected and obvious effect of thalidomide as an anti-restenotic agent for the treatment and prevention of arterial stenosis, the present invention was completed.

Disclosure of the Invention Technical Problem

It is therefore an object of the present invention to provide a method and compositions to prevent and reduce vascular stenosis after revascularization.

Technical Solution

To attain the object mentioned above, the present invention provides a method and compositions to treat and prevent vascular stenosis after revascularization.

A pharmaceutical composition of the present invention for the treatment and prevention of vascular stenosis, is characterized by- containing thalidomide as an active ingredient.

Thalidomide, originally synthesized as a sedative, has since been shown to have potent anti-inflammatory, anti-angiogenic and immunomodulatory properties. Presently, the use of it has expanded to various clinical areas where inflammation is thought to play an important role, such as refractory cutaneous lupus, rheumatoid arthritis, Crohn's disease, multiple myeloma and chronic graft versus host disease.

Especially, thalidomide has been proved to show less toxicity in animal experiments even at 10g/kg.

Advantageous Effects

A pharmaceutical composition of the present invention for the treatment and prevention of vascular stenosis, inhibiting significantly neointimal hyperplasia due to its anti-inflammatory effect and its anti-proliferative action, can be applied as a useful anti-restenotic agent for the treatment and prevention of vascular stenosis.

Brief Description of the Drawings

Figures 1 show low andhighmagnificationphotomicrographs of arterial section from the control and fromthalidomide-treated groups (NI: Neointima, M: Media, Ad: Adventitia) . A: Carotid artery of control group prior to procedure B: Carotid artery of thalidomide group prior to procedure C: Carotid artery of control group 3 days after procedure D: Carotid artery of thalidomide group 3 -days after procedure E: Carotid artery of control group 2 weeks after procedure F: Carotid artery of thalidomide group 2 weeks after procedure G: Carotid artery of control group 2 weeks after procedure H: Carotid artery of thalidomide group 2 weeks after procedure (A-F: low magnigication, G and H: high magnification) Figures 2a are the histomorphometric data showing luminal, neointimal and medial area collected from both control and thalidomide group 2 weeks after balloon injury.

Figures 2b are the graphs representing neointimal area to medial area

(N/M) ratio of control and thalidomide groups 2 weeks after balloon injury.

(*: p<0.001)

Figures 3a are the ELISA data used to determine serum TNF-αproduction in control and thalidomide-treated groups (*: p=0.001), while Figures 3b shows a strong positive correlation between the serum TNF-αlevel and the

N/M ratio on the 14 days after balloon injury.

Figures 4a and 4b are the results of western blot anaylsis used to ascertain the effect of thalidomide treatment on the local expressions of

TNF-α and bFGF respectively in an injured vessel.

Figures 5 represent effect of thalidomide treatment on the local expression of bFGF in an injured vessel using immunohistocherciical analysis. A: Carotid artery of control group prior to procedure

B: Carotid artery of thalidomide group prior to procedure

C: Carotid artery of control group 3 days after procedure

D: Carotid artery of thalidomide group 3 days after procedure

E: Carotid artery of control group 2 weeks after procedure

F: Carotid artery of control group 2 weeks after procedure

Figures 6 are the graphs of bFGF positive indices in control and thalidomide-treated groups (*: p<0.001) .

Figures 7 are the immunohistochemical analysis of infiltration of macrophages in carotid arteries using ED-I staining (NI: Neointima, M:

Media)

A: Uninjured carotid artery

B: Carotid artery of control group 2 weeks after procedure

C: Carotid artery of thalidomide group 2 weeks after procedure

Figures 8 are the graphs of ED-I positive indices used to assess the infiltration of macrophages in carotid arteries (*: p=0.021) .

Figures 9 are the immunohistochemical analysis of carotid arteries used to determine the proliferation of VSMCs in control and thalidomide-treated groups (NI: Neointima, M: Media, i: PCNA positive cell) .

A: Carotid artery of control group prior to procedure

B: Carotid artery of thalidomide group prior to procedure

C: Carotid artery of control group 3 days after procedure

D: Carotid artery of thalidomide group 3 days after procedure E: Carotid artery of control group 2 weeks after procedure F: Carotid artery of control group 2 weeks after procedure Figures 10 are the graphs of PCNA positive indices used to determine proliferative activity of VSMCs in control and thalidomide-treated groups

(*: p<0.001) .

Best Mode

In one embodiment of this invention, a model of de-novo neointimal growth in response to vascular injury is used to confirm whether thalidomide has a suppressive effect on neointimal proliferation. As is shown in Figure 1 and Figure 2b, neointimal growth was significantly inhibited in the thalidomide-treated groups, which resulted in larger luminal area in that groups compared with control groups.

In another embodiment of this invention, serum TNF-αwas measured by ELISA to investigate the effects of thalidomide on TNF-αproduction. TNF-α, as a central mediator of inflammatory response, is known to be expressed by activatedmacrophages or VSMCs after balloon angioplasty. As is presented in Figure 3a, thalidomide significantly decreased serum TNF-α, which is a marker of systemic inflammation, after balloon injury. Moreover, Figure 3b shows a strong positive correlation between the serum TNF-α level and the degree of neointimal growth. Therefore, the lower the serum TNF-α level, the smaller the N/M ratio. The lower the level of inflammation, the lower development of neointimal hyperplasia is. In yet another embodiment of this invention, to determine whether thalidomide treatment suppresses local inflammatory response at the site of balloon angioplasty, western blot analysis and immunohistochemistry were conducted to evaluate the tissue expression of TNF-αand bFGF as well as macrophages infiltration in the injured artery. As is put forward in Figure 4a and Figure 4b, western blotting showed attenuated expression of TNF-αand bFGF inthe thalidomide-treated arteries. Furthermore, immunohistochemical analysis confirmed decreased bFGF staining in nuclei and cytoplasms in the thalidomide group compared with the control group (Figure 5, Figure 6) . Figure 7 and Figure 8 shows that thalidomide administration reduced significantly macrophage infiltration. In addition, thalidomide treatment suppressed not only the systemic release of TNF-α but also its local expression in injured arteries, which suggest that thalidomide therapy inhibits the development of neointimal overgrowth via its inhibitory effect on systemic and local inflammatory response to vascular injury. Therefore, thalidomide administration induced the suppressed macrophage infiltration and the reduced expression of TNF-α and bFGF, which led to the decreased proliferative activity of VSMCs and then to the attenuated neointimal hyperplasia.

Thalidomide described above can be directly synthesized by anymethod known in the art. Certain of this compound such as Thalidomid™ and Sauramide™, are commercially available for use. In a specific embodiment of the present invention, thalidomide purchased from Alan Pharmaceuticals (Sauramide™) was employed. Therapeutic dose of the compound of the present invention can be determined variably according to the kind of target vessel, general condition of the individual patient and the duration of treatment as previously designed. For example, daily practical doses of the compound can vary to obtain a plasma concentration of 2.0ug/mL or more. In a specific embodiment of the present invention, administration of thalidomide at 100mg/kg/day for 2 weeks, can achieve a significant suppression of the neointimal hyperplasia.

The compounds of the present invention are highly effective for the treatment and prevention of vascular restenosis. The arterial stenosis mentioned above may be due to neointimal hyperplasia. This stenosis may also be induced by percutaneous transluminal coronary angioplasty, atherectomy, stent implantation, coronary artery bypass grafting and arteriovenous • anatomosis.

For preparing pharmaceutical compositions from the compounds of the present invention, many pharmaceutically acceptable carriers may be employed. Pharmaceutically acceptable carriers encompass but are not limited to normal saline, buffered normal saline, sterile water, glycerol, ethanol, etc. The compositions described above may contain pharmacologically acceptable auxiliary substances as required, such as excipient, stabilizer, dispersing agent, lubricating agent, preserving agent, suspending agent, sweetening agent, flavoring agent and binder.

The compounds of the present invention can be manufactured as pharmaceutically acceptable formulation using preparation method known to those of ordinary skill in the art. Thus, composition can be prepared in the form of tablets, pills, capsules, granules, powders, elixirs, suspensions, emulsions, syrups, solutions, oils, ointments, creams. Routes of administration^ of the present invention include oral and parenteral route.

Furthermore, the compounds of the present invention can be coated alone or in combination with other agent, on a stent or a synthetic vascular graft, which may be coated by preferable methods known in the art, such as dip coating and polymer-based coating.

The optimal dose to be administered will vary in consideration of other associated conditions. For example, the daily dose range of the compound administered orally is between lOOmg to 1600mg. However, not limited to the dose range mentioned above, the dose of the compounds can be adjusted reasonably according to the age, sex, body weight, general condition of patient, diet, administration route and interval, absorption rate, bioavailability, the severity of disease, other drugs in use, etc.

The following preferred examples of the present invention are described below in detail. However, they will only serve to illustrate formulation which can be made according to the invention but should not to be construed as a limitation in the scope of the present invention.

Mode of invention

[Examples] Preparation Of Thalidomide

Thalidomide (Sauramide™, Alan Pharmaceutical, London, U.K.) was dissolved in corn oil (Sigma-Aldrich Korea) to give a 50mg/ml suspension.

Experimental Animals

Male Sprague-Dawley rats, 17 weeks old, weighing 417± Hg from Daehan Biolink (Chungbuk, South Korea) , were used as the appropriate test system for the purposes of this study.

Procedures involving animals were in accordance with the "Guide for Experimental Animal Research"from the Laboratory for Experimental Animal Research, Clinical Research Institute, Seoul National University- Hospital.

Vascular Injury Model With Following Treatment

As a pretreatment, thalidomide (100mg/kg qd) was administered to the rats in the study by gavage from 3days prior to balloon injury.

Blinded of the above-mentionedpretreatment details, balloon injury was performed as follows; under Xylazine (5 mg/kg IP; Yuhan Corp, Bayer Korea) and ketamine hydrochloride (50 mg/kg IP; Yuhan Corp, Bayer Korea) induced anesthesia, the right external carotid arteries were exposed and the common carotid arteries were denuded of endothelium by the intraluminal passage of a 2F arterial catheter (Baxter Healthcare Corp) , which was passed to the proximal common carotid artery and withdrawn in the, inflated state 5 times .

Then thalidomide of the same dose (100 mg/kg qd) was administered by gavage to the rats that underwent the vascular injury for 2 weeks.

Control animals were given 100mg/kg of sucrose in the same manner.

Statistical Analysis

Data from the following exmaples are presented as mean± SD. Comparisons between the thalidomide-treated group and control group were performed using an unpaired, 2-tailed t test. SPSS 11.0 was used for all statistical calculations and p<0.05 was considered significant.

Example 1: Effect of thalidomide on neointimal hyperplasia After balloon injury, histomorphometric analysis was performed as follows. Three days and 14 days after balloon injury, rats (n=8 per time point per group) were euthanized with a lethal dose of pentobarbital.

Bilateral common carotid arterial segments, about lcm long,were harvested and perfusion fixed with 10% neutral buffered formalin at physiological pressure. Tissues were then embedded in paraffin, and 4 to 6 sections 4 μm thick were cut from 4 equally spaced locations from the harvested arterial segments. The sections were stained with hematoxylin and eosin or Verhoeff-van Gieson stain. The luminal, neointimal, and medial areas were calculated using the Image-Pro Plus 4.5 software (Media Cybernetics Inc.) . Figure 1 shows low and high magnification photomicrographs of arterial section from the control and thalidomide-treated groups before and after the procedure (NI: Neointima, M; Media, Ad: Adventitia) . As is shown Figure 1 A and B, arteries not injured by the balloons revealed no histological differences between the control and thalidomide-treated animals .

Morphometric analysis 3 days after balloon injury revealed no significant differences in the neointimal or medial areas of the thalidomide-treated and control groups (C, D, n=8 per group) . No neointima could be found in either group.

However, two weeks after injury, bulky concentric neointimal hyperplasia was detected in the control animals (E and G) , whereas neointimal growth was significantly inhibited in the thalidomide-treated rats (F and H) .

Figure 2a is the histomorphometric data showing luminal, neointimal and medial area collected from both control and thalidomide group 2 weeks after balloon injury. Figure 2b is the graphs representing neointimal area to medial area (N/M) ratio of control and thalidomide groups 2 weeks after balloon injury. In the thalidomide-treated animals, there was a 71%

reduction in the neointimal area (control versus thalidomide, 0.17+ 0.04 versus 0.05± 0.02 mm², n=16, p<0.001) , and a 72% reduction in the N/M ratio (control versus thalidomide, 1.26± 0.29 versus 0.35+ 0.13, p<0.001) compared with the control group. Thus, lumen area at 2 weeks was significantly greater in the thalidomide treated group (luminal area; control versus thalidomide, 0.24+ 0.04 versus 0.36+ 0.06 mm², p<0.001) . Therefore, thalidomide treatment led to larger luminal area through the suppression of neointimal formation.

Example 2: Determination of anti-inflammatory effect To understand the mechanism of thalidomide treatment in vivo, inflammatory and proliferative activities of the injured carotid arteries were examined. Injured vessel segments and arterial blood were collected at 3days and 14 days after the injury for the following measurements.

2-1) Measurement of systemic inflammation by serum TNF-αELISA

TNF-α, as a central mediator of inflammation,is known to be expressed by activated leukocytes and VSMCs in injured arteries after balloon angioplasty(Tanaka H, Sukhova G, Schwartz D, Libby P., Arterioscler Thromb Vase Biol 1996_/16:12-8, Clausell N, de Lima VC, Molossi S, Liu P, TurleyE, GotliebAI, AdelmanAG, Rabinovitch M. , Br Heart J 1995;73:534-9) . Moreover most of neointimal VSMCs were demonstrated to express TNF-α along with its iriRNA and to sustain proliferation even IOdays after injury(Tanaka H, Sukhova G, Schwartz D, Libby P., Arterioscler Thromb Vase Biol 1996/16:12-8) , and the local blockade of TNF-α with TNF-α antibody eluting stentreduced VSMC proliferation in saphenous vein organ culture (Javed Q, SwansonN, Vohra H, ThurstonH, GershlickAH. , ExpMoI Pathol 2002/73:104-11) . In other studies, tissue macrophage infiltration in the neointima andmedia, which is a major sources of TNF-α, was ,shown to be significantly increased and sustained till 2 to 4 weeks after injury (Mori E, Komori K, Yamaoka T, Tanii M, Kataoka C, Takeshita A, Usui M, Egashira K, Sugimachi K., Circulation 2002/105: 2905-10, Bishop GG, McPherson JA, Sanders JM, Hesselbacher SE, Feldman MJ, McNamara CA, Gimple LW, Powers ER, Mousa SA, Sarembock IJ., Circulation 2001_/103:1906-11) .

To investigate the effects of thalidomide on systemic inflammation,

serum TNF-α was measured as follows on days3 and 14 after the balloon injury.

Arterial blood was collected into calcium-containing tubes from the abdominal aorta via an 18G intravenous cannula at the time of vessel

harvest. Serum was separated at 4^°C and stored at -70"C until further analysis. After it had thawed, TNF-α level was measured in duplicate with a commercial enzyme-linked immunosorbent assay (ELISA) kit purchased from BD PharMingen (CA, USA) . The sensitivity of TNF-α ELISA was 13pg/ml.

As is shown in Figure 3, thalidomide significantly decreased serum TNF-α, which is a marker of systemic inflammation, by 48% and 51% on 3 and 14 days after balloon injury respectively, (control" versus thalidomide 856

± 213 versus 449± 68 pg/ml on day 3, n=16, P=O.001 129+ 34 versus 63+ 18 pg/ml on day 14, n=lβ, P=O.001) . Furthermore, a strong positive correlation was found between the serum TNF-αlevel and the N/M ratio 14 days after the injury (Figure 2 B) . Therefore the lowet the serum TNF-αlevel, the smaller the N/M ratio and vice versa (Pearson correlation coefficient 0.915, n=16, P<0.001, two-tailed) . Therefore, these results show that thalidomide treatment inhibits systemic inflammatory response, which is associated with neointimal formation.

2-2) Assessment of local tissue inflammation by Western blot analysis and immunohistochemistry

Western blot analysis:

To determine whether thalidomide treatment suppresses not only systemic inflammation but also local tissue inflammation, western blot analysis was conducted to evaluate the tissue expression of TNF-α and bFGF in the harvested injured artery.

Vessel tissues were homogenized in lysis buffer and protein concentrations were determined using a Micro BCA Protein Assay kit (Pierce) . Twenty micrograms of protein per specimen were separated on a SDS-polyacrylamide gel, blotted onto nitrocellulose membranes, and probed

with specific antibodies against TNF-α (Santa Cruz Biotechnology) and bFGF (Santa Cruz Biotechnology) .

Immunohistochemical staining:

Macrophage recruitment and infiltration occurs at sites of vascular injury, which is a major source of various cytokines and growth factor (Danenberg HD, Welt FG, Walker M 3rd, Seifert P, Toegel GS, Edelman ER., Circulation 2002;105:2917-22, Bishop GG, McPherson JA, Sanders JM, Hesselbacher SE, Feldman MJ, McNamara CA, Gimple LW, Powers ER, Mousa SA, Sarembock IJ., Circulation 2001;103:1906-11) . Species-specific antibodies were used to immunohistochemically identifymacrophages infiltration (ED-I, Serotec) and bFGF expression (Santa Cruz Biotechnology) .

Standard peroxidase-antiperoxidase staining procedures (avidin-biotin-peroxidase kit, Dako) were used in conjunction with heat-induced epitope retrieval. Sections were counterstained with Mayer's hematoxylin, dehydrated and mounted. The ED-1-positive cells were divided by the total number of nucleated intimal and medial cells in 4 sectors per vessel section at 14 days after injury (n=4 per group) . Through which • staining, inhibitory effect of thalidomide on the migration of the inflammatory cells was confirmed.

Results:

The suppressive effect of thalidomide on the local expressions of

TNF-α and bFGF in the injured vessels was confirmed by western blot analysis

(Figure 4a and 4b) . Arteries not injured by balloon angioplasty produced only faint signals indicating that these inflammatory molecules were expressed at low levels in the basal condition, and balloon injury induced

high levels of TNF-α andbFGF expression on 3 and 14 days after balloon injury in the control group. However, in thalidomide-treated animals, the

expression of TNF-α and bFGF was significantly reduced at both time points.

In Figure 5, the anti-bFGF effect of thalidomide was also demonstrated by immunohistochemistry. bFGF staining was significantly increased in the nucleus as well as the cytoplasm of the control animals (C and E) following angioplasty. But this was definitely attenuated in thalidomide treated rats (D and F) .

Figures 6 are the graphs of bFGF-positive indices in control and thalidomide-treated groups. The indeces were measure by counting bFGF-positive nuclei against the total nuclei in the neointimal and medial smooth muscle cells. Specifically, the bFGF-labeling index was significantly lower in the thalidomide-treated group both at 3 days (43.3 + 6.9 versus 9.0+ 2.8%, n=3, p<0.01 for control versus thalidomide) and at 2 weeks (39.8+ 4.2 versus 15.0+ 5.8%, n=3, p<0.01 for control versus thalidomide) after balloon injury.

Figure 1 is the immunohistochemical staining of the glycoprotein ED-I (HOkD) expressed predominantly on the lysosomal membrane of tissue macrophages, which cells are major sources of TNF-α andmany other cytokine. Brown cytoplasmic stain indicates ED-I positive tissue macrophages.

Macrophage recruitment and infiltration into the injured arteries of thalidomide-treated animals was dramatically reduced (C) versus the control group (B) two weeks after balloon injury. Specifically, the number of recruited macrophages was decreased by 79 % (Figure 8, control versus

thalidomide, 19.2+ 3.5 versus 4.0+ 2.1 %, P=O.021) .

Therefore, Data from the Figures 3-8 indicate that thalidomide inhibited local inflammatory response in the injured vessel as well as systemic inflammation. Example 3; Determination of anti-proliferative activity To determine whether suppressed inflammation is accompanied by the reduced proliferative activity of VSMCs, we performed immunohistochemical staining against proliferating cell nuclear antigen (PCNA) (PClO, DAKO) . To quantify VSMC proliferation after balloon injury, we counted the percentage of PCNA-positive cells against total nucleated cells in 4 different sectors per vessel section (n=3 per time point per group) .

Figure 9 is the immunohistochemical analysis of the proliferation of VSMCs of rat carotid arteries in control and thalidomide-treated animals before and after the angioplasty, which figure showed that suppressed inflammation was accompanied by a reduction in the proliferative activity of VSMCs. A remarkable fall-off in the number of PCNA-positive VSMCs was demonstrated in the medial layers of arteries of thalidomide-treated rats 3 days after injury (D) . Moreover, this anti-proliferative effect of thalidomide was sustained till 14 days after the injuries in the neointimal and medial layers of thalidomide-treated animals (F) . Thus, definite reductions were observed in the number of nuclei stained with PCNA in animals treated with thalidomide on 3 days (D) and 14 days (F) after balloon injury, respectively, compared with the controls (C and E) . Few PCNA-labeled cells were found in the uninjured arteries of both groups (A and B) .

In Figure 10, the arrows indicate typical PCNA-positive cells. The percentages of PCNA-positive cells in the vessel wall at days 3 and 14, were 31.0+ 7.3 and 43.l± 2.9% in controls, and 9.4± 1.9 and 7.4± 1.7% in thalidomide-treated group, respectively (p< 0.01) . PCNA-positive cells in the media and neointima were counted and are presented as mean ± SD.

Claims

1 . What is claimed is :

Pharmaceutical compositions containing thalidomide as an active ingredient for the treatment and prevention of vascular stenosis .

2 . Pharmaceutical compositions according to claim 1 , wherein vascular stenosis may be due to neointimal hyperplasia .

3 . Pharmaceutical compositions according to claims 1 or 2 , wherein vascular stenosis may be induced by percutaneous transluminal coronary angioplasty, atherectomy, stent implantation, coronary artery bypass grafting and arteriovenous anatomosis .