WO2006112597A1

WO2006112597A1 - A stent coated with anti-integrin antibody and process for preparing the same

Info

Publication number: WO2006112597A1
Application number: PCT/KR2006/000492
Authority: WO
Inventors: Yang Su Jang; Yong Sun Ryu; Ki Chul Hwang; Kwang Hoe Chung; Dong Lyun Cho; Myung Ho Jeong
Original assignee: Humed Co., Ltd.
Priority date: 2005-02-12
Filing date: 2006-02-11
Publication date: 2006-10-26
Also published as: KR100759130B1; KR20060091028A

Abstract

The present invention relates to a stent whose surface is coated with anti-integrin antibody, thereby preventing restenosis after surgical operation of a stent and process for preparing the same. The process for preparing a stent coated with anti-integrin antibody comprises the steps of binding amine group to surface of a stent by plasma polymerization and linking anti-integrin antibody with the amine group on the surface of the stent. The stent coated with anti-integrin antibody of the present invention can be practically applied for the safe surgical operation of the stent, since it can effectively prevent the restenosis after surgical operation of a stent while promoting the regeneration of vascular wall damaged with the operated stent and inhibiting the hyperplasia of neointima and lowers the rate of inflammation in the vicinity of a stent.

Description

A STENT COATED WITH ANTI-INTEGRIN ANTIBODY AND PROCESS FOR PREPARING THE SAME

Technical Field

[1] The present invention relates to a stent coated with anti-integrin antibody and process for preparing the same, more specifically, a stent whose surface is coated with anti-integrin antibody, thereby preventing restenosis after surgical operation of a stent while promoting the regeneration of vein wall damaged with the operated stent and inhibiting hyperplasia of neointima, and process for preparing the same.

[2]

Background Art

[3] To treat stenosis, which is one of coronary artery diseases, angiography using a stent is being widely performed, but restenosis which occurs after the surgical operation of stent has been considered as a major detrimental factor in the surgical operation. Restenosis is caused by various causes such as glycosuria, chronic inf lammation, microbial infection, restenosis of other vein, increase of angiotensin converting enzyme activity, instable heart attack, surgical operation of a stent which is discordant with the diameter and length of vein, pus plaque, changes in coronary ostial, excessive operation of stents, etc., whose symptom appears depending on hyperplasia of neointima by barotrauma of vein wall and continuous stimulation of foreign body( see: Hoffman R., et al, Circulation, 94:1247-1254, 1966; Dussaillant G.R., et al, J. Am. Coll. Cardiol., 26:720-724, 1995).

[4] To prevent restenosis by the surgical operation of a stent, various attempts have been made as follows: WO 05/115405 discloses a method for preventing and treating restenosis by using an inhibitor for ribonucleotide reductase; WO 06/003331 suggests a medicament for prevention and treatment of restenosis, which comprises an active ingredient of a flavonol family chemical; and, US Patent No. 6,958,147 teaches a method for preventing and treating restenosis by using vascular endothelial growth factor C(VEGF-C).

[5] In case of using the medicaments for prevention and treatment of restenosis caused by the stent operation, the occurrence of restenosis, however, can be controlled only for a limited period of time that the medicaments act, which essentially requires continuous administration of the medicaments. In this regard, various stents which can continuously release the medicaments have been developed as follows: WO 05/092244 discloses a double layered stent in which lumen is positioned in the central part to contain a medicament for prevention and treatment of restenosis, WO 05/039443 suggests a stent whose surface is coated with tocopherols that can prevent restenosis, WO 04/064911 proposes a stent whose surface is provided with many struts containing wells in which medicaments for prevention and treatment of restenosis are included.

[6] The stents releasing the medicaments continuously, though they do not require continous administration of the medicaments, are proven to be less satisfactory in a sense that the use of complicate structure and use of a high molecular linker for coating the medicaments such as polyethyleneglycol(PEG), etc. cause the abnormal aggregation of blood platelet in bloodstream, which, in turn, requires the administration of a strong inhibitor for blood platelet aggregation more than 6 months. However, the solution for the said problem has not been considered in the art.

[7] If a stent improved in a point that a medicament is continuously released therefrom and blood platelet in bloodstream is not aggregated therewith is developed, the surgical operation of a stent can be made more safely with a wide application, however, any significant progress has not been reported until now.

[8]

Disclosure of Invention Technical Problem

[9] The present inventors have made an effort to develop an improved stent that continuously releases a medicament and is not aggregated with blood platelet in bloodstream, and discovered that surgical operation of a stent whose surface is coated with anti-integrin antibody could effectively inhibit the restenosis after surgical operation of a stent.

[10]

[11] A primary object of the invention is, therefore, to provide a stent whose surface is coated with anti-integrin antibody.

[12]

[13] The other object of the invention is to provide a process for preparing the stent coated with anti-integrin antibody.

[14]

Technical Solution

[15] The present inventors have tried to explore and develop a way of preventing restenosis after surgical operation of medical instruments for treating the diseases in vascular vein such as stents, coils, grafts for blood vein, artificial blood veins, etc., and paid attention to integrin, a biologically active protein which exists in human body.

[16] The integrin is known as a protein that mediates the attachment of various cells such as hematopoietic stem cell and leukocyte to the endothelial cells and ectoplasm proteins, which are generally classified as α β , α β , α β , α β , α β , α β , α β , α β , ^r ° ^J I¹ 1 2¹ I 3¹ I 4¹ I 5¹ I 6¹ I 8¹ I 9 1 α β , α β , α β , α β and α β (see: Springer TA, Cell, 76:301-314, 1994). It has been v l v 3 v 5 6 4 v 8 also reported that the integrin is associated with vascularization, and plays a crucial role in the migration and differentiation of hematopoietic progenitor cells(see: Vajkoczy P., et al., J. Exp. Med., 197: 1755-1765, 2003; Kollet O., et al., Blood, 97:3283-3291, 2001; Bowden R. A., et al., Circ. Res., 90:562-569, 2002). Moreover, it has been known that vascularization, migration and differentiation of hematopoietic progenitor cells do not occur without integrin, implying that the said protein is closely related to the migration of cell through the blood stream(see: Scott L. M., et al., MoI. Cell. Biol., 23:9349-9360, 2003).

[17] In this regard, the present inventors made an assumption that restenosis after surgical operation of a stent which is caused by the over-proliferation of vascular endothelial cells, can be efficiently prevented by inhibiting the biological activity of integrin in blood stream, and tried to develop a stent coated with anti-integrin antibody.

[18] However, a stent coated with a medicament, as stated above, has revealed a shortcoming that a polymeric compound used as a linker for coating the medicament leads to the abnormal aggregation of blood platelet in bloodstream. The said problem necessarily motivated the inventors to employ plasma polymerization for binding a low molecular hydrocarbon having an amine group as an alternative to the high molecular polymeric linker to the surface of a stent.

[19] According to the plasma polymerization method, a target compound of gas phase is changed to a plasma state under a vacuum condition, which is then coated a to metal surface. The principle of the plasma polymerization is understood as follows: both a metal having even surface and a target compound of gas phase are put into a plasma chamber under a vacuum condition, and the target compound is changed to a plasma state having a high energy level by which the chemical bondages in metal surface are broken and the metal surface thus broken is combined with the target compound of plasma state to lower the energy level, finally to give a metal whose surface is coated with the target compound(see: P. I. John, Plasma Science and the Creation of Wealth, Tata-McGraw-Hill, New Delhi, 2005).

[20] The present inventors employed the plasma polymerization method for binding a linker for coating anti-integrin antibody to a stent made of metal: that is, they used a low molecular hydrocarbon compound, i.e., methane and a low molecular compound having amine group which can be easily linked with carboxyl group of anti-integrin antibody, i.e., ammonia, to obtain a stent whose surface has reactive amine groups, and performed amide bond formation with carboxyl groups of anti-integrin antibody to prepare a stent whose surface is coated with anti-integrin antibody. Since the stent coated with anti-integrin antibody uses low molecular compounds an as a linker, it can effectively prevents blood platelet in blood stream from abnormal aggregation with the linker of a stent, which is basically different from the prior art using a linker of high molecular compounds for coating a medicament to a stent. [21] The process for preparing a stent coated with anti-integrin antibody, therefore, comprises the steps of: (i) binding amine group to surface of a stent by plasma polymerization; and, (ii) linking anti-integrin antibody with the amine group on the surface of the stent: The binding of amine group in step (i) is, not limited thereto, carried out by sequential binding a gas phase of methane and ammonia to the surface of the stent. The anti-integrin antibody is, not limited thereto, a monoclonal antibody or a polyclonal antibody against an integrin protein selected from the group consisting of α β , α β , α β , α β , α β , α β , α β , α β , α β , α β , α β , α β , α β and α β , most

1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 1 v 1 v 3 v 5 6 4 v 8 preferably, a polyclonal antibody against α β integrin. And, an amide bond formation v 3 between carboxyl group of the anti-integrin antibody and amine group on the surface of the stent in step (ii) is preferably carried out by using a catalyst which is, not limited thereto, l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride(EDAC), N- hydroxysuccinimide(NHS) or a mixture thereof. The stent prepared by the process of the invention is a stent whose surface is linked with amine group which is bound with anti-integrin antibody. [22]

Advantageous Effects

[23] The present inventors prepared two types of stents each of which is coated with anti-integrin antibody by carrying out the sequential binding of methane and ammonia or binding DACH to the surface of a stent by plasma polymerization method, and then linked anti-integrin antibodies with the amine groups on the surface of the stent, and prepared a stent which is not coated with anti-integrin antibody as well. Then, surgical operation of the stents were performed on the pigs, and compared the thickness of operated coronary artery, neointima area, etc. after 4 weeks of surgical operation. As a result, it was examined that blood vessels of coronary arteries operated with the stents coated with anti-integrin antibody have thinner wall, smaller neointima area than those operated with the stents which is not coated with anti-integrin antibody. Further, it was found that blood vessels of coronary arteries operated with the stents coated with anti- integrin antibody, which were prepared by sequential binding with methane and ammonia, has thinner wall and smaller neointima area than those operated with the stents coated with anti-integrin antibody, which were prepared by binding DACH.

[24] Moreover, the use of a catalyst promoting the amide bond formation between the carboxyl groups of anti-integrin antibody and amine groups of the surface of a stent made the coating of the antibodies with a high efficiency and stability.

[25] Brief Description of the Drawings

[26] Objects and features of the present invention will become apparent from the following descriptions given in conjunction with the accompanying drawings, in which: [27] [28] Figure 1 is a schematic diagram showing a plasma polymerization method for binding amine groups to the surface of a stent by sequential binding of methane and ammonia in accordance with the present invention. [29] Figure 2 is a schematic diagram showing a plasma polymerization method for binding amine group of DACH to the surface of a stent. [30] Figure 3 is a microscopic photograph showing the cell adhesion ability of the stent of the invention to vascular endothelial cells of pig. [31] Figure 4 is a microscopic photograph showing the cell adhesion ability of the stent of the invention to smooth muscle cells of pig. [32] Figure 5 is a microscopic photograph showing the cell adhesion ability of the stent of the invention to vascular endothelial cells of human. [33] Figure 6 is a microscopic photograph showing the cell adhesion ability of the stent of the invention to blood platelet. [34]

Best Mode for Carrying Out the Invention [35] The present invention is further illustrated in the following examples, which should not be taken to limit the scope of the invention. [36]

[37] Example 1: Preparation of a coated stent by plasma coating method

[38]

[39] Example 1-1: Preparation of anti-integrin antibody

[40] [41] A mixture of 2 D(IO D/D) offkxintegrin(Centocor Inc., USA) and 2 □ of adjuvant was v 3 injected into 7 weeks old rabbit three times at an interval of 1 week, and two times of injection was followed at an interval of 2 weeks. Then, small amount of blood was collected from the immunized rabbit and measured antibody titer of α β integrin, and v 3 whole blood was collected from the immunized rabbit by cardiac puncture. The collected blood was left to stand for 24 hrs at room temperature to obtain blood serum as a supernatant.

[42] And then, the blood serum was electrophoresed on 12 %(w/v) acrylamide gradient gel, and subjected to Western blot analysis using α β integrin to determine molecular weight of antibody in blood serum. [43] Then, the blood serum was applied on a gel-filtration chromatography which is capable of separating proteins in a range of molecular weight determined as above, to prepare anti-α β integrin antibody. v 3

[44]

[45] Example 1-2: Preparation of stents bound with amine groups by using various plasma polymerization methods

[46]

[47] Stents bound with amine groups were prepared by way of plasma polymerization methods using methane and ammonia, and diaminocyclohexane(DACH), respectively.

[48]

[49] Example 1-2-1: Preparation of a stent bound with amine group by a plasma polymerization method using methane and ammonia gases

[50]

[51] A stent was loaded into a plasma reactor(MPS-60R, Woosin Cry o vac Co., Ltd.,

Korea), and subjected to vacuum condition of 1 mtorr. Then, methane and ammonia gases were sequentially infused into the plasma reactor, and plasma was treated with a radiofrequency of 13.56 Hz, to prepare a stent whose surface is bound with amine groups. In the course of plasma polymerization, methane gas was infused at a fluid speed of 5 seem to maintain the pressure of methane gas in the plasma reactor as 20 mtorr, which was followed by the treatment of plasma with an electrical power of 100 W for 10 min, and ammonia gas was infused at a fluid speed of 5 seem to maintain the pressure of ammonia gas in the plasma reactor as 20 mtorr, which was followed by the treatment of plasma with an electrical power of 300 W for 10 min (see: Fig. 1). Figure 1 is a schematic diagram showing a plasma polymerization method to bind amine group to the surface of a stent by sequential treatment of methane and ammonia gases. As can be seen in Fig. 1, a hydrocarbon layer was first formed in the surface of a stent by using methane gas, and plasma treatment was performed by ammonia gas to prepare a stent whose surface is bound with amine groups.

[52]

[53] Example 1-2-2: Preparation of a stent bound with amine group by a plasma polymerization using DACH

[54]

[55] A stent was loaded into a plasma reactor, and subjected to vacuum condition of 1 mtorr. Then, DACH gas was infused into the plasma reactor, and plasma was treated with a radiofrequency of 13.56 Hz, to prepare a stent whose surface is bound with amine groups. In the course of plasma poymerization, DACH gas was infused at a fluid speed of 10 seem to maintain the pressure of methane gas in the plasma reactor as 20 mtorr, which was followed by the treatment of plasma with an electrical power of 100 W for 5 min(see: Fig. 2). Figure 2 is a schematic diagram showing a plasma polymerization method to bind amine group of DACH to the surface of a stent. As can be seen in Fig. 2, DACH gas was infused into the plasma reactor to give a plasma state to produce ion or radical leading to a sequential polymerization, which forms a layer on the surface of a stent bound with amine groups.

[56]

[57] Example 1-3: Preparation of a stent coated with anti-integrin antibody

[58]

[59] With the surface of a stent bound with amine groups prepared in Examples 1-2- 1 and 1-2-2, was linked with anti-α β antibodies prepared in Example 1-1, to prepare a v 3 stent coated with anti-integrin antibody: 2.5 D of anti-αβ antibodies prepared in v 3

Example 1-1 was dissolved in 20 D of a buffer solution for coating(0.1 M sodium citrate, pH 5.0), added carbodiimide in a concentration of 250 ppm, and reacted at 4 ⁰C for 2 hrs to activate carboxyl groups in anti-integrin antibody molecule. Then, to the buffer solution was immersed a stent bound with amine groups prepared in Examples 1-2-1 and 1-2-2, reacted at 10 ⁰C for 5 hrs, and washed and dried at room temperature to prepare a stent coated with anti-integrin antibody.

[60]

[61] Example 2: Surgical operation of a stent coated with anti-integrin antibody and determination of a restenosis level

[62]

[63] Thirty of the stents coated with anti-α β integrin antibody, which were prepared by v 3 linking anti-integrin antibodies with the amine groups on the surface of the stent in Example 1-3 (Experimental group 1), thirty of the stents coated with anti-integrin antibody, which were prepared by binding DACH to the surface of the stent(Experimental group 2), and thirty of the stents free of anti-α β of integrin v 3 antibody(Control group) were interposed into the blood vessels of coronary arteries of pigs, respectively, to perform angiography. Then, the stent-interposed coronary arteries were collected from the pigs, and tissue pathological features of the blood vessels of coronary arteries and the pigs before and after stent operation were examined and compared with one another. The examination was carried out in the test items of the number of operated stents, the number of dead population, weight(D) of unoperated pigs, weight(D) of operated pigs after 4 weeks, ratio of inner diameter, inner diameter (mm) of unoperated coronary arteries, inner diameter(mm) of operated coronary arteries after 4 weeks, restenosis rate(%) after 4 weeks, area of neointima(mm ²) after 4 weeks, and the number of inflammatary cells in the vicinity of a stent after 4 weeks, where the restenosis rate(%) was calculated as the ratio of pigs in which restenosis was examined among the total operated pigs(see: Table 1). [64] Table 1

Comparison of tissue pathological features of blood vessels of coronary arteries and pigs before and after stent operation

[65] [66] As can be seen in Table 1 above, it was clearly demonstrated that there was no dead pigs after 4 weeks of surgical operation, and the weight of pigs remained the same, indicating that the stent operation was successfully performed in the Control and Experimental groups.

[67] On the other hand, it was examined that blood vessels of coronary arteries operated with the stents coated with anti-α β integrin antibody(Experimental groups 1 and 2), v 3 after 4 weeks of surgical operation, has larger inner diameter of coronary artery, lower restenosis rate, smaller neointima area, and fewer inflammatary cells than those operated with the stents unbound with anti-α β integrin antibody(Control group). v 3

[68] Furthermore, among the stents coated with anti-α β integrin antibody, blood v 3 vessels of coronary arteries operated the stents coated with anti-α β integrin antibody, v 3 which were prepared by binding with methane and ammonia(Experimental group 1), after 4 weeks of surgical operation, has larger inner diameter of coronary artery, lower restenosis rate, smaller neointima area, and fewer inflammatary cells than those operated the stents coated with anti-α β integrin antibody, which were prepared by v 3 binding DACH(Experimental group 2).

[69] Since the outer diameter of blood vessels, upon the surgical operation of the stent, is maintained by the muscle layer around the blood vessels, the smaller inner diameter of blood vessels implies that the wall of blood vessels is relatively thick. As a result, it was formed that: the wall of coronary arteries operated with the stents coated with anti- integrin antibody are thinner than those operated with the stents which is not coated with anti-integrin antibody; and, the wall of coronary arteries operated with the stents coated with anti-integrin antibody, which were prepared by sequential binding with methane and ammonia, are thinner than those operated with the stents coated with anti- integrin antibody, which were prepared by binding DACH.

[70] Therefore, it was demonstrated that the stent coated with anti-α β integrin antibody v 3 prepared by binding with methane and ammonia, was the most superior among the Experimental groups, in terms of inner diameter, restenosis rate, neointima area and the number of inflammatory cells of coronary artery after 4 weeks of surgical operation. In particular, it was assumed that the stent coated with anti-α β integrin antibody, which was prepared by binding with methane and ammonia inhibited the hyperplasia of neointima, so that it can prevent the hypertrophy of inner diameter of coronary artery.

[71]

[72] Example 3: Preparation of a stent coated with anti-integrin antibody by using a catalyst

[73]

[74] Coated amount, coating stability and cell adhesion ability were determined in a stent coated with anti-integrin antibody prepared by using catalysts of l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride(EDAC) and N- hydroxysuccinimide(NHS) which promotes an amide bond formation between carboxyl group of the anti-integrin antibody and amine group on the surface of the stent, and a stent coated with anti-integrin antibody prepared without a catalyst, respectively, and compared with each other.

[75]

[76] Example 3- 1 : Preparation of a stent coated with anti-integrin antibody

[77]

[78] First, a stent coated with anti-integrin antibody was prepared without using a catalyst: anti-α β integrin antibodies prepared in Example 1-1 were dissolved in a v 3 buffer solution for coating(0.1 M sodium citrate, pH 5.0), added 250 ppm of carbodiimide, and reacted at 4 ⁰C for 2 hours to activate carboxyl group in the anti- integrin antibody. And, a stent bound with amine group prepared in Example 1-2-1 was immersed in the buffer solution, reacted at 24 ⁰C for 24 hours, washed and dried at room temperature to prepare a stent coated with anti-α β integrin antibody(Experimental group 11).

[79] Then, a stent coated with anti-integrin antibody was prepared by using a catalyst: a stent coated with anti-α β integrin antibody(Experimental group 12) was prepared v 3 similarly as in Experimental group 11, with an exception of using a buffer solution for coating in which anti-α β integrin antibody, EDAC and NHS are dissolved in a molar v 3 ratio of 4:4: 10, and a stent coated with anti-α β integrin antibody(Experimental group v 3

13) was prepared similarly as in Experimental group 11, with an exception of using a buffer solution for coating in which anti-α β integrin antibody, EDAC and NHS are v 3 dissolved in a molar ratio of 4:20:50, respectively.

[80]

[81] Example 3-2: Comparison of coated amount of anti-integrin antibody in a stent coated with anti-integrin antibody prepared by using a catalyst

[82]

[83] Each of the stents prepared in Example 3- 1 was immersed in 500 D of a surfactant solution(l % CHAPS, 500 mM NaCl in PBS), reacted at room temperature for 2 min by using a rotary reactor(Rotamix, Seourin Co., Korea) to solubilize the anti-integrin antibody coated in the stents, and separated the stents from the reactant. Protein concentration in the reactant was determined to quantitate the amounts of antibodies coated in the stents. Then, the stents separated from the reactant was immersed in 500 D of IN KOH solution, heated at 100 ⁰C for 10 min and chilled, and quantitated the amounts of antibodies solubilized in the solution. By adding the one determined as the above, the total amounts of antibodies coated in the stents were determined, re- spectively(see: Table 2).

[84]

[85] Table 2

Comparison of the coated amounts of anti-integrin antibodies in the stents coated with anti-integrin antibody prepared without or by using a catalyst

[86]

[87] As can be seen in Table 2 above, it was clearly demonstrated that: the coated amounts of anti-integrin antibodies in the stents coated with anti-integrin antibody prepared by using a catalyst(Experimental Groups 12 and 13) were much larger than a stent coated with anti-integrin antibody prepared without a catalyst(Experimental Group 11); and, the coated amounts of anti-integrin antibodies in the stents coated with anti-integrin antibody prepared by using a relatively large amount of catalyst(Experimental Group 13) were larger than the stents coated with anti-integrin antibody prepared by using relatively small amount of catalyst(Experimental Group 12), indicating that the coated amount of antibody is proportional to the used amount of catalyst.

[88] [89] Example 3-3: Comparison of stability of coated anti-integrin antibody in a stent prepared by using a catalyst

[90] [91] Stability of the coated anti-integrin antibodies in each of the stents prepared in Example 3-1 was examined as follows: each of the stents prepared in the above Example 3-1 (Experimental Groups 11 to 13) and the stents sterilized by the treatment of EO(ethylene oxide) gas(Experimental Groups 14 to 16) were immersed in 20 D of a surfactant solution (0.1 %(v/v) Tween 20 in PBS), and reacted at room temperature for 14 days by using a rotary reactor(Rotamix, Seourin Co., Korea) to solubilize the anti- integrin antibodies coated in the stents, and determined protein concentrations in the solutions after 2, 4, 6 and 14 days to compare the stability of antibodies coated in each of the stents(see: Table 3).

[92] [93] Table 3

Changes in the antibodies released from the stents by the treatment of a surfactant solution (unit: D)

[94] [95] As can be seen in Table 3 above, it was clearly demonstrated that: the stents coated with anti-integrin antibody which was prepared without a catalyst(Experimental Group 11) released the antibodies from the stents continuously for 6 days and did not release those after 6 days, and the stents coated with anti-integrin antibody which was prepared by using a catalyst(Experimental Groups 12 and 13) released the antibodies from the stents continuously for 4 days and did not release those after 4 days.

[96] Further, it was found that the stents coated with anti-integrin antibody prepared by using a relatively large amount of catalyst(Experimental Group 13) released relatively small amount of antibody than a stent coated with anti-integrin antibody prepared by using relatively small amount of catalyst(Experimental Group 12). Accordingly, it was clearly determined that: the antibodies were much more stably coated in the stents coated with anti-integrin antibody prepared by using a catalyst than those prepared without a catalyst.

[97] Further, the results of Experimental Groups 14-16 have revealed that the anti- integrin antibody coated stent of the invention, even though it was sterilized, maintained the same antibody binding stability.

[98] [99] Example 3-4: Cell adhesion ability of a stent coated with anti-integrin antibody prepared by using a catalyst

[100] [101] Surgical operation of a stent in the corony arteries leads to a lesion in the blood vessel by the operated stent, whose remedy should be accompanied by the aggregation of vascular endothelial cells in the damaged region. In this regard, it was examined on whether the coated stent of the invention has a specific cell adhesion ability on the vascular endothelial cells: Each of the stents prepared in the above Example 3-1 was immersed in a 48-well plate, added 500 D of PBS and incubated at 37 ⁰C for 30 min. Then, PBS was removed therefrom, added trypsin-treated vascular endothelial cells from pig, cells from pig smooth muscles, vascular endothelial cells from human and blood platelet were added with an amount of 1 x 10 cells/well, and incubated at 37 ⁰C for 30 min to lead an adhesion of the said cells to each of the stents. And then, all of the components except for the stents were removed from each of the wells, and the stents were washed with PBS carefully, and added 80 % formic acid to fix the cells adhered to the stents and examined with a microscope of x 1000 magnifications (see: Figs. 3 to 6). Figure 3 is a microscopic photograph showing the cell adhesion ability of the stent of the invention to the vascular endothelial cells of pig, Figure 4 is a microscopic photograph showing the cell adhesion ability of the stent of the invention to the smooth muscle cells from pig, Figure 5 is a microscopic photograph showing the cell adhesion ability of the stent of the invention to the vascular endothelial cells of human, and Figure 6 is a microscopic photograph showing the cell adhesion ability of the stent of the invention to the blood platelet, where "Control" shows a microscopic photograph of blood platelets.

[102] As can be seen in Figs. 3 and 5, vascular endothelial cells from pig and human were adhered to the stents of Experimental Groups 11 to 13, however Fig. 4 showed that all of pig smooth muscle cells were not adhered to the stents of Experimental Groups 11 to 13. Further, it was examined that vascular endothelial cells from pig and human were adhered to the stents of Experimental Groups 12 and 13 with relatively large amount than Experimental Group 11, implying that the stent of the invention possesses a specific adhesion ability to the vascular endothelial cells.

[103] On the other hand, the blood platelets, as can be seen in Fig. 6, were not adhered to the stents of Experimental Groups 11 to 13, which surely implies that restenosis caused by blood platelet aggregation does not possibly occur.

[104]

[105] From the results of Examples 3-1 to 3-4, it was clearly demonstrated that the use of a catalyst promoting the amide bond formation between the carboxyl groups of anti- integrin antibody and amine groups of the surface of a stent makes possible the coating of the antibodies with a high efficiency and stability.

[106]

Industrial Applicability

[107] As clearly explained and demonstrated as above, the present invention provides a stent coated with anti-integrin antibody which prevents restenosis after surgical operation of a stent and process for preparing the same. The stent coated with anti- integrin antibody of the present invention can be practically applied for the safe surgical operation of the stent, since it can effectively prevent the restenosis after surgical operation of a stent by promoting the regeneration of vascular wall damaged with the operated stent while inhibiting the hyperplasia of neointima and lowers the rate of inflammation in the vicinity of a stent.

Claims

[1] A process for preparing a stent coated with anti-integrin antibody, which comprises the steps of: (i) binding amine group to surface of a stent by plasma polymerization; and, (ii) linking anti-integrin antibody with the amine group on the surface of the stent.

[2] The process for preparing a stent coated with anti-integrin antibody of claim 1, wherein the binding of amine group in step (i) is carried out by sequential binding a gas phase of methane and ammonia to the surface of the stent.

[3] The process for preparing a stent coated with anti-integrin antibody of claim 1, wherein the anti-integrin antibody is a monoclonal antibody or a polyclonal antibody against an integrin protein selected from the group consisting of α β , α β α β α β α β α β α β α β α β α β α β α β α β and α β

2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 1 v l v 3 v 5 6 4 v 8

[4] The process for preparing a stent coated with anti-integrin antibody of claim 1, wherein the anti-integrin antibody is a polyclonal antibody against α β integrin. v 3

[5] The process for preparing a stent coated with anti-integrin antibody of claim 1, wherein an amide bond formation between carboxyl group of the anti-integrin antibody and amine group on the surface of the stent in step (ii) is carried out by using a catalyst.

[6] The process for preparing a stent coated with anti-integrin antibody of claim 5, wherein the catalyst is l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hy- drochloride(EDAC), N-hydroxysuccinimide(NHS) or a mixture thereof.

[7] A stent prepared by the process of claim 1, whose surface is coated with anti- integrin antibody.

[8] The stent coated with anti-integrin antibody of claim 7, wherein the anti-integrin antibody is a monoclonal antibody or a polyclonal antibody against an integrin protein selected from the group consisting ofαβ,αβ,αβ,αβ,αβ,αβ,α β,αβ,αβ,αβ,αβ,αβ,αβ andαβ.

¹ 1 8¹ 1 9¹ 1 v¹ 1 v¹ 3 v¹ 5 6¹ 4 v' 8