NZ517188A - Radioactively coated device and method of making same for preventing restenosis - Google Patents

Abstract

The present invention relates to a rapid and reproducible electrochemical method leading to the production of radioactive angioplastic device such as stents, based on rapid and effective deposition or electrodeposition of charged radioactively coated molecule on oppositely charged surfaces (stainless or gold).

Description

517188 PCT/CAO0/OO974 Radioactivelv coated device and method of maiHng for preventing restenosis BACKGROUND OF THE INVENTION (a) Field of the Invention The invention relates to a radioactively coated device and to a method of making same by deposition of a radioisotope-containing molecule on the device, (b) Description of Prior Art 10 Although coronary angioplasty procedure reduces anginal symptoms, a high incidence of restenosis (30 to 40% within 6 months) is the "Achilles' heel" of interventional cardiology. With over one million coronary procedures performed annually around the 15 world, the economic effect of restenosis is substantial. It is estimated that an effective strategy to prevent restenosis, which would have to be applied after all coronary procedures, would represent a market of at least one billion U.S. dollars (US$) 20 per year. Pharmacological approaches to prevent restenosis have failed to be effective and only coronary stenting procedure reduced restenosis rates (STRESS and BENESTENT trials). Stent deployment, however, frequently induces a new coronary occlusion 25 known as in-stent restenosis. About 20% of stented patients develop in-stent restenosis. To prevent occurrence of stenosis, new therapeutic strategies on the basis of ionizing radiation have recently been proposed. Intracoronary radiation therapy was reported to prevent intimal hyperplasia in various animal models (Raizner et al., Chap 3: 287-296, WO 01/14617 PCT/CA0O/OO974 Vascular Brachytherapy, Second Edition. Armonk, NY, 1999) . In clinical development, endovascular radiotherapy (wire- and stent-based) in patients was reported to be safe and effective in preventing 5 restenosis post-angioplasty (Condado et al., Circulation, 90 (3) -.121-122, 1997; Teirstein et al., N. Engl. J. Med., 336 (24) : 1697-1703, 1997; King et al. , Circulation, 97:2025-2030, 1998; Waksman et al. , Circulation, 101:1895-1898, 2000) . To date, there is 10 no consensus on the use of beta- or gamma-sources and on the choice of medium-energy or higher beta energy (Coursey and Ravinder, Physics Today, vol. 53(4):25-30, 2000) to prevent restenosis. However, beta-emitter source (i.e., 32P, 90Y, 90Sr/Y) significantly reduces 15 operator exposure compared with previous trials with the gamma-emitter isotopes (192Ir) . Coirpared to brachytherapy approach, stent-based radiotherapy acts by preventing both vessel shrinkage and excessive neointimal proliferation.

One of the main limitations of the extensive use of radioactive stent in interventional cardiology is the complex clinical prescription of the metallic prosthesis (diameter, length, type, etc.) associated with the choice of the radioisotope and the activity 25 in function of the physical half-life. Regarding those specifications, the production of an active inventory of such device in a daily practice can be difficult and problematic. A major difficulty to overcome is the need to load any pre-manufactured 30 stents with defined amounts of radioactivity at the time of use. Using stents that are preloaded by the CA000097- manufacturer is not ideal because the stent specifications (specific radioactivity, length diameter, etc.) may differ from.the need.

Hafeli et al. (Biomaterials 19:925-933, 1998) suggested a method for electrodepositing Rhenium (186Re or 188Re) on a stent. However, Hafeli et al. teach that rhenium alone do not electroporate well by itself, and that they had to co-deposit the rhenium with cobalt. Again co-deposition with cobalt caused cracking and flaking of the deposited layer. To overcome these problems, Hafeli et al. deposited over the layer of cobalt rhenium previously deposited a second layer of gold to overlay cobalt and thus prevent cracking. Hafeli et al. also teach that gold, being a noble metal compete with rhenium during the deposition such that gold is deposited preferentially over rhenium.

In International Publication WO 98/17331, there is disclosed an implantable medical device on which a bioactive material may be deposited thereon and retained with a porous layer deposited over the bioactive material layer. However, such procedure is complicated and may not in every cases be reliable and reproducible.

International Publication WO 98/23299 only discloses the preparation .and use of a radiolabeled DNA oligonucleotide, without further providing any method for preparing an angioplastic device as described jLn the_present application.

Furthermore, International Publication WO 99/02195 describes a stent with a radioactive, AMENDED SHEET 13-11-2001 CA000097- -3a- radiopague coating. However, the radioactivity needs to be deposited on the radiopaque material, which itself is deposited on the stent, rendering the method more complicated than the one disclosed hereinafter in 5 the present application.

Consequently, it would be highly desirable to be provided with a strong and rapid deposition process of radioactivity emitting source (such as 32P-oligonucleotide based) on the surface of a device such 10 as a stent to prevent restenosis post-angioplasty, and ^ that would not crack or flake. The ability of 32P- labeled oligonucleotide to inhibit neointimal hyperplasia was already demonstrated in an in vitro model (Fareh et al.. Circulation, 99:1477-1484, 1999).

IS SUMMARY OP THE INVENTION One aim of the present invention is to provide a strong and rapid deposition process of radioactive AMENDED SHEET WO 01/14617 PCT/CA00/OO974 molecule on the surface of an angioplastic device for preventing restenosis post-angioplasty.

In accordance with the present invention there is provided a method for depositing a charged molecule 5 on an angioplastic device. This methods comprises the step of contacting the angioplastic device with a solution containing the charged molecule under suitable conditions for deposition of the charged molecule on the angioplastic device. The charged 10 molecule is preferably a radioactive charged molecule.

In particular, the invention provides a method for coating an angioplastic device, said method comprising the step of: contacting an angioplastic device with a solution containing charged molecules for deposition of the charged molecules on the ^ angioplastic device; wherein no subsequent porous coating is further applied over said charged molecules.

The invention also provides an angioplastic device for preventing restenosis in a coronary and/or peripheral artery, said device comprising charged molecules deposited on its surface, said charged molecules not being chelated.

The deposition can be passive or active. By active deposition, it is meant to comprise e1ectrodeposit ion.

In passive deposition, the angioplastic device 25 has preferably stainless steel or gold on its surface. For gold surface, the charged molecule preferably comprises a thiol-containing group for attaching to the gold on the angioplastic device. For stainless steel, the surface is preferably coated with silicon 30 oxyde (Si02) or silicon (Si) to be modified with (followed by page 4a) INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 8 AUG 2003 RECEIVED - 4a - chemical or electrochemical treatments for its functionnalization. Stainless steel surface can be also directly used for-— electrochemical functionnalization.

Also in accordance with the present invention, there is provided a method for immobilizing a charged molecule on an angioplastic device using passive deposition or electrodeposition. For the electric approach (electrodeposition), the method comprises the step of applying an electric potential difference between the angioplastic device and a solution (followed by page 5) 13-11-2001 CA0000974 "5" containing the charged molecule, said charged molecule having a charge opposite to the electric potential difference and being thereby electrodeposited on the angioplastic device.

The electric potential difference can be made positive or negative, depending on the charge of the molecule to be coated on the device.

Preferably the radioactive molecule comprises a 0-emitter. Preferred p-emitters are selected from the 10 group consisting of Antimony-124, Cesium-134, Cesium-137, Calcium-45, Calcium-47, Cerium 141, Chlorine-36, Cobalt-60, Europium-152, Gold-19'8, Hafnium-181, Holmiun-166, Iodine-131, Iridium-192, Iron-59, Lutetium-177, Mercury-203, Neodymium-147, Nickel-63, 15 Phosphorus-32, Phosphorus-33, Rhenium-186, Rhodium-106, Rubidium-86, Ruthenium-106, Samarium-153, Scandium-46, Silver-llOm, Strontium-89, Strontium-90, Sulfur-35, Technetium-99, Terbium-160, Thulium-170, Tungsten-188, Yttrium-90 and Xenon-133. 20 When the electric potential difference applied is positive, the radioactive molecule is preferably selected from the group consisting of a radioactive DNA or an analog thereof, a radioactive RNA, a radioactive nucleotide, a radioactive oligonucleotide, 25 radiactive H3PO4, radioactive diethylenetriaminepenta-acetic acid, and a radiactive polyanionic complex. More preferably the radioactive molecule is a radioactive oligonucleotide. The oligonucleotide is preferably—a 8j=.— to 35-mer oligonucleotide, more 30 preferably a 8- to 20-mer oligonucleotide, and most preferably a 15-mer oligonucleotide. These molecules.

AMENDED SHEET WO 01/14617 PCT/CA0Q/00974 form negative ions in solutions and are therefore attracted onto the angioplastic device.

When the electric potential difference applied is negative, molecules are preferably selected from 5 the "group consisting of conjugated cationic polypeptides, cationic peptides, dextran, polyamines and chitosan. These molecules are preferably radioactive molecules. These molecules form positive ions in solutions and are therefore attracted onto the 10 angioplastic device.

The angioplastic device may be for example a stent. Preferably the angioplastic device has a metallic surface, such as stainless steel, gold, tantalum, nickel and titanium or any alloy thereof. 15 The method of the present invention may further comprise before the step of applying an electric potential difference, a step of surface cleaning of the angioplastic device with a solvent, electrochemical or argon-ion sputtering treatments for 20 removing impurities at the surface of said angioplastic device, or, after the step of applying an electric potential difference, a further step of rinsing the angioplastic device for removing free molecule at the surface of said angioplastic device. 25 In a preferred embodiment of the present invention, the surface of the angioplastic device is functionnalized for molecule coating. The angioplastic device may be functionnalized for example with a diazonium treatment.

Still in accordance with the present invention, there is provided an angioplastic device for WO 01/14617 PCT/CAOO/00974 preventing restenosis in a coronary and/or peripheral artery, said device comprising a radioactive charged molecule deposited on its surface.

Further in accordance with the present 5 invention, there is provided a method for preventing restenosis in a coronary and/or peripheral artery comprising implanting an angioplastic device as defined above at a site of potential restenosis such as coronary and/or peripheral artery in a patient in 10 need of such a treatment.

The method of the present invention is rapid and allows obtaining a radioactively coated device, on which a radioisotope-containing molecule is effectively and uniformly deposited. No adverse 15 effects of deposition treatment are observed in coated stent in vitro (mechanical and colorless properties) and in vivo (clotting, thrombogenicity). Strong and effective binding of 32P-oligonucleotides on metallic surface was obtained.

Since the method of the present invention is rapid, it also allows to use simultaneously a stent with radiotherapy for preventing restenosis. It is now possible with the method of the present invention to attach a radioisotope-carrying molecule on a device 25 such as a stent, according to a simple method. The simplicity of the method allows for that method to prepare a radioactively coated stent to be used for implantation just moments after its preparation.

By the term functionalization, it is intended 30 to mean the application of a reagent to a solid surface that will permit molecule coating. By the WO 01/14617 PCT/CA00/00974 term radioactively coated device, it ' is intended to mean any device used in the art for treating restenosis. Such device can be without limitation a stent or a radioactive filament for radiotherapy at 5 the site of restenosis or at the site of angioplasty for preventing restenosis in coronary or peripheral vessels.

By the term angioplastic device, it is intended to mean any device used for angioplasty for which 10 radiotherapy would be beneficial. Such device may be without limitation a stent or a wire or any other device to which a person of the art may think of for the prevention of an uncontrolled proliferative lesion. The term angioplastic device is also meant to 15 include any prosthesis to be itrplanted within a vessel or within other body conduit such as, but not restricted to, the bile duct or urethra for the purpose of endovascular treatment.

By the term analog of DNA, it is intended to 20 mean nucleic acid sequences such as double-strand DNA sequences, single-strand DNA sequences, RNA or any combination thereof.

By the term radioactive polyanionic complex, it is intended to means a molecule carrying at least one 25 radioactive element and bearing at least one negative charge.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, 30 that is to say, in the sense of "including, but not limited to". (followed by page 8a) 2 s AUj 21 BECElVFn - 8a - BRIEF DESCRIPTION OF THE DRAWINGS Having thus generally -described the nature of the invention, reference will now be made to the (followed by page 9) INTELLECTUAL PROPERTY OFFICE OF N.Z. 2 8 AUG 2G03 RECEIVED WO 01/14617 PCT/C AOG/00974 accompanying drawings, showing by way of illustration a preferred embodiment thereof, and wherein: Fig. 1 illustrates a schematic electrodeposition set-up in accordance with a 5 preferred embodiment of the present invention; Fig. 2 is a schematic reaction chamber for glicidoxy-propyltriethoxy silane (GPTS) modification for passive deposition; Fig. 3 illustrates a schematic electrodeposition set-up used for diazonium functionnalization of silicon and stainless steel surfaces for passive deposition; Fig. 4 shows the effect of duration of passive deposition of 32P-oligonucleotide on bromobenzenediazonium coated stainless steel surface; Fig. 5 is a line graph of electrodeposition of 15-mer oligonucleotide on gold electrode as a function of potential; Fig. 6 illustrates the adsorption isotherm of 20 15-mer oligonucleotide on gold electrode at different pH of the electrolyte solutions; Fig. 7 illustrates the adsorption isotherm of 8-mer oligonucleotide at different concentrations on gold electrode; Fig. 8 illustrates the effect of duration of polarization on the level of coating of radioactive 15-mer oligonucleotide onto gold plated stent; Fig. 9 illustrates the effect of increasing activity of radioactive 15-mer oligonucleotide on 30 coating onto gold plated stent; WO 01/14617 PCT/CA0Q/00974 Fig. 10 illustrates the effect of duration of polarization on the level of coating of radioactive 15-mer oligonucleotide onto stainless steel stent; Fig. 11 illustrates the effect of increasing 5 activity of radioactive 15-mer oligonucleotide on coating onto stainless steel stent; Fig. 12 is a scan graph of gold plated stents coated with the electrochemical method of the present invention illustrating the distribution of the 10 radioactive molecules onto the metallic surface along the length of the stent; Fig. 13 is a scan graph of stainless steel stents coated with the electrochemical method of the present invention illustrating the distribution of the 15 radioactive molecules onto the metallic surface along the length of the stent; Fig. 14 is a line graph of the in vitro retention profile of 32P-oligonucleotide coated onto the surface of a gold plated stent; Fig. 15 is a line graph of the in vitro retention profile of 32P-oligonucleotide coated onto the surface of a stainless steel stent; Fig. 16 is a line graph of the retention profile of 32P-oligonucleotide-coated gold stent (16 25 mm) when implanted in porcine coronary; and Fig. 17 is a line graph of the retention profile of 32P-oligonucleotide-stainless steel stent (18 mm) when implanted in porcine coronary artery.

DETAILED DESCRIPTION OF THE INVENTION WO 01/14617 PCT/CAOO/00974 In accordance with the present invention, there is provided a method for electrodepositing a radioactive molecule on a device for preventing restenosis.

In a preferred embodiment of the invention, the deposition is an electrodeposition as illustrated in Fig. 1 with the potentiostat/Galvanostat (EG&G model 273A) 20, hereinafter referred to as the potentiostat. In fact, Fig. 1 illustrates the Schematic drawing of 10 the electrochemical cell and angioplastic device used for radioactive molecule coating onto gold and stainless steel surfaces.

In this embodiment, electrodeposition is effected under a nitrogen atmosphere (Na) , in a glass 15 cell 22. The stent 24, which acts as the working electrode, is submerged in the electrolyte 26 with a reference electrode 28 {preferably a PdH2 electrode) and a counter electrode 30 (Pt plate) . The three electrodes are connected to the potentiostat 20, which 20 is itself connected to a computer 32 for recording the working conditions. The cell 22 is provided with a - cover 34 provided with holes for allowing the wires of the electrodes to pass through. The cover 34 is also provided with a gas inlet 36 and a gas outlet 38 for 25 allowing nitrogen to be circulated.

In another preferred embodiment of the invention, the deposition is a passive deposition in which case the set up is similar to the one illustrated in Fig. 1, with the exception that no 30 potentiostat 20 is needed. In such an embodiment, the alternate method of depositing a radioactive WO 01/14617 PCT/CAOO/00974 polyanionic complex, such as a radioactive oligonucleotide, comprises the step of modifying the oligonucleotide by adding a thiol-containing group. The thiol-containing group may be for example a Cs 5 chain carrying a thiol function at its extremity and which is added at the 5' end of the oligonucleotide. The so-modified oligonucleotide may be labeled with 3aP or other radioactive elements. A gold or gold-coated stent is incubated in either 0.1M potassium phosphate 10 buffer (KH2P04 pH 7.0) or pure tetrahydrofuran containing the radiolabeled oligonucleotide. After a 60 minute incubation period at room temperature, the stent is rinsed with distilled water. The radioactive oligonucleotide attaches to gold by the thiol group, 15 producing a radioactively coated stent. This preferred embodiment is only an example (refer to example I) of passive deposition caused by the high affinity of gold for thiol group.

Another example of passive deposition is based 20 on the surface coating with silicon (Si) or silicon oxyde (Si02j followed by surface functionnalization with substrates. In this other preferred embodiment of the invention, the Si02-treated surface is then modified with glicidoxy-propyltriethoxy silane (GPTS), 25 whereas the Si-treated surface is functionnalized with 4-bromobenzenediazonium tetrafluoroborate (diazonium). Stainless steel surface can be directly activated with 4-bromobenzenediazonium tetrafluoroborate without Si/Si02 pre-treatment. The GPTS modification is 30 passive (Fig. 2), whereas the diazonium deposition is an electrochemical functionalization, in which case WO 01/14617 PCT/CAOO/00974 the set up is similar to the one illustrated in Fig. 3.

Fig. 2 illustrates a Schematic drawing of the reaction chamber for glicidoxy-propyltriethoxy silane (GPTS) 5 modification of silicon oxyde treated surfaces.

In Fig. 2, the substrates are taken out of the oven they are placed in the various slots of the 2 glass holders 50. Each holder is hooked to the reaction chamber 52 were the silanization will take 10 place. The whole lot is then placed inside a glove box which is under dry N2 atmosphere. Once inside the glove box the GPTS reaction compounds were then added, in sequence, to the reaction chamber. A magnetic stirring bar 54 is added to the reaction mixture, the 15 reaction chamber is then closed and removed from the glove box. The reaction chamber is connected to a water circulator 56 with temperature control. Stirring is initiated and the reaction is allowed to proceed for 4 hours at 70°C, under continuous N2 flow 20 58 originating from a gas tank.

Fig. 3 illustrates Schematic drawing of the electrochemical cell used for bromobenzenediazonium functionnalization of silicon and stainless steel surfaces.

In Fig. 3, the electrochemical cell 22 was a standard three-electrode setup. The reference electrode 28 used was a saturated Calomel electrode (SCE) and the counter electrode 30 was platinum foil (1 cm2) . The bromo-aryldiazonium solution was used as 30 the electrolyte for cyclic voltammetry in order to attach the bromo-aryldiazonium to the surface (0.5 cm2 WO 01/14617 PCT/CAOO/00974 area) of the Si or 316L substrates acting as working electrode 24. A scanning potentiostat was used to apply dc potentials to the working electrodes. The current-voltage response was recorded on an XY 5 recorder.

In this preferred embodiment of the invention, the alternate method of depositing a radioactive polyanionic complex, such as a radioactive oligonucleotide, comprises the step of modifying the 10 oligonucleotide by adding an amine-containing group. The amine-containing group may be for example a C« chain carrying an amine function at its extremity and which is added at the 5' end of the oligonucleotide. The so-modified oligonucleotide may be labeled with 32P 15 or other radioactive elements.

This preferred embodiment is only an example of passive deposition caused by the high affinity of GPTS and diazonium substrates for amine group.

In another embodiment of the invention, the 20 radioisotope can be attached to other radioisotope-carrying molecule.

For instance, in the preferred embodiment of an electrodeposition set-up (Fig. 1) where the stent plays the role of the anode (positively charged), a 25 negatively charged molecule can be used for an effective electrodeposition onto the stent surface. Preferred negatively charged molecules can be for example without limitation labeled DNA or RNA, or labeled analogs thereof, labeled nucleotides, 30 radioactive h3po4, labeled diethylene triamine pentaacetic acid (DTPA) or labeled polyanionic complexes.

In another preferred embodiment of an electrodeposition set-up where the stent plays the 5 role of the cathode (negatively charged), a positively charged molecule can be used for an effective electrodeposition onto the stent surface. Such positively charged molecule can be for example without limitation labeled conjugated polypeptides, labeled 10 cationic peptides, labeled dextran, labeled chitosan or labeled polyamines.

In accordance with one embodiment of the invention, there is provided a process that can be performed in a daily practice moments prior to the 15 implantation of the device in a catheterization laboratory or in the radiation oncology department, and administered to the patient according to the specification desired. The vehicle carrying the radioisotopic source such as a beta-source (32P) is 20 preferably a short DNA sequence (15 mer oligonucleotides linked together by 11 phosphorothioate bounds), rendering the molecule stable over a long time. Strong binding of DNA-oligonucleotides was reported on gold (Sellergren et 25 al., Anal. Chem., 68(2):402-407, 1996).

When double-stranded nucleic acid is used to be coated on the stent, a first non-radiolabeled strand of this double-stranded nucleic acid can be coated on the stent in accordance with one embodiment of the 30 invention. The second complementary strand of the double-stranded nucleic acid can be labeled and WO 01/14617 PCT/CA00/00974 annealed to the first strand. Such embodiment is also envisioned by the present invention, and is also encompassed in the term a radioactively coated device.

While a P-emitter source of radioisotope is 5 preferred, other sources of radioisotope can also be used in accordance with the present invention.

The radioisotopic source is determined according to the treatment determined. Depending on the cases, the radiotherapy might vary from one 10 patient to another. Accordingly, the radioisotopic source will be determined based on the half-life of the radioisotopic source, its energy and the specific activity of the radioisotopic source desired. The determination of the radioisotopic source is within 15 the skill of a person of the art.

Preferably the radioactive molecule comprises a p-emitter. Preferred p-emitters are selected from the group consisting of Antimony-124, Cesium-134, Cesium-137, Calcium-45, Calcium-47, Cerium 141, Chlorine-36, 20 Cobalt-60, Europium-152, Gold-198, Hafnium-181, Holmiun-166, Iodine-131, Iridium-192, Iron-59, Lutetium-177, Mercury-203, Neodymium-147, Nickel-63, Phosphorus-32, Phosphorus-33,. Rhenium-186, Rhodium-106, Rubidium-86, Ruthenium-106, Samarium-153, 25 Scandium-46, Silver-llOm, Strontium-89, Strontium-90, Sulfur-35, Technetium-99, Terbium-160, Thulium-170, Tungsten-188, Yttrium-90 and Xenon-133.

WO 01/14617 PCT/CAO0/00974 Electrodeposition Stainless steel stent characteristics and surface pre-treatment In a preferred embodiment, ACS multi-link RX 5 DUET™ stents (Guidant Vascular Intervention, Santa Clara, CA) of 13 to 23 mm of length were used in accordance with the present invention. Commercial. 316L stainless steel samples, in the form of 1 cm diameter discs, 0.2 mm thick (Goodfellow Cambridge Ltd., 10 Huntingdon, England) were also used.

Deposition or electrodeposition is more effective when the surface to be coated is cleaned to remove contaminants. To do so, stents to be coated were first washed with organic solvents (acetone or 15 methanol) for removing contaminants and then air-dried. Another example of surface cleaning is argon-ion sputtering. The sputtering of stents or discs was carried out under the following conditions : Initial chamber pressure 1,3x10-8 torr Pressure after argon introduction 1,3x10-5 torr Energy 2 keV Focus voltage 1 keV Current 4 jzA Time 20 min (discs) 5 min (stents) Again, transfer of discs and stents was carried out under vacuum.

An electrochemical method can also be used for cleaning stainless steel surface (stents or discs). 30 Electropolishing was carried out in the glove box using a voltage generator. The cleaning solution was composed of 1 M oxalic acid 15% hydrogen peroxide.

WO 01/14617 PCT/CA00/00974 Only two electrodes were used : the sample was one and the other, a Pt disk. A potential of 10 V was applied for 10 minutes between. these electrodes, followed by extensive rinsing and transferred to the 5 electrochemical deposition cell of Fig. 1.

Gold stent characteristics and surface pre-treatment In a preferred embodiment, NIROYAL™ 24 ct gold plated stents (Boston Scientific Ireland Ltd. Ballybrit Business Park, Galway, Ireland) of 13 to 23 10 mm of length were used in accordance with the present invention. Gold-coated 316L discs in the form of 1 cm diameter discs (Goodfellow) were also used.

Gold surface can be directly used for electrodeposition or cleaned with argon-ion sputtering 15 in conditions as previously described for stainless steel metal. 3SP-oligonucleotide compounds In one embodiment of the invention, the vehicle chosen for carrying the beta-source (32P) is a short 20 DNA sequence (15 mer oligonucleotides linked together by 11 phosphorothioate bounds, patent No. 5,821,354). This short DNA sequence was reported to be highly stable and effective in the prevention of cell proliferation with no side effects (Fareh et al., 25 Circulation, 99:1477-1484, 1999).

For the embodiment of passive deposition, the radioactive molecule has at its 51 end either an amine-containing group as for example a C6 chain carrying an amine fvinetion or a C6 chain carrying a 30 thiol function. The amine- and thiol-modified WO 01/14617 PCT/CA00/00974 oligonucleotides may be labeled with 32P or other radioactive elements.

Electrodeposition of 32P-oligonucleotides Electrodeposition is effected in an 5 electrochemical cell containing the 32P-oligonucleotides (75 jaCi/50 |iL of water) diluted in 250 jiL of acetate sodium buffer, (CH3CH2C02Na. 3H20 at 0.2 M) at pH 8.5. In the electrochemical cell containing both electrolyte and 32P-oligonucleotide solutions, a 10 metallic stent was fixed to the anode and the cathode was composed of a platinum wire of 2 mm diameter and 5 cm length or a Pt plate.

Electrodeposition is performed by applying a voltage of 1 Volt (50-60 mA) for 15 minutes using a 15 standard potentiostat 20 at room temperature.

Electrodeposition succeeds in binding 2.5% of initial 32P-oligonucleotides on the stent surface, when any post-treatments were applied.

Another example of electrolytes for effective 20 electrodeposition is aqueous phosphate solutions.

To evaluate the electrodeposition of the 15-mer oligonucleotide onto the gold surface (electrodes and plated stents) in aqueous phosphate solutions as electrolytes, a method for studying the adsorption of 25 DNA was used. Briefly, cyclic voltammetry (CV) coupled to electrochemical quartz crystal nanobalance system was used to study the adsorption of organic molecules on gold surface. Since the frequency variation of the crystal and the cyclic voltammogram are recorded 30 simultaneously, this method allows to measure the quantity of molecules adsorbed on gold in the whole 13-11-2001 CA000097* potential window and in only one cycle. Fig. 5 illustrates a surface concentration (t) of 15-mer oligonucleotide (3.8 |jM) on gold electrodes as a function of potential in the pH-6.98-7.0 phosphate 5 buffered solution. The scan rate is lOO mV/s. An arrow indicates the beginning of the scan.

As illustrated in Fig. 5, the electrosorption of 15-mers increases as the polarization potential is increased and reaches a maximum E - 1.1-1.2 V vs. SCE 10 (calomel reference electrode) (see Fig. 5). At potential higher than 1.1-1.2 V, the surface concentration of the molecule starts to decrease. This • phenomenon can be explained by the oxidation of gold occurring at these potentials when using phosphate 15 buffer as electrolyte solution.

After repeating the same procedure for several different concentrations- of 15-mer molecule, the adsorption isotherm at constant potential was obtained in those conditions. For that example, gold plated 20 stents (NIROYAL™) and commercial electrodes of gold (0.1684 cm3, Aldrich Canada) were used. Gold wires were inserted in a Kel-F rod in order to have only one tip of the wirer in contact with the solution. Kel-F was chosen as the support material because it is inert in 25 acidic and basic aqueous media. The electrode was polished with a 0.5 ^m alumina suspension. Aqueous phosphate solutions were prepared from a Na2HP04,7H20 (17.8897 g/L) and a KH2P04 (9.0725.g/L) solutions.

Fi-g-i—6 illustrates the adsorption isotherm of 30 15-mer oligonucleotide on gold electrodes at E-l.l V, SCE (calomel reference electrode) in phosphate AMENDED SHEET WO 01/14617 PCT/CAOO/00974 buffered solutions at pH=6.98-7.0, pH=8.04 and pH=5.59. In Fig. 6, the adsorption isotherm of nonradioactive 15-mer oligonucleotide on gold at 1.1 V vs. SCE is presented for the three buffered solutions 5 studied. One may note that at pH=6.98-7.0, an increase of the concentration of oligonucleotide leads to an increase of the surface concentration, until a plateau is attained at a concentration of about 20 jiM. Beyond this point, an increase in the concentration of 10 15-mer oligonucleotide does not enhance the surface concentration. Similar experiments were performed at pH=5.59 and pH=8.04 showing that electrosorption of 15-mer oligonucleotide on gold is more effective at pH=6.98-7.0. Higher electrosorption was obtained when 15 polarization was performed at 60°C.

Fig. 7 illustrates the adsorption isotherm of 8-mer oligonucleotide on gold electrodes at E=l.l V, SCE (calomel reference electrode) in phosphate buffered solutions at pH=6.98-7.0. As shown in Fig. 20 7, electrosorption of 8-mer oligonucleotides is effective onto gold electrode surface, when applying a voltage of" 1.2 V during 15 minutes at room temperature. Higher electrosorption was obtained when polarization was performed at. 60°C. Similar adsorption 25 isotherm of a 35-mer oligonucleotide was reported.

When gold stents (16 mm) were polarized at 1.2 V during 15 to 30 minutes in presence of 32P-oligonucleotide (800 jiCi) at room temperature, 2.5 to 3 fiCi of radioactivity were detected onto the stent 30 surface (corresponding to 0.3% of efficacy coating) WO 01/14617 PCT/CA00/00974 and no alteration of the surface integrity was reported.

Other electrolyte useful for the present invention 32P-oligonucleotide depositions were carried out 5 in 0,1 M HC104 under nitrogen (bubbler), at a potential of 1,45 V vs. SCE (saturated calomel electrode) and at a temperature of 60 ± 10 °C. For that preferred embodiment, higher coating was obtained at 60°C. However, coating of 32P-oligonucleotide onto stainless 10 steel or gold surfaces is also feasible and effective at room temperature.

The electrochemical cell (Fig. 1) was composed of three electrodes : i) the working electrode (our sample); ii) the counter electrode (Pt disk); and the 15 reference electrode (Pd/PdH2) , calibrated before each measurement.

The reference electrode is made by flowing hydrogen on a Pd disk in 0,1 M HC104 for 30 minutes.

The effects of polarization duration and the 2.0 initial activity were assessed with native gold stents of 16 mm, where no surface cleaning was performed. Similarly, stainless steel stent of 18 mm, previously cleaned with 1M oxalic acid 15% hydrogen peroxide were also used. A series of time of electrodeposition (5, 25 15, 30 and 60 min) were used.

Fig. 8 illustrates the effect of coating duration on electrodeposition level (16 mm-gold plated stents). As illustrated on Fig. 8, the maximal coating was reached at 5 to 15 min on gold surface, 30 underlying the rapid and effective electrodeposition of 32P-oligonucleotide onto the gold stent (average of WO 01/14617 PCT/CAOO/00974 1.6%). Fig. 9 reports the activity-dependent coating onto gold surface when increasing activity of 32P-oligonucleotide (0.25, 0.5, 1 and 2 mCi) were tested during 5 minutes. However, higher effective coating 5 was obtained at low initial activity (1.9%, 1.2%, 0.8% and 0.5% for 0.250, 0.500, 1.0 and 2.0 mCi respectively). In those conditions (5 min of coating), an effective coating of 0.5% (average) was obtained, corresponding, for example, of an activity of 10 jiCi 10 onto a 16 mm-gold stent. Similar levels were obtained when the gold surface was cleaned with argon-ion sputtering.

Fig. 10 ilustrates the effect of coating duration on electrodeposition level (18 mm-stainless steel 15 stents) . As illustrated in Fig. 10, a similar coating of 0.5% was obtained at 5 to 15-20 min to reach a maximal coating (1.0%) at 60 min on stainless steel surface, underlying the rapid and effective electrodeposition of 32P-oligonucleotide onto the 20 stainless steel stent. When increasing activity of 32P-oligonucleotide (0.25, 0.5, 1 and 2 mCi) were tested, similar coating with activity of 0.25 to 1.0 mCi (average of 2.5-3.0 H-Ci) were obtained, whereas higher activity (2.0 mCi) led to significant amount of 32P-25 oligonucleotide onto the stainless steel stent surface (Fig. 11). In those conditions (15 min of coating), an effective coating of 0.5% (average) was obtained, corresponding, for example, of an activity of 10 |iCi onto a 18 mm-stainless steel stent. Fig. 11 30 illustrates the effect of increasing activity of 32P-oligonucleotide on coating efficiency (18 mm-stainless WO 01/14617 PCT/CA00/00974 steel stents). Similar levels were obtained when the stainless steel surface was cleaned with argon-ion sputtering. 32P-oligonucleotide distribution onto the surface Coated stents (n=6 gold plated and n=6 stainless steel stents), using HC104 as electrolytes, were scanned for 4 hours to visualize the distribution of 32P-oligonucleotides onto the metallic surface along the length of the stent. The stent radiation uniformity 10 was measured using a 0.5 mm slit in front of a Geiger counter which was moved over the stent in 0.5 mm steps by a computer-controlled stepping motor.

Regarding the scan graph of the coated stent, the electrodeposition was highly uniform on the 15 metallic surface of gold plated (Fig. 9) and stainless steel stents (Figs. 12 and 13). Figs 12 and 13 illustrate scan graphs of a gold plated stent or of a stainless steel stent, respectively, coated with 32P-oligonucleot ide.

Similar uniform distribution of radioactivity was also obtained when acetate sodium buffer as electrolytes was used to perform electrodeposition in the set-up of Fig. 1.

Post-treatment of the radioactive stents (in vitro 25 retention) Following electrodeposition in the acetate sodium buffer electrolyte, radioactive stents were rinsed in distilled water for 24 hours at room temperature and air-dried or sonicated for 3 0 minutes. 30 Biological treatments were investigated by incubating radioactive stents with DMEM supplemented with an enzyme solution consisting of 5 pi of Nuclease Si WO 01/14617 PCT/CA0G/00974 (332 JJ/fil) , 1 ill of Exonuclease III (E. coli; 100 U/^il) , and 1 ijlI of phosphodiesterase (0.5 U//il) in presence of 10% Fetal Bovine Serum (FBS, Gibco) overnight at 37°C. Following incubation of coated 5 stents in water for 24 hours, 80% of initial coating solution remained on the metallic surface, whereas additional sonication procedure (30 minutes) reduced to 50% the retention rate. Following a biological treatment (blood mimicking enzyme solution) of coated 10 stents at 37°C during 14 to 16 hours, 12% of the amount of radioactivity remained on the stent, when compared to the initial electrodeposition level.

Following electrodeposition in the HC104 0.1M electrolyte, radioactive coated stents (n=8 gold 15 plated stents of 16 mm) were incubated in biological medium composed of DMEM in presence of 20% Fetal Bovine Serum (FBS, Gibco) at 37°C with constant agitation. Those physical and biological conditions were used to mimic in vivo conditions. A sample of 20 medium (50 jiL) was counted following 15, 30, 60, 120, 240 min and 24 hours of incubation. Fig. 14 illustrates the retention profile of coated 32P-oligonucleotide onto 16 mm-gold plated stent surface in in vitro conditions (blood mimicking conditions) . 25 As illustrated in Fig. 14, following incubation of gold coated stents at 37°C, a progressive elution of the 32P-oligonucleotide was reported, corresponding to a remaining activity of an average of 50, 40 and 35% after 60, 120 and 240 min respectively. A significant 30 sustained activity of 10-12% is reported up to 8 days of treatment in blood mimicking conditions, when WO 01/14617 PCT/CA0Q/00974 compared to the initial electrodeposition level {Fig. 14) .

Similarly, radioactive coated stents (n=8 stainless steel stents of 18 mm) were incubated in 5 biological medium composed of DMEM in presence of 20% Fetal Bovine Serum (FBS, Gibco) at 37°C with constant agitation. A sample of medium (50 (iL) was counted following 15, 30, 60, 120, 240 min and 18 hours of incubation. Fig. 15 illustrates the retention profile 10 of coated 32 P-oligonucleotide onto 18 mm-stainless steel stent surface in in vitro conditions (blood mimicking conditions). As illustrated in Fig. 15, following incubation of stainless steel coated stents at 37°C, a progressive elution of the 32P-15 oligonucleotide was reported, corresponding to a remaining activity of an average of 45 to 37-40% after 60 to 240 min. A significant sustained activity of 40% is reported following 1 day of treatment in blood mimicking conditions; an average of less than 10% of 20 initial electrodeposition level remained up to 7 days of incubat ion.

Regarding the combination of~a simple method to produce radioactive stent and a well defined release of the radioactive molecule from the angioplastic 25 device, a classical stent-based radiation as well as a stent-based pharmacological approach can be envisaged to prevent restenosis.

To reinforce the strength of the proposed radioactive coating, the metallic surface can be 30 embedded in a simple manner. A series of biostable coatings and agar solution of 1 to 2% were tested and WO 01/14617 PCT/CA00/00974 shown to improve the molecule retention by reducing the elimination of the 32P-oligonucleotide from the metallic surface. Polymer coating (such as parylene) already used for medical application is proposed to 5 embed the angioplastic device.

To support the pharmacological approach, the well-defined elution from the coated stents can serve as a local drug delivery device to prevent restenosis, based on data obtained on intra-arterial sustained-10 release of beta particles. In that case no device embedding is performed.

Mechanical properties of the radioactive coated stents General observations were done on the coated stents such as determination of color and rigidity. 15 Mechanical properties were estimated by mimicking in vivo stent deployment. After mounting the stent on deflated balloon, the balloon was inflated to 10-14 atm and the capability of stent deployment was evaluated. No physical alteration (color and deployment ability, surface deterioration, cracking and flaking of the surface) was observed in coated stents according to the present invention. Under fluoroscopy, the visibility of the coated stent was not modified.

Implantation of the radioactive coated Btent in porcine coronary arteries Domestic pigs were sedated with intramuscular injection of ketamin, azaperon and atropine to undergo anesthesia with thiopental sodium (iv). The pigs were 30 intubated and ventilated with a mix of isoflurane 2% and oxygen during the procedure. An 8 Fr. guiding catheter was advanced through a femoral sheath with a WO 01/14617 PCT/CA00/00974 0.035 J guide-wire, under fluoroscopic monitoring in the ascending aorta. The guide wire was then removed, allowing the guiding catheter to be positioned in the ostium of the target vessel. Prior to performing the 5 angiography, a bolus of 1 ml of nitroglycerin solution with a concentration of 0.3 mg/mL is injected intra-coronary. The angiography was then performed in at least two near orthogonal views that visualize the target site of right coronary artery (RCA) or left 10 circumflex artery (LCX) of the pig. A quantitative coronary angiography (QCA) measure was done to assess the vessel size for adequate stent implantation. Stent was advanced to the target site and balloon inflation at 10 to 12 atm for 30 seconds was performed 15 to adequately deploy the stent (2 stents per pig) . Following stent implantation, the balloon was deflated and the catheter withdrawn. Control angiography was then performed to document any residual luminal stenosis or vessel wall dissection. If spasm was 20 documented, 1 ml of nitroglycerin solution at a concentration of 0.3 mg/mL was injected intra-coronary.

Hacroscopical observations After stent implantation, treated pigs were 25 maintained for 6 hours under observation. Following pig euthanasia with a lethal dose of KCl, myocardium was dissected to remove stented arteries. A macroscopical observation of the heart and stented artery was performed to explore the potential side 30 effects of coating stent implantation (thrombogenicity, clotting, etc.). Stents were then WO 01/14617 PCT/CAOO/00974 removed from the artery to be counted to assess the in vivo retention of 32P-oligonucleotides onto the stent surface. For that example, coated-stents generated with acetate sodium buffer as electrolytes and Fig. 1 5 as electrochemical set-up were used for coronary implantation Following fluoroscopy and macroscopical observations, no side effects related to the implantation of a radioactive treated stent according 10 to the present invention were observed either in the myocardial tissue or in the implanted artery. Measurements of radioactivity level of coated stents revealed that 6 hours following stent implantation 45% of initial coated activity remained on the stent 15 surface, whereas low radioactivity was detected in the target artery (less than 3%) , suggesting that coronary wash-out eliminates more than 44% of the drug from the stent surface within 6 hours. The biological half-life of coated 32P-oligonucleotides on the surface stent in 20 porcine coronary arteries was estimated to be approximately 5.5 to 6 hours. The residence time of the coated 32P-oligonucleotides is 11- to 12-fold higher than direct intra-mural administration of liquid 32P-oligonucleotides using the Infiltrator® 25 catheter (0.51 hours).

In vivo follow-up of 32P-oligonucleotide elution from coated stents The catheter-based radiation detection via the endovascular detector permits the fine and continuous determination of the elution profile of the radioactive molecule from the stent. For that issue, gold-plated (16 mm) and stainless steel (18 mm) stents WO 01/14617 PCT/CAOO/00974 were used. 32P-coated stents, generated with HCL04 as electrolytes, were implanted in porcine coronary arteries (LCX and RCA) for 3 hours as previously described.

Using the endovascular detector, measurements of radioactivity levels were done every 30 seconds to follow local 32P-oligonucleotide elution from the stent. At the end of continuous endovascular monitoring (up to 3 hours) , the pig was sacrificed 10 with a lethal dose of KC1, myocardium was dissected to remove stented arteries. Blood was collected during the experiment.

Figs. 16 and 17 illustrate the retention profile of coated 32P-oligonucleotide gold-plated stent 15 (16 mm) and coated 32P-oligonucleotide stainless steel stent (18 mm), respectively, when implanted in porcine coronary artery. As illustrated in Figs. 16 and 17, the elution profile of gold-plated and stainless steel stents, electrocoated with 32P-oligonucleotide, is 20 characterized by two components: a rapid elution during the first 30 min. and a significant sustained radioactivity, which is maintained up to 3 hours. Few radioactivity was detected in blood samples, stented coronary and the adjacent myocardium. 25 The present invention will be more readily un derstood by referring to the following examples, which are given to illustrate the invention rather than to limit its scope. 13-11-2001 CA0000974 EXAMPLE I Passive Deposition Using Thiol-modified Oligonucleotide Coating of gold-plated stents with 33P-oligonucleotide 5 containing a 5'-end thiol moiety.

NIROYAL™ gold-stents (6 mm) were placed in a piranha solution (3:7 v/v, 30% H202 : 98% H2S04) at 70°C for 20 min. Stents were then washed with H20, acetone, ethanol and H2O and dried under a stream o£ N2 gas. The 10 pre-cleaned stents were then placed either in potassium phosphate buffer (K2HP04 - KH2P04; pH 7.0) or in tetrahydrofuran (THF) containing 100 y.Ci of 32P-oligonucleotide containing a 5'-end thiol moiety to be incubated 60 min. .at room temperature. Radioactive 15 stents were then rinsed 3 times with 50 ml of H20.

Radioactivity levels of NIROYAL™ gold stents following passive deposition was 1.15 |iCi when incubated in pure tetrahydrofuran and 0.02 jiCi when incubated in potassium phosphate buffer, corresponding 20 to an efficiency of passive deposition of 1.15% and 0.02% respectively. Following immobilization, stents were incubated 2 days in pig blood at 37°C with constant agitation. Stents were then removed from biological conditions to be rinsed with water and 25 remaining radioactivity was assessed by scintillation counting. NIROYAL™ gold stents incubated in the tetrahydrofuran solution supplemented with 32P-oligonucleotide lost 33% (0.80 jiCi residual activity) and 66% (0.34 nCi residual activity) of its initial 30 activity after" 1 and 2 days of incubation, respectively. Stents incubated in potassium phosphate AMENDED SHEET WO 01/14617 PCT/CA0O/OO974 buffer lost 100% of their initial activity after 1 day of incubation.

EXAMPLE II Passive Deposition Using GPTS Modification Functionalization of Si/Si02 substrates using glicidoxy-propyltriethoxy silane (GPTS) - Substrates: The Si/Si02 substrates were 1 cm x 1 cm plates 10 taken from diced 4 inch wafers (Tronics Microsystems, Grenoble). The Si (100) is n-type, phosphorous doped to a density of 1015 cm"3, and has a thickness of 0.3 Jim. The Si is covered with a thermally grown Si02 layer which is 150 A thick. The back of the Si plates was 15 covered with a Cr/Au ohmic contact.

- Cleaning: The substrates were placed in boiling acetone (Sprectrograde, Aldrich) for 5 minutes, followed by another 5 minutes in boiling methanol (Sprectrograde, 20 Aldrich). The substrates were then dipped in sulfochromic acid (prepared by adding 95 mL of concentrated sulfuric acid (H2S04) to 5 mL of a saturated aqueous solution of potassium dichromate (K2Cr207)) for 4 minutes at room temperature. 25 The substrates were rinsed for 15 seconds with distilled-deionized (d-d) water, and then placed in boiling d-d water for 10 minutes. Following this, the substrates were dried with N2 flow and placed in a clean oven (ambiant atmosphere) at 140°C for 1 hour.

WO 01/14617 PCT/CA00/00974 - GPTS modification: The substrates, with the reaction chamber illustrated in Fig. 2, are placed inside a glove box which is under dry N2 atmosphere. Once inside the glove 5 box, the substrates were placed in the reaction chamber and the GPTS reaction compounds were then added, in sequence, to the reaction chamber. The reaction mixture consisted of 111 mL of o-xylene (98% sealed under nitrogen, Aldrich), followed by 12.5 mL 10 of GPTS (98% purity, Fluka) , and then 1.5 mL of diisopropyl-ethyl amine (99.5% purity sealed under nitrogen, Aldrich) (for a batch of 8 substrates). A magnetic stirring bar is added to the reaction mixture, the reaction chamber is then closed and 15 removed from the glove box.

The reaction chamber is connected to a water circulator with temperature control. Stirring is initiated and the reaction is allowed to proceed for 4 hours at 70°C, under continuous N2 flow. 20 The substrates are removed from the reaction chamber, dipped in ethanol (Spectrograde, Aldrich) for 5 minutes (at room temperature) , and allowed to dry under ambiant atmosphere. The substrates are finally stored individually in glass vials containing 5 mL of 25 ethyl ether (99.9% purity HPLC grade, Aldrich).

WO 01/14617 PCT/CA00/00974 Tiwmfth< 1 ^ nation of 32P-oliqoniicleotide onto GPTS modified Si/SiOj substrates 32P-oligonucleotide (40 fiCi, with or without a C6 amino linker at the 5' end) is directly deposited onto 5 the surface of a GPTS modified substrate. The 32P-oligonucleotide solution was left to react for 2 hours on the GPTS surface in 0.01M in KOH, under humid atmosphere. The substrate surface was then rinsed with d-d water.

Results When passive deposition was performed on Si/Si02 substrates functionnalized with GPTS, a 5-fold increase of coating was obtained with the 32P-oligonucleotide with amino linker, when compared to 15 simple 32P-oligonucleotide (0.10% vs 0.02% of initial activity, respectively), corresponding to a better affinity of 32P-oligonucleotide with amino linker to the GPTS surface than the non-modified 32P-oligonucleotide. Moreover, the radioactivity level due 20 to immobilized 32P-oligonucleotide with amino linker increases with initial 32P-oligonucleotide concentration up to 300 ^Ci, at which point it appears to level off. Immobilization efficiency was better at a reaction temperature of 52°C (2.19% of initial 25 activity), when compared to 22°C (0.16% of initial activity), 37°C (0.19% of initial activity) and 70°C (1.0% of initial activity) . A 12 to 13 fold-increase of coating was reported when deposition was performed at 52°C, when compared to room temperature conditions. - 35 -EXAMPLE III Paaslve Deposition Using pi«»rm->nTn Modification Electrochemical functionalization of Si and stainless steel substrates (discs and stents) with 5 bromobenzenediazonium, and 3aP"oligonucleotide immobilization The procedure used to electrochemically. modify the Si and the 316L Stainless Steel substrates is described in C. Henry de Villeneuve et al., (J". Phys.

Chem. B, 101, 2415-2419 (1997)).

Purity of chemicals and solvents Chemicals Source Purity/Concentration Trichloroethylene Fisher Scientific Reagent Grade Ammonium Fluoride J. T. Baker Chem. Co. 40% Solution Methanol EM Scientific HPLC Grade Acetone Fisher Scientific HPLC Grade Hydrofluoric Acid Fisher Scientific 49% 4-Bromobenzene- diazonium Tet raf1uoroborat e Aldrich Chem. Co. 96% Sulfuric Acid Mallinckrodt 96% Substratess The silicon (Si, 100) substrates were lxl cm2, taken from a diced wafer purchased from Tronics Microsystems (Grenoble, France). The Si was phosphorous doped (n-type) to a density of 1015cm"3. A gold/chromium film was deposited under vacuum at the 20 backside of the Si substrate providing an ohmic contact. The stainless steel substrates were 316L type WO 01/14617 PCT/CA00/00974 (Fe/Crl8/Nil0/Mo3) , 10 ram in diameter and 0.2 mm thick, from Goodfellow Cambridge Ltd. (Huntingdon, England). In a preferred embodiment, ACS multi-link RX DUET™ stents (Guidant Vascular Intervention, Santa 5 Clara, CA) of 18 mm of length were used in accordance with the present invention. Stents were cut to have a 9 mm of length for experiments.

Prior to the electrochemical functionalization, both types of substrates were submitted to a 10 cleaning/etching procedure. The Si substrates were cleaned by immersing in trichloroethylene, acetone, and methanol for 1 minute each, respectively. They were rinsed in distilled-deionized (d-d) water and dried with N2 flow. The Si substrates were then 15 chemically etched for one minute in hydrofluoric acid and six minutes in buffered ammonium fluoride, rinsed once again and dried using N2. The 316L substrates (discs and stents) were immersed in 50 mL of aqua regia (concentrated HC1:HNC>3, 4:1 (v/v)) for 1 minute, 20 rinsed with d-d water and dried with N2 flow.

Bromo-aryldiazonium salt solution A 20mM aqueous solution of" 4-bromobenzenediazonium tetrafluoroborate in 0.1M H2S04 and 2% HF was prepared by dissolving 0.54g of 4-25 bromobenzenediazonium tetrafluoroborate, 0.56 mL of concentrated H2S04 and 4mL of concentrated HF in 100 mL of d-d water. The solution was deaerated by bubbling N2 for approximately 20 minutes.

Electrochemical functionalization: The electrochemical cell was a standard three- electrode setup. The reference electrode used was a WO 01/14617 PCT/CA00/00974 saturated Calomel electrode (SCE) purchased from Fisher Scientific and the counter electrode was platinum foil (1 cm2) . The electrochemical cell is illustrated in Fig. 3.

The bromo-aryldiazonium solution was used as the electrolyte for cyclic voltammetry in order to attach the bromo-aryldiazonium to the surface of the Si or 316L substrates acting as working electrode. A scanning potentiostat (EG&G Princeton Applied Research 10 Model 362) was used to apply dc potentials to the working electrodes. The current-voltage response was recorded on an XY recorder (Phillips, Model PM 8143).

A single-cycle voltammogram was run on each substrate. The current range was set at 1mA. The 15 reductive scan was run from an initial potential of -0.3 V to a final potential of - 1.9 V vs. SCE, and back. The scan rate was set at 100 mV/s. A typical reductive wave (at - -1.5 V) was observed for modification of a Si substrate. The current density is 20 greater for the 316L substrate because of its greater conductivity and the reduction wave is observed at - -0.95 vs. SCE.

Results In that series of experiments, all stainless 25 steel surface (discs and stents) were functionnalized with diazonium and then coated in presence of 50 jiL (50 jiCi) of 32P-oligonucleotide/amino linker solution. They were rinsed as previously described. 32P-oligonucleotide with a C6 amino linker at the 5' end 30 was used for that embodiment.

WO 01/14617 PCT/CA00/00974 Using the discs surface, immobilization efficiency reached a level of 9.5 nCi/cm2 with initial activity of 50 nCi of 32P-oligonucleotide/amine linker solution (9.5% of efficiency). Increasing initial 5 activity to 300 jiCi improved the coating efficiency to 15.8 fxCi/cm2. Coating was better at a reaction temperature of 52°C, when compared to 22 and 70°C. A 2 to 3 fold-increase of coating was reported when deposition was performed at 52°C (8 to 18 |xCi/cm2) , 10 when compared to room temperature conditions. As shown in Fig. 4, the level of coating increased with the reaction time (5, 15, 30, 60 and 120 minutes) . The radioactivity undergoes a gradual increase with reaction time, going from approximately 6 nCi/cm2 at 5 15 minutes to 17.5 fxCi/cm2 at 120 minutes. When compared to disk functionnalization, immobilization efficiency was increased by 1.4 fold on stainless steel stent surface. An average of 2.93 ^.Ci of 32P-oligonucleotide/amino linker solution was coated on 20 a stainless steel stent of 9 mm, corresponding to a level of 24.5"|iCi/ cm2 or an activity of 5.9 p.Ci for a stent of 18 mm. Those experimental conditions underlined the rapidity of the coating of 32P-oligonucleotide/amino linker solution of the stent 25 surface.

Fig. 4 illustrates the effect of duration of passive deposition on 32P-oligonucleotide coating onto bromobenzenediazonium-treated stainless steel surface.

While the invention has been described in 30 connection with specific embodiments thereof, it will WO 01/14617 PCI7CAOO/00974 be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the 5 invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the 10 scope of the appended claims. 40

Claims (37)

Claims

1. A method for coating an angioplastic device, said method comprising the step of: contacting an angioplastic device with a solution containing charged molecules for deposition of the charged molecules on the angioplastic device; wherein no subsequent porous coating is further applied over said charged molecules.

2. The method of Claim 1, wherein the deposition is a passive deposition.

3. The method of Claim 2, wherein the angioplastic device has gold on its surface, and wherein the charged molecule comprises a thiol-containing group for attaching to the gold1 on the angioplastic device.

4. The method of Claims 1 or 3, wherein the deposition is an electrodeposition.

5. The method of Claim 4, wherein a charge is applied to said angioplastic device for depositing the charged molecule, said charged molecule having a charge opposite to the charge of the angioplastic device to thereby electrodeposit the charged molecule on the angioplastic device.

6. The method of Claim 5, wherein the charge of the angioplastic device is positive.

7. The method of Claim 5, wherein the charge of the angioplastic device is negative. ^i'ULLUUML^HUPtHTY 0FF(Ce] 2 8 AUG 2003 RECEIVED 41 9. 10. 14.

The method of Claim 7, wherein the charged molecule is selected from the group consisting of conjugated polypeptides, conjugated cationic peptides, dextran, polyamines and chitosan.

The method of any one of Claims 7 to 8, wherein the angioplastic device is a stent.

The method of Claim 9, wherein the angioplastic device has a metallic surface.

The method of Claim 10, wherein the metallic surface is selected from the group consisting of stainless steel, gold, tantalum, nickel and titanium or any alloy thereof.

The method of Claim 5, further comprising before the step of applying a charge to the angioplastic device, a step of washing the angioplastic device with a solvent for removing impurities at the surface of said angioplastic device.

The method of Claim 5, further comprising after the step of applying a charge to the angioplastic device, a step of rinsing the angioplastic device for removing free molecules at the surface of said angioplastic device.

The method of Claims 1 to 13, wherein said charged molecule is a radioactive charged molecule.

The method of Claim 14, wherein the angioplastic device has gold on its surface, and wherein the radioactive charged molecule comprises a 42 thiol-containing group for attaching to the gold on the angioplastic device.

16. The method of Claims 14 or 15, wherein the radioactive molecule comprises a (3-emitter.

17. The method of Claim 16, wherein the p-emitter is Antimony-124, Cesium-134, Cesium-137, Calcium-45, Calcium-47, Cerium-141, Chlorine-36, Cobalt-60, Europium-152, Gold-198, Hafiiium-181, Holmiun-166, Iodine-131, Iridium-192, Iron-59, Lutetium-177, Mercury-203, Neodymium-147, Nickel-63, Phosphorus-32, Phosphorus-33, Rhenium-186, Rhodium-106, Rubidium-86, Ruthenium-106, Samarium-153, Scandium-46, Silver-llOm, Strontium-89, Strontium-90, Sulfur-35, Technetium-99, Terbium-160, Thulium-170, Tungsten-188, Yttrium-90 and Xenon-133.

18. The method of Claim 6, wherein the radioactive molecule is selected from the group consisting of a radioactive DNA, a radioactive RNA, a radioactive nucleotide, a radioactive oligonucleotide, radioactive H3PO4, and a molecule carrying at least one radioactive element and bearing at least one negative charge.

19. The method of Claim 18, wherein the radioactive molecule is a radioactive oligonucleotide.

20. The method of Claim 19, wherein the oligonucleotide is an 8- to 35-mer oligonucleotide. 43

21. The method of Claim 19, wherein the oligonucleotide is an 8- to 20-mer oligonucleotide.

22. The method of Claim 19, wherein the oligonucleotide is a 15-mer oligonucleotide.

23. The method of any one of Claims 5 to 22, further comprising after the step of applying a charge to the angioplastic device, a step of rinsing the angioplastic device for removing free radioactive molecules at the surface of said angioplastic device.

24. The method of any one of Claims 5 to 23, wherein before the step of applying a charge to the angioplastic device, the surface of the angioplastic device is treated for molecule coating.

25. The method of Claim 24, wherein the angioplastic device is treated with a diazonium treatment.

26. An angioplastic device for preventing restenosis in a coronary and/or peripheral artery, said device comprising charged molecules deposited on its surface, said charged molecules not being chelated.

27. The angioplastic device of Claim 26, wherein the angioplastic device is a stent or a microcatheter wire.

28. The angioplastic device of any one of Claims 26 to 27, wherein the charged molecule comprises a P-emitter.

29. The angioplastic device of Claim 28, wherein the (3-emitter is selected from the group consisting of Antimony-124, Cesium-134, Cesium-137, intellectual property office OF N.Z. 2 8 AUG 2003 RECEIVFD 44

30.

31.

32. #

33.

34.

35. Calcium-45, Calcium-47, Cerium-141, Chlorine-36, Cobalt-60, Europium-152, Gold-198, Hafirium-181, Holmiun-166, Iodine-131, Iridium-192, Iron-59, Lutetium-177, Mercury-203, Neodymium-147, Nickel-63, Phosphorus-32, Phosphorus-33, Rhenium-186, Rhodium-106, Rubidium-86, Ruthenium-106, Samarium-153, Scandium-46, Silver-llOm, Strontium-89, Strontium-90, Sulfur-35, Technetium-99, Terbium-160, Thulium-170, Tungsten-188, Yttrium-90 and Xenon-133. The angioplastic device of any one of Claims 26 to 29, wherein said charged molecule is being selected from the group consisting of a radioactive DNA, a radioactive RNA, a radioactive nucleotide, a radioactive oligonucleotide, radioactive H3PO4, and a molecule carrying at least one radioactive element and bearing at least one negative charge. The angioplastic device of any one of Claims 26 to 30, wherein the charged molecule is a radioactive oligonucleotide. The angioplastic device of Claim 31, wherein the oligonucleotide is a 10- to 30-mer oligonucleotide. The angioplastic device of Claim 31, wherein the oligonucleotide is an 8- to 20-mer oligonucleotide. The angioplastic device of Claim 31, wherein the oligonucleotide is a 15-mer oligonucleotide. The angioplastic device of any one of Claims 26 to 29, wherein the radioactive molecule is selected from the group consisting of radioactive 45 conjugated polypeptides, radioactive cationic peptides, radioactive dextran, radioactive polyamines and radioactive chitosan.

36. A method for coating an angioplastic device substantially as herein described with reference to the accompanying Examples.

37. An angioplastic device for preventing restenosis in a coronary and/or peripheral artery substantially as herein described with reference to the accompanying Examples. END OF CLAIMS -1 0 c: 2003 RECEIVED 100248816_1.DOC