Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D153/00—Coating compositions based on block copolymers containing at least one sequence of a polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds; Coating compositions based on derivatives of such polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/28—Materials for coating prostheses
  • A61L27/34—Macromolecular materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/54—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
  • A61L29/08—Materials for coatings
  • A61L29/085—Macromolecular materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
  • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
  • A61L29/16—Biologically active materials, e.g. therapeutic substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/08—Materials for coatings
  • A61L31/10—Macromolecular materials
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L31/16—Biologically active materials, e.g. therapeutic substances
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
  • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
  • C08F220/10—Esters
  • C08F220/22—Esters containing halogen
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
  • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F297/00—Macromolecular compounds obtained by successively polymerising different monomer systems using a catalyst of the ionic or coordination type without deactivating the intermediate polymer
  • C08F297/02—Macromolecular compounds obtained by successively polymerising different monomer systems using a catalyst of the ionic or coordination type without deactivating the intermediate polymer using a catalyst of the anionic type
  • C08F297/026—Macromolecular compounds obtained by successively polymerising different monomer systems using a catalyst of the ionic or coordination type without deactivating the intermediate polymer using a catalyst of the anionic type polymerising acrylic acid, methacrylic acid or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L53/00—Compositions of block copolymers containing at least one sequence of a polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
  • A61L2300/606—Coatings

Definitions

• the present invention relates to a new class of fluorinated copoiymers which, due to their good combination of low or negligible cytotoxicity, biocompatibility (especially blood compatibility), infection and encrustation resistance and due to their interaction with tissue cells leading to integration, are useful for biomedical applications.
• the present invention also relates to a process for manufacturing such fluorinated copoiymers.
• this invention also relates to implantable and invasive biomedical devices such as implants, stents, catheters, staples and the like which are coated with a layer of such a fluorinated copolymer. Another use of such polymers is as a substrate for seeding endothelial cells.
• the present invention relates to a method for treating atherosclerosis by making use of a coronary stent coated with a layer of such a fluorinated copolymer, the said layer being optionally loaded with biologically-active ingredients.
• the invention is in the field of biomaterials, more particularly for stenting of coronary arteries and for other implant therapies.
• a coronary stent made from a metal tubing which is brought to the place of obstruction, i.e. inserted into a vessel, for instance via percutanic transluminal coronary angioplasty and then dilatated, e.g.
• Stents provide a permanent support of the vessel wall and ensure an efficient flow therethrough.
• acute occlusion due to thrombogenecity of the metal and restenosis due to neointimal proliferation of smooth muscle cells tend to impede the success of the presently existing stents.
• their thrombotic character induces the need for a simultaneous anti-coagulation therapy which adds to the discomfort of the treatment. Restenosis of blood vessels occurs after narrowed or occluded arteries are forcibly dilated by balloon catheters, drills, lasers and the like in an angioplastic procedure.
• stents are expected to reduce restenosis by preventing recoil of the treated blood vessel to its original dimensions.
• Various stents are known in the art, including those which are expandable by balloon catheters, heat expandable or self-expandable.
• stents alone cannot prevent restenosis caused by neointimal hyperplasia of the tissues of the vessel wall.
• the stent material itself may accelerate such hyperplasia, since it is foreign to the body tissues. Therefore there is a need in the art for coating stents with a material which is not recognized as a foreign body.
• stent coating material which can be applied onto the stent body as a flat thin layer, because during stenting, expansion usually leads to a rough surface which will cause more tissue damage, e.g. more restenosis. Solving these problems would open the way to a number of further medical applications since stents are not only used for coronary arteries, but also in the oesophagus for strictures or cancer, the urether for maintaining drainage from the kidneys, or the bile duct for pancreatic cancer or cholangiocarcinoma.
• - X is hydrogen or methyl
• - Y is a single bond or a radical of the formula CH 2 NRS0 2 , R being a C ⁇ - 6 alkyl group,
• - Z is hydrogen or fluorine
• - n is 0 to 12 and moieties derived from at least one second non-fluorinated comonomer, the glass transition temperature of the homopolymer of said second comonomer being lower than the glass transition temperature of the homopolymer of said first comonomer.
• the present invention further provides terpolymers and tetrapolymers additionally comprising moieties derived from at least one hydrophiiic, e.g. cationic, third comonomer and/or from at least one non-hydrophilic fourth comonomer copolymerizable with the first comonomer and the second comonomer.
• the present invention provides such copoiymers, terpolymers and tetrapolymers in the form of a layer preferably having a thickness in the range of about 0.1 to 15 ⁇ m and optionally further comprising a biologically active ingredient.
• the present invention provides biomedical devices, e.g. implantable and invasive biomedical devices such as implants, catheters (both cardiovascular and urinary), artificial veins and arteries, valves, stents (especially coronary stents comprising a stent body having a metal surface) and the like coated with at least one such layer and optionally coated with at least one barrier layer.
• the copoiymers, terpolymers and tetrapolymers of the invention are capable of providing a sustained or controlled release of the biologically active ingredient over an extended period of time.
• the present invention provides use of such a copolymer, terpolymer or tetrapolymer as a substrate for seeding of endothelial cells.
• the present invention further provides a method for treating atherosclerosis by making use of a coronary stent coated with at least one layer of such a copolymer, terpolymer or tetrapolymer.
• Figure 1 shows the rate of conversion and the proportions of comonomers, versus time, incorporated into a copolymer of the invention.
• Figure 2 shows the sensorgrams of human serum albumin-antibody responses in a surface plasmon resonance study of a copolymer of the invention.
• Figure 3 shows antibody binding of various proteins in a surface plasmon resonance study of a copolymer of the invention.
• Figure 4 shows radioactive counts for various copoiymers and terpolymers of the invention in an in vitro bacterial adhesion study via radiolabelling.
• Figure 5 shows the detection of adenosine triphosphate for various copoiymers and terpolymers of the invention in a bacterial resistance study.
• the present invention is based on the unexpected discovery that the polymeric combination of a fluorinated monomer and a non-fluorinated monomer able to decrease the glass transition temperature of the polymer sequence derived from the fluorinated monomer results in useful biomaterials. More specifically, it was observed that this polymeric combination, in the form of a copolymer optionally comprising additional hydrophilic and non-hydrophiiic comonomers, provides some generally useful biocompatibility properties.
• biocompatibility as used herein comprises in particular a low or negligible cytotoxicity, as well as anti- thrombogenic, anti-inflammatory and anti-immunological properties (i.e.
• - X is hydrogen or methyl
• - Y is a single bond or a radical of the formula CH 2 NRSO 2 or NRSO 2 , R being a C- ⁇ - 6 alkyl group, - Z is hydrogen or fluorine, and n is 0 to 12.
• the comonomer of formula (I) is a fluorinated acrylate or methacrylate optionally (i.e. when Y is not a single bond) bearing a sulfonamido group.
• the comonomer of formula (I) is not necessarily a perfluoro acrylate or methacrylate.
• C-i- ⁇ alkyl as used herein, unless otherwise stated, means a linear or branched alkyl group having from 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n- hexyi and the like.
• Non limiting examples of comonomers of formula (I) include trifluoroethyl methacry.ate, octafluoropentyl methacrylate, dodecafluoroheptyl methacrylate, pentadecafluorooctyl methacrylate, heptadecafluorooctyl ethylsulfonamidoethyl acrylate and heptadecafluorooctyl butylsulfonamidoethyl acrylate.
• the homopolymer of the fluorinated monomer of formula (I) usually (i.e. for a few possible exceptions) usually exhibits a relatively high glass transition temperature (T g ), i.e. a T g of at least about 25°C, it is generally not entirely suitable for biomedical applications, most of which require some degree of flexibility and elastomeric character which is able to be maintained after sterilization.
• T g glass transition temperature
• the said first fluorinated monomer of formula (I) be admixed in a polymeric composition together with at least one second non-fluorinated comonomer, the glass transition temperature of the homopolymer of said second non-fluorinated comonomer being lower than the glass transition temperature of the homopolymer of said first fluorinated comonomer.
• the second non-fluorinated comonomer must be copolymerizable in some way with the first fluorinated monomer of formula (I), so that a copolymer comprising moieties from each comonomer species can be obtained.
• the second non- fluorinated comonomer be an acrylic monomer.
• a non limiting example of such a non-fluorinated commercial comonomer is 2-ethylhexyl acrylate. Keeping in mind the desirable T g of the homopolymer, those skilled in the art will readily be able to access additional suitable comonomers, in particular acrylic comonomers.
• copoiymers of the present invention exhibit a glass transition temperature in the range of about -20°C to 20°C.
• T g above about 20°C will generally exhibit some kind of brittleness which will make them inappropriate for most biomedical uses, namely for use in implants such as stents and catheters.
• copoiymers with a T g below about -20°C will generally exhibit some substantial tendency to stickiness which will make them uneasy to handle in manufacturing processes and/or in application of biomedical devices.
• Tailoring the glass transition temperature of the copolymer of the invention in the range of about -20°C to 20°C is easily available to those skilled in the art and can be achieved by a number of different ways such as, for instance : - selecting the first fluorinated comonomer within the frame of formula (I),
• the average composition of the copoiymers of the present invention may thus be from about 1 to 99 mole %, preferably from about 25 to 75 mole %, of the first fluorinated comonomer and from about 99 to 1 mole %, preferably from about 75 to 25 mole. %, of the second non-fluorinated comonomer.
• this average composition does not exclude the presence in the copolymer of some fragments or sequences or blocks exclusively constituted from either comonomer moieties.
• the detailed structure of the copoiymers of the present invention may in some respect play a role in their suitability for biomedical applications and was therefore investigated.
• the copoiymers of the present invention include at least one block derived from the homopolymer of the first fluorinated comonomer and/or at least one block derived from the homopolymer of the second non-fluorinated comonomer, i.e. they may include a sequence from either or both homopolymers of the building comonomers. This may be derived from the fact that one of the comonomers, usually the fluorinated comonomer of the formula (I), is substantially consumed at the very beginning of the copolymerization reaction (i.e.
• the copoiymers of the present invention may conveniently be obtained via a free radical polymerization process. Therefore another object of the present invention is a process for making the said copoiymers, comprising providing a mixture comprising :
• a solvent for the said first fluorinated comonomer and the said second non-fluorinated comonomer there can be used a halogenated aliphatic or aromatic hydrocarbon or a mixture thereof.
• the said solvent may suitably be selected from 1 ,1 ,1-trichioroethane, trichlorotoluene, trifluorotoluene and their mixtures.
• the temperature conditions used in performing the process of the invention include a temperature not exceeding the boiling point of the solvent.
• the pressure in the reactor may be either raised above or reduced below the normal pressure, although this is in general not necessary.
• the free radical initiator to be used in the said process may be selected from azo ⁇ c compounds such as 2,2'- azobisisobutyronitrile peroxides (e.g. benzoyl peroxide) or conventional alternatives) and redox initiators such as benzoyl peroxide combined with N,N- dimethyltoluidine. Selection of an appropriate free-radical initiator as a function of the selected copolymerization temperature and of the effective amount of such initiator for performing the process of the invention is within the general knowledge of those skilled in the art of polymer synthesis.
• An alternative method for preparing the copoiymers of the present invention involves anionically copolymerizing a mixture comprising :
• the glass transition temperature of the homopolymer of said second comonomer being lower than the glass transition temperature of the homopolymer of said first comonomer, at a temperature below about 30°C in the presence of an aprotic solvent, for instance tetrahydrofuran or the like, and further in the presence of at least an anionic polymerization catalyst such as an alcaline metal alkoxide of a tertiary alcohol (e.g. potassium or cesium tertiary butoxide) and/or a crown-ether such as 1 ,4,7,10,13,16-hexaoxocyclooctane (18-crown-6).
• an alcaline metal alkoxide of a tertiary alcohol e.g. potassium or cesium tertiary butoxide
• a crown-ether such as 1 ,4,7,10,13,16-hexaoxocyclooctane (18-crown-6).
• anionic polymerization can take place at very low temperatures, down to -78°C. Selection of an appropriate anionic polymerization catalyst and of its effective amount for performing such a process is within the knowledge of those skilled in the art of polymer synthesis.
• additional moieties derived from other comonomers in particular from comonomers which are able to impart some specific physical and /or chemical properties to the resulting copoiymers.
• the latter may further comprising moieties derived from at least one hydrophilic third comonomer copolymerizable with the first comonomer and the second comonomer.
• the ability of the third comonomer to copolymerize with the other comonomers is easily determinable by those skilled in the art.
• the hydrophilic third comonomer is an acrylic monomer. Many such acrylic monomers may be contemplated for this purpose.
• the hydrophilic comonomer may be selected from acrylic acid, methacrylic acid and their alcali or alcaiine-earth salts, N,N-dialkylaminoalkyl acrylates and methacrylates (e.g. N,N- dimethylaminoethylmethacryiate), quaternized salts of N,N-dialkylaminoalkyl acrylates and methacrylates (e.g. N,N-dimethylaminoethylmethacrylate benzalkonium chloride useful for its aseptic properties) and polyalkyleneglycol- containing monomers wherein the polyalkyleneglycol sequence (e.g.
• polyethyleneglycol or polypropyleneglycol preferably has a molecular weight in the range of about 400 to 10,000.
• the latter monomers include, among others, monoacrylates and monomethacrylates as well as monomers derived from polyalkyleneglycol-containing macro-initiators.
• hydrophilic third comonomer results in the formation of a terpolymer with hydrophilic properties. It may be achieved, when the said hydrophilic third comonomer is not a quaternized N,N-dialkylaminoalkyl acrylate or methacrylate, directly copolymerizing the said hydrophilic third comonomer in a reactor together with the first fluorinated comonomer and the second non- fluorinated comonomer under the conditions stated hereinabove.
• the hydrophilic third comonomer is a quaternized N,N-dialkylaminoalkyl acrylate or methacrylate
• hydrocarbon groups such as arylalkyl groups may be covalently linked to the main terpolymer chain via the nitrogen atom of the N,N-dialkylaminoalkyl acrylate or methacrylate.
• hydrophilic third comonomer is acrylic acid or methacrylic acid or their salts and/or a quaternized N,N-dialkylaminoalkyl acrylate or methacrylate
• copolymerization may result in an ionic terpolymer, i.e. a so-called ionomer, obtainable in the form of an aqueous solution following procedures well known to those skilled in the art.
• ionomer i.e. a so-called ionomer
• the hydrophilic third comonomer is a polyalkyleneglycol-containing monomer
• free-radical copolymerization results in an amphiphilic terpolymer which is especially useful for its protein- and blood platelet- repelling properties.
• the polyalkyleneglycol-containing monomer derives from a polyalkyleneglycol-containing macro-initiator
• the latter may be prepared in a two-steps reaction involving, in a first step, reacting an azo ⁇ c compound having terminal carboxylic acid groups, such as 4,4- azobiscyanopentanoic acid, with for instance a N-hydroxyimide in a solvent (such as tetrahydrofuran) and further in the presence of a coupling agent such as cyclohexylcarbodiimide, and in a second step reacting the resulting azo ⁇ c compound having terminal imido-ester groups with an ⁇ -amino- ⁇ -methoxy- polyalkyleneglycol, the polyalkyleneglycol sequence (e.g.
• polyethyleneglycol or polypropyleneglycol preferably having a molecular weight in the range of about 400 to 10,000.
• a polyalkyleneglycol-containing macro-initiator can then be used as an initiator-comonomer in a free-radical copolymerization process together with the first fluorinated and second non-fluorinated comonomers, thus leading to a resulting terpolymer with a terminal moiety having e.g. the formula :
• hydrophilic third comonomer incorporated into the terpolymers is not critical to the present invention. It usually needs not to be a high proportion in order to impart useful hydrophilic properties to the resulting terpolymer.
• a proportion of the hydrophilic third comonomer of up to about 40 mole %, preferably from about 1 to 30 mole %, with respect to the combined amounts of the first. comonomer and the second comonomer is usually a sufficient amount for the purpose of the present invention. This proportion will also depend upon the nature of the hydrophilic third comonomer ; for instance a proportion of about 1 to 10 mole % should be sufficient when acrylic acid or methacrylic acid constitutes the said hydrophilic third comonomer.
• the copoiymers of the present invention may further comprise moieties derived from at least one additional non-hydrophilic comonomer (hereinafter the « fourth comonomer ») copolymerizable with the first fluorinated comonomer and the second non-fluorinated comonomer.
• additional non-hydrophilic comonomer hereinafter the « fourth comonomer »
• the said non-hydrophilic fourth comonomer may be selected for its ability to modify the copolymer surface properties by bearing at least one functional group which is able to react with the copolymer's environment in medical applications, such as the amino groups present in protein.
• the said non-hydrophilic fourth comonomer should preferably be an acrylic monomer.
• succinimidyl methacrylate is succinimidyl methacrylate.
• incorpororation of such a non-hydrophilic fourth comonomer results in the formation of a ter- or tetrapolymer with additional specific physical or chemical properties, including a suitable reactivity with amino groups.
• the succinimidyl methacrylate moieties are able to react with an ⁇ -amino- ⁇ -methoxy polyethyleneglycol in order to introduce polyethyleneglycol methacrylamido groups into the polymeric chain.
• the amount or proportion of the non-hydrophilic fourth comonomer is not critical to the present invention.
• a proportion of the non-hydrophilic fourth comonomer of up to about 15 mole %, preferably from about 1 to 10 mole %, with respect to the combined amounts of the first and second comonomers - and optionally third comonomer - is usually a sufficient amount for the purpose of the present invention.
• the copoiymers and terpolymers of this invention most often exhibit a number average molecular weight in the range of about 25,000 to 200,000, more preferably about 30,000 to 130,000 and/or a molecular weight polydispersity in the range of about 1.3 to 5.5, more preferably about 1.5 to 3.5, i.e. a relatively narrow distribution of molecular weights.
• the copoiymers of this invention may be sterilized. This may conveniently be effected by means of irradiation, for instance via electron-beam or X-rays.
• the copoiymers of the present invention may be processed in the form of flat thin layers,.
• the term « thin layer » as used herein, unless otherwise stated, means a layer that can obtained by either of the conventional dip coating and spray coating techniques. This usually corresponds to a thickness in the range of about 0.1 to 15 ⁇ m.
• the need for a flat layer is especially crucial for the coating of stents.
• stents coated with a copolymer layer according to the invention will help reducing restenosis and therefore improve the success of angioplasty.
• copolymer layers may further comprise a biologically effective amount of at least one biologically active ingredient such as a therapeutic, diagnostic or prophylactic agent.
• a biologically active ingredient such as a therapeutic, diagnostic or prophylactic agent.
• the said therapeutic agent can be selected for its specific properties such as for instance its anti-thrombotic, anti- inflammatory, anti-proliferative or anti-microbial efficiency.
• anti-microbial agents such as broad spectrum antibiotics for combating clinical and sub-clinical infections.
• proteins and peptides produced either by isolation from natural sources or recombinantly
• hormones including proteins and peptides (produced either by isolation from natural sources or recombinantly), hormones, carbohydrates, antineoplastic agents or anti-proliferative agents, anti- inflammatory agents, antiangiogenic agents, vasoactive agents, anticoagulants, immunomodulators, cytotoxic agents, antiviral agents, antibodies, neurotransmitters,
• Therapeutically active proteins which can additionally be present in the formulations of this invention include fibroblast growth factors, epidermal growth factors, platelet-derived growth factors, macrophage-derived growth factors such as granulocyte macrophage colony stimulating factors, ciliary neurotrophic factors, cystic fibrosis regulator genes, tissue plasminogen activator, B cell stimulating factors, cartilage induction factor, differentiating factors, growth hormone releasing factors, human growth hormone, hepatocyte growth factors, immunoglobulins, insulin-like growth factors, interleukins, cytokines, interferons, tumor necrosis factors, nerve growth factors, endothelial growth factors, non-steroidal anti- inflammatory drugs, osteogenic factor extract, T cell growth factors, tumor growth inhibitors, enzymes and the like, as well as fragments thereof.
• macrophage-derived growth factors such as granulocyte macrophage colony stimulating factors, ciliary neurotrophic factors, cystic fibrosis regulator genes, tissue plasminogen activator, B cell stimulating factors, cartilage in
• Diagnostic agents which can be present in the copolymer layers of this invention include conventional imaging agents (for instance as used in tomography, fluoroscopy, magnetic resonance imaging and the like) such as transition metal chelates. Such agents should be incorporated into the layers of the invention in an effective amount for performing the relevant diagnostic.
• the at least one biologically active ingredient may be formulated together with additional pharmaceutically acceptable excipients and carriers, as well as together with a biological delivery system able to perform a specific pharmacological function, such as for instance a composition for providing a controlled or sustained release of the said biologically active ingredient into the body of a mammal, in particular of a human patient in need of a specific therapy.
• the nature and amount of the at least one biologically active ingredient incorporated in the copolymer layers of the present invention depends on the specific biological action to be performed when implanting a device coated with such a layer into the body of a patient in need of a specific therapy.
• Another embodiment of the present invention consists of a biomedical device, for instance an implantable or invasive biomedical device, coated with at least one layer comprising a copolymer as hereinabove described (i.e. including ter- and tetrapolymers possibly incorporating a third and/or fourth comonomer) and possibly further comprising a biologically effective amount of at least one biologically active ingredient.
• a biomedical device for instance an implantable or invasive biomedical device, coated with at least one layer comprising a copolymer as hereinabove described (i.e. including ter- and tetrapolymers possibly incorporating a third and/or fourth comonomer) and possibly further comprising a biologically effective amount of at least one biologically active ingredient.
• a copolymer as hereinabove described (i.e. including ter- and tetrapolymers possibly incorporating a third and/or fourth comonomer) and possibly further comprising a biologically effective amount
• Non limiting examples of implantable and invasive biomedical devices which fall within the framework of this invention include, for instance, catheters, stents, staples, threads, needles, pacemakers, valves, artificial veins and arteries, electronic pumps and sensors, tubings, prostheses, i.e. implants and materials of all kinds designed for use in the body of a mammal, and more specifically for the human body.
• implantable and invasive biomedical devices are well known to those skilled in the art of surgery making use of implants. If necessary for some end uses, they may additionally be coated with at least one barrier layer.
• the biomedical devices of this invention have a bio-inert surface, for instance a hydrophobic surface with low surface energy and possibly with amphiphilic heterogeneous microdomains.
• the biomedical device of this invention may also usefully exhibit antagonist properties namely against protein activation, cell activation (e.g. anti-inflammatory), bacteria, and/or anti-thrombotic properties, encrustation repellency and the like.
• the biomedical device of this invention may be a stent, for example a coronary stent, comprising an expandable stent body having a plastic or metal surface and wherein the copolymer layer is coated onto at least a part of the said surface, for instance onto at least one side thereof.
• a stent is particularly useful for the treatment of atherosclerosis, consequently the present invention also provides a method for such treatment by making use of, i.e. by implanting into the body of a patient in need of such treatment, a coronary stent coated with a copolymer layer (such as disclosed hereinbefore) on at least a part of its surface.
• another embodiment of the present invention consists of a process for making a coated stent, comprising coating a stent body having a non- biocompatible surface, for instance a metal or plastic surface, with at least one polymer, the said polymer comprising a copolymer as hereinabove described (i.e. including ter- and tetrapolymers possibly incorporating a third and/or fourth comonomer) and possibly further comprising a biologically effective amount of at least one biologically active ingredient.
• Coating may be effected by any of the conventional dip coating or spray coating techniques and is preferably effected in a manner such as to provide a thin copolymer layer such as hereinabove described.
• the use of the copolymer layer of this invention in biomedical applications, especially in implantable and invasive biomedical devices, is among others based on a satisfactory adhesion to the copolymer of those proteins which are abundantly present in plasma and which fulfill transport and passivating functions in the last step of the coagulation cascade in immune reactions such as albumin, fibrinogen and immunoglobulin G. Adherence of such proteins is believed to facilitate endothelial cell adherence.
• a thin natural endothelial layer will substantially prevent the surface, for instance the metal surface, of the said implantable or invasive biomedical device to be recognized as a foreign body by the cells of the patient in which the device is implanted. Tissue reactions such as inflammation, severe immunological reactions, thrombus formation, accute occlusion or restenosis are thereby minimized or suppressed.
• a steady or controlled release of restenosis-suppressing agents over a period of about 10 to 30 days would also allow to inhibit the start of the restenosis process.
• Protein adhesion was found to be especially satisfactory for those terpolymers of the invention which include moieties derived from a cationic third comonomer, such as a quaternized N,N-dialkyiaminoalkyl acrylate or methacrylate or a (meth)acryiic acid or acid salt.
• a cationic third comonomer such as a quaternized N,N-dialkyiaminoalkyl acrylate or methacrylate or a (meth)acryiic acid or acid salt.
• reaction mixture 100 ml methylene chloride. After addition of 60 ml triethylamine (0.43 mole), the solution was cooled down to 0°C. Next, 65 ml methacrylic acid anhydride (0.43 mole) was added dropwise. After 4 hours reaction at 40°C, the reaction mixture was further stirred overnight at room temperature. In order to neutralize traces of unreacted methacrylic acid anhydride, the reaction mixture was reacted during 20 minutes with 5.2 ml (0.086 mole) aminoethanol and then diluted with 100 ml CH 2 CI 2 and 100 ml water and additionally stirred for 1 hour.
• the glass transition temperature of this copolymer is 14°C. Its average number molecular weight (determined by gel permeation chromatography while using a Styragel mixed B column, 10 ⁇ , 2 x 30 cm from Polymer Laboratories on N-methylpyrrolidone and using polystyrene as a standard) is 89,000 and its molecular weight polydispersity M w /M n is 1.7.
• EXAMPLE 4 preparation of a poly(pentadecafluorooctylmethacrylate-co-2- ethylhexylacrylate)
• the procedure of example 3 is repeated, except that 0.65 mole of pentadecafluorooctylmethacrylate, either prepared in accordance with a procedure similar to example 1 or originating from Polyscience (Germany), is copolymerized with 0.35 mole 2-ethylhexyl acrylate while using trichlorotrifluoroethane or trifluorotoluene as the reaction solvent.
• the copolymer is obtained with a yield of 87%.
• the glass transition temperature (measured as in example 3) of this copolymer is 13°C. Its average number molecular weight (determined as in eample 3) is 123,000 and its molecular weight polydispersity M w /M n is 1.8.
• EXAMPLE 5 preparation of a poly(trifluoroethylmethacrylate-co-2- ethylhexylacrylate)
• the procedure of example 3 is repeated, except that 0.5 mole of trifluoroethylmethacrylate, either prepared in accordance with a procedure similar to example 1 or originating from Polyscience (Germany), is copolymerized with 0.5 mole 2-ethylhexyl acrylate.
• the copolymer was obtained with a yield of 95%.
• the glass transition temperature of this copolymer (measured as in example 3) is 14°C. Its average number molecular weight (determined as in example 3) is 40,000 and its molecular weight polydispersity M w /M n is 3.0.
• EXAMPLE 6 preparation of a poly(octafluoropentylmethacrylate-co-2- ethylhexylacrylate-co-dimethylaminoethylmethacrylate) Terpolymerization is effected according to a procedure similar to that of example 3, except that 0.37 g dimethylaminoethylmethacrylate is added into the polymerization tube. A yield of 80% of terpolymer was obtained.
• EXAMPLE 7 preparation of a quaternized poly(octafluoropentylmethacry- late-co-2-ethylhexylacrylate-co-dimethylaminoethylmethacrylate
• terpolymer obtained in example 6 comprising 0.0238 mole dimethylaminoethylmethacrylate
• CH 2 CI 2 30% (weight/volume)
• EXAMPLE 8 preparation of a poly(octafluoropentylmethacrylate-co-2- ethylhexylacrylate-co-succinimidyl methacrylate)
• a procedure similar to that of example 3 is used, except that 0.7125 mole of octafluoropenttylmethacrylate prepared according to example 1 is copolymerized with 0.2375 mole of 2-ethylhexylacrylate and 0.05 mole of succinimidyl methacrylate prepared according to example 2, while using methylene chloride as the reaction solvent.
• the copolymer was obtained with a yield of 90%.
• EXAMPLE 9 preparation of a poly(octafluoropentylmethacrylate-co-2- ethylhexylacrylate-co-polyethyleneglycolmethacrylamide)
• the dichloromethane phase was dried by means of MgSO 4 and evaporated and the resulting terpolymer, bearing polyethyleneglycol methacrylamido groups, was further vacuum dried and characterized by 1 H-NMR in CDCI 3 providing the following spectrum (monomer abbreviations are as in example 6, with PEG designating polyethyleneglycol): ⁇ (OCH 3 -EHA) 3.38 ppm (s), ⁇ (OCH 2 - PE G) 3.65 ppm, 6(OCH 2 -EHA) 3.90 ppm, ⁇ (OCH 2 . F M P) 4.45 ppm, ⁇ (CF 2 H) 5.85-6.35 ppm (t).
• Infrared spectroscopy (by means of Perkin-Elmer 1600 FTIR) additionally shows the appearance of polyether groups at 1272 and 1259 cm “1 and therefor confirms the resulting structure. Similar syntheses were performed with ⁇ -amino- ⁇ -methoxy polyethyleneglycols having molecular weights from 400 to 4,000 and the resulting terpolymers were used for protein adhesion tests via surface plasmon resonance in example 14 and for sterilization tests in example 17 below.
• EXAMPLE 10 Determination of the degree of conversion in the radical copolymerisation of octafluoropentylmethacrylate and 2-ethylhexylacrylate by means of gas chromatography. 0.01 mole of a monomer mixture of octafluoropentylmethacrylate and 2- ethylhexylacrylate.was weighed in a 10 ml flask. 8.2 mg 2,2'-azobisisobutyronitrile (free radical initiator) and 0.67 g n-decane (internal standard) were added.
• the mixture was further diluted with CHCI3 to a total volume of 10 ml and brought to a two-neck flask, which was sealed with a septum and a valve. Degassing was done by bringing the reaction mixture several times briefly under vacuum. During copolymerization, effected at 65°C like in example 3, an inert nitrogen atmosphere was maintained. During 28 hours, at regular time intervals, a 50 ⁇ l sample was taken and diluted chloroform to 300 ⁇ l. 0.5 ⁇ l was injected on a polydiphenyldimethylsiloxane column (RSL-200 bonded FSOT, 30 m x 0.25 mm, split injection).
• RSL-200 bonded FSOT polydiphenyldimethylsiloxane column
• EXAMPLE 11 determination of copolymer compositions by H-NMR.
• Copolymer of example 3 contains 81 mole% octafluoropentylmethacrylate and 19 mole% 2-ethylhexylacrylate.
• Copolymer of example 4 contains 67 mole% pentadecafluorooctylmethacrylate and 33 moie% 2-ethylhexylacrylate.
• Copolymer of example 5 contains 51 mole% trifluoroethylmethacryiate and 49 mole% 2- ethylhexylacrylate.
• Copolymer of example 10 contains 63 mole% octafluoropentylmethacrylate and 37 mole% 2-ethylhexylacrylate.
• a polyethyleneglycol macro-initiator was prepared following a procedure similar to that described by A.Ueda et al. in J.Polym.Sci., Part A, Polym. Chem. (1986) 24:405.
• 1 g of 4,4-azo-bis (4-cyanopentanoic acid) and 0.83g N-hydroxysuccinimide were dissolved in 10 ml tetrahydrofurane.
• 1.48 g dicyclohexylcarbodiimide in 5 ml tetrahydrofurane was slowly added to the reaction medium at 0°C. After 24 hours stirring, dicyclohexylurea formed by the reaction was removed by filtering.
• the polyethyleneglycol-containing macro-initiator formed was precipitated in pentane, vacuum dried and further characterized by infrared spectroscopy (linking amide at 1716 cm “1 , polyethyleneglycol ether at 1113 cm “1 , methylene at 2885 cm “1 ) and 1 H-NMR in CDCI 3 : ⁇ (CH 3 ) 1.7 ppm, ⁇ (OCH 2 ) 2.4 ppm and 2.5 ppm, ⁇ (OCH 3 ) 3.4 ppm, ⁇ (OCH 2 ) 3.65 ppm.
• EXAMPLE 13 preparation of poly(octafluoropentylmethacrylate-co-2- ethylhexylacrylate-co-polyethyleneglycol derived monomer).
• a polyethyleneglycol macro-initiator according to example 12 was brought, in a proportion (from 0.5 to 2% by mole of the total monomers) specified in the following table 1 , to a polymerization tube, then 1 g octafluoropentylmethacrylate (3.33 mmole) and 0.205 g of 2-ethylhexylacrylate (1.11 mmole) were added and toluene was used as the polymerization solvent.
• the polymerization mixture was frozen in liquid nitrogen and degassed under vacuum. This was repeated trice, after which the polymerization tube was sealed. After 24 hours of polymerization in a thermostatic bath at 65 °C, the resulting terpolymer was precipitated twice in pentane and vacuum dried.
• a thin film was prepared from the said terpolymer via solvent casting from a CH 2 CI 2 /acetone (80/20) solution and was extracted during 24 hours at 55 °C with different portions of water, the water fraction was freeze dried and the isolated product identified by means of 1 H-NMR-spectroscopy in CDCI 3 as follows: 6(OCH 3 -PEG) 3.4 ppm (s), ⁇ (OCH 2 -p EG ) 3.6 ppm, 6(OCH 2 -EHA) 4.0 ppm, ⁇ (OCH 2 . F p) 4.7 ppm, ⁇ (CF 2 H) 5.3-6.3 ppm.
• EXAMPLE 15 protein adhesion to copolymer surfaces measured through surface plasmon resonance.
• SPR Surface plasmon resonance
• Laser light incides at the underside of a prism optically coupled to a microscopy glass covered by a thin gold or silver layer. Total internal reflection takes place if light goes to a medium with smaller refractive index and if the angle of incidence is bigger than the critical angle.
• the resonance angle ⁇ spr resonance takes place between the extinguishing wave of the photon and the free oscillating surface electrons from the metal layer (plasmons).
• a minimal intensity of the reflected light beam arises and is registered by a photodiode array detector.
• a sensogram is obtained by recording the shift in resonance angle ( ⁇ spr ) versus time, the slope of the curve being a measure for the amount of adhered material.
• Protein adsorption takes place immediately after introduction of a foreign body into the body of a mammal and this initially adsorbed protein layer further influences physiological and cellular processes such as blood coagulation, fibrinolysis, thrombogenesis, leukocyte activation, immunological and inflammatory responses.
• the driving interaction forces, such as H-bridges, electrostatic and hydrophobic interactions, for protein adhesion are non-covalent.
• Protein adsorption is a complex, competitive process whereby some proteins can desorb again and be replaced by other proteins. Many factors, such as affinity of the protein for the surface, contact time, concentration and flow speed of the protein play a role in this substitution process.
• affinity of the protein for the surface When a protein has a high affinity for the surface, it will undergo conformational changes so that multiple bonds with the surface are possible. The longer a protein remains attached, the more it will spread and the stronger the bond is. An irreversible protein monolayer is then formed. Affinity itself is determined in part by the protein, in part by the material surface properties.
• Vroman effect is based on the substitution of proteins and suggests that proteins present in high concentrations bind first and are later replaced by other proteins present in smaller amounts, although with high affinity for the surface.
• Plasma proteins such as albumin, immunoglobulin G, fibrinogen, fibronectin, Hageman factor, high molecular weight kininogen and high density lipoprotein (HDL) will adhere sequentially.
• Material surfaces entail a differential humoral and cellular activity, depending on the protein composition and conformation in the adsorbed layer. A change in conformation can entail activation but also inhibition of the biological function of the protein. Generally speaking, extremely hydrophilic materials impede protein adsorption whereas extremely hydrophobic materials adhere a strongly attached monolayer. Micro-heterogeneous materials usually adsorb proteins in an ordered way, whereby changes in protein structure and eel! activation are avoided. In the following experiment, it will be shown to which extent human serum albumin (HSA) and fibrinogen (HFB) adhere to the copolymer surface and to which extent binding is irreversible. 0.5% copolymer solutions and 0.05 % (weight/volume) protein solutions were used in this study.
• HSA human serum albumin
• HFB fibrinogen
• EXAMPLE 16 protein adhesion and substitution to a copolymer surface measured through surface plasmon resonance.
• the copolymer of example 3 was tested as follows for protein adsorption and substitution upon subsequent contact with single component solutions of albumin, fibrinogen and immunoglobulin G (available from Sigma Immunochemicals).
• albumin, fibrinogen and immunoglobulin G available from Sigma Immunochemicals.
• the ⁇ spr for maximal amounts of adhered protein were determined.
• the protein layer was reacted with its corresponding monoclonal antibody.
• the second shift in resonance angle, due to the binding of the antibody, is also a measure for the amount of protein adhered.
• HSA solution was followed by a human immunoglobulin-G (HigG) solution.
• HigG human immunoglobulin-G
• the sensorgram of the various HSA-antibody responses is provided in figure 2 and the data from the substitution experiments ( ⁇ spr being expressed in mdA) are presented in the following table 3.
• HFB and HigG possess a high affinity for the hydrophobic fluorinated copolymer surface and that they replace HSA reversibly.
• substitution an equilibrium with both HFB and HigG present at the surface is obtained.
• the substituted protein fraction depends on the extent to which the first adhered protein is able to relax and bind irreversibly.
• EXAMPLE 17 competitive adsorption of plasma proteins to a copolymer surface measured through surface plasmon resonance.
• EXAMPLE 18 Effect of irradiation sterilization on the macromolecular characteristics of terpolymers.
• Staphylococcus epidermis the most frequent pathogen of infested implants, was studied via radiolabelling (e.g. 3 H-adenin).
• the "bacterial adhesion to hydrocarbons” (BATH) test was used for this purpose since it is widely recognized for its accuracy to test the relative hydrophobicity of micro-organisms, based on the adhesion to n-decane.
• the samples were prepared by coating silanated scintillation bottles with a 2% (weight/volume) copolymer solution.
• polymer plates 2.5 x 3 cm
• polyurethane Cholethane
• LDPE low density polyethylene
• PS polystyrene
• Glass and stainless steel (SS, Inox 64 K°, Metaplast) plates were also used as reference materials.
• Staphylococcus epidermis (LMG 10474, ATCC 11775) was obtained from the Culture Collection of the University of Gent and cultured on a nutrient agar (pH 7.4) supplemented with 1% glucose. Growth was followed by means of absorbancy measurements at 550 nm (Vitalab 10 photometer, Vital Scientific). Only Staphylococcus epidermis in the stationary growth phase (after more than 10 hours of incubation) was used for the experiments. The hydrophobicity of the bacterial surface was determined following the method of Rosenberg. Several volumes of n- octane (0.1 -1 ml) were added to 3 ml of the bacterial suspension in phosphate buffer (9.5 x 10 8 ceils). After two minutes shaking, two separated phases were obtained again after 15 minutes, then the aqueous phase was removed. The difference in absorbancy between the original and the final suspension is a measure for the bacterial hydrophobicity.
• Staphylococcus epidermis was radioactively labelled with (2,8- 3 H)-adenine (Dupont, Belgium, specific activity 288,000 Ci/mol, cone. 10,000 Ci/ml).
• the radioactive suspension was brought during one hour into contact with the polymer coatings and the reference plates.
• the scintillation bottles were mounted in a rotating disc and rotated at 60 rpm through a water bath at 37°C. A dynamic bacterial flow was secured and the contact with the materials was favoured. The materials were washed twice with phosphate buffer in order to remove reversibly adhered bacteria (3 minutes at 60 rpm).
• EXAMPLE 21 determination of the bacterial resistance of a copolymer via adenosine triphosphate (ATP) intracellular concentration.
• ATP adenosine triphosphate
• Staphylococcus epidermis was cultured on nutrient agar plates overnight at 37°C, harvested, centrifuged (Sorvall RT 6000B, 20 min, 3500 rpm) and washed twice. The pellet was resuspended in phosphate buffer until a dense suspension of 1.8 x 10 9 cells/ml was obtained (Spectrophotometer Pharmacia LKB, Novaspec II, 550 nm). The coated Eppendorf tubes were brought into contact with the bacterial suspension for 1 hour at 37°C (Innova incubator 4230, 150 rpm) then washed five times by adding 1 ml of phosphate buffer and suctioning this again.
• the adhering micro-organisms were lysed by 5 to 10 minutes incubation with 1 ml of a trichloroacetic acid (TCA) solution (TCA 1 %, EDTA 2mM, xylenol blue 0.002% in distilled water).
• TCA trichloroacetic acid
• a 20 ⁇ l sample was pipetted in a 96-well microtiter plate (Dynatech Microlite 1 ).
• the pink lytic solution was neutralized with 180 ⁇ l tris acetate buffer, as a consequence of which a change in colour (pink to yellow) took place.
• the microtiter plate was kept on ice. Measurements were effected using an Amerlite illuminometer (Amersham) at a maximal emission wavelength of 562 nm.
• Urine pH was measured and uropathogenic micro-organisms determined.
• a polymer disc was sutured with vicryl suture thread into the mucus wall, in order to impair obstruction of the passage to the urether. After suture, the wound was rinsed thoroughly with an isobetadin solution.
• a long-acting analgesic (brupenorphin, 0.3 mg/kg) was administered subcutanously. After 9 weeks, the rats were killed via an overdose CO 2 . Again, urine pH was determined and microbiological analyses performed.
• the polymeric discs were collected and the deposited calcium salts dissolved with 10 ml of an acidic lanthanum chloride solution. The amounts of calcium were determined via atomic absorption spectroscopy for each type of disc and expressed in ⁇ mole. Results were as follows: 2.51 ⁇ mole for the copolymer of example 3
• Methylprednisolon (MP) Sigma Chemicals
• VAL valsartan
• the stents were mounted on a rotary platform and spray-dusted via capillary atomisation under air pressure.
• a barrier coating was applied by, after 2 hours drying of the first polymer layer, atomizing for a second time 10 ml of the 1% (weight/volume) solution of the copolymer over the stents. Scanning electron microscopy by means of an SEM 505 apparatus showed the presence of a 5 ⁇ m thick copolymer layer.
• Balloon expandable, stainless steal stents 16 mm long and with specific zigzag shape were folded out of a 0.18 mm diameter thread.
• the stents were provided with a coating of the copolymer of example 3 by dipping or spraying according to the methods of examples 23 and 24. Dust-free drying was effected at room temperature.
• the stents were sterilized by X-ray irradiation at 25kGy.
• Cross-bred pigs (20-25 kg) were used as laboratory animals, a standard grain diet without any fat or cholesterol supplements being fed to them. The animals were treated according to the standards of the 'National Institute of Health Guide for the care and use of laboratory animals'. Under full narcosis, the stents were implanted in the crown/coronary artery of the animals. Heparin and acetylsalicylic acid were administered during the operation. Via the thigh artery, per pig, one (coated or non-coated) stent with a conventional 3.0 mm coronary angioplastic balloon catheter was placed in the right coronary and expanded under a pressure of 8 atm. during 60 seconds. By means of coronary angiography the procedure was being followed.
• the stented segment (from 1 cm proximal to 1 cm distal from the stent) was carefully dissected and fixed in a 2% formalin solution.
• the stent thread was removed with a stereomicroscope, avoiding thereby deformation of or damage to the artery.
• the segments were imbedded in a cold-polymerising holder (Technovit 710-Heraeus Kulzer GmbH, Germany) and 5 ⁇ m thick sections were prepared with a microtome (HM 360, Microm, Germany). The microscopic sections were stained with haemalaun-eosin, Masson's trichome, elastica von Gieson and phophotungstic acid heamatoxylin (PTAH).
• tissue sections/samples were studied with a light microscope by an experienced pathologist, who was unaware of the type of coating. Morphometric analyses were performed via a computer steered program (Leitz CBA 8000). For the statistical processing of the angiographic measurements, a paired t-test was used. For comparison of two different groups (coated-not coated, with-without drug), the non-paired t-test was used. The data were presented as mean values ⁇ standard deviations. Measurements with a p-value below 0.05 were considered to differ significantly.

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