Classifications

• • 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
  • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
  • A61L33/06—Use of macromolecular materials
  • A61L33/08—Polysaccharides
• • 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
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
• • 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
  • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
  • A61L33/0005—Use of materials characterised by their function or physical properties
  • A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
  • A61L33/0041—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate characterised by the choice of an antithrombatic agent other than heparin

Definitions

• the invention relates generally to a coating composition for a polymeric surface, methods for coating a polymeric surface, methods for preparing coated medical devices, polymeric surfaces coated with the coating 5 composition, and medical devices comprising the coating.
• Clotting is a significant clinical issue for many devices including hemodialysis catheters, central venous access catheters, endoluminal grafts, coronary and peripheral stents, extracorporeal devices, etc.
• the physical consequences of clotting in a device can be very serious and can ultimately lead to pulmonary 0 embolism if the clot dislodges and travels to the lung.
• various types of surface coatings have been developed for medical devices that are exposed to blood in order to prevent clotting.
• the most common anticoagulant used for this purpose is heparin.
• a covalent complex of antithrombin (AT) and heparin (ATH) has been developed that has significant anticoagulant activities (Chan et al. Journal of Biological Chemistry 272:22111-22117, 1997; Chan et al. Blood Coagulation and Fibrinolysis 9:587-595, 1998; Berry et al. Journal of Biological Chemistry 273:34730- 34736, 1998; Berry et al. Journal of Biochemistry, 132: 167-176, 2002; Klement et al. Biomaterials 23:527- 535, 2002; Chan et al. Thrombosis and Haemostasis 87:606-613, 2002; Chan et al.
• ATH was shown to react rapidly with Ila (Chan et al. Journal of Biological Chemistry 272:22111- 22117, 1997; Berry et al. Journal of Biological Chemistry 273:34730-34736, 1998; Chan et al. Circulation 106:261-265, 2002) forming a stable covalent ATH-IIa complex (Chan et al. Journal of Biological Chemistry 272.221 1 1-221 17, 1997) Furthermoi e, ATH also possesses a potent ability to eataly/e inhibition of factor ?'a or Ila by added AT (Chan et al. Journal of Biological Chemistry 272:22111-22117, 1997; Chan et al.
• ATH has a more rapid onset of action than heparin or antithrombin alone.
• antithrombin For antithrombin to bind to, and inactivate thrombin, it must first be rendered active tlirough the binding of heparin through a specific pentasaccharide sequence.
• antithrombin In the ATH molecule, antithrombin is already in the active conformation, ready to bind to and inactivate thrombin, thereby inhibiting clot formation.
• ATH has improved potency over heparin because all of the heparin chains in ATH 0 are active (Berry et al. Journal of Biological Chemistry 273:34730-34736, 1998).
• ATH coated devices and processes for coating medical devices with ATH are described in U.S. Patent No. 6,491,965, in Klement et al. Biomaterials 23:527-535, 2002 and in Berry L., Andrew M. and Chan A. K. C. Antithrombin-Heparin Complexes (Chapter 25). In: Polymeric Biomaterials. Part II: Medical and Pharmaceutical Applications of Polymers. (Second Edition) Ed. S. Dumitriu. Marcel Dekker Inc., New York, 5 pp. 669-702, 2001.
• the present invention provides improved coating compositions and methods for coating polymeric surfaces.
• the invention provides improved coated polymeric surfaces, in particular, medical devices. More specifically, the present invention provides improved medical devices, and methods of manufacturing same.
• the advantages achieved by the present invention include a simple and readily controlled process that can provide improved coating compositions that can be used with suitable substrates, in particular medical devices.
• a method of the invention provides a stable attachment of a coating to a polymeric surface, in particular, a polymeric surface of a medical device.
• the invention can provide a permanent coating technique that assures more uniform coverage of a polymeric surface. It also allows surface modification of devices to provide advantageous properties such as anti-thrombogenic properties.
• the invention can provide greater exposure of active or therapeutic compounds in the coating to biological fluids or surfaces that are in contact with the coating. For example, it can provide greater exposure of anticoagulants in the coating to blood.
• the invention relates to a coating composition for association with a polymeric surface, preferably a polymeric surface of a suitable substrate, in particular a medical device, comprising a cross-linked basecoat displaying a plurality of active groups in association with se ⁇ ins or serpin derivatives, wherein the serpins or serpin derivatives are not substantially cross-linked with other serpins or serpin derivatives.
• the coating composition may be in association or combination with a polymeric surface.
• the invention provides a method for coating a polymeric surface with a serpin or serpin derivative which comprises the following steps: (i) introducing monomers, preferably heterofunctional monomers, with active groups on the polymeric surface; and (ii) reacting with a preparation comprising the serpin or serpin derivative so that the se ⁇ in or serpin derivative associates with the active groups.
• the invention also provides a polymeric surface that is coated with a basecoat displaying a plurality of active groups associated with serpins or serpin derivatives.
• the invention also contemplates a coated polymeric surface prepared by a method of the invention.
• the invention also relates to a suitable substrate for inco ⁇ orating a coating composition of the invention, in particular a medical device.
• the invention contemplates a medical device comprising a polymeric surface that is coated with a coating composition comprising a cross-linked basecoat displaying a plurality of active groups in association with se ⁇ ins or serpin derivatives, wherein se ⁇ ins or serpin derivatives are not substantially cross-linked with serpins or se ⁇ in derivatives.
• the invention further contemplates a method for preparing a coated medical device comprising coating a polymeric surface of the medical device with a composition of the invention.
• the invention also relates to a kit for preparing a coating composition, a coated polymeric surface, or a coated medical device according to the invention.
• the present invention additionally provides methods of rendering a blood- or tissue- contacting surface of a medical device resistant to fibrin accumulation and/or clot formation which method comprises coating at least a portion of a polymeric surface of the medical device with a coating composition of the invention.
• the invention further contemplates a method of rendering a polymeric surface of a preformed medical material or device anti-thrombogenic comprising coating the polymeric surface with a coating composition of the invention.
• the invention contemplates a method of rendering a polymeric surface of a preformed medical material or device anti-thrombogenic comprising coating the polymeric surface with a coating composition of the invention.
• a coating composition of the invention may be used to reduce clotting in a medical device used in a patient. Therefore, the invention provides a method of treating a patient comprising introducing into the patient a medical device comprising a polymeric surface coated with a coating composition of the invention in an amount sufficient to prevent or inhibit thrombosis.
• the present invention additionally provides methods of using or uses of a medical device coated with a coating composition of the invention.
• the use or method comprises providing to a patient in need thereof a medical device comprising a body and at least a portion of the body coated with a coating composition comprising a cross-linked basecoat displaying a plurality of active groups capable of associating with se ⁇ ins or serpin derivatives, wherein the se ⁇ ins or serpin derivatives are not substantially cross-linked with other serpins or serpin derivatives.
• Figure 1 shows a schematic diagram of a method for covalent linkage to a polymeric surface of a basecoat displaying a plurality of active groups in association with an antithrombin-heparin complex.
• Figure 2 shows a schematic diagram of a method for non-covalent linkage to a polymeric surface of a basecoat displaying a plurality of active groups in association with an antithrombin-heparin complex
• Figure 3 shows immunoblots of proteins eluted from the inner (I) and outer (O) surfaces of PU-ATH catheters following in vivo experiments.
• Figure 4 shows the effects of monomer composition and total monomer concentration on ATH graft density of coated catheters during washing with saline (0.8 g NaCl/100 ml H 2 0). Saline washing solution was replaced every 24 hours with fresh saline washing solution and the catheters analyzed for 125 I-ATH. Codes are for monomer composition and total concentration.
• 112-20 is an experiment in which the ratio of volume of poly(ethyleneglycol) diacrylate monomer to volume of isocyanato-ethylmethacrylate monomer to volume of glycidyl mefhacrylate monomer in the basecoat is 1 to 1 to 2 and the percent of total volume of all monomers in the total volume of monomers + solvent is 20.
• 103-20 is an experiment in which the ratio of volume of glycidyl methacrylate to polyethyleneglycol methacrylate in the basecoat is 3:1.
• Figure 5 shows the effects of monomer composition and total monomer concentration on ATH graft density of coated catheters during washing with sodium dodecyl sulfate (2 g SDS/100 ml H 2 0). SDS washing solution was replaced every 24 hours with fresh SDS washing solution and the catheters analyzed for 125 I- ATH. Codes are for monomer composition and total concentration.
• 112-20 is an experiment in which the ratio of volume of poly(ethyleneglycol) diacrylate monomer to volume of isocyanato- ethylmethacrylate monomer to volume of glycidyl methacrylate monomer in the basecoat is 1 to 1 to 2 and the percent of total volume of all monomers in the total volume of monomers + solvent is 20.
• the experiment designated as contl was a control experiment in which a catheter that was not coated with a basecoat was incubated with 125 I-ATH, followed by the same washing procedure.
• Figure 6 shows the effects of monomer composition and total monomer concentration on ATH graft density of coated catheters 0.1 mg of protease/ml. Protease solution was replaced with fresh protease solution every 24 hours and the catheters analyzed for 125 I-ATH. Codes are for monomer composition and total concentration.
• 112-20 is an experiment in which the ratio of volume of poly(ethyleneglycol) diacrylate monomer to volume of isocyanato-ethylmethacrylate monomer to volume of glycidyl methacrylate monomer in the basecoat is 1 to 1 to 2 and the percent of total volume of all monomers in the total volume of monomers + solvent is 20.
• Coating or “coated” in the context of a method, of the invention refers to complete, substantially complete,- or partial coverage of a polymeric surface with a cross-linked basecoat displaying a plurality of active groups associated with a serpin or se ⁇ in derivative. In embodiments of the invention at least a portion of the polymeric surface is covered with a basecoat.
• the tenn "associate”, “association” or “associating” refers to a condition of proximity between an active group and a se ⁇ in or se ⁇ in derivative, or parts or fragments thereof, or between a polymeric surface and a cross-linked basecoat displaying a plurality of active groups associated with a serpin or serpin derivative or a coating composition of the invention.
• the association may be non-covalent i.e. where the juxtaposition is energetically favored by for example, hydrogen-bonding, van der Waals, or electrostatic or hydrophobic interactions, or it may be covalent.
• Polymeric surface refers to a surface that is capable of being coated with a se ⁇ in or se ⁇ in derivative or coating composition in accordance with a method of the invention.
• a polymeric surface may be natural or synthetic.
• a polymeric surface may be composed of a synthetic polymer.
• a synthetic polymer may be composed of urethanes, acrylates, acrylamides, (for example, polyurethanes, polyacrylates, and polymethacrylates), and combinations thereof.
• particular polymers include but are not limited to poly 2-hydroxyethyl methacrylate, polyacrylamide, polyether polyurethane urea (PEUU), polyurethane, silicone, polyethylene, polypropylene, polytetrafluoroethylene, poly(vinylchloride), polydimethylsiloxane, an ethylene-acrylic acid copolymer, Dacron, polyester-polyurethane, polycarbonate-polyurethane, urethane acrylate, epoxy acrylate, polyamide (Nylon) and polystyrene.
• a polymeric surface may be a surface of a suitable substrate, in particular a medical device.
• medical device refers to any material comprising a polymeric surface that is used in the treatment, monitoring, or prophylaxis of a condition in a patient.
• the device is preferably one that is implanted into a patient or otherwise comes into contact with blood and for which it would be desirable to reduce blood coagulation.
• the device is suited for introduction into the coronary and peripheral vascular systems.
• Examples of medical devices include catheters, multilumen catheters, drip chamber filter meshes for blood circuits employed for extraco ⁇ oreal circulation, film or hollow fibre oxygen-exchanging membranes for artificial lungs and connectors for tube connections, film or hollow fibre dialysis membranes for artificial kidneys, endovascular tubing, arterial and central venous lines, cardiac catheters, cardiopulmonary bypass circuits, dialysis circuits, wound drains, guide wires, nerve-growth guides, chest tubes, septums, hemodialysis catheters, central venous access catheters, endoluminal grafts, stents including coronary and peripheral stents, AV shunts for artificial kidneys and artificial blood vessels, sheath introducers, canulas, by-pass tubes, extracorporeal devices or other external blood contacting instruments, as well as pacemaker leads, arterial and venous catheters for cannulation of large vessels thrombectomy catheters, sutures, blood filters, intravenous lines, mechanical valves, stents, prosthetics,
• Polymeric surfaces of medical devices may comprise Ioplex materials and other hydrogels such as those based on 2-hydroxyethyl methacrylate or acrylamide, and polyether polyurethane ureas (PEUU) including Biomer (Ethicon Corp.) and Avcothane (Avco-Everrett Laboratories).
• PEUU polyether polyurethane ureas
• Materials used most frequently for tubular applications are polyethylene, poly 2-hydroxyethyl methacrylate, polypropylene, silicone, polytetrafluoroethylene (Gore-T.ex), poly(vinylchloride), polydimethylsiloxane, an ethylene-acrylic acid copolymer, polycarbonate, polyester, polyamide, polyacrylate, polyvinyl alcohol, polycaprolactonc, polylactide, polyglycolide, knitted or woven Dacron, polyester-polyurethane, polyurethane, polycarbonate- polyurethane (Corethane.TM.), vinyl acrylate, allyl compounds, polyamide (Nylon) and polystyrene, and copolymers of any two or more of the foregoing, siloxanes, natural and artificial rubbers, glass, and metals, including steel and graphite.
• a polymeric surface may be associated with a biological tissue such as vascular grafts, heart valve tissues, or synthetic membranes made from various hydrophobic or hydrophilic polymers.
• a polymeric surface may be associated with a matrix employed in the fractionation of cells, in particular blood cells.
• a matrix may be a packing material contained within a column or fibrous material compressed into a filter and held in a housing of conventional design and construction.
• Se ⁇ in(s) refers to a serine protease inhibitor and is exemplified by species comprising antithrombin III and heparin cofactor II. The term includes a se ⁇ in derivative.
• Se ⁇ in derivative refers to a serpin that possesses a biological activity (either functional or structural or both) that is substantially similar to the biological activity of a se ⁇ in.
• derivative is intended to include “variants” “analogs” or “chemical derivatives” of a serpin.
• variant is meant to refer to a molecule substantially similar in structure and/or function to a serpin or a part thereof.
• a molecule is "substantially similar” to a se ⁇ in if both molecules have substantially similar structures or if both molecules possess similar biological activity.
• the term “analog” refers to a molecule substantially similar in function to a serpin.
• the term “chemical derivative” describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule.
• a se ⁇ in may be obtained from natural or non-natural sources (e.g. recombinant or transgenic) and it may be obtained from commercial sources.
• Se ⁇ in(s) also refers to conjugates or complexes comprising a serpin, in particular a conjugate or complex comprising a se ⁇ in associated with a glycosaminoglycan.
• glycosaminoglycan refers to linear chains of largely repeating disaccharide units containing a hexosamine and an uronic acid. The precise identity of the hexosamine and uronic acid may vary widely.
• the disaccharide may be optionally modified by alkylation, acylation, sulfonation (O- or N-sulfated), sulfonylation, phosphorylation, phosphonylation and the like.
• the degree of such modification can vary and may be on a hydroxyl group or an amino group. Most usually the C6 hydroxyl and the C2 amino are sulfated.
• the length of the chain may vary and the glycosaminoglycan may have a molecular weight of greater than 200,000 daltons, typically up to 100,000 daltons, and more typically less than 50,000 daltons. Glycosaminoglycans are typically found as mucopolysaccharides.
• glycosaminoglycans include, heparin, dermatan sulfate, heparan sulfate, chondroitin-6-sulfate, chondroitin-4- sulfate, keratan sulfate, chondroitin, hyaluronic acid, polymers containing N-acetyl monosaccharides (such as N-acetyl neuraminic acid, N-acetyl glucosamine, N-acetyl galactosamine, and N-acetyl muramic acid) and the like and gums such as gum arabic, gum Tragacanth and the like. See Heinegard, D. and Sommarin Y. (1987) Methods in Enzymology 144:319-373.
• the glycosaminoglycan is heparin.
• the serpin is antithi ombin associated with heparin.
• the methods, coating compositions, devices and kits of the present invention preferably use an antithrombin and heparin covalent conjugate (i.e. ATH) as described in U.S. Patent No. 6,491,965, Klement et al. Biomaterials 23:527-535, 2002 and in Berry L., Andrew M. and Chan A. K. C. Antithrombin-Heparin Complexes (Chapter 25). In: Polymeric Biomaterials. Part II: Medical and Pharmaceutical Applications of Polymers. (Second Edition) Ed. S. Dumitriu. Marcel Dekker Inc., New York, pp. 669-702, 2001.
• ATH antithrombin and heparin covalent conjugate
• the antithrombin in ATH may be derived from plasma (see for example, U.S. Patent No. 4,087,415), it may be transgenic (see for example, U.S. Patent 6,441,145), or recombinant (see for example, U.S. Patent No. 4,632,981).
• Heparin may be obtained from pig intestine or bovine lung or it may be obtained from commercial sources.
• the heparin is a "high affinity" heparin enriched for species containing more than one copy of the pentasaccharide.
• “Not substantially cross-linked” in the context of se ⁇ in and se ⁇ in derivatives in a coating composition, device, kit, or method of the invention means that the degree of cross-linking of the se ⁇ in or se ⁇ in derivatives with other se ⁇ ins or serpin derivatives is less than 1-5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, and 40%.
• a “basecoat” refers to polymers comprising monomers after the monomers have been polymerized.
• the degree of polymerization of the monomers is typically 50% or more, 60% or more, and particularly 80% and more, and further 90% or more.
• the degree of polymerization can be substantially 100%.
• “Monomers” refers to any compounds with active groups that are capable of polymerizing and associating with a serpin or serpin derivative, in particular compounds with unsaturated double bonds.
• the monomers may have active groups such as epoxide or epoxy groups.
• a preparation of the same or different monomers can be used to prepare a coating composition or coat a polymeric surface in accordance with the invention.
• the monomers may be heterofunctional monomers of the Formula I: A n -(R)-B n (Formula I)
• a basecoat may comprise a polymer containing heterofunctional monomers of the Formula I.
• the "A” group used in the context of a monomer of the Formula I is a group capable of polymerizing.
• the "B” group used in the context of the Formula I is an active group capable of associating with a serpin or se ⁇ in derivative.
• Suitable "A” and “B” groups include but are not limited to acryloyl, mefhacryloyl, N-succinimidyl, sulfonylsuccinimidyl, glycidyl ether, 1,2-epoxy, chlorocarbonyl and anhydride.
• the B group is glycidyl ether.
• "n" is an integer, preferably 1-40,' more preferably 1-20, still more preferably 1-10, most preferably 1-5, 1-3, or 1.
• R is an optional linker.
• R is a hydrocarbyl group, preferably a Cj -C 50 divalent hydrocarbyl group.
• a hydrocarbyl moiety may be substituted with at least one atom other than carbon, including moieties in which a carbon chain atom is substituted with a hetero atom such as nitrogen, oxygen, silicon, phosphorous, boron, sulfur, or a halogen atom.
• Exemplary substituted hydrocarbyl moieties include, heterocyclo, alkoxyalkyl, alkenyloxyalkyl, alkynyloxyalkyl, aryloxyalkyl, hydroxyalkyl, protected hydroxyalkyl, keto, acyl, nitroalkyl, aminoalkyl, cyano, alkylthioalkyl, arylthioalkyl, ketals, acetals, amides, acids, esters, anhydrides, and the like.
• R is a polyethylene oxide group.
• Suitable monomers that can be used in the invention include but are not limited to one or more compounds with unsaturated double bonds such as methyl methacrylate, styrene, methyl methacrylate, methyl acrylate, ethylene diacrylate, ethylmethacrylate, acrylamide, diurethane dimethacrylate, poly-isoprene-graft- maleic acid monoethyl ester, glycidyl methacrylate, isocyanato-ethylmefhacrylate, polyethylene glycol methacrylate, polyethylene glycol diacrylate, and/or polyethylene glycol dimethacrylate, preferably polyethylene glycol dimethacrylate.
• the monomers comprise one or more of isocyanato-ethylmethacrylate, glycidyl methacrylate, and polyethylene glycol diacrylate.
• Polymerizing agent refers to a compound that is capable of initiating polymerization of monomers, preferably a radical polymerization initiator, to form a basecoat.
• Suitable polymerizing agents include but are not limited to azobis (cyanovaleric acid), azobiscyclohexanecarbonitrile, azobisisobutyronitrile (AIBN), benzoyl peroxide, iron (II) sulphate, and ammonium persulfate.
• the polymerizing agent maybe designated A', which in the context of a heterofunctional monomer of the Formula I, is capable of initiating a polymerization reaction with the A group of the Formula I.
• Portion in reference to the coating of a polymeric surface, in particular a substrate, more particularly a medical device, means at least about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the polymeric surface is associated with a coating composition of the invention.
• Adhesive molecule refers to a molecule that promotes cellular attachment or growth.
• Suitable adhesive molecules that may be used in the invention include fibronectin, laminin, vitronectin, thrombospondin, heparin-binding domains, and heparin sulfate binding domains, and synthetic polymers of amino acids containing adhesive sequences from one or more of the foregoing.
• Other suitable adhesive molecules include lectins that bind to heparin and carbohydrate moieties on the cell surface.
• the invention provides a coating composition for association with a polymeric surface comprising a basecoat displaying a plurality of active groups in association with se ⁇ ins or se ⁇ in derivatives, wherein the serpins or se ⁇ in derivatives are not substantially cross-linked with other se ⁇ ins or se ⁇ in derivatives.
• the association may involve non-covalent interactions such as electrostatic or hydrophobic interactions, van der
• the invention provides a coating composition for association with a polymeric surface comprising a basecoat comprising a polymer of heterofunctional monomers displaying a plurality of active groups in association with serpins or serpin derivatives, wherein the se ⁇ ins or se ⁇ in derivatives are not substantially cross- linked with other serpins or seipin derivatives.
• the invention provides a coating composition for a polymeric surface of a medical device which composition comprises a basecoat displaying a plurality of active groups in association with serpins or serpin derivatives, wherein the serpins or se ⁇ in derivatives are not substantially cross-linked with other se ⁇ ins or se ⁇ in derivatives.
• the se ⁇ in or se ⁇ in derivative is a complex or conjugate of heparin and antithrombin, in particular ATH.
• a coating composition of the invention can alter the surface properties of a coated product, in particular, the serpin or se ⁇ in derivative can be an anticoagulant that provides anti-thrombogenic properties.
• the coating provides a single layer of a serpin or serpin derivative on the surface of a medical device or product.
• the coating allows the production of a non-inflammatory material.
• the invention contemplates an anti-thrombogenic coating composition.
• a coating composition of the invention may additionally comprise an adhesive molecule.
• the se ⁇ in is a conjugate or complex comprising a se ⁇ in and heparin, and the adhesive molecule is bound to heparin.
• the coating composition comprises a basecoat displaying a plurality of active groups in association with ATH molecules, wherein the heparin of the ATH molecules is associated with an adhesive molecule.
• Such coating composition is non-thrombogenic, and may in some applications promote cellular attachment and cell growth. The association between the active groups and the se ⁇ in or se ⁇ in derivative (e.g.
• ATH and optionally adhesive molecule in a coating composition of the invention may involve non-covalent interactions such as electrostatic or hydrophobic interactions, van der Waal's forces, hydrogen bonding, or it may be a covalent interaction.
• a coating composition of the invention may include therein various conventional additives, including stabilizers, pH adjustment agents, and cosolvents. Additives are generally selected that are compatible with the intended use of the coating composition. Suitable additives to employ in coating compositions of the invention include benzalkonium, 4-dimethylaminopyridinium, tetrabutylammonium halides and the like.
• the coating composition may be used to deliver other pharmaceutical and therapeutic agents including antibiotics and analgesics.
• the invention also provides a method for coating a polymeric surface with a serpin or serpin derivative which comprises the following steps:
• monomers with active groups are introduced on the polymeric surface by applying a basecoat that displays a plurality of active groups on the polymeric surface.
• the active groups are epoxy or epoxide groups.
• the monomers are covalently attached to the polymeric surface, and become part of the polymeric surface.
• the invention also provides a method for coating a polymeric surface with a serpin or serpin derivative, which comprises the following steps:
• a method for coating a polymeric surface with a se ⁇ in or serpin derivative which comprises the following steps:
• the method substantially provides a single layer of a se ⁇ in or se ⁇ in derivative on the basecoat where the serpin or serpin derivatives are not substantially cross- linked to other se ⁇ in or se ⁇ in derivatives.
• one embodiment of the present invention contemplates the attachment of one active group moiety to each single se ⁇ in molecule.
• the invention provides a method for coating a polymeric surface with a se ⁇ in or se ⁇ in derivative, which comprises the following steps:
• the invention provides a method for coating a substrate, in particular a medical device with a serpin or se ⁇ in derivative, which comprises the following steps:
• a method of the invention for coating a polymeric surface in particular a polymeric surface of a substrate, more particularly a medical device may further comprise recovering any su ⁇ lus serpin or serpin derivative that is not associated with the active groups on the cross-linked basecoat. Conventional techniques may be used to recover su ⁇ lus se ⁇ in or serpin derivative.
• the invention provides a method of applying a uniform coating to a medical device comprising providing a medical device comprising a polymeric surface, and applying a coating composition of the invention to a portion of the polymeric surface.
• the monomers are heterofunctional monomers of the Formula I: A n -(R)-B n (Formula I) wherein A is a group capable of polymerizing; R is an optional linker;
• B is an active group which, when the monomer has polymerized to form the cross linked basecoat, is capable of associating with the se ⁇ in or se ⁇ in derivative
• n is an integer, preferably 1-40, more preferably 1-20, still more preferably 1-10, most preferably 1-5, 1-3, and 1.
• R is a -Cso divalent hydrocarbyl group, more preferably a polyethylene oxide group.
• a basecoat is made by applying heterofunctional monomers of Formula I in combination with at least one polymerizing agent A', wherein A' is capable of initiating a polymerization reaction with the A-group of the heterofunctional monomer to form a cross-linked basecoat.
• the B group is capable of forming a non-covalent association with a se ⁇ in or serpin derivative.
• the association involves one or more of electrostatic or hydrophobic interactions, van der Waal's forces, and hydrogen bonding.
• the B group is capable of forming a covalent bond with a serpin or se ⁇ in derivative.
• the covalent linkage involves a primary amino group on the serpin or se ⁇ in derivative.
• a and B are derived from one or more compounds, 5 which are the same or different, including but not limited to acryloyl, methacryloyl, N-succinirnidyl, sulfonylsuccinimidyl, glycidyl, 1,2-epoxy, chlorocarbonyl, and an anhydride functional group.
• the monomers (basecoat) and preparation comprising a se ⁇ in or se ⁇ in derivative may be applied simultaneously, separately, sequentially in any order, and at different points in time, to a polymeric surface.
• the methods of the invention are carried out under suitable conditions to provide the coating 10 composition, or coated polymeric surface or medical device. It will be within the ordinary skill of a person skilled in the art to determine suitable reaction conditions including temperatures, amounts of monomers, serpin or serpin derivatives, reagents, and reaction times.
• the coating reaction can be performed at temperatures between about 0° to 80°C, in particular 20° to 60°C, and the reaction time can vary from about 5 minutes to 48 hours, in particular 15 20 min to 2 hours.
• Monomers are preferably selected that provide a coating composition with a desirable graft density and/or durability.
• the monomers are glycidyl methacrylate monomers.
• the monomers are glycidyl methacrylate and polyethyleneglycol diacrylate, 20 and preferably the volume of glycidyl methacrylate monomer to the volume of polyethyleneglycol methacrylate monomer in the basecoat is 3: 1.
• a coating composition prepared using these monomers may be further characterized as providing a desirable graft density.
• the concentration of the monomers may be from 2% to 80% by volume, and preferably from about 10% to 50% by volume.
• the concentration of the se ⁇ in or serpin derivative may be from O.Olmg/ml to 20mg/ml by weight, and preferably from about 0.3 mg/ml to 8 mg/ml by weight. 30 In methods of the invention, the percent of total volume of all monomers in the total volume of monomers + solvent is 5-50%, 10-30%, or 20%.
• the cross-linking of monomers can be achieved using a polymerizing agent.
• concentration of polymerizing agent may be in the range of about 0. 01% to about 5% by weight, and preferably in the range from about 0.05% to about 0.2% by weight.
• An annealing step under suitable 35 conditions e.g. 50°C for 30 minutes may follow the cross-linking of the monomers.
• a serpin or se ⁇ in derivative can be applied in a solvent that is selected depending on the nature of the association between the active groups and se ⁇ in or se ⁇ in derivative. Suitable solvents are those that do not interfere with the activity of the se ⁇ in or se ⁇ in derivative. Examples of solvents include water (e.g.
• organic solvents including but not limited to dichloromethane, chloroform, ethyl acetate, acetyl acetate, 1,4-dioxane, dimethylformamide, formamide, dimethyl sulf oxide, tetrahydrofuran, acetone, methanol, ethanol, or a mixture of water and solvents including but not limited to dimethyl sulfoxide (DMSO), acetonitrile, alcohols such as methanol, ethanol, propanol, and ethylene glycol.
• DMSO dimethyl sulfoxide
• alcohols such as methanol, ethanol, propanol, and ethylene glycol.
• a method of the invention may also comprise attaching an adhesive molecule or pharmaceutic or therapeutic agent to a se ⁇ in or se ⁇ in derivative before or after applying the serpin or serpin derivative to the basecoat.
• the serpin or se ⁇ in derivative is a conjugate or complex of heparin and antithrombin, in particular an ATH molecule, and the adhesive molecule is bound to heparin in the conjugate/complex or in the ATH molecule.
• a method of the invention may further comprise analyzing the coating composition.
• Antithrombogenic properties may be determined by measuring the anti-factor Xa activity and anti-IIa activity.
• Coating uniformity may be analysed using conventional immunoassay procedures with antibodies specific for a se ⁇ in or se ⁇ in derivative. Coating stability and density may also be analyzed using standard methods such as those described in the Examples.
• a method of the invention may also comprise the step of sterilizing a coated polymeric surface.
• Standard sterilization techniques can be employed in the invention (e.g. ethylene oxide).
• the invention also contemplates a surface modification method based on single layer coating of a se ⁇ in or se ⁇ in derivative on a basecoat associated with a polymeric surface.
• a coating composition of the invention can be applied to polymeric surfaces, in particular the blood- contacting, tissue-containing, or cell contacting surfaces of any of a wide variety of medical devices, to provide the medical devices with one or more non-thrombogenic surfaces.
• Coating compositions comprising adhesive molecules may also provide medical devices with one or more surfaces that promote cellular adhesion and attachment.
• a coating composition of the invention comprising an adhesive molecule can be used to attach cells to implantable medical devices such as prostheses, including vascular grafts, bone and cartilage implants, nerve guides and the like.
• the invention also provides a polymeric surface that is coated with a cross-linked basecoat displaying a plurality of active groups associated with a se ⁇ in or se ⁇ in derivative, wherein the se ⁇ in or se ⁇ in derivative is associated with the plurality of active groups on the cross-linked basecoat.
• a polymeric surface is provided which is coated with a se ⁇ in or se ⁇ in derivative, wherein the serpin or se ⁇ in derivative is associated with a plurality of active groups on the cross-linked basecoat and serpin or se ⁇ in derivatives are not substantially cross-linked with other serpin or serpin derivatives.
• the invention also contemplates a polymeric surface prepared by a method of the invention.
• the invention provides a coated polymeric surface prepared by a method comprising: (i) introducing monomers with active groups on a polymeric surface; and
• a polymeric surface of the invention may additionally comprise an adhesive molecule associated with the se ⁇ in or se ⁇ in derivative.
• the invention also contemplates a suitable substrate comprising a polymeric surface that includes on a portion thereof a coating comprising a cross-linked basecoat displaying a plurality of active groups capable of associating with a se ⁇ in or serpin derivative, wherein the se ⁇ in or se ⁇ in derivatives are not substantially cross-linked with other se ⁇ in or se ⁇ in derivatives.
• a medical device or product comprising a polymeric surface that includes on a portion thereof a coating comprising a cross-linked basecoat displaying a plurality of active groups capable of associating with a se ⁇ in or se ⁇ in derivative, wherein the serpin or serpin derivatives are not substantially cross-linked with other se ⁇ in or se ⁇ in derivatives.
• the medical device is designed to be at least partially inserted into a patient.
• a medical device of the invention may be sterilized using conventional methods known in the art (e.g. ethylene oxide).
• the invention provides an antithrombotic medical material or device characterized in that it is a medical material or device having on a polymeric surface thereof, a coating composition of the invention.
• the medical material or device additionally comprises an adhesive molecule, or a pharmaceutic or therapeutic agent.
• a medical material or device is contemplated that is non-thrombogenic and promotes cellular adhesion.
• the invention provides a medical device for the treatment of vascular disease comprising: a scaffold structure with a polymeric surface, and a coating composition associated with at least a portion of the polymeric surface.
• a catheter comprising a substantially tubular body comprising a polymeric surface and a coating composition of the invention on a portion of the polymeric surface.
• the invention provides an intraco ⁇ eal medical device comprising a polymeric surface coated with a coating composition of the invention.
• the invention also contemplates an implantable vascular device comprising a catheter or stent structure adapted for introduction into a vascular system of a patient the structure comprising a polymeric surface coated with a coating composition of the invention.
• the invention also relates to a kit for preparing a coating composition or polymeric surface according to the invention.
• the kit comprises:
• kits of the invention may additionally comprise a preparation of the serpin or se ⁇ in derivative.
• a method for rendering a tissue- or blood- contacting surface of a medical device resistant to fibrin accumulation and clot formation comprises coating the surfaces with a non-thrombogenic coating composition of the invention.
• the invention contemplates a method of rendering a polymeric surface of a preformed medical material or device anti-thrombogenic comprising coating the polymeric surface with a coating composition of the invention.
• a coating composition of the invention may be used to reduce clotting in a medical device used in a patient.
• the coating compositions can be used, to reduce the thrombogenicity of internal and extraco ⁇ oral devices that contact blood, and finds special use for coating thrombogenic medical devices including prosthetic surfaces.
• the invention provides a method of treating a patient comprising introducing into the patient a medical device comprising a polymeric surface coated with a coating composition of the invention in an 5 amount sufficient to prevent or inhibit thrombosis.
• the present invention additionally provides methods of using a medical device coated with a coating composition of the invention.
• the method comprises the steps of providing to a patient in need thereof a medical device comprising a body and at least a portion of the body coated with a coating composition comprising a cross-linked basecoat displaying a plurality of active groups capable of associating 10 with a se ⁇ in or se ⁇ in derivative, wherein the se ⁇ in or serpin derivatives are not substantially cross-linked with other se ⁇ in or serpin derivatives.
• the coating composition of the invention may have particular application in reducing or preventing vascular thrombosis associated with intravascular catheters.
• a catheter coated with a coating composition of the invention comprising ATH is provided, wherein the patency of the catheter is at 15 least 50, 75, or 100 days.
• the invention provides a method for fractionating cells, in particular blood cells comprising applying the cells to a matrix coated with a coating composition of the invention.
• ATH was prepared using the method described in Chan et al, Journal of Biological Chemistry 25 272:22111-22117, 1997. In general, antithrombin and heparin in pH 7.3 phosphate buffered saline (PBS) are mixed and incubated at 40°C for 13 days. Sodium cyanoborohydride is added at the end of this incubation to ensure the covalent stability of any Shiff base that has not undergone an Amidori rearrangement. ATH and unreacted AT are then bound to a butyl hydrophobic interaction column to allow removal of unreacted heparin. After suitable high salt washes, the ATH and AT are released and bound to a DEAE anion exchange 30 column.
• PBS pH phosphate buffered saline
• ATH-sparing coating of polyurethane devices using a basecoat 35 This example describes the procedure for coating ATH (antithrombin-heparin covalent complex) on polyurethane catheters via a basecoat that is attached to the polyurethane ( Figure 1).
• An improved chemistry is used that allows reuse of unbound coating ATH stock.
• the ATH and basecoat are linked to each other to form an ATH single-layer through. a covalent linkage.
• This coating is considerably less expensive, inherently more uniform, more easily controlled, and the AT linker has greater exposure to blood.
• Polyurethane catheters are dip-coated in isocyanato-ethylmethacrylate, reacted with the coating at 60°C for 20 minutes, and then dip-coated in allyl glycidyl ether (epoxide) and the free radical initiator AIBN. Free radical polymerization to form the cross-linked basecoat occurs when the catheters are heated at 80 C C for 2 hours. This is followed by an annealing step carried out by lowering the temperature to 50°C over a 30 minute period. The catheters are then immersed and incubated in a solution of ATH to generate the link between ATH and the basecoat.
• allyl glycidyl ether epoxide
• AIBN free radical initiator
• Unreacted ATH is recovered, the catheters are washed with 2%SDS in saline, and the catheters are sterilized with ethylene oxide. They are then spot-check analysed for AT content, anti-Xa activity, and for coating uniformity.
• the acrylate double bond is reactive in the presence of heat or light, thus all reactants and products must be protected from light.
• the epoxide is reactive to water, thus ' this group must be kept dry until it is exposed to ATH.
• the final graft density of ATH is dependant on the reaction concentration of ATH.
• the concentration of ATH that can be used is lmg/ml. Detailed Procedures lml isocyanato-ethylmethacrylate is added to 49ml acetone and mixed.
• Uncoated catheters are immersed in the mixture, and, immediately after removal from the isocyanato-ethylmethacrylate acetone solution, incubated at 60°C for 20 minutes. After incubation, any remaining solution (i.e. excess isocyanato-ethylmethacrylate) is discarded. Allyl glycidyl ether (20 ml) and 2,2'-azobis isobutyronitrile (AIBN) (0.025gm) in acetone (30ml) are added, mixed by inversion, and then incubated for 10 minutes at room temperature with inversion mixing. The liquid is discarded and the catheters are dried in a vacuum chamber for at least 4 hours at room temperature. The coating is polymerized at 80°C for 40 minutes, and the temperature is reduced to 50°C over a 20 minute period to anneal the coating.
• AIBN 2,2'-azobis isobutyronitrile
• the base coated catheters are immersed in ATH diluted with PBS to 50ml of l.Omg (AT)/ml, and incubated at room temperature for at least 4 hours.
• the ATH solution is removed and is available to be used for coating other catheters.
• the catheters are washed with 0.125 saline +2% SDS at room temperature for 3 ⁇ minutes. The solution is discarded and the wash step is repeated.
• the catheters are washed with PBS and Milli-Q water, and dried with a clean nitrogen gas stream.
• ATH antithrombin-heparin covalent complex
• polyurethane catheters via a non- covalently attached basecoat sheath using a chemistry that allows reuse of unbound coating ATH stock ( Figure 2).
• the ATH and sheatli are covalently linked to each other as ATH (single-layer) on a basecoat (cross-linked complex). This coating is considerably less expensive, inherently more uniform, more easily controlled, and the AT linker has greater exposure to blood.
• Polyurethane catheters are dip-coated in a mixture of glycidyl methacrylate, polyethylene glycol diacrylate, and the free radical initiator AIBN in acetone.
• the coating is dried in place and polymerized into a cross-linked basecoat by heating at 80°C for 40 minutes, followed by cooling to 50°C over 20 minutes.
• the catheters are then immersed and incubated in a solution of ATH at room temperature for 20 hours to generate the covalent (epoxide-primary amine) link. Unreacted ATH is recovered, the catheters are thoroughly sequentially washed with several solutions, and the catheters are sterilized with ethylene oxide.
• the catheters can be analyzed for AT content, anti-Xa activity, and coating uniformity.
• a basecoat solution is prepared by mixing glycidyl methacrylate (10.5 ml), polyethylene glycol diacrylate
• Uncoated catheters are totally immersed in the basecoat solution, incubated at room temperature for 20 minutes, and the liquid is discarded.
• the catheters are dried in a vacuum chamber for 2 hours at room temperature, and the coating is polymerized by heating at 80°C for 40 minutes. The temperature is reduced to 50 C C over a 20 minute period to anneal the coating.
• Base-coated catheters are immersed in ATH diluted with Milli-Q water to 70ml of l.Omg (AT)/ml, and incubated at room temperature with stirring (overnight or for at least 4 hours).
• the ATH solution is removed for use in coating other catheters.
• the catheters are flushed with 0.15 M phosphate buffer, buffer + 2M NaCl , buffer + 0.1% SDS, PBS, and Milli-Q water.
• the catheters are dried with clean nitrogen gas stream, and sterilized (e.g. by ethylene oxide).
• Example 4 ATH-coating of polyurethane catheters
• This example describes the procedure for coating ATH (antithrombin-heparin covalent complex) on polyurethane catheters via a basecoat that is covalently attached to the polyurethane.
• An improved chemistry is used that allows reuse of unbound coating ATH stock.
• the ATH and basecoat are covalently linked to each other to form an ATH single-layer. This coating is considerably less expensive, inherently more uniform, more easily controlled, and the AT linker has greater exposure to blood.
• Polyurethane catheters are immersed for 60 minutes at 40°C in isocyanato-ethylmethacrylate
• allyl glycidyl ether epoxide, dissolved in acetone
• free radical initiator AIBN free radical initiator
• Free radieal polymerisation to form progressive cross-links between vinyl groups occurs when the catheters are heated to 80°C for 2 hours, followed by an annealing step at 50°C for 30 minutes.
• the catheters are then immersed and incubated in a solution of ATH to generate the covalent link between ATH primary amines and basecoat epoxide groups. Unreacted ATH is recovered.
• the catheters are washed with several solutions and sterilized with ethylene oxide. Catheters are then spot-check analysed for AT content, anti-Xa activity, and for coating uniformity.
• the acrylate double bond is reactive in the presence of heat or light, thus all reactants and products are protected from light.
• the epoxide is reactive to water, thus this group is kept dry until it is exposed to ATH.
• the final graft density of ATH correlates with the reaction concentration of ATH (in particular, lmg/ml is used).
• Catheter occlusion and vascular thrombosis are common problems associated with use of intravascular catheters.
• the types of proteins adsorbed onto biomaterials affects thrombus formation at the blood-material interface. Since the outer surface of an implanted catheter is exposed to flowing blood and the inner surface of the catheter is exposed to different fluids (including, saline, drugs being infused as well as blood), protein adso ⁇ tion on the outer and inner surface of a catheter is potentially different, with associated different effects on thrombogenicity. Therefore, it is important to establish whether protein adsorption is indeed different on the inside and outside of a catheter in vivo.
• ATH coated surfaces have previously been shown to be resistant to thrombus formation (Klement P, et al, Biomaterials, 23, 527, 2000), but in this study the ATH coated catheters were tested for a long period of time and the resulting effects on outer and inner catheter surfaces were compared.
• ATH prepared according to protocols published previously (Chan AKC, et al, J Biol Chem, 272, 22111, 1997), was purified by hydrophobic chromatography on butyl Sepharose followed by anion exchange chromatography on DEAE Sepharose. ATH was concentrated at 4°C by pressure dialysis under nitrogen.
• Polyurethane catheters were coated on both the inner and outer surfaces by polymerization of an activated monomer. ATH was then covalently linked to the surface by incubation with the polymerized, activated monomer on the catheters.
• the coated catheters were rinsed sequentially with buffer (pH 8.0), followed by 2M NaCl in buffer, then SDS in buffer and finally PBS.
• Catheters were allowed to drain, placed in semi permeable bags, sterilized with ethylene oxide, and stored dry at room temperature prior to implantation into the animal.
• the graft density of the ATH on the catheters was 5 - 10 pmol/cm 2 .
• New Zealand white male rabbits were anaesthetized, and the coated catheters inserted into the right jugular vein and advanced to the edge of the right atrium. The rabbits were allowed to recover. Catheter patency was examined by withdrawing 0.5 mL blood samples through the catheter twice daily, followed by a 2 mL saline flush of the catheter. At the end of the experiment (determined by catheter . occlusion or predetermined time), the coated catheter was removed from the animal and rinsed with saline. Adsorbed proteins were eluted from the inner and outer surfaces of the catheters with 2% SDS. Initially only the inner surfaces were exposed to the SDS solution.
• Poly(ethyleneglycol) diacrylate, isocyanato-ethylmethacrylate and glycidyl methacrylate were mixed with 2,2'-azobis isobutyronitrile (AIBN) polymerizing agent in acetone solvent and incubated with polyurethane catheter segments for 20 minutes at room temperature.
• AIBN 2,2'-azobis isobutyronitrile
• the segments were then gravity drained of monomer solution, vacuum dried and incubated at 80°C for 40 minutes, followed by cooling to 50°C over 20 minutes.
• the catheters are then immersed in a solution of ATH (containing ATH labelled with 125 I) and incubated at room temperature for 20 hours to generate the covalent link between ether groups on the basecoat and amino groups on ATH.
• the volume ratio of the 3 monomers was varied and the total concentration of the monomers in acetone was varied to determine the effect of monomer composition in the basecoat on density of ATH grafted onto the polymeric surface (polyurethane catheter segment). Detection of ATH present on the surface was by gamma counting of the catheter segments to measure remaining surface-bound 125 I-ATH. Stability of ATH coating was assessed by multiple washes and protease treatment. Detailed Procedure Various volume ratios and total concentrations of monomers were tested to determine the effect of basecoat composition on ATH attachment on polyurethane catheter surfaces.
• a basecoat solution was prepared by mixing poly(ethyleneglycol) diacrylate (0.2 ml), isocyanato- ethylmethacrylate (0.2 ml), glycidyl methacrylate (0.4 ml), 0.002 g 2,2"-azobisisobutyronitrile (AIBN), and acetone (3.2 ml).
• Uncoated polyurethane catheter segments (7 French, 1 cm 2 surface area, approximately 1 cm in length) were totally immersed in the basecoat, and incubated at room temperature for 20 minutes. The liquid was discarded. The catheters were dried in a vacuum chamber for 2 hours at room temperature, and the coating polymerized by heating at 80°C for 40 minutes. The temperature was reduced to 50°C over a 20 minute period to anneal the coating. Base coated catheter segments were immersed in ATH solution and incubated for 20 hours at room temperature. The ATH solution contained ATH that had been labelled using Na 125 I (New England Nuclear) and iodobeads (Pierce Chemical Company) according to the method by the manufacturer of the iodobeads.
• ATH solution contained ATH that had been labelled using Na 125 I (New England Nuclear) and iodobeads (Pierce Chemical Company) according to the method by the manufacturer of the iodobeads.
• the 125 I-ATH incubation solution was l.Omg (AT)/ml of PBS and had a gamma radioactivity of 26300 counts per minute per ml.
• the ATH solution was removed for use in coating of other catheters.
• the catheter segments were washed by agitation. In some cases, the catheters were each washed with 5 ml of 0.8 g NaCl/100 ml H 2 0 for 24 hours. After 24 hours of NaCl wash, the wash solution was replaced every 24 hours with another 5 ml 0.8 g NaCl/100 ml H 2 0 and washing continued. After every change of wash solution, the catheter segment was gamma counted for remaining bound 125 I-ATH.
• the catheters were given 3 short washes with 5 mL of 0.8 g NaCl/100 ml H 2 0, followed by a wash withl ml of 2% SDS in H 2 0 for 24 hours. After 24 hours of SDS wash, the wash solution was replaced every 24 hours with another 1 ml of 2% SDS, along with a gamma count of remaining bound 2 I-ATH. Stability of coating to protease treatment of surfaces washed with 0.8 g NaCl/100 ml was evaluated.
• Catheter segments washed with NaCl solution were agitated with 1 ml solution of a general protease (P-5147 from Sigma) at 0.1 mg protease/ml of H 2 0 for 24 hours at room temperature. Every 24 hours, the protease solution was replaced with a fresh 1 ml of 0.1 mg protease/ml of H 2 0 and the catheter segment gamma counted to determine remaining bound 125 I-ATH. Given the gamma radioactivity per mg ATH in the original incubation mixtures and the surface area of the catheter segments, the graft density of ATH on eaeh catheter in pmoles/enr was calculated.
• P-5147 general protease

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