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WO1999049907A1 - Dispositifs medicaux traites de façon a susciter un refus de coagulation du sang - Google Patents

Dispositifs medicaux traites de façon a susciter un refus de coagulation du sang Download PDF

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Publication number
WO1999049907A1
WO1999049907A1 PCT/GB1999/001008 GB9901008W WO9949907A1 WO 1999049907 A1 WO1999049907 A1 WO 1999049907A1 GB 9901008 W GB9901008 W GB 9901008W WO 9949907 A1 WO9949907 A1 WO 9949907A1
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WIPO (PCT)
Prior art keywords
protein
apc
stent
blood
stents
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PCT/GB1999/001008
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English (en)
Inventor
Ian Garner
Anthony Harvey Gershlick
David Paul DE BONO
Original Assignee
Ppl Therapeutics (Scotland) Ltd.
DE BONO, Anne
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Publication date
Application filed by Ppl Therapeutics (Scotland) Ltd., DE BONO, Anne filed Critical Ppl Therapeutics (Scotland) Ltd.
Priority to AU33386/99A priority Critical patent/AU3338699A/en
Publication of WO1999049907A1 publication Critical patent/WO1999049907A1/fr

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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
    • 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
    • A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
    • A61L17/005—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters containing a biologically active substance, e.g. a medicament or a biocide
    • 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
    • A61L29/14—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
    • 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
    • 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
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • 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
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/42—Anti-thrombotic agents, anticoagulants, anti-platelet agents

Definitions

  • the present invention relates to medical devices, the surfaces of which are treated so as to discourage blood coagulation. Specifically, the invention relates to the use of certain proteins in the preparation of such devices.
  • Plasma proteins adsorb themselves on depending on plasma concentration of the protein, surface charge, wetability and geometry of the material, collision probability and, most importantly, the affinity constant of the individual proteins with the foreign body.
  • Fibrinogen displaces other 'bland' proteins increasingly because of its higher affinity constant and plays a key role in further activation of other important plasma proteins and the coagulation cascade.
  • Fibrinogen is also responsible for starting a cellular phase of events involving activation of platelets in conjunction with von Willebrand factor, thrombospondin and fibronectin. This dynamic interplay of plasma proteins and cells lead on to the ensuing process of thrombosis and restenosis.
  • the coagulation cascade described above can lead to difficulties during and after surgery if that involves devices (such as surgical instruments, prostheses, etc) coming directly in contact with blood, either temporarily or permanently. If the problem of thrombogenicity is not addressed, such devices could easily lead to formation of blood clots, especially as many such devices (eg stents) are metallic.
  • Balloon angioplasty by achieving plaque compression and non atheromatous stretching, produces deep injury to the vessel wall. Endothelial denudation, intimal disruption and medial damage accompany plaque splitting and plaque haemorrhage.
  • the repair processes that result involve complex inflammatory and thrombotic pathways. Activated platelets adhere to the damaged wall within seconds and what follows is a vicious cycle of platelet deposition and thrombus formation.
  • the coagulation pathways chiefly extrinsic, lead to the activation of clotting factors and thrombin.
  • a BENESTENT-type lesion is one of native vessel, of stable angina, single lesion reference size >3mm, ⁇ 15mm length, non-ostial, non-bifurcational, non-restenotic with no thrombus.
  • Post-stent MLD for STRESS and BENESTENT II were 2.49mm and 2.48mm respectively. It is fairly certain now, given the close relation between initial post-procedural MLD (acute again) and follow up MLD (late Loss), that the value of stenting lies in the 'bigger is better' principle.
  • Acute/subacute stent occlusion is a very significant problem indeed in bailout angioplasty stenting and in small vessels. Less established risk factors include periprocedural thrombus at angiography, residual dissection and the use of multiple stents. The outcome looks very serious indeed against the background of high vascular complication rates due to deep anticoagulation treatment.
  • Immobilising drugs to the surface of stents is another possibility.
  • heparin as an example-drug of choice, it has been shown to adsorb well onto stent surfaces from a benzalkonium alcohol-heparin solution.
  • the downside was the erratic rate of release of heparin (Breckwoldt, 1991).
  • Negatively charged heparin molecules have also been used to bind electrostatically to surfaces with positive charges (Grode et al, 1969; Tanazawa et al, 1973).
  • Heparin has also been incorporated into a permanent component of polymer coating the stent (Jozefowicz, 1986). However, the results of these attempts have not been satisfactory.
  • Angiopeptin is a synthetic cyclic octapeptide analogue of somatostatin that reduces neointimal hyperplasia.
  • De Scheerder (1995) reported that local angiopeptin delivery using drug-loaded polymer-coated metallic stents resulted in a sigmficant reduction in luminal narrowing in porcine coronaries.
  • Forskolin an adenylate cyclase activating drug which causes platelet disaggregation and vasodilation, incorporated into the polyurethane coating of a heat-activated temporary stent showed delay time in thrombosis (Lambert, 1994). However, these methods still leave scope for improvement.
  • the present invention relates to the use of Protein C in addressing the problem of blood coagulation caused by contact with foreign bodies.
  • the invention is based on the observation that Protein C can be efficiently delivered locally by attachment to medical devices that come into direct contact with blood.
  • a medical device to at least part of the surface of which a protein having the activity of Protein C or Activated Protein C is directly or indirectly attached.
  • Protein C was first described as a vitamin K-dependant, phospholipid-binding zymogen of a serum protease anticoagulant (StenfloJ976; Esmon et al, 1976; Seegers et al, 1976). It circulates in human plasma at a concentration of 2-4 ⁇ g ml-1. Protein C is a glycoprotein of approximate molecular weight 62,000. Fully processed Protein C molecules contain 9-carboxyglutamic acid residues. Two molecular forms of Protein C are readily detectable in plasma, a single-chain [5- 15%] and a two-chain molecule [85-95%]. The latter consists of a heavy chain [260 amino acids] and a light chain [155 amino acids] linked by a single disulphide bond.
  • the conformation of thrombin alters as does its activity towards fibrinogen and factor V.
  • the preferred substrate of the complexed thrombin becomes Protein C which it activates through specific proteolysis and liberation of a 12 amino acid activation peptide from the amino terminus of the heavy chain (Kisiel, 1979).
  • the resulting Activated Protein C or APC functions as an anticoagulant by degradation of the active forms of factors V and VIII (Comp and Esmon, 1979; Walker et al, 1979; Dahlback and Stenflo, 1980).
  • the anticoagulation effect of APC is substantially increased by another vitamin K- dependant protein, the co-factor Protein S.
  • Both factors V and VIII are large, single- chain glycoproteins which are converted to active co-factors by the proteolytic action of thrombin. Following the formation of factor VILtissue factor complex, they are involved in the activation of factor X and the subsequent prothrombin to thrombin transition during the coagulation cascade of zymogenic activation, leading to clot formation. Their inactivation by APC prevents the de novo formation of thrombin and the continued generation/expansion of clots.
  • Protein C may be useful clinically in the treatment of thrombosis.
  • the anticoagulant properties of APC have been demonstrated in primate, rabbit and dog thrombosis models (Gruber et al, 1989; Gruber et al, 1990; Gruber et al, 1991; Arnljots et al, 1994; Sakamoto et al, 1994).
  • Protein C has also been proposed to possess profibrinolytic activity by indirectly promoting the activation of plasminogen to plasmin. Infusion of Activated Protein C into dogs was shown to enhance fibrinolysis (Comp and Esmon, 1981).
  • the present invention is based on the finding that Protein C, Activated Protein C and other molecules having the activity of Protein C or Activated Protein C can be directly or indirectly attached on the surface of a device without undue loss of activity and can exert the biological function when the device comes into direct contact with blood.
  • the delivery of such Protein C molecules via such devices has a number of advantages. Systemic toxic effects can be avoided.
  • the active agent can be given in doses that will not produce side-effects but still have a high effective local concentration. Effective treatment is targeted to the site required and treatment is tailored to the pathological process more easily and precisely.
  • Any protein having the activity of Protein C may be used in this invention. This includes the whole, glycoprotein form of Protein C. Variants and derivatives having the activity of Protein C are also included.
  • Variants of Protein C or Activated Protein C may consist of the particular natural amino acid sequence of Protein C or Activated Protein C, or may have an additional N- terminal and/or an additional C-terminal amino acid sequence.
  • N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art. These include using gene-cloning techniques to ligate together nucleic acid molecules encoding polypeptides or parts thereof, followed by expressing a polypeptide encoded by the resultant recombinant nucleic acid molecule.
  • Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems.
  • an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone somatostatin by fusing it at its N-terminus to part of the ⁇ -galactosidase enzyme (Itakwa et al, 1977).
  • Additional sequences can also be useful in altering the properties of a polypeptide to aid in identification or purification.
  • a signal sequence may be present to direct the transport of the polypeptide to a particular location within a cell or to export the polypeptide from the cell. Different signal sequences can be used for different expression systems.
  • Another example of the provision of an additional sequence is where a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an antigen or an epitope and the affinity column 10
  • the fusion protein may comprise immobilised antibodies or immobilised antibody fragments that bind to said antigen or epitope (desirably with a high degree of specificity).
  • the fusion protein can usually be eluted from the column by addition of an appropriate buffer.
  • N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain the protein of the present invention and need not provide any particular advantageous characteristic.
  • proteins having one or more amino acid substitutions, deletions or insertions compared to the natural sequence of Protein C or Activated Protein C are also within the scope of the invention.
  • variants include allelic and non-allelic variants.
  • An example of a variant within the scope of the present invention is a Protein C or Activated Protein C which has one or more amino acids substituted for the natural residues.
  • amino acids have similar properties.
  • One or more such amino acids of a polypeptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that polypeptide.
  • amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
  • amino acids having aliphatic side chains amino acids having aliphatic side chains.
  • glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic).
  • amino acids having aromatic side chains include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains).
  • Amino acid deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced. For example, dosage levels can be reduced.
  • Amino acid insertions relative to the natural Protein C or Activated Protein C sequence can also be made. This may be done to alter the properties of the polypeptide (e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins). 12
  • Polypeptides incorporating above amino acid changes can be provided using any suitable techniques.
  • a nucleic acid sequence incorporating a desired sequence change can be provided by site directed mutagenesis. This can then be used to allow the expression of a polypeptide having a corresponding change in its amino acid sequence.
  • One way of determining amino acid sequence identity is to align a given amino acid sequence with the amino acid sequence of natural Protein C or Activated Protein C in a manner which achieves the maximum number of matches of amino acids over the full length of the natural amino acid sequence (or a single chain of it).
  • the percentage sequence identity will then be (m/t) x 100 , where m is the number of matches between the two aligned sequences over the length of the amino acid sequence in the natural sequence and t is the total number of amino acids present in the natural amino acid sequence.
  • sequence identity allows for the introduction of gaps when matching two amino acid sequences (0,1,2,3 or even more gaps may be allowed for in each sequence). This can be done, for example, by using the "Gap” program, which is available from Genetics Computer Group as part of "The Wisconsin Package” . This program is based upon an algorithm provided by Smith and Waterman (Advances in Applied Mathematics, 482-489 (1981)).
  • the natural form of Protein C or Activated Protein C is used.
  • any variants or derivatives having the activity of Protein C may also be used.
  • preferred proteins of the present invention have at least 50% sequence identity with the natural amino acid sequence of Protein C; more preferably the degree of sequence identity is at least 75%. Sequence identities of at least 90% or of at least 95% are most preferred. Where high degrees of sequence identity are present there may be relatively few differences in amino acid sequence. Thus for example there may be fewer than 20, fewer than 10, or even fewer than 5 differences.
  • a medical device of the present invention includes any device used in medicine but is especially a device which comes into direct contact with blood.
  • the contact with blood may be within a patient or extracorporeal, temporary or permanent.
  • examples of such devices include, but are not limited to: vascular prostheses including stents, catheters, dialysis membranes, artificial hearts or components thereof, artificial heart valves and surgical suture material.
  • the invention finds particular use in stents used in vascular angioplasty.
  • Such devices may be used in humans, or may be used in general veterinary industry, in particular domestic pets such as dogs and cats and farm animals such as horses, pigs, cattle, sheep, etc.
  • Stents which have been treated with Protein C according to the current invention gradually elute Protein C that retains potent anticoagulant activity.
  • Such stents will obviate stent thrombosis following a variety of clinical thrombogenic situations, eg elective percutaneous coronary artery angioplasty.
  • Local delivery of an inherently safe anticoagulant molecule in this way provides a safer alternative to currently used anticoagulant therapies.
  • a drug-eluting stent provides the further advantage of not causing additional vascular injury or myocardial ischaemia. It may also allow longer local drug retention (Linkoff et al, 1994)
  • Protein C or Activated Protein C is attached to at least part of the surface of the device.
  • the area covered is all or substantially all of the area that comes into contact with blood.
  • Such a device is also within the scope of the present invention.
  • Protein C (in various forms described above) is directly or indirectly attached to the surface of a device.
  • the protein can be attached onto the surface of the device directly, or indirectly through an intermediate layer.
  • the intermediate layer can be of any suitable material known to the person skilled in the art, depending on the construction of the device.
  • polymeric coating eg. using a nitrocellulose polymer, may be suitable for stents.
  • the attachment of the protein to the surface, or an intermediate layer, of a device can be by any method.
  • the form of attachment is not critical as long as the biological function of the protein is not unduly affected. Accordingly, it includes physical attachment such as adsorption or absorption, as well as chemical bonding, ionic interaction, Van der Waals forces and other conventional means of attaching biological materials to objects.
  • the protein is directly adsorbed onto an intermediate polymeric coating on the surface of a device.
  • the scope of the present invention also extends to devices, the surface to which one or more other molecules, which do not have the activity of Protein C but which may, for example, aid the activity of Protein C, are directly or indirectly attached.
  • cofactors such as Protein S can also be attached to the surface of the device, along with proteins having the activity of Protein C.
  • Other possible molecules are angiopeptin and forskolin.
  • Devices with more than one type of Protein C molecule attached are also included in the scope of the present invention.
  • the present invention also provides a method of reducing the tendency of a medical device to cause blood coagulation when it is in direct contact with blood, the method comprising directly or indirectly attaching a protein having the activity of Protein C onto at least part of the surface of the device.
  • the method of attaching the protein may be physical or chemical.
  • the attachment is by adsorption of the protein onto the surface of a device.
  • the attachment may also be by chemical bonding.
  • Figure 2 shows the elution of APC from APC-adsorbed wires (the quantity of APC remaining on stent-wires when placed in a perfusing circuit is shown);
  • Figure 3 shows the effect of APC-eluting wires on the deposition of fibrinogen/fibrin in a recalcified plasma system, wherein the black columns represent results obtained after 2 hours and the grey columns represent results obtained after 4 hours;
  • Figure 4 shows 125 I-Fibrinogen/fibrin deposition on stent-wires;
  • Figure 5 shows the effect of APC in an activated partial thromboplastin time assay;
  • Figure 6 shows the effect of APC-eluting wires on an activated partial thromboplastin time assay, wherein the diamonds represent group 1 results, the squares represent group 2 results and the triangles represent the averaged values;
  • Figure 7 shows activated partial thromboplastin time assay tested on ambient buffer containing stent-wires;
  • Figure 8 shows the flow
  • Cellulose polymer-coated stainless steel stent wires and stents (20 mm long x 3 mm expanded diameter) were provided by Cook Inc. Radioactive iodine ( I25 I) and indium ( m In) was purchased from Amersham Life Science (Bucks, UK). Citrated rabbit plasma for in vitro work was obtained from Harlan-Sera-Lab Ltd. (Crawley Down, UK). All other products were purchased from Sigma Chemicals Co (St Louis, MO) 17
  • APC and rabbit fibrinogen were labeled with 125 I by the lodo-gen method.
  • Autologous rabbit blood was drawn on the same day of operation and platelets l "Indium-labelled as previously described by Aggarwal RK et al [Aggarwal RK, Ireland DC, Azrin MA, Ezekowitz MD, de Bono D, Gershlick AH . Antithrombotic potential of polymer-coated stents eluting platelet glycoprotein Ilb/IIIa receptor antibody. Circulation. 1996;94:3311-3317]. All radioactivity was measured using the automated Cobra-II auto-gamma (Packard, CT). Animal procedures were conducted in accordance with the Animals [Scientific Procedures] Act 1986 under a licence from the Home Office, London.
  • Protein C was isolated from citrated rabbit plasma using DEAE anion exchange chromatography. Bound proteins were eluted with calcium chloride. Following this, immunoaffmity chromatography was performed using the protein C-specific monoclonal antibody, HC2 from Sigma Chemicals Co (St Louis, MO). Elution of bound protein C was effected using alkaline pH.
  • ACC-C activator protease purified from the venom of the Southern Copperhead snake (Agkistrodon contortrix contortrix) (ACC-C), which was a gift from Dr. Walter Kisiel (University of New Mexico, Alberquerque). ACC- C was purified away from protein C after activation using anion exchange chromatography on a Resource Q column (Amersham Pharmacia Biotech, Bucks, UK).
  • the rabbit protein C preparation was shown by Western blotting to be at least 95% activated and free of ACC-C.
  • Results are shown as either mean ⁇ SD or as proportions. Differences between groups are analysed by one-way ANOVA. Where values could not be assumed as normal distribution, Kruskal-Wallis test was used. Where there was a significant overall difference between groups, multiple comparisons were made by Mann- Whitney U test. Differences in platelet deposition were analysed by one-sample t test. Significance was defined as E ⁇ 0.05.
  • Polymer-coated stent wire segments (10-mm lengths) were immersed in a 0.542 mg/mL solution of APC containing an iodinated-APC spike (specific activity 4.7 cpm/ng (or 4.46 ⁇ Ci/ ⁇ g may be used) in 20 mM TBS/1 %BSA buffer (pH 7.2 (or pH 7.4 may be used) at 37°C.
  • APC solutions were contained in 1.5 mL polypropylene (Eppendorf) tubes. Total and uniform immersion of wire segments was ensured by placing the tubes on a Magic Roundabout Wheel.
  • wires were removed from each solution rinsed 10 times in 5 mL PBS and gamma counted for 15 seconds (15-75 keV window for 125 Iodine; Cobra II auto-gamma counting system, Packard Instruments, Meridien, CT) to determine APC binding onto each wire. Six wires were assessed for each time point.
  • Stent wire segments were immersed in 0.542 mg/mL solutions of APC in buffer at 37°C for 60 minutes as described above. Baseline protein binding to wires was determined by counting the radioactivity of each wire. The wires were then placed in individual housings in a closed-loop circuit continuously perfusing PBS/1 %BSA at a rate of 10 mL/min (or a rate of 20ml/min may be used). Housings were made from glass chambers, diameter 2.0 mm, connected to a manifold device (modified from a multiple manifold dispenser; Lablndustries, CA) to ensure equal flow through across all channels.
  • a manifold device modified from a multiple manifold dispenser; Lablndustries, CA
  • the PBS/1 %BSA was pumped through the circuit with a peristaltic pump (Watson-Marlow 302S, Falmouth, UK).
  • Sterile silicone tubing (3-mm bore, Fisons, Loughborough, UK) was used to carry the perfusate to the chambers housing the stent wires.
  • All wires were removed from the circuit and individually gamma counted to quantify the amount of protein remaining bound to each. After counting, wires were returned to the circuit until the next time point. Six wires were used in the perfusing circuit.
  • Stent wire segments were immersed in 0.542 mg/mL solutions of APC in buffer at 37°C for 60 minutes as described above. Following ten times of rinsing with PBS/1 %BSA, wires were placed in 200 ⁇ L of recalcified rabbit plasma containing an iodinated-fibrinogen spike (specific activity »2.32 ⁇ Ci/ ⁇ g). After incubation at 37°C for 4 hours, wires were removed and counted to determine 125 fibrinogen/fibrin deposition.
  • Relative 125 fibrinogen/ fibrin deposition ratios after 4 hours were 1:5.27 (ANOVA, P ⁇ 0.001), 1:53.8 (/ ⁇ ⁇ 0.001), 1 :4.7 (P ⁇ 0.001) and 1:2.63 (P ⁇ 0.001) for APC-eluting wire:plain base-polymer wire, APC:stainless steel wire, APC:wire preloaded with 10%BSA and APC:wire preloaded with inhibited-APC respectively.
  • APC-eluting wire plain base-polymer wire
  • APC stainless steel wire
  • APC wire preloaded with 10%BSA
  • APC wire preloaded with inhibited-APC respectively.
  • the activity of the rabbit APC was tested in an activated partial thromboplastin time (APTT) assay using a rabbit plasma system.
  • 100 ⁇ L citrated rabbit plasma, 100 ⁇ L PTT Automat 5® (Diagnostica Stago, Asnieres, Fr) and 50 ⁇ L 20mM pH7.2 TBS/1 %BSA buffer containing increasing concentrations of APC were mixed in a 1- mL aggregometer tube containing a metal stirrer and incubated at 37°C for 3 min. Clotting was initiated with 100 ⁇ L 20 mM CaCl 2 . ( Figure 5) 22
  • Stent wire segments were immersed in 0.542 mg/mL solutions of APC in buffer at 37°C for 60 minutes as described above. Wires were then placed separately into tubes containing 80 ⁇ L TBS/1 %BSA buffer. At 0, 2, 5, 10, 15, 45, 60, 90 and 120 minutes, 50 ⁇ L buffer was removed and tested in the APTT assay described above. Plain polymer-coated wires were used as negative controls. Four wire segments were used for each time point.
  • Example 6 Efficacy of the APC-eluting stents in vivo
  • Each rabbit received aspirin 6 mg.kg ⁇ * .d "* and Ticlopidine 8 mg.kg d " ' administered in drinking water for 5 days and 3 days respectively prior to operation. 17 mL of blood was collected from an ear artery 2 hours before operation into 3 mL of acid-citrate-dextrose for 11 'Indium-labeling of platelets. All animals were anaesthetised with Hypnorm 0.3 mL/kg IM (fentanyl citrate, 0.315 mg/mL and fluanisone, 10 mg/mL) and inhaled halothane and oxygen. Animals were placed on a heating pad (38°C) and had continuous intraoperative monitoring of rectal temperature, heart rate and respiratory rate.
  • Hypnorm 0.3 mL/kg IM (fentanyl citrate, 0.315 mg/mL and fluanisone, 10 mg/mL) and inhaled halothane and oxygen. Animals were placed on a heating pad (38
  • APC-eluting stents were prepared by immersion of stents into either 1.24 mg/mL or 0.62 mg/mL of APC solutions (under the in vitro conditions described above). Plain polymer-coated stents and stents similarly loaded with 1.2mg/mL BSA were used for controls. The type of stent deployed was assigned with the operator blinded.
  • each animal was given the autologous injection of lu In-labeled platelets (3-mL suspension, 0.15 to 0.27 mCi) via an ear vein.
  • bilateral femoral and common iliac arteries and the abdominal aorta were exposed.
  • baseline blood flow was recorded for 2 to 3 minutes in each vessel using two separate perivascular transit time flowprobes (T206 small animal blood flow meter with 2.5SB probes; Transonic Ine, Ithaca, NY).
  • a superficial femoral artery arteriotomy was fashioned as distally as possible on each side. Through the arteriotomy, a 3-mm-diameter noncompliant coronary angioplasty balloon catheter 24
  • APC eluting stents (3 x 20mm, balloon-mounted) were prepared for the study by complete immersion of plain polymer-coated stents in APC solution [either 1.24 mg/mL or 0.62 mg/mL] at 37°C pH 7.5 for 60 minutes. Each immersion was undertaken just prior to stent deployment.
  • the average percentage of flow remaining in APC-eluting stents was 45.81 ⁇ 8.8% for 1.24 mg/mL and 44.86 ⁇ 13.9% for 0.62 mg/mL.
  • the average percentage of flowrate remaining in albumin-loaded stents was 3.10 ⁇ 3.7%; and that for plain polymer coated stents was 2.53 + 3.4% (Figure 9).
  • Albumin-loaded stents were prepared by immersion of polymer-coated stents in BSA [1.2 mg/mL] 37°C for 60 minutes. Flowrate changes in arteries deployed with albumin loaded stents were similar to that in plain polymer coated stents. Flowrates were dramatically reduced within 30 minutes of stent deployment and remained non- pulsatile and non-biphasic in the arteries that did not occlude (Figure 8d).
  • Example 7 Platelet deposition in stented arteries
  • Platelet deposition was determined by counting n 'In-labeled platelet accumulation on the stented artery. For each animal relative platelet deposition was determined by the ratio of radioactivity on the study stented vessel compared to control stented vessel. The results reflected closely thrombosis that was observed in the arteries. APC-eluting stents had significantly reduced platelet deposition as compared to both albumin-loaded stents and plain polymer coated stents.
  • 3-mL venous blood was removed at 30-minute interval during the 2-hour monitoring period, collected into 0.3-mL acid-citrate, centrifuged immediately and tested in the APTT assay.
  • the stent was pre-immersed expanded in an APC solution [1.2 mg/mL] containing an iodinated-APC spike.
  • the animal was killed 2 hours after stenting and tissue/organs including the heart, aorta, liver, spleen, left lung, left kidney, urine, blood, stented artery and contralateral unstented artery were removed and gamma-counting to determine the systemic distribution of APC eluting off the stent. Each specimen was weighed and counts were normalised for weights.
  • organs removed after 2 hours of monitoring including the heart, aorta, liver, spleen, left lung, left kidney, urine, blood and the contralateral unstented artery, showed only background radioactivity as compared to the high radioactivity found on the stented artery.
  • Nan Beusekom et al [1993] Circulation, 88(Suppl 1), pp 1-645. Van der Giessen et al, [1992] J Interven Cardiol, 5, pp 175- 185.

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Abstract

La présente invention concerne des dispositifs médicaux comportant une protéine sur tout ou partie de leur surface. Cette protéine est soit une protéine présentant une activité de protéine C, soit une protéine C activée. Les dispositifs de l'invention peuvent être des prothèses vasculaires telles que des stents. Cette protéine peut se rattacher indirectement à la surface du dispositif via une couche intermédiaire telle qu'un revêtement polymère. Le rattachement peut être de type mécanique. Les dispositifs en de tels matériaux ont une moindre propension à provoquer la coagulation sanguine lorsqu'ils sont en contact avec le sang. Les dispositifs de l'invention conviennent pour de nombreux systèmes, et notamment le système cardio-vasculaire du mammifère, lorsque leur utilisation implique que de tels appareils viennent au contact direct du sang.
PCT/GB1999/001008 1998-03-31 1999-03-31 Dispositifs medicaux traites de façon a susciter un refus de coagulation du sang WO1999049907A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
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EP1259223A1 (fr) * 2000-02-28 2002-11-27 Gel-Del Technologies, Inc. Materiaux de matrice proteique, dispositifs correspondants et procedes pour leur production et leur utilisation
US7488444B2 (en) 2005-03-03 2009-02-10 Icon Medical Corp. Metal alloys for medical devices
US7662409B2 (en) 1998-09-25 2010-02-16 Gel-Del Technologies, Inc. Protein matrix materials, devices and methods of making and using thereof
US8465537B2 (en) 2003-06-17 2013-06-18 Gel-Del Technologies, Inc. Encapsulated or coated stent systems
US9034245B2 (en) 2010-03-04 2015-05-19 Icon Medical Corp. Method for forming a tubular medical device
US9107899B2 (en) 2005-03-03 2015-08-18 Icon Medical Corporation Metal alloys for medical devices
US11766506B2 (en) 2016-03-04 2023-09-26 Mirus Llc Stent device for spinal fusion
US11779685B2 (en) 2014-06-24 2023-10-10 Mirus Llc Metal alloys for medical devices
US11890371B2 (en) 2007-12-26 2024-02-06 Petvivo Holdings, Inc. Biocompatible protein-based particles and methods thereof

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JPH0268039A (ja) * 1988-09-05 1990-03-07 Kuraray Co Ltd 血液成分測定装置
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7662409B2 (en) 1998-09-25 2010-02-16 Gel-Del Technologies, Inc. Protein matrix materials, devices and methods of making and using thereof
US8871267B2 (en) 1998-09-25 2014-10-28 Gel-Del Technologies, Inc. Protein matrix materials, devices and methods of making and using thereof
EP1259223A1 (fr) * 2000-02-28 2002-11-27 Gel-Del Technologies, Inc. Materiaux de matrice proteique, dispositifs correspondants et procedes pour leur production et leur utilisation
EP1259223A4 (fr) * 2000-02-28 2006-11-29 Gel Del Technologies Inc Materiaux de matrice proteique, dispositifs correspondants et procedes pour leur production et leur utilisation
US8465537B2 (en) 2003-06-17 2013-06-18 Gel-Del Technologies, Inc. Encapsulated or coated stent systems
US7488444B2 (en) 2005-03-03 2009-02-10 Icon Medical Corp. Metal alloys for medical devices
US9107899B2 (en) 2005-03-03 2015-08-18 Icon Medical Corporation Metal alloys for medical devices
US11890371B2 (en) 2007-12-26 2024-02-06 Petvivo Holdings, Inc. Biocompatible protein-based particles and methods thereof
US9034245B2 (en) 2010-03-04 2015-05-19 Icon Medical Corp. Method for forming a tubular medical device
US11779685B2 (en) 2014-06-24 2023-10-10 Mirus Llc Metal alloys for medical devices
US11766506B2 (en) 2016-03-04 2023-09-26 Mirus Llc Stent device for spinal fusion

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