WO1999006054A1 - Compositions pour le traitement de lesions et de blessures et utilisation de ces compositions - Google Patents

Compositions pour le traitement de lesions et de blessures et utilisation de ces compositions Download PDF

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Publication number
WO1999006054A1
WO1999006054A1 PCT/AU1998/000608 AU9800608W WO9906054A1 WO 1999006054 A1 WO1999006054 A1 WO 1999006054A1 AU 9800608 W AU9800608 W AU 9800608W WO 9906054 A1 WO9906054 A1 WO 9906054A1
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Prior art keywords
growth factor
perlecan
active fragment
heparin binding
adhesive protein
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PCT/AU1998/000608
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English (en)
Inventor
John Michael Whitelock
Patricia Anne Underwood
Original Assignee
Cardiac Crc Nominees Pty. Ltd.
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Publication date
Application filed by Cardiac Crc Nominees Pty. Ltd. filed Critical Cardiac Crc Nominees Pty. Ltd.
Priority to AU86173/98A priority Critical patent/AU8617398A/en
Publication of WO1999006054A1 publication Critical patent/WO1999006054A1/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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0052—Mixtures of macromolecular compounds
    • 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
    • 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
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • 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
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow

Definitions

  • the present invention relates to compositions and methods suitable for the treatment of wounds and for the promotion of wound healing.
  • the invention also relates to methods for lessening restenosis of body lumens by way of application of intraluminal stents, vascular biomaterials or grafts having anti-thrombosis and/or anti-restenosis properties.
  • the present invention is particularly concerned with the treatment of endothelial cells although it may be used to treat other cells.
  • Perlecan is the major heparan sulfate (HS) proteoglycan of basement membranes and other connective tissues.
  • the human molecule has a protein core of molecular weight approximately 470,000, which has a well described domain structure.
  • Domain I is the N-terminal domain that carries the 3 sites for heparan sulfate attachment.
  • Domain II has homology to the LDL receptor with strict conservation of the cysteine residues.
  • Domain III shares homology with the short arm of the laminin ⁇ chain.
  • Domain IV is the largest domain with a molecular weight in excess of 200.000. and has immunoglobulin-like repeats similar to those found in the neural cell adhesion molecule.
  • Domain V is the C-terminal domain and has three regions with homology to the globular domains of laminin ⁇ chain which are interdispersed with four regions showing homology to epidermal growth factor (Murdoch et al., 1992). These domains have been expressed in bacterial cells and more recently in eucaryotic expression systems (Costell et al., 1997; Groffen et al, 1996; Costell et al. 1996: Chakravarti et al, 1995; Schulz et al., 1996). Recombinant domain III of mouse perlecan was shown to be involved in the attachment of mammary tumour cells via an RGD dependant pathway (Chakravarti et al, 1995).
  • Domain I of mouse perlecan expressed by transfected human kidney cells was present as a pool of molecules, some of which had no glycosaminoglycan decoration and others which had either HS, chondroitin sulfate / dermatan sulfate (CS/DS) or a mix of both attached to them.
  • the glycosaminoglycans chains that were coupled to the recombinant protein were approximately one-sixth the length of those found on native perlecan (Costell et al., 1997). The study of the functional role of recombinant domain I, as well as those of the other domains is of intense interest.
  • Native perlecan has been purified from the mouse Englebroth Holm Swarm (EHS) tumour (Hassell et al., 1980), porcine kidneys (Gauer et al.,1996), bovine kidneys (Hagen et al, 1993), human placenta (Isemura et al., 1987), from the conditioned medium of bovine aortic endothelial cells (Saku and Furthmayr, 1989), and from the extracellular matrix of cultured human fetal lung fibroblasts (Heremans et al., 1988; Aviezer et al., 1997).
  • the perlecan has been isolated using very stringent chaotropic and denaturing agents such as guanidine and urea.
  • the protein core isolated has had a molecular weight of approximately 400,000 for the mouse and around 450,000 for the human, however, the type and amount of glycosaminoglycan attached to this protein core has varied.
  • the protein core is decorated with HS only and on other occasions it may be either unglycosylated or have CS/DS present (Iozzo et al.,1994; Hassell et al., 1980; Isemura et al., 1987).
  • Bovine endothelial cells have been shown to attach to the protein core of native mouse perlecan via ⁇ l and ⁇ 3 integrins, however, if the HS is left on the protein core, the cells did not attach.
  • the binding to the protein core was independent of the RGD sequence (which is present in domain III of mouse perlecan) and was inhibited by exogenous heparin (Hayashi et al., 1992).
  • Rugli cells a rat glioma derived cell line
  • HS a rat glioma derived cell line
  • mesenchymal and epithelial derived tumour cell lines mirrored that of the Rugli cells.
  • cells derived from mouse endothelioma, human melanoma or human astrocytoma did not adhere at all, to the intact proteoglycan (Battaglia et al., 1993).
  • heparin binding growth factor bFGF binds to the HS on domain I of perlecan (Aviezer et al., 1994) and that it is released by biologically relevant enzymes, svich as plasmin, collagenase and heparinases which may have a role in the regulation of the growth factor activity (Whitelock et al., 1996).
  • Other heparin binding growth factors may also bind perlecan in a similar fashion.
  • a major aim of a study which led to the present invention was to demonstrate the biological relevance of the bFGF/perlecan interaction in a vascular setting. This was achieved by studying the growth of endothelial and smooth muscle cells on a matrix of perlecan and fibronectin to which had been attached bFGF. We have shown that vascular cells grow better on a matrix that contains perlecan to bind and present the bFGF. If the perlecan was absent, the cells did not grow as well. Presumably because bFGF could not be made available to the cells. These data suggest a role for perlecan in the growth of vascular cells in the response to injury, and further suggest that this proteoglycan may have a wider role in wound healing generally.
  • the present invention provides a wound and injury treatment composition including perlecan or active fragment thereof, and a pharmaceutically acceptable carrier of diluent.
  • a wound and injury treatment composition including perlecan or active fragment thereof, and a pharmaceutically acceptable carrier of diluent.
  • the composition of the first aspect further includes a heparin binding growth factor or active fragment thereof and/or a cell adhesive protein or an active fragment thereof.
  • composition of the first aspect further includes both a heparin binding growth factor or active fragment thereof and a cell adhesive protein or an active fragment thereof
  • the perlecan is human perlecan or a biologically active fragment thereof.
  • the cell adhesive protein may be fibronectin or vitronectin or a combination thereof.
  • the heparin binding growth factor may be selected from one or more of the group consisting of basic fibroblast growth factor (bFGF), transforming growth factor ⁇ , transforming growth factor ⁇ , epidermal growth factor, platelet derived growth factor, heparin binding epithelial growth factor, granulocyte colony stimulating factor, vascular endothelial growth factor (VEGF), VEGF-related growth factor, acidic FGF and other members of the FGF family.
  • bFGF basic fibroblast growth factor
  • transforming growth factor ⁇ transformed growth factor ⁇
  • epidermal growth factor epidermal growth factor
  • platelet derived growth factor platelet derived growth factor
  • heparin binding epithelial growth factor granulocyte colony stimulating factor
  • VEGF vascular endothelial growth factor
  • VEGF-related growth factor acidic FGF and other members of the FGF family.
  • the heparin binding growth factor is bFGF or VEGF.
  • composition of the invention may include one or more other agents that promote the healing of wounds or treatment of symptoms accompanying wounds.
  • the other agent may be, for example, an antibacterial agent, anti-viral agent, anti-fungal agent or local anaesthetic.
  • composition may be in the form of a liquid, semi-solid or solid.
  • the composition may be in the form providing controlled or sustained release.
  • the carrier may be a biomaterial.
  • the biomaterial may be formed in whole or in part from any suitable material, for example, polymers including biodegradable polymers.
  • the carrier may be a bandage.
  • the carrier may be a catheter or the like, for example, an angioplastic catheter or other intraluminal catheter.
  • the active component(s) of the composition of the invention may be present in any suitable amount.
  • the component(s) may be present in an amount of about 0.0001 to about 10% of the composition.
  • the active components are present in an amount of about 0.0001 to 1% of the composition.
  • the present invention provides a method of treatment of an injury in a subject, the method including administering to the subject, perlecan or active fragment thereof.
  • the method of the second aspect includes administration of a heparin binding growth factor or active fragment thereof, and optionally a source of cell adhesive protein or active fragment thereof.
  • the heparin binding growth factor may be selected from the group set out above.
  • the heparin binding growth factor is bFGF.
  • the source of cell adhesive protein may be fibronectin or vitronectin or a combination thereof.
  • the source of cell adhesive protein may be plasma or serum.
  • the plasma or serum is of human origin.
  • the plasma or serum may originate from the treated subject at the time of the procedure or injury.
  • the injury may be in the form of cellular damage such as that resulting from burns, sores, incisions or wounds.
  • the injury may be cellular injury or trauma occasioned by a surgical procedure.
  • the cells may be vascular cells.
  • Restenosis is the reclosure of a peripheral or coronary artery following trauma resulting from the opening of a stenosed portion of the artery by angioplasty procedures such as coronary angioplasty or by procedures such as bypass grafting, removal of material by atherectomy, coronary artery bypass surgery or insertion of stents. Restenosis is believed to result from an extension of the natural healing reaction to the injury of the arterial wall that is caused by angioplasty procedures, or to result from localised tissue reaction to the foreign material in an implanted stent or synthetic vascular graft. The healing reaction begins with the thrombotic reaction at the site of the injury. The final result of the complex steps of the healing process can be the uncontrolled migration and proliferation of medial smooth muscle cells, combined with their extracellular matrix production (ECM), until the artery or vascular graft is again stenosed or occluded.
  • ECM extracellular matrix production
  • the present invention provides a method for inhibiting or reducing restenosis and/or thrombosis in a subject, the method including treating the subject with perlecan or active fragment thereof.
  • the method of the third aspect may include treatment of the subject with heparin binding growth factor or active fragment thereof, and/or a source of cell adhesive protein or active fragment thereof.
  • the method of the third aspect includes treatment of the subject with a heparin binding growth factor or active fragment thereof, and optionally a source of cell adhesive protein or active fragment thereof.
  • the growth factor and source of cell adhesive protein may be that set out above.
  • the growth factor is bFGF.
  • the method of the third aspect may involve treatment before, during or following a surgical procedure such as corony angioplasty, atherectomy, arterial bypass graft surgery, biomaterial implantation or the like.
  • a surgical procedure such as corony angioplasty, atherectomy, arterial bypass graft surgery, biomaterial implantation or the like.
  • One means of preventing restenosis in angioplasty procedures is by the use of metallic intra vascular stents, which are permanently implanted in the coronary or peripheral vessel.
  • the stent may be inserted by catheter into the vascular lumen and expanded into contact with the diseased portion of the arterial wall, thus providing a mechanical support of the lumen.
  • early and late complications such as thrombotic closure and restenosis are common.
  • the present invention further provides a means by which the occurrence of restenosis and/or thrombosis following angioplasty procedures or the like may be prevented or lessened.
  • the present invention provides a method for preventing or reducing restenosis and/or thrombosis in a body lumen treated to open a luminal restriction, the method including the steps of providing a stent body, vascular graft implant or the like, treating the stent body, vascular graft or implant with perlecan or active fragment thereof and delivering the treated stent body, vascular graft or implant percutaneously or transluminally into a body lumen.
  • the body lumen is a vascular lumen.
  • the method of the fourth aspect may include treatment of the stent body, vascular graft, implant or the like with a heparin binding growth factor or active fragment thereof and/or a source of cell adhesive protein or active fragment thereof.
  • the method of the fourth aspect includes treatment of the stent body or vascular graft implant or the like with a heparin binding growth factor or active fragment thereof, and optionally a source of cell adhesive protein or active fragment thereof.
  • the body lumen is a vascular lumen.
  • the present invention provides a surgical device such as a stent, vascular graft, implant or the like treated on at least a portion thereof with perlecan or active fragment thereof.
  • the device may further be treated with a heparin binding growth factor or active fragment thereof and/or a source of cell adhesive protein or active fragment thereof.
  • the device has been further treated with a heparin binding growth factor or active fragment thereof, and optionally a source of cell adhesive protein or active fragment thereof.
  • the device may be formed from any suitable material.
  • the device may be formed from a suitable metal, ceramic or polymer.
  • the device may be formed from a biomaterial.
  • the perlecan or active fragment thereof, and/or cell adhesive protein or active fragment thereof, may be coated on, or chemically bound to, a surface of the device.
  • the heparin binding growth factor is bound to the SLirface by its interaction with perlecan or an active fragment thereof.
  • the device may be exposed to the plasma or serum.
  • the source of cell adhesive protein may be plasma or serum.
  • the plasma or serum is of human origin.
  • the plasma or serum may originate from the treated subject at the time of the procedure.
  • Figure 1 shows Western blots and SDS -PAGE(la) and COA- PAGE(lb) of purified human endothelial perlecan
  • FIG. 2 is a graph showing adhesion of human arterial smooth muscle cells (HASMC) and human umbilical arterial endothelial cells (HUAEC) in response to an increasing concentration of purified perlecan:
  • Figure 3 is a graph showing cell adhesion of HASMC cells or HUAEC cells to either intact perlecan or the protein core of perlecan. Fibronectin is shown as the control:
  • Figure 4 is a graph showing the binding affinity between bFGF and surface coated perlecan
  • Figures 5 shows growth of HUAEC cells (5 a) and of HASMC cells (5b) in the presence of bFGF and combinations thereof with perlecan and fibronectin fetal calf serum.
  • the cells are grown in 10% fetal calf serum which has been depleted of fibronectin, vitronectin and heparin binding growth factor;
  • Figure 6 shows HUAEC cell giowth on a perlecan coated surface which has been exposed to plasma with bFGF in the gi owth medium (6A), HUAEC cell growth on a perlecan coated surface which has been exposed to plasma with surface bound bFGF only (6B), HUAEC cell growth on a perlecan coated surface which has been exposed to plasma with vascular endothelial cell growth factor (VEGF) in the medium (6C), and HUAEC cell growth on a perlecan coated surface which has been exposed to plasma with surface bound VEGF (6D),
  • VEGF vascular endothelial cell growth factor
  • Figure 7 is a graph showing relative platelet attachment to different coating treatment compositions as shown.
  • Heparin H3149
  • Tris Triton X-100
  • ABTS 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid)
  • Heparitinase I (heparinase III) was from Seikagaku Corporation Co. All other chemicals were of analytical grade. Tissue culture plastic ware was from either Nunc or Corning. Medium 199 containing Earles salts was from Gibco. All solutions for endothelial cell culture were prepared using pyrogen free water (Baxters) and small aliquots were filtered through sterile Zetapore 0.2 ⁇ m pore nylon membranes (Cuno Pacific).
  • Recombinant [ 125 I]labeled bFGF, peroxidase conjugated streptavidin and biotin conjugated rabbit anti-mouse IgG were purchased from Amersharn.
  • Peroxidase conjugated to rabbit anti mouse IgG, unconjugated rabbit anti-mouse IgG and alkaline phosphatase conjugated rabbit anti-mouse IgG were purchased from Dako.
  • Bromochloroindolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) substrate for alkaline phosphatase was purchased from Promega.
  • Cn-Br activated Sepharose 4B, heparin Sepharose, DEAE Sepharose, Protein A-Sepharose and Gelatin Sepharose were from AMRAD-Pharmacia Biotech.
  • Foetal calf serum was from P. A. Biologicals.
  • Anti-Heparin antibody 10E4 was purchased from Seikagaku Corporation Co. Depleted sera was prepared according to the method of Underwood and Steele, (1991).
  • Culture medium was Medium 199 with Earle's Salts containing 20% pyrogen free foetal calf serum (FCS), 100 units/ml penicillin, 100 ⁇ g/ml streptomycin sulfate, 100 ⁇ g/ml heparin and 2% bovine brain extract prepared as described (Maciag et al., (1979)). Cells were passaged at a 1:3 split ratio after disaggregation with 0.125% trypsin, 0.02% EDTA, and used between passages 6 and 10. Conditioned medium was prepared by culturing endothelial cells in complete medium for up to 72 h. The medium was collected, centrifuged at 2000 g to remove cellular debris and stored at - 20°C.
  • FCS pyrogen free foetal calf serum
  • the CllSTH cell line is derived from a spontaneously transformed human umbilical venous endothelial cell culture and has been characterised elsewhere (Cockerill et al., 1994). These cells were grown under the same conditions that were used for the HUAECs.
  • HASMCs were cultured by explant outgrowth from pieces of internal mammary arteries discarded at surgery as described previously (Whitelock et al., 1997). Smooth muscle cells were maintained in medium 199 containing 20% foetal calf serum (FCS), 4mM glutamine, 100 units/ml penicillin and 100 mg/ml streptomycin. Cells were passaged at a 1:2 or 1:3 split ratio after disaggregation with 0.125% trypsin 0.02% EDTA, and used between passage 4 and 6.
  • FCS foetal calf serum
  • the anti-perlecan monoclonal antibodies were the result of a fusion using Sp2/0 myeloma cells and spleen cells from mice immunized with whole bovine corneal endothelial cell ECM, described previously (Underwood and Bennett, 1989). The specificity of these antibodies has been described elsewhere including identification of perlecan domain specificity for some of them (Whitelock et al., 1996). Monoclonal antibodies A 71. A74, and A76 were purified from ascites fluid by affinity chromatography on protein A-Sepharose.
  • Conditioned medium from confluent CLiltures of human endothelial cells was batched (approx. 2 litres) and passed over (1 ml/min) a DEAE Sepharose column (100ml bed volume) which had been equilibrated with 20mM Tris, 250mM NaCl. 10m M EDTA, 1 mM PMSF, 1 mM benzamidine, pH 7.5. The column was washed with the start buffer until a baseline was achieved and the bound proteins containing the perlecan were eluted with a step gradient of 1M NaCl in 20mM Tris, lOmM EDTA. 1 mM PMSF, 1 mM benzamidine, pH 7.5.
  • the peak from the 1M NaCl elution was then passed over an immunoaffinity column of A71 antibodies conjugated to CnBr activated Sepharose 4B (5mg A71 per ml of Sepharose 4B: 5 ml bed volume).
  • the eluate was cycled over the column for four - five hours at a flow rate of approx. 1 ml/min.
  • the column was washed with 20mM Tris, lOmM EDTA, 1M NaCl, 1 mM PMSF, 1 mM benzamidine, pH 7.5 to achieve a baseline and the bound perlecan was eluted with 6M urea in PBS.
  • SDS-PAGE was performed using 4-15 % polyacrylamide gradient gels as described by Laemmli (1970).
  • Composite agarose- polyacrylamide gel electrophoresis (COA-PAGE) was performed as described by McDevitt and Muir (1971) except that the gels did not contain ⁇ -dimethyl aminopropionitrile.
  • COA-PAGE Composite agarose- polyacrylamide gel electrophoresis
  • McDevitt and Muir (1971) except that the gels did not contain ⁇ -dimethyl aminopropionitrile.
  • electrophoresis some of the SDS-PAGE gels were stained with 0.25% w/v Coomassie Blue R250 in 45% v/v methanol, 10% v/v acetic acid in H2O for 30 minutes at room temperature, and destained in the same buffer.
  • SDS-PAGE and COA-PAGE gels were transferred to nitrocellulose or PVDF membranes (Towbin et al.. 1979) and probed with the antibodies directed against either the protein core or heparan sulfate of perlecan.
  • Immunoreactive bands were detected using alkaline phosphatase conjugated rabbit anti-mouse IgG as secondary antibody and bromochloroindolyl phosphate (BCIP) with nitro blue tetrazolium (NBT) as substrate.
  • BCIP bromochloroindolyl phosphate
  • NBT nitro blue tetrazolium
  • fibronectin 10 ⁇ g/ml
  • perlecan perlecan at the concentrations indicated, or a mix of the two for 2 hours at 37 ⁇ C plus 16 hours at 4°C.
  • fibronectin 10 ⁇ g/ml
  • perlecan perlecan
  • a mix of the two for 2 hours at 37 ⁇ C plus 16 hours at 4°C.
  • heparitinase I diluted in 1% BSA (bovine serum albumin) in PBS for 2 hours at 37° C.
  • BSA bovine serum albumin
  • ⁇ /(l - ⁇ ) was plotted against ⁇ L where ⁇ is the proportion of bindable 125 I-labeled bFGF bound to the wells (CPM) and L is the molar concentration of unlabeled bFGF.
  • the slope of the straight line obtained is equal to - 1/K where K is the dissociation constant and the intercept on the y axis is P/K where P is the molar concentration of bFGF binding sites on perlecan bound to the well.
  • Human perlecan was purified from the conditioned medium of endothelial cells by sequential DEAE Sepharose and A71 Sepharose immunoaffinity chromatography. The presence of perlecan core protein along with the presence of HS was monitored throughout the purification schedule by an ELISA using A76 and 10E4 antibodies, respectively. The perlecan from the conditioned medium bound the DEAE column and was released by washing the column with 1M NaCl. If sequential fractions were taken throughout the wash, the perlecan eluted later suggesting that it was released from the column with a relatively high concentration of NaCl (data not shown). The peak containing the perlecan was applied directly to the immunoaffinity column and eluted with urea.
  • Fractions containing purified perlecan were screened in an ELISA for the presence of other ECM molecules and found to be free of contaminating fibronectin, vitronectin, laminin, collagen types I, , III, IV and V, versican, decorin, CS and elastin (data not shown).
  • Perlecan was adsorbed onto the wells of a tissue culture plate (polystyrene) and either human endothelial (HUAEC) or smooth muscle cells (HASMC) were plated onto it in the absence of the serum proteins, fibronectin and vitronectin.
  • the number of cells attached to perlecan coated wells was compared to that which attached to wells coated with fibronectin.
  • a coating concentration of 20 ⁇ g/ml both cell types adhered to perlecan with the HASMCs adhering better (80%) than the HUAECs (35%) (Fig.2).
  • the cells attached to perlecan at 2 hours were not as well spread as those cells plated onto fibronectin.
  • Perlecan purified from primary endothelial cells was less effective at attaching cells to polystyrene than that purified from the endothelial cell line (CllSTH).
  • CllSTH endothelial cell line
  • Perlecan was shown to bind bFGF with a Kd of approx 50 nM which is similar to previously published dissociation constants for the growth factor and perlecan. The aim of these studies was to assess the functional role of perlecan in the growth of vascular cells in response to bound bFGF. Both human arterial smooth muscle cells (HASMC) and endothelial cells (HUAEC) adhered to perlecan coated on tissue culture polystyrene at about 80% and 35%, respectively when compared to the level of cell attachment to fibronectin.
  • HASMC human arterial smooth muscle cells
  • UOAEC endothelial cells
  • CELL PROUFERATION ASSAYS The wells of a 96-well plate were coated with either fibronectin (10 ⁇ g/ml) or perlecan (10 ⁇ g/ml) for 2 hours at 37°C, followed by 16 hours at 4°C.
  • the growth factor of choice either bFGF or VEGF
  • bFGF or VEGF were added to the wells at a concentration of 100 ng/ml diluted in medium 199 containing 10% human plasma followed by incubation for 4 h at 37°C in a humidified atmosphere containing 5% C0 2 .
  • Wells were washed two times with either medium 199 or phosphate buffered saline.
  • Endothelial cells were added to the wells (3 x 10 cells/well) in medium 199 containing 10% serum depleted of fibronectin and vitronectin. In some treatments, the growth factor was omitted from the plasma treatment, but was included in the growth medium. At days 1, 3 and 6, an MTS assay (Promega) was performed which gives a measure of cell proliferation.
  • Platelets were harvested from human blood collected from healthy donors by venous phlebotomy. Platelet rich plasma (PRP) was prepared by centrifugation of the blood at 187 g for 20 minutes at room temperature followed by careful removal of the upper platelet-rich layer. To separate platelets from plasma proteins, PRP was centrifuged at 500 g for 15 minutes at room temperature. After washing the pellet with tyrodes buffer they were counted in a haemocytometer and the concentration adjusted to 1 x 10 8 /ml in tyrodes plus 0.1 U/ml apyrase.
  • PRP Platelet rich plasma
  • both Ca 2+ and Mg 2+ were added to the platelet suspension to a final concentration 1 mM.
  • the wells were aspirated and 1 x 10 8 platelets/ml added to each well (100 ⁇ l/well) and allowed to settle for 45 minutes at 37°C.
  • the wells were then rinsed 8 times with PBS, and platelet adhesion was determined by a LDH (Lactate Dehydrogenase) method.
  • the adhered platelets were lysed by adding 200 ⁇ l/well 1% Triton X-100 at 37°C for 30 minutes.
  • LDH Lignotoxicity Detection Kit
  • a cell adhesive protein was used in combination with perlecan to obtain cell growth (see figs 5 A and 5B).
  • the extra cell adhesive protein is provided by exposing the surface to human plasma. This step is performed in conjunction with binding the heparin binding growth factor.
  • This is the methodology used to generate the data shown in Figs 6A, B, C and D.
  • Endothelial cell growth on a perlecan coated surface which has been exposed to plasma, with basic fibroblast growth factor (bFGF) in the culture medium is shown in Fig. 6A.
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial cell growth factor
  • a perlecan coated surface was compared to a non-adhesive surface (albumin coated) and an adhesive surface (plasma coated) for its ability to adhere platelets. These results demonstrate that perlecan does not adhere platelets (Fig. 7). Furthermore, we have demonstrated that when a perlecan coated surface is exposed to plasma, it reduces the amount of platelets which adhere to the plasma proteins.
  • Recombinant domain III of perlecan promotes cell attachment through its RGDS sequence / Biol Chem, 270, 404-409.

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Abstract

La présente invention concerne des compositions et des procédés convenant au traitement des lésions et favorisant la guérison des blessures. L'invention concerne également, non seulement des procédés permettant de limiter la resténose des lumières anatomiques, mais aussi des stents intraluminaux ainsi que des biomatériaux vasculaires pour greffes présentant des propriétés antithrombotiques et/ou antiresténotiques. L'invention concerne plus particulièrement le traitement de cellules endothéliales, bien qu'elle puisse s'utiliser pour le traitement d'autres cellules. Le principe de l'invention est d'utiliser une combinaison de perlecan ou de l'un de ses fragments actifs, et éventuellement d'un facteur de croissance ou de l'un de ses composants actifs et une source de protéine d'adhérence cellulaire. La source de protéine d'adhérence cellulaire peut être un plasma ou un sérum.
PCT/AU1998/000608 1997-07-30 1998-07-30 Compositions pour le traitement de lesions et de blessures et utilisation de ces compositions WO1999006054A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001037853A2 (fr) * 1999-11-23 2001-05-31 North Shore - Long Island Jewish Health System Procedes et agents d'inhibition de la proliferation des cellules du muscle lisse
WO2003048333A2 (fr) * 2001-12-04 2003-06-12 Thomas Jefferson University Compositions d'endorepelline et techniques d'inhibition de l'angiogenese
WO2008143863A1 (fr) * 2007-05-14 2008-11-27 Agenta Biotechnologies Cicatrisation de plaies et de blessures cutanées avec un acide nucléique codant un polypeptide de protéoglycane
US7488719B1 (en) 2002-04-22 2009-02-10 Agenta Biotechnologies, Inc. Wound and cutaneous injury healing with a nucleic acid encoding perlecan
US7671018B2 (en) 2000-08-30 2010-03-02 University Of Delaware Delivery system for heparin-binding growth factors

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US4963146A (en) * 1989-04-20 1990-10-16 Colla-Tec Incorporated Multi-layered, semi-permeable conduit for nerve regeneration
WO1993005167A1 (fr) * 1991-09-06 1993-03-18 Children's Medical Center Corporation Proteoglycanes a chaines heparane-sulfate specifiques de types de cellules, et leurs utilisations
US5206023A (en) * 1991-01-31 1993-04-27 Robert F. Shaw Method and compositions for the treatment and repair of defects or lesions in cartilage
AU4236297A (en) * 1996-08-26 1998-03-19 University Of Washington Therapeutic and diagnostic applications of perlecan domain splice variants
WO1998024466A1 (fr) * 1996-12-05 1998-06-11 Alcon Laboratories, Inc. UTILISATION D'INHIBITEURS DES FONCTIONS DU TGF-β, AFIN D'AMELIORER LA PATHOLOGIE OCULAIRE

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Publication number Priority date Publication date Assignee Title
US4963146A (en) * 1989-04-20 1990-10-16 Colla-Tec Incorporated Multi-layered, semi-permeable conduit for nerve regeneration
US5206023A (en) * 1991-01-31 1993-04-27 Robert F. Shaw Method and compositions for the treatment and repair of defects or lesions in cartilage
WO1993005167A1 (fr) * 1991-09-06 1993-03-18 Children's Medical Center Corporation Proteoglycanes a chaines heparane-sulfate specifiques de types de cellules, et leurs utilisations
AU4236297A (en) * 1996-08-26 1998-03-19 University Of Washington Therapeutic and diagnostic applications of perlecan domain splice variants
WO1998024466A1 (fr) * 1996-12-05 1998-06-11 Alcon Laboratories, Inc. UTILISATION D'INHIBITEURS DES FONCTIONS DU TGF-β, AFIN D'AMELIORER LA PATHOLOGIE OCULAIRE

Non-Patent Citations (2)

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Title
CELL, (1994), Vol. 79, D. AVIEZER et al., "Perlecan, Basal Lamina Proteoglycan, Promotes Basic Fibroblast Growth Factor-Receptor Binding, Mitogenesis and Angiogenesis", pages 1005-1013. *
J. OF BIOL. CHEM., (1996), Vol. 271(17), J.M. WHITELOCK et al., "The Degradation of Human Endothelial Cell-Derived Perlecan and Release of Bound Basic Fibroblast Growth factor by Stromelysin, Collagenase, Plasmin and Heparanases", pages 10079-10086. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001037853A2 (fr) * 1999-11-23 2001-05-31 North Shore - Long Island Jewish Health System Procedes et agents d'inhibition de la proliferation des cellules du muscle lisse
WO2001037853A3 (fr) * 1999-11-23 2001-11-01 Long Island Jewish Res Inst Procedes et agents d'inhibition de la proliferation des cellules du muscle lisse
US7671018B2 (en) 2000-08-30 2010-03-02 University Of Delaware Delivery system for heparin-binding growth factors
US7875591B2 (en) 2000-08-30 2011-01-25 University Of Delaware Delivery system for heparin-binding growth factors
WO2003048333A2 (fr) * 2001-12-04 2003-06-12 Thomas Jefferson University Compositions d'endorepelline et techniques d'inhibition de l'angiogenese
WO2003048333A3 (fr) * 2001-12-04 2004-02-26 Univ Jefferson Compositions d'endorepelline et techniques d'inhibition de l'angiogenese
US6821947B2 (en) * 2001-12-04 2004-11-23 Thomas Jefferson University Endorepellin: methods and compositions for inhibiting angiogenesis
US7488719B1 (en) 2002-04-22 2009-02-10 Agenta Biotechnologies, Inc. Wound and cutaneous injury healing with a nucleic acid encoding perlecan
US7666852B2 (en) 2002-04-22 2010-02-23 Agenta Biotechnologies, Inc. Wound and cutaneous injury healing with a nucleic acid encoding a proteoglycan polypeptide
WO2008143863A1 (fr) * 2007-05-14 2008-11-27 Agenta Biotechnologies Cicatrisation de plaies et de blessures cutanées avec un acide nucléique codant un polypeptide de protéoglycane
JP2010534621A (ja) * 2007-05-14 2010-11-11 アジエンタ・バイオテクノロジーズ・インコーポレーテツド プロテオグリカンポリペプチドをコードする核酸による創傷および皮膚損傷の治癒

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