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WO1999047070A1 - Biomateriaux implantables ameliores, compositions et leurs procedes de preparation et d'utilisation - Google Patents

Biomateriaux implantables ameliores, compositions et leurs procedes de preparation et d'utilisation Download PDF

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Publication number
WO1999047070A1
WO1999047070A1 PCT/US1999/005967 US9905967W WO9947070A1 WO 1999047070 A1 WO1999047070 A1 WO 1999047070A1 US 9905967 W US9905967 W US 9905967W WO 9947070 A1 WO9947070 A1 WO 9947070A1
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WIPO (PCT)
Prior art keywords
protein
modified
biomaterial
platelet
mutated
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PCT/US1999/005967
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English (en)
Inventor
David C. Sane
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Wake Forest University
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Wake Forest University
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Filing date
Publication date
Application filed by Wake Forest University filed Critical Wake Forest University
Priority to AU31922/99A priority Critical patent/AU3192299A/en
Publication of WO1999047070A1 publication Critical patent/WO1999047070A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to improved implantable biomaterials which are used in contact with blood or blood components containing platelets, and which are characterized, inter alia, by reduced thrombogenicity and incidence of infection as a result of applying to at least a portion of the surface thereof a modified form of an adhesive, platelet - binding protein, which is altered at the platelet receptor binding site.
  • biomaterials include stents, catheters, guide wires, arterial grafts, cardiac pacer leads, automatic implantable cardiodefibrilator (AICD) leads, ventricular assist devices, artificial hearts and the like.
  • AICD automatic implantable cardiodefibrilator
  • ventricular assist devices artificial hearts and the like.
  • these biomaterials provide many notable health benefits, they are not without certain limitations.
  • a limitation of particular concern is the tendency of implantable biomaterials to give rise to thrombosis. Thrombus formation on biomaterials has the potential to cause embolization, which may result in strokes, myocardial infarctions and other end-organ damage.
  • thrombus formation on the biomaterial surface may interfere with its proper function and possibly cause it to fail altogether.
  • Another limitation is the incidence of infection attributable to thrombus formation on biomaterials, the thrombus serving as a nidus for infection.
  • a composition for imparting reduced thrombogenicity and incidence of infection to implantable biomaterials adapted for use in contact with blood or blood components containing platelets.
  • the composition comprises a modified, platelet -binding adhesive plasma protein which is altered at the platelet receptor binding site preferably by site- directed mutagenesis. This mutation effectively provides an anti-stick coating on the biomaterial surface, thus rendering it resistant to platelet binding.
  • a method for reducing the thrombogenicity of an implantable biomaterial adapted for use in contact with blood or blood components containing plasma.
  • the method involves applying the composition of the invention, described above, to at least a portion of the surface of the biomaterial.
  • implantable materials include but are not limited to stents, catheters, guide wires, arterial grafts, cardiac pacer leads, prosthetic heart valves, cardiopulmonary bypass membranes, automatic implantable cardiodefibrilator leads, ventricular assist devices or artificial hearts.
  • the resulting implantable biomaterial constitutes another aspect of the present invention.
  • a method for reducing the occurrence of thrombosis and infection following implantation of a biomaterial in a patient which comprises the steps of :
  • kits which contain reagents and materials which may be used to advantage in practicing the methods described herein.
  • kits include for example, a composition of the invention in lyophilized form, a suitable sterile buffer for reconstitution of the composition and an implantable material or device to which the composition is to be applied.
  • the kits may include protocols for preparing the biomaterial of reduced thrombogenicity according to the present invention and an information sheet pertaining to the storage, handling and/or reconstitution of the lyophilized composition.
  • the implantable biomaterials of the invention exhibit a good biocompatability with blood and blood components including platelets, and serve to maintain the proper functioning of the biomaterial.
  • fibrinogen refers to such proteins as vitronectin, fibronectin, thrombospondin, fibrinogen, von Willebrand's factor, collagen and the like, which exhibit platelet binding activity. All of these proteins also contain at least one Arg-Gly-Asp platelet binding sequence.
  • fibrinogen contains another platelet binding site (HHLGGAKQAGDV) on the C-terminus of the gamma chain. This binding site on fibrinogen could also be mutated using the methods of the present invention to abrogate platelet binding mediated by this molecule.
  • vitronectin is an adhesive glycoprotein present in the plasma and extracellular matrix that is involved in cell adhesion, spreading and migration (Tomasini, B.R. et al . , (1990) Prog. Haemostasis Thromb . 10:269-305). Vitronectin also has important roles in the regulation of the complement, coagulation and fibrinolytic systems (Tomasini et al . , supra; Preissner, K.T and D. Jenne (1991) Thromb. Haemostas 66:123-132). Vitronectin regulates fibrinolysis by binding and stabilizing plasminogen activator inhibitor-I (PAI-I) (Tomasini et al .
  • PAI-I plasminogen activator inhibitor-I
  • modification of the protein may impair its capacity to become bound by the microorganism.
  • cells such as smooth muscle cells and fibroblasts, will not migrate into the modified vitronectin-bearing surface.
  • mutants of vitronectin were prepared in which the platelet receptor binding site was mutated, the resulting mutants having the sequences Arg-Ala-Asp and Arg-Gly-Glu. These two mutants are hereafter referred to as RAD- rVN and RGE-rV ⁇ , respectively. This mutation converted the platelet binding sequence to inactive forms.
  • a similar effect may be achieved by chemically modifying adhesive, platelet-binding proteins to abrogate platelet adhesion activity.
  • chemically modified protein in the manner described herein is contemplated to be within the scope of the invention.
  • these mutant forms of vitronectin have been expressed and purified using a baculovirus-based system.
  • vitronectin or other adhesive, platelet -binding plasma proteins
  • the mutated form of vitronectin may be applied to a biomaterial surface in various ways, including, without limitation, chemical coupling or cross-linking, or surface adsorption via immersion, padding, brushing, roller coating, spray coating or the like.
  • the protein may be applied using a suitable carrier vehicle such as phosphate-buffered saline (PBS) .
  • PBS phosphate-buffered saline
  • Procedures for chemically coupling proteinaceous substances to biomaterial surfaces are well known to those skilled in the art. See, for example, U.S. Patents Nos . 5,061,750, and 5,098,977.
  • the benefit of chemical coupling is the development of a longer lasting anti-thrombogenic effect.
  • kits Preparation of the biomaterials of the invention can be facilitated by providing the constituents in kit form.
  • a preferred kit includes lyophilized recombinant mutant or chemically modified protein and a suitable sterile buffer for reconstitution. Such a kit would facilitate the wide application of the invention to any blood-contacting biomaterial that may potentially be implanted into a test subject.
  • a particular benefit of the present invention is derived from an improvement in arterial stents .
  • stents which are coated with the modified forms of VN described herein are expected to be less prone to restenosis, since thrombus formation is likely a key element in the restenotic process and since the above-described RGE and RAD mutants fail to support (or do so only weakly) the haptotaxis, or migration of smooth muscle cells.
  • the modified protein-coated stents may have an attentuating effect on the process. Restenosis has been called the "Achilles' heel" of interventional cardiology, occurring in 30-50% of all procedures, i.e., angioplasties, atherectomies, and the like. Although stent implantation has reduced the incidence of restenosis to perhaps 20%, this is still a serious problem. Another added benefit of the present invention is that biomaterials, e.g., stents, coated with the compositions of the invention would be less likely to induce platelet-fibrin thrombus formation.
  • treated or coated stents or the like may reduce the need for the administration of platelet inhibiting agents (e.g., ticlopidine, abciximab, tirofiban, eptifibitide) to patients receiving implants. While the foregoing agents have shown benefit in the setting of stents, they are quite expensive. Coating such implantable biomaterials may effectively reduce medical costs associated with such procedures .
  • platelet inhibiting agents e.g., ticlopidine, abciximab, tirofiban, eptifibitide
  • plasmid pMel Bac transfer vector Sf9 cells and Hi -5 cells were purchased from Invitrogen, San Diego, CA.
  • pFast Bac Dual expression vector, recombinant hTGF- ⁇ l, the synthetic RGD peptide GRGDSP, monoclonal antibody to human integrin o.v ⁇ 5 (clone P1F6) , E. coli competent cells DH 10 Bac and cell culture medium SF 900II SF were from GIBOO BR Life
  • a monoclonal antibody to vitronectin (clone VIT-2), and protease inhibitor cocktail were from Sigma Chemicals, St. Louis, MO.
  • the humanized form of monoclonal antibody LM609 to human v ⁇ 3 (Vitaxin) was provided by IXSYS, Inc., San Diego, CA. Sheep anti-mouse IgG horseradish peroxidase conjugate was purchased from Amersham.
  • Human aortic smooth muscle cells (CRL 1999) were purchased from ATCC, Rockville, MD. Rabbit aortic smooth muscle cells were a gift from Dr. Maurice Nachtigal, University of South Carolina School of Medicine, Columbia, SC. All chemicals were of analytical or molecular biological grade.
  • the plasmid pGEMHVN containing a full length cDNA for human vitronectin is available from the ATCC.
  • Expression vectors or recombinant hVN and hVN mutants were constructed as described previously (Zhao, Y, and D.C. Sane (1993) Biochem. Biophys . Res. Comm. 192:575-582) .
  • the cDNA for human vitronectin and its RGD mutants (RAD-rVN and RGE-rV ⁇ ) were cloned into the Bam HI site of plasmid pMelBac B.
  • the recombinant plasmids (pMelbac B rV ⁇ , pMelbac E RGE-rV ⁇ or pMelbac B RAD-rV ⁇ ) were co-transfected into Sf9 cells with linear AcM ⁇ PV D ⁇ A. Recombinant virus was isolated and plaque-purified on X-gal plates using the plaque assay, in accordance with the manufacturer' s recommended protocol .
  • Oligonucleotide primers were synthesized on an ABI 394 DNA/RNA synthesizer using standard cyanoethyl phosphoramide procedure in the DNA synthesis Core
  • the modified cDNAs for ⁇ S-rVN and ⁇ H-rVN were then cloned into the Rsr II- Hind III site of the pFastBac dual vector.
  • Recombinant plasmids with cDNAs for ⁇ S -rVN and ⁇ H-rVM were sequenced to eliminate any PCR-induced errors.
  • the recombinant deletion mutants pFastBac dual ⁇ S-rVN and pFastBac dual ⁇ H-rVN were transformed in competent E. coli DH 10 Bac cells and the recombinant bacmids were isolated from these cells, in accordance with the manufacturer's recommended protocol. Sf9 cells were then infected to produce high titer viral stocks, which were stored at 4oc.
  • Hi -5 insect cells were grown as a monolayer at 27oc in serum-free SF900II SFM medium. Cells were infected with baculoviruses for recombinant wild type human vitronectin or VN mutants. The medium was harvested at 48 hours post-infection, protease inhibitor cocktail was added and the harvested medium was frozen at -20°C until purification.
  • vitronectin Infection with the recombinant baculovirus resulted in the secretion of vitronectin into the medium.
  • the maximum expression of vitronectin was - 11 -
  • vitronectin expression decreased, and different sizes of vitronectin proteolytic products were observed by immunoblotting (data not shown) .
  • Recombinant hVN and its mutants were purified on FPLC .
  • Medium was harvested at 48 hours post infection and filtered through a 0.22 ⁇ m filter.
  • a protease inhibitor cocktail for general use was added and loaded onto the anion exchange column (Mono Q 10/10 or source Q 10/10), which had been previously equilibrated in 20 mM Tris-HCl pH 7.0 (buffer A) .
  • the column was washed with 10 column volumes of buffer A and a linear gradient of buffer B (0.75M NaCl in Buffer A) was applied to elute the bound proteins from the column.
  • the flow rate of the column was maintained at 0.5 ml/min.
  • Absorbance was read at 280 nm and 2.0 ml fractions were collected by an online fraction collector.
  • Recombinant VN and its mutants were obtained as described in Example IV, above, and transferred to a nitrocellulose membrane, which had been equilibrated for 5-10 minutes in IX transfer buffer (25 mM Tris, 192 M glycine, 20% methanol, pH 8.3) .
  • the proteins were transferred for 90 minutes in cold transfer buffer at 0.6 amp constant current .
  • the membrane was blocked for 60 minutes in 5% nonfat dry milk in IX TBS (Tris-buffered saline) containing 0.1% Tween 20 (TBST) . After blocking, the membrane was washed twice in IX TBST for 15-20 minutes.
  • the membrane was placed in a heat sealable bag with primary antibody, VN mab, clone VIT-2, at a 1:1000 dilution.
  • the primary antibody was added in blocking solution and the membrane incubated for 60 minutes at room temperature.
  • the membrane was then washed twice in IX TBST buffer for 15-20 minutes at room temperature, followed by the addition of the secondary antibody (anti -mouse IgG- horse radish peroxidase conjugate at a 1:5000 dilution) .
  • the membrane was incubated at room temperature for an additional 60 minutes.
  • the membrane was then washed twice in IX TBST for 15-20 minutes.
  • Two different substrates were used for the detection of bound secondary antibody.
  • Amersham' s ECL detection system was used.
  • the membrane was exposed to Amersham' s Hyperfilm for 10-120 seconds.
  • 1 tablet 5 mg
  • Electroblotting for protein sequencing was performed as previously described (LeGendre, N., and P.T. Matsudara, (1989) Purification of proteins and peptides by SDS-PAGE in "A Practical Guide to Protein and Peptide Purification for Microsequencing” pgs. 52- 66, Editor, P.T Matsudaira, Academic Press, Inc) . Purified recombinant hVN and mutants of VN (7-8 ⁇ g) were separated by 10% SDS PAGE, as described previously. A PVDF membrane (BioRAD) was washed sequentially with the following: 1) 100% methanol for 2-3 seconds and 2) water for 2-3 seconds.
  • the membrane was then equilibrated in IX transfer buffer (10 mM CAPS, 10% methanol, pH 11.0) for 15-20 minutes at room temperature. See Current Protocols in Molecular Biology (eds. Ausubel, F.M.) 10.8.16 (John Wiley and Sons, New York, 1997) for CAPS. After electrophoresis was completed, the gel was equilibrated in IX transfer buffer for 5-10 minutes and the proteins were transferred at 0.6 amp for 90 minutes. After transfer the membrane was washed several times in deionized water (5 minutes each) at room temperature. VN and its mutants were visualized by staining with Coomassie blue R-250 (0.1% in 50% methanol) for 5 minutes, and destained in 50% methanol, 10% acetic acid. The membrane was rinsed - 14 -
  • Electroblotted VN and VN mutants were directly sequenced using an Applied Biosystems 477A Protein sequencer in Protein Core Laboratory of the Comprehensive Cancer Center of Wake Forest University, School of Medicine.
  • the derivatives of phenylthiohydantoin (PTH) amino acids were analyzed by an on-line PTH analyzer. Ten cycles were sequenced for VN and VN mutants .
  • the adhesion assay was performed as described (Technical Bulletin (1995) Focus on Applications, GIBCOBRL Life Technology "Attachment of cells to ECM components" with little modification. Briefly, polystyrene, non-tissue culture treated 96 well plates (Nunc) were coated for 16 hours at 4°c with 10 ⁇ g/ml vitronectin or VN mutants in phosphate buffer saline pH 7.4 (137 mM ⁇ aCl , 2.7 mM Kcl, 4.3 mM ⁇ a 2 HP0 4 .7H 2 0 and 1.4 mM KH 2 P0 4 ) . After coating, residual proteins were removed and the plate was washed with IX PBS.
  • cells were washed with serum free DMEM/0.5% BSA, and a cell suspension (0.1 ml) containing 10,000 cells was added to each well and incubated at 37°C for 2 hours.
  • the wells were initially washed with IX PBS. Buffered formalin (10%; 0.1 ml) was added to each well and the plates incubated for 30 minutes at room temperature. After fixing the cells, 50 ⁇ l of toluidine blue (1%) in 10% buffered formalin was added to each well and incubated for 1 hour at room temperature. After incubation, the wells were extensively washed with deionized water, then air dried overnight.
  • Gore-Tex vascular graft material (5 cm in length) was left uncoated or coated overnight with RGE-rVN (10 ug/ml) .
  • the graft tubing was then perfused with 20 mis of citrated rabbit blood at a flow rate of 5ml/min and subsequently rinsed with 100 mis of phosphate buffered saline. After rinsing, adherent platelets were fixed in 2.5% glutaraldehyde and submitted for scanning EM. Scanning EM demontsrated a 10 -fold reduction in the density of adherent platelets onto the coated graft as compared to the uncoated material .

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  • Chemical & Material Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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Abstract

Selon cette invention, on applique sur la surface de biomatériaux implantables, destinés à être mis en contact avec le sang ou des composants sanguins contenant des plaquettes, une protéine de recombinaison adhésive, ayant une activité de liaison plaquettaire, telle qu'une vitronectine, cette protéine étant mutée au niveau du site de liaison du récepteur des plaquettes. Les biomatériaux obtenus présentent une thrombose réduite du fait de l'adhésion et de l'agrégation plaquettaire.
PCT/US1999/005967 1998-03-18 1999-03-18 Biomateriaux implantables ameliores, compositions et leurs procedes de preparation et d'utilisation Ceased WO1999047070A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU31922/99A AU3192299A (en) 1998-03-18 1999-03-18 Improved implantable biomaterials, compositions and methods for their preparation and uses thereof

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US7839898P 1998-03-18 1998-03-18
US60/078,398 1998-03-18

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024219A1 (fr) 2000-09-22 2002-03-28 Queensland University Of Technology Complexe du facteur de croissance
WO2005012508A1 (fr) 2003-07-28 2005-02-10 Queensland University Of Technology Systeme de regeneration de la peau
WO2011063477A1 (fr) 2009-11-30 2011-06-03 Queensland University Of Technology Chimères de fibronectine/facteur de croissance
EP2357194A1 (fr) 2003-02-05 2011-08-17 Queensland University Of Technology Complexe de facteur de croissance et modulation de la migration et de la croissance cellulaire
WO2013134822A1 (fr) 2012-03-13 2013-09-19 James Cook University Procédé de traitement de l'inflammation
WO2015039188A1 (fr) 2013-09-18 2015-03-26 James Cook University Protéines inflammatoires et procédés d'utilisation
US9090706B2 (en) 2003-02-05 2015-07-28 Queensland University Of Technology Fibronectin: growth factor chimeras
EP4509188A2 (fr) 2013-09-18 2025-02-19 James Cook University Proteines anti-inflammatoires modifiees et procede d'utilisation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5492890A (en) * 1990-12-03 1996-02-20 The Scripps Research Institute Polypeptides for promoting cell attachment
US5756452A (en) * 1991-04-05 1998-05-26 Genentech, Inc. Platelet aggregation inhibitors having high specificity for GP IIb IIIa
US5786344A (en) * 1994-07-05 1998-07-28 Arch Development Corporation Camptothecin drug combinations and methods with reduced side effects

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5492890A (en) * 1990-12-03 1996-02-20 The Scripps Research Institute Polypeptides for promoting cell attachment
US5756452A (en) * 1991-04-05 1998-05-26 Genentech, Inc. Platelet aggregation inhibitors having high specificity for GP IIb IIIa
US5786344A (en) * 1994-07-05 1998-07-28 Arch Development Corporation Camptothecin drug combinations and methods with reduced side effects

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024219A1 (fr) 2000-09-22 2002-03-28 Queensland University Of Technology Complexe du facteur de croissance
EP2385064A2 (fr) 2000-09-22 2011-11-09 Queensland University of Technology Agents qui séparent un complexe du facteur de croissance
EP2385063A2 (fr) 2000-09-22 2011-11-09 Queensland University of Technology Complexe d'un facteur de croissance
EP2357194A1 (fr) 2003-02-05 2011-08-17 Queensland University Of Technology Complexe de facteur de croissance et modulation de la migration et de la croissance cellulaire
EP2357195A1 (fr) 2003-02-05 2011-08-17 Queensland University Of Technology Complexe de facteur de croissance et modulation de la migration et de la croissance cellulaire
US9090706B2 (en) 2003-02-05 2015-07-28 Queensland University Of Technology Fibronectin: growth factor chimeras
WO2005012508A1 (fr) 2003-07-28 2005-02-10 Queensland University Of Technology Systeme de regeneration de la peau
WO2011063477A1 (fr) 2009-11-30 2011-06-03 Queensland University Of Technology Chimères de fibronectine/facteur de croissance
WO2013134822A1 (fr) 2012-03-13 2013-09-19 James Cook University Procédé de traitement de l'inflammation
WO2015039188A1 (fr) 2013-09-18 2015-03-26 James Cook University Protéines inflammatoires et procédés d'utilisation
EP4509188A2 (fr) 2013-09-18 2025-02-19 James Cook University Proteines anti-inflammatoires modifiees et procede d'utilisation

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Publication number Publication date
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