EP0983096A2 - Polymeres du type collagene a activite de liaison cellulaire - Google Patents

Polymeres du type collagene a activite de liaison cellulaire

Info

Publication number
EP0983096A2
EP0983096A2 EP98928267A EP98928267A EP0983096A2 EP 0983096 A2 EP0983096 A2 EP 0983096A2 EP 98928267 A EP98928267 A EP 98928267A EP 98928267 A EP98928267 A EP 98928267A EP 0983096 A2 EP0983096 A2 EP 0983096A2
Authority
EP
European Patent Office
Prior art keywords
collagen
polymer
polymer compounds
bulk material
compounds
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98928267A
Other languages
German (de)
English (en)
Inventor
John Gerard Steele
Graham Johnson
Murray Goodman
Hans Jörg GRIESSER
Keith Michael Mclean
Gerrit Jan Beumer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Biocure Inc
Original Assignee
Commonwealth Scientific and Industrial Research Organization CSIRO
Novartis AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AUPO6862A external-priority patent/AUPO686297A0/en
Priority claimed from AUPO6842A external-priority patent/AUPO684297A0/en
Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO, Novartis AG filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Publication of EP0983096A2 publication Critical patent/EP0983096A2/fr
Withdrawn legal-status Critical Current

Links

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
    • 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
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • 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
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
    • 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

Definitions

  • the present invention relates to "collagen-like polymers" which exhibit cell binding activity, to bulk materials having a coating of the polymers applied thereto and to methods of covalently binding the polymers to bulk materials.
  • the present invention relates to implants such as wound repair materials, implanted contact lenses, synthetic epikeratopiasties, synthetic skin or connective tissue, orthopaedic implants, prosthetic joints, synthetic arterial surfaces, synthetic neural tissue, prosthetic organs or other synthetic bioactive apparatus or materials which comprise the collagen-like polymers.
  • the present invention relates to methods of treatment involving the implants which comprise the cell binding collagen-like polymers.
  • collagen relates to a family of native proteins present in the extracellular matrix, and particularly connective tissues, ligaments, tendons and bones of animals including humans.
  • Native collagen has a primary structure of repeating trimeric amino acid sequences. Within a helical region, which constitutes about 95% of collagen molecules, the amino acid glycine (Gly) occurs at every third position of a peptide trimer. Imino residues (I), either proiine (Pro) or hydroxyproline (Hyp), occur in 56% of the trimers, 20% as Gly-X-I; 27% as Gly-I-Y; and 9% as Gly-I-I (where X and Y are amino acid residues other than Pro or Hyp).
  • Tripeptide sequences (Gly-X-Y) wherein X and Y are amino acid residues other than proiine (Pro) or hydroxyproline (Hyp) make up 44% of the collagen amino acid trimers.
  • Glutamic acid, leucine and phenylaianine occur mostly in the X position and threonine, glutamine, methionine, arginine and lysine occur mostly at the Y position.
  • glutamine, methionine, arginine and lysine occur mostly at the Y position.
  • the X position amino acids have bulky side chains.
  • synthetic polymers which have collagen-like properties which can be used within biomaterials for clinical applications.
  • collagen-like synthetic polymers For example use of collagen-like synthetic polymers has been contemplated in drug delivery devices, occular devices and wound healing materials. With these goals in mind, many synthetic peptides composed of the trimeric amino acid sequences Gly-Pro-Xaa and Gly-Xaa-Pro (where Xaa is any natural amino acid residue) have been prepared to mimic the collagen structures.
  • Native collagens have a characteristic tertiary, secondary and primary structure and are made up of three polypeptide chains comprising repeating amino acid trimers. These chains are arranged in three extended left-handed spirals of about 3 residues per turn: the polyproline ll-like chains, as described in Ridge (1955) Nature 176: 915, which is also included herein by way of reference, in its entirety.
  • the polyproline ll-like chains are arranged in a parallel direction and intertwined to adopt a supercoiled, right-handed triple helix conformation (Bella et al (1994) Science 266: 75-81).
  • this triple helical conformation can be effectively mimicked, as collagen-like activity is dependent upon this conformation.
  • Implants for human and animal bodies are currently being developed at a dramatic rate.
  • implants may vary from a simple synthetic skin for treating burn, wound or aesthetic surgery patients, to synthetic ligaments, articular surfaces or prosthetic joints for orthopaedic patients.
  • bioactive apparatus such as artificial organs which will fulfil the function of the heart or lungs etc.
  • Materials which assist the repair of wounds, implantabie contact lenses and synthetic epikeratopiasties, as well as other synthetic connective tissues, orthopaedic implants, synthetic arterial surfaces, synthetic neural tissues or other synthetic bioactive apparatus or materials are also being increasingly implanted into human or animal patients.
  • implanted materials which are generally manufactured from metals or metal alloys, plastics polymers or ceramics will not encourage cell binding and tissue colonisation.
  • peptoid residue containing polymers are not as susceptible to enzymatic attack, which can adversely affect the activity of natural peptides when used to stimulate cell binding and tissue colonisation.
  • the present inventors have deiermined that some collagen-like polymer compounds which comprise peptoid residues are capable of initiating cell binding and tissue colonisation.
  • the present inventors have identified that collagen-like polymers comprising at least nine adjacent repeats of the sequence Gly-Pro-Nleu have the ability to initiate cell binding and tissue colonisation.
  • Gly represents the amino acid glycine
  • Pro represents the amino acid proiine
  • Nleu represents the peptoid residue N- isobutylglycine, as described in Simon et al Proc. Natl. Acad. Sci. U.S.A. 1992, 89(20), 9367-9371. which is included herein by reference in its entirety.
  • the present inventors have also determined means by which the collagen-like polymers according to the invention can be bound to implantabie bulk materials, namely by covalent binding of terminal primary amine groups on the collagen-like polymer compounds with either aldehyde or carboxyl groups on a surface or surfaces of the implantabie bulk material.
  • Such covalent bonds are in the form of either Schiff base, amine or amide linkages.
  • the latter two linkages are preferred as they enable irreversible immobilisation of the collagen-like polymers with respect to the bulk material.
  • collagen-like polymer compounds capable of initiating cellular attachment thereto, said polymer compounds comprising one or more peptoid residues.
  • collagen-like polymer compounds capable of initiating cellular attachment thereto, said polymer compounds comprising the sequence of formula I, wherein (y) denotes ⁇ 9: -(Giy-Pro-Nleu) (y) - (I).
  • an implantabie bulk material comprising one or more collagen-like polymer compounds as described above, covIERly bound to a surface or surfaces thereof.
  • biopolymer compounds comprise Kemp triacid adducts of biopolymer compounds comprising one or more peptoid residues, the method comprising the steps of:
  • a method of binding collagen-like polymer compounds to an implantabie bulk material wherein said poiymer compounds are capable of initiating celiular attachment, which polymer compounds comprise the sequence of formuia I, as well as primary amine groups, the method comprising the steps of: (a) providing aldehyde or carboxyl groups on a surface or surfaces of said implantabie bulk material;
  • implantabie bulk materials comprising a copolymer which includes collagen-like polymer compounds that comprise one or more peptoid residues.
  • implarilable bulk materials comprising a copolymer which includes collagen-like polymer compounds that comprise the sequence of formula I.
  • a method of treatment of a human or animal patient requiring an implantation which comprises obtaining an appropriate implantabie bulk material as described above and implanting said implantabie bulk material into said patient.
  • the essential feature of the polymer compounds is that they are collagen-like, exhibit cellular binding initiation characteristics and include one or more peptoid units.
  • the collagen-like polymer compounds according to the present invention include at least nine consecutive repeats of the sequence "-(Gly-Pro-Nleu)-" wherein Giy represents glycine, Pro represents proiine and Nleu represents the peptoid residue N-isobutylglycine as disclosed in the Simon et al paper referred to above.
  • a feaiure of polymers according to the invention is that they exhibit the triple helical tertiary conformation analogous to that of naturally occurring collagen proteins.
  • the polymer compounds according to the present invention are considered to be "collagen-like".
  • Collagen- ⁇ ke polymers containing peptoid residues are disclosed in WO 97/19106. The disclosure of these specifications is included herein in its entirety by way of reference.
  • the collagen-like polymer compounds according to the present invention may additionally include one or more or many other chemical constituents.
  • other naturally occurring or non-naturally occurring amino or imino acid groups or other synthetic polymers eg. hydrogel constituents
  • the collagen-like polymer compounds are terminated by an amine group or by the Kemp triacid (KTA) (cis,cis-1 ,3,5-trimethylcyclohexane-1 ,3,5-tricarboxylic acid, as referred to in Kemp and Petrakis J. Org. Chem. 1981 , 46, 5140-5143, which is included herein in its entirety by way of reference).
  • KTA Kemp triacid
  • the collagen-like polymer compounds according to the present invention By the term “capable of initiating cellular attachment”, it is intended to convey that the collagen-like polymer compounds according to the present invention, and impiantable bulk materials having the collagen-like polymer compounds bound thereto, will encourage cellular binding and tissue colonisation thereto when located in an environment containing human or animal cells and/or tissues. It is also specifically envisaged that the collagen-like polymer compounds according to the present invention may include an extra terminal primary amine group, which can be utilised for the purposes of binding these collagen-like polymer compounds to materials to be used for implantation purposes. As a result, what can take place is that a biomateria! surface which comprises the collagen-like polymer compounds according to the present invention can be applied to an implantabie bulk material in order that the implantabie material will be appropriately established within the human or animal organism by attachment of cells and tissues thereto.
  • the Kemp triacid adduct of three collagen-like polymer compounds of suitable composition and length is particularly advantageous because when the Kemp triacid is condensed with glycine or alanine it conveniently acts as a template for inducing ⁇ -helicity of an attached polymer (Muller et al., 1993, Chapter 33 in Perspectives in Medicinal Chemistry, Testa et al. Editors, Basel).
  • peptoid residues are a relatively new class of unnatural imino acids which comprise N-substituted glycine residues, wherein the substituents on the nitrogen atom are the ⁇ -positioned side chains of amino acids.
  • peptoid residues do not occur in nature, peptoid residues and the collagen-like polymers comprising them have improved resistance to enzymatic attack relative to naturally occurring amino acids and peptides.
  • the present invention includes within iis scope collagen-like polymer compounds which include one or more of the same or different peptoid residues. Collagen-like polymer compounds that include any of the peptoid residues are comprehended by the invention, as long as they exhibit the requisiie cellular binding.
  • the impiantable bulk materials are many and varied and include the following which are listed here by way of example: wound repair materials, synthetic skin or connective tissue, ocular implants such as implanted contact lenses and synthetic epikeratoplasties or corneal grafts, orthopaedic implants such as prosthetic joints or synthetic arterial surfaces, synthetic tendon or ligament tissues or materials used to secure bone or ligament in surgical procedures, synthetic neural tissue, prosthetic organs such as apparatus which will carry out the function of the heart, lungs, etc, components of blood contacting devices, immunoassays, antigen/antibody detection kits, affinity matrices etc., other synthetic bioactive apparatus such as heart pacemakers or other synthetic implantabie materials.
  • wound repair materials synthetic skin or connective tissue
  • ocular implants such as implanted contact lenses and synthetic epikeratoplasties or corneal grafts
  • orthopaedic implants such as prosthetic joints or synthetic arterial surfaces
  • synthetic tendon or ligament tissues or materials used to secure bone or ligament in surgical procedures synthetic neural tissue
  • prosthetic organs such as
  • Another important aspect of the present invention relates to the means by which the collagen-like polymers according to the present invention can be bound to impiantable bulk materials.
  • this binding relates to covalent binding which will result in a permanent interaction between the collagen-like polymer compounds and the impiantable bulk material. That is, whereas polymers or peptides simply physisorbed to an impiantable bulk material would be likely to undergo exchange with other biomolecules within a cellular or tissue system, covendingly bound polymer compounds will not be so affected.
  • active groups on both of these entities it is necessary for active groups on both of these entities to be either inserted or exploited.
  • active groups for the purposes of binding to impiantable bulk materials will be present at or near a terminus of the collagen-like polymer compound. In this way it is less likely that binding of the collagen-like polymer compound to the implantabie bulk material will adversely affect the confirmation of the collagen-like polymer compound.
  • a mechanically and optionally optically suitable bulk material does not inherently contain on its surface chemical groups capable of reacting with reactive groups on the collagen-like polymer compounds, then a range of suitable chemical groups can be introduced into the surface of the bulk material by application of surface modification techniques known in the art.
  • surface amine groups can be provided by a plasma polymerization treatment using alkylamine vapours (Griesser and Chatelier, Journal of Applied Poiymer Science, Vol. 48, pp 361 -384, 1990).
  • Particularly useful examples of chemical linkage between the collagen-like polymer compounds and the impiantable bulk materials involve the exploitation of terminal primary amine groups on the collagen-like polymer compound which can be reacted with either aldehyde or carboxyl groups on a surface or surfaces of the impiantable bulk material. Binding between terminal amine groups and surface aldehyde groups can be used to result in a Schiff base linkage whereas binding between surface carboxyl groups and terminal amine groups can be used to form an amide linkage between the collagen-like polymer and the implantabie bulk materials.
  • Schiff base linkages are of a reversible nature and it may therefore be advantageous for them to be reduced to amine linkages, by treatment with an appropriate reducing agent such as, for example, cyanoborohydride.
  • collagen-like polymers according to the invention it is also possible for collagen-like polymers according to the invention to be copolymerised with other units in order to produce impiantable bulk materials. Naturally, it would be necessary for these other units to be carefully chosen on the basis of properties which would make them suitable for use in the particular bulk material concerned. For example, in the case of impiantable contact lenses the units chosen to be combined with the collagenlike polymers of the invention would need to exhibit satisfactory stability, permeability, strength, flexibility and optical charactistics for ocular implantation.
  • the seeding culture medium was then replaced with fresh culture media containing serial dilutions of each of the collagen-like polymers, from a final concentration of 1.0 mg/ml down to zero.
  • the cells were incubated at 37°C for forty hours in the collagen-like poiymer-containing culture media.
  • the culture media were then removed and MTS solution added to each well and incubated for a further four hours at 37°C to determine the viability of the attached cells.
  • the relative numbers of cells per well was determined colorimetrically on an ELISA plate reader at an absorbance wavelength of 490 nm. The results are shown in Tables 1 (a)-(i).
  • Tables 1 (a)-(i) show assays of attachment of bovine stromal fibroblasts seeded onto tissue culture polystyrene, then exposed to polymers #1 -4 and 95120-95124, after four hours incubation. Polymer concentration (mg/ml) is plotted against absorbance at 490nm.
  • the synthetic collagen-like polymer sequences were assayed for a specific binding reaction at the cell surface, consistent with the specific sequence having cell-binding activity.
  • This assay involves using the collagen-like polymer in solution and adding the collagen-like polymer at the time of inoculating the cells, to determine whether the collagen-like polymer acts as an inhibitor of the initial adhesion of cells to the culture substratum. If a collagen-like polymer has a cell-binding activity that enables it to effectively compete for cell membrane receptor sites then the collagen-like polymer may inhibit cell attachment, and so a reduced cell attachment is evidence of that collagen-like polymer having cell-binding activity.
  • the inhibitory effect was reduced at collagen-like polymer concentrations iower that 0.5 mg/ml.
  • the inhibitory effect was specific to this sequence as other collagen-like polymers tested which contained long or short chains of the alternative sequence -(Gly-Pro-Hyp) n -did not reduce cell attachment.
  • none of the synthetic collagen-like polymers tested interfered with normal metabolic processes (see Section b above) but two of them demonstrated a cell- binding activity (based on their ability to interfere with the attachment of bovine corneal fibroblasts to a TCPS cell culture substrate) when present at collagen-like polymer concentrations > 0.25 mg/ml; these two collagen-like polymers containing the sequence
  • Tables 2(a)-(i) show assays of attachment of bovine stromal fibroblasts seeded onto tissue culture polystyrene in the presence of polymers #1 -4 and 95120-95124. Polymer concentration (mg/ml) is plotted against absorbance at 490nm.
  • the number of adherent cells was determined colorimetrically using an MTS-based method and then measuring absorbance at 490 nm on a plate reader.
  • Corneal epithelial cell attachment was reduced by 85% in medium containing 1.0 mg/ml of Polymer #95121 (single chain with the sequence Ac-(Gly-Pro-Nleu) 9 -NH 2 -) as compared to serum-free medium alone. Similarly, this collagen-like polymer reduced by 50% the attachment of corneal epithelial cells to TCPS, when cultured in medium containing 20% (v/v) depleted serum (Table 3(b)). Serum-free medium containing 0.5 mg/ml of polymer #95121 reduced cell attachment by 20%.
  • Polymer #95122 comprised of a KTA template linked to three [Gly-(Gly-Pro-Nleu) 9 -NH 2 ] chains, was also found to inhibit attachment of corneal epithelial cells, when employed in serum-free culture medium and at the highest concentrations.
  • Serum-free culture medium containing 1 .0 mg/ml of poiymer #95122 reduced corneal epithelial cell attachment by 27%, whilst a collagen-like polymer concentration of 0.5 mg/ml resulted in a 19% reduction. The effect was diminished at the lower concentrations (Table 3(c)).
  • Poiymer #95106 containing the single sequence Ac-Gly-Pro-Nleu-NH 2 , was included as a short sequence control for the other two collagen-like polymers. There was no inhibition of cell attachment at any concentration of this collagen-like polymer, in either culture medium (Table 3(a)).
  • Tables 3(a)-(c) show assays of attachment of bovine corneal epithelial cells to tissue culture polystyrene after seeding in the presence of Ac-Gly-Pro-Nleu-NH 2 (a), Ac-(Gly-Pro-Nleu) 10 -NH 2 (b) and KTA-[Gly-(Gly-Pro-Nleu) 9 -NH 2 ] 3 .
  • Polymer concentration (mg/ml) is plotted against absorbance at 490nm.
  • Gly-Arg-Gly-Asp-Ser-Pro-Cys (RGDSPC, cat #PO11), obtained from Telios Pharmaceuticals, and ii) Gly-Arg-Gly-Asp-Ser-Pro-Lys (RGDSPK, cat#12145-017), obtained from Life Technologies Inc.
  • Corneal epithelial cell attachment to tissue culture polystyrene (TCPS) was not inhibited by the presence of any of the -(Gly-Nleu-Pro) n - collagen-like polymers (Tables 4(a)-(c)).
  • Corneal epithelial cell attachment was not inhibited by either short sequence repeats (Ac-(G!y-Nleu-Pro) 3 -NH 2 , polymer #96127, Table 4(a), or longer ones (Ac-(Gly-Nleu-Pro) 10 - NH 2 (Bioresearch commercia! version), Table 4(b), either under serum-free or serum- containing conditions.
  • Tables 4(a)-(c) show assays of attachment of bovine cornea! epithelial cells to tissue culture polystyrene after seeding in the presence of Ac-(Gly-Nleu-Pro) 3 -NH 2 (a), Ac-(Gly-Nleu-Pro) 10 -NH2 (b) and Gly-Arg-Gly-Asp-Ser-Pro-Lys. Polymer concentration (mg/ml) is plotted against absorbance at 595nm.
  • Tables 5(a)-(c) show assays of attachment of bovine corneal epithelial cells to tissue culture polystyrene after seeding in the presence of H-(Gly-Pro-Nleu) 3 -NH 2 (a), H-(Gly-Pro-Nleu) 5 -NH 2 (b) and Gly-Arg-Gly-Asp-Ser-Pro-Lys. Polymer concentration (mg/ml) is plotted against absorbance at 595nm.
  • n is up to ten repeats long, have no effect on corneal epithelial cell attachment in vitro.
  • Collagen-like polymers or the Kemp triacid adduct of collagen-like polymers can be covalently immobilized permanently onto synthetic surfaces by a number of covalent interfacial linking strategies which are known in the art. For instance, it is practical to utilize collagen-like poiymer molecules that have been equipped with a terminal primary amine group (Structure 1),
  • Structure 1 Structure of amine-endfu ⁇ ctionalized collagen-like poiymer molecule.
  • amine-functionalized collagen-like polymer molecules can be immobilized by a number of interfacial covalent reaction schemes onto surfaces that possess various chemical groups capable of reacting with primary amine groups.
  • surfaces that can covalently immobilize amine-endfunctionalized collagen-like polymers comprise:
  • such aldehyde groups can be provided using established surface modification techniques, e.g., reacting surface hydroxyl groups with glutaraldehyde (Gotoh et al., Journal of Membrane Science, 41 , 1989, 291 -303 ; Zhuang and Butterfield, Journal of Membrane Science, 66, 1992, 247-257), or providing surface aldehyde groups by gas plasma methods.
  • this linkage is then optionally stabilized with sodium cyanoborohydride, to result in a -NH-CH 2 - linkage, to confer better resistance to slow, thermally induced reversion back to amine and aldehyde groups.
  • a mechanically and optionally optically suitable implantabie bulk material is coated with a thin plasma polymer containing amine groups (Griesser and Chatelier, Journal of Applied Poiymer Science: Applied Polymer Symposium, 46, 1990, 361 -384); intermediate reagent molecules containing at least two carboxylic acid groups, or at least two functional groups capable of forming carboxylic acid groups upon e.g.
  • interfacial linking reaction schemes for covalently attaching the Kemp triacid adduct can be designed on the basis of general knowledge of established chemical reactions.
  • the KTA adduct can form amide linkages with an alkylamine plasma modified surface or a bisamino-PEG spacer attached to an aldehyde containing surface.
  • Such linkage reaction schemes between collagen-like polymer and the bulk material have been illustrated in Figures 1 to 8 of the priority document AU 6842/97 of the present invention, the disclosure of said Figures being incorporated herein by reference.
  • This section describes the procedures used for coupling the synthetic collagen-like polymer sequences onto a surface that does not itself support cell colonisation: fluorinated ethylene propylene, FEP.
  • Surface analyses results that demonstrate that the collagen-like polymers were immobilised onto the surface are described.
  • Biological assessment of the surface containing the immobilised synthetic co ⁇ age ⁇ -like polymer sequences was performed by measuring the attachment and growth of bovine corneal epithelial cells on the surfaces over a seven days period.
  • Acetaldehyde plasma poiymer AApp or heptylamine plasma polymer (Happ), deposited on FEP, were used to achieve linkage of synthetic collagen-like polymer sequences through the presence of the free amine group on the collagen-like polymer molecule.
  • Acetaldehyde Plasma Polymer Surface activation was undertaken by exposing the FEP to acetaldehyde monomer in a RF plasma. The process results in the formation of aldehyde groups on the surface which can then react to form a collagen-like polymer-surface linkage. Sodium cyanoboro hydride (NaCNBH 3 ), in excess, was used as a reducing agent.
  • NaCNBH 3 Sodium cyanoboro hydride
  • Heptylamine Plasma Polymer Surface activation using heptylamine process vapour in a RF plasma produces amine groups on the surface, which are then succinylated to generate carboxyl groups on the surface. The amine containing collagen-like poiymer is coupled to the carboxyl groups using EDC/NHS.
  • amines on the heptylamine plasma polymer treated FEP surface are exposed to starburst dendrimers as intermediates in the collagen-like polymer coupling methodology.
  • the multiple (32) carboxylic acid groups on each dendrimer molecule provide a higher surface density of carboxylic acid groups than the succinylation approach and provide a high degree of in-built redundancy.
  • Acetaldehyde Plasma Polymer deposition Fluorinated ethylene propylene (FEP) Teflon 100 Type A DuPont (12.7mm in width), was used as received and plasma polymer deposited in a custom-built plasma apparatus, employing a Javac DDL 300 rotary high vacuum pump and an ENI HPG-2 High Frequency plasma generator. Acetaldehyde monomer was used as purchased from Aldrich, 99%, cat# 11 ,007-8, Mw 44.05).
  • P b 0.015 Torr
  • P m 0.130 Torr
  • P 20 Watts
  • F 200 kHz
  • T 20 sec.
  • To succinylate the HApp surface freshly prepared samples ( ⁇ 2 hours old) were placed in 25ml of a 10% (v/v) solution of succinyl chloride in acetone for 1 hour, rinsed twice in acetone then soaked in acetone for 1 hour. The samples were then rinsed two times in water and soaked for 1 hour in MilliQ water.
  • Collagen-like polymer coating solution was prepared by dissolving synthetic collagen-like polymer sequence in sterile phosphate buffered saline (PBS), pH 7.4, to either 50 ⁇ g/ml end concentration for acetaldehyde linkage; or in sterile MilliQ water for heptylamine linkage (phosphate interferes with the EDC/NHS reaction).
  • the polymer #96123 was used as a model polymer for attachment studies. FEP samples with freshly prepared AApp surfaces ( ⁇ 2 hrs old) were left to react with this coating solution (overnight at 4°C and subsequently 2 hr at room temperature). Excess NaCNBH 3 (Sigma, 90%, Cat# S-8628, lot# 33H3669, Mw 62.84) was added. Thereafter samples were washed (3 x 5 minutes) in sterile MilliQ water and air-dried prior to analysis by XPS.
  • samples were prepared with and without the use of NaCNBH 3 or EDC/NHS and analysed (XPS) before and after autoclaving (15 minutes, 120 PSI). Samples were prepared and are identified under the following codes:
  • the collagen (Vitrogenl OO, Collagen Corp, Palo Alto, CA, USA) coupled surfaces were included as positive controls in the cell colonization assay as this combination has been previously shown to support good BCEp cell attachment and growth.
  • XPS X-ray photoelectron spectroscopy
  • BCEp Bovine corneal epithelial
  • the culture medium contained 20 % (v/v) foetal bovine serum (FBS) depleted of the cell adhesive glycoproteins, fibronectin and vitronectin (Underwood and Bennett, 1989). Removal of fibronectin and vitronectin from the serum ensured that exogenous cell attachment factors would not play a significant role in the assay, or mask any effects contributed by the coupled collagen-like polymers.
  • FBS foetal bovine serum
  • Acetaldehyde Plasma XPS analysis (Table A) of acetaldehyde plasma polymers on FEP showed the absence of a fluorine signal, indicating that the Aapp film coating was at least 10-15nm thick (sampling depth of XPS).
  • the incorporation of oxygen and carbonyls is a further indication of surface activation by AApp deposition.
  • a solution containing the polymer # 96123 50 ⁇ g/ml
  • incorporation of nitrogen indicative of the polymer was detected. This incorporation occurs in both the presence and absence of the NaCNBH 3 from the coating solution.
  • coatings with and without the NaCNBH 3 were autoclaved.
  • Table A Results of XPS analysis of attachment of polymer 96123 to acetaldehyde plasma polymer.
  • Heptylamine Plasma Following heptylamine plasma polymer treatment, the fluorine signal from the FEP was not detected and nitrogen was detected, indicative of the presence of the heptylamine plasma polymer with a thickness >10nm. Following succinylation and incubation of HA coated FEP in a solution of polymer # 96123, an increase in the nitrogen signal is observed both in the presence and absence of EDC/NHS (Table B). Following autociaving, nitrogen signal is lost from surfaces, both plus and minus EDC/NHS, (removal of bound polymer) but a higher nitrogen signal is retained in the presence of EDC/NHS.
  • Table B Results of XPS analysis of attachment of poiymer 96123 to heptylamine plasma polymer.
  • BCEp Celi Growth on Immobilised Collagen-like polymers BCEp cells were seeded onto plasma-modified FEP surfaces that had covalently coupled collagen-like polymers attached. The cells were cultured for seven days and relative numbers determined by either metabolically labelling the cells at day six with 35 S-methionine and measuring the amount of incorporated 3o S, or by colorimetrically measuring the amount of formazan produced by the cells after exposure to MTT tetrazolium salt (Promega cell proliferation assay). Table 6 shows that the surfaces containing the covalently attached synthetic collagen-like polymer sequences supported cell colonisation at levels similar to that of the positive control surface, tissue culture polystyrene (TCPS). Based on these results, immobilization of collagen-like polymers onto an FEP surface through a strategy that involves activation of the FEP surface through deposition of a plasma film does produce a surface that provides enhanced BCEp cell attachment.
  • TCPS tissue culture polystyrene
  • Example 4 Corneal epithelial ceil attachment and growth on covalently immobilised -Gly-Pro-Nleu- and -Gly-Nieu-Pro- sequences
  • the ability of the collagen-like peptoid sequences to support the attachment and growth of cells, when these sequences were covalently attached to a surface was tested as follows. Disaggregated bovine corneal epithelial cells (at cell culture passage 2) were seeded onto a series of surfaces. These surfaces were constructed on top of fluoroethylene propylene (FEP) film, by the deposition of a crossiinked polymer (by RF plasma treatment using heptylamine, HApp, monomer during the plasma treatment).
  • FEP fluoroethylene propylene
  • CMD carboxymethyldextran
  • GPNI GPNI
  • Other surfaces included the following control surfaces: bovine collagen type I, bovine plasma fibronectin, polylysine, bovine serum albumin.
  • the intermediate surface construct, the CMD alone surface, was also included as a control.
  • a tissue culture poiystyrene (TCPS) surface was used as a positive control surface, as this surface supports the attachment and growth of cells.
  • the corneal epithelial cells were cultured in a culture medium supplemented with 20% (v/v) of a treated foetal calf serum.
  • This serum had previously been depleted of fibronectin and vitronectin, which are cell attachment giycoproteins.
  • This serum that does not in itself support cell attachmentand so any cell attachment activity that is observed in the test can be ascribed to an activity of the material surface itself with the cells.
  • the culture medium was replenished every second day, and so any unattached cells were removed at days 2, 4 and 6 of culture. After seven days culture, the number of cells attached to the various surfaces was measured using a formazan-dye based assay (using MTT). Cell numbers were expressed as a mean percentage ( ⁇ standard deviation of the mean) of that number found attached to the TCPS control surface, which was set as 100%.
  • the CMD alone surface provided minimal (4 ⁇ 1 %) support of cell attachment and growth.
  • the control surface that contained coupled bovine serum albumin or coupled polylysine supported only a low level of activity (22 ⁇ 6% for the albumin surface; 27 ⁇ 12% for the polylysine surface).
  • the surface that contained coupled fibronectin supported effective cell attachment and growth (71 ⁇ 14%) as expected, and the coupled collagen I surface supported cell attachment and growth (61 ⁇ 3%).
  • the immobilised collagen-like peptoid sequence H-(Gly-Pro-Nleu) ⁇ 0 -Gly-Pro-NH 2 very effectively supported the attachment and growth of corneal epithelial cells, at the level of 52 ⁇ 4 % of that found on the TCPS control surface.

Abstract

L'invention concerne essentiellement une matière biocompatible comprenant une matière en vrac qui a lié par covalence à une ou plusieurs de ses surfaces un ou plusieurs composés biopolymères, lesdits composés biopolymères comprenant au moins un reste peptoïde. Les composés biopolymères sont de préférence des composés polymères comprenant la séquence de la formule (I): -(Gly-Pro-Nleu)(y)-, dans laquelle (y) à la signification ≥ 9. Un procédé de liaison de composés biopolymères à une matière en vrac fait aussi l'objet de cette invention. Lesdits composés biopolymères comprennent au moins un reste peptoïde, ainsi que des groupes amino primaires. Le procédé consiste (a) à placer des groupes aldéhyde ou carboxyle sur une ou plusieurs surfaces de ladite matière en vrac; (b) à faire réagir lesdits groupes amino primaires avec des groupes aldéhyde ou carboxyle pour former des liaisons covalentes.
EP98928267A 1997-05-16 1998-05-14 Polymeres du type collagene a activite de liaison cellulaire Withdrawn EP0983096A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
AUPO684297 1997-05-16
AUPO6862A AUPO686297A0 (en) 1997-05-16 1997-05-16 Collagen-like polymers with cell binding activity
AUPO686297 1997-05-16
AUPO6842A AUPO684297A0 (en) 1997-05-16 1997-05-16 Biocompatible materials
PCT/EP1998/002857 WO1998052620A2 (fr) 1997-05-16 1998-05-14 Polymeres du type collagene a activite de liaison cellulaire

Publications (1)

Publication Number Publication Date
EP0983096A2 true EP0983096A2 (fr) 2000-03-08

Family

ID=25645423

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98928267A Withdrawn EP0983096A2 (fr) 1997-05-16 1998-05-14 Polymeres du type collagene a activite de liaison cellulaire

Country Status (5)

Country Link
EP (1) EP0983096A2 (fr)
JP (1) JP2001527451A (fr)
AU (1) AU8017498A (fr)
CA (1) CA2286687A1 (fr)
WO (1) WO1998052620A2 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR021240A1 (es) * 1998-11-13 2002-07-03 Commw Scient Ind Res Org Peptidos acoplados
JP2004501784A (ja) * 2000-06-23 2004-01-22 バイオイラスティックス　リサーチ，リミティド バイオエラストマーのナノマシンとバイオセンサー
DE60232880D1 (de) 2001-05-24 2009-08-20 Neuren Pharmaceuticals Ltd Gpe-analoga und peptidomimetika
US7605177B2 (en) 2001-05-24 2009-10-20 Neuren Pharmaceuticals Limited Effects of glycyl-2 methyl prolyl glutamate on neurodegeneration
US7714020B2 (en) 2001-05-24 2010-05-11 Neuren Pharmaceuticals Limited Treatment of non-convulsive seizures in brain injury using G-2-methyl-prolyl glutamate
US20030045942A1 (en) * 2001-09-05 2003-03-06 Lai Wen-Fu T. Regenerative bone implants
EP1364663A1 (fr) * 2002-05-21 2003-11-26 Commonwealth Scientific And Industrial Research Organisation Dispositifs oculaires avec surface fonctionalisée conférant des propriétés adhésives
JP4705319B2 (ja) * 2002-07-19 2011-06-22 三洋化成工業株式会社 創傷被覆材
EP2183280B1 (fr) 2007-08-01 2011-10-26 Ethicon, Inc. Peptides associés à du collagène et leurs utilisations
JP5388090B2 (ja) * 2008-05-29 2014-01-15 国立大学法人東北大学 強膜透明化による角膜移植材料調製方法
US20100021527A1 (en) 2008-07-25 2010-01-28 Chunlin Yang Collagen-related peptides and uses thereof and hemostatic foam substrates

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3241589A1 (de) * 1982-11-10 1984-05-17 Pfaudler-Werke Ag, 6830 Schwetzingen Implantate und verfahren zu deren herstellung
DE3788062D1 (de) * 1987-05-12 1993-12-09 Christian Dr Med Mittermayer Verfahren zur Besiedlung einer Polymeroberfläche mit menschlichen Gefässinnenhautzellen.
US5354736A (en) * 1989-08-14 1994-10-11 Regents Of The University Of California Synthetic compounds and compositions with enhanced cell binding
ATE180681T1 (de) * 1991-03-29 1999-06-15 Vascular Graft Research Center Künstliches blutgefäss aus komposit-material
US6096710A (en) * 1995-11-17 2000-08-01 The Regents Of The University Of California Collagen-like peptoid residue-containing structures

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9852620A3 *

Also Published As

Publication number Publication date
JP2001527451A (ja) 2001-12-25
AU8017498A (en) 1998-12-11
CA2286687A1 (fr) 1998-11-26
WO1998052620A2 (fr) 1998-11-26
WO1998052620A3 (fr) 1999-02-25

Similar Documents

Publication Publication Date Title
US10265436B2 (en) Immobilized bioactive hydrogel matrices as surface coatings
EP0494216B1 (fr) Surfaces presentant des effets desirables d'adherence de cellules
US5278063A (en) Chemical modification of promote animal cell adhesion on surfaces
US20090149673A1 (en) Synthetic non-fouling amino acids
US9631190B2 (en) Cell attachment coatings and methods using phosphorous-containing photoreagent
US20120201858A1 (en) Multiple-arm peptide compounds, methods of manufacture and use in therapy
JP2009526862A (ja) 非滲出性抗菌ペプチドを含む医療デバイスおよびコーティング
EP0983096A2 (fr) Polymeres du type collagene a activite de liaison cellulaire
JP2010088933A (ja) ペプチド被覆インプラントおよびその調製方法
US20070009568A1 (en) Methods for immobilizing molecules on surfaces
AU776564B2 (en) Surface modification of substrates
US7820172B1 (en) Laminin-derived multi-domain peptides
Zhang Biocompatibility of materials
US20120171769A1 (en) Cell attachment coatings and methods
US20030008397A1 (en) Coupled peptides
Dubs et al. Dextran‐based coating system for the immobilization of cell adhesion promoting molecules on titanium surfaces
Ito 5.1 Cell Adhesion Factor Immobilized Materials
EP0594592B1 (fr) Facteur de croissance utilise en rapport avec des transplants artificiels

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19991029

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

17Q First examination report despatched

Effective date: 20001110

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: COMMONWEALTH SCIENTIFIC & INDUSTRIAL RESEARCH ORG

Owner name: BIOCURE, INC.

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20010827