Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
  • C08F289/00—Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds not provided for in groups C08F251/00 - C08F287/00
• • 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
• • 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
  • A61L27/24—Collagen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
  • C07K17/02—Peptides being immobilised on, or in, an organic carrier
  • C07K17/06—Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
  • A61F2310/00005—The prosthesis being constructed from a particular material
  • A61F2310/00365—Proteins; Polypeptides; Degradation products thereof

Definitions

• the invention relates to a process for obtaining copolymers by grafting vinyl units and the resulting bioactive copolymers.
• copolymers comprising at least one molecule having a biological activity, associated by irreversible bond to a macromolecular material, by means of grafted units of one or more vinyl monomers.
• copolymer has been studied to develop, for example, vascular prostheses or even active phases usable in affinity chromatography.
• some of the co-inventors of the present application have attempted to combine heparin by radiochemical grafting, using vinyl monomers on a polymer material constituted by a polyester, to form a vascular prosthesis (Baquey et al. Innov. Tech. Biol. Med. vol 2, n ° 4 1981, 379-389).
• the object of the invention is therefore to provide a process for obtaining bioactive copolymers ensuring better maintenance of the conformational freedom of the biological molecule and, thereby, of its biological activity.
• the invention further relates to the biological applications of these copolymers for the preparation, for example, of vascular prostheses or of active phases usable in affinity chromatography.
• the process for obtaining the bioactive copolymers of the invention is characterized in that at least one compound A chosen from a protein, a peptide, a protein or peptide derivative and a synthetic or natural material B is subjected simultaneously to radiochemical, chemical or photochemical treatment, in the presence of one or more vinyl monomers, of at least one homopolymerization inhibitor and of a crosslinking agent, so as to obtain the grafting of the units of vinyl origin, by irreversible association on A and B and the crosslinking of the vinyl motifs.
• at least one compound A chosen from a protein, a peptide, a protein or peptide derivative and a synthetic or natural material B is subjected simultaneously to radiochemical, chemical or photochemical treatment, in the presence of one or more vinyl monomers, of at least one homopolymerization inhibitor and of a crosslinking agent, so as to obtain the grafting of the units of vinyl origin, by irreversible association on A and B and the crosslinking of the vinyl motifs.
• a protein In general, use is made of a protein, a peptide or one of their derivatives endowed with a biological activity having a particular affinity for a given molecular or cellular ligand, for example a cellular receptor.
• collagen is advantageously used as protein A.
• collagen of bovine origin containing a preponderant proportion of type I collagen is used.
• coagulation proteases such as antithrombin III (AT III), or pro-activator of fibrinolysis, an inhibitory action on platelet adhesion such as albumin, or alternatively a mediating action of fibrinolysis or a mediating action of cell adhesion and growth.
• urokinase tissue plasminogen activator (tpA) streptokinase.
• tpA tissue plasminogen activator
• Proteins promoting cell attachment and / or intervening in the organization of the extracellular matrix of tissues such as fibronectin or laminin, used where appropriate in conjunction with other proteins, for example collagen, make it possible to have copolymers which promote cell attachment.
• proteins include elastin or growth factors.
• the proteins used according to the invention can be in admixture with other compounds, for example with proteoglycans and / or glycosaminoglycans.
• the protein derivatives are for example glycoproteins or else lipoproteins.
• proteins which can be used in affinity chromatography techniques such as specific immunoglobulins or even antithrombin III.
• affinity chromatography techniques such as specific immunoglobulins or even antithrombin III.
• peptides suitable for implementing the invention mention will be made of those of interest in controlling cell adhesion and having or corresponding to particular sequences playing a role in the adhesion of cells to the extracellular matrix. In view of their properties, such peptides are advantageously incorporated into the structure of surfaces intended to be colonized by cells.
• One such peptide sequence recognized by cell adhesion receptors is of the arg-gly-asp type.
• This tripeptide sequence can be used alone, in a synthetic peptide or in a repeating sequence.
• the macromolecular material B is of synthetic or natural origin.
• REPLACEMENT SHEET Synthetic materials which are particularly advantageous for the biological applications of these copolymers include polymers such as polyesters, polyurethanes, fiber-forming polymers.
• the material B linked to A constitutes a coating on the surface of another type of material. The covalent bond therefore only involves the macromolecular network of the surface coating.
• Natural macromolecular materials include, for example, cellulose, agarose, dextran, starch.
• the vinyl monomer is chosen from acrylic or methacrylic acid or acrylates and methacrylates of alkyl and hydroxyalkyl, in particular from C 1 to C 4, in particular methyl, derivatives of these compounds such as acrylamide or acrylonitrile .
• the vinyl monomer can be formed by multivinyl compounds.
• the grafting is carried out by any suitable radiochemical, chemical or photochemical means.
• the grafting is carried out according to a radiochemical process.
• radioactive sources emitting gamma rays allows easy grafting.
• the dose delivered is at least 1 kilogray.
• the dose rate is approximately 3 kilograys / hour.
• the grafting rates can be limited, which makes it possible to better maintain the conformational freedom of A and therefore its biological activity.
• the crosslinking agent allows the branching and crosslinking of growing homopolymer chains. The probability of bridging between compounds A and B is thereby increased.
• crosslinking agents As suitable crosslinking agents, mention will be made of agents comprising at least two vinyl double bonds such as ethylene dimethacrylate or bis-acrylamide.
• the grafting is preferably carried out in the liquid phase.
• the macromolecular material B is immersed in an aqueous solution containing the compound A, a vinyl or multivinyl monomer, the homopolymerization inhibitor and the crosslinking agent.
• the proportions of reagents making it possible to obtain the satisfactory mechanical properties of the macromolecular material and an unchanged macroscopic appearance correspond to the use, as a percentage by weight, of the vinyl derivative at a rate of 15 to 25%, of the homopolymerization inhibitor to from 5 to 10% approximately, of the crosslinking agent at 0.001% to 0.25%, compound A representing approximately 30%, the remainder being water.
• the homopolymerization inhibitor is a salt, for example a copper or iron salt, such as CuSO4 or Fe SO4.
• the grafting can be carried out photochemically or alternatively chemically, for example using redox systems such as the Ce III + / Ce IV + pair.
• the invention also relates, as new products, to the graft copolymers as obtained by implementing the process defined above.
• copolymers are characterized in that they comprise a compound A chosen from a protein, a peptide, a protein derivative or a peptide derivative, associated by covalent bond to a macromolecular material B via polymeric links such as resulting grafting of vinyl type monomers or of a mixture of such monomers, forming a network by means of a vinyl crosslinking agent.
• copolymers of the invention are ternary copolymers of protein A type / links of crosslinked vinyl origin / macromolecular material B.
• Protein A is formed by a protein of a given type or comprises several proteins chosen according to the applications envisaged.
• the macromolecular material can be formed of a single type of material or of composite materials or of complex structure in which only the surface serves as a support for protein A.
• compound A is a protein derivative, a peptide or a protein or peptide derivative as already defined.
• copoly ⁇ mers of the invention are specially suitable for the development of vascular prosthesis substitutes.
• copolymers of the invention include the development of active phases which can be used in affinity chromatography, in particular for the purification of biological molecules or for carrying out cell sorting and including heparin Sepharose® and Protein A Sepharose represent particularly well-known examples.
• active phases which can be used in affinity chromatography, in particular for the purification of biological molecules or for carrying out cell sorting and including heparin Sepharose® and Protein A Sepharose represent particularly well-known examples.
• heparin Sepharose® and Protein A Sepharose represent particularly well-known examples.
• REPLACEMENT SHEET Similarly, the fixing of ATIII on a support B. makes it possible to have a material for separating heparin or heparin fragments from a given medium or products having an activity modeled on that of heparin.
• the ability of compound A to be recognized by the molecules of the medium to be analyzed makes it possible to carry out separations with high yields.
• the molecules of A linked to the support B to which they bring a specific affinity for a given molecule, or a given cell type retain their functional integrity better than when they are linked according to the chemical processes of the prior art.
• polyester samples Preparation of polyester samples The prostheses are cut into samples, weighed, washed with boiling distilled water in a soxhlet apparatus until constant mass.
• the acrylic acid supplied by MERCK contains 200 ppm of hydroquinone as a polymerization inhibitor. This inhibitor can be removed by distillation of the monomer which is then stored under vacuum in ampoules at -80 ° C.
• Collagen of bovine origin is presented in lyophilized form, supplied by ORGANOTECHNICS.
• a 10-2 mol / l aqueous solution of copper sulphate is produced. Added to the reaction medium, it makes it possible to avoid excessive homopolymerization of the monomer (s) in solution during the irradiation.
• the crosslinking agent is stored at + 4 ° C. and used as it is at very low concentration. .
• a grafting solution containing 15 to 30% of acrylic acid, 5 to 10% of a 10 ⁇ 2M solution of CuSO 4, 5H20, 40% of collagen solution, 0.01% to 0.25% of is used.
• crosslinking agent with 100% water is used.
• the polyester samples are placed in a glass enclosure containing the homogenized grafting solution.
• the contents of the cell are degassed by 4 successive cycles of freezing - pumping - thawing.
• Freezing is carried out by immersion of the enclosure in liquid nitrogen (-196 ° C).
• the vacuum is achieved using a vane pump.
• the cell is thawed in a water bath at • 37 ° C.
• the number of cycles performed depends on the amount of solution to be degassed.
• the quality of degassing is assessed visually.
• Cellophane 1 / acrylic acid / albumin or collagen.
• the affinity of the treated material, for the platelets on the one hand, and for fibrinogen on the other hand is modified compared to that of the starting polyester.
• a first series of samples (series A) is rinsed with the solvent buffer until a constant value of their residual radioactivity is obtained; then, the rinsing is continued with an albumin solution of the same concentration as the solution used during the incubation, but free of radioactive tracer. There is then a decrease in the residual radioactivity of the samples, proving an exchange of pre-adsorbed albumin molecules, and possibly labeled, with cold albumin molecules.
• Example 2 The samples are then rinsed with water and then with an albumin solution without radioactive tracer like the samples of series A. There is practically no reduction in their residual radioactivity, which leads to the conclusion that the irreversible association of
• EXAMPLE 4 BIOLOGICAL CHARACTERIZATION. Culture of human endothelial cells (CEH) in the presence of extracts of copolymers of the invention (collagen / acrylic acid / Dacron®).
• grafted samples according to the invention and ungrafted samples are sterilized with 25 kilogray gamma rays.
• the extracts of tested materials are prepared from samples immersed for 120 hours at 37 ° C in physiological saline (0.9% NaCl).
• the volume of serum used is such that the ratio of the surface area of the sample to this volume is equal to 5 cm2 / ml.
• the final concentration of the extract in the culture medium is 10% (vol / vol).
• the CEH are isolated from umbilical cord veins according to the method described by Jaffe in Transplantation proceedings 1980 (Sept) XII, n ° 3 Suppl-1, 49-53.
• the cells are inoculated into containers containing several wells at a starting density of 7.5 ⁇ 10 3 cells / cm 2.
• the cells are maintained at 37 "C under 5% C02 and the culture medium is renewed every day.
• 37 ° C under 5% CO 2 and the culture medium is renewed every day e.
• the biocompatibility of the copolymer is evaluated by cell attachment (after 3 to 6 hours), proliferation and the protein content of the cells.
• the cells are rinsed and then lysed in distilled water using five freeze-thaw cycles.
• the total protein content (in ug / 105 cells) is determined according to the technique known as Pierce BCA protein assay (Interchim, Paris). For specific biocompatibility, the intracellular presence of factor VIII (FVIII) is revealed by immunofluorescence in the controls and the series tested on the 6th day of proliferation.
• FVIII factor VIII
• the CEH express and maintain their characteristics, namely the synthesis of FVIII antigen, even in the presence of extracts of copolymer materials according to the invention.
• phase chosen is SEPHADEX ® G 25 (crosslinked Dextran) and the protein albumin.
• Sephadéx ® (1 g) is swelled in an albumin solution (7.0 mg / ml) accompanied by albumin labeled with iodine 125 in physiological phosphate buffer (PBS)
• the final suspension is degassed under a vacuum of 5.102 torr, then irradiated until absorption of 1 Mrad at a dose rate of 0.3 Mrad.h-1 approximately.
• the Sephadex® gel is recovered by decantation and then extracted with a soxhlet using a 1: 1 mixture (methanol / water) for 5 days.
• the radioactivity measurements make it possible to calculate a retention rate of 1.03 mg / g of dry Sephadex®, which is 8 times more than the quantity retained when the Sephadex® exposed to the albumin solution is not irradiated and rinsed with 100 ml of water only (no hot extraction).
• the invention therefore provides graft copolymers of high stability, the structure of which makes it possible to take full advantage of the biological activity of the compound of protein or peptide type, which is of major interest in the various biological applications where these copolymers are found. appropriate.

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• Public Health (AREA)
• Transplantation (AREA)
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• Proteomics, Peptides & Aminoacids (AREA)
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• 1989
  • 1989-11-27 FR FR8915575A patent/FR2655048A1/fr active Granted
• 1990
  • 1990-11-27 WO PCT/FR1990/000858 patent/WO1991008242A1/fr not_active Ceased

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