Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
• • 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
  • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
  • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
  • A61L15/325—Collagen
• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
  • C08H1/00—Macromolecular products derived from proteins
  • C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09H—PREPARATION OF GLUE OR GELATINE
  • C09H7/00—Preparation of water-insoluble gelatine
• • 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 present invention relates to new derivatives of biopolymers:
• Such derivatives are capable of being used, in particular, in the preparation of biomaterials, from which it is possible to obtain articles which are applicable, for example in medicine, and more particularly in surgery or in cosmetics.
• articles we can cite: - artificial tissues or organs, such as artificial skin, prostheses or bone, ligament, cardiovascular, intraocular implants, etc.,
• bioencapsulation systems implants, microspheres, microcapsules, allowing the controlled release of active ingredients.
• the invention also relates to one of the processes for obtaining these new biopolymer derivatives.
• Biopolymers must be biocompatible materials and capable of assimilating as best as possible to biological materials, particularly mechanically, so that they can be replaced.
• biopolymers considered in this presentation are all the macromolecules of natural or synthetic origin and of protein and / or carbohydrate and / or lipid nature.
• Collagen is a known protein, present at all levels of the organization of connective tissue: this is the main protein in the skin and connective tissue. By nature, it has biochemical and physicochemical characteristics relatively well suited for uses as biomaterials. Within the meaning of the present invention, it should be noted that the term “collagen” also designates any peptide of collagenic nature, such as native collagen with or without telopeptides, or any species of more or less denatured collagen, including gelatin.
• graft a monofunctional reagent
• amino acid composition may vary depending on the origin of the collagen (pig skin or bone) and the method of extraction (digestion in an acidic or basic medium) of the protein, the average amino acid composition can be determined functional function (expressed as the number of amino acids per hundred amino acids in the protein): glutamic acid ⁇ 7, aspartic acid "4, serine” 2, threonine ⁇ 3, hydroxyproline "8, arginine ⁇ 5, lysine ⁇ 3.
• the chemical crosslinking techniques known in the art involve toxic reagents (dialdehydes, glutaraldehydes, diacid chloride and diisocyanates) and / or generate residues affected by the same defect.
• the crosslinking agent used is a cystine derivative, in which the two amino functions of cystine have been blocked by a protective group, of the benzyloxycarbonyl type and in which the two acid functions of cystine have been activated by esterification with using nitrophenol.
• the grafting of this cystine derivative on the collagen takes place in a neutral medium based on dimethylformamide and water. After grafting on the collagen, the disulfide bridges were reduced, then reoxidized by air oxygen (self-crosslinking factor) in basic medium.
• 4-carboxy-L-thiazolidine is known, capable of being obtained by condensation of aldehyde or ketone with cysteine and used in synthesis peptide as an amino acid substituent.
• the present invention aims to provide a biopolymer, and in particular a modified collagen, a precursor of crosslinkable forms by the formation of disulphide bridges in the presence of mild oxidants providing excellent control of the kinetics and of the crosslinking rate.
• Another object of the invention is to provide a biopolymer, in particular a collagen, "thiolated” i. e crosslinkable and from a stable "pre-crosslinkable” form.
• Another object of the invention is to provide a biopolymer, and in particular a modified collagen, which is in the form of a reticulate comprising S - S bridges.
• Another object of the invention is to provide a process for the preparation of modified biopolymers, in particular collagens, which is simple to use and which leads to precursors of crosslinkable forms, which precursors being stable and easily convertible into crosslinkable products, without 'it is necessary to go through reticulated forms.
• the present invention which relates, first of all, to a biopolymer, in particular collagen, modified using grafts consisting of residues of cysteine or of its derivatives ("residues cysteics ”) linked to reactive functions, of which said biopolymer is carrying and each comprising at least one sulfur group and at least one nitrogen group, both protected by a single protective group.
• cysteic residues mentioned above are compounds comprising at least one sulfur functionality of the thiol type and at least one nitrogen functionality of the amino type. It can be, for example, residues of cysteine, homocysteine, penicillamine, etc.
• One of the original features of the invention resides in the fact of having found a protective group, constituted by a single and same molecular entity, and matching particularly well both with the sulfur groups and with the nitrogen groups of the cysteic residue.
• this graft corresponds to the following general formula (I)
• R 1 and R 2 are integer and when this integer is greater than 1, R 1 and R 2 may be identical or different from carbon to carbon,
• R 1 , R 2 , R 3 , R 4 are similar or different and represent hydrogen or a hydrocarbon radical, preferably chosen from linear and / or cyclic and / or branched alkyls, aryls, aralkyls, alkenyls , aralkenyls, alkynes or aralcynes, all these radicals being optionally substituted, and lower C 1 -C 6 alkyls being more particularly preferred,
• R 5 is hydrogen or a hydrocarbon radical, preferably chosen from acyl, alkyloxycarbonyl, aryloxycarbonyl or aralkyloxycarbonyl radicals, hydrogen being more particularly preferred.
• This graft is directly linked to biopolymers via its carboxylic function, which is capable of reacting with reactive sites of the NH 2 and OH type of the biopolymer, when it has them. And this is the case with collagen.
• the graft can consist of y repeating units, which gives it a polygreffon character, linked to at least one substitution site for the biopolymer via carbonyl.
• x and y are equal to 1 or 2, preferably 1, independently of one another.
• the substituents R 1 , R 2 , R 3 and R 4 of graft I according to the invention correspond to hydrogen or to the following lower alkyl radicals: methyl, ethyl, propyl, butyl.
• the products thus transformed constitute precursors of those in crosslinkable form.
• These precursors have the advantage of being particularly stable in the open air and of being able to be used directly for obtaining crosslinkable biomaterials, in a first step, and crosslinked biomaterials in a second step.
• the first step is simply to put the biopolymer linked to graft I in an aqueous solution, within which the release of the protective group masking the thiol functions, which are capable of self-crosslinking in a second step and this, so spontaneous or by implementing an oxidation, preferably gentle.
• the subject of the present invention is therefore also: - on the one hand, the biopolymer, in particular collagen, thiol obtained from the precursor defined above, - and, on the other hand, the biopolymer, in particular collagen, crosslinked by formation of disulphide bridges and obtained directly from the thiolated product and, in fact, indirectly from the precursor.
• the biopolymer in particular collagen, thiol obtained from the precursor defined above
• the biopolymer in particular collagen, crosslinked by formation of disulphide bridges and obtained directly from the thiolated product and, in fact, indirectly from the precursor.
• These three types of modified biopolymers or collagens can be easily shaped and handled industrially.
• the simple and economical nature of their synthesis should be emphasized.
• they make it possible to obtain medical articles of the type implants, prostheses or artificial skins, non-toxic, non-immunogenic and whose mechanical and biological properties can be perfectly adapted to the intended application.
• this substituent R 5 corresponds to hydrogen.
• This characteristic is particularly original and advantageous because it reflects the fact that, in accordance with the invention and in a su ⁇ renant and unexpected manner, it is not necessary to provide protection on the nitrogen (amino) group of the graft 1 This removes a whole series of operations and reagents more or less easy to eliminate and liable to pollute the biomaterial. It goes without saying that this advantage cannot be considered as a limitation of the invention.
• the substituent R 5 can be constituted by any protective group known in itself and suitable for peptide synthesis, e. g: benzyloxycarbonyl, nitrobenzyloxycarbonyl, butoxycarbonyl, etc.
• the grafting rate of this modified biopolymer, in particular of this collagen can vary within a wide range of values. This is particularly interesting with regard to the adaptability, in particular in terms of mechanical properties, of the biomaterial in its crosslinked form, to various end applications.
• the graft I is fixed to the biopolymer, in a concentration less than or equal to about 3.0 mmol of (I) / g of collagen.
• Collagen is a biopolymer, which is particularly well, but not limited to, within the scope of the invention. It can be native collagen, with or without telopeptides, or even collagen in denatured form (unwound). According to another of its aspects, the present invention relates to a process for the preparation of a biopolymer, in particular of modified collagen, consisting essentially:
• a solvent preferably an organic solvent
• a graft consisting of at least one cysteic residue comprising at least one sulfur group and at least one nitrogen group, both protected by a single protective group
• the graft responds to the general formula (F) below
• R 1 and R 2 are integer and when this integer is greater than 1, R 1 and R 2 may be identical or different from carbon to carbon,
• R 1 , R 2 , R 3 , R 4 are similar or different and represent hydrogen or a hydrocarbon radical, preferably chosen from linear and / or cyclic and / or branched alkyls, aryls, aralkyls, alkenyls , aralkenyls, alkynes or aralcynes, all these radicals being optionally substituted, and lower C 1 -C 6 alkyls being more particularly preferred,
• R 5 is hydrogen or a hydrocarbon radical, preferably chosen from acyl, alkyloxycarbonyl, aryloxycarbonyl or aralkyloxycarbonyl radicals, hydrogen being more particularly preferred,
• the principle of this process therefore consists in reacting the graft F with the reactive sites of the polymer chains (NH 2 , OH in the case of collagen).
• the bonds thus formed are therefore ester or peptide bonds which make it possible to provide, through compound V, nitrogen and sulfur functionalization, the latter conferring on the biopolymers a capacity for crosslinking.
• x and y correspond, advantageously, to 1.
• R 5 represents, for its part, hydrogen or a protective group for conventional amines, hydrogen being more commonly retained.
• Z a hydroxyl (acid form of the molecule) or conventional activation radicals in peptide synthesis are advantageously selected, as will be described below.
• grafts Y examples include: 4-carboxy-2,2-dimethylthiazolidine, 4-carboxy-thiazolidine or 4-carboxy-2,2,5,5-tetramethylthiazolidine.
• the starting biopolymer is a native collagen or an atelocollagen, denatured or not, or alternatively a mixture of at least two of these different collagens.
• the method according to the invention allows a simple and economical grafting of compound Y on the biopolymer.
• the graft Y is preferably formed from 4-carboxythiazolidine
• the starting biopolymer is a native collagen or an atelocollagen, denatured or not, or alternatively a mixture of at least two of these different collagens.
• the starting collagen biopolymer is dissolved or dispersed in the solvent medium, preferably anhydrous.
• this solvent is selected from organic solvents and, preferably, from the following nonlimiting list: N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolydone, dimethylsulfoxide, methanol, ethanol , propanol or other alcohol as well as the analogues of all these solvents and their mixtures.
• This solubilization is preferably carried out separately and before bringing said starting polymer into contact with graft I '.
• Such activation is well known in the field of peptide synthesis. It consists in coupling the acid function with compounds capable of promoting the formation of an amide between the COOH of graft Y and the reactive sites of primary amide type of the biopolymer.
• a suitable coupling system there may be mentioned:
• DCC dicyclohexylcarbodiimide
• CDI - carbonyldiimidazole
• DMAP N, N-dimethylpyridine
• the reaction between the solvated biopolymer and the graft is carried out by mixing its compounds and keeping the reaction medium under stirring for several hours.
• solvent to precipitate collagen, mix with a suitable additive. Acetone and ethyl acetate are two examples of this type of additive.
• This methodology makes it possible to obtain a biopolymer, in particular a collagen, modified by reaction with the graft Y, the latter corresponding to formula (I) once bound to the polymer.
• crosslinkable biopolymer precursor i. thiolated
• this deprotection can consist of a hydrolysis of the protective group, intervening during the implementation of said biomaterial or during a purification, such as dialysis.
• an acid medium at a pH, preferably, less than or equal to 4.
• the biopolymer (collagen) thus prepared is functionalized by SH groups according to variable grafting rates: between 80-90 grafts (I) (deprotected cysteic residues) per 1000 amino acids of the collagen, in the case where the biopolymer considered is formed by this protein.
• the mild oxidation conditions recommended in accordance with the invention consist in using: air, O 2 , iodinated compounds (I 2 , betadine or the like), H 2 O 2 , sodium persulfate , etc.
• this process makes it possible to treat numerous biopolymers having reactive OH, NH 2 functions .
• collagen in its native form t without causing denaturation of its triple helix structure, which can be particularly advantageous in certain applications.
• the modified collagen according to the invention is not affected in its structural integrity and can be subjected to the known treatment of fibrillogenesis at isoelectric pH (phosphate pH approximately 7.4). According to this treatment, an alignment of the triple helices of collagen is caused by the interaction of the charged radicals NH 3 + and COO " which collagen carries at this pH.
• the graft (F) is attached to collagen, by forming an amide bond with the amino acids, collagenic amino acids.
• the amines thus neutralized are replaced by the NR 5 amines of the grafts (Y).
• the modified collagen always has a amino functionality allowing fibrillogenesis.
• the present invention therefore also relates to a modified collagen characterized in that it is in the form of non-denatured triple helices and in that these triple helices are organized and aligned in a bundle, so as to form a fibrous structure.
• the non-hydrolyzed intermediate products which it allows to obtain are stable in the open air.
• reaction conditions] es and the reagents used within the framework of the process according to the invention are not toxic, nor aggressive in themselves with respect to living tissues, any more than they are convertible into by-products having these characters.
• these reagents are not numerous and can be easily removed by non-degrading methods, such as dialysis for example.
• Obtaining thiolated biopolymers according to this process does not involve the formation of biopolymers in crosslinked form, which constitutes an additional and troublesome step on the industrial level.
• This method according to the invention also offers the very appreciable possibility of being able to control the kinetics and the rate of crosslinking of the collagen, which subsequently makes it possible to modulate its mechanical properties.
• crosslinkable products according to the invention find immediate applications, on the one hand, in human medicine and, on the other hand, in the field of biology.
• these may be implants, for example ophthalmological, prostheses, for example bone, dressings in the form of films or felts, artificial tissues (epidermis, vessels, ligaments, bones), systems of bioencapsulation (microspheres, microcapsules) allowing the controlled release of active ingredients in vivo, biocompatibilization coatings for implantable medical articles or, even, sutures.
• implants for example ophthalmological, prostheses, for example bone, dressings in the form of films or felts, artificial tissues (epidermis, vessels, ligaments, bones), systems of bioencapsulation (microspheres, microcapsules) allowing the controlled release of active ingredients in vivo, biocompatibilization coatings for implantable medical articles or, even, sutures.
• the materials according to the invention constitute excellent supports for two-dimensional cell cultures (films) and three-dimensional (felts).
• the crosslinked collagen according to the invention can be used alone or as a mixture with modified or unmodified biological polymers or synthetic polymers.
• atelocollagen is carried out in 150 ml of methanol.
• a DMT solution is prepared (according to the method of J. KEMP, "Org. Chem.”, 1989, 54, 3640).
• An activation step is then carried out by reaction of 1.18 g of DMT-COOH and 1.17 g of carbonyldiimidazole (CDI) in 10 ml of N-methylpyrrolidine (NMP) for 60 minutes at room temperature.
• CDI carbonyldiimidazole
• NMP N-methylpyrrolidine
• Collagen precipitation is carried out by the addition of one liter of ethyl acetate. Filtration and drying finally give a white product (1.5 g).
• graft I (cysteic residue) is determined by reaction with dithionitrobenzoic acid (DTNB), then spectrophotometric assay.
• DTNB dithionitrobenzoic acid
• the graft I content (cysteic residue) is determined by reaction with DTNB, then spectrophotometric assay.
• an activated DMT solution is prepared by reacting 1.0 g of 4-carboxy-thiazolidine (Aldrich) with 1.17 g of CDI in 10 ml of for 60 minutes at room temperature. The two solutions are then mixed together for 16 hours. After adding 30 ml of water at pH 2, the solution is dialyzed against water at pH 2, then lyophilized. Finally, a deprotected white product (1.5 g) is recovered.
• the content of cysteic residues is determined by reaction with DTNB, then spectrophotometric assay.
• Example 1 The product obtained in Example 1 is dissolved in an aqueous medium pH 3 at a concentration of 1% and the solution is poured into a mold. Hydrogen peroxide is added to obtain a final concentration of 0.3% and left to stand for 24 hours. The soft gel is then recovered, washed with water and stored in a humid enclosure. The calorimetric analysis reveals that the denaturation temperature in buffer solution pH 7.4 is 48 ° C.
• Example 2 The product obtained in Example 1 is dissolved in an aqueous medium pH 3 at a concentration of 1% and an iodine solution (3% I 2 -KI) is added. The disappearance of the characteristic color of the iodine is observed and the formation of a gel identical to that obtained in Example 2.
• Example 1 The product obtained in Example 1 is dissolved at + 4 ° C in an aqueous medium of pH 2 at a concentration of 3.5 g / 1 for 24 hours. The solution thus obtained is maintained at 4 ° C. A quantity of 0.2 M of potassium phosphate (dibasic) is added thereto to reach a pH of 7.4. The solution is then heated to 24 ° C. It is maintained at 24 ° C for 4 hours. The medium is then centrifuged to recover the pieces of hydrogel, which are washed with phosphate buffer (pH 7.4) then recentrifuged. The calorimetric analysis reveals that the denaturation temperature in buffer solution pH 7.4 is 48 ° C.
• the products obtained in Examples 2 and 3 can be dissolved in an aqueous medium pH 3-10 at a concentration of 10%.
• a physical gel is obtained by cooling the solutions. Then, the object can be removed from the mold and placed in a hydrogen peroxide bath (at 1%) cooled to 4 ° C for 24 hours. The crosslinked gels obtained are then recovered from the bath, washed with water and stored in a humid enclosure.

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• 1994
  • 1994-08-29 FR FR9410539A patent/FR2723957A1/fr active Granted
• 1995
  • 1995-08-24 WO PCT/FR1995/001117 patent/WO1996006880A1/fr active Application Filing

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