Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1858—Platelet-derived growth factor [PDGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

• the present invention relates to novel Platelet-Derived Growth Factor (PDGF) complexes associated with amphiphilic polymers for improving the in vitro and in vivo physical and chemical stability of the therapeutic protein for pharmaceutical applications.
• PDGF Platelet-Derived Growth Factor
• PDGFs are glycoproteins of approximately 30,000 Daltons, composed of two polypeptide chains linked together by two disulfide bridges. Four types of chains have been identified as A, B, C and D.
• the native protein exists in the form of a homodimer or a type AB heterodimer
• PDGF was isolated for the first time in platelets. These are growth factors released during blood coagulation, capable of promoting the growth of different types of cells (Ross R. et al., Proc Natl Acad Sci USA, 1974, 71, 1207, Kohler N. & Lipton A., Exp. CeII Res., 1974, 87, 297). It is now known that PDGF is produced by a number of cells other than platelets and that it is mitogenic for most cells derived from mesenchyme, ie blood, muscle, bone and cartilage cells, as well as cells of the brain. connective tissue (Raines EW, in "Biology of Platelet-De ⁇ vated Growth Factor", 1993, Westermark, B. and C.
• the activities of PDGF also include, in particular, the stimulation of the release of granules by neutrophil monocytes (Tzeng DY et al., Blood, 1985, 66, 179), the facilitation of steroid synthesis by Leydig cells (Risbridger GP Mol., CeII, Endocri ⁇ ol., 1993, 97, 125), the stimulation of neutrophil phagocytosis (Wilson E. et al., Proc Natl Acad Sci USA, 1987, 84, 2213), the modulation of Expression and secretion of thrombospondin (Majak RA et al., J. Biol Chem., 1987, 262, 8821), and post-regulation of the ICAM-1 gene in smooth muscle cells vascular (Morisaki N. et al., Biochem Biophys Res Commun, 1994, 200, 612).
• ulcers like skin scarring in general, is a complex phenomenon that requires the coordinated intervention in time and space of many cell types, which can be summed up in three phases: an inflammation phase, a proliferation phase and a remodeling phase.
• macrophages kill bacteria, debride damaged tissue, and regenerate tissue.
• macrophages secrete collagenases, cytokines and growth factors.
• the 3rd day to 3 weeks for normal healing three events follow one another.
• the wound is filled with granulation tissue, angiogenesis develops, and the wound becomes covered with epithelial cells.
• the granulation tissue grows from the edges to the center. Fibroblasts produce abundant type III collagen.
• Type III collagen is replaced by type I collagen, which is organized into tension lines and cross-links.
• the PDGF plays a key role.
• the platelets aggregate and release the PDGF.
• PDGF attracts neutrophils, macrophages and fibroblasts to the wound and is a potent mitogen. Macrophages and endothelial cells synthesise and secrete PDGF in turn.
• PDGF stimulates the production of the new extracellular matrix by fibroblasts, essentially non-collagenic compounds such as glycosaminoglycans and adhesion proteins (J. F. Norton et al., Essentiai Practice of Surgery, Springer, 2003, Chapter 7, 77-89).
• the healing process is in fact a delicate balance between a necessary destruction process to remove the damaged tissue and the repair process that leads to the formation of new tissue.
• Proteases and growth factors play a crucial role in this process by regulating this balance. In the case of chronic wounds, this balance is broken in favor of degradation which explains their delay in healing. While there are different types of chronic wounds, biochemically they are relatively similar in the sense that they are characterized by prolonged phases of inflammation leading to high levels of proteases and thus decreasing the activity of growth factors (G. Lauer et al J. Invest Dermatol 115 (2000) 12-18). This degradation of growth factors contributes to an overall loss of tissue associated with these chronic wounds not promoting healing (D. R. Yager et al J. Invest Dermatol 107 (1996) 743-748).
• Human recombinant PDGF-BB corresponding to the international nonproprietary name "becaplermin”, sold under the trade name Regranex ®.
• This medicine is indicated for the treatment of ulcers of the lower limbs of diabetics. It is in the form of a topically applied gel and promotes the healing of ulcers. In particular, it allows, just like endogenous PDGF, to promote cell proliferation and thus the formation of new tissues.
• the Regranex product that contains PDGF-BB has been shown to be effective in increasing the cure rate to 50% of treated patients compared to only 36% for patients who have received only standard wound treatment. Despite this significant improvement in the treatment of diabetic foot ulcers, it is clear that only 50% of patients recover after long and costly treatment. In cases of non-healing, the consequences can be extremely serious and lead in many cases to amputation of the lower limb. It should be added that the average duration of treatment is very long, about 20 weeks and its application is expensive and restrictive due to a cleaning of the wound and an application of Regranex in the morning followed 12 hours later by a cleaning of the wound. These two interventions most often require the care of a nurse. In addition, the average cost of a treatment lasting twenty weeks is excessively high (about US $ 1,400).
• PDGF on the wound to be treated. This degradation results in the case of a chronic wound of a prolonged state of inflammation generating, at the wound level, a hostile environment to PDGF by stimulating an overproduction of proteases.
• the growth factors like the PDGF, the TGF /? or bFGF are key elements in the healing process by their ability to induce cell migration, proliferation, protein synthesis, matrix formation and more generally by controlling the repair process.
• these growth factors are protein molecules, and therefore sensitive to proteolytic degradation.
• Several studies show that degradation of growth factors such as PDGF is much faster when brought into contact with fluids from chronic wounds because they contain high concentrations of metalloproteinases (DR Yager et al., J. Invest Dermatol. 107 (1996) 743-748).
• PDGF is particularly sensitive to post-translational proteolysis (Hart et al., Biochemistry 29: 166-172, 1990 and US Serial No. 07 / 557,219) and in particular at the level of the binding between the acid. amine arginine at position 32 and threonine amino acid at position 33 of the mature chain of the protein. Other sites are sensitive to proteolysis such as the binding between arginine at position 79 and lysine at position 80 or the binding between arginine at position 27 and arginine at position 28 of the B chain of PDGF.
• the solution proposed by Cardium is to introduce the PDGF-BB expression gene into the wound cells to overexpress it locally.
• This gene therapy using an adenovector has healed nearly 80% of diabetic foot ulcers resistant to standard treatments, on a cohort of 15 patients.
• This therapeutic solution is promising.
• pharmaceutical developments based on gene therapy are still very risky for security reasons related to the use of viral vectors of the adenovirus type.
• the problem to be solved is therefore essentially a protection of the PDGF on the chronic wound.
• U.S. Patent 5,905,142 discloses a means of overcoming these PDGF proteolysis problems by generating mutants of the protein having increased resistance to proteolytic attack by substituting or suppressing one or more amino acids lysine or arginine close thereto potential cleavage sites. This strategy to make the protein more resistant to proteases is unsatisfactory.
• This genetic modification of PDGF can lead to changes in biological activity with different affinities to these different receptors which can also induce toxicological problems.
• such a modification of PDGF requires a new pharmaceutical development, which is extremely expensive and hazardous.
• PDGF is "a very sticky protein" because of its cationic and hydrophobic properties
• PDGF is indeed a highly cationic protein whose isoelectric point is between 9.8 and 10.5. Other authors confirm this behavior as Wei et al.
• the authors show that by combining several parameters, a polymer that does not interact with the protein, a buffer with a slightly acidic pH that makes it possible to limit the deamidation reaction, and a neutral preservative with respect to the protein, it is possible to formulate PDGF in order to obtain a pharmaceutically stable formulation.
• the invention therefore relates to the stabilization of PDGF with respect to chemical or physical degradations that can occur at physiological pH in vitro and in vivo by developing a complex between an amphiphilic polymer and a PDGF.
• the invention therefore relates to the formation of a complex between an amphiphilic polymer and a PDGF (amphiphilic polymer-PDGF), this complex providing a chemical and physical stabilization to the protein with respect to degradation at physiological pH in vitro and in vitro. vivo.
• the present invention therefore relates to a water-soluble, physically and chemically stable, amphiphilic-PDGF polymer complex, characterized in that:
• amphiphilic polymers consist of a hydrophilic polymer skeleton functionalized with hydrophobic substituents and hydrophilic groups according to the following general formula.
• R, R ' being a bond or a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated comprising one or more heteroatoms, such as O, N and / or S,
• X being a hydrophilic group that can be: o a carboxylate o a sulphate o a sulphonate o a phosphate o a phosphonate
• Y being a hydrophilic group that can be: o a sulphate o a sulphonate o a phosphate o a phosphonate
• Hy is a hydrophobic group that may be: linear or branched C8 to C30 alkyl, optionally unsaturated and / or containing one or more heteroatoms, such as O, N or S. o linear or branched C8 alkylaryl or arylalkyl at C18, optionally unsaturated and / or possibly containing a heteroatom or an optionally unsaturated C8 to C30 polycycle.
• n and o are between 1 and 3
• h represents the mole fraction of hydrophobic unit with respect to a monomeric unit of between 0.01 and 0.5
• x represents the mole fraction of hydrophilic groups with respect to a monomeric unit, between 0 and 2.0.
• y represents the mole fraction of hydrophilic groups with respect to a monomeric unit, between 0 and 0.5.
• PDGF is chosen from the group of PDGFs (Platelet-Derivated Growth Factors).
• the PDGF is selected from the group consisting of recombinant human PDGFs comprising two B chains (rhPDGF-BB).
• the substituents of the amphiphilic polymers are distributed in a controlled or statistical manner.
• the polymers having a controlled distribution substituents include, for example, block copolymers and alternating copolymers.
• the invention also relates to an amphiphilic-PDGF polymer complex characterized in that the polymer is chosen from polymers whose substituents are randomly distributed.
• the invention also relates to an amphiphilic polymer-PDGF complex, characterized in that the amphiphilic polymer is chosen from polyamino acids.
• the polyamino acids are selected from the group consisting of polyglutamates or polyaspartates.
• the polyamino acids are homopolyglutamates.
• the polyamino acids are homopolyaspartates. In one embodiment, the polyamino acids are copolymers of aspartate and glutamate. These copolymers are either blocky or random.
• the invention also relates to an amphiphilic-PDGF polymer complex characterized in that the polymer is chosen from among polysaccharides.
• the polysaccharides are selected from the group consisting of hyaluronans, alginates, chitosans, galacturonans, chondroitin sulfate, dextrans, celluloses.
• the group of celluloses consists of celluloses functionalized with acids such as carboxymethylcellulose.
• the group of dextrans consists of dextrans functionalized with acids such as carboxymethyldextran.
• the polysaccharide is a soluble dextran derivative having the following formula (I):
• D represents a polysaccharide chain, preferably constituted by chains of glucosidic units
• MC represents methylcarboxylic groups
• - Su represents sulfate groups (sulfation of free hydroxyl functions, carried by glucoside units), - a, b and c represent the degree of substitution (ds), respectively of groups MC, B and Su with i) a strictly greater than 0; Ii) b is such that:
• either b is greater than or equal to 0.3 and c is between 0.1 and 0.5; . either b is strictly less than 0.3 and c corresponds to the following equation (1): c> 8.5 b 2 - 5.41 b + 0.86 (1).
• dextran derivatives of formula (I), as well as their process of preparation are described more generally in the patent application WO 99/29734.
• These dextran derivatives of formula (I) are trivially called DMCBSu and are considered to be copolymers consisting of R-OH and R-OX subunits, X being able to be a methyicarboxylic (MC), benzylamide (B) or sulfate ( Su).
• a methyicarboxylic dextran (DMC) with a degree of substitution (ds) of 0.6 to methylcarboxylic groups contains 0.6 substituted group (R-MC) and 2.4 hydroxyl groups (R-OH), per glucosidic unit. .
• D has a molar mass of between 1000 and 2000,000 Da, and in one embodiment less than 70,000 Da.
• the dextran derivatives are chosen from the compounds of formula (I) in which b is greater than or equal to 0.35.
• the dextran derivatives are chosen from compounds of formula (I) in which a is between 0.5 and 0.8, and c is between 0.1 and 0, 5.
• the polysaccharides are selected from the group consisting of hyaluronans, alginates, chitosans.
• R CH 2 COOH or H, Carboxymethyl Dextran
• the polysaccharide may have an average degree of polymerization m of between 10 and 10,000.
• it has an average degree of polymerization m of between 10 and 5000.
• the invention also relates to an amphiphilic polymer-PDGF complex characterized in that the hydrophobic group Hy is chosen from the group consisting of fatty acids, fatty alcohols, fatty amines, benzyl amines, derivatives cholesterol and phenols.
• the cholesterol derivative is cholic acid.
• the phenol is alpha-tocopherol.
• amphiphilic polymer-PDGF complex according to the invention is reversible.
• the polymers used are synthesized according to the techniques known to those skilled in the art or purchased from suppliers such as, for example, Sigma-Aldrich, NOF Corp. or CarboMer Inc.
• the PDGFs are chosen from human recombinant PDGFs, obtained according to the techniques known to those skilled in the art or purchased from suppliers, for example from the companies Gentaur (USA) or Research Diagnostic Inc. (USA).
• a test for the detection of the amphiphilic polymer-PDGF complex according to the invention carried out by gel electrophoresis (Gel Mobility Shift Assay); a test for slowing down the enzymatic degradation of an amphiphilic-PDGF polymer complex according to the invention; performed by contacting with a protease
• the invention also relates to an amphiphilic polymer-PDGF complex, characterized in that it satisfies the tests for demonstrating the chemical and physical stabilization, namely a test of detection of the complex (Gel Mobility Shift Assay), the test slowing of enzymatic degradation by contact with a protease and the physical stabilization test at physiological pH performed by SDS-Page.
• the Gel Mobility Shift Assay complex highlighting test based on the displacement of ions under the effect of an electric field.
• the anionic complexes migrate towards the anode and the cationic complexes move towards the cathode.
• the proteins are transferred by capillarity onto a PVDF membrane and revealed by an antibody specific for the protein recognized by a second antibody coupled to peroxidase.
• the protein alone does not migrate or little, the protein complexed with the amphiphilic polymer migrates to the anode or cathode as a function of the overall charge of the complex.
• the slowing down test of the enzymatic degradation is based on the verification of the integrity of the protein after contacting the amphiphilic-PDGF polymer complex according to the invention with a protease.
• a solution of a protease (trypsin, chymotrypsin, etc.) is added to the complex solution and kinetics are performed.
• the reaction is stopped by adding a specific inhibitor of the enzyme (indole, benzamidine).
• the integrity of the protein is then analyzed by polyacrylamide gel electrophoresis (SDS-Page).
• the physical stabilization test of a PDGF is based on the verification of the integrity of the protein by comparison of a solution of the amphiphilic-PDGF polymer complex according to the invention with a solution of PDGF alone at pH 7.4 in terms of concentration. in protein in the solution. These two solutions are placed on a stirring bench for 48 hours at room temperature and then centrifuged. The concentration of PDGF in each of the solutions is evaluated by SDS-Page.
• the amphiphilic polymer-PDGF complex according to the invention is formed by the aqueous solution of a PDGF and an amphiphilic polymer at physiological pH without using an organic solvent which may denature the protein.
• the formation of the amphiphilic polymer-PDGF complex is spontaneous and does not involve a covalent bond between PDGF and the amphiphilic polymer. This association is by weak bonds which are essentially hydrophobic interactions and ionic interactions.
• the invention also relates to the process for preparing the amphiphilic polymer-PDGF complex according to the invention, characterized in that a solution at physiological pH is brought into contact with a PDGF and an amphiphilic polymer.
• the invention also relates to an amphiphilic-PDGF polymer complex characterized in that the PDGF / amphiphilic polymer ratio is between 1/5 and 1/5000.
• it is between 1/100 and 1/5000.
• it is between 1/300 and 1/700.
• the invention also relates to a therapeutic composition characterized in that it comprises an amphiphilic polymer-PDGF complex according to the invention.
• composition that can be used in human or veterinary medicine.
• the pharmaceutical composition according to the invention is preferably a composition for topical application which may be in the form of a gel, cream, sprays or a paste, or a patch.
• composition according to the invention when in the form of a gel, it is for example a gel made from polymers such as carboxymethylcelluloses (CMC) vinyl polymers, copolymers of PEO-PPO type, polysaccharides, PEOs, acrylamides or acrylamide derivatives.
• CMC carboxymethylcelluloses
• excipients may be used in this invention to adjust the formulation parameters such as a pH adjusting buffer, an isotonicity adjusting agent, preservatives such as methyl parahydroxybenzoate, propyl parahydroxybenzoate, m-cresol, or phenol or an antioxidant such as L-lysine hydrochloride.
• the therapeutic composition is characterized in that it allows an administration of about 100 ⁇ g per ml of PDGF.
• the present invention also relates to the use of an amphiphilic-PDGF polymer complex according to the invention for the preparation of a therapeutic composition with healing action intended for the treatment of ulcers by the topical route.
• the amphiphilic polymer is synthesized from a carboxymethyl dextran having a degree of carboxymethyl substitution per saccharide unit of 1.0 and an average molar mass of 40000 g / mol.
• the benzylamine is grafted on the acids of this polymer according to a conventional method of coupling in water in the presence of a water-soluble carbodiimide.
• the degree of substitution of benzylamine per saccharide unit is 0.4, determined by 1 H NMR.
• This polymer is then sulfated with a SO 3 / pyridine complex.
• the degree of sulfate substitution per saccharide unit is 0.3.
• a solution of PDGF-BB at 0.1 mg / ml is added to 90 ⁇ l of a solution of DMCBSu at 50 mg / ml.
• the PDGF-BB and DMCBSu solutions are buffered at pH 7.4 and 300 mOsm. This solution is stirred gently for two hours at room temperature and stored at 4 ° C.
• the concentration of PDGF-BB is measured every 30 minutes by Elisa after having stopped the enzymatic reaction by the addition of 10 .mu.l of an indole solution at 10 .mu.g / ml.
• a primary culture of human dermal fibroblasts (Human Dermal Fibroblast Adult (HDFa)) is carried out at a temperature of 37 ° C. in ⁇ MEM medium with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin in an atmosphere saturated with moisture and enriched with CO2 (5%) The medium is renewed every 4 days A dilution of the cell suspension in the culture medium was then carried out to seed the culture dishes at a density of 5,000 cells / well for 96 well plates (company Nunc). For each batch of cells, the stabilizing effect of PDGF-BB by the complex at different concentrations was verified by incorporation of tritiated thymidine (5000 cells / well in 100 ⁇ l).
• the fibroblasts are stimulated by the addition of PDGF-BB, at different concentrations ranging from 0.1 to 100 ng / ml, in the presence or absence of the amphiphilic polymer, at a concentration of 1 ⁇ g / ml.
• the incorporation of the tritiated thymidine is carried out 18 hours after stimulation with PDGF-BB in the presence or absence of the complex, by addition of a 50 ⁇ Ci / ml solution, ie 0.5 ⁇ Ci / well, the radioactivity is recovered. in counting flasks, the wells were rinsed with 100 ⁇ l of 100 mM NaOH and the radioactivity was counted after addition of 1 ml of scintillation liquid (Zinsser Analytic), on an automatic counter.
• scintillation liquid Zinsser Analytic
• the solid line curve represents the results of the complex according to the invention at a concentration of 1 ⁇ g / ml of dextran derivative and the curve in broken lines the results in the absence of the dextran derivative.
• L ⁇ D50 is the concentration of PDGF-BB to have 50% human fibroblast proliferation.
• the ratio R is the ratio of the ED50 calculated as follows:
• the amphiphilic polymer is synthesized from a carboxymethyl dextran having a degree of carboxymethyl substitution per saccharide unit of 1.0 and an average molar mass of 40000 g / mol.
• the methyl ester of tryptophan is grafted onto the acids of this polymer according to a conventional method of coupling in water in the presence of a water-soluble carbodiimide.
• the degree of substitution with tryptophan per saccharide unit is 0.3 determined by 1 H NMR.
• a solution of PDGF-BB at 0.1 mg / ml is added to 90 ⁇ l of a solution of DMCTrpOMe at 50 mg / ml.
• the PDGF-BB and DMCTrpOMe solutions are buffered at pH 7.4 and 300 mOsm. This solution is stirred gently for two hours at room temperature and stored at 4 ° C.
• the PDGF-BB / DMCTrpOMe complex migrates to the anode. Its negative charge is explained by a composition much richer in DMCTrpOMe than in PDGF-BB.
• the control consisting solely of PDGF-BB, does not migrate. Demonstration of the stability of the PDGF-BB / DMCTrpOMe complex
• the amphiphilic polymer is synthesized from a carboxymethyl cellulose having a degree of carboxymethyl substitution per saccharide unit of 1.2 and an average molar mass of 30000 g / mol.
• the benzylamine is grafted on the acids of this polymer according to a conventional method of coupling in water in the presence of a water-soluble carbodiimide.
• the degree of substitution of benzylamine per saccharide unit is 0.2 determined by 1 H NMR. Sulfation is carried out in the presence of a SO 3 -Pyridine complex, the degree of substitution in sulphate is 0.30.
• a solution of PDGF-BB at 0.1 mg / ml is added to 90 ⁇ l of a solution of CMCB at 50 mg / ml.
• the PDGF-BB and CMCBSu solutions are buffered at pH 7.4 and 300 mOsm. This solution is stirred gently for two hours at room temperature and stored at 40C .
• the amphiphilic polymer is synthesized from a carboxymethyl cellulose having a degree of carboxymethyl substitution per saccharide unit of 1.2 and an average molar mass of 30000 g / mol.
• the benzylamine is grafted on the acids of this polymer according to a conventional method of coupling in water in the presence of a water-soluble carbodiimide.
• the degree of substitution of benzylamine per saccharide unit is 0.2 determined by 1 H NMR.
• a solution of PDGF-BB at 0.1 mg / ml is added to 90 ⁇ l of a solution of CMCB at 50 mg / ml.
• the PDGF-BB and CMCB solutions are buffered at pH 7.4 and 300 mOsm. This solution is stirred gently for two hours at room temperature and stored at 4 ° C.

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