US20090221805A1 - Complex polymere amphiphile-PDGF - Google Patents

Complex polymere amphiphile-PDGF Download PDF

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US20090221805A1
US20090221805A1 US11/526,735 US52673506A US2009221805A1 US 20090221805 A1 US20090221805 A1 US 20090221805A1 US 52673506 A US52673506 A US 52673506A US 2009221805 A1 US2009221805 A1 US 2009221805A1
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pdgf
complex according
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Latifa Dahri-correia
Jose Correia
Guy Dubreucq
David Duracher
Remi Soula
Olivier Soula
Gerard Soula
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Adocia SAS
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    • 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 complexes of platelet-derived growth factor (PDGF) associated with amphiphilic polymers for improving the physical and chemical stability, in vitro and in vivo, of the therapeutic protein for pharmaceutical applications.
  • PDGF platelet-derived growth factor
  • PDGFs are glycoproteins of approximately 30 000 daltons, made up of two polypeptide chains linked to one another via two disulphide bridges. Four types of chains have been identified, A, B, C and D.
  • the native protein exists in the form of a homodimer or of an AB-type heterodimer (Oefner C. EMBO J. 11, 3921-2926, 1992).
  • PDGF was isolated for the first time from platelets.
  • PDGFs are growth factors released during blood clotting, capable of promoting the growth of various cell types (Ross R. et al., Proc. Natl. Acad. Sci. USA, 1974, 71, 1207; Kohler N. & Lipton A., Exp. Cell Res., 1974, 87, 297). It is known that PDGF is produced by a certain number of cells other than platelets, and that it is mitogenic with respect to most of the cells derived from the mesenchyma, i.e. blood, muscle, bone and cartilaginous cells, and also connective tissue cells (Raines E.
  • PDGF recombinant PDGFs in the pharmaceutical field
  • the use of PDGF has in particular been approved for the treatment of diabetic foot ulcers (Regranex, J&J) and for periodontal repair (GEM 21S, Biomimetic).
  • Ulcer healing just like cutaneous healing in general, is a complex phenomenon that requires the coordinated intervention, over time and in space, of numerous cell types, that can be summarized in three phases: an inflammation phase, a proliferation phase and a remodeling phase.
  • macrophages kill the bacteria, debride damaged tissues and regenerate the tissues. To do this, the macrophages secrete collagenases, cytokines and growth factors.
  • the granulation tissues mature, and the fibroblasts produce less collagen.
  • the blood vessels formed during the granulation that are of no use are eliminated by apoptosis.
  • the collagen type III is replaced with collagen type I, which organizes according to lines of tension and crosslinks.
  • PDGF plays an essential role. During the formation of a wound, platelets aggregate and release PDGF. The PDGF attracts neutrophils, macrophages and fibroblasts to the wound and is a potent mitogen. The macrophages and the endothelial cells in turn synthesize and secrete PDGF. The PDGF stimulates the production of the new extracellular matrix by the fibroblasts, essentially the non-collagen compounds such as glycosaminoglycans and the adhesion proteins (J. F. Norton et al, Essential practice of surgery, Springer, 2003, chapter 7, 77-89).
  • the healing process is in fact a delicate balance between a process of destruction necessary in order to eliminate the damage to tissues and the repair process that results in the formation of new tissues.
  • Proteases and growth factors play an essential role in this process by regulating this balance. In the case of chronic wounds, this balance is disturbed in favour of degradation, therefore these wounds are slow to heal.
  • different types of chronic wounds exist, they are biochemically relatively similar in the sense that they are characterized by sustained inflammation phases that result in high levels of proteases and thus decrease growth factor activity (G. Lauer et al. J. Invest. Dermatol. 115 (2000) 12-18). This growth factor degradation contributes to an overall loss of tissues associated with these chronic wounds that does not favour healing (D. R. Yager et al., J. Invest. Dermatol. 107 (1996) 743-748).
  • PDGF-BB-based medicament corresponding to the international non-proprietory name “becaplermin”, sold under the trade name Regranex®.
  • This medicament is indicated for the treatment of lower limb ulcers in diabetics. It is in the form of a gel for topical application and makes it possible to promote ulcer healing. It makes it possible in particular, just like endogenous PDGF, to promote cell proliferation and therefore the formation of new tissues.
  • the partial efficacy can be explained by a rapid degradation of the PDGF on the wound to be treated. This degradation results, in the case of a chronic wound, from a sustained inflammation state generating, at the wound, an environment hostile to the PDGF due to the stimulation of an overproduction of proteases.
  • growth factors such as PDGF, TGF ⁇ or bFGF are key elements in the healing process due to their abilities to induce cell migration, proliferation, protein synthesis and matrix formation and, more generally, due to the fact that they control the repair process.
  • these growth factors are protein molecules and, consequently, are sensitive to proteolytic degradation.
  • Several studies show that the degradation of growth factors such as PDGF is much more rapid when they are brought into contact with fluids originating from chronic wounds since they contain high concentrations of metalloproteinases (D. R. 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 U.S. patent Ser. No. 07/557, 219) and especially at the level of the bond between the arginine amino acid at position 32 and the threonine amino acid at position 33 of the mature chain of the protein.
  • Other sites are sensitive to proteolysis, such as the bond between the arginine at position 79 and the lysine at position 80, or else the bond between the arginine at position 27 and the arginine at position 28 of the B chain of PDGF.
  • the problem to be solved is therefore essentially that of protecting PDGF on chronic wounds.
  • U.S. Pat. No. 5,905,142 describes a means of remedying these proteolysis problems concerning PDGF by generating mutants of the protein that have an increased resistance with respect to proteolytic attacks, by substituting or by deleting one or more lysine or arginine amino acids close to the potential cleavage sites.
  • This strategy for making the protein more resistant to proteases is not satisfactory.
  • This genetic modification of PDGF can lead to modifications of the biological activity, with affinities that are different with respect to its various receptors, which can also induce toxicological problems.
  • such a modification of PDGF requires a new pharmaceutical development, which is extremely expensive and risky.
  • PDGF is “a very sticky protein” due to its cationic and hydrophobic properties (Heldin, C. H. EMBO J. 11:4251-4259, 1992; Raines and Ross, J. Biol. Chem. 257(9):5154-5160, 1982; Antoniades, PNAS 78:7314, 1981; Deuel et al. J. Biol. Chem. 256:8896, 1981).
  • PDGF is in fact a highly cationic protein, the isoelectric point of which is between 9.8 and 10.5. Other authors confirm this behaviour, such as Wei et al.
  • SDS anionic surfactant
  • the authors show that, by combining several parameters, a polymer that does not exhibit any interactions with the protein, a buffer at slightly acidic pH making it possible to limit the deamidation reaction and a preserving agent that is neutral with respect to the protein, it is possible to formulate the PDGF in order to obtain a formulation that is stable from a pharmaceutical point of view.
  • the invention therefore relates to the stabilization of PDGF with respect to chemical or physical degradations that may 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 the protein with chemical and physical stabilization with respect to degradations at physiological pH in vitro and in vivo.
  • the present invention therefore relates to a physically and chemically stable, water-soluble, amphiphilic polymer-PDGF complex, characterized in that:
  • n and o are between 1 and 3
  • h represents the molar fraction of hydrophobic unit relative to a monomeric unit of between 0.01 and 0.5
  • x represents the molar fraction of hydrophilic groups relative to a monomeric unit, of between 0 and 2.0
  • y represents the molar fraction of hydrophilic groups relative to a monomeric unit, of between 0 and 0.5;
  • the PDGF is chosen from the group consisting of human recombinant PDGFs containing two B chains (rhPDGF-BB).
  • PDGF PDGF-BB.
  • the substituents of the amphiphilic polymers are distributed in a controlled manner or randomly.
  • the invention also relates to an amphiphilic polymer-PDGF complex characterized in that the polymer is chosen from polymers in which the substituents are distributed randomly.
  • the invention also relates to an amphiphilic polymer-PDGF complex characterized in that the amphiphilic polymer is chosen from polyamino acids.
  • polyamino acids are chosen from the group consisting of polyglutamates or polyaspartates.
  • the polyamino acids are homopolyglutamates.
  • the polyamino acids are homopoly-aspartates.
  • the polyamino acids are copolymers of aspartate and glutamate. These copolymers are either block copolymers or random copolymers.
  • the invention also relates to an amphiphilic polymer-PDGF complex characterized in that the polymer is chosen from polysaccharides.
  • the polysaccharides are chosen from the group consisting of hyaluronans, alginates, chitosans, galacturonans, chondroitin sulphate, dextrans and celluloses.
  • the group of celluloses consists of celluloses functionalized with acids, such as carboxymethyl-cellulose.
  • the group of dextrans consists of dextrans functionalized with acids, such as carboxymethyl-dextran.
  • the polysaccharide is a soluble dextran derivative corresponding to formula (I) below:
  • dextran derivatives of formula (I), and also the process for preparing them, are described more generally in 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, it being possible for X to be a methylcarboxylic (MC), benzylamide (B) or sulphate (Su) group.
  • a methylcarboxylic dextran (DMC) with a degree of substitution (ds) of 0.6 in terms of methylcarboxylic groups contains 0.6 substituted group (R-MC) and 2.4 hydroxyl groups (R—OH), per glucoside unit.
  • D has a molar mass of between 1000 and 2 000 000 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 the 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 chosen from the group consisting of hyaluronans, alginates and chitosans.
  • 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.
  • it has an average degree of polymerization m of between 10 and 500.
  • 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, cholesterol derivatives 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 are 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 such as, for example, from the companies Gentaur (USA) or Research Diagnostic Inc. (USA).
  • the invention also relates to an amphiphilic polymer-PDGF complex characterized in that it passes the tests for demonstration of the chemical and physical stabilization, i.e. a test for demonstration of the complex (Gel Mobility Shift Assay), the test for slowing down of the enzymatic degradation by bringing into contact with a protease, and the test for physical stabilization at physiological pH carried out by SDS-Page.
  • a test for demonstration of the complex Gel Mobility Shift Assay
  • the test for slowing down of the enzymatic degradation by bringing into contact with a protease the test for physical stabilization at physiological pH carried out by SDS-Page.
  • the test for demonstration of the complex carried out by Gel Mobility Shift Assay is based on the displacement of ions under the effect of an electric field.
  • the anionic complexes migrate to the anode and the cationic complexes are displaced to the cathode.
  • the proteins are transferred by capillary action onto a PVDF membrane and visualized by means of an antibody specific for the protein, that is recognized by a peroxidase-coupled second antibody.
  • the test for slowing down of the enzymatic degradation is based on verification of the integrity of the protein after the amphiphilic polymer-PDGF complex according to the invention has been brought into contact with a protease.
  • a solution of a protease (trypsin, chymotrypsin, etc.) is added to the solution of complex and kinetics are determined.
  • the reaction is stopped through the addition of an inhibitor specific for the enzyme (indole, benzamidine).
  • the integrity of the protein is then analysed by polyacrylamide gel electrophoresis (SDS-Page).
  • the test for physical stabilization of a PDGF is based on verification of the integrity of the protein by comparison of a solution of the amphiphilic polymer-PDGF complex according to the invention with a solution of PDGF alone at pH 7.4, in terms of protein concentration in the solution. These two solutions are placed on a shaking bench for 48 h at ambient temperature and then centrifuged. The PDGF concentration in each of the solutions is evaluated by SDS-Page.
  • the amphiphilic polymer-PDGF complex according to the invention is formed by solubilizing a PDGF and an amphiphilic polymer in an aqueous solution at physiological pH without using any organic solvent that may denature the protein.
  • the formation of the amphiphilic polymer-PDGF complex is spontaneous and does not involve any covalent bond between the PDGF and the amphiphilic polymer. This association takes place via weak bonds that are essentially hydrophobic interactions and ionic interactions.
  • the invention also relates to the method of preparing the amphiphilic polymer-PDGF complex according to the invention, characterized in that a PDGF and an amphiphilic polymer are brought into contact in solution at physiological pH.
  • the invention also relates to an amphiphilic polymer-PDGF 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 is intended to mean a composition that can be used in human or veterinary medicine.
  • the pharmaceutical composition according to the invention is preferably a composition for topical application that may be in the form of a gel, a cream, a spray, a paste or a patch.
  • composition according to the invention when in the form of a gel, the latter is, for example, a gel made from polymers such as carboxymethylcelluloses (CMCs), vinyl polymers, copolymers of PEO-PPO type, polysaccharides, PEOs, acrylamides or acrylamide derivatives.
  • CMCs carboxymethylcelluloses
  • vinyl polymers vinyl polymers
  • copolymers of PEO-PPO type polysaccharides
  • PEOs polysaccharides
  • PEOs polysaccharides
  • acrylamides acrylamide derivatives.
  • excipients can be used in this invention in order to adjust the parameters of the formulation, such as a buffer for adjusting the pH, an agent for adjusting the isotonicity, preserving agents such as methyl para-hydroxybenzoate, propyl para-hydroxybenzoate, m-cresol or phenol, or else an anti-oxidant such as L-lysine hydrochloride.
  • the therapeutic composition is characterized in that it allows an administration of approximately 100 ⁇ g per ml of PDGF.
  • the present invention also relates to the use of an amphiphilic polymer-PDGF complex according to the invention, for the preparation of a therapeutic composition with healing action, for use in the treatment of ulcers by topical application.
  • It also relates to a method of therapeutic treatment for human or veterinary use, characterized in that it consists in administering, at the site of treatment, a therapeutic composition comprising the amphiphilic polymer-PDGF complex according to the invention.
  • the amphiphilic polymer is synthesized from a carboxymethyl dextran having a degree of substitution, in terms of carboxymethyl per saccharide unit, of 1.0 and an average molar mass of 40 000 g/mol.
  • the benzylamine 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, in terms of benzylamine per saccharide unit is 0.4, determined by 1 H NMR.
  • This polymer is then sulphated with an SO 3 /pyridine complex.
  • the degree of substitution, in terms of sulphate 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 gently stirred for two hours at ambient temperature and then stored at 4° C.
  • the PDGF-BB/DMCBSu complex migrates to the anode. Its negative charge can be explained by the fact that it has a composition much more rich in DMCBSu than in PDGF-BB.
  • the control consisting only of PDGF-BB, does not migrate.
  • a 10 ⁇ l sample is taken every 30 minutes and the concentration of PDGF-BB is measured by Elisa after having stopped the enzymatic reaction by adding 10 ⁇ l of a solution of indole at 10 ⁇ g/ml.
  • a primary culture of human dermal fibroblasts (Human Dermal Fibroblast adult (HDFa)) is realized at a temperature of 37° C. in ⁇ MEM medium with 10% of foetal calf serum (FCS) and 1% of penicillin-streptomycin in an atmosphere saturated with humidity and enriched in CO 2 (5%). The medium is renewed every 4 days. A dilution of the cell suspension in the culture medium was then realized in order to seed the culture dishes at a density of 5000 cells/well for 96-well plates (the company Nunc).
  • the PDGF-BB-stabilizing effect by means of the complex of various concentrations was verified by incorporation of tritiated thymidine (5000 cells/well in 100 ⁇ l).
  • the fibroblasts are stimulated by the addition of PDGF-B, at various concentrations ranging from 0.1 to 100 ng/ml, in the absence or presence of the amphiphilic polymer, at a concentration of 1 ⁇ g/ml.
  • the tritiated thymidine incorporation is carried out 18 hours after the stimulation with PDGF-BB in the presence or absence of the complex, by the addition of a solution at 50 ⁇ Ci/ml, i.e. 0.5 ⁇ Ci/well.
  • the radioactivity is recovered in counting vials, the wells are rinsed with 100 ⁇ l of 100 mM NaOH and the radioactivity is counted after the addition of 1 ml of scintillation fluid (Zinsser Analytic), on an automatic counter.
  • results obtained are represented in the attached FIG. 1 , in which the amount of tritiated thymidine incorporated by the fibroblasts (in Dpm ⁇ 10 3 ) is expressed as a function of the concentration of PDGF-BB in ⁇ g/ml.
  • the solid-line curve represents the results of the complex according to the invention at a concentration of 1 ⁇ g/ml in terms of dextran derivative, and the dashed-line curve represents the results in the absence of the dextran derivative.
  • the ED50 corresponds to the concentration of PDGF-BB so as to have 50% proliferation of the human fibroblasts.
  • the ratio R is the ratio of the ED50 values, calculated as follows:
  • the amphiphilic polymer is synthesized from a carboxymethyl dextran having a degree of substitution, in terms of carboxymethyl per saccharide unit, of 1.0 and an average molar mass of 40 000 g/mol.
  • the tryptophan methyl ester 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, in terms of tryptophan per saccharide unit, is 0.3 determined by 1 H NMR.
  • PDGF-BB 10 ⁇ l of 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 gently stirred for two hours at ambient temperature and then stored at 4° C.
  • the PDGF-BB/DMCTrpOMe complex migrates towards the anode. Its negative charge can be explained by the fact that its composition is much richer in DMCTrpOMe than in PDGF-BB.
  • the control consisting only of PDGF-BB, does not migrate.
  • 10 ⁇ l of the solution of the PDGF-BB/DMCTrpOMe complex described above are incubated with 90 ⁇ l of a solution of trypsin at 10 ng/ml at 37° C. 10 ⁇ l samples are taken every 30 minutes and the structural integrity of the PDGF-BB is evaluated by polyacrylamide gel electrophoresis (SDS-Page) after having stopped the enzymatic reaction by adding 10 ⁇ l of a solution of indole at 10 ⁇ g/ml.
  • SDS-Page polyacrylamide gel electrophoresis
  • the amphiphilic polymer is synthesized from a carboxy-methylcellulose having a degree of substitution, in terms of carboxymethyl per saccharide unit, of 1.2 and an average molar mass of 30 000 g/mol.
  • the benzylamine 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, in terms of benzylamine per saccharide unit is 0.2 determined by 1 H NMR.
  • the sulphation is carried out in the presence of an SO 3 -pyridine complex, the degree of substitution, in terms of sulphate, is 0.30.
  • PDGF-BB 10 ⁇ l of 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 gently stirred for two hours at ambient temperature and then stored at 4° C.
  • the PDGF-BB/CMCBSu complex migrates towards the anode. This negative charge can be explained by the fact that its composition is much richer in CMCBSu than in PDGF-BB.
  • the control consisting only of PDGF-BB, does not migrate.
  • 10 ⁇ l of the solution of the PDGF-BB/CMCBSu complex described above are incubated with 90 ⁇ l of a solution of trypsin at 10 ng/ml at 37° C. 10 ⁇ l samples are taken every 30 minutes and the structural integrity of the PDGF-BB is evaluated by polyacrylamide gel electrophoresis (SDS-Page) after having stopped the enzymatic reaction by adding 10 ⁇ l of a solution of indole at 10 ⁇ g/ml.
  • SDS-Page polyacrylamide gel electrophoresis
  • the amphiphilic polymer is synthesized from a carboxy-methylcellulose having a degree of substitution, in terms of carboxymethyl per saccharide unit, of 1.2 and an average molar mass of 30 000 g/mol.
  • the benzylamine 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, in terms of benzylamine per saccharide unit, is 0.2 determined by 1 H NMR.
  • PDGF-BB 10 ⁇ l of a solution of PDGF-BB at 0.1 mg/ml are added to 90 ⁇ l of a solution of CMCB at 50 mg/ml.
  • the PDGF-BB CMCB solutions are buffered at pH 7.4 and 300 mOsm.
  • the visualization does not show any migration of the PDGF-BB alone with the amphiphilic polymer. There has been no formation of complex between the CMCB and the PDGF-BB.

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US10583175B2 (en) 2012-11-13 2020-03-10 Adocia Rapid-acting insulin formulation comprising a substituted anionic compound
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US9700599B2 (en) 2012-11-13 2017-07-11 Adocia Rapid-acting insulin formulation comprising a substituted anionic compound
US9795678B2 (en) 2014-05-14 2017-10-24 Adocia Fast-acting insulin composition comprising a substituted anionic compound and a polyanionic compound
US10525133B2 (en) 2014-05-14 2020-01-07 Adocia Aqueous composition comprising at least one protein and one solubilizing agent, preparation thereof and uses thereof
US10792335B2 (en) 2015-11-16 2020-10-06 Adocia Rapid-acting insulin composition comprising a substituted citrate

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FR2891149B1 (fr) 2007-11-30
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JP5438968B2 (ja) 2014-03-12
EP1940449A2 (fr) 2008-07-09
PL1940448T3 (pl) 2013-08-30
CN103203023B (zh) 2016-06-01
RU2424824C2 (ru) 2011-07-27
EP1940448A2 (fr) 2008-07-09
CA2623529A1 (fr) 2007-03-29
CN101631804B (zh) 2012-11-28
WO2007034321A3 (fr) 2007-10-04
FR2891149A1 (fr) 2007-03-30
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DK1940448T3 (da) 2013-06-03
US20070254828A1 (en) 2007-11-01
CN101631804A (zh) 2010-01-20
US8241620B2 (en) 2012-08-14
US20110301086A1 (en) 2011-12-08
WO2007034321A2 (fr) 2007-03-29
WO2007034320A3 (fr) 2007-10-04
ZA200902008B (en) 2010-09-29
RU2008116585A (ru) 2009-11-10
ES2406229T3 (es) 2013-06-06
PT1940448E (pt) 2013-04-15
WO2007034320A2 (fr) 2007-03-29
KR101506593B1 (ko) 2015-03-30
AU2006293613B2 (en) 2012-05-17
BRPI0616439A2 (pt) 2011-06-21
JP2009509952A (ja) 2009-03-12
KR20080080278A (ko) 2008-09-03
CN103203023A (zh) 2013-07-17
AU2006293613A1 (en) 2007-03-29

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