EP2349338A2 - Complexe constitue d'un polysaccharide et d'une hpb - Google Patents

Complexe constitue d'un polysaccharide et d'une hpb

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Publication number
EP2349338A2
EP2349338A2 EP09740953A EP09740953A EP2349338A2 EP 2349338 A2 EP2349338 A2 EP 2349338A2 EP 09740953 A EP09740953 A EP 09740953A EP 09740953 A EP09740953 A EP 09740953A EP 2349338 A2 EP2349338 A2 EP 2349338A2
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EP
European Patent Office
Prior art keywords
polysaccharide
tryptophan
acid
growth factor
function
Prior art date
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Application number
EP09740953A
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German (de)
English (en)
French (fr)
Inventor
Olivier Soula
Rémi SOULA
Martin Gaudier
Gérard Soula
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Adocia SAS
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Adocia SAS
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Publication of EP2349338A2 publication Critical patent/EP2349338A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • 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
    • A61K38/1825—Fibroblast growth factor [FGF]
    • 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
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • 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]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • 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
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • 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
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • 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
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein

Definitions

  • the present invention relates to the formulation of therapeutic proteins and more particularly the formulation of Heparin-Binding Proteins, HBPs, heparin-binding proteins, which are associated, in vivo, with heparin in the form of a complex, which allows their stabilization and maintenance of their activity in vivo.
  • Heparin is a natural polysaccharide carrying carboxylate and sulphate functions whose overall charge is anionic. But this polysaccharide is known for its anti-coagulant activity since it is involved in the formation of a complex between thrombin and anti-thrombin III and this anti-coagulant activity is not compatible with a therapeutic use, for example , in healing treatments, growth control, bone reconstruction.
  • HBPs have been the subject of pharmaceutical development.
  • these proteins are known to be physically unstable (aggregation) and chemically (enzymatic or chemical degradation). This instability may occur in the formulations and / or at the site of administration. It can cause immunoglogic reactions or loss of efficacy.
  • solutions such as increasing doses or the frequency of administrations are used. They are unsatisfactory, particularly because of the high price of these proteins.
  • Genentech has developed a VEGF formulation for the healing of diabetic foot ulcers. A first clinical study in which the treatment was administered every other day took place. The second study was performed with daily administration, probably to answer the problem of a VEGF action time too short for one application every other day.
  • HBPs belong to eight families of proteins, they have very different sizes, biochemical properties and activities, but they are all likely to associate with heparin complex form, Bernfield M. et al., Annu. Rev. Biochem., 1999, 68, 729-777.
  • Hormones are messenger proteins produced by the endocrine system. These proteins then act at a distance after being carried throughout the body by the blood or lymph or outside of it. Hormones include growth hormone (hGH) and parathyroid hormone (PTH). [00015]. By growth factors is meant proteins normally produced in the body stimulating the proliferation of cells or their differentiation.
  • TGF- ⁇ transforming growth factor beta
  • IGF-2 Insulin-like growth factor 2
  • HB-EGF Heparin Binding EGF-like Growth Factor
  • FGF fibroblast growth factors type 1 to 14
  • KGF Keratinocyte Growth Factor
  • NGF-beta Nerve Growth Factor beta
  • CGF Connective Tissue Growth Factor
  • PIGF Placental Growth Factor
  • R-spondines 1 to 4 vascular endothelial growth factors A and B
  • VEGF Vascular Endothelial Growth Factors
  • the invention therefore relates to the use of a polysaccharide substituted with a tryptophan or a tryptophan derivative, said tryptophan or tryptophan derivative being salifiable or salified for the stabilization of HBPs. [00023]. It relates more particularly to the use of said polysaccharide for the preparation of a pharmaceutical formulation of stable HBPs.
  • the polysaccharides according to the invention consist of glycoside bonds of (1,6) and / or (1,4) and / or (1,3) and / or (1,2) type. They can be neutral, that is to say they can not carry acidic or anionic functions, that is to say they carry acid functions. They are functionalized by at least one residue of tryptophan or a derivative of tryptophan, noted Trp:
  • said tryptophan derivative being grafted or bound to the polysaccharides by coupling with an acid function, said acid function possibly being an acid function of an anionic polysaccharide and / or an acid function carried by a linker R linked to the polysaccharide by a function F, said function F resulting from the coupling between the linker R and a function -OH of the neutral or anionic polysaccharide,
  • F being either an ester, thioester, amide, carbonate, carbamate, ether, thioether or amine
  • - R being a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated, comprising one or more heteroatoms, such as O, N or / and S, and having at least one acid function
  • Trp being a residue of tryptophan or a tryptophan derivative, L or D, produces coupling between the amine of tryptophan and at least one acid carried by the R group and / or an acid carried by the anionic polysaccharide, said tryptophan or derivative of tryptophan being salifiable or salified.
  • Trp is a residue of tryptophan or a tryptophan derivative of configuration D.
  • Trp is a residue of tryptophan or a tryptophan derivative of configuration L.
  • HPB is selected from the group consisting of: - hormones such as growth hormone (hGH) or parathyroid hormone (PTH: parathyroid hormone). growth factors such as transforming growth factor beta (TGF- ⁇ ) and transforming growth factors ⁇ 1 and 2, type 2 insulin-like growth factor (IGF-2: Insulin-like growth factor 2), heparin-binding growth factor and epidermal growth factor-like analogue
  • - hormones such as growth hormone (hGH) or parathyroid hormone (PTH: parathyroid hormone).
  • growth factors such as transforming growth factor beta (TGF- ⁇ ) and transforming growth factors ⁇ 1 and 2, type 2 insulin-like growth factor (IGF-2: Insulin-like growth factor 2), heparin-binding growth factor and epidermal growth factor-like analogue
  • HB-EGF Heparin Binding EGF-like Growth Factor
  • FGF fibroblast growth factors
  • KGF keratinocyte growth factor
  • the functionalized polysaccharides may correspond to the following general formulas:
  • R being a chain comprising between 1 and 18 carbons, optionally branched and / or unsaturated, comprising one or more heteroatoms, such as O, N or / and S, and having at least one acid function
  • - Trp being a residue of a tryptophan derivative, L or D, produced from the coupling between the amine of tryptophan or of a tryptophan derivative and at least one acid carried by the R group and / or an acid carried by the anionic polysaccharide, said tryptophan or derivative of tryptophan being salifiable or salified.
  • n represents the molar fraction of the Rs substituted with Trp and is between 0.2 and 0.7,
  • 0 represents the mole fraction of the acid functions of the polysaccharides substituted with Trp and is between 0.2 and 0.7,
  • j represents the mole fraction of acid functions carried by the anionic polysaccharide per saccharide unit and is between 0 and 1
  • (i + j) represents the mole fraction of acid functions per saccharide unit and is between 0.5 and 2, when R is not substituted by Trp, then the acid or acids of the R group are alkali metal cation carboxylates. preferably as Na + or K +, when the polysaccharide is an anionic polysaccharide, when one or more acid functions of the polysaccharide are not substituted by Trp, then they are salified by a cation, alkaline preferably as Na or K, said polysaccharides being neutral pH.
  • F is either an ester, a carbonate, a carbamate or an ether.
  • the polysaccharide consists predominantly of glycoside bonds of (1,6) type.
  • the polysaccharide consisting predominantly of glycoside bonds of (1,6) type is a dextran.
  • the polysaccharide consists predominantly of glycoside bonds of (1,4) type.
  • the polysaccharide consisting predominantly of (1,4) type glycosidic linkages is selected from the group consisting of pullulan, alginate, hyaluronan, xylan, galacturonan or a cellulose soluble in the water.
  • the polysaccharide is a pullulan.
  • the polysaccharide is an alginate.
  • the polysaccharide is a hyaluronan.
  • the polysaccharide is a xylan.
  • the polysaccharide is a galacturonan.
  • the polysaccharide is a water-soluble cellulose.
  • the polysaccharide consists predominantly of glycoside bonds of (1,3) type. [00042]. In one embodiment, the polysaccharide consisting predominantly of glycoside bonds of (1,3) type is a curdlane. [00043]. In one embodiment, the polysaccharide consists predominantly of glycoside bonds of (1,2) type.
  • the polysaccharide consisting predominantly of glycoside bonds of (1,2) type is an inulin. [00045]. In one embodiment, the polysaccharide consists predominantly of glycoside bonds of (1,4) and (1,3) [00046] type. In one embodiment, the polysaccharide consisting predominantly of glycoside bonds of (1,4) and (1,3) type is a glucan. [00047]. In one embodiment, the polysaccharide consists predominantly of glycoside bonds of (1,4), and (1,3) and (1,2) type.
  • the polysaccharide consisting predominantly of (1,4) and (1,3) and (1,2) glycosidic linkages is mannan.
  • the polysaccharide according to the invention is characterized in that the group R is chosen from the following groups:
  • the polysaccharide according to the invention is characterized in that the tryptophan or tryptophan derivative is selected from the group consisting of tryptophan, tryptophanol, tryptophanamide, 2-indole ethylamine and their alkaline cation salts. .
  • This tryptophan or tryptophan derivative substituent is a salifiable or salified substituent, that is to say it has at least one hydrophilic, anionic or cationic function, namely acid, alcohol or amine capable of giving carboxylates, alcoholates or amine salts.
  • the polysaccharide may have a degree of polymerization m of between 10 and 10,000.
  • it has a degree of polymerization m of between 10 and 1000. [00054]. In another embodiment, it has a degree of polymerization m of between 10 and 500.
  • the complex according to the invention is characterized in that the mass ratio polysaccharide / HBP is between 0.5 and 500, preferably between 1 and 300.
  • the complex according to the invention is characterized in that the mass ratio polysaccharide / HBP is between 0.5 and 100, preferably between 1 and 10.
  • BPH is selected from the group of hormones such as growth hormone (hGH: human growth hormone) or parathyroid hormone (PTH: parathyroid hormone ). [00058].
  • the complex according to the invention is characterized in that HBP is selected from the group of growth factors such as proteins such as transforming growth factors ⁇ 1 and 2 (TGF- ⁇ : transforming growth factor beta), type 2 insulin-like growth factor (IGF-2), heparin-binding growth factor and epidermal growth factor analogue (HB-EGF: Heparin Binding EGF-like Growth Factor), fibroblast growth factors type 1 to 14 (FGF: fibroblast growth factors), keratinocyte growth factor (KGF: Keratinocyte Growth Factor), ⁇ nerve growth factor (NGF- ⁇ : Nerve Growth Factor ⁇ ), Connective Tissue Growth Factor (CTGF), Placental Growth Factor (PIGF), R-spondines 1 to 4, and Growth Factors vascular endothelium e A and B (VEGF: Vascular Endothelial Growth Factors).
  • TGF- ⁇ transforming growth factor beta
  • IGF-2 insulin-like growth factor
  • HB-EGF Heparin Binding EGF-
  • the polysaccharides according to the invention are obtained by grafting a tryptophan derivative as defined above on an acid function of an anionic polysaccharide, by coupling between the amine function of tryptophan or a tryptophan derivative.
  • R groups may be grafted onto the polysaccharide alcohol functions and the grafting of tryptophan or a tryptophan derivative may be carried out:
  • the invention also relates to a pharmaceutical composition comprising a complex according to the invention as described above.
  • the invention also relates to a pharmaceutical composition according to the invention as described above, characterized in that it is obtained by drying and / or lyophilization.
  • BPH can be exogenous, that is to say that it is provided by the composition according to the invention. It may also be endogenous, for example the growth factors that will be secreted in a wound during the first phase of healing and which may be stabilized by the formation of the complex according to the invention in vivo in the wound. [00066]. According to the targeted pathologies, it is intended for local or systemic treatment. [00067].
  • the proposed modes of administration are intravenous, subcutaneous, intradermal, transdermal, intramuscular, oral, nasal, vaginal, ocular, oral, pulmonary etc. [00068].
  • the pharmaceutical compositions according to the invention are either in liquid form, in aqueous solution, or in powder, implant or film form. They further comprise conventional pharmaceutical excipients well known to those skilled in the art. [00069].
  • the pharmaceutical compositions may advantageously comprise, in addition, excipients for formulating them in the form of gel, sponges, injectable solution, oral solution, lyoc, etc. [00070].
  • the invention also relates to a pharmaceutical composition according to the invention as described above, characterized in that it is administrable in stent form, film or "coating" of implantable biomaterials implant.
  • Example 1 Synthesis of a sodium dextranemethylcarboxylate modified with the sodium salt of tryptophan, polymer 1.
  • the reaction medium is diluted with 200 ml of water, neutralized with acetic acid and purified by ultrafiltration on PES membrane 5 kD against 6 volumes of water.
  • the sodium dextranmethylcarboxylate solution is passed through a Purolite (anionic) resin to obtain the acidic dextranethylcarboxylic acid which is then lyophilized for 18 hours.
  • the reaction medium is then diluted with 70 ml of water. , then filtered on sintered glass porosity 1 then on glass frjtté porosity 3, it is then clear.
  • the solution is ultrafiltered on a 10 kD PES membrane against 10 volumes of 0.9% NaCl solution and then 6 volumes of water.
  • the concentration of the polymer solution 1 is determined by dry extract.
  • Example 2 Synthesis of sodium tryptophan sodium salt modified sodium tripulanemethylcarboxylate, polymer 2.
  • 8 g (148 mmol of hydroxyl functional groups) of Pullulane with a weight average molar mass of approximately 100 kg / mol (Fluka) are solubilized in water at 42 g / l.
  • 15 ml of 10 N NaOH 148 mmol NaOH
  • the mixture is brought to 35 ° C and then 23 g (198 mmol) of sodium chloroacetate are added.
  • the temperature of the reaction medium is brought to 60 ° C. at 0.5 ° C./min and then maintained at 60 ° C. for 100 minutes.
  • the reaction medium is diluted with 200 ml of water, neutralized with acetic acid and purified by ultrafiltration on PES membrane 5 kD against 6 volumes of water.
  • the sodium pullulanemethylcarboxylate solution is passed through a Purolite resin (anionic) to obtain the acidic pullulanemethylcarboxylic acid which is then lyophilized for 18 hours.
  • the reaction medium is then diluted with 70 ml of water, then filtered on sintered glass porosity 1 then on sintered glass porosity 3, it is then clear.
  • the solution is ultrafiltered on a 10 kD PES membrane against 10 volumes of 0.9% NaCl solution and then 6 volumes of water.
  • the concentration of the polymer solution 2 is determined by dry extract.
  • a solution fraction is lyophilized and analyzed by 1 H NMR in D 2 O to determine the DS grafted tryptophan.
  • Polymer 3 is a sodium dextranethyl carboxylate modified with ethyl ester of L-tryptophan obtained from a dextran of average molecular weight by weight of 40 kg / mol (Pharmacosmos) according to the process described in Example 1 using the ethyl ester of L-tryptophan instead of the sodium salt of L-tryptophan.
  • EXAMPLE 4 Synthesis of a Sodium Dextran Methylcarboxylate Modified by Phenylalanine Ethyl Ester, Polymer 4
  • Polymer 3 is a sodium dextranethyl carboxylate modified with ethyl ester of L-phenylalanine obtained from a dextran with a weight average molar mass of 40 kg / mol (Pharmacosmos) according to the process described in Example 1 in employing the ethyl ester of L-phenylalanine instead of the sodium salt of L-tryptophan.
  • the protein / polymer solution (10 ⁇ l) is mixed with 3 ⁇ l of loading buffer (glycerol, tris-acetate and bromophenol blue in water). These 13 .mu.l containing 50 ng of protein and 25 .mu.g of polymer are deposited in a well of a 0.8% agarose gel. Control solutions (protein or polymer alone) are deposited in a similar manner.
  • the electrophoresis tank is closed and the generator is set at 30V. The migration lasts 1 hour. [00087]. After migration, the gel is transferred to a PVDF membrane by capillarity with an Apelex system for 2 hours at room temperature.
  • the membrane is then saturated with skimmed milk for 1 hour at room temperature and then incubated with primary rabbit antibodies directed against the protein (overnight at 4 ° C) and finally incubated with secondary antibodies, rabbit anti goat HRP (1 hour). at room temperature).
  • the revelation is by reaction of HRP on Opti-4CN.
  • the Revelation is stopped by rinsing in water when the staining is sufficient since the product of the reaction absorbs in the visible. [00088].
  • the protein is alone or does not form a complex with the polymer, it can migrate if it is anionic or remain at the point of the deposit if it is cationic.
  • the protein is then detected either at the loading wells or in the form of a single spot 0.3-0.4 cm from the deposit.
  • the complex is driven more strongly by the charges of the polymer and moves towards the anode. It is detected as a single spot at 0.7 cm
  • polymer 1, obtained in example 1 polymer 2, obtained in example 2, and proteins selected from the groups of cell adhesion molecules, hormones, coagulation proteins, factors Heparin-binding growth, proteins are
  • 1.5 ⁇ g of protein are added to 1.5 ⁇ g of polymer, to 3 ⁇ l of 10X migration buffer (tris acetate pH 7).
  • the solution is completed at 60 ⁇ l with I ⁇ 20. This solution is incubated at room temperature for 20 minutes. 2 ⁇ l of this second solution containing 50 ng of protein and 50 ng of polymer are diluted in 8 ⁇ l of migration buffer IX.
  • the protein / polymer solution (10 ⁇ l) is mixed with 3 ⁇ l of loading buffer (glycerol, tris-acetate and bromophenol blue in water). These 13 .mu.l containing 50 ng of protein and 50 ng of polymer are deposited in a well of a 0.8% agarose gel.
  • the electrophoresis tank is closed and the generator is set at 30V.
  • the migration lasts 1 hour. [00095].
  • the gel is transferred to a PVDF membrane by capillarity with an Apelex system for 2 hours at room temperature.
  • the membrane is then saturated with skimmed milk for 1 hour at room temperature and then incubated with primary rabbit antibodies directed against the protein (overnight at 4 ° C) and finally incubated with secondary antibodies, rabbit anti goat HRP (1 hour). at room temperature).
  • the revelation is by reaction of HRP on Opti-4CN. The revelation is stopped by rinsing in water when the coloration is sufficient since the reaction product absorbs in the visible. [00096].
  • the protein is alone or does not complex with the polymer, it can migrate if it is anionic or remain at the deposition site if it is cationic.
  • the protein is then detected either at the loading wells or in the form of a single spot 0.3-0.4 cm from the deposit.
  • the complex is driven more strongly by the charges of the polymer and moves towards the anode. It is detected as a single spot 0.7 cm from the deposit.
  • Solution 3 is filtered through a 0.22 ⁇ m filter to obtain a sterile solution.
  • the formation of the complex in solution 3 can be demonstrated by co-electrophoresis. [000103].
  • the complex between a polymer according to the invention and a heparin binding protein is formed by simply mixing aqueous solutions at room temperature without addition of organic solvent.
  • Example 7 Obtaining a Polymer / Protein Complex Binding to Heparin
  • Example 6 The operations described in Example 6 are carried out in such a way as to obtain a 1/10 ratio 1 / VEGF polymer complex, by using 1 ⁇ l of solution 1 supplemented with 5 ⁇ l of water and 5 ⁇ l of solution 2. 10 ⁇ l of a solution 4 containing polymer 1 at a concentration of 10 mg / ml and VEGF at a concentration of 1 mg / ml are obtained.
  • Example 8 Obtaining a Heparin-binding Polymer / Protein Complex
  • Example 6 The operations described in Example 6 are carried out so as to obtain a 1 / VEGF polymer complex of ratio 1/2, by using 0.2 ⁇ l of solution 1 supplemented with 5 ⁇ l of water and 5 ⁇ l of water. solution 2. 10 ⁇ l of a solution containing polymer 1 at a concentration of 2 mg / ml and VEGF at a concentration of 1 mg / ml are obtained.
  • Example 9 Obtaining a Polymer / Protein Complex Binding to Heparin [000106] The operations described in Example 6 are carried out with the polymer described in Example 2, Polymer 2, and FGF-2 as heparin-binding protein. Different aqueous solutions of Polymer 2 / FGF-2 complex are obtained whose Polymer 2 / FGF-2 ratios vary from 1 to 100.
  • the complex between a polymer according to the invention and a heparin-binding protein can be formed by simply adding a lyophilizate of polymer or protein to an aqueous solution of protein or polymer at room temperature without adding of organic solvent.

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FR2914305B1 (fr) * 2007-03-29 2009-07-03 Proteins & Peptides Man Dextran fonctionnalise par des amino-acides hydrophobes.
US20120041079A1 (en) * 2006-09-26 2012-02-16 Adocia Dextran functionalized by hydrophobic amino acids
WO2012119004A2 (en) * 2011-03-01 2012-09-07 Sloan-Kettering Institute For Cancer Research Parathyroid hormone analogs, compositions and uses thereof
BR112015010799B1 (pt) 2012-11-13 2023-01-17 Adocia Composição em solução aquosa, e, formulação farmacêutica
CN103755838B (zh) * 2013-12-30 2016-01-20 烟台东诚药业集团股份有限公司 一种肝素钠的制备方法
FR3020952B1 (fr) * 2014-05-14 2017-09-08 Adocia Formulation a action rapide d'insuline comprenant un compose anionique substitue et un compose polyanionique
FR3020947B1 (fr) 2014-05-14 2018-08-31 Adocia Composition aqueuse comprenant au moins une proteine et un agent solubilisant, sa preparation et ses utilisations
US9795678B2 (en) 2014-05-14 2017-10-24 Adocia Fast-acting insulin composition comprising a substituted anionic compound and a polyanionic compound
FR3043557B1 (fr) 2015-11-16 2019-05-31 Adocia Composition a action rapide d'insuline comprenant un citrate substitue
CN114605570B (zh) * 2022-04-15 2022-11-15 南京泛成生物科技有限公司 一种天然阳离子普鲁兰多糖调理剂及其制备方法和应用

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US6277828B1 (en) * 1993-08-20 2001-08-21 Syntex (U.S.A.) Inc. Pharmaceutical formulations of nerve growth factor
WO2002058718A2 (en) 2001-01-26 2002-08-01 Genetix Pharmaceuticals, Inc. Use of compositions containing pdgf-bb for promoting angiogenesis
FR2891149B1 (fr) * 2005-09-26 2007-11-30 Biodex Sarl Composition pharmaceutique a action cicatrisante comprenant un derive de dextrane soluble et un facteur de croissance derive des plaquettes.
FR2914305B1 (fr) * 2007-03-29 2009-07-03 Proteins & Peptides Man Dextran fonctionnalise par des amino-acides hydrophobes.
EP2007816A1 (fr) * 2006-04-07 2008-12-31 Adocia Polysaccharides bifonctionnalises
FR2914191A1 (fr) 2007-03-29 2008-10-03 Proteins & Peptides Man Composition angiogenique.
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JP2012503643A (ja) 2012-02-09
IL211903A0 (en) 2011-06-30
RU2011116418A (ru) 2012-11-10
WO2010035122A2 (fr) 2010-04-01
US8367640B2 (en) 2013-02-05
RU2498820C2 (ru) 2013-11-20
AU2009295590A1 (en) 2010-04-01
KR20110061639A (ko) 2011-06-09
US20100184965A1 (en) 2010-07-22

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