MX2008004085A - Pdgf amphiphilic polymer complex - Google Patents

Abstract

The invention concerns novel platelet-derived growth factor (PDGF) complexes associated with amphiphilic polymers for enhancing the physical and chemicalin vitroandin vivostability of the therapeutic protein for pharmaceutical applications. The invention also concerns a method for preparing the PDGF-amphiphilic polymer complex characterized in that it consists in preparing said polymer/PDGF-BB complex in an aqueous medium and in the absence of organic solvent likely to cause denaturation of the protein and the use of said PDGF-amphiphilic polymer complex for preparing a therapeutic wound-healing composition for topically treating ulcers.

Description

POLYMER COMPLEX ANFIF LO-GROWTH FACTOR DERIVED FROM PLATELETS DESCRIPTION OF THE INVENTION The present invention refers to the new complexes of platelet-derived growth factor (PDGF) associated with amphiphilic polymers that improve the physical and chemical stability in vi tro and in vivo of the protein Therapeutics for pharmaceutical applications. PDGFs are approximately glycoproteins 30000 Daltons, composed of two polypeptide chains linked together by two disulfide bridges. Four types of chains have been identified, namely, A, B, C and D. The native protein exists in the form of homodimer or heterodimer of the type AB (Oefner C. EMBO J. 11, 3921-2926, 1992) . PDGF has been isolated for the first time in platelets. These are growth factors released at the time of blood coagulation, capable of promoting the growth of different cell types (Ross R. et al., Proc. Nati, Acad. Sci. USA, 1974, 71, 1207; Kohler N. Y Lipton A., Exp. Cell Res., 1974, 87, 297). It is now known that PDGF is produced by a number of cells other than platelets, and that it is a mitogen for most cells derived from mesenchyme, for example, blood, muscle, bone or cartilage cells, REF. : 191423 as well as connective tissue cells (Raines E., in "Biology of Platelet-Derivative Growth Factor", 1993, estermark, B. and C. Sorg, Ed. Basel, Kerger, p.74). Numerous articles also tend to demonstrate that PDGF from macrophages behaves as a chemotactic and mitogenic agent for lysed muscle cells, which contribute to the myointimal thickening of the arterial walls, characteristic of arteriosclerosis (Ross R. et al., Science, 1990, 248, 1009). PDGF activities also include and mainly stimulation of granule release by neutrophilic monocytes (Tzeng DY et al., Blood, 1985, 66, 179), facilitation of spheroid synthesis by Leydig cells (Risbridger GP, Mol. Cell, Endocrol., 1993, 97, 125), the stimulation of neutrophil phagocytosis (Wilson E. et al., Proc. Nati, Acad. Sci. United States, 1987, 84, 2213), modulation of the expression and secretion of thrombospondin (Majak RA et al., J. Biol. Chem., 1987, 262, 8821), and post-regulation of the ICAM-1 gene in vascular lysed muscle cells (Morisaki N et al., Biochem. Biophys., Res. Commun., 1994, 200, 612). Taking into account these various properties, the use of recombinant PDGFs in the pharmaceutical domain has already been considered. The use of PDGF has been tested mainly for the treatment of diabetic foot ulcers (Regranex, J &J) and for periodontal repair (GEM 21S, Biomimetic). The healing of ulcers, as well as general cutaneous scarring, is a complex phenomenon that requires the coordinated intervention in time and space of numerous cell types, which can be summarized in three phases; an inflammation phase, a proliferation phase and a remodeling phase. In the inflammatory phase, about 7 days for normal healing, macrophages kill bacteria, debride damaged tissues and regenerate tissues. To do this, macrophages secrete collagenases, cytokines, and growth factors. In the course of the proliferation phase, from the 3rd day to the 3rd week for normal healing, three events occur. The wound is filled with granulation tissue, angiogenesis develops and the wound is covered with epithelial cells. The granulation tissue grows from the edges towards the center. Fibroblasts produce abundant type III collagen. In the course of remodeling, from the 3rd week to 1 or even 2 years, the granulation tissues mature, the fibroblasts produce less collagen. The blood vessels in the course of granulation that are useless are eliminated by apoptosis. Collagen type III is replaced by type I collagen that is organized according to the tension and reticule lines. In these processes, the PDGF plays a key role. At the time of wound formation, platelets aggregate and release PDGF. PDGF attracts neutrophils, macrophages and fibroblasts on the wound and is a powerful mitogen. The macrophages, the endothelial cells synthesize and secrete all the PDGF. PGDF stimulates the production of the new extracellular matrix by fibroblasts, essentially non-collagenic compounds such as glycosaminoglycans and adhesion proteins (JF Norton et al, Essentiai practice of surgery, Springer, 2003, chapter 7, 77-89) . Chronic wounds such as diabetic foot ulcers, venous ulcers and pressure ulcers present the peculiarity of healing very slowly and always incompletely because the healing processes do not develop normally (R. Lobmann et al, J. of Diabetes and its complications, 2006, 20, 329-335). The healing process is in fact a delicate balance between a necessary destruction process in order to eliminate the damaged tissues, and the repair process that leads to the formation of new tissues. Proteases and growth factors play a crucial role in this process by regulating this balance. At In the case of chronic wounds, this balance is broken in favor of degradation, which explains its delay in healing. When there are different types of chronic wounds biochemically these are relatively similar in the sense that they are characterized by prolonged phases of inflammation, which leads to high levels of proteases and thus decreases the activity of growth factors (G. Lauer et al. J. Invest. Dermatol 115 (2000) 12-18). This degradation of the growth factors contributes to an overall loss of the tissues associated with these chronic wounds, not favoring healing (D. R. Yager et al., J. Invest Dermatol 107 (1996) 743-748). There is currently a medicine on the market of human recombinant PDGF-BB corresponding to the international common denomination "becaplermin", sold under the trade name Regranex®. This medicine is indicated for the treatment of ulcers of the lower limbs of diabetics. These are presented in the form of a gel for topical application, and can promote the healing of ulcers. This allows in particular, always like the endogenous PDGF, to favor cell proliferation and therefore the formation of new tissues. This treatment has limited efficacy (Cullen et al., The international journal of biochemistry & Cell Biology 34, 1544-1556, 2002) even if clinical studies have shown improvements in healing and the duration needed for healing (Greenhalgh et al., American Journal of Pathology, 136, 1235-1246 1990; Ladin Plástic and Reconstructive Surgery, 105, 1230-1231 2000; Holloway et al., Wounds .5 / 4, 198-206; Mandracchia et al., Clinics in Podiatric Medecine and Surgery, 18, 189-209 2001; Wieman, TJ. American Journal of Surgery, 176, 74S-79S 1998). The Regranex product containing PDGF-BB, marketed by J &J, has demonstrated its effectiveness by increasing the cure rate to 50% of treated patients versus only 36% for patients who have received only a standard wound treatment. Despite this significant improvement in the treatment of diabetic foot ulcers, it is necessary to confirm that only 50% of patients are cured after a long and expensive treatment. In cases of non-healing, the consequences can be extremely serious and lead in many cases to an amputation of the lower limb. It should be added that the average duration of treatment is very long, approximately 20 weeks and its application is expensive and annoying, by virtue of a wound cleaning and an application of Regranex in the morning followed by 12 hours after a cleaning of the wound. These two interventions most often need the care of a nurse.

In addition, the average cost of a twenty-week treatment is excessively high (of the order of 1400 US dollars). The partial efficacy can be explained by a rapid degradation of PDGF on the wound to be treated. This degradation results in the case of a chronic wound of a state of prolonged annoying inflammation, at the level of the wound, an environment hostile to PDGF by stimulation of an overproduction of proteases. While control of degradation is necessary for wound healing, excessive proteolytic activity is detrimental leading to the degradation of the extracellular matrix (F. Grinnell et al., J. Dermatol 106 (1996) 335-341 et al. CN Rao et al., J. Invest Dermatol 105 (1995) 572-578) and molecules that have a key functional role as growth factors (V. Falanga et al., J. Derm. Surg. (1992) 604-606, DR Yager et al., Wound Rep. Reg. 5 (1997) 23-32, and M. Wlaschek et al., Br. J. Dermatol., 137 (1997) 646-647). Indeed, growth factors such as PDGF, TGFβ or bFGF are key elements in the healing process due to their ability to induce cell migration, proliferation, protein synthesis, matrix formation and more generally controlling the repair processes. However, these growth factors are protein molecules, and consequently sensitive to proteolytic degradation. Several studies show that the degradation of growth factors such as PDGF is much faster when they are put in contact with fluids that come from chronic wounds, since they contain high concentrations of metalloproteinases (DR Yager et al., J. Invest. Dermatol 107 (1996) 743-748). For the treatment of venous ulcers, Regranex, after a pilot clinical study reported in the publication (TJ Wieman, Wounds, 2003, vol 15, n ° 8, 257-264) has shown only a minor improvement of the treatments current based on a regular cleaning of the wound with a compression therapy. The problem of PDGF instability for example has been revealed at the time of the production of the protein. It is known that PDGF is particularly sensitive to post-translational proteolysis (Hart et al., Biochemistry 29: 166-172, 1990 and U.S. Patent No. Serial No. 07/557, 219) and primarily to level of the linkage of the amino acid arginine at position 32 and the amino acid threonine at position 33 of the mature chain of the protein, Other sites are sensitive to proteolysis as the link between arginine at position 79 and the plant at position 80 or even the link between arginine at position 27 and arginine at position 28 of the B chain of PDGF. This proteolytic instability imposes a major problem in the framework of obtaining this protein, which is produced recombinantly in the yeast, according to the procedure described in US Patent No. 4,845,075. Indeed, U.S. Patent No. 7,084,262 teaches us that the analysis and purification of PDGF-BB leads to the obtaining of 21 isoforms resulting from post-translational endoproteolytic cleavage. This great structural heterogeneity has the consequence of involving a 50% decrease in the activity of the protein produced by genetic engineering, in relation to the intact protein in a mature form. On the other hand, recent clinical results of Cardium, after a press release dated August 14, 2006 (www.prnewswire.com) about unhealed diabetic foot ulcers, after 14 weeks show the potential that can offer a treatment with PDGF-BB. The solution proposed by Cardium consists of introducing the expression gene of PDGF-BB in the cells of the wound to overexpress them locally. This gene therapy with the help of an adenovector has allowed to heal almost 80% of these diabetic foot ulcers resistant to common treatments, on a group of 15 patients. This therapeutic solution is promising. But the developments Pharmacists of gene therapy-based treatments are nowadays even very dangerous for the safety reasons linked to the use of viral vectors of the adenovirus type.There is therefore a need and a possibility of improvement of the current treatments of diabetic foot ulcer With PDGF, in the case of treatment of diabetic foot ulcer, the ultimate goal is threefold: accelerate healing> increase the cure rate> simplify the treatment protocol There is also the case of venous and ulcer ulcers Pressure ulcers, causes of major pain and very serious medical complications The problem to be solved is therefore essentially a protection of the PDGF over the chronic wound Various solutions have been proposed. 5,905,142 describes a means to remedy these proteolytic problems concerning PDGF by generating mutants of the protein that have increased resistance against proteolytic attacks by substituting or deleting one or more amino acids, usina or arginine, near the sites of potential cleavage. This strategy to make the protein more resistant to proteases is not satisfactory. This genetic modification of PDGF may involve changes in biological activity with different affinities towards these different receptors, which may also lead to toxicological problems. In addition, such a modification of PDGF again requires pharmaceutical development, which is extremely expensive and dangerous. In the 1970s when this protein had been extensively studied, it was revealed that purification would be extremely delicate since PDGF is "a very sticky protein" due to its cationic and hydrophobic properties (Heldin.CH 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). The PDGF is in effect a strongly cationic protein where the isoelectric point is between 9.8 and 10.5. Other authors confirm this behavior as Wei et al. (Journal of controlled release 112: 103-110, 2006) that explain that PDGF is easily adsorbed on the surfaces that contain it, in which a solution is found. The authors solve the problem in adding either 0.1% BSA or a mixture of 0.1% BSA / Tween 20 in the mixture. These solutions solve the problem to a large extent since up to 95% of the protein is found in solution. But these solutions are not satisfactory from a pharmaceutical point of view given the animal origin of BSA and the risks linked to bovine spongiform encephalopathy. Another solution proposed by the same authors, is to add a more powerful anionic surfactant (SDS) that allows to maintain the PDGF in solution. Unfortunately, SDS also induces a partial denaturation of the protein, leading to a loss of bioactivity. This solution is therefore not satisfactory for stabilizing the protein. In WO93 / 08825, the inventors have shown that the purified PDGF presents a strong instability when formulated in the form of a gel for a topical application. These give, for example, the incompatibility of PDGF with a certain number of products conventionally used to formulate pharmaceutical products such as methylcellulose or hydroxypropylcellulose, as well as certain conventional preservatives such as benzyl alcohol. The authors impose the problem by explaining that there is a need to formulate the PDGF in the form of a gel for topical administration always having a good long-term stability. The same authors show that the PDGF in solution is degraded by a process of deamidation at neutral pH and that the protein is more stable at a slightly pH acid. The authors show that combining several parameters, a polymer does not present interactions with the protein, a buffer at slightly acid pH allows to limit the deamidation reaction, and a neutral preservative against the protein, it is possible to formulate the PDGF in order to obtain a stable formulation from a point of view pharmacist. The authors show that it is possible to obtain a storage stable formulation by adding a polymer that does not interact with the protein to the conditions to keep the formulation at a slightly acid pH, in order to avoid degradation reactions by deamidation of the protein. This solution is not always satisfactory since it does not allow to protect the growth factor from proteolytic degradations in vivo at physiological pH. In World Patent WO97 / 12601 which describes the formulations of PDGF under the gel form, the authors explain that the cellulose derivative they use is able to stabilize the growth factors towards eventual loss of activity at the time of storage. . For this, they are based on the results obtained above on EGF in U.S. Patent No. 4,717,717. However, they also explain that the stability of the cellulose gel containing PDGF can be greatly improved by adding to the formulation a charged chemical species such as charged amino acids or metal ions. Even there, this solution allows to stabilize the growth factors in the formulation at the time of storage of the product, but does not allow a stabilization of these growth factors against the proteases present at the level of chronic wounds at physiological pH. There is thus a therapeutic interest in stabilizing and protecting the growth factors, mainly the PDGF, in order to increase its effectiveness in the framework of a treatment of chronic wounds, and more particularly that of the wounds of diabetic foot ulcers. The invention thus relates to the stabilization of PDGF against chemical or physical degradations that can intervene at physiological pH in vi tro and in vivo, by putting a complex between an amphiphilic polymer and a PDGF to the point. 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 against degradation at physiological pH, in vi tro e in vivo The present invention therefore relates to a complex of amphiphilic polymer-PDGF, physically and chemically stable, soluble in water, characterized in that: the amphiphilic polymers are constituted of a hydrophilic polymeric skeleton functionalized with hydrophobic substituents and hydrophilic groups according to the following general formula.

DP = m monomer units • R, R 'are a bond or chain comprising between 1 and 18 carbon atoms, optionally branched and / or unsaturated, including one or more heteroatoms, such as oxygen, nitrogen and / or sulfur, R and R' are identical or different from each other • F, F 'are an ester, a thioester, an amide, a carbonate, a carbamate, an ether, a thioether, an amine, F and F' are identical or different from each other • X is a group hydrophilic which can be: either a carboxylate or a sulfate or a sulfonate or a phosphate or a phosphonate And is a hydrophilic group that can be: or a sulphate or a sulfonate or a phosphate or a phosphonate • Hy is a hydrophobic group which can be: or a linear or branched alkyl of 8 to 30 carbon atoms, optionally unsaturated and / or containing one or more heteroatoms such as oxygen nitrogen or sulfur . or a linear or branched alkylaryl or arylalkyl of 8 to 18 carbon atoms, optionally unsaturated and / or optionally containing a heteroatom or a polycyclic group of 8 to 30 carbon atoms optionally unsaturated. n and o are comprised between 1 and 3, h represents the mole fraction of the hydrophobic portion relative to a monomer unit comprised between 0.01 and 0.5. x represents the molar fraction of the hydrophilic groups relative to a monomeric unit, comprised between 0 and 2.0. and represents the molar fraction of the hydrophilic groups relative to a monomeric unit, comprised between 0 and 0.5. PDGF is chosen from the group of PDGFs (Growth Factors Derived from Platelets).

This refers to a complex characterized in that the PDGF is chosen by the group consisting of human recombinant PDGFs that include two B chains (rhPDGF-BB). This refers to a complex characterized in that the PDGF is PDGF-BB. The substituents of the amphiphilic polymers are distributed in a controlled or statistical manner. Among the polymers having a controlled distribution of the substituents, for example, block copolymers and alternating copolymers can be mentioned.

Statistical copolymer * - Monomer - Monomer - Monomer - Monomer - Monomer - Monomer - * Hydrophobic Hydrophobic Hydrophobic Block copolymer * - Monomer - Monomer - Monomer - Monomer - Monomer - Monomer - * I I I Hydrophobic Hydrophobic Hydrophobic Alternating Copolymer * - Monomer - Monomer - Monomer - Monomer - Monomer - Monomer - * Hydrophobic Hydrophobic Hydrophobic Thus, in one embodiment, the invention also relates to an amphiphilic-PDGF polymer complex characterized in that the polymer is chosen from among the polymers where the substituents are distributed in a statistical manner. In one embodiment, the invention also relates to an amphiphilic-PDGF polymer complex characterized in that the amphiphilic polymer is chosen from polyamino acids. In one embodiment, the polyamino acids are selected from the group consisting of polyglutamates or polyaspartates. In one embodiment, polyamiano acids are homopolyglutamates. In one embodiment, the polyamiano acids are omopoliaspores. In one embodiment, the polyamino acids are copolymers of aspartate and glutamate. These copolymers are either block or statistical. In one embodiment, the invention also relates to an amphiphilic-PDGF polymer complex characterized in that the polymer is chosen from polysaccharides. In one embodiment, the polysaccharides are chosen from the group consisting of hyaluronans, alginates, chitosans, galacturonans, chondroitin sulfate, dextrans, celluloses. The group of celluloses consists of celluloses functionalized by acids such as carboxymethylcellulose. The group of dextrans is made up of dextrans functionalized by acids such as carboxymethyldextran. In one embodiment, the polysaccharide is a soluble dextran derivative which corresponds to the following formula (I): DMCaBbSuc (I) In which: - D represents a polysaccharide chain, preferably constituted by chains of glucosidic units, MC represents the methylcarboxylic groups, B represents the N-benzylmethylenecarboxamide groups, - Su represents the sulfate groups (sulfation of the free hydroxyl functional groups, carried by the glycosidic units), a, b and c represent the degree of substitution (ds), respectively of the groups MC, B and Su with i) a strictly higher 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; or b is strictly less than 0.3 and e responds to equation (1) below: c > 8.5 b2 - 5.41 b + 0.86 (1). These dextran derivatives of the formula (I), as well as their process of operation, are described more generally in the patent application WO 99/29734. These dextran derivatives of the formula (I) are trivially named DMCBSu and are considered as copolymers constituted of subunits R-OH and R-OX, where X can be a methylcarboxylic (MC), benzylamide (B) or sulfate (Su ).

Thus, a methylcarboxylic dextran (DMC) with a degree of substitution (ds) of 0.6 in methylcarboxylic groups, contains 0.6 substituted groups (R-MC) and 2.4 of the hydroxyl groups (R-OH), per glucosidic unit. In one embodiment, D has a molar mass between 1 000 and 2 000 000 Da, and in one modality, less than 70 000 Da. In one embodiment, the dextran derivatives are chosen from the compounds of the formula (I) in which b is greater than or equal to 0.35. In this case, and according to one embodiment, the dextran derivatives are chosen from the compounds of the formula (I) in which a is between 0.5 and 0.8, and c is between 0.1 and 0.5. In one embodiment, the polysaccharides are chosen from the group consisting of hyaluronans, alginates, and chitosans. These different polysaccharides can be represented as follows: Galacturonan Hyaluronan R = H, dextran R = CH2COOH or H, carboxymethyl-dextran Chitosan Alginate The polysaccharide can have an average degree of polymerization m comprised between 10 and 10000. In one embodiment, there is an average degree of polymerization m comprised between 10 and 5000. In another embodiment, there is an average degree of polymerization m comprised between 10 and 500 In one embodiment, the invention also relates to a complex of amphiphilic polymer-PDGF, characterized in that the hydrophobic group Hy is selected from the group consisting of fatty acids, fatty alcohols, fatty amines, benzylic amines, derivatives of cholesterol and phenols. In one embodiment, the cholesterol derivative is cholic acid. In another embodiment, phenol is alpha-tocopherol. In one embodiment, the polymer complex amphiphilic-PDGF according to the invention, is reversible. The polymers used are synthesized according to techniques known to those skilled in the art or purchased from suppliers such as, for example, Sigma-Aldrich, NOF Corp. or CarboMer Inc. PDGFs are chosen from human recombinant PDGFs, obtained in accordance to the techniques known to the person skilled in the art, or purchased by suppliers such as, for example, the companies Gentaur (United States) or Research Diagnostic Inc. (USA). The demonstration of the stabilization at the same time chemical and physical can be carried out mainly by the putting into operation of the following tests: • an evidence of the evidence of the amphiphilic polymer complex-PDGF according to the invention, made by electrophoresis on gel (Mobility Mobility Displacement Test) • a test of the enhancement of the enzymatic degradation of a complex amphiphilic polymer-PDGF, according to the invention, carried out by contag a protease • a physical stabilization test of a amphiphilic polymer-PDGF complex according to the invention, at physiological pH, carried out on SDS-Page. The invention also relates to a complex of Amphiphilic polymer-PDGF, characterized in that it satisfies the evidence of the demonstration of chemical and physical stabilization, namely a test of the evidence of the complex (Gel Mobility Displacement Test), the test of encouraging enzymatic degradation by contact with a protease, and the physical stabilization test at physiological pH performed by SDS-Page. The evidence of the complex is shown by the Gel Mobility Displacement Test, based on the displacement of the ions under the effect of an electric field. The anionic complexes migrate towards the anode and the cationic complexes move towards the cathode. After migration, the proteins are transferred by capillarity on the PVDF membrane and revealed by an antibody specific for the protein recognized by a second antibody coupled to the peroxidase. The protein alone does not migrate or migrates very little, the protein formed in complex with the amphiphilic polymer migrates towards the anode or the cathode, depending on the overall charge of the complex. The test for encouraging enzymatic degradation is based on the verification of the integrity of the protein after the contact of the amphiphilic polymer complex-PDGF according to the invention, with a protease. A solution of a protease (trypsin, chymotrypsin, etc.) is added to the solution of the complex and a kinetic is performed. The rean is stopped by the addition of a specific inhibitor of the enzyme (indole, benzamidine). The integrity of the protein is then analyzed by electrophoresis on polyacrylamide gel (SDS-Page). The physical stabilization test of a PDGF is based on the verification of the integrity of the protein by comparing a solution of the amphiphilic polymer complex-PDGF according to the invention, to a PDGF solution only at pH 7.4, in terms of the protein concentration in the solution. These two solutions are placed on a shaking 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 the organic solvent capable of denaturing the protein. The formation of the amphiphilic polymer complex PDGF is spontaneous and does not involve covalent linkage between the PDGF and the amphiphilic polymer. This association is made by weak links that are essentially hydrophobic interans and interans ionic The invention also relates to the process for the preparation of the amphiphilic polymer-PDGF complex according to the invention, characterized in that a PDGF and an amphiphilic polymer are contacted in solution at physiological pH. Other tests can therefore be performed to improve the demonstration of the formation of the amphiphilic-PDGF complex according to the invention. • a maintenance test of the tertiary structure of PDGF determined by circular dichroism • a stability test of a PDGF in the amphiphilic polymer complex-PDGF according to the invention at physiological pH under stress. Stress can be a particular mode of agitation, the presence of salts, etc. The invention also relates to a complex amphiphilic polymer-PDGF, characterized in that the PDGF / amphiphilic polymer ratio is between 1/5 and 1/5000. In one mode, it is between 1/100 and 1/5000. In another mode, this is between 1/300 and 1/700. The invention also relates to a Therapeutic composition characterized in that it comprises a complex amphiphilic polymer-PDGF according to the invention. By "therapeutic composition" is meant a composition usable 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, a cream, a spray or a paste or a patch. The nature of the excipients that can be formulated with the amphiphilic complex-PDGF according to the invention, is chosen according to the form of presentation according to the general knowledge of the doctor. Thus, when the composition according to the invention is in the form of a gel, it is for example a gel made from polymers such as carboxymethylcelluloses (CMC), vinyl polymers, copolymers of the PEO-PPO type. , polysaccharides, PEOs, acrylamides or acrylamide derivatives. Other excipients can be used in this invention in order to adjust the parameters of the formulation as a buffer to adjust the pH, an agent that allows to adjust isotonicity, conservatives such as methyl parahydroxybenzoate, propyl parahydroxybenzoate, cresol, or phenol or even an anti- oxidant such as L-lysine hydrochloride. According to the invention, the therapeutic composition is characterized in that it allows an administration of approximately 100 μg per ml of PDGF. The present invention also relates to the use of a complex amphiphilic polymer-PDGF according to the invention, for the preparation of a therapeutic composition with healing action for the treatment of ulcers by topical route. This also refers to a method of therapeutic treatment for human or veterinary use, characterized in that it consists of administering at the treatment site a therapeutic composition comprising the amphiphilic polymer-PDGF complex according to the invention.

EXAMPLES Example 1: PDGF-BB / DMCBSu complex Synthesis of sulfated carboxymethyldextran, modified with benzylamine (DMCBSu) The amphiphilic polymer is synthesized from a carboxymethyldextran having a degree of carboxymethyl substitution per saccharide unit of 1.0 and an average molar mass of 40000 g / mol. Benzylamine is grafted onto the acids of this polymer according to a classical method of coupling in water, in the presence of a carbodiimide water soluble The degree of substitution in benzylamine per saccharide unit is 0.4, determined by 1H NMR. This polymer is then sulphated with an S03 / pyridine complex. The degree of substitution in sulfate per saccharide unit is 0.3.

Preparation of the PDGF-BB / DMCBSu complex 10 μl of a PDGF-BB solution at 0.1 mg / ml is added to 90 μl of a 50 mg / ml DMCBSu solution. The PDGF-BB and DMCBSu solutions are buffered at pH 7.4 and 300 mOsm. This solution is placed under gentle stirring for two hours at room temperature and then stored at 4 ° C.

Evidence of the formation of a PDGF-BB / DMCBSu complex 10 μl of the solution of the PDGF-BB / DMCBSu complex described above are deposited on an agarose gel. The migration of the compounds is carried out under the effect of an electric field (200 mA-4 hours). After migration, the PDGF-BB is transferred onto a PVDF membrane overnight, then developed by immunoblotting with the goat anti-PDGF-BB antibodies, on which the goat anti-goat IgG secondary antibodies are fixed. HRP peroxidase revealed by a substrate (5-bromo-4-chloro-3-phosphate) indolyl phosphate / nitroblue tetrazolium). The PDGF-BB / DMCBSu complex migrates towards the anode. Its negative charge is explained by a composition much richer in DMCBSu than in PDGF-BB. The control, consisting only of PDGF-BB does not migrate.

Evidence of the stability of the solution of the PDGF-DMCBSu complex 10 μl of a solution of PDGF-BB at 0.01 mg / ml at pH 7.4, and 10 μl of the solution of the PDGF-BB / DMCBSu complex at pH 7. 4 described above, are placed on a shaking bench for 48 hours at room temperature. After centrifugation, the concentration of PDGF-BB in each of the solutions is evaluated by SDS-Page. It appears that the concentration of PDGF-BB in solution in the case of the PDGF-BB / DMCBSu complex is not changed, whereas that of the PDGF-BB solution alone has decreased. Evidence of the protection of PDGF-BB against trypsin in this complex PDGF-BB / DMCBSu 10 μl of the BMP-2 / DMCTrpOMe complex solution described above are poured into 90 μl of a trypsin solution at 10 ng / ml at 37 ° C. Using a 10 μl sample, the concentration of PDGF-BB is measured for 30 minutes by ELISA after stopping the enzymatic reaction by the addition of μl of an indole solution at 10 μg / ml. This kinetics reveals that PDGF-BB alone is completely degraded in 1 hour 30 minutes while it is not completely degraded in the PDGF-BB / DMCBSu complex.

Validation of the biological activity of the PDGF-BB / DMCBSu complex A primary culture of human dermal fibroblasts (adult human dermal fibroblasts (FDA)) is performed at a temperature of 37 ° C in the aMEM medium with 10% fetal calf serum (SVF), and 1% penicillin-streptomycin in an atmosphere saturated with moisture, and enriched in C02 (at 5%). The medium is renewed every 4 days. A dilution of the cell suspension in the culture medium was then performed to plant the culture boxes at a density of 5,000 cells / well for the 96-well plates (company Nunc). For each batch of cells, the stabilizing effect of PDGF-BB by the complex at different concentrations has been verified by incorporation of tritiated thymidine (5000 cells / well in 100 μl). After 24 hours of sowing, 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 μg / ml. The incorporation of thymidine tritiated is performed 18 hours after stimulation by PDGF-BB in the presence or absence of the complex, by adding a solution of 50 μCi / ml, or 0.5 μCi / well. The radioactivity is recovered in the counting bottles, the wells are rinsed with 100 μl of 100 mM NaOH and the radioactivity is counted after the addition of 1 ml of scintillation fluid (Zinsser Analytic), on an automatic counter. The results obtained are represented in the attached Figure 1 in which the amount of tritiated thymidine incorporated by the fibroblasts (in Dpm x 103), is expressed as a function of the concentration of PDGF-BB in μg / ml. The continuous curve represents the results of the complex according to the invention at a concentration of 1 μg / ml of the dextran derivative and the dotted curve shows the results in the absence of the dextran derivative. The ED50 corresponds to the concentration of PDGF-BB to have 50% proliferation of human fibroblasts. The ratio R is the ratio of ED50 calculated as follows: R = ED50 (PDGF-BB / ED50 (PDGF-BB + DMCBSu) These results show that when PDGF-BB is used alone, it is necessary to use 6 μg / ml to obtain 50% proliferation, whereas when the PDGF-BB is formed in complex with a dextran derivative of the formula (I), it is enough 2 μg / ml to reach 50% of the proliferation. The proportion R, in this case, is equal to 3.

Example 2: PDGF-BB / DMCTrpOMe Complex Synthesis of triptophan methyl ester modified carboxymethyldextran (DMCTrpOMe) Amphiphilic polymer is synthesized from a carboxymethyldextran having a degree of carboxymethyl substitution per saccharide unit of 1.0 and a mean molar mass of 40000 g / ml. The methyl ester of tryptophan is grafted onto the acids of this polymer according to a classical method of coupling in water, in the presence of a water-soluble carbodiimide. The degree of substitution of tryptophan per saccharide unit is 0.3, determined by 1H NMR.

Preparation of the PDGF-BB / DMCTrpOMe complex 10 μl of a PDGF-BB solution at 0.1 mg / ml are added to 90 μl of a 50 mg / ml DMCTrpOMe solution. The PDGF-BB and DMCTrpOMe solutions are buffered at pH 7.4 and 300 mOsm. This solution is placed under gentle stirring for two hours at room temperature and then stored at 4 ° C.

Evidence of the formation of a PDGF-BB / DMCTrpOMe complex 10 μl of the PDGF-BB / DMCTrpOMe complex solution described above are deposited on an agarose gel for gel electrophoresis with an immunological disclosure. The migration of the compounds takes place under the effect of an electric field (200 mA-4 hours). After migration, the PDGF-BB is transferred onto a PVDF membrane overnight, then developed by immunoblotting with goat anti-PDGF-BB antibodies on which goat anti-goat IgG antibodies, coupled in the peroxidase revealed by a substrate (5-bromo-4-chloro-3-indolyl / nitro blue tetrazolium phosphate). The PDGF-BB / DMCTrpOMe complex migrates towards the anode.

Its negative charge is explained by a composition much richer in DMCTrpOMe than in PDGF-BB. The control, consisting only of PDGF-BB, does not migrate.

Evidence of the stability of the PDGF-BB / DMCTrpOMe complex 10 μl of a solution of PDGF-BB at 0.01 mg / ml at pH 7.4 and 10 μl of the solution of the PDGF-BB / DMCTrpOMe complex at pH 7.4 described above are placed on a shaking bench for 48 hours at room temperature. The The concentration of PDGF-BB in each of the solutions is evaluated by SDS-Page. It seems that the concentration of PDGF-BB in solution in the case of the PDGF-BB / DMCTrpOMe complex has not changed, whereas that of the PDGF-BB solution alone has decreased.

Demonstration of the protection of PDGF-BB against trypsin in the PDGF-BB / DMCTrpOMe complex 10 μl of the PDGF-BB / DMCTrpOMe complex solution described above are incubated with 90 μl of a 10 ng / ml trypsin solution at 37 ° C. Using a 10 μl dose every 30 minutes, the structural integrity of PDGF-BB is evaluated by polyacrylamide gel electrophoresis (SDS-Page) after the enzymatic reaction has been stopped by the addition of 10 μl of an indole solution to 10 μl. μg / ml. This kinetics reveals that PDGF-BB alone is completely degraded in 1 hour 30 minutes, whereas this one is not degraded in the PDGF-BB / DMCTrpOMe complex.

Example 3: PDGF-BB / CMCBSu Complex Synthesis of sulfated carboxymethylcellulose, modified with benzylamine (CMCBSu) The amphiphilic polymer is synthesized from a carboxymethylcellulose having a degree of carboxymethyl substitution per saccharide unit of 1.2, and a molar mass average of 30000 g / mol. The benzylamine is grafted onto the acids of this polymer according to a classical 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 1H NMR. Sulfation is carried out in the presence of an S03-pyridine complex, the degree of substitution in sulfate is 0.30.

Preparation of the PDGF-BB / CMCBSu complex 10 μl of a 0.1 mg / ml PDGF-BB solution are added to 90 μl of a 50 mg / ml CMCB solution. The PDGF-BB and CMCBSu solutions are buffered at pH 7.4 and 300 mOsm. This solution is placed under gentle stirring for two hours at room temperature and then stored at 4 ° C.

Evidence of the formation of a PDGF-BB / CMCBSu complex 10 μl of the solution of the PDGF-BB / CMCBSu complex described above are deposited on an agarose gel for gel electrophoresis, with an immunological disclosure. The migration of the compounds takes place under the effect of an electric field (200 mA-4 hours). After migration, PDGF-BB is transferred onto a PVDF membrane overnight, then developed by immunoblotting with goat anti-PDGF-BB antibodies on which goat anti-IgG secondary antibodies coupled to HRP peroxidase revealed by a substrate (5-bromo-4-chloro-3-indolyl / nitroblue tetrazolium phosphate) were fixed. The PDGF-BB / CMCBSu complex migrates towards the anode.

Its negative charge is explained by a composition much richer in CMCBSu than in PDGF-BB. The control, consisting only of PDGF-BB, does not migrate.

Evidence of the stability of the PDGF-BB / CMCBSu complex 10 μl of a solution of PDGF-BB at 0.01 mg / ml at pH 7.4 and 10 μl of the solution of the PDGF-BB / CMCBSu complex at pH 7.4 described above, place on a shaking bench for 48 hours at room temperature. After centrifugation, the concentration in PDGF-BB in each of the solutions is evaluated by SDS-Page. It seems that the concentration of PDGF-BB in solution in the case of the PDGF-BB / CMCBSu complex does not change, whereas that of the PDGF-BB solution alone has decreased.

Evidence of the protection of PDGF-BB against trypsin in this complex PDGF-BB / CMCBSu 10 μl of the solution of the PDGF-BB / CMCBSu complex described above, are incubated with 90 μl of a solution of trypsin at 10 ng / ml at 37 ° C. Through a 10 μl shot every 30 minutes, the structural integrity of the PDGF-BB by electrophoresis on polyacrylamide gel (SDS-Page) after stopping the enzymatic reaction by the addition of 10 μl of an indole solution at 10 μg / ml. This kinetics reveals that PDGF-BB alone is completely degraded in 1 hour 30 minutes, whereas this one is not degraded in the PDGF-BB / DMCBSu complex.

EXAMPLE 1: PDGF-BB / CMCB Complex Synthesis of benzylamine modified carboxymethyl cellulose (CMCB) 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 onto the acids of this polymer according to a classical 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.

Preparation of the PDGF-BB / CMCB complex 10 μl of a 0.1 mg / ml PDGF-BB solution is added to 90 μl of a 50 mg / ml CMCB solution. The PDGF-BB and CMCB solutions are buffered at pH 7.4 and 300 mOsm. This solution is placed under gentle stirring for two hours at room temperature and then stored at 4 ° C.

Evidence of non-formation of the PDGF-BB / CMCB complex 10 μl of the solution of the PDGF-BB / CMCB complex described above are deposited on an agarose gel for gel electrophoresis, with an immunological revelation. The migration of the compounds takes place under the effect of an electric field (200 mA-4 hours). After migration, PDGF-BB is transferred onto a PVDF membrane overnight, then developed by immunoblotting with goat anti-PDGF-BB antibodies on which goat anti-goat IgG antibodies coupled to the HRP peroxidase revealed by a substrate (5-bromo-4-chloro-3-indolyl / nitro blue tetrazolium phosphate). The disclosure does not show migration of PDGF-BB alone with the amphiphilic polymer. There is no complex formation between the CMCB and the PDGF-BB. It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (26)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A complex of amphiphilic polymer-PDGF, physically and chemically stable, soluble in water, characterized in that: the amphiphilic polymers are constituted by a polymer skeleton functionalized hydrophilic with hydrophobic substituents and hydrophilic groups according to the following general formula. DP = m monomer units • R, R 'are a bond or chain comprising between 1 and 18 carbon atoms, optionally branched and / or unsaturated, including one or more heteroatoms, such as oxygen, nitrogen and / or sulfur, R and R' are identical or different from each other • F, F 'are an ester, a thioester, an amide, a carbonate, a carbamate, an ether, a thioether, an amine, F and F' are identical or different from each other X is a hydrophilic group that can be: either a carboxylate or a sulfate or a sulfonate or a phosphate or a phosphonate • Y is a hydrophilic group that can be: either a sulfate or a sulfonate or a phosphate or a phosphonate • Hy is a hydrophobic group which can be: or a linear or branched alkyl of 8 to 30 carbon atoms, optionally unsaturated and / or containing one or more heteroatoms such as oxygen nitrogen or sulfur. or a linear or branched alkylaryl or arylalkyl of 8 to 18 carbon atoms, optionally unsaturated and / or optionally containing a heteroatom or a polycyclic group of 8 to 30 carbon atoms optionally unsaturated. n and o are comprised between 1 and 3, h represents the mole fraction of the hydrophobic portion relative to a monomer unit comprised between 0.01 and 0.5. x represents the molar fraction of the hydrophilic groups relative to a monomeric unit, comprised between 0 and 2.0. and represents the molar fraction of the hydrophilic groups relative to a monomeric unit, comprised between 0 and 0.5. PDGF is chosen from the group of PDGFs (Growth Factors Derived from Platelets).
2. 2. The complex according to claim 1, characterized in that the PDGF is chosen from the group consisting of the human recombinant PDGFs, which include two B chains (rhPDGF-BB).
3. 3. The complex according to any of the preceding claims, characterized in that the polymer is chosen among the polymers where the substituents are distributed in a statistical manner.
4. 4. The complex according to any of the preceding claims, characterized in that the amphiphilic polymer is chosen from the polyamino acids.
5. 5. The complex according to claim 4, characterized in that the polyamino acids are selected from the group consisting of polyglutamates or polyaspartates.
6. 6. The complex according to claim 5, characterized in that the polyamino acids they are homopoliglutamatos.
7. 7. The complex according to claim 6, characterized in that the polyamino acids are homopolysaccharides.
8. The complex according to claim 7, characterized in that the polyamino acids are the copolymers of aspartate and glutamate.
9. 9. The complex according to any of claims 1 to 5, characterized in that the polymer is chosen among the polysaccharides.
10. 10. The complex according to claim 9, characterized in that the polysaccharides are chosen from the group consisting of hyaluronans, alginates, chitosans, galacturonans, chondroitin sulfate, dextrans, celluloses.
11. 11. The complex according to claim 10, characterized in that the group of celluloses consists of celluloses functionalized with acids such as carboxymethylcellulose.
12. The complex according to claim 10, characterized in that the group of dextrans consists of dextrans functionalized with acids such as carboxymethyldextran.
13. 13. The complex according to claim 9, characterized in that the polysaccharides are chosen from the group consisting of hyaluronans, alginates, chitosans.
14. The complex according to claim 9, characterized in that the polysaccharides are chosen from the group consisting of the soluble dextran derivatives, which correspond to the following formula (I): DMCaBbSuc (I) wherein: D represents a polysaccharide chain, preferably constituted by chains of glucosidic units, MC represents the methylcarboxylic groups, B represents the N-benzylmethylenecarboxamide groups, Su represents the sulfate groups (sulfation of the free hydroxyl functional groups, carried by the glycosidic units), a, b and c represent the degree of substitution (ds), respectively of the groups MC, B and Su with i) a strictly higher 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; . or b is strictly less than 0.3 and c responds to equation (1) below: c > 8.5 b2 - 5.41 b + 0.86 (1).
15. 15. The complex according to any of the preceding claims, characterized in that the hydrophobic group Hy is selected from the group consisting of fatty acids, fatty alcohols, fatty amines, benzylic amines, cholesterol derivatives and phenols.
16. 16. The complex according to any of the preceding claims, characterized in that the formation of the amphiphilic polymer-PDGF complex is reversible.
17. 17. The complex according to any of the preceding claims, characterized in that it satisfies the evidence of evidence of chemical and physical stabilization.
18. 18. The complex according to claim 17, characterized in that the evidence of the demonstration of the chemical and physical stabilization are chosen from the group constituted by the evidence of evidence of the complex (Gel Mobility Displacement Test), of the test for encouraging enzymatic degradation by contacting a protease and the physical stabilization test at physiological pH, carried out by SDS-Page.
19. 19. The complex according to any of the preceding claims, characterized in that the PDGF / amphiphilic polymer ratio is comprised between 1/5 and 1/5000.
20. The complex according to any of the preceding claims, characterized in that the PDGF / amphiphilic polymer ratio is between 1/100 and 1/5000.
21. 21. The complex according to any of the preceding claims, characterized in that the PDGF / amphiphilic polymer ratio is between 1/300 and 1/700.
22. 22. The process for preparing the amphiphilic polymer complex PDGF according to any of the preceding claims, characterized in that this complex polymer / PDGF-BB is prepared in aqueous medium and in the absence of the organic solvent capable of denaturing the protein.
23. 23. The therapeutic composition characterized in that it comprises an amphiphilic polymer-PDGF complex according to any of claims 1 to 22.
24. 24. The therapeutic composition according to claim 20, characterized in that it allows an administration of 100 μg per ml of PDGF.
25. 25. The use of a complex amphiphilic polymer-PDGF according to any of claims 1 to 22, for the preparation of a therapeutic composition for healing action, intended for the treatment of ulcers by topical route.
26. 26. The method of therapeutic treatment for human or veterinary use, characterized in that it consists in administering at the treatment site a therapeutic composition comprising a amphiphilic-PDGF polymer complex according to any of claims 1 to 22.