FR2933305A1 - Open implant having osteogenic composition comprising osteogenic growth factor, soluble divalent cation salt and organic support, useful e.g. to fill bone defects, to make spinal fusions, or to treat absence of fracture healing - Google Patents

Abstract

Open implant constituted of an osteogenic composition comprising at least: an osteogenic growth factor; a soluble divalent cation salt; and an organic support, where the organic support does not comprise demineralized bone matrix, is claimed. An independent claim is included for a process for the preparation of the implant comprising first placing a solution comprising the osteogenic growth factor, second placing the organic matrix and/or polymer forming the hydrogel, first adding the solution containing the growth factor to the organic matrix and/or the hydrogel, and optionally homogenizing the mixture, second adding a solution of a soluble divalent cation salt to the obtained implant, and optionally processing freeze-drying of the obtained implant. ACTIVITY : Osteopathic. MECHANISM OF ACTION : None given.

Description

The present invention relates to the field of osteogenic formulations and more particularly to osteogenic protein formulations belonging to the family of Bone Morphogenetic Proteins, BMPs. Bone Morphogenetic Proteins (BMPs) are growth factors involved in osteoinduction mechanisms. BMPs also referred to as Osteogenic Proteins (OPs) were initially characterized by Urist in 1965 (Urist MR Science 1965; 150,893). These proteins isolated from cortical bone have the ability to induce bone formation in a large number of animals (Urist MR Science 1965, 150, 893).

BMPs are expressed as propeptides which, after post-translational processing, have a length of between 104 and 139 residues. They have a great homology of sequences between them and have similar three-dimensional structures. In particular, they have 6 cysteine residues involved in intramolecular disulfide bonds forming a cysteine knot (Scheufler C. 2004 J. Mol Biol 1999, 287, 103, Schlunegger MP, J. Mol Biol 1993, 231, 445). . Some of them have a 7th cysteine also involved in an intermolecular disulfide bridge at the origin of dimer formation (Scheufler C. 2004 J. Mol.

Biol. 1999; 287: 103)..

In their active form, BMPs assemble into homodimers or even heterodimers as described by Israel et al. (Israel DI, Growth Factors, 1996, 13 (3-4), 291). Dimeric BMPs interact with BMPR transmembrane receptors (Mundy et al., Growth Factors, 2004, 22 (4), 233). This recognition is at the origin of a cascade of intracellular signaling involving Smad proteins in particular resulting in the activation or repression of target genes.

BMPs, with the exception of BMPs 1 and 3, play a direct and indirect role in the differentiation of mesenchymal cells causing their differentiation into osteoblasts (Cheng H., J Bone and Joint Surgery, 2003, 85A 1544-1552). They also possess chemotaxis properties and induce proliferation and differentiation.

Certain human recombinant BMPs, and in particular rhBMP-2 and rhBMP-7, have clearly demonstrated an ability to induce bone formation in vivo in humans and have been approved for certain medical applications. Thus, recombinant human BMP-2, dibotermin alfa according to the international nonproprietary name, is formulated in the products marketed under the name InFUSE in the United States and InductOs in Europe. This product is prescribed in the fusion of the lumbar vertebrae and bone regeneration of the tibia for so-called non-union fractures. In the case of InFUSE for fusion of the lumbar vertebrae, the surgical procedure consists first of all, to soak a collagen sponge with a solution of rhBMP-2, then to place the sponge in a hollow cage, LT Cage , previously implanted between the vertebrae.

Human recombinant BMP-7, eptotermin alpha according to the international nonproprietary name, has the same therapeutic indications as BMP-2 and is the basis of two products: OP-1 Implant for open fractures of the tibia and OP-1 Putty for the fusion of the lumbar vertebrae. OP-1 Implant consists of a powder containing rhBMP-7 and collagen to be taken up in 0.9% saline solution. The paste obtained is then applied to the fracture during a surgical procedure.

OP-1 Putty comes in the form of two powders: one containing rhBMP-7 and collagen, the other carboxymethylcellulose (CMC). During surgery, CMC is reconstituted with 0.9% saline and mixed with rhBMP-7 and collagen. The paste thus obtained is applied to the site to be treated.

However, it remains essential to find a formulation to improve the performance of these growth factors BMPs so as to reduce, for example, the quantities to be administered.

This problem is common to many growth factor formulations, since these proteins are generally used in doses that are several orders of magnitude higher than the physiological doses.

It is the merit of the Applicant to have found a formulation of osteogenic growth factors to improve their activity by adding a solution of a soluble salt of an at least divalent cation to the matrix containing the growth factors. .

Surprisingly, this new formulation makes it possible to produce the same osteogenic effect with lesser amounts of growth factors.

The invention relates to an osteogenic composition comprising at least: • an osteogenic growth factor, • a soluble salt of at least divalent cation • an organic matrix.

In one embodiment, the composition is in the form of a lyophilizate. In one embodiment, the at least one divalent cationic soluble salt is a divalent cation soluble salt selected from calcium, magnesium or zinc cations. In one embodiment, the divalent cation soluble salt is a calcium salt whose counterion is selected from chloride, D-gluconate, oxalate, hydroxide, formate, D-saccharate, phosphate, carbonate, acetate, sulfate, L-ascorbate, L-tartrate, L-lactate, glutamate, aspartate.

In one embodiment, the divalent cation soluble salt is a magnesium salt whose counterion is selected from chloride, D-gluconate, oxalate, hydroxide, formate, D-saccharate, phosphate Carbonate, acetate, sulfate, L-ascorbate, L-tartrate, L-lactate, glutamate, aspartate.

In one embodiment, the divalent cation soluble salt is a zinc salt whose counterion is selected from chloride, D-gluconate, oxalate, hydroxide, formate, D-saccharate, phosphate , carbonate, acetate, sulfate, L-ascorbate, L-tartrate, L-lactate, glutamate, aspartate.

In one embodiment, the divalent cation soluble salt is calcium chloride. In one embodiment, the soluble cation salt is a soluble multivalent cation salt.

Multivalent cations are understood to mean species bearing more than two known positive charges, iron, aluminum, cationic polymers such as polylysine, spermine, protamine, fibrin.

By osteogenic growth factor or BMP alone or in combination is meant a BMP selected from the group of therapeutically active BMPs (Bone Morphogenetic Proteins).

More particularly, the osteogenic proteins are selected from the group consisting of BMP-2 (Dibotermin-alpha), BMP-4, BMP-7 (Eptotermin-alpha), BMP-14 and GDF-5.

The BMPs used are recombinant human BMPs, obtained according to the techniques known to those skilled in the art or purchased from suppliers such as, for example, Research Diagnostic Inc. (USA).

In one embodiment, the organic matrix is chosen from matrices based on purified, sterilized natural collagen which is preferably crosslinked.

Indeed, natural polymers such as collagen are components of the extracellular matrix that promote attachment, migration and cell differentiation. They have the advantage of being extremely biocompatible and are degraded by enzymatic digestion mechanisms. Collagen-based matrices are obtained from type I or IV fibrillar collagen extracted from tendon or bones of beef or pork. These collagens are first purified before being crosslinked and then sterilized.

It can also be obtained by acid extraction of autologous bone, leading to the loss of the majority of the mineralized components but to the preservation of collagenic or non-collagenic proteins, including growth factors. These demineralized matrices can also be prepared in inactive form after extraction with chaotropic agents. These matrices are essentially composed of insoluble and highly crosslinked Type I collagen.

Mixed materials can also be used, for example a matrix which combines collagen and inorganic particles and which can be in the form of a composite material with enhanced mechanical properties or even in the form of a putty or collagen plays a role of binder.

Usable inorganic materials include essentially calcium phosphate ceramics such as hydroxyapatite (HA), tricalcium calcium phosphate (TOP), biphasic calcium phosphate (BCP) or amorphous calcium phosphate (ACP) which have as their main interest a chemical composition very close to that of the bone. These materials have good mechanical properties and are immunologically inert. These materials can be in various forms such as powders, aggregates or blocks. These materials have very different degradation rates depending on their compositions and hydroxyapatite is degraded very slowly (several months) while tricalcium calcium phosphate degrades more quickly (several weeks). It is for this purpose that biphasic calcium phosphates have been developed because they have intermediate resorption rates. These inorganic materials are known to be primarily osteoconductive.

Lyophilisate is the product or composition resulting from a freeze-drying operation.

Lyophilization is a water sublimation technique allowing dehydration of the composition. This technique is commonly used for protein storage and stabilization.

The rehydration of a lyophilizate is very rapid and makes it possible to easily obtain a ready-to-use formulation, said formulation being rehydrated before implantation or implanted in its dehydrated form, the rehydration then taking place, after implantation, by contact with biological fluids.

In addition, it is possible to add to these osteogenic growth factors, other proteins and in particular angiogenic growth factors such as PDGF, VEGF or FGF.

The invention therefore relates to a composition according to the invention characterized in that it further comprises angiogenic growth factors selected from the group consisting of PDGF, VEGF or FGF.

The osteogenic compositions according to the invention are used by implantation, for example, to fill bone defects, to perform vertebral fusions or maxillofacial repairs or for the treatment of the absence of fracture consolidation (nonunion).

In these different therapeutic uses, the size of the matrix and the amount of osteogenic growth factor are a function of the volume of the site to be filled.

In one embodiment, for a vertebral implant, the osteogenic growth factor doses will be between 0.05 mg and 8 mg, preferably between 0.1 mg and 4 mg, more preferably between 0.1 mg and 2 mg. mg, while commonly accepted doses in the literature are between 8 and 12 mg.

For uses in maxillofacial repair or in the treatment of pseudarthrosis, for example, the doses administered will be of the order of ten micrograms.

In one embodiment, the divalent cation solutions have concentrations between 0.01 and 1 M, preferably between 0.05 and 0.2 M.

In one embodiment, for a vertebral implant the angiogenic growth factor doses will be between 0.05 mg and 8 mg, preferably between 0.1 mg and 4 mg, more preferably between 0.1 mg and 15 mg. mg.

The invention also relates to the process for preparing the compositions according to the invention which comprises at least the following steps: a) an organic matrix is available, b) said matrix is impregnated with a solution comprising an osteogenic growth factor c) adding to the matrix obtained in step b) a solution of a soluble salt of at least divalent cation d) the lyophilization of the matrix obtained in step c) is optionally carried out.

In one embodiment in step c), the at least divalent cation soluble salt solution is a divalent cation solution.

In one embodiment, the soluble salts of divalent cation are calcium salts whose counterion is selected from chloride, D-gluconate, oxalate, hydroxide, formate, D-saccharate, phosphate, carbonate, acetate, sulfate, L-ascorbate, L-tartrate, L-lactate, glutamate, aspartate. In one embodiment, the soluble salts of divalent cation are magnesium salts whose counterion is selected from chloride, D-gluconate, oxalate, hydroxide, formate, D-saccharate, phosphate, carbonate, acetate, sulfate, L-ascorbate, L-tartrate, L-lactate, glutamate, aspartate.

In one embodiment, the soluble salts of divalent cation are zinc salts whose counterion is chosen from chloride, D-gluconate, oxalate, hydroxide, formate, D-saccharate and phosphate. , carbonate, acetate, sulfate, L-ascorbate, L-tartrate, L-lactate, glutamate, aspartate.

In one embodiment, the divalent cation soluble salt is calcium chloride. In one embodiment in step c), the matrix is impregnated with a solution of a soluble salt of multivalent cation.

In one embodiment the multivalent cations are selected from the group consisting of multivalent cations of iron, aluminum, cationic polymers such as polylysine, spermine, protamine, fibrin

In one embodiment in step a), the matrix is also impregnated with a solution of non-osteogenic growth factor.

The invention also relates to the use of the composition according to the invention as a bone implant.

In one embodiment, said composition may be used in combination with a prosthetic device of the type of vertebral prosthesis or vertebral fusion cage.

It also relates to therapeutic and surgical methods using said composition in bone reconstruction. The invention is illustrated by the following examples.

Example 1: Preparation of collagen sponge / rhBMP-2 implants

Implant 1: 40 μl of a solution of rhBMP-2 at 0.05 mg / ml is sterilely introduced into a Helistat-type sterile 200 mm3 cross-linked collagen sponge (Integra LifeSciences, Plainsboro, New Jersey). The solution is allowed to incubate for 30 minutes in the collagen sponge before use. The dose of BMP-2 is 2 μg.

Implant 2: It is prepared as implant 1 with 40 μl of a solution of rhBMP-2 at 0.5 mg / ml. The dose of BMP-2 is 20 μg. Example 2 Preparation of Implants Collagen Sponge / RhBMP-2 with Calcium Chloride

Implant 3: 40 μl of a 0.5 mg / ml rhBMP-2 solution is sterilely introduced into a Helistat-type sterile 200 mm3 crosslinked collagen sponge (Integra LifeSciences, Plainsboro, New Jersey). The solution is allowed to incubate for 30 minutes in the collagen sponge before adding 100 μl of a solution of calcium chloride at a concentration of 18.3 mg / ml. The sponge is ready for use after 15 minutes. The dose of BMP-2 is 20 μg.

EXAMPLE 3 Preparation of Implants Collagen Sponge / RhBMP-2 with Calcium Chloride, Freeze Dried

Implant 4: 40 μl of a solution of rhBMP-2 0.05 mg / ml at 0.5 mg / ml is sterilized in a Helistat-type sterile 200 mm3 cross-linked collagen sponge (Integra LifeSciences, Plainsboro, UK). New Jersey). The solution is allowed to incubate for 30 minutes in the collagen sponge before adding 100 μl of a calcium chloride solution at a concentration of 18.3 mg / ml. The sponge is then frozen and sterilized lyophilized. The dose of BMP-2 is 2 μg. Implant 5: It is prepared as implant 4 with 40 μl of a solution of rhBMP-2 at 0.5 mg / ml. The dose of BMP-2 is 20 μg.

Example 4 Evaluation of the Osteoinductive Power of the Different Formulations

The objective of this study is to demonstrate the osteoinductive power of different formulations in a model of ectopic bone formation in rats. Male rats of 150 to 250 g (Sprague Dawley OFA-SD, Charles River Laboratories France, B.P. 109, 69592 l'Arbresle) are used for this study. Analgesic treatment (buprenorphine, Temgesic, Pfizer, France) is given before surgery. Rats are anesthetized by inhalation of a 02 isoflurane (1-4%) mixture. The fur is removed by shaving over a wide dorsal area. The skin of this dorsal zone is disinfected with a solution of povidone iodine (Vetedine solution, Vetoquinol, France). Paravertebral incisions of about 1 cm are made to clear the left and right paravertebral dorsal muscles. Access to the muscles is performed by transfacial incision. Each of the implants is placed in a pocket in such a way that no compression on them can be exerted. Four implants are implanted per rat (two implants per site). The opening of the implants is then sutured using a polypropylene wire (Prolene 4/0, Ethicon, France). The skin is closed with a non-absorbable suture. The rats are then returned to their respective cages and kept under observation during their recovery. At 21 days, the animals are anesthetized by an injection of tiletamine-zolazepam (ZOLETIL 25-50 mg / kg, IM, VIRBAC, France).

The animals are then euthanized by injection of a pentobarbital dose (DOLETHAL, VETOQUINOL, France). A macroscopic observation of each site is then carried out, any sign of local intolerance (inflammation, necrosis, haemorrhage) and the presence of bone and / or cartilage tissue is recorded and scored according to the following scale: 0: absence, 1: low, 2: moderate, 3: marked, 4: important.

Each of the implants is removed from its implantation site and macroscopic photographs are taken. The size and weight of the implants are then determined. Each implant is then stored in a 10% buffered formalin solution.

Results: This in vivo experiment measures the osteoinductive effect of BMP-2 by placing the implant in a muscle of the back of a rat. This non-osseous site is said to be ectopic.

Macroscopic observations of explants allow us to evaluate the presence of bone tissue and to determine the mass of implants. Implant Presence of bone tissue Mass of implants (mg) Implant 1 Implants not found Implant 2 3.6 38 Implant 3 4 75 Implant 4 3.2 26 Implant 5 3.3 171 A dose of 2 μg of BMP-2 in a collagen sponge (Implant 1) does not have sufficient osteoinductive power to be able to find the collagen implants after 21 days.

A 20 μg dose of BMP-2 in a collagen sponge (Implant 2) has sufficient osteoinductive power to achieve 21-day ossified implants with an average weight of 38 mg.

For the same 20 microgram dose of BMP-2, the addition of calcium salts to the collagen sponge containing BMP-2 increases the osteogenic activity of BMP-2. The average mass of the implants 3 is twice as large as that of the implants 2 with an equivalent ossification score.

Also for the BMP-2 dose of 20 μg, freeze-drying makes it possible to increase the effect of the calcium salt on the osteogenic activity of BMP-2 (Implant 5). The average mass of lyophilized implants containing BMP-2 and CaCl2 is approximately four times that of implants containing only BMP-2 (implant 2). In addition, the bone score is equivalent between these implants. For a dose of BMP-2 of 2 μg, BMP-2 in the presence of freeze-dried CaCl 2 in the collagen sponge (Implant 4) makes it possible to generate ossified implants in contrast to BMP-2 alone at the same dose.

The new formulation can greatly reduce the doses of BMP-2 to be administered without reducing osteogenic activity.

Claims (18)

CLAIMS1 Osteogenic composition comprising at least: • an osteogenic growth factor, • a soluble salt of cation at least divalent • an organic matrix. 2. Composition according to claim 1, characterized in that said composition is in lyophilizate form. 3. Composition according to any one of the preceding claims, characterized in that the osteogenic growth factor is selected from the group of therapeutically active BMPs (Bone morphogenetic Proteins). 4. Composition according to any one of the preceding claims, characterized in that the osteogenic growth factor is selected from the group consisting of BMP-2 (Dibotermine-alpha), BMP-4, BMP-7 (Eptotermin). -alpha), BMP-14 and GDF-5. 5. A composition according to any one of the preceding claims characterized in that it further comprises angiogenic growth factors selected from the group consisting of PDGF, VEGF or FGF. 6. Composition according to any one of the preceding claims, characterized in that the cation is a divalent cation selected from the group consisting of calcium, magnesium or zinc cations. 30 7. Composition according to any one of the preceding claims characterized in that the soluble salt of divalent cation is a calcium salt whose counterion is selected from chloride, D-gluconate, oxalate, hydroxide, formate, D-saccharate, phosphate, carbonate, acetate, sulfate, L-ascorbate, L-tartrate, L-lactate, glutamate, aspartate. 8. Composition according to any one of the preceding claims, characterized in that the soluble salt of divalent cation is calcium chloride. 9. Composition according to any one of claims 1 to 5, characterized in that the cation is a multivalent cation selected from the group consisting of iron cations, aluminum or cationic polymers selected from polylysine, spermine, protamine and fibrin, alone or in combination. 10. Composition according to any one of the preceding claims, characterized in that the matrix is chosen from matrices based on natural collagen. 11. Composition according to any one of the preceding claims, characterized in that the organic matrix further comprises inorganic particles. 20 12. Process for preparing the compositions according to any one of claims 1 to 10 comprising at least the following steps: a) an organic matrix is available, b) said matrix is impregnated with a solution comprising an osteogenic growth factor. C) adding to the matrix obtained in step b) a solution of a soluble salt of at least divalent cation d) the lyophilization of the matrix obtained in step c) is optionally carried out. 30 13. The method of claim 12 characterized in that in step a), the matrix is also impregnated with a solution of an angiogenic growth factor selected from the group consisting of PDGF, VEGF or FGF. 15 14. Process according to any one of claims 12 to 13 characterized in that in step c) the soluble salt solution of at least divalent cation is a divalent cation solution. 15. Method according to any one of claims 12 to 14 characterized in that in step c) the soluble salt of divalent cation is a calcium salt whose counterion is selected from chloride, D-gluconate , oxalate, hydroxide, formate, D-saccharate, phosphate, carbonate, acetate, sulfate, L-ascorbate, L-tartrate, L-lactate, glutamate, aspartate. 16. Process according to any one of claims 14 to 15 characterized in that in step c) the soluble salt of divalent cation is calcium chloride. 17. Method according to any one of claims 12 to 13 characterized in that in step c) the matrix is impregnated with a solution of a soluble salt of multivalent cation. 18. Use of a composition according to any one of claims 1 to 11 as a bone implant.