WO2006072613A2 - Preparation comprising microparticles of an insoluble polysaccharide resin with a biopolymeric active ingredient and method for production thereof - Google Patents

Abstract

The invention relates to a preparation for the sustained in-vivo release of an active ingredient, comprising microparticles of an insoluble polymer salt, dispersed in a liquid medium and having functional groups forming an ionic bond with the active ingredient and the use thereof as a medicament. In a preferred embodiment, the microparticles are encapsulated by coacervation in aqueous medium. The invention further relates to a method for production of said preparation.

Description

 PREPARATION CONTAINING MICROPARTICLES OF AN INSOLUBLE POLYMER SALT CARRYING AN ACTIVE INGREDIENT, AND METHOD OF MANUFACTURING THE SAME

The invention relates to a preparation for the sustained in vivo release of an active ingredient, which comprises microparticles of an insoluble polymer dispersed in a liquid medium and carrying functional groups forming an ionic bond with the active ingredient, as well as its use as a medicine. In a preferred embodiment, the microparticles are encapsulated by coacervation in an aqueous medium. The invention also relates to a method of manufacturing such a preparation.

The use of very poorly soluble or insoluble polypeptide salts for the production of programmed release forms of peptides and proteins has already been described in the literature (US Pat. No. 4,010,125, Schalty et al. March 1977, US 5,776,885, Orsolini et al, July 1998, US 6,087,324, Igari et al, July 2000). Patent applications for ionic complexes between polypeptides and macromolecules have been filed. One of the examples relates to a composition comprising a polyester, preferably a homo- or copolymer of lactic and glycolic acid, containing one or more free COOH groups forming an ionic bond with a polypeptide comprising an amine in which at least 50% by weight of the The polypeptide present in the composition is ionically conjugated to polyester (US Patent No. 5,672,659, Shalaby et al, September 1997).

The second example relates to water-insoluble programmed release forms comprising a peptide-binding complex, preferably an LHRH analogue, and an anionic carboxymethylcellulose (CMC) macromolecule, wherein the peptide content in the complex is between 57% and 80% by weight (US Pat. No. 6,180,608, Gefter et al. January 2001). The method of manufacturing the complex necessarily implies that the macromolecule is soluble in an aqueous solvent. It is based on a coprecipitation of the peptide and the macromolecule.

Similarly, the international patent application published under the number WO 00/47234 (Asta Medica, August 2000) describes the obtaining of an insoluble ionic complex by coprecipitation of a solubilized macromolecule and a peptide. This application also describes the addition of an ion exchange resin to control the chemical reaction. This resin is then filtered and removed. In the end, there remains only the complex, or coprecipitate. In these examples, the macromolecule / peptide / complex ratio in the final formulation is not known.

On the other hand, the physicochemical interactions between polypeptides and ion exchange resins have been widely studied in the field of ion exchange chromatography, gel chromatography and high pressure liquid chromatography for the assay and the determination of peptide purities. Preparative-type chromatography techniques also contributed to a better understanding of these interactions. In chromatography, a column filled with a resin has certain characteristics which make it possible, thanks to the use of different solvents, to separate the active principles that pass through it. The different ways of interacting with the polypeptides are as follows: adsorption, partitioning, ion exchange, affinity, gel filtration. Ion exchange chromatography (IEC) is applicable to the separation of most charged molecules from large proteins to small nucleotides and amino acids. It is frequently used for peptides and proteins under various conditions.

The use as active ingredient or excipient of ion exchange resins administered orally is known. Advantages, such as the stabilization of fragile substances (perfumes), the protection of the gastric mucosa against aggressive substances such as nonsteroidal anti-inflammatory drugs (NSAIDs), the modification of the release of drugs by an effect gastro-resistant delay or taste masking for bitter active ingredients, have been described in the literature. A non-exhaustive list of commercial applications of ion exchange resins is given below:

- As active ingredients: sodium polystyrene sulfonate for the treatment of hyperkalemia and cholestyramine for the treatment of hypercholesterolemia,

As excipients: nicotine gum (Nicorette, Pfizer, France), stabilization of the vitamin B 12, taste-masking paroxetine (Paxil ^®, GlaxoSmithKline, UK), sustained release diclofenac (Voltaren ^®, Novartis, France), chlorpheniramine (Tussionex ^® , Celltech

Pharmaceuticals, USA), dextrometorphan (Delsym ^® , Celltech Pharmaceuticals, USA), betaxolol (Betoptic ^® , Alcon Laboratories, USA) and repaglinide (Novonorm ^® , Novo Nordisk, Denmark).

The majority of the resinase-based drugs described in the literature are made with "small molecule" chemical active substances that act at relatively high doses and are administered orally. The notion of delayed release with resinates is not new. It has been described for example by charging active ingredients on ion exchange resins (see

Remington's Pharmaceutical Sciences, 15th Ed. 1975). This prolongation of action is presumed to be the result of a decrease in the rate of displacement of the ions by other ions when the complex is in contact with the physiological fluids of the digestive tract. The attachment of an active ingredient to an ion exchange resin is considered as the result of an electrostatic interaction (Jenquin et al,

Int. J. of Pharmaceutics 101 (1994) 23-34). However, the complexes obtained cause a relatively short delay effect and a low control of the level of release of the drug because the control parameters are limited to the variation in size of the resin particles and the degree of crosslinking of the resin used.

Attempts to reduce the release rate by coating techniques have been investigated, but have shown relative inefficiency because the complex formed a gel very rapidly after contact with the physiological media. This property, currently underutilized, is particularly interesting parenterally. Numerous proposals such as the incorporation of solvents such as polyethylene glycol, aliphatic alcohols or cellulose ethers incorporated directly into the polymeric matrix have not given more convincing results (see US Pat. Nos. 4,221,778 and US Pat.

4,861,598) and the publication of Feely et al. (Int J. Pharmaceutics 44 (1988) 131-139). Other US patent publication numbers which describe improvements in drug release rates are given below: 4,996,047; 5,186,930; 4,894,239; 4,859,462; 4,959,219; 4,762,709; 4,999,189; 4,859,461; and 5,368,852.

Controlled release of an active ingredient for use in the gastrointestinal tract has been described in US Patent 4,221,778 (Raghunathan, September 1980). The method used to prepare the modified release forms is based on a three-step process (i) preparation of the resin-drug complex; (ii) treating this complex with a suitable impregnating agent; and (iii) coating the particles of the treated complex with a water permeable diffusion barrier. The use of impregnating agents is made to prevent swelling or breaking of the coating barrier.

The use of an enteric coating to delay absorption until the product outlet of the stomach is well known, and is described in particular in US Patent No. 5,851,579. However, the nature of the chemical active substances included in the resinous composition, in particular, the amount of active ingredient that is relatively important for a tablet or capsule, is responsible for a rapid release of the active ingredient from the resinate, followed by a phase of gradual reduction of the joint release to a decrease in the concentration of active substance. Because of this, it has been difficult in the various formulations described in the literature to provide constant, prolonged or delayed release. Therefore, applications of resin technology for the delivery of conventional oral chemicals have remained relatively limited in the pharmaceutical industry. Parenterally, ion exchange resins have been used as vectors of active principles (doxorubicin, cisplatin) for anti-tumor chemoembolization treatments (Chan et al., Pharm Phaπnacol 1992; 44: 211-7 and Codde JP et al. al, Anticancer Res 1990; 10: 1715-8). The effect sought in this case is a local diffusion of anti-cancer agents doubled by anoxia of the tumor by blocking the blood flow feeding it.

The matrix concepts of the modified release of an active ingredient have been developed for many years, and can be very beneficial for the delivery of polypeptides. By protecting the polypeptide, it is possible to increase the time during which the plasma concentration of the active substance is between a high limit defined by the toxicological properties of the molecule and a low limit defined by its effectiveness.

In the case of polypeptides administered primarily parenterally in the form of implants or microspheres, action times of several months are now controlled after a single injection, subcutaneously or intramuscularly. Formulations based on biodegradable polymers of the polyester or polyanhydride type are marketed in various therapeutic areas such as prostate cancer, breast cancer, endocrine digestive tumors, acromegaly, precocious puberty. The concept of releasing these first generation forms is related to the incorporation of particles or polypeptide solutions into a polymeric matrix which progressively degrades with time in the physiological fluids. It Examples of products marketed leuprolide (Lupron Depot ^10, TAP Pharmaceutical Products, USA), the octréoide (Sandostatin LAR ^®, Novartis, France), somatropin (Nutropin Depot ', Genentech, USA) or triptorelin ( Trelstar

Depot ^® , Pharmacia & Upjohn, USA) which are in the form of microspheres in which the biodegradable polymer is the copolymer of lactic and glycolic acid

(PLGA), or carmustine (Gliadel ^® in "wafer" form poly [bis (p-carboxyphenoxy) propane: sebacic acid] in a 20:80 molar ratio, Guilford

Pharmaceuticals, USA)), goserelin (Zoladex ^® under foπne "rod", PLGA, Astra Zeneca, USA) and doxycycline (Atridox gel, PLGA, CollaGenex Pharmaceuticals).

In the present invention, water-insoluble polypeptide salts have been developed which allow sustained in vivo release of polypeptides into physiological media. These salts are constituted by microparticles of an ion-exchange polymer to which a polypeptide is reversibly attached by an ionic bond. They induce a better protection of the polypeptide, a better bioavailability and a controlled release effect when the formulation is administered to mammals parenterally or by any conventional route of administration.

Compared to the matrix forms of the state of the art, the preparation according to the invention, also referred to herein as "polypeptide salts" or "polypeptide resinates," can be used directly for sustained release. in vivo and has a number of advantages that are summarized as follows:

Resinates (of polypeptides) are applicable to all peptides and ionized proteins. The physical and / or macroscopic appearance of the polypeptide is not a critical manufacturing parameter; Resinates allow a simple, economic and aseptic manufacture of galenic forms applicable to all types of peptides or ionized proteins;

- The use of radiation sterilization can be avoided;

- Resinates allow sustained release after parenteral administration over a period of time ranging from a few days to a few months depending on the specific therapeutic needs related to each peptide or protein. Indeed, the preparation according to the present invention makes it possible to avoid the effect of initial burst effect inherent in matrix encapsulation technology in polymers;

- Resinates provide protection and programmed release in the gastrointestinal tract, ensuring high bioavailability and reproducibility, which provides a therapeutic benefit in terms of safety and efficacy for patients. Indeed, the preparation according to the invention makes it possible to protect the peptide or the protein which it is desired to administer enzymatic and chemical attacks on the stomach, and to release the peptide in the intestine where it can be absorbed;

The composition of the preparation according to the invention is different from that of the prodrugs (in a "prodrug", there is a valid bond between the polypeptide and the macromolecule) which promote the stimulation of the immune system and the formation of neutralizing antibodies deleterious for the therapeutic activity of the polypeptide.

In contrast to the insoluble salts of the prior art, the present preparation corresponds to an insoluble salt which forms a single entity: the polymer and the polypeptide, or any other active principle as defined below, are mixed in stoichiometric quantities during the preparation salt ; after fixing the active ingredient on the polymer by ionic bonds, the non-fixed amount of active ingredient that remains in solution is removed. In addition, the present inventor has discovered that if the polypeptide salts according to the invention are injected directly, the release time of the polypeptides is increased. It would appear that in the body, a progressive ion exchange between ions present in vivo (sodium ions, chloride, ...) and ionized (or "charged") polypeptides occurs, the ions present in vivo "moving Polypeptides. Indeed, the polymers used can not be degraded in this period of time because of their high molecular weight. Moreover, the use of a very small amount of polypeptide attached to the polymer (of the order of milligrams or even nanograms) induces after parenteral administration, a protection effect of the polypeptide vis-à-vis fluids physiological and results in a prolonged release effect.

Thus, according to a first aspect, the subject of the invention is a preparation for the in vivo prolonged release of an active principle, said preparation comprising microparticles of an insoluble polymer, biologically acceptable and bearing functional groups forming an ionic bond with the active ingredient, said microparticles being dispersed in a biologically acceptable liquid. Preferably, the subject of the invention is a preparation for parenteral administration for the in vivo prolonged release of a biopolymer, said preparation comprising microparticles of an insoluble polysaccharide resin, biologically acceptable and carrying functional groups forming an ionic bond with the biopolymer, said microparticles being dispersed in a biologically acceptable liquid at a pH between 6.0 and 7.8.

By "pH between 6.0 and 7.8" it should be understood that the pH of the biologically acceptable liquid in which the microparticles are dispersed is between about 6.0 and about 7.8, those skilled in the art being accustomed to variations of ± 10 ^"2 to 10 ^" 'of the pH of a solution. Advantageously, the pH is between about 6.5 and about 7.5, particularly preferably about 7.2. It should be understood again that these pH values can vary very slightly, from ± 10 ^" to 10 ^" .

The polymer that can be used in the context of the present invention must meet two criteria: it must be insoluble and biologically acceptable.

By the term "biologically acceptable polymer" is meant in the present application a biocompatible polymer, that is to say, well tolerated in vivo and which does not cause rejection reaction, toxic reaction, or lesion on the skin. biological functions of a living organism, and biodegradable, that is to say degradable in vivo in simple elements that do not accumulate and participate in natural cycles. Preferably, the polymer is pharmacologically acceptable, i.e. it consists of a biologically acceptable polymer suitable for use for pharmaceutical purposes as an agent for administering an active ingredient. The term "insoluble" means in the present application that the polymer is not soluble or very slightly soluble both in the preparation buffer of the polypeptide salt according to the invention and in the biologically acceptable liquid with which it is associated in the final preparation. The solubility of a polymer depends on several criteria, in particular the type of molecules (monomer) of which it consists of its degree of polymerization (monomers repeated n times), and its molecular mass (MM). The higher the MM of the polymer, the less soluble the polymer. Similarly, when the polymer is crosslinked, its solubility will be a function of the degree of crosslinking. Examples of insoluble and biologically acceptable polymers include, but are not limited to, polysaccharides (dextran and derivatives, poly dextrose, agarose, carbohydrate-based polymers, chitin derivatives, chitosan, starch and its derivatives), polyaliphatic alcohols, such as polyethylene oxide and derivatives including PEG (polyethylene glycol), PEG-acrylates, polyethylene imines, polyvinyl polymers, polyvinyl alcohols, polyvinyl pyrrolidone, poly vinyl phosphate, polyvinylphosphonic acid and derivatives, and polyvinyl acetates and derivatives, polyacrylic and derivatives acids, polyacrylamides, poly esters and polyorthoesters, polyanhydrides, polyorganic acids such as as poly maleic acids and derivatives, poly amino acids and poly imino acids and derivatives, poloxamer 407 or Pluronic ^® L-IOL TM, tert-polymers and derivatives thereof, poly ethers such as poly (tetra methylene ether glycol) and derivatives, natural polymers such as zein and pullulan and derivatives, polyimides, such as poly tris (hydroxyethyl) methyl methacrylates and derivatives, surfactants, such as polyoxyethylene sorbitan and derivatives, branched or cyclic polymers such as branched PEGs and cyclodextrins and derivatives, polyaldehydes, such as poly (perfluoropropylene oxide-b-perfluoroformaldehyde) and derivatives. Poly styrene polymers (Amberlite ^® type, etc.) are not suitable for use in the context of the present invention. Indeed, such polymers are not biocompatible. The same goes for some poly vinyls such as polyvinyl chlorides.

As an example of polyester, there may be mentioned homo or copolymers of poly lactic acid or glycolic acid (PLGA) which, although they are not crosslinked, are insoluble in water when they have a sufficient MM. Preferably, the PLGA MM is greater than or equal to 5 Kdaltons.

It may also be mentioned as polysaccharide polymer insoluble in water, derivatives of cellulose (EC: ethyl cellulose, HERCULES, Germany), which, like PLGA, are non-crosslinked polymers.

Another example of a polysaccharide polymer preferentially used in the context of the present invention is dextran, a generic name given to a large family of polysaccharides of microbial origin which are assembled or polymerized outside cells by enzymes called dextrans. sucrases. This class of polysaccharides is composed of glucose monomers and is stored as a source of energy by yeasts and bacteria. Dextrans are produced by fermentation or enzymatic conversion of sucrose, a product of the sugar cane industry or beets. Dextrans and drifts are generally soluble in aqueous buffers at at least 10 mg / ml. Their solubility decreases when the MM increases. The maximum solubility is 1 mg / ml for a dextran of 3000 daltons, 50 mg / ml for a dextran of 10,000 daltons, 25 mg / ml for a dextran of 70,000 daltons and 5-10 mg / ml for dextrans included between 500000 and 2000000 daltons. For a higher rate of biodegradation, MM less than 2000000 Daltons are preferred for use of the parenteral polypeptide resins.

Thus, according to a particular embodiment, the preparation according to the invention is characterized in that the polymer is a polymeric resin of a polysaccharide nature. Preferably, the polymeric resin of polysaccharide nature is of the dextran type. More preferably, the dextran has a molecular weight of between 3 Kdaltons and 2000 Kdaltons, preferably between 50 Kdaltons and 1000 Kdaltons, even more preferably between 100 Kdaltons and 500 Kdaltons, and most preferably between 100 Kdaltons. Kdaltons and 200 Kdaltons.

The insoluble and biologically acceptable polymer as defined in this invention carries functional groups. In the present application, these functional groups are ionizable. Thus, when the polymer carrying these groups, or "modified" polymer, is brought into contact with a preparation buffer such as water for injection (water ppi), physiological saline or pH-controlled buffer ( Tris (hydroxy methyl) amino methane, ...), these groups will ionize ("salif"), but the polymer does not will not be solubilized for as much. Thus, for example, modified dextran, modified agarose, modified PLGA, modified starch, or modified EC will be discussed. The ionized functional groups can be strongly acidic, for example sulphonic acid or phosphoric acid, weakly acidic, for example carboxylic acid, weakly basic, such as, for example, highly basic primary amines, for example a quaternary ammonium or a combination of acidic and basic groups.

In the present application, the term "ion exchange resin" or "polymeric ion exchange resin" will be indifferently referred to as the polymer according to the invention which carries these groups, or polymer

"Modified". Indeed, the "modified" polymer or ion exchange resin, is characterized by its ability to exchange ions. This characteristic is quantified by the "ion exchange capacity" measured by the equivalent number of an ion that can be exchanged and which is expressed relative to the mass of polymer "bulk capacitance" or in relation to the volume of resin "volume capacity". A unit frequently used for bulk capacities is the milliequivalence of exchange capacity per gram of dry resin, in abbreviation "meq / g".

The ion exchange resin, when crosslinked, forms a porous matrix, and consists of a tri-dimensional network. This type of resin is particularly insoluble.

Thus, preferably, the ion exchange resin is crosslinked; may be mentioned as an example of cross-linked ion exchange resin cross-linked dextran available under the tradename Sephadex ^®, usually used as highly specialized gel filtration and chromatography medium, in particular, ion exchange resins composed of beads microscopic. In this case, the organic polymer chains are crosslinked to give a three-dimensional network having ionic functional groups covalently attached to the glucose units of the polysaccharide chains.

We can also mention the specialty called Deflux ^® (Q-MED, Sweden), approved in the USA by the FDA (Deflux ^® was also approved in Europe in 1998). This specialty is a gel containing microparticles that is injected into the bladder wall where the ureters enter the bladder. The gel consists of two well known polysaccharides, dextranomer and hyaluronic acid. Dextranomers are crosslinked dextran molecules presented as microparticles with a diameter of 40 to 120 μm. The dextranomer is 2- (diethylamino) ethyl Sephadex ^®, and can be considered as a weak anionic exchange resin. Both substances are perfectly tolerated by the tissues and are biodegradable over a period of several months. Dextranomer has been used for more than 30 years as a wound and ulcer treatment agent (Debrisan ^® , Pharmacia & Upjohn, USA) and has no side effects. The dextranomer or DEAE-Sephadex ^® is an ion exchange resin positively charged which is capable of reacting with an ionized polypeptide. The size of the microparticles of the dextranomer evolves from 40 μm to 120 μm. Sephadex ^®, in particular SP Sephadex ^® C50 and QAE (Aldrich Chemical, USA), are part of the crosslinked ion exchange resins which are preferably used in the context of the present invention. The ion exchange resins may be acidic in nature (in this case the resin is a cation exchange) or basic (anion exchange). In the resinates of this invention, the insoluble charged (ionized) resin particles are reversibly bound to the polypeptides, or any other active ingredient, which are positively or negatively charged depending on the pH and the amino acids that compose them. Small strongly charged ions are used to make the elutions, that is to say the capacity to exchange one ion by another: CF, HO ^" , Na ⁴ , H ^1" ...

The cationic or anionic ion exchange resins may be classified as weak or strong ion exchange resins depending on the nature of the charged functional groups bonded (covalently) to the optionally crosslinked polymeric support of the resin.

The microparticles of the polymer according to the invention must be of appropriate size for their administration in vivo. Preferably, the diameter of the microparticles is between about 0.1 μm and about 2000 μm, preferably between about 20 μm and 200 μm, and preferably between about 0.5 μm and 200 μm. By "active ingredient" is meant in the present application any ionizable compound having curative properties (antibiotics, anti-cancer, anti-HIV, ...) or preventive (vaccines, ...) with respect to human diseases or animal, as well as any compound that can be administered to humans or animals, in order to establish a medical diagnosis or to restore, correct, or modify their organic functions. The active ingredient may be a polypeptide, a nucleic acid, or any other organic, chemical or biochemical molecule, as well as derivatives and analogues thereof.

By "biopolymer" is meant in this application a polypeptide or polynucleotide.

The terms peptide, polypeptide and protein are used indifferently in the present application to designate an amino acid sequence or, for their derivatives (glycoproteins, lipoproteins), containing an amino acid sequence. By way of example and without limitation are cytokines, cell receptors, antibodies, antigens, toxins and allergens.

The length of the amino acid sequence can be from two to several hundred amino acids. Preferably, the length is between 2 and 100 amino acids, preferably between 8 and 50 amino acids, and more preferably between 8 and 25 amino acids.

By nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, which are used interchangeably in the present description, is meant a nucleotide sequence, modified or not, to define a fragment or a region of a nucleic acid, with or without unnatural nucleotides, and which may be as well a double-stranded DNA, a single-stranded DNA or a

CDNA only to transcripts of said DNA (RNA, interference RNA or siRNA for "small interfering RNA", etc.). Examples of other organic molecules include, but are not limited to, carbohydrates, lipid compounds and all natural or chemically synthesizable compounds.

For example, the following active principles may be mentioned (this list of active principles is not limiting and may be supplemented by other active ingredients which have sufficient biological activity):

insulin, proinsulin, glucagon, parathyroid hormone, calcitonin, vasopressin, erythropoietin (EPO), renin, prolactin, human growth hormone (hGH), thyroid stimulating hormone (TSH), corticotropin, follicle stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin, atrial peptides, interferon, tissue plasminogen activator ( tPA), gamma globulin, urokinase, streptokinase, lymphokines such as interleukins and colony stimulating factors (CSF), growth hormone releasing factor (GHRF), corticotropin (CRGHRF), luteinizing hormone releasing hormone (LHRH), somatostatin, calcitonin, thyrotropin releasing hormone (TRH), calcitonin gene-related peptide (CGRP) , "glucagon-like peptide" (GLP), parathyroid hormone and peptide bound with parathyroid hormone (PHRP), their agonists, antagonists and the like, a transferase, a hydrolase, an isomerase, a protease, a ligase, an oxidoreductase, an esterase, and a phosphatase, streptokinase, fibrinolysin, deoxyribonuclease, and asparaginase, an endogenous opioid agonist such as enkephalin or endorphin, a hypothalamic hormone such as gonadotropin releasing hormone, melanostatin, melanoliberin, somatostatin, thyrotropin releasing hormone, substance P, and neurotensin, a hormone adenohypophyseal such as corticotropin, lipotropin, melanotropin, lutropin, thyrotropin, prolactin and somatotropin, a neurohypophyseal hormone, a calcitropic hormone such as parathyrin and calcitonin, a thymic factor such as thymosin, thymopoietin, thymic circulation factor (CTF) and thymic humoral factor (THF), a pancreatic hormone such as somatostatin, a gastrointestinal hormone such as gastrin, cholecystokinin, secretin, gastric inhibitory polypeptide (GIP), vaso-intestinal peptide and motillin, a chorionic hormone such as choriogonadotropin and choriomammotropin, an ovarian hormone such as relaxin, a vasoactive hormone such as angiotensin and bradykinin, a neurotrophic factor such as cystic neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF), a growth factor such as nerve growth factor (NGF), epidermal growth (EGF), the fibroblast growth factor

(FGF), insulin-like growth factor (IGF), tumor necrosis factor (TNF) and transforming growth factor (TGF), somatomedin and urogastrone, a coagulation factor such as factors VIII and IX an artificial peptide or pseudo-peptide such as deferoxamine, and a somatostatin analogue such as octreotide and lanreotide, an LHRH analogue such as nafarelin, buserelin, leuprorelin, goserelin, deslorelin, gonadorelin, triptorelin, and histrelin, artificial peptides or pseudo-peptides, such as deferoxamine, more generally, the peptides and proteins suitable for the present invention are described in the book by Johannes Meienhofer, "Peptide and Protein Hormones ", Burger's Medicinal Chemistry, 4th ed., (Part II), Wolff, Ed., John Wiley and Sons (1979).

According to a preferred embodiment, the preparation according to the invention is characterized in that the active principle is a polypeptide. Preferably, the polypeptide is a hormone, a coagulation factor, a neurotrophic factor or a growth factor. More preferably, the polypeptide is luteinizing hormone releasing hormone (LFIRH) or an analog thereof. In the present application, the term "analogue" of a polypeptide means an agonist or an antagonist of this polypeptide.

According to another preferred embodiment, the polypeptide is an interferon, the polysaccharide resin being advantageously an anion exchange resin.

Preferably, the sustained release in vivo is a release in a mammal, preferably the human. The present preparation may be administered in any convenient manner such as oral, vaginal, rectal, or parenteral administration, such as subcutaneous or intramuscular injection.

According to a particular embodiment, the preparation according to the invention is intended to be used as a medicament.

In general, the active ingredients of the present invention will be administered in a therapeutically effective amount by any of the accepted modes of administration. The appropriate dosage ranges are typically 1 to 500 mg per day, preferably 1 to 100 mg per day, and more preferably 1 to 30 mg per day. However, the preparation according to the invention also makes it possible to very advantageously administer very small quantities of active principle, of the order of one microgram or even one picogram.

In another aspect, the present invention relates to the use of the preparation according to the invention for the manufacture of a medicament for preventing or treating a disease selected from the group consisting of cancer, in particular cancer of the prostate cancer, ovarian cancer or breast cancer, diabetes, multiple sclerosis, viral hepatitis, cachexia, osteoporosis, stunting, endocrine digestive tumors, or any other be treated with an active ingredient according to the invention. The use according to the invention comprises the particular embodiments and advantages of the invention as defined above.

The present invention relates to a method for treating or preventing a disease by administering the preparation according to the invention. Disease may be cancer, including prostate cancer, ovarian cancer or breast cancer, diabetes, stunted growth, endocrine digestive tumors, or any other disease that may be treated with an active ingredient according to the invention.

The preparation according to the invention is generally in hydrated form. In some cases it forms an aqueous gel. Indeed, after it has been prepared, it is in the preparation buffer. In general, the preparation buffer is also the biologically acceptable liquid in which the preparation is located when it is to be administered. The preparation is therefore directly administrable, in particular parenterally.

Thus, the preparation buffer or the biologically acceptable liquid may be any liquid which allows the ionization of the functional groups carried by the polymer and which is biocompatible, that is to say, well tolerated in vivo and which does not cause a reaction. of rejection, toxic reaction, or injury to the biological functions of a living organism. As an example of preparation buffer, or biologically acceptable liquid, mention may be made of, but not limited to, water for injection (water ppi, COOPER, France), physiological saline (COOPER, France) or a pH buffer controlled (the buffers are made to order), for example 0.5M tris (hydroxy methyl) amino methane buffer or 0.5M sodium acetate buffer.

However, it is conceivable to replace the preparation buffer at the end of the manufacturing process with another biologically acceptable buffer. This is particularly the case when the preparation is dehydrated for storage and subsequently rehydrated in a biologically acceptable liquid for its administration.

When the preparation, initially in gel form (dispersed microparticles), is dehydrated, it is generally in solid form. Dehydration can be partial or total. The preparation according to the invention in dehydrated form can also be administered.

Thus, according to a particular embodiment, the preparation is either hydrated or dehydrated. Dehydration is preferably carried out by lyophilization or drying in the oven.

Preferably, the preparation according to the invention is administered parenterally, orally, rectally or vaginally.

The preparation according to the invention may be in the form of a powder, a tablet, a pill, a capsule, a cachet, a suppository or a dispersible granule. The processes for obtaining these galenic forms are well known to those skilled in the art.

According to another embodiment, the microparticles are incorporated into a matrix of a biodegradable and biocompatible polymer of the microsphere or implant type. Preferably, the preparation is administered parenterally

(injection).

According to an alternative embodiment, the microparticles are covered with an envelope of a biodegradable and biocompatible polymer and form microcapsules. Preferably, the preparation is administered parenterally (injection).

More preferably, the microcapsules are obtained by aqueous coacervation.

The biodegradable and biocompatible polymer making it possible to obtain the preparation according to the invention in the form of microspheres, implants or microcapsules may be any type of suitable biocompatible polymer such as poly esters or polyanhydrides, which is well known to the skilled person. Preferably, the polymer is a poly ester of homo type or copolymer of lactic and / or glycolic acid, more preferably a copolymer of lactic and glycolic acid (PLGA), and even more preferably, the PLGA 50/50 (BOEHRINGER INGELHEIM, Germany).

The person skilled in the art knows the processes for manufacturing the microsphere forms, implants or microcapsules, and knows how to adapt them to the preparation according to the invention.

According to another aspect, the subject of the present invention is a preparation for the in vivo prolonged release of an active principle, said preparation comprising microparticles of an insoluble polymer, biologically acceptable, and carrying functional groups forming an ionic bond with the active ingredient, said microparticles being dispersed in a biologically acceptable liquid, characterized in that it is obtainable by the process comprising the following steps: optionally, sterilization of the polymer, - addition of a preparation buffer to a polymer, said polymer being insoluble in the preparation buffer, ionization of an active principle in distilled water or in an aqueous buffer, formation of the preparation by placing the active ingredient in the presence of the polymer when the pH of the preparation buffer is below the point isoelectric (IpH) of the active ingredient, by fixing the active ingredient whose overall surface charge is positive on the polymer whose functional groups are negative, or when the pH of the preparation buffer is greater than the isoelectric point (IpH) of the active ingredient by fixing the active ingredient whose overall surface charge is negative on the polymer whose Functional issues are positive, and optionally, washing of the preparation. Preferably, the present invention relates to a preparation for parenteral administration for the in vivo prolonged release of a biopolymer, said preparation comprising microparticles of an insoluble polysaccharide resin, biologically acceptable, and carrying functional groups forming an ionic bond with the biopolymer, said microparticles being dispersed in a biologically acceptable liquid at a pH of between 6.0 and

7.8, characterized in that it is obtainable by the process comprising the following steps: optionally, sterilization of the polysaccharide resin, addition to a pH of between 6.0 and 7.8 of a preparation buffer a polysaccharide resin, said polysaccharide resin being insoluble in the preparation buffer, ionizing a biopolymer in distilled water or in an aqueous buffer, forming the preparation by placing the active ingredient in contact with the polysaccharide resin when the pH of the preparation buffer is lower than the isoelectric point

(IpH) of the biopolymer, by fixing the biopolymer whose overall surface charge is positive on the polysaccharide resin whose functional groups are negative, or when the pH of the preparation buffer is greater than the isoelectric point (IpH) of the biopolymer, by fixing biopolymer whose overall surface charge is negative on the polysaccharide resin whose functional groups are positive, and optionally washing the preparation.

The preparation according to the invention obtainable by the process as defined above comprises the particular embodiments and advantages of the invention as defined above.

Preferably, the method further comprises, after the step of forming the preparation, an aqueous coacervation step by placing the preparation in the presence of a polyester of homo- or copolymer type of lactic acid and / or glycolic, preferably dissolved in a polar solvent of the PEG 400 type.

Preferably, the homopolymer or copolymer type of lactic acid and / or glycolic acid is a copolymer of lactic acid and glycolic acid (PLGA).

According to yet another aspect, the subject of the present invention is a process for the manufacture of a preparation for the in vivo prolonged release of an active principle, said preparation comprising microparticles of an insoluble, biologically acceptable polymer carrying groups. functional groups forming an ionic bond with the active ingredient, said microparticles being dispersed in a biologically acceptable liquid, said process comprising the following steps: optionally, sterilization of the polymer, adding a preparation buffer to a polymer, said polymer being insoluble in the preparation buffer, ionizing an active ingredient in distilled water or in an aqueous buffer, forming the preparation (or "salification") by placing the active ingredient in contact with the polymer when the pH of the preparation buffer is lower than the isoelectric point (IpH) of the active principle, by fixing the active ingredient whose overall surface charge is positive on the polymer whose functional groups are negative, or when the pH of the preparation buffer is greater than the isoelectric point (IpH) of the active principle, by fixing the active ingredient whose overall surface charge is negative on the polymer whose functional groups are positive, and - optionally, washing of the preparation.

Preferably, the subject of the present invention is a process for the manufacture of a preparation for parenteral administration for the in vivo prolonged release of a biopolymer, said preparation comprising microparticles of an insoluble polysaccharide resin, biologically acceptable, and carrying groups. functional forming an ionic bond with the biopolymer, said microparticles being dispersed in a biologically acceptable liquid at a pH of between 6.0 and 7.8, said process comprising the following steps: optionally, sterilizing the polysaccharide resin, adding to a pH between 6.0 and 7.8 of a polysaccharide resin preparation buffer, said polysaccharide resin being insoluble in the preparation buffer, ionization of a biopolymer in distilled water or in an aqueous buffer; formation of the preparation (or "salification") by placing the biopolymer in the presence of and polysaccharide resin when the pH of the preparation buffer is lower than the isoelectric point (IpH) of the biopolymer, by fixing the biopolymer whose overall surface charge is positive on the polysaccharide resin whose functional groups are negative, or when the pH of the preparation buffer is greater than the isoelectric point (IpH) of the biopolymer, by fixing the biopolymer whose overall surface charge is negative on the polysaccharide resin whose functional groups are positive, and optionally washing the preparation.

The manufacturing method according to the invention comprises the particular embodiments and advantages of the invention as defined above.

The sterilization of the polymer can be obtained by any appropriate method known to those skilled in the art, in particular by autoclaving. Preferably, the sterilization of the polymer is obtained by autoclaving at a temperature of between 100 ^° C. and 150 ^° C., preferably between 110 ^° C. and 130 ^° C., and more preferably at 121 ° C. for about 30 minutes.

On the other hand, the active principle solution is optionally previously filtered on a porous membrane, preferably having a porosity of between 0.1 μm and 2 μm, more preferably between 0.2 μm and 0.5 μm. and most preferably 0.22 μm.

A polypeptide possesses a net charge due to charged amino acids that compose depending on the pH of the medium (see Table N ⁰ I below). IpH is the pH for which the polypeptide has a net charge of 0. For example, at pH 7.0, the charge of the amino acid aspartate (pK: 3.9) is negative, the cysteine charge (pK: 8.3 ) is neutral and the charge of arginine (pK: 12.5) is positive. Table No. I: Charge of ionizable amino acids at pH 7

With regard to the ionic charges of existing polypeptides, 85% of the polypeptides at pH 7.0 are negatively charged and conversely 15% of the polypeptides at pH 7.0 are positively charged.

In general, a polypeptide will bind to a negatively charged cation exchange resin if the pH of the preparation medium is lower than the isoelectric point (IpH) of the polypeptide, and bind to a charged anion exchange resin. positively if the pH is higher than IpH. The optimum pH stability of a polypeptide is at about pH 7.0.

When the preparation according to the invention (or "polypeptide resinate") is exposed to physiological fluids, the active ingredient is released from the resinate by an ion exchange mechanism. The rate of release of the active principle of the preparation according to the invention depends on several factors, as for example, but the list is not limiting, the size of the microparticles, the pK of the ionized functional groups of the polymer, if appropriate, the nature and degree of crosslinking of the polymeric chains of the ion exchange resin, the solubility of the active ingredient in the physiological fluids, the ionic bond (which may be partial or total), the pH of the physiological fluids, the pK of the principle active, the MM of the polymer and that of the active ingredient.

The property of a polypeptide that governs its adsorption on an ion exchange resin is its net surface charge (see Figure 2). Since the surface charge is the resultant of the acidic and basic functional groups of the active ingredient, the adsorption on the resin is highly sensitive pH. Starting from a low pH to a high pH, the net charge of the polypeptide varies from a positive charge to a negative charge. The knowledge of the isoelectric point (IpH) of the The polypeptide is therefore absolutely necessary in the design of the methods for preparing the resinates of polypeptides.

The polypeptide resinates thus formed are washed and packaged for use. At no time in the process, the ion exchange resins are solubilized in the preparation medium.

Preferably, the microparticles of the polymer are pre-sterilized by autoclaving at 121 ^° C. for 30 minutes in distilled water or in a buffer. Choice of the ion exchange resin depending on the nature of the polypeptide: The polypeptides are attached to the ion exchange resin when the functional groups of the resin carry charges opposite to the polypeptide. Fixation of the polypeptide on the resin is reversible. For example, the IpH of the ovalbumin protein is 4.5.

For the binding reaction, a positively charged anion exchange resin should be used at a pH greater than IpH, i.e. from 5, where the protein is negatively charged. In this case, a Sephadex QAE-50 type anion exchange resin is usable. The Sephadex QAE-50 resin has [ ⁺ NH] / [-N] amino functional groups characterized by a high pKa of 12. At a pH lower than the pKa (acidic pH), the functional groups of the resin will be predominantly under ionized form [-NH ⁺ ]. At a fold greater than pKa the functional groups of the resin are un-ionized.

The choice of the resin is made according to the stability of the polypeptide at the pH fixation conditions and the type of controlled release forms desired. With this same protein, it is possible to use a negatively charged cation exchange resin but at a reaction pH lower than IpH, that is to say below 4. In this case a cation exchange resin of the type Sephadex

SP C-50 is indicated. The Sephadex SP C-50 resin has sulfonate functional groups [-SO ₃ ^" ] / [-SO ₃ H] characterized by a very low pKa of the order of 2.5 at a pH below the pKa (acidic pH). the functional groups of the resin will predominantly be in non-ionized acid [-SO ₃ H] form at a pH greater than pKa the functional groups of the resin are ionized.

In summary : - If the polypeptide is more stable below its IpH, it will be necessary to use in formulation a cation exchange resin.

- If the polypeptide is more stable above its IpH, it will be necessary to use in formulation an anion exchange resin. If the polypeptide is stable over a wide pH range, all types of resins can be used.

Choice of the type of buffer for the reaction of fixation of the polypeptide on the resin: If the ions of the buffer used as a resinate preparation medium have a charge opposite to that of the functional groups of the resin, they will participate in the exchange phenomenon of ions and will cause local variations in pH. It is therefore necessary to use buffers containing ions of the same charge, positive or negative, as the functional groups of the resin. Gel capacity of ion exchange resins:

Sephadex ion exchange resins are insoluble in most solvents. They are stable in water, saline solutions, organic solvents, in acidic and basic solutions. In strongly basic solutions, hydrolysis of glycosidic bonds can occur and therefore in formulation, a pH of use above 2 is recommended. Exposure to highly oxidizing agents or dextranases should be avoided. The microparticles of resins alone or the resinates of polypeptides are able to swell in the presence of water to form gels. This property is directly applied for parenteral formulations. Because of the presence of charged functional groups, the gelation capacity varies according to the ionic strength and the pH of the preparation solvent. Sephadex ion exchange resins should not be swollen in distilled water because the structure of the beads can be broken by strong ionic interactions between the molecules. Sephadex QAE and SP resins have pH-independent gelling properties because they are ionized over a very wide pH range. The degree of gelation decreases as the ionic strength increases. The injection of these gels through conventional systems of syringes and needles is particularly easy, even subcutaneously. The ion exchange resins can be sterilized by autoclaving at 121 ⁰ C for 30 minutes at neutral pH without questioning the physical integrity of the microscopic beads.

Fixing capacity of the polypeptide according to the nature of the ion exchange resin:

The ion exchange resins derived from Sephadex G-25 are more finely crosslinked than those based on Sephadex G-50 and will therefore be less swollen and will be more rigid. Resins derived from Sephadex G-50 are more porous and have better binding capacity for polypeptides of significant molecular mass (greater than 20,000 daltons). In the literature, an exclusion limit of 1 million daltons is allowed for Sephadex-type ion exchange resins.

Industrial application of polypeptide resinases: in vivo release of the polypeptide from a resinate: Case of parenteral administration of a peptide (IpH: 4, 5)

The pharmaceutical form is a more or less compact sterile aqueous gel or a lyophilisate, injected subcutaneously or intramuscularly. The peptide is formed of about ten amino acids whose IpH is about 4.5. The ion exchange resin used for the preparation of the resinate is a QAE - 50 type anion exchange resin which has amino functional groups. The peptide resinate was prepared in an aqueous buffer solution of Tris (hydroxy methyl) amino methane at 0.05 M ionic strength at a pH of between 6 and 7.5 and preferably at physiological pH 7.2. After injection into the muscle, the gel will gradually become hydrated. The pH of the medium is greater than the IpH of the peptide. The peptide is negatively charged and remains strongly attached to the positively charged anion exchange resin. The gelled polypeptide resinate remains in the form of an insoluble salt in the medium. Over time, the microparticles of resins which act as a reservoir of active principle will allow dissociation of the resinate which will release the peptide by an ion exchange mechanism in a programmed manner as a function of the foπnulation parameters (average diameter, mass molecular, degree of crosslinking, ....). According to a particular embodiment, the manufacturing method according to the invention is characterized in that it further comprises, after the step of forming the preparation, an aqueous coacervation step by placing the preparation in the presence of the preparation with a poly ester homo- or copolymer type of lactic acid and / or glycolic, preferably dissolved in a polar solvent PEG 400 type.

Preferably, the homopolymer or copolymer type of lactic acid and / or glycolic acid is a copolymer of lactic acid and glycolic acid (PLGA). Indeed, the present inventor has discovered that it is possible to form microcapsules extemporaneously just after obtaining the preparation. Due to the insolubility of the preparation according to the invention ("polypeptide resinate"), coacervation in aqueous medium is possible (see Example 5). Microencapsulation techniques by solvent evaporation or coacervation for peptides and proteins usually very soluble in water are carried out in organic medium using an apolar solvent of the acetone, methylene chloride, acetonitrile type (Okada H. et al., J Pharm Exp

Tech, 1988, 244: 744-750; U.S. Patent 4,673,595, Debiopharm, 1991, Orsolini et al.).

In addition to being difficult to transpose industrially and the presence of residual solvents, these techniques can affect the quality of peptides and encapsulated proteins (Sah H., "Protein instability to organic / water emulsification: implications for protein microencapsulated into microspheres", PDA, J Pharm Sci Technology, 53: 3-10, 1999).

In the present invention, homo or copolymers of lactic acid and / or glycolic acid (PLGA) are solubilized in a biocompatible polar solvent of the PEG 400 type. By simple mixing of the aqueous gel microparticles carrying functional groups forming an ionic bond with the active ingredient and the PLGA solution in PEG 400, microcapsules are formed by aqueous coacervation of PLGA, the PEG 400 being miscible with water.

The advantages of this technique are numerous: Because of the strong ionic bond between the active ingredient and the functional groups of the microparticles, the effectiveness of microencapsulation in water is very significant (by more than 80%).

The "burst effect" conventionally observed with the matrix systems is reduced because of the ionic bond.

The preparation of the microcapsules is extemporaneous. There is no need for specific microencapsulation equipment or reactor. Good injectability and tolerance of the preparation mainly composed of aqueous solvent. - Aseptic manufacture after sterilization of raw materials.

The figures and examples which follow make it possible to better describe the invention, but in no way limit its scope.

FIGURES

Figures IA and IB: Loads of ion exchange resins as a function of the pH of the medium. The ionic properties of the resins are dependent on the pH of the medium. The resins must be sufficiently charged to operate in ion exchange and allow reversible binding in the pH range of 4 to 8 where the majority of the polypeptides are stable.

Figure 2: Influence of pH on the net charge of a polypeptide.

A polypeptide will bind to a negatively charged cationic ion exchange resin in a buffer if the pH of the buffer is lower than the isoelectric point (IpH) of the polypeptide, and will bind to a positively charged anion exchange resin if the pH is higher than the IpH.

Figure 3: Measurement of testosterone level in the rat in mmol / l after subcutaneous administration of an LHRH analogue resinate. Chemical castration of the rats is observed over a period of one week for animals treated with a rebound phenomenon after D 15.

Figure 4: Manufacture of Polypeptide Resinates Negatively charged polypeptides replace the counterions and bind to the positively charged polymer particles. The counterions are removed in the buffer. There is thus formation of a stable insoluble salt.

Figures 5A and 5B: Photographs of resin particles embedded in the coacervate of PLGA 50/50.

See Example 5.

Figure 6: Principle of aqueous coacervation

EXAMPLES

Example 1

It is important to show that the polypeptide binds to the ion exchange resin as a function of pH conditions. The following method based on a UV / visible spectrophotometry technique has been developed. The protocol for binding the polypeptide to an ion exchange resin has made it possible to select an optimum pH for preparing the polypeptide resinates. This protocol is as follows: - Selection of a series of 10 glass tubes of 15 ml,

Add in each tube, 100 mg of ion exchange resin and then 10 ml of 0.5M buffer solution,

The gel formed is equilibrated in each tube by washing with the 0.5M buffer solution. The ion exchange resin is left standing for 1 hour (hydration phase of the resin). The gel formed is again equilibrated in each tube by washing with a 0.05M buffer solution.

The pH is adjusted in each tube to obtain a pH range compatible with the formation of the polypeptide resinate. A known amount of polypeptide is added to each tube.

The tubes are stirred in the same manner according to a determined protocol and the polypeptide is determined by UV / visible spectrophotometry at 280 nm in the supernatant after the salification reaction.

The following assay method was applied: The solution to be analyzed is placed in a 1 cm quartz vat and placed in a dual-beam UV / visible spectrophotometer (PERKIN ELMER). The samples were previously filtered on cellulose acetate pre-filters. Absorbances are used to calculate the polypeptide concentration as a function of a standard range prepared from known amounts of samples. The results of different assays performed with a model protein, ovalbumin, are given in Examples 2 and 3 below.

The results of the experiments clearly show that the ion exchange resins bind ovalbumin according to the pH conditions of the medium. The results are expressed in percentages of ovalbumin attached to the ion exchange resin.

Example 2

Ovalbumin fixation on a Sephadex SP C-50 cation exchange resin. The protocol for binding ovalbumin to the ion exchange resin is that described in Example 1.

0.1g of Sephadex SP C-50 are deposited in each 15ml glass tube. 10 ml of 0.5M sodium acetate buffer solution (pKa 4.6) are added to each tube. The Sephadex SP C-50 ion exchange resin is allowed to stand for 1 hour (hydration phase of the resin). The gel formed is equilibrated in each tube by removing the supernatant and washing with a 0.05 M sodium acetate buffer solution. The pH is then adjusted in each tube to obtain a range of pH from 3.0 for the tube N ⁰ I to 7.5 for the tube N ⁰ IO and varying from

0.5 in 0.5. The same quantity of ovalbumin, ie 4 mg, is added to each tube. The final volume of solution in each tube is of the order of 8 ml. The tubes are vortexed 1 minute at 1400rpm on an IKA MS2.

A fluffy gel is observed in tubes No. 1, 2, 3 and to a lesser extent in tube No. 4. The supernatant of each tube after decantation of the gel is taken by a 5ml syringe system (TERUMO) equipped with a 21 G needle, 0.8 × 50.

The samples, supplemented with the filtered solution of starting ovalbumin at 2 mg / ml in the buffer (tube N ⁰ 1 1) and the filtered sodium acetate buffer solution (tube

No. 12) are determined by UV / visible spectrophotometry according to the method described in Example No. 1.

The results are summarized in Table II below.

N ⁰ Table II: Percentage of ovalbumin attached to the cation exchange resin

Sephadex SP C-50.

Fixation of ovalbumin in aqueous solution is observed for the pH where the protein is positively charged (pH less than IpH: 4.5) and the ion exchange resin is negatively charged (pH greater than the pKa of the resin is 2 , 5).

Example 3

Fixing ovalbumin on exchange resin am ^'ons Sephadex QAE-50. The protocol for binding ovalbumin to the ion exchange resin is that written in Example 1.

0.1 g of Sephadex QAE-50 are deposited in each 15 ml glass tube. 10 ml of 0.5M tris (hydroxy methyl) amino methane buffer solution (pKa: 8.3) are added to each tube. The Sephadex QAE-50 ion exchange resin is allowed to stand for 1 hour (hydration phase of the resin). The gel formed is equilibrated in each tube by removing the supernatant and washing with a 0.05 M tris (hydroxy methyl) amino methane buffer solution. The pH is then adjusted in each tube to obtain a pH range starting from 6.0. for tube No. 1 at 10.5 for tube No. 10 and ranging from 0.5 to 0.5. The same quantity of ovalbumin, ie 4 mg, is added to each tube. The final volume of solution in each tube is of the order of 8 ml. The tubes are vortexed 1 minute at 1400rpm on an IKA MS2. A fluffy gel is observed in all the tubes. The supernatant of each tube after decantation of the gel is taken by a 5ml syringe system (TERUMO) equipped with a 21 G needle, 0.8 × 50. The samples, supplemented with the filtered buffer solution of tris (hydroxy methyl) amino methane (tube N ⁰ I 1) and the filtered solution of ovalbumin starting at 2 mg / ml in the buffer (tube N ⁰ 12) are determined spectrophotometrically. UV / visible according to the method described in Example No. 1. The results are summarized in Table III N ⁰ below. N ⁰ Table III: Percentage of ovalbumin attached to the cation exchange resin Sephadex QAE-50.

A fixation of ovalbumin in aqueous solution is observed for the pH range where the protein is negatively charged (pH greater than IpH: 4.5) and the ion exchange resin is positively charged (pH lower than the pKa of the resin ie 12).

Example 4

An in vivo rat study was conducted to validate the concept of release from a polypeptide resinate. An insoluble salt based on Sephadex QAE and an LHRH analogue was injected into male rats. A significant decrease in testosterone levels of the animals was recorded over a period of one week.

10 male Sprague-Dawley rats, CrI CD® (SD) IGS BR, Caesarian Obtained,

Barrier Sustained-Virus Antibody Free (COBS-VAF®), were purchased at Charles River Laboratories, France. The age of the animals at the beginning of the treatment is 12 weeks. The rats are acclimated for 5 days and grouped by 2 or 3 by cage. Normal conditions for feeding water and food are applied. The number of groups of animals corresponds to a placebo control group and a treated group. The number of animals is 5 in each group.

The formulations (sterile LHRH analogue resinate suspensions) were prepared the day before the injection and injected subcutaneously at a dose of 5 ml / kg. The LHRH analogue content in the decapeptide resinate is 2% (w / w). Mortality, morbidity, clinical signs at D1 and once a week were monitored as well as weight loss and food consumption. Blood samples for each animal (0.5 ml) are taken from the orbital sinus under anesthesia with isoflurane in heparinized tubes at the following times: pre-dose and D 1, 2, 3, 7, 14, 21, 28. The samples of plasma are frozen at -2O ⁰ C before sending to the National Veterinary School of Lyon, for analysis by radio immuno assay (Sorin). At the end of the observation period, the animals are killed by cervical dislocation under Isofiurane anesthesia and the injection site is removed and the carcasses are examined.

The results are grouped in the following table (the average testosterone level recorded for the placebo lot is 8.83 nmol / L):

Day O Day 1 Day 2 Day 3 Day 7 Day 14 Day 21 Day 28

GOOD m 9.5 12.3 4.1 4.4 8.5 14.0 11.3 10.0

SEM 1.15 2.88 1.20 0.57 2.24 4.17 2.47 2.55

Testosterone dosage (nmol / L)

Chemical castration of the rats is observed over a period of one week for animals treated with a rebound phenomenon after D (see Figure 3). Example 5

Extemporaneous microencapsulation assay of a peptide-free SEPHADEX SP C50 resin (placebo) using a 50/50 PLGA lactic and glycolic acid copolymer

(1) Preparation of a solution of 50/50 PLGA, PCAS, lot: 33101N009 in PEG 400, ACROS ORGANICS, lot: A020487801 at a concentration of 10% (w / v). at. Weighing PLGA 50/50: 1.01g b. Dissolution of the PLGA in 10 ml of hot PEG 400 with magnetic stirring until complete dissolution of the PLGA 50/50 which forms a transparent and viscous clear yellow liquid. vs. Stirring time: 30 minutes, temperature below 120 ⁰ C

(2) Preparation of SEPHADEX SP C50, ALDRICH CHEMICAL hydrated ion exchange resin gel, lot 08402DF. at. Weighing the resin: 0.046g b. Hydration of the resin in 4ml of MILLIQ purified water to form a homogeneous colloidal solution.

The theoretical amount of dry ion exchange resin for salifying an LHRH analog is at least 4 mg (assuming a resin loading rate of 100% and a salt content of 50%). The dose of pure peptide to be injected is 4 mg for one month of treatment.

Three placebo microencapsulation trials were performed with two different volumes of hydrated gel with or without surfactant:

(1) With 0.4 ml of hydrated resin gel added to 2 ml of the 50/50 PLGA solution in PEG 400. There is foπnation of a latex (small particles suspended in the mixture of milky appearance. No massive precipitate but injection test with a syringe and needle TERUMO 2OG xl, 0,9x25 is no satisfactory. Under an optical microscope, observation of more or less important agglomerates.

(2) With 0.4 ml of hydrated resin gel added to 2 ml of 50/50 PLGA solution in PEG 400 mixed with polysorbate 80 (TWEEN 80). A homogeneous and stable latex is formed (very small particles in suspension in the milky mixture) No massive precipitate The injectability test with a syringe and needle TERUMO 2OG xl, 0,9x25 is correct .

(3) With 0.8 ml of hydrated resin gel added to 2 ml of the 50/50 PLGA solution in PEG 400. Latex is formed (very small particles suspended in the milky-looking mixture No massive precipitate The injection test with a syringe and needle TERUMO 2OG xl, 0.9x25 is good but more difficult with a needle TERUMO 21Gx2 / 0.8x50 finer.The transfer of the latex in a bath of water MILLIQ and TWEEN 80 (volume 20ml) results in the formation of microbeads of resins coated with PLGA 50/50.

These tests demonstrate that it is possible to form microcapsules extemporaneously directly in the final packaging just before injection into SC or IM. The latex observed is due to a desolvation of PLGA 50/50 from PEG 400 which forms a coacervate suspended in the mixture of the two solvents (the PEG 400 gradually mixes with the purified water). The resin particles are fully coated with the 50/50 PLGA coacervate (see Figures 5A and 5B). It is possible to inject the latex directly SC or IM.

Claims

claims 1. Preparation for parenteral administration for the in vivo prolonged release of a biopolymer, said preparation comprising microparticles of an insoluble polysaccharide resin, biologically acceptable and carrying functional groups forming an ionic bond with the biopolymer, said microparticles being dispersed in a biologically acceptable liquid at a pH between 6.0 and 7.8. 2. Preparation according to claim 1, characterized in that the polysaccharide resin is of the dextran type. 3. Preparation according to claim 2, characterized in that the dextran has a molecular mass of between 3 Kdaltons and 2000 Kdaltons. 4. Preparation according to any one of the preceding claims, characterized in that the biopolymer is a polypeptide. 5. Preparation according to claim 4, characterized in that the polypeptide is a hormone, a coagulation factor, a neurotrophic factor or a growth factor. 6. Preparation according to claim 5, characterized in that the polypeptide is the luteinizing hormone releasing hormone (LHRH) or one of its analogues. 7. Preparation according to claim 4, characterized in that the polypeptide is an interferon, the polysaccharide resin being advantageously an anion exchange resin. 8. Preparation according to any one of the preceding claims, characterized in that the preparation is either hydrated or dehydrated. 9. Preparation according to any one of the preceding claims, characterized in that the microparticles are incorporated in a matrix of a biodegradable and biocompatible polymer microsphere type or implant. 10. Preparation according to one of claims 1 to 8, characterized in that the microparticles are covered with a shell of a biodegradable and biocompatible polymer and form microcapsules. 1. Preparation according to claim 9 or 10, characterized in that the biodegradable and biocompatible polymer is a poly ester homopolymer type or copolymer of lactic and / or glycolic acid. The preparation according to any one of the preceding claims for use as a medicament. A method of making a preparation for parenteral administration for in vivo sustained release of a biopolymer, said preparation comprising microparticles of an insoluble polysaccharide resin, biologically acceptable, and bearing functional groups forming an ionic bond with the biopolymer said microparticles being dispersed in a biologically acceptable liquid at a pH between 6.0 and 7.8, said process comprising the following steps: optionally, sterilizing the polysaccharide resin, adding to a pH of between 6.0 and 7, 8 of a polysaccharide resin preparation buffer, said polysaccharide resin being insoluble in the preparation buffer, - ionization of a biopolymer in distilled water or in an aqueous buffer, forming the preparation by bringing the biopolymer and the polysaccharide resin into contact when the pH of the preparation buffer is lower than the isoelectric point (IpH) of the biopolymer, by fixing the biopolymer whose overall surface charge is positive on the polysaccharide resin of which the functional groups are negative, or when the pH of the preparation buffer is greater than the isoelectric point (IpH) of the biopolymer, by fixing the biopolymer whose overall surface charge is negative on the polysaccharide resin whose functional groups are positive, and optionally , washing of the preparation. 14. The manufacturing method according to claim 13, characterized in that it further comprises, after the step of forming the preparation, an aqueous coacervation step by placing the preparation in the presence of a homo type poly ester. or copolymer of lactic and / or glycolic acid. 15. The manufacturing method according to claim 14, characterized in that the lactic and / or glycolic acid type homo- or copolymer poly ester is a copolymer of lactic and glycolic acid (PLGA).