MICROPARTICLES BASED ON HYBRID POLYMERIC MATERIALS FOR CONTROLLED RELEASE OF BIOLOGICALLY ACTIVE MOLECULES, A PROCESS FOR PREPARING THE SAME AND THEIR USES FOR IN VIVO AND IN VITRO THERAPY, PROPHYLAXIS AND DIAGNOSTICS

Abstract: Chemically homogeneous microparticles consisting of a proteic component and of a natural, synthetic or semisynthetic polymer or a mixture of them are described, as well as a process for preparing such microparticles and their uses for therapy, prophylaxis and diagnostics.

Description: MICROPARTICLES BASED ON HYBRID POLYMERIC MATERIALS FOR CONTROLLED RELEASE OF BIOLOGICALLY ACTIVE MOLECULES, A PROCESS FOR PREPARING THE SAME AND THEIR USES FOR IN VIVO AND IN VITRO THERAPY, PROPHYLAXIS AND DIAGNOSTICS This invention relates to microparticles based on hybrid polymeric materials for controlled release of biologically active molecules, to a process for preparing said microparticles and to their uses for in vi tro and in vivo therapy, prophylaxis and diagnostics . More particularly, this invention relates to the preparation of chemically homogeneous microparticles consisting of a component of proteic kind and of a natural, synthetic, semisynthetic polymer. Prior art mentions numerous examples of preparation (and of employment) of macromolecular aggregates and of microparticles which are made up of polymeric materials of proteic or non-proteic nature, synthetic and semisynthetic, for instance cf. Carth W. Hastings and Paul Ducheyne, Macromolecular Biomaterials, CRC Press Inc. (1984) . Prior art also mentions examples of preparation of macroaggregates and microparticles containing both proteins (and polypeptides) and natural, synthetic, semisynthetic polymers as well. Among proteins, a particular emphasis has been put on albumin, as it interacts with many compounds, and among them the compounds containing electronegative functional groups or acid groups. In addition to its reaction with long-chain at acids, Carr C.W., Proc . Soc Expl . Biol. Med. 89: 546 (1959), Bears A.G., Am. J. Med. 22: 747 (1957), albumin also interacts with: anionic polyelectrolytes such as heparin, H.F.M. Cremers et al . , J. Controlled Rel. 11, 167:179 (1990), polyelectrolytes deriving from copolymerization of maleic anhydride and vinyl ethers or vinyl esters, H. Ohno et al., Makromol. Chem. 182: 1253 (1981); M. Brissova, Makromol. Rapid Comm. 15: 211 - 216 (1994) , with cationic polyelectrolytes such as chitosan, Y. Murat Elcin, Art. Cells Blood. Subs. & Immob. Biotech. 24 (3) 257 - 271 (1996) . Prior art also mentions numerous examples of incorporation of proteins and polypeptides into natural and artificial polymeric matrices. However, the incorporation of albumin (human, bovine) has been finalized exclusively to analytical purposes, T. Atkins, J. Biomat. Sci. Polymer Edn. 7: 1065 - 1073 (1996), for modulating the release of bioactive protein molecules, U.S. 5.102.872, or as a stabilizing agent for proteins, ' M.A. Hanson, Introduction to Formulation of Protein Pharmaceutical, Plenum Press 1992, pg. 209, as a compound suitable for stabilizing microparticles as to phagocytosis exerted by polymorphonucleate cells, T. Verrecchia, J. Control Rel. 36: 49 - 51 (1995). However, in all cases mentioned above the microspheres are obtained by interaction of proteins with polymers of hydrophobic nature. This brings about the use of organic solvents for dissolving the synthetic polymer, with the consequent at least partial denaturation of proteins. Moreover, microspheres so obtained are made up of a heterogeneous aggregation of proteic material and synthetic polymer, in general of a proteic mass coated by a hydrophobic synthetic polymer shell. The Authors of this invention have discovered that by suitably mixing solutions containing at least a water soluble protein and water solutions containing variable amounts of organic solvents that contain at least one anionic, natural, synthetic, semisynthetic polyelectrolyte under particular and definite mechanical and environmental conditions, chemically homogeneous microparticles are obtained, which are advantageously employable for controlled release of biologically active molecules, for both in vi tro and in vivo therapy, prophylaxis and diagnostics, and in the cosmetic and dietetic fields as well. Numerous methods for the preparation of microparticles are known. J. Ogawa, J. Biomater. Sci. Polym. EDN 8: 391 - 409 (1997). All known methods are based essentially on phase separation, solvent evaporation, spray-drying, emulsification, interface polymerisation. However, no methods already known provide the employment of analogous techniques similar or equal to that which is the object of this invention, and they do not result in chemically homogeneous particles . Accordingly, it is the object of this invention a microparticle which is chemically homogeneous and is made up of a proteic component and a natural, synthetic, or semisynthetic polymer or a mixture of such polymers. Preferably, the proteic components comprises at least one protein from albumin, alpha 1-globulin, alpha 2-globulin, collagen, fibrinogen, gelatin, more preferably albumin is human albumin, natural or recombinant. Preferably the polymer comprises polyanionic polymers or their mixtures, more preferably those containing carboxyl and/or phosphoric and/or sulfuric groups . Preferably the polymers comprise semiesters of alternate copolymers of aleic anhydride with oligoethyleneglycol vinyl ethers whose oligomerization degree is 1 < n < 100, more preferably 1 < n < 10. Preferably the microparticle is spheroidal in shape, with a diameter between 50 n and 2,000 nm, more preferably between 100 nm and 300 nm. It is a further object of this invention a process for the preparation of microparticles according to this invention, which process comprises the step of mixing a first solution containing the natural polymer at a concentration between 5 g and 200 g/1, with a mixture of hydrophilic, protic and aprotic organic solvents, with a second solution containing the proteic component at a concentration between 1 g and 40 g/1, in which the weight ratio of the polymer to the proteic component is between 100 and 1, at a pH between 2 and 9, and at a temperature between 0°C and 60°C, under stirring conditions so as to prevent aggregation phenomena from occurring and so as to favor coprecipitation of the polymer and of the proteic component . Preferably the organic solvent of the first solution is made up of ethanol, alternatively of a water/ethanol mixture with an ethanol content between 0% and 80%. Preferably the proteic component in the first solution is at a concentration between 2 g and 6 g/1. Preferably the polymer in the second solution is at a concentration between 40 g and 60 g/1. Preferably the weight ratio of the polymer to the proteic component is between 10 and 1, and more preferably between 5 and 1. Preferably the pH is between 4 and 7. Preferably, the temperature is comprised between 4°C and 25°C. It is a further object of this invention the employment of the microparticles of this invention for therapeutic, prophylactic, dietetic, analytic, cosmetic and preparative objectives of systems featuring controlled release of active molecules. This invention will be disclosed now by means of examples of the same, which are not limitative of its object and scope. Example 1 Preparation of PAM14/HSA microspheres A solution of 60 mg of methyl hemiester of the alternate copolymer of aleic anhydride/methoxyethyl vinyl ether (PAM14) in 2.0 ml of water was dropped by means of a syringe supplied with a 22G needle into a solution of 24 mg of human seroalbumin (HSA) in 2.0 ml of bidistilled water kept under magnetic stirring. When the addition was complete, magnetic stirring was kept for 60 minutes. Dimensional analysis by means of dynamic light scattering showed that the microspheres were characterized by an average diameter of 140 nm and by a polydispersion index of 0.1 - 0.3. Example 2 Preparation of PAM11/MYO/HSA microspheres A solution of 100 mg of the methyl hemiester of the alternate copolymer of maleic anhydride/methoxyethyl vinyl ether (PAM11) in 3.0 ml of a mixture 1:3 of ethanol/water was dropped by means of a syringe fitted with a 22G needle into a solution of 50 mg of a mixture 1:4 of myoglobin/hu an seroalbumin (MYO/HSA) in 8 ml of bidistilled water under magnetic stirring. After completing the addition, magnetic stirring was kept for a further time of 60 minutes. Dimensional analysis by means of dynamic light scattering showed that the microspheres were characterized by an average diameter of 450 nm and a polydispersion index of 0.1 - 0.3. Example 3 Preparation of PAM14/HSA/HSA-FITC/MAN microspheres A solution of 210 mg of the butyl hemiester of the alternate copolymer maleic anhydride/methoxyethyl vinyl ether (PAM14) in 4.0 ml of a 1:4 mixture of ethanol/water was dropped by means of a syringe fitted with a 22G needle into a solution of 40 mg of human seroalbumin (HSA) and 600 mg of human seroalbumin fluorescein isothiocyanate (HSA-FITC) in 10 ml of bidistilled water containing 10% wt. of mannitol (MAN). When the addition was finished, magnetic stirring was kept for 90 minutes. Dimensional analysis by means of dynamic light scattering showed that the microspheres were characterized by an average diameter of 160 nm and a polydispersion index of 0.1 - 0.3. Example 4 Preparation of PAM14/HSA/GDA-bCD microspheres A solution of 200 mg of butyl hemiester of the alternate copolymer maleic anhydride/methoxyethyl vinyl ether (PAM14) in 4.0 ml of a 3:2 ethanol/water mixture was dropped by means of a syringe fitted with a 22G needle into a solution of 40 mg of human seroalbumin (HSA) in 10.0 ml of bidistilled water containing 5% wt. glycidyldiisd-propylidenarabitol-betacyclodextrin (GDA- bCD) . When the addition was finished, the dispersion was kept magnetically stirred for 60 minutes. Dimensional analysis by means of dynamic light scattering showed that the microspheres were characterized by an average diameter of 190 nm and by a polydispersion index of 0.1 - 0.2. Example 5 Preparation of PAM14/HSA/ASFET microspheres A solution of 100 mg of butyl hemiester of the alternate copolymer of maleic anhydride/methoxy vinyl ether (PAM14) in 2.0 ml of a 3:2 mixture of ethanol/water was dropped by means a syringe fitted with a 22G needle into a solution of 31 mg of human seroalbumin (HSA) and 11 mg of asialofetuin (ASFET) in 5.0 ml of bidistilled water. The dispersion of microsphere was left under magnetic stirring for 60 minutes. Dimensional analysis by means of dynamic light scattering showed that the microspheres were characterized by an average diameter of 1450 nm and a polydispersion index of 0.6. Example 6 Preparation of microspheres of AA/HSA/GDX-bCD A solution of 100 mg of polyacrylic acid (AA) in 4 ml of water was added by means of a syringe fitted with a 22G needle into a solution of 60 mg of human seroalbumin (HSA) in 10 ml of bidistilled water con- taining 5% by weight of glycidyldiisopropylidenxylitol- betacyclodextrin (GDA-bCD) , under magnetic stirring. When the addition was finished, magnetic stirring was kept for a further time of 60 minutes. Dimensional analysis by means of dynamic light scattering showed that the microspheres were characterized by an average diameter of 350 nm and a polydispersion index of 0.3 - 0.5. Example 7 Preparation of microspheres of AAL/HSA/GLU A solution of 200 mg of alginic acid (AAL) in 4 ml of water was dropped by means of a syringe fitted with a 22G needle into a solution of 60 mg of human seroalbumin (HSA) in 12 ml of bidistilled water containing 7% by weight of glucose, under magnetic stirring. Magnetic stirring was kept going on for 60 minutes. Dimensional analysis by means of dynamic light scattering showed that the microspheres were characterized by an average diameter of 210 nm and a polydispersion index of 0.2 - 0.4.

Claims: CLAIMS 1. A chemically homogeneous microparticle consisting of a proteic component and of a natural, synthetic or semisynthetic polymer or of a mixture of them. 2. A microparticle according to claim 1, wherein the proteic component comprises at least a protein chosen from among albumin, alpha 1-globulin, alpha 2-globulin, collagen, fibrinogen, gelatin. 3. A microparticle according to claim 2, wherein said protein is natural or recombinant human albumin. 4. A microparticle according to anyone of the preceding claims, wherein said polymer comprises poly- anionic polymers or their mixtures. 5. A microparticle according to claim 4, wherein said polyanionic polymers contain carboxyl and/or phosphoric and/or sulfuric groups. 6. A microparticle according to claim 5, wherein said polymers comprise hemiesters of alternate copolymers of maleic anhydride with vinyl ethers of oligoethylene-glycols having oligomerization degree l≤n≤lOO. 7. A microparticle according to claim 6, wherein the oligomerization degree of said polymers is l≤n≤lO. 8. A microparticle according to anyone of the preceding claims, which is spheroidal in shape and has a diameter between 50 nm and 2,000 nm. 9. A microparticle according to claim 8, wherein the diameter is between 100 nm and 300 nm. 10. A process for the preparation of microparticles according to anyone of the preceding claims, said process comprising the step of mixing a first solution containing the natural polymer at a concentration between 5 g and 200 g per litre with a mixture of hydrophilic protic and aprotic organic solvents, with a second solution containing the proteic component at a concentration between 1 g and 40 g per litre, wherein the weight ratio of the polymer to the proteic component is between 100 and 1, at a pH between 2 and 9, and at a temperature between 0°C and 60°C, under such conditions of stirring as to prevent aggregation phenomena from occurring and as to favor the coprecipitation of the polymer and of the proteic component . 11. A process according to claim 10, wherein the organic solvent of the first solution is ethanol. 12. A process according to claim 10, wherein the organic solvent of the first solution is made up of a water/ethanol mixture with an ethanol content between 0% and 80%. 13. A process according to claim 10, wherein the proteic component in the first solution is at a concentration between 2 g and 6 g per litre. 14. A process according to claim 10, wherein the polymer in the second solution is at a concentration between 40 g and 60 g per litre. 15. A process according to claim 10, wherein the weight ratio of the polymer to the proteic component is between 10 and 1. 16. A process according to claim 15, wherein the weight ratio of the polymer to the proteic component is between 5 and 1. 17. A process according to claim 10, wherein the pH is between 4 and 7. 18. A process according to claim 10, wherein the temperature is between 4°C and 25°C. 19. Employment of the microparticles according to anyone of the preceding claims 1 - 9, for therapeutic, diagnostic, prophylactic, dietetic, analytic and cosmetic objectives, as well as for preparation of ' active molecule controlled-release systems .

Inventors: CARLSSON ANDERS, CHIELLINI ELISABETTA, CHIELLINI EMO, COWDALL JENNY, DAVIES JOHN, MAZZANTI GIUSEPPE, ROBERTS MARK, SOEDERLIND ERIK, SOLARO ROBERTO, CARLSSON, ANDERS, CHIELLINI, ELISABETTA, CHIELLINI, EMO, COWDALL, JENNY, DAVIES, JOHN, MAZZANTI, GIUSEPPE, ROBERTS, MARK, SOEDERLIND, ERIK, SOLARO, ROBERTO
Applicants: CARLSSON ANDERS, CHIELLINI ELISABETTA, CHIELLINI EMO, COWDALL JENNY, DAVIES JOHN, ISI IST SIEROVACCINOGENO ITAL, MAZZANTI GIUSEPPE, ROBERTS MARK, SOEDERLIND ERIK, SOLARO ROBERTO, CARLSSON, ANDERS, CHIELLINI, ELISABETTA, CHIELLINI, EMO, COWDALL, JENNY, DAVIES, JOHN, ISTITUTO SIEROVACCINOGENO ITALIANO I.S.I. S.P.A., MAZZANTI, GIUSEPPE, ROBERTS, MARK, SOEDERLIND, ERIK, SOLARO, ROBERTO
Application year: 1998
Main classification: A61K9
IPC codes: A61K9