WO2001068059A1 - Method and device for the production of microparticles for controlled release of water-soluble pharmaceuticals and viral vectors. application to the administration of plasmid dna and defective recombinant adenovirus - Google Patents

Method and device for the production of microparticles for controlled release of water-soluble pharmaceuticals and viral vectors. application to the administration of plasmid dna and defective recombinant adenovirus Download PDF

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Publication number
WO2001068059A1
WO2001068059A1 PCT/ES2001/000100 ES0100100W WO0168059A1 WO 2001068059 A1 WO2001068059 A1 WO 2001068059A1 ES 0100100 W ES0100100 W ES 0100100W WO 0168059 A1 WO0168059 A1 WO 0168059A1
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phase
microparticles
encapsulated
release
emulsion
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PCT/ES2001/000100
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Spanish (es)
French (fr)
Inventor
Guillermo Garcia Del Barrio
Francisco Javier Novo Villaverde
Félix Juan RECARTE FLAMARIQUE
María Jésus RENEDO OMAECHEVERRIA
Juan Manuel Irache Garreta
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Instituto Cientifico Y Tecnologico De Navarra, S.A.
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Priority to AU40700/01A priority Critical patent/AU4070001A/en
Publication of WO2001068059A1 publication Critical patent/WO2001068059A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient

Definitions

  • the present invention is part of the field of continuous manufacturing and production processes of pharmaceutical forms or microparticle type conveyors.
  • the manufacturing takes place in an apparatus called 11 TROMS "(Total Recirculation One-Machine System or Monoblock Complete Recirculation System) based on the turbulent injection of two phases, of different polarity, into a reactor to form a double emulsion.
  • 11 TROMS Total Recirculation One-Machine System or Monoblock Complete Recirculation System
  • This new procedure allows obtaining microparticles of a predetermined size and with a very uniform distribution.
  • this new process allows the incorporation of biologically active materials and / or molecules of a hydrophilic nature and not very resistant to conventional techniques of agitation and / or emulsification.
  • DNA plasmid deoxyribbonucleic acid
  • adenovirus-like viral vectors for gene therapy.
  • its administration in the form of microparticles can allow (i) to significantly increase its stability, both physical-chemical (during storage) and biological and (n) offer new formulations with profiles of sustained release over time of the material Built-in microparticles.
  • State of the art state of the art
  • vectors among which microparticles, nanoparticles and liposomes, have had a great development in recent years.
  • These pharmaceutical forms allow designing a more rational and better adapted therapy, by increasing the efficacy and specificity of the biologically active drug or molecule that they incorporate.
  • these vectors or drug carriers provide the following advantages (Couvreur & Mrieux. Adv. Drug Del. Rev., 10 (1993) 141-162):
  • microparticle type systems stand out in size greater than or equal to the micrometer (generally accepted between 1 and 250 mm), also called microparticles that may have porous or vesicular matrix structure (Orecchioni et Irache, Formes pharmaceutiques pour application lócale. Lavoisier Tech S. Doc., Paris, 1996, 441- 457). According to the manufacturing techniques used and depending on the morphology of the particles, it is possible to distinguish three categories of microparticles (Benoit et al., Microencapsulation methods and industrial application ⁇ , Marcel Dekker, New York, 1996, 35-71):
  • microcapsules or spherical particles constituted by a solid coating that contains in its interior a solid, liquid or pasty substance.
  • Each microcapsule constitutes a reservoir system that gives rise to a state of maximum heterogeneity.
  • microspheres spherical particles constituted by a continuous network of support or polymeric material in which the substance to be encapsulated is dispersed to the molecular state (solid solution) or to the particular state (solid dispersion). This structure, in a state of maximum homogeneity, constitutes a material system.
  • homogeneous microcapsules multinuclear forms
  • heterogeneous microspheres partial dispersions: they are intermediate systems between the two possible states of heterogeneity (microcapsules) and homogeneity (microspheres). They are identified by the presence of rich and poor areas in active principle and by having an internal structure of crystalline dispersion type.
  • microparticles offer protection to the material and / or encapsulated drug from its eventual degradation in storage and / or biological conditions and to allow profiles of sustained release over time, without the need for repeated administrations.
  • the microparticles can be obtained from natural or synthetic materials.
  • the former include proteins (albumin, collagen, gelatin) and polysaccharides (hyaluronic acid, alginic acid, chitosan).
  • Synthetic materials include hydroxy acid polyesters, poly orthoesters, polycarbonates, polyamino acids, poly anhydrides, poly acrylamides and poly-alkyl l- ⁇ -cyanoacrylates.
  • biodegradable polymers of the poly-ester type the following polymers can be mentioned: polylactic acid (PLA), copolymers of lactic acid and glycolic acid (PLAGA), poly (hydroxybutyl) acid and poly ( ⁇ -caprolactone).
  • microparticle manufacturing procedures these can be classified into three main groups: physical-chemical, chemical and mechanical
  • the selection of a micro-particle manufacturing process depends on the physical and chemical properties of the principle couple active / polymer.
  • properties of the microparticles that want to be prepared granulometry, internal structure, loading in active principle, release profile, wettability, etc.
  • microencapsulation by coacervation microcapsulation by evaporation of the solvent and microcapsulation by fusion can be highlighted.
  • microencapsulation by coacervation or phase separation Microencapsulation by coacervation or phase separation.
  • Coacervation allows the separation of a colloid in solution in a phase rich in colloid (called coacervate) and another poor in this substance (Nairn, Advances in Pharmaceutical Sciences. Vol. 7, Academic Press Ltd., London, 1995, 93 -219). This procedure generally leads to the formation of microcapsules, although sometimes microspheres can also be obtained.
  • simple coacervation refers to the procedures used with a single colloid (proteins, celluloses, poly esters). This is dissolved in an appropriate solvent where the active ingredient is dispersed.
  • Phase separation is induced by the addition of a second compound (electrolytes, organic solvent) incompatible with the presence in colloid solution ( ⁇ ner & Groves, J “ . Pharm. Pharmacol., 45 (1993) 866-870; Nihant et al ., “ . Controlled Re ⁇ ., 35 (1995) 117-125) or by modification of the physicochemical characteristics of the solution (Ezpeleta et al., Eur. J. Pharm. Biophar., 42 (1996) 36-4).
  • the coacervate is deposited on the suspended particles by coating them and finally, it is consolidated.
  • Complex coacervation refers to the techniques that are applied to coacervation simultaneously of two different colloids.
  • the phenomenon occurs mainly by modifying the pH of the initial solution and is applied to the coacervation of gelatin with gum arabic, polyacrylic acid, sodium alginate or polyphosphate (Tirkkonen et al., J. Microencaps., 11 (1994) 615- 626). Generally, and for both techniques, a supplementary stage consisting of the consolidation or stabilization of the obtained coacervate is necessary.
  • the drug to be encapsulated is incorporated into the lipophilic phase (0), so that it will be trapped within the m formed croparticles (Bodmeier & McGmity, Pharm. Res., 4 (1987) 465-471). This method is ideal for incorporating liposoluble drugs.
  • This group includes techniques for preparing microparticles by polymerization reactions and by gelation phenomena (i) Micro-encapsulation by mfacial polycondensation
  • the mtfacial polycondensation is the reaction that occurs between two chemically different monomers at the interface of two non-irascible liquids. This reaction leads to the formation of a polymethyl film and to the obtaining of microcapsules.
  • an emulsion of the hydrophilic type is carried out in lipophilic (A / O), in which the internal aqueous phase contains the substance to be encapsulated and one of the monomers, and the dispersing lipophilic phase is formed by an organic solvent, the second monomer and a surfactant.
  • the monomers diffuse to the mterfase where they will react to form a single polymer that encapsulates the dispersed phase (Whateley Microencapsulation. Methods and Industrial Applications, Marcel Dekker, New York, 1996, 349-375).
  • All polymers form gels under particular hydration conditions. When it is possible to stabilize the gel formed, this property can be used to encapsulate an active substance.
  • Sodium alginate for example, can give rise to gels in the presence of bivalent cations (Ca 2+ ) and even organize themselves in semi-permeable membranes that are stabilized by polycations (Dupuy et al., Artif. Blood Subst. Immobil. Biotech. , 22 (1994) 71-82).
  • This group can include microencapsulation in a fluid bed, by spraying, by means of fluids under supercritical conditions and or turbulent coaxial injection. The biggest advantage of all These procedures are to be used on an industrial scale.
  • This technique is intended for encapsulation of solid particles (granules and crystals) through the use of celluloses, methacrylic derivatives, etc.
  • the active substance particles are suspended in a chamber crossed by a strong upward air current.
  • the coating solution containing the polymer is sprayed into the chamber and covers the surface of the particles (Vervaet et al., Int. J. Pharm., 116 (1995) 131-146).
  • Spray microencapsulation is performed in chambers at the upper end of which a liquid phase is finely dispersed through a sprayer.
  • the droplets formed pass through a stream of air at a certain temperature, whereby evaporation of the solvent occurs. This allows the blood cells to solidify and be recovered at the base of the spray chamber.
  • two main procedures are distinguished: - spray and drying ("* " * Spray-drymg ").
  • the most used method is the evaporation of the solvent after the formation of a multiple emulsion of the hydrophilic / lipophilic / hydrophilic type or water / oil / water ⁇ A 1 / Q / A 2 ).
  • the formation of the emulsion A 1 / ⁇ / A 2 requires the use of very violent agitation or homogenization systems.
  • the most commonly used are the Ultra-turrax ® system and the ultrasound probes.
  • the present invention is based on a new method and apparatus that allows obtaining a multiple emulsion, of very homogeneous size, suitable for the encapsulation of drugs, biologically active molecules and materials of a water-soluble nature or to be handled in aqueous solutions.
  • multiple emulsion is obtained through a continuous homogenization system much less violent and aggressive than traditional emulsification systems. Under these conditions, the stability of the drug, biologically active molecule or material to be encapsulated is much greater and structural damage is minimized. Logically, by increasing the structural stability of the material, preventing its rupture, its activity is preserved. On the other hand, the absence of energy homogenization methods allows the use of this technique for the microencapsulation of labile materials or of low physical-chemical stability.
  • RNA ribonucleic acid
  • antisense oligonucleotides molecules derived from spike and pyrimidine bases
  • peptides and related molecules molecules that originate in biotechnology
  • RNA ribonucleic acid
  • antisense oligonucleotides molecules derived from spike and pyrimidine bases
  • peptides and related molecules molecules that originate in biotechnology
  • adenoviruses are good vectors for the administration of therapeutic genes and have a significant number of advantages.
  • adenoviruses are very efficient in transducing a large number of different cell types, they can be purified and obtained with high titers (approx.
  • the conditions of applicability that allow us to think about the industrial interest of this procedure and apparatus for the production of microparticles from synthetic polymers and their application to the administration of drugs of a water-soluble nature and viral vectors of defective adenovirus recornbinant type may be the following :
  • microparticles of very homogeneous size are of the type homogeneous microcapsules or heterogeneous microspheres.
  • Working conditions at room temperature that prevent the possible degradation of thermo-labile active substances of a water-soluble nature, of chemical and / or biotechnological origin, or of temperature sensitive materials that are to be incorporated into the microparticles .
  • microparticles capable of protecting against premature inactivation of drugs, biologically active molecules and encapsulated materials. This protection increases the stability of the encapsulated material during (i) storage and conservation processes and (n) in biological media.
  • microparticles with viral vectors inside that can prevent the use, for these viral particles, of transport, storage and conservation systems of very high cost.
  • the encapsulation of viral vectors within micro-particles should be able to increase the efficiency of gene transfer, decrease the number of doses (and therefore systemic toxicity) and minimize host immune responses.
  • the present invention proposes a new microparticle manufacturing system based on the turbulent two-phase injection and subsequent evaporation of the organic solvent used. This manufacturing method takes place in a device called , TROMS "or ⁇ ** Total Recirculation One-Machine System. Among the main advantages of this new procedure, it is possible to highlight: (i) easily reproducible method ;
  • the apparatus object of this patent is represented in Figure 1. It consists of a pumping system (1) that is connected to a Rheodyne TM valve (2) from which two needles (3) and (4) of different internal diameter. The first one is inside the first mixing vessel (5) and the other in the second mixing vessel (6). The organic phase is incorporated into the system using a glass syringe with a Teflon plunger (7). The mixing vessels are connected to the pump through two valves (8 and 9).
  • phase A ⁇ aqueous solvent, drug or biologically active material, and possibly surfactant or viscosizer
  • Phase O water-immiscible organic solvent, polymer soluble in the organic solvent
  • Phase A 2 aqueous solvent and possibly a surfactant
  • Biodegradable are used as a polymer (generally polyesters such as poly-epsilon caprolactone, polylactic acids, PLA, or copolymers between lactic acid and glycolic acid, PLAGA).
  • Polyvinylpyrrolidone (PVP) at concentrations of the order of 0-20% w / v, gelatin (0-5% w / v) or xanthan gum (0-1% p) are generally used as internal surfactant or within the gallbladder / v).
  • PVA polyvinyl alcohol
  • a copolymer of ethylene oxide and propylene oxide is also incorporated as a lyophilization aid.
  • a cryoprotectant mannitol and sucrose
  • the pump flow rate is adjusted and the needles are selected according to the desired particle size.
  • the aqueous phase A. which contains the drug or the material to be encapsulated, is introduced.
  • the pumping system is operated keeping the valves connecting the mixing vessels with the pump closed, so that phase 0 is introduced through the syringe (7), which contains the microparticle-forming polymer dissolved in a suitable solvent.
  • Rheodyne TM is in the position that allows turbulent injection through the needle (3). Phase O is therefore injected turbulently over phase A :.
  • an O / A emulsion is formed which is quickly invested in an A / 0 emulsion since the volume of the O phase is substantially greater than that of the L- phase.
  • the valve (8) is opened and the formed emulsion is allowed to recirculate through the system for a period of time sufficient for the emulsion to be homogeneous (this period depends on the volume of emulsion formed).
  • the position of the Rheodyne TM valve (2) for the mixture to pass through the needle (4) of internal diameter greater than the (3) is changed.
  • the A ⁇ O emulsion is thus injected into the vessel (6) containing the aqueous phase A 2 .
  • Turbulent injection makes the emulsion 1/0/2 into the vessel (6) is formed.
  • the valve (8) is closed and the (9) is opened to allow total recirculation of the mixture, for a period of time sufficient for the emulsion to have a homogeneous droplet size.
  • the emulsion is stirred at room temperature until complete evaporation of the phase O solvent by means of a propeller stirrer (10).
  • This operation can be carried out in the same apparatus, incorporating a propeller stirrer in the vessel (6), or by means of a stirring system outside the apparatus.
  • the evaporation of the organic solvent leads to the formation of the microparticles, which contain its interior is phase A., where the drug or biologically active material is found.
  • the following examples illustrate the method and apparatus objects of the invention. In no way are the examples described herein intended to limit the scope of the invention.
  • the materials used were: Polyvinylpyrrolidone (PVP, molecular mass 42,500), Lactic-glycolic acid copolymer (Resomer® 756, Resomer® 502), sodium fluorescema, polyvinyl alcohol (molecular mass 115,000), dichloromethane, glycerol, copolymer of ethylene oxide (80%) and propylene oxide (20%) with a molecular mass of 8400 (Pluronic ® F-68, Sigma).
  • Plasmid DNA containing the luciferase gene under the control of the cytomegalovirus promoter (CMV), was prepared by ammonium exchange chromatography.
  • Defective recombinant adenoviruses contained the E. coli ⁇ -galactosidase (lacZ) gene, ba or CMV promoter control.
  • HeLa cells were grown in DMEM (Doubelco's modified Eagle medium) supplemented with 10% a fetal bovine serum, 100 U / mL penicillin and 100 mg / mL streptomycin.
  • DMEM Doubelco's modified Eagle medium
  • the size of the microparticles was determined by laser diffraction spectroscopy, with a Mastersizer S (Malvern Instruments).
  • Example 1 Manufacture of empty microparticles. Modification of the average size of the microparticles according to the recirculation time of the emulsion h 1/0/2.
  • Phase A x 300 ⁇ L of water
  • Viscosizer Polyvinyl rrolidone (PM 42,500) at 5% w / v
  • Phase O 4 mL dichloromethane
  • Phase A 2 40 mL of water
  • Emulsion recirculation time A j / O 1 minute
  • Figure 2 shows how increasing the recirculation time of the double emulsion decreases the size of the microparticles obtained.
  • Example 2 Preparation of microparticles that incorporate a water-soluble model molecule (sodium fluorescein) inside.
  • the phases used in the method were formed by: Phase A ⁇ : 300 ⁇ L of water
  • Viscos zante Polyvinylpyrrolidone (PM 42,500) at 5% w / v
  • the recirculation time of the A./O emulsion was 1 minute.
  • the micro- particles obtained, once purified, were collected and dispersed in a 2% w / v aqueous solution of Pluronic ® F-68, and lyophilized.
  • sucrose or mamtol was used as a cryoprotectant at a concentration of 5% w / v.
  • the average size of the particles obtained is 3.7 ⁇ m.
  • the internal structure of the microparticles can be observed by means of optical microscopy with UV light, with the Al-phase vesicles - where the fluorescein is - inside ( Figure 3).
  • Example 3 Preparation of microparticles incorporating plasmid DNA (7 b).
  • Phase A 225 ⁇ L of Viscosizing water: Polyvinylpyrrolidone (PM 42,500) at 5% w / v at 10% w / v, Gelatin 1% w / v xanthan gum at 0, 1% w / v Plasmid DNA (7kb) 675 ⁇ g Phase O: 3 mL dichloromethane Poly (lactic / glycolic acid) copolymer
  • the recirculation time of the A ⁇ O emulsion was 1 minute.
  • the obtained micro particles, once purified, were collected and dispersed in a 2% w / v aqueous solution of Pluronic ® F-68, and lyophilized.
  • the encapsulation efficiency was determined by ultraviolet (UV) spectroscopy after dissolution of the microparticles in dimethylsulfoxide (DMSO).
  • UV ultraviolet
  • DMSO dimethylsulfoxide
  • the integrity of the DNA was determined by gel electrophoresis in the supernatants of the preparations.
  • lyophilized microparticles were incubated in 0.5 mL of phosphate buffered buffer (PBS) at 37 ° C or stirring. At different times, the microparticles were centrifuged and the supernatants collected to determine the amount of plasmid DNA released and replaced by new buffer. The amount of DNA released was determined by UV spectroscopy, using controls for all formulations.
  • PBS phosphate buffered buffer
  • Table 1 shows the main physicochemical characteristics of the different batches of microparticles obtained by TROMS.
  • Figure 4 shows an SEM photomicrograph of a batch of MSP-D. The release profiles are shown in Figure 5. After 4 weeks, only the MSP-A formulation had released the total amount of the encapsulated DNA. The lower release rate corresponds to the MSP-D formulation, which suggests that xanthan gum is a good excipient to delay the outflow of encapsulated material. All formulations showed an effect , x burst "or rapid release during the first day of the experience. This initial release value varied between 50% for the MSP-C formulation and 20% for the MSP-D. Subsequently, slower and continuous releases over time can be observed for all formulations.
  • Example 4 Preparation of defective recombinant Adenovirus microparticles inside.
  • the supernatants obtained during the purification steps were titrated. For this, the supernatants were mixed with serum-free DMEM and added to HeLa cells (approx. 60% confluence). Two hours after the addition of the supernatants, an aliquot of DMEM (modified Eagle medium from Doubelco) with serum was added. The cells were incubated at 37 ° C and under C0 2 atmosphere for 4 Oh. The cells were fixed with a solution of glutaraldehyde (1 mL per well) and incubated on ice for 15 mm.
  • DMEM modified Eagle medium from Doubelco
  • the average size of the microparticles obtained was 10.5 ⁇ m and the amount of encapsulated recombinant adenoviruses was calculated at 3.84xl0 8 u
  • the release of the virus from the particles corresponds to its own sustained release kinetics, as can be seen in Figure 7.
  • This figure represents the time against the number of blue cells (infected by the recombinant adenovirus).
  • the adenoviruses encapsulated in the microparticles of the invention remain active for up to 5 days, compared to 24 hours at 20-25 ° C that the viral particles last without encapsulating in an aqueous medium.
  • xanthan gum allows an initial release in the first hours of approximately 20%. Subsequently, a slow and sustained release in time is observed until the end of the experiment, reaching 40% of the initial dose.
  • RNA ribonucleic acid
  • DMEM Doubelco X-Gal modified Eagle medium: 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside
  • SEM scanning electron microscopy
  • TROMS Total Recirculation One-machine System, Monobloc Complete Recirculation System
  • Figure 1 Diagram of the TROMS device (Total Recirculation One-Machine System). (1) Pumping system (2) Multi-way valve (3), (4) Needles (5), (6) Mixing vessels (7) Syringe (8), (9) Valves (10) Propeller shaker
  • Figure 2 Relationship between the time of recirculation of the emulsion and the size of the microparticles. Abcisas: time in minutes; Ordered: size in myras.
  • Figure 3 Microparticles manufactured by TROMS with sodium fluorescein in the inner vesicles (magnification x400).
  • FIG. 1 Plasmid DNA release profiles from the microparticles prepared by TROMS. Abcisas: time in days; Sorted:% DNA released; 0 PVP 5%, D PVP 10%, O gelatin 1%, ⁇ xanthan gum 0.1%.
  • Figure 6A Gel electrophoresis of the supernatant obtained after the manufacture of the microparticles by TROMS (lane 1) and of the washes of the microparticles after their centrifugation (lanes 2-3).
  • P Street original plasmid. Unmarked street: control of DNA of different sizes.
  • Figure 6B Gel electrophoresis of the supernatant obtained after the manufacture of the microparticles using Ultra-turrax as a method of agitation (lane 1). Unmarked street: control of DNA of different sizes.
  • Figure 7 Infection of HeLa cells by adenoviruses released from microparticles as a function of time. Abcisas: time in h; Ordered: number of infected cells.

Abstract

The method involves obtaining a multiple emulsion by injecting immiscible liquid phases under high turbulence. The device used for said purpose, which is called TROMS or 'Total Recirculation One-Machine System', comprises a pumping system (1) that is connected to a Rehodine valve (2) from which two needles (3, 4) having different inner diameters stick out. The first needle is inserted into the first mixing vessel (5) and the other needle is inserted into the second mixing vessel (6). The organic phase is introduced into the system by means of a glass syringe with a Teflon plunger (7). The mixing vessels are connected to the pump by two valves (8 and 9). The method and device used are suitable for the production of homogenous microcapsule or heterogeneous microsphere type microparticles that are suitable for the encapsulation of water-soluble pharmaceuticals including plasmid DNA, RNA, genes, oligonucleotides, peptides, proteins and viral vectors used in genic therapy and defective recombinant adenovirus.

Description

Procedimiento y aparato para la producción de micropartículas para la liberación controlada de fármacos hidrosolubles y vectores virales. Aplicación a la administración de ADN plas ídico y de adenovirus recombinantes defectivos. Procedure and apparatus for the production of microparticles for the controlled release of water-soluble drugs and viral vectors. Application to the administration of recombinant recombinant adenovirus and plasic DNA.
Campo técnico de la invenciónTechnical Field of the Invention
La presente invención se adscribe al ámbito de los procedimientos de fabricación y producción en continuo de formas farmacéuticas o transportadores de tipo micropar- tícula. La fabricación tiene lugar en un aparato denominado 11 TROMS" (Total Recirculation One-Machine System o Sistema Monobloque de Recirculación Completa) basado en la inyección turbulenta de dos fases, de polaridad diferente, en el seno de un reactor para formar una doble emulsión.The present invention is part of the field of continuous manufacturing and production processes of pharmaceutical forms or microparticle type conveyors. The manufacturing takes place in an apparatus called 11 TROMS "(Total Recirculation One-Machine System or Monoblock Complete Recirculation System) based on the turbulent injection of two phases, of different polarity, into a reactor to form a double emulsion.
Este nuevo procedimiento permite la obtención de micropartículas de un tamaño prefijado y con una distribución muy uniforme. Por otra parte, este nuevo procedimiento permite incorporar materiales y/o moléculas biológi- camente activas de naturaleza hidrofílica y poco resistentes a las técnicas convencionales de agitación y/o emulsi- ficación. Entre ellas es de destacar la aplicación de este nuevo procedimiento y aparato TROMS a la administración de ácido desoxirπbonucleico (ADN) plasmídico y de vectores virales de tipo adenovirus destinados a terapia génica. En ambos casos, su administración bajo forma de micropartículas puede permitir (i) incrementar de manera notable su estabilidad, tanto físico-química (durante el almacenamiento) como biológica y (n) ofrecer nuevas formulaciones con perfiles de liberación sostenida en el tiempo del material incorporado en las micropartículas. Estado de la técnicaThis new procedure allows obtaining microparticles of a predetermined size and with a very uniform distribution. On the other hand, this new process allows the incorporation of biologically active materials and / or molecules of a hydrophilic nature and not very resistant to conventional techniques of agitation and / or emulsification. Among them, it is worth highlighting the application of this new TROMS procedure and apparatus to the administration of plasmid deoxyribbonucleic acid (DNA) and of adenovirus-like viral vectors for gene therapy. In both cases, its administration in the form of microparticles can allow (i) to significantly increase its stability, both physical-chemical (during storage) and biological and (n) offer new formulations with profiles of sustained release over time of the material Built-in microparticles. State of the art
Los nuevos sistemas de administración de medicamentos llamados vectores, entre los que destacan las micropartículas, las nanopartículas y los liposomas, han tenido un gran desarrollo en los últimos años. Estas formas farmacéuticas permiten diseñar una terapéutica más racional y mejor adaptada, al aumentar la eficacia y especificidad del fármaco o molécula biológicamente activa que incorporan. En general , se puede afirmar que estos vectores o transpor- tadores de fármacos aportan las siguientes ventajas (Couvreur & Puisieux. Adv. Drug Del . Rev. , 10 (1993) 141- 162) :The new drug delivery systems called vectors, among which microparticles, nanoparticles and liposomes, have had a great development in recent years. These pharmaceutical forms allow designing a more rational and better adapted therapy, by increasing the efficacy and specificity of the biologically active drug or molecule that they incorporate. In general, it can be affirmed that these vectors or drug carriers provide the following advantages (Couvreur & Puisieux. Adv. Drug Del. Rev., 10 (1993) 141-162):
(i) proteger al material que incorporan frente a su inactivación química, enzimática o mmunológica, (n) mejorar el transporte de la molécula biológicamente activa hasta lugares difíciles de alcanzar y de su penetración en la célula;(i) protect the material they incorporate against its chemical, enzymatic or immunological inactivation, (n) improve the transport of the biologically active molecule to hard-to-reach places and its penetration into the cell;
(m) aumentar la especificidad de acción por concentración selectiva, eficaz y regular del fármaco en el blanco celular y/o molecular. Así, con dosis más pequeñas, la actividad terapéutica obtenida es, al menos, idéntica y los efectos secundarios menores;(m) increase the specificity of action by selective, effective and regular concentration of the drug in the cell and / or molecular target. Thus, with smaller doses, the therapeutic activity obtained is at least identical and the minor side effects;
(ív) disminuir la toxicidad para ciertos órganos mediante modificación de la distribución tisular de la molécula biológicamente activa transportada;(iv) decrease toxicity for certain organs by modifying the tissue distribution of the transported biologically active molecule;
(v) prolongar el tiempo de residencia del fármaco en el organismo (interesante para aquellas moléculas con aclara- miento elevado y se ivida biológica baja) y, control de su liberación. Todo ello implica disminuir la frecuencia de tomas e, indirectamente, aumentar la observancia del tratamiento por parte del paciente.(v) prolong the residence time of the drug in the organism (interesting for those molecules with high clearance and low biological quality) and, control of its release. All this implies reducing the frequency of takings and, indirectly, increasing the observance of treatment by the patient.
Dentro del grupo de los vectores no biológicos, destacan los sistemas de tipo partícula sólida de tamaño superior o igual al micrómetro (generalmente aceptado entre 1 y 250 mm) , también llamadas micropartículas que pueden tener estructura matricial porosa o vesicular (Orecchioni et Irache, Formes pharmaceutiques pour application lócale . Lavoisier Tech S. Doc . , París, 1996, 441-457). Según las técnicas de fabricación utilizadas y en función de la morfología de las partículas, es posible distinguir tres categorías de micropartículas (Benoit et al., Microencapsulation methods and industrial applicationε , Marcel Dekker, New York, 1996, 35-71) :Within the group of non-biological vectors, solid particle type systems stand out in size greater than or equal to the micrometer (generally accepted between 1 and 250 mm), also called microparticles that may have porous or vesicular matrix structure (Orecchioni et Irache, Formes pharmaceutiques pour application lócale. Lavoisier Tech S. Doc., Paris, 1996, 441- 457). According to the manufacturing techniques used and depending on the morphology of the particles, it is possible to distinguish three categories of microparticles (Benoit et al., Microencapsulation methods and industrial applicationε, Marcel Dekker, New York, 1996, 35-71):
(i) microcápsulas o partículas esféricas constituidas por un recubrimiento sólido que contiene en su interior una sustancia sólida, líquida o pastosa. Cada microcápsula constituye un sistema reservorio que da lugar a un estado de heterogeneidad máximo.(i) microcapsules or spherical particles constituted by a solid coating that contains in its interior a solid, liquid or pasty substance. Each microcapsule constitutes a reservoir system that gives rise to a state of maximum heterogeneity.
(n) microesferas : partículas esféricas constituidas por una red continua de material soporte o polimérico en el cual la sustancia a encapsular está dispersada al estado molecular (solución sólida) o al estado particular (disper- sión sólida) . Esta estructura, en estado de homogeneidad máximo, constituye un sistema matπcial .(n) microspheres: spherical particles constituted by a continuous network of support or polymeric material in which the substance to be encapsulated is dispersed to the molecular state (solid solution) or to the particular state (solid dispersion). This structure, in a state of maximum homogeneity, constitutes a material system.
(m) microcápsulas homogéneas (formas multinucleares) o microesferas heterogéneas (dispersiones particulares) : son sistemas intermedios entre los dos estados posibles de heterogeneidad (microcápsulas) y homogeneidad (microes- feras) . Se identifican por la presencia de zonas ricas y pobres en principio activo y por tener una estructura interna de tipo dispersión cristalina.(m) homogeneous microcapsules (multinuclear forms) or heterogeneous microspheres (particular dispersions): they are intermediate systems between the two possible states of heterogeneity (microcapsules) and homogeneity (microspheres). They are identified by the presence of rich and poor areas in active principle and by having an internal structure of crystalline dispersion type.
En general, las ventajas principales de las micropar- tículas son el ofrecer una protección al material y/o fármaco encapsulado de su eventual degradación en las condiciones de almacenamiento y/o biológicas y el permitir perfiles de liberación sostenida en el tiempo, sin necesidad de administraciones repetidas.In general, the main advantages of microparticles are to offer protection to the material and / or encapsulated drug from its eventual degradation in storage and / or biological conditions and to allow profiles of sustained release over time, without the need for repeated administrations.
Las micropartículas se pueden obtener a partir de materiales naturales o sintéticos. Los primeros incluyen proteínas (albúmina, colágeno, gelatina) y polisacáπdos (ácido hialurónico, ácido algínico, quitosano) . Entre los materiales sintéticos cabe destacar los poli-ésteres de hidroxiácidos, los poli-ortoésteres, los poli-alquilcarbo- natos, los poli -aminoácidos, los poli-anhídridos, las poli- acrilamidas y los poli-alqu l-α-cianoacπlatos De todas formas, los materiales más utilizados en la fabricación de micropartículas son los polímeros biodegradables de tipo poli-éster. Entre ellos se pueden citar los siguientes polímeros: ácido poliláctico (PLA) , copolímeros del ácido láctico y del ácido glicólico (PLAGA) , ácido poli (hidroxi- butíπco) y la poli- (ε-caprolactona) .The microparticles can be obtained from natural or synthetic materials. The former include proteins (albumin, collagen, gelatin) and polysaccharides (hyaluronic acid, alginic acid, chitosan). Synthetic materials include hydroxy acid polyesters, poly orthoesters, polycarbonates, polyamino acids, poly anhydrides, poly acrylamides and poly-alkyl l-α-cyanoacrylates. However, the most commonly used materials in the manufacture of microparticles are biodegradable polymers of the poly-ester type. Among them, the following polymers can be mentioned: polylactic acid (PLA), copolymers of lactic acid and glycolic acid (PLAGA), poly (hydroxybutyl) acid and poly (ε-caprolactone).
Por lo que respecta a los procedimientos de fabricación de micropartículas, estos se pueden clasificar en tres grandes grupos: físico-químicos, químicos y mecánicos La selección de un procedimiento de fabricación de micro- partículas depende de las propiedades físicas y químicas de la pareja principio activo/polímero. Además, es necesario tener en cuenta las propiedades de las micropartículas que quieren ser preparadas (granulometría, estructura interna, carga en principio activo, perfil de liberación, moja- bilidad, etc . ) .With regard to microparticle manufacturing procedures, these can be classified into three main groups: physical-chemical, chemical and mechanical The selection of a micro-particle manufacturing process depends on the physical and chemical properties of the principle couple active / polymer. In addition, it is necessary to take into account the properties of the microparticles that want to be prepared (granulometry, internal structure, loading in active principle, release profile, wettability, etc.).
Procedimientos físico-químicos . En este grupo se puede destacar la microencapsulación por coacervación, la micro- encapsulación por evaporación del disolvente y la micro- encapsulación por fusión. (i) Microencapsulación por coacervación o separación de fases .Physical-chemical procedures In this group, microencapsulation by coacervation, microcapsulation by evaporation of the solvent and microcapsulation by fusion can be highlighted. (i) Microencapsulation by coacervation or phase separation.
La coacervación permite la separación de un coloide en solución en una fase rica en coloide (llamado coacervato) y otra pobre en esta sustancia (Nairn, Advances in Pharmaceu - tical Sciences . Vol . 7, Academic Press Ltd., London, 1995, 93-219) . Este procedimiento conduce generalmente a la formación de microcápsulas, aunque a veces también pueden ser obtenidas microesferas . Se distinguen dos técnicas pπn- cipales: la coacervación simple y la coacervación compleja. La coacervación simple hace referencia a los procedimientos utilizados con un único coloide (proteínas, celulosas, poli-ésteres ) . Este se disuelve en un disolvente apropiado donde el principio activo se encuentra disper- sado. La separación de fases se induce por adición de un segundo compuesto (electrolitos, disolvente orgánico) incompatible con la presencia en solución del coloide (Óner & Groves, J". Pharm . Pharmacol . , 45 (1993) 866-870; Nihant et al., ". Controlled Reí . , 35 (1995) 117-125) o mediante modificación de las características fisicoquímicas de la disolución (Ezpeleta et al., Eur. J. Pharm . Biophar . , 42 (1996) 36-4) . El coacervato se deposita sobre las partículas en suspensión recubriéndolas y finalmente, se consolida. La coacervación compleja hace referencia a las técnicas que se aplican a la coacervación de forma simultánea de dos coloides diferentes. El fenómeno tiene lugar fundamentalmente por modificación del pH de la solución inicial y se aplica a la coacervación de la gelatina con goma arábiga, ácido poliacrílico, alginato sódico o polifosfato (Tirkkonen et al., J. Microencaps . , 11 (1994) 615-626) . Generalmente, y para ambas técnicas, es necesaria una etapa suplementaria consistente en la consolidación o estabilización del coacervato obtenido.Coacervation allows the separation of a colloid in solution in a phase rich in colloid (called coacervate) and another poor in this substance (Nairn, Advances in Pharmaceutical Sciences. Vol. 7, Academic Press Ltd., London, 1995, 93 -219). This procedure generally leads to the formation of microcapsules, although sometimes microspheres can also be obtained. There are two main techniques: simple coacervation and complex coacervation. Simple coacervation refers to the procedures used with a single colloid (proteins, celluloses, poly esters). This is dissolved in an appropriate solvent where the active ingredient is dispersed. Phase separation is induced by the addition of a second compound (electrolytes, organic solvent) incompatible with the presence in colloid solution (Óner & Groves, J " . Pharm. Pharmacol., 45 (1993) 866-870; Nihant et al ., " . Controlled Reí., 35 (1995) 117-125) or by modification of the physicochemical characteristics of the solution (Ezpeleta et al., Eur. J. Pharm. Biophar., 42 (1996) 36-4). The coacervate is deposited on the suspended particles by coating them and finally, it is consolidated. Complex coacervation refers to the techniques that are applied to coacervation simultaneously of two different colloids. The phenomenon occurs mainly by modifying the pH of the initial solution and is applied to the coacervation of gelatin with gum arabic, polyacrylic acid, sodium alginate or polyphosphate (Tirkkonen et al., J. Microencaps., 11 (1994) 615- 626). Generally, and for both techniques, a supplementary stage consisting of the consolidation or stabilization of the obtained coacervate is necessary.
(11) Microencapsulación por evaporación del disolvente.(11) Microencapsulation by evaporation of the solvent.
Es la técnica más utilizada y está basada en la evaporación por agitación de la fase interna de una emulsión. En una primera etapa, el polímero y el fármaco o material a encapsular se disuelven en un disolvente orgánico volátil Esta fase orgánica se emulsiona bajo agitación en un medio dispersante constituido por un líquido incapaz de disolver el polímero ni de mezclarse con él (solución acuosa de un tensioactivo) . Una vez obtenida la emulsión lipófilo en hidrófilo (O/A) . el disolvente orgánico presente en las gotículas de la emulsión se elimina por agitación dando lugar a microesferas sólidas (Watts et al., Cπt. Rev . Ther. Drug Carrier Syst . , 7 (1990) 235-259). El fármaco a encapsular se incorpora en la fase lipófila (0) , de tal forma que quedará atrapado dentro de las m cropartículas formadas (Bodmeier & McGmity, Pharm . Res . , 4 (1987) 465- 471) . Este método es ideal para incorporar fármacos liposo- lubles .It is the most used technique and is based on the evaporation by agitation of the internal phase of an emulsion. In a first stage, the polymer and the drug or material to be encapsulated are dissolved in a volatile organic solvent. This organic phase is emulsified under stirring in a dispersing medium consisting of a liquid unable to dissolve the polymer or mix with it (aqueous solution of a surfactant). Once the lipophilic emulsion is obtained in hydrophilic (O / A). The organic solvent present in the droplets of the emulsion is removed by stirring resulting in solid microspheres (Watts et al., Cπt. Rev. Ther. Drug Carrier Syst., 7 (1990) 235-259). The drug to be encapsulated is incorporated into the lipophilic phase (0), so that it will be trapped within the m formed croparticles (Bodmeier & McGmity, Pharm. Res., 4 (1987) 465-471). This method is ideal for incorporating liposoluble drugs.
En el caso de fármacos o materiales hidrosolubles, se propuso una variante consistente en la preparación de una emulsión múltiple de tipo hidrófilo en lipófilo en hidrófilo (A1/θ/A2) . Para ello, el principio activo a encapsular se incorpora en solución acuosa (Ax ) en la solución orgánica del polímero (O) con el fin de obtener una primera emulsión de tipo hidrófilo en lipófilo (A2/0) . Posteriormente, esta emulsión primaria o interna se emulsiona a su vez en una fase acuosa externa (A2) obteniéndose la emulsión múltiple A/0/A2. Finalmente, la evaporación del disolvente orgánico dará lugar a micropartículas sólidas (microcápsulas homo- géneas) que contienen vesículas acuosas en su interior y donde está el principio activo en solución (Okada et al , U. S . Patent 4,652,441 (1987); Ogawa et al., Chem . Pharm . Bull . 36 (1988) 1095-1103; Esposito et al., Int . J. Pharm . , 129 (1996) 263-273) . Los mayores inconvenientes de esta técnica son la necesidad de tener que utilizar cantidades elevadas de tensioactivos y el uso de sistemas de agitación, para emulsionar las distintas fases, muy energéticos como el Ultra-turrax® o las sondas de ultrasonidos. Estos sistemas de agitación pueden causar daños estructurales importantes en ciertas macromoléculas biológicas de gran interés terapéutico (ADN plasmídico, ol gonucleótidos, pép- tidos, proteínas, etc.) .In the case of water-soluble drugs or materials, a variant consisting of the preparation of a hydrophilic multiple emulsion in lipophilic in hydrophilic (A 1 / θ / A 2 ) was proposed. For this, the active substance to be encapsulated is incorporated in aqueous solution (A x) in the organic solution of the polymer (O) in order to obtain a first emulsion type hydrophilic lipophilic (A 2/0). Subsequently, this primary or internal emulsion is in turn emulsified in an external aqueous phase (A 2 ) obtaining the multiple emulsion A / 0 / A 2 . Finally, evaporation of the organic solvent will result in solid microparticles (homogenous microcapsules). genera) containing aqueous vesicles inside and where the active substance is in solution (Okada et al, U. S. Patent 4,652,441 (1987); Ogawa et al., Chem. Pharm. Bull. 36 (1988) 1095-1103 ; Esposito et al., Int. J. Pharm., 129 (1996) 263-273). The major drawbacks of this technique are the need to use high amounts of surfactants and the use of agitation systems, to emulsify the different, very energetic phases such as Ultra-turrax® or ultrasound probes. These agitation systems can cause significant structural damage in certain biological macromolecules of great therapeutic interest (plasmid DNA, ol gonucleotides, peptides, proteins, etc.).
(ni) Microencapsulación por fusión en caliente del polímero(ni) Hot melt microencapsulation of the polymer
Esta técnica conduce a la obtención de microesferas a partir de materiales con ba o punto de fusión (algunos lí- pidos como la cera de carnauba o el alcohol cetílico) . El fármaco o material a encapsular se disuelve o se dispersa en el excipiente fundido. Posteriormente, el conjunto se emulsiona en una fase dispersante por la cual el principio activo no tenga afinidad (agua para moléculas lipofílicas; aceite de silicona para moléculas hidrofílicas) y, final- mente, los glóbulos se solidifican por enfriamiento brusco del medio reaccionante (Akiyama et al., J. Controlled Reí . , 26 (1993) 1-10; Domb & Bergelson, Microencapsulation . Methods and Indus trial Appli ca tions, Marcel Dekker, New York, 1996, 377-410) .This technique leads to the obtaining of microspheres from materials with ba or melting point (some lipids such as carnauba wax or cetyl alcohol). The drug or material to be encapsulated is dissolved or dispersed in the molten excipient. Subsequently, the assembly is emulsified in a dispersing phase whereby the active ingredient has no affinity (water for lipophilic molecules; silicone oil for hydrophilic molecules) and, finally, the globules are solidified by abrupt cooling of the reaction medium (Akiyama et al., J. Controlled Reí., 26 (1993) 1-10; Domb & Bergelson, Microencapsulation. Methods and Indus trial Appli cations, Marcel Dekker, New York, 1996, 377-410).
2. Procedimientos químicos. En este grupo se incluyen las técnicas de preparación de micropartículas por reacciones de polimerización y mediante fenómenos de gelificación (i) Microencapsulación por policondensación mterfacial2. Chemical procedures. This group includes techniques for preparing microparticles by polymerization reactions and by gelation phenomena (i) Micro-encapsulation by mfacial polycondensation
La policondensación mterfacial es la reacción que se produce entre dos monómeros químicamente diferentes en la interfase de dos líquidos no irascibles Esta reacción con- duce a la formación de una película poliméπca y a la obtención de microcápsulas En la práctica se realiza una emulsión de tipo hidrófilo en lipófilo (A/O) , en la que la fase acuosa interna contiene la sustancia a encapsular y uno de los monómeros, y la fase lipófila dispersante está formada por un disolvente orgánico, el segundo monómero y un tensioactivo . Los monómeros difunden hacia la mterfase donde reaccionarán para formar un único polímero que encap- sula la fase dispersada (Whateley Microencapsulation . Methods and Industrial Applications, Marcel Dekker, New York, 1996, 349-375) .The mtfacial polycondensation is the reaction that occurs between two chemically different monomers at the interface of two non-irascible liquids. This reaction leads to the formation of a polymethyl film and to the obtaining of microcapsules. In practice, an emulsion of the hydrophilic type is carried out in lipophilic (A / O), in which the internal aqueous phase contains the substance to be encapsulated and one of the monomers, and the dispersing lipophilic phase is formed by an organic solvent, the second monomer and a surfactant. The monomers diffuse to the mterfase where they will react to form a single polymer that encapsulates the dispersed phase (Whateley Microencapsulation. Methods and Industrial Applications, Marcel Dekker, New York, 1996, 349-375).
(n) Microencapsulación por gelificación del material soporte .(n) Microencapsulation by gelation of the support material.
Todos los polímeros forman geles en condiciones parti- culares de hidratación. Cuando es posible estabilizar el gel formado, esta propiedad puede ser utilizada para encapsular una sustancia activa. El algmato sódico, por ejemplo, puede dar lugar a geles en presencia de cationes bivalentes (Ca2+) e incluso organizarse en membranas semí- permeables que son estabilizadas mediante policationes (Dupuy et al., Artif . Blood Subst . Immobil . Biotech . , 22 (1994) 71-82) .All polymers form gels under particular hydration conditions. When it is possible to stabilize the gel formed, this property can be used to encapsulate an active substance. Sodium alginate, for example, can give rise to gels in the presence of bivalent cations (Ca 2+ ) and even organize themselves in semi-permeable membranes that are stabilized by polycations (Dupuy et al., Artif. Blood Subst. Immobil. Biotech. , 22 (1994) 71-82).
3. Procedimientos mecánicos. En este grupo se pueden incluir la microencapsulación en lecho fluido, por pulverización, mediante fluidos en condiciones supercríticas y or inyección coaxial turbulenta. La mayor ventaja de todos estos procedimientos es el de poder ser utilizados a escala industrial .3. Mechanical procedures. This group can include microencapsulation in a fluid bed, by spraying, by means of fluids under supercritical conditions and or turbulent coaxial injection. The biggest advantage of all These procedures are to be used on an industrial scale.
(i) Microencapsulación en lecho fluido.(i) Microencapsulation in fluid bed.
Esta técnica está destinada a la encapsulación de par- tículas sólidas (granulados y cristales) mediante la utilización de celulosas, derivados metacrílicos , etc. Las partículas de principio activo se ponen en suspensión dentro de una cámara atravesada por una corriente de aire ascendente de fuerte potencia. La solución de recubrimiento que contiene el polímero se pulveriza dentro de la cámara y va recubriendo la superficie de las partículas (Vervaet et al., Int . J. Pharm . , 116 (1995) 131-146).This technique is intended for encapsulation of solid particles (granules and crystals) through the use of celluloses, methacrylic derivatives, etc. The active substance particles are suspended in a chamber crossed by a strong upward air current. The coating solution containing the polymer is sprayed into the chamber and covers the surface of the particles (Vervaet et al., Int. J. Pharm., 116 (1995) 131-146).
(n) Microencapsulación por pulverización. La microencapsulación por pulverización se realiza en cámaras en cuyo extremo superior una fase líquida es finamente dispersada a través de un pulverizador. Las gotículas formadas atraviesan una corriente de aire a una temperatura determinada, con lo que se produce la evaporación del di- solvente. Esto permite que los glóbulos se solidifiquen y sean recuperados en la base de la cámara de pulverización. Según la técnica de secado utilizada y de las características de la fase líquida inicial se distingue entre dos procedimientos principales : - pulverización y secado ( "* "* Spray-drymg" ) . Permite la en- capsulación de sólidos o aceites dispersados ínicialmente en una solución de recubrimiento adecuada. Para ello, el polímero disuelto en un disolvente adecuado junto con el fármaco o material a encapsular se pulveriza en una cámara de aire caliente a vacío. En esas condiciones, el disolvente se evapora y el polímero precipita en forma de micro- partículas (Thoma & Schlutermann, Pharmazi e, A l (1992) 115- 119) . Al igual que el método de la evaporación del disol- vente, es especialmente apto para atrapar sustancias lipo- solubles . En el caso de sustancias hidrosolubles es necesario formar, antes de la nebulización, una primera emulsión A/O ( agenaar & Múller, Biomaterials, 15 (1994) 49-54) .(n) Spray microencapsulation. Spray microencapsulation is performed in chambers at the upper end of which a liquid phase is finely dispersed through a sprayer. The droplets formed pass through a stream of air at a certain temperature, whereby evaporation of the solvent occurs. This allows the blood cells to solidify and be recovered at the base of the spray chamber. Depending on the drying technique used and the characteristics of the initial liquid phase, two main procedures are distinguished: - spray and drying ("* " * Spray-drymg "). It allows the encapsulation of solids or oils initially dispersed in a suitable coating solution.To do this, the polymer dissolved in a suitable solvent together with the drug or material to be encapsulated is sprayed in a vacuum hot air chamber.In these conditions, the solvent evaporates and the polymer precipitates in the form of a micro - particles (Thoma & Schlutermann, Pharmazi e, A l (1992) 115-119), as well as the solvent evaporation method It is especially suitable for trapping lipo-soluble substances. In the case of water-soluble substances, it is necessary to form, before nebulization, a first A / O emulsion (agenaar & Múller, Biomaterials, 15 (1994) 49-54).
- pulverización y congelación ( Λ Spray congealmg" ) . Este procedimiento se parece a la fusión en caliente descrito anteriormente. El material de recubrimiento fundido, que contiene el principio activo dispersado, es pulverizada en la cámara que, en este caso, está atravesada por una corriente de aire frío (Wichert & Rohdewald, J. Controlled Reí . , 14 (1990) 269-283) .- spraying and freezing ( Λ Spray congealmg "). This procedure resembles the hot melt described above. The molten coating material, which contains the dispersed active substance, is sprayed into the chamber which, in this case, is traversed by a stream of cold air (Wichert & Rohdewald, J. Controlled Reí., 14 (1990) 269-283).
(ni) Microencapsulación mediante fluidos en condiciones supercríticas .(ni) Microencapsulation by means of fluids under supercritical conditions.
En este caso, una solución del polímero en un disolvente orgánico que contiene el principio activo, es pulverizada dentro de una columna que contiene un fluido en condiciones supercríticas, obteniéndose de manera instan- tánea las micropartículas (Knutson et al., Mi croparticulate syste s for the delivery of protems and vaccines, Marcel Dekker Inc., New York, 1996, 89-125).In this case, a solution of the polymer in an organic solvent containing the active substance is sprayed into a column containing a fluid under supercritical conditions, the microparticles being obtained instantly (Knutson et al., My croparticulate syste s for the delivery of protems and vaccines, Marcel Dekker Inc., New York, 1996, 89-125).
(ív) Microencapsulación por inyección coaxial turbulenta. Este procedimiento se basa en la inyección coaxial, a alta presión, de dos fases inmiscibles en el seno de un reactor. La primera fase contiene el polímero y el fármaco o material a encapsular, disuelto en un disolvente orgánico apolar y la segunda es una disolución acuosa de un tensio- activo. El régimen turbulento que se alcanza permite obtener una emulsión muy homogénea que, tras la evaporación del disolvente orgánico, dará lugar a microesferas (Recarte et al., Patente Española ES2134130, 1997). Sin embargo, este procedimiento no puede utilizarse para la encapsu- lación de fármacos o materiales hidrosolubles(ív) Microencapsulation by turbulent coaxial injection. This procedure is based on the coaxial injection, at high pressure, of two immiscible phases within a reactor. The first phase contains the polymer and the drug or material to be encapsulated, dissolved in a nonpolar organic solvent and the second is an aqueous solution of a surfactant. The turbulent regime that is achieved allows a very homogeneous emulsion to be obtained which, after evaporation of the organic solvent, will give rise to microspheres (Recarte et al., Spanish Patent ES2134130, 1997). Nevertheless, This procedure cannot be used for encapsulation of drugs or water-soluble materials.
Para la encapsulación de las sustancias hidrosolubles , a las que se refiere esta invención, el método más empleado es el de la evaporación del disolvente tras la formación de una emulsión múltiple del tipo hidrófílo/lipófílo/hidróflio o agua/aceite/agua {A1/Q/A2 ) . Por este procedimiento, la formación de la emulsión A1/θ/A2 requiere la utilización de sistemas de agitación o homogeneización muy violentos. Entre ellos, los más utilizados son el sistema Ultra- turrax® y las sondas de ultrasonidos.For the encapsulation of the water-soluble substances, to which this invention refers, the most used method is the evaporation of the solvent after the formation of a multiple emulsion of the hydrophilic / lipophilic / hydrophilic type or water / oil / water {A 1 / Q / A 2 ). By this procedure, the formation of the emulsion A 1 / θ / A 2 requires the use of very violent agitation or homogenization systems. Among them, the most commonly used are the Ultra-turrax ® system and the ultrasound probes.
La presente invención se basa en un nuevo procedimiento y aparato que permite la obtención de una emulsión múltiple, de tamaño muy homogéneo, apta para la encapsu- lación de fármacos, moléculas biológicamente activas y materiales de naturaleza hidrosoluble o que deben manipularse en soluciones acuosas. Además, la emulsión múltiple se obtiene mediante un sistema de homogeneización en continuo mucho menos violento y agresivo que los sistemas de emulsificación tradicionales. En esas condiciones, la estabilidad del fármaco, molécula biológicamente activa o material a encapsular es mucho mayor y se minimiza el daño estructural. Lógicamente, al aumentar la estabilidad estructural del material, evitando su ruptura, se preserva su actividad. Por otra parte, la ausencia de métodos de homo- geneización energéticos, permite la utilización de esta técnica para la microencapsulación de materiales lábiles o de baja estabilidad físico-química . Entre ellos cabe destacar las moléculas originarias de la biotecnología como ADN, ácido ribonucleico (ARN) , oligonucleótidos antisen- tido, moléculas derivadas de bases puncas y pirimidínicas, péptidos y moléculas relacionadas. Por otra parte, uno de los vectores virales más utilizados en la actualidad en el diseño de nuevos tratamientos basados en la terapia génica, lo constituyen los adenovirus recombmantes defectivos. Estos adenovirus recombinantes son buenos vectores para la administración de genes terapéuticos y poseen un número importante de ventajas Así, los adenovirus son muy eficientes para trasducir un número elevado de tipos celulares diferentes, pueden ser purificados y obtenidos con elevados títulos (aprox. 1013 partículas/mL) y sobreviven bastante bien en el organismo (Beer et al., Adv Drug Deliv Rev. , 27 (1997) 59-66; Kelley & Sukhatme Am J Physiol . , 276 (Renal Physiol 45), (1999) F1-F9) Sin embargo, también presentan ciertos inconvenientes. Entre ellos, destaca su baja estabilidad a tempe- ratura ambiente. Así, por ejemplo, 24h a temperatura de 20- 25 °C inactiva la práctica totalidad de las partículas dispersas en una solución acuosa. Este fenómeno implica que el almacenamiento, transporte y conservación de estos vectores se realice a muy bajas temperaturas (congeladores de -80°C, nieve carbónica, etc..) . Por último, es de destacar que la incorporación de ADN plasmídico, como vectores virales u oro tipo de materiales en el seno de micropartículas, permite su liberación sostenida en el tiempo.The present invention is based on a new method and apparatus that allows obtaining a multiple emulsion, of very homogeneous size, suitable for the encapsulation of drugs, biologically active molecules and materials of a water-soluble nature or to be handled in aqueous solutions. In addition, multiple emulsion is obtained through a continuous homogenization system much less violent and aggressive than traditional emulsification systems. Under these conditions, the stability of the drug, biologically active molecule or material to be encapsulated is much greater and structural damage is minimized. Logically, by increasing the structural stability of the material, preventing its rupture, its activity is preserved. On the other hand, the absence of energy homogenization methods allows the use of this technique for the microencapsulation of labile materials or of low physical-chemical stability. Among them, it is worth mentioning the molecules that originate in biotechnology such as DNA, ribonucleic acid (RNA), antisense oligonucleotides, molecules derived from spike and pyrimidine bases, peptides and related molecules. On the other hand, one of the most commonly used viral vectors in the design of new treatments based on gene therapy, is the defective recombinant adenovirus. These recombinant adenoviruses are good vectors for the administration of therapeutic genes and have a significant number of advantages. Thus, adenoviruses are very efficient in transducing a large number of different cell types, they can be purified and obtained with high titers (approx. 10 13 particles / mL) and survive quite well in the body (Beer et al., Adv Drug Deliv Rev., 27 (1997) 59-66; Kelley & Sukhatme Am J Physiol., 276 (Renal Physiol 45), (1999) F1- F9) However, they also have certain drawbacks. Among them, its low stability at room temperature stands out. Thus, for example, 24 hours at a temperature of 20-25 ° C inactivates almost all the particles dispersed in an aqueous solution. This phenomenon implies that the storage, transport and conservation of these vectors is carried out at very low temperatures (freezers of -80 ° C, carbonic snow, etc.). Finally, it is noteworthy that the incorporation of plasmid DNA, such as viral vectors or gold type of materials within microparticles, allows their sustained release over time.
Las condiciones de aplicabilidad que permiten pensar en el interés industrial de este procedimiento y aparato para la producción de micropartículas a partir de polímeros sintéticos y su aplicación a la administración de fármacos de naturaleza hidrosoluble y vectores virales de tipo adenovirus recornbinante defectivo, pueden ser las siguientes:The conditions of applicability that allow us to think about the industrial interest of this procedure and apparatus for the production of microparticles from synthetic polymers and their application to the administration of drugs of a water-soluble nature and viral vectors of defective adenovirus recornbinant type, may be the following :
1. Obtención y producción en continuo de micropartículas de tamaño muy homogéneo. Estas micropartículas son del tipo microcápsulas homogéneas o microesferas heterogéneas. 2. Utilización de polímeros preformados ampliamente aceptados en el campo químico- farmacéutico para su utilización en el diseño y fabricación de medicamentos y productos de suturas . 3. Condiciones de trabajo a temperatura ambiente que eviten la posible degradación de las sustancias activas termo- lábiles de naturaleza hidrosoluble, y de origen químico y/o biotecnológico, o de materiales sensibles a la temperatura que van a ser incorporados al interior de las micropartículas.1. Collection and continuous production of microparticles of very homogeneous size. These microparticles are of the type homogeneous microcapsules or heterogeneous microspheres. 2. Use of preformed polymers widely accepted in the chemical-pharmaceutical field for use in the design and manufacture of medications and suture products. 3. Working conditions at room temperature that prevent the possible degradation of thermo-labile active substances of a water-soluble nature, of chemical and / or biotechnological origin, or of temperature sensitive materials that are to be incorporated into the microparticles .
4. Minimizar la intervención humana durante la etapa de producción de las micropartículas .4. Minimize human intervention during the microparticle production stage.
5. Posibilidad de controlar, en todo momento, todos los parámetros de fabricación (temperatura, volúmenes de mez- cía, cantidades inyectadas...). Posibilidad de trabajar en condiciones estériles .5. Possibility of controlling, at all times, all manufacturing parameters (temperature, mixing volumes, injected quantities ...). Possibility of working in sterile conditions.
6. Obtención de micropartículas capaces de proteger contra la inactivación prematura de los fármacos, moléculas biológicamente activas y materiales encapsulados . Esta protección aumenta la estabilidad del material encap- sulado durante (i) los procesos de almacenamiento y conservación y (n) en los medios biológicos.6. Obtaining microparticles capable of protecting against premature inactivation of drugs, biologically active molecules and encapsulated materials. This protection increases the stability of the encapsulated material during (i) storage and conservation processes and (n) in biological media.
7. Obtención de micropartículas con vectores virales en su interior que pueden evitar la utilización, para estas partículas virales, de sistemas de transporte, almacenamiento y conservación de coste muy elevado. Además, la encapsulación de vectores virales en el seno de micro- partículas debe poder aumentar la eficacia de la transferencia génica, disminuir el número de dosis (y por tanto la toxicidad sistémica) y minimizar las respuestas mmunitaπas del huésped.7. Obtaining microparticles with viral vectors inside that can prevent the use, for these viral particles, of transport, storage and conservation systems of very high cost. In addition, the encapsulation of viral vectors within micro-particles should be able to increase the efficiency of gene transfer, decrease the number of doses (and therefore systemic toxicity) and minimize host immune responses.
8. Obtención de micropartículas que permiten la liberación controlada del fármaco, molécula biológicamente activa o material encapsulado en su interior. Esto permite no tener que recurrir a administraciones repetidas cada poco tiempo Además, esta liberación controlada junto con la protección ofrecida frente al medio biológico del mate- nal incluido en su interior permite optimizar y facilitar los tratamientos terapéuticos.8. Obtaining microparticles that allow controlled release of the drug, biologically active molecule or encapsulated material inside. This allows not having to resort to repeated administrations every little time. In addition, this controlled release together with the protection offered against the biological environment of the material included in its interior allows the therapeutic treatments to be optimized and facilitated.
Descripción de la invenciónDescription of the invention
La presente invención propone un nuevo sistema de fabricación de micropartículas basado en la inyección turbulenta de dos fases y posterior evaporación del disolvente orgánico utilizado. Este método de fabricación tiene lugar en un aparato denominado , TROMS" o λ ** Total Recirculation One-Machine System (Sistema Monobloque de Recirculación Completa) . Entre las principales ventajas de este nuevo procedimiento, es posible destacar: (i) método fácilmente reproducible ;The present invention proposes a new microparticle manufacturing system based on the turbulent two-phase injection and subsequent evaporation of the organic solvent used. This manufacturing method takes place in a device called , TROMS "or λ ** Total Recirculation One-Machine System. Among the main advantages of this new procedure, it is possible to highlight: (i) easily reproducible method ;
(n) gran uniformidad en los tamaños de las micropartículas obtenidas ; (m) posibilidad de selección del tamaño de las micro- partículas a obtener;(n) great uniformity in the sizes of the microparticles obtained; (m) possibility of selecting the size of the micro particles to be obtained;
(ív) evita el uso de técnicas agresivas de emulsificación, agitación y/o homogeneización; (v) apto para la encapsulación de fármacos o moléculas bio- lógicamente activas de naturaleza hidrosoluble, incluyendo las macromoléculas de origen biotecnológico (ADN plasmí- dico, ARN, genes, oligonucleótidos , péptidos, proteínas); (vi) apto para la encapsulación de materiales sensibles y que deben estar disueltos o dispersos en soluciones acuosas (vectores virales utilizados en terapia génica) .(iv) avoids the use of aggressive emulsification, agitation and / or homogenization techniques; (v) suitable for the encapsulation of biologically active drugs or molecules of a water-soluble nature, including macromolecules of biotechnological origin (plasmid DNA, RNA, genes, oligonucleotides, peptides, proteins); (vi) suitable for encapsulation of sensitive materials and that must be dissolved or dispersed in aqueous solutions (viral vectors used in gene therapy).
El aparato objeto de esta patente está representado en la Figura 1. Consta de un sistema de bombeo (1) que está conectado a una válvula Rheodyne™ (2) de la que salen dos agujas (3) y (4) de distinto diámetro interno. La primera de ellas se encuentra dentro de la primera vasija de mezclado (5) y la otra en la segunda vasija de mezclado (6) . Mediante una jeringa de vidrio con émbolo de teflon (7) se incorpora al sistema la fase orgánica. Las vasijas de mezclado están conectadas a la bomba a través de dos válvulas (8 y 9) .The apparatus object of this patent is represented in Figure 1. It consists of a pumping system (1) that is connected to a Rheodyne ™ valve (2) from which two needles (3) and (4) of different internal diameter. The first one is inside the first mixing vessel (5) and the other in the second mixing vessel (6). The organic phase is incorporated into the system using a glass syringe with a Teflon plunger (7). The mixing vessels are connected to the pump through two valves (8 and 9).
El proceso de producción de las micropartículas es como sigue: - Se preparan las fases según la formulación deseada [Fase Aλ : disolvente acuoso, fármaco o material biológicamente activo, y eventualmente tensioactivo o viscosizante; Fase O: disolvente orgánico inmiscible con el agua, polímero soluble en el disolvente orgánico; Fase A2 : disolvente acuoso y eventualmente un tensioactivo] . Como polímero se utilizan los biodegradables (generalmente poli- ésteres como la poli-épsilon caprolactona, los ácidos poli-lácticos, PLA, o los copolímeros entre el ácido láctico y el ácido glicólico, PLAGA) . Como tensioactivo interno o dentro de la vesícula se emplean generalmente la polivinilpirrolidona (PVP) a concentraciones del orden del 0-20% p/v, la gelatina (0-5% p/v) o la goma xantano (0-1% p/v) . Como tensioactivo/viscosizante externo se emplea generalmente el alcohol polivmílico, PVA. También se incorpora como adyuvante de la liofílización un copolímero de óxido de etileno y óxido de propileno. Eventualmente también puede utilizarse un crio- protector (manitol y sacarosa) . Se ajusta el caudal de la bomba y se procede a la selección de las agujas según el tamaño de partícula deseado. En la primera vasija de mezclado (5) se introduce la fase acuosa A., que contiene el fármaco o el material a encapsular. Se acciona el sistema de bombeo manteniendo cerradas las válvulas que conectan las vasijas de mezcla con la bomba, de tal forma que a través de la jeringa (7) se introduce la fase 0, que contiene el polímero formador de las micropartículas disuelto en un disolvente adecuado. La válvulaThe production process of the microparticles is as follows: - The phases are prepared according to the desired formulation [Phase A λ : aqueous solvent, drug or biologically active material, and possibly surfactant or viscosizer; Phase O: water-immiscible organic solvent, polymer soluble in the organic solvent; Phase A 2 : aqueous solvent and possibly a surfactant]. Biodegradable are used as a polymer (generally polyesters such as poly-epsilon caprolactone, polylactic acids, PLA, or copolymers between lactic acid and glycolic acid, PLAGA). Polyvinylpyrrolidone (PVP) at concentrations of the order of 0-20% w / v, gelatin (0-5% w / v) or xanthan gum (0-1% p) are generally used as internal surfactant or within the gallbladder / v). As an external surfactant / viscosizer polyvinyl alcohol, PVA, is generally used. A copolymer of ethylene oxide and propylene oxide is also incorporated as a lyophilization aid. Eventually a cryoprotectant (mannitol and sucrose) can also be used. The pump flow rate is adjusted and the needles are selected according to the desired particle size. In the first mixing vessel (5) the aqueous phase A., which contains the drug or the material to be encapsulated, is introduced. The pumping system is operated keeping the valves connecting the mixing vessels with the pump closed, so that phase 0 is introduced through the syringe (7), which contains the microparticle-forming polymer dissolved in a suitable solvent. The valve
Rheodyne™ está en la posición que permite la inyección turbulenta a través de la aguja (3) . La fase O es por tanto inyectada de forma turbulenta sobre la fase A: . Inicialmente se forma, dentro de la vasija (3) , una emulsión O/A que rápidamente se in-vierte en una emulsión A/0 dado que el volumen de la fase O es sustancialmente mayor que el de la fase -L . En ese momento se abre la válvula (8) y se permite que la emulsión formada recircule a través del sistema durante un periodo de tiempo suficiente para que la emulsión sea homogénea (este periodo depende del volumen de emulsión formado) . Una vez la emulsión A1/0 es homogénea, se cambia la posición de la válvula Rheodyne™ (2) para que la mezcla pase a través de la aguja (4) , de mayor diámetro interno que la (3) . Se inyecta así la emulsión A^O en la vasija (6) que contiene la fase acuosa A2. La inyección turbulenta hace que se forme la emulsión A1/0/A2 dentro de la vasija (6) . Se cierra la válvula (8) y se abre la (9) para permitir la recirculación total de la mezcla, durante un periodo de tiempo suficiente para que la emulsión tenga un tamaño de gota homogéneo. Tras ese tiempo, la emulsión se de a agitando a temperatura ambiente hasta la completa evaporación del disolvente de la fase O mediante un agitador de hélice (10) . Esta operación puede realizarse en el mismo aparato, incorporando en la vasija (6) un agitador de hélice, o bien mediante un sistema de agitación ajeno al aparato. La evaporación del disolvente orgánico lleva consigo la formación de las micropartículas, que contienen en su interior la fase A., donde se encuentra el fármaco o el material biológicamente activo.Rheodyne ™ is in the position that allows turbulent injection through the needle (3). Phase O is therefore injected turbulently over phase A :. Initially, within the vessel (3), an O / A emulsion is formed which is quickly invested in an A / 0 emulsion since the volume of the O phase is substantially greater than that of the L- phase. At that time the valve (8) is opened and the formed emulsion is allowed to recirculate through the system for a period of time sufficient for the emulsion to be homogeneous (this period depends on the volume of emulsion formed). Once the emulsion 1/0 is uniform, the position of the Rheodyne ™ valve (2) for the mixture to pass through the needle (4) of internal diameter greater than the (3) is changed. The A ^ O emulsion is thus injected into the vessel (6) containing the aqueous phase A 2 . Turbulent injection makes the emulsion 1/0/2 into the vessel (6) is formed. The valve (8) is closed and the (9) is opened to allow total recirculation of the mixture, for a period of time sufficient for the emulsion to have a homogeneous droplet size. After that time, the emulsion is stirred at room temperature until complete evaporation of the phase O solvent by means of a propeller stirrer (10). This operation can be carried out in the same apparatus, incorporating a propeller stirrer in the vessel (6), or by means of a stirring system outside the apparatus. The evaporation of the organic solvent leads to the formation of the microparticles, which contain its interior is phase A., where the drug or biologically active material is found.
Ejemplos no limitativos de realización de la invención.Non-limiting examples of embodiment of the invention.
Los siguientes ejemplos ilustran el procedimiento y aparato objetos de la invención. En modo alguno los ejemplos aquí descritos pretenden limitar el alcance de la invención. Los materiales empleados fueron los siguientes- Polivinilpirrolidona (PVP, masa molecular 42.500), Copolí- mero de ácido láctico-glicólico (Resomer® 756, Resomer® 502), fluorescema sódica, poliviml alcohol (masa molecular 115.000), diclorometano, glicerol, copolímero de óxido de etileno (80%) y óxido de propileno (20%) con una masa molecular de 8400 (Pluronic® F-68, Sigma) . Se preparó ADN plasmídico, conteniendo el gen de la luciferasa bajo el control del promotor citomegalovirus (CMV) , mediante cromatografía de intercambio amónico. Los adenovirus recombi- nantes defectivos contenían el gen de la β-galactosidasa (lacZ) de E. coli , ba o control del promotor del CMV.The following examples illustrate the method and apparatus objects of the invention. In no way are the examples described herein intended to limit the scope of the invention. The materials used were: Polyvinylpyrrolidone (PVP, molecular mass 42,500), Lactic-glycolic acid copolymer (Resomer® 756, Resomer® 502), sodium fluorescema, polyvinyl alcohol (molecular mass 115,000), dichloromethane, glycerol, copolymer of ethylene oxide (80%) and propylene oxide (20%) with a molecular mass of 8400 (Pluronic ® F-68, Sigma). Plasmid DNA, containing the luciferase gene under the control of the cytomegalovirus promoter (CMV), was prepared by ammonium exchange chromatography. Defective recombinant adenoviruses contained the E. coli β-galactosidase (lacZ) gene, ba or CMV promoter control.
Las células HeLa crecieron en DMEM (medio Eagle modificado de Doubelco) suplementado con 10% un de suero fetal bovino, 100 U/mL penicilina y 100 mg/mL estreptomicina. El tamaño de las micropartículas se determinó mediante espectroscopia por difracción de láser, con un Mastersizer S (Malvern Instruments) .HeLa cells were grown in DMEM (Doubelco's modified Eagle medium) supplemented with 10% a fetal bovine serum, 100 U / mL penicillin and 100 mg / mL streptomycin. The size of the microparticles was determined by laser diffraction spectroscopy, with a Mastersizer S (Malvern Instruments).
Ejemplo 1: Fabricación de micropartículas vacías. Modificación del tamaño medio de las micropartículas según el tiempo de recirculación de la emulsión h1/0/A2 .Example 1: Manufacture of empty microparticles. Modification of the average size of the microparticles according to the recirculation time of the emulsion h 1/0/2.
Fases empleadas:Phases used:
Fase Ax : 300 μL de agua Viscosizante : Polivimlp rrolidona (PM 42.500) al 5% p/v Fase O: 4 mL de diclorometanoPhase A x : 300 μL of water Viscosizer: Polyvinyl rrolidone (PM 42,500) at 5% w / v Phase O: 4 mL dichloromethane
Copolímero de ácido poli (láctico/glicólico) 75:25 (PM 98.000) al 5% p/vCopolymer of poly (lactic / glycolic acid) 75:25 (PM 98,000) at 5% w / v
Fase A2 : 40 mL de aguaPhase A 2 : 40 mL of water
Tensioactivo: Poliviml alcohol (PM: 115 000) al 0,5% p/v Aguja (3) : 0, 12 mm Aguja (4) : 0,17 mmSurfactant: Polyvinyl alcohol (PM: 115 000) at 0.5% w / v Needle (3): 0.12 mm Needle (4): 0.17 mm
Tiempo de recirculación de la emulsión Aj/O: 1 minutoEmulsion recirculation time A j / O: 1 minute
La Figura 2 muestra como al aumentar el tiempo de recirculación de la emulsión doble disminuye el tamaño de las micropartículas obtenidas.Figure 2 shows how increasing the recirculation time of the double emulsion decreases the size of the microparticles obtained.
Ejemplo 2: Preparación de micropartículas que incorporan una molécula hidrosoluble modelo (fluoresceína sódica) en su interior. Las fases empleadas en el método estaban formadas por: Fase Aλ : 300 μL de aguaExample 2: Preparation of microparticles that incorporate a water-soluble model molecule (sodium fluorescein) inside. The phases used in the method were formed by: Phase A λ : 300 μL of water
Viscos zante : Polivinilpirrolidona (PM 42.500) al 5% p/vViscos zante: Polyvinylpyrrolidone (PM 42,500) at 5% w / v
Fluoresceína sódica al 0,2% p/v Fase O: 4 mL de diclorometano Copolímero de ácido poli (láctico/glicólico)0.2% w / v sodium fluorescein Phase O: 4 mL dichloromethane Poly (lactic / glycolic acid) copolymer
50:50 (PM 40-75.000) al 5% p/v Fase A2 : 40 mL de agua50:50 (PM 40-75,000) at 5% w / v Phase A 2 : 40 mL of water
Tensioactivo: Polivinil alcohol (PM: 115.000) al 0,5% p/v Aguja (3) : 0, 12 mmSurfactant: Polyvinyl alcohol (PM: 115,000) at 0.5% w / v Needle (3): 0.12 mm
Aguja (4) : 0,17 mmNeedle (4): 0.17 mm
El tiempo de recirculación de la emulsión A./O fue de 1 minuto. El de la emulsión A-/0/A2 de 3 minutos. Las micro- partículas obtenidas, una vez purificadas, se recogieron y dispersaron en una solución acuosa de Pluronic® F-68 2% p/v, y se liofílizaron . Eventualmente, como crioprotector se utilizó sacarosa o mamtol a una concentración 5% p/v. El tamaño medio de las partículas obtenidas es de 3,7 μm. Mediante microscopía óptica con luz UV puede observarse la estructura interna de las micropartículas, con las vesículas de la fase Al -donde está la fluoresceína- en su interior (Figura 3) .The recirculation time of the A./O emulsion was 1 minute. The emulsion A- / 0 / A 2 of 3 minutes. The micro- particles obtained, once purified, were collected and dispersed in a 2% w / v aqueous solution of Pluronic ® F-68, and lyophilized. Eventually, sucrose or mamtol was used as a cryoprotectant at a concentration of 5% w / v. The average size of the particles obtained is 3.7 μm. The internal structure of the microparticles can be observed by means of optical microscopy with UV light, with the Al-phase vesicles - where the fluorescein is - inside (Figure 3).
Ejemplo 3: Preparación de micropartículas que incorporan ADN plasmídico (7 b) .Example 3: Preparation of microparticles incorporating plasmid DNA (7 b).
Las fases empleadas en el método estaban formadas por: Fase A. : 225 μL de agua Viscosizante : Polivinilpirrolidona (PM 42.500) al 5% p/v o al 10% p/v, Gelatina al 1% p/v o goma xantano al 0,1% p/v ADN plasmídico (7kb) 675 μg Fase O: 3 mL de diclorometano Copolímero de ácido poli (láctico/glicólico)The phases used in the method were formed by: Phase A.: 225 μL of Viscosizing water: Polyvinylpyrrolidone (PM 42,500) at 5% w / v at 10% w / v, Gelatin 1% w / v xanthan gum at 0, 1% w / v Plasmid DNA (7kb) 675 μg Phase O: 3 mL dichloromethane Poly (lactic / glycolic acid) copolymer
50:50 (PM 80.000) al 5% p/v Fase A2 : 30 mL de agua50:50 (PM 80,000) at 5% w / v Phase A 2 : 30 mL of water
Tensioactivo: Polivinil alcohol (PM 115.000) al 0,5% p/v Aguja (3) : 0, 12 mm Aguja (4) : 0, 17 mmSurfactant: Polyvinyl alcohol (PM 115,000) 0.5% w / v Needle (3): 0.12 mm Needle (4): 0.17 mm
El tiempo de recirculación de la emulsión A^O fue de 1 minuto. El de la emulsión Aj/O/A- de 2 minutos. Las micro- partículas obtenidas, una vez purificadas, se recogieron y dispersaron en una solución acuosa de Pluronic® F-68 2% p/v, y se liofílizaron. La eficacia de encapsulación se determinó por espectroscopia de ultravioleta (UV) tras disolución de las micropartículas en dimetilsulfoxido (DMSO) . Por otra parte la integridad del ADN se determinó por electroforesis en gel en los sobrenadantes de las preparaciones . Para la realización de las cinéticas de liberación, 10 mg de micropartículas liofilizadas se incubaron en 0,5 mL de tampón salmo fosfato (PBS) a 37°C ba o agitación A diferentes tiempos, las micropartículas se centrifugaron y los sobrenadantes recogidos para determinar la cantidad de ADN plasmídico liberado y reemplazados por tampón nuevo. La cantidad de ADN liberada se determinó por espectroscopia UV, usando controles para todas las formulaciones.The recirculation time of the A ^ O emulsion was 1 minute. The emulsion A j / O / A- of 2 minutes. The obtained micro particles, once purified, were collected and dispersed in a 2% w / v aqueous solution of Pluronic ® F-68, and lyophilized. The encapsulation efficiency was determined by ultraviolet (UV) spectroscopy after dissolution of the microparticles in dimethylsulfoxide (DMSO). On the other hand the integrity of the DNA was determined by gel electrophoresis in the supernatants of the preparations. To carry out the release kinetics, 10 mg of lyophilized microparticles were incubated in 0.5 mL of phosphate buffered buffer (PBS) at 37 ° C or stirring. At different times, the microparticles were centrifuged and the supernatants collected to determine the amount of plasmid DNA released and replaced by new buffer. The amount of DNA released was determined by UV spectroscopy, using controls for all formulations.
Tabla 1. Características físico-químicas de las diferentes formulaciones .Table 1. Physicochemical characteristics of the different formulations.
Figure imgf000022_0001
Figure imgf000022_0001
* Tamaño medio de tres determinaciones . La Tabla 1 recoge las principales características físico-químicas de los distintos lotes de micropartículas obtenidos por TROMS. Igualmente, en la Figura 4 se recoge una microfotografía SEM de un lote de MSP-D. Los perfiles de liberación se muestran en la Figura 5. Tras 4 semanas, únicamente la formulación MSP-A había liberado la cantidad total del ADN encapsulado. La menor velocidad de liberación se corresponde con la formulación MSP- D, lo que sugiere que la goma xantano es un buen excipiente para retrasar la salida del material encapsulado. Todas las formulaciones mostraron un efecto ,xburst" o de liberación rápida durante el primer día de la experiencia. Este valor de liberación inicial variaba entre el 50% para la formulación MSP-C y el 20% para la MSP-D. Posteriormente, se puede observar liberaciones más lentas y continuadas en el tiempo para todas las formulaciones.* Average size of three determinations. Table 1 shows the main physicochemical characteristics of the different batches of microparticles obtained by TROMS. Similarly, Figure 4 shows an SEM photomicrograph of a batch of MSP-D. The release profiles are shown in Figure 5. After 4 weeks, only the MSP-A formulation had released the total amount of the encapsulated DNA. The lower release rate corresponds to the MSP-D formulation, which suggests that xanthan gum is a good excipient to delay the outflow of encapsulated material. All formulations showed an effect , x burst "or rapid release during the first day of the experience. This initial release value varied between 50% for the MSP-C formulation and 20% for the MSP-D. Subsequently, slower and continuous releases over time can be observed for all formulations.
Mediante la electroforesis en gel del ADN presente en los sobrenadantes podemos ver en qué estado está el plás- mido: superenrrollado (gran capacidad para la transfec- ción) , abierto circular (menor capacidad de transíección) o lineal (evidencia de rotura del ADN) . En este caso es importante que se encuentre mayoritaπamente en su estado superenrrollado ya que de ello depende su eficacia terapéutica. La figura 6 muestra el estado del ADN tras la fabricación de micropartículas utilizando como sistema de agitación un Ultra-turrax (6A) o TROMS (6B) . Se puede observar como el estado del DNA tras la utilización del Ultra-turrax da lugar a una rotura importante del ADN. Sin embargo, la fabricación de micropartículas por TROMS consigue que la práctica totalidad del ADN se encuentre en estado superenrollado o abierto circular.By means of the gel electrophoresis of the DNA present in the supernatants we can see in what state the plasmid is: supercoiled (large capacity for transfection), open circular (less transiection capacity) or linear (evidence of DNA breakage) . In this case it is important that it is mostly in its supercoiled state since its therapeutic efficacy depends on it. Figure 6 shows the state of the DNA after the manufacture of microparticles using as an agitation system an Ultra-turrax (6A) or TROMS (6B). It can be seen how the state of the DNA after the use of Ultra-turrax results in a significant DNA breakage. However, the manufacture of microparticles by TROMS ensures that almost all of the DNA is in a supercoiled or circular open state.
Ejemplo 4: Preparación de micropartículas con Adenovirus recombinantes defectivos en su interior.Example 4: Preparation of defective recombinant Adenovirus microparticles inside.
Las fases empleadas en el método estaban formadas por: Fase Ax .- 225 μL de aguaThe phases used in the method were formed by: Phase A x .- 225 μL of water
Polivimlpirrolidona (PM 42.500) al 5% p/v Adenovirus defectivos (AdCMVlacZ) en glicerol (glicerol al 6.6%) Fase O: 3 mL de diclorometanoPolyvinylpyrrolidone (PM 42,500) at 5% w / v Defective adenovirus (AdCMVlacZ) in glycerol (6.6% glycerol) Phase O: 3 mL dichloromethane
Copolímero de ácido poli (láctico/glicólico) 50:50 (PM 12.000) al 5% p/vCopolymer of poly (lactic / glycolic acid) 50:50 (PM 12,000) at 5% w / v
Fase A2 : 21 mL de aguaPhase A 2 : 21 mL of water
Poliviml alcohol (PM 115.000) al 0,5% p/v Agu a (3) : 0, 12 mm Aguj a ( 4 ) : 0 , 17 mm El tiempo de recirculación de la emulsión A /O fue de 1 minuto, con un flujo de 15 mL/mm. El de la emulsión A./θ/A2 de 2 minutos, con un flujo de 20 mL/mm. Las micropartículas obtenidas, una vez purificadas, se recogieron y dispersaron en una solución acuosa de Pluronic® F-68 2% p/v, y se liofílizaron .Polyvinyl alcohol (PM 115,000) at 0.5% w / v Agu a (3): 0.12 mm Needle (4): 0.17 mm The recirculation time of the A / O emulsion was 1 minute, with a flow of 15 mL / mm. The emulsion A./θ/A 2 of 2 minutes, with a flow of 20 mL / mm. The obtained microparticles, once purified, were collected and dispersed in a 2% w / v aqueous solution of Pluronic ® F-68, and lyophilized.
Para determinar la eficacia de encapsulación, los sobrenadantes obtenidos durante las etapas de purificación se titularon. Para ello, los sobrenadantes se mezclaron con DMEM libre de suero y se añadieron a células HeLa (aprox. al 60% de confluencia) . Dos horas después de la adición de los sobrenadantes, se añadió una alícuota de DMEM (medio Eagle modificado de Doubelco) con suero. Las células se incubaron a 37°C y en atmósfera de C02 durante 4 Oh. Las células se fijaron con una solución de glutaraldehído (1 mL por pocilio) y se incubaron en hielo durante 15 mm. Posteriormente, tras lavar las células con PBS , éstas se tiñeron con una solución de X-Gal (5-bromo-4-cloro-3 - indolil-β-D-galactósido) . Finalmente, las células azules se contaron con ayuda de un microscopio invertido. Las cinéticas de liberación se realizaron en "transwell mserts". Las micropartículas se colocaron sobre las membranas de los "• λ transwell " y éstos se colocaron enci-ma de pocilios que contenían DMEM con 1% de suero (1 mL) como medio de liberación, pero que no contenían células A diferentes tiempos, alícuotas del medio de liberación se transfirieron a pocilios que contenían células HeLa (0,5xl06 células por pocilio), y se dejaron 8 h a 37°C. Finalmente las células se fijaron con glutaraldehído, se tiñeron con X-Gal y la expresión de lacZ se determinó por contaje del número de células con color azul .To determine the encapsulation efficacy, the supernatants obtained during the purification steps were titrated. For this, the supernatants were mixed with serum-free DMEM and added to HeLa cells (approx. 60% confluence). Two hours after the addition of the supernatants, an aliquot of DMEM (modified Eagle medium from Doubelco) with serum was added. The cells were incubated at 37 ° C and under C0 2 atmosphere for 4 Oh. The cells were fixed with a solution of glutaraldehyde (1 mL per well) and incubated on ice for 15 mm. Subsequently, after washing the cells with PBS, they were stained with a solution of X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). Finally, the blue cells were counted with the help of an inverted microscope. Release kinetics were performed in "transwell mserts". The microparticles were placed on the membranes of the " λ transwell" and these were placed on top of wells containing DMEM with 1% of serum (1 mL) as a release medium, but not containing cells At different times, aliquots of the release medium were transferred to wells containing HeLa cells (0.5 x 10 6 cells per well), and left 8 h at 37 ° C . Finally the cells were fixed with glutaraldehyde, stained with X-Gal and lacZ expression was determined by counting the number of cells with blue color.
El tamaño medio de las micropartículas obtenidas fue de 10,5 μm y la cantidad de adenovirus recombinantes encap- sulados fue calculada en 3,84xl08 íu La liberación de los virus desde las partículas corresponde a una cinética propia de liberación sostenida, como puede verse en la Figura 7. En esta figura se representa el tiempo frente al número de células azules (infectadas por el adenovirus recom- binante) . En esta figura puede observarse como los adenovirus encapsulados en las micropartículas de la invención permanecen activos hasta 5 días, frente a las 24 h a 20-25°C que duran las partículas virales sin encapsular en un medio acuoso. In vitro se observa, en cuanto a la liberación sostenida de ADN plasmídico que: la utilización de polivinilpirrolidona como viscosizante en la fase A., permite la liberación del ADN encapsulado durante periodos de tiempo mayores a 30 días. En todos los casos estudiados se observó una liberación rápida de aproximadamente el 40% de la dosis encapsulada. A partir de ese momento, y dependiendo de la concentración de agente viscosizante, el ADN se liberó de distinta forma. A concentraciones del 5% p/v, la totalidad del fármaco restante se fue liberando de forma constante hasta el final de los 28 días. A concentraciones del 10% p/v, hasta el día 20 no se observa liberación, para después observar otro pulso de salida del ADN. La utilización de gelatina permite, igualmente, la liberación de una dosis inicial importante de aproximadamente el 50% durante las primeras horas, seguido por un periodo de ausencia de liberación (hasta el día 14) y posteriormente una liberación constante hasta el final del experimento (casi el 80% de la dosis inicial liberada) .The average size of the microparticles obtained was 10.5 μm and the amount of encapsulated recombinant adenoviruses was calculated at 3.84xl0 8 u The release of the virus from the particles corresponds to its own sustained release kinetics, as can be seen in Figure 7. This figure represents the time against the number of blue cells (infected by the recombinant adenovirus). In this figure it can be seen how the adenoviruses encapsulated in the microparticles of the invention remain active for up to 5 days, compared to 24 hours at 20-25 ° C that the viral particles last without encapsulating in an aqueous medium. In vitro, it is observed, as regards the sustained release of plasmid DNA that: the use of polyvinylpyrrolidone as a viscosifier in phase A., allows the release of encapsulated DNA for periods of time greater than 30 days. In all the cases studied, a rapid release of approximately 40% of the encapsulated dose was observed. From that moment, and depending on the concentration of the viscosifying agent, the DNA was released differently. At concentrations of 5% w / v, all of the remaining drug was released constantly until the end of the 28 days. At concentrations of 10% w / v, no release is observed until day 20, and then another pulse of DNA output is observed. The use of gelatin also allows the release of an important initial dose of approximately 50% during the first hours, followed by a period of absence of release (until day 14) and subsequently a constant release until the end of the experiment ( almost 80% of the initial dose released).
La utilización de la goma de xantano permite una liberación inicial en las primeras horas de aproximadamente el 20%. Posteriormente, se observa una liberación lenta y sostenida en el tiempo hasta el final del experimento, alcanzando el 40% de la dosis inicial.The use of xanthan gum allows an initial release in the first hours of approximately 20%. Subsequently, a slow and sustained release in time is observed until the end of the experiment, reaching 40% of the initial dose.
Abreviaturas empleadasAbbreviations used
ADN: ácido desoxirribonucleico ARN: ácido ribonucleicoDNA: deoxyribonucleic acid RNA: ribonucleic acid
DMEM: medio Eagle modificado de Doubelco X-Gal : 5-bromo-4-cloro-3-indolil-β-D-galactósido SEM: microscopía electrónica de barridoDMEM: Doubelco X-Gal modified Eagle medium: 5-bromo-4-chloro-3-indolyl-β-D-galactoside SEM: scanning electron microscopy
TROMS: Total Recirculation One-machine System, Sistema Monobloque de Recirculación CompletaTROMS: Total Recirculation One-machine System, Monobloc Complete Recirculation System
Descripción de las FigurasDescription of the Figures
Figura 1: Esquema del aparato TROMS (Total Recirculation One-Machine System) . (1) Sistema de bombeo (2) Válvula multivía (3) , (4) Agujas (5), (6) Vasijas de mezcla (7) Jeringa (8) , (9) Válvulas (10) Agitador de héliceFigure 1: Diagram of the TROMS device (Total Recirculation One-Machine System). (1) Pumping system (2) Multi-way valve (3), (4) Needles (5), (6) Mixing vessels (7) Syringe (8), (9) Valves (10) Propeller shaker
Figura 2 : Relación entre el tiempo de recirculación de la emulsión y el tamaño de las micropartículas. Abcisas : tiempo en minutos; Ordenadas: tamaño en mieras.Figure 2: Relationship between the time of recirculation of the emulsion and the size of the microparticles. Abcisas: time in minutes; Ordered: size in myras.
Figura 3: Micropartículas fabricadas por TROMS con fluores- ceína sódica en las vesículas interiores (aumento x400) .Figure 3: Microparticles manufactured by TROMS with sodium fluorescein in the inner vesicles (magnification x400).
Figura 4. Fotografía SEM de un lote de MSP-D.Figure 4. SEM photograph of a batch of MSP-D.
Figura 5. Perfiles de liberación del ADN plasmídico desde las micropartículas preparadas por TROMS. Abcisas: tiempo en días; Ordenadas: % ADN liberado; 0 PVP 5%, D PVP 10%, O gelatina 1%, Δ goma xantano 0,1%.Figure 5. Plasmid DNA release profiles from the microparticles prepared by TROMS. Abcisas: time in days; Sorted:% DNA released; 0 PVP 5%, D PVP 10%, O gelatin 1%, Δ xanthan gum 0.1%.
Figura 6A: Electroforesis en gel del sobrenadante obtenidos tras la fabricación de las micropartículas por TROMS (calle 1) y de los lavados de las micropartículas tras su centri- fugación (calles 2-3) . Calle P: plásmido original. Calle sin marcar: control de ADN de distintos tamaños.Figure 6A: Gel electrophoresis of the supernatant obtained after the manufacture of the microparticles by TROMS (lane 1) and of the washes of the microparticles after their centrifugation (lanes 2-3). P Street: original plasmid. Unmarked street: control of DNA of different sizes.
Figura 6B : Electroforesis en gel del sobrenadante obtenido tras la fabricación de las micropartículas utilizando Ultra-turrax como método de agitación (calle 1) . Calle sin marcar: control de ADN de distintos tamaños.Figure 6B: Gel electrophoresis of the supernatant obtained after the manufacture of the microparticles using Ultra-turrax as a method of agitation (lane 1). Unmarked street: control of DNA of different sizes.
Figura 7: Infección de células HeLa por los adenovirus liberados desde las micropartículas en función del tiempo. Abcisas: tiempo en h; Ordenadas: número de células infectadas . Figure 7: Infection of HeLa cells by adenoviruses released from microparticles as a function of time. Abcisas: time in h; Ordered: number of infected cells.

Claims

REIVINDICACIONES
1.- Aparato de producción de micropartículas por un método de evaporación del disolvente tras la formación en continuo de una emulsión múltiple que consta de un sistema de bombeo (1) conectado a una válvula muítivia (2) de la que parten al menos 2 sistemas de inyección turbulenta (3) y (4) caracterizados esencialmente por consistir en al menos 2 agujas de diferentes diámetros internos, agujas que vierten su contenido, particularmente fases acuosas, a sendas vasijas de mezcla (5, 6), dichas vasijas se conectan al sistema de bombeo (1) por sendas válvulas (8) y (9) y al menos a un tercer sistema de inyección turbulenta (7) de una fase orgánica.1.- Microparticle production apparatus by a solvent evaporation method after the continuous formation of a multiple emulsion consisting of a pumping system (1) connected to a muitic valve (2) from which at least 2 systems start of turbulent injection (3) and (4) characterized essentially by consisting of at least 2 needles of different internal diameters, needles that pour their contents, particularly aqueous phases, into two mixing vessels (5, 6), said vessels are connected to the pumping system (1) by two valves (8) and (9) and at least a third turbulent injection system (7) of an organic phase.
2. - Aparato según la reivindicación 1 que opcionalmente comprende además al menos un sistema de agitación (10) en al menos una de las vasijas de mezcla. 2. - Apparatus according to claim 1 which optionally further comprises at least one stirring system (10) in at least one of the mixing vessels.
3.- Aparato según las reivindicaciones 1 y 2 caracterizado porque el sistema de inyección turbulenta (7) de la fase orgánica es una jeringa de vidrio con émbolo de teflón. 3. Apparatus according to claims 1 and 2 characterized in that the turbulent injection system (7) of the organic phase is a glass syringe with a Teflon piston.
4.- Aparato según las reivindicaciones 1 a 3 caracterizado porque los diámetros internos de las agujas (3) y (4) se seleccionan en función del tamaño de micropartícula que se desea obtener.4. Apparatus according to claims 1 to 3, characterized in that the internal diameters of the needles (3) and (4) are selected according to the microparticle size to be obtained.
5.- Aparato según las reivindicaciones 1 a 4 caracterizado porque la aguja (3) tiene un diámetro de 0,12 mm mientras que la aguja (4) tiene un diámetro de 0,17 mm. 5. Apparatus according to claims 1 to 4, characterized in that the needle (3) has a diameter of 0.12 mm while the needle (4) has a diameter of 0.17 mm.
6.- Procedimiento de preparación de micropartículas por evaporación del disolvente tras la formación en continuo de una emulsión múltiple caracterizado por llevarse a cabo en el aparato de las reivindicaciones 1 a 5. 6. Method of preparing microparticles by evaporation of the solvent after the continuous formation of a multiple emulsion characterized by being carried out in the apparatus of claims 1 to 5.
7 - Procedimiento según la reivindicación 6 caracterizado porque al menos una primera fase acuosa Al se bombea e inyecta a través de la aguja (3) a la primera vasija (5) , inyectándose a su vez, en régimen turbulento, en dicha misma vasija al menos una fase orgánica procedente de la jeringa (7), formándose una emulsión A1/0 que se recircula a través de la válvula (8) por el sistema hasta su homogemzación para, a continuación, inyectar, por medio de la válvula multivía (2), la emulsión Ax/0 a la segunda vasija (6) , a través de la aguja (4) de mayor diámetro interno que la aguja (3) , también en régimen turbulento, vasija (6) donde se ha bombeado previamente al menos una segunda fase acuosa A2, formándose en su interior una emulsión
Figure imgf000029_0001
recirculando posteriormente a través de la válvula (9) dicha emulsión A./0/A2 por el sistema hasta conseguir un tamaño homogéneo de gota y evaporándose posteriormente el disolvente de la fase orgánica O. 7 - Method according to claim 6 characterized in that at least a first aqueous phase Al is pumped and injected through the needle (3) to the first vessel (5), in turn injected, in turbulent regime, into said same vessel at least one organic phase from the syringe (7), forming an emulsion 1/0 which is recirculated through the valve (8) by the system until homogemzación to then injected, via the multiway valve ( 2), the emulsion A x / 0 to the second vessel (6), through the needle (4) of greater internal diameter than the needle (3), also in turbulent regime, vessel (6) where it has been previously pumped at least a second aqueous phase A 2 , forming inside an emulsion
Figure imgf000029_0001
subsequently recirculating through the valve (9) said emulsion A./0/A 2 through the system until a homogeneous droplet size is achieved and the solvent of the organic phase O. is subsequently evaporated.
8.- Procedimiento según las reivindicaciones 6 y 7 caracterizado porque la evaporación del disolvente se realiza mediante un agitador de hélice (10) dispuesto en el interior de la vasija (6) .8. Method according to claims 6 and 7, characterized in that the evaporation of the solvent is carried out by means of a propeller stirrer (10) arranged inside the vessel (6).
9.- Procedimiento según las reivindicaciones 6 y 7 caracterizado porque la evaporación del disolvente se hace mediante un agitador ajeno al aparato de las reivm- aleaciones 1 a 5.9. Method according to claims 6 and 7, characterized in that the evaporation of the solvent is carried out by means of an agitator outside the apparatus of claims 1 to 5.
10.- Procedimiento según las reivindicaciones 6 a 9 caracterizado porque las micropartículas formadas se extraen mediante medios convencionales, entre otros, evaporación, centrifugación o filtración. 10. Method according to claims 6 to 9, characterized in that the microparticles formed are removed by conventional means, among others, evaporation, centrifugation or filtration.
11.- Procedimiento según las reivindicaciones 6 a 10 caracterizado porque las micropartículas formadas opcionalmente se liofilizan una vez extraídas. 11. Method according to claims 6 to 10, characterized in that the optionally formed microparticles are lyophilized once removed.
12.- Procedimiento según las reivindicaciones 6 a 11 caracterizado porque las micropartículas obtenidas son formas farmacéuticas de tipo microcápsula homogénea o microesfera heterogénea. 12. Method according to claims 6 to 11, characterized in that the microparticles obtained are pharmaceutical forms of the homogeneous microcapsule or heterogeneous microsphere type.
13. - Procedimiento según las reivindicaciones 6 a 12 caracterizado porque las fases que se inyectan se componen de:13. - Method according to claims 6 to 12 characterized in that the phases that are injected are composed of:
Fase Al: fase acuosa, en la que se incorpora el fármaco, molécula biológicamente activa o material a encapsular y, opcionalmente, un tensioactivo y/o un viscosizante . Fase 0: fase inmiscible con las fases A1 y A2 constituida, preferentemente, por un disolvente orgánico volátil en la que se incorpora el polímero formador de las micropartículas, preferentemente un polímero biodegradable tipo poliéster y más preferentemente poliépsilon caprolactona, ácidos poli- lácticos (PLA) o copolímeros entre el ácido láctico y el ácido glicólico (PLAGA) . Fase A2 : fase acuosa que puede contener opcionalmente un tensioactivo o viscosizante. Al phase: aqueous phase, in which the drug, biologically active molecule or material to be encapsulated and, optionally, a surfactant and / or a viscosifier are incorporated. Phase 0: immiscible phase with phases A 1 and A 2 , preferably constituted by a volatile organic solvent in which the microparticle forming polymer is incorporated, preferably a biodegradable polyester type polymer and more preferably polypropylene caprolactone, polylactic acids (PLA) or copolymers between lactic acid and glycolic acid (PLAGA). Phase A 2 : aqueous phase that may optionally contain a surfactant or viscosizer.
14. - Procedimiento según las reivindicaciones 6 a 13 caracterizado porque la concentración de polímero en la fase O está comprendida entre el 1 y el 20% p/v. 14. - Method according to claims 6 to 13, characterized in that the concentration of polymer in phase O is between 1 and 20% w / v.
15.- Procedimiento según las reivindicaciones 6 a 14 caracterizado porque la concentración de tensioactivo y/o viscosizante en la fase Aτ está comprendida entre el 0 y el 20% p/v.15. Process according to claims 6 to 14, characterized in that the concentration of surfactant and / or viscosizer in phase A τ is between 0 and 20% w / v.
16.- Procedimiento según las reivindicaciones 6 a 15 caracterizado porque la fase A2 comprende un tensioactivo y/o viscosizante en una concentración entre el 0 y el 20% p/v. 16. Method according to claims 6 to 15, characterized in that phase A 2 comprises a surfactant and / or viscosizer in a concentration between 0 and 20% w / v.
17.- Procedimiento según las reivindicaciones 6 a 16 caracterizado porque las micropartículas resultantes se liofilizan utilizando, como adyuvante, un copolímero de óxido de etileno (80%) y óxido de propileno (20%) , a una concentración comprendida entre el 0 y el 10% p/v y, opcionalmente se añade un cπoprotector tal como sacarosa o manitol, a una concentración comprendida entre el 0 y el 5% p/v. 17. Method according to claims 6 to 16, characterized in that the resulting microparticles are lyophilized using, as an adjuvant, a copolymer of ethylene oxide (80%) and propylene oxide (20%), at a concentration between 0 and 10% w / v and, optionally, a protective cap such as sucrose or mannitol is added, at a concentration between 0 and 5% w / v.
18.- Procedimiento según las reivindicaciones 6 a 17 caracterizado porque el volumen de la fase O es de 2 a 20 veces mayor que el de la fase A± .18. Method according to claims 6 to 17, characterized in that the volume of phase O is 2 to 20 times greater than that of phase A ± .
19.- Procedimiento según las reivindicaciones 6 a 18 caracterizado porque el volumen de la fase A2 es de 2 a 20 veces mayor que el de la fase O. 19. Method according to claims 6 to 18, characterized in that the volume of phase A 2 is 2 to 20 times greater than that of phase O.
20.- Procedimiento según cualquiera de las reivindicaciones 6 a 19 caracterizado porque la fase A es una solución viscosizante de polívinilpirrolidona, gelatina o goma xantano, en agua y que contiene opcionalmente la molécula biológicamente activa o material a encapsular, la fase 0 es una solución de un copolímero de ácido poli (láctico/glicólico) en diclorometano y la fase A2 es una solución tensioactiva de polivinilalcohol en agua. 20. Method according to any of claims 6 to 19 characterized in that phase A is a viscous solution of polyvinylpyrrolidone, gelatin or xanthan gum, in water and optionally containing the biologically active molecule or material to be encapsulated, phase 0 is a solution of a copolymer of poly (lactic / glycolic acid) in dichloromethane and phase A 2 is a surfactant solution of polyvinyl alcohol in water.
21.- Procedimiento según cualquiera de las reivindicaciones 6 a 20 caracterizado porque el tamaño medio de las micropartículas obtenidas está comprendido entre 1 y 200 μm.21. Method according to any of claims 6 to 20, characterized in that the average size of the microparticles obtained is between 1 and 200 μm.
22.- Procedimiento según las reivindicaciones 6 a 21 caracterizado porque los fármacos o moléculas biológicamente activas encapsulados en las micropartículas son de naturaleza hidrofílica.22. Method according to claims 6 to 21, characterized in that the biologically active drugs or molecules encapsulated in the microparticles are hydrophilic in nature.
23. - Procedimiento según las reivindicaciones 6 a 22 caracterizado porque la molécula biológicamente activa o fármaco de naturaleza hidrofílica encapsulado en el seno de las micropartículas es de elevada masa molecular, preferentemente con un peso molecular superior a 1000. 23. - Method according to claims 6 to 22 characterized in that the biologically active molecule or hydrophilic drug encapsulated in the breast of the microparticles is of high molecular mass, preferably with a molecular weight greater than 1000.
24. - Procedimiento según las reivindicaciones 6 a 23 caracterizado porque las micropartículas obtenidas pueden ser utilizadas para la encapsulación de cualquier tipo de materiales dispersos en soluciones acuosas, con independencia de que sean de origen biológico o de síntesis química. 24. - Method according to claims 6 to 23, characterized in that the microparticles obtained can be used for encapsulation of any type of materials dispersed in aqueous solutions, regardless of whether they are of biological origin or of chemical synthesis.
25.- Procedimiento según las reivindicaciones 6 a 24 caracterizado porque la molécula biológicamente activa o fármaco encapsulado en las micropartículas es ADN, particularmente plasmídico, ARN, oligonucleótidos , péptidos o vectores virales. 25. Method according to claims 6 to 24, characterized in that the biologically active molecule or drug encapsulated in the microparticles is DNA, particularly plasmid, RNA, oligonucleotides, peptides or viral vectors.
26.- Procedimiento según las reivindicaciones 6 a 25 caracterizado porque el material encapsulado en las micro- partículas son vectores de tipo viral destinados a tratamientos de terapia génica, tales como los adenovirus recom- b nantes defectivos . 26.- Method according to claims 6 to 25, characterized in that the material encapsulated in the micro-particles are viral type vectors intended for gene therapy treatments, such as defective recombinant adenoviruses.
27.- Procedimiento según las reivindicaciones 6 a 26 caracterizado porque las micropartículas obtenidas aumentan la vida media y la estabilidad a temperatura ambiente de los vectores de tipo viral encapsulados, facilitando su transporte, conservación y almacenamiento. 27.- Method according to claims 6 to 26, characterized in that the microparticles obtained increase the half-life and stability at room temperature of the encapsulated viral type vectors, facilitating their transport, conservation and storage.
28.- Procedimiento según las reivindicaciones 6 a 27 caracterizado porque las micropartículas obtenidas permiten una liberación sostenida del fármaco, molécula biológicamente activa o material biológico o sintético, encapsulado al producirse una primera fase de liberación inmediata seguida, inmediatamente o tras un periodo sin liberación, por una segunda fase de liberación más lenta.28.- Method according to claims 6 to 27, characterized in that the microparticles obtained allow a sustained release of the drug, biologically active molecule or biological or synthetic material, encapsulated when a first phase of immediate release occurs followed, immediately or after a period without release, for a second phase of slower release.
29.- Procedimiento según la reivindicación 28 caracterizado porque la liberación sostenida en el caso de ADN plasmídico se prolonga como mínimo 28 días. 29.- Method according to claim 28, characterized in that the sustained release in the case of plasmid DNA is prolonged for at least 28 days.
30.- Procedimiento según la reivindicación 29 caracterizado porque en la fase de liberación rápida hasta el 40% del ADN plasmídico se libera durante las primeras 24 horas, seguido, a continuación o tras un periodo de no liberación de 13-19 días, de una liberación constante del resto del ADN plasmídico hasta alcanzar el mínimo de 28 días. 30.- Method according to claim 29 characterized in that in the rapid release phase up to 40% of the plasmid DNA is released during the first 24 hours, followed, after or after a non-release period of 13-19 days, of a constant release of the rest of the plasmid DNA until reaching the minimum of 28 days.
31.- Micropartículas obtenidas según el procedimiento de las reivindicaciones 6 a 30 caracterizadas por presentar un tamaño medio comprendido entre 1 y 200 μm y llevar encap- sulado una molécula biológicamente activa, fármaco o material de origen biológico o sintético, de naturaleza hidro- fílica y de masa molecular elevada, preferentemente con un peso molecular superior a 1000. 31.- Microparticles obtained according to the method of claims 6 to 30 characterized by having an average size between 1 and 200 μm and carrying a biologically active molecule, drug or material of biological or synthetic origin, of hydrophilic nature. and of high molecular mass, preferably with a molecular weight greater than 1000.
32.- Micropartículas según la reivindicación 31 caracte- rizadas porque el material encapsulado es, entre otros, ADN, particularmente plasmídico, ARN, oligonucleótidos, péptidos o vectores virales.32.- Microparticles according to claim 31 characterized in that the encapsulated material is, among others, DNA, particularly plasmid, RNA, oligonucleotides, peptides or viral vectors.
33.- Micropartículas según la reivindicación 32 caracterizadas porque el vector viral encapsulado en un adenovirus recombinante defectivo.33.- Microparticles according to claim 32 characterized in that the viral vector encapsulated in a defective recombinant adenovirus.
34.- Uso de las micropartículas de las reivindicaciones 31 a 33 en la dosificación del material encapsulado mediante liberación sostenida con una primera fase de liberación inmediata seguida de una segunda fase de liberación más lenta.34. Use of the microparticles of claims 31 to 33 in the dosing of the encapsulated material by sustained release with a first phase of immediate release followed by a second phase of slower release.
35.- Uso de las micropartículas de las reivindicaciones 31 a 34 en la preparación de composiciones de terapia génica. 35. Use of the microparticles of claims 31 to 34 in the preparation of gene therapy compositions.
PCT/ES2001/000100 2000-03-15 2001-03-15 Method and device for the production of microparticles for controlled release of water-soluble pharmaceuticals and viral vectors. application to the administration of plasmid dna and defective recombinant adenovirus WO2001068059A1 (en)

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