EP2029119A2 - Nanoparticules polymères solides fonctionnalisées pour applications diagnostiques et thérapeutiques - Google Patents

Nanoparticules polymères solides fonctionnalisées pour applications diagnostiques et thérapeutiques

Info

Publication number
EP2029119A2
EP2029119A2 EP07726022A EP07726022A EP2029119A2 EP 2029119 A2 EP2029119 A2 EP 2029119A2 EP 07726022 A EP07726022 A EP 07726022A EP 07726022 A EP07726022 A EP 07726022A EP 2029119 A2 EP2029119 A2 EP 2029119A2
Authority
EP
European Patent Office
Prior art keywords
polymer
polymer nanoparticles
nanoparticles according
particles
nanoparticles
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07726022A
Other languages
German (de)
English (en)
Inventor
Katrin Claudia Fischer
Sascha General
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer Pharma AG
Original Assignee
Bayer Schering Pharma AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer Schering Pharma AG filed Critical Bayer Schering Pharma AG
Publication of EP2029119A2 publication Critical patent/EP2029119A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention describes cationic surface potential polymer nanoparticles in which both hydrophobic and hydrophilic pharmaceutically active substances can be included.
  • the hydrophilic and thus water-soluble substances are entrapped by ionic complexation with a charged polymer in the core of the particle by co-precipitation.
  • both therapeutics and diagnostics can be used as pharmaceutically active substances.
  • the cationic particle surface provides stable, electrostatic surface modification with partially oppositely charged compounds that may contain target-specific ligands to enhance passive and active targeting.
  • nanoparticulate drug delivery systems are mainly due to their small size, thereby overcoming different physiological barriers is possible [Fahmy T.M., Fong P.M. et al., Mater. Today, 2005; 8 (8): 18-26].
  • the associated altered distribution in the organism can, for. B. for the diagnosis as well as for the therapy of various tumor diseases can be used advantageously.
  • the associated therapeutic monitoring will enable faster detection of treatment resistance in the future and by timely use of alternative Therapies significantly improve the patient's cure [Emerich DF, Thanos CG, Curr. Nanosci., 2005; 1: 177-188].
  • a substance class used very successfully in tumor therapy is the group of cytostatic drugs. All rapidly dividing cells of the body, including tumor cells, are damaged by these substances. However, this not only leads to a death of the tumor cells, but often other vital organs and tissues such as the bone marrow, mucous membranes or heart vessels are affected.
  • EPR effect for short
  • This EPR effect was already described by Matsumura and Maeda in 1986 as a strategy for targeted drug accumulation in solid tumors [Matsumura Y, Maeda H., Cancer Res., 1986; 46: 6387-6392] [Maeda H., Adv. Enzyme Regul., 2001; 41: 189-207].
  • This is a passive enrichment mechanism that exploits the structural peculiarities of tumorous or inflamed tissue [Ulbrich K., Subr V., Adv. Drug Deliv. Rev., 2004; 56 (7): 1023-1050].
  • tumor tissue is characterized by its fast growth and various messenger substances usually by a fenestr Understand "holey" tissue structure as well as a lack of lymphatic drainage
  • holey tissue structure As well as a lack of lymphatic drainage
  • this area is also referred to as nanosize window [Hobbs SK, Monsky WL et al., Proc. Natl. Acad. Sci., USA, 1998; 95: 4607-4612] [Brigger I., Dubernet C. et al., Adv. Drug Deliv. Rev., 2002; 54 (5): 631-651].
  • the nanoparticles must circulate in the blood stream for a sufficient period of time.
  • This requires particle sizes between approx. 10 nm and 380 nm as well as suitable particle surfaces.
  • pegylated particle surfaces can prevent endogenous proteins from identifying the particles as foreign and rapid elimination via the organs of the reticulo-endothelial system (short RES) [Otsuka H. et al., Adv. Drug DeNv. Rev., 2003; 55 (3): 403-419].
  • active ligands on the particle surface e.g., antibodies
  • tissue-specific enrichment can be further optimized [Nobs L. et al. Pharm. Sci., 2004; 93: 1980-1992] [Yokoyama M., J. Artif. Organs, 2005; 8: 77-84].
  • the cell membrane For a recording of the active ingredients in the cell, another physiological barrier, the cell membrane, must be overcome.
  • the active substance is introduced into the cell via endocytosis with the aid of nanoparticles, it is possible to circumvent ejecting transporters and to prevent multi-drug resistance (MDR) [Bharadwaj V., J. Biomed. Nanotechnol., 2005; 1: 235-258] [Huwyler J. et al., J. Drug Target., 2002; 10 (1): 73-79].
  • MDR multi-drug resistance
  • the release properties of the active substance from the nanoparticle can additionally be controlled by targeted selection of the polymer.
  • a nanoparticulate formulation can thus minimize the frequency of application and lead to a reduction of the therapeutically necessary dose. Furthermore, unwanted plasma peak levels can be avoided by encapsulation in nanoparticles and a delayed release can be achieved.
  • a nanoparticulate system which already fulfills all the described advantages, has not yet been developed according to the current state of knowledge.
  • the variety of nanoparticulate carrier systems described in the literature also makes it clear that there is currently no optimal nano-formulation for all problems.
  • the overall structure of the particles, matrix-forming substances, and especially their surface are of crucial importance for in vivo behavior [Choi SW, Kim WS, Kim JH, Journal of Dispersion Science and Technology, 2003; 24 (3 & 4): 475-487].
  • the physicochemical properties of various drugs differ greatly. Accordingly, there remains a need in the development of colloidal drug carrier systems with improved properties.
  • optical imaging techniques such as sonography, X-ray diagnostics, cross-sectional imaging (CT, MRI) and nuclear medicine (PET, SPECT) are available for in vivo detection.
  • CT cross-sectional imaging
  • PET nuclear medicine
  • SPECT nuclear medicine
  • Another and relatively new method is optical imaging, whose detection principle is based on the use of near-infrared fluorescence. It is a non-invasive procedure that works without ionizing radiation and is very inexpensive and inexpensive compared to methods such as MRI.
  • N I R dyes such as indocyanine green developed for such an application are very soluble in water, and therefore it is difficult to efficiently encapsulate them in a hydrophobic polymer matrix.
  • the reason for this is the rapid change of the hydrophilic substance into the aqueous phase, for example during production by means of nanoprecipitation.
  • hydrophilic substances in nanoparticles only a few technologies are available that have different disadvantages.
  • the amphiphilic nature of liposomes or polymerosomes allows the inclusion of hydrophilic substances in the aqueous interior of the particles, whereas hydrophobic compounds can be incorporated in the membrane. Due to the localization in the core or in the shell of the particles, the loading is very limited and thus usually insufficient.
  • a further disadvantage is that, above all, hydrophilic substances in an aqueous environment are rapidly washed out of such systems.
  • both water-soluble dyes for diagnosis and therapeutic substances which are usually poorly water-soluble due to their hydrophobic properties, should be able to be encapsulated effectively and with sufficient stability to wash out.
  • a technological challenge remains, on the one hand, through the use of suitable surfaces on the one hand a sufficient Particle stability and on the other hand to ensure a specific accumulation in the target tissue.
  • the whereabouts at the site of the enrichment depends, among other things, on how well the particles are absorbed into the tissue and the cell.
  • nanoparticulate systems with cationic surface properties should be prepared without the potential toxicologically harmful properties hindering an in vivo application.
  • the particle surface must be inconspicuous to the body's own defense mechanisms (Opsonine, RES), allowing only a sufficiently long circulation time, which is a prerequisite for a corresponding accumulation of particles from the blood stream in the target tissue.
  • the nanoparticulate systems should continue to support uptake into the target cell and endolysosomal release.
  • Another difficulty in the production of nanoparticulate systems is to apply suitable substances or target structures to the particle surface.
  • the surface of the particles is modified by means of covalent coupling reactions.
  • Prerequisite for this are functional groups on the polymer backbone or on the particle surface, which can be irreversibly linked by appropriate chemical coupling reactions with the target molecule [Nobs L. et al., J. Pharm. Sei., 2004; 93: 1980-1992]. Since the stability of colloidal dispersions is often greatly reduced by the reagents or under the reaction conditions, the chemical implementation is usually complicated and problematic [Koo OM et al., Nanomedicine, 2005; 1 (3): 193-212] [Choi SW et al., J. Dispersion Sei. Technol., 2003; 24 (3 & 4): 475-487].
  • the covalent attachment of molecules and particle surfaces must be particularly suitable for any new molecule to be applied to the surface in order to avoid possible unwanted chemical reactions. Avoiding organic solvents, often used for covalent linkages, is also desirable because of the reduced burden on the environment and the simplification of the implementation of the reaction. Ideally, therefore, a non-covalent, easy-to-perform, and thus flexible, yet stable surface modification should be possible.
  • the known from the literature colloidal systems are usually only suitable for encapsulation of hydrophobic substances or hydrophilic substances. In the case of the frequently used covalent surface modification of the particles, there is little flexibility with regard to the use of a wide variety of surface structures on one and the same core particle.
  • the ligands for specific enrichment often adversely affect the uptake in the actual tumor tissue and in particular on the cell uptake. Insofar as the particles ensure adequate circulation and passively or actively accumulate well in the target tissue, internalization and endolysosomal release are usually not optimal [van Osdol W., Cancer Res., 1991; 51: 4776-4784] [Weinstein JN et al., Cancer Res., 1992; 52 (9): 2747-2751].
  • An object of the invention was therefore to provide an improved pharmaceutical formulation in which on the one hand hydrophilic as well as hydrophobic drugs can be encapsulated.
  • a flexible and sufficiently stable surface modification should allow optimal accumulation in the diseased tissue.
  • such a colloidal system must also be efficiently incorporated into the target tissue as well as into the individual cells where endolysosomal release can occur.
  • they should be practicable manufacturing methods to enable production in a timely and cost-effective manner.
  • the invention relates to polymer nanoparticles having a cationic surface potential containing a cationic polymer and a polymer which is sparingly soluble in water, characterized in that said polymer nanoparticles contain diagnostic and / or therapeutic agents.
  • substances can be encapsulated in the polymer particles which are suitable for the diagnosis and / or the therapy of various diseases.
  • the cationically functionalized particle surface can be electrostatically stably and flexibly surface-modified with a partially oppositely charged compound.
  • the described invention is therefore suitable for detecting diseases (diagnosis), for the treatment of diseases (therapy) as well as for monitoring a therapy.
  • the invention includes a suitable drug form using pharmaceutically acceptable excipients, which are necessary for the respective dosage form.
  • the drug form developed in the context of the invention can be applied to humans or animals via various routes of administration. Necessary application systems are also part of the invention described herein.
  • the composition of the nanoparticles includes a poorly water-soluble polymer, which is preferably a biodegradable polymer or else a mixture of various biodegradable polymers.
  • a biodegradable polymer can be described via individual monomer units which form said polymer by means of polymerization or other processes.
  • the polymer can be defined by its name.
  • the poorly water-soluble polymer is derived from the group of natural and / or synthetic polymers or from homo- and co-polymers of corresponding monomers.
  • the polymer is derived from the group of alkyl cyanoacrylates such as, for example, butyl cyanoacrylates and isobutyl cyanoacrylates, acrylates such as methacrylates, lactides, for example L-lactides or DL-lactides, glycolides, caprolactones such as ⁇ -caprolactones and others ,
  • said polymer or part of the polymer is selected from the group of polycyanoacrylates and polyalkylcyanoacrylates (PACA) such as polybutylcyanoacrylate (PBCA), polyesters such as poly (DL-lactides), poly (L-lactides), polyglycolides, polydioxanones, polyoxazolines , Poly (glycolide-co-trimethylene-carbonate), polylactide-co-glycolide (PLGA) such as poly (L-lactide-co-glycolide) or poly (DL-lactide-co-glycolide), poly (L-lactide) co-DL-lactides), poly (glycolide-co-trimethylene), poly (carbonates-co-dioxanones), alginic acid, hyaluronic acid, polysialic acid, acidic cellulose derivatives, acidic starch derivatives, polysaccharides such as dextrans, alginates, cycl
  • the poorly water-soluble polymer is selected from the group of polyalkyl cyanoacrylates (PACA).
  • PPA polyalkyl cyanoacrylates
  • polyalkylcyanoacrylates shows the structure indicated (formula 1), wherein the radical R given is preferably linear and branched alkyl groups having 1 to 16 carbon atoms, a cyclohexyl, benzyl or a phenyl group.
  • the poorly water-soluble polymer is a polybutyl cyanoacrylate (PBCA) (Formula 2).
  • PBCA polybutyl cyanoacrylate
  • the poorly water-soluble polymer forms the larger proportion of the polymer matrix of the particles.
  • the cationic polymer is derived from the group of natural and / or synthetic polymers or from homo- and co-polymers.
  • Suitable cationic polymers in the context of this invention are polymers having free primary, secondary or tertiary amino groups which can form salts with any low molecular weight acid, the salts being soluble in aqueous-organic solvents. Also suitable are polymers or their
  • Solvents are soluble.
  • the following groups of cationic polymers, polycations and polyamine compounds or polymers of homopolymers and copolymers of corresponding monomers are particularly suitable: modified natural cationic polymers, cationic proteins, synthetic cationic polymers, aminoalkanes of different chain lengths, modified cationic dextrans, cationic polysaccharides , cationic starch or cellulose derivatives, chitosans, guar derivatives, cationic cyanoacrylates, methacrylates and methacrylamides and those monomers and comonomers which can be used to form suitable compounds and the corresponding salts which can form with suitable inorganic or low molecular weight organic acids.
  • These include in particular: diethylaminoethyl-modified dextrans, hydroxymethylcellulosetrimethylamine, polylysine, protamine sulfate,
  • the amino group-bearing compound in particular a cationic polymer
  • an organic solvent which is infinitely miscible with water, preferably acetone, methanol, ethanol, propanol, dimethylsulfoxide (DMSO), or in a mixture of these solvents with water ,
  • the polymethylene particles contain, as the amino-containing compound, a cationically modified polyacrylate (poly-N, N-dimethylaminoethyl methacrylate, P (DMAEMA)) (formula 3).
  • a cationically modified polyacrylate poly-N, N-dimethylaminoethyl methacrylate, P (DMAEMA)
  • the biodegradable, cationically modified polyacrylate P (DMAEMA) is encapsulated in the polymer matrix, in particular the PBCA polymer matrix, by nanoprecipitation.
  • the surface of the resulting nanoparticles has a positive (cationic) surface potential (zeta potential) due to the amino groups of the cationic polymer.
  • the cationic particle surface ensures good cell uptake and enables flexible electrostatic surface modification with partially anionically charged compounds.
  • the polymer nanoparticles contain as cationic polymer a modified polyacrylate
  • the polymer nanoparticles contain as cationic polymer polyethyleneimine (PEI) of various molecular weights, in particular 1, 8 kDa, 10 kDa, 70 kDa and 750 kDa, (formula 4).
  • PEI polyethyleneimine
  • PEI is a polycation commonly used in the field of non-viral gene therapy for DNA polyplexes (PEK) and accordingly much studied [Remy J.-S. et al., Adv. Drug DeNv. Rev., 1998; 30 (1-3): 85-95].
  • the particle shell Due to the encapsulation of the cationic polyelectrolyte PEI in the PBCA polymer matrix, the particle shell consists of PEI polymer chains, which produce a cationic surface potential.
  • diagnostic as well as therapeutic substances can be included in the polymer matrix by means of nanoprecipitation.
  • the following substance classes for different molecular imaging methods can be used as diagnostic substances for encapsulation, in particular contrast agents or tracers for the following method for molecular imaging (Molecular Imaging) are to be mentioned: optical imaging, such as DOT (diffuse optical ultrasound imaging, OPT (optical projection tomography), near-infrared fluorescence imaging, fluorescence protein imaging and bioluminescence imaging (BLI) and magnetic resonance imaging (MRI, MRI) and X-ray imaging (X-ray imaging) ). But there are also other methods conceivable.
  • optical imaging such as DOT (diffuse optical ultrasound imaging, OPT (optical projection tomography), near-infrared fluorescence imaging, fluorescence protein imaging and bioluminescence imaging (BLI) and magnetic resonance imaging (MRI, MRI) and X-ray imaging (X-ray imaging)
  • DOT diffuse optical ultrasound imaging
  • OPT optical projection tomography
  • near-infrared fluorescence imaging fluorescence protein imaging and bioluminescence imaging (BLI)
  • the diagnostic agent is a dye, in particular selected from the following group: fluorescein,
  • Difluorofluorescein such as Oregon GreenTM 488, Oregon GreenTM 500 or
  • Polymethine dyes coumarin dyes, e.g. Coumarin 6,7-amino-4-methyl coumarin, metal complexes of DTPA or tetraazamacrocycles (Cyclen,
  • the diagnostic substance is a fluorescence-active dye.
  • the diagnostic agent is a fluorescent near-infrared (NIR) dye.
  • NIR dyes preferably used for optical imaging, absorb and emit light in the NIR range between 650 nm and 1000 nm.
  • the preferred dyes belong to the class of polymethine dyes and are selected from the following groups: carbocyanines such as diethyloxacarbocyanine (DOC), diethyloxadicarbocyanine (DODC ) Diethyloxatricarbocyanine (DOTC), indodi or indotricarbocyanines, tricarbocyanines, merocyanines, oxonol dyes (WO 96/17628), rhodamine dyes, phenoxazine or phenothiazine dyes,
  • carbocyanines such as diethyloxacarbocyanine (DOC), diethyloxadicarbocyanine (DODC ) Diethyloxatricarbocyanine (DOTC), in
  • Tetrapyrrole dyes especially benzoporphyrins, chorine and phthalocyanines.
  • Suitable inorganic cations or counterions for these dyes are, for example, the lithium ion, the potassium ion, the hydrogen ion and in particular the sodium ion.
  • Suitable cations of organic bases are, among others, those of primary, secondary or tertiary amines, such as ethanolamine, diethanolamine, morpholine, glucamine, N 1 N- dimethylglucamine and especially N-methylglucamine, and polyethyleneimine.
  • Suitable cations of amino acids are, for example, those of lysine, arginine and ornithine and the amides of otherwise acidic or neutral amino acids.
  • the preferred dyes may be used as bases or as salts thereof.
  • the diagnostic substance is a carbocyanine dye.
  • the general structure of the carbocyanines is described as follows (Formula 8).
  • R 20 to R 29 , R 32 and R 33 independently of one another represent a hydrogen atom, a hydroxy group, a carboxy, a sulfonic acid radical or a carboxyalkyl, alkoxycarbonyl or alkoxyoxoalkyl radical having up to 10 C atoms or a sulfoalkyl radical up to 4 carbon atoms, or for a non-specific binding macromolecule, or for a radical of general formula VI
  • R 20 to R 29 corresponds to a non-specific binding macromolecule or the general formula VI, where o and s are 0 or independently of one another for an integer from 1 to 6, q and v are independently 0 or 1,
  • R 34 represents a hydrogen atom or a methyl radical
  • R 35 is an alkyl radical having 3 to 6 C atoms, which has 2 to n-1 hydroxyl groups, where n is the number of C atoms, or a substituted with 1 to 3 carboxy groups alkyl radical having 1 to 6 carbon atoms, aryl radical 6 to 9 carbon atoms or arylalkyl radical having 7 to 15 carbon atoms, or a radical of the general formula IMd or MIe
  • anionic, readily water-soluble substances such as certain carbocyanines can be stably entrapped in the hydrophobic polymer matrix of the described nanoparticles.
  • an anionic water-soluble substance is encapsulated by ion complexation and co-precipitation with a cationic polymer in a sparingly water-soluble polymer matrix by nanoprecipitation, resulting in particles of defined size.
  • the carbocyanine dye is the readily water-soluble anionic tetrasulfocyanine (TSC) (Formula 9).
  • the carbocyanine dye is IDCC (indodicarbocyanine) (Formula 10).
  • the carbocyanine dye is ICG (indocyanine green) (Formula 11).
  • the encapsulated pharmaceutically active substance is a therapeutic agent.
  • the therapeutic agent is a substance for the treatment of tumor diseases, in particular vascularized tumors and metastases, or diseases with inflammatory reactions.
  • the latter can be, for example, diseases of the rheumatic type, such as, for example, Rheumatoid arthritis, psoriatic arthritis, collagenosis, vasculitis, and infectious diseases.
  • Other diseases with inflammatory processes and possible tissue changes include chronic inflammatory bowel disease (Crohn's disease, ulcerative colitis), multiple sclerosis, atopic dermatitis as well as certain erythema disorders.
  • the following groups can be used as therapeutic substances: immunogenic peptides or proteins, chemotherapeutic agents, toxins, radiotherapeutic agents, radiosensitizers, angiogenesis inhibitors and antiinflammatory substances such as NSAIDs or a combination of these.
  • the therapeutic agents for the treatment of tumor diseases can be selected from the group of alkylating agents, in particular bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, ifosfamide, lomustine, Melphalan, nimustine, thiotepa, treosulfan and trofosfamide, the group of antimetabolites, in particular cytarabine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate and tioguanine, the group of alkaloids and diterpenes, in particular vinblastine, vincristine, vindesine, vinorelbine, etoposide, and docetaxel , Paclitaxel, the group of taxanes, the group of antibiotics, especially aclubicin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, mito
  • the therapeutic agent is a substance which is suitable for the treatment of inflammation.
  • They are in particular selected from the group of nonsteroidal anti-inflammatory drugs (NSAIDs), in particular salicylates (inter alia acetylsalicylic acid), arylacetic acid derivatives (inter alia acemetacin, diclofenac), propionic acid derivatives (inter alia ibuprofen, ketoprofen, naproxen, tiaprofen), indole derivatives (inter alia Indomethacin, acemetacin, lonazolac, proglumetacin), oxicams (including piroxicam, tenoxicam), alkalons (including nabumetone), pyrazolones (including azapropazone, pyrazinobutazone, phenylbutazone, oxyphenbutazone), anthranilic acid derivatives (including mefenamic acid, niflumic acid), COX 2 inhibitors (including melaminophen
  • the polymer nanoparticles are precipitation aggregates which are produced by nanoprecipitation.
  • the following production methods are available for this purpose:
  • Direct precipitation in a test tube by introducing the dissolved polymer-substance mixture into a surfactant-containing aqueous solution which is mixed by means of a magnetic stirrer.
  • Substance mixture in the surfactant-containing aqueous solution In the production of nanoparticles by nanoprecipitation, the organic solvent is abruptly withdrawn from the matrix polymer and the substances dissolved with it, when the polymer-containing organic solution is added to a significantly larger volume of an aqueous solution.
  • compounds dissolved in the polymer phase with amino groups water-soluble as well as water-insoluble
  • the condition for this is the unlimited miscibility of the organic solvent (eg acetone, ethanol) with water as well as the insolubility of the matrix polymer in the aqueous phase.
  • the surface of the polymer nanoparticles is electrostatically modified.
  • the electrostatic modification of the cationic nanoparticle surface is an outstanding advantage of the present invention.
  • the particle surface can be modified with a suitable substance without a chemical coupling reaction.
  • the prerequisite for this is that the modifying agent has partial charges which are opposite to the particle surface charge.
  • this method electrostatic surface modification by charge titration
  • the enrichment (active and passive targeting) of nanoparticles from the bloodstream into the target tissue requires that the particles circulate in the blood stream for a sufficient period of time.
  • the circulation time in the body can be adapted individually, in particular by using polyethylene oxides or polyethylene glycols (see Example 5).
  • Another outstanding advantage is that the electrostatic surface modification described here can be carried out quickly and without problems directly before use. This is done by simply mixing appropriate amounts of the nanoparticle dispersion with the modifying agent.
  • the separation of core particles and modifying agent further allows a surface modification according to the individual requirements of the patient.
  • the surface modification on a modular principle offers maximum flexibility for diagnosis, therapy and monitoring, whereby the uncomplicated implementation of the modification is carried out directly by the user.
  • the partially anionically charged moiety fulfills the function of an anchor on the positively charged particle surface through electrostatic interactions.
  • the neutral, aligned to the surrounding aqueous medium moiety consists of polyethylene glycol and / or polyethylene oxide units (PEG units) of different lengths. Preference is given here to PEG chains having a molecular weight of from 100 to 30,000 daltons and more preferably from 3,000 to 5,000 daltons. This moiety may alternatively be made of other suitable structures, such as. As hydroxyethyl starch (HES) and all possible polymeric compounds exist.
  • the radical R is preferably hydrogen or a methyl unit.
  • the surface of the polymer nanoparticle is modified with Glu (10) -b-PEG (110) (Formula 6).
  • the negative moiety (anchor) is the carboxylate groups of the glutamic acid subunits of the block copolymer.
  • Formula 6 Structural Formula of GIu (10) -b-PEG (110);
  • a target structure is present.
  • This target structure has at least one negatively charged moiety which is applied to the cationic particle surface by electrostatic interactions.
  • the partially anionically charged moiety fulfills the function of an anchor on the positively charged particle surface through electrostatic interactions.
  • the middle, neutral part of the molecule consists of polyethylene glycol units and / or polyethylene oxide units (PEG units) of different lengths. Preference is given here to PEG chains having a molecular weight of from 100 to 30,000 daltons and more preferably from 3,000 to 5,000 daltons. This moiety may alternatively be made of other suitable structures, such as. As hydroxyethyl starch (HES) and all possible polymeric compounds exist.
  • HES hydroxyethyl starch
  • the ligand X of the surface-modifying agent also referred to as target structure, serves to improve passive and active accumulation mechanisms of the polymer nanoparticles.
  • Suitable ligands as target structures may be antibodies, peptides, receptor ligands of ligand mimetics or an aptamer. Structures include amino acids, peptides, CDR (compementary determining regions), antigens, haptens, enzyme substances, enzyme cofactors, biotin, carotenoids, hormones, vitamins, growth factors, lymphokines, carbohydrates, oligosaccharides, lecithins, dextrans, lipids, nucleosides such as DNA or an RNA molecule containing native, modified or artificial nucleosides, nucleic acids, oligocucleotides, polysaccharides, B, A, Z-HeNx or hairpin structure (hairpin), a chemical entity, modified polysaccharides as well as receptor binding substances or fragments thereof consideration. Target structures may also be transferrin or folic acid or parts thereof, or all possible combinations of the foregoing.
  • ligands are bound to the nanoparticles via electrostatic interactions, but it is also possible to bind the ligands via covalent bonds to the particle surface. Furthermore, it is possible to incorporate a linker between ligand and nanoparticles.
  • the electrostatic attachment of the target structures takes place via charge interactions with at least one negatively charged moiety on the cationic particle surface.
  • compounds such as acetate, carbonate, citrate, succinate, nitrate, carboxylate, phosphate, sulfonate or sulfate groups, as well as salts and free acids of these groups, are suitable as the negatively charged moiety (anchor).
  • the size of the nanoparticles is between 1 nm and 800 nm.
  • the size of the nanoparticles is between 5 nm and 800 nm.
  • the size of the nanoparticles is between 1 nm and 500 nm.
  • the size of the nanoparticles is between 1 nm and 300 nm.
  • the size of the nanoparticles is between 5 nm and 500 nm.
  • the size of the nanoparticles is between 5 nm and 300 nm.
  • the size of the nanoparticles is between 10 nm and 300 nm.
  • the size of the resulting polymer nanoparticles is determined by photon correlation spectroscopy (PCS).
  • PCS photon correlation spectroscopy
  • the polymer nanoparticles are characterized by carrying out the following process steps:
  • the water-insoluble polymer is dissolved in a suitable organic solvent which is infinitely miscible with water, preferably acetone, methanol, ethanol, propanol, dimethylsulfoxide (DMSO), or in a mixture of these solvents with water.
  • a suitable organic solvent which is infinitely miscible with water, preferably acetone, methanol, ethanol, propanol, dimethylsulfoxide (DMSO), or in a mixture of these solvents with water.
  • the cationic polymer is dissolved in a suitable solvent which is immiscible with water indefinitely, preferably acetone,
  • the active ingredient (diagnostic or therapeutic) is dissolved in an organic solvent which is infinitely miscible with water, preferably acetone, methanol, ethanol, propanol, dimethylsulfoxide
  • a completely homogeneous solution of cationic polymer, water-insoluble polymer and active ingredient is produced. • By introducing the dissolved polymer-substance mixture in a surfactant-containing solution, as a surfactant in particular Pluronic F68, Triton X-100 and Synperonic T707, the spontaneous formation of a colloidal precipitation unit is brought about.
  • the organic solvent is then removed either under atmospheric pressure or negative pressure, via lyophilization or heat completely or other suitable methods.
  • the aqueous, stable nanoparticle dispersion is mixed in a suitable ratio with the modifying agent dissolved in water.
  • the determination of the appropriate quantitative ratio is carried out by stepwise titration of
  • Particle dispersion with the modifying agent The degree of electrostatic surface modification (charge titration) is controlled by determining the zeta potential.
  • the described nanoparticles can be further processed using suitable pharmaceutical additives to various dosage forms which are suitable for application to humans or animals. These include in particular aqueous dispersions, lyophilisates, solid oral dosage forms such as fast-dissolving tablets, capsules and others.
  • suitable pharmaceutical additives may be: sugar alcohols for lyophilization (eg sorbitol, mannitol), tableting excipients, polyethylene glycols, etc.
  • aqueous nanoparticle dispersion or of a further developed pharmaceutical form can be applied orally, parenterally (intravenously), subcutaneously, intramuscularly, intraocularly, intrapulmonary, nasally, intraperitoneally, dermally as well as on all other possible human or animal routes of administration.
  • the invention relates to a process for the preparation of a polymer nanoparticle, characterized in that the following process steps are carried out:
  • active ingredient includes therapeutically and diagnostically effective compounds. Also included are compounds that are effective in animals other than humans and plants.
  • matrix polymer as used herein describes the polymer which quantitatively constitutes the major proportion of the particle mass, wherein further encapsulated substances (both arbitrary additives and pharmaceutically active substances) may be uniformly and / or non-uniformly embedded.
  • (nano) precipitation describes the formation of a colloidal precipitate by precipitating a sparingly water-soluble polymer when placed in an aqueous phase, with mixing of the solvents taking place Community precipitation of several substances which may be both water-soluble and poorly water-soluble in the context of the invention.
  • this precipitating aggregate consists of a matrix polymer in which further polymeric substances as well as pharmaceutically active substances can be partially or completely embedded in. A uniform or uneven distribution of the coagulum may be present. encapsulated substances in the matrix polymer.
  • an “anchor”, as used herein, describes an ionic moiety of the modifying agent that allows immobilization and thus localization of the modifying agent on the charged particle surface by ionic interactions between oppositely charged compounds.
  • Charge titration describes the process of electrostatic coupling of the armature on the particle surface that can be traced by zeta potential measurement. The charged anchor changes the zeta potential of the particle towards the charge of the anchor.
  • Surfactants according to the invention are, on the one hand, surface-active substances which lower the interfacial tension between two immiscible phases, which makes it possible to stabilize colloidal dispersions. Furthermore, according to the invention, surfactants can be any type of substances which are capable of sterically and / or electrostatically stabilizing colloidal dispersions.
  • active targeting is used when tissue- or cell-specific ligands are used for targeted enrichment, and active ligands can be coupled both directly to drugs (ligand-drug conjugates) and to the surface of colloidal carrier systems.
  • passive targeting is used when the distribution of the active ingredient is due to (nonspecific) physical, biochemical or immunological processes, primarily due to the enhanced permeation and retention effect (EPR effect) this is a passive enrichment mechanism that exploits the structural peculiarities of tumorous or inflamed tissue [Ulbrich K., Subr V., Adv. Drug Deliv. Rev., 2004; 56 (7): 1023-1050].
  • surface potential also referred to as surface charge
  • zeta potential is synonymous with the term “zeta potential.”
  • This zeta potential is determined by the method of laser Doppler anemometry (LDA).
  • LDA laser Doppler anemometry
  • the surface potential also referred to as zeta potential, indicates the potential of a migrating particle at the shear plane, ie when most of the diffuse layer has been sheared off by movement of the particle.
  • the surface potential was determined by the method of laser Doppler anemometry using a "Zetasizer 3000" (Malvern Instruments).
  • the migration speed of the particles in the electric field is determined. Particles with a charged surface migrate in an electric field to the oppositely charged electrode, wherein the
  • Migration rate of the particles depends on the amount of surface charges and the applied field strength.
  • particles traveling in the electric field are irradiated with a laser and the scattered laser light is detected.
  • a frequency shift in the reflected light is measured in comparison to the incident light.
  • the amount of this frequency shift is dependent on the rate of migration and is referred to as the so-called Doppler frequency (Doppler effect).
  • Doppler frequency Doppler effect
  • the electrophoretic mobility results from the quotient of the migration speed and the electric field strength.
  • the product of electrophoretic mobility and factor 13 corresponds to the zeta potential, the unit of which is [mV].
  • the software used was PCS V1.41 / PCS V1.51 Rev.
  • the control measurements of the zeta potential were made with standard latex particles of Company Malvern Instruments Ltd. (-50 mV ⁇ 5 mV). The measurements were made under the standard settings of Malvern Instruments Ltd. carried out.
  • the size of the nanoparticles was determined by Dynamic Light Scattering (DLS) using a "Zetasizer 3000" (Malvern Instruments). In addition, images were taken in the scanning electron microscope (SEM), as shown by way of example in FIG. Figure 12 ( Figure 12) also confirms the spherical shape of the nanoparticles.
  • the determination of the particle size by DLS is based on the principle of Photon Correlation Spectroscopy (PCS).
  • PCS Photon Correlation Spectroscopy
  • the mean particle diameter is calculated from the slope of the correlation function.
  • the particles should have a spherical shape, which can be checked by SEM images (see above), do not sediment or float.
  • the measurements were carried out with samples in appropriate dilution, at a constant temperature of 25 ° C and a defined viscosity of the solution.
  • the measuring device was calibrated with standard latex particles of different sizes from Malvern Instruments Ltd.
  • the scanning electron micrographs (SEM images) for determining the particle size were prepared using a field emission scanning electron microscope of the type XL-30-SFEG from FEI (Kassel, Germany). In advance, the samples were sputtered in a high vacuum sputter 208 HR from Cressington (Watford, England) with a 5 nm gold-palladium layer.
  • solubility of a substance indicates whether and to what extent a pure substance can be dissolved in a solvent. It thus designates the property of a substance under homogeneous distribution (as atoms, molecules or
  • the solubility of a compound is determined to be the concentration of a saturated solution that is in equilibrium with the undissolved sediment as a function of temperature (space temperature).
  • a poorly soluble compound has a
  • Sicomet 6000 is used for PBCA production by anionic polymerization of butyl cyanoacrylate (BCA).
  • BCA butyl cyanoacrylate
  • the polymerization process is carried out by slow, permanent dropping of a total of 2.5% [m / v] BCA into a 1% [m / v] Triton X-100 solution at pH 2.2.
  • the pH is previously adjusted by means of a 0.1 N HCl solution.
  • the resulting dispersion is stirred while cooling in an ice bath (about 4 ° C) for 4 hours constantly at 450 U / min. Subsequently, larger agglomerates are separated by filtration through a paper-pleated filter.
  • the BCA polymerized to PBCA is precipitated and the filter residue obtained is washed several times with purified water (MiIIiQ system). After drying of the filter residue PBCA in a drying oven at 40 0 C for 24 h has an average molecular weight is determined (Mn ⁇ 2000 Da) by GPC. Polystyrene standards are used.
  • the mixed dye-polymer mixture is taken up with a 2.5 ml Eppendorf pipette and pipetted into 10 ml of an intensely stirred 1% [m / v] Synperonic T707 solution.
  • the nanoparticle dispersion is stirred for 2 h at 600 rpm (standard magnetic stirrer) and for a further 16 h at 100 rpm for complete evaporation of the solvent.
  • the workup is carried out by centrifugation in Eppendorf Caps.
  • PLGA-P (DMAEMA) nanoparticles 500 ⁇ l of a 2% acetone PLGA solution [m / v] with 100 ⁇ l of P (DMAEMA) in acetone (2% [m / v]) are used.
  • 100 ⁇ l of the dye solutions a-d mentioned under i) are used.
  • the mixed dye-polymer mixture is taken up in a 2.5 ml Eppendorf pipette and pipetted into 10 ml of an intensively stirred 1% Synperonic T707 solution.
  • the nanoparticle dispersion is stirred for 2 h at 600 rpm (standard magnetic stirrer) and for a further 16 h at 100 rpm for complete evaporation of the solvent.
  • the workup is carried out by centrifugation in Eppendorf Caps.
  • Example 3 Influence of Nanoprecipitation by Changing the Polymer Content in the Surfactant Phase
  • FIG. 1 shows that the particle size of the PBCA-P (DMAEMA) nanoparticles can be controlled during production by varying the polymer concentration.
  • the cationically functionalized particles are prepared according to Example 2.
  • FIG. 2 shows the particle diameter as well as the zeta potential of PBCA [PEI-I DCC] nanoparticles, which are stabilized by either the Triton X-100 or Pluronic F 68 surfactant. Due to the encapsulation of the polycation polyethylenimine in the PBCA matrix, the particles have a positive zeta potential between 30 mV and 40 mV. Both the particle size and the zeta potential are constant before and after processing of the particles (washing process), evidence of a good stability of the particles.
  • PBCA [PEI-IDCC] nanoparticles used here are prepared according to Example 2.
  • the aqueous, stable nanoparticle dispersion is mixed in a suitable quantitative ratio with the modifying agent dissolved in water.
  • the determination of the appropriate quantitative ratio is carried out by stepwise titration of the particle dispersion with the modifying agent.
  • the degree of electrostatic surface modification (charge titration) is controlled by determining the zeta potential.
  • the change in the zeta-potential from +25 mV to about -30 mV is illustrated in FIG. 3 by stepwise addition of the modifying agent (Glu (10) -b-PEG (HO)) for particle dispersion (charge titration).
  • Example 6 R E M images of PBCA-P (DMAEMA) nanoparticles loaded with different fluorescent dyes
  • FIG. 4 shows an SEM image of DODC-loaded PBCA-P (DMAEMA) nanoparticles.
  • FIG. 5 shows an SEM micrograph of coumarin-6-loaded PBCA-P (DMAEMA) nanoparticles.
  • FCS fetal calf medium
  • Additives penicillin / streptamycin
  • the cells are passaged regularly and sowed for experimental purposes 24 hours before the start of the investigations.
  • the cells are in 96-well plates of the company
  • FCS-containing medium is aspirated and replaced with 50 .mu.l of serum-free medium.
  • CMXRos dye previously diluted in medium, from Molecular Probes Europe BV, Leiden (NL) (0.25 ⁇ l / ml) is used. Incubation with 50 ⁇ l of the dye solution takes place in the incubator for 15 min (37 ° C., 5% CO 2). The dye solution is then filtered off with suction and the cells are washed 2-3 times with PBS.
  • the fixation of the cells is carried out with 100 ul of 1, 37% formaldehyde for 10 min at room temperature. After aspirating the fixing solution, the cells are washed 2-3 times with PBS. Nuclear staining takes place on the already fixed cells with a maximum of 33342. For this purpose, 100 ⁇ l of the dye solution diluted in PBS (2 ⁇ g / ml) are incubated for 10 min at room temperature. After removal of the dye solution, the cells are washed with 100 ⁇ l PBS 2-3 times. The fixed plates are stored in the refrigerator until the fluorescence microscopic examination with 200 .mu.l PBS / Well protected from light in the refrigerator at 8 ° C.
  • Example 8 Influence of functionalized particle surfaces on cell uptake
  • the nanoparticles used in Example 8 are prepared according to Example 2. Unmodified or after electrostatic surface modification with folic acid or Glu (10) -b-PEG (110), the particles have the properties listed in Table 1. In the 96-well plate used for the experiment all wells have the same cell density (sowing 24 h before experiment: 1x10 4 cells). A constant particle concentration of the particles listed in the table (Table 1) is incubated in the incubator over a period of 60 minutes. Subsequently, the cells are washed, fixed and measured on the following day. The uptake of the fluorescent cells is carried out with an automatic fluorescence microscope at 20 ⁇ magnification and constant exposure time (see FIG. 6).
  • Example 9 Cell uptake behavior of GIu (10) -b-PEG (110) modified PBCA P (DMAEMA) nanoparticles
  • the brightly fluorescent dots, which are endosomes or endolysosomes, are evidence of efficient uptake of the nanoparticles into the cell by endocytosis ( Figure 7).
  • the scale of the magnification proves that in this photograph individual particles can not be visible due to their size of less than 200 nm. A large number of particles within these vesicles (endosomes / endolysosomes) cause strong, punctate fluorescence contrast in the cytoplasm.
  • FIG. 11 shows in relation to Fig. 10 an increased particle uptake with incubation of a higher particle concentration.
  • Example 12 Characterization of the PBCA [P (DMAEMA) ICG] nanoparticles
  • FIG. 12 Based on the SEM image (FIG. 12), it can additionally be shown that they are spherical nanoparticles with a size of about 200 nm.
  • Charge titration is used to modify the cationic surface of the PBCA-P (DMAEMA) nanoparticles with the block copolymer Glu (10) -b-PEG (110) (see FIG. 14).
  • the surface charge measured as zeta potential is correspondingly titrated from approx. +3 ohms above the neutral point until reaching the dissociation equilibrium at approx. -3 oMV.
  • the surface-modified PBCA [P (DMAEMA) -ICG] particles show no change in the zeta potential over the investigated period of 7 days after titration. In connection with the unchanged particle size and the constant low PI, a good particle stability can be proven.
  • FIG. 15 shows the UV-Vis absorption spectra of an aqueous ICG solution as well as the ICG nanoparticle dispersion (washed and unwashed).
  • Indocyanine green is a near-infrared fluorescent dye whose absorption and emission spectrum is in the wavelength range between 650-900 nm.
  • DMAEMA cationic polyacrylate P
  • the animals used are supplied by Taconic M & B. These are female albino nude mice of the type NMRI nude. The adult animals have a weight of 22-24 g after about 8 weeks.
  • Five female nude mice are inoculated with 2x10 6 cells of a F9 teratoma in the right posterior flank. The cells are obtained from the company ATCC / LGC Promochem GmbH. It is mouse-derived embryonic cells of a testinal teratocarcinoma, which is used as a tumor model for cancer research purposes in mice. After 18 days, four of the five mice had tumors with an average size of about 0.5-1 cm in diameter.
  • the animals are permanently anesthetized with a rompun-ketavet injection in a dose of 100 ⁇ l / 10 g of animal for the first hour of the experiment.
  • the solution for injection consists of a 1: 1 mixture of 1:10 Dilution Rompun or 1: 5 dilution Ketavet with physiological saline.
  • 200 ⁇ l of the nanoparticle dispersion are injected iv into the tail vein.
  • the following anesthetics are carried out with Rompunketavet via the lungs as an inhalation anesthetic in order to minimize the circulation of the animals.
  • the animals are examined by fluorescence optics.
  • GIu (10) -b-PEG (110) -modified PBCA [P (DMAEMA) -ICG] nanoparticles after intravenous administration are capable of passive enrichment mechanisms (EPR effect) in tumor tissue.
  • Examination of the tumors ex vivo shows a clear enhancement of the fluorescence contrast in treated compared to untreated tumor tissue (compare Fig. 18 b with a and c with a).
  • Multiple, time-shifted detection of fluorescence in one and the same animal is possible after 24 h and 48 h (FIG. 17). Consequently, the particles can circulate in vivo for a sufficiently long time and accumulate accordingly in the tumor.
  • the electrostatically pegylated surface is thus stably connected to the particle surface.
  • the device used for the animal experiment was set up by the company LMTB (Berlin, Germany). As individual components were used:
  • Fig. 1 control of the particle diameter by changing the
  • FIG. 1 shows that the particle size of the PBCA
  • P (DMAEMA) nanoparticles can be controlled during production by varying the polymer concentration.
  • Fig. 3 Zeta potential Glu (10) -b-PEG (1 10) modified PBCA [PEI-IDCC] -
  • Fig. 4 SEM (Scanning Electron Microscope) image DODC-loaded
  • the figure shows an SEM image of DODC-loaded PBCA-P (DMAEMA) nanoparticles.
  • FIG. 6 Influence of functionalized particle surfaces on the
  • Cell uptake a) Comparison of cell uptake behavior after surface modification; Row 1: unmodified particles; Row 2:
  • NP with folic acid Row 3: NP with Glu (10) -b-PEG (110); b) Section: Row 3 / Well 1 / Site 15; Arrows indicate marked fluorescence enhancement in the nucleus.
  • Fig. 7 Nanoparticle uptake in HeLa cells; Fluorescence of
  • the figure shows the cell uptake behavior of GIu (10) -b-PEG (HO) -modified PBCA P (DMAEMA) nanoparticles in HeLa cells.
  • P (DMAEMA) nanoparticles surface-modified with Glu (10) -b-PEG (HO); Abbreviations used PEG-NP: pegylated coumarin-containing
  • PBCA-P (DMAEMA) nanoparticles
  • NP coumarin-loaded PBCA-P (DMAEMA) nanoparticles
  • CP clathrin-coated pits
  • ES endosomes
  • LS lysosomes
  • ELS endolysosomes
  • ZK cell nucleus
  • H + H + ATPase
  • PEG-Glu free Glu (10) -b-PEG (110) block copolymer; Size relations do not correspond to reality.
  • Particle concentration of 0.21 mg / ml was incubated.
  • GIu (10) -b-PEG (110) surface-modified PBCA-P (DMAEMA) particles were used.
  • Fig. 11 Increased particle uptake with incubation of higher particle concentration: 0.85 mg / ml; Fluorescence of the NP as grayscale image;
  • the figure shows more strongly fluorescent HeLa cells after a higher particle concentration of 0.85 mg / ml was incubated.
  • GIu (10) -b-PEG (110) surface-modified PBCA-P (DMAEMA) particles were used for this purpose.
  • Fig. 12 SEM image of PBCA [P (DMAEMA) ICG] nanoparticles
  • FIG. 13 particle diameter d hyd of the PBCA [P (DMAEMA) ICG] nanoparticles, surface-modified with Glu (10) -b-PEG (110); Shown is the particle size of the surface-modified PBCA [P (DMAEMA) -ICG] nanoparticles used for the animal experiment over a period of 7 days after preparation for the animal experiment.
  • Figure 14 Zeta potential of the untitrated (washed / unwashed) and titrated PBCA [P (DMAEMA) ICG] nanoparticles;
  • the abbreviation shows the zeta potential measured surface charge of the PBCA-P (DMAEMA) nanoparticles modified with the block copolymer Glu (10) -b-PEG (110). This was accordingly about + 3OmV beyond the neutral point titrated until reaching the dissociation equilibrium at about -3OmV.
  • Fig. 15 UV-Vis absorption spectra: a) aqueous ICG solution, b) PBCA [P (DMAEMA) -ICG] -NP unwashed; c) PBCA- [P (DMAEMA) -ICG]
  • This figure shows the UV-Vis absorption spectra of an aqueous ICG solution as well as the ICG nanoparticle dispersion (washed and unwashed).
  • Fig. 16 Emission spectrum of the PBCA [P (DMAEMA) ICG] nanoparticles and an aqueous ICG solution;
  • the figure represents the corresponding emission spectra of the aqueous ICG solution compared to the nanoparticle dispersion.
  • Fig. 17 Detection of NIR fluorescence in vivo
  • the images show the NIR fluorescence in a time frame of 24 and 48 h after substance injection (a) ventrally 24 h, b) 24 h laterally, c) 48 h laterally, d) ventrally empty).
  • Fig. 18 NIR fluorescence contrast of the tumor tissue ex vivo 48 h after
  • the figure shows NIR fluorescence contrasts a) an untreated tumor without NIR fluorescence contrast, b) a large, treated tumor and c) a median, treated tumor ex vivo 48 h after treatment.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Nanotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne des nanoparticules polymères possédant un potentiel superficiel cationique, dans lesquelles des substances pharmaceutiquement actives hydrophobes comme hydrophiles peuvent être encapsulées. Les substances hydrophiles et donc solubles dans l'eau sont incluses dans le cœur de la particule par complexation ionique avec un polymère chargé en utilisant un procédé de co-précipitation. Comme substances pharmaceutiquement actives à encapsuler, on peut utiliser aussi bien des agents thérapeutiques que des agents de diagnostic. La surface cationique des particules permet une modification de surface électrostatique stable avec des composés de charge en partie opposée, qui peuvent contenir des ligands spécifiques à une cible afin d'améliorer le ciblage passif et actif.
EP07726022A 2006-06-08 2007-06-07 Nanoparticules polymères solides fonctionnalisées pour applications diagnostiques et thérapeutiques Withdrawn EP2029119A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US81175606P 2006-06-08 2006-06-08
PCT/EP2007/005258 WO2007141050A2 (fr) 2006-06-08 2007-06-07 Nanoparticules polymères solides fonctionnalisées pour applications diagnostiques et thérapeutiques

Publications (1)

Publication Number Publication Date
EP2029119A2 true EP2029119A2 (fr) 2009-03-04

Family

ID=38441504

Family Applications (1)

Application Number Title Priority Date Filing Date
EP07726022A Withdrawn EP2029119A2 (fr) 2006-06-08 2007-06-07 Nanoparticules polymères solides fonctionnalisées pour applications diagnostiques et thérapeutiques

Country Status (5)

Country Link
US (1) US20100196280A1 (fr)
EP (1) EP2029119A2 (fr)
JP (1) JP2009539793A (fr)
CA (1) CA2654593A1 (fr)
WO (1) WO2007141050A2 (fr)

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102007059752A1 (de) * 2007-12-10 2009-06-18 Bayer Schering Pharma Aktiengesellschaft Funktionalisierte, feste Polymernanopartikel enthaltend Epothilone
US20110318267A1 (en) * 2008-01-25 2011-12-29 President And Fellows Of Harvard College Ph-responsive nanostructures
EP2113257A1 (fr) * 2008-04-30 2009-11-04 Consorzio per il Centro di Biomedica Moleculare Scrl Polyélectrolyte avec chargement net positif à utiliser en tant que médicament et pour le diagnostic du cancer
US7973096B2 (en) * 2008-05-07 2011-07-05 3M Innovative Properties Company Antimicrobial nanoparticles
JP5663741B2 (ja) * 2009-03-03 2015-02-04 株式会社ナノカム アミノ酸抱合シアノアクリレートポリマー粒子
EP2608781A4 (fr) * 2010-08-04 2014-01-29 Tixupharma Complexes d'inclusion composés de cyclodextrines et de spermidine, et compositions de prolifération/réparation contenant ces complexes
FR2968993B1 (fr) * 2010-12-17 2012-12-28 Flamel Tech Sa Nanoparticules comportant au moins un actif et au moins deux polyelectrolytes
EP2750662A4 (fr) 2011-08-31 2015-06-24 Univ Georgia Nanoparticules ciblant l'apoptose
CN102977413B (zh) * 2011-09-07 2015-05-13 江南大学 聚合物复合制备胶束的一种新方法
TWI462752B (zh) * 2011-09-21 2014-12-01 Univ Nat Cheng Kung 包覆疏水性藥物之膠囊粒子製造方法
CN103083222B (zh) * 2011-10-28 2015-08-19 江南大学 一锅法制备三组分聚合物胶束
ES2669561T3 (es) 2012-02-17 2018-05-28 University Of Georgia Research Foundation, Inc. Nanopartículas para el transporte mitocondrial de agentes
JP2015510824A (ja) * 2012-03-19 2015-04-13 ネオメンド、インク． 共沈法
KR101900080B1 (ko) * 2012-11-29 2018-09-18 한국전자통신연구원 전기영동 입자 및 이를 이용한 전기영동 디스플레이
CN105764491A (zh) 2013-12-09 2016-07-13 度瑞公司 药物活性剂复合物、聚合物复合物，以及包括其的组合物和方法
US10398663B2 (en) 2014-03-14 2019-09-03 University Of Georgia Research Foundation, Inc. Mitochondrial delivery of 3-bromopyruvate
AU2015269068A1 (en) * 2014-06-06 2016-12-22 The Board Of Regents Of The University Of Texas System Library of pH responsive polymers and nanoprobes thereof
EP3242690B1 (fr) * 2015-01-06 2020-09-16 De Haas, Anthony H. Marqueurs colorants chirurgicaux fluorescents dans l'infrarouge proche
CN106866902A (zh) * 2017-01-11 2017-06-20 华南理工大学 一种可降解双响应聚合物及其负载药物和金纳米粒子胶束的制备方法与应用
JP2020523430A (ja) * 2017-05-30 2020-08-06 エリューム リミテッド ナノ粒子凝集体
JP2021519748A (ja) * 2018-01-29 2021-08-12 ザ・ジョンズ・ホプキンス・ユニバーシティ タンパク質治療薬をカプセル製剤化および徐放性製剤化を可能にするポリマーナノ粒子組成物
EP4026859A4 (fr) * 2019-09-05 2022-12-28 Dalian Heyuan Medical Equipments Co., Ltd. Hydrolysat de poly[alpha-cyanoacrylate], et procédé de préparation et application de ce dernier
CN114569740A (zh) * 2021-07-13 2022-06-03 浙江大学 一种靶向免疫生发中心抑制抗体介导的排斥反应的新型纳米缓释载药材料、制备方法及应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1021168A4 (fr) * 1997-10-09 2006-08-30 Univ Vanderbilt Dispositif d'administration de microparticules ou de nanoparticules de polymeres
ATE228883T1 (de) * 1998-03-19 2002-12-15 Max Planck Gesellschaft Herstellung von mit mehrlagen gestrichenen partikeln und hohlen schalen durch elektrostatische selbstorganisierung von nanokompositmehrlagen auf zersetzbaren schablonen
ATE408398T1 (de) * 2002-07-29 2008-10-15 Nanodel Technologies Gmbh Nanopartikele für dna verabreichung zu einem zielorgan
GB2407501A (en) * 2003-11-03 2005-05-04 Ist Superiore Sanita Nanoparticles for delivery of a pharmacologically active agent, comprising water insoluble (co)polymer core & hydrophilic acrylate-based (co)polymer shell
JP2006028041A (ja) * 2004-07-13 2006-02-02 Ltt Bio-Pharma Co Ltd 核酸含有ナノ粒子

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007141050A2 *

Also Published As

Publication number Publication date
CA2654593A1 (fr) 2007-12-13
US20100196280A1 (en) 2010-08-05
JP2009539793A (ja) 2009-11-19
WO2007141050A3 (fr) 2008-07-31
WO2007141050A2 (fr) 2007-12-13

Similar Documents

Publication Publication Date Title
EP2029119A2 (fr) Nanoparticules polymères solides fonctionnalisées pour applications diagnostiques et thérapeutiques
DE102007059752A1 (de) Funktionalisierte, feste Polymernanopartikel enthaltend Epothilone
Voigt et al. Surfactants, not size or zeta-potential influence blood–brain barrier passage of polymeric nanoparticles
Yang et al. Chitosan mediated solid lipid nanoparticles for enhanced liver delivery of zedoary turmeric oil in vivo
US20090148384A1 (en) Functionalized, solid polymer nanoparticles comprising epothilones
US10307372B2 (en) Rapid diffusion of large polymeric nanoparticles in the mammalian brain
US20070148074A1 (en) Nanoparticle based stabilization of ir fluorescent dyes
Schädlich et al. Accumulation of nanocarriers in the ovary: a neglected toxicity risk?
US20140213641A1 (en) Polymeric nanoparticles for drug delivery
Conte et al. Multi-component bioresponsive nanoparticles for synchronous delivery of docetaxel and TUBB3 siRNA to lung cancer cells
EA020753B1 (ru) Терапевтические полимерные наночастицы, содержащие алкалоиды vinca, и их применение
EP2848262B1 (fr) Ciblage spécifique à des cellules par des systèmes porteurs nanostructurés
Mattu et al. Alternating block copolymer-based nanoparticles as tools to modulate the loading of multiple chemotherapeutics and imaging probes
JP2017530941A (ja) 脳内に蓄積する治療用ナノ粒子
Huang et al. Polymeric nanoparticles functionalized with muscle-homing peptides for targeted delivery of phosphatase and tensin homolog inhibitor to skeletal muscle
US20210353758A1 (en) Microcarrier for embolization and preparation method therefor
EP3950007A1 (fr) Procédé de préparation d'un nano-agent de diagnostic apte à colorer sélectivement des tissus anormaux inflammatoires ou des tissus tumoraux
US20220031869A1 (en) Nanocarrier systems for imaging and delivery of active agents
Zhang et al. Exploring the systemic delivery of a poorly water-soluble model drug to the retina using PLGA nanoparticles
JP6824535B2 (ja) 脳間質内のナノ粒子分布を改善するための組成物および方法
WO2022066636A1 (fr) Nanoparticules et procédés d'utilisation associés
KR20130088081A (ko) 수난용성 약물을 내부에 포함하는 알부민 나노입자 제조방법
CN113499318A (zh) 红细胞膜包被载药纳米粒/探针及其在脑胶质母细胞瘤诊疗中的应用
Negron et al. Strategies to Enhance the Distribution of Therapeutic Nanoparticles in the Brain by Convection Enhanced Delivery
North Poly (Lactic-co-Glycolic Acid)-Polyethylene Glycol Nanoparticles for Therapeutic Delivery to Traumatic Brain Injury

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL BA HR MK RS

17P Request for examination filed

Effective date: 20090202

RBV Designated contracting states (corrected)

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20100723