DE102007059752A1 - Functionalized solid polymer nanoparticles containing epothilones - Google Patents

Abstract

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. For encapsulation, 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.

Description

The The present invention describes polymer nanoparticles with cationic Surface potential, in which neutral, hydrophobic and hydrophilic pharmaceutically active substances are included can. The hydrophilic and thus water-soluble Substances are charged by ionic complexation with a Polymer in the core of the particle by co-precipitation locked in. For encapsulation are as pharmaceutically active Substans are both therapeutics, especially epothilones, as well Diagnostics usable. The cationic particle surface allows a stable, electrostatic surface modification with partially oppositely charged compounds that improve of passive and active targeting include target-specific ligands can.

Background of the invention

The special properties of nanoparticulate drug delivery systems are based on their small size, which makes it possible to overcome different physiological barriers [ Fahmy ™, Fong PM 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.

Nanoparticular systems, which can be used both for the detection and for the treatment of diseases, are referred to as so-called theranostics (= therapeutics + diagnostics). The associated therapeutic monitoring will enable a faster recognition of therapy resistance in the future and significantly improve the healing success of the patient by the timely use of alternative therapies [ 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. The associated undesirable toxicity is often the dose limiting factor of therapy [ Silacci D., Neri M., Modern Biopharmaceuticals: Design, Development and Optimization, Volume 3, Part V, Wiley-VCH, Weinheim, 2005; 1271-1299 ].

It has been shown that, for example, by encapsulating cytotoxic substances such as doxorubicin in nanoparticulate systems, healthy tissue is less damaged and a locally higher concentration of active substance in the tumor tissue is achieved [ Silacci D., Neri M., Modern Biopharmaceuticals: Design, Development and Optimization, Volume 3, Part V, Wiley-VCH, Weinheim, 2005; 1271-1299 ].

In the case of the liposomal formulation of doxorubicin, the cardiotoxicity of the substance can be significantly reduced. By reducing the dose-limiting cardiotoxicity, in turn, a higher therapeutic efficiency can be achieved. Due to the demonstrable clinical benefit, the liposome-encapsulated doxorubicin was successfully approved under the name Doxil ^® or Cealyx for tumor therapy.

Epothilones are a new class of antitumor compounds that cause apoptosis. Various publications and reports of study results ( eg IDrugs, 2002, 5 (10): 949-954 ) have proven their effectiveness against cancer. The dose for her dose is from study reports or from other publications, eg. B. for the epothilones A and B from WO 99/43320 also known. Using a nanoparticulate formulation, the therapeutic effect or the side-effect profile of this new substance class can be positively influenced by using special distribution mechanisms.

First and foremost, the enhanced permeation and retention effect (EPR effect for short) is held responsible. This EPR effect was described as early as 1986 by Matsumura and Maeda 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 ]. It is a passive enrichment mechanism that exploits the structural features of tumorous or inflamed tissue [ Ulbrich K., Subr V., Adv. Drug Deliv. Rev., 2004; 56 (7): 1023-1050 ]. In particular tumor tissue is characterized by its fast growth and various messenger substances usually by a fenestrierte "holey" tissue structure as well as a lack of lymphatic drainage Depending on the type of tumor, the size of the fenestrations between 380 nm and 780 nm indicated, which is why 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 ]. In contrast, normal tissues such as the heart, brain, or lung have so-called tight junctions, which are less than 10 nm (usually 2 nm to 4 nm) in diameter impermeable to colloidal drug carriers [ Hughes GA, Nanomedicine, 2005; 1 (1): 22-30 ].

in the Bloodstream circulating nanoparticles are thus able to become Passively enriched by diffusion from the bloodstream in the tumor tissue. A lack of lymphatic drainage favors the permanent Enrichment in the tumor or prevents a quick washout the nanoparticles (EPR effect).

For this enrichment mechanism to be possible, 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. For example, pegylated particle surfaces can prevent the body's own proteins from identifying the particles as foreign and rapid elimination via the organs of the reticulo-endothelial system (RES for short) [ Otsuka H. et al., Adv. Drug Deliv. Rev., 2003; 55 (3): 403-419 ]. By using active ligands on the particle surface (eg antibodies), the tissue-specific accumulation can be further optimized [ Nobs L. et al., Pharm. Sci., 2004; 93: 1980-1992 ] [ Yokoyama M., J. Artif. Organs, 2005; 8: 77-84 ].

For a recording of the active ingredients in the cell, another physiological barrier, the cell membrane, must be overcome. The difficulty for many drugs is that the cell has very effective transport mechanisms (eg P-glycoprotein) for the removal of foreign or toxic substances. If, however, the active substance is introduced into the cell via endocytosis with the help of nanoparticles, it is possible to bypass escaping transporters and 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 ].

The internalization into the cell takes place with nanoparticles mostly by means of endocytosis. For this reason, the particles are present in endosomes or endolysosomes after the uptake process [ Koo OM et al., Nanomedicine, 2005; 1 (3): 193-212 ]. If there is no release of the particles from the endolysosomes, an enzymatic degradation of active ingredient and colloidal carrier system occurs within the vesicles. An endolysosomal release of the particles and therefore of the active substance is therefore essential for the intracellular therapeutic effect.

The Release properties of the drug from the nanoparticle can additionally controlled by targeted selection of the polymer become. A nanoparticulate formulation can thus the Minimize frequency of application as well as a reduction the therapeutically necessary dose. Furthermore you can unwanted plasma mirror tips by encapsulation in Nanoparticles avoided and delayed release be achieved.

• (i) gezielte Anreicherung der Wirkstoffe
• (a) passiv mittels EPR-Effekt,
• (b) aktiv mittels gewebs- oder zellspezifischer Liganden, z. B. Antikörper,
• (ii) steuerbare Wirkstoff-Freisetzung durch gezielte Polymerauswahl,
• (iii) Vermeidung starker Schwankungen der Plasmaspiegel,
• (iv) Senkung der Dosis bzw. Steigerung der Effektivität bei gleicher Dosis,
• (v) geringere Nebenwirkungen bei erhöhtem Sicherheitsprofil,
• (vi) verringerte Applikationshäufigkeit mit verbesserter Compliance und
• (vii) Umgehung von Resistenzmechanismen (P-Gycoprotein) [ Rosen H., Abribat T., Nature Reviews Drug Discovery, 2005 May; 4(5): 381–5 ][ McLennan D. N., Porter C. J. H. et al., Drug Discovery Today: Technologies, 2005 Spring; 2(1): 89–96 ].

In summary, the following advantages are crucial for the development of polymer nanoparticles:

• (i) targeted enrichment of the active ingredients
• (a) passively by EPR effect,
• (b) actively by means of tissue or cell specific ligands, e.g. B. antibodies,
• (ii) controllable drug release through targeted polymer selection,
• (iii) avoiding large fluctuations in plasma levels,
• (iv) lowering the dose or increasing the effectiveness at the same dose,
• (v) fewer side effects with increased safety profile,
• (vi) reduced frequency of application with improved compliance and
• (vii) Bypassing of resistance mechanisms (P-glycoprotein) [ Rosen H., Abribat T., Nature Reviews Drug Discovery, 2005 May; 4 (5): 381-5 ] [ McLennan DN, Porter CJH et al., Drug Discovery Today: Technologies, 2005 Spring; 2 (1): 89-96 ].

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. In addition to the size, the overall structure of the particles, the matrix-forming substances and above all their surface are of decisive importance for the behavior in vivo [ Choi SW, Kim WS, Kim JH, Journal of Dispersion Science and Technology, 2003; 24 (3 & 4): 475-487 ]. In addition, the physicochemical properties of various drugs, in particular Active substance classes, differ greatly. Accordingly, there remains a need in the development of colloidal drug carrier systems with improved properties.

For For example, future therapeutic approaches will be necessary by a diagnostic proof of distribution the particle in the organism to demonstrate that an enrichment before especially in diseased tissue (eg in the tumor).

For a detection in vivo include optical imaging techniques such as sonography, X-ray diagnostics, cross-sectional imaging (CT, MRI) and Nuclear Medicine (PET, SPECT). A Another and relatively new method is Optical Imaging, whose detection principle based on the use of near-infrared fluorescence. It is about a non-invasive procedure that does not involve ionizing radiation works and very cost-effective compared to procedures such as MRI and is not very expensive. The for such an application developed NIR dyes such as indocyanine green are very good water soluble, which is why it is difficult to do this efficiently to encapsulate in a hydrophobic polymer matrix. Reason is the fast Change of the hydrophilic substance in the aqueous phase for example, in the production by means of nanoprecipitation.

For the encapsulation of hydrophilic substances in nanoparticles is only possible few technologies available that are different Disadvantage. The amphiphilic character of liposomes or For example, polymerosomes allow inclusion hydrophilic substances in the aqueous interior of the particles, whereas hydrophobic compounds are incorporated in the membrane can. Due to the localization in the nucleus or in the Shell of the particles, the loading is very limited and usually inadequate. Another disadvantage is that, above all hydrophilic substances in an aqueous environment quickly such systems are washed out.

An alternative encapsulation of water-soluble substances in Polyelektrolytkomplexe is limited, since dyes such as indocyanine green (ICG) are small molecules with only a few charged groups, which are insufficient charges for electrostatic complexation available. In addition, polyelectrolyte complexes in aqueous solution are very dynamic systems, which usually have insufficient colloidal stability in plasma [ Thünemann AF et al., Adv. Polym. Sci., 2004; 166: 113-171 ].

As previously described, it will be necessary in the future by means of a diagnostic nanoparticle to prove that the enrichment the particle mainly at the destination, such as the tumor takes place. Becomes If this proof is provided, then a therapeutically active substance may be used be encapsulated in one and the same system and a maximum achieve therapeutic effect at the site of action, since the desired Distribution of nanoparticles already by means of the diagnostic system was detected. To the distribution properties of the particles It is therefore important to not change for the diagnostic proof and the subsequent treatment to use one and the same nanoparticulate system. As described above, there are a number of different nanoparticulate Systems, however, either only for the encapsulation hydrophilic or hydrophobic substances are suitable. From the literature It is known that only small changes in nanoparticle properties like particle size, surface material, Type of matrix polymer or else the use of another surfactant a huge impact on the distribution of particles in the organism to have. That's why it's important to have both diagnosis, therapy as well a possible monitoring of treatment with one and the same System to perform.

Ideally should therefore be in one and the same nanoparticulate System both water-soluble dyes for diagnosis as also therapeutic substances, which are mostly due to their hydrophobic Properties are difficult to dissolve, effectively and with water encapsulated in sufficient stability to wash out can be.

A The technological challenge continues to be through the Use of suitable surfaces on the one hand a sufficient Particle stability and on the other hand a specific enrichment in the target tissue. The whereabouts at the place of Enrichment (target tissue) in turn is u. a. depends on, how well the particles are absorbed into the tissue and the cell.

It is known from the literature that cationic particle surfaces promote uptake into the cell [ Mounkes LC et al., J. Biol. Chem., 1998; 273 (40): 26164-26170 ] [ Mislick KA, Baldeschwieler JD, Proc. Natl. Acad. Sci. USA, 1996; 93: 12349-12354 ]. Reason are electrostatic interactions between the negatively charged cell membrane (sulfated proteoglycans) and the cationic particle surface (usually protonated amine functions). In addition, polymers or substances carrying amino groups are known to have endo-osmotic activity, ie they promote intracellular release of the particles from the endolysosomes by damaging the endolysosomal membrane [ DeDuve C. et al., Biochem. Pharmacol., 1974; 23: 2495-2531 ]. If the particles remain within the cell in the endolysosomes, a degradation of the particle matrix and the substances contained therein takes place via cell-specific enzymes. The endolysosomal release of the encapsulated drugs is therefore essential for the therapeutic effect.

However, the problem is that partially serious toxicological effects have been described in in vivo investigations of polyplexes and lipids with highly cationic charged surfaces [ Kircheis S. et al., J. Gene Med., 1999; 1: 111-120 ] [ Ogris M. et al., Gene Ther., 1999; 6: 595-605 ]. The reason is that cationic particles aggregate with negatively charged erythrocytes resulting in blockage of the blood vessels. In addition, this usually leads to a strong accumulation in the lungs, which pass the particles as the first capillary bed after iv application [ Kircheis R. et al., Drug Deliv. Rev., 2001; 53 (3): 341-58 ]. Here there is a risk of pulmonary embolism, favored by agglomerates of particles and erythrocytes or other blood components [ Kircheis S. et al., J. Gene Med., 1999; 1: 111-120 ] [ Ogris M. et al., Gene Ther., 1999; 6: 595-605 ].

Ideally should therefore use nanoparticulate systems with cationic Surface properties are produced without that possible toxicologically questionable properties an in vivo Hinder application. In addition, the particle surface must be towards the body's own defense mechanisms (Opsonine, RES) inconspicuous, whereby only a sufficiently long circulation time allows for which condition for a corresponding enrichment of the particles from the bloodstream in the target tissue is. The nanoparticulate systems should continue to do so uptake into the target cell and endolysosomal release support.

A further difficulty in the production of nanoparticulate Systems is to detect suitable substances or target Apply structures on the particle surface.

Frequently, 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-recognizing molecule [ Nobs L. et al., J. Pharm. Sci., 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 Sci. Technol., 2003; 24 (3 & 4): 475-487 ]. In addition, 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 should therefore be a non-covalent, easy to perform and thus flexible yet stable surface modification to 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. In addition, the ligands for specific enrichment often adversely affect the uptake in the actual tumor tissue and in particular on the cell uptake. If 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 ].

consequently There is still a need for pharmaceutical, nanoparticulate Formulations which: (i) both water-soluble and heavy water-soluble pharmaceutically active substances, effective and encapsulate with sufficient stability to wash out (ii) a non-covalent, easy-to-perform (flexible) and still allow stable surface modification, (iii) allow sufficient circulation time (iv), be effectively absorbed into the target tissue and (v) there intracellularly released from the endolysosomes.

An object of the invention was therefore to provide an improved pharmaceutical formulation for Ver In addition to which hydrophilic as well as hydrophobic agents can be encapsulated. On the other hand, a flexible and sufficiently stable surface modification should allow optimal accumulation in the diseased tissue. To achieve maximum diagnostic or therapeutic effect, 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. Likewise, they should be practicable manufacturing methods to enable production in a timely and cost-effective manner.

Description of the invention

The This invention relates to polymer nanoparticles having a cationic surface potential. containing a cationic polymer and a sparingly soluble in water Polymer, characterized in that these polymer nanoparticles diagnostic and therapeutic agents, in particular epothilones, encapsulated together or separately, or contain only epothilones.

Surprisingly was found by co-precipitation of a water-soluble cationic polymer with a sparingly water-soluble Polymer stable polymer nanoparticles can be produced which have a cationically functionalized surface. Furthermore, it was surprising in the sense of the invention that both hydrophilic, highly water-soluble and hydrophobic, poorly water-soluble substances in the polymer matrix of previously described nanoparticles could be encapsulated. unexpectedly led the ionic complexation of water-soluble Low molecular weight substances with the charged cationic Polymer for a successful encapsulation in the polymer matrix the particle by means of nanoprecipitation. Within the meaning of the invention substances can be encapsulated in the polymer particles, which are used for the diagnosis and / or therapy of different Diseases are suitable.

Farther was found to be the cationically functionalized particle surface electrostatically with a partially oppositely charged compound stable and flexible surface modification can be.

One The subject of the described invention is therefore the use the nanoparticles according to the invention for detecting of diseases (diagnosis), for the treatment of diseases (therapy) as well as to monitor a therapy.

• (a) ein in einen Partikel verkapseltes Diagnostikum und (b) ein in einen Partikel verkapseltes Epothilon, wobei die Partikel gemeinsam oder getrennt, gegebenenfalls verdünnt, verabreicht werden können.

Another aspect of the invention is a kit consisting of separately prepared nanoparticulate systems (a) and (b) of the same composition containing

• (a) a diagnostic encapsulated in a particle; and (b) an epothilone encapsulated in a particle, which particles may be administered together or separately, optionally diluted.

One Another aspect of the invention is a kit as described above. wherein the components (a) and (b) are in the solid state and additionally, if appropriate, an agent (c) is contained, which is suitable for the nanoparticulate systems (a) and (b) optionally optionally separately or together to solve or to disperse.

Farther For example, the invention includes a suitable dosage form using of pharmaceutically acceptable excipients suitable for the respective dosage form are necessary. The in the sense of the invention developed drug form can be administered to humans or animals via various Application routes are applied.

To necessary application systems are also part of the ones described here Invention.

The Composition of nanoparticles contains a poorly water-soluble Polymer, which is preferably a biodegradable polymer or also a mixture of various biodegradable polymers. The biodegradable polymer can be via individual monomer units which can be described by means of a polymerization or other Processes said polymer form. Furthermore, the polymer can over its Be defined name.

In In one embodiment, the sparingly water-soluble Polymer from the group of natural and / or synthetic Polymers or homo- and co-polymers of corresponding monomers from. In particular, the polymer is derived from the group of alkyl cyanoacrylates such as the butyl cyanoacrylates and the isobutyl cyanoacrylates, the acrylates, such as the methacrylates, the lactides, for example the L-lactide or DL-lactide, the glycolide, the caprolactone like of ε-caprolactones and others.

In a further embodiment, said polymer or part of the polymer is selected from the group of polycyanoacrylates and polyalkylcyanoacrylates (PACA), such as, for example, polybutylcyanoacrylate (PBCA), polyesters, for example poly (DL-lactides), poly (L-lactides), polyglycolides, polydioxanones, Polyoxazolines, poly (glycolide-co-trimethylene carbonates), polylactide-co-glycolides (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-dioxanone),
Alginic acid, hyaluronic acid, polysialic acid, acidic cellulose derivatives, acidic starch derivatives,
Polysaccharides such as dextrans, alginates, cyclodextrins, hyaluronic acid, chitosans,
acidic polyamino acids, polymeric proteins such as collagen, gelatin or albumin,
Polyamides such as poly (aspartic acid), poly (glutamic acid),
Polylysine, poly (iminocarbonates) (poly (carbonates) derived from tyrosine, poly (β-hydroxybutyrate),
Polyanhydrides such as polysebacidic acid (poly (SA)),
Poly (adipic acid), poly (CPP-SA), poly (CPH), polyCPM) aromatic polyanhydrides,
polyorthoesters,
Polycaprolactones such as poly-ε- or γ-caprolactones, polyphosphoric acid such as polyphosphates, polyphosphonates, polyphosphazenes, polyamide-enamines), azopolymers, polyurethanes, dendrimers, Pseudopolymaminosäuren as well as all mixtures and co-polymers of the same compounds.

In a preferred embodiment, the sparingly water-soluble polymer is selected from the group of the following polymers:
Polyacrylates, polylactides and Polygylcolide, or their co-polymers.

In In a particularly preferred embodiment, this is difficult water-soluble polymer from the group of polyalkylcyanoacrylates (PACA).

Formel (I): Strukturformel PACA, n = 5–20000, bevorzugt n = 5–6000, bzw. n = 5–100The structure of these polyalkylcyanoacrylates shows the structure indicated (formula I), wherein the radical R given is preferably a linear and branched alkyl group having 1 to 16 carbon atoms, a cyclohexyl, benzyl or a phenyl group.

Formula (I): Structural formula PACA, n = 5-20,000, preferably n = 5-6000, or n = 5-100

Formel II: Strukturformel PBCA; n = 5–20000 bevorzugt n = 5–6000, bzw. n = 5–100 In a further particularly preferred embodiment, the sparingly water-soluble polymer is a polybutyl cyanoacrylate (PBCA) (formula II).

Formula II: Structural Formula PBCA; n = 5-20,000, preferably n = 5-6000, or n = 5-100

The poorly water-soluble polymer forms in the context of the invention the larger proportion of the polymer matrix of the particles.

Surprisingly could be found that by incorporating compounds with Amino groups, especially a cationic polymer, into one heavy water-soluble, solid polymer matrix nanoparticles with a cationically charged surface potential (zeta potential) be generated.

In In one embodiment, the cationic polymer is derived from the group of natural and / or synthetic polymers or from homo- and co-polymers of corresponding monomers.

When Cationic polymers are suitable for the purposes of this invention polymers with free primary, secondary or tertiary Amino groups with any low molecular weight acids Salts can form, the salts in aqueous-organic Solvents are soluble. Also suitable Polymers or their salts, the quaternary ammonium groups and soluble in organic solvents are.

In a preferred embodiment are the following groups cationic polymers, polycations and polyamine compounds or Polymers of homo- and co-polymers of corresponding monomers especially suitable: modified natural cationic polymers, cationic proteins, synthetic cationic polymers, aminoalkanes different chain length, modified cationic Dextrans, cationic polysaccharides, cationic starch or cellulose derivatives, chitosans, guar derivatives, cationic cyanoacrylates, Methacrylates and methacrylamides and such monomers and comonomers which are used to form appropriate appropriate compounds can and the corresponding salts, which are suitable with inorganic or low molecular weight organic acids can form.

To include in particular: diethylaminoethyl-modified dextrans, Hydroxymethylcellulosetrimethylamine, polylysine, protamine sulfate, Hydroxyethylcellulosetrimethylamine, polyallylamines, protamine chloride, Polyallylamine hydrosalts, polyamines, polyvinylbenzyltrimethylammonium salts, Polydiallyldimethylammonium salts, polyimidazoline, polyvinylamine and polyvinylpyridine, polyethyleneimine (PEI), putrescine (butane-1,4-diamine), Spermidine (N- (3-aminopropyl) butane-1,4-diamine), spermine (N, N'-bis (3-aminopropyl) butane-1,4-diamine) Dimethylaminoethyl acrylate, poly-N, N-dimethylaminoethyl methacrylate = P (DMEAMA), dimethylaminopropylacrylamide, dimethylaminopropylmethacrylamide, Dimethylaminostyrene, vinylpyridine and methyldiallylamine, poly-DADMAC, guar, deacetylated chitin and the corresponding salts which are with suitable inorganic or low molecular weight organic Sowing can form (Claim 5). Suitable acids for salt formation are z. B.: Hydrochloric acid, sulfuric acid, but especially acetic acid, glycolic acid or lactic acid.

In In one embodiment, the amino group-bearing compound, in particular a cationic polymer, in an organic solvent, which is infinitely miscible with water, preferably acetone, Methanol, ethanol, propanol, Dimetylsulfoxid (DMSO), or in one Mixture of these solvents dissolved with water become.

Formel (III): Strukturformel P(DMAEMA), n = 5–20000, bevorzugt n = 5–6000, bzw. n = 5–100 oder P(DMAPMAM) = Poly(N,N-Dimethylaminopropylmethacrylamid)

In a preferred embodiment, the polymer nanoparticles contain, as the amino group-bearing compound, a cationically modified polyacrylate (poly-N, N-dimethylaminoethyl methacrylate, P (DMAEMA)) (Formula 3).

Formula (III): Structural formula P (DMAEMA), n = 5-20,000, preferably n = 5-6000, or n = 5-100 or P (DMAPMAM) = poly (N, N-dimethylaminopropylmethacrylamide)

The biodegradable, cationically modified polyacrylate, e.g. B. P (DMAEMA), P (DMAPMAM) is incorporated into the polymer matrix, in particular the PBCA polymer matrix, encapsulated by nanoprecipitation. The surface of the resulting nanoparticles is due to the amino groups of the cationic polymer have a positive (cationic) Surface potential (zeta potential). The cationic Particle surface ensures good cell uptake and allows a flexible electrostatic surface modification with partially anionically charged compounds.

In In another preferred embodiment, the Polymer nanoparticles as cationic polymer a modified polyacrylate Poly (dimethylaminopropylmethacrylamide (P (DMAPMAM)).

In In another embodiment, the polymer nanoparticles contain as a cationic polymer polyethyleneimine (PEI) of different molecular weights, in particular 1.8 kDa, 10 kDa, 70 kDa and 750 kDa, (formula 4).

Formel (IV): Allgemeine Strukturformel für verzweigtes Polyethylenimin, wobei x, y und z = 10–50%, bevorzugt x, y und z = 20–40% sind und in die Geamtheit der Anteile 100% ergibt PEI is a polycation frequently used in the field of non-viral gene therapy for DNA polyplexes (PEK) and accordingly much studied [ Remy J. -S. et al., Adv. Drug Deliv. Rev., 1998; 30 (1-3): 85-95 ].

Formula (IV): General structural formula for branched polyethylenimine, wherein x, y and z = 10-50%, preferably x, y and z = 20-40%, giving 100% in the entirety of the proportions

by virtue of the encapsulation of the cationic polyelectrolyte PEI in the PBCA polymer matrix the particle shell consists of PEI polymer chains, which create a cationic surface potential.

additionally can according to the invention in addition to the previously mentioned cationic polymers or compounds with amino groups diagnostic but also therapeutic substances in the polymer matrix be encapsulated by nanoprecipitation.

When Diagnostic substances for encapsulation may be the following Substance classes for different molecular imaging methods In particular, here are contrast agents or Tracer for the following method for molecular imaging (Molecular Imaging): optical imaging (optical Imaging), such. DOT (diffuse optical imaging), US (ultrasound imaging), OPT (optical projection tomography), near-infrared fluorescence imaging, Fluorescence protein imaging and BLI (bioluminescence imaging) and magnetic resonance imaging (MRI, MRI) or X-ray imaging (X-ray). But there are also other methods conceivable. The encapsulation a suitable diagnostic substance from named substance groups allows detection of the particles in vitro and / or in vivo.

In a preferred embodiment, the diagnostic agent is a dye, in particular selected from the following group: fluorescein, fluorescein isothiocyanate, carboxyfluorescein or calcein, tetrabromfluoresceins or eosines, tertaiodefluorescein or erythrosines, difluorofluorescein, such as Oregon Green ^™ 488, Oregon Green ^™ 500 or Oregon Green ^™ 514, Carboxyrhodol (Rhodol Green ^™ ) dyes ( US 5,227,487 ; US 5,442,045 ), Carboxyrodamine dyes (Rhodamine Green ^™ Dyes) ( US 5,366,860 ), 4,4-difluoro-4-bora-3a, 4a-diaza-indacenes, such as. Dodipy FL, Bodipy 493/503 or Bodipy 530/550 and derivatives thereof ( US 4,774,339 ; US 5,187,288 ; US 5,248,782 ; US 5,433,896 ; US 5,451,663 ), Polymethine dyes, coumarin dyes such. As coumarin 6, 7-amino-4-methylcoumarin, metal complexes of DTPA or Tetraazamacrozyclen (Cyclen, Pyvlen) with terbium or europium or Tetrapyrrolfarbstoffe, especially porphyrins.

In one embodiment is the diagnostic Substance about a fluorescent dye.

In a further embodiment is in the diagnostic agent to a fluorescent near-infrared (NIR) dye.

These 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 (DOTC) WO 96/17628 ), Rhodamine dyes, phenoxazine or phenothiazine dyes, tetrapyrrole dyes, especially benzoporphyrins, chorine and phthalocyanines.

The Above mentioned dyes can be used either as acids or used as salts. 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 under such as primary, secondary or tertiary Amines, such as ethanolamine, diethanolamine, morpholine, glucamine, N, N-dimethylglucamine and especially N-methylglucamine and polyethyleneimine. Suitable cations of amino acids are, for example those of lysine, arginine and ornithine, as well as amides otherwise acidic or neutral amino acids.

As well The dyes can be used as bases or as Salts of these are used.

wobei Q ein Fragment

wobei
R³⁰ für ein Wasserstoffatom, eine Hydroxygruppe, eine Carboxygruppe, einen Alkoxyrest mit 1 bis 4 Kohlenstoffatomen oder ein Chloratom steht,
R³¹ für ein Wasserstoffatom oder einen Alkylrest mit 1 bis 4 Kohlenstoffatomen steht,
X und Y unabhängig voneinander für ein Fragment -O-, -S-, -CH=CH- oder -C(CH₂R³²)(CH₂R³³)- stehen,
R²⁰ bis R²⁹, R³² und R³³ unabhängig voneinander für ein Wasserstoffatom, eine Hydroxygruppe, einen Carboxy-, einen Sulfonsäure-Rest oder einen Carboxyalkyl –, Alkoxycarbonyl- oder Alkoxyoxoalkyl- Rest mit bis zu 10 C-Atomen oder ein Sulfoalkylrest mit bis zu 4 C-Atomen, oder für ein nicht spezifisch bindendes Makromolekül, oder für einen Rest der allgemeinen Formel VI -(O)v-(CH2)o-CO-NR34-(CH2)s-(NH-CO)q-R35 (VI)steht,
mit der Massgabe, dass bei der Bedeutung von X und Y gleich O, S, -CH=CH- oder -C(CH₃)₂- mindestens einer der Reste R²⁰ bis R²⁹ einem nicht spezifisch bindenden Makromolekül oder der allgemeinen Formel VI entspricht,
wobei
o und s gleich 0 sind oder unabhängig voneinander für eine ganze Zahl von 1 bis 6 stehen,
q und v unabhängig voneinander für 0 oder 1 stehen,
R³⁴ ein Wasserstoffatom oder einen Methylrest darstellt,
R³⁵ ein Alkylrest mit 3 bis 6 C-Atomen, welcher 2 bis n-1 Hydroxygruppen aufweist, wobei n die Anzahl der C-Atome ist, oder ein mit 1 bis 3 Carboxygruppen substituierter Alkylrest mit 1 bis 6 C-Atomen, Arylrest mit 6 bis 9 C-Atomen oder Arylalkylrest mit 7 bis 15 C-Atomen, oder ein Rest der allgemeinen Formel IIId oder IIIe

ist, unter der Massgabe, dass q für 1 steht,
oder ein nicht spezifisch bindendes Makromolekül bedeutet,
R²⁰ und R²¹, R²¹ und R²², R²² und R²³, R²⁴ und R²⁵, R²⁵ und R²⁶, R²⁶ und R²⁷ zusammen mit den zwischen ihnen liegenden Kohlenstoffatomen einen 5- oder 6-gliedrigen aromatischen oder gesättigten annelierten Ring bilden, sowie deren physiologisch verträgliche Salze.In a most preferred embodiment, the diagnostic substance is a carbocyanine dye. The general structure of the carbocyanines is described as follows (Formula V).

where Q is a fragment

in which
R ^{30 represents} a hydrogen atom, a hydroxy group, a carboxy group, an alkoxy radical having 1 to 4 carbon atoms or a chlorine atom,
R ^{31 is} a hydrogen atom or an alkyl radical having 1 to 4 carbon atoms,
X and Y independently of one another represent a fragment -O-, -S-, -CH = CH- or -C (CH ₂ R ³² ) (CH ₂ R ³³ ) -,
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 - (O) v- (CH 2 ) O-CO-NR 34 - (CH2) s- (NH-CO) q 35 (VI) stands,
with the proviso that in the meaning of X and Y is O, S, -CH = CH- or -C (CH ₃ ) ₂ - at least one of R ²⁰ to R ^{29 is} a non-specifically binding macromolecule or the general formula VI corresponds,
in which
o and s are 0 or independently of one another are 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 hydroxy groups, where n is the number of Is C atoms, or an alkyl radical having 1 to 6 C atoms substituted with 1 to 3 carboxy groups, aryl radical having 6 to 9 C atoms or arylalkyl radical having 7 to 15 C atoms, or a radical of the general formula IIId or IIIe

is, with the proviso that q stands for 1,
or a non-specific binding macromolecule means
R ²⁰ and R ²¹ , R ²¹ and R ²² , R ²² and R ²³ , R ²⁴ and R ²⁵ , R ²⁵ and R ²⁶ , R ²⁶ and R ²⁷ together with the carbon atoms between them a 5- or 6-membered aromatic or form a saturated fused ring, and their physiologically acceptable salts.

Formula 8: General structure of carbocyanines

In the case of carbocyanines, reference is made to the applications DE 44 45 065 and DE 69 91 1034 taken, the content of which should be the subject of this application.

unexpectedly can be anionic, readily water-soluble substances how certain carbocyanins in the hydrophobic polymer matrix described Stably included nanoparticles.

in the According to the invention is an anionic water-soluble Substance by ion complex formation and co-precipitation with a cationic polymer in a slightly water-soluble Polymer matrix encapsulated by nanoprecipitation, wherein Particles of defined size arise.

By the incorporation of an NIR-active fluorescent dye in the polymer matrix The particles are non-invasive by means of optical imaging Tissue detectable via fluorescence. It exists with it in vivo the possibility of distribution or enrichment fluorescently labeled nanoparticles to detect.

Formel (VIII): Tetrasulfocyanin/TSC In a particularly preferred embodiment, the carbocyanine dye is the readily water-soluble anionic tetrasulfocyanine (TSC) (formula VIII).

Formula (VIII): Tetrasulfocyanine / TSC

Formel (IX): Indodicarbocyanin/IDCC In another preferred embodiment, the carbocyanine dye is IDCC (indodicarbocyanine) (Formula IX).

Formula (IX): Indodicarbocyanine / IDCC

Formel (X): Indocyaningrün (ICG) In another preferred embodiment, the carbocyanine dye is ICG (indocyanine green) (Formula 11).

Formula (X): Indocyanine Green (ICG)

In the embodiment of the invention it is the encapsulated pharmaceutically active substance around an epothilone.

worin
R^1a, R^1b unabhängig voneinander Wasserstoff, C₁-C₁₀-Alkyl, Aryl, Aralkyl, oder zusammen eine Gruppe -(CH₂)_m
wobei m 2 bis 5 bedeutet;
R^2a, R^2b unabhängig voneinander Wasserstoff, C₁-C₁₀-Alkyl, C₂-C₁₀-Alkenyl C₂-C₁₀-Alkinyl, Aryl, Aralkyl, oder zusammen eine Gruppe -(CH₂)_n- wobei n 2 bis 5 bedeutet,
R³ Wasserstoff, C₁-C₁₀ Alkyl, Aryl, Aralkyl;
R^4a, R^4b unabhängig voneinander Wasserstoff, C₁-C₁₀-Alkyl, Aryl, Aralkyl, oder zusammen eine Gruppe -(CH₂)_p-
wobei p 2 to 5 bedeutet;
R⁵ Wasserstoff, C₁-C₁₀-Alkyl, Aryl, Aralkyl, CO₂H, CO₂Alkyl, CH₂OH, CH₂O-C₁-C₅-Alkyl, CH₂OAcyl, CN, CH₂NH₂, CH₂N((C₁-C₅-Alkyl), Acyl)_1,2, or CH₂Hal, CHal₃;
R⁶, R⁷ unabhängig voneinander Wasserstoff, oder zusammen eine weitere Bindung oder eine Epoxid Funktion;
G O oder CH₂;
D–E zusammen die Gruppe -H₂C-CH₂-, -HC=CH-, -C≡C-, -CH(OH)-CH(OH)-, -CH(OH)-CH₂-, -CH₂-CH(OH)-, -CH₂-O-, -O-CH₂- or

wobei wenn G Sauerstoff bedeutet, D-E nicht CH₂-O sein kann; oder
D-E-G zusammen die Gruppe H₂C-CH=CH
W die Gruppe C(=X)R⁸, oder ein bi- or trizyklischer aromatischer oder heteroaromatischer Rest;
X O oder die Gruppe CR⁹R¹⁰;
R⁸ Wasserstoff, C₁-C₁₀ Alkyl, Aryl, Aralkyl, Halogen, CN;
R⁹, R¹⁰ unabhängig voneinander Wasserstoff, C₁-C₂₀-Alkyl, Aryl, Aralkyl, oder zusammen mit dem Methylenkohlenstoffatom einen 5- bis 7-gliedrigen carbozyklischen Ring;
Z O oder Wasserstoff und die Gruppe OR¹¹;
R¹¹ Wasserstoff der eine Schutzgruppe PG^Z;
A-Y eine Gruppe O-C(=O), O-CH₂, CH₂-C(=O), NR¹²-C(=O), NR¹²-SO₂;
R¹² Wasserstoff, oder C₁-C₁₀ Alkyl;
PG^Z C₁-C₂₀ Alkyl, eine C₄-C₇ Cycloalkylgruppe, die ein oder mehrere Sauerstoffatome im Ring enthalten kann, Aryl, Aralkyl, C₁-C₂₀ Acyl, Aroyl, C₁-C₂₀ Alkylsulfonyl, Arylsulfonyl, Tri(C₁-C₂₀alkyl)silyl, Di(C₁-C₂₀alkyl)Arylsilyl, (C₁-C₂₀Alkyl)diarylsilyl, or Tri(aralkyl)silyl;
als einzelnes Stereoisomer oder als Mischung aus verschiedenen Stereoisomeren und/oder als pharmazeutisch akzeptables Salz verkapselt ist.In further embodiments of the present invention, the epothilone is defined by the general formula (XI)

wherein
R ^1a , R ^1b independently of one another are hydrogen, C ₁ -C ₁₀ -alkyl, aryl, aralkyl, or together a group - (CH ₂ ) _m
where m is 2 to 5;
R ^2a , R ^2b independently of one another are hydrogen, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl C ₂ -C ₁₀ -alkynyl, aryl, aralkyl, or together a group - (CH ₂ ) _n - where n is 2 to 5 means
R ^{3 is} hydrogen, C ₁ -C ₁₀ alkyl, aryl, aralkyl;
R ^4a , R ^4b independently of one another are hydrogen, C ₁ -C ₁₀ -alkyl, aryl, aralkyl, or together a group - (CH ₂ ) _p -
where p is 2 to 5;
R ^{5 is} hydrogen, C ₁ -C ₁₀ alkyl, aryl, aralkyl, CO ₂ H, CO ₂ alkyl, CH ₂ OH, CH ₂ OC ₁ -C ₅ alkyl, CH ₂ O acyl, CN, CH ₂ NH ₂ , CH ₂ N ((C ₁ -C ₅ alkyl), acyl) _1,2, or CH ₂ Hal, CHal _3;
R ⁶ , R ^{7 are} independently hydrogen, or together another bond or an epoxide function;
G is O or CH _2;
D-E together form the group -H ₂ C-CH ₂ -, -HC = CH-, -C≡C-, -CH (OH) -CH (OH) -, -CH (OH) -CH ₂ -, - CH ₂ -CH (OH) -, -CH ₂ -O-, -O-CH ₂ - or

where G is oxygen, DE can not be CH ₂ -O; or
DEG together the group H ₂ C-CH = CH
W is the group C (= X) R ⁸ , or a bi- or tricyclic aromatic or heteroaromatic radical;
XO or the group CR ⁹ R ¹⁰ ;
R ^{8 is} hydrogen, C ₁ -C ₁₀ alkyl, aryl, aralkyl, halogen, CN;
R ⁹ , R ¹⁰ independently of one another are hydrogen, C ₁ -C ₂₀ -alkyl, aryl, aralkyl or together with the methylene carbon atom a 5- to 7-membered carbocyclic ring;
ZO or hydrogen and the group OR ¹¹ ;
R ^{11 is} hydrogen of a protective group PG ^Z ;
AY is a group OC (= O), O-CH ₂ , CH ₂ -C (= O), NR ¹² -C (= O), NR ¹² -SO ₂ ;
R ^{12 is} hydrogen, or C ₁ -C ₁₀ alkyl;
PG ^Z C ₁ -C ₂₀ alkyl, a C ₄ -C ₇ cycloalkyl group which may contain one or more oxygen atoms in the ring, aryl, aralkyl, C ₁ -C ₂₀ acyl, aroyl, C ₁ -C ₂₀ alkylsulfonyl, arylsulfonyl, tri (C ₁ -C ₂₀ alkyl) silyl, di (C ₁ -C ₂₀ alkyl) arylsilyl, (C ₁ -C ₂₀ alkyl) diarylsilyl, or tri (aralkyl) silyl;
is encapsulated as a single stereoisomer or as a mixture of different stereoisomers and / or as a pharmaceutically acceptable salt.

For another embodiment of the present invention, the epothilone is defined by the formula (XI)
wherein
R ^1a , R ^1b independently of one another are hydrogen, C ₁ -C ₄ -alkyl, or together the group - (CH ₂ ) _m - where m is 2 to 5;
R ^2a , R ^2b independently of one another are hydrogen, C ₁ -C ₅ -alkyl, or together form the group - (CH ₂ ) _m - where m is 2 to 5, or C ₂ -C ₆ -alkenyl, or C ₂ -C ₆ alkynyl;
R ^{3 is} hydrogen,
R ^4a , R ^4b independently of one another are hydrogen, C ₁ -C ₄ -alkyl,
R ^{5 is} hydrogen, C ₁ -C ₄ -alkyl, C (Hal) ₃
R ⁶ , R ^{7 are} both hydrogen, or together another bond, or together an epoxide function,
G CH ₂ ;
DE is the group H ₂ C-CH ₂ , or
DEG together the group H ₂ C-CH = CH
W is the group C (= X) R ⁸ , or a bi- or tricyclic aromatic or heteroaromatic radical;
X is the group CR ⁹ R ¹⁰ ;
R ^{8 is} hydrogen, C ₁ -C ₄ -alkyl, halogen,
R ⁹ , R ¹⁰ are each, independently of one another, hydrogen, C ₁ -C ₄ -alkyl, aryl, aralkyl,
Z oxygen
AY is the group OC (= O) or the group NR ¹² -C (= O)
R ^{12 is} hydrogen, or C ₁ -C ₄ alkyl;
encapsulated as a single stereoisomer or as a mixture of different stereoisomers and / or as pharmaceutically acceptable salts.

In a further embodiment, epothilones of the general formula (XI), in which
R ^1a , R ^1b are each independently hydrogen, are each independently hydrogen, C ₁ -C ₂ alkyl, or together form a group - (CH ₂ ) _m - where m is 2 to 5,
R ^2a , R ^2b are each independently hydrogen, C ₁ -C ₅ alkyl, or together the group a - (CH ₂ ) _n - wherein n is 2 to 5, or C ₂ -C ₆ alkenyl, or C ₂ -C ₆ alkynyl;
R ^{3 is} hydrogen,
R ^4a , R ^{4b are} both independently hydrogen, C ₁ -C ₂ alkyl,
R ^{5 is} hydrogen or methyl or trifluoromethyl,
R ⁶ , R ⁷ together form another bond, or together an epoxide function;
G CH ₂ ;
DE is the group H ₂ C-CH ₂ ,
DEG together the group H ₂ C-CH = CH
W The group C (= X) R ⁸ , or thiazolyl, oxazolyl, pyridyl, N-oxo-pyridyl; Benzothiazolyl, benzoxazolyl, benzimidazolyl, which may be optionally substituted by C ₁ -C ₃ alkyl, C ₁ -C ₃ hydroxyalkyl, C ₁ -C ₃ aminoalkyl, C ₁ -C ₃ alkylsulfonyl
X is the group CR ⁹ R ¹⁰ ;
R ^{8 is} hydrogen, methyl, chlorine, fluorine,
R ⁹ , R ¹⁰ are each, independently of one another, hydrogen, C ₁ -C ₄ -alkyl, thiazolyl, oxazolyl, pyridyl, N-oxo-pyridyl, which may optionally be substituted by C ₁ -C ₃ -alkyl, C ₁ -C ₃ - Hydroxyalkyl, aralkyl,
Z oxygen
AY is the group OC (= O), or the group NR ¹² -C (= O)
R ^{12 is} hydrogen or C ₁ -C ₄ -alkyl;
encapsulated as a single stereoisomer or as a mixture of different stereoisomers and / or as a pharmaceutically acceptable salt.

In another embodiment, epothilones of general formula (XI) wherein AY is OC (= O); DE is H ₂ C-CH ₂ ; G CH ₂ ; ZO; R ^1a , R ₁ ^{b is} C ₁ -C ₁₀ -alkyl or together a - (CH ₂ ) _p - group, wherein p is 2 to 3; R ^2a , R ^2b independently of one another are hydrogen, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl, or C ₂ -C ₁₀ -alkynyl; R ^{3 is} hydrogen; R ^4a , R ^{4b are} independently hydrogen or C ₁ -C ₁₀ alkyl and R ^{5 is} C ₁ -C ₁₀ alkyl encapsulated.

In another embodiment, epothilones of general formula (XI) wherein R ^2a , R ^{2b are} independently hydrogen, C ₂ -C ₁₀ alkenyl or C ₂ -C ₁₀ alkynyl; R ⁶ , R ⁷ together represents an epoxide function and W represents a 2-methylbenzothiazol-5-yl group or a 2-methylbenzoxazol-5-yl group or a quinoline-7-yl group.

In a preferred embodiment, epothilones of general formula (XI) are encapsulated, which are listed in the following list:
(4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (2-methyl-benzoxazol-5-yl) -1-oxa-5,5,9,13-tetramethyl- 7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione;
(1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzoxazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione;
(4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-oxa-5,5,9,13-tetramethyl- 7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione;
(1S / R, 3S, 7S, 10R, 11S, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione; (1S, 3S, 7S, 10R, 11S, 12S, 16R) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5-yl) - 8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione;
(1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecanes-5,9-dione;
(4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-oxa-9,13-dimethyl-5,5- (1,3-trimethylene) -7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione;
(1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -12.16-dimethyl-8,8- (1,3-trimethylene) -4.17-dioxabicyclo [14.1.0] heptadecane-5,9-dione;
(4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-oxa-5,5,9,13-tetramethyl- 7- (prop-2-in-1-yl) -cyclohexadec-13-ene-2,6-dione;
(1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-in-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione;
(4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-dihydroxy-16- (quinolin-7-yl) -1-oxa-5,5,9,13-tetramethyl-7- (prop -2-en-1-yl) -cyclohexadec-13-ene-2,6-dione;
(1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (quinolin-7-yl) - 8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione;
(4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (1,2-dimethyl-1H-benzoimidazol-5-yl) -1-oxa-5,5,9, 13-tetramethyl-7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione;
(1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-ihydroxy-10- (prop-2-en-1-yl) -3- (1,2-dimethyl-1H -benzoimidazol-5-yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione;
(4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-aza-5,5,9,13-tetramethyl- 7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione;
(1S / R, 3S, 7S, 10R, 11S, 12S, 16R / S) -7,11-dihydroxy-L0 (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4-aza-17-oxabicyclo [14.1.0] heptadecane-5,9-dione,
(1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4-aza-17-oxabicyclo [14.1.0] heptadecane-5,9-dione,
(1S, 3S (E), 7S, 10R, 11S, 12S, 16R) -7,11-dihydroxy-8,8,10,12,16-pentamethyl-3- [1- (2-methyl-1,3 -thiazol-4-yl) prop-1-en-2-yl] -4.17-dioxabicyclo [14.1.0] heptadecane-5,9-dione,
(1S, 3S (E), 7S, 10R, 11S, 12S, 16R) -7,11-dihydroxy-8,8,10,12,16-pentamethyl-3- [1- (2-methyl-1,3 -thiazol-4-yl) prop-1-en-2-yl] -17-oxa-4-azabicyclo [14.1.0] heptadecane-5,9-dione,
(4S, 7R, 8S, 9S, 13Z, 16S (E)) - 4,8-dihydroxy-5,5,7,9,13-pentamethyl-16- [1- (2-methyl-1,3-thiazol -4-yl) prop-1-en-2-yl] -oxacyclohexadec-13-ene-2,6-dione,
(4S, 7R, 8S, 9S, 10E, 13Z, 16S (E)) - 4,8-dihydroxy-5,5,7,9,13-pentamethyl-16- [1- (2-methyl-1,3 -thiazol-4-yl) prop-1-en-2-yl] -oxacyclohexadec-10,13-diene-2,6-dione,
(4S, 7R, 8S, 9S, 10E, 13Z, 16S (E)) - 4,8-dihydroxy-5,5,7,9-tetramethyl-13-trifluoromethyl-16- [1- (2-methyl-1 , 3-thiazol-4-yl) prop-1-en-2-yl] oxacyclohexadec-10,13-diene-2,6-dione,
(1S, 3S (E), 7S, 10R, 11S, 12S, 16R) -7,11-dihydroxy-8,8,10,12,16-pentamethyl-3- [1- (2-methylsulfanyl-1,3 -thiazol-4-yl) prop-1-en-2-yl] -4.17-dioxabicyclo [14.1.0] heptadecane-5,9-dione
as a single stereoisomer or as a mixture of different stereoisomers and / or pharmaceutically acceptable salt

Particularly preferred are the epothilones
(4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-oxa-5,5,9,13-tetramethyl- 7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione;
(1S / R, 3S, 7S, 10R, 11S, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione as a single stereoisomer or as a mixture of different stereoisomers and / or pharmaceutically acceptable salt.

All particular preference is given to (1S, 3S, 7S, 10R, 11S, 12S, 16R) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methylbenzothiazole-5- yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecanes-5,9-dione. as a single stereoisomer or as a mixture of different stereoisomers and / or pharmaceutically acceptable salt.

In In another embodiment, the diagnostic agent and including the epothilone together in the particle.

In Another embodiment is the diagnostic and the epothilone in separate same nanoparticulate Systems, in particular, have the same structure.

The Invention thus also relates to a kit consisting of the inventive Particles containing a diagnostic agent and an epothilone in common and a kit comprising (a) the particles of the invention containing a diagnostic agent and (b) the inventive Contains particles containing an epothilone separately, as well as the use of the kit for therapy and diagnosis / monitoring.

• • Direkte Fällung (Präzipitation) in einem Reagenzglas durch Einbringen des gelösten Polymer-Substanz-Gemisches in eine tensidhaltige wässrige Lösung, welche mittels Magnetrührer durchmischt wird.
• • Fällung des Polymer-Substanz-Gemisches in der tensidhaltigen wässrigen Lösung durch Zusammenführung der beiden Lösungen mittels eines Micro-Mischer-Systems.
• • Verwendung von Ultraschall zur gleichmäßigen Verteilung des Polymer-Substanz-Gemisches in der tensidhaltigen wässrigen Lösung.

In a preferred embodiment, the polymer nanoparticles are precipitation aggregates which are produced by nanoprecipitation. In particular, the following production methods are available for this purpose:

• Direct precipitation (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.
• • Precipitation of the polymer-substance mixture in the surfactant-containing aqueous solution by combining the two solutions by means of a micro-mixer system.
• Use of ultrasound for uniform distribution of the polymer-substance mixture in the surfactant-containing aqueous solution.

at the production of nanoparticles by nanoprecipitation The organic solvent is abruptly the matrix polymer and removed the substances dissolved with it, if the polymer-containing organic solution in a much larger Volume of an aqueous solution is given. Surprisingly become dissolved in the polymer phase compounds with amino groups (water-soluble as well as water-insoluble) in the sparingly soluble polymer during the precipitation coverkapselt. Condition for this is the unlimited miscibility of the organic solvent (particularly suitable z. As acetone, ethanol) with water and the insolubility of the matrix polymer in the aqueous phase.

In an embodiment of the invention Particles, the diagnostic agent is negatively charged and as an ion pair enclosed with the cationic polymer in the particle.

In a preferred embodiment for all the preceding Polymer nanoparticles is the surface of the polymer nanoparticles electrostatically modified.

The electrostatic modification of the cationic nanoparticle surface is an outstanding advantage of the present invention. On the Basis of ionic interactions can be without a chemical coupling reaction modified the particle surface with a suitable substance become. The prerequisite for this is that the modifying Agent partially has charges, which opposite the particle surface charge are. This allows this Method (electrostatic surface modification by means of Charge titration) a simple, flexible and versatile modification the particle surface.

additionally is it possible to have unstable active ingredients on the particle surface to adsorb, which thereby protected from degradation by enzymes are and therefore a higher therapeutic Effect.

The Enrichment (active and passive targeting) of nanoparticles From the bloodstream into the target tissue requires that the particles circulate in the bloodstream for a sufficient period of time. According to the invention by means of the surface modification described above Circulation time in the body can be adjusted individually in particular by using polyethylene oxides or polythylene glycols (see Example 5).

One Another outstanding advantage is that the one described here electrostatic surface modification directly in front of Application can be carried out quickly and without problems can. This is done by simply mixing appropriate amounts nanoparticle dispersion with the modifying agent.

In order to There is also the option of removing the core particles separately from the surface-modifying agent and store. This is for a particularly advantageous for the colloidal long-term stability. For another extremely labile surface-modifying substances such as Peptides or antibodies under suitable conditions until for use.

The Separation of core particles and modifying agent allows furthermore a surface modification according to individual requirements in the patient. The surface modification according to a modular principle offers maximum flexibility for diagnosis, therapy and monitoring, with the uncomplicated Implementation of the modification directly by the user he follows.

One preferred construction of the surface-modifying agent for cationically functionalized polymer nanoparticles, in particular The described PBCA nanoparticles are shown in formula 5. The partially anionically charged moiety satisfies the function of an anchor on the positively charged particle surface electrostatic interactions. The neutral, to the surrounding aqueous medium aligned molecule part consists from polyethylene glycol and / or polyethylene oxide units (PEG units) different length. Preferred here are PEG chains with a molecular weight of 100 to 30,000 daltons and especially preferably with 3000 to 5000 daltons. This part of the molecule may alternatively also from other suitable structures, such. B. Hydroxyethyl starch (HES) and all of them possible polymeric compounds exist. The rest R is preferably hydrogen or a methyl unit.

Formula 5: General structural formula of a surface-modifying agent (R = hydrogen, C ₁ -C ₃ -alkyl or an uncharged amino acid, preferably H, CH ₃ , wherein mixtures of R in a polymer are also included), n = 5-700 , preferably n = 5-200

The anionic anchor may be, for example, a polymer of 5 to 50 units of glutamic acid (Glu) or aspartic acid (Asp) or the salts of the acids. It may also be a mixed polymer of the named subunits. Furthermore, uncharged subunits such. B. neutral amino acids in the anionic block (= remainder R) regularly or irregularly incorporated. Generally, as a negatively charged moiety (anchor) are compounds or polymeric structures having groups such as acetate, carbonate, citrate, succinate, nitrate, carboxylate, phosphate, sulfonate or sulfate groups, as well as Sal ze and free acids of these groups.

In In one embodiment, the anionic anchor may preferably be a polyamino acid.

In In one embodiment, the surface is Glu (10) -b-PEG (110). Glu (10) -b-PEG (114) or Asp (15) -b-PEG (114).

In In one embodiment, the surface is Glu (10) -b-PEG (110) or Asp (15) -b-PEG (114).

In a particularly preferred embodiment is the surface of the polymer nanoparticle is modified with Glu (10) -b-PEG (110) (Formula 6). As a negative part of the molecule (anchor) serve the carboxylate groups of Glutamic acid subunits of the block copolymer.

Formel 6: Strukturformel von Glu(10)-b-PEG(110);

Formula 6: Structural Formula of Glu (10) -b-PEG (110);

In In another embodiment, a target is recognizable Structure available.

These Target recognizing structure has at least one negatively charged one Part of the molecule caused by electrostatic interactions is applied to the cationic particle surface.

One Another particularly preferred structure of the surface-modifying Agent for cationically functionalized polymer nanoparticles, in particular the described PBCA nanoparticles, is in formula 7 is shown.

Formel 7: Allgemeine Strukturformel eines oberflächenmodifizierenden Agens, n = 5–700, bevorzugt n = 5–200, wobei X eine mögliche Ziel erkennende Struktur und der anionische Anker ein Polymerblock sein kann. Der anionische Anker kann entsprechend der zuvor benannten Beschreibung von Formel 5 aufgebaut sein.

Formula 7: General structural formula of a surface modifying agent, n = 5-700, preferably n = 5-200, wherein X may be a possible target recognizing structure and the anionic anchor may be a polymer block. The anionic anchor may be constructed according to the above-described description of Formula 5.

Of the partially fulfilled anionic charged moiety 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 100 to 30000 daltons, and more preferably from 3000 to 5000 daltons. This moiety may alternatively be made of other suitable ones Structures, such. As hydroxyethyl starch (HES) and all from this possible polymeric compounds exist.

Of the Ligand X of the surface-modifying agent, hereinafter also called target cognitive structure, serves to improve passive and active enrichment mechanisms of polymer nanoparticles.

Suitable ligands X as targeting structures may be antibodies, peptides, receptor ligands of ligand mimetics or an aptamer. Structures are 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 a DNA or an RNA molecule containing native, modified or artificial nucleosides, nucleic acids, oligocucleotides, polysaccharides, B, A, Z helix or hairpin structure (hairpin), a chemical moiety, modified polysaccharides as well as receptor binding substances or fragments thereof. Target-recognizing structures may also be transferrin or folic acid or parts thereof, or all possible combinations of the foregoing.

These Ligands are according to the invention electrostatic interactions bound to the nanoparticles, It is also possible, however, for the ligands to be covalent Bind bonds to the particle surface. Farther it is possible to have a linker between ligand and nanoparticles install.

The Electrostatic deposition of the target-recognizing structures takes place via Charge interactions with at least one negatively charged moiety to the cationic particle surface. As negatively charged Part of the molecule (anchor) are compounds or polymers Structures with groups such as acetate, carbonate, citrate, succinate, Nitrate, carboxylate, phosphate, sulfonate or sulfate groups, as well as salts and free acids of these groups.

In In one embodiment, the size is of nanoparticles between 1 nm-800 nm. (Claim 23)

In In another embodiment, the size is of the nanoparticles between 5 nm-800 nm.

In In one embodiment, the size is of the nanoparticles between 1 nm and 500 nm. (Claim 24)

In a preferred embodiment is the Size of nanoparticles between 1 nm-300 nm. (Claim 25)

In a particularly preferred embodiment the size of the nanoparticles is between 5 nm-500 nm.

In Another particularly preferred embodiment the size of the nanoparticles is between 5 nm-300 nm.

In Another particularly preferred embodiment the size of the nanoparticles between 10 nm-300 nm.

The Size of the resulting polymer nanoparticles is determined by photon correlation spectroscopy (PCS).

• • Das wasserunlösliche Polymer wird in einem geeigneten organischen Lösungsmittel, welches unbegrenzt mit Wasser mischbar ist, vorzugsweise Aceton, Methanol, Ethanol, Propanol, Isopropanol Dimetylsulfoxid (DMSO), oder in einem Gemisch aus diesen Lösungsmitteln mit Wasser gelöst.
• • Das kationische Polymer wird in einem geeigneten Lösungsmittel gelöst, welches unbegrenzt mit Wasser mischbar ist, vorzugsweise Aceton, Methanol, Ethanol, Propanol, Dimetylsulfoxid (DMSO), oder in einem Gemisch aus diesen Lösungsmitteln mit Wasser gelöst.
• • Der Wirkstoff (Diagnostikum und/oder Epothilon) wird in einem organischen Lösungsmittel, welches unbegrenzt mit Wasser mischbar ist, vorzugsweise Aceton, Methanol, Ethanol, Propanol, Dimetylsulfoxid (DMSO), Isopropanol oder in einem Gemisch aus diesen Lösungsmitteln mit Wasser gelöst.
• • Es wird eine vollständig homogene Lösung aus kationischem Polymer, wasserunlöslichem Polymer und Wirkstoff hergestellt durch Zusammengeben der vorher getrennt hergestellten Lösungen.
• • Durch ein Einbringen des gelösten Polymer-Substanz-Gemisches in eine tensidhaltige Lösung, als Tensid insbesondere Pluronic F68, Triton X-100 und Synperonic T707, wird die spontane Bildung eines kolloidalen Fällungsaggregats herbeigeführt.
• • Das organische Lösungsmittel wird anschließend entweder unter Atmosphärendruck oder Unterdruck, über Lyophilisation oder Hitze vollständig oder andere geeignete Methoden entfernt.
• • Gegebenenfalls wird zur Modifikation der Partikeloberfläche die oben hergestellte wässrige, stabile Nanopartikeldispersion in einem geeigneten Mengenverhältnis mit dem in Wasser gelösten modifizierenden Agens vermischt. Die Bestimmung des geeigneten Mengenverhältnisses erfolgt durch schrittweise Titration der Partikeldispersion mit dem modifizierenden Agens. Das Ausmaß der elektrostatischen Oberflächenmodifikation (Ladungstitration) wird durch Bestimmung des Zetapotentials kontrolliert. Der Zielwert des Zetapotentials hängt von dem geplanten Applikationsweg ab. Für i. v. Anwendungen wäre beispielsweise ein neutrales bis negatives Zetapotential bevorzugt, bei oraler und buccaler Applikation wird eher ein neutrales oder kationisches Zetapotential bevorzugt, usw.
• • Gegebenenfalls erneut Entfernen des Dispergens.

In a particularly preferred embodiment, the preparation of the polymer nanoparticles is characterized by carrying out the following process steps:

• The water-insoluble polymer is dissolved in water in a suitable organic solvent which is infinitely miscible with water, preferably acetone, methanol, ethanol, propanol, isopropanol, dimethylsulfoxide (DMSO), or in a mixture of these solvents.
• The cationic polymer is dissolved in a suitable solvent which is infinitely miscible with water, preferably acetone, methanol, ethanol, propanol, dimethylsulfoxide (DMSO), or dissolved in a mixture of these solvents with water.
• The active ingredient (diagnostic agent and / or epothilone) is dissolved in an organic solvent which is infinitely miscible with water, preferably acetone, methanol, ethanol, propanol, dimethylsulfoxide (DMSO), isopropanol or in a mixture of these solvents with water.
• A completely homogeneous solution of cationic polymer, water-insoluble polymer and active ingredient is prepared by combining the previously separately prepared solutions.
• • 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.
• Optionally, to modify the particle surface, the aqueous stable nanoparticle dispersion prepared above is mixed in a suitable proportion 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 target value of the zeta potentials depends on the planned route of application. For iv applications, for example, a neutral to negative zeta potential would be preferred, for oral and buccal administration, a neutral or cationic zeta potential is more preferred, etc.
• • If necessary, remove the dispersant again.

In Another embodiment is after removal of the organic solvent, a purification step, for example by washing the particles with a suitable solution, carried out.

suitable Solutions for the washing process are for example aqueous surfactant solutions 0.1-2%, but also pure water.

In In another embodiment, after removal of the organic solvent and optionally purification or lyophilized after surface modification become. The lyophilized nanoparticles can then be used as Kit and reconstituted and administered for use become.

Therefore Another object of the invention is a kit that particles containing a diagnostic agent and / or an epothilone as lyophilisate contains. This kit may additionally be a suitable Means for reconstitution of the lyophilisate z. B. physiological Saline or water for injection / infusion contain.

One Another object of the invention is a kit that does not surface-modified Particles, means for preparing a solution of the surface-modifying agent.

In In another embodiment, the described Nanoparticles using suitable pharmaceutical additives be further processed to various dosage forms, which are to Application to humans or animals are suitable. These include in particular aqueous dispersions, lyophilisates, solid oral dosage forms like fast dissolving tablets, capsules and others. suitable Pharmaceutical additives may be: sugar alcohols for lyophilization (eg sorbitol, mannitol), tabletting excipients, Polyethylene glycols, etc.

The Application of the aqueous nanoparticle dispersion or a further developed dosage form can be administered orally, parenterally (intravenously), subcutaneous, intramuscular, intraocular, intrapulmonary, nasal, Intraperitoneal, dermal, and on all others for human or animal possible application routes.

• • Lösen des kationischen Polymers in einem organischen Lösungsmittel oder einem Lösungsmittelgemisch mit Wasser
• • Lösen des wasserunlöslichen Polymers in einem organischen Lösungsmittel
• • Lösen des Wirkstoffes (Diagnostikum oder Therapeutikum) in einem organischen Lösungsmittel oder einem Lösungsmittelgemisch mit Wasser,
• • Herstellen eines vollständig gelösten Gemisches aus kationischem Polymer, wasserunlöslichem Polymer und Wirkstoff
• • Einbringen des Gemisches in eine tensidhaltige Lösung, wobei es zur spontanen Bildung von Fällungsaggregaten kommt,
• • Entfernen des Lösungsmittels.
• • Gegebenenfalls Aufreinigung der Partikeldispersion
• • Gegebenenfalls Lyophilisieren
• • Elektrostatische Oberflächenmodifikation der Partikel durch Zusammenfügen von Nanopartikeldispersion und modifizierendem Agens in geeigneten Mengen (Fakultativ)
• • Gegebenenfalls lyophilisieren.

The invention relates to a process for the preparation of a polymer nanoparticle, characterized in that the following process steps are carried out:

• Dissolving the cationic polymer in an organic solvent or a solvent mixture with water
• Dissolving the water-insoluble polymer in an organic solvent
• Dissolving the active ingredient (diagnostic or therapeutic agent) in an organic solvent or a solvent mixture with water,
• • Make a completely dissolved mixture of cationic polymer, water-insoluble polymer and active ingredient
• Introducing the mixture into a surfactant-containing solution, which leads to the spontaneous formation of precipitation aggregates,
• • Remove the solvent.
• • If necessary, clean up the particle dispersion
• • Lyophilize if necessary
• Electrostatic surface modification of the particles by combining nanoparticle dispersion and modifying agent in appropriate amounts (optional)
• • If necessary, lyophilize.

definitions

Of the The term "active ingredient" as used herein includes therapeutically and diagnostically active compounds. Also included are compounds, effective in animals other than humans and in plants are.

The term "epothilone or epothilone" includes all naturally occurring epothilones and their derivatives, epothilone derivatives are known, for example, from WO 93/10102 . WO 93/10121 and DE 41 38 042 A2 . WO 97/19086 and WO 98/25929 . WO 99/43320 . WO 2000/066589 . WO 00/49021 WO 00/71521 . WHERE 2001027308 . WO 99/02514 . WO 2002080846 ,

The epothilones suitable for use in the present invention, for example, and their preparation are disclosed in U.S. Patent Nos. 3,729,769 and 4,405,847 DE 19907588 . WO 98/25929 . WO 99/58534 . WO 99/2514 . WO 99/67252 . WO 99/67253 . WO 99/7692 . EP 99/4915 . WO 00/1333 . WO 00/66589 . WO 00/49019 . WO 00/49020 . WO 00/49021 . WO 00/71521 . WO 00/37473 . WO 00/57874 . WO 01/92255 . WO 01/81342 . WO 01/73103 . WO 01/64650 . WO 01/70716 . US 6,204,388 . US 6387927 . US 6380394 . US 02/52028 . US 02/58286 . US 02/62030 . WO 02/32844 . WO 02/30356 . WO 02/32844 . WO 02/14323 , and WO 02/8440 , Particularly suitable are the compounds which are in WO 00/66589 are disclosed.

Prefers For the purposes of the present invention, epothilones, as used by Formula II are included.

Of the Term epothilone also excludes the possibility with that in a preparation different epothilone derivatives selected from the disclosed list of claim 21 become.

Of the The term "matrix polymer" as used herein describes the polymer which quantitatively the larger Particle mass fraction forms, with other encapsulated substances (any additives as well as pharmaceutically active substances) even and / or uneven can be embedded.

Of the Term "(nano) precipitation" as used herein describes the formation of a colloidal precipitate by precipitation a poorly water-soluble polymer when introduced into an aqueous phase, wherein a thorough mixing of the solvent takes place. In the case of co-precipitation occurs a common precipitation of several substances, which in the context of the invention both water-soluble and difficult to dissolve in water.

A "precipitation aggregate", as used here arises in the course of nanoprecipitation. This precipitator consists according to the invention a matrix polymer, wherein further polymeric substances as well as pharmaceutically active substances partially or completely embedded could be. It can be a uniform or uneven distribution of the co-encapsulated Substances are present in the matrix polymer.

One Anchor, as used herein, describes an ionic substructure of the modifying agent which immobilizes and thus Localization of the modifying agent on the charged particle surface by ionic interactions between oppositely charged Connections possible.

charge titration describes the traceable via Zetapotentialmessung Process of electrostatic coupling of the anchor on the particle surface. The charged anchor changes the zeta potential of the Particles towards the charge of the anchor.

surfactants For the purposes of the invention are on the one hand surface-active Substances which show the interfacial tension between two lower immiscible phases, thereby stabilizing colloidal dispersions is possible. It can continue in the case of surfactants according to the invention by any type of substances which are capable of colloidal dispersions to stabilize sterically and / or electrostatically.

With The term "target structure" is a target that recognizes the target Structure meant. In the case of "passive targeting" can the target structure indirectly supports an enrichment at the destination, recordable by an extension of the circulation time the particle in the bloodstream increases the likelihood an enrichment via the fenestrations in the tumor endothelium.

Of the Term "active targeting" is used when referring to tissue or cell-specific ligands for targeted enrichment be used. Active ligands can both act on drugs directly (ligand-drug conjugates) as well as on the surface coupled colloidal carrier systems.

The term "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 features of tumorous or inflamed tissue [ Ulbrich K., Subr V., Adv. Drug Deliv. Rev., 2004; 56 (7): 1023-1050 ].

The term "surface potential", also referred to as surface charge, is synonymous with the term "zeta potential". This zeta potential is determined by the method of laser Doppler anemometry (LDA) determined.

The Surface potential, also referred to as zeta potential, gives the potential of a migratory particle at the shear plane on, d. H. if by movement of the particle the largest Part of the diffuse layer has been sheared off. The surface potential was using the method of laser Doppler anemometry using of a "Zetasizer 3000" (Malvern Instruments).

through The method of laser Doppler anemometry is the migration rate determines the particle in the electric field. Particles with a charged Surface migrate in an electric field to the opposite charged electrode, wherein the migration speed of the particles from the amount of surface charges and applied Field strength is dependent. To determine the migration speed For example, particles migrating in the electric field are irradiated with a laser and detects the scattered laser light. Through the movement of Particle becomes a frequency shift in the reflected light measured in comparison to the incident light. The amount of this Frequency shift depends on the migration speed and is referred to as the so-called Doppler frequency (Doppler effect). From the Doppler frequency, the scattering angle and the wavelength the migration speed of a particle can be deduced. The electrophoretic mobility results from the quotient the migration speed and the electric field strength. The product of electrophoretic mobility and the factor 13 corresponds to the zeta potential whose unit is [mV].

The Measurements (n = 5) were taken with a Zetasizer Advanced 3000 and a Zetamaster from Malvern Instruments Ltd. (Worcestershire, England) after dilution in low-electrolyte dispersion medium (MilliQ Water: resistance 18.2 MΩ · cm, 25 ° C and TOC content (total organic carbon) <10 ppb) and below defined pH value (pH 6.8-7.0) performed. The software used was PCS V1.41 / PCS V1.51 Rev. The control measurements of the zeta potential were made with latex standard particles from Malvern Instruments Ltd. (-50 mV ± 5 mV). The measurements were under the default settings of the company 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 in 12 is shown by way of example. The 12 ( 12 ) also confirms the spherical shape of the nano-particles.

The Determination of particle size by DLS based on the principle of photon correlation spectroscopy (Photon Correlation Spectroscopy, PCS). This method is suitable for surveying of particles with a size in the range of 3 nm to 3 μm. The particles are in solution an undirected motion triggered by the collision with liquid molecules of the dispersant, whose driving force is Brownian motion. The resulting movement of particles is faster, the smaller you are Particle diameter is. Will a sample in a cuvette irradiated with laser light, it is up to the undirected moving Particles for scattering the light. Through this movement of the particles if the dispersion is not constant, but fluctuates over the time. The detected at 90 ° angle fluctuations of Intensity of the scattered laser light are all the stronger, the faster the particles move, d. H. the smaller they are. On the basis of these intensity fluctuations one can using an autocorrelation function on the particle size shut down. The average particle diameter is from the Decreases in the correlation function. For correct calculation of the average particle diameter, the particles should be spherical Have shape what to check by SEM images leaves (see above), do not sediment or float. The measurements were carried out with suitable samples Dilution, at a constant temperature of 25 ° C and a defined viscosity of the solution. The instrument was calibrated with standard latex particles different size of the company Malvern Instruments Ltd.

The Scanning electron micrographs (SEM images) for determination Particle size was determined using a field emission scanning electron microscope made of the type XL-30-SFEG FEI (Kassel, Germany). In advance, the samples were placed in a high vacuum sputter 208 HR Cressington (Watford, England) sputtered with a 5 nm gold-palladium layer.

The solubility of a substance indicates whether and to what extent a pure substance can be dissolved in a solvent. It thus describes the property of a substance to mix with the solvent under homogeneous distribution (as atoms, molecules or ions). 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 points a solubility <0.1 mol / l, a moderately soluble between 0.1-1 mol / l and a slightly soluble compound> 1 mol / l.

The Invention will now be further described below in the examples without being limited to it.

Examples

Example 1: Preparation of PBCA by means of anionic polymerization

For the PBCA preparation by anionic polymerization of butyl cyanoacrylate (BCA) Sicomet 6000 is used. The polymerization process takes place by slow, permanent dropping of a total of 2.5% [m / v] BCA in a 1% [m / v] Triton X-100 solution in an acidic Solution (pH 1.5-pH 2.5, ideal pH 2.2). The pH is pre-adjusted using a 0.1N HCl solution. The resulting dispersion is cooled in an ice bath (about 4 ° C) over 4 hours constant at 450 U / min touched. Subsequently, larger ones Agglomerates by filtration through a paper-fold filter separated. By addition of methanol (or other suitable alcohols such as ethanol) precipitates the BCA polymerized to PBCA and the filter residue obtained with it several times washed purified water (MilliQ system). After drying of PBCA filter residue in the oven at 40 ° C An average molecular weight is determined by means of GPC for 24 h (Mn ~ 2000 Da). Polystyrene standards are used.

to special purification of precipitated in methanol PBCAs this can be dissolved again in tetrahydrofuran and then be precipitated again with heptane. The by filtration (Folded filter, suction filter o. Ä.) Residue is rinsed with heptane 2-n times. The residue is up to constant weight in the drying oven at 40-50 ° C. dried (about 4 days).

Example 2: Production of epothilone loaded PBCA-P (DMAEMA) nanoparticles by nanoprecipitation

a) Preparation of the polymer-substance mixture (= Mixture 1) and the surfactant solution

In a screw vial (50 ml) is added 30 ml of a 4% acetone PBCA solution (m / v), 3 ml of 4% acetonic PDMAEMA solution (m / v) and 3 ml of an acetone epothilone solution (concentration about 60 mg / ml) and after closure with a screw cap mix thoroughly with shaking (= mixture 1). The used PBCA is prepared by Example 1. 10 ml each of 1% Synperonic T707 solution (m / v) is placed in a 20 ml screw-cap submitted with magnetic stirrer.

b) Preparation of the particle dispersion by nanoprecipitation

at high stirring speed (600 rpm), 1.2 ml of Rapidly pipette 1 to 10 ml of surfactant solution. After 2-3 h at lower agitation (100 Rpm) for a further 18-24 h the remaining acetone evaporated under the hood.

c) workup of the particle approaches

20 Approaches according to manufacturing instructions a, b become one Approach united and worked up. Crystalline, not encapsulated Epothilone is removed by means of a 1 μm glass fiber filter (PALL, 25 mm, 1 μm P / N 4523T). The purification of the Filtrates are carried out using an Amicon ultrafiltration cell 8050 using a polyethersulfone filter membrane (100 kDa). For purification, the filtrate is concentrated to about 1/3 of the volume and then the concentrate by adding a 1% Synperonic T707 solution brought back to its original volume. It follows a renewed narrowing. This process is repeated twice and the target concentration of the epothilone particle dispersion is over adjusted the final volume of the solution (epothilone content 0.1-2 mg / ml).

Example 3: Production of epothilone loaded PBCA-P (DMAPMAM) nanoparticles by nanoprecipitation

a) Preparation of the polymer-substance mixture (= Mixture 1) and the surfactant solution

Into a screw vial (50 ml), 30 ml of a 4% acetone PBCA solution (m / v), 3 ml of a 4% acetone PDMAPMAM solution (m / v) and 3 ml of an acetone epothilone solution (concentration approx 60 mg / ml) and, after capping with a screw cap, shaking thoroughly mixes (= mixture 1). The PBCA used is prepared by Example 1. 10 ml of a 1% Synperonic T707 solution (m / v) are placed in a 20 ml screw-capped magnetic stirrer tube.

b) Preparation of the particle dispersion by nanoprecipitation

at high stirring speed (600 rpm), 1.2 ml of Rapidly pipette 1 to 10 ml of surfactant solution. After 2-3 h at lower agitation (100 Rpm) for a further 18-24 h the remaining acetone evaporated under the hood.

c) workup of the particle approaches

20 Approaches according to manufacturing instructions a, b become one Approach united and worked up. Crystalline, not encapsulated Epothilone is removed by means of a 1 μm glass fiber filter (PALL, 25 mm, 1 μm P / N 4523T). The purification of the Filtrates are carried out using an Amicon ultrafiltration cell 8050 using a polyethersulfone filter membrane (100 kDa). For purification, the filtrate is concentrated to about 1/3 of the volume and then the concentrate by adding a 1% Synperonic T707 solution brought back to its original volume. It follows a renewed narrowing. This process is repeated twice and the target concentration of the epothilone particle dispersion is over adjusted the final volume of the solution (epothilone content 0.1-2 mg / ml).

Example 4: Preparation of epothilone loaded PLGA-P (DMAEMA) nanoparticles by nanoprecipitation

a) Preparation of the polymer-substance mixture (= Mix1)

In a screw vial (50 ml) is added 30 ml of a 4% acetone PLGA solution (m / v) (PLGA RG 752S), 3 ml of a 4% acetamide PDMAEMA solution (m / v) and 3 ml of an acetone epothilone solution (concentration about 60 mg / ml) and after closure with a screw cap mixed well with shaking.

b) Preparation of the surfactant solution

ever 10 ml of a 1% Synperonic T707 solution (m / v) placed in a 20 ml screw-top glass with magnetic stirrer core.

c) Preparation of the particle dispersion by nanoprecipitation

Under high stirring speed (600 rpm), 1.2 ml of Mix 1 to 10 ml of surfactant solution quickly. After 2-3 h at lower agitation (100 Rpm) for a further 18-24 h the remaining acetone evaporated under the hood.

d) workup of the particle mixtures

20 Approaches according to manufacturing instructions a-c united and worked up to one approach. Crystalline, not encapsulated epothilone is by means of a 1 micron glass fiber filter (PALL, 25 mm, 1 μm P / N 4523T) separated. The purification the filtrate is carried out using an Amicon ultrafiltration cell 8050 using a polyethersulfone filter membrane (100 kDa). For purification, the filtrate is concentrated to about 1/3 of the volume and then the concentrate by adding a 1% Synperonic T707 solution brought back to its original volume. It follows a renewed narrowing. This process is repeated twice and the target concentration of the epothilone particle dispersion is over set the final volume of the solution.

Example 5: Preparation of dye-loaded PBCA nanoparticles by nanoprecipitation

i) PBCA-P (DMAEMA) nanoparticles (with ICG, DODC, IDCC or Coumarin 6)

It 500 μl of a 2% acetone PBCA solution [m / v] with 100 μl of a 2% acetone P (DMAEMA) solution [m / v] under occlusion (to prevent evaporation of the acetone) thoroughly mixed using a standard laboratory shaker. The PBCA used for this purpose is as in the example 1 produced. To this polymer mixture are each 100 ul added to the dye solutions described below.

dye solution a: 3 mg of indocyanine green are purified in 300 μl Pre-dissolved water in an ultrasonic bath and then with 700 ul Acetone added. Dye solution b, c, d: the dyes DODC, IDCC and coumarin 6 are in 0.02% acetone Solution [m / v] used.

The mixed dye-polymer mixture is mixed with a 2.5 ml Eppendorf pipette and in 10 ml of an intensely stirred 1% [m / v] Synperonic T707 solution pipetted. The Nanoparticle dispersion is carried out for 2 h at 600 rpm (standard Magnetic stirrer) and for a further 16 h at 100 rpm for complete evaporation of the solvent touched. The workup is carried out by centrifugation in Eppendorf caps. 1 ml each of the particle dispersion and 0.5 ml a 1% [m / v] CETAC solution (cetyltrimethylammonium chloride solution) After mixing for 10 min at 14000 rpm (on a Sigma 2 K 15 laboratory centrifuge). The supernatant is removed, the particles in the 1% CETAC solution redispersed and centrifuged again. This washing process will repeated three times, finally the particles in a 1% Solution Synperonic T707 be included.

ii) PBCA [PEI-IDCC] nanoparticles

It 500 μl of a 2% acetone PBCA solution [m / v] with PEI 1.8 kDa in isopropanol (2% [m / v]). It will 100 μl each of the dye solutions mentioned under i) a-d used.

The mixed dye-polymer mixture is mixed with a 2.5 ml Eppendorf pipette and in 10 ml of an intensely stirred 1% Triton X-100 solution pipetted. The nanoparticle dispersion becomes for 2 h at 600 rpm (standard magnetic stirrer) and for a further 16h at 100rpm to complete Evaporation of the solvent stirred. The workup by centrifugation in Eppendorf caps. 1 ml each of the Particle dispersion and 0.5 ml of a 1% [m / v] CETAC solution (Cetyltrimethylammonium chloride solution) after mixing centrifuged for 10 min at 14000 rpm (on a Sigma 2 K 15 laboratory centrifuge). The supernatant is removed, the particles in the 1% CETAC solution redispersed and centrifuged again. This Washing process is repeated three times, ending with the particles in a 1% solution of Triton X-100.

Example 6: Influence of Nanoprecipitation by changing the polymer content in the surfactant phase

In 4 It is shown that the particle size of the PBCA-P (DMAEMA) nanoparticles can be controlled during production by varying the polymer concentration. The stabilization of the PBCA-P (DMAEMA) nanoparticles prepared according to Example 2 (but without epothilone) is carried out with the Synperonic T707 surfactant. During particle production (nanoprecipitation), the volume of the organic polymer solution injected into the surfactant phase is kept constant and only the polymer concentration is changed accordingly. All other production conditions (surfactant concentration, polymer ratio PBCA: P (DMAEMA) = 10: 1, dye concentration, temperature, stirring speed / stirring fish, vessel, type of injection) remain constant. The use of a lower polymer concentration in the surfactant phase during the precipitation results in smaller particle diameters. Over the period studied, no change in particle size was observed for the same polymer content.

Example 7: Electrostatic surface modification epothilone-loaded PBCA-PDMAEMA nanoparticles with Glu (10) -b-PEG (110)

The Epothilone PBCA-PDMAEMA nanoparticles used here are as described in Example 2 produced.

to Modification of the particle surface becomes the aqueous, stable nanoparticle dispersion in a suitable ratio with the modifying agent (Glu (10) -b-PEG (110) / Glu (10) -b-PEG (114) dissolved in water) mixed. The determination of the appropriate quantity ratio takes place by stepwise titration of the particle dispersion with the modifying Agent. The extent of electrostatic surface modification (Charge titration) is controlled by determining the zeta potential.

Is shown in 3a ) the change of the Zetaptentials from (+) 25 mV to approx. (-) 30 mV, resp. 3b ) from +35 mV to -10 mV, by stepwise addition of the modifying agent (Glu (10) -b-PEG (110)) or Glu (10) -b-PEG (114) for particle dispersion (charge titration).

Example 8: SEM images of epothilone loaded PBCA-P (DMAEMA) nanoparticles

The Epothilone-loaded PBCA-P (DMAEMA) are prepared according to the example 2 produced.

In 5 is a SEM image of epothilone-loaded PBCA-P (DMAEMA) nanoparticles shown.

Example 9: Cell culture experiments

Cultivation of the HeLa cell line is carried out in 225 cm ² culture flasks at 37 ° C. and 5% CO ₂ in Dulbecco's modified Eagles medium (DMEM) with the addition of 10% fetal calf medium (FCS) and 2 mM L-glutamine. Antibiotic additives (penicillin / streptamycin) are dispensed with in order to influence the cell processes as little as possible. The cells are passaged regularly and sowed for experimental purposes 24 hours before the start of the investigations. For the investigations, the cells are seeded in 96-well plates from Falcon / Becton Dickinson.

In front At the beginning of the test, a visual check is carried out the vitality or typical morphology of the cells. Subsequently the FCS-containing medium is aspirated and replaced by 50 .mu.l serum-free Medium replaced.

After a maximum of 60 minutes incubation time of a nanoparticle dispersion, which is prepared according to Example 5 (Coumarin 6 loaded nanoparticles), the supernatant particle dispersion is aspirated and the cells washed 2-3 times with PBS. For staining the mitochondria, the MitoTracker Red CMXRos dye, previously diluted in medium, from Molecular Probes Europe BV, Leiden (NL) (0.25 μl / ml) is used. Incubation with 50 μl 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 μl of the dye solution diluted in PBS (2 μg / ml) are incubated for 10 min at room temperature. After removing the dye solution, the cells are washed with 100 μl 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 10 Influence of Functionalized Particle Surfaces on Cell Acquisition Tab. 1: Particle _{diameter dhyd} , polydispersity index and zeta potential of (non) functionalized coumarin 6 loaded PBCA-P (DMAEMA) nanoparticles (NP) Size d _hyd [nm] Polydispersity Index [PI] Zeta potential [mV] 1.) Unmodified NP 191 0.13 +31.5 ± 1.5 2.) NP with folic acid 195 0.06 +8.1 ± 3.7 3.) NP with Glu-PEG 208 0.08 -28.4 ± 1.2

The Nanoparticles used in Example 9 are prepared according to Example 5 produced. Unmodified or after electrostatic surface modification with folic acid or Glu (10) -b-PEG (110) have the particles the properties listed in Table 1 on.

In the 96-well plate used for the experiment, all wells have the same cell density (seeding 24 h before experiment: 1 × 10 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 20x magnification and constant exposure time (see 6 ). Based on 6 It is shown how different surface properties of one and the same batch of nanoparticles influence cell uptake behavior. Unmodified particles in series 1.) with a cationic surface potential show a higher affinity for the cell surface, recognizable by the stronger Fluorescence contrast on or in the cells. The equally effective internalization of particles with negative surface potential after titration with Glu (10) -b-PEG (110) can be shown by the enlarged section of the cells from row 3.).

Example 11: Cell uptake behavior Glu (10) -b-PEG (110) modified PBCA P (DMAEMA) nanoparticle loaded with coumarin 6 (Fluorescent dye for in vitro detection)

The nanoparticles used in Example 9 are prepared according to Example 5. After electrostatic surface modification with Glu (10) -b-PEG (110), the cell uptake behavior of the particles (d _hyd = 171 nm, ZP = -33 mV) is investigated.

The brightly fluorescent dots, which are endosomes or endolysosomes, are evidence of efficient uptake of the nanoparticles into the cell by endocytosis ( 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. Cell uptake of PBCA-P (DMAEMA) nanoparticles surface-modified with Glu (10) -b-PEG (110) is reported in 8th shown schematically.

Example 12: Enrichment of Glu (10) -b-PEG (110) modified PBCA P (DMAEMA) nanoparticles in the nucleus

Based on the recording of the middle cell level with the aid of the confocal laser scanning microscope ( 9 ) it can be shown that a partial enrichment of the particles takes place in the cell nucleus.

Example 13: Increased particle uptake with incubation of higher particle concentrations

Coumarin 6-loaded, Glu (10) -b-PEG (110) surface-modified PBCA-P (DMAEMA) particles are prepared according to Example 5. A low particle concentration of 0.21 mg / ml ( 10 ) and a higher particle concentration of 0.85 mg / ml ( 11 ) incubated for the same period on the cells according to Example 9 incubated. 11 shows in relation to 10 an increased particle uptake with incubation of a higher particle concentration.

Example 14: Characterization of the PBCA (P (DMAEMA) ICG] nanoparticles

Shown is the particle size ( 13 ) 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. The constant particle size and the consistently low polydispersity index (PI <0.1) as a feature of a very narrow particle size distribution are evidence of a good stability of the surface-modified particles.

Based on the SEM image ( 12 ) can be additionally demonstrated that they are spherical nanoparticles with a size of 200 nm.

By charge titration, the cationic surface of the PBCA-P (DMAEMA) nanoparticles is modified with the block copolymer Glu (10) -b-PEG (110) (see 14 ). The surface charge measured as zeta potential is accordingly titrated from approx. +30 mV above the neutral point until reaching the dissociation equilibrium at approx. -30 mV. 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.

In 15 the UV-Vis absorption spectra of an aqueous ICG solution as well as the ICG nanoparticle dispersion (washed and unwashed) are shown. Indocyanine green is a near-infrared fluorescent dye whose absorption and emission spectrum is in the wavelength range between 650-900 nm. The complexation and encapsulation of ICG using the cationic polyacrylate P (DMAEMA) leads to a minimal bathochromic shift of the two wavelength maxima.

Example 15: Animal experiment

The animals used are supplied by Taconic M & B. These are female Al bino 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 2 × 10 ⁶ cells of a teratoma-F9 in the right rear 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 μl / 10 g of animal for the first hour of the experiment. The solution for injection consists of a 1: 1 mixture of a 1:10 dilution Rompun or 1: 5 dilution Ketavet with physiological saline. Subsequently, 200 μl of the nanoparticle dispersion are injected iv into the tail vein. The subsequent anesthesia is carried out with Rompun-Ketavet via the lungs as an inhalation anesthetic in order to minimize the circulation of the animals. In a time frame of 24 and 48 h after substance injection, the animals are examined by fluorescence optics.

With the help of 17 It is shown that Glu (10) -b-PEG (110) -modified PBCA- [P (DMAEMA) -ICG] nanoparticles after intravenous administration (tail vein) are able to adapt to passive enhancement mechanisms (EPR effect) To enrich tumor tissue. Examination of the tumors ex vivo shows a clear enhancement of the fluorescence contrast in treated compared to untreated tumor tissue (comparison 18 b with a or c with a). Multiple, time-shifted fluorescence detection in one and the same animal is possible after 24 h and 48 h ( 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. There is a rapid biliary elimination of non-tumor associated particles from the liver. Indication for this is a lack of NIR fluorescence contrast in the liver after 24 or 48 h. Rapid elimination of non-tumor-enriched particles from the organism (eg liver) allows good tumor contrast with minimal stress on other organs, a prerequisite for a low-contrast contrast agent system.

The device used for the animal experiment was set up by the company LMTB (Berlin, Germany). As individual components were used: Laser: Diode laser (742 nm), model Ceralas PDT 742 / 1.5W; CeramOptec (Bonn, Germany) Excitation filter: 1xLCLS-750 nm-F; 1x740 nm interference filter (bandpass) Emission filter: 1xbk 802.5 to 22--C1; 1xbk-801-15-C1 Camera: Peltier air-cooled CCD camera, model C4742-95 12ER, Fa. Hamamatsu (Herrsching, Germany) Software: Simple PCI 5.0, Compix / Hamamatsu

characters

1 : Short term stability PBCA-PDMAEMA nanoparticles with epothilone (not surface modified).

2 : Short-term Stability PBCA-PDMAEMA nanoparticles with epothilone surface-modified with Glu (10) -b-PEG (110)
a) Hydrodynamic particle _diameter d _hyd and polydispersity index PI / # EpoPD19Ak2konz PEG-Glu
b) Hydrodynamic particle diameter dhyd and zeta potential

3 : Titration profile (zeta potential) in the surface modification of epothilone-loaded PBCA-PDMAEMA nanoparticles Shown in this figure is the change in the zetapotential of 3a) +25 mV to approx. -30 mV or from 3b) +35 mV to -10 mV by stepwise addition of the modifying agent (Glu (10) -b-PEG (110)) or (Glu (10) -b-PEG (114)) for particle dispersion (charge titration).

4 : Control of the particle diameter by changing the polymer concentration;
In 1 It is shown that the particle size of the PBCA-P (DMAEMA) nanoparticles can be controlled during production by varying the polymer concentration.

5 : The figure shows an SEM image of epothilone-loaded PBCA-P (DMAEMA) nanoparticles.

6 : Influence of functionalized particle surfaces on 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.

7 : Nanoparticle uptake in HeLa cells; Fluorescence of the nanoparticles as grayscale image;
The figure shows the cell uptake behavior of Glu (10) -b-PEG (110) modified PBCA P (DMAEMA) nanoparticles in HeLa cells.

8th : Schematic representation of cell uptake of PBCA-P (DMAEMA) nanoparticles surface-modified with Glu (10) -b-PEG (110);
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.

9 a) fluorescence imaging in the middle cell level (CLSM, Confocal Laser Scanning Microscope), b) computer-based 3D fluorescence imaging;
The plot shows the accumulation of the Glu (10) -b-PEG (110) modified PBCA-P (DMAEMA) nanoparticles in the nucleus. This is possible by loading with the fluorescence-active dye coumarin 6.

10 : Lower particle uptake when incubating lower particle concentrations: 0.21 mg / ml; Fluorescence of the NP as grayscale image;
The figure shows fluorescent HeLa cells after a particle concentration of 0.21 mg / ml was incubated. Glu (10) -b-PEG (110) surface-modified PBCA-P (DMAEMA) particles were used for this purpose.

11 : Increased particle uptake on incubation of higher particle concentrations: 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. Glu (10) -b-PEG (110) surface-modified PBCA-P (DMAEMA) particles were used for this purpose.

12 : SEM image of PBCA [P (DMAEMA) -ICG] nanoparticles

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.

14 : Zeta potential of the titrated (washed / unwashed) and titrated PBCA [P (DMAEMA) ICG] nanoparticles;
The abbreviation shows the surface charge of the PBCA-P (DMAEMA) nanoparticles, measured as zeta potential, modified with the block copolymer Glu (10) -b-PEG (110). This was correspondingly titrated from about +30 mV beyond the neutral point until reaching the dissociation equilibrium at about -30 mV.

15 : UV-Vis absorption spectra: a) aqueous ICG solution, b) PBCA [P (DMAEMA) -ICG] -NP unwashed; c) PBCA- [P (DMAEMA) -ICG] nanoparticles washed;
This figure shows the UV-Vis absorption spectra of an aqueous ICG solution as well as the ICG nanoparticle dispersion (washed and unwashed).

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.

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).

18 : NIR fluorescence contrast of tumor tissue ex vivo 48 h after treatment;
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.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

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Claims (32)

Polymer nanoparticles having a cationic surface potential containing a cationic polymer and a sparingly water-soluble polymer, characterized in that said polymer nanoparticles contain diagnostic agents and epothilones or only epothilones. Polymer nanoparticles according to claim 1, characterized in that it is a precipitation unit is. Polymer nanoparticles according to claim 1 or 2, characterized in that the sparingly soluble in water Polymer a polycyanoacrylate, polyalkylcyanoacrylate (PACA), polyester Alginic acid, hyaluronic acid, polysialic acid, acidic cellulose derivatives, acidic starch derivatives, polysaccharides, polymeric proteins, polyamides, polyanhydrides, polyorthoesters, polycaprolactones, Polyphosphoric acid, poly (amideeneamines), azopolymers, polyurethanes, Polyorthoesters, dendrimers, pseudopolymamic acids or all mixtures and co-polymers of the same compounds is. Polymer nanoparticles according to claim 3, characterized in that the sparingly soluble in water polymer a polybutyl cyanoacrylate (PBCA). Polymer nanoparticles, according to one of claims 1 or 2, containing a cationically modified Polyacrylate P (DMAEMA), diethylaminoethyl-modified dextrans, hydroxymethylcellulosetrimethylamine, Polylysine, protamine sulfate, hydroxyethylcellulosetrimethylamine, polyallylamines, Protamine chloride, polyallylamine hydrosalts, polyamines, polyvinylbenzyltrimethylammonium salts, Polydiallyldimethylammonium salts, polyimidazoline, polyvinylamine and polyvinylpyridine, polyethyleneimine (PEI), putrescine (butane-1,4-diamine), Spermidine (N- (3-aminopropyl) butane-1,4-diamine), spermine (N, N'-bis (3-aminopropyl) butane-1,4-diamine) Dimethylaminoethyl acrylate, poly-N, N-dimethylaminoethyl methacrylate Dimethylaminopropylacrylamide, dimethylaminopropylmethacrylamide, Dimethylaminostyrene, vinylpyridine and methyldiallylamine, poly-DADMAC, Guar, or deacetylated chitin and the corresponding salts which themselves with suitable inorganic or low-molecular organic Sowing can form. Polymer nanoparticles according to claim 5, containing a cationically modified polyacrylate P (DMAEMA), Polymer nanoparticles according to claim 5, containing a polyethyleneimine. Polymer nanoparticles according to one of claims 1-7, characterized in that the surface is electrostatically modified. The polymer nanoparticle according to claim 8, wherein the surface-modifying agent is a compound of the formula ()

wherein n is 5 to 700 R is hydrogen, C ₁ -C ₃ alkyl or a neutral amino acid and anionic anchor is a polymer of up to 10 units glutamic acid (Glu) or aspartic acid (Asp) or their salts, or their copolymers, wherein the Residues R can also occur mixed. Polymer nanoparticles according to claim 9, characterized in that the surface with Glu (10) -b-PEG (110), Glu (10) -b-PEG (114) or Asp (15) -b-PEG (114). Polymer nanoparticles according to one of claims 1-10, characterized in that the diagnostic agent is negatively charged and as an ion pair with the cationic polymer is included in the particle. Polymer nanoparticles according to one of claims 1-11, characterized in that the diagnostic agent is a fluorescent dye is. Polymer nanoparticles according to one of claims 12, characterized in that the fluorescent Dye is a fluorescent NIR dye. Polymer nanoparticles according to one of claims 1-11, characterized in that the diagnostic agent is a carbocyanine dye is. Polymer nanoparticles according to claims 14, characterized in that it is in the diagnostic Agent to TSC (tetrasulfocyanine) acts. Polymer nanoparticles according to claims 14, characterized in that it is in the diagnostic Agent is IDCC (Indodicarbocyanin). Polymer nanoparticles according to claims 14, characterized in that it is the diagnostic agent is ICG (indocyanine green). Polymer nanoparticles according to claim 1 or 2, wherein the epothilone is a compound of general formula (II)

wherein R ^1a , R ^{1b are} independently hydrogen, C ₁ -C ₁₀ alkyl, aryl, aralkyl, or together a group - (CH ₂ ) _m wherein m is 2 to 5; R ^2a , R ^2b independently of one another are hydrogen, C ₁ -C ₁₀ -alkyl, C ₂ -C ₁₀ -alkenyl C ₂ -C ₁₀ -alkynyl, aryl, aralkyl, or together a group - (CH ₂ ) _n - where n is 2 to 5, R ^{3 is} hydrogen, C ₁ -C ₁₀ alkyl, aryl, aralkyl; R ^4a , R ^4b are each independently hydrogen, C ₁ -C ₁₀ alkyl, aryl, aralkyl, or together a group - (CH ₂ ) _p wherein p is 2 to 5; R ^{5 is} hydrogen, C ₁ -C ₁₀ alkyl, aryl, aralkyl, CO ₂ H, CO ₂ alkyl, CH ₂ OH, CH ₂ OC ₁ -C ₅ alkyl, CH ₂ O acyl, CN, CH ₂ NH ₂ , CH ₂ N ((C ₁ -C ₅ alkyl), acyl) _1,2, or CH ₂ Hal, CHal _3; R ⁶ , R ^{7 are} independently hydrogen, or together another bond or an epoxide function; G is O or CH _2; DE together form the group -H ₂ C-CH ₂ -, -HC = CH-, -C≡C-, -CH (OH) -CH (OH) -, -CH (OH) -CH ₂ -, -CH ₂ -CH (OH) -, -CH ₂ -O-, -O-CH ₂ - or

where G is oxygen, DE can not be CH ₂ -O; or DEG together the group H ₂ C-CH = CH W the group C (= X) R ⁸ , or a bi- or tricyclic aromatic or heteroaromatic radical; XO or the group CR ⁹ R ¹⁰ ; R ^{8 is} hydrogen, C ₁ -C ₁₀ alkyl, aryl, aralkyl, halogen, CN; R ⁹ , R ¹⁰ independently of one another are hydrogen, C ₁ -C ₂₀ -alkyl, aryl, aralkyl or together with the methylene carbon atom a 5- to 7-membered carbocyclic ring; ZO or hydrogen and the group OR ¹¹ ; R ^{11 is} hydrogen of a protective group PG ^Z ; AY is a group OC (= O), O-CH ₂ , CH ₂ -C (= O), NR ¹² -C (= O), NR ¹² -SO ₂ ; R ^{12 is} hydrogen, or C ₁ -C ₁₀ alkyl; PG ^Z C ₁ -C ₂₀ alkyl, a C ₄ -C ₇ cycloalkyl group which may contain one or more oxygen atoms in the ring, aryl, aralkyl, C ₁ -C ₂₀ acyl, aroyl, C ₁ -C ₂₀ alkylsulfonyl, arylsulfonyl, tri (C ₁ -C ₂₀ alkyl) silyl, di (C ₁ -C ₂₀ alkyl) arylsilyl, (C ₁ -C ₂₀ alkyl) diarylsilyl, or tri (aralkyl) silyl; is encapsulated as a single stereoisomer or as a mixture of different stereoisomers and / or as a pharmaceutically acceptable salt. Polymer nanoparticles according to claim 18, wherein the epothilone is selected from the list containing (4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (2-methyl-benzoxazol-5-yl) -1-oxa-5,5,9,13-tetramethyl- 7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione; (1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzoxazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione; (4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-oxa-5,5,9,13-tetramethyl- 7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione; (1S / R, 3S, 7S, 10R, 11S, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione; (1S, 3S, 7S, 10R, 11S, 12SA, 16R) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5-yl) - 8,8,12,16-tetramethyl-4,17-dioxablcyclo [14.1.0] heptadecane-5,9-dione; (1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecanes-5,9-dione; (4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-oxa-9,13-dimethyl-5,5- (1,3-trimethylene) -7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione; (1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -12.16-dimethyl-8,8- (1,3-trimethylene) -4.17-dioxabicyclo [14.1.0] heptadecane-5,9-dione; (4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-oxa-5,5,9,13-tetramethyl- 7- (prop-2-in-1-yl) -cyclohexadec-13-ene-2,6-dione; (1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-in-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione; (4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-dihydroxy-16- (quinolin-7-yl) -1-oxa-5,5,9,13-tetramethyl-7- (prop -2-en-1-yl) -cyclohexadec-13-ene-2,6-dione; (1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (quinolin-7-yl) - 8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione; (4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-Dihydroxy-16- (1,2-dimethyl-1H-benzoimidazol-5-yl) -1-oxa-5,5,9, 13-tetramethyl-7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione; (1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-ihydroxy-10- (prop-2-en-1-yl) -3- (1,2-dimethyl-1H -benzoimidazol-5-yl) -8,8,12,16-tetramethyl-4,17-dioxabicyclo [14.1.0] heptadecane-5,9-dione; (4S, 7R, 8S, 9S, 13E / Z, 16S) -4,8-dihydroxy-16- (2-methyl-benzothiazol-5-yl) -1-aza-5,5,9,13-tetramethyl- 7- (prop-2-en-1-yl) -cyclohexadec-13-ene-2,6-dione; (1S / R, 3S, 7S, 10R, 11S, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4-aza-17-oxabicyclo [14.1.0] heptadecane-5,9-dione, (1S / R, 3S, 7S, 10R, 11R, 12S, 16R / S) -7,11-dihydroxy-10- (prop-2-en-1-yl) -3- (2-methyl-benzothiazol-5 -yl) -8,8,12,16-tetramethyl-4-aza-17-oxabicyclo [14.1.0] heptadecane-5,9-dione, (1S, 3S (E), 7S, 10R, 11S, 12S, 16R) -7,11-dihydroxy-8,8,10,12,16-pentamethyl-3- [1- (2-methyl-1,3 -thiazol-4-yl) prop-1-en-2-yl] -4.17-dioxabicyclo [14.1.0] heptadecane-5,9-dione, (1S, 3S (E), 7S, 10R, 11S, 12S, 16R) -7,11-dihydroxy-8,8,10,12,16-pentamethyl-3- [1- (2-methyl-1,3 -thiazol-4-yl) prop-1-en-2-yl] -17-oxa-4-azabicyclo [14.1.0] heptadecane-5,9-dione, (4S, 7R, 8S, 9S, 13Z, 16S (E)) - 4,8-dihydroxy-5,5,7,9,13-pentamethyl-16- [1- (2-methyl-1,3-thiazol -4-yl) prop-1-en-2-yl] -oxacyclohexadec-13-ene-2,6-dione, (4S, 7R, 8S, 9S, 10E, 13Z, 16S (E)) - 4,8-dihydroxy-5,5,7,9,13-pentamethyl-16- [1- (2-methyl-1,3 -thiazol-4-yl) prop-1-en-2-yl] -oxacyclohexadec-10,13-diene-2,6-dione, (4S, 7R, 8S, 9S, 10E, 13Z, 16S (E)) - 4,8-dihydroxy-5,5,7,9-tetramethyl-13-trifluoromethyl-16- [1- (2-methyl-1 , 3-thiazol-4-yl) prop-1-en-2-yl] oxacyclohexadec-10,13-diene-2,6-dione, (1S, 3S (E), 7S, 10R, 11S, 12S, 16R) -7,11-dihydroxy-8,8,10,12,16-pentamethyl-3- [1- (2-methylsulfanyl-1,3 -thiazol-4-yl) prop-1-en-2-yl] -4.17-dioxabicyclo [14.1.0] heptadecane-5,9-dione when single stereoisomer or as a mixture of different stereoisomers and / or pharmaceutically acceptable salt Polymer nanoparticles according to claim 8, characterized in that the surface-modifying Agent contains a target-recognizing structure. Polymer nanoparticles according to claim 18, where the target-recognizing structure is a negatively charged moiety possesses and by electrostatic interactions on the cationic Particle surface is applied. Polymer nanoparticles according to claim 20 or 21, characterized in that the target recognizing Sruktur is selected from a list containing an antibody, a protein, a polypeptide, a polysaccharide, a DNA molecule, an RNA molecule, a chemical entity, a nucleic acid, a lipid, a carbohydrate or combinations of the foregoing. Polymer nanoparticles according to one of claims 1-22, characterized in that the size of the particles between 1-800 nm is. Polymer nanoparticles according to one of claims 1-22, characterized in that the size of the particles is between 5-500 nm is. Polymer nanoparticles according to one of claims 1-22, characterized in that the size of the particles is between 10-300 nm is. Use of a polymer nanoparticle according to of claims 1- 27 for the therapy of tumors or diseases with inflammatory reactions go along and / or for the diagnosis. Process for the preparation of a polymer nanoparticle according to any one of claims 1-26, characterized in that the following method steps performed become: a) dissolving the cationic polymer in one organic solvent or a solvent mixture with water b) dissolving the water-insoluble polymer in an organic solvent c) Solve one or more active ingredients in an organic solvent or a solvent mixture with water, d) manufacture a completely dissolved mixture of cationic Polymer, water-insoluble polymer and active ingredient Combining the manufactured individual solutions e) Introducing the mixture into a surfactant-containing solution, f) Remove the solvent. g) If necessary, electrostatic Surface modification of the particles by assembly of nanoparticle dispersion and modifying agent in suitable ratio h) If necessary, remove again of the solvent Use of a nanoparticle according to a of claims 1-25 for the preparation of a pharmaceutical Preparation / dosage form using pharmaceutically acceptable Excipients. Use of a nanoparticle according to a of claims 1-25, wherein the pharmaceutical Preparation via a suitable application system to humans or animal administered via a suitable route of administration becomes. Kit consisting of the particles according to claim 1-25, which is a diagnostic and an epothilone in common encapsulated included. Kit consisting of separately prepared nanoparticulate Systems (a) and (b) of the same composition according to one of claims 1-25, containing (a) one in one Particles encapsulated diagnostic agent and (b) one in a particle encapsulated epothilone, where the particles are shared or separated, optionally diluted, can be administered. A kit according to claim 31, wherein said Ingredients (a) and (b) are in the solid state and in addition, if necessary an agent (c) is included which is suitable for the nanoparticulate Systems (a) and (b) optionally optionally separately or together to dissolve or disperse.