EP1965769A2

EP1965769A2 - Protein-based delivery system for overcoming resistance in tumour cells

Info

Publication number: EP1965769A2
Application number: EP06841159A
Authority: EP
Inventors: Sebastian Dreis; Klaus Langer; Jörg KREUTER; Martin Michaelis; Jindrich Cinatl
Original assignee: LTS Lohmann Therapie Systeme AG
Current assignee: LTS Lohmann Therapie Systeme AG
Priority date: 2005-12-27
Filing date: 2006-12-22
Publication date: 2008-09-10
Also published as: US20090181090A1; ZA200804572B; WO2007073932A2; JP2009521515A; RU2008130167A; WO2007073932A3; DE102005062440A1; CN101346131A; BRPI0620800A2; CA2631003A1; KR20080081080A; DE102005062440B4; NZ569898A; IL192343A0; AU2006331030A1; RU2404916C2

Abstract

The invention relates to nanoparticles, the particle matrix of which is based on at least one protein in which at least one active agent is embedded, a method of production of the nanoparticles with at least one active agent embedded in t the protein matrix and the use of said nanoparticles for the treatment of tumours, in particular, for the treatment of tumours which are resistant to chemical medicaments.

Description

Protein-based carrier system for the resistance of tumor cells

The development of resistance in the treatment of solid tumors is a major problem in oncology. Resistance is often due to increased excretion of the chemotherapeutic agents by the tumor cells. The mechanism of this resistance development is related to the overexpression of P-glycoprotein (Pgp) [Krishna et al. , (2000), Eur. J. Pharm. 11, 265]. Pgp is an ATP-dependent efflux pump that can actively deliver drugs from tumor cells. As a result of its overexpression, there is a reduced accumulation of the chemotherapeutic agent in the cells, so that its intracellular concentration is insufficient for an antineoplastic effect. In order to compensate for the decreased accumulation of the chemotherapeutic agent, a dose adjustment of the cytostatic agent is necessary, i. H. an increase in the dose, which is limited due to the associated toxic side effects of the cytostatic. The overexpression of Pgp leads to the so-called multidrug resistance (MDR), in which the cell is not only resistant to the original substance, but in addition to a variety of cytotoxic agents. This phenomenon significantly limits the success of tumor chemotherapy.

In the past, various approaches have been developed to counter the resistance of tumor cells The most recently investigated approaches involve the use of active substances which act as inhibitors of Pgp. As early as 1981, the inhibitory effect of calcium antagonists on Pgp was determined [Tsuruo et al., (1981), Cancer Res. 41, 1967]. In these investigations, an increased

Accumulation of vincristine and doxorubicin in vincristine-resistant P388 tumor cells was observed when the tumor cells were additionally incubated with a calcium antagonist. As a promising representative of the drug group of calcium antagonists has become

Verapamil exposed. However, other drugs such as cyclosporin A are potent inhibitors of Pgp, as has been shown [Slater et al., (1986) J. Clin. Invest. 77, 1405]. In these studies, the resistance of acute lymphocytic leukemia cells to vincristine and daunorubicin was overcome by the concomitant administration of cyclosporin A.

Since both verapamil and cyclosporin A have a considerable side effect potential, further Pgp inhibitors were sought. Thus, the two Pgp inhibitors MS-209 and SDZ PSC 833 were able to overcome the multidrug resistance of the P388 / ADM and K562 / ADM cells in in vitro experiments [Naito et al., (1997), Cancer Chemother. Pharmacol. 40, Suppl. S20].

Another strategy for overcoming multidrug resistance is chemical modification of drugs. This strategy seeks to overcome the resistance of tumor cells by conjugating antineoplastic agents to various macromolecules. Serve the macromolecules as a carrier for the active ingredient. One speaks also of a carrier system.

As early as 1992, doxorubicin-loaded polyisohexylcyanoacrylate (PIHCA) nanospheres were shown to overcome Pgp-mediated resistance in various cancer cell lines [Cuvier et al., (1992) Biochem. Pharmacol. 44, 509]. These studies were confirmed on doxorubicin-resistant C6 cells in which the inhibitory concentration 50 (IC50) of doxorubicin-loaded polyisohexylcyanoacrylate nanospheres was significantly lower than for nonconjugated doxorubicin [Bennis et al., (1994), Eur. J. Cancer 3OA, 89]. With corresponding doxorubicin-loaded PIHCA nanoparticles, this result was also confirmed in hepatocellular carcinoma cells [Barraud et al. , (2005) J. Hepatol. 42, 736].

The mechanism of resistance by colloidal carrier systems was initially the cause for

Speculation. A widespread opinion was that such carrier systems would be taken up by an endocytic process of the target cells and thereby would pass the Pgp-mediated resistance mechanisms. This opinion has been refuted with respect to polyisohexylcyanoacrylate nanoparticle [Henry-Toulme et al., (1995), Biochem. Pharmacol. 50, 1135]. In fluorescence microscopic studies of resistant tumor cell lines after incubation with PIHCA nanoparticles no accumulation of the particles was observed in the cells, whereas their accumulation in was to detect phagocytic cells such as macrophages. The overcoming of the multi-resistance by PIHCA nanoparticles was therefore discussed as synergism of products of the polymer matrix and the active ingredient. This hypothesis is supported by studies showing that doxorubicin-loaded polyisobutylcyanoacrylate (PIBCA) nanoparticles have an increased cytotoxic effect on resistant P388 / Adr cells [Colin de Verdiere et al. , (1994), Cancer Chemother. Pharmacol. 33, 504]. The

Incubation of the cells with PIBCA nanopatics resulted in a 5-fold higher drug concentration in the target cells. As a mechanism underlying this phenomenon, a nanoparticle / cell interaction was discussed in contrast to an endocytotic uptake of the nanoparticles.

In 1993, Ohkawa et al. [Cancer Res. 53, 4238-4242] a study on the effect of doxorubicin-bovine serum albumin conjugates on resistant rat

Hepatoma cells (AH66DR) published. The doxorubicin-bovine serum albumin conjugates showed an increased cytotoxic effect compared to unmodified drug control. The cause of this effect was an increased accumulation of conjugates due to decreased efflux. Treatment of peritoneal tumor bearing rats showed that the doxorubicin-bovine serum albumin conjugates increased the median survival from 30 days in the control group to 50 days. The preparation of the Ohkawa et al. Doxorubicin-bovine serum albumin conjugates described by dissolving the active ingredient and bovine serum albumin in a suitable solvent and then adding glutaraldehyde. The glutaraldehyde reacts with functional groups of the drug and the target protein, in this case amino groups, resulting in a covalent linkage of the molecules. For the doxorubicin-bovine albumin conjugates, a transport capacity of three to four drug molecules per carrier unit is given.

In the case of Ohkawa et al. Thus, the doxorubicin-bovine serum albumin conjugates described are covalent chemical bonds of doxorubicin to bovine serum albumin. By such a chemical modification of the active ingredient, the physicochemical properties of the active ingredient are changed. New active ingredients (NCI: new chemical entities) are emerging that have other and new effects in biological systems.

For antineoplastic action of the doxorubicin-bovine serum albumin conjugates, it is necessary that the covalent drug-protein binding in the target tissue be cleaved. Only then is a release of the therapeutically effective agent achieved.

Despite these disadvantages, the use of colloidal "drug delivery systems" or drug-conjugated carrier systems such as nanoparticles or nanospheres is one of the promising strategies for overcoming the resistance of tumor cells. The object of the present invention was therefore to provide a colloidal "drug delivery system" for overcoming resistances in tumor cells, which does not have the disadvantages of the known conjugates of active ingredients covalently bound to a carrier material.

This object is achieved by the provision of nanoparticles in which at least one active ingredient is included in a matrix of protein but is not covalently coupled to the protein.

The invention relates to nanoparticles whose particle matrix is based on at least one protein and in which at least one active substance is embedded, to processes for producing such nanoparticles and to the use of such nanoparticles for the treatment of tumors or for the production of medicaments for the treatment of tumors, in particular for treatment tumors that are resistant to chemotherapeutic agents.

The nanoparticles according to the invention comprise at least one protein on which the particle matrix is based and at least one active substance embedded in the particle matrix.

As protein or proteins, which (s) form the matrix of the nanoparticles, in principle all physiologically compatible, pharmacologically acceptable proteins which are soluble in an aqueous medium are suitable. Particularly preferred proteins are gelatin and

Albumin, which is derived from different animal species (cattle, Pig, etc.), as well as the milk protein casein. In principle, other proteins can also be used as starting material for the preparation of the nanoparticles according to the invention, eg. B. immunoglobulins.

Basically, any intracellular acting drug can be embedded in the particle matrix. Preferably, however, cytostatics and / or other antineoplastic agents are used to be administered with the aid of nanoparticles according to the invention for the treatment of tumors, in particular for the treatment of tumors which are resistant to cytostatics or other antineoplastic agents. Particularly preferred nanoparticles have anthracyclines such as doxorubicin, daunorubicin, epirubicin or idarubicin embedded in their protein matrix.

Suitable antineoplastic agents which may be embedded in the protein matrix of the nanoparticles are, for example:

- cytostatic drugs,

- herbal cytostatics, z. As mistletoe preparations,

- chemically defined cytostatics,

- alkaloids and podophyllotoxins, - vinca alkaloids and analogs, e.g. Vinblastine, vincristine, vindesine, vinorelbine,

- Podophyllotoxin derivatives, eg. Etoposide, teniposide,

- alkylating agents,

- Nitrosoureas, z. Nismutin, Carustin, Lomustin,

Nitrogen-free Analbga, e.g. B. cyclophosphamide, Estramustine, melphalan, ifosfamide, trofosfamide, chlorambucil, bendamustine,

- Other alkylating agents, eg. B. dacarbazine, busulfan, procarbazine, treosulfan, temozolomide, thiotepa, - cytotoxic antibiotics,

- substances related to anthracyclines, eg. Mitoxantrone,

- other cytotoxic antibiotics, eg. Bleomycin, mitomycin, dactinomycin, antimetabolites,

Folic acid analogs, e.g. B. methotrexate

Purine analogs, e.g. Fludarabine, cladribine, mercaptopurine, thioguanine

- Pyrimidine analogs, z. Cytarabine, gemcitabine, fluorouracil, capecitabine,

- Other cytotoxic agents such. Paclitaxel, docetaxel - other antineoplastic agents,

- Platinum compounds, z. Carboplatin, cisplatin, oxaliplatin, - other antineoplastic agents such as amsacrine,

Irinotecan, hydroxycarbamide, pentostatin, porfimer sodium, aldesleukin, tretinoin and asparaginase.

It is possible to include any of the active ingredients listed in the previous list of active ingredients in the

Embed particle matrix of the protein-based carrier system. Due to the different physicochemical properties of the active ingredients (eg solubility, adsorption isotherms, plasma protein binding, pKs values), however, it may be necessary to delay the manufacturing process for the active ingredients Optimize nanoparticles for the respective active ingredient.

The nanoparticles according to the invention are thus a protein-based carrier system with at least one active substance embedded in the protein matrix of the particles, preferably for the treatment of tumors, in particular for the treatment of resistant tumors.

The nanoparticles according to the invention preferably have a size of from 100 to 600 .mu.m, more preferably from 100 to 400 .mu.m. In a very particularly preferred embodiment, the nanoparticles have a size of 100 to 200 μm.

The nanoparticles according to the invention are able to overcome the resistance of tumor cells to chemotherapeutics.

Figure 1 is a graph illustrating the influence of doxorubicin nanoparticles (Dxr-NP), doxorubicin solution (Dxr-Lsg) and doxorubicin liposomes (Dxr-Lip) on the cell viability of parental neuroblastoma cells.

Figure 2 is a graph illustrating the influence of doxorubicin nanoparticles (Dxr-NP), doxorubicin solution (Dxr-Lsg) and doxorubicin liposomes (Dxr-Lip) on cell viability of resistant neuroblastoma cells. The nanoparticles of the invention may have a modified surface. For example, the surface may be PEGylated, ie polyethylene glycols may be bound to the surface of the nanoparticles through covalent bonds. By modifying the surface with polyethylene glycols (PEGs), the properties of the nanoparticles can be changed so that their stability, half-life in the organism, water solubility, immunological properties and / or bioavailability can be improved.

However, the nanoparticles may also have "drug-targeting ligands" on their surface, by means of which targeted enrichment of the nanoparticles in a specific organ or in specific cells is possible. <br /> <br /> Preferred drug-targeting ligands are tumor-specific protein-recognizing ligands, for example from Group selected, the tumor-specific proteins recognizing antibodies such as trastuzumab and cetuximab, and transferrin as well

Galactose. The drug-targeting ligands may also be coupled to the surface of the nanoparticles via bifunctional PEG derivatives.

In connection with modifications of the surface of nanoparticles according to the invention, reference is made to the publication WO 2005/089797 A2, the content of which is fully incorporated into the disclosure of the present invention by this reference. The preparation of the nanoparticles according to the invention is preferably carried out by first bringing the active ingredient (s) and the protein (s) together in solution, preferably in water or an aqueous medium. Subsequently, the protein is precipitated by simple desolvation by controlled addition of a non-solvent for the protein, preferably an organic solvent, more preferably ethanol, slowly and in a controlled manner from the solution. Here, the colloidal carrier system (nanoparticles) forms around the drug molecules in solution. The active ingredient is thereby embedded unmodified in the matrix of the carrier system.

In the preparation of the drug-loaded nanoparticles, the active ingredient is preferably used in a molar excess, based on the protein. The molar ratio of active ingredient to protein is particularly preferably 5: 1 to 50: 1. Also, loading in molar ratios of more than 50: 1 is possible.

Subsequent crosslinking of the protein matrix by adding a crosslinking agent, preferably glutaraldehyde, stabilizes the matrix of the nanoparticles.

By varying the amount of crosslinker, a different degree of stabilization of the particle matrix can be achieved. Preferably, nanoparticles are prepared which are stabilized to 50% to 200%. These Percentages refer to the molar ratios of the amino groups present on the protein used to the aldehyde functions of glutaraldehyde. A molar ratio of 1: 1 corresponds to 10% stabilization.

In addition to the bifunctional aldehyde glutaraldehyde, other bifunctional substances that can form covalent bonds with the protein are suitable for stabilizing the protein matrix. These

Substances can, for example, react with amino groups or sulfhydryl groups of the proteins. Examples of suitable crosslinking agents are formaldehyde, bifunctional succinimides, isothiocyanates, sulfonyl chlorides, maleimides and pyridyl sulfides.

However, a stabilization of the protein matrix can also be effected by the action of heat. Preferably, the protein matrix is stabilized by a two-hour incubation at 70 ⁰ C or a one-hour incubation at 80 ⁰ C.

The carrier system according to the invention is not a chemically covalent bond of an active substance to the protein because of the crosslinking which takes place after precipitation of the nanoparticles. Rather, the active ingredient is embedded in the matrix of the carrier system. Therefore, the incorporation of the drug is largely independent of the nature of the drug and universally applicable. In contrast to covalently bound drug conjugates, where it is necessary that the drug-protein binding can be cleaved in the target tissue to achieve a release of the drug, the drug release takes place in the inventive colloidal carrier system by the degradation of the Protein structure by lysosomal enzymes that are present in all tissues. The direct cleavage of a drug-protein binding is not necessary.

The present particle system for overcoming

Resistance in tumor cells has the following advantages:

1. Overcoming the resistance of tumor cells. 2. Increased cytotoxicity on tumor cells, compared to liposomal preparations and the solution of an active ingredient.

3. The protein-based nanoparticles consist of physiological material. 4. No additional medication with Pgp inhibitors is necessary.

5. The active ingredient is protected from external influences inside the particle matrix.

6. Modifications of particle surfaces are easily possible.

By chemical reaction of the functional surface located on the particle surface (amino groups, carboxyl groups, hydroxyl groups) with suitable chemical reagents, for example, polyethylene glycol chains (PEG) of different chain length to the Nanopartikθl be bound. In this method, called pegylation or protein pegylation, the surface modification of the nanoparticles is essentially brought about by stable, covalent bonds between an amino or sulfhydryl group on the protein and a chemically reactive group (carbonate, ester, aldehyde or tresylate) on the PEG. The resulting structures can be linear or branched. The PEGylation reaction is determined by factors such as mass of the PEG, type of protein, concentration of the protein in the reaction mixture,

Reaction time, temperature and pH. Therefore, the appropriate PEGs must be determined for each carrier system.

In addition to the PEGylation of the particle surface in the narrower sense, ie the reaction of the protein particles with monofunctional PEG derivatives, bifunctional PEG derivatives can also be bound to the particle surface in order to couple so-called "drug-targeting ligands ^w to the particles. Other surface modifications include, for example, the reaction of functional groups on the particle surface with acetic anhydride or iodoacetic acid to attach acetyl or acetic acid groups, respectively.

The surface of the nanoparticles according to the invention can also be modified by protein-chemical reactions with a corresponding drug-targeting ligand, whereby enrichment of the nanoparticles in certain organs or cells can be achieved without prior adaptation of the carrier system. Suitable receptors for the drug-targeting ligands are all tumor-specific proteins. Particularly preferred antibodies recognizing tumor-specific proteins are used as "drug-targeting ligands", for example the antibodies trastuzumab and cetuximab Trastuzumab (Herceptin®) recognizes HER2 receptors that are overexpressed by a large number of tumor cells and is used for the treatment of breast cancer Cetuximab (Erbitux®) recognizes the epidermal receptor

Growth factor on a variety of tumor cells and is approved for the treatment of colorectal carcinoma. In addition to antibodies, "drug targeting" can also be carried out via ligands bound to the particles, such as transferrin, which recognizes the transferrin receptor overexpressed by tumor cells, or can be achieved via low-molecular receptor ligands such as galactose, which is bound to hepatocytes by the asialoglycoprotein receptor.

embodiment

To prepare nanoparticles according to the invention, 20.0 mg of human serum albumin and 1.0 mg doxorubicin hydrochloride were dissolved in 1.0 ml ultrapure water, which corresponds to a molar ratio of 5: 1 (active ingredient to protein), and incubated for 2 h with stirring. Addition of 3.0 ml of 96% ethanol through a pump system (1.0 ml / min) precipitated serum albumin as nanoparticles. These were obtained by adding different amounts of glutaraldehyde, 8% (Table 1). cross-linked in different amounts for 24 h. The stabilized nanoparticles were divided into 2.0 ml aliquots and purified by centrifugation and redispersion in an ultrasonic bath for 3 cycles. The supernatants of the individual washing steps were collected and determined the proportion of unbound doxorubicin in them by RPl8-HPLC. To determine the nanoparticle concentration 50.0 ul of the preparation were applied to a balanced metal boat and dried at 80 ⁰ C for 2 h. After cooling, the mixture was weighed again and the nanoparticle concentration was calculated.

The loading efficiency with doxorubicin was determined by quantitation of the unbound fraction by RP18-HPLC. Depending on the degree of cross-linking, the absolute loading was 35.0-48.0 μg of active ingredient per mg of the carrier system. The transport capacity of the carrier system is thus about 10 active ingredient molecules per carrier unit (= nanoparticles).

Table 1: Stabilization of doxorubicin-containing nanoparticles based on human serum albumin

(. Dxr soln) to test the cytotoxicity of doxorubicin IN Nanopartikθl (Dxr NP) produced compared to a doxorubicin liposomal doxorubicin and a preparation (Caelyx ^®), following Zeil- lines were used:

• Parental cells of a human neuroblastoma cell line of the University Hospital Frankfurt (UKF-NB3 Par.) • Doxorubicin-resistant cells of the human

Neuroblastoma cell line of the University Hospital Frankfurt (UKF-NB3 Dxr-R.)

The MTT test was used to determine cytotoxicity. With this test, the viability of the cells in the presence of different concentrations of a substance is determined and compared with a cell control. From the results it is possible to calculate the IC50 value (inhibitory concentration 50), the concentration of a substance at which 50% of the cells die off. The assay is based on the reduction of 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide in the mitochondria of vital cells. The yellow tetrazolium salt is reduced to formazan, which precipitates as blue crystals. After dissolution of the crystals with SDS / DMF solution, the color intensity can be measured photometrically. Herein, high absorption means high cell viability.

For testing cytotoxicity in parental and resistant neuroblastoma cells, the cells were evenly divided into a 96-well microtiter plate. One column of the wells contained pure medium and corresponded to the blank, in a second column the cells for growth control (100% value) were cultured. The other wells were pipetted with doxorubicin-containing preparations (Dxr-NP, Dxr-Lsg and Dxr-Lip) with increasing from right to left (0.75, 1.5, 3.0, 6.0, 12.5 25.0, 50.0, 100.0 ng / ml). The microtiter plate was then incubated for 5 days in the incubator at 37 ° C, 5% CO ₂ . 25 μl of MTT solution were pipetted into each well and incubated again for 4 h at 37 ° C. in the incubator. The reduction of the tetrazolium bromide to the blue formazan crystals was stopped by the addition of 100 μl of SDS / DMF solution. After a further incubation at 37 ⁰ C overnight, the color crystals had completely dissolved and there was measured the color intensity in each depression photometrically at 620/690 order. By subtracting the blank from the measurements and referring to the control, cell viability can be expressed as a percentage.

The cytotoxicity of various preparations containing doxorubicin was performed on a parental neuroblastoma cell line (UKF-NB3 par.) Without resistance mechanisms and a doxorubicin-resistant neuroblastoma cell line (UKF-NB3 Dxr-R.). Testing of the parental cell line (Figure 1) revealed that both the Dxr-Lsg and the Dxr-NP with a 100% stabilization have a strong cytotoxic effect on parental neuroblastoma cells. Already at a low concentration of 3 ng / ml doxorubicin cell viability decreased to below 50%. The liposomal Dxr preparation (Caelyx®) showed a clear lower cytotoxic effect on the cells. Here higher concentrations of drug were required (25.0 ng / ml). The calculation of the IC50 values for the individual formulations confirms this result (Table 2). Dxr-NP and Dxr-Lsg killed 50% of the cells already at concentrations of 2.4 ng / ml and 1.6 ng / ml, respectively, whereas the Dxr-liposomes with an IC50 of 25.8 ng / ml significantly had to be dosed higher.

To investigate a possible resistance overcome, the doxorubicin-containing preparations were also tested on doxorubicin-resistant neuroblastoma cells. Here, a significant difference in the various preparations showed (Figure 2). The highest cytotoxicity is shown by the nanoparticulate Dxr preparation with an IC50 of 14.4 ng / ml. A significantly weaker influence on cell viability was found in the Dxr solution. In her case the IC50 increased to 53.46 ng / ml compared to the test in the parent UKF-NB3 cells. The liposomal Dxr preparation had no effect on the growth of the UKF-NB3 Dxr-R. Cells. Even concentrations of 100 ng / ml doxorubicin showed no cytotoxic effect.

Table 2: IC50 values of Dxr-NP, Dxr-Lsg, Dxr-liposomes in parental and resistant UKF-NB3 cells.

UKF-NB3 par. UKF-NB3 Dxr-R.

Dxr-NP 2, 4 ng / ml 14, 4 ng / ml

Dxr-Lsg 1, 6 ng / ml 53, 5 ng / ml

Dxr liposomes 25, 8 ng / ml> 100, 0 ng / ml The results of the cytotoxicity test make it clear that doxorubicin strongly inhibits cell growth of tumor cells in various preparations. In non-resistant cells, the Dxr nanoparticles and the Dxr solution showed a comparable effect. But it comes during a

Cytostatic therapy for the development of drug resistance, the nanoparticulate Dxr preparation is superior to a drug solution. By contrast, liposomal Dxr preparations are unable to overcome resistance mechanisms of tumor cells.

Claims

claims

1. Nanoparticulate for treating resistant tumor cells, comprising a matrix of at least one protein, in which at least one active ingredient is embedded.

2. Nanoparticulate according to claim 1, characterized in that the protein is selected from the group comprising albumin, gelatin, casein and immunoglobulins, wherein human serum albumin is particularly preferred.

3. Nanoparticle according to one of the preceding claims, characterized in that the active substance is an antineoplastic agent.

4. Nanoparticles according to one of the preceding claims, characterized in that the active ingredient is selected from the group of cytostatics, the cytostatic agents, chemically defined cytostatics from the groups of alkaloids, in particular the vinca alkaloids, the podophyllotoxins, Podophyllotoxinderivate, alkylating agents, in particular Nitrosoureas, nitrogen mustard analogs, cytotoxic antibiotics, preferably anthracyclines, antimetabolites, particularly folic acid analogs, purine analogs and pyrimidine analogs.

5. Nanoparticles according to one of the preceding claims, characterized in that the active substance is selected from the group consisting of mistletoe preparations, vinblastine, vincristine, vindesine, vinorelbine, etoposide, teniposide,

Nismuthine, Carustin, Lomustine, Cyclophosphamide, Estramutin, Melphalan, ifosfamide, trofosfamide, chlorambucil, bendaiαustin, dacarbazine, busulfan, procarbazine, treosulfan, temozolomide, thiotepa, daunorubicin, doxorubicin, epirubicin, mitoxantrone, isarubicin, bleomycin, mitomycin, dactinomycin, methotrexate, fludarabine, cladribine, mercaptopurine, thioguanine, cytarabine, Gemcitabine, fluorouracil, capecitabine, paclitaxel, docetaxel, carboplatin, cisplatin and oxaliplatin.

6. Nanoparticles according to claim 3, characterized in that the antineoplastic agent is selected from the group comprising platinum compounds, amsacrine, irinotecan, hydroxycarbamide, pentostatin, porfimer sodium, aldesleukin, tretinin and asparaginase.

7. Nanoparticles according to one of the preceding claims, characterized in that their surface has polyethylene glycol molecules or drug-targeting ligands.

8. nanoparticles according to claim 7, characterized in that it is mono- or bifunctional polyethylene glycol derivatives in the polyethylene glycol molecules.

9. Nanoparticles according to claim 7 or 8, characterized in that the drug-targeting ligands are selected from the group comprising trastuzumab, cetuximab, tumor-specific proteins recognizing antibodies, transferrin and galactose.

10. nanoparticles according to any one of the preceding claims, characterized in that they have a size of 100 to 600 microns, preferably from 100 to 400 microns, and more preferably from 100 to 200 nm.

11. A method of producing nanoparticles for treating resistant tumor cells comprising the steps of:

Dissolving at least one protein and at least one active ingredient in an aqueous medium; Precipitation of the protein in the form of nanoparticles by controlled addition of a non-solvent for the protein, preferably an organic solvent, more preferably ethanol;

Stabilizing the precipitated nanoparticles by adding a crosslinking agent or by heat treatment;

Purify the nanoparticles by washing / centrifuging.

12. The method according to claim 11, characterized in that the molar ratio of active ingredient to protein 5: 1 to 50: 1.

13. The method according to claim 11 or 12, characterized in that the crosslinking agent is selected from the group of substances containing glutaraldehyde,

Formaldehyde, bifunctional succinimides, isothiocyanates, sulfonyl chlorides, maleimides and pyridyl disulfides.

14. The method according to claim 11 or 12, characterized in that the heat treatment for 1 hour at 80 ⁰ C or for 2 hours at 70 ⁰ C.

15. The method according to any one of claims 11 to 14, characterized in that the surface of the nanoparticles is modified by covalent bonding of PEG derivatives and / or drug-targeting ligands.

A method according to claim 15, characterized in that the drug-targeting ligand is selected from the group comprising tumor-specific proteins, antibodies, trastuzumab, cetuximab, transferrin and galactose.

17. Use of nanoparticles according to one of claims 1 to 10 for the manufacture of a medicament for the treatment of tumors, in particular for the treatment of resistant tumors.

18. Use of nanoparticles according to one of claims 1 to 10 for the treatment of tumors, in particular for the treatment of resistant tumors.