Method for carrying therapeutic substances into cells

Abstract

The present invention relates to compositions containing nanoparticies and uses of said composition for transferring therapeutically active substances into cells by means of specifically coated nanoparticles. The chemical design of the particles is such that a large amount thereof is absorbed into the cells. No direct bond between nanoparticle and the therapeutically active substance is required for the transfer into the cells. Thanks to said transfer, an increased efficacy of the substance and simultaneously reduced systemic toxicity is achieved, i.e. an increase in the efficacy while the side effects are reduced.

Claims

1. A method for increasing the activity of an anti-cancer drug comprising the steps of administering to a patient in need thereof a pharmaceutical composition comprising magnetic nanoparticles having an affinity to degenerated cells, at least one pharmaceutical composition comprising an anticancer drug and at least one pharmaceutically acceptable carrier, excipient and/or solvent, wherein the magnetic nanoparticles have a positive surface charge, and comprise a coating of polycondensed aminosilane, wherein the pharmaceutical compositions comprising the magnetic nanoparticles and the at least one anti-cancer drug are administered separately, wherein the magnetic nanoparticles and the at least one anti-cancer drug are present at the same time in the patient, and wherein the increase in activity of the anti-cancer drug occurs without hyperthermia.

2. The method for increasing the activity of an anti-cancer drug according to claim 1, further comprising administering radiation therapy.

3. The method according to claim 1, wherein the nanoparticles comprise iron oxide, magnetite, maghemite or M(II)Fe.sub.2O.sub.4, wherein M represents Zn, Cu, Co, Ni, Cd, Ba or Mn.

4. The method according to claim 1, wherein the at least one cancer drug is a cytostatic agent, an antiproliferative agent, an antiangiogenic agent, or a microtubule inhibitor.

5. The method according to claim 1, wherein the at least one cancer drug is selected from the group comprising: actinomycin D, aminoglutethimide, amsacrine, anastrozole, antagonists of purine and pyrimidine bases, anthracyclines, aromatase inhibitors, asparaginase, antiestrogens, bexarotene, bleomycin, buserelin, busulfan, camptothecin derivatives, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, alkylating cytostatics, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin (adriamycin), doxorubicin lipo, epirubicin, estramustine, etoposide, exemestane, fludarabine, fluorouracil, folic acid antagonists, formestane, gemcitabine, goserelin, hormones and hormone antagonists, hycamtin, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, letrozole, leuprorelin, lomustine, melphalan, mercaptopurine, methotrexate, mitomycins, mitosis inhibitors, mitoxantrone, nimustine, oxaliplatin, pentostatin, procarbazine, tamoxifen, temozolomide, teniposide, testolactone, thiotepa, thioguanine, topoisomerase inhibitors, topotecan, treosulfan, tretinoin, triptorelin, trofosfamide, vinblastine, vincristine, vindesine, vinorelbine, cytostatically active antibiotics, somatostain, bafilomycin, 4-hydroxyoxycyclophosphamide, bendamustine, thymosin α-1, aclarubicin, fludarabine-5′-dihydrogen phosphate, hydroxycarbamide, aldesleukin, pegaspargase, adriamycin, cepharanthine, epothilone A and B, c myc antisense, b-myc antisense, betulinic acid, camptothecin, melanocyte stimulating hormone (α-MSH), lapachol, β-lapachone, podophyllotoxin, podophyllinic acid 2-ethyl hydrazide, molgramostim (rhuGM-CSF), peginterferon α-2b, lenograstim (r-HuG-CSF), filgrastim, macrogol, cephalomannine, trastuzumab, daclizumab, angiopeptin, fluroblastin, bFGF antagonists, probucol, 1,11-dimethoxyeanthin-6-one, 1-hydroxy-11-methoxycanthin-6-one, scopoletin, colchicine, staurosporine, β-estradiol, α-estradiol, estriol, estrone, ethinyl estradiol, fosfestrol, medroxyprogesterone, estradiol cypionates, estradiot benzoates, tranilast, kamebakaurin, tyrosine kinase inhibitors (tyrphostins), ciclosporin A, paclitaxel and derivatives thereof comprising 6-α-hydroxy paclitaxel, baccatin, elipticine, D-24851, colcemid, cytochalasin A-E, indanocine, nocodazole, bacitracin, vitronectin receptor antagonists, free nucleic acids, nucleic acids incorporated into virus transmitters, DNA and RNA fragments, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, antisense oligonucleotide, VEGF inhibitors, thioprotease inhibitors, interferon α, β and γ, NF-kB or Bcl-xL antisense oligonucleotides, halofuginone, nifedipine, tocopherol, tea polyphenols, epicatechin gallate, epigallocatechin gallate, boswellic acids and derivatives thereof, mutamycin, retinoic acid, natural and synthetically obtained steroids comprising bryophyllin A, inotodiol, maquiroside A, ghalakinoside, mansonine, strebloside, hippocaesculin, barringtogenol-C21-angelate 14-dehydroagrostistachin, agroskerin, agrostistachin, 17-hydroxyagrostistachin, ovatodiolids, 4,7-oxycycloanisomelic acid, baccharinoids B1, B2, B3 and B7, tubeimoside, bruceanol A, B and C, bruceantinoside C, yadanziosides N and P, isodeoxyelephantopin, tomenphantopin A and B, coronarin A, B, C and D, ursolic acid, hyptatic acid A, zeorin, iso-iridogermanal, maytenfoliol, effusantin A, excisanin A and B, longikaurin B, sculponeatin C, kamebaunin, leukamenin A and B, 13,18-dehydro-6-alpha-senecioyloxychaparrine, taxamairin A and B, regenilol, triptolide, anopterin, hydroxyanopterin, berberine, cheliburin chloride, cicutoxin, sinococuline, combrestatin A and B, cudraisoflavone A, curcumin, dihydronitidine, nitidine chloride, 12-beta-hydroxypregnadiene-3,20-dione bilobol, helenalin, indicine, indicine-N-oxide, lasiocarpine, justicidin A and B, larreatin, malloterin, mallotochromanol, isobutyrylmallotochromanol, marchantin A, maytansine, lycoridicin, margetine, pancratistatin, liriodenine, bisparthenolidine, oxoushinsunine, aristolactam-AII, deoxypsorospermin, psychorubin, ricin A, sanguinarine, manwu wheat acid, methylsorbifolin, chromones of spathelia, stizophyllin, akagerine, dihydrousambaraensine, hydroxyusambarine, strychnopentamine, strychnophylline, usambarine, usambarensine, daphnoretin, lariciresinol, methoxylariciresinol, syringaresinol, umbelliferone, afromoson, acetylvismione B, desacetylvismione A, vismione A and B.

6. The method according to claim 1, wherein the pharmaceutical compositions are present in formulations which are suitable for injection or infusion.

7. The method of claim 1, wherein the coating consists of polycondensed monomeric aminosilanes selected from the group of 3-aminopropyltriethoxysilane, 2-aminoethyl-3-aminopropyltrimethoxysilane, trimethoxysilyl-propyl-diethylentriamine, and N-(6-aminohexyl)-3-aminopropyltrimethoxysilane.

8. The method of claim 1, wherein the coating consists of polycondensed N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows RUSIRS1 cells 3 hrs after the addition of mitomycin in 0.9% NaCl

(2) FIG. 2 shows RUSIRS1 cells 3 hrs after the addition of mitomycin in 0.9% NaCl+nanoparticle

(3) FIG. 3 shows RUSIRS1 cells 24 hrs after the addition of mitomycin in 0.9% NaCl

(4) FIG. 4 shows RUSIRS1 cells 24 hrs after the addition of mitomycin in 0.9% of NaCl+nanoparticle

(5) FIG. 5 shows RUSIRS1 cells 48 hrs after the addition of mitomycin in 0.9% NaCl

(6) FIG. 6 shows RUSIRS1 cells 48 hrs after the addition of mitomycin in 0.9% NaCl+nanoparticle

(7) FIG. 7 shows RUSIRS1 cells after 48 hrs (control)

(8) FIG. 8 shows BT20 cells after 72 hrs as control with cefamandole but without nanoparticles

(9) FIG. 9 shows BT20 cells after 72 hrs of incubation with nanoparticles and cefamandole

(10) FIG. 10 shows BT20 cells after 72 hrs of incubation with nanoparticles and cefamandole

(11) FIG. 11 shows WiDr cells after 72 hrs as control with cefamandole but without nanoparticles

(12) FIG. 12 shows WiDr cells after 72 hrs of incubation with nanoparticles and cefamandole

(13) FIG. 13 shows WiDr cells after 72 hrs of incubation with nanoparticles and cefamandole

EXAMPLES

Example 1

(14) Increase in the Efficacy of the Cytostatic Mitomycin (In Vitro)

(15) The increase in the efficacy of mitomycin for the treatment of tumor cells could be proved by tests in vitro. The tests in vitro were performed with the glioblastoma human cell line RUSIRS 1 (brain tumor). The glioblastoma cells were taken from tumor tissue of a patient and cultivated as described in DE 199 12 798 C1. 2×10.sup.6 RUSIRS 1 cells, respectively, were prepared in a 75 cm.sup.3 cell culture bottle with 25 ml of cell culture medium (D-MEM+20% FBS+1.2 ml of pyruvate) for testing the efficacy of the mitomycin/nanoparticle mixture. 136 pl of magnetic fluid MFL AS M01 (iron oxide nanoparticle coated with polycondensed N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine, manufacturer: Mag Force Nanotechnologies GmbH, Berlin, Germany) (c.sub.Fe=2 mol/l) and 390 μl of mitomycin solution (1 mg/ml in 0.9% NaCl) were added to said cell suspension. Before being added to the cells, the samples of the nanoparticles were heated to 37° C. for 15 minutes and allowed to rest at RT for 10 minutes. A control sample with mitomycin but without nanoparticles was prepared in the same way.

(16) The influence of the nanoparticles on the efficacy of mitomycin can be illustrated by means of FIGS. 1-6. Cells to which nothing but a mitomycin solution had been added only showed significant damage after 48 hrs of incubation. By contrast, cells which were incubated with the cytostatic and the particles already showed significant damage after 3 hrs. The absorption of the iron oxide nanoparticles into the cells can be proven by a brown coloration of the cell. Control experiments showed that the nanoparticles alone (without mitomycin) are also absorbed, but do not cause a similarly high cell damage. Rapid cell damage (after 3 hrs) occurs only if particle and mitomycin are present at the same time. Consequently, mitomycin was also transferred by the endocytosis of the particles, thereby causing significant cell damage.

Example 2

(17) Increase in the Efficacy of the Antibiotic Cefamandole (In Vitro)

(18) Cefamandole (CAS No 30034-03-8) is used to combat bacterial infections. In 2000, an efficacy against cancer cells was surprisingly found on biopsy material of liver metastases (MagForce Nanotechnologies). This substance's potential to combat cancer cells, however, is to be considered to be rather low. Our experiments showed that a destruction of tumor cells (in vitro), usually, can only be achieved by using a concentration of 0.5 mg/ml (concentration in the cell culture medium) or more. It is, however, possible to drastically increase the efficacy of cefamandole in the treatment of tumor cells by the simultaneous application of nanoparticles.

(19) The experiments in vitro were carried out with the cell lines BT20 (breast carcinoma) and WiDr (colon carcinoma). The tumor cells were taken from tumor tissue of a patient and cultivated as described in DE 199 12 798 C1. 2×10.sup.6 cells, respectively, were prepared in a 75 cm.sup.3 cell culture bottle with 25 ml of cell culture medium (RPMI+10% FBS+1.2 ml of pyruvate for WiDr cells, or respectively BME+10% FBS+pyruvate+5 ml of non-essential amino acids+5 ml glutamine for BT20 cells) for testing the efficacy of the cefamandole/nanoparticle mixture. 136 μl of magnetic fluid MFL AS M01 (iron oxide nanoparticle coated with polycondensed N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine, manufacturer: MagForce Nanotechnologies GmbH, Berlin, Germany) (c.sub.Fe=2 mol/l) and 390 μl of cefamandole solution (stock solution 1 mg/ml in 0.9% NaCl) were added to said cell suspension. Therefore, the concentration of cefamandole in the cell culture medium (25 ml) was 0.016 mg/ml and thus significantly below the threshold of efficacy of pure cefamandole.

(20) After 72 hrs of incubation, significant cell damage could be observed, as evidenced by FIGS. 7-12. After 72 hrs, 30.5% of the BT20 cells and 24% of the WiDr cells had died. Neither cefamandole in the selected concentration, nor the nanoparticle alone, are capable of causing cell death (0% of dead cells). Only the combination of cefamandole and nanoparticles leads to said significant damage of the tumor cells which is due to the transfer of cefamandole into the cells.

Example 3

(21) Preparation of a Pharmaceutical Composition Consisting of Nanoparticles, Pharmacological Active Ingredient and Solvent

(22) About 1 mg of a cytostatic (or 1 to 10 mmol, preferably 2 to 6 mmol of a cytostatic) is added to one ml of an aqueous dispersion of superparamagnetic iron oxide nanoparticles (iron concentration of 2 ml/l).

(23) In the case that the cytostatic is not sufficiently soluble in water, cosolvents in a quantity of up to 20 volume % of the solution can be used. DMSO, DMS, ethanol, acetic acid ethyl ester or other physiologically acceptable solvents may be used as cosolvents.

Example 4

(24) 1 mg of carmustine or 1 mg of cisplatin or 1 mg of epirubicin or 1.5 mg of gemcitabine or 1 mg of imatinib or 0.8 mg of paclitaxel or 1.2 mg of vinblastine or 1 mg of vincristine or 1.5 mg of adriamycin or 1 mg of oxacillin or 1 mg of tetracycline or 1 mg of temozolomide are added to one ml of the magnetic fluid MFL AS M01 (iron oxide nanoparticle coated with polycondensed N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine, manufacturer: MagForce Nanotechnologies AG, Berlin, Germany) (c.sub.Fe=2 mol/l) and thoroughly mixed.

Example 5

(25) Increase in the Efficacy of the Cytostatic Mitoxantrone (In Vitro)

(26) Primary prostate carcinoma cells were cultivated as described in DE 199 12 798 C1. 2×10.sup.6 cells, respectively, were prepared in a 75 cm.sup.3 cell culture bottle with 25 ml of cell culture medium for testing the efficacy of the mitoxantrone/nanoparticle mixture. 136 μl of magnetic fluid MFL AS M01 (iron oxide nanoparticle coated with polycondensed N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine, manufacturer: MagForce Nanotechnologies GmbH, Berlin, Germany) (c.sub.Fe=2 mol/l) and 390 μl of mitoxantrone solution (stock solution 1 mg/ml in 0.9% NaCl) were added to said cell suspension. Significant cell damage could be observed after 72 hrs of incubation. There was evidence for a similar effect when the cytostatics epirubicin and docetaxel (dissolved in polyoxyethylated sorbitol; polysorbate 80) were used.

Example 6

(27) Increase in the Efficacy of the Cytostatic 5-fluorouracil (In Vitro)

(28) Primary rectal carcinoma cells were cultivated as described in DE 199 12 798 C1. 2×10.sup.6 cells, respectively, were prepared in a 75 cm.sup.3 cell culture bottle with 25 ml of cell culture medium for testing the efficacy of the 5-fluorouracil/nanoparticle mixture. 136 μl of magnetic fluid MFL AS M01 (iron oxide nanoparticle coated with polycondensed N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine, manufacturer: MagForce Nanotechnologies GmbH, Berlin, Germany) (c.sub.Fe=2 mol/l) and 390 μl of 5-fluorouracil solution (stock solution 1 mg/ml in 0.9% NaCl) were added to said cell suspension. Significant cell damage could be observed after 72 hrs of incubation. There was evidence for a similar effect when the cytostatics irinotecan and oxaliplatin were used.

Example 7

(29) Increase in the Efficacy of the Cytostatic Carboplatin (In Vitro)

(30) Primary bronchial carcinoma cells (non-small cell lung cancer; NSCLC) were cultivated as described in DE 199 12 798 C1. 2×10.sup.6 cells, respectively, were prepared in a 75 cm.sup.3 cell culture bottle with 25 ml of cell culture medium for testing the efficacy of the carboplatin/nanoparticle mixture. 136 μl of magnetic fluid MFL AS M01 (iron oxide nanoparticle coated with polycondensed N-(2-aminoethyl)-3-(trimethoxysilyl)propylamine, manufacturer: MagForce Nanotechnologies GmbH, Berlin, Germany) (c.sub.Fe=2 mol/l) and 390 μl of carboplatin solution (stock solution 1 mg/ml in 0.9% NaCl) are added to said solution. Significant cell damage could be observed after 72 hrs of incubation. There was evidence for a similar effect when the cytostatics epirubicin and docetaxel (dissolved in polyoxyethylated sorbitol; polysorbate 80) were used.

Examples 8-196

(31) Corresponding to the experiment procedure according to example 2, the following 7 cell lines were tested in vitro with the active ingredients listed in table 1: a) glioblastoma human cell line RUSIRS 1; b) breast carcinoma cell lines BT20; c) colon carcinoma cell line WiDR; d) bronchial carcinoma cells NSCLC, e) rectal carcinoma cells and f) prostate carcinoma cell line DU 145.

(32) In all cases, an increased activity of the cytostatic could be observed. The increase in activity is indicated in parentheses after the respective cytostatic, wherein (+) means an increase of about 5% to 80% and (++) means an increase of 80% to 500%.

(33) TABLE-US-00001 TABLE 1 increase in the activity of cytostatics glioblastoma breast colon prostate human cell carcinoma carcinoma bronchial rectal carcinoma neuroglioma line cell lines cell line carcinoma carcinoma cell line cell line RUSIRS1 BT20 WiDr cells NSCLC cells DU 145 H4 letrozole letrozole letrozole letrozole letrozole letrozole letrozole (+) (+) (+) (+) (+) (+) (+) tamoxifen tamoxifen tamoxifen tamoxifen tamoxifen tamoxifen (+) (+) (++) (+) (+) (+) somatostatin somatostatin somatostatin somatostatin somatostatin somatostatin (+) (+) (+) (+) (++) (+) tacrolimus tacrolimus tacrolimus tacrolimus tacrolimus tacrolimus (+) (+) (+) (+) (+) (++) ascomycin ascomycin ascomycin ascomycin ascomycin ascomycin (++) (+) (+) (+) (+) (+) cerivastatin cerivastatin cerivastatin cerivastatin cerivastatin cerivastatin cerivastatin (+) (+) (+) (+) (++) (+) (+) simvastatin simvastatin simvastatin simvastatin simvastatin simvastatin simvastatin (+) (+) (+) (++) (+) (+) (+) bendamustine bendamustine bendamustine bendamustine (+) (+) (+) (+) tobramycin tobramycin tobramycin tobramycin tobramycin tobramycin tobramycin (+) (+) (+) (+) (+) (+) (+) ganciclovir ganciclovir ganciclovir ganciclovir ganciclovir ganciclovir ganciclovir (++) (+) (+) (+) (+) (+) (+) acyclovir acyclovir acyclovir acyclovir acyclovir acyclovir acyclovir (+) (+) (+) (+) (++) (+) (+) ibuprofen ibuprofen ibuprofen ibuprofen ibuprofen ibuprofen ibuprofen (+) (+) (++) (+) (+) (+) (+) paclitaxel paclitaxel paclitaxel paclitaxel paclitaxel paclitaxel paclitaxel (+) (++) (+) (+) (+) (+) (+) diclofenac diclofenac diclofenac diclofenac diclofenac diclofenac (+) (+) (+) (++) (+) (+) azelastine azelastine azelastine azelastine azelastine (+) (+) (+) (+) (+) oxacillin oxacillin oxacillin oxacillin oxacillin oxacillin oxacillin (+) (+) (++) (+) (+) (++) (+) nifedipine nifedipine nifedipine nifedipine nifedipine nifedipine (+) (+) (+) (+) (+) (+) leflunomide leflunomide leflunomide leflunomide leflunomide leflunomide leflunomide (+) (+) (+) (+) (+) (+) (+) tetracycline tetracycline tetracycline tetracycline tetracycline tetracycline tetracycline (+) (+) (+) (+) (++) (++) (+) rapamycin rapamycin rapamycin rapamycin rapamycin rapamycin rapamycin (+) (++) (+) (+) (++) (+) (+) imatinib imatinib imatinib imatinib imatinib imatinib imatinib (+) (+) (++) (+) (+) (+) (+) vincristine vincristine vincristine vincristine vincristine vincristine vincristine (+) (++) (+) (++) (++) (+) (+) gemcitabine gemcitabine gemcitabine gemcitabine gemcitabine gemcitabine gemcitabine (+) (+) (+) (++) (+) (+) (+) cladribine cladribine cladribine cladribine cladribine cladribine (+) (+) (++) (++) (+) (+) idarubicin idarubicin idarubicin idarubicin idarubicin idarubicin idarubicin (++) (+) (+) (++) (+) (+) (+) busulfan busulfan busulfan busulfan busulfan (+) (+) (++) (+) (+) chlorambucil chlorambucil chlorambucil chlorambucil chlorambucil chlorambucil chlorambucil (+) (+) (++) (+) (+) (+) (++) carmustine carmustine carmustine carmustine carmustine carmustine carmustine (+) (+) (+) (++) (+) (+) (+) cisplatin cisplatin cisplatin cisplatin cisplatin cisplatin cisplatin (+) (+) (+) (++) (+) (+) (++)