TARGETED ANTHRACYCLINE DELIVERY SYSTEM FOR CANCER TREATMENT

Abstract

The present invention relates to a drug delivery system at least comprising a drug encapsulated in a polymeric nano vesicle (polymersome), wherein the drug component is an anthracycline derivative according to the formula I wherein R.sup.1 is selected from the group consisting of H, F, —OMe or —OEt; R.sup.2 is selected from the group consisting of H, —OMe, methyl or ethyl; R.sup.3 is selected from the group consisting of H, methyl or ethyl, and R.sup.4 is H or a protecting group; the polymersome is formed by polymers comprising PEG, PEA, PCL, PTMC or PTMB building blocks or combinations thereof, wherein the polymersome polymers are, at least in part, functionalized by chemically attaching via a linker group L a targeting moiety, wherein the targeting moiety is selected from the group consisting of antibodies, peptides, aptamers or mixtures thereof. In addition, the present invention relates to process for the production of an anthracycline derivative loaded, targeted polymersome drug delivery system, a pharmaceutical composition comprising said drug delivery system and the use of said pharmaceutical composition for the treatment of cancer.

Claims

1) Drug delivery system at least comprising a drug encapsulated in a polymeric nanovesicle, characterized in that the drug component is an anthracycline derivative according to the formula I ##STR00005## wherein R.sup.1 is selected from the group consisting of H, F, —OMe and —OEt; R.sup.2 is selected from the group consisting of H, —OMe, methyl and ethyl; R.sup.3 is selected from the group consisting of H, methyl and ethyl, and R.sup.4 is H or a protecting group; wherein the polymersome is formed by polymers comprising PEG, PLA, PCL, PTMC or PTMB building blocks or combinations thereof, wherein the polymersome polymers are, at least in part, functionalized by chemically attaching via a linker group L a targeting moiety, and wherein the targeting moiety is selected from the group consisting of antibodies, peptides, aptamers or mixtures thereof.

2) The drug delivery system according to claim 1, wherein the targeting moiety is a peptide and the peptide is a tumor penetrating peptide selected from the group of consisting of CendR peptides, iRGD (CRGDKGPDC), LyP-1 (CGNKRTRGC), RPAR (RPARPAR), TT1 (CKRGARSTC), LinTT1 (AKRGARSTA), iNGR (CRNGRGPDC), tLyp-1 (CGNKRTR) or precursors thereof.

3) The drug delivery system according to claim 1, wherein the polymersome comprises di-block PEG-copolymers, wherein the second block is selected from the group consisting of PLA, PCL, PTMC and PTMBP.

4) The drug delivery system according to claim 1, wherein the polymersome consists of PEG-PCL diblock-copolymers, and wherein the different polymer blocks have a weight ratio of calculated as PEG-segment weight divided by PCL-segment weight, is larger or equal 0.1 and smaller or equal 5.

5) The drug delivery system according to claim 1, wherein in formula I R.sup.1 = H, R.sup.2 is CH.sub.3, R.sup.3 = H, and R.sup.4 is a protection group.

6) The drug delivery system according to claim 5, wherein R.sup.4 is acetyloxymethyl carbamate.

7) The drug delivery system according to claim 1, wherein the linker group L is maleimide.

8) The drug delivery system according to claim 1, wherein the polymersome has a molar ratio of peptide modified polymer chains to the total number of polymers chains calculated as number of peptide-modified polymer chains divided by total number of polymer chains, is larger or equal to 0.01 and smaller or equal to 0.4.

9) The drug delivery system according to claim 1, wherein the polymersome has a molar ratio of peptide modified polymer chains to the total number of polymers chains calculated as number of peptide-modified polymer chains divided by total number of polymer chains, is larger or equal to 0.05 and smaller or equal to 0.1.

10) The drug delivery system according to claim 1, wherein the polymeric nanovesicle has a concentration of the drug which is larger or equal to 20 .Math.M and smaller or equal to 500 .Math.M.

11) The drug delivery system according to claim 1, wherein the drug loaded polymeric nanovesicle has a polydispersity index which is larger or equal to 0.01 and smaller or equal to 0.25.

12) A process for the production of an anthracycline derivative loaded, targeted polymersome drug delivery system according to claim 1, characterized in that the drug according to formula I is encapsulated in the polymeric nanovesicle by a thin-film hydration step.

13) The process according to claim 12, wherein the drug loaded polymeric nanovesicle is subjected in a further step to a size-exclusion chromatographic step.

14) A pharmaceutical composition comprising the drug delivery system according to claim 1, in a pharmaceutically acceptable solvent.

15) A method of treating cancer comprising the pharmaceutical composition according to claim 14.

Description

EXPERIMENTAL EXAMPLES

[0040] A possible mode of action of the inventive drug delivery system is schematically displayed in FIG. 1.

[0041] For all experiments the following anthracycline derivative (UTO) was used:

##STR00004##

[0042] The anthracycline derivative comprises with respect to formula I the following substitution pattern: R.sup.1 = H; R.sup.2 = CH.sub.3; R.sup.3 = H and R.sup.4 = protecting group (i.e. acetyloxymethyl carbamate).

1. Drug Synthesis

[0043] The UTO synthesis is performed according to the scheme as depicted in FIG. 8. A possible mechanism of in vivo de-protection of the protected drug is shown in FIG. 9.

[0044] Amrubicinone (1) was glycosylated with 1,4-di-O-acetyl-N-trifluoroacetyl-β-L-daunosamine (2) in presence of trimethylsilyl trifluoromethanesulfonate. After quenching of the reaction, the product was purified by column chromatography on silica gel (eluent diethyl ether/ethyl acetate). The carbonyl group of compound 3 was reduced using 2.1 equivalents of sodium triacetoxyborohydride in ethanol, the crude product was extracted in diethyl ether and purified by column chromatography on silica gel (eluent dichloromethane/methanol). Compound 4 was deprotected - N-trifluoroacetyl and O-acetyl groups from the L-daunosamine part were cleaved using lithium hydroxide (10 equivalents) in tetrahydrofuran/methanol/water mixture. The reaction mixture was neutralized to pH 8.2 and the crude product was separated by extraction. The crude product was further purified by column chromatography using lower phase of chloroform/methanol/aqueous ammonia mixture and chloroform as eluents. The purified compound 5 was reacted with 1.9 equivalents of paraformaldehyde in dry chloroform for 3 days. Unreacted compound 5 was separated by filtration through 0.45 .Math.m pore filter, the obtained solution was concentrated and triturated with diethyl ether to obtain compound 6. The product was characterized by Nuclear Magnetic Resonance (NMR).

[0045] Synthesis of compound 8: 100 mg of compound 6 (0.19 mmol) was dissolved in 6 mL of dry dimethyl formamide and 49 mg (1 equivalent, 0.19 mmol) of 4-nitrophenyl-(acetyloxy)-methylcarbonate was added. The mixture was stirred for 26 h at room temperature under argon atmosphere. After this period, reaction mixture was partially concentrated (to ~1 mL) at room temperature under reduced pressure, mixed with 6 mL of solution of acetic acid (1%) in acetonitrile:water (1:1) and stirred for 2 h to hydrolyze the unreacted oxazolidine cycle. The obtained mixture was purified by preparative HPLC (Column Luna C18(2) Axia 27.2×250 mm, eluent system water/acetonitrile). A total of 22 mg of conjugate 8 was separated. The structure of the product was confirmed by NMR and High Resolution Mass Spectrometry (HRMS, calculated MW 627.2185, found MW 627.2182).

2. Synthesis of Drug Delivery Systems

[0046] Polyethylene glycol-polycaprolactone (PEG.sub.5000-PCL.sub.10000; M.sub.w 5,000 and 10,000 respectively) (PEG-PCL), Fluorescein-PEG-PCL (FAM-PEG-PCL), and maleimide-PEG.sub.5000-PCL.sub.10000 (mal-PEG-PCL) were mixed and dissolved in 0.5 mL acetone. The total amount of polymer was 5 mg. Different percentages of mal-PEG-PCL were used (0; 2; 5; 10; and 20%), and all the polymersomes (PS) samples contained 5% of FAM-PEG-PCL polymer. The acetone was evaporated with nitrogen flow forming a thin polymeric film on the wall of the glass vial (Sigma-Aldrich, Germany). Next, the film was hydrated with 0.4 mL of PBS pH 7.4 previously purged with nitrogen flow, heated for 30 seconds in 65° C. water bath and sonicated for 30 seconds. The heating and sonication steps were repeated until the PS were formed and polymer aggregates were not observed in the suspension. After that, 4 equivalents of Cys-RPAR peptide with respect the mal-PEG-PCL were dissolved in 0.1 mL of PBS and added to the PS suspension. The sample was sonicated for additional 10 minutes, mixed in the shaker for 3 h at RT and kept overnight at 4° C. The final volume of PS samples was 0.5 mL and total polymer concentration 10 mg/mL.

[0047] For the encapsulation of the drug inside PS, 50 nmols of drug were dissolved in 100 .Math.L of acetone and added to the polymers dissolved in acetone (total amount of polymer was 5 mg). The acetone was evaporated to form the polymer/drug film and the PS were formed as described above.

[0048] PS were purified by size exclusion chromatography. Agarose beads with a diameter of 45-165 .Math.m (Sephadex 4B gel) were used as stationary phase. The height of the Sephadex gel in a column was 8 cm (volume 25.13 mL). The PS sample was eluted with PBS pH 7.4.

[0049] The average hydrodynamic diameter of PS was measured with dynamic light scattering (DLS) by using a Zetasizer Nano ZSP (Malvern, USA). PS samples were diluted with PBS pH 7.4 to 1 mg/mL. Samples were scanned for 10 seconds at 173°. The results represent and average over 10 runs. Measurements were repeated 3 times and averaged. Zeta potential was measured using Zetasizer Nano ZSP (Malvern, USA) at 0.2 mg of polymer/mL in NaCl 10 mM, performing 50 runs per sample. For transmission electron microscopy (TEM), PS samples were diluted in mQ water (0.5 mg/mL) and transferred onto copper grids for 1 min, stained with 0.75% phosphotungstic acid (pH 7) for 20 sec, air-dried, and visualized using Tecnai 10 TEM (Philips, Netherlands).

[0050] The amount of encapsulated drug was quantified using a Nanodrop 2000c UV-VIS spectrophotometer (Thermo Scientific, USA). For drug quantification, serial dilutions of drug in MeOH:water 1:1 were prepared and the absorbance at 490 nm was measured. Using gathered data, the linear trend line was constructed in MS Excel program and the formula was further used to evaluate the concentration of drug inside PS.

[0051] The percentage of FAM-PEG-PCL in the PS samples was quantified by fluorimetry. First, a calibration curve of FAM-Cys was prepared in MeOH:PBS 1:1 and the fluorescence at 480 nm/535 nm was measured using Victor X5 Multilabel Microplate Reader (Perkin Elmer, USA). PS samples (25 .Math.L) were mixed with 25 .Math.L of MeOH and the fluorescence was measured to calculate the percentage of FAM-PEG-PCL in the PS composition. The FAM-PEG-PCL percentage in the PS composition was 4.9 ± 0.3.

[0052] To estimate the peptide amount on the PS with optimum peptide density, PS were formed using 20% of Mal-PEG-PCL and 80% of PEG-PCL and FAM-Cys-RPAR peptide was conjugated to the PS as described above. The standard curve of FAM-Cys-RPAR was prepared in PBS and the fluorescence was measured by fluorimetry at 480 nm/535 nm. PS functionalized with FAM-Cys-RPAR peptide (25 .Math.L) were mixed with 25 .Math.L of MeOH and the fluorescence was measured to calculate the percentage of FAM-RPAR-PEG-PCL in the PS composition. The FAM-peptide-PEG-PCL percentage with respect the total polymer amount was 6%.

3. Characterization of the Drug Delivery System

3.1 Peptide Density

[0053] For testing the effect of the overall peptide density, polymersomes comprising different RPAR densities on their surface were prepared. The density was varied by using different proportions of maleimide-PEG-PCL (0; 2; 5; 10 and 20%) relative to whole amount of co-polymer used for synthesis. The maleimide group amount determines the maximum achievable peptide density, as peptide conjugation occurs through formation of thioether bond between the cysteine thiol group of the peptide and the maleimide group of the copolymer. All PS were prepared by the method described above. PS were functionalized with Cys-RPAR peptide and contained 5% of FAM-PEG-PCL as a fluorescent-label.

[0054] The hydrodynamic diameter of the different PS samples was measured by dynamic light scattering. The average PS diameter was 105 ± 12 nm and the polydispersity index (PDI) was 0.19 ± 0.02. Transmission electron microscopy showed that all the FAM-labeled RPAR-PS (RPAR-FAM-PS) samples were homogeneous comprising spherical vesicles (FIG. 2).

[0055] In addition, the RPAR density effect on the PS surface was evaluated with respect to the best tumor cell targeting using PPC-1 and M21 tumor cells. PPC-1 cells, derived from human primary prostate cancer cells, comprise, in comparison to healthy cells, elevated expression of NRP-1 receptors. On the contrary, M21 cells, derived from human melanoma cells, are lacking NRP-1. These cell lines provide a useful tool for studying the specific binding and internalization of RPAR-targeted PS to NRP-1 expressing cells.

[0056] Both cell lines were incubated with the different RPAR-FAM-PS samples for 1 h and the cellular binding and internalization were measured using flow cytometry. There was specific binding of RPAR-FAM-PS to PPC-1 cells and the peptide increase on PS surface resulted in higher internalization in PPC-1 cells (FIG. 3). PS formed with 20% maleimide-PEG-PCL showed the highest uptake by PPC-1 cells, approximately 100% of the labeled cells. In contrast, the binding of RPAR-FAM-PS to M21 cells was very low and independent from peptide density, confirming the dependency of cell binding and internalization on the peptide-receptor interaction.

[0057] In addition, the RPAR-FAM-PS uptake by PPC-1 and M21 cells was tested with fluorescence confocal microscopy. After 1 h incubation, the signal representing RPAR-FAM-PS was detected only in PPC-1 cells whereas M21 cells did not show PS uptake. Confirming the results from flow cytometry, significantly higher RPAR-FAM-PS signal was detected in PPC-1 cells incubated with PS formed using 20% maleimide-PEG-PCL. Notably, when PPC-1 cells were incubated with PS having 20% of maleimide-PEG-PCL, the corresponding fluorescent signal was seen inside the cells, indicating the successful cell penetration of RPAR-FAM-PS. Based on these findings, further PS were synthesized using 20% of maleimide-PEG-PCL.

3.2. Encapsulation Efficiency

[0058] For assessment of the UTO encapsulation efficiency the above depicted anthracycline derivative was encapsulated in a PS using a thin-film hydration method. After encapsulation the sample was purified by size exclusion chromatography to remove non-encapsulated drug. The morphology and hydrodynamic diameter of loaded RPAR-functionalized PS (RPAR-UTO-PS), UTO-loaded non-targeted PS (UTO-PS), and “empty” PS (PS) were similar for all samples and in the range of 100 +- 12 nm and a PDI of 0.15 +- 0.06 indicating a very homogeneous PS diameter. The UTO concentration in UTO-PS samples was approx. 50 .Math.M with an encapsulation efficiency (EE) of 80%. The retention of UTO in the PS membrane, probably based on its matching hydrophobic character, resulted in a higher EE compared to encapsulation efficiency of Doxorubicin.Math.HCl (1% EE).

3.3. Cytotoxicity

[0059] For assessment of the UTO cytotoxicity the cytotoxicity of RPAR-UTO-PS, UTO-PS, PS, free UTO, and free DOX in cultured PPC-1 (FIG. 4) and M21 (FIG. 5) cells after 30 min treatment over a range of drug concentrations was tested. Free UTO was significantly more toxic compared to free DOX in PPC-1 cells at concentrations of 2.5 .Math.M (cell viability 68% vs 87% respectively) and 25 .Math.M (14% vs 56% respectively). In M21 cells, free UTO also showed a higher toxicity compared to DOX at concentration of 25 .Math.M (15% vs 58%). In NRP-1 positive PPC-1 cells RPAR-UTO-PS were significantly more toxic than UTO-PS at a concentration of 2.5 .Math.M of UTO (41% of cell viability versus 70%). NRP-1 negative M21 cells showed significantly less viability when treated with free UTO at a concentration of 25 .Math.M, confirming that TPP-PS penetration and thus toxicity in tumor cells depends on the peptide binding to NRP-1. Moreover, RPAR-UTO-PS showed higher toxicity compared to free UTO in PPC-1 cells at 2.5 .Math.M drug concentration, demonstrating that the internalization triggered by the CendR peptide is more efficient compared to cell internalization of the free drug at that concentration.

3.4. In Vivo Testing - Homing Capabilities

[0060] To evaluate the ability of PS targeted with TPPs for specific drug delivery to tumors in vivo, the tumor accumulation of PS labeled with the dye DiR in an orthotopic TNBC model was used. DiR is a hydrophobic molecule with near-infrared (NIR) absorption and emission spectrum, providing a useful tool for whole-body imaging. NIR-light is able to penetrate into tissues whilst having minimal background interference in that region.

[0061] LinTT1-, RPAR-targeted, and nontargeted PS encapsulating DiR (LinTT1-DiR-PS, RPAR-DiR-PS, and DiR-PS) were prepared. The PS were spherical with an average hydrodynamic diameter similar to the previous PS formulations (average size: 116 ± 8 nm, PDI ~0.15), demonstrating that the dye presence in the PS’s membrane is not affecting the structure of the nanovesicles.

[0062] For assessing tumor internalization MCF10CA1a cancer cells - an aggressive human derived TNBC cell line - were used. These cells are known to overexpress surface p32 and NRP-1 proteins, thus making them a good target for LinTT1 and RPAR CendR peptides. TNBC model was used in vivo because LinTT1 peptide has already been used for early detection and treatment of breast tumors.

[0063] LinTT1-DiR-PS, RPAR-DiR-PS, and DiR-PS were injected i.v. into TNBC mice and live imaging was carried out at 1; 3; 6; 24; and 48 h post-injection (FIG. 6). Targeting with LinTT1 and RPAR peptides increased tumor homing of DiR-PS. LinTT1- and RPAR-DiR-PS were detected in the tumor at 3 h after administration, already, while untargeted DiR-PS were visible starting from 24 h post-injection, only. The highest tumor homing was observed 24 and 48 h after injection for LinTT1-DiR-PS. The integral intensity, assessed by the area under the curve (AUC, FIG. 7), in the tumor at 24 h is approx. 42% higher compared to DiR-PS. The AUC for RPAR-DiR-PS was also significantly higher compared to DiR-PS (approx. 25% higher). After 24 and 48 h LinTT1-, RPAR-, and non-targeted DiR-PS were also observed in the liver as well as in the spleen. This can be explained by the crucial role of these organs in the body clearance of drugs and NPs. 48 h after LinTT1-DiR-PS injection, breast tumors and heart were excised and the microscopic localization of PS was analyzed by fluorescence confocal microscopy. The accumulation of LinTT1-DiR-PS deep inside the tumor parenchyma was visible. Cardiotoxicity is one of the drawbacks of anthracyclines, and, therefore, also the accumulation of LinTT1-DiR-PS in the heart of TNBC-bearing mice was assessed. Significantly lower PS signal levels were observed in the heart tissue in comparison to the tumor cells. The LinTT1 receptor, p32, is also overexpressed in activated macrophages, which play an important role in tumor progression.

[0064] In addition, also the LinTT1-DiR-PS co-localization with the CD206 receptor, expressed in pro-tumoral M2 macrophages was measured. It was observed that LinTT1-DiR-PS targeted M2 macrophages in the tumor.

3.5. In Vivo Testing - Drug Accumulation in the Tumor

[0065] Encouraged by the enhanced LinTT1-targeted PS accumulation effect, the drug accumulation after i.v. administration of LinTT1-UTO-PS, UTO-PS, and free DOX in orthotopic MCF10CA1a tumor bearing mice was studied. The samples were injected, allowed to circulate for 24 h, tumors were collected and analyzed by confocal immunoanalysis.

[0066] A significantly higher UTO tumor accumulation in mice injected with LinTT1-UTO-PS in comparison with other samples was found. The UTO fluorescence observed with low co-localization with blood vessels (CD31 staining), suggested that UTO loaded in LinTT1-PS have extravasated and penetrated into the tumor tissue. This result demonstrates that the encapsulation of UTO in LinTT1-PS enhanced the tumor accumulation and penetration of the drug, showing the potential application of our formulation for efficient TNBC treatment.

4. Comparison PS-UTO vs. PS-DOX

[0067] In order to quantitatively evaluate the differences of DOX and UTO in a PS encapsulation system a direct comparison was performed. UTO and DOX were both encapsulated in PEG-PCL-PS using the film hydration method. The encapsulation and PS formation for this test was performed as described below:

4.1 PS Formation

[0068] Polymersomes were prepared by dissolving 5 mg of PEG-PCL in 0.3 mL acetone. The acetone was evaporated with nitrogen flow forming a thin polymeric film on the wall of the glass vial. The film was hydrated with 0.5 mL of PBS pH 7.4 previously purged with nitrogen flow, heated for 30 seconds in a 65° C. water bath, and sonicated for 30 seconds. The heating and sonication steps were repeated until the PS were formed and polymer aggregates were not observed in the suspension. The final volume of PS samples was 0.5 mL and the total polymer concentration 10 mg/mL.

4.2 Drug Encapsulation

[0069] For the PS encapsulation of UTO 50 nM of UTO were dissolved in 100 .Math.L of acetone (0.5 mM concentration) and added to the polymers dissolved in acetone (total amount of polymer - 5 mg, UTO concentration 0.125 mM). The acetone was evaporated to form the polymer/drug film and the PS were formed as described above.

[0070] For DOX encapsulation the polymeric film was hydrated with 2 mM DOX solution in PBS pH 7.4 and the PS were formed as described above. The higher DOX concentration is necessary because the DOX encapsulation efficiency is approximately 20 times lower compared to UTO. The used difference leads to a comparable UTO and DOX concentration in the PS.

4.3 Purification

[0071] UTO-PS and DOX-PS samples were purified using size exclusion chromatography. As a stationary phase agarose beads were used with a diameter of 45-165 .Math.m (Sephadex 4B gel). The height of the Sephadex gel in a column was 8 cm (volume 25.13 mL) giving the dead volume of ~2.5 mL. The PS fraction (0.5 mL) was collected until the turbidity of the solution, indicating the presence of PS, was not observed.

4.3 Encapsulation Efficiency (EE)

[0072] The UTO and DOX PS-encapsulated amounts were quantified using UV-VIS spectrometry (Thermo Scientific, USA). For UTO quantification, serial dilutions of UTO in MeOH:water 1:1 were prepared and the absorbance at 485 nm was used for calibration. For DOX quantification, serial dilutions of DOX in PBS were prepared and also the absorbance at 485 nm was measured. A linear fit of the data was further used to evaluate the UTO and DOX concentration.

[0073] The quantitative results reveal, that after purification the encapsulation efficiency (EE) of UTO-loaded PS was 85% and dramatically higher compared to the 2.3% achievable for doxorubicin. HCl (DOX). This is very surprising based on the similar chemical structures of the different drugs. Consequently, much higher drug loadings can be achieved by using UTO instead of DOX.

[0074] FIG. 10 displays the hydrodynamic diameter of DOX-PS and UTO-PS measured by Dynamic Light Scattering (DLS). Very similar PS sizes are achievable using DOX or UTO. The mean particle size is 92 nm (+- 33) for DOX-PS and 87 nm (+- 37) for UTO-PS.

4.3 Drug Release

[0075] To further evaluate the UTO and DOX cumulative release behavior from polymersomes, UTO-and DOX-loaded PS were incubated at 37° C. in PBS (0.25 mL) for different time periods (0; 1; 4; 24; and 48 h). The samples were centrifuged using Amicon Ultra centrifugal filters (MWCO 100 kDa) for 20 min at 6,000 g at RT. The fluorescence of the filtrates was measured at 485 nm/535 nm (0.1 s) using a Victor X5 Multilabel Microplate Reader (Perkin Elmer, USA) to quantify the released drug amount.

[0076] The results of the drug release are displayed in FIG. 11. It is shown that after 48 h less than 3% of UTO was released from the PS. In comparison to UTO approximately 10% DOX was released from the PS. This finding is a further indicator that UTO is surprisingly the “better” drug in a PS encapsulation system. The drug is encapsulated in higher amounts and the drug is better retained in PS compared to DOX. Such a behavior is very surprising, considering the structural similarities of UTO and DOX. Nevertheless, the better in-vivo efficacy might also be based, at least in part, on the better encapsulation and higher storage stability compared to DOX.