Targeted anthracycline delivery systems for cancer therapy

Abstract

The present invention relates to a drug delivery system comprising at least a drug encapsulated in a polymer nanovesicle, wherein the drug component is an anthracycline derivative according to formula I, 1 is selected from the group consisting of H, F, -OMe, or -OEt; R 2 is selected from the group consisting of H, -OMe, methyl or ethyl; R 3 is selected from the group consisting of H, methyl or ethyl, and R 4 is H or a protecting group; the polymersome is formed by a polymer comprising a PEG, PLA, PCL, PTMC, or PTMB building block, or a combination thereof, wherein the polymersome polymer is functionalized, at least in part, by chemically attaching a targeting moiety via a linker group L, the targeting moiety being selected from the group consisting of an antibody, a peptide, an aptamer, or a mixture thereof. Furthermore, the present invention relates to a method for producing a targeted polymersome drug delivery system loaded with an anthracycline derivative, a pharmaceutical composition comprising said drug delivery system, and the use of said pharmaceutical composition for the treatment of cancer.

Description

[Technical Field]

[0001] The present invention relates to a drug delivery system comprising at least a drug encapsulated in a polymer nanovesicle (polymersome), wherein the drug component is an anthracycline derivative according to Formula I.

[0002] [ka]

[0003] R 1 is selected from the group consisting of H, F, -OMe, or -OEt; R 2 is selected from the group consisting of H, -OMe, methyl, or ethyl; R 3 is selected from the group consisting of H, methyl, or ethyl, and R 4 is H or a protecting group; the polymersome is formed by a polymer comprising a PEG, PLA, PCL, PTMC, or PTMB building block, or a combination thereof; the polymersome polymer is functionalized, at least in part, by chemically attaching a targeting moiety via a linker group L, the targeting moiety being selected from the group consisting of an antibody, a peptide, an aptamer, or a mixture thereof. Furthermore, the present invention relates to a method for producing a targeted polymersome drug delivery system loaded with an anthracycline derivative, a pharmaceutical composition comprising said drug delivery system, and the use of said pharmaceutical composition for the treatment of cancer. [Background technology]

[0004] Anthracyclines, such as doxorubicin and daunorubicin, have historically been the most widely used and effective anticancer drugs in cancer treatment and have been used for over 30 years. Their mechanism of action is based on DNA intercalation, which causes interference with DNA synthesis and repair and RNA production, leading to inhibition of cell replication and subsequent cell death.

[0005] Structurally, anthracyclines are tetracyclic molecules with an anthraquinone backbone linked to a sugar via a glycosidic bond. The tetracyclic structure intercalates into DNA, with the sugar interacting with adjacent base pairs in the minor groove. Intercalation into DNA forms a stable complex between the anthracycline, DNA, and topoisomerase II, thus inhibiting the action of topoisomerase II and preventing DNA repair. The mechanism of action of anthracyclines also involves the formation of toxic reactive oxygen species generated by the quinone moiety.

[0006] Although anthracyclines are effective against several cancer types, their use is limited by cardiotoxicity. Extensive research has been conducted to develop synthetic anthracyclines with a wide therapeutic index and delivery systems that reduce side effects. PEGylated liposomal doxorubicin (Caelyx) has been used to treat breast and ovarian cancer, multiple myeloma, and Kaposi's sarcoma, demonstrating improved cardiac safety without compromising the anticancer efficacy of doxorubicin. Despite the reduced cardiotoxicity of liposomal anthracyclines compared with the free drug, their anticancer efficacy is not significantly higher in some cancer types, such as metastatic breast cancer. Furthermore, skin toxicity and mucositis, among others, are common side effects of liposomal doxorubicin.

[0007] Therefore, there is an urgent need to develop more potent chemotherapeutic agents and tumor-specific delivery systems that can reduce the side effects of drug administration. One approach involves the use of drug nanocarriers, such as liposomes, whose increased accumulation in tumors is primarily due to the enhanced vascular permeability and retention (EPR) effect. Unfortunately, the EPR effect is highly heterogeneous and depends on the carcinoma and stage of disease.

[0008] The topic of drug delivery is also addressed in the patent literature.

[0009] For example, US 2008 181 939 A1 discloses a hydrolysis-triggered controlled-release polymersome nanodelivery system for delivering cytotoxic anticancer therapeutic active agents to cells, the system comprising: at least one hydrolytically degradable hydrophobic copolymer that, when combined with hydrophilic PEO, controls the hydrolysis of polyester chains in the membrane such that the PEO volume fraction (fEO) and chemical chains control the encapsulant release kinetics from the copolymer vesicle and membrane destabilization of the polymersome carrier; stable, purely synthetic, released PEO-based polymersome vesicles having a semipermeable, thin-walled, amphiphilic, high-molecular-weight polyethylene oxide (PEO)-based block copolymer encapsulation membrane with a desired controlled release rate for releasing the anticancer therapeutic encapsulation agent; and when prepared in aqueous solution, at least one hydrophilic PEO block copolymer and at least one inert hydrophobic PEG-block copolymer form amphiphilic high-molecular-weight PEO-based polymersomes with a desired controlled release rate for the at least one anticancer active agent encapsulation agent contained therein and the cytotoxic anticancer therapeutic active agent encapsulated therein.

[0010] Furthermore, US 2011 027 347 A1 describes polymersomes containing one or more biologically active agents, which are derived from specific polymers having the formula XY2, where X contains a hydrophilic group and Y contains a hydrophobic group.

[0011] In addition, US 2017 002 7868 A1 discloses a liposome composition for the treatment of cancer, comprising targeted PEGylated liposomes, the targeted PEGylated liposomes being targeted by 25 to 100 P15 molecules, the P15 molecules being a P15 peptide having the sequence H-Cys-Gly-Gly-Gly-Pro-Pro-Leu-Ser-Gln-Glu-Thr-Phe-Ser-Asp-Leu-Trp-Lys-Leu-Leu-OH, and the targeted PEGylated liposomes being loaded with doxorubicin.

[0012] Nevertheless, in addition to existing solutions in the field of targeted drug delivery systems, there is still a need for further solutions that can provide efficient targeted delivery of potent drugs to tissues of interest.

[0013] It is therefore an object of the present invention to at least partially overcome the drawbacks of the state of the art, in particular to provide a potent drug delivery system in which drug cytotoxicity is improved, undesirable systemic side effects are minimized, and high drug levels in cancer cells are achieved.

[0014] The above-mentioned problem is solved by a drug delivery system comprising the features of independent claim 1. Moreover, the problem is further solved by a method for the preparation of a targeted drug delivery system loaded with an anthracycline derivative, a pharmaceutical composition comprising said delivery system and an inventive use of the delivery system in the treatment of cancer, according to the features of the respective independent claims.

[0015] Preferred embodiments of the invention are also defined by the features disclosed in the description and drawings and by the features of the dependent claims, and unless expressly excluded, collections of features of separate parts are within the scope of the invention. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 illustrates a possible mechanism of action of the drug delivery system of the present invention. [Figure 2] FIG. 2 shows the results of observation of FAM-labeled RPAR-PS (RPAR-FAM-PS) using a transmission electron microscope. [Figure 3] FIG. 3 shows the uptake of PS into PPC-1 cells or M21. [Figure 4] FIG. 4 shows the cytotoxicity of RPAR-UTO-PS, UTO-PS, PS, free UTO, or free DOX in PPC-1 cells. [Figure 5]FIG. 5 shows the cytotoxicity of RPAR-UTO-PS, UTO-PS, PS, free UTO, or free DOX in M21 cells. [Figure 6] Figure 6 shows the results of live imaging of TNBC mice intravenously injected with LinTT1-DiR-PS, RPAR-DiR-PS, or DiR-PS. [Figure 7] Figure 7 shows the area under the curve (AUC) 24 hours after intravenous injection of LinTT1-DiR-PS, RPAR-DiR-PS, or DiR-PS into TNBC mice. [Figure 8] FIG. 8 shows a scheme for UTU synthesis. [Figure 9] FIG. 9 shows a possible deprotection mechanism of the protected drug in vivo. [Figure 10] FIG. 10 shows the hydrodynamic diameters of DOX-PS and UTO-PS measured by dynamic light scattering (DLS). [Figure 11] FIG. 11 shows the cumulative release behavior of UTO and DOX from polymersomes. Summary of the Invention

[0017] The above problem is solved by a drug delivery system comprising at least a drug encapsulated in a polymer nanovesicle (polymersome, PS), wherein the drug component is an anthracycline derivative according to formula I.

[0018] [ka]

[0019] R 1 is selected from the group consisting of H, F, -OMe, or -OEt; R 2 is selected from the group consisting of H, -OMe, methyl, or ethyl; R 3 is selected from the group consisting of H, methyl, or ethyl, and R 4is H or a protecting group; the polymersome is formed by a polymer comprising a PEG, PLA, PCL, PTMC, or PTMB building block, or a combination thereof, wherein the polymer of the polymersome is functionalized, at least in part, by chemically attaching a targeting moiety via a linker group L, wherein the targeting moiety is selected from the group consisting of an antibody, a peptide, an aptamer, or a mixture thereof.

[0020] Surprisingly, anthracyclines according to Formula I have been found to exhibit several synergistic advantages when combined with the targeted polymersome (PS) delivery system described above. Certain anthracyclines exhibit enhanced anticancer efficacy against prostate cancer and melanoma cells compared to doxorubicin. Furthermore, encapsulation results in biocompatible and biodegradable polymersomes, and encapsulation is achieved at high encapsulation rates. The delivery system can be tailored to target cells by selecting targeting moieties, such as tumor-penetrating peptides (TPPs), or other classes of affinity targeting ligands, and the efficacy of the system can be flexibly tailored, for example, by varying the density of tumor-penetrating peptides on the PS. Homing systems generally increase drug toxicity in cells expressing the correct receptor, thus increasing anticancer efficacy and reducing systemic drug side effects. In particular, targeted delivery can avoid undesirable side effects, such as anthracycline cardiotoxicity, preventing unwanted drug accumulation in the heart in vivo. Furthermore, the homing-functionalized delivery system exhibited extremely fast tumor penetration, suggesting applications in tumor detection and imaging. Such synergistic benefits are surprising because in vivo and in vitro efficacy depends on the complex interplay between drug chemistry and the delivery system. Drug encapsulation at high loading levels and the resulting stability of loaded delivery systems during storage and in vivo environments are difficult to predict. Furthermore, the stability and targeting performance of the delivery system must be tailored to ensure minimal drug release during delivery, rapid and specific targeting, complete internalization in target cells, and rapid degradation at the target site. Therefore, at some stage during delivery, subtle but significant changes in the surrounding chemistry must be able to disrupt the stability equilibrium between the drug and PS, resulting in the desired drug action.

[0021] The drug delivery system includes at least a drug encapsulated in a polymer nanovesicle. The disclosed system includes at least two different types of molecules: one is a drug, i.e., a substance that, when administered, causes a physiological or psychological change in a human or animal organism, and the other is a vesicle that surrounds or encapsulates the drug. This means that the drug is embedded inside the vesicle or forms part of the vesicle bilayer, with the bilayer wall formed by the polymer. The size of the vesicle is in the submicron range; for example, the vesicle can be spherical in shape and have a diameter ranging from 50 nm to 500 nm.

[0022] The drug component of the delivery system is an anthracycline derivative according to Formula I.

[0023] [ka]

[0024] R 1 is selected from the group consisting of H, F, -OMe, or -OEt; R 2 is selected from the group consisting of H, -OMe, methyl, or ethyl; R 3 is selected from the group consisting of H, methyl, or ethyl, and R 4are H or protecting groups. This novel 9-aminoanthracycline derivative contains a hydroxyl group at C13 and an oxazolidine ring at C-3' and C-4' of the daunosamine moiety. These 9-aminoanthracyclines are less cardiotoxic than other anthracyclines, and the cytotoxic effect of the anthracyclines can be further enhanced by attaching a methylene or ether group to the amino and hydroxyl groups (between the respective nitrogen and oxygen atoms) of the 1,2-aminoalcohol moiety of daunosamine. The five-membered ring covalently binds to DNA in target cells, forming drug-DNA adducts. The stability of the oxazolidine ring in aqueous media can be adjusted by attaching a protecting group to the nitrogen atom of the five-membered ring. Usable protecting groups can be linked by carbamate bonds. Suitable protecting groups can be acetyloxyalkylcarbamates or similar protecting groups. Under certain physiological conditions, for example, by pH shifts or induced esterases, the protecting group is hydrolyzed, exposing the reactive oxazolidine ring. The oxazolidine ring can react with DNA to form anthracycline-DNA adducts.

[0025] Anthracyclines are among the most effective anticancer drugs, effective against a wide range of cancer types; however, cardiotoxicity limits their dosing and exposes patients to cardiovascular events and potential death. The development of drugs and delivery systems with greater tumor cell toxicity and fewer side effects is necessary to increase the therapeutic index of current cancer treatments. 9-Aminoanthracyclines, such as amrubicin, are less cardiotoxic than other anthracyclines. The metabolism of most anthracyclines involves enzymatic reduction of the C-13 carbonyl group to a hydroxyl group. In the case of amrubicin, the corresponding metabolite, amrubicinol, is 5 to 50 times more potent than the parent drug. The intracellular reduction of anthracyclines also forms free radicals capable of oxidizing other molecules in the cell, producing formaldehyde, which then reacts with the amino group present in the anthracycline to form drug-DNA adducts. To produce the adduct, formaldehyde first reacts with the 3'-amino of daunosamine in the anthracycline to form an activated Schiff base, which can then form an aminal (NCN) linkage with the exocyclic amino group of the guanine residue. This mechanism of cytotoxicity can be facilitated by the formation of an oxazolidine ring. The proposed design of anthracycline derivatives is based on the following: 1) Reduced cardiotoxicity of 9-aminoanthracyclines; 2) increased potency by reducing the C-13 carbonyl group to a hydroxy group; and 3) Increased cytotoxicity due to the formation of an oxazolidine ring.

[0026] When anthracycline derivatives penetrate cells, the protecting group is hydrolyzed by esterases in the cytosol, exposing a reactive oxazolidine ring. The four-ring structure of anthracycline derivatives can intercalate into DNA, and the oxazolidine ring covalently binds to guanine via the methylene carbon, thereby blocking the molecular processes of DNA. Esterases such as carboxylesterases are overexpressed in some cancer types, making selected anthracycline derivatives more tumor-selective than other anthracyclines, such as DOX.

[0027] PS is formed by polymers containing PEG, PLA, PCL, PTMC, or PTMB building blocks, or combinations thereof. PS formed from or containing the above-mentioned polymer blocks has been found to exhibit favorable properties in terms of drug loading capacity, stability, and drug release kinetics. The hydrophilic / hydrophobic nature of the block is assumed to be in a range appropriate for interaction with the functional groups or ring structures of the anthracycline derivatives used in the present invention. The block may contain two or more of the above-mentioned monomers. The meanings of abbreviations are known to those skilled in the art, for example, PEG stands for polyethylene glycol, PLA stands for polylactic acid or polylactide, PCL stands for polycaprolactone, and PTMC stands for polytrimethylene carbonate. The PEG block may contain, for example, 2,000 to 10,000 repeating units, and the other blocks may contain, for example, 5,000 to 40,000 repeating units, preferably, the PEG block may contain 4,000 to 7,000 repeating units, and the other blocks may contain, preferably, 8,000 to 20,000 repeating units.

[0028] The polymersome polymer is functionalized, at least in part, by chemically attaching a targeting moiety via a linker group L, where the targeting moiety is selected from the group consisting of antibodies, peptides, aptamers, or mixtures thereof. To generate a homing or targeted PS, all or part of the polymer contains a chemical moiety, which is covalently attached to the polymer that constitutes the PS via the linker group L. The moiety is generally biologically active in the sense that it interacts biologically with the target cell, for example, by binding to the target cell or by inducing internalization into the target cell. Thus, the chemical moiety induces a favorable interaction between the drug delivery system and the target cell type. Possible moieties can be selected from the list above, where the correct moiety type can be selected as a function of the target. Mixtures can contain two or more different targeting moieties on the same polymersome. Aptamers are, for example, oligonucleotide or peptide molecules that bind to specific target molecules or cells. The target molecule can be, for example, a cell surface receptor. Antibodies are proteins, and the structure of the protein also allows for specific binding of the protein to the target structure. Suitable peptides can be, for example, TPP.Targeting moiety can be linked to polymer by suitable functional group of targeting moiety.Specific functional group is known to those skilled in the art.The suitable degree of polymer functionalization depends on the size of targeting moiety and the stability of PS as a whole.The possible ratio of functionalized polymer to non-functionalized polymer can be 1% or more and 100% or less.

[0029] In a preferred embodiment of the drug delivery system, the polymersome polymer is functionalized, at least in part, by chemically attaching a tumor-penetrating peptide selected from the group consisting of CendR peptide, iRGD (CRGDKGPDC), LyP-1 (CGNKRTRGC), RPAR (RPARPAR), TT1 (CKRGARSTC), LinTT1 (AKRGARSTA), iNGR (CRNGRGPDC), tLyp-1 (CGNKRTR), or their precursors via a linker group L. To generate a homing or targeting PS, all or a portion of the polymer contains a peptide group, which is covalently attached to the polymer constructing the PS via the linker group L. For example, the CendR peptide enhances the permeability of tumor blood vessels and tumor tissue through binding to the transmembrane glycoprotein neuropilin-1 (NRP-1). The CendR peptide contains the sequence (R / KXXR / K), and the iRGD peptide targets tumor fibroblasts or tumor cells and contains the sequence CRGDKGPDC. The LyP-1 peptide targets tumor endothelial cells, macrophages, tumor lymphatic tissue, and tumor cells and contains the sequence CGNKRTRGC. The RPAR peptide targets NRP-1-expressing cells (tumor endothelial cells, macrophages, tumor lymphatic vessels, and tumor cells) and contains the sequence RPARPAR. The TT1 peptide targets tumor endothelial cells, macrophages, tumor lymphatic tissue, and tumor cells and contains the sequence CKRGARSTC. The LinTT1 peptide targets tumor endothelial cells, macrophages, tumor lymphatic tissue, and tumor cells and contains the sequence AKRGARSTA. The iNGR peptide targets tumor endothelial cells or other cells in tumors and contains the sequence CRNGRGPDC. The tLyp-1 peptide targets tumor endothelial cells or other NRP-positive cells in tumors and contains the sequence CGNKRTR. In addition to a given sequence, peptides can be bound to PS in the formation of peptide precursors, which may contain additional functional or non-functional groups, which may be removed from the peptide fragment in vivo prior to binding to target cells.

[0030] In a preferred embodiment of the drug delivery system, the polymersome may contain a diblock PEG copolymer, with the second block selected from the group consisting of PLA, PCL, PTMC, and PTMBP. It has been found useful to construct PS from a diblock copolymer containing at least a PEG block for PS stability, drug loading capacity, and enhanced in vivo half-life. The other block can be selected from the above groups, and the diblock copolymer can be a biodegradable polymer, which provides increased stability in combination with the anthracyclines of the present invention under in vivo conditions and accelerated degradability in tumor cells.

[0031] In a preferred embodiment of the drug delivery system, the polymersome may be composed of a PEG-PCL diblock copolymer, in which the weight ratio of the different polymer blocks, calculated as the weight of the PEG portion divided by the weight of the PCL portion, is 0.1 or more and 5 or less. Encapsulation of anthracycline derivatives in the above-mentioned PS has proven highly effective in in vivo and in vitro tests on tumor cells. Without being bound by theory, it is believed that the interaction between the drug and the diblock chains results in improved stability, reducing the risk of drug release before entering the target cells. Furthermore, in addition to improved stability, favorable drug-polymer interactions also allow for high drug loading in the vesicles. Lower ratios may be disadvantageous because they may reduce PS stability, while higher ratios may be disadvantageous because they may make the PS hydrophobic, thereby reducing PS flexibility and solubility / stability in in vivo media.

[0032] In a preferred embodiment of the drug delivery system of Formula I, R 1 is H and R 2 , R 3 is H and R 4This substitution pattern of anthracyclines has been found to exhibit improved cytotoxicity and improved PS encapsulation stability. The latter may be due to the favorable interaction of the methyl groups with the polymer PS block, while the physiological effect may be due to the substitution and overall ring structure containing the oxazolidine ring, which exhibits better interaction with the DNA structure of target cells.

[0033] In a further preferred feature of the drug delivery system, R 4 The oxazolidine ring may be an acetyloxymethyl carbamate. It has been found useful to incorporate anthracyclines containing a carbamate-protected oxazolidine ring into PS. The protection not only alters the stability of the oxazolidine ring itself, but also appears to favor favorable interactions with the delivery system by increasing interactions with the polymer block. 4 may have the general formula RR'N-CO-O-CHR''-O-CO-R''', where R'' may be H or Me and COR'''' may be acyl. Based on the substitution pattern of the protecting groups, the hydrolysis rate can also be tuned in surrounding tumor cells.

[0034] Alternatively, in a preferred embodiment of the drug delivery system, the group L can be a maleimide. Maleimides have been found to be useful for achieving functional attachment of peptides or peptide precursors to PS. The attachment can be highly selective, and the overall PS structure, drug loading capacity, and integrity are not affected. Attachment of the peptide to the maleimide is preferably effected via a thioether bond between the maleimide and the cysteine ​​amino acid of the peptide.

[0035] In a preferred embodiment of the drug delivery system, the molar ratio of peptide-modified polymer chains to the total number of polymer chains in the polymersome, calculated as the number of peptide-modified polymer chains divided by the total number of polymer chains, can be 0.01 or more and 0.4 or less. The above molar ratio has been found to be preferable for enhancing PS stability and the efficiency of PS internalization into tumor target cells. The overall PS solubility and stability, conferred by the block polymer structure, do not change significantly within this range. Furthermore, a lower ratio may be disadvantageous due to insufficient targeted interaction with tumor cell surface receptors. The ratio can be assessed by fluorescence measurement of fluorescent dye-labeled peptides.

[0036] In a preferred embodiment of the drug delivery system, the molar ratio of peptide-modified polymer chains to the total number of polymer chains in the polymersome, calculated as the number of peptide-modified polymer chains divided by the total number of polymer chains, is 0.05 or more and 0.1 or less. This ratio has been found to be useful for maintaining the basic stability, density, and size characteristics of the anthracycline-loaded PS while also maintaining the desired dissolution and drug release behavior of the target PS. The PS exhibits improved cell binding, internalization efficiency, and size compared to PS with ratios outside this range.

[0037] In a more preferred embodiment of the drug delivery system, the drug concentration in the polymer vesicle can be 20 μM or more and 500 μM or less. The combination of anthracycline and PS allows for a higher drug loading compared to other encapsulation systems. The stability of PS, especially that of the block copolymer described above, allows for such a large amount of drug loading with or without a small amount of release before internalization of PS in target cells. Such stability is unusual because drug loading also affects the polymer loading and polymer interaction in the PS wall.

[0038] Another desirable feature of the drug delivery system is that the average hydrodynamic diameter of the drug-loaded polymer nanovesicles can be greater than 80 nm and less than 125 nm. To achieve the best balance between PS volume and drug loading, stability under in vivo conditions and internalization efficiency, the above-mentioned PS size ranges have been found to be beneficial. Larger sizes can reduce the internalization efficiency and overall stability of the drug in the PS, while smaller PS sizes can negatively affect the available drug content in the PS. The hydrodynamic diameter can be measured by dynamic light scattering, as described in the Examples.

[0039] In a more preferred embodiment of the drug delivery system, the polydispersity index of the drug-loaded polymer nanovesicles can be 0.01 or more and 0.25 or less. The use of PS with the above polydispersity has been found to be beneficial to the internalization efficiency and stability of the drug-loaded PS. Based on the uniform and narrow PS size distribution, systemic drug release can be reduced.

[0040] In addition, it is within the scope of the present invention to disclose a method for preparing an anthracycline derivative-loaded targeted polymersome drug delivery system according to any of the preceding claims, characterized in that a drug according to Formula I is encapsulated in a polymer nanovesicle by a thin film hydration process. In particular, thin film hydration has been found to be useful for constructing the system of the present invention containing a specific anthracycline drug and a specific polymer selection. It is possible to achieve a polymer size distribution in the "right" polymer size range, containing only a low level of polydispersity. In addition, the anthracycline derivative appears to be stably incorporated into the PS, particularly without a significant amount of "loosely" incorporated or only bound drug molecules.

[0041] In a preferred embodiment of this method, the drug-loaded polymer nanovesicles can be subjected to a size-exclusion chromatography step in a further step. This size-exclusion step has been found to be very useful for achieving a very narrow size distribution and ensuring very low levels of improperly incorporated drug. A very stable and narrow size distribution is achieved, in which the drug has a very uniform elution profile upon changes in chemical environment.

[0042] It is within the scope of the present invention to disclose a pharmaceutical composition comprising the drug delivery system of the present invention in a pharmaceutically acceptable solvent. The drug delivery system of the present invention can be easily dissolved in some pharmaceutically acceptable solvents, thus forming a stable suspension. The advantages of the pharmaceutical composition are explicitly mentioned in the advantages of the drug delivery system of the present invention. Suitable acceptable solvents may be selected from the group consisting of, for example, PBS, saline, or a mixture thereof.

[0043] Furthermore, it is within the scope of the present invention to disclose the use of the pharmaceutical composition of the present invention for the treatment of cancer. Pharmaceutical compositions of the present invention, including the drug delivery system of the present invention and the anthracycline drug of the present invention, exhibit excellent antitumor efficacy in in vitro and in vivo experiments. The better efficacy is at least partly due to the higher toxicity of the drug compared to the state-of-the-art anthracyclines. Based on the synergistic combination of the drug and the delivery system, it is possible to provide better biocompatibility and fewer side effects compared to the state-of-the-art delivery systems. Therefore, a safe and highly effective targeted antitumor vehicle is provided by the present invention. [Example]

[0044] The possible mechanism of action of the drug delivery system of the present invention is shown schematically in FIG.

[0045] For all experiments, the following anthracycline derivatives (UTO) were used:

[0046] [ka]

[0047] The anthracycline derivatives, with respect to Formula I, include the following substitution patterns: 1 =H;R 2 =CH3;R 3 =H and R 4 = protecting group (i.e., acetyloxymethyl carbamate).

[0048] 1. Drug Synthesis The synthesis of UTO is carried out according to the scheme shown in Figure 8. Figure 9 shows a possible mechanism for the in vivo deprotection of the protected drug.

[0049] Amrubicinone (1) was glycosylated with 1,4-di-O-acetyl-N-trifluoroacetyl-B-L-daunosamine (2) in the presence of trimethylsilyl trifluoromethanesulfonate. After quenching the reaction, the product was purified by silica gel column chromatography (eluent: diethyl ether / ethyl acetate). The carbonyl group of compound 3 was reduced with 2.1 equivalents of sodium triacetoxyborohydride in ethanol, and the crude product was extracted with diethyl ether and purified by column chromatography on silica gel (eluent: dichloromethane / methanol). Compound 4 was obtained by deprotection of the N-trifluoroacetyl group, and the O-acetyl group from the L-daunosamine moiety was cleaved with lithium hydroxide (10 equivalents) in a tetrahydrofuran / methanol / water mixture. The reaction mixture was neutralized to pH 8.2, and the crude product was isolated by extraction. The crude product was further purified by column chromatography using a chloroform / methanol / aqueous ammonia mixture as the lower phase and chloroform as the eluent. 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 a 0.45 μM pore filter, and the resulting solution was concentrated and triturated with diethyl ether to give compound 6. The product was characterized by nuclear magnetic resonance (NMR).

[0050] Synthesis of compound 8: 100 mg of compound 6 (0.19 mmol) was dissolved in 6 mL of dry dimethylformamide, and 49 mg (1 equivalent, 0.19 mmol) of 4-nitrophenyl-(acetyloxy)-methyl carbonate was added. The mixture was stirred at room temperature under an argon atmosphere for 26 h. After this, the reaction mixture was partially concentrated (to ≤1 mL) under reduced pressure at room temperature and mixed with 6 mL of a 1% solution of acetic acid in acetonitrile:water (1:1) and stirred for 2 h to hydrolyze the unreacted oxazolidine ring. The resulting mixture was purified by preparative HPLC (Luna C18(2) Axia 27.2x250 mm column, eluent water / acetonitrile). A total of 22 mg of conjugate 8 was isolated. The structure of the product was confirmed by NMR and high-resolution mass spectrometry (HRMS, calculated MW 627.2185, observed MW 627.2182).

[0051] 2. Synthesis of Drug Delivery Systems Polyethylene glycol-polycaprolactone (PEG 5,000 -PCL 10,000 MW 5,000 and 1,0000, respectively) (PEG-PCL), fluorescein-PEG-PCL (FAM-PEG-PCL), and maleimide-PEG 5,000 -PCL 10,000The MAL-PEG-PCL was mixed and dissolved in 0.5 mL of 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 polymersome (PS) samples contained 5% FAM-PEG-PCL polymer. The acetone was evaporated with a nitrogen stream to form a thin polymer film on the wall of a glass vial (Sigma-Aldrich, Germany). The film was then hydrated with 0.4 mL of PBS (pH 7.4) previously purged with a nitrogen stream, heated in a 65°C water bath for 30 seconds, and sonicated for 30 seconds. The heating and sonication steps were repeated until PS was formed and no polymer aggregates were observed in the suspension. Then, 4 equivalents of Cys-RPAR peptide relative to MAL-PEG-PCL were dissolved in 0.1 mL of PBS and added to the PS suspension. The sample was sonicated for an additional 10 minutes, mixed in a mechanical mixer at room temperature for 3 hours, and then maintained at 4°C overnight. The final volume of the PS sample was 0.5 mL, and the total polymer concentration was 10 mg / mL.

[0052] For drug encapsulation within PS, 50 nmol of drug was dissolved in 100 μL of acetone and added to the polymer dissolved in acetone (total amount of polymer was 5 mg). The acetone was evaporated to form a polymer / drug film, and PS was prepared as described above.

[0053] PS was purified by size-exclusion chromatography. Agarose beads (Sephadex 4B gel) with diameters of 45–165 μM were used as the stationary phase. The height of the Sephadex gel in the column was 8 cm (volume: 25.13 mL). PS samples were eluted with PBS at pH 7.4.

[0054] The average hydrodynamic diameter of PS was measured by dynamic light scattering (DLS) using a Zetasizer Nano ZSP (Malvern, USA). PS samples were diluted to 1 mg / mL in PBS at pH 7.4. Samples were scanned at 173° for 10 seconds. Results represent the average of 10 runs. Measurements were repeated three times and averaged. Zeta potential was measured at 0.2 mg / mL of polymer in 10 mM NaCl using a Zetasizer Nano ZSP (Malvern, USA), with 50 runs per sample. For transmission electron microscopy (TEM), PS samples were diluted in mQ water (0.5 mg / mL), transferred to a copper grid for 1 minute, stained with 0.75% phosphotungstic acid (pH 7) for 20 seconds, air-dried, and visualized using a Tecnai 10 TEM (Philips, Netherlands).

[0055] The amount of encapsulated drug was quantified using a Nanodrop 2000c UV-VIS spectrophotometer (Thermo Scientific, USA). For drug quantification, serial dilutions of the drug were prepared in 1:1 MeOH:water and the absorbance was measured at 490 nM. The collected data were used to generate a linear trend line in MS Excel, and the resulting equation was used to further evaluate the drug concentration inside the PS.

[0056] The percentage of FAM-PEG-PCL in the PS samples was quantified by fluorescence measurement. First, a calibration curve of FAM-Cys was prepared in 1:1 MeOH:PBS, and fluorescence was measured at 480 nm / 535 nm using a Victor X5 Multilabel Microplate Reader (Perkin Elmer, USA). The PS sample (25 μL) was mixed with 25 μL of MeOH, and the fluorescence was measured to calculate the percentage of FAM-PEG-PCL in the PS composition. The percentage of FAM-PEG-PCL in the PS composition was 4.9 ± 0.3.

[0057] To evaluate the amount of peptide on PS with optimal peptide density, PS was formed using 20% ​​MAL-PEG-PCL and 80% PEG-PCL, and FAM-Cys-RPAR peptide was conjugated to PS as described above. A standard curve of FAM-Cys-RPAR in PBS was generated, and fluorescence was measured by fluorimetry at 480 nm / 535 nm. PS functionalized with FAM-Cys-RPAR peptide (25 μL) was mixed with 25 μL of MeOH, and fluorescence was measured to calculate the percentage of FAM-RPAR-PEG-PCL in the PS composition. The percentage of FAM-peptide-PEG-PCL relative to the total polymer amount was 6%.

[0058] 3. Characterization of Drug Delivery Systems 3.1 Peptide density To test the effect of overall peptide density, polymersomes containing various RPAR densities on the surface were prepared. Density was varied by using various percentages of maleimide-PEG-PCL (0, 2, 5, 10, and 20%) relative to the total amount of copolymer used in the synthesis. Because peptide conjugation occurs via the formation of thioether bonds between the cysteine ​​thiol groups of the peptide and the maleimide groups of the copolymer, the amount of maleimide groups determines the maximum achievable peptide density. All PS were prepared by the method described above. The PS was functionalized with Cys-RPAR peptide and contained 5% FAM-PEG-PCL as a fluorescent label.

[0059] The hydrodynamic diameters of the different PS samples were measured by dynamic light scattering. The average PS diameter was 105 ± 12 nm, with a polydispersity index (PDI) of 0.19 ± 0.02. Transmission electron microscopy showed that all FAM-labeled RPAR-PS (RPAR-FAM-PS) samples were homogeneous, containing spherical vesicles (Figure 2).

[0060] Furthermore, we evaluated the effect of RPAR density on the PS surface for optimal tumor cell targeting using PPC-1 and M21 tumor cells. PPC-1 cells, derived from human primary prostate cancer cells, contain elevated expression of the NRP-1 receptor compared to normal cells. Conversely, M21 cells, derived from human melanoma cells, lack 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.

[0061] Both cell lines were incubated with different RPAR-FAM-PS samples for 1 hour, and cell binding and internalization were measured using flow cytometry. There was specific binding of RPAR-FAM-PS to PPC-1 cells, and increasing the amount of peptide on the PS surface resulted in higher internalization in PPC-1 cells (Figure 3). PS formed with 20% maleimide-PEG-PCL showed the highest uptake by PPC-1 cells, approximately 100% of labeled cells. In contrast, binding of RPAR-FAM-PS to M21 cells was very low and independent of peptide density, confirming the dependence of cell binding and internalization on peptide-receptor interaction.

[0062] Furthermore, RPAR-FAM-PS uptake by PPC-1 and M21 cells was examined by fluorescence confocal microscopy. After 1 hour of incubation, signals representing RPAR-FAM-PS were detected only in PPC-1 cells, whereas M21 cells showed no PS uptake. Flow cytometry confirmed that significantly higher RPAR-FAM-PS signals were detected in PPC-1 cells incubated with PS formed using 20% ​​maleimide-PEG-PCL. Notably, when PPC-1 cells were incubated with PS containing 20% ​​maleimide-PEG-PCL, corresponding fluorescent signals were observed within the cells, indicating successful cellular penetration of RPAR-FAM-PS. Based on these findings, further PS was synthesized using 20% ​​maleimide-PEG-PCL.

[0063] 3.2. Encapsulation efficiency To evaluate the UTO encapsulation efficiency, the above-mentioned anthracycline derivatives were encapsulated in PS using the thin-film hydration method. After encapsulation, the samples were purified by size-exclusion chromatography to remove unencapsulated drug. The morphology and hydrodynamic diameter of the loaded RPAR-functionalized PS (RPAR-UTO-PS), UTO-loaded nontargeted PS (UTO-PS), and "empty" PS (PS) were similar for all samples, ranging from 100 to 12 nm and PDIs of 0.15 to 0.06, indicating highly uniform PS diameters. The UTO concentration in the UTO-PS sample was approximately 50 μM, and the encapsulation efficiency (EE) was 80%. The retention of UTO in the PS membrane resulted in a higher EE compared to the encapsulation efficiency of doxorubicin HCl (1% EE), likely due to its matching hydrophobicity.

[0064] 3.3.Cytotoxicity To evaluate UTO cytotoxicity, we tested the cytotoxicity of RPAR-UTO-PS, UTO-PS, PS, free UTO, and free DOX in cultured PPC-1 cells (Figure 4) and M21 cells (Figure 5) after 30 minutes of treatment across a range of drug concentrations. In PPC-1 cells, free UTO was significantly more cytotoxic than free DOX at concentrations of 2.5 μM (cell viability 68% vs. 87%) and 25 μM (cell viability 14% vs. 56%). In M21 cells, free UTO was also more cytotoxic than DOX at 25 μM (15% vs. 58%). In NRP-1-positive PPC-1 cells, RPAR-UTO-PS was significantly more cytotoxic than UTO-PS at 2.5 μM UTO (cell viability 41% vs. 70%). NRP-1-negative M21 cells showed significantly reduced viability when treated with free UTO at 25 μM, confirming that TPP-PS penetration and tumor cell cytotoxicity depend on peptide binding to NRP-1. Furthermore, in PPC-1 cells, RPAR-UTO-PS showed higher cytotoxicity compared with free UTO at a drug concentration of 2.5 μM, demonstrating that internalization induced by the CendR peptide is more efficient than that of the free drug at that concentration.

[0065] 3.4. In vivo testing - homing ability To evaluate the ability of TPP-targeted PS for specific drug delivery to tumors in vivo, we used tumor accumulation of PS labeled with the dye DiR in an orthotopic TNBC model. DiR is a hydrophobic molecule with near-infrared (NIR) absorption and emission spectra, providing a useful tool for whole-body imaging. NIR light can penetrate tissues while having minimal background interference in that region.

[0066] We prepared LinTT1-, RPAR-targeted, and non-targeted PS encapsulating DiR (LinTT1-DiR-PS, RPAR-DiR-PS, and DiR-PS). The PS were spherical with an average hydrodynamic diameter similar to previous PS formulations (average size: 116 ± 8 nm, PDI ≤ 0.15), demonstrating that the presence of dye in the PS membrane did not affect the nanovesicle structure.

[0067] To assess tumor internalization, we used MCF10CA1a cancer cells, an aggressive human-derived TNBC cell line. These cells overexpress surface p32 and NRP-1 proteins, making them good targets for LinTT1 and RPAR CendR peptides. Because LinTT1 peptides have already been used for early detection and treatment of breast tumors, we used an in vivo TNBC model.

[0068] LinTT1-DiR-PS, RPAR-DiR-PS, and DiR-PS were intravenously injected into TNBC mice, and live imaging was performed at 1, 3, 6, 24, and 48 h postinjection (Figure 6). Targeting with the LinTT1 and RPAR peptides increased tumor homing of DiR-PS. LinTT1- and RPAR-DiR-PS were detected in tumors 3 h after injection, whereas untargeted DiR-PS only became visible 24 h after injection. The highest tumor homing was observed 24 and 48 h after injection of LinTT1-DiR-PS. The integrated intensity, as assessed by the area under the curve (AUC, Figure 7), in tumors at 24 h was approximately 42% higher than that of DiR-PS. The AUC of RPAR-DiR-PS was also significantly higher (approximately 25% higher) than that of DiR-PS. After 24 and 48 hours, LinTT1-, RPAR-, and nontargeted DiR-PS were also observed in the liver and spleen. This may be explained by the important role of these organs in the systemic clearance of drugs and NPs. 48 hours after LinTT1-DiR-PS injection, breast tumors and hearts were excised, and the microscopic localization of PS was analyzed by fluorescence confocal microscopy. LinTT1-DiR-PS accumulation was observed deep within the tumor parenchyma. Because cardiotoxicity is one of the drawbacks of anthracyclines, we also evaluated LinTT1-DiR-PS accumulation in the hearts of TNBC mice. Significantly lower PS signal levels were observed in cardiac tissue compared with tumor cells. The LinTT1 receptor, p32, is also overexpressed in activated macrophages, which play an important role in tumor progression.

[0069] In addition, we also measured the colocalization of LinTT1-DiR-PS with the CD206 receptor, which is expressed on pro-tumorigenic M2 macrophages, and observed that LinTT1-DiR-PS targeted M2 macrophages in tumors.

[0070] 3.5. In vivo testing - drug accumulation in tumors We investigated drug accumulation after intravenous administration of LinTT1-UTO-PS, UTO-PS, and free DOX in orthotopic MCF10CA1a tumor-bearing mice, driven by the enhanced effect of LinTT1-targeted PS accumulation. Samples were injected, allowed to circulate for 24 hours, and tumors were harvested and analyzed by confocal immunoassay.

[0071] Mice injected with LinTT1-UTO-PS showed significantly higher UTO tumor accumulation compared to other samples. Low colocalization with blood vessels (CD31 staining) was observed for UTO fluorescence, suggesting that UTO loaded in LinTT1-PS extravasated and penetrated into tumor tissue. This result demonstrates that encapsulation of UTO in LinTT1-PS enhances tumor accumulation and penetration of the drug, indicating the potential application of our formulation for efficient TNBC treatment.

[0072] 4. Comparison of PS-UTO and PS-DOX To quantitatively evaluate the differences between DOX and UTO in PS encapsulation systems, a direct comparison was performed. Both UTO and DOX were encapsulated in PEG-PCL-PS using the film hydration method. The encapsulation and PS formation for this study were performed as described below:

[0073] 4.1 PS formation Polymersomes were prepared by dissolving 5 mg of PEG-PCL in 0.3 mL of acetone. The acetone was evaporated with a nitrogen stream to form a thin polymer film on the wall of a glass vial. The film was hydrated with 0.5 mL of PBS (pH 7.4) previously purged with a nitrogen stream, heated in a 65°C water bath for 30 seconds, and sonicated for 30 seconds. The heating and sonication steps were repeated until PS was formed and no polymer aggregates were observed in the suspension. The final volume of the PS sample was 0.5 mL, and the total polymer concentration was 10 mg / mL.

[0074] 4.2 Drug Encapsulation For UTO encapsulation in PS, 50 nM UTO was dissolved in 100 μL of acetone (0.5 mM concentration) and added to the polymer dissolved in acetone (5 mg total polymer, 0.125 mM UTO concentration). The acetone was evaporated to form a polymer / drug film, and PS was formed as described above.

[0075] For DOX encapsulation, the polymer film was hydrated with a 2 mM DOX solution in PBS at pH 7.4, and PS was formed as described above. Because the DOX encapsulation efficiency is approximately 20-fold lower than that of UTO, a higher DOX concentration is required. The difference used results in comparable concentrations of UTO and DOX in the PS.

[0076] 4.3 Purification UTO-PS and DOX-PS samples were purified using size-exclusion chromatography. Agarose beads with diameters of 45–165 μm were used as the stationary phase (Sephadex 4B gel). The Sephadex gel height in the column was 8 cm (volume 25.13 mL), with a dead volume of 2.5 mL or less. PS fractions (0.5 mL) were collected until no turbidity, indicating the presence of PS, was observed.

[0077] 4.3 Encapsulation Efficiency (EE) The amounts of UTO and DOX encapsulated in PS were quantified using UV-VIS spectroscopy (Thermo Scientific, USA). For UTO quantification, serial dilutions of UTO were prepared in 1:1 MeOH:water, and the absorbance at 485 nm was used as a calibration. For DOX quantification, serial dilutions of DOX were prepared in PBS, and the absorbance at 485 nm was also measured. Linear fitting of the data was further used to estimate the concentrations of UTO and DOX.

[0078] Quantitative results revealed that after purification, the encapsulation efficiency (EE) of UTO-loaded PS was 85%, dramatically higher than the 2.3% achievable for doxorubicin·HCl (DOX). This is quite surprising given the similar chemical structures of the different drugs. Therefore, much higher drug loading can be achieved by using UTO instead of DOX.

[0079] Figure 10 shows the hydrodynamic diameters of DOX-PS and UTO-PS as measured by dynamic light scattering (DLS). Very similar PS sizes are achievable using DOX or UTO. The mean particle diameter is 92 nm (+33) for DOX-PS and 87 nm (+37) for UTO-PS.

[0080] 4.3 Drug release To further evaluate the cumulative release behavior of UTO and DOX from polymersomes, UTO- and DOX-loaded PS were incubated in PBS (0.25 mL) at 37°C for various periods (0, 1, 4, 24, and 48 h). Samples were centrifuged at 6,000 g for 20 min at room temperature using Amicon Ultra centrifugal filters (MWCO 100 kDa). The fluorescence of the filtrate was measured at 485 nm / 535 nm (0.1 s) using a Victor X5 Multilabel Microplate Reader (Perkin Elmer, USA) to quantify the amount of released drug.

[0081] The drug release results are shown in Figure 11. After 48 hours, less than 3% of UTO was released from PS. Approximately 10% of DOX was released from PS compared to UTO. This finding is a further indication that UTO is surprisingly the "better" drug in PS-encapsulated systems. The drug is encapsulated in higher amounts, and the drug is better retained in PS compared to DOX. Given the structural similarity between UTO and DOX, this behavior is quite surprising. Nevertheless, the better in vivo efficacy may also be due, at least in part, to better encapsulation and higher storage stability compared to DOX.

Claims

1. A drug delivery system comprising at least a drug encapsulated in a polymer nanovesicle, wherein the drug component is an anthracycline derivative according to formula I: 【Chemical 1】 R 1 is selected from the group consisting of H, F, -OMe, or -OEt; R 2 is selected from the group consisting of H, -OMe, methyl, or ethyl; R 3 is selected from the group consisting of H, methyl, or ethyl, and R 4 is H or a protecting group; wherein the protecting group is a group that forms a carbamate bond via the N to which R 4 is attached; 1. A drug delivery system, characterized in that the polymersome is formed by a polymer comprising a PEG, PLA, PCL, PTMC or PTMB building block or a combination thereof, wherein the polymersome polymer is functionalized, at least in part, by chemically attaching a targeting moiety via a linker group L, wherein the targeting moiety is selected from the group consisting of an antibody, a peptide, an aptamer or a mixture thereof.

2. The drug delivery system of claim 1, wherein R 4 is -CO-O-CHR''-O-CO-R''', where R'' is H or Me and COR''' is acyl.

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

4. The drug delivery system of any one of claims 1 to 3, wherein the polymersome comprises a diblock PEG-copolymer, the second block being selected from the group consisting of PLA, PCL, PTMC, PTMBP.

5. 5. The drug delivery system according to claim 1, wherein the polymersome consists of a PEG-PCL diblock copolymer, and the weight ratio of the different polymer blocks, calculated as the weight of the PEG-moiety divided by the weight of the PCL-moiety, is ≧0.1 and ≦5.

6. In Formula I, R 1 is H and R 2 , R 3 is H and R 4 The drug delivery system according to any one of claims 1 to 5, wherein is a protecting group.

7. R 4 The drug delivery system of any one of claims 1 to 6, wherein is -CO-O-CH2-O-CO-CH3.

8. The drug delivery system according to any one of claims 1 to 7, wherein the group L is maleimide.

9. The drug delivery system according to any one of claims 1 to 8, wherein the molar ratio of peptide-modified polymer chains to the total number of polymer chains in the polymersome, calculated as the number of peptide-modified polymer chains divided by the total number of polymer chains, is 0.01 or more and 0.4 or less.

10. The drug delivery system according to any one of claims 1 to 9, wherein the molar ratio of peptide-modified polymer chains to the total number of polymer chains in the polymersome, calculated as the number of peptide-modified polymer chains divided by the total number of polymer chains, is 0.05 or more and 0.1 or less.

11. The drug delivery system according to any one of claims 1 to 10, wherein the concentration of the drug in the polymer nanovesicle is 20 µM or more and 500 µM or less.

12. The drug delivery system according to any one of claims 1 to 11, wherein the polydispersity index of the drug-loaded polymer nanovesicles is 0.01 or more and 0.25 or less.

13. A method for preparing the anthracycline derivative-loaded targeted polymersome drug delivery system according to any one of claims 1 to 12, characterized in that the drug according to formula I is encapsulated in the polymer nanovesicles by a thin film hydration process.

14. The method of claim 13, wherein the drug-loaded polymer nanovesicles are subjected to a size exclusion chromatography step in a further step.

15. A pharmaceutical composition comprising the drug delivery system of any one of claims 1 to 12 in a pharmaceutically acceptable solvent.

16. 16. The pharmaceutical composition of claim 15 for the treatment of cancer.

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