Self-assembled diblock copolymers composed of pegmema and drug bearing polymeric segments

Abstract

To provide a polymer-drug conjugate, a polymer micelle, and a method for preparing a polymer-drug conjugate.SOLUTION: Provided is a polymer-drug conjugate of Formula I in the form of a block copolymer for delivering a therapeutic agent, wherein D is an anticancer agent which is combretastatin, 5-fluorouracil (5-FU), or gemcitabine.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to polymer-drug conjugates according to formula I, aggregates consisting of the polymer-drug conjugates of formula I, methods for preparing said polymer-drug conjugates and aggregates, and their use for treating diseases such as cancer.

[0002] Chemotherapeutic agents used in the treatment of cancer are mostly cytotoxic. These agents can accumulate in the body's tissues in addition to the targeted area, which in turn causes a decrease in therapeutic benefit and an undesirable distribution of the drug throughout healthy body tissues. The uncontrolled distribution of these agents throughout the body causes serious side effects in patients.

[0003] To address the above problems, drug delivery systems that can deliver drugs to targeted areas of the body have been developed. For example, in some approaches to cancer treatment, these systems utilize the enhanced permeability and retention (EPR) effect, which implies that drug carriers with high molecular weight and large hydrodynamic volume accumulate in solid tumors, which in turn results in passive targeting of drug molecules to tumor tissue and minimizes damage to healthy tissue by chemotherapeutic agents.

[0004] Since the discovery of the enhanced permeability and retention (EPR) effect, macromolecules with long blood circulation have become a major substance for drug delivery due to improved accumulation in tumors through fenestrated blood vessels.

[0005] Regarding polymer aggregates in drug delivery systems for cancer diseases, great interest has been mainly focused on their body distribution characteristics. The present invention also relates to aggregates suitable for use in drug delivery, specifically, anti-cancer agents for cancer treatment.

[0006] There are various types of techniques for preparing polymer aggregates known in the current state of the art. For example, the self-assembly of polymer compounds is an attractive method for polymer aggregate formation. Eisenberg and his co-workers demonstrated the first example of the self-assembly of asymmetric copolymers in solution in the 1990s. Subsequently, amphiphilic block copolymers having polymer blocks with different physical and chemical properties have been attracting attention for generating polymer structures by self-assembly.

[0007] Drug loading into such polymer aggregates can be achieved by physical encapsulation via covalent bonding or hydrophobic interactions. Considering that the total volume of these aggregates in a colloidal suspension is very small, a high drug loading capacity is extremely important for achieving an effective drug formulation. The low drug loading capacity of polymer aggregates mainly in micelle formulations remains a major drawback in this field.

[0008] Some researchers have addressed this problem by utilizing π-π interactions by modifying drug molecules or using additive compounds rather than providing covalent bonds. However, unencapsulated drug aggregates in micelle-type polymer nanoparticle formulations are another drawback for providing a safe formulation for their clinical use.

[0009] In another approach, Stenzel and his co-workers utilized drug-binding amphiphilic block copolymers to form the hydrophobic segments of block copolymers for forming polymer aggregates. This strategy has a lack of complete conjugation of drugs to the polymer backbone due to steric hindrance resulting in an ill-defined final structure. Furthermore, residual reactive groups on the polymer backbone due to incomplete conjugation can be the most important drawback of this type of post-polymerization conjugation strategy as there is a possibility of undesirable biological interactions in vivo.

[0010] In light of the current state of the art summarized above, there is a need for block copolymers and assemblies formed therefrom that provide efficient conjugation of drugs with a high drug loading capacity, clearly defined by the drug content and having no remaining reactive sites.

[0011] The inventors have found that the amphiphilic block copolymers according to the invention have a high drug content and provide the formation of micelle-type assemblies that are clearly defined by drug binding.

[0012] The present invention relates to a polymer-drug conjugate in the form of a block copolymer for delivering a therapeutic agent as shown in formula I [Chemical formula]

[0013] In other words, the present invention relates to a polymer-drug conjugate of formula I in the form of a block copolymer, and an assembly made from the polymer-drug conjugate of formula I, wherein · R1 and R2 are independently selected from H or -CH3 · R3 is selected from -H or -CH3 · x is a natural number from 1 to 100 · y is a natural number from 1 to 100 · n is a natural number from 1 to 50 · L is a cleavable linker or L may be null · D is a therapeutic agent selected from the group comprising combretastatin, 5-fluorouracil, gemcitabine, chloroquine, and doxorubicin · A is a terminal group or A may be null · B is a terminal group or B may be null

[0014] In addition to overcoming the drawbacks of prior art polymer aggregates, the inventors of the present invention have also found that the polymer aggregates of the present invention comprising a polymer-drug conjugate of formula I reduce the amount of polymer administered to a patient for high drug loading.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0016] The term "polymer-drug conjugate" refers to a polymer structure having a therapeutic agent covalently attached to the polymer.

[0017] The term "polymer aggregate" refers to a structure having a diameter of 1 to 100 nm. The aggregates consist of self-assembled polymer chains, which may or may not have hollow cavities of various sizes.

[0018] The terms "polymer backbone" and "polymer backbone" can be used interchangeably and refer to a polymer chain having side chains or pendant groups. For example, the side chain may have oligoethylene glycol units, and the pendant group may carry one therapeutic agent or any other group that can be used to bind a therapeutic agent and / or a diagnostic agent or a target group.

[0019] Throughout this document, the term "polymer-drug conjugate of the present invention" should be construed to mean "polymer-drug conjugate according to Formula I" or "polymer-drug conjugate of Formula I", or "Formula I", and these terms can be used interchangeably.

[0020] Throughout this document, the term "polymer aggregate" should be construed to mean "nanoparticles composed of the polymer-drug conjugate of Formula I", or "nanoparticles composed of the copolymer shown in Formula I", or "nanoparticles formed as a result of the self-assembly of the polymer-drug conjugate of Formula I", or "micelle aggregates composed of the copolymer shown in Formula I", and these terms can be used interchangeably.

[0021] The term "PEG" refers to a polyether compound having the structure of H-(O-CH2-CH2)n-OR3, where n is a natural number from 1 to 200, and R3 is selected from H or -CH3. PEG is defined as an oligomer or polymer of ethylene oxide. The terms "PEG", "polyethylene glycol", "polyethylene oxide", "PEO", "polyoxyethylene", and "POE" refer to the same structure and can be used interchangeably within this document.

[0022] In an embodiment of the present invention, R1, R2, and R3 are independently selected from H or -CH3.

[0023] In one embodiment, R1 = H, R2 = H, R3 = H, or R1 = H, R2 = H, R3 = -CH3, or R1 = H, R2 = -CH3, R3 = H, or R1 = H, R2 = -CH3, R3 = -CH3, or R1 = -CH3, R2 = H, R3 = H, or R1 = -CH3, R2 = H, R3 = -CH3, or R1 = -CH3, R2 = -CH3, R3 = H, or R1 = -CH3, R2 = -CH3, R3 = -CH3.

[0024] The term "block copolymer" refers to a copolymer in which all of one type of monomer are grouped together and all of another type of monomer are grouped together. The polymer-drug conjugate of the present invention is in the form of a block copolymer. The fact that the polymer-drug conjugate of the present invention is in the form of a block copolymer enables the formation of the polymer aggregates of the present invention.

[0025] The therapeutic agent is bound to the polymer via a cleavable linker such that the therapeutic agent can be released, for example, under reducing conditions, oxidizing conditions, or hydrolysis of esters, amides, or hydrazides.

[0026] The cleavable linker can be any hydrocarbon or substituted hydrocarbon-based compound that can dissociate under physiological conditions. In a preferred embodiment, the linker is a compound that is cleaved under the acidic conditions of a tumor or with the aid of overexpressed enzymes present in the extracellular or intracellular matrix of tumor cells (any C1-C including acetal or ester functional groups, substituted or unsubstituted and / or linear and / or cyclic hydrocarbons, etc.). 10 It can be selected from substituted or unsubstituted and / or linear and / or cyclic hydrocarbons, etc.

[0027] The linker can be any kind of substance that can form a covalent bond with the polymer backbone at one end and with the therapeutic agent at the other end, such that one end can form a covalent bond with the polymer backbone and the other end can form a covalent bond with the therapeutic agent. The linker can also be a short peptide having a specific peptide sequence that is unstable to cathepsin B, such as Gly-Phe-Leu-Gly (SEQ ID NO: 1), which is also shown as GFLG or Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu (SEQ ID NO: 2).

[0028] The linker can also contain a functional group that dissociates under physiological conditions, such as an acetal, ester, imine, amide, disulfide, carbonate, carbamate, hydrazone, etc., such as a C1-C 10 hydrocarbon or a C1-C 10 substituted or hetero-substituted hydrocarbon.

[0029] In an embodiment of the present invention, the linker (L) is GFLG or Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu.

[0030] In an embodiment of the present invention, the linker is a C1-C 10 hetero-substituted hydrocarbon containing at least one disulfide functional group, or a C1-C 10 hetero-substituted hydrocarbon containing at least one acetal functional group, or a C1-C 10 hetero-substituted hydrocarbon containing at least one ester functional group, or a C1-C 10 hetero-substituted hydrocarbon containing at least one imine functional group, or a C1-C 10 hetero-substituted hydrocarbon containing at least one amide functional group, or a C1-C 10 hetero-substituted hydrocarbon containing at least one carbonate functional group, or a C1-C 10A hetero-substituted hydrocarbon, or a C1-C containing at least one hydrazone functional group 10 is a hetero-substituted hydrocarbon.

[0031] In another embodiment of the present invention, the linker is a C1-C containing two or more functional groups selected from the group consisting of acetal, ester, imine, amide, disulfide, carbonate, carbamate, hydrazone 10 and may contain a substituted or hetero-substituted hydrocarbon.

[0032] In another embodiment of the present invention, the linker can be null, which means that the therapeutic agent D is directly attached to the polymer chain. The direct attachment of the therapeutic agent to the polymer chain may also be through an ester, imine, amide, disulfide, carbonate, carbamate, hydrazine bond.

[0033] In a preferred embodiment of the present invention, the drug molecule is attached to the polymer via a direct ester bond that dissociates under the physiological conditions of the tumor. In another embodiment of the present invention, the linker is a C1-C containing at least one functional group selected from the group consisting of acetal, ester, imine, amide, disulfide, carbonate, carbamate, hydrazone 10 and can be a combination of a substituted hydrocarbon and a peptide chain selected from the group consisting of GFLG, Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu. Therapeutic agents selected from combretastatin, 5-fluorouracil, gemcitabine, chloroquine, and doxorubicin can be present in an amount of 5 wt% to 50 wt% of the polymer-drug conjugate, preferably in an amount of 6 wt% to 48 wt% of the drug-polymer conjugate, most preferably in an amount of 10 wt% to 45 wt% of the drug-polymer conjugate. The therapeutic agent can be present, for example, in an amount in the range of 10 wt% to 48 wt%, or 15 wt% to 46 wt%, or 20 wt% to 45 wt%, or 25 wt% to 44 wt% of the drug-polymer conjugate.

[0034] The term "end group" refers to a functional group or a structural unit at the end of a polymer. The end groups denoted as A and B may be the same as or different from each other.

[0035] In an embodiment, A is an end group that is optionally a fragment of a chain transfer agent or an initiator, or a chain transfer agent or an initiator fragment conjugated to a target moiety, or a chain transfer agent or an initiator fragment having a reactive functional group.

[0036] In an embodiment, B is an end group that is optionally a fragment of a chain transfer agent or an initiator.

[0037] The term "chain transfer agent" refers to a chemical compound that can regulate the molecular weight of a polymer by preventing the uncontrolled growth of the polymer chain. Chain transfer agents are well established in the literature and their structures are well known to those skilled in the art. The terms "chain transfer agent" and its abbreviation "CTA" refer to the same group of compounds and can be used interchangeably throughout this document.

[0038] A and B can optionally be fragments of a chain transfer agent. In particular, when a CTA is used in the polymerization reaction, the CTA fragment can remain as a terminal group on the polymer. The CTA used herein can be any material suitable for initiating polymerization reactions known in the art. In one embodiment, A and / or B is 3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, 3-butenyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, 2-cyanobutan-2-yl 4-chloro-3,5-dimethyl-1H-pyrazole-1-carbodithioate, 2-cyanobutan-2-yl 3,5-dimethyl-1H-pyrazole-1-carbodithioate, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol, cyanomethyl (3,5-dimethyl-1H-pyrazole)-carbodithioate, cyanomethyldodecyltrithiocarbonate, cyanomethyl [3-(trimethoxysilyl)propyl]trithiocarbonate, 2-cyano-2-propyldodecyltrithiocarbonate, S,S-dibenzyltrithiocarbonate, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid N-hydroxysuccinimide ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiocarbonothioylthio)propionic acid, methyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate], phthalimidomethylbutyltrithiocarbonate, 1,1,Selected from the group consisting of 1-tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane, benzyl 1H-pyrrole-1-carbodithioate, cyanomethyl diphenylcarbamodithioate, cyanomethyl methyl(phenyl)carbamodithioate, cyanomethyl methyl(4-pyridyl)carbamodithioate, 2-cyanopropan-2-yl N-methyl-N-(pyridin-4-yl)carbamodithioate, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate, benzyl benzodithioate, cyanomethyl benzodithioate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 2-cyano-2-propyl benzodithioate, 2-cyano-2-propyl-4-cyanobenzodithioate, ethyl 2-(4-methoxyphenylcarbonothioylthio)acetate, ethyl 2-methyl-2-(phenylthiocarbony lthio)propionate, ethyl 2-(phenylcarbonothioylthio)-2-phenylacetate, ethyl 2-(phenylcarbonothioylthio)propionate, 1-(methoxycarbonyl)ethyl benzodithioate, 2-(4-methoxyphenylcarbonothioylthio)ethanoic acid, 2-nitro-5-(2-propynyloxy)benzyl 4-cyano-4-(phenylcarbonothioylthio)pentanoate 2-(phenylcarbonothioylthio)propanoic acid, 2-phenyl-2-propyl benzodithioate, cyanomethyl methyl(4-pyridyl)carbamodithioate, 2-cyanopropan-2-yl N-methyl-N-(pyridin-4-yl)carbamodithioate, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate, or any fragment of the initiators listed herein, either as such or conjugated to a target moiety before and after a polymerization reaction or modified with a reactive group.,

[0039] As used herein, the term "fragment" refers to a compound formed by the cleavage of one or more of the covalent bonds that form the initiator molecule.

[0040] The fragmentation of the CTA listed herein and the structures of the fragments formed are well established in the art, and thus the structures of A and B can be determined without using inventive step by utilizing the state of the art.

[0041] A and / or B are optionally null (i.e., non-existent).

[0042] In an embodiment of the invention, A is null and B is a fragment of the CTA.

[0043] In another embodiment, B is null and A is a fragment of the CTA.

[0044] In another embodiment, A and B are both fragments of the CTA, but they are structurally different from each other. In other words, A and B are different fragments of the same CTA.

[0045] In another embodiment, A and B are both fragments of the CTA and they have the same chemical structure.

[0046] The term "target moiety" refers to a molecule that has a tendency to bind to a specific target site in the body. In other words, the target moiety is a molecule that specifically binds to cells having complementary receptors.

[0047] The target moiety can be selected from the group including antibodies; antibody fragments, or peptides such as Cyclo(Arg-Gly-Asp-D-Phe-Lys) (SEQ ID NO: 3) (cRGDfK). In a preferred embodiment, cRGDfK is used as the target moiety.

[0048] The term "reactive group" refers to an atom or related atomic group in a chemical substance that is intended or reasonably expected to undergo an easy chemical reaction.

[0049] Reactive groups can be acetals, hemiacetals, carboxylic acids, alcohols, amides, imides, anhydrides, aryl halides, azo compounds, diazo compounds, hydrazines, azides, carbonates, chlorosilanes, cyanides, esters, sulfate esters, phosphate esters, thiophosphate esters, isocyanates, isothiocyanates, thiocarbamate esters, dithiocarbamate esters. Preferably, the reactive group is an ester, imide, or carbonate. The reactive group can be, for example, N-hydroxysuccinimide.

[0050] Depending on the polymerization technique used to prepare the polymer conjugate of the present invention, A and / or B can be fragments of an initiator.

[0051] As used herein, an initiator refers to a chemical compound that reacts with a monomer to form an intermediate compound that can be continuously linked to a number of other monomers to form a polymer compound. The terms "initiator" and "polymerization initiator" can be used interchangeably within the context of this application.

[0052] Depending on the polymerization technique used to prepare the polymer conjugate of the present invention, different initiators can be used.

[0053] In an embodiment, A and / or B is 4,4'-azobis(4-cyanovaleric acid), 4,4'-azobis(4-cyanovaleric acid), 1,1'-azobis(cyclohexanecarbonitrile), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionitrile) (also known as AIBN), ammonium persulfate, sodium hydroxymethanesulfinate dihydrate, potassium persulfate, sodium persulfate, tert-butyl hydroperoxide, tert-butyl peracetate, cumene hydroperoxide, 2,5-di(tert-butylperoxy)-2,5-dimethyl-3-hexyne, dicumyl peroxide, 2,5-bis(tert-butylperoxy)-2,5-dimethylhexane, 1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane, 1,1-bis(tert-amylperoxy)cyclohexane, benzoyl peroxide, 2-butanone peroxide, tert-butyl peroxide, di-tert-amyl peroxide, lauroyl peroxide, tert-butyl peroxybenzoate, tert-butyl peroxy-2-ethylhexyl carbonate, tert-butyl hydroperoxide, 2-azidoethyl-2-bromoisobutyrate, bis[2-(2-bromoisobutyryloxy)undecyl] disulfide, bis[2-(2'-bromoisobutyryloxy)ethyl] disulfide, N-hydroxysuccinimide ester of 2-bromoisobutanoic acid, 2-bromoisobutyric anhydride, α-bromoisobutyryl bromide, 2-(2-bromoisobutyryloxy)ethyl methacrylate, tert-butyl α-bromoisobutyrate, 3-butynyl-2-bromoisobutyrate, dipentaerythritol hexakis(2-bromoisobutyrate), dodecyl 2-bromoisobutyrate, ethyl α-bromoisobutyrate, ethylene bis(2-bromoisobutyrate), 2-hydroxyethyl 2-bromoisobutyrate, 1-(DL-1,(2-Isopropylidene glycerol) 2-bromoisobutyrate, methyl α-bromoisobutyrate, octadecyl 2-bromoisobutyrate, pentaerythritol tetrakis(2-bromoisobutyrate), 1-(phthalimidomethyl) 2-bromoisobutyrate, poly(ethylene glycol) bis(2-bromoisobutyrate), propargyl 2-bromoisobutyrate, 1,1,1-tris(2-bromoisobutyryloxymethyl)ethane, 10-undecenyl 2-bromoisobutyrate, N-tert-butyl-O-[1-[4-(chloromethyl)phenyl]ethyl]-N-(2-methyl-1-phenylpropyl)hydroxylamine, N-tert-butyl-N-(2-methyl-1-phenylpropyl)-O-(1-phenylethyl)hydroxylamine, TEMPO, TEMPO methacrylate, 2,2,5-trimethyl-4-phenyl-3-azahexane-3-nitroxide, 3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, 3-butenyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanol, cyanomethyl dodecyl, cyanomethyl [3-(trimethoxysilyl)propyl]trithiocarbonate, 2-cyano-2-propyl dodecyl trithiocarbonate, S,S-dibenzyl trithiocarbonate, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid N-hydroxysuccinimide ester, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiocarbonothioylthio)propionic acid, methyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate], phthalimidomethyl butyl trithiocarbonate, 1,1,Fragments of initiators that can be selected from the group consisting of 1-tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane, benzyl benzodithioate, cyanomethyl benzodithioate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 2-cyano-2-propyl benzodithioate, 2-cyano-2-propyl 4-cyanobenzodithioate, ethyl 2-(4-methoxyphenylcarbonothioylthio)acetate, ethyl 2-methyl-2-(phenylthiocarbonylthio)propionate, ethyl 2-(phenylcarbonothionoylthio)-2-phenylacetate, ethyl 2-(phenylcarbonothioylthio)propionate, 1-(methoxycarbonyl)ethyl benzodithioate, 2-(4-methoxyphenylcarbonothioylthio)ethanoic acid, 2-nitro-5-(2-propynyloxy)benzyl, 4-cyano-4-(phenylcarbonothioylthio)pentanoate, 2-(phenylcarbonothioylthio)propanoic acid, 2-phenyl-2-propyl benzodithioate, cyanomethyl methyl(4-pyridyl)carbamodithioate, cyanopropan-2-yl N-methyl-N-(pyridin-4-yl)carbamodithioate, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate, 1-succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate, or combinations thereof, either as themselves or conjugated to a target moiety before and after a polymerization reaction or modified with a reactive group.,

[0054] The definition and examples of the target moiety are as described above.

[0055] The definition and examples of the reactive group are as described above.

[0056] The chain transfer agents and initiators listed within this application are provided as examples for the description of the present invention, and any chemical compound published before and after the filing of this document that can act as a CTA and / or initiator is included within the scope of the present invention.

[0057] The measurement of the amount of drug in the polymer conjugate of the present invention is carried out by using conventional techniques well known in the art, for example, by calculating the drug ratio from the 1 1H-NMR of the polymer-drug conjugate or by determining the amount by forced release of the drug.

[0058] In another embodiment, the polymer-drug conjugate of the present invention has an average molecular weight of 5 kDa to 60 kDa. In a preferred embodiment, the polymer-drug conjugate of the present invention has an average molecular weight of 6 kDa to 50 kDa, and in the most preferred embodiment, the polymer-drug conjugate of the present invention has an average molecular weight of 7 kDa to 40 kDa.

[0059] The molecular weight of the polymer-drug conjugate of the present invention is determined by using conventional techniques known in the art, for example, by using gel permeation chromatography (GPC).

[0060] Another embodiment of the present invention is a polymer aggregate formed using the polymer-drug conjugate represented by Formula I, wherein;

Chemical formula

[0061] Another embodiment of the present invention is a polymeric micelle formed using a polymer-drug conjugate represented by formula I, wherein;

Chemical formula

[0062] In another embodiment, the assembly according to the present invention is used for encapsulating a therapeutic molecule, and in that sense, a polymeric assembly consisting of a polymer-drug conjugate of formula I that encapsulates a therapeutic agent other than those attached to the polymer chain is another embodiment of the present invention.

[0063] The term "encapsulating" refers to confining a therapeutic agent, e.g., a guest molecule, inside the cavity of a host molecule, e.g., a polymeric assembly consisting of a polymer-drug conjugate of formula I. Encapsulation preferably occurs by non-covalent interaction between the therapeutic molecule and the polymeric assembly of the present invention.

[0064] The term "therapeutic agent" refers to any compound suitable for use in the treatment of a disease. The terms "therapeutic agent", "chemotherapeutic agent", "anticancer agent", and "antineoplastic agent" all refer to compounds suitable for use in the treatment of a disease, and these terms can be used interchangeably. In one embodiment, the disease is cancer.

[0065] Furthermore, "therapeutic agent" also refers to any agent suitable for use in the treatment of a disease, such as cancer. Any therapeutic agent that can be directly or indirectly conjugated to the polymer-drug conjugate of the present invention can be used. U.S. Patent No. 6,342,221 also describes agents related to anticancer agents, and this document is incorporated herein by reference. Anticancer agents can be classified as chemotherapeutic agents, cytotoxins, antimetabolites, alkylating agents, protein kinase inhibitors, anthracyclines, antibiotics, antimitotic agents (e.g., antitubulin agents), corticosteroids, radiopharmaceuticals, and proteins (e.g., cytokines, enzymes, or interferons), but are not limited thereto. Specific examples of anticancer agents are, for example, docetaxel, gemcitabine, imatinib, 5-fluorouracil, 9-aminocamptothecin, amine-modified geldanamycin, doxorubicin, paclitaxel, procarbazine, hydroxyurea, meso-chlorin, cisplatin, and radionuclides (e.g., I-131, Y-90, In-111, and Tc-99m). There are many other anticancer agents known in the art, and many are still being developed, and those agents are also included within the scope of the present invention.

[0066] The therapeutic agent can also be selected from the subgroup consisting of, but not limited to, nucleoside analogs, antifolate agents, other metabolites, topoisomerase I inhibitors, anthracyclines, podophyllotoxins, taxanes, vinca alkaloids, alkylating agents, platinumates, antihormone agents, radiopharmaceuticals, monoclonal antibodies, tyrosine kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors, retinoids, immunomodulators, histone deacetylase inhibitors, and other agents.

[0067] Nucleoside analogs can be selected from the group consisting of, but not limited to, azacitidine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, mercaptopurine, nelarabine, pentostatin, thioguanine, trifluridine, tipiracil.

[0068] Antifolate agents can be selected from the group consisting of, but not limited to, methotrexate, pemetrexed, pralatrexate, raltitrexed.

[0069] Other metabolites can be selected from the group consisting of, but not limited to, hydroxycarbamide.

[0070] Topoisomerase I inhibitors can be selected from the group consisting of, but not limited to, irinotecan and topotecan.

[0071] Anthracyclines can be selected from the group consisting of, but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin.

[0072] Podophyllotoxins can be selected from the group consisting of, but not limited to, etoposide and teniposide.

[0073] Taxanes can be selected from the group consisting of, but not limited to, cabazitaxel, docetaxel, paclitaxel.

[0074] Vinca alkaloids can be selected from the group consisting of, but not limited to, vinblastine, vincristine, vindesine, vinflunine, vinorelbine.

[0075] Alkylating agents can be selected from the group consisting of, but not limited to, bendamustine, chlorambucil, dacarbazine, melphalan, streptozotocin, trabectedin.

[0076] The antihormone compound can be selected from the group including, but not limited to, abiraterone, bicalutamide, cyproterone, degarelix, exemestane, fulvestrant, goserelin, histrelin, leuprolide, mifepristone, and triptorelin.

[0077] The tyrosine kinase inhibitor can be selected from the group including, but not limited to, afatinib, axitinib, bosutinib, cobimetinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, osimertinib, pazopanib, luxolitinib, sunitinib, and vandetanib.

[0078] The mammalian target of rapamycin (mTOR) inhibitor can be selected from the group including, but not limited to, everolimus and temsirolimus.

[0079] The retinoid can be selected from the group including, but not limited to, alitretinoin, bexarotene, isotretinoin, tamibarotene, and tretinoin.

[0080] The immunomodulator can be selected from the group including, but not limited to, lenalidomide, pomalidomide, and thalidomide.

[0081] The histone deacetylase inhibitor can be selected from the group including, but not limited to, belinostat, panobinostat, valproate, and vorinostat.

[0082] The platinum complex can be selected from the group including, but not limited to, cisplatin, carboplatin, oxaliplatin, and nedaplatin.

[0083] Other agents can be selected from the group including, but not limited to, anagrelide, ceritinib, dabrafenib, idelalisib, ibrutinib, palbociclib, vemurafenib, bleomycin, bortezomib, dactinomycin, eribulin, estramustine, ixabepilone, mitomycin, procarbazine, alectinib, fluoxymesterone, ioguanine, imiquimod, interferon, ixazomib, lanreotide, lentinan, octreotide, omacetaxine, tegafur, gimeracil, oteracil, uracil, combretastatin, chloroquine.

[0084] In a preferred embodiment of the present invention, the therapeutic agent is selected from taxanes, antifolates, tyrosine kinase inhibitors, anthracyclines, nucleoside analogs, or other agents. Most preferably, the therapeutic agent is selected from the group including docetaxel, pemetrexed, chloroquine, combretastatin, gemcitabine, doxorubicin, fluorouracil (5-FU), 5'-deoxy-5-fluorocytidine (5'-DFCR), lapatinib.

[0085] In an embodiment of the present invention, the therapeutic agent is docetaxel.

[0086] In an embodiment of the present invention, the therapeutic agent is carboplatin.

[0087] In an embodiment of the present invention, the therapeutic agent is doxorubicin.

[0088] Another embodiment of the present invention is a method (Method I) for preparing the polymer-drug conjugate (Formula I) of the present invention, which includes the polymerization of PEG (meth) acrylate monomer (Formula II).

Chemical formula

Chemical formula

[0089] Then, (ii) the polymer of formula IIa is further reacted with a (meth)acrylate L-D monomer (formula IIIa),

Chemical formula

[0090] In another aspect, a method (Method II) for preparing the polymer-drug conjugate (formula I) of the present invention comprises (i) the polymerization of a PEG (meth)acrylate monomer (formula II),

Chemical formula

Chemical formula

[0091] Then, (ii) the compound of formula IIa is further reacted with a (meth)acrylate L monomer (formula IIIb),

Chemical formula

Chemical formula

[0092] Then, (iii) the compound of formula IIb is reacted with a therapeutic agent (D) selected from the group consisting of combretastatin, 5-FU, gemcitabine, chloroquine, doxorubicin to obtain a polymer conjugate as shown in formula I.

[0093] Another embodiment of the present invention is a method (Method III) for preparing a polymer-drug conjugate (formula I) of the present invention, which comprises the polymerization of a PEG (meth)acrylate monomer (formula II),

Chemical formula

Chemical formula

[0094] Then, (ii) Formula IIa is further reacted with a (meth)acrylate L-D monomer (Formula IIIa),

Chemical formula

[0095] The term "acrylate" refers to derivatives of acrylic acid. These derivatives include the parent acid (CH2CHCO2H) and esters. Thus, the term "acrylate-based" defines a functional group having any of the above acrylate derivatives.

[0096] The term "methacrylate" refers to derivatives of methacrylic acid. These derivatives include the parent acid (CH2C(CH3)CO2H) and esters. Thus, the term "methacrylate-based" defines a functional group having any of the above methacrylate derivatives.

[0097] The term "(meth)acrylate" refers to the terms "acrylate" and "methacrylate". Thus, the term "(meth)acrylate" can be used interchangeably with "acrylate" and "methacrylate" and includes all the features of these terms as described above. The term "(meth)acrylate" should be construed to mean "methacrylate and / or acrylate".

[0098] Step (i) of Methods I, II, and III may further comprise the use of a chain transfer agent and / or an initiator.

[0099] In another aspect, the present invention relates to a polymer-drug conjugate of Formula I prepared by any one of Methods I, II, or III.

[0100] In another embodiment, the present invention relates to a polymer-drug conjugate of Formula I prepared by Method I because this method provides a polymer-drug conjugate having a very well-defined polymer structure and drug content. Also, the polymer-drug conjugate prepared by this method does not have free reactive groups that remain unconjugated to the drug molecule on the side chains.

[0101] In one embodiment, PEG (meth)acrylate (Formula II) is preferably selected from the group consisting of polyethylene glycol methyl ether methacrylate (CAS number: 26915-72-0), polyethylene glycol methacrylate (CAS number: 25736-86-1), polyethylene glycol methyl ether acrylate (CAS number: 32171-39-4), and polyethylene glycol acrylate (CAS number: 9051-31-4). For the structure of the compounds, refer to Table 1. In a preferred embodiment of the present invention, polyethylene glycol methyl ether methacrylate in which both R1 and R3 are -CH3 is used.

[0102] In embodiments of the present invention, PEG (meth) acrylate (Formula II) having an average molecular weight of 50 to 2000 g / mol is used. In a preferred embodiment, PEG (meth) acrylate (Formula II) has an average molecular weight of 100 to 1500 g / mol, and in a most preferred embodiment, PEG (meth) acrylate (Formula II) has an average molecular weight of 150 to 1000 g / mol. The PEG (meth) acrylate (Formula II) of the present invention can have an average molecular weight of, for example, 60, 70, 80, 80, 100, 150, 200 - 250, 300, 400, 500, 600, 700, 800, 900, 1000 g / mol.

[0103] The cleavable linker according to the present invention can be obtained from commercial sources or can be prepared by known methods provided in the literature.

[0104] In another aspect, the present invention relates to a block copolymer of Formula IIb for use in the synthesis of a polymer - drug conjugate of Formula I,

Chemical formula

[0105] As disclosed above, the methods (Methods I, II, and III) for preparing the polymer-drug conjugates of Formula I involve at least two polymerization steps in which the monomers of Formula II and the monomers of Formula IIIa or IIIb are polymerized. In embodiments of the invention, these polymerization steps may further include an initiator and / or a polymerizing agent and / or a solvent.

[0106] The polymer-drug conjugates of the present invention can be prepared by any of the known polymerization methods. Any suitable initiator and / or catalyst known in the art can be used in the preparation of the polymer-drug conjugates of the present invention. When a polymerization initiator is used, the initiator or a fragment thereof may be present in the resulting polymer-drug conjugate.

[0107] The polymer backbone of the polymer-drug conjugates of the present invention can be obtained, for example, by bulk polymerization, solution polymerization, and / or suspension polymerization techniques known in the art.

[0108] The polymerization techniques used in the preparation of the polymer-drug conjugates of the present invention may proceed by free radical polymerization or controlled / living free radical polymerization. As used herein, the term "controlled / living free radical polymerization" refers to atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, iodine transfer polymerization (ITP), selenium-centered radical-mediated polymerization, telluride-mediated polymerization (TERP), nitroxide-mediated polymerization (NMP). In a preferred embodiment of the invention, RAFT polymerization is used to prepare the polymer-drug conjugates of the present invention.

[0109] Suitable polymerization initiators can be selected from the group described in this document.

[0110] When using RAFT polymerization, the CTA and the initiator can be used together to prepare the polymer-drug conjugates of the present invention according to Method I, II, or III provided herein.

[0111] In embodiments of the present invention, the polymer-drug conjugates of the present invention and the polymer aggregates made therefrom may further comprise a targeting group. As used herein, the term "targeting group" refers to a tumor-specific ligand that specifically binds to a cell, preferably a tumor cell having a complementary receptor.

[0112] The term "targeting group" means a molecule that serves to deliver the polymer-drug conjugates of the present invention to a specific site for a desired activity, i.e., that results in the localization of the compound. Localization is mediated by specific recognition of molecular determinants, the molecular size of the targeting agent or conjugate, ionic interactions, hydrophobic interactions, etc. Other mechanisms for targeting a drug to a specific tissue or region are known to those skilled in the art. Examples of targeting ligands include molecules that bind to molecules on the surface of the targeted cell. Exemplary targeting ligands include antibodies, antibody fragments, organic small molecules, peptides, peptoids, proteins, polypeptides, oligosaccharides, transferrin, HS-glycoproteins, coagulation factors, serum proteins, β-glycoproteins, G-CSF, GM-CSF, M-CSF, EPO, and the like. In exemplary embodiments of the present invention, the targeting system comprises covalently attaching a targeting ligand such as RGDfK, EPPT1 peptide, bisphosphonic acid, or folate to a carrier molecule or linker.

[0113] In certain embodiments, the present invention is characterized by polymer-drug conjugates that do or do not contain a targeting ligand. In some embodiments, the targeting ligand can be RGDfK, EPPT1, bisphosphonic acid, or folate.

[0114] Another embodiment of the present invention provides a method for delivering a therapeutic agent, comprising administering to a subject an effective amount of a polymer-drug conjugate represented by Formula I.

[0115] Another embodiment of the present invention provides a method for delivering a therapeutic agent, comprising administering to a subject an effective amount of a polymer aggregate consisting of a polymer-drug conjugate represented by Formula I.

[0116] Another embodiment of the present invention is the polymer-drug conjugate of the present invention for use in the treatment of various disorders that require the delivery of an anti-cancer agent or a similar drug.

[0117] Another embodiment of the present invention is a polymer aggregate composed of the polymer-drug conjugate of the present invention for use in the treatment of various disorders that require the delivery of an anti-cancer agent or a similar drug.

[0118] In a preferred embodiment, the present invention relates to a polymer-drug conjugate represented by Formula I for use as a medicament for cancer treatment.

[0119] In yet another preferred embodiment, the present invention relates to a polymer aggregate composed of the polymer-drug conjugate of Formula I for use as a medicament for cancer treatment.

[0120] As used herein, "treating" or "treatment" means inhibiting, reducing, modulating, ameliorating, or arresting at least one symptom that characterizes a pathological condition in a subject threatened by or suffering from that condition. A non-limiting list of various types of cancer is as follows: carcinoma, solid tissue carcinoma, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, high-grade glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, metastatic cancer, or general cancer.

[0121] Specific examples of cancers for which the disclosed compositions can be used in treatment include B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, bladder cancer, brain tumor, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, oral squamous cell carcinoma, laryngeal cancer, colorectal cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer; testicular cancer; colorectal and rectal cancer, prostate cancer, or pancreatic cancer.

[0122] The polymer-drug conjugates of the present invention and / or polymer aggregates comprising the polymer-drug conjugates of the present invention can also be used for the treatment of precancerous conditions such as cervical and anal dysplasia, other dysplasia, severe dysplasia, hyperplasia, atypical hyperplasia, and neoplasia.

[0123] As used herein, the terms "cancer" and "cancerous" refer to malignant tumors or represent physiological states characterized by disordered cell growth.

[0124] As contemplated herein, the polymer-drug conjugates of the present invention and / or polymer aggregates comprising the polymer-drug conjugates of the present invention find use in the treatment and / or prevention of cancer. For such use, the polymer-drug conjugates of the present invention and / or polymer aggregates comprising the polymer-drug conjugates of the present invention are generally administered in the form of pharmaceutical compositions.

[0125] Accordingly, the present invention provides a pharmaceutical composition comprising a polymer-drug conjugate according to formula I, and at least one pharmaceutically acceptable diluent, excipient, and / or carrier. The term "treatment" includes either therapeutic or prophylactic therapy.

[0126] Furthermore, the present invention provides a pharmaceutical composition comprising a polymer aggregate consisting of a polymer-drug conjugate according to formula I, and at least one pharmaceutically acceptable diluent, excipient, and / or carrier.

[0127] The composition comprising the polymer-drug conjugate of the present invention and / or the polymer aggregate composed of the polymer-drug conjugate of the present invention can be in any suitable form depending on the desired method of administering it to a patient. The composition comprising the polymer-drug conjugate of the present invention and / or the polymer aggregate composed of the polymer-drug conjugate of the present invention can be formulated, for example, in the form of a liquid dispersion or an aqueous or oily suspension for oral administration, or they can be formulated for parenteral administration, such as subcutaneous, intravenous, intramuscular, intrasternal, intraperitoneal, intradermal, transdermal, or other injection techniques.

[0128] The composition comprising the polymer-drug conjugate of the present invention and / or the polymer aggregate composed of the polymer-drug conjugate of the present invention can also be formulated in the form of an aerosol or a solution for administration with an inhaler or nebulizer for administration by inhalation. The polymer-drug conjugate of the present invention and / or the polymer aggregate composed of the polymer-drug conjugate of the present invention is preferably administered to a subject via transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, or inhalation. The most suitable route of administration in any given case depends on the particular therapeutic agent present in the polymer-drug conjugate of the present invention, the subject, and the nature and severity of the subject's disease and health condition.

[0129] The polymer-drug conjugate of the present invention and / or the polymer aggregate composed of the polymer-drug conjugate of the present invention can be administered in combination with one or more other therapeutically active compounds, for example, simultaneously, sequentially, or separately, and the therapeutically active compound can be an anti-cancer agent, or it can be an immunomodulatory agent, an antiviral agent, an anti-infective agent, an antibacterial agent, an anti-infective agent or an anesthetic, or a combination thereof.

[0130] The polymer aggregate of the present invention may encapsulate a second therapeutic agent selected from the list provided above and may be further administered in combination with one or more other therapeutically active compounds, for example, simultaneously, sequentially, or separately, and the therapeutically active compound may be an anti-cancer agent or it may be an immunomodulatory agent, an antiviral agent, an anti-infective agent, an antibacterial agent, an anti-infective agent or an anesthetic, or a combination thereof.

[0131] The second therapeutic agent can be selected from the therapeutic agents listed above, provided that it is different from the therapeutic agent present in the polymer-drug conjugate of the present invention.

[0132] As used in the context of this specification, it is intended to mean including.

[0133] Where technically appropriate, embodiments of the present invention may be combined.

[0134] Embodiments are described herein as including certain features / elements. The present disclosure also extends to separate embodiments consisting of or consisting essentially of said features / elements.

[0135] Technical references such as patents and applications are incorporated herein by reference.

[0136] Any embodiment specifically and explicitly recited herein may form the basis of an exemption, either alone or in combination with one or more further embodiments.

[0137] The present invention will be described with reference to the following examples, which are illustrative only and should in no way be construed as limiting the scope of the present invention.

[0138] Example The following examples provide a stepwise preparation of the polymer-drug conjugates of formula I and their polymer aggregates.

[0139] Example 1A: Synthesis of Combretastatin-A4 Monomer (CombMA) Combretastatin-A4 (300 mg, 0.95 mmol), triethylamine (TEA, 191 mg, 1.89 mmol), and methacryloyl chloride (198 mg, 1.89 mmol) were dissolved in dry dichloromethane (DCM, 10 mL) in a 25 mL round-bottom flask under N2. The reaction solution was stirred at room temperature for 16 h. The crude product was extracted with saturated NaHCO3 (20 mL × 2) and distilled water (20 mL × 2). The organic layer was dried over Na2SO4 and the solvent was evaporated. The CombMA monomer was purified using silica column chromatography with hexane.

[0140] Example 1B: Preparation of Chain Transfer Agent (CTA) with Reactive Functional Group 4-Cyanopentanoic acid dithiopentyl ester (CPDB) is a CTA, and this CTA was modified with N-hydroxysuccinimide (NHS), a reactive functional group, according to the following procedure.

[0141] Briefly, CPADB (200 mg, 0.72 mmol) and N-hydroxysuccinimide (125 mg, 1.07 mmol) were dissolved in anhydrous DCM (4 mL). Dicyclohexylcarbodiimide (DCC) (177 mg, 0.86 mmol) was dissolved in anhydrous DCM (1 mL). Then, the two solution mixtures were combined and the reaction mixture was stirred at room temperature in the dark for 16 h. The insoluble white byproduct dicyclohexylurea (DCU) was removed by filtration. The resulting solution was dried under vacuum and the crude product was purified by silica column chromatography using hexane and EtOAc.

[0142] Example 1C: Synthesis of POEGMEMA and NHS-POEGMEMA Homopolymers Reversible addition-fragmentation chain transfer (RAFT) polymerization was used for the synthesis of POEGMEMA and NHS-activated POEGMEMA (NHS-POEGMEMA) homopolymers. To synthesize the POEGMEMA polymer, AIBN (1.31 mg, 0.008 mmol) was added to a solution of OEGMA (600 mg, 2.0 mmol) and CPADB (20.12 mg, 0.072 mmol) in DMF (3 mL). The mixture was purged with N2 to remove O2 and the polymerization was stirred at 70 °C. The polymerization was stopped by cooling and air exposure. The POEGMEMA polymer was purified by precipitation in diethyl ether. The polymer precipitate was dried under vacuum to obtain approximately 35 OEGMEMA repeating units (460 mg, 77% yield). For the synthesis of the NHS-POEGMEMA homopolymer, the same procedure was applied using SCPDB (27.10 mg, 0.072 mmol) as the chain transfer agent to obtain approximately 35 OEGMEMA repeating units (480 mg, 80% yield).

[0143] Example 1D: Synthesis of POEGMEMA-co-CombMA and NHS-POEGMEMA-co-CombMA Block Copolymers RAFT polymerization was used for the synthesis of POEGMEMA-b-PCombMA and NHS-POEGMEMA-b-PCombMA block copolymers. AIBN (0.15 mg, 0.00092 mmol) was added to a solution of CombMA (100 mg, 0.26 mmol) and POEGMEMA polymer (50 mg, 0.005 mmol) as the macro chain transfer agent in DMF (0.75 mL). The mixture was purged with N2 to remove O2 and the polymerization was stirred at 65 °C. The polymerization was stopped by cooling and air exposure. The crude product was precipitated in diethyl ether. The polymer precipitate was dried under vacuum to obtain approximately 32 CombMA repeating units (98 mg, 65% yield). The same procedure was applied to synthesize the NHS-POEGMEMA-b-PCombMA block copolymer using NHS-POEGMEMA (50 mg, 0.005 mmol) as the macro chain transfer agent to obtain approximately 33 CombMA repeating units (105 mg, 70% yield).

[0144] Example 1E: Synthesis of Targeted Block Copolymer NHS-POEGMEMA-b-PCombMA (50 mg, 0.002 mmol) and cRGDfK (6.4 mg, 0.01 mmol) were co-dissolved in DMF (0.25 mL), and N,N-diisopropylethylamine (6.5 mg, 0.05 mmol) was added to this reaction mixture. The reaction mixture was stirred at 30 °C for 24 hours. The crude product was precipitated in diethyl ether. The polymer precipitate was dried under reduced pressure to obtain cRGDfK-POEGMEMA-b-CombMA (41 mg, yield 82%). An overall view showing the preparation of the cRGDfK-POEGMEMA-b-CombMA block copolymer is provided as Figure 1.

[0145] Example 1F: Preparation of Targeted and Non-Targeted Polymer Aggregates As used herein, "targeted" refers to a polymer aggregate composed of the cRGDfK-POEGMEMA-b-CombMA block copolymer, and "non-targeted" refers to a polymer aggregate composed of the POEGMEMA-b-PCombMA block copolymer.

[0146] Two solutions were prepared to obtain targeted and non-targeted aggregates. For the formation of non-targeted aggregates, 2.3 mg of the POEGMEMA-b-PCombMA block copolymer was dissolved in 500 μL of THF in a glass vial, and 3 mL of water was added to this solution. Then, the THF in the vial was evaporated at room temperature in an open atmosphere for 24 hours to obtain micelles. Targeted aggregates were prepared in the same manner using a cRGDfK-POEGMEMA-b-PCombMA and POEGMEMA-b-PCombMA mixture (1 / 5, w / w). Schematic diagrams of the preparation of non-targeted and targeted aggregates are provided as Figures 2 and 3.

[0147] Example 1G: Critical Micelle Concentration (CMC) Measurement The fluorescence probe method was utilized to determine the CMC values of the targeted and non-targeted aggregates. A block copolymer solution (450 μL) was prepared in THF in vials using serial dilution as described above. A 50 μL solution of Nile Red in THF (0.03 mg / mL) was then added to each vial, followed by 3 mL of water. The THF was allowed to evaporate completely in an open atmosphere, and final concentration values were obtained for 16 samples in the range of 1×10 -9 ~1×10 -5 M. Fluorescence measurements were recorded with a fluorescence spectrophotometer at an excitation wavelength of 550 nm, and emission was monitored at 580 - 660 nm.

[0148] To determine the minimum required concentration of the amphiphilic polymer to form a micelle-type polymer structure by self-assembly, the CMC value of the block copolymer was calculated by tracking the fluorescence intensity of Nile Red as a function of polymer concentration.

[0149] Fluorescence emission spectra of Nile Red in the non-targeted and targeted aggregates were obtained at an excitation wavelength of 550 nm, and emission was monitored at 580 - 660 nm (Figure 4 and Figure 5, respectively).

[0150] To determine the CMC value, a plot of the emission intensity at 612 nm versus the logarithm of the copolymer concentration was obtained. The CMC values for non-targeted and targeted aggregate formation were calculated by the intersection of the trend line belonging to the intensity ratio with a relatively constant value and the rapidly increasing intensity ratio (Figure 6 and Figure 7, respectively). The CMC values for non-targeted and targeted aggregate formation were found to be 1.796×10 -6 M and 1.566×10 -6 M, respectively.

[0151] Example 2A: Synthesis of Polymerizable 5FU Monomer (5FU-MA) 5-Fluorouracil (250 mg, 1.92 mmol) and 37% formalin (244 μL) were reacted at 60 °C for 2 hours. The mixture was then cooled and completely lyophilized to obtain a viscous compound. As a second step, this product (315 mg, 1.97 mmol) was dissolved in anhydrous acetonitrile (5.8 mL), and then triethylamine (422 μL, 3.03 mmol) was added to this solution. After cooling the reaction mixture to 0 °C, methacryloyl chloride (232 μL, 2.38 mmol) was added dropwise. The reaction was allowed to proceed overnight at room temperature, and then the white precipitate was filtered off. All volatiles were evaporated under vacuum, and then the crude product was dissolved in dichloromethane (20 mL) and subsequently extracted with 1 M HCl (2 × 10 mL), 1 M NaHCO3 (10 mL), and brine (10 mL). After concentrating the organic portion, the product was isolated in pure form by silica column chromatography eluting with an ethyl acetate:hexane mixture (30:70 v / v). The product was obtained as a viscous colorless oil (305 mg, 68% yield).

[0152] Example 2B: Synthesis of POEGMEMA Homopolymer Reversible addition-fragmentation chain transfer (RAFT) polymerization was used for the synthesis of the POEGMEMA homopolymer. For the synthesis of the POEGMEMA polymer, AIBN (0.78 mg, 4.76 μmol) was added to a solution of OEGMA (500 mg, 1.66 mmol) and CPADB (11.97 mg, 42.85 μmol) in DMF (2.5 mL). The mixture was purged with N2 to remove O2, and the polymerization was stirred at 70 °C for 16 hours. The polymerization was stopped by cooling and exposure to air. The POEGMEMA polymer was purified by precipitation in diethyl ether. The polymer precipitate was dried under vacuum to obtain 95 mg of polymer in 19% yield.

[0153] Example 2C: Synthesis of POEGMEMA-co-5FU-MA Block Copolymer RAFT polymerization was used for the synthesis of the POEGMEMA-b-P5FU-MA block copolymer. AIBN (0.13 mg, 0.786 μmol) was added to a solution of 5FU-MA (17.9 mg, 78.6 μmol) and the POEGMEMA polymer (40 mg, 3.93 μmol) as a macro chain transfer agent in DMF (0.30 mL). The mixture was purged with N2 to remove O2 and the polymerization was stirred at 70 °C for 16 h. The polymerization was stopped by cooling and air exposure. The crude product was precipitated in diethyl ether. The polymer precipitate was dried in vacuo to obtain 16 mg of the polymer in 28% yield.

[0154] Example 3A: Synthesis of the polymerizable combretastatin-A4 monomer (Comb- SS -MA) 2-Hydroxyethyl methacrylate (500 mg, 3.8 mmol), 4,4'-dithiobutyric acid (1.83 g, 7.6 mmol), N,N'-dicyclohexylcarbodiimide (1.59 mg, 7.8 mmol), and 4-(dimethylamino)pyridine (281 mg, 2.3 mmol) were dissolved in anhydrous dichloromethane (DCM) (54 mL) under N2 in a 100 mL round-bottom flask. The reaction solution was stirred at room temperature for 16 h. The crude product was precipitated in cold diethyl ether and left at -20 °C for 20 min. The product was purified by silica column chromatography using ethyl acetate and hexane (20:80 v / v) (426 mg, 32% yield).

[0155] Next, the obtained product (300 mg, 0.85 mmol) and combretastatin-A4 (270 mg, 0.85 mmol) were dissolved in anhydrous dichloromethane (DCM) (6 mL) with DIPC (129 mg, 1.02 mmol) and DMAP (104 mg, 0.85 mmol) under N2 in a 25 mL round-bottom flask. The reaction solution was stirred at room temperature for 16 h. The Comb- SS -MA monomer was purified by silica column chromatography using ethyl acetate and hexane (30:70 v / v) (195 mg, 35% yield).

[0156] Example 3B: Synthesis of POEGMEMA and Comb- SS -MA-POEGMEMA Homopolymer Reversible addition-fragmentation chain transfer (RAFT) polymerization was used for the synthesis of POEGMEMA. For the synthesis of the POEGMEMA polymer, AIBN (2.17 mg, 0.0013 mmol) was added to a solution of OEGMA (1.0 g, 3.3 mmol) and CPADB (36.88 mg, 0.133 mmol) in DMF (5 mL). The mixture was purged with N2 to remove O2, and the polymerization was stirred at 70 °C for 16 h. The polymerization was terminated by cooling and air exposure. The POEGMEMA polymer was purified by precipitation in diethyl ether. The polymer precipitate was dried under vacuum to obtain 715 mg of the polymer with a yield of 69%.

[0157] Example 3C: Synthesis of POEGMEMA-co-Comb- SS -MA Block Copolymer POEGMEMA-b-PComb- SS RAFT polymerization was used for the synthesis of the POEGMEMA-b-PComb- SS -MA block copolymer. AIBN (0.68 mg, 0.004 mmol) was added to Comb-

[0158] The above examples show that the present invention is applicable to a range of various linkers such as disulfides and acetals, as well as various drug molecules such as combretastatin and 5-FU.

[0159] [Claim 1] A polymer-drug conjugate of formula I in the form of a block copolymer for delivering a therapeutic agent, [Chemical formula] In the formula, ·R1, R2, and R3 are independently selected from H or -CH3 ·x is a natural number from 1 to 100 ·y is a natural number from 1 to 100 ·n is a natural number from 1 to 50 ·L is a cleavable linker ·D is a therapeutic agent, and is combretastatin, or 5-fluorouracil (5-FU), or gemcitabine, or chloroquine, or doxorubicin ·A is a terminal group or A may be null ·B is a terminal group or B may be null. [Claim 2] The polymer-drug conjugate according to claim 1, wherein the linker is selected from the group consisting of poly(ethylene glycol), amino acids, poly(amino acids), and short-chain peptides. [Claim 3] The polymer-drug conjugate according to claim 1 or 2, wherein the linker is a short peptide unstable to cathepsin B. [Claim 4] The polymer-drug conjugate according to any one of claims 1 to 3, wherein the short peptide is selected from the group consisting of Gly-Phe-Leu-Gly (SEQ ID NO: 1), Val-Cit, Phe-Lys, Val-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 2). [Claim 5] The polymer-drug conjugate according to any one of claims 1 to 4, wherein the linker is a C1-C hydrocarbon or a C1-C substituted or hetero-substituted hydrocarbon containing a functional group that dissociates under physiological conditions. 10 hydrocarbon or C1-C 10 substituted or hetero-substituted hydrocarbon. [Claim 6] The polymer-drug conjugate according to claim 5, wherein the functional group is selected from acetal, ester, imine, amide, disulfide, carbonate, hydrazine, carbamate. [Claim 7] The polymer-drug conjugate according to any one of claims 1 to 6, wherein no linker is present. [Claim 8] The polymer-drug conjugate according to any one of claims 1 to 7, wherein the therapeutic agent is present in an amount of 5% to 50% by weight of the polymer-drug conjugate. [Claim 9] The polymer-drug conjugate according to any one of claims 1 to 10, wherein the polymer-drug conjugate of the formula has an average molecular weight of 5 kDa to 60 kDa. [Claim 10] The polymer-drug conjugate according to any one of claims 1 to 13, wherein A is a terminal group that is a chain transfer agent (CTA) or a fragment of an initiator, or a CTA or a fragment of an initiator conjugated to a target moiety, or a CTA or a fragment of an initiator having a reactive functional group. [Claim 11] The polymer-drug conjugate according to any one of claims 1 to 14, wherein B is a terminal group that is a chain transfer agent or a fragment of an initiator. [Claim 12] The polymer-drug conjugate according to claim 10, wherein the target moiety is selected from the group consisting of an antibody, an antibody fragment, or a peptide such as Cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGDfK) (SEQ ID NO: 3). [Claim 13] The polymer-drug conjugate according to claim 10, wherein the reactive group is selected from the group consisting of acetal, hemiacetal, carboxylic acid, alcohol, amide, imide, anhydride, aryl halide, azo compound, diazo compound, hydrazine, azide, carbonate, chlorosilane, cyanide, ester, sulfate ester, phosphate ester, thiophosphate ester, isocyanate, isothiocyanate, thiocarbamate ester, dithiocarbamate ester. [Claim 14] A polymer aggregate (nanoparticle or micelle) formed using the polymer-drug conjugate of formula I according to any one of claims 1 to 13.

Chemical formula

[0160] Sequence Listing 1 <223>Sequence Number 1 Sequence Listing 2 <223>Sequence Number 2 Sequence Listing 3 <223>Period

Claims

1. A polymer-drug conjugate of formula I in the form of a block copolymer for delivering a therapeutic agent, 【Chemical 1】 wherein, ・R １ , R ２ , and R ３ are -CH ３ respectively - x is a natural number from 1 to 100 - y is a natural number from 1 to 100 - n is a natural number from 1 to 50 - L is a cleavable linker and contains GFLG - D is an anti-cancer agent, combretastatin, or 5-fluorouracil (5-FU), or gemcitabine - A is a terminal group and contains cRGDfK - B is a fragment of a polymerization initiator and is part of S,S-dibenzyl trithiocarbonate.

2. A polymeric micelle comprising the polymer-drug conjugate of formula I according to claim 1, [Chemical 2] wherein, ・R １ 、R ２ 、and R ３ is -CH ３ as follows - x is a natural number from 1 to 100 - y is a natural number from 1 to 100 - n is a natural number from 1 to 50 - L is a cleavable linker and contains GFLG - D is an anti-cancer agent, combretastatin, or 5-fluorouracil, or gemcitabine - A is a terminal group and is a target moiety containing cRGDfK - B is a fragment of a polymerization initiator and is part of S,S-dibenzyl trithiocarbonate.

3. The polymeric micelle according to claim 2, further comprising an anti-cancer agent encapsulated as a second anti-cancer agent and bound to the polymer-drug conjugate of formula I.

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