EA044119B1 - SELF-ASSEMBLED DIBLOCK COPOLYMERS CONSISTED OF PEGMEMA AND DRUG-CARRIING POLYMER SEGMENTS - Google Patents

Description

This invention relates to polymer-drug conjugates of formula I, agglomerates composed of polymer-drug conjugates of formula I, methods for preparing said polymer-drug conjugates and agglomerates, and their use in the treatment of diseases such as cancer.

BACKGROUND OF THE INVENTION

Chemotherapeutic agents used to treat cancer are mostly cytotoxic. These drugs can accumulate in body tissues other than the target area, which in turn causes reduced therapeutic benefit and undesirable distribution of the drug to healthy body tissues. Uncontrolled distribution of these drugs throughout the body causes severe side effects in the patient.

To solve the above problems, drug delivery systems are being developed that can deliver the drug to target areas of the body. For example, in some cancer treatment approaches, these systems take advantage of the enhanced permeability and retention (EPR) effect, which means that drug carriers having a high molecular weight and high hydrodynamic volume accumulate in solid tumors, which in turn leads to the passive direction of the drug molecule to the tumor tissue and minimizes the destruction of healthy tissue by chemotherapeutic agents.

Since the discovery of the enhanced permeability and retention (EPR) effect, long-circulating macromolecules have become the main agents for drug delivery due to improved accumulation in tumors through porous-walled blood vessels.

There is great interest in polymer agglomerates in drug delivery systems for the treatment of cancer, mainly due to their body distribution properties. The present invention also relates to agglomerates that are suitable for use in drug delivery, in particular anticancer agents for the treatment of cancer.

Field of technology to which the invention relates

There are various types of methods for producing polymer agglomerates known in the art. For example, self-assembly of polymer compounds is an attractive way to obtain a polymer agglomerate. Eisenberg and colleagues demonstrated the first example of self-assembly of asymmetric copolymers in solution in the 1990s. Next, attention was focused on amphiphilic block copolymers, having polymer blocks with different physical and chemical properties that produce polymer structures through self-assembly.

Drug incorporation into such polymer agglomerates can be achieved through covalent attachment or physical encapsulation through hydrophobic interactions. Given the very small total volume of these agglomerates in their colloidal suspensions, high drug loading rates are critical to obtaining effective drug formulations. Low drug loading into polymer agglomerates, mainly in micellar formulations, continues to be a major drawback in the field.

Several researchers have addressed this issue by using π-π interactions to modify drug molecules or by using an additional compound instead of providing covalent attachment. However, unencapsulated drug aggregates in micellar-type polymer nanoparticle formulations constitute another drawback in providing a safe formulation for their clinical applications.

In another approach, Stenzel and colleagues used amphiphilic drug-attached block copolymers to produce the hydrophobic segment of the block copolymers to produce polymer agglomerates. This strategy did not allow complete conjugation of the drug to the polymer scaffold due to steric hindrance, resulting in an unclear final structure. In addition, remaining reactive groups on the polymer backbone due to incomplete conjugation may represent the most important disadvantage of this kind of post-polymerization conjugation strategies due to possible unwanted biological interactions in vivo.

In light of the current state of the art summarized above, there is a need for block copolymers and agglomerates derived therefrom that can be well defined by drug content, that provide efficient drug conjugation without any remaining reactive sites, and that have high degree of drug administration.

The inventors have discovered that the amphiphilic block copolymers of the present invention provide the formation of micellar-type agglomerates that have a high drug content and that are well defined by drug attachment.

Brief description of the invention

The present invention relates to a polymer-drug conjugate in the form of a block copolymer for the delivery of therapeutic agents, which is shown in Formula I

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In other words, the invention relates to a polymer-drug conjugate of formula I in the form of a block copolymer and agglomerates derived from polymer-drug conjugates of formula I, where

_R1 and R2 are independently selected from H or -CH3;

R3 is selected from -H or -CH3;

x means a natural number between 1-100;

y means a natural number between 1-100;

n means a natural number between 1-50;

L is a cleavable linker and

D is a therapeutic agent that is selected from the group consisting of combretastatin or 5-fluorouracil (5-FU), or gemcitabine, or chloroquine or doxorubicin, and which is present in an amount of from 5 to 50 wt.%, polymer-drug conjugate;

A represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or an initiator conjugated to a directing moiety, or an initiator having a reactive functional group, or A may be absent;

B represents a terminal group that is a fragment of a chain transfer agent (CTA) or initiator, or B may be absent.

The polymer-drug conjugate has an average molecular weight of 5 to 60 kDa. The inventors have discovered that in addition to overcoming the disadvantages of prior art polymer agglomerates; The polymer agglomerates of the present invention, which are composed of polymer-drug conjugates of Formula I, also make it possible to reduce the amount of polymer that is given to the patient due to high drug loading.

Detailed Description of the Invention

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

The term polymer agglomerate refers to a structure having a diameter between 1-100 nm. Said agglomerates are composed of self-assembled polymer chains and may or may not have hollow cavities of varying sizes.

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 an oligo-ethylene glycol unit, and the pendant group may carry one therapeutic agent or any other group that can be used to attach a therapeutic and/or diagnostic agent or targeting group.

Throughout the text, the term polymer-drug conjugate of the invention is intended to mean a polymer-drug conjugate of Formula I or a polymer-drug conjugate of Formula I or Formula I, and these terms may be used interchangeably.

Throughout the text, the term polymer agglomerates should be considered to mean a nanoparticle consisting of a polymer-drug conjugate of Formula I or a nanoparticle consisting of a copolymer shown by Formula I or a nanoparticle that is formed by self-assembly of a polymer-drug conjugate of Formula I or micellar an agglomerate consisting of a copolymer shown by Formula I, and these terms can be used interchangeably.

The term PEG refers to a polyester compound having the structure H-(O-CH2-CH2)nOR3, where n is a natural number between 1-200, 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 may be used interchangeably throughout this text.

In one embodiment, R1, R2 and R3 are independently selected from H or -CH3.

In one embodiment, R1=H, R ₂ =H, R3=H, or R1=H, R ₂ =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.

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The term block copolymer refers to a copolymer where all of one type of monomer is grouped together, and all of the other type of monomer is grouped together. The polymer-drug conjugates of the invention are in the form of a block copolymer. The fact that the polymer-drug conjugates of the invention are in the form of a block copolymer allows the formation of a polymer agglomerate of the invention.

The therapeutic agent is attached to the polymer via a cleavable linker so that the therapeutic agent can be released, for example, under reducing conditions, under oxidative conditions, or by hydrolysis of an ester, amide, hydrazide.

Said cleavable linker may be any hydrocarbon or substituted hydrocarbon based compound that is capable of dissociating under physiological conditions. In a preferred embodiment, the linker may be selected from compounds that are degraded under tumor oxidative conditions (such as any C ₁ -C ₁ o-substituted or unsubstituted and/or linear and/or cyclic hydrocarbon containing an acetal or ester functionality) or at with the help of overexpressed enzymes present in the intercellular or intracellular matrix of tumor cells.

The linker can be any type of structural unit capable of binding to both the polymer backbone and the drug, such as poly(ethylene glycol), amino acid, poly(amino acid) (eg, peptide or oligopeptide), or polypeptide (eg, protein ) so 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. Linkers can also include short peptides with specific peptide sequences that are cathepsin labile (disintegrated by cathepsin B), such as Gly-Phe-Leu-Gly (SEQ ID NO: 1), also referred to as GFLG, or Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu (SEQ ID NO: 2).

The linker may also be a C ₁ -C ₁₀ hydrocarbon or a C ₁ -C ₁₀ substituted or heterosubstituted hydrocarbon so long as it contains a functional group that dissociates under physiological conditions, such as an acetal, ester, imine, amide , disulfide, carbonate, carbamate, hydrazone.

In one embodiment of the invention, the linker (L) is GFLG or Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu.

In one embodiment, the linker is a C ₁ -C ₁₀ heterosubstituted hydrocarbon containing at least one disulfide functional group or a C ₁ -C ₁₀ heterosubstituted hydrocarbon containing at least one acetal functional group or C ₁ -C ₁₀ -heterosubstituted hydrocarbon containing at least one ester functional group or C ₁ -C ₁₀ -heterosubstituted hydrocarbon containing at least one imine functional group or C ₁ -C ₁₀ -heterosubstituted hydrocarbon containing at least one amide functional group or C ₁ -C ₁ o-heterosubstituted hydrocarbon containing at least one carbonate functional group or C ₁ -C ₁₀ -heterosubstituted hydrocarbon containing at least one carbamate functional group or C ₁ -C ₁₀ -heterosubstituted hydrocarbon containing at least one hydrazone functional group.

In yet another embodiment, the linker may be a C1-C ₁₀ substituted or heterosubstituted hydrocarbon containing two or more functional groups selected from the group consisting of acetal, ester, imine, amide, disulfide, carbonate, carbamate, hydrazone.

In another embodiment of the invention, the linker may be absent, which means that therapeutic agent D is attached directly to the polymer chain. Direct attachment of the therapeutic agent to the polymer chain can be accomplished through ester, imine, amide, disulfide, carbonate, carbamate, or hydrazine linkages.

In a preferred embodiment of the invention, the drug molecule is attached to the polymer via a direct ester linkage, which dissociates in the physiological environment of the tumor. In another embodiment, the linker may be a combination of a C ₁ -C ₁₀ substituted hydrocarbon containing at least one functional group selected from the group consisting of acetal, ester, imine, amide, disulfide, carbonate, carbamate, hydrazone , and a peptide chain selected from the group consisting of GFLG, Val-Cit or Phe-Lys or Val-Ala or Ala-Leu-Ala-Leu. The therapeutic agent selected from combretastatin, 5-fluorouracil, gemcitabine, chloroquine and doxorubicin may be present in an amount of from 5 to 50% by weight of the polymer-drug conjugate, preferably in an amount of from 6 to 48% by weight of the polymer-drug conjugate and most preferably in an amount of from 10 to 45% by weight of the polymer-drug conjugate. The therapeutic agent may be present in an amount ranging from, for example, 10 to 48% or 15 to 46% or 20 to 45% or 25 to 44% by weight of the polymer-drug conjugate.

The term end group refers to functional groups or structural (elementary) groups

- 3 044119 links that are located at the end of the polymer. The end groups shown as A and B may be the same or may be different from each other.

In one embodiment, A represents a terminal group that is optionally a fragment of a chain transfer agent or initiator, or a fragment of a chain transfer agent or initiator conjugated to a directing moiety, or a fragment of a chain transfer agent or initiator having a reactive functional group.

In one embodiment, B represents a terminal group that is optionally part of a chain transfer agent or initiator.

The term chain transfer agent refers to chemical compounds that are capable of adjusting the molecular weight of polymers by interrupting uncontrolled polymer chain growth. Chain transfer agents are well described 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 may be used interchangeably throughout the text.

A and B may optionally be a fragment of a chain transfer agent. In particular, when CTA is used in a polymerization reaction, a CTA moiety may be attached to the polymer as a terminal group. CTA, as used herein, can be any substance known in the art that is suitable for initiating a polymerization reaction. In one embodiment, A and/or B is selected from the group consisting of 3,5-bis(2dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, 3-butenyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, 2-cyanobutane-2- yl 4-chloro-3,5-dimethyl-1H-pyrazole-1-carbodithioate, 2-cyanobutanyl-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,Sdibenzyltrithiocarbonate, 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid, 3-azido-1propanol 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid ester, 2-(dodecyl) Nhydroxysuccinimide ester thiocarbonothioylthio)-2-methylpropionic acid , 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiocarbonothioylthio)propionic acid, methyl 2-(dodecylthiocarbonothioylthio)-2methylpropionate, tetrakis[2-(dodecylthiocarbonothioylthio)-2- methylpropionate]pentaerythritol, phthalimidomethyl-butyltrithiocarbonate, 1,1,1-tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane, benzyl 1H-pyrrole-1-carbodithioate, cyanomethyldiphenylcarbamodithioate, cyanomethylmethyl(phenyl)carbamodithioate, cyanomethylmethyl(4-pyridyl)carbamodithioate, 2-cyanopropan-2-yl N-methyl-N(pyridin-4-yl)carbamodithioate, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate, 1succinimidyl-4-cyano-4-[N-methyl-N-(4-pyridyl)carbamothioylthio ]pentanoate, benzylbenzodithioate, cyanomethylbenzodithioate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, 2-cyano-2-propylbenzodithioate, 2-cyano-2- propyl 4-cyanobenzodithioate, ethyl 2-(4-methoxyphenylcarbonothioylthio)acetate, ethyl 2-methyl-2-(phenylthiocarbonylthio)propionate, ethyl 2-(phenylcarbonothioylthio)2-phenylacetate, ethyl 2-(phenylcarbonothioylthio)propionate, 1-(methoxycarbonyl) )ethylbenzodithioate, 2(4-methoxyphenylcarbonothioylthio)ethanoic acid, 2-nitro-5-(2-propynyloxy)benzyl 4-cyano-4(phenylcarbonothioylthio)pentanoate, 2-(phenylcarbonothioylthio)propanoic acid, 2-phenyl-2-propylbenzodithioate, cyanomethylmethyl(4-pyridyl)carbamodithioate, 2-Cyanopropan-2-yl N-methyl-N(pyridin-4-yl)carbamodithioate, methyl 2-[methyl(4-pyridinyl)carbamothioylthio]propionate, 1succinimidyl-4-cyano-4 -[N-methyl-N-(4-pyridyl)carbamothioylthio]pentanoate or any moiety of the initiators provided herein, alone or conjugated to a directing moiety before or after the polymerization reaction or modified with a reactive group.

The term fragment as used herein refers to compounds that are formed by the cleavage of one or more covalent bonds forming the initiator molecule.

The fragmentation of the CTA compounds provided herein and the structure of the resulting fragments are well established in the art, so the structure of A and B can be determined without the use of inventive skill by using the current state of the art.

A and/or B are not necessarily missing.

In one embodiment of the invention, A is absent and B is a fragment of CTA.

In yet another embodiment, B is absent and A is a CTA fragment.

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

In yet another embodiment, A and B are both CTA fragments and they have the same chemical structure.

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The term targeting moiety refers to a molecule that typically binds to specific target sites in the body. In other words, the targeting fragment is a molecule that binds specifically to a cell that has a complementary receptor.

The targeting moiety may be selected from the group consisting of antibodies; antibody fragments, or peptides, such as cyclo(Arg-Gl·y-Asp-D-Phe-Lys) (amino acid sequence SEQ ID NO: 3) (cRGDfK). In a preferred embodiment, cRGDfK is used as a targeting moiety.

The term reactive group refers to an atom or associated group of atoms in a chemical substance that is implied or reasonably expected to readily undergo a chemical reaction.

The reactive group may be an 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. Preferably the reactive group is an ester, imide or carbonate. The reactive group, for example, may be N-hydroxysuccinimide.

Depending on the polymerization methods used in the preparation of the polymer conjugates of the invention, A and/or B may be an initiator moiety.

An initiator as used herein refers to a chemical compound that reacts with a monomer to form an intermediate capable of combining sequentially with a large number of other monomers to form a polymeric compound. The terms initiator and polymerization initiator may be used interchangeably within the context of this application.

Depending on the polymerization methods used to prepare the polymer conjugates of the invention, various initiator agents may be used.

In one embodiment, A and/or B is an initiator moiety that may be selected from the group consisting of 4,4'-azobis(4-cyanovaleric acid), 4,4'-azobis(4cyanovaleric acid), 1,1 '-azobis(cyclohexanecarbonitrile), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionitrile) (also called AIBN), ammonium persulfate, hydroxymethanesulfinic acid monosodium dihydrate, potassium persulfate, sodium persulfate, tert-butyl hydroperoxide, tert-butyl peracetate, cumene hydroperoxide, 2,5-di(tert-butylperoxy)-2,5-dimethyl-3-hexine, 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-butylperoxybenzoate, tert-butylperoxy 2-ethylhexyl carbonate, tert-butyl hydroperoxide, 2-azidoethyl-2bromoisobutyrate, bis[2-(2-bromoisobutyryloxy)undecyl]disulfide, bis[2-(2'-bromoisobutyryloxy)ethyl ]disulfide, 2-bromoisobutanoic acid N-hydroxysuccinimide ester, 2-bromoisobutyric anhydride, α-bromoisobutyryl bromide, 2-(2-bromoisobutyryloxy)ethyl methacrylate, tert-butyl-a-bromoisobutyrate, 3-butynyl 2-bromoisobutyrate, dipentaerythritol hexakis(2-bromoisobutyrate), up to decile 2-bromoisobutyrate, ethyl α-bromoisobutyrate, ethylene bis(2-bromoisobutyrate), 2-hydroxyethyl 2-bromoisobutyrate, 1-(DL-1,2-isopropylideneglyceryl)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, 1,1,1tris(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-3azahexane-3-nitroxide, 3,5-bis(2-dodecylthiocarbonothioylthio-1-oxopropoxy)benzoic acid, 3-butenyl 2- (Dodeciltocyltiocarbonotioiltio) -2-methylpropionate, 4-social-4-[(predecil sulfinylthycarbonil) sulfannil] penthane acid, 4-social-4-[(dodecyl sulfinyltycarbonyl) sulfanil] pentanol, cyanomethyldodecil, cyanomethyl [3- (trimetoxisylil) carbonate, 2-all-2-prophyldeeciltricocarbonate, S, S-dibenzyltrittiocarbonate, 2- (Dodeciltocyltiocarbonotioiltio) -2-methylpropionic acid, 2- (predecylthicocarbonotioiltio) -2-methylpropionic acid, 3-azido-1propanol complex ether, n-gydroxycsuccation Nimed complex ether 2 (dodecylthiocarbonothioylthio)-2-methylpropionic acid, 2(dodecylthiocarbonothioylthio)-2-methylpropionic acid pentafluorophenyl ester, 2-(dodecylthiocarbonothioylthio)propionic acid, methyl 2-(dodecylthiocarbonothioylthio)-2-methylpropionate, pentaer ytrite tetrakis[2(dodecylthiocarbonothioylthio)-2 -methylpropionate], phthalimidomethyl-butyltrithiocarbonate, 1,1,1tris[(dodecylthiocarbonothioylthio)-2-methylpropionate]ethane, benzylbenzodithioate, cyanomethylbenzodithioate, 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, N-succinimidyl ester 4cyano-4- ( phenylcarbonothioylthio)pentanoic acid, 2-cyano-2-propylbenzodithioate, 2-cyano-2-propyl 4-cyanobenzodithioate, ethyl 2-(4-methoxyphenylcarbonothioylthio)acetate, ethyl 2-methyl-2-(phenylthiocar

-5 044119 Boiltio) Propionate, ethyl 2- (phenylkarbonotioiltio) -2-phenylacetate, ethyl 2- (phenelcarbonotioiltio) propionate, 1-(methoxiccarbonyl) ethylbenzoditioate, 2- (4-methoxyphencarbonotioiltio) ethanic acid, 2-5-5- 5- 2-propynyloxy)benzyl, 4-cyano-4-(phenylcarbonothioylthio)pentanoate, 2-(phenylcarbonothioylthio)propanoic acid, 2-phenyl-2-propylbenzodithioate, 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-K-(4pyridyl)carbamothioylthio]pentanoate or a combination thereof, as such or conjugated to a targeting moiety before or after the polymerization reaction or modified with a reactive group.

The definition and examples of a guide fragment are as described above.

The definition and examples of a reactive group are as described above.

The chain transfer agents and initiators provided in this application are given by way of example to illustrate the invention, and any chemical compounds published before or after the filing of this document that may act as a CTA and/or initiator are included within the scope of the present invention.

Measuring the amount of drug in the polymer conjugate of the invention is performed using commonly used methods well known in the art, for example, by calculating the drug fraction based on 1H NMR data for the polymer-drug conjugate or by determining the amount during accelerated release of the drug.

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

The molecular weight of the polymer-drug conjugate of the invention is determined using commonly used methods known in the art, for example, using gel permeation chromatography (GPC).

Another embodiment of the invention is polymer agglomerates in the form of nanoparticles formed by polymer-drug conjugates shown by formula I, where

Formula.

R ₁ and R 2 are independently selected from H or -CH ₃ ;

R ₃ is selected from -H or -CH ₃ ;

x means a natural number between 1-100;

y means a natural number between 1-100;

n means a natural number between 1-50;

L is a cleavable linker and

D is a therapeutic agent that is combretastatin or 5fluorouracil or gemcitabine or chloroquine or doxorubicin, and which is present in an amount of from 5 to 50% by weight of the polymer-drug conjugate;

A represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or an initiator conjugated to a directing moiety, or an initiator having a reactive functional group, or A may be absent;

B represents a terminal group that is a fragment of a chain transfer agent (CTA) or initiator, or B may be absent.

Another embodiment of the invention provides polymer micelles formed by polymer-drug conjugates shown by formula I, where

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R ₂

Formula I

R1 and R2 are independently selected from H or -CH3;

R3 is selected from -H or -CH3;

x means a natural number between 1-100;

y means a natural number between 1-100;

n means a natural number between 1-50;

L is a cleavable linker or L may be absent and

D is a therapeutic agent that is combretastatin or 5fluorouracil or gemcitabine or chloroquine or doxorubicin, and which is present in an amount of from 5 to 50% by weight of the polymer-drug conjugate;

A represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or an initiator conjugated to a directing moiety, or an initiator having a reactive functional group, or A may be absent;

B represents a terminal group that is a fragment of a chain transfer agent (CTA) or initiator, or B may be absent.

In yet another embodiment, the agglomerates of the present invention are used to encapsulate therapeutic molecules and, in that aspect, polymer agglomerates composed of polymer-drug conjugates of Formula I encapsulating therapeutic agents in addition to the therapeutic agents attached to the polymer chain represent another embodiment of this invention .

The term encapsulation refers to the enclosure of a guest molecule, such as a therapeutic agent, within the cavity of a host molecule, such as a polymer agglomerate composed of a polymer-drug conjugate of Formula I. Encapsulation preferably occurs through non-covalent interactions of the therapeutic molecule with the polymer agglomerate of the invention.

The term therapeutic agent refers to any compound that is 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 may be used interchangeably. In one embodiment, the disease is cancer.

In addition, a therapeutic agent also refers to any agent that is suitable for use in treating a disease, such as cancer. Any therapeutic agent that can be directly or indirectly attached to the polymer-drug conjugate of the invention can be used. US Patent No. 66342221 also describes agents related to anti-cancer agents and is incorporated herein by reference. Anticancer agents may be classified as chemotherapeutic agents, cytotoxins, antimetabolites, alkylating agents, protein kinase inhibitors, anthracyclines, antibiotics, antimitotic agents (eg, antitubulin agents), corticosteroids, radiopharmaceuticals, and proteins (eg, cytokines, enzymes, or interferons), but are not limited to this. Specific examples of anticancer agents include, for example, docetaxel, gemcitabine, imatinib, 5-fluorouracil, 9-aminocamptothecin, amine-modified geldanamycin, doxorubicin, paclitaxel, procarbazine, hydroxyurea, meso e-chlorin, cisplatin, and radionuclides (eg, 1131, Y-90 , In-111 and Ts-99m). There are many other anti-cancer agents known in the art, and many continue to be developed, all of which are also included within the scope of this invention.

The therapeutic agent may also be selected from the subgroup including nucleoside analogs, antifolates, other metabolites, topoisomerase I inhibitors, anthricyclines, podophyllotoxins, taxanes, vinca alkaloids, alkylating agents, platinates, antihormones, radiopharmaceuticals, monoclonal antibodies, tyrosine kinase inhibitors, inhibitors mammalian target of rapamycin (mTOR), retinoids, immunomodulatory agents, histone deacetylase inhibitors and other agents, but not limited to.

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Nucleoside analogues may be selected from the group including, but not limited to, azacitidine, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, gemcitabine, mercaptopurine, nelarabine, pentostatin, thioguanine, trifluridine, tipiracil.

Antifolates may be selected from the group including, but not limited to, methotrexate, pemetrexed, pralatrexed, raltitrexed.

Other metabolites may be selected from the group including, but not limited to, hydroxyurea.

Topoisomerase I inhibitors may be selected from the group including, but not limited to, irinotecan and topotecan.

Anthracyclines may be selected from the group including, but not limited to, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin.

Podophyllotoxins may be selected from the group including, but not limited to, etoposide and teniposide.

Taxanes may be selected from the group including, but not limited to, cabazitaxel, docetaxel, paclitaxel.

Vinca alkaloids may be selected from the group including, but not limited to, vinblastine, vincristine, vindesine, vinflunine, vinorelbine.

Alkylating agents may be selected from the group including, but not limited to, bendamustine, chlorambucil, dacarbazine, melphalan, streptozotocin, trabectedin.

Antihormone compounds may be selected from the group including, but not limited to, abiraterone, bicalutamide, cyproterone, degarelix, exemestane, fulvestrant, goserelin, histrelin, leuprolide, mifepristone, triptorelin.

Tyrosine kinase inhibitors may be selected from the group including, but not limited to, afatinib, axitinib, bosutinib, cobimetinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, osimertinib, pazopanib, ruxolitinib, sunitinib, vandetanib.

Mammalian target of rapamycin (mTOR) inhibitors may be selected from the group including, but not limited to, everolimus, temsirolimus.

Retinoids may be selected from the group including, but not limited to, alitretinoin, bexarotene, isotretinoin, tamibarotene, tretinoin.

Immunomodulatory agents may be selected from the group including, but not limited to, lenalidomide, pomalidomide, thalidomide.

Histone deacetylase inhibitors may be selected from the group including, but not limited to, belinostat, panobinostat, valproate, vorinostat.

Platinates may be selected from the group including, but not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin.

Other agents may be selected from the group consisting of anagrelide, ceritinib, dabrafenib, idelalisib, ibrutinib, palbociclib, vemurafenib, bleomycin, bortezomib, dactinomycin, eribulin, estramustine, ixabepilone, mitomycin, procarbazine, alectinib, fluximesterone, iobenguane, them quimod, interferon , ixazomib, lanreotide, lentinan, octreotide, omacetaxine, tegafur, gimerazil, oteracil, uracil, combretastatin, chloroquine, but not limited to.

In a preferred embodiment of the 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 consisting of docetaxel, pemetrexed, chloroquine, combretastatin, gemcitabine, doxorubicin, fluorouracil (5-FU), 5'deoxy-5-fluorocytidine (5'-DFCR), lapatinib.

In one embodiment of the invention, the therapeutic agent is docetaxel.

In one embodiment of the invention, the therapeutic agent is carboplatin.

In one embodiment of the invention, the therapeutic agent is doxorubicin.

Another embodiment of the invention is a method (Method I) for preparing the polymer-drug conjugate of the invention (Formula I), which includes (i) polymerizing a PEG-(meth)acrylate monomer (Formula II)

- 8 044119

Where

R1 is selected from H or -CH3

R3 is selected from -H or -CH3 n is a natural number between 1-50, giving the polymer of formula IIa

Where

A represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or an initiator conjugated to a directing moiety, or an initiator having a reactive functional group, or A may be absent,

B represents a terminal group that is a fragment of a chain transfer agent (CTA) or initiator, or B may be absent.

And then (ii) further reacting the compound of formula IIa with the (meth)acrylate-L-D monomer (formula IIIa)

Fl,

Formula where R2 is selected from H or -CH3,

L is a cleavable linker or L may be absent,

D is a therapeutic agent selected from the group consisting of combretastatin, 5-FU, gemcitabine, chloroquine, doxorubicin, resulting in a polymer-drug conjugate of formula I.

In another aspect, the method (Method II) for preparing the polymer-drug conjugate of the invention (Formula I) comprises (i) polymerizing a PEG-(meth)acrylate monomer (Formula II)

Where

R1 is selected from H or -CH3;

R3 is selected from -H or -CH3;

n means a natural number between 1-50;

What does a polymer of formula IIa produce?

- 9 044119

Where

A represents a terminal group or A may be absent,

B represents a terminal group or B may be absent.

And then (ii) further reacting the compound of formula Ha with the (meth)acrylate-L monomer (formula IIIb)

R?

Formula |||h where R2 is selected from H or -CH3;

L is a cleavable linker or L may be absent, resulting in a copolymer as shown by Formula IIb

V

About η Re

Formula ||ь where x means a natural number between 1-100;

y is a natural number between 1-100 and then (iii) reacting a compound of formula IIb with a therapeutic agent (D) selected from the group consisting of combretastatin, 5-FU, gemcitabine, chloroquine, doxorubicin, giving the polymeric conjugate shown formula I.

Another embodiment of the invention is a method (Method III) for preparing the polymer-drug conjugate of the invention (Formula I), which includes (i) polymerizing a PEG-(meth)acrylate monomer (Formula II)

Where

R1 is selected from H or -CH3;

R3 is selected from -H or -CH3;

n is a natural number between 1-50, giving the polymer of formula IIa

- 10 044119

Formula Ila where

A represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or an initiator conjugated to a directing moiety, or an initiator having a reactive functional group, or A may be absent,

B represents a terminal group that is a fragment of a chain transfer agent (CTA) or initiator, or B may be absent.

And then (ii) further reacting the compound of formula IIa with the (meth)acrylate-L-D monomer (formula IIIa)

So

L' ь

Formulyoshs!

where R2 is selected from H or -CH3,

L is a cleavable linker or L may be absent,

D is a therapeutic agent selected from the group consisting of combretastatin, 5-FU, gemcitabine, chloroquine, doxorubicin, resulting in a block copolymer.

And then (iii) reacting the resulting block copolymer with a targeting moiety to give the polymer-drug conjugate shown by Formula I.

The term acrylate refers to derivatives of acrylic acids. These derivatives include the parent acid (CH2CHCO2H) and esters, so the term acrylate-based defines functional groups having any of the above acrylate derivatives.

The term methacrylate refers to derivatives of methacrylic acids. These derivatives include the parent acid (CH2C(CH3)CO2H) and esters. Thus, the term methacrylate-based defines functional groups having any of the above methacrylate derivatives.

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 described above. The term (meth)acrylate should be considered to mean methacrylate and/or acrylate.

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

In another aspect, the invention relates to a polymer-drug conjugate of formula I prepared by any of methods I, II or III.

In yet another embodiment, the invention relates to polymer-drug conjugates of Formula I prepared by Method I, since this method provides a polymer-drug conjugate with a very distinct polymer structure and a very well-defined drug content. Also, polymer-drug conjugates produced by this method do not have free reactive groups on the side chain, which remain unconjugated to the drug molecule.

In one embodiment, the PEG-(meth)acrylate (Formula II) is preferably selected from the group consisting of polyethylene glycol methyl ether methacrylate (CAS No.: 26915-72-0), polyethylene glycol methacrylate (CAS No.: 25736-86-1) , polyethylene glycol methyl ether acrylate (CAS No.: 32171-394), and polyethylene glycol acrylate (CAS No.: 9051-31-4), see table. 1 for information on the structures of these compounds. In a preferred embodiment of the invention, polyethylene glycol methyl ether methacrylate is used wherein R1 and R3 are both -CH ₃ .

In one embodiment of the invention, PEG(meth)acrylate (Formula II) is used, which has an average molecular weight between 50-2000 g/mol. In a preferred embodiment, PEG(meth)acrylate (Formula II) has an average molecular weight between 100-1500 g/mol and in a most preferred embodiment, PEG(meth)acrylate (Formula II) has an average molecular weight between 150-1000 g/mol . The PEG(meth)acrylate (formula II) of the invention may have

- 11 044119 average molecular weight, for example from 60, 70, 80, 80, 100, 150, 200, to 250, 300, 400, 500, 600, 700,

800, 900, 1000 g/mol.

The cleavable linker of the present invention can be purchased commercially or can be prepared according to known methods in the literature.

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

Re

The formula is where x means a natural number between 1-100, y means a natural number between 1-100,

R1 and R2 are independently selected from H or -CH3,

R3 is selected from -H or -CH3, n is a natural number between 1-50,

L is a cleavable linker or L may be absent,

A means end group or A may be absent,

B is a terminal group or B may be absent.

As disclosed above, methods for producing a polymer-drug conjugate of formula I (method I, II and III) include at least two polymerization steps where monomers of formula II and monomers of formula IIIa or IIIb are polymerized. In one embodiment of the invention, these polymerization steps may further include an initiator and/or a polymerization agent and/or a solvent.

The polymer-drug conjugate of the invention can be prepared by any of the known polymerization methods. Any suitable initiators and/or catalysts known in the art can be used to prepare the polymer-drug conjugate of the present invention. When a polymerization initiator is used, the resulting polymer-drug conjugate may contain the initiator or a fragment thereof.

The polymer backbone of the polymer-drug conjugate of the invention can be prepared, for example, by bulk polymerization, solution polymerization, and/or suspension polymerization methods known in the art.

The polymerization method used to prepare the polymer-drug conjugate of the present invention may be 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 polymerization (RAFT), iodine-assisted chain transfer polymerization (ITP), centered radical-mediated polymerization. on selenium, 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 invention.

A suitable polymerization initiator may be selected from the group given herein.

When RAFT polymerization is used, CTA and the initiator can be used together to prepare the polymer-drug conjugates of the invention according to Methods I, II or III provided herein.

In one embodiment of the invention, the polymer-drug conjugate of the invention and the polymer agglomerates derived therefrom may further include directing groups. As used herein, the term targeting group refers to specific tumor ligands that specifically bind to a cell, preferably a tumor cell, that has a complementary receptor.

The term targeting group means a molecule that facilitates the delivery of the polymer-drug conjugate of the invention to a specific site to produce the desired effect, that is, it provides localization of the compound. Localization is determined by specific

- 12 044119 recognition of molecular determinants, molecular size of the targeting agent or conjugate, ionic interactions, hydrophobic interactions, and the like. Other mechanisms for targeting the agent to a specific tissue or area are known to those skilled in the art. Targeting ligands include, for example, molecules that bind to molecules on the surface of a target cell. Exemplary targeting ligands include antibodies, antibody fragments, organic small molecules, peptides, peptoids, proteins, polypeptides, oligosaccharides, transferrins, heparan sulfate (HS)-glycoprotein, coagulation factors, serum proteins, betaglycoprotein, G-CSF, GM-CSF , M-CSF, EPO, and the like. In exemplary embodiments of the present invention, the targeting system includes covalently attaching a targeting ligand, such as RGDfK, EPPT1 peptide, bisphosphonic acid, or folate, to a carrier molecule or linker.

In some embodiments, the present invention is characterized by polymer-drug conjugates with or without a targeting ligand. In some embodiments, the targeting ligand may be RGDfK, EPPT1, bisphosphonic acid, or folate.

Another embodiment of the present invention provides a method of delivering a therapeutic agent comprising administering to a subject an effective amount of a polymer-drug conjugate shown by Formula I.

Another embodiment of the present invention provides a method of delivering a therapeutic agent comprising administering to a subject an effective amount of a polymer agglomerate composed of a polymer-drug conjugate illustrated by Formula I.

Another embodiment of the present invention provides a polymer-drug conjugate of the invention for use in the treatment of a variety of disorders that require the delivery of anticancer or the like agents.

Another embodiment of the invention are polymer agglomerates composed of polymer-drug conjugates of the invention for use in the treatment of a variety of disorders that require the delivery of anticancer or the like agents.

In a preferred embodiment, the invention relates to a polymer-drug conjugate illustrated by Formula I for use as a drug for the treatment of cancer.

In yet another preferred embodiment, the invention relates to a polymer agglomerate composed of a polymer-drug conjugate of formula I, for use as a drug for the treatment of cancer.

As used herein, to treat or treat means to inhibit, reduce, modulate, ameliorate, or block at least one symptom that characterizes a pathological condition in a subject at risk of or suffering from the condition. A non-limiting list of various types of cancer is the following: carcinomas, solid tissue carcinomas, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, high-grade gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas, metastatic cancers, or cancerous diseases in general. Specific examples of cancers for which the disclosed compositions may be used include B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's disease, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, kidney cancer, lung cancers 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, squamous cell carcinomas of the oral cavity, pharynx, larynx, colon cancer, cervical cancer, cervical carcinoma, breast and epithelial cancer, renal cancer, urogenital cancer, lung cancer, esophageal carcinoma, head and neck carcinoma, colon cancer, hematopoietic cancers; testicular cancer; colorectal cancer, prostatic cancer, or pancreatic cancer.

The polymer-drug conjugates of the invention and/or polymer agglomerates composed of the polymer-drug conjugates of the invention can also be used for the treatment of precancerous conditions such as cervical and anal dysplasia, other types of dysplasia, severe dysplasia, hyperplasia, atypical hyperplasia and neoplasia.

The terms cancer and cancerous as used herein refer to malignant tumors or describe a physiological condition characterized by uncontrolled cellular growth.

As discussed herein, the polymer-drug conjugates of the invention and/or polymer agglomerates composed of the polymer-drug conjugates of the present invention find use in the treatment and/or prevention of cancer. For such use, the polymer-drug conjugate of the invention and/or polymer agglomerates composed of the polymer-drug conjugates of the present invention will generally be

- 13 044119 is administered in the form of a pharmaceutical composition.

Thus, according to the invention, there is provided a pharmaceutical composition containing 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.

In addition, according to the invention there is provided a pharmaceutical composition containing a polymer agglomerate consisting of a polymer-drug conjugate according to Formula I, and at least one pharmaceutically acceptable diluent, excipient and/or carrier.

The composition comprising the polymer-drug conjugate of the invention and/or polymer agglomerates consisting of the polymer-drug conjugates of the present invention may be in any suitable form depending on the desired route of administration to the patient. The composition containing the polymer-drug conjugates of the invention and/or polymer agglomerates consisting of the polymer-drug conjugates of the invention may be formulated for oral administration, for example, in the form of liquid dispersions or aqueous or oily suspensions, or they may be formulated for parenteral administration, for example, for subcutaneous, intravenous, intramuscular, intrasternal, intraperitoneal, intradermal, transdermal or other infusion techniques. A composition comprising the polymer-drug conjugates of the invention and/or polymer agglomerates consisting of the polymer-drug conjugates of the present invention may also be formulated for inhalation administration in the form of an aerosol or solution for administration by an inhaler or nebulizer. The polymer-drug conjugates of the invention and/or polymer agglomerates consisting of the polymer-drug conjugates of the invention are preferably administered to a subject transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally or by inhalation. The most appropriate route of administration in any of the above cases will depend on the particular therapeutic agent present in the polymer-drug conjugate of the present invention, the subject, and the nature and severity of the disease and physical condition of the subject.

The polymer-drug conjugates of the invention and/or polymer agglomerates consisting of the polymer-drug conjugates of the invention may be administered in combination, for example, simultaneously, sequentially or separately, with one or more other therapeutically active compounds, which may be an anticancer agent, or they may be an immunomodulatory, antiviral, anti-infective, antimicrobial, anti-infective or anesthetic agent or combinations thereof.

The polymer agglomerates of the invention may encapsulate a second therapeutic agent selected from the list above and may further be administered in combination, for example simultaneously, sequentially or separately, with one or more other therapeutically active compounds, which may be an anticancer agent, or they may be an immunomodulatory, antiviral, anti-infective, antimicrobial, anti-infective or anesthetic agent or combinations thereof.

Said second therapeutic agent may be selected from the therapeutic agents listed above, provided that it is different from the agent present in the polymer-drug conjugate of the invention.

Comprising, as used herein, is intended to mean including.

Embodiments are described herein as including certain features/elements. The disclosure also extends to individual embodiments consisting or substantially consisting of the mentioned features/elements.

Referenced technical materials, such as patents and applications, are incorporated herein by reference.

Any embodiments specifically or expressly listed herein may constitute a basis for disclaiming an embodiment, alone or in combination with one or more additional embodiments.

The invention will now be described with reference to the following examples, which are illustrative only and which should not be construed as limiting the scope of the present invention in any way.

Examples

The following examples provide stepwise preparation of polymer-drug conjugates of Formula I and polymer agglomerates thereof.

Example 1A. Synthesis of polymerizable monomer combretastatin-A4 (CombMA)

Combretastatin-A4 (300 mg, 0.95 mmol), triethylamine (TEA, 191 mg, 1.89 mmol), methacryloyl chloride (198 mg, 1.89 mmol) are dissolved in anhydrous dichloromethane (DCM, 10 ml) under N2 in a round bottom bottle 25 ml flask. The reaction mixture was stirred for 16 hours at room temperature.

- 14 044119

The crude product is extracted with saturated NaHCO ₃ solution (20 mlx2) and distilled water (20 mlx2). The organic layer is dried with Na ₂ SO ₄ and the solvent is evaporated. The CombMA monomer is purified using silica gel column chromatography through hexanes.

Example 1B. Preparation of a chain transfer agent (CTA) having a reactive functional group

4-Cyanopentanoic acid dithiopentanoate (CPDB) is a CTA, and this CTA is modified with N-hydroxysuccinimide (NHS), which is a reactive functional group, according to the following procedure;

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). The two solution mixtures are then mixed and the reaction mixture is stirred at room temperature in the dark for 16 hours. The insoluble white byproduct dicyclohexylurea (DCU) is removed by filtration. The resulting solution is dried under vacuum and the crude product is purified by column chromatography on silica gel with hexane and EtOAc.

Example 1C. Synthesis of homopolymers POEGMEMA and NHS-POEGMEMA

Reversible addition-fragmentation polymerization (RAFT) is used to synthesize homopolymers POEGMEMA and NHS-activated POEGMEMA (NHSPOEGMEMA). To synthesize the polymer POEGMEMA, AIBN (1.31 mg, 0.008 mmol) is 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 is purged with N2 to remove _O2 and the polymerization mixture is stirred for 16 hours at 70°C. Polymerization is stopped by cooling and exposure to air. The POEGMEMA polymer is purified by precipitation into diethyl ether. The polymer precipitate is dried under vacuum to give approximately 35 OEGMEMA repeating units (4-60 mg, 77% yield). To synthesize the NHS-POEGMEMA homopolymer, the same procedure was followed using SCPDB (27.10 mg, 0.072 mmol) as the chain transfer agent, yielding approximately 35 OEGMEMA repeat units (480 mg, 80% yield).

Example 1. Synthesis of block copolymers POEGMEMA-co-CombMA and NHS-POEGMEMA-co-CombMA

RAFT polymerization is used to synthesize the block copolymers POEGMEMA-b-PCombMA and NHS-POEGMEMA-b-PCombMA. To a solution of CombMA (100 mg, 0.26 mmol) and POEGMEMA macrochain transfer agent (50 mg, 0.005 mmol) in DMF (0.75 ml), add AIBN (0.15 mg, 0.00092 mmol). The mixture is purged with N2 to remove _O2 and the polymerization mixture is stirred at 65°C. Polymerization is stopped by cooling and exposure to air. The crude product is precipitated in diethyl ether. The polymer cake is dried under vacuum to give approximately 32 CombMA repeat units (98 mg, 65% yield). The same procedure is followed to synthesize the block copolymer NHS-POEGMEMA-b-PCombMA using NHS-POEGMEMA (50 mg, 0.005 mmol) as the macrochain transfer agent, yielding approximately 33 CombMA repeat units (105 mgd, 70% yield).

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,Ndiisopropylethylamine (6 .5 mg, 0.05 mmol). The reaction mixture is stirred at 30°C for 24 hours. The crude product is precipitated in diethyl ether. The polymer residue is dried under reduced pressure to give cRGDfK-POEGMEMA-b-CombMA (41 mg, 82% yield). A general scheme showing the preparation of the cRGDfK-POEGMEMA-b-CombMA block copolymer is provided as FIG. 1.

Example 1F. Preparation of polymer agglomerates of directional and non-directional effects

As used herein, directional refers to polymer agglomerates composed of cRGDfK-POEGMEMA-b-CombMA block copolymers, and non-directional refers to polymer agglomerates composed of POEGMEMA-b-PCombMA block copolymers.

Two solutions are prepared to obtain agglomerates of directional impact and non-directional impact. To prepare non-directional agglomerates, 2.3 mg of POEGMEMA-b-PCombMA block copolymer is dissolved in 500 μl of THF in a glass vial and 3 ml of water is added to this solution. The THF in the vials is then evaporated at room temperature for 24 h in open air to produce micelles. Targeting agglomerates are prepared in the same manner using a mixture of cRGDfK-POEGMEMA-b-PCombMA and POEGMEMA-b-PCombMA (1/5, w/w). A schematic representation of the production of agglomerates of non-directional impact and directional impact is presented in Fig. 2 and 3.

Example 1G. Measurement of critical micelle concentration (CMC)

The fluorescent probe method is used to determine the CMC values of directional and non-directional impact agglomerates. Solutions of block copolymers (450 μl) in THF are prepared using serial dilutions in the above vials. In every bubble

- 15 044119 inject 50 μl of a solution of Nile Red dye in THF (0.03 mg/ml) and then 3 ml of water. THF was evaporated completely in open air and final concentrations were obtained for 16 samples ranging from ^1x10'9 to 1x10'5 M in 3 ml of water. Fluorescence measurement data is recorded using a fluorescence spectrophotometer at an excitation wavelength of 550 nm and emission is monitored at a wavelength from 580 to 660 nm.

To determine the minimum required concentration of amphiphilic polymers to obtain micellar-type polymer structures via self-assembly, CMC values for block copolymers are calculated by monitoring Nile Red fluorescence intensity as a function of polymer concentration.

Fluorescence emission spectra of Nile Red in non-directional and directional agglomerates were obtained at an excitation wavelength of 550 nm, and emission was monitored at wavelengths from 580 to 660 nm (Figs. 4 and 5, respectively).

To determine the CMC values, plot the emission intensity at 612 nm as a function of the logarithm (log) of the copolymer concentration. The CMC values for the formation of non-directional and directional agglomerates are calculated from the intersection of straight lines belonging to relative intensity values with relatively constant values and relative intensity values with rapidly increasing values (Figs. 6 and 7, respectively). The CMC values for non-directional and directional agglomerate formation were found to be 1.796 x 10'6 M and 1.566 x 10'6 M, respectively.

Example 2A. Synthesis of Polymerizable Monomer 5FU (5FU-MA)

5-Fluorouracil (250 mg, 1.92 mmol) and 37% formaldehyde (244 μl) are reacted at 60°C for 2 hours. The mixture is then cooled and lyophilized completely, giving a viscous compound. In the second step, this product (315 mg, 1.97 mmol) is dissolved in anhydrous acetonitrile (5.8 ml) and then triethylamine (422 μl, 3.03 mmol) is added to this solution. After cooling the reaction mixture to 0°C, methacryloyl chloride (232 μl, 2.38 mmol) was added dropwise. The reaction is allowed to proceed overnight at room temperature and the white precipitate is then filtered off. All volatile compounds are evaporated under vacuum and the crude product is then dissolved in dichloromethane (20 ml), followed by extraction with 1M HCl (2x10 ml), 1M NaHCO ₃ (10 ml) and brine (10 ml). After concentration of the organic part, the product is isolated in pure form by column chromatography on silica gel, eluting with ethyl acetate:hexane (30:70 v/v). The product is obtained as a viscous colorless oil (305 mg, 68% yield).

Example 2B. Synthesis of the homopolymer POEGMEMA

Reversible addition-fragmentation transfer (RAFT) polymerization is used to synthesize the POEGMEMA homopolymer. To synthesize the polymer POEGMEMA, AIBN (0.78 mg, 4.76 µmol) is 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 is purged with N ₂ to remove O ₂ and the polymerization mixture is stirred at 70° C. for 16 hours. The polymerization is stopped by cooling and exposure to air. The POEGMEMA polymer is purified by precipitation into diethyl ether. The polymer precipitate is dried under vacuum, which gives 95 mg of polymer with a yield of 19%.

Example 2C. Synthesis of block copolymer POEGMEMA-co-5FU-MA

RAFT polymerization is used to synthesize the block copolymer POEGMEMA-b-P5FU-MA. AIBN (0.13 mg, 0.786 µmol). The mixture is purged with N2 to remove _O2 and the polymerization mixture is stirred at 70° C. for 16 hours. The polymerization is stopped by cooling and exposure to air. The crude product is precipitated into diethyl ether. The polymer precipitate is dried under vacuum, which gives 16 mg of polymer with a yield of 28%.

Example 2A. Synthesis of polymerizable monomer combretastatin-A4 (Comb-ss-MA)

2-hydroxyethyl methacrylate (500 mg, 3.8 mmol), 4,4'-dithiodibutyric acid (1.83 g, 7.6 mmol), N,N'-dicyclohexylcarbodiimide (1.59 mg, 7.8 mmol ), 4-(dimethylamino)pyridine (281 mg, 2.3 mmol) was dissolved in anhydrous dichloromethane (DCM) (54 mL) under N2 in a 100 mL round bottom flask. The reaction mixture was stirred for 16 hours at room temperature. The crude product is precipitated into cold diethyl ether and left for 20 minutes at -20°C. The product was purified using silica gel column chromatography with ethyl acetate and hexane (20:80 v/v) (426 mg, 32% yield).

The resulting product (300 mg, 0.85 mmol) and combretastatin-A4 (270 mg, 0.85 mmol) were then dissolved in anhydrous dichloromethane (DCM) with DIPC (129 mg, 1.02 mmol) and DMAP (104 mg, 0 .85 mmol) under N ₂ atmosphere in a 25 ml round bottom flask. The reaction mixture was stirred for 16 hours at room temperature. The Comb-ss-MA monomer was purified by silica gel column chromatography with ethyl acetate and hexane (30:70 v/v) (195 mg, 35% yield).

Example 3B. Synthesis of homopolymers POEGMEMA and Comb-ss-MA-POEGMEMA

Reversible addition-fragmentation chain transfer (RAFT) polymerization

-

Claims (9)

used for the synthesis of POEGMEMA. To synthesize the polymer POEGMEMA, 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 is purged with N ₂ to remove O ₂ and the polymerization mixture is stirred at 70° C. for 16 hours. The polymerization is stopped by cooling and exposure to air. The POEGMEMA polymer is purified by precipitation into diethyl ether. The polymer precipitate is dried under vacuum, which gives 715 mg of polymer with a yield of 69%. Example 3C. Synthesis of block copolymer POEGMEMA-co-Comb-ss-MA RAFT polymerization is used to synthesize the block copolymer POEGMEMA-b-PComb-ss-MA. To a solution of Comb-ss-MA (80 mg, 0.20 mmol) and POEGMEMA macrochain transfer agent (134 mg, 0.020 mmol) in DMF (1.07 ml) add AIBN (0.68 mg, 0.004 mmol) . The mixture is purged with N ₂ to remove O ₂ and the polymerization mixture is stirred at 65°C. Polymerization is stopped by cooling and exposure to air. The crude product is precipitated into diethyl ether. The polymer precipitate is dried under vacuum, which gives 98 mg of polymer with a yield of 65%. The examples above show that the invention is applicable to a number of different linkers, such as disulfide and acetal, and to various drug molecules, such as combretastatin and 5-FU. Brief description of the figures Fig. 1: Schematic representation showing the synthesis of an NHS-modified polymer-drug conjugate (a polymer-drug conjugate of formula I having a reactive functional group) and a cRGDfK-modified polymer-drug conjugate (a polymer-drug conjugate of formula I having a directing group). Fig. 2: Schematic representation showing the preparation of non-directional polymer agglomerates of the invention from polymer-drug conjugates of formula I. Fig. 3: Schematic representation showing the preparation of targeted polymer agglomerates of the invention from polymer-drug conjugates of formula I. Fig. 4: Fluorescence emission spectra of Nile Red in non-targeted polymer agglomerates according to the invention. Fig. 5: Fluorescence emission spectra of Nile Red in targeted polymer agglomerates according to the invention. Fig. 6: Plot of emission intensity at 612 nm versus logarithm (log) of POEGMEMA-b-PCombMA concentration. Fig. 7: Plot of emission intensity at 612 nm versus logarithm (log) of total concentration of POEGMEMA-b-PCombMA and cRGDfK-POEGMEMA-b-PCombMA. CLAIM 1. Polymer-drug conjugate of formula I in the form of a block copolymer for the delivery of therapeutic agents \=o 'd formula I where R1, _R2 and _R3 are independently selected from H or _-CH3 ; x means a natural number between 1-100; y means a natural number between 1-100; n means a natural number between 1-50; L is a cleavable linker and D is a therapeutic agent that is combretastatin or 5-fluorouracil (5-FU) or gemcitabine or chloroquine or doxorubicin and which is present in an amount of from 5 to 50 wt.% of the polymer-drug conjugate; A represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or an initiator conjugated to a directing moiety, or an initiator having a reactive functional group, or A may be absent; B represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or B may be absent; the polymer-drug conjugate has an average molecular weight of 5 to 60 kDa. - 17 044119 2. The polymer-drug conjugate of claim 1, wherein the linker is selected from the group consisting of poly(ethylene glycol), amino acid, poly(amino acid) and short peptides, preferably the short peptide is degraded by cathepsin B, and most preferably the short peptide is selected from group including Gly-Phe-Leu-Gly (sequence (SEQ) ID NO: 1), Val-Cit, PheLys, Val-Ala, Ala-Leu-Ala-Leu (sequence (SEQ) ID NO: 2) . 3. The polymer-drug conjugate according to claims 1, 2, where the linker is a C1-C10 hydrocarbon or a C ₁ -C ₁₀ -substituted or heterosubstituted hydrocarbon, which contains a functional group that dissociates under physiological conditions, preferably said functional group is selected from acetal, ester, imine, amide, disulfide, carbonate, hydrazine, carbamate. 4. The polymer-drug conjugate according to claims 1-3, where A represents an end group that is a fragment of CTA or an initiator having a reactive functional group, where the reactive group is selected from an acetal, a hemiacetal, a carboxylic acid, an alcohol, an amide, an 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. 5. The polymer-drug conjugate according to claims 1-4, where the targeting fragment is selected from the group consisting of antibodies; antibody fragments, or peptides, such as cyclo(Arg-Gly-Asp-D-PheLys) (cRGDfK) (sequence (SEQ) ID NO: 3). 6. Polymer agglomerate in the form of nanoparticles or micelles obtained from polymer-drug conjugates of formula I according to claims 1-5 formula I where R _b R2 and R ₃ are independently selected from H or -CH ₃ ; x means a natural number between 1-100; y means a natural number between 1-100; n means a natural number between 1-50; L is a cleavable linker or L may be absent; And D is a therapeutic agent that is combretastatin or 5fluorouracil or gemcitabine or chloroquine or doxorubicin and which is present in an amount of from 5% to 50% by weight of the polymer-drug conjugate; A represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or an initiator conjugated to a directing moiety, or an initiator having a reactive functional group, or A may be absent; B represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or B may be absent; the polymer-drug conjugate has an average molecular weight of 5 to 60 kDa. 7. A polymer agglomerate in the form of nanoparticles or micelles) according to claim 6, encapsulating therapeutic agents in addition to the therapeutic agents attached to the polymer-drug conjugate of formula I, wherein said therapeutic agents may be selected from the group consisting of nucleoside analogs, antifolates, topoisomerase inhibitors I, helium, dusk, dasins, barvinka alkaloids, alkylating agents, platinals, antighormones, radiopharmaceutical compounds, monoclonal antibodies, thyroosinkinase inhibitors, rapamicin targets in mammalian cells (MTOR), retinoids, immunomodulating agents, INGIBICE INGIBICS, INGIBITIONS. Stondacilasis and hydroxicarbamide, anagrelid, ceritinib , dabrafenib, idelalisib, ibrutinib, palbociclib, vemurafenib, bleomycin, bortezomib, dactinomycin, eribulin, estramustine, ixabepilone, mitomycin, procarbazine, alectinib, fluximesterone, iobenguane, imiquimod, interferon, ixazomib, lanreotide, lentinan , octreotide, omacetaxin, tegafur, gimerazil , oteracil, uracil, combretastatin, chloroquine. 8. A method for producing a polymer-drug conjugate of formula I according to one of claims 1 to 4, including the polymerization of a PEG-(meth)α-acrylate monomer (formula II) - 18 044119 Where R ₁ and R 3 are independently selected from H or -CH 3 ; n is a natural number between 1-50, yielding a polymer of formula IIa Where A represents a terminal group that is a fragment of a chain transfer agent (CTA) or an initiator, or an initiator conjugated to a directing moiety, or an initiator having a reactive functional group, or A may be absent, B represents an end group that is a fragment of a chain transfer agent (CTA) or initiator, or B may be absent, and then (ii) additional interaction of a compound of formula IIa with a (meth)acrylate-LD monomer (formula IIIa) Where R2 is selected from H or -CH3, L is a cleavable linker or L may be absent, D is a therapeutic agent selected from combretastatin or 5-FU or gemcitabine or chloroquine or doxorubicin to produce a polymer-drug conjugate of formula I. 9. A method for producing a polymer-drug conjugate of formula I according to any one of claims 1 to 3, comprising (i) polymerization of a PEG-(meth)acrylate monomer (formula II) Where R ₁ and R 3 are independently selected from H or -CH 3 n is a natural number between 1-50 to yield a polymer of formula IIa -