Novel copolymers

Abstract

The technical problem to be solved by the present invention is to provide a novel copolymer that can be used in a drug delivery technique. The present invention pertains to a copolymer obtained by binding a targeting affinity molecule to a copolymer X containing structural units represented by formulae (A), (B), and (C). [In the formula, R1, R2 and R3 may be the same or different and each represents a hydrogen atom or a C1-3 alkyl group; r4 represents a C1-3 alkyl group; r5 represents a hydrogen atom, a C1-18 alkyl group, a 3-to 8-membered cycloalkyl group which may have a substituent, an adamantyl group, a C6-18 aryl group which may have a substituent, or a 5-to 10-membered heteroaryl group which may have a substituent; x1, X2, and X3 may be the same or different and each represents an oxygen atom, a sulfur atom, or N-R7; r6 represents a hydrogen atom, a leaving group or a linking group; r7 represents a hydrogen atom or a C1-3 alkyl group; m represents an integer of 1 to 100; and n represents an integer of 0-3. ] # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a novel copolymer that can be used in drug delivery technology. More specifically, it relates to a copolymer used as a drug delivery carrier that targets tumors, a pharmaceutical composition obtained by making the copolymer carry a physiologically active substance such as an anticancer agent, and a pharmaceutical product containing the composition. Background Art

[0002] In recent years, research on drug delivery systems (DDS) has been actively conducted as a technology for effectively and safely delivering drugs to disease sites. Among them, as a technology that utilizes the structural characteristics of disease sites to improve the selectivity of drug aggregation, the demand for DDS using nanoparticles as drug delivery carriers is increasing.

[0003] For example, in solid cancer tissue, since the structure of new blood vessels (tumor blood vessels) is not yet mature compared to normal blood vessels, a cell gap of about hundreds of nm is generated in the vascular endothelium, and the permeability of the substance is high. It is known that through this structural feature, high molecular weight bodies containing nanoparticles selectively penetrate tumor blood vessels and gather in solid cancer tissue. Furthermore, in solid cancer tissue, the lymphatic system involved in the discharge of macromolecules is dysfunctional, so the infiltrated nanoparticles are continuously retained in the tissue (enhanced permeability and retention effect, Enhanced permeability and retention effect, EPR effect). General low molecular drugs leak out of the blood vessels due to the membrane penetration of vascular cells, so they are non-selectively distributed in the tissue and will not gather in solid cancer tissue. According to the methodology of the EPR effect, the distribution of drug delivery using nanoparticles in the tissue is controlled by the permeability of the intercellular gap of the vascular endothelial cell, which will lead to the improvement of the tissue selectivity of drug distribution to solid cancer. Therefore, the EPR effect has become a powerful academic basis for the development of nanotechnology-based pharmaceuticals (nano drugs) targeting solid cancer.

[0004] It is believed that the drug delivery process in the EPR effect is via the blood flow, and the blood vessel leakage process of nanoparticles is passive. Therefore, in order to maximize the aggregation of nanoparticles in solid cancer, it is important to give the constituent components of the nanoparticles as drug delivery carriers a molecular design that can withstand long-term retention in the blood. Therefore, for drug delivery carriers, it is required to have the ability to avoid the following obstacles, that is, non-specific interactions with blood constituents; foreign body recognition of the reticuloendothelial system (RES) in the liver, spleen and lungs; glomerular filtration in the kidneys, etc. In addition, it is known that these obstacles can be overcome by optimizing the particle characteristics such as particle size or surface modification using biocompatible polymers. For example, the particle size of the drug delivery carrier is ideally greater than the threshold of kidney clearance, i.e., about 6nm, and less than 200nm that can be exempted from being recognized by RES.

[0005] In addition, the particle size of known drug delivery carriers also has an impact on the tissue permeability of disease sites. For example, a comparative study has been conducted on the anticancer activity of drug-encapsulated nanoparticles with a particle size of 30nm, 50nm, 70nm and 100nm that show equal retention in the blood, and it is known that the drug-encapsulated nanoparticles with a particle size of 30nm have a high therapeutic effect (non-patent literature 1) due to reaching the deep part of the disease site. Therefore, it is considered that the particle size of the nanoparticles used in drug delivery carriers targeted with solid cancer is preferably small as much as possible within the range of avoiding kidney clearance.

[0006] As nanoparticles for drug delivery carriers, methods using liposomes, emulsifiers or colloidal dispersions of nanoparticles, methods using biological raw materials such as albumin, methods using natural polymers such as natural polysaccharides, or methods using synthetic polymers have been developed. Among them, synthetic polymers can prepare nanoparticles with precisely controlled particle sizes by appropriately selecting monomers as constituent components and synthesis methods, so they can be widely used as constituent components of drug delivery carriers.

[0007] For example, the method of utilizing the amphiphilic block copolymer comprising a hydrophilic segment and a hydrophobic segment as a drug delivery carrier has been disclosed. The block copolymer spontaneously associates in an aqueous medium with the hydrophobic interaction between molecules as a driving force to form core-shell nanoparticles (polymer micelles). It is known that the obtained polymer micelles containing drugs can be enclosed or combined with low-molecular drugs in the hydrophobic segments of the polymer micelles, and the obtained polymer micelles containing drugs show higher stability in blood, and selectively aggregate in solid cancers due to the EPR effect, thus compared with the solution administration of low-molecular drugs, bringing higher anticancer activity (patent document 1). However, polymer micelles are the associations of multiple molecules, so the particle diameter of about 30nm is the lower limit that can be prepared, and the fine size control of the particle diameter of about 10nm that can avoid the influence of kidney clearance is then difficult.

[0008] On the other hand, in the nanoparticles formed by the known synthetic polymer, the material (hereinafter referred to as single chain nanoparticle (SCNP)) that forms particles using chemical crosslinking, hydrophobic interaction, ionic bonding, etc. in the single chain as driving force forms small diameter nanoparticles with a particle diameter of less than 20nm (non-patent literature 2). Therefore, although the usefulness of SCNP as a drug delivery carrier is expected, the technology that can accurately control its particle diameter has not yet been found.

[0009] As a delivery technology for other drugs, there is a drug delivery system that utilizes the combination of avidin / biotin. Avidin / streptavidin has a very high affinity for biotin, and its interaction is widely used in the fields of biochemistry, molecular biology or medicine (non-patent literature 3, 4). Furthermore, a method for drug delivery and a pre-targeting method (non-patent literature 5) have been studied that combine its higher binding energy with antibody molecules.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: Japanese Patent No. 3270592

[0013] Non-patent literature

[0014] Non-patent document 1: H. Cabral et al., Nat. Nanotechnol. 6 815-823 (2011)

[0015] Non-patent document 2: Jose A. Pomposo, Single-Chain Polymer Nanoparticles: Synthesis, Characterization, Simulations, and Applications (2017)

[0016] Non-patent document 3: NS Green, Adv. Protein Chem., 29: 85-133 (1975)

[0017] Non-patent document 4: NS Green, Methods Enzymol. 184: 51-67 (1990)

[0018] Non-patent document 5: DJ Hnatowich et al., J. Nucl. Med. 28, 1294-1302 (1987) Summary of the invention

[0019] Technical problem to be solved by the invention

[0020] The technical problem to be solved by the present invention is to provide a copolymer for drug delivery carriers targeting tumors. More specifically, the technical problem to be solved by the present invention is to provide a copolymer for drug delivery carriers that can be used to improve the blood retention and / or tumor aggregation of drugs.

[0021] Technical solutions for solving technical problems

[0022] The inventors of the present invention have conducted in-depth research to solve the above problems and found that the terpolymer of acrylic acid derivatives forms SCNPs in water. In addition, in addition to the precise particle size control of SCNPs of about 10nm below 20nm, they have also successfully created a new polymer for drug delivery carriers with high tumor aggregation. Furthermore, the polymer was made to carry anticancer agents and applied to subcutaneous transplantation model mice of colorectal cancer, and the results confirmed that there was an excellent antitumor effect. Furthermore, the above-mentioned terpolymer derivatives that form biotin-bound SCNPs that can be applied to pre-targeting methods have also been successfully created.

[0023] The present invention relates to the following inventions.

[0024] [1] A copolymer in which a targeting affinity molecule is bonded to a copolymer X comprising structural units represented by the following formulae (A), (B) and (C).

[0025]

[0026] [Where R 1 , R2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 Alkyl; m represents an integer of 1 to 100; n represents an integer of 0 to 3.]

[0027] [2] The copolymer according to [1] above, wherein the copolymer X is a copolymer formed by polymerization of three monomers represented by the following general formulas (1) to (3).

[0028]

[0029] [Where R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 Alkyl; m represents an integer of 1 to 100; n represents an integer of 0 to 3.]

[0030] [3] The copolymer according to [1] or [2], wherein R 1 A hydrogen atom.

[0031] [4] The copolymer according to any one of [1] to [3] above, wherein R2 A hydrogen atom.

[0032] [5] The copolymer described in any one of [1] to [4] above, wherein R 3 A hydrogen atom.

[0033] [6] The copolymer according to any one of [1] to [5], wherein R 4 It is methyl.

[0034] [7] The copolymer according to any one of [1] to [6], wherein R 5 is a C which may have a substituent 6-18 Aryl.

[0035] [8] The copolymer described in any one of [1] to [7] above, wherein R 5 It is phenyl.

[0036] [9] The copolymer described in any one of [1] to [8] above, wherein R 6 A hydrogen atom.

[0037]

[10] The copolymer described in any one of [1] to [8] above, wherein R 6 The leaving group is a group of the following formula (4).

[0038]

[0039]

[11] The copolymer described in any one of [1] to [8] above, wherein R 6 The linking group is a group selected from the following formulae (5) to (7).

[0040]

[0041]

[12] The copolymer according to any one of [1] to

[11] , wherein X 1 For oxygen atoms.

[0042]

[13] The copolymer according to any one of [1] to

[12] , wherein X 2 For oxygen atoms.

[0043]

[14] The copolymer according to any one of [1] to

[13] , wherein X 3 For oxygen atoms.

[0044]

[15] The copolymer according to any one of [1] to

[14] above, wherein m is an integer of 4 to 22.

[0045]

[16] The copolymer according to any one of [1] to

[15] above, wherein n is 1.

[0046]

[17] The copolymer described in [1] to

[16] above, wherein the ratio of the structural units (A), (B) and (C) is 0.01 to 100 parts by mass of (B) and 0.1 to 100 parts by mass of (C) per 1 part by mass of (A).

[0047]

[18] The copolymer according to any one of [2] to

[16] above, which is obtained by polymerizing 0.01 to 100 parts by mass of the monomer (2) and 0.1 to 100 parts by mass of the monomer (3) per 1 part by mass of the monomer (1).

[0048]

[19] The copolymer according to any one of [1] to

[18] above, which has a number average molecular weight of 5,000 to 150,000.

[0049]

[20] The copolymer described in any one of [1] to

[19] above, wherein the targeting affinity molecule is biotin or a derivative thereof.

[0050]

[21] The copolymer described in any one of [1] to

[19] above, wherein the targeting affinity molecule is a molecule having a biotin-based residue represented by the following formula (a).

[0051]

[0052] (In the formula, Y represents O or NH.)

[0053]

[22] The copolymer according to any one of [1] to

[19] above, wherein the targeting affinity molecule is any one of the molecules represented by the following formulas (8) to (11).

[0054]

[0055]

[23] The copolymer as described in any one of [1] to

[22] above, wherein the binding of the targeting affinity molecule to the copolymer X is covalent binding or non-covalent binding.

[0056]

[24] A single-chain nanoparticle comprising the copolymer described in any one of [1] to

[23] above.

[0057]

[25] A pharmaceutical composition comprising the copolymer according to any one of [1] to

[24] above.

[0058]

[26] The pharmaceutical composition according to

[25] above, which is used for pretargeting method.

[0059] Effects of the Invention

[0060] As can be seen from the examples described below, the substance formed by carrying an anticancer agent on the SCNP obtained by the self-association of the copolymer of the present invention shows a tumor growth inhibitory effect in a mouse cancer-bearing model, so it can be applied to a therapeutic agent for malignant tumors. The substance formed by carrying an anticancer agent on the SCNP obtained by the self-association of the copolymer of the present invention has a high tumor growth inhibitory effect at a low dose, so it is possible to provide a therapeutic agent for malignant tumors that can take into account both enhanced pharmacological effects and suppressed side effects. In addition, the copolymer of the present invention combined with a targeting affinity molecule (hereinafter, sometimes also referred to as a targeting affinity copolymer) is extremely useful as a drug delivery and pre-targeting agent using the combination of biotin / avidin. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The copolymer obtained in Example 1 is measured by nuclear magnetic resonance spectroscopy (NMR). 1 Figure 1 H-NMR spectrum.

[0062] Figure 2 This is a diagram showing a chromatogram obtained by gel permeation chromatography (GPC) of the copolymer obtained in Example 1.

[0063] Figure 3 It is a graph showing the particle size measurement results (scattering intensity distribution) of the copolymer before encapsulation of DACHPt (Example 69) and the SCNP encapsulating DACHPt (Example 70) by dynamic light scattering (DLS).

[0064] Figure 4 is the value obtained by nuclear magnetic resonance spectroscopy (NMR) for the biotin-bound SCNP obtained in Example 73. 1 Figure 1 H-NMR spectrum.

[0065] Figure 5 This is a diagram showing a chromatogram obtained by gel permeation chromatography (GPC) for the biotin-bound SCNP obtained in Example 73.

[0066] Figure 6 This is a graph showing the particle size measurement results (scattering intensity distribution) of the biotin-conjugated SCNP obtained in Example 73 using the dynamic light scattering (DLS) method.

[0067] Figure 7 is the value obtained by nuclear magnetic resonance spectroscopy (NMR) for the biotin-bound SCNP obtained in Example 75. 1 Figure 1 H-NMR spectrum.

[0068] Figure 8 This is a diagram showing a chromatogram obtained by gel permeation chromatography (GPC) for the biotin-bound SCNP obtained in Example 75.

[0069] Fig. 9 This is a graph showing the particle size measurement results (scattering intensity distribution) of the biotin-conjugated SCNP obtained in Example 75 using the dynamic light scattering (DLS) method.

[0070] Fig.10 is the value obtained by nuclear magnetic resonance spectroscopy (NMR) for the biotin-bound SCNP obtained in Example 77. 1 Figure 1 H-NMR spectrum.

[0071] Fig.11 This is a diagram showing a chromatogram obtained by gel permeation chromatography (GPC) for the biotin-bound SCNP obtained in Example 77.

[0072] Fig.12 This is a graph showing the particle size measurement results (scattering intensity distribution) of the biotin-conjugated SCNP obtained in Example 77 using the dynamic light scattering (DLS) method.

[0073] Fig.13 is the value obtained by nuclear magnetic resonance spectroscopy (NMR) for the biotin-bound SCNP obtained in Example 79. 1 Figure 1 H-NMR spectrum.

[0074] Fig.14 This is a diagram showing a chromatogram obtained by gel permeation chromatography (GPC) for the biotin-bound SCNP obtained in Example 79.

[0075] Fig.15This is a graph showing the particle size measurement results (scattering intensity distribution) of the biotin-conjugated SCNP obtained in Example 79 using the dynamic light scattering (DLS) method.

[0076] Fig.16 This is a graph showing the change in relative tumor volume when an oxaliplatin solution or DACHPt-encapsulated SCNP (Example 70) was administered three times every other day to a dorsal subcutaneous transplantation model mouse of a mouse colon cancer cell line (C26). DETAILED DESCRIPTION

[0077] Unless otherwise specified, the terms used in this specification are used with the meanings commonly used in the field. Hereinafter, the present invention will be described in more detail.

[0078] In this specification, the term "nanoparticle" refers to a structure having a particle size of 100 nm or less.

[0079] In this specification, "single-chain nanoparticles (SCNPs)" refer to nanoparticles formed by chemical crosslinking, hydrophobic interaction, ionic bonding, etc. within a single chain as a driving force. 20 nm or less also means a relatively small particle size in most nanoparticles.

[0080] In this specification, the "initiator" means an initiator for thermal radical polymerization such as an azo compound or a peroxide.

[0081] In the present specification, the "chain transfer agent" refers to a compound that causes a chain transfer reaction during radical polymerization, and is preferably a compound having a thiocarbonyl group.

[0082] In this specification, the term “C 1-3 The term "alkyl group" refers to a linear or branched alkyl group having 1 to 3 carbon atoms, and examples thereof include methyl, ethyl, n-propyl, and isopropyl.

[0083] In this specification, the term “C 1-18 The term "alkyl" refers to a linear or branched alkyl group having 1 to 18 carbon atoms, and examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl.

[0084] In the present specification, the term "3- to 8-membered cycloalkyl group which may have a substituent" means a cyclic alkyl group having 3 to 8 carbon atoms, and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The substituent is not particularly limited, and examples thereof include halogen atoms, alkyl groups having 1 to 6 carbon atoms, alkenyl groups having 2 to 6 carbon atoms, alkynyl groups having 2 to 6 carbon atoms, hydroxyl groups, alkoxy groups having 1 to 6 carbon atoms, amino groups, alkylamino groups having 1 to 6 carbon atoms, two alkylamino groups having 1 to 6 carbon atoms in which the alkyl groups are the same or different, thiol groups, alkylthio groups having 1 to 6 carbon atoms, carboxyl groups, alkoxycarbonyl groups having 1 to 6 carbon atoms, and carbamoyl groups.

[0085] In this specification, the term "C 6-18 "Aryl" means a monocyclic or condensed polycyclic aromatic hydrocarbon group, examples of which include phenyl, naphthyl, anthracenyl, phenanthrenyl, terphenyl, pyrene, In addition, the term "C optionally substituted" refers to 6-14 The term "aryl" means a monocyclic or condensed polycyclic aromatic hydrocarbon group, and examples thereof include phenyl, naphthyl, anthryl, phenanthryl, and the like. The substituent is not particularly limited, and examples thereof include halogen atoms, alkyl groups having 1 to 6 carbon atoms, alkenyl groups having 2 to 6 carbon atoms, alkynyl groups having 2 to 6 carbon atoms, hydroxyl groups, alkoxy groups having 1 to 6 carbon atoms, amino groups, alkylamino groups having 1 to 6 carbon atoms, two alkylamino groups having 1 to 6 carbon atoms in which the alkyl groups are the same or different, thiol groups, alkylthio groups having 1 to 6 carbon atoms, carboxyl groups, alkoxycarbonyl groups having 1 to 6 carbon atoms, and carbamoyl groups.

[0086] In the present specification, the term "5- to 10-membered heteroaryl group which may have a substituent" means a 5- to 10-membered monocyclic aromatic heterocyclic group or a condensed aromatic heterocyclic group in which the atoms constituting the ring include, in addition to carbon atoms, 1 to 4 heteroatoms selected from nitrogen atoms, oxygen atoms and sulfur atoms. Examples of the monocyclic aromatic heterocyclic group include furanyl, thienyl, pyrrolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, isoxazolyl, 1,3,4-thiadiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, tetrazolyl and the like. Examples of the fused aromatic heterocyclic group include benzofuranyl, benzothienyl, quinoxalinyl, indolyl, isoindolyl, isobenzofuranyl, chromanyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolyl, isoquinolyl, etc. In addition, the term "6- to 10-membered heteroaryl group which may have a substituent" means a 6- to 10-membered monocyclic aromatic heterocyclic group or a fused aromatic heterocyclic group in which the atoms constituting the ring include, in addition to carbon atoms, 1 to 4 heteroatoms selected from nitrogen atoms, oxygen atoms, and sulfur atoms. Examples of the monocyclic aromatic heterocyclic group include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, etc. Examples of the condensed aromatic heterocyclic group include benzofuranyl, benzothienyl, quinoxalinyl, indolyl, isoindolyl, isobenzofuranyl, chromanyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolyl, isoquinolyl, etc. The substituent is not particularly limited, and examples thereof include halogen atoms, alkyl groups having 1 to 6 carbon atoms, alkenyl groups having 2 to 6 carbon atoms, alkynyl groups having 2 to 6 carbon atoms, hydroxyl groups, alkoxy groups having 1 to 6 carbon atoms, amino groups, alkylamino groups having 1 to 6 carbon atoms, alkylamino groups having 1 to 6 carbon atoms, two alkylamino groups having 1 to 6 carbon atoms in which the alkyl groups are the same or different, thiol groups, alkylthio groups having 1 to 6 carbon atoms, carboxyl groups, alkoxycarbonyl groups having 1 to 6 carbon atoms, and carbamoyl groups.

[0087] In the present specification, examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0088] In this specification, the so-called "targeting affinity molecule" is biotin or its derivatives that have a specific binding function to avidin / streptavidin or its variant (modified) or functional fragment. The targeting affinity molecule can be carried on the copolymer X of the present invention through electrostatic interaction, hydrogen bonding, hydrophobic interaction or covalent bonding.

[0089] In this specification, the so-called "pre-targeting method" is a drug delivery method that allows drugs to be reliably accumulated in target cells such as cancer cells. It includes the following steps: first, a fusion of a molecular probe such as a cancer antigen-specific antibody molecule and avidin / streptavidin or its variant (modified) body or functional fragment is prepared and administered to a patient, thereby causing the fusion to be accumulated in cancer cells; then, a conjugate of a targeting affinity molecule such as biotin having affinity for the above-mentioned avidin / streptavidin or its variant (modified) body or functional fragment and a pharmaceutical composition is administered to the patient, thereby causing the drug to be reliably accumulated in cancer cells.

[0090] Alternatively, a complex may be prepared in advance and administered to a patient. The complex is formed by combining a molecular probe such as a cancer antigen-specific antibody molecule with a conjugate of avidin / streptavidin or a variant (modified) thereof or a functional fragment thereof, and a conjugate of the targeting affinity molecule with a pharmaceutical composition.

[0091] In this specification, the so-called "pharmaceutical composition" means a substance in which an active ingredient (drug, physiologically active substance) that can be used for diagnosis, prevention or treatment of a disease is supported on the copolymer X or the targeting affinity copolymer of the present invention through electrostatic interaction, hydrogen bonding, hydrophobic interaction or covalent bonding. As the supported form, when the copolymer X or the targeting affinity copolymer forms nanoparticles, the form in which the drug is present on the surface of the particle or the form in which the drug is enclosed in the nanoparticle, or a combination of these forms can be cited.

[0092] One embodiment of the present invention is a copolymer (targeting affinity copolymer) obtained by bonding a targeting affinity molecule to a copolymer X including structural units represented by the following formulae (A), (B), and (C).

[0093]

[0094] [Where R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 Alkyl; m represents an integer of 1 to 100; n represents an integer of 0 to 3.]

[0095] In the copolymer X of the present invention, the structural unit (A) functions as a unit that imparts hydrophilicity, and the structural unit (B) functions as a unit that imparts hydrophobicity. In addition, the structural unit (C) functions as a scaffold for binding the effective ingredient (drug, physiologically active substance) to the copolymer X or the targeting affinity copolymer. The copolymer X or the targeting affinity copolymer of the present invention has the three structural units, and therefore has the property of forming SCNPs in water, and can precisely control the particle size of the formed SCNPs to a microscale of less than 20 nm, and functions as a drug delivery carrier with high tumor aggregation.

[0096] R in the structural unit (A) 1 Represents a hydrogen atom or C 1-3 An alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0097] X 1 represents an oxygen atom, a sulfur atom or NR 7 , but is preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0098] m represents an integer of 1 to 100, preferably an integer of 3 to 100, and preferably 3 to 80, more preferably 4 to 60, further preferably 4 to 40, further more preferably 4 to 22 from the viewpoint of imparting good hydrophilicity.

[0099] R 4 Represents C 1-3 Specifically, the alkyl group is a methyl group, an ethyl group, an n-propyl group or an isopropyl group, preferably a methyl group or an ethyl group, and more preferably a methyl group.

[0100] R in the structural unit (B) 2 Represents a hydrogen atom or C 1-3 An alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0101] X 2 represents an oxygen atom, a sulfur atom or NR 7 , but is preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0102] n represents an integer of 0 to 3, and is preferably an integer of 1 to 3, and more preferably 1.

[0103] R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 An aryl group or a 5- to 10-membered heteroaryl group which may have a substituent, but from the viewpoint of imparting hydrophobicity to the structural unit (B), C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; more preferably C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; more preferably C 1-18 Alkyl, 3-8 membered cycloalkyl, adamantyl or C 6-18 On the other hand, a 3- to 8-membered cycloalkyl group which may have a substituent, an adamantyl group, a C 6-14 An aryl group or a 6- to 10-membered heteroaryl group which may have a substituent.

[0104] Here, the substituent is preferably one or more selected from a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, and an alkynyl group having 2 to 6 carbon atoms.

[0105] R in the structural unit (C) 3 Represents a hydrogen atom or C 1-3 An alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0106] X 3 represents an oxygen atom, a sulfur atom or NR 7 , but is preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0107] R 6 represents a hydrogen atom, a leaving group or a linking group. The leaving group is a group that can leave when the structural unit (C) is combined with the drug (physiologically active substance), and the linking group is a group that can be used for crosslinking when the structural unit (C) is combined with the drug (physiologically active substance). As these leaving groups or linking groups, C which may have a substituent is preferred. 1-18 Alkyl, 3- to 8-membered cycloalkyl which may have a substituent, C 7-19Aralkyl. Here, as a substituent, for example, there can be mentioned a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an amino group, an alkylamino group having 1 to 6 carbon atoms, two alkylamino groups having 1 to 6 carbon atoms in which the alkyl groups are the same or different, a thiol group, an alkylthio group having 1 to 6 carbon atoms, a carboxyl group, an alkoxycarbonyl group having 1 to 6 carbon atoms, a carbamoyl group, etc. As a linking group among these groups, a group having a functional group such as a hydroxyl group, an amino group, a thiol group, a carboxyl group, etc. as a substituent is preferred.

[0108] As R 6 Preferred specific examples of the leaving group include a group represented by the following formula (4).

[0109]

[0110] As R 6 Preferred specific examples of the linking group include groups selected from the following formulae (5) to (7).

[0111]

[0112] The copolymer X of the present invention is a copolymer having structural units shown in formulas (A), (B) and (C). The copolymer X may be a random copolymer or a block copolymer, preferably a random copolymer. The composition ratio of each structural unit in one molecule is preferably a ratio of 0.01 to 100 parts by mass of (B) and 0.1 to 100 parts by mass of (C) when (A) is 1 part by mass; more preferably, a ratio of 0.05 to 18 parts by mass of (B) and 0.1 to 20 parts by mass of (C) when (A) is 1 part by mass; particularly preferably, a ratio of 0.05 to 4 parts by mass of (B) and 0.1 to 16 parts by mass of (C) when (A) is 1 part by mass.

[0113] The degree of polymerization of the copolymer X of the present invention is not particularly limited, but the number average molecular weight is preferably 5,000 to 150,000, more preferably 8,000 to 150,000.

[0114] In the copolymer of the present invention, as described above, the monomer represented by the general formula (1) functions as a unit imparting hydrophilicity, and the monomer represented by the general formula (2) functions as a unit imparting hydrophobicity. In addition, the monomer represented by the general formula (3) functions as a scaffold for combining drugs with the copolymer. The monomer that functions as the hydrophobic unit represented by the general formula (2) can be exemplified by the monomer represented by the following formula.

[0115]

[0116]

[0117] In the general formula (1), R 1 Represents a hydrogen atom or C 1-3 An alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0118] In the general formula (2), R 2 Represents a hydrogen atom or C 1-3 An alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0119] In the general formula (3), R 3 Represents a hydrogen atom or C 1-3 An alkyl group is preferably a hydrogen atom or a methyl group, and is preferably a hydrogen atom, an ethyl group or a propyl group, and is more preferably a hydrogen atom.

[0120] In the general formula (1), R 4 Represents C 1-3 Specifically, the alkyl group is a methyl group, an ethyl group, an n-propyl group or an isopropyl group, preferably a methyl group or an ethyl group, and more preferably a methyl group.

[0121] In the general formula (1), X 1 represents an oxygen atom, a sulfur atom or NR 7 , but is preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0122] In the general formula (1), m represents an integer of 1 to 100, preferably an integer of 3 to 100, and preferably 3 to 80, more preferably 4 to 60, further preferably 4 to 40, further more preferably 4 to 22 from the viewpoint of imparting good hydrophilicity.

[0123] In the general formula (2), R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 The aryl group or the 5- to 10-membered heteroaryl group which may have a substituent is preferably C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent, more preferably C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 An aryl group or an optionally substituted 5- to 10-membered heteroaryl group, preferably a C 1-18 Alkyl, 3-8 membered cycloalkyl, adamantyl or C 6-18On the other hand, a 3- to 8-membered cycloalkyl group which may have a substituent, an adamantyl group, a C 6-14 Aryl or 6- to 10-membered heteroaryl group which may have a substituent. Here, the substituent is preferably one or more selected from the group consisting of a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, and an alkynyl group having 2 to 6 carbon atoms.

[0124] In the general formula (2), X 2 represents an oxygen atom, a sulfur atom or NR 7 , preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0125] In the general formula (2), n represents an integer of 0 to 3, preferably an integer of 1 to 3, and more preferably 1.

[0126] In the general formula (3), R 6 represents a hydrogen atom, a leaving group or a linking group. As these leaving groups or linking groups, preferably C 1-18 Alkyl, 3- to 8-membered cycloalkyl which may have a substituent, C 7-19 Aralkyl. Here, as a substituent, for example, there can be mentioned a halogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an amino group, an alkylamino group having 1 to 6 carbon atoms, two alkylamino groups having 1 to 6 carbon atoms in which the alkyl groups are the same or different, a thiol group, an alkylthio group having 1 to 6 carbon atoms, a carboxyl group, an alkoxycarbonyl group having 1 to 6 carbon atoms, a carbamoyl group, etc. As a linking group among these groups, a group having a functional group such as a hydroxyl group, an amino group, a thiol group, a carboxyl group, etc. as a substituent is preferred.

[0127] As R 6 Preferred specific examples of the leaving group include a group represented by the following formula (4).

[0128]

[0129] As R 6 Preferred specific examples of the linking group include groups selected from the following formulae (5) to (7).

[0130]

[0131] In the general formula (3), X 3 represents an oxygen atom, a sulfur atom or NR 7 , but is preferably an oxygen atom, a sulfur atom or NH, more preferably an oxygen atom.

[0132] The copolymer X of the present invention is formed by copolymerizing three monomers represented by general formulas (1) to (3). The copolymerization may be random copolymerization or block copolymerization, and is preferably formed by random copolymerization. Regarding the mixing ratio of the three monomers, it is preferred that 0.01 to 100 parts by mass of the monomer (2) and 0.1 to 100 parts by mass of the monomer (3) are polymerized when the mass part of the monomer (1) is set to 1, more preferably 0.05 to 18 parts by mass of the monomer (2) and 0.1 to 20 parts by mass of the monomer (3) are polymerized, and it is particularly preferred that 0.05 to 4 parts by mass of the monomer (2) and 0.1 to 16 parts by mass of the monomer (3) are polymerized.

[0133] In addition, "solvates" coordinated with various solvents are also included in the copolymer X of the present invention. In the present specification, examples of "solvates" include hydrates and ethanolates. Any number of solvents may be coordinated with the copolymer X of the present invention.

[0134] The copolymer X of the present invention can be produced by various known methods. The production method is not particularly limited, and for example, it can be produced according to the basic polymer synthesis method described below.

[0135]

[0136] [wherein, R' represents a hydrogen atom or C 1-3 Alkyl, R" represents the above R 4 , R 5 or R 6 The groups shown.]

[0137] This reaction represents a step of making a chain transfer agent (II) and an initiator react with a monomer (I) to produce a polymer (III). This reaction can be carried out without a solvent, or in an alcohol such as methanol, ethanol, 1-propanol, 2-propanol; an ether such as diethyl ether, tetrahydrofuran, 1,4-dioxane; an aromatic hydrocarbon such as benzene, toluene, xylene; a halogenated hydrocarbon such as dichloromethane, chloroform, 1,2-dichloroethane; N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, ethyl acetate, etc., preferably using an aromatic hydrocarbon such as toluene and xylene as a solvent. The chain transfer agent may be 2-(dodecylthiocarbonylthio)-2-methylpropionic acid (DDMAT), cyanomethyldodecyl trithiocarbonate (CDTTC), 2-cyano-2-propyldodecyl trithiocarbonate (CPDTTC), 4-cyano-4-[(dodecylsulfanyl-thiocarbonyl)sulfanyl]pentanoic acid (CDSPA), 2-(dodecylsulfanylthiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester (N3-CTA), etc., preferably DDMAT or N3-CTA, more preferably N3-CTA. When a chain transfer agent is used for polymerization, the copolymer X of the present invention adopts a structure in which a part or all of the structure of the chain transfer agent is partially bonded. When the copolymer X contains the structure of the chain transfer agent, the structure can be removed by an appropriate method. The initiator may be an azo polymerization initiator such as 2,2'-azobisisobutyronitrile (AIBN), 1,1'-azobis(cyclohexanecarbonitrile) (ACHN), 2,2'-azobis-2-methylbutyronitrile (AMBN), 2,2'-azobis-2,4-dimethylvaleronitrile (ADVN), etc., preferably AIBN. The reaction temperature is 0 to 300°C, preferably 0 to 150°C, more preferably 1 to 100°C; the reaction time is 1 minute to 48 hours, preferably 5 minutes to 24 hours. In this reaction, by reacting in the presence of monomers (I) having different structures, a copolymer X obtained by random copolymerization can be produced.

[0138] For example, when N3-CTA having an azide group is used as a chain transfer agent, a copolymer X having an azide group at the terminal can be obtained, which is advantageous for producing a targeting affinity copolymer through the click reaction described below.

[0139] The targeting affinity molecule bound to the copolymer X is biotin or its derivatives having a specific binding function to avidin / streptavidin or its variant (modified) or functional fragment.

[0140] As biotin or its derivatives, for example, biotin, norbiotin, homobiotin, iminobiotin, oxybiotin, thiobiotin, ethylbiotin, desthiobiotin, diaminobiotin, biotin carbamate, biotin carbonate, trinorbiotin sulfoxide, bisnorbiotin sulfoxide, norbiotin sulfoxide, biotin sulfoxide, homobiotin sulfoxide, bishomobiotin sulfoxide, trihomobiotin sulfoxide, biotin sulfone, reduced biotin (biotinol), or compounds described in Japanese Patent Application Laid-Open No. 58-154508, International Publication No. 2014 / 129446, etc. In addition, biocytin or compounds having a spacer as described in Avidin-Biotin Technical Handbook of ThermoFisher can also be used. Furthermore, as bibiotin derivatives having a higher affinity for streptavidin variants having reduced affinity for natural biotin, compounds described in International Publication Nos. 2015 / 125820, 2018 / 151301, 2019 / 189867, 2019 / 230905, and 2021 / 210573 can be listed, but are not limited to these.

[0141] Among them, biotin or its derivative is preferably a molecule having a biotin-based residue represented by the following formula (a).

[0142]

[0143] (In the formula, Y represents O or NH.)

[0144] Preferred examples include compounds represented by the following formulas (8) to (11).

[0145]

[0146] More preferably, there is mentioned a compound represented by the following formula (8).

[0147]

[0148] When geometrical isomers or optical isomers exist in the compounds of the present invention, mixtures or separated products of these isomers are also included in the scope of the present invention. Separation of isomers can be carried out by conventional methods.

[0149] The targeting affinity copolymer of the present invention can be produced by various known methods. The production method is not particularly limited, and for example, it can be produced according to the click reaction synthesis method described below.

[0150]

[0151] "Click chemistry" has the following properties, is classified as a reaction similar to natural biochemical reactions, and has the following characteristics. It is a very efficient reaction, that is, it proceeds rapidly until a high yield is reached. In addition, it is a very selective reaction, that is, no by-products are generated at all (or almost), and it tolerates multiple functional groups. Furthermore, it is a reaction carried out under mild reaction conditions such as low temperature (or nearby), or in an aqueous solution. This reaction can be carried out in water or alcohols such as methanol, ethanol, 1-propanol, and 2-propanol; ethers such as diethyl ether, tetrahydrofuran, and 1,4-dioxane; aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as dichloromethane, chloroform, and 1,2-dichloroethane; N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, acetonitrile, ethyl acetate, etc. Solvents such as ethyl acetate are preferably carried out in water or using N,N-dimethylformamide as a solvent. In some embodiments, cyclooctyne is dibenzocyclooctyne (DBCO), difluorobenzocyclooctyne (DIFBO), biaryl azacyclooctyne ketone (BARAC), dibenzocyclooctyne (DIBO), difluorinated cyclooctyne (DIFO), monofluorinated cyclooctyne (MOFO), dimethoxy azacyclooctyne (DIMAC) or aryl-lessoctyne (ALO), preferably dibenzocyclooctyne (DBCO). In some embodiments, the alkyne is an aliphatic alkyne, and the step of reacting is carried out in the presence of a copper (I) catalyst. In some embodiments, the alkyne is cyclooctyne, and the step of reacting is carried out under copper-free conditions. The reaction temperature is 0 to 300°C, preferably 0 to 150°C, more preferably 1 to 100°C, and the reaction time is 1 minute to 48 hours, preferably 5 minutes to 24 hours.

[0152] The reaction in the above reaction formula is a Huisgen cycloaddition reaction, which is a type of click reaction and is a 1,3-dipolar addition cyclization reaction of an azide group and an alkyne to form a 1,2,3-triazole.

[0153] The produced polymer X and targeting affinity copolymer of the present invention can be purified by a method for separating and purifying polymers generally known in the field of polymer chemistry. Specifically, for example, the methods include extraction, recrystallization, salting out using ammonium sulfate or sodium sulfate, centrifugal separation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, or a combination thereof.

[0154] The copolymer X and the targeting affinity copolymer of the present invention can be used as carriers for the delivery of various physiologically active substances (drugs). For example, the pharmaceutical composition in which the tumor therapeutic drug is supported (encapsulated) in the copolymer X or the targeting affinity copolymer of the present invention can inhibit the proliferation of tumors as confirmed in the following test examples, and can therefore be used as a preventive and / or therapeutic agent for various cancers such as colorectal cancer, duodenal cancer, gastric cancer, pancreatic cancer, liver cancer, lung cancer, uterine cancer, and ovarian cancer. In addition, due to the high tumor aggregation ability, it can be used as a diagnostic agent and contrast agent for tumors.

[0155] When the copolymer and the targeting affinity copolymer of the present invention are used as drug delivery carriers, the dosage and the number of administrations can be appropriately selected in consideration of the dosage form, the patient's age, weight, the nature or severity of the symptoms to be treated, etc. The dosage and the number of administrations are not limited. When the polymer containing the drug is intravenously injected by injection, it is preferred to administer 0.12 mg to 12,000,000 mg per adult (60 kg), for example, once, more preferably 1.2 mg to 1,200,000 mg, and particularly preferably 12 to 120,000 mg.

[0156] The pharmaceutical composition of the present invention can be prepared by mixing the copolymer X of the present invention or the targeting affinity copolymer with a drug. Preferably, the copolymer of the present invention or the targeting affinity copolymer is mixed with a drug to prepare single-chain nanoparticles, or the drug is mixed after preparing the single-chain nanoparticles of the copolymer X of the present invention or the targeting affinity copolymer. The single-chain nanoparticles can be prepared by a known method.

[0157] In the pharmaceutical composition of the present invention, the drug may be supported on the copolymer X or the targeting affinity copolymer by electrostatic interaction, hydrogen bonding, hydrophobic interaction, covalent bonding, or the like.

[0158] The drug may be used for various cancer diseases, and is preferably an anticancer agent that acts on cancer cells to inhibit the proliferation of cancer cells, and examples thereof include metabolic antagonists, alkylating agents, anthracyclines, antibiotics, mitotic inhibitors, topoisomerase inhibitors, proteasome inhibitors, and antihormonal agents.

[0159] Examples of metabolic antagonists include azathioprine, 6-mercaptopurine, 6-thioguanine, fludarabine, pentostatin, cladribine, 5-fluorouracil (5FU), floxuridine (FUDR), cytosine arabinoside (cytarabine), methotrexate, trimethoprim, pyrimethamine, and pemetrexed. Examples of alkylating agents include cyclophosphamide, methyldichloroethylamine, uramustine, melphalan, chlorambucil, thiotepa / chlorambucil, ifosfamide, carmustine, lomustine, streptozotocin, busulfan, dibromomannitol, cisplatin, carboplatin, nedaplatin, oxaliplatin, miplatin, satraplatin, triplatin tetranitrate, procarbazine, hexamethylmelamine, dacarbazine, mitozolamide, trabectedin, or temozolomide. Examples of anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, aclarubicin, amrubicin, and pirarubicin. Examples of the antibiotic include actinomycin, bleomycin, mithramycin, anthramycin, streptozotocin, gramicidin D, staurosporine, mitomycins (eg, mitomycin C), duocarmycins (eg, CC-1065), and calicheamicins. Examples of the mitotic inhibitor include maytansine alkaloids (e.g., DM0, maytansine (also known as DM1), DM2, DM3, DM4, or emtansine), auristatins (e.g., auristatin E, auristatin phenylalanine phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin D, and monomethyl auristatin F), dolastatins, nostocins, vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinorelbine), taxanes (e.g., paclitaxel, docetaxel), and colchicine. Examples of topoisomerase inhibitors include irinotecan, topotecan, nogitecan, amsacrine, etoposide, teniposide, mizantrone, mitoxantrone, SN-38, exatecan, deruxtecan, etc. Examples of proteasome inhibitors include peptidylboronic acid, carfilzomib, bortezomib, etc. Examples of antihormonal agents include fulvestrant, tamoxifen, toremifene, etc.When these drugs are used to prepare the pharmaceutical composition of the present invention, one type may be used or a combination of two or more types may be used. The drug may be supported in the copolymer X or the targeting affinity copolymer in the form of a free body.

[0160] The administration route of the pharmaceutical composition of the present invention is preferably the most effective way for treatment, and can be administered in the form of oral administration preparations, injections, or non-oral administration preparations such as transdermal administration preparations, for example, preferably intra-arterial injection, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection and other non-oral administration, more preferably intra-arterial injection and intravenous injection. The number of administrations should not be limited, for example, it can be listed as: an average of 1 to several administrations per week.

[0161] Various preparations suitable for the route of administration can be prepared by appropriately selecting pharmaceutical additives such as excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavoring agents, analgesics, stabilizers, isotonic agents, etc., which are commonly used in pharmaceutical preparations, and by conventional methods.

[0162] The preparation additives that can be included in the above-mentioned various preparations are not particularly limited as long as they are additives allowed in medicine. As the example of this preparation additive, it is possible to enumerate: purified water, water for injection, distilled water for injection, organic solvents allowed in medicine, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerol, propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, etc. The additive used can be appropriately selected according to various preparations, and can be used alone or in combination.

[0163] In addition, the injection can also be prepared into a non-aqueous diluent (for example, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), a suspending agent or an emulsion. The injection can be sterilized by filtering and sterilizing with a filter, or by using a bactericide. In addition, the injection can be manufactured into a form modulated when used. That is, a sterile solid composition can be prepared by freeze drying, etc., and dissolved in water for injection, distilled water for injection, or other solvents before use.

[0164] The therapeutic method using the targeting affinity copolymer of the present invention comprises the following steps: first, a fusion of a molecular probe such as a cancer antigen-specific antibody molecule and avidin / streptavidin or a variant (modified) thereof or a functional fragment thereof is prepared and administered to a patient, thereby causing the fusion to aggregate to cancer cells; then, a conjugate of the targeting affinity copolymer of the present invention and an anticancer agent is administered to the patient, thereby causing the drug to reliably aggregate to cancer cells.

[0165] In addition, a complex can be prepared in advance and administered to a patient. The complex is formed by combining a molecular probe such as a cancer antigen-specific antibody molecule with a conjugate of avidin / streptavidin or its variant (modified) or functional fragment, and a conjugate of the targeting affinity copolymer of the present invention and an anticancer agent.

[0166] [Example]

[0167] The present invention will be described in more detail below using examples. These examples are provided for illustrative purposes and are not intended to limit the embodiments of the present invention.

[0168] [Example 1] Preparation of Poly[(Benzyl acrylate)-co-(Poly(ethylene glycol) methyl ether acrylate)-co-(1-ethoxyethyl acrylate)]

[0169] (1) Synthesis of 1-ethoxyethyl acrylate (EEA)

[0170] Under an argon atmosphere, weigh ethyl vinyl ether (28.725 mL), add phosphoric acid (50 mg) while cooling in an ice bath. Then, add acrylic acid (17.15 mL) and stir at room temperature for 48 hours. Add hydrotalcite (3 g) and stir for another 2 hours to terminate the reaction. After filtering with diatomaceous earth, remove the unreacted ethyl vinyl ether by evaporation. Add phenothiazine as a polymerization inhibitor in a manner to reach 500 ppm, and perform vacuum distillation with calcium hydroxide to purify (distillation temperature is 28-32°C). The obtained 1-ethoxyethyl acrylate is divided into a glass vial and stored at -30°C.

[0171] 13 C NMR (400MHz, CDCl3), δ, ppm: 15.29(-OCH2CH3), 21.16(-COOCH(CH3)), 64.98(-OCH2-), 96.73(-COOCH(CH3)), 128.84(CH2CH-), 131.43(CH2CH-), 166.00(-COO).

[0172] (2) Synthesis of poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(1-ethoxyethyl acrylate)]

[0173] 100 mg of 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid (DDMAT) was weighed and dissolved in 17.3 mL of toluene to prepare a DDMAT / toluene stock solution (DDMAT concentration was 5.78 mg / mL). Similarly, 22 mg of 2,2'-azobis(2-methylpropionitrile) (AIBN) was weighed and dissolved in 17.3 mL of toluene to prepare an AIBN / toluene stock solution (AIBN concentration was 1.27 mg / mL). In addition, 1.296 g of poly(ethylene glycol) methyl ether acrylate (mPEGA, the average value (n) of the number of repeats of ethylene glycol was 9), 0.394 g of benzyl acrylate (BnA), 0.039 g of 1-ethoxyethyl acrylate, 1.73 mL of the DDMAT / toluene stock solution, and 1.73 mL of the AIBN / toluene stock solution were added, and polymerization was carried out in an oil bath at 70°C. After 90 minutes, the polymerization was terminated, and the copolymer was recovered by reprecipitation or methanol dialysis of the reaction solution. The obtained copolymer was basically a viscous body, so regarding the reprecipitation method, the reaction solution was added dropwise to a centrifuge tube added with a poor solvent (hexane / ethyl acetate = 7 / 3 [v / v]), and recovered by centrifugation (2,000×g, 5 min). The above operation was repeated 3 times, and finally vacuum dried to obtain 1.223 g of poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(1-ethoxyethyl acrylate)].

[0174] The obtained copolymer was measured by NMR. 1 H-NMR spectroscopy analysis of the polymerization degree and number average molecular weight (M n,NMR ), the results show that the degree of polymerization of mPEGA (n = 9) is 102, the degree of polymerization of BnA is 94, the degree of polymerization of EEA is 9, and the degree of polymerization of M n,NMR The molecular weight dispersion (M w / M n ), the result is 1.53.

[0175]

[0176] [Measurement equipment and conditions, etc.]

[0177] (1) 1 H-NMR measurement

[0178] Device: JNM-ECX400 (400MHz) / JEOL

[0179] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0180] Sample concentration: 20mg / mL

[0181] Measurement temperature: 25℃

[0182] Total times: 256 times

[0183] result: Figure 1

[0184] (2) GPC measurement

[0185] Equipment: HPLC-Prominence system / Shimadzu Corporation

[0186] Detector: RID-10A refractive index detector / Shimadzu Corporation

[0187] Column: TSKgelα-2500 column / Tosoh

[0188] (column size: 7.8 mm × 300 mm, particle size: 7 μm, exclusion limit molecular weight: 5 × 10 3 )

[0189] TSKgelα-4000 column / Tosoh

[0190] (column size: 7.8 mm × 300 mm, particle size: 10 μm, exclusion limit molecular weight: 4 × 10 5 )

[0191] TSKgel Guard Column / Tosoh

[0192] Mobile phase: N,N-dimethylformamide (DMF) containing 10 mmol / L lithium bromide

[0193] Temperature: 40℃

[0194] Flow rate: 0.5mL / min

[0195] Sample concentration: 6mg / mL

[0196] Standard material: Poly(methyl methacrylate) Standard ReadyCal Kit, M p 800-2,200,000Da / SIGMA

[0197] result: Figure 2

[0198] [Table 1]

[0199]

[0200] [Examples 2 to 68]

[0201] The same method as in Example 1 was used by appropriately changing the types of monomers (mPEGA, BnA, EEA), feed amounts, reaction temperatures, and polymerization times, thereby producing polymers having different composition ratios or average molecular weights as shown in the following table.

[0202] [Table 2]

[0203]

[0204]

[0205]

[0206] [Table 3]

[0207]

[0208]

[0209] [Example 69]

[0210] Preparation of poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(acrylic acid)]

[0211] The poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(1-ethoxyethyl acrylate)] obtained in Example 1 was treated with 0.5N hydrochloric acid (HCl) at room temperature to obtain 1.176 g of a terpolymer having carboxyl groups after the ethoxyethyl groups were removed. The Z average particle size and polydispersity index of the obtained terpolymer in water were measured by dynamic light scattering (DLS), and the result was 8.5 nm (polydispersity index was 0.14).

[0212]

[0213] [Measurement equipment and conditions, etc.]

[0214] (1) DLS assay

[0215] Device: Zetasizer NanoZS / Malvern Instruments Ltd.

[0216] Measurement temperature: 25℃

[0217] Sample concentration: 10mg / mL

[0218] result: Figure 3

[0219] [Example 70]

[0220] Method for producing SCNP containing (1,2-diaminocyclohexane) platinum (II)

[0221] 65.28 mg of (1,2-diaminocyclohexane) platinum (II) (hereinafter referred to as DACHPt) Cl(H2O) body (DACHPt·Cl·H2O) was dissolved in 20 mL of purified water and stirred at 70°C for 2 hours. 287.4 mg of the terpolymer obtained in Example 69 was added to 5 mL of the solution and stirred at 50°C overnight. After the stirring, the reaction solution was dialyzed and purified using purified water as the external liquid to obtain 5 mL of SCNPs containing DACHPt. The Pt content of the SCNPs containing DACHPt obtained after purification was measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES), which was 720 μg / mL (DACHPt was 1.14 mg / mL). In addition, 200 μL of SCNPs containing DACHPt were freeze-dried, and the solid content concentration was calculated. The ratio with the Pt content was taken to calculate the Pt binding amount of each polymer, and the result was 3.4 mol / mol. In addition, the Z average particle size and polydispersity index of the obtained SCNPs containing DACHPt were measured by dynamic light scattering (DLS), and the result was 8.7 nm (polydispersity index was 0.14). The particle size of the SCNPs before and after the inclusion of DACHPt is shown in Figure 3 The particle size of SCNPs hardly changed before and after the inclusion of DACHPt. The results are summarized in the table below.

[0222] [Measurement equipment and conditions, etc.]

[0223] (1) ICP-AES measurement

[0224] Device: Sequential high-frequency plasma luminescence device ICPE-9000 / Shimadzu Corporation

[0225] Pre-treatment device: Microwave sample pre-treatment device ETHOS EASY / Milestone General

[0226] Measurement wavelength: 214nm

[0227] Standard solution: Platinum standard solution (Pt1000) for ICP analysis / Fujifilm Wako Pure Chemical Industries, Ltd.

[0228] (2) DLS assay

[0229] Device: Zetasizer NanoZS / Malvern Instruments Ltd.

[0230] Measurement temperature: 25℃

[0231] Sample concentration: 10mg / mL

[0232] result: Figure 3

[0233] [Table 4]

[0234]

[0235] [Example 71]

[0236] Preparation of N3-poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(1-ethoxyethyl acrylate)]

[0237] N3-CTA was used to replace the chain transfer agent DDMAT used in Example 1 for synthesis. 100 mg of 2-(dodecylthiothiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester (N3-CTA) was weighed and dissolved in 17.3 mL of toluene to prepare an N3-CTA / toluene stock solution (N3-CTA concentration was 5.78 mg / mL). Similarly, 10 mg of 2,2'-azobis(2-methylpropionitrile) (AIBN) was weighed and dissolved in 7.87 mL of toluene to prepare an AIBN / toluene stock solution (AIBN concentration was 1.27 mg / mL). Separately, 2.592 g of poly(ethylene glycol) methyl ether acrylate (mPEGA, average number of repeats (n) of ethylene glycol is 9), 0.684 g of benzyl acrylate (BnA), 0.172 g of 1-ethoxyethyl acrylate, 4.15 mL of N3-CTA / toluene stock solution, and 3.46 mL of AIBN / toluene stock solution were added, and polymerization was carried out in an oil bath at 70°C. After 90 minutes, the polymerization was terminated, and the reaction solution was subjected to a reprecipitation method or methanol dialysis to recover the copolymer. The obtained copolymer was basically a viscous body, so regarding the reprecipitation method, the reaction solution was added dropwise to a centrifuge tube added with a poor solvent (hexane / ethyl acetate = 7 / 3 [v / v]), and recovered by centrifugal separation (2,000×g, 5 min). The above operation was repeated 3 times, and finally vacuum dried to obtain 2.643 g of N 3 -poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(1-ethoxyethyl acrylate)]. The obtained copolymer was determined by NMR. 1 H-NMR spectroscopy analysis of the polymerization degree and number average molecular weight (Mn,NMR ), the results show that the polymerization degree of mPEGA (n = 9) is 70, the polymerization degree of BnA is 56, the polymerization degree of EEA is 15, and the polymerization degree of M n,NMR The molecular weight dispersion (M w / M n ), the result is 1.32.

[0238] [Example 72]

[0239] Preparation of N3-poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(acrylic acid)]

[0240] The N obtained in Example 71 was treated with 0.5N HCl at room temperature. 3 -poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(1-ethoxyethyl acrylate)] was treated to obtain 2.455 g of a terpolymer having carboxyl groups after the ethoxyethyl groups were removed. The Z average particle size and polydispersity index of the obtained terpolymer in water were measured by dynamic light scattering (DLS) and the result was 7.5 nm (polydispersity index was 0.18).

[0241] [Example 73]

[0242] Fabrication of biotin-conjugated SCNPs

[0243] Weigh 275 mg of N3-poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(acrylic acid)] and 19.5 mg of ((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(22-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)-9-(2- ((4-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)butyl)amino)-2-oxoethyl)-11,18-dioxo-3,6-dioxa-9,12,17-triazacosyl)carbamate was added to a glass vial, 5 mL of DMF was added and dissolved, and stirred at room temperature overnight. The reaction solution was dialyzed against methanol, and after the solvent was distilled off, 265.7 mg of biotin-bound SCNPs were recovered by vacuum drying. The Z-average particle size and polydispersity index of biotin-bound SCNPs were measured by dynamic light scattering (DLS), and the result was 8.6 nm (polydispersity index was 0.17).

[0244]

[0245] [Measurement equipment and conditions, etc.]

[0246] (1) 1 H-NMR measurement

[0247] Device: JNM-ECX400 (400MHz) / JEOL

[0248] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0249] Sample concentration: 20mg / mL

[0250] Measurement temperature: 25℃

[0251] Cumulative times: 6000 times

[0252] result: Figure 4

[0253] (2) GPC measurement

[0254] Equipment: HPLC-Prominence system / Shimadzu Corporation

[0255] Detector: RID-10A refractive index detector / Shimadzu Corporation

[0256] Column: TSKgelα-2500 column / Tosoh

[0257] (column size: 7.8 mm × 300 mm, particle size: 7 μm, exclusion limit molecular weight: 5 × 10 3 )

[0258] TSKgelα-4000 column / Tosoh

[0259] (column size: 7.8 mm × 300 mm, particle size: 10 μm, exclusion limit molecular weight: 4 × 10 5 )

[0260] TSKgel Guard Column / Tosoh

[0261] Mobile phase: N,N-dimethylformamide (DMF) containing 10 mmol / L lithium bromide

[0262] Temperature: 40℃

[0263] Flow rate: 0.5mL / min

[0264] Sample concentration: 6mg / mL

[0265] Standard material: Poly(methyl methacrylate) Standard ReadyCal Kit, M p 800-2,200,000Da / SIGMA

[0266] result: Figure 5

[0267] (3) DLS measurement

[0268] Device: Zetasizer NanoZS / Malvern Instruments Ltd.

[0269] Measurement temperature: 25℃

[0270] Sample concentration: 10mg / mL

[0271] result: Figure 6

[0272] [Example 74]

[0273] Synthesis of ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2-(2-(2-(3,5-bis(6-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)benzamide)ethoxy)ethoxy)ethyl)carbamate di(2,2,2-trifluoroacetate)

[0274] In 415 mg of (3aS,3a'S,4S,4'S,6aR,6a'R)-4,4'-{5,5'-[6,6'-(5-(2-(2-(2-aminoethoxy)ethoxy)ethylcarbonyl)-1,3-phenylene)bis(nitrogendiyl)bis(6-oxohexane-6,1-diyl)]bis(nitrogendiyl)bis(5-oxopentan-5,1-diyl)}bis 15 mL of DMF, 445 μL of triethylamine and 93.0 mg of (1R, 8S, 9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (BCN-NHS) were added to (tetrahydro-1H-thieno[3,4-d]imidazol-2(3H)-imide)tri(2,2,2-trifluoroacetate), and the mixture was stirred at room temperature for 2 hours. After concentration, the mixture was purified by reverse phase silica gel column chromatography (0.1% aqueous TFA solution / MeOH=90 / 10→30 / 70) to obtain 302 mg of ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2-(2-(2-(3,5-bis(6-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanamido)benzamide)ethoxy)ethoxy)ethyl)carbamate di(2,2,2-trifluoroacetate) (Compound (9)).

[0275]

[0276] 1 H NMR (400MHz, CD3OD) δ: 8.02 (brs, 1H), 7.72 (d, J = 1.6Hz, 2H), 4.71 (dd, J = 4.4, 7.6Hz, 2H), 4.52 (dd, J=4.4,7.6Hz,2H),4.09(d,J=8.4Hz,2H),3.70-3.62(m,6H),3.59-3.52(m,4H),3.29-3.24(m,4H), 3.19(t,J=7.2Hz,4H),2.98(dd,J=4.8,13.2Hz,2H),2.81(d,J=13.2Hz,2H),2.40(t,J=7.2Hz,4H), 2.26-2.10(m,10H),1.79-1.51(m,18H),1.47-1.38(m,8H),1.38-1.30(m,1H),0.90(t,J=9.6Hz,2H)

[0277] [Example 75]

[0278] Preparation of compound (9) combined with SCNP

[0279] 200 mg of N3-poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(acrylic acid)] and 30 mg of compound (9) obtained in Example 74 were weighed into a glass vial, 5 mL of DMF was added and dissolved, and stirred overnight at room temperature. The reaction solution was dialyzed with methanol, and after the solvent was distilled off, 197.4 mg of compound (9) bound to SCNP was recovered by vacuum drying. The Z average particle size and polydispersity index of compound (9) bound to SCNP were measured by dynamic light scattering (DLS), and the result was 7.2 nm (polydispersity index was 0.18).

[0280] [Measurement equipment and conditions, etc.]

[0281] (1) 1 H-NMR measurement

[0282] Device: JNM-ECX400 (400MHz) / JEOL

[0283] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0284] Sample concentration: 20mg / mL

[0285] Measurement temperature: 25℃

[0286] Cumulative times: 6000 times

[0287] result: Figure 7

[0288] (2) GPC measurement

[0289] Equipment: HPLC-Prominence system / Shimadzu Corporation

[0290] Detector: RID-10A refractive index detector / Shimadzu Corporation

[0291] Column: TSKgelα-2500 column / Tosoh

[0292] (column size: 7.8 mm × 300 mm, particle size: 7 μm, exclusion limit molecular weight: 5 × 10 3 )

[0293] TSKgelα-4000 column / Tosoh

[0294] (column size: 7.8 mm × 300 mm, particle size: 10 μm, exclusion limit molecular weight: 4 × 10 5 )

[0295] TSKgel Guard Column / Tosoh

[0296] Mobile phase: N,N-dimethylformamide (DMF) containing 10 mmol / L lithium bromide

[0297] Temperature: 40℃

[0298] Flow rate: 0.5mL / min

[0299] Sample concentration: 6mg / mL

[0300] Standard material: Poly(methyl methacrylate) Standard ReadyCal Kit, M p 800-2,200,000Da / SIGMA

[0301] result: Figure 8

[0302] (3) DLS measurement

[0303] Device: Zetasizer NanoZS / Malvern Instruments Ltd.

[0304] Measurement temperature: 25℃

[0305] Sample concentration: 10mg / mL

[0306] result: Fig. 9

[0307] [Example 76]

[0308] Synthesis of ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl(2-(2-(2-(3-(3,5-bis((2-(2-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethyl)carbamoyl)phenyl)ureido)ethoxy)ethoxy)ethyl)carbamate di(2,2,2-trifluoroacetate)

[0309] In 450 mg of 5-(3-(2-(2-(2-aminoethoxy)ethoxy)ethyl)ureido)-N 1 ,N 315 mL of DMF, 475 μL of Et3N and 99.3 mg of BCN-NHS were added to -bis(2-(2-(5-((3aS,3a'S,4S,4'S,6aR,6a'R)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoylamido)ethoxy)ethyl)isophthalimide tris(2,2,2-trifluoroacetate), and the mixture was stirred at room temperature for 2 hours. After concentration, the mixture was purified by reverse phase silica gel column chromatography (0.1% aqueous TFA solution / MeOH=90 / 10→30 / 70) to obtain 327 mg of ((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)methyl (2-(2-(2-(3-(3,5-bis((2-(2-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)ethoxy)ethyl)carbamoyl)phenyl)ureido)ethoxy)ethoxy)ethyl)carbamate di(2,2,2-trifluoroacetate) (Compound (10)).

[0310]

[0311] 1 H NMR(400MHz,CD3OD)δ:8.00(d,J=1.2Hz,2H),7.88(t,J=1.6Hz,1H),4.71(dd,J=4.4,7.6Hz,2H),4.50(dd ,J=4.0,7.6Hz,2H),4.17(d,J=8.4Hz,2H),3.67-3.63(m,8H),3.62-3.51(m,12H),3.42(t,J=5.2Hz,2H),3 .38(t,J=5.2Hz,4H),3.29-3.27(m,2H),3.26-3.19(m,2H),2.98(dd,J=4.8,12.8Hz,2H),2.81(d,J=12.8H z,2H),2.26-2.10(m,10H),1.73-1.62(m,2H),1.61-1.46(m,8H),1.41-1.28(m,5H),0.90(t,J=9.6Hz,2H)

[0312] [Example 77]

[0313] Preparation of compound (10) combined with SCNP

[0314] 200 mg of N3-poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(acrylic acid)] and 30 mg of compound (10) obtained in Example 76 were weighed into a glass vial, 5 mL of DMF was added and dissolved, and stirred overnight at room temperature. The reaction solution was dialyzed with methanol, and after the solvent was distilled off, 195.8 mg of compound (10) bound to SCNP was recovered by vacuum drying. The Z average particle size and polydispersity index of compound (10) bound to SCNP were measured by dynamic light scattering (DLS), and the result was 8.3 nm (polydispersity index was 0.19).

[0315] [Measurement equipment and conditions, etc.]

[0316] (1) 1 H-NMR measurement

[0317] Device: JNM-ECX400 (400MHz) / JEOL

[0318] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0319] Sample concentration: 20mg / mL

[0320] Measurement temperature: 25℃

[0321] Cumulative times: 6000 times

[0322] result: Fig.10

[0323] (2) GPC measurement

[0324] Equipment: HPLC-Prominence system / Shimadzu Corporation

[0325] Detector: RID-10A refractive index detector / Shimadzu Corporation

[0326] Column: TSKgelα-2500 column / Tosoh

[0327] (column size: 7.8 mm × 300 mm, particle size: 7 μm, exclusion limit molecular weight: 5 × 10 3 )

[0328] TSKgelα-4000 column / Tosoh

[0329] (column size: 7.8 mm × 300 mm, particle size: 10 μm, exclusion limit molecular weight: 4 × 10 5 )

[0330] TSKgel Guard Column / Tosoh

[0331] Mobile phase: N,N-dimethylformamide (DMF) containing 10 mmol / L lithium bromide

[0332] Temperature: 40℃

[0333] Flow rate: 0.5mL / min

[0334] Sample concentration: 6mg / mL

[0335] Standard material: Poly(methyl methacrylate) Standard ReadyCal Kit, M p 800-2,200,000Da / SIGMA

[0336] result: Fig.11

[0337] (3) DLS measurement

[0338] Device: Zetasizer NanoZS / Malvern Instruments Ltd.

[0339] Measurement temperature: 25℃

[0340] Sample concentration: 10mg / mL

[0341] result: Fig.12

[0342] [Example 78]

[0343] Synthesis of dimethyl 6,6'-((5-(3-(1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10-trioxa-4-azadodec-12-yl)ureido)isophthaloyl)bis(ureadiyl))(2S,2'S)-bis(2-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoate)bis(2,2,2-trifluoroacetate)

[0344] To 165 mg of (R,S,S,2S,2'S)-dimethyl 6,6'-((5-(3-(2-(2-(2-aminoethoxy)ethoxy)ethyl)ureido)isophthaloyl)bis(azadiyl))bis(2-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoate)tri(2,2,2-trifluoroacetate) were added 10 mL of DMF, 160 μL of Et3N and 33.6 mg of BCN-NHS, and the mixture was stirred at room temperature for 2 hours. After concentration, the mixture was purified by reverse phase silica gel column chromatography (0.1% aqueous TFA solution / MeOH=90 / 10→30 / 70) to obtain 120 mg of dimethyl 6,6'-((5-(3-(1-((1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-yl)-3-oxo-2,7,10-trioxa-4-azadodec-12-yl)ureido)isophthaloyl)bis(ureadiyl))(2S,2'S)-bis(2-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoate)di(2,2,2-trifluoroacetate) (Compound (11)).

[0345]

[0346] 1 H NMR(400MHz,CD3OD)δ:7.95(d,J=1.6Hz,2H),7.81(t,J=1.6Hz,1H),4.72(dd,J=4.4,7.6Hz,2H),4.52(dd ,J=4.4,7.6Hz,2H),4.41(dd,J=4.8,9.6Hz,2H),4.11(d,J=7.6Hz,2H),3.71(s,6H),3.65(s,4H),3.60(t ,J=5.6Hz,2H),3.55(t,J=5.6Hz,2H),3.44-3.37(m,6H),3.29-3.25(m,4H),2.99(dd,J=4.8,12.8Hz,2H) ,2.81(d,J=13.2Hz,2H),2.28-2.10(m,10H),1.94-1.84(m,2H),1.79-1.32(m,25H),0.90(t,J=9.6Hz,2H)

[0347] [Example 79]

[0348] Preparation of compound (11) combined with SCNP

[0349] 200 mg of N3-poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(acrylic acid)] and 30 mg of compound (11) obtained in Example 78 were weighed into a glass vial, 5 mL of DMF was added and dissolved, and stirred overnight at room temperature. The reaction solution was dialyzed with methanol, and after the solvent was distilled off, 198.5 mg of compound (11) bound to SCNP was recovered by vacuum drying. The Z average particle size and polydispersity index of compound (11) bound to SCNP were measured by dynamic light scattering (DLS), and the result was 8.6 nm (polydispersity index was 0.25).

[0350] [Measurement equipment and conditions, etc.]

[0351] (1) 1 H-NMR measurement

[0352] Device: JNM-ECX400 (400MHz) / JEOL

[0353] Solvent: Dimethyl sulfoxide-d6 containing 0.03% tetramethylsilane / Kanto Chemical

[0354] Sample concentration: 20mg / mL

[0355] Measurement temperature: 25℃

[0356] Cumulative times: 6000 times

[0357] result: Fig.13

[0358] (2) GPC measurement

[0359] Equipment: HPLC-Prominence system / Shimadzu Corporation

[0360] Detector: RID-10A refractive index detector / Shimadzu Corporation

[0361] Column: TSKgelα-2500 column / Tosoh

[0362] (column size: 7.8 mm × 300 mm, particle size: 7 μm, exclusion limit molecular weight: 5 × 10 3 )

[0363] TSKgelα-4000 column / Tosoh

[0364] (column size: 7.8 mm × 300 mm, particle size: 10 μm, exclusion limit molecular weight: 4 × 10 5 )

[0365] TSKgel Guard Column / Tosoh

[0366] Mobile phase: N,N-dimethylformamide (DMF) containing 10 mmol / L lithium bromide

[0367] Temperature: 40℃

[0368] Flow rate: 0.5mL / min

[0369] Sample concentration: 6mg / mL

[0370] Standard material: Poly(methyl methacrylate) Standard ReadyCal Kit, M p 800-2,200,000Da / SIGMA

[0371] result: Fig.14

[0372] (3) DLS measurement

[0373] Device: Zetasizer NanoZS / Malvern Instruments Ltd.

[0374] Measurement temperature: 25℃

[0375] Sample concentration: 10mg / mL

[0376] result: Fig.15

[0377] [Comparative Example 1]

[0378] Preparation of oxaliplatin solution

[0379] 1 mL of Elplat intravenous injection 50 mg (Yakult, Ltd.) was added to 5.58 mL of 5.9 (w / v) % glucose solution to prepare a 5 (w / v) % glucose solution containing 760 μg of oxaliplatin.

[0380] [Test example] Drug efficacy test

[0381] The mouse colon cancer cell line C26 (American Type Culture Collection) was subcutaneously transplanted into female nude mice (BALB / c-nu nu / nu, 7 weeks old; Charles River Laboratories Japan, Inc.) to obtain a cancer-bearing model, which was used for drug efficacy testing. The subcultured mouse colon cancer cell line C26 was suspended in a liquid culture medium (Double's modified Eagle's medium-high glucose, Sigma-Aldrich) in a CO2 incubator to a cell number of 1×10 per mouse. 6After the nude mice were fed for about a week, the tumors grew to an average volume of about 30 mm. 3 The drug administration was started at 1:00 p.m. The SCNPs containing DACHPt (SCNPs containing DACHPt prepared using the copolymer of Example 70) were administered intravenously in the tail vein (3 times every other day), and the anti-tumor effect was evaluated based on the tumor volume (4 to 5 mice in 1 group). For comparison, an oxaliplatin solution (Comparative Example 1) was used and administered in the same manner. Regarding the dosage of each preparation, the maximum dose that can be administered for the oxaliplatin solution is 8 mg / kg (3.9 mg / kg in terms of Pt), and the SCNPs containing DACHPt are 3 mg / kg in terms of Pt.

[0382] The changes in tumor volume over time are shown in Fig.16 . In the case of SCNPs containing DACHPt, T / C = 0.4 [T / C: ratio of tumor volume of drug administration group (T) to control group (C)] 14 days after administration. In the case of oxaliplatin solution (Comparative Example 1), T / C = 1.1 14 days after administration. In addition, it was confirmed that SCNPs containing DACHPt significantly inhibited tumor growth compared with the control group 14 days after administration (Student t test). The above results show that SCNPs containing DACHPt have excellent anti-tumor effects compared with oxaliplatin solution.

Claims

1. A copolymer obtained by bonding a targeting affinity molecule to a copolymer X comprising structural units represented by the following formulae (A), (B) and (C), wherein: In the formula, R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 An alkyl group; m represents an integer of 1 to 100; and n represents an integer of 0 to 3.

2. The copolymer according to claim 1, characterized in that: The polymer X is a copolymer formed by polymerization of three monomers represented by the following general formulas (1) to (3), In the formula, R 1 , R 2 and R 3 Same or different, indicating hydrogen atoms or C 1-3 Alkyl; R 4 Represents C 1-3 Alkyl; R 5 represents hydrogen atom, C 1-18 alkyl, 3- to 8-membered cycloalkyl which may have a substituent, adamantyl, C 6-18 Aryl or 5- to 10-membered heteroaryl which may have a substituent; X 1 , X 2 and X 3 The same or different, representing oxygen atoms, sulfur atoms or NR 7 ; R 6 represents a hydrogen atom, a leaving group or a connecting group; R 7 Represents a hydrogen atom or C 1-3 An alkyl group; m represents an integer of 1 to 100; and n represents an integer of 0 to 3.

3. The copolymer according to claim 1 or 2, characterized in that: R 1 A hydrogen atom.

4. The copolymer according to claim 1 or 2, characterized in that: R 2 A hydrogen atom.

5. The copolymer according to claim 1 or 2, characterized in that: R 3 A hydrogen atom.

6. The copolymer according to claim 1 or 2, characterized in that: R 4 It is methyl.

7. The copolymer according to claim 1 or 2, characterized in that: R 5 is a C which may have a substituent 6-18 Aryl.

8. The copolymer according to claim 1 or 2, characterized in that: R 5 It is phenyl.

9. The copolymer according to claim 1 or 2, characterized in that: R 6 A hydrogen atom.

10. The copolymer according to claim 1 or 2, characterized in that: R 6 The leaving group is a group represented by the following formula (4):

11. The copolymer according to claim 1 or 2, characterized in that: R 6 The connecting group is a group selected from the following formulas (5) to (7):

12. The copolymer according to claim 1 or 2, characterized in that: X 1 For oxygen atoms.

13. The copolymer according to claim 1 or 2, characterized in that: X 2 For oxygen atoms.

14. The copolymer according to claim 1 or 2, characterized in that: X 3 For oxygen atoms.

15. The copolymer according to claim 1 or 2, characterized in that: m is an integer from 4 to 22.

16. The copolymer according to claim 1 or 2, characterized in that: n is 1.

17. The copolymer according to claim 1 or 2, characterized in that: The ratio of the structural units (A), (B), and (C) is 0.01 to 100 parts by mass of (B) and 0.1 to 100 parts by mass of (C) per 1 part by mass of (A).

18. The copolymer according to claim 2, characterized in that: The monomer (2) is prepared by polymerizing 0.01 to 100 parts by mass of the monomer (2) and 0.1 to 100 parts by mass of the monomer (3) with respect to 1 part by mass of the monomer (1).

19. The copolymer according to claim 1 or 2, characterized in that: The number average molecular weight is 5000 to 150000.

20. The copolymer according to claim 1 or 2, characterized in that: The targeting affinity molecule is biotin or its derivatives.

21. The copolymer according to claim 1 or 2, characterized in that: The targeting affinity molecule is a molecule having a biotin residue as shown in the following formula (a): In the formula, Y represents O or NH.

22. The copolymer according to claim 1 or 2, characterized in that: The targeting affinity molecule is any one of the following molecules represented by formula (8) to (11):

23. The copolymer according to claim 1 or 2, characterized in that: The binding of the targeting affinity molecule to the copolymer X is covalent binding or non-covalent binding.

24. A single-chain nanoparticle, characterized in that: The copolymer comprising claim 1 or 2.

25. A pharmaceutical composition, characterized in that: The copolymer comprising claim 1 or 2.

26. The pharmaceutical composition according to claim 25, wherein: It is used in pretargeting methods.

Images