Polymeric micelle comprising single polymer chain of self-aggregating type

Abstract

The present invention addresses the problem that many conventional nano-sized contrast agents are aggregates of a plurality of constituent molecules and, under diluted conditions resulting from prolonged blood circulation, dissociate by reaching a critical micelle concentration (CMC) or critical aggregate concentration (CAC) or lower, such that it is difficult to control in vivo kinetics thereof up to the discharge phase. The present invention provides a polymeric micelle comprising a single polymer chain of a self-aggregating type, said single polymer chain containing one or more gadolinium complexes and having a particle size of not more than 10 nm in water.

Description

Polymeric micelles composed of self-aggregating single polymer chains

[0001] The present invention relates to novel polymeric micelles, specifically self-aggregated polymeric micelles formed by folding of a single polymer chain, and their use and production in magnetic resonance imaging and radiotherapy such as neutron capture therapy. Furthermore, the polymeric micelles of the present invention can maintain high longitudinal relaxivity and positive effects of gadolinium (Gd) complexes during magnetic resonance imaging due to the self-aggregation of the single polymer chain, and enable tumor treatment by radiotherapy such as neutron capture therapy.

[0002] Magnetic resonance imaging (MRI) is an important diagnostic method for tumor imaging, enabling noninvasive assessment of tumors in patients while providing visual contrast [Non-Patent Documents 1 and 2]. Contrast agents (CAs), such as paramagnetic metal complexes, are generally useful for controlling the longitudinal (T1) or transverse (T2) nuclear relaxation rates (relaxation times) of protons in water molecules. Polymers bound to such metal complexes are known to enhance molecular relaxivity by promoting an increase in rotational correlation time [Non-Patent Documents 3 and 4]. While the additivity of relaxivity changes upon incorporation of complexes into polymers and the resulting relaxivity have not generally been established in current molecular design, the incorporation of contrast agents into polymers is known as a versatile method for increasing image contrast [Non-Patent Document 5].

[0003] In systemic drug delivery systems (DDS), nanosized DDS carriers are known to exhibit stealth function, resulting in long-term blood circulation and selective accumulation in solid tumors [Non-Patent Documents 6 and 7]. Furthermore, many DDS carriers, such as liposomes, polymeric micelles, and nanoparticles, have been considered effective vehicles for delivering contrast agents to cancer [Non-Patent Documents 8-10]. Because the signal-to-noise ratio (S / N) in MRI depends on the concentration balance between blood and target tissue and the contrast ratio between the lesion and normal tissue, increasing the S / N ratio is also crucial when using nanosized contrast agents (NCAs) [Non-Patent Document 11]. Therefore, when administering NCAs, controlling their blood clearance and accumulation in target tissues affects the accuracy of MRI diagnostics. In addition, the contrast ratio between the lesion and normal tissue upon accumulation in target tissue, i.e., the positive effect, also affects diagnostic accuracy. However, most of the nano-sized contrast agents known to date are aggregates of multiple structural molecules, and under dilution conditions caused by prolonged blood circulation, they dissociate below their critical micelle concentration (CMC) or critical aggregation concentration (CAC) [12 and 13], making it difficult to control their pharmacokinetics up to the excretion stage. In addition, many nano-sized contrast agents suffer from negative effects such as increased hydrophobicity and T2 shortening in vivo due to disturbances in the local magnetic field, resulting in a decrease in the contrast ratio between lesions and normal tissue.

[0004] Stabile, A. et al., Nat. Rev. Urol., 17, 41-61 (2020)Sclafani, F. et al., Br. J. Cancer, 117, 1478-1485 (2017)Wahsner, J. et al., Chem. Rev., 119, 957-1057 (2018)Jeon, M. et al. al., Adv. Mater., 33, 1906539 (2021)Tang, J. et al., Prog. Polym. Sci., 38, 462-502 (2013)Wang, AZ et al., Annu. Rev. Med., 63, 185-198 (2012)Cheng, CJ & Saltzman, WM, Nat. Nanotechnol., 7, 346-347 (2012) Kanasty, R. et al., Nat. Mater., 12, 967-977 (2013) Nishiyama, N. et al., Cancer Sci., 107, 867-874 (2016) Cho, K. et al., Clin. Cancer Res., 14, 1310-1316 (2008)Versilst, P. et al., Chem. Soc. Rev., 44, 1791-1806 (2015)Cabral, H. et al., Nat. Nanotechnol., 6, 815-823 (2011)Lu, Y. et al., Nat. Biomed. Eng., 2, 318-325 (2018)

[0005] To date, no molecular design has been reported that can improve relaxivity and further enhance positive effects by incorporating a contrast agent into a polymer. Many conventional nano-sized contrast agents are aggregates of multiple structural molecules, and under dilution conditions caused by prolonged blood circulation, they dissociate when they reach or fall below their critical micelle concentration (CMC) or critical aggregation concentration (CAC), making it difficult to control their pharmacokinetics up to the excretion stage. The present invention aims to solve this problem.

[0006] Definitions Unless otherwise specified, the terms used in this specification have the same meanings as technical and scientific terms commonly used by those skilled in the art. The terms defined below have meanings that are more appropriate for specifying the present invention when used in this specification.

[0007] As used herein, the term "single-chain polymer" refers to a polymer consisting of a single chain, which is a polymer in which different types of monomer segments are linked, and is used synonymously with and interchangeable with "single polymer." According to the present invention, the polymer of the present invention can spontaneously fold in water to form micelles with a particle size of 10 nm or less.

[0008] As used herein, the term "association number" refers to the number of single polymer chains according to the present invention that form one micelle in water. In this specification, the term "association number" can be used synonymously with the term "aggregation number." Furthermore, according to the present invention, the DA (degree of association) value, which indicates the degree of molecular association, can be used as the association number of single polymer chains according to the present invention. The association number can be calculated, for example, by the following formula: N ａｇｇ = M ｗ，ＭＡＬＳ (PBS) / M ｗ，ＭＡＬＳ It can be calculated using the following formula (chloroform). DA = 1.0 indicates ideal intramolecular association, i.e., SMDC formation, but in the present invention, DA < 1.5 is defined as SMDC. A high DA (DA ≥ 1.5) indicates multiple intermolecular association. A low DA (DA < 1.0) also indicates SMDC formation in water, but these form reverse micelle-type SMDC in organic solvents.

[0009] In order to solve the above problems, the present inventors have conducted extensive research and have found that a polymer having a predetermined structure can achieve high longitudinal relaxivity by restricting the molecular motion of a Gd complex during magnetic resonance imaging through its self-aggregation, while at the same time maintaining hydrophilicity during self-aggregation and avoiding T2 shortening, thereby maintaining positive effects, and enabling the treatment of tumors by neutron capture therapy, thereby completing the present invention.

[0010] That is, aspects of the present invention include, but are not limited to, the following: [1] A polymeric micelle comprising a self-aggregating single-chain polymer, which contains one to multiple gadolinium complexes and spontaneously folds in water to form micelles with a particle size of 10 nm or less. [2] A polymeric micelle comprising a self-aggregating single-chain polymer according to [1], in which (A) a side chain comprising polyethylene glycol (PEG) or a PEG derivative; (B) a side chain comprising a hydrophobic group; and (C) a side chain comprising a gadolinium complex are bonded to the main backbone via a linker or directly. [3] A polymeric micelle comprising a self-aggregating single-chain polymer according to [2], in which (D) a side chain comprising a carboxylic acid group is bonded to the main backbone via a linker or directly. [4] The PEG or PEG derivative in the side chain (A) has the following structure:

[0011]

[0012] (In the formula, R ７ is a methoxy group, an ethoxy group, an acetyl group, or a benzyl group; R ８ is an amide bond, an ether bond, or an ester bond, which is a moiety that is bonded to the main skeleton; R ９is a biodegradable linker; and n is 1 to 10. [5] A polymeric micelle comprising a self-aggregating single-chain polymer according to any one of [2] to [4], wherein the hydrophobicity (clog P value) of the hydrophobic group in the side chain (B) is 1 to 10. [6] A polymeric micelle comprising a self-aggregating single-chain polymer according to any one of [2] to [5], wherein the hydrophobic group in the side chain (B) is selected from the group consisting of a phenyl group, a naphthyl group, a pyrenyl group, anthracene, cholesterol, a steroid derivative, 1-pyrenemethylamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-naphthylmethylamine, DL-α-tocopherol, nerol, farnesol, and an anticancer drug. [7] The polymeric micelle comprising a self-aggregating single polymer chain according to any one of [1] to [6], wherein the gadolinium complex is at least one selected from the group consisting of gadoteridol, meglumine gadoterate, and gadobutrol.[8] When the numbers of repeating units of the side chains (A) to (D) in a single polymer chain are a, b, c, and d, respectively, the following conditions are met: (I) When the clog P value is less than 1.5 and the total degree of polymerization (DP) of a+b+c+d is 100 or more and 500 or less, a has 50% or more of the total DP; b has 40% or less of the total DP; c has 10% or less of the total DP; and d has 10% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP; (II) When the clog P value is 1.5 or more and less than 4.5 and the total degree of polymerization (DP) of a+b+c+d is less than 200, a has 50% or more of the total DP; b has 30% or less of the total DP; c has 5% or less of the total DP; and (III) When the clogP value is 1.5 or greater but less than 4.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or greater but less than 500, then a has 50% or greater of the total DP; b has 40% or less of the total DP; c has 5% or less of the total DP; and d has 15% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP; (IV) When the clogP value is 4.5 or greater but less than 5.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or greater but less than 500, then a has 50% or greater of the total DP; and b has 30% or less of the total DP; (V) When the clogP value is 5.5 or more and the total degree of polymerization (DP) of a+b+c+d is 200 or more and 500 or less, a has 60% or more of the total DP; b has 20% or less of the total DP; c has 2% or less of the total DP; and d has 8% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦10% of the total DP. [9] M. ｗ，ＭＡＬＳ (PBS) / M ｗ，ＭＡＬＳThe polymeric micelle comprising the self-aggregating single polymer chain according to any one of [1] to [8], wherein the number of associations of the single polymer chain in water or a buffer solution is less than 1.5 when calculated from the (chloroform) ratio.

[10] A polymeric micelle comprising a self-aggregating single polymer chain according to any one of [1] to [9], wherein the main chain in the single polymer chain is selected from the group consisting of polyether, polyester, polyalkylene, polycarbonate, poly(dioxanone), polyacid anhydride, polyhydroxy acid, polyfumarate, polycaprolactone, polyamide, polyacetal, poly(orthoester), polyhydroxybutyrate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, poly(arylate), polycarbonate, poly(propylene fumarate), polyhydroxyalkanoate, polyketal, polyesteramide, polyhydroxyvalerate, polyorthocarbonate, poly(vinylpyrrolidone), polyalkylene oxalate, polyalkylene succinate, polylactic acid, poly(malic acid), poly(methyl vinyl ether), and poly(maleic anhydride).

[11] A compound having the following structure, which contains one or more gadolinium complexes and spontaneously folds in water to form micelles with a particle size of 10 nm or less:

[0013]

[0014] or

[0015]

[0016] (In the formula, R １ and R ６ are independently a methoxy group, an ethoxy group, an acetyl group, or a benzyl group; R ２ is PEG or a PEG derivative; R ３ is a hydrophobic group; R ４ is a gadolinium complex; R ５ is a carboxylic acid group; L １ ~L ６are each independently absent or a linker; a, b, c, and d satisfy any of the following conditions (I) to (V): (I) when the clogP value is less than 1.5 and the total degree of polymerization (DP) of a+b+c+d is 100 or greater and 500 or less, a has 50% or greater of the total DP; b has 40% or less of the total DP; c has 10% or less of the total DP; and d is 10% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP; (II) when the clogP value is 1.5 or greater and less than 4.5 and the total degree of polymerization (DP) of a+b+c+d is less than 200, a has 50% or greater of the total DP; b has 30% or less of the total DP; c has 5% or less of the total DP; and (III) When the clogP value is 1.5 or greater but less than 4.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or greater but less than 500, then a has 50% or greater of the total DP; b has 40% or less of the total DP; c has 5% or less of the total DP; and d has 15% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP; (IV) When the clogP value is 4.5 or greater but less than 5.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or greater but less than 500, then a has 50% or greater of the total DP; and b has 30% or less of the total DP; (V) When the clogP value is 5.5 or more and the total degree of polymerization (DP) of a+b+c+d is 200 or more and 500 or less, a has 60% or more of the total DP; b has 20% or less of the total DP; c has 2% or less of the total DP; and d has 8% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦10% of the total DP.

[12] PEG or PEG derivatives include, but are not limited to, polymeric micelles consisting of a self-aggregating single polymer chain having the following structure:

[0017]

[0018] (In the formula, R ７is a methoxy group, an ethoxy group, an acetyl group, or a benzyl group; R ８ is an amide bond, an ether bond, or an ester bond, which is a moiety that is bonded to the main skeleton; R ９ is a biodegradable linker; and n is 1 to 10.

[13] The self-aggregating single-chain polymer micelle according to

[11] or

[12] , wherein the hydrophobicity (clogP value) of the hydrophobic group in the side chain (B) is 1 to 10.

[14] The self-aggregating single-chain polymer micelle according to any one of

[11] to

[13] , wherein the hydrophobic group is selected from the group consisting of a phenyl group, a naphthyl group, a pyrenyl group, anthracene, cholesterol, a steroid derivative, 1-pyrenemethylamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-naphthylmethylamine, DL-α-tocopherol, nerol, farnesol, and an anticancer drug.

[15] The polymeric micelle comprising the self-aggregating single polymer chain according to any one of

[11] to

[14] , wherein the gadolinium complex is at least one selected from the group consisting of gadoteridol, gadoterate meglumine, and gadobutrol.

[16] M ｗ，ＭＡＬＳ (PBS) / M ｗ，ＭＡＬＳ

[17] A polymeric micelle comprising a self-aggregating single polymer chain according to any one of

[11] to

[15] , wherein the number of associations of the single polymer chain in water or a buffer solution is less than 1.5 when calculated from the (chloroform) ratio.

[18] When used in magnetic resonance imaging (MRI) for detecting or diagnosing cancer or tumors, the longitudinal relaxivity (r1) / transverse relaxivity (r2) ratio is maintained at r1 of 20 s or less while maintaining r1 / r2 of 0.70 or more. －１ mM －１

[18] A pharmaceutical composition for treating cancer or tumors by neutron capture therapy, comprising the polymeric micelle comprising the self-aggregating single polymer chain according to any one of [1] to

[16] .

[0019] The high longitudinal relaxivity of the Gd complexes can simultaneously maintain positive effects during magnetic resonance imaging, and allow tumor treatment with neutron capture therapy.

[0020] The outline of the formation of a polymeric drug carrier (Single-Molecular Drug-Carrier; SMDC) is shown. The boundary of monomolecular association of poly-2a in aqueous solution is shown. ａｇｇ ≦1.0, black circle; 1.0<N ａｇｇ ≦1.5, open circle; 1.5<N ａｇｇ ≦2.0, black square; 2.0<N ａｇｇ ≦5.0, half black and white square; 5.0<N ａｇｇ , white square; x, N ａｇｇ The dotted line indicates the threshold between intermolecular and intramolecular self-assembly. It shows the boundary of monomolecular association of poly-2b in aqueous solution. N ａｇｇ ≦1.0, black circle; 1.0<N ａｇｇ ≦1.5, open circle; 1.5<N ａｇｇ ≦2.0, black square; 2.0<N ａｇｇ ≦5.0, half black and white square; 5.0<N ａｇｇ , white square; x, N ａｇｇ The dotted line indicates the threshold between intermolecular and intramolecular self-assembly. It shows the boundary of monomolecular association of poly-2c in aqueous solution. N ａｇｇ ≦1.0, black circle; 1.0<N ａｇｇ ≦1.5, open circle; 1.5<N ａｇｇ ≦2.0, black square; 2.0<N ａｇｇ ≦5.0, half black and white square; 5.0<N ａｇｇ , white square; x, N ａｇｇ The dotted line indicates the threshold between intermolecular and intramolecular self-assembly. It shows the boundary of monomolecular association of poly-2d in aqueous solution. N ａｇｇ ≦1.0, black circle; 1.0<N ａｇｇ ≦1.5, open circle; 1.5<N ａｇｇ ≦2.0, black square; 2.0<N ａｇｇ ≦5.0, half black and white square; 5.0<N ａｇｇ , white square; x, N ａｇｇThe dotted line indicates the threshold between intermolecular and intramolecular self-assembly. It shows the boundary of monomolecular association of poly-2e in aqueous solution. N ａｇｇ ≦1.0, black circle; 1.0<N ａｇｇ ≦1.5, open circle; 1.5<N ａｇｇ ≦2.0, black square; 2.0<N ａｇｇ ≦5.0, half black and white square; 5.0<N ａｇｇ , white square; x, N ａｇｇ The dotted line indicates the threshold between intermolecular and intramolecular self-assembly. It shows the boundary of monomolecular association of poly-2f in aqueous solution. N ａｇｇ ≦1.0, black circle; 1.0<N ａｇｇ ≦1.5, open circle; 1.5<N ａｇｇ ≦2.0, black square; 2.0<N ａｇｇ ≦5.0, half black and white square; 5.0<N ａｇｇ , white square; x, N ａｇｇ The dotted line indicates the threshold between intermolecular and intramolecular self-assembly. It shows the boundary of monomolecular association in aqueous solution of poly-2g. ａｇｇ ≦1.0, black circle; 1.0<N ａｇｇ ≦1.5, open circle; 1.5<N ａｇｇ ≦2.0, black square; 2.0<N ａｇｇ ≦5.0, half black and white square; 5.0<N ａｇｇ , white square; x, N ａｇｇ The SEC chromatogram of TP1 is shown. The NMR spectrum of TP4 is shown. A diagram defining the boundary conditions for unimolecular association of universal poly-2 in aqueous solution is shown. N ａｇｇ ≦1.0, black ball; 1.0<N ａｇｇ ≦1.5, gray sphere; 1.5<N ａｇｇ ≦2.0, black cube; 2.0<N ａｇｇ ≦5.0, dark gray cube; 5.0<N ａｇｇ, light gray cube. The curved surface indicates the threshold between intermolecular and intramolecular self-assembly. Kratky plot in aqueous solution. Kratky plot in DMF. SMDC-Gd ４ The scale bar indicates 100 nm. ４ TEM images of SMDC-Gd, scale bar indicates 100 nm. An example of particle size analysis by DLS is shown. Zeta potential measurement is shown. Comparison of blood retention is shown. Comparison of tumor accumulation is shown. Comparison of kidney accumulation is shown. Comparison of liver accumulation is shown. Comparison of spleen accumulation is shown. Comparison of brain accumulation is shown. Comparison of muscle accumulation is shown. Comparison of pancreas accumulation is shown. Cytotoxicity evaluation is shown. Changes in WBC after administration of SMDC-Gd are shown. Changes in RBC after administration of SMDC-Gd are shown. Changes in HGB after administration of SMDC-Gd are shown. Changes in HCT after administration of SMDC-Gd are shown. Changes in PLT after administration of SMDC-Gd are shown. Changes in BUN after administration of SMDC-Gd are shown. Changes in CRE after administration of SMDC-Gd are shown. Changes in GPT after administration of SMDC-Gd are shown. Changes in GOT after administration of SMDC-Gd are shown. This shows the change in ALP after administration of SMDC-Gd. This shows the change in TP after administration of SMDC-Gd. This shows the time-dependent change in quantitative mapping images of longitudinal relaxivity (Gd conc. 0.1 mmol / kg). This shows the time-dependent change in tumor / muscle ratio of longitudinal relaxivity (Gd conc. 0.1 mmol / kg). This shows the time-dependent change in longitudinal relaxivity of tumor (Gd conc. 0.1 mmol / kg). This shows the quantitative mapping images of longitudinal relaxivity 24 hours after administration. This shows the time-dependent change in longitudinal relaxivity of tumor 24 hours after administration. T １ Signal changes in the bladder and kidneys are shown in the weighted image. １ The graph shows the time course of MR signal changes in the kidney in weighted images. １ The graph shows the time course of MR signal changes in the bladder in weighted images. ４ Comparison of organ distribution between three doses and a single dose. Comparison of organ distribution between three doses and a single dose of Gd-DOTA. Antitumor effect of Gd-NCT. Change in body weight after Gd-NCT.

[0021] The present invention provides a self-aggregating polymeric micelle containing a drug or a gadolinium complex. This polymeric micelle is characterized by the folding of a single polymer chain in water, and can be used as an MRI contrast agent and a gadolinium formulation for neutron capture therapy. The present invention provides a polymeric micelle made of a single polymer chain whose relaxivity is improved by the folding of the single polymer chain. The present application will be described in detail below.

[0022] The polymeric micelles of the present invention are random copolymers that have hydrophilic and hydrophobic groups in their side chains that act as the driving force for folding, and to which gadolinium complexes, which function as MRI contrast agents and neutron capture therapy agents, are chemically bonded to the side chains. Due to their self-folding ability in water, these polymers become polymeric drug carriers (Single-Molecular Drug-Carriers; SMDCs) with particle sizes of 10 nm or less (see Figure 1).

[0023] In water, the polymeric micelles fold into a structure with a shell made of hydrophilic side chains and a core made of hydrophobic side chains, encapsulating the gadolinium complex. This folding allows the gadolinium complex to exist in a more crowded environment than when simply incorporated into a comb-shaped polymer. As a result, the polymeric micelles increase the rotational correlation time of the gadolinium complex and function as polymeric micelles that improve relaxivity.

[0024] These polymeric micelles exhibit behavior similar to the enhanced permeability and retention (EPR) effect, enabling tumor-selective drug delivery. Furthermore, because their size can be precisely controlled to less than 10 nm, their ex vivo clearance rate, such as renal excretion, can be adjusted. This improves tumor diagnosis, therapeutic efficacy, and therapeutic benefits. In particular, when used as a diagnostic agent, such as a nanocontrast agent, they can reduce background signals from target organs and blood, enabling the acquisition of data with a favorable signal-to-noise ratio.

[0025] The polymeric micelle of the present invention will be specifically described below. The polymeric micelle of the present invention contains one to a plurality of (e.g., 1 to 10) gadolinium complexes, and spontaneously forms particles of 10 nm or less in size in water by folding a single polymer chain.

[0026] In one embodiment, the polymer of the present invention is a self-assembling micelle having the following attached to the main backbone via a linker or directly: (A) a side chain comprising polyethylene glycol (PEG) or a PEG derivative; (B) a side chain comprising a hydrophobic group; (C) a side chain comprising a gadolinium complex; and (D) a side chain comprising a carboxylic acid group.

[0027] The side chain (A) is a moiety having PEG or a PEG derivative that is located on the outer shell of the particle when a polymeric micelle is formed and is expected to have biocompatibility and inhibit interaction with blood proteins, etc., and the PEG or PEG derivative has the following structure:

[0028]

[0029] (In the formula, R ７ is a methoxy group, an ethoxy group, an acetyl group, or a benzyl group; R ８ is an amide bond, an ether bond, or an ester bond, which is a moiety that is bonded to the main skeleton; R ９ is a biodegradable linker; and n is 1 to 10.

[0030] The side chain (B) is a moiety having a hydrophobic group that serves as a driving force for folding in water. The hydrophobicity (clogP value) of the hydrophobic group in the side chain (B) is 1 to 10. Examples of the hydrophobic group include, but are not limited to, a phenyl group, a naphthyl group, a pyrenyl group, anthracene, cholesterol, a steroid derivative, 1-pyrenemethylamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-naphthylmethylamine, DL-α-tocopherol, nerol, farnesol, and an anticancer drug.

[0031] The side chain (C) is a moiety having a gadolinium complex that contributes to MR imaging or neutron capture therapy. Examples of gadolinium complexes include gadoteridol, gadoterate meglumine, and gadobutrol.

[0032] The side chain (D) is composed of a moiety having a carboxylic acid for making the overall charge negative when a polymer micelle is formed.

[0033] The polymer of the present invention is characterized by being controlled by the number of side chains (A), the hydrophobicity (clogP value) of the side chains (B), and the total number of repeating molecules of the side chains (A) to (D), and is a composition in which the association number in water or a buffer solution is less than 1.5.

[0034] Here, when the numbers of repeating units of the side chains (A) to (D) in a single polymer chain are a, b, c, and d, respectively, it is preferable that any one of the following conditions (I) to (V) be satisfied:

[0035] (I) When the clogP value is less than 1.5 and the total degree of polymerization (DP) of a + b + c + d is 100 or greater and 500 or less, then: a comprises 50% or greater of the total DP; b comprises 40% or less of the total DP; c comprises 10% or less of the total DP; and d comprises 10% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP.

[0036] (II) When the clogP value is 1.5 or greater but less than 4.5, and the total degree of polymerization (DP) of a + b + c + d is less than 200, then: a comprises 50% or greater of the total DP; b comprises 30% or less of the total DP; c comprises 5% or less of the total DP; and d comprises 15% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP.

[0037] (III) When the clogP value is 1.5 or greater but less than 4.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or greater but less than 500, then: a comprises 50% or greater of the total DP; b comprises 40% or less of the total DP; c comprises 5% or less of the total DP; and d comprises 15% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP.

[0038] (IV) When the clogP value is 4.5 or greater but less than 5.5, and the total degree of polymerization (DP) of a + b + c + d is 200 or greater but less than 500, then a comprises 50% or greater of the total DP; b comprises 30% or less of the total DP; c comprises 5% or less of the total DP; and d comprises 15% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP.

[0039] (V) When the clogP value is 5.5 or greater and the total degree of polymerization (DP) of a + b + c + d is 200 or greater but 500 or less, a has 60% or greater of the total DP; b has 20% or less of the total DP; c has 2% or less of the total DP; and d has 8% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦10% of the total DP.

[0040] The polymeric micelle specified by any of the above has an association number of single polymer chains of less than 1.5 in water or a buffer solution.

[0041] The polymeric micelle of the present invention is characterized by side chains (A) to (D) bonded to the main skeleton via a linker or directly. The main skeleton (main chain) is not particularly limited, and examples thereof include polyether, polyester, polyalkylene (e.g., polyethylene), polycarbonate, poly(dioxanone), polyacid anhydride, polyhydroxy acid, polyfumarate, polycaprolactone, polyamide, polyacetal, poly(orthoester), polyhydroxybutyrate, polyvinyl alcohol, polyurethane, and polyphosphazene. Examples of the polymer include olefins, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, poly(arylates), polycarbonates, poly(propylene fumarates), polyhydroxyalkanoates, polyketals, polyesteramides, polyhydroxyvalerates, polyorthocarbonates, poly(vinylpyrrolidone), polyalkylene oxalates, polyalkylene succinates, polylactic acids, poly(malic acids), poly(methyl vinyl ether), and poly(maleic anhydride). Note that the polymer of the present invention specified in the above embodiment may be a single molecule of a random copolymer.

[0042] In another embodiment, the polymer of the present invention is a polymeric micelle containing a gadolinium complex (optionally a pharmaceutical agent) consisting of one molecule of a random copolymer, and the main chain of the random copolymer is preferably, but not limited to, a highly flexible main chain skeleton such as a hydrocarbon or polyether skeleton having no unsaturated bond, as shown in the following formula (1) or formula (2):

[0043]

[0044]

[0045] R １ and R ６ are independently a methoxy group, an ethoxy group, an acetyl group, or a benzyl group.

[0046] R ２ is a moiety having PEG or a PEG derivative that is located on the outer shell of the particle when a polymeric micelle is formed and is expected to have biocompatibility and inhibit interactions with blood proteins, etc. For example, the PEG or PEG derivative may have, but is not limited to, the following structure:

[0047]

[0048] (In the formula, R ７ is a methoxy group, an ethoxy group, an acetyl group, or a benzyl group; R ８ is an amide bond, an ether bond, or an ester bond, which is a moiety that is bonded to the main skeleton; R ９ is a biodegradable linker; and n is 1 to 10.

[0049] R ３ is a portion having a hydrophobic group that acts as a driving force for folding in water. ４ is a moiety having a gadolinium complex that contributes to MR imaging or neutron capture therapy.

[0050] R ５ is composed of a moiety having a carboxylic acid that makes the overall charge negative when a polymeric micelle is formed.

[0051] The polymer of the present invention is R ２ The number of and R ３The hydrophobicity (clogP value) of R ２ ~R ５ and the association number in water or a buffer solution is less than 1.5.

[0052] In one embodiment, the polymer of the present invention comprises R ３ When the clogP value of a + b + c + d is less than 1.5 and the total degree of polymerization (DP) of a + b + c + d is 100 or greater but 500 or less, then: a has 50% or greater of the total DP; b has 40% or less of the total DP; c has 10% or less of the total DP; and d is 10% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP.

[0053] In one embodiment, the polymer of the present invention comprises R ３ is greater than or equal to 1.5 and less than 4.5, and the total DP of a+b+c+d is less than 200: a has 50% or more of the total DP; b has 30% or less of the total DP; c has 5% or less of the total DP; and d has 15% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP.

[0054] In one embodiment, the polymer of the present invention comprises R ３ is greater than or equal to 1.5 and less than 4.5, and the total DP of a+b+c+d is greater than or equal to 200 and less than or equal to 500, then: a has 50% or more of the total DP; b has 40% or less of the total DP; c has 5% or less of the total DP; and d is 15% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP.

[0055] In one embodiment, the polymer of the present invention comprises R ３ is 4.5 or more and less than 5.5, and the total DP of a + b + c + d is 200 or more and 500 or less: a has 50% or more of the total DP; b has 30% or less of the total DP; c has 5% or less of the total DP; and d has 15% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP.

[0056] In one embodiment, the polymer of the present invention comprises R ３is 5.5 or more, and the total DP of a + b + c + d is 200 or more and 500 or less, then a has 60% or more of the total DP; b has less than 20% of the total DP; c has less than 2% of the total DP; and d is less than 8% of the total DP, with the proviso that c and d satisfy 1%≦c+d≦10% of the total DP.

[0057] In any of the above embodiments, L １ ~L ６ are each independently absent or a linker. Examples of linkers include, but are not limited to, preferably cleavable linkers or biodegradable linkers.

[0058] In any of the above embodiments, when the polymeric micelle of the present invention is used in magnetic resonance imaging (MRI) for detecting or diagnosing cancer or tumors, the longitudinal relaxivity (r1) / transverse relaxivity (r2) ratio is maintained at r1 of 20 s or less while maintaining r1 / r2 of 0.70 or more. －１ mM －１ That's all.

[0059] According to the present invention, the polymeric micelles of the present invention are characterized by having a gadolinium complex chemically bound to the molecule, and the presence of a corresponding drug enables the diagnosis and / or treatment of cancers or tumors, including, but not limited to, neuroblastoma, liver cancer, malignant melanoma, uterine cancer, bladder cancer, biliary tract cancer, esophageal cancer, osteosarcoma, testicular tumors, thyroid cancer, acute myeloid leukemia, brain tumors, prostate cancer, pancreatic cancer, head and neck squamous cell carcinoma, mesothelioma, lung cancer, colorectal cancer, renal cancer, ovarian cancer, and breast cancer.

[0060] According to the present invention, there is provided a pharmaceutical composition for treating cancer or tumors by neutron capture therapy, which comprises the polymeric micelle of the present invention.

[0061] <Contrast Agent> The polymeric micelle according to the present invention can contain at least one gadolinium contrast agent shown in Table 1. Two or more gadolinium contrast agents may be used in combination, but preferably only one is contained.

[0062]

[0063]

[0064] The polymeric micelle containing the gadolinium contrast agent of the present invention preferably has the following structure:

[0065]

[0066] It has.

[0067] The polymeric micelles containing the gadolinium contrast agent according to the present invention may contain salts derived from inorganic or organic acids and bases, such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, and 2-naphthalenesulfonate. Examples of suitable salts include salts with nicotinates, oxalates, pamoates, pectinates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, succinates, tartrates, thiocyanates, tosylates, undecanoates, ammonium salts, alkali metal salts (e.g., sodium and potassium salts), alkaline earth metal salts (e.g., calcium, magnesium, and zinc salts), salts with organic bases (e.g., dicyclohexylamine salts, N-methyl-D-glucamine), and salts with amino acids (e.g., arginine, lysine).

[0068] The polymeric micelles containing the gadolinium contrast agent of the present invention can contain any carrier, adjuvant, or vehicle, including, but not limited to, ion exchange materials, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances (e.g., phosphates), glycine, sorbic acid, potassium sorbate, TRIS (tris(hydroxymethyl)aminomethane), a partial glyceride mixture of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0069] The polymeric micelles containing the gadolinium contrast agent according to the present invention may be in the form of a sterile injectable preparation (e.g., a sterile injectable aqueous or oleaginous suspension). Such suspensions may be formulated using suitable dispersing and / or wetting agents and / or suspending agents according to techniques known in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent (e.g., as a solution in 1,3-butanediol). Acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic saline. Additionally, sterile, fixed oils are employed as solvents or suspending media. For this purpose, any bland, fixed oil may be employed, including synthetic mono- or diglycerides. Fatty acids (e.g., oleic acid and its glyceride derivatives), as well as natural pharmaceutically acceptable oils (e.g., olive oil or castor oil), are useful in the preparation of injectables, particularly in their polyoxyethylated versions.

[0070] When the polymeric micelle containing the gadolinium contrast agent according to the present invention is used as a contrast agent for MRI, parenteral administration, specifically intravenous administration, is preferred. When the polymeric micelle is administered intravenously, the administration concentration is preferably 5 mg / kg (body weight), for example.

[0071] On the other hand, the gadolinium contrast agent of the present invention can also be used as a gadolinium preparation for neutron capture therapy for cancer or tumors. For a general overview of gadolinium preparations, see Ichikawa et al., "Frontline - Creation of Gadolinium Preparations for Neutron Capture Therapy for Malignant Tumors" (Pharmacia, Vol. 56, No. 5, pp. 396-400, 2020). The applicability of the polymeric micelles of the present invention for neutron capture therapy is demonstrated in Example 4 (FIG. 14).

[0072] The dosage (amount or concentration) for imaging depends on the sensitivity of the diagnostic imaging device and the composition of the contrast agent. For example, for MRI imaging, gadolinium contrast agents of the present invention generally require lower dosages than contrast agents containing paramagnetic substances with lower magnetic moments, such as iron(III). Preferably, the dosage is about 0.001 to 1 mmol / kg of body weight per dose.

[0073] It should be further understood that the specific dosage regimen for any particular patient will also depend on a variety of factors, including the subject's age, weight, health, sex, drug combination, and the judgment of the treating physician.

[0074] In accordance with the present invention, MRI imaging is performed following appropriate administration of the gadolinium contrast agent of the present invention. The selection of pulse sequence (inversion recovery (IR); spin echo (SE); echo tomography (EPI); time of flight (TOF); turboflash; gradient echo (GE)) and imaging parameter values ​​(echo time (TE); inversion time (TI); repetition time (TR); flip angle, etc.) are dictated by the diagnostic information desired. Generally, if it is desired to obtain T1-weighted images, TE should be less than (or a minimum of) 30 milliseconds to maximize T1 weighting. Conversely, if T2 measurements are desired, TE should be greater than 30 milliseconds to minimize competing T1 effects. TI and TR are kept approximately the same for both T1-weighted and T2-weighted images. Typically, TI and TR are on the order of about 5-1000 milliseconds and 2-1000 milliseconds, respectively.

[0075] <Production Example> A method for producing the gadolinium contrast agent according to the present invention will be described below.

[0076] Synthesis of poly{[poly(ethylene glycol) monomethyl ether acrylate]-stat-(2-carboxyethyl acrylate)} (poly-1) In a glove box (Miwa Seisakusho, DBO-1.5KH-TSO, argon atmosphere), a 10 mL recovery flask was charged with poly(ethylene glycol) methyl ether acrylate (PEGA, average number of ethylene glycol repeats = 9) (3.01 g, 6.27 mmol, 55.8 equivalents), 2-carboxyethyl acrylate (CEA) (990 mg, 6.87 mmol, 61.1 equivalents), dehydrated and deoxygenated THF (6.3 mL), 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) (41.0 mg, 112 μmol, 1.0 equivalents), and 2,2′-azobis(isobutyronitrile) (AIBN) (51.2 mM). A THF solution (436 μL, 22.3 μmol, 0.2 equivalents) was added. The flask was removed from the glove box, and argon gas was bubbled into the reaction solution for 30 minutes. The reaction vessel was then placed in a 60°C oil bath and stirred for 2 hours. The polymerization was terminated by freezing the reaction solution with liquid nitrogen, and the reaction solution was added dropwise to a large amount of ice-cold diethyl ether. The precipitate was separated and washed three times with ice-cold diethyl ether before being dissolved in methanol. The methanol was then completely removed by distillation under reduced pressure and vacuum drying. The nonvolatile components were redissolved in distilled water and freeze-dried to obtain 1.58 g of a yellow viscous liquid, poly-1-1.

[0077] The resulting copolymer was １ The degree of polymerization of each monomer and the number average molecular weight (M ｎ，ＮＭＲ ) was calculated, the degree of polymerization of PEG was 45, the degree of polymerization of CEA was 65, and M ｎ，ＮＭＲ The molecular weight dispersity (M ｗ / M ｎ ) was calculated by GPC to be 1.46.

[0078]

[0079] [Measurement equipment and conditions] (1) １H-NMR measurement Apparatus: Bruker Avance III (400 MHz) / Bruker Solvent: 0.03% tetramethylsilane-containing chloroform-d / Kanto Scientific Measurement temperature: Room temperature Number of accumulations: 256 Result: δ (ppm) 4.71-4.18 (-COOCH ２ CH ２ O-, COOCH ２ CH ２ COOH), 3.88-3.32(-CH ２ (OCH ２ CH ２ ) ８ CH ３ , -CH ２ (CH ２ ) １０ CH ３ ), 2.75-2.65 (-COOCH ２ CH ２ COOH), 2.32-1.64 (main chain), 1.30-1.09 (DDMAT), 0.88 (3H, -CH ２ (CH ２ ) １０ CH ３ )

[0080] (2) GPC Measurement Apparatus: JASCO Extrema HPLC system / JASCO (CO-4060, AS-4050, PU-4180) Detector: RI-4030 differential refractive index detector / JASCO Column: TSKgel SuperAW-L guard column / Tosoh Corporation (4.6 mm x 35 mm), TSKgel SuperAW3000 / Tosoh Corporation (6.0 mm x 150 mm; particle size, 4 μm), TSKgel SuperAW4000 / Tosoh Corporation (6.0 mm x 150 mm; particle size, 6 μm) Mobile phase: N,N-dimethylformamide (DMF) containing 50 mmol / L lithium bromide Temperature: 40°C Flow rate: 0.2 mL / min Standard substance: Shodex STANDARD M-75 / Resonac

[0081] Polymers having various copolymer compositions and average molecular weights shown in the table below were synthesized using the same method as above, while appropriately changing the amounts of the reagents (PEGA, CEA, DDMAT, AIBN) used in poly-1-1, the solvent, the reaction temperature, and the polymerization time.

[0082]

[0083]

[0084] Synthesis of poly-2a-i: In a glove box, poly-1-1 (183 mg, 5.90 μmol, 1.0 equivalent), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC) (140 mg, 732 μmol, 124 equivalent), DMF (4.74 mL), 4-dimethylaminopyridine (DMAP) (753 mM DMF solution, 760 μL, 572 μmol, 97 equivalent), and benzylamine (BnA) (2.02 M DMF solution, 284 μL, 575 μmol, 98 equivalent) were added to a test tube, capped, and the test tube was removed from the glove box and stirred at room temperature for 3 days. The reaction was terminated by dialysis in methanol and purified (MWCO = 3.5 kDa). The solution in the dialysis membrane was evaporated under reduced pressure, and the methanol was completely removed by vacuum drying. Thereafter, the non-volatile components were redissolved in distilled water and freeze-dried to obtain 188 mg of a yellow viscous liquid poly-2a-1.

[0085] The resulting copolymer was １ The degree of polymerization of each monomer unit and the number average molecular weight (M ｎ，ＮＭＲ ) was calculated, the degree of polymerization of PEG was 45, that of CEA was 20, that of CEA-BnA was 45, and M ｎ，ＮＭＲ were calculated to be 35,000.

[0086] [Measurement equipment and conditions] (1) １ H-NMR measurement Apparatus: Bruker Avance III (400 MHz) / Bruker Solvent: 0.03% tetramethylsilane-containing chloroform-d / Kanto Scientific Measurement temperature: Room temperature Number of accumulations: 256 Results: poly-2a δ (ppm) 7.53-7.03 (Aromatic), 4.71-3.89 (-COOCH ２ CH ２ O-, -COOCH ２ CH ２ CO-, -CONHCH ２ Ph), 3.87-3.37(-CH２ (OCH ２ CH ２ ) ８ CH ３ , -CH ２ (CH ２ ) １０ CH ３ ), 3.02-1.32 (main chain, -COOCH ２ CH ２ CO-), 1.26-1.10 (DDMAT), 0.90-0.86 (-CH ２ (CH ２ ) １０ CH ３ )

[0087] Polymers having various copolymer compositions and average molecular weights, as shown in the table below, were synthesized in the same manner as above, except that the reagent BnA used in poly-2-1 was replaced with 1-naphthylmethylamine, 1-pyrenemethylamine, 2-ethylhexylamine, nerol, farnesol, DL-α-tocopherol, losartan, or ferphenazine, and the amounts of WSC and DMAP charged were 1.5 equivalents relative to the carboxyl groups in poly-1.

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] Determination of boundary conditions for monomolecular association of poly-2a to poly-2g in aqueous solution To determine the boundary conditions for monomolecular association of poly-2a to poly-2g in aqueous solution, the results of evaluating the association behavior of poly-2a to poly-2g in aqueous solution were used. The association behavior of poly-2a to poly-2g in aqueous solution was determined by the absolute weight-average molecular weight M calculated from a system using size exclusion chromatography and a multi-angle light scattering detector (SEC-MALS) in aqueous solution (PBS) and organic solvent (chloroform) of poly-2a to poly-2g obtained by the above procedure. ｗ，ＭＡＬＳ The number of associations N calculated from the ratio ａｇｇ The evaluation was carried out using N ａｇｇ is calculated by the following formula: N ａｇｇ = M ｗ，ＭＡＬＳ (PBS) / M ｗ，ＭＡＬＳ (chloroform), where N ａｇｇ When the value of N is less than 1.5, the corresponding polymer forms a micelle in an aqueous solution. ａｇｇ When the value of N is 1.5 or more, it indicates that the corresponding polymer is multi-molecularly associated in an aqueous solution. ａｇｇ From the degree of polymerization of the polymer and the proportion of hydrophobic side chains in the polymer, the boundary conditions for the degree of polymerization and the proportion of hydrophobic side chains at which the polymers can associate as monomolecules in aqueous solution were determined for each of poly-2a to poly-2g. These boundary conditions are represented by the dashed curves in Figures 2 to 8.

[0097] [Measurement equipment and conditions] (1) SEC-MALS measurement (PBS) Equipment: JASCO Extrema HPLC system / JASCO (CO-4060, AS-4050, PU-4180) Detector: RI-4030 differential refractive index detector / JASCO, DAWN 8 multi-angle light scattering detector / Wyatt Technology Corporation Column: Shodex OHpak SB-G 6B / Resonac (6.0 mm × 50 mm), Shodex OHpak SB-806M HQ / Resonac (8.0 mm × 300 mm; particle size, 13 μm), Shodex OHpak SB-804 HQ / Resonac (8.0mm x 300mm; particle size, 10μm) Mobile phase: 137mM NaCl, 2.7mM KCl, 11.9mM phosphate buffer (PBS), pH 7.4 Temperature: 40°C Flow rate: 1mL / min Sample concentration: 3mg / mL Results: Tables 12 to 19

[0098] (2) SEC-MALS Measurement (Chloroform) Apparatus: High-speed GPC apparatus / Tosoh Corporation (HLC-8420GPC) Detector: DAWN-HELEOS II multi-angle light scattering detector / Wyatt Technology Corporation, Optilab T-rEX differential refractive index detector / Wyatt Technology Corporation Column: Shodex LF-G / Resonaq (4.6 mm × 10 mm; particle size, 6 μm), Shodex LF-804 / Resonaq (8.0 mm × 300 mm; particle size, 6 μm) Mobile phase: Chloroform Temperature: 40 ° C. Flow rate: 1 mL / min Sample concentration: 3 mg / mL Results: Tables 12 to 19

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(2-carboxyethyl acrylate), BZA ｍ -PEGA ｎ -CEA ｋ Synthesis of

[0108] Typical Polymerization Methods

[0109]

[0110] Synthesis of TP1: 146 mg of benzyl acrylate (BZA), 389 mg of poly(ethylene glycol) methyl ether acrylate (PEGA, Mn = 480 g / mol), and 13 mg of 2-carboxyethyl acrylate were weighed out to prepare a monomer solution. Separately, 0.5 mg of 2,2'-azobis(2-methylpropionitrile) (AIBN) and 2.2 mg of 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT) were weighed out and dissolved in 1 mL of distilled and purified N,N-dimethylformamide (DMF). This solution was then added to the monomer solution, and polymerization was carried out in an oil bath at 70 °C. After 20 minutes, the reaction was stopped by cooling the reaction solution with liquid nitrogen. The reaction solution was then dialyzed (MWCO = 6-8 kD) against methanol and ultrapure water to purify the target compound. Finally, freeze-drying was carried out to obtain poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(acrylic acid 2-carboxyethyl acrylate)]. The conversion rate, degree of polymerization, and number average molecular weight (M ｎＮＭＲ )teeth １ The conversion rates were 73.3% for BZA, 65.7% for PEGA, and 57.1% for CEA, respectively. ｎ ＮＭＲ = 62,700 g / mol, and the molecular weight dispersity (M ｗ / M ｎ ) was measured using GPC, ｗ / M​ｎ By changing the amounts of monomers (PEGA, BZA, CEA) charged under the same conditions, polymers (TP1-TP3) with different composition ratios and a copolymer without BZA (PEG １２４ -CEA １０ , PEGA １０４ -CEA ３０ ) was synthesized.

[0111] Measurement equipment and conditions (1) １ H-NMR measurement apparatus: Bruker biospin AVANCE III (400 MHz) / Bruker Solvent: Dimethyl sulfoxide-d ６ containing 0.03% tetramethylsilane / Kanto Kagaku Measurement temperature: Room temperature Result: δ (ppm) = 0.88 (t, 3H, -S-CH ２ - (CH ２ ) １０ -CH ３ ), 1.10-1.29 (m, 20H, -S-CH ２ - (CH ２ ) １０ -CH ３ ), 1.33-1.99 (m, 406H, -(CH ２ -CH(BZAsegment)) ｍ -, -(CH ２ -CH (PEGA segment) ｎ -, -(CH ２ -CH (CEA segment) ｋ -, (CH ３ ) ２ -C(COOH)-), 2.19-2.53(m, 200H, -(CH ２ -CH(BZA segment) ｍ -, -(CH ２ -CH(PEGA segment) ｎ -, -(CH ２ -CH(CEA segment) ｋ -), 2.60-2.75 (m, 20H, -COO-CH ２ -CH ２ -COOH), 3.31-3.40(m, 272H, -CH ２ -CH２ -O-(CH ２ -CH ２ -O) ８ -CH ３ , -S-CH ２ - (CH ２ ) １０ -CH ３ ), 3.42-3.74 (m, 3060H, -CH ２ -CH ２ -O-(CH ２ -CH ２ -O) ８ -CH ３ ), 3.90-4.40 (m, 200H, -CH ２ -CH ２ -O-(CH ２ -CH ２ -O) ８ -CH ３ , -COO-CH ２ -CH ２ -COOH), 4.81-5.16 (m, 200H, -COO-CH ２ -C ６ H ５ ), 7.17-7.40 (m, 500H, -COO-CH ２ -C ６ H ５ ).

[0112] (2) GPC Measurement Apparatus: JASCO Extrema HPLC system (LC-Net II / ADC, Co-4060, AS-4050, PU-4180 / JASCO) Detector: UV-4070 / JASCO, RI-4030 / JASCO Column: TSKgel α-2500 column / Tosoh (column size 7.8 mm × 300 mm, pore size 2.5 nm, particle size 7 μm, exclusion limit 1 × 10 ４ g / mol) TSKgel α-4000 column / Tosoh (column size 7.8 mm × 300 mm, pore size 45 nm, particle size 10 μm, exclusion limit 1 × 10 ６g / mol) TSKgel guard column-α / Tosoh Mobile phase: N,N-dimethylformamide (DMF) containing 10 mmol / L lithium bromide Temperature: 40°C Flow rate: 1 mL / min Sample concentration: 5 mg / mL Standard substance: Shodex STANDARD M-75 / Showa Denko Results: Figure 9

[0113] (5) Poly[(benzyl acrylate)-co-(poly(ethylene glycol) methyl ether acrylate)-co-(2-carboxyethyl acrylate)-(Gd-s-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecanetetraacetic acid), BZA ｍ -PEGA ｎ -CEA ｋ (DOTA) ｊ Synthesis of

[0114]

[0115] Synthesis of TP4 The copolymer (2449 mg, 0.04 mmol) and DMT-MM (132.8 mg, 0.48 mmol) were dissolved in a methanol / DMF mixture (20 mL, methanol:DMF=1:1) and stirred at 0°C for 1 hour. Subsequently, s-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetra-tert-butyl acetate (p-NH ２ The reaction solution was dialyzed (MWCO = 6-8 kD) against methanol and ultrapure water to purify the target compound. Finally, BZA was obtained by lyophilization. １００ -PEGA ９０ -CEA １０ (DOTA-tBu) ４ The compound obtained was dissolved in a trifluoroacetic acid (TFA) / chloroform mixture (20 mL, TFA:chloroform = 1:1) and stirred overnight at room temperature. The reaction solution was dialyzed (MWCO = 6-8 kD) against methanol and ultrapure water to purify the target compound. Finally, the compound was lyophilized to obtain BZA. １００ -PEGA ９０ -CEA １０ (DOTA)４ The obtained polymer was １ The copolymers were analyzed by H-NMR. The copolymers with different compositions were also reacted and analyzed under the same conditions.

[0116] Measurement equipment and conditions (1) １ H-NMR measurement apparatus: Bruker biospin AVANCE III (400 MHz) / Bruker Solvent: Dimethyl sulfoxide-d ６ containing 0.03% tetramethylsilane / Kanto Chemical Measurement temperature: room temperature Result: δ (ppm) = 0.88 (t ， 3H ， -S-CH ２ - (CH ２ ) 10-CH ３ ) ， 0.93-1.27(m ， 20H ， -S-CH ２ - (CH ２ ) １０ -CH ３ ) ， 1.30-1.98 (m ， 406H ， - (CH ２ -CH(BZA segment) ｍ - ， - (CH ２ -CH(PEGA segment) ｎ - ， - (CH ２ -CH(CEA segment) ｋ - ， (CH ３ ) ２ -C(COOH)-) ， 2.05-2.43 (m ， 200H ， - (CH ２ -CH(BZA segment) ｍ - ， - (CH ２ -CH(PEGA segment) ｎ - ， - (CH ２ -CH(CEA segment) ｋ-) ， 2.54-2.68(m ， 20H ， -COO-CH ２ -CH ２ -COOH ， -COO-CH ２ -CH ２ -CO-DOTA segment) ， 3.18-3.27(m ， 422H ， -CH ２ -CH ２ -O-(CH ２ -CH ２ -O) ８ -CH ３， -CH ２ -CH ２ -N(CH ２ COOH)- ， -S-CH ２ -(CH ２ ) １０ -CH ３ ) ， 3.28-3.71(m ， 3100H ， -CH ２ -CH ２ -O-(CH ２ -CH ２ -O) ８ -CH ３， -CH ２ -CH ２ -N(CH ２ COOH)- ， -NH-C ６ H ４ -CH ２ -) ， 3.85-4.35(m ， 200H ， -CH ２ -CH ２ -O-(CH ２ -CH ２ -O) ８ -CH ３， -COO-CH ２ -CH ２ -COOH ， -COO-CH ２ -CH ２ -CO-DOTA segment) ，4.77-5.15 (m ， 200H ， -COO-CH ２ -C ６ H ５ ) ， 7.00-7.60 (m ， 540H ， -COO-CH ２ -C ６ H ５， -NH-C ６ H ４ -CH ２ -) (Figure 10)

[0117] The copolymer synthesized in the previous section and gadolinium chloride hexahydrate (2973.6 mg, 8.0 mmol) were dissolved in ultrapure water and stirred overnight in a water bath at 60°C. The reaction solution was dialyzed (MWCO = 6-8 kD) against ultrapure water, then mixed with PBS(-) (100 mL) and stirred overnight at room temperature. The reaction solution was then filtered and purified by dialysis (MWCO = 6-8 kD) against ultrapure water again. Finally, the target compound was obtained by lyophilization. The amount of Gd introduced into each polymer was confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The same procedure was used to diagnose the copolymer without BZA by adding p-NH ２ -Bn-DOTA-tBu and Gd were introduced and purified. Five final products were obtained from the above reaction. TP4 = BZA １００ -PEGA ９０ -CEA １０ (DOTA) ４ , TP5=BZA １００ -PEGA ８０ -CEA ２０ (DOTA) ９ , TP6=BZA １００ -PEGA ７０ -CEA ３０ (DOTA) １７ , PEG-Gd ４ = PEGA １２４ -CEA １０ (DOTA) ４ , PEGA １０４ -CEA ３０ (DOTA) １２ .

[0118] The present invention will be described in more detail below based on examples, although the present invention is not limited to the following examples.

[0119] Example 1: Evaluation of hydrophobicity of hydrophobic side chains of poly-2a-i The degree of hydrophobicity of the hydrophobic side chains introduced into poly-2a-i was evaluated by inputting the structure of the introduced compound into molinspiration [https: / / molinspiration.com]. Results: Table 20

[0120]

[0121] Example 2: Determination of universal boundary conditions for monomolecular association of poly-2 in aqueous solution Based on the boundary conditions for monomolecular association of poly-2a to poly-2g in aqueous solution described above, boundary conditions for monomolecular association of poly-2 in aqueous solution that can accommodate various hydrophobic side chain structures were determined. Specifically, in a three-dimensional space with the clogP value of the introduced hydrophobic side chains described above on the first axis, the degree of polymerization of the polymer on the second axis, and the proportion of hydrophobic side chains in the polymer on the third axis, the boundary conditions for monomolecular association of poly-2a to poly-2g in solution described above were connected to depict a curved surface to determine the boundary conditions. Below this curved surface are the degree of polymerization of the polymer that can associate with poly-2 in aqueous solution as a monomolecule, the proportion of hydrophobic side chains, and the clogP value of the hydrophobic side chains. Results: Figure 11

[0122] Example 3: Evaluation of SMDC formation BZA obtained by the above procedure ｍ -PEGA ｎ -CEA ｋ (DOTA) ｊ The folding ability of the water-soluble polymer was measured by the absolute weight-average molecular weight M calculated from size exclusion chromatography and multi-angle light scattering detector (SEC-MALS) in aqueous solution (PBS) and organic solvent (chloroform or DMF). ｗ The data obtained from SEC-MALS was analyzed using a Zimm plot, and the dn / dc value was calculated by measuring the refractive index of four samples with different concentrations (4, 3, 2, 1 mg / mL) in each solvent. DA was calculated using the following formula: DA = M ｗＰＢＳ / MｗＣＨＣｌ３

[0123] (1) GPC Measurement (Aqueous System) Apparatus: JASCO Extrema HPLC system (LC-Net II / ADC, Co-4060, AS-4050, PU-4180 / JASCO) Detector: UV-4070 / JASCO, RI-4030 / JASCO, DAWN 8 MALS detector / Wyatt Technology Corporation Column: OHpak SB-804HQ column / Showa Denko (column size: 8 mm x 300 mm, pore size: 20 nm, particle size: 10 μm, exclusion limit: 1 x 10 ６ g / mol) OHpak SB-806MHQ column / Showa Denko (column size 8 mm x 300 mm, pore size 1.5 μm, particle size 13 μm, exclusion limit 2 × 10 ７ g / mol) OHpak SB-G 6B guard column / Showa Denko Mobile phase: 137 mM NaCl, 2.7 mM KCl, 11.9 mM phosphate buffer (PBS), pH 7.4 Temperature: 40°C Flow rate: 1 mL / min Sample concentration: 5 mg / mL Results: Table 21

[0124] (2) GPC measurement (chloroform system) Apparatus: TOSOH HLC-8220 GPC system / Tosoh Detector: DAWN HELEOS II MALS detector / Wyatt Column: LF-804 column / Showa Denko (column size 8 mm x 300 mm, pore size 300 nm, particle size 6 μm, exclusion limit 2 x 10 ６ g / mol) LF-G guard column / Showa Denko Mobile phase: chloroform Temperature: 40°C Flow rate: 1 mL / min Sample concentration: 5 mg / mL Results: Table 21

[0125]

[0126] As mentioned above, a DA of 1.0 indicates intramolecular association, i.e., SMDC formation, whereas a high DA (DA > 1.0) indicates intermolecular self-assembly, and a low DA (DA < 1.0) indicates SMDC formation in water, potentially resulting in micelle formation in organic solvents. The DA values ​​of TP4, TP5, and TP6 from the table above were 0.91, 0.88, and 0.53, respectively, suggesting SMDC formation in water.

[0127] Example 4: Design and Characterization of SMDC-Gd BZA obtained in the previous section ｍ -PEGA ｎ -CEA ｋ (DOTA) ｊ and PEGA ｎ -CEA ｋ (DOTA) ｊ The particle size was confirmed by Guinier plot of small angle X-ray scattering (SAXS), and SMDC formation was confirmed by Kratky plot. Furthermore, the particle size was measured by transmission electron microscope (TEM) and dynamic light scattering. Furthermore, the surface potential was measured.

[0128] (1) SAXS Measurement Apparatus: nano-viewer SAXS system / Rigaku Solvent: ultrapure water or DMF Sample concentration: 10 mg / ml Measurement temperature: room temperature Results: Figures 12 and 13

[0129] (2) TEM Measurement Apparatus: JEM-2100F TEM system / JEOL Solvent: ultrapure water Pretreatment: stained on formvar / carbon supported copper grids Measurement temperature: room temperature Results: Figures 14 and 15

[0130] (3) DLS Apparatus: Malvern Zetasizer Nano ZSP / Malvern Panalytical Solvent: Ultrapure water Sample concentration: 10 mg / ml Measurement temperature: Room temperature Results: Figures 16 and 17

[0131] The peaks in the Kratky plots (Figure 12) in aqueous solution indicate particle formation for TP4, TP5, and TP6 in aqueous solution. On the other hand, the absence of peaks in the Kratky plots in DMF indicates the absence of particle formation in DMF. TEM images and particle size analysis (Figures 14 and 15) and SAXS analysis (Figures 12 and 13) of TP4 and TP6 indicated diameters of approximately 5-7 nm with narrow particle size distributions. This was consistent with the DLS analysis results. Many polymeric micelles have been used as drug delivery carriers, and studies comparing polymeric micelles with CMC / CAC diameters of 30-100 nm have reported that smaller carriers exhibit better accumulation, permeability, and extravascular infiltration in malignant tumors. However, few carriers without a CMC / CAC of 10 nm or less have been reported. Renal excretion, one of the major excretion routes after administration, requires a threshold for filtration by the renal glomerulus. Accumulation in the liver can also be controlled by setting the particle size to 8-9 nm. TP4 and TP6 analyzed in this example are slightly larger than the renal excretion threshold. TP4, TP6, PEG-Gd ４ The zeta potential of the polymer was measured (Figure 17), and the surface charge was found to be negative due to the influence of the carboxyl groups remaining on the polymer side chains. This prevents nonspecific interactions with plasma proteins and suppresses excretion from the body by the mononuclear phagocyte system.

[0132] Example 5: Evaluation of relaxivity of SMDC-Gd Various polymer samples (Gd conc. 1 mM, 0.5 mM, 0.25 mM, 0.125 mM) were prepared, and the longitudinal relaxation time T １ and the transverse relaxation time T ２ The results were plotted against the Gd concentration and the longitudinal relaxivity r was calculated from the slope of the fitted curve based on the following equation: １ , transverse relaxivity r ２ Also, (1 / T ｉ ) ｏｂｓ is the measured value of the relaxation time, (1 / T ｉ ) ｄ indicates the relaxation time of the solvent. (1 / T ｉ ) ｏｂｓ= (1 / T ｉ ) ｄ +r ｉ [Gd]. i=1,2

[0133] (1) Relaxivity Measurement Apparatus: Bruker minispec 0.47T NMR / Bruker Solvent: Ultrapure water or ultrapure water containing BSA (10 mg / mL) Measurement conditions: Temperature 37°C, receiver gain = 80-82, recycle delay = 2. Results: Table 22

[0134]

[0135] Regarding the relaxivity measurement results (Table 22), PEG-Gd ４ , PEG-Gd １２ , TP4, TP5, and TP6 have higher longitudinal relaxivities (r １ ), transverse relaxivity (r ２ ) was observed. This is because the covalent bonding of the Gd-DOTA complex to the polymer caused a slow tumbling effect, improving its relaxivity. However, the effect of increasing the amount of Gd-DOTA introduced per polymer molecule was negligible. This is due to the fast mobility of Gd-DOTA within the polymer, or the mobility of the entire polymer. Interestingly, the folding of the polymer due to SMDC formation created a crowded environment, suppressing the movement of Gd-DOTA and improving relaxivity. This phenomenon has not been reported before and was first revealed in this example. In addition, the small effect of increasing the amount of Gd-DOTA introduced into SMDC suggested that SMDC-Gd may exhibit high relaxivity with a low Gd loading. Even in the presence of bovine serum albumin (BSA), there was almost no effect on the relaxivity, indicating that there is almost no interaction between SMDC-Gd and PEG-Gd and proteins. １ / r ２ These findings demonstrate that SMDC-Gd can be used as a systemically administered MRI contrast agent.

[0136] Example 6: Pharmacokinetics test of SMDC-Gd A tumor-bearing model was prepared by subcutaneously transplanting mouse colon cancer cells CT26 into a mouse (Balb / c, 6-week-old, female, SLC) and used for the pharmacokinetics test. The tumor-bearing model was subcultured (5% CO) in DMEM medium (containing 10% FBS and 1% penicillin streptomycin). ２ The cell suspension (1 × 10 ６ Cells / mouse, DMEM). 10 days after administration, tumor size was randomized, and various samples were administered via the tail vein of mice (Gd conc. 5 mg / kg, n = 6). After administration, blood was collected at predetermined times (1, 4, 8, 24, and 72 hours), and the mice were dissected and organs (tumor, liver, spleen, kidney, pancreas, skin, muscle, and brain) were collected. However, after blood collection, plasma was collected by centrifugation and used as a sample. The collected organs were pretreated (70% nitric acid, heated at 90°C for at least 1 hour, and then diluted with ultrapure water), and the Gd concentration was measured using ICP-MS.

[0137] (1) ICP-MS Measurement Apparatus: Agilent 7700x ICP-MS / Agilent Pretreatment device: EYELA MG-2300 / Tokyo Rikakikai Standard solution: Gadolinium standard solution (Gd1000) for ICP analysis / Fujifilm Wako Pure Chemical Industries Results: Figures 18 to 25

[0138] Comparison of blood retention (Figure 18) shows that SMDC-Gd ４ and PEG-Gd ４ The blood retention and clearance from blood of SMDC-Gd were similar, but １７ Regarding tumor accumulation (Fig. 19), SMDC-Gd ４ The Gd concentration 24-48 hours after administration of SMDC-Gd １７ and PEG-Gd ４ In the tumor model used in this example, Gd-DOTA was rapidly eliminated from the blood and showed low tumor accumulation. These results suggest that the intramolecular folding provides a PEG layer around SMDC, suppressing aggregation and protein binding. Regarding accumulation in multiple organs (Figures 20 to 25), SMDC-Gd ４was comparable to that of typical nanomedicines, but SMDC-Gd １７ was accumulated in the liver, spleen, and kidneys. １７ The data potential of SMDC-Gd ４ , PEG-Gd ４ Generally, if the surface charge is neutral or slightly negative, it can prevent nonspecific interactions with plasma proteins and avoid excretion by the mononuclear phagocyte system. １７ Since the data potential of was as low as -37 mV, it was taken up by non-parenchymal cells of the liver such as Kupffer cells and macrophages through endocytosis mediated by scavenger receptors.

[0139] Example 7: Toxicity test of SMDC-Gd The cytotoxicity of SMDC-Gd was evaluated using cell counting kit-8 (CCK8). The cells were subcultured (5% CO) in DMEM medium (containing 10% FBS and 1% penicillin streptomycin). ２ CT26 cells were incubated at 37°C (50 μL, 10,000 cells / well) in a 96-well plate and cultured for 24 hours. After incubation for 48 hours, each sample was added, followed by the addition of CCK8 solution (10 μL), and the incubation was continued for another hour. The absorbance at 450 nm was measured using a plate reader.

[0140] (1) Absorbance measurement Apparatus: iMark microplate reader / Bio-Rad Laboratories Results: Figure 26

[0141] Toxicity tests of SMDC-Gd were conducted using mice (Balb / c, 6 weeks old, female, SLC). Each sample was administered to the mice via the tail vein at a Gd concentration of 0.1 mmol / kg (n=5). Blood was collected 4 and 48 hours after administration, and 90 μL of the collected blood was mixed with 10 μL of ethylenediaminetetraacetic acid (EDTA) solution (EDTA concentration = 12 mg / mL, solvent: ultrapure water). White blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), and platelets (PLT) were measured using a multiparameter automated animal hematology analyzer. Plasma was obtained from the remaining blood by centrifugation, and blood urea nitrogen (BUN), creatinine (CRE), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), alkaline phosphatase (ALP), and total protein (TP) were measured using a dry clinical chemistry analyzer.

[0142] (1) WBC, RBC, HGB, HCT, PLT measurement Equipment: automatic multiple blood cell counter pocH-100iV Diff / Sysmex Results: Figures 27 to 31

[0143] (2) Measurement of BUN, CRE, GPT, GOT, ALP, and TP. Apparatus: Dri-Chem 7000IZ / Fujifilm Wako Pure Chemical Industries. Results: Figures 32 to 37.

[0144] Evaluation of the cytotoxicity (FIG. 26) and physiological parameters (FIGS. 27 to 37) of SMDC-Gd showed no acute effects at a clinically applicable dose (0.1 mmol / kg of Gd).

[0145] Example 8: MRI experiment of SMDC-Gd. A tumor-bearing model was created by subcutaneously transplanting mouse colon cancer cells CT26 into mice (BALB / c nu / nu mice, 5 weeks old, female, SLC) and used for MRI. The tumor-bearing model was subcultured (5% CO) using DMEM medium (containing 10% FBS and 1% penicillin streptomycin). ２ , 37°C) was injected subcutaneously into the back of the mouse (1 × 10 ６One week after administration, tumor size was randomized, and then various samples were administered to the mice via the tail vein (Gd conc. 0.1, 0.05, 0.03, 0.01 mmol / kg). MRI imaging (R １ Mapping, T １ Enhanced imaging was performed.

[0146] (1) R １ Mapping imaging device: horizontal 1.0 T Bruker ICON MRI system / Bruker Pulse sequence: rapid acquisition with relaxation enhancement (RARE)-based inversion recovery sequence Measurement conditions: Echo time (TE) = 10 ms; Repetition time (TR) = 10,000 ms; Inversion time (T) = 10,000 ms Time (TI) = 100, 300, 500, 700, 900, 1300, 1700, 1900, 2100, 2500, 2900, 3300, 3700 ms RARE Factor = 4; Field of View (FOV) = 28.0 × 16.0 mm ２ Slice thickness = 2.0 mm; Number of pixels = 70 x 40; In-plane resolution = 0.4 x 0.4 mm ２ Number of additions = 1; Fat suppression mode: Off; FOV saturation: On; Measurement time: Before administration, 20, 40, 60 minutes, and 24 hours after administration; Results: Figures 38 to 42

[0147] In the quantitative mapping image of the longitudinal relaxation rate (Fig. 38), SMDC-Gd ４ The superiority of Gd-DOTA was confirmed. １The values ​​are adipose tissue, and can be reduced or eliminated by using fat suppression techniques during MRI imaging. The longitudinal relaxation rates of the tumor and those of the tumor and muscle were compared (Figures 39 and 40). ４ showed a high contrast effect even in tumor tissue, with the tumor / muscle ratio (T / M ratio) of relaxation rate at 1 hour after administration being 1.17. On the other hand, the T / M ratio at 1 hour after administration of Gd-DOTA was 0.77, and SMDC-Gd ４ The T / M ratio of SMDC-Gd was significantly higher than that of Gd-DOTA. ４ The T / M ratio of PEG-Gd4 increased to 1.36 within 24 hours after administration. １ The values ​​tended to be dose-dependent, and within 24 hours, the R １ The value is always SMDC-Gd ４ was lower than

[0148] (1) T １ Weighted image Apparatus: Permanent magnet type 1.0T Bruker ICON MRI system / Bruker Pulse sequence: Spin-echo sequence Measurement conditions: Echo time (TE) = 7 ms; Repetition time (TR) = 400 ms Field of view (FOV) = 30.0 × 30.0 mm ２ Slice thickness = 1.0 mm; Number of slices = 20; Slice spacing = 1.0 mm; Number of pixels = 150 x 150; In-plane resolution = 0.2 x 0.2 mm ２ Number of additions = 4; Fat suppression mode: None; FOV saturation: None; Measurement time: Before administration, 0-60 minutes after administration; Results: Figures 43 to 45

[0149] The time course of MR signals in the kidney and bladder was evaluated up to 1 hour after administration (Figs. 43 to 45). The MR signals in the kidney showed a similar tendency to the amount of accumulation in the kidney (Fig. 20). However, the difference in clearance time between the kidney and the bladder was due to the administration of SMDC-Gd. ４ , PEG-Gd４ This reflects the difference in permeability of SMDC-Gd to the negatively charged glomerular basement membrane (GBM). ４ and PEG-Gd ４ had similar surface charges, but SMDC-Gd ４ was designed to have a diameter of approximately 6 nm upon folding, which was within the threshold particle size for penetration through the GBM. ４ In the SAXS analysis example (Fig. 12), no peaks were observed in the Kratky plots, indicating that the polymer was in a flexible polymer state. Therefore, even within the threshold particle size for GBM penetration, such properties of a single polymer chain may significantly affect its behavior in the GBM.

[0150] Example 9: Pharmacokinetics Test Experiment Aimed at Predicting the Therapeutic Efficacy of Gd-Based Neutron Capture Therapy (NCT) Following the same procedure as in the SMDC-Gd pharmacokinetics test, a tumor-bearing model was prepared by subcutaneously implanting mouse colon cancer cells CT26 into mice (Balb / c, 6-week-old, female, SLC), and a pharmacokinetics test for Gd-NCT experiments was performed. However, various samples were administered once or three times via the tail vein of the mice (0, 24, and 48 hours later, Gd conc. 0.1 mmol / kg, n = 5). 24 hours after the final administration, blood was collected, and the mice were dissected to recover organs (tumor, liver, and kidney). Each organ was pretreated using the same procedure as described above, and Gd concentrations were also measured using ICP-MS.

[0151] (1) ICP-MS measurement Apparatus: Agilent 7700x ICP-MS / Agilent Pretreatment device: EYELA MG-2300 / Tokyo Rikakikai Standard solution: Gadolinium standard solution (Gd1000) for ICP analysis / Fujifilm Wako Pure Chemical Results: Figures 46 and 47

[0152] １５７ Gd can be used in neutron capture therapy, but it has not yet been applied to tumor treatment in clinical practice. One of the reasons for this is that in order to achieve therapeutic effects, １５７ The Gd concentration needs to be high. ４ ​The Gd concentration in the tumor was evaluated when the number of administrations of SMDC-Gd was changed. ４ A single dose of Gd( １５７ When administered three times at 24-hour intervals, the Gd concentration was 91.2 ppm ( １５７ It was confirmed that the concentration of HCl increased to 14.3 ppm based on Gd.

[0153] Example 10: Gd-NCT experiment of SMDC-Gd A tumor-bearing model in which mouse colon cancer cells CT26 were subcutaneously transplanted into mice (Balb / c, 6-week-old, female, SLC) was used for the Gd-NCT experiment. A CT26 cell suspension was injected into the right thigh of the mouse (2 × 10 ５ Approximately two weeks later, various samples were administered three times via the tail vein of the mice (0, 24, and 48 hours later, Gd conc. 0.1 mmol / kg, n=5). A PBS-administered group was used as a control. 72 hours after the first administration, the holder containing the mice was placed in a 40 wt% thermal neutron blocking solution. ６ LiF (96% ６ The tumor was placed on a 5 mm thick thermoplastic plate containing Li, and only the tumor site on the right thigh was irradiated with neutrons for 10 minutes (5 MW, Kyoto University Research Reactor, fluence: 2.87 × 10 １２ ~3.29 x 10 １２ Thermal neutron / cm ２ , 5.10 x 10 １１ ~5.86 x 10 １１ Epithermal neutrality / cm ２ The antitumor effect was confirmed by measuring the change in tumor volume over time, and the toxicity was confirmed by measuring the change in mouse body weight.

[0154] (1) Gd-NCT Regarding the antitumor effect and body weight change of Gd-NCT (Fig. 48 and Fig. 49), the three groups of Gd-DOTA, PBS, and no administration + no irradiation did not show sufficient antitumor activity, whereas SMDC-Gd ４ The SMDC-Gd suppressed tumor growth without causing weight loss. ４ is sufficient to achieve a therapeutic effect with NCT. １５７This is because Gd was delivered to the tumor tissue.

[0155] All publications and patents cited herein are incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, those skilled in the art will readily understand that various modifications may be made therein without departing from the spirit and scope of the invention.

[0156] References

[0157]

[0158]

Claims

1. Polymeric micelles consisting of a self-aggregating single polymer chain that contains one or more gadolinium complexes and spontaneously folds in water to form micelles with a particle size of 10 nm or less.

2. A polymeric micelle comprising a self-aggregating single polymer chain according to claim 1, in which: (A) a side chain containing polyethylene glycol (PEG) or a PEG derivative; (B) a side chain containing a hydrophobic group; and (C) a side chain containing a gadolinium complex; are bonded to the main backbone via a linker or directly.

3. A polymeric micelle comprising a self-aggregating single polymer chain according to claim 2, further comprising (D) a side chain containing a carboxylic acid group bonded to the main backbone via a linker or directly.

4. The PEG or PEG derivative in the side chain (A) has the following structure: (In the formula, R ７ is a methoxy group, an ethoxy group, an acetyl group, or a benzyl group; R ８ is an amide bond, an ether bond, or an ester bond, which is a moiety that is bonded to the main skeleton; R ９ is a biodegradable linker; and n is 1 to 10. The polymeric micelle comprising the self-aggregating single polymer chain according to claim 2 .

5. A polymeric micelle comprising a self-aggregating single polymer chain according to claim 2, wherein the hydrophobicity (clogP value) of the hydrophobic group in the side chain (B) is 1 to 10.

6. A polymeric micelle comprising a self-aggregating single polymer chain according to claim 2, wherein the hydrophobic group in the side chain (B) is selected from the group consisting of a phenyl group, a naphthyl group, a pyrenyl group, anthracene, cholesterol, a steroid derivative, 1-pyrenemethylamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-naphthylmethylamine, DL-α-tocopherol, nerol, farnesol, and an anticancer drug.

7. A polymeric micelle comprising a self-aggregating single polymer chain according to any one of claims 1 to 6, wherein the gadolinium complex is at least one selected from the group consisting of gadoteridol, meglumine gadoterate, and gadobutrol.

8. When the numbers of repeating units of side chains (A) to (D) in a single chain of polymer are a, b, c, and d, respectively, the following conditions are met: (I) When the clogP value is less than 1.5 and the total degree of polymerization (DP) of a+b+c+d is 100 to 500, a has 50% or more of the total DP; b has 40% or less of the total DP; c has 10% or less of the total DP; and d is 10% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP; (II) When the clogP value is 1.5 to 4.5 and the total degree of polymerization (DP) of a+b+c+d is less than 200, a has 50% or more of the total DP; b has 30% or less of the total DP; (III) when the clogP value is 1.5 or more and less than 4.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or more and 500 or less, a has 50% or more of the total DP; b has 40% or less of the total DP; c has 5% or less of the total DP; and d has 15% or less of the total DP, and c and d have 1% or less of the total DP; (IV) when the clogP value is 4.5 or more and less than 5.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or more and 500 or less, a has 50% or more of the total DP; (V) when the clogP value is 5.5 or more and the total degree of polymerization (DP) of a+b+c+d is 200 or more and 500 or less, a has 60% or more of the total DP; b has 20% or less of the total DP; c has 2% or less of the total DP; and d has 8% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦10% of the total DP. The polymeric micelle comprising the self-aggregating single polymer chain according to any one of claims 2 to 6.

9. M ｗ，ＭＡＬＳ (PBS) / M ｗ，ＭＡＬＳ A polymer micelle comprising the self-aggregating single polymer chain according to any one of claims 1 to 6, wherein the number of associations of the single polymer chain in water or a buffer solution is less than 1.5, as calculated from the (chloroform) ratio.

10. A polymeric micelle comprising a self-aggregating single polymer chain according to any one of claims 1 to 6, wherein the main chain in the single polymer chain is selected from the group consisting of polyether, polyester, polyalkylene, polycarbonate, poly(dioxanone), polyanhydride, polyhydroxy acid, polyfumarate, polycaprolactone, polyamide, polyacetal, poly(orthoester), polyhydroxybutyrate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, poly(arylate), polycarbonate, poly(propylene fumarate), polyhydroxyalkanoate, polyketal, polyesteramide, polyhydroxyvalerate, polyorthocarbonate, poly(vinylpyrrolidone), polyalkylene oxalate, polyalkylene succinate, polylactic acid, poly(malic acid), poly(methyl vinyl ether), and poly(maleic anhydride).

11. A compound having the following structure, which contains one or more gadolinium complexes and spontaneously folds in water to form micelles with a particle size of 10 nm or less: or (In the formula, R １ and R ６ are independently a methoxy group, an ethoxy group, an acetyl group, or a benzyl group; R ２ is PEG or a PEG derivative; R ３ is a hydrophobic group; R ４ is a gadolinium complex; R ５ is a carboxylic acid group; L １ ~L ６ are each independently absent or a linker; a, b, c, and d satisfy any of the following conditions (I) to (V): (I) when the clogP value is less than 1.5 and the total degree of polymerization (DP) of a+b+c+d is 100 or more and 500 or less, a has 50% or more of the total DP; b has 40% or less of the total DP; c has 10% or less of the total DP; and d is 10% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦20% of the total DP; (II) when the clogP value is 1.5 or more and less than 4.5 and the total degree of polymerization (DP) of a+b+c+d is less than 200, a has 50% or more of the total DP; b has 30% or less of the total DP; c has 5% or less of the total DP; and (III) when the clogP value is 1.5 or more and less than 4.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or more and 500 or less, a has 50% or more of the total DP; b has 40% or less of the total DP; c has 5% or less of the total DP; and d has 15% or less of the total DP, and c and d have 1% or less of the total DP; (IV) when the clogP value is 4.5 or more and less than 5.5, and the total degree of polymerization (DP) of a+b+c+d is 200 or more and 500 or less, a has 50% or more of the total DP; b has 30% or less of the total DP; (V) When the clogP value is 5.5 or more and the total degree of polymerization (DP) of a+b+c+d is 200 or more and 500 or less, a has 60% or more of the total DP; b has 20% or less of the total DP; c has 2% or less of the total DP; and d has 8% or less of the total DP, with the proviso that c and d satisfy 1%≦c+d≦10% of the total DP.

12. PEG or a PEG derivative has the following structure: (In the formula, R ７ is a methoxy group, an ethoxy group, an acetyl group, or a benzyl group; R ８ is an amide bond, an ether bond, or an ester bond, which is a moiety that is bonded to the main skeleton; R ９ is a biodegradable linker; and n is 1 to 10. The polymeric micelle comprising the self-aggregating single polymer chain according to claim 11 .

13. A polymeric micelle comprising a self-aggregating single polymer chain according to claim 11 or 12, wherein the hydrophobicity (clogP value) of the hydrophobic group is 1 to 10.

14. A polymeric micelle comprising a self-aggregating single polymer chain according to claim 11 or 12, wherein the hydrophobic group is selected from the group consisting of a phenyl group, a naphthyl group, a pyrenyl group, anthracene, cholesterol, a steroid derivative, 1-pyrenemethylamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-naphthylmethylamine, DL-α-tocopherol, nerol, farnesol, and an anticancer drug.

15. A polymeric micelle comprising a self-aggregating single polymer chain according to claim 11 or 12, wherein the gadolinium complex is one or more selected from the group consisting of gadoteridol, meglumine gadoterate, and gadobutrol.

16. M ｗ (PBS) / M ｗ 13. A polymer micelle comprising a self-aggregating single polymer chain according to claim 11 or 12, wherein the number of associations of the single polymer chain in water or a buffer solution is less than 1.5, as calculated from the (chloroform) ratio.

17. When used in magnetic resonance imaging (MRI) to detect or diagnose cancer or tumors, the longitudinal relaxivity (r1) / transverse relaxivity (r2) is maintained at r1 of 20 s or less while maintaining r1 / r2 of ≥ 0.

70. －１ M.M. －１ The polymer micelle comprising the self-aggregating single polymer chain according to any one of claims 1 to 6, 11, and 12.

18. A pharmaceutical composition for treating cancer or tumors by neutron capture therapy, comprising a polymeric micelle consisting of a self-aggregating single polymer chain according to any one of claims 1 to 6, 11 and 12.

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