Micelle for hydrophobic molecule, method for delivering hydrophobic molecule using said micelle, and composition containing micelle encapsulating hydrophobic molecule for use in said method

Abstract

The present disclosure discloses a composition for intracellularly delivering hydrophobic molecules having a molecular weight of 500 or more. The composition of the present disclosure contains: amphiphilic block copolymers including an uncharged hydrophilic block and a hydrophobic block; and molecules that form a hydrophobic bond with the hydrophobic blocks in an aqueous solution, the block copolymers being crosslinked with each other. The hydrophobic molecules are released from micelles in the absence of crosslinking. Therefore, the crosslinking can be cleaved in response to pH.

Description

A micelle for hydrophobic molecules, a method for delivering hydrophobic molecules using the micelle, and a composition containing a micelle containing a hydrophobic molecule for use in the method.

[0001] This disclosure relates to micelles for hydrophobic molecules, a method for delivering hydrophobic molecules using such micelles, and a composition comprising micelles containing hydrophobic molecules for use in such method.

[0002] Patent Document 1 discloses micelles for delivering JQ-1, which has antitumor activity, and a polymer composition for forming the micelles. Patent Document 1 discloses that a hydrazide group is introduced into JQ-1, and the hydrazide is reacted with an aldehyde of the polymer, linking the two by a hydrazone bond.

[0003] Non-patent documents 1 and 2 provide an overview of PROTAC, a protein degradation inducer that is expected to have various clinical applications.

[0004] Non-patent documents 3 and 4 disclose ARV-825-encapsulated micelles, which are PROTACs that target BRD4. In non-patent documents 3 and 4, the micelles are formed from a polymer having the structure of methoxy-polyethylene glycol-polylactic acid. In non-patent documents 3 and 4, the therapeutic effect of these micelles on gliomas was confirmed by in vivo administration.

[0005] Non-patent document 5 discloses that ARV-825 exhibits antifibrotic activity against pulmonary fibrosis. In addition, PROTAC is expected to have antitumor activity against cancer and therapeutic effects against neurodegenerative diseases (see Non-patent document 6).

[0006] JP2019-142822A

[0007] Pharmacia, Vol. 57 No. 7 2021MEDCHEM NEWS 31(1) 36-40(2021)Acta Pharmaceutica Sinica B 2022;12(6):2658e2671Nanoscale, 2024, 16, 4378Respiratory Investigation, Volume 61, Issue 6, 2023, Pages 781-792 Antibodies (Basel). 2023 Jun 26;12(3):43.

[0008] This disclosure provides micelles for hydrophobic molecules, a method for delivering hydrophobic molecules using such micelles, and a composition comprising micelles encapsulating hydrophobic molecules for use in such method. This disclosure is particularly suitable for the encapsulation and intracellular delivery of hydrophobic molecules having large molecular weights. In a preferred embodiment of this disclosure, the polymer in the micelle is crosslinked, and the crosslinking of the polymer cleaves in the low pH environment of endosomes and lysosomes, releasing the encapsulated molecule. The molecule is bound to the polymer by hydrophobic bonds, but does not need to be covalently bonded. This disclosure also provides lyophilized formulations for the preparation of such compositions. In particular, the hydrophobic delivery difficulties and / or toxicity in non-target tissues that pose problems when administering PROTAC compounds to living organisms can be mitigated by encapsulation in micelles for hydrophobic molecules of this disclosure.

[0009] The present disclosure provides the following inventions: (1) A composition comprising (i) an amphiphilic block copolymer comprising an uncharged hydrophilic block and a hydrophobic block, and (ii) a micelle comprising a molecule (e.g., a hydrophobic molecule) that forms a hydrophobic bond with the hydrophobic block in an aqueous solution, wherein the hydrophobic block is a block copolymer having hydrophobic side chains, and the micelle has interpolymer crosslinks. (2) The composition according to (1) above, wherein the interpolymer crosslinks are pH-responsive to cleavage. (3) The composition according to (1) or (2) above, wherein the hydrophobic side chain comprises a hydrophobic moiety and a carbonyl group, the crosslinks are formed by a crosslinking agent having two hydrazide groups or amino groups, and a spacer (also called a linker) between the two groups. (4) The composition according to any one of (1) to (3) above, wherein the hydrophobic side chain comprises a hydrophobic moiety and a carbonyl group, the hydrophobic moiety comprises a phenyl group, the crosslinks are formed by a crosslinking agent having two hydrazide groups or amino groups, and a hydrophobic spacer between the two groups. (5) The hydrophobic block is given by the following formula: {In the formula, n1 is a numerical value from 1 to 3, n2 is a numerical value from 1 to 3, n1 and n2 may be the same or different, m1 is from 5 to 100, and has a number average of 5 to 100 formyl groups, one of *1 and *2 is linked to an uncharged hydrophilic polymer block, the other of *1 and *2 is hydrogen, a methoxy group, an acetyl group, an acetoxy group, a protecting group, a polymerizable group, or a hydrophobic group, and at least a portion of the formyl groups forms a hydrazone bond with the hydrazide or amino group of the crosslinking agent, as described in any of (1) to (4) above. (6) The composition according to any of (1) to (5) above, wherein the uncharged hydrophilic polymer block comprises polyalkylene glycol. (7) The composition according to any of (1) to (6) above, wherein the uncharged hydrophilic polymer block comprises polyethylene glycol. (8) The composition according to any of (1) to (7) above, wherein the molecule has a logP value of 1 or more. (9) The composition according to any one of (1) to (8) above, wherein the molecule has a molecular weight of 500 Da or more. (10) The composition according to (8) above, wherein the molecule has a molecular weight of 500 Da or more. (11) The composition according to any one of (1) to (10) above, wherein the molecule is an anticancer agent or a fluorescent dye. (12) The composition according to any one of (1) to (11) above, wherein the molecule comprises a target protein ligand and an E3 ubiquitin ligase ligand, and the target protein ligand and the E3 ubiquitin ligase ligand are linked via a linker or directly without a linker. (13) A lyophilized formulation comprising a lyophilized version of the composition according to any one of (1) to (12) above (which may be a pharmaceutical composition). (14) A ready-to-use kit comprising the lyophilized formulation according to (13) above and reconstituted water for preparing the composition according to any one of (1) to (12) above (which may be a pharmaceutical composition).

[0010] This disclosure describes the concept of one invention relating to crosslinked micelles as illustrated in this disclosure. It explains the concept of one invention relating to crosslinking as illustrated in this disclosure. Polymers can be crosslinked using the linkage reaction between aldehyde groups and hydrazide groups. NMR spectra of polymers having 4-formylbenzyl groups when crosslinked with various crosslinking agents are shown. The particle size distribution by dynamic light scattering (DLS) of a micelle formed using one polymer illustrated in this disclosure, and the estimated molecular structure and particle size distribution of the crosslinked portion after crosslinking with a crosslinking agent are shown, respectively. NMR spectra of micelles formed using one polymer illustrated in this disclosure before and after crosslinking are shown. NMR spectra of PROTAC-encapsulated micelles formed by one polymer illustrated in this disclosure before and after crosslinking, as well as NMR spectra of PROTAC alone and the polymer alone are shown. NMR spectra of ARV-825-encapsulated micelles formed by one polymer illustrated in this disclosure and micelles after crosslinking with various amounts of crosslinking agents are shown. The blood retention properties of micelles before and after crosslinking (i.e., uncrosslinked or crosslinked micelles) are shown. This document shows the time-course observation results of residual micelles in the ear vessels after intravenous administration of cross-linked micelles in vivo. It also shows the toxicity to the administration site after administration of bare ARV-825 or ARV-825-encapsulated micelles formed from one polymer as exemplified in this disclosure. A scheme for a therapeutic experiment using ARV-825-encapsulated micelles (ARV825 / m) formed from one polymer as exemplified in this disclosure is shown. The results of measuring luminescence of gliomas by IVIS after administration of bare ARV-825 or ARV-825-encapsulated micelles are shown. The changes in glioma volume after administration of bare ARV-825 or ARV-825-encapsulated micelles in a glioma orthotopic transplant model are shown. The Kaplan-Meier curves after administration of bare ARV-825 or ARV-825-encapsulated micelles in a glioma orthotopic transplant model are shown. This document outlines the treatment scheme for a glioma orthotopic transplant model. Treatment involved the combined use of ARV-825-encapsulated micelles and temozolomide (TMZ). The results of glioma luminescence measurement by IVIS 23 days after ARV-825-encapsulated micelle administration, and the results of glioma detection by MRI are shown.The Kaplan-Meier curves after administration of ARV-825-encapsulated micelles and after combination therapy with TMZ in a glioma orthotopic transplantation model are shown. Reporter assay results are shown after treating fibroblasts containing DNA with the luciferase gene operably linked downstream of the Smad binding element (SBE) and minimum promoter with bare ARV-825 or ARV-825-encapsulated micelles and culturing them in the presence and absence of TGF-β (upper panel shows measured values, lower panel shows relative trend intensity). The results of immunohistochemical staining confirming collagen I expression levels after treating fibroblasts with bare ARV-825 or ARV-825-encapsulated micelles and culturing them in the presence and absence of TGF-β are shown. This document shows the results of immunohistochemical staining to confirm the expression level of α-smooth muscle actin (αSMA) after treating fibroblasts with bare ARV-825 or ARV-825-encapsulated micelles and culturing them in the presence and absence of TGF-β. It also shows collagen deposition in lung tissue sections of a bleomycin-treated idiopathic pulmonary fibrosis (IPF) model. A treatment scheme for the IPF model is shown. Micro-CT images and lung volume after treatment of the IPF model are shown. The formation of IR780-encapsulated micelles and the NMR spectra of the formed mice are shown. These NMR spectra are compared with the NMR spectra of the polymer alone and IR780 alone. The dynamics of IR780 in mice after tail injection of IR780-encapsulated micelles are shown. NCL represents uncrosslinked mice, and CL represents crosslinked mice. The data shows the accumulation of free IR780 and IR780-encapsulated micelles (with and without crosslinking) in tumor-bearing models after thrombus administration. In the upper panel, the left bar represents data for mice with and without crosslinking, and the right bar represents data for mice with crosslinking. In the lower panel, the left bar represents the control, and the right bar represents data for free dye. Kaplan-Meier survival curves for mice administered BLM at various doses are shown. Micro-CT images of IPF model mice 2 weeks after administration of various treatments are shown. Hounsfield unit density (panel a) and quantitative analysis of lung air volume (panel b) in IPF model mice 2 weeks after administration of various treatments are shown. Kaplan-Meier survival curves for mice up to 21 days after BLM induction are shown.The survival curves up to day 21 of surviving mice that underwent therapeutic intervention and received IV administration of 10 mg / kg ARV-825 (maximum dissolved dose) and 10, 20, and 30 mg / ml ARV-825 micelles are shown. 2D and 3D μCT images of the lungs of mice that survived to day 21 after receiving ARV twice weekly or ARV / m at each dose starting one week after BLM (5 mg / kg) administration are shown. Representative IHC staining of type I collagen (panel a), α-SMA (panel b), and F4 / 8 (panel c) in lung sections of therapeutically intervened IFP model mice are shown. β-Gal (panel a) and P16 in lung sections of therapeutically intervened IFP model mice are also shown. INK4a+ Representative IHC staining for (Panel b) and PGDFRβ (Panel c) is shown. Hematological analysis results of blood cells collected from normal mice administered with PBS, ARV-825, and ARV-825 micelles (20 mg / kg), respectively, are shown. Biochemical analysis results of key markers for evaluating potential organ toxicity induced by administration of PBS, ARV-825, and ARV-825 micelles to normal mice are shown.

[0011] In this specification, "block copolymer" refers to a polymer comprising multiple polymer blocks having different monomer structures. A block copolymer, for example, includes hydrophilic polymer blocks and hydrophobic polymer blocks and is amphiphilic. While hydrophilicity and hydrophobicity are relative, in this specification, hydrophilic polymer blocks and hydrophobic polymer blocks have a difference in hydrophilicity to the extent that they can form micelles in aqueous solution. An amphiphilic block copolymer having a concentration exceeding the critical micelle concentration forms a core-shell structure in aqueous solution, with the hydrophobic polymer block located in the center and the hydrophilic polymer block located on the outer surface. For example, by causing micelles to form in the presence of hydrophobic molecules, the hydrophobic molecules can be encapsulated in the core of these micelles. Crosslinking between block copolymers stabilizes the micelles. Decomposition of crosslinking between block copolymers destabilizes the micelles and can release the hydrophobic molecules from the micelles.

[0012] In this specification, “targeting means” refers to means for actively targeting micelles to a target antigen in vivo. The targeting means can bind to the target antigen in vivo. The targeting means may be attached to the uncharged hydrophilic polymer block side, preferably the end, of a block copolymer having an uncharged hydrophilic polymer block and a hydrophobic polymer block. This allows the targeting means to be exposed in the micelles when the block copolymer forms micelles, and to link the micelles to the target antigen. Examples of targeting means include aptamers (e.g., DNA aptamers, RNA aptamers, modified nucleic acid aptamers), peptides (RGD, cyclic RGD), receptors that bind to a target, ligands for target receptors, antibodies, and their antigen-binding fragments and other antigen-binding substances (affimers, nanobodies, VHH antibodies, affibodies, DARPin, monobodies, scFv and other antibody fragments).

[0013] In this specification, "aptamer" refers to a nucleotide chain (including DNA and RNA) or peptide that exhibits affinity and selectivity for a specific molecule or target. Peptide aptamers can be chemically synthesized. Aptamers can also be obtained using methods that involve artificial molecular evolution. One such method is the SELEX method. The SELEX method selects aptamers with high selectivity and affinity for a specific molecule by repeating a cycle of selecting aptamers with affinity for a specific molecule from an oligonucleotide library, removing those that do not bind, amplifying the selected aptamers using an erupron DNA polymerase, and further selecting aptamers with affinity for the specific molecule several to several dozen times.

[0014] In this specification, "Affimer" is a protein having a biologically inactive and physically stable variant of stephin A or cystatin as its basic skeleton, and having antigen-binding sites in two loops presented on the same side from the four β-sheet structures of the basic skeleton. The amino acid sequence of the loop portion can be made diverse. By making the amino acid sequence of the loop portion diverse, it is possible to obtain affimers with binding affinity to various antigens by phage display. As the basic skeleton of an affimer, for example, a variant of stephin A having the amino acid sequence corresponding to SEQ ID NO: 1 disclosed in WO2009 / 136182 can be used, and a variant of stephin A in which the fourth glycine of stephin A may be replaced with arginine is a protein having heterologous amino acid sequences in two loops presented on the same side from the four β-sheet structures of the basic skeleton (for example, amino acid sites 46-54 and 67-84 of stephin A). The affimer has an α-helix structure at its N-terminus. Affimers may also be proteins that have a plant-derived cystatin as their basic backbone, and may have antigen-binding sites on two loops presented on the same side from the four β-sheet structures of the basic backbone. Examples of cystatin-based affimers include any one of the sequences SEQ ID NOs. 1 to 6 disclosed in WO2014 / 125290. The molecular weight of the affimer may be approximately 12 to 14 kDa.

[0015] In this invention, a "nanobody" is an antigen-binding protein based on the variable region domain of an antibody consisting solely of a heavy chain, as discovered in certain animals, in contrast to antibodies which consist of heavy and light chains. This antibody, consisting solely of a heavy chain, is commonly found in dromedary camels, Bactrian camels, llamas, and alpacas, and can bind to antigens solely through the variable region domain of the heavy chain. In recent years, similar antibodies consisting solely of heavy chains have also been discovered in cartilaginous fish (such as sharks). The nanobody has three complementarity-determining regions (CDRs), and binds to the antigen through these three CDRs. The nanobody can be obtained by immunizing animals that produce the above-mentioned antibody consisting solely of a heavy chain with an antigen, isolating B cells from the immunized animals, obtaining a cDNA library containing the variable region, incorporating it into a phage display library using M13 phage, and screening it with the antigen. The nanobody has a β-sheet structure at its N-terminus and C-terminus to which a fluorescent substance can be attached in this invention. In this invention, the β-sheet structure at the N-terminus and / or C-terminus of a nanobody, for example, the β-sheet structure at the C-terminus, can be linked to the β-sheet structure of a fluorescent protein or a fluorescently labeled tag.

[0016] In this specification, "scFv" is a single-chain antigen-binding protein in which the heavy chain variable region (VH) and light chain variable region (VL) of an antibody are linked by a flexible peptide linker. The flexible linker connects the heavy chain variable region and the light chain variable region to promote their association, while maintaining the antigen specificity of the original antibody by allowing antigen recognition between the two regions. The flexible linker can freely change its structure depending on the association state between the scFv and the antigen; for example, a glycine-rich sequence of approximately 15 amino acids (for example, serine may be inserted to ensure hydrophilicity) is preferably used. As a flexible linker, for example, a linker having the amino acid sequence -(GGGGGS)3- may be used. scFv can be obtained by selecting from a phage library in which the antigen-binding sites of scFv with framework sequences (for example, framework sequences of mammals such as humans) have been randomized, using the desired antigen-binding ability as an indicator. The scFv has β-sheet structures at its N-terminus and C-terminus to which a fluorescent substance can be attached according to the present invention. In the present invention, the β-sheet structures at the N-terminus and / or C-terminus of the scFv, for example, the β-sheet structure at the C-terminus, can be linked to the β-sheet structure of a fluorescent protein or a fluorescently labeled tag.

[0017] In this specification, “DARPin” refers to an antigen-binding protein developed by Molecular Partners AG. DARPin is an artificial protein having ankyrin repeat units (typically 2 to 30 units), each repeat unit containing a skeletal residue and a target interaction residue (see, e.g., WO2002 / 020565). The ankyrin repeat unit has a common folding structure consisting of two antiparallel α-helices followed by a β-hairpin {where the β-pair pin has a loop that binds to the next repeat unit}. The stacking of ankyrin repeat units forms a curved structure in DARPin. Target interaction residues may be present in the β-hairpin and the exposed portion of the first α-helix of the ankyrin repeat unit. DARPin can be obtained by selection based on its binding affinity to antigens. DARPin can be obtained using methods such as phage display, ribosome display, and plasmid display. DARPin has an α-helix at one end and a β-sheet at the other, and in the present invention, it provides a site for connecting a fluorescent substance.

[0018] In this specification, "monobody," also known as FingR or Adnectin, is a scaffold protein whose basic structure is the tenth domain of human fibronectin (fibronectin type III domain). This domain has a structure similar to the variable domain of an antibody, namely, it has seven β-sheet structures forming a β-sandwich and three loops on both sides corresponding to three complementarity-determining regions. Antigen binding specificity can be modified by the amino acid sequences of loop BC between the second and third β-sheets, loop DE between the fourth and fifth β-sheets, and loop FG between the sixth and seventh β-sheets. Alternatively, it can be modified by the amino acid sequences of the third, fourth, sixth, and seventh β-sheets in addition to loop CD between the third and fourth β-sheets and loop FG between the sixth and seventh β-sheets. The monobody has β-sheets at the N-terminus and C-terminus, which can provide sites for attaching fluorescent substances.

[0019] In this specification, "Affibody" refers to a small artificial protein molecule developed by Affibody AB, a Swedish biotechnology company (Experimental & Molecular Medicine volume 49, e306 (2017)). Affibody originally derives from a small triple-helix subdomain of the Z domain (IgG-binding domain) of protein A, which has been modified to serve as a targeting scaffold. To obtain Affibody, a large library has been created by randomizing 13 amino acids from two of the three α-helices. Affibody is very small, with a length of approximately 58 amino acids and a size of approximately 6 kDa. Known Affibody molecules that bind to HER2 include ABY-025, ZHER2:342, ZHER2:477, and ZHER2:2891. Furthermore, Z-j1 and Z-j2 are known as affibodies that bind to PD-L1. ZEGFR:1907 and ZEGFR:2377 are known as affibodies that bind to EGFR. ZHER3:8698 is known as an affibodies that bind to HER3. ZPDGFRb_3 and ZPDGFRβ:1 are known as affibodies that bind to PDGFRβ. ZAbeta3 is known as an affibo that binds to Aβ. ZVEGFR2 is known as an affibodies that bind to VEGFR2. These can be attached to the uncharged hydrophilic polymer block of the block copolymer, preferably to its ends, to form micelles, thereby enabling the delivery of these antigens.

[0020] In this specification, "uncharged hydrophilic polymer block" refers to a block made of a polar but uncharged polymer. Polarity can be provided, for example, by a group having an O atom or an N atom, thereby conferring hydrophilicity to the polymer block. By linking a hydrophilic polymer block with a hydrophobic polymer block, an amphiphilic block copolymer containing both hydrophilic and hydrophobic polymer blocks is formed. Uncharged hydrophilic polymer blocks are also known for their high biocompatibility. Examples of uncharged hydrophilic polymer blocks include polyalkylene glycol blocks and polyoxazoline blocks.

[0021] In this specification, “hydrophobic block” refers to an uncharged polymer block. A hydrophobic block is hydrophobic overall (compared to an uncharged hydrophilic block) and may have, for example, rings (e.g., aromatic rings, cycloalkyl groups) in its side chains. The aromatic ring is not particularly limited, but is preferably phenyl. The phenyl is substituted with an aldehyde group such as a formyl group for crosslinking. 1-6 Alkyl alkyl groups (for example, C 1-3 It may be substituted with an alkyl group.

[0022] In this specification, a "hydrophobic molecule" is a molecule having a logP value of 1 or more. The logP value is log 10 This is an index of hydrophobicity determined by ([Compound] Octanol phase / [Compound] Aqueous phase). The [Compound] Octanol phase and [Compound] Aqueous phase represent the concentration of the compound in the octanol phase and the concentration of the compound in the aqueous phase, respectively, when the compound is dissolved in a two-phase solution system of water and octanol. The higher the logP value, the more hydrophobic the molecule.

[0023] In this specification, "micelle" refers to a particle formed by the self-assembly of the amphiphilic block copolymer in an aqueous solution. Micelles have, for example, a size on the order of submicrometers. In micelles in an aqueous solution, the amphiphilic block copolymer is oriented so that the uncharged hydrophilic polymer block faces outward and the hydrophobic polymer block faces inward. Micelles can encapsulate hydrophobic molecules, which are encapsulated by forming hydrophobic interactions with the hydrophobic polymer block inside the micelle. Micelles can be stabilized by crosslinking. When the crosslinking is broken, the micelle disintegrates in vivo. This is because, as shown in the examples described later, amphiphilic block copolymers do not form micelles at concentrations below the critical micelle concentration (CMC), or the micelles formed in an environment containing amphiphilic block copolymers at concentrations below the CMC are prone to disintegration.

[0024] In this specification, "ligand" refers to a molecule that binds to a target protein. A molecule that binds to protein X is called a protein X ligand.

[0025] The disclosure provides a composition comprising (i) an amphiphilic block copolymer comprising an uncharged hydrophilic block and a hydrophobic block, and (ii) a micelle comprising a molecule that forms a hydrophobic bond with the hydrophobic block in an aqueous solution, wherein the hydrophobic block is a block copolymer having hydrophobic side chains, and the micelle has interpolymer crosslinks. In the disclosure, the amphiphilic block copolymer and the molecule are not covalently bonded.

[0026] The molecule is hydrophobic and may have logP values ​​of, for example, 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more. The molecule is hydrophobic and may have logP values ​​of, for example, 7 or less, 6 or less, 5 or less, or 4 or less. The molecule is hydrophobic and may have logP values ​​of, for example, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 7, 4 to 6, 4 to 5, 5 to 7, 5 to 6, or 6 to 7.

[0027] Further, the molecule may have a molecular weight of 200 or more, 300 or more, 400 or more, preferably 500 or more. In a preferred embodiment, the molecule may have a molecular weight of 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, 1000 or more, 1100 or more, 1200 or more, 1300 or more, 1400 or more, 1500 or more. In a preferred embodiment, the molecule may have a molecular weight of 5000 or less, 4000 or less, 3000 or less, 2000 or less, 1900 or less, 1800 or less, 1700 or less, 1600 or less, 1500 or less, 1400 or less, 1300 or less, 1200 or less, 1100 or less, or 1000 or less. In a certain preferred embodiment, the molecule may have a molecular weight of 500 - 5000, 500 - 4000, 500 - 3000, 500 - 2000, 500 - 1900, 500 - 1800, 500 - 1700, 500 - 1600, 500 - 1500, 500 - 1400, 500 - 1300, 500 - 1200, 500 - 1100, 500 - 1000.

[0028] In a certain preferred embodiment, the molecule is hydrophobic, has a logP value of 1 - 7, and may have a molecular weight of 500 - 5000. In a certain preferred embodiment, the molecule is hydrophobic, has a logP value of 1 - 7, and may have a molecular weight of 500 - 2000. The molecule may have, for example, a logP value of 1 - 7, 1 - 6, 1 - 5, 1 - 4, 1 - 3, 1 - 2, 2 - 7, 2 - 6, 2 - 5, 2 - 4, 2 - 3, 3 - 7, 3 - 6, 3 - 5, 3 - 4, 4 - 7, 4 - 6, 4 - 5, 5 - 7, 5 - 6, or 6 - 7 and may have a molecular weight of 500 - 5000, 500 - 4000, 500 - 3000, 500 - 2000, 500 - 1900, 500 - 1800, 500 - 1700, 500 - 1600, 500 - 1500, 500 - 1400, 500 - 1300, 500 - 1200, 500 - 1100, 500 - 1000.

[0029] Polymer crosslinking is preferably cleavable and environmentally responsive. For example, in late endosomes and lysosomes after being taken up by cells, the pH decreases. Therefore, the polymer crosslinking preferably cleaves in a pH-responsive manner. Examples of pH-responsive crosslinks include hydrazone bonds and crosslinks having a pH-responsive amide bond. Hydrazone bonds and pH-responsive amides cleave in response to a low pH environment (e.g., the late endosomal environment inside cells).

[0030] In one embodiment, the hydrophobic side chain contains a hydrophobic moiety and a carbonyl group. The crosslinking is preferably carried out between carbonyl groups between polymers. For example, in one embodiment, the hydrophobic side chain contains a hydrophobic moiety and a carbonyl group, the crosslinking agent has two hydrazide groups, one hydrazide group and one amino group, or two amino groups, the crosslinking agent has a spacer between the two groups, and the crosslinking is formed by the reaction of a carbonyl group with the hydrazide group or amino group.

[0031] In one embodiment, the hydrophobic moiety of the hydrophobic side chain contains an aromatic ring, preferably a phenyl group. In one embodiment, the hydrophobic side chain is substituted with a functional group having a carbonyl group. In one embodiment, the hydrophobic side chain contains formylphenyl.

[0032] In one embodiment, the hydrophobic side chain is -(CH2) n2 -CO-NH-(CH2) n3 -phenyl-formyl group may be included. n2 and n3 are each independently a natural number from 1 to 6, preferably 1 each.

[0033] In one embodiment, the hydrophobic polymer block has the following formula: {In the formula, n1 is a numerical value between 1 and 3, n2 is a numerical value between 1 and 3, n1 and n2 may be the same or different, m1 is between 5 and 100, and has a number average of 5 to 100 formyl groups, one of *1 and *2 is linked to an uncharged hydrophilic polymer block, and the other of *1 and *2 may be hydrogen, an alkoxy group (e.g., a methoxy group), an acetyl group, an acetoxy group, a protecting group, a polymerizable group, or a hydrophobic group, or a targeting means}. Originally, this polymer contains acid-degradable peptide bonds in its side chains and can be degraded in a low pH environment (in endosomes / lysosomes), but in a preferred embodiment, in the micelle, at least some of the aldehyde groups in the formyl groups of the hydrophobic polymer block form hydrazone bonds with the hydrazide or amino groups of the crosslinking agent, conferring pH dependence at the crosslinking portion, which cleaves the crosslink when the micelle is taken up by a cell, promoting the release of the encapsulated molecules.

[0034] In one embodiment, the targeting means targets, for example, a tumor antigen. In one embodiment, the tumor antigen is not particularly limited, but examples include α-integrin, α-fetoprotein (AFP), ALK, B7-H3, B7-H4, B-cell melanoma antigen (BAGE), BCMA (B-cell maturation) Examples include antigens, CA-125, CA19-9, CD19, CD20, CD22, CD30, CD33, cancer embryo antigen (CEA), CTLA-4, EGFR, EpCAM, folate receptor α (FRα), GD2, glypican 3 (GPC3), HER2, ICAM1, IGF-1R, IL-13, IL-17A, IL-6, IL-6R, L1CAM, MAGE-A1, mesothelin (MSLN), MUC1, NY-ESO-1, nectin-4, PDGFR, PD-1, PD-L1, PSA, PSMA, SLAMF7, tissue factor, TRIPO-2, TMEM180, TMEM132A, TRIPO-2, VEGF, VEGFFR, VEGFFR2, and WT1. Insoluble fibrin is not a surface antigen of cancer cells, but it is produced by bleeding that inevitably occurs in solid tumor tissue and can be an effective target for ADCs. Other examples include α-integrin, α-fetoprotein (AFP), ALK, B7-H3, B7-H4, B-cell melanoma antigen (BAGE), and BCMA (B-cell maturation). Examples include antigens, CA-125, CA19-9, CD19, CD20, CD22, CD30, CD33, cancer embryo antigen (CEA), CTLA-4, EGFR, EpCAM, folate receptor α (FRα), GD2, glypican 3 (GPC3), HER2, ICAM1, IGF-1R, IL-13, IL-17A, IL-6, IL-6R, L1CAM, MAGE-A1, mesothelin (MSLN), MUC1, NY-ESO-1, nectin-4, PD-1, PD-L1, PSA, PSMA, SLAMF7, tissue factor, TRIPO-2, TMEM180, TMEM132A, TRIPO-2, VEGF, VEGFR, and WT1. Insoluble fibrin is not a surface antigen of cancer cells, but it is produced by bleeding that inevitably occurs in solid tumor tissue and can be an effective target for ADCs. The targeting means can bind to amidroids.Examples of amyloids, though not particularly limited, include amyloid-beta, α-synuclein, huntingtin, TDP-43 or its aggregates, tau, and tau oligomers.

[0035] Examples of targeting methods for cancer antigens include ibritumomab, tocitumomab, catumakisomab, blinatumomab, moxetumomab, rituximab, cetuximab, dinutuximab, isatuximab, margetuximab, daclizumab, trastuzumab, gemtuzumab, alemtuzumab, bevacizumab, natalizumab, mogamulizumab, pertuzumab, obinutuzumab, pembrolizumab, elotuzumab, atezolizumab, ocrelizumab, inotuzumab, polatuzumab, sacituzumab, tafacitamab, and be Examples include lantamab, naxitamab, roncasutuximab, dostalimab, panitumumab, ofatumumab, ipilimumab, ramucirumab, nivolumab, nesitumumab, daratumumab, olaratumumab, avelumab, durvalumab, enfotumab, amivantamab, tisotumab, semiprimab, teventafusp, as well as antibodies having a heavy chain variable region with heavy chain CDR1-3 and a light chain variable region with light chain CDR1-3 of the same antibody, and antigen-binding fragments of these antibodies.

[0036] The presumed cross-linked polymer may have the following substructures: {In the formula, *5 and *6 are linked to the backbone or side chains of different polymer chains, and L1 is a linker, which may be derived from a crosslinking agent.}. L1 may be a non-cleavable, inert linker. L1 is not particularly limited, but may be one or more selected from the group consisting of a single bond, alkylene (e.g., methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, and decylene), phenyl, alkylenephenyl, alkylenephenylalkylene, polyethylene glycol (PEG), alkylene-PEG, PEG-alkylene, and alkylene-PEG-alkylene.

[0037] Examples of polymers, crosslinking agents, and hydrophobic molecules preferred in this disclosure are given below.

[0038] (1) Crosslinkable amphiphilic polymer methoxy-poly(ethylene glycol)-b-poly[N-((4-formylbenzyl)-asparagineamide]

[0039] {In the formula, n = 12, m is the number of aldehydes, and m = 12 to 35.}

[0040] This polymer can be prepared by introducing aromatic aldehyde functional groups to methoxy-poly(ethylene glycol)-b-poly(β-benzyl-aspartamide) (see Figure 1 in JP2019142822A for details of the polymer and its preparation method). In the above, m is defined as the number of aldehydes (number of formyl groups) because there is significance in defining the number of aromatic aldehyde functional groups. It is clear from the examples described later that the number of aromatic aldehyde functional groups contributes to the formation of a hydrophobic environment, as well as to the stabilization effect of micelles by π-π stacking, and the stabilization effect of micelles and the promotion of environmentally responsive disintegration by crosslinking between polymers utilizing aldehyde groups. In one embodiment, the polymer has acid-degradable peptide bonds in its side chains. Acid hydrolysis resistance is known to increase along the series aromatic aldehyde < aromatic ketone < aliphatic ketone < aliphatic aldehyde, which means that aromatic aldehydes and aromatic ketones are more susceptible to acid than those prepared from aliphatic ketones. This property of aromatic aldehydes and aromatic ketones promotes the degradation of the aforementioned binding at low pH (pH 3.5–5.5) within endosomes / lysosomes, while remaining stable in the extracellular environment of tumor cells (pH 6.5–7), thus reducing or avoiding the release of encapsulated substances outside the target. This is expected to reduce toxicity, for example, when delivering anticancer drugs.

[0041] (2) Crosslinking agent The crosslinking agent can be various reagents that can crosslink aldehydes. The crosslinking agent may be non-cleavable, but preferably it may be cleavable and cleave in response to the environment. To impart environmental responsiveness, for example, the hydrophobic portion of the polymer may have an aromatic aldehyde functional group, one of the crosslinking agents may be a hydrazide, and the other may be a group that reacts with the aromatic aldehyde functional group (hydrazide group, amino group, hydroxyl group, thiol group, phenol group, carbonyl group, etc.). In the crosslinking agent, one group and the other group are linked directly or via a linker. The linkage with the aromatic aldehyde functional group is stable, but the linker may have an acid-degradable group (hydrazone bond, amide bond, ester bond, acetal bond, ketal bond, Schiff base bond).

[0042] 1. ADH: Dihydrazide adipic acid (CAS: 1071-93-8) ADH is a highly versatile compound widely used as a crosslinking agent in various industrial applications, particularly in the formulation of coatings, adhesives, and sealants. A bifunctional molecule, ADH has two hydrazide (-NH-NH2) groups bonded to a flexible adipic acid skeleton. This structure allows it to react with various functional groups, especially carbonyl-containing compounds such as aldehydes and ketones, to form stable covalent bonds. Bifunctional molecules typically have a structure in which two different monofunctional molecules are linked, with or without a linker.

[0043] 2. IDH: Isophthalic acid dihydrazide (CAS: 2760-98-7) The central structure of IDH is based on an isophthalic acid moiety consisting of a benzene ring with two carboxylic acid groups (-COOH) bonded at the 1,3 position (meta position). In IDH, these carboxylic acid groups are converted to hydrazide groups, which are responsible for the crosslinking ability.

[0044] 3. ODH: Oxalyl dihydrazide (CAS: 996-98-5) The central oxalyl group (C(=O)-C(=O)) is a short, rigid linker consisting of two carbonyl groups (C=O) linked by a single carbon-carbon bond. This rigidity allows for more controlled crosslinking reactions because the molecule has less flexibility to rotate or bend.

[0045] 4. PEG-DH: Hydrazide-PEG4-Hydrazide (PurePEG, 233804-250, CAS: 2752050-42-1)

[0046] 5. PDH: Pentanedihydrazide (Cosmo Bio Co., Ltd., SC-296037, CAS: 1508-67-4)

[0047] 6. SDH: Dihydrazide succinate (CAS: 4146-43-4) SDH is a crosslinking agent known for its straightforward yet effective structure, which can enhance the performance of linkers. The succinic acid skeleton of SDH is composed of flexible aliphatic chains (-CH2-CH2-), providing a degree of flexibility to the crosslinking network. This flexibility is advantageous in applications where a balance between strength and elasticity is required.

[0048] 7. TDH: Dihydrazide terephthalate (CAS: 136-64-1)

[0049] 8. EDA: Ethylenediamine (CAS: 107-15-3) Ethylenediamine (EDA) is a simple yet highly reactive organic linker.

[0050] Diamines: Due to their high nucleophilicity caused by the amine group, they readily react with carboxylic acids, isocyanates, epoxides, and other electrophilic compounds, making them useful in a wide range of chemical synthesis processes, such as the production of polyamides and epoxy curing. In crosslinking, they form amine-based bonds within the polymer, such as amide bonds in reactions with carboxylic acids and urethane bonds in reactions with isocyanates.

[0051] While hydrazide groups are also nucleophilic, they tend to react more selectively with carbonyl-containing compounds (aldehydes, ketones, etc.) to form hydrazone bonds. Therefore, dihydrazides are particularly effective as crosslinking agents in applications where such carbonyl compounds are present. Reactions with aldehydes and ketones form hydrazone bonds, resulting in a strong and stable crosslinking network within the polymer. For example, the reaction between a dihydrazide and an aldehyde produces the following reaction:

[0052]

[0053] The hydrazone bonds formed by crosslinking are pH-responsive and cleave when the pH decreases. For example, when taken up into cellular endosomes, they cleave in response to a decrease in the pH of the endosomes and lysosomes. Hydrophobic molecules of this disclosure would be released from micelles if crosslinking were absent, but this disclosure utilizes this property to effectively release hydrophobic molecules within cells by cleaving the crosslinks in a pH-responsive manner. Furthermore, the hydrazone bonds are stable at normal pH levels in the blood and in vivo.

[0054] (3) Hydrophobic compounds Below, we will give examples of numerous compounds, including PROTAC, as examples of hydrophobic compounds.

[0055] Proteolytically Targeted Chimeras (PROTACs) PROTACs generally have a site that binds to a target protein (site A) and a site that binds to an E3 ubiquitin ligase (site B), and sites A and B are linked, with or without a linker. The linker of a PROTAC expected to be active in cells is non-cleavable. That is, it does not cleave in vivo until it expresses its intended function. Examples of target E3 ubiquitin ligases include von Hippel-Lindou (VHL), Cereblon (CRBN), MDM2, IAP, and KEAP1. Target proteins include kinases (e.g., BTK, CDK, EGFR), transcription factors (e.g., androgen receptor (AR), estrogen receptor (ER), Myc), apoptosis regulatory proteins (e.g., Bcl family (especially Bcl-2), MCL-1), proteases (e.g., PSMA), cell cycle regulators (e.g., CDK4 / 6, Aurora kinase), immune checkpoints (e.g., PD-1, PD-L1, and CTLA-4), viral proteins (e.g., Tat protein, HBx protein), abnormal proteins (e.g., p52, tau, huntingtin protein, TDP-43, α-synuclein), and signaling molecules (e.g., RAF, STAT3, and mTOR) (including normal and mutant forms of each). Because it is possible to target and induce degradation of such a wide variety of molecules, a diverse range of diseases such as cancer, infectious diseases, fibrosis, neurodegenerative diseases, inflammatory diseases, and immune diseases are targets for treatment in this disclosure.

[0056] The site that binds to E3 ubiquitin ligase (site B) may have, for example, any of the structures shown below or derivatives thereof (many known derivatives exist). {In the formula, "*" indicates the linker or binding site to site A. The lower right is known as a VHL ligand, and the others are known as CRBN ligands.}

[0057] Furthermore, the site that binds to E3 ubiquitin ligase (site B) may have, for example, any of the structures shown below or derivatives thereof (many known derivatives exist). {In the formula, "*" indicates the linker or binding site to site A. From top to bottom, these represent bestatin and VH032.} Bestatin targets cIAP1, and VH032 targets VHL. Bestatin methyl ester is also a ligand that targets cIAP1. In addition, MV1 and LCL161 are also cIAP1 ligands (see Pharmacia, Vol. 57 No. 7 2021).

[0058] Furthermore, the site that binds to E3 ubiquitin ligase (site B) may have, for example, a nutrin or a derivative thereof (many known derivatives exist) having the structure shown below. {In the formula, "*" indicates the linker or the binding site to site A.} Nutrin is an MDM2 ligand.

[0059] The site that binds to the E3 ubiquitin ligase (site B) can be, for example, a peptide. For example, ALAPYIP (SEQ ID NO: 1) binds to VHL. Also, for example, LDPETGEYL (SEQ ID NO: 2) binds to Keap1. RRRG (SEQ ID NO: 3) is also an E3 ligase ligand. These can also be effectively used as site B. A cell membrane permeable peptide (CPP) may be linked to the peptide ligand. Examples of CPPs include polyarginine (preferably octaarginine). Also, for example, RAEDSGNESEGE (SEQ ID NO: 4) {where both serines are phosphorylated} can also be used as an E3 ligase ligand.

[0060] PROTAC comprises site A, site B, and a linker, each of which is often a large molecule, and the overall molecule tends to be large as well. Therefore, PROTAC may have problems with membrane permeability. In one embodiment, the linker contains polyethylene glycol. In a preferred embodiment, the linker contains polyethylene glycol and has a logP value of 1 or more.

[0061] As a guideline, molecules (including PROTAC) having a molecular weight of 500 or more and a logP value of 1 or more can be suitably encapsulated in the micelles of this disclosure.

[0062] 1. ARV-825 (CAS: 1818885-28-7) ARV-825 is a potent and selective protac designed to target and degrade BET proteins such as BRD4, binding to BET proteins via a targeting ligand. It also recruits CRBN E3 ligase via a ligase-binding domain. Therefore, the PEG segment of the above structure has the BET protein ligand on the left and the CRBN ligand on the right. In preclinical trials, ARV-825 has shown promise as a treatment for cancers in which BET proteins play a crucial role, such as certain glioblastomas, leukemias, and lymphomas. By degrading BET proteins, ARV-825 inhibits the transcription of oncogenes driven by these proteins, thereby killing cancer cells. Because ARV-825 causes degradation of BET proteins rather than simply inhibiting them, it is more effective than conventional BET inhibitors, especially when resistance to these inhibitors has developed. PROTACs having other CRBN ligands instead of the CRBN ligand, or PROTACs having E3 ubiquitin ligase ligands such as VHL ligand, IAP ligand, Keep1 ligand, and MDM2 ligand instead of the CRBN ligand, can also be preferably used. Thus, PROTACs may have other ligands as appropriate as the E3 ubiquitin ligase ligand.

[0063] 2. LC2: (BLD Pharmatec, BD01255712, CAS: 2502156-03-6)

[0064] LC-2 is a small molecule drug that functions as a PROTAC specifically designed to target and degrade KRAS, a known oncogene that frequently mutates in various cancers, including lung, colorectal, and pancreatic cancer. Because KRAS mutations are difficult to target with conventional small molecule inhibitors, PROTACs like LC-2 represent a promising new approach. LC-2 has a ligand that selectively binds to the KRAS protein, and the other end is designed to recruit the E3 ubiquitin ligase von Hippel-Lindau (VHL). By degrading KRAS, LC-2 may be able to inhibit the proliferation and survival of cancer cells that depend on this oncogene. One of the challenges with targeting KRAS with conventional inhibitors is the development of drug resistance. LC-2's mechanism of action offers a way to overcome resistance arising from mutations and compensatory pathways by degrading the protein rather than simply inhibiting its function.

[0065] 3. Fulvestrant (CAS: 129453-61-8) Fulvestrant is a selective estrogen receptor downregulator (SERD) primarily used to treat estrogen receptor-positive breast cancer. It is a potent anti-estrogen drug that inhibits the activity of estrogen receptors by degrading them, thereby affecting cancer cell proliferation. Fulvestrant has a modified steroid-like structure that enhances its ability to bind to estrogen receptors. Its structure is similar to estradiol, but significantly altered to make it an effective antagonist. This structural change triggers the degradation of estrogen receptors. Fulvestrant promotes receptor ubiquitination, leading to proteasome-mediated destruction.

[0066] 4. ABT-263 (CAS: 923564-51-6) ABT-263, also known as Navitoclax, is a molecule that targets Bcl family proteins. ABT-263 targets Bcl family proteins such as Bcl-2, Bcl-XL, and Bcl-w, inducing their degradation.

[0067] 5. DT2216 (CAS: 2365172-42-3) DT2216 is a compound in which Navitrax and the von Lippel-Lindau (VHL) E3 ubiquitin ligase targeting site are linked by a linker.

[0068] 6. KT-474 (CAS: 2432994-31-3) KT474 is a PROTAC that targets IRAK4.

[0069] 7. ARV-110 (CAS: 2222112-77-6)

[0070] 8. NX-2127 (CAS: 2416131-46-7) NX-2127 is a PROTAC that targets BTK. NX-2127 is also effective against the BTKC481S BTK variant.

[0071] 9. KT-333 (CAS: 2502186-79-8) KT-333 is a protaproyltransmitter that targets STAT3. It is expected to be used in the treatment of hematological malignancies such as large granular lymphocytic leukemia (LGL-L), peripheral T-cell lymphoma (PTCL), and cutaneous T-cell lymphoma (CTCL).

[0072] 10. CTF7455 (CAS: 2504235-67-8) CTF7455 is a PROTAC that targets the zinc finger transcription factors IKZF1 and Aeolus (IKZF3).

[0073] 11. MRT-2359 (CAS: 2803881-11-8) MRT-2359 targets GSPT1. It exhibits antitumor effects against MYC-driven cancers such as non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) accompanied by N-Myc and L-Myc expression.

[0074] 12. ARV-471 (CAS: 2229711-68-4) ARV-471 is a protac that targets the estrogen receptor (ER). ARV-471 can induce degradation of ER variants as well, and is expected to have applications in the treatment of ER-positive breast cancer.

[0075] 12. FHD-609 (CAS: 2676211-64-4) FHD-609 is a PROTAC that targets BRD9. FHD-609 has antitumor effects and is undergoing clinical trials as a treatment for synovial sarcoma.

[0076] Other potentially useful PROTACs include AC682, which targets ER; CC-94676, which targets AR; KT-413, which targets IRAK4; and NX-2127 and NX-5948, which target BTK (see Antibodies 2023, 12, 43).

[0077] The following are examples of anticancer drugs: 1. JQ1 (CAS: 1268524-70-4) JQ1 is a small molecule compound that selectively inhibits bromodomain and extraterminal (BET) family proteins, including BRD2, BRD3, BRD4, and BRDT. It selectively inhibits BET family proteins, including BRD2, BRD3, BRD4, and BRDT, and is often used to study the role of BET proteins in various biological processes and diseases, particularly cancer. Preclinical studies have shown its potential for the treatment of various cancers, including leukemia, lymphoma, and solid tumors.

[0078] 2. MRTX 849 (CAS: 2326521-71-3) MRTX849 is designed to specifically bind to the KRAS G12C mutant, which has a specific cysteine ​​residue at position 12. This mutation is characterized by the substitution of glycine with cysteine. The drug forms a covalent bond with the cysteine ​​residue of the KRAS G12C protein. The drug is currently in various clinical trials to evaluate its safety and efficacy against KRAS G12C-mutated cancers. Early results are promising, indicating the potential for significant clinical benefits.

[0079] 3. Lenalidomide (CAS: 191732-72-6) Lenalidomide is an immunomodulatory drug (IMiD) used to treat various cancers, including multiple myeloma and certain types of lymphoma. Lenalidomide is known to modulate the immune system and affect the proliferation and survival of cancer cells. It modulates the immune system by affecting the activity of T cells, natural killer (NK) cells, and macrophages. Lenalidomide enhances the immune response against cancer cells, helping the body better recognize and destroy them.

[0080] 4. Gemcitabine (CAS 95058-81-4) Gemcitabine is a modified nucleoside analog of deoxycytidine. Its chemical structure involves the replacement of the 2' and 3' hydroxyl groups of the ribose sugar in the deoxycytidine skeleton with fluorine atoms. This modification makes it a potent inhibitor of DNA synthesis. It is particularly effective against cancers such as pancreatic cancer, non-small cell lung cancer (NSCLC), breast cancer, and bladder cancer.

[0081] 5. Etoposide (CAS: 33419-42-0) Etoposide is a chemotherapeutic agent primarily used to treat various cancers, including lung cancer, testicular cancer, and leukemia. It is classified as a topoisomerase II inhibitor and plays a crucial role in its mechanism of action against cancer cells.

[0082] 6. Nintedanib (CAS: 656247-17-5) Nintedanib is a multi-kinase inhibitor primarily used to treat certain types of cancer and fibrous diseases. Because nintedanib targets various tyrosine kinases involved in tumor growth and fibrosis, it is a useful drug for conditions in which these pathways are dysregulated.

[0083] 7. Crizotinib (CAS: 877399-52-5) Crizotinib is a small molecule tyrosine kinase inhibitor primarily used to treat certain cancers. It is particularly effective in treating non-small cell lung cancer (NSCLC) with specific genetic mutations. Crizotinib has a complex chemical structure containing a pyridine ring, a cyclopropyl group, and a benzyl group. Its structure is designed to specifically inhibit target kinases involved in the proliferation and survival of cancer cells.

[0084] 8. Epirubicin (CAS: 56420-45-2) Epirubicin is an anthracycline antibiotic used as a chemotherapeutic agent in the treatment of various cancers. Epirubicin is a derivative of doxorubicin and has a similar mechanism of action and therapeutic applications. Epirubicin possesses a modified sugar moiety and contains a planar anthraquinone ring system that is crucial for DNA interaction. Epirubicin is widely used in the treatment of breast cancer, lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma, ovarian cancer, and other solid tumors, and is effective against a broad range of cancers, making it a versatile component in chemotherapy regimens.

[0085] 9. Temozolomide (CAS: 85622-93-1) Temozolomide (TMZ) is an oral alkylating agent used to treat certain cancers, particularly brain tumors. Temozolomide is gaining attention as a treatment for glioblastoma multiforme and other types of gliomas.

[0086] Furthermore, a substance having physiological activity against target cells can be encapsulated in the micelles. In a preferred embodiment, the physiologically active substance is a cytotoxin. Examples of cytotoxins include chemotherapeutic agents. Examples of chemotherapeutic agents include chemotherapeutic agents having antitumor effects such as mitotic inhibitors, DNA topoisomerase 1 inhibitors, alkylating agents, antimetabolites, hormone suppressants, immune checkpoint inhibitors, molecular targeted therapeutic agents, microtubule inhibitors, DNA minor groove binders, etc., and each can be preferably used. In addition, examples of chemotherapeutic agents include, for example, anticancer agents such as commercially available anticancer drugs, for example, auristatins (auristatin E, auristatin F, auristatin F phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin F, and their derivatives), maytansinoids DM1 and DM4 and their derivatives), camptothecins (SN-38, irinotecan, rutotecan, DB67, BMP1350, ST1481, CKD602, topotecan, and exatecan, and their derivatives), DNA minor groove binders (engein, lexitropsin, duocarmycin and their derivatives), taxanes (paclitaxel and docetaxel and their derivatives), polyketides (discodermolide and its derivatives), anthraquinones (mitoxantrone and its derivatives), benzodiazepines (pyrrolobenzodiazepines, indolinobenzodiazepines, and oxazolidinobenzodiazepines and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin and their derivatives), cardenolides (digitoxin and its derivatives), calicheamicin, epothilone, cryptophycin, semadotin, semadotin, lysocine, netropsin, combretastatin, erythrobins, etoposide, T67 (tulirik), and nocodazole), radioisotopes (for example, 32 P, 60 C, 90 Y, 111 In, 131 I, 125 I, 153 Sm, 186 Re,188 Re, and 212 Examples include complexes of Bi with a chelating agent, toxins (e.g., diphtheria toxin A, pseudomonas endotoxin, lysine, saporin, etc.), halichondrin B such as mytansine and eribulin, and their derivatives, as well as nucleic acids that exhibit antitumor effects (e.g., siRNA, shRNA, gapmers, mixmers, microRNA, etc.). As cytotoxic agents, pharmaceutically acceptable salts, solvates (e.g., hydrates), esters, or prodrugs of the above cytotoxic agents may be used.

[0087] The following are examples of dyes: 1. IR 780 (CAS: 207399-07-3) IR-780 iodide is a near-infrared lipophilic cation heptametine fluorescent dye. It exhibits good fluorescence intensity and can be used as a photothermal therapy agent. It shows a light absorption spectrum at 780 nm and is useful as a medical sonodynamic therapy agent.

[0088] 2. Indocyanine Green (CAS: 3599-32-4) Indocyanine green (ICG) is a dye primarily used in medical imaging. Due to its unique properties and behavior within the body, it is known for its use in fluorescence imaging and liver function tests. It is used to measure cardiac output, liver function, hepatic and gastric blood flow, and in ophthalmic and cerebral angiography. Its spectral absorption peak is approximately 800 nm. ICG is used in fluorescence-guided surgery to visualize blood vessels and tissues during surgery. ICG helps to identify tumor margins and assess the extent of cancerous tissue during surgery.

[0089] 3. Nile Red (CAS: 7385-67-3) Nile Red is a synthetic dye primarily used for staining and imaging lipids in biological samples. Known for its fluorescence properties, Nile Red is a valuable tool in cell biology and biochemistry. It is widely used to stain and visualize intracellular lipid droplets and lipid-rich structures. It is particularly useful for studying adipocytes (lipid cells) and understanding lipid metabolism.

[0090] Other fluorescent dyes include, for example, dialkylcarbocyanine dyes (e.g., 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), 3,3'-dioctadecyloxycarbocyanine perchlorate (DiO), and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiD)), borondipyrmethene (BODIPY) (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581 / 591, BODIPY TR, BODIPY 630 / 650, BODIPY Examples include 650 / 665), rhodamine dyes (e.g., rhodamine 123, rhodamine B, rhodamine 6G), pyrroline Y (pyrometene 546, pyrrometene 597, azadipyrometene), indocyanine green (ICG, ICG-EG4-sulfo-OSu, ICG-EG8-sulfo-OSu, ICG-sulfo-OSu), fluorescent molecules having a polymethine skeleton (e.g., O0574, C3693), curcumin, tetramethylrhodamine (e.g., tetramethylrhodamine methyl ester, 5-carboxytetramethylrhodamine), fluorescent derivatives thereof, and salts thereof. As fluorescent dyes, those having a logP value of 1 or more and a molecular weight of 500 or more are particularly preferred.

[0091] In one preferred embodiment, the hydrophobic polymer block is The structure is represented by {wherein n1 is a numerical value between 1 and 3, n2 is a numerical value between 1 and 3, n1 and n2 may be the same or different, m1 is between 5 and 100, and has a number average of 5 to 100 formyl groups, one of *1 and *2 is linked to an uncharged hydrophilic polymer block, the other of *1 and *2 is hydrogen, a methoxy group, an acetyl group, an acetoxy group, a protecting group, a polymerizable group, or a hydrophobic group, and at least some of the aldehyde groups in the formyl group form a hydrazone bond with the hydrazide or amino group of the crosslinking agent}, and the crosslinking agent may be SDH.

[0092] In one preferred embodiment, the non-charged hydrophilic polymer block may be a polyethylene glycol block.

[0093] In a preferred embodiment, the molecule may be a compound having a molecular weight of 500 or more and a logP value of 1 or more (particularly a protein-targeting chiller (PROTAC)). PROTAC is a bifunctional molecule that binds to a target protein with one arm and to an E3 ubiquitin ligase with the other arm, leading to ubiquitin-dependent degradation of the target protein. Because PROTAC is a bifunctional molecule, it has a large molecular weight. In a preferred embodiment, the molecule may be a fluorescent dye having a molecular weight of 500 or more and a logP value of 1 or more. In a preferred embodiment, the molecule may be an anticancer agent having a molecular weight of 500 or more and a logP value of 1 or more. In a preferred embodiment, the molecule may be a protein having a molecular weight of 500 or more and a logP value of 1 or more.

[0094] Micelles can be obtained by mixing the block copolymer and molecules of the present disclosure. Micelles may preferably have an average particle size of 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, or 30 nm or less, as measured by DLS. Micelles may preferably have an average particle size of 20 nm or more, more preferably 30 nm or more, as measured by DLS. Micelles may have a polydispersity index (PDI) of 0.3 or less, 0.25 or less, 0.2 or less, 0.15 or less, or 0.1 or less. Micelles may have a PDI of 0.1 or more. In a preferred embodiment, the micelles have an average particle size of 30 nm to 120 nm as determined by DLS and a PDI of 0.1 to 0.3, and in a more preferred embodiment, they have an average particle size of 30 nm to 80 nm as determined by DLS and a PDI of 0.1 to 0.3.

[0095] In one preferred embodiment, the micelles of the Disclosure circulate in the blood for 10 hours after tail vein administration to mice. In one preferred embodiment, the circulating blood volume of the mice at 10 hours may be 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the circulating blood volume at 1 hour after tail vein administration to mice. The circulating blood volume of the micelles can be measured in the blood vessels of a depilated ear. The micelles may be labeled with a fluorescent dye, for example, so that the circulating blood volume of the mice can be estimated by fluorescence intensity using a fluorescence microscope (such as a confocal fluorescence microscope).

[0096] In one preferred embodiment, the micelles of the present disclosure may have a particle size of 50 to 100 nm by dynamic light scattering (DLS), and a polydispersity index of 0.1 to 0.3, preferably 0.2 or less.

[0097] In one preferred embodiment, a method for treating fibrosis (e.g., pulmonary fibrosis) in a subject having fibrosis (e.g., pulmonary fibrosis) is provided, comprising administering to the subject a therapeutically effective amount of micelles of the Disclosure encapsulating an antifibrotic agent, and a pharmaceutical composition comprising such micelles for use in the method may be provided. Examples of antifibrotic agents include ARV-825, pirfenidone, and nintedanib. Examples of fibrosis include idiopathic pulmonary fibrosis, secondary fibrosis, pulmonary fibrosis, hepatic fibrosis, myocardial fibrosis, renal fibrosis, cutaneous fibrosis, collagen disease-related fibrosis, chronic inflammatory disease-related fibrosis, acute fibrosis, and chronic fibrosis. Examples of pulmonary fibrosis include idiopathic pulmonary fibrosis, drug-induced pulmonary fibrosis such as bleomycin-induced pulmonary fibrosis, occupational pulmonary fibrosis, radiation-induced pulmonary fibrosis, collagen disease-related pulmonary fibrosis, pulmonary fibrosis with chronic hypersensitivity pneumonitis, and pulmonary fibrosis due to interstitial pneumonia.

[0098] In one preferred embodiment, a method for treating a tumor (particularly cancer) in a subject having a tumor (particularly cancer) is provided, comprising administering to the subject a micelle of the Disclosure containing a therapeutically effective amount of an anticancer agent, and a pharmaceutical composition comprising the micelle for use in such method.

[0099] Tumors may be, but are not limited to, hematopoietic tumors or solid cancers. Tumors may be malignant. Tumors may be benign. Tumors may include, but are not limited to, solid cancers such as lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, stomach cancer, colorectal cancer, uterine cancer, ovarian cancer, skin cancer, thyroid cancer, thymic cancer, kidney cancer, testicular cancer, penile cancer, liver cancer, biliary tract cancer, brain tumors, bone and soft tissue tumors, retroperitoneal tumors, angiovascular and lymphangiosarcomas, and metastatic cancers thereof; and hematopoietic tumors such as myeloproliferative neoplasms ( For example, chronic myeloid leukemia (e.g., BCR-ABL1 fusion gene positive), chronic neutrophilic leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic eosinophilic leukemia, mastocytosis, and unclassifiable myeloproliferative neoplasms), eosinophilia and myelolymphoid neoplasms with PGDFRA, PDGFRB, or FGFR gene abnormalities, myelodysplastic / myeloproliferative neoplasms (e.g., chronic myelomonocytic leukemia, atypical Chronic myeloid leukemia (e.g., BCR-ABL1 fusion gene negative), juvenile myelomonocytic leukemia, unclassifiable myelodysplastic / myeloproliferative neoplasm), myelodysplastic syndromes (e.g., refractory anemia with dysplasia in a single system, sideroblastic anemia, refractory anemia with dysplasia in multiple systems, refractory anemia with blast cell proliferation, myelodysplastic syndrome with deletion of the long arm of chromosome 5 alone, unclassifiable myelodysplastic syndrome, pediatric myelodysplastic syndrome), acute bone Myelin leukemia and related progenitor cell neoplasms, and acute leukemia of unknown origin; as well as lymphomas, such as follicular lymphoma, MALT lymphoma / marginal zone lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma (nasal type), and myeloma (e.g., multiple myeloma).

[0100] Anticancer agents include, but are not limited to, auristatins (auristatin E, auristatin F, auristatin F phenylenediamine (AFP), monomethyl auristatin E, monomethyl auristatin F, and their derivatives), meitansinoids DM1 and DM4 and their derivatives), camptothecines (SN-38, irinotecan, ruthecan, DB67, BMP1350, ST1481, CKD602, topotecan, and exatecan, and their derivatives), DNA ligatures (endiyne, lexitropsin, duocalmycin and their derivatives), taxanes (paclitaxel and docetaxel and their derivatives), polyketides (discodermolide and its derivatives) ), anthraquinones (mitoxantrone and its derivatives), benzodiazepines (pyrrolobenzodiazepine, indolinobenzodiazepine, and oxazolidinobenzodiazepine and their derivatives), vinca alkaloids (vincristine, vinblastine, vindesine, and vinorelbine and their derivatives), doxorubicins (doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin and their derivatives), cardiac glycosides (digitoxin and its derivatives), kalechiamycin, epothyron, cryptophycin, semadin, rhizoxin, netropsin, combretastatin, eryuterobin, etoposide, T67 (Churalic), and nocodazole), radioisotopes (e.g., 32 P, 60 C, 90 Y, 111 In, 131 I, 125 I, 153 Sm, 186 Re, 188 Re, and 212 Examples include Bi), toxins (e.g., diphtheria toxin A, pseudomonas endotoxin, lysine, saporin, etc.), mytansine, halichondrin B such as eribulin, and derivatives thereof. Among these anticancer agents, those having a logP value of 1 or more and a molecular weight of 500 or more are particularly preferred.

[0101] In one preferred embodiment, a method for delivering a molecule to the brain in a subject is provided, comprising administering to the subject a micelle of the Disclosure containing an effective amount of the molecule, and a composition comprising the micelle for use in the method is provided. As will be described later, the micelles of the Disclosure have a hydrophobic core and have successfully delivered large hydrophobic molecules to the brain. This is evident from the examples described later, for example, in which intravenous administration of micelles of the Disclosure containing PROTAC was performed to obtain a therapeutic effect on an orthotopic glioma transplant model. The brain is protected from blood flow by the blood-brain barrier (BBB) ​​and is normally one of the organs to which delivery of substances (especially molecules with large molecular weights, e.g., molecules with a molecular weight of 500 Da or more) is difficult. The micelles of the Disclosure have the effect of transferring even such molecules from the blood flow to the brain. The micelles of the Disclosure may be particularly effective in delivering bifunctional molecules (e.g., PROTAC), preferably molecules with a molecular weight of 500 Da or more and having 1 or more logP, to the brain.

[0102] In one preferred embodiment, a method for treating a brain disease (e.g., a neurodegenerative disease) in a subject having the brain disease (e.g., a neurodegenerative disease) is provided, comprising administering to the subject a micelle of the Disclosure containing a therapeutically effective amount of a therapeutic agent for the brain disease (e.g., a neurodegenerative disease), and a composition comprising the micelle for use in such a method may be provided. Examples of neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, multiple system atrophy, frontotemporal dementia, progressive supranuclear palsy, Lewy body dementia, and spinocerebellar degeneration, and a person skilled in the art can appropriately select and use a therapeutic agent for the neurodegenerative disease suitable for each treatment.

[0103] In Alzheimer's disease, deposition of Aβ such as Aβ1-40 or Aβ1-42, and accumulation of tau and phosphorylated tau neurofibrils are observed and are considered to be contributing factors to dementia. PROTACs targeting tau have been developed, including PROTACs that bind to one or all of tau (wild-type, A152T variant, P301L variant), such as QC-01-175, T807, and C004019 that binds to phosphorylated S214 and phosphorylated S404. In addition, PROTACs containing THK5105 or its derivatives as tau ligands (e.g., I3) can also be used (see Antibodies 2023, 12, 43). Examples of PROTACs whose functional molecules are peptides include TH006 and Peptide1. For more information on these PROTACs, see Acta Mater Med. 2022; 1(1): 24-41. doi:10.15212 / amm-2021-0001. HyT-Tau-CPP may also be an effective PROTAC (see MEDCHEM NEWS 31(1) 36-40(2021)). In some embodiments, HyT-Tau-CPP has octaarginine at the C-terminus and a hydrophobic morph at the N-terminus, and has, for example, the following structure.

[0104] In addition, various PROTACs are exemplified in Life 2021, 11(7), 607, all of which may be preferred in this disclosure.

[0105] As a Tau ligand, for example, YQQYQDATADEQG (SEQ ID NO: 5) is known. This peptidolytic Tau ligand may be linked to a peptidolytic VHL ligand or Keep1 ligand via a peptide linker (for example, a GS linker, for example, (GSGS)n {where n is a natural number from 1 to 4} (SEQ ID NO: 6)). For example, peptides containing YQQYQDATADEQGGGSSALAPYIP (SEQ ID NO: 7) (for example, 30 amino acid lengths or less, 29 amino acid lengths or less, 28 amino acid lengths or less, 27 amino acid lengths or less, 26 amino acid lengths or less, or 25 amino acid lengths or less) function as PROTACs that degrade tau. Furthermore, peptides containing, for example, YQQYQDATADEQGGSLLDPETGEYL (SEQ ID NO: 8) (e.g., 30 amino acid lengths or less, 29 amino acid lengths or less, 28 amino acid lengths or less, 27 amino acid lengths or less, 26 amino acid lengths or less, or 25 amino acid lengths or less) function as tau-degrading PROTACs. These peptides may also have octaarginine further added to their N-terminus or C-terminus. For more information on peptide PROTACs, see Antibodies 2023, 12, 43.

[0106] In several neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, α-synuclein filaments are deposited, leading to a state called synucleopathy. Representative PROTACs include a site that binds to α-synuclein protein and a site that targets VHL (see, for example, WO 2020 / 041331 and ACS Med Chem Lett. 2020 Jun 11; 11(6): 1086-1087 and its Compound 1, 4, 5, 7, 30, 34). Sery308 is known as a site that binds to α-synuclein filaments and has the following structure. sery308 can be linked to a linker or E3 ligase binding site via an amino group. sery308 can be incorporated into PROTAC linked, for example, to a VHL ligand and a linker (e.g., PEG) (J. Med. Chem. 2023, 66, 7926-7942). sery308 can also be incorporated into PROTAC linked, for example, to a CRBN ligand (e.g., pomalidomide) and a linker (e.g., PEG) (J. Med. Chem. 2023, 66, 7926-7942). In this way, α-synuclein fibers can be degraded (J. Med. Chem. 2023, 66, 7926-7942). Examples of peptide-type α-synuclein ligands include GVLYVGSKTR (SEQ ID NO: 9) (see Antibodies 2023, 12, 43). As PROTAC, for example, peptides containing GVLYVGSKTRRRRRG (SEQ ID NO: 10) (with lengths of 20 amino acids or less, 19 amino acids or less, 18 amino acids or less, 17 amino acids or less, 16 amino acids or less, or 15 amino acids or less) can be used.

[0107] Abnormalities in TDP-43 (especially highly phosphorylated aggregates) can be a cause of ALS. EDLIKGISV (SEQ ID NO: 11) can be used as the TDP-43 binding site.

[0108] The extraterminal domain (BET) proteins consist of four members (BRD2, BRD3, BRD4, and BRDT), which have been suggested to play important roles in Alzheimer's disease and other neuroinflammatory diseases (see Acta Mater Med. 2022; 1(1): 24-41. doi:10.15212 / amm-2021-0001). PROTACs targeting these proteins may be therapeutic agents for Alzheimer's disease and neuroinflammatory diseases. Examples of BRD4-targeting PROTACs, though not limited to them, include ARV-825, SNIPER(BRD4)-1, ARC-766, ARV-771, and dBET-1 (see MEDCHEM NEWS 31(1) 36-40 (2021)).

[0109] Huntintin is a therapeutic target in Huntington's disease. Examples of PROTACs targeting huntingtin include compounds with the following structures (see Acta Pharmaceutica Sinica B Volume 10, Issue 2, 2020, Pages 207-238). {In the formula, "Ph" represents phenyl.}

[0110] Furthermore, targeting factors in any of the signaling pathways consisting of PI3K signaling, mTOR signaling, and AKT signaling, and suppressing any of these signaling mechanisms, could provide a significant treatment option for Alzheimer's disease or tumors (see Acta Mater Med. 2022; 1(1): 24-41. doi:10.15212 / amm-2021-0001).

[0111] Treatment for Alzheimer's disease may involve the administration of acetylcholinesterase inhibitors such as donepezil, rivastigmine, and galantamine, and NMDA receptor antagonists such as adamantane derivatives such as memantine. Treatment for Parkinson's disease may involve dopamine replacement therapy such as levodopa and carbidopa; dopamine agonists such as pramipexole and robinirole; MAO-B inhibitors such as selegiline and rasagiline; COMT inhibitors such as entacapone; anticholinergics such as trihexyphenidyl; and NMDA receptor antagonists such as amantadine. Treatment for ALS may involve riluzole and edaravone. Treatment for Huntington's disease may involve tetrabenazine, as well as antipsychotics, antidepressants, and anxiolytics such as risperidone and olanzapine. Treatment for multiple system atrophy may involve midodrine and levodopa. Frontotemporal dementia is treated with antidepressants and antipsychotics such as SSRIs. Progressive supranuclear palsy may be treated with levodopa and antidepressants. Multiple sclerosis may be treated with immunomodulators such as interferon-beta and glatiramer acetate, fingolimod, teriflunomide, and steroids. Lewy body dementia may be treated with acetylcholinesterase inhibitors such as dobepezil, quetiabine, and levodopa. Spinocerebellar degeneration may be treated with amantadine and antioxidants.

[0112] Examples of brain disorders include mental illnesses, such as mood disorders, anxiety disorders, psychotic disorders, personality disorders, eating disorders, trauma and stress-related disorders, developmental disorders, impulse control and behavioral disorders, dissociative disorders, somatoform disorders, sleep disorders, and substance use disorders.

[0113] Mood disorders include depression, bipolar disorder, persistent depressive disorder (dysthymia), and seasonal affective disorder (SAD). Anxiety disorders include generalized anxiety disorder (GAD), panic disorder, social anxiety disorder, specific phobias, separation anxiety disorder, and obsessive-compulsive disorder (OCD). Psychotic disorders include schizophrenia, schizoaffective disorder, brief psychotic disorder, and delusional disorder. Personality disorders include borderline personality disorder, narcissistic personality disorder, antisocial personality disorder, avoidant personality disorder, obsessive-compulsive personality disorder, and paranoid personality disorder. Eating disorders include anorexia nervosa, bulimia nervosa, and binge-eating disorder.Trauma and stress-related disorders include post-traumatic stress disorder (PTSD), acute stress disorder, and adjustment disorders. Developmental disorders include autism spectrum disorder (ASD), attention-deficit / hyperactivity disorder (ADHD), and learning disorders. Impulse control and behavioral disorders include intermittent explosive disorder, oppositional defiant disorder, and conduct disorder. Dissociative disorders include dissociative identity disorder (DID), dissociative amnesia, and depersonalization / derealization disorder. Somatoform disorders include somatic symptom disorder, illness anxiety disorder, and conversion disorder. Sleep disorders include insomnia disorder, hypersomnolence disorder, narcolepsy, and sleep apnea. Substance use disorders include alcohol use disorder, substance use disorders, and nicotine dependence. Those skilled in the art can select appropriate medications for each of these conditions.

[0114] According to this disclosure, the pharmaceutical composition may contain pharmaceutically acceptable additives (i.e., medicinal additives). Examples of pharmaceutically acceptable additives include water, physiological saline, salt, pH adjusters, isotonic agents, stabilizers, dispersants, solubilizers, antioxidants, preservatives, and analgesics. After filling a solution containing these additives into a container, the formulation for use can be prepared as a solid dosage form by freeze-drying or other means. For this purpose, the pharmaceutical composition may contain cryoprotective agents. Furthermore, a single dose may be filled into one container, or multiple doses may be filled into one container.

[0115] The present disclosure provides a composition comprising micelles encapsulating hydrophobic molecules such as anticancer agents or antifibrotic agents (particularly hydrophobic anticancer agents, e.g., PROTAC compounds), which can be administered to a subject (particularly intravenously) in a manner that preferably delivers an effective amount of the anticancer agent to a target organ and / or target cells, and preferably does not significantly increase the concentrations of one or more or all of the group selected from creatine phosphokinase (CPK), creatine kinase MB isozyme (CKMB), aspartate aminotransferase (GOT / AST), alanine aminotransferase (ALT), creatinine (CRE-P), and lactate dehydrogenase (LDH) in the subject's blood after administration compared to before administration.

[0116] A. Materials and Methods Solvents: Dimethylacetamide (DMAc), Acetonitrile (ACN), Dimethyl sulfoxide (DMSO)

[0117] (1) Preparation of cross-linked micelles Following the technical concept shown in Figure 1A, release-controlled micelles were constructed that release drugs into intracellular environments, particularly acidic environments such as endosomes.

[0118] 1. Non-crosslinked polymer micelles encapsulating ARV 825: a. 1.2 mg of mPEG-b-poly[N-((4-formylbenzyl)-asparagineamide](PEGPAsp (Ald)) was mixed with 0.3 mg of ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 5.1 mL of water was added dropwise to the solution, and stirring was continued for 1 hour. The micelle solution was dialyzed against water to remove the organic solvent.

[0119] 2. Crosslinking was introduced between the polymer molecules constituting the micelles containing ARV 825, as shown in Figure 1B. The polymer molecules were crosslinked using a crosslinking agent that has an aldehyde group and hydrazides at both ends.

[0120] ADH crosslinking (r=0.5): b. 1.2 mg (PEGPAsp (Ald)) was mixed with 0.3 mg ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 0.16 mg of ADH was added to the solution and the mixture was stirred for 48 hours. The micelle solution was dialyzed against water to remove the organic solvent.

[0121] IDH crosslinking (r=0.5): c. 1.2 mg (PEGPAsp (Ald)) was mixed with 0.3 mg ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 0.17 mg of IDH was added to the solution and stirred for 48 hours. The micelle solution was dialyzed against water to remove the organic solvent.

[0122] ODH crosslinking (r=0.5): d. 1.2 mg (PEGPAsp (Ald)) was mixed with 0.3 mg ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 5.1 mL of water was added dropwise to the solution and stirred for 1 hour. 0.106 mg of ODH was added to the solution and stirred for 48 hours. The micelle solution was dialyzed against water to remove the organic solvent.

[0123] PEG-DH crosslinking (r=0.5): e. 1.2 mg (PEGPAsp (Ald)) was mixed with 0.3 mg ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 0.33 mg of PEG-DH was added to the solution and stirred for 48 hours. The micelle solution was dialyzed against water to remove the organic solvent.

[0124] PDH crosslinking (r=0.5): f. 1.2 mg (PEGPAsp (Ald)) was mixed with 0.3 mg ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 5.1 mL of water was added dropwise to this solution, and stirring continued for 1 hour. 0.14 mg of PDH was added to the solution, and stirring was continued for 48 hours. The micelle solution was dialyzed against water to remove the organic solvent.

[0125] SDH crosslinking (r=0.5): 1.2 mg (PEGPAsp (Ald)) was mixed with 0.3 mg ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 5.1 mL of water was added dropwise to the solution and stirred for 1 hour. 0.16 mg of SDH was added to the solution and stirred for 48 hours.

[0126] TDH crosslinking (r=0.5): h. 1.2 mg (PEGPAsp (Ald)) was mixed with 0.3 mg ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 5.1 mL of water was added dropwise to the solution and stirred for 1 hour. 0.17 mg of TDH was added to the solution and stirred for 48 hours.

[0127] 3. Diamine-crosslinked micelles encapsulating ARV 825: EDA crosslinking (r=0.5): 1. 1.2 mg (PEGPAsp (Ald)) was mixed with 0.3 mg ARV 825, and 170 μL of organic solvent was added to dissolve the carrier and PROTAC. The mixture was stirred at 40°C for 12 hours. 0.05 mg of EDH was added to the solution and the mixture was stirred for 48 hours. The micelle solution was dialyzed against water to remove the organic solvent.

[0128] The micelles in steps (II and III) are concentrated by membrane filtration. The NMR spectra of the micelles with different crosslinking agents are shown in Figure 2.

[0129] Model cross-linked hydrazide micelles (empty micelles) without encapsulating the drug were prepared, and nanomicelles with monodisperse particle size peaks were obtained (see Figure 3). The obtained empty micelles were freeze-dried. When the NMR spectra of the freeze-dried micelles were compared with and without cross-linking, as shown in Figure 4, the peaks derived from carbonyl groups almost disappeared after cross-linking. In addition, the peaks derived from phenyl groups also disappeared after cross-linking, which is thought to be due to the difficulty in detecting the peaks derived from phenyl groups by the PEG covering the micelles during micelle formation. Therefore, the disappearance of the peaks derived from phenyl groups suggests that the micelle structure was stably maintained even after freeze-drying after cross-linking, and that interpolymer cross-linking in the micelles stabilizes the freeze-dried formulation. According to the 1H NMR spectra of the drug (ARV-825), the polymer, drug-encapsulated non-crosslinked micelles, and drug-encapsulated crosslinked micelles, the drug exhibited two typical peaks at 9.8 ppm and 10.8 ppm, originating from ARV-825, and these peaks were observed in both the non-crosslinked and crosslinked micelles (see Figure 5).

[0130] The tests were conducted by changing the mixing ratio of the polymer and the crosslinking agent. The mixing ratio was defined as the ratio of the number of aldehyde groups in the polymer to the number of hydrazide groups in the crosslinking agent (A / H ratio) (see Table 1).

[0131]

[0132] The results are shown in Table 2 and Figure 6. The results showed that increasing the crosslinking ratio reduced the intensity of the 9.8 ppm free aldehyde peak compared to the 10.8 ppm PROTAC peak.

[0133] We investigated the relationship between changes in the ratio of drug to polymer, the amount of crosslinking agent, and the drug retention rate.

[0134]

[0135] As shown in Table 2, cross-linked micelles exhibit high drug loading efficiency, and experimental results revealed that micelles can load up to 40% of their total mass with drugs (various PROTACs). Cross-linked micelles are a versatile and effective platform for encapsulating and delivering various drugs (e.g., PROTACs), and as a result, can be applied to the delivery and performance improvement of diverse therapeutics, from ARV-825 targeted protein degradation to fulvestrant hormone receptor modulation and experimental compounds such as LC2. LC2 is a PROTAC that targets Kras, and fulvestrant is a PROTAC that targets the estrogen receptor (ER).

[0136]

[0137] The effect of the crosslinking ratio on the PROTAC loading effect in nanomicelles was evaluated, and as shown in Table 3, the drug (PROTAC) was effectively loaded into the micelles even in non-crosslinked micelles. Furthermore, in contrast to non-crosslinked micelles, which initially decomposed and may have limited drug (PROTAC) retention, crosslinked micelles maintained a robust structure and supported a higher drug (PROTAC) capacity even with the same initial ratio. As the A / H ratio increased from 0 to 0.5, the PROTAC loading capacity gradually increased. As the ratio of crosslinking agent increased, the micelle structure became increasingly stable and rigid. This is thought to be because more free aldehyde groups crosslink the dihydrazide, forming a high-density network that provides a stronger and more stable hydrophobic environment in the core, allowing for greater drug (PROTAC) retention within the core. At an A / H ratio of 0, the micelles have a relatively loose structure, resulting in minimal drug (PROTAC) loading. However, as the A / H ratio is gradually increased from 0.1, 0.2, 0.3, 0.4, and finally to 0.5, the cross-linking network within the micelle becomes significantly stronger, resulting in increased drug (PROTAC) loading at each stage. This improvement in drug (PROTAC) loading capacity is thought to be due to improved structural integrity and reduced micelle degradation, which may lead to increased drug retention and more effective delivery of therapeutic agents to the target site.

[0138]

[0139] The diameter of uncrosslinked micelles carrying various aldehydes and hydrazides tends to vary due to the lack of stabilizing interactions. However, as shown in Table 4, the diameter of these micelles becomes more consistently stable as the crosslinking ratio increases from 0 to 0.5. Without crosslinking (0), micelles exhibit greater size variation. At a crosslinking ratio of 0.1, slight stabilization begins, and the variation in diameter decreases. At ratios of 0.2 and 0.3, micelles show remarkable size uniformity, reflecting improved structural integrity. By the time crosslinking ratios reach 0.4 and 0.5, the variation in diameter of micelles decreases significantly, maintaining a more consistent and stable size, ideal for efficient drug delivery.

[0140] The selection of dihydrazide crosslinking agents such as SDH, ADH, IDH, and ODH can affect both the size of the crosslinked micelles and their pH-responsive release characteristics. The choice of a specific dihydrazide crosslinking agent not only determines the size of the micelles but also profoundly influences their pH-responsive release behavior, providing versatility for drug delivery applications.

[0141]

[0142] Table 5 shows the diameters of non-crosslinked micelles (NCLs) and crosslinked micelles (with the type of crosslinking agent indicated) using different linkers. SDH, known for its short and flexible chains, tends to produce small micelles with strong crosslinking, and its stable structure may result in a slower release rate of encapsulated drugs at high pH levels. ADH, characterized by longer and more flexible chains, typically produces larger micelles. Degradation of the crosslinking agent ADH may result in a moderately pH-responsive release effect. IDH, with its rigid aromatic structure, tends to form micelles with a rigider skeleton. TDH, due to its unique chemical properties, can influence micelle size and pH-responsive release behavior. ODH, due to its very short chain length, cannot fully promote the aldehyde group, resulting in an unstable structure.

[0143] II. Based on the results of screening for the formation of cross-linked micelles containing various drugs, the following process was used as a general method for preparing micelles using various PROTACs, low molecular weight drugs, drugs, and fluorescent dyes. As shown in Table 6, 1.2 mg (PEGPAsp (Ald)) was mixed with 0.5 mg of drug molecules, 170 μL of organic solvent was added to dissolve the carrier and molecules, and the mixture was stirred at 40°C for 12 hours. 5.1 mL of water was added dropwise to the solution and the mixture was stirred for 1 hour. 0.32 mg of SDH was added to the solution and the mixture was stirred for 48 hours.

[0144]

[0145] III. Scale-up of ARV 825 micelle formation for in vivo experiments 75 mg (PEGPAsp (Ald)) was mixed with 22.5 mg of ARV 825, and 9.75 mL of organic solvent was added to dissolve the carrier and ARV 825. The mixture was stirred at 40°C for 12 hours. 292.5 mL of water was added dropwise to the solution and stirred for 1 hour. 12.2 mg of SDH was added to the solution and stirred for 48 hours. The micelle solution was dialyzed against water to remove the organic solvent.

[0146] IV. Dissolve 1.2 mg of empty micelles (PEGPAsp (Ald)) in 170 μL of organic solvent and stir at 40°C for 12 hours. Add 5.1 mL of water dropwise to the solution and stir for 1 hour. Add 0.32 mg of SDH to the solution and stir for 48 hours.

[0147] A catheter was inserted into the tail vein of mice anesthetized with isoflurane. The ears of these mice were dehaired, immersion oil was applied, and a coverslip was placed over them. The blood vessels of the ears were observed using a confocal microscope. Ten seconds after the start of measurement, a fluorescently labeled sample (crosslinked micelles, non-crosslinked micelles, etc., labeled with Alexa Fluor 467 polymer) was administered via the tail vein through the catheter. Images were taken at 30 frames / sec for the first 3 minutes, and then every 1 minute thereafter. After imaging, an ROI was set, and the fluorescence intensity at each time point in the ROI was obtained. The relative fluorescence intensity was calculated using the following formula: Relative fluorescence intensity (%) = 100 × [(fluorescence intensity at each time point) - (fluorescence intensity before administration)] / [(maximum fluorescence intensity) - (fluorescence intensity before administration)]

[0148] The results are shown in Figures 7 and 8. As shown in Figure 7, when the intravenous fluorescence intensity was observed for cross-linked micelles (CL) and non-cross-linked micelles (non-CL), cross-linked micelles clearly showed higher blood retention than non-cross-linked micelles. According to Figure 7, cross-linked micelles maintained a nearly constant blood concentration (over 90%) even 10 hours after administration. Figure 8 shows a venous fluorescence image, showing that cross-linked micelles were still stably present in the blood even after 8 hours.

[0149] In vitro cytotoxicity test mouse glioma cell line (GL261) is cultured in DMEM. 50 μL of cells are plated into a 96-well plate (5,000 cells / well) and cultured for 24 hours to allow cell adhesion. Cells are treated with 50 μL of various concentrations of ARV825 for 96 hours. MTT solution (5 mg / mL) is prepared in PBS, and 10 μL of MTT solution is added to each well, and incubated at 37°C for 3–4 hours. SDS-HCl solution is prepared by adding 10 mL of 0.01 M HCl to 1 g of SDS. 100 μL of SDS-HCl solution is added to each well and mixed thoroughly by pipetting. Microplates are incubated in a chamber at 37°C for 4–18 hours, and the absorbance at 570 nm is read.

[0150]

[0151] As shown in Table 7, the prepared ARV-825-encapsulated cross-linked micelles exhibited favorable cytotoxicity against GL261 cells. Since they were more cytotoxic than free ARV-825, it is suggested that micelle formation improves the efficiency of ARV-825 uptake into cells.

[0152]

[0153] Similar tests were conducted using the pancreatic cancer cell line (MiaPaca 2). LC2 was encapsulated as the drug. As shown in Table 8, the prepared LC2-encapsulated cross-linked micelles exhibited favorable cytotoxicity against MiaPaca 2 cells. Since they were more cytotoxic than free LC2, it is suggested that micelle formation improves the efficiency of LC2 uptake into cells.

[0154] In vivo toxicity study: Cross-linked mice containing ARV-825 were administered via the tail vein of mice, and tail toxicity was evaluated. The experimental groups were: A: PBS administration group, B: ARV-825 administration group dissolved in 70% ethanol (EtOH), C: ARV-825 administration group dissolved in water and polyethylene glycol 400 (PEG400), and D: ARV-825-encapsulated cross-linked mice administration group. The dose for all ARV-825 administration groups was 20 mg / kg body weight.

[0155] The results are shown in Figure 9. As shown in Figure 9, free ARV-825 caused severe ulceration (Group B) or ulceration or amputation (Group C) in the tail, whereas no such toxicity was observed in the ARV-825-encapsulated cross-linked micelle administration group (Group D), and continuous administration was possible.

[0156] Orthotopic brain tumor (monotherapy): Following the scheme shown in Figure 10A, GL261 luc cells (1 × 10⁶ cells in 2 μl injection volume) were injected into the skull of C57BL / 6J mice. 5 Cells were inoculated 1 mm anteriorly, 2 mm to the right of the forehead, and 3 mm deep. Six days after inoculation, 150 mg / kg body weight of luciferin was injected, and bioluminescence signals from GL261-luc tumors were observed using an in vivo imaging system (IVIS). Mice were divided into groups (n=5) and intravenously injected with a PROTAC preparation (10 mg / kg body weight) via the tail vein. Injections were performed on days 7, 10, 13, 16, 19, and 21 until the end of the experiment. Mouse survival rates were tracked, and the significance of prolongation was determined by a log-rank test.

[0157] The results are shown in Figures 10B and 10C. As shown in Figures 10B and 10C, no significant antitumor effect was observed in the group administered with free ARV-825, while a significant antitumor effect was observed in the group administered with ARV-825-encapsulated cross-linked micelles. The micelles of this disclosure were considered suitable for crossing the blood-brain barrier (BBB) ​​and delivering the drug to brain tumors. In contrast, since free ARV-825 showed almost no antitumor effect, it was considered that ARV-825 could not cross the BBB on its own or was not delivered to tumor cells. Furthermore, when overall survival (OS) was plotted (see Figure 10D), the survival time of the orthotopic brain tumor model was significantly extended in the group administered with ARV-825-encapsulated cross-linked micelles. It should be noted that naked ARV-825 could not be administered repeatedly intravenously according to the above schedule, which is consistent with the result in Figure 9.

[0158] Orthotopic brain tumors (with temozolomide) Following the scheme shown in Figure 11A, GL261 luc cells (1 × 10⁵ cells in a 2 μl injection volume) were inoculated intracranially into C57BL / 6J mice at a location 1 mm anterior, 2 mm to the right of the frontal lobe, and 3 mm deep. Six days after inoculation, 150 mg of luciferin (1 mg / kg body weight) was injected, and bioluminescence signals from the GL261-luc tumors were observed using an in vivo imaging system (IVIS). Mice were divided into groups (n=5), and PROTAC preparations (10 mg / kg body weight) and temozolomide (TMZ) (50 mg / kg body weight) were intravenously administered via the tail vein, either alone or in combination. Injections were given on days 7, 10, 13, 16, 19, and 21 until the end of the experiment. Mouse survival rates were tracked, and the significance of prolongation was determined by a log-rank test.

[0159] As a result, the group receiving combination therapy with ARV-825-encapsulated cross-linked micelles and TMZ showed the most superior antitumor effect (see Figures 11B and 11C). This demonstrated a synergistic antitumor effect achieved by combining a BRD4 inhibitor with an alkylating agent.

[0160] Anti-fibrotic effect: A construct containing DNA with a luciferase gene operably linked downstream of a Smad-binding element (SBE) and a minimum promoter was created and introduced into a fibroblast cell line (NIH / 3T3). Cells were 20 × 10⁶ 3 Cells were seeded in plates at a rate of one cell per well and cultured in the presence of ARV-825-encapsulated cross-linked micelles, free ARV-825 (ARV-825f), or a negative control (PBS or irinotecan). Cells were harvested, cell lysates were obtained, and chemiluminescence was induced using luciferin, with the luminescence intensity being confirmed. The results are shown in Figure 12A, and the ARV-825-encapsulated cross-linked micelle treatment group showed an inhibitory effect on the Smad signal. In the free ARV-825 treatment group, luciferase activity was observed to be lower due to more cell death than in the micelle treatment group.

[0161] Next, NIH / 3T3 100 x 10 3 Cells were seeded in a 6-well plate at a rate of cells / well and cultured for 24 hours. Cells were further cultured in the presence of TGF-β (10 ng / mL) and ARV-825 (ARVf) or ARV-825-encapsulated cross-linked micelles (ARVm) (400 μg / mL of ARV concentration for each). Cells were harvested, fixed with 4% paraformaldehyde, washed with TNT buffer, and blocked with 1% BSA. Cells were stained by incubation overnight with Alexa Fluoro488-labeled anti-collagen I antibody (1:500). As shown in Figure 12B, collagen I production was significantly suppressed (approximately 90%) in the ARV-825-encapsulated cross-linked micelle-treated group. DAPI staining in Figure 12B shows that cells are sparser in the ARVf group, indicating cell death.

[0162] The above experiment was carried out using an anti-α-smooth muscle actin (α-SMA) antibody instead of an anti-collagen I antibody. As a result, as shown in Figure 12C, α-SMA expression was significantly suppressed (approximately 66%) in the ARV-825 encapsulated cross-linked micelle treatment group. Evaluation of DAPI staining in Figure 12C shows that the cells in ARVf are sparse, indicating that cell death has occurred.

[0163] As shown in Figures 12A-12C, free ARV-825 exhibited high cytotoxicity and a tendency to induce cell death. In contrast, ARV-825, through micelle formation, suppressed the induction of cell death and showed remarkable anti-fibrotic effects.

[0164] The above is an in vitro experiment, but in vivo, while free ARV-825 is rapidly cleared and excreted from the body, micellar ARV-825 circulates in the body for a long period, and it is thought that its antifibrotic effect is exerted over a long period while its cytotoxicity is limited (see, for example, Figure 7). Therefore, it is thought that micellar enhances the antifibrotic effect of ARV-825 even in vivo.

[0165] Establishment of an Idiopathic Pulmonary Fibrosis Model and In Vivo Treatment Trial: C57BL / 6 mice were administered a single intratracheal dose of bleomycin (BLM) (1.28 mg / kg). Thirty days after bleomycin administration, the lungs of these mice were removed and collagen staining was performed, revealing collagen deposition in the tissue (see Figure 13A). This demonstrated the establishment of a pulmonary fibrosis model.

[0166] Using this model, ARV-825-encapsulated cross-linked micelles (indicated as "ARV825 / m" or "ARV / m" in Figures 13B-C) (10 mg / kg body weight) or free ARV-825 (indicated as "ARV825" or "ARV" in Figures 13B-C) were administered via the tail vein twice a week for 5 weeks, starting 11 days after bleomycin administration. Fibrosis was monitored weekly using micro-CT.

[0167] As a result, as shown in Figure 13C, lung volume decreased significantly in the PBS-administered group (negative control), while lung volume in the ARV-825-encapsulated cross-linked micelle-administered group (ARV825 / m or ARV / m) was almost the same as that of normal lungs, showing a significant increase in volume. In contrast, as also shown in Figure 13C, lung volume in the free ARV-825-administered group (ARV825 or ARV) did not differ significantly from lung volume in the PBS-administered group (negative control). This suggests that ARV-825-encapsulated cross-linked micelles exhibit a higher anti-fibrotic effect in vivo than free ARV-825. Furthermore, this result was consistent with the results of the in vitro study on "anti-fibrotic effect" described above.

[0168] Immobilization of fluorescent dye into micelles: IR780 was micellarized, and the micelles were subjected to crosslinking. The NMR spectrum of the crosslinked micelles is shown in Figure 14A. The reduction of the 9.8 ppm peak originating from the aldehyde group due to crosslinking suggests that micelle formation and crosslinking were successful.

[0169] Free IR780, non-crosslinked mice (NCLs) containing IR780, and crosslinked mice (CLs) containing IR780 were prepared and administered via tail injection to tumor-bearing mice. Tumor-bearing mice were prepared by adding 2 × 10⁶ of nude mice. 6 It was created by subcutaneous transplantation of human medulloblastoma cells ONS-76 (JCRB cell bank: IFO50355).

[0170] As a result, as shown in Figure 14B, the group administered with cross-linked micelles (CLs) containing IR780 showed the longest duration of high systemic fluorescence intensity. The accumulation of IR780 in tumors was compared. As shown in Figure 15, based on fluorescence intensity, the cross-linked micelles (CLs) containing IR780 resulted in the greatest accumulation of IR780 in tumors.

[0171] Further development of a bleomycin-induced pulmonary fibrosis model and therapeutic experiments. 20-week-old C57BL / 6 mice (female) were administered a single intratracheal dose of bleomycin (BLM) (1.28, 3, 4, 5 mg / kg). Survival rates were observed up to 2 months, and fibrosis was monitored by micro-CT on day 14. The results are shown in Figure 16. As shown in Figure 16, in the 30-day survival study of IPF-induced mice with a single BLM dose, a clear dose-dependent mortality pattern was observed in the tested concentration range of 3, 4, and 5 mg / kg. The highest dose group of 5 mg / kg showed the most severe results, with all individuals dying on day 11. This suggests that it does not produce a model of sustained fibrosis, but rather induces acute and lethal toxicity. At the intermediate dose of 4 mg / kg, mortality was progressive, and survival rates steadily decreased over time, with only 40% of individuals surviving at day 30. This reflects a more severe but still manageable disease course. In contrast, at the lowest dose tested, 3 mg / kg, 60% of mice survived up to day 30, demonstrating the highest overall survival rate. This suggests that sustained disease progression is possible at this dose without immediate lethality. This survival trend highlights the importance of dose selection when establishing an experimental fibrosis model for IPF treatment using ARV-825 micelles.

[0172] 1. Low-Dose BLM Model 20-week-old C57BL / 6 mice were given a single intratracheal dose of BLM (1.28 mg / kg). On day 11, various PROTAC preparations (10, 20, 30 mg / kg) were administered twice weekly via the tail vein for two weeks. Fibrosis was monitored using micro-CT (μ-CT). The results are shown in Figures 17 and 18. As shown in Figure 17, the results of the micro-CT images at week 2 of treatment clearly show progressive changes in lung structure consistent with the fibrosis score. To evaluate how and to what extent pneumonia and fibrosis induce changes in the lungs, the fibrotic area was quantified from micro-CT scans acquired weekly until week 5 after BLM administration. In the IPF model, micro-CT images of mice administered 10 mg / kg of ARV-825 mice showed a significant reduction in fibrotic lesions and improvement in lung structure accompanied by improved air permeability. Furthermore, as shown in Figure 18, quantitative analysis of lung density and air volume using Hounsfield units (HU) revealed clear differences between the untreated and IPF-treated groups. In untreated mice (naive), the lungs showed low HU values ​​and high air volume, reflecting normal ventilation tissue structure. In contrast, in IPF mice (PBS), HU density was significantly increased and air volume decreased, consistent with fibrous remodeling, extracellular matrix deposition, and lung dysfunction. In the free ARV-825 administration group (ARVF), mild improvements were observed compared to untreated IPF mice, including a slight decrease in HU density and partial recovery of air volume. In particular, in the ARV-825 micelle administration group (ARV / m), a remarkable recovery to normal lung parameters was observed, with HU density and air volume approaching those of untreated mice. These results demonstrate that micelle-mediated ARV-825 delivery more effectively maintains lung structure and function, highlighting its excellent therapeutic potential in reversing BLM-induced IPF.

[0173] 2. High-Dose BLM Model 20-week-old C57BL / 6 mice were given a single intratracheal dose of BLM (5 mg / kg). On day 3, various PROTAC formulations (10, 20, 30 mg / kg) were administered twice weekly via the tail vein for 2 weeks. Survival rates were monitored within 2 months. The results are shown in Figure 19. As shown in Figure 19, the therapeutic effects of ARV-825 in its free form and in its delivery form via a micelle-based nanocarrier system were evaluated in a BLM pulmonary fibrosis mouse model established with a high loading dose of 5 mg / kg. Significant differences were observed between the two formulations in terms of survival results. The Kaplan-Meier survival curves showed a clear preventive effect of ARV-825 (ARVF) and micelle-encapsulated ARV-825 (ARVm) against the BLM effect under a high BLM dose of 5 mg / kg. In the PBS-treated group, all mice died on day 11, while the ARV-825-treated group showed good survival, particularly in the 20 mg / kg micelle-treated ARV-825 group, where 3 out of 8 mice survived (there was no statistically significant difference compared to PBS). Free ARV-825 administration had a limited protective effect, with only 1 out of 8 mice surviving by day 21. This indicates that while the compound exerted some therapeutic effect, its bioavailability and stability were insufficient to provide a sustained effect under severe bleomycin-induced fibrotic stress. In contrast, micelle-delivered ARV-825 improved survival rates, reaching 12.5% ​​(ARV-F, 30 mg / kg), 25% (10 mg / kg), and 37.5% (20 mg / kg) by day 21, with 20 mg / kg being the optimal dose. μ-CT scans were performed to assess the degree of pulmonary fibrosis in surviving mice. The results are shown in Figure 20. As shown in Figure 20, μ-CT images of mice administered ARV-825 micelles in the IPF model showed a significant reduction in fibrous lesions and improvement in lung structure compared to mice injected with ARV-825.

[0174] Immunohistochemical (IHC) staining 1. Fixation of lung tissue Mice were euthanized using isoflurane, and approximately 10-20 mL (mouse) of PBS was perfused into the right ventricle, removing blood until the drainage was clear. After perfusion, the lungs were removed, and 4% paraformaldehyde (PFA) in PBS was injected into the lungs via a 25G catheter through the trachea to preserve the alveolar structure. The lungs were then fixed in 4% PFA at 4°C for 12 hours. After washing with PBS, the tissue was transferred to a 10% sucrose solution in PBS until it settled (approximately 4-6 hours). It was then transferred to 20% and 30% sucrose solutions and held until it settled (approximately overnight at 4°C). Excess sucrose was removed, and the tissue was embedded in a cryomold. After adding OCT to determine the orientation, it was frozen in isopentane cooled with liquid nitrogen as described above. Stored at -80°C. Sections were prepared at -20°C using a cryostat. 2. After leaving the sections on IHC glass slides at room temperature (RT) for 30 minutes, gently wash with TBS three times for 5 minutes each to remove free OCT. Inactivate the hydrogen peroxide enzyme with 3% hydrogen peroxide solution in methanol for 35 minutes, then wash with PBS / TBS three times for 5 minutes each. Then, immerse in serum blocking solution in a humid chamber at room temperature (RT) for 60 minutes. Wash with PBS / TBS three times for 5 minutes each. Dilute the primary antibody with blocking buffer (starting dilution 1:400, range 1:200–1:800), drop it onto the sections, and incubate overnight at 4°C. Wash with TBS (three times for 5 minutes each). Dilute the HRP-labeled secondary antibody and drop it onto the sections, incubating at room temperature in the dark for 1 hour. Wash with TBS (three times for 5 minutes each), add fluorescently labeled Tyramid SuperBoost reagent and incubate for 10 minutes. Wash (three times for 5 minutes each), add diluted DAPI solution (0.5–1 μg / mL) and incubate for 5 minutes. Finally, the specimens were washed with PBS, mounted on glass using an anti-bleeding fixative, and observed under a fluorescence microscope.

[0175] As shown in Figure 21, collagen I (COL I) is a major pathological factor that promotes excessive deposition of the extracellular matrix (ECM) and the formation of fibrous scars in idiopathic pulmonary fibrosis (IPF). Immunohistochemical staining (IHC) analysis of COL I revealed clear differences between healthy mice, IPF model mice, and the ARV-825 treatment group (Figure 21 panel a). In healthy lung tissue, COL I expression was extremely low, consistent with normal alveolar structure and the absence of fibrous remodeling. In contrast, extensive and severe COL I deposition was observed in the IPF model, indicating progression of fibrosis. In the group treated with free ARV-825, a partial decrease in COL I expression was observed compared to the untreated IPF group, but residual fibrous deposition was significant, indicating a limited anti-fibrotic effect. On the other hand, ARV-825 delivered by a micelle formulation significantly suppressed COL I expression and greatly inhibited the progression of fibrosis. Significant differences were also observed among the groups in α-smooth muscle actin (α-SMA), another indicator of fibrosis (Figure 21, panel b). In healthy lungs, α-SMA expression was mainly limited to vascular smooth muscle, and no abnormal staining was observed in the interstitium or alveolar regions. On the other hand, in the IPF model, widespread and strong α-SMA-positive staining originating from activated myofibroblasts was observed, suggesting persistent fibrotic remodeling. Although α-SMA expression was partially suppressed by administration of free ARV-825, it remained at a high level compared to the healthy group. In contrast, the ARV-825 micelle formulation significantly suppressed α-SMA expression and effectively suppressed fibroblast activity. Furthermore, clear differences were observed among the groups in F4 / 80, an indicator of macrophage infiltration (Figure 21, panel c). In healthy mice, F4 / 80-positive cells were scattered, but in the IPF model, their number increased significantly, indicating macrophage accumulation and enhanced inflammatory activity associated with the progression of fibrosis. Administration of free ARV-825 moderately reduced F4 / 80-positive cells and partially suppressed macrophage infiltration, but the persistence of inflammatory cells remained significant.In contrast, delivery of ARV-825 via micelle formulations resulted in a significant reduction in F4 / 80-positive cells, suggesting enhanced therapeutic effects through the suppression of macrophage accumulation and the inflammatory environment.

[0176] Beta-galactosidase (β-Gal) and p16 are indicators related to aging and fibrosis. INK4 Immunohistochemical staining (IHC) of positive cells and platelet-derived growth factor receptor β (PDGFRβ) showed consistent trends across all experimental groups, as shown in Figure 22. In healthy mouse lungs, the expression of these markers was extremely low, indicating normal cell turnover, the absence of senescent cells, and fibroblast inactivity. In contrast, in the bleomycin-induced IPF model, β-Gal and p16 INK4 A significant increase in positive cells was observed, suggesting extensive accumulation of senescent cells. Furthermore, PDGFRβ expression was remarkably elevated in the fibrotic regions, indicating enhanced extracellular matrix (ECM) deposition through increased activated fibroblasts and expansion of mesenchymal cell populations. Treatment with free ARV-825 moderately suppressed the expression of these three markers and achieved partial suppression of senescent cell accumulation and fibroblast activity, but many pathological features remained. On the other hand, ARV-825 delivered via micelle formulations showed β-Gal, p16 INK4 Both significantly suppressed PDGFRβ expression and potently inhibited the accumulation of senescent cells and activated fibroblasts. This simultaneous suppression of aging and fibroblast activity demonstrates that micellar formulations can effectively control both the drivers of cellular pathology and tissue remodeling in pulmonary fibrosis, supporting their therapeutic advantage.

[0177] Toxicity study: 20-week-old C57BL / 6 mice were administered a single dose of ARV-825 and ARV-825 micelles (20 mg / kg). After 24 hours, the mice were euthanized, and blood was collected by cardioplasty. 45 μL of blood was mixed with 5 μL of EDTA, and the sample was stored on ice for blood analysis. The remaining blood was centrifuged at 5000 rpm for 15 minutes, and the serum was recovered and stored on ice for quantification of various analytes.

[0178]

[0179] Hematological analyses were performed to evaluate the systemic toxicity of ARV-825 and its micelle formulations in healthy mice. The measured parameters are shown in Table 9. All values ​​were evaluated after administration of PBS, free ARV-825, and ARV-825 micelles. As shown in Figure 23, no significant differences were observed between groups, and all values ​​remained within the normal physiological range. These findings indicate that both free ARV-825 and its micelle formulations have minimal hematological toxicity, supporting the safety of systemic administration in vivo.

[0180] Serum biochemical analysis of healthy mice was performed using a Dri-Chem chip to evaluate potential organ toxicity after administration of PBS, free ARV-825, or ARV-825 micelles. The primary markers measured are shown in Table 10.

[0181]

[0182] As shown in Figure 24, all parameters remained within the normal physiological range in PBS-treated mice, serving as a baseline control. Interestingly, mice treated with free ARV-825 showed significant elevations in several markers, including CPK, GPT-P, CK-MB, LDH, and GOT / AST, suggesting potential stress or damage to myocardial and hepatic tissue. These elevations suggest that free ARV-825 (not encapsulated in micelles) may induce off-target toxicity, particularly affecting the liver and heart, even in healthy animals. In contrast, mice treated with ARV-825 micelles showed similar values ​​to the PBS-treated control group, with no significant increases in any of the measured parameters. This suggests that encapsulation of ARV-825 in micelles effectively mitigates systemic toxicity by improving drug delivery specificity, reducing off-target exposure, and enhancing biocompatibility. Overall, these findings highlight the safety advantages of micelle formulations over free ARV-825 and support the potential for therapeutic applications with reduced organ toxicity risks.

[0183] The hydrophobicity of the encapsulated molecules (e.g., logP) is related to the micelle-forming ability.

[0184]

[0185] As shown in Table 11, renelidomide and etoposide, which had logP values ​​of 1 or less, did not form micelles. Additionally, gemcitabine and temozolimide had logP values ​​of 0 or less and did not form micelles. It was clear that higher logP values ​​were associated with better micelle formation. Even molecules like DT2216, with a Da exceeding 1500 Da, could be successfully encapsulated in micelles.

[0186] Any reference cited herein is incorporated herein in its entirety by citation.

[0187]

Claims

1. A composition comprising (i) an amphiphilic block copolymer containing an uncharged hydrophilic block and a hydrophobic block, and (ii) a micelle containing a molecule that forms a hydrophobic bond with the hydrophobic block in an aqueous solution, wherein the hydrophobic block is a block copolymer having hydrophobic side chains, and the micelle has interpolymer crosslinks.

2. The composition according to claim 1, wherein the interpolymer crosslinks cleave in a pH-responsive manner.

3. The composition according to claim 1 or 2, wherein the hydrophobic side chain comprises a hydrophobic moiety and a carbonyl group, and the crosslinking is formed by a crosslinking agent having two hydrazide groups or amino groups, with a spacer between the two groups.

4. The composition according to any one of claims 1 to 3, wherein the hydrophobic side chain comprises a hydrophobic moiety and a carbonyl group, the hydrophobic moiety comprises a phenyl group, and the crosslink is formed by a crosslinking agent having two hydrazide groups or amino groups, with a hydrophobic spacer between the two groups.

5. The hydrophobic block has the following formula: {In the formula, n1 is a numerical value of 1 to 3, n2 is a numerical value of 1 to 3, n1 and n2 may be the same or different, m1 is 5 to 100, has a formyl group with a number average of 5 to 100, and one of *1 and *2 is linked to a non-charged hydrophilic polymer block, and the other of *1 and *2 is hydrogen, a methoxy group, an acetyl group, an acetoxy group, a protecting group, a polymerizable group, or a hydrophobic group, and at least a part of the aldehyde group in the formyl group forms a hydrazone bond with a hydrazide or amino group of a crosslinking agent. The composition according to any one of claims 1 to 4.

6. The composition according to any one of claims 1 to 4, wherein the uncharged hydrophilic polymer block comprises polyalkylene glycol.

7. The composition according to any one of claims 1 to 6, wherein the uncharged hydrophilic polymer block comprises polyethylene glycol.

8. The composition according to any one of claims 1 to 7, wherein the molecule has a logP value of 1 or more.

9. The composition according to any one of claims 1 to 8, wherein the molecule has a molecular weight of 500 Da or more.

10. The composition according to claim 8, wherein the molecule has a molecular weight of 500 Da or more.

11. The composition according to any one of claims 1 to 10, wherein the molecule is an anticancer agent or a fluorescent dye.

12. The composition according to any one of claims 1 to 11, wherein the molecule comprises a target protein ligand and an E3 ubiquitin ligase ligand, and the target protein ligand and the E3 ubiquitin ligase ligand are linked either via a linker or directly without a linker.

13. A lyophilized formulation comprising a lyophilized version of the composition for preparing the composition according to any one of claims 1 to 12.

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