Biocompatible composite polymer suitable as carrier for drug delivery to cancer tissue

Abstract

To provide a substance that exhibits accumulation in cancer tissue and that can more efficiently accumulate in cancer tissue even at a second or later administration, or even when administered to a subject possessing anti-PEG immunity.SOLUTION: A composite polymer comprises a conjugate of a branched polymer and a plurality of zwitterionic polymers, wherein the zwitterionic polymers are linked to termini of the branched polymer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to biocompatible composite polymers and the like that are useful as carriers for drug delivery to cancer tissues. [Background technology]

[0002] Nanoparticles with high blood retention are useful for cancer drug delivery systems (DDS) because they are known to accumulate in cancer tissues due to the Enhanced Permeability and Retention (EPR) effect. Polyethylene glycol (PEG)-modified nanoparticles have been widely used in cancer DDS, but they present a problem of accelerated blood clearance (ABC), where antibodies are produced after the first administration, leading to recognition by the immune system and subsequent excretion from the blood after multiple administrations (Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] A. Abu Lila et al., J. Control Release, 172, 38-47 (2013) Summary of the Invention [Problem to be solved by the invention]

[0004] An object of the present disclosure is to provide a substance that has the ability to accumulate in cancer tissue and can accumulate in cancer tissue more efficiently even when administered for the second or subsequent time, or when administered to a subject with anti-PEG immunity. [Means for solving the problem]

[0005] The structure of branched polymers such as dendrimers can be precisely controlled, and the number of terminals and particle size can be controlled by the generation number (G). For example, the number of terminals of fourth-generation and fifth-generation (G4 and G5) polyamidoamine (PAMAM) dendrimers is 64 and 128, respectively, and the particle size is 4.4 nm and 5.7 nm. Furthermore, branched polymers can retain drugs by utilizing the terminal functional groups and internal voids, so they can be used as drug delivery vehicles. Therefore, the present inventors focused on the use of branched polymers.

[0006] As a result of extensive research, the present inventors have found that the above-mentioned problems can be solved by a conjugated polymer comprising a branched polymer and a plurality of zwitterionic polymers linked together, wherein the zwitterionic polymers are linked to the ends of the branched polymers.

[0007] Item 1. A composite polymer comprising a linker between a branched polymer and a plurality of zwitterionic polymers, wherein the zwitterionic polymers are linked to the ends of the branched polymers.

[0008] Item 2. The conjugated polymer according to Item 1, wherein the branched polymer is a dendrimer, a dendron, a dendrigraft, or a hyperbranched polymer.

[0009] Item 3. The conjugated polymer according to Item 1 or 2, wherein the number of bonds of the zwitterionic polymer is 10 or more.

[0010] Item 4. The composite polymer according to any one of Items 1 to 3, wherein the degree of polymerization of the zwitterionic polymer is 5 or more.

[0011] Item 5. The composite polymer according to any one of Items 1 to 4, wherein the zwitterionic polymer is a polymer of a betaine-type zwitterionic monomer.

[0012] Item 6. The conjugated polymer according to any one of Items 1 to 5, wherein the zwitterionic polymer is a living radical polymer of a zwitterionic monomer.

[0013] Item 7. The conjugated polymer according to any one of Items 1 to 6, wherein the molecular weight of the linker is 30,000 to 1,000,000.

[0014] Item 8. The conjugated polymer according to any one of Items 1 to 7, which holds a drug.

[0015] Item 9. The conjugated polymer according to Item 8, wherein the drug is an anticancer drug.

[0016] Item 10. A carrier for drug delivery to cancer tissue, comprising the composite polymer according to any one of Items 1 to 7.

[0017] Item 11. A medicine comprising the conjugated polymer according to item 8 or 9. [Effects of the Invention]

[0018] According to the present disclosure, it is possible to provide a substance that has the ability to accumulate in cancer tissue and can accumulate in cancer tissue more efficiently even when administered for the second or subsequent time, or when administered to a subject with anti-PEG immunity. [Brief explanation of the drawings]

[0019] [Figure 1] H NMR assignments for [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (DMAPS) (a) and poly([2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide) (PDMAPS) (b) are shown. [Figure 2] The 1H NMR spectra of DMAPS (a), PDMAPS (crude) (b), and PDMAPS (in DO) (c) are shown. [Figure 3] The partial structure of polyamidoamine (PAMAM) dendrimer (den(G4)) is shown. [Figure 4] 1H NMR spectra of PAMAM dendrimer (den(G4)) (a), PDMAPS (b), and composite polymer (c) are shown. [Figure 5] This shows a photograph of in vivo fluorescence imaging when a fluorescently labeled conjugated polymer (den(G4)-PDMAPS4.5k53) was administered in Test Example 1. The time elapsed since administration of the fluorescently labeled conjugated polymer is shown at the bottom of the photograph. The area surrounded by the oval is the tumor site. [Figure 6] This shows a photograph of in vivo fluorescence imaging when a fluorescently labeled conjugated polymer (den(G4)-PDMAPS5.0k30) was administered in Test Example 1. The time elapsed since administration of the fluorescently labeled conjugated polymer is shown at the bottom of the photograph. The area surrounded by the oval is the tumor site. [Figure 7] This shows a photograph of in vivo fluorescence imaging when a fluorescently labeled conjugated polymer (den(G4)-PDMAPS5.9k38) ​​was administered in Test Example 1. The time elapsed since administration of the fluorescently labeled conjugated polymer is shown at the bottom of the photograph. The area surrounded by the oval is the tumor site. [Figure 8] This shows a photograph of in vivo fluorescence imaging when a fluorescently labeled conjugated polymer (den(G4)-PDMAPS5.0k17) was administered in Test Example 1. The time elapsed since administration of the fluorescently labeled conjugated polymer is shown at the bottom of the photograph. The area surrounded by the oval is the tumor site. [Figure 9] The tumor / liver accumulation ratio and spleen / liver accumulation ratio were calculated from the amounts of fluorescently labeled conjugated polymer accumulated in the tumor and each organ measured in an ex vivo fluorescence imaging experiment in Test Example 1. The legend indicates the molecular weight of PDMAPS and the number of PDMAPS units bound in the conjugated polymer used. The columns are numbered 4.5kJ53, 5.0kJ17, and 5.0kJ30 from left to right. [Figure 10] These figures show photographs of in vivo fluorescence imaging when fluorescently labeled conjugated polymers ((a)(b): den(G4)-PDMAPS4.5k53, (c)(d): den(G4)-PDMAPS5.0k30) were administered in Test Example 2. The area surrounded by the oval is the tumor site. [Figure 11]The tumor / liver accumulation ratio and spleen / liver accumulation ratio were calculated from the accumulation amounts of fluorescently labeled conjugated polymer in tumors and each organ measured in ex vivo fluorescence imaging experiments. On the horizontal axis, "single" indicates the case of a single-dose test (Test Example 1), and "double" indicates the case of a multiple-dose test (Test Example 2). The horizontal axis indicates the conjugated polymer used. [Figure 12] These figures show photographs of in vivo fluorescence imaging when fluorescently labeled conjugated polymers ((a): den(G5)-SB, (b): den(G4)-PSB4.5k53) were administered in Test Example 3. The area surrounded by the oval is the tumor site. DETAILED DESCRIPTION OF THE INVENTION

[0020] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0021] In this specification, when multiple upper and lower limits are given for a certain parameter, a range formed by combining any upper limit and any lower limit is also uniquely disclosed.

[0022] In one aspect, the present disclosure relates to a conjugated polymer (sometimes referred to herein as the "conjugated polymer of the present disclosure") comprising a linker between a branched polymer and a plurality of zwitterionic polymers, wherein the zwitterionic polymers are linked to the ends of the branched polymers.

[0023] The branched polymer preferably has, as an end group, one or more functional groups capable of bonding to the zwitterionic polymer described below. Examples of such functional groups include amino groups, carboxyl groups, hydroxyl groups, and sulfone groups. Among these, amino groups are preferred.

[0024] Examples of branched polymers include dendrimers, dendrons, "hyperbranched polymers" with low regularity in branching in a dendritic structure, "dendrigrafts" (also called random dendritic polymers), and star polymers.

[0025] Dendrimers are compounds with a highly three-dimensionally branched tree-like structure and are known to have an almost spherical shape. Dendrimers generally consist of a core, several generations of branches, and terminal groups. The dendrimer core is derived from a compound having one or more functional groups. Functional groups include primary amino groups, secondary amino groups, hydroxy groups, carboxylic acid groups, thiol groups, ester groups, amide groups, ketone groups, and aldehyde groups. Primary amino groups and secondary amino groups are preferred. Examples of compounds that constitute the core include ammonia, ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 1,10-diaminodecane, and 1,12-diaminododecane. An alkyldiamine containing an S-S bond (cystamine) may also be used.

[0026] The branching portion of a dendrimer is composed of repeating branching structural units containing atoms with a valence of three or more. Examples of atoms with a valence of three or more include carbon, nitrogen, silicon, and phosphorus. For example, the following structure is known as the branching portion of a dendrimer.

[0027] [ka]

[0028] (wherein n is an integer of 1 or more, preferably an integer of 60 to 300) The dendrimers having the branched structures (A) to (C) above are described in the following documents: The branched portion of the dendrimer may contain two or more types of repeating units as described above. (A) Tomalia DA et al., Polym. J. 17, 117 (1985) and Tomalia DA et al., Angew. Chem. Int. Ed. Engl. 29, 138 (1990) (B) de Brabander-van den Berg EMM et al., Angew. Chem. Int. Ed. Engl. 32, 1308 (1993); de Brabander-van den Berg EMM et al., Macromol. Symp. 77, 51 (1994); Hummelen JC et al., Chem. Eng. J. 3, 1489 (1997) and Waner C. et al., Angew. Chem. Int. Ed. Engl. 32, 1300 (1993) (C) Ihre HR et al., Bioconjugate Chem. 13, 443-452 (2002) and Goodwin AP et al., J. Am. Chem. Soc., 129, 6994 (2007).

[0029] The structure of the dendrimer's terminal group can be appropriately selected. For example, the terminal group may have the structure of the last repeating unit of the branched portion, or may have a structure different from that of the branched portion. The dendrimer's terminal group preferably has one or more functional groups capable of bonding to the zwitterionic polymer described below.

[0030] A dendron is a partial structure that constitutes a dendrimer, and has a structure in which at least one functional group in the core portion is not branched.

[0031] Hyperbranched polymers are generally polymers in which branches are developed by polymerizing AB2 type monomers. Here, A and B respectively represent a combination of functional groups capable of polymerization, such as a hydroxyl group and a carboxyl group, or an amino group and a carboxyl group. Alternatively, a substance that functions as a branching core may be used in combination. Hyperbranched polymers may also be produced by ring-opening polymerization of glycidol, ethyleneimine, or the like.

[0032] Dendrigrafts are polymers synthesized by reacting oligomers, which are composed of multiple monomers, in a stepwise manner. These are collectively called random dendritic polymers. Random dendritic polymers, like dendrimers, have branched portions, but do not necessarily have a core. Furthermore, the branched portions of random dendritic polymers may have irregular or discontinuous portions due to partial defects.

[0033] The branching units of the random dendritic polymer may be a polylysine skeleton, a polyglycerin skeleton, or various sugar skeletons. Examples of random dendritic polymers in which the branching unit is lysine (—NH—CH(CHNH—)CO—) include PolyLysine-Dendri-graft (PLD: COLCOM). Examples of random dendritic polymers in which the branching unit is polyglycerin represented by the following formula include PGL X and PGL 10 (Daicel Chemical Industries).

[0034] [ka]

[0035] (wherein n and m are independently integers of 10 or more, preferably 20 or more). Star polymers are known as polymers in which three or more polymer chains radiate from a center. Star polymers are broadly classified into regular star polymers, in which the polymer chains are of the same type, and asymmetric star polymers, in which the polymer chains are of different types or molecular weights. Regular star polymers can be obtained by living anionic polymerization of a living anionic polymer of styrene or 1,3-dienes with a polyfunctional silyl chloride. Currently, polybutadiene star polymers with 3 to 128 polymer chains and star polystyrenes with 33 polymer chains have been synthesized. Furthermore, asymmetric star polymers, four-component star polymers composed of polystyrene, polyisoprene, polybutadiene, and poly(4-methylstyrene) are known.

[0036] It is preferable that the branched polymer does not contain PEG as a partial structure, so that it can accumulate in cancer tissues more efficiently even when administered to an anti-PEG immune patient.

[0037] As the branched polymer, preferably, dendritic polymers such as dendrimers, dendrons, dendrigrafts, and hyperbranched polymers are used, and particularly preferably, dendrimers are used.

[0038] The generation number of the dendrimer can be appropriately selected depending on the structure of the core and branched portions of the dendrimer used. In this technology, dendrimers of generations 0 to 10 are generally available. The generation number is preferably 3 to 6, and more preferably 4 to 5.

[0039] Methods for producing dendrimers are known in the art and are described in, for example, the above-mentioned documents. For example, polyamidoamine (PAMAM) dendrimers can be produced by using diamine as a raw material, and then subjecting it to a Michael addition reaction with an acrylic ester, followed by an ester-amido exchange reaction using a diaminoalkane to obtain a first-generation amide compound, and then repeating the Michael addition reaction and the ester-amido exchange reaction (see Tomalia, D. et al., Polym. J. 17, 117-132 (1985); Fréchet, JMJ, Tomalia, DA (eds.), (2001) Dendrimers and other dendritic polymers, J. Wiley & Sons, West Sussex). The above-mentioned diamines are generally available.

[0040] Polyamidoamine dendrimers are commercially available under the trade name Starburst (registered trademark) and are generally available.

[0041] The molecular weight of the branched polymer is preferably 2,000 to 1,000,000, more preferably 5,000 to 200,000, even more preferably 8,000 to 100,000, still more preferably 10,000 to 50,000, and particularly preferably 10,000 to 20,000.

[0042] Examples of the functional group at the end of the branched polymer include an amino group, a carboxyl group, and a hydroxyl group, and an amino group is preferred.

[0043] The number of functional groups at the terminal of the branched polymer is, for example, 5 to 1,000, preferably 10 to 500, more preferably 20 to 300, even more preferably 30 to 200, and still more preferably 40 to 100.

[0044] The zwitterionic polymer is a polymer of zwitterionic monomers, and is not particularly limited in this respect.

[0045] The zwitterionic monomer is not particularly limited as long as it has a cationic moiety and an anionic moiety in the molecule and can be polymerized to form a polymer.

[0046] The cationic moiety may be, for example, a quaternary amine moiety. Specific examples of the quaternary amine moiety include the following partial structures:

[0047] [ka]

[0048] In the above partial structure, R independently represents a hydrocarbon group in each occurrence. The hydrocarbon group is, for example, an alkyl group or an aryl group (preferably an alkyl group), and has, for example, 1 to 15 carbon atoms (preferably 1 to 6, more preferably 1 to 4, and particularly preferably 1 to 2).

[0049] The anion moiety may be, for example, a carboxyl group (-COO - ), sulfo group (-S(=O)2-O -) and a phosphate group moiety. Specific examples of the phosphate group moiety include the following partial structures:

[0050] [ka]

[0051] The zwitterionic monomer preferably has the same charge at the cationic moiety as the charge at the anionic moiety, for example, if the zwitterionic monomer has only one quaternary amine moiety as the cationic moiety, it preferably has only one carboxyl, sulfo, or phosphate moiety as the anionic moiety.

[0052] The zwitterionic monomer has a polymerizable functional group that allows polymerization. The polymerizable functional group is not particularly limited as long as it can form a polymer, but is preferably a radically polymerizable functional group, such as an acryloyl group or a methacryloyl group.

[0053] The molecular weight of the zwitterionic monomer is, for example, 70 to 1,000, preferably 120 to 500, and more preferably 150 to 400.

[0054] The zwitterionic monomer is preferably a betaine-type zwitterionic monomer, more preferably a phosphobetaine-type zwitterionic monomer, a sulfobetaine-type zwitterionic monomer, or a carboxybetaine-type zwitterionic monomer.

[0055] Various zwitterionic monomers are commercially available; for example, products manufactured by Tokyo Chemical Industry Co., Ltd. (https: / / www.tcichemicals.com / JP / ja / product / pick / Zwitterionic_Monomers_Suitable_for_Biocompatible_Polymer_Synthesis) and products manufactured by Fujifilm Corporation (https: / / labchem-wako.fujifilm.com / jp / category / 03158.html) can be used.

[0056] The polymerization method of zwitterionic monomers to form zwitterionic polymers is not particularly limited, and examples include radical polymerization and ionic polymerization. Among these, living radical polymerization is preferred from the viewpoint of uniformity of the molecular weight of the resulting zwitterionic polymer. Furthermore, among these, RAFT polymerization is preferred from the viewpoint of suitability of the resulting zwitterionic polymer for biological use. The zwitterionic polymer is preferably a living radical polymer of zwitterionic monomers, and particularly preferably a RAFT polymer.

[0057] The polymerization reaction can be carried out according to a known method. RAFT polymerization can be carried out, for example, by reacting a solution containing a zwitterionic monomer, an initiator (e.g., an azo polymerization initiator), and a RAFT agent (e.g., a dithioester, a trithiocarbonate, a dithiocarbamate, a dithiocarbonate, etc.) at an appropriate temperature (e.g., 50 to 75°C) for a certain period of time (e.g., 5 to 24 hours).

[0058] From the viewpoints of accumulation in cancer tissues, inhibition of the ABC phenomenon, etc., the number of zwitterionic monomers constituting the zwitterionic polymer (degree of polymerization of the zwitterionic polymer) is preferably 5 or more, more preferably 10 or more, and even more preferably 12 or more. There is no particular upper limit, and it is, for example, 100, 70, 50, or 30.

[0059] The number is determined according to or in accordance with the method described in Reference Example 1. 1 It can be calculated from the integrated value of the peak in the H NMR spectrum. It can also be measured according to or in accordance with the GPC method in Example 1.

[0060] The zwitterionic polymer does not include PEG as a moiety.

[0061] From the viewpoints of accumulation in cancer tissues, inhibition of the ABC phenomenon, etc., the molecular weight of the zwitterionic polymer is preferably 1000 or more, more preferably 2000 or more, even more preferably 3000 or more, still more preferably 4000 or more, and particularly preferably 4800 or more. There is no particular upper limit, and it is, for example, 30000, 20000, 10000, or 7000.

[0062] A linker between a branched polymer and multiple zwitterionic polymers refers to a linker between one branched polymer and each of multiple zwitterionic polymers via a covalent bond. In the conjugated polymers of the present disclosure, the zwitterionic polymers are linked to the ends of the branched polymers.

[0063] The conjugate is preferably obtained by first preparing a branched polymer and a plurality of zwitterionic polymers and then linking them by a linking reaction. The linking reaction is not particularly limited and can be carried out by reacting a reactive functional group (e.g., an amino group, a carboxyl group, a hydroxyl group, a thiol group, a halogen atom, an alkene group, an alkyne group, an azide group, etc.) at the end of the branched polymer with a reactive functional group (e.g., an amino group, a carboxyl group, a hydroxyl group, a halogen atom, an alkene group, an alkyne group, an azide group, etc.) on the zwitterionic polymer. Thus, the conjugate can have a structure in which a group resulting from the removal of multiple reactive functional groups at the end of the branched polymer and a group resulting from the removal of one reactive functional group on the zwitterionic polymer are linked via a post-reaction linking structure (e.g., an amide bond, an ester bond, an ether bond, a disulfide bond, an ene-thiol bond, or a click bond).

[0064] Alternatively, the linker can be prepared by polymerizing a zwitterionic monomer from the end of a branched polymer. For example, by introducing a RAFT agent (e.g., dithioester, trithiocarbonate, dithiocarbamate, dithiocarbonate, etc.) into the end of a branched polymer, a zwitterionic polymer can be introduced by RAFT polymerization. Alternatively, by introducing chlorine or bromine into the end of a branched polymer, a zwitterionic polymer can be introduced by atom transfer radical polymerization (ATRP).

[0065] The number of zwitterionic polymers linked to the branched polymer (the number of zwitterionic polymer bonds) is preferably 5 or more, more preferably 10 or more, even more preferably 10 to 100, still more preferably 10 to 70, particularly preferably 10 to 50, especially more preferably 12 to 40, and particularly preferably 15 to 35, from the viewpoints of accumulation in cancer tissues, suppression of the ABC phenomenon, etc.

[0066] From the viewpoints of accumulation in cancer tissues, inhibition of the ABC phenomenon, etc., the number of zwitterionic polymers linked to a branched polymer is preferably 5% or more, more preferably 12% or more, even more preferably 12 to 80%, even more preferably 15 to 65%, and particularly preferably 20 to 55%, relative to the number of reactive functional groups at the ends of the branched polymer (100%).

[0067] The number of zwitterionic polymers linked to the branched polymer can be determined according to or in accordance with the method described in Example 1. 1 It can be measured by 1 H NMR or fluorescamine assay.

[0068] From the viewpoints of accumulation in cancer tissues, inhibition of the ABC phenomenon, etc., the molecular weight of the linker is preferably 20,000 to 1,000,000, more preferably 30,000 to 700,000, even more preferably 50,000 to 500,000, still more preferably 70,000 to 400,000, particularly preferably 80,000 to 300,000, and particularly preferably 90,000 to 240,000.

[0069] The molecular weight of the linker can be measured according to or in accordance with the GPC method of Example 1.

[0070] The composite polymer of the present disclosure can be used as a carrier for drug delivery to cancer tissue. From this perspective, in one aspect, the present disclosure relates to a carrier for drug delivery to cancer tissue, comprising the composite polymer of the present disclosure. Furthermore, the composite polymer of the present disclosure preferably holds a drug.

[0071] The manner in which the drug is retained is not particularly limited, and examples thereof include a manner in which the drug is linked to the linker via a covalent bond, a manner in which the drug is encapsulated within the linker, etc. In the former case, examples include a manner in which the drug is linked to the end of a branched polymer, a manner in which the drug is linked to a zwitterionic polymer, etc.

[0072] When the branched polymer is a dendrimer as described below, methods for encapsulating a drug in the internal space of the dendrimer are known in the art and are described, for example, in Kojima C. et al., Bioconjuge Chem 11: 910-7 (2000).

[0073] The drug is not particularly limited as long as it can be retained in the conjugate polymer of the present disclosure. Examples of the drug include peptides, nucleic acids, sugars, low molecular weight compounds, high molecular weight compounds, inorganic substances, and complexes thereof. The drug is preferably an anticancer drug.

[0074] Examples of anticancer agents include platinum agents, metabolic antagonists, alkylating agents, microtubule inhibitors, antibiotic anticancer agents, topoisomerase inhibitors, molecular targeted drugs, hormone agents, and biological agents.

[0075] Examples of platinum preparations include cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, miriplatin, lobaplatin, spiroplatin, tetraplatin, ormaplatin, and iproplatin.

[0076] Examples of antimetabolites include enocitabine, carmofur, capecitabine, tegafur, tegafur-uracil, tegafur-gimeracil-oteracil potassium, gemcitabine, cytarabine, cytarabine ocfosfate, nelarabine, fluorouracil, fludarabine, pemetrexed, pentostatin, methotrexate, cladribine, doxifluridine, hydroxycarbamide, and mercaptopurine.

[0077] Examples of alkylating agents include cyclophosphamide, ifosfamide, nitrosourea, dacarbazine, temozolomide, nimustine, busulfan, melphalan, procarbazine, and ranimustine.

[0078] Examples of microtubule inhibitors include alkaloid anticancer drugs such as vincristine, and taxane anticancer drugs such as docetaxel and paclitaxel.

[0079] Examples of antibiotic anticancer agents include mitomycin C, doxorubicin, epirubicin, daunorubicin, bleomycin, actinomycin D, aclarubicin, idarubicin, pirarubicin, peplomycin, mitoxantrone, amrubicin, and zinostatin stimalamer.

[0080] Examples of topoisomerase inhibitors include CPT-11, irinotecan, and nogitecan, which have a topoisomerase I inhibitory effect, and etoposide and sobuzoxane, which have a topoisomerase II inhibitory effect.

[0081] Examples of hormone agents include dexamethasone, finasteride, tamoxifen, astrozole, exemestane, ethinylestradiol, chlormadinone, goserelin, bicalutamide, flutamide, prednisolone, leuprorelin, letrozole, estramustine, toremifene, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, and mepitiostane.

[0082] Examples of biological agents include interferon α, β and γ, interleukin 2, ubenimex, and dried BCG.

[0083] The conjugated polymer of the present disclosure can be used in medicines, reagents, etc. (hereinafter, these may be collectively referred to as "agents of the present disclosure.") In particular, the conjugated polymer of the present disclosure can be used as an active ingredient in medicines for preventing or treating cancer.

[0084] The pharmaceutical is one used for administration to animals, and is not particularly limited in this respect. The pharmaceutical can be a pharmaceutical composition containing other components described below. The reagent is one used for use in experiments (particularly, in the present disclosure, one used for introduction into cells), and is not particularly limited in this respect. The reagent can be a reagent composition containing other components described below.

[0085] The agent of the present disclosure is not particularly limited as long as it contains the conjugated polymer of the present disclosure, and may further contain other components as needed. The other components are not particularly limited as long as they are pharmaceutically acceptable, and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0086] The mode of use of the agent of the present disclosure is not particularly limited, and an appropriate mode of use can be adopted depending on the type of agent. The agent of the present disclosure can be used, for example, in vitro (for example, by adding it to a culture medium for cultured cells) or in vivo (for example, by administering it to an animal (preferably an animal with cancer)).

[0087] The target of application of the agent of the present disclosure is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, etc.; animal cells, etc. The type of cell is also not particularly limited, and examples thereof include blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells, etc.

[0088] The dosage form of the agent of the present disclosure is not particularly limited, and can be an appropriate dosage form depending on its mode of use.For example, when administered to animals, examples include injectable preparations such as drip infusion, intravenous injection, intramuscular injection, subcutaneous injection, and intradermal injection; oral preparations such as tablets, capsules, granules, powders, fine granules, syrups, enteric-coated preparations, sustained release capsules, chewable tablets, drops, pills, oral liquid preparations, confectionery tablets, sustained-release preparations, and sustained-release granules; external preparations such as nasal drops, inhalants, rectal suppositories, inserts, enemas, and jellies.In addition, the agent of the present disclosure may be any of solid preparations, semi-solid preparations, and liquid preparations.

[0089] The content of the composite polymer of the present disclosure in the agent of the present disclosure depends on the mode of use, the target of application, the condition of the target of application, etc., and is not limited, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0090] The dosage of the agent of the present disclosure administered to an animal is not particularly limited as long as it is an effective amount that exerts a medicinal effect, and is usually 0.1 to 1000 mg / kg body weight per day in terms of the weight of the conjugated polymer of the present disclosure, which is the active ingredient. The dosage is preferably administered once a day or in two or three divided doses, and can be increased or decreased as appropriate depending on the age, pathological condition, and symptoms. [Example]

[0091] The present disclosure will be described in detail below based on examples, but the present disclosure is not limited to these examples.

[0092] (reagent) PAMAM dendrimer ethylenediamine core generation 2.0 solution (den(G2), SIGMA-ALDRICH), PAMAM dendrimer ethylenediamine core generation 4.0 solution (den(G4), SIGMA-ALDRICH), 4-cyano-(4-phenylcarbonothioylthio)pentanoic acid (CTP, Nacalai Tesque), N,N'-dimethylformamide (DMF, Watanabe Industries), N-hydroxysuccinimide (NHS, Tokyo Chemical Industry), N,N'-dicyclohexylcarbodiimide (DCC, Tokyo Chemical Industry), triethylamine (TEA, Nacalai Tesque), dimethyl sulfoxide (DMSO, SIGMA-ALDRICH), 3-((2-acryloyloxy)ethyl)-dimethylammonio)propane-1-sulfonate (DMAPS, Tokyo Chemical Industry), The following reagents were used: 4,4-azobis(4-cyanovaleric acid) (ACVA, Wako Pure Chemical Industries, Ltd.), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorphonium chloride (DMT-MM, Fujifilm Wako Pure Chemical Industries, Ltd.), Fluorescamine (Nacalai Tesque), boric acid (Nacalai Tesque), acetone (Nacalai Tesque), methanol (MeOH, Nacalai Tesque), ethanol (EtOH, Nacalai Tesque), dichloromethane (DCM, Nacalai Tesque), ICG-NHS (Nacalai Tesque), dimethyl sulfoxide (DMSO, SIGMA-ALDRICH), deuterated methanol (CD3OD, Nacalai Tesque), heavy water (DO, SIGMA-ALDRICH), and DMSO-d6 (Nacalai Tesque). Phosphate buffered saline (PBS), 100 mM NaH2PO4 aqueous solution, 100 mM Na2HPO4 aqueous solution, NaHCO3 aqueous solution, and 0.5 M NaOH aqueous solution were prepared by ourselves.

[0093] (Laboratory equipment) Centrifugation was performed using a centrifuge (Eppendorf 5804-R, Greiner J804701). Cells were incubated at 37°C in a 5% CO2 incubator (Forma Direct Heat CO2 Incubator 310 Thermo Fisher Scientific). 1H NMR spectra were measured at room temperature using a Bruker NMR Ascend 400 (Nanobey). Gel permeation chromatography (GPC) was performed using an intelligent column oven (JASCO CO-2065) equipped with a refractive index (RI) detector, ultraviolet (UV) detector, and pump. The columns were an α-3000 column and a TSK gel GMPW column. XL The columns were connected and used. Fluorescamine assays were performed using a FP-6200 spectrofluorometer (JASCO) at an excitation wavelength of 390 nm, an emission wavelength of 480 nm, and low sensitivity to measure fluorescence intensity. UV-Vis measurements were performed using a UV / Vis Spectrophotometer V-630 (JASCO) at wavelengths of 700–900 nm and a temperature of 25°C. Fluorescence imaging experiments were performed using an IVIS Lumina Series III (PerkinElmer, Inc., Shelton, CT, USA).

[0094] Reference Example 1. Synthesis of Zwitterionic Polymer Zwitterionic polymers (PDAMPS) were synthesized according to the following scheme.

[0095] [ka]

[0096] The equivalent ratio of DMAPS / CTP / ACVA was adjusted and dissolved in methanol, and the solution was subjected to four freeze-degassing-thaw cycles. After polymerization in an oil bath, the supernatant was removed by vacuum drying, and the solution was dissolved in water. PDMAPS was obtained by reprecipitation using a large amount of ethanol, centrifugation, and freeze-drying. A similar synthesis method was also performed using DMSO and 0.5 M phosphate buffer solution. 1H NMR of PDMAPS in D2O:δ 0.90-1.60 (m, A and a ), 1.60-2.60 (m, b and g), 3.00 (br, h), 3.25 (br, e), 3.62 (br, f), 3.84 (br, d), 4.53 (br, c), 7.55 (m, E), 7.74 (m, F), 8.03 (m, D).

[0097] Figure 1 shows the results of DMAPS and PDMAPS. 1 The H NMR assignments are shown in Figure 2. 1 The H NMR spectrum is shown.

[0098] The conversion was calculated by comparing the integral of the peak b around 2.2 ppm with the integral of the sum of the peaks c and c' around 4.40-4.75 ppm. The degree of polymerization of the polymer was calculated by comparing the integral of the peak c around 4.53 ppm with the integral of the peak D around 8.03 ppm.

[0099] The equivalent ratio of materials, reaction conditions, polymerization rate, degree of polymerization, and yield are shown in Table 1. In the table, values ​​that were not measured or could not be measured are indicated with a -. When 0.5 M phosphate buffer solution was used, the product disappeared during dialysis, which is thought to be why polymerization did not proceed.

[0100] [Table 1]

[0101] Example 1. Synthesis of composite polymer 1 A composite polymer (den(G4)-PDMAPS) of zwitterionic polymer (PDMAPS) and PAMAM dendrimer (den(G4)) was synthesized according to the following scheme.

[0102] [ka]

[0103] After removing the solvent by vacuum drying, the equivalent ratio of den(G4) / PDMAPS / DMT-MM was adjusted and dissolved in 3 mL of PBS and stirred at room temperature. After ultrafiltration (MWCO: 30k, 25 °C, 10,000 rcf, 3 PBSs, 3 purified waters), den(G4)-PDMAPS was obtained by lyophilization. The same synthesis was also performed using water as the solvent. 1 H NMR of den(G4)-PDMAPS in D2O:δ 0.70-4.20 (br, a, b, d, e, f, g, A, B, C and (A), (A'), (B), (B'), (C), (C'),(D), (D') for PAMAM dendrimer), 4.53 (br, c), 7.54 (br, E), 7.74 (br, F), 8.00 (br, D).

[0104] Figure 3 shows the partial structure of the PAMAM dendrimer. 1 The H NMR spectrum is shown.

[0105] 1 The PDMAPS bond was confirmed by H NMR and GPC measurements. The number of PDMAPS bonds was calculated using equation (1) based on the fluorescamine assay.

[0106] (GPC measurement) Five mg of the synthesized sample was dissolved in 1 mL of 80% 0.1 M ammonium sulfate aqueous solution and 20% acetonitrile. The molecular weight of the compound was measured using an intelligent column oven (JASCO, CO-2065) equipped with a refractive index (RI) detector, ultraviolet (UV) detector, and pump, and using an α-3000 column and a TSK gel GMPWXL column at room temperature, a flow rate of 0.5 mL / min, and a measurement time of 50 minutes.

[0107] (Fluorescamine assay) 1.55 g (0.025 mol) of boric acid was dissolved in 50 mL of pure water, and the pH was adjusted to 8-9 with 5 M NaOH solution. 0.9 mg of fluorescamine was dissolved in 3 mL of acetone. 1 mg of den(G4) (7.035 × 10 -5 mol) was dissolved in 1 mL of pure water, and then diluted with 44.02 mL of pure water to prepare a 100 μM NH2-terminated solution. This was then diluted to prepare solutions of various concentrations (Table 2).

[0108] [Table 2]

[0109] 4 mg of sample was dissolved in 1 mL of pure water, then diluted with 9 mL of pure water to obtain the sample solution. 400 μL of the sample solution was taken, and 4 mL of 0.5 M borate buffer solution (pH 8-9) was added. 200 μL of a 0.3 mg / mL acetone solution of fluorescamine was then added and vortexed. Fluorescence intensity was measured using an FP-6200 spectrofluorometer (JASCO) under the following conditions: excitation light: 390 nm, emission wavelength: 480 nm, sensitivity: low. The binding number was calculated by substituting into equation (2). C sample is the concentration of the sample solution, M poymer is the molecular weight of the polymer to be bonded, C NH2 indicates the amino terminal concentration obtained from the calibration curve, and x indicates the desired bond number.

[0110]

number

[0111] The synthesis results under various conditions are shown in Table 3. The molecular weight was calculated using the molecular weight and bond number of PDMAPS. In the name column of the composite polymers, 4.5k, 5.9k, and 5.0k indicate the molecular weight of PDMAPS (calculated from the degree of polymerization (number in parentheses in the polymer column)), and the numbers on the far right (53, 3, 17, 30, 36, and 39) indicate the bond number of PDMAPS (numbers in the bond number column). The molecular weight column shows the molecular weight of the composite polymer.

[0112] [Table 3]

[0113] Comparative Example 1. Synthesis of Composite Polymer 2 We synthesized a sulfobetaine monomer-modified dendrimer (den(G5)-SB) by reacting a fifth-generation PAMAM dendrimer (den(G5)) with the sulfobetaine monomer 3-[(3-acrylamidopropyl)dimethylammonio]propane-1-sulfonic acid (DMAAPS). This is a hybrid polymer of branched polymers and multiple zwitterionic monomers. The structure is shown below.

[0114] [ka]

[0115] Test Example 1. Single-dose test of composite polymer 1 The conjugated polymer (1 eq) from Example 1 was dissolved in 1 mL of 125 mM NaHCO3 aqueous solution + PBS aqueous solution (pH approx. 8), and 120 μL of DMSO was added. Then, 10 eq of a 10 mM DMSO solution of ICG-NHS ester was added and stirred for 24 hours in the dark. Ultrafiltration (MWCO: 10k, 25°C, 10,000 rcf, 2 PBSs, 3 pure waters) was performed, followed by lyophilization to obtain the fluorescently labeled conjugated polymer.

[0116] To investigate the tumor accumulation of the fluorescently labeled conjugated polymer, we administered the fluorescently labeled sample to tumor-bearing mice and performed in vivo and ex vivo fluorescence imaging experiments.

[0117] (4T1 cell culture) 4T1 cells, derived from mouse breast cancer cells, were used. 4T1 cells were cultured in DMEM medium at 37°C in a 5% CO2 incubator. 4T1 cells were cultured in DMEM medium containing 10% FBS and a penicillin-streptomycin mixture at 37°C in a Forma Direct Heat CO2 Incubator 310 (Thermo Fisher Scientific) in a 5% CO2 atmosphere.

[0118] (Fluorescence imaging experiment) BALB / c mice (8 weeks old, female, 20 g, Japan SLC Co., Ltd.) were used. 4T1 cells (0.5-1.0 × 10 6 Cells (ATCC) were dissolved in 100 μL of PBS and administered subcutaneously to BALB / C mice. Two weeks later, a fluorescently labeled conjugated polymer solution (18 nmol ICG / mL) was administered via the tail vein. After 3, 6, 9, 12, and 24 hours, the mice were anesthetized and subjected to in vivo imaging. Afterwards, the mice were euthanized and subjected to ex vivo imaging of the tumor, liver, kidneys, lungs, heart, and spleen.

[0119] The results of the in vivo fluorescence imaging experiments are shown in Figures 5 to 8. In addition, the tumor / liver accumulation ratio and spleen / liver accumulation ratio were calculated from the accumulation amounts of the fluorescently labeled conjugated polymer in the tumor and each organ measured in the ex vivo fluorescence imaging experiments. The results are shown in Figure 9.

[0120] Test Example 2: Multiple administration test of composite polymer The experiment was conducted in the same manner as in Test Example 1, except that one week after administration of 4T1 cells, a solution of the conjugated polymer (fluorescently unlabeled) (0.02 mg / mL) was administered via the tail vein, and one week later, a solution of the same conjugated polymer fluorescently labeled (18 nmol ICG / mL) was administered via the tail vein.

[0121] The results of the in vivo fluorescence imaging experiment are shown in Figure 10. In addition, the tumor / liver and spleen / liver accumulation ratios were calculated from the accumulation amounts of the fluorescently labeled conjugated polymer in the tumor and each organ measured in the ex vivo fluorescence imaging experiment. The results compared with those of single administration are shown in Figure 11.

[0122] Test Example 3. Composite Polymer Single-Dose Administration Test 2 The test was carried out in the same manner as in Test Example 1, except that the conjugated polymer of Comparative Example 1 was used and in vivo imaging was carried out 24 hours after administration of the fluorescently labeled conjugated polymer solution.

[0123] The results of the in vivo fluorescence imaging experiment are shown in FIG.

Claims

1. A conjugated polymer comprising a branched polymer and a plurality of zwitterionic polymers linked together, the zwitterionic polymers being linked to the ends of the branched polymers.

2. The conjugated polymer of claim 1 , wherein the branched polymer is a dendrimer, a dendron, a dendrigraft, or a hyperbranched polymer.

3. The conjugated polymer of claim 1 , wherein the zwitterionic polymer has a bond number of 10 or more.

4. 10. The conjugated polymer of claim 1, wherein the degree of polymerization of the zwitterionic polymer is 5 or greater.

5. 2. The conjugated polymer of claim 1, wherein the zwitterionic polymer is a polymer of betaine-type zwitterionic monomers.

6. 10. The conjugated polymer of claim 1, wherein the zwitterionic polymer is a living radical polymer of zwitterionic monomers.

7. The conjugated polymer of claim 1, wherein the molecular weight of the linker is 30,000 to 1,000,000.

8. The conjugated polymer of any one of claims 1 to 7, which carries a drug.

9. The conjugated polymer of claim 8 , wherein the drug is an anti-cancer drug.

10. A carrier for drug delivery to cancer tissue, comprising the composite polymer according to any one of claims 1 to 7.

11. A medicine comprising the conjugated polymer of claim 8.

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