Conjugates and Cancer Therapeutics

Abstract

A conjugate, wherein a deferoxamine is bound to a biocompatible polymer, wherein the deferoxamine is at least one compound selected from the group consisting of deferoxamine or its ions or salts, and their derivatives, and the biocompatible polymer comprises a first biocompatible polymer chain and a second biocompatible polymer chain different from the first biocompatible polymer chain.

Description

Technical Field

[0001] The present invention relates to a conjugate with deferoxamine and a cancer therapeutic agent.

[0002] This case claims priority based on Tokugawa Published Application No. 2019-083250 filed in Japan on April 24, 2019, the contents of which are incorporated herein by reference. Background Art

[0003] The required amount of iron required for cancer cell proliferation is high, and the iron chelation therapy that iron chelators are applied to cancer therapeutic drugs as a new method for cancer treatment is receiving attention. Among numerous iron chelators, deferoxamine (DFO) that demonstrates specific chelation to iron (III) ions is widely used as a therapeutic drug for iron overload, so its research is the most prevalent so far, and it is reported that deferoxamine demonstrates the effect of inhibiting cell proliferation by cell cycle arrest to various cancer cell strains (non-patent literature 1-3). In addition, clinical studies have also been carried out, demonstrating certain antitumor effects (non-patent literature 4 and 5).

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent literature 1: Becton DL., Bryles P. Deferoxamine Inhibition of Human Neuroblastoma Viability and Proliferation. Cancer Research 48, 7189-7192, (1988);

[0007] Non-patent literature 2: Yang Y., Xu Y., Su A., Yang D., Zhang X. Effects of Deferoxamine on Leukemia In Vitro and Its Related Mechanism. Med Sci Monit. 24, 6735-6741, (2018);

[0008] Non-Patent Document 3: Bajbouj K., Shafarin J., Hamad M. High-Dose Deferoxamine Treatment Disrupts Intracellular Iron Homeostasis, Reduces Growth, and Induces Apoptosis in Metastatic and Nonmetastatic Breast Cancer Cell Lines. Technol Cancer Res Treat 17, 1-11, (2018);

[0009] Non-Patent Document 4: Yamasaki T., Terai S., Sakaida I. Deferoxamine for Advanced Hepatocellular Carcinoma. The New England Journal of Medicine 365, 576-578, (2011);

[0010] Non-Patent Document 5: Donfrancesco A., Deb G., Dominici C., Pileggi D., Castello M A., Helson L. Effects of a Single Course of Deferoxamine in Neuroblastoma Patients. Cancer Research 50, 4929-4930, (1990);

[0011] Non-Patent Document 6: Hamilton J L., Kizhakkedathu J N. Polymeri c nanocarriers for the treatment of systemic iron overload. Mol. Cell. Ther. 3, 3, (2015);

[0012] Non-Patent Document 7: Hallaway P E., Eaton J W., Panter S S., Hedlund B E. Modulation of deferoxamine toxicity and clearance by covalent attachment to biocompatible polymers. Proc Natl Acad Sci USA 86, 10108-10112, (1989);

[0013] Non-patent literature 8: Blatt J., Boegel F., Hedlund BE., Arena VC., ShadduckRK. Failure to alter the course of acute myelogenous leukemia in the rat with subcutaneous deferoxamine. Leukemia Research 15, 391-394, (1991);

[0014] Non-patent literature 9: Wang W., Tabu K., Hagiya Y., Sugiyama Y., Kokubu Y., Murota Y., Ogura S., Taga T. Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by ironchelation. Scientific Reports 7:42070, (2017). Summary of the invention

[0015] Problems to be solved by the invention

[0016] However, the half-life of DFO in the blood is extremely short, less than 20 minutes, and in order to obtain a sufficient therapeutic effect, it must be administered for a long time (8 hours to) by intravenous drip (Non-patent Documents 4-6). In addition, since it does not have selective accumulation in tumors, there is also concern that it may have side effects on the whole body.

[0017] The aforementioned early disappearance problem can be improved by combining DFO with a polymer such as dextran or hydroxyethyl starch (HES) (Non-Patent Document 7), and there are reports that the survival time of leukemia rats administered with DFO combined with HES (HES-DFO) is prolonged (Non-Patent Document 8). However, the tumor accumulation of HES-DFO has not been studied, and there are no reports of polymer-modified DFO showing an antitumor effect on solid cancer.

[0018] The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a conjugate and a cancer therapeutic agent which have excellent blood retention and tumor accumulation and exhibit excellent anti-tumor effects.

[0019] Technical means to solve the problem

[0020] That is, the present invention has the following aspects.

[0021] <1> A conjugate, wherein a deferoxamine is bound to a biocompatible polymer, wherein the deferoxamine is at least one compound selected from the group consisting of deferoxamine or its ion or salt, and their derivatives, and the biocompatible polymer comprises a first biocompatible polymer chain and a second biocompatible polymer chain different from the first biocompatible polymer chain.

[0022] <2> The conjugate according to <1> above, wherein the deferoxamine is bound to the second biocompatible polymer chain.

[0023] <3> The conjugate according to <1> or <2> above, wherein the second biocompatible polymer chain is a polyamino acid.

[0024] <4> The conjugate according to any one of <1> to <3> above, wherein the first biocompatible polymer chain is polyethylene glycol.

[0025] <5> The conjugate according to any one of <1> to <4> above, comprising a structure represented by the following general formula (1) or (1-1):

[0026] [Chemistry 1]

[0027]

[0028] (In formulas (1) to (1-1),

[0029] A represents the first biocompatible polymer chain,

[0030] L represents a linker portion,

[0031] B represents the second biocompatible polymer chain, and includes the repeating structure represented by (b2) below, or the repeating structure represented by (b1) and the repeating structure represented by (b2) below.

[0032] [Chemistry 2]

[0033]

[0034] (In formulas (b1) to (b2),

[0035] R 1 represents the amino acid side chain,

[0036] R 2 It is formed by combining the amino acid side chain with the deferoxamine.

[0037] n represents the total number of (b1) and (b2), n is an integer from 1 to 1000, m is an integer from 1 to 1000 (where m≤n), and when n-m is 2 or more, a plurality of R 1 They may be the same or different from each other. When m is 2 or more, multiple R 2 can be the same or different from each other).

[0038] <6> The conjugate according to <5>, comprising a structure represented by the following general formula (1-2):

[0039] [Chemistry 3]

[0040]

[0041] (In formula (1-2),

[0042] B represents the second biocompatible polymer chain, and includes the repeating structure represented by the following (b2-1), or the repeating structure represented by (b1-1) and the repeating structure represented by (b2-1)

[0043] [Chemistry 4]

[0044]

[0045] (In formulas (b1-1) to (b2-1),

[0046] R 11 represents the amino acid side chain,

[0047] R 12 The carboxyl group in the aspartic acid side chain represented by -CH2-COOH or the glutamic acid side chain represented by -CH2-CH2-COOH is bonded to the deferoxamine,

[0048] n1 represents the total number of (b1-1) and (b2-1), n1 is an integer from 1 to 1000, m1 is an integer from 1 to 1000 (where m1≤n1), and when n1-m1 is 2 or more, multiple R 11 They may be the same or different from each other. When m1 is 2 or more, multiple R 12 can be the same or different from each other).

[0049] <7> The conjugate according to any one of <1> to <6> above, which has a number average molecular weight of 2,000 to 200,000.

[0050] <8> A cancer therapeutic agent comprising the conjugate according to any one of <1> to <7> above as an active ingredient.

[0051] Effects of the Invention

[0052] According to the present invention, a conjugate and a cancer therapeutic agent which have excellent blood retention and tumor accumulation and exhibit excellent anti-tumor effects can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 PEG-PBLA prepared in Example 35 of 1 H NMR spectrum.

[0054] Figure 2 PEG-PBLA 35 GPC curve of .

[0055] Figure 3 PEG-PBLA prepared in Example 78 of 1 H NMR spectrum.

[0056] Figure 4 PEG-PBLA 78 GPC curve of .

[0057] Figure 5 PEG-PAsp prepared in the example 35 of 1 H NMR spectrum.

[0058] Figure 6 PEG-PAsp 35 GPC curve of .

[0059] Figure 7 PEG-PAsp prepared in the example 78 of 1 H NMR spectrum.

[0060] Figure 8 PEG-PAsp 78 GPC curve of .

[0061] Fig. 9 PEG-P[Asp(DFO) prepared in the example 10 ] 35 of 1 H NMR spectrum.

[0062] Fig.10 PEG-P[Asp(DFO) 10 ] 35 GPC curve of .

[0063] Fig.11 PEG-P[Asp(DFO) prepared in the example 26 ]78 of 1 H NMR spectrum.

[0064] Fig.12 PEG-P[Asp(DFO) 26 ] 78 GPC curve of .

[0065] Fig.13 This is a schematic diagram for explaining the principle of the Calcein-AM method.

[0066] Fig.14 It is DFO / Fe and PEG-P[Asp(DFO) m ] n UV-Vis spectrum of (n=35, 78) / Fe.

[0067] Fig.15A This is a graph showing the fluorescence intensity of calcein fluorescence acquired by flow cytometry.

[0068] Fig. 15B This is a graph showing the fluorescence intensity of calcein fluorescence acquired by flow cytometry.

[0069] Fig.16 The images observed using a confocal microscope show the state of DLD-1 cells after adding Cy5-PEG-PAsp (DFO) and culturing for 24 hours.

[0070] Fig.17A It represents DFO and PEG-P[Asp(DFO) 10 ] 35 Graph of cellular uptake.

[0071] Fig. 17B It represents DFO and PEG-P[Asp(DFO) 10 ] 35 Graph of cellular uptake.

[0072] Fig.18A It indicates that the addition of DFO and PEG-P[Asp(DFO) 10 ] 35 This is a graph showing the results of cell cycle analysis of cultured DLD-1 cells.

[0073] Fig.18B It indicates that the addition of DFO and PEG-P[Asp(DFO) 10 ] 35 This is a graph showing the results of cell cycle analysis of cultured DLD-1 cells.

[0074] Fig.19It means that after adding PEG-P[Asp(DFO) m ] n (n=35, 78) Graph showing inhibition of cell proliferation in cultured DLD-1 cells.

[0075] Fig. 20 It is a comparison of DFO and PEG-P[Asp(DFO) in a CT26 subcutaneous tumor mouse model. m ] n This is a graph showing the results of blood retention of (n=35, 78).

[0076] Fig.21 It is a comparison of DFO and PEG-P[Asp(DFO) in a CT26 subcutaneous tumor mouse model. m ] n Figure 2 is a graph showing the results of tumor accumulation of (n=35, 78).

[0077] Fig. 22 It indicates that in the DLD-1 subcutaneous tumor mouse model, administration of DFO or PEG-P[Asp(DFO) m ] n (n=35, 78) Graph showing the time course changes in the size of the tumor induced by the cytokine.

[0078] Fig.23 It indicates that in the DLD-1 subcutaneous tumor mouse model, administration of DFO or PEG-P[Asp(DFO) m ] n (n=35, 78) is a graph showing the changes in body weight over time caused by

[0079] Fig.24 It indicates that in the CT26 subcutaneous tumor mouse model, administration of DFO or PEG-P[Asp(DFO) m ] n (n=35, 78) Graph showing the time course changes in the size of the tumor induced by the cytokine.

[0080] Fig.25 It indicates that in the CT26 subcutaneous tumor mouse model, administration of DFO or PEG-P[Asp(DFO) m ] n (n=35, 78) is a graph showing the changes in body weight over time caused by

[0081] Fig.26 1 is a schematic diagram illustrating the principle of photodynamic diagnosis and / or therapy using 5-aminolevulinic acid.

[0082] Fig.27A It means adding DFO or PEG-P[Asp(DFO) 10] 35 , and confocal microscopy images of DLD-1 cells after culture with FerroOrange.

[0083] Fig.27B It means that DFO or PEG-P[Asp(DFO) 10 ] 35 This is a graph showing the results of analyzing the amount of free iron in the cytoplasm of DLD-1 cells after culture.

[0084] Fig.28A It means adding DFO or PEG-P[Asp(DFO) 10 ] 35 , and confocal microscopy images of DLD-1 cells after FITC-anti-BrdU antibody culture.

[0085] Fig.28B It means that DFO or PEG-P[Asp(DFO) 10 ] 35 This is a graph showing the results of cell cycle analysis of cultured DLD-1 cells.

[0086] Fig.29A It means that DFO or PEG-P[Asp(DFO) 10 ] 35 This is a graph showing the results of FCM analysis of the apoptosis-inducing status in cultured DLD-1 cells.

[0087] Fig.29B It means that DFO or PEG-P[Asp(DFO) 10 ] 35 This is a graph showing the results of converting cultured DLD-1 cells into viable cells, early-stage apoptotic cells, or late-stage apoptotic / necrotic cells.

[0088] Fig.30 It indicates that in the CT26 subcutaneous tumor mouse model, administration of DFO or PEG-P[Asp(DFO) 10 ] 35 A graph showing the temporal changes in protoporphyrin IX accumulation in induced tumors.

[0089] Fig.31 It means that vitamin C and PEG-P[Asp(DFO) 36 ] 76 A graph showing the cancer cell-killing effect.

[0090] Fig.32It indicates that in the CT26 subcutaneous tumor mouse model, administration of vitamin C, or vitamin C and PEG-P[Asp(DFO) 36 ] 76 Graph of the changes in size of induced tumors over time. DETAILED DESCRIPTION

[0091] Hereinafter, the conjugate and cancer therapeutic agent according to one embodiment of the present invention will be described.

[0092] "Couplet"

[0093] The conjugate of the embodiment is formed by combining a deferoxamine compound with a biocompatible polymer, wherein the biocompatible polymer comprises a first biocompatible polymer chain and a second biocompatible polymer chain different from the first biocompatible polymer chain.

[0094] The conjugates of the embodiments have iron chelation from deferoxamines, preferably specific chelation for iron (III) ions.

[0095] The conjugate of the embodiment is formed by combining deferoxamine with a polymer. The polymer may include a first polymer chain and a second polymer chain different from the first polymer chain. The polymer may be a biocompatible polymer.

[0096] <Deferoxamine>

[0097] In the present specification, the term "deferoxamine-type compound" refers to at least one compound selected from the group consisting of deferoxamine, its ion or salt, and derivatives thereof.

[0098] Deferoxamine is known as a compound represented by the following formula (I) (hereinafter referred to as "compound (I)"). Among various iron chelators, deferoxamine exhibits a specific chelating action on iron (III) ions and is widely used as a therapeutic drug for iron overload.

[0099] [Chemistry 5]

[0100]

[0101] Deferoxamine can be provided in the form of an ion or a salt. As the ion of deferoxamine in the compound (I), the compound (I) may be a cation or an anion.

[0102] When compound (I) is used as a cation, examples include the addition of a proton to the group represented by "-NH2" in compound (I) to convert it into "-NH3 + "The cationic part represented by

[0103] Examples of the ion of compound (I) include compounds represented by the following formula (I-1).

[0104] [Chemistry 6]

[0105]

[0106] Examples of the salt of deferoxamine include pharmacologically acceptable salts, and deferoxamine mesylate represented by the following formula (I-2) is preferred.

[0107] [Chemistry 7]

[0108]

[0109] As pharmacologically acceptable salts, pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, etc. can be cited. As acid addition salts, for example, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, formate, acetate, propionate, toluenesulfonate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, maleate, malate, etc. can be exemplified. As metal salts, alkali metal salts such as lithium salt, sodium salt, potassium salt, alkaline earth metal salts such as magnesium and calcium salt, aluminum, zinc, etc. can be exemplified. As ammonium salts, alkylammonium salts such as ammonium salt and tetramethylammonium salt can be exemplified. As organic amine salts, triethylamine salts, piperidine salts, morpholine salts, toluidine salts, etc. can be exemplified. In addition, these salts can also be used as solutions when used.

[0110] As a derivative of deferoxamine, there is no particular limitation as long as it has the iron chelating effect. As a derivative of deferoxamine, there can be cited derivatives obtained by replacing one or more hydrogen atoms or groups with groups (substituents) other than deferoxamine or its ions or salts. In addition, hydrogen atoms can be added or removed from deferoxamine or its ions or salts. Here, as substituents, there can be cited: hydroxyl, amino, monovalent chain saturated hydrocarbon groups with 1 to 4 carbon atoms, halogen atoms, etc. As monovalent chain saturated hydrocarbon groups with 1 to 4 carbon atoms, for example, there can be cited: methyl, ethyl, propyl, butyl, pentyl, etc. As halogen atoms, there can be cited: fluorine atoms, chlorine atoms, etc.

[0111] It is believed that the iron chelation effect of deferoxamine is exerted through the three hydroxamic acid structural parts shown in the following formula (I-3). Therefore, from the aspect of being difficult to affect the iron chelation effect, for example, it is preferred that the group represented by the terminal "-NH2" in the above-mentioned compound (I-1) (the terminal "-NH3 + ”) is substituted with other groups (the above-mentioned substituents).

[0112] [Chemistry 8]

[0113]

[0114] <Biologically compatible polymers>

[0115] In the conjugate of the present embodiment, the biocompatible polymer refers to a polymer that does not or is unlikely to cause significant harmful effects or adverse effects such as strong inflammatory response or damage when administered to a living body.

[0116] As the biocompatible polymer, there is no particular limitation as long as the effects of the present invention can be obtained, and examples thereof include polyethylene glycol (PEG), acrylic resins (resins containing structural units derived from (meth)acrylates), polyamino acids, polynucleotides, polyacrylamides, polyethers, polyesters, polyurethanes, polysaccharides, and copolymers thereof. The biocompatible polymer may have an arbitrary group introduced in a part thereof during its synthesis. As such a group, for example, a part of a polymerization initiator may be mentioned.

[0117] The biocompatible polymer involved in this embodiment includes a first biocompatible polymer chain and a second biocompatible polymer chain. In addition, the first biocompatible polymer chain is different from the second biocompatible polymer chain, and the biocompatible polymer involved in this embodiment can be provided in the form of a block copolymer of a block including the first biocompatible polymer chain and a block including the second biocompatible polymer chain. In addition, the biocompatible polymer involved in this embodiment can further include other polymer chains in addition to the first biocompatible polymer chain and the second biocompatible polymer chain.

[0118] In the present embodiment, "block copolymer" refers to a polymer formed by combining multiple types of blocks (partial constituent components obtained by repeated combination of structural units of the same type). The blocks constituting the block copolymer may be 2 or 3 or more types.

[0119] The number average molecular weight (Mn) of the biocompatible polymer can also be appropriately determined according to the molecular weight of the conjugate of this embodiment.

[0120] Utilization of the first biocompatible polymer chain 1 The number average molecular weight calculated by H NMR is preferably 700 to 100,000, more preferably 2,000 to 50,000, and even more preferably 7,000 to 30,000.

[0121] Utilization of the second biocompatible polymer chain 1The number average molecular weight calculated by H NMR is preferably 700 to 100,000, more preferably 2,000 to 50,000, and even more preferably 7,000 to 30,000.

[0122] The number average molecular weight ratio of the first biocompatible polymer chain to the second biocompatible polymer chain (first biocompatible polymer chain: second biocompatible polymer chain) may be, for example, 10:1 to 1:10, or 10:3 to 3:10.

[0123] The dispersity (Mw / Mn) of the biocompatible polymer is preferably 1.0 or more and less than 2.0, more preferably 1.0 to 1.5, further preferably 1.0 to 1.3, and particularly preferably 1.0 to 1.2. From the perspective that the conjugate of the embodiment can more effectively exert excellent tumor accumulation, the dispersity of the biocompatible polymer is preferably within the above range.

[0124] In this specification, the number average molecular weight of the polymer can be expressed as 1 The value calculated by the ratio of the peak integral value obtained from the H NMR spectrum. As a calculation method, for example, as shown in the following examples, the degree of polymerization of the monomer is calculated from the ratio of the peak integral value of the structure derived from the initiator present at the polymer chain terminal to the peak integral value of the structure derived from the monomer of the calculation object portion, and the total molecular weight derived from the polymerized monomer structure and the molecular weight derived from the initiator structure are added to calculate the number average molecular weight.

[0125] The number average molecular weight of the following conjugates can also be partially adopted according to 1 The value is calculated from the ratio of the peak integration value obtained from the H NMR spectrum. As a calculation method, for example, as shown in the following examples, the number of DFO bonds is calculated from the ratio of the peak integration value of the structure derived from the initiator present at the end of the polymer chain to the peak integration value of the structure derived from DFO in the calculation target portion, and the total molecular weight of the structure derived from the bound DFO is added to the number average molecular weight of the polymer chain.

[0126] From the viewpoint of excellent biocompatibility and versatility, the first biocompatible polymer chain or the second biocompatible polymer chain is preferably polyethylene glycol.

[0127] From the viewpoint of excellent biocompatibility and a balance between biostability and biodegradability, the first biocompatible polymer chain or the second biocompatible polymer chain is preferably a polyamino acid.

[0128] As a combination of the first biocompatible polymer chain and the second biocompatible polymer chain included in the biocompatible polymer, for example, preferably, the first biocompatible polymer chain is polyethylene glycol (PEG) and the second biocompatible polymer chain is a combination of polyamino acids.

[0129] In the conjugate of this embodiment, the biocompatible polymer is preferably biodegradable.

[0130] Biodegradability means the property of being absorbed or decomposed in a living body. The biodegradable biocompatible polymer is not particularly limited as long as the effects of the present invention can be obtained, and examples thereof include polyamino acids, polyesters, polynucleotides, polysaccharides, and the like.

[0131] In this specification, the biocompatible polymer being biodegradable means that at least a portion of the biocompatible polymer is biodegradable. Therefore, block copolymers of polyamino acids, polyesters, polynucleotides, polysaccharides, etc. with PEG, acrylic resins (resins containing structural units derived from (meth)acrylates), polyacrylamides, polyethers, polyurethanes, etc., also belong to biodegradable biocompatible polymers.

[0132] By using a biodegradable polymer, accumulation of the conjugate in the body can be suppressed, and side effects can be reduced.

[0133] In this specification, biostability means that the polymer can exist in the body without being immediately absorbed or decomposed. When the biocompatible polymer has biodegradability and biostability, it means that the polymer can exist in the body before being absorbed or decomposed in the body.

[0134] In this specification, the biocompatible polymer being biostable means that at least a portion of the biocompatible polymer is biostable. Therefore, block copolymers of polyamino acids, polyesters, polynucleotides, polysaccharides, etc. with PEG, acrylic resins (resins containing structural units derived from (meth)acrylates), polyacrylamides, polyethers, polyurethanes, etc., also belong to biostable biocompatible polymers.

[0135] The method for producing the biocompatible polymer comprising the first biocompatible polymer chain and the second biocompatible polymer chain is not particularly limited. For example, it can be produced by the following method: after synthesizing the first biocompatible polymer chain by a known polymerization reaction, the monomers of the second biocompatible polymer chain are polymerized to the first biocompatible polymer chain. The polymer chains obtained by the polymerization reaction can be in the state of precursors (for example, having protective groups), and the precursors obtained by the polymerization reaction can be subjected to a common treatment selected by a person skilled in the art to produce the first biocompatible polymer chain and the second biocompatible polymer chain.

[0136] Alternatively, the first biocompatible polymer chain or its precursor provided in advance as a polymer can be combined with the second biocompatible polymer chain or its precursor using a known reaction. In this case, the two can be combined by combining reactive functional groups. When using a precursor, the first biocompatible polymer chain and the second biocompatible polymer chain can be manufactured by appropriately treating them in the same manner.

[0137] <Conjugate>

[0138] The conjugate of this embodiment is formed by combining deferoxamine with a biocompatible polymer.

[0139] In addition, regarding the binding of the biocompatible polymer and the deferoxamine, the biocompatible polymer and the deferoxamine may be bound directly or via any linker, and the binding method is not particularly limited as long as the effects of the present invention can be obtained.

[0140] In order to ensure binding stability in vivo, it is preferred that the biocompatible polymer and the deferoxamine are bound via a covalent bond.

[0141] For example, the biocompatible polymer and the deferoxamines can be bound by utilizing the mutually reactive functional groups of the biocompatible polymer and the deferoxamines. The reactive functional groups may be inherent in the biocompatible polymer and / or the deferoxamines, or may be modified or introduced.

[0142] In the combination of a biocompatible polymer and deferoxamine, as long as deferoxamine and the biocompatible polymer can each achieve the effects of the present invention, structural changes required for their combination may be accepted.

[0143] In the conjugate of the embodiment, only one deferoxamine compound may be bound to the biocompatible polymer, or two or more deferoxamine compounds may be bound to the biocompatible polymer.

[0144] The number of deferoxamines in the conjugate of this embodiment may be an integer greater than 1, and may be an integer from 1 to 1000, an integer from 3 to 100, or an integer from 5 to 50.

[0145] When the number is equal to or greater than the lower limit, the iron chelating effect of the deferoxamine is well exerted, and when the number is equal to or less than the upper limit, the hydrophilicity of the conjugate is appropriately improved, which is preferred.

[0146] In the conjugate of this embodiment, the deferoxamine can be bound to any site in the biocompatible polymer. The deferoxamine can be bound to the first biocompatible polymer chain and / or the second biocompatible polymer chain.

[0147] For example, deferoxamine can be bound to a functional group of the first biocompatible polymer chain and / or the second biocompatible polymer chain.

[0148] The binding mode is not particularly limited. For example, in the deferoxamine of the compound (I), if it is bound to a group (amino group) represented by "-NH2", it is not easy to affect the iron chelation effect of deferoxamine, and there is no need to modify deferoxamine or make it into a derivative. Therefore, for example, deferoxamine can be bound to the "group capable of binding to the amino group" of the first biocompatible polymer chain and / or the second biocompatible polymer chain through the amino group of deferoxamine.

[0149] Examples of the group that can be bonded to the amino group include a carboxyl group, a hydroxyl group, an aldehyde group, and a carbonyl group. These groups may be groups possessed by the side chains of the polymer chains.

[0150] Among them, from the viewpoint of binding stability with an amino group and ease of synthesis, a carboxyl group is preferably used as a group capable of binding with an amino group. The first biocompatible polymer chain and / or the second biocompatible polymer chain may have a carboxyl group. By forming an amide bond between the carboxyl group of the first biocompatible polymer chain and / or the second biocompatible polymer chain and the amino group of the deferoxamine, the first biocompatible polymer chain and / or the second biocompatible polymer chain can be bound to the deferoxamine.

[0151] Examples of the biocompatible polymer chain having a carboxyl group in the molecule include polyamino acids, polyacrylic acid, polymethacrylic acid, carboxymethylcellulose, and hyaluronic acid.

[0152] The carboxyl group may be protected by a protecting group.

[0153] In the case of a biocompatible polymer chain having no carboxyl group or a carboxyl group protected by a protecting group, a carboxyl group can be introduced by a known method using chloroacetic acid, succinic anhydride, p-nitrophenyl chloroformate, or the like.

[0154] For example, when the biocompatible polymer is polyacrylamide, a biocompatible polymer having a carboxyl group or a carboxyl group protected by a protecting group can be produced by using acrylic acid or benzyl acrylate as a raw material by a known production method such as free radical polymerization or living radical polymerization.

[0155] As a method for forming an amide bond using a carboxyl group and an amino group, for example, there can be mentioned: a biocompatible polymer chain having a carboxyl group and a deferoxamine having an amino group are subjected to a condensation reaction in the presence of a condensing agent such as N,N'-dicyclohexylcarbodiimide. In addition, in the case of a biocompatible polymer chain having a carboxyl group protected by a protecting group, the protecting group can be deprotected by a known reaction to obtain a biocompatible polymer chain having a carboxyl group, and then a condensation reaction can be similarly carried out.

[0156] In addition, the deferoxamines may be bound to only one of the first biocompatible polymer chain or the second biocompatible polymer chain. For example, the deferoxamines may be bound to the second biocompatible polymer chain. For example, the second biocompatible polymer chain has a side chain, and the deferoxamines may be bound to the side chain of the second biocompatible polymer chain.

[0157] An example of the conjugate according to the present embodiment is a conjugate comprising a structure represented by the following general formula (1) or (1-1).

[0158] [Chemistry 9]

[0159]

[0160] (In formulas (1) to (1-1), A represents the first biocompatible polymer chain, L represents a linker, and B represents the second biocompatible polymer chain bonded to the deferoxamine.)

[0161] The linker part is preferably an alkylene group having 1 to 20 carbon atoms, preferably a straight-chain alkylene group having 1 to 20 carbon atoms, and more preferably a straight-chain alkylene group having 1 to 5 carbon atoms. One or more -CH2- in the alkylene group may be independently substituted with -CH=CH-, -O-, -CO-, -S-, -NH-, or -CONH-. Examples of the alkylene group include methylene, ethylene, trimethylene, tetramethylene, pentamethylene, and the like.

[0162] In the conjugate of this embodiment, the second biocompatible polymer chain to which the deferoxamine compound is bound is preferably a polyamino acid.

[0163] When the second biocompatible polymer chain is a polyamino acid, B in the general formula (1) or (1-1) is preferably the following.

[0164] B represents the second biocompatible polymer chain bonded to the deferoxamine, and the second biocompatible polymer chain preferably includes a repeating structure represented by (b2) below, or a repeating structure represented by (b1) and a repeating structure represented by (b2).

[0165] [Chemistry 10]

[0166]

[0167] (In formulas (b1) to (b2),

[0168] R 1 represents the amino acid side chain,

[0169] R 2It is formed by combining the amino acid side chain with the deferoxamine.

[0170] n represents the total number of (b1) and (b2), n is an integer from 1 to 1000, m is an integer from 1 to 1000 (where m≤n), and when n-m is 2 or more, a plurality of R 1 They may be the same or different from each other. When m is 2 or more, multiple R 2 can be the same or different from each other).

[0171] The amino acid side chain is used in the usual sense in this field to refer to structures other than the amino group and the carboxyl group involved in the amide bond of the polypeptide, for example, a hydrogen atom in the case of glycine, a methyl group in the case of alanine, and an isopropyl group in the case of valine.

[0172] When the second biocompatible polymer chain includes a repeating structure represented by (b1) and a repeating structure represented by (b2), the arrangement of (b1) and (b2) may be random. m represents the total number of (b2) in the second biocompatible polymer chain, and n-m represents the total number of (b1) in the second biocompatible polymer chain. n-m may be 0 (i.e., the second biocompatible polymer chain may have only (b2) bound to deferoxamine among (b1) and (b2).

[0173] The second biocompatible polymer chain may be composed of the repeating structure represented by (b2) or the repeating structure represented by (b1) and the repeating structure represented by (b2).

[0174] In addition, R 1 The amino acid side chains and R 2 The amino acid side chains may be the same as or different from each other.

[0175] In formulae (b1) and (b2), n is an integer of 1 to 1000, and may be an integer of 10 to 500 or an integer of 20 to 100. It is preferred that the value of n is within the above range because the molecular weight of the second biocompatible polymer chain becomes appropriate.

[0176] In formulas (b1) to (b2), m is an integer of 1 to 1000, and may be an integer of 3 to 100, or an integer of 5 to 50. When the value of m is greater than or equal to the lower limit, the iron chelating effect of deferoxamine is well exerted, and when the value of m is less than or equal to the upper limit, the hydrophilicity of the conjugate is improved, which is preferred.

[0177] In addition, although the numerical range in the case where n is greater than m is also exemplified here, n and m may be the same number.

[0178] The binding mode of polyamino acid and deferoxamine is not particularly limited, and the amino acid side chain of polyamino acid is preferably combined with deferoxamine. As a method for combining deferoxamine with the amino acid side chain of polyamino acid, it can be enumerated as follows: forming an amide bond with the amino group of lysine side chain, forming a disulfide bond with the thiol group of cysteine ​​side chain, etc. As described above, for example, in the deferoxamine of the compound (I), if combined with the group (amino group) represented by "-NH2", it is not easy to affect the iron chelation of deferoxamine, and there is no need to make deferoxamine into a derivative, so it is preferred to form an amide bond with the amino group of aspartic acid side chain or glutamic acid side chain.

[0179] When the second biocompatible polymer chain is a polyamino acid and contains aspartic acid and / or glutamic acid as a structural unit, B in the general formula (1) or (1-1) is preferably the following structure.

[0180] B represents the second biocompatible polymer chain bonded to the deferoxamine, and the second biocompatible polymer chain preferably includes the repeating structure represented by (b2-1) below, or the repeating structure represented by (b1-1) and the repeating structure represented by (b2-1).

[0181] [Chemistry 11]

[0182]

[0183] (In formulas (b1-1) to (b2-1),

[0184] R 11 represents the amino acid side chain,

[0185] R 12 The carboxyl group in the aspartic acid side chain represented by -CH2-COOH or the glutamic acid side chain represented by -CH2-CH2-COOH is bonded to the deferoxamine,

[0186] n1 represents the total number of (b1-1) and (b2-1), n1 is an integer from 1 to 1000, m1 is an integer from 1 to 1000 (where m1≤n1), and when n1-m1 is 2 or more, multiple R 11 They may be the same or different from each other. When m1 is 2 or more, multiple R 12 can be the same or different from each other).

[0187] When the second biocompatible polymer chain includes a repeating structure represented by (b1-1) and a repeating structure represented by (b2-1), the arrangement of (b1-1) and (b2-1) may be random. The relationship between n1 and m1 is the same as that between n and m, and the numerical values ​​described for n and m may also be applied to n1 and m1.

[0188] The second biocompatible polymer chain may be composed of the repeating structure represented by (b2-1) or the repeating structure represented by (b1-1) and the repeating structure represented by (b2-1).

[0189] In addition, R 11 The amino acid side chains and R 12 The amino acid side chains may be the same as or different from each other.

[0190] The first biocompatible polymer chain is preferably polyethylene glycol.

[0191] When the first biocompatible polymer chain is polyethylene glycol and the second biocompatible polymer chain is polyamino acid and contains aspartic acid and / or glutamic acid as structural units, the structure represented by the general formula (1) is preferably the structure represented by the following general formula (1-2).

[0192] [Chemistry 12]

[0193]

[0194] (In formula (1-2),

[0195] I is an integer of 1 to 1500, B represents the second biocompatible polymer chain bonded to the deferoxamine, and the second biocompatible polymer chain comprises the repeating structure represented by the following (b2-1), or the repeating structure represented by (b1-1) and the repeating structure represented by (b2-1)).

[0196] In formula (1-2), I is an integer of 1 to 1500, may be an integer of 10 to 1000, or may be an integer of 100 to 500.

[0197] [Chemistry 13]

[0198]

[0199] (In formulas (b1-1) to (b2-1), R 11 , R 12 , n1, and m1 have the same meanings as above).

[0200] The conjugate of this embodiment preferably has a number average molecular weight (Mn) of 2,000 to 200,000, for example, 5,000 to 100,000, 10,000 to 50,000, 17,000 to 45,000, or 20,000 to 40,000.

[0201] By setting the number average molecular weight of the conjugate within the above range, the blood retention and accumulation of the conjugate in tumor tissues can be appropriately improved, and accumulation in normal tissues such as the liver can be prevented. As a result, deferoxamine can be efficiently delivered to tumor tissues.

[0202] It is believed that the tumor accumulation of the conjugate is achieved by selectively accumulating in the tumor by utilizing the enhanced vascular permeability of the tumor, that is, by the enhanced permeability and retention effect (EPR (enhanced permeability and retention) effect), and by selectively removing iron in the tumor, thereby achieving a more excellent anti-tumor effect.

[0203] In addition, the conjugate can be in the form of polymer micelles or polymer vesicles.

[0204] The conjugate of this embodiment is excellent in blood retention and tumor accumulation, and exhibits excellent anti-tumor effects, which is very outstanding.

[0205] Conventionally, DFO lacks blood retention and thus requires continuous administration by drip. On the other hand, the conjugate of this embodiment has excellent blood retention and can be administered in a form with longer intervals, making it easy to prescribe.

[0206] The conjugate of this embodiment has excellent tumor accumulation, so it does not induce the accumulation of drugs in normal tissues, but can only enhance the accumulation of drugs in tumor tissues. As a result, the conjugate of this embodiment can achieve high drug efficacy and low side effects.

[0207] Conventionally, although DFO has been conjugated to a polymer such as dextran or hydroxyethyl starch (HES), there has been no report of polymer-modified DFO showing an antitumor effect on solid cancers.

[0208] The reason why the conjugate of the present embodiment is believed to have excellent blood retention and tumor accumulation is that the biocompatible polymer includes a first biocompatible polymer chain and a second biocompatible polymer chain different from the first biocompatible polymer chain. It is believed that by making the conjugate include different types of polymer chains, when it is administered into a living body, the different types of chains will not mix with each other, and can be bound to other molecules in the living body and stabilized. It is believed that the conjugate of the embodiment, for example, the conjugate of the present embodiment, has an improved affinity with serum albumin, which is a cancer-accumulating protein, and exerts a more excellent tumor accumulation and anti-tumor effect.

[0209] Cancer therapeutic agents

[0210] As one embodiment of the present invention, a pharmaceutical composition containing the conjugate of the embodiment as an active ingredient is provided.

[0211] As one embodiment of the present invention, there is provided a cancer therapeutic agent comprising the conjugate of the embodiment as an active ingredient.

[0212] Since the conjugate of the above embodiment has iron chelation, it is expected to have therapeutic effects on various diseases by utilizing iron chelation.

[0213] Examples of target diseases for which therapeutic effects are expected include iron overload and iron-requiring cancers, such as solid cancers, etc. Examples of human solid cancers include brain cancer, head and neck cancer, esophageal cancer, thyroid cancer, small cell cancer, non-small cell cancer, breast cancer, stomach cancer, gallbladder / bile duct cancer, lung cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, choriocarcinoma, uterine endometrial cancer, cervical cancer, renal pelvis / ureter cancer, bladder cancer, prostate cancer, penile cancer, testicular cancer, embryonal carcinoma, Wilms' cancer, skin cancer, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's tumor, and soft tissue sarcoma.

[0214] The pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient may further contain other anticancer agents, etc. With this configuration, a synergistic effect on cancer treatment can be expected.

[0215] The pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient can be used for photodynamic diagnosis and / or therapy using 5-aminolevulinic acid or its derivatives, salts or esters (hereinafter, also referred to as "ALAs"; details of ALAs are described below). The principle of photodynamic diagnosis and / or therapy using 5-aminolevulinic acid is described below.

[0216] like Fig.26 As shown, 5-aminolevulinic acid (5-ALA) is taken into cells and used in the heme biosynthesis pathway. It is known that protoporphyrin IX, which is a metabolic intermediate, selectively accumulates in cancer cells. Protoporphyrin IX is a photosensitive substance, so by irradiating light of a specific wavelength, it is possible to produce harmful reactive oxygen species in cancer cells and kill cancer cells (photodynamic therapy). In addition, by irradiating protoporphyrin IX with light of a specific wavelength (e.g., 400-410nm), red fluorescence can be emitted, thereby effectively making cancer cells visible (photodynamic diagnosis).

[0217] However, for example, cancer stem cells, which are deeply involved in cancer recurrence, have a particularly high iron intake, so the metabolism of protoporphyrin IX is enhanced. Since protoporphyrin IX is not accumulated in cancer stem cells, there is a concern that the therapeutic effect will be reduced.

[0218] Therefore, as shown in Non-Patent Document 9, it is considered that by using the cancer therapeutic agent of the embodiment to chelate iron ions in cancer cells, the accumulation of protoporphyrin IX selectively in cancer cells is increased, thereby improving the effect of photodynamic diagnosis and / or treatment.

[0219] The pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient can be provided alone as a pharmaceutical composition or cancer therapeutic agent for photodynamic diagnosis and / or treatment, or can be provided in a dosage form such as a mixture or combined preparation with the above-mentioned ALAs.

[0220] As one embodiment, a method is provided, which comprises administering the ALAs and the conjugates of the embodiments to a subject in need of treatment.

[0221] As one embodiment, a method for treating cancer is provided, which comprises administering the ALAs and the conjugates of the embodiments to a subject in need of treatment.

[0222] As one embodiment, a pharmaceutical composition or cancer therapeutic agent is provided, which contains the conjugate of the embodiment as an active ingredient, for administration to a subject to which the ALAs are administered or being administered.

[0223] As one embodiment, a pharmaceutical composition or cancer therapeutic agent is provided, which contains the ALAs as an active ingredient and is used for administration to a subject to whom the conjugate of the embodiment has been or is being administered.

[0224] As one embodiment, a conjugate according to the embodiment is provided for use in photodynamic diagnosis and / or treatment of cancer.

[0225] As an embodiment, the ALAs and conjugates of the embodiments are provided for use in photodynamic diagnosis and / or treatment of cancer.

[0226] As one embodiment, provided is the use of the conjugate of the embodiment for photodynamic diagnosis and / or treatment of cancer.

[0227] As an embodiment, the present invention provides the use of the ALAs and the conjugates of the embodiments for photodynamic diagnosis and / or treatment of cancer.

[0228] As one embodiment, a pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient is provided, which is used for photodynamic diagnosis and / or treatment of cancer.

[0229] As one embodiment, a pharmaceutical composition or cancer therapeutic agent containing the ALAs and the conjugates of the embodiments as active ingredients is provided, which is used for photodynamic diagnosis and / or treatment of cancer.

[0230] As one embodiment, a kit for photodynamic diagnosis and / or treatment of cancer is provided, wherein the kit comprises: a pharmaceutical composition or a cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient, and a pharmaceutical composition or a cancer therapeutic agent containing the ALAs as an active ingredient.

[0231] As one embodiment, provided is use of the conjugate of the embodiment in preparing a medicament for photodynamic diagnosis and / or treatment of cancer.

[0232] As one embodiment, provided is the use of the ALAs and the conjugates of the embodiments in preparing a drug for photodynamic diagnosis and / or treatment of cancer.

[0233] As the subject, a cancer patient is exemplified.

[0234] The photodynamic diagnosis and / or treatment for cancer includes irradiating light to cells of a subject to which the ALAs and / or the conjugates of the embodiments have been administered. The cells are a concept including tumors. The wavelength of the light is well known, and any wavelength is acceptable as long as it can generate reactive oxygen species in cancer cells.

[0235] In this specification, ALA means 5-aminolevulinic acid. ALA is also called δ-aminolevulinic acid and is a type of amino acid.

[0236] Examples of ALA derivatives include compounds represented by the following formula (II). 21 represents a hydrogen atom or an acyl group, R 22 represents a hydrogen atom, a straight-chain or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group. 21 and R 22 For the case of hydrogen atoms.

[0237] [Chemistry 14]

[0238] R 21 -NHCH2COCH2CH2COOR 22 (II)

[0239] ALAs can function as active ingredients in the form of ALA of formula (II) or a derivative thereof in vivo, or can be administered as prodrugs (proderivatives) decomposed by enzymes in the body.

[0240] As R of formula (II) 21The acyl group in the alkyl group may be a linear or branched alkanoyl group having 1 to 8 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, octanoyl, benzylcarbonyl, or an aromatic acyl group having 7 to 14 carbon atoms, such as benzoyl, 1-naphthoyl, 2-naphthoyl.

[0241] As R of formula (II) 22 The alkyl group in includes linear or branched alkyl groups having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl and octyl.

[0242] As R of formula (II) 22 Examples of the cycloalkyl group include cycloalkyl groups having 3 to 8 carbon atoms and which may partially have an unsaturated bond, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclododecyl and 1-cyclohexenyl.

[0243] As R of formula (II) 22 The aryl group in ' includes aryl groups having 6 to 14 carbon atoms, such as phenyl, naphthyl, anthracenyl, and phenanthryl.

[0244] As R of formula (II) 22 The aralkyl group in the above may be exemplified as the aryl group for the aryl portion and as the alkyl group for the alkyl group, and specifically may be aralkyl groups having 7 to 15 carbon atoms, such as benzyl, phenethyl, phenylpropyl, phenylbutyl, diphenylmethyl, triphenylmethyl, naphthylmethyl and naphthylethyl.

[0245] Preferred ALA derivatives include R 21 ALA derivatives preferably include the following: 22 ALA derivatives preferably include the following: 21 With R 22 The combinations of the compounds are (formyl and methyl), (acetyl and methyl), (propionyl and methyl), (butyryl and methyl), (formyl and ethyl), (acetyl and ethyl), (propionyl and ethyl), and (butyryl and ethyl).

[0246] Among the ALA class, as salts of ALA or its derivatives, pharmacologically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, etc. can be listed. As acid addition salts, for example, inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, phosphate, nitrate, sulfate, formates, acetates, propionates, toluenesulfonates, succinates, oxalates, lactates, tartrates, glycolates, methanesulfonates, butyrates, valerates, citrates, fumarates, maleates, malate and other organic acid addition salts can be exemplified. As metal salts, alkali metal salts such as lithium salts, sodium salts, potassium salts, alkaline earth metal salts such as magnesium and calcium salts, aluminum, zinc and other metal salts can be exemplified. As ammonium salts, alkylammonium salts such as ammonium salts and tetramethylammonium salts can be exemplified. As organic amine salts, triethylamine salts, piperidine salts, morpholine salts, toluidine salts and other salts can be exemplified. In addition, these salts can also be used as solutions when used.

[0247] Examples of ALA esters include methyl ester, ethyl ester, propyl ester, butyl ester, and pentyl ester, but are not limited thereto.

[0248] Among the above ALAs, the most preferred are ALA and various esters such as ALA methyl ester, ALA ethyl ester, ALA propyl ester, ALA butyl ester, and ALA pentyl ester, and their hydrochlorides, phosphates, and sulfates. In particular, ALA hydrochloride and ALA phosphate can be exemplified as the most suitable ALAs.

[0249] The ALAs can be produced by known methods such as chemical synthesis, production using microorganisms, and production using enzymes. In addition, the ALAs can form hydrates or solvates, and the ALAs can be used alone or in combination of two or more.

[0250] When preparing the ALAs as aqueous solutions, care must be taken to prevent the aqueous solutions from becoming alkaline in order to prevent the ALAs from decomposing. If the aqueous solutions have become alkaline, decomposition can be prevented by removing oxygen.

[0251] The pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient can also be used for vitamin C therapy using ascorbic acid or its derivatives or salts thereof (hereinafter, also referred to as "ascorbic acid"; details of ascorbic acid will be described below). The principle of vitamin C therapy using ascorbic acid is described below.

[0252] It is known that if pharmacological concentrations of vitamin C (ascorbic acid) are intravenously injected, hydrogen peroxide (H2O2) is generated in the extracellular domain of the tumor, and the hydrogen peroxide penetrates into the tumor cells to produce an anti-tumor effect (vitamin C therapy) [Q. Chen, MG Espey, MC Krishna, JBMitchell, CP Corpe, GR Buettner, E. Shacter, M. Levine, Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a prodrug to deliver hydrogen peroxide to tissues. Proc. Natl. Acad. Sci. USA 102, 13604-13609 (2005)., Q. Chen, MG Espey, AY Sun, JHL Lee, MC Krishna, E. Shacter, PL Choyke, C. Pooput, KL Kirk, GR Buettner, M. Levine, Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo.Proc.Natl.Acad.Sci.USA104,8749-8754(2007).,Q.Chen,MGEspey,AYSun,C.Pooput,KLKirk,MCKrishna,DSKhosh,J.Drisko,M.Levine,Pharmacologic doses of ascorbate actas a prooxidant and decrease growth of aggressive tumor xenografts inmice.Proceedings of the National Academy of Sciences of the United States of America 105,11105-11109(2008)].

[0253] On the other hand, according to recent studies [M. Mojic, J. Bogdanovic Pristov, D. Maksimovic-Ivanic, DR Jones, M. Stanic, S. Mijatovic, I. Spasojevic, Extracellular iron diminishes anticancer effects of vitamin C: an in vitro study. Sci. Rep. 4, 5955 (2014)], it is reported that excess iron ions in the extracellular domain of tumors convert hydrogen peroxide into hydroxyl radicals through the Fenton reaction, and the hydroxyl radicals have low intracellular permeability, so the cell-killing effect is reduced. Therefore, it is expected that if the excess iron ions in the tumor can be inactivated, the anti-tumor effect produced by vitamin C can be improved.

[0254] In this specification, "ascorbic acid" is a concept including ascorbic acid or its derivatives or salts thereof, and is also called vitamin C. In addition, although L-ascorbic acid exists naturally, L-ascorbic acid and D-ascorbic acid obtained by chemical synthesis can be used preferably.

[0255] Examples of the salt of ascorbic acid include the salts of the types exemplified as the salts of ALA.

[0256] The pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient may be provided alone as a pharmaceutical composition or cancer therapeutic agent for vitamin C therapy, or may be provided in a dosage form such as a mixture or combined preparation with the ascorbic acid.

[0257] As one embodiment, a method is provided, which comprises administering the ascorbic acid and the conjugate of the embodiments to a subject in need of treatment.

[0258] As one embodiment, a method for treating cancer is provided, which comprises administering the conjugate of the ascorbic acid and the embodiment to a subject in need of treatment.

[0259] As one embodiment, provided is a pharmaceutical composition or cancer therapeutic agent comprising the conjugate of the embodiment as an active ingredient, which is used for administration to a subject to which the ascorbic acid is administered or is being administered.

[0260] As one embodiment, provided is a pharmaceutical composition or cancer therapeutic agent containing the ascorbic acid as an active ingredient, which is used for administration to a subject to which the conjugate of the embodiment has been or is being administered.

[0261] As one embodiment, a conjugate according to the embodiment is provided for use in vitamin C therapy for cancer.

[0262] As an embodiment, the ascorbic acids and conjugates of the embodiments are provided for use in vitamin C therapy for cancer.

[0263] As one embodiment, use of the conjugate according to one embodiment is provided for vitamin C therapy for cancer.

[0264] As one embodiment, provided is the use of the ascorbic acids and the conjugates of the embodiments for vitamin C therapy for cancer.

[0265] As one embodiment, a pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient is provided, which is used for vitamin C therapy for cancer.

[0266] As one embodiment, a pharmaceutical composition or cancer therapeutic agent containing the ascorbic acid and the conjugate of the embodiment as an active ingredient is provided, which is used for vitamin C therapy for cancer.

[0267] As one embodiment, a kit is provided for vitamin C therapy for cancer, the kit comprising: a pharmaceutical composition or a cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient, and a pharmaceutical composition or a cancer therapeutic agent containing the ascorbic acid as an active ingredient.

[0268] As one embodiment, provided is the use of the conjugate of the embodiment for preparing a medicament for vitamin C therapy against cancer.

[0269] As one embodiment, provided is the use of the ascorbic acid and the conjugate of the embodiment for preparing a drug for vitamin C therapy for cancer.

[0270] As the subject, a cancer patient is exemplified.

[0271] The vitamin C therapy comprises administering ascorbic acid to a subject, preferably by intravenous injection. The subject is a concept including patients with tumors.

[0272] The concentration of ascorbic acid in the tumor during vitamin C therapy is not particularly limited as long as it has an anti-tumor effect. As an example, it can be 1 mM or more, 2 mM or more, or 3 mM or more. As the upper limit of the concentration of ascorbic acid in the tumor during vitamin C therapy, there is no particular limitation. As an example, it can be 20 mM or less, 15 mM or less, or 10 mM or less. As an example of the numerical range of the concentration of ascorbic acid in the tumor, for example, it can be 1 mM or more and 20 mM or less, 2 mM or more and 15 mM or less, or 3 mM or more and 10 mM or less.

[0273] The pharmaceutical composition or cancer therapeutic agent of the embodiment can be administered to a subject or a patient by, for example, intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, transdermal, or oral methods, using methods known to those skilled in the art. The dosage varies depending on the patient's weight or age, the method of administration, etc., and those skilled in the art can appropriately select an appropriate dosage. The dosage and the method of administration vary depending on the patient's weight or age, symptoms, etc., and those skilled in the art can appropriately select.

[0274] The dosage form of the pharmaceutical composition or cancer therapeutic agent of the embodiment can be appropriately determined according to the administration route and is not particularly limited. Examples include injections, drips, tablets, capsules, fine granules, powders, liquids, aqueous solutions dissolved in syrups, patches, suppositories, etc.

[0275] The dosage form of the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient is preferably an oral preparation, an injection, or an intravenous drip.

[0276] The dosage form of the pharmaceutical composition or cancer therapeutic agent containing the ALAs as an active ingredient is preferably an oral preparation, an injection, or an intravenous drip.

[0277] The dosage form of the pharmaceutical composition or cancer therapeutic agent containing the ascorbic acid as an active ingredient is preferably an injection or a drip, and more preferably an injection or a drip for intravenous injection.

[0278] The pharmaceutical composition or cancer therapeutic agent may contain other pharmaceutically effective ingredients, nutrients, carriers and other optional ingredients as needed. As optional ingredients, for example, various formulation ingredients for preparation such as crystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetable and animal fats, oils, glues, polyalkylene glycols and other pharmaceutically acceptable common carriers, binders, stabilizers, solvents, dispersion media, extenders, excipients, diluents, pH buffers, disintegrants, solubilizers, solubilizers, isotonic agents and the like may be added.

[0279] Examples of the preparations in the pharmaceutical composition or cancer therapeutic agent of the embodiment include oral preparations used in the form of tablets, capsules, elixirs, and microcapsules coated with sugars as necessary.

[0280] Alternatively, there can be mentioned a preparation for parenteral use in the form of an injection of a sterile solution or suspension with water or a pharmaceutically acceptable liquid other than water. In addition, there can be mentioned a preparation prepared by appropriately combining with a pharmacologically acceptable carrier or medium, specifically sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, carrier, preservative, adhesive, etc., and mixing in a unit dosage form required by a generally recognized pharmaceutical practice.

[0281] As additives that can be mixed in tablets and capsules, for example, adhesives such as gelatin, corn starch, tragacanth gum, gum arabic, excipients such as crystalline cellulose, swelling agents such as corn starch, gelatin, alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as mint, balsam oil or cherry can be used. In the case where the dosage unit form is a capsule, the above materials may further contain a liquid carrier such as oil. The sterile composition for injection can use a carrier such as distilled water for injection and be prescribed according to the usual dosage form.

[0282] As aqueous solutions for injection, for example, physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be mentioned, and they can be used together with appropriate cosolvents, such as alcohols, specifically ethanol, polyols, such as propylene glycol, polyethylene glycol, and nonionic surfactants, such as polysorbate 80 (TM), HCO-50.

[0283] As the oily liquid, sesame oil and soybean oil can be mentioned, which can be used together with benzyl benzoate and benzyl alcohol as a cosolvent. In addition, it can be formulated with a buffer such as a phosphate buffer and a sodium acetate buffer, an analgesic such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, and an antioxidant. The prepared injection is usually filled into a suitable ampoule.

[0284] Depending on the height, weight, age, and symptoms of the subject, the dosage of the conjugate to be administered via a pharmaceutical composition or cancer therapeutic agent can be in the range of 0.1 mg to 1,000 mg, preferably 0.1 mg to 200 mg, more preferably 0.5 mg to 50 mg, and even more preferably 1 mg to 20 mg per 1 kg of the subject's body weight, calculated as DFO.

[0285] The dosage of ALA to be administered via the pharmaceutical composition or cancer therapeutic agent is calculated as ALA per kg of the subject's body weight (i.e., in formula (II), R 21 and R 22 The dosage is preferably in the range of 1 mg to 1,000 mg, preferably 5 mg to 100 mg, more preferably 10 mg to 30 mg, and even more preferably 15 mg to 25 mg.

[0286] Depending on the height, weight, age, and symptoms of the subject, the dosage of ascorbic acid to be administered via the pharmaceutical composition or cancer therapeutic agent may be 5 mg or more, or 20 mg or more, for example, 5 to 1,000 mg, or 20 to 1,000 mg per kg of the subject's body weight, in terms of ascorbic acid. When investigating the dosage of ascorbic acid at a higher concentration, the dosage of ascorbic acid may be 50 to 1,000 mg, 250 to 1,000 mg, 400 to 1,000 mg, or 500 to 800 mg per kg of the subject's body weight, in terms of ascorbic acid.

[0287] The number of administrations and the frequency of administration of the ALAs or ascorbic acids and the conjugates of the embodiments when administered to a subject can be appropriately determined by a person skilled in the art in view of the respective administration conditions (administration interval, number of administrations, and administration period) of the ALAs or ascorbic acids and the conjugates used together. In one embodiment of the present invention, a pharmaceutical composition or a cancer therapeutic agent containing the conjugate of the embodiments as an active ingredient can be administered before, during, or after the administration of the ALAs or ascorbic acids.

[0288] The pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient and the ALAs or ascorbic acids can be administered to the subject at the same time or at different times, continuously or at intervals in their respective effective amounts. The pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient and the ALAs or ascorbic acids can be administered to the tumor patient in the same administration cycle or in different administration cycles.

[0289] In one embodiment of the present invention, the administration of the ALA or ascorbic acid to the subject is started before the start of administration of the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient. For example, the ALA or ascorbic acid can be administered more than once or continuously for several days between one week before and the day of the start of administration of the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient. There is no particular limitation on the start time of administration of the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient. The pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient is preferably administered within 1 week, more preferably within 3 days, and further preferably within 1 day from the start time of administration of the ALA or ascorbic acid.

[0290] In another embodiment of the present invention, the administration of the ALA or ascorbic acid to the subject starts on the same day as the administration of the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient. For example, the ALA or ascorbic acid can be administered from the day the administration of the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient starts. When the ALA or ascorbic acid is administered simultaneously with the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient, they can be prepared as a single preparation for administration, or they can be administered simultaneously using different administration routes.

[0291] In another embodiment of the present invention, the administration of the ALA or ascorbic acid to the subject is initiated after the administration of the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient. For example, the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient may be administered more than once or continuously for several days from 1 week before to the day of the start of administration of the ALA or ascorbic acid. There is no particular limitation on the start time of administration of the ALA or ascorbic acid. The ALA or ascorbic acid is preferably administered within 1 week, more preferably within 3 days, and further preferably within 1 day from the start time of administration of the pharmaceutical composition or cancer therapeutic agent containing the conjugate of the embodiment as an active ingredient.

[0292] Unless otherwise defined, the terms used in this specification are for describing specific embodiments and are not intended to limit the invention.

[0293] In addition, the term "comprising" used in this specification means that the described matters (components, steps, elements, numbers, etc.) exist, and does not exclude the existence of other matters (components, steps, elements, numbers, etc.), except where it is obvious that it should be understood differently depending on the context.

[0294] [Example]

[0295] Hereinafter, the present invention will be described in further detail with reference to Examples, but the present invention is not limited to the following Examples.

[0296] 1. PEG 10k -Poly[aspartic acid (deferoxamine) m ] n (PEG 10k -Poly[Aspartic acid(Deferoxamine) m ] n )

[0297] <1.1. Summary>

[0298] The iron chelator PEG prepared in the examples is described. 10k -Poly[aspartic acid (deferoxamine) m ] n (hereinafter referred to as PEG-P[Asp(DFO) m ] n ). n represents the degree of polymerization of Asp, and m represents the number of DFO introduced.

[0299] [Chemistry 15]

[0300]

[0301] PEG 10k -NH2 as initiator, BLA-NCA as monomer, and N-carboxyanhydride (NCA) polymerization to synthesize PEG-PBLA n PEG-PAsp was obtained by deprotecting the carboxyl group of the side chain under alkaline conditions. n Then, the amino group of deferoxamine was bound to PEG-PAsp n The carboxyl group of PEG-P[Asp(DFO) m ] n .

[0302] <1.2. Reagents>

[0303] Commercially available reagents and solvents not otherwise specified were used as they are.

[0304] ·α-Methoxy-ω-amino-poly(ethylene glycol) (PEG-NH2) [Mw: 10K]: NOF Co., Inc.

[0305] Benzene: Nacalai Tesque Inc.

[0306] β-Benzyl L-aspartic acid N-carboxyanhydride (BLA-NCA): Chuo Kaseihin Co., Inc.

[0307] Dichloromethane (DCM): Wako Pure Chemical Industries Co., Ltd.

[0308] Used in distillation under argon atmosphere. (bp40℃)

[0309] ·N,N-Dimethylformamide (DMF): Wako Pure Chemical Industries Co., Ltd.

[0310] Used in distillation under argon atmosphere. (bp135℃)

[0311] Hexane: Kanto Chemical CO., Inc.

[0312] Ethyl acetate: Kanto Chemical CO., Inc.

[0313] ·5mol / L HCl: Nacalai Tesque Inc.

[0314] ·5mol / L NaOH: Wako Pure Chemical Industries Co.,Ltd.

[0315] Dimethyl sulfoxide (DMSO): Nacalai Tesque Inc.

[0316] ·Deferoxamine mesylate (DFO): Sigma Aldrich Co.,llc.

[0317] ·N,N'-Dicyclohexylcarbodiimide (DCC): Wako Pure Chemical Industries Co., Ltd.

[0318] · N,N-Dimethyl-4-aminopyridine (DMAP): Wako Pure Chemical Industries Co., Ltd.

[0319] Sodium chloride (NaCl): Nacalai Tesque Inc.

[0320] Sodium dihydrogen phosphate (NaH2PO3): Wako Pure Chemical Industries Co., Ltd.

[0321] <1.3. Measuring instruments>

[0322] NMR (Nuclear Magnetic Resonance): BRUKER AVANCEIII400 (400MHz, BRUKER BioSpin)

[0323] Rotation: Off

[0324] Total times: 24 times

[0325] Temperature: 25℃

[0326] ·GPC (Gel Permeation Chromatography): Jasco International Co., Ltd.

[0327] Column: TSK-gel superAW3000 (Tosoh Corporation),

[0328] Superdex 200Increase 10 / 300GL(GE Healthcare)

[0329] Detector: RI-2031

[0330] <1.4. Synthesis method>

[0331] [PEG-PBLA n Synthesis of

[0332] Weigh 1.00 g (0.100 mmol) of PEG-NH2 into a 300 mL two-necked eggplant flask, dissolve it in 10.0 mL of benzene, and then freeze-dry it. Under an argon environment, weigh 1.20 g (4.81 mmol) (n=35) and 2.40 g (9.62 mmol) (n=78) of BLA-NCA into a 100 mL two-necked eggplant flask. Add 1.36 mL of DMF and 13.6 mL of DCM to PEG-NH2. In addition, add 1.63 mL (n=35) and 3.26 mL (n=78) of DMF to BLA-NCA, and add 16.3 mL (n=35) and 32.6 mL (n=78) of DCM, and dissolve them. Add the BLA-NCA solution to the PEG-NH2 solution, and stir at room temperature under an argon environment for 48 hours. The reaction solution was added dropwise to 600 mL (n=35) and 750 mL (n=78) of a mixed solvent of hexane and ethyl acetate (hexane:ethyl acetate=3:2), respectively, and purified by reprecipitation. Thereafter, it was dried under reduced pressure to obtain white solid PEG-PBLA with a yield of 1.71 g (n=35) and 2.54 g (n=78) and a yield of 97% (n=35) and 98% (n=78). n .Will 1 The H-NMR spectrum is shown in Figure 1 and Figure 3 The GPC curve (obtained under the conditions of column: TSK-gel superAW3000, eluent: NMP (50 mM LiBr), flow rate: 0.30 mL / min, measurement temperature: 40°C) is shown in Figure 2 and Figure 4 .

[0333] PEG-PBLA 35 of 1 H NMR spectrum

[0334] 1H NMR (400MHz, DMSO-d6): δ2.53-2.69,2.76-2.92(br,-CH2-C=OO-),3.42-3.67(br,-CH2-CH2-O-),4.95-5.13(br,-O-CH2-Ph),7.20-7.39(br,Ph)

[0335] PEG-PBLA 78 of 1 H NMR spectrum

[0336] 1 H NMR (400 MHz, DMSO-d6): Properties similar to those of the above PEG-PBLA 35 of 1 H NMR spectra are identical

[0337] [PEG-PBLA n Deprotection of

[0338] Weigh PEG-PBLA 35 500mg (0.0284mmol) and PEG-PBLA 78 500 mg (0.0194 mmol) was added to a 50 mL eggplant flask, 5 mL of 0.1 M NaOH was added, and the mixture was stirred at room temperature overnight. The reaction solution was placed in a dialysis membrane (MWCO = 6-8 kDa) and dialyzed twice using 2 L of 0.01 M HCl and then 2 L of pure water. The solution was freeze-dried to obtain white solid PEG-PAsp with a yield of 367 mg (n = 35) and 331 mg (n = 78) and a yield of 92% (n = 35) and 90% (n = 78). n .Will 1 The H-NMR spectrum is shown in Figure 5 and Figure 7 The GPC curve (obtained under the conditions of column: Superdex 200increase 10 / 300GL, eluent: 10mM NaH2PO4, 140mM NaCl (pH 7.4), flow rate: 0.75mL / min, measurement temperature: room temperature) is shown in Figure 6 and Figure 8 .

[0339] PEG-PAsp 35 of 1 H NMR spectrum

[0340] 1 H NMR (400MHz, D2O): δ2.73-2.97(br,-CH2-COOH),3.62-3.82(br,-CH2-CH2-O-)

[0341] PEG-PAsp 78 of 1 H NMR spectrum

[0342] 1 H NMR (400 MHz, D2O): Properties similar to those of the above PEG-PAsp 35 of 1 H NMR spectra are identical

[0343] [DFO and PEG-PAsp n Combination of

[0344] Weigh PEG-Asp 35 100mg (7.14μmol) and PEG-Asp 78 100 mg (5.26 μmol) was added to a 50 mL eggplant flask and dissolved in 30 mL of DMSO. 256 mg (1.24 mmol) (n=35) and 407 mg (1.91 mmol) (n=78) of DCC, 30.3 mg (0.248 mmol) (n=35) and 48.1 mg (0.394 mmol) (n=78) of DMAP, 163 mg (0.248 mmol) (n=35) and 259 mg (0.394 mmol) (n=78) of DFO were added thereto, and stirred at room temperature overnight. The reaction solution was placed in a dialysis membrane (MWCO=3.5 kD), dialyzed three times with 300 mL of acetone, and then dialyzed twice with 2 L of 0.01 M NaOH and then with 2 L of pure water. The obtained solution was filtered through a 0.45 μm filter and then freeze-dried to obtain a white solid PEG-P[Asp(DFO)] with a yield of 120 mg (n=35) and 139 g (n=78) and a yield of 87% (n=35) and 80% (n=78). m ] n .Will 1 The H-NMR spectrum is shown in Fig. 9 and Fig.11 , the GPC curve is shown in Fig.10 and Fig.12 .

[0345] ·PEG-P[Asp(DFO) 10 ] 35 of 1 H NMR spectrum

[0346] 1H NMR (400MHz, D2O, NaOD): δ1.13-1.96(br,-CH2-CH2-CH2-),1.98-2.09(br,-C=O-CH3),2.43-2.55(br,-C H2-C=O-NH-),3.08-3.33(br,-C=O-NH-CH2-),3.52-3.62(br,-CH2-NOH-),3.66-3.90(br,-CH2-CH2-O-)

[0347] ·PEG-P[Asp(DFO) 26 ] 78 of 1 H NMR spectrum

[0348] 1 H NMR (400MHz, D2O, NaOD): Properties similar to those of the above PEG-P[Asp(DFO) 10 ] 35 of 1 H NMR spectra are identical

[0349] <1.5. Analysis>

[0350] [PEG-PBLA n ]

[0351] according to 1 From the ratio of the integrated values ​​of the peaks generated by the initiator [3.42-3.67 ppm (-CH2-CH2-O-)] and the peaks generated by BLA-NCA [2.53-2.69, 2.76-2.92 ppm (-CH2-C=OO-), 4.95-5.13 (-O-CH2-Ph), 7.20-7.39 ppm (Ph)] in the H NMR spectrum, the polymerization degrees of PBLA DP=35 (n=35) and DP=77 (n=78), the number average molecular weight Mn=17,600 (PEG:PBLA=10,000:7,600) (n=35) and Mn=25,800 (PEG:PBLA=10,000:15,800) (n=78) were calculated. Furthermore, the molecular weight distribution of the obtained polymer was determined from the GPC curve to be Mw / Mn=1.12 (n=35) and Mw / Mn=1.11 (n=78), confirming that the polymer had a narrow molecular weight distribution.

[0352] [PEG-PAsp n ]

[0353] according to 1The ratio of the integral value of the peak [3.62-3.82 ppm (-CH2-CH2-O-)] generated by the initiator to the peak (2.73-2.97 ppm) of the β-hydrogen of aspartic acid in the H NMR spectrum was used to determine the degree of polymerization of Asp: DP = 35 (n = 35) and DP = 78 (n = 78), the number average molecular weight: Mn = 14,000 (PEG: PAsp = 10,000: 4,000) (n = 35) and Mn = 19,000 (PEG: PBLA = 10,000: 9,000) (n = 78). In addition, the GPC curve confirmed that the obtained polymer had a unimodal narrow molecular weight distribution.

[0354] [PEG-P[Asp(DFO) m ] n ]

[0355] according to 1 The ratio of the integral values ​​of the peaks [3.66-3.90 ppm (-CH2-CH2-O-)] generated by the initiator and the peaks [1.13-1.96 ppm (-CH2-CH2-CH2-), 1.98-2.09 ppm (-C=O-CH3), 2.43-2.55 ppm (-CH2-C=O-NH-), 3.08-3.33 ppm (-C=O-NH-CH2-), 3.52-3.62 ppm (-CH2-NOH-)] generated by the H NMR spectrum showed that the number of DFO introduced was 10 (n=35) and 26 (n=78), and the number average molecular weight was Mn=19,300 (n=35) and Mn=32,900 (n=78). In addition, the GPC curve confirmed that the obtained polymer had a unimodal narrow molecular weight distribution.

[0356] In addition, PEG-P[Asp(DFO) 36 ] 76 (Mn=45,000).

[0357] use 1 H NMR (400 MHz, D2O, NaOD) analysis confirmed that the properties were similar to those of the above-mentioned PEG-P[Asp(DFO) 10 ] 35 of 1 The H NMR spectra were identical.

[0358] 2.PEG-P[Asp(DFO) m ] n Evaluation of the iron chelating capacity of

[0359] <2.1. Summary>

[0360] The DFO / Fe(III) complex has a characteristic absorption peak around 420 nm. Here, the absorption spectrum was measured to evaluate the PEG-P[Asp(DFO) m ] n In addition, the calcein-AM method, which is established as an indirect method for quantifying the amount of iron ions in cells, was used to evaluate the iron chelating ability of PEG-P[Asp(DFO) m ] n Iron chelating capacity. Fig.13 The principle of the Calcein-AM method is briefly shown.

[0361] [Principle of Calcein-AM Method]

[0362] Calcein-AM is a compound of cell membrane permeability, which itself does not show fluorescence, but is hydrolyzed by intracellular esterase to become calcein. This calcein is a compound showing strong yellow-green fluorescence, and is retained in the cell due to being non-permeable. In addition, calcein has iron chelating ability, and if it forms a complex with iron, the fluorescence disappears, so by measuring the fluorescence of calcein, the iron ion concentration in the cell can be indirectly measured.

[0363] <2.2. Reagents>

[0364] Commercially available reagents and solvents not otherwise specified were used as they are.

[0365] ·Deferoxamine mesylate (DFO): Sigma Aldrich Co.,llc.

[0366] ·PEG-P[Asp(DFO) 10 ] 35 (Mn=19,300)

[0367] ·PEG-P[Asp(DFO) 26 ] 78 (Mn=32,900)

[0368] ·FeCl3·6H2O: Wako Pure Chemical Industries Co., Ltd.

[0369] ·D-PBS(-): Wako Pure Chemical Industries Co., Ltd.

[0370] Roswell Park Memorial Institute medium (RPMI): Sigma Aldrich Co.,llc.

[0371] Fetal bovine serum (FBS): Biosera Inc.

[0372] Trypsin-EDTA solution: Sigma life science Co., Ltd.

[0373] Penicillin / Streptomycin: Sigma life science Co., Ltd.

[0374] Calcein-AM: Dojindo Molecular Technologies Inc.

[0375] CT26 cells (mouse colon cancer cell line): American Type Culture Collection

[0376] DLD-1 cells (human colon adenocarcinoma cell line): American Type Culture Collection

[0377] <2.3. Measuring instruments>

[0378] ·NanoDrop One: Thermo Fisher Scientific Inc..

[0379] ·Countess: Thermo Fischer Scientific Inc.

[0380] ·LSM710: Carl Zeiss Co.,Ltd.

[0381] Guava (registered trademark) easyCyte flow cytometer (FCM): Merck Millipore

[0382] <2.4. Evaluation by absorption spectrum measurement>

[0383] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution, PEG-P[Asp(DFO) 26 ] 78 , Preparation of FeCl3 solution]

[0384] DFO / D-PBS solution: 0.625 mM

[0385] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 0.0635 mM (DFO concentration = 0.625 mM)

[0386] ·PEG-P[Asp(DFO) 26 ] 78 / D-PBS solution: 0.0245 mM (DFO concentration = 0.625 mM)

[0387] FeCl3 / D-PBS solution: 2.50 mM

[0388] 800 μL of DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution and PEG-P[Asp(DFO) 26 ] 78 200 μL of FeCl3 solution was added to the solution. After 2-fold dilution with D-PBS, the mixture was stirred thoroughly and the absorption spectrum was measured with Nanodrop. The results are shown in Fig.14 .

[0389] Since PEG-P[Asp(DFO) 10 ] 35 / Fe and PEG-P[Asp(DFO) 26 ] 78 / Fe all showed the characteristic absorption peak of DFO / Fe, thus confirming that they had excellent iron chelating ability.

[0390] <2.5. Evaluation by Calcein-AM method>

[0391] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution, PEG-P[Asp(DFO) 26 ] 78 Solution Preparation]

[0392] DFO / D-PBS solution: 2.00 mM

[0393] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 0.203 mM (DFO concentration = 2.00 mM)

[0394] ·PEG-P[Asp(DFO) 26 ] 78 / D-PBS solution: 0.0714 mM (DFO concentration = 2.00 mM)

[0395] DLD-1 or CT26 cells were cultured at a volume of 5.0 × 10 4The cells were inoculated into a 24-well plate at 10 cells / well and pre-cultured at 37°C and 5% CO2 for 24 hours. The prepared solution was diluted 10 times with RPMI, and 500 μL was added to each well and cultured for 24 hours. After washing with 500 μL of D-PBS, 0.100 μM calcein-AM solution was added, and DLD-1 cells were cultured for 30 minutes and CT26 cells were cultured for 15 minutes in the dark. After washing twice with 500 μL of D-PBS, 150 μL of trypsin-EDTA solution was added, and DLD-1 cells were cultured for 10 minutes and CT26 cells were cultured for 3 minutes. After confirming cell detachment with an optical microscope, 300 μL of D-PBS containing 10% FBS was added to fully suspend them. The suspension was filtered with a cell strainer, and calcein fluorescence was measured by FCM. The results obtained are shown in Figure 15. Fig.15A The results of the DLD-1 cell assay are shown in Figure 2. Fig. 15B The results are shown for CT26 cells. The results are mean ± SD (n = 3), and statistically significant differences were evaluated by t-test (*p < 0.05, **p < 0.01).

[0396] Due to the use of PEG-P[Asp(DFO) 10 ] 35 and PEG-P[Asp(DFO) 26 ] 78 The fluorescence intensity of calcein was increased, indicating that the iron concentration in the cells was reduced, confirming that PEG-P[Asp(DFO) 10 ] 35 and PEG-P[Asp(DFO) 26 ] 78 It also has the ability to chelate iron in the biological environment.

[0397] 3. Evaluation of cultured cells

[0398] <3.1. Summary>

[0399] The fluorescence of PEG-P[Asp(DFO) m ] n (Cy5-PEG-P[Asp(DFO) m ] n ) in the cells. m ] n Next, to investigate the cellular uptake of PEG-P[Asp(DFO) m ] nFinally, CCK-8 analysis was used to evaluate the cancer cell proliferation inhibition effect.

[0400] <3.2. Reagents and cell lines>

[0401] Commercially available reagents were used as they were unless otherwise specified.

[0402] ·PEG-P[Asp(DFO) 10 ] 35 (Mn=19,300)

[0403] ·PEG-P[Asp(DFO) 26 ] 78 (Mn=32,900)

[0404] ·Cy5-NHS: Thermo Fisher Scientific Inc.

[0405] Used in the form of 10 mg / mL Cy5-NHS / DMSO.

[0406] Dimethyl sulfoxide (DMSO): Nacalai Tesque Inc.

[0407] ·GaCl3: Tokyo Chemical Industry Co.,Ltd.

[0408] ·5mol / L NaOH: Wako Pure Chemical Industries Co.,Ltd.

[0409] ·HNO3: Wako Pure Chemical Industries Co.,Ltd.

[0410] Roswell Park Memorial Institute Medium (RPMI): Sigma Aldrich Co.,llc.

[0411] ·D-PBS(-): Wako Pure Chemical Industries Co., Ltd.

[0412] Fetal bovine serum (FBS): Biosera Inc.

[0413] Trypsin-EDTA solution: Sigma life science Co., Ltd.

[0414] Penicillin / Streptomycin: Sigma life science Co., Ltd.

[0415] LysoTracker (registered trademark) red DND-99: Thermo Fisher Scientific Inc.

[0416] ·Hoechst 33342: Thermo Fisher Scientific Inc.

[0417] ·RNase A:Macherey nagel Inc.

[0418] Propidium iodide: Dojindo Molecular Technologies Inc.

[0419] Cell Counting Kit-8: Dojindo Molecular Technologies Inc.

[0420] CT26 cells (mouse colon cancer cell line): American Type Culture Collection

[0421] DLD-1 cells (human colon adenocarcinoma cell line): American Type Culture Collection

[0422] <3.3. Measuring instruments>

[0423] ·Countess: Thermo Fischer Scientific Inc.

[0424] ·LSM710: Carl Zeiss Co.,Ltd.

[0425] Guava (registered trademark) easyCyte flow cytometer (FCM): Merck Millipore

[0426] ·Agilent 7900ICP-MS: Agilent Technology Co.,Ltd.

[0427] <3.4. PEG-P[Asp(DFO) m ] n Observation of the intracellular distribution of

[0428] Introduction of fluorescent dye (Cy5) into PEG-P[Asp(DFO) m ] n , Cy5-PEG-P[Asp(DFO) m ] n intracellular distribution.

[0429] [Cy5-PEG-P[Asp(DFO) 10 ] 35 Synthesis of

[0430] Cy5-NHS was conjugated to PEG-P[Asp(DFO) m ] n The amino group at the end of PEG-P[Asp(DFO) 10 ] 35 20.0 mg (0.99 μmol) was weighed into a 6 mL vial and dissolved in 2.00 mL of pure water. Then, relative to PEG-P[Asp(DFO) 10 ] 35 1 equivalent of Cy5-NHS (10.0 mg / mL Cy5-NHS / DMSO solution 107 μL) was dissolved in 0.200 mL of pure water and added to PEG-P[Asp(DFO) 10 ] 35 The solution was stirred at room temperature overnight. The reaction solution was placed on an ultrafiltration membrane (MWCO = 10 kD) and ultrafiltered five times. Thereafter, the PD-10 column was used for further purification, and the polymer solution was freeze-dried to obtain a blue solid Cy5-PEG-P[Asp(DFO) 10 ] 35 18.0mg.

[0431] [Observation using a confocal microscope]

[0432] DLD-1 cells were cultured at a rate of 5.0 × 10 4 Cells / dish were seeded on 35 mm 2 The glass bottom dish was pre-cultured for 24 hours at 37°C and 5% CO2. 23.3 μM Cy5-PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution (DFO concentration = 200μM) was diluted 10-fold with RPMI, and 1 mL was added to each dish and cultured for 24 hours. After washing with 1 mL of D-PBS, 1 mL of 100nM LysoTracker (registered trademark) red DND-99 / (D-PBS:RPMI=1:9) solution was added and cultured for 30 minutes. After washing with 1 mL of D-PBS, 1 mL of 5.0μg / mL Hoechst / D-PBS solution was added and cultured for 5 minutes. After washing twice with 1 mL of D-PBS, 2 mL of RPMI was added and observed using CLSM. The results obtained are shown in Fig.16 .

[0433] The iron chelators used in this example were localized in endosomes / lysosomes, thus suggesting that they entered the cell via endocytosis.

[0434] <3.5.PEG-P[Asp(DFO) m ] n Evaluation of cellular uptake>

[0435] To compare DFO and PEG-P[Asp(DFO) m ] n The cellular uptake of DFO / Ga(III) complex and PEG-P[Asp(DFO) 10 ] 35 After adding the Ga(III) / Ga(III) complex to cells, the Ga content in the cells was measured by ICP-MS.

[0436] [DFO / Ga solution, PEG-P[Asp(DFO) 10 ] 35 / Preparation of Ga solution]

[0437] DFO / Ga solution

[0438] 1 mL of 19.9 mM GaCl3 / D-PBS solution was added to 9 mL of 2.22 mM DFO / D-PBS solution, and stirred overnight. The pH was adjusted to 7.4 using NaOH, and then filtered using a filter. Finally, 90 mL of RPMI medium (DFO concentration = 200 μM) was added.

[0439] ·PEG-P[Asp(DFO) 10 ] 35 / Ga solution

[0440] 0.226 mM PEG-P[Asp(DFO) 10 ] 35 1 mL of 19.9 mM GaCl3 / D-PBS solution was added to 9 mL of D-PBS solution (DFO concentration = 2.22 mM) and stirred overnight. The reaction solution was placed on an ultrafiltration membrane (MWCO = 10 kD) and ultrafiltered 5 times. 10 mL of D-PBS was added to the purified solution and filtered using a filter. Finally, 90 mL of RPMI medium (DFO concentration = 200 μM) was added.

[0441] [Determination of intracellular Ga content]

[0442] DLD-1 or CT-26 cells were cultured at a volume of 5.0 × 10 6 cells / flask to 75 mm 2Flasks were pre-cultured at 37°C and 5% CO2 for 24 hours. 10 mL of the above-prepared solution was added to each flask and cultured for 3 hours or 24 hours. After washing twice with 5 mL of D-PBS, 2.5 mL of trypsin-EDTA solution was added, and DLD-1 cells were cultured for 10 minutes and CT-26 cells were cultured for 5 minutes. After confirming cell detachment using an optical microscope, 2.5 mL of RPMI medium was added for suspension. Centrifuge at 1200 rpm for 3 minutes and remove the supernatant. 5 mL of RPMI medium was added to fully suspend it and the number of cells was measured. Centrifuge again under the same conditions and remove the supernatant. Thereafter, 200 μL of HNO3 was added and cultured at 90°C for 1 hour. The volume was made up to 2 mL with pure water, and after filtering with a hydrophobic filter, the Ga content of the cells was determined by ICP-MS. The results obtained are shown in Figure 17. Fig.17A This is the result of the measurement of DLD-1 cells. Fig. 17B The results are the measurement results of CT26 cells. The results are mean ± SD (n = 3), and the statistically significant difference was evaluated by t-test (**p < 0.01).

[0443] PEG-P[Asp(DFO) 10 ] 35 There was no significant difference in the intake of DFO as a small molecule. 10 ] 35 It efficiently enters cells through endocytosis.

[0444] <3.6. By PEG-P[Asp(DFO) m ] n Cell cycle arrest caused by

[0445] In order to investigate the m ] n The cell cycle was analyzed by propidium iodide (PI) staining.

[0446] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution Preparation]

[0447] DFO / D-PBS solution: 45.0 μM

[0448] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 61.0 μM (DFO concentration = 600 μM)

[0449] DLD-1 cells were cultured to a size of 2.0 × 10 5 Cells were seeded into 6-well plates at 10 cells / well and pre-cultured at 37°C and 5% CO2 for 24 hours. The prepared solution was diluted 10-fold with RPMI, and 3 mL was added to each well and cultured for 72 hours. After washing with 3 mL of D-PBS, 700 μL of trypsin-EDTA solution was added and cultured for 10 minutes. After confirming cell detachment with an optical microscope, 700 μL of D-PBS containing 10% FBS was added to fully suspend it. The suspension was centrifuged at 1200 rpm for 3 minutes and the supernatant was removed. 500 μL of D-PBS was added to the cell mass to suspend it. The suspension was added dropwise to 4.5 mL of 70% ethanol and fixed in a -20°C freezer overnight. 500 mL of D-PBS containing 10.0 μg / mL propidium iodide and 100 μg / mL RNase was added and cultured in the dark for 30 minutes. Finally, the suspension was filtered using a cell strainer, and the fluorescence of propidium iodide was measured using FCM. The measurement data was analyzed using ModFit LT. The obtained results are shown in FIG18 . Fig.18A is a graph showing the distribution of fluorescence obtained by FCM. Fig.18B The results converted to cell cycle are shown. The results show the value of mean ± SD (n = 3).

[0450] DFO and PEG-P[Asp(DFO) 10 ] 35 Induces cell cycle arrest in S phase.

[0451] <3.7.PEG-P[Asp(DFO) m ] n Cell proliferation inhibition effect>

[0452] Evaluation of PEG-P[Asp(DFO) by CCK-8 assay m ] n The inhibitory effect on cancer cell proliferation.

[0453] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution, PEG-P[Asp(DFO) 26 ] 78 Solution Preparation]

[0454] DFO / D-PBS solution: 3.00 mM

[0455] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 0.305 mM (DFO concentration = 3.00 mM)

[0456] ·PEG-P[Asp(DFO) 26 ] 78 / D-PBS solution: 0.118 mM (DFO concentration = 3.00 mM)

[0457] DLD-1 cells were cultured at a rate of 1.0 × 10 3 The cells were seeded into a 96-well plate at 10 cells / well and pre-cultured for 24 hours at 37°C and 5% CO2. 10 μL of the above-prepared solution was added to each well and cultured for 72 hours. 10 μL of CCK-8 solution was added to each well and cultured for 2 hours, and then the absorbance at 450 nm was measured. The results are shown in Fig.19 The results show the mean±SD (n=5), and statistically significant differences were evaluated by t-test (****p<0.0001).

[0458] DFO, PEG-P[Asp(DFO) 10 ] 35 and PEG-P[Asp(DFO) 26 ] 78 Significantly inhibited the proliferation of DLD-1 cells.

[0459] 4. Effects on subcutaneous tumor mouse model (blood retention / tumor accumulation / anti-tumor effect)

[0460] <4.1. Summary>

[0461] Evaluation of DFO and PEG-P[Asp(DFO) in a CT26 (mouse colorectal cancer cell) subcutaneous tumor mouse model m ] n Evaluation of DFO and PEG-P[Asp(DFO) in DLD-1 (human colorectal cancer cell) and CT26 subcutaneous tumor mouse models m ] n anti-tumor effect.

[0462] <4.2. Reagents, cells, and animals>

[0463] ·Deferoxamine mesylate (DFO): Sigma Aldrich Co.,llc.

[0464] ·PEG-P[Asp(DFO) 10 ] 35 (Mn=19,300)

[0465] ·PEG-P[Asp(DFO) 26 ] 78 (Mn=32,900)

[0466] ·D-PBS(-):Nacalai Tesque Inc.

[0467] ·GaCl3: Tokyo Chemical Industry Co.,Ltd.

[0468] ·5mol / l NaOH: Wako Pure Chemical Industries Co.,Ltd.

[0469] ·HNO3: Wako Pure Chemical Industries Co.,Ltd.

[0470] CT-26 cells (mouse colon cancer cell line): American Type Culture Collection

[0471] DLD-1 cells (human colon adenocarcinoma cell line): American Type Culture Collection

[0472] BALB / c nude mice: Charles River Japan Inc.

[0473] BALB / c mice: Charles River Japan Inc.

[0474] <4.3. Instruments / Equipment>

[0475] ·Countess: Thermo Fischer Scientific Inc.

[0476] ·Agilent 7900ICP-MS: Agilent Technology Co.,Ltd.

[0477] <4.4. In vivo dynamics of DFO and PEG-PAsp (DFO)>

[0478] In order to evaluate the m ] n The blood retention and tumor accumulation of DFO / Ga(III) complex and PEG-P[Asp(DFO) m ] n The Ga(III) / Ga(III) complex was intravenously injected into the CT26 subcutaneous tumor mouse model, and the Ga content in the blood and tumor was measured by ICP-MS after a certain period of time.

[0479] [DFO / Ga, PEG-P[Asp(DFO) 10 ]35 / Ga、PEG-P[Asp(DFO) 26 ] 78 / Preparation of Ga solution]

[0480] DFO / Ga solution

[0481] Add 300 μL of 26.8 mM GaCl3 / D-PBS solution to 2.70 mL of 16.9 mM DFO / D-PBS solution and stir overnight. Adjust pH to 7.4 with NaOH and filter through a filter. (DFO concentration = 15.2 mM)

[0482] ·PEG-P[Asp(DFO) 10 ] 35 / Ga solution

[0483] 1.71 mM PEG-P[Asp(DFO) 10 ] 35 2.70 mL of 26.8 mM GaCl3 / D-PBS solution (DFO concentration = 16.9 mM) was added to 300 μL of 26.8 mM GaCl3 / D-PBS solution and stirred overnight. The reaction solution was placed on an ultrafiltration membrane (MWCO = 10 kD) and ultrafiltered 5 times. 3 ml of D-PBS was added to the purified solution and filtered using a filter. (DFO concentration = 15.2 mM)

[0484] ·PEG-P[Asp(DFO) 26 ] 78 / Ga solution

[0485] 0.662 mM PEG-P[Asp(DFO) 26 ] 78 2.70 mL of 26.8 mM GaCl3 / D-PBS solution (DFO concentration = 16.9 mM) was added to 300 μL of 26.8 mM GaCl3 / D-PBS solution and stirred overnight. The reaction solution was placed on an ultrafiltration membrane (MWCO = 10 kD) and ultrafiltered 5 times. 3 ml of D-PBS was added to the purified solution and filtered using a filter. (DFO concentration = 15.2 mM)

[0486] [Establishment of CT26 subcutaneous tumor mouse model]

[0487] BALB / c mice were subcutaneously injected with 100 μl of CT26 cell suspension (1.0×10 6 cells / ml).

[0488] [Evaluation of in vivo dynamics]

[0489] For tumors up to 200 mm in size 3100 μl of the prepared solution (DFO 1.52 μmol / mouse) was administered to the tail vein of model mice. Dissection was performed 1, 3, 6, and 24 hours after the sample administration, and the blood and various organs were recovered in 10 mL test tubes (ファルコンチューブ). 1 mL of HNO3 was added, and the mixture was incubated at 50°C for 15 min, 70°C for 15 min, and 90°C for 1 hour. The volume was made up to 10 ml with pure water, and after filtering with a hydrophobic filter, the gallium content in the blood and various organs was measured by ICP-MS. The results are shown in Fig. 20 and Fig.21 The results show the values ​​of mean±SD (n=3).

[0490] The blood concentration of DFO was 0.25% dose / mL 1 hour after administration, indicating that it disappeared rapidly from the blood. On the other hand, PEG-P[Asp(DFO) 10 ] 35 and PEG-P[Asp(DFO) 26 ] 78 Six hours after administration, the blood concentrations were 10% and 8.6% of the dose / mL, respectively. 10 ] 35 and PEG-P[Asp(DFO) 26 ] 78 The drug accumulated in the tumor through the EPR effect, and the intratumoral concentrations were 3.9% and 4.1% of the dose / g, respectively, at 6 hours after administration. In addition, the intratumoral concentrations were maintained at 2.0% and 3.4% of the dose / g, respectively, after 24 hours of administration. These results show that PEG-P[Asp(DFO) 10 ] 35 and PEG-P[Asp(DFO) 26 ] 78 In addition to improving the blood retention of DFO, it also achieves the ability to accumulate and retain in tumors.

[0491] <4.5. Anti-tumor effect>

[0492] DLD-1 and CT26 subcutaneous tumor mouse models were treated with intravenous injection of DFO and PEG-P[Asp(DFO) m ] n , to evaluate the anti-tumor effect.

[0493] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution, PEG-P[Asp(DFO) 26 ] 78 Solution Preparation]

[0494] DFO / D-PBS solution: 15.2 mM

[0495] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 1.54 mM (DFO concentration = 15.2 mM)

[0496] ·PEG-P[Asp(DFO) 26 ] 78 / D-PBS solution: 0.596 mM (DFO concentration = 15.2 mM)

[0497] [Establishment of DLD-1 and CT26 subcutaneous tumor mouse models]

[0498] BALB / c mice and BALB / c nude mice were subcutaneously injected with 100 μl of DLD-1 cell suspension (1.0×10 7 cells / ml), CT26 cell suspension (1.0×10 6 cells / ml).

[0499] [Evaluation of anti-tumor effects]

[0500] For tumors 50 to 100 mm in size 3 100 μl of the above prepared solution (DFO 1.52 μmol / mouse) was administered into the tail vein of mice. As for the control group, the same amount of PBS as that of the treatment group was injected into the tail vein. The administration schedule is as follows.

[0501] DLD-1 subcutaneous tumor mouse model: once a day, 5 times a week, a total of 15 times

[0502] CT26 subcutaneous tumor mouse model: 1 dose per day for 14 times

[0503] During the administration period, the tumor diameter and body weight were measured every 1 to 2 days. The measured tumor diameter was substituted into the elliptical volume approximation formula (ab 2 ×1 / 2, a: long side, b: short side) to obtain the tumor volume. The changes in tumor size and body weight over time are shown in Fig. 22 and Fig.24 , Fig.23 and Fig.25 . Fig. 22 and Fig.23 The results of the DLD-1 subcutaneous tumor mouse model are as follows. Fig.24 and Fig.25 Results are from the CT26 subcutaneous tumor mouse model. Results are shown as mean ± SD.

[0504] In DLD-1 and CT26 subcutaneous tumor mouse models, PEG-P[Asp(DFO) 10 ] 35 and PEG-P[Asp(DFO) 26 ] 78 It produces an anti-tumor effect superior to that of DFO. This is believed to be due to the fact that PEG-P[Asp(DFO) 10 ] 35 and PEG-P[Asp(DFO) 26 ] 78 Tumor accumulation was achieved. In addition, since no weight loss was observed during the treatment, it is believed that no serious side effects occurred.

[0505] 5. Evaluation of cultured cells

[0506] <5.1. Summary>

[0507] The absorption spectrometry and calcein-AM method confirmed that PEG-P[Asp(DFO) m ] n To investigate the iron chelating ability of PEG-P[Asp(DFO) m ] n FerroOrange was used to detect the free iron in the cytoplasm of the treated cells. m ] n The cell proliferation inhibition mechanism of PEG-P[Asp(DFO)] is cell cycle arrest at the S phase, which is believed to be caused by DNA synthesis inhibition. Therefore, DNA synthesis inhibition was evaluated by BrdU staining. m ] n Whether cell apoptosis is induced, apoptotic cells are detected by PI and annexin V staining.

[0508] <5.2. Reagents and cell lines>

[0509] Commercially available reagents were used as they were unless otherwise specified.

[0510] ·DFO: Sigma Aldrich Co.,llc.

[0511] ·PEG-P[Asp(DFO) 10 ] 35 (Mn=19,300)

[0512] Roswell Park Memorial Institute Medium (RPMI): Sigma Aldrich Co.,llc.

[0513] ·D-PBS(-):Nacalai Tesque Inc.

[0514] Fetal bovine serum (FBS): Biosera Inc.

[0515] Trypsin-EDTA solution: Sigma life science Co., Ltd.

[0516] Penicillin / Streptomycin: Sigma life science Co., Ltd.

[0517] ·FerroOrange: Goryo Chemical, Inc.

[0518] ·BrdU: Sigma Aldrich Co.,llc.

[0519] ·5M HCl: Nacalai Tesque Inc.

[0520] ·Sodium borate: Nacalai Tesque Inc.

[0521] ·Paraformaldehyde (PFA): Nacalai Tesque Inc.

[0522] ·TritonX-100: Tokyo Chemical Industry Co.,Ltd.

[0523] Bovine serum albumin (BSA): Nacalai Tesque Inc.

[0524] FITC-anti-BrdU antibody: Thermo Fisher Scientific, Inc.

[0525] ·Hoechst 33342: Thermo Fisher Scientific Inc.

[0526] ·Tween20:Sigma life science Co.,Ltd.

[0527] ·Apoptosis kit (Annexin V-FITC kit): Medical & Biological Laboratories Co., Ltd

[0528] DLD-1 cells (human colon adenocarcinoma cell line): American Type Culture Collection

[0529] <5.3. Measuring instruments>

[0530] ·Countess: Thermo Fischer Scientific Inc.

[0531] ·LSM710: Carl Zeiss Co.,Ltd.

[0532] Guava (registered trademark) easyCyte flow cytometer (FCM): Merck Millipore

[0533] Spark (registered trademark): Tecan Japan Co., Ltd.

[0534] <5.4.PEG-P[Asp(DFO) m ] n Evaluation of iron chelating capacity

[0535] In order to investigate the m ] n Whether it reduces the free iron in the cytoplasm was detected by FerroOrange and DFO and PEG-P[Asp(DFO) m ] n Cytoplasmic free iron in treated cells.

[0536] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution Preparation]

[0537] DFO / D-PBS solution: 2.00 mM

[0538] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 200 μM (DFO concentration = 2.00 mM)

[0539] [Observation using a confocal microscope]

[0540] DLD-1 cells were cultured to a size of 3.0 × 10 5 Cells / well were seeded into 35 mm 2Pre-cultured in glass bottom dishes at 37°C, 5% CO2 for 24 hours. After removing the solution, add 2 mL of the above prepared solution diluted 10 times with RPMI to each dish and culture for 24 hours. After washing with 2 mL of serum-free RPMI, add 1 mL of serum-free RPMI containing 1 μM FerroOrange and culture in the dark for 30 minutes. Thereafter, observe the fluorescence of FerroOrange using CLSM. The results obtained are shown in Fig.27A .

[0541] [Assay using a plate reader]

[0542] DLD-1 cells were cultured at a rate of 1.0 × 10 4 Cells were seeded into a 96-well plate at 10 cells / well and pre-cultured at 37°C and 5% CO2 for 24 hours. After removing the solution, 100 μL of the above-prepared solution diluted 10 times with RPMI was added to each well and cultured for 24 hours. After washing with 100 μL of serum-free RPMI, 100 μL of serum-free RPMI containing 1 μM FerroOrange was added and cultured in the dark for 30 minutes. Thereafter, the fluorescence intensity of FerroOrange was measured using a plate reader. The results obtained are shown in Fig.27B The results show the values ​​of mean±SD (n=5). Statistically significant differences were evaluated by Tukey's multiple comparison test (****p<0.0001).

[0543] DFO and PEG-P[Asp(DFO) 10 ] 35 The free iron in the cytoplasm is significantly reduced.

[0544] <5.5. By PEG-P[Asp(DFO) m ] n Inhibition of DNA synthesis caused by

[0545] In order to investigate the m ] n The mechanism of cell cycle arrest at S phase was evaluated by BrdU staining to assess DNA synthesis inhibition.

[0546] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution Preparation]

[0547] DFO / D-PBS solution: 450 μM

[0548] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 60.0 μM (DFO concentration = 600 μM)

[0549] [Observation using a confocal microscope]

[0550] DLD-1 cells were cultured at a rate of 1.0 × 10 5 Cells / well were seeded into 35 mm 2 The glass bottom dish was pre-cultured at 37°C and 5% CO2 for 24 hours. After removing the solution, 3 mL of the above prepared solution diluted 10 times with RPMI was added to each well and cultured for 72 hours. Thereafter, 330 μL of 10 mM BrdU / D-PBS solution was added and cultured for 1 hour. After removing the solution, 1 mL of 1.0 M HCl solution was added and cultured for 10 min. After removing the solution, 1 mL of 0.1 M sodium tetraborate buffer (pH 8.5) was added and cultured for 30 min. After washing with 2 mL of D-PBS, 1 mL of 4% PFA / D-PBS solution was added and cultured for 10 min. After removing the solution, 1 mL of 0.2% Triton X-100 / D-PBS solution was added and cultured for 5 min. After washing with 2 mL of D-PBS, 2 mL of 5% BSA / D-PBS solution was added and cultured for 1 hour. After removing the solution, add FITC-anti-BrdU antibody / 1% BSA solution and incubate in the dark for 30 minutes. After washing with 2 mL of D-PBS, add 1 mL of 5.0 μg / mL Hoechst / D-PBS solution and incubate in the dark for 5 minutes. After washing with 2 mL of D-PBS, observe using CLSM. The results obtained are shown in Fig.28A .

[0551] [Measurement by flow cytometry]

[0552] DLD-1 cells were cultured to a size of 2.0 × 10 5Cells were inoculated into 6-well plates at 10 cells / well and pre-cultured at 37°C and 5% CO2 for 24 hours. After removing the solution, 3 mL of the above-prepared solution diluted 10 times with RPMI was added to each well and cultured for 72 hours. Thereafter, 330 μL of 10mM BrdU / D-PBS solution was added and cultured for 1 hour. After washing with 3 mL of D-PBS, 700 μL of trypsin-EDTA solution was added and cultured for 10 minutes. After confirming that the cells were detached using an optical microscope, 700 μL of RPMI was added to fully suspend them. The suspension was centrifuged at 1200 rpm for 3 minutes and the supernatant was removed. 500 μL of D-PBS was added to the cell cluster and suspended. The number of cells was measured and the number of cells for each sample was unified. The suspension was added dropwise to 4.5 mL of 70% ethanol and fixed in a freezer at -20°C overnight. The suspension was centrifuged at 1200 rpm for 3 minutes and the supernatant was removed. Add 500μL of 2.0M HCl / 0.5% TritonX-100 solution and incubate for 30 minutes. Centrifuge the suspension at 1200rpm for 3 minutes and remove the supernatant. Add 500μL of 0.1M sodium tetraborate buffer (pH 8.5) and incubate for 2 minutes. Centrifuge the suspension at 1200rpm for 3 minutes and remove the supernatant. After washing with 1mL of 1% BSA / 0.5% Tween20 / D-PBS solution, add 95μL of 1% BSA / 0.5% Tween20 / D-PBS solution. Add 5μL of FITC-anti-BrdU antibody and incubate for 1 hour. After washing with 1mL of 1% BSA / 0.5% Tween20 / D-PBS solution, add 500μL of 1% BSA / 0.5% Tween20 / D-PBS solution. Finally, filter the suspension with a cell strainer and measure the fluorescence of BrdU using FCM. The obtained results are shown in Fig.28B .

[0553] Display DFO and PEG-P[Asp(DFO) 10 ] 35 Since BrdU incorporation is significantly inhibited, inhibition of DNA synthesis is induced, and it is considered that as a result, the cell cycle is arrested at the S phase.

[0554] <5.6.PEG-P[Asp(DFO) m ] n Evaluation of apoptosis induction

[0555] In order to investigate the m ] n Whether cell apoptosis is induced, apoptotic cells are detected by PI and annexin V staining.

[0556] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution Preparation]

[0557] DFO / D-PBS solution: 1.00 mM

[0558] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 100 μM (DFO concentration = 1.00 mM)

[0559] DLD-1 cells were cultured at a rate of 1.0 × 10 5 Cells / well were inoculated into 6-well plates and pre-cultured at 37°C and 5% CO2 for 24 hours. After removing the solution, 3 mL of the above-prepared solution diluted 10 times with RPMI was added to each well and cultured for 24 hours. After washing with 3 mL of D-PBS, 700 μL of trypsin-EDTA solution was added and cultured for 10 minutes. After confirming cell detachment with an optical microscope, 700 μL of RPMI was added to fully suspend it. The suspension was centrifuged at 1200 rpm for 3 minutes and the supernatant was removed. 500 μL of RPMI was added, the number of cells was measured, and the number of cells for each sample was unified. The suspension was centrifuged at 1200 rpm for 3 minutes and the supernatant was removed. 85 μL of binding buffer, 10 μL of annexin V solution, and 5 μL of PI solution attached to the apoptosis kit were added and cultured in the dark for 15 minutes. After adding 400 μL of binding buffer, the suspension was filtered using a cell strainer, and the fluorescence of PI and annexin V was measured using FCM. The obtained results are shown in FIG29 . Fig.29A is a graph showing the distribution of fluorescence acquired by FCM. Fig.29B The results are converted into live cells, early apoptotic cells, and late apoptotic / necrotic cells. The results show the mean ± SD (n = 3). Statistically significant differences were evaluated by Tukey's multiple comparison test (****p < 0.0001).

[0560] DFO and PEG-P[Asp(DFO) 10 ] 35 Significantly increased the ratio of apoptotic cells and necrotic cells.

[0561] 6. Effect on protoporphyrin IX accumulation in tumors of subcutaneous tumor model mice

[0562] <6.1. Summary>

[0563] Evaluation of the efficacy of 5-aminolevulinic acid hydrochloride combined with DFO or PEG-P[Asp(DFO)] in a CT26 (mouse colorectal cancer) subcutaneous tumor mouse model m ] n Accumulation of protoporphyrin IX in tumors at the time of onset.

[0564] <6.2. Reagents, cells, and animals>

[0565] ·Deferoxamine mesylate (DFO): Sigma Aldrich Co.,llc.

[0566] ·PEG-P[Asp(DFO) 10 ] 35 (Mn=19,300)

[0567] 5-Aminolevulinic acid hydrochloride: COSMO OIL Co., Ltd.

[0568] ·D-PBS(-):Nacalai Tesque Inc.

[0569] ·PBS(-):Wako Pure Chemical Industries Co.,Ltd

[0570] ·N,N-Dimethylformamide (DMF): Wako Pure Chemical Industries Co.,Ltd

[0571] Protoporphyrin IX: Frontier Scientific, Inc.

[0572] CT-26 cells (mouse colon cancer cell line): American Type Culture Collection

[0573] BALB / c mice: Charles River Japan Inc.

[0574] <6.3. Instruments / Equipment>

[0575] ·infinite M200 PRO:Tecan Group Ltd.

[0576] <6.4. Evaluation of protoporphyrin IX accumulation in tumors>

[0577] [DFO solution, PEG-P[Asp(DFO) 10 ] 35 Solution Preparation]

[0578] DFO / D-PBS solution: 15.2 mM

[0579] ·PEG-P[Asp(DFO) 10 ] 35 / D-PBS solution: 1.54 mM (DFO concentration = 15.2 mM)

[0580] [Preparation of 5-aminolevulinic acid hydrochloride solution]

[0581] 5-Aminolevulinic acid hydrochloride was dissolved in physiological saline to a concentration of 25 mg / ml.

[0582] [Establishment of CT26 subcutaneous tumor mouse model]

[0583] BALB / c mice were subcutaneously injected with 100 μl of CT26 cell suspension (1.0×10 6 cells / ml).

[0584] [Evaluation of protoporphyrin IX in tumors]

[0585] For tumors up to 200 mm in size 3 Model mice were injected with DFO or PEG-P[Asp(DFO) 10 ] 35 Solution 100μl (DFO 1.52μmol / mouse). The control group was injected with 100μl of physiological saline through the tail vein in the same manner as the DFO administration group. Immediately thereafter, 100μl of 5-aminolevulinic acid hydrochloride solution (250mg / kg body weight, converted to 195.6mg / kg body weight as ALA) was orally administered. Dissection was performed 0, 1, 2, 3, and 5 hours after the administration of 5-aminolevulinic acid hydrochloride solution, and the tumor was recovered. Add 9 times the weight of the tumor in PBS (-), homogenize and perform ultrasonic disruption. 300μl of the disrupted liquid was mixed with 700μl of DMF and centrifuged. The fluorescence intensity of the centrifuged supernatant was measured (excitation wavelength 405nm), and a fluorescence spectrum with a peak at 635nm was obtained. The amount of protoporphyrin IX was calculated using the fluorescence intensity at an excitation wavelength of 405nm and a fluorescence wavelength of 630nm. Fig.30 The results of protoporphyrin IX measurement in tumors are shown. The results show the values ​​of mean ± SD (n = 3).

[0586] In the CT26 subcutaneous tumor mouse model, PEG-P[Asp(DFO) 10 ] 35 Protoporphyrin accumulation in tumors was superior to that of controls and DFO.

[0587] 7. Effects of Vitamin C Therapy

[0588] <7.1. Background>

[0589] It was investigated whether the therapeutic effect of vitamin C therapy could be enhanced by using the iron chelator used in this example together with vitamin C.

[0590] <7.2. In vitro activity evaluation>

[0591] [Experimental Materials]

[0592] ·PEG-P[Asp(DFO) 36 ] 76 (Mn=45,000)

[0593] ·L-ascorbic acid sodium salt (Wako Pure Chemical Industries, Ltd.)

[0594] ·FeCl3(Wako Pure Chemical Industries, Ltd.)

[0595] CT26 cells (ATCC)

[0596] RPMI medium (Sigma-Aldrich) supplemented with 10% FBS + 1% penicillin / streptomycin for cell culture

[0597] Cell Counting Kit-8 (Tongren Chemical)

[0598] [Experimental methods]

[0599] CT26 cells were plated in 96-well plates at 5 × 10 3 Cells / well were cultured for 24 hours.

[0600] The cells were cultured in the presence of various concentrations of PEG-P[Asp(DFO) 36 ] 76 5 mM L-ascorbic acid sodium salt and 15 μM Fe 3+ culture medium for 2 hours.

[0601] After replacing the medium, culture was continued for 24 hours.

[0602] After removing the medium, 100 μL of medium containing 10% Cell Counting Kit-8 was added and cultured for 1.5 hours.

[0603] ·Measure the absorbance at 450 nm and calculate the cell survival rate.

[0604] The obtained results are shown in Fig.31 .

[0605] Fig.31 It means PEG-P[Asp(DFO) 36 ] 76Graph showing the effect of vitamin C treatment in vitro. Concentrations in the graph represent the equivalent of PEG-P[Asp(DFO) 36 ] 76 The results are expressed as mean ± standard deviation (n = 9).

[0606] [result]

[0607] The cytocidal effect of vitamin C was enhanced by adding PEG-P[Asp(DFO) 36 ] 76 , the cancer cell killing effect is greatly improved ( Fig.31 ).

[0608] <7.3. In vivo activity evaluation>

[0609] [Experimental Materials]

[0610] ·PEG-P[Asp(DFO) 36 ] 76 (Mn=45,000)

[0611] ·L-ascorbic acid sodium salt (Wako Pure Chemical Industries, Ltd.)

[0612] CT26 cells (ATCC)

[0613] RPMI medium (Sigma-Aldrich) supplemented with 10% FBS + 1% penicillin / streptomycin for cell culture

[0614] BALB / c mice (female, purchased from Japan SLC)

[0615] [Experimental methods]

[0616] BALB / c mice were injected subcutaneously with 3×10 5 The subcutaneous tumor model was established by injecting CT26 cells into mice.

[0617] When the tumor size reaches 50 mm 3 At the time point, PEG-P[Asp(DFO) 36 ] 76 Intravenous injection (PEG-P[Asp(DFO) 36 ] 76 Administration amount: 0.9 mg / mouse = 45 mg / kg body weight or 1.8 mg / mouse = 90 mg / kg body weight).

[0618] ·PEG-P[Asp(DFO) 36 ] 76Six hours after the administration, L-ascorbic acid sodium salt dissolved in pure water was intravenously injected at 10 mg / mouse = 500 mg / kg body weight.

[0619] The above injections were performed for 5 consecutive days, and the changes in tumor size over time were examined.

[0620] The obtained results are shown in Fig.32 .

[0621] Fig.32 PEG-P[Asp(DFO) 36 ] 76 Graph of the in vivo effects of treatment with vitamin C. Results are presented as mean ± SD.

[0622] [result]

[0623] By adding PEG-P[Asp(DFO) 36 ] 76 It shows high therapeutic effect when used together with high concentration of vitamin C. In addition, in PEG-P[Asp(DFO) 36 ] 76 When the dosage was 0.9 mg / mouse and when it was 1.8 mg / mouse, the dosage of 0.9 mg / mouse showed a tendency to have a higher effect.

[0624] 8. Summary

[0625] The content of the above embodiment is summarized. In this embodiment, an iron chelator delivery system is constructed by combining the terminal amino group of DFO that does not participate in chelating iron with a biocompatible polymer having a polycarboxylic acid structure. By absorption spectrum measurement, it is confirmed that the DFO of the side chain of the iron chelator used in this embodiment has the same iron chelating ability as free DFO. It is confirmed that the iron chelator used in this embodiment maintains chelating ability even in a biological environment, chelates the iron ions in cultured colorectal cancer cells, and reduces the free iron in the cytoplasm of cultured colon adenocarcinoma cells. In addition, the effect on the cell cycle in the cell was investigated, and the results confirmed that the application of the iron chelator used in this embodiment inhibited DNA synthesis, and the iron chelator used in this embodiment stopped the cell cycle in the S phase. By cell proliferation analysis, it was confirmed that it has proliferation inhibition and apoptosis induction.

[0626] DFO and the iron chelator used in this example were intravenously injected into a subcutaneous tumor mouse model, and the blood retention was compared. The results showed that DFO disappeared from the blood early, but the iron chelator used in this example still maintained a very high blood concentration 6 hours after administration. In addition, the iron chelator used in this example accumulated in the tumor, and its accumulation increased over time. The anti-tumor effect was studied using this subcutaneous tumor model, and the results showed that a more significant therapeutic effect was obtained than DFO.

[0627] In addition, administration of the iron chelator and 5-aminolevulinic acid used in this example confirmed that the accumulation of protoporphyrin IX in the tumor increased, indicating that the iron chelator used in this example is suitable for photodynamic diagnosis and / or therapy using 5-aminolevulinic acid.

[0628] Furthermore, the administration of the iron chelator used in this example and vitamin C demonstrated an excellent antitumor effect, indicating that the iron chelator used in this example is suitable for vitamin C therapy.

[0629] Each configuration and combination thereof in each embodiment is merely an example, and addition, omission, substitution, and other changes may be made to the configuration without departing from the gist of the present invention. In addition, the present invention is not limited to each embodiment but only to the scope of the claims.

Claims

1. A conjugate, wherein a deferoxamine is bound to a biocompatible polymer, wherein the deferoxamine is deferoxamine or an ion thereof, wherein The biocompatible polymer includes a first biocompatible polymer chain which is polyethylene glycol and a second biocompatible polymer chain which is different from the first biocompatible polymer chain. The conjugate comprises a structure represented by the following general formula (1-2): [Chemistry 2] In formula (1-2), I is an integer from 1 to 1500, B represents the second biocompatible polymer chain, which includes the repeating structure represented by (b1-1) and the repeating structure represented by (b2-1), [Chemistry 3] In formulas (b1-1) to (b2-1), R 11 represents the aspartic acid side chain, R 12 The carboxyl group in the aspartic acid side chain represented by -CH2-COOH is combined with the deferoxamine, n1 represents the total of (b1-1) and (b2-1), n1 is 35, m1 is 10; or n1 is 78, m1 is 26; or n1 is 76, m1 is 36, Its number average molecular weight is 2,000 to 200,000.

2. A cancer therapeutic agent comprising the conjugate according to claim 1 as an active ingredient.

Images