JPWO2018038240A1 - Method for stabilizing micelles containing complexes of metal and block copolymer and stabilized micelles, and method for controlling metal release from micelles - Google Patents

Abstract

The present invention provides controlled release micelles that are stable in vivo and can release drugs encapsulated in cancer cells. The present invention also provides an active release control technique of the drug from the micelle. According to the present invention, there is provided a transition metal complex-containing micelle formed from a block copolymer of a hydrophilic polymer and an anionic polymer and a transition metal complex or an amphoteric metal complex, wherein the transition metal or the amphoteric metal in the complex is Micelles that are in oxidized form or heat treated micelles are provided. According to the invention there is provided a combination of said micelles and a reducing agent.

Description

Cross-reference of applications

  This application is an application that enjoys the benefit of the priority of the patent application (Japanese Patent Application No. 2016-165298) filed on Aug. 26, 2016 in Japan, the entire content of which is incorporated herein by reference. It is a thing.

  The present invention relates to a method for stabilizing micelles comprising a complex of a metal and a block copolymer, a stabilized micelle, and a method for controlling metal release from micelles.

  Platinum complexes have shown efficacy in cancer treatment, and cisplatin and carboplatin have been marketed as anti-cancer agents. In general, stabilization of the metal complex often results in no metal being released from the formed complex or no metal being released efficiently or at an appropriate rate. For example, chloride ion ligands of cisplatin (cis-diammine dichloroplatinum (II), also referred to as CDDP) are required to be side chain residues such as cysteine, methionine and histidine in proteins (these are much stronger than carboxyl anions). When exchanging at the nucleus, a very stable complex is formed, and even in saline, re-exchange to a further chloride ion ligand does not occur, and CDDP may even show no activity in vivo.

  In addition, techniques for delivering platinum complexes to cancer using micelles including platinum complexes have also been developed (Patent Document 1 and Non-patent Document 1). Particularly, in Patent Document 1, a complex of a block copolymer comprising a poly (ethylene glycol) segment and a poly (α-glutamic acid) segment and CDDP has a stable micelle structure, and when administered intravenously, it is more effective than CDDP alone. It has also been shown to exhibit excellent anti-cancer activity. Patent Document 2 discloses a micelle containing dahaplatin as a platinum complex as an active ingredient.

WO 2002/26241 WO2005 / 056641

H. Cabral et al., J. Control. Release 101: 223-232, 2005

  If it is possible to provide micelles with higher in vivo stability and reduced risk of drug release due to decomposition in blood, it is possible to treat cancer with fewer side effects. The present invention provides a method of stabilizing micelles comprising a complex of a metal and a block copolymer, a stabilized micelle, and a controlled release of transition metal from the micelle.

  The present inventors have found that the micelles are significantly stabilized in solution (eg, blood) by oxidizing or heat treating micelles containing a complex of transition metal and block copolymer. The inventors also found that reduction of the micelles obtained by the oxidation treatment destabilizes the micelles and efficiently releases internal transition metals. For example, it has been found that when micelles stabilized by the above method are introduced into a reducing environment equivalent to cancer cytoplasm, the micelles are destabilized and internal transition metals are efficiently released. The present inventors also found that when ascorbic acid and micelles stabilized by the above method are used in combination, the anticancer effect of the micelles is increased immediately after administration of the reducing agent ascorbic acid. The present inventors further found that when micelles were subjected to both oxidation treatment and heat treatment, they exhibited a higher stabilization effect than micelles subjected to each treatment alone. The present invention is an invention based on these findings.

The present invention provides the following inventions.
(1) A transition metal-containing micelle comprising a block copolymer of a hydrophilic polymer and an anionic polymer and a complex of a transition metal, wherein the transition metal is in an oxidized form.
(2) The micelle according to (1) above, wherein the transition metal is platinum or copper.
(3) The micelle according to (1) or (2) above, wherein the hydrophilic polymer is polyethylene glycol and the anionic polymer is an anionic amino acid polymer.
(4) The micelle according to (3) above, wherein the anionic amino acid polymer forms a coordination bond with the transition metal by performing ligand exchange with the ligand of the transition metal complex as a monomer unit.
(5) The micelle according to (3) above, wherein the anionic amino acid polymer contains glutamic acid as a monomer unit.
(6) The micelle according to the above (1), wherein the hydrophilic polymer is polyethylene glycol, the anionic polymer is polyglutamic acid or polyaspartic acid, and the transition metal complex contains oxidized platinum or oxidized copper.
(7) A pharmaceutical composition comprising the micelle according to any one of (1) to (6) above.
(8) The pharmaceutical composition according to (7) above, for use in combination with a reducing agent.
(9) A combined medicament of a reducing agent and the pharmaceutical composition according to (7) above.
(10) The pharmaceutical composition according to (8) or the combined drug according to (9), wherein the reducing agent is ascorbic acid or a pharmaceutically acceptable salt thereof.
(11) A pharmaceutical composition for use in treating cancer, comprising ascorbic acid or a pharmaceutically acceptable salt thereof, which is a pharmaceutical composition according to any one of (1) to (8) above. Pharmaceutical composition for use in combination with

(12) A transition metal-containing micelle comprising a block copolymer of a hydrophilic polymer and an anionic polymer and a complex of a transition metal, wherein the micelle is heat-treated at 40 ° C to 85 ° C.
(13) The micelle according to (12) above, wherein the transition metal is platinum or copper.
(14) The micelle according to (12) or (13) above, wherein the hydrophilic polymer is polyethylene glycol and the anionic polymer is an anionic amino acid polymer.
(15) The micelle according to the above (14), wherein the anionic amino acid polymer performs ligand exchange with the ligand of the transition metal complex as a monomer unit to form a coordination bond with the transition metal.
(16) The micelle according to (14) above, wherein the anionic amino acid polymer contains glutamic acid as a monomer unit.
(17) The micelle according to (12) above, wherein the hydrophilic polymer is polyethylene glycol, the anionic polymer is polyglutamic acid or polyaspartic acid, and the transition metal complex contains oxidized platinum or oxidized copper.
(18) A pharmaceutical composition comprising the micelle according to any one of (12) to (17) above.

(A1) A transition metal comprising a block copolymer of a hydrophilic polymer and a polymer containing a monomer unit having a metal coordinating functional group in a side chain as a monomer unit and a complex of a transition metal or an amphoteric metal Or an amphoteric metal-containing micelle, wherein the transition metal or the amphoteric metal is in an oxidized form.
(A2) The micelle according to the above (A1), wherein the polymer containing a monomer unit having a metal coordinating functional group in a side chain is an anionic polymer.
(A3) The micelle according to any of (A1) or (A2) above, wherein the hydrophilic polymer is polyethylene glycol and the anionic polymer is an anionic amino acid polymer.
(A4) The micelle according to (A3) above, wherein the anionic amino acid polymer contains glutamic acid as a monomer unit.
(A5) Any of the above (A1) to (A4), wherein the hydrophilic polymer is polyethylene glycol, the anionic polymer is polyglutamic acid or polyaspartic acid, and the transition metal complex contains oxidized platinum or oxidized copper Micelle as described in.
(A6) A pharmaceutical composition comprising the micelle according to any one of (A1) to (A5) above.

(B1) A transition metal containing a block copolymer of a hydrophilic polymer and a polymer containing a monomer unit having a metal coordinating functional group in a side chain as a monomer unit and a complex of a transition metal or an amphoteric metal Or an amphoteric metal-containing micelle, which is heat-treated at a temperature of 40 ° C. to 85 ° C. during or after formation of the micelle.
(B2) The micelle according to (B1) above, wherein the polymer containing a monomer unit having a metal coordinating functional group in a side chain is an anionic polymer.
(B3) The micelle according to (B2) above, wherein the anionic amino acid polymer contains glutamic acid as a monomer unit.
(B4) Any one of the above (B1) to (B3), wherein the hydrophilic polymer is polyethylene glycol, the anionic polymer is polyglutamic acid or polyaspartic acid, and the transition metal complex contains oxidized platinum or oxidized copper Micelle as described in.
(B5) A pharmaceutical composition comprising the micelle according to any one of (B1) to (B4) above.

(C1) The micelle according to any one of (A1) to (A5) above, which has been heat-treated at a temperature of 40 ° C. to 85 ° C. during or after micelle formation.
(C2) A pharmaceutical composition comprising the micelle as described in (C1) above.

FIG. 1 shows X-ray absorption spectra, a / b ratios, and relative amounts of tetravalent platinum (Pt (IV)) of dahaplatin-containing micelles and oxidized dahaplatin-containing micelles. FIG. 2 shows the effect of oxidation treatment on micellar stabilization in dachaplatin-incorporated micelles having various molecules as ligands. FIG. 3 shows that treatment of oxidized transition metal complex micelles with glutathione (GSH) as a reducing agent results in release of platinum from the micelles and dissociation of the micelles immediately after addition of the reducing agent. FIG. 4 shows that when oxidized transition metal complex micelles are treated with sodium ascorbate (vitamin C sodium salt or NaAsc) as a reducing agent, platinum is released from the micelles immediately after adding the reducing agent, and the micelles are dissociated. Indicates to do. 4-2 shows that the stability of oxidized transition metal complex micelles is improved using a transition metal different from the transition metal of FIG. 4, and when the micelles are treated with sodium ascorbate, immediately after the treatment, It shows that the micelles dissociate. FIG. 5 shows that sodium ascorbate (NaAsc) has a higher reducing potential than GSH, but has a high reducing ability, but has a high ability to destabilize oxidized transition metal complex micelles. FIG. 6 shows the retention amount of oxidized dahaplatin micelles in blood and the abundance of tetravalent platinum (Pt (IV)) at each time after administration in blood. Fig. 7 shows that oxidized dahaplatin micelles can be administered with many anticancer agents by increasing the maximum tolerated dose (MTD) compared to reduced dahaplatin (A), and the antitumor effect is enhanced. It shows that the antitumor effect is significantly enhanced by the combination with an acid (B). FIG. 7-2 shows that oxidized dahaplatin micelles show stronger antitumor activity over a long period of time in the tumor support model than reduced micelles (upper panel). FIG. 7-2 also shows that oxidized dahaplatin micelles have lower biotoxicity than reduced as shown by weight change of mice after administration (lower panel). FIG. 7-3 shows the hepatotoxicity (changes in plasma GOT concentration and plasma GPT concentration) of oxidized dahaplatin micelles and reduced dahaplatin micelles. FIG. 7-4 shows bone marrow toxicity (white blood cell count, red blood cell count, and platelet count) of oxidized dahaplatin micelles and reduced dahaplatin micelles. FIGS. 7-5 show the stability (left panel) in the plasma of oxidized dahaplatin micelles and reduced dahaplatin micelles and the accumulation to tumors (right panel). FIGS. 7-6 show accumulation of oxidized dahaplatin micelles and reduced dahaplatin micelles in non-tumor tissues (liver, spleen and kidney). FIGS. 7-7 show the tumor accumulation selectivity (ratio of accumulation to tumor to liver, spleen, or kidney) of oxidized dahaplatin micelles and reduced dahaplatin micelles as a ratio of accumulated platinum amount. 7-8 shows AUC (0 to 96 hours) of the ratio of accumulation to tumor to liver, spleen, or kidney in oxidized dahaplatin micelles and reduced dahaplatin micelles. FIG. 8 shows micelles that have been subjected to heat treatment at 70 ° C. for 24 hours during micelle formation (hereinafter, micelles subjected to heat treatment at the time of micelle formation are referred to as “heat-treated micelles (I)”) It is a figure which shows comparing and stabilizing. FIG. 9 shows the particle size, polydispersity (pdl) and scattered light intensity of heat-treated micelles (I) heated at various temperatures. FIG. 9-2 is a diagram showing the relationship between the heating temperature at the time of micelle formation, the average particle size of the micelles, the degree of polydispersity, and the scattered light intensity. FIG. 10 shows the concentration ratio of Pt to glutamic acid (A) in heat-treated micelles (I) heated at various temperatures, the incorporation yield of Pt into micelles (B), and the incorporation yield of polymers into micelles (C) Indicates FIG. 10-2 shows the concentration ratio of Pt to glutamic acid in heat-treated micelles (I) heated at various temperatures, the incorporation yield of Pt into micelles, and the incorporation yield of polymer into micelles. FIG. 11 is a view showing that heat-treated micelle (I) heated in a temperature range of 50 ° C. to 80 ° C. centering on 70 ° C. is stable for a long time. FIG. 11-2 is a diagram showing the relationship between heating time and stability of heated micelles in solution. FIG. 11-3 shows the relationship between the heating temperature at the time of formation of the cisplatin-containing micelle and the stability in the solution. FIG. 12 is a view showing the release behavior of heat-treated heat-treated micelle (I) at a pH representing blood and a pH representing endosomes in cells. FIG. 12-2 shows the relationship between the stability of cisplatin internal micelle and pH. FIG. 13 shows that the heat-treated micelles (I) and (II) subjected to the oxidation treatment have higher stability than the oxidation treatment or the heat treatment alone. FIG. 14 shows the relationship between heating temperature and maximum tolerated dose (MTD) during micelle formation. FIG. 15 shows the antitumor effect of a heated dahaplatin micelle (heat-treated micelle (I)) in a tumor-supporting model (left panel). FIG. 15 also shows the side effects of the micelle administration by the weight of the administered mice (right panel). FIG. 16 is a Kaplan-Meier curve showing the antitumor effect of a heated dahaplatin micelle (heat-treated micelle (I)) in a tumor-supporting model. FIG. 17 shows the hepatotoxicity of heated dahaplatin micelles (heat-treated micelles (I)).

Detailed Description of the Invention

  As used herein, "treatment" refers to an intervention that is performed with the intention of preventing the onset of symptoms, suppressing development, or altering the medical condition. As used herein, "treatment" is used to include both therapeutic and prophylactic treatment. As used herein, "treatment" is used in the sense of reducing the rate of progression of the condition, preventing the progression, reducing the condition and eliminating the condition.

  As used herein, the term "subject" means a mammal, for example, used to include human subjects.

As used herein, "transition metal" is an element of Groups 3-11. The transition metal may have an electron configuration in which the electron orbit (usually, the d orbit) inside the outermost shell of the atom is partially filled. The transition metal is not particularly limited, and examples thereof include platinum, copper, chromium, manganese, cobalt, nickel and molybdenum. The transition metal can have unstable unpaired electrons in the inner shell d-orbit, and thus can have multiple valences. As used herein, "amphoteric metal" refers to elements of Groups 13-15. Examples of amphoteric metals include zinc, gallium, germanium and antimony.
The transition metal can be chemically bonded to various atomic groups and can form a complex. In the complex, the transition metal and the atomic group are bound by coordination bond. In the present specification, an atomic group coordinated with a transition metal is referred to as a ligand (ligand). Also, in the present specification, exchange of a ligand for another ligand is referred to as ligand exchange.

As used herein, "dahaplatin" (also referred to as "DACHPt") means platinum (II) having a diaminocyclohexane ligand. In the present specification, in order to clarify the distinction between dahaplatin and oxidized dahaplatin described later, dahaplatin may be referred to as reduced dahaplatin. As used herein, "oxidized dahaplatin" (also referred to as "O-DACHPt") means diaminocyclohexane platinum (IV).
Dahaplatin is known to react with carboxyl groups by ligand exchange of the complex. In particular, when dahaplatin is mixed with a polymer containing a monomer unit having a carboxyl group, dahaplatin binds with the carboxyl group on the polymer by ligand exchange, thereby neutralizing the charge of the polymer and turning the polymer into hydrophobicity. It is known that micelles of the polymer and dahaplatin (hereinafter, also referred to as "dahaplatin micelles", "dahaplatin containing micelles" or "DACHPt / m") are formed. The polymer can be preferably a block copolymer of polyethylene glycol (hereinafter also referred to as "PEG") and a monomer unit having a carboxyl group.

As used herein, a ligand (ligand) refers to a functional group or the entire molecule that is a metal ligand. For example, in the case of dahaplatin, the ligand means the molecule to which Pt in dahaplatin coordinates. The ligand of dahaplatin is not particularly limited, and examples thereof include Cl, NO ₃ , H ₂ O and AcO in addition to the diaminocyclohexane ligand. The acceptable combination of dahaplatin as a ligand is not particularly limited. For example, in addition to a diaminocyclohexane ligand, a combination of Cl and NO ₃ , a combination of Cl and H ₂ O, an AcO and an AcO Combinations and combinations of H ₂ O and H ₂ O can be mentioned. Dahaplatin can be coordinated with the carboxyl group (carboxylate) in the polymer by ligand exchange as described above.

As used herein, "carboplatin" refers to platinum having a 1,1-cyclobutanedicarboxylic acid ligand. Carboplatin may have NH ₃ as a ligand in addition to the 1,1-cyclobutanedicarboxylic acid ligand. Carboplatin can be coordinated with a carboxyl group (carboxylate) in the polymer by ligand exchange.

  As used herein, "cisplatin" means cis-diamminedichloroplatinum (II). However, in the present specification, one in which one or two of chlorine atoms of cis-diamminedichloroplatinum (II) are ligand-exchanged with the side chain of the copolymer by ligand exchange can be conveniently co-administered with cisplatin Expressed as a complex with a polymer. Also, micelles containing such complexes are conveniently expressed as cisplatin-containing micelles. Cisplatin can be coordinated with the carboxyl group (carboxylate) in the polymer by ligand exchange.

In general, stabilization of the metal complex often results in no metal being released from the formed complex or no metal being released efficiently or at an appropriate rate. For example, chloride ion ligands of cisplatin (cis-diammine dichloroplatinum (II), also referred to as CDDP) are required to be side chain residues such as cysteine, methionine and histidine in proteins (these are much stronger than carboxyl anions). When exchanging at the nucleus, a very stable complex is formed, and even in saline, re-exchange to a further chloride ion ligand does not occur, and CDDP may even show no activity in vivo. However, in addition to having a stable micellar structure, a complex of a block copolymer comprising a poly (ethylene glycol) segment and a poly (α-glutamic acid) segment and CDDP has superior anti-CDDP performance over CDDP alone. It has been shown to exhibit cancer action (WO 2002/26241).
However, if it is possible to provide micelles with higher in vivo stability and reduced risk of drug release due to decomposition in blood, it is possible to treat cancer with fewer side effects. .

  According to the present invention, the oxidized transition metal or amphoteric metal has an oxidized form and can stabilize micelles (the obtained micelles are oxidized micelles or oxidized transition metal inclusion micelles or oxidized amphoteric) Called metal encapsulated micelles). While not wishing to be bound by theory, increasing the oxidation number of the transition metal or amphoteric metal slows down the ligand exchange reaction of the complex, resulting in coordination between the transition metal or amphoteric metal and the polymer that constitutes the micelle. Stabilization was considered to be a factor. On the other hand, the resulting oxidized micelles may be reduced in a reducing environment to release the destabilized, encapsulated drug from the micelles. In vivo, the inside of cancer cells has a reducing environment that is sufficient to destabilize oxidized micelles. Thus, micelles encapsulating an oxidized transition metal or amphoteric metal according to the present invention provide a drug delivery system that releases the encapsulated drug in cancer cells. In addition, the above-mentioned oxidized micelle can release the drug which has been reduced and contained by a reducing agent such as ascorbic acid (vitamin C) from the micelle. Thus, the oxidized transition metal-encapsulated micelles according to the present invention can be controlled in drug release by reducing agent administration. Thus, the present invention provides a drug delivery system and controlled release technology of the drug encapsulated therein.

In the present invention, for example, platinum can be used as the transition metal. Platinum is a II metal in a reducing environment but becomes a IV metal when exposed to an oxidizing environment. In the present invention, when the transition metal is platinum, the oxidized form means platinum having a valence of IV, and the reduced form means platinum having a valence of II. Furthermore, in the present invention, when the transition metal is platinum, the oxidized micelle means a micelle containing platinum of IV valence in the complex, and the reduced micelle means a micelle containing platinum of valence II in the complex. Means Also, in this specification, oxidized transition metals can be obtained by oxidation, and reduced transition metals can be obtained by reduction. Examples of platinum complexes include so-called cisplatin (CAS number: 15663-27-1), dahaplatin, carboplatin (CAS number: 41575-94-4), and oxaliplatin (CAS number: 63121-00-6). It can be used to make the micelles of the invention.
For example, iron and copper may be used as the transition metal. In one embodiment, for example, copper is in a reduced form of + I, a oxidized form of + II, an iron is in a reduced form of + II, and an oxidized form of + III. Oxidation of vanadium, manganese, cobalt (reduced by + II and oxidized by + III), molybdenum, ruthenium, and palladium can also enhance bonding between a metal and a ligand. It is a relative matter whether the transition metal or the amphoteric metal is in the oxidized form or in the reduced form, but it can be understood by those skilled in the art as the valence increases to stabilize the micelles. Also, it can be easily determined by those skilled in the art whether it is oxidized or reduced.

  According to the present invention, there is provided a transition metal or amphoteric metal containing micelle comprising a block copolymer of a hydrophilic polymer and an anionic polymer and a transition metal or amphoteric metal complex, wherein the transition metal or amphoteric metal is in an oxidized form. Some micelles are provided.

  In the present invention, the block copolymer of a hydrophilic polymer and an anionic polymer may be a free form or a salt of a block copolymer of a hydrophilic polymer and an anionic polymer. The salts include, for example, pharmaceutically acceptable salts. As used herein, a block copolymer of a hydrophilic polymer and an anionic polymer is a block copolymer of a hydrophilic polymer and an anionic polymer, or a linker between the hydrophilic polymer and the anionic polymer. It can be a block copolymer having The block copolymer of a hydrophilic polymer and an anionic polymer is a group (eg, methyl group) which does not affect the micellization of the block copolymer at the end on the hydrophilic polymer side and / or at the end on the anionic polymer side. It may have a methoxy group etc.).

Examples of hydrophilic polymer blocks include polyethylene glycol and can be used in the present invention. The polyethylene glycol block is represented by-(CH ₂ -CH ₂ -O) n-, n is an integer of 5-20,000, preferably an integer of 10-5,000, more preferably an integer of 40-500, More preferably, it can be an integer of 10 to 500.
In one embodiment of the present invention, hyperbranched polyethylene glycol, polyvinyl pyriridone (PVP), polyvinyl alcohol (PVA), polyacrylamide, N- (2-hydroxypropyl) methacrylamide (HPMA), divinyl ether as a hydrophilic polymer block -Maleic anhydride (DIVEMA), polyoxazoline, polyphosphoric acid, polyphosphazene, and 2-methacryloyloxyethyl phosphoryl choline (MPC).

  As a metal coordinating functional group, a carboxyl group (including a deprotonated state), an amino group (regardless of primary, secondary, tertiary or aromatic), a thiol group (including a deprotonated state), Amide, thioamide, imine, cyano group, urethane, carbonyl, hydroxyl group (including deprotonated state), thioether (sulfide), phosphino group, ester, thioester, phosphate ester, sulfo group, chelate group containing the above functional group The ligands (ethylenediamine, oxalic acid, glycine, 2,2′-bipyridine etc.) can be mentioned, and those skilled in the art can appropriately select and use a combination of a metal and a metal coordinating functional group.

  A polymer block containing a monomer unit having a metal coordinating functional group is a polymer having the above metal coordinating functional group, such as xanthan gum, pectin, chitosan, dextran, carrageenan, guar gum, cellulose Ether, sodium carboxymethylcellulose, hyaluronic acid, albumin and starch are included. The polymer having the metal coordinating functional group may be, for example, a peptide, and may be, for example, polyglutamic acid or polyaspartic acid.

  As the anionic polymer, for example, a polymer of anionic amino acid, for example, a polymer containing glutamic acid and aspartic acid such as a polymer of glutamic acid and aspartic acid, a polymer containing glutamic acid such as polyglutamic acid, aspartic acid such as polyaspartic acid Included are polymers comprising, polymers comprising acrylic acid, and polymers comprising methacrylic acid, which may be used in the present invention. In these anionic amino acid polymers, the monomer unit is an integer of 2 to 20,000, preferably an integer of 2 to 5,000, more preferably an integer of 10 to 500, and still more preferably an integer of 10 to 200. can do.

  A block copolymer of a hydrophilic polymer and an anionic polymer can be made as follows. For example, a PEG-polyglutamic acid block copolymer can be obtained by polymerizing .gamma.-benzyl-L-glutamic acid N-carboxyanhydride to PEG and deprotecting the protecting group. PEG-polyaspartic acid block copolymers can also be made in a similar manner.

  According to the present invention, a transition metal or amphoteric metal containing micelle comprising a block copolymer of a hydrophilic polymer and an anionic polymer and a complex of a transition metal or an amphoteric metal (hereinafter referred to as “transition metal containing micelle” or The amphoteric metal-containing micelles are obtained by a ligand exchange reaction by mixing a block copolymer of a hydrophilic polymer and an anionic polymer with a transition metal complex or an amphoteric metal. By mixing, the block copolymer is considered to form a coordination bond by ligand exchange with the transition metal complex or the amphoteric metal to form a micelle. In this sense, many of the transition metal or amphoteric metal-containing micelles are considered to be ligand-exchanged and in a different chemical bonding state from the transition metal complex to be mixed or the amphoteric metal.

  According to the present invention, the oxidizing agent can oxidize the transition metal encapsulated in the obtained micelle. In the present specification, the micelle thus obtained is referred to as an oxidized transition metal-containing micelle. According to the present invention, the oxidizing agent can oxidize the amphoteric metal encapsulated in the obtained micelle. In the present specification, the micelle thus obtained is referred to as an oxidized amphoteric metal-containing micelle. In the oxidized transition metal-containing micelles or the oxidized amphoteric metal-containing micelles, as a result of the increase in the oxidation number of the transition metal, the coordination bond with the block copolymer as a ligand is strengthened, and further ligand exchange is performed. It was considered to be resistant. Moreover, it was also considered that oxidation promoted ligand exchange and the number of coordination bonds with the block copolymer increased. The oxidized transition metal or amphoteric metal encapsulated micelle of the present invention is stable from decomposition as long as it is oxidized, and the release of the encapsulated transition metal or amphoteric metal is suppressed. According to the invention, oxidized transition metals or amphoteric metals may be encapsulated in micelles.

  In certain aspects of the invention, the transition metal encapsulated micelles are selected from the group consisting of block copolymers of hydrophilic and anionic polymers, and platinum complexes such as cisplatin, dahaplatin, carboplatin and oxaliplatin. It may be a micelle obtained by mixing with one or more platinum complexes. In one aspect of the present invention, the oxidized transition metal-containing micelle may be obtained by further oxidizing the micelle thus obtained. The oxidation of the transition metal-containing micelle can be performed using an oxidizing agent. In one embodiment of the present invention, for example, hydrogen peroxide can be used as the oxidizing agent, although it is not particularly limited.

  In one aspect of the present invention, there is provided a pharmaceutical composition comprising the oxidized transition metal-containing micelle or the oxidized amphoteric metal-containing micelle of the present invention. In one embodiment of the present invention, in the oxidized transition metal-encapsulated micelle or amphoteric metal-encapsulated micelle, the hydrophilic polymer is polyethylene glycol, the anionic polymer is polyglutamic acid or polyaspartic acid, and the transition metal complex is oxidized platinum. Including dahaplatin or cisplatin.

The pharmaceutical composition containing the oxidized transition metal or amphoteric metal encapsulated micelle of the present invention can be used in combination with a reducing agent (pharmaceutically acceptable reducing agent such as ascorbic acid or a pharmaceutically acceptable salt thereof). The reducing agent may reduce the transition metal or amphoteric metal in the oxidized transition metal-containing or amphoteric metal micelle, but the micelle may destabilize and release the encapsulated transition metal or amphoteric metal from the micelle. Thus, for example, a pharmaceutical composition comprising the oxidized transition metal or amphoteric metal encapsulated micelle of the present invention is reduced after its administration (a pharmaceutically acceptable reducing agent such as ascorbic acid or a pharmaceutically acceptable salt thereof) Can be administered. Pharmaceutically acceptable salts of ascorbic acid include, for example, sodium ascorbate.
In the present invention, ascorbic acid exhibits two aspects, anti-cancer action and reducing action, so that even at a concentration at which ascorbic acid has an anti-cancer action, it exerts a reducing action alone. Ascorbic acid can also be used at concentrations that do not have anticancer activity. Therefore, the pharmaceutical composition containing the oxidized transition metal or amphoteric metal encapsulated micelle of the present invention can be used in combination with ascorbic acid at a dose that does not exert anti-cancer effect alone.

  In one aspect of the present invention, there is provided a pharmaceutical composition for use in treating cancer, comprising ascorbic acid or a pharmaceutically acceptable salt thereof, which comprises the oxidized transition metal or amphoteric metal encapsulated micelle of the present invention. A pharmaceutical composition for use in combination with the pharmaceutical composition is provided. Ascorbic acid is known to have anti-cancer activity, but in the present invention, the property of ascorbic acid as a reducing agent is used to convert oxidized transition metal or oxidized amphoteric metal encapsulated in micelles from micelles. It is possible to obtain a new effect of releasing. These cells are in a reduced form in cells, since the cells are in a reducing environment.

  Ascorbic acid or a salt thereof can be administered after the administration of the oxidized transition metal-encapsulated micelles or the oxidized amphoteric metal-encapsulated micelles of the present invention, but not limited thereto, for example, after 0 to 24 hours. Ascorbic acid or a salt thereof can also be administered after sufficient time has passed for the oxidized transition metal-encapsulated micelles or the oxidized amphoteric metal-encapsulated micelles of the present invention to spread to cancer tissues. In this aspect, ascorbic acid or a salt thereof is administered, for example, after 1 hour to 336 hours, after 24 to 240 hours, or after 24 to 168 hours after administration of the oxidized transition metal-containing micelle or the oxidized amphoteric metal-containing micelle of the present invention It can be administered after hours.

  According to the present invention, it comprises a block copolymer of a hydrophilic polymer and a polymer containing a monomer unit having a metal coordinating functional group in a side chain as a monomer unit and a complex of a transition metal or an amphoteric metal A transition metal or amphoteric metal-containing micelle is provided, which is heat-treated at a temperature of 40 to 85 ° C. when or after formation of the micelle. The transition metal or amphoteric metal can be the metal listed in Table 1. The hydrophilic polymer can be a polymer as described above, and the monomer unit can be a monomer unit as described above. The transition metal or amphoteric metal can be a metal as described above.

  The temperature of the heat treatment can be a temperature in the temperature range of 40 ° C. to 85 ° C., preferably 40 ° C. to 75 ° C., more preferably 50 ° C. to 75 ° C., still more preferably 60 ° C. to 70 ° C. Or it may be 50 ° C-70 ° C.

  The heat treatment time is not particularly limited, but is 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 12 hours or more, 1 day or more , 2 days or more, 3 days or more, 4 days or more, or 5 days or more. Moreover, the upper limit at the time of heating may be, for example, within 3 days, within 2 days, within 24 hours, or within 15 hours.

  The heat treatment may be performed during micelle formation or may be performed after micelle formation.

  In the present invention, the micelles may be heat-treated in addition to the oxidation treatment. For example, in one aspect of the present invention, the micelles can be heat treated upon micelle formation and oxidized after micelle formation. In one aspect of the present invention, micelles can be heat-treated and oxidized after micelle formation.

  According to the present invention, it comprises a block copolymer of a hydrophilic polymer and a polymer containing a monomer unit having a metal coordinating functional group in a side chain as a monomer unit and a complex of a transition metal or an amphoteric metal C., a method for producing transition metal or amphoteric metal-containing micelles (or pharmaceutical preparations containing the micelles), comprising heating at 40.degree. C. to 85.degree. C. during micelle formation or after micelle formation, or oxidizing the micelles And the micelles obtained by this method are provided. Here, the transition metal or the amphoteric metal may be oxidized.

  In the present invention, a transition comprising a block copolymer of a hydrophilic polymer and a polymer containing a monomer unit having a metal coordinating functional group in a side chain as a monomer unit, and a complex of a transition metal or an amphoteric metal A method for stabilizing metal or amphoteric metal-containing micelles in blood, or a method for improving the stability in blood, comprising heating at 40 ° C. to 85 ° C. during micelle formation or after micelle formation, or A method is provided, comprising oxidizing the micelles.

Example 1: Preparation of micelles containing oxidized transition metal complex

1. Synthesis of polyethylene glycol-polyglutamic acid block copolymer With α-methoxy-ω-amino polyethylene glycol (manufactured by NOF) as an initiator, γ-benzyl-L-glutamic acid N-carboxy anhydride (manufactured by Chuo Kasei Co., Ltd.) in DMSO The resulting solution was dialyzed against methanol to obtain polyethylene glycol-poly (γ-benzyl-L-glutamate) (PEG-PBLG). Subsequently, 5 equivalents of 0.5 N aqueous solution of sodium hydroxide is reacted with BLG to give polyethylene glycol-poly (sodium L-glutamate) (PEG-PGlu; M _{w, PEG} = 12000; DP _PGlu = 40) Obtained.

2. Synthesis of Transition Metal Complex In this example, dahaplatin was synthesized as an example.

200 mg of aquachloro (trans-1,2-diaminocyclohexane-N, N ') nitrate DACHPtCl ₂ (Hereus, Germany) is suspended in 50 ml of pure water, 59.74 mg of silver nitrate is added, and the mixture is stirred for 16 hours at 25 ° C. in the dark did. The precipitated silver chloride was filtered and the filtrate was lyophilized to give the title complex. Hereinafter, the title complex is also referred to as [Pt ^II Cl (dach) (H ₂ O)] (NO ₃ ).

Bis (acetate) (trans-1,2-diaminocyclohexane-N, N ') platinum (II)
300 mg of DACHPtCl ₂ was suspended in 75 ml of pure water, 175.55 mg of silver oxide and 86.73 μl of acetic acid were added, and the mixture was stirred at 60 ° C. in the dark for 16 hours. The precipitated silver chloride was filtered off and the filtrate was evaporated to dryness at 60 ° C. After cooling at room temperature, the product was washed twice with ethanol and twice with diethyl ether, and dried under reduced pressure to give the title complex. Alternatively, hereinafter, the title complex is also referred to as [Pt ^II (AcO) ₂ (dach)].

  Moreover, in the following, when referring to both of the above-mentioned complexes, it is written as DACHPt.

3-1. Preparation of oxidized micelles The platinum complex (DACHPt) and PEG-PGlu obtained above were mixed in an aqueous solution so that the concentration was [Pt] = [Glu] = 5 mM, and reacted at 37 ° C. for 120 hours . The resulting solution was purified by dialysis and ultrafiltration to give DACHPt / m. The micelles obtained here are called micelles before oxidation treatment. Subsequently, 100 equivalents of hydrogen peroxide was reacted at room temperature with respect to the platinum concentration of micelles, and thereafter excess hydrogen peroxide was removed by dialysis to obtain oxidized micelle O-DACHPt / m. The micelles obtained here are referred to as micelles after oxidation treatment.
When cisplatin was used as the platinum complex, oxidized micelles were similarly obtained.
3-2. Preparation of Heated Micelle The platinum complex (DACHPt or cisplatin) obtained in 2 above and PEG-PGlu are mixed in an aqueous solution so that the concentration is [Pt] = [Glu] = 5 mM, and various temperatures (25 , 37, 50, 60, 70, 80, 90 ° C.) for 120 hours. The resulting solution was purified by dialysis and ultrafiltration to obtain heat treated micelles (I). In addition, although the above is heating during micelle formation, heat treatment micelles (II) are prepared by reacting micelles at a temperature of 37 ° C. and then reacting at a temperature of 70 ° C. for 24 hours after micelle formation. Got). In the present specification, micelles obtained by heat treatment during micelle formation are referred to as heat treatment micelles (I), and micelles obtained by heat treatment after micelle formation are referred to as heat treatment micelles (II).

4. Structural analysis of micelles
Analysis of particle size distribution Analysis of particle size distributions of oxidized micelles and heat treated micelles (II) was performed by a dynamic light scattering apparatus (DLS 8000; Otsuka Electronics) at a temperature condition of 25 ° C. The results were as shown in Tables 1-1 and 1-2.

  As shown in Table 1-2, it can be seen that heat treatment (70 ° C., 24 hours) does not change the micelle particle size but reduces the polydispersity.

Quantitative Determination of Endogenous Platinum Concentration Concentrated nitric acid was added to the oxidized micelles and thermally decomposed, and the remaining residue was dissolved in 1% HNO ₃ , and the platinum concentration was quantified by inductively coupled plasma mass spectrometry (Agilent 7700x).
Analysis of platinum oxidation number The oxidized micelle solution was evaporated to dryness to obtain an X-ray absorption fine structure (XANES) spectrum (SPring-8 BL37 XU beam line). From the ratio (a / b) of the maximum absorbance (a) around 11.57 keV to the minimum absorbance (b) around 11.57 keV appearing in the XANES spectrum, the ratio of the tetravalent complex contained in the oxidized micelle was calculated.

  The results were as shown in FIG. As shown in the upper right panel of FIG. 1, the ratio of a to b (a / b) can be used to calculate the degree of oxidation of platinum. When the ratio was calculated, as shown in FIG. 1 upper right panel, it was found that platinum was oxidized from divalent to tetravalent in both the cisplatin-containing micelle and the dahaplatin-containing micelle by hydrogen peroxide treatment. Further, as shown in the lower panel of FIG. 1, it was found that about 60% of the contained platinum was oxidized to tetravalent in O-DACHPt / m.

Example 2 Evaluation of Stability of Oxidized Micelle In this example, the stability of the oxidized micelle obtained by Example 1 was evaluated.

Dissociation behavior of oxidized micelles under physiological conditions Oxidized micelles (concentration 0.050 based on platinum complex) in phosphate buffered saline (10 mM PB + 150 mM NaCl, pH 7.4, 37 ° C) mg / ml) was incubated, and the scattered light intensity (632.8 nm, 90 °, 37 ° C.) of the solution was measured continuously. The scattered light intensity at each time obtained was normalized to the scattered light intensity at the start of the experiment and compared. In the case of oxidized micelles encapsulating cisplatin, the concentration of oxidized micelles was 0.040 mg / ml based on the platinum complex. The results were as shown in FIG.

  As shown in FIG. 2, in the micelles not subjected to the oxidation treatment (Normal), it is understood that the relative scattered light intensity (SLI) decreases with the passage of time, and the micelles are dissociated. There was no observed dissociation with time. From this, it became clear that the dissociation of the transition metal complex micelle can be almost completely suppressed by the oxidation treatment.

Release behavior of platinum complex of oxidized micelle under physiological conditions: 1 ml of oxidized micelle dialyzed in 50 ml of phosphate buffered saline (10 mM PB + 150 mM NaCl, pH 7.4, 37 ° C) (micelle in dialysis membrane Initial concentration was 0.25 mg / ml based on the platinum complex), and the amount of platinum complex released to the external solution was analyzed by ICP-MS. In the case of oxidized micelles encapsulating cisplatin, the initial concentration of micelles in the dialysis membrane was 0.50 mg / ml based on the platinum complex.
Behavior of oxidized micelles under reducing conditions Glutathione (GSH) or sodium ascorbate (NaAsc) after a certain time (0 h, 72 h, 240 h) against the same phosphate buffered saline as above Was added to reduce platinum, and the dissociation behavior and platinum complex release behavior of micelles were investigated. The concentration of reducing agent added was 5 mM, which is equivalent to that in cancer cells. The results were as shown in FIGS. 3 and 4.

  As shown in FIG. 3, the oxidized micelles release a platinum complex into solution with the addition of glutathione (FIG. 3 left) while the micelles themselves dissociate with the addition of glutathione (FIG. 3 right) It became. This suggests that oxidized micelles are stable in plasma and can release platinum complexes as soon as they enter cancer cells.

  Also, as shown in FIG. 4, oxidized micelles release a platinum complex into solution with the addition of sodium ascorbate (VC) (FIG. 4 right) while the micelle itself is sodium ascorbate (VC). It became clear that it dissociated with addition (FIG. 4 left). This suggests that oxidized micelles are stable in plasma and release of platinum complexes upon addition of ascorbic acid.

  As a result of this, as the oxidation number of the transition metal or amphoteric metal increases, the ligand exchange reaction of the complex becomes slower and the coordination bond between the transition metal or amphoteric metal and the polymer constituting the micelle is stabilized. Was considered to be a cause. On the other hand, the resulting oxidized micelles may be reduced in a reducing environment to release the destabilized, encapsulated drug from the micelles. In vivo, the inside of cancer cells has a reducing environment that is sufficient to destabilize oxidized micelles. Therefore, it is considered that micelles encapsulating an oxidized transition metal or amphoteric metal according to the present invention provide a drug delivery system that releases the encapsulated drug in cancer cells.

  Next, stability in a buffer solution or salt solution in micelles in which an oxidized copper complex, specifically a copper (II) sulfate complex or an iron (III) sulfate complex, is changed to a platinum complex, and The effect of sodium ascorbate reduction was investigated. HEPES buffer (10 mM) was used as a buffer solution, and 150 mM NaCl solution was used as a salt solution. Copper (II) sulfate complex-containing micelles or iron (III) complex-encapsulating micelles were prepared as follows.

The copper (II) sulfate complex-containing micelles are prepared by mixing 5 mM copper (II) sulfate and PEG-PGlu synthesized in Example 1 in an aqueous solution so that the concentration is [Cu] = [Glu] = 5 mM. It was prepared by reacting at room temperature for 24 hours.
Ferric sulfate (III) complex-encapsulating micelles mix ferric sulfate and PEG-PGlu in an aqueous solution so that the concentration becomes [Fe] = 2 mM, [Glu] = 5 mM, and allow 24 hours at room temperature It was prepared by reaction. As another report, iron (II) sulfate and PEG-PGlu are mixed in an aqueous solution so that the concentration becomes [Fe] = 2 mM, [Glu] = 5 mM, and reacted at room temperature for 24 hours It was prepared by adding 100 equivalents of hydrogen peroxide solution to promote oxidation.

  The stability of the micelles was estimated by the scattered light intensity as described above. After 24 hours, sodium ascorbate (VC) was added to reduce the copper sulfate complex encapsulated in the micelles. The results were as shown in Figure 4-2. As shown in Figure 4-2, oxidized copper (II) complex-containing micelles are stable in HEPES buffer solution and NaCl solution, and reduction of the complex by sodium ascorbate addition destabilizes the micelle and collapses the micelles. It was found that (ie, the internal complex was released). From these results, it was revealed that the copper complex micelles have the property of being stabilized in solution by being converted to an oxidized form, destabilizing in solution by being reduced, and releasing the content of the copper complex. . Also, the oxidized iron complex-containing micelles prepared by the above two methods were also more stable in solution than the reduced form.

  These results show that the relative stability of transition metal or amphoteric metal complex micelles is changed by relatively increasing or decreasing the oxidation number.

  Furthermore, the dissociation rates of micelles were compared between glutathione and ascorbic acid. The results were as shown in FIG.

  As shown in FIG. 5, with regard to the dissociation rate of the oxidized micelles in solution, the dissociation rate after adding ascorbic acid was faster than the dissociation rate after adding glutathione. Ascorbic acid is considered to be smaller in reducing power than glutathione, so it was surprising that the dissociation rate of oxidized micelles by ascorbic acid with small reducing power was faster than that by glutathione.

Example 3 Evaluation of Retaining Properties of Oxidized Micelles in Blood In this example, the retentivity of oxidized micelles in blood was evaluated in vivo.

  Oxidized micelles are intravenously administered at a dose of 100 μg / mouse to BALB / c mice (6 weeks old, n = 3, female, Charles River), and blood is collected after 24 hours and 48 hours, Plasma was obtained by centrifugation. A portion of the obtained plasma sample was subjected to heat treatment with concentrated nitric acid, and the amount of platinum complex was quantified by ICP-MS. Moreover, after freeze-drying a part of the plasma sample, XANES spectra were measured to analyze the oxidation number of platinum atoms. The results were as shown in FIG.

As shown in FIG. 6, 49% of the administered micelles were detected in the blood even after 24 hours of administration, and 33% of the administered micelles were detected in blood even after 48 hours of administration. In addition, when the valence number of the platinum complex was evaluated, as shown in FIG. 6, 59% of the micelles were 28% even after 24 hours relative to oxidized micelles (that is, contained Pt (IV)). Remained as oxidized micelles.
According to H. Cabral et al., J. Control. Release 101, 223, (2005), reduced dahaplatin micelles remain less than 20% at 24 hours after administration and less than 8% at 48 hours after administration In consideration of not performing (see FIG. 6), the oxidized micelles show high blood stability even in vivo.

Example 4: Antitumor effect of oxidized micelle In this example, the antitumor effect of the oxidized micelle obtained in the above example was confirmed. Also in this example, after administration of the oxidized micelle obtained in the above example, the antitumor effect was confirmed when ascorbic acid was administered to dissociate the micelle.

1 × 10 ⁶ C26 cells were implanted subcutaneously in BALB / c mice. One week later, PBS (control), 15 mg / kg of dahaplatin micelles and 50 mg / kg of oxidized dahaplatin micelles were administered through the tail vein, and changes in tumor volume were followed every two days. In addition, in order to evaluate the effect of high concentration vitamin C, 4 g / kg of sodium ascorbate was intraperitoneally administered on the second day after drug administration. C26 cells were cancer cells provided by the National Cancer Center, and were cultured in RPMI 1640 medium containing 10% FBS and 100 U / ml penicillin-streptomycin under conditions of 5% CO ₂ and 37 ° C. The results were as shown in FIG.

  As shown in panel A of FIG. 7, administration of 15 mg / kg resulted in a significant change in the body weight of mice with dahaplatin micelles, and died at 8 days, but administration of 50 mg / kg with oxidized dahaplatin micelles But weight change was small. In addition, as shown in panel B of FIG. 7, it was found that the anti-tumor effect was larger when the oxidized dahaplatin micelle was administered than the dahaplatin micelle. In particular, oxidized dahaplatin micelles were able to reduce tumor volume. Next, it was also revealed that in the group where ascorbic acid was administered following oxidized dahaplatin micelles, this antitumor effect was enhanced.

2 × 10 ⁵ C26 cells were implanted subcutaneously into BALB / c mice (6 weeks old; female). Five days later, PBS (control), oxaliplatin 15 mg / kg, dahaplatin micelles 10 mg / kg, oxidized dahaplatin micelles 10 mg / kg, oxidized dahaplatin micelles 20 mg / kg are administered from the tail vein, I followed the change in volume. Mice with an average tumor diameter greater than 15 mm were euthanized at that time. C26 cells were cancer cells provided by the National Cancer Center and were cultured at 37 ° C. in RPMI 1640 medium containing 10% FBS and 100 U / ml penicillin-streptomycin until subcutaneous implantation. . The results were as shown in FIG. 7-2.

As shown in the upper panel of FIG. 7-2, oxidized dahaplatin micelles (O-DACHPt / m) have a more pronounced antitumor effect than equivalent amounts of reduced dahaplatin micelles (DACHPt / m), It strongly suppressed the increase of relative tumor volume. Also, the strength of the effect was dose dependent. As shown in the lower panel of FIG. 7-2, oxidized dahaplatin micelles had less or no effect on the body weight of mice compared to oxaliplatin and reduced dahaplatin micelles.
Plasma GOT and GPT concentrations were determined by standard methods. The results are as shown in FIG. 7-3. As shown in FIG. 7-3, oxidized dahaplatin micelles showed lower GOT concentrations and lower GPT concentrations compared to reduced dahaplatin micelles. This indicates that oxidized platinum micelles have almost no hepatotoxicity.

  Next, the bone marrow toxicity of oxidized platinum micelles was also confirmed. PBS (control), oxaliplatin, dahaplatin micelles, and oxidized dahaplatin micelles were administered to BALB / c mice (6 weeks old; female) from 10 mg / kg tail vein respectively. Seven days later, blood was collected, and white blood cell count, red blood cell count, and platelet count were calculated by a hemocytometer. The results were as shown in FIG. 7-4. As shown in FIG. 7-4, in the case of oxidized dahaplatin micelles, no significant decrease by administration was confirmed for any of white blood cells, red blood cells and platelets. This result was in contrast to oxaliplatin and reduced dahaplatin micelles, which affect them by administration (see FIG. 7-4). The results show that oxidized platinum micelles have almost no bone marrow toxicity.

  Furthermore, the organ accumulation of oxidized platinum micelles was examined. Specifically, to a BALB / c mouse (6 weeks old; female), dahaplatin micelles or oxidized dahaplatin micelles were administered from the tail vein at a dose of 5 mg / kg. After 24 hours, 48 hours, 72 hours and 96 hours, blood, tumor, liver, spleen and kidney were collected, and blood was obtained by centrifugation to obtain plasma. The obtained sample was heat-treated with concentrated nitric acid, and the amount of platinum complex was quantified by ICP-MS. The results were as shown in FIGS. 7-5 to 7-8.

As shown in the left panel of FIG. 7-5, oxidized dahaplatin micelles were shown to be significantly higher in blood retention after intravenous administration as compared to reduced dahaplatin micelles. In addition, as shown in the right panel of FIG. 7-5, oxidized dahaplatin micelles maintain high accumulation in the tumor regardless of the passage of time, and this is a reduced dahaplatin micelle and the accumulated amount over time. This is in contrast to the large decrease.
As shown in FIG. 7-6, the accumulation amount by organ was significantly lower in oxidized liver, spleen, and kidney respectively than oxidized dahaplatin micelles than reduced dahaplatin micelles. Also, in FIGS. 7-7, these accumulations were expressed as tumor / liver, tumor / spleen, and tumor / kidney ratios. As a result, it was revealed that oxidized dahaplatin micelles show significantly higher tumor accumulation selectivity than reduced dahaplatin micelles. In Figures 7-8, the 96 hour area under the curve (AUC) of the tumor / liver, tumor / spleen, and tumor / renal ratios are presented. As shown in FIG. 7-8, it was revealed that oxidized dahaplatin micelles have a higher tumor / organ accumulation ratio than reduced dahaplatin micelles.

  Thus, oxidized platinum micelles have high stability in vivo. This high stability was considered to be due to the decreased accumulation in organs such as liver and kidney, that is, the decreased clearance contributed to the accumulation selectivity to the tumor. That is, micelles are less likely to be taken up by normal tissues, and exhibit accumulation selectivity to tumors by the EPR effect. On the other hand, when the micelle is broken, the anticancer drug is released into the blood, resulting in the reduction of the selectivity. If the micelles were stable in blood, it was thought that the concentration of free anti-cancer drug released into the blood decreased, nonspecific accumulation in non-tumor was suppressed, and as a result, tumor selectivity was improved. .

  Also, due to its stability, oxidized dahaplatin micelles can be administered in large amounts as the maximum tolerated dose (MTD) increases. This makes it possible to obtain low toxicity and high antitumor effects. By administering ascorbic acid, it is considered that oxidized dahaplatin micelles are dissociated in the body and dahaplatin is released. As a result, by using ascorbic acid in combination, a new controlled release technology has been established in which oxidized micelles stably staying in the blood are disrupted to exert an antitumor effect.

Example 5: Evaluation of stability of heat-treated micelles In this example, the stability of the heat-treated micelles (I) obtained above was evaluated in the same manner as in Example 2.

  The micelles used were micelles heated at 70 ° C. for 24 hours. The results were as shown in FIG. As shown in FIG. 8, the heat treated micelles had improved stability compared to the micelles that were not heat treated. The stabilizing effect was also confirmed in the heat-treated micelle (II).

  Next, for micelles subjected to heat treatment at various temperatures, the particle size, polydispersity (pdl) and scattered light intensity were measured in the same manner as in Example 1. The results are as shown in FIG. As shown in FIG. 9, the particle size increased as the heating temperature increased. The degree of polydispersity was not significantly changed by the increase of temperature. The scattered light intensity increased as the heating temperature increased. However, all measured values showed numerical values significantly different from other temperatures when heated at 90 ° C. It was considered that heating at 90 ° C. might cause aggregation and the like and the generation of abnormal micelles.

The amount of incorporated platinum ([Pt] / [Glu]) was measured in the same manner as in Example 1. The results were as shown in FIG. As shown in panel A of FIG. 10, the amount of incorporated platinum of the heat-treated micelle (I) obtained showed a tendency to increase as the heating temperature was increased. This indicates that the heat treatment increased the coordination of platinum to the carboxylate which is the side chain of glutamic acid. However, at the time of heating at 90 ° C., the amount of contained platinum showed a value which was significantly different from the others.
Confirming the efficiency (yield) of incorporation of Pt into micelles, the incorporation yield of Pt decreased at 80 ° C. and 90 ° C., as shown in panel B of FIG. Also, as shown in panel C of FIG. 10, the incorporation yield of glutamate polymer into micelles was greatly reduced at 80 ° C. and 90 ° C.
Furthermore, also in the cisplatin-incorporated micelles, the incorporated platinum amount ([Pt] / [Glu]) of heat-treated micelles (I), the incorporation yield of Pt into micelles, and the incorporation yield of glutamate polymer into micelles were measured. The results were as shown in FIG. As shown in FIG. 10-2, the amount of contained platinum showed a tendency to increase as the heating temperature increased. Further, as shown in FIG. 10-2, the incorporation yield of Pt into micelles also tended to increase as the heating temperature was increased. In addition, the incorporation yield of polymer into micelles decreased slightly at 70 ° C., but was generally constant regardless of temperature.

  Furthermore, the stability of heat-treated micelle (I) in phosphate buffered saline was evaluated under the same conditions as in Example 2. The results were as shown in FIG. As shown in FIG. 11, the heat-treated micelle (I) showed a tendency to become stable when the temperature was high in the temperature range of 25 ° C. to 70 ° C. The effect of improving the stability was limited at 90 ° C. as compared to micelles formed at 25 ° C. or 37 ° C. In particular, the stabilizing effect of micelles by heat treatment at 50 ° C. to 80 ° C. was remarkable.

  Next, the influence of heating time on the stability of heat-treated micelles was investigated. In the above, micelles heated for 24 hours were used, but the stability of micelles heated for 3 hours, 6 hours, or 9 hours was evaluated in the same manner as in Example 2. The results were as shown in FIG. 11-2. As shown in FIG. 11-2, it became clear that the stability in the solution was improved depending on the heating time.

  In addition, the solution stability of the heated cisplatin-containing micelle obtained in Example 1 was evaluated. Specifically, heating was carried out at 25 ° C., 37 ° C., 50 ° C. or 70 ° C. for 24 hours, respectively, and the stability in the solution was evaluated by the method described in Example 2. The results were as shown in FIG. 11-3. As shown in FIG. 11-3, heating at 70 ° C. was effective for stabilization of micelles even for cisplatin encapsulated micelles.

  Furthermore, as in Example 2, the release behavior of platinum from heat treated micelle (I) in phosphate buffered saline (pH 5.5 and pH 7.4) was examined. The results were as shown in FIG. As shown in FIG. 12, it was found that the heat-treated micelle (I) had higher stability of the micelle as the heat-treatment temperature was higher, and the release of Pt was suppressed. On the other hand, as shown in FIG. 12 below, heat-treated micelle (I) prepared by heating at 70 ° C. in particular has the property of being more likely to release Pt under an environment of pH 5.5. It became clear. This indicates that the heat-treated micelle (I) prepared by heating at 70 ° C. is stable in blood (pH 7.4) and has a tendency to release platinum in late endosomes when taken into cells. It is shown that the drug delivery system is excellent as a drug delivery system in that it does not release the drug in blood but releases the drug in cells.

  Next, the effect of pH on the stability of heated cisplatin-encapsulating micelles was confirmed. The results were as described in Figure 12-2. As shown in FIG. 12-2, it was confirmed that the higher the heating temperature, the slower the release rate of cisplatin from the micelles. Further, as shown in FIG. 12-2, according to the Pt release ratio of pH 5.5 / pH 7.4, it was found that this ratio is particularly large when the heating temperature is 50 ° C.

  Thus, the heat-treated platinum complex micelle is suitable as a drug that improves the amount of ligand exchange with the polymer, stabilizes the micelle and reduces cytotoxicity, while being easy to release in the intracellular environment It became clear that it has a property.

  Furthermore, the relationship between the heat treatment and the maximum tolerated dose (MTD) was investigated. For the BALB / c mice (6 weeks old; female), dahaplatin micelles prepared at 37 ° C, dahaplatin micelles prepared at 50 ° C, dahaplatin micelles prepared at 70 ° C are administered from the tail vein, The weight change was followed to determine the maximum tolerated dose. The results are as shown in FIG. FIG. 14 shows that dead mice show weight until death, and non-death mice show weight change after administration. As shown in FIG. 14, the MTD of dahaplatin micelles prepared at 37 ° C. is at least 12 mg / kg, and the MTD of dahaplatin micelles prepared at 50 ° C. is at least 15 mg / kg, daha prepared at 70 ° C. The MTD of platin micelles was shown to be at least 20 mg / kg. When the weight change in MTD of dahaplatin micelles prepared at each temperature is superimposed and displayed, the transition of the weight change is almost the same (see FIG. 14).

Furthermore, dahaplatin micelles prepared at 37 ° C., dahaplatin micelles prepared at 50 ° C., dahaplatin micelles prepared at 70 ° C. were administered from the tail vein, and the inhibitory effect on the increase of the transplanted tumor was examined. Specifically, 2 × 10 ⁵ C26 cells were subcutaneously implanted into BALB / c mice (6 weeks old; female). After 5 days 3 times every 2 days, PBS (control), 8 mg / kg of oxaliplatin (oxaliplatin), prepared at 37 ° C. dahaplatin micelles (H37 / m) 3 mg / kg, prepared at 50 ° C. dahaplatin Administration of 3 mg / kg of micelles (H50 / m), 3 mg / kg of dahaplatin micelles prepared at 70 ° C, 4 mg / kg of dahaplatin micelles (H70 / m) prepared at 70 ° C from the tail vein Follow the change of Mice with an average tumor diameter greater than 15 mm were euthanized at that time. C26 cells were cancer cells provided by the National Cancer Center, and were cultured in RPMI 1640 medium containing 10% FBS and 100 U / ml penicillin-streptomycin under conditions of 5% CO ₂ and 37 ° C. The results were as shown in FIGS.

As shown in FIG. 15, the dahaplatin micelles prepared at 70 ° C. have less effect on the body weight of mice than the micelles prepared at 37 ° C. or 50 ° C., and the antitumor effect is 4 mg / kg. It was the highest with micelles prepared at 70 ° C that could be administered. In particular, for the dahaplatin micelles prepared at 70 ° C, the MTD reaches 4 mg / kg, which is higher than 3 mg / kg, which is the MTD for dahaplatin micelles prepared at 37 ° C or 50 ° C, resulting in the strongest antitumor effect I was able to get The MTD in FIG. 15 is the MTD in the administration method administered three times every two days, and shows a value different from the MTD in FIG. 14 in a single administration.
FIG. 16 shows the Kaplan-Meier curve of the above experiment. As shown in FIG. 16, the number of dead mice was the lowest in the dahaplatin micelles prepared at 70 ° C., and the tumors completely disappeared in 2 out of 8 mice.
In this experiment, hepatotoxicity was estimated by measuring plasma GOT and GPT concentrations. Then, the dahaplatin micelles prepared at 50 ° C. and the dahaplatin micelles prepared at 70 ° C. had lower hepatotoxicity and the same level as PBS-administered mice than the dahaplatin micelles prepared at 37 ° C.

Example 6 Stability of Oxidized Heat-Treated Micelles In this example, the stability of micelles subjected to both oxidation treatment and heat treatment was evaluated.

  Micelles were made as described in Example 1. Thereafter, heat treatment and oxidation treatment of the obtained micelles (DACHPt / m) were performed as follows. Oxidized DACHPt / m (O-DACHPt / m) was obtained by mixing micelles and 100 equivalents of hydrogen peroxide and reacting at room temperature for 24 hours. The heat-treated micelle (I) (H-DACHPt / m) was obtained by heating the micelles at 70 ° C. for 24 hours. The heat treated and oxidized treated micelles (HO-DACHPt / m) were obtained by treating the micelles at 70 ° C. for 24 hours, then mixing with 100 equivalents of hydrogen peroxide and reacting at room temperature for 24 hours. Heat treated micelles after oxidation treatment (OH-DACHPt / m) were obtained by mixing the micelles and 100 equivalents of hydrogen peroxide, reacting for 24 hours, and then treating at 70 ° C. for 24 hours.

  The stability of the micelles was assessed by scattered light intensity in phosphate buffered saline as described in Example 1. The scattered light intensity was measured over time by incubating 0.050 mg / mL based on the platinum complex, and the scattered light intensity at the start of the measurement was standardized as 1. Seventy two hours after the start of measurement, 5 mM ascorbic acid was added. The results were as shown in FIG.

As shown in FIG. 13, micelles (OH-DACHPt / m) subjected to heat treatment following oxidation treatment are oxidized micelles (O-DACHPt / m), heat-treated micelles (I) (H-DACHPt / m) And heat treatment followed by oxidation treatment (HO-DACHPt / m) showed higher stability.
In addition, after the addition of ascorbic acid, the property of releasing Pt simultaneously with the addition was observed. However, the heat-treated OH-DACHPt / m showed a more stable micelle structure.

  From the results of this example, it is clear that performing both the heat treatment and the oxidation treatment shows a higher stabilization effect of micelles than performing each alone. In particular, it was revealed that heat treatment was carried out following oxidation treatment to show a high stabilization effect of micelles.

Claims (20)

  A transition metal-containing micelle comprising a block copolymer of a hydrophilic polymer and an anionic polymer and a complex of a transition metal, wherein the transition metal is in an oxidized form.   The micelle according to claim 1, wherein the transition metal is platinum or copper.   3. The micelle according to claim 1 or 2, wherein the hydrophilic polymer is polyethylene glycol and the anionic polymer is an anionic amino acid polymer.   The micelle according to claim 3, wherein the anionic amino acid polymer performs ligand exchange with the ligand of the transition metal complex as a monomer unit to form a coordination bond with the transition metal.   The micelle according to claim 3, wherein the anionic amino acid polymer comprises glutamic acid as a monomer unit.   The micelle according to claim 1, wherein the hydrophilic polymer is polyethylene glycol, the anionic polymer is polyglutamic acid or polyaspartic acid, and the transition metal complex comprises oxidized platinum or oxidized copper.   Pharmaceutical composition containing the micelle as described in any one of Claims 1-6.   The pharmaceutical composition according to claim 7 for use in combination with a reducing agent.   A combined medicament of a reducing agent and the pharmaceutical composition according to claim 7.   The pharmaceutical composition according to claim 8 or the combination drug according to claim 9, wherein the reducing agent is ascorbic acid or a pharmaceutically acceptable salt thereof.   A pharmaceutical composition for use in treating cancer, comprising ascorbic acid or a pharmaceutically acceptable salt thereof, for use in combination with the pharmaceutical composition according to any one of claims 1 to 8, Pharmaceutical composition.   A transition metal-containing micelle comprising a block copolymer of a hydrophilic polymer and an anionic polymer and a complex of a transition metal, wherein the micelle is heat-treated at 40 ° C to 85 ° C.   13. The micelle according to claim 12, wherein the transition metal is platinum or copper.   14. The micelle according to claim 12 or 13, wherein the hydrophilic polymer is polyethylene glycol and the anionic polymer is an anionic amino acid polymer.   15. The micelle according to claim 14, wherein the anionic amino acid polymer performs ligand exchange with a ligand of the transition metal complex as a monomer unit to form a coordination bond with the transition metal.   15. The micelle according to claim 14, wherein the anionic amino acid polymer comprises glutamic acid as monomer unit.   13. The micelle according to claim 12, wherein the hydrophilic polymer is polyethylene glycol, the anionic polymer is polyglutamic acid or polyaspartic acid, and the transition metal complex comprises oxidized platinum or oxidized copper.   A pharmaceutical composition comprising the micelle according to any one of claims 12-17.   A transition metal or amphoteric metal containing a block copolymer of a hydrophilic polymer and a polymer containing a monomer unit having a metal coordinating functional group in a side chain as a monomer unit and a complex of a transition metal or an amphoteric metal An incorporated micelle, wherein the transition metal or the amphoteric metal is in an oxidized form.   A transition metal or amphoteric metal containing a block copolymer of a hydrophilic polymer and a polymer containing a monomer unit having a metal coordinating functional group in a side chain as a monomer unit and a complex of a transition metal or an amphoteric metal An incorporated micelle, which is heat-treated at a temperature of 40 ° C. to 85 ° C. during or after formation of the micelle.

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