Hydrophilic-hydrophobic copolymers carrying dichloroacetic acid on side chain and medical use thereof

Abstract

The present invention discloses copolymers which are represented by general formulae (I), (II-a), and (II-b). (The variable groups or moieties in the formulae are defined in the specification. In particular, R1 or R2 includes a dichloroacetyl group.) These copolymers or polymeric micelles which are formed from these copolymers in an aqueous medium have a tumor-specific radiation enhancement effect and enhance the therapeutic effect of radiation therapy on tumors. Consequently, the present invention provides a medical use of these copolymers or polymeric micelles for exerting such an effect.

Description

Hydrophilic-hydrophobic copolymers bearing dichloroacetic acid in the side chains and their medical applications

[0001] The present invention relates to a hydrophilic-hydrophobic copolymer having a block having dichloroacetic acid in the hydrophobic segment side chain via an ester or amide bond and poly(ethylene glycol) as a hydrophilic segment block, self-assembled nanoparticles (or polymeric micelles) of the copolymer, and medical uses of the copolymer or nanoparticles, more specifically, uses as a tumor-specific radiosensitizer and a pharmaceutical preparation for enhancing the therapeutic effect of tumor radiotherapy when used in combination.

[0002] Cancer cells are increasingly dependent on glycolysis for ATP production, even in the presence of oxygen (the Warburg effect), and the application of glycolysis inhibitors in cancer therapy has attracted interest. Dichloroacetic acid (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), has undergone clinical trials as a glycolysis inhibitor, but has not yet been approved as a cancer treatment. This is due in part to DCA's inability to demonstrate the expected antitumor effects in vitro and in vivo. Therefore, attempts have been made to combine DCA with certain chemotherapeutic agents or radiation therapy in cancer treatment. These studies have demonstrated the radiosensitization of certain tumor cells by DCA (see F. Zwicker et al., Strahlenther Onkol. 2013; 189(8): 684-692 (hereinafter referred to as Non-Patent Document 1)). Incidentally, the reason why DCA has not been approved as a cancer treatment drug as mentioned above is that, due to its simple structure, it is metabolized quickly and does not accumulate sufficiently in solid tumors. Therefore, although it is effective in cell tests, it is not sufficiently effective in animal experiments. Furthermore, there are problems such as the toxicity of DCA itself having an effect when the dosage is increased.

[0003] As a measure to solve such problems, if it can be effectively applied to DCA, one possible approach would be to modify DCA to exhibit an effect of enhancing vascular permeability and retention (EPR effect) by covalently binding DCA to a certain polymer compound to form a polymer, as first reported by Maeda (see Hiroshi Maeda, Cancer Science, 2013; 104(7): 779-789 (hereinafter referred to as Non-Patent Document 2)).

[0004] On the other hand, some of the present inventors have previously demonstrated that by introducing a chain transfer group or the like to the end of poly(ethylene glycol) and radically polymerizing a short-chain vinyl fatty acid, short-chain fatty acids such as propionic acid and butyric acid are introduced via an ester bond to the side chain of a hydrophobic segment, and that the resulting core-shell nanoparticles function as drugs for treating diabetes, non-alcoholic steatohepatitis (NASH), liver fibrosis, and anticancer (see WO 2021 / 024906 A1 (hereinafter referred to as Patent Document 1)).

[0005] However, dichloroacetic acid esters are highly hydrolyzable and highly prone to chain transfer, unlike short-chain fatty acid esters such as propionic acid and butyric acid, and even if attempts were made to polymerize DCA using techniques similar to those described in Non-Patent Document 2 and Patent Document 1, it was difficult to synthesize the desired polymer compound.

[0006] Therefore, an object of the present invention is to provide an invention that effectively polymerizes dichloroacetic acid (DCA) while at the same time providing the resulting polymeric compound or any structure formed therefrom with properties that overcome or correct the drawbacks associated with the use of DCA itself, such as rapid metabolism and insufficient accumulation in solid tumors.

[0007] The present inventors have found that, rather than introducing a chain transfer group or the like to the end of poly(ethylene glycol) (PEG) and directly radically polymerizing a short-chain fatty acid vinyl, the desired polymer compound can be effectively obtained by hydrolyzing PEG-b-poly(short-chain fatty acid vinyl) obtained as described in Patent Document 1 and then exchanging it with a dichloroacetic acid ester. Furthermore, the nano-sized polymeric micelles or nanoparticles formed from the thus obtained polymer compound through self-assembly in an aqueous medium (e.g., water, an aqueous solution of a water-miscible organic solvent, etc.) eliminate or correct the above-mentioned drawbacks associated with the use of DCA itself and at least exhibit tumor-specific radiosensitivity. Alternatively, the present inventors have found that the desired polymer compound can be effectively obtained by reacting dichloroacetic anhydride with the side-chain amino groups (at the δ- or ε-position, respectively) of pre-prepared PEG-b-poly(ornithine) or PEG-b-poly(lysine) to introduce dichloroacetyl groups. Furthermore, it was found that polymeric micelles or nanoparticles formed from the polymer compound thus obtained also have properties substantially equivalent to those of the PEG-b-poly(short-chain fatty acid vinyl)-derived copolymer.

[0008] Therefore, the main subject matter or features disclosed in this specification can include the following:

[0009] Subject 1: A hydrophilic-hydrophobic copolymer comprising a block containing a poly(ethylene glycol) segment and a block containing a polymer segment carrying dichloroacetic acid in the side chain, the copolymer being represented by formula (I) or formula (II-a) or (II-b).

[0010] Formula (I): In the formula, A １ (i) unsubstituted or substituted C １ -C １２ The substituents when substituted are formyl, ａ R ｂ CH- (where R ａ and R ｂ is independent, C １ -C ４ Alkyloxy or R ａ and Rｂ Let's get together -OCH ２ CH ２ O-, -O(CH ２ ) ３ O- or -O(CH ２ ) ４ (ii) a group of the formula: is a group represented by the formula: １ teeth, or (CH ２ ) ｃ S, CO(CH ２ ) ｃ S, (CH ２ ) ｃ NH, (CH ２ ) ｃ R is selected from the group consisting of CO, CO, OCOO, and CONH, wherein b is an integer from 2 to 6, and each c is independently an integer from 1 to 5; １ is a hydrogen atom, C １ -C ６ Alkylcarbonyl or C(=O)CHCl ２ and each R １ may be different from each other, but 20% or more of m1 are C(=O)CHCl ２ Y is a hydrogen atom, SH, S(C=S)-Ph, S(=S)OCH ２ CH ３ , hydroxyl group, C １ -C ６ Alkyloxy group or aryl-C １ -C ２ an alkyloxy group; m1 is an integer of 5 to 1000; and n1 is an integer of 5 to 500.

[0011] Formula (II-a): In the formula, A ２ is A of formula (I). １ is the same as defined for L ２ａ is (CH ２ ) ａ -NH or (CH ２ ) ａ —O, where each a is independently an integer of 1 to 6; ２represents a hydrogen atom, benzyloxycarbonyl (Cbz), t-butoxycarbonyl, C １ -C ６ Alkylcarbonyl or C(=O)CHCl ２ and each R ２ may be different from each other, but 20% or more of m2a is C(=O)CHCl ２ and Za is H, C １ -C ２１ Alkylcarbonyl, substituted C １ -C ４ Alkylcarbonyl, unsubstituted or substituted C ３ -C ７ represents a cycloalkylcarbonyl, an unsubstituted or substituted arylcarbonyl, or an unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl, wherein the substituents, when substituted, are one or more C １ -C ４ m2a may be alkyloxy, phenyl, or a fluorine atom; m2a is an integer of 5 to 500; n2a is an integer of 5 to 500; and q is an integer of 1 or 2.

[0012] Formula (II-b): In the formula, A ２ is A of formula (I). １ is the same as defined for L ２ｂ is (CH ２ ) ｂ -C(=O), where b is an integer from 1 to 6; R ２ represents a hydrogen atom, benzyloxycarbonyl (Cbz), t-butoxycarbonyl, C １ -C ６ Alkylcarbonyl or C(=O)CHCl ２ and each R ２ may be different from each other, but 20% or more of m2b is C(=O)CHCl ２ Zb is OH, unsubstituted or substituted C １ -C ２１ Alkyloxy, unsubstituted or substituted C ３ -C ７represents cycloalkyloxy, unsubstituted or substituted aryloxy, or unsubstituted or substituted 5- or 6-membered heteroaryloxy, wherein the substituents, when substituted, are one or more C １ -C ４ m2b may be alkyloxy, phenyl, or a fluorine atom; m2b is an integer of 5 to 500; n2b is an integer of 5 to 500; and q is an integer of 1 or 2.

[0013] Subject 2: The copolymer according to Subject 1, wherein the copolymer is represented by formula (I):

[0014] Subject 3: The copolymer according to Subject 1, wherein the copolymer is represented by formula (II-a) or formula (II-b).

[0015] Subject 4: Nano-sized polymeric micelles formed in an aqueous medium from the copolymer described in Subject 1.

[0016] Subject 5: A tumor-specific radiosensitizer comprising, as an active ingredient, the copolymer described in Subject 1 or nano-sized polymeric micelles formed from the copolymer in an aqueous medium.

[0017] Subject 6: A pharmaceutical preparation for enhancing therapeutic effects in tumor radiation therapy by combined use, comprising as an active ingredient the copolymer described in Subject 1 or nano-sized polymeric micelles formed from said copolymer in an aqueous medium.

[0018] Subject 7: The copolymer described in Subject 1 or the polymeric micelle described in Subject 4 for use as a tumor-specific radiosensitizer.

[0019] Subject 8: The copolymer according to Subject 1 or the polymeric micelle according to Subject 4 for use in a pharmaceutical preparation for enhancing the therapeutic effect of tumor radiotherapy by combining the copolymer with the polymeric micelle.

[0020] Subject 9: A method for enhancing the therapeutic effect of a tumor, comprising administering to a patient or individual who is scheduled to undergo or is currently undergoing tumor radiation therapy an effective amount of the copolymer described in Subject 1 or nanosized polymeric micelles formed from said copolymer in an aqueous medium.

[0021] According to the present invention, there are provided copolymers represented by formula (I), (II-a), or (II-b), and nano-sized polymeric micelles formed therefrom, which can be dramatically more rapidly accumulated in tumors than by administration of dichloroacetic acid itself. The polymeric micelles accumulate in tumors upon administration, thereby specifically sensitizing tumors to radiation irradiated thereto (tumor-specific radiosensitization). Therefore, the combined use of the copolymers or nano-sized polymeric micelles derived therefrom in tumor radiotherapy can enhance the therapeutic efficacy of tumors. Furthermore, these copolymers and polymeric micelles have low toxicity and are amphiphilic, exhibiting hydrophilic-hydrophobic properties. Therefore, similar to hydrophilic-hydrophobic copolymers known in the art, they are also useful as carrier materials for encapsulating poorly water-soluble drugs and the like in polymeric micelles.

[0022] PEG-b-PVA (N1223) in Production Example 1 １ 1H-NMR spectrum of PEG-b-PVCl obtained in Production Example 2. ２ 1 is a size distribution diagram showing the results of dynamic light scattering measurement of polymeric micelles derived from Ac(N1228). ２ Ac(N1228) １ 1H-NMR spectrum of PEG-b-POrnOC(=O)CHCl obtained in Production Example 3. ２ 1 is a size distribution diagram showing the results of dynamic light scattering measurement of polymeric micelles derived from (N1224). ２ (N1224) １ 1 is a schematic diagram of the experimental schedule for the colony formation method in Test Example 1. In Test Example 1, EL-Nano ＤＣＡ , AL-Nano ＤＣＡ 1 is a graph showing the effect of DCA on radiosensitivity and a numerical table summarizing the sensitization. 2 is a schematic diagram of the schedule of the animal experiment in Test Example 2(a). 3 is a graph showing the results of measuring the change in tumor volume associated with DCA alone or in combination with X-rays in Test Example 2(a), with the p-value after statistical significance test associated with the graph shown at the bottom. 4 is a graph showing the effect of AL-Nano in Test Example 2(a) on radiosensitivity and a numerical table summarizing the sensitization. ＤＣＡThe graph shows the results of measuring the change in tumor volume associated with the administration of EL-Nano alone or in combination with X-ray, and the p-value after statistical significance test associated with the graph is shown at the bottom. ＤＣＡ The graphs show the results of measuring the changes in tumor volume associated with the administration of DCA alone or in combination with X-ray, and the p-values ​​after statistical significance tests associated with the graphs are shown at the bottom. ＤＣＡ and EL-Nano ＤＣＡ 1 shows the survival curves of tumor-bearing mice treated with X-rays in combination with DCA, and the p-values ​​after statistical significance tests associated with the graphs are shown. This is a schematic diagram of the schedule of the animal experiment in Test Example 2(b). In Test Example 2(b), the survival curves of tumor-bearing mice treated with X-rays alone, DCA alone, and AL-Nano ＤＣＡ Alone and EL-Nano ＤＣＡ 1 is a graph showing the results of measuring the weight change of test animals after single administration. ＤＣＡ Fluorescently labeled EL-Nano localized in tumors after administration ＤＣＡ 1 is a photograph of a fluorescent microscope image of EL-Nano in Test Example 4. ＤＣＡ 1 is a graph showing the results of measuring the amount of intracellular ATP after administration and X-ray treatment. Detailed Description of the Invention and Best Mode for Carrying Out the Invention

[0023] Terms and the like used in connection with the subject matter or the present invention disclosed in this specification are used as having the meanings or contents commonly used in the art unless otherwise specified.

[0024] Certain terms and the like disclosed herein may be further explained below, either generally or specifically.

[0025] In this specification, a poly(ethylene glycol) segment or a polymer segment carrying dichloroacetic acid in a side chain is disclosed, but the terms "poly" and "polymer" used herein are used as concepts that include "oligo" and "oligomer," which are polymers formed by the bonding of a relatively small number of monomers.

[0026] In formula (I) or formula (II-a) or formula (II-b), the variable groups or moieties are defined, for example, C １ -C １２ Alkyl or C １ -C ２１ Alkylcarbonyl or C １ -C ４ Alkylcarbonyl or C １ -C ２１ The alkyl moiety in alkylcarbonyl and the like is, for example, C １ -C ２１ Alkyl includes straight chain, branched or cyclic alkyl having 1 to 21 carbon atoms, and may typically be, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, isooctyl, 2-ethylhexyl, nonyl, isononyl, decyl, isodecyl, undecyl, dodecyl, hexadecyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, cyclohexylmethyl, and the like.

[0027] Similarly, for example, the aryl moiety in arylcarbonyl or aryloxy has 6 to 30 carbon atoms, and specific examples include phenyl, tolyl, xylyl, ethylphenyl, naphthyl, anthryl, etc. The heteroaryl in heteroarylcarbonyl or heteroaryloxy may be, for example, pyridyl, pyrimidyl, furyl, tetrahydrofuryl, dioxolanyl, imidazolidyl, oxazolidyl, piperidyl, morpholinyl, etc.

[0028] In particular, R in formula (I) １ C included in the definition of １ -C ６The alkylcarbonyl in the short-chain fatty acid in the PEG-b-poly(short-chain fatty acid vinyl) obtained according to the description of Patent Document 1 is preferably a group derived from, for example, acetic acid, propionic acid, butyric acid (or butanoic acid), isobutyric acid, or isovaleric acid.

[0029] R in formula (I) or formula (II-a) or formula (II-b) １ or R ２ In relation to the definition of m1, m2a or m2b, 20% or more of m1 or m2a or m2b are C(=O)CHCl ２ However, the 20% or more may be 40% or more or 60% or more, respectively. ２ Each group other than the above may be present within the range that does not adversely affect the subject matter disclosed herein or the achievement of the object of the present invention.

[0030] L in formula (I) or formula (II-a) or formula (II-b) １ or L ２ａ Or L ２ｂ Regarding the definitions of m1 and m2a, when the overall structure of each copolymer differs depending on the direction in which each group is bonded in each formula, it is understood that m1 is incorporated into each formula in the direction of the bond. m1 is an integer of 5 to 1,000, but may be an integer of 10 to 500, or an integer of 20 to 250, or an integer of 50 to 160. m2a or m2b is an integer of 5 to 500, but may be an integer of 10 to 500, or an integer of 20 to 250, or an integer of 50 to 160.

[0031] On the other hand, n1 is an integer of 5 to 500, but may be an integer of 10 to 300, or an integer of 20 to 200, or an integer of 50 to 160, and n2a or n2b is an integer of 5 to 500, but may be an integer of 10 to 300, or an integer of 20 to 200, or an integer of 50 to 160.

[0032] The fourth aspect provides for nano-sized polymeric micelles formed in an aqueous medium from the copolymer described in the first aspect. In another embodiment, the nano-sized polymeric micelles can be defined, for example, as follows: A nano-sized polymeric micelle comprising the copolymer described in the first aspect, the polymeric micelle comprising a poly(ethylene glycol) segment in the shell of the polymeric micelle and a polymer segment carrying dichloroacetic acid in a side chain in the core of the polymeric micelle.

[0033] In this specification, nano-sized polymeric micelles and nanoparticles are used interchangeably, and the term "nano-sized" or "nano" used herein means that when dynamic light scattering (DLS) measurements of polymeric micelles or nanoparticles are performed in water, the average diameter is on the order of nanometers, and the average diameter is about 10 nm to about 2000 nm, or about 10 nm to about 500 nm, or about 25 nm to about 200 nm.

[0034] <Production of Copolymer Represented by Formula (I)> A hydrophilic-hydrophobic copolymer containing a block of a polymer segment having dichloroacetic acid in the hydrophobic segment side chain via an ester bond and a block of a hydrophilic segment containing poly(ethylene glycol), for example, a copolymer represented by formula (I), can be conveniently produced by a copolymer represented by formula (BC): In particular, R is a part of a block copolymer represented by -(C=O)R １ and R １ However, this can be carried out, for example, by treating a copolymer having alkyl groups having 1 to 6 carbon atoms under known hydrolysis conditions to convert all or part of the OR groups to OH groups, and then reacting the copolymer thus produced having OH groups with dichloroacetic anhydride to dichloroacetylate all or part of the OH groups.

[0035] <Preparation of Copolymer Represented by Formula (II-a) or Formula (II-b)> The hydrophilic-hydrophobic copolymer represented by formula (II-a), which comprises a block of a polymer segment having dichloroacetic acid in the hydrophobic segment side chain via an amide bond and a block of poly(ethylene glycol) as a hydrophilic segment, can be conveniently prepared by reacting a copolymer obtained based on the synthesis method of PEG-b-P(L-Orn) or PEG-b-P(L-Lys), which is a precursor for preparing a copolymer containing a poly(L-arginine) segment or a poly(L-lysine-Gua) segment (here, Gua means a guanidine group), described in WO 2016 / 167333 A1, with dichloroacetic anhydride to form a copolymer having an NH at the δ-position of Orn. ２ or ε-position NH of Lys ２ This can be carried out by dichloromethylating the compound.

[0036] Briefly, the precursor PEG-bP(L-Orn) or PEG-bP(L-Lys) can be prepared, for example, according to the following reaction scheme:

[0037] In the above formula, q is an integer of 1 or 2, Cbz represents a benzyloxycarbonyl protecting group for an amino group, and A and R ２ , m, and n are each A defined for formula (II-a). ２ , R ２ , m2a, n2a.

[0038] Alternatively, for example, the α-terminal of PEG represented by reaction scheme 1 has an oxy protected by a protecting group, and the ω-terminal oxy is connected to a linking group L in formula (II-a). ２ａ Equivalent to -(CH ２ ) ａ -NH ２ or -(CH ２ ) ａA compound having —OH (some of which are commercially available) is prepared, and a polymer having a repeating unit of m represented by 2, a carboxyl group at the C-terminus, and a Za group at the N-terminus (for example, a polymer obtained by ring-opening polymerization of the corresponding amino acid N-carboxyanhydride (NCA) using an initiator that is easily eliminated) is prepared. These are coupled in the presence of a suitable dehydrating condensing agent, and then, after optionally eliminating the Cbz group, a δ-NH group at the Orn is added. ２ or ε-position NH of Lys ２ It can also be produced by dichloromethylating

[0039] The copolymer represented by formula (II-b) is, for example, a copolymer of PEG represented by the above 1, in which the α-terminal thereof has an oxy protected by a protecting group, and the ω-terminal oxy is connected to the linking group L in formula (II-b). ２ｂ Equivalent to -(CH ２ ) ｂ A compound having —COOH is prepared, and a polymer having a repeating unit of m represented by 2, an N-terminal free amino group, and a C-terminal Zb is prepared. These are coupled in the presence of a suitable dehydration condensation agent, and then, after optionally removing the Cbz group, a δ-position NH of Orn is formed. ２ or ε-position NH of Lys ２ can be produced by dichloromethyloylating

[0040] <Formation of Polymeric Micelles or Nanoparticles> The copolymers represented by formula (I) or formula (II-a) or formula (II-b) disclosed herein are amphiphilic, and if necessary, after preparing an aqueous solution containing a water-soluble organic solvent, such as N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), unreacted raw materials and the like are removed from the reaction mixture. In order to associate and self-assemble a plurality of the copolymers, the mixture is dialyzed against water through a dialysis membrane with a certain molecular weight cut-off, thereby providing the desired nano-sized polymeric micelles or nanoparticles. The polymeric micelles or nanoparticles thus provided can be obtained as a separated solid by, for example, freeze-drying, centrifugation, or the like. When forming polymeric micelles from the copolymer represented by formula (II-a) or formula (II-b), free NH２ In the presence of the copolymer, some ingenuity is required to stably form polymeric micelles. For example, the pH of an aqueous solution containing the copolymer may be adjusted to an acidic level, or, as described in WO 2016 / 167333 A1, a polyanionic polymer selected from the group consisting of, but not limited to, polyanionic polysaccharides, polyanionic polypeptides, poly(acrylic acid), and poly(methacrylic acid) may be coexisted to form a polyion complex.

[0041] The copolymer represented by formula (II-a) or formula (II-b) is preferably produced using L-type Orn or Lys as a starting material, but may also be produced using a racemic mixture of L-type and D-type.

[0042] <Pharmaceutical Formulations> The nanoparticles prepared as described above can be used as active ingredients to prepare tumor-specific radiosensitizers. Because the nanoparticles can be dissolved or uniformly dispersed in aqueous media (including aqueous solutions that may contain physiological salts and pH adjusters, if necessary) and reconstituted with water, they can be provided as formulations in a variety of forms. Therefore, a wide variety of formulations can be prepared, including parenteral formulations (including subcutaneous administration, intravenous infusions, etc.) and various oral formulations. When preparing oral formulations or compositions, conventional pharmaceutical vehicles (e.g., water, glycols, oils, alcohols, flavorings, preservatives, colorings, etc.; for oral liquid formulations, e.g., suspensions, elixirs, and solutions) or carriers (e.g., starches, sugars, microcrystalline cellulose, diluents, granules, lubricants, binders, disintegrants, etc.; for oral solid formulations, e.g., powders, hard and soft capsules, and tablets) can be used. For example, when the oral dosage form is a tablet, it may contain binders (e.g., gum tragacanth, acacia, corn starch, or gelatin), excipients (e.g., disodium phosphate), disintegrants (e.g., corn starch, potato starch, alginic acid), lubricants (e.g., sodium stearate), sweeteners (e.g., sucrose, lactose, or saccharin).

[0043] The optimal dose of such pharmaceutical preparations varies depending on the disease to be treated and the administration method, so a uniform dose cannot be specified. However, the dose can be determined by a specialist based on data obtained through small-scale clinical trials, etc. For example, but not limited to, the nanoparticles may be contained as an active ingredient in an amount of at least 5% by weight. The proportion of the active ingredient in these preparations or compositions may vary and may conveniently be between about 30% and about 90% by weight based on the total weight of the preparation or composition. Since the nanoparticles may be effective over a wide range of administration doses, for example, a dose of about 0.1 to about 10,000 mg / day, preferably about 1 to about 1,000 mg / day, can be used for the treatment of an adult. The optimal dose depends on the desired therapy, the administration form, the subject to be treated, the subject's weight, and the experience of the attending physician.

[0044] <Medicinal Uses> As can be seen from the test examples described below, pharmaceutical preparations containing nanoparticles formed from the copolymers represented by Formula (I) or Formula (II-a) or (II-b) as an active ingredient can exert the aforementioned physiological activity of dichloroacetic acid itself in tumor tissues to which the nanoparticles are delivered or in the local living body of a mammal used in the test. Therefore, the patient or individual referred to in Subject 9 is not limited to humans but also includes non-human mammals, particularly pet animals. Furthermore, the tumor-specific sensitizers and pharmaceutical preparations described in Subjects 5 and 6, for example, are not limited to humans but also include non-human mammals as subjects of use.

[0045] In particular, according to Test Examples 1 and 2, the nanoparticles derived from the copolymer of formula (I) (containing an ester bond) are more likely to be derived from the copolymer of formula: PEG-b-POrn(-NH at the δ position)C(=O)Cl synthesized in Production Example 2. ２It can be understood that the nanoparticles derived from the latter formula exhibit a superior effect than those derived from the former formula. While not intending to limit the scope of the present invention by theory, it is presumed that this is because the former nanoparticles were used in the test example in a tumor in which inflow from broken blood vessels (EPR effect) is more likely to occur than the latter nanoparticles. Furthermore, without being bound by theory, it is presumed that in the test, the nanoparticles derived from the latter formula reached the tumor tissue before releasing DCA, where DCA was released by enzymes from cancer cells, such as matrix metalloproteinase. Thus, depending on the type of tumor, the EPR effect may be weak. In such cases, the time the nanoparticles remain stably in the bloodstream becomes important, and the amide bond may exhibit a superior effect.

[0046] Therefore, according to the present invention, there is provided a pharmaceutical preparation for enhancing the therapeutic effect of tumor radiation therapy by combining it with a tumor-specific sensitizer, the preparation comprising, as an active ingredient, the copolymer described in Subject 1 or the polymeric micelle described in Subject 4; in other words, a preparation comprising, as an active ingredient, nanoparticles derived from the copolymer represented by formula (I) or the copolymer represented by formula (II-a) or formula (II-b).

[0047] Furthermore, if DCA acts as a PDK inhibitor and shifts the energy metabolism of cancer from glycolysis to the mitochondrial electron transport system, it can be inferred that the nanoparticles (which can release DCA in vivo) can be used widely in other cancer treatments, not just in combination with radiation therapy. In fact, it has been reported that DCA in combination with cisplatin (see Cancer Lett. 2016; 371(1): 20-29) and in combination with 5-ALA photodynamic therapy (see PLoS One. 2018; 13(10): e0206182) demonstrates certain effects. It can be reasonably interpreted that, in these combinations, it would also be beneficial to use nanoparticles derived from the copolymers represented by Formula (I), (II-a), or (II-b) as the active ingredient in the combinations or photodynamic therapy, instead of DCA.

[0048] The subject matter disclosed in this specification or the present invention will be explained in more detail below by giving specific examples, but the subject matter disclosed in this specification or the present invention is not limited to these in any way.

[0049] Reference Production Example 1: Synthesis of poly(ethylene glycol)-b-poly(vinyl butyrate) (PEG-b-PVBu) The title copolymer was produced in the same manner as described in "Example 5" of Patent Document 1. The obtained copolymer is designated N1193. The PEG used in this production method had a MW of 5000. If necessary, the properties of the copolymer may be further described in Patent Document 1, for example, １ See the H NMR spectrum etc.

[0050] The following Production Examples 1 and 2 are each carried out according to the following simplified reaction scheme. <Simplified Reaction Scheme> In the above formula, R ０ are C(=O)R or a hydrogen atom, at least one of which is a hydrogen atom, and R １ is a hydrogen atom, C １ -C ６ or C(=O)CHCl ２ and each R １ may be different from each other, but at least one is C(=O)CHCl ２ m and n correspond to m1 and n1, respectively, as defined for formula (I).

[0051] Here, the term "simplicity" refers to, for example, the ω-terminal group (S(=S)OCH ２ CH ３ ) etc. are omitted.

[0052] Preparation Example 1: Synthesis of PEG-b-polyvinyl alcohol (PEG-b-PVA; N1223) 5 g of PEG-b-PVBu (N1193) was weighed into a 100 mL flask, and 50 mL of methanol and potassium carbonate (K ２ CO ３) was added and stirred at room temperature for 3 hours. After the reaction, the reaction solution was placed in a dialysis membrane tube (molecular weight cutoff MWCO = 3.5 kDa) and dialyzed against water to remove butyric acid, methanol, and calcium carbonate (for 1 day). The solution was concentrated to 60 mL and recovered by freeze-drying. The resulting PEG-b-PVA (N1223) １ The H-NMR spectrum is shown in FIG.

[0053] Preparation Example 2: PEG-b-poly(vinyl dichloroacetate) (PEG-b-PVCl ２ Synthesis of PEG-b-PVA (N1223) (N1223) 2 g and 50 mL of DMF were added to a 100 mL flask and dissolved. After that, 5 mL of dichloroacetic anhydride was added and the mixture was allowed to react at room temperature for 3 hours. The solution was then placed in a dialysis membrane tube (MWCO = 3.5 kDa) and dialyzed against water to purify the target copolymer and form polymeric micelles from the copolymer. The solution was freeze-dried to recover the resulting polymeric micelles derived from the copolymer. Figure 2 shows the resulting polymeric micelles (N1228-derived nanoparticles, hereinafter referred to as EL-Nano). ＤＣＡ The results of dynamic light scattering measurements of the recovered copolymer are shown in Figure 3. １ The H-NMR spectrum is shown.

[0054] Reference Production Example 2: Synthesis of poly(ethylene glycol)-b-poly(ornithine) (PEG-b-P(L-Orn)) The synthesis was carried out according to the method described in Examples 1 and 2 of WO2016 / 167333 A1 (N1182).

[0055] Preparation Example 3: Poly(ethylene glycol)-b-dichloromethyloylated poly(ornithine) (PEG-b-POrn(NH at the δ position)-C(═O)CHCl ２Synthesis of PEG-b-P(L-Orn) (N1182) (2.5 g) and 50 mL of DMF were added to a 100 mL flask and dissolved. After that, 5 mL of dichloroacetic anhydride was added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was then dialyzed (MWCO = 3.5 kDa) to obtain a purified solution. The solution was concentrated and freeze-dried to obtain polymeric micelles derived from the copolymer. The resulting polymeric micelles (N1224-derived nanoparticles, hereinafter referred to as AL-Nano) ＤＣＡ The results of dynamic light scattering measurement of the recovered copolymer are shown in Figure 4. １ The H-NMR spectrum is shown in FIG.

[0056] Test Example 1: Confirmation of radiosensitizing effect In this test, EL-Nano ＤＣＡ and AL-Nano ＤＣＡ To verify the radiosensitizing effect of each compound, a colony formation assay was performed using spheroids. For comparison, a drug-free group and a dichloroacetic acid (DCA)-treated group were also prepared.

[0057] SCCVII cells derived from mouse squamous cell carcinoma were seeded at 5,000 cells / well in a U-shaped 96-well plate and incubated at 4°C and 5% CO ２ The cells were cultured under the conditions for 3 days to form spheroids. ＤＣＡ , AL-Nano ＤＣＡ 3 mM of each was added and X-ray irradiation was performed. After dispersing the spheroids using Accumax, an appropriate number were seeded on a 60 mm plastic dish and cultured for 5 days. The cells were fixed with methanol for 10 minutes and stained with 2% Giemsa stain. Colonies containing 50 or more cells were counted, and the cell survival rate was calculated by standardizing it with the colony formation rate of the non-irradiated control group. An outline of the experimental schedule for the colony formation method is shown in Figure 6. The change in cell survival rate relative to the irradiation dose is shown in Figure 7. The cell survival rate was as follows: non-drug administration group > DCA > AL-Nano ＤＣＡ >EL-Nano ＤＣＡ The radiosensitization ratio compared to the non-administered group was 1.00 for DCA and 1.00 for AL-Nano. ＤＣＡ 1.04, EL-Nano ＤＣＡ It becomes 1.18, and EL-Nano ＤＣＡThe sensitization ratio was calculated based on the dose (D １０ The calculation was based on the value.

[0058] Test Example 2: In vivo radiosensitization (a) An animal experiment was carried out using mice (C3H / HeJYok S1c, male, 6 weeks old). 1.0 × 10 SCCVII cells were implanted into the right thigh of the mouse. ５ The animals were randomly divided into the following eight groups (n=5), and animal experiments were conducted according to the schedule shown in Figure 8. Tumor volumes were measured daily during the experiment. (1) Non-drug administration group (2) DCA (200 mg / kg) administration group (3) AL-Nano ＤＣＡ (200 mg / kg) administration group (4) EL-Nano ＤＣＡ (200 mg / kg) administration group (5) No drug administration + 5 Gy group (6) DCA (200 mg / kg) administration + 5 Gy group (7) AL-Nano ＤＣＡ (200 mg / kg) administration + 5 Gy group (8) EL-Nano ＤＣＡ (200 mg / kg) + 5 Gy group

[0059] The changes in tumor volume before and after each treatment are shown in Figure 9, and the results for DCA alone or in combination with X-rays are shown in Figure 10. ＤＣＡ The results related to the use of EL-Nano alone or in combination with X-rays are shown in Figure 10. ＤＣＡ The results related to DCA, AL-Nano alone or in combination with X-rays are shown in Figure 11. The arrows in these figures indicate the treatment days, and the p-values ​​after statistical significance tests on the 10th day from the start of the experiment are shown at the bottom of each graph. ＤＣＡ Regarding EL-Nano, no significant antitumor effect was observed either with the drug alone or in combination with X-rays. ＤＣＡ Regarding , a significant antitumor effect was observed in the combined treatment with X-rays, and a tendency to show a sensitizing effect was also observed in the treatment with each alone.

[0060] Furthermore, DCA, AL-Nano ＤＣＡ or EL-Nano ＤＣＡThe survival curves of tumor-bearing mice treated with X-rays are shown in Figure 12. ３ The survival curve using the time point when the survival rate exceeds 100% as an index also shows that EL-Nano ＤＣＡ A significant survival time was observed only in the combined use of X-rays. Here, the tumor endpoint is not the death of the mouse, but the time when the tumor volume reaches 1500 mm ３ Any point exceeding this threshold is considered to be death as a humane endpoint.

[0061] (b) In (a) above, the changes in the body weight of the mice were recorded over time as well as the tumor volume (for reference, please refer to the schedule of the animal experiment shown in Figure 13). The results of recording the changes in body weight due to the treatment with the drug alone in addition to the irradiation with X-rays alone are shown in Figure 14. The changes in body weight of the DCA and AL-Nano groups were compared with the changes in body weight of the drug-untreated group. ＤＣＡ , and EL-Nano ＤＣＡ No significant weight loss was observed in any of the animals administered, confirming the safety of the drug at a dose of 200 mg / kg.

[0062] Test Example 3: In vivo tumor tissue distribution after administration of nanoparticle DCA TAMRA-EL-Nano labeled with fluorescent dye TAMRA (carboxy tetramethylrhodamine) ＤＣＡ Using this, EL-Nano was injected into the tumor. ＤＣＡ The accumulation of TAMRA-EL-Nano was evaluated from the tail vein of SCCVII-implanted tumor mice. ＤＣＡ Tumor tissues were collected at random times thereafter (see Figure 15 for an outline of the animal experiment schedule). Cell nuclei in the tissue sections were stained with the fluorescent dye DAPI and observed under a fluorescence microscope. The observed images are shown in Figure 16. ＤＣＡ At 1 hour, 3 hours, 6 hours, and 12 hours after administration, TAMRA-EL-Nano was observed around the blood vessels indicated by the white arrows. ＤＣＡ An image of accumulation is observed.

[0063] Test Example 4: Effect of nanoparticle DCA and X-ray treatment on energy production EL-NanoＤＣＡ To investigate the mechanism by which EL-Nano exerts its radiosensitizing effect, its effect on cellular energy metabolism was investigated using ATP production as an indicator. ＤＣＡ The cells were treated for 1 hour before X-ray irradiation, and the amount of intracellular ATP was measured 24 hours later. (See FIG. 17 for an outline of the schedule of the experiment using the cells.) The results of the measurement are shown in FIG. 18. From FIG. 18, it can be seen that the amount of intracellular ATP was ＤＣＡ The tendency for this to decrease with treatment, and also with X-ray treatment and EL-Nano ＤＣＡ The combined treatment with EL-Nano and X-rays showed a significant decrease. ＤＣＡ The reduction was significant compared to when both drugs were used alone, suggesting that the combined use has a strong inhibitory effect on energy metabolism.

Claims

1. A hydrophilic-hydrophobic copolymer comprising a block containing a poly(ethylene glycol) segment and a block containing a polymer segment carrying dichloroacetic acid in the side chain, said copolymer being represented by formula (I) or formula (II-a) or (II-b). Formula (I): In the formula, A １ (i) unsubstituted or substituted C １ -C １２ The substituents when substituted are formyl, ａ R ｂ CH- (where R ａ and R ｂ is independent, C １ -C ４ Alkyloxy or R ａ and R ｂ Let's get together - OCH ２ CH ２ O-, -O(CH ２ ) ３ O- or -O(CH ２ ) ４ (ii) a group of the formula: L is a group represented by the formula: １ teeth, or (CH ２ ) ｃ S, CO (CH ２ ) ｃ S, (CH ２ ) ｃ NH, (CH ２ ) ｃ R is selected from the group consisting of CO, CO, OCOO, and CONH, where b is an integer from 2 to 6, and each c is independently an integer from 1 to 5; １ is a hydrogen atom, C １ -C ６ Alkylcarbonyl or C(=O)CHCl ２ Wherein each R １ may be different, but 20% or more of m1 are C(=O)CHCl ２ Y is a hydrogen atom, SH, S(C=S)-Ph, S(=S)OCH ２ CH ３ , hydroxyl group, C １ -C ６ Alkyloxy group or aryl-C １ -C ２ m1 is an integer from 5 to 1000, and n1 is an integer from 5 to 500. Formula (II-a): In the formula, A ２ is A of formula (I). １ is the same as defined for ２ａ (CH ２ ) ａ -NH or (CH ２ ) ａ —O, where each a is independently an integer from 1 to 6; ２ is a hydrogen atom, benzyloxycarbonyl (Cbz), t-butoxycarbonyl, C １ -C ６ Alkylcarbonyl or C(=O)CHCl ２ Wherein each R ２ may be different, but 20% or more of m2a is C(=O)CHCl ２ Za is H, C １ -C ２１ Alkylcarbonyl, substituted C １ -C ４ Alkylcarbonyl, unsubstituted or substituted C ３ -C ７ represents a cycloalkylcarbonyl, an unsubstituted or substituted arylcarbonyl, or an unsubstituted or substituted 5- or 6-membered heteroarylcarbonyl, wherein the substituents, when substituted, are one or more C １ -C ４ m2a may be an alkyloxy, phenyl, or fluorine atom, m2a is an integer of 5 to 500, n2a is an integer of 5 to 500, and q is an integer of 1 or 2. Formula (II-b): In the formula, A ２ is A of formula (I). １ is the same as defined for ２ｂ (CH ２ ) ｂ represents -C(=O); ｂ is an integer from 1 to 6; R ２ is a hydrogen atom, benzyloxycarbonyl (Cbz), t-butoxycarbonyl, C １ -C ６ Alkylcarbonyl or C(=O)CHCl ２ Wherein each R ２ may be different, but 20% or more of m2b is C(=O)CHCl ２ Zb is OH, unsubstituted or substituted C １ -C ２１ Alkyloxy, unsubstituted or substituted C ３ -C ７ cycloalkyloxy, unsubstituted or substituted aryloxy, or unsubstituted or substituted 5- or 6-membered heteroaryloxy, where the substituents, if substituted, are one or more C １ -C ４ m2b may be an alkyloxy, phenyl, or fluorine atom; m2b is an integer of 5 to 500; n2b is an integer of 5 to 500; and q is an integer of 1 or 2.

2. The copolymer of claim 1, wherein the copolymer is represented by formula (I).

3. The copolymer according to claim 1, which is represented by formula (II-a) or formula (II-b).

4. Nano-sized polymeric micelles formed in an aqueous medium from the copolymer according to claim 1.

5. A tumor-specific radiosensitizer comprising as an active ingredient the copolymer according to claim 1 or nano-sized polymeric micelles formed from said copolymer in an aqueous medium.

6. A pharmaceutical preparation for enhancing therapeutic effects in tumor radiation therapy by combination use, comprising as an active ingredient the copolymer described in claim 1 or nano-sized polymeric micelles formed from said copolymer in an aqueous medium.

7. A copolymer according to claim 1 or a polymeric micelle according to claim 4 for use as a tumor-specific radiosensitizer.

8. A copolymer according to claim 1 or a polymeric micelle according to claim 4 for use in a pharmaceutical preparation for enhancing therapeutic effects by combined use in tumor radiation therapy.

9. A method for enhancing the efficacy of tumor treatment, comprising administering to a patient or individual scheduled to undergo or currently undergoing tumor radiation therapy an effective amount of the copolymer described in claim 1 or nano-sized polymeric micelles formed from said copolymer in an aqueous medium.

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